Immunostaining Of Cells Research Articles

Plasma kallikrein-kinin system contributes to peripheral inflammation in temporal lobe epilepsy.

Temporal lobe epilepsy (TLE) is a chronic disease, characterized by severe and refractory seizures, triggered in the hippocampus and/or amygdala, disrupting the blood-brain barrier. This disruption can sustain, or aggravate, the epileptic condition. The aim of this study was to evaluate the activation of the kallikrein-kinin system in patients with TLE, as it relates to the maintenance of blood-brain barrier. Human hippocampal sclerotic tissues removed after surgery for seizure control, plasma, and serum were used in the following assays: immunostaining for white blood cells in the TLE hippocampus, C-reactive protein in serum, quantification of plasma kallikrein (PKal) and cathepsin B (CatB) activity in serum and plasma, quantification of C1-inhibitor, analysis of high-molecular-weight kininogen (H-kininogen) fragments, and activation of plasma prekallikrein for comparison with healthy controls. Infiltration of white blood cells in the sclerotic hippocampus and a significant increase in the neutrophil/lymphocyte ratio in the blood of TLE patients were observed. High levels of C-reactive protein (TLE=1.4±0.3µg/mL), PKal (TLE=5.4±0.4U/mL), and CatB (TLE=4.9±0.4U/mL) were also evident in the serum of TLE patients comparing to controls. A strong linear correlation was observed between active CatB and PKal in the serum of TLE patients (r=0.88). High levels of cleaved H-kininogen and free PKal, and low levels of C1-inhibitor (TLE=188 ± 12µg/mL) were observed in the serum of TLE patients. Our data demonstrated that the plasma kallikrein-kinin system is activated in patients with TLE. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/.

In Vitro Generation of Heart Field-specific Cardiac Progenitor Cells.

Pluripotent stem cells offer great potential for understanding heart development and disease and for regenerative medicine. While recent advances in developmental cardiology have led to generating cardiac cells from pluripotent stem cells, it is unclear if the two cardiac fields - the first and second heart fields (FHF and SHF) - are induced in pluripotent stem cells systems. To address this, we generated a protocol for in vitro specification and isolation of heart field-specific cardiac progenitor cells. We used embryonic stem cells lines carrying Hcn4-GFP and Tbx1-Cre; Rosa-RFP reporters of the FHF and the SHF, respectively, and live cell immunostaining of the cell membrane protein Cxcr4, a SHF marker. With this approach, we generated progenitor cells which recapitulate the functional properties and transcriptome of their in vivo counterparts. Our protocol can be utilized to study early specification and segregation of the two heart fields and to generate chamber-specific cardiac cells for heart disease modelling. Since this is an in vitro organoid system, it may not provide precise anatomical information. However, this system overcomes the poor accessibility of gastrulation-stage embryos and can be upscaled for high-throughput screens.

On-chip immunofluorescence analysis of single cervical cells using an electroactive microwell array with barrier for cervical screening.

Several specific tests for cervical screening have been developed recently, including p16/Ki67 dual immunostaining for diagnosing high-risk human papillomavirus positive squamous intraepithelial lesion in the cervix. However, manual screening of cells in an entire glass slide is currently a standard clinical procedure for quantification and interpretation of immunocytochemical features of the cells. Here, we developed a microfluidic device containing an electroactive microwell array with barriers (EMAB) for highly efficient single-cell trapping followed by on-chip immunofluorescence analysis with minimum loss of the sample. EMAB utilizes patterned electrodes at the bottom of cell-sized microwells to trap single cells using dielectrophoresis (DEP) and cell-holding structures behind the microwells to stabilize the position of trapped cells even without DEP. Using the device, we evaluated the performance of p16/Ki67 dual immunostaining of HeLa cells on the chip. The device shows 98% cell-trapping efficiency as well as 92% cell-holding efficiency against the fixed HeLa cells, and we successfully demonstrated high-efficiency on-chip immunofluorescence analysis with minimal loss of sample. p16/Ki67 dual immunostaining using EMAB may be useful for complementary tests for cervical screening in confirming the histopathological diagnosis.

Abstract LB-039: Chronic inflammation-mediated contribution of bone marrow-derived epithelial cells and hair follicle stem cells to development of cutaneous neoplasms

Abstract Bone marrow-derived epithelial cells (BMDECs) are recruited to sites of chronic inflammation; however, their clinical significance has not been determined. Here, we used gender-mismatched allogeneic bone marrow transplantation (BMT) in the context of the classical multistage model for murine cutaneous carcinogenesis to probe the recruitment of BMDECs in skin tumors initiated with the carcinogen, dimethylbenz[a]anthracene (DMBA), and promoted with the phorbol ester, 12-O-tetradecanolyphorbol-13-acetate (TPA). We detected clusters of bone marrow-derived cells (BMDCs) in over 40% of papillomas, where they occupied 25% or more of the epithelial lesional area. In the dysplastic ulcers which arose in some irradiated DMBA/TPA treated mice, the magnitude of the recruitment was greater. The BMDCs clustered in the cutaneous epithelium where they became immunoreactive to epidermal keratins, and proliferated and stratified, thereby contributing to the lesions comparably with the progeny of hair follicle stem cells in engrafted Krt1-15Cre;R26R mice. Moreover, cytokeratins were detected by immunostaining and Q-PCR in plastic adherent bone marrow cells (BMCs) cultured in the presence of filter-separated epidermal keratinocytes (KCs). Cytokeratin production was enhanced by bone morphogenetic protein 5 (BMP5). Moreover, BMCs migrated towards the alarmin protein, High Mobility Group Box 1 (HMGB1), as well as towards epidermal KCs in ex vivo invasion assays. Furthermore, when naïve female mice received BMTs from donors previously treated with DMBA, several papillomas and a squamous cell carcinoma were observed after TPA promotion alone. We conclude that surprisingly large numbers of BMDECs are recruited to a subset of cutaneous papillomas and dysplastic ulcers and reflect a previously unrecognized systemic contribution to these lesions. We also conclude that carcinogen-exposed cells originating in the BMCs are sufficient to initiate a subset of benign and malignant lesions upon tumor promotion. Ultimately, these findings may provide novel targets for treatment of non-melanoma skin cancers as well as other cancers. Citation Format: Heuijoon Park, Sonali Lad, Kelsey Boland, Kelly Johnson, Nyssa Readio, Guangchun Jin, Samuel A sfaha, Kelly S. Patterson, Ashok Singh, Xiangdong Yang, Douglas Londono, Anupama Singh, Carol Trempus, Derek Gordon, Timothy C. Wang, Rebecca J. Morris. Chronic inflammation-mediated contribution of bone marrow-derived epithelial cells and hair follicle stem cells to development of cutaneous neoplasms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-039.

MicroRNA-7 directly targets Reg1 in pancreatic cells.

Regenerating islet-derived (Reg) proteins, which were first discovered in the pancreas, are associated with increased proliferation, prevention of apoptosis, and enhanced differentiation in normal and disease states, but very little is known about the regulation of their expression. We hypothesized that Reg expression is influenced by microRNAs. Bioinformatic analysis predicted Reg1 to be a target of microRNA-7 (miR-7), which influences pancreatic β-cell function. To this end, we investigated the effects of miR-7 on Reg1 expression in pancreatic acinar and islet β-cells. High levels of Reg1 were noted by immunostaining and Western blotting in acinar cells in contrast to islet cells. A reciprocal expression pattern was observed for miR-7. Overexpression of miR-7 resulted in Reg1 mRNA suppression and reduction of secreted Reg1 protein. Conversely, miR-7 knockdown led to increases in Reg1. Targeting of Reg1 by miR-7 was confirmed via luciferase activity assays. In contrast, miR-7 did not directly repress the human ortholog of Reg1 REG1A as well as REG1B indicating species differences in the regulation of Reg expression. This is the first account of microRNA modulation of any Reg member warranting studies to fill gaps in our knowledge of Reg protein biology, particularly in disease contexts.

A micro-fabricated in vitro complex neuronal circuit platform

Developments in micro-manufacture as well as biofabrication technologies are driving our ability to create complex tissue models such as ‘organ-on-a-chip’ devices. The complexity of neural tissue, however, requires precisely specific cellular connectivity across many neuronal populations, and thus there have been limited reports of complex ‘brain-on-a-chip’ technologies modelling specific cellular circuit function. Here we describe the development of a model of in vitro brain circuitry designed to accurately reproduce part of the complex circuitry involved in neurodegenerative diseases; using segregated co-culture of specific basal ganglia (BG) neuronal subtypes to model central nervous system circuitry. Lithographic methods and chemical modification were used to form structured micro-channels, which were populated by specifically cultured neuronal sub-types to represent parts of the inter-communicating neural circuit. Cell morphological assessment and immunostaining showed connectivity, which was supported by electrophysiology measurements. Electrical activity of cells was measured using patch-clamp, showing voltage dependant Na+ and K+ currents, and blocking of Na+ current by TTX, and calcium imaging showing TTX-sensitive slow Ca2+ oscillations resulting from action potentials. Monitoring cells across connected ports post-TTX addition demonstrated both upstream and downstream changes in activity, indicating network connectivity. The model developed herein provides a platform technology that could be used to better understand neurological function and dysfunction, contributing to a growing urgency for better treatments of neurodegenerative disease. We anticipate the use of this advancing technology for the assessment of pharmaceutical and cellular therapies as a means of pre-clinical assessment, and further for the advancement of neural engineering approaches for tissue engineering.

Pneumolysin and the bacterial capsule of Streptococcus pneumoniae cooperatively inhibit taxis and motility of microglia

BackgroundStreptococcus pneumoniae is the cause of a highly lethal form of meningitis in humans. Microglial cells in the brain represent the first line of defense against pathogens, and they participate in the inflammatory response. The cholesterol-dependent cytolysin pneumolysin and the bacterial capsule are key pathogenic factors, known to exacerbate the course of pneumococcal meningitis.MethodsWe utilized live imaging and immunostaining of glial cells in dissociated and acute brain slice cultures to study the effect of pneumococcal factors, including the cholesterol-dependent cytolysin pneumolysin and the pneumococcal capsule, on microglial motility and taxis.ResultsIn brain tissue, primary microglia cells showed an enhanced response towards lysates from bacteria lacking capsules and pneumolysin as they moved rapidly to areas with an abundance of bacterial factors. The presence of bacterial capsules and pneumolysin cumulatively inhibited microglial taxis. In mixed cultures of astrocytes and microglia, the motility of microglia was inhibited by capsular components within minutes after exposure. The reduced motility was partially reversed by mannan, a mannose receptor inhibitor. The effects on microglia were not mediated by astrocytes because pure microglial cells responded to various pneumococcal lysates similarly with distinct cell shape changes as seen in mixed cultures.ConclusionsOur data indicate that microglia possess the capacity for a very agile response towards bacterial pathogens, but key pathogenic factors, such as pneumococcal capsules and pneumolysin, inhibited this response shortly after a bacterial challenge. Furthermore, we demonstrate for the first time that the bacterial capsule affects cellular behaviors such as motility and taxis.

Clostridium perfringens Delta-Toxin Damages the Mouse Small Intestine.

Clostridium perfringens strains B and C cause fatal intestinal diseases in animals. The secreted pore-forming toxin delta-toxin is one of the virulence factors of the strains, but the mechanism of intestinal pathogenesis is unclear. Here, we investigated the effects of delta-toxin on the mouse ileal loop. Delta-toxin caused fluid accumulation and intestinal permeability to fluorescein isothiocyanate (FITC)-dextran in the mouse ileal loop in a dose- and time-dependent manner. Treatment with delta-toxin induced significant histological damage and shortening of villi. Delta-toxin activates a disintegrin and metalloprotease (ADAM) 10, leading to the cleavage of E-cadherin, the epithelial adherens junction protein, in human intestinal epithelial Caco-2 cells. In this study, E-cadherin immunostaining in mouse intestinal epithelial cells was almost undetectable 1 h after toxin treatment. ADAM10 inhibitor (GI254023X) blocked the toxin-induced fluid accumulation and E-cadherin loss in the mouse ileal loop. Delta-toxin stimulated the shedding of intestinal epithelial cells. The shedding cells showed the accumulation of E-cadherin in intracellular vesicles and the increased expression of active caspase-3. Our findings demonstrate that delta-toxin causes intestinal epithelial cell damage through the loss of E-cadherin cleaved by ADAM10.

Microscopic structural alterations in response to mesenchymal stem cells injection in experimentally induced oligospermia in adult rat testis (Histological and immunohistochemical study)

Background: Lead is one of the heavy metals that has many side effects on different organs. Recently, infertility among both men and women has become a major concern. Transplantation of mesenchymal stem cells to the male reproductive organ is used for curing various diseases.Aim of the work: The aim of the present work is to study the microscopic structural variations and immunohistochemical appearance of the effects of mesenchymal stem cells transplantation (MSCs) on oligospermia induced in adult male albino rats.Material and methods: Thirty-two adult male rats were divided into; Group I (control group-ten rats), group II, twelve rats injected intravenously by a single dose of lead nitrate (LN, 23.3 mg/kg body weight) and group III (L N with MSCs) ten rats injected intravenously for six weeks. Sperm analysis, hormonal assay and testicular samples were taken for histological, immunohistochemical and statistical analysis at the end of the study.Results: The results revealed that the mean percentage of sperm count, sperm motility and testosterone levels were significantly affected after LN toxicity with apparent changes in the histological pattern of the seminiferous tubules which showed distortion, abnormal distribution, vacuolation, deeply stained nuclei (apoptotic nuclei) and detachment of spermatogenic cells from the germinal epithelium in comparison to the control group. Immunohistochemical study showed that a significant elevation of the intensity of caspase-3 immunostaining in Sertoli, Leydig and peritubular myoid cells in comparison to that of the control group. MSCs injection after lead toxicity showed significant improvement in sperm analysis (count, motility, abnormality and morphology) with marked improvement in histological changes as well as decrease in the mean area% of caspase -3 immune reaction.Conclusion: MCSs partially prevent testicular degenerative changes induced by lead toxicity, suggesting that the MSCs may give hope in the treatment of male infertility.

Expression of bovine interleukin 15 in Pichia pastoris and study on its biological activity: a T-cell activator

Interleukin 15 (IL-15) plays crucial role as stimulatory cytokine in generation and proliferation of CD8+ T memory cells. The present study was initiated to analyze the role of yeast-expressed bovine IL-15 in inducing the CD8+ T memory cells. The bIL-15 was cloned in pPICZαA expression vector. The expressed bovine IL-15 was purified by anion exchange chromatography and further characterized using SDS-PAGE and Western blotting. Biological activity of the purified protein was evaluated in vitro through induction of Bcl2 in IL-15 stimulated PBMCs was measured using qPCR and the phosphorylation of STAT3 was confirmed by immuno-staining of HEK273 cells. From the recent research studies, it was evident that the fatty acid oxidation catalyzed by Carnitine Palmitoyl Transferase 1a (Cpt1a) is a critical step during the conversion of effector CD8+ T cells to memory CD8+ T cells which is induced by IL-15. There were no research studies revealed the role of IL-15 on activating memory CD8+ T cells by influencing the Cpt1a in Bovines was documented; yet and in the present study, we evaluated that bovine IL-15 could induce the expression of Cpt1a in IL-15 treated bovine PBMCs and helps in memory cell induction.

Aliskiren Effect on Kidney Renin‐Prorenin Stained Cells in a Rat Model for Fat Embolism

IntroductionThe renin angiotensin system (RAS) is involved in the control of blood pressure through its action on the kidney and the treatment by the direct renin inhibitor Aliskiren (Ali) reduces it. We have previously shown in a rat model of fat embolism (FE) that triolein (T) injection leads to a severe inflammatory pulmonary reaction that causes fibrosis in lungs and Ali rescues it. We extended this study to the kidney by evaluating the renin/prorenin (R/P) presence in the kidney with and without Ali in a rat model of fat embolism.MethodsSprague Dawley rats (280–300 grams) were injected with T (0.2 ml IV, n=18) or saline (n=4) through tail vein. The T treated rats were divided into three groups of 6 rats each and one hour later injected with 0.2 ml saline, Ali 50mg/kg or Ali 100mg/kg while 4 controls received saline. After 48 hours, rats were euthanized, the kidneys were dissected out and fixed in 10 % formalin, sectioned and later immunostained for R/P specific antibody (from Abcam). Two pathologists unaware of the slide identity took 10 random images. R/P immunostained cells were quantified by using ImageJ program on medulla and cortical regions where the glomeruli were evident.ResultsIn contrast to our previous results in the lungs of the same animals, the renal sections showed a minimal inflammatory damage without significant vasculitis and fibrosis. There are multiple cells cortical, tubular and glomerular showing the R/P cells immunostaining. It was evident by quantification of these cells by Image J that treatment with saline, T + saline, and T+ Ali resulted in an increase of R/P positive cells versus the saline + saline group but there was no statistically significant difference. However, in the glomeruli of the cortical region, R/P staining was mostly confined in the juxtaglomeruli of saline + saline and T+ saline groups and treatment with both doses of Ali reduced it and was statistically significant.ConclusionAs previously reported for lungs and hearts, R/P positive cells were also present in kidneys. However in kidney, marked differences in several regions were seen after Ali administration. Furthermore, by reducing the R/P evaluation only to glomeruli, the study confirms that Ali pharmacological activity is interacting with the RAS system, and the drug has a potential use for the treatment of the respiratory distress syndrome consequent to the occurrence of fat embolism.Support or Funding InformationSupported by Mary Katherine Geldmacher Research Foundation, St. Louis MO.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The Influence of Chemesthesis on Taste Bud Signal Transduction

Chemesthesis, the general chemical sensitivity of the skin and mucus membranes in the oronasal cavities and being perceived as pungency, irritation or heat, impacts taste responsiveness. Previously, capsaicin‐mediated modulation of taste nerve responses was thought to be produced indirectly, via stimulation of peripheral sensory nerve terminals that have ability to release stored transmitters, e.g., calcitonin gene‐related peptide (CGRP) and substance P (SP), onto taste cells. this implies a role in gustatory signaling, by efferent signals from somatosensory neurons. The underlying assumption has been that neuropeptides exert their effects on taste transmitter secretion in taste buds of mice, a well‐accepted model for studying taste bud functions, as theoretically may relate to the processing of the gustatory information. We reported that applying CGRP elicited Type III cells to secrete serotonin (5‐HT), which reduced ATP secretion from Type II cells during the gustatory stimulation. The complex interplay between taste cells and chemosensory neurons, via transmitters, may play an important role during the processing of the complex stimuli involving both spicy and tasty components. Furthermore, to test this assumption, using a combination of Ca2+ imaging with cellular biosensors – genetically‐engineered Chinese Hamster Ovary (CHO) cells, transgenic mice expressing green fluorescent protein (GFP) in the specific taste cell type and immunostaining, we investigated the net effect of SP reduced taste‐evoked ATP secretion from mouse taste buds. Specifically, SP elicited phospholipase C (PLC) activation‐dependent intracellular Ca2+ transients in taste cells via neurokinin 1 (NK1) receptors, most likely on glutamate‐aspartate transporter (GLAST)‐expressing Type I cells. Recently, γ‐aminobutyric acid (GABA) as well as the associated synthetic enzyme have been identified in Type I cells. Consequently, Type I cells secrete GABA in respond to SP. Combined with the recent findings that GABA provides negative paracrine feedback onto Type II cells by activating GABAA and/or GABAB receptors and reducing taste‐evoked ATP secretion, our results showed that taste‐evoked ATP secretion from intact taste buds, which remain cell‐cell communication intact, was reduced when SP was co‐applied with taste stimuli in the bath medium. We conclude that SP plays a role as an inhibitory transmitter that shapes peripheral taste signals via GABAergic signaling during processing gustatory information in taste buds. This seemingly tangled complex represents a new concept of taste signaling and that neuropeptides released from afferent sensory terminals play a regulatory role in processing taste signals.Support or Funding InformationAAA FGAP (AAA‐3419) and SIUSOM Research Grants to AYH.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Clinical impact of a cytological screening system using cyclin D1 immunostaining and genomic analysis for the diagnosis of thyroid nodules

BackgroundFine-needle aspiration (FNA) is the most reliable method for diagnosing thyroid nodules; however, some features such as atypia of undetermined significance or follicular lesion of undetermined significance can confound efforts to identify malignancies. Similar to BRAF, cyclin D1 may be a strong marker of cell proliferation.MethodsOne hundred two patients with thyroidal nodule were enrolled in this prospective study.Expression of cyclin D1 in thyroid nodules was determined by immunohistochemistry using both surgical specimens and their cytological specimens. The identification of the optimal cut off points for the diagnosis of malignancy were evaluated using the receiver operating characteristic (ROC) curves and the assessment of the area under the ROC curve (AUC). The specificity, sensitivity, positive predictive value (PPV) of markers were evaluated from crosstabs based on cut off points and significance were calculated. We also analyzed genetic variants by target NGS for thyroid nodule samples.ResultsThe positive predictive value (PPV) and median stain ratio (MSR) of cyclin D1 nuclear staining was determined in papillary thyroid carcinoma (PPV = 91.5%, MSR = 48.5%), follicular adenoma (PPV = 66.7%, MSR = 13.1%), and adenomatous goiter and inflammation controls (MSR = 3.4%). In FNA samples, a threshold of 46% of immunolabelled cells allows to discriminate malignant lesions from benign ones (P < 0.0001), with 81% sensitivity and 100% specificity. A 46% cutoff value for positive cyclin D1 immunostaining in thyroid cells demonstrated 81% sensitivity and 100% specificity. In surgical specimens, ROC curve analysis showed a 5.8% cyclin D1 immunostaining score predicted thyroid neoplasms at 94.4% sensitivity and 92.3% specificity (P = 0.003), while a 15.7% score predicted malignancy at 86.4% sensitivity and 80.5% specificity (P < 0.0001). Finally, three tested clinico-pathological variables (extra thyroidal extension, intraglandular metastasis, and lymph node metastasis) were significant predictors of cyclin D1 immunostaining (P < 0.001).ConclusionOur cytological cyclin D1 screening system provides a simple, accurate, and convenient diagnostic method in precision medicine enabling ready determination of personalized treatment strategies for patients by next generation sequencing using cytological sample.

Expression of BK channels and Na+-K+ pumps in the apical membrane of lacrimal acinar cells suggests a new molecular mechanism for primary tear-secretion

PurposePrimary fluid secretion in secretory epithelia relies on the unidirectional transport of ions and water across a single cell layer. This mechanism requires the asymmetric apico-basal distribution of ion transporters and intracellular Ca2+ signaling. The primary aim of the present study was to verify the localization and the identity of Ca2+-dependent ion channels in acinar cells of the mouse lacrimal gland. MethodsWhole-cell patch-clamp-electrophysiology, spatially localized flash-photolysis of Ca2+ and temporally resolved digital Ca2+-imaging was combined. Immunostaining of enzymatically isolated mouse lacrimal acinar cells was performed. ResultsWe show that the Ca2+-dependent K+-conductance is paxilline-sensitive, abundant in the luminal, but negligible in the basal membrane; and co-localizes with Cl−-conductance. These data suggest that both Cl− and K+ are secreted into the lumen and thus they account for the high luminal [Cl−] (∼141 mM), but not for the relatively low [K+] (<17 mM) of the primary fluid. Accordingly, these results also imply that K+ must be reabsorbed from the primary tear fluid by the acinar cells. We hypothesized that apically-localized Na+-K+ pumps are responsible for K+-reabsorption. To test this possibility, immunostaining of lacrimal acinar cells was performed using anti-Na+-K+ ATP-ase antibody. We found positive fluorescence signal not only in the basal, but in the apical membrane of acinar cells too. ConclusionsBased on these results we propose a new primary fluid-secretion model in the lacrimal gland, in which the paracellular pathway of Na+ secretion is supplemented by a transcellular pathway driven by apical Na+-K+ pumps.

Isolation and Culture of Single Myofiber and Immunostaining of Satellite Cells from Adult C57BL/6J Mice.

Myofiber isolation followed with ex vivo culture could recapitulate and visualize satellite cells (SCs) activation, proliferation, and differentiation. This approach could be taken to understand the physiology of satellite cells and the molecular mechanism of regulatory factors, in terms of the involvement of intrinsic factors over SCs quiescence, activation, proliferation and differentiation. Single myofiber culture has several advantages that the traditional approach such as FASC and cryosection could not compete with. For example, myofiber isolation and culture could be used to observe SCs activation, proliferation and differentiation at a continuous manner within their physiological "niche" environment while FACS or cryosection could only capture single time-point upon external stimulation to activate satellite cells by BaCl2, Cardiotoxin or ischemia. Furthermore, in vitro transfection with siRNA or overexpression vector could be performed under ex vivo culture to understand the detailed molecular function of a specific gene on SCs physiology. With these advantages, the physiological state of SCs could be analyzed at multiple designated time-points by immunofluorescence staining. In this protocol, we provide an efficient and practical protocol to isolate single myofiber from EDL muscle, followed with ex vivo culture and immunostaining.

Isolation and Culture of Individual Myofibers and Their Adjacent Muscle Stem Cells from Aged and Adult Skeletal Muscle.

The isolation and culture of single floating myofibers with their adjacent muscle stem cells allow the analysis and comparison of muscle stem cells from aged and young mice. This method has the advantage that muscle stem cells are cultured on the myofiber, thereby culturing them in conditions as close to their endogenous niche as possible. Here we describe the isolation, culture, transfection with siRNA, and subsequent immunostaining for muscle stem cells on their adjacent myofibers from aged and young mice.

Novel preparation of Au nanoparticles loaded Laponite nanoparticles/ECM injectable hydrogel on cardiac differentiation of resident cardiac stem cells to cardiomyocytes

Cardiac tissue engineering offers a facile biomedical technology to develop a cardiac tissue regeneration or tissue repair by involving combinations of effective biomaterials and nanomaterials with engineering strategies. The cardiac regenerative materials were fabricated by electrically active nanoparticles onto the biocompatible matrix to inspire low-resistance electrical signal in the native heart tissues. In the current report, we investigated on the improvement of cardiac functionally inspired conductive injectable hydrogel fabricated from the electroactive gold (Au) and laponite (Lap) nanoparticles loaded myocardial extracellular matrix (ECM) to enhance the functional and biological properties of cardiomyocytes. The incorporation of nanoformulations into the ECM was maintained the functionality behaviors and maintenance of electrical conductivity into cardiomyocytes. The effects of nanoparticles onto the ECM were provided decreasing porous structure and interconnected pores in the hydrogel structure for the favorable environment of cardiac tissues. The biological analysis of cell survival and immunostaining studies of cardiomyocytes established that Au loaded Lap/ECM hydrogel improve cell compatibility and phenotypes maturation of cardiac specific proteins. The combination of electrically active nanoformulations and biologically active ECM was enhanced the cell expression of cardiac specific markers (SAC, cTnl and Cx43), indicating the potential role of nanoparticles loaded ECM hydrogel as an appropriate cardiac regenerative material for the repair of infarcted myocardium.

The Diagnostic Utility of p16 Immunostaining in Differentiating Cancer and HSIL from LSIL and Benign in Cervical Cells

Cervical liquid-based cytology plays an important role in the diagnosis of cervical squamous intraepithelial lesion (SIL). However, cytological evaluation alone has a relatively low sensitive. To overcome this problem, HPV DNA testing or HPV DNA combined with cytology has been applied. HPV DNA testing significantly improved the sensitivity, but the specificity is low, especially in cancer and high-grade SIL (HSIL) cases. The aim of this study was to evaluate the diagnostic utility of p16 overexpression in cervical cells of patients with HSIL and cancer. The expression of p16 was detected by immunostaining in liquid-based cells from cervical brushing in 278 patients which including: Cancer (n = 13), HSIL (n = 112), low-grade SIL (LSIL) (n = 45), and Benign (n = 108). The expression levels of p16 were significantly higher in the cancer and HSIL groups when compared with the LSIL and Benign groups (P < 0.01). The accurate diagnostic rates of cancer and HSIL were significantly increased by p16 immunostaining plus cytology than that by cytology alone (P < 0.01). The false negative or false positive of p16 immunostaining occurred with a unicellular pattern. With sensitivity of 96.0% and accuracy of 91.7%, the diagnostic performance of p16 immunostaining was much better than that of cytology alone with sensitivity of 36.0% and accuracy of 70.9% (P < 0.01). p16 immunostaining in cervical brushing cells may not only be used as an ancillary tool to cytological diagnosis of cervical neoplasia but also help to distinguish HSIL from LSIL and the triage of transient infection.

Leptin and IGF1 receptors in alpaca (Vicugna pacos) ovaries

Folliculogenesis and ovulation are regulated by gonadotrophins and other factors such as Insulin like growth factor 1 (IGF1) and leptin. In various species the presence of IGF1 receptor (IGF1R) and leptin receptor (ObR) has been detected in the ovary, but not in the alpaca. Thus, the aim of the present study was to evaluate the presence of these receptors in this tissue and analyze if the presence of these receptors in the ovary is related to the presence of a corpus luteum (CL) and if abundances, as determined by immunostaining intensity vary with follicle size. The IGF1R and ObR were identified in primary and secondary follicles, granulosa and theca interna cells of tertiary follicles and in CL. There were greater abundances of IGF1R in granulosa cells of tertiary follicles of ovaries without compared with those with CL. In both groups, the immunostaining of granulosa cells was greater than in theca interna cells. The abundance of ObR was greater in primary and secondary follicles, and theca interna cells of tertiary follicles in ovaries with than those without CL. Immunostaining of granulosa cells was greater than theca interna cells only in ovaries without CL. There were no differences in the abundance of ObR and IGF1R between primary and secondary follicles and granulosa cells of tertiary follicles, neither in ovaries with or without CL. The abundance of IGF1R was not correlated with abundance of ObR neither in ovaries with or without CL. These results indicate a possible role for IGF and leptin in ovarian function. Furthermore, these receptors could be regulated by ovarian steroid hormones because abundance of these receptors in ovaries varies depending on whether there is a CL present in the ovary.

Amelioration of Titanium Dioxide nanoparticles induced injury on the cerebellum of the adult mice by a hydromethanolic root extract of Withania Somnifera

Introduction: Titanium Dioxide (TiO2) nanoparticles have potential risks to human health. They were able to enter brain to be detected in the cerebral cortex, cerebellum and hippocampus. Withania somnifera (WS) improve rotenone induced damage in cerebellum.Aim of the work: To study the effects of oral administration of TiO2 nanoparticles on the cerebellum and the protective role of hydromethanolic root extract of WS (Egyptian Ashwaghandha).Material and Methods: Seventy five adult male albino mice were divided into five groups (N=15): group 1 (control), group 2 (gum acacia group), and group 3 (WS root extract group) recieved orally hydromethanolic WS root extract (500 mg/kg) once daily, group 4 (TiO2 nanoparticles group) recieved orally TiO2 nanoparticles (150 mg/ Kg), dissolved in gum acacia solution once daily, group 5 (TiO2 nanoparticles + WS root extract group). After sixty days, Sections from the cerebellum were prepared and stained with HandE, Cresyl violet stain as well as Immunohistological stains for nNOS, iNOS, eNOS and GFAP. Morphometrical and statistical analyses were performed.Results: In TiO2 nanoparticles group, cerebellum showed disrupted purkinje cells with marked degenerative changes, decreased Nissl granules in Purkinje cells, vacuolations in all layers of the cerebellar cortex and dilated congested capillaries in white matter. The number of the Purkinje cell were decreased while GFAP positive astrocytes were increased. Decreased nNOS immunostaining in cortical layers, while the iNOS immunostaining increased and increased eNOS immunostaining in capillary endothelial cells were detected. Administration of the hydromethanolic WS root extract improved the altered cerebellar morphology with significant statistical improvement in purkinje cell and astrocyte count.Conclusion: TiO2 nanoparticles oral administration induced toxic effects and WS contains active ingredients that counteract these effects.