Discovery of a Potent Deubiquitinase (DUB) Small-Molecule Activity-Based Probe Enables Broad Spectrum DUB Activity Profiling in Living Cells.

Deubiquitinases (DUBs) are a family of >100 proteases that hydrolyze isopeptide bonds linking ubiquitin to protein substrates, often leading to reduced substrate degradation through the ubiquitin proteasome system. Deregulation of DUB activity has been implicated in many diseases, including cancer, neurodegeneration and auto-inflammation, and several have been recognized as attractive targets for therapeutic intervention. Ubiquitin-derived covalent activity-based probes (ABPs) provide a powerful tool for DUB activity profiling, but their large recognition element impedes cellular permeability and presents an unmet need for small molecule ABPs which can account for regulation of DUB activity in intact cells or organisms. Here, through comprehensive chemoproteomic warhead profiling, we identify cyanopyrrolidine (CNPy) probe IMP-2373 (12) as a small molecule pan-DUB ABP to monitor DUB activity in physiologically relevant live cells. Through proteomics and targeted assays, we demonstrate that IMP-2373 quantitatively engages more than 35 DUBs across a range of non-toxic concentrations in diverse cell lines. We further demonstrate its application to quantification of changes in intracellular DUB activity during pharmacological inhibition and during MYC deregulation in a model of B cell lymphoma. IMP-2373 thus offers a complementary tool to ubiquitin ABPs to monitor dynamic DUB activity in the context of disease-relevant phenotypes.

Body of evidence: integrating Eduard Pernkopf's Atlas into a librarian-led medical humanities seminar.

BACKGROUND: Anatomical subjects depicted in Eduard Pernkopf's richly illustrated Topographische Anatomie des Menschen may be victims of the Nazi regime. Special collections librarians in the history of medicine can use this primary resource to initiate dialogs about ethics with medical students. CASE PRESENTATION: Reported here is the authors' use of Pernkopf's Atlas in an interactive medical humanities seminar designed for third-year medical students. Topical articles, illustrations, and interviews introduced students to Pernkopf, his Atlas, and the surrounding controversies. We aimed to illustrate how this controversial historical publication can successfully foster student discussion and ethical reflection. CONCLUSIONS: Pernkopf's Atlas and our mix of contextual resources facilitated thoughtful discussions about history and ethics amongst the group. Anonymous course evaluations showed student interest in the subject matter, relevance to their studies, and appreciation of our special collection's space and contents.

Neurognostics question. Gordon Morgan Holmes.

Gordon Morgan Holmes, MD, MRCP was an Irish born neurologist who received his medical education at Trinity College, Dublin, Ireland. He was trained in neuroanatomy and neuropathology at the Senckenberg Institute, Frankfort-Am-Main by Ludwig Edinger. He then returned to serve as a Registrar (House Officer) mentored by Richard Gowers and John Hughlings Jackson at the National Hospital, Queen Square, London. He collaborated with Thomas Granger Stewart in describing the loss of recoil in patients with cerebellar hemispheric tumors in 1904. Volunteering in 1914 for frontline hospital duty, he examined soldiers who had injuries to their occipital area causing hypotonia, dysmetria, staggering gait, and falling to the side ipsilateral to their injured cerebellar hemisphere. Holmes discovered that increasing the pace of the finger-nose manuever and applying slight resistance to a moving limb attenuated the dysmetria. Continuing observation of these patients afforded him to describe the evolution of their injuries to include increasing tremor and decreasing hypotonia. Holmes first attached levers to the limbs of hispatients to record their movements on a moving smoked paper kymograph. In 1939 he published photograh tracings made by low mass minature light bulbs attached to ataxic limbs that showed thehpometira and hypometria of their movements ipsilateral to their damaged cerebellar lobes. Holmes made sigficant contributions to understanding of the physiology of the human cerebellum.

Identification of low-level degradants from low dose tablets.

A multifaceted approach was successfully used to identify three of four unknown degradants in degraded low dose tablets. Accelerated solvent extraction (ASE) was found to be an invaluable tool in this multifaceted approach. ASE was capable of extracting four individual degradants of an active pharmaceutical component from 10 tablets into 15 mL of solvent with approximately 100% recovery for each degradant. Using ASE instead of manual extraction led to the extraction and isolation of the degradants in 1 day instead of 7 days. One of the degradants was extracted by ASE, isolated by semi-prep HPLC, and identified by LC-MS and NMR spectroscopy. The structures of two of the remaining three degradants were confirmed by synthesis of authentic samples, while the fourth degradant is yet to be identified.

Pharmaceutical impurity identification: a case study using a multidisciplinary approach.

A multidisciplinary team approach to identify pharmaceutical impurities is presented in this article. It includes a representative example of the methodology. The first step is to analyze the sample by LC-MS. If the structure of the unknown impurity cannot be conclusively determined by LC-MS, LC-NMR is employed. If the sample is unsuitable for LC-NMR, the impurity needs to be isolated for conventional NMR characterization. Although the technique of choice for isolation is preparative HPLC, enrichment is often necessary to improve preparative efficiency. One such technique is solid-phase extraction. For complete verification, synthesis may be necessary to compare spectroscopic characteristics to those observed in the original sample. Although not widely practiced, an effective means of getting valuable structural information is to conduct a degradation study on the purified impurity itself. This systematic strategy was successfully applied to the identification of an impurity in the active pharmaceutical ingredient 1-(1,2,3,5,6,7-hexahydro-s-indacen-4-yl)-3-[4-(1-hydroxy-1-methyl-ethyl)-furan-2-sulphonylurea. Identification required the use of all of the previously mentioned techniques. The instability of the impurity under acidic chromatographic conditions presented an additional challenge to purification and identification. However, we turned this acidic instability to an advantage, conducting a degradation study of the impurity, which provided extensive and useful information about its structure. The following discussion describes how the information gained from each analytical technique was brought together in a complementary fashion to elucidate a final structure.

The history of the development of the cerebellar examination.

The cerebellar examination evolved from observations of experimental lesions made by neurophysiologists and clinical descriptions of patients with trauma to the cerebellum. At the beginning of the 19th century, neurophysiologists such as Luigi Rolando, Marie-Jean-Pierre Flourens, and John Call Dalton, Jr. ablated portions of the cerebellum of a variety of animals and observed staggering gait, clumsiness, and falling from side to side without loss of strength. They concluded that the cerebellum coordinated voluntary movements. In 1899, Joseph Francois Félix Babinski observed that patients with cerebellar lesions could not execute complex movements without breaking down into their elemental movements and described the defect as dysmetria. In 1902, Babinski coined the term dysdiodochokinesis to describe the inability to perform rapid execution of movements requiring alternate contractions of agonist and antagonist muscles. Gordon Holmes in 1904 described the phenomena of rebound, noting that if a limb ipsilateral to a cerebellar lesion is suddenly released from tension, the appendage will flail. In 1917, Gordon Holmes reported hypotonia and dysmetria in men wounded by gunshot wounds to their cerebellum. These observations were rapidly included in descriptions of the cerebellar examination in popular contemporaneous textbooks of neurology. Modern observations have demonstrated that the cerebellum influences such cognitive functions such as planning, verbal fluency, abstract reasoning, prosody, and use of correct grammar.