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The primo vascular system (PVS), floating in lymph ducts, was too transparent to be observed by using a stereomicroscope. It was only detectable with the aid of staining dyes, for instance, Alcian blue, which was injected into the lymph nodes. Some dyes were absorbed preferentially by the PVS than the lymph wall. It remains a standing problem to know what dyes are absorbed better by the PVS than the lymph walls. Such information would be useful to unravel the biochemical properties of the PVS that are badly in need for obtaining large amount of PVS specimens. In the current work we tried two other familiar dyes which were used in PVS research before. We found that Trypan blue and toluidine blue did not visualize the PVS. Trypan blue was cleared by the natural washing. Toluidine blue did not stain the PVS, but it did leave stained spots in the lymph wall and its surrounding tissues, and it leaked out of the lymph wall to stain surrounding connective tissues. These completely different behaviors of the three dyes were found for the first time in the current work and provide valuable information to elucidate the mechanism through which some special dyes stained the PVS preferentially compared to the lymphatic wall.
Glycocalyx (GCX) is a thin layer of negatively charged glycoproteins that covers the vascular endothelial surface and regulates various biological processes. Because of the delicate and fragile properties of this structure, it is difficult to detect GCX morphologically. We established a simple method for a three-dimensional visualization of endothelial GCX using low-vacuum scanning electron microscopy (LVSEM) on formalin-fixed paraffin-embedded (FFPE) sections. Mouse kidney tissue was fixed with 10% buffered formalin containing 1% Alcian blue (ALB) via perfusion and immersion. FFPE sections were observed by light microscopy (LM) and LVSEM, and formalin-fixed epoxy resin-embedded ultrathin sections were observed by transmission electron microscopy (TEM). The endothelial GCX from various levels of kidney blood vessels was stained blue in LM and confirmed as a thin osmiophilic layer in TEM. In LVSEM, the sections stained by periodic acid methenamine silver (PAM) revealed the endothelial GCX as a layer of dense silver-enhanced particles, in both the samples fixed via perfusion and immersion. Correlative light and electron microscopy (CLEM) revealed the fine visible structure of endothelial GCX. This simple method using FFPE samples with ALB will enable the three-dimensional evaluation of endothelial GCX alterations in various human diseases associated with endothelial injury in future studies.
The main step in the assessment of nanomaterial safety and suitability for biomedical use is the location and the dynamic tracking of nanoparticles (NPs) inside cells or tissues. To precisely investigate the uptake mechanisms and intracellular fate of NPs, transmission electron microscopy is the technique of choice; however, the detection of NPs may sometimes be problematic. In fact, while NPs containing strongly electron dense (e.g., metal) components do not require specific detection methods at the ultrastructural level, organic NPs are hardly detectable in the intracellular environment due to their intrinsic moderate electron density. In this study, the critical-electrolyte-concentration Alcian Blue method set up by Schofield et al. in 1975 was applied to track hyaluronic-acid-based NPs in muscle cells in vitro. This long-established histochemical method proved to be a powerful tool allowing to identify not only whole NPs while entering cells and moving into the cytoplasm, but also their remnants following lysosomal degradation and extrusion.
Astrocytes play an essential role in the normal functioning of the nervous system and are active contributors to the pathogenesis of neurodegenerative diseases such as Alzheimer's disease (AD). Therefore, to comprehend the astrocytes and amyloid plaques relationship there is a need for imaging techniques providing simultaneous visualization of astrocytes using fluorescence and amyloid plaques revealed by transmitted light microscopy.
In more than half a century, exploring the biological connotation of the meridians was one of the core components of scientific research studies in traditional Chinese medicine (TCM). Based on the previous works of low hydraulic resistance channel (LHRC) along meridians (LHRCM), the differential proteomics between the Alcian blue track (ABT) on LHRC along the conception vessel (CV) and nonmeridians tissue next to the CV were investigated in this study to explore the material basis and biological function of LHRCM. Proteomics based on LC-MS was introduced into the subcutaneous connective tissues (SCT) of ABT along the CV and the adjacent nonmeridian (1 cm from the CV). A total of 2328 proteins were identified from ABT along the CV and adjacent nonmeridian based on data-dependent acquisition (DDA) mode. In total, 1970 proteins were quantified based on the SWATH (sequential window acquisition of all theoretical fragment ions) label-free model, and the nonstandard and quantitative methods of MSALL were applied to analyze the data. There were 468 proteins differentially expressed. GO analytic results showed that the differential proteins were of varieties in molecular function and biological process. Most of differential proteins were involved in metabolic process, cellular process, response to hormone, and response to wounding. Further analysis showed that the upregulated differential proteins involved in ATP metabolism (ATP5E, GAPDH), redox reactions (Gpx-3), and Ca2+ transmembrane transport (CACNA2D1) were closely related to meridian phenomenon and acupuncture effect. These differential proteins would be potential characteristic proteins of the LHRC along the CV in rats which may be useful to deepen the knowledge on LHRCM.
The use of enzyme-linked immunosorbent assay for the detection of aminoglycosides has been hindered due to low molecular weight compound adsorption to solid phases. Here, we describe an enzyme-linked immunosorbent assay based on the treatment of polystyrene microtiter plates with Alcian blue prepared in acetic acid prior to coating with the antibiotic. Whereas no detection of tobramycin was possible on commercially treated or untreated enzyme-linked immunosorbent assay plates, the Alcian blue treatment permitted detection of 0.025 and 0.05 microg ml(-1) of tobramycin respectively using 0.05 and 0.1% of Alcian blue with a coefficient of variation of 1.85 and 7.69%, respectively. Comparative studies of five tobramycin samples of unknown quantity using enzyme-linked immunosorbent assay and high-performance liquid chromatography gave equivalent results while those done via microbiological agar-diffusion assay were an overestimation of the actual quantity. The use of the Alcian blue pretreatment enzyme-linked immunosorbent assay procedure has permitted, in previous studies, the measure of antibodies against synthetic peptides and phospholipids. Subsequently, our demonstration of the sensitivity and reliability of this method in the quantification of tobramycin strongly suggests that the use of Alcian blue pretreatment in enzyme-linked immunosorbent assay can be applied universally to avert molecule immobilization problems on solid phases.
The RUNX2 transcription factor is a key regulator for the development of cartilage and bone. Global or resting chondrocyte-specific deletion of the Runx2 gene results in failure of chondrocyte hypertrophy, endochondral ossification, and perinatal lethality. The terminally mature hypertrophic chondrocyte regulates critical steps of endochondral ossification. Importantly, expression of the Runx2 gene starts in the resting chondrocyte and increases progressively, reaching the maximum level in hypertrophic chondrocytes. However, the RUNX2 role after chondrocyte hypertrophy remains unknown. To answer this question, we deleted the Runx2 gene specifically in hypertrophic chondrocytes using the Col10-Cre line. Mice lacking the Runx2 gene in hypertrophic chondrocytes (Runx2HC/HC ) survive but exhibit limb dwarfism. Interestingly, the length of the hypertrophic chondrocyte zone is doubled in the growth plate of Runx2HC/HC mice. Expression of pro-apoptotic Bax decreased significantly while anti-apoptotic Bcl2 remains unchanged leading to a four-fold increase in the Bcl2/Bax ratio in mutant mice. In line with this, a significant reduction in apoptosis of Runx2HC/HC hypertrophic chondrocyte is noted. A large amount of cartilage matrix is present in the long bones that extend toward the diaphyseal region of Runx2HC/HC mice. This is not due to enhanced synthesis of the cartilage matrix as the expression of both collagen type 2 and aggrecan were comparable among Runx2HC/HC and WT littermates. Our qPCR analysis demonstrates the increased amount of cartilage matrix is due to impaired expression of cartilage degrading enzymes such as metalloproteinase and aggrecanase as well as tissue inhibitor of metalloproteinases. Moreover, a significant decrease of TRAP positive chondroclasts was noted along the cartilage islands in Runx2HC/HC mice. Consistently, qPCR data showed an 81% reduction in the Rankl/Opg ratio in Runx2HC/HC littermates, which is inhibitory for chondroclast differentiation. Finally, we assess if increase cartilage matrix in Runx2HC/HC mice serves as a template for bone and mineral deposition using micro-CT and Von Kossa. The mutant mice exhibit a significant increase in trabecular bone mass compared to littermates. In summary, our findings have uncovered a novel role of Runx2 in apoptosis of hypertrophic chondrocytes and degradation of cartilage matrix during endochondral ossification.
Mechanistic target of rapamycin complex 1 (mTORC1) plays a pivotal role in controlling cell growth and metabolism in response to nutrients and growth factors. The activity of mTORC1 is dually regulated by amino acids and growth factor signaling, and amino acid-dependent mTORC1 activity is regulated by mTORC1 interaction with the Ragulator-Rag GTPase complex, which is localized to the surface of lysosomes via a membrane-anchored protein, p18/Lamtor1. However, the physiological function of p18-Ragulator-dependent mTORC1 signaling remains elusive. The present study evaluated the function of p18-mediated mTORC1 signaling in the intestinal epithelia using p18 conditional knockout mice. In p18 knockout colonic crypts, mTORC1 was delocalized from lysosomes, and in vivo mTORC1 activity was markedly decreased. Histologically, p18 knockout crypts exhibited significantly increased proliferating cells and dramatically decreased mucin-producing goblet cells, while overall crypt architecture and enteroendocrine cell differentiation were unaffected. Furthermore, p18 knockout crypts normally expressed transcription factors implicated in crypt differentiation, such as Cdx2 and Klf4, indicating that p18 ablation did not affect the genetic program of cell differentiation. Analysis of colon crypt organoid cultures revealed that both p18 ablation and rapamycin treatment robustly suppressed development of mucin-producing goblet cells. Hence, p18-mediated mTORC1 signaling could promote the anabolic metabolism required for robust mucin production in goblet cells to protect the intestinal epithelia from various external stressors.Key words: mTORC1, p18/lamtor1, intestinal epithelium, goblet cells, mucin.
The distribution of mucous secreting goblet cells was examined in the gastrointestinal tracts of three insectivores namely: Acomys spinosissimus (Southern African spiny mouse), Crocidura cyanea (Reddish gray musk shrew) and Amblysomus hottentotus (Hottentot golden mole) in order to improve our understanding of the quality and composition of the protective intestinal biofilm. Intestinal tracts were fixed and processed to wax for histology. Serial transverse sections were stained using alcian blue-periodic acid Schiff, alcian blue-aldehyde fuchsin and alcian blue-high iron diamine techniques. Photomicrographs of the stained sections were analyzed by quantifying the number of goblet cells containing mucins per mm(2) in the surface epithelial or crypt areas. Neutral mucins predominated in the gastric epithelium of all three insectivores, while sialomucins were absent in the stomach of C. cyanea. In all three species, goblet cells producing a mixture of neutral and acid mucins were most abundant throughout the intestinal tract as were cells secreting a mixture of sulfomucins and sialomucins. However, differences between the insectivore species were observed in the qualitative expression and distribution of mucins throughout the intestinal tract. Similarities between the insectivores of this study and other distantly related species suggest that mixed mucin goblet cells are essential for the formation of the biofilm, irrespective of their diet or taxonomy.
Proteasome activity is significantly increased in advanced cancers, but its role in cancer initiation is not clear, due to difficulties in monitoring this process in vivo. We established a line of transgenic mice that carried the ZsGreen-degron(ODC) (Gdeg) proteasome reporter to monitor the proteasome activity. In combination with Pdx-1-Cre;LSL-Kras(G12D) model, proteasome activity was investigated in the initiation of precancerous pancreatic lesions (PanINs). Normal pancreatic acini in Gdeg mice had low proteasome activity. By contrast, proteasome activity was increased in the PanIN lesions that developed in Gdeg;Pdx-1-Cre;LSL-Kras(G12D) mice. Caerulein administration to Gdeg;Pdx-1-Cre;LSL-Kras(G12D) mice induced constitutive elevation of proteasome activity in pancreatic tissues and accelerated PanIN formation. The proteasome inhibitor markedly reduced PanIN formation in Gdeg;Pdx-1-Cre;LSL-Kras(G12D) mice (P = 0.001), whereas it had no effect on PanIN lesions that had already formed. These observations indicated the significance of proteasome activity in the initiation of PanIN but not the maintenance per se. In addition, the expressions of pERK and its downstream factors including cyclin D1, NF-κB, and Cox2 were decreased after proteasome inhibition in PanINs. Our studies showed activation of proteasome is required specifically for the initiation of PanIN. The roles of proteasome in the early stages of pancreatic carcinogenesis warrant further investigation.
Various miRNAs have been reported to regulate the chondrogenic differentiation of bone marrow mesenchymal stem cells (BMSCs); however, whether miR-134 plays a role in this biological process remains undetermined. In the present study, we first evaluated the chondrogenic differentiation of BMSCs by Alcian blue staining, and examined the miR-134 expression by quantitative real-time PCR (qRT-PCR) during this process. And miR-134 inhibitor was used to investigate the functions of miR-134 in chondrogenic differentiation of BMSCs by Alcian blue staining, qRT-PCR, and Western blot. Subsequently, the correlation between miR-134 and SMAD6 was assessed via bioinformatics analysis and dual-luciferase reporter assay. Finally, the role of SMAD6 in chondrogenic differentiation of BMSCs was also determined through Alcian blue staining, qRT-PCR, and Western blot. As results showed that miR-134 expression was significantly down-regulated during chondrogenic differentiation, and inhibition of miR-134 obviously promoted chondrogenic differentiation. Dual-luciferase reporter assay indicated that miR-134 could directly target the 3'-UTRs of SMAD6, inhibit miR-134 expression in BMSCs, and up-regulate SMAD6 expression. Moreover, we found that overexpression of SMAD6 significantly promoted chondrogenic differentiation, and that SMAD6-induced promotion of chondrogenic differentiation could be reversed by miR-134 mimics. In conclusion, our findings suggest that miR-134 may act as a negative regulator during chondrogenic differentiation of BMSCs by interacting with SMAD6.
Stem/stromal cell-based therapies are a branch of regenerative medicine and stand as an attractive option to promote the repair of damaged or dysfunctional tissues and organs. Olfactory mucosa mesenchymal stem/stromal cells have been regarded as a promising tool in regenerative therapies because of their several favorable properties such as multipotency, high proliferation rate, helpful location, and few associated ethical issues. These cells are easily accessible in the nasal cavity of most mammals, including the rat, can be easily applied in autologous treatments, and do not cope with most of the obstacles associated with the use of other stem cells. Despite this, its application in preclinical trials and in both human and animal patients is still limited because of the small number of studies performed so far and to the nonexistence of a standard and unambiguous protocol for collection, isolation, and therapeutic application. In the present work a validation of a protocol for isolation, culture, expansion, freezing, and thawing of olfactory mucosa mesenchymal stem/stromal cells was performed, applied to the rat model, as well as a biological characterization of these cells. To investigate the therapeutic potential of OM-MSCs and their eventual safe application in preclinical trials, the main characteristics of OMSC stemness were addressed.
The immunotoxicity of Pseudomonas aeruginosa exotoxin A (ETA) on macrophages was evaluated by incubating rat peritoneal macrophages (RPM) with 1-100 ng/ml ETA for 3-60 h. Although the overall changes in cell viability and DNA, RNA, and protein synthesis of the ETA-treated RPM (E-RPM) were reduced in a dose- and time-dependent manner, there was a transient but evident rebound in RNA and/or protein synthesis at 24-36 h post-incubation (HPI) at 1-50 ng/ml ETA. However, a more apparent enhancement appeared in RNA and protein synthesis at 36-48 HPI in 10 and 50 ng/ml E-RPM after normalized on the basis of viable cell. Most 50-100 ng/ml E-RPM underwent necrosis/apoptosis before 24 HPI. By 36 HPI, 41% of 10 ng/ml E-RPM remained viable but were full of cytoplasmic granules due to the accumulation of glycoprotein in segmentally dilated endoplasmic reticulum. Immunological staining of the granules revealed strong IL-1alpha but weak or no signals for IL-1beta, IL-1 receptor antagonist, IL-6, and TNF-alpha. A time-dependent increase in IL-1alpha but no IL-1beta was detected in cell lysate of 10 ng/ml E-RPM; however, neither IL-1alpha nor IL-1beta was detected in culture supernatant. Thus, besides cytopathic and functional effects, ETA could induce a unique selective production and endoplasmic reticular accumulation of IL-1alpha in RPM.
Knee osteoarthritis (KOA) refers to a common disease in orthopaedics, whereas effective treatments have been rarely developed. As indicated from existing studies, chondrocyte death, extracellular matrix degradation and subchondral bone injury are recognized as the pathological basis of KOA. The present study aimed to determine the therapeutic effect of decellularized extracellular matrix-chitosan (dECM-CS) compound on KOA. In this study, rat knee cartilage was decellularized, and a satisfactory decellularized extracellular matrix was developed. As suggested from the in vitro experiments, the rat chondrocytes co-cultured with allogeneic dECM grew effectively. According to the results of the alamar blue detection, dECM did not adversely affect the viability of rat chondrocytes, and dECM could up-regulate the genes related to the cartilage synthesis and metabolism. As reported from the animal experiments, dECM-CS compound could protect cartilage, alleviate knee joint pain in rats, significantly delay the progress of KOA in rats, and achieve high drug safety. In brief, dECM-CS compound shows a good therapeutic effect on KOA.
Bottom-up methods for the fabrication of nanoporous polymer membranes have numerous advantages. However, it remains challenging to fabricate nanoporous membranes that are mechanically robust and have aligned pores, that is, with a low tortuosity. Here, a mechanically robust thin-film composite membrane was fabricated consisting of a two-dimensional (2D) porous smectic liquid crystalline polymer network inside an anisotropic, microporous polymer scaffold. The polymer scaffold allows for relatively straightforward planar alignment of the smectic liquid crystalline mixture, which consisted of a diacrylate cross-linker and a dimer forming benzoic acid-based monoacrylate. Polymerized samples displayed a smectic A (SmA) phase, which formed the eventual 2D porous channels after base treatment. The aligned 2D nanoporous membranes showed a high rejection of anionic solutes bigger than 322 g/mol. Cleaning and reusability of the system were demonstrated by intentionally fouling the porous channels with a cationic dye and subsequently cleaning the membrane with an acidic solution. After cleaning, the membrane properties were unaffected; this, combined with numerous pressurizing cycles, demonstrated reusability of the system.
Dynamic regulation of intestinal epithelial cell (IEC) proliferation and differentiation is crucial for maintaining mucosa homeostasis and the response to helminth infection. O-GlcNAc transferase (OGT), an enzyme catalyzing the transfer of GlcNAc from the donor substrate UDP-GlcNAc onto acceptor proteins, has been proposed to promote intestinal epithelial remodeling for helminth expulsion by modifying and activating epithelial STAT6, but whether the IEC intrinsic OGT-STAT6 axis is involved in anti-helminth responses has not been tested in vivo. Here, we show that the inducible deletion of Ogt in IECs of adult mice leads to reduced tuft and goblet cell differentiation, increased crypt cell proliferation, and aberrant Paneth cell localization. By using a mouse model with concurrent Ogt deletion and STAT6 overexpression in IECs, we provide direct in vivo evidence that STAT6 acts downstream of OGT to control tuft and goblet cell differentiation in IECs. However, epithelial OGT regulates crypt cell proliferation and Paneth cell differentiation in a STAT6-independent pathway. Our results verify that protein O-GlcNAcylation in IECs is crucial for maintaining epithelial homeostasis and anti-helminthic type 2 immune responses.
The effective separation of dyes and inorganic salts is highly desirable for recycling inorganic salts and water resource in printing and dyeing wastewater treatment. In this work, tannic acid (TA) and polyethyleneimine (PEI) were grafted on the PES/Fe ultrafiltration membrane via the coordination assembly and Michael addition strategy to fabricated a loose nanofiltration membrane (LNM). The effect of PEI concentration on membrane morphologies and properties was systematically investigated. The membrane surface becomes more hydrophilic and transforms into positive charge after the PEI grafting. The optimized PES/Fe-TA-PEI membrane possesses high pure water flux (124.6 L·m-2·h-1) and excellent dye rejections (98.5%, 99.8%, 98.4%, and 86.4% for Congo red, Eriochrome black T, Alcian blue 8GX, and Bromophenol blue, respectively) under 2 bar operation pressure. Meanwhile, the LNM showed a high Alcian blue 8GX rejection (>98.4%) and low NaCl rejection (<5.3%) for the dye/salt mixed solutions separation. Moreover, the PES/Fe-TA-PEI LNM exhibited good antifouling performance and long-term performance stability. These results reveal that such LNM shows great potential for effective fractionation of dyes and salts and recycling of textile wastewater.
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