Primary cilia are found on nearly every mammalian cell, including osteocytes, fibroblasts, and chondrocytes. However, the functions of primary cilia have not been extensively studied in these cells, particularly chondrocytes. Interestingly, defects in the primary cilium result in skeletal defects such as polydactyly in Bardet-Biedl Syndrome (BBS), a ciliary disorder that also results in obesity, retinopathy, and cognitive impairments (1–4). Wild-type mice and mutant mice of the ciliary proteins Bbs1, Bbs2, and Bbs6 were evaluated with respect to histological and biochemical differences in chondrocytes from articular cartilage and xiphoid processes. Using immunofluorescence microscopy, chondrocytic cilia were visualized from the load-bearing joints and non-load-bearing xiphoid processes. Significant differences in ciliary morphology were not identified between mutant and wild-type mice. However, after expanding chondrocytes in cell culture and implanting them in solid agarose matrix, it was seen that the fraction of ciliated cells in cultures from mutant mice was significantly lower than in the wild-type cultures (p<.05). In addition, in Safranin-O-stained whole joint sections, Bbs mutant mice had significantly lower articular joint thickness (p<.05) and lower proteoglycan content saturation (p<.05) than wild-type mice. Moreover, there were statistically significant differences of cell distribution between Bbs mutant and wild-type mice (p<.05), indicating that mutant articular cartilage had changes consistent with early signs of osteoarthritis. These data indicate that Bbs genes and their functions in the chondrocytic primary cilium are important for normal articular cartilage maintenance.
chondrocyte; primary cilium; articular cartilage; Bardet-Biedl syndrome; osteoarthritis
A microRNA regulates the expression of a network of genes in the heart to ensure that progenitor cells develop into strongly contractile cardiac muscle.
microRNA-1; cardiac; sarcomere; Telokin; genetics; smooth muscle gene expression; Mouse
The objective of this study was to determine if acute cartilage impact damage could be predicted by a quantification of the frequency content of the impact force signal.
Osteochondral specimens excised from bovine lateral tibial plateaus were impacted with one of six impact energies. Each impact force signal underwent frequency analysis, with the amount of higher-frequency content (percent of frequency spectrum above 1 KHz) being registered. Specimens were histologically evaluated to assess acute structural damage (articular surface cracking and cartilage crushing) resulting from the impact.
Acute histologic structural damage to the cartilage had higher concordance with the high-frequency content measure than with other mechanical impact measures (delivered impact energy, impact maximum stress, and impact maximum stress rate of change).
This result suggests that the frequency content of an impact force signal, specifically the proportion of higher-frequency components, can be used as a quick surrogate measure for acute structural cartilage injury. Taking advantage of this relationship could reduce the time and expense of histological processing needed to morphologically assess cartilage damage, especially for purposes of initial screening when evaluating new impaction protocols.
Articular cartilage; histology; impact testing; impact injury; post-traumatic osteoarthritis
Among the most common human congenital anomalies, cleft lip and palate (CL/P) affects up to 1 in 700 live births. MicroRNA (miR)s are small, non-coding RNAs that repress gene expression post-transcriptionally. The miR-17-92 cluster encodes six miRs that have been implicated in human cancers and heart development. We discovered that miR-17-92 mutant embryos had severe craniofacial phenotypes, including incompletely penetrant CL/P and mandibular hypoplasia. Embryos that were compound mutant for miR-17-92 and the related miR-106b-25 cluster had completely penetrant CL/P. Expression of Tbx1 and Tbx3, the DiGeorge/velo-cardio-facial (DGS) and Ulnar-mammary syndrome (UMS) disease genes, was expanded in miR-17-92 mutant craniofacial structures. Both Tbx1 and Tbx3 had functional miR seed sequences that mediated gene repression. Analysis of miR-17-92 regulatory regions uncovered conserved and functional AP-2α recognition elements that directed miR-17-92 expression. Together, our data indicate that miR-17-92 modulates expression of critical T-box transcriptional regulators during midface development and is itself a target of Bmp-signaling and the craniofacial pioneer factor AP-2α. Our data are the first genetic evidence that an individual miR or miR cluster is functionally important in mammalian CL/P.
CL/P are very common birth defects in humans. The genetic mechanism underlying CL/P pathogenesis is poorly understood. MiRs, small non-coding RNAs that function to post-transcriptionally regulate gene expression, have been identified as pivotal modulators of various developmental events and diseases. To date, there is no individual miR or miR cluster that has been identified as functionally essential in mammalian CL/P. Here, we have discovered that deletion of miR-17-92 cluster in mice results in craniofacial malformations including CL/P. Importantly, MIR-17-92 is located on a critical human chromosome region associated with 13q deletion syndrome, a chromosomal disorder that presents with defects including CL/P, suggesting the advantages of our animal model to study human disease. Moreover, our work demonstrated that miR-17-92 cluster directly repressed T-box factors, which have critical functions during craniofacial development. We further showed that miR-17-92 was directly activated by Bmp-signaling and transcription factor AP-2α. Together, our work identified a novel miR-mediated transcriptional network underlying CL/P, providing new insights into craniofacial developmental biology.
Donor site morbidity, limited numbers of cells, loss of phenotype during ex vivo expansion, and age-related decline in chondrogenic activity present critical obstacles to the use of autologous chondrocyte implantation for cartilage repair. Chondrocytes from juvenile cadaveric donors may represent an alternative to autologous cells.
The authors hypothesized that juvenile chondrocyte would show stronger and more stable chondrogenic activity than adult cells in vitro and that juvenile cells pose little risk of immunologic incompatibility in adult hosts.
Controlled laboratory study.
Cartilage samples were from juvenile (<13 years old) and adult (> 13 years old) donors. The chondrogenic activity of freshly isolated human articular chondrocytes and of expanded cells after monolayer culture was measured by proteoglycan assay, gene expression analysis, and histology. Lymphocyte proliferation assays were used to assess immunogenic activity.
Proteoglycan content in neocartilage produced by juvenile chondrocytes was 100-fold higher than in neocartilage produced by adult cells. Collagen type II and type IX mRNAs in fresh juvenile chondrocytes were 100- and 700-fold higher, respectively, than in adult chondrocytes. The distributions of collagens II and IX were similar in native juvenile cartilage and in neocartilage made by juvenile cells. Juvenile cells grew significantly faster in monolayer cultures than adult cells (p = 0.002) and proteoglycan levels produced in agarose culture was significantly higher in juvenile cells than in adult cells after multiple passages (p < 0.001). Juvenile chondrocytes did not stimulate lymphocyte proliferation.
These results document a dramatic age related decline in human chondrocyte chondrogenic potential and show that allogeneic juvenile chondrocytes do not stimulate an immunologic response in vivo.
Juvenile human chondrocytes have greater potential to restore articular cartilage than adult cells, and may be transplanted without the fear of rejection, suggesting a new allogeneic approach to restoring articular cartilage in older individuals.
juvenile human chondrocytes; neocartilage; agarose culture; immunogenicity; serial expansion; cartilage repair; chondrocyte transplantation; aging
Structural cell migration plays a central role in the pathophysiology of several diseases, including asthma. Previously, we established that IL-17–induced (CXCL1, CXCL2, and CXCL3) production promoted airway smooth muscle cell (ASMC) migration, and consequently we sought to investigate the molecular mechanism of CXC-induced ASMC migration. Recombinant human CXCL1, CXCL2, and CXCL3 were used to assess migration of human primary ASMCs from normal and asthmatic subjects using a modified Boyden chamber. Neutralizing Abs or small interfering RNA (siRNA) knockdown and pharmacological inhibitors of PI3K, ERK1/2, and p38 MAPK pathways were used to investigate the receptors and the signaling pathways involved in CXC-induced ASMC migration, respectively. We established the ability of CXCL2 and CXCL3, but not CXCL1, to induce ASMC migration at the tested concentrations using normal ASMCs. We found CXCL2-induced ASMC migration to be dependent on p38 MAPK and CXCR2, whereas CXCL3-induced migration was dependent on p38 and ERK1/2 MAPK pathways via CXCR1 and CXCR2. While investigating the effect of CXCL2 and CXCL3 on asthmatic ASMC migration, we found that they induced greater migration of asthmatic ASMCs compared with normal ones. Interestingly, unlike normal ASMCs, CXCL2- and CXCL3-induced asthmatic ASMC migration was mainly mediated by the PI3K pathway through CXCR1. In conclusion, our results establish a new role of CXCR1 in ASMC migration and demonstrate the diverse mechanisms by which CXCL2 and CXCL3 mediate normal and asthmatic ASMC migration, suggesting that they may play a role in the pathogenesis of airway remodeling in asthma.
The use of allogenic juvenile chondrocytes or autologous chondral fragments has shown promising laboratory results for the repair of chondral lesions.
The purpose of the study was to evaluate in vitro the extracellular matrix production of mixed adult/juvenile cultures of both chondrocytes (part 1) and minced cartilage fragments (part 2). The authors hypothesized that juvenile chondrocytes would not affect matrix production when mixed with adult chondrocytes or cartilage fragments.
Controlled laboratory study.
Cartilage sources consisted of three adult and three juvenile (human) donors. In part 1, per each donor, juvenile chondrocytes were mixed with adult chondrocytes in five different proportions: 100, 50, 25, 12.5 and 0 %. Three-dimensional cultures in low melt agarose were performed. At 6 weeks, biochemical and histological analyses were performed. In part 2, isolated adult, isolated juvenile, and mixed three-dimensional cultures (1:1) were performed with chondral fragments (<1mm), both with low melt agarose and a hyaluronic acid scaffold. At 2 and 6 weeks, cultures were evaluated with biochemical and histological analyses.
Part 1: biochemical and histological analyses showed that isolated juvenile cultures performed significantly better than mixed and isolated adult cultures. No significant differences were noted between mixed cultures (1:1) and isolated adult cultures. Part 2: biochemical and histological results at 6 weeks showed that mixed cartilage fragment cultures performed better than isolated adult cultures in terms of PG/DNA ratio (p=0.014), percentage of safranin-O positive cells (p=0.012), Bern score (p=0.001), and Collagen type II. No statistical difference was noted between juvenile and mixed cultures.
Extracellular matrix production of juvenile chondrocytes is inhibited by adult chondrocytes. The addition of juvenile cartilage fragments to adult fragments improves matrix production, with a positive interaction between the two sources.
Even if the underlying mechanisms are still unknown, this study describes the behavior of juvenile/adult co-cultures using both chondrocytes and cartilage fragments, with potential for new research and clinical applications.
juvenile; adult; chondrocytes; cartilage fragments; co-culture; cartilage repair
Articular cartilage degeneration in osteoarthritis has been linked to abnormal mechanical stresses that are known to cause chondrocyte apoptosis and metabolic derangement in in vitro models. Evidence implicating oxidative damage as the immediate cause of these harmful effects suggests that the anti-oxidant defenses of chondrocytes might influence their tolerance for mechanical injury. Based on evidence that anti-oxidant defenses in many cell types are stimulated by moderate oxidant exposure, we hypothesized that oxidant pre-conditioning would reduce acute chondrocyte death and proteoglycan depletion in cartilage explants after exposure to abnormal mechanical stresses. Porcine cartilage explants were treated every 48 hours with tert-butyl hydrogen peroxide (tBHP) at non-lethal concentrations (25, 100, 250, 500 µM) for a varying number of times (1, 2 or 4) prior to a bout of unconfined axial compression (5 MPa, 1 Hz, 1800 cycles). When compared with untreated controls, tBHP had significant positive effects on post-compression viability, lactate production, and proteoglycan losses. Overall, the most effective regime was 100 µM tBHP applied 4 times. RNA analysis revealed significant effects of 100 µM tBHP on gene expression. Catalase, hypoxia-inducible factor-1alpha (HIF-1α), and glyceraldehyde 6-phosphate dehydrogenase (GAPDH) were significantly increased relative to untreated controls in explants treated 4 times with 100 µM tBHP, a regime that also resulted in a significant decrease in matrix metalloproteinase-3 (MMP-3) expression. These findings demonstrate that repeated exposure of cartilage to sub-lethal concentrations of peroxide can moderate the acute effects of mechanical stress, a conclusion supported by evidence of peroxide-induced changes in gene expression that could render chondrocytes more resistant to oxidative damage.
Cartilage; mechanical stress; oxidant
Irregular bone remodeling is associated with a number of bone diseases such as osteoporosis and multiple myeloma. Computational and mathematical modeling can aid in therapy and treatment as well as understanding fundamental biology. Different approaches to modeling give insight into different aspects of a phenomena so it is useful to have an arsenal of various computational and mathematical models. Here we develop a mathematical representation of bone remodeling that can effectively describe many aspects of the complicated geometries and spatial behavior observed.
There is a sharp interface between bone and marrow regions. Also the surface of bone moves in and out, i.e. in the normal direction, due to remodeling. Based on these observations we employ the use of a level-set function to represent the spatial behavior of remodeling. We elaborate on a temporal model for osteoclast and osteoblast population dynamics to determine the change in bone mass which influences how the interface between bone and marrow changes.
We exhibit simulations based on our computational model that show the motion of the interface between bone and marrow as a consequence of bone remodeling. The simulations show that it is possible to capture spatial behavior of bone remodeling in complicated geometries as they occur in vitro and in vivo.
By employing the level set approach it is possible to develop computational and mathematical representations of the spatial behavior of bone remodeling. By including in this formalism further details, such as more complex cytokine interactions and accurate parameter values, it is possible to obtain simulations of phenomena related to bone remodeling with spatial behavior much as in vitro and in vivo. This makes it possible to perform in silica experiments more closely resembling experimental observations.
Bone remodeling; level-set equation; cytokines; osteoclast; osteoblast
The processes of whole-joint osteoarthritis development following localized joint injuries are not well understood. To demonstrate this local-to-global linkage, it was hypothesized that a localized osteoarticular injury in the rabbit knee would not only cause biomechanical and histological abnormalities in the involved compartment, but also concurrent histological changes in the non-involved compartment. Twenty rabbits had an acute osteoarticular injury that involved localized joint incongruity (a 2 mm osteochondral defect created in the weight-bearing area of the medial femoral condyle), while another twenty received control sham surgery. At the time of sacrifice at eight or sixteen weeks post-surgery, the experimental knees were subjected to sagittal-plane laxity measurement, followed by cartilage histo-morphological evaluation using the Mankin score. The immediate effects of defect creation on joint stability and contact mechanics were explored in concomitant rabbit cadaver experimentation. The injured animals had cartilage histological scores significantly higher than in the sham surgery group (p<0.01) on the medial femoral, medial tibial, and lateral femoral surfaces (predominantly on the medial surfaces), accompanied by slight (mean 20%) increase of sagittal-plane laxity. Immediate injury-associated alterations in the medial compartment contact mechanics were also demonstrated. Localized osteoarticular injury in this survival animal model resulted in global joint histological changes.
joint injury; osteoarthritis; cartilage histology; rabbit knee; osteochondral defect
This report summarises the case of a 19-year-old male, with a history of gastro-oesophageal reflux disease, who presented to hospital with an acute chest pain. An electrocardiographic and biochemical diagnosis of ST elevation myocardial infarction was made; however, subsequent coronary angiography and echocardiography were both normal. In the week preceding the admission, the patient had consumed large quantities of a popular energy drink and the authors believe this may have implicated the development of his coronary event. This is an association that has been suggested previously and this report briefly summarises the evidence supporting the connection.
To develop a method for repeated same-site measurement of mechanical properties suitable for the detection of degenerative changes in a biologically-active explant model after a single blunt impact injury.
Focal blunt impact injuries to articular surfaces lead to local cartilage degeneration and loss of mechanical properties. We employed a repeated measurement methodology to determine variations in mechanical same-site properties before and after injury in living cartilage with the hypothesis that normalization with initial mechanical properties may provide a clearer evaluation of impact effects and improve our understanding of the biologic responses to impact injury.
Materials and Methods
Bovine osteochondral explants were cultured for up to 14 days after impact injury. Indentation tests were performed before and after impact injury to assess relative changes in mechanical properties.
Creep strain increased significantly in impacted explants after 7 days and in both impacted and control explants after 14 days. Further analysis at 14 days revealed decreases in stretch factor β, creep time constant and local compressive modulus.
A repeated measures methodology reliably detected changes in mechanical behavior viable osteochondral explants after a single impact injury.
Histologic assessment of cartilage degradation has traditionally involved semiquantitative techniques, the most commonly utilized being the Mankin scale. Such assessments depend on human observer subjectivity, and thus have drawn criticism on the basis of associated inter- and intraobserver variability. We report a newly developed computational image analysis procedure for fully automated and fully objective assessment of the Mankin scale. Image processing routines were developed in a widely used programming environment (Matlab®) to analyze cartilage degradation. One hundred and twenty-five histology images incorporating a wide range of degradation features were analyzed by the algorithm and by seven observers experienced in cartilage histologic assessment. Based on random effects linear statistical models, the computer program performed well, showing a correlation of 0.88 between its Mankin scores and latent (average of human observers’) image scores. Regarding the four subcomponents of the Mankin scale, computer program correlations with observer scores were best for surface defect and proteoglycan depletion, but less favorable for cellularity and tidemark invasion. While limitations exist with image processing techniques, the new algorithm provides an objective and automated method for analyzing cartilage histology sections, consistent with human observer grading.
osteoarthritis; cartilage; image processing; comparative histology
Although the fibroblast growth factor (FGF) signaling axis plays important roles in heart development, the molecular mechanism by which the FGF regulates cardiogenesis is not fully understood.
To investigate the mechanism by which FGF signaling regulates cardiac progenitor cell differentiation.
Methods and results
Using mice with tissue-specific ablation of FGF receptors and FGF receptor substrate 2α (Frs2α) in heart progenitor cells, we demonstrate that that disruption of FGF signaling leads to premature differentiation of cardiac progenitor cells in mice. Using embryoid body (EB) cultures of mouse embryonic stem cells (ESCs), we reveal that FGF signaling promotes mesoderm differentiation in ESCs, but inhibits cardiomyocyte differentiation of the mesoderm cells at later stages. Furthermore, we also report that inhibiting FRS2α-mediated signals increases autophagy and that activating autophagy promotes myocardial differentiation and vice versa.
The results indicate that the FGF/FRS2α-mediated signals prevent premature differentiation of heart progenitor cells through suppressing autophagy. The findings provide the first evidence that autophagy plays a role in heart progenitor differentiation.
FGF; autophagy; heart development; second heart field; premature differentiation; heart defect
Until recently many studies of bone remodeling at the cellular level have focused on the behavior of mature osteoblasts and osteoclasts, and their respective precursor cells, with the role of osteocytes and bone lining cells left largely unexplored. This is particularly true with respect to the mathematical modeling of bone remodeling. However, there is increasing evidence that osteocytes play important roles in the cycle of targeted bone remodeling, in serving as a significant source of RANKL to support osteoclastogenesis, and in secreting the bone formation inhibitor sclerostin. Moreover, there is also increasing interest in sclerostin, an osteocyte-secreted bone formation inhibitor, and its role in regulating local response to changes in the bone microenvironment. Here we develop a cell population model of bone remodeling that includes the role of osteocytes, sclerostin, and allows for the possibility of RANKL expression by osteocyte cell populations. We have aimed to give a simple, yet still tractable, model that remains faithful to the underlying system based on the known literature. This model extends and complements many of the existing mathematical models for bone remodeling, but can be used to explore aspects of the process of bone remodeling that were previously beyond the scope of prior modeling work. Through numerical simulations we demonstrate that our model can be used to explore theoretically many of the qualitative features of the role of osteocytes in bone biology as presented in recent literature.
Due to the widespread prevalence of resistant parasites, chloroquine (CQ) was removed from front-line antimalarial chemotherapy in the 1990s despite its initial promise of disease eradication. Since then, resistance-conferring mutations have been identified in transporters such as the PfCRT, that allow for the efflux of CQ from its primary site of action, the parasite digestive vacuole. Chemosensitizing/chemoreversing compounds interfere with the function of these transporters thereby sensitizing parasites to CQ once again. However, compounds identified thus far have disappointing in vivo efficacy and screening for alternative candidates is required to revive this strategy. In this study, we propose a simple and direct means to rapidly screen for such compounds using a fluorescent-tagged CQ molecule. When this screen was applied to a small library, seven novel chemosensitizers (octoclothepin, methiothepin, metergoline, loperamide, chlorprothixene, L-703,606 and mibefradil) were quickly elucidated, including two which showed greater potency than the classical chemosensitizers verapamil and desipramine.
Although the majority of the ATP in chondrocytes is made by glycolysis rather than by oxidative phosphorylation in mitochondria there is evidence to suggest that reactive oxygen species produced by mitochondrial electron transport help to maintain cellular redox balance in favor of glycolysis. The objective of this study was to test this hypothesis by determining if rotenone, which inhibits electron transport and blocks oxidant production inhibits glycolytic ATP synthesis.
Bovine osteochondral explants were treated with rotenone, an electron transport inhibitor; or oligomycin an ATP synthase inhibitor; or 2-fluoro-2-deoxy-D-glucose, a glycolysis inhibiter; or peroxide, an exogenous oxidant; or mitoquinone, a mitochondria-targeted anti-oxidant. Cartilage extracts were assayed for ATP, NAD+, and NADH, and culture medium was assayed for pyruvate and lactate after 24 hours of treatment. Imaging studies were used to measure superoxide production in cartilage.
Rotenone and 2-fluoro-2-deoxy-D-glucose caused a significant decline in cartilage ATP (p < 0.001). In contrast, ATP levels were not affected by oligomycin. Peroxide treatment blocked rotenone effects on ATP, while treatment with MitoQ significantly suppressed ATP levels. Rotenone and 2-fluoro-2-deoxy-D-glucose caused a significant decline in pyruvate, but not in lactate production. NADH:NAD+ ratios decreased significantly in both rotenone and 2-fluoro-2-deoxy-D-glucose-treated explants (p < 0.05). Rotenone also significantly reduced superoxide production
These findings showing a link between glycolysis and electron transport are consistent with previous reports on the critical need for oxidants to support normal chondrocyte metabolism. They suggest a novel role for mitochondria in cartilage homeostasis that is independent of oxidative phosphorylation.
cartilage; glycolysis; mitochondrial electron transport; adenosine triphosphate; oxidants
asthma pathogenesis; autophagy; lung function; polymorphism; SNP; ATG5; autophagosome
To create annotated clinical narratives with layers of syntactic and semantic labels to facilitate advances in clinical natural language processing (NLP). To develop NLP algorithms and open source components.
Manual annotation of a clinical narrative corpus of 127 606 tokens following the Treebank schema for syntactic information, PropBank schema for predicate-argument structures, and the Unified Medical Language System (UMLS) schema for semantic information. NLP components were developed.
The final corpus consists of 13 091 sentences containing 1772 distinct predicate lemmas. Of the 766 newly created PropBank frames, 74 are verbs. There are 28 539 named entity (NE) annotations spread over 15 UMLS semantic groups, one UMLS semantic type, and the Person semantic category. The most frequent annotations belong to the UMLS semantic groups of Procedures (15.71%), Disorders (14.74%), Concepts and Ideas (15.10%), Anatomy (12.80%), Chemicals and Drugs (7.49%), and the UMLS semantic type of Sign or Symptom (12.46%). Inter-annotator agreement results: Treebank (0.926), PropBank (0.891–0.931), NE (0.697–0.750). The part-of-speech tagger, constituency parser, dependency parser, and semantic role labeler are built from the corpus and released open source. A significant limitation uncovered by this project is the need for the NLP community to develop a widely agreed-upon schema for the annotation of clinical concepts and their relations.
This project takes a foundational step towards bringing the field of clinical NLP up to par with NLP in the general domain. The corpus creation and NLP components provide a resource for research and application development that would have been previously impossible.
Gold Standard Annotations; UMLS; Treebank; Propbank; Natural Language Processing; cTAKES
Citrus byproducts (CBPs) are utilized as a low cost nutritional supplement to the diets of cattle and have been suggested to inhibit the growth of both Escherichia coli O157:H7 and Salmonella. The objective of this study was to examine the effects in vitro that varying concentrations of CBP in the powdered or pelleted variety have on the survival of Shiga-toxin Escherichia coli (STEC) serotypes O26:H11, O103:H8, O111:H8, O145:H28, and O157:H7 in bovine ruminal microorganism media. The O26:H11, O111:H8, O145:H28, and O157:H7 serotypes did not exhibit a change in populations in media supplemented with CBP with either variety. The O103:H8 serotype displayed a general trend for an approximate 1log10 reduction in 5% powdered CBP and 20% pelleted CBP over 6 h. There was a trend for reductions in populations of a variant form of O157:H7 mutated in the stx1 and stx2 genes in higher concentrations of CBP. These results suggest that variations exist in the survival of these serotypes of STEC within mixed ruminal microorganism fluid media when supplemented with CBP. Further research is needed to determine why CBPs affect STEC serotypes differently.
Hindbrain rhombomere 1 (r1) is located caudal to the isthmus, a critical organizer region, and rostral to rhombomere 2 in the developing mouse brain. Dorsal r1 gives rise to the cerebellum, locus coeruleus, and several brainstem nuclei, whereas cells from ventral r1 contribute to the trochlear and trigeminal nuclei as well as serotonergic and GABAergic neurons of the dorsal raphe. Recent studies have identified several molecular events controlling dorsal r1 development. In contrast, very little is known about ventral r1 gene expression and the genetic mechanisms regulating its formation. Neurons with distinct neurotransmitter phenotypes have been identified in ventral r1 including GABAergic, serotonergic, and cholinergic neurons. Here we show that PITX2 marks a distinct population of GABAergic neurons in mouse embryonic ventral r1. This population appears to retain its GABAergic identity even in the absence of PITX2. We provide a comprehensive map of markers that places these PITX2-positive GABAergic neurons in a region of r1 that intersects and is potentially in communication with the dorsal raphe.
hindbrain; development; transcription factor
Norcantharidin (NCTD) has been reported to induce tumor cell apoptosis. However, the underlying mechanism behinds its antitumor effect remains elusive. We have previously shown that TR3 expression is significantly decreased in metastatic melanomas and involved in melanoma cell apoptosis. In this study, we showed that NCTD inhibited melanoma cell proliferation and induced apoptosis in a dose related manner. NCTD induced translocation of TR3 from nucleus to mitochondria where it co-localized with Bcl-2 in melanoma cells. NCTD also increased cytochome c release from mitochondria to the cytoplasm. These changes were accompanied by increased expression of Bax and cleaved caspase-3 along with decreased expression of Bcl2 and NF-κB2. The effects of NCTD were inhibited by knockdown of TR3 expression using TR3 specific shRNA in melanoma cells. Furthermore, NCTD significantly decreased tumor volume and improved survival of Tyr::CreER; BRAFCa/+; Ptenlox/lox transgenic mice. Our data indicates that NCTD inhibits melanoma growth by inducing tumor cell apoptosis via activation of a TR3 dependent pathway. These results suggest that NCTD is a potential therapeutic agent for melanoma.
norcantharidin; apoptosis; TR3; melanoma
Preeclampsia is considered a disease of immunological origin associated with abnormalities in inflammatory cytokines, tumor necrosis factor-α (TNF-α), and activated lymphocytes secreting AT1-AA. Recent studies have also demonstrated that an imbalance of angiogenic factors, soluble fms-like tyrosine kinase (sFlt-1), and sEndoglin, exists in preeclampsia; however, the mechanisms that initiate their overproduction are unclear.
To determine the role of immune regulation of these factors, circulating and placental sFlt-1 and/or sEndoglin was examined from pregnant rats chronically treated with TNF-α or AT1-AA. On day 19 of gestation blood pressure was analyzed and serum and tissues were collected. Placental villous explants were excised and cultured on matrigel coated inserts for 24 h and sFlt-1 and sEndoglin was measured from media.
In response to TNF-α-induced hypertension, sFlt-1 increased from 180 ± 5 to 2,907 ± 412 pg/ml. sFlt-1 was also increased from cultured placental explants of TNF-α induced hypertensive pregnant rats (n = 12) (2,544 ± 1,132 pg/ml) vs. explants from normal pregnant (NP) rats (n = 12) (2,189 ± 586 pg/ml) where as sEng was undetectable. Circulating sFlt-1 increased from 245 ± 38 to 3,920 ± 798 pg/ml in response to AT1-AA induced hypertension. sFlt-1 levels were higher (3,400 ± 350 vs. 2,480 ± 900 pg/ml) in placental explants from AT1-AA infused rats (n = 12) than NP rats (n = 7). In addition, sEndoglin increased from 30 ± 2.7 to 44 ± 3.3 pg/ml (P < 0.047) in AT1-AA infused rats but was undetectable in the media of the placental explants.
These data suggest that immune factors may serve as an important stimulus for both sFlt-1 and sEndoglin production in response to placental ischemia.
antiangiogenic factors; blood pressure; hypertension; immune activation; pregnancy
Noneosinophilic asthma has been regarded as a distinct phenotype characterized by a poor response to inhaled corticosteroids (ICS).
To determine whether noneosinophilic, steroid-naive asthmatic subjects show an improvement in asthma control, asthma symptoms and spirometry after four weeks of treatment with ICS, and whether they further benefit from the addition of a long-acting beta-2 agonists to ICS.
A randomized, double-blind, placebo-controlled, multicentre study comparing the efficacy of placebo versus inhaled fluticasone propionate 250 μg twice daily for four weeks in mildly uncontrolled, steroid-naive asthmatic subjects with a sputum eosinophil count ≤2%. This was followed by an open-label, four-week treatment period with fluticasone propionate 250 μg/salmeterol 50 μg, twice daily for all subjects.
After four weeks of double-blind treatment, there was a statistically significant and clinically relevant improvement in the mean (± SD) Asthma Control Questionnaire score in the ICS-treated group (n=6) (decrease of 1.0±0.5) compared with the placebo group (n=6) (decrease of 0.09±0.4) (P=0.008). Forced expiratory volume in 1 s declined in the placebo group (−0.2±0.2 L) and did not change in the ICS group (0.04±0.1 L) after four weeks of treatment (P=0.02). The open-label treatment with fluticasone propionate 250 μg/salmeterol 50 μg did not produce additional improvements in those who were previously treated for four weeks with inhaled fluticasone alone.
A clinically important and statistically significant response to ICS was observed in mildly uncontrolled noneosinophilic asthmatic subjects.
Asthma; Asthma Control Questionnaire; Eosinophils; Sputum cell counts
Even with current treatments of acute joint injuries, more than 40% of people who suffer significant ligament or meniscus tears, or articular surface injuries, will develop osteoarthritis. Correspondingly, 12% or more of all patients with lower extremity osteoarthritis have a history of joint injury. Recent research suggests that acute joint damage that occurs at the time of an injury initiates a sequence of events that can lead to progressive articular surface damage. New molecular interventions, combined with evolving surgical methods, aim to minimize or prevent progressive tissue damage triggered by joint injury. Seizing the potential for progress in the treatment of joint injuries to forestall OA will depend on advances in: (1) quantitative methods of assessing the injury severity, including both structural damage and biologic responses, (2) understanding of the pathogenesis of post-traumatic OA, taking into account potential interactions among the different tissues and the role of post-traumatic incongruity and instability, and (3) application of engineering and molecular research to develop new methods of treating injured joints. This paper highlights recent advances in understanding of the structural damage and the acute biological response following joint injury, and it identifies important directions for future research.