Antioxidant, anti-amylase, anti-lipase, and efficiency of Satureja fatty acid on the anti-inflammatory parameters in lipopolysaccharide-stimulated macrophage through Nrf2/NF-kB/NADH oxidase pathway.

Satureja is an aromatic plant that is used for flavoring, perfume, and food manufacturing due to its pleasant essential oil. Modern medicine research revealed several biological activities of Satureja essential oil, including antifungal, antibacterial, antiviral, antioxidant, anticancer, and anti-inflammatory. However, the functional properties of Satureja fatty acid have not been explored. This study examined the fatty acid profile, lipid nutritional quality, antioxidant, anti-amylase, and anti-lipase capacities of Satureja. The efficiency of Satureja fatty acid on the anti-oxidative and anti-inflammatory parameters in LPS-induced macrophage through the Nrf2/NF-kB/NADH oxidase pathway was examined. The whole lipid extract was prepared with chloroform/methanol/water solution. Fatty acids methyl ester from whole lipid extract were prepared with methanol/sulfuric acid reagent. The fatty acid profile was analyzed using gas chromatography-mass spectrometry. Total antioxidant was determined by ABTS decolorization. Lipase and amylase activities were determined by monitoring the decomposition of p-nitrophenyl butyrate and starch. The macrophage cell line was grown in DMEM media in the presence of fatty acid. The hydrogen peroxide production in treated cells was monitored using the FOX reagent. NADH oxidase activity was measured by monitoring NADH breakdown. The expression of NOX, NF-kB, and NRF2, were tested in the treated cells by real-time PCR. The main components of the Satureja fatty acid were linolenic acid (24.67-37.32%), palmitic acid (10.65-20.29%), linoleic acid (8.31-13.39%), oleic acid (4.42-14.35%), stearic acid (2.76-8.77%) and palmitoleic acid (1.77-4.95%). Given the nutritional quality, omega-3 PUFA (23.58-37.32%), SFA (21.53-26.70%), omega-6 PUFA (10.86-16.14%), omega-9 MUFA (4.42-14.35%), and omega-7 MUFA (1.77-4.95%) comprise the majority of fatty acids. Satureja fatty acid has a promising unsaturation index (120.77-164.27), PUFA/MUFA (2.07-6.41), hypocholesterolemic index (2.44-3.47), health-promoting index (2.03-2.42), PUFA/SFA (1.37-1.94), nutritive value index (0.53-1.71), MUFA/SFA (0.30-0.80) omega-6/omega-3 (0.34-0.65), atherogenicity index (0.41-0.49), and thrombogenicity index (0.17-0.27). Satureja fatty acid displayed strong antioxidant capacity (with IC50 ranging from 354 to 428 µg/mL), anti-lipase capacity (with IC50 ranging from 354 to 428 µg/mL), and anti-amylase capacity (with IC50 ranging from 370 to 390 µg/mL). LPS induced the expression of NOX, NRF2, and NF-kB and the synthesis of hydrogen peroxide in macrophage cells. In LPS-stimulated macrophages, Satureja fatty acid reduced NOX expression, hydrogen peroxide, and NF-kB expression and increased NRF2 at 0.04 mg/mL. In conclusion, Satureja fatty acids have potent antioxidant, anti-amylase, anti-lipase, and anti-inflammatory activities. The mechanisms in lowering oxidative stress markers depended on down-regulating superoxide-producing enzymes at gene and protein levels. Satureja polyunsaturated omega-3 fatty acids could be recommended for healthy products combined with dietary therapy to treat obesity, diabetes, and oxidative stress.

[Ginsenoside Rg_1 ameliorates OGD/R-induced PC12 cell injury by inhibiting autography via IRE1-JNK-CHOP pathway].

This study investigated the protective effect of ginsenoside Rg_1(GRg_1) on oxygen and glucose deprivation/reoxygenation(OGD/R)-injured rat adrenal pheochromocytoma(PC12) cells and whether the underlying mechanism was related to the regulation of inositol-requiring enzyme 1(IRE1)-c-Jun N-terminal kinase(JNK)-C/EBP homologous protein(CHOP) signaling pathway. An OGD/R model was established in PC12 cells, and PC12 cells were randomly classified into control, model, OGD/R+GRg_1(0.1, 1, 10 µmol·L~(-1)), OGD/R+GRg_1+rapamycin(autophagy agonist), OGD/R+GRg_1+3-methyladenine(3-MA,autophagy inhibitor), OGD/R+GRg_1+tunicamycin(endoplasmic reticulum stress agonist), OGD/R+GRg_1+4-phenylbutyric acid(4-PBA, endoplasmic reticulum stress inhibitor), and OGD/R+GRg_1+3,5-dibromosalicylaldehyde(DBSA, IRE1 inhibitor) groups. Except the control group, the other groups were subjected to OGD/R treatment, i.e., oxygen and glucose deprivation for 6 h followed by reoxygenation for 6 h. Cell viability was detected by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl tetrazolium bromide(MTT) assay. Apoptosis was detected by Hoechst 33342 staining, and the fluorescence intensity of autophagosomes by the monodansylcadaverine(MDC) assay. Western blot was employed to determine the expression of autophagy-related proteins(Beclin1, LC3-Ⅱ, and p62) and the pathway-related proteins [IRE1, p-IRE1, JNK, p-JNK, glucose-regulated protein 78(GRP78), and CHOP]. The results showed that GRg_1 dose-dependently increased the viability of PC12 cells and down-regulated the expression of Beclin1, LC3-Ⅱ, p-IRE1, p-JNK, GRP78, and CHOP, compared with the model group. Furthermore, GRg_1 decreased the apoptosis rate and MDC fluorescence intensity and up-regulated the expression of p62 protein. Compared with the OGD/R+GRg_1(10 µmol·L~(-1)) group, OGD/R+GRg_1+rapamycin and OGD/R+GRg_1+tunicamycin groups showed increased apoptosis rate and MDC fluorescence intensity, up-regulated protein levels of Beclin1, LC3-Ⅱ, p-IRE1, p-JNK, GRP78, and CHOP, decreased relative cell survival rate, and down-regulated protein level of p62. The 3-MA, 4-PBA, and DBSA groups exerted the opposite effects. Taken together, GRg_1 may ameliorate OGD/R-induced PC12 cell injury by inhibiting autophagy via the IRE1-JNK-CHOP pathway.

In Silico Comparison of Bioactive Compounds Characterized from Azadirachta indica with an FDA-Approved Drug against Schistosomal Agents: New Insight into Schistosomiasis Treatment.

The burden of human schistosomiasis, a known but neglected tropical disease in Sub-Saharan Africa, has been worrisome in recent years. It is becoming increasingly difficult to tackle schistosomiasis with praziquantel, a drug known to be effective against all Schistosoma species, due to reports of reduced efficacy and resistance. Therefore, this study seeks to investigate the antischistosomal potential of phytochemicals from Azadirachta indica against proteins that have been implicated as druggable targets for the treatment of schistosomiasis using computational techniques. In this study, sixty-three (63) previously isolated and characterized phytochemicals from A. indica were identified from the literature and retrieved from the PubChem database. In silico screening was conducted to assess the inhibitory potential of these phytochemicals against three receptors (Schistosoma mansoni Thioredoxin glutathione reductase, dihydroorotate dehydrogenase, and Arginase) that may serve as therapeutic targets for schistosomiasis treatment. Molecular docking, ADMET prediction, ligand interaction, MMGBSA, and molecular dynamics simulation of the hit compounds were conducted using the Schrodinger molecular drug discovery suite. The results show that Andrographolide possesses a satisfactory pharmacokinetic profile, does not violate the Lipinski rule of five, binds with favourable affinity with the receptors, and interacts with key amino acids at the active site. Importantly, its interaction with dihydroorotate dehydrogenase, an enzyme responsible for the catalysis of the de novo pyrimidine nucleotide biosynthetic pathway rate-limiting step, shows a glide score and MMGBSA of -10.19 and -45.75 Kcal/mol, respectively. In addition, the MD simulation shows its stability at the active site of the receptor. Overall, this study revealed that Andrographolide from Azadirachta indica could serve as a potential lead compound for the development of an anti-schistosomal drug.

Riboswitch and small RNAs modulate btuB translation initiation in Escherichia coli and trigger distinct mRNA regulatory mechanisms.

Small RNAs (sRNAs) and riboswitches represent distinct classes of RNA regulators that control gene expression upon sensing metabolic or environmental variations. While sRNAs and riboswitches regulate gene expression by affecting mRNA and protein levels, existing studies have been limited to the characterization of each regulatory system in isolation, suggesting that sRNAs and riboswitches target distinct mRNA populations. We report that the expression of btuB in Escherichia coli, which is regulated by an adenosylcobalamin (AdoCbl) riboswitch, is also controlled by the small RNAs OmrA and, to a lesser extent, OmrB. Strikingly, we find that the riboswitch and sRNAs reduce mRNA levels through distinct pathways. Our data show that while the riboswitch triggers Rho-dependent transcription termination, sRNAs rely on the degradosome to modulate mRNA levels. Importantly, OmrA pairs with the btuB mRNA through its central region, which is not conserved in OmrB, indicating that these two sRNAs may have specific targets in addition to their common regulon. In contrast to canonical sRNA regulation, we find that OmrA repression of btuB is lost using an mRNA binding-deficient Hfq variant. Together, our study demonstrates that riboswitch and sRNAs modulate btuB expression, providing an example of cis- and trans-acting RNA-based regulatory systems maintaining cellular homeostasis.

Adrenal-Permissive Germline HSD3B1 Allele and Prostate Cancer Outcomes.

Importance: The adrenal androgen-metabolizing 3ß-hydroxysteroid dehydrogenase-1 enzyme, encoded by the HSD3B1 gene, catalyzes the rate-limiting step necessary for synthesizing nontesticular testosterone and dihydrotestosterone production. The common adrenal-permissive HSD3B1(1245C) allele is responsible for encoding the 3ß-HSD1 protein with decreased susceptibility to degradation resulting in higher extragonadal androgen synthesis. Retrospective studies have suggested an association of the HSD3B1 adrenal-permissive homozygous genotype with androgen deprivation therapy resistance in prostate cancer. Objective: To evaluate differences in mortality outcomes by HSD3B1 genetic status among men with prostate cancer. Design, Setting, and Participants: This cohort study of patients with prostate cancer who were enrolled in the Million Veteran Program within the Veterans Health Administration (VHA) system between 2011 and 2023 collected genotyping and phenotyping information. Exposure: HSD3B1 genotype status was categorized as AA (homozygous adrenal-restrictive), AC (heterozygous adrenal-restrictive), or CC (homozygous adrenal-permissive). Main Outcomes and Measures: The primary outcome of this study was prostate cancer-specific mortality (PCSM), defined as the time from diagnosis to death from prostate cancer, censored at the date of last VHA follow-up. Secondary outcomes included incidence of metastases and PCSM in predefined subgroups. Results: Of the 5287 participants (median [IQR] age, 69 [64-74] years), 402 (7.6%) had the CC genotype, 1970 (37.3%) had the AC genotype, and 2915 (55.1%) had the AA genotype. Overall, the primary cause of death for 91 patients (1.7%) was prostate cancer. Cumulative incidence of PCSM at 5 years after prostate cancer diagnosis was higher among men with the CC genotype (4.0%; 95% CI, 1.7%-6.2%) compared with the AC genotype (2.1%; 95% CI, 1.3%-2.8%) and AA genotype (1.9%; 95% CI, 1.3%-2.4%) (P = .02). In the 619 patients who developed metastatic disease at any time, the cumulative incidence of PCSM at 5 years was higher among patients with the CC genotype (36.0%; 95% CI, 16.7%-50.8%) compared with the AC genotype (17.9%; 95% CI, 10.5%-24.7%) and AA genotype (18.5%; 95% CI, 12.0%-24.6%) (P = .01). Conclusions and Relevance: In this cohort study of US veterans undergoing treatment for prostate cancer at the VHA, the HSD3B1 CC genotype was associated with inferior outcomes. The HSD3B1 biomarker may help identify patients who may benefit from therapeutic targeting of 3ß-hydroxysteroid dehydrogenase-1 and the androgen-signaling axis.

Cancer cell-specific and pro-apoptotic SMAC peptide-doxorubicin conjugated prodrug encapsulated aposomes for synergistic cancer immunotherapy.

BACKGROUND: Immunogenic cell death (ICD) is a crucial approach to turn immunosuppressive tumor microenvironment (ITM) into immune-responsive milieu and improve the response rate of immune checkpoint blockade (ICB) therapy. However, cancer cells show resistance to ICD-inducing chemotherapeutic drugs, and non-specific toxicity of those drugs against immune cells reduce the immunotherapy efficiency. METHODS: Herein, we propose cancer cell-specific and pro-apoptotic liposomes (Aposomes) encapsulating second mitochondria-derived activator of caspases mimetic peptide (SMAC-P)-doxorubicin (DOX) conjugated prodrug to potentiate combinational ICB therapy with ICD. The SMAC-P (AVPIAQ) with cathepsin B-cleavable peptide (FRRG) was directly conjugated to DOX, and the resulting SMAC-P-FRRG-DOX prodrug was encapsulated into PEGylated liposomes. RESULTS: The SMAC-P-FRRG-DOX encapsulated PEGylated liposomes (Aposomes) form a stable nanostructure with an average diameter of 109.1 ± 5.14 nm and promote the apoptotic cell death mainly in cathepsin B-overexpressed cancer cells. Therefore, Aposomes induce a potent ICD in targeted cancer cells in synergy of SMAC-P with DOX in cultured cells. In colon tumor models, Aposomes efficiently accumulate in targeted tumor tissues via enhanced permeability and retention (EPR) effect and release the encapsulated prodrug of SMAC-P-FRRG-DOX, which is subsequently cleaved to SMAC-P and DOX in cancer cells. Importantly, the synergistic activity of inhibitors of apoptosis proteins (IAPs)-inhibitory SMAC-P sensitizing the effects of DOX induces a potent ICD in the cancer cells to promote dendritic cell (DC) maturation and stimulate T cell proliferation and activation, turning ITM into immune-responsive milieu. CONCLUSIONS: Eventually, the combination of Aposomes with anti-PD-L1 antibody results in a high rate of complete tumor regression (CR: 80%) and also prevent the tumor recurrence by immunological memory established during treatments.

Enhancement of progesterone biosynthesis via kisspeptin stimulation: Upregulation of steroidogenic transcripts and phosphorylated extracellular signal-regulated kinase (p-ERK1/2) expression in the buffalo luteal cells.

The presence of Kisspeptin (Kp) and its receptors in the corpus luteum (CL) of buffalo has recently been demonstrated. In this study, we investigated the role of Kp in the modulation of progesterone (P4) synthesis in vitro. The primary culture of bubaline luteal cells (LCs) was treated with 10, 50, and 100 nM of Kp and Kp antagonist (KpA) alongside a vehicle control. The combined effect of Kp and KpA was assessed at 100 nM concentration. Intracellular response to Kp treatment in the LCs was assessed by examining transcript profiles (LHR, STAR, CYP11A1, HSD3B1, and ERK1/2) using quantitative polymerase chain reaction (qPCR). In addition, the immunolocalization of ERK1/2 and phosphorylated ERK1/2 (p-ERK1/2) in the LCs was studied using immunocytochemistry. Accumulation of P4 from the culture supernatant was determined using enzyme-linked immunosorbent assay (ELISA). The results indicated that LCs had a greater p-ERK1/2 expression in the Kp treatment groups. A significant increase in the P4 concentration was recorded at 50 nM and 100 nM Kp, while KpA did not affect the basal concentration of P4. However, the addition of KpA to the Kp-treated group at 100 nM concentration suppressed the Kp-induced P4 accumulation into a concentration similar to the control. There was significant upregulation of ERK1/2 and CYP11A1 expressions in the Kp-treated LCs at 100 nM (18.1 and 37fold, respectively, p < 0.01). However, the addition of KpA to Kp-treated LCs modulated ERK1/2, LHR, STAR, CYP11A1, and HSD3B1 at 100 nM concentration. It can be concluded that Kp at 100 nM stimulated P4 production, while the addition of KpA suppressed Kp-induced P4 production in the buffalo LCs culture. Furthermore, an increment in p-ERK1/2 expression in the LCs indicated activation of the Kp signaling pathway was associated with luteal steroidogenesis.

The Conjugates of Indolo[2,3-b]quinoline as Anti-Pancreatic Cancer Agents: Design, Synthesis, Molecular Docking and Biological Evaluations.

New amide conjugates of hydroxycinnamic acids (HCAs) and the known antineoplastic 5,11-dimethyl-5H-indolo[2,3-b]quinoline (DiMIQ), an analog of the natural alkaloid neocryptolepine, were synthesized and tested in vitro for anticancer activity. The compound 9-[((2-hydroxy)cinnamoyl)amino]-5,11-dimethyl-5H-indolo[2,3-b]quinoline (2), which contains the ortho-coumaric acid fragment, demonstrated dose-dependent effectiveness against both normal BxPC-3 and metastatic AsPC-1 pancreatic cancer cells. The IC50 values for AsPC-1 and BxPC-3 were 336.5 nM and 347.5 nM, respectively, with a selectivity index of approximately 5 for both pancreatic cancer cells compared to normal dermal fibroblasts. Conjugate 2 did not exhibit any hemolytic activity against human erythrocytes at the tested concentration. Computational studies were performed to predict the pharmacokinetic profile and potential mechanism of action of the synthesized conjugates. These studies focused on the ADME properties of the conjugates and their interactions with DNA, as well as DNA-topoisomerase alpha and beta complexes. All of the conjugates studied showed approximately one order of magnitude stronger binding to DNA compared to the reference DiMIQ, and approximately two orders of magnitude stronger binding to the topoisomerase II-DNA complex compared to DiMIQ. Conjugate 2 was predicted to have the strongest binding to the enzyme-DNA complex, with a Ki value of 2.8 nM.

Efficient production of peptidylglycine α-hydroxylating monooxygenase in yeast for protein C-terminal functionalization.

Peptidylglycine α-hydroxylating monooxygenase (PHM) is pivotal for C-terminal amidation of bioactive peptides in animals, offering substantial potential for customized protein synthesis. However, efficient PHM production has been hindered by the complexity of animal cell culture and the absence of glycosylation in bacterial hosts. Here, we demonstrate the recombinant expression of Caenorhabditis elegans PHM in the yeast Pichia pastoris, achieving a remarkable space-time yield of 28.8 U/L/day. This breakthrough surpasses prior PHM production rates and eliminates the need for specialized cultivation equipment or complex transfection steps. Mass spectrometry revealed N-glycosylation at residue N182 of recombinant CePHM, which impacts the enzyme's activity as indicated by biochemical experiments. To showcase the utility of CePHM, we performed C-terminal amidation on ubiquitin at a substrate loading of 30 g/L, a concentration meeting the requirements for pharmaceutical peptide production. Overall, this work establishes an efficient PHM production method, promising advancements in scalable manufacturing of C-terminally modified bioactive peptides and probe proteins.

Novel insight on IRE1 in the regulation of chondrocyte dedifferentiation through ER stress independent pathway.

Inositol-requiring enzyme-1 (IRE1) is the master regulator of the unfolded protein response pathway, associated with the endoplasmic reticulum (ER) in sensing and regulating cell stress. The activity of IRE1 is highly explored and well-characterized in cancer and other cells. However, the IRE1 molecular mechanism in chondrocytes is poorly understood. The present study explored the effect of IRE1 on chondrocytes regarding its chondrogenic gene expression and its correlation with different cellular pathways and cell behavior. Chondrocytes transfected with the cDNA of IRE1 reduced the expression of type II collagen, disrupting chondrocyte differentiation as confirmed by western blotting and immunofluorescence. Upon siRNA treatment, the influence of IRE1 on chondrocyte differentiation is restored by reviving the normal expression of type II collagen. Different molecular pathways were explored to investigate the role of IRE1 in causing chondrocyte dedifferentiation. However, we found no significant correlation, as IRE1 induces dedifferentiation through independent pathways. In response to various endoplasmic reticulum (ER) agonists (2-deoxy-D-glucose), and ER stress antagonists (tauroursodeoxycholic acid and salubrinal), IRE1 overexpression did not affect GRP78/94, as implicated in the pathogenesis of ER stress. Moreover, when IRE1 overexpression was correlated with the inflammation pathway, nuclear factor-kappa B (NFκB), IRE1 substantially increased the expression of p50 while decreasing the expression of nuclear factor kappa light polypeptide alpha (IκBα). These results suggest that IRE1 induces dedifferentiation in chondrocytes by modulating inflammatory pathways that cause dedifferentiation by disrupting type II collagen expression.

Formyl phloroglucinol meroterpenoids from the leaves of Eucalyptus globulus subsp. maidenii and their ATP-citrate lyase inhibitory activities.

Three new formyl phloroglucinol meroterpenoids, eumaidials A-C (1-3), were isolated from the leaves of Eucalyptus globulus subsp. maidenii, along with ten known analogues (4-13). Their chemical structures were determined by various spectral data and electronic circular dichroism calculations. Eumaidial A (1) is the first ß-caryophyllene-based formyl phloroglucinol meroterpenoids from the genus Eucalyptus. Compounds 1-4 and 10 exhibited ATP-citrate lyase inhibitory activities, and compounds 2 and 3 suppressed the hepatocyte lipogenesis.

Intratumoral androgen biosynthesis associated with 3ß-hydroxysteroid dehydrogenase 1 promotes resistance to radiotherapy in prostate cancer.

Half of all men with advanced prostate cancer (PCa) inherit at least 1 copy of an adrenal-permissive HSD3B1 (1245C) allele, which increases levels of 3ß-hydroxysteroid dehydrogenase 1 (3ßHSD1) and promotes intracellular androgen biosynthesis. Germline inheritance of the adrenally permissive allele confers worse outcomes in men with advanced PCa. We investigated whether HSD3B1 (1245C) drives resistance to combined androgen deprivation and radiotherapy. Adrenally permissive 3ßHSD1 enhanced resistance to radiotherapy in PCa cell lines and xenograft models engineered to mimic the human adrenal/gonadal axis during androgen deprivation. The allele-specific effects on radiosensitivity were dependent on availability of DHEA, the substrate for 3ßHSD1. In lines expressing the HSD3B1 (1245C) allele, enhanced expression of DNA damage response (DDR) genes and more rapid DNA double-strand break (DSB) resolution were observed. A correlation between androgen receptor (AR) expression and increased DDR gene expression was confirmed in 680 radical prostatectomy specimens. Treatment with the nonsteroidal antiandrogen enzalutamide reversed the resistant phenotype of HSD3B1 (1245C) PCa in vitro and in vivo. In conclusion, 3ßHSD1 promotes prostate cancer resistance to combined androgen deprivation and radiotherapy by upregulating DNA DSB repair. This work supports prospective validation of early combined androgen blockade for high-risk men harboring the HSD3B1 (1245C) allele.

Approaches to assessing 3ß-hydroxysteroid dehydrogenase-1.

The enzyme 3ß-hydroxysteroid dehydrogenase-1 (3ßHSD1), encoded by the gene HSD3B1, plays an essential role in the peripheral conversion of 3ß-OH, Δ5-steroids to 3-keto, Δ4-steroids. In human physiology, the adrenal produces dehydroepiandrosterone (DHEA) and DHEA-sulfate, which are major precursors for the biosynthesis of potent androgens and estrogens. DHEA is converted by 3ßHSD1 and subsequently is converted by steroid-5α-reductase to potent androgens or by aromatase to estrogens. Assessment of 3ßHSD1 is therefore critical under various conditions. In this chapter, we detail several approaches to assessing 3ßHSD1. First, we describe a genotyping protocol for the identification of a common missense-encoding variation that regulates 3ßHSD1 cellular metabolic activity. This protocol distinguishes between the HSD3B1(1245A) and the HSD3B1(1245C) allele which have lower and higher metabolic activity, respectively. Second, we detail mass spectrometry approaches to determining 3ßHSD1 activity using stable isotope dilution. Third, we describe methods for using tritiated DHEA and high performance liquid chromatography coupled with a beta-RAM to also determine 3ßHSD1 activity. Together, we provide multiple methods of directly assessing 3ßHSD1 activity or anticipated 3ßHSD1 activity.

Elucidation of the Active Form and Reaction Mechanism in Human Asparaginase Type III Using Multiscale Simulations.

l-asparaginases catalyze the asparagine hydrolysis to aspartate. These enzymes play an important role in the treatment of acute lymphoblastic leukemia because these cells are unable to produce their own asparagine. Due to the immunogenic response and various side effects of enzymes of bacterial origin, many attempts have been made to replace these enzymes with mammalian enzymes such as human asparaginase type III (hASNaseIII). This study investigates the reaction mechanism of hASNaseIII through molecular dynamics simulations, quantum mechanics/molecular mechanics methods, and free energy calculations. Our simulations reveal that the dimeric form of the enzyme plays a vital role in stabilizing the substrate in the active site, despite the active site residues coming from a single protomer. Protomer-protomer interactions are essential to keep the enzyme in an active conformation. Our study of the reaction mechanism indicates that the self-cleavage process that generates an N-terminal residue (Thr168) is required to activate the enzyme. This residue acts as the nucleophile, attacking the electrophilic carbon of the substrate after a proton transfer from its hydroxyl group to the N-terminal amino group. The reaction mechanism proceeds with the formation of an acyl-enzyme complex and its hydrolysis, which turns out to be the rate-determining step. Our proposal of the enzymatic mechanism sheds light on the role of different active site residues and rationalizes the studies on mutations. The insights provided here about hASNaseIII activity could contribute to the comprehension of the disparities among different ASNases and might even guide the design of new variants with improved properties for acute lymphoblastic leukemia treatment.

Steroidogenic activity of liposomal methylated resveratrol analog 3,4,5,4'-tetramethoxystilbene (DMU-212) in human luteinized granulosa cells in a primary three-dimensional in vitro model.

PURPOSE: Steroid hormone secretion is one of the key functions of granulosa cells (GCs). Resveratrol is a natural polyphenol, known for its beneficial health effects, such as improving reproductive health. However, its application is limited due to poor bioavailability. The methoxy derivative of resveratrol (DMU-212) was demonstrated to be more lipophilic, and therefore of greater bioavailability. However, since the addition of methoxy groups to the stilbene scaffold was found to make the molecule insoluble in water, DMU-212 was loaded into liposomes. This study aimed to evaluate how the liposomal formulation of DMU-212 (lipDMU-212) alters estradiol and progesterone secretion of human ovarian GCs in a primary three-dimensional cell culture model. METHODS: DMU-212-loaded liposomes were prepared by thin film hydration followed by extrusion. Cell viability was measured after exposure of GCs spheroids to the liposomal formulation of DMU-212 using CellTiter-Glo® 3D Cell Viability Assay. The secretion of estradiol and progesterone was determined using commercial ELISA kits. RT-qPCR was conducted to analyze the expression of steroidogenesis-related genes. Finally, the western blot technique was used to analyze the effect of lipDMU-212 and FSH treatments on CYP11A1 and HSD3B1 protein levels. RESULTS: lipDMU-212 was found to significantly increase estradiol and progesterone secretion in a dose-dependent manner by enhancing the expression of CYP11A1, HSD3B1, StAR, CYP17A1, CYP19A1, and HSD17B1 genes. We have also shown that lipDMU-212, used alone and in combination with FSH, significantly increased the expression of the HSD3B1 and CYP11A1 proteins in GCs. Furthermore, our study suggests that lipDMU-212 increases FSH activity. CONCLUSIONS: This is the first study to describe the steroidogenic activity of liposomal formulation of DMU-212, possibly through increasing the StAR and CYP19A1 expression. These findings suggest that lipDMU-212 might have a beneficial effect in the treatment of disorders related to estrogen deficiency and hyperandrogenism, such as PCOS.

NR5A2/HSD3B1 pathway promotes cellular resistance to second-generation antiandrogen darolutamide.

This study investigated cellular mechanisms in steroidogenesis responsible for treatment resistance to the novel antiandrogen agent darolutamide in prostate cancer. HSD3B1 was overexpressed in darolutamide-resistant cells and induced by darolutamide treatment and AR knockdown. Inversely, HSD3B1 knockdown increased cellular sensitivity to darolutamide. Similarly, its upstream regulator NR5A2 was up-regulated in darolutamide-resistant cells and induced by darolutamide treatment and AR knockdown. Inversely, NR5A2 knockdown and NR5A2 inhibitor ML180 decreased expression of various steroidogenic enzymes including HSD3B1, leading to increased cellular sensitivity to darolutamide. The NR5A2/HSD3B1 pathway promoted cellular resistance to darolutamide and targeting NR5A2/HSD3B1 pathway is a promising therapeutic strategy to overcome darolutamide resistance.

Cyanocobalamin (VB12) Bionic Enzyme-Assisted Photocatalytic Chain-Growth Polymerization for Detection of Lung Cancer Biomarkers.

Cobalt-mediated radical polymerization is noted for its great level of control over the polymerization of acrylic and vinyl esters monomers, even at high molar mass. Vitamin B12, a natural bionic enzyme cobalt complex, involves the conversion of organic halides to olefins through chain-growth polymerization. In this work, the notion of R-Co(III) free radical persistent free radical effect and vitamin B12 circulation were first reported for the perception of ultralow abundance of microRNA-21, a lung cancer biomarker. Indeed, most Co-containing catalytic reactions can occur under mild conditions due to their minimal bond dissociation of the C-Co bond, with blue light irradiation. Based on the intrinsic stability of the vitamin B12 framework and recycling of the catalyst, it is evident that this natural catalytic scheme has potential applications in medicinal chemistry and biomaterials. In addition, this strategy, combined with highly specific recognition probes and vitamin B12 circulation-mediated chain-growth polymerization, has a detection limit as low as 910 aM. Furthermore, it is sensitive for sensing in serum samples containing biomarkers and shows great potential for RNA selection and amplification sensing in clinical samples.

Progesterone modulates HSD11B1-mediated cortisol production in luteinized bovine granulosa cells.

Progesterone (P4) and cortisol production increase in luteinized granulosa cells (LGCs) during the periovulatory period, but their interaction is not well established. Therefore, we investigated their interaction in cultured bovine LGCs. Granulosa cells were collected from follicles of 2-5 mm in diameter and cultured in DMEM/F-12 supplemented with 10% fetal calf serum for up to 14 days. P4 production and the expression of steroidogenic acute regulatory protein (STAR), cholesterol side-chain cleavage enzyme (CYP11A1), and 3ß-hydroxysteroid dehydrogenase type 1 (HSD3B1) rapidly increased until day 10 and remained high thereafter. No de novo production of cortisol from P4 was detected during the culture period. The expression of 11ß-hydroxysteroid dehydrogenase type 1 (HSD11B1), which converts cortisone to cortisol, increased dramatically on day two, decreased until day 8, and remained relatively constant. To investigate how P4 and cortisol influence each other's production, LGCs were treated with trilostane (a P4 synthesis inhibitor), nomegestrol acetate (NA, a synthetic progestogen), P4, and/or cortisol for 24 h on days 6 and 12 of culture. Trilostane suppressed P4 and STAR expression while elevating HSD11B1 and HSD3B1 expression and cortisol production. Concomitant treatment with NA or P4 dose-dependently decreased cortisol production and HSD11B1 and HSD3B1 expression but elevated STAR expression in both days 6 and 12. Conversely, cortisol treatment increased HSD11B1 and HSD3B1 expression and decreased STAR expression without influencing P4 production. These results indicate that progestogens suppress cortisol production by modulating HSD11B1 expression and that progestogens and cortisol differentially regulate STAR, HSD3B1, and HSD11B1 expression in bovine LGCs.

Control of Reversible Formation and Dispersion of the Three Enzyme Networks Integrating DNA Computing.

The majority of biological reactions in the cytoplasm of living cells occur via enzymatic cascade reactions. To achieve efficient enzyme cascade reactions mimicking the proximity conditions of enzymes in the cytoplasm, the proximity of each enzyme, creating a high local concentration of proteins, has been recently investigated using the conjugation of synthetic polymer molecules, proteins, and nucleic acids. Although there have been methodologies reported for the complex formation and enhanced activity of cascade reactions due to the proximity of each enzyme using DNA nanotechnology, one pair of the enzyme (GOx and HRP) complex is only assembled by the mutual independence of various shapes of the DNA structure. This study reports the network formation of three enzyme complexes assembled by a triple-branched DNA structure as a unit, thus enabling the reversible formation and dispersion of the three enzyme complex networks using single-stranded DNA, RNA, and enzymes. It was found that the activities of the three enzyme cascade reactions in the enzyme-DNA complex network were controlled by formation and dispersion of the three enzyme complex networks, due to the proximity of each enzyme with the enzyme-DNA complex network. Furthermore, three micro RNA sequences for breast cancer biomarkers were successfully detected using an enzyme-DNA complex network integrated with DNA computing. Overall, the reversible formation and dispersion of the enzyme-DNA complex network through the external stimulation of biomolecules and DNA computing provide a novel platform for controlling the production amount, diagnosis, theranostics, and biological or environmental sensing.