Disease-causing mutations in cartilage oligomeric matrix protein cause an unstructured Ca2+ binding domain.

Chondrocytes from pseudoachondroplasia (PSACH) and multiple epiphyseal dysplasia (EDM1) patients display an enlarged rough endoplasmic reticulum that accumulates extracellular matrix proteins, including cartilage oligomeric matrix protein (COMP). Mutations that cause PSACH and EDM1 are restricted to a 27-kDa Ca(2+) binding domain (type 3 repeat). This domain has 13 Ca(2+)-binding loops with a consensus sequence that conforms to Ca(2+)-binding loops found in EF hands. Most disease-causing mutations are found in the 11-kDa C-terminal region of this domain. We expressed recombinant native and mutant forms of the type 3 repeat domain (T3) and its 11-kDa C-terminal region (T3-Cterm). T3 and T3-Cterm bind approximately 13 and 8 mol of Ca(2+)/mol of protein, respectively. CD, one-dimensional proton, and two-dimensional (1)H-(15)N HSQC spectra of Ca(2+)-bound T3-Cterm indicate a distinct conformation that has little helical secondary structure, despite the presence of 13 EF hand Ca(2+)-binding loops. This conformation is also formed within the context of the intact T3. 19 cross-peaks found between 9.0 and 11.4 ppm are consistent with the presence of strong hydrogen bonding patterns, such as those in beta-sheets. Removal of Ca(2+) leads to an apparent loss of structure as evidenced by decreased dispersion and loss of all down field resonances. Deletion of Asp-470 (a mutation found in 22% of all PSACH and EDM1 patients) decreased the Ca(2+)-binding capacity of both T3 and T3-Cterm by about 3 mol of Ca(2+)/mol of protein. Two-dimensional (1)H-(15)N HSQC spectra of mutated T3-Cterm showed little evidence of defined structure in the presence or absence of Ca(2+). The data demonstrate that Ca(2+) is required to nucleate folding and to maintain defined structure. Mutation results in a partial loss of Ca(2+)-binding capacity and prevents Ca(2+)-dependent folding. Persistence of an unstructured state of the mutated Ca(2+) binding domain in COMP is the structural basis for retention of COMP in the rough endoplasmic reticulum of differentiated PSACH and EDM1 chondrocytes.     

A simple non-invasive protocol to establish primary cell lines from tail and toe explants for cytogenetic studies in Australian dragon lizards (Squamata: Agamidae)

Primary cell lines were established from cultures of tail and toe clips of five species of Australian dragon lizards: Tympanocryptis pinguicolla, Tympanocryptis sp., Ctenophorus fordi, Amphibolurus norrisi and Pogona vitticeps. The start of exponential cell growth ranged from 1 to 5 weeks. Cultures from all specimens had fibroblastic morphology. Cell lines were propagated continuously up to ten passages, cryopreserved and recovered successfully. We found no reduction in cell viability after short term (<6 months) storage at −80 °C. Mitotic metaphase chromosomes were harvested from these cell lines and used in differential staining, banding and fluorescent in situ hybridisation. Cell lines maintained normal diploidy in all species. This study reports a simple non-invasive method for establishing primary cell lines from Australian dragon lizards without sacrifice. The method is likely to be applicable to a range of species. Such cell lines provide a virtually unlimited source of material for cytogenetic, evolutionary and genomic studies.     

Toll-like receptor expression in normal ovary and ovarian tumors

Recent studies have implicated inflammation in the initiation and progression of ovarian cancer, though the mechanisms underlying this effect are still not clear. Toll-like receptors (TLRs) allow immune cells to recognize pathogens and to trigger inflammatory responses. Tumor cell expression of TLRs can promote inflammation and cell survival in the tumor microenvironment. Here we sought to characterize the expression of TLRs in normal human ovaries, benign and malignant ovarian tumors from patients, and in established ovarian tumor cell lines. We report that TLR2, TLR3, TLR4, and TLR5 are strongly expressed on the surface epithelium of normal ovaries. In contrast to previous studies of uterus and endocervix, we found no cyclic variation in TLR expression occurred in murine ovaries. TLR2, TLR3, TLR4, and TLR5 are expressed in benign conditions, epithelial tumors, and in ovarian cancer cell lines. Variable expression of TLR6 and TLR8 was seen in benign and malignant epithelium of some patients, while expression of TLR1, TLR7, and TLR9 was weak. Normal and malignant ovarian stroma were negative for TLR expression. Vascular endothelial cells, macrophages, and occasional fibroblasts in tumors were positive. Functional activity for TLRs was demonstrated by stimulation of cell lines with specific ligands and subsequent activation and translocation of NFκB and release of the proinflammatory cytokines interleukin-6 and CCL-2. These studies demonstrate expression of multiple TLRs in the epithelium of normal ovaries and in ovarian tumor cells, and may indicate a mechanism by which epithelial tumors manipulate inflammatory pathways to facilitate tumor progression.     

Chemoirradiation for Glioblastoma Multiforme: The National Cancer Institute Experience

Purpose

Standard treatment for glioblastoma (GBM) is surgery followed by radiation (RT) and temozolomide (TMZ). While there is variability in survival based on several established prognostic factors, the prognostic utility of other factors such as tumor size and location are not well established.

Experimental Design

The charts of ninety two patients with GBM treated with RT at the National Cancer Institute (NCI) between 1998 and 2012 were retrospectively reviewed. Most patients received RT with concurrent and adjuvant TMZ. Topographic locations were classified using preoperative imaging. Gross tumor volumes were contoured using treatment planning systems utilizing both pre-operative and post-operative MR imaging.

Results

At a median follow-up of 18.7 months, the median overall survival (OS) and progression-free survival (PFS) for all patients was 17.9 and 7.6 months. Patients with the smallest tumors had a median OS of 52.3 months compared to 16.3 months among patients with the largest tumors, P = 0.006. The patients who received bevacizumab after recurrence had a median OS of 23.3 months, compared to 16.3 months in patients who did not receive it, P = 0.0284. The median PFS and OS in patients with periventricular tumors was 5.7 and 17.5 months, versus 8.9 and 23.3 months in patients with non-periventricular tumors, P = 0.005.

Conclusions

Survival in our cohort was comparable to the outcome of the defining EORTC-NCIC trial establishing the use of RT+TMZ. This study also identifies several potential prognostic factors that may be useful in stratifying patients.     

Macrophages are crucial for epithelial cell death and adipocyte repopulation during mammary gland involution

Mammary gland development is dependent on macrophages, as demonstrated by their requirement during the expansion phases of puberty and pregnancy. Equally dramatic tissue restructuring occurs following lactation, when the gland regresses to a state that histologically resembles pre-pregnancy through massive programmed epithelial cell death and stromal repopulation. Postpartum involution is characterized by wound healing-like events, including an influx of macrophages with M2 characteristics. Macrophage levels peak after the initial wave of epithelial cell death, suggesting that initiation and execution of cell death are macrophage independent. To address the role of macrophages during weaning-induced mammary gland involution, conditional systemic deletion of macrophages expressing colony stimulating factor 1 receptor (CSF1R) was initiated just prior to weaning in the Mafia mouse model. Depletion of CSF1R(+) macrophages resulted in delayed mammary involution as evidenced by loss of lysosomal-mediated and apoptotic epithelial cell death, lack of alveolar regression and absence of adipocyte repopulation 7 days post-weaning. Failure to execute involution occurred in the presence of milk stasis and STAT3 activation, indicating that neither is sufficient to initiate involution in the absence of CSF1R(+) macrophages. Injection of wild-type bone marrow-derived macrophages (BMDMs) or M2-differentiated macrophages into macrophage-depleted mammary glands was sufficient to rescue involution, including apoptosis, alveolar regression and adipocyte repopulation. BMDMs exposed to the postpartum mammary involution environment upregulated the M2 markers arginase 1 and mannose receptor. These data demonstrate the necessity of macrophages, and implicate M2-polarized macrophages, for epithelial cell death during normal postpartum mammary gland involution.     

HIV-1 integrase preassembled on donor DNA is refractory to activity stimulation by LEDGF/p75

LEDGF/p75 is known to enhance the integrase strand transfer activity in vitro, but the underlying mechanism is unclear. Using an integrase assay with a chemiluminescent readout adapted to a 96-well plate format, the effect of LEDGF/p75 on both the 3'-processing and strand transfer steps was analyzed. Integrase inhibitors of the strand transfer reaction remained active in the presence of LEDGF/p75, but displayed 3- to 7-fold higher IC50 values. Our analyses indicate that, in the presence of 150 nM LEDGF/p75, active integrase/donor DNA complexes were increased by 5.3-fold during the 3'-processing step. In addition, these integrase/donor DNA complexes showed a 4.5-fold greater affinity for the target DNA during the subsequent strand transfer step. We also observed a 3.7-fold increase in the rate constant of catalysis of the strand transfer step when 150 nM LEDGF/p75 was present during the 3'-processing step. In contrast, when LEDGF/p75 was added at the beginning of the strand transfer step, no increase in either the concentration of active integrase/donor DNA complex or its rate constant of strand transfer catalysis was observed. This observation suggested that the integrase/donor DNA formed in the absence of LEDGF/p75 became refractory to the stimulatory effect of LEDGF/p75. Instead, this LEDGF/p75 added at the start of the strand transfer step was able to promote the formation of a new cohort of active integrase/donor DNA complexes which became functional with a delay of 45 min after LEDGF/p75 addition. We propose a model whereby LEDGF/p75 can only bind integrase before the latter binds donor DNA whereas donor DNA can engage either free or LEDGF/p75-bound integrase.     

Relationships between four novel ceratomyxid parasites from the gall bladders of labrid fishes from Heron Island, Queensland, Australia

Ceratomyxid parasites from the gall bladder of fishes from the family Labridae were examined for their taxonomic identity and their relatedness to other species of myxozoans. This paper describes four novel species Ceratomyxa choerodonae n. sp. from Choerodon cyanodus; Ceratomyxa cheilinae n. sp. from Cheilinus chlorourus; Ceratomyxa oxycheilinae n. sp. from Oxycheilinus digramma; and Ceratomyxa thalassomae n. sp. from Thalassoma lunare. Each species is characterised morphologically and small subunit (18 S) rDNA sequences were used in molecular phylogenetic analyses. Comparison of the small subunit rDNA sequences revealed four distinct species even though morphological differences indicated the presence of only two morphologically distinctive spore types. Phylogenetic analyses revealed that the ceratomyxids from labrid fishes did not form a clade to the exclusion of all other myxosporeans and provides evidence that host relatedness is not a good character to discriminate between species within the genus Ceratomyxa.     

Discovery of thieno[2,3-c]pyridines as potent COT inhibitors

Evaluation of hit chemotypes from high throughput screening identified a novel series of 2,4-disubstituted thieno[2,3-c]pyridines as COT kinase inhibitors. Structural modifications exploring SAR at the 2- and 4-positions resulting in inhibitors with improved enzyme potency and cellular activity are disclosed.     

The Use of a Respiratory Rate Biofeedback Device to Reduce Dental Anxiety: An Exploratory Investigation

Anxiety experienced by individuals visiting the dental office to receive treatment is common. Evidence has shown biofeedback to be a useful modality of treatment for numerous maladies associated with anxiety. The purpose of the current pilot study was to investigate the use of a novel biofeedback device (RESPeRATE™) to reduce patients’ pre-operative general anxiety levels and consequently reduce the pain associated with dental injections. Eighty-one subjects participated in this study, forty in the experimental group and forty-one in the control group. Subjects in the experimental group used the biofeedback technique, while those in the control group were not exposed to any biofeedback. All subjects filled out a pre-injection anxiety survey, then received an inferior alveolar injection of local anesthetic. Post-injection, both groups were given an anxiety survey and asked to respond to four questions regarding the injection experience using a Visual Analog Scale (VAS). With the use of the respiratory rate biofeedback device, there was a significant reduction of negative feelings regarding the overall injection experience, as measured by a VAS. Our findings demonstrate that this novel biofeedback technique may be helpful in the amelioration of dental anxiety, and may help produce a more pleasant overall experience for the patient.     

Highly selective c-Jun N-terminal kinase (JNK) 3 inhibitors with in vitro CNS-like pharmacokinetic properties II. Central core replacement

In this Letter, we describe the evolution of selective JNK3 inhibitors from 1, that routinely exhibit >10-fold selectivity over JNK1 and >1000-fold selectivity over related MAPKs. Strong SAR was found for substitution of the naphthalene ring, as well as for inhibitors adopting different central scaffolds. Significant potency gains were appreciated by inverting the polarity of the thione of the parent triazolothione 1, resulting in potent compounds with attractive pharmacokinetic profiles.