Cellular Enzyme Activity Research Articles

Role of sumoylation modification of sirtuin 3 in hypertension-associated

Abstract Objective The present study was designed to investigate whether mitochondrial dysfunction and oxidative stress in HTN-injured cardiomyocytes are associated with SIRT3 SUMOylation and whether SIRT3 deSUMOylation can ameliorate the related cardiomyocyte injury. Design and method The human cardiomyocyte AC16 cell line was used for the study. The SIRT3-K288R cell line was constructed by lentiviral transfection;The expression of SOD2, acetyl-SOD2 (MnSOD), protein acetylation level, inflammatory vesicle NLRP3, etc. in different subgroups were detected by Western Blot, and the roles of SUMOylation modification and SIRT3 deacetylation in HTN-associated cell injury were investigated by flow cytometry. Results (1) Western Blot results showed that compared to the HTN cell injury model, the cardiomyocytes in the SIRT3-K288R intervention group had increased Acetyl- SOD2/SOD2 levels were increased (p < 0.05) and protein acetylation levels were decreased in the SIRT3-K288R intervention group compared with the HTN cell injury model, indicating that SIRT3 increased the deacetylation effect on mitochondria and increased cellular antioxidant stress enzyme activity; (2) Western Blot results showed that compared with the HTN cell injury model, cardiomyocytes in the SIRT3-K288R intervention group NLRP3 and NOX4 levels were significantly reduced, indicating that SIRT3 deSUMOylation could improve the inflammatory response to Ang-II-related cell injury. (3) Flow cytometry results showed that K288R transfection ameliorated the reduction of cellular membrane potential, suggesting that deSUMOylation modification of SIRT3 reduces Ang-II damage to mitochondrial membrane potential. Conclusions In the HTN-associated cell injury model, the removal of SUMOylation modification by viral transfection could improve oxidative stress, protein acetylation levels and associated inflammatory responses in HTN-injured cardiomyocytes, indicating that SIRT3 SUMOylation may play an important mechanism in HTN-associated cell injury, and reducing SIRT3 SUMOylation modification may be a possible direction for future research.

Novel Silybin-Conjugated Chitosan Polymeric Micelles for Improving the Oral Absorption of Doxorubicin Based on the Inhibition of P-gp and CYP3A4.

A drug delivery system based on silybin-conjugated chitosan (CS-SB) polymeric micelles was developed to improve the oral absorption of doxorubicin (DOX). SB was grafted to CS via succinic acid, and CS-SB was identified by 1H NMR and FT-IR. The DOX-loaded micelles were prepared by self-assembly, and the characteristics of micelles, including a small particle size of 167.8 ± 2.3 nm, a high drug loading capacity of 8.59%, and a low critical micelle concentration of 1.3 × 10-5 g/mL, were demonstrated. The micelles showed oral bioavailability of up to 193% versus DOX·HCl. The cytotoxicity test showed the biosafety of CS-SB and the potential of reductive DOX-induced cardiotoxicity. The inhibition of P-gp efflux and CYP3A4 enzyme in CS-SB micelles was confirmed by cellular uptake and enzyme activity inhibition tests. The endocytosis process of micelles was revealed by an endocytosis inhibition test. The findings exhibited the potential of CS-SB micelles in drug delivery.

Kynurenic Acid Modulates the Expression of Genes and the Activity of Cellular Antioxidant Enzymes in the Hypothalamus and Hippocampus in Sheep.

Kynurenic acid (KYNA), a tryptophan metabolite, is believed to exert neuromodulatory and neuroprotective effects in the brain. This study aimed to examine KYNA's capacity to modify gene expression and the activity of cellular antioxidant enzymes in specific structures of the sheep brain. Anestrous sheep were infused intracerebroventricularly with two KYNA doses-lower (4 × 5 μg/60 μL/30 min, KYNA20) and higher (4 × 25 μg/60 μL/30 min, KYNA100)-at 30 min intervals. The abundance of superoxide dismutase 2 (SOD2), catalase (CAT), and glutathione peroxidase 1 (GPx1) mRNA, as well as enzyme activities, were determined in the medial-basal hypothalamus (MBH), the preoptic (POA) area of the hypothalamus, and in the hippocampal CA1 field. Both doses of KYNA caused a decrease (p < 0.01) in the expression of SOD2 and CAT mRNA in all structures examined compared to the control group (except for CAT in the POA at the KYNA100 dose). Furthermore, lower levels of SOD2 mRNA (p < 0.05) and CAT mRNA (p < 0.01) were found in the MBH and POA and in the POA and CA, respectively, in sheep administered with the KYNA20 dose. Different stimulatory effects on GPx1 mRNA expression were observed for both doses (p < 0.05-p < 0.01). KYNA exerted stimulatory but dose-dependent effects on SOD2, CAT, and GPx1 activities (p < 0.05-p < 0.001) in all brain tissues examined. The results indicate that KYNA may influence the level of oxidative stress in individual brain structures in sheep by modulating the expression of genes and the activity of at least SOD2, CAT, and GPx1. The present findings also expand the general knowledge about the potential neuroprotective properties of KYNA in the central nervous system.

Enhancing Liver Transplant Outcomes through Liver Precooling to Mitigate Inflammatory Response and Protect Mitochondrial Function.

Transplanted organs experience several episodes of ischemia and ischemia-reperfusion. The graft injury resulting from ischemia-reperfusion (IRI) remains a significant obstacle to the successful survival of transplanted grafts. Temperature significantly influences cellular metabolic rates because biochemical reactions are highly sensitive to temperature changes. Consequently, lowering the temperature could reduce the degradative reactions triggered by ischemia. In mitigating IRI in liver grafts, the potential protective effect of localized hypothermia on the liver prior to blood flow obstruction has yet to be explored. In this study, we applied local hypothermia to mouse donor livers for a specific duration before stopping blood flow to liver lobes, a procedure called "liver precooling". Mouse donor liver temperature in control groups was controlled at 37 °C. Subsequently, the liver donors were preserved in cold University of Wisconsin solution for various durations followed by orthotopic liver transplantation. Liver graft injury, function and inflammation were assessed at 1 and 2 days post-transplantation. Liver precooling exhibited a significant improvement in graft function, revealing more than a 47% decrease in plasma aspartate transaminase (AST) and alanine aminotransferase (ALT) levels, coupled with a remarkable reduction of approximately 50% in liver graft histological damage compared to the control group. The protective effects of liver precooling were associated with the preservation of mitochondrial function, a substantial reduction in hepatocyte cell death, and a significantly attenuated inflammatory response. Taken together, reducing the cellular metabolism and enzymatic activity to a minimum level before ischemia protects against IRI during transplantation.

Green synthesis of graphene-based derivatives from Ulva lactuca extract: Characterization and biomedical applications

Graphene-based materials have been extensively researched due to its biocompatibility, unique conjugated structure, and relatively low cost in the realm of biology. Graphene is increasingly being explored for many medical uses, including as DNA-hybridization devices, cancer treatment, and medicine administration. Furthermore, it exhibits substantial potential in several fields of biological applications, such as antibacterial interventions, therapy, and biosensing. The synthesis of GO and rGO was achieved in this work for the first-time using macroalgae, Ulva lactuca as a reducing agent. The antibacterial activity of graphene-based derivatives was evaluated against two different human pathogenic bacteria. Bacterial growth inhibition increased in proportion to the higher concentration. Higher concentration of nanosheets could enhance the cellular enzyme activity. Different scavenging assays are adapted to assess the antioxidant activity of GO and rGO. Significant in-vitro antioxidant activity was observed in a concentration-dependent manner. Furthermore, the antitumor activity of synthesized nanosheets was tested against human breast cancer cells (MCF-7) at various concentrations. The test results show that the graphene derivatives in the MCF-7 cell line exhibit an increase in apoptotic behaviour as sample concentration rises. This study could offer novel insights into the antibacterial, antioxidant, and anticancer activities of the green synthesized graphene-based derivatives.

Combining CD38 antibody with CD47 blockade is a promising strategy for treating hematologic malignancies expressing CD38.

CD38 and CD47 are expressed in many hematologic malignancies, including multiple myeloma (MM), B-cell non-Hodgkin lymphoma (NHL), B-cell acute lymphoblastic leukemia (ALL), and B-cell chronic lymphocytic leukemia (CLL). Here, we evaluated the antitumor activities of CD38/CD47 bispecific antibodies (BsAbs). Five suitable anti-CD38 antibodies for co-targeting CD47 and CD38 BsAb were developed using a 2 + 2 "mAb-trap" platform. The activity characteristics of the CD38/CD47 BsAbs were evaluated using in vitro and in vivo systems. Using hybridoma screening technology, we obtained nine suitable anti-CD38 antibodies. All anti-CD38 antibodies bind to CD38+ tumor cells and kill tumor cells via antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). Five anti-CD38 antibodies (4A8, 12C10, 26B4, 35G5, and 65A7) were selected for designing CD38/CD47 BsAbs (IMM5605) using a "mAb-trap" platform. BsAbs had higher affinity and binding activity to the CD38 target than those to the CD47 target, decreasing the potential on-target potential and off-tumor effects. The CD38/CD47 BsAbs did not bind to RBCs and did not induce RBC agglutination; thus, BsAbs had much lower blood toxicity. The CD38/CD47 BsAbs had a greater ability to block the CD47/SIRPα signal in CD38+/CD47+ tumor cells than IMM01 (SIRPα Fc fusion protein). Through Fc domain engineering, CD38/CD47 BsAbs were shown to kill tumors more effectively by inducing ADCC and ADCP. IMM5605-26B4 had the strongest inhibitory effect on cellular CD38 enzymatic activity. IMM5605-12C10 had the strongest ability to directly induce the apoptosis of tumor cells. The anti-CD38 antibody 26B4 combined with the SIRPα-Fc fusion proteins showed strong antitumor effects, which were better than any of the mono-therapeutic agents used alone in the NCI-H929 cell xenograft model. The CD38/CD47 BsAbs exhibited strong antitumor effects; specifically, IMM5605-12C10 efficiently eradicated all established tumors in all mice. A panel of BsAbs targeting CD38 and CD47 developed based on the "mAb-tarp" platform showed potent tumor-killing ability in vitro and in vivo. As BsAbs had lower affinity for binding to CD47, higher affinity for binding to CD38, no affinity for binding to RBCs, and did not induce RBC agglutination, we concluded that CD38/CD47 BsAbs are safe and have a satisfactory tolerability profile.

PSV-3 Effects of maternal early gestational weight gain on multigenerational skeletal muscle gene regulation in offspring

Abstract Maternal nutrition during the periconceptional period may have multigenerational effects on the gene regulation of fetal skeletal muscle. This study aimed to investigate the impact of maternal rate of body weight gain (F0) during early gestation on the differential gene expression in the skeletal muscle of newborn (F1) and second-generation fetuses (F2) female offspring. To this end, crossbred Angus heifers (F0) were assigned to either a low gain (LG; 0.28 kg/d; n = 8) or moderate gain (MG; 0.79 kg/d; n = 8) group during the first 84 d of gestation. Subsequently, heifers (F0) were managed as a single group on a forage-based diet until the weaning of the F1 offspring (8 mo). The F1 heifers were estrus synchronized and bred via AI at 15 mo, and F2 fetuses were harvested on d 84 of gestation. Longissimus dorsi muscle was collected at birth (F1) and the 84-d harvest in F1 heifers and their F2 fetuses for multigenerational RNA-Seq analysis. Differentially expressed genes (DEGs) were identified using the DESeq2 R-package. Significant genes were retrieved based on P - value ≤ 0.05 and log2 fold change|0.5|. We previously reported that F1 heifers from MG dams at birth were ~2 kg heavier (P &amp;lt; 0.03), and herein we report the impact on the gene regulation of skeletal muscle in the F1 and F2 generations. At birth, F1 heifers from MG cows exhibited 275 DEGs, of which 98 were downregulated, and 177 genes were upregulated, related to the immune system (TNFAIP3, TLR5), cellular signaling and transduction (RASGRP2, NFE2), and extracellular matrix (TGM3, MMP17). Changes in gene expression persisted in F1 heifers through development, breeding, and until the evaluation at d 84 of gestation. At this time point, we identified 75 upregulated genes out of 177 DEGs. These genes were underlying cellular functions (USP43, PASK), hematopoiesis (THPO), metabolism, and enzymatic activity (SMOX, OAT) as possible metabolic adaptations. Similarly, 102 genes were downregulated, mainly associated with immune and inflammatory response (CHI3L1, LDAF1). Regarding the F2 fetuses, 151 DEGs were differentially expressed. Among them, 63 were upregulated in the MG group and involved with biological functions such as ion channels and transport (KCNAB1, SLC9A3), transcriptional regulation, and signaling (ZNF75D, TMEM106B, DPP6), enzymatic activity, and metabolism (B3GNT4, FAM20A, ALDH5A1, and PRSS23). Likewise, 88 downregulated genes were associated with the cell cycle and proliferation (APOOL, CIR1, and STIL). In conclusion, periconceptional maternal nutrition aimed at moderate weight gains can have multigenerational effects on the gene regulation of fetal muscle tissue.

Rapid Extraction of Prefrontal Cortex and Hippocampus for Molecular Analysis in Rats

Memory functions in laboratory animals are a crucial area of research in neuroscience, with a particular focus on identifying drugs that enhance various aspects of memory, particularly long-term declarative memory. This field involves studying cognition and memory from behavioral and molecular perspectives. Researchers evaluate changes in genes, enzymes, and proteins to discover the neurobiological mechanisms underlying memory formation and retrieval. The hippocampus and prefrontal cortex are key brain regions associated with memory and cognition, undergoing synaptogenesis following the acquisition of new information or skills. The production of specific proteins plays a crucial role in consolidating and storing memories, necessitating precise analysis of these brain regions to understand the molecular mechanisms involved in memory processes fully. However, the rapid degradation of these proteins by cellular enzymatic activities highlights the need for efficient and timely extraction techniques to ensure precise and reliable analysis. This methodological study aimed to introduce a rapid and accurate method for extracting the prefrontal cortex and hippocampus in rats.

(2-Methylbutyryl)shikonin Naturally Occurring Shikonin Derivative Ameliorates the α-MSH-Induced Melanogenesis via ERK1/2 and p38 MAP Kinase-Mediated Down-Regulation of the MITF Transcription Factor.

Cutaneous pigmentation is an important phenotypic trait whose regulation, despite recent advances, has yet to be completely elucidated. Melanogenesis, a physiological process of melanin production, is imperative for organism survival as it provides protection against the environmental insults that majorly involve sunlight-induced skin photodamage. However, immoderate melanin synthesis can cause pigmentation disorders associated with a psychosocial impact. In this study, the hypopigmentation effect of (2-methylbutyryl)shikonin, a natural product present in the root extract of Lithospermum erythrorhizon, and the underlying mechanisms responsible for the inhibition of melanin synthesis in α-MSH-stimulated B16F10 cells and C57BL/6J mice was studied. Non-cytotoxic concentrations of (2-methylbutyryl)shikonin significantly repressed cellular tyrosinase activity and melanin synthesis in both in vitro and in vivo models (C57BL/6J mice). (2-Methylbutyryl)shikonin remarkably abolished the protein expression of MITF, tyrosinase, tyrosinase-related protein 1, and tyrosinase-related protein 2, thereby blocking the production of pigment melanin via modulating the phosphorylation status of MAPK proteins, viz., ERK1/2 and p38. In addition, specific inhibition of ERK1/2 attenuated the inhibitory effects of (2-methylbutyryl)shikonin on melanin synthesis, whereas selective inhibition of p38 augmented the inhibitory effect of BSHK on melanin synthesis. Moreover, topical application of (2-methylbutyryl)shikonin on C57BL/6J mouse tails remarkably induced tail depigmentation. In conclusion, with these findings, we, for the first time, report the hypopigmentation effect of (2-methylbutyryl)shikonin via inhibition of cellular tyrosinase enzyme activity, subsequently ameliorating the melanin production, thereby indicating that (2-methylbutyryl)shikonin is a potential natural therapy for hyperpigmentation disorders.

Cleistocalyx nervosum var. paniala berry extract and cyanidin-3-glucoside inhibit hepatotoxicity and apoptosis.

Excessive oxidative toxicity in liver cells is a significant risk factor that can cause cellular injury, leading to the development of chronic liver disease (CLD). Natural anthocyanins have been shown to prevent the harmful effects of oxidative toxicity in mammalian cells. Ripe Cleistocalyx nervosum var. paniala berry fruits are rich in anthocyanins, which have been reported to possess many health benefits. Therefore, this study examined the protective effect of ethanolic fruit extract of C. nervosum var. paniala (CNPE) against hydrogen peroxide (H2O2)-induced oxidative damage and cell death in human hepatoma HepG2 cells. Results showed that CNPE had strong antioxidant capabilities and high amounts of total phenolics and anthocyanins. HPLC analysis showed that CNPE consists of cyanidin-3-glucoside (C3G). Our investigations found that HepG2 cells pretreated with CNPE or anthocyanin C3G inhibited H2O2-induced cellular damage and apoptosis by increasing the viability of cells, the expression of antiapoptotic Bcl-2 protein, and the activities of cellular antioxidant enzymes, namely SOD, CAT, and GPx. Moreover, both CNPE and C3G significantly suppressed expression of apoptotic proteins (Bax and cytochrome c) and the activities of cleaved caspase-9 and caspase-3 caused by H2O2. Our results indicate that CNPE and C3G can suppress H2O2-induced hepatotoxicity and cell death through stimulation of endogenous antioxidant enzyme activities and inhibition of apoptosis pathway in HepG2 cells. These findings might support development of CNPE as an alternative natural product for preventing CLD.

DIA-based quantitative proteomics analysis of plasma exosomes in rat model of allergic rhinitis

Allergic rhinitis (AR) is a common chronic inflammatory disease characterized by symptoms such as itching, rhinorrhea, sneezing, and nasal obstruction. Despite being classified as an IgE-mediated typeⅠ allergy for many years, the complex pathophysiological mechanism of AR continues to present a challenge in clinical management. The objective of this study was to quantify the proteomics of plasma exosomes using data independent acquisition (DIA) in combination with liquid chromatography-mass spectrometry (LC-MS/MS) to identify the key proteins involved in the development and progression of AR. In the AR rat model, a total of 41 proteins demonstrated significant up-regulation, while 51 proteins were found to be significantly down-regulated. Gene ontology (GO) analysis results indicated that the altered proteins were highly enriched in cellular regulatory processes and enzymatic activity in AR rats. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis and protein-protein interaction (PPI) network results revealed that the pivotal proteins C4b, C1qa, C1qc, and Mbl1 might be involved in the metabolic pathways of the immune system in AR through the activation of the complement and coagulation cascades pathway. These proteins could serve as diagnostic markers and therapeutic targets for AR, which is of great significance in understanding the role of exosome proteins in AR.

Modeling Peptide-Protein Interactions by a Logo-Based Method: Application in Peptide-HLA Binding Predictions.

Peptide-protein interactions form a cornerstone in molecular biology, governing cellular signaling, structure, and enzymatic activities in living organisms. Improving computational models and experimental techniques to describe and predict these interactions remains an ongoing area of research. Here, we present a computational method for peptide-protein interactions' description and prediction based on leveraged amino acid frequencies within specific binding cores. Utilizing normalized frequencies, we construct quantitative matrices (QMs), termed 'logo models' derived from sequence logos. The method was developed to predict peptide binding to HLA-DQ2.5 and HLA-DQ8.1 proteins associated with susceptibility to celiac disease. The models were validated by more than 17,000 peptides demonstrating their efficacy in discriminating between binding and non-binding peptides. The logo method could be applied to diverse peptide-protein interactions, offering a versatile tool for predictive analysis in molecular binding studies.

Real-time fluorescent monitoring of phase I xenobiotic-metabolizing enzymes.

This article explores the intricate landscape of advanced fluorescent probes crafted for the detection and real-time monitoring of phase I xenobiotic-metabolizing enzymes. Employing state-of-the-art technologies, such as fluorescence resonance energy transfer, intramolecular charge transfer, and solid-state luminescence enhancement, this article unfolds a multifaceted approach to unraveling the dynamics of enzymatic processes within living systems. This encompassing study involves the development and application of a diverse range of fluorescent probes, each intricately designed with tailored mechanisms to heighten sensitivity, providing dynamic insights into phase I xenobiotic-metabolizing enzymes. Understanding the role of phase I xenobiotic-metabolizing enzymes in these pathophysiological processes, is essential for both medical research and clinical practice. This knowledge can guide the development of approaches to prevent, diagnose, and treat a broad spectrum of diseases and conditions. This adaptability underscores their potential clinical applications in cancer diagnosis and personalized medicine. Noteworthy are the trifunctional fluorogenic probes, uniquely designed not only for fluorescence-based cellular imaging but also for the isolation of cellular glycosidases. This innovative feature opens novel avenues for comprehensive studies in enzyme biology, paving the way for potential therapeutic interventions. The research accentuates the selectivity and specificity of the probes, showcasing their proficiency in distinguishing various enzymes and their isoforms. The sophisticated design and successful deployment of these fluorescent probes mark significant advancements in enzymology, providing powerful tools for both researchers and clinicians. Beyond their immediate applications, these probes offer illuminating insights into disease mechanisms, facilitating early detection, and catalyzing the development of targeted therapeutic interventions. This work represents a substantial leap forward in the field, promising transformative implications for understanding and addressing complex biological processes. In essence, this research heralds a new era in the development of fluorescent probes, presenting a comprehensive and innovative approach that not only expands the understanding of cellular enzyme activities but also holds great promise for practical applications in clinical settings and therapeutic endeavors.

Modulation of Growth and Antioxidative Defense Mechanism in Wheat (Triticum aestivum L.) Mediated by Plant-Beneficial Strain Pseudomonas veronii MR-15 under Drought Conditions

Drought stress severely influences plants in various ways and is considered an alarming threat to sustainable crop production worldwide. However, plant-growth-promoting rhizobacteria (PGPRs) have the natural ability to tolerate drought and enable plants to induce stress resistance by altering critical metabolic pathways. In this study, we isolated and characterized a drought-tolerant rhizobacterium from the ground nut (Arachis hypogaea). Sequencing of the 16S rRNA gene traced its lineage to Pseudomonas veronii, named MR-15. The strain exhibited natural capabilities to solubilize phosphate, produce indole acetic acid, and grow a drought medium containing PEG (polyethylene glycol). The seeds of two wheat varieties (Triticum aestivum) inoculated with MR-15 were grown under drought and fully hydrated conditions and showed a significant increase in plant biomass, enhanced cellular antioxidant enzyme activity, and reduced reactive oxygen species. The MR-15 strain also significantly increased pigmentation and protein contents compared to plants raised from seeds grown without inoculation. These beneficial effects were consistent under drought stress conditions, indicating that MR-15 effectively alleviated wheat plants from drought-induced cellular oxidative damage. The findings suggest that MR-15 has the potential to serve as a biofertilizer, and further experiments should be conducted to explore its role in promoting plant growth and yield under drought conditions, particularly in semi-arid and arid zones. This is the first study reporting Pseudomonas veronii as a potential PGPR strain.

MiR-630 Promotes Radioresistance by Induction of Anti-Apoptotic Effect via Nrf2-GPX2 Molecular Axis in Head-Neck Cancer.

Head and neck cancer (HNC) ranks among the top ten prevalent cancers worldwide. Radiotherapy stands as a pivotal treatment component for HNC; however, radioresistance in cancerous cells often leads to local recurrence, becoming a substantial factor in treatment failure. MicroRNAs (miRNAs) are compact, non-coding RNAs that regulate gene expression by targeting mRNAs to inhibit protein translation. Although several studies have indicated that the dysregulation of miRNAs is intricately linked with malignant transformation, understanding this molecular family's role in radioresistance remains limited. This study determined the role of miR-630 in regulating radiosensitivity in HNC. We discovered that miR-630 functions as an oncomiR, marked by its overexpression in HNC patients, correlating with a poorer prognosis. We further delineated the malignant function of miR-630 in HNC cells. While it had a minimal impact on cell growth, the miR-630 contributed to radioresistance in HNC cells. This result was supported by decreased cellular apoptosis and caspase enzyme activities. Moreover, miR-630 overexpression mitigated irradiation-induced DNA damage, evidenced by the reduced levels of the γ-H2AX histone protein, a marker for double-strand DNA breaks. Mechanistically, the overexpression of miR-630 decreased the cellular ROS levels and initiated Nrf2 transcriptional activity, resulting in the upregulation of the antioxidant enzyme GPX2. Thus, this study elucidates that miR-630 augments radioresistance by inducing an anti-apoptotic effect via the Nrf2-GPX2 molecular axis in HNC. The modulation of miR-630 may serve as a novel radiosensitizing target for HNC.

Probiotic and postbiotic analytical methods: a perspective of available enumeration techniques.

Probiotics are the largest non-herbal/traditional dietary supplements category worldwide. To be effective, a probiotic strain must be delivered viable at an adequate dose proven to deliver a health benefit. The objective of this article is to provide an overview of the various technologies available for probiotic enumeration, including a general description of each technology, their advantages and limitations, and their potential for the future of the probiotics industry. The current "gold standard" for analytical quantification of probiotics in the probiotic industry is the Plate Count method (PC). PC measures the bacterial cell's ability to proliferate into detectable colonies, thus PC relies on cultivability as a measure of viability. Although viability has widely been measured by cultivability, there has been agreement that the definition of viability is not limited to cultivability. For example, bacterial cells may exist in a state known as viable but not culturable (VBNC) where the cells lose cultivability but can maintain some of the characteristics of viable cells as well as probiotic properties. This led to questioning the association between viability and cultivability and the accuracy of PC in enumerating all the viable cells in probiotic products. PC has always been an estimate of the number of viable cells and not a true cell count. Additionally, newer probiotic categories such as Next Generation Probiotics (NGPs) are difficult to culture in routine laboratories as NGPs are often strict anaerobes with extreme sensitivity to atmospheric oxygen. Thus, accurate quantification using culture-based techniques will be complicated. Another emerging category of biotics is postbiotics, which are inanimate microorganisms, also often referred to as tyndallized or heat-killed bacteria. Obviously, culture dependent methods are not suitable for these products, and alternative methods are needed for their quantification. Different methodologies provide a more complete picture of a heterogeneous bacterial population versus PC focusing exclusively on the eventual multiplication of the cells. Alternative culture-independent techniques including real-time PCR, digital PCR and flow cytometry are discussed. These methods can measure viability beyond cultivability (i.e., by measuring cellular enzymatic activity, membrane integrity or membrane potential), and depending on how they are designed they can achieve strain-specific enumeration.

Commentary] Will this novel stimulus be the catalyst for transforming biomedical shape memory polymers?

Shape memory polymers (SMPs) circumscribe materials exhibiting responsive behavior to diverse external stimuli, ranging from heat and light to electric and magnetic fields. Originating in mid-20th-century studies, the realm of SMPs has expanded swiftly, tapping into the domains of biomedical applications, revolutionizing biomaterials, and finding applications from medical devices to regenerative medicine. Evolving beyond biocompatibility, the emphasis shifted to cytocompatible SMPs for cell mechanobiology, paving the way for direct cell-responsive SMPs. Recent breakthroughs in enzymatically triggered SMPs, particularly those influenced by cellular enzymatic activity, signify a transformative leap. This opens avenues for precision drug delivery and biosensors, merging SMPs with biomedical applications. This commentary celebrates the discovery of polymers responding to cells, unfolding the promising future of SMPs in personalized medicine, and seamlessly integrating advanced materials with cellular therapies for groundbreaking medical solutions.

PhosBoost: Improved phosphorylation prediction recall using gradient boosting and protein language models.

Protein phosphorylation is a dynamic and reversible post-translational modification that regulates a variety of essential biological processes. The regulatory role of phosphorylation in cellular signaling pathways, protein-protein interactions, and enzymatic activities has motivated extensive research efforts to understand its functional implications. Experimental protein phosphorylation data in plants remains limited to a few species, necessitating a scalable and accurate prediction method. Here, we present PhosBoost, a machine-learning approach that leverages protein language models and gradient-boosting trees to predict protein phosphorylation from experimentally derived data. Trained on data obtained from a comprehensive plant phosphorylation database, qPTMplants, we compared the performance of PhosBoost to existing protein phosphorylation prediction methods, PhosphoLingo and DeepPhos. For serine and threonine prediction, PhosBoost achieved higher recall than PhosphoLingo and DeepPhos (.78, .56, and .14, respectively) while maintaining a competitive area under the precision-recall curve (.54, .56, and .42, respectively). PhosphoLingo and DeepPhos failed to predict any tyrosine phosphorylation sites, while PhosBoost achieved a recall score of .6. Despite the precision-recall tradeoff, PhosBoost offers improved performance when recall is prioritized while consistently providing more confident probability scores. A sequence-based pairwise alignment step improved prediction results for all classifiers by effectively increasing the number of inferred positive phosphosites. We provide evidence to show that PhosBoost models are transferable across species and scalable for genome-wide protein phosphorylation predictions. PhosBoost is freely and publicly available on GitHub.

Influence of the Cellular Ultrastructure and Enzyme Activity of the Leaf Sheath on Spontaneous Defoliation in Sugarcane

Spontaneous defoliation improves the harvesting efficiency and yield of sugarcane. Here, we investigated the ultrastructural changes and pectinase and cellulase activity in the third, fifth, and seventh leaf sheaths in four sugarcane varieties with varying spontaneous defoliation performance during maturation. At the early and middle stages of spontaneous defoliation, the cells in the abscission zones of the third, fifth, and seventh leaf sheaths were degrading. At the late stage, no complete organelles and hollow or broken spots in the cell walls were observed in the abscission zone cells of defoliation-prone varieties, while complete organelles and intact cell walls were present in the abscission zone cells at the same leaf positions in defoliation-resistant varieties. From the early to late stages, defoliation-prone varieties had higher pectinase activity in the abscission zones of the fifth and seventh leaf sheaths. At the early stage of defoliation, defoliation-prone varieties had significantly higher cellulase activity in the abscission zones of the third, fifth, and seventh leaf sheaths. Correlation analysis showed that the spontaneous defoliation rate was significantly positively correlated with pectinase activity in the leaf sheaths. In conclusion, the spontaneous defoliation of sugarcane was closely related to changes in cell morphology and pectinase activites in leaf sheaths.

Curcumin Protects Pheochromocytoma Cells Against Hydrogen Peroxide-Induced Oxidative Stress

Objective: Oxidative stress represents a characteristic of neurodegenerative diseases, which may be alleviated by the functional food component—curcumin. However, the underlying mechanism remains unclear. The current study aimed to address how curcumin protects variant differentiated pheochromocytoma (PC12) cells against hydrogen peroxide (H2O2)-induced cell stress, using low- and high-differentiated PC12 cells, and validated in old-aged mice. Methods: The MTT assay was first carried out to test the cell viability upon H2O2 and/or curcumin treatment and cellular lipid peroxidation was assessed as cellular damages by quantifying the amount of cellular malondialdehyde. Cellular oxidative stress was determined by measuring the cellular and mitochondrial ROS levels, and total cellular antioxidant capacities were determined as the ABTS free radical scavenging ability (ABTS) and Fe3+ reduction ability of cells. The activities and expression of major antioxidant enzymes, including glutathione peroxidase (GPx), superoxidase dismutase (SOD) and catalase (CAT), were also tested. Furthermore, oxidative phosphorylation of mitochondria was assessed by measuring the ATP level and the ADP/ATP ratio. Finally, the antioxidant activity of curcumin was determined in 6-month-old mice by measuring cellular antioxidant capacity and antioxidant enzyme activities. Results: H2O2 caused oxidative damage and decreased cell viability, which was reversed by curcumin with different dosed effects in variant differentiated PC12 cells. Moreover, curcumin's protective effect was attributed to an enhanced antioxidant enzyme system. In consistent, curcumin recovered H2O2-deteriorated oxidative phosphorylation. Further studies verified that curcumin significantly improved the antioxidant capacity of old-aged mice, which may have elevated oxidative stress. Conclusion: Taken together, curcumin improved the antioxidant capacity of variant differentiated PC12 cells and old-aged mice, which was closely associated with the modulation of mitochondrial function and antioxidant enzyme system. This study may shed light on the molecular mechanism underlying the alleviation of neurodegenerative diseases by natural polyphenols.