Search Results (1,094)

High-risk human papillomavirus (HPV) is the leading etiological factor in cervical cancer, creating a pressing need for less invasive and more objective diagnostic tools. This pilot study pioneers the application of Raman spectroscopy to cell-free cervicovaginal lavage (CVL) for distinguishing between low-grade and high-grade squamous intraepithelial lesions (LSIL and HSIL) in HPV-positive patients. Raman spectra were acquired at 532-nm excitation from cell-free CVL samples of 20 patients with histologically confirmed LSIL (n = 9) or HSIL (n = 11). Comparative analysis of Raman bands revealed a significant biochemical shift in HSIL, presumably characterized by reduced glycogen and lactate/lactic acid levels alongside substantially elevated heme proteins. A diagnostic model based on key spectral intensity ratios achieved differentiation between LSIL and HSIL with 80% sensitivity and 86% specificity. These findings demonstrate that Raman spectroscopy of cell-free CVL effectively captures profound metabolic and microvascular alterations characteristic of neoplastic progression, showcasing its strong potential as a rapid, cost-effective, non-invasive, and objective tool for cervical lesion risk stratification. Full article

Horseradish peroxidase (HRP) is a heme-containing oxidoreductase with extensive applications in biotechnology, medical diagnostics, and environmental protection. In this study, Pichia pastoris was utilized to produce HRP. Successfully, expression strains with 1–5 copies of HRP-C were constructed, and the strain with the highest expression level and activity of HRP-C was obtained. Different molecular chaperones (PDI1, HAC1, BIP1) were selected, and co-expression was carried out through co-induction and separate induction methods. The results showed that the yield of HRP increased approximately 1.4 times with the assistance of PDI1 and HAC1 molecular chaperones in the 3-copy Pichia pastoris expression strain, with enzyme activities increasing by 1.2-fold and 1.3-fold, respectively. High-density fermentation of the recombinant strain transformed with BDM-PDI1-HRP-C-3C was carried out in a 50 L fermenter, and after methanol induction for 72 h, a target protein expression level of up to 200 mg/L was achieved. The enzyme activity reached 1796 U/mL, which is nearly three times higher than that of shake-flask fermentation and is the highest reported in the literature to date. Full article

Background/Objectives: Molnupiravir (MOV) and nirmatrelvir (NMV) are antiviral drugs that were FDA-approved under the emergency use authorization (EUA) for coronavirus disease-2019 (COVID-19) treatment. MOV and NMV target the viral RNA-dependent RNA polymerase and main protease, respectively. Paxlovid is a combination of NMV and ritonavir (RTV), an inhibitor of the human cytochrome P450-3A4 (hCYP3A4). In this study, the structural consequences in the hCYP3A4 caused by MOV-induced mutations (MIM) were evaluated using in silico tools. Methods: MOV-induced mutations (MIM) were inserted into all the possible hotspots in the active site region of the hCYP3A4 gene, and mutant protein models were built. Structural changes in the heme-porphyrin ring of hCYP3A4 were analyzed in the presence and absence of substrates/inhibitors, including RTV. Molecular dynamics (MD) simulations were performed to analyze the effect of MIM-induced structural changes in hCYP3A4 on drug binding. Results: MD simulations confirm that MIMs, R375G and R440G in hCYP3A4 severely affect the heme-porphyrin ring stability by causing a tilt that in turn affects RTV binding, suggesting a possible inefficiency in the function of hCYP3A4. Similar results were seen for amlodipine, atorvastatin, sildenafil and warfarin, which are substrates of hCYP3A4. Conclusions: The current in silico studies indicate that hCYP3A4 containing MIMs can create complications in the treatment of COVID-19 patients, particularly with co-morbidities due to its functional inefficiency. Hence, clinicians must be vigilant when using MOV in combination with other drugs. Further in vitro studies focused on hCYP3A4 containing MIMs are currently in progress to support our current in silico findings. Full article

Dehydroandrographolide (DA), a bioactive diterpenoid from Andrographis paniculata with diverse biological activity, was investigated for its antioxidant and anti-inflammatory effects in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages and dextran sulfate sodium (DSS)-induced murine colitis. In vitro, DA inhibited the inflammatory response by modulating extracellular Signal-Regulated Kinase (Erk), c-Jun N-terminal Kinase (Jnk), p38 Mitogen-Activated Protein Kinase (P38), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) p65 activation, and downregulated interleukin-6 (il-6) and interleukin-1β (il-1β) mRNA. It also had antioxidant effects by upregulating Nuclear Factor Erythroid 2-Related Factor 2 (Nrf2), NAD(P)H quinone dehydrogenase 1 (Nqo-1) and heme oxygenase-1 (Ho-1), promoting protein kinase B (Akt) and 5′-adenosine monophosphate-activated protein kinase-α1 (Ampk-α1) phosphorylation. DA decreased cyclooxygenase-2 (Cox-2) and inducible nitric oxide synthase (iNos) levels and alleviated intracellular reactive oxygen species (ROS) accumulation. In vivo, DA alleviated DSS-induced colitis in wild type (WT) mice by improving weight loss, disease activity index, colonic inflammation, and oxidative stress. The beneficial effects were linked to inhibiting Erk, Jnk, and P38 activation and enhancing Nrf2 signaling pathway. DA inhibited NOD-like receptor family pyrin domain-containing 3 (Nlrp3) inflammasome-mediated pryoptosis. However, DA’s protective effects were abolished in DSS-induced nrf2^−/− mice, suggesting its efficacy depends on Nrf2 signaling. Overall, DA alleviates oxidative stress, inflammatory responses, and pyroptosis in experimental colitis mice mainly by activating Nrf2 signaling pathway, highlighting its potential as a promising therapeutic option for inflammatory bowel disease. Full article

Aspergillus flavus is both an agricultural and clinical pathogen, notable for its ability to contaminate crops with aflatoxins and cause invasive aspergillosis. The increasing emergence of azole resistance in A. flavus poses a serious challenge to food safety and human health. Although mutations in ergosterol biosynthesis genes have been reported in resistant isolates, their functional contributions remain largely unvalidated. In this study, we investigated the role of the CYP51A Y119F mutation in azole resistance. Site-directed mutants were generated using PCR-based gene editing, and their susceptibility to antifungal agents was assessed through Clinical and Laboratory Standards Institute broth microdilution and agar diffusion assays. The Y119F mutation reduced susceptibility specifically to voriconazole and isavuconazole, while susceptibility to itraconazole and posaconazole remained unchanged. To explore the structural basis of this phenotype, molecular dynamics simulations were performed. The mutant protein exhibited greater fluctuations and reduced conformational stability compared to the wild-type enzyme. Tunnel analysis further indicated that the Y119F substitution caused narrowing and shortening of the main access tunnels to the heme-binding pocket, likely impairing azole access and binding. The combined biochemical and structural analyses suggest that Y119F represents a primary resistance-conferring mutation that modifies the structural dynamics of CYP51A. Full article

Ishige okamurae Yendo (I. okamurae) is a brown macroalga with diverse biological activities. Recently, its ameliorative effects against dementia progression have been demonstrated in various in vitro and in vivo models of Alzheimer’s disease (AD), glutamate excitotoxicity, and bacterial-driven neuroinflammation. I. okamurae extract (IOE) inhibited AD progression by regulating amyloid beta–induced neuronal death and cognitive impairments. Moreover, IOE attenuated glutamate-induced neurodegeneration by modulating the mitogen-activated protein kinases/Nrf2/heme oxygenase-1 signaling pathway. Furthermore, IOE effectively suppressed lipopolysaccharide-mediated neuroinflammation and memory deficits by downregulating the Toll-like receptor 4/MyD88-dependent signaling pathway. Collectively, these findings highlight the potential of IOE as a natural multi-target, anti-dementia agent. In this review, we summarize the neuroprotective and cognition-enhancing properties of IOE, discuss the molecular mechanisms underlying its action, and consider the advantages of I. okamurae as a promising natural resource for effective therapeutic developments that are capable of targeting multiple pathogenic causes of dementia. Full article

Heme proteins are central to metabolism and stress responses but remain challenging to express recombinantly due to cytotoxicity and folding constraints. Phytoglobins (Pgbs) exemplify these difficulties, as expression protocols often fail to translate across protein species. Here, we used a cell-free protein synthesis (CFPS) platform powered by digital microfluidics to screen expression determinants for sugar beet Pgb 1.2 (BvPgb 1.2), its C86A variant, and three of eight newly identified oat Pgbs (AsPgbs), including their cysteine-to-alanine substituted variants. Benchmarking with multiple solubility tags and cell-free blends revealed protein- and variant-specific preferences, with alanine substitutions frequently improving expression and purification yields. Oxidative additives such as glutathione disulfide, alone or combined with protein disulfide isomerase, consistently enhanced production, underscoring the importance of redox environments for Pgb stability. Two selected variants were scaled up and yielded putative soluble apo-form proteins. The results highlight how CFPS enables rapid, parallelized identification of expression requirements while uncovering the role of conserved cysteines and redox conditions in Pgb biogenesis. Full article

An overproduction of reactive oxygen species (ROS) creates oxidative stress that disrupts neuronal activity and contributes to the pathogenesis of neurodegenerative diseases. Arecoline, the predominant alkaloid component of Areca catechu L., is known for multiple biological activities, yet its involvement in neuronal oxidative injury has not been fully clarified. This study investigated arecoline’s effect on hydrogen peroxide (H₂O₂)-induced toxicity in SH-SY5Y human neuroblastoma cells (SH-SY5Y). Arecoline pretreatment significantly improved cell viability and preserved plasma membrane integrity, accompanied by reduced lipid peroxidation and restoration of cellular antioxidant enzyme activities. Moreover, arecoline maintained mitochondrial membrane potential and suppressed apoptotic progression. At the molecular level, Arecoline stimulated nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) protein expression, concurrently diminishing Kelch-like ECH-associated protein 1 (Keap1) levels. In parallel, it altered the apoptosis profile by increasing B-cell lymphoma 2 (Bcl2) levels and decreasing Bcl-2-associated X protein (Bax) and total cysteine aspartate protease-3 (Caspase-3) protein expression. Collectively, the findings suggest that arecoline safeguards neurons against oxidative stress by simultaneously activating antioxidant defenses and restraining apoptosis. This study adds novel molecular evidence supporting the potential neuroprotective relevance of arecoline in oxidative stress-related neuropathology. Full article

Myoglobin (Mb), a heme-containing protein, plays crucial roles in storing and transporting oxygen in muscle cells. Various Mb structures have been extensively determined using conventional cryogenic crystallography, providing valuable information for understanding the molecular mechanisms of the protein. However, this approach has limitations attributable to cryogenic temperatures and radiation damage. Serial femtosecond crystallography (SFX) using X-ray free-electron lasers is an emerging technique that enables the determination of biologically relevant room-temperature structures without causing radiation damage. In this study, we assessed the crystallization, collection, and processing of SFX diffraction data of Mb from equine skeletal muscle. Needle- and needle cluster-shaped Mb crystals were obtained using the microbatch method. Fixed-target SFX data collection was performed at the Pohang Accelerator Laboratory X-ray Free Electron Laser, yielding 1389 indexed diffraction patterns. The phase problem was solved by molecular replacement. The preliminary Mb structure determined at 2.3-Å resolution in this study exhibited subtle structural differences in the heme environment compared with previously reported Mb structures determined by SFX. These results both confirm the feasibility of myoglobin SFX experiments and establish a foundation for future time-resolved studies aiming to visualize ligand binding and oxygen transport. Full article

Dermatan sulfate (DS) is an animal glycosaminoglycan with significant structural heterogeneity and a high, but variable density of negative electric charge. Owing to these characteristics DS displays a high degree of biological reactivity that is subject to regulation. We previously demonstrated that structural variants of DS rapidly induce moderate necroptosis in luminal breast cancer cells. In the present study, we investigated the intracellular molecular mechanism(s) that may underlie this effect, focusing on the expression of key regulators of intrinsic (BCL-2A1) and extrinsic (cFLIP) apoptosis, autophagy (Beclin-1), and oxidative stress protection (heme oxygenase-1 (HO-1)). Using RT-qPCR, Western blotting, immunofluorescence, and pharmacological inhibition, we have shown for the first time that DS, depending on its structure and the cancer cell line, can rapidly, albeit transiently, upregulate either the long or short cFLIP splicing variant and also reduce the level of HO-1. These effects are mediated via DS-triggered PI3K and/or NFκB signaling. Moreover, DS can also influence the intracellular distribution of these proteins. In contrast, this glycan did not affect the expression of BCL-2A1 and BECN1. These findings indicate that DS induces coordinated molecular remodeling in luminal breast cancer cells that creates an intracellular environment favorable for necroptosis induction. Full article

Due to their prevalence, diabetes and its complications continue to pose a significant challenge in modern medicine. It is particularly important to identify and develop new biomarkers that would enable faster and more effective detection of specific diseases, including the most common complication of diabetes—diabetic kidney disease (DKD). This review presents the current knowledge on two proteins—fetuin-A and heme oxygenase 1 (HO-1)—whose biological functions and involvement in the pathophysiology of the discussed disease make them potentially useful biomarkers. Moreover, there are studies indicating an association of polymorphisms in the genes encoding fetuin-A and HO-1 with the risk of developing diabetes or DKD. Based on the available literature, both proteins appear promising for use in the diagnosis of diabetes and its complications or assessing the risk of these diseases. However, this requires confirmation in large-scale studies and the development and standardization of detection methods. Full article

Background: This study aimed to investigate rapid oxidation in poultry and fish myoglobin compared to livestock myoglobin using protein structural differences and bioinformatics tools. Methods: Myoglobins from beef (Bos taurus), bison (Bos bison), sheep (Ovis aries), goat (Capra hircus), red deer (Cervus elaphus), pork (Sus scrofa), chicken (Gallus gallus), turkey (Meleagris gallopavo), yellowfin tuna (Thunnus albacares), and tilapia (Oreochromis niloticus) were analyzed to understand differences in structure and function that may influence oxidative behavior. Results: Fish and poultry had shorter or absent D-helix in their myoglobin structure than other species. Tilapia showed the largest heme cavity surface area, indicating significant internal void space, while yellowfin tuna had the largest heme cavity volume, which could affect ligand binding dynamics compared with poultry and other livestock species. However, the heme solvent-accessible area was greater in chicken and turkey than in fish and other livestock species. Tuna myoglobin contains a cysteine and fish myoglobins have fewer amino acids compared to other species. Limited knowledge is currently available on the effects of proteoform, especially post-translational modifications, on the oxidation of myoglobin from different species. Conclusions: The bioinformatics approach used in this study suggests that, in addition to physiological reasons, shorter D-helix, larger heme cavity in tilapia and yellowfin tuna, and greater solvent-accessible area in poultry contribute to increased oxidation in myoglobin from poultry and fish compared with myoglobin from livestock species. Full article

It was hypothesized that differences in production system and muscle type may influence the formation of lipid oxidation products (LOP) as well as protein oxidation (protein carbonyl compounds, PCC) during the in vitro gastrointestinal digestion of chicken meat. To test our hypothesis, we investigated the formation of LOP and PCC after heating and in vitro gastrointestinal digestion of conventional and organic chicken breast and thigh meat and Wooden Breast meat. Prior to the in vitro digestion, thigh and breast meat was minced and heated. Digests of organic thigh meat had significantly higher levels of all LOP measured compared to conventional thigh meat (between +37% and +173%). Lower levels of LOP were found in digests of breast meat regardless of the production system and Wooden Breast phenotype. LOP correlated positively with heme-Fe and polyunsaturated fatty acids, negatively with anserine, and not with carnosine and α-tocopherol. PCC levels were significantly higher in thigh meat than in breast meat after heating (+43%) and digestion (+25%), irrespective of the production system. Overall, organic thigh meat exhibited the highest oxidative sensitivity during digestion. The cut-dependent differences in composition and oxidative susceptibility between organic and conventional chicken highlight the need for further research to assess potential health implications. Full article

Oxidative stress and mitochondrial dysfunction play a key role in the early stage of Doxorubicin (Doxo)-induced cardiotoxicity. Our study investigated the potential cardioprotective role of Simvastatin (Sim), widely known for its antioxidant properties, in an in vitro model of Doxo-induced acute cardiotoxicity. Human Cardiomyocytes (HCMs) were treated with Sim (10 µM, 4 h) and then co-exposed to Doxo (1 µM) and Sim for 20 h. Our data showed that Sim co-treatment significantly (p < 0.05) reduced both cytosolic and mitochondrial Doxo-induced reactive oxygen species overproduction. In Sim co-treated cells, significant reductions in nuclear factor erythroid 2-related factor 2 (Nrf2) gene expression (p < 0.01) and catalase (CAT), heme-oxygenase 1 (HO-1), and superoxide dismutase 2 (SOD2) levels (p < 0.05) compared to Doxo-treated cells were also demonstrated, suggesting a decreased need for compensatory antioxidant defense responses. Moreover, significant reductions in Doxo-induced mitochondrial calcium overload, mitochondrial membrane depolarization (p < 0.005), and apoptosis (p < 0.005) confirmed the protective effects of Sim co-treatment on cardiomyocytes. These data confirm that Sim could be a valuable therapeutic strategy for reducing Doxo-induced HCM damage, preventing the development of dilated cardiomyopathy and long-term heart damage, which are the main limitations of anthracycline use. Finally, real-time PCR analysis revealed that Sim co-treatment significantly reduced (p < 0.001) the Doxo-induced overexpression of MAP4K4, a mitogen-activated protein kinase kinase kinase kinase-4 (MAP4K4) involved in oxidative stress-induced cell death, thus suggesting the involvement of other molecular mechanisms in Sim-mediated cardioprotection. Full article

Class 1 phytoglobins (Pgbs) are known for their multifunctional roles in plant stress responses, with recent studies suggesting broader interactions involving metabolic and transcriptional regulation. Interestingly, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) moonlights in many roles in colocalized spaces during cellular stress that are strikingly suitable for supporting Pgb function. This study investigates the molecular interactions of class 1 Pgb from sugar beet (Beta vulgaris), BvPgb 1.2, and an alanine-substituted mutant (C86A), focusing on their ability to bind GAPDH and DNA. Using dual-emission isothermal spectral shift (SpS) analysis, we report strong binding interactions with GAPDH, with dissociation constants (K_D) of 260 ± 50 nM for the recombinant wild-type protein (rWT) and a significantly stronger affinity for C86A (120 ± 40 nM), suggesting that the cysteine residue limits the interaction. Remarkably strong DNA-binding affinities were also observed for both variants, displaying biphasic binding. This behavior is characteristic of hexacoordinated globins and reflects the presence of two distinct species: a fast-reacting open pentacoordinated form and a slow-reacting closed hexacoordinated form with high apparent affinity. Here, the K_D in the open configuration was 120 ± 50 nm and 50 ± 20 nM for rWT and C86A, respectively. In the closed configuration, however, the cysteine appears to support the interaction, as the K_D was measured at 100 ± 10 pM and 230 ± 60 pM for rWT and C86A, respectively. Protein–protein docking studies reinforced these findings, revealing electrostatically driven interactions between BvPgb 1.2 and GAPDH, characterized by a substantial buried surface area indicative of a stable, biologically relevant complex. Protein–DNA docking similarly confirmed energetically favorable binding near the heme pocket without obstructing ligand accessibility. Together, these findings indicate a potential regulatory role for BvPgb 1.2 through its interaction with GAPDH and DNA. Full article