Circadian rhythms are deeply rooted in the biology of virtually all organisms. The pervasive use of artificial lighting in modern society disrupts circadian rhythms and can be detrimental to our ...health. To investigate the relationship between disrupting circadian rhythmicity and disease, we exposed mice to continuous light (LL) for 24 weeks and measured several major health parameters. Long-term neuronal recordings revealed that 24 weeks of LL reduced rhythmicity in the central circadian pacemaker of the suprachiasmatic nucleus (SCN) by 70%. Strikingly, LL exposure also reduced skeletal muscle function (forelimb grip strength, wire hanging duration, and grid hanging duration), caused trabecular bone deterioration, and induced a transient pro-inflammatory state. After the mice were returned to a standard light-dark cycle, the SCN neurons rapidly recovered their normal high-amplitude rhythm, and the aforementioned health parameters returned to normal. These findings strongly suggest that a disrupted circadian rhythm reversibly induces detrimental effects on multiple biological processes.
•Long-term exposure to LL impairs rhythms in the central clock•Muscle strength, bone structure, and immune function are reduced by LL exposure•Robust environmental rhythms can rescue major health parameters
Lucassen et al. describe that exposure to continuous light, commonly present in intensive care units, chronically attenuates internal 24-hr rhythms leading to a deterioration in muscle strength, bone microstructure, and innate immune response. Upon re-exposure to robust light-dark cycles, these health parameters restore.
Background Near-infrared (NIR) fluorescence imaging using indocyanine green (ICG) is a promising technique to obtain real-time assessment of the extent and number of colorectal liver metastases ...during surgery. The current study aims to optimize dosage and timing of ICG administration. Materials and Methods Liver tumors were induced in 18 male WAG/Rij rats by subcapsular inoculation of CC531 rat colorectal cancer cells into three distinct liver lobes. Rats were divided in two groups: imaging after 24 and 48 h or 72 and 96 h after intravenous ICG administration. In each time group, rats were allocated to three dose groups: 0.04, 0.08, or 0.16 mg ICG. Intraoperative imaging and ex vivo measurements were performed using the Mini-FLARE imaging system and confirmed by fluorescence microscopy. Fluorescence intensity was quantified using the Mini-FLARE software and the difference between tumor signal and liver signal (tumor-to-liver ratio; TLR) was calculated. Results In all 18 rats, all colorectal liver metastases ( n = 34), some as small as 1.2 mm, were identified using ICG and the Mini-FLARE imaging system. Average tumor-to-liver ratio (TLR) over all groups was 3.0 ± 1.2. TLR was significantly higher in the 72 h time group compared with other time points. ICG dose did not significantly influence TLR, but a trend was found favoring the 0.08 mg dose group. Fluorescence microscopy demonstrated a clear fluorescent rim around the tumor. Conclusions This study demonstrates that colorectal cancer liver metastases can be clearly identified during surgery using ICG and the Mini-FLARE imaging system, with optimal timing of 72 h post-injection and an optimal dose of 0.08 mg (0.25 mg/kg) ICG. NIR fluorescence imaging has the potential to improve intraoperative detection of micrometastases and, thus, the completeness of resection.
Background
Irradical tumor resections and iatrogenic ureteral injury remain a significant problem during lower abdominal surgery. The aim of the current study was to intraoperatively identify both ...colorectal tumors and ureters in subcutaneous and orthotopic animal models using cRGD-ZW800-1 and near-infrared (NIR) fluorescence.
Methods
The zwitterionic fluorophore ZW800-1 was conjugated to the tumor specific peptide cRGD (targeting integrins) and to the a-specific peptide cRAD. One nmol cRGD-ZW800-1, cRAD-ZW800-1, or ZW800-1 alone was injected in mice bearing subcutaneous HT-29 human colorectal tumors. Subsequently, cRGD-ZW800-1 was injected at dosages of 0.25 and 1 nmol in mice bearing orthotopic HT-29 tumors transfected with luciferase2. In vivo biodistribution and ureteral visualization were investigated in rats. Fluorescence was measured intraoperatively at several time points after probe administration using the FLARE imaging system.
Results
Both subcutaneous and orthotopic tumors could be clearly identified using cRGD-ZW800-1. A significantly higher signal-to-background ratio was observed in mice injected with cRGD-ZW800-1 (2.42 ± 0.77) compared with mice injected with cRAD-ZW800-1 or ZW800-1 alone (1.21 ± 0.19 and 1.34 ± 0.19, respectively) when measured at 24 h after probe administration. The clearance of cRGD-ZW800-1 permitted visualization of the ureters and also generated minimal background fluorescence in the gastrointestinal tract.
Conclusions
This study appears to be the first to demonstrate both clear tumor demarcation and ureteral visualization after a single intravenous injection of a targeted NIR fluorophore. As a low dose of cRGD-ZW800-1 provided clear tumor identification, clinical translation of these results should be possible.
•We validate a simple and fast method of quantification of in vitro mineralization.•Fluorescently labeled agents can detect calcium deposits in the mineralized matrix of cell cultures.•Fluorescent ...signals of the probes correlated with Alizarin Red S staining.
Alizarin Red S staining is the standard method to indicate and quantify matrix mineralization during differentiation of osteoblast cultures. KS483 cells are multipotent mouse mesenchymal progenitor cells that can differentiate into chondrocytes, adipocytes and osteoblasts and are a well-characterized model for the study of bone formation. Matrix mineralization is the last step of differentiation of bone cells and is therefore a very important outcome measure in bone research. Fluorescently labelled calcium chelating agents, e.g. BoneTag and OsteoSense, are currently used for in vivo imaging of bone. The aim of the present study was to validate these probes for fast and simple detection and quantification of in vitro matrix mineralization by KS483 cells and thus enabling high-throughput screening experiments.
KS483 cells were cultured under osteogenic conditions in the presence of compounds that either stimulate or inhibit osteoblast differentiation and thereby matrix mineralization. After 21days of differentiation, fluorescence of stained cultures was quantified with a near-infrared imager and compared to Alizarin Red S quantification. Fluorescence of both probes closely correlated to Alizarin Red S staining in both inhibiting and stimulating conditions. In addition, both compounds displayed specificity for mineralized nodules. We therefore conclude that this method of quantification of bone mineralization using fluorescent compounds is a good alternative for the Alizarin Red S staining.
Expression of serotonin receptors in bone Westbroek, I; van der Plas, A; de Rooij, K E ...
The Journal of biological chemistry,
08/2001, Letnik:
276, Številka:
31
Journal Article
Recenzirano
Odprti dostop
The 5-hydroxytryptamine (5-HT) receptors 5-HT(2A), 5-HT(2B), and 5-HT(2C) belong to a subfamily of serotonin receptors. Amino acid and mRNA sequences of these receptors have been published for ...several species including man. The 5-HT(2) receptors have been reported to act on nervous, muscle, and endothelial tissues. Here we report the presence of 5-HT(2B) receptor in fetal chicken bone cells. 5-HT(2B) receptor mRNA expression was demonstrated in osteocytes, osteoblasts, and periosteal fibroblasts, a population containing osteoblast precursor cells. Pharmacological studies using several agonists and antagonists showed that occupancy of the 5-HT(2B) receptor stimulates the proliferation of periosteal fibroblasts. Activity of the 5-HT(2A) receptor could however not be excluded. mRNA for both receptors was shown to be equally present in adult mouse osteoblasts. Osteocytes, which showed the highest expression of 5-HT(2B) receptor mRNA in chicken, and to a lesser extent osteoblasts, are considered to be mechanosensor cells involved in the adaptation of bone to its mechanical usage. Nitric oxide is one of the signaling molecules that is released upon mechanical stimulation of osteocytes and osteoblasts. The serotonin analog alpha-methyl-5-HT, which preferentially binds to 5-HT(2) receptors, decreased nitric oxide release by mechanically stimulated mouse osteoblasts. These results demonstrate that serotonin is involved in bone metabolism and its mechanoregulation.
It is generally thought that class III β-tubulin expression is limited to cells of the neural lineage and is therefore often used to identify neurons amongst other cell types, both in vivo and in ...vitro. Melanocytes are derived from the neural crest and share both morphological features and functional characteristics with peripheral neurons. Here, we show that these similarities extend to class III β-tubulin (TUBB3) expression, and that human melanocytes express this protein both in vivo and in vitro. In addition, we studied the expression of class III β-tubulin in two murine melanogenic cell lines and show that expression of this protein starts as melanoblasts mature into melanocytes. Melanin bleaching experiments revealed close proximity between melanin and TUBB3 proteins. In vitro stimulation of primary human melanocytes by α-MSH indicated separate regulatory mechanisms for melanogenesis and to TUBB3 expression. Together, these observations imply that human melanocytes express TUBB3 and that this protein should be recognized as a wider marker for multiple neural crest-derived cells.
•Class III β-tubulin is expressed by human melanocytes, in vivo and in vitro.•Expression was found both in epidermal and hair follicle melanocytes.•Expression starts upon differentiation of melanoblasts into mature melanocytes.•TUBB3 expression and melanogenesis are separately regulated.•This data adds to the debate over similarity between melanocytes and neurons.
Osteocytes are the predominant cells in bone, where they form a cellular network and display important functions in bone homeostasis, phosphate metabolism and mechanical transduction. Several ...proteins strongly expressed by osteocytes are involved in these processes, e.g., sclerostin, DMP-1, PHEX, FGF23 and MEPE, while others are upregulated during differentiation of osteoblasts into osteocytes, e.g., osteocalcin and E11. The receptor-type protein tyrosine phosphatase µ (RPTPμ) has been described to be expressed in cells which display a cellular network, e.g., endothelial and neuronal cells, and is implied in mechanotransduction. In a capillary outgrowth assay using metatarsals derived from RPTPμ-knock-out/LacZ knock-in mice, we observed that the capillary structures grown out of the metatarsals were stained blue, as expected. Surprisingly, cells within the metatarsal bone tissue were positive for LacZ activity as well, indicating that RPTPμ is also expressed by osteocytes. Subsequent histochemical analysis showed that within bone, RPTPμ is expressed exclusively in early-stage osteocytes. Analysis of bone marrow cell cultures revealed that osteocytes are present in the nodules and an enzymatic assay enabled the quantification of the amount of osteocytes. No apparent bone phenotype was observed when tibiae of RPTPμ-knock-out/LacZ knock-in mice were analyzed by μCT at several time points during aging, although a significant reduction in cortical bone was observed in RPTPμ-knock-out/LacZ knock-in mice at 20 weeks. Changes in trabecular bone were more subtle. Our data show that RPTPμ is a new marker for osteocytes.
Osteocytes are the most abundant cells in bone; however, relatively little is known about their properties and functions. The development of monoclonal antibody MAb OB7.3 directed against chicken ...osteocytes enabled us to purify osteocytes from enzymatically isolated bone cells. Cultures of purified osteocytes were used to gain better insight into the role of osteocytes in bone metabolism. Until now, the antigen of MAb OB7.3 has not been elucidated. In this study, we examined the antigen to which this osteocyte‐specific antibody is directed. Immunoprecipitation and purification of the protein, followed by amino acid sequence analysis of two isolated peptides, revealed that the antigen has high homology to human and murine PHEX/Phex protein sequences (PHosphate‐regulating gene with homology to Endopeptidases on the X chromosome). The OB7.3 antigen was therefore identified as chicken Phex protein. In addition, using suppression subtractive hybridization, we obtained a complementary DNA (cDNA) sequence of 502 base pairs (bp) with high homology to the human and murine PHEX/Phex genes. This method was applied to identify genes, which are differentially expressed in osteocytes compared with osteoblasts. The results also suggest that Phex is expressed at higher levels in chicken osteocytes compared with osteoblasts. Reverse‐transcription polymerase chain reaction (RT‐PCR) and Northern blot analyses supported these findings. The function of Phex is not completely understood. However, it is known that the gene is preferentially expressed in bone and that mutations in PHEX/Phex lead to X‐linked hypophosphatemia and bone mineralization abnormalities. Our findings suggest that osteocytes play an important role in the Phex‐regulated phosphate handling in the kidney and in bone.
Objective Previously, we identified Serpina1 as a potent inhibitor of hematopoietic stem and progenitor cell (HSC/HPC) mobilization. Serpina1 protein is found in the bone marrow (BM) extracellular ...fluid and concentrations are decreased during granulocyte colony-stimulating factor–induced HSC/HPC mobilization in mice. In addition, administration of exogenous Serpina1 protein inhibits HSC/HPC mobilization. BM cells responsible for production and secretion of Serpina1 remain unknown. Here, we examined the expression of Serpina1 in order to identify cell populations of the BM that synthesize Serpina1. Materials and Methods Osteoblast (OB) and hematopoietic BM cell fractions were isolated from femurs, tibias, and humeri obtained from untreated mice. Subsequently, each BM fraction was examined for the production of Serpina1 messenger RNA and protein by quantitative real-time polymerase chain reaction, Western blotting, and immunohistochemistry. Results Quantitative real-time polymerase chain reaction analysis showed that Serpina1 messenger RNA is produced at high levels by OB compared to hematopoietic BM cells. Furthermore, Western blot analysis indicated that Serpina1 protein was secreted by OB. In contrast, no Serpina1 protein could be detected in the supernatant obtained from overnight cultured hematopoietic BM cells. Finally, in BM sections obtained from the femurs of untreated mice, Serpina1 protein was detected in OB cells lining the bone. Conclusion Serpina1 protein in the BM extracellular fluid is predominantly produced by OB. This indicates that Serpina1 may play a regulatory role in the maintenance of HSC in the OB stem cell niche.
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