Substrate Preference Research Articles

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.

Discovery, characterization, and comparative analysis of new UGT72 and UGT84 family glycosyltransferases

Glycosylated derivatives of natural product polyphenols display a spectrum of biological activities, rendering them critical for both nutritional and pharmacological applications. Their enzymatic synthesis by glycosyltransferases is frequently constrained by the limited repertoire of characterized enzyme-catalyzed transformations. Here, we explore the glycosylation capabilities and substrate preferences of newly identified plant uridine diphosphate (UDP)-dependent glycosyltransferases (UGTs) within the UGT72 and UGT84 families, with particular focus on natural polyphenol glycosylation from UDP-glucose. Four UGTs are classified according to their phylogenetic relationships and reaction products, identifying them as biocatalysts for either glucoside (UGT72 enzymes) or glucose ester (UGT84 members) formation from selected phenylpropanoid compounds. Detailed kinetic evaluations expose the unique attributes of these enzymes, including their specific activities and regio-selectivities towards diverse polyphenolic substrates, with product characterizations validating the capacity of UGT84 family members to perform di-O-glycosylation on flavones. Sequence analysis coupled with structural predictions through AlphaFold reveal an unexpected absence of a conserved threonine residue across all four enzymes, a trait previously linked to pentosyltransferases. This comparative analysis broadens the understood substrate specificity range for UGT72 and UGT84 enzymes, enhancing our understanding of their utility in the production of natural phenolic glycosides. The findings from this in-depth characterization provide valuable insights into the functional versatility of UGT-mediated reactions.

In-depth mapping of DNA-PKcs signaling uncovers noncanonical features of its kinase specificity

DNA-PKcs is a DNA damage sensor kinase with established roles in DNA double-strand break repair via nonhomologous end joining. Recent studies have revealed additional roles of DNA-PKcs in the regulation of transcription, translation, and DNA replication. However, the substrates through which DNA-PKcs regulates these processes remain largely undefined. Here, we utilized quantitative phosphoproteomics to generate a high coverage map of DNA-PKcs signaling in response to ionizing radiation and mapped its interplay with the ATM kinase. Beyond the detection of the canonical S/T-Q phosphorylation motif, we uncovered a noncanonical mode of DNA-PKcs signaling targeting S/T-ψ-D/E motifs. Sequence and structural analyses of the DNA-PKcs substrate recognition pocket revealed unique features compared to closely related phosphatidylinositol 3-kinase-related kinases that may explain its broader substrate preference. These findings expand the repertoire of DNA-PKcs and ATM substrates while establishing a novel preferential phosphorylation motif for DNA-PKcs.

High-throughput screening carbon and nitrogen sources to promote growth and sporulation in Rhizopus arrhizus

Rhizopus arrhizus is a saprotrophic, sometimes clinically- and industrially-relevant mold (Mucorales) and distributed worldwide, suggesting it can assimilate a broad spectrum of substrates. Here, 69 strains of R. arrhizus were investigated by using the Biolog FF MicroPlate for the profiles of utilizing 95 carbon and nitrogen substrates. The study showed that most R. arrhizus strains were similar in average well color development (AWCD) and substrate richness (SR). Nevertheless, 13 strains were unique in principal component analyses, heatmap, AWCD, and SR analyses, which may imply a niche differentiation within R. arrhizus. The species R. arrhizus was able to utilize all the 95 carbon and nitrogen substrates, consistent with the hypothesis of a great metabolic diversity. It possessed a substrate preference of alcohols, and seven substrates were most frequently utilized, with N-acetyl-d-galactosamine and l-phenylalanine ranking at the top of the list. Eight substrates, especially l-arabinose and xylitol, were capable of promoting sporulation and being applied for rejuvenating degenerated strains. By phenotyping R. arrhizus strains in carbon and nitrogen assimilation capacity, this study revealed the extent of intra-specific variability and laid a foundation for estimating optimum substrates that may be useful for industrial applications.

Dietary protein from different sources escapes host digestion and is differentially modified by the microbiota.

Protein is an essential macronutrient and variations in its source and quantity have been shown to impact long-term health outcomes. Differential health impacts of dietary proteins from various sources are likely driven by differences in their digestibility by the host and subsequent availability to the intestinal microbiota. However, our current understanding regarding the fate of dietary proteins from different sources in the gut, specifically how component proteins within these sources interact with the host and the gut microbiota, is limited. To determine which dietary proteins are efficiently digested by the host and which proteins escape host digestion and are used by the gut microbiota, we used high-resolution mass spectrometry to quantify the proteins that make up different dietary protein sources before and after digestion in germ-free and conventionally raised mice. Contrary to expectation, we detected proteins from all sources in fecal samples of both germ-free and conventional mice suggesting that even protein sources with a high digestive efficiency make it in part to the colon where they can serve as a substrate for the microbiota. Additionally, we found clear patterns where specific component proteins of the dietary protein sources were used as a preferred substrate by the microbiota or were not as accessible to the microbiota. We found that specific proteins with functions that could impact host health and physiology were differentially enriched in germ-free or conventionally raised mice. These findings reveal large differences in the fate of dietary protein from various sources in the gut that could explain some of their differential health impacts.

Shy is a proteobacterial steroid hydratase which catalyzes steroid side chain degradation without requiring a catalytically inert partner domain

Shy (side chain hydratase) and Sal (side chain aldolase), are involved in successive reactions in the pathway of bile acid side chain catabolism in Proteobacteria. Untagged Shy co-purified with His-tagged Sal indicating that the two enzymes form a complex. Shy contains a MaoC and a DUF35 domain. When co-expressed with Sal, the DUF35 domain but not the MaoC domain of Shy was observed to co-purify with Sal, indicating Sal interacts with Shy through its DUF35 domain. The MaoC domain of Shy (ShyMaoC) remained catalytically viable and could hydrate cholyl-enoyl-CoA with similar catalytic efficiency as in the Shy-Sal complex. Sal expressed with the DUF35 domain of Shy (Sal-ShyDUF35) was similarly competent for the retroaldol cleavage of cholyl-3-OH-CoA. ShyMaoC showed a preference for C5 side chain bile acid substrates, exhibiting low activity towards C3 side chain substrates. The ShyMaoC structure was determined by X-ray crystallography, showing a hot dog fold with a short central helix surrounded by a twisted anti-parallel β-sheet. Modeling and mutagenesis studies suggest that the bile acid substrate occupies the large open cleft formed by the truncated central helix and repositioning of the active site housing. ShyMaoC therefore contains two substrate binding sites per homodimer, making it distinct from previously characterized MaoC steroid hydratases that are (pseudo)-heterodimers with one substrate binding site per dimer. The characterization of Shy provides insight into how MaoC family hydratases have adapted to accommodate large polycyclic substrates that can facilitate future engineering of these enzymes to produce novel steroid pharmaceuticals.

Structure-Function Insights into the Dual Role in Nucleobase and Nicotinamide Metabolism and a Possible Use in Cancer Gene Therapy of the URH1p Riboside Hydrolase.

The URH1p enzyme from the yeast Saccharomyces cerevisiae has gained significant interest due to its role in nitrogenous base metabolism, particularly involving uracil and nicotinamide salvage. Indeed, URH1p was initially classified as a nucleoside hydrolase (NH) with a pronounced preference for uridine substrate but was later shown to also participate in a Preiss-Handler-dependent pathway for recycling of both endogenous and exogenous nicotinamide riboside (NR) towards NAD+ synthesis. Here, we present the detailed enzymatic and structural characterisation of the yeast URH1p enzyme, a member of the group I NH family of enzymes. We show that the URH1p has similar catalytic efficiencies for hydrolysis of NR and uridine, advocating a dual role of the enzyme in both NAD+ synthesis and nucleobase salvage. We demonstrate that URH1p has a monomeric structure that is unprecedented for members of the NH homology group I, showing that oligomerisation is not strictly required for the N-ribosidic activity in this family of enzymes. The size, thermal stability and activity of URH1p towards the synthetic substrate 5-fluoruridine, a riboside precursor of the antitumoral drug 5-fluorouracil, make the enzyme an attractive tool to be employed in gene-directed enzyme-prodrug activation therapy against solid tumours.

Hydroxysteroid 17-β dehydrogenase 14 (HSD17B14) is an L-fucose dehydrogenase, the initial enzyme of the L-fucose degradation pathway

L-Fucose (6-deoxy-L-galactose), a monosaccharide abundant in glycolipids and glycoproteins produced by mammalian cells, has been extensively studied for its role in intracellular biosynthesis and recycling of GDP-L-fucose for fucosylation. However, in certain mammalian species, L-fucose is efficiently broken down to pyruvate and lactate in a poorly understood metabolic pathway. In the 1970s, L-fucose dehydrogenase, an enzyme responsible for the initial step of this pathway, was partially purified from pig and rabbit livers and characterized biochemically. However, its molecular identity remained elusive until recently. This study reports the purification, identification, and biochemical characterization of the mammalian L-fucose dehydrogenase. The enzyme was purified from rabbit liver approximately 340-fold. Mass spectrometry analysis of the purified protein preparation identified mammalian hydroxysteroid 17-β dehydrogenase 14 (HSD17B14) as the sole candidate enzyme. Rabbit and human HSD17B14 were expressed in HEK293T and Escherichia coli, respectively, purified and demonstrated to catalyze the oxidation of L-fucose to L-fucono-1,5-lactone, as confirmed by mass spectrometry and NMR analysis. Substrate specificity studies revealed that L-fucose is the preferred substrate for both enzymes. The human enzyme exhibited a catalytic efficiency for L-fucose that was 359-fold higher than its efficiency for estradiol. Additionally, recombinant rat HSD17B14 exhibited negligible activity towards L-fucose, consistent with the absence of L-fucose metabolism in this species.The identification of the gene encoding mammalian L-fucose dehydrogenase provides novel insights into the substrate specificity of enzymes belonging to the 17-β-hydroxysteroid dehydrogenase family. This discovery also paves the way for unraveling the physiological functions of the L-fucose degradation pathway, which remains enigmatic.

Bioengineering secreted proteases converts divergent Rcr3 orthologs and paralogs into extracellular immune co-receptors.

Secreted immune proteases "Required for Cladosporium resistance-3" (Rcr3) and "Phytophthora-inhibited protease-1" (Pip1) of tomato (Solanum lycopersicum) are both inhibited by Avirulence-2 (Avr2) from the fungal plant pathogen Cladosporium fulvum. However, only Rcr3 acts as a decoy co-receptor that detects Avr2 in the presence of the Cf-2 immune receptor. Here, we identified crucial residues in tomato Rcr3 that are required for Cf-2-mediated signaling and bioengineered various proteases to trigger Avr2/Cf-2-dependent immunity. Despite substantial divergence in Rcr3 orthologs from eggplant (Solanum melongena) and tobacco (Nicotiana spp.), minimal alterations were sufficient to trigger Avr2/Cf-2-mediated immune signaling. By contrast, tomato Pip1 was bioengineered with 16 Rcr3-specific residues to initiate Avr2/Cf-2-triggered immune signaling. These residues cluster on one side of the protein next to the substrate-binding groove, indicating a potential Cf-2 interaction site. Our findings also revealed that Rcr3 and Pip1 have distinct substrate preferences determined by two variant residues and that both are suboptimal for binding Avr2. This study advances our understanding of Avr2 perception and opens avenues to bioengineer proteases to broaden pathogen recognition in other crops.

Formation of the pyruvoyl-dependent proline reductase Prd from Clostridioides difficile requires the maturation enzyme PrdH.

Stickland fermentation, the coupled oxidation and reduction of amino acid pairs, is a major pathway for obtaining energy in the nosocomial bacterium Clostridioides difficile. D-proline is the preferred substrate for the reductive path, making it not only a key component of the general metabolism but also impacting on the expression of the clostridial toxins TcdA and TcdB. D-proline reduction is catalyzed by the proline reductase Prd, which belongs to the pyruvoyl-dependent enzymes. These enzymes are translated as inactive proenzymes and require subsequent processing to install the covalently bound pyruvate. Whereas pyruvoyl formation by intramolecular serinolysis has been studied in unrelated enzymes, details about pyruvoyl generation by cysteinolysis as in Prd are lacking. Here, we show that Prd maturation requires a small dimeric protein that we have named PrdH. PrdH (CD630_32430) is co-encoded with the PrdA and PrdB subunits of Prd and also found in species producing similar reductases. By producing stable variants of PrdA and PrdB, we demonstrate that PrdH-mediated cleavage and pyruvoyl formation in the PrdA subunit requires PrdB, which can be harnessed to produce active recombinant Prd for subsequent analyses. We further created PrdA- and PrdH-mutants to get insight into the interaction of the components and into the processing reaction itself. Finally, we show that deletion of prdH renders C. difficile insensitive to proline concentrations in culture media, suggesting that this processing factor is essential for proline utilization. Due to the link between Stickland fermentation and pathogenesis, we suggest PrdH may be an attractive target for drug development.

Seasonal dynamics and habitat preferences of juvenile fish assemblages in the Wanquan Estuary, South China: Implications for conservation and management

Tropical estuaries encompass diverse habitats, which are crucial for supporting juvenile fish by providing nurseries, refuges, and migration routes. In the present study, we investigated the seasonal and spatial dynamics of juvenile fish assemblages in the Wanquan Estuary, Hainan Island, China, examining variations during the wet and dry seasons across four types of habitats. Our research aimed to test the hypotheses: (1) the wet season supports a greater abundance and species richness of juvenile fish compared to the dry season, and (2) juvenile fish showed varying habitat preferences across seasons. We recorded a total of 3617 individuals belonging to 33 families, 50 genera, and 78 taxa. Our findings revealed significant seasonal fluctuations in the assemblage structure, with peak abundance during the wet season in August, primarily consisting of diadromous species. A notable shift towards marine species was observed in March. Spatially, juvenile fish exhibited significant habitat preferences, with a strong preference for sand substrates for most species. Contrary to common expectations, mangrove fringe habitats were not identified as the primary nursery grounds for juvenile fish. Instead, our study highlights the importance of habitat diversity and connectivity in enhancing nursery functions. Water temperature and salinity were the key drivers influencing the seasonal and temporal patterns of juvenile fish assemblages in the Wanquan River estuary The pronounced temporal dynamics of these assemblages, driven by seasonal environmental alterations affecting water variations and habitat connectivity, were more significant than spatial differences. The findings emphasize the need for focused connectivity of various habitats to support the conservation of diverse fish communities in tropical estuarine ecosystems.

Comparative characterisation of an ecto-5'-nucleotidase (CD73) in non-tumoral MCF10-A breast cells and triple-negative MDA-MB-231 breast cancer cells.

Ecto-5'-nucleotidase (CD73) hydrolyses 5'AMP to adenosine and inorganic phosphate. Breast cancer cells (MDA-MB-231) express high CD73 levels, and this enzyme has been found to play a tumour-promoting role in breast cancer. However, no studies have sought to investigate whether CD73 has differential affinity or substrate preferences between noncancerous and cancerous breast cells. In the present study, we aimed to biochemically characterise ecto-5'-nucleotidase in breast cancer cell lines and assess whether its catalytic function and tumour progression are correlated in breast cancer cells. The results showed that compared to nontumoral breast MCF-10A cells, triple-negative breast cancer MDA-MB-231 cells had a higher ecto-5'-nucleotidase expression leveland enzymatic activity. Although ecto-5'-nucleotidase activity in the MDA-MB-231 cell line showed no selectivity among monophosphorylated substrates, 5'AMP was preferred by the MCF-10A cell line. Compared to the MCF-10A cell line, the MDA-MB-231 cell line has better hydrolytic ability, lower substrate affinity, and high inhibitory potential after treatment with a specific CD73 inhibitor α,β‑methylene ADP (APCP). Therefore, we demonstrated that a specific inhibitor of the ecto-5-nucleotidase significantly reduced the migratory and invasive capacity of MDA-MB-231 cells, suggesting that ecto-5-nucleotidase activity might play an important role in metastatic progression.