Loss Of Enzyme Activity Research Articles

PLGA nanoparticle-encapsulated lysostaphin for the treatment of Staphylococcus aureus infections

Staphylococcus aureus possesses the ability to become pathogenic, leading to severe and life-threatening infections. Its methicillin-resistant variant MRSA has garnered high-priority status due to its increased morbidity and associated mortality. This emphasizes the urgency for novel anti-staphylococcal agents. The bacteriocin lysostaphin stands out for its remarkable bactericidal activity against S. aureus, including MRSA, outperforming conventional antibiotics. However, the clinical application of lysostaphin faces challenges, including enzymatic activity loss under physiological conditions and potential immunogenicity. This study introduces a novel approach by encapsulating lysostaphin within polylactic-co-glycolic acid (PLGA) nanoparticles, a biodegradable copolymer known for its biocompatibility and sustained drug release ability. The study assesses the antimicrobial activity of lysostaphin-loaded PLGA nanoparticles against different S. aureus strains, and we also used GFP-expressing S. aureus for facilitating its traceability in planktonic, biofilm, and intracellular infection models. The results showed the significant reduction in bacteria viability both in planktonic and biofilm states. The in vitro intracellular infection model demonstrated the significantly enhanced efficiency of the developed nanoparticles compared to the treatment with the free bacteriocin. This research presents lysostaphin encapsulation within PLGA nanoparticles and offers promising avenues for enhancing lysostaphin's therapeutic efficacy against S. aureus infections.

GAPDH inhibition mediated by thiol oxidation in human airway epithelial cells exposed to an environmental peroxide

Intracellular redox homeostasis in the airway epithelium is closely regulated through adaptive signaling and metabolic pathways. However, inhalational exposure to xenobiotic stressors such as secondary organic aerosols (SOA) can alter intracellular redox homeostasis. Isoprene hydroxy hydroperoxide (ISOPOOH), a ubiquitous volatile organic compound derived from the atmospheric photooxidation of biogenic isoprene, is a major contributor to SOA. We have previously demonstrated that exposure of human airway epithelial cells (HAEC) to ISOPOOH induces oxidative stress through multiple mechanisms including lipid peroxidation, glutathione oxidation, and alterations of glycolytic metabolism. Using dimedone-based reagents and copper catalyzed azo-alkynyl cycloaddition to tag intracellular protein thiol oxidation, we demonstrate that exposure of HAEC to micromolar levels of ISOPOOH induces reversible oxidation of cysteinyl thiols in multiple intracellular proteins, including GAPDH, that was accompanied by a dose-dependent loss of GAPDH enzymatic activity. These results demonstrate that ISOPOOH induces an oxidative modification of intracellular proteins that results in loss of GAPDH activity, which ultimately impacts the dynamic regulation of the intracellular redox homeostatic landscape in HAEC.

Biochemical characterization of a novel hyperthermophilic chitinase from a deep-sea Thermotogae bacterium

Chitin represents the most abundant source of renewable biomass in marine ecosystems, and its degradation by chitinase holds potential for numerous industrial applications. In this study, a novel hyperthermophilic chitinase, TbChi52, derived from a deep-sea bacterium of the Thermotogae phylum was introduced. TbChi52 exhibits optimal enzymatic activity at 90 ℃ and pH 5.5. Furthermore, it shows exceptional thermal stability characterized by a prolonged thermal denaturation half-life and a significant melting temperature. Functionally, TbChi52 displays dual activities, acting as both an exo-chitinase and a β-N-acetylaminoglucosidase (NAGase). It yields N-acetyl-D-glucosamine (GlcNAc) as the primary product during the hydrolysis of colloidal chitin. Leveraging the robust thermo-stability of TbChi52, a streamlined, one-step purification protocol was developed. A mere 10-minute thermal treatment at 75 ℃ suffices to yield a highly pure TbChi52 preparation with minimal enzyme activity loss in chitin hydrolysis, achieving an 87.4 % hydrolysis rate of colloidal chitin and a 92.7 % purity of GlcNAc. Collectively, the outstanding thermal properties and the simple preparation of TbChi52 highlight its potential for industrial applications. It provides a sustainable and eco-friendly approach to harnessing the world's most abundant marine biomass, along with serving as a potential enzyme-engineered template tailored for specific applications.

Conserved proline residues prevent dimerization and aggregation in the β-lactamase BlaC.

Evolution leads to conservation of amino acid residues in protein families. Conserved proline residues are usually considered to ensure the correct folding and to stabilize the three-dimensional structure. Surprisingly, proline residues that are highly conserved in class A β-lactamases were found to tolerate various substitutions without large losses in enzyme activity. We investigated the roles of three conserved prolines at positions 107, 226, and 258 in the β-lactamase BlaC from Mycobacterium tuberculosis and found that mutations can lead to dimerization of the enzyme and an overall less stable protein that is prone to aggregate over time. For the variant Pro107Thr, the crystal structure shows dimer formation resembling domain swapping. It is concluded that the proline substitutions loosen the structure, enhancing multimerization. Even though the enzyme does not lose its properties without the conserved proline residues, the prolines ensure the long-term structural integrity of the enzyme.

Structural Impact Assessment of Cytochrome P450 2A13 Polymorphisms Using Molecular Dynamics Simulations.

One of the members of CYP, a monooxygenase, CYP2A13 is involved in the metabolism of nicotine, coumarin, and tobacco-specific nitrosamine. Genetic polymorphisms have been identified in CYP2A13, with reported loss or reduction in enzymatic activity in CYP2A13 allelic variants. This study aimed to unravel the mechanism underlying the diminished enzymatic activity of CYP2A13 variants by investigating their three-dimensional structures through molecular dynamics (MD) simulations. For each variant, MD simulations of 1000 ns were performed, and the obtained results were compared with those of the wild type. The findings indicated alterations in the interaction with heme in CYP2A13.4, .6, .8, and .9. In the case of CYP2A13.5, observable effects on the helix structure related to the interaction with the redox partner were identified. These conformational changes were sufficient to cause a decrease in enzyme activity in the variants. Our findings provide valuable insights into the molecular mechanisms associated with the diminished activity in the CYP2A13 polymorphisms.

Abstract B035: Evidence for CD73 loss promoting cancer cell stemness via metabolic reprogramming in endometrial cancer

Abstract Cancer stem cells (CSCs) are a subpopulation of cancer cells that are responsible for disease recurrence and chemoresistance. The mechanisms by which CSCs arise in endometrial cancer are not fully understood and elucidating key molecular events may provide insight into therapeutic targets. CD73, a cell surface 5’nucleotidase that generates adenosine, is frequently downregulated in endometrial tumorigenesis, and CD73 loss is associated with aggressive disease and poor patient outcomes. In developing and characterizing CRISPR/Cas-9 models for CD73, we discovered CD73 deletion (CD73−/−) in HEC-1-A cells causes growth arrest and prolonged survival which are features suggestive of a stemness phenotype. Thus, we hypothesized that loss of CD73 is critical for promoting CSCs in endometrial cancer. To evaluate CSC characteristics, we performed MTT and spheroid assays and interrogated CSC marker protein expression (e.g., CD44 and NOTCH3). CD73−/− HEC-1-A cells formed larger spheroids and expressed high levels of CD44 and NOTCH3 compared to wild-type (CD73+/+) cells. To assess whether loss of CD73 enzymatic activity is responsible for the enrichment of CSCs, CD73+/+ HEC-1-B and HEC-50 cells were treated with AoPCP, a catalytic inhibitor of CD73. Similar to genetic deletion of CD73 in HEC-1-A cells, AoPCP treatment resulted in HEC-1-B and HEC-50 cells forming larger spheroids and expressing high CD44 and NOTCH3. These data indicate that loss of CD73 enzyme activity is important to the enrichment of CSCs. In contrast, HEYA8 ovarian cancer cells showed the opposite effects, wherein treatment with AoPCP decreased spheroid size. These differences support the emerging paradigm that CD73 has tissue- and cancer-specific roles. As CD73 is a major node in purine metabolic signaling, we further hypothesized that loss of CD73 activity may be promoting CSC enrichment via changes to metabolic pathways. Mass spectrometry metabolomics revealed significantly different profiles of CD73+/+ and CD73−/− HEC-1-A cells. Conditioned media experiments showed CD73−/−-conditioned media induces growth arrest in HEC-1-A wild-type cells, whereas the growth arrest and CSC marker expression enrichment of CD73−/− cells was abrogated by CD73+/+-conditioned media or daily media replenishment. Metabolomic profiling of CD73+/+ and CD73−/− HEC-1-A cells with/without daily media replenishment revealed metabolic pathways, such as NAD+ biosynthesis, lipid metabolism, and sugar alcohol synthesis, are up-regulated by CD73 deficiency and parallels the enrichment of CSCs. Current studies are dissecting these metabolic dependencies in supporting endometrial cancer cell stemness. Altogether, these studies provide novel insight into how CSCs arise in endometrial cancer and why CD73 loss is detrimental in this disease. Citation Format: Emily M. Rabjohns, Blake R. Rushing, Sayali Joseph, Cyrus Vaziri, Jessica L. Bowser. Evidence for CD73 loss promoting cancer cell stemness via metabolic reprogramming in endometrial cancer [abstract]. In: Proceedings of the AACR Special Conference on Endometrial Cancer: Transforming Care through Science; 2023 Nov 16-18; Boston, Massachusetts. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(5_Suppl):Abstract nr B035.

Iron-Confined CRISPR/Cas9-Ribonucleoprotein Delivery System for Redox-Responsive Gene Editing.

Clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein 9 (Cas9) is a promising gene editing tool to treat diseases at the genetic level. Nonetheless, the challenge of the safe and efficient delivery of CRISPR/Cas9 to host cells constrains its clinical applicability. In the current study, a facile, redox-responsive CRISPR/Cas9-Ribonucleoprotein (RNP) delivery system by combining iron-coordinated aggregation with liposomes (Fe-RNP@L) is reported. The Fe-RNP is formed by the coordination of Fe3+ with amino and carboxyl groups of Cas9, which modifies the lipophilicity and surface charge of RNP and alters cellular uptake from primary endocytosis to endocytosis and cholesterol-dependent membrane fusion. RNP can be rapidly and reversibly released from Fe-RNP in response to glutathione without loss of structural integrity and enzymatic activity. In addition, iron coordination also improves the stability of RNP and substantially mitigates cytotoxicity. This construct enabled highly efficient cytoplasmic/nuclear delivery (≈90%) and gene-editing efficiency (≈70%) even at low concentrations. The high payload content, high editing efficiency, good stability, low immunogenicity, and ease of production and storage, highlight its potential for diverse genome editing and clinical applications.

Positive epistasis drives clavulanic acid resistance in double mutant libraries of BlaC β-lactamase

Phenotypic effects of mutations are highly dependent on the genetic backgrounds in which they occur, due to epistatic effects. To test how easily the loss of enzyme activity can be compensated for, we screen mutant libraries of BlaC, a β-lactamase from Mycobacterium tuberculosis, for fitness in the presence of carbenicillin and the inhibitor clavulanic acid. Using a semi-rational approach and deep sequencing, we prepare four double-site saturation libraries and determine the relative fitness effect for 1534/1540 (99.6%) of the unique library members at two temperatures. Each library comprises variants of a residue known to be relevant for clavulanic acid resistance as well as residue 105, which regulates access to the active site. Variants with greatly improved fitness were identified within each library, demonstrating that compensatory mutations for loss of activity can be readily found. In most cases, the fittest variants are a result of positive epistasis, indicating strong synergistic effects between the chosen residue pairs. Our study sheds light on a role of epistasis in the evolution of functional residues and underlines the highly adaptive potential of BlaC.

Evaluation of packaging materials for enhancing the storage life of marigold flowers (Tagetes erecta)

Among the various loose flower crops, marigold (Tagetes erecta L.) holds a prominent place among commercial crops. Its vibrant blooms are in high demand, but flowers are perishable, rendering them susceptible to significant post-harvest losses. Efforts to mitigate these losses will enhance the overall flower production, distribution to maximize its economic potential and to sustain valuable contribution for the industry. So, the present experiment on storage of marigold flowers was conducted during 2021 and 2022 at the ICAR- Indian Agricultural Research Institute, New Delhi with an objective to improve the shelf life of flowers by using different packaging materials. The results revealed that in marigold var. Pusa Basanti Gainda, maximum shelf life, high carotenoid content, minimum ion leakage, physiological loss in weight and enzyme activity were achieved in flowers packed in shrink-wrap and stored under low-temperature (6±20C) conditions.

Oleander attenuates hepatic inflammation in a TLR4-independent manner and by favorable modulation of hepatocellular global metabolome that supports cytoprotection

Ethnopharmacological relevanceNerium oleander is used to treat liver-associated chronic metabolic diseases in traditional medicinal systems across the globe. The hepatoprotective effects of oleander are mentioned in Indian and Chinese traditional medicinal literature. Aim of the studyThe present study aimed to investigate the cellular mechanisms behind the hepatoprotective effects of a non-toxic dose of oleander (NO). Materials and methodsThe hepatoprotective effects of NO were tested against lipopolysaccharide (LPS)-treated HepG2 cells. Oxidative stress response was studied using cellular enzymatic assays, and gene expression was analyzed using qRT-PCR. HepG2 cells were pretreated with TAK-242 (pharmacological inhibitor of TLR4) to decipher the anti-inflammatory mechanisms of NO. Cell-free metabolites were analyzed using GCMS and were subjected to pathway enrichment analysis. ResultsNO reduced systemic inflammation, serum lipid peroxidation byproducts, and glucose without affecting serum transaminase levels and hepatic histopathological features. NO attenuated the inflammation-induced loss of antioxidant enzyme activities and mRNA expressions of toll-like receptor-4 (TLR4)/nuclear factor κβ (NFκβ)-dependent inflammatory genes. In TAK-242 pretreated cells, LPS was unable to induce inflammatory and oxidative responses. However, NO treatment in TAK-242 pretreated cells with LPS stimulation further reduced the signs of inflammation and improved hepatoprotective activities. A comparative analysis of the intracellular global metabolome from HepG2 cells with and without NO treatment indicated NO-mediated favorable modulation of intracellular metabolic pathways that support cytoprotective activities. ConclusionNO protects HepG2 cells from LPS-induced oxidative and inflammatory injury. The hepatoprotective effects of NO are mediated by a TLR4-independent process and through a favorable modulation of the intracellular global metabolome that supports cytoprotection.

Mechanisms of photoisomerization of the prenylated flavin mononucleotide cofactor: a theoretical study.

The enzymatic decarboxylation of α,β-unsaturated acids using the ferulic acid decarboxylase (Fdc1) enzyme and prenylated flavin mononucleotide (prFMN) cofactor is a potential, environmentally friendly reaction for the biosynthesis of styrene and its derivatives. However, experiments showed that the enzyme activity of Fdc1 depends on the ring structure of prFMN, namely, the iminium and ketimine forms, and the loss of enzyme activity results from prFMNim → prFMNket photoisomerization. To obtain insight into this photochemical process and to improve the enzyme efficiency of Fdc1, two proposed photoisomerization mechanisms with different proton sources for the acid-base reaction were studied herein using theoretical methods. The potential energy surfaces calculated using the density functional theory method with the Becke, 3-parameter, and Lee-Yang-Parr hybrid functionals and DZP basis set (DFT/B3LYP/DZP) and TD-DFT/B3LYP/DZP methods confirmed that the light-dependent reaction occurs in the rate-determining proton transfer process and that the mechanism involving intermolecular proton transfer between prFMNim and Glu282 (external base) is energetically more favorable than that involving intramolecular proton transfer in prFMNim (internal base). The thermodynamic results obtained from the transition state theory method suggested that the exothermic relaxation energy in the photo-to-thermal process can promote the spontaneous formation of a high-energy-barrier transition state, and an effective enzymatic decarboxylation could be achieved by slowing down the formation of the undesirable thermodynamically favorable product (prFMNket). Because the rate constant for formation of the high-energy-barrier transition state varies exponentially over the temperature range of 273-298 K, and experimental results have shown that incubating Fdc1 on ice results in a complete loss of enzyme activity, it is recommended to perform the decarboxylation reaction at 285 K to strike a balance between minimizing enzyme stability loss at 273 K and mitigating the effects of UV irradiation. The computational strategy and fundamental insights obtained in this study could serve as guidelines for future theoretical and experimental investigations on the same and similar photochemical systems.

Crystal structure of a thiolase from Archaeal Pyrococcus furiosus and its in silico functional annotation

In most of the eukaryotes and archaea, isopentenyl pyrophosphate (IPP) and dimethyl allyl pyrophosphate (DMAPP) essential building blocks of all isoprenoids synthesized in the mevalonate pathway. Here, the first enzyme of this pathway, acetoacetyl CoA thiolase (PFC_04095) from an archaea Pyrococcus furiosus is structurally characterized. The crystal structure of PFC_04095 is determined at 2.7 Å resolution, and the crystal structure reveals the absence of catalytic acid/base cysteine in its active site, which is uncommon in thiolases. In place of cysteine, His285 of HDAF motif performs both protonation and abstraction of proton during the reaction. The crystal structure shows that the distance between Cys83 and His335 is 5.4 Å. So, His335 could not abstract a proton from nucleophilic cysteine (Cys83), resulting in the loss of enzymatic activity of PFC_04095. MD simulations of the docked PFC_04095-acetyl CoA complex show substrate binding instability to the active site pocket. Here, we have reported that the stable binding of acetyl CoA to the PFC_04095 pocket requires the involvement of three protein complexes, i.e., thiolase (PFC_04095), DUF35 (PFC_04100), and HMGCS (PFC_04090).

Biallelic NUDT2 variants defective in mRNA decapping cause a neurodevelopmental disease.

Dysfunctional RNA processing caused by genetic defects in RNA processing enzymes has a profound impact on the nervous system, resulting in neurodevelopmental conditions. We characterized a recessive neurological disorder in 18 children and young adults from 10 independent families typified by intellectual disability, motor developmental delay and gait disturbance. In some patients peripheral neuropathy, corpus callosum abnormalities and progressive basal ganglia deposits were present. The disorder is associated with rare variants in NUDT2, a mRNA decapping and Ap4A hydrolysing enzyme, including novel missense and in-frame deletion variants. We show that these NUDT2 variants lead to a marked loss of enzymatic activity, strongly implicating loss of NUDT2 function as the cause of the disorder. NUDT2-deficient patient fibroblasts exhibit a markedly altered transcriptome, accompanied by changes in mRNA half-life and stability. Amongst the most up-regulated mRNAs in NUDT2-deficient cells, we identified host response and interferon-responsive genes. Importantly, add-back experiments using an Ap4A hydrolase defective in mRNA decapping highlighted loss of NUDT2 decapping as the activity implicated in altered mRNA homeostasis. Our results confirm that reduction or loss of NUDT2 hydrolase activity is associated with a neurological disease, highlighting the importance of a physiologically balanced mRNA processing machinery for neuronal development and homeostasis.

Tyrosine hydroxylase variants influence protein expression, cellular localization, stability, enzymatic activity and the physical interaction between tyrosine hydroxylase and GTP cyclohydrolase 1.

Tyrosine hydroxylase (TH) is the rate-limiting enzyme in dopamine biosynthesis catalyzing the tetrahydrobiopterin (BH4)-dependent hydroxylation of tyrosine to L-DOPA. Here, we analyzed 25 TH variants associated with various degrees of dopa-responsive dystonia and evaluate the effect of each variant on protein stability, activity and cellular localization. Furthermore, we investigated the physical interaction between TH and human wildtype (wt) GTP cyclohydrolase 1 (GTPCH) and the effect of variants on this interaction. Our in vitro results classify variants according to their resistance to proteinase K digestion into three groups (stable, intermediate, unstable). Based on their cellular localization, two groups of variants can be identified, variant group one with cytoplasmic distribution and variant group two forming aggregates. These aggregates do not correlate with loss of enzymatic activity but nevertheless might be a good target for molecular chaperones. Unfortunately, no obvious correlation between the half-life of a variant and its enzymatic activity or between solubility, stability and enzymatic activity of a given variant could be found. Excitingly, some variants disrupt the physical interaction between TH and human wildtype GTPCH, thereby interfering with enzymatic activity and offering new druggable targets for therapy. Taken together, our results highlight the importance of an in-depth molecular analysis of each variant in order to be able to classify groups of disease variants and to find specific therapies for each subgroup. Stand-alone in silico analyses predict less precise the effect of specific variants and should be combined with other in vitro analyses in cellular model systems.

Bienzyme co-immobilized on Zr-Based MOF with hierarchical porous structure for visual detection of glucose

Metal-organic frameworks (MOFs) are considered as promising carriers for immobilized enzymes. Whereas, some interrelated and restrictive problems exist in the construction of MOFs immobilized enzymes with excellent catalytic performance, resulting in reduced catalytic activity of the enzymes. In this work, a novel hierarchical porous structure of Zr-based metal-organic frameworks (Zr-MOF, HPU) was accurately constructed for co-immobilizing horseradish peroxidase (HRP) and glucose oxidase (GOx). The prepared HPU carrier can enrich the substrate by using the abundant micropore structure around the mesopore, thus reducing the mass transfer resistance and making up for the loss of enzyme activity during enzyme immobilization. Notably, GOx-HRP@HPU demonstrated outstanding catalytic efficiency and excellent stability, as well as ideal reusability, maintaining nearly 95% initial activity at 60 ℃ after 1 h incubation and above 65% initial activity after 10 reuses. Both the immobilization process and interaction of substrates with co-immobilized bienzyme reactor were investigated by means of microcalorimetry, which demonstrated HPU was an excellent platform for co-immobilized HRP and GOx. Furthermore, GOx-HRP@HPU was used for the detection of glucose with excellent detection performance.

A Model-Based 13C-Sucrose Breath Test Diagnostic for Gut Function Disorders Characterized by a Loss of Sucrase-Isomaltase Enzymatic Activity

BackgroundEnvironmental enteric dysfunction (EED) causes malnutrition in children in low-resource settings. Stable-isotope breath tests have been proposed as noninvasive tests of altered nutrient metabolism and absorption in EED, but uncertainty over interpreting the breath curves has limited their use. The activity of sucrose-isomaltase, the glucosidase enzyme responsible for sucrose hydrolysis, may be reduced in EED. We previously developed a mechanistic model describing the dynamics of the 13C-sucrose breath test (13C-SBT) as a function of underlying metabolic processes. ObjectivesThis study aimed to determine which breath test curve dynamics are associated with sucrose hydrolysis and with the transport and metabolism of the fructose and glucose moieties and to propose and evaluate a model-based diagnostic for the loss of activity of sucrase-isomaltase. MethodsWe applied the mechanistic model to 2 sets of exploratory 13C-SBT experiments in healthy adult participants. First, 19 participants received differently labeled sucrose tracers (U-13C fructose, U-13C glucose, and U-13C sucrose) in a crossover study. Second, 16 participants received a sucrose tracer accompanied by 0, 100, and 750 mg of Reducose, a sucrase-isomaltase inhibitor. We evaluated a model-based diagnostic distinguishing between inhibitor concentrations using receiver operator curves, comparing with conventional statistics. ResultsSucrose hydrolysis and the transport and metabolism of the fructose and glucose moieties were reflected in the same mechanistic process. The model distinguishes these processes from the fraction of tracer exhaled and an exponential metabolic process. The model-based diagnostic performed as well as the conventional summary statistics in distinguishing between no and low inhibition [area under the curve (AUC): 0.77 vs. 0.66–0.79] and for low vs. high inhibition (AUC 0.92 vs. 0.91–0.99). ConclusionsCurrent summary approaches to interpreting 13C breath test curves may be limited to identifying only gross gut dysfunction. A mechanistic model-based approach improved interpretation of breath test curves characterizing sucrose metabolism.

Enzymatic Activity and Its Relationships with the Total Phenolic Content and Color Change in the High Hydrostatic Pressure-Assisted Curing of Vanilla Bean (Vanilla planifolia).

Diverse enzymatic reactions taking place after the killing of green vanilla beans are involved in the flavor and color development of the cured beans. The effects of high hydrostatic pressure (HHP) at 50-400 MPa/5 min and blanching as vanilla killing methods were evaluated on the total phenolic content (TPC), polyphenoloxidase (PPO), and peroxidase (POD) activity and the color change at different curing cycles of sweating-drying (C0-C20) of vanilla beans. The rate constants describing the above parameters during the curing cycles were also obtained. The TPC increased from C1 to C6 compared with the untreated green beans after which it started to decrease. The 400 MPa samples showed the highest rate of phenolic increase. Immediately after the killing (C0), the highest increase in PPO activity was observed at 50 MPa (46%), whereas for POD it was at 400 MPa (25%). Both enzymes showed the maximum activity at C1, after which the activity started to decrease. As expected, the L* color parameter decreased during the entire curing for all treatments. An inverse relationship between the rate of TPC decrease and enzymatic activity loss was found, but the relationship with L* was unclear. HHP appears to be an alternative vanilla killing method; nevertheless, more studies are needed to establish its clear advantages over blanching.

Tafazzin mediates tamoxifen resistance by regulating cellular phospholipid composition in ER-positive breast cancer.

Tamoxifen is the frontline therapeutic agent for the estrogen receptor-positive (ER + ) subtype of breast cancer patients, which accounts for 70-80% of total breast cancer incidents. However, clinical resistance to tamoxifen has become increasingly common, highlighting the need to identify the underlying cellular mechanisms. In our study, we employed a genome-scale CRISPR-Cas9 loss-of-function screen and validation experiments to discover that Tafazzin (TAZ), a mitochondrial transacylase, is crucial for maintaining the cellular sensitivity of ER+ breast cancer cells to tamoxifen and other chemotherapies. Mechanistically, we found that cardiolipin, whose synthesis and maturation rely on TAZ, is required to maintain cellular sensitivity to tamoxifen. Loss of metabolic enzymatic activity of TAZ causes ERα downregulation and therapy resistance. Interestingly, we observed that TAZ deficiency also led to the upregulation of lysophosphatidylcholine (LPC), which in turn suppressed ERα expression and nuclear localization, thereby contributing to tamoxifen resistance. LPC is further metabolized to lysophosphatidic acid (LPA), a bioactive molecule that supports cell survival. Thus, our findings suggest that the depletion of TAZ promotes tamoxifen resistance through an LPC-LPA phospholipid synthesis axis, and targeting this lipid metabolic pathway could restore cell susceptibility to tamoxifen treatment.

Nattokinase: Insights into Biological Activity, Therapeutic Applications, and the Influence of Microbial Fermentation

Nattokinase, a serine protease that originates from the traditional food natto, has garnered widespread attention due to its pharmacological functions and therapeutic potential. This review aims to delve into the major advancements of nattokinase across various domains, particularly its emerging roles in Alzheimer’s disease prevention and the treatment of retinal diseases, thereby seeking to usher in a newfound hope in the fields of neurology and ophthalmology. However, the production and preservation of nattokinase present a multitude of challenges, including issues of unstable yield and enzyme activity loss. To address these challenges, we explore potential solutions such as the heterologous expression of the nattokinase gene, the optimization of microbial fermentation strategies, and innovative purification methods. Furthermore, we focus on enhancing the stability and protection of nattokinase through encapsulation and immobilization techniques, thus ensuring its sustainability across a wide array of applications. This review provides readers with the opportunity to gain an in-depth understanding of the diverse prospects for nattokinase applications. Future research directions will encompass a deeper exploration of its biological mechanisms, the development of novel nattokinase derivatives, and the extension of its applications into a broader spectrum of disease treatments and health maintenance.

In vitro functional study of fifteen SRD5A2 variants found in Chinese patients and the relation between the SRD5A2 genotypes and phenotypes

The 5α-reductase type 2 (5α-RD2) deficiency is one of the most common etiology of 46, XY disorders of sex development and is caused by pathogenic variants in SRD5A2. Massively parallel sequencing contributes to identification of numerous novel SRD5A2 variants, in vitro functional study could help to determine their pathogenicity. In this study, we aim to present the functional study of fifteen SRD5A2 variants found in Chinese patients and explore the genotype–phenotype association. We collected the clinical manifestation and genotype of 38 patients with 5α-RD2 deficiency who visited our center between 2009 and 2021. The pathogenicity of seven missense SRD5A2 variants, were predicted by in-silico tools. Furthermore, fifteen SRD5A2 variants without reported functional assay were studied in vitro to analyze the role of these variants in enzymatic activity. Twenty-four SRD5A2 rare variants were identified in 38 patients with 5α-RD2 deficiency. Fifteen variants without reported functional assay decreased the conversation of testosterone (T) to dihydrotestosterone(DHT) and caused the almost complete loss of enzyme activity (<8 %) in our in-vitro functional study. Thirty-eight patients with three different external genital phenotypes (complete female, clitoromegaly and hypospadias) were found to have same variants. Patients with different testicular position (scrotum/clitoris and cryptorchidism) were found to have same variants. Our study showed 15 SRD5A2 variants caused complete loss of 5α-RD2 enzyme activity by functional study. Patients with different clinical phenotypes can have the same genotypes and no obvious genotype–phenotype association exist in our series patients.