Glycosylphosphatidylinositol (GPI) anchored protein deficiency serves as a reliable reporter of Pig‐a gene Mutation: Support from an in vitro assay based on L5178Y/Tk+/− cells and the CD90.2 antigen

Lack of cell surface glycosylphosphatidylinositol (GPI)‐anchored protein(s) has been used as a reporter of Pig‐a gene mutation in several model systems. As an extension of this work, our laboratory initiated development of an in vitro mutation assay based on the flow cytometric assessment of CD90.2 expression on the cell surface of the mouse lymphoma cell line L5178Y/Tk^+/?. Cells were exposed to mutagenic and nonmutagenic compounds for 24 hr followed by washout and incubation for an additional 7 days. Following this mutant manifestation time, cells were labeled with fluorescent antibodies against CD90.2 and CD45 antigens. These reagents indicated the presence of GPI‐anchored proteins and general cell surface membrane receptor integrity, respectively. Instrument set‐up was aided by parallel processing of a GPI anchor‐deficient subclone. Results show that the mutagens reproducibly caused increased frequencies of mutant phenotype cells, while the nonmutagens did not. Further modifications to the method, including application of a viability dye and an isotype control for instrument set‐up, were investigated. As a means to verify that the GPI‐anchored protein‐negative phenotype reflects bona fide Pig‐a gene mutation, sequencing was performed on 38 CD90.2‐negative L5178Y/Tk^+/? clones derived from cultures treated with ethyl methanesulfonate. All clones were found to have mutation(s) within the Pig‐a gene. The continued investigation of L5178Y/Tk^+/? cells, CD90.2 labeling, and flow cytometric analysis as the basis of an in vitro mutation assay is clearly supported by this work. These data also provide evidence of the reliability of using GPI anchor‐deficiency as a valid reporter of Pig‐a gene mutation. Environ. Mol. Mutagen. 59:18–29, 2018. © 2017 Wiley Periodicals, Inc.     

The Maladaptive Pursuit of Consumption: the Impact of Materialism,Pain of Paying,Social Anxiety,Social Support,and Loneliness on Compulsive Buying

The current research examined the capability of materialism, pain of paying, social anxiety, social support, and loneliness to predict compulsive buying. A sample of students attending a public university located in the northeast USA were surveyed. A multiple regression indicated materialism, pain of paying, anhedonia, coping with substances, and social support received from family were predictors of compulsive buying. Pain of paying was the strongest predictor of compulsive buying. Understanding factors that affect compulsive buying aids the identification of compulsive buying and informs the treatment of compulsive buying. Treatment models may be more effective if additional attention is given to addressing and developing social support networks of compulsive buyers. Such social support may act as a buffer against the social anxiety compulsive buyers experience and may help reduce feelings of anhedonia and use of substances to control social anxiety.

     

Laparoscopic robotic pyeloplasty using the Zeus Telesurgical System

We present the initial clinical experience using a robot to perform a laparoscopic dismembered pyeloplasty at a Canadian centre. Five patients were confirmed to have ureteropelvic junction obstructions through nuclear renography, cross sectional imaging and intravenous pyelography. After performing a retrograde ureteropyelography and double J stent placement, laparoscopic dismembered pyeloplasty was performed by a single surgeon at a remote workstation using the ZeusTM Telepresence Surgery System (Intuitive Surgicala). The mean total operative time was 225+/-48 minutes, anastomotic time was 71+/-16 minutes, and the mean time required to set-up the robot was 30+/-17 minutes. The estimated blood loss was less than 100 ml in each case. A mean total of 22+/-10 mg of morphine sulfate equivalents were used for analgesia, and the patients were discharged home after a mean of 58+/-10 hrs. There were no robotic failures, and all evaluable patients are free of pain and demonstrable obstruction. One patient developed a delayed urine leak, which resolved with percutaneous drainage. The robot provides the ability to perform complicated operations with precision through elimination of tremor, scaling of motion, and through the use of 'wristed' instruments that enhance the freedom of movement normally limited by straight-shafted laparoscopic needle drivers. The development of robotic telesurgery is still in its infancy, and the significance of its role in urologic surgery continues to be evaluated.     

Preclinical to Clinical Translation of CNS Transporter Occupancy of TD-9855, a Novel Norepinephrine and Serotonin Reuptake Inhibitor

Background:

Monoamine reuptake inhibitors exhibit unique clinical profiles that reflect distinct engagement of the central nervous system (CNS) transporters.

Methods:

We used a translational strategy, including rodent pharmacokinetic/pharmacodynamic modeling and positron emission tomography (PET) imaging in humans, to establish the transporter profile of TD-9855, a novel norepinephrine and serotonin reuptake inhibitor.

Results:

TD-9855 was a potent inhibitor of norepinephrine (NE) and serotonin 5-HT uptake in vitro with an inhibitory selectivity of 4- to 10-fold for NE at human and rat transporters. TD-9855 engaged norepinephrine transporters (NET) and serotonin transporters (SERT) in rat spinal cord, with a plasma EC₅₀ of 11.7ng/mL and 50.8ng/mL, respectively, consistent with modest selectivity for NET in vivo.Accounting for species differences in protein binding, the projected human NET and SERT plasma EC₅₀ values were 5.5ng/mL and 23.9ng/mL, respectively. A single-dose, open-label PET study (4–20mg TD-9855, oral) was conducted in eight healthy males using the radiotracers [¹¹C]-3-amino-4- [2-[(di(methyl)amino)methyl]phenyl]sulfanylbenzonitrile for SERT and [¹¹C]-(S,S)-methylreboxetine for NET. The long pharmacokinetic half-life (30–40h) of TD-9855 allowed for sequential assessment of SERT and NET occupancy in the same subject. The plasma EC₅₀ for NET was estimated to be 1.21ng/mL, and at doses of greater than 4mg the projected steady-state NET occupancy is high (>75%). After a single oral dose of 20mg, SERT occupancy was 25 (±8)% at a plasma level of 6.35ng/mL.

Conclusions:

These data establish the CNS penetration and transporter profile of TD-9855 and inform the selection of potential doses for future clinical evaluation.     

Wnt signaling in rheumatoid synoviocyte activation

Rheumatoid arthritis (RA) is a joint-specific disease with complex pathogenesis. It is characterized by synovial inflammation, cartilage loss, and joint destruction. The reasons why joint damage recurs when therapy is discontinued are not clearly understood. Several lines of evidence suggest that cartilage damage is promoted by the transformed and invasive fibroblast-like synoviocytes (FLS) of the rheumatoid joint. It has been demonstrated in several systems that aberrant wnt-mediated signaling causes blockade of cartilage differentiation and malformation of joints. In this review, we have discussed the importance of wnt–frizzled-mediated signaling in the autonomous activation of FLS in patients with RA. Anti-wnt/anti-frizzled antibodies, frizzled receptor antagonists, or small molecule inhibitors of wnt–frizzled signaling might be useful for therapeutic interventions in RA. Received: May 15, 2001/Accepted: September 28, 2001