Showing papers in "Tissue & Cell in 2019"

A comparative study of deep learning architectures on melanoma detection.

Abstract: Melanoma is the most aggressive type of skin cancer, which significantly reduces the life expectancy. Early detection of melanoma can reduce the morbidity and mortality associated with skin cancer. Dermoscopic images acquired by dermoscopic instruments are used in computational analysis for skin cancer detection. However, some image quality limitations such as noises, shadows, artefacts exist that could compromise the robustness of the skin image analysis. Hence, developing an automatic intelligent system for skin cancer diagnosis with accurate detection rate is crucial. In this paper, we evaluate the performance of several state-of-the-art convolutional neural networks in dermoscopic images of skin lesions. Our experiment is conducted on a graphics processing unit (GPU) to speed up the training and deployment process. To enhance the quality of images, we employ different pre-processing steps. We also apply data augmentation methodology such as horizontal and vertical flipping techniques to address the class skewness problem. Both pre-processing and data augmentation could help to improve the final accuracy.

Comparative assessment of CNN architectures for classification of breast FNAC images.

Abstract: Fine needle aspiration cytology (FNAC) entails using a narrow gauge (25-22 G) needle to collect a sample of a lesion for microscopic examination. It allows a minimally invasive, rapid diagnosis of tissue but does not preserve its histological architecture. FNAC is commonly used for diagnosis of breast cancer, with traditional practice being based on the subjective visual assessment of the breast cytopathology cell samples under a microscope to evaluate the state of various cytological features. Therefore, there are many challenges in maintaining consistency and reproducibility of findings. However, the advent of digital imaging and computational aid in diagnosis can improve the diagnostic accuracy and reduce the effective workload of pathologists. This paper presents a comparison of various deep convolutional neural network (CNN) based fine-tuned transfer learned classification approach for the diagnosis of the cell samples. The proposed approach has been tested using VGG16, VGG19, ResNet-50 and GoogLeNet-V3 (aka Inception V3) architectures of CNN on an image dataset of 212 images (99 benign and 113 malignant), later augmented and cleansed to 2120 images (990 benign and 1130 malignant), where the network was trained using images of 80% cell samples and tested on the rest. This paper presents a comparative assessment of the models giving a new dimension to FNAC study where GoogLeNet-V3 (fine-tuned) achieved an accuracy of 96.25% which is highly satisfactory.

Serial block face scanning electron microscopy in cell biology: Applications and technology.

Abstract: Three-dimensional electron microscopy (3DEM) is an imaging field containing several powerful modalities such as serial section transmission electron microscopy and electron tomography. However, large-scale 3D studies of biological ultrastructure on a cellular scale have historically been hampered by the difficulty of available techniques. Serial block face scanning electron microscopy (SBFSEM) is a 3DEM technique, developed in 2004, which has greatly increased the reliability, availability and throughput of 3DEM. SBFSEM allows for 3D imaging at resolutions high enough to resolve membranes and small vesicles whilst having the capability to collect data with a large field of view. Since its introduction it has become a major tool for ultrastructural investigation and has been applied in the study of many biological fields, such as connectomics, cellular and matrix biology. In this review, we will discuss biological SBFSEM from a technical standpoint, with a focus on cellular applications and also subsequent image analysis techniques.

Volume microscopy in biology: FIB-SEM tomography.

Abstract: Volume microscopy has become an important method in cellular biology. In contrast to tedious serial sectioning volumes can now far more conveniently be obtained with serial-block face and focussed ion beam scanning electron microscopy. Serial-block face scanning electron microscopy is the instrument of choice for large volumes whereas focussed ion beam scanning electron microscopy has its merits in high voxel resolution. These aspects are discussed along with some specific applications of a focussed ion beam scanning electron microscope.

Coenzyme Q10 protects hepatocytes from ischemia reperfusion-induced apoptosis and oxidative stress via regulation of Bax/Bcl-2/PUMA and Nrf-2/FOXO-3/Sirt-1 signaling pathways.

Abstract: Coenzyme Q10 (CoQ10) is a component of the mitochondrial electron transport chain and regarded as a strong anti-oxidant agent. In this study, we focused on the mechanistic insights involved in the hepato-protective effects of CoQ10 against hepatic ischemia reperfusion (IR) injury. Our results revealed that CoQ10 significantly improved hepatic dysfunctions and oxidative stress caused by IR injury. Interestingly, as compared to IR subjected rat, CoQ10 inhibited apoptosis by marked down-regulation of both Bax and PUMA genes while the level of Bcl-2 gene was significantly increased. Moreover, CoQ10 up-regulated PI3K, Akt and mTOR protein expressions while it inhibited the expression of both GSK-3β and β-catenin. Additionally, CoQ10 restored oxidant/antioxidant balance via marked activated Nrf-2 protein as well as up-regulation of both Sirt-1 and FOXO-3 genes. Moreover, CoQ10 strongly inhibited inflammatory response through down-regulation of NF-κB-p65 and decrease both JAK1 and STAT-3 protein expressions with a subsequent modulating circulating inflammatory cytokines. Furthermore, histopathological analysis showed that CoQ10 remarkably ameliorated the histopathological damage induced by IR injury. Taken together, our results suggested and proved that CoQ10 provided a hepato-protection against hepatic IR injury via inhibition of apoptosis, oxidative stress, inflammation and their closed related pathways.

Caffeic acid reduces oxidative stress and microglial activation in the mouse hippocampus

Abstract: A number of studies have indicated the benefits of coffee consumption on physical and mental health; however, scientific evidence on these effects, in particular of the benefits to brain function, has not been determined. In the present study, we aimed to determine the benefits of caffeic acid in the nervous system. For this purpose, we administered doses of 0 or 300 mg/kg for 30 days to mice that were not otherwise affected. We analyzed survival of newly born cells, oxidative stress, inflammatory marker expression, and microglial activation in the hippocampus. We found that caffeic acid had no effect on the expression levels of neurotrophic factors and inflammatory or anti-inflammatory cytokines. However, caffeic acid-treated mice exhibited significantly lower levels of 4-hydroxynonenal, an oxidative stress marker, in the hippocampus, as well as significantly fewer activated microglia. Abnormally high oxidative stress, as well as activated microglia accumulation are both considered to relate to the pathophysiology of neurological and psychiatric disorders. The present study demonstrates the physiological effects of caffeic acid and may explain the suggested benefits of coffee consumption on brain health.

Notch signaling regulates osteosarcoma proliferation and migration through Erk phosphorylation

Abstract: We used a murine spontaneous osteosarcoma cell line with high metastatic potential, the K7M2 cell line to study the role of Notch signaling in the biological manifestations of osteosarcoma, to understand its underlying mechanism in the regulation of cell proliferation and migration, and to improve patient prognosis in cases of osteosarcoma through the discovery of novel therapeutic targets, First, Notch expression in K7M2 was determined by immunostaining, and the γ-secretase inhibitor N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT) was used to inhibit proteolytic cleavage of the Notch intracellular domain (NICD), resulting in the inhibition of Notch activation. By using the Sulforhodamine B assay, colony-forming units assay, Brdu and Ki67 staining, and flow cytometry assays of apoptosis and cell cycle stage, DAPT was found to inhibit K7M2 proliferation in a dose-dependent manner. By using wound healing and transwell migration assays, DAPT was found to inhibit K7M2 migration in a dose-dependent manner as well. By using a combination of micro-Raman spectroscopy and K-means clustering analysis, we found that DAPT inhibit a variety of important cell metabolism-related components in most K7M2 cell structures. Then, DAPT was found to inhibit Notch1ICD expression in a concentration-dependent manner, and this expression was directly correlated with Phospho-Erk1/2 (p-Erk) by using Western blotting. To confirm this finding, we used the Notch signaling ligand Jagged1 to activate the Notch signaling pathway, which in turn up-regulated p-Erk, resulting in increased proliferation and migration of K7M2. Using the Erk pathway inhibitor U0126, we showed that p-Erk was downregulated and the proliferation and migration of K7M2 decreased along with it. Finally, we constructed a K7M2 mouse para-tibial tumor model and lung metastatic model. We found DAPT inhibits p-Erk in vivo, effectively controls tumor growth, reduces angiogenesis, reduces metastasis to the lungs, and improves overall survival. In summary, Notch signaling plays an oncogene role and promotes metastasis in osteosarcoma through p-Erk. DAPT effectively inhibits osteosarcoma proliferation and metastasis in vivo and in vitro by inhibiting Erk phosphorylation. Therefore, the inhibition of Notch activation resulted the down-regulation of phosphorylation of Erk pathway can be used as potential therapeutic targets in clinical treatment to improve osteosarcoma prognosis.

Immunophenotypic characterization, multi-lineage differentiation and aging of zebrafish heart and liver tissue-derived mesenchymal stem cells as a novel approach in stem cell-based therapy.

Abstract: Mesenchymal stem cells (MSCs) are a good model for preclinical and clinical investigations, and alternative sources of MSCs are subject to intensive experiments. In this study, mesenchymal stem cells (MSCs) were isolated from heart and liver tissue of Zebrafish (Danio rerio). The flow-cytometry as well as RT-PCR were used to analyze the expression of a panel of cell surface markers CD44, CD90, CD31 and CD34. In the following, alizarin red, oil red-O and toluidine blue staining were carried out to evaluate the multi-lineage differentiation of zebrafish heart and liver tissue-derived MSCs. Subsequently, the gene and protein expression of Oct4, Sox2 and Nanog as pluri-potent markers were analyzed by RT-PCR and western blotting, respectively. In addition, MTT assay was used for cell proliferation potential and population doubling time (PDT) assessment. Also, the aging of cells was investigated by β-galactosidase activity assay. The results showed that, like other MSCs, zebrafish heart and liver tissue-derived MSCs were positive for mesenchymal, negative for hematopoietic markers and expressed pluripotent markers Oct4, Sox2 and Nanog. Moreover, these cells were differentiated to osteocyte, adipocyte, and chondrocyte lineages following directed differentiation. It was found that PDT of zebrafish heart and liver tissue-derived MSCs were 50.67 and 46.61 h, respectively. These cells had significantly more rapid growth on day 4. Our results show that zebrafish heart and liver tissue-derived MSCs exhibited typical MSC characteristics including fibroblast morphology, multi-lineage differentiation capacity, pluripotency potential and expression of a typical set of classic MSC surface markers.

A biomimetic cartilage gradient hybrid scaffold for functional tissue engineering of cartilage.

Abstract: Osteochondral tissue has a complex layered structure that is not self-repairing after a cartilage defect. Therefore, constructing a biomimetic gradient scaffold that meets the specific structural requirements of osteochondral tissue is a major challenge in the field of cartilage tissue engineering. In this study, chitosan/Sodium β-glycerophosphate/Gelatin (Cs/GP/Gel) biomimetic gradient scaffolds were prepared by regulating the mass ratio of single layer raw materials. The same ratio of Cs/GP/Gel hybrid scaffold material was used as the control. Physical properties such as water absorption, porosity and the degradation rate of the material were compared to optimize the proportion of scaffold materials. P3 Bone Mesenchymal Stem Cells (BMSCs) were inoculated on the gradient and the control scaffolds to investigate its biocompatibility. Scanning electron microscopy (SEM) results show that 3:1:2, 6:1:3.5, 9:1:5, 12:1:6.5, 15:1:8 Cs/GP/Gel gradient scaffolds had excellent three-dimensional porous structures. Channels were also shown to have been interconnected, and the walls of the pores were folded. In the longitudinal dimension, gradient scaffolds had an obvious stratified structure and pore gradient gradualism, that effectively simulated the natural physiological stratified structure of real cartilage. The diameter of the pores in the control scaffold was uniform and without any pore gradient. Gradient scaffolds had good water absorption (584.24 ± 3.79˜677.47 ± 1.70%), porosity (86.34 ± 5.10˜95.20 ± 2.86%) and degradation (86.09 ± 2.46˜92.48 ± 3.86%). After considering the physical properties assessed, the Cs/GP/Gel gradient scaffold with a ratio of 9:1:5 was found to be the most suitable material to support osteochondral tissue. BMSCs were subsequently inoculated on the proportional gradient and hybrid scaffolds culture. These cells survived, distributed and extended well on the gradient and hybrid scaffold material. The biomimetic gradient scaffold designed and prepared in this study provides an important foundation for the development of new gradient composite biomedical materials for osteochondral repair.

Effect of royal jelly on testicular antioxidant enzymes activity, MDA level and spermatogenesis in rat experimental Varicocele model

Abstract: Varicocele is one of the most prevalent causes of infertility. It causes induction of oxidative stress, increases lipid peroxidation in the testis and disrupts spermatogenesis cycle. The aim of the study was to investigate the possible protective effects of royal jelly against varicocele induced oxidative stress, biochemical and histological alterations in the experimental varicocele model in rat. Twenty-one adult Wistar rats were divided into three groups. The control group (I), Varicocele and administration of normal saline (II), varicocele and treatment with RJ (III). At the end of the experiment, all the animals were sacrificed and testes excised. The activity of catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx) and MDA levels were measured. Also, histopathological examinations, Johnsen scores and sperm parameters were determined. There was a significant (p<0.05) increase in the activity level of CAT (0.223±0.005), SOD (0.177±0.0062), GPx (9.575±0.318) and a significant (p<0.05) reduction in the MDA level (2.674±0.336) of the experimental varicocele treated with royal jelly when compared to the activity of CAT (0.011±0.004), SOD (0.035±0.0096), GPx (8.864±0.397) and MDA level (4.630±0.579) of the experimental varicocele and administration of normal saline. Results of the Johnsen score showed a significant increase (p<0.05) in the mean score of the RJ group (7.94±1.5) when compared to the normal saline group (6.04±1.4). Therefore, RJ is a potential area for further studies and improving in spermatogenesis cycle after varicocele.

Sugar and ice: Immunoelectron microscopy using cryosections according to the Tokuyasu method.

Abstract: Since the pioneering work of Kiyoteru Tokuyasu in the 70ths the use of thawed cryosections prepared according to the "Tokuyasu-method" for immunoelectron microscopy did not lose popularity We owe this method a whole subcellular world described by discrete gold particles pointing at cargo, receptors and organelle markers on delicate images of the inner life of a cell Here we explain the procedure of sample preparation, sectioning and immunolabeling in view of recent developments and the reasoning behind protocols including some historical perspective Cryosections are prepared from chemically fixed and sucrose infiltrated samples and labeled with affinity probes and electron dense markers These sections are ideal substrates for immunolabeling, since antigens are not exposed to organic solvent dehydration or masked by resin Instead, the structures remain fully hydrated throughout the labeling procedure Furthermore, target molecules inside dense intercellular structural elements, cells and organelles are accessible to antibodies from the section surface For the validation of antibody specificity several approaches are recommended including knock-out tissue and reagent controls Correlative light and electron microscopy strategies involving correlative probes are possible as well as correlation of live imaging with the underlying ultrastructure By applying stereology, gold labeling can be quantified and evaluated for specificity

In vivo articular cartilage regeneration through infrapatellar adipose tissue derived stem cell in nanofiber polycaprolactone scaffold.

Abstract: In this study, we report the development of a nanofiber polycaprolactone scaffold that can act as a stem cell carrier to induce chondrogenesis and promote cartilage repair in vivo. Infrapatellar fat pads were obtained from sheep knee and the stem cells were isolated and characterized by flow cytometry. Defects were created in sheep knee, two defects received adipose tissue derived stem cells (ASCs)-polycaprolactone construct, second group received polycaprolactone (PCL), the third group was chosen as the ASCs group and the fourth group was control group. Morphological evaluation showed that defects treated with ASCs-scaffold constructs were completely filled with cartilage-like tissue, while other groups revealed the formation of a thin layer of cartilage-like tissue in the defects. Real-Time RT-PCR showed the increase in collagen type 2 mRNA levels, aggrecan and Sox9 in ASCs/PCL groups in comparison with the other groups. Immunofluorescence and toluidine blue staining results showed the protein expression of collagen type 2 and formation of round and polygonal clusters of chondrocytes in ASCS/PCL group. According to our results nanofiber polycaprolactone promoted the chondrogenesis of infrapatellar adipose tissue derived stem cells in vivo and could offer significant promise in the biological functionality of stem cell tissue engineering in clinical practice.

Non-integrin laminin receptors in epithelia.

Abstract: The interactions between cells and the extracellular matrix (ECM) play a major role in normal and pathological conditions. The ECM can modulate several biological functions including cell proliferation, adhesion, differentiation and survival through its interactions with cell receptors. Laminins are one of the most important glycoproteins present in basement membranes, a type of ECM. The pattern of expression of its different isoforms depends on the spatiotemporal organization of each tissue. While integrins are the most studied laminin receptors, other non-integrin laminin receptors are also involved. This review focuses on two particular non-integrin laminin receptors in the epithelial context: dystroglycan and 37/67 laminin receptor (37/67LR). Dystroglycan is a two-subunit protein discovered in the muscle as part of the dystrophin-associated glycoprotein complex. This protein can also be found in many epithelia where its roles are variable. The 37/67LR is a still incompletely understood laminin receptor that is important to regulate intestinal epithelial cell function and could be involved in various pathological conditions.

Protective effects of exercise on heart and aorta in high-fat diet-induced obese rats

Abstract: We investigated the protective effects of swimming exercise on high-fat diet-induced heart and aorta damage by evaluating oxidative stress and the endothelial nitric oxide (NO) system. Sprague Dawley rats were fed either standard chow (STD, 6% fat) or high-fat diet (HFD; 45% fat) for 18 weeks, with half of the animals trained by daily swimming sessions (EXC; 1 h per day for 5 days/week) for the last 6 weeks of the experimental period and half kept sedentary (SED). Heart and aorta tissues were prepared for routine light and electron microscopy evaluation. Endothelial NOS (eNOS) and inducible NOS (iNOS) distribution in the tissue samples were examined by immunohistochemistry. Biochemical examinations, including blood serum lipid profiles, malondialdehyde (MDA), glutathione (GSH), superoxide dismutase (SOD), and tissue NO levels were measured. Deteriorated heart and aorta morphology, increased MDA levels and iNOS-immunoreactivity (iNOS-ir), as well as decreased GSH, NO, SOD, and eNOS-ir parameters were observed in the HFD + SED group. These morphological and biochemical parameters were ameliorated in the HFD + EXC group. Our study revealed that obesity-induced iNOS activation and increased oxidative stress in cardiac and aorta tissues. Exercise protected the obesity-induced cardiac and aortic tissue damage by modulating oxidant/antioxidant balance via involvement of the NO system.

Pre-embedding labeling for subcellular detection of molecules with electron microscopy

Abstract: Labeling of specific molecules for electron microscopy provides an extremely powerful tool to investigate subcellular distribution of proteins, lipids and sugars with resolution of a few nanometers. Moreover, this technology offers a unique opportunity to see how the pattern of these molecules changes in different physiological or pathological conditions. Pre-embedding labeling evolved as one of the easiest method for molecule detection in electron microscopy. It was named "pre-embedding" because the procedure involves a labeling step before the embedding of the specimen in the resin, its subsequent sectioning and analysis of sections under the electron microscope. Here we review different strategies and technical tips of the pre-embedding procedure, the potential of this method for detection and quantification of molecular components at the ultrastructural level, and the integration of the pre-embedding approach with rapidly developing light and electron microscopy technologies.

PLLA scaffolds with controlled architecture as potential microenvironment for in vitro tumor model.

Abstract: The "microenvironment" where a tumor develops plays a fundamental role in determining its progression, the onset of metastasis and, eventually, its resistance to therapies. Tumor cells can be considered more or less invasive depending both on the nature of the cells and on the site where they are located. Commonly adopted laboratory culture protocols for the investigation of tumor cells take usually place on standard two-dimensional supports. However, such cultures do not allow for reproduction of the biophysical properties of the tumor’s microenvironment, thus causing the cells to lose most of their relevant characteristics. In this work MDA-MB 231 breast cancer cells were cultivated within Poly- l -Lactic Acid (PLLA) scaffolds produced via Thermally Induced Phase Separation (TIPS). Starting from a ternary solution (polymer-solvent-nonsolvent) we produced scaffolds with different morphologies, porosities and pore architectures. The influence of porosity and average pore size upon cell adhesion and growth were investigated by using Cell Counting Kit-8 (CCK-8) as cell viability test, a fluorescence assay staining cell with DAPI and Scanning Electron Microscopy (SEM). Our study demonstrates that the average pore size of the polymeric scaffolds influences both the cell adhesion and resulting morphology of the growing breast cancer cells. In particular, the reported data corroborate the evidence that an average pore size ranging from 40 to 50 μm induces tumor cell aggregation and the formation of the irregular tumor masses typically observed in-vivo. In addition, TIPS proved to be a suitable manufacturing technique for finely tuning the scaffolds’ architecture, relevant to developing the most effective microenvironment for an in-vitro tumor cells growth closely mimicking in-vivo conditions.

Leydig cell tumorigenesis - implication of G-protein coupled membrane estrogen receptor, peroxisome proliferator-activated receptor and xenoestrogen exposure. In vivo and in vitro appraisal.

Abstract: The etiology and molecular characteristics of Leydig cell tumor (LCT) are scarcely known. From the research data stems that estrogen can be implicated in LCT induction and development, however it is not investigated in detail. Considering the above, herein we analyzed the relation between G-protein coupled membrane estrogen receptor, peroxisome proliferator-activated receptor and insulin-like family peptides (insulin-like 3 peptide; INSL3 and relaxin; RLN) expressions as well as estrogen level with impact of xenoestrogen (bisphenol A; BPA, tetrabromobisphenol A; TBBPA, and tetrachlorobisphenol A; TCBPA). While in our previous studies altered GPER-PPAR partnership was found in human LCT being a possible cause and/or additionally effecting on LCT development, here mouse testes with experimentally induced LCT and mouse tumor Leydig cell (MA-10) treated with BPA chemicals were examined. We revealed either diverse changes in expression or co-expression of GPER and PPAR in mouse LCT as well as in MA-10 cells after BPA analogues when compared to human LCT. Relationships between expression of INSL3, RLN, including co-expression, and estrogen level in human LCT, mouse LCT and MA-10 cells xenoestrogen-treated were found. Moreover, involvement of PI3K-Akt-mTOR pathway or only mTOR in the interactions of examined receptors and hormones was showed. Taken together, species, cell of origin, experimental system used and type of used chemical differences may result in diverse molecular characteristics of LCT. Estrogen/xenoestrogen may play a role in tumor Leydig cell proliferation and biochemical nature but this issue requires further studies. Experimentally-induced LCT in mouse testis and MA-10 cells after BPA exposure seem to be additional models for understanding some aspects of human LCT biology.

Characterisation and 3D structure of melanomacrophage centers in shorthorn sculpins (Myoxocephalus scorpius).

Abstract: Melanomacrophage centres (MMCs) are distinct aggregations of pigment-containing cells in internal organs of fish, amphibians and reptiles. Although MMCs are commonly used as biomarkers for anthropogenic exposure in many environmental monitoring programs, a substantial knowledge on characteristics of MMCs is required prior to the assessment of MMC responses. The present study was the first to determine the 3D structure of splenic MMCs of a fish from a number of consecutive histology sections by use of the Fiji and AutoCad software. Most splenic MMCs of shorthorn sculpins (Myoxocephalus scorpius) had spherical shape and limited variation in size (maximum diameter). We confirmed the close relationship between MMCs and blood vessels in spleen of shorthorn sculpins as 97% of investigated MMCs (60 whole MMCs over 510 μm thickness of the samples) were closely associated with splenic blood capillaries (mainly ellipsoids) at least once in a set of consecutive sections. In this paper, we describe variations in morphology, density, size, area, distribution, pigments and response to pathogens of MMC populations from different organs (spleen, kidney, liver, pancreas and gills). Additionally, we provide evidence suggesting the presence and dominance of pheomelanin in MMCs of shorthorn sculpins.

Allogeneic platelet rich plasma serves as a scaffold for articular cartilage derived chondroprogenitors.

Abstract: Limited self-restorative ability of the cartilage has necessitated the use of cell and tissue engineering based therapies. Recent advances in the isolation, expansion and characterization of articular cartilage derived chondroprogenitors(CPs) has gained popularity in its role for cartilage repair. Platelet rich plasma (PRP) is a reliable biological scaffold for in-vitro and in-vivo studies with reported therapeutic applications in cartilage and bone pathologies. The aim of this study was to evaluate whether human allogeneic PRP could serve as a biological scaffold for chondroprogenitors (CPs) in cartilage repair. CPs were isolated from the superficial layer of three osteoarthritic knee joints by fibronectin adhesion assay and characterized using flow cytometric analysis. Allogeneic citrated blood was harvested from three subjects to obtain PRP. CPs at a concentration of one million cells per ml were gelled with PRP using calcium chloride. The PRP-CP scaffolds were subjected for adipogeneic, osteogenic, chondrogeneic differentiation and processed for post differentiation-staining studies (Oil Red O, Von Kossa, Alcian blue staining), immunofluorescence (collagen II) and live dead assays (Calcein AM-Ethidium Homodimer). We show that PRP was able to sustain CP cell viability and differentiate towards adipogenic, osteogenic and chondrogenic lineage under appropriate culture conditions. We also noted positive extracellular matrix production in PRP-CP scaffolds cultured without chondrogenic supplementation. Our results suggest that PRP could be a promising bio-active scaffold due to its synergistic effect in supporting cell proliferation, maintaining cell viability and favoring extracellular matrix production. PRP can be used as biological scaffold for the delivery of CPs in cartilage healing.

Dimethyl fumarate up-regulates expression of major neurotrophic factors in the epidermal neural crest stem cells

Abstract: There is an agreement that combining treatments can lead to substantial improvement, therefore the present study assessed the effects of different concentrations of dimethyl fumarate (DMF) on viability of epidermal neural crest stem cells (EPI-NCSCs). In addition, this investigation was designed to evaluate the effects of DMF on relative expression of major trophic factors mainly the ones with neurotrophic effects, expressed in EPI-NCSCs in order to enhance their therapeutic potential. To determine the appropriate concentration of DMF for EPI-NCSCs treatment, the MTT assay was employed and based on the obtained data, EPI-NCSCs treated with 10μM DMF for 6, 24, 72 or 168 h. In each time point, quantitative RT-PCR technique was used to evaluate NGF, NT-3, BDNF, GDNF and VEGF transcripts. The acquired data showed that 10μM DMF significantly increased the mRNA expression of NGF, NT-3 and BDNF, 72 h following treatment; however, DMF inhibitory effect on GDNF mRNA expression was observed in various time points. No significant changes were detected for VEGF transcript. Our findings reveled that expression of major neurotrophic factors were up-regulated by dimethyl fumarate treatment. Therefore, combining EPI-NCSCs with DMF treatment might be a valuable strategy to improve their therapeutic functions in vivo.

Reactivity of microglia and astrocytes after an excitotoxic injury induced by kainic acid in the rat spinal cord.

Abstract: After injury of the nervous system glial cells react according to the stimuli by modifying their morphology and function. Glia activation was reported in different kainic acid (KA)-induced neurodegeneration models. Here, we describe glial morphometric changes occurring in an excitotoxic KA-induced cervical spinal cord injury model. Concomitant degenerative and apoptotic processes are also reported. Male rats injected at the spinal cord C5 segment either with KA or saline were euthanized at post-injection (PI) days 1, 2, 3 or 7. Anti-IBA-1 and anti-GFAP antibodies were used to identify microglia and activated astrocytes, respectively, and to morphometrically characterized them. Fluoro-Jade B staining and TUNEL reaction were used to determine neuronal and glial degeneration and apoptosis. KA-injected group showed a significant increase in microglia number at the ipsilateral side by PI day 3. Different microglia reactive phenotypes were observed. Reactive microglia was still present by PI day 7. Astrocytes in KA-injected group showed a biphasic increase in number at PI days 1 and 3. Degenerative and apoptotic events were only observed in KA-injected animals, increasing mainly by PI day 1. Understanding the compromise of glia in different neurodegenerative processes may help to define possible common or specific therapeutic approaches directed towards neurorestorative strategies.

The prognostic value of sex hormone receptors expression in laryngeal carcinoma.

Abstract: Laryngeal cancer was identified as the second most common respiratory system malignancy with squamous cell carcinoma being the most common malignant tumor of the larynx. Larynx being a secondary sex organ showing physiological changes during puberty, raises inquiry about the relationship between sex hormones receptors as estrogen receptors (ER), progesterone receptors (PR), androgen receptors (AR) and the development of laryngeal carcinoma. This study was carried out in cancer tissue samples from 50 patients with laryngeal squamous cell carcinoma. Immunohistochemical staining using ER-β, PR, and AR was carried out. The immunohistochemical expression of ER-β, PR and AR was positive in 56%, 50% and 64% of cases respectively. ER-β, and PR expression were significantly higher in poorly differentiated cases and cases with lymphatic invasion while AR expression was significantly lower in poorly differentiated cases and with lymphatic invasion. In conclusion, ER-β and PR may be considered as markers for poor biological behavior of laryngeal carcinoma.

Comparison of human articular chondrocyte and chondroprogenitor cocultures and monocultures: To assess chondrogenic potential and markers of hypertrophy

Abstract: Background and Objective In the field of cartilage repair, use of two or more cell populations such as mesenchymal stem cells with chondrocytes in an in-vitro co-culture synergistic environment has been attempted to evade limitations of monoculture systems and promote/induce chondrogenesis. Articular cartilage-derived chondroprogenitors (CPs), considered to have stem-cell like characteristics have been proposed as a potential contender for neocartilage development. Our objective was to assess whether co-cultures using different ratios of chondrocytes(C) and CPs would demonstrate better results in terms of growth kinetics, surface marker expression, chondrogenic potential, tendency for hypertrophy and glycosaminoglycan deposition than monocultures. Study design Human chondrocytes and CPs (fibronectin adhesion assay) from the same cartilage source were isolated. Passage 2 cells were subjected to monolayer/pellet cultures and were grown as monocultures and cocultures at the following percentage ratios(C:CP) 80:20, 65:35, 50:50, 35:65 and 20:80. Results Analysis of data acquired from population doubling, flow cytometry, RT-PCR and Safranin O uptake demonstrated similar results in all monoculture and co-culture groups with no significant inter-group variation, even when reported specific markers of identification (CD54 and CD44:chondrocyte markers) and isolation (CD29 and CD49e: forming heterodimeric fibronectin receptor for CP sorting) were examined. Conclusion In conclusion, this study suggests the need for improved sorting techniques based on a characteristic differentiating biomarker for selection of cells which are true representatives of CPs possessing properties of enhanced chondrogenesis and reduced hypertrophy.

Canine amniotic membrane mesenchymal stromal/stem cells: Isolation, characterization and differentiation.

Abstract: The amniotic membrane can be considered as one of the sources of isolation of these cells, since it is found in the fetal maternal interface and has low immunogenicity. Mesenchymal stromal/stem cells (MSCs) have not been identified in canine amniotic membrane (AMC). Therefore, our objective was to isolate, culture, characterize and differentiate cells derived from canine amniotic membrane (AMC) and to verify its immunological and tumorigenic potential. For this, 12 dogs fetuses of each gestational age 32, 43 and 55 days were used, and the isolation and culture of the AMC were performed. We observed that the cells presented fibroblastoid morphology and high confluence even after freezing. We also observed that, when induced, they were able to differentiate into osteogenic, adipogenic, and chondrogenic cells, as well as being CD34- and CD105+. Regarding the immunological markers, we found that IL-1, IL-2, IL-6, IL-10 and MHC II were not expressed, whereas MHC I was expressed. After application of AMC cells in nude mice we can verify that there was no tumor formation. Based on this, we conclude that canine amniotic membrane is a good and accessible source for obtaining MSCs of low immunogenic and tumorigenic potential for veterinary therapeutic applications.

Expression of miR-210 mediated by adeno-associated virus performed neuroprotective effects on a rat model of acute spinal cord injury.

Abstract: Acute spinal cord injuries (ASCI) are common neural disorders in traumatology medicine. MicroRNA-210 (miR-210) plays a crucial role in cell survival, endothelial cell migration and cell regeneration. This paper is aim to validate the pathophysiological function of miR-210 on ASCI. We built a rat model of ASCI and utilized an adeno-associated virus (rAAV)-expressing miR-210 for stable over-expression of miR-210. We tested in vivo miR-210 gain of function on ASCI by microinjected rAAV-miR-210 into the rat spinal cord. We further screened the targeting genes of miR-210 by PCR array and detected related signal proteins by Western Blot and qPCR. Over-expression of miR-210 protected neurons while neurologic function scores were improved. We further identified less TUNEL-positive cells, few features of apoptosis under electron microscopy, decreased activities of caspase-3 and 8 and increased vessel count in the spinal cord from rAAV-miR-210 group. We also found rAAV-miR-210 promoted expression of angiogenesis and metastasis-related protein (VEGF and Glut1) and regulated serum levels of inflammation-related cytokines. PCR screen array showed PTP1B, target of miR-210, was significantly down-regulated and Akt phosphorylation was significantly increased in rAAV-miR-210 group. The current data suggest that over-expression of miR-210 may target PTP1B and plays a neuroprotective role on rats after ASCI.

KIF18B as a regulator in microtubule movement accelerates tumor progression and triggers poor outcome in lung adenocarcinoma.

Abstract: KIF18B is involved in several tumor progression and exerts critical effects on microtubule growth during mitosis, but its role in lung adenocarcinoma still remains rare. Hence, we attempted to explore the biological function of KIF18B in lung adenocarcinoma. We first analyzed the expressional pattern of KIF18B in lung adenocarcinoma, and detected the correlation between KIF18B expression and clinical characteristics in lung adenocarcinoma based on The Cancer Genome Atlas (TCGA) database and Oncomine dataset. Subsequently, cell counting kit-8 (CCK-8) assay, wound-healing analysis, and transwell method were performed to assess the effects of KIF18B in lung adenocarcinoma cells. Quantitative real-time reverse transcription-PCR (qRT-PCR) and western blotting were utilized to measure the mRNA and protein expression levels. Our results illustrated that KIF18B expression was significantly up-regulated in lung adenocarcinoma samples compared to normal specimens. High levels of KIF18B were associated with unfavorable prognosis of lung adenocarcinoma patients. Down-regulation of KIF18B in lung adenocarcinoma cells inhibited cell prolifartion, migration, and invasion. Western blot assay demonstrated that KIF18B knockdown markedly decreased Rac1-GTP expression, an important marker of migration and invasion in tumors. Moreover, the phosphorylation of AKT and mTOR expression levels were attenuated after KIF18B knockdown. Taken together, these data enhanced the point that KIF18B might promote lung adenocarcinoma cell proliferation, migration, and invasion by activating Rac1 and mediating the AKT/mTOR signaling pathway.

Oral recombinant methioninase combined with oxaliplatinum and 5-fluorouracil regressed a colon cancer growing on the peritoneal surface in a patient-derived orthotopic xenograft mouse model.

Abstract: The aim of this study was to determine the efficacy of oral recombinant methioninase (o-rMETase) on a model of colon cancer growing on the peritoneal surface using a patients-derived orthotopic xenograft (PDOX) nude mouse model. Forty PDOX mouse models with colon cancer growing on the peritoneum were divided into 4 groups of 10 mice each by measuring the tumor size and fluorescence intensity: untreated control; 5-fluorouracil (5-FU) (50 mg/kg, once a week for two weeks, ip) and oxaliplatinum (OXA) (6 mg/kg, once a week for two weeks, ip); o-rMETase (100 units/day, oral 14 consecutive days); combination 5-FU + OXA and o-rMETase. All treatments inhibited tumor growth compared to the untreated control. The combination of 5-FU + OXA plus o-rMETase was significantly more efficacious than the control and each drug alone and was the only treatment that caused tumor regression. The present study is the first demonstrating the efficacy of o-rMETase combination therapy on a PDOX model of peritoneal colon cancer, suggesting potential clinical development of o-rMETase in a recalcitrant cancer.

Adipose derived mesenchymal stem cells improve the structure and function of autografted mice ovaries through reducing oxidative stress and inflammation: A stereological and biochemical analysis.

Abstract: The aim was to investigate the effect of injecting adipose derived mesenchymal stem cells (ADSCs) on reducing oxidative stress, inflammation and improving the structure and function of the autografted ovaries through stereological and biochemical evaluations. Mice (4–5 weeks old) were divided into three groups: control, autograft, and autograft + ADSCs (six mice per group). 7 days after ovary autografting and ADSCs injection, serum concentrations of IL-6, TNF-α and IL-10, malondialdehyde and superoxide dismutase activity were measured. On day 28, ovary histology and CD31 expression was assessed. Serum concentrations of progesterone and estradiol were also estimated. in the autograft + ADSCs group, the total volume of the ovary and the volume of the cortex, the number of follicles, the serum concentrations of IL10, estradiol and SOD activity significantly increased compared to the autograft group(P ≤ 0.05). Serum concentrations of IL6, TNFα and MDA in the autograft + ADSCs group were significantly lower than the autograft group (P ≤ 0.05). The localization of CD31-positive cells in the theca layer of follicles improved to the control level following ADSCs transplantation. The ability of ADSCs to reduce oxidative stress and inflammation probably plays a considerable role in improving the structure and function of the autografted ovaries.

Metalloproteinase inhibitor GM6001 delays regeneration in holothurians.

Abstract: The effect of the GM6001 metalloproteinase inhibitor on the regeneration of ambulacral structures in Eupentacta fraudatrix has been investigated. Inhibition of proteinase activity exerts a marked effect on regeneration, being dependent on the time when GM6001 is injected. When administration of the inhibitor begins on day 3 post-injury, regeneration is completely abolished, and the animals die. This means that early activation of proteinases is crucial for triggering the regenerative process in holothurians. When GM6001 in first injected on day 7 post-injury, the regeneration rate decreases. However, this effect has proven to be reversible: when inhibition ceases, the regeneration resumes. The effect of the inhibitor is manifested as a retarded degradation of the extracellular matrix, the lack of cell dedifferentiation, and, probably, a slower cell migration. The gelatinase activity is detected in all the regenerating organs of E. fraudatrix. In the holothurian Cucumaria japonica, which is not capable of healing skin wounds and ambulacrum reparation, no gelatinase activity was observed at the site of damage. A suggestion is made that proteinases play an important role in regeneration in holothurians. The most probable morphogenesis regulators are matrix metalloproteinases with gelatinase activity.

Autophagy, lysosome dysfunction and mTOR inhibition in MNU-induced photoreceptor cell damage.

Abstract: Progressive photoreceptor death is the main cause of retinal degeneration diseases. Determining the underlying mechanism of this process is essential for therapy improvement. Autophagy has long been considered to be involved in neuronal degeneration diseases, and the regulation of autophagy is thought to have potential implications for neurodegenerative disease therapies. However, whether autophagy is protective or destructive varies among diseases and is controversial. In the present study, we established an N-methyl-N-nitrosourea (MNU)-induced photoreceptor cell damage model in vitro that faithfully replicated photoreceptor cell death in retinal degeneration diseases. Cell viability was tested by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxy-methoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assays. Reactive oxygen species (ROS) levels were assessed through 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) fluorescence. Autophagy was confirmed by observing autophagosomes using transmission electron microscopy (TEM). A lysosome tracker was used to identify acidic lysosomes in cells. We also measured the expression of some proteins related to autophagy, apoptosis and lysosomal degradation by western blot and immunofluorescence assays. We found that MNU could decrease photoreceptor cell viability in a time- and dose-dependent manner, and this change was accompanied by concomitant increases in ROS and the expression of the apoptosis-inducing protein cleaved caspase-3. Moreover, autophagy was activated by MNU treatment during this process. Inhibition of autophagy with 3-methyladenine accelerated cell damage. Lysosome dysfunction was confirmed by autophagosome enlargement and increased cathepsin expression, which was accompanied by mTOR dephosphorylation. In conclusion, autophagy was activated through inhibition of the PI3K/mTOR pathway in the context of MNU-induced photoreceptor cell death. Prolonged mTOR dephosphorylation and autophagy activation resulted in autophagic vacuole accumulation, as indicated by inefficient degradation in lysosomes, and further led to apoptosis.