Expression Levels Of Enzymes Research Articles

Engineered β1-3-N-Acetylglucosaminyltransferase Facilitating the One-Pot Multienzyme Synthesis of Human Milk Oligosaccharides.

β1-3-linked N-acetylglucosaminide is a prevalent carbohydrate motif found in oligosaccharides, polysaccharides, glycoproteins, and glycolipids. It is a crucial component of human milk oligosaccharides (HMOs). Neisseria meningitidis β1-3-N-acetylglucosaminyltransferase (NmLgtA) catalyzes the formation of a glycosidic bond and has the potential for use in synthesizing HMOs. However, this application is hindered by challenges such as low levels of enzyme expression, poor stability, and significant aggregation. Since there is no available crystal structure for NmLgtA, we used its AlphaFold 2 predicted structure to identify potential unfavorable factors. We then modified the enzyme by removing the 17 N-terminal amino acids and substituting nine specific residues. The engineered NmLgtA-Opti exhibited improved thermal stability, increased soluble protein expression, complete relief from aggregation, and enhanced catalysis while maintaining its catalytic specificity and substrate promiscuity. Furthermore, NmLgtA-Opti maximizes substrate utilization and can be employed in a sequential one-pot multienzyme platform for high-yield production of HMOs.

Versican Proteolysis by ADAMTS: Understanding Versikine Expression in Canine Spontaneous Mammary Carcinomas

Background: The present study investigates VKINE, a bioactive proteolytic fragment of the proteoglycan VCAN, as a novel and significant element in the tumor extracellular matrix (ECM). Although VKINE has been recognized for its immunomodulatory potential in certain tumor types, its impact on ECM degradation and prognostic implications remains poorly understood. Objectives: This study aimed to evaluate VCAN proteolysis and its association with ADAMTS enzymes involved in extracellular matrix remodeling in spontaneous canine mammary gland cancer. Methods: The expression levels of VKINE, ADAMTS enzymes, and collagen fibers were comparatively analyzed in situ and in invasive areas of carcinoma in mixed tumor (CMT) and carcinosarcoma (CSS) with different prognoses. Results: VKINE was notably expressed in the stroma adjacent to the invasion areas in CMT, whereas ADAMTS-15 was identified as the enzyme associated with VCAN proteolysis. Inverse correlations were observed between type III collagen and VCAN expression in in situ areas. Conclusions: Our findings suggest that VKINE and ADAMTS-15 play crucial roles in the tumor microenvironment, influencing invasiveness and type III collagen deposition. This study contributes to a better understanding of the dynamics within the ECM, paving the way for potential new tools in diagnosing and treating human and canine mammary tumors.

Activation of the kynurenine pathway identified in individuals with covert hepatic encephalopathy

Background: HE is a neuropsychiatric complication of liver disease characterized by systemic elevation in ammonia and proinflammatory cytokines. These neurotoxins cross the blood-brain barrier and cause neuroinflammation, which can activate the kynurenine pathway (KP). This results in dysregulated production of neuroactive KP metabolites, such as quinolinic acid, which is known to cause astrocyte and neuronal death. Our aim was to compare KP activity between patients with covert HE (CHE), patients without encephalopathic cirrhosis (NHE), and healthy controls (HCs). Methods: This was a single-center prospective cohort study conducted between 2018 and 2021 at St Vincent’s Hospital, Sydney. Overall, 13 patients with CHE, 10 patients with NHE, and 12 with HC were recruited. Patients with cirrhosis were diagnosed with CHE if they scored ≤−4 on the Psychometric Hepatic Encephalopathy Score. KP metabolite levels were quantified on plasma samples via HPLC and gas chromatography/mass spectrometry. One-way Kruskal-Wallis test was used to compare the expression levels of KP enzymes. Results: KP was highly activated in patients with cirrhosis, demonstrated by higher levels of activity in the rate-limiting enzymes, indoleamine 2,3-dioxygenase, and tryptophan-2,3-dioxygenase in both CHE (65.04±20.72, p=0.003) and patients with NHE (64.85±22.10, p=0.015) compared to HC (40.95±7.301). Higher quinolinic acid concentrations were demonstrated in CHE (3726 nM±3385, p<0.001) and patients with NHE (1788 nM±632.3, p=0.032) compared to HC (624 nM±457). KP activation was positively correlated with inflammatory marker C-reactive protein in patients with CHE (Rs=0.721, p≤0.01). Conclusions: KP is highly activated in patients with CHE, resulting in heightened production of neurotoxic metabolites. Dysregulation of the pathway is demonstrable in patients who do not yet show clinical signs of neurocognitive impairment. Therapeutic agents that modulate KP activity may be able to alleviate symptoms of patients with CHE.

Diammonium glycyrrhizinate alleviates iron overload-induced liver injury in mice via regulating the gut-liver axis

BackgroundEvidence indicates a close association between iron overload (IO) and the pathogenesis of chronic liver diseases, highlighting the potential for interventions targeted at IO to impede or decelerate the progression of chronic liver diseases. Diammonium glycyrrhizinate (DG), the medicinal form of glycyrrhizic acid, a principal constituent of licorice, has been clinically employed as a hepatoprotective agent; however, its protective effect against IO-induced liver injury and underlying molecular mechanisms remain elusive. PurposeThe aim of the present study is to investigate the hepatoprotective effect of DG against IO-induced liver injury with a focus on the gut-liver axis. Study design and methodsAnimal models of IO-induced liver injury and DG treatment have been established in vivo. Iron deposition, liver injury, intestinal barrier damage, and liver inflammation were assessed in mice treated with iron dextran or DG. The microbiome composition in feces was analyzed using 16S rRNA full-length sequencing. Bile acids (BAs) profiles in feces were detected by UPLC-Q-TOF-MS technique, and the expression levels of receptors, enzymes or transporters involved in BAs metabolism were also determined. ResultsDG partially reduced the iron deposition and the levels of ferrous ion in the livers of mice with IO, thereby mitigating oxidative damage. DG also improved gut microbiota dysbiosis, repaired intestinal barrier damage, inhibited endotoxin translocation to the liver, and subsequently suppressed TLR4/NF-κB/NLRP3 pathway-mediated liver inflammation caused by IO. Moreover, DG modulated BAs metabolism disorder in IO mice, reducing the accumulation of BAs in the liver. ConclusionDG alleviates IO-induced liver injury in mice by regulating the gut-liver axis. This study provides novel insights into the underlying mechanisms through which DG ameliorates liver injury caused by IO.

SIRT2-mediated deacetylation of glutathione transferase alleviates oxidative damage and increases the heat tolerance of Pleurotus ostreatus

High-temperature stress (HS) severely threatens agricultural production. Pleurotus ostreatus is cultivated in many parts of the world, and its growth is strongly affected by HS. We previously reported that metabolic rearrangement occurred in HS, but the gene expression levels of several key enzymes remained unchanged. Therefore, in this study, we investigated the contribution of posttranslational modifications of proteins to HS resistance in P. ostreatus. We found that the level of acetylation of P. ostreatus decreased under short-term HS treatment and increased as the duration of HS treatment increased. Acetylation omics revealed that almost all metabolic enzymes were acetylated. We found that deacetylation under HS can improve the growth recovery ability of mycelia, the activity of matrix-degrading enzyme, and the contents of antioxidants, such as nicotinamide adenine dinucleotide phosphate (NADPH) and glutathione (GSH), but can decreased H2O2 levels. In vitro acetylation experiments and point mutations revealed that the deacetylase SIRT2 increased the activity of glutathione transferases (GSTs) by deacetylating GST1 66K, GST2 206K, and GST2 233K. Together, SIRT2 is activated by short-term HS and improves its antioxidant activity by deacetylating GSTs, thereby improving the resistance of P. ostreatus to HS. In this study, we identified new non-histone substrate proteins and new lysine acetylation sites of SIRT2 under HS. We also discovered the role of non-histone acetylation in the adaptation of organisms to HS.

Larvicidal and enzyme inhibition effects of Phoenix pusilla derived Methyl oleate and malathion on Aedes aegypti strains

This study explores the larvicidal potential of methanolic flower extracts from Phoenix pusilla (Pp-Fe), its major compound, and malathion (MLT), against laboratory strain (LS) and field strain (FS) of Aedes aegypti, the dengue mosquito vector. We identified thirty-one derivatives, with methyl oleate (MO) comprising 28.5% of Pp-Fe. Comparative efficacy evaluations were performed using peak dosages of Pp-Fe (500 ppm), MO (5 ppm), and MLT (5 ppm) on LS and FS larvae. Both LS and FS second instars showed higher susceptibility to Pp-Fe (95% and 93%, respectively) and MO (85% and 83%, respectively). MLT resulted in significant mortality rates among LS larvae (98%) and notable reductions among FS larvae (71%). The expression levels of key biomarker enzymes (carboxylesterase, GST, and CYP450) exhibited a consistent decrease and subsequent upregulation in LS and FS larvae following exposure to Pp-Fe and MO, contrasting with the significant expression variations observed in LS and FS larvae exposed to MLT. LS larvae demonstrated heightened susceptibility and evident midgut cell damage following all treatments, suggesting potential disparities in susceptibility and adaptive responses between LS and FS strains towards MLT. These observations underscore the promising larvicidal attributes of Pp-Fe and MO, emphasizing the need for further exploration of their mechanisms of action in the development of environmentally sustainable mosquito control strategies and resistance management.

Phenylpropanoid pathway mediated the defense response of ‘Korla’ fragrant pear against Alternaria alternata infection

Alternaria alternata has been found to be the dominating pathogenic fungus of harvested ‘Korla’ fragrant pear, and the resulting blackhead disease is a significant factor affecting the storage quality of pears. The present study explored the specific mechanisms by which the phenylpropanoid pathway mediates the defense response to A. alternata infection in pear fruit. In the A. alternata-inoculated group, the fruit exhibited increased activity and gene expression levels of key enzymes (PAL, C4H, and 4CL) as well as higher content of phenolic acids (trans-cinnamic acid, ferulic acid, caffeic acid, p-coumaric acid, and sinapic acid) and total phenol in the general phenylpropanoid pathway. In the mid-to-late storage period, the activity and gene expression levels of key enzymes in the lignin biosynthetic pathway (CCR and CAD) were suppressed, and the content of lignin monomers (sinapyl alcohol, coniferyl alcohol, and p-coumaryl alcohol) and lignin was reduced. Notably, the activity and gene expression levels of flavonoid biosynthetic pathway-related enzymes (CHS and CHI) as well as the content of various flavonoids (naringenin, apigenin, rutin, quercetin, and epicatechin) and total flavonoids continuously increased in response to A. alternata infection in the early to middle stages of storage but declined in the late storage period. In summary, the initial infection of A. alternata infection activated the stress response of pear fruit, particularly the phenylpropanoid–flavonoid branch pathway, to enhance the fruit’s defense against pathogens, but with the prolongation of the infestation time, the fruit could not continuously resist the invasion of pathogens, ultimately leading to the outbreak of disease. The present findings furnish a theoretical foundation further elucidating the interaction between ‘Korla’ fragrant pear and A. alternata and for developing an effective strategy to control the blackhead disease.

The Role of PI3K/AKT/HIF-1α Pathway in the Effect of Nano-TiO2 on Lactate Production in TM4 Cells.

Titanium dioxide nanoparticles (nano-TiO2) can cause a reduction in sperm counts, and lactate production in Sertoli cells plays a key role in spermatogenesis. The aim of this study was to evaluate the effect of nano-TiO2 on lactate production in mouse Sertoli cell line (TM4 cells) and to investigate whether the effect is mediated through the PI3K/AKT/HIF-1α pathway. After TM4 cells were treated with different concentrations of nano-TiO2 for 48 h, cell viability, contents of glucose and lactate, and the expression levels of GLUT3 and key enzymes (HK1, HK2, PFKM, ENO1, LDH) during lactate production were detected. PI3K/AKT/HIF-1α pathway proteins were measured. In addition, PI3K agonist (IGF-1) was added to explore whether nano-TiO2 regulates HIF-1α through PI3K/AKT pathway, thereby affecting the production of lactate in TM4 cells. The results showed that nano-TiO2 significantly inhibited TM4 cell viability, increased glucose content, decreased lactate content, and downregulated the expression levels of GLUT3 and key enzymes during lactate production. Meanwhile, nano-TiO2 decreased the expression of PI3K/AKT pathway phosphorylated proteins and HIF-1α, and IGF attenuated this effect of nano-TiO2. Collectively, nano-TiO2 downregulated the expression level of proteins and enzymes related to lactate production in TM4 cells by inhibiting PI3K/AKT/HIF-1α pathway, resulting in the decrease of lactate production in TM4 cells.

Light-Induced Nanobody-Mediated Targeted Protein Degradation for Metabolic Flux Control.

In metabolic engineering, increasing chemical production usually involves manipulating the expression levels of key enzymes. However, limited synthetic tools exist for modulating enzyme activity beyond the transcription level. Inspired by natural post-translational mechanisms, we present targeted enzyme degradation mediated by optically controlled nanobodies. We applied this method to a branched biosynthetic pathway, deoxyviolacein, and observed enhanced product specificity and yield. We then extend the biosynthesis pathway to violacein and show how simultaneous degradation of two target enzymes can further shift production profiles. Through the redirection of metabolic flux, we demonstrate how targeted enzyme degradation can be used to minimize unwanted intermediates and boost the formation of desired products.

Optimizing HMG-CoA Synthase Expression for Enhanced Limonene Production in Escherichia coli through Temporal Transcription Modulation Using Optogenetics.

Overexpression of a single enzyme in a multigene heterologous pathway may be out of balance with the other enzymes in the pathway, leading to accumulated toxic intermediates, imbalanced carbon flux, reduced productivity of the pathway, or an inhibited growth phenotype. Therefore, optimal, balanced, and synchronized expression levels of enzymes in a particular metabolic pathway is critical to maximize production of desired compounds while maintaining cell fitness in a growing culture. Furthermore, the optimal intracellular concentration of an enzyme is determined by the expression strength, specific timing/duration, and degradation rate of the enzyme. Here, we modulated the intracellular concentration of a key enzyme, namely HMG-CoA synthase (HMGS), in the heterologous mevalonate pathway by tuning its expression level and period of transcription to enhance limonene production in Escherichia coli. Facilitated by the tuned blue-light inducible BLADE/pBad system, we observed that limonene production was highest (160 mg/L) with an intermediate transcription level of HMGS from moderate light illumination (41 au, 150 s ON/150 s OFF) throughout the growth. Owing to the easy penetration and removal of blue-light illumination from the growing culture which is hard to obtain using conventional chemical-based induction, we further explored different induction patterns of HMGS under strong light illumination (2047 au, 300 s ON) for different durations along the growth phases. We identified a specific timing of HMGS expression in the log phase (3-9 h) that led to optimal limonene production (200 mg/L). This is further supported by a mathematical model that predicts several periods of blue-light illumination (3-9 h, 0-9 h, 3-12 h, 0-12 h) to achieve an optimal expression level of HMGS that maximizes limonene production and maintains cell fitness. Compared to moderate and prolonged transcription (41 au, 150 s ON/150 s OFF, 0-73 h), strong but time-limited transcription (2047 au, 300 s ON, 3-9 h) of HMGS could maintain its optimal intracellular concentration and further increased limonene production up to 92% (250 mg/L) in the longer incubation (up to 73 h) without impacting cell fitness. This work has provided new insight into the "right amount" and "just-in-time" expression of a critical metabolite enzyme in the upper module of the mevalonate pathway using optogenetics. This study would complement previous findings in modulating HMGS expression and potentially be applicable to heterologous production of other terpenoids in E. coli.

Transcriptomic and physiological analyses reveal the response mechanism of the liver under acute ammonia stress in the Chinese soft-shelled turtle (Pelodiscus sinensis)

Greenhouse intensive aquaculture is a common culture mode for juvenile Chinese soft-shelled turtles (CSST). Under this culture mode, the juvenile CSST are vulnerable to the stress of high ammonia‑nitrogen levels in the water, resulting in a suboptimal health state that may lead to disease susceptibility. However, few studies have reported the response mechanism of the CSST in the ammonia‑nitrogen environment. In this study, we detected the lethal range concentration of non-ionic ammonia‑nitrogen in CSST and examined the impact of acute ammonia nitrogen exposure for durations of 0 h, 12 h, 24 h, 48 h, 96 h, and 144 h on the blood indices and liver function in CSST. The results show that the lethal concentration range of non-ionic ammonia‑nitrogen is 23.0 mg/mL to 70.5 mg/mL. In the blood, blood ammonia, urea nitrogen and aspartate aminotransferase activity increased significantly, suggesting that the high ammonia‑nitrogen environment caused hyperammonemia, increased urea synthesis and liver damage in the CSST. The impact of acute ammonia nitrogen on the liver function of the CSST was assessed through analysis of liver histopathology, antioxidant defense, ammonia metabolism pathway, and transcriptome data. The results showed that the hepatic cord structure within the liver tissue of the CSST was destroyed, with the presence of vacuoles and edema. In addition, hepatic superoxide dismutase (SOD) and catalase (CAT) enzyme activities and Malondialdehyde (MDA) content were significantly increased, and the mRNA expression levels of Mn-SOD, Cu/Zn-SOD, and CAT were significantly increased, suggesting that the hepatic antioxidant system was activated to resist the environmental stresses caused by ammonia‑nitrogen. Moreover, there was a significant increase in the enzyme activity and expression levels of glutamate dehydrogenase (GDH), and a significant decrease in the enzyme activity and no significant change in the expression levels of glutamine synthetase (GS), suggesting that NH4+ is synthesized from glutamate by GDH in the liver to enter the urea metabolic cycle, and that glutamine is not synthesized for storage. Analysis of transcriptome data revealed potential effects on energy metabolism and lipid metabolism in response to short-term (48 h) ammonia‑nitrogen stress. In contrast, prolonged (96 h) exposure to ammonia nitrogen stress may increase the susceptibility of CSST to pathogens, and trigger the tissue repair mechanisms in the liver. These findings provide new insights into the potential risks and hazards of ammonia‑nitrogen to CSST and provide a scientific foundation for managing water quality in CSST aquaculture.

Investigation of the acute effect of the synthetic hemodialysis membrane on the expression of XRCC1 and PARP1 in chronic hemodialysis patients.

The interaction between blood from end-stage renal failure patients undergoing hemodialysis treatment and the hemodialysis (HD) membranes used may lead to DNA damage, contingent upon the biocompatibility of the membranes. Given that this process could impact the disease's course, it is crucial to assess the efficacy of DNA repair mechanisms. In our study, we investigated the gene expression levels of XRCC1 and PARP1 enzymes, which are involved in the base excision repair (BER) repair mechanism crucial for repairing oxidative DNA damage, in 20 end-stage renal disease (ESRD) patients undergoing HD treatment both before and after dialysis sessions. Additionally, we compared our findings with those from 20 healthy controls. We assessed gene expression levels using real-time polymerase chain reaction (qRT-PCR). We observed that the HD process utilizing a polysulfone membrane did not impact the expression levels of genes. However, we noted a lower expression level of the PARP1 gene in ESRD patients undergoing HD compared to the control group (0.021 ± 0.005 vs 0.0019 ± 0.0013, p = 0.0001). Although our study findings indicate that HD membranes do not affect gene expression overall, the specific decrease in PARPI gene expression suggests that the effectiveness of the BER DNA repair mechanism is impaired in ESRD patients, which may play a significant role in the progression of the disease.

Recovery of protein-rich biomass from surimi rinsing wastewater by using a sustainable cold plasma treatment

Surimi rinsing wastewater is typically discarded, causing waste of protein resources and environmental pollution. This study investigated the technology of a cold atmospheric plasma jet (CAPJ) for the recovery of protein-rich biomass (PRB), including myofibrillar proteins (MPs) and sarcoplasmic proteins (SPs), from surimi rinsing wastewater. The protein recovery yield was up to 59.84 %. CAPJ induced an increase in carbonyl and decreased sulfhydryl in protein content. Furthermore, the secondary structure of the protein was unfolded, particularly the transition from α-helix to β-sheet. The formation of disulfide bonds and increased hydrophobic interactions promoted protein aggregation (the particle size from 185.76 nm to 1869.07 nm, P < 0.05) and reduced solubility. The proteomic results indicated that CAPJ increased the expression level of antioxidant enzymes. Overall, the CAPJ technology could recover proteins from surimi rinsing wastewater for industrial application, which will promote the sustainable development of the surimi industry.

Population Pharmacokinetic Quantification of CYP2D6 Activity in Codeine Metabolism in Ambulatory Surgical Patients for Model-Informed Precision Dosing.

Codeine metabolism in humans is complex due to the involvement of multiple cytochrome P450 (CYP) enzymes, and has a strong genetic underpinning, which determines the levels of relevant CYP450 enzyme expression in vivo. Polymorphic CYP2D6 metabolises codeine to morphine via O-demethylation, while a strong correlation between CYP2D6 phenotype and opioidergic adverse effects of codeine is well documented. The aim of this study was to quantify the effect of CYP2D6 genotype on the biotransformation of codeine. We conducted a prospective clinical trial with 1000 patients, during which ambulatory patients were administered 60 mg of codeine preoperatively and the association between CYP2D6 activity and morphine exposure across various CYP2D6 genotypes was quantified using a population pharmacokinetic model. Plasma concentration data for codeine and its primary metabolites were obtained from 997 patients and CYP2D6 genotype was screened for study subjects, and respective sums of activity scores assigned for each CYP2D6 allele were used as covariates in model development. Our final model predicts the disposition of codeine and the formation of morphine, codeine-6-glucuronide and morphine-3-glucuronide adequately while accounting for variability in morphine exposure on the basis of CYP2D6 genotype. In agreement with previous results, patients with decreased function alleles (CYP2D6*10 and *41) showed varying levels of decrease in CYP2D6 activity that were inconsistent with increasing activity scores. Model simulations demonstrate that morphine concentrations in ultrarapid CYP2D6 metabolisers reach systemic concentrations that can potentially cause respiratory depression (over 9.1 ng/mL), and have 218% higher exposure (19 versus 8.7 µg · h/L, p < 0.001) to morphine than normal metabolisers. Similarly, poor and intermediate metabolisers had significantly reduced morphine exposure (1.0 and 3.7 versus 8.7 µg · h/L, p < 0.001) as compared with normal metabolisers. Our final model leads the way in implementing model-informed precision dosing in codeine therapy and identifies the use of genetic testing as an integral component in the effort to implement rational pharmacotherapy with codeine.

AKH/AKHR signalling system induced antioxidant response mediated by entomopathogenic fungi in Nilaparvata lugens (Stål)

The brown planthopper Nilaparvata lugens is one of the most economically important rice crop pests in Asia. Entomopathogenic fungi (EPF) have been developed as a biological control of N. lugens. Insect adipokinetic hormones (AKHs) are pleiotropic hormones that play a protective role in response to oxidative stress. However, the role of AKH in the anti-oxidative response of N. lugens to EPFs (Metarhizium anisopliae and Beauveria bassiana) infection remains largely unexplored. In this study, the results of relative enzyme activities and expression levels of antioxidant enzymes demonstrated the response of the antioxidant system of N. lugens to EPF infection. Additionally, the expression of AKH/adipokinetic hormone receptor (AKHR) also induced responding to the infection of EPF in N. lugens. Silencing NlAKH or NlAKHR significantly increased mortality in nymphs treated with fungi compared with controls, whereas the injection of AKH peptide decreased mortality. Further research indicated that the AKH/AKHR system positively influenced antioxidant enzymes, potentially involving the transcription factors forkhead-box O and Cap’ n’ collar C. These findings provide an important theoretical basis for developing new pest control agents targeting the neuropeptide AKH and offer new insights for mitigating brown planthopper resistance and promoting green control strategies.

Thioredoxin reductase inhibition and glutathione depletion mediated by glaucocalyxin A promote intracellular disulfide stress in gastric cancer cells.

Thioredoxin reductase 1 (TXNRD1) has been identified as one of the promising chemotherapeutic targets in cancer cells. Therefore, a novel TXNRD1 inhibitor could accelerate chemotherapy in clinical anticancer research. In this study, glaucocalyxin A (GlauA), a natural diterpene extracted from Rabdosia japonica var. glaucocalyx, was identified as a novel inhibitor of TXNRD1. We found that GlauA effectively inhibited recombinant TXNRD1 and reduced its activity in gastric cancer cells without affecting the enzyme's expression level. Mechanistically, the selenocysteine residue (U498) of TXNRD1 was irreversibly modified by GlauA through a Michael addition. Additionally, GlauA formed a covalent adduct with glutathione (GSH) and disrupted cellular redox balance by depleting cellular GSH. The inhibition of TXNRD1 and depletion of GSH by GlauA conferred its cytotoxic effects in spheroid culture and Transwell assays in AGS cells. The disulfide stress induced cytotoxicity of GlauA could be mitigated by adding reducing agents, such as DTT and β-ME. Furthermore, the FDA-approval drug auranofin, a TXNRD1 inhibitor, triggered oligomerization of the cytoskeletal protein Talin-1 in AGS cells, indicating that inhibiting TXNRD1 triggered disulfide stress. In conclusion, this study uncovered GlauA as an efficient inhibitor of TXNRD1 and demonstrated the potential of TXNRD1 inhibition as an effective anticancer strategy by disrupting redox homeostasis and inducing disulfide stress.

PYGB targeted by androgen receptor contributes to tumor progression and metabolic reprogramming in esophageal squamous carcinoma

BackgroundThe incidence and mortality rates of esophageal squamous cell carcinoma (ESCC) are conspicuously augmented in men in contrast to women. The androgen receptor (AR), prevalently associated with the manifestation of male characteristics, is regarded as a pivotal determinant in tumor progression. Nevertheless, its exact role in ESCC remains insufficiently delineated. MethodsIn this study, we probed the expression levels of AR and glucose metabolism enzymes in ESCC tissues by means of immunohistochemistry. We exploited chromatin immunoprecipitation and dual luciferase reporter assays to delve into the transcriptional regulatory interrelationships between AR and these enzymes. A gamut of molecular techniques—including multi-omics sequencing, colony formation assays, cell counting kit 8 (CCK8), 5-Ethynyl-2′-deoxyuridine (EdU) incorporation assays, wound-healing assays, transwell migration assays, extracellular acidification rate (ECAR) measurements, lipid droplet fluorescence imaging, and xenograft models—were enlisted to illuminate the functions of these enzymes within ESCC cells. ResultsOur discoveries manifested that AR expression was strikingly higher in male ESCC tissues than in their female counterparts. Significantly, we discerned that glycogen phosphorylase B (PYGB), a cardinal enzyme implicated in glucose metabolism, demonstrated not only a positive correlation with AR expression but also an association with adverse prognostic outcomes for ESCC patients. Moreover, AR directly binds to the promoter region of the PYGB gene, thereby potentiating its transcriptional activity. This upregulation of PYGB was ascertained to facilitate proliferation, invasion, and metastasis among ESCC cells while intensifying glycolysis and modifying lipid metabolism pathways within these cells. In animal models employing nude mice, elevated PYGB levels were witnessed to expedite subcutaneous tumor growth as well as lung metastasis. ConclusionsCollectively, our study establishes PYGB as a direct target of AR that assumes an indispensable role in both tumor progression and metabolic reprogramming affiliated with ESCC, thus paving novel avenues for therapeutic strategies centered on metabolic intercessions.

CRISPR associated enzymes are mislocalized to the cytoplasm in iPSC-derived neurons resulting in KRAB-specific degradation.

The use of CRISPR-associated enzymes in iPSC-derived neurons for precise gene targeting and high-throughput gene perturbation screens offers great potential but presents unique challenges compared to dividing cell lines. CRISPRi screens in iPSC-derived neurons and glia have already been successful in relating gene function to neurological phenotypes; however, loss of dCas9-KRAB expression after differentiation has been observed by many labs and has been largely ascribed to transgene silencing after differentiation. Here, we investigated the expression levels of different CRISPR enzymes in iPSC and Ngn2-derived neurons using piggybac delivery. We found that the commonly used dCas9-KRAB (using the KOX1 domain) displayed dramatic reduction in protein expression levels following neuronal differentiation, yet surprisingly, nCas9 constructs retained comparable protein expression between iPSCs and neurons. We further found that CRISPR constructs, primarily relying on the SV40 Nuclear Localization Signal (NLS), fail to efficiently localize to the nuclei of neurons, despite having robust nuclear levels in iPSCs, leading to KRAB-specific cytoplasmic degradation. By adding a neuronal-specific NLS, we were able to correct neuronal nuclear localization and protein expression, confirming the contribution of mislocalization to the instability of dCas9-KRAB in neurons. As the lack of nuclear localization can have a profound impact on editing and gene perturbation efficiency, we suggest further investigation across both cultured and in-vivo postmitotic cell models.

Chitosan, Methyl Jasmonate, and Silicon Induce Resistance to Angular Leaf Spot in Common Bean, Caused by Pseudocercospora griseola, with Expression of Defense-Related Genes and Enzyme Activities

This study assessed the efficacy of chitosan, methyl jasmonate, and silicon in the reduction of disease severity and the induction of defense responses in common bean plants against angular leaf spot caused by Pseudocercospora griseola. The expression level of several pathogenesis-related (PR) proteins, PR1, PR2 (β-1,3-glucanase), and PR3 (chitinase), and defense-related enzymes, phenylalanine ammonia-lyase, peroxidase, and lipoxygenase, was analyzed at different time points in common bean plants after different treatments. Elicitor treatments significantly reduced disease severity 21 days after inoculation, with silicon at a 2 mM concentration proving most effective with 38.93% disease control, followed by 1 mM MeJA and 2% chitosan, respectively. Treatments with chitosan, methyl jasmonate, and silicon, regardless of pathogen infection, significantly elevated PR1, PR2, and PR3 gene expressions at 48 h after inoculation (hpi). PAL and POD activities were similarly increased following elicitor treatments and pathogen infection, especially at 48 hpi. Chemical elicitors applied post-inoculation induced PR proteins, PAL, and POD enzyme activities at 48 hpi, while LOX activity exhibited a variable fluctuation with treatments. These findings suggested that chemical elicitors, especially silicon, were effective in reducing ALS disease severity in common beans, with improved resistance associated with the expression of pathogen-responsive genes. This study is the first to analyze the expression profiles of defense-related genes in common beans treated with chemical elicitors prior to P. griseola infection.

PBPK modeling: What is the role of CYP3A4 expression in the gastrointestinal tract to accurately predict first-pass metabolism?

Gastrointestinal first-pass metabolism plays an important role in bioavailability and in drug-drug interactions. Physiologically-based pharmacokinetic (PBPK) modeling is a powerful tool to integrate these processes mechanistically. However, a correct bottom-up prediction of GI first-pass metabolism is challenging and depends on various model parameters like the level of enzyme expression and the basolateral intestinal mucosa permeability (Pmucosa). This work aimed to investigate if cytochrome P450 (CYP) 3A4 expression could help predict the first-pass effect using PBPK modeling or whether additional factors like Pmucosa do play additional roles using PBPK modeling. To this end, a systematic review of the absolute CYP3A expression in the human gastrointestinal tract and liver was conducted. The resulting CYP3A4 expression profile and two previously published profiles were applied to PBPK models of seven CYP3A4 substrates (alfentanil, alprazolam, felodipine, midazolam, sildenafil, triazolam, and verapamil) built-in PK-Sim®. For each compound, it was assessed whether first-pass metabolism could be adequately predicted based on the integrated CYP3A4 expression profile alone or whether an optimization of Pmucosa was required. Evaluation criteria were the precision of the predicted interstudy bioavailabilities and area under the concentration-time curves. It was found that none of the expression profiles provided upfront an adequate description of the extent of GI metabolism and that optimization of Pmucosa as a compound-specific parameter improved the prediction of most models. Our findings indicate that a pure bottom-up prediction of gastrointestinal first-pass metabolism is currently not possible and that compound-specific features like Pmucosa must be considered as well.