Characterizing Glycosylation of Adeno-Associated Virus Serotype 9 Capsid Proteins Generated from HEK293 Cells through Glycopeptide Mapping and Released Glycan Analysis.

Recombinant adeno-associated viral (AAV) vectors have emerged as prominent gene delivery vehicles for gene therapy. AAV capsid proteins determine tissue specificity and immunogenicity and play important roles in receptor binding, the escape of the virus from the endosome, and the transport of the viral DNA to the nuclei of target cells. Therefore, the comprehensive characterization of AAV capsid proteins is necessary for a better understanding of the vector assembly, stability, and transduction efficiency of AAV gene therapies. Glycosylation is one of the most common post-translational modifications (PTMs) and may affect the tissue tropism of AAV gene therapy. However, there are few studies on the characterization of the N- and O-glycosylation of AAV capsid proteins. In this study, we identified the N- and O-glycosylation sites and forms of AAV9 capsid proteins generated from HEK293 cells using liquid chromatography-tandem mass spectrometry (LC-MS)-based glycopeptide mapping and identified free N-glycans released from AAV9 capsid proteins by PNGase F using hydrophilic interaction (HILIC) LC-MS and HILIC LC-fluorescence detection (FLD) methods. This study demonstrates that AAV9 capsids are sprinkled with sugars, including N- and O-glycans, albeit at low levels. It may provide valuable information for a better understanding of AAV capsids in supporting AAV-based gene therapy development.

Cul3 substrate adaptor SPOP targets Nup153 for degradation.

SPOP is a Cul3 substrate adaptor responsible for degradation of many proteins related to cell growth and proliferation. Because mutation or misregulation of SPOP drives cancer progression, understanding the suite of SPOP substrates is important to understanding regulation of cell proliferation. Here, we identify Nup153, a component of the nuclear basket of the nuclear pore complex, as a novel substrate of SPOP. SPOP and Nup153 bind to each other and colocalize at the nuclear envelope and some nuclear foci in cells. The binding interaction between SPOP and Nup153 is complex and multivalent. Nup153 is ubiquitylated and degraded upon expression of SPOPWT but not its substrate binding-deficient mutant SPOPF102C. Depletion of SPOP via RNAi leads to Nup153 stabilization. Upon loss of SPOP, the nuclear envelope localization of spindle assembly checkpoint protein Mad1, which is tethered to the nuclear envelope by Nup153, is stronger. Altogether, our results demonstrate SPOP regulates Nup153 levels and expands our understanding of the role of SPOP in protein and cellular homeostasis.

Human Protein-l-isoaspartate O-Methyltransferase Domain-Containing Protein 1 (PCMTD1) Associates with Cullin-RING Ligase Proteins.

The spontaneous l-isoaspartate protein modification has been observed to negatively affect protein function. However, this modification can be reversed in many proteins in reactions initiated by the protein-l-isoaspartyl (d-aspartyl) O-methyltransferase (PCMT1). It has been hypothesized that an additional mechanism exists in which l-isoaspartate-damaged proteins are recognized and proteolytically degraded. Herein, we describe the protein-l-isoaspartate O-methyltransferase domain-containing protein 1 (PCMTD1) as a putative E3 ubiquitin ligase substrate adaptor protein. The N-terminal domain of PCMTD1 contains l-isoaspartate and S-adenosylmethionine (AdoMet) binding motifs similar to those in PCMT1. This protein also has a C-terminal domain containing suppressor of cytokine signaling (SOCS) box ubiquitin ligase recruitment motifs found in substrate receptor proteins of the Cullin-RING E3 ubiquitin ligases. We demonstrate specific PCMTD1 binding to the canonical methyltransferase cofactor S-adenosylmethionine (AdoMet). Strikingly, while PCMTD1 is able to bind AdoMet, it does not demonstrate any l-isoaspartyl methyltransferase activity under the conditions tested here. However, this protein is able to associate with the Cullin-RING proteins Elongins B and C and Cul5 in vitro and in human cells. The previously uncharacterized PCMTD1 protein may therefore provide an alternate maintenance pathway for modified proteins in mammalian cells by acting as an E3 ubiquitin ligase adaptor protein.

Yeast grown in continuous culture systems can detect mutagens with improved sensitivity relative to the Ames test.

Continuous culture systems allow for the controlled growth of microorganisms over a long period of time. Here, we develop a novel test for mutagenicity that involves growing yeast in continuous culture systems exposed to low levels of mutagen for a period of approximately 20 days. In contrast, most microorganism-based tests for mutagenicity expose the potential mutagen to the biological reporter at a high concentration of mutagen for a short period of time. Our test improves upon the sensitivity of the well-established Ames test by at least 20-fold for each of two mutagens that act by different mechanisms (the intercalator ethidium bromide and alkylating agent methyl methanesulfonate). To conduct the tests, cultures were grown in small, inexpensive continuous culture systems in media containing (potential) mutagen, and the resulting mutagenicity of the added compound was assessed via two methods: a canavanine-based plate assay and whole genome sequencing. In the canavanine-based plate assay, we were able to detect a clear relationship between the amount of mutagen and the number of canavanine-resistant mutant colonies over a period of one to three weeks of exposure. Whole genome sequencing of yeast grown in continuous culture systems exposed to methyl methanesulfonate demonstrated that quantification of mutations is possible by identifying the number of unique variants across each strain. However, this method had lower sensitivity than the plate-based assay and failed to distinguish the different concentrations of mutagen. In conclusion, we propose that yeast grown in continuous culture systems can provide an improved and more sensitive test for mutagenicity.

Phospho-regulation of mitotic spindle assembly.

The assembly of the bipolar mitotic spindle requires the careful orchestration of a myriad of enzyme activities like protein posttranslational modifications. Among these, phosphorylation has arisen as the principle mode for spatially and temporally activating the proteins involved in early mitotic spindle assembly processes. Here, we review key kinases, phosphatases, and phosphorylation events that regulate critical aspects of these processes. We highlight key phosphorylation substrates that are important for ensuring the fidelity of centriole duplication, centrosome maturation, and the establishment of the bipolar spindle. We also highlight techniques used to understand kinase-substrate relationships and to study phosphorylation events. We conclude with perspectives on the field of posttranslational modifications in early mitotic spindle assembly.

Phase Separation in Cell Division.

Cell division requires the assembly and organization of a microtubule spindle for the proper separation of chromosomes in mitosis and meiosis. Phase separation is an emerging paradigm for understanding spatial and temporal regulation of a variety of cellular processes, including cell division. Phase-separated condensates have been recently discovered at many structures during cell division as a possible mechanism for properly localizing, organizing, and activating proteins involved in cell division. Here, we review how these condensates play roles in regulating microtubule density and organization and spindle assembly and function and in activating some of the key players in cell division. We conclude with perspectives on areas of future research for this exciting and rapidly advancing field.

Burnout and Resilience After a Decade in Palliative Care: What Survivors Have to Teach Us. A Qualitative Study of Palliative Care Clinicians With More Than 10 Years of Experience.

CONTEXT: Burnout is common among palliative care clinicians (PCCs). Resilience helps to reduce burnout, compassion fatigue, and is associated with longevity in palliative care. OBJECTIVES: We aimed to study PCCs who have remained in the field for longer than 10 years to deepen our understanding on their views on burnout and resilience. METHODS: We conducted a qualitative study using semistructured interviews and purposive sampling on 18 PCCs - five doctors, 10 nurses, and three social workers who worked in various palliative care settings (hospital palliative care team, home hospice, and inpatient hospice). The mean age of the interviewees was 52 years, and the mean number of years practicing palliative care was 15.7 years (range 10-25). The interviews were recorded verbatim, transcribed, and analyzed using a grounded theory approach. RESULTS: Four major themes emerged from our analysis - struggling, changing mindset, adapting, and resilience. Intervening conditions, such as self-awareness, reflection, and evolution, were also important factors. The core phenomenon of our study was that of transformational growth - a process that PCCs have to go through before they achieve resilience. We also further classified resilience into both personal and collective resilience. CONCLUSION: Our findings highlight the evolving process of transformational growth that PCCs must repeatedly undergo as they strive toward sustained resilience and longevity. It also stresses the importance of taking individual and collective responsibility toward building a culture of personal and team resilience.

Dissecting the mechanisms of cell division.

Cell division is a highly regulated and carefully orchestrated process. Understanding the mechanisms that promote proper cell division is an important step toward unraveling important questions in cell biology and human health. Early studies seeking to dissect the mechanisms of cell division used classical genetics approaches to identify genes involved in mitosis and deployed biochemical approaches to isolate and identify proteins critical for cell division. These studies underscored that post-translational modifications and cyclin-kinase complexes play roles at the heart of the cell division program. Modern approaches for examining the mechanisms of cell division, including the use of high-throughput methods to study the effects of RNAi, cDNA, and chemical libraries, have evolved to encompass a larger biological and chemical space. Here, we outline some of the classical studies that established a foundation for the field and provide an overview of recent approaches that have advanced the study of cell division.

Leukemia Cell Cycle Chemical Profiling Identifies the G2-Phase Leukemia Specific Inhibitor Leusin-1.

Targeting the leukemia proliferation cycle has been a successful approach to developing antileukemic therapies. However, drug screening efforts to identify novel antileukemic agents have been hampered by the lack of a suitable high-throughput screening platform for suspension cells that does not rely on flow-cytometry analyses. We report the development of a novel leukemia cell-based high-throughput chemical screening platform for the discovery of cell cycle phase specific inhibitors that utilizes chemical cell cycle profiling. We have used this approach to analyze the cell cycle response of acute lymphoblastic leukemia CCRF-CEM cells to each of 181420 druglike compounds. This approach yielded cell cycle phase specific inhibitors of leukemia cell proliferation. Further analyses of the top G2-phase and M-phase inhibitors identified the leukemia specific inhibitor 1 (Leusin-1). Leusin-1 arrests cells in G2 phase and triggers an apoptotic cell death. Most importantly, Leusin-1 was more active in acute lymphoblastic leukemia cells than other types of leukemias, non-blood cancers, or normal cells and represents a lead molecule for developing antileukemic drugs.

Gamma Knife thalamotomy for essential tremor.

OBJECTIVES: The purpose of this study was to evaluate the results following Gamma Knife thalamotomy (GKT) for medically refractory essential tremor in a series of patients in whom open surgical techniques were not desirable. METHODS: Thirty-one patients underwent GKT for disabling essential tremor after medical therapy had failed. Their mean age was 77 years. Most patients were elderly or had concomitant medical illnesses. A single 4-mm isocenter was used to target a maximum dose of 130 or 140 Gy to the nucleus ventralis intermedius. Items from the Fahn-Tolosa-Marin clinical tremor rating scale were used to grade tremor and handwriting before and after radiosurgery. RESULTS: The median follow-up was 36 months. In the group of 26 evaluable patients, the mean tremor score (+/- standard deviation) was 3.7 +/- 0.1 preoperatively and 1.7 +/- 0.3 after radiosurgery (p < 0.000015). The mean handwriting score was 2.8 +/- 0.2 before GKT and 1.7 +/- 0.2 afterward (p < 0.0002). After radiosurgery, 18 patients (69%) showed improvement in both action tremor and writing scores, 6 (23%) only in action tremor scores, and 3 (12%) in neither tremor nor writing. Permanent mild right hemiparesis and speech impairment developed in 1 patient 6 months after radiosurgery. Another patient had transient mild right hemiparesis and dysphagia. CONCLUSIONS: Gamma Knife thalamotomy is a safe and effective therapy for medically refractory essential tremor. Its use is especially valuable for patients ineligible for radiofrequency thalamotomy or deep brain stimulation. Patients must be counseled on potential complications, including the low probability of a delayed neurological deficit.

In vivo evaluation of biomechanical anterior cervical plate failure.

Anterior cervical plate (ACP) failure is rarely addressed in the literature. In this retrospective, observational, longitudinal, cohort study, the objectives were to (1) identify incidences of in vivo biomechanical failure in commercially available, US Food and Drug Administration-approved ACP systems, (2) describe modes of failure, (3) suggest structural explanations for system failure, and (4) discuss complications and treatment of patients with failed ACP systems. Investigators retrospectively identified patients who underwent anterior cervical procedures followed by use of ACP as a fusion adjunct and showed evidence of ACP failure on plain radiographs. Components of the ACP system that led to failure were identified and examined. A total of 240 patients received ACP supplementation of anterior cervical fusion constructs during the 9.5-y study period. Evidence of ACP failure was noted in 7 patients (3.3%), and an eighth patient was referred for evaluation after ACP failure. Screw-plate interface failure occurred in all 8 cases. The biomechanical method by which the bone screw head was secured into the vertebral body, or against the ACP, the use of hybrid systems, the surgical technique selected, and host factors were used to determine the rate of failure. Concern for esophageal or other tissue injury often necessitated ACP removal. Screw-plate interface failure, which was found to be the most common mode of biomechanical ACP failure, may occur in hybrid constructs and in systems that do not create a rigid interface between the screw head and the ACP. Surgical technique and patient host factors may also influence the rate of biomechanical construct failure.

Prophylaxis for deep venous thrombosis in craniotomy patients: a decision analysis.

OBJECTIVE: We sought to determine the most efficient perioperative prophylactic strategy for deep venous thrombosis (DVT) in craniotomy patients by use of a decision analysis model. METHODS: We conducted a structured review of the relevant literature and compiled the reported incidences of DVT, pulmonary embolism, and postoperative intracranial hemorrhage (ICH) in craniotomy patients. We also obtained from the literature estimates of the likelihood and the impact of various outcomes of these complications. Data from 810 craniotomies performed at our own institution were also examined. The decision analytic model was then used to compare the effectiveness of pneumatic compression boots with pneumatic compression boots combined with either unfractionated or low-molecular-weight heparin. The model dealt with variability by using both sensitivity analysis and Monte Carlo simulation. RESULTS: As expected, the addition of heparin lowered the incidence of both DVT and pulmonary embolism, but at the cost of increasing ICH. Because the deleterious effects of ICH were so much greater than the benefits from heparinization, overall outcomes were best with mechanical prophylaxis alone. This was especially true for low-molecular-weight heparin, which is associated with a relatively high risk of ICH. Our own institutional data support the findings in the literature. Although the differences are modest, they reach statistical significance in the case of low-molecular-weight heparin. CONCLUSION: Using decision analytic modeling, we have shown that mechanical prophylaxis yields outcomes in craniotomy patients superior to those of either unfractionated or low-molecular-weight heparin.

Conus medulla-cauda compression from nerve root hypertrophy in a child with Dejerine-Sottas syndrome: improvement with laminectomy and duraplasty. Case report.

This 7-year-old boy with Dejerine-Sottas syndrome caused by a mutation in the myelin protein zero gene began to suffer rapid deterioration with increasing leg weakness, loss of the ability to ambulate, and bowel and bladder incontinence. Magnetic resonance imaging of the spine revealed nerve root hypertrophy resulting in compression of the conus medullaris and cauda equina. Decompressive surgery was successful in reversing some of his deficits.

Converging three-dimensional Stokes flow of two fluids in a T-type bifurcation.

Studies of three-dimensional Stokes flow of two Newtonian fluids that converge in a T-type bifurcation have important applications in polymer coextrusion, blood flow through the venous microcirculation, and other problems of science and technology. This flow problem is simulated numerically by means of the finite element method, and the solution demonstrates that the viscosity ratio between the two fluids critically affects flow behaviour. For the parameters investigated, we find that as the viscosity ratio between the side branch and the main branch increases, the interface between the merging fluids bulges away from the side branch. The viscosity ratio also affects the velocity distribution: at the outlet branch, the largest radial gradients of axial velocity appear in the less-viscous fluid. The distribution of wall shear stress is non-axisymmetric in the outlet branch and may be discontinuous at the interface between the fluids.