Substrate Preference Research Articles

Fermentability assessment of selected oligosaccharides by Lactiplantibacillus plantarum BIOTECH 1074 towards short-chain organic acid production with anti-vibrio efficacy

Vibriosis, caused by Vibrio harveyi and Vibrio parahaemolyticus, poses a significant threat to aquaculture, resulting in substantial economic losses. Oligosaccharides are known for their ability to improve gut health and prevent vibriosis in aquaculture species. This study explored the fermentative utilization of galacto-oligosaccharides (GOS), manno-oligosaccharides (MOS), and inulin by Lactiplantibacillus plantarum BIOTECH 1074. GOS and glucose were the preferred substrates for L. plantarum growth, producing high levels of lactic acid, propanoic acid, and acetic acid. These compounds showed substantial anti-Vibrio activities, having inhibitory concentration values of 0.252 ± 0.005 and 0.656 ± 0.015 mg total organic acid/mL against V. harveyi, V. cholerae, and V. parahaemolyticus from GOS fermented supernatant. The fermentative capabilities of L. plantarum BIOTECH 1074, in utilizing specific oligosaccharide substrates to produce organic acids with anti-Vibrio potential, offers a promising strategy for enhancing disease management and overall efficiency in aquaculture systems.

Intramuscular administration of fractalkine modulates mitochondrial properties and promotes fast glycolytic phenotype.

A newly categorized myokine called fractalkine (CX3CL1) has been associated with divergent conditions such as obesity, tissue inflammation, and exercise. CX3CL1 works through specific membrane-bound receptors (CX3CR1) found in various tissues including skeletal muscles. Studies indicate CX3CL1 induces muscles to uptake energy substrates thereby improving glucose utilization and countering diabetes. Here, we tested if the administration of purified CX3CL1 directly into mice skeletal muscles affects its histoarchitecture, mitochondrial activity, and expression of metabolic proteins. We analyzed four muscles: two upper-limb (quadriceps, hamstrings) and two lower-limb (tibialis anterior, gastrocnemius), contralateral leg muscles were taken as controls. The effects of CX3CL1 treatment on histoarchitecture, mitochondrial activity, and expression of metabolic proteins in muscles were characterized. We used histochemical staining succinate dehydrogenase (SDH)/cytochrome c oxidase (COX), myosin ATPase, alkaline phosphatase (ALP) to evaluate the mitochondrial activity, fiber types, and vascularization in the muscles, respectively. Western blotting was used to evaluate the expression of proteins associated with mitochondrial metabolism (OXPHOS), glycolysis, and vascularization. Overall, this study indicates CX3CL1 primarily modulates mitochondrial metabolism and shifts substrate preference toward glucose in the skeletal muscle. Evidence also supports that CX3CL1 stimulates the relative composition of fast fiber types, influencing selection of energy substrates in the skeletal muscle.

Structure-based reshaping of a new ketoreductase from Sphingobacterium siyangense SY1 toward α-haloacetophenones

Ketoreductases play an indispensable role in the asymmetric synthesis of chiral drug intermediates, and an in-depth understanding of their substrate selectivity can improve the efficiency of enzyme engineering. In this endeavor, a new short-chain dehydrogenase/reductase (SDR) SsSDR1 identified from Sphingobacterium siyangense SY1 by gene mining method was successfully cloned and functionally expressed in Escherichia coli. Its activity against halogenated acetophenones has been tested and the results illustrated that SsSDR1-WT exhibits high activity for 3,5-bis(trifluoromethyl)acetophenone (1f), an important precursor in the synthesis of aprepitant. In addition, SsSDR1-WT showed obvious substrate preference for acetophenones without α-halogen substitution compared to their α-halogen analogs. To explore the structural basis of substrate selectivity, the X-ray crystal structures of SsSDR1-WT in its apo form and the complex structure with NAD were resolved. Taking 2-chloro-1-(3, 4-difluorophenyl) ethanone (1i) as the representative α-haloacetophenone, the key sites affecting substrate selectivity of SsSDR1-WT were identified and through the rational remodeling of the cavities C1 and C2 of SsSDR1, an excellent mutant I144A/S153L with significantly improved activity against α-halogenated acetophenones was obtained. The asymmetric catalysis of 1f and 1i was performed at the scale of 50 mL, and the space-time yields (STY) of the two were 1200 and 6000 g/L∙d, respectively. This study not only provides valuable biocatalysts for halogenated acetophenones, but also yields insights into the relationship between the substrate-binding pocket and substrate selectivity.

Functional knockout of the Oatp1d1 membrane transporter affects toxicity of diclofenac in zebrafish embryos

Organic anion transporting polypeptides (OATPs) facilitate the cellular uptake of a large number of compounds. Zebrafish Oatp1d1 matches the functional capabilities of human OATP orthologs, particularly in hormone and drug transport. It is highly expressed in the liver and later stages of embryonic development, indicating its critical role in zebrafish physiology and development. Data from previous in vitro analyses have shown a high affinity of zebrafish Oatp1d1 for pharmaceuticals and xenobiotics, providing the basis for further in vivo studies on its defence and developmental functions. Using CRISPR-Cas9 technology, we have generated an Oatp1d1 zebrafish mutant that has highly reduced Oatp1d1 expression in embryos and adult tissues compared to wild type (WT). The absence of Oatp1d1 was confirmed using custom-made antibodies. To evaluate its ecotoxicological relevance, mutant and WT embryos were exposed to increasing concentrations of diclofenac, an NSAID known for its wide and frequent use, environmental pseudo-persistence and ecological implications. WT embryos showed developmental delays and malformations such as spinal curvature, cardiac edema and blood pooling at higher diclofenac concentrations, whereas the Oatp1d1 mutant embryos showed marked resilience, with milder developmental defects and delayed toxic effects. These observations suggest that the absence of Oatp1d1 impedes the efficient entry of diclofenac into hepatocytes, thereby slowing its biotransformation into potentially more toxic metabolites. In addition, the changes in transcript expression of other uptake transporters revealed a highly probable and complex network of compensatory mechanisms. Therefore, the results of this study point to the importance of Oatp1d1-mediated transport of diclofenac, as demonstrated for the first time in vivo using an Oatp1 deficient zebrafish line. Finally, our data indicates that the compensatory role of other transporters with overlapping substrate preferences needs to be considered for a reliable understanding of the physiological and/or defensive role(s) of membrane transporters.

A comparative study of the substrate preference of the sialidases, CpNanI, HpNanH, and BbSia2 towards 2-Aminobenzamide-labeled 3′-Sialyllactose, 6′-Sialyllactose, and Sialyllacto-N-tetraose-b

Sialidases catalyze the removal of terminal sialic acids from sialylated biomolecules, and their substrate preference is frequently indicated in terms of the glycosidic linkages cleaved (α2-3, α2-6, and α2-8) without mention of the remaining sub-terminal reducing-end saccharide moieties. Many human gut commensal and pathogenic bacteria secrete sialidases to forage for sialic acids, which are then utilized as an energy source or assimilated into membrane/capsular structural components. Infant gut commensals similarly utilize sialylated human milk oligosaccharides containing different glycosidic linkages. Here, we have studied the preference of the bacterial sialidases, BbSia2 from Bifidobacterium bifidum, CpNanI from Clostridium perfringens, and HpNanH from Glaesserella parasuis, for the glycosidic linkages, Siaα2-3Gal, Siaα2-6Gal, and Siaα2-6GlcNAc, by employing 2-Aminobenzamide-labeled human milk oligosaccharides, 3′-Sialyllactose (3′-SL), 6′-Sialyllactose (6′-SL), and Sialyllacto-N-tetraose-b (LSTb), respectively, as proxies for these glycosidic linkages. BbSia2, CpNanI, and HpNanH hydrolyzed these three oligosaccharides with the glycosidic linkage preferences, 3′-SL (Siaα2-3Gal) ≥ LSTb (Siaα2-6GlcNAc) ≥ 6′-SL (Siaα2-6Gal), 3′-SL (Siaα2-3Gal) ≥ 6′-SL (Siaα2-6Gal) > LSTb (Siaα2-6GlcNAc), and 3′-SL (Siaα2-3Gal) ≥ 6′-SL (Siaα2-6Gal) > LSTb (Siaα2-6GlcNAc), respectively. Our finding suggests that sub-terminal reducing-end saccharide moieties can profoundly influence the substrate preference of sialidases, and advocates for the characterization and indication of the substrate preference of sialidases in terms of both the glycosidic linkage and the sub-terminal reducing-end saccharide moiety.

Substrate Characterization for Hydrolysis of Multiple Types of Aromatic Esters by Promiscuous Aminopeptidases.

Synthetic aromatic esters, widely employed in agriculture, food, and chemical industries, have become emerging environmental pollutants due to their strong hydrophobicity and poor bioavailability. This study attempted to address this issue by extracellularly expressing the promiscuous aminopeptidase (Aps) from Pseudomonas aeruginosa GF31 in B. subtilis, achieving an impressive enzyme activity of 13.7 U/mg. Notably, we have demonstrated, for the first time, the Aps-mediated degradation of diverse aromatic esters, including but not limited to pyrethroids, phthalates, and parabens. A biochemical characterization of Aps reveals its esterase properties and a broader spectrum of substrate profiles. The degradation rates of p-nitrobenzene esters (p-NB) with different side chain structures vary under the action of Aps, showing a preference for substrates with relatively longer alkyl side chains. The structure-dependent degradability aligns well with the binding energies between Aps and p-NB. Molecular docking and enzyme-substrate interaction elucidate that hydrogen bonding, hydrophobic interactions, and π-π stacking collectively stabilize the enzyme-substrate conformation, promoting substrate hydrolysis. These findings provide new insights into the enzymatic degradation of aromatic ester pollutants, laying a foundation for the further development and modification of promiscuous enzymes.

Shifting Mycobacterial Serine Hydrolase Activity Visualized Using Multi-Layer In-Gel Activity Assays.

The ability of Mycobacterium tuberculosis to derive lipids from the host, store them intracellularly, and then break them down into energy requires a battery of serine hydrolases. Serine hydrolases are a large, diverse enzyme family with functional roles in dormant, active, and reactivating mycobacterial cultures. To rapidly measure substrate-dependent shifts in mycobacterial serine hydrolase activity, we combined a robust mycobacterial growth system of nitrogen limitation and variable carbon availability with nimble in-gel fluorogenic enzyme measurements. Using this methodology, we rapidly analyzed a range of ester substrates, identified multiple hydrolases concurrently, observed functional enzyme shifts, and measured global substrate preferences. Within every growth condition, mycobacterial hydrolases displayed the full, dynamic range of upregulated, downregulated, and constitutively active hydrolases independent of the ester substrate. Increasing the alkyl chain length of the ester substrate also allowed visualization of distinct hydrolase activity likely corresponding with lipases most responsible for lipid breakdown. The most robust expression of hydrolase activity was observed under the highest stress growth conditions, reflecting the induction of multiple metabolic pathways scavenging for energy to survive under this high stress. The unique hydrolases present under these high-stress conditions could represent novel drug targets for combination treatment with current front-line therapeutics. Combining diverse fluorogenic esters with in-gel activity measurements provides a rapid, customizable, and sensitive detection method for mycobacterial serine hydrolase activity.

Heterologous expression, characterization, and application of recombinant thermostable α-amylase from Geobacillus sp. DS3 for porous starch production

Novel Geobacillus sp. DS3, isolated from the Sikidang Crater in Dieng, exhibits promising characteristics for industrial applications, particularly in thermostable α-amylase production. Recombinant technology was used to express thermostable α-amylase in E. coli BL21(DE3) to overcome high-temperature production challenges. The study aimed to express, purify, characterize, and explore potential applications of this novel enzyme. The enzyme was successfully expressed in E. coli BL21(DE3) at 18 °C for 20 h with 0.5 mM IPTG induction. Purification with Ni-NTA column yielded 69.23 % from the initial crude enzyme, with a 3.6-fold increase in specific activity. The enzyme has a molecular weight of ±70 kDa (±58 kDa enzyme+11 kDa SUMO protein). It exhibited activity over a wide temperature range (30–90 °C) and pH range (6–8), with optimal activity at 70 °C and pH 6 with great stability at 60 °C. Kinetic analysis revealed Km and Vmax values of 324.03 mg/ml and 36.5 U/mg, respectively, with dextrin as the preferred substrate without cofactor addition. As a metalloenzyme, it showed the best activity in the presence of Ca2+. The enzyme was used for porous starch production and successfully immobilized with chitosan, exhibiting improved thermal stability. After the fourth reuse, the immobilized enzyme maintained 62 % activity compared to the initial immobilization.

Crystallographic fragment-binding studies of the Mycobacterium tuberculosis trifunctional enzyme suggest binding pockets for the tails of the acyl-CoA substrates at its active sites and a potential substrate-channeling path between them.

The Mycobacterium tuberculosis trifunctional enzyme (MtTFE) is an α2β2 tetrameric enzyme in which the α-chain harbors the 2E-enoyl-CoA hydratase (ECH) and 3S-hydroxyacyl-CoA dehydrogenase (HAD) active sites, and the β-chain provides the 3-ketoacyl-CoA thiolase (KAT) active site. Linear, medium-chain and long-chain 2E-enoyl-CoA molecules are the preferred substrates of MtTFE. Previous crystallographic binding and modeling studies identified binding sites for the acyl-CoA substrates at the three active sites, as well as the NAD binding pocket at the HAD active site. These studies also identified three additional CoA binding sites on the surface of MtTFE that are different from the active sites. It has been proposed that one of these additional sites could be of functional relevance for the substrate channeling (by surface crawling) of reaction intermediates between the three active sites. Here, 226 fragments were screened in a crystallographic fragment-binding study of MtTFE crystals, resulting in the structures of 16 MtTFE-fragment complexes. Analysis of the 121 fragment-binding events shows that the ECH active site is the `binding hotspot' for the tested fragments, with 41 binding events. The mode of binding of the fragments bound at the active sites provides additional insight into how the long-chain acyl moiety of the substrates can be accommodated at their proposed binding pockets. In addition, the 20 fragment-binding events between the active sites identify potential transient binding sites of reaction intermediates relevant to the possible channeling of substrates between these active sites. These results provide a basis for further studies to understand the functional relevance of thelatter binding sites and to identify substrates for which channeling is crucial.

Diversity and seasonal variability of Heterobranchs in Tulamben waters, Bali, Indonesia

This study examines the ecological traits of heterobranchs, which are found in various habitats associated with reef regions. We use data on heterobranchs abundance, depth, substrates, and seasonal observations to analyze their ecological traits, as these factors are often under studied. The species diversity of heterobranch species found in Tulamben, Bali is exceptionally rich. From a total of 41 surveys carried out at depths between 0 and 25 m every week in a one-year period, more than 144 species of heterobranchs belonging to 63 genera were recorded. A total of 1001 individuals were recorded during our survey efforts with 645 individuals in the dry season and 356 during the wet season. Hypselodoris roo and Mexichromis trilineata are the most abundant species of heterobranch recorded in this study. Despite these numbers, no significant difference was found between the abundance of heterobranchs between the dry and wet seasons; however, depth was a significant factor in determining heterobranchs abundance and species richness. The highest abundance was recorded for zone C (between 15 and 20 m depth) where 437 individual heterobranchs were recorded. The lowest abundance of heterobranchs was recorded for zone A (5–9 m depth) where only 72 individuals were recorded. Substrate preference was also observed among the individuals in which the highest abundance of heterobranchs were found on sponges in both dry (47.9 %) and wet (41 %) seasons. The second most dominant substrate was recorded as sand, accounting for 25 % relative abundance in both dry and wet seasons. To learn more about the seasonality of heterobranchs, further research considering tides, temperature, and other environmental factors needs to be considered.

Optimizing luminous efficiency in polymer light-emitting diodes through anode oxygen-plasma treatment under diverse pressure conditions

This study investigates the impact of oxygen-plasma (O2-plasma) treatment on the microscale electrical properties of indium tin oxide (ITO) surfaces for enhancement of the luminous efficiency of polymer light-emitting diodes (PLEDs). Significant effects on conductivity distribution were observed when treatment was performed at pressures exceeding 0.4 Torr. ITO-6, the ITO treated with O2-plasma at 0.6 Torr, emerged as the preferred anode substrate for PLEDs, displaying a 72.5 % coverage of stable-conductive regions. When utilized as the anode in PLED, it exhibited a remarkable 209 % increase in average current efficiency and a 184 % enhancement in electroluminescent intensity compared to the reference PLED. These findings provide valuable insights for optimizing PLEDs through surface modification of ITO.

Sirtuin insights: bridging the gap between cellular processes and therapeutic applications.

The greatest challenges that organisms face today are effective responses or detection of life-threatening environmental changes due to an obvious semblance of stress and metabolic fluctuations. These are associated with different pathological conditions among which cancer is most important. Sirtuins (SIRTs; NAD+-dependent enzymes) are versatile enzymes with diverse substrate preferences, cellular locations, crucial for cellular processes and pathological conditions. This article describes in detail the distinct roles of SIRT isoforms, unveiling their potential as either cancer promoters or suppressors and also explores how both natural and synthetic compounds influence the SIRT function, indicating promise for therapeutic applications. We also discussed the inhibitors/activators tailored to specific SIRTs, holding potential for diseases lacking effective treatments. It may uncover the lesser-studied SIRT isoforms (e.g., SIRT6, SIRT7) and their unique functions. This article also offers a comprehensive overview of SIRTs, linking them to a spectrum of diseases and highlighting their potential for targeted therapies, combination approaches, disease management, and personalized medicine. We aim to contribute to a transformative era in healthcare and innovative treatments by unraveling the intricate functions of SIRTs.

Integrative genomic reconstruction of carbohydrate utilization networks in bifidobacteria: global trends, local variability, and dietary adaptation.

Bifidobacteria are among the earliest colonizers of the human gut, conferring numerous health benefits. While multiple Bifidobacterium strains are used as probiotics, accumulating evidence suggests that the individual responses to probiotic supplementation may vary, likely due to a variety of factors, including strain type(s), gut community composition, dietary habits of the consumer, and other health/lifestyle conditions. Given the saccharolytic nature of bifidobacteria, the carbohydrate composition of the diet is one of the primary factors dictating the colonization efficiency of Bifidobacterium strains. Therefore, a comprehensive understanding of bifidobacterial glycan metabolism at the strain level is necessary to rationally design probiotic or synbiotic formulations that combine bacterial strains with glycans that match their nutrient preferences. In this study, we systematically reconstructed 66 pathways involved in the utilization of mono-, di-, oligo-, and polysaccharides by analyzing the representation of 565 curated metabolic functional roles (catabolic enzymes, transporters, transcriptional regulators) in 2973 non-redundant cultured Bifidobacterium isolates and metagenome-assembled genomes (MAGs). Our analysis uncovered substantial heterogeneity in the predicted glycan utilization capabilities at the species and strain level and revealed the presence of a yet undescribed phenotypically distinct subspecies-level clade within the Bifidobacterium longum species. We also identified Bangladeshi isolates harboring unique gene clusters tentatively implicated in the breakdown of xyloglucan and human milk oligosaccharides. Predicted carbohydrate utilization phenotypes were experimentally characterized and validated. Our large-scale genomic analysis considerably expands the knowledge of carbohydrate metabolism in bifidobacteria and provides a foundation for rationally designing single- or multi-strain probiotic formulations of a given bifidobacterial species as well as synbiotic combinations of bifidobacterial strains matched with their preferred carbohydrate substrates.

Fatty acid preference for beta-oxidation in mitochondria of murine cultured astrocytes.

The brain utilizes glucose as a primary energy substrate but also fatty acids for the β-oxidation in mitochondria. The β-oxidation is reported to occur mainly in astrocytes, but its capacity and efficacy against different fatty acids remain unknown. Here, we show the fatty acid preference for the β-oxidation in mitochondria of murine cultured astrocytes. Fatty acid oxidation assay using an extracellular flux analyzer showed that saturated or monosaturated fatty acids, palmitic acid and oleic acid, are preferred substrates over polyunsaturated fatty acids like arachidonic acid and docosahexaenoic acid. We also report that fatty acid binding proteins expressed in the astrocytes contribute less to fatty acid transport to mitochondria for β-oxidation. Our results could give insight into understanding energy metabolism through fatty acid consumption in the brain.

Structural and mechanistic analysis of Ca2+-dependent regulation of transglutaminase 2 activity using a Ca2+-bound intermediate state

Mammalian transglutaminases, a family of Ca2+-dependent proteins, are implicated in a variety of diseases. For example, celiac disease (CeD) is an autoimmune disorder whose pathogenesis requires transglutaminase 2 (TG2) to deamidate select glutamine residues in diet-derived gluten peptides. Deamidation involves the formation of transient γ-glutamyl thioester intermediates. Recent studies have revealed that in addition to the deamidated gluten peptides themselves, their corresponding thioester intermediates are also pathogenically relevant. A mechanistic understanding of this relevance is hindered by the absence of any structure of Ca2+-bound TG2. We report the X-ray crystallographic structure of human TG2 bound to an inhibitory gluten peptidomimetic and two Ca2+ ions in sites previously designated as S1 and S3. Together with additional structure-guided experiments, this structure provides a mechanistic explanation for how S1 regulates formation of an inhibitory disulfide bond in TG2, while also establishing that S3 is essential for γ-glutamyl thioester formation. Furthermore, our crystallographic findings and associated analyses have revealed that i) two interacting residues, H305 and E363, play a critical role in resolving the thioester intermediate into an isopeptide bond (transamidation) but not in thioester hydrolysis (deamidation); and ii) residues N333 and K176 stabilize preferred TG2 substrates and inhibitors via hydrogen bonding to nonreactive backbone atoms. Overall, the intermediate-state conformer of TG2 reported here represents a superior model to previously characterized conformers for both transition states of the TG2-catalyzed reaction.

Coupling the Isopentenol Utilization Pathway and Prenyltransferase for the Biosynthesis of Licoflavanone in Recombinant Escherichia coli.

Prenylflavonoids are promising candidates for food additives and functional foods due to their diverse biological activities and potential health benefits. However, natural prenylflavonoids are generally present in low abundance and are limited to specific plant species. Here, we report the biosynthesis of licoflavanone from naringenin and prenol by recombinant Escherichia coli. By investigating the activities of seven different sources of prenyltransferases overexpressed in E. coli toward various flavonoid substrates, the prenyltransferase AnaPT exhibits substrate preference when naringenin serves as the prenyl acceptor. Furthermore, licoflavanone production was successfully achieved by coupling the isopentenol utilization pathway and AnaPT in recombinant E. coli. In addition, the effects of fermentation temperatures, induction temperatures, naringenin concentrations, and substrate feeding strategies were investigated on the biosynthesis of licoflavanone in recombinant E. coli. Consequently, the recombinant E. coli strain capable of improved dimethylallyl diphosphate (DMAPP) supply and suitable for prenylflavonoid biosynthesis increased licoflavanone titers to 142.1 mg/L in a shake flask and to 537.8 mg/L in a 1.3 L fermentor, which is the highest yield for any prenylflavonoids reported to date. These strategies proposed in this study provide a reference for initiating the production of high-value prenylflavonoids.

DNA-encoded probe-based assay for profiling plant kinase activities.

Elucidating kinase-substrate relationships is pivotal for deciphering cellular signaling mechanisms, yet it remains challenging due to the complexity of kinase networks. Herein, we report the development of a versatile DNA-based kinase assay platform for high-throughput profiling of plant protein kinase activities and substrate preferences. Our approach employs DNA-linked peptide substrates, facilitating quantitative and specific kinase activity detection through next-generation DNA sequencing. Leveraging DNA barcodes as quantitative readouts, our approach establishes a high-throughput, sensitive, and specific platform for dissecting kinase-substrate networks in plants, representing a powerful tool for elucidating signaling mechanisms in plants.

Effects of substrate availability and mitochondrial disruption on oxidative metabolism and sperm motility in fertile dogs.

In several mammalian species, the measurement of mitochondrial oxygen consumption (MITOX) under different metabolic conditions has demonstrated a positive correlation with sperm motility and may be a sensitive indicator of mitochondrial health. In general, the maintenance of sperm motility and many key sperm functions and fertilizing events are heavily energy-dependent processes, and some species-specific substrate preferences exist. Although canine sperm have been known to undergo capacitation and maintain motility with supplementation of a wide range of energy substrates, the relationship between mitochondrial function, and the maintenance of oxidative metabolism and sperm motility remain unclear. The objective of this study was to explore the metabolic flexibility of canine sperm, and to investigate the relationship between mitochondrial function, and maintenance of motility under differing nutrient conditions. We explored substrate preferences and the bioenergetics underlying maintenance of canine sperm motility by monitoring mitochondrial oxidative function and sperm kinematics in the presence of mitochondrial effector drug treatments: FCCP, antimycin (ANTI), and oligomycin (OLIGO). We hypothesized that canine sperm possess the ability to use compensatory pathways and utilize diverse nutrient sources in the maintenance of motility. Oxygen consumption (change in pO2, oxygen partial pressure) and sperm kinematics (CASA) were measured concurrently (t0-t30) to assess the relationship between oxidative metabolism and maintenance of sperm motility in dogs. Four media were tested: containing glucose, lactate, and pyruvate (GLP), containing glucose (G), fructose (F), or lactate and pyruvate (LP). In the absence of pharmacological inhibition of the electron transport chain, energetic substrate had no effect on sperm kinematics in fertile dogs. Following mitochondrial disruption by ANTI and OLIGO, mitochondrial oxygen consumption was negatively correlated with several sperm motility parameters in GLP, G, F, and LP media. In every media, FCCP treatment quickly induced significantly higher oxygen consumption than in untreated sperm, and spare respiratory capacity, the maximal inducible oxidative metabolism, was high. With respiratory control ratios RCR >1 there was no indication of bioenergetic dysfunction in any media type, indicating that sperm mitochondria of fertile dogs have a high capacity for substrate oxidation and ATP turnover regardless of substrate. Our results suggest MITOX assessment is a valuable tool for assessing mitochondrial functionality, and that canine sperm employ flexible energy management systems which may be exploited to improve sperm handling and storage.

1‐Haloalkynes: Privileged Substrates for Gold Catalysis

AbstractAlkynes have been the preferred substrates since the early development of gold catalysis at the dawn of this century. Amongst them, 1‐haloalkynes have gained increased interest in recent years due to the versatility offered by these bifunctional entities as well as the identification of unique reaction modes in this already mature research field. Herein, the seminal contributions that resulted in the observed growth in recent years and the most recent developments will be discussed.

The specificity of endogenous fatty acid nitration: only conjugated substrates support the in vivo formation of nitro-fatty acids

Through multiple pathways, nitrogen dioxide (•NO2) is the main species involved in endogenous nitration reactions. Early studies in the field primarily explored tyrosine nitration, a dominant reaction in the field. It was later shown that lipids are also nitration targets and generate an array of reaction products. Conjugated fatty acids are the preferential substrates of lipid nitration in vivo, generating electrophilic nitro-fatty acids (NO2–FAs), which serve as pleiotropic signaling modulators. In contrast, exposure of bisallylic fatty acids, including linoleic, linolenic and arachidonic acid, to •NO2 does not lead, under biological conditions, to the formation of nitrated species. This review focuses on the reaction mechanisms and products of lipid nitration and substrate specificity, focusing on the differential reactivity of conjugated dienes and bisallylic alkenes.