Mathew P. Estey

Distinct roles of septins in cytokinesis: SEPT9 mediates midbody abscission

SEPT9 localizes the vesicle-tethering exocyst complex to the midbody to promote daughter cell separation.

SEPT9 occupies the terminal positions in septin octamers and mediates polymerization-dependent functions in abscission

Mammalian SEPT9 is positioned at the end of septin octamers and is necessary for octamer assembly into polymers necessary for abscission during cytokinesis.

Metal release from hip prostheses: cobalt and chromium toxicity and the role of the clinical laboratory

Abstract Individuals with advanced hip disease suffer from pain, impaired hip function, and decreased quality of life. Roughly one million metal-on-metal (MoM) hip prostheses have been implanted worldwide in order to ameliorate these issues. While most MoM hip replacements are successful, some patients suffer from serious adverse effects secondary to the release of metal debris due to implant wear and corrosion. MoM hip prostheses are comprised predominantly of cobalt and chromium, and the serum concentration of these metal ions has been shown to correlate with both implant wear and the accumulation of metal debris in the periprosthetic tissue. Consequently, measurement of cobalt and chromium concentrations may be useful in the assessment of implant function and the potential for adverse effects in the follow-up of patients with MoM hip prostheses. The purpose of this Mini Review is to describe the adverse biological consequences of metal release from hip prostheses, provide an overview of the clinical utility of cobalt and chromium measurement and the current recommendations for testing, and alert laboratorians and physicians to the many challenges associated with measuring these metal ions.

Characterization of a SEPT9 interacting protein, SEPT14, a novel testis-specific septin

Septins are a highly conserved family of GTP-binding cytoskeletal proteins implicated in multiple cellular functions, including membrane transport, apoptosis, cell polarity, cell cycle regulation, cytokinesis, and oncogenesis. Here we describe the characterization of a novel interacting partner of the septin family, initially cloned from a human testis expression library following yeast two-hybrid isolation to identify SEPT9 binding partners. Upon further genomic characterization and bioinformatics analyses it was determined that this novel septin-interacting partner was also a new member of the mammalian septin family, named SEPT14. SEPT14 maps to 7p11.2 in humans and includes a conserved GTPase domain and a predicted carboxy-terminus coiled-coil domain characteristic of other septins. Three potential translational start methionines were identified by 5′ RACE-PCR encoding proteins of 432-, 427-, and 425-residue peptides, respectively. SEPT14 shares closest homology to SEPT10, a human dendritic septin, and limited homology to SEPT9 isoforms. SEPT14 colocalized with SEPT9 when coexpressed in cell lines, and epitope-tagged forms of these proteins coimmunoprecipitated. Moreover, SEPT14 was coimmunoprecipitated from rat testes using SEPT9 antibodies, and yeast two-hybrid analysis suggested SEPT14 interactions with nine additional septins. Multitissue Northern blotting showed testis-specific expression of a single 5.0-kb SEPT14 transcript. RT-PCR analysis revealed that SEPT14 was not detectable in normal or cancerous ovarian, breast, prostate, bladder, or kidney cell lines and was only faintly detected in fetal liver, tonsil, and thymus samples. Interestingly, SEPT14 was expressed in testis but not testicular cancer cell lines by RT-PCR, suggesting that further investigation of SEPT14 as a testis-specific tumor suppressor is necessary.

Characterization of presynaptic septin complexes in mammalian hippocampal neurons.

Abstract Septins are GTPases that form heteromeric complexes and are linked to neurological disorders. Although several septin subcomplexes have been reported in various mammalian tissues, the cellular and subcellular distribution of these complexes is largely unexplored. Using antibodies against ten mammalian septins, we show that septins diverge with respect to their tissue distribution implying that septin complexes in various tissues have unique composition. Although all ten septins examined were expressed in brain tissue, we describe septin complex(es) including SEPT3, SEPT5, SEPT6, SEPT7 and SEPT11 that could be functional within the presynapse because, unlike other septins they: (1) showed an increase in expression from embryonic day 15 to post-natal day 70, (2) were abundantly expressed in axons and growth cones of developing hippocampal neurons, (3) were found in presynaptic terminals of mature synapses, (4) were enriched in a preparation of synaptic vesicles and (5) immunoprecipitated together from brain tissue and cultured nerve cells. Knockdown of SEPT5 or SEPT7 in developing hippocampal neurons impaired axon growth. Because septins are functionally linked to the cytoskeleton and vesicle traffic, presynaptic neuronal septin complexes could be important for ensuring proper axon development and neurotransmitter release.

Mitotic Regulation of SEPT9 Protein by Cyclin-dependent Kinase 1 (Cdk1) and Pin1 Protein Is Important for the Completion of Cytokinesis

Background: Evidence is lacking to explain how septin function is controlled during the cell cycle. Results: Phosphorylation of SEPT9 by Cdk1 creates a binding site for Pin1 that is required for cytokinesis. Conclusion: Septin interaction with Pin1 is controlled by Cdk1 and is necessary for timely cytokinesis. Significance: Improper cell division results in cancer, in which both SEPT9 and Pin1 have been implicated. Precise cell division is essential for multicellular development, and defects in this process have been linked to cancer. Septins are a family of proteins that are required for mammalian cell division, but their function and mode of regulation during this process are poorly understood. Here, we demonstrate that cyclin-dependent kinase 1 (Cdk1) phosphorylates septin 9 (SEPT9) upon mitotic entry, and this phosphorylation controls association with the proline isomerase, Pin1. Both SEPT9 and Pin1 are critical for mediating the final separation of daughter cells. Expression of mutant SEPT9 that is defective in Pin1 binding was unable to rescue cytokinesis defects caused by SEPT9 depletion but rather induced dominant-negative defects in cytokinesis. However, unlike SEPT9 depletion, Pin1 was not required for the accumulation of the exocyst complex at the midbody. These results suggest that SEPT9 plays multiple roles in abscission, one of which is regulated by the action of Cdk1 and Pin1.

Hemoglobin Hirose: A rare beta chain variant causing falsely low HbA1c by HPLC

OBJECTIVES To investigate the underlying cause of unexpectedly low HbA1c results (3.3-3.5%) obtained by HPLC in three siblings undergoing routine screening for type 2 diabetes mellitus. DESIGN AND METHODS HbA1c was measured using an alternate method based on a different analytical principle (the Siemens DCA 2000+ immunoassay). Hemoglobin fractionation was performed by HPLC on the BioRad Variant II, gel electrophoresis at acid and alkaline pH on the Sebia Hydrasys 2, and capillary electrophoresis on the Sebia Capillarys 2. Sequencing of the beta globin gene was also conducted. RESULTS HbA1c analysis by immunoassay gave significantly higher results, ranging from 5.2-5.5%. Hemoglobin fractionation by HPLC showed an abnormal peak comprising approximately 43% of total hemoglobin, suggesting the presence of a beta chain hemoglobin variant. Gel electrophoresis at alkaline pH revealed a very unusual pattern, with 3 abnormal bands migrating with Hb F, between Hb F and Hb S, and slightly cathodal to Hb S. A single band in the Hb A position was seen on gel electrophoresis at acid pH. Capillary electrophoresis revealed two abnormal peaks, comprising 42% and 5% of total hemoglobin. Sequencing of the beta globin gene showed heterozygosity for Hb Hirose (beta 37(C3) Trp>Ser), an extremely rare variant with a substitution at the α1β2 interface. CONCLUSIONS We describe the chromatographic and electrophoretic properties of Hb Hirose, and demonstrate that this rare variant causes falsely low HbA1c results on the BioRad variant II Turbo 2.0. Recognition of this interference is crucial in order to prevent reporting erroneous results.

Evaluation of thyroid test utilization through analysis of population-level data

Abstract: Background: Inappropriate laboratory test utilization can result in unnecessary patient testing and increased healthcare costs. While several thyroid function tests are available, thyroid-stimulating hormone (TSH) is recommended as the first-line test for investigating and monitoring thyroid dysfunction. We evaluate thyroid test utilization in Northern Alberta in terms of testing patterns, frequencies, and reflex cutpoints. Methods: This retrospective study analyzed thyroid test requests from January to December 2014. Each request was designated as appropriate or potentially inappropriate as per clinical practice guidelines and Choosing Wisely recommendations, and the frequencies of each testing pattern were calculated. Sub-analysis was performed to categorize testing patterns based on physician specialty. The number of test requests per patient was determined to assess the appropriateness of testing frequency. Receiver operating characteristic (ROC) curves were generated to define optimal TSH cutpoints for automatic reflex to FT4 testing. Results: Of 752,217 test requests, approximately 10% were potentially inappropriate in terms of testing patterns. Free thyroxine (FT4) and free triiodothyronine (FT3) requested with TSH accounted for 59% of all potentially inappropriate test requests, and 49% of requests from endocrinologists (ENDO) were potentially inappropriate, occurring most frequently among those with less experience. Excessive testing frequencies were observed in 869 patients, accounting for 9382 test requests. Adjustment of our TSH reflex cutpoint would significantly increase specificity for identifying a low FT4 without compromising sensitivity. Conclusions: This study suggests that questionable testing patterns, excessive testing frequencies, and suboptimal reflexive testing cutpoints contribute to inappropriate thyroid test utilization.

Hemoglobin C in a patient with sickle cell anemia: transfusion-acquired SC disease.

• We report a sickle cell anemia patient who had low amounts of hemoglobin C following routine red cell exchange transfusion