TUBB3 Research Articles

Acute and Delayed Effects of Mechanical Injury on Calcium Homeostasis and Mitochondrial Potential of Primary Neuroglial Cell Culture: Potential Causal Contributions to Post-Traumatic Syndrome.

In vitro models of traumatic brain injury (TBI) help to elucidate the pathological mechanisms responsible for cell dysfunction and death. To simulate in vitro the mechanical brain trauma, primary neuroglial cultures were scratched during different periods of network formation. Fluorescence microscopy was used to measure changes in intracellular free Ca2+ concentration ([Ca2+]i) and mitochondrial potential (ΔΨm) a few minutes later and on days 3 and 7 after scratching. An increase in [Ca2+]i and a decrease in ΔΨm were observed ~10 s after the injury in cells located no further than 150–200 µm from the scratch border. Ca2+ entry into cells during mechanical damage of the primary neuroglial culture occurred predominantly through the NMDA-type glutamate ionotropic channels. MK801, an inhibitor of this type of glutamate receptor, prevented an acute increase in [Ca2+]i in 99% of neurons. Pathological changes in calcium homeostasis persisted in the primary neuroglial culture for one week after injury. Active cell migration in the scratch area occurred on day 11 after neurotrauma and was accompanied by a decrease in the ratio of live to dead cells in the areas adjacent to the injury. Immunohistochemical staining of glial fibrillary acidic protein and β-III tubulin showed that neuronal cells migrated to the injured area earlier than glial cells, but their repair potential was insufficient for survival. Mitochondrial Ca2+ overload and a drop in ΔΨm may cause delayed neuronal death and thus play a key role in the development of the post-traumatic syndrome. Preventing prolonged ΔΨm depolarization may be a promising therapeutic approach to improve neuronal survival after traumatic brain injury.

Characteristics of Whale Müller Glia in Primary and Immortalized Cultures.

Müller cells are the principal glial cells in the retina and they assume many of the functions carried out by astrocytes, oligodendrocytes and ependymal cells in other regions of the central nervous system. Müller cells express growth factors, neurotransmitter transporters and antioxidant agents that could fulfill important roles in preventing excitotoxic damage to retinal neurons. Vertebrate Müller cells are well-defined cells, characterized by a common set of features throughout the phylum. Nevertheless, several major differences have been observed among the Müller cells in distinct vertebrates, such as neurogenesis, the capacity to reprogram fish Müller glia to neurons. Here, the Müller glia of the largest adult mammal in the world, the whale, have been analyzed, and given the difficulties in obtaining cetacean cells for study, these whale glia were analyzed both in primary cultures and as immortalized whale Müller cells. After isolating the retina from the eye of a beached sei whale (Balaenoptera borealis), primary Müller cell cultures were established and once the cultures reached confluence, half of the cultures were immortalized with the simian virus 40 (SV40) large T-antigen commonly used to immortalize human cell lines. The primary cell cultures were grown until cells reached senescence. Expression of the principal molecular markers of Müller cells (GFAP, Vimentin and Glutamine synthetase) was studied in both primary and immortalized cells at each culture passage. Proliferation kinetics of the cells were analyzed by time-lapse microscopy: the time between divisions, the time that cells take to divide, and the proportion of dividing cells in the same field. The karyotypes of the primary and immortalized whale Müller cells were also characterized. Our results shown that W21M proliferate more rapidly and they have a stable karyotype. W21M cells display a heterogeneous cell morphology, less motility and a distinctive expression of some typical molecular markers of Müller cells, with an increase in dedifferentiation markers like α-SMA and β-III tubulin, while they preserve their GS expression depending on the culture passage. Here we also discuss the possible influence of the animal’s age and size on these cells, and on their senescence.

Electrochemically differentiated human MSCs biosensing platform for quantification of nestin and β-III tubulin as whole-cell system

Polydimethylsiloxane (PDMS) on ITO substrate was used to create a well with conducting surface to adhere human mesenchymal stem cells (hMSCs) and provide electrochemical stimulation for inducing their differentiation into neural-like cells. The cells that received electrochemical stimulation did not show any noticeable change in their viability and proliferation. The cell morphology of the differentiated hMSCs adherent on ITO showed outgrowth and elongation in one dimension, resembling neural-like cells. Immunocytochemistry assessment by quantifying the expression of nestin and β-III tubulin also confirmed the differentiation of hMSCs. These differentiated hMSCs adherent on ITO were used as electrochemical biosensing platform for differential pulse voltammetry (DPV) measurement for selectively quantifying cell surface markers expressed by neural stem cells and mature neurons. The variation of nestin antibodies concentrations from 9 μU to 27 μU showed a linear increase in DPV current with a detection sensitivity of ∼28 nA/μU of antibody. Varying concentrations of β-III tubulin antibodies from 30 μU to 210 μU showed a linear increase in DPV current with a detection sensitivity of ∼2.0 nA/μU of antibody. The highest expression level of cell surface marker corresponding to β-III tubulin in total adherent cells on ITO was calculated. It was in the order of 10−8 U of antibodies/cell, representing the total population of mature neuron cells. This new way of detection may rapidly assess the quantitative expression of cell surface markers/antigens.

814 Differences in Human, Pig, and Rat Spinal Cord Stem Cells in Response to Inflammatory and Regenerative Factors In Vitro

INTRODUCTION: The translation of preclinical science to human applications, including regenerative strategies for spinal cord injury, has been poor. Historically, animal models, such as rats and pigs, have been used to model human spinal cord given the difficulty in obtaining live human spinal cord. METHODS: To mimic post-injury inflammation, primary-derived NSPCs from adult humans (n=8), rats (n=6), and pigs (n=5) were treated with interleukin-6 (IL-6), tumor necrosis factor- α (TNFα), or transforming growth factor- β (TGFβ). To direct regeneration, NSPCs were treated with retinoic acid (RA), platelet-derived growth factor (PDGFα), or bone morphogenic protein-4 (BMP4) to induce neurons, oligodendrocytes or astrocytes, respectively. NSPCs were treated for 7 or 14 days and characterized by immunocytochemistry (GFAP, β-iii tubulin, O4). RESULTS: IL-6, TNFα and TGFβ induced astrogenesis of rat NSPCs (3.9±0.7, 5.0±0.9, and 4.0±0.6 fold, respectively) and reduced neurogenesis (0.14±0.90, 0.07±0.04, 0.07±0.05 fold, respectively) after 7 days. Pig NSPCs increased astrogenesis (1.38±0.04, 1.26±0.05, and 1.45±0.04 fold, respectively) after 14 days. Human NSPCs had reduced astrogenesis (0.14±0.07, 0.6±0.2, and 0.12±0.07 fold, respectively) over 14 days, but generated more neurons with IL-6 and TGFβ treatments (1.23±0.05 and 1.34±0.04 fold, respectively). RA increased neurogenesis of human and rat NSPCs, PDGFα increased oligodendrocyte differentiation of rat NSPCs, and BMP4 increased astrogenesis of human and rat NSPCs at low (40ng/mL) and high (100ng/mL) concentrations, respectively. CONCLUSIONS: For the first time, we have compared primary-derived adult spinal cord NSPCs from humans, pigs, and rats and identified differences in their response to pathophysiological and regenerative factors which would impact successful translation of stem cell-based therapies.

Dissection of molecular and histological subtypes of papillary thyroid cancer using alternative splicing profiles

Despite growing evidence of the relevance of alternative splicing (AS) to cancer development and progression, the biological implications of AS for tumor behaviors, including papillary thyroid cancer (PTC), remain elusive. With the aim of further understanding the molecular and histological subtypes of PTC, we in this study explored whether AS events might act as new molecular determinants. For this purpose, AS profiles were analyzed in RNA-sequencing data from The Cancer Genome Atlas (TCGA) and from a Korean patient dataset. A total of 23 distinct exon-skipping (ES) events that correlated significantly with PTC oncogenic activity and differentiation scores were identified. The two top-ranked ES events, NUMA1_17515 in exon 18 of NUMA1 and TUBB3_38175 in exon 6 of TUBB3, showed high correlations with oncogenic activities and discriminated histological and molecular subtypes of PTC. Furthermore, two novel intron-retention (IR) events for TUBB3 were uncovered. All ES and IR events for the TUBB3 gene were predicted to induce nonsense-mediated mRNA decay. The relative abundances of intron reads in the PTC dataset from TCGA showed IR levels to differ significantly among PTC subtypes, possibly reflecting their different tumor behaviors. This study provides a landscape of AS changes among PTC subtypes and identified two significant AS events, NUMA1_17515 and TUBB3_38175, as potential AS biomarkers for PTC subclassification and characterization. The AS events identified in this study may be involved in the development of phenotypic differences underlying the functional characteristics and histological differentiation of PTCs.

Olfactory Stem Cells for the Treatment of Spinal Cord Injury—A New Pathway to the Cure?

Because full functional recovery after spinal cord injury (SCI) remains a major challenge, stem cell therapies represent promising strategies to improve neurologic functions after SCI. The olfactory mucosa (OM) displays an attractive source of multipotent cells for regenerative approaches and is easily accessible by biopsies because of its exposed location. The regenerative capacity of the resident olfactory stem cells (OSCs) has been demonstrated in animal as well as clinical studies. This study aims to demonstrate the feasibility of isolation, purification and cultivation of OSCs. OM specimens were taken dorso-posterior from nasal middle turbinate. OSCs were isolated and purified using the neurosphere assay. Differentiation capacity of the OSCs in neural lineage and their behavior in a plasma clot matrix were investigated. Our study demonstrated that OSCs differentiated into neural lineage and were positive for GFAP as well as β-III tubulin. Furthermore, OSCs were viable and proliferated in a plasma clot matrix. Because there are no standard methods for purification, characterization, and delivery of OSCs to the injury site, which is a prerequisite for the clinical approval, this study focuses on the establishment of appropriate methods and underlines the high potential of the OM for autologous cell therapeutic approaches.

Predictive and Prognostic Significance of mRNA Expression and DNA Copies Aberrations of ERCC1, RRM1, TOP1, TOP2A, TUBB3, TYMS, and GSTP1 Genes in Patients with Breast Cancer.

Increasingly, many researchers are focusing on the sensitivity in breast tumors (BC) to certain chemotherapy drugs and have personalized their research based on the assessment of this sensitivity. One such personalized approach is to assess the chemotherapy’s gene expression, as well as aberrations in the number of DNA copies—deletions and amplifications with the ability to have a significant effect on the gene’s activity. Thus, the aim of this work was to study the predictive and prognostic significance of the expression and chromosomal aberrations of eight chemosensitivity genes in breast cancer patients. Material and methods. The study involved 97 patients with luminal B breast cancer IIB–IIIB stages. DNA and RNA were isolated from samples of tumor tissue before and after treatment. Microarray analysis was performed for all samples on high-density microarrays (DNA chips) of Affymetrix (USA) CytoScanTM HD Array and Clariom™ S Assay, human. Detection of expression level of seven chemosensitivity genes—RRM1, ERCC1, TOP1, TOP2a, TUBB3, TYMS, and GSTP1—was performed using PCR real-time (RT-qPCR). Results. The expression of the RRM1 (AC scheme), TOP2α, TYMS, and TUBB3 genes in patients with an objective response to treatment (complete and partial regression) is higher than in patients with stabilization and progression (p < 0.05). According to our results, the presence of a high level of GSTP1 in a tumor biopsy is associated with the low efficiency of the NAC CP scheme (p = 0.05). The presence of RRM1 deletion is associated with complete and partial regression, as for the TOP1 and TUBB3 genes (p < 0.05). Higher rates of metastatic survival are associated with a high level of expression and amplification of the GSTP1 gene (log-rank test p = 0.02 and p = 0.05). Conclusion. Thus, a complex assessment of the chemotherapy’s gene expression is important not only for understanding the heterogeneity and molecular biology of breast cancer but also to obtain a more accurate disease prognosis.

Epichlo\xeb scottii sp. nov., a new endophyte isolated from Melica uniflora is the missing ancestor of Epichlo\xeb disjuncta

Here we describe a new, haploid and stroma forming species within the genus Epichloë, as Epichloë scottii sp. nov. The fungus was isolated from Melica uniflora growing in Bad Harzburg, Germany. Phylogenetic reconstruction using a combined dataset of the tubB and tefA genes strongly support that E. scottii is a distinct species and the so far unknown ancestor species of the hybrid E. disjuncta. A distribution analysis showed a high infection rate in close vicinity of the initial sampling site and only two more spots with low infection rates. Genetic variations in key genes required for alkaloid production suggested that E. scottii sp. nov. might not be capable of producing any of the major alkaloids including ergot alkaloid, loline, indole-diterpene and peramine. All isolates and individuals found in the distribution analysis were identified as mating-type B explaining the lack of mature stromata during this study. We further release a telomere-to-telomere de novo assembly of all seven chromosomes and the mitogenome of E. scottii sp. nov.

Novel colchicine derivative CR42-24 demonstrates potent anti-tumor activity in urothelial carcinoma.

Bladder cancers, and specifically urothelial carcinoma, have few effective treatment options, and tumors typically develop resistance against standard of care chemotherapies leading to significant mortality. The development of alternative therapies with increased selectivity and improved tolerability would significantly impact this patient population. Here, we investigate a novel colchicine derivative, CR42-24, with increased selectivity for the βIII tubulin subtype as a treatment for urothelial carcinoma. βIII tubulin is a promising target due to its low expression in healthy tissues and its clinical association with poor prognosis. This study demonstrated that CR42-24 is selectively cytotoxic to several cancer cell lines at low nanomolar IC50, with high activity in bladder cancer cell lines both in vitro and in vivo. CR42-24 monotherapy in an aggressive urothelial carcinoma xenograft model results in effective control when treated early. We observed significant ablation of large tumors and patient-derived xenografts at low doses with excellent tolerability. CR42-24 was highly synergistic in combination with the standard of care chemotherapies gemcitabine and cisplatin, further increasing its therapeutic potential as a novel treatment for urothelial carcinoma.

Human Sensory Neuron-like Cells and Glycated Collagen Matrix as a Model for the Screening of Analgesic Compounds.

Increased collagen-derived advanced glycation end-products (AGEs) are consistently related to painful diseases, including osteoarthritis, diabetic neuropathy, and neurodegenerative disorders. We have recently developed a model combining a two-dimensional glycated extracellular matrix (ECM-GC) and primary dorsal root ganglion (DRG) that mimicked a pro-nociceptive microenvironment. However, culturing primary cells is still a challenge for large-scale screening studies. Here, we characterized a new model using ECM-GC as a stimulus for human sensory-like neurons differentiated from SH-SY5Y cell lines to screen for analgesic compounds. First, we confirmed that the differentiation process induces the expression of neuron markers (MAP2, RBFOX3 (NeuN), and TUBB3 (β-III tubulin), as well as sensory neuron markers critical for pain sensation (TRPV1, SCN9A (Nav1.7), SCN10A (Nav1.8), and SCN11A (Nav1.9). Next, we showed that ECM-GC increased c-Fos expression in human sensory-like neurons, which is suggestive of neuronal activation. In addition, ECM-GC upregulated the expression of critical genes involved in pain, including SCN9A and TACR1. Of interest, ECM-GC induced substance P release, a neuropeptide widely involved in neuroinflammation and pain. Finally, morphine, the prototype opiate, decreased ECM-GC-induced substance P release. Together, our results suggest that we established a functional model that can be useful as a platform for screening candidates for the management of painful conditions.

The protective effects of miR-128-3p on sevoflurane-induced progressive neurotoxicity in rats by targeting NOVA1.

MiR-128 is highly expressed in the central nervous system and may regulate the directional differentiation of bone marrow stromal stem cells into nerve cells. However, its role and mechanism in sevoflurane-induced progressive neurotoxicity in rats are rarely reported. Therefore, this study aims to explore the protection of miR-128-3p on sevoflurane-induced neurotoxicity. Hippocampal neurons were isolated and sevoflurane was used to treat the cells. Cell counting kit-8 (CCK-8) was used to detect cell viability. Immunofluorescence was used to detect enrichment of GFAP or βIII tubulin to identify nerve cells. Dual luciferase assay was used to identify the targeted binding relationship between miR-128-3p and NOVA1. The effect of miR-128-3p and sevoflurane on cells regarding apoptosis was detected by flow cytometry. The expression of apoptosis-related protein and oxidative stress-related proteins were detected by western blot. Enzyme-linked immuno-sorbent assay (ELISA) was used to measure inflammatory cytokine levels. Hippocampal neurons' cell viability was significantly decreased by treatment with sevoflurane. MiR-128-3p was down-regulated after sevoflurane treatment in cells. Overexpressed miR-128-3p partially reversed the role of sevoflurane treatment in triggering cell apoptosis, enhancing the expression of Bax and cleaved caspase-3 and inhibiting Bcl-2 expression obviously. Overexpressed miR-128-3p partially reversed the role of sevoflurane treatment in promoting the expression of NOX1and NOX4, and inflammatory cytokine levels by targeting with NOVA1. MiR-128-3p might be a potential therapeutic target for the prevention or treatment of sevoflurane-induced neurotoxicity by targeting with NOVA1.

Generation of Induced Pluripotent Stem Cells from Turner Syndrome (45XO) Fetal Cells for Downstream Modelling of Neurological Deficits Associated with the Syndrome.

Chromosomal aneuploidies cause severe congenital malformations including central nervous system malformations and fetal death. Prenatal genetic screening is purely diagnostic and does not elucidate disease mechanism. Although cells from aneuploid fetuses are valuable biological material bearing the chromosomal aneuploidy, these cells are short lived, limiting their use for downstream research experiments. Generation of induced pluripotent stem cell (iPSC) models is an effective method of cell preparation for perpetual conservation of aneuploid traits. They are self-renewing and differentiate into specialized cells reminiscent of embryonic development. Thus, iPSCs serve as excellent tools to study early developmental events. Turner syndrome (TS) is a rare condition associated with a completely or partially missing X chromosome. The syndrome is characterized by infertility, short stature, endocrine, metabolic, autoimmune and cardiovascular disorders and neurocognitive defects. The following protocol describes isolation and culturing of fibroblasts from TS (45XO) fetal tissue, generation of integration free TSiPSCs through delivery of episomal reprogramming plasmids by nucleofection followed by characterization. The reprogramming TSiPSCs were initially screened by live cell alkaline phosphatase staining followed by extensive probing for pluripotency biomarkers. Selected colonies were mechanically dissected, passaged several times and stable self-renewing cells were used for further experiments. The cells expressed pluripotency transcription factors OCT4, NANOG, SOX2, cell surface markers SSEA 4 and TRA1-81 typical of pluripotent stem cells. The original 45XO karyotype was retained post reprogramming. The TSiPSCs were able to form embryoid bodies and differentiate into cells of endoderm, mesoderm and ectoderm expressing lineage specific biomarkers ((SRY BOX17), (MYOSIN VENTRICULAR HEAVY CHAINα/β), (βIII TUBULIN)). The exogenous episomal plasmids were lost spontaneously and not detected after passage 15 in cells. These TSiPSCs are a valuable cellular resource for modelling defective molecular and cellular neurodevelopment causing neurocognitive deficits associated with Turner syndrome.

TREM2-induced activation of microglia contributes to synaptic resilience in non-demented individuals with Alzheimer's neuropathology.

The existence of individuals who remain cognitively intact despite presenting histopathological signs of Alzheimer's disease (AD), here referred to as "Non demented with AD neuropathology" (NDAN), suggests that some unknown mechanisms are triggered to resist cognitive impairment. Synaptic dysfunction has been identified as one of the major AD causes and it is established that microglia, attracted to plaques, engulf damaged synapses. In this context, exposed phosphatidylserine (ePS) represents a neuronal "eat-me" signal involved in microglial-mediated phagocytosis of damaged synapses. A possible candidate mediator of this process is represented by TREM2, a microglial surface receptor activated by ligands including PS. Based on TREM2 role in the scavenging function of microglia, we hypothesize that an efficient microglial phagocytosis of damaged synapses underlies synaptic resilience in NDAN, thus protecting from memory deficits. Using immunofluorescence microscopy, a comparative study of human post-mortem frontal cortices of aged-matched individuals, AD and NDAN individuals has been performed. The distribution of activated microglia (IBA1 and IBA1/CD68 positive cells) and the expression of phagocytic microglia-related proteins (TREM2 and DAP12) were evaluated. To test the efficacy of microglia in removing debris and damaged synapses, preservation of synapses around plaques was assessed using MAP2 and tubulin βIII as dendritic and axonal markers respectively, and PSD95 as a post synaptic marker. Furthermore, taking advantage of flow cytometry techniques, a study of the ePS using Annexin V assay has been performed. Immunofluorescence analyses indicate higher microglial activation and TREM2 expression in NDAN individuals, as well as preserved axonal and dendritic structure around plaques vs. AD. High levels of PSD95 around NDAN plaques and the colocalization of PSD95 and CD68 may suggest a prompt removal of damaged synapses by hyperactive phagocytic microglia. Annexin V assay on synaptosomes isolated from aged-matched, AD and NDAN individuals indicates changes in the ePS in NDAN individuals, confirming the engulfment of damaged synapses. Our results suggest a higher efficiency of TREM2-induced phagocytic microglia in removing damaged synapses, underlying synaptic resilience in NDAN individuals.

Optimizing fibrin hydrogel toward effective neural progenitor cell delivery in spinal cord injury

Transplantation of neural progenitor cell (NPC) possessing the potential to differentiate into neurons may guard against spinal cord injury (SCI)- associated neuronal trauma. We propose that autologous-like NPC may reduce post-transplant immune response. The study used the rat SCI model to prove this concept. For isolation and expansion of rat NPC for cell-based SCI therapy, the in vitro protocol standardized with human NPC seemed suitable. The primary aim of this study is to select a cell/neural tissue-compatible biomaterial for improving NPC survival in vivo. The composition of the fibrin hydrogel is adjusted to obtain degradable, porous, and robust fibrin strands for supporting neural cell attachment, migration, and tissue regeneration. This study employed NPC culture to evaluate the cytocompatibility and suitability of the hydrogel, composed by adding graded concentrations of thrombin to a fixed fibrinogen concentration. The microstructure evaluation by scanning electron microscope guided the selection of a suitable composition for delivering the embedded cells. On adding more thrombin, fibrinogen clotted quickly but reduced porosity, pore size, and fiber strand thickness. The high activity of thrombin also affected NPC morphology and the in vitro cell survival. The selected hydrogel carried viable NPC and retained them at the injury site post-transplantation. The fibrin hydrogel played a protective role throughout the transfer process by providing cell attachment sites and survival signals. The fibrin and NPC together regulated the immune response at the SCI site reducing ED1+ve/ED2+ve macrophages in the early period of 8–16 d after injury. Migration of β-III tubulin+ve neural-like cells into the fibrin-injected control SCI is evident. The continuous use of a non-neurotoxic fibrin matrix could be a convenient strategy for in vitro NPC preparation, minimally invasive cell delivery, and better transplantation outcome.

Mouse Neural Stem Cell Differentiation and Human Adipose Mesenchymal Stem Cell Transdifferentiation Into Neuron- and Oligodendrocyte-like Cells With Myelination Potential.

Stem cell therapy is an interesting approach for neural repair, once it can improve and increase processes, like angiogenesis, neurogenesis, and synaptic plasticity. In this regard, adult neural stem cells (NSC) are studied for their mechanisms of proliferation, differentiation and functionality in neural repair. Here, we describe novel neural differentiation methods. NSC from adult mouse brains and human adipose-derived stem cells (hADSC) were isolated and characterized regarding their neural differentiation potential based on neural marker expression profiles. For both cell types, their capabilities of differentiating into neuron-, astrocyte- and oligodendrocytes-like cells (NLC, ALC and OLC, respectively) were analyzed. Our methodologies were capable of producing NLC, ALC and OLC from adult murine and human transdifferentiated NSC. NSC showed augmented gene expression of NES, TUJ1, GFAP and PDGFRA/Cnp. Following differentiation induction into NLC, OLC or ALC, specific neural phenotypes were obtained expressing MAP2, GalC/O4 or GFAP with compatible morphologies, respectively. Accordingly, immunostaining for nestin+ in NSC, GFAP+ in astrocytes and GalC/O4+ in oligodendrocytes was detected. Co-cultured NLC and OLC showed excitability in 81.3% of cells and 23.5% of neuron/oligodendrocyte marker expression overlap indicating occurrence of in vitro myelination. We show here that hADSC can be transdifferentiated into NSC and distinct neural phenotypes with the occurrence of neuron myelination in vitro, providing novel strategies for CNS regeneration therapy. Superior Part: Schematic organization of obtaining and generating hNSC from hADSC and differentiation processes and phenotypic expression of neuron, astrocyte and oligodendrocyte markers (MAP2, GFAP and O4, respectively) and stem cell marker (NES) of differentiating hNSC 14days after induction. The nuclear staining in blue corresponds to DAPI. bar = 100μm. Inferior part: Neural phenotype fates in diverse differentiation media. NES: nestin; GFAP: Glial fibrillary acidic protein. MAP2: Microtubule-associated protein 2. TUJ1: β-III tubulin. PDGFRA: PDGF receptor alpha. Two-way ANOVA with Bonferroni post-test with n = 3. * p < 0.05 and ** p < 0.01: (NSCiM1 NSC induction medium 1) vs differentiation media.

Effects of amyloid precursor protein peptide APP96-110, alone or with human mesenchymal stromal cells, on recovery after spinal cord injury.

Delivery of a peptide (APP96-110), derived from amyloid precursor protein (APP), has been shown to elicit neuroprotective effects following cerebral stroke and traumatic brain injury. In this study, the effect of APP96-110 or a mutant version of this peptide (mAPP96-110) was assessed following moderate (200 kdyn, (2 N)) thoracic contusive spinal cord injury (SCI) in adult Nude rats. Animals received a single tail vein injection of APP96-110 or mAPP96-110 at 30 minutes post-SCI and were then assessed for functional improvements over the next 8 weeks. A cohort of animals also received transplants of either viable or non-viable human mesenchymal stromal cells (hMSCs) into the SC lesion site at one week post-injury to assess the effect of combining intravenous APP96-110 delivery with hMSC treatment. Rats were perfused 8 weeks post-SCI and longitudinal sections of spinal cord analyzed for a number of factors including hMSC viability, cyst size, axonal regrowth, glial reactivity and macrophage activation. Analysis of sensorimotor function revealed occasional significant differences between groups using Ladderwalk or Ratwalk tests, however there were no consistent improvements in functional outcome after any of the treatments. mAPP96-110 alone, and APP96-110 in combination with both viable and non-viable hMSCs significantly reduced cyst size compared to SCI alone. Combined treatments with donor hMSCs also significantly increased βIII tubulin+, glial fibrillary acidic protein (GFAP+) and laminin+ expression, and decreased ED1+ expression in tissues. This preliminary study demonstrates that intravenous delivery of APP96-110 peptide has selective, modest neuroprotective effects following SCI, which may be enhanced when combined with hMSC transplantation. However, the effects are less pronounced and less consistent compared to the protective morphological and cognitive impact that this same peptide has on neuronal survival and behaviour after stroke and traumatic brain injury. Thus while the efficacy of a particular therapeutic approach in one CNS injury model may provide justification for its use in other neurotrauma models, similar outcomes may not necessarily occur and more targeted approaches suited to location and severity are required. All animal experiments were approved by The University of Western Australia Animal Ethics Committee (RA3/100/1460) on April 12, 2016.

Prospective Clinical Study of Postoperative Individualized Adjuvant Chemotherapy for Patients with Non-Small-Cell Lung Cancer Based on mRNA Expression of the Molecular Markers RRM1, TUBB3, and ERCC1

Objective To investigate the clinical significance of the mRNA expression of RRM1, TUBB3, and ERCC1 in non-small-cell lung cancer (NSCLC) tissues for the selection of adjuvant/postoperative chemotherapy regimens. Methods Patients diagnosed with stage Ib-IIIa NSCLC were enrolled and randomly divided into a control group (undetected group) and an experimental group (detected group) after radical operation. The control group randomly received chemotherapy with gemcitabine plus cisplatin or paclitaxel plus cisplatin. The mRNA expression of RRM1, TUBB3, and ERCC1 was detected in the experimental group before chemotherapy, and based on the detected expression, the chemotherapy regimen of cisplatin plus gemcitabine or cisplatin plus paclitaxel was chosen. The disease-free survival (DFS) of the control group and experimental group was compared. Results Pathological type, stage, gene expression detection, and treatment method were not significantly correlated with DFS (P > 0.05). In the subgroups treated with gemcitabine, the median DFS was 17 months in the detected group and 10.5 months in the undetected group (hazard ratio = 0.2147, 95% confidence interval: 0.07909–0.5827, P=0.0025). Multivariate regression analysis was performed to analyse whether gene expression detection was independently correlated with DFS in the subgroups treated with gemcitabine (P=0.025). In the detected group, the prognosis of patients with low expression of RRM1 was better than that of patients with high expression of RRM1 after paclitaxel treatment (P=0.0039). Conclusions The selection of chemotherapy regimen based on mRNA expression of the RRM1, TUBB3, and ERCC1 genes may improve selection of candidate patients to receive clinical chemotherapy. However, large-scale prospective clinical studies are needed for in-depth investigation.

Identification of novel microtubule inhibitors effective in fission yeast and human cells and their effects on breast cancer cell lines.

Microtubules are critical for a variety of cellular processes such as chromosome segregation, intracellular transport and cell shape. Drugs against microtubules have been widely used in cancer chemotherapies, though the acquisition of drug resistance has been a significant issue for their use. To identify novel small molecules that inhibit microtubule organization, we conducted sequential phenotypic screening of fission yeast and human cells. From a library of diverse 10 371 chemicals, we identified 11 compounds that inhibit proper mitotic progression both in fission yeast and in HeLa cells. An in vitro assay revealed that five of these compounds are strong inhibitors of tubulin polymerization. These compounds directly bind tubulin and destabilize the structures of tubulin dimers. We showed that one of the compounds, L1, binds to the colchicine-binding site of microtubules and exhibits a preferential potency against a panel of human breast cancer cell lines compared with a control non-cancer cell line. In addition, L1 overcomes cellular drug resistance mediated by βIII tubulin overexpression and has a strong synergistic effect when combined with the Plk1 inhibitor BI2536. Thus, we have established an economically effective drug screening strategy to target mitosis and microtubules, and have identified a candidate compound for cancer chemotherapy.

Neuroprotective role of epigallocatechin-3-gallate in acute glaucoma via the nuclear factor-κB signalling pathway.

Glaucoma is a disease involving impaired visual function accompanied by degeneration and necrosis of the optic nerve. Epigallocatechin-3-gallate (EGCG) exerts a neuroprotective effect against the degeneration of retinal ganglion cells. However, whether EGCG can relieve glaucoma and the possible mechanisms remain unclear. In order to determine the function of EGCG in glaucoma, an acute glaucoma rat model was established. Optic neuropathology was examined by haematoxylin-eosin staining and immunofluorescence staining for class III-β tubulin. The levels of inflammation-associated cytokines, such as interleukin (IL)-4, IL-6, TNF-α, IL-1β, IL-13 and IFN-γ were measured by flow cytometry. T cell proliferation was assessed by the carboxyfluorescein diacetate succinimidyl ester method. Finally, the functional role of EGCG in glaucoma was explored. The levels of the inflammation-associated proteins p-IκBα and p-p65 were measured by western blot analysis. The results showed that optic nerve injury occurred, and elevated levels of the inflammatory cytokines IL-4, IL-6, TNF-α, IL-1β, IL-13 and IFN-γ were observed in the rat model of acute glaucoma. In addition, an increased T lymphocyte proliferation rate and imbalance of Th1/Th2 cytokines were present in the models. Importantly, treatment with EGCG significantly alleviated optic nerve injury. At the molecular level, EGCG decreased the levels of inflammation-associated cytokines, decreased the proliferation rate of T lymphocyte cells, and repaired the imbalance of Th1/Th2 cytokines. Moreover, EGCG inhibited the increase in the phosphorylation of IκBα and p65 caused by modelling and thus suppressed the activation of the nuclear factor (NF)-κB signalling pathway. The findings of the present study indicate that EGCG could attenuate the symptoms of glaucoma and inhibit inflammatory responses by suppressing the NF-κB signalling pathway in a rat glaucoma model.

Characterization of neuronal precursors obtained from human adipose-derived mesenchymal stem cells

Abstract Background Mesenchymal stem cells (MSCs) can be isolated from any tissue derived from the mesoderm and have as main characteristics: high plasticity, the ability to originate mesodermal and non-mesodermal tissues, acting in the modulation of the inflammatory response, and the tissue repair. When grown in microenvironments with elasticity comparable to the human brain, these cells can differentiate efficiently in neural cells due to the mechanism related to the YAP protein, which can mediate responses to substrate stiffness in mesenchymal stem cells. Methods Human adipose-derived MSCs were isolated*, then it was done the trilineage test into adipocytes, osteocytes and, chondrocytes. Besides that, differentiation to neural precursor cells was through neurospheres after seeding the cells over a natural biopolymer matrix as NFBX. Those cells were analyzed using flow cytometry for the surface markers CD13, CD34, CD45, CD73, CD90, CD105, HLA-DR, HLA-ABC, immunocytochemistry for the proteins Nestina, ß-tubulin III, YAP and AMOT and RT-PCR for the NEFM and TUBB3 genes. Results Isolated cells demonstrated characteristics of MSCs. Those cells were differentiated in neural precursors, expressing the proteins Nestina and ß-tubulin III on immunocytochemistry and, the NEFM and TUBB3 genes in RT-PCR. Regarding the YAP and AMOT proteins, it was possible to observe the translocation of the YAP protein in response to the regulation of AMOT out of the cell nucleus, proving neurodifferentiation. Conclusions Human adipose-derived MSCs seeded in a natural biopolymer matrix were able to differentiate into neural precursors expressing characteristic neural markers without adding any neural growth factors or genetic induction.