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Supplementary Figure Legends 1-2 from Receptor Channel TRPC6 Is a Key Mediator of Notch-Driven Glioblastoma Growth and Invasiveness

Glioblastoma multiforme (GBM) is the most frequent and incurable type of brain tumor of adults. Hypoxia has been shown to direct GBM toward a more aggressive and malignant state. Here we show that hypoxia increases Notch1 activation, which in turn induces the expression of transient receptor potential 6 (TRPC6) in primary samples and cell lines derived from GBM. TRPC6 is required for the development of the aggressive phenotype because knockdown of TRPC6 expression inhibits glioma growth, invasion, and angiogenesis. Functionally, TRPC6 causes a sustained elevation of intracellular calcium that is coupled to the activation of the calcineurin-nuclear factor of activated T-cell (NFAT) pathway. Pharmacologic inhibition of the calcineurin-NFAT pathway substantially reduces the development of the malignant GBM phenotypes under hypoxia. Clinically, expression of TRPC6 was elevated in GBM specimens in comparison with normal tissues. Collectively, our studies indicate that TRPC6 is a key mediator of tumor growth of GBM in vitro and in vivo and that TRPC6 may be a promising therapeutic target in the treatment of human GBM. Cancer Res; 70(1); 418–27

Supplementary Table 1 from Receptor Channel TRPC6 Is a Key Mediator of Notch-Driven Glioblastoma Growth and Invasiveness

Supplementary Figure 2 from Receptor Channel TRPC6 Is a Key Mediator of Notch-Driven Glioblastoma Growth and Invasiveness

Supplementary Figure 1 from Receptor Channel TRPC6 Is a Key Mediator of Notch-Driven Glioblastoma Growth and Invasiveness

Glioblastoma multiforme (GBM) possesses a small but significant population of cancer stem cells (CSCs) thought to play a role in its invasiveness, recurrence, and metastasis. The CSCs display transcriptional profiles for multipotency, self-renewal, tumorigenesis, and therapy resistance. There are two possible theories regarding the origin of CSCs in the context of neural stem cells (NSCs); i.e., NSCs modify cancer cells by conferring them with cancer-specific stemness, or NSCs themselves are transformed into CSCs due to the tumor environment created by cancer cells. To test the theories and to investigate the transcriptional regulation of the genes involved in CSC formation, we cocultured NSC and GBM cell lines together. Where genes related to cancer stemness, drug efflux, and DNA modification were upregulated in GBM, they were downregulated in NSCs upon coculture. These results indicate that cancer cells shift the transcriptional profile towards stemness and drug resistance in the presence of NSCs. Concurrently, GBM triggers NSCs differentiation. Because the cell lines were separated by a membrane (0.4 µm pore size) to prevent direct contact between GBM and NSCs, cell-secreted signaling molecules and extracellular vesicles (EVs) are likely involved in reciprocal communication between NSCs and GBM, causing transcription modification. Understanding the mechanism of CSC creation will aid in the identification of precise molecular targets within the CSCs to exterminate them, which, in turn, will increase the efficacy of chemo-radiation treatment.

Mitochondrial dysfunction is a hallmark of neurodegeneration. The expression level of Tom40, a crucial mitochondrial membrane protein, is significantly reduced in neurodegenerative disease subjects. Tom40 overexpression studies have shown to protect the neurons against oxidative stress by improving mitochondrial function. Thus, successful delivery of Tom40 protein to the brain could lead to a novel therapy for neurodegenerative diseases. However, delivering protein to the cell may be difficult. Especially the blood-brain barrier (BBB) is a big hurdle to clear in order to deliver the protein to the brain. In the current study, we engineered exosomes, which are the extracellular vesicles of endosomal origin, and able to cross BBB as delivery vehicles packing human Tom40. We found Tom40 protein delivery by the exosome successfully protected the cells against hydrogen peroxide-induced oxidative stress. This result suggests that exosome-mediated delivery of Tom40 may potentially be useful in restoring mitochondrial functions and alleviating oxidative stress in neurodegenerative diseases, such as Alzheimer’s and Parkinson’s diseases.

Exosomes, nanovesicles secreted by all cells, carry out intercellular communication by transmitting biologically active cargo comprising DNA, RNA, and proteins. These biomolecules reflect the status of their parent cells and can be altered by pathological conditions. Therefore, the researchers have been investigating differential sequences and quantities of DNA associated with exosomes as valuable biomarkers of diseases. Exosomes carry different types of DNA molecules, including genomic, cytoplasmic, and mitochondrial (mtDNA). The mtDNA aberrations are reported to be a hallmark of diseases involving oxidative stress, such as cancer and neurodegenerative diseases. Establishing robust in vitro models comprising appropriate cell lineages is the first step towards investigating disease-specific anomalies and testing therapeutics. Induced pluripotent stem (iPS) cells from patients with diseases have been used for this purpose since they can differentiate into various cells. The current study investigated mtDNA aberrations in exosomes secreted by primary cancer cells and neural stem cells (NSCs) differentiated from iPS cells. The primary cancer cells were isolated from surgically removed glioblastoma multiforme (GBM) tissue, and the iPS cells were produced from control and Alzheimer’s disease (AD) subjects’ B lymphocytes. We detected aberrations in mtDNA associated with exosomes secreted from GBM cells but not from the NSCs. This result indicates that the cells may not secrete exosomes carrying mtDNA aberration without exposure to a pathological condition. Thus, we may need to consider this fact when we use iPS cell-derived cells as an in vitro disease model.

The size of our pupils changes continuously in response to variations in ambient light levels, a process known as the pupillary light reflex (PLR). The PLR is not a simple reflex as its function is modulated by cognitive brain function and any long-term changes in brain function secondary to injury should cause a change in the parameters of the PLR. We performed a retrospective clinical review of the PLR of our patients using the BrightLamp Reflex iPhone app. The PLR variables of latency, maximum pupil diameter (MaxPD), minimum pupil diameter (MinPD), maximum constriction velocity (MCV), and the 75% recovery time (75% PRT) were associated with significant differences between subjects who had suffered a concussion and those that had not. There were also significant differences in PLR metrics over the life span and between genders and those subjects with and without symptoms. The differences in PLR metrics are modulated not only by concussion history but also by gender and whether or not the person has symptoms associated with a head injury. A concussive injury to the brain is associated with changes in the PLR that persist over the life span, representing biomarkers that might be used in clinical diagnosis, treatment, and decision making.

Introduction: Wounds are associated with ranges of simple to complex disruption or damage to anatomical structure and function. They are also associated with enormous economic and social costs, increasing yearly, resulting in a severe impact on the wellbeing of individuals and society. Technology that might accelerate wound healing is associated with many benefits to injured people. Methods: BALBc mice underwent symmetrical excisional wounds through the panniculus carnosus. They were divided into a treatment group placed on an autonomous ceramic far-field infrared blanket (cIFRB) and a control group maintained under standard conditions. We also expanded and cultured adipose tissue-derived mesenchymal stem cells (MSCs) on cIFRB and compared them to standard conditions subjected to a scratch injury to compare survival, proliferation, and wound healing. Results: The wound healing of the cIRFB treatment group was significantly faster than the control group of mice. The wound-healing effect of mesenchymal stem cells on cIRFB was also increased and associated with significant migration to the wound area. Conclusions: Wound healing is improved in a mouse model exposed to cFIRB. The ceramic blanket also promotes survival, proliferation, increased migration, and wound healing of MSCs without affecting their survival and proliferation. The utilization of cFIRB in cellular biology and medical applications may be promising in many situations currently explored in animal and human models. This technology needs no direct or battery power source and is entirely autonomous and noninvasive, making its application possible in any environment.

Pseudogenes, once considered the "junk remnants of genes," are found to significantly affect the regulatory network of healthy and cancer cells, as well as to be highly specific markers of cancer cell identity. Qualitative and quantitative analysis of pseudogenes has a diagnostic and prognostic value in cancer research via the detection of cell-free pseudogenic DNA circulating throughout the body. Exosomes, nanoparticles with a lipid membrane secreted by almost all types of cells, carry cellular-blueprint molecules, including pseudogenic DNA, as cancer-specific cargo. Therefore, it is vital to develop better laboratory techniques and protocols to identify exosome-associated pseudogenes.

Pseudogenes, once considered the "junk remnants of genes," are found to significantly affect the regulatory network of healthy and cancer cells, as well as to be highly specific markers of cancer cell identity. Qualitative and quantitative analysis of pseudogenes has a diagnostic and prognostic value in cancer research via the detection of cell-free pseudogenic DNA circulating throughout the body. Exosomes, nanoparticles with a lipid membrane secreted by almost all types of cells, carry cellular-blueprint molecules, including pseudogenic DNA, as cancer-specific cargo. Therefore, it is vital to develop better laboratory techniques and protocols to identify exosome-associated pseudogenes.

Inflammation is considered an important target for stroke therapy because it induces secondary brain damage after the initial ischemic insult. Peripheral monocytes migrate to the brain parenchyma after a central insult. They then differentiate to macrophages in a positive feedback fashion contributing to damage instead of ischemic resolution and inflammation control. A cyclic diterpenoid, (1S,2E,4R,6R,7E,11E)-cembra-2,7,11-triene-4,6-diol (4R), decreases neurodegeneration after ischemia with central anti-inflammatory activity. This study aims to determine whether the central anti-inflammatory effect of 4R is effective against peripheral inflammation triggered by brain ischemia. To investigate the anti-inflammatory effect of 4R, we treated macrophages with lipopolysaccharide (LPS) as an inflammatory model, followed by treatment with 4R. Microarray transcriptome analysis of over 30,000 genes identified the differential expression of 393 genes. Genes related to inflammation, cell adhesion, and transcription were validated with qPCR, and reduced expression was determined. Quantification of NF-kB phosphorylation served as a marker for the modulation of inflammation through gene transcription. Our results show that 4R was associated with a reduction in NFKB1 and ITGB5 gene expression, increased phosphorylation of NF-kB, and a decrease in macrophage adhesion in a blood-brain barrier model. These results indicate that 4R can partially modulate the peripheral immune response, making 4R a potential drug against post-ischemic inflammation.

Human neuroblastoma cells, SH-SY5Y, are often used as a neuronal model to study Parkinson's disease and dopamine release in the substantia nigra, a midbrain region that plays an important role in motor control. Using amperometric single-cell recordings of single vesicle release events, we can study molecular manipulations of dopamine release and gain a better understanding of the mechanisms of neurological diseases. However, single-cell analysis of neurotransmitter release using traditional techniques yields results with very low throughput. In this paper, we will discuss a monolithically-integrated CMOS sensor array that has the low-noise performance, fine temporal resolution, and 1024 parallel channels to observe dopamine release from many single cells with single-vesicle resolution. The measured noise levels of our transimpedance amplifier are 415, 622, and 1083 ${\text{fA}}_{{\text{RMS}}}$ , at sampling rates of 10, 20, and 30 kS/s, respectively, without additional filtering. Post-CMOS processing is used to monolithically integrate 1024 on-chip gold electrodes, with an individual electrode size of 15 μm × 15 μm, directly on 1024 transimpedance amplifiers in the CMOS device. SU-8 traps are fabricated on individual electrodes to allow single cells to be interrogated and to reject multicellular clumps. Dopamine secretions from 76 cells are simultaneously recorded by loading the CMOS device with SH-SY5Y cells. In the 42-s measurement, a total of 7147 single vesicle release events are monitored. The study shows the CMOS device's capability of recording vesicle secretion at a single-cell level, with 1024 parallel channels, to provide detailed information on the dynamics of dopamine release at a single-vesicle resolution.

Sample preparation is an essential process that precedes nucleic acid detections which use quantitative polymerase chain reaction (qPCR). However, sample preparation is a labor-intensive process and requires skilled labor, thus limiting the public's access in low-resource settings to many high-quality nucleic acid-based detection mechanisms. In this paper, we present a simple, handheld, battery-operated sample preparation device to minimize user's involvement. The device uses a simple pouring method to process the DNA sample without pipetting or using disposable pipette tips. The developed device has a size of 12 × 8 × 8 cm3 and mass of only 364 g. The device is compared to gold standard methods, including magnetic bead-based and silica filter-based DNA extractions. For a short segment DNA target of 68 bp, the presented device captured 8.67× more DNA compared to that of the manual magnetic bead-based method. Because of automation, the measured capture efficiency is more consistent and has a smaller deviation between multiple repetitions than the manual method. To present a comprehensive, portable, battery-operated diagnostic system, the sample preparation device is tested in conjunction with a 3D-manufactured qPCR device. The test using three diluted target DNA samples, each spiked in whole blood (1×, 0.1×, and 0.01×), revealed a quantitative detection with ideal cycle threshold separations between the measurements. The combination of two devices will aid in resource-limited settings to promptly and accurately diagnose infections of patients.

Cancerous and non-cancerous cells secrete exosomes, a type of nanovesicle known to carry the molecular signature of the parent for intercellular communications. Exosomes secreted by tumor cells carry abnormal DNA, RNA, and protein molecules that reflect the cancerous status. DNA is the master molecule that ultimately affects the function of RNA and proteins. Aberrations in DNA can potentially lead a cell to malignancy. Deviant quantities and the differential sequences of exosomal DNA are useful characteristics as cancer biomarkers. Since these alterations are either associated with specific stages of cancer or caused due to a clinical treatment, exosomal DNA is valuable as a diagnostic, prognostic, predictive, and therapeutic-intervention response biomarker. Notably, the exosomes can cross an intact blood–brain barrier and anatomical compartments by transcytosis. As such, the cancer-specific trademark molecules can be detected in systemic blood circulation and other body fluids, including cerebrospinal fluid, with non-invasive or minimally invasive procedures. This comprehensive review highlights the cancer-specific modulations of DNA associated with circulating exosomes that are beneficial as glioma biomarkers.

Neural stem cells (NSCs), capable of self-renew and differentiate into neural cells, hold promise for use in studies and treatments for neurological diseases. However, current approaches to obtain NSCs from a live brain are risky and invasive, since NSCs reside in the subventricular zone and the in the hippocampus dentate gyrus. Alternatively, mesenchymal stem cells (MSCs) could be a more available cell source due to their abundance in tissues and easier to access. However, MSCs are committed to producing mesenchymal tissue and are not capable of spontaneously differentiating into neural cells. Thus, the process of reprogramming of MSCs into neural cells to use in clinical and scientific settings has significantly impacted the advancement of regenerative medicine. Previously, our laboratory reported trans-differentiation of MSCs to neural cells through the induced pluripotent stem (iPS) cells state, which was produced by overexpression of the embryonic stem cell gene NANOG. In the current study, we demonstrate that treatment with exosomes derived from NSCs makes MSCs capable of expressing neural cell markers bypassing the generation of iPS cells. An epigenetic modifier, decitabine (5-aza-2'-deoxycytidine), enhanced the process. This novel Xeno and transgene-free trans-differentiation technology eliminates the issues associated with iPS cells, such as tumorigenesis. Thus, it may accelerate the development of neurodegenerative therapies and in vitro neurological disorder models for personalized medicine.

Neuroblastoma cells are regularly used to study Parkinson's disease, as they are suitable cell models. Specifically, the release of dopamine from neurssoblastoma cells is studied, as Parkinson's disease lowers the amount of dopamine released within the sustantia nigra, a structure within the midbrain that is important for controlling movement. To further the ability to study dopamine release from neuroblastoma cells, we developed a monolithic CMOS sensor array containing 1024 on-chip electrodes. Each electrode is 15 μm × 15 μm and is paired with a dedicated transimpedance amplifier capable of measuring sub-pA current. The channels throughout the array have both low-noise performance and spatiotemporal resolution enabling the study of dopamine release from a single vesicle. The presented device is used to record dopamine release from the SH-SY5Y neuroblastoma cell line to demonstrate its efficacy. The device's characteristics enable the study of the detailed molecular dynamics that occur during dopamine secretion.

Neuroblastoma cells are often used as a cell model to study Parkinson's disease, which causes reduced dopamine release in substantia nigra, the midbrain that controls movements. In this paper, we developed a 1024-ch monolithic CMOS sensor array that has the spatiotemporal resolution as well as low-noise performance to monitor single vesicle release of dopamine from neuroblastoma cells. The CMOS device integrates 1024 on-chip electrodes with an individual size of $15 \mu \mathrm{m}\times 15 \mu \mathrm{m}$ and 1024 transimpedance amplifiers for each electrode, which are each capable of measuring sub-pA current. Thus, this device can be used to study the detailed molecular dynamics of dopamine secretion at single vesicle resolution.

Stem cell therapies have been proposed as a treatment option for neurodegenerative diseases, but the best stem cell source and therapeutic efficacy for neuroregeneration remain uncertain. Embryonic stem cells (ESCs) and neural stem cells (NSCs), which can efficiently generate neural cells, could be good candidates but they pose ethical and practical issues. Not only difficult to find the good source of those cells but also they alway pose immunorejection problem since they may not be an autologous cells. Even if we overcome the immunorejection problem, it has also been reported that transplantation of ESCs develop teratoma. Although adult stem cells are more accessible, they have a limited developmental potential. We developed technologies to increase potency of mesenchymal stem cells, which allow them to develop into neural cells, by over expression of the ESC gene, nanog. We also developed a small molecule compound, which significantly increases endogenous NSCs by peripheral administration, eliminating even the necessity of stem cell injection to the brain. These novel technologies may offer neuroregenerative therapies for Alzheimers disease (AD). However, we found that AD pathological condition prevent neurogenesis from NSCs. This chapter discusses how to overcome the problem associated stem cell therapy under AD pathology and introduces exosome as a tool to improve the modification of adult stem cells. These new technologies may open a door for the new era for AD therapy.

Stem cell therapies have been proposed as a treatment option for neurodegenerative diseases, but the best stem cell source and therapeutic efficacy for neuroregeneration remain uncertain. Embryonic stem cells (ESCs) and neural stem cells (NSCs), which can efficiently generate neural cells, could be good candidates but they pose ethical and practical issues. Not only difficult to find the good source of those cells but also they alway pose immunorejection problem since they may not be an autologous cells. Even if we overcome the immunorejection problem, it has also been reported that transplantation of ESCs develop teratoma. Although adult stem cells are more accessible, they have a limited developmental potential. We developed technologies to increase potency of mesenchymal stem cells, which allow them to develop into neural cells, by over expression of the ESC gene, nanog. We also developed a small molecule compound, which significantly increases endogenous NSCs by peripheral administration, eliminating even the necessity of stem cell injection to the brain. These novel technologies may offer neuroregenerative therapies for Alzheimers disease (AD). However, we found that AD pathological condition prevent neurogenesis from NSCs. This chapter discusses how to overcome the problem associated stem cell therapy under AD pathology and introduces exosome as a tool to improve the modification of adult stem cells. These new technologies may open a door for the new era for AD therapy.

Articular cartilage defects often progress to osteoarthritis, which negatively impacts quality of life for millions of people worldwide and leads to high healthcare expenditures. Tissue engineering approaches to osteoarthritis have concentrated on proliferation and differentiation of stem cells by activation and suppression of signaling pathways, and by using a variety of scaffolding techniques. Recent studies indicate a key role of environmental factors in the differentiation of mesenchymal stem cells to mature cartilage-producing chondrocytes. Therapeutic approaches that consider environmental regulation could optimize chondrogenesis protocols for regeneration of articular cartilage. This review focuses on the effect of scaffold structure and composition, mechanical stress and hypoxia in modulating mesenchymal stem cell fate and the current use of these environmental factors in tissue engineering research.

Alzheimer’s disease is investigated by scientists broadly. Although there is no definitive treatment, a better medical intervention can be assumed as a preventive medicine which postpones the onset of disease and delays its progression. Understanding that the sleep changes affect the brain which it will be stabilized later as a cardinal manifestation of disease leads us to review recent studies and theories. In this review, we show that the interruption of circadian rhythm along with disruption of immune and endocrine systems can cause neuronal physiology changes. These changes can enhance neuro inflammation pathology by the time through Aβ deposition forming plaques in the brain tissue. Besides higher level of Aβ in CSF, immune system dysfunction occurred by aging will change solubility of Aβ. Furthermore, the defect in insulin and subsequently decreases in insulin receptor impair glucose metabolism. The dysregulation of glucose metabolism expedites the degenerative pathway and contributes to other oxidative stresses in neurons. The cortex of brain gradually will be damaged extremely and the brain size will shrink intensely and atrophy. In Review Article Khorgami and Sugaya; BJMMR, 9(7): 1-12, 2015; Article no.BJMMR.19333 2 summary, we found that improving of sleep quality can reduce the disease progression and delay its symptoms by having effects in neuropathology.

Cellular motion along substrate channels confines the cellular motion linearly. The addition of an optical field linearly polarized parallel to the substrate channel enhances this motion due to optical torques applied to subcellular elements.

NANOG has been demonstrated to play an essential role in the maintenance of embryonic stem cells, and its pseudogene, NANOGP8, is suggested to promote the cancer stem cell phenotype. As the roles of these genes are intimately involved with glioblastoma multiforme progression and exosomes are critical in intercellular communication, we conducted a detailed analysis of the association of the NANOG gene family with exosomes to identify diagnostic markers for cancer. Exosomes were precipitated from conditioned culture media from various cell lines, and NANOG gene fragments were directly amplified without DNA isolation using multiple primer sets. The use of the enzymes AlwNI and SmaI with restriction fragment length polymorphism analysis functioned to distinguish NANOGP8 from other NANOG family members. Collectively, results suggest that the NANOG DNA associated with exosomes is not full length and that mixed populations of the NANOG gene family exist. Furthermore, sequence analysis of exosomal DNA amplified with a NANOGP8 specific primer set frequently showed an insertion of a 22 bp sequence into the 3’ UTR. The occurrence rate of this insertion was significantly higher in exosomal DNA clones from cancer cells as compared to normal cells. We have detected mixed populations of NANOG DNA associated with exosomes and have identified preferential modulations in the sequences from cancer samples. Our findings, coupled with the properties of exosomes, may allow for the detection of traditionally inaccessible cancers (i.e. GBM) through minimally invasive techniques. Further analysis of exosomal DNA sequences of NANOG and other embryonic stemness genes (OCT3/4, SOX2, etc.) may establish a robust collection of exosome based diagnostic markers, and further elucidate the mechanisms of cancer formation, progression, and metastasis.