Role of the LPA2 Receptor in Protecting Against Apoptosis

Lysophosphatidic acid (LPA) is a naturally occurring lipid mediator. It exists abundantly in biological fluids such as serum, saliva, follicular fluid, seminal fluid and malignant effusions and induces a vast array of biological responses affecting cell growth, survival, differentiation, migration and morphology. We recently identified lysophosphatidic acid (LPA) as a potent antiapoptotic agent for the intestinal epithelium. Based on computational modeling octadecenyl thiophosphate (OTP) was synthesized: a novel rationally designed, metabolically stabilized LPA mimic. OTP was more efficacious than LPA in reducing g-irradiation-, camptothecin-, or TNF-a/cycloheximide-induced apoptosis and caspase 3, 8 and 9 activity in the IEC-6 cell line. The OTP- and LPA-elicited antiapoptotic effects were completely blocked by the MEK inhibitor PD98059 and the PI3K inhibitor LY294002. Pertussis toxin partially abolished OTP-induced ERK1/2 and apoptotic protection. In RH7777 cells lacking LPA receptors, OTP selectively protected LPA2 but not LPA1 and LPA3 transfectants. In C57BL/6 and LPA1 knockout mice exposed to 15 Gy g-irradiation, orally applied OTP reduced the number of apoptotic bodies and activated caspase 3 positive cells but was ineffective in LPA2 knockouts. OTP, with higher efficacy than LPA, enhanced intestinal crypt survival in C57BL/6 mice but had no effect in LPA2 knockouts. Intraperitonealy administered OTP reduced death caused by LD100/30 radiation by 50%. Our data indicate that OTP is a highly effective antiapoptotic agent that engages similar prosurvival pathways to LPA through the LPA2 receptor subtype. Unique sequence motifs in the LPA2 carboxyl-terminal (CT) enable it to form macromolecular complexes with PDZ and LIM domain proteins, which link it to G protein-independent signaling networks. Using deletion and site-specific mutagenesis, we mapped out C311xxC314 motif of LPA2-CT that is required for interaction with LIM domain proteins thyroid hormone receptor interactive protein 6 (TRIP6) and the proapoptotic molecule Siva-1 in vitro and in vivo. Palmitoylation that occurs on these cysteine residues, however, did not affect the association with TRIP6 or Siva-1. The L351A mutation in the PDZ motif weakened but did not abolish interaction with LIM proteins. Alanine mutation of the LIM binding motif or PDZ domain binding motif attenuated LPA-induced activation of the prosurvival ERK1/2 and Akt pathways in mouse embryonic fibroblasts (MEF) derived from LPA1 and LPA2 double knockout mice and reconstituted with mutants of LPA2. Neither of these mutations alone nor in combination had a detectable effect on G-protein-linked activation of Ca2+ mobilization. Triple alanine mutations modifying residues Cys311, Cys314, and Leu351 abolished the antiapoptotic effect of LPA. Together, these findings suggest the macromolecular complex formed between LPA2, TRIP6/Siva-1, and PDZ domain proteins plays an important role in mediating the anti-apoptotic effects of LPA2

Comparative analysis and integrative classification of NCI60 cell lines and primary tumors using gene expression profiling data

BACKGROUND: NCI60 cell lines are derived from cancers of 9 tissue origins and have been invaluable in vitro models for cancer research and anti-cancer drug screen. Although extensive studies have been carried out to assess the molecular features of NCI60 cell lines related to cancer and their sensitivities to more than 100,000 chemical compounds, it remains unclear if and how well these cell lines represent or model their tumor tissues of origin. Identification and confirmation of correct origins of NCI60 cell lines are critical to their usage as model systems and to translate in vitro studies into clinical potentials. Here we report a direct comparison between NCI60 cell lines and primary tumors by analyzing global gene expression profiles. RESULTS: Comparative analysis suggested that 51 of 59 cell lines we analyzed represent their presumed tumors of origin. Taking advantage of available clinical information of primary tumor samples used to generate gene expression profiling data, we further classified those cell lines with the correct origins into different subtypes of cancer or different stages in cancer development. For example, 6 of 7 non-small cell lung cancer cell lines were classified as lung adenocarcinomas and all of them were classified into late stages in tumor progression. CONCLUSION: Taken together, we developed and applied a novel approach for systematic comparative analysis and integrative classification of NCI60 cell lines and primary tumors. Our results could provide guidance to the selection of appropriate cell lines for cancer research and pharmaceutical compound screenings. Moreover, this gene expression profile based approach can be generally applied to evaluate experimental model systems such as cell lines and animal models for human diseases

A Regional Bolus Tracking and Real-time B1_1 Calibration Method for Hyperpolarized 13^{13}C MRI

Purpose: Acquisition timing and B$_1$ calibration are two key factors that affect the quality and accuracy of hyperpolarized $^{13}$C MRI. The goal of this project was to develop a new approach using regional bolus tracking to trigger Bloch-Siegert B$_1$ mapping and real-time B$_1$ calibration based on regional B$_1$ measurements, followed by dynamic imaging of hyperpolarized $^{13}C$ metabolites in vivo. Methods: The proposed approach was implemented on a system which allows real-time data processing and real-time control on the sequence. Real-time center frequency calibration upon the bolus arrival was also added. The feasibility of applying the proposed framework for in vivo hyperpolarized $^{13}$C imaging was tested on healthy rats, tumor-bearing mice and a healthy volunteer on a clinical 3T scanner following hyperpolarized [1-$^{13}$C]pyruvate injection. Multichannel receive coils were used in the human study. Results: Automatic acquisition timing based on either regional bolus peak or bolus arrival was achieved with the proposed framework. Reduced blurring artifacts in real-time reconstructed images were observed with real-time center frequency calibration. Real-time computed B$_1$ scaling factors agreed with real-time acquired B$_1$ maps. Flip angle correction using B$_1$ maps results in a more consistent quantification of metabolic activity (i.e, pyruvate-to-lactate conversion, k$_{PL}$). Experiment recordings are provided to demonstrate the real-time actions during the experiment. Conclusion: The proposed method was successfully demonstrated on animals and a human volunteer, and is anticipated to improve the efficient use of the hyperpolarized signal as well as the accuracy and robustness of hyperpolarized $^{13}$C imaging

A Metabolite Specific 3D Stack-of-Spiral bSSFP Sequence for Improved Lactate Imaging in Hyperpolarized [1-13^{13}C]Pyruvate Studies on a 3T Clinical Scanner

Purpose: The balanced steady-state free precession sequence has been previously explored to improve the efficient use of non-recoverable hyperpolarized $^{13}$C magnetization, but suffers from poor spectral selectivity and long acquisition time. The purpose of this study was to develop a novel metabolite-specific 3D bSSFP ("MS-3DSSFP") sequence with stack-of-spiral readouts for improved lactate imaging in hyperpolarized [1-$^{13}$C]pyruvate studies on a clinical 3T scanner. Methods: Simulations were performed to evaluate the spectral response of the MS-3DSSFP sequence. Thermal $^{13}$C phantom experiments were performed to validate the MS-3DSSFP sequence. In vivo hyperpolarized [1-$^{13}$C]pyruvate studies were performed to compare the MS-3DSSFP sequence with metabolite specific gradient echo ("MS-GRE") sequences for lactate imaging. Results: Simulations, phantom and in vivo studies demonstrate that the MS-3DSSFP sequence achieved spectrally selective excitation on lactate while minimally perturbing other metabolites. Compared with MS-GRE sequences, the MS-3DSSFP sequence showed approximately a 2.5-fold SNR improvement for lactate imaging in rat kidneys, prostate tumors in a mouse model and human kidneys. Conclusions: Improved lactate imaging using the MS-3DSSFP sequence in hyperpolarized [1-$^{13}$C]pyruvate studies was demonstrated in animals and humans. The MS-3DSSFP sequence could be applied for other clinical applications such as in the brain or adapted for imaging other metabolites such as pyruvate and bicarbonate

Vascular adhesion protein-1 defines a unique subpopulation of human hematopoietic stem cells and regulates their proliferation

Although the development of hematopoietic stem cells (HSC) has been studied in great detail, their heterogeneity and relationships to different cell lineages remain incompletely understood. Moreover, the role of Vascular Adhesion Protein-1 in bone marrow hematopoiesis has remained unknown. Here we show that VAP-1, an adhesin and a primary amine oxidase producing hydrogen peroxide, is expressed on a subset of human HSC and bone marrow vasculature forming a hematogenic niche. Bulk and single-cell RNAseq analyses reveal that VAP-1+ HSC represent a transcriptionally unique small subset of differentiated and proliferating HSC, while VAP-1− HSC are the most primitive HSC. VAP-1 generated hydrogen peroxide acts via the p53 signaling pathway to regulate HSC proliferation. HSC expansion and differentiation into colony-forming units are enhanced by inhibition of VAP-1. Contribution of VAP-1 to HSC proliferation was confirmed with mice deficient of VAP-1, mice expressing mutated VAP-1 and using an enzyme inhibitor. In conclusion, VAP-1 expression allows the characterization and prospective isolation of a new subset of human HSC. Since VAP-1 serves as a check point-like inhibitor in HSC differentiation, the use of VAP-1 inhibitors enables the expansion of HSC.Peer reviewe

Continued Treatment With Tirzepatide for Maintenance of Weight Reduction in Adults With Obesity: The SURMOUNT-4 Randomized Clinical Trial.

IMPORTANCE: The effect of continued treatment with tirzepatide on maintaining initial weight reduction is unknown. OBJECTIVE: To assess the effect of tirzepatide, with diet and physical activity, on the maintenance of weight reduction. DESIGN, SETTING, AND PARTICIPANTS: This phase 3, randomized withdrawal clinical trial conducted at 70 sites in 4 countries with a 36-week, open-label tirzepatide lead-in period followed by a 52-week, double-blind, placebo-controlled period included adults with a body mass index greater than or equal to 30 or greater than or equal to 27 and a weight-related complication, excluding diabetes. INTERVENTIONS: Participants (n = 783) enrolled in an open-label lead-in period received once-weekly subcutaneous maximum tolerated dose (10 or 15 mg) of tirzepatide for 36 weeks. At week 36, a total of 670 participants were randomized (1:1) to continue receiving tirzepatide (n = 335) or switch to placebo (n = 335) for 52 weeks. MAIN OUTCOMES AND MEASURES: The primary end point was the mean percent change in weight from week 36 (randomization) to week 88. Key secondary end points included the proportion of participants at week 88 who maintained at least 80% of the weight loss during the lead-in period. RESULTS: Participants (n = 670; mean age, 48 years; 473 [71%] women; mean weight, 107.3 kg) who completed the 36-week lead-in period experienced a mean weight reduction of 20.9%. The mean percent weight change from week 36 to week 88 was -5.5% with tirzepatide vs 14.0% with placebo (difference, -19.4% [95% CI, -21.2% to -17.7%]; P \u3c .001). Overall, 300 participants (89.5%) receiving tirzepatide at 88 weeks maintained at least 80% of the weight loss during the lead-in period compared with 16.6% receiving placebo (P \u3c .001). The overall mean weight reduction from week 0 to 88 was 25.3% for tirzepatide and 9.9% for placebo. The most common adverse events were mostly mild to moderate gastrointestinal events, which occurred more commonly with tirzepatide vs placebo. CONCLUSIONS AND RELEVANCE: In participants with obesity or overweight, withdrawing tirzepatide led to substantial regain of lost weight, whereas continued treatment maintained and augmented initial weight reduction

Evaluation of Petrifilm™ Select E. coli Count Plate medium to discriminate antimicrobial resistant Escherichia coli

<p>Abstract</p> <p>Background</p> <p>Screening and enumeration of antimicrobial resistant <it>Escherichia coli </it>directly from samples is needed to identify emerging resistant clones and obtain quantitative data for risk assessment. Aim of this study was to evaluate the performance of 3M™ Petrifilm™ Select <it>E. coli </it>Count Plate (SEC plate) supplemented with antimicrobials to discriminate antimicrobial-resistant and non-resistant <it>E. coli</it>.</p> <p>Method</p> <p>A range of <it>E. coli </it>isolates were tested by agar dilution method comparing the Minimal Inhibitory Concentration (MIC) for eight antimicrobials obtained by Mueller-Hinton II agar, MacConkey agar and SEC plates. Kappa statistics was used to assess the levels of agreement when classifying strains as resistant, intermediate or susceptible.</p> <p>Results</p> <p>SEC plate showed that 74% of all strains agreed within ± 1 log₂dilution when comparing MICs with Mueller-Hinton II media. High agreement levels were found for gentamicin, ampicillin, chloramphenicol and cefotaxime, resulting in a kappa value of 0.9 and 100% agreement within ± 1 log₂dilution. Significant variances were observed for oxytetracycline and sulphamethoxazole. Further tests showed that the observed discrepancy in classification of susceptibility to oxytetracycline by the two media could be overcome when a plate-dependent breakpoint of 64 mg/L was used for SEC plates. For sulphamethoxazole, SEC plates provided unacceptably high MICs.</p> <p>Conclusion</p> <p>SEC plates showed good agreement with Mueller-Hinton II agar in MIC studies and can be used to screen and discriminate resistant <it>E. coli </it>for ampicillin, cephalothin, streptomycin, chloramphenicol, cefotaxime and gentamicin using CLSI standardized breakpoints, but not for sulphamethoxazole. SEC plates can also be used to discriminate oxytetracycline-resistant <it>E. coli </it>if a plate-dependent breakpoint value of 64 mg/L is used.</p