Extensive protein pyrophosphorylation revealed in human cell lines.

Reversible protein phosphorylation is a central signaling mechanism in eukaryotes. Although mass-spectrometry-based phosphoproteomics has become routine, identification of non-canonical phosphorylation has remained a challenge. Here we report a tailored workflow to detect and reliably assign protein pyrophosphorylation in two human cell lines, providing, to our knowledge, the first direct evidence of endogenous protein pyrophosphorylation. We manually validated 148 pyrophosphosites across 71 human proteins, the most heavily pyrophosphorylated of which were the nucleolar proteins NOLC1 and TCOF1. Detection was consistent with previous biochemical evidence relating the installation of the modification to inositol pyrophosphates (PP-InsPs). When the biosynthesis of PP-InsPs was perturbed, proteins expressed in this background exhibited no signs of pyrophosphorylation. Disruption of PP-InsP biosynthesis also significantly reduced rDNA transcription, potentially by lowering pyrophosphorylation on regulatory proteins NOLC1, TCOF1 and UBF1. Overall, protein pyrophosphorylation emerges as an archetype of non-canonical phosphorylation and should be considered in future phosphoproteomic analyses.

Cannabicitran: Its unexpected racemic nature and potential origins.

Cannabicitran is a cannabinoid found in levels up to ~10% in commercial "purified" cannabidiol (CBD) extracts. The structure of this natural product was first reported more than 50 years ago. However, few studies have investigated cannabicitran or its origin despite the rapidly increasing interest in the use of cannabinoids for the treatment of a wide range of physiological conditions. Following on a recent detailed NMR and computational characterization of cannabicitran, our group initiated ECD and TDDFT studies aimed at unequivocally determining the absolute configuration of cannabicitran present in Cannabis sativa extracts. To our surprise, we discovered the natural product was racemic, raising questions around its presumed enzymatic origin. Herein, we report the isolation and absolute configuration of (-)-cannabicitran and (+)-cannabicitran. Several possible scenarios for production of the racemate in the plant and/or during extract processing are discussed.

Calculated and experimental 1 H and 13 C NMR assignments for cannabicitran.

Cannabicitran is an important cannabinoid natural product produced by Cannabis sativa and is often found at surprisingly high levels (up to ~10%) in "purified" commercial cannabidiol (CBD) extract preparations. Despite the prevalence of this molecule in CBD oil and other cannabinoid-related products, and the rapidly expanding interest in cannabinoids for treatment of a wide range of physiological conditions, only unassigned 1 H NMR data and partial unambiguous 13 C assignments have been published. Herein, we report the complete 1 H and 13 C NMR assignments of cannabicitran and comparatively evaluate the performance of several density functional theory (DFT) methods with varying levels of theory for the calculation of NMR chemical shifts.

Palladium-Catalyzed Construction of Quaternary Stereocenters by Enantioselective Arylation of γ-Lactams with Aryl Chlorides and Bromides.

Herein, we report the first Pd-catalyzed enantioselective arylation of α-substituted γ-lactams. Two sets of conditions were developed for this transformation, allowing for the use of either aryl chlorides or bromides as electrophiles. Utilizing a highly electron-rich dialkylphosphine ligand we have been able to construct α-quaternary centers in good yields (up to 91 % yield) and high enantioselectivities (up to 97 % ee).

Quinine-Promoted, Enantioselective Boron-Tethered Diels-Alder Reaction by Anomeric Control of Transition-State Conformation.

Diels-Alder reactions of tethered vinyl-metal species offer the opportunity to fashion highly functionalized diol intermediates for synthesis. We have developed the first enantioselective boron-tethered Diels-Alder reaction using quinine as a chiral promoter. Quinine recovery, enantioselectivity enhancement, and manipulation of the cyclohexene core are also investigated. DFT modeling calculations confirm the role of quinine as a bidentate ligand enhancing reaction rates. The enantioselectivity of the cycloaddition is proposed to originate from a boron-centered anomeric effect.

Detection of Biosynthetic Precursors, Discovery of Glycosylated Forms, and Homeostasis of Calcitonin in Human Cancer Cells.

The peptide hormone calcitonin is intimately connected with human cancer development and proliferation. Its biosynthesis is reasoned to proceed via glycine-, α-hydroxyglycine-, glycyllysine-, and glycyllysyllysine-extended precursors; however, as a result of the limitations of current analytical methods, until now, there has been no procedure capable of detecting these individual species in cell or tissue samples. Therefore, their presence and dynamics in cancer had not been established. Here, we report the first methodology for the separation, detection, and quantification of calcitonin and each of its precursors in human cancer cells. We also report the discovery and characterization of O-glycosylated calcitonin and its analogous biosynthetic precursors. Through direct and simultaneous analysis of the glycosylated and nonglycosylated species, we interrogate the hormone biosynthesis. This shows that the cellular calcitonin level is maintained to mitigate effects of biosynthetic enzyme inhibitors that substantially change the proportions of calcitonin-related species released into the culture medium.

Cornelia de Lange syndrome and molecular implications of the cohesin complex: Abstracts from the 7th biennial scientific and educational symposium 2016.

Cornelia de Lange Syndrome (CdLS) is due to mutations in the genes for the structural and regulatory proteins that make up the cohesin complex, and is considered a cohesinopathy disorder or, more recently, a transcriptomopathy. New phenotypes have been recognized in this expanding field. There are multiple clinical issues facing individuals with all forms of CdLS, particularly in the neurodevelopmental system, but also gastrointestinal, cardiac, and musculoskeletal. Aspects of developmental and cell biology have found common endpoints in the biology of the cohesin complex, with improved understanding of the mechanisms, easier diagnostic tests, and the possibility of potential therapeutics, all major clinical implications for the individual with CdLS. The following abstracts are the presentations from the 7th Cornelia de Lange Syndrome Scientific and Educational Symposium, June 22-23, 2016, in Orlando, FL, in conjunction with the Cornelia de Lange Syndrome Foundation National Meeting. In addition to the scientific and clinical discussions, there were talks related to practical aspects of behavior including autism, transitions, communication, access to medical care, and databases. At the end of the symposium, a panel was held, which included several parents, affected individuals and genetic counselors, and discussed the greatest challenges in life and how this information can assist in guiding future research. The Research Committee of the CdLS Foundation organizes this meeting, reviews, and accepts abstracts, and subsequently disseminates the information to the families through members of the Clinical Advisory Board and publications. AMA CME credits were provided by Greater Baltimore Medical Center, Baltimore, MD.

Synthesis of ß-substituted tryptamines by regioselective ring opening of aziridines.

Functionalized tryptamines are targets of interest for development as small molecule therapeutics. The ring opening of aziridines with indoles is a powerful method for tryptamine synthesis if site selectivity can be controlled. 4-Nitrobenzyl carbamate (PNZ)-protected aziridines undergo regioselective ring opening to produce ß-substituted tryptamines for a series of indoles. The PNZ-protected tryptamines can be further manipulated by PNZ removal under mild conditions.

The Asfora Bullet Cage System Shows Comparable Fusion Rate Success Versus Control Cage in Posterior Lumbar Interbody Fusion in a Randomized Clinical Trial.

BACKGROUND: Low back pain and degeneration of the intervertebral disc are an integrated malady that affects millions of Americans. Cage devices used in association with posterior lumbar interbody fusion (PLIF) have been shown to be an effective approach in the treatment of a number of lower spine disorders attributed to degenerative disc disease (DDD). OBJECTIVE: This study was undertaken as part of a U.S. Food and Drug Administration (FDA) Investigational Device Exemption (IDE) study and compares the effectiveness of the Asfora Bullet Cage System (ABCS) to successfully fuse vertebra at one or two levels between L2 and S1 in patients with DDD to an FDA approved comparison device, the Medtronic-Sofamor Danek Inter Fix Threaded Fusion Device (MSDIFD). METHODS: A total of 257 randomized participants were implanted with either the ABCS device (n = 132) or the MSDIFD device (n = 125) through an open posterior approach using autogenous local bone graft without the use of pedicle screws. Patients were evaluated prior to surgery and at the 24 month (24-M) visit for fusion status, deep tendon reflex status, sensory function, motor function, straight leg raise status, pain, disability, and device safety. Radiological evaluation and statistical analysis were performed by independent professionals. RESULTS: Evaluation of device success was performed at 24-M visit. From the original group of 257 patients, 59 were lost to follow-up. Primary measures of success at the 24-M visit involved pain and function, fusion, neurological status, and device-related adverse events measures. Pain and function improved in both (MSDIFD: 75.7 percent; ABCS: 82.6 percent). Fusion success with all radiographic points at 24-M visits was 79.4 percent MSDIFD and 88.2 percent ABCS. Neurological improvement was seen in both (MSDIFD: 77.0 percent; ABCS: 87.8 percent). One device-related grade 1 adverse event was reported in the MSDIFD group. Disc height preservation was equivalent for single level fusions (MSDIFD: 16.1 percent; ABCS: 20.0 percent) and second level fusions (MSDIFD: 10.7 percent; ABCS: 14.3 percent). General health and well-being improvement was the same (MSDIFD: 37.0 percent; ABCS: 40.0 percent). Subsequent fusion, up to 10 years, was equivalent (MSDIFD: 83.8 percent; ABCS: 91.2). Results for both devices were considered to be satisfactory, with a slight non-significant superiority for the ABCS. CONCLUSION: From the ABCS device FDA IDE sanctioned study and the review of the literature, we concluded that the Asfora Bullet Cage System is safe, effective and comparable to other interbody fusion devices which are used stand-alone or in conjunction with pedicle screws, rhBMP-2, or autogenous bone harvested from the iliac crest inserted through anterior, lateral or posterior approaches.

Scalable synthesis of N-acylaziridines from N-tosylaziridines.

N-Acylaziridines are important starting materials for the synthesis of chiral amine derivatives. The traditional methods for producing these activated aziridines have significant drawbacks. The gram scale synthesis of N-acylaziridines by deprotection of N-tosylaziridines and reprotection with N-hydroxysuccinimide derivatives is described. Mono- and disubstituted aziridines perform well, with complete retention of stereochemical purity. The consistently moderate yields are linked to the N-tosylaziridine deprotection step, while acylation with N-hydroxysuccinimide derivatives is highly efficient.

Successful fusion of remote type II odontoid fracture using anterior screw fixation of the odontoid and rhBMP-2: report of two cases.

Anterior screw fixation of the odontoid is contraindicated in remote type II fractures. The alternative surgical treatment consists of a posterior C1 to C2 or an occiput to C3 fusion, which is met with much resistance by patients as this option limits head motion, especially rotational movement. Furthermore, elderly patients may not be medically fit to undergo surgery of this magnitude. This report presents two remote type II odontoid fractures in elderly patients (67 and 73 years of age) who were successfully treated by means of anterior screw fixation of the odontoid along with an injection of recombinant human bone morphogenic protein (rhBMP-2) (Medtronic Inc.) into the fracture line with infiltration of the fibrous union tissue and adjacent anterior longitudinal ligament. To our knowledge, this is the first documented report of solid fusion of remote type II odontoid fracture treated with rhBMP-2 and anterior screw fixation. The authors believe that this technique may be a viable alternative for the treatment of failed odontoid fractures older than six months.

Extreme multi-level anterior cervical discectomy and fusion.

The use of anterior cervical discectomy and fusion (ACDF) is a common procedure used to treat those who suffer from degenerative disc disease (DDD) of the cervical spine which may result in spondylolisthesis, spinal stenosis, disc herniation, nerve root and/or cord compression. ACDF is regularly used for the fusion of one to four cervical spine levels; however, the literature documenting the fusion of 5 or 6 levels is surprisingly lacking. In this retrospective review of our case series, we document two 5-level and two 6-level ACDF in elderly patients using custom made titanium fusion plates and patellar allograft bone for interbody placement without posterior fixation. The documentation of these long anterior constructs without supplemental posterior fixation or the use of a halo vest apparatus, but a simple neck collar for three months, is an important contribution to the literature illustrating that with utilization of appropriate technique, a high number of levels can be safely fused in the elderly patients necessitating this particular procedure.

Investigating the activity of quinine analogues versus chloroquine resistant Plasmodium falciparum.

Plasmodium falciparum, the deadliest malarial parasite species, has developed resistance against nearly all man-made antimalarial drugs within the past century. However, quinine (QN), the first antimalarial drug, remains efficacious worldwide. Some chloroquine resistant (CQR) P. falciparum strains or isolates show mild cross resistance to QN, but many do not. Further optimization of QN may provide a well-tolerated therapy with improved activity versus CQR malaria. Thus, using the Heck reaction, we have pursued a structure-activity relationship study, including vinyl group modifications of QN. Certain derivatives show good antiplasmodial activity in QN-resistant and QN-sensitive strains, with lower IC(50) values relative to QN.

Enantioselective synthesis and stereoselective ring opening of N-acylaziridines.

Kinetic resolution of N-acylaziridines by nucleophilic ring opening was achieved with (R)-BINOL as the chiral modifier under boron-catalyzed conditions (see scheme; Ar=3,5-dinitrophenyl). The consumed enantiomer of aziridine can be further converted to an enantioenriched 1,2-chloroamide with recovery of (R)-BINOL.

Palladium-Catalyzed, Enantioselective Desymmetrization of N-Acylaziridines with Indoles.

Ring opening reactions of meso-aziridines generate chiral amine derivatives where the control of stereochemistry is possible through enantioselective catalysis. We report the use of a diphosphine-palladium(II) catalyst for the highly enantioselective desymmetrization of N-acylaziridines with indoles. The ß-tryptamine products are isolated in moderate to high yield across a range of indole and aziridine substitution patterns. The synthetic utility of ß-tryptamine products is demonstrated by conversion to the brominated pyrroloindoline derivative.

Structural revision of a Wnt/ß-catenin modulator and confirmation of cannabielsoin constitution and configuration.

In this report, we revise the structure for a previously reported synthetic product proposed to be the 1R,2S-cannabidiol epoxide and reassign it as cannabielsoin using anisotropic NMR and synthetic chemistry methods. These results provide a direct link to the first known biological target and function of cannabielsoin.

Treatment of colloid cyst of the third ventricle by stereotactic aspiration followed by radiosurgery: Report of four cases.

BACKGROUND: Colloid cysts are rare benign lesions with potentially devastating results. Complications intra and posttreatment can result in high rates of recurrence. Stereotactic radiosurgery may present an attractive option for decreasing the rate of recurrence in conjunction with stereotactic aspiration. CASE DESCRIPTION: Here, we report four cases of colloid cyst of the third ventricle managed by stereotactic aspiration with the use of the Leksell frame followed by stereotactic radiosurgery with the BrainLab/Novalis linear accelerator. Follow-up ranged from 7 to 10 years. There were no complications related to the treatment and no recurrence of the cysts, to date. All four patients remain asymptomatic. CONCLUSIONS: The combination of stereotactic aspiration and radiosurgery may have a role in the management of colloid cysts of the third ventricle.

Pyrophosphorylation via selective phosphoprotein derivatization.

An important step in elucidating the function of protein post-translational modifications (PTMs) is gaining access to site-specifically modified, homogeneous samples for biochemical characterization. Protein pyrophosphorylation is a poorly characterized PTM, and here a chemical approach to obtain pyrophosphoproteins is reported. Photo-labile phosphorimidazolide reagents were developed for selective pyrophosphorylation, affinity-capture, and release of pyrophosphoproteins. Kinetic analysis of the reaction revealed rate constants between 9.2 × 10-3 to 0.58 M-1 s-1, as well as a striking proclivity of the phosphorimidazolides to preferentially react with phosphate monoesters over other nucleophilic side chains. Besides enabling the characterization of pyrophosphorylation on protein function, this work highlights the utility of phosphoryl groups as handles for selective protein modification for a variety of applications, such as phosphoprotein bioconjugation and enrichment.

Enantiospecific Synthesis of ß-Substituted Tryptamines.

Functionalized tryptamines are targets of interest for development as small molecule therapeutics. The ring opening of aziridines with indoles is a powerful method for tryptamine synthesis where isomer formation can be controlled. 3,5-Dinitrobenzoyl (DNB)-protected aziridines undergo regioselective, enantiospecific ring opening to produce ß-substituted tryptamines for a series of indoles. Attack at the more substituted aziridine carbon occurs in an SN2-like fashion to generate DNB-tryptamine products as synthetic precursors.

Palladium-Catalyzed, Enantioselective Heine Reaction.

Aziridines are important synthetic intermediates for the generation of nitrogen-containing molecules. N-Acylaziridines undergo rearrangement by ring expansion to produce oxazolines, a process known as the Heine reaction. The first catalytic, enantioselective Heine reaction is reported for meso-N-acylaziridines where a palladium(II)-diphosphine complex is employed. The highly enantioenriched oxazoline products are valuable organic synthons and potential ligands for transition-metal catalysis.