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The role of transient receptor potential melastatin 7 (TRPM7) in cell viability: a potential target to suppress breast cancer cell cycle

The divalent cation-selective channel transient receptor potential melastatin 7 (TRPM7) channel was shown to affect the proliferation of some types of cancer cell. However, the function of TRPM7 in the viability of breast cancer cells remains unclear. Here we show that TRPM inhibitors suppressed the viability of TRPM7-expressing breast cancer cells. We first demonstrated that the TRPM7 inhibitors 2-aminoethyl diphenylborinate (2-APB), ginsenoside Rd (Gin Rd), and waixenicin A preferentially suppressed the viability of human embryonic kidney HEK293 overexpressing TRPM7 (HEK-M7) cells over wildtype HEK293 (WT-HEK). Next, we confirmed the effects of 2-APB on the TRPM7 channel functions by whole-cell currents and divalent cation influx. The inhibition of the viability of HEK-M7 cells by 2-APB was not mediated by the increase in cell death but by the interruption of the cell cycle. Similar to HEK-M7 cells, the viability of TRPM7-expressing human breast cancer MDA-MB-231, AU565, and T47D cells were also suppressed by 2-APB by arresting the cell cycle in the S phase. Furthermore, in a novel TRPM7 knock-out MDA-MB-231 (KO-231) cell line, decreased divalent influx and reduced proliferation were observed compared to the wildtype MDA-MB-231 cells. 2-APB and Gin Rd preferentially suppressed the viability of wildtype MDA-MB-231 cells over KO-231 by affecting the cell cycle in wildtype but not KO-231 cells. Our results suggest that TRPM7 regulates the cell cycle of breast cancers and is a potential therapeutic target.

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Development of an in-situ forming, self-healing scaffold for dermal wound healing: in-vitro and in-vivo studies

The importance of the extra-cellular matrix (ECM) for wound healing has been extensively researched. Under-standing its importance, multiple ECM mimetic scaffolds have been developed. However, the majority of such scaffolds are prefabricated. Due to their stiffness, prefabricated scaffolds cannot come into direct contact with the basal skin cells at the wound bed, limiting their efficacy. We have developed a unique wound dressing, using chitosan (CH) and chondroitin sulfate (CS), that can form a porous scaffold (CH-CS PEC) in-situ, at the wound site, by simple mixing of the polymer solutions. As CH is positively and CS is negatively charged, mixing these two polymer solutions would lead to electrostatic cross-linking between the polymers, converting them to a porous, viscoelastic scaffold. Owing to the in-situ formation, the scaffold can come in direct contact with the cells at the wound bed, supporting their proliferation and biofunction. In the present study, we confirmed the cross- linked scaffold formation by solid-state NMR, XRD, and TGA analysis. We have demonstrated that the scaffold had a high viscoelastic property, with self-healing capability. Both keratinocyte and fibroblast cells exhibited significantly increased migration and functional markers expression when grown on this scaffold. In the rat skin- excisional wound model, treatment with the in-situ forming CH-CS PEC exhibited enhanced wound healing ef-ficacy. Altogether, this study demonstrated that mixing CH and CS solutions lead to the spontaneous formation of a highly viscoelastic, porous scaffold, which can support epidermal and dermal cell proliferation and bio- function, with an enhanced in-vivo wound healing efficacy.

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Bionanomachine diagnostics and nanonetwork therapeutic in brain malignancies with bionanodevice interfaces

Molecular Communication is evolving as a supradiscipline of bioinspired information and communication technologies. The next breakthroughs in molecular communication will greatly depend on the deep understanding of complex diseases, epitomized by brain malignancies. This will be achieved by delineating underlying cellular and sub-cellular processes in the context of interactive bionanomachines and their comprehensive management with diagnostic and therapeutic interventions controlled externally. In this paper, we present our recent progress in the fields of Externally Controllable Molecular Communication and brain malignancies, aiming towards drastic transformation in the way we diagnose and treat such disorders. Promising recent data involve targeting cell- and exosome-mediated theranostic systems as well as phenotypic switching and network formation. With the aim to transform Externally Controllable Molecular Communication from a theoretical concept to a clinically applicable technology, we identify gaps and challenges and share our views and thoughts on how advancements in diagnostics and therapeutics based on bionanomachine, nanonetwork and bionanodevice interfaces can bring a paradigm shift in the way we manage malignant disorders, via targeted breakthroughs.

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Chemical composition, biological properties and bioinformatics analysis of two Caesalpina species: A new light in the road from nature to pharmacy shelf

Caesalpinia bonduc and C. decapeleta var. japonica have great importance in traditional medicine systems but scientific information’s are still lacking for their potentials. To explore their bioactivity, we assessed the antioxidant, enzyme inhibitory abilities of the dichloromethane (DCM), ethyl acetate, methanol, and water extracts prepared from the leaves and bark. The cytotoxicity and anticancer properties of the extracts were also assessed in vitro. The water extract of C. decapeleta leaves possessed highest phenolic content (108.16 mg gallic acid equivalent (GAE)/g extract), while the highest flavonoid content was recorded for the C. bonduc leaf methanolic extract (27.89 mg rutin equivalent (RE)/g extract). In general, C. decapeleta extracts possessed higher radical scavenging potential compared to C. bonduc extracts. C. decapeleta DCM leaves extract (10.20 mg galantamine equivalent (GALAE)/g extract) showed highest inhibition against butyrylcholinesterase. The cytotoxicity of the most potent methanolic and aqueous extracts were assessed against four cell lines. The chemical profiles of both species appeared to be different. C. bonduc was abundant in organic and phenolic acids as well as their esters. Flavonoid glycosides, bonducellin and its derivatives and caesalminaxins were identified. Whereas, C. decalpetala possessed many galloylated compounds. The cytotoxicity of C. bonduc and C. decapetala extracts was tested using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) based assay on VERO (kidney of an adult African Green monkey cells), HeLa (human cervical adenocarcinoma cells), RKO (human colon carcinoma cells), FaDu (human hypopharyngeal squamous carcinoma cells) cell lines. C. bonduc bark water extract exhibited the highest cytotoxicity towards HeLa (50 % cytotoxic concentration (CC50): 28.5 μg/mL) cancer cell line, as compared to normal VERO cells (CC50:35.87 μg/mL). For C. decapetala, the highest cytotoxicity was found for bark methanol extract on the HeLa cells with CC50 of 46.08 μg/mL and selectivity index of 3.33. In the gene ontology analysis, prostate cancer, nuclear factor kappa B (NF-kappa B) signaling, proteoglycans in cancer pathways might support the results of the cytotoxic assays. These results showed that the tested Caesalpinia species, showing potent inhibitory action against butyrylcholinesterase, might represent novel phytotherapeutic avenues for the management of Alzheimer’s disease.

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Neither gonadotropin nor cumulus cell expansion is needed for the maturation of competent porcine oocytes in vitro

In vitro maturation of oocytes from immature females is widely used in assisted reproductive technologies. Here we illustrate that cumulus cell (CC) expansion, once considered a key indicator of oocyte quality, is not needed for oocytes to mature to the metaphase II (MII) stage and to gain nuclear and cytoplasmic competence to produce offspring. Juvenile pig oocytes were matured in four different media: 1) Basal (-gonadotropins (GN)-FLI); 2) -GN+FLI (supplement of FGF2, LIF, and IGF1); 3) +GN-FLI; 4) +GN+FLI. There was no difference in maturation to MII or progression to the blastocyst stage after fertilization of oocytes that had been matured in -GN+FLI medium and oocytes matured in +GN+FLI medium. Only slight CC expansion occurred in the two media lacking GN compared to the two where GN was present. The cumulus-oocytes-complexes (COC) matured in +GN+FLI exhibited the greatest expansion. We conclude that FLI has a dual role. It is directly responsible for oocyte competence, a process where GN are not required, and, when GN are present, it has a downstream role in enhancing CC expansion. Our study also shows that elevated phosphorylated MAPK may not be a necessary correlate of oocyte maturation and that the greater utilization of glucose by COC observed in +GN+FLI medium probably plays a more significant role to meet the biosynthetic needs of the CC to expand than to attain oocyte developmental competence. Gene expression analyses have not been informative in providing a mechanism to explain how FLI medium enhances oocyte competence without promoting CC expansion.

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Proteomics of resistance to Notch1 inhibition in acute lymphoblastic leukemia reveals targetable kinase signatures

Notch1 is a crucial oncogenic driver in T-cell acute lymphoblastic leukemia (T-ALL), making it an attractive therapeutic target. However, the success of targeted therapy using γ-secretase inhibitors (GSIs), small molecules blocking Notch cleavage and subsequent activation, has been limited due to development of resistance, thus restricting its clinical efficacy. Here, we systematically compare GSI resistant and sensitive cell states by quantitative mass spectrometry-based phosphoproteomics, using complementary models of resistance, including T-ALL patient-derived xenografts (PDX) models. Our datasets reveal common mechanisms of GSI resistance, including a distinct kinase signature that involves protein kinase C delta. We demonstrate that the PKC inhibitor sotrastaurin enhances the anti-leukemic activity of GSI in PDX models and completely abrogates the development of acquired GSI resistance in vitro. Overall, we highlight the potential of proteomics to dissect alterations in cellular signaling and identify druggable pathways in cancer.

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Cutting-edge microscopy systems as remote teaching and research tools for undergraduate students

Whether due to illness, weather, safety, or other concerns, it is very difficult for biology students to gather meaningful and timely data without access to campus. This has been especially evident during the COVID-19 pandemic, in which most laboratory exercises have been conducted as a simulation. Simulated experiments provide a stopgap for certain courses, but for upper-level and research courses, they are often insufficient. Many new microscopy tools now on the market can be adapted to allow students to generate and analyze novel data with little aid from instructors. Remote brightfield-based systems like the CytoSMART Lux2 can be used to gather real-time insight into the progression of cell growth, cell migration, and cell viability over time. The data from these systems can be viewed via the Internet or downloaded for later analysis. Confocal microscopy also offers unique remote-learning opportunities. Because these fluorescence-based microscopes are controlled almost exclusively by a computer, free “remote desktop” software can allow students to learn how to use this cutting-edge technology and can also allow for the generation and analysis of novel data. While these systems can be expensive, they offer a variety of benefits for undergraduate students and researchers, whether they are in the laboratory or working remotely.

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Filopodium-derived vesicles produced by MIM enhance the migration of recipient cells

Extracellular vesicles (EVs) are classified as large EVs (l-EVs, or microvesicles) and small EVs (s-EVs, or exosomes). S-EVs are thought to be generated from endosomes through a process that mainly depends on the ESCRT protein complex, including ALG-2 interacting protein X (ALIX). However, the mechanisms of l-EV generation from the plasma membrane have not been identified. Membrane curvatures are generated by the bin-amphiphysin-rvs (BAR) family proteins, among which the inverse BAR (I-BAR) proteins are involved in filopodial protrusions. Here, we show that the I-BAR proteins, including missing in metastasis (MIM), generate l-EVs by scission of filopodia. Interestingly, MIM-containing l-EV production was promoted by in vivo equivalent external forces and by the suppression of ALIX, suggesting an alternative mechanism of vesicle formation to s-EVs. The MIM-dependent l-EVs contained lysophospholipids and proteins, including IRS4 and Rac1, which stimulated the migration of recipient cells through lamellipodia formation. Thus, these filopodia-dependent l-EVs, which we named as filopodia-derived vesicles (FDVs), modify cellular behavior.

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Potentiation of electrochemotherapy effectiveness by immunostimulation with IL-12 gene electrotransfer in mice is dependent on tumor immune status

Electrochemotherapy (ECT) exhibits high therapeutic effectiveness in the clinic, achieving up to 80% local tumor control but without a systemic (abscopal) effect. Therefore, we designed a combination therapy consisting of ECT via intratumoral application of bleomycin, oxaliplatin or cisplatin with peritumoral gene electrotransfer of a plasmid encoding interleukin-12 (p. t. IL-12 GET). Our hypothesis was that p. t. IL-12 GET potentiates the effect of ECT on local and systemic levels and that the potentiation varies depending on tumor immune status. Therefore, the combination therapy was tested in three immunologically different murine tumor models. In poorly immunogenic B16F10 melanoma, IL-12 potentiated the antitumor effect of ECT with biologically equivalent low doses of cisplatin, oxaliplatin or bleomycin. The most pronounced potentiation was observed after ECT using cisplatin, resulting in a complete response rate of 38% and an abscopal effect. Compared to B16F10 melanoma, better responsiveness to ECT was observed in more immunogenic 4 T1 mammary carcinoma and CT26 colorectal carcinoma. In both models, p. t. IL-12 GET did not significantly improve the therapeutic outcome of ECT using any of the chemotherapeutic drugs. Collectively, the effectiveness of the combination therapy depends on tumor immune status. ECT was more effective in more immunogenic tumors, but GET exhibited greater contribution in less immunogenic tumors. Thus, the selection of the therapy, namely, either ECT alone or combination therapy with p. t. IL-12, should be predominantly based on tumor immune status.

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A novel ultra‐low conductivity electromanipulation buffer improves cell viability and enhances dielectrophoretic consistency

Cell separation has become a critical diagnostic, research, and treatment tool for personalized medicine. Despite significant advances in cell separation, most widely used applications require the use of mulitple, expensive antibodies to known markers in order to identify subpopulations of cells for separation. Diaelectrophoresis (DEP) provides a biophysical separation technique that can target cell subpopulations based on phenotype without labels and return native cells for downstream analysis. One challenge in employing any DEP device is the sample being separated must be transferred into an ultra-low conductivity medium, which can be detrimental in retaining cells' native phenotypes for separation. Here, we measured properties of traditional DEP reagents and determined that after just 1-2 hours of exposure and subsequent culture, cell's viaiblity was significantly reduced below 50%. We developed and tested a novel buffer (CytoBuffer) that achieved 6 weeks of stable shelf life and demonstrated significantly imporved viability and physiologicla properties. We then dtermined the impmact of CytoBuffer on cell's dielectric properties and morphology and found that cells retained properties more similar to that of their native media. Finally, we vetted CytoBuffer's usability on a cell separation platform (Cyto R1) to determine combined efficacy for cell seperations. Here, more than 80% of cells from different cell lines were recovered and were dtermined to be >70% viable following exposure to CytoBuffer, flow stimulation, electromanipulation, and downstream collection and growth. The developed buffer demonstrated opportunities for electrical manipulation, enrichment, and recovery for next generation cell separations.

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