Melissa S. Gildenberg

Patients compliant with prescribed amphetamine (AMPH) should not have detectable methamphetamine (METH) in their urine; detectable METH typically indicates illicit use. However, we have identified patients with results suggestive of METH as an impurity in prescribed AMPH. Derive a METH:AMPH ratio cut-off from a training set of patients compliant with AMPH prescriptions to differentiate METH as an impurity from illicit use. Retrospective review of AMPH and METH-positive cases by liquid chromatography-tandem mass spectrometry (LC-MS/MS) at Brigham and Women's Hospital (BWH) and Luxor Scientific. Correlated results with clinical and medication history and compliance with prescribed medications. The median ± interquartile range (IQR) METH:AMPH ratio for the Adderall training sets was 0.43 ± 0.31 % and 0.05 ± 0.040 %, with a maximum ratio of 1.125 % and 0.125 % at BWH and Luxor, respectively. The median ± IQR METH:AMPH ratio for the Luxor d-AMPH training set was 0.039 ± 0.028 %, with a maximum ratio of 0.09 %; not statistically different from the Adderall training set. Assessment of the BWH test set where METH < AMPH (n = 22) revealed that METH was likely due to an impurity (n = 10), distant METH mis/use (n = 11), or requiring further analysis (n = 1). METH was also detected by LC-MS/MS in a commercial AMPH calibrator and in Adderall XR. METH may represent an impurity in the AMPH formulation. Laboratories are encouraged to define a METH:AMPH ratio below which an impurity is the likely explanation for METH and/or to increase the METH positivity cut-off to 50 or 100 ng/mL to reduce potential false-accusations of illicit METH use.

Translesion synthesis polymerases efficiently incorporate nucleotides opposite DNA lesions. Pol ι, for example, bypasses minor-groove and exocyclic purine adducts. Conventional X-ray crystallography showed that this enzyme incorporates nucleotides by forming Hoogsteen base pairs with the incoming nucleotide rather than Watson-Crick base pairs. While this revealed the structural basis of nucleotide selection during nucleotide binding, it did not allow the visualization of the process of phosphodiester bond formation or the detection of reaction intermediates that form during nucleotide incorporation. Here, we use a combination of time-lapse crystallography and molecular dynamics simulations to examine the mechanism of pol ι-catalyzed nucleotide incorporation. We show that this enzyme maintains Hoogsteen base pairing with the incoming dNTP during the entire reaction. We also show that pol ι possesses a pyrophosphatase activity that generates two monophosphates within its active site. Our findings provide insights into the features of pol ι’s active site that allow it to translocate along DNA and catalyze processive DNA synthesis.

PURPOSE: RET rearrangements and RET activating point mutations represent targetable genomic alterations in advanced solid tumors. However, the frequency and clinicopathologic characteristics of wild-type RET amplification in cancer and its potential role as a targetable oncogenic driver are not well-characterized. METHODS: In two institutional cohorts of patients with solid cancers from the Dana-Farber Cancer Institute (DFCI) and Memorial Sloan Kettering Cancer Center (MSKCC) whose tumors underwent next-generation sequencing (NGS), the frequency and clinicopathologic features of wild-type RET amplification in the absence of RET rearrangements or activating mutations was assessed. The findings were validated using merged data from The Cancer Genome Atlas (TCGA), Genomics Evidence Neoplasia Information Exchange (GENIE), and China Pan-Cancer data sets. RESULTS: The frequency of wild-type RET amplification across all solid cancers was 0.08% (26 of 32,505) in the DFCI cohort, 0.05% (26 of 53,152) in the MSKCC cohort, and 0.25% (71 of 28,623) in the cohort from TCGA, GENIE, and China Pan-Cancer. Cancer types with RET amplification included non–small-cell lung cancer (NSCLC), hepatobiliary cancer, prostate cancer, breast cancer, and others. The median RET copy number in RET-amplified cases was 7.5 (range, 6-36) in the DFCI cohort and 5.7 (range, 4-27.7) in the MSKCC cohort. Among 11 RET-amplified NSCLCs, eight had no other concurrent driver mutations. Finally, we report on a 69-year-old man with recurrent NSCLC harboring high-level wild-type RET amplification (22-28 copies) as the only identified putative genomic driver who experienced both a systemic and intracranial confirmed response to the RET inhibitor selpercatinib. CONCLUSION: Amplification of wild-type RET represents a novel, targetable molecular subset of cancer.

DNA damage leads to genome instability by interfering with DNA replication. Cells possess several damage bypass pathways that mitigate the effects of DNA damage during replication. These pathways include translesion synthesis and template switching. These pathways are regulated largely through post-translational modifications of proliferating cell nuclear antigen (PCNA), an essential replication accessory factor. Mono-ubiquitylation of PCNA promotes translesion synthesis, and K63-linked poly-ubiquitylation promotes template switching. This article will discuss the mechanisms of how these post-translational modifications of PCNA control these bypass pathways from a structural and biochemical perspective. We will focus on the structure and function of the E3 ubiquitin ligases Rad18 and Rad5 that facilitate the mono-ubiquitylation and poly-ubiquitylation of PCNA, respectively. We conclude by reviewing alternative ideas about how these post-translational modifications of PCNA regulate the assembly of the multi-protein complexes that promote damage bypass pathways.

Several pathways exist to bypass DNA damage during replication. One such pathway is template switching. The Rad5 protein plays two important roles in template switching: it is an E3 ubiquitin ligase that catalyzes PCNA poly-ubiquitylation and it is a helicase that converts replication forks to chicken foot structures. To understand the structure, conformational flexibility, and mechanism of Rad5, we used a full-ensemble hybrid method combining Langevin dynamics simulations and small-angle X-ray scattering. From these studies, we generated the first experimentally validated, high-resolution structural model of Rad5. We found that Rad5 is more compact and less extended than is suggested by its large amount of predicted intrinsic disorder. Thus, Rad5 likely has a novel intra-molecular interaction that limits the range of conformational space it can sample. We provide evidence for a novel interaction between the HIRAN and the helicase domains of Rad5, and we discuss the biological and mechanistic implications of this.

Proteins and protein complexes with high conformational flexibility participate in a wide range of biological processes. These processes include genome maintenance, gene expression, signal transduction, cell cycle regulation, and many others. Gaining a structural understanding of conformationally flexible proteins and protein complexes is arguably the greatest problem facing structural biologists today. Over the last decade, some progress has been made toward understanding the conformational flexibility of such systems using hybrid approaches. One particularly fruitful strategy has been the combination of small-angle X-ray scattering (SAXS) and molecular simulations. In this article, we provide a brief overview of SAXS and molecular simulations and then discuss two general approaches for combining SAXS data and molecular simulations: minimal ensemble approaches and full ensemble approaches. In minimal ensemble approaches, one selects a minimal ensemble of structures from the simulations that best fit the SAXS data. In full ensemble approaches, one validates a full ensemble of structures from the simulations using SAXS data. We argue that full ensemble models are more realistic than minimal ensemble searches models and that full ensemble approaches should be used wherever possible.

Though the importance of high-resolution structure and dynamics of membrane proteins has been well recognized, optimizing sample conditions to retain the native-like folding and function of membrane proteins for Nuclear Magnetic Resonance (NMR) or X-ray measurements has been a major challenge. While bicelles have been shown to stabilize the function of membrane proteins and are increasingly utilized as model membranes, the loss of their magnetic-alignment at low temperatures makes them unsuitable to study heat-sensitive membrane proteins like cytochrome-P450 and protein-protein complexes. In this study, we report temperature resistant bicelles that can magnetically-align for a broad range of temperatures and demonstrate their advantages in the structural studies of full-length microsomal cytochrome-P450 and cytochrome-b5 by solid-state NMR spectroscopy. Our results reveal that the N-terminal region of rabbit cytochromeP4502B4, that is usually cleaved off to obtain crystal structures, is helical and has a transmembrane orientation with ~17° tilt from the lipid bilayer normal.