Determination of Endovaginal Ultrasound Proficiency and Learning Curve Among Emergency Medicine Trainees.

OBJECTIVES: Performing and interpreting endovaginal ultrasound is an important skill used during the evaluation of obstetric and gynecologic emergencies. This study aims to describe the level of proficiency and confidence achieved after performing 25 endovaginal examinations. METHODS: This is a prospective study at a single urban academic emergency department. Participants performed a minimum of 25 endovaginal ultrasounds under the supervision of a point-of-care ultrasound expert. Anatomical structures were identified by the expert under ultrasound prior to each session. Each examination was scored for agreement of findings between the participant and expert. The data were used to develop a performance curve identifying when proficiency was achieved, where experiential benefit diminished, and when participants felt confident. RESULTS: A total of 1117 endovaginal ultrasound examinations were performed by 50 participants. Agreement after 25 examinations was highest (>95%) for probe insertion and preparation, bladder and uterus identification, and directionality. Agreement was lowest for identification of the ovaries (76%). Experiential benefit plateaus occurred earliest (10 exams) for preparation and insertion followed by bladder identification and directionality. Surprisingly, ovarian experiential benefit plateaued at 16 exams. Participant confidence improved overall and was lowest for the identification of ovaries and abnormal pelvic anatomy. CONCLUSIONS: There is a significant learning curve when performing endovaginal ultrasound. Our data do not support the use of 25 examinations as a minimum standard for identification of the ovaries or abnormal ovarian pathology.

The Effect of Head Rotation on the Relative Vascular Anatomy of the Neck: Implications for Central Venous Access.

CONTEXT: Previous studies have shown that safe venous cannulation is difficult when the internal jugular vein (IJV) overlies the carotid artery (CA) as the probability of inadvertent arterial penetration is greatly increased. AIMS: The goal of this study was to examine the anatomical relationships of the IJV and CA as a function of the degree of head rotation in order to minimize the risk for CA puncture. SETTINGS AND DESIGN: Our study was a prospective study using a sample of 496 Emergency Department patients. METHODS AND MATERIAL: The anatomic relationships of the right and left IJVs and CAs were recorded with head rotation at three different positions. Patients who had the IJV in a 45 to 135 degree relationship to the CA were deemed to be in the high-risk zone for arterial puncture. STATISTICAL ANALYSIS: Chi square, ANOVA. RESULTS: Right IJVs were in the high risk zone for 39.5%, 47.8% and 60.9% of cases at 0, 45 and 80 degrees of head rotation, respectively (P < 0.001). Left IJVs were in the high risk zone for 59.1%, 69.2% and 80.0% at 0, 45 and 80 degrees of head rotation, respectively. (P < 0.001). CONCLUSIONS: Head rotation should be minimized during IJV cannulation to decrease the overlap of CA by IJV. Cannulation of the left IJV appears to carry a higher degree of risk as compared to the right IJV. Placing the head in neutral position, avoiding rotation, and using ultrasound guidance are recommended to minimize complications during central venous access.

Structural Basis for the Failure of the C1 Domain of Ras Guanine Nucleotide Releasing Protein 2 (RasGRP2) to Bind Phorbol Ester with High Affinity.

The C1 domain represents the recognition module for diacylglycerol and phorbol esters in protein kinase C, Ras guanine nucleotide releasing protein (RasGRP), and related proteins. RasGRP2 is exceptional in that its C1 domain has very weak binding affinity (Kd = 2890 ± 240 nm for [(3)H]phorbol 12,13-dibutyrate. We have identified four amino acid residues responsible for this lack of sensitivity. Replacing Asn(7), Ser(8), Ala(19), and Ile(21) with the corresponding residues from RasGRP1/3 (Thr(7), Tyr(8), Gly(19), and Leu(21), respectively) conferred potent binding affinity (Kd = 1.47 ± 0.03 nm) in vitro and membrane translocation in response to phorbol 12-myristate 13-acetate in LNCaP cells. Mutant C1 domains incorporating one to three of the four residues showed intermediate behavior with S8Y making the greatest contribution. Binding activity for diacylglycerol was restored in parallel. The requirement for anionic phospholipid for [(3)H]phorbol 12,13-dibutyrate binding was determined; it decreased in going from the single S8Y mutant to the quadruple mutant. The full-length RasGRP2 protein with the mutated C1 domains also showed strong phorbol ester binding, albeit modestly weaker than that of the C1 domain alone (Kd = 8.2 ± 1.1 nm for the full-length protein containing all four mutations), and displayed translocation in response to phorbol ester. RasGRP2 is a guanyl exchange factor for Rap1. Consistent with the ability of phorbol ester to induce translocation of the full-length RasGRP2 with the mutated C1 domain, phorbol ester enhanced the ability of the mutated RasGRP2 to activate Rap1. Modeling confirmed that the four mutations helped the binding cleft maintain a stable conformation.

Myosin IIIB uses an actin-binding motif in its espin-1 cargo to reach the tips of actin protrusions.

Myosin IIIA (MYO3A) targets actin protrusion tips using a motility mechanism dependent on both motor and tail actin-binding activity [1]. We show that myosin IIIB (MYO3B) lacks tail actin-binding activity and is unable to target COS7 cell filopodia tips, yet is somehow able to target stereocilia tips. Strikingly, when MYO3B is coexpressed with espin-1 (ESPN1), a MYO3A cargo protein endogenously expressed in stereocilia [2], MYO3B targets and carries ESPN1 to COS7 filopodia tips. We show that this tip localization is lost when we remove the ESPN1 C terminus actin-binding site. We also demonstrate that, like MYO3A [2], MYO3B can elongate filopodia by transporting ESPN1 to the polymerizing end of actin filaments. The mutual dependence of MYO3B and ESPN1 for tip localization reveals a novel mechanism for the cell to regulate myosin tip localization via a reciprocal relationship with cargo that directly participates in actin binding for motility. Our results are consistent with a novel form of motility for class III myosins that requires both motor and tail domain actin-binding activity and show that the actin-binding tail can be replaced by actin-binding cargo. This study also provides a framework to better understand the late-onset hearing loss phenotype in patients with MYO3A mutations.

Myosin IIIa boosts elongation of stereocilia by transporting espin 1 to the plus ends of actin filaments.

Two proteins implicated in inherited deafness, myosin IIIa, a plus-end-directed motor, and espin, an actin-bundling protein containing the actin-monomer-binding motif WH2, have been shown to influence the length of mechanosensory stereocilia. Here we report that espin 1, an ankyrin repeat-containing isoform of espin, colocalizes with myosin IIIa at stereocilia tips and interacts with a unique conserved domain of myosin IIIa. We show that combined overexpression of these proteins causes greater elongation of stereocilia, compared with overexpression of either myosin IIIa alone or espin 1 alone. When these two proteins were co-expressed in the fibroblast-like COS-7 cell line they induced a tenfold elongation of filopodia. This extraordinary filopodia elongation results from the transport of espin 1 to the plus ends of F-actin by myosin IIIa and depends on espin 1 WH2 activity. This study provides the basis for understanding the role of myosin IIIa and espin 1 in regulating stereocilia length, and presents a physiological example where myosins can boost elongation of actin protrusions by transporting actin regulatory factors to the plus ends of actin filaments.

CLAMP, a novel microtubule-associated protein with EB-type calponin homology.

Microtubules (MTs) are polymers of alpha and beta tubulin dimers that mediate many cellular functions, including the establishment and maintenance of cell shape. The dynamic properties of MTs may be influenced by tubulin isotype, posttranslational modifications of tubulin, and interaction with microtubule-associated proteins (MAPs). End-binding (EB) family proteins affect MT dynamics by stabilizing MTs, and are the only MAPs reported that bind MTs via a calponin-homology (CH) domain (J Biol Chem 278 (2003) 49721-49731; J Cell Biol 149 (2000) 761-766). Here, we describe a novel 27 kDa protein identified from an inner ear organ of Corti library. Structural homology modeling demonstrates a CH domain in this protein similar to EB proteins. Northern and Western blottings confirmed expression of this gene in other tissues, including brain, lung, and testis. In the organ of Corti, this protein localized throughout distinctively large and well-ordered MT bundles that support the elongated body of mechanically stiff pillar cells of the auditory sensory epithelium. When ectopically expressed in Cos-7 cells, this protein localized along cytoplasmic MTs, promoted MT bundling, and efficiently stabilized MTs against depolymerization in response to high concentration of nocodazole and cold temperature. We propose that this protein, designated CLAMP, is a novel MAP and represents a new member of the CH domain protein family.

Expression of prestin, a membrane motor protein, in the mammalian auditory and vestibular periphery.

Hair cells are specialized mechanoreceptors common to auditory and vestibular sensory organs of mammalian and non-mammalian species. Different hair cells are believed to share common features related to their mechanosensory function. It has been shown that hair cells possess various forms of motile properties that enhance their receptor function. Membrane-based electromotility is a form of hair cell motility observed in isolated outer hair cells (OHCs) of the cochlea. A novel membrane protein, prestin, recently cloned from gerbil and rat tissues, is presumably responsible for electromotility. We cloned prestin from mouse organ of Corti and confirmed strong homology of this protein among different rodent species. We explored whether or not prestin is present in hair cells of the vestibular system. Using reverse transcription-polymerase chain reaction, we demonstrated that prestin is expressed in mouse and rat auditory and vestibular organs, but not in chicken auditory periphery. In situ hybridization and immunolocalization studies confirmed the presence of prestin in OHCs as well as in vestibular hair cells (VHCs) of rodent saccule, utricle and crista ampullaris. However, in the VHCs, staining of varying intensity with anti-prestin antibodies was observed in the cytoplasm, but not in the lateral plasma membrane or in the stereociliary membrane. Whole-cell patch-clamp recordings showed that VHCs do not possess the voltage-dependent capacitance associated with membrane-based electromotility. We conclude that although prestin is expressed in VHCs, it is unlikely that it supports the form of somatic motility observed in OHCs.