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Preclinical characterization of the JAK/STAT inhibitor SGI-1252 on skeletal muscle function, morphology, and satellite cell content

"Recent studies have highlighted the JAK/STAT signaling pathway in the regulation of muscle satellite cell behavior. Herein we report preclinical studies designed to characterize the effects of a novel JAK/STAT inhibitor on plantar flexor skeletal muscle function, morphology, and satellite cell content. The compound, SGI-1252, was administered orally (400mg/kg) in a 10% dextrose solution to wild type mice (n = 6) 3 times per week for 8 weeks. A control group (n = 6) received only the dextrose solution. SGI-1252 was well tolerated, as animals displayed similar weight gain over the 8-week treatment period. Following treatment, fatigue in the gastrocnemius-soleus-plantaris complexwas greater in the SGI-1252 mice during a 300 second tetanic contraction bout (p = 0.035),though both the rate of fatigue and maximal force production were similar. SGI-1252 treated mice had increased type II myofiber cross-sectional area (1434.8 ± 225.4 vs 1754.7 ±138.5 μm2), along with an increase in wet muscle mass (125.45 ± 5.46 vs 139.6 ± 12.34 mg,p = 0.032) of the gastrocnemius relative to vehicle treated mice. SGI-1252 treatment reduced gastrocnemius STAT3 phosphorylation 53% (94.79 ± 45.9 vs 44.5 ± 6.1 MFI) and significantly increased the concentration of Pax7+ satellite cells (2589.2 ± 105.5 vs 2859.4 ±177.5 SC/mm3) in the gastrocnemius. SGI-1252 treatment suppressed MyoD (p = 0.013) and Myogenin (p<0.0001) expression in human primary myoblasts, resulting in reduced myogenic differentiation (p = 0.039). Orally delivered SGI-1252 was well tolerated, attenuates skeletal muscle STAT3 activity, and increases satellite cell content in mouse gastrocnemius muscle, likely by inhibiting myogenic progression."

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The cytosolic N-terminus motif of MAL2 promotes changes in the cell membrane morphology and decreases cell proliferation, migration and invasion in liver-derived cancer cells

Myelin and Lymphocyte Protein 2 (MAL2), a lipid raft associated protein that is involved in the basolateral-to-apical transcytotic machinery of polarized epithelial cells, has been found to be highly up-regulated in a variety of human carcinomas including cholangiocarcinomas, renal carcinomas, metaplasias of the stomach, and cancers of the breast, ovary and pancreas. Recent work has also correlated MAL2 up-regulation with poor survival in patients with pancreatic cancer. However, it is still unknown how MAL2 up-regulation relates to tumorigenesis and cancer progression. One of the hallmarks of cancer is the loss of cell polarity; in fact, most human carcinomas derive from polarized epithelial cells and are AASLD Abstracts AASLD Abstracts characterized by the loss of or failure to achieve polarity. This is of particular interest to us because MAL2 is thought to be involved with the establishment and/or maintenance of a polarized phenotype. Therefore, it is important to consider how loss or dysregulation of polarized protein trafficking contributes to malignant transformation. To identify possible mechanisms linking MAL2 up-regulation to malignancy, we overexpressed wild-type MAL2 in hepatoma-derived Clone9 cells that lack endogenous MAL2. Our results showed that MAL2 overexpression induced actin-based protrusions formation with MAL2 localized to their tips.

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A novel quinazoline derivative, MJ-56, exhibits phototoxicity toward human bladder cancer cells

Background: Quinazolines, which process a wide spectrum of biological properties such as antibacterial, antifungal, antivirus, and anticancer activities, are considered one of the most important heterocycles in medicinal chemistry. Here, we described for the first time the novel quinazoline derivative MJ-56 (6-pyrrolidinyl-2-(3-bromostyryl) quinazoline-4-one) which emits green fluorescent in the cytosol and exhibits phototoxicity toward human bladder cancer (BC) cells under blue-light exposure. Materials and Methods: Human BC cells (5637 and T24) and immortalized uroepithelial cell (SV-HUC1) were utilized in this study. To trace the localization of MJ-56, MitoTracker and LysoTracker were applied. The cell viability with or without blue light exposure were monitored by WST-1 reagent, direct recording, and clonogenic assays. The apoptosis induction in MJ-56 treated cells was detected. Results: MJ-56 emits green fluorescent in the cytosol. Vital staining of mitochondria or lysosomes demonstrated that the MJ-56 fluorescent was not located in either organelles. MJ-56 treatment for 24 h did not cause significant loss of cell viability in BC cells. However, treatment of 0.125 μM MJ-56 for 1 h and exposed to blue light for 15 mins significantly reduced cell viability. Interestingly, our results showed that MJ-56 has minimal impact on SV-HUC1 even with the blue-light exposure. The caspase 3/7 activities in BC cells treated with MJ-56 and exposed to blue light were significantly increased 1 h post-treatment. However, the DNA fragmentation cannot be detected at 1, 6, or 24 h posttreatment due to the loss of viable cells. Conclusions: MJ-56 exhibits phototoxicity toward BC cells with minimal impact on uroepithelial cells, indicating a novel therapeutic agent against BC. The mechanism underlying MJ-56-induced cell death as well as the translational studies warrants further investigation.

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Neuroregeneration improved by ketones

Ketone bodies serve as alternative fuel for the brain when glucose availability is limited. The ketone body beta‐hydroxybutyrate (BHB) is synthesized in the liver from acetoacetate, the first ketone produced in the fasting state. Several studies show that being in the state of ketosis can be neuroprotective, but the exact mechanism is not known. It is also not known whether ketones would aid regeneration of a nervous system injury. In this study we investigated on cell cultures whether ketone bodies improve neuroregeneration after injury or trauma. The effect of ketone bodies on cell regeneration was tested in 3 weeks old rat (Sprague Dawley) primary neuronal cell cultures. Scratch assay was performed for 24 hours, while the cells were either exposed to 2mM R, S‐sodium‐3‐hydroxibutyrate (BHB) or not (control). One hundred snapshots were taken with the CYTOSMART system to document the cell migration, changes in cell density and the process of cell regeneration into the damaged area over the 24 hours. Immunofluorescence reaction was performed to label beta III tubulin and synapsin I in the regenerating areas. Cell cultures treated with BHB showed more intense cell migration and regeneration of the damaged area over the 24 hour period. Significantly more DAPI stained cell nuclei were found in the damaged area in cell cultures with 2mM BHB at 20× (p=0.01)and at 10× (p=0.0004) magnification photos as well, and the cell coverage was higher than in control cell cultures. Markers that are associated with flagellar movement, cytoskeletal functions and axonogenesis, synaptogenesis were denser in treated neuronal cultures at the regeneration site as revealed by fluorescence microscopy. These above results show potential applications for ketogenic compounds for treating traumatic brain injury or other injury to the central nervous system.

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Random and oriented electrospun fibers based on a multicomponent, in situ clickable elastin-like recombinamer system for dermal tissue engineering

Herein we present a system to obtain fibers from clickable elastin-like recombinamers (ELRs) that crosslink in situ during the electrospinning process itself, with no need for any further treatment to stabilize them. These ELR-click fibers are completely stable under in vitro conditions. A wrinkled fiber morphology is obtained. In addition to a random fiber orientation, oriented fibers with a high degree of alignment and coherence can also be obtained by using a rotational electrode. The production of multicomponent fibers means that different functionalities, such as cell-adhesion domains (RGD peptides), can be incorporated into them. In a subsequent study, two main cell lines present in the dermis and epidermis, namely keratinocytes and fibroblasts, were cultured on top of the ELR-click fibers. Adhesion, proliferation, fluorescence, immunostaining and histology studies showed the cytocompatibility of these scaffolds, thus suggesting their possible use for wound dressings in skin tissue engineering applications.

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Robot-scientists will lead tomorrow's biomaterials discovery

Biomaterials engineering is tightly linked with progress in its underlying sciences and technologies, such as biology, chemistry, physics, and engineering. Current establishment of high throughput screening platforms has warranted the need for data analysis as part of biomaterials engineering. We believe that current advancement in artificial intelligence, miniaturization of materials fabrication and application of robotics will eventually lead to the emergence of autonomous, intelligent systems able to perform biomaterials research on their own. In this manuscript, we describe the state of the art in the triangle of material engineering, biology, and data science, and sketch their integration to yield the biomaterials conveyer belt of tomorrow.

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Dose intensification of TRAIL-inducing ONC201 inhibits metastasis and promotes intratumoral NK cell recruitment

ONC201 is a first-in-class, orally active antitumor agent that upregulates cytotoxic TRAIL pathway signaling in cancer cells. ONC201 has demonstrated safety and preliminary efficacy in a first-in-human trial in which patients were dosed every 3 weeks. We hypothesized that dose intensification of ONC201 may impact antitumor efficacy. We discovered that ONC201 exerts dose- and schedule-dependent effects on tumor progression and cell death signaling in vivo. With dose intensification, we note a potent anti-metastasis effect and inhibition of cancer cell migration and invasion. Our preclinical results prompted a change in ONC201 dosing in all open clinical trials. We observed accumulation of activated NK+ and CD3+ cells within ONC201-treated tumors and that NK cell depletion inhibits ONC201 efficacy in vivo, including against TRAIL/ONC201-resistant Bax–/– tumors. Immunocompetent NCR1-GFP mice, in which NK cells express GFP, demonstrated GFP+ NK cell infiltration of syngeneic MC38 colorectal tumors. Activation of primary human NK cells and increased degranulation occurred in response to ONC201. Coculture experiments identified a role for TRAIL in human NK-mediated antitumor cytotoxicity. Preclinical results indicate the potential utility for ONC201 plus anti–PD-1 therapy. We observed an increase in activated TRAIL-secreting NK cells in the peripheral blood of patients after ONC201 treatment. The results offer what we believe to be a unique pathway of immune stimulation for cancer therapy.

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Chloroquine induces lysosomal membrane permeability-mediated cell death in bladder cancer cells

Chloroquine (CQ) is recognized as a potent adjuvant when combined with other chemotherapies to treat cancers. However, the effects of a single treatment of CQ on bladder cancer (BC) cells have not been investigated. The growth and viability of CQ-treated BC cells were examined. The lysosomal morphology was detected using LysoTracker. The induction of lysosomal membrane permeability (LMP) was detected by acridine orange (AO) translocation, and cathepsin B and D release. The expression of the bid, caspase-3, and cytosolic cytochrome C (Cyto. C) in CQ-treated cells was detected by the Western blot. The pepstatin A and E64d were used to attenuate CQ-induced LMP. A single dose of CQ treatment induced BC cell death, and attenuated by pepstatin A and E64d. The diminishing of fluorescent in CQ-treated cells stained with LysoTracker, suggesting that CQ targets lysosomal functions. This was further supported by increased AO translocation and the releasing of CatB and CatD into the cytosol. The increased level of cleavage bid and cytosolic Cyto. C indicated mitochondrial outer membrane permeabilization and subsequently leading to apoptosis induction judged by the increased level of activated caspase 3. CQ-induced LMP that enhances apoptosis and ultimately leading to BC cell death. The study results demonstrated for the first time that single CQ treatment against BC cells by inducing LMP and subsequent mitochondria membrane permeability that trigger apoptosis, making it a potential treatment for BC therapy in the future.

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Quantitative assessment of cancer cell morphology and motility using telecentric digital holographic microscopy and machine learning

The noninvasive, fast acquisition of quantitative phase maps using digital holographic microscopy (DHM) allows tracking of rapid cellular motility on transparent substrates. On two-dimensional surfaces in vitro, MDA-MB-231 cancer cells assume several morphologies related to the mode of migration and substrate stiffness, relevant to mechanisms of cancer invasiveness in vivo. The quantitative phase information from DHM may accurately classify adhesive cancer cell subpopulations with clinical relevance. To test this, cells from the invasive breast cancer MDA-MB-231 cell line were cultured on glass, tissue-culture treated polystyrene, and collagen hydrogels, and imaged with DHM followed by epifluorescence microscopy after staining F-actin and nuclei. Trends in cell phase parameters were tracked on the different substrates, during cell division, and during matrix adhesion, relating them to F-actin features. Support vector machine learning algorithms were trained and tested using parameters from holographic phase reconstructions and cell geometric features from conventional phase images, and used to distinguish between elongated and rounded cell morphologies. DHM was able to distinguish between elongated and rounded morphologies of MDA-MB-231 cells with 94% accuracy, compared to 83% accuracy using cell geometric features from conventional brightfield microscopy. This finding indicates the potential of DHM to detect and monitor cancer cell morphologies relevant to cell cycle phase status, substrate adhesion, and motility.

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The transcription factor ZEB1 promotes an aggressive phenotype in prostate cancer cell lines

It has been reported that one of the factors that promotes tumoral progression is the abnormal activation of the epithelial-mesenchymal transition program. This process is associated with tumoral cells acquiring invasive and malignant properties and has the transcription factor zinc finger E-box-binding homeobox 1 (ZEB1) as one of its main activators. However, the role of ZEB1 in promoting malignancy in prostate cancer (PCa) is still unclear. Here, we report that ZEB1 expression correlates with Gleason score in PCa samples and that expression of ZEB1 regulates epithelial-mesenchymal transition and malignant characteristics in PCa cell lines. The results showed that ZEB1 expression is higher in samples of higher malignancy and that overexpression of ZEB1 was able to induce epithelial-mesenchymal transition by upregulating the mesenchymal marker Vimentin and downregulating the epithelial marker E-Cadherin. On the contrary, ZEB1 silencing repressed Vimentin expression and upregulated E-Cadherin. ZEB1 expression conferred enhanced motility and invasiveness and a higher colony formation capacity to 22Rv1 cells whereas DU145 cells with ZEB1 silencing showed a decrease in those same properties. The results showed that ZEB1 could be a key promoter of tumoral progression toward advanced stages of PCa.

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