Search Results (38)

Neutrophil extracellular traps (NETs) critically influence thrombosis by promoting platelet aggregation, fibrin formation, and thrombus stabilisation. However, primary human neutrophils present experimental limitations, including short lifespan ex vivo and ethical concerns. In this article, we discuss the available data on PLB-985 cells, a neutrophil-like model with potential to replace human neutrophils in research. Additionally, we present novel structural comparisons showing that both PLB-985- and human neutrophil-derived NETs significantly increased fibrin fibre thickness compared to thrombin-only controls, with similar fibre morphology across conditions. Notably, we also see spherical particles resembling microvesicles within PLB-985-derived NETs, suggesting potential additional procoagulant effects via microvesicle-associated tissue factor level in these cells. New and existing data presented in this article suggest that differentiated PLB-985 cells are able to effectively replicate key structural and functional aspects of human neutrophil NETs. These observations support the use of PLB-985 cells as an ethical, reproducible, and practical alternative for in vitro studies of NETs. Further characterisation is required to determine differences between human neutrophils and neutrophil-like models in macrovesicle formation and implication in NET-related thrombosis research. Full article

Male pattern baldness (MPB) is a highly prevalent condition with a complex, poorly understood molecular basis that limits therapeutic innovation. This study aimed to bridge the gap between statistical genetic associations and biological function by identifying and prioritizing causal proteins and pathways involved in MPB. Using data from 24,069 men in the UK Biobank, we performed a proteome-wide association study of 2911 plasma proteins with self-reported MPB severity via multivariable ordinal logistic regression, adjusting for age, Body Mass Index (BMI), ethnicity, lifestyle, socioeconomic factors, and testosterone levels. Significant proteins underwent pathway enrichment analysis. Genetic integration included MAGMA for gene-level aggregation and tissue prioritization, transcriptome-wide association studies (TWAS) with GTEx models, conditional fine-mapping, and validation in an independent scalp biopsy transcriptomics dataset (GSE90594). Druggability and pleiotropy were evaluated using databases and phenome-wide association studies. Forty-seven proteins were significantly associated with MPB severity, enriched in pathways involving epidermis development, hair cycle regulation, and cell adhesion. Multi-omic integration prioritized five independent candidate genes: CD38, FGF5, TACSTD2, DPEP1, and PLB1. Transcriptomic validation confirmed differential expression in balding scalp for CD38 (upregulated) and TACSTD2, PLB1 (downregulated). CD38 was identified as druggable with low pleiotropic risks. This study elucidates the molecular architecture of MPB, revealing novel biological pathways beyond canonical androgen signaling. By prioritizing promising non-hormonal targets like CD38, our findings provide a robust, evidence-based framework to guide the development of future therapeutic interventions for this common condition. Full article

Trimeric intracellular cation channel A (TRIC-A) provides counter-ion support for sarcoplasmic reticulum (SR) Ca²⁺ release, yet its physiological role in the intact heart under stress remains poorly defined. Here, we demonstrate that TRIC-A is essential for maintaining balanced SR Ca²⁺ release, mitochondrial integrity, and cardiac resilience during β-adrenergic stimulation. Tric-a^−/− cardiomyocytes exhibited Ca²⁺ transients evoked by electrical stimuli and exaggerated isoproterenol (ISO)-evoked Ca²⁺ release, consistent with SR Ca²⁺ overload. These defects were accompanied by selective upregulation of protein kinase A (PKA)-dependent phosphorylation of ryanodine receptor 2 (RyR2) (S2808) and phospholamban (PLB) (S16). Acute ISO challenge induced mitochondrial swelling, cristae disruption, and Evans Blue Dye uptake, and elevated circulating troponin T in Tric-a^−/− hearts, hallmarks of necrosis-like cell death. Mitochondrial Ca²⁺ uptake inhibition with Ru360 markedly reduced membrane injury, establishing mitochondrial Ca²⁺ overload as the proximal trigger of cardiac cell death. With sustained β-adrenergic stimulation by ISO, Tric-a^−/− hearts developed extensive interstitial and perivascular fibrosis without exaggerated hypertrophy. Cardiac fibroblasts lacked TRIC-A expression and displayed normal Ca²⁺ signaling and activation, indicating that fibrosis arises secondarily from cardiomyocyte injury rather than fibroblast-intrinsic abnormalities. These findings identify TRIC-A as a critical regulator of SR-mitochondrial Ca²⁺ coupling and a key molecular safeguard that protects the heart from catecholamine-induced injury and maladaptive remodeling. Full article

This study aims to investigate the protective effect of the natural phthalide compound Levistolide A (LA) against myocardial ischemia–reperfusion injury (MIRI) and to elucidate its underlying mechanisms. Utilizing network pharmacology, potential targets of LA in the treatment of MIRI were predicted. Subsequently, a hypoxia/reoxygenation (H/R) model was established using rat H9C2 cardiomyocytes to simulate MIRI, and the mechanisms of action were validated through cellular experiments. Network pharmacology analysis indicated that the potential targets of LA in treating MIRI were significantly enriched in calcium signaling pathways, with the adenosine A2B receptor (ADORA2B), a G protein-coupled receptor (GPCR), identified as a key protein. Cellular experiments demonstrated that 24 μM LA significantly alleviated H/R-induced damage in H9C2 cells, enhanced cell viability, and reduced the release of lactate dehydrogenase (LDH), creatine kinase isoenzyme MB (CK-MB), and cardiac troponin I (cTnI). Pre-treatment with LA significantly activated the ADORA2B/Cyclic adenosine monophosphate (cAMP)/Protein kinase A (PKA) signaling axis, promoting the phosphorylation of phospholamban (PLB), enhancing the activity and protein expression of sarco/endoplasmic reticulum Ca²⁺-ATPase 2 alpha (SERCA2α), and effectively mitigating intracellular calcium overload induced by H/R. However, the ADORA2B antagonist MRS 1754 partially reverses the aforementioned protective effects of LA. The findings of this study reveal a novel mechanism by which LA exerts cardioprotective effects through the ADORA2B/cAMP/PKA/PLB/SERCA2α signaling axis, preventing calcium overload and improving calcium homeostasis, and identify potential candidate compounds and precise targets for the treatment of MIRI. Full article

Background/Objectives: The hippocampus is an essential part of the central nervous system (CNS); it plays a significant role in social–cognitive memory processing. Prenatal exposure to valproic acid (VPA) can lead to impaired hippocampal functions. In this study, we evaluated the effect of plumbagin (PLB) as a natural product on spatial learning and memory, neuro-morphological changes, and inflammation levels in a VPA-induced autism model during adolescence. Methods: Pregnant Wistar rats received a single intraperitoneal (i.p.) injection of VPA (600 mg/kg) or saline on gestational day 12.5. The male offspring were then categorized and assigned to five groups: Saline+DMSO-, VPA+DMSO-, and VPA+PLB-treated groups at doses of 0.25, 0.5, or 1 mg/kg. Spatial learning and memory were evaluated using the Morris water maze. Histopathological evaluations of the hippocampus were performed using Nissl and hematoxylin–eosin staining, as well as immunofluorescence. The pro-inflammatory cytokine levels were also quantified by quantitative real-time PCR. Results: The findings revealed that a VPA injection on gestational day 12.5 is associated with cognitive impairments in male pups, including a longer escape latency and traveled distance, as well as decreased time spent in the target quadrant. Treatment with PLB significantly enhanced the cognitive function, reduced dark cells, and ameliorated neuronal–morphological alterations in the hippocampus of VPA-exposed rats. Moreover, PLB was found to reduce astrocyte activation and the expression levels of pro-inflammatory cytokines. Conclusions: These findings suggest that PLB partly mitigates VPA-induced cognitive deficits by ameliorating hippocampal inflammation levels. Full article

Background/Objectives: Leukemia is a common cancer that arises in both children and adults when bone marrow’s hematopoietic stem cells proliferate unrestrained because of anomalies in normal cell regulatory systems. The present study focused on biological evaluation of oxazole-based oxadiazole scaffolds to evaluate the anti-proliferative effect on leukemic cancer cell lines. Methods: All novel oxazole-based oxadiazole scaffolds were synthesized and structurally characterized via ¹³C NMR, ¹H NMR, and HREI-MS. In order to identify an efficient anti-leukemia agent, the biological profiles of each compound were evaluated in comparison to the reference drug, Etoposide (IC₅₀ = 10.50 and 15.20 μM). Results: Analog 6 substituted with p-CF₃ at phenyl ring was identified with excellent inhibition against the HL-60 and PLB-985 cancer cell lines, with IC₅₀ of 8.50 and 12.50 μM. Through hydrogen bond formation, the trifluoromethyl moiety of analog 6 interacts with target tyrosine kinase enzyme (PDB-ID:4CSV). The interactive character of active ligands with target enzyme was demonstrated by molecular docking. The rate of inhibition in contrast with the drug concentration was also tested to check the inhibition percentage and inhibitor type via enzyme kinetics. Furthermore, the enzyme–ligand complex was also investigated via MD simulation along with pharmacophore modeling. DFT calculations were used to estimate the lead compounds’ relative stability and reactivity. According to ADMET investigation, there is safe toxicological profile for these compounds. Conclusions: The current study suggests that the potent compounds have significant anti-proliferative potential, and with further in vivo validation, hold promise for future optimization as potential leukemia treatments. Full article

Liparis loeselii (L.) Rich, an endangered member of the Orchidaceae family, is found in alkaline fens. With the declining populations of L. loeselii, there is a pressing need to reintroduce this species in Central Europe. As in vitro germination is a crucial tool for obtaining plants for introduction into the environment, we looked at the morphological changes occurring during the early stages of L. loeselii development in vitro. As the early stages of orchid development, especially the protocorm stage, are thought to be responsible for SAM formation and the initiation of symbiotic association, we focused on cell wall elements whose epitopes have been found in similar processes in other species: the extensin and pectin rhamnogalacturonan I (RG-I) side chain epitopes. We addressed the following questions: Does the cell wall of L. loeselii change its composition during the early stages of development, as noted in other species? Are there noticeable similarities in the cell wall to organs of different species whose function is to contact microorganisms? Are there regularities that allow the recognition of individual structures on this basis? Immunolocalization revealed changes in the distribution of certain extensins (JIM11 and JIM20) and RG-I (LM5 and LM6) side chain epitopes. Extensins, a type of cell wall protein, were observed during the initial stages of the formation of PLB and the shoot apical meristem of protocorms and PLBs. RG-I, on the other hand, was found to play a significant role in the development of the protocorm and PLB. In pseudobulbs, which appeared on the protocorms, extensins occurred in their storage part. However, RG-I side chains (1→4)-β-galactans (LM5), and (1→5)-α-L-arabinans (LM6) were not found in pseudobulbs. We revealed that a common feature of protocorms and PLBs was an increased amount of extensins, which were detected with the JIM11 antibody, and pectins, which were detected with the LM5 antibody, that were present together, which may prove helpful in determining the identity of the induced structures and distinguishing them from pseudobulbs. Thus, our study unveiled the role of extensins and RG-I during the growth of protocorms and PLBs. We suggest that PLBs may mimic the wall remodelling that occurs in protocorms, which indicates that using cell wall components is an invitation to be colonised by a fungal partner. However, this needs to be tested in future research. The findings of this research can help interpret future studies on the propagation, acclimatisation, and introduction of L. loeselii into the natural environment. Full article

Plumbagin (PLB) is a naphthoquinone extracted from Plumbago indica. In recent times, there has been a growing body of evidence suggesting the potential importance of naphthoquinones, both natural and artificial, in the pharmacological world. Numerous studies have indicated that PLB plays a vital role in combating cancers and other disorders. There is substantial evidence indicating that PLB may have a significant role in the treatment of breast cancer, brain tumours, lung cancer, hepatocellular carcinoma, and other conditions. Moreover, its potent anti-oxidant and anti-inflammatory properties offer promising avenues for the treatment of neurodegenerative and cardiovascular diseases. A number of studies have identified various pathways that may be responsible for the therapeutic efficacy of PLB. These include cell cycle regulation, apoptotic pathways, ROS induction pathways, inflammatory pathways, and signal transduction pathways such as PI3K/AKT/mTOR, STAT3/PLK1/AKT, and others. This review aims to provide a comprehensive analysis of the diverse pharmacological roles of PLB, examining the mechanisms through which it operates and exploring its potential applications in various medical conditions. In addition, we have conducted a review of the various formulations that have been reported in the literature with the objective of enhancing the efficacy of the compound. However, the majority of the reviewed data are based on in vitro and in vivo studies. To gain a comprehensive understanding of the safety and efficacy of PLB in humans and to ascertain its potential integration into therapeutic regimens for cancer and chronic diseases, rigorous clinical trials are essential. Finally, by synthesizing current research and identifying gaps in knowledge, this review seeks to enhance our understanding of PLB and its therapeutic prospects, paving the way for future studies and clinical applications. Full article

Secretory phospholipase B1 (PLB1) and biofilms act as microbial virulence factors and play an important role in pulmonary cryptococcosis. This study aims to formulate the ethanolic extract of propolis-loaded niosomes (Nio-EEP) and evaluate the biological activities occurring during PLB1 production and biofilm formation of Cryptococcus neoformans. Some physicochemical characterizations of niosomes include a mean diameter of 270 nm in a spherical shape, a zeta-potential of −10.54 ± 1.37 mV, and 88.13 ± 0.01% entrapment efficiency. Nio-EEP can release EEP in a sustained manner and retains consistent physicochemical properties for a month. Nio-EEP has the capability to permeate the cellular membranes of C. neoformans, causing a significant decrease in the mRNA expression level of PLB1. Interestingly, biofilm formation, biofilm thickness, and the expression level of biofilm-related genes (UGD1 and UXS1) were also significantly reduced. Pre-treating with Nio-EEP prior to yeast infection reduced the intracellular replication of C. neoformans in alveolar macrophages by 47%. In conclusion, Nio-EEP mediates as an anti-virulence agent to inhibit PLB1 and biofilm production for preventing fungal colonization on lung epithelial cells and also decreases the intracellular replication of phagocytosed cryptococci. This nano-based EEP delivery might be a potential therapeutic strategy in the prophylaxis and treatment of pulmonary cryptococcosis in the future. Full article

Dendrobium loddigesii has long been used in traditional folk medicine. The purpose of this study was to optimize the culture conditions for its protocorm-like bodies (PLBs) and explore their biological activities. The use of an air-lift bioreactor demonstrated superior PLB proliferation compared to agitated and solid culture methods. The optimal inoculum quantity of 30 g/vessel, cultured for 28 days in the bioreactor, yielded the highest PLB biomass production. Analysis of PLB extracts revealed that the ethyl acetate (EtOAc) extract exhibited the highest levels of flavonoids and alkaloids, as well as potent antioxidant activity demonstrated by DPPH free radical scavenging assay and reducing power. Furthermore, the antiproliferative effects of the PLB extracts were assessed using MTT assays, and the EtOAc extract showed significant efficacy by reducing cell viability by over 60% in the human colon carcinoma cell line SW620 at the highest tested concentration (200 μg/mL). Mechanistic analysis revealed the downregulation of key regulatory apoptosis genes, including survivin, p53, caspase-3, and caspase-9. These results demonstrate the potential of the bioreactor culture method for the efficient production of D. loddigesii PLBs and the biological activities of the EtOAc extract, suggesting its therapeutic potential. Full article

The potential of cold atmospheric plasma (CAP) in biomedical applications has received significant interest, due to its ability to generate reactive oxygen and nitrogen species (RONS). Upon exposure to living cells, CAP triggers alterations in various cellular components, such as the cell membrane. However, the permeation of RONS across nitrated and oxidized membranes remains understudied. To address this gap, we conducted molecular dynamics simulations, to investigate the permeation capabilities of RONS across modified cell membranes. This computational study investigated the translocation processes of less hydrophilic and hydrophilic RONS across the phospholipid bilayer (PLB), with various degrees of oxidation and nitration, and elucidated the impact of RONS on PLB permeability. The simulation results showed that less hydrophilic species, i.e., NO, NO₂, N₂O₄, and O₃, have a higher penetration ability through nitro-oxidized PLB compared to hydrophilic RONS, i.e., HNO₃, s-cis-HONO, s-trans-HONO, H₂O₂, HO₂, and OH. In particular, nitro-oxidation of PLB, induced by, e.g., cold atmospheric plasma, has minimal impact on the penetration of free energy barriers of less hydrophilic species, while it lowers these barriers for hydrophilic RONS, thereby enhancing their translocation across nitro-oxidized PLB. This research contributes to a better understanding of the translocation abilities of RONS in the field of plasma biomedical applications and highlights the need for further analysis of their role in intracellular signaling pathways. Full article

Plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone, PLB), a naturally occurring naphthoquinone mainly isolated from the plant Plumbago zeylanica L., has been proven to possess anticancer activities towards multiple types of cancer. Although there has been an increasing amount of research regarding its anticancer effects, the association between oxidative stress, genotoxicity and the cell cycle arrest induced by PLB still remains unclear. Therefore, it is important to investigate their potential connections and the involvement of DNA damage and the ataxia telangiectasia mutated protein (ATM)-p53 signaling pathway in PLB’s anticancer mechanism. The present study showed that PLB exposure significantly reduced HCC cell viability and colony formation. In addition, PLB-induced G2/M cell cycle arrest, oxidative stress, and DNA damage was detected, which could be almost blocked by NAC pretreatment. PLB could trigger a DNA damage response by activating cell cycle checkpoints such as ATM, checkpoint kinase 1 (Chk1), checkpoint kinase 2 (Chk2) and p53. Meanwhile, the key modulator of the G2/M transition factor, Cell Division Cycle 25C (cdc25C), was significantly downregulated in an ROS-dependent manner. Furthermore, pretreatment with ATM and p53 inhibitors (KU55933 and Pifithrin-α) could reduce the occurrence of G2/M cell cycle arrest by inhibiting the activation of the ATM-p53 pathway. Taken together, these results indicate that ROS-mediated oxidative stress plays a key role in PLB-induced G2/M cell cycle arrest mediated by the ATM-p53 pathway. Full article

Cadmium (Cd²⁺) exposure induces chronic kidney disease and renal cancers, which originate from injury and cancerization of renal tubular cells. Previous studies have shown that Cd²⁺ induced cytotoxicity by disrupting the intracellular Ca²⁺ homeostasis that is physically regulated by the endoplasmic reticulum (ER) Ca²⁺ store. However, the molecular mechanism of ER Ca²⁺ homeostasis in Cd²⁺-induced nephrotoxicity remains unclear. In this study, our results firstly revealed that the activation of calcium-sensing receptor (CaSR) by NPS R-467 could protect against Cd²⁺ exposure-induced cytotoxicity of mouse renal tubular cells (mRTEC) by restoring ER Ca²⁺ homeostasis through the ER Ca²⁺ reuptake channel sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA). Cd²⁺-induced ER stress and cell apoptosis were effectively abrogated by SERCA agonist CDN1163 and SERCA2 overexpression. In addition, in vivo, and in vitro results proved that Cd²⁺ reduced the expressions of SERCA2 and its activity regulator phosphorylation phospholamban (p-PLB) in renal tubular cells. Cd²⁺-induced SERCA2 degradation was suppressed by the treatment of proteasome inhibitor MG132, which suggested that Cd²⁺ reduced SERCA2 protein stability by promoting the proteasomal protein degradation pathway. These results suggested that SERCA2 played pivotal roles in Cd²⁺-induced ER Ca²⁺ imbalance and stress to contribute to apoptosis of renal tubular cells, and the proteasomal pathway was involved in regulating SERCA2 stability. Our results proposed a new therapeutic approach targeting SERCA2 and associated proteasome that might protect against Cd²⁺-induced cytotoxicity and renal injury. Full article

Extracellular traps are released by neutrophils and other immune cells as part of the innate immune response to combat pathogens. Neutrophil extracellular traps (NETs) consist of a mesh of DNA and histone proteins decorated with various anti-microbial granule proteins, such as elastase and myeloperoxidase (MPO). In addition to their role in innate immunity, NETs are also strongly linked with numerous pathological conditions, including atherosclerosis, sepsis and COVID-19. This has led to significant interest in developing strategies to inhibit NET release. In this study, we have examined the efficacy of different antioxidant approaches to selectively modulate the inflammatory release of NETs. PLB-985 neutrophil-like cells were shown to release NETs on exposure to phorbol myristate acetate (PMA), hypochlorous acid or nigericin, a bacterial peptide derived from Streptomyces hygroscopicus. Studies with the probe R19-S indicated that treatment of the PLB-985 cells with PMA, but not nigericin, resulted in the production of HOCl. Therefore, studies were extended to examine the efficacy of a range of antioxidant compounds that modulate HOCl production by MPO to prevent NETosis. It was shown that thiocyanate, selenocyanate and various nitroxides could prevent NETosis in PLB-985 neutrophils exposed to PMA and HOCl, but not nigericin. These results were confirmed in analogous experiments with freshly isolated primary human neutrophils. Taken together, these data provide new information regarding the utility of supplementation with MPO inhibitors and/or HOCl scavengers to prevent NET release, which could be important to more specifically target pathological NETosis in vivo. Full article

In phagocytes, cytoskeletal and membrane remodeling is finely regulated at the phagocytic cup. Various smaFll G proteins, including those of the Arf family, control these dynamic processes. Human neutrophils express AGAP2, an Arf GTPase activating protein (ArfGAP) that regulates endosomal trafficking and focal adhesion remodeling. We first examined the impact of AGAP2 on phagocytosis in CHO cells stably expressing the FcγRIIA receptor (CHO-IIA). In unstimulated CHO-IIA cells, AGAP2 only partially co-localized with cytoskeletal elements and intracellular compartments. In CHO-IIA cells, AGAP2 transiently accumulated at actin-rich phagocytic cups and increased Fcγ receptor-mediated phagocytosis. Enhanced phagocytosis was not dependent on the N-terminal GTP-binding protein-like (GLD) domain of AGAP2. AGAP2 deleted of its GTPase-activating protein (GAP) domain was not recruited to phagocytic cups and did not enhance the engulfment of IgG-opsonized beads. However, the GAP-deficient [R618K]AGAP2 transiently localized at the phagocytic cups and enhanced phagocytosis. In PLB-985 cells differentiated towards a neutrophil-like phenotype, silencing of AGAP2 reduced phagocytosis of opsonized zymosan. In human neutrophils, opsonized zymosan or monosodium urate crystals induced AGAP2 phosphorylation. The data indicate that particulate agonists induce AGAP2 phosphorylation in neutrophils. This study highlights the role of AGAP2 and its GAP domain but not GAP activity in FcγR-dependent uptake of opsonized particles. Full article