Target Proteins Research Articles

Detection of TurboID fusion proteins by fluorescent streptavidin outcompetes antibody signals and visualises targets not accessible to antibodies.

Immunofluorescence localises proteins via fluorophore-labelled antibodies. However, some proteins evade detection due to antibody-accessibility issues or because they are naturally low abundant or antigen density is reduced by the imaging method. Here, we show that the fusion of the target protein to the biotin ligase TurboID and subsequent detection of biotinylation by fluorescent streptavidin offers an 'all in one' solution to these restrictions. For all proteins tested, the streptavidin signal was significantly stronger than an antibody signal, markedly improving the sensitivity of expansion microscopy and correlative light and electron microscopy. Importantly, proteins within phase-separated regions, such as the central channel of the nuclear pores, the nucleolus, or RNA granules, were readily detected with streptavidin, while most antibodies failed. When TurboID is used in tandem with an HA epitope tag, co-probing with streptavidin and anti-HA can map antibody-accessibility and we created such a map for the trypanosome nuclear pore. Lastly, we show that streptavidin imaging resolves dynamic, temporally, and spatially distinct sub-complexes and, in specific cases, reveals a history of dynamic protein interaction. In conclusion, streptavidin imaging has major advantages for the detection of lowly abundant or inaccessible proteins and in addition, provides information on protein interactions and biophysical environment.

Computational assessment of the reactivity and anticancer activity of 1,2,3-triazole-thiazolidinones derivatives: An approach combining DFT calculations, molecular dynamics simulations, molecular docking, and ADMET

Cancer is a global health burden with escalating incidence and mortality rates, necessitating the exploration of novel therapeutic strategies. 1,2,3-Triazoles, recognized for their diverse biological activities, have emerged as attractive candidates in drug discovery targeting key proteins associated with various cancers. This study presents the synthesis of a library of 1,2,3-triazole hybrids incorporating thiazolidinone and isoxazoline moieties. The antitumor potential of these compounds was evaluated using both in-silico and in-vitro assays. Structure-activity relationship (SAR) analysis revealed that strategic substitution of the para position of the phenyl ring, particularly with a nitro (NO2) group, is promising to enhance cytotoxic activity. The binding potential of the synthesized compounds with the XIAP BIR2 domain, the specific receptor of caspase-3/7 activators, was explored through molecular docking. The stability of the protein-compound complexes procured from the docking was assessed through molecular dynamics (MD) simulation. The docking study demonstrated that the synthesized compounds had the potential to bind to the target protein. Among the compounds and their stereoisomers, compounds (R)-6d and (S)-6e exhibited a better binding potential to the protein. The MD simulation study revealed that the protein-(R)-6d complex was relatively stable.

The genetic landscape of neuro-related proteins in human plasma.

Understanding the genetic basis of neuro-related proteins is essential for dissecting the molecular basis of human behavioural traits and the disease aetiology of neuropsychiatric disorders. Here the SCALLOP Consortium conducted a genome-wide association meta-analysis of over 12,000 individuals for 184 neuro-related proteins in human plasma. The analysis identified 125 cis-regulatory protein quantitative trait loci (cis-pQTL) and 164 trans-pQTL. The mapped pQTL capture on average 50% of each protein's heritability. At the cis-pQTL, multiple proteins shared a genetic basis with human behavioural traits such as alcohol and food intake, smoking and educational attainment, as well as neurological conditions and psychiatric disorders such as pain, neuroticism and schizophrenia. Integrating with established drug information, the causal inference analysis validated 52 out of 66 matched combinations of protein targets and diseases or side effects with available drugs while suggesting hundreds of repurposing and new therapeutic targets.

Morinda Officinalis Polysaccharides Inhibit Osteoclast Differentiation by Regulating miR-214-3p/NEDD4L in Postmenopausal Osteoporosis Mice.

To investigate the potential mechanism of Morinda officinalis F. C. How polysaccharides (MOPs) in regulating osteoclast differentiation and apoptosis through miR-214-3p and its target protein. Ovariectomy was performed in 8-week female C57BL6 mice to establish the postmenopausal osteoporosis (PMOP) model. Mice were treated immediately with 500mg/kg of MOPs (prevention group); others were treated 2weeks after operation (treatment group). Left femur bone mineral density (BMD) was examined. RAW264.7 cells were administered with receptor activator of NF-κB ligand (RANKL) to establish the osteoclast (OC) model and treated with serum containing 1 or 2g/kg of MOPs. Apoptosis-related indexes, miR-214-3p, and Expressed Developmentally Down-regulated 4-Like (NEDD4L) were detected by western blot, quantitative real-time-reverse transcription polymerase chain reaction (qRT-PCR), and flow cytometry. OC received a miR-214-3p inhibitor or NEDD4L small interfering RNA (siRNA). MOPsreversed the PMOP-induced changes in bones. Compared with the RANKL group, MOPs increased the apoptosis and related markers in OCs. MOPs decreased the femur miR-214-3p of PMOP mice (P < 0.001). Higher concentrations of MOPs reversed the upregulation of miR-214 mRNA in OCs (P < 0.001). miR-214-3p inhibitor increased the expression of Bax and CC3 (P < 0.01) and decreased the expression of Bcl-2 (P < 0.05). NEDD4L is targeted by miR-214. NEDD4L was upregulated in the RANKL + MOPs group (P < 0.01). miR-214-3p inhibitor increased the upregulation of NEDD4L induced by MOPs (P < 0.05). siRNA NEDD4L significantly reversed the inhibition of MOPs on osteoclast differentiation with miR-214-3p inhibitor (P < 0.01). MOPs effectively prevent PMOP by inhibiting osteoclastogenesis and inducing OC apoptosis through the miR-214-3p/NEDD4L pathway.

HERC5: a comprehensive in silico analysis of its diagnostic, prognostic, and therapeutic potential in cancer.

HERC5, a vital protein in the HERC family, plays crucial roles in immune response, cancer progression, and antiviral defense. This bioinformatic study comprehensively assessed HERC5's significance across various malignancies by analyzing its gene expression, immune and molecular subtype expressions, target proteins, biological functions, and prognostic and diagnostic values in pan-cancer. We further examined its correlation with clinical features, co-expressed and differentially expressed genes, and prognosis in clinical subgroups, focusing on endometrial cancer (UCEC). Our findings showed that HERC5 RNA is expressed at low levels in most cancers and significantly differs across immune and molecular subtypes. HERC5 accurately predicts cancer and correlates with most cancer prognoses. In UCEC, HERC5 was significantly associated with age, hormonal status, clinical stage, treatment status, and metastasis. Elevated HERC5 expression was linked to worse progression-free interval, disease-specific survival, and overall survival in UCEC, particularly in diverse clinical subgroups. Significant differences in HERC5 expression were also observed in various human cancer cell line validations. In summary, HERC5 may be a critical biomarker for pan-cancer prognosis, progression, and diagnosis, as well as a promising new target for cancer therapy.

Application of genetic code expansion to regulate the synthesis of poly(lactate-co-3-hydroxybutyrate) in Escherichia coli

Genetic codon expansion has the potential to introduce a variety of unnatural amino acids to specific sites within target proteins. In this study, genetic codon expansion was employed to regulate the enzyme expression in metabolic pathways. Firstly, a purple protein from Actinia tenebrosa was selected as the candidate to be engineered. Bringing in UAG stop codon caused premature termination of translation, while expressing orthogonal aminoacyl-tRNA synthetase and tRNA from Methanococcus jannaschii restored translation at UAG site. However, leakage expression was observed without addition of unnatural amino acids, still it can be decreased by increasing numbers of UAG mutations. Subsequently, poly(lactate-co-3-hydroxyburyrate) [P(LA-3HB)] biosynthesis pathway was constructed in Escherichia coli, and propionyl-CoA transferase was mutated to harboring one or two more stop codons. With genetic codon expansion tools, the function of propionyl-CoA transferase was restored, promoting cells to synthesize P(LA-3HB) copolymer. Moreover, the lactate monomer content was regulated ranging from 0 to 33.42 mol% by altering the addition time of inducers. Finally, the strain accumulated 27.09 g/L P(25.1 mol% LA-3HB) in 5-L bioreactor cultivation. This is the first report on metabolic engineering of polyhydroxyalkanoate biosynthesis through genetic codon expansion and would provide helpful strategies to achieve dynamic regulation of multiple metabolic pathways.

Anti-Neuroinflammatory Potential of Areca Nut Extract and Its Bioactive Compounds in Anthracene-Induced BV-2 Microglial Cell Activation.

Particulate matter (PM2.5) containing polycyclic aromatic hydrocarbons (PAHs) is of considerable environmental importance worldwide due to its adverse effects on human health, which are associated with neurodegenerative diseases (NDDs). Areca catechu L. (AC) fruit is known to possess various pharmacological properties; however, the anti-neuroinflammatory roles of AC on the suppression of PAH-induced neuroinflammation are still limited. Thus, we focused on the effects and related signaling cascades of AC and its active compounds against anthracene-induced toxicity and inflammation in mouse microglial BV-2 cells. Phytochemicals in the ethanolic extract of AC (ACEE) were identified using LC-MS, and molecular docking was conducted to screen the interaction between compounds and target proteins. Significant bioactive compounds in ACEE such as arecoline, (-)-epicatechin, and syringic acid were evinced through the LC-MS spectrum. The docking study revealed that (-)-epicatechin showed the highest binding affinities against NF-κB. For cell-based approaches, anthracene induced intracellular ROS, mRNA levels of TNF-α, IL-1β, and IL-6, and the release of TNF-α through enhancing JNK, p38, and NF-κB signaling pathways. However, the co-treatment of cells with ACEE or (-)-epicatechin could reverse those anthracene-induced changes. The overall study suggested that ACEE-derived bioactive compounds such as (-)-epicatechin may be developed as a potential anti-neuroinflammatory agent by preventing inflammation-mediated NDDs.

Multi-omics combined to investigate potential druggable therapeutic targets for stroke: A systematic Mendelian randomization study and transcriptome verification

ObjectiveStroke is a highly prevalent and disabling disease whose disease mechanisms are not fully understood. The discovery of disease-associated proteins with genetic evidence of pathogenicity provides an opportunity to identify new therapeutic targets. MethodWe examined the observed and causal associations of thousands of plasma and inflammatory proteins that were measured using affinity-based proteomic assays. First, we pooled >3000 relevant proteins using a fixed-effects meta-analysis of 2 population-based studies involving 48,383 participants, then investigated the causal effects of stroke and its subtype-associated proteins by forward Mendelian randomization using cis-protein quantitative locus genetic tools identified from genome-wide association studies of these >48,000 individuals. To improve the accuracy of causal estimation, we implemented a systematic Mendelian randomization model that accounts for cascading imbalances between instruments and tested the robustness of causal estimation through multi-method analyses. To further validate the hypothesis that ginsenoside Rg1 monomer acts on the five protein targets screened for drug-targeted regulation, we conducted a comparative analysis of the mRNA (gene) expression levels of a limited number of genes in the brain tissues of different groups of SD rats. The druggability of the candidate proteins was investigated and the mechanism of action and potential targeting side effects were explored by Phenome-wide MR. ResultsSix circulating proteins were identified to have a significant genetic association with stroke (PFDR < 0.05). For example, in patients with cardioembolic stroke, higher genetically predicted APRT was associated with a lower risk of cardioembolic stroke (ORivw [95 % CI] = 0.641 [0.517, 0.795]; P = 5.25 × 10−5, ORSMR [95 % CI] = 0.572, [0.397, 0.825], PSMR = 0.003). Mediation analyses suggested that atrial fibrillation, angina pectoris, and heart failure may mediate the association of CD40L, LIFR, and UPA with stroke. Molecular docking revealed promising interactions between the identified proteins and glycosides. Transcriptomic sequencing in animal models indicated that ginsenoside Rg1 may act through APRT, IL15RA, and VSIR pathways, with APRT showing significant variability in mRNA sequencing expression. Phenome-wide MR of the six target proteins showed an overwhelming predominance of PFDR > 0.05, indicating less toxicity. ConclusionsThe present study provides genetic evidence to support the potential efficacy of targeting the three druggable protein targets for the treatment of stroke. This is achieved by triangulating population genomic and proteomic data. Furthermore, the study validates the pathway mechanisms by which APRT, IL15RA, and VSIR dock ginsenoside Rg1 in animal models. This will help to prioritize stroke drug development.

Development and clinical implementation of an LC-HRMS method for ivacaftor, lumacaftor, tezacaftor and elexacaftor in human plasma and breast milk.

The four cystic fibrosis (CF) transmembrane conductance regulator (CFTR) modulators, ivacaftor, lumacaftor, tezacaftor, and elexacaftor, have revolutionised the treatment of CF by direct action on the protein target behind the disease's development. The aim was to develop and validate a quantification method for these CFTR modulators in plasma and breast milk to better understand inter-patient variability in pharmacokinetics and treatment outcome, including the risk of adverse drug reactions. The ability to monitor CFTR modulators in breast milk enables the estimation of the exposure of breastfed infant, with a potential concern for CFTR modulator-induced liver injury. The analysis was performed on a Thermo Vanquish Flex Binary UHPLC system coupled to a high-resolution mass spectrometer (HRMS), Thermo Q Exactive. The analytes were detected using positive electrospray ionisation in full scan mode. After sample preparation by protein precipitation, the supernatant was injected onto the LC system and the analytes were separated using a Zorbax SB-C18 Rapid Res HPLC column (3.5µm, 4.6 × 75mm). This is the first published method for CFTR modulators in breast milk. The validated quantification range for ivacaftor is 0.0050-10µg/mL with a coefficient of variation < 6% and a mean accuracy of 97-106%; for lumacaftor, tezacaftor, and elexacaftor, the validated quantification range is 0.050-100µg/mL with a coefficient of variation < 8% and a mean accuracy 93-106%. A simple and sensitive quantification method for CFTR modulators has been developed and used for routine analysis of human plasma and breast milk samples since 2022.

Application of Proteomics Technology Based on LC-MS Combined with Western Blotting and Co-IP in Antiviral Innate Immunity.

As an interferon-stimulating factor protein, STING plays a role in the response and downstream liaison in antiviral natural immunity. Upon viral invasion, the immediate response of STING protein leads to a series of changes in downstream proteins, which ultimately leads to an antiviral immune response in the form of proinflammatory cytokines and type I interferons, thus triggering an innate immune response, an adaptive immune response in vivo, and long-term protection of the host. In the field of antiviral natural immunity, it is particularly important to rigorously and sequentially probe the dynamic changes in the antiviral natural immunity connector protein STING caused by the entire anti-inflammatory and anti-pathway mechanism and the differences in upstream and downstream proteins. Traditionally, proteomics technology has been validated by detecting proteins in a 2D platform, for which it is difficult to sensitively identify changes in the nature and abundance of target proteins. With the development of mass spectrometry (MS) technology, MS-based proteomics has made important contributions to characterizing the dynamic changes in the natural immune proteome induced by viral infections. MS analytical techniques have several advantages, such as high throughput, rapidity, sensitivity, accuracy, and automation. The most common techniques for detecting complex proteomes are liquid chromatography (LC) and mass spectrometry (MS). LC-MS (Liquid Chromatography-Mass Spectrometry), which combines the physical separation capability of LC and the mass analysis capability of MS, is a powerful technique mainly used for analyzing the proteome of cells, tissues, and body fluids. To explore the combination of traditional proteomics techniques such as Western blotting, Co-IP (co-Immunoprecipitation), and the latest LC-MS methods to probe the anti-inflammatory pathway and the differential changes in upstream and downstream proteins induced by the antiviral natural immune junction protein STING.

Exploring the environmental contamination toxicity and potential carcinogenic pathways of perfluorinated and polyfluoroalkyl substances (PFAS): An integrated network toxicology and molecular docking strategy

The objective of this study was to investigate the potential carcinogenic toxicity and mechanisms of PFAS in thyroid, renal, and testicular cancers base on network toxicology and molecular docking techniques. Structural modeling was performed to predict relevant toxicity information, and compounds and cancer-related targets were screened in multiple databases. The interaction of PFAS with three cancers and their key protein targets were explored by combining protein network analysis, enrichment analysis and molecular docking techniques. PFOA, PFOS, and PFHXS exhibited significant carcinogenic and cytotoxic effects. These compounds may induce cancer by mediating active oxygen metabolism and the transduction of phosphatidylinositol 3-kinase/protein kinase B signaling pathway through genes such as ALB, mTOR, MDM2, and ERBB2. Furthermore, the underlying toxic mechanisms may be linked to the pathways in cancer, chemical carcinogenesis through reactive oxygen species/receptor activation, and the FoxO signaling pathway. The results contribute to a comprehensive understanding of the effects of these environmental pollutants on genes, proteins, and metabolic pathways in living organisms. It revealed their toxicity mechanisms in inducing thyroid, renal, and testicular cancers, and provided a solid theoretical foundation for designing new environmental control strategies and drug screening initiatives. Additionally, the integrated application of network toxicology and molecular docking technology can enhance our understanding of the toxicity and mechanisms of unknown environmental pollutants, which is beneficial for protecting the environment and human health.

REPRESSOR OF UV-B PHOTOMORPHOGENESIS proteins target ABA INSENSITIVE 5 for degradation to promote early plant development.

REPRESSOR OF UV-B PHOTOMORPHOGENESIS 1 (RUP1) and RUP2 are WD-40 domain-containing proteins that have been extensively characterized for their role in UV-B signaling. However, the roles of the RUP proteins outside the canonical UV-signaling pathway are less known. Here, we identify that RUP1 and RUP2 play important roles in ABA signaling to regulate seed germination and early seedling development in Arabidopsis thaliana. Our protein interaction studies confirmed that RUP1 and RUP2 physically interact with ABA INSENSITIVE 5 (ABI5). In the presence of abscisic acid, rup1, rup2 and rup1rup2 exhibited reduced germination and seedling establishment compared to the wild type. Germination and seedling establishment in rup1rup2abi5-8 was similar to abi5-8, suggesting that RUP1 and RUP2 suppress ABA-mediated inhibition of germination and early seedling development in an ABI5-dependent manner. The DDB1-binding WD40 (DWD) protein RUP2 promoted the ubiquitination of ABI5 to regulate its degradation. ABI5, in turn, establishes a negative feedback loop to inhibit the expression of RUP1/RUP2. ABI5 also inhibited the direct binding of ELONGATED HYPOCOTYL 5 (HY5) to the promoters of RUP1 and RUP2 under ABA. This study highlights the coordinated action of RUP1, RUP2, ABI5, and HY5 in regulating early plant development.

Pharmacogenomic discovery of genetically targeted cancer therapies optimized against clinical outcomes

Despite the clinical success of dozens of genetically targeted cancer therapies, the vast majority of patients with tumors caused by loss-of-function (LoF) mutations do not have access to these treatments. This is primarily due to the challenge of developing a drug that treats a disease caused by the absence of a protein target. The success of PARP inhibitors has solidified synthetic lethality (SL) as a means to overcome this obstacle. Recent mapping of SL networks using pooled CRISPR-Cas9 screens is a promising approach for expanding this concept to treating cancers driven by additional LoF drivers. In practice, however, translating signals from cell lines, where these screens are typically conducted, to patient outcomes remains a challenge. We developed a pharmacogenomic (PGx) approach called “Clinically Optimized Driver Associated-PGx” (CODA-PGX) that accurately predicts genetically targeted therapies with clinical-stage efficacy in specific LoF driver contexts. Using approved targeted therapies and cancer drugs with available real-world evidence and molecular data from hundreds of patients, we discovered and optimized the key screening principles predictive of efficacy and overall patient survival. In addition to establishing basic technical conventions, such as drug concentration and screening kinetics, we found that replicating the driver perturbation in the right context, as well as selecting patients where those drivers are genuine founder mutations, were key to accurate translation. We used CODA-PGX to screen a diverse collection of clinical stage drugs and report dozens of novel LoF genetically targeted opportunities; many validated in xenografts and by real-world evidence. Notable examples include treating STAG2-mutant tumors with Carboplatin, SMARCB1-mutant tumors with Oxaliplatin, and TP53BP1-mutant tumors with Etoposide or Bleomycin.

In vivo and in vitro study on the regulatory mechanism of XiaoChaiHu decoction on PANoptosis in sepsis-induced cardiomyopathy

Ethnopharmacological relevanceIn accordance with the tenets of traditional Chinese medicine, sepsis is categorized into three distinct syndromes: heat syndrome, blood stasis syndrome, and deficiency syndrome. Xiaochaihu decoction (XCHD) has many functions, including the capacity to protect the liver, cholagogue, antipyretic, anti-inflammatory, and anti-pathogenic microorganisms. XCHD exerts the effect of clearing heat and reconciling Shaoyang. The XCHD contains many efficacious active ingredients, yet the mechanism of sepsis-induced cardiomyopathy (SIC) remains elusive. Aim of the studyTo investigate the molecular mechanisms underlying the protective effects of XCHD against SIC using an integrated approach combining network pharmacology and molecular biology techniques. Materials and methodsNetwork pharmacology methods identified the active ingredients, target proteins, and pathways affected by XCHD in the context of SIC. We conducted in vivo experiments using mice with lipopolysaccharide-induced SIC, evaluating cardiac function through echocardiography and histology. XCHD-containing serum was analyzed to determine its principal active components using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The effects of XCHD-containing serum on SIC were further tested in vitro in LPS-treated H9c2 cardiac cells. Protein expression levels were quantified via Western blotting and enzyme-linked immunosorbent assay (ELISA). Additionally, molecular docking was performed between the active components and ZBP1, a potential target protein. Overexpression of ZBP1 in H9c2 cells allowed for a deeper exploration of its role in modulating SIC-associated gene expression. ResultsUPLC-MS/MS identified 31 shared XCHD and XCHD-containing serum components. These included organic acids, terpenoids, and flavonoids, which have been identified as the active components of XCHD. Our findings revealed that XCHD alleviated LPS-induced myocardial injury, improved cardiac function, and preserved cardiomyocyte morphology in mice. In vitro studies, we demonstrated that XCHD-containing serum significantly suppressed the expression of inflammatory cytokines (IL-6, IL-1β, and TNF-α) in LPS-induced H9c2 cells. Mechanistic investigations showed that XCHD downregulated genes associated with PANoptosis, a novel cell death pathway, suggesting its protective role in sepsis-damaged hearts. Conversely, overexpression of ZBP1 abolished the protective effects of XCHD and amplified PANoptosis-related gene expression. ConclusionsOur study provides the first evidence supporting the protective effects of XCHD against SIC, both in vitro and in vivo. The underlying mechanism involves the inhibition of ZBP1-initiated PANoptosis, offering new insights into treating SIC using XCHD.

Inhibition of mitochondrial over-division by (+)-14,15-Dehydrovincamine attenuates cisplatin-induced acute kidney injury via the JNK/Mff pathway

Cisplatin-induced acute kidney injury (AKI) is characterized by mitochondrial damage and apoptosis, and safe and effective therapeutic agents are urgently needed. Renal tubular epithelial cells, the main site of AKI, are enriched with a large number of mitochondria, which are crucial for the progression of AKI with an impaired energy supply. Vincamine has anti-inflammatory and antioxidant effects in mouse AKI models. As a natural compound derived from Tabernaemontana pandacaqui, (+)-14, 15-Dehydrovincamine and Vincamine differ in structure by only one double bond, and the role and exact mechanism of (+)-14, 15-Dehydrovincamine remains to be elucidated in AKI. The present study demonstrated that (+)-14,15-Dehydrovincamine significantly ameliorated mitochondrial dysfunction and maintained mitochondrial homeostasis in a cisplatin-induced AKI model. Furthermore, (+)-14,15-Dehydrovincamine ameliorates cytochrome C-dependent apoptosis in renal tubular epithelial cells. c-Jun NH2-terminal kinase (JNK) was identified as a potential target protein of (+)-14,15-Dehydrovincamine attenuating AKI by network pharmacological analysis. (+)-14,15-Dehydrovincamine inhibited cisplatin-induced JNK activation, mitochondrial fission factor (Mff) phosphorylation, and dynamin-related protein 1 (Drp1) translocation to the mitochondria in renal tubular epithelial cells. Meanwhile, the JNK activator anisomycin restored Mff phosphorylation and Drp1 translocation, counteracting the protective effect of (+)-14,15-Dehydrovincamine on mitochondrial dysfunction in cisplatin-induced TECs injury. In conclusion, (+)-14,15-Dehydrovincamine reduced mitochondrial fission, maintained mitochondrial homeostasis, and attenuated apoptosis by inhibiting the JNK/Mff/Drp1 pathway, which in turn ameliorated cisplatin-induced AKI.

Human induced pluripotent stem cell based hepatic-modeling of lipid metabolism associated TM6SF2 E167K variant.

TM6SF2 rs58542926 (E167K) is related to increased prevalence of metabolic dysfunction-associated steatotic liver disease (MASLD). Conflicting mouse study results highlight the need for a human model to understand this mutation's impact. This study aims to create and characterize a reliable human in vitro model to mimic the effects of the TM6SF2-E167K mutation for future studies. We used gene editing on human human-induced pluripotent stem cells (iPSC) from a healthy individual to create cells with the TM6SF2-E167K mutation. After hepatocyte directed differentiation, we observed decreased TM6SF2 protein expression, increased intracellular lipid droplets and total cholesterol in addition to reduced VLDL secretion. Transcriptomics revealed upregulation of genes involved in lipid, fatty acid, and cholesterol transport, flux, and oxidation. Global lipidomics showed increased lipid classes associated with ER stress, mitochondrial dysfunction, apoptosis, and lipid metabolism. Additionally, the TM6SF2-E167K mutation conferred a pro-inflammatory phenotype with signs of mitochondria and ER stress. Importantly, by facilitating protein folding within the ER of hepatocytes carrying TM6SF2-E167K mutation, VLDL secretion and ER stress markers improved. Our findings indicate that induced hepatocytes generated from iPSCs carrying the TM6SF2-E167K recapitulate the effects observed in human hepatocytes from individuals with the TM6SF2 mutation. This study characterizes an in vitro model that can be used as a platform to identify potential clinical targets and highlights the therapeutic potential of targeting protein misfolding to alleviate ER stress and mitigate the detrimental effects of the TM6SF2-E167K mutation on hepatic lipid metabolism.

On the mechanism of miR-29b enhancement of etoposide toxicity in vitro

MicroRNA hsa-miR-29 was connected to a number of malignancies. Its target genes are many, among them Mcl-1 that is expressed in three possible isoforms, one of which is anti-apoptotic and another one pro-apoptotic. Ratio of these two isoforms appears to affect cell response to external stimuli. We have demonstrated that miR-29b enhanced etoposide toxicity in HeLa cell line by modulating this ratio of Mcl-1 isoforms. However, it is not known whether the described miR-29 effect is common to various cancer types or even have the opposite effect. This represents a significant problem for possible future applications. In this report, we demonstrate that miR-29b affects toxicity of 60 μM etoposide in cell lines derived from selected malignancies. The mechanism, however, differs among the cell lines tested. Hep G2 cells demonstrated similar effect of miR-29b on etoposide toxicity as was described in HeLa cells, i.e. modulation of Mcl-1 expression. Target protein down-regulated by miR-29b resulting in enhanced etoposide toxicity in Caco-2 cells was, however, Bcl-2 protein. Moreover, H9c2, Hek-293 and ARPE-19 cell lines selected as a representatives of non-malignant cells, showed no effect of miR-29b on etoposide toxicity. Our data suggest that miR-29b could be a common enhancer of etoposide toxicity in malignant cells due to its modulation of Bcl family proteins.

The potential of aptamers for the analysis of ceramic bound proteins found within pottery

Archaeological pottery are the most numerous objects found during excavations and reflect the culinary practices of the past. However, their functionality for cooking/storing specific foods or drinks cannot be deduced solely from comparing their shapes and sizes. Analysis of protein residues bound to ceramics can reveal the protein/animal type through their amino acid sequence, thus enabling direct identification of food types. Therefore, the aim of our experimental study was to test sixteen aptamers for the analysis of proteinaceous organic residues found within the porous structure of pottery. Traditionally prepared archaeological ceramic replicas were cooked for 5 days in various food/protein suspensions, were UV aged, buried for a year, excavated, and extensively cleaned. Their shards were analysed using immunofluorescence microscopy with aptamers. Results show that eight aptamers (Clone1 and Kirby for egg residuals; seqU5 and BLG14 for milk residuals; HA for blood residuals; Gli4 for gluten residuals; Par1 for fish residuals; and D1 for collagen residuals) produced a successful/specific immunofluorescence microscopy result when they were hybridised to shards containing target protein residuals. Interestingly, on whole egg control samples, when the egg lysozyme-targeting aptamer Kirby was used, fluorescence intensity was 3.1 times greater compared to that observed with anti-ovalbumin antibodies.

Potential mechanism prediction of indole-3-propionic acid against diminished ovarian reserve via network pharmacology, molecular docking and experimental verification

BackgroundOxidative stress (OS) is one of the major causes of ovarian aging and dysfunction. Indole-3-propionic acid (IPA) is an indole compound derived from tryptophan with free radical scavenging and antioxidant properties, and thus may have potential applications in protecting ovarian function, although the exact mechanisms are unknown. This study aims to preliminarily elucidate the potential mechanisms of IPA that benefit ovarian reserve function through network pharmacology, molecular docking, and experimental verification.MethodsThe related protein targets of IPA were searched on SwissTargetPrediction, TargetNet, BATMAN-TCM, and PharmMapper databases. The potential targets of diminished ovarian reserve (DOR) were identified from OMIM, GeneCards, DrugBank, and DisGeNET databases. The common targets were uploaded directly to the STRING database to construct PPI networks. We then performed GO and KEGG enrichment analysis on the targets. Subsequently, molecular docking and molecular dynamics simulation were used to validate the binding conformation of IPA to candidate targets. Furthermore, we carried out in vitro experiments to validate the prediction results of network pharmacology.ResultsWe identified a total of 61 potential targets for the interaction of IPA with DOR. The PPI network topological parameter analysis yielded 13 hub genes for DOR treatment. The GO biological process enrichment analysis identified 293 entries, mainly enriched in aging, signal transduction, response to hypoxia, negative regulation of apoptotic process, and positive regulation of cell proliferation. The KEGG enrichment analysis mainly included lipid and atherosclerosis, progesterone-mediated oocyte maturation, AGE-RAGE, relaxin, estrogen, and other signaling pathways. The molecular docking further revealed the direct binding of IPA with six hub proteins including NOS3, AKT1, EGFR, PPARA, SRC, and TNF. In vitro experiments showed that IPA pretreatment attenuated H2O2-induced cellular oxidative stress damage, while IPA exerted cytoprotective and antioxidant damage effects by regulating the six hub genes and antioxidant proteins.ConclusionWe systematically illustrated the potential protective effects of IPA against DOR through multiple targets and pathways using network pharmacology, and further verified the cytoprotective effect and antioxidant properties of IPA through in vitro experiments. These findings provide new insights into the targets and molecular mechanisms whereby IPA improves DOR.

BMS345541 is predicted as a repurposed drug for the treatment of TMZ-resistant Glioblastoma using target gene expression and virtual drug screening

Glioblastoma (GBM) is one of the most aggressive and fatal cancers, for which Temozolomide (TMZ) chemo drug is commonly used for its treatment. However, patients gradually develop resistance to this drug, leading to tumor relapse. In our previous study, we have identified lncRNAs that regulate chemoresistance through the competing endogenous RNA (ceRNA) mechanism. In this study, we tried to find FDA-approved drugs against the target proteins of these ceRNA networks through drug repurposing using differential gene expression profiles, which could be used to nullify the effect of lncRNAs and promote the sensitivity of TMZ in GBM. We performed molecular docking and simulation studies of predicted repurposed drugs and their targets. Among the predicted repurposed drugs, we found BMS345541 has a higher binding affinity towards its target protein - FOXG1, making it a more stable complex with FOXG1-DNA. The ADMET analysis of this drug BMS345541 shows a higher half-life and lower cytotoxicity level than other predicted repurposed drugs. Hence, we conjecture that this could be a better drug for increasing the sensitivity of TMZ for treating GBM patients.