The activity of human TREX2-catalyzed 3 3 5-deoxyribonuclease has been analyzed in steady-state and single turnover kinetic assays and in equilibrium DNA binding studies. These kinetic data provide evidence for cooperative DNA binding within TREX2 and for coordinated catalysis between the TREX2 active sites supporting a model for communication between the protomers of a TREX2 dimer. Mobile loops positioned adjacent to the active sites provide the major DNA binding contribution and facilitate subsequent binding into the active sites. Mutations of three arginine residues on these loops cause decreased TREX2 activities by up to 60-fold. Steady-state kinetic assays of these arginine to alanine TREX2 variants result in increased K m values for DNA substrate with no effect on k cat values indicating contributions exclusively to DNA binding by all three of the loop arginines. TREX2 heterodimers were prepared to determine whether exonuclease activity in one protomer is communicated to the opposing protomer. Evidence for communication across the dimer interface is provided by the 7-fold lower catalytic activity measured in the TREX2 WT/H188A heterodimer compared with the TREX2 WT homodimer, contrasting the 2-fold lower activity measured in the TREX2 WT/R163A,R165A,R167A heterodimer. The measured activity in TREX2WT/H188A heterodimer indicates that defective catalysis in one protomer reduces activity in the opposing protomer. A DNA binding analysis of TREX2 and the heterodimers indicates a cooperative binding effect within the TREX2 protomer. Finally, single turnover kinetic assays identify DNA binding as the rate-limiting step in TREX2 catalysis.The 3Ј 3 5Ј-deoxyribonucleases play fundamental roles in a variety of DNA metabolic pathways. These enzymes process DNA 3Ј ends by removing nucleotides one at a time to remodel 3Ј termini for subsequent molecular events in cells (1, 2). TREX1 is the most active 3Ј-deoxyribonuclease purified from mammalian cells (3, 4). TREX2 has not been purified from cells, but recombinant TREX1 and TREX2 generated in Escherichia coli confirm the robust nature of these deoxyribonucleases with TREX1 exhibiting about a 10-fold greater activity than TREX2(5-7). The high catalytic efficiencies measured for TREX1 and TREX2 can be attributed in part to the nature of the active sites. The TREX gene sequences and Mg 2ϩ ion dependence suggested that these deoxyribonucleases might be members of the DnaQ-like exonuclease family that utilize a two Mg 2ϩ ion-dependent mechanism for catalysis (8 -12). The DnaQ-like family contains both deoxyribo-and riboexonucleases and has been divided into two subfamilies characterized by the presence of four conserved carboxylate residues and a histidine (DEDD-h) or a tyrosine (DEDD-y) positioned in the active site (13). The TREX enzymes prefer excision of deoxynucleotide polymers over ribonucleotide polymers by about 1000-fold (5) 2 with specificity for unmodified 3Ј termini (14, 15). The four carboxylate residue side chains in the DnaQ-like enzymes coordinate two divalen...
Perivascular epithelioid cell tumors (PEComas) are mesenchymal neoplasms with characteristic epithelioid or spindled cytomorphology that typically grow around blood vessels. These tumors are phenotypically and immunohistochemically distinct, expressing markers of both melanocytic and smooth muscle differentiation. Herein, we describe a case of histopathologically malignant cutaneous PEComa without metastatic spread, with review of the pertinent literature. Telescoping punch biopsy demonstrated an epithelioid neoplasm with marked atypia, hypercellularity, and increased mitotic activity. Immunohistochemical stains for HMB‐45, NK1‐C3, PGP9.5, MiTF, CD10, and CD68 were positive within tumor cells. In addition, there was diffuse expression of caldesmon and focal cytoplasmic staining for smooth muscle actin on the excision specimen. The patient underwent treatment with surgical excision with adjuvant radiation and surveillance computed tomography (CT). The patient remains free of recurrence or metastatic disease after 10 months of follow‐up. To our knowledge, this is only the third reported case of a malignant cutaneous PEComa reported in the literature to date.
Background A previous single-center randomized trial suggested that patients with stable International Normalized Ratio (INR) values could safely receive INR testing as infrequently as every 12 weeks. Objective To test the success of implementation of an extended INR testing interval for stable warfarin patients in a practice-based, multicenter collaborative of anticoagulation clinics. Methods At six anticoagulation clinics, patients were identified as being eligible for extended INR testing on the basis of prior INR value stability and minimal warfarin dose changes between 2014 and 2016. We assessed the frequency with which anticoagulation clinic providers recommended an extended INR testing interval (> 5 weeks) to eligible patients. We also explored safety outcomes for eligible patients, including next INR values, bleeding events, and emergency department visits. Results At least one eligible period for extended INR testing was identified in 890 of 3362 (26.5%) warfarin-treated patients. Overall, the use of extended INR testing in eligible patients increased from 41.8% in the first quarter of 2014 to 69.3% in the fourth quarter of 2016. The number of subsequent out-of-range next INR values were similar between eligible patients who did and did not have an extended INR testing interval (27.3% versus 28.4%, respectively). The numbers of major bleeding events were not different between the two groups, but rates of clinically relevant non-major bleeding (0.02 per 100 patient-years versus 0.09 per 100 patient-years) and emergency department visits (0.07 per 100 patient-years versus 0.19 per 100 patient-years) were lower for eligible patients with extended INR testing intervals than for those with non-extended INR testing intervals. Conclusions Extended INR testing for stable warfarin patients can be successfully and safely implemented in diverse, practice-based anticoagulation clinic settings.
Our review included 32 articles and focused primarily on the programmed death (PD) pathway. We found that endocrine side effects after anti-PD-1/PD-L1 therapy are relatively rare, with hypothyroidism (range < 1 to 40%) and hypophysitis (range < 1 to 10%) being the two most common. None of the studies specifically commented on the infertility or sexual side effects of these drugs. However, two studies evaluating biochemical profiles of patients undergoing therapy with ipilimumab (a CTLA-4 inhibitor) or combination therapy (CTLA-4 + PD-1/PD-L1 inhibitors) noted that about < 1 to ~ 60% of the patients developed hypogonadotropic hypogonadism. None of the studies provided information regarding clinically meaningful sexual health endpoints such as libido, erectile function assessments, or sexual function-related quality of life. Endocrine side effects, although uncommon, are important and unique side effects of immune checkpoint therapy because they are often complex and can be life threatening. While side effects on sexual health may not be life threatening, they are lifestyle limiting. Thus, long-term follow-up, post-marketing surveillance, and future studies will need to elucidate the true rates of endocrine/sexual side effects and the mechanisms underlying them. This will aid in better counseling of the patients, as more of them undergo these novel immune checkpoint inhibitor therapies.
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