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The neurotrophin growth factors bind and activate two types of cell surface receptors: the tropomyosin receptor kinase (Trk) family and p75. TrkA, TrkB, and TrkC are bound preferentially by nerve growth factor, brain-derived neurotrophic factor, and neurotrophin 3 (NT3), respectively, to activate neuroprotective signals. The p75 receptors are activated by all neurotrophins, and paradoxically in neurodegenerative disease p75 is upregulated and mediates neurotoxic signals. To test neuroprotection strategies, we engineered NT3 to broadly activate Trk receptors (mutant D) or to reduce p75 binding (mutant RK). We also combined these features in a molecule that activates TrkA, TrkB, and TrkC but has reduced p75 binding (mutant DRK). In neurodegenerative disease mouse models in vivo, the DRK protein is a superior therapeutic agent compared with mutant D, mutant RK, and wild-type neurotrophins and protects a broader range of stressed neurons. This work rationalizes a therapeutic strategy based on the biology of each type of receptor, avoiding activation of p75 toxicity while broadly activating neuroprotection in stressed neuronal populations expressing different Trk receptors. SIGNIFICANCE STATEMENT The neurotrophins nerve growth factor, brain-derived neurotrophic factor, and neurotrophin 3 each can activate a tropomyosin receptor kinase (Trk) A, TrkB, or TrkC receptor, respectively, and all can activate a p75 receptor. Trks and p75 mediate opposite signals. We report the engineering of a protein that activates all Trks, combined with low p75 binding, as an effective therapeutic agent in vivo.

Prolonged skin exposure to ultraviolet radiation can lead to development of several acute and chronic diseases, with UVA exposure considered a primary cause of dermal photodamage. We prepared a wild ginseng adventitious root extract (ARE) that could alleviate UVA irradiation-induced NIH-3T3 cell viability decline. After employing a series of purification methods to isolate main active components of ARE, adventitious root protein mixture (ARP) was identified then tested for protective effects against UVA irradiation-induced NIH-3T3 cell damage. The results showed that ARP treatment significantly reduced UVA-induced cell viability decline and confirmed that the active constituent of ARP was the protein, since proteolytic hydrolysis and heat treatment each eliminated ARP protective activity. Moreover, ARP treatment markedly inhibited UVA-induced apoptosis, cell cycle arrest and DNA fragmentation, while also significantly reversing UVA effects (elevated Bax levels, reduced Bcl-2 expression) by reducing Bax levels and increasing Bcl-2 expression. Mechanistically, ARP promoted Akt phosphorylation regardless of UVA exposure, thus confirming ARP resistance to inactivation by UVA light. Notably, in the presence of Akt inhibitor SC0227, ARP could no longer counteract UVA-induced cell viability decline and DNA fragmentation. Additionally, our results demonstrated that ARP treatment protected UVA-irradiated NIH-3T3 cells by preventing UVA-induced reduction of collagen-I expression. Taken together, these results suggest that ARP treatment of NIH-3T3 cells effectively mitigated UVA-induced cell viability decline by activating intracellular Akt to reduce UVA-induced DNA damage, leading to reduced rates of apoptosis and cell cycle arrest after UVA exposure and restoring collagen expression to normal levels.

NF‐κB, a critical cytokine of inflammatory bowel diseases (IBD), is a viable marker to reflect the inflammatory activity of the intestine. We aimed to develop NF‐κB‐targeted microbubbles (MBs) and perform molecular contrast‐enhanced ultrasound (CEUS) to quantify NF‐κB expressions on the intestinal wall in IBD mice in vivo. In this study, NF‐κB‐targeted MBs were fabricated by connecting biotin‐loaded NF‐κB antibodies and avidin‐loaded MBs. NF‐κB‐targeted MBs presented as transparent and round bubbles with an average diameter of 1.03/μm±0.01. The specific binding of targeted MBs and inflammatory cells was validated by in vitro experiments, including flow cytometry, Western blot and immunofluorescence, which revealed the specific binding of targeted MBs and inflammatory cells. Subsequently, NF‐κB‐targeted CEUS imaging was performed on mice with chemical‐induced colitis, and the peak intensity (PI) and time‐to‐peak (TTP) were quantified. Pathological and immunohistochemical (IHC) examinations were further implemented. For the target CEUS group, fast enhancement followed by slow subsiding was observed. The PI of target CEUS of the IBD mice was significantly higher than that of non‐target CEUS of the IBD mice, healthy controls and target CEUS of the treated IBD mice (34835%[13379–73492%] VS 437%[236–901%], 130%[79–231%], 528%[274–779%], p

Carbene-based macromolecules are an emerging new stimuli-sensitive class of biomaterials that avoid the impediments of free radical polymerization but maintain a rapid liquid-to-biorubber transition. Activation of diazirine-grafted polycaprolactone polyol (CaproGlu) is limited to UVA wavelengths that have tissue exposure constraints and limited light intensities. For the first time, UVA is circumvented with visible light-emitting diodes at 445 nm (blue) to rapidly activate diazirine-to-carbene covalent cross-linking. Iridium photocatalysts serve to initiate diazirine, despite having little to no absorption at 445 nm. CaproGlu's liquid organic matrix dissolves the photocatalyst with no solvents required, creating a light transparent matrix. Considerable differences in cross-linking chemistry are observed in UVA vs visible/photocatalyst formulations. Empirical analysis and theoretical calculations reveal a more efficient conversion of diazirine directly to carbene with no diazoalkane intermediate detected. Photorheometry results demonstrate a correlation between shear moduli, joules light dose, and the lower limits of photocatalyst concentration required for the liquid-to-biorubber transition. Adhesion strength on ex vivo hydrated tissues exceeds that of cyanoacrylates, with a fixation strength of up to 20 kg·f·cm2. Preliminary toxicity assessment on leachates and materials directly in contact with mammalian fibroblast cells displays no signs of fibroblast cytotoxicity. Agency for Science, Technology and Research (A*STAR) Ministry of Education (MOE) Nanyang Technological University Accepted version T.W.J.S. and I.D. are co-inventors of patent application Hygroscopic, Crosslinking Coatings and Bioadhesives; PCT/ SG2018/050452. The project was supported by the Ministry of Education Tier 1 Grant RG17/18 (S): Novel light activated, diazo protecting groups; Ministry of Education Tier 2 Grant (MOE2018-T2-2-114): CaproGlu, double-sided wet tissue adhesives; NTUitive POC (Gap) Fund NGF/2018/05: Aesthetic Applications of CaproGlu Bioadhesives; and A*Star IAF PP Grant (H19/01/a0/0II9): CathoGlu Bioadhesives-preventing catheter extravasation and skin infections. L.B. acknowledges funding from the Spanish Ministry of Science and Innovation (MCIU), project PID2019- 104654GB-I00.

CRISPR-based cancer dependency maps are accelerating advances in cancer precision medicine, but adequate functional maps are limited to the most common oncogenes. To identify opportunities for therapeutic intervention in other rarer subsets of cancer, we investigate the oncogene-specific dependencies conferred by the lung cancer oncogene, RIT1. Here, genome-wide CRISPR screening in KRAS, EGFR, and RIT1-mutant isogenic lung cancer cells identifies shared and unique vulnerabilities of each oncogene. Combining this genetic data with small-molecule sensitivity profiling, we identify a unique vulnerability of RIT1-mutant cells to loss of spindle assembly checkpoint regulators. Oncogenic RIT1M90I weakens the spindle assembly checkpoint and perturbs mitotic timing, resulting in sensitivity to Aurora A inhibition. In addition, we observe synergy between mutant RIT1 and activation of YAP1 in multiple models and frequent nuclear overexpression of YAP1 in human primary RIT1-mutant lung tumors. These results provide a genome-wide atlas of oncogenic RIT1 functional interactions and identify components of the RAS pathway, spindle assembly checkpoint, and Hippo/YAP1 network as candidate therapeutic targets in RIT1-mutant lung cancer., RIT1 mutations are mutually exclusive with other lung cancer drivers and lack targeted therapies. Here the authors examine genetic dependencies of mutant RIT1 with genome-wide CRISPR screens, revealing synergy between RIT1 and YAP1, and increased sensitivity to Aurora kinase inhibitors.

Summary During development, cells respond rapidly to intra- and intercellular signals, which induce signaling cascades regulating the activity of transcription factors at the transcriptional and/or post-translational level. The transcription factor ISL1 plays a key role in second heart field development and cardiac differentiation, and its mRNA levels are tightly regulated during cardiogenesis. Here, we show that a BMP-p38 MAPK signaling axis controls ISL1 protein function at the post-translational level. BMP-mediated activation of p38 MAPK leads to ISL1 phosphorylation at S269 by p38, which prevents ISL1 degradation and ensures its transcriptional activity during cardiogenesis. Interfering with p38 MAPK signaling leads to the degradation of ISL1 by the proteasome, resulting in defects in cardiomyocyte differentiation and impaired zebrafish and mouse heart morphogenesis and function. Given the critical role of the tight control of ISL1 activity during cardiac lineage diversification, modulation of BMP4-p38 MAPK signaling could direct differentiation into specific cardiac cell subpopulations., Highlights • ISL1 is phosphorylated by p38 MAPK at serine 269 • A BMP4-p38 MAPK signaling axis controls ISL1 protein stability • Phosphorylation of ISL1 by p38 regulates its activity during cardiogenesis • p38 Inhibition in vivo results in ISL1 degradation and second heart field defects, In this article, Jing and colleagues describe a key role of BMP4-p38 MAPK signaling in controlling ISL1 protein stability and activity during cardiogenesis through p38-mediated ISL1 phosphorylation at S269. Interfering with p38 MAPK signaling leads to the degradation of ISL1 by the proteasome, resulting in defects in ISL1+ cardiac progenitor cell differentiation and impaired heart morphogenesis and function.

Calponin 2 is an actin cytoskeleton-associated protein and plays a role in regulating cell motility-related functions such as phagocytosis, migration, and division. We previously reported that overexpression of calponin 2 inhibits the rate of cell proliferation. To investigate the underlying mechanism, our present study found that the levels of endogenous calponin 2 in NIH3T3 and HEK293 cells rapidly decreased before cell division characterized by an absence at the actin contractile ring. In cells lacking endogenous calponin 2, transfective expression of GFP-fusion calponin 2 inhibited cell proliferation similar to that of nonfusion calponin 2. Fluorescent imaging studies of mitotic cells indicated that a proper level of calponin 2 expression and effective degradation during cytokinesis are necessary for normal cell division. Computer-assisted dynamic image analysis of dividing cells revealed that overexpression of calponin 2 significantly affects motility and shape behaviors of cells only on the interval from the start of anaphase to the start of cytokinesis, i.e., the pre-cytokinesis phase, but not on the interval from the start of cytokinesis to 50% completion of cytokinesis. The pre-cytokinesis degradation of calponin 2 was attenuated by MG132 inhibition of the ubiquitin proteasome and inhibitor of protein kinase C (PKC), suggesting that PKC phosphorylation-triggered degradation of calponin 2 could determine the rate of cytokinesis. The novel role of calponin 2 in regulating the rate of cytokinesis may be targeted for therapeutic applications such as in an inhibition of malignant tumor growth.

The bacterial exopolysaccharide Curdlan has a unique collagen-like triple helical structure and immune-modulation activities. Although there have been several types of Curdlan gels reported for antibacterial or wound healing purposes, none of them exhibit favorable mechanical properties for clinically applicable wound healing materials. Herein, we present a two-step approach for preparing Ag-embedded Curdlan hydrogels that are highly soft but are very stretchable compared with common polysaccharide-based hydrogels. Ag ions were first reduced in a diluted Curdlan solution to form AgNP-decorated triple helices. Then, the aqueous solution consisting of Curdlan/Ag nanoparticles was mixed with a dimethyl sulfoxide solution consisting of a high concentration of Curdlan. This mixing triggered the conformation transformation of Curdlan random coils into triple helices, and then the helices were further packed into semicrystalline nanofibrils of ∼20 nm in diameter. Due to the presence of semicrystalline fibrils, this novel Curdlan hydrogel exhibits a fracture strain of ∼350% and fracture stress of ∼0.2 MPa at a water content of ∼97%. This nanofibril hydrogel supported the attachment, spreading, and growth of fibroblasts and effectively inhibited the growth of Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus. Moreover, the hydrogels downregulated NO production and proinflammatory gene expression levels in lipopolysaccharide (LPS)-stimulated macrophages but did not change the anti-inflammatory gene expression levels in IL-4-stimulated macrophages. In an animal study, these hydrogels accelerated wound healing in a bacteria-infected mice skin wound model. These results validate the further development of Curdlan/AgNPs nanofibril hydrogels in clinical wound management.

A green biocompatible route for the deposition and simultaneous assembly, by pH increment, of collagen/chitin composites was proposed. Both assembled and unassembled samples with different collagen/chitin ratios were synthesized, maintaining the β-chitin polymorph. The first set showed a microfibrous organization with compositional submicron homogeneity. The second set presented a nanohomogeneous composition based on collagen nanoaggregates and chitin nanofibrils. The sets were tested as scaffolds for fibroblast growth (NIH-3T3) to study the influence of composition and assembly. In the unassembled scaffolds, the positive influence of collagen on cell growth mostly worn out in 48 h, while the addition of chitin enhanced this effect for over 72 h. The assembled samples showed higher viability at 24 h but a less positive effect on viability along the time. This work highlighted critical aspects of the influence that composition and assembly has on fibroblast growth, a knowledge worth exploiting in scaffold design and preparation.

Hypoxia, a common characteristic of bacterial infections, is known to be closely associated with the emergence of multidrug-resistant bacteria, which hastens the need to develop advanced microbicides and antibacterial techniques. Photodynamic therapy is a promising strategy to reduce bacterial antibiotic resistance and employs photosensitizers, excitation light sources, and sufficient oxygen to generate toxic reactive oxygen species (ROS). The inherent limitation of PDT is that the generation of ROS is restricted by the hypoxic microenvironment in infection sites. Here, an oxygen self-supplying nanotherapeutic is developed to enhance antibacterial activity against multidrug-resistant bacteria on the basis of fluorinated boron dipyrromethene (BODIPY)-based glycomimetics. The nanotherapeutic not only could capture the bacteria efficiently but also was able to act as an oxygen carrier to relieve the hypoxic microenvironment of bacterial infections, thus achieving enhanced PDT efficacy. In a Pseudomonas aeruginosa infection of a rat cornea, typical administration of the nanotherapeutic decreased the infiltrate and showed a faster healing capacity in comparison with BODIPY-based glycomimetics. Self-supplying oxygen nanotherapeutics that relieve the hypoxic microenvironment and interfere with bacterial colonization have been shown to be a promising candidate for the management of drug-resistant microbial keratitis.

In recent years, an increasing number of studies have shown that fibroblast growth factor 12 (FGF12) plays important roles in regulating neural development and function. Importantly, changes of FGF12 expression are thought to be related to the pathophysiology of many neurological diseases. However, little research has been performed to explore the protective effect of FGF12 on nerve damage. This study aims to explore its neuroprotective effects using our recombinant humanized FGF12 (rhFGF12). The hFGF12 gene was cloned and ligated into an expression vector to construct a recombinant plasmid pET-3a-hFGF12. Single colonies were screened to obtain high expression engineering strains, and fermentation and purification protocols for rhFGF12 were designed and optimized. The biological activities and related mechanisms of rhFGF12 were investigated by MTT assay using NIH3T3 and PC12 cell lines. The in vitro neurotoxicity model of H2O2-induced oxidative injury in PC12 cells was established to explore the protective effects of rhFGF12. The results indicate that the beneficial effects of rhFGF12 were most likely achieved by promoting cell proliferation and reducing apoptosis. Moreover, a transgenic zebrafish (islet) with strong GFP fluorescence in the motor neurons of the hindbrain was used to establish a central injury model caused by mycophenolate mofetil (MMF). The results suggested that rhFGF12 could ameliorate central injury induced by MMF in zebrafish. In conclusion, we have established an efficient method to express and purify active rhFGF12 using an Escherichia coli expression system. Besides, rhFGF12 plays a protective effect of on nerve damage, and it provides a promising therapeutic approach for nerve injury. KEY POINTS: • Effective expression and purification of bioactive rhFGF12 protein in E. coli. • ERK/MAPK pathway is involved in rhFGF12-stimulated proliferation on PC12 cells. • The rhFGF12 has the neuroprotective effects by inhibiting apoptosis.

Burn injury has posed devastating burdens on the public health due to its inevitable damage to the skin structure resulting in the increased risk of infection. Therefore, it is highly demanding to develop efficacious antibacterial wound-healing dressing. Despite the favourable wound-healing activities, the curative efficacy of phytochemical compounds of quercetin (Qe) and its derivatives is limited by their poor water solubility. Here, we have fabricated a novel electrospun nanofiber membrane (ENM) consisting of polycaprolactone (PCL), chitosan oligosaccharides (COS), and Qe/Rutin (Ru) as the potential bioactive dressing for wound healing. The incorporation of chitosan oligosaccharides (COSs) in the PCL scaffold at the optimized molar ratio not only contributed to the improved hydrophilicity and water absorption performance of the ENM but effectively increased the specific surface area of the formed nanofibers. In particular, the antioxidant and antibacterial activities of the Qe/rutin-loaded nanofiber membranes were tested, which revealed that the PCL-COS-Qe membrane exhibited superior performance among all nanofiber membranes. Therefore, the developed PCL-COS-Qe/Ru nanofiber membranes hold enormous potential as healthcare products, such as wound dressings for burn injuries.

Background Rupture of atherosclerotic plaque can cause acute malignant heart and cerebrovascular events, such as acute coronary heart disease, stroke and so on, which seriously threaten the safety of human life and property. Therefore, the early diagnosis and inhibition of atherosclerotic plaque progress still be a vital task. Results In this study, we presented the development of composite mesoporous silica nanoparticle (Ru(bpy)3@SiO2-mSiO2, CMSN)-based nanomedicines (NMs) (Ru(bpy)3@SiO2-mSiO2@SRT1720@AntiCD36, CMSN@SRT@Anti) for accurate diagnosis and treatment of atherosclerosis (AS). In vitro cell experiments showed that both RAW264.7 and oxidized low density lipoprotein (ox-LDL)-stimulated RAW264.7 cells could significantly uptake CMSN@SRT@Anti. Conversely, little fluorescence signal could be observed in CMSN@SRT group, showing the excellent targeting ability of CMSN@SRT@Anti to Class II scavenger receptor, CD36 on macrophage. Additionally, such fluorescence signal was significantly stronger in ox-LDL-stimulated RAW264.7 cells, which might benefit from the upregulated expression of CD36 on macrophages after ox-LDL treatment. For another, compared with free SRT1720, CMSN@SRT@Anti had a better and more significant effect on the inhibition of macrophage foaming process, which indicated that drug-carrying mesoporous silicon with targeting ability could enhance the efficacy of SRT1720. Animal experimental results showed that after the abdominal injection of CMSN@SRT@Anti, the aortic lesions of ApoE-/-mice could be observed with obvious and persistent fluorescence signals. After 4 weeks post-treatment, the serum total cholesterol, aortic plaque status and area were significantly improved in the mouse, and the effect was better than that in the free SRT1720 group or the CMSN@SRT group. Conclusions The designed CMSN@SRT@Anti with excellent biocompatibility, high-performance and superior atherosclerosis-targeting ability has great potential for accurate identification and targeted therapy of atherosclerotic diseases. Graphic abstract

Biologically active scaffolds with tunable mechano- and bio-performance remain desirable for soft tissue engineering. Previously, highly elastic and robust silk fibroin (SF) scaffolds were prepared via cryogelation. In order to get more insight into the role of ethylene glycol diglycidyl ether (EGDE) on the structure and properties of SF scaffolds, we investigated the fate of SF scaffolds with different usages of the crosslinking agent in vitro and in vivo. Although SF scaffolds with varied EGDE contents showed similar micro-morphology, increasing EGDE from 1 mmol/g to 5 mmol/g resulted in firstly increased and later decreased content of β-sheet conformation, and linearly increased tensile modulus and decreased elasticity. The dual-crosslinked SF scaffolds with EGDE up to 5 mmol/g did not show in vitro cytotoxicity for NIH3T3 fibroblasts. In vivo subcutaneous implantation of SF scaffolds with

In embryonic stem (ES) cells, oxidative stress control is crucial for genomic stability, self-renewal and cell differentiation. Heme oxygenase-1 (HO-1) is a key player of the anti-oxidant system, and is also involved in stem cell differentiation and pluripotency acquisition. We found that the HO-1 gene is expressed in ES cells and induced after promoting differentiation. Moreover, downregulation of the pluripotency transcription factor (TF) OCT4 increased HO-1 mRNA levels in ES cells, and analysis of ChIP-seq public data revealed that this TF binds to the HO-1 gene locus in pluripotent cells. Finally, ectopic expression of OCT4 in heterologous systems repressed a reporter carrying the HO-1 gene promoter and the endogenous gene. Hence, this work highlights the connection between pluripotency and redox homeostasis.

Withanolide derivatives have anticancer, anti-inflammatory, and other functions and are components of Indian traditional Ayurvedic medicine. Here, we found that 2,3-dihydro-3β-methoxy withaferin-A (3βmWi-A), a derivative of withaferin-A (Wi-A) belonging to a class of withanolides that are abundant in Ashwagandha (Withania somnifera), lengthened the period of the circadian clock. This compound dose-dependently elongated circadian rhythms in Sarcoma 180 cancer cells and in normal fibroblasts including NIH3T3 and spontaneously immortalized mouse embryonic fibroblasts (MEF). Furthermore, 3βmWi-A dose-dependently upregulated the mRNA expression and promoter activities of Bmal1 after dexamethasone stimulation and of the nuclear orphan receptors, Rora and Nr1d1, that comprise the stabilization loop for Bmal1 oscillatory expression. We showed that 3βmWi-A functions as an inverse agonist for RORa with an IC50 of 11.3 μM and that 3βmWi-A directly, but weakly, interacts with RORa (estimated dissociation constant [Kd], 5.9 μM). We propose that 3βmWi-A is a novel modulator of circadian rhythms.

In the present study, we report a human-inherited, impaired, adaptive immunity disorder, which predominantly manifested as a B cell differentiation defect, caused by a heterozygous IKZF3 missense variant, resulting in a glycine-to-arginine replacement within the DNA-binding domain of the encoded AIOLOS protein. Using mice that bear the corresponding variant and recapitulate the B and T cell phenotypes, we show that the mutant AIOLOS homodimers and AIOLOS–IKAROS heterodimers did not bind the canonical AIOLOS–IKAROS DNA sequence. In addition, homodimers and heterodimers containing one mutant AIOLOS bound to genomic regions lacking both canonical motifs. However, the removal of the dimerization capacity from mutant AIOLOS restored B cell development. Hence, the adaptive immunity defect is caused by the AIOLOS variant hijacking IKAROS function. Heterodimeric interference is a new mechanism of autosomal dominance that causes inborn errors of immunity by impairing protein function via the mutation of its heterodimeric partner. The zinc-finger transcription factor IKAROS is essential for B cell development. Taniuchi, Morio and colleagues identify a human kindred presenting with B cell immunodeficiency that was caused by a heterozygous missense mutation in IKZF3 encoding the related AIOLOS protein. AIOLOSG159R is a mutant protein that interferes with both wild-type AIOLOS and IKAROS by forming heterodimers that bind to aberrant DNA-binding sites and prevent normal expression of IKAROS-dependent genes.

Wound infection can cause a delay in wound healing or even wound deterioration, threatening patients' lives. The excessive accumulation of reactive oxygen species (ROS) in infected wounds activates a strong inflammatory response to delay wound healing. Therefore, it is highly desired to develop hydrogels with inherent antimicrobial activity and antioxidant capability for infected wound healing. Herein, a dopamine-substituted multidomain peptide (DAP) with inherent antimicrobial activity, strong skin adhesion, and ROS scavenging has been developed. DAP can form bilayer β-sheets with dopamine residues on the surface of nanofibers. The enhanced rheological properties of DAP-based hydrogel can be achieved not only through UV irradiation but also by incorporation of multivalent ions (e.g., PO43-). Furthermore, the DAP hydrogel shows a broad spectrum of antimicrobial activity due to the high positive charges of lysine residues and the β-sheet formation. When applied to full-thickness dermal wounds in mice, the DAP hydrogel results in a significantly shortened inflammatory stage of the healing process because of its remarkable antimicrobial activity and antioxidant capability. Accelerated wound closure with thick granulation tissue, uniform collagen arrangement, and dense vascularization can be achieved. This work suggests that the DAP hydrogel can serve as antimicrobial coating and ROS-scavenging wound dressing for bacterial-infected wound treatment.

Single-cell RNA-sequencing analysis is one of the most effective tools for understanding specific cellular states. The use of single cells or pooled cells in RNA-seq analysis requires the isolation of cells from a tissue or culture. Although trypsin or more recently cold-active protease (CAP) has been used for cell dissociation, the extent to which the gene expression changes are suppressed has not been clarified. To this end, we conducted detailed profiling of the enzyme-dependent gene expression changes in mouse skeletal muscle progenitor cells, focusing on the enzyme treatment time, amount, and temperature. We found that the genes whose expression was changed by the enzyme treatment could be classified in a time-dependent manner, and that there were genes whose expression was changed independently of the enzyme treatment time, amount, and temperature. This study will be useful as reference data for genes that should be excluded or considered for RNA-seq analysis using enzyme isolation methods.

Extracellular vesicles (EVs) are secreted from most cells and play important roles in cell-cell communication by transporting proteins, lipids, and nucleic acids. As the involvement of EVs in diseases has become apparent, druggable regulators of EV secretion are more desirable. However, the lack of a highly sensitive EV detection system has made the development of EV regulators difficult. We developed an ELISA system to detect EVs using TIM4 proteins, which have high affinity to phosphatidylserine and screened a 1,567-compound library. Consequently, we identified one inhibitor and three activators of EV secretion in a variety of cells. The inhibitor, apoptosis activator 2, suppressed EV secretion via a different mechanism and had a broader cellular specificity than GW4869. The three activators, cucurbitacin B, gossypol, and obatoclax, also had broad cellular specificity, including HEK293T cells and human mesenchymal stem cells (hMSCs). In vitro bioactivity assays revealed that some regulators control EV secretion from glioblastoma and hMSCs, which induces angiogenesis and protects cardiomyocytes against apoptosis, respectively. In conclusion, we developed a high-throughput method to detect EVs with high sensitivity and versatility, and identified three compounds that can regulate the bioactivity of EVs.

Haw-Ming Huang,1,2 Ping-Han Wu,3 Po-Chien Chou,4 Wen-Tien Hsiao,5 Hsin-Ta Wang,4 Hsin-Pei Chiang,3 Chi-Ming Lee,6 Shwu-Huey Wang,6 Yu-Cheng Hsiao2,7 1School of Dentistry, College of Oral Medicine, Taipei Medical University, Taipei, Taiwan; 2Graduate Institute of Biomedical Optomechatronics, College of Biomedical Engineering, Taipei Medical University, Taipei, Taiwan; 3Graduate Institute of Biomedical Materials and Tissue Engineering, College of Biomedical Engineering, Taipei Medical University, Taipei, Taiwan; 4School of Organic and Polymeric, National Taipei University of Technology, Taipei, Taiwan; 5Department of Medical Imaging and Radiological Technology, Yuanpei University of Medical Technology, Hsinchu, Taiwan; 6Core Facility Center, Office of Research and Development, Taipei Medical University, Taipei, Taiwan; 7Cell Physiology and Molecular Image Research Center, Taipei Municipal Wan Fang Hospital, Taipei Medical University, Taipei, TaiwanCorrespondence: Yu-Cheng Hsiao; Ping-Han WuTaipei Medical University, No. 250, Wu-Hsing Street, Taipei, 11031, TaiwanTel +886-291-937-9783Fax +886 2 27362295Email ychsiao@tmu.edu.tw; gahwclbjwph@gmail.comIntroduction: It has been reported that low-molecular-weight hyaluronic acid (LMWHA) exhibits a potentially beneficial effect on cancer therapy through targeting of CD44 receptors on tumor cell surfaces. However, its applicability towards tumor detection is still unclear. In this regard, LMWHA-conjugated iron (Fe3O4) nanoparticles (LMWHA-IONPs) were prepared in order to evaluate its application for enhancing the T2* weighted MRI imaging sensitivity for tumor detection.Methods: LMWHA and Fe3O4 NPs were produced using γ-ray irradiation and chemical co-precipitation methods, respectively. First, LMWHA-conjugated FITC was prepared to confirm the ability of LMWHA to target U87MG cells using fluorescence microscopy. The hydrodynamic size distribution and dispersion of the IONPs and prepared LMWHA-IONPs were analyzed using dynamic light scattering (DLS). In addition, cell viability assays were performed to examine the biocompatibility of LMWHA and LMWHA-IONPs toward U87MG human glioblastoma and NIH3T3 fibroblast cell lines. The ability of LMWHA-IONPs to target tumor cells was confirmed by detecting iron (Fe) ion content using the thiocyanate method. Finally, time-of-flight secondary ion mass spectrometry (TOF-SIMS) imaging and in vitro magnetic resonance imaging (MRI) were performed to confirm the contrast enhancement effect of LMWHA-IONPs.Results: Florescence analysis results showed that LMWHA-FITC successfully targeted the surfaces of both tested cell types. The ability of LMWHA to target U87MG cells was higher than for NIH3T3 cells. Cell viability experiments showed that the fabricated LMWHA-IONPs possessed good biocompatibility for both cell lines. After co-culturing test cells with the LMWHA-IONPs, detected Fe ion content in the U87MG cells was much higher than that of the NIH3T3 cells in both thiocyanate assays and TOF-SIMs images. Finally, the addition of LMWHA-IONPs to the U87MG cells resulted in an obvious improvement in T2* weighted MR image contrast compared to control NIH3T3 cells.Discussion: Overall, the present results suggest that LMWHA-IONPs fabricated in this study provide an effective MRI contrast agent for improving the diagnosis of early stage glioblastoma in MRI examinations.Keywords: hyaluronic acid, Fe3O4 nanoparticles, TOF-SIMS, MRI

Dual specificity tyrosine phosphorylation regulated kinase 1A, DYRK1A, functions in multiple cellular pathways, including signaling, endocytosis, synaptic transmission, and transcription. Alterations in dosage of DYRK1A leads to defects in neurogenesis, cell growth, and differentiation, and may increase the risk of certain cancers. DYRK1A localizes to a number of subcellular structures including vesicles where it is known to phosphorylate a number of proteins and regulate vesicle biology. However, the mechanism by which it translocates to vesicles is poorly understood. Here we report the discovery of TRAF2, an E3 ligase, as an interaction partner of DYRK1A. Our data suggest that TRAF2 binds to PVQE motif residing in between the PEST and histidine repeat domain (HRD) of DYRK1A protein, and mediates K63-linked ubiquitination of DYRK1A. This results in translocation of DYRK1A to the vesicle membrane. DYRK1A increases phosphorylation of Sprouty 2 on vesicles, leading to the inhibition of EGFR degradation, and depletion of TRAF2 expression accelerates EGFR degradation. Further, silencing of DYRK1A inhibits the growth of glioma cells mediated by TRAF2. Collectively, these findings suggest that the axis of TRAF2–DYRK1A-Sprouty 2 can be a target for new therapeutic development for EGFR-mediated human pathologies.

Profiling studies have revealed considerable phenotypic heterogeneity in cancer-associated fibroblasts (CAFs) present within the tumour microenvironment, however, functional characterisation of different CAF subsets is hampered by the lack of specific markers defining these populations. Here we show that genetic deletion of the Endo180 (MRC2) receptor, predominantly expressed by a population of matrix-remodelling CAFs, profoundly limits tumour growth and metastasis; effects that can be recapitulated in 3D co-culture assays. This impairment results from a CAF-intrinsic contractility defect and reduced CAF viability, which coupled with the lack of phenotype in the normal mouse, demonstrates that upregulated Endo180 expression by a specific, potentially targetable CAF subset is required to generate a supportive tumour microenvironment. Further, characterisation of a tumour subline selected via serial in vivo passage for its ability to overcome these stromal defects provides important insight into, how tumour cells adapt to a non-activated stroma in the early stages of metastatic colonisation., Endo180, a collagen binding receptor, is highly expressed in a subset of cancer-associated fibroblasts. The authors show, using knockout mice and 3D in vitro assays, that Endo180 depletion impairs tumour fibroblast contractility and viability resulting in reduced tumour growth and metastasis.

Exosomes are extracellular vesicles derived from the endosomal compartment with potential involvement in intercellular communication. We found that frequently used biomarkers of exosomes are heterogeneous and do not exhibit universal utility across different cell types. To uncover ubiquitous and abundant proteins that can serve as biomarkers of exosomes, we employed an unbiased and quantitative proteomic approach based on Super-SILAC coupled to high-resolution mass spectrometry. In total, 1,212 proteins were consistently quantified in the proteome of exosomes irrespective of the cellular source or isolation method. A cohort of 22 proteins were universally enriched, and 15 proteins consistently depleted in the proteome of exosomes when compared to cells. Among the enriched proteins, we identified biogenesis-related, GTPases, and membrane proteins, such as CD47 and ITGB1. The cohort of depleted proteins in exosomes was predominantly composed of nuclear proteins. We identified Syntenin-1 as the highest consistently abundant protein in exosomes from distinct cellular origins, which was also present in exosomes across different species and biofluids, with potential utility as a putative universal biomarker candidate for exosomes. Our study provides a comprehensive quantitative atlas of core proteins ubiquitous to exosomes that can serve as a resource for the scientific community dedicated to the study of exosomes.

Necroptosis is a lytic, inflammatory form of cell death that not only contributes to pathogen clearance but can also lead to disease pathogenesis. Necroptosis is triggered by RIPK3-mediated phosphorylation of MLKL, which is thought to initiate MLKL oligomerisation, membrane translocation and membrane rupture, although the precise mechanism is incompletely understood. Here, we show that K63-linked ubiquitin chains are attached to MLKL during necroptosis and that ubiquitylation of MLKL at K219 significantly contributes to the cytotoxic potential of phosphorylated MLKL. The K219R MLKL mutation protects animals from necroptosis-induced skin damage and renders cells resistant to pathogen-induced necroptosis. Mechanistically, we show that ubiquitylation of MLKL at K219 is required for higher-order assembly of MLKL at membranes, facilitating its rupture and necroptosis. We demonstrate that K219 ubiquitylation licenses MLKL activity to induce lytic cell death, suggesting that necroptotic clearance of pathogens as well as MLKL-dependent pathologies are influenced by the ubiquitin-signalling system., Necroptosis is a form of cell death characterized by membrane rupture via MLKL oligomerization, although mechanistic details remain unclear. Here, the authors show that MLKL ubiquitylation of K219 facilitates high-order membrane assembly and subsequent rupture, promoting cytotoxicity.

Fibroblast growth factor receptors (FGFRs) are integral membrane proteins involved in various biological processes including proliferation, migration and apoptosis. There are a number of regulatory mechanisms of FGFR signaling, which tightly control the specificity and duration of transmitted signals. The effect of the FGFRs spatial distribution in the plasma membrane on receptor-dependent functions is still largely unknown. We have demonstrated that oligomerization of FGF1 with coiled-coil motifs largely improves FGF1 affinity for FGFRs and heparin. Set of developed FGF1 oligomers evoked prolonged activation of FGFR1 and receptor-downstream signaling pathways, as compared to the wild type FGF1. The majority of obtained oligomeric FGF1 variants showed increased stability, enhanced mitogenic activity and largely improved internalization via FGFR1-dependent endocytosis. Importantly, FGF1 oligomers with the highest oligomeric state exhibited reduced ability to stimulate FGFR-dependent glucose uptake, while at the same time remained hyperactive in the induction of cell proliferation. Our data implicate that oligomerization of FGF1 alters the biological activity of the FGF/GFR1 signaling system. Furthermore, developed FGF1 oligomers, due to improved stability and proliferative potential, can be applied in the regenerative medicine or as drug delivery vehicles in the ADC approach against FGFR1-overproducing cancers.

Background The echinoderm microtubule-associated protein-like-4 anaplastic lymphoma kinase (EML4-ALK) fusion gene was identified in a subset of non-small cell lung cancer (NSCLC) patients. They responded positively to ALK inhibitors. This study aimed to characterize the mechanisms triggered by EML4-ALK to induce NSCLC transformation. Methods HEK293 and NIH3T3 cells were transfected with EML4-ALK variant 3 or pcDNA3.1-NC. H2228 cells were transfected with siRNA-EML4-ALK or siRNA-NC. Cell viability and proliferation were measured by the CCK-8 and EdU methods, respectively. Flow cytometry revealed apoptosis. Gene expression profiles were generated from a signaling pathway screen in EML4-ALK-regulated lung cancer cells and verified by qPCR and Western blotting. The co-immunoprecipitation and immunohistochemistry/ immunofluorescence determined the interaction and colocalization of JAK2-STAT pathway components with EML4-ALK. Results Microarray identified several genes involved in the JAK2-STAT pathway. JAK2 and STAT6 were constitutively phosphorylated in H2228 cells. EML4-ALK silencing downregulated phosphorylation of STAT6. Expression of EML4-ALK in HEK293 and NIH3T3 cells activated JAK2, STAT1, STAT3, STAT5, and STAT6. In EML4-ALK-transfected HEK293 cells and EML4-ALK-positive H2228 cells, activated STAT6 and JAK2 colocalized with ALK. STAT3 and STAT6 were phosphorylated and translocated to the nucleus of H2228 cells following IL4 or IL6 treatment. Apoptosis increased, while cell proliferation and DNA replication decreased in H2228 cells following EML4-ALK knockdown. In contrast, HEK293 cell viability increased following EML4-ALK overexpression, while H2228 cell viability significantly decreased after treatment with ALK or JAK-STAT pathway inhibitors. Conclusions Our data suggest that the aberrant expression of EML4-ALK leads to JAK2-STAT signaling pathway activation, which is essential for the development of non-small cell lung cancer.

Theranostic liposomes have recently found a broad range of applications in nanomedicine due to stability, the high solubility of biomacromolecules, bioavailability, efficacy, and low adverse effects. However, the limitations of liposomes concerning the short systemic circulation in the body, limited controllability of the release rate, and the inability of in vivo imaging remain challenging. Herein, the development of novel hybrid ultrasound-activated piezoelectric nanoparticles based on a hybrid liposome nanocarrier composed of poly(vinylidene fluoride-trifluoroethylene), graphene quantum dots (GQDs), and Silibinin (a hydrophobic drug) is presented. The hybrid nanoparticles are an acoustically sensitive drug delivery platform that releases the biomacromolecules in a specific tissue area (through surface labeling with PD-1 antibody) in a non-invasive and controlled manner. We show that the developed hybrid nanoparticles (with an average outer diameter of ∼ 230 ± 20 nm) enable piezoelectric-stimulated drug delivery combined with simultaneous fluorescent imaging of cancer cells in vivo. Significant enhancement (80 % up to 240 h) and tunable drug release from the nanocarrier through enhanced diffusion from the liposome membrane are demonstrated. Cytotoxicity assays using MCF-7, 4T1, and NIH3T3 cell lines exhibit no confrontational influence of nanoparticles on cell viability up to 125 µg/ml. The PD-1 antibody on the surface of the hybrid nanocarrier allows for selective delivery to breast cancer tumors and low biodistribution to other tissues. Our results affirm that the developed ultrasound-activated piezoelectric nanoparticles have great potential as multifunctional platforms with sustainable release profiles for the delivery of hydrophobic drugs to breast cancer, especially when the ability for adequate labeling and cell monitoring is valued.

Cell senescence is one of the most important forms of injury induced by cardiovascular and other ischemic diseases. Fibroblasts are important participants in tissue repair after ischemic injury and the main source of IL11 secretion. However, the roles of oxygen–glucose deprivation (OGD) and IL11 in promoting fibroblast senescence and their regulatory mechanisms remain unclear. This study selected the NIH3T3 and L929 fibroblast cell lines as research objects. We found that OGD could induce the expression of p53, P16, p21, and collagen in fibroblasts. In the condition of OGD, when IL11 intervened, fibroblasts’ senescence and collagen expression were changed. Some studies have found that changes in kynurenine (KYN) metabolism are related to aging diseases, and indoleamine 2,3-dioxygenase 1 (IDO1) is a key rate-limiting enzyme in the KYN metabolic pathway. We found that KYN secretion decreased after OGD increased fibroblast senescence, and inhibition of IL11 promoted IDO1 and increased KYN secretion. These results suggest that OGD may promote fibroblast senescence and collagen expression via IL11 inhibition of the IDO1/KYN metabolic pathway. Therefore, the revealed mechanism of OGD-promoted fibroblast senescence could provide an effective theoretical basis for the clinical treatment of aging-related ischemic diseases.

Oregano infusions have traditionally been used to treat some diseases related to inflammation and cancer; also, some species have shown antiproliferative activity on cancer cell lines, for example, colon and liver, and this has been attributed to its phytochemical profile, mainly its phenolic compounds. This study aimed to evaluate the cytotoxicity and antiproliferative potential of the polyphenols-rich extracts (PRE) of the oregano species

Chronic wounds are associated with infectious microbial complex communities called biofilms. The management of chronic wound infection is limited by the complexity of selecting an appropriate antimicrobial dressing with antibiofilm activity due to antimicrobial resistance in biofilms. Herein, the in situ developed bacterial cellulose/poly(vinyl alcohol) (BC-PVA) composite is ex situ modified with genipin-crosslinked silk sericin (SS) and azithromycin (AZM) (SSga). The composite is evaluated as a wound dressing material for preventing the development, dispersion, and/or eradication of microbial biofilm. Fourier transform infrared spectroscopy confirms the intermolecular interactions between the components of BC-PVA@SSga scaffolds. The addition of PVA during BC production significantly increases the porosity from 53.5% ± 2.3% to 83.5% ± 2.9%, the pore size from 2.3 ± 1.9 to 16.8 ± 4.5 µm, the fiber diameter from 35.5 ± 10 to 120 ± 27.4 nm, and improves the thermal stability and flexibility. Studies using bacteria and fungi indicate high inhibition and disruption of biofilms upon AZM addition. In vitro biocompatibility analysis confirms the nontoxic nature of BC-PVA@SSga toward HaCaT and NIH3T3 cells, whereas the addition of SS enhances cell proliferation. The developed BC-PVA@SSga accelerates wound healing in the infected mouse model, thus can be a promising wound dressing biomaterial.

Angiosarcomas are rare, malignant soft tissue tumors in children that arise in a wide range of anatomical locations and have limited targeted therapies available. Here, we report a rare case of a pediatric angiosarcoma (pAS) with Li-Fraumeni syndrome (LFS) expressing a novel

Genetic mutations in the MYBPC3 gene encoding cardiac myosin binding protein C (cMyBP-C) are the most common cause of hypertrophic cardiomyopathy (HCM). Myocardial fibrosis (MF) plays a critical role in the development of HCM. However, the mechanism for mutant MYBPC3-induced MF is not well defined. In this study, we developed a R495Q mutant pig model using cytosine base editing and observed an early-onset MF in these mutant pigs shortly after birth. Unexpectedly, we found that the “cardiac-specific” MYBPC3 gene was actually expressed in cardiac fibroblasts from different species as well as NIH3T3 fibroblasts at the transcription and protein levels. CRISPR-mediated disruption of Mybpc3 in NIH3T3 fibroblasts activated nuclear factor κB (NF-κB) signaling pathway, which increased the expression of transforming growth factor beta (TGF-β1) and other pro-inflammatory genes. The up-regulation of TGF-β1 promoted the expression of hypoxia-inducible factor-1 subunit α (HIF-1α) and its downstream targets involved in glycolysis such as GLUT1, PFK and LDHA. Consequently, the enhanced aerobic glycolysis with higher rate of ATP biosynthesis accelerated the activation of cardiac fibroblasts, contributing to the development of HCM. This work reveals an intrinsic role of MYBPC3 in maintaining cardiac fibroblast homeostasis and disruption of MYBPC3 in these cells contributes to the disease pathogenesis of HCM.

In the last decade, DNA-based tension sensors have made significant contributions to the study of the importance of mechanical forces in many biological systems. Albeit successful, one shortcoming of these techniques is their inability to reversibly measure receptor forces in a higher regime (that is, >20 pN), which limits our understanding of the molecular details of mechanochemical transduction in living cells. Here, we developed a reversible shearing DNA-based tension probe (RSDTP) for probing molecular piconewton-scale forces between 4 and 60 pN transmitted by cells. Using these probes, we can easily distinguish the differences in force-bearing integrins without perturbing adhesion biology and reveal that a strong force-bearing integrin cluster can serve as a ‘mechanical pivot’ to maintain focal adhesion architecture and facilitate its maturation. The benefits of the RSDTP include a high dynamic range, reversibility and single-molecule sensitivity, all of which will facilitate a better understanding of the molecular mechanisms of mechanobiology. Li et al. develop reversible shearing DNA-based tension probes to quantify molecular piconewton-scale forces, estimate the number of mechanically active receptors with single-molecule sensitivity and study mechanisms of force transduction in live cells.

Rationale: Cardiomyopathy is characterized by the deposition of extracellular matrix by activated resident cardiac fibroblasts called myofibroblasts. There are currently no therapeutic approaches to blunt the development of pathological fibrosis and ventricle chamber stiffening that ultimately leads to heart failure. Objective: We undertook a high-throughput screen to identify small molecule inhibitors of myofibroblast activation that might limit the progression of heart failure. We evaluated the therapeutic efficacy of the polyether ionophore salinomycin in patient-derived cardiac fibroblasts and preclinical mouse models of ischemic and nonischemic heart failure. Methods and Results: Here, we demonstrate that salinomycin displays potent anti-fibrotic activity in cardiac fibroblasts obtained from heart failure patients. In preclinical studies, salinomycin prevents cardiac fibrosis and functional decline in mouse models of ischemic and nonischemic heart disease. Remarkably, interventional treatment with salinomycin attenuates preestablished pathological cardiac remodeling secondary to hypertension and limits scar expansion when administered after a severe myocardial infarction. Mechanistically, salinomycin inhibits cardiac fibroblast activation by preventing p38/MAPK (mitogen activated protein kinase) and Rho signaling. Salinomycin also promotes cardiomyocyte survival and improves coronary vessel density, suggesting that cardioprotection conferred by salinomycin occurs via the integration of multiple mechanisms in multiple relevant cardiac cell types. Conclusions: These data establish salinomycin as an antifibrotic agent that targets multiple cardioprotection pathways, thereby holding promise for the treatment of heart failure patients.

Live cell imaging using fluorescent DNA markers are an indispensable molecular tool in various biological and biomedical fields. It is a challenge to develop DNA probes that avoid UV light photo-excitation, have high specificity for DNA, are cell-permeable and are compatible with cutting-edge imaging techniques such as super-resolution microscopy. Herein, we present N-aryl pyrido cyanine (N-aryl-PC) derivatives as a class of long absorption DNA markers with absorption in the wide range of visible light. The high DNA specificity and membrane permeability allow the staining of both organelle DNA as well as nuclear DNA, in various cell types, including plant tissues, without the need for washing post-staining. N-aryl-PC dyes are also highly compatible with a separation of photon by lifetime tuning method in stimulated emission depletion microscopy (SPLIT-STED) for super-resolution imaging as well as two-photon microscopy for deep tissue imaging, making it a powerful tool in the life sciences.

Elevated plasma levels of hyaluronic acid (HA) is a disease marker in liver pathology and other inflammatory disorders. Inhibition of HA synthesis with coumarin 4-methylumbelliferone (4MU) has a beneficial effect in animal models of fibrosis, inflammation, cancer and metabolic syndrome. 4MU is an active compound of approved choleretic drug hymecromone with low bioavailability and a broad spectrum of action. New, more specific and efficient inhibitors of hyaluronan synthases (HAS) are required. We have tested several newly synthesized coumarin compounds and commercial chitin synthesis inhibitors to inhibit HA production in cell culture assay. Coumarin derivative compound VII (10′-methyl-6′-phenyl-3′H-spiro[piperidine-4,2′-pyrano[3,2-g]chromene]-4′,8′-dione) demonstrated inhibition of HA secretion by NIH3T3 cells with the half-maximal inhibitory concentration (IC50) = 1.69 ± 0.75 μΜ superior to 4MU (IC50 = 8.68 ± 1.6 μΜ). Inhibitors of chitin synthesis, etoxazole, buprofezin, triflumuron, reduced HA deposition with IC50 of 4.21 ± 3.82 μΜ, 1.24 ± 0.87 μΜ and 1.48 ± 1.44 μΜ, respectively. Etoxazole reduced HA production and prevented collagen fibre formation in the CCl4 liver fibrosis model in mice similar to 4MU. Bioinformatics analysis revealed homology between chitin synthases and HAS enzymes, particularly in the pore-forming domain, containing the proposed site for etoxazole binding.

The sensing and generation of cellular forces are essential aspects of life. Traction force microscopy (TFM) has emerged as a standard broadly applicable methodology to measure cell contractility and its role in cell behavior. While TFM platforms have enabled diverse discoveries, their implementation remains limited in part due to various constraints, such as time-consuming substrate fabrication techniques, the need to detach cells to measure null force images, followed by complex imaging and analysis, and the unavailability of cells for postprocessing. Here we introduce a reference-free technique to measure cell contractile work in real time, with commonly available substrate fabrication methodologies, simple imaging, and analysis with the availability of the cells for postprocessing. In this technique, we confine the cells on fluorescent adhesive protein micropatterns of a known area on compliant silicone substrates and use the cell deformed pattern area to calculate cell contractile work. We validated this approach by comparing this pattern-based contractility screening (PaCS) with conventional bead-displacement TFM and show quantitative agreement between the methodologies. Using this platform, we measure the contractile work of highly metastatic MDA-MB-231 breast cancer cells that is significantly higher than the contractile work of noninvasive MCF-7 cells. PaCS enables the broader implementation of contractile work measurements in diverse quantitative biology and biomedical applications.

Mouse telomerase and the DNA polymerase alpha-primase complex elongate the leading and lagging strands of telomeres, respectively. To elucidate the molecular mechanism of lagging strand synthesis, we investigated the interaction between DNA polymerase alpha and two paralogs of the mouse POT1 telomere-binding protein (POT1a and POT1b). Yeast two-hybrid analysis and a glutathione S-transferase pull-down assay indicated that the C-terminal region of POT1a/b binds to the intrinsically disordered N-terminal region of p180, the catalytic subunit of mouse DNA polymerase alpha. Subcellular distribution analyses showed that although POT1a, POT1b, and TPP1 were localized to the cytoplasm, POT1a-TPP1 and POT1b-TPP1 coexpressed with TIN2 localized to the nucleus in a TIN2 dose-dependent manner. Coimmunoprecipitation and cell cycle synchronization experiments indicated that POT1b-TPP1-TIN2 was more strongly associated with p180 than POT1a-TPP1-TIN2, and this complex accumulated during the S phase. Fluorescence in situ hybridization and proximity ligation assays showed that POT1a and POT1b interacted with p180 and TIN2 on telomeric chromatin. Based on the present study and a previous study, we propose a model in which POT1a/b-TPP1-TIN2 translocates into the nucleus in a TIN2 dose-dependent manner to target the telomere, where POT1a/b interacts with DNA polymerase alpha for recruitment at the telomere for lagging strand synthesis.

γ-Glutamylcyclotransferase (GGCT) is involved in glutathione homeostasis, in which it catalyzes the reaction that generates 5-oxoproline and free amino acids from γ-glutamyl peptides. Increasing evidence shows that GGCT has oncogenic functions and is overexpressed in various cancer tissues, and that inhibition of GGCT activity exerts anticancer effects in vitro and in vivo. Here, we demonstrate that U83836E ((2R)-2-[[4-(2,6-dipyrrolidin-1-ylpyrimidin-4-yl)piperazin-1-yl]methyl]-3,4-dihydro-2,5,7,8,-tetramethyl-2H-1-benzopyran-6-ol, dihydrochloride), a lazaroid that inhibits lipid peroxidation, inhibits GGCT enzymatic activity. U83836E was identified from a high-throughput screen of low molecular weight compounds using a fluorochrome-conjugated GGCT probe. We directly quantified that U83836E specifically inhibited GGCT by measuring the product of a fluorochrome-conjugated GGCT substrate assay, and showed that U83836E inhibited GGCT activity in extracts of NIH3T3 cells overexpressing GGCT. Moreover, U83836E significantly inhibited tumor growth in a xenograft model that used immunodeficient mice orthotopically inoculated with MCF7 human breast cancer cells. These results indicate that U83836E may be a useful GGCT inhibitor for the development of potential cancer therapeutics.

Targeting the dimer interface for the epidermal growth factor receptor (EGFR) that is highly conserved in the structure and directly involved in dimerization may solve the resistance problem that plagues anti-EGFR therapy. Heavy chain single domain antibodies have promising prospects as therapeutic antibodies. A bispecific nanobody was constructed based on previously screened humanized nanobodies that target the β-loop at the EGFR dimer interface, an anti-FcγRIIIa (CD16) of natural killer cells (NK) nanobodies and anti-human serum albumin (HSA) nanobodies. The target gene was effectively expressed and secreted while controlled by promoter GAP in Pichia pastoris X33, and the expressed product was purified with a cation exchange and nickel chelation chromatography. The bispecific nanobody specifically bound to the surfaces of EGFR-overexpressed human epidermal carcinoma A431 cells and effectively inhibited tumor cell growth both in vitro and in vivo. In the A431 cell nude mouse xenograft model, the growth inhibition effect from the bispecific nanobody was significantly increased with the assistance of peripheral blood mononuclear cells (PBMCs), which was consistent with the results obtained in vitro, suggesting that there was an antibody-dependent cell-mediated cytotoxicity (ADCC) effect. In addition, the intraperitoneal administration of bispecific nanobodies effectively reached tumor tissues in the shoulder dorsal region, but in significantly less distributed quantities than EGFR Dimer Nb77. To conclude, a bispecific nanobody targeting the EGFR dimer interface with ADCC effect was successfully constructed.

Developing a new family of hydrogel-based wound dressings that could have a dual biofunctionality of antibacterial and biological responses is highly desirable. In this study, an inherently effective antibacterial and biodegradable hydrogel dressing without the need for impregnated antibiotics was designed, synthesized, characterized, and examined for its effect on macrophages, which initiated inflammatory activity and activated both NO and TNF-α production for the purpose of achieving a better and faster wound healing. The purposes of this research was to develop a novel family of cationic biodegradable hydrogels based on arginine-based poly(ester urea urethane) (Arg-PEUU) and glycidyl methacrylate-modified chitosan (CS-GMA) that has both inherent antibacterial and bioactive functionality as a wound healing dressing for accelerated healing of contaminated or infected wounds. These hybrid hydrogels present a well-defined three-dimensional microporous network structure and have a high water absorption ability, and their biodegradation is effectively accelerated in the presence of lysozymes. The hemolytic activity test, MTT assay, and live/dead assay of these hybrid hydrogels indicated that they had no cytotoxicity toward red blood cells, NIH-3T3 fibroblast cells, and human vascular endothelial cells, thus corroborating their cytocompatibility. Furthermore, these hybrid hydrogels could elevate the release of both produced NO and TNF-α by stimulating and activating RAW 264.7 macrophages, augmenting their antibacterial biological response. The antibacterial assay of these hybrid hydrogels demonstrated their excellent antibacterial activity without the need for impregnated antibacterial agents. Taken together, this new family of biodegradable, antibacterial, and biologically responsive hybrid hydrogels exhibits great potential as biofunctional antibacterial wound dressing candidates for wound healing.