Mfge8 is critical for mammary gland remodeling during involution

Apoptosis is a critical process in normal mammary gland development and the rapid clearance of apoptotic cells prevents tissue injury associated with the release of intracellular antigens from dying cells. Milk fat globule-EGF-factor 8 (Mfge8) is a milk glycoprotein that is abundantly expressed in the mammary gland epithelium and has been shown to facilitate the clearance of apoptotic lymphocytes by splenic macrophages. We report that mice with disruption of Mfge8 had normal mammary gland development until involution. However, abnormal mammary gland remodeling was observed postlactation in Mfge8 mutant mice. During early involution, Mfge8 mutant mice had increased numbers of apoptotic cells within the mammary gland associated with a delay in alveolar collapse and fat cell repopulation. As involution progressed, Mfge8 mutants developed inflammation as assessed by CD45 and CD11b staining of mammary gland tissue sections. With additional pregnancies, Mfge8 mutant mice developed progressive dilatation of the mammary gland ductal network. These data demonstrate that Mfge8 regulates the clearance of apoptotic epithelial cells during mammary gland involution and that the absence of Mfge8 leads to inflammation and abnormal mammary gland remodeling.     

Discovery of pectin-degrading enzymes and directed evolution of a novel pectate lyase for processing cotton fabric

There is a growing need in the textile industry for more economical and environmentally responsible approaches to improve the scouring process as part of the pretreatment of cotton fabric. Enzymatic methods using pectin-degrading enzymes are potentially valuable candidates in this effort because they could reduce the amount of toxic alkaline chemicals currently used. Using high throughput screening of complex environmental DNA libraries more than 40 novel microbial pectate lyases were discovered, and their enzymatic properties were characterized. Several candidate enzymes were found that possessed pH optima and specific activities on pectic material in cotton fibers compatible with their use in the scouring process. However, none exhibited the desired temperature characteristics. Therefore, a candidate enzyme was selected for evolution. Using Gene Site Saturation Mutagenesistrade mark technology, 36 single site mutants exhibiting improved thermotolerance were produced. A combinatorial library derived from the 12 best performing single site mutants was then generated by using Gene Reassemblytrade mark technology. Nineteen variants with further improved thermotolerance were produced. These variants were tested for both improved thermotolerance and performance in the bioscouring application. The best performing variant (CO14) contained eight mutations and had a melting temperature 16 degrees C higher than the wild type enzyme while retaining the same specific activity at 50 degrees C. Optimal temperature of the evolved enzyme was 70 degrees C, which is 20 degrees C higher than the wild type. Scouring results obtained with the evolved enzyme were significantly better than the results obtained with chemical scouring, making it possible to replace the conventional and environmentally harmful chemical scouring process.     

Endotoxin responsiveness and subchronic grain dust-induced airway disease

Endotoxin is one of the principal components of grain dust that causes acute reversible airflow obstruction and airway inflammation. To determine whether endotoxin responsiveness influences the development of chronic grain dust-induced airway disease, physiological and airway inflammation remodeling parameters were evaluated after an 8-wk exposure to corn dust extract (CDE) and again after a 4-wk recovery period in a strain of mice sensitive to (C3H/HeBFeJ) and one resistant to (C3H/HeJ) endotoxin. After the CDE exposure, both strains of mice had equal airway hyperreactivity to a methacholine challenge; however, airway hyperreactivity persisted only in the C3H/HeBFeJ mice after the recovery period. Only the C3H/HeBFeJ mice showed significant inflammation of the lower airway after the 8-wk exposure to CDE. After the recovery period, this inflammatory response completely resolved. Lung stereological measurements indicate that an 8-wk exposure to CDE resulted in persistent expansion of the airway submucosal cross-sectional area only in the C3H/HeBFeJ mice. Collagen type III and an influx of cells into the subepithelial area participated in the expansion of the submucosa. Our findings demonstrate that subchronic inhalation of grain dust extract results in the development of chronic airway disease only in mice sensitive to endotoxin but not in mice that are genetically hyporesponsive to endotoxin, suggesting that endotoxin is important in the development of chronic airway disease.     

Determination of a novel substance P inhibitor in human plasma by high-performance liquid chromatography with atmospheric pressure chemical ionization mass spectrometric detection using single and triple quadrupole detectors

Methods based on high-performance liquid chromatography (HPLC) with atmospheric-pressure chemical ionization (APCI) mass spectrometric (MS) detection using either single (MS) or triple (MS/MS) quadrupole mass spectrometric detection for the determination of (2R)-[1(R)-(3,5-bis-trifluoromethylphenyl)ethoxy]-3(S)-(4-fluoro-phenyl)morpholin-4-ylmethyl]-5-oxo-4,5-dihydro-[1,2,4]triazol)methyl morpholine (Aprepitant, Fig. 1) in human plasma has been developed. Aprepitant (I) and internal standard (II, Fig. 1) were isolated from the plasma matrix buffered to pH 9.8 using a liquid-liquid extraction with methyl-t-butyl ether (MTBE). The analytes were separated on a Keystone Scientific's Javelin BDS C-8 2 mm x 4.6 mm 3 microm guard column coupled to BDS C-8 50 mm x 4.6 mm 3 microm analytical column, utilizing a mobile phase of 50% acetonitrile and 50% water containing 0.1% formic acid and 10 mM ammonium acetate delivered at a flow rate of 1 ml/min. The single quadrupole instrument was operated in a single ion monitoring (SIM) mode analyzing the protonated molecules of Aprepitant and II at m/z 535 and 503, respectively. The triple quadrupole mass spectrometer was operated in multiple reaction monitoring mode (MRM) monitoring the precursor --> ion combinations of m/z 535 --> 277 and 503 --> 259 for Aprepitant and II, respectively. The linear calibration range for both single and triple quadrupole detectors was from 10 to 5000 ng/ml of plasma with coefficients of variation less than 8% at all concentrations. Both single and triple quadrupole instruments yielded similar precision and accuracy results. Matrix effect experiments performed on both instruments demonstrated the absence of any significant change in ionization of the analytes when comparing neat standards to analytes in the presence of plasma matrix. Both instruments were used successfully to support numerous clinical trials of Aprepitant.     

Rapid one-step characterization of recombinant vectors by direct analysis of transformed Escherichia coli colonies

We have developed a polymerase chain reaction (PCR) based procedure for rapidly analyzing recombinant vectors in whole bacterial cells. No purification, restriction mapping or sequencing of vectors is required and the results are available within 6 hours. Whole cells are added to a PCR mix that is designed to amplify DNA only if the correct insert is present in the required orientation. The presence of an appropriately sized band on an agarose gel is indicative of a correct clone.     

Sixty simple sequence repeat markers for use in the Festuca–Lolium complex of grasses

So far only very few simple sequence repeat (SSR) markers developed from grass species have had their primer sequences published. To make more markers available to the scientific community, we isolated and sequenced 256 microsatellite‐containing clones from four genome libraries of a Lolium multiflorum×Festuca glaucescens F₁ hybrid following enrichment in (TC)_n, (TG)_n, or both repeats. In this work, we report the primer sequences of 60 SSRs including preliminary results of polymorphism for mapping.     

Preclinical evaluation of the imipridone family,analogs of clinical stage anti-cancer small molecule ONC201, reveals potent anti-cancer effects of ONC212

Anti-cancer small molecule ONC201 upregulates the integrated stress response (ISR) and acts as a dual inactivator of Akt/ERK, leading to TRAIL gene activation. ONC201 is under investigation in multiple clinical trials to treat patients with cancer. Given the unique imipridone core chemical structure of ONC201, we synthesized a series of analogs to identify additional compounds with distinct therapeutic properties. Several imipridones with a broad range of in vitro potencies were identified in an exploration of chemical derivatives. Based on in vitro potency in human cancer cell lines and lack of toxicity to normal human fibroblasts, imipridones ONC206 and ONC212 were prioritized for further study. Both analogs inhibited colony formation, and induced apoptosis and downstream signaling that involves the integrated stress response and Akt/ERK, similar to ONC201. Compared to ONC201, ONC206 demonstrated improved inhibition of cell migration while ONC212 exhibited rapid kinetics of activity. ONC212 was further tested in >1000 human cancer cell lines in vitro and evaluated for safety and anti-tumor efficacy in vivo. ONC212 exhibited broad-spectrum efficacy at nanomolar concentrations across solid tumors and hematological malignancies. Skin cancer emerged as a tumor type with improved efficacy relative to ONC201. Orally administered ONC212 displayed potent anti-tumor effects in vivo, a broad therapeutic window and a favorable PK profile. ONC212 was efficacious in vivo in BRAF V600E melanoma models that are less sensitive to ONC201. Based on these findings, ONC212 warrants further development as a drug candidate. It is clear that therapeutic utility extends beyond ONC201 to include additional imipridones.