Cellular Viability Research Articles

AM404 Analogs as Activators of the 20S Isoform of the Human Proteasome.

The proteasome is a multisubunit protease system responsible for the majority of the protein turnover in eukaryotic organisms. Dysregulation of this enzymatic complex leads to protein accumulation, subsequent aggregation, and ultimately diseased states; for that reason, positive modulation of its activity has been recently investigated as a therapeutic strategy for neurodegenerative and age-related diseases. The small molecule AM404 was recently identified as an activator of the 20S isoform of the proteasome and further exploration of the scaffold revealed the importance of the polyunsaturated fatty acid chain to elicit activity. Herein, we report the investigation of the aromatic region of the scaffold and the evaluation of the small molecules in a variety of proteasome activity and protein degradation assays. We found that derivatives A22 and A23, compared to AM404, exhibit enhanced proteasome activity in biochemical and cellular proteasome assays and more favorable cellular viability profiles. Additionally, these compounds demonstrate the ability to degrade intrinsically disordered proteins, regardless of their molecular weight, and the ability to restore the proteasome activity in the presence of toxic oligomeric α-Syn species in a biochemical setting.

Humanin’s impact on pain markers and neuronal viability in diabetic neuropathy model

Objective This study investigates the impact of chronic humanin (HN) treatment on pain-related markers (NMDA, substance P, TRPV1, and IL-1β) in diabetic mice’s dorsal root ganglia (DRG). Additionally, we assess the effects of HN on cellular viability in DRG neurons. Methods In vivo experiments involved 15 days of HN administration (4 mg/kg) to diabetic mice (n = 10). Protein levels of NMDA, IL-1β, TRPV1, and substance P were measured in diabetic DRG. In vitro experiments explored HN’s impact on apoptosis and cellular viability, focusing on the JAK2/STAT3 pathway. Results Humanin significantly reduced the elevated expression of NMDA, IL-1β, TRPV1, and substance P induced by diabetes (p < .05). Furthermore, HN treatment increased cellular viability in DRG neurons through JAK2/STAT3 pathway activation (p < .05). Conclusion These findings highlight the significance of understanding mitochondrial function and pain markers, as well as apoptosis in diabetes. The study provides insights for managing the condition and its complications.

Protective effect of avicularin against lung cancer via inhibiting inflammation, oxidative stress, and induction of apoptosis: an in vitro and in vivo study.

The purpose of this research was to investigate whether or not avicularin (AVL) possesses any anticancer properties when tested against lung cancer. In the beginning, the effect that it had on the cellular viability of A549 cells was investigated, and it was discovered that AVL has a considerable negative impact on cellular viability. Following that, an investigation using flow cytometry was carried out to investigate its function in the process of apoptosis and the cell cycle of A549 cells. It has been discovered that AVL significantly promotes apoptosis and stops the cell cycle at the G2/M phase. The colony-forming capacity of A549 cells was observed to be greatly suppressed as the AVL concentration increased compared to the group that received no treatment. In addition to this, the benzo(a)pyrene in vivo model was established in order to investigate the pharmacological value of AVL. The findings revealed that AVL greatly prevented the formation of pro-inflammatory cytokines, in addition to the reduction in oxidative stress, which was evidenced by a reduction in the concentration of TNF-α, IL-1β, IL-6, and MDA with an improvement in the concentration of SOD and GPx, respectively. Our results successfully demonstrated the pharmacological benefit of avicularin against lung cancer, and it has been suggested that it showed a multifactorial effect.

Abstract ND07: BBO-8520, a first-in-class, direct inhibitor of KRASG12C (ON), locks GTP-bound KRASG12C in the state 1 conformation resulting in rapid and complete blockade of effector binding

Abstract The glycine to cysteine mutation on codon 12 of KRAS (KRASG12C) is found in ~15% of non-small cell lung cancers and in a low percentage of colorectal and pancreatic adenocarcinomas. This activating mutation pushes the balance of cellular KRAS towards its active, GTP-bound (ON) state that signals downstream and drives cellular proliferation. Recently approved inhibitors of KRASG12C that bind and sequester the oncogenic protein in its inactive GDP-bound (OFF) state, have demonstrated clinical efficacy; however, median duration of response has been &amp;lt; 9 months. Rapid tumor cell adaptation to KRASG12C (OFF)-only inhibitors like sotorasib and adagrasib has been attributed to reactivation of MAPK signaling through increased RTK flux and KRASG12C gene amplification that result in increased KRASG12C (ON) presence and activity. To block rapid KRASG12C (ON)-driven adaptation to KRASG12C (OFF) inhibitors, we have developed a first-in-class, direct, small molecule covalent inhibitor of both KRASG12C (ON) and (OFF) states. BBO-8520 binds in the switch II pocket and covalently modifies both the (ON) and (OFF) forms of KRASG12C independently of any other partner proteins. Mass spectrometry analysis of KRASG12C covalent engagement by BBO-8520 shows complete modification of both KRASG12C states within 15 minutes, while sotorasib and adagrasib, only modify the (OFF) state. 31P NMR studies demonstrate that BBO-8520 inhibits KRASG12C (ON) by locking the GTP-bound protein in state 1, a conformation where it is unable to bind effectors. The ability to directly bind KRASG12C (ON) leads to potent (~30 nM) inhibition in an effector (Raf1) binding assay where inactive (OFF) inhibitors demonstrate no measurable potency. BBO-8520 displays highly significant binding to KRASG12C in a global cysteine proteome analysis and is 100x more selective for KRASG12C than for WT KRAS and other mutant isoforms, with no measurable activity against N- or H-RAS. Cellular signaling and viability assays show that BBO-8520 has sub-nanomolar potency against KRASG12C mutant cell lines. In effector binding assays, BBO-8520 rapidly and completely blocks the RAS-RAF1 interaction, clearly differentiating it from inactive (OFF) inhibitors that require longer times to allow for cycling to the inactive (OFF) state. Additionally, long-term clonogenic assays that detect the emergence of resistance in the presence of inhibitors show that BBO-8520 is at least 30x more potent than sotorasib and adagrasib at preventing outgrowth. Importantly, the presence of growth factors (e.g., EGF) that push KRASG12C into its (ON) state, and significantly lower the potency of inactive (OFF) inhibitors (&amp;gt;20x), have only minor effects on BBO-8520’s potency. Drug-like pharmacokinetic properties allow BBO-8520 to achieve strong dose- and time-dependent pharmacodynamic effects (&amp;gt;80% inhibition of pERK) following a single, oral dose in KRASG12C mutant tumor bearing mice. In vivo target engagement and pERK inhibition in the MIAPaCa-2 and H358 KRASG12C mutant tumor models resulted in durable tumor regressions at 10 mg/kg. Similarly, daily dosing of 10 mg/kg of BBO-8520 in the KrasG12C-p53 driven GEMM model for 6 weeks resulted in better than 50% lung tumor volume regression. BBO-8520’s potent activity against KRASG12C (ON) presents, for the first time, the potential opportunity to directly target all mutant KRASG12C in cells, including the active form of KRASG12C, enabling efficacy following complete or near-complete target inhibition. Citation Format: Anna E. Maciag, James Stice, Bin Wang, Alok Sharma, Albert Chan, Ken Lin, Devansh Singh, Marcin Dyba, Yue Yang, Saman Setoodeh, Brian P. Smith, Dana Rabara, Zuhui Zhang, Erik K. Larsen, Dom Esposito, John Paul Denson, Michela Ranieri, Mary Meynardie, Sadaf Mehdizadeh, Patrick Alexander, Maria Abreu Blanco, David Turner, Rui Xu, Felice C. Lightstone, Kwok Kin Wong, Dhirendra Simanshu, Keshi Wang, Andrew G. Stephen, Kerstin Sinkevicius, Dwight V. Nissley, Eli Wallace, Frank McCormick, Pedro J. Beltran. BBO-8520, a first-in-class, direct inhibitor of KRASG12C (ON), locks GTP-bound KRASG12C in the state 1 conformation resulting in rapid and complete blockade of effector binding [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr ND07.

Abstract LB028: Discovery and characterization of selective heterobifunctional degraders of EP300 in hematopoietic malignancies

Abstract The paralogous histone acetyltransferases EP300 and CREBBP are two of the master regulators of transcription by controlling both enhancer and promoter activity through histone and non-histone acetylation. EP300 and CREBBP activity is primarily driven by their acetylation of K27 and K18 on histone H3 leading to open and active chromatin state. Targeting the EP300/CREBBP axis of chromatin regulation may be a way to disrupt the signaling pathways downstream of key oncogenic signaling networks like MYC/IRF4 in hematopoietic cancers. Successful development of dual EP300/CREBBP BRDi and HATi has led to promising pre-clinical efficacy, but due to observed tolerability issues may potentially limit their utility in the clinic. We hypothesized, based upon published mouse KO phenotypes, that a selective EP300 compound may improve tolerability. Due to the high sequence homology between EP300 and CREBBP in both the BRD and HAT domains, developing selective inhibitors has proven to be challenging. To combat this, we have utilized heterobifunctional protein degrader technology (ProDegs, also known as PROTACS) to develop EP300 selective ProDegs with greater than 100-fold selectivity over CREBBP. We observe rapid targeted degradation of EP300 with maximal degradation occurring within the first 2-4 hours after treatment. Concomitant with the loss of EP300, we see modulation of the associated H3K27ac and H3K18ac biomarkers as well as alterations in key oncogenic transcription networks. In both in vitro and in vivo assays looking at impact to platelet maturation, we observed that selective loss of EP300 largely abrogated observed thrombocytopenia compared to dual EP300/CREBBP inhibitors. Upon treatment, MM and NHL models show strong downregulation of MYC/IRF4 oncogenic signaling pathways, both of which culminate in a loss of cellular viability and cell death. Additionally, we observe in vivo dose responsive tumor growth inhibition in OPM2 and Pfeiffer xenograft models. Together, the data demonstrate the potential utility of EP300 selective ProDegs for the treatment of hematopoietic malignancies. Citation Format: Frederick A. Derheimer, Ninvita Givarkes, Natalie Miller, Rita Grantner, Casey Quinlan, Sonja Brun, Thomas Paul, Astrid Ruefli-Brasse, Stefan Steyn, Derek Bartlett, Matthew Hayward, Matthew Brown, Daniel Uccello, Benjamin J. Burke, Kris Borzilleri, Adam Gilbert, Michelle Hemkens. Discovery and characterization of selective heterobifunctional degraders of EP300 in hematopoietic malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB028.

Investigating the individual and mixture cytotoxicity of co-occurring aflatoxin B1, enniatin B, and sterigmatocystin on gastric, intestinal, hepatic, and renal cellular models

This study investigates the individual and combined effects of the mycotoxins, Aflatoxin B1 (AFB1), Enniatin B (ENNB) and Sterigmatocystin (STG), on the cellular viability of gastric (NCI–N87), intestinal (Caco-2), hepatic (Hep-G2) and renal (Hek-293) cells, shedding light on synergistic or antagonistic effects using a constant ratio combination design proposed by Chou-Talalay. These toxins are prevalent in cereal-based foods, frequently consumed by children which raises concerns about their exposure to these mycotoxins. This population is particularly vulnerable to the effects of these toxins due to their underdeveloped organs and incompletely structured physiological processes. Results showed that ENB was the most toxic of the three mycotoxins across all cell lines, while STG and AFB1 showed lower toxicity. The combination of ENNB + STG was found to be the most potent in terms of binary combinations. In regard to ternary combinations, Caco-2 cells are more sensitive to the tested mycotoxins, whereas NCI–N87 cells show lower levels of cell damage. Worrying dose reduction values (>10-fold) were found for ENNB in binary and ternary combinations at low exposure levels. These findings are significant for establishing initial reference values, which play a pivotal role in estimating reference doses that are subsequently incorporated into the broader risk assessment process.

Abstract LB029: Degraders of TEAD transcription factors based on interface 3 binders

Abstract TEAD transcription factors have emerged as clinically validated targets for Hippo-altered cancers, e.g. mesothelioma driven by NF2 inactivation/deficiency. We have developed a series of novel small molecule targeted protein degraders of TEAD based on pan-TEAD interface 3 ligands. In cells, the compounds induce degradation of TEAD by formation of a ternary complex with Cereblon, leading to ubiquitination of TEAD and subsequent proteasomal degradation. In a cell-based luciferase reporter assay the degraders show low nanomolar activity. The downstream effects of TEAD degradation were further investigated by qPCR and WB analyses of bona fide YAP-TEAD target genes such as CTGF, Cyr61 and AMOTL2. The effectiveness of the TEAD degraders were compared to other classes of TEAD modulators such as palmitoylation inhibitors and YAP-TEAD protein-protein inhibitors by means of cellular viability assays using various mesothelioma cell lines. Finally, we performed an unbiased, quantitative high-throughput drug combination screening by combining a TEAD degrader with a library of approximately 2,800 oncology-focused drugs. This research was supported in part by the Intramural/Extramural research program of the NCATS, NIH. Citation Format: Rajiv Sawant, Matthis Geitmann, Thomas Gossas, Wei B. Emond, Ulf Bremberg, Konrad Koehler, Michele Ceribelli, Craig J. Thomas, Peter Brandt. Degraders of TEAD transcription factors based on interface 3 binders [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB029.

Enhancing 7-dehydrocholesterol suppresses brain ferroptosis and tissue injury after neonatal hypoxia–ischemia

Neonatal hypoxic-ischemic brain injury (HIBI) results in part from excess reactive oxygen species and iron-dependent lipid peroxidation (i.e. ferroptosis). The vitamin D precursor 7-dehydrocholesterol (7-DHC) may inhibit iron-dependent lipid peroxidation. Primary neurons underwent oxygen and glucose deprivation (OGD) injury and treatment with 7-DHC-elevating medications such as cariprazine (CAR) or vehicle. Postnatal day 9 mice underwent sham surgery or carotid artery ligation and hypoxia and received intraperitoneal CAR. In neurons, CAR administration resulted in significantly increased cell survival compared to vehicle controls, whether administered 48 h prior to or 30 min after OGD, and was associated with increased 7-DHC. In the mouse model, malondialdehyde and infarct area significantly increased after HIBI in the vehicle group, which were attenuated by post-treatment with CAR and were negatively correlated with tissue 7-DHC concentrations. Elevating 7-DHC concentrations with CAR was associated with improved cellular and tissue viability after hypoxic-ischemic injury, suggesting a novel therapeutic avenue.

Honokiol regulates ovarian cancer cell malignant behavior through YAP/TAZ pathway modulation.

Ovarian cancer (OVCA) stands as one of the most fatal gynecological malignancies. Honokiol (HNK) has been substantiated by numerous studies for its anti-tumor activity against malignancies including OVCA. Consequently, this work was designed to elucidate the impact of HNK-mediated modulation of the YAP/TAZ pathway on the biological functions of OVCA cells. OVCA cells were subjected to treatment with varying concentrations (0, 25, 50, 75, and 100 μM) of HNK, concomitant with the administration of YAP agonist (XMU). Assessment of cellular viability was executed employing the CCK-8 assay, while quantification of cellular proliferation transpired via colony formation assays. Apoptosis was ascertained using flow cytometry, and expression of apoptosis-related proteins (caspase-3, Bcl-2, Bax), EMT-related proteins (E-cadherin, N-cadherin), migration-associated proteins (MMP-2, MMP-9), and YAP/TAZ pathway-related proteins was evaluated by western blot. Transwell experiments were conducted to assess cellular migratory and invasive propensities. Xenograft tumor models were built to observe tumor growth (volume and weight), apoptosis was assessed by TUNEL staining, and Ki67 expression was evaluated through IHC. HNK exerted inhibitory effects on the viability and proliferative capacity of OVCA cells, elicited apoptotic responses, curtailed the migratory and invasive tendencies of cells, and downregulated the YAP/TAZ pathway. Stimulation with YAP agonist (XMU-MP-1) partially attenuated the impacts of HNK on OVCA cell biology. Experiments in vivo confirmed that HNK inhibited OVCA tumor growth. The outcomes of this investigation conclusively established that HNK orchestrated the modulation of the YAP/TAZ pathway, thereby exerting control over the malignant phenotypic manifestations of OVCA cells. The ascertained function of HNK in restraining cellular proliferation and tumor progression provided novel evidence of its anti-proliferative activity within OVCA cells.

Multi-omic dissection of the cold resistance traits of white water lily

Abstract The white water lily (Nymphaea candida), exemplifying nature's resilience, thrives in the high-altitude terrains of Xinjiang, China, serving as an ideal model for investigating cold adaptation mechanisms in aquatic plants. This study meticulously elucidates the complex cold adaptation mechanisms of the white water lily through a comprehensive and integrated methodological approach. We discovered that the water lily undergoes ecodormancy in winter, retaining high cellular viability and growth potential. During overwintering, the white water lily demonstrates effective resource reallocation, a process facilitated by morphological adjustments, thereby strengthening its resistance to cold temperatures. This enhancement is achieved particularly through the compartmentalization of large vacuoles, the accumulation of osmoregulatory substances, and an increased antioxidant capacity. We established the first exhaustive full-length transcriptome for the white water lily. A subsequent comprehensive analysis of the transcriptome, phytohormones, and metabolome uncovered a multifaceted regulatory network orchestrating cold adaptation. Our research spotlights phytohormone signaling, amino acid metabolism, and circadian rhythms as key elements in the water lily's defense against cold. The results emphasize the critical role of nitrogen metabolism, especially amino acid-related pathways, during cold stress. Metabolite profiling revealed the importance of compounds like myo-inositol and L-proline in enhancing cold tolerance. Remarkably, our study demonstrates that the white water lily notably diminishes the utilization of unsaturated fatty acids in its temperature regulation strategies. In conclusion, this research substantially enriches our understanding of the white water lily’s intricate cold adaptation mechanisms, offering new perspectives on the adaptive strategies of aquatic plants and potential applications in agricultural advancement.

The protective effect and mechanism of glycosides of cistanche deserticola on rats in middle cerebral artery occlusion (MCAO) model

ABSTRACT Cerebral ischemia-reperfusion injury (CIRI) occurs frequently clinically as a complication following cardiovascular resuscitation resulting in neuronal damage specifically to the hippocampal CA1 region with consequent cognitive impairment. Apoptosis and oxidative stress were proposed as major risk factors associated with CIRI development. Previously, glycosides obtained from Cistanche deserticola (CGs) were shown to play a key role in counteracting CIRI; however, the underlying mechanisms remain to be determined. This study aimed to investigate the neuroprotective effect of CGs on subsequent CIRI in rats. The model of CIRI was established for 2 hr and reperfusion for 24 hr by middle cerebral artery occlusion (MCAO) model. The MCAO rats were used to measure the antioxidant and anti-apoptotic effects of CGs on CIRI. Neurological function was evaluated by the Longa neurological function score test. 2,3,5-Triphenyltetrazolium chloride (TTC) staining was used to detect the area of cerebral infarction. Nissl staining was employed to observe neuronal morphology. TUNEL staining was used to detect neuronal apoptosis, while Western blot determined protein expression levels of factors for apoptosis-related and PI3K/AKT/Nrf2 signaling pathway. Data demonstrated that CGs treatment improved behavioral performance, brain injury, and enhanced antioxidant and anti-apoptosis in CIRI rats. In addition, CGs induced activation of PI3K/AKT/Nrf2 signaling pathway accompanied by inhibition of the expression of apoptosis-related factors. Evidence indicates that CGs amelioration of CIRI involves activation of the PI3K/AKT/Nrf2 signaling pathway associated with increased cellular viability suggesting these glycosides may be considered as an alternative compound for CIRI treatment.

In Vitro Immunomodulatory Activity of Sagalanoi Chooranam (Poly Herbal Formulation) in RAW Macrophage Cell Line

Within the realm of Siddha medicine, an ancient and venerable traditional healing system hailing from India, a comprehensive methodology is deployed to combat liver disorders. This approach is characterised by the utilisation of herbal remedies, accentuating the reinstatement of equilibrium in the body's vital life force, known as "Vatham," "Pitham," and "Kapham." The utilisation of cell line studies is crucial in assessing the immunomodulatory properties of herbal medicine, offering invaluable insights into their capacity to influence the immune system and advancing our comprehension of their therapeutic benefits. The primary objective of this investigation was to elucidate the potential immunomodulatory properties of sagalanoi chooranam, a polyherbal Siddha formulation, through the execution of anti-proliferative assays conducted. On the RAW 264.7 macrophage cell line. Various test solutions were precisely prepared, encompassing a spectrum of concentrations (50, 100 and 200 μg/ml). The RAW 264.7 cells were diligently cultured and then carefully subjected to stimulation with lipopolysaccharide (LPS) to initiate cellular activation. Following this activation, the test formulation was introduced at varying concentrations, after which the cells underwent a 24-hour incubation period. The resultant nitrite levels, serving as indicators of immunomodulatory response, were evaluated within the cell lysate. The outcomes unveiled a notable decline in nitrite levels, correlating with the dosage of the test formulation during the incubation with RAW 264.7 cells. The outcomes of the study elucidate a conspicuous decline in nitrite levels in direct correlation with escalating concentrations of the test formulation, contrasting starkly with the control group exclusively subjected to LPS. Furthermore, the investigation meticulously probed the ramifications of the test formulation on cellular viability. The vitality of RAW 264.7 cells exhibited a discernible downward trajectory as the concentration of the test formulation ascended. Noteworthy is the fact that, at the concentration of 200 μg/ml, cellular viability registered at a mere 55.53 ± 3.567%. Collectively, these findings lend credence to the hypothesis that the test formulation possesses a dose-dependent capacity to attenuate both nitrite levels and cellular viability within the RAW 264.7 cell milieu, underscoring its auspicious immunomodulatory attributes. This research expands our insights into the potential immunological ramifications of the test formulation and underscores its plausible utility within the realm of immunomodulation. 

Parthenolide induces gallbladder cancer cell apoptosis via MAPK signalling.

Parthenolide (PTL) has a wide range of clinical applications owing to its anti-inflammatory and antitumor effects. To date, the antitumor effect of PTL on gallbladder cancer (GBC) remains largely unknown. Therefore, we aimed to investigate the biological effects of PTL on GBC. The cellular viability and proliferation of GBC-SD and NOZ cell lines after treatment with different concentrations of PTL were analyzed using the Cell Counting Kit-8 (CCK8)assay and colony formation assay. Apoptosis analysis was performed using flow cytometry. Hoechst staining was performed. RNA sequencing (RNA-seq) was performed to identify PTL-related genes and signalling pathways. Furthermore, we confirmed the involvement of these signalling pathways by qRT-PCR and western blotting. For the in-vivo experiments, a xenograft model was used to evaluate the effects of PTL on the proliferation of NOZ cells. PTL significantly inhibited GBC cell growth in vitro and induced apoptosis in the GBC-SD and NOZ cell lines in a dose-dependent manner. RNA sequencing data showed that the immune response and mitogen-activated protein kinase (MAPK) signalling pathways are closely associated with PTL-induced gallbladder cancer cell apoptosis. PTL upregulated BAX, cleaved PARP-1, cleaved caspase-3, cleaved caspase-9, P53 and decreased the expression of BCL-2, phosphorylated ERK, and phosphorylated MEK in vitro. Tumour volume and weight were also suppressed by PTL in vivo. Moreover, the effects of PTL on GBC cells might be mediated by the MAPK pathway. PTL significantly inhibits gallbladder cancer cell proliferation and induces apoptosis through the MAPK pathway, which is a potential molecular reagent for treating GBC. However, further exploration is needed to verify the antitumor effects of PTL and its intracellular signalling mechanism.

Evaluation of the antimicrobial and antioxidant properties of synthetic phenolipids

Functionalization of phenolic compounds by lipophilization allowing the synthesis of new compounds with improved biological properties. This study aims to investigate the antimicrobial effects of eight synthesized phenolipids, derived from bioactive coumaric and ferulic acid esters, against Bacillus subtilis, Escherichia coli, and Candida albicans. Antioxidant activity, as well as antimicrobial capacity were evaluated through DPPH assays and classical disc agar diffusion, respectively, concomitantly with the evaluation of cellular viability using flow cytometry assay. Antioxidant activities results showed that ferulic acid esters exhibit higher antiradical activity than coumaric acid esters. Results of agar diffusion assay revealed that, 1-methylpropyl coumarate and 1-methylpropyl ferulate (1-MPF) have an effective antimicrobial activity with Minimum Inhibitory Concentrations (MICs) values between 0.9 – 3.5mM against all tested microorganisms. Also, flow cytometry showed that 1-MPF and decyl coumarate exhibited significant bactericidal activity (p < 0.05), with almost 63% of dead Bacillus subtilis cells. Compared to the commonly used agar method, flow cytometry proved to be a fast and straightforward analytical tool to assess antimicrobial activity while providing information on inhibition type. These results suggest the applicability of phenolipids as potent lipophilic bioactive molecules to protect food products and cosmetic formulations from oxidative stress and microbial spoilage.

Phytochemical Characterization and Multifaceted Bioactivity Assessment of Essential Oil from Ptychotis verticillata Duby: Anti-Diabetic, Anti-Tyrosinase, and Anti-Inflammatory Activity

The aim of this study is to explore the pharmacological properties of the essential oil derived from Ptychotis verticillata Duby (PVEO), a medicinal plant native to Morocco, focusing on its antidiabetic, anti-tyrosinase, and anti-inflammatory effects. Additionally, the study aims to characterize the phytochemical composition of PVEO and evaluate its potential as a natural therapeutic alternative for various health conditions. To achieve this, phytochemical analysis was conducted using gas chromatography-mass spectrometry (GC-MS). Furthermore, in vitro assessments were conducted to investigate PVEO's antidiabetic activity by inhibiting α-amylase, xanthine oxidase, and α-glucosidase. Tests were also undertaken to evaluate the anti-inflammatory effect of PVEO on RAW 264.7 cells stimulated by lipopolysaccharide (LPS), as well as its efficacy as an anti-tyrosinase agent and its lipoxygenase inhibition activity. The results of the phytochemical analysis revealed that PVEO is rich in terpene compounds, with percentages of 40.35% γ-terpinene, 22.40% carvacrol, and 19.77% β-cymene. Moreover, in vitro evaluations demonstrated that PVEO exhibits significant inhibitory activity against α-amylase, xanthine oxidase, and α-glucosidase, indicating promising antidiabetic, and anti-gout potential. Furthermore, PVEO showed significant anti-tyrosinase activity, with an IC50 of 27.39 ± 0.44 μg/mL, and remarkable lipoxygenase inhibition (87.33 ± 2.6%), suggesting its candidacy for dermatoprotection. Additionally, PVEO displayed a dose-dependent capacity to attenuate the production of NO and PGE2, two inflammatory mediators implicated in various pathologies, without compromising cellular viability. The findings of this study provide a solid foundation for future research on natural therapies and the development of new drugs, highlighting the therapeutic potential of PVEO in the treatment of gout, diabetes, pigmentation disorders, and inflammation.

Astragaloside IV combined with ligustrazine ameliorates abnormal mitochondrial dynamics via Drp1 SUMO/deSUMOylation in cerebral ischemia-reperfusion injury.

Astragaloside IV (AST IV) and ligustrazine (Lig), the main ingredients of Astragali Radix and Chuanxiong Rhizoma respectively, have demonstrated significant benefits in treatment of cerebral ischemia -reperfusion injury (CIRI); however, the mechanisms underlying its benificial effects remain unclear. SUMO-1ylation and deSUMO-2/3ylation of dynamin-related protein 1 (Drp1) results in mitochondrial homeostasis imbalance following CIRI, which subsequently aggravates cell damage. This study investigates the mechanisms by which AST IV combined with Lig protects against CIRI, focusing on the involvement of SUMOylation in mitochondrial dynamics. Rats were administrated AST IV and Lig for 7 days, and middle cerebral artery occlusion was established to mimic CIRI. Neural function, cerebral infarction volume, cerebral blood flow, cognitive function, cortical pathological lesions, and mitochondrial morphology were measured. SH-SY5Y cells were subjected to oxygen-glucose deprivation/reoxygenation (OGD/R) injury. Mitochondrial membrane potential and lactic dehydrogenase (LDH), reactive oxygen species (ROS), and adenosine triphosphate (ATP) levels were assessed with commercial kits. Moreover, co-immunoprecipitation (Co-IP) was used to detect the binding of SUMO1 and SUMO2/3 to Drp1. The protein expressions of Drp1, Fis1, MFF, OPA1, Mfn1, Mfn2, SUMO1, SUMO2/3, SENP1, SENP2, SENP3, SENP5, and SENP6 were measured using western blot. In rats with CIRI, AST IV and Lig improved neurological and cognitive functions, restored CBF, reduced brain infarct volume, and alleviated cortical neuron and mitochondrial damage. Moreover, in SH-SY5Y cells, the combination of AST IV and Lig enhanced cellular viability, decreased release of LDH and ROS, increased ATP content, and improved mitochondrial membrane potential. Furthermore, AST IV combined with Lig reduced the binding of Drp1 with SUMO1, increased the binding of Drp1 with SUMO2/3, suppressed the expressions of Drp1, Fis1, MFF, and SENP3, and increased the expressions of OPA1, Mfn1, Mfn2, SENP1, SENP2, and SENP5. SUMO1 overexpression promoted mitochondrial fission and inhibited mitochondrial fusion, whereas SUMO2/3 overexpression suppressed mitochondrial fission. AST IV combined with Lig could reverse the effects of SUMO1 overexpression while enhancing those of SUMO2/3 overexpression. This study posits that the combination of AST IV and Lig has the potential to reduce the SUMO-1ylation of Drp1, augment the SUMO-2/3ylation of Drp1, and thereby exert a protective effect against CIRI.

3D printed β-tricalcium phosphate versus synthetic bone mineral scaffolds: A comparative in vitro study of biocompatibility

BACKGROUND: β-tricalcium phosphate (β-TCP) has been successfully utilized as a 3D printed ceramic scaffold in the repair of non-healing bone defects; however, it requires the addition of growth factors to augment its regenerative capacity. Synthetic bone mineral (SBM) is a novel and extrudable carbonate hydroxyapatite with ionic substitutions known to facilitate bone healing. However, its efficacy as a 3D printed scaffold for hard tissue defect repair has not been explored. OBJECTIVE: To evaluate the biocompatibility and cell viability of human osteoprecursor (hOP) cells seeded on 3D printed SBM scaffolds via in vitro analysis. METHODS: SBM and β-TCP scaffolds were fabricated via 3D printing and sintered at various temperatures. Scaffolds were then subject to qualitative cytotoxicity testing and cell proliferation experiments utilizing (hOP) cells. RESULTS: SBM scaffolds sintered at lower temperatures (600 °C and 700 °C) induced greater levels of acute cellular stress. At higher sintering temperatures (1100 °C), SBM scaffolds showed inferior cellular viability relative to β-TCP scaffolds sintered to the same temperature (1100 °C). However, qualitative analysis suggested that β-TCP presented no evidence of morphological change, while SBM 1100 °C showed few instances of acute cellular stress. CONCLUSION: Results demonstrate SBM may be a promising alternative to β-TCP for potential applications in bone tissue engineering.

Biotechnological Effects of Advanced Smart-Bile Acid Cyclodextrin-Based Nanogels for Ear Delivery and Treatment of Hearing Loss.

Inner ear delivery requires safe and effective drug delivery vehicles incorporating high-viscosity formulations with permeation enhancers. This study designs novel thermoresponsive-smart polymer-bile acid and cyclodextrin-based nanogels for inner ear delivery. Nanogels are examined for their rheological and physical properties. The biocompatibility studies will be assessed on auditory and macrophage cell lines by investigating the impact of nanogels on cellular viability, mitochondrial respiration, glycolysis, intracellular oxidative stress, inflammatory profile, and macrophage polarization. Novel ther nanogels based on bile acid and beta-cyclodextrin show preserved porous nanogels' inner structure, exhibit non-Newtonian, shear-thinning fluid behavior, have fast gelation at 37°C and minimal albumin adsorption on the surface. The nanogels have minimal impact on cellular viability, mitochondrial respiration, glycolysis, intracellular oxidative stress, and inflammatory profile of the auditory cell line House Ear Institute-Organ of Corti 1 after 24h incubation. Nanogel exposure of 24h to macrophage cell line RAW264.7 leads to decreased viability, mitochondrial dysfunction, and increased intracellular ROS and inflammatory cytokines. However, polarization changes from M2 anti-inflammatory to M1 pro-inflammatory macrophages are minimal, and inflammatory products of RAW264.7 macrophages do not overly disrupt the survivability of HEI-OC1 cells. Based on these results, thermoresponsive bile acid and cyclodextrin nanogels can be potential drug delivery vehicles for inner ear drug delivery.

Fabrication of a Novel 3D Extrusion Bioink Containing Processed Human Articular Cartilage Matrix for Cartilage Tissue Engineering

Cartilage damage presents a significant clinical challenge due to its intrinsic avascular nature which limits self-repair. Addressing this, our study focuses on an alginate-based bioink, integrating human articular cartilage, for cartilage tissue engineering. This novel bioink was formulated by encapsulating C20A4 human articular chondrocytes in sodium alginate, polyvinyl alcohol, gum arabic, and cartilage extracellular matrix powder sourced from allograft femoral condyle shavings. Using a 3D bioprinter, constructs were biofabricated and cross-linked, followed by culture in standard medium. Evaluations were conducted on cellular viability and gene expression at various stages. Results indicated that the printed constructs maintained a porous structure conducive to cell growth. Cellular viability was 87% post printing, which decreased to 76% after seven days, and significantly recovered to 86% by day 14. There was also a notable upregulation of chondrogenic genes, COL2A1 (p = 0.008) and SOX9 (p = 0.021), suggesting an enhancement in cartilage formation. This study concludes that the innovative bioink shows promise for cartilage regeneration, demonstrating substantial viability and gene expression conducive to repair and suggesting its potential for future therapeutic applications in cartilage repair.

Evaluation of cellular viability in chitosan/L-arginine hydrogels.

There is a lack of studies evaluating the toxicity of nitric oxide (NO) precursors in chitosan/L-arginine hydrogels and their topical administration. However, clarifying the characteristics of these elements is essential for their possible use in non-surgical techniques of tooth movement acceleration. Such characteristics include interaction with different cell types, metabolism and drug safety. This in vitro study aimed to assess the cytotoxicity of chitosan hydrogels on human HeLa cells using different concentrations of L-arginine. The hydrogels were synthesized in a materials engineering laboratory, with a controlled environment, using 4 different L-arginine concentrations of 0%, 10%, 15%, and 20%. Once the hydrogels were prepared, their physical and chemical properties were characterized, and viability analysis was performed using 2 different methods, including a 48-h assay with Artemia salina nauplii and a 24-h cell culture with human HeLa cells followed by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) proliferation assay. Data analysis was performed using a Mann-Whitney U test to evaluate positive and negative controls in the cell culture, with a significance level of 0.01. A Wilcoxon paired test contrasted the 24-h compared to 48-h Artemia salina assays, with a Kruskal-Wallis and post hoc Dunn test used to compare groups using a significance level of 0.05. In the more viscous hydrogels, Artemia salina nauplii decreased drastically in 24 h, while the 15% and 20% hydrogels had no statistical differences from the negative control. The 10% and 20% hydrogels were statistically different from the negative control when comparing cell culture data. Our findings suggest that chitosan/L-arginine hydrogels could be used in humans without toxic effects. However, more trials and tests are needed to evaluate tooth movement rate during orthodontic treatment.