Mechanistic Insights Research Articles

Antifungal Synergy: Mechanistic Insights into the R-1-R Peptide and Bidens pilosa Extract as Potent Therapeutics against Candida spp. through Proteomics.

Previous reports have demonstrated that the peptide derived from LfcinB, R-1-R, exhibits anti-Candida activity, which is enhanced when combined with an extract from the Bidens pilosa plant. However, the mechanism of action remains unexplored. In this research, a proteomic study was carried out, followed by a bioinformatic analysis and biological assays in both the SC5314 strain and a fluconazole-resistant isolate of Candida albicans after incubation with R-1-R. The proteomic data revealed that treatment with R-1-R led to the up-regulation of most differentially expressed proteins compared to the controls in both strains. These proteins are primarily involved in membrane and cell wall biosynthesis, membrane transport, oxidative stress response, the mitochondrial respiratory chain, and DNA damage response. Additionally, proteomic analysis of the C. albicans parental strain SC5314 treated with R-1-R combined with an ethanolic extract of B. pilosa was performed. The differentially expressed proteins following this combined treatment were involved in similar functional processes as those treated with the R-1-R peptide alone but were mostly down-regulated (data are available through ProteomeXchange with identifier PXD053558). Biological assays validated the proteomic results, evidencing cell surface damage, reactive oxygen species generation, and decreased mitochondrial membrane potential. These findings provide insights into the complex antifungal mechanisms of the R-1-R peptide and its combination with the B. pilosa extract, potentially informing future studies on natural product derivatives.

Global analysis of the RNA-RNA interactome in Acinetobacter baumannii AB5075 uncovers a small regulatory RNA repressing the virulence-related outer membrane protein CarO.

Acinetobacter baumannii is an opportunistic Gram-negative pathogen that infects critically ill patients. The emergence of antimicrobial resistant A. baumannii has exacerbated the need to characterize environmental adaptation, antibiotic resistance and pathogenicity and their genetic regulators to inform intervention strategies. Critical to adaptation to changing environments in bacteria are small regulatory RNAs (sRNAs), however, the role that sRNAs play in the biology of A. baumannii is poorly understood. To assess the regulatory function of sRNAs and to uncover their RNA interaction partners, we employed an RNA proximity ligation and sequencing method (Hi-GRIL-seq) in three different environmental conditions. Forty sRNAs were ligated to sRNA-RNA chimeric sequencing reads, suggesting that sRNA-mediated gene regulation is pervasive in A. baumannii. In-depth characterization uncovered the sRNA Aar to be a post-transcriptional regulator of four mRNA targets including the transcript encoding outer membrane protein CarO. Aar initiates base-pairing with these mRNAs using a conserved seed region of nine nucleotides, sequestering the ribosome binding sites and inhibiting translation. Aar is differentially expressed in multiple stress conditions suggesting a role in fine-tuning translation of the Aar-target molecules. Our study provides mechanistic insights into sRNA-mediated gene regulation in A. baumannii and represents a valuable resource for future RNA-centric research endeavours.

Mechanistic insights into ethylene catalytic combustion and CO2 formation in vinyl acetate synthesis

Vinyl acetate is a crucial monomer for the production of polyvinyl alcohol and polyvinyl acetate, with extensive applications in the photovoltaic and pharmaceutical industries. CO2 is the primary by-product in the synthesis of vinyl acetate, significantly burdening the separation process and impacting the ecological environment. Reducing CO2 generation at the source is an effective solution. In the vinyl acetate synthesis process, CO2 is primarily produced through the catalytic combustion of ethylene. However, the reaction mechanism of ethylene catalytic combustion on Pd-based catalysts in vinyl acetate synthesis remains incomplete, with existing mechanisms presenting certain limitations. Therefore, further investigation into the reaction mechanism of ethylene catalytic combustion leading to CO2 formation on Pd-based catalysts is necessary.This study employs density functional theory to calculate the reaction mechanism of ethylene catalytic combustion on the Pd(100) surface, obtaining elementary reaction kinetics data. Based on these data, the kinetic Monte Carlo method was used to investigate the reaction process of ethylene catalytic combustion, revealing the predominant pathway for CO2 formation. This research provides targeted strategies for catalyst modification. It aims to improve the understanding of the reaction mechanism of ethylene catalytic combustion in vinyl acetate synthesis, reduce the consumption of ethylene in by-product formation, enhance vinyl acetate yield and ethylene utilization, and ultimately decrease CO2 production, thereby lowering carbon emissions.

Revisiting iodide species transformation in peracetic acid oxidation: unexpected role of radicals in micropollutants decontamination and iodate formation

Peracetic acid (PAA) is an alternative disinfectant for saline wastewaters, and hypohalous acids are typically regarded as the reactive species for oxidation and disinfection. However, new results herein strongly suggest that reactive radicals instead of HOI primarily contributed to decontamination during PAA treatment of iodine-containing wastewater. The presence of I- could greatly accelerate the micropollutants (e.g., sulfamethoxazole (SMX)) transformation by PAA. Chemical probes experiments and electron paramagnetic resonance analysis demonstrate acetylperoxyl radical rather than reactive iodine species primarily responsible for SMX degradation. The kinetic model was developed to further distinguish and quantify the contribution of radicals and iodine species, as well as to elucidate the transformation pathways of iodine species. Density functional theory calculations indicated that the nucleophilic attack of I- on the peroxide bond of PAA could form unstable O−I bond, with the transition state energy barrier for radical generation lower than that for HOI formation. The transformation of iodine species was regulated by acetylperoxyl radical to generate nontoxic IO3-, greatly alleviating the iodinated DBPs formation in saline wastewaters. This work provides mechanistic insights in radical-regulated iodine species transformation during PAA oxidation, paving the way for the development of viable and eco-friendly technology for iodide containing water treatment.

Structural and Mechanistic Insights into a Novel Monooxygenase for Poly(acrylic acid) Biodegradation.

Polyacrylamide (PAM) is a high-molecular-weight polymer with extensive applications. However, the inefficient natural degradation of PAM results in environmental accumulation of the polymer. Biodegradation is an environmentally friendly approach in the field of PAM treatment. The first phase of PAM biodegradation is the deamination of PAM, forming the product poly(acrylic acid) (PAA). The second phase of PAM biodegradation involves the cleavage of PAA into small molecules, which is a crucial step in the degradation pathway of PAM. However, the enzyme that catalyzes the degradation of PAA and the molecular mechanism remain unclear. Here, a novel monooxygenase PCX02514 is identified as the key enzyme for PAA degradation. Through biochemical experiments, the monooxygenase PCX02514 oxidizes PAA with the participation of NADPH, causing the cleavage of carbon chains and a decrease in the molecular weight of PAA. In addition, the crystal structure of the monooxygenase PCX02514 is solved at a resolution of 1.97 Å. The active pocket is in a long cavity that extends from the C-terminus of the TIM barrel to the protein surface and exhibits positive electrostatic potential, thereby causing the migration of oxygen-negative ions into the active pocket and facilitating the reaction between the substrates and monooxygenase PCX02514. Moreover, Arg10-Arg125-Ser186-Arg187-His253 are proposed as potential active sites in monooxygenase PCX02514. Our research characterizes the molecular mechanism of this monooxygenase, providing a theoretical basis and valuable tools for PAM bioremediation.

Flow Behavior and Mechanism Insights into Nanoparticle-Surfactant-Stabilized Nitrogen Foam for Enhanced Oil Recovery in the Mature Waterflooding Reservoir.

To solve the problem of poor stability and low enhanced oil recovery efficiency of conventional foam, nanoparticle-surfactant-stabilized nitrogen foam was prepared, and the influence of temperature, salinity, oil content, and pressure on foam performance was systematically investigated. Then, the flow behavior of conventional foam and nanoparticle-surfactant-stabilized foam in porous media was studied. Parallel sand pack flooding and visualization microflooding experiments were performed to investigate the enhanced oil recovery ability of nanoparticle-surfactant-stabilized foam from core-scale to pore-scale. Results showed that the nanoparticles can improve foam performance. When the temperature increases from 60 to 100 °C, the foam volume and foam half-life of nanoparticle-surfactant-stabilized foam decrease by 20 and 36%, respectively. The nanoparticle-surfactant-stabilized foam has a good salt resistance. The oil content limit value of the foam performance is 15%. With the increase of pressure, the foaming performance and foam stability are enhanced obviously. Compared with conventional surfactant-stabilized foam, the nanoparticle-surfactant-stabilized foam can have better plugging and expansion of the swept volume capacity. The micromodel flooding results are consistent with the parallel sand pack flooding results. Compared with conventional surfactant stabilized foam, nanoparticle-surfactant-stabilized foam has better enhanced oil recovery ability than conventional surfactant-stabilized foam due to its higher foaming ability, foam stability, and sweep efficiency improvement ability.

20(S)-ginsenoside Rg3 protects against diabetic muscle atrophy by promoting myoblastic differentiation and protecting mitochondrial function

BackgroundHigh glucose levels are a primary cause of diabetes-associated cellular dysfunction and tissue damage. Muscles are the key insulin target organ and therefore, have a high level of sensitivity to hyperglycemia. Our previous study revealed that 20(S)-ginsenoside Rg3 (S-Rg3) is a monomer with a good myogenic differentiation effect in ginsenoside. Furthermore, it can alleviate dexamethasone-induced muscle atrophy by protecting mitochondrial function. However, whether S-Rg3 is effective for diabetic-induced muscle atrophy has not been reported. PurposeThis study aimed to investigate the protective effect of S-Rg3 on diabetic-induced muscle atrophy. MethodsC2C12 myoblasts, Drosophila, and mice were used as model systems, and the protective effect of S-Rg3 on diabetes was evaluated by assessing the levels of glucose and lipids. Furthermore, H&E, toluidine blue, Giemsa, and immunofluorescence staining were performed to detect the effects of S-Rg3 on muscle atrophy and myogenic differentiation. Moreover, the effects of S-Rg3 on mitochondrial morphology and function were also evaluated by electron microscopy, flow cytometry, and Seahorse. In addition, the underlying pathways of S-Rg3 effects were detected by Western blot. The related inhibitors and gene mutations in Drosophila were used for validation. ResultsThe analysis of diabetic mice model fed with a high-fat diet (HFD) and high glucose (HG) revealed that in the injured C2C12 myoblasts, S-Rg3 treatment significantly reduced the levels of triglycerides and glucose. Furthermore, it promoted the differentiation of myoblasts and inhibited mitochondrial dysfunction. In the Drosophila HG and HFD diabetic model, S-Rg3 reduced triglyceride and trehalose levels, increased climbing distance values, promoted myoblasts differentiation, preserved mitochondrial function, and inhibited muscle atrophy. Mechanistically, the beneficial effects of S-Rg3 were at least partially associated with the phosphorylation of AMPK and FoxO3 together with the inhibition of Smad3 phosphorylation, this pathway was validated by the UAS-AMPKα-RNAi Drosophila model. ConclusionIn summary, this study revealed mechanistic insights into how S-Rg3 protects against diabetes-associated muscle atrophy in cells, Drosophila, and mice.

Gene expression-phenotype association study reveals the dual role of TNF-α/TNFR1 signaling axis in confined breast cancer cell migration

AimsWhile enhanced tumor cell migration is a key process in the tumor dissemination, mechanistic insights into causal relationships between tumor cells and mechanical confinement are still limited. Here we combine the use of microfluidic platforms to characterize confined cell migration with genomic tools to systematically unravel the global signaling landscape associated with the migratory phenotype of breast cancer (BC) cells. Meterials and methodsThe spontaneous migration capacity of seven BC cell lines was evaluated in 3D microfluidic devices and their migration capacity was correlated with publicly available molecular signatures. The role of identified signaling pathways on regulating BC migration capacity was determined by receptor stimulation through ligand binding or inhibition through siRNA silencing. Downstream effects on cell migration were evaluated in microfluidic devices, while the molecular changes were monitored by RT-qPCR. Key findingsExpression of 715 genes was correlated with BC cells migratory phenotype, revealing TNF-α as one of the top upstream regulators. Signal transduction experiments revealed that TNF-α stimulates the confined migration of triple negative, mesenchymal-like BC cells that are also characterized by high TNFR1 expression, but inhibits the migration of epithelial-like cells with low TNFR1 expression. TNFR1 was strongly associated with the migration capacity and triple-negative, mesenchymal phenotype. Downstream of TNF/TNFR1 signaling, transcriptional regulation of NFKB seems to be important in driving cell migration in confined spaces. SignificanceTNF-α/TNFR1 signaling axis reveals as a key player in driving BC cells confined migration, emerging as a promising therapeutic strategy in targeting dissemination and metastasis of triple negative, mesenchymal BC cells.

Antibiotics Trigger Host Innate Immune Response via Microbiota-Brain Communication in C. elegans.

Besides their direct bactericidal effect, antibiotics have also been suggested to stimulate the host immune response to defend against pathogens. However, it remains unclear whether any antibiotics may stimulate the host immune response by affecting bacterial activity. In this study, reasoning that genetic mutations inhibit bacterial activities and, thereby, may mimic the effects of antibiotics, we performed genome-wide screening and identified 77 E. coli genes whose inactivation induces C. elegans cyp-14A4, representing an innate immune and detoxification response. Further analyses reveal that this host immune response can clearly be induced through either inactivating the E. coli respiratory chain via the bacterial cyoB mutation or using the antibiotic Q203, which is able to enhance host survival when encountering the pathogen Pseudomonas aeruginosa. Mechanistically, the innate immune response triggered by both the cyoB mutation and Q203 is found to depend on the host brain response, as evidenced by their reliance on the host neural gene unc-13, which is required for neurotransmitter release in head neurons. Therefore, our findings elucidate the critical involvement of the microbiota-brain axis in modulating the host immune response, providing mechanistic insights into the role of antibiotics in triggering the host immune response and, thus, facilitating host defense against pathogens.

Constructing heterointerfaces to enhance electron transfer of CoFe/CoFe2O4 mediated electro-Fenton process for bisphenol A degradation: Mechanistic insights and performance evaluation

The inefficient electron transfer and Fe(III)/Fe(II) cycle remain significant obstacles in the electro-fenton process. Herein, a nitrogen-doped graphite felt (NGF)-supported CoFe/CoFe2O4 heterostructure is synthesized that achieves highly efficient interfacial electron transfer and stable Co/Fe redox cycle. Theoretical calculations demonstrate that the CoFe/CoFe2O4 heterointerfaces induce local charge redistribution benefiting from the electronic communication between electron acceptor (CoFe2O4) and electron donor (CoFe). Analysis of the Fermi level indicates that the introduction of CoFe alloy enhances the electron density of the catalyst. The CoFe/CoFe2O4@NGF cathode can remove bisphenol A (BPA) completely in 90 min, and the pseudo-first-order kinetic constant (0.0625 min−1) is 23 times higher than the GF system. Notably, the CoFe/CoFe2O4@NGF electrode maintains excellent catalytic capability and stability within a broad pH range (3–11). The construction of the CoFe/CoFe2O4 heterostructure reduces the adsorption energy of oxygen, which ultimately increases the yield of reactive oxygen species (ROS). Quenching experiments revealed that hydroxyl radical (·OH) played a dominant role in the degradation of BPA, and significantly reduced the biotoxicity of intermediate products. This work offers a dependable approach for developing bimetallic catalysts with high catalytic activity.

Mechanistic insights into molybdenum immobilization within geopolymer waste form

Molybdenum (Mo) tends to form highly mobile, non-sorbing complex redox species, which complicates its immobilization and encapsulation in cement and glass matrices. This study explores the potential of incorporating Mo into an alternative green cementitious material known as geopolymer (GP). By employing X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS), morphological analysis, and nitrogen adsorption-desorption techniques, we obtained comprehensive insights into the chemical behavior of Mo and its impact on the morphology and structure of the GP matrix. XRD and TGA analysis demonstrated that increasing Mo content significantly enhances the crystallinity and thermal stability of the GP matrix, attributed to Mo field effects. High-resolution SEM, TEM, and EDS mapping revealed the structural encapsulation of Mo-rich cavities within the GP waste form. A decrease in leaching rates with higher Mo content, alongside reduced pore volume and pore size, underscores the material's chemical durability. Post-leaching characterizations confirmed that the GP retains excellent structural strength with minimal changes in mesoporosity and surface area. This resilience highlights the effectiveness of GPs in immobilizing non-sorbing species like MoO32−, making them ideal for long-term waste management.

Non-specific yet selective interactions contribute to small molecule condensate partitioning behavior.

Biomolecular condensates are essential in various cellular processes, and their misregulation has been demonstrated to be underly disease. Small molecules that modulate condensate stability and material properties offer promising therapeutic approaches, but mechanistic insights into their interactions with condensates remain largely lacking. We employ a multiscale approach to enable long-time, equilibrated all-atom simulations of various condensate-ligand systems. Systematic characterization of the ligand binding poses reveals that condensates can form diverse and heterogeneous chemical environments with one or multiple chains to bind small molecules. Unlike traditional protein-ligand interactions, these chemical environments are dominated by non-specific hydrophobic interactions. Nevertheless, the chemical environments feature unique amino acid compositions and physicochemical properties that favor certain small molecules over others, resulting in varied ligand partitioning coefficients within condensates. Notably, different condensates share similar sets of chemical environments but at different populations. This population shift drives ligand selectivity towards specific condensates. Our approach can enhance the interpretation of experimental screening data and may assist in the rational design of small molecules targeting specific condensates.

OPA1 promotes ferroptosis by augmenting mitochondrial ROS and suppressing an integrated stress response

Ferroptosis, an iron-dependent form of nonapoptotic cell death mediated by lipid peroxidation, has been implicated in the pathogenesis of multiple diseases. Subcellular organelles play pivotal roles in the regulation of ferroptosis, but the mechanisms underlying the contributions of the mitochondria remain poorly defined. Optic atrophy 1 (OPA1) is a mitochondrial dynamin-like GTPase that controls mitochondrial morphogenesis, fusion, and energetics. Here, we report that human and mouse cells lacking OPA1 are markedly resistant to ferroptosis. Reconstitution with OPA1 mutants demonstrates that ferroptosis sensitization requires the GTPase activity but is independent of OPA1-mediated mitochondrial fusion. Mechanistically, OPA1 confers susceptibility to ferroptosis by maintaining mitochondrial homeostasis and function, which contributes both to the generation of mitochondrial lipid reactive oxygen species (ROS) and suppression of an ATF4-mediated integrated stress response. Together, these results identify an OPA1-controlled mitochondrial axis of ferroptosis regulation and provide mechanistic insights for therapeutically manipulating this form of cell death in diseases.

Unveiling the mechanistic insights into the potassium resistance of 3.5 W-V-1 %K NH3-SCR catalysts: The dual functionality of V-O-W structure as acid and redox sites

The poisoning effect of alkali metals over Vanadium-based catalysts still faced a significant challenge in the selective catalytic reduction of NOx with NH3 (NH3-SCR). In order to improve the alkali (mainly potassium resistance) resistance of the Vanadium-based catalysts, WO3 was introduced into the vanadium oxide catalysts. The obtained 3.5WO3-V2O5(3.5 W-V) catalyst exhibited the excellent low-temperature NH3-SCR performance owing to the formation of V-O-W structures. Noteworthily, when 1 wt% K was introduced into the 3.5 W-V catalyst, vanadium oxide (V-O, V=O) species preferentially bonded to alkali metal K as an acidic sacrifice site, prompting more active V-O-W species to remain. Thus, the 3.5 W-V catalyst also possessed excellent alkali resistance. The NH3-TPD results showed that the 3.5 W-V-1 %K catalyst had a considerable acid site to ensure the adsorption capacity of NH3, while the XPS results showed that the 3.5 W-V-1 %K catalyst had excellent redox performance to ensure the activation capacity of the reactant NH3. Moreover, in-situ DRIFTS transient experiments revealed that the 3.5 W-V and 3.5 W-V-1 %K catalysts were more capable of adsorbing and activating NH3 species to rapidly react with gaseous NO, obeying the Eley-Rideal (E-R) pathway in NH3-SCR reaction. This work provides new insights into poisoning resistant of vanadium-based catalysts.

Cover Feature: Mechanistic Insight into Heteroatom Removal from Vacuum Gas Oil Blended with PMMA or PET Waste (ChemSusChem 15/2024)

Cover Feature: Mechanistic Insight into Heteroatom Removal from Vacuum Gas Oil Blended with PMMA or PET Waste (ChemSusChem 15/2024)

Safflower Yellow Injection Alleviates Myocardial Ischemia/Reperfusion Injury by Reducing Oxidative and Endoplasmic Reticulum Stress.

Safflower yellow is an extract of the famous Chinese medicine Carthamus tinctorious L, and safflower yellow injection (SYI) is widely used clinically to treat angina pectoris. However, there are few studies on the anti-myocardial ischemia/reperfusion (I/R) injury effect of SYI, and its mechanisms are unclear. In the present study, we aimed to investigate the protective effect of SYI on myocardial I/R injury and explore its underlying mechanisms. Male Sprague Dawley rats were randomly divided into a control group, sham group, model group, and SYI group (20 mg/kg, femoral vein injection 1 h before modeling). The left anterior descending coronary artery was ligated to establish a myocardial I/R model. H9c2 cells were exposed to oxygen-glucose deprivation/reoxygenation (OGD/R) after incubation with 80 μg/mL SYI for 24 h. In vivo, TsTC, HE, and TUNEL staining were performed to evaluate myocardial injury and apoptosis. A kit was used to detect superoxide dismutase (SOD) and malondialdehyde (MDA) to assess oxidative stress. In vitro, flow cytometry was used to detect the reactive oxygen species (ROS) content and apoptosis rate. Protein levels were determined via Western blotting. Pretreatment with SYI significantly reduced infarct size and pathological damage in rat hearts and suppressed cardiomyocyte apoptosis in vivo and in vitro. In addition, SYI inhibited oxidative stress by increasing SOD activity and decreasing MDA content and ROS production. Myocardial I/R and OGD/R activate endoplasmic reticulum (ER) stress, as evidenced by increased expression of activating transcription factor 6 (ATF6), glucose-regulated protein 78 (GRP78), cysteinyl aspartate-specific proteinase caspase-12, and C/EBP-homologous protein (CHOP), which were all inhibited by SYI. SYI ameliorated myocardial I/R injury by attenuating apoptosis, oxidative damage, and ER stress, which revealed new mechanistic insights into its application.

Computational simulations uncover enantioselective metabolism of chiral triazole fungicides by human CYP450 enzymes: A case study of tebuconazole

Tebuconazole (TEB), a prominent chiral triazole fungicide, has been extensively utilized for plant pathogen control globally. Despite experimental evidence of TEB metabolism in mammals, the enantioselectivity in the biotransformation of R- and S-TEB enantiomers by specific CYP450s remains elusive. In this work, integrated in silico simulations were employed to unveil the binding interactions and enantioselective metabolic fate of TEB enantiomers within human CYP1A2, 2B6, 2E1, and 3A4. Molecular dynamics (MD) simulations clearly delineated the binding specificity of R- and S-TEB to the four CYP450s, crucially determining their differences in metabolic activity and enantioselectivity. The primary driving force for robust ligand binding was identified as van der Waals interactions with CYP450s, particularly involving the hydrophobic residues. Mechanistic insights derived from quantum mechanics/molecular mechanics (QM/MM) calculations established C2-methyl hydroxylation as the predominant route of R-/S-TEB metabolism, while C6-hydroxylation and triazol epoxidation were deemed kinetically infeasible pathways. Specifically, the resulting hydroxy-R-TEB metabolite primarily originates from R-TEB biotransformation by 1A2, 2E1 and 3A4, whereas hydroxy-S-TEB is preferentially produced by 2B6. These findings significantly contribute to our comprehension of the binding specificity and enantioselective metabolic fate of chiral TEB by CYP450s, potentially informing further research on human health risk assessment associated with TEB exposure.

Fabrication of micropatterned PCL-collagen nanofibrous scaffold for cellular confinement induced early osteogenesis

The intricate interaction of the scaffold's architecture/geometry and with the cells is essential for tissue engineering and regenerative medicine. Cells sense their surrounding dynamic cues such as biophysical, biomechanical, and biochemical, and respond to them differently. Numerous studies have recently explored and reported the effect of contact guidance by culturing various types of cells on different types of micropatterned substrates such as microgrooves, geometric (square and triangle) micropattern, microstrips, micropatterned nanofibers. Amongst all of these micropatterned polymeric substrates; electrospun nanofibers have been regarded as a suitable substrate as it mimics the native ECM architectures. Therefore, in the present study; stencil-assisted electrospun Grid-lined micropatterned PCL-Collagen nanofibers (GLMPCnfs) were fabricated and its influence on the alignment and differentiation of pre-osteoblast cells (MC3T3-E1) was investigated. The randomly orientated Non-patterned PCL-Collagen nanofibers (NPPCnfs) were used as control. The patterns were characterized for their geometrical features such as area and thickness of deposition using surface profiler and scanning electron microscopy. A 61 % decrease in the overall area of GLMPCnfs as compared to the stencil area demonstrated the potential of electrofocusing phenomenon in the process of patterning electrospun nanofibers into various micron-scale structures. The MC3T3-E1 cells were confined and aligned in the direction of GLMPCnfs as confirmed by a high cellular aspect ratio (AR = 5.41), lower cellular shape index (CSI = 0.243), and cytoskeletal reorganization assessed through the F-actin filament immunocytochemistry (ICC) imaging. The aligned cells along the GLMPCnfs exhibited elevated alkaline phosphatase activity and enhanced mineralization. Furthermore, the gene expression profiling revealed upregulation of key osteogenic markers, such as ALP, OCN, OPN, COL1A1, and osteocyte markers DMP1, and SOST. Consequently, the research highlights the impact of GLMPCnfs on the cellular behaviour that results to the pre-osteoblast differentiation and the potential for stimulant-free early osteogenesis. These results offer an extensive understanding and mechanistic insight into how scaffold topography can be modified to influence cellular responses for effective bone regeneration strategies.

Acetylation of Steroidogenic Acute Regulatory Protein Sensitizes 17β-Estradiol Regulation in Hormone-Sensitive Breast Cancer Cells.

An imbalance in estrogen signaling is a critical event in breast tumorigenesis. The majority of breast cancers (BCs) are hormone-sensitive; they majorly express the estrogen receptor (ER+) and are activated by 17β-estradiol (E2). The steroidogenic acute regulatory protein (StAR) mediates the rate-limiting step in steroid biosynthesis. The dysregulation of the epigenetic machinery, modulating E2 levels, is a primary occurrence for promoting breast tumorigenesis. StAR expression, concomitant with E2 synthesis, was reported to be aberrantly high in human and mouse hormone-dependent BC cells compared with their non-cancerous counterparts. However, the mechanism of action of StAR remains poorly understood. We discovered StAR as an acetylated protein and have identified a number of lysine (K) residues that are putatively acetylated in malignant and non-malignant breast cells, using LC-MS/MS (liquid chromatography-tandem mass spectrometry), suggesting they differently influence E2 synthesis in mammary tissue. The treatment of hormone-sensitive MCF7 cells with a variety of histone deacetylase inhibitors (HDACIs), at therapeutically and clinically relevant doses, identified a few additional StAR acetylated lysine residues. Among a total of fourteen StAR acetylomes undergoing acetylation and deacetylation, K111 and K253 were frequently recognized either endogenously or in response to HDACIs. Site-directed mutagenesis studies of these two StAR acetylomes, pertaining to K111Q and K253Q acetylation mimetic states, resulted in increases in E2 levels in ER+ MCF7 and triple negative MB-231 BC cells, compared with their values seen with human StAR. Conversely, these cells carrying K111R and K253R deacetylation mutants diminished E2 biosynthesis. These findings provide novel and mechanistic insights into intra-tumoral E2 regulation by elucidating the functional importance of this uncovered StAR post-translational modification (PTM), involving acetylation and deacetylation events, underscoring the potential of StAR as a therapeutic target for hormone-sensitive BC.

Anti-inflammatory potential of Piper betleoides C. DC., a promising Piper species of Northeast India: in vitro and in vivo evidence and mechanistic insight.

The present study aims to investigate the anti-inflammatory potential of the leaf hydroalcoholic extract of Piper betleoides C. DC., also known as "Jangli Paan" in Northeast India, using lipopolysaccharide (LPS)-treated both cell culture (RAW264.7, macrophage cells) and animal (albino rat) model of inflammation. Treatment with leaf hydroalcoholic extract of Piper betleoides (PBtE) dose-dependently (5, 10, and 20µg/mL) decreased the secretion of pro-inflammatory (TNF-α, IL-6, and MCP-1) and increased anti-inflammatory (IL-4 and IL-10) cytokines in LPS-treated macrophages. Similarly, treatment with PBtE also prevented the alternation in mRNA expression of inflammatory markers (TNF-α, CCL-2, IL-6, and IL-10) in LPS-treated macrophages. Dose-dependent supplementation with PBtE further reduced the production of intracellular ROS and increased the phagocytosis efficacies in LPS-treated cells. Further in vivo studies demonstrated that treatment with PBtE dose-dependently (50, 100, and 200mg/kg body weight) prevented the dysregulation of the secretion of inflammatory cytokines (TNF-α, IL-4, IL-6, and IL-10) and reduced the circulatory levels of prostaglandin (PGE2) and nitric oxide products (nitrite) in LPS-treated animals. In addition, alternation of blood cell profiling and the liver as well as kidney dysfunctions were also prevented by the treatment with PBtE in LPS-treated rats. The anti-inflammatory potential of PBtE was comparable to those seen in sodium diclofenac (positive control) treated group. LC-MS analyses showed piperine, piperlongumine, piperolactam-A, and dehydropipernonaline and GC-MS analyses demonstrated phytol, caryophyllene, and falcarinol as the phytochemicals present in Piper betleoides, which might play an important role in preventing inflammation and associated pathophysiology. Different treatments didn't cause any toxicity in cell culture and animal models. This study for the first time demonstrated the promising anti-inflammatory potential of the leaf hydroalcoholic extract of Piper betleoides.