Conotoxin κM-RIIIJ, a tool targeting asymmetric heteromeric Kv1 channels.

The vast complexity of native heteromeric K+ channels is largely unexplored. Defining the composition and subunit arrangement of individual subunits in native heteromeric K+ channels and establishing their physiological roles is experimentally challenging. Here we systematically explored this "zone of ignorance" in molecular neuroscience. Venom components, such as peptide toxins, appear to have evolved to modulate physiologically relevant targets by discriminating among closely related native ion channel complexes. We provide proof-of-principle for this assertion by demonstrating that κM-conotoxin RIIIJ (κM-RIIIJ) from Conus radiatus precisely targets "asymmetric" Kv channels composed of three Kv1.2 subunits and one Kv1.1 or Kv1.6 subunit with 100-fold higher apparent affinity compared with homomeric Kv1.2 channels. Our study shows that dorsal root ganglion (DRG) neurons contain at least two major functional Kv1.2 channel complexes: a heteromer, for which κM-RIIIJ has high affinity, and a putative Kv1.2 homomer, toward which κM-RIIIJ is less potent. This conclusion was reached by (i) covalent linkage of members of the mammalian Shaker-related Kv1 family to Kv1.2 and systematic assessment of the potency of κM-RIIIJ block of heteromeric K+ channel-mediated currents in heterologous expression systems; (ii) molecular dynamics simulations of asymmetric Kv1 channels providing insights into the molecular basis of κM-RIIIJ selectivity and potency toward its targets; and (iii) evaluation of calcium responses of a defined population of DRG neurons to κM-RIIIJ. Our study demonstrates that bioactive molecules present in venoms provide essential pharmacological tools that systematically target specific heteromeric Kv channel complexes that operate in native tissues.

Mapping of voltage sensor positions in resting and inactivated mammalian sodium channels by LRET.

Voltage-gated sodium channels (Navs) play crucial roles in excitable cells. Although vertebrate Nav function has been extensively studied, the detailed structural basis for voltage-dependent gating mechanisms remain obscure. We have assessed the structural changes of the Nav voltage sensor domain using lanthanide-based resonance energy transfer (LRET) between the rat skeletal muscle voltage-gated sodium channel (Nav1.4) and fluorescently labeled Nav1.4-targeting toxins. We generated donor constructs with genetically encoded lanthanide-binding tags (LBTs) inserted at the extracellular end of the S4 segment of each domain (with a single LBT per construct). Three different Bodipy-labeled, Nav1.4-targeting toxins were synthesized as acceptors: ß-scorpion toxin (Ts1)-Bodipy, KIIIA-Bodipy, and GIIIA-Bodipy analogs. Functional Nav-LBT channels expressed in Xenopus oocytes were voltage-clamped, and distinct LRET signals were obtained in the resting and slow inactivated states. Intramolecular distances computed from the LRET signals define a geometrical map of Nav1.4 with the bound toxins, and reveal voltage-dependent structural changes related to channel gating.

Extremely Potent Block of Bacterial Voltage-Gated Sodium Channels by µ-Conotoxin PIIIA.

µ-Conotoxin PIIIA, in the sub-picomolar, range inhibits the archetypal bacterial sodium channel NaChBac (NavBh) in a voltage- and use-dependent manner. Peptide µ-conotoxins were first recognized as potent components of the venoms of fish-hunting cone snails that selectively inhibit voltage-gated skeletal muscle sodium channels, thus preventing muscle contraction. Intriguingly, computer simulations predicted that PIIIA binds to prokaryotic channel NavAb with much higher affinity than to fish (and other vertebrates) skeletal muscle sodium channel (Nav 1.4). Here, using whole-cell voltage clamp, we demonstrate that PIIIA inhibits NavBac mediated currents even more potently than predicted. From concentration-response data, with [PIIIA] varying more than 6 orders of magnitude (10-12 to 10-5 M), we estimated an IC50 = ~5 pM, maximal block of 0.95 and a Hill coefficient of 0.81 for the inhibition of peak currents. Inhibition was stronger at depolarized holding potentials and was modulated by the frequency and duration of the stimulation pulses. An important feature of the PIIIA action was acceleration of macroscopic inactivation. Docking of PIIIA in a NaChBac (NavBh) model revealed two interconvertible binding modes. In one mode, PIIIA sterically and electrostatically blocks the permeation pathway. In a second mode, apparent stabilization of the inactivated state was achieved by PIIIA binding between P2 helices and trans-membrane S5s from adjacent channel subunits, partially occluding the outer pore. Together, our experimental and computational results suggest that, besides blocking the channel-mediated currents by directly occluding the conducting pathway, PIIIA may also change the relative populations of conducting (activated) and non-conducting (inactivated) states.

NCCN Guidelines as a Model of Extended Criteria for Lung Cancer Screening.

Background: This review assessed the performance of patients in NCCN high-risk group 2 in a clinical CT lung screening (CTLS) program. Methods: We retrospectively reviewed screening results for all patients from our institution undergoing clinical CTLS from January 2012 through December 2016, with follow-up through June 2017. To qualify for screening, patients had to meet the NCCN Guidelines high-risk criteria for CTLS, have a physician order for screening, be asymptomatic, be lung cancer-free for 5 years, and have no known metastatic disease. We compared demographics and screening performance of NCCN high-risk groups 1 and 2 across >4 rounds of screening. Screening metrics assessed included rates of positive and suspicious examinations, significant incidental and infectious/inflammatory findings, false negatives, and cancer detection. We also compared cancer stage and histology detected in each NCCN high-risk group. Results: A total of 2,927 individuals underwent baseline screening, of which 698 (24%) were in NCCN group 2. On average, group 2 patients were younger (60.6 vs 63.1 years), smoked less (38.8 vs 50.8 pack-years), had quit longer (18.1 vs 6.3 years), and were more often former smokers (61.4% vs 44.2%). Positive and suspicious examination rates, false negatives, and rates of infectious/inflammatory findings were equivalent in groups 1 and 2 across all rounds of screening. An increased rate of cancer detection was observed in group 2 during the second annual (T2) screening round (2.7% vs 0.5%; P=.005), with no difference in the other screening rounds: baseline (T0; 2% vs 2.3%; P=.61), first annual (T1; 1.2% vs 1.7%; P=.41), and third annual and beyond (≥T3; 1.2% vs 1.1%; P=1.00). Conclusions: CTLS appears to be equally effective in both NCCN high-risk groups.

Local anesthetic and antiepileptic drug access and binding to a bacterial voltage-gated sodium channel.

Voltage-gated sodium (Nav) channels are important targets in the treatment of a range of pathologies. Bacterial channels, for which crystal structures have been solved, exhibit modulation by local anesthetic and anti-epileptic agents, allowing molecular-level investigations into sodium channel-drug interactions. These structures reveal no basis for the "hinged lid"-based fast inactivation, seen in eukaryotic Nav channels. Thus, they enable examination of potential mechanisms of use- or state-dependent drug action based on activation gating, or slower pore-based inactivation processes. Multimicrosecond simulations of NavAb reveal high-affinity binding of benzocaine to F203 that is a surrogate for FS6, conserved in helix S6 of Domain IV of mammalian sodium channels, as well as low-affinity sites suggested to stabilize different states of the channel. Phenytoin exhibits a different binding distribution owing to preferential interactions at the membrane and water-protein interfaces. Two drug-access pathways into the pore are observed: via lateral fenestrations connecting to the membrane lipid phase, as well as via an aqueous pathway through the intracellular activation gate, despite being closed. These observations provide insight into drug modulation that will guide further developments of Nav inhibitors.

Gating transitions in the selectivity filter region of a sodium channel are coupled to the domain IV voltage sensor.

Voltage-dependent ion channels are crucial for generation and propagation of electrical activity in biological systems. The primary mechanism for voltage transduction in these proteins involves the movement of a voltage-sensing domain (D), which opens a gate located on the cytoplasmic side. A distinct conformational change in the selectivity filter near the extracellular side has been implicated in slow inactivation gating, which is important for spike frequency adaptation in neural circuits. However, it remains an open question whether gating transitions in the selectivity filter region are also actuated by voltage sensors. Here, we examine conformational coupling between each of the four voltage sensors and the outer pore of a eukaryotic voltage-dependent sodium channel. The voltage sensors of these sodium channels are not structurally symmetric and exhibit functional specialization. To track the conformational rearrangements of individual voltage-sensing domains, we recorded domain-specific gating pore currents. Our data show that, of the four voltage sensors, only the domain IV voltage sensor is coupled to the conformation of the selectivity filter region of the sodium channel. Trapping the outer pore in a particular conformation with a high-affinity toxin or disulphide crossbridge impedes the return of this voltage sensor to its resting conformation. Our findings directly establish that, in addition to the canonical electromechanical coupling between voltage sensor and inner pore gates of a sodium channel, gating transitions in the selectivity filter region are also coupled to the movement of a voltage sensor. Furthermore, our results also imply that the voltage sensor of domain IV is unique in this linkage and in the ability to initiate slow inactivation in sodium channels.

Novel radiology PRIMER course enhances medical student perception of radiology and key concept comprehension.

RATIONALE AND OBJECTIVES: A novel three-day radiology course, PRIMER, directly preceding medical students' clinical year, was created and assessed. The required course consisted of large group lecture sessions, small group breakout sessions, and individual assignments. Though early exposure to radiology has been described in preclinical anatomy curricula, few schools offer immersive experiences to radiology as a direct predecessor to the wards. MATERIALS AND METHODS: An identical survey was distributed prior to and at the completion of the PRIMER course. Students' perceptions of radiology were assessed through Likert-style questions. Students' knowledge of radiological concepts was assessed through multiple choice questions (MCQs) related to key concepts, MCQs in which students selected the most likely diagnosis, and hotspot questions in which learners had to select the area of greatest clinical importance. Mean pre- and post-course student perception scores were compared using a T-test. For knowledge-based questions, each student received an exam score, and mean pre- and post-exam scores were compared using a T-test. RESULTS: Students' opinions of radiology changed significantly in a favorable direction across all tested questions between inception and conclusion of PRIMER (p < 0.01). Students demonstrated superior knowledge of radiological concepts after course completion (posttest mean 52% vs pretest mean 26.3%, p < 0.01). CONCLUSION: The novel radiology PRIMER course promoted a positive impression of radiology and increased medical students' knowledge of key concepts. These results suggest that a condensed introductory radiology curriculum delivered at a key moment in the overarching curriculum can have a significant impact on medical students' perceptions and knowledge.

Near-Peer Paradigms in Medical School: Integrating Student Teaching Assistants Into a Core Radiology Clerkship.

RATIONALE AND OBJECTIVES: Near-peer paradigms have been demonstrated effective in supporting how students navigate novel clinical environments. In this study, we describe an innovative model of incorporating teaching assistants (TAs) into a core radiology clerkship and investigate both its perceived educational value by clinical-year learners and its perceived impact on professional growth by TAs. MATERIALS AND METHODS: At one U.S. medical school, the core clinical year includes a clerkship in radiology incorporating both reading room exposure and a didactic curriculum. Radiology faculty deliver a variety of traditional and interactive, "dynamic" lectures, while medical student TAs deliver additional dynamic sessions, including a final TA-created review session. The educational value of each didactic session by clerkship students was assessed using a five-point scale survey, and the professional value of the experience by TAs was assessed using a five-point Likert survey. RESULTS: Spanning from 2020 to 2023, 268 (74.4%) clinical-year students submitted the post-clerkship survey, with the didactic review sessions created and led by TAs receiving the highest ratings of any didactic session. Of 16 former TAs, 12 (75%) completed the post-service survey, with all respondents agreeing or strongly agreeing that they enjoyed and would recommend their experience, and that it enhanced their interest in radiology and in academic medicine. CONCLUSION: Near-peer education in a core radiology clerkship enhances the experience of the learner through peer guidance and the experience of the teacher through professional development. These findings may increase student interest in pursuing academic radiology as a career and invite opportunities for broadening medical school education in radiology.

Medical student perspectives on radiology subspecialties prior to core clerkship.

RATIONALE AND OBJECTIVES: Radiology remains underrepresented in U.S. medical school clinical curricula, and preconceived opinions about the field may impact whether students pursue elective exposure. A core radiology clerkship at one academic institution presents an opportunity to evaluate students' early preferences for different radiology subspecialties, thus informing curricular design and inspiring student recruitment. MATERIALS AND METHODS: At a single allopathic medical school, a required, four-week, clinical-year radiology clerkship that occurs in the second year includes immersive one-week experiences in two subspecialties. Prior to their clerkship, students rank their immersion preferences by distributing 100 points across eight fields, the values of which were analyzed as a proxy for interest. A secondary survey was distributed to active first- and second-year medical students to further investigate the factors drawing them to radiology. RESULTS: Immersive experiences in musculoskeletal, body, and breast imaging were most preferred, earning ≥20 points from 41.6 %, 34.3 %, and 31.9 % of students, respectively. Women were significantly more likely than men to express interest in breast imaging (35.8 % vs. 24.8 %, p = 0.037) and pediatric radiology (28.8 % vs. 12.8 %, p = 0.001). Men were significantly more likely than women to prefer body imaging (41.9 % vs. 30.2 %, p = 0.034), neuroradiology (29.1 % vs. 19.5 %, p = 0.048), and nuclear medicine (11.1 % vs. 5.1 %, p = 0.044). CONCLUSION: Early in their clinical education, medical students prefer certain subspecialties, especially musculoskeletal, body, and breast imaging. Women expressed significantly stronger interest in breast imaging and pediatric radiology, while men indicated significantly stronger interest in body imaging, neuroradiology, and nuclear medicine.

Comparison of Low-Dose Computed Tomography Versus Conventional-Dose Computed Tomography in the Evaluation of Distal Radius Fractures.

BACKGROUND: Distal radius fractures (DRFs) are common upper extremity fractures and often require surgical fixation when they are intraarticular. Preoperative computed tomography (CT) has emerged as a surgical planning tool to evaluate intraarticular DRFs. Although CT affords additional details, patients receive higher radiation doses than standard radiographs. We aim to develop a low-dose CT (LDCT) protocol, relative to the institutional standard-dose CT wrist for intraarticular DRFs although providing adequate detail for surgical decision-making. METHODS: A single-institution prospective study was conducted on patients with intraarticular DRFs who underwent closed reduction and below-elbow splinting who otherwise would undergo wrist CT. Observations were defined as total measurements taken, with each view undergoing 44 measurements. Patients underwent 2 scans with a standard dose and a 10× dose reduction. Articular step and gap measurements were recorded in the sagittal and coronal images. RESULTS: A total of 11 patients were enrolled (7 women and 4 men). The mean age was 55 years (SD = 20.1). There were a total of 4 reviewers: 1 attending surgeon, 2 resident physicians, and 1 student. When comparing LDCT and conventional-dose CT (CDCT), there were no significant differences in step and gap measurements across all reviewers. CONCLUSION: This study demonstrated that LDCT provides comparable imaging quality for surgical planning as a CDCT without significant diagnostic decay in the setting of DRFs. This comes with the added benefit of a 10-fold reduction in radiation exposure. These results suggest that LDCT is an opportunity to reduce effective radiation in patients although providing beneficial preoperative imaging.

The Use of Structured Oral Exams for the Assessment of Medical Students in their Radiology Clerkship.

RATIONALE & OBJECTIVES: There is increasing interest in narrative feedback and competency-based evaluation in medical student education. This study evaluates the implementation of a structured oral exam for a required radiology clerkship in furtherance of these aims. MATERIALS & METHODS: A structured oral exam was instituted in academic year (AY) 20-21. Students prepared to discuss 5 varied imaging cases as they would to a medical colleague and as to a patient. For AY 20-21, students took the oral and a written exam. In AY 21-22, students took the oral exam alone and the written exam was discontinued. The perceived educational value of clerkship components, including the oral and written exam, were scored by the students on a 5-point Likert scale. RESULTS: All students in AY 20-21 received a passing score on the written (mean 89.0, SD 4.59) and oral exams. All students in AY 21-22 received a passing score on the oral exam. In AY 20-21, the educational value of the oral exam was rated significantly higher than that of the written exam (4.30 vs 4.02, P = 0.021). There was no significant difference in rating of the oral exam between AY 20-21 and AY 21-22 (4.30 vs 4.38; P = 0.499). CONCLUSION: The implementation of a structured final oral exam for a required radiology clerkship was felt to be successful in the aims of delivering educational value while evaluating students for competency. Further evaluation of oral exams for radiology medical student education are warranted to optimize the career preparation of future physicians.

Association and Trends in Medicare Denials and Utilization for Brain CT: Indirect Impacts by Targeted Policy Intervention?

Twenty-five years of annual Medicare Physician/Supplier Procedure Summary (PSPS) Master File data were used to assess trends in normalized volume and claim denial rates for brain computerized tomography. Alongside growth in utilization of brain computerized tomography (services, denial rates, fell from 1999-2005 and with relatively leveled growth and less denial rate volatility thereafter. More recent trends in denial rates may be related to policy interventions initially aimed at cost and volume reduction.

Comparison of Simulated Low-Dose and Conventional-Dose CT for Preoperative Planning in Shoulder Arthroplasty.

BACKGROUND: Shoulder computed tomography (CT) is commonly utilized in preoperative planning for total shoulder arthroplasty. Conventional-dose shoulder CT may expose patients to more ionizing radiation than is necessary to provide high-quality images for this procedure. The purpose of this study was to evaluate the utility of simulated low-dose CT images for preoperative planning using manual measurements and common preoperative planning software. METHODS: Eighteen shoulder CT scans obtained for preoperative arthroplasty planning were used to generate CT images as if they had been acquired at reduced radiation dose (RD) levels of 75%, 50%, and 25% using a simulation technique that mimics decreased x-ray tube current. This technique was validated by quantitative comparison of simulated low-dose scans of a cadaver with actual low-dose scans. Glenoid version, glenoid inclination, and humeral head subluxation were measured using 2 commercially available software platforms and were also measured manually by 3 physicians. These measurements were then analyzed for agreement across RD levels for each patient. Tolerances of 5° of glenoid version, 5° of glenoid inclination, and 10% humeral head subluxation were used as equivalent for preoperative planning purposes. RESULTS: At all RD levels evaluated, the preoperative planning software successfully segmented the CT images. Semiautomated software measurement of 25% RD images was within tolerances in 99.1% of measurements; for 50% RD images, within tolerances in 96.3% of measurements; and for 75% RD images, within tolerances in 100% of measurements. Manual measurements of 25% RD images were within these tolerances in 95.1% of measurements; for 50% RD images, in 98.8% of measurements; and for 75% RD images, in 99.4% of measurements. CONCLUSIONS: Simulated low-dose CT images were sufficient for reliable measurement of glenoid version, glenoid inclination, and humeral head subluxation by preoperative planning software as well as by physician-observers. These findings suggest the potential for substantial reduction in RD in preoperative shoulder CT scans without compromising surgical planning. CLINICAL RELEVANCE: The adoption of low-dose techniques in preoperative shoulder CT may lower radiation exposure for patients undergoing shoulder arthroplasty, without compromising image quality.

Prospective Multireader Evaluation of Photon-counting CT for Multiple Myeloma Screening.

Purpose To determine whether photon-counting CT (PCCT) acquisition of whole-body CT images provides similar quantitative image quality and reader satisfaction for multiple myeloma screening at lower radiation doses than does standard energy-integrating detector (EID) CT. Materials and Methods Patients with monoclonal gammopathy of undetermined significance prospectively underwent clinical noncontrast whole-body CT with EID and same-day PCCT (August-December 2021). Five axial scan locations were evaluated by seven radiologists, with 11% (eight of 70) of images including osteolytic lesions. Images were shown in randomized order, and each reader rated the following: discernibility of the osseous cortex and osseous trabeculae, perceived image noise level, and diagnostic confidence. Presence of lytic osseous lesions was indicated. Contrast-to-noise ratio (CNR) and signal-to-noise ratio (SNR) were calculated. Comparisons were made using paired t tests and mixed linear effects models. Results Seven participants (four women) were included (mean age, 66 years ± 9 [SD]; body mass index, 30.1 kg/m2 ± 5.2). Mean cortical definition, trabecular definition, image noise, and image quality scores were 83, 67, 75, and 78 versus 84, 66, 74, and 76 for EID and PCCT, respectively (P = .65, .11, .26, and .11, respectively). PCCT helped identify more lesions (79% [22 of 28]) than did EID (64% [18 of 28]). CNRs and SNRs were similar between modalities. PCCT had lower radiation doses than EID (volume CT dose index: EID, 11.37 ± 2.8 vs PCCT, 1.8 ± 0.6 [P = .06]; dose-length product: EID, 1654.1 ± 409.6 vs PCCT, 253.4 ± 89.6 [P = .05]). Conclusion This pilot investigation suggests that PCCT affords similar quantitative and qualitative scores as EID at significantly lower radiation doses. Keywords: CT, CT-Spectral, Skeletal-Axial, Spine, Hematologic Diseases, Whole-Body Imaging, Comparative Studies Supplemental material is available for this article. © RSNA, 2022.

Multiple, distributed interactions of µ-conotoxin PIIIA associated with broad targeting among voltage-gated sodium channels.

The first µ-conotoxin studied, µCTX GIIIA, preferentially blocked voltage-gated skeletal muscle sodium channels, Na(v)1.4, while µCTX PIIIA was the first to show significant blocking action against neuronal voltage-gated sodium channels. PIIIA shares >60% sequence identity with the well-studied GIIIA, and both toxins preferentially block the skeletal muscle sodium channel isoform. Two important features of blocking by wild-type GIIIA are the toxin's high binding affinity and the completeness of block of a single channel by a bound toxin molecule. With GIIIA, neutral replacement of the critical residue, Arg-13, allows a residual single-channel current (~30% of the unblocked, unitary amplitude) when the mutant toxin is bound to the channel and reduces the binding affinity of the toxin for Na(v)1.4 (~100-fold) [Becker, S., et al. (1992) Biochemistry 31, 8229-8238]. The homologous residue in PIIIA, Arg-14, is also essential for completeness of block but less important in the toxin's binding affinity (~55% residual current and ~11-fold decrease in affinity when substituted with alanine or glutamine). The weakened dominance of this key arginine in PIIIA is also seen in the fact that there is not just one (R13 in GIIIA) but three basic residues (R12, R14, and K17) for which individual neutral replacement enables a substantial residual current through the bound channel. We suggest that, despite a high degree of sequence conservation between GIIIA and PIIIA, the weaker dependence of PIIIA's action on its key arginine and the presence of a nonconserved histidine near the C-terminus may contribute to the greater promiscuity of its interactions with different sodium channel isoforms.

Imaging of Patellofemoral Instability.

Patellar instability is a broad term that encompasses patellar dislocation, patellar subluxation, and patellar instability. Although both functional and anatomic considerations contribute to symptoms of patellar instability, the most important are thought to be patella alta, trochlear dysplasia, and lateralization of the tibial tubercle. In patients with a history suspicious for prior patellar dislocation, careful evaluation of MRI and radiographic studies can reveal characteristic findings. The most common methods to address patellofemoral instability are medial patellofemoral ligament reconstruction and tibial tubercle osteotomy with either anteromedialization or medialization. Less commonly trochleoplasty is indicated as well. Patients may be treated with one of or a combination of these techniques, each of which has specific indications and complications.

Changes in Current Procedural Terminology Coding and Its Effect on Specialty-Level Utilization of Musculoskeletal Ultrasound.

PURPOSE: Concerns regarding increasing utilization of non-vascular extremity ultrasound (US) imaging led to the Current Procedural Terminology (CPT) Editorial Panel separating a singular billing code into distinct comprehensive and focused examination codes with differential reimbursement. We explore this policy change's temporal association with utilization. METHODS: Using Physician/Supplier Procedure Summary Master Files, we identified all nonvascular extremity US services billed for Medicare fee-for-service beneficiaries between 1994 and 2017. These included generic (CPT code 76880 from 1994 to 2010), complete (code 76881 from 2011 to 2017), and limited (code 76882 from 2011 to 2017) examinations. Annual utilization per 100,000 beneficiaries was computed and stratified by billing specialty. Compound annual growth rates were calculated. RESULTS: Radiologists and podiatrists were the top 2 billing specialties for nonvascular extremity US examinations. From 1994 to 2010, radiologist services increased 6.1% annually. Following the 2011 code separation, radiologists' utilization increased 2.7% annually for complete and 12.3% for limited exams. Between 1994 and 2017, radiologists' market share decreased 72.8% to 40.4%. From 1994 to 2010, podiatrist services increased 87.1% annually. Following the code separation, podiatrists' annual utilization growth stabilized 0.4% for complete and 0.6% for limited exams. Podiatrists' market share was 9.1% in 2001, peaked at 31.3% in 2009, and declined to 14.3% in 2017. CONCLUSIONS: Prior rapid growth in extremity nonvascular US for podiatrists slowed considerably following CPT code separation in 2011. Subsequent service growth has largely been related to less costly, focused examinations performed by radiologists. Further study may help better understand how CPT coding changes alter imaging utilization more broadly.

Phenotypic Evaluation and Genetic Analysis of Seedling Emergence in a Global Collection of Wheat Genotypes (Triticum aestivum L.) Under Limited Water Availability.

The challenge in establishing an early-sown wheat crop in southern Australia is the need for consistently high seedling emergence when sowing deep in subsoil moisture (>10 cm) or into dry top-soil (4 cm). However, the latter is strongly reliant on a minimum soil water availability to ensure successful seedling emergence. This study aimed to: (1) evaluate 233 Australian and selected international wheat genotypes for consistently high seedling emergence under limited soil water availability when sown in 4 cm of top-soil in field and glasshouse (GH) studies; (2) ascertain genetic loci associated with phenotypic variation using a genome-wide association study (GWAS); and (3) compare across loci for traits controlling coleoptile characteristics, germination, dormancy, and pre-harvest sprouting. Despite significant (P < 0.001) environment and genotype-by-environment interactions within and between field and GH experiments, eight genotypes that included five cultivars, two landraces, and one inbred line had consistently high seedling emergence (mean value > 85%) across nine environments. Moreover, 21 environment-specific quantitative trait loci (QTL) were detected in GWAS analysis on chromosomes 1B, 1D, 2B, 3A, 3B, 4A, 4B, 5B, 5D, and 7D, indicating complex genetic inheritance controlling seedling emergence. We aligned QTL for known traits and individual genes onto the reference genome of wheat and identified 16 QTL for seedling emergence in linkage disequilibrium with coleoptile length, width, and cross-sectional area, pre-harvest sprouting and dormancy, germination, seed longevity, and anthocyanin development. Therefore, it appears that seedling emergence is controlled by multifaceted networks of interrelated genes and traits regulated by different environmental cues.

Modulation of KvAP unitary conductance and gating by 1-alkanols and other surface active agents.

The actions of alcohols and anesthetics on ion channels are poorly understood. Controversy continues about whether bilayer restructuring is relevant to the modulatory effects of these surface active agents (SAAs). Some voltage-gated K channels (Kv), but not KvAP, have putative low affinity alcohol-binding sites, and because KvAP structures have been determined in bilayers, KvAP could offer insights into the contribution of bilayer mechanics to SAA actions. We monitored KvAP unitary conductance and macroscopic activation and inactivation kinetics in PE:PG/decane bilayers with and without exposure to classic SAAs (short-chain 1-alkanols, cholesterol, and selected anesthetics: halothane, isoflurane, chloroform). At levels that did not measurably alter membrane specific capacitance, alkanols caused functional changes in KvAP behavior including lowered unitary conductance, modified kinetics, and shifted voltage dependence for activation. A simple explanation is that the site of SAA action on KvAP is its entire lateral interface with the PE:PG/decane bilayer, with SAA-induced changes in surface tension and bilayer packing order combining to modulate the shape and stability of various conformations. The KvAP structural adjustment to diverse bilayer pressure profiles has implications for understanding desirable and undesirable actions of SAA-like drugs and, broadly, predicts that channel gating, conductance and pharmacology may differ when membrane packing order differs, as in raft versus nonraft domains.

Isolation and characterization of a high affinity peptide inhibitor of ClC-2 chloride channels.

The ClC protein family includes voltage-gated chloride channels and chloride/proton exchangers. In eukaryotes, ClC proteins regulate membrane potential of excitable cells, contribute to epithelial transport, and aid in lysosomal acidification. Although structure/function studies of ClC proteins have been aided greatly by the available crystal structures of a bacterial ClC chloride/proton exchanger, the availability of useful pharmacological tools, such as peptide toxin inhibitors, has lagged far behind that of their cation channel counterparts. Here we report the isolation, from Leiurus quinquestriatus hebraeus venom, of a peptide toxin inhibitor of the ClC-2 chloride channel. This toxin, GaTx2, inhibits ClC-2 channels with a voltage-dependent apparent K(D) of approximately 20 pm, making it the highest affinity inhibitor of any chloride channel. GaTx2 slows ClC-2 activation by increasing the latency to first opening by nearly 8-fold but is unable to inhibit open channels, suggesting that this toxin inhibits channel activation gating. Finally, GaTx2 specifically inhibits ClC-2 channels, showing no inhibitory effect on a battery of other major classes of chloride channels and voltage-gated potassium channels. GaTx2 is the first peptide toxin inhibitor of any ClC protein. The high affinity and specificity displayed by this toxin will make it a very powerful pharmacological tool to probe ClC-2 structure/function.