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Targeted delivery of drugs and hyperthermia in cardiovascular disease demand the accurate delivery of nanoparticles in complex arterial geometries. This paper introduces combined hybrid computational model that concomitantly examines the combined impact of nanoparticle radius and interparticle spacing on the thermal and mass transport characteristics of ternary bio-nanofluid flow under magnetohydrodynamic (MHD) effect. The ternary fluid is composed of blood fluid with suspended nanoparticles such as gold (Au), silver (Ag) silica (SiO2). The mathematical model accounts for geometric properties of nanoparticles such as nanoparticles radius and interparticle spacing for their practical utility for several medical interventions. The numerical analysis is based on hybrid computational strategy, where the solutions are determined through the bvp4c numerical solver and then a novel supervised multi hidden layers Artificial neural network (ANN) is integrated. The proposed model has a high predictive capability with an exceptionally high accuracy with the lowest Mean squared error and ideal regression coefficient MSE=9.6327×10-11, Gradient=9.5681e-08, Mu=1e-09, and R2=1.0. Some of the main findings indicate that less spacing between particles (h=0.1) leads to continuous networks of thermal percolation, which enhance the thermal conductivity by up to 35% to improve the efficiency of hyperthermia, whereas the larger nanoparticles (radius ≥1.5) offer a higher drug-loading capacity, yet the rate of heat transfer decreases by 15-20%. Optimization of the magnetic parameter (M=0.1-0.7) also decreases flow velocity by 28% and extends the nanoparticle residence time at the stenosis by 35% which allows sustained drug delivery, results directly applicable to clinical-strength (1.5-3T) MRI-guided interventions. Radiation parameter (Rd=0.5-2.5) increases temperature of the arteries by 15-20% giving controllable thermal modulation to applications of hyperthermia. The proposed model predicts that optimal nanoparticle preparations (50 nm radius, 20 nm spacing) have to potential to lower the rate of restenosis by 30-40% in relation to traditional drug-eluting stents. The purpose of such an integrated computational-machine learning systems is to give quantitative advice to stent coating design, nanoparticle formulation, and optimization of treatment protocols, and has been directly used in biomedical interventions. The results can be used to offer practical advice to stent manufactures, interventional radiologist and pharmaceutical developers to create evidence-based cardiovascular therapy of the next generation.

Biotherapeutic antibodies are increasingly being developed and, various strategies have recently been used to maximize their potential therapeutic efficacy. The crystallizable fragment (Fc) region of therapeutic monoclonal antibodies (mAbs) is often engineered to tailor their effector functions and pharmacokinetic (PK) properties by introducing point mutations. Notably, most of these mutations are in the hinge and constant domains of the heavy chain, which may silence antibody effector functions. Several liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods have been published to quantify biotherapeutics with a canonical human Fc portion. This work presents a rapid and sensitive hybrid immunocapture liquid chromatography-tandem mass spectrometry (IC-LC-MS/MS) method for quantifying total antibody concentration, specifically targeting the LALA-mutated peptide (L234A/L235A). The sample preparation process, which includes immunocapture, as well as trypsin and Glu-C digestion, is efficiently completed within two days through automation. The developed method was validated according to the ICH M10 guideline and white papers recommendation, focusing on the following parameters - accuracy, precision, dilution linearity, selectivity, stability, recovery- and using a humanized IgG1 LALA-mutated antibody, teplizumab, as analytical standard. The method demonstrated linearity for total antibody detection in mouse plasma samples, with a dynamic range from 150 ng/mL (lower limit of quantification, LLOQ) to 15,000 ng/mL (upper limit of quantitation, ULOQ). All validation parameters tested in mouse plasma met the predefined acceptance criteria, demonstrating the method's reliability and robustness. Additionally, the qualified method was successfully used to characterize the pharmacokinetic profile in mice of an antibody-drug conjugate (ADC-1) containing the LALA mutation in its Fc region. This work provides a valuable foundation for the quantification of new biological entities (NBEs) and antibody-drug conjugates (ADCs) in pharmaceutical development, as it enables the measurement of engineered Fc biotherapeutics using a unique and highly selective peptide, irrespective of the type of biological matrix and even in the presence of other biomolecules of similar IgG isotype.

Medicinal plants are widely used for applications in agriculture, food, medicine, and cosmetics due to their abundant bioactive secondary metabolites (SMs) such as terpenoids, phenylpropanoids, and alkaloids. The biosynthesis and accumulation of SMs are highly associated with multiple environmental factors. Among these abiotic stresses, drought plays a pivotal role in regulating the quality of medicinal plants. Understanding the regulatory mechanisms of medicinal plants in response to drought is beneficial for (i) cultivating high-quality traditional Chinese medicinal plants via targeted water management strategies; (ii) screening candidate marker genes to breed high-quality novel cultivars with enhanced bioactive compound accumulation under drought conditions, thereby addressing the adverse impacts of drought induced by global climate change; (iii) mining dual-functional genes that confer drought tolerance while maintaining high bioactive compound content, thus ensuring both the yield and quality of medicinal plants. To summarize the latest advances in the transcriptional regulation of SM biosynthesis with a focus on terpenoids, phenylpropanoids, and alkaloids in medicinal plants under drought conditions. A comprehensive literature search was conducted in three electronic databases including PubMed, Scopus, and Web of Science using the search terms "regulatory mechanism", "secondary metabolites", "medicinal plants", "drought stress", "transcription factor", "bioactive compound", "synthetic biology", "smart irrigation", "terpenoid biosynthesis", "phenylpropanoid biosynthesis", "phenolic biosynthesis" and "alkaloid biosynthesis". All the retrieved data were then critically reviewed and summarized. Drought affects secondary metabolite biosynthesis via a complex molecular regulatory network, including shifts in microbial community composition, epigenetic remodeling, changes in global gene expression profiles, altered catalytic activity of core biosynthetic enzymes, as well as modifications of transcription factors. This review offers novel insights into unraveling the underlying transcriptional regulatory networks, and practical implications for researchers in the fields of medicinal plant biology, natural product chemistry, and crop stress physiology.

Dandelion (Taraxacum officinale) is an edible medicinal herb having an extended history for its traditional usage owing to the health promoting benefits associated with this plant. Nevertheless, traditional extraction methods limit the recovery of bioactive compounds from different parts of dandelion and insufficient research is available on process optimization. Hence, current research developed an effective ultrasound-assisted extraction method for maximum recovery of total phenolics contents (TPC), total flavonoids contents (TFC), antioxidant activities (DPPH, ABTS, FRAP assays), and bioactive compounds (HPLC) from dandelion plant using response surface methodology in combination with Box-Behnken design (BBD). The optimal extraction conditions determined by RSM were as follows: sonication time, 30 min; ultrasound amplitude, 70%, and ultrasound temperature, 40 °C. At these conditions, the recovery of total phenolics and total flavonoids reached 40.77 mg GAE/g and 22.68 mg RE/g, respectively. Moreover, the antioxidant activities determined based on DPPH-scavenging, ABTS+-scavenging and FRAP were reported as 88.55%, 445.39 µM TE/mg, and 30.64 mg TE/g, respectively. Additionally, a total of 11 major bioactive compounds were quantified using HPLC, including 6 phenolic acids and 5 flavonoids. Under optimized conditions major bioactive compounds identified and quantified were chlorogenic acid, quercetin, apigenin, luteolin-7-O-glycoside, luteolin, p-coumaric acid, caffeic acid, ferulic acid, cichoric acid, isoetin, and caftaric acid. Conclusively, results of present study give a comprehensive insight into optimized ultrasound-assisted extraction method for recovery of maximum antioxidants and bioactive compounds from dandelion using a combination of BBD and RSM. Furthermore, this study may provide a reference in utilizing optimized extraction process for dandelion bioactive compounds in food and pharmaceutical industry.

Obsessive-compulsive disorder (OCD) is a debilitating neuropsychiatric disorder that remains chronic unless intervened with evidence-based intervention. Cognitive behavioral therapy (CBT) with exposure and response prevention is the gold-standard psychological intervention for OCD, but many individuals do not receive this intervention due to barriers to accessing treatment. Mental health technology tools such as telehealth, computerized programs, internet-delivered CBT, and mobile applications have been adopted to expand the accessibility of CBT. An up-to-date summary of the evidence base of technology-mediated formats of CBT for OCD treatment is provided. Clinical benefits offered by such approaches, current limitations, and future research directions are discussed.

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The optimal integration of immunotherapy and surgery in the management of advanced clear cell renal cell carcinoma (ccRCC) remains under investigation. Deferred cytoreductive nephrectomy (dCN) following immune checkpoint inhibitor (IO)-based therapy may provide clinical benefit in selected patients, but radiologic and pathologic predictors of response are not well defined. We conducted a retrospective analysis of patients with advanced or metastatic ccRCC treated with perioperative IO-IO or IO-tyrosine kinase inhibitor (IO-TKI) regimens followed by dCN. Radiographic response, IVC thrombus changes, pathological necrosis, immune-related adverse events, and progression-free survival (PFS) were evaluated. A total of 55 patients were eligible and included in this analysis. Regimens with IO-TKI were associated with greater tumor size reduction, higher rates of IVC thrombus downstaging, and increased tumor necrosis compared to IO-IO. Tumor necrosis ≥ 20% and ≥ 80% on final pathology correlated with improved PFS. Immune-related toxicities were more common in patients receiving IO-IO. Preoperative IO-TKI combinations are associated with meaningful radiologic and pathologic responses in advanced ccRCC and may be considered when the goal is cytoreduction. Tumor necrosis may serve as a prognostic marker of response. These findings support the ongoing evaluation of dCN in prospective trials and highlight the importance of multidisciplinary patient selection.

Covalent Bruton tyrosine kinase (BTK) inhibitors have advanced the treatment of Waldenström macroglobulinaemia; however, the occurrence of progression, intolerance, and acquired resistance are not fully understood. We aim to report on the safety and activity of pirtobrutinib (a highly selective, non-covalent BTK inhibitor) in patients with relapsed or refractory Waldenström macroglobulinaemia, including those who received previous covalent BTK inhibitors as part of the phase 1/2 BRUIN trial. The BRUIN study was an open-label, multicentre, phase 1/2 trial that enrolled patients with relapsed or refractory B-cell malignancies from 29 sites across eight countries. Patients aged 18 years or older who previously received BTK inhibitor-containing regimens, had an Eastern Cooperative Oncology Group performance status of 0-2, and histologically confirmed Waldenström macroglobulinaemia were eligible. In phase 1, patients received 100-300 mg oral pirtobrutinib once a day in 28-day cycles and the recommended phase 2 dose (RP2D) of 200 mg pirtobrutinib once a day was determined. The phase 2 primary endpoint was antitumour activity of pirtobrutinib based on objective response rate as assessed by an investigator in patients with chronic lymphocytic leukaemia, small lymphocytic leukaemia, or mantle cell lymphoma. In patients with Waldenström macroglobulinaemia, response was evaluated using the Sixth International Workshop on Waldenström Macroglobulinemia (IWWM-6) criteria. BRUIN is registered with ClinicalTrials.gov, NCT03740529 (completed). BRUIN recruited patients from Aug 12, 2019, to March 14, 2022, and 778 patients received pirtobrutinib. 80 patients had relapsed or refractory Waldenström macroglobulinaemia (n=18 in phase 1 and n=62 in phase 2), with a median age of 68·5 years (IQR 61·0-75·0). 52 (65%) patients were male and 28 (35%) were female. The median number of previous lines of systemic therapy was 3·0 (2·0-5·0). 63 (79%) patients received previous covalent BTK inhibitors. 73 (91%) received 200 mg pirtobrutinib once per day (the RP2D). Using IWWM-6 criteria, the objective response rate was 82·5% (95% CI 72·4-90·1), with one (1·3%) patient reaching complete response, eight (10·0%) reaching very good partial response, 49 (61·3%) reaching partial response, and eight (10·0%) reaching minor response. The median study follow-up was 35·0 months (17·7-47·7). The objective response rate was 81·0% (69·1-89·8) for those who received previous covalent BTK inhibitors and 88·2% (63·6-98·5) for covalent BTK inhibitor-naive patients. Grade 3 or higher treatment-emergent adverse events occurred in 57 (71%) patients, with the most common being neutropenia or neutrophil count decreased (15 [19%]) and anaemia (19 [24%]). Treatment-emergent deaths were reported in five (6%) patients (bacterial sepsis, intracranial haemorrhage, COVID-19 pneumonia, hypertensive cardiomegaly and pneumonia [n=1 each unrelated to treatment], and treatment-related necrotising pneumonia [n=1]). Treatment-emergent adverse events leading to dose reductions occurred in four (5%) patients and pirtobrutinib discontinuation in 12 (15%). Pirtobrutinib was highly active and well tolerated, regardless of previous exposure to covalent BTK inhibitors, and might be a promising new therapeutic option for patients with relapsed or refractory Waldenström macroglobulinaemia, particularly in those previously exposed to covalent BTK inhibitors, for whom durable and effective treatments are needed. Eli Lilly and Company.

Extracellular vesicles (EVs) are cell-secreted phospholipid bilayer vesicles that play a key role in intercellular communication by transporting molecular cargo and engaging in surface-level signaling. Due to their intrinsic biological features, EVs not only reflect the functional attributes of their originating cells but also hold promise as both therapeutic agent and natural carriers for targeted delivery. In recent years, plant-derived nanovesicles (PDNVs) containing bioactive molecules have attracted the attention of researchers because of their better biocompatibility, low immunogenicity, wide range of sources, and ability to act as natural therapeutic agents for diseases. PDNVs play an increasingly important role in human-plant interactions, as they are able to enter the human system and deliver effector molecules to cells, which in turn modulate cellular signaling pathways. PDNVs play a critical role in human health and disease. This review provides a comprehensive overview of PDNVs, encompassing their biogenesis, methods of isolation and purification, physicochemical characterization, stability, and storage strategies. It further explores their routes of administration, internalization, and biodistribution as therapeutic agents, highlighting their potential in the treatment of conditions such as inflammation, cancer, tissue regeneration, viral infections, liver and brain disorders, and osteoporosis. Lastly, the review examines current clinical applications of PDNVs and the key challenges hindering their broader implementation. We look forward to further exploration of the functions of PDNVs to facilitate their clinical translation and increase their benefits in humans.

When androgen receptor (AR) signaling is suppressed, prostate cancer progression is inhibited; however, many patients eventually relapse, developing castration-resistant prostate cancer (CRPC). Recently, the incidence of double-negative CRPC (DNPC)-which lacks AR and neuroendocrine activity-has increased, yet effective treatments remain unavailable. Our research demonstrated that KRAS has minimal influence during the AR-dependent stages of prostate cancer but significantly activates cancer cells when AR signaling is suppressed. Further investigation revealed that AR inhibition modifies fibroblast growth factor receptor expression in prostate cancer cells. Additionally, CCL2, secreted by AR-inhibited prostate cancer cells, induces FGF8b secretion from stromal cells within the tumor microenvironment, which in turn enhances KRAS activation. A pan-KRAS inhibitor effectively suppressed AR-independent prostate cancer cells by disrupting KRAS-mediated cell survival signaling. This inhibition led to the significant induction of programmed cell death, characterized by the downregulation of the anti-apoptotic protein BCL-xL and the promotion of apoptosis as evidenced by increased cleaved caspase-3 in vivo. These findings highlight KRAS activation, driven by the CRPC microenvironment, as a critical factor in DNPC progression and identify the induction of KRAS-targeted cell death as a promising therapeutic strategy for DNPC.

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A simple suspension method has been widely used in Japan, however, chemical interactions among drugs in co-suspensions have not been fully clarified. Magnesium oxide (MgO), which is frequently prescribed as a laxative, can interact with various drugs. Eplerenone and spironolactone, potassium-sparing diuretics, are sometimes co-prescribed with MgO to older patients. In this study, we investigated the chemical stability of eplerenone and spironolactone in a co-suspension with MgO and characterized the structures of the degradation products that were formed under the conditions. An eplerenone or spironolactone tablet was soaked with or without an MgO tablet in warm water in a tube according to a standard simple suspension method. The contents in the tube were mixed by inversion after 10 min, 1 h or 5 h to prepare a simple suspension. In separate experiments, the suspension prepared after 10 min soaking was allowed to stand for 50 min or 4.8 h at room temperature. The suspensions were immediately analyzed by high-performance liquid chromatography. The recovery rates of the diuretics from the suspensions were calculated relative to the labeled amounts. The degradation products were isolated and the structures analyzed by high-resolution mass spectrometry. The high-performance liquid chromatographic analysis showed that the diuretics were stable in their simple suspensions without MgO under any of the conditions. When co-suspended with MgO, a slight degradation was observed for eplerenone after just 10 min soaking and the degradation was statistically significant after 5 h soaking, whereas spironolactone was stable even after 5 h soaking. On the other hand, when the co-suspensions with MgO were left alone after mixing, eplerenone significantly degraded in 50 min, and spironolactone slightly degraded in the same period. Based on the mass spectra from the degradation products, hydrolysis of the lactone ring was shown to have occurred in both diuretics co-suspended with MgO. For spironolactone, hydrolysis and elimination of the thioester were also shown to have occurred in the co-suspensions. Eplerenone is more unstable than spironolactone in the simple co-suspension with MgO. As such, the simple co-suspensions of eplerenone are preferably prepared immediately before administration.

Ectonucleotidases, including NTPDases and ecto-5'-nucleotidase (e-5'NT/CD73), regulate extracellular purinergic signaling by converting ATP to adenosine, a pathway critically involved in immune response, inflammation, and cancer progression. In this study, a novel library of 22 N-propylsulfonyl-substituted indole-based hydrazinecarbothioamides (5a-5v) was synthesized and structurally characterized. Biological evaluation against human e-5'NT and NTPDase1, -2, -3, and - 8 revealed that several compounds exhibited low micromolar inhibitory activity, with 5n (IC50 = 1.7 µM), 5o (IC50 = 1.7 µM), 5f (IC50 = 1.0 µM), and 5i (IC50 = 1.6 µM) emerging as the most promising derivatives, showing strong potency and isoform selectivity. Structure-activity relationship analysis indicated that both electronic and steric features of substituents significantly influence activity and enzyme preference. Molecular docking studies performed on e-5'NT demonstrated that active compounds adopt consistent binding modes within the catalytic pocket, stabilized by key residues such as Asp-506, Phe-500, Phe-417 and Arg-395. Binding free energy calculations (MM-GBSA) supported strong ligand-protein interactions ( ~ - 70 kcal/mol). The docking protocol was validated by redocking, yielding an RMSD value well below the accepted threshold. Molecular dynamics simulations (500 ns) confirmed stable complex formation, with low RMSD values (~ 1-3 Å), limited residue fluctuations, and persistent interactions with catalytic residues. Surface and compactness parameters (rGyr, SASA) remained stable, indicating consistent ligand accommodation. In silico ADME analysis suggested favorable drug-like properties for most compounds, particularly for the lead candidates. Overall, these findings identify 5n and 5o as the most promising lead compounds, supported by both experimental and computational results, and highlight this scaffold as a valuable platform for the development of selective ectonucleotidase inhibitors.

Cbl-b, an E3 ubiquitin ligase, is a critical negative regulator of T-cell activation and an attractive target for cancer immunotherapy. Current small-molecule inhibitors largely rely on hydrophobic π-π stacking interactions with the gatekeeper residue Tyr363, which restricts the structural diversity of Cbl-b inhibitors and hinders the discovery of inhibitors with novel scaffolds. This study reports the stepwise optimization of the cyclic carbamate lead compound 5, eventually leading to the discovery of novel, representative alkylamine-based Cbl-b inhibitors. Our optimization process comprised three stages: (1) conformational restriction via lactamization, which yielded initial hit 12 (IC50 = 31.99 ± 3.88 μM); (2) hydrophobic cavity filling, which provided the improved analog 22 (IC50 = 8.58 ± 0.25 μM); and (3) SeeSAR-guided scaffold hopping, which ultimately identified the representative lead compound 27 (IC50 = 6.83 ± 0.51 μM). Molecular docking and molecular dynamics (MD) simulations confirmed that 27 binds to the TKB-LH interface and stabilizes the inactive conformation of Cbl-b. Notably, MD simulations revealed that 27 engages Tyr363 through a unique polar interaction mode dominated by hydrogen bonds and water bridges, a distinct departure from traditional hydrophobic stacking. This novel alkylamine scaffold provides a new approach for developing structurally diverse Cbl-b inhibitors.

The umbilical cord and the amniotic membrane are a precious source of human mesenchymal stem cells (hMSCs), even though they are often discarded after the delivery. Due to their immunomodulatory and anti-inflammatory properties, hMSCs could be part of relevant strategies in the field of regenerative medicine. Additionally, they can be obtained from these tissues via a non-invasive and cost-effective process, overcoming ethical controversies. This study aims to propose protocol refinement to obtain hMSCs from the amnion and umbilical cord of healthy donors (utilizing limited and defined laboratory resources), and to compare and characterize these cells, thereby enabling future research on their properties. 30 women from "Paolo Giaccone" University Hospital of Palermo (Italy) were enrolled, according to the inclusion and exclusion criteria approved by the local Ethics Committee. A sample of umbilical cord and amnion was obtained from every patient and processed via an enzymatic or mechanical method. After refining the isolation protocol, hMSCs were characterized using flow cytometry, RT-qPCR, inducing a trilinear differentiation, and testing the formation of spheroids. This research shows reliable and practical methods to isolate hMSCs from birth tissues, validating them with extensive cell characterization. No direct association was observed between mothers' age and newborns' sex and the success rate in establishing hMSCs primary cultures, while a possible association between neonatal weight and the successful establishment of umbilical cord-derived cultures was found. Moreover, a difference in the adipogenic potential of the two hMSCs sources was highlighted. hMSCs have a relevant role in biomedicine, along with their derivatives, for their promising regenerative properties: this study aims to explore new insights to promote further research in this field.

Restoring the tumor-suppressor function of p53 by inhibiting its negative regulator, MDM2, represents a significant therapeutic avenue for cancers that maintain wild-type p53. This research aimed to identify new MDM2 inhibitors through a phylogenetically guided strategy that involved the construction of a focused virtual library of metabolites derived from the Penicillium genus. A comprehensive computational framework was developed, employing machine learning-based quantitative structure-activity relationship (ML-QSAR) modeling, ensemble molecular docking, network pharmacology, molecular dynamics (MD) simulations, and ADMET profiling. The gradient boosting ML-QSAR model achieved a test set R2 of 0.80 and was externally validated against 39 known MDM2 inhibitors (R2 = 0.82, RMSE = 0.80 pIC50 units), confirming its predictive reliability. Ensemble docking studies against 13 conformations of MDM2 highlighted three leading candidates (CNP0147553.1, CNP0154476.3, and CNP0154476.4) demonstrating binding affinities comparable to the known control inhibitor Nutlin-3a, with docking scores validated against experimental binding data. Further investigations through 500 ns MD simulations provided insights into the stability of the CNP0147553.1-MDM2 complex, which maintained a mean ligand RMSD of 0.039 nm and a complex RMSD of 0.176 nm, alongside a favorable binding free energy of -25.82 kcal/mol. Key residue analysis revealed that CNP0147553.1 achieved pronounced stabilization of critical binding pocket residues, including an 81.6% reduction in flexibility of HIS96. Network pharmacology analysis revealed a polypharmacology potential, indicating that the hub genes related to the identified compounds predominantly converged on the PI3K-AKT-mTOR and RAS-RAF-MAPK signaling pathways. ADMET profiling suggested promising pharmacokinetic and safety profiles for the lead candidates, establishing the basis for future experimental validation.

β-Sitosterol is a bioactive phytosterol with recognized anti-inflammatory, hypocholesterolemic, and immunomodulatory properties. While Zygophyllum fabago L. is known to contain steroidal compounds, systematic studies on the isolation of pure β-sitosterol from this species are limited, particularly regarding the use of green extraction technologies and the achievement of high-purity yields for industrial applications. This study aimed to isolate and identify β-sitosterol from the herb of Z. fabago L. utilizing subcritical CO2 extraction as an environmentally sustainable and selective methodology for nonpolar compounds. β-Sitosterol was isolated from the herb of Zygophyllum fabago L. using subcritical CO2 extraction (57-65 atm, 18-27 °C, 6 L/h). The crude extract was purified via vacuum liquid chromatography (VLC) on silica gel 60 and recrystallized from methanol, with purity monitored by TLC (UV 254/366 nm and H2SO4 visualization). Identification was performed using 1H and 13C NMR spectroscopy and the structure was confirmed by comparison with literature data. The subcritical CO2 extraction yielded 20 g of crude extract (2.3% yield). The purification process resulted in 1.0 g of pure β-sitosterol, representing a 5% yield relative to the crude extract. TLC analysis showed a single characteristic spot, and NMR data definitively confirmed the structure, identifying key signals such as the C-3 hydroxyl group, the Δ5 double bond, and the characteristic branched aliphatic side chain. These results demonstrate that Z. fabago L. is a high-yielding natural source of β-sitosterol. Furthermore, the study validates subcritical CO2 extraction as a highly effective and selective approach for the preparative isolation of phytosterols from plant matrices, offering significant advantages in terms of purity and environmental impact.

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The production of monoclonal antibodies (mAbs) in Chinese hamster ovary (CHO) cells is often affected by position-effect variegation and the gradual loss of transgene expression over time. Hence, we have designed a dual-promoter IgG expression vector and compared versions that either contained or lacked a CHO-derived matrix-attachment region (MAR). Stable CHO-S pools, cultured in serum-free conditions, revealed that the MAR-containing construct produced higher and more consistent antibody levels across ten passages, as confirmed by Western blot and Protein A Octet analysis. Product-quality analysis by size-exclusion chromatography and reducing SDS-PAGE confirmed formation of properly assembled, mainly monomeric antibodies in both cases. Quantitative PCR indicated greater transgene copy numbers in MAR pools (+ 48% for the light chain and + 71% for the heavy chain), and RT-qPCR showed roughly fourfold higher transcript levels for both chains relative to controls. Bioinformatic analysis revealed several SATB1 binding motifs within the MAR sequence, and ChIP-qPCR demonstrated SATB1 association with the MAR-linked transgene locus. Overall, the data suggested that a CHO-native MAR could enhance transgene dosage and transcriptional activity, while preserving product integrity, possibly through SATB1-mediated chromatin organization. Ongoing work includes chromatin-mark profiling and process-level productivity measurements to better define the impact of MAR-based vector design on biomanufacturing performance.