A Novel In Vitro Simulator to Investigate Promotion of Reconstruction of Damaged Neuronal Cell Colony Differentiated from iPS Cells with the Aid of Micro Dynamic Stimulation

Neuronal cells are equipped with the function of a sensor that senses stimulation and elongates neurites to connect nearby neuronal cells in forming a neuronal network, as they are generally said to be hard to recover from physical damage, such as in the case of a spinal cord injury. Therefore, in this study, a novel in vitro simulator in which micro dynamic stimulations are applied to a damaged neuronal cell colony artificially is proposed to investigate the possibility of promoting the reconstruction of damaged neuronal cells on a colony basis. A neuronal cell colony differentiated from iPS cells is physically damaged by cutting off treatment, and micro dynamic stimulations are applied to the colony by utilizing a developed mini-vibration table system. NeuroFluor NeuO is used to establish a method for fluorescent staining of the living neuronal cells, and morphologies of the reconstructing neurons are analysed, revealing a relationship between the stimulation and the reconstructing process of the damaged neurons. It is found that significant differences are observed in the reconstructing efficiency between the statically cultured damaged neuronal cell colony and the dynamically stimulated one. The results suggest that applying appropriate micro dynamic stimulations is a promising approach to promote the reconstruction of a damaged neuronal cell colony.

The Evidence of the Bystander Effect after Bleomycin Electrotransfer and Irreversible Electroporation

One of current applications of electroporation is electrochemotherapy and electroablation for local cancer treatment. Both of these electroporation modalities share some similarities with radiation therapy, one of which could be the bystander effect. In this study, we aimed to investigate the role of the bystander effect following these electroporation-based treatments. During direct CHO-K1 cell treatment, cells were electroporated using one 100 µs duration square wave electric pulse at 1400 V/cm (for bleomycin electrotransfer) or 2800 V/cm (for irreversible electroporation). To evaluate the bystander effect, the medium was taken from directly treated cells after 24 h incubation and applied on unaffected cells. Six days after the treatment, cell viability and colony sizes were evaluated using the cell colony formation assay. The results showed that the bystander effect after bleomycin electrotransfer had a strong negative impact on cell viability and cell colony size, which decreased to 2.8% and 23.1%, respectively. On the contrary, irreversible electroporation induced a strong positive bystander effect on cell viability, which increased to 149.3%. In conclusion, the results presented may serve as a platform for further analysis of the bystander effect after electroporation-based therapies and may ultimately lead to refined application of these therapies in clinics.

Naturally occurring Girinimbine alkaloid inhibits the proliferation, migration, and invasion of human breast cancer cells via induction of apoptosis and inhibition of MEK/ERK and STAT3 signalling pathways

The currently used anticancer drugs against breast cancer possess serious side effects, have limited efficacy, and often lead to recurrence of the malignancy. The main aim of this research was to investigate the anticancer potential of Girinimbine, a naturally occurring carbazole alkaloid, against ER-negative breast cancer cells (MDA-MB-453) along with its effects on cell migration and invasion, apoptosis, and MEK/ERK/STAT3 pathway. MTT assay was used to evaluate antiproliferative effects of Girinimbine while a clonogenic assay was used to study cell colony formation. Transwell migration and invasion assays were involved to study its effects on cell migration and invasion. Fluorescence microscopy using acridine orange/ethidium bromide was used to study apoptotic effects of girinimbine which was quantified by annexin v-FITC assay. Effects of girinimbine on MEK/ERK and STAT3 signalling pathways were evaluated by western blot assay. Results showed that girinimbine exhibited dose-dependent as well as time-dependent antiproliferative effects in MDA-MB-453 cells along with strongly inhibiting the cell colony potency of these cancerous cells. Girinimbine can inhibit both cancer cell migration as well as invasion. Girinimbine has induced potent chromatin condensation and nuclear fragmentation. The percentage of both early and late apoptotic cells increased significantly after girinimbine exposure. The anticancer effects of girinimbine were shown to be mediated via inhibition of the MEK/ERK as well as STAT3 signalling pathways. In conclusion, it may be proposed that girinimbine could be a promising anticancer agent against breast cancer, provided further in-depth studies are carried out.

Early megakaryocyte lineage-committed progenitors in adult mouse bone marrow

Hematopoietic stem cells (HSCs) have been considered to progressively lose their self-renewal and differentiation potentials prior to the commitment to each blood lineage. However, recent studies have suggested that megakaryocyte progenitors are generated at the level of HSCs. In this study, we newly identified early megakaryocyte lineage-committed progenitors (MgPs) in CD201-CD48- cells and CD48+ cells separated from the CD150+CD34-Kit+Sca-1+Lin- HSC population of the bone marrow in C57BL/6 mice. Single-cell transplantation and single-cell colony assay showed that MgPs, unlike platelet-biased HSCs, had little repopulating potential in vivo, but formed larger megakaryocyte colonies in vitro (on average eight megakaryocytes per colony) than did previously reported megakaryocyte progenitors (MkPs). Single-cell RNA-sequencing supported that these MgPs lie between HSCs and MkPs along the megakaryocyte differentiation pathway. Single-cell colony assay and single-cell RT-PCR analysis suggested the coexpression of CD41 and Pf4 is associated with megakaryocyte colony-forming activity. Single-cell colony assay of a small number of cells generated from single HSCs in culture suggested that MgPs are not direct progeny of HSCs. In this study, we propose a differentiation model in which HSCs give rise to MkPs through MgPs.

CircASXL1 knockdown represses the progression of colorectal cancer by downregulating GRIK3 expression by sponging miR-1205

Background Colorectal cancer (CRC) is a common aggressive tumor that poses a heavy burden to human health. An increasing number of studies have reported that circular RNA (circRNA) is involved in the progression of CRC. In this study, the special profiles of circASXL1 (circ_0001136) in CRC progression were revealed. Methods The expression of circASXL1, microRNA-1205 (miR-1205), and glutamate ionotropic receptor kainate type subunit 3 (GRIK3) mRNA was detected by quantitative real-time polymerase chain reaction (qRT-PCR). The protein expression was determined by Western blot or immunohistochemistry. Cell colony-forming ability was investigated by colony formation assay. Cell cycle and apoptosis were demonstrated using cell-cycle and cell-apoptosis analysis assays, respectively. Cell migration and invasion were detected by wound-healing and transwell migration and invasion assays, respectively. The binding sites between miR-1205 and circASXL1 or GRIK3 were predicted by circBank or miRDB online database, and identified by dual-luciferase reporter assay. The impact of circASXL1 on tumor formation in vivo was investigated by in vivo tumor formation assay. Results CircASXL1 and GRIK3 expression were apparently upregulated, and miR-1205 expression was downregulated in CRC tissues and cells relative to control groups. CircASXL1 knockdown inhibited cell colony-forming ability, migration and invasion, whereas induced cell arrest at G0/G1 phase and cell apoptosis in CRC cells; however, these effects were attenuated by miR-1205 inhibitor. Additionally, circASXL1 acted as a sponge for miR-1205, and miR-1205 was associated with GRIK3. Furthermore, circASXL1 silencing hindered tumor formation by upregulating miR-1205 and downregulating GRIK3 expression. Conclusion CircASXL1 acted an oncogenic role in CRC malignant progression via inducing GRIK3 through sponging miR-1205. Our findings provide a theoretical basis for studying circASXL1-directed therapy for CRC.

Cell/Colony Motion of Oral Keratinocytes Determined by Non-Invasive and Quantitative Measurement Using Optical Flow Predicts Epithelial Regenerative Capacity

Cell/colony motion determined by non-invasive, quantitative measurements using the optical flow (OF) algorithm can indicate the oral keratinocyte proliferative capacity in early-phase primary cultures. This study aimed to determine a threshold for the cell/colony motion index to detect substandard cell populations in a subsequent subculture before manufacturing a tissue-engineered oral mucosa graft and to investigate the correlation with the epithelial regenerative capacity. The distinctive proliferating pattern of first-passage (passage 1 (p1)) cells reveals the motion of p1 cells/colonies, which can be measured in a non-invasive, quantitative manner using OF with fewer full-screen imaging analyses and cell segmentations. Our results demonstrate that the motion index lower than 40 μm/hour reflects cellular damages by experimental metabolic challenges and can be used as the threshold to determine the quality of cultured cells. Because the p1 cell/colony motion index is correlated with epithelial regenerative capacity, it is a reliable index for quality control of oral keratinocytes.

Anti-Austerity Activity of Thai Medicinal Plants: Chemical Constituents and Anti-Pancreatic Cancer Activities of Kaempferia parviflora

Human pancreatic tumor cells have an intrinsic ability to tolerate nutrition starvation and survive in the hypovascular tumor microenvironment, the phenomenon termed as “austerity”. Searching for an agent that inhibits such tolerance to nutrient starvation and kills the pancreatic cancer cells preferentially in nutrient-starvation is a unique anti-austerity strategy in anti-cancer drug discovery. In this strategy, plant extracts and compounds are tested against PANC-1 human pancreatic cancer cell line under the conditions of nutrient-deprived medium (NDM) and nutrient-rich medium (DMEM), to discover the compounds that show selective cytotoxicity in NDM. Screening of twenty-five Thai indigenous medicinal plant extracts for their anti-austerity activity against the PANC-1 human pancreatic cancer cell line in nutrient deprived medium (NDM) resulted in the identification of four active plants, Derris scandens, Boesenbergia pandurata, Citrus hystrix, and Kaempferia parviflora, with PC50 values 0.5–8.9 µg/mL. K. parviflora extract also inhibited PANC-1 cancer cell colony formation. Phytochemical investigation of K. parviflora extract led to the isolation of fourteen compounds, including two polyoxygenated cyclohexanes (1 and 2), eleven flavonoids (3–13), and β-sitosterol (14). Stereochemical assignment of compound 1 was confirmed through X-ray analysis. All isolated compounds were tested for their preferential cytotoxicity against PANC-1 cells. Among them, 5-hydroxy-7-methoxyflavone (3) displayed the most potent activity with a PC50 value of 0.8 µM. Mechanistically, it was found to induce apoptosis in PANC-1 cell death in NDM as evident by caspase cleavage. It was also found to inhibit PANC-1 cancer cell colony formation in DMEM. Therefore, compound 3 can be considered as a potential lead compound for the anticancer drug development based on the anti-austerity strategy.