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Oncogenic mutations in RAS provide a compelling yet intractable therapeutic target. Using co-immunoprecipitation mass spectrometry, we uncovered an interaction between RAS and Argonaute 2 (AGO2). Endogenously, RAS and AGO2 co-sediment and co-localize in the endoplasmic reticulum. The AGO2 N-terminal domain directly binds the Switch II region of KRAS, agnostic of nucleotide (GDP/GTP) binding. Functionally, AGO2 knockdown attenuates cell proliferation in mutant KRAS-dependent cells and AGO2 overexpression enhances KRAS(G12V)-mediated transformation. Using AGO2-/- cells, we demonstrate that the RAS-AGO2 interaction is required for maximal mutant KRAS expression and cellular transformation. Mechanistically, oncogenic KRAS attenuates AGO2-mediated gene silencing. Overall, the functional interaction with AGO2 extends KRAS function beyond its canonical role in signaling.

Epoxyeicosatrienoic acids (EETs), the metabolites of arachidonic acid derived from the cytochrome P450 (CYP450) epoxygenases, are mainly metabolized by soluble epoxide hydrolase (sEH) to their corresponding diols. EETs but not their diols, have anti-inflammatory properties and inhibition of sEH might provide protective effects against inflammatory fibrosis. We test the effects of a selected sEH inhibitor, 1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) urea (TPPU), on bleomycin-induced pulmonary fibrosis (PF) in mice. A mouse model of PF was established by intratracheal injection of bleomycin and TPPU was administered for 21 days after bleomycin injection. We found TPPU treatment improved the body weight loss and survival rate of bleomycin-stimulated mice. Histological examination showed that TPPU treatment alleviated bleomycin-induced inflammation and maintained the alveolar structure of the pulmonary tissues. TPPU also decreased the bleomycin-induced deposition of collagen and the expression of procollagen I mRNA in lung tissues of mice. TPPU decreased the transforming growth factor-β1 (TGF-β1), interleukin-1β (IL-1β) and IL-6 levels in the serum of bleomycin-stimulated mice. Furthermore, TPPU inhibited the proliferation and collagen synthesis of mouse fibroblasts and partially reversed TGF-β1-induced α-smooth muscle actin expression. Our results indicate that the inhibition of sEH attenuates bleomycin-induced inflammation and collagen deposition and therefore prevents bleomycin-induced PF in a mouse model.

UnlabelledBipolar disorder (BD) is associated with mood episodes and low amplitude circadian rhythms. Previously, we demonstrated that fibroblasts grown from BD patients show weaker amplification of circadian rhythms by lithium compared to control cells. Since calcium signals impact upon the circadian clock, and L-type calcium channels (LTCC) have emerged as genetic risk factors for BD, we examined whether loss of function in LTCCs accounts for the attenuated response to lithium in BD cells. We used fluorescent dyes to measure Ca(2+) changes in BD and control fibroblasts after lithium treatment, and bioluminescent reporters to measure Per2::luc rhythms in fibroblasts from BD patients, human controls, and mice while pharmacologically or genetically manipulating calcium channels. Longitudinal expression of LTCC genes (CACNA1C, CACNA1D and CACNB3) was then measured over 12-24 h in BD and control cells. Our results indicate that independently of LTCCs, lithium stimulated intracellular Ca(2+) less effectively in BD vs. control fibroblasts. In longitudinal studies, pharmacological inhibition of LTCCs or knockdown of CACNA1A, CACNA1C, CACNA1D and CACNB3 altered circadian rhythm amplitude. Diltiazem and knockdown of CACNA1C or CACNA1D eliminated lithium's ability to amplify rhythms. Knockdown of CACNA1A or CACNB3 altered baseline rhythms, but did not affect rhythm amplification by lithium. In human fibroblasts, CACNA1C genotype predicted the amplitude response to lithium, and the expression profiles of CACNA1C, CACNA1D and CACNB3 were altered in BD vs.ControlsWe conclude that in cells from BD patients, calcium signaling is abnormal, and that LTCCs underlie the failure of lithium to amplify circadian rhythms.

Cytoplasmic dyneins 1 and 2 are related members of the AAA+ superfamily (ATPases associated with diverse cellular activities) that function as the predominant minus-end-directed microtubule motors in eukaryotic cells. Dynein 1 controls mitotic spindle assembly, organelle movement, axonal transport, and other cytosolic, microtubule-guided processes, whereas dynein 2 mediates retrograde trafficking within motile and primary cilia. Small-molecule inhibitors are important tools for investigating motor protein-dependent mechanisms, and ciliobrevins were recently discovered as the first dynein-specific chemical antagonists. Here, we demonstrate that ciliobrevins directly target the heavy chains of both dynein isoforms and explore the structure-activity landscape of these inhibitors in vitro and in cells. In addition to identifying chemical motifs that are essential for dynein blockade, we have discovered analogs with increased potency and dynein 2 selectivity. These antagonists effectively disrupt Hedgehog signaling, intraflagellar transport, and ciliogenesis, making them useful probes of these and other cytoplasmic dynein 2-dependent cellular processes.

The construction of synthetic straightforward, biocompatible and biodegradable targeted drug delivery system with fluorescent tracking abilities, high anticancer activities and low side effects is still a challenge in the field of biochemistry and material chemistry. In this work, we constructed targeted paclitaxel (Taxol) delivery nanoparticles composed of permethyl-β-cyclodextrin modified hyaluronic acid (HApCD) and porphyrin modified paclitaxel prodrug (PorTaxol), through host-guest and amphiphilic interactions. The obtained nanoparticles (HATXP) were biocompatible and enzymatic biodegradable due to their hydrophilic hyaluronic acid (HA) shell and hydrophobic Taxol core, and exhibited specific targeting internalization into cancer cells via HA receptor mediated endocytosis effects. The cytotoxicity experiments showed that the HATXP exhibited similar anticancer activities to, but much lower side effects than commercial anticancer drug Taxol. The present work would provide a platform for targeted paclitaxel drug delivery and a general protocol for the design of advanced multifunctional nanoscale biomaterials for targeted drug/gene delivery.

A new micropallet array platform for adherent cell colony sorting has been developed. The platform consisted of thousands of square plastic pallets, 270 μm by 270 μm on each side, large enough to hold a single colony of cells. Each pallet included a magnetic core, allowing them to be collected with a magnet after being released using a microscope mounted laser system. The micropallets were patterned from 1002F epoxy resist and were fabricated on translucent, gold coated microscope slides. The gold layer was used as seed for electroplating the ferromagnetic cores within every individual pallet. The gold layer also facilitated the release of each micropallet during laser release. This array allows for individual observation, sorting and collection of isolated cell colonies for biological cell colony research. In addition to consistent release and recovery of individual colonies, we demonstrated stable biocompatibility and minimal loss in imaging quality compared to previously developed micropallet arrays.

The Peroxisome proliferator-activated receptor-gamma coactivator 1 alpha (PGC-1α) is a transcriptional co-activator that plays a central role in adapted metabolic responses. PGC-1α is dynamically methylated and unmethylated at the residue K779 by the methyltransferase SET7/9 and the Lysine Specific Demethylase 1A (LSD1), respectively. Interactions of methylated PGC-1α[K779me] with the Spt-Ada-Gcn5-acetyltransferase (SAGA) complex, the Mediator members MED1 and MED17, and the NOP2/Sun RNA methytransferase 7 (NSUN7) reinforce transcription, and are concomitant with the m(5)C mark on enhancer RNAs (eRNAs). Consistently, loss of Set7/9 and NSun7 in liver cell model systems resulted in depletion of the PGC-1α target genes Pfkl, Sirt5, Idh3b, and Hmox2, which was accompanied by a decrease in the eRNAs levels associated with these loci. Enrichment of m(5)C within eRNA species coincides with metabolic stress of fasting in vivo. Collectively, these findings illustrate the complex epigenetic circuitry imposed by PGC-1α at the eRNA level to fine-tune energy metabolism.

Gonadotropin-releasing hormone (GnRH), a neuropeptide released from a small population of neurons in the hypothalamus, is the central mediator of the hypothalamic-pituitary-gonadal axis, and is required for normal reproductive development and function. Evolutionarily conserved regulatory elements in the mouse, rat, and human Gnrh1 gene include three enhancers and the proximal promoter, which confer Gnrh1 gene expression specifically in GnRH neurons. In immortalized mouse hypothalamic GnRH (GT1-7) neurons, which show pulsatile GnRH release in culture, RNA sequencing and RT-qPCR revealed that expression of a novel long noncoding RNA at Gnrh1 enhancer 1 correlates with high levels of GnRH mRNA expression. In GT1-7 neurons, which contain a transgene carrying 3 kb of the rat Gnrh1 regulatory region, both the mouse and rat Gnrh1 enhancer-derived noncoding RNAs (GnRH-E1 RNAs) are expressed. We investigated the characteristics and function of the endogenous mouse GnRH-E1 RNA. Strand-specific RT-PCR analysis of GnRH-E1 RNA in GT1-7 cells revealed GnRH-E1 RNAs that are transcribed in the sense and antisense directions from distinct 5' start sites, are 3' polyadenylated, and are over 2 kb in length. These RNAs are localized in the nucleus and have a half-life of over 8 hours. In GT1-7 neurons, siRNA knockdown of mouse GnRH-E1 RNA resulted in a significant decrease in the expression of the Gnrh1 primary transcript and Gnrh1 mRNA. Over-expression of either the sense or antisense mouse GnRH-E1 RNA in immature, migratory GnRH (GN11) neurons, which do not express either GnRH-E1 RNA or GnRH mRNA, induced the transcriptional activity of co-transfected rat Gnrh1 gene regulatory elements, where the induction requires the presence of the rat Gnrh1 promoter. Together, these data indicate that GnRH-E1 RNA is an inducer of Gnrh1 gene expression. GnRH-E1 RNA may play an important role in the development and maturation of GnRH neurons.

The Hedgehog pathway is a potent regulator of cellular growth and plays a central role in the development of many cancers including basal cell carcinoma (BCC). The majority of BCCs arise from mutations in the Patched receptor resulting in constitutive activation of the Hedgehog pathway. Secondary driver mutations promote BCC oncogenesis and occur frequently due to the high mutational burden resulting from sun exposure of the skin. Here, we uncover novel secondary mutations in Suppressor of Fused (SUFU), the major negative regulator of the Hedgehog pathway. SUFU normally binds to a Hedgehog transcriptional activator, GLI1, in order to prevent it from initiating transcription of Hedgehog target genes. We sequenced tumor-normal pairs from patients with early sporadic BCCs. This resulted in the discovery of nine mutations in SUFU, which were functionally investigated to determine whether they help drive BCC formation. Our results show that four of the SUFU mutations inappropriately activate the Hedgehog pathway, suggesting they may act as driver mutations for BCC development. Indeed, all four of the loss of function SUFU variants were found to disrupt its binding to GLI, leading to constitutive pathway activation. Our results from functional characterization of these mutations shed light on SUFU's role in Hedgehog signaling, tumor progression, and highlight a way in which BCCs can arise.

Cell membrane is made up of a complex structure of lipids and proteins that diffuse laterally giving rise to what we call membrane fluidity. During cellular development, such as differentiation cell membranes undergo dramatic fluidity changes induced by proteins such as ARC and Cofilin among others. In this study we used the generalized polarization (GP) property of fluorescent probe Laurdan using two-photon microscopy to determine membrane fluidity as a function of time and for various cell lines. A low GP value corresponds to a higher fluidity and a higher GP value is associated with a more rigid membrane. Four different cell lines were monitored such as hN2, NIH3T3, HEK293 and L6 cells. Membrane fluidity was measured at 12h, 72h and 92 h. Our results show significant changes in membrane fluidity among all cell types at different time points. GP values tend to increase significantly within 92 h in hN2 cells and 72 h in NIH3T3 cells and only at 92 h in HEK293 cells. L6 showed a marked decrease in membrane fluidity at 72 h and starts to increase at 92 h. As expected, NIH3T3 cells have more rigid membrane at earlier time points. On the other hand, neurons tend to have the highest membrane fluidity at early time points emphasizing its correlation with plasticity and the need for this malleability during differentiation. This study sheds light on the involvement of membrane fluidity during neuronal differentiation and development of other cell lines.

Here we describe a spontaneous mutation in the Zdhhc13 (zinc finger, DHHC domain containing 13) gene (also called Hip14l), one of 24 genes encoding palmitoyl acyltransferase (PAT) enzymes in the mouse. This mutation (Zdhhc13luc) was identified as a nonsense base substitution, which results in a premature stop codon that generates a truncated form of the ZDHHC13 protein, representing a potential loss-of-function allele. Homozygous Zdhhc13luc/Zdhhc13luc mice developed generalized hypotrichosis, associated with abnormal hair cycle, epidermal and sebaceous gland hyperplasia, hyperkeratosis, and increased epidermal thickness. Increased keratinocyte proliferation and accelerated transit from basal to more differentiated layers were observed in mutant compared with wild-type (WT) epidermis in untreated skin and after short-term 12-O-tetradecanoyl-phorbol-13-acetate treatment and acute UVB exposure. Interestingly, this epidermal phenotype was associated with constitutive activation of NF-κB (RelA) and increased neutrophil recruitment and elastase activity. Furthermore, tumor multiplicity and malignant progression of papillomas after chemical skin carcinogenesis were significantly higher in mutant mice than WT littermates. To our knowledge, this is the first report of a protective role for PAT in skin carcinogenesis.

BackgroundClinical therapy for metastatic colorectal cancer (CRC) remains limited, especially when the tumor harbors a KRAS mutation. This study aimed to identify prognostic biomarkers in CRC that are accessible for therapeutic inhibition.MethodsConditioned media from human CRC epithelial cells and myofibroblasts were screened by cytokine arrays for tumorigenic factors. The protein and mRNA expressions of these factors were determined by immunohistochemistry and gene microarrays in human CRC tissues. Prognostic biomarkers were determined by correlation of mRNA expression to overall survival in stage IV CRC patients. Inhibition of CXCL1 was performed with specific neutralizing antibody and lentiviral shRNAs. Malignant growth was assessed by soft agar growth assays and xenograft tumor growth in immunocompromised mice.ResultsCXCL1 was highly secreted by KRAS mutant human CRC cells and myofibroblasts in a complementary adaptive response to serum deprivation. Elevated CXCL1 level promoted anchorage-independent growth of murine fibroblasts and human CRC cells. Inhibition of CXCL1 by neutralizing antibody and specific shRNAs decreased CRC tumor growth. Highly elevated CXCL1 expression significantly correlated with decreased overall survival in stage IV CRC patients (hazard ratio 0.28; 95% CI 0.11-0.72).ConclusionsHigh CXCL1 expression is a poor prognostic biomarker in metastatic CRC. CXCL1 inhibition suppressed tumorigenic growth of KRAS mutant CRC cells.

Tissue surface tension influences cell sorting and tissue fusion. Earlier mechanical studies suggest that multicellular spheroids actively reinforce their surface tension with applied force. Here we study this open question through high-throughput microfluidic micropipette aspiration measurements on cell spheroids to identify the role of force duration and spheroid deformability. In particular, we aspirate spheroid protrusions of mice fibroblast NIH3T3 and human embryonic HEK293T homogeneous cell spheroids into micron-sized capillaries for different pressures and monitor their viscoelastic creep behavior. We find that larger spheroid deformations lead to faster cellular retraction once the pressure is released, regardless of the applied force. Additionally, less deformable NIH3T3 cell spheroids with an increased expression level of alpha-smooth muscle actin, a cytoskeletal protein upregulating cellular contractility, also demonstrate slower cellular retraction after pressure release for smaller spheroid deformations. Moreover, HEK293T cell spheroids only display cellular retraction at larger pressures with larger spheroid deformations, despite an additional increase in viscosity at these larger pressures. These new insights demonstrate that spheroid viscoelasticity is deformation-dependent and challenge whether surface tension truly reinforces at larger aspiration pressures., (© 2024. The Author(s).)

Wound healing represents a complex biological process crucial for tissue repair and regeneration. In recent years, biomaterial-based scaffolds loaded with bioactive compounds have emerged as promising therapeutic strategies to accelerate wound healing. In this study, we investigated the properties and wound healing effects of cryogels loaded with calcium peroxide (CP) and berberine (BB). The cryogels were synthesized through a cryogenic freezing technique and displayed pore diameters of 83 ± 39 μm, with porosity exceeding 90%. Following 20 days of degradation, the percentage of remaining weight for GPC and GPC-CP-BB cryogels was determined to be 12.42 ± 2.45% and 10.78 ± 2.08%, respectively. Moreover, the swelling ratios after 3 minutes for GPC and GPC-CP-BB were found to be 22.10 ± 0.05 and 21.00 ± 0.07, respectively. In vitro investigations demonstrated the cytocompatibility of the cryogels, with sufficient adhesion and proliferation of fibroblast (NIH-3T3) cells observed on the scaffolds, along with their hemocompatibility. Furthermore, the cryogels exhibited sustained release kinetics of both calcium peroxide and berberine, ensuring prolonged therapeutic effects at the wound site. In vivo assessment using a rat model of full-thickness skin wounds demonstrated accelerated wound closure rates in animals treated with the GPC-CP-BB scaffold compared to controls. Histological analysis revealed enhanced granulation tissue formation, re-epithelialization, and collagen deposition in the GPC-CP-BB group. Overall, our findings suggest that the scaffold loaded with CP and BB holds great promise as a therapeutic approach for promoting wound healing. Its multifaceted properties offer a multifunctional platform for localized delivery of therapeutic agents while providing mechanical support and maintaining a favorable microenvironment for tissue regeneration.

Introduction: This study presents a Mammalian Linear Expression System (MLES), a linear covalently closed (LCC) vector based on pVAX-1. The purpose of this system was to improve gene expression in mammalian cells and to test the efficacy of MLES in transient transfection and transgene expression using in vitro and in vivo models. Additionally, we aimed to evaluate potential inflammatory responses in vivo., Materials and Methods: MLES was developed by modifying pVAX-1, and the construct was confirmed by gel electrophoresis. Lipofectamine ® 2000 was used to assess the transfection efficiency and expression of MLES in various cell lines. In vivo studies were conducted in mice injected with MLES/EGFP, and the resulting transfection efficiency, gene expression, and inflammatory responses were analyzed., Results: MLES exhibited higher transfection efficiency and expression levels compared to pVAX-1 when tested on HEK-293, CHO-K1, and NIH-3T3 cells. When tested in vivo, MLES/EGFP showed elevated expression in the heart, kidney, liver, and spleen compared with pVAX-1/EGFP. Minimal changes are observed in the lungs. Additionally, MLES induced a reduced inflammatory response in mice compared with pVAX-1/EGFP., Conclusions: MLES offer improved transfection efficiency and reduced inflammation, representing a significant advancement in gene therapy and recombinant protein production. Further research on MLES-mediated gene expression and immune modulation will enhance gene therapy strategies., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Polymer adhesives play an important role in many medical, consumer, and industrial products. Polymers of α-lipoic acid (αLA) have the potential to fulfill the need for versatile and environmentally friendly adhesives, but their performance is plagued by spontaneous depolymerization. We report a family of stabilized αLA polymer adhesives that can be tailored for a variety of medical or nonmedical uses and sustainably sourced and recycled in a closed-loop manner. Minor changes in monomer composition afforded a pressure-sensitive adhesive that functions well in dry and wet conditions, as well as a structural adhesive with strength equivalent to that of conventional epoxies. αLA surgical superglue successfully sealed murine amniotic sac ruptures, increasing fetal survival from 0 to 100%.

Porous carbon materials from food waste have gained growing interest worldwide for multiple applications due to their natural abundance and the sustainability of the raw materials and the cost-effective synthetic processing. Herein, orange waste-derived porous carbon (OWPC) was developed through a freeze-drying method to prevent the demolition of the original biomass structure and then was pyrolyzed to create a large number of micro, meso and macro pores. The novelty of this work lies in the fact of using the macro-channels of the orange waste in order to create a macroporous network via the freeze-drying method which remains after the pyrolysis steps and creates space for the development of different types of porous in the micro and meso scale in a controlled way. The results showed the successful preparation of a porous carbon material with a high specific surface area of 644 m 2 g -1 without any physical or chemical activation. The material's cytocompatibility was also investigated against a fibroblast cell line (NIH/3T3 cells). OWPC triggered a mild intracellular reactive oxygen species production without initiating apoptosis or severely affecting cell proliferation and survival. The combination of their physicochemical characteristics and high cytocompatibility renders them promising materials for further use in biomedical and pharmaceutical applications.

Renal fibrosis is a representative pathological feature of various chronic kidney diseases, and efficient treatment is needed. Interstitial myofibroblasts are a key driver of kidney fibrosis, which is dependent on the binding of TGF-β1 to type I TGF-β receptor (TβRI) and TGF-β1-related signaling pathways. Therefore, attenuating TGF-β1 activity by competing with TGF-β1 in myofibroblasts is an ideal strategy for treating kidney fibrosis. Recently, a novel TβRI-mimicking peptide RIPΔ demonstrated a high affinity for TGF-β1. Thus, it could be speculated that RIPΔ may be used for anti-fibrosis therapy. Platelet-derived growth factor β receptor (PDGFβR) is highly expressed in fibrotic kidney. In this study, we found that target peptide Z-RIPΔ, which is RIPΔ modified with PDGFβR-specific affibody Z PDGFβR , was specifically and highly taken up by TGF-β1-activated NIH3T3 fibroblasts. Moreover, Z-RIPΔ effectively inhibited the myofibroblast proliferation, migration and fibrosis response in vitro. In vivo and ex vivo experiments showed that Z-RIPΔ specifically targeted fibrotic kidney, improved the damaged renal function, and ameliorated kidney histopathology and renal fibrosis in UUO mice. Mechanistic studies showed that Z-RIPΔ hold the stronger inhibition of the TGF-β1/Smad and TGF-β1/p38 pathways than unmodified RIPΔ in vitro and in vivo. Furthermore, systemic administration of Z-RIPΔ to UUO mice led to minimal toxicity to major organs. Taken together, RIPΔ modified with Z PDGFβR increased its therapeutic efficacy and reduced its systemic toxicity, making it a potential candidate for targeted therapy for kidney fibrosis., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Prolonged or excessive oxidative stress can lead to premature cellular and body aging. Mannan-binding lectin (MBL) is synthesized by the liver and plays an important role in innate immunity, anti-inflammation, and anti-oxidation, and has a positive impact on health and longevity. To date, few studies investigated the role of MBL in attenuating oxidative stress-induced senescence. In this study, we evaluated the role of MBL in oxidative stress-induced premature aging and explored its underlying mechanism in C57BL/6 mice and mouse embryonic fibroblasts (NIH/3T3). First, we established an oxidative premature senescence model induced by D-galactose in C57BL/6 mice. We found that MBL-deficient mice had a marked aging-like appearance, reduced learning and spatial exploration abilities, severe liver pathological damage, and significantly upregulated expression of Senescence-associated proteins (p53 and p21), inflammatory kinesins (IL-1β and IL-6), and the senescence β-galactosidase (SA-β-Gal) positive rate as compared with WT mice. In the H 2 O 2 -induced oxidative senescence model of NIH/3T3 cells, consistent results were obtained after MBL intervention. In addition, MBL effectively inhibited G1 phase arrest, ROS levels, DNA damage, and mitochondrial dysfunction in premature senescent cells. Mechanistically, we found that oxidative stress inhibited the nicotinamide adenine dinucleotide (NAD+)/ silent information regulator 1 (Sirt1) signaling pathway, while MBL activated the NAD+/Sirt1 signaling pathway inhibited by oxidative stress. In addition, MBL could activate the NAD+/Sirt1 pathway by upregulating NAMPT, which in turn inhibited p38 phosphorylation by activating the NAD+/Sirt1 pathway. In conclusion, MBL inhibits oxidative aging, which may facilitate the development of therapeutics to delay oxidative aging., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Zn-containing TiO 2 -based coatings with Na, Ca, Si, and K additives were obtained by plasma electrolytic oxidation (PEO) of Ti in order to achieve an effective and broad bactericidal protection without compromising biocompatibility. A protocol has been developed for cleaning the coating surface from electrolyte residues, ensuring the preservation of the microstructure and composition of the surface layer. Using high-resolution transmission electron microscopy, three characteristic microstructural zones in the PEO-Zn coating are well documented: zone 1 with a TiO 2 -based nanocrystalline structure, zone 2 with an amorphous structure, and zone 3 around pores with an amorphous-nanocrystalline structure. The excellent cytocompatibility of PEO-Zn samples was confirmed by three different methods: monitoring the proliferation of MC3T3-E1 cells, assessing the viability of sheep osteoblast cells using calcein-AM staining and fluorescence microscopy, and incubation with spheroids based on primary osteoblast cells and mouse embryonic fibroblast NIH3T3 cells. The PEO-Zn coatings absorb >60% of the incident light over the UV and Vis-NIR spectral ranges. After 24 h, the PEO-Zn coatings completely inactivate four types of strains: Gram-positive Staphylococcus aureus CSA154 and ATCC29213 and Gram-negative Escherichia coli K261 and U20, and also prevent E. coli U20 and K261 biofilm formation. The superior antibacterial activity is associated with the synergistic effect of Zn 2+ ions in safe concentration and reactive oxygen species (ROS) generated in response to either UV irradiation or soft short-term X-ray irradiation. The X-ray irradiation-induced ROS formation by a PEO coating is reported for the first time. The enhanced bactericidal activity after X-ray irradiation compared to UV illumination is attributed to the more intense ROS generation in the first few hours. The results obtained significantly expand the possibilities of using PEO coatings on the surfaces of titanium implants.

Efforts in researching the efficient anti-tumor properties of three novel arene ruthenium(II) complexes incorporating thiophene-based aroylhydrazone ligands have been undertaken. The complexes' elemental composition was [( η 6 - p -cymene)Ru(L)Cl]. They were comprehensively characterized through elemental and spectroscopic analyses (FT-IR, UV-vis, NMR, and HR-MS). Single crystal X-ray diffraction studies revealed a pseudo-octahedral geometry with bidentate coordination of the ligands in a representative complex. The in vitro assessment of the complexes' cancer cell growth inhibition was conducted using the MTT assay against A549 (human lung carcinoma), HeLa (human cervical carcinoma), HuH-7 (hepatocellular carcinoma), and NIH-3T3 (mouse fibroblast non-cancerous cell line). Results indicated significant cytotoxicity across all cancer cell lines, with IC 50 concentrations of complex 2 being 6.8 μM for A549, 11.6 μM for HeLa, and 9.4 μM for HuH-7, compared to cisplatin with IC 50 values of 18.9 μM, 17.68 μM, and 24 μM respectively. Notably, complex 2 demonstrated particularly promising cytotoxicity against all tested cancerous cell lines. Fluorescent staining analysis such as acridine orange/ethidium bromide (AO-EB) and HOECHST 33342 revealed cell death mechanisms involving membrane disintegration and nuclear condensation following treatment with complex 2. Further studies were conducted to measure reactive oxygen species (ROS) levels using the dichlorodihydrofluorescein diacetate (DCFH-DA) assay, and mitochondrial membrane potential (MMP) was assessed using the JC-1 dye assay. These studies demonstrated that complex 2 increased ROS levels, decreased membrane potential, and promoted mitochondrial dysfunction-mediated cell death pathways. Additionally, flow cytometry analysis, utilizing dual staining of Annexin V-FITC and propidium iodide (PI), was employed to quantitatively study apoptosis induction.

Fibrosis is a complex pathological process that can lead to the permanent loss of biological function, with P2ry2 playing a crucial role in this process. Long non-coding RNAs (lncRNAs) have been reported to play an critically important role in the fibrotic process. However, it remains unclear whether lncRNAs can regulate fibrosis through P2ry2. In this study, we detected the expression of the long non-coding RNA metastasis-associated lung adenocarcinoma transcript 1 (lnc-MALAT1). We investigated the expression patterns of lnc-MALAT1 and P2ry2 in denervated skeletal muscle, a classical model of fibrosis. Additionally, we utilized a TGF-β-mediated fibrosis model in NIH/3T3 cells to examine the effects of lnc-MALAT1 and P2ry2 on fibroblast activation and the underlying regulatory mechanisms in vitro. Our results demonstrated that the expression levels of lnc-MALAT1 and P2ry2 were consistently elevated in denervated skeletal muscle, correlating with the degree of fibrosis. In vitro experiments confirmed the regulatory effect of lnc-MALAT1 on P2ry2. Furthermore, we identified miR-335-3p as a potential key molecule in the regulatory relationship of lnc-MALAT1/P2ry2. Dual luciferase reporter assays and AGO2-RIP verified the molecular sponging effect of lnc-MALAT1 on miR-335-3p. Additionally, we validated the regulation of the lnc-MALAT1/miR-335-3p/P2ry2 axis through experimental approaches. In conclusion, our study identified a crucial role of lnc-MALAT1/miR-335-3p/P2ry2 axis in fibroblast activation, providing a promising treatment option against the fibrosis., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Chen et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Transformation of a fibrous mat into a three-dimensional (3D) scaffold opens up abundant innovative prospects in biomedical research, particularly for studying both soft as well as hard tissues. Electrospun nanofibers, which mimic the extracellular matrix have attracted significant attention in various studies. This research focuses on rapidly converting a fibrous mat made of polycaprolactone (PCL)/pluronic F-127 (PF-127) with different percentages of monetite calcium phosphate (MCP) into desirable 3D matrix cotton using a unique gas foaming technology. These matrix cottons possess biomimetic properties and have oriented porous structures. Using this innovative technique, various shapes of 3D matrix cotton, such as squares, hollow tubes, and other customizable forms, were successfully produced. Importantly, these 3D matrix cottons showed a consistent distribution of monetite particles with total porosity ranging from 90% to 98%. The structure of the 3D matrix cotton, its water/blood absorption capacity, the potential for causing non-hemolysis, and rapid hemostatic properties were thoroughly investigated. Additionally, periodontal cells were cultured on the 3D matrix cotton to assess their viability and morphology, revealing promising results. Furthermore, a coculture study involving NIH-3T3 and MG-63 cells on the 3D matrix cotton showed spheroidal formation within 24 h. Notably, in vitro assessments indicated that the matrix cotton containing 15% monetite (PCL-MMC15%) exhibited superior absorbent capabilities, excellent cell viability, and rapid hemostatic characteristics. Subsequently, the effectiveness of PCL-MMC15% in promoting mandibular bone regeneration was evaluated through an in vivo study on rabbits using a mandibular injury model. The results demonstrated that PCL-MMC15% facilitated the resolution of defects in the mandibular region by initiating new bone formation. Therefore, the presented 3D matrix cotton (PCL-MMC15%) shows significant promise for applications in both mandibular bone regeneration and hemostasis.

The viscosity that ensures the controlled diffusion of biomolecules in cells is a crucial biophysical parameter. Consequently, fluorescent probes capable of reporting viscosity variations are valuable tools in bioimaging. In this field, red-shifted probes are essential, as the widely used and gold standard probe remains green-emitting molecular rotors based on BODIPY. Here, we demonstrate that pyrrolyl squaraines, red-emissive fluorophores, exhibit high sensitivity over a wide viscosity range from 30 to 4890 mPa·s. Upon alkylation of the pyrrole moieties, the probes improve their sensitivity to viscosity through an enhanced twisted intramolecular charge transfer phenomenon. We utilized this scaffold to develop a plasma membrane probe, pSQ-PM, that efficiently stains the plasma membrane in a fluorogenic manner. Using fluorescence lifetime imaging, pSQ-PM enabled efficient sensing of viscosity variations in the plasma membrane under various conditions and in different cell lines (HeLa, U2OS, and NIH/3T3). Moreover, upon incubation, pSQ-PM stained the membrane of intracellular vesicles and suggested that the lysosomal membranes displayed enhanced fluidity.

Colorectal cancer (CRC) is the third most common cancer in the world. Despite considerable improvements in the treatment of this cancer, further research to discover novel and more effective agents is ongoing. In this study, possible cytotoxic and apoptotic properties of six benzothiazolopyrimidine derivatives were studied. To assess the IC 50 values of these agents, MTT assay was performed on HCT 116, CT26, and NIH/3T3 cells. Moreover, cell death mechanism induced by studied compounds was evaluated by PI/annexin V staining. Then, based on molecular docking results and in vitro experiments, the compounds with the highest anticancer properties were further analyzed in vivo in a mouse model of CRC. MTT results indicated that BTP(1) and BTP(4) had the highest selective cytotoxicity on colorectal cancer cells. Furthermore, flow cytometry results demonstrated a considerable increase in the percentage of the early apoptotic cells in BTP(1)- and BTP(4)-treated groups. In vivo studies confirmed the antitumor properties of the two compounds by a significant regression in tumor size of BTP(1)- and BTP(4)-treated mice compared to control groups. Histopathological examination of tumor tissues showed an increased number of apoptotic cells in these two groups compared to the control animals. Additionally, hematoxylin and eosin staining of the spleen and liver of treated mice did not exhibit considerable tissue damage. Thus, BTP(1) and BTP(4) can be considered promising agents in the treatment of colorectal cancer, although further experiments are required to assess their mechanism of action before their application in clinical studies., (© 2024 Wiley Periodicals LLC.)

This study investigated the fabrication of gallic acid-loaded chitosan nanoparticles (Gal-Chi-NPs) that enhanced the DNA damage and apoptotic features by inhibiting FEN-1 expressions in MDA-MB 231 cells. Gal-Chi-NPs were fabricated by the ionic gelation method, and it was characterized by several studies such as dynamic light spectroscopy, Fourier-transforms infrared spectroscopy, x-ray diffraction, scanning electron microscopy, energy-dispersive x-ray, atomic force microscopy, and thermogravimetric analysis. We have obtained that Gal-Chi-NPs displayed 182.2 nm with crystal, smooth surface, and heat stability in nature. Gal-Chi-NPs induce significant toxicity in MDA-MB-231 cells that compared with normal NIH-3T3 cells. A significant reactive oxygen species (ROS) overproduction was observed in Gal-Chi-NPs treated MDA-MB-231. Flap endonuclease-1 (FEN-1) is a crucial protein involved in long patch base excision repair that is involved in repairing the chemotherapeutic mediated DNA-damaged base. Therefore, inhibition of FEN-1 protein expression is a crucial target for enhancing chemotherapeutical efficacy. In this study, we have obtained that Gal-Chi-NPs treatment enhanced the DNA damage by observing increased p-H2AX, PARP1; and suppressed the expression of FEN-1 in MDA-MB-231 cells. Moreover, Gal-Chi-NPs inhibited the expression of tumor proliferating markers p-PI3K, AKT, cyclin-D1, PCNA, and BCL-2; induced proapoptotic proteins (Bax and caspase-3) in MDA-MB 231 cells. Thus, Gal-Chi-NPs induce DNA damage and apoptotic features and inhibit tumor proliferation by suppressing FEN-1 expression in triple-negative breast cancer cells., (© 2024 Wiley Periodicals LLC.)

The biologically produced gold nanoparticles (AuNPs) are novel carriers with promising use in targeted tumor therapy. Still, there are no studies regarding the efficacy of nanoparticle internalization by cancer and noncancer cells. In this study, AuNPs were produced by Fusarium oxysporum and analyzed by spectrophotometry, transmission electron microscopy (TEM), energy dispersive x-ray spectroscopy (EDS), and Zetasizer. Obtained AuNPs were about 15 nm in size with a zeta potential of -35.8 mV. The AuNPs were added to cancer cells (4T1), noncancer cells (NIH/3T3), and macrophages (RAW264.7). The viability decreased in 4T1 (77 ± 3.74%) in contrast to NIH/3T3 and RAW264.7 cells (89 ± 4.9% and 90 ± 3.5%, respectively). The 4T1 cancer cells also showed the highest uptake and accumulation of Au (∼80% of AuNPs was internalized) as determined by graphite furnace atomic absorption spectroscopy. The lowest amount of AuNPs was internalized by the NIH/3T3 cells (∼30%). The NIH/3T3 cells exhibited prominent reorganization of F-actin filaments as examined by confocal microscopy. In RAW264.7, we analyzed the release of proinflammatory cytokines by flow cytometry and we found the AuNP interaction triggered transient secretion of tumor necrosis factor alpha (TNF-α) and interferon gamma (IFN-γ). In summary, we proved the biologically produced AuNPs entered all the tested cell types and triggered cell-specific responses. High AuNP uptake by tumor cells was related to decreased cell viability, while low nanoparticle uptake by fibroblasts triggered F-actin reorganization without remarkable toxicity. Thus, the biologically produced AuNPs hold promising potential as cancer drug carriers and likely require proper surface functionalization to shield phagocytizing cells., (© 2024 The Authors. Biotechnology and Applied Biochemistry published by Wiley Periodicals LLC on behalf of International Union of Biochemistry and Molecular Biology.)

Objective: This study aimed to investigate the use of 5,7,3',4'-tetramethoxyflavone (TMF) to treat pulmonary fibrosis (PF), a chronic and fatal lung disease. In vitro and in vivo models were used to examine the impact of TMF on PF., Methods: NIH-3T3 (Mouse Embryonic Fibroblast) were exposed to transforming growth factor‑β1 (TGF-β1) and treated with or without TMF. Cell growth was assessed using the MTT method, and cell migration was evaluated with the scratch wound assay. Protein and messenger ribonucleic acid (mRNA) levels of extracellular matrix (ECM) genes were analyzed by western blotting and quantitative reverse transcription-polymerase chain reaction (RT-PCR), respectively. Downstream molecules affected by TGF-β1 were examined by western blotting. In vivo , mice with bleomycin-induced PF were treated with TMF, and lung tissues were analyzed with staining techniques., Results: The in vitro results showed that TMF had no significant impact on cell growth or migration. However, it effectively inhibited myofibroblast activation and ECM production induced by TGF-β1 in NIH-3T3 cells. This inhibition was achieved by suppressing various signaling pathways, including Smad, mitogen-activated protein kinase (MAPK), phosphoinositide 3-kinase/AKT (PI3K/AKT), and WNT/β-catenin. The in vivo experiments demonstrated the therapeutic potential of TMF in reducing PF induced by bleomycin in mice, and there was no significant liver or kidney toxicity observed., Conclusion: These findings suggest that TMF has the potential to effectively inhibit myofibroblast activation and could be a promising treatment for PF. TMF achieves this inhibitory effect by targeting TGF-β1/Smad and non-Smad pathways.

This work reports the production of biocompatible thin layers for biomedical applications based on a graphene-like material (GL), a graphene-related material (GRM) obtained from carbon black. GL was combined in a hybrid fashion with polydopamine (pDA), a mussel-inspired water-resistant wet adhesive bonding obtained by the oxidative polymerization of dopamine (DA), and polyvinyl pyrrolidinone (PVP), a nontoxic synthetic polymer with intrinsic adhesion properties, to obtain a tighter adhesion of the thin layer to the substrate (silicone slices). Matrix-assisted pulsed laser evaporation (MAPLE) was used to coat PDMS slices with thin films of GL-pDA and GL-PVP directly from their frozen suspensions in water. The results indicate that the relevant chemical-physical characteristics of both thin films (evidenced by FTIR and AFM) were maintained after MAPLE deposition and that the films exhibit uniformity also at the nanometric level. After deposition, the GL-pDA and GL-PVP films underwent a biological survey toward murine fibroblasts (NIH3T3), human keratinocytes (HaCAT), and human cervical adenocarcinoma epithelial-like (HeLa) cells to assess the feasibility of this approach. Results indicate that both the GL-pDA and GL-PVP films did not perturb the biological parameters evaluated, including cytoskeleton alterations. Both hybrid films enhanced the effects of GL on cellular vitality across all cell lines. Specifically, the GL-pDA film exhibited a more stable effect over time (up to 72 h), whereas the GL-PVP film behaved similarly to the GL film in NIH3T3 and HeLa cell lines after long-term exposure. These promising results make the GL-pDA and GL-PVP films potential candidates for the manufacture of coated flexible devices for biomedical applications.

Background: Aldehyde dehydrogenase 2 (ALDH2) is critical for alcohol metabolism by converting acetaldehyde to acetic acid. In East Asian descendants, an inactive genetic variant in ALDH2, rs671, triggers an alcohol flushing response due to acetaldehyde accumulation. As alcohol flushing is not exclusive to those of East Asian descent, we questioned whether additional ALDH2 genetic variants can drive facial flushing and inefficient acetaldehyde metabolism using human testing and biochemical assays., Methods: After IRB approval, human subjects were given an alcohol challenge (0.25 g/kg) while quantifying acetaldehyde levels and the physiological response (heart rate and skin temperature) to alcohol. Further, by employing biochemical techniques including human purified ALDH2 proteins and transiently transfected NIH 3T3 cells, we characterized two newly identified ALDH2 variants for ALDH2 enzymatic activity, ALDH2 dimer/tetramer formation, and reactive oxygen species production after alcohol treatment., Results: Humans heterozygous for rs747096195 (R101G) or rs190764869 (R114W) had facial flushing and a 2-fold increase in acetaldehyde levels, while rs671 (E504K) had facial flushing and a 6-fold increase in acetaldehyde levels relative to wild type ALDH2 carriers. In vitro studies with recombinant R101G and R114W ALDH2 enzyme showed a reduced efficiency in acetaldehyde metabolism that is unique when compared to E504K or wild-type ALDH2. The effect is caused by a lack of functional dimer/tetramer formation for R101G and decreased V max for both R101G and R114W. Transiently transfected NIH-3T3 cells with R101G and R114W also had a reduced enzymatic activity by ~ 50% relative to transfected wild-type ALDH2 and when subjected to alcohol, the R101G and R114W variants had a 2-3-fold increase in reactive oxygen species formation with respect to wild type ALDH2., Conclusions: We identified two additional ALDH2 variants in humans causing facial flushing and acetaldehyde accumulation after alcohol consumption. As alcohol use is associated with a several-fold higher risk for esophageal cancer for the E504K variant, the methodology developed here to characterize ALDH2 genetic variant response to alcohol can lead the way precision medicine strategies to further understand the interplay of alcohol consumption, ALDH2 genetics, and cancer., (© 2024. The Author(s).)

Background: Silicosis is an irreversible fibrotic disease of the lung caused by chronic exposure to silica dust, which manifests as infiltration of inflammatory cells, excessive secretion of pro-inflammatory cytokines, and pulmonary diffuse fibrosis. As the disease progresses, lung function further deteriorates, leading to poorer quality of life of patients. Currently, few effective drugs are available for the treatment of silicosis. Bicyclol (BIC) is a compound widely employed to treat chronic viral hepatitis and drug-induced liver injury. While recent studies have demonstrated anti-fibrosis effects of BIC on multiple organs, including liver, lung, and kidney, its therapeutic benefit against silicosis remains unclear. In this study, we established a rat model of silicosis, with the aim of evaluating the potential therapeutic effects of BIC., Methods: We constructed a silicotic rat model and administered BIC after injury. The FlexiVent instrument with a forced oscillation system was used to detect the pulmonary function of rats. HE and Masson staining were used to assess the effect of BIC on silica-induced rats. Macrophages-inflammatory model of RAW264.7 cells, fibroblast-myofibroblast transition (FMT) model of NIH-3T3 cells, and epithelial-mesenchymal transition (EMT) model of TC-1 cells were established in vitro. And the levels of inflammatory mediators and fibrosis-related proteins were evaluated in vivo and in vitro after BIC treatment by Western Blot analysis, RT-PCR, ELISA, and flow cytometry experiments., Results: BIC significantly improved static compliance of lung and expiratory and inspiratory capacity of silica-induced rats. Moreover, BIC reduced number of inflammatory cells and cytokines as well as collagen deposition in lungs, leading to delayed fibrosis progression in the silicosis rat model. Further exploration of the underlying molecular mechanisms revealed that BIC suppressed the activation, polarization, and apoptosis of RAW264.7 macrophages induced by SiO 2 . Additionally, BIC inhibited SiO 2 -mediated secretion of the inflammatory cytokines IL-1β, IL-6, TNF-α, and TGF-β1 in macrophages. BIC inhibited FMT of NIH-3T3 as well as EMT of TC-1 in the in vitro silicosis model, resulting in reduced proliferation and migration capability of NIH-3T3 cells. Further investigation of the cytokines secreted by macrophages revealed suppression of both FMT and EMT by BIC through targeting of TGF-β1. Notably, BIC blocked the activation of JAK2/STAT3 in NIH-3T3 cells required for FMT while preventing both phosphorylation and nuclear translocation of SMAD2/3 in TC-1 cells necessary for the EMT process., Conclusion: The collective data suggest that BIC prevents both FMT and EMT processes, in turn, reducing aberrant collagen deposition. Our findings demonstrate for the first time that BIC ameliorates inflammatory cytokine secretion, in particular, TGF-β1, and consequently inhibits FMT and EMT via TGF-β1 canonical and non-canonical pathways, ultimately resulting in reduction of aberrant collagen deposition and slower progression of silicosis, supporting its potential as a novel therapeutic agent., (© 2024. The Author(s).)

Background: Idiopathic pulmonary fibrosis (IPF) is a debilitating interstitial lung disorder characterized by its limited therapeutic interventions. Macrophages, particularly the alternatively activated macrophages (M2 subtype), have been acknowledged for their substantial involvement in the development of pulmonary fibrosis. Hence, targeting macrophages emerges as a plausible therapeutic avenue for IPF. Icariside II (ISE II) is a natural flavonoid glycoside molecule known for its excellent anti-tumor and anti-fibrotic activities. Nevertheless, the impact of ISE II on pulmonary fibrosis and the intricate mechanisms through which it operates have yet to be elucidated., Objective: To scrutinize the impact of ISE II on the regulation of M2 macrophage polarization and its inhibitory effect on pulmonary fibrosis, as well as to delve deeper into the underlying mechanisms of its actions., Methods: The effect of ISE II on proliferation and apoptosis in RAW264.7 cells was assessed through the use of EdU-488 labeling and the Annexin V/PI assay. Flow cytometry, western blot, and qPCR were employed to detect markers associated with the M2 polarization phenotype. The anti-fibrotic effects of ISE II in NIH-3T3 cells were investigated in a co-culture with M2 macrophages. Si-Ctnnb1 and pcDNA3.1(+)-Ctnnb1 plasmid were used to investigate the mechanism of targeted intervention. The murine model of pulmonary fibrosis was induced by intratracheal administration of bleomycin (BLM). Pulmonary function, histopathological manifestations, lung M2 macrophage infiltration, and markers associated with pulmonary fibrosis were evaluated. Furthermore, in vivo transcriptomics analysis was employed to elucidate differentially regulated genes in lung tissues. Immunofluorescence, western blot, and immunohistochemistry were conducted for corresponding validation., Results: Our investigation demonstrated that ISE II effectively inhibited the proliferation of RAW264.7 cells and mitigated the pro-fibrotic characteristics of M2 macrophages, exemplified by the downregulation of CD206, Arg-1, and YM-1, Fizz1, through the inhibition of the PI3K/Akt/β-catenin signaling pathway. This impact led to the amelioration of myofibroblast activation and the suppression of nuclear translocation of β-catenin of NIH-3T3 cells in a co-culture. Consequently, it resulted in decreased collagen deposition, reduced infiltration of profibrotic macrophages, and a concurrent restoration of pulmonary function in mice IPF models. Furthermore, our RNA sequencing results showed that ISE II could suppress the expression of genes related to M2 polarization, primarily by inhibiting the PI3K/Akt and β-catenin signaling pathway. In essence, our findings suggest that ISE II holds potential as an anti-fibrotic agent by orchestrating macrophage polarization. This may have significant implications in clinical practice., Conclusion: This study has provided evidence that ISE II exerts a significant anti-fibrotic effect by inhibiting macrophage M2 polarization through the suppression of the PI3K/Akt/β-catenin signaling pathway. These findings underscore the potential of ISE II as a promising candidate for the development of anti-fibrotic pharmaceuticals in the future., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier GmbH. All rights reserved.)

In situ-forming biocompatible hydrogels have great potential in various medical applications. Here, we introduce a pH-responsive, self-healable, and biocompatible hydrogel for cell scaffolds and the development of a tumor spheroid phantom for magnetic resonance imaging. The hydrogel (pMAD) was synthesized via amino-yne click chemistry between poly(2-methacryloyloxyethyl phosphorylcholine- co -2-aminoethylmethacrylamide) and dialkyne polyethylene glycol. Rheology analysis, compressive mechanical testing, and gravimetric analysis were employed to investigate the gelation time, mechanical properties, equilibrium swelling, and degradability of pMAD hydrogels. The reversible enamine and imine bond mechanisms leading to the sol-to-gel transition in acidic conditions (pH ≤ 5) were observed. The pMAD hydrogel demonstrated potential as a cellular scaffold, exhibiting high viability and NIH-3T3 fibroblast cell encapsulation under mild conditions (37 °C, pH 7.4). Additionally, the pMAD hydrogel also demonstrated the capability for in vitro magnetic resonance imaging of glioblastoma tumor spheroids based on the chemical exchange saturation transfer effect. Given its advantages, the pMAD hydrogel emerges as a promising material for diverse biomedical applications, including cell carriers, bioimaging, and therapeutic agent delivery.

The design of adhesive and conductive soft hydrogels using biopolymers with tunable mechanical properties has received significant interest in the field of wearable sensors for detecting human motions. These hydrogels are primarily fabricated through the modification of biopolymers to introduce cross-linking sites, the conjugation of adhesive components, and the incorporation of conductive materials into the hydrogel network. The development of a multifunctional copolymer that integrates adhesive and conductive properties within a single polymer chain with suitable cross-linking sites eliminates the need for biopolymer modification and the addition of extra conductive and adhesive components. In this study, we synthesized a copolymer based on poly([2-(methacryloyloxy)ethyl] trimethylammonium chloride- co -dopamine methacrylamide) (p(METAC-DMA)) using a controlled radical polymerization, allowing for the efficient conjugation of both adhesive and conductive units within a single polymer chain. Subsequently, our multifunctional hydrogel named Gel-MD was fabricated by mixing the p(METAC-DMA) copolymer with non-modified gelatin in which cross-linking took place in an oxidative environment. We confirmed the biocompatibility of the Gel-MD hydrogel through in vitro studies using NIH 3T3 cells as well as in vivo subcutaneous implantation in rats. Furthermore, the Gel-MD hydrogel was effective and sensitive in detecting various human motions, making it a promising wearable sensor for health monitoring and diagnosis.

Epidermal growth factor (EGF) protein is a crucial biomolecule involved in regulating cell growth, proliferation, migration and differentiation, which is used in various therapeutic applications, such as wound healing and tissue regeneration. The production of recombinant EGF is essential for studying its biological function and for its clinical translation. However, EGF protein expressed in prokaryotic cells often occurs in inclusion bodies, and co-expression with soluble tag protein is an effective method to prepare recombinant EGF. In this study, we expressed recombinant human EGF (rhEGF) fused to a HaloTag (Halo-rhEGF) and a large portion of Halo-rhEGF was found in the soluble fraction. Cell growth assay showed that the purified Halo-rhEGF protein could promote the proliferation of fibroblasts (NIH 3T3) and epithelial cells (HaCaT), and significantly increased their viability. Phosphorylation of the intracellular signaling proteins, ERK1/2 and c-Jun, was stimulated by treatment with Halo-rhEGF and the expression levels of proteins regulating cell proliferation were significantly increased. RNA sequencing analysis revealed that rhEGF could increase the transcription of genes enriched in ribosome generation and cell proliferation. Moreover, Halo-rhEGF can be labelled by HaloTag ligand for fluorescence imaging and can be slowly released in tissue repair by binding to anion biomaterials. In conclusion, HaloTag is an efficient fusion tag for rhEGF protein expression, purification and controlled release, and Halo-rhEGF can promote the proliferation and viability of epithelial and fibroblast cells., Competing Interests: The authors declare that they have no competing interests. Yezhuo Liu is employed by Shanghai Hongdu New Material Science and Technology and Favorsun Medical Group. Changye Sun is the part-time technical director of Shanghai Hongdu New Material Science., (© 2024 Bai et al.)

Lunatic Fringe (LFNG) is required for spinal development. Biallelic pathogenic variants cause spondylocostal dysostosis type-III (SCD3), a rare disease generally characterized by malformed, asymmetrical, and attenuated development of the vertebral column and ribs. However, a variety of SCD3 cases reported have presented with additional features such as auditory alterations and digit abnormalities. There has yet to be a single, comprehensive, functional evaluation of causative LFNG variants and such analyses could unveil molecular mechanisms for phenotypic variability in SCD3. Therefore, nine LFNG missense variants associated with SCD3, c.564C>A, c.583T>C, c.842C>A, c.467T>G, c.856C>T, c.601G>A, c.446C>T, c.521G>A, and c.766G>A, were assessed in vitro for subcellular localization and protein processing. Glycosyltransferase activity was quantified for the first time in the c.583T>C, c.842C>A, and c.446C>T variants. Primarily, our results are the first to satisfy American College of Medical Genetics and Genomics PS3 criteria (functional evidence via well-established assay) for the pathogenicity of c.583T>C, c.842C>A, and c.446C>T, and replicate this evidence for the remaining six variants. Secondly, this work indicates that all variants that prevent Golgi localization also lead to impaired protein processing. It appears that the FRINGE domain is responsible for this phenomenon. Thirdly, our data suggests that variant proximity to the catalytic residue may influence whether LFNG is improperly trafficked and/or enzymatically dysfunctional. Finally, the phenotype of the axial skeleton, but not elsewhere, may be modulated in a variant-specific fashion. More reports are needed to continue testing this hypothesis. We anticipate our data will be used as a basis for discussion of genotype-phenotype correlations in SCD3., (© 2024 The Author(s). The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

Calcium Hydroxide-based endodontic sealer loaded with antimicrobial agents have been commonly employed in conventional root canal treatment. These sealers are not effective against E. faecalis due to the persistent nature of this bacterium and its ability to evade the antibacterial action of calcium hydroxide. Therefore, endodontic sealer containing Carbon nanodots stabilized silver nanoparticles (CD-AgNPs) was proposed to combat E. faecalis. The therapeutic effect of CD-AgNPs was investigated and a new cytocompatible Calcium Hydroxide-based endodontic sealer enriched with CD-AgNPs was synthesized that exhibited a steady release of Ag+ ions and lower water solubility at 24 hours, and enhanced antibacterial potential against E. faecalis. CD-AgNPs was synthesized and characterized morphologically and compositionally by Scanning Electron Microscopy, Fourier Transform Infrared Spectroscopy (FTIR), and UV-Vis Spectroscopy, followed by optimization via minimum inhibitory concentration (MIC) determination against E. faecalis by broth microdilution technique and Cytotoxicity analysis against NIH3T3 cell lines via Alamar Blue assay. Calcium hydroxide in distilled water was taken as control (C), Calcium hydroxide with to CD-AgNPs (5mg/ml and 10mg/ml) yielded novel endodontic sealers (E1 and E2). Morphological and chemical analysis of the novel sealers were done by SEM and FTIR; followed by in vitro assessment for antibacterial potential against E. faecalis via agar disc diffusion method, release of Ag+ ions for 21 days by Atomic Absorption Spectrophotometry and water solubility by weight change for 21 days. CD-AgNPs were 15-20 nm spherical-shaped particles in uniformly distributed clusters and revealed presence of constituent elements in nano-assembly. FTIR spectra revealed absorption peaks that correspond to various functional groups. UV-Vis absorption spectra showed prominent peaks that correspond to Carbon nanodots and Silver nanoparticles. CD-AgNPs exhibited MIC value of 5mg/ml and cytocompatibility of 84.47% with NIH3T3 cell lines. Novel endodontic sealer cut-discs revealed irregular, hexagonal particles (100-120 nm) with aggregation and rough structure with the presence of constituent elements. FTIR spectra of novel endodontic sealers revealed absorption peaks that correspond to various functional groups. Novel endodontic sealers exhibited enhanced antibacterial potential where E-2 showed greatest inhibition zone against E. faecalis (6.3±2 mm), a steady but highest release of Ag+ ions was exhibited by E-1 (0.043±0.0001 mg/mL) and showed water solubility of <3% at 24 hours where E-2 showed minimal weight loss at all time intervals. Novel endodontic sealers were cytocompatible and showed enhanced antibacterial potential against E. faecalis, however, E2 outperformed in this study in all aspects., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Ali et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The self-assembly of peptides and peptide analogues may be exploited to develop platforms for different biomedical applications, among which CEST-MRI (chemical exchange saturation transfer magnetic resonance imaging) represents one of the most attractive techniques to be explored as a novel metal-free contrast approach in imaging acquisitions. A lysine-containing peptide sequence (LIVAGK-NH 2 , named K2) was thus modified by insertion, at the N-terminus, of a peptide nucleic acid (PNA) base, leading to a primary amine suitable for the signal generation. a-K2, c-K2, g-K2 and t-K2 peptides were synthesized and characterized. The c-K2 sequence displayed gelling properties and the Watson and Crick pairing, arising from its combination with g-K2, allowed a significant increase in the mechanical responsivity of the hydrogel. These matrices were able to generate a CEST signal around 2.5 ppm from water and, after assessing their cytocompatibility on GL261 (murine glioma), TS/a (murine breast carcinoma), and 3T3-NIH (murine fibroblasts) cell lines, their capability to work as implants for in vivo detection, was proved by intratumor injection in Balb/c mice inoculated with TS/a murine breast cancer cells.

Objective: This study was aimed to develop a cationic lipoplex formulation loaded with Nintedanib and miR-29b (LP-NIN-miR) as an alternative approach in the combination therapy of idiopathic pulmonary dibrosis (IPF) by proving its additive anti-fibrotic therapeutic effects through in vitro lung fibrosis model., Significance: This is the first research article reported that the LP-NIN-MIR formulations in the treatment of IPF., Methods: To optimize cationic liposomes (LPs), quality by design (QbD) approach was carried out. Optimized blank LP formulation was prepared with DOTAP, CHOL, DOPE, and DSPE-mPEG 2000 at the molar ratio of 10:10:1:1. Nintedanib loaded LP (LPs-NIN) were produced by microfluidization method and were incubated with miR-29b at room temperature for 30 min to obtain LP-NIN-miR. To evaluate the cellular uptake of LP-NIN-miR, NIH/3T3 cells were treated with 20 ng.mL -1 transforming growth factor-β1 (TGF-β1) for 96 h to establish the in vitro IPF model and incubated with LP-NIN-miR for 48 h., Results: The hydrodynamic diameter, polydispersity index (PDI), and zeta potential of the LP-NIN-miR were 87.3 ± 0.9 nm, 0.184 ± 0.003, and +24 ± 1 mV, respectively. The encapsulation efficiencies of Nintedanib and miR-29b were 99.8% ± 0.08% and 99.7% ± 1.2%, respectively. The results of the cytotoxicity study conducted with NIH/3T3 cells indicated that LP-NIN-miR is a safe delivery system., Conclusions: The outcome of the transfection study proved the additive anti-fibrotic therapeutic effect of LP-NIN-miR and suggested that lipoplexes are effective delivery systems for drug and nucleic acid to the NIH/3T3 cells in the treatment of IPF.

Severe corneal cryoinjury can cause permanent corneal swelling and bullous keratopathy, one of the main reason for loss of sight. Mouse amniotic fluid mesenchymal stem cells (mAF-MSCs) can repair corneal damage caused by freezing; however, whether the exosomes derived from mAF-MSCs have the same repair effect is unknown. In this study, the mAF-MSC-exosomes were transplanted into the eyeballs of corneal cryoinjured mice. Histopathological examination showed that the mAF-MSC-exosomes improved the corneal structure and status of corneal epithelial cells in corneal cryoinjured mice. RRBS-sequencing showed that compared with the control group, four genes (Rpl13-ps6, miR-33, Hymai, and Plagl1), underwent DNA hypermethylation modification after mAF-MSC-exosomes treatment. The result of FISH indicated that miR-33-3p hybridization signals were enhanced in corneal epithelial cells from mice treated with mAF-MSC-exosomes. Semi-quantitative PCR and western blotting indicated that mAF-MSC-exosomes contained high levels of DNMT1 mRNA and protein. Additionally, luciferase report assays indicated that miR-33-3p overexpression in NIH-3T3 mouse embryonic fibroblast cells inhibited the activity of luciferase carrying a sequence from the 3' untranslated region of Bcl6. Moreover, BCL6 mRNA and protein levels in corneal tissues from mice treated with mAF-MSC-exosomes were higher than those in the control group. Therefore, our results suggested that mAF-MSC-exosomes could repair corneal cryoinjury by releasing DNMT1, which induced hypermethylation of the miR-33 promoter in corneal epithelial cells. Consequent downregulated miR-33 transcription upregulated Bcl6 expression, ultimately achieving the repair of corneal cryoinjury in mice., (© 2024. The Author(s) under exclusive licence to Japan Human Cell Society.)

Wound healing involves several cellular and molecular pathways. Tridax procumbens activates genetic pathways with antibacterial, antioxidant, anticancer, and anti-inflammatory properties, aiding wound healing. This study purified Procumbenase, a serine protease from T. procumbens extract, using gel filtration (Sephadex G-75) and ion exchange (CM-Sephadex C-50) chromatography. Characterization involved analyses of protease activity, RP-HPLC, SDS-PAGE, gelatin zymogram, PAS staining, mass spectrometry, and circular dichroism. Optimal pH and temperature were determined. Protease type was identified using inhibitors. Wound-healing potential was evaluated through tensile strength, wound models, hydroxyproline estimation, and NIH 3T3 cell scratch analysis. In incision wound rat models, Procumbenase increased tensile strength on day 14 more than saline and Povidone‑iodine. It increased wound contraction by 89 % after 10 days in excision wound models, attaining full contraction by day 15 and closure by day 21. Scarless wound healing was enhanced by 18 days of epithelialization against 22 and 21 days for saline and povidone‑iodine. Procumbenase increased hydroxyproline concentration 2.53-fold (59.93 ± 2.89 mg/g) compared to saline (23.67 ± 1.86 mg/g). In NIH 3 T3 cell scratch assay, Procumbenase increased migration by 60.93 % (50 μg) and 60.57 % (150 μg) after 48 h. Thus, Procumbenase is the primary bioactive molecule in T. procumbens, demonstrates scar-free wound healing properties., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Genetic changes in the ERBB family of receptor tyrosine kinases serve as oncogenic driver events and predictive biomarkers for ERBB inhibitor drugs. ERBB3 is a pseudokinase member of the family that, although lacking a fully active kinase domain, is well known for its potent signaling activity as a heterodimeric complex with ERBB2. Previous studies have identified few transforming ERBB3 mutations while the great majority of the hundreds of different somatic ERBB3 variants observed in different cancer types remain of unknown significance. Here, we describe an unbiased functional genetics screen of the transforming potential of thousands of ERBB3 mutations in parallel. The screen based on a previously described iSCREAM (in vitro screen of activating mutations) platform, and addressing ERBB3 pseudokinase signaling in a context of ERBB3/ERBB2 heterodimers, identified 18 hit mutations. Validation experiments in Ba/F3, NIH 3T3, and MCF10A cell backgrounds demonstrated the presence of both previously known and unknown transforming ERBB3 missense mutations functioning either as single variants or in cis as a pairwise combination. Drug sensitivity assays with trastuzumab, pertuzumab and neratinib indicated actionability of the transforming ERBB3 variants., (© 2024. The Author(s).)

Self-healing hydrogels possess an ability to recover their functionality after experiencing damage by regenerating cross-links. The main challenge in making self-healing hydrogels based on host-guest (HG) interactions is their limited mechanical strength, which can be solved using beta-cyclodextrin dimers (β-CDsD). Here, β-CDsD as a host cross-linker was used to increase the mechanical property of the HG interactions. Alginate with acceptable biocompatibility was modified by dopamine (ALG-DOP) and employed as a guest polymer. Self-healing hydrogel was developed between them, and Ag nanoparticles were added to create an antibacterial activity. Dopamine with appropriate size and suitable adhesiveness established HG interactions with β-CDsD, and cells were able to grow well on hydrogel. This hydrogel showed an impressive self-healing capability <5 min. These hydrogels revealed a respectable porosity from 15 to 55 μm essential for exchanging the substances required for cell growth and cell waste elimination. Biocompatibility was investigated against NIH 3 T3 fibroblasts cells, and the results showed that the cells grew well. The in vitro release of curcumin from the hydrogel was examined in PBS at pH of 7.4. The hydrogel can be a perfect candidate for controlled drug release, and wound-dressing due to self-healing property, antibacterial activity, adhesion, and biocompatibility., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Introduction: Nucleoporin 98 (NUP98) fusion proteins are recurrently found in leukemia and are associated with unfavorable clinical outcomes. They are distributed to the nucleus and contribute to leukemogenesis via aberrant transcriptional regulation. We previously identified NUP98-BPTF (NB) fusion in patients with T-cell acute lymphoblastic leukemia (T-ALL) using next-generation sequencing. The FG-repeat of NUP98 and the PHD finger and bromodomain of bromodomain PHD finger transcription factor (BPTF) are retained in the fusion. Like other NUP98 fusion proteins, NB is considered to regulate genes that are essential for leukemogenesis. However, its target genes or pathways remain unknown., Materials and Methods: To investigate the potential oncogenic properties of the NB fusion protein, we lentivirally transduced a doxycycline-inducible NB expression vector into mouse NIH3T3 fibroblasts and human Jurkat T-ALL cells., Results: NB promoted the transformation of mouse NIH3T3 fibroblasts by upregulating the proto-oncogene Pim1, which encodes a serine/threonine kinase. NB transcriptionally regulated Pim1 expression by binding to its promoter and activated MYC and mTORC1 signaling. PIM1 knockdown or pharmacological inhibition of mTORC1 signaling suppressed NB-induced NIH3T3 cell transformation. Furthermore, NB enhanced the survival of human Jurkat T-ALL cells by inactivating the pro-apoptotic protein BCL2-associated agonist of cell death (BAD)., Conclusion: We demonstrated the pivotal role of NB in cell transformation and survival and identified PIM1as a key downstream target of NB. These findings propose a promising therapeutic strategy for patients with NB fusion-positive leukemia., (© 2024 The Author(s). Cancer Medicine published by John Wiley & Sons Ltd.)

Altering the route of uptake by the cells is an attractive strategy to overcome drug-receptor adaptation problems. Carbon nanoparticles (CNPs) with emission beyond tissue autofluorescence for imaging biological tissues were used to study the phenomenon of uptake by the cells. In this regard, red-emitting carbon nanoparticles (CNPs) were synthesized and incorporated onto lipid microbubbles (MBs). The CNPs showed red emissions in the range of 640 nm upon excitation with 480 nm wavelength of light. Atomic force microscopic and confocal microscopic images showed the successful loading of CNPs onto the MB. Carbon nanoparticle loaded microbubbles (CNP-MBs) were treated with NIH 3 T3 cells at different concentrations. Confocal microscopic imaging studies confirm the presence of CNPs inside the treated cells. Cytotoxicity studies revealed that the CNPs showed minimal toxicity towards cells after loading onto MBs. The CNPs are usually taken up by the cells through the clathrin-mediated (CME) pathway, but when loaded onto MBs, the mechanism of uptake of CNPs is altered, and the uptake by the cells was observed even in the presence of inhibitors for the CME pathway. Loading CNPs onto MBs resulted in the uptake of CNPs by the cell through micropinocytosis and sonophoresis in the presence of ultrasound. The in vivo uptake CNP-MBs were performed in Danio rerio (Zebrafish larvae). This study provides insights into altering the uptake pathway through reformulation by loading nanoparticles onto MBs., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Aberrant activation of hedgehog (Hh) signaling has been implicated in various cancers. Current FDA-approved inhibitors target the seven-transmembrane receptor Smoothened, but resistance to these drugs has been observed. It has been proposed that a more promising strategy to target this pathway is at the GLI1 transcription factor level. GANT61 was the first small molecule identified to directly suppress GLI-mediated activity; however, its development as a potential anti-cancer agent has been hindered by its modest activity and aqueous chemical instability. Our study aimed to identify novel GLI1 inhibitors. JChem searches identified fifty-two compounds similar to GANT61 and its active metabolite, GANT61-D. We combined high-throughput cell-based assays and molecular docking to evaluate these analogs. Five of the fifty-two GANT61 analogs inhibited activity in Hh-responsive C3H10T1/2 and Gli-reporter NIH3T3 cellular assays without cytotoxicity. Two of the GANT61 analogs, BAS 07019774 and Z27610715, reduced Gli1 mRNA expression in C3H10T1/2 cells. Treatment with BAS 07019774 significantly reduced cell viability in Hh-dependent glioblastoma and lung cancer cell lines. Molecular docking indicated that BAS 07019774 is predicted to bind to the ZF4 region of GLI1, potentially interfering with its ability to bind DNA. Our findings show promise in developing more effective and potent GLI inhibitors.

Bacterial infection can lead to various complications, such as inflammations on surrounding tissues, which can prolong wound healing and thus represent a significant clinical and public healthcare problem. Herein, a report on the fabrication of a novel genipin/quaternized chitosan (CS) hydrogel for wound dressing is presented. The hydrogel was prepared by mixing quaternized CS and genipin under 35 °C bath. The hydrogels showed porous structure (250-500 μm) and mechanical properties (3000-6000 Pa). In addition, the hydrogels displayed self-healing ability and adhesion performance on different substrates. Genipin crosslinked quaternized CS hydrogels showed antibacterial activities against E. coli and S. aureus . The CCK-8 and fluorescent images confirmed the cytocompatibility of hydrogels by seeding with NIH-3T3 cells. The present study showed that the prepared hydrogel has the potential to be used as wound dressing., (© 2024 IOP Publishing Ltd.)

The emergence of antimicrobial resistance, exemplified by methicillin-resistant Staphylococcus aureus (MRSA), poses a grave threat to public health globally. Over time, MRSA has evolved resistance to multiple antibiotics, challenging conventional treatment strategies. The relentless adaptability of MRSA underscores the urgent need for innovative and targeted antimicrobial approaches to combat this resilient pathogen. Ancient knowledge and practices, along with scientific evidence, have established that metallic copper, and its organic coordination complexes can act as potential antibacterial substances. In search of a smart and effective antimicrobial against MRSA, we designed, synthesized, and characterized a bidentate copper(II) ligand complex (SG-Cu) utilizing a comprehensive array of analytical techniques, including ESI-MS, elemental analysis, X-ray photoelectron spectroscopy, electron paramagnetic resonance spectroscopy, and others. Antibacterial efficacy and mechanism of action of the complex were assessed through bacterial growth analyses, bacterial membrane perturbation assays, ROS elicitation assays, and field emission scanning electron microscopy. SG-Cu was found to maintain robust biocompatibility against the mammalian cell lines HEK-293, WI-38, and NIH/3T3. Remarkably, SG-Cu demonstrated significant biofilm disruptive tendency evidenced by the retardation of sliding motility, reduction in slime production, reduction in biofilm viability, and enhanced biofilm eradication, both in vitro and in urinary catheters. In vivo studies on murine excisional wounds, with SG-Cu impregnated in a palmitic acid conjugated NAVSIQ hexapeptide (PA-NV) hydrogel, revealed the sustained release of SG-Cu from the gel matrix, facilitating accelerated wound healing and effective wound disinfection. This multifaceted investigation highlights the potential of SG-Cu as a versatile option for combating MRSA infections and promoting wound healing, solidifying its claim to be developed into a viable therapeutic.

Accelerating wound healing, as well as preventing infection and scar formation are among the most important medical challenges. This study aims to examine the antimicrobial, immunomodulatory, and anticancer properties of Morus nigra. The antimicrobial activities of ripe and unripe M. nigra fruit (MNF) extracts were tested. HPLC was employed to measure the components in the extract. Oserin ointment was made with 8 % extract. To test the ointment, 48 Wistar rats were randomly assigned into eight groups. The ointment was used daily by treating the wounds. Tissue histology and wound healing were assessed over nine days. Comparative evaluation of wound healing was conducted by analyzing TGF-β, TNF-α, and IL-1 mRNA levels. Finally, cytotoxic effects on AGS cancer and NIH-3 T3 fibroblast cells were examined. The ANOVA test and Prsim program were used for statistical analysis. Unripe MNF extract had good antimicrobial properties in standard and nosocomial strains. The most abundant compound in the extract was ascorbic acid (0.0441 mg/10 mg extract), followed by naringenin and gallic acid. In all groups treated with MNF extract ointment, a significant reduction in wound area was observed compared to other groups (p < 0.05). After six days of treatment, the microbial load was uncountable. In the microscopic studies of the wounds, a significant increase was observed in fibroblasts, angiogenesis, and in neutrophils in the first days as well as a decrease in the final days. The treatment caused a significant decline in the expression of IL-1 and TNF-α genes, as well as an increase in the expression of TGF-β (p < 0.05). This extract had no significant cytotoxic effects on human fibroblast cells (p > 0.05). In general, it can be concluded that the unripe MNF extract ointment can be a suitable option for the treatment of infectious and non-infectious skin wounds., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Nutraceuticals, that include food ingredients and bioactives from natural products, confer physiological health benefits and protection against chronic diseases. Annatto is a tropical shrub grown in Central and South America and parts of India. Its seeds are rich in the edible carotenoid-derived apocarotenoid pigment, bixin, which is used as a natural colorant in food, textiles, and cosmetics, and is now gaining attention for its potential health-promoting attributes. Here, we compared a green solvent (ethyl lactate) based extraction of bixin and associated metabolites in annatto seeds (crushed and seed coat) with two other conventional solvents (acetone and acid-base). Bixin was characterized in the extracts using UV-visible- and FTIR-spectroscopy and thin-layer chromatography. The bixin-containing solvent extracts were then profiled for other co-existing metabolites using GC-MS analysis, which were found to be sesquiterpenes, terpenes, terpenoids, phytosterols, and tocotrienols. Their bioactivity was evaluated based on antioxidant and wound-healing efficacies and compared with pure bixin, using NIH-3T3 fibroblast cells in-vitro. Pure bixin, as well as the annatto solvent extracts, showed strong antioxidant and wound healing properties, wherein pure bixin and green solvent extract (ethyl lactate coat) exhibited higher levels of antioxidant activity, achieving 46.00% and 44.60% reduction in MDA levels, respectively, as well as enhanced wound-healing activity, with 54.09% and 53.60% wound closure within 24 h. The green solvent extracts of annatto seeds revealed: (a) differential bioactive profiles in annatto seeds (crushed and seed coat) in comparison with other solvents, and (b) strong antioxidant and wound healing properties. Thus, ethyl lactate extraction shows strong potential for sustainable environmental friendly production of functional foods/nutraceuticals from annatto seeds.