The Illustration of Altered Glucose Dependency in Drug-Resistant Cancer Cells.

A chemotherapeutic approach is crucial in malignancy management, which is often challenging due to the development of chemoresistance. Over time, chemo-resistant cancer cells rapidly repopulate and metastasize, increasing the recurrence rate in cancer patients. Targeting these destined cancer cells is more troublesome for clinicians, as they share biology and molecular cross-talks with normal cells. However, the recent insights into the metabolic profiles of chemo-resistant cancer cells surprisingly illustrated the activation of distinct pathways compared with chemo-sensitive or primary cancer cells. These distinct metabolic dynamics are vital and contribute to the shift from chemo-sensitivity to chemo-resistance in cancer. This review will discuss the important metabolic alterations in cancer cells that lead to drug resistance.

Dysfunctional Glucose Metabolism in Alzheimer's Disease Onset and Potential Pharmacological Interventions.

Alzheimer's disease (AD) is the most common age-related dementia. The alteration in metabolic characteristics determines the prognosis. Patients at risk show reduced glucose uptake in the brain. Additionally, type 2 diabetes mellitus increases the risk of AD with increasing age. Therefore, changes in glucose uptake in the cerebral cortex may predict the histopathological diagnosis of AD. The shifts in glucose uptake and metabolism, insulin resistance, oxidative stress, and abnormal autophagy advance the pathogenesis of AD syndrome. Here, we summarize the role of altered glucose metabolism in type 2 diabetes for AD prognosis. Additionally, we discuss diagnosis and potential pharmacological interventions for glucose metabolism defects in AD to encourage the development of novel therapeutic methods.

Azoxystrobin Impairs Neuronal Migration and Induces ROS Dependent Apoptosis in Cortical Neurons.

Fungicides often cause genotoxic stress and neurodevelopmental disorders such as autism (ASD). Fungicide-azoxystrobin (AZOX) showed acute and chronic toxicity to various organisms, and remained a concern for ill effects in developing neurons. We evaluated the neurotoxicity of AZOX in developing mouse brains, and observed prenatal exposure to AZOX reduced neuronal viability, neurite outgrowth, and cortical migration process in developing brains. The 50% inhibitory concentration (IC50) of AZOX for acute (24 h) and chronic (7 days) exposures were 30 and 10 µM, respectively. Loss in viability was due to the accumulation of reactive oxygen species (ROS), and inhibited neurite outgrowth was due to the deactivation of mTORC1 kinase activity. Pretreatment with ROS scavenger- N-acetylcysteine (NAC) reserved the viability loss and forced activation of mTORC1 kinase revived the neurite outgrowth in AZOX treated neurons. Intra-amniotic injection of AZOX coupled with in utero electroporation of GFP-labelled plasmid in E15.5 mouse was performed and 20 mg/kg AZOX inhibited radial neuronal migration. Moreover, the accumulation of mitochondria was significantly reduced in AZOX treated primary neurons, indicative of mitochondrial deactivation and induction of apoptosis, which was quantified by Bcl2/Bax ratio and caspase 3 cleavage assay. This study elucidated the neurotoxicity of AZOX and explained the possible cure from it.

Acori tatarinowii rhizoma extract ameliorates Alzheimer's pathological syndromes by repairing myelin injury and lowering Tau phosphorylation in mice.

It has been shown that Acori tatarinowii rhizoma (ATR) extract can improve cognitive functions in Alzheimer Diseas (AD) patients or animal models. In this study, we have examined the activity of ATR in 3×Tg-AD model mice with different comprehensive behavioral tests like the Morris water maze and Y-maze test assay for behavior. Moreover, we performed LFB staining for myelin determination in the AD model mouse. By analyzing different pathways, we determined key proteins that are beneficial for ameliorating AD syndrome in the mouse. Periluminally, ATR treatment improved the learning and memory ability that was determined by comprehensive behavioral tests. Moreover, treatment reduces the p-Tau accumulation in the 3×Tg-AD mouse and the level of p-Tau accumulation was at per with the wildtype control mouse and improves the myelin lining in 3×Tg-AD mouse. In conclusion, our results indicate that ATR-treatment can improve the learning ability of AD model mice and the hyperphosphorylation of Tau protein was decreased. ATR can protect myelin lining from damage in AD syndrome.

Targeting the Difficult-to-Drug CD71 and MYCN with Gambogic Acid and Vorinostat in a Class of Neuroblastomas.

BACKGROUND/AIMS: Although neuroblastoma is a heterogeneous cancer, a substantial portion overexpresses CD71 (transferrin receptor 1) and MYCN. This study provides a mechanistically driven rationale for a combination therapy targeting neuroblastomas that doubly overexpress or have amplified CD71 and MYCN. For this subset, CD71 was targeted by its natural ligand, gambogic acid (GA), and MYCN was targeted with an HDAC inhibitor, vorinostat. A combination of GA and vorinostat was then tested for efficacy in cancer and non-cancer cells. METHODS: Microarray analysis of cohorts of neuroblastoma patients indicated a subset of neuroblastomas overexpressing both CD71 and MYCN. The viability with proliferation changes were measured by MTT and colony formation assays in neuroblastoma cells. Transfection with CD71 or MYCN along with quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting were used to detect expression changes. For pathway analysis, gene ontology (GO) and Protein-protein interaction analyses were performed to evaluate the potential mechanisms of GA and vorinostat in treated cells. RESULTS: For both GA and vorinostat, their pathways were explored for specificity and dependence on their targets for efficacy. For GA-treated cells, the viability/proliferation loss due to GA was dependent on the expression of CD71 and involved activation of caspase-3 and degradation of EGFR. It relied on the JNK-IRE1-mTORC1 pathway. The drug vorinostat also reduced cell viability/proliferation in the treated cells and this was dependent on the presence of MYCN as MYCN siRNA transfection led to a blunting of vorinostat efficacy and conversely, MYCN overexpression improved the vorinostat potency in those cells. Vorinostat inhibition of MYCN led to an increase of the pro-apoptotic miR183 levels and this, in turn, reduced the viability/proliferation of these cells. The combination treatment with GA and vorinostat synergistically reduced cell survival in the MYCN and CD71 overexpressing tumor cells. The same treatment had no effect or minimal effect on HEK293 and HEF cells used as models of non-cancer cells. CONCLUSION: A combination therapy with GA and vorinostat may be suitable for MYCN and CD71 overexpressing neuroblastomas.

Oxyresveratrol activates parallel apoptotic and autophagic cell death pathways in neuroblastoma cells.

BACKGROUND: Drug resistance from apoptosis is a challenging issue with different cancer types, and there is an interest in identifying other means of inducing cytotoxicity. Here, treatment of neuroblastoma cells with oxyresveratrol (OXYRES), a natural antioxidant, led to dose-dependent cell death and increased autophagic flux along with activation of caspase-dependent apoptosis. METHODS: For cell viability, we performed the CCK-8 assay. Protein expression changes were with Western blot and immunocytochemistry. Silencing of proteins was with siRNA. The readouts for cell cycle, mitochondria membrane potential, caspase-3, autophagy and apoptosis were performed with flow cytometry. RESULTS: Phosphorylation of p38 MAPK increased with OXYRES treatment and inhibition of p38 reduced autophagy and cell death from OXYRES. In contrast, PI3K/AKT/mTOR signaling decreased in the target cells with OXYRES and inhibition of PI3K or mTOR enhanced OXYRES-mediated cytotoxicity with increased levels of autophagy. Modulation of either of the apoptosis and autophagy flux pathways affected the extent of cell death by OXYRES, but did not affect the indicators of these pathways with respect to each other. Both pathways were independent of ROS generation or p53 activation. CONCLUSION: OXYRES led to cell death from autophagy, which was independent of apoptosis induction. The OXYRES effects were due to changes in the activity levels of p38 MAPK and PI3K/AKT/mTOR. GENERAL SIGNIFICANCE: With two independent and parallel pathways for cytotoxicity induction in target cells, this study puts forward a potential utility for OXYRES or the pathways it represents as novel means of inducing cell death in neuroblastoma cells.

Graveoline isolated from ethanolic extract of Ruta graveolens triggers apoptosis and autophagy in skin melanoma cells: a novel apoptosis-independent autophagic signaling pathway.

Anti-cancer drugs generally kill cancer cells by apoptosis but fail to do so when they become resistant and escape apoptosis signals. But these resistant cells can still be killed by autophagy. Therefore, drugs having both apoptotic and autophagic abilities are solicited in effective cancer management. In search of such a drug, we examined the efficacy of graveoline, a bioactive compound isolated from Ruta graveolens on skin melanoma A375 cells through the use of specific signaling cascades and their inhibitors. Cytotoxicity of graveoline was tested by conducting MTT assay. Induction of autophagy and apoptosis was checked. Expression of related proteins and their localization were studied by conducting immunoblot assay and through confocal microscopy, respectively. We found graveoline-induced Beclin-1 associated autophagy in A375 cells and 3-methyladenine, an inhibitor of autophagy did not affect apoptosis. Conversely, caspase inhibitor that blocked apoptosis did not affect autophagic cell death, suggesting thereby that these two were independent events. Use of reactive oxygen species (ROS) scavengers inhibited cell death, but blocking autophagy did not affect graveoline-induced ROS generation, suggesting that ROS generation ensued autophagy. Thus, graveoline-induced both apoptotic and autophagic cell death in skin melanoma cells, a desirable quality in effective anti-cancer drug design.

Condurango-glycoside-A fraction of Gonolobus condurango induces DNA damage associated senescence and apoptosis via ROS-dependent p53 signalling pathway in HeLa cells.

Gonolobus condurango plant extract is used as an anticancer drug in some traditional systems of medicine including homeopathy, but it apparently lacks any scientific validation. Further, no detailed study is available to suggest whether condurango-glycoside-A (CGA), a major ingredient of condurango serves as a potent anticancer compound. Therefore, we investigated apoptosis-inducing ability of CGA against cervix carcinoma cells (HeLa). ß-galactosidase-activity and DNA damage were critically studied at different time points; while induced DNA-damage was observed at 9-12th hours, senescence of cells appeared at a later stage (18th hour after CGA treatment), implicating thereby a possible role of DNA damage in inducing pre-mature cell senescence. Concurrently, the number of cells undergoing apoptosis increased along with increase in reactive oxygen species (ROS) generation. Expression of p53 was also up-regulated, indicating that apoptosis could have been mediated through p53 pathway. DCHFDA (4',6-Diamidino-2-phenylindole dihydrochloride) assay, acridine orange/ethidium bromide staining and annexin V/PI assay results collectively confirmed that apoptosis was induced by increased ROS generation. Reduction in proliferation of cells was further evidenced by the cell cycle arrest at G0/G1 stage. Expression profiles of certain relevant genes and proteins like p53, Akt, Bcl-2, Bax, cytochrome c and caspase 3 also provided evidence of ROS mediated p53 up-regulation and further boost in Bax expression and followed by cytochrome c release and activation of caspase 3. Overall results suggest that CGA initiates ROS generation, promoting up-regulation of p53 expression, thus resulting in apoptosis and pre-mature senescence associated with DNA damage.

5-lipoxygenase antagonist therapy: a new approach towards targeted cancer chemotherapy.

Leukotrienes are the bioactive group of fatty acids and major constituents of arachidonic acid metabolism molded by the catalytic activity of 5-lipoxygenase (5-LOX). Evidence is accumulating in support of the direct involvement of 5-LOX in the progression of different types of cancer including prostate, lung, colon, and colorectal cancers. Several independent studies now support the correlation between the 5-LOX expression and cancer cell viability, proliferation, cell migration, invasion through extracellular matrix destruction, metastasis, and activation of anti-apoptotic signaling cascades. The involvement of epidermal growth factor receptor and 5-oxo-ETE receptor (OXER1) is the major talking point in the downstream of the 5-LOX pathway, which relates the cancer cells to the proliferative pathways. Antisense technology approaches and use of different kinds of blocker targeted to 5-LOX, FLAP (5-LOX-activating protein), and OXER1 have shown a greater efficiency in combating different cancer cell types. Lastly, suppression of 5-LOX activity that reduces the cell proliferation activity also induces intrinsic mitochondrial apoptotic pathway in either p53-dependent or independent manner. Pharmacological agents that specifically inhibit the LOX-mediated signaling pathways have been used during last few years to treat inflammatory diseases such as asthma and arthritis. Studies of these well-characterized agents are therefore warranted for their use as possible candidates for chemotherapeutic studies against the killer disease cancer.

Oxidative stress in cerebrovascular disease and associated diseases.

Cellular aging is the most severe risk factor for neurodegenerative disease. Simultaneously, oxidative stress (OS) is a critical factor in the aging process, resulting from an imbalance between reactive oxygen and nitrogen species and the antioxidant defense system. Emerging evidence indicates that OS is a common cause of several age-related brain pathologies, including cerebrovascular diseases. Elevated OS disrupts endothelial functional ability by diminishing the bioavailability of nitric oxide (a vascular dilator), induces atherosclerosis, and impairs vasculature, which are all common characteristics of cerebrovascular disease. In this review, we summarize evidence supporting an active role of OS in cerebrovascular disease progression, focusing primarily on stroke pathogenesis. We briefly discuss hypertension, diabetes, heart disease, and genetic factors that are often linked to OS and are considered associated factors influencing stroke pathology. Finally, we discuss the current pharmaceutics/therapeutics available for treating several cerebrovascular diseases.

Ethanolic extract of Thuja occidentalis blocks proliferation of A549 cells and induces apoptosis in vitro.

OBJECTIVE: To study the possible anticancer and antiproliferative activities of ethanolic leaf extract of Thuja occidentalis (TO) on A549 non-small lung carcinoma cells in vitro. METHODS: Cell viability was ascertained through 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay after deployment of TO in different doses. The half maximal inhibitory concentration (IC50) dose (282 µg/mL) was determined, and two other doses for dose-dependence study, one below the IC50 dose (IC35=188 µg/mL) and one above the IC50 dose (IC65=376 µg/mL) were selected. Bromodeoxyuridine (BrdU) incorporation assay and migration studies were performed to elucidate antiproliferative activity of the drug, if any. Fluorescence-activated cell sorting analysis after annexin V-fluorescein isothiocyanate and propidium iodide (annexin V-FITC-PI) dual staining method was done to ascertain early stage of apoptosis, if any. DNA fragmentation assay was done through Hoechst 33258 and acridine orange-ethidium bromide staining. DNA damage was quantified through comet assay. Bax-Bcl2 regulation and expression studies were performed through indirect enzyme-linked immunosorbent assay (ELISA). Caspase 3 activity was measured at gene level through reverse transcription-polymerase chain reaction (RT-PCR) analysis. Its activation at protein level was analyzed through indirect ELISA and Western blot analysis. RESULTS: TO demonstrated a dose-dependent decrease in viability of A549 cells after 24 h of exposure. Cell proliferation was reduced in a time-dependent manner of drug exposure as revealed from BrdU incorporation and migration studies. Annexin-V-FITC positivity of cells up to 11.72% as compared to the untreated control revealed early state of TO-induced apoptosis. Occurrence of comet tail and increased fluorescence of Hoechst after 24 h of drug exposure revealed significant DNA nick generation and chromatin condensation. Bax up-regulation and Bcl-2 down-regulation suitably altered ratio of Bax/Bcl-2 in favor of apoptosis. From RT-PCR, indirect ELISA and Western blot studies, caspase 3 activity was also found to be significantly increased along with cleaved poly ADP-ribose polymerase expression. CONCLUSION: Ethanolic leaf extract of TO demonstrated apoptotic and antiproliferative potentials against A549 cell line.

Chelidonine isolated from ethanolic extract of Chelidonium majus promotes apoptosis in HeLa cells through p38-p53 and PI3K/AKT signalling pathways.

OBJECTIVE: To evaluate the role of chelidonine isolated from ethanolic extract of Chelidonium majus in inducing apoptosis in HeLa cells and to assess the main signalling pathways involved. METHODS: Cells were initially treated with different concentrations of chelidonine for 48 h and the median lethal dose (LD50) value was selected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Morphological analysis of nuclear condensation and DNA damage and fragmentation were measured by 4',6-diamidino-2-phenylindole staining and comet assay. Further, reactive oxygen species (ROS) generation, cell cycle arrest and change in mitochondrial membrane potential were also examined and analyzed by flow cytometry. Evaluation of interaction of drug with CT DNA was investigated by circular dichroism (CD) spectral analysis to find any possible drug-CT DNA interaction. The mRNA and protein expressions of major signal proteins like p38, p53, protein kinase B (AKT), phosphatidylinositol 3-kinases (PI3K), Janus kinase 3 (JAK3), signal transducer and activator of transcription 3 (STAT3) and E6 and E7 oncoproteins as well as the pro-apoptotic genes and antiapoptotic genes were also estimated by reverse transcriptase-polymerase chain reaction and Western blotting. RESULTS: Based on LD(50) value (30 µg/mL) of chelidonine, three doses were selected, namely, 22.5 µg/mL (D1), 30.0 µg/mL (D2) and 37.5 µg/mL (D3). Results showed that chelidonine inhibited proliferation and induced apoptosis in HeLa cells through generation of ROS, cell cycle arrest at sub-G1 and G0/G1 stage, change in mitochondrial membrane potential and fragmentation of DNA. Results of CD spectra showed effective interaction between chelidonine and calf thymus DNA. Studies of signalling pathway revealed that chelidonine could efficiently induce apoptosis through up-regulation of expressions of p38, p53 and other pro-apoptotic genes and down-regulation of expressions of AKT, PI3K, JAK3, STAT3, E6, E7 and other antiapoptotic genes. CONCLUSION: Chelidonine isolated from Chelidonium majus efficiently induced apoptosis in HeLa cells through possible alteration of p38-p53 and AKT/PI3 kinase signalling pathways.

RNA Binding Protein Rbms1 Enables Neuronal Differentiation and Radial Migration during Neocortical Development by Binding and Stabilizing the RNA Message for Efr3a.

Various RNA-binding proteins (RBPs) are key components in RNA metabolism and contribute to several neurodevelop-mental disorders. To date, only a few of such RBPs have been characterized for their roles in neocortex development. Here, we show that the RBP, Rbms1, is required for radial migration, polarization and differentiation of neuronal progenitors to neurons in the neocortex development. Rbms1 expression is highest in the early development in the developing cortex, with its expression gradually diminishing from embryonic day 13.5 (E13.5) to postnatal day 0 (P0). From in utero electroporation (IUE) experiments when Rbms1 levels are knocked down in neuronal progenitors, their transition from multipolar to bipolar state is delayed and this is accompanied by a delay in radial migration of these cells. Reduced Rbms1 levels in vivo also reduces differentiation as evidenced by a decrease in levels of several differentiation markers, meanwhile having no significant effects on proliferation and cell cycle rates of these cells. As an RNA binding protein, we profiled the RNA binders of Rbms1 by a cross-linked-RIP sequencing assay, followed by quantitative real-time polymerase chain reaction verification and showed that Rbms1 binds and stabilizes the mRNA for Efr3a, a signaling adapter protein. We also demonstrate that ectopic Efr3a can recover the cells from the migration defects due to loss of Rbms1, both in vivo and in vitro migration assays with cultured cells. These imply that one of the functions of Rbms1 involves the stabilization of Efr3a RNA message, required for migration and maturation of neuronal progenitors in radial migration in the developing neocortex.

mTORC1-Inhibition Potentiating Metabolic Block by Tyrosine Kinase Inhibitor Ponatinib in Multiple Myeloma.

Studies in targeting metabolism in cancer cells have shown the flexibility of cells in reprogramming their pathways away from a given metabolic block. Such behavior prompts a combination drug approach in targeting cancer metabolism, as a single compound may not address the tumor intractability. Overall, mammalian target of rapamycin complex 1 (mTORC1) signaling has been implicated as enabling metabolic escape in the case of a glycolysis block. From a library of compounds, the tyrosine kinase inhibitor ponatinib was screened to provide optimal reduction in metabolic activity in the production of adenosine triphosphate (ATP), pyruvate, and lactate for multiple myeloma cells; however, these cells displayed increasing levels of oxidative phosphorylation (OXPHOS), enabling them to continue generating ATP, although at a slower pace. The combination of ponatinib with the mTORC1 inhibitor, sirolimus, blocked OXPHOS; an effect also manifested in activity reductions for hexokinase 2 (HK2) and glucose-6-phosphate isomerase (GPI) glycolysis enzymes. There were also remarkably higher levels of reactive oxygen species (ROS) produced in mouse xenografts, on par with increased glycolytic block. The combination of ponatinib and sirolimus resulted in synergistic inhibition of tumor xenografts with no overt toxicity in treated mice for kidney and liver function or maintaining weight.

Reversing the HDAC-inhibitor mediated metabolic escape in MYCN-amplified neuroblastoma.

In MYCN-amplified neuroblastoma (NB), we noticed that the single compound treatment with the HDAC inhibitor vorinostat led to a reprogramming of the glycolytic pathway in these cells. This reprogramming was upregulation of fatty acid oxidation (FAO) and oxidative phosphorylation (OXPHOS), allowing the cells to generate ATP, albeit at a reduced rate. This behavior was dependent on reduced levels of MYCN and a corresponding increase in the levels of PPARD transcription factors. By integrating metabolic and functional studies in NB cells and mouse xenografts, we demonstrate a compensatory upregulation of FAO/OXPHOS metabolism that promotes resistance to HDAC inhibitors. From the additional compounds that could reverse this metabolic reprogramming, the mTORC1 inhibitor sirolimus was selected. Besides both a block of glycolysis and OXPHOS, the HDAC/mTORC1 inhibitor combination produced significantly higher levels of reactive oxygen species (ROS) in the treated cells and in xenograft tumor samples, also a consequence of increased glycolytic block. The lead compounds were also tested for changes in the message levels of the glycolytic enzymes and their pathway activity, and HK2 and GPI glycolytic enzymes were most affected at their RNA message level. This combination was seen with no overall toxicity in treated mice in terms of weight loss or liver/kidney function.

RNA binding protein HuD promotes autophagy and tumor stress survival by suppressing mTORC1 activity and augmenting ARL6IP1 levels.

BACKGROUND: Neuronal-origin HuD (ELAVL4) is an RNA binding protein overexpressed in neuroblastoma (NB) and certain other cancers. The RNA targets of this RNA binding protein in neuroblastoma cells and their role in promoting cancer survival have been unexplored. In the study of modulators of mTORC1 activity under the conditions of optimal cell growth and starvation, the role of HuD and its two substrates were studied. METHODS: RNA immunoprecipitation/sequencing (RIP-SEQ) coupled with quantitative real-time PCR were used to identify substrates of HuD in NB cells. Validation of the two RNA targets of HuD was via reverse capture of HuD by synthetic RNA oligoes from cell lysates and binding of RNA to recombinant forms of HuD in the cell and outside of the cell. Further analysis was via RNA transcriptome analysis of HuD silencing in the test cells. RESULTS: In response to stress, HuD was found to dampen mTORC1 activity and allow the cell to upregulate its autophagy levels by suppressing mTORC1 activity. Among mRNA substrates regulated cell-wide by HuD, GRB-10 and ARL6IP1 were found to carry out critical functions for survival of the cells under stress. GRB-10 was involved in blocking mTORC1 activity by disrupting Raptor-mTOR kinase interaction. Reduced mTORC1 activity allowed lifting of autophagy levels in the cells required for increased survival. In addition, ARL6IP1, an apoptotic regulator in the ER membrane, was found to promote cell survival by negative regulation of apoptosis. As a therapeutic target, knockdown of HuD in two xenograft models of NB led to a block in tumor growth, confirming its importance for viability of the tumor cells. Cell-wide RNA messages of these two HuD substrates and HuD and mTORC1 marker of activity significantly correlated in NB patient populations and in mouse xenografts. CONCLUSIONS: HuD is seen as a novel means of promoting stress survival in this cancer type by downregulating mTORC1 activity and negatively regulating apoptosis.

Identification and prevention of heterotopias in mouse neocortical neural cell migration incurred by surgical damages during utero electroporation procedures.

In utero electroporation (IUE) is a useful technique for gene delivery in embryonic mouse brain. IUE technique is used to investigate the mammalian brain development in vivo. However, according to recent studies, IUE methodology has some limitations like the formation of artificial ectopias and heterotopias at the micro-injection site. Thus far, the artificial heterotopias generated by physical trauma during IUE are rarely reported. Here, we reported the artificial heterotopias and ectopias generated from surgical damages of micropipette in detail, and moreover, we described the protocol to avoid these phenotypes. For the experimental purpose, we transferred empty plasmids (pCAGIG-GFP) with green fluorescent-labelled protein into the cortical cortex by IUE and then compared the structure of the cortex region between the injected and un-injected cerebral hemispheres. The coronary section showed that ectopias and heterotopias were appeared on imperfect-injected brains, and layer maker staining, which including Ctip2 and TBR1 and laminin, can differentiate the physical damage, revealing the neurons in artificial ectopic and heterotopic area were not properly arranged. Moreover, premature differentiation of neurons in ectopias and heterotopias were observed. To avoid heterotopias and ectopias, we carefully manipulated the method of IUE application. Thus, this study might be helpful for the in utero electroporator to distinguish the artificial ectopias and heterotopias that caused by the physical injury by microneedle and the ways to avoid those undesirable circumstances.

Machine-Learning Based Automatic and Real-time Detection of Mouse Scratching Behaviors.

Scratching is a main behavioral response accompanied by acute and chronic itch conditions, and has been quantified as an objective correlate to assess itch in studies using laboratory animals. Scratching has been counted mostly by human annotators, which is a time-consuming and laborious process. It has been attempted to develop automated scoring methods using various strategies, but they often require specialized equipment, costly software, or implantation of device which may disturb animal behaviors. To complement limitations of those methods, we have adapted machine learning-based strategy to develop a novel automated and real-time method detecting mouse scratching from experimental movies captured using monochrome cameras such as a webcam. Scratching is identified by characteristic changes in pixels, body position, and body size by frame as well as the size of body. To build a training model, a novel two-step J48 decision tree-inducing algorithm along with a C4.5 post-pruning algorithm was applied to three 30-min video recordings in which a mouse exhibits scratching following an intradermal injection of a pruritogen, and the resultant frames were then used for the next round of training. The trained method exhibited, on average, a sensitivity and specificity of 95.19% and 92.96%, respectively, in a performance test with five new recordings. This result suggests that it can be used as a non-invasive, automated and objective tool to measure mouse scratching from video recordings captured in general experimental settings, permitting rapid and accurate analysis of scratching for preclinical studies and high throughput drug screening.