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In vitro 3D culture is an important model for tissues in vivo. Cells in different locations of 3D tissues are physiologically different, because they are exposed to different concentrations of oxygen, nutrients, and signaling molecules, and to other environmental factors (temperature, mechanical stress, etc). The majority of high-throughput assays based on 3D cultures, however, can only detect the average behavior of cells in the whole 3D construct. Isolation of cells from specific regions of 3D cultures is possible, but relies on low-throughput techniques such as tissue sectioning and micromanipulation. Based on a procedure reported previously (“cells-in-gels-in-paper” or CiGiP), this paper describes a simple method for culture of arrays of thin planar sections of tissues, either alone or stacked to create more complex 3D tissue structures. This procedure starts with sheets of paper patterned with hydrophobic regions that form 96 hydrophilic zones. Serial spotting of cells suspended in extracellular matrix (ECM) gel onto the patterned paper creates an array of 200 micron-thick slabs of ECM gel (supported mechanically by cellulose fibers) containing cells. Stacking the sheets with zones aligned on top of one another assembles 96 3D multilayer constructs. De-stacking the layers of the 3D culture, by peeling apart the sheets of paper, “sections” all 96 cultures at once. It is, thus, simple to isolate 200-micron-thick cell-containing slabs from each 3D culture in the 96-zone array. Because the 3D cultures are assembled from multiple layers, the number of cells plated initially in each layer determines the spatial distribution of cells in the stacked 3D cultures. This capability made it possible to compare the growth of 3D tumor models of different spatial composition, and to examine the migration of cells in these structures., Chemistry and Chemical Biology

The subset of pro-inflammatory B cells, called late memory, tissue-like or double negative (DN), accumulates in the blood of elderly individuals. Here we show that DN B cells do not proliferate and do not make antibodies to influenza antigens, but they secrete antibodies with autoimmune reactivity, in agreement with their membrane phenotype (CD95+CD21-CD11c+) and their spontaneous expression of the transcription factor T-bet. These cells also increase in the blood of individuals with obesity and autoimmune diseases, but causative mechanisms and signaling pathways involved are known only in part. In the present paper we compare frequencies and metabolic requirements of these cells in the blood of healthy individuals of different ages and in the blood and the subcutaneous adipose tissue (SAT) of individuals with obesity. Results show that DN B cells from young individuals have minimal metabolic requirements, DN B cells from elderly and obese individuals utilize higher amounts of glucose to perform autoimmune antibody production and enroll in aerobic glycolysis to support their function. DN B cells from the SAT have the highest metabolic requirements as they activate oxidative phosphorylation, aerobic glycolysis and fatty acid oxidation. DN B cells from the SAT also show the highest levels of ROS and the highest levels of phosphorylated AMPK (5’-AMP activated kinase) and Sestrin 1, both able to mitigate stress and cell death. This metabolic advantage drives DN B cell survival and function (secretion of autoimmune antibodies). [ABSTRACT FROM AUTHOR]

Organelle selective imaging can reveal structural and functional characters of cells undergoing external stimuli, and is considered critical in revealing biological fundamentals, designing targeted delivery system, and screening potential drugs and therapeutics. This paper describes the nucleoli targeting ability of nanoscale carbon dots (including nanodiamond) that are hydrothermally made with controlled surface charges. The surface charges of carbon dots are controlled in the range of -17.9 to -2.84 mV by changing the molar ratio of two precursors, citric acid (CA) and ethylenediamine (EDA). All carbon dots samples show strong fluorescence under wide excitation wavelength, and samples with both negative and positve charges show strong fluorescent contrast from stained nucleoli. The nucleoli selective imaging of live cell has been confirmed with Hoechst staining and nucleoli specific staining (SYTO RNA-select green), and is explained as surface charge heterogeneity on carbon dots. Carbon dots with both negative and positive charges have better ability to penetrate cell and nucleus membranes, and the charge heterogeneity helps carbon dots to bind preferentially to nucleoli, where the electrostatic environment is favored. [ABSTRACT FROM AUTHOR]

Plasma cell segmentation is the first stage of a computer assisted automated diagnostic tool for multiple myeloma (MM). Owing to large variability in biological cell types, a method for one cell type cannot be applied directly on the other cell types. In this paper, we present PCSeg Tool for plasma cell segmentation from microscopic medical images. These images were captured from bone marrow aspirate slides of patients with MM. PCSeg has a robust pipeline consisting of a pre-processing step, the proposed modified multiphase level set method followed by post-processing steps including the watershed and circular Hough transform to segment clusters of cells of interest and to remove unwanted cells. Our modified level set method utilizes prior information about the probability densities of regions of interest (ROIs) in the color spaces and provides a solution to the minimal-partition problem to segment ROIs in one of the level sets of a two-phase level set formulation. PCSeg tool is tested on a number of microscopic images and provides good segmentation results on single cells as well as efficient segmentation of plasma cell clusters. [ABSTRACT FROM AUTHOR]

This paper presents the design and fabrication of a multi-layer and multi-chamber microchip system using thiol-ene ‘click chemistry’ aimed for drug transport studies across tissue barrier models. The fabrication process enables rapid prototyping of multi-layer microfluidic chips using different thiol-ene polymer mixtures, where porous Teflon membranes for cell monolayer growth were incorporated by masked sandwiching thiol-ene-based fluid layers. Electrodes for trans-epithelial electrical resistance (TEER) measurements were incorporated using low-melting soldering wires in combination with platinum wires, enabling parallel real-time monitoring of barrier integrity for the eight chambers. Additionally, the translucent porous Teflon membrane enabled optical monitoring of cell monolayers. The device was developed and tested with the Caco-2 intestinal model, and compared to the conventional Transwell system. Cell monolayer differentiation was assessed via in situ immunocytochemistry of tight junction and mucus proteins, P-glycoprotein 1 (P-gp) mediated efflux of Rhodamine 123, and brush border aminopeptidase activity. Monolayer tightness and relevance for drug delivery research was evaluated through permeability studies of mannitol, dextran and insulin, alone or in combination with the absorption enhancer tetradecylmaltoside (TDM). The thiol-ene-based microchip material and electrodes were highly compatible with cell growth. In fact, Caco-2 cells cultured in the device displayed differentiation, mucus production, directional transport and aminopeptidase activity within 9–10 days of cell culture, indicating robust barrier formation at a faster rate than in conventional Transwell models. The cell monolayer displayed high TEER and tightness towards hydrophilic compounds, whereas co-administration of an absorption enhancer elicited TEER-decrease and increased permeability similar to the Transwell cultures. The presented cell barrier microdevice constitutes a relevant tissue barrier model, enabling transport studies of drugs and chemicals under real-time optical and functional monitoring in eight parallel chambers, thereby increasing the throughput compared to previously reported microdevices. [ABSTRACT FROM AUTHOR]

Rottlerin is a natural polyphenolic compound that was initially indicated as a PKCδ inhibitor. However, it was recently revealed that it may target a number of molecules and have biological effects on various cell types and is considered as a possible agent for tumor and cell proliferative diseases. Psoriasis is a chronic inflammatory cutaneous disorder with undefined etiology and is characterized by abnormal cellular proliferation, angiogenesis, and inflammation. Therefore, this paper investigates the regulatory effects of rottlerin on normal human epidermal keratinocytes (NHEKs) and imiquimod (IMQ)-induced psoriasiform (IPI) lesions. In vitro results showed that rottlerin inhibited cell proliferation in NHEKs through growth arrest and NFκB inhibition. It may also induce apoptosis in an autophagy-dependent pathway. We found that rottlerin inhibited human microvascular endothelial cells tube formation on matrigel. Rottlerin also decreased the cell senescence of keratinocytes and intracellular ROS generation, which indicated its antioxidant effect. We also showed that rottlerin affects the expression of keratinocyte proliferation biomarkers. In 12-O-tetradecanoylphorbol13-acetate (TPA)–induced keratinocytes, rottlerin significantly inhibited the expression of the induced pro-inflammatory cytokines in keratinocytes. An animal experiment provided the corresponding evidence based on this evidence in vitro, by using IPI model, we found that rottlerin could relieve the psoriasiform of BALB/c mice by inhibiting keratinocyte proliferation, inflammatory cell infiltration, and vascular proliferation. In conclusion, our results suggest that rottlerin may prove useful in the development of therapeutic agents against psoriasis. However, the deep mechanism still requires further study. [ABSTRACT FROM AUTHOR]

Although leishmaniases are responsible for high morbidity and mortality all over the world, no really satisfying treatment exists. Furthermore, the corresponding parasite Leishmania undergoes a very characteristic form of programmed cell death. Indeed, different stimuli can induce morphological and biochemical apoptotic-like features. However, the key proteins involved in mammal apoptosis, such as caspases and death receptors, are not encoded in the genome of this parasite. Currently, little is known about Leishmania apoptosis, notably owing to the lack of specific tools for programmed cell death analysis in these parasites. Furthermore, there is a need for a better understanding of Leishmania programmed cell death in order (i) to better understand the role of apoptosis in unicellular organisms, (ii) to better understand apoptosis in general through the study of an ancestral eukaryote, and (iii) to identify new therapeutic targets against leishmaniases. To advance understanding of apoptosis in Leishmania, in this study we developed a new tool based on the quantification of calcein and propidium iodide by flow cytometry. This double labeling can be employed to distinguish early apoptosis, late apoptosis and necrosis in Leishmania live cells with a very simple and rapid assay. This paper should, therefore, be of interest for people working on Leishmania and related parasites. [ABSTRACT FROM AUTHOR]

This paper summarizes some of our results on the application of oligothiophenes as fluorescent markers for biological studies. The oligomers of thiophene, widely known for their semiconductor properties in organic electronics, are also fluorescent compounds characterized by chemical and optical stability, high absorbance and quantum yield. Their fluorescent emission can be easily modulated via organic synthesis by changing the number of thiophene rings and the nature of side-chains. This review shows how oligothiophenes can be derivatized with active groups such as phosphoramidite, N-hydroxysuccinimidyl and 4-sulfotetrafluorophenyl esters, isothiocyanate and azide by which the (bio)molecules of interest can be covalently bound. This paper also describes how molecules such as oligonucleotides, proteins and even nanoparticles, tagged with oligothiophenes, can be used in experiments ranging from hybridization studies to imaging of fixed and living cells. Finally, a few multilabeling experiments are described. [ABSTRACT FROM AUTHOR]

Cell interaction with biomaterials is one of the keystones to developing medical devices for tissue engineering applications. Biomaterials are the scaffolds that give three-dimensional support to the cells, and are vectors that deliver the cells to the injured tissue requiring repair. Features of biomaterials can influence the behaviour of the cells and consequently the efficacy of the tissue-engineered product. The adhesion, distribution and motility of the seeded cells onto the scaffold represent key aspects, and must be evaluated in vitro during the product development, especially when the efficacy of a specific tissue-engineered product depends on viable and functional cell loading. In this work, we propose a non-invasive and non-destructive imaging analysis for investigating motility, viability and distribution of Mesenchymal Stem Cells (MSCs) on silk fibroin-based alginate microcarriers, to test the adhesion capacity of the fibroin coating onto alginate which is known to be unsuitable for cell adhesion. However, in depth characterization of the biomaterial is beyond the scope of this paper. Scaffold-loaded MSCs were stained with Calcein-AM and Ethidium homodimer-1 to detect live and dead cells, respectively, and counterstained with Hoechst to label cell nuclei. Time-lapse Light Sheet Fluorescent Microscopy (LSFM) was then used to produce three-dimensional images of the entire cells-loaded fibroin/alginate microcarriers. In order to quantitatively track the cell motility over time, we also developed an open source user friendly software tool called Fluorescent Cell Tracker in Three-Dimensions (F-Tracker3D). Combining LSFM with F-Tracker3D we were able for the first time to assess the distribution and motility of stem cells in a non-invasive, non-destructive, quantitative, and three-dimensional analysis of the entire surface of the cell-loaded scaffold. We therefore propose this imaging technique as an innovative holistic tool for monitoring cell-biomaterial interactions, and as a tool for the design, fabrication and functionalization of a scaffold as a medical device. [ABSTRACT FROM AUTHOR]

Background: Liver fibrosis was viewed as a reversible process. The activation of hepatic stellate cells (HSCs) is a key event in the process of liver fibrosis. The induction of senescence of HSCs would accelerate the clearance of the activated HSCs. Previously, we demonstrated that soluble egg antigens (SEA) of Schistosoma japonicum promoted the senescence of HSCs via STAT3/P53/P21 pathway. In this paper, our study was aimed to explore whether there are other signaling pathways in the process of SEA-induced HSCs aging and the underlying effect of SKP2/P27 signal on senescent HSCs. Methodology/Principal findings: Human hepatic stellate cell line, LX-2 cells, were cultured and stimulated with SEA. Western blot and cellular immunofluorescence analysis were performed to determine the expression of senescence-associated protein, such as P27, SKP2 and FoxO3a. Besides, RNA interfering was applied to knockdown the expression of related protein. The senescence of HSCs was determined by senescence-associated β-gal staining. We found that SEA increased the expression of P27 protein, whereas it inhibited the expression of SKP2 and FoxO3a. Knockdown of P27 as well as overexpression of SKP2 both suppressed the SEA-induced senescence of HSCs. In addition, the nuclear translocation of FoxO3a from the nucleus to the cytoplasm was induced by SEA stimulation. Conclusions/Significance: The present study demonstrates that SEA promotes HSCs senescence through the FoxO3a/SKP2/P27 pathway. [ABSTRACT FROM AUTHOR]

Huntington’s disease (HD) cellular pathology is characterised by the aggregation of mutant huntingtin (mHTT) protein into inclusion bodies. The present paper compared the sensitivity of five widely used mHTT antibodies (S830; MW8; EM48; 1C2; ubiquitin) against mice from five commonly used HD mouse models (R6/1; YAC128; HdhQ92; B6 HdhQ150; B6 x129/Ola HdhQ150) at two ages to determine: the most sensitive antibodies for each model; whether mHTT antibody binding differed depending on aggregation stage (diffuse versus frank inclusion); the role of ubiquitin during aggregation as the ubiquitin proteosome system has been implicated in disease development. The models demonstrated unique profiles of antibody binding even when the models varied only by background strain (HdhQ150). MW8 was highly sensitive for detecting frank inclusions in all lines whereas EM48, ubiquitin and 1C2 demonstrated consistent staining in all models irrespective of age or form of mHTT. MW8 and S830 were the most sensitive antibodies with 1C2 the least. Ubiquitin levels were stable for each model regardless of age. Ubiquitin was particularly sensitive in young YAC128 mice that demonstrate an absence of inclusions until ~12 months of age suggesting high affinity to mHTT in its diffuse form. The data indicate that generalisations across models regarding the quantification of aggregations may not be valid and that mHTT antibody binding is unique to the mouse model and sensitive to changes in inclusion development. [ABSTRACT FROM AUTHOR]

Multiple in vitro tests are widely applied to assess the anticancer activity of new compounds, including their combinations and interactions with other drugs. The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay is one of the most commonly used assays to assess the efficacy and interactions of anticancer agents. However, it can be significantly influenced by compounds that modify cell metabolism and reaction conditions. Therefore, several assays are sometimes used to screen for potential anticancer drugs. However, the majority of drug interactions are evaluated only with this single method. The aim of our studies was to verify whether the choice of an assay has an impact on determining the type of interaction and to identify the source of discrepancies. We compared the accuracy of MTT and CVS (crystal violet staining) assays in the interaction of two compounds characterized by similar anticancer activity: isothiocyanates (ITCs) and Selol. Confocal microscopy studies were carried out to assess the influence of these compounds on the reactive oxygen species (ROS) level, mitochondrial membrane potential, dead-to-live cell ratio and MTT-tetrazolium salt reduction rate. The MTT assay was less reliable than CVS. The MTT test of Selol and 2-oxoheptyl ITC, which affected the ROS level and MTT reduction rate, gave false negative (2-oxoheptyl ITC) or false positive (Selol) results. As a consequence, the MTT assay identified an antagonistic interaction between Selol and ITC, while the metabolism-independent CVS test identified an additive or synergistic interaction. In this paper, we show for the first time that the test assay may change the interpretation of the compound interaction. Therefore, the test method should be chosen with caution, considering the mechanism of action of the compound. [ABSTRACT FROM AUTHOR]

Human parechovirus 1 (HPeV-1) (family Picornaviridae) is a global cause of pediatric respiratory and CNS infections for which there is no treatment. Although biochemical and in vitro studies have suggested that HPeV-1 binds to αVβ1, αVβ3 and αVβ6 integrin receptor(s), the actual cellular receptors required for infectious entry of HPeV-1 remain unknown. In this paper we analyzed the expression profiles of αVβ1, αVβ3, αVβ6 and α5β1 in susceptible cell lines (A549, HeLa and SW480) to identify which integrin receptors support HPeV-1 internalization and/or replication cycle. We demonstrate by antibody blocking assay, immunofluorescence microscopy and RT-qPCR that HPeV-1 internalizes and replicates in cell lines that express αVβ1 integrin but not αVβ3 or αVβ6 integrins. To further study the role of β1 integrin, we used a mouse cell line, GE11-KO, which is deficient in β1 expression, and its derivate GE11-β1 in which human integrin β1 subunit is overexpressed. HPeV-1 (Harris strain) and three clinical HPeV-1 isolates did not internalize into GE11-KO whereas GE11-β1 supported the internalization process. An integrin β1-activating antibody, TS2/16, enhanced HPeV-1 infectivity, but infection occurred in the absence of visible receptor clustering. HPeV-1 also co-localized with β1 integrin on the cell surface, and HPeV-1 and β1 integrin co-endocytosed into the cells. In conclusion, our results demonstrate that in some cell lines the cellular entry of HPeV-1 is primarily mediated by the active form of αVβ1 integrin without visible receptor clustering. [ABSTRACT FROM AUTHOR]

The development and progression of systemic lupus erythematosus is mediated by the complex interaction of genetic and environmental factors. To decipher the genetics that contribute to pathogenesis and the production of pathogenic autoantibodies, our lab has focused on the generation of congenic lupus-prone mice derived from the New Zealand Black (NZB) strain. Previous work has shown that an NZB-derived chromosome 4 interval spanning 32 to 151 Mb led to expansion of CD5+ B and Natural Killer T (NKT) cells, and could suppress autoimmunity when crossed with a lupus-prone mouse strain. Subsequently, it was shown that CD5+ B cells but not NKT cells derived from these mice could suppress the development of pro-inflammatory T cells. In this paper, we aimed to further resolve the genetics that leads to expansion of these two innate-like populations through the creation of additional sub-congenic mice and to characterize the role of IL-10 in the suppression of autoimmunity through the generation of IL-10 knockout mice. We show that expansion of CD5+ B cells and NKT cells localizes to a chromosome 4 interval spanning 91 to 123 Mb, which is distinct from the region that mediates the majority of the suppressive phenotype. We also demonstrate that IL-10 is critical to restraining autoantibody production and surprisingly plays a vital role in supporting the expansion of innate-like populations. [ABSTRACT FROM AUTHOR]

Fluorescent dyes are used in biological systems, because they are highly sensitive and selective. In this work, we investigated the fluorescent probe properties of 2‐(5‐(pyridin‐2‐yl)‐1H‐pyrazol‐3‐yl) phenol (PYDP) in two media [sodium dodecyl sulfate (SDS) and human serum albumin (HSA)]. Energy transfer parameters, photophysical and thermodynamic parameters of probe were determined. We investigated cytotoxicity of PYDP against colorectal adenocarcinoma cell lines (HT‐29), breast cancer cell lines (MCF‐7) and 3T3‐L1 adipocytes (3T3 L1) cells. The cell staining property of PYDP was monitored using a confocal microscope. The results showed that PYDP binds to HSA, bindings are due to electrostatic/ionic interactions, and the binding process is spontaneous. PYDP was found to exhibit negligible cytotoxicity at high concentrations, and confocal microscope images showed that PYDP stained the cytoplasm of MCF‐7 cells. [ABSTRACT FROM AUTHOR]

1,3,4-Oxadiazole pharmacophore is still considered a viable biologically active scaffold for the synthesis of more effectual and broad-spectrum antimicrobial agents. Therefore, the present study is based on five 1,3,4-oxadiazole target structures, viz., CAROT, CAROP, CARON (D-A-D-A systems) and NOPON and BOPOB (D-A-D-A-D systems) bearing various bioactive heterocyclic moieties relevant to potential biological activities. Three of the compounds, CARON, NOPON and BOPOB were assessed in-vitro for their efficacy as antimicrobial agents against gram positive (Staphylococcus aureus and Bacillus cereus) and gram negative (Escherichia coli and Klebsiella pneumonia) bacteria; and two fungi, Aspergillus niger and Candida albicans; also, as an anti-tuberculosis agent against Mycobacterium tuberculosis. Most of the tested compounds displayed promising antimicrobial activity, especially CARON which was then analyzed for the minimum inhibitory concentration (MIC) studies. Similarly, NOPON portrayed the highest anti-TB activity among the studied compounds. Consequently, to justify the detected anti-TB activity of these compounds and to recognize the binding mode and important interactions between the compounds and the ligand binding site of the potential target, these compounds were docked into the active binding site of cytochrome P450 CYP121 enzyme of Mycobacterium tuberculosis, 3G5H. The docking results were in good agreement with the result of in-vitro studies. In addition, all the five compounds were tested for their cell viability and have been investigated for cell labeling applications. To conclude, one of the target compounds, CAROT was used for the selective recognition of cyanide ion by 'turn-off' fluorescent sensing technique. The entire sensing activity was examined by spectrofluorometric method and MALDI spectral studies. The limit of detection obtained was 0.14 µM. [ABSTRACT FROM AUTHOR]

The spread of KPC-type carbapenemases is mainly attributed to the global dissemination of Klebsiella pneumoniae (KP) strains belonging to the clonal group (CG) 258, including sequence type (ST) 258 and other related STs. Two distinct clades of CG258-KP have evolved, which differ mainly for the composition of their capsular polysaccharides, and recent studies indicate that clade 1 evolved from an ancestor of clade 2 by recombination of a genomic fragment carrying the capsular polysaccharide (cps) locus. In this paper, we investigated the ability of two ST258-KP strains, KKBO-1 and KK207-1, selected as representatives of ST258-KP clade 2 and clade 1, respectively, to activate an adaptive immune response using ex vivo-stimulation of PBMC from normal donors as an experimental model. Our data showed that KKBO-1 (clade 2) induces a Th17 response more efficiently than KK207-1 (clade 1): the percentage of CD4+IL17+ cells and the production of IL-17A were significantly higher in cultures with KKBO-1 compared to cultures with KK207-1. While no differences in the rate of bacterial internalization or in the bacteria-induced expression of CD86 and HLA-DR by monocytes and myeloid dendritic cells were revealed, we found that the two strains significantly differ in inducing the production of cytokines involved in the adaptive immune response, as IL-1β, IL-23 and TNF-α, by antigen-presenting cells, with KKBO-1 being a more efficient inducer than KK207-1. The immune responses elicited by KK207-1 were comparable to those elicited by CIP 52.145, a highly virulent K. pneumoniae reference strain known to escape immune-inflammatory responses. Altogether, present results suggest that CG258-KP of the two clades are capable of inducing a different response of adaptive immunity in the human host. [ABSTRACT FROM AUTHOR]

This paper summarizes some of our results on the application of oligothiophenes as fluorescent markers for biological studies. The oligomers of thiophene, widely known for their semiconductor properties in organic electronics, are also fluorescent compounds characterized by chemical and optical stability, high absorbance and quantum yield. Their fluorescent emission can be easily modulated via organic synthesis by changing the number of thiophene rings and the nature of side-chains. This review shows how oligothiophenes can be derivatized with active groups such as phosphoramidite, N-hydroxysuccinimidyl and 4-sulfotetrafluorophenyl esters, isothiocyanate and azide by which the (bio)molecules of interest can be covalently bound. This paper also describes how molecules such as oligonucleotides, proteins and even nanoparticles, tagged with oligothiophenes, can be used in experiments ranging from hybridization studies to imaging of fixed and living cells. Finally, a few multilabeling experiments are described.

125I seeds coated with titanium are considered a safe and effective interstitial brachytherapy for tumors, while the cost of 125I seeds is a major problem for the patients implanting lots of seeds. The aim of this paper was to develop a novel silicone coating for 125I seeds with a lower cost. In order to show the radionuclide utilization ratio, the silicone was coated onto the seeds using the electro-spinning method and the radioactivity was evaluated, then the anti-tumor efficacy of silicone 125I seeds was compared with titanium 125I seeds. The seeds were divided into four groups: A (control), B (pure silicone), C (silicone 125I), D (titanium 125I) at 2 Gy or 4 Gy. Their anti-tumour activity and mechanism were assessed in vitro and in vivo using a human extrahepatic cholangiocarcinoma cell line FRH-0201 and tumor-bearing BALB/c nude mice. The silicone 125I seeds showed higher radioactivity; the rate of cell apoptosis in vitro and the histopathology in vivo demonstrated that the silicone 125I seeds shared similar anti-tumor efficacy with the titanium 125I seeds for the treatment of extrahepatic cholangiocarcinoma, while they have a much lower cost. [ABSTRACT FROM AUTHOR]

Transforming growth factor‑β1 (TGF-β1) is a versatile cytokine. It has context-dependent pro- and anti-cell proliferation functions. Activation of latent TGF-β1 requires release of the growth factor from pro-complexes and is regulated through TGF-β binding proteins. Two types of TGF-β binding partners, latent TGF-β-binding proteins (LTBPs) and leucine-rich-repeat-containing protein 32 (LRRC32), have been identified and their expression are cell specific. TGF-β1 also plays important roles in acute myeloid leukemia (AML) cells. However, the expression of LTBPs and LRRC32 are lacking in myeloid lineage cells and the binding protein of TGF-β1 in these cells are unknown. Here we show that a novel leucine-rich-repeat-containing protein family member, LRRC33, with high mRNA level in AML cells, to be the binding and regulating protein of TGF-β1 in AML cells. Using two representative cell lines MV4-11 and AML193, we demonstrate that the protein expression of LRRC33 and TGF-β1 are correlated. LRRC33 co-localizes and forms complex with latent TGF-β1 protein on the cell surface and intracellularly in these cells. Similar as in other cell types, the activation of TGF-β1 in MV4-11 and AML193 cells are also integrin dependent. We anticipate our study to be a starting point of more comprehensive research on LRRC33 as novel TGF-β regulating protein and potential non-genomic based drug target for AML and other myeloid malignancy. [ABSTRACT FROM AUTHOR]

RNA contains various chemical modifications, among which N6-methyladenosine (m6A) is the most prevalent modified nucleotide in eukaryotic mRNA. Emerging evidence suggests that m6A plays an important role in regulating a variety of cellular functions by controlling mRNA processing, translation and degradation. Because m6A is not detectable by standard chemical modification-based approaches, immunological methods, such as ELISA, immunoblotting, immunohistochemistry, m6A RNA immunoprecipitation sequencing and m6A individual-nucleotide resolution cross-linking and immunoprecipitation, have been employed to detect m6A in RNA. Although the most important factor determining the success of these methods is the integrity of highly specific antibodies against m6A, the development of m6A-specific monoclonal antibodies has been challenging. We developed anti-m6A monoclonal antibodies using our recently developed single cell-based monoclonal antibody production system. The binding of one selected antibody, #B1-3, to RNA oligoribonucleotide containing a single m6A had an equilibrium dissociation constant of 6.5 nM, and this antibody exhibited negligible binding to oligoribonucleotides containing a single N1-methyladenosine and unmodified adenosine. The binding was competed by the addition of increasing concentrations of N6-methyl-ATP but not N1-methyl-ATP or ATP. Furthermore, this mAb specifically crosslinked m6A-containing oligoribonucleotide by ultraviolet light, resulting in the induction of cDNA truncation at m6A position. These results show the feasibility of using the validated m6A monoclonal antibody for the specific detection of m6A in RNA. [ABSTRACT FROM AUTHOR]

Calreticulin (CRT) is a high-capacity Ca2+ protein whose expression is up-regulated during cellular transformation and is associated with disease progression in multiple types of malignancies. At the same time, CRT has been characterized as an important stress-response protein capable of inducing immunogenic cell death (ICD) when translocated to the cell surface. It remains unclear why CRT expression is preserved by malignant cells during the course of transformation despite its immunogenic properties. In this study, we identify a novel, critical function of CRT as a cell survival factor in multiple types of human solid-tissue malignancies. CRT knockdown activates p53, which mediates cell-death response independent of executioner caspase activity and accompanied full-length poly ADP ribose polymerase (PARP) cleavage. Mechanistically, we show that down-regulation of CRT results in mitochondrial Ca2+ overload and induction of mitochondria permeability transition pore (mPTP)-dependent cell death, which can be significantly rescued by the mPTP inhibitor, Cyclosporin A (CsA). The clinical importance of CRT expression was revealed in the analysis of the large cohort of cancer patients (N = 2,058) to demonstrate that high levels of CRT inversely correlates with patient survival. Our study identifies intracellular CRT as an important therapeutic target for tumors whose survival relies on its expression. [ABSTRACT FROM AUTHOR]

Compounds belonging to the group of 5-substituted 4-(1,3,4-thiadiazol-2-yl) benzene-1,3-diols exhibit a broad spectrum of biological activity, including antibacterial, antifungal, and anticancer properties. The mechanism of the antifungal activity of compounds from this group has not been described to date. Among the large group of 5-substituted 4-(1,3,4-thiadiazol-2-yl) benzene-1,3-diol derivatives, the compound 4-(5-methyl-1,3,4-thiadiazole-2-yl) benzene-1,3-diol, abbreviated as C1, was revealed to be one of the most active agents against pathogenic fungi, simultaneously with the lowest toxicity to human cells. The C1 compound is a potent antifungal agent against different Candida species, including isolates resistant to azoles, and molds, with MIC100 values ranging from 8 to 96 μg/ml. The antifungal activity of the C1 compound involves disruption of the cell wall biogenesis, as evidenced by the inability of cells treated with C1 to maintain their characteristic cell shape, increase in size, form giant cells and flocculate. C1-treated cells were also unable to withstand internal turgor pressure causing protoplast material to leak out, exhibited reduced osmotic resistance and formed buds that were not covered with chitin. Disturbances in the chitin septum in the neck region of budding cells was observed, as well as an uneven distribution of chitin and β(1→3) glucan, and increased sensitivity to substances interacting with wall polymerization. The ATR-FTIR spectral shifts in cell walls extracted from C. albicans cells treated with the C1 compound suggested weakened interactions between the molecules of β(1→3) glucans and β(1→6) glucans, which may be the cause of impaired cell wall integrity. Significant spectral changes in the C1-treated cells were also observed in bands characteristic for chitin. The C1 compound did not affect the ergosterol content in Candida cells. Given the low cytotoxicity of the C1 compound to normal human dermal fibroblasts (NHDF), it is possible to use this compound as a therapeutic agent in the treatment of surface and gastrointestinal tract mycoses. [ABSTRACT FROM AUTHOR]

Introduction: Genome-Wide Association Studies suggest glutathione S transferase C terminal domain (GSTCD) may play a role in development of Chronic Obstructive Pulmonary Disease. We aimed to define the potential role of GSTCD in airway inflammation and contraction using precision cut lung slice (PCLS) from wild-type (GSTCD+/+) and GSTCD knockout mice (GSTCD-/-). Methods: PCLS from age and gender matched GSTCD+/+ and GSTCD-/- mice were prepared using a microtome. Contraction was studied after applying either a single dose of Methacholine (Mch) (1 μM) or different doses of Mch (0.001 to 100 μM). Each slice was then treated with lipopolysaccharide (LPS) or vehicle (PBS) for 24 hours. PCLS contraction in the same airway was repeated before and after stimulation. Levels of TNFα production was also measured. Results: There were no differences in contraction of PCLS from GSTCD+/+ and GSTCD-/- mice in response to Mch (EC50 of GSTCD+/+ vs GSTCD-/- animals: 100.0±20.7 vs 107.7±24.5 nM, p = 0.855, n = 6 animals/group). However, after LPS treatment, there was a 31.6% reduction in contraction in the GSTCD-/- group (p = 0.023, n = 6 animals). There was no significant difference between PBS and LPS treatment groups in GSTCD+/+ animals. We observed a significant increase in TNFα production induced by LPS in GSTCD-/- lung slices compared to the GSTCD+/+ LPS treated slices. Conclusion: GSTCD knockout mice showed an increased responsiveness to LPS (as determined by TNFα production) that was accompanied by a reduced contraction of small airways in PCLS. These data highlight an unrecognised potential function of GSTCD in mediating inflammatory signals that affect airway responses. [ABSTRACT FROM AUTHOR]

Cord blood (CB) mononuclear cells (MNC) are being tested in clinical trials to treat hypoxic-ischemic (HI) brain injuries. Although early results are encouraging, mechanisms underlying potential clinical benefits are not well understood. To explore these mechanisms further, we exposed mouse brain organotypic slice cultures to oxygen and glucose deprivation (OGD) and then treated the brain slices with cells from CB or adult peripheral blood (PB). We found that CB-MNCs protect neurons from OGD-induced death and reduced both microglial and astrocyte activation. PB-MNC failed to affect either outcome. The protective activities were largely mediated by factors secreted by CB-MNC, as direct cell-to-cell contact between the injured brain slices and CB cells was not essential. To determine if a specific subpopulation of CB-MNC are responsible for these protective activities, we depleted CB-MNC of various cell types and found that only removal of CB CD14+ monocytes abolished neuroprotection. We also used positively selected subpopulations of CB-MNC and PB-MNC in this assay and demonstrated that purified CB-CD14+ cells, but not CB-PB CD14+ cells, efficiently protected neuronal cells from death and reduced glial activation following OGD. Gene expression microarray analysis demonstrated that compared to PB-CD14+ monocytes, CB-CD14+ monocytes over-expressed several secreted proteins with potential to protect neurons. Differential expression of five candidate effector molecules, chitinase 3-like protein-1, inhibin-A, interleukin-10, matrix metalloproteinase-9 and thrombospondin-1, were confirmed by western blotting, and immunofluorescence. These findings suggest that CD14+ monocytes are a critical cell-type when treating HI with CB-MNC. [ABSTRACT FROM AUTHOR]

This article proposes the coupling of the recombinant protein avidin to the polysaccharide gellan gum to create a modular hydrogel substrate for 3D cell culture and tissue engineering. Avidin is capable of binding biotin, and thus biotinylated compounds can be tethered to the polymer network to improve cell response. The avidin is successfully conjugated to gellan gum and remains functional as shown with fluorescence titration and electrophoresis (SDS-PAGE). Self-standing hydrogels were formed using bioamines and calcium chloride, yielding long-term stability and adequate stiffness for 3D cell culture, as confirmed with compression testing. Human fibroblasts were successfully cultured within the hydrogel treated with biotinylated RGD or biotinylated fibronectin. Moreover, human bone marrow stromal cells were cultured with hydrogel treated with biotinylated RGD over 3 weeks. We demonstrate a modular and inexpensive hydrogel scaffold for cell encapsulation that can be equipped with any desired biotinylated cell ligand to accommodate a wide range of cell types. [ABSTRACT FROM AUTHOR]

Direct protein delivery into intact plants remains a challenge for the agricultural and plant science fields. Cell-penetrating peptide (CPP)-mediated protein delivery requires the binding of CPPs to a protein to carry the protein into the cell through the cell wall and lipid bilayer. Thus, we prepared ionic complexes of a CPP-containing carrier peptide and a cargo protein, namely, Citrine yellow fluorescent protein, and subsequently studied their physicochemical properties. Two types of carrier peptides, BP100(KH)9 and BP100CH7, were investigated for delivery efficiency into rice callus. Both BP100(KH)9 and BP100CH7 successfully introduced Citrine protein into rice callus cells under pressure and vacuum treatment. Moreover, delivery efficiency varied at different growth stages of rice callus; 5-day rice callus was a more efficient recipient for Citrine than 21-day callus. [ABSTRACT FROM AUTHOR]

In humans and mice, the detailed phenotypic and functional characterization of peripheral blood monocytes allows for identification of three monocyte subsets. There are also evidences of monocyte phenotypic heterogeneity in other species, including cattle, sheep, pig and horse. However, little is known about such variability in dogs. The aim of the study was to determine whether and how peripheral blood monocytes of healthy dogs differ in the presence of MHCII and CD4 and in the basal production of reactive oxygen species (ROS). Three distinct subsets of CD11b+CD14+ monocytes were found in peripheral blood samples of healthy dogs, based on the variations in the density of MHCII and CD4 surface molecules: MHCII+CD4– (Mo1), MHCII+CD4+ (Mo2) and MHCII–CD4+ (Mo3). The Mo2 and Mo3 were significantly lower in percentage than Mo1 but their basal ROS production was higher. Within the Mo2 and Mo3 subsets, the percentage of cells producing ROS was significantly higher comparing to cells lacking this activity. Canine peripheral blood monocytes vary in the expression of MHCII and CD4 and in the activity suggesting that cells within the three identified subsets carry out different functions. The higher production of ROS in non-activated cells within small subsets of Mo2 and Mo3 monocytes might indicate their immunomodulatory potential. [ABSTRACT FROM AUTHOR]

Mung bean (Vigna radiata), an important legume crop, has the property of desiccation tolerance (DT), which is lost in the final stage of germination (preimbibition, 18 h-24 h). We compared parameters related to the programmed cell death (PCD) of mung bean seeds before and after dehydration at different imbibition stages through various detection methods. The results of Evans blue and TTC staining methods showed that the dehydration process could lead to cell death. The results of optical and subcellular morphology showed that PCD occurred after dehydration. The destruction of DNA integrity and the activity changes in caspase and total nuclease in mung bean seeds after dehydration treatment indicated that the loss of desiccation tolerance was related to PCD. Dehydration resulted in the destruction of the mitochondrial structure, reversal of the membrane potential, and the entrance of cytochrome C into the cytoplasm. These processes all indicate that the mitochondrial apoptosis pathway was the main form of dehydration-induced PCD. The results of cytoplasmic Ca2+ concentration showed that Ca2+ signaling also played a role in inducing PCD, with the upstream signal being dehydration-induced changes in water potential and the downstream signal being the ROS and mitochondrial PT channel, according to the order in which these signals happened. The mitochondrial apoptosis pathway can be considered the main mechanism of dehydration-induced PCD based on our analysis of the sequence of major events in PCD. The main processes include dehydration induction, changes in Ca2+ and mitochondrial respiratory electron transport, the reversal of mitochondrial membrane potential induced by ROS and Ca2+, and the transmission and execution of PCD downstream signals induced by cytochrome C release. [ABSTRACT FROM AUTHOR]

ASAP1 is a multi-domain adaptor protein that regulates cytoskeletal dynamics, receptor recycling and intracellular vesicle trafficking. Its expression is associated with poor prognosis for a variety of cancers, and promotes cell migration, invasion and metastasis. Little is known about its physiological role. In this study, we used mice with a gene-trap inactivated ASAP1 locus to study the functional role of ASAP1 in vivo, and found defects in tissues derived from mesenchymal progenitor cells. Loss of ASAP1 led to growth retardation and delayed ossification typified by enlarged hypertrophic zones in growth plates and disorganized chondro-osseous junctions. Furthermore, loss of ASAP1 led to delayed adipocyte development and reduced fat depot formation. Consistently, deletion of ASAP1 resulted in accelerated chondrogenic differentiation of mesenchymal cells in vitro, but suppressed osteo- and adipogenic differentiation. Mechanistically, we found that FAK/Src and PI3K/AKT signaling is compromised in Asap1GT /GT MEFs, leading to impaired adipogenic differentiation. Dysregulated FAK/Src and PI3K/AKT signaling is also associated with attenuated osteogenic differentiation. Together these observations suggest that ASAP1 plays a decisive role during the differentiation of mesenchymal progenitor cells. [ABSTRACT FROM AUTHOR]

Semiconductor nanowires are increasingly used in optoelectronic devices. However, their effects on human health have not been assessed fully. Here, we investigate the effects of gallium phosphide nanowires on human lung adenocarcinoma cells. Four different geometries of nanowires were suspended in the cell culture for 48 hours. We show that cells internalize the nanowires and that the nanowires have no effect on cell proliferation rate, motility, viability and intracellular ROS levels. By blocking specific internalization pathways, we demonstrate that the nanowire uptake is the result of a combination of processes, requiring dynamin and actin polymerization, which suggests an internalization through macropinocytosis and phagocytosis. [ABSTRACT FROM AUTHOR]

Eukaryotic elongation factor 1 alpha 2 (eEF1A2) is a translation factor selectively expressed by heart, skeletal muscle, nervous system and some specialized cells. Its ectopic expression relates with tumorigenesis in several types of human cancer. No data are available about the role of eEF1A2 in Triple Negative Breast Cancers (TNBC). This study investigated the relation between eEF1A2 protein levels and the prognosis of TNBC. A total of 84 TNBC diagnosed in the period 2002–2011 were included in the study. eEF1A2 protein level was measured in formalin-fixed paraffin-embedded tissues by immunohistochemistry in a semi-quantitative manner (sum of the percentage of positive cells x staining intensity) on a scale from 0 to 300. Cox regression assessed the association between eEF1A2 levels and disease-free survival (DFS) and breast cancer-specific survival (BCSS). Elevated values of eEF1A2 were associated with older age at diagnosis (p = 0.003), and androgen receptors positivity (p = 0.002). At univariate Cox analysis, eEF1A2 levels were not significantly associated with DFS and BCSS (p = 0.11 and p = 0.08, respectively) whereas adjusting for stage of disease, elevated levels of eEF1A2 protein resulted associated with poor prognosis (HR = 1.05, 95% CI: 1.01–1.11, p = 0.04 and HR = 1.07, 95% CI: 1.01–1.14, p = 0.03 for DFS and BCSS, respectively). This trend was confirmed analyzing negative versus positive samples by using categorized scores. Our data showed a negative prognostic role of eEF1A2 protein in TNBC, sustaining further investigations to confirm this result by wider and independent cohorts of patients. [ABSTRACT FROM AUTHOR]

In this study, we report on the synthesis of silver nanoparticles (AgNPs) from the leaf extracts of Cynara scolymus (Artichoke) using microwave irradiation and the evaluation of its anti-cancer potential with photodynamic therapy (PDT). Silver nanoparticles formation was characterized by scanning electron microscopy with energy dispersive x-ray spectroscopy and Fourier transform infrared (FTIR) spectroscopy. Silver nanoparticles formation was also investigated the surface charge, particle size and distribution using zetasizer analysis. The cytotoxic effect of AgNPs and/or PDT was studied by MTT assay and migration by the scratch assay. The apoptotic inducing ability of the AgNPs and/or PDT was investigated by intracellular ROS analysis, antioxidant enzyme levels (SOD, CAT, GPx and GSH), Hoechst staining and Bax/Bcl-2 analysis using western blotting. The mean particle size of produced AgNPs was found 98.47±2.04 nm with low polydispersity (0.301±0.033). Zeta potential values of AgNPs show -32.3± 0.8 mV. These results clearly indicate the successful formation of AgNPs for cellular uptake. Mitochondrial damage and intracellular ROS production were observed upon treatment with AgNPs (10μg/mL) and PDT (0.5 mJ/cm2) showed significant reducing cell migration, expression of Bax and suppression of Bcl-2. Significantly, biosynthesized AgNPs showed a broad-spectrum anti-cancer activity with PDT therapy and therefore represent promoting ROS generation by modulating mitochondrial apoptosis induction in MCF7 breast cancer cells. [ABSTRACT FROM AUTHOR]

During viral infection, tight regulation of CD8+ T-cell functions determines the outcome of the disease. Recently, others and we determined that the natural killer (NK) cells kill hyperproliferative CD8+ T cells in the context of viral infection, but molecules that are involved in shaping the regulatory capability of NK cells remain virtually unknown. Here we used mice lacking the Fc-receptor common gamma chain (FcRγ, FcεRIγ, Fcer1g–/–mice) to determine the role of Fc-receptor and NK-receptor signaling in the process of CD8+ T-cell regulation. We found that the lack of FcRγ on NK cells limits their ability to restrain virus-specific CD8+ T cells and that the lack of FcRγ in Fcer1g–/–mice leads to enhanced CD8+ T-cell responses and rapid control of the chronic docile strain of the lymphocytic choriomeningitis virus (LCMV). Mechanistically, FcRγ stabilized the expression of NKp46 but not that of other killer cell–activating receptors on NK cells. Although FcRγ did not influence the development or activation of NK cell during LCMV infection, it specifically limited their ability to modulate CD8+ T-cell functions. In conclusion, we determined that FcRγ plays an important role in regulating CD8+ T-cell functions during chronic LCMV infection. [ABSTRACT FROM AUTHOR]

Hyperthermia (HT) has shown feasibility and potency as an anticancer therapy. Administration of HT in the chemotherapy has previously enhanced the cytotoxicity of drugs against pancreatic cancer. However, the drugs used when conducting these studies are substantially conventional chemotherapeutic agents that may cause unwanted side effects. Additionally, the thermal dosage in the treatment of cancer cells could also probably harm the healthy cells. The purpose of this work was to investigate the potential of the two natural polyphenolic compounds, epigallocatechin gallate (EGCG) and chlorogenic acid (CGA), as heat synergizers in the thermal treatment of the PANC-1 cells. Furthermore, we have introduced a unique strategy entitled the thermal cycling-hyperthermia (TC-HT) that is capable of providing a maximum synergy and minimal side effect with the anticancer compounds. Our results demonstrate that the combination of the TC-HT and the CGA or EGCG markedly exerts the anticancer effect against the PANC-1 cells, while none of the single treatment induced such changes. The synergistic activity was attributed to the cell cycle arrest at the G2/M phase and the induction of the ROS-dependent mitochondria-mediated apoptosis. These findings not only represent the first in vitro thermal synergistic study of natural compounds in the treatment of pancreatic cancer, but also highlight the potential of the TC-HT as an alternative strategy in thermal treatment. [ABSTRACT FROM AUTHOR]

In this report we demonstrate the effect of a novel electron emission-based cell culture device on the proliferation and differentiation of pre-osteoblastic MC3T3-E1 cells. Our device has an electron emission element that allows, for the first time, stable emission of electrons into an atmosphere. Atmospheric electrons react with gas molecules to generate radicals and negative ions, which induce a variety of biochemical reactions in the attached cell culture system. In this study, we demonstrated the effect of this new electron emission-based cell culture device on cell proliferation and differentiation using pre-osteoblastic MC3T3-E1 cells. Electron emission stimulation (EES) was applied directly to culture medium containing plated cells, after which the number of living cells, the mRNA levels of osteogenesis-related genes, and the alkaline phosphatase (ALP) activity were evaluated. The growth rate of EES-exposed cells increased by approximately 20% in comparison with unexposed control cells. We also found the mRNA levels of osteogenic specific genes such as collagen type I α-1, core-binding factor α-1, and osteocalcin to be up-regulated following EES. ALP activity, a marker for osteogenic activity, was significantly enhanced in EES-treated cells. Furthermore, reactive oxygen species generated by EES were measured to determine their effect on MC3T3-E1 cells. These results suggest that our new electron emission-based cell culture device, while providing a relatively weak stimulus in comparison with atmospheric plasma systems, promotes cell proliferation and differentiation. This system is expected to find application in regenerative medicine, specifically in relation to bone regeneration. [ABSTRACT FROM AUTHOR]

Cadmium (Cd) is a heavy and highly toxic metal that contaminates air, food and water. Cadmium accumulates in several organs altering normal functions. The kidney is the major organ at risk of damage from chronic exposure to cadmium as a contaminant in food and water. This study aims to investigate the beneficial effects of OLE in renal collecting duct MCD4 cells exposed to a low dose cadmium (1 μM). In MCD4 cells cadmium caused an increase in ROS production, as well as generation of lipid droplets and reduced cell viability. Moreover, cadmium exposure led to a remarkable increase in the frequency of micronuclei and DNA double-strand breaks, assessed using the alkaline comet assay. In addition, cadmium dramatically altered cell cytoskeleton architecture and caused S-glutathionylation of actin. Notably, all cadmium-induced cellular deregulations were prevented by co-treatment with OLE, possibly due to its antioxidant action and to the presence of bioactive phytocompounds. Indeed, OLE treatment attenuated Cd-induced actin S-glutathionylation, thereby stabilizing actin filaments. Taken together, these observations provide a novel insight into the biological action of OLE in renal cells and support the notion that OLE may serve as a potential adjuvant against cadmium-induced nephrotoxicity. [ABSTRACT FROM AUTHOR]

The enzymes GALNTs add GalNAc sugar to Ser and Thr residues, forming the Tn glycan. GALNTs are activated by trafficking from Golgi to ER, a process driven by the Src kinase and negatively regulated by ERK8. This GALNTs activation (aka GALA) pathway induces high Tn levels and is a key driver of liver tumor growth. Recently, Tabak and colleagues have contested our previous data that EGF stimulation can induce GALNTs relocation. Here, we show that relocation induced by EGF is actually detectable in the very images acquired by Tabak et al. Furthermore, we show that over-expression of EGFR strongly enhances EGF-induced relocation and that EGFR appears required to drive relocation induced by ERK8 depletion. Direct co-localisation of GALNT with the ER marker Calnexin is observed after EGF stimulation. We furthermore propose that quantification of O-glycosylation of the ER resident protein PDIA4 provides a mean to quantify GALA independently of imaging. In sum, we demonstrate that the claimed non-reproducibility was due to experimental imaging conditions, that EGFR is indeed a driver of GALA and propose additional markers to facilitate the study of this pathway. [ABSTRACT FROM AUTHOR]

The Ki-67 labeling index (LI) is an important prognostic factor in breast carcinoma. The Ki-67 LI is traditionally calculated via unaided microscopic estimation; however, inter-observer and intra-observer variability and low reproducibility are problems with this visual assessment (VA) method. For more accurate assessment and better reproducibility with Ki-67 LI, digital image analysis was introduced recently. We used both VA and automated digital image analysis (ADIA) (Ventana Virtuoso image management software) to estimate Ki-67 LI for 997 cases of breast carcinoma, and compared VA and ADIA results. VA and ADIA were highly correlated (intraclass correlation coefficient 0.982, and Spearman’s correlation coefficient 0.966, p<0.05). We retrospectively analyzed cases with a greater than 5% difference between VA and ADIA results. The cause of these differences was: (1) tumor heterogeneity (98 cases, 56.0%), (2) VA interpretation error (32 cases, 18.3%), (3) misidentification of tumor cells (26 cases, 14.9%), (4) poor immunostaining or slide quality (16 cases, 9.1%), and (5) Estimation of non-tumor cells (3 cases, 1.7%). There were more discrepancies between VA and ADIA results in the group with a VA value of 10–20% compared to groups with <10% and ≥20%. Although ADIA is more accurate than VA, there are some limitations. Therefore, ADIA findings require confirmation by a pathologist. [ABSTRACT FROM AUTHOR]

To use human limbal explants as an alternative source for generating conjunctival epithelium and to determine the effect of interleukin-13 (IL-13) on goblet cell number, mucin expression, and stemness. Human limbal explants prepared from 17 corneoscleral rims were cultured with or without IL-13 (IL-13+ and IL-13-, respectively) and followed up to passage 2 (primary culture [P0]–P2). Cells were characterized by alcian blue/periodic acid–Schiff (AB/PAS) staining (goblet cells); immunofluorescent staining for p63α (progenitor cells), Ki-67 (proliferation), MUC5AC (mucin, goblet cells), and keratin 7 (K7, conjunctival epithelial and goblet cells); and by quantitative real-time polymerase chain reaction for expression of the p63α (TP63), MUC5AC, MUC4 (conjunctival mucins), K3, K12 (corneal epithelial cells), and K7 genes. Clonogenic ability was determined by colony-forming efficiency (CFE) assay. Using limbal explants, we generated epithelium with conjunctival phenotype and high viability in P0, P1, and P2 cultures under IL-13+ and IL-13- conditions, i.e., epithelium with strong K7 positivity, high K7 and MUC4 expression and the presence of goblet cells (AB/PAS and MUC5AC positivity; MUC5AC expression). p63α positivity was similar in IL-13+ and IL-13- cultures and was decreased in P2 cultures; however, there was increased TP63 expression in the presence of IL-13 (especially in the P1 cultures). Similarly, IL-13 increased proliferative activity in P1 cultures and significantly promoted P0 and P1 culture CFE. IL-13 did not increase goblet cell number in the P0–P2 cultures, nor did it influence MUC5AC and MUC4 expression. By harvesting unattached cells on day 1 of P1 we obtained goblet cell rich subpopulation showing AB/PAS, MUC5AC, and K7 positivity, but with no growth potential. In conclusion, limbal explants were successfully used to develop conjunctival epithelium with the presence of putative stem and goblet cells and with the ability to preserve the stemness of P0 and P1 cultures under IL-13 influence. [ABSTRACT FROM AUTHOR]

It is demonstrated that cells can be classified by pattern recognition of the subcellular structure of non-stained live cells, and the pattern recognition was performed by machine learning. Human white blood cells and five types of cancer cell lines were imaged by quantitative phase microscopy, which provides morphological information without staining quantitatively in terms of optical thickness of cells. Subcellular features were then extracted from the obtained images as training data sets for the machine learning. The built classifier successfully classified WBCs from cell lines (area under ROC curve = 0.996). This label-free, non-cytotoxic cell classification based on the subcellular structure of QPM images has the potential to serve as an automated diagnosis of single cells. [ABSTRACT FROM AUTHOR]

Adhesion formation that occurred after alkali-induced injury of the cecum was used as a novel adhesion model in rats, and it was compared with that of a common adhesion model after abrading the cecum. Using the novel adhesion model, inhibition of adhesion formation by a chymase inhibitor, Suc-Val-Pro-PheP(OPh)2, and by sodium hyaluronate/carboxymethylcellulose (Seprafilm) was evaluated, and their mechanisms were assessed. The degree of adhesion formation was more severe and more stable in the alkali-induced injury model than in the abrasion-induced injury model. Both the chymase inhibitor and Seprafilm showed significant attenuation of the degree of adhesion 14 days after alkali-induced injury. Chymase activity in the cecum was significantly increased after alkali-induced injury, but it was significantly attenuated by the chymase inhibitor and Seprafilm. Myeloperoxidase and transforming-growth factor (TGF)-β levels were significantly increased after alkali-induced injury, but they were attenuated by both the chymase inhibitor and Seprafilm. At the level of the adhesions, the numbers of both chymase-positive cells and TGF-β-positive cells were significantly increased, but their numbers were reduced by the chymase inhibitor and Seprafilm. In conclusion, a chymase inhibitor attenuated the degree of adhesions to the same degree as Seprafilm in a novel peritoneal adhesion model that was more severe and more stable than the common adhesion model, and not only the chymase inhibitor, but also Seprafilm reduced the chymase increase at the adhesions. [ABSTRACT FROM AUTHOR]

Type 1 diabetes is an autoimmune disease where autoreactive T lymphocytes destroy pancreatic beta cells. We previously reported a defect in CD4+ Tregs cell proliferation and reduced CD4+ Tregs PD-1 expression in patients. Another ‘memory-like’ regulatory subset, CD8+ Tregs, evaluated as CD8+CD25+FOXP3+, has recently raised interest for their effective suppressive activity. Different CD8+ T cell populations, their proliferation capacity and expression of PD-1 molecule were evaluated by flow-cytometer analysis in newly diagnosed, long-term Type 1 diabetes patients compared to healthy normal donors. Under basal conditions, CD8+ Tregs and CD8+ Teffs were seemingly represented among study groups while there was evidence of diminished expression of PD-1 in Teff subsets of long-term patients. After 3 days of PMA/ionomycin stimulation, patients CD8+ Tregs showed decreased percentage in respect to control group. CD8+ Teffs were instead increased in long-term diabetics versus controls. PD-1+CD8+ Tregs were represented at a much lower percentage in long-term diabetic patients, in respect to controls. Importantly, patients CD8+ Tregs and CD8+ Teffs presented a significant proliferation defect in respect to the control group. In conclusion, our study indicates that a defect of CD8+ Tregs is observed in diabetics. This subset could thus represent a novel target of immunotherapy in patients. [ABSTRACT FROM AUTHOR]

The Rab family of small GTPases regulate various aspects of cellular dynamics in eukaryotic cells. Membrane trafficking has emerged as central to the functions of leucine-rich repeat kinase 2 (LRRK2), which is associated with inherited and sporadic forms of Parkinson’s disease (PD). Rabs act as both regulators of the catalytic activity and targets for serine/threonine phosphorylation by LRRK2. Rab32, Rab38 and Rab29 have been shown to regulate LRRK2 sub-cellular localization through direct interactions. Recently, Rab29 was shown to escort LRRK2 to the Golgi apparatus and activate the phosphorylation of Rab8 and Rab10. Rab32 is linked to multiple cellular functions including endosomal trafficking, mitochondrial dynamics, and melanosome biogenesis. A missense mutation in Rab32 has also recently been linked to PD. Here, we demonstrate that Rab32 directly interacts with sorting nexin 6 (SNX6). SNX6 is a transient subunit of the retromer, an endosome-Golgi retrieval complex whose Vps35 subunit is strongly associated with PD. We could further show that localization of cation-independent mannose-6-phosphate receptors, which are recycled to the trans-Golgi network (TGN) by the retromer, was affected by both Rab32 and SNX6. These data imply that Rab32 is linked to SNX6/retromer trafficking at the Golgi, and also suggests a possible connection between the retromer and Rab32 in the trafficking and biological functions of LRRK2. [ABSTRACT FROM AUTHOR]

The ability to resist copper toxicity is important for microbial pathogens to survive attack by innate immune cells. A sur7Δ mutant of the fungal pathogen Candida albicans exhibits decreased virulence that correlates with increased sensitivity to copper, as well as defects in other stress responses and morphogenesis. Previous studies indicated that copper kills sur7Δ cells by a mechanism distinct from the known resistance pathways involving the Crp1 copper exporter or the Cup1 metallothionein. Since Sur7 resides in punctate plasma membrane domains known as MCC/eisosomes, we examined overexpression of SUR7 and found that it rescued the copper sensitivity of a mutant that fails to form MCC/eisosomes (pil1Δ lsp1Δ), indicating that these domains act to facilitate Sur7 function. Genetic screening identified new copper-sensitive mutants, the strongest of which were similar to sur7Δ in having altered plasma membranes due to defects in membrane trafficking, cortical actin, and morphogenesis (rvs161Δ, rvs167Δ, and arp2Δ arp3Δ). Consistent with the mutants having altered in plasma membrane organization, they were all more readily permeabilized by copper, which is known to bind phosphatidylserine and phosphatidylethanolamine and cause membrane damage. Although these phospholipids are normally localized to the intracellular leaflet of the plasma membrane, their exposure on the surface of the copper-sensitive mutants was indicated by increased susceptibility to membrane damaging agents that bind to these phospholipids. Increased copper sensitivity was also detected for a drs2Δ mutant, which lacks a phospholipid flippase that is involved in maintaining phospholipid asymmetry. Copper binds phosphatidylserine with very high affinity, and deleting CHO1 to prevent phosphatidylserine synthesis rescued the copper sensitivity of sur7Δ cells, confirming a major role for phosphatidylserine in copper sensitivity. These results highlight how proper plasma membrane architecture protects fungal pathogens from copper and attack by the immune system, thereby opening up new avenues for therapeutic intervention. [ABSTRACT FROM AUTHOR]

Platelet-derived growth factor (PDGF) has been implicated in the pathogenesis of pulmonary fibrosis. Nintedanib, a multi-kinase inhibitor that targets several tyrosine kinases, including PDGF receptor (PDGFR), was recently approved as an anti-fibrotic agent to reduce the deterioration of FVC in patients with idiopathic pulmonary fibrosis (IPF). However, the effects of PDGFR-α or -β on pulmonary fibrosis remain unclear. In an attempt to clarify their effects, we herein used blocking antibodies specific for PDGFR-α (APA5) and -β (APB5) in a bleomycin (BLM)-induced pulmonary fibrosis mouse model. The effects of these treatments on the growth of lung fibroblasts were examined using the 3H-thymidine incorporation assay in vitro. The anti-fibrotic effects of these antibodies were investigated with the Ashcroft score and collagen content of lungs treated with BLM. Their effects on inflammatory cells in the lungs were also analyzed using bronchoalveolar lavage fluid. We investigated damage to epithelial cells and the proliferation of fibroblasts in the lungs. APA5 and APB5 inhibited the phosphorylation of PDGFR-α and -β as well as the proliferation of lung fibroblasts induced by PDGF-AA and BB. The administration of APB5, but not APA5 effectively inhibited BLM-induced pulmonary fibrosis in mice. Apoptosis and the proliferation of epithelial cells and fibroblasts were significantly decreased by the treatment with APB5, but not by APA5. The late treatment with APB5 also ameliorated fibrosis in lungs treated with BLM. These results suggest that PDGFR-α and -β exert different effects on BLM-induced pulmonary fibrosis in mice. A specific approach using the blocking antibody for PDGFR-β may be useful for the treatment of pulmonary fibrosis. [ABSTRACT FROM AUTHOR]

The specific role of polyamines in the testis physiology is not fully understood. Antizymes (OAZs) and antizyme inhibitors (AZINs) are modulators of ornithine decarboxylase (ODC), a key enzyme in polyamine biosynthesis and polyamine uptake. Although the three known OAZs are expressed in the testis, only OAZ3 is testis specific and has been proven to have an essential role in male fertility. Regarding the two existing AZINs, AZIN2 is the most abundantly expressed member in this gonad. Whereas previous studies suggested that AZIN2 might participate in mouse spermatogenesis, immunohistological analysis of human testicular sections revealed that AZIN2 is also detected in the steroidogenic Leydig cells but not in the germinal epithelium. In the present study, we found a close ontogenic similarity in the mRNA levels of OAZs and AZINs between mice and rats, but an opposite expression pattern of ODC activity. Further analysis of AZIN2 and OAZ3 in the testis of mice with different alterations in spermatogenesis and fertility, induced either genetically or pharmacologically, corroborated that both AZIN2 and OAZ3 are mainly expressed in the haploid germinal cells. Finally, by using transgenic mice with a truncated Azin2 gene fused to the bacterial lacZ gene, we studied the expression of Azin2 in testes, epididymides and spermatozoa. AZIN2 was detected in spermatids and spermatozoa, as well as in Leydig cells, and in epithelial epidydimal cells. Azin2 knock-out male mice were fertile; however, they showed marked decreases in testicular putrescine and plasma and testicular testosterone levels, and a dramatic reduction in the sperm motility. These results suggest an important role for AZIN2 in testicular cells by modulating polyamine concentrations, testosterone synthesis and sperm function. Overall, our data corroborate the relevance of polyamine regulation in testis functions, where both AZIN2 and OAZ3 play fundamental roles. [ABSTRACT FROM AUTHOR]

Introduction: Antibiotic-loaded poly(methyl methacrylate) bone cements (ALBCs) are widely used in total joint replacement (TJR), for local delivery of antibiotics to provide prophylaxis against prosthetic joint infections (PJI). One of the shortcomings of the current generation of ALBCs is that the antibiotic release profile is characterized by a burst over the first few hours followed by a sharp decrease in rate for the following several days (often below minimum inhibitory concentration (MIC)), and, finally, exhaustion (after, typically, ~ 20 d). This profile means that the ALBCs provide only short-term antimicrobial action against bacterial strains involved PJI. Rationale: The purpose of the present study was to develop an improved antibiotic delivery system for an ALBC. This system involved using a layer-by-layer technique to load the antibiotic (gentamicin sulphate) (GEN) on silica nanoparticles, which are then blended with the powder of the cement. Then, the powder was mixed with the liquid of the cement (NP-GEN cement). For controls, two GEN-loaded brands were used (Cemex Genta and Palacos R+G). Gentamicin release and a host of other relevant properties were determined for all the cements studied. Results: Compared to control cement specimens, improved GEN release, longer antimicrobial activity (against clinically-relevant bacterial strains), and comparable setting time, cytocompatibility, compressive strength (both prior to and after aging in PBS at 37 oC for 30 d), 4-point bend strength and modulus, fracture toughness, and PBS uptake. Conclusions: NP-GEN cement may have a role in preventing or treating PJI. [ABSTRACT FROM AUTHOR]

Human amniotic membrane (AM) has a long history of clinical use for wound treatment. AM serves as a wound protective barrier maintaining proper moisture. AM is anti-inflammatory, anti-microbial and antifibrotic, and supports angiogenesis, granulation tissue formation and wound re-epithelialization. These properties of AM are attributed to its native extracellular matrix, growth factors, and endogenous cells including mesenchymal stem cells. Advances in tissue preservation have helped to overcome the short shelf life of fresh AM and led to the development of AM products for clinical use. Viable cryopreserved amnion (VCAM), which retains all native components of fresh AM, has shown positive outcomes in clinical trials for wound management. However, cryopreservation requires ultra-low temperature storage and shipment that limits widespread use of VCAM. We have developed a lyopreservation technique to allow for ambient storage of living tissues. Here, we compared the structural, molecular, and functional properties of a viable lyopreserved human amniotic membrane (VLAM) with properties of VCAM using in vitro and in vivo wound models. We found that the structure, growth factors, and cell viability of VLAM is similar to that of VCAM and fresh AM. Both, VCAM and VLAM inhibited TNF-α secretion and upregulated VEGF expression in vitro under conditions designed to mimic inflammation and hypoxia in a wound microenvironment, and resulted in wound closure in a diabetic mouse chronic wound model. Taken together, these data demonstrate that VLAM structural and functional properties are equivalent to VCAM but without the constraints of ultra-low temperature storage. [ABSTRACT FROM AUTHOR]