Cardiac resynchronization using fusion pacing during exercise.

INTRODUCTION: Fusion pacing requires correct timing of left ventricular pacing to right ventricular activation, although it is unclear whether this is maintained when atrioventricular (AV) conduction changes during exercise. We used cardiopulmonary exercise testing (CPET) to compare cardiac resynchronization therapy (CRT) using fusion pacing or fixed AV delays (AVD). METHODS: Patients 6 months post-CRT implant with PR intervals < 250 ms performed two CPET tests, using either the SyncAV™ algorithm or fixed AVD of 120 ms in a double-blinded, randomized, crossover study. All other programming was optimized to produce the narrowest QRS duration (QRSd) possible. RESULTS: Twenty patients (11 male, age 71 [65-77] years) were recruited. Fixed AVD and fusion programming resulted in similar narrowing of QRSd from intrinsic rhythm at rest (p = .85). Overall, there was no difference in peak oxygen consumption (V̇O2 PEAK , p = .19), oxygen consumption at anaerobic threshold (VT1, p = .42), or in the time to reach either V̇O2 PEAK (p = .81) or VT1 (p = .39). The BORG rating of perceived exertion was similar between groups. CPET performance was also analyzed comparing whichever programming gave the narrowest QRSd at rest (119 [96-136] vs. 134 [119-142] ms, p < .01). QRSd during exercise (p = .03), peak O2 pulse (mL/beat, a surrogate of stroke volume, p = .03), and cardiac efficiency (watts/mL/kg/min, p = .04) were significantly improved. CONCLUSION: Fusion pacing is maintained during exercise without impairing exercise capacity compared with fixed AVD. However, using whichever algorithm gives the narrowest QRSd at rest is associated with a narrower QRSd during exercise, higher peak stroke volume, and improved cardiac efficiency.

Effective magnetic susceptibility of 3D-printed porous metal scaffolds.

PURPOSE: 3D-printed porous metal scaffolds are a promising emerging technology in orthopedic implant design. Compared to solid metal implants, porous metal implants have lower magnetic susceptibility values, which have a direct impact on imaging time and image quality. The purpose of this study is to determine the relationship between porosity and effective susceptibility through quantitative estimates informed by comparing coregistered scanned and simulated field maps. METHODS: Five porous scaffold cylinders were designed and 3D-printed in titanium alloy (Ti-6Al-4V) with nominal porosities ranging from 60% to 90% using a cellular sheet-based gyroid design. The effective susceptibility of each cylinder was estimated by comparing acquired B0 field maps against simulations of a solid cylinder of varying assigned magnetic susceptibility, where the orientation and volume of interest of the simulations was informed by a custom alignment phantom. RESULTS: Magnitude images and field maps showed obvious decreases in artifact size and field inhomogeneity with increasing porosity. The effective susceptibility was found to be linearly correlated with porosity (R2  = 0.9993). The extrapolated 100% porous (no metal) magnetic susceptibility was -9.9 ppm, closely matching the expected value of pure water (-9 ppm), indicating a reliable estimation of susceptibility. CONCLUSION: Effective susceptibility of porous metal scaffolds is linearly correlated with porosity. Highly porous implants have sufficiently low effective susceptibilities to be more amenable to routine imaging with MRI.

In vivo reverse total shoulder arthroplasty contact mechanics.

BACKGROUND: Several in vitro studies have investigated the biomechanics of reverse total shoulder arthroplasty (RTSA); however, few in vivo studies exist. The purpose of this study was to examine in vivo RTSA contact mechanics in clinically relevant arm positions. Our hypothesis was that contact would preferentially occur in the inferior region of the polyethylene liner. METHODS: Forty patients receiving a primary RTSA were recruited for a prospective cohort study. All patients received the same implant design with a nonretentive liner. Stereo radiographs were taken at maximal active range of motion. Model-based radiostereometric analysis was used to identify implant position. Contact area between the polyethylene and glenosphere was measured as the geometric intersection of the 2 components and compared with respect to polyethylene liner size, arm position, and relative position within the liner. RESULTS: There were no differences in the proportion of contact area in any arm position between polyethylene liner sizes, ranging from 30% ± 17% to 38% ± 23% for 36-mm liners and 32% ± 21% to 41% ± 25% for 42-mm liners. Contact was equally distributed between the superior and inferior halves of the liner at each arm position (P = .06-.79); however, greater contact area was observed in the outer radius of the liner when the arm was flexed (P = .002). CONCLUSION: This study highlights that contact mechanics are similar between 36- and 42-mm liners. Contact area is generally equally distributed throughout the liner across the range of motion and not preferentially in the inferior region as hypothesized.

Vibration of osteoblastic cells using a novel motion-control platform does not acutely alter cytosolic calcium, but desensitizes subsequent responses to extracellular ATP.

Low-magnitude high-frequency mechanical vibration induces biological responses in many tissues. Like many cell types, osteoblasts respond rapidly to certain forms of mechanostimulation, such as fluid shear, with transient elevation in the concentration of cytosolic free calcium ([Ca2+ ]i ). However, it is not known whether vibration of osteoblastic cells also induces acute elevation in [Ca2+ ]i . To address this question, we built a platform for vibrating live cells that is compatible with microscopy and microspectrofluorometry, enabling us to observe immediate responses of cells to low-magnitude high-frequency vibrations. The horizontal vibration system was mounted on an inverted microscope, and its mechanical performance was evaluated using optical tracking and accelerometry. The platform was driven by a sinusoidal signal at 20-500 Hz, producing peak accelerations from 0.1 to 1 g. Accelerometer-derived displacements matched those observed optically within 10%. We then used this system to investigate the effect of acceleration on [Ca2+ ]i in rodent osteoblastic cells. Cells were loaded with fura-2, and [Ca2+ ]i was monitored using microspectrofluorometry and fluorescence ratio imaging. No acute changes in [Ca2+ ]i or cell morphology were detected in response to vibration over the range of frequencies and accelerations studied. However, vibration did attenuate Ca2+ transients generated subsequently by extracellular ATP, which activates P2 purinoceptors and has been implicated in mechanical signaling in bone. In summary, we developed and validated a motion-control system capable of precisely delivering vibrations to live cells during real-time microscopy. Vibration did not elicit acute elevation of [Ca2+ ]i , but did desensitize responses to later stimulation with ATP.

Hip capsular strain varies between ligaments dependent on both hip position- and applied rotational force.

PURPOSE: To noninvasively characterize the ligament strain in the hip capsule using a novel CT-based imaging technique. METHODS: The superior iliofemoral ligament (SIFL), inferior iliofemoral ligament (IIFL), ischiofemoral ligament (IFL) and pubofemoral ligament (PFL) were identified and beaded in seven cadavers. Specimens were mounted on a joint motion simulator within an O-arm CT scanner in - 15°, 0°, 30°, 60°, and 90° of flexion. 3 Nm of internal rotation (IR) and external rotation (ER) were applied and CT scans obtained. Strains were calculated by comparing bead separation in loaded and unloaded conditions. Repeated-measures ANOVA was used to evaluate differences in strain within ligaments between hip positions. RESULTS: For the SIFL, strain significantly decreased in IR at 30° (p = 0.045) and 60° (p = 0.043) versus 0°. For ER, there were no significant position-specific changes in strain (n.s.). For the IIFL, strain decreased in IR and increased in ER with no significant position-specific differences. For the IFL, strain increased with IR and decreased with ER with no significant position-specific differences. Finally, in the PFL there was a significant flexion angle-by-load interaction (p < 0.001; ES = 0.566), with peak strains noted at 60˚, however pair-wise comparisons failed to identify significant differences between positions (n.s.). Strain decreased in ER, with no significant position-specific differences. CONCLUSION: The SIFL and IIFL limit hip external rotation with greater effect in higher flexion angles, while the IFL and PFL limit hip internal rotation. Following hip arthroscopy, consideration should be given to restricting external rotation as traditional capsulotomies cause injury to the SIFL and IIFL.

Development of a Wireless Telemetry Sensor Device to Measure Load and Deformation in Orthopaedic Applications.

Due to sensor size and supporting circuitry, in-vivo load and deformation measurements are currently restricted to applications within larger orthopaedic implants. The objective of this study is to repurpose a commercially available low-power, miniature, wireless, telemetric, tire-pressure sensor (FXTH87) to measure load and deformation for future use in orthopaedic and biomedical applications. The capacitive transducer membrane was modified, and compressive deformation was applied to the transducer to determine the sensor signal value and the internal resistive force. The sensor package was embedded within a deformable enclosure to illustrate potential applications of the sensor for monitoring load. To reach the maximum output signal value, sensors required compressive deformation of 350 ± 24 µm. The output signal value of the sensor was an effective predictor of the applied load on a calibrated plastic strain member, over a range of 35 N. The FXTH87 sensor can effectively sense and transmit load-induced deformations. The sensor does not have a limit on loads it can measure, as long as deformation resulting from the applied load does not exceed 350 µm. The proposed device presents a sensitive and precise means to monitor deformation and load within small-scale, deformable enclosures.

Echocardiographic changes following active heat acclimation.

Heat adaption through acclimatisation or acclimation improves cardiovascular stability by maintaining cardiac output due to compensatory increases in stroke volume. The main aim of this study was to assess whether 2D transthoracic echocardiography (TTE) could be used to confirm differences in resting echocardiographic parameters, before and after active heat acclimation (HA). Thirteen male endurance trained cyclists underwent a resting blinded TTE before and after randomisation to either 5 consecutive daily exertional heat exposures of controlled hyperthermia at 32°C with 70% relative humidity (RH) (HOT) or 5-days of exercise in temperate (21°C with 36% RH) environmental conditions (TEMP). Measures of HA included heart rate, gastrointestinal temperature, skin temperature, sweat loss, total non-urinary fluid loss (TNUFL), plasma volume and participant's ratings of perceived exertion (RPE). Following HA, the HOT group demonstrated increased sweat loss (p = 0.01) and TNUFL (p = 0.01) in comparison to the TEMP group with a significantly decreased RPE (p = 0.01). On TTE, post exposure, there was a significant comparative increase in the HOT group in left ventricular end diastolic volume (p = 0.029), SV (p = 0.009), left atrial volume (p = 0.005), inferior vena cava diameter (p = 0.041), and a significant difference in mean peak diastolic mitral annular velocity (e') (p = 0.044). Cardiovascular adaptations to HA appear to be predominantly mediated by improvements in increased preload and ventricular compliance. TTE is a useful tool to demonstrate and quantify cardiac HA.

Cardiac MRI improves cardiovascular risk stratification in hazardous occupations.

BACKGROUND: The benefit of cardiovascular magnetic resonance Imaging (CMR) in assessing occupational risk is unknown. Pilots undergo frequent medical assessment for occult disease, which threatens incapacitation or distraction during flight. ECG and examination anomalies often lead to lengthy restriction, pending full investigation. CMR provides a sensitive, specific assessment of cardiac anatomy, tissue characterisation, perfusion defects and myocardial viability. We sought to determine if CMR, when added to standard care, would alter occupational outcome. METHODS: A retrospective review was conducted of all personnel attending the RAF Aviation Medicine Consultation Service (AMCS) for assessment of a cardiac anomaly, over a 2-year period. Those undergoing standard of care (history, examination, exercise ECG, 24 h-Holter and transthoracic echocardiography), and those undergoing a CMR in addition, were identified. The influence of CMR upon the final decision regarding flying restriction was determined by comparing the diagnosis reached with standard of care plus CMR vs. standard of care alone. RESULTS: Of the ~ 8000 UK military aircrew, 558 personnel were seen for cardiovascular assessment. Fifty-two underwent CMR. A normal TTE did not reliably exclude abnormalities subsequently detected by CMR. Addition of CMR resulted in an upgraded occupational status in 62% of those investigated, with 37% returning to unrestricted duties. Only 8% of referrals were undiagnosed following CMR. All these were cases of borderline chamber dilatation and reduction in systolic function in whom diagnostic uncertainty remained between physiological exercise adaptation and early cardiomyopathy. CONCLUSIONS: CMR increases the likelihood of a definitive diagnosis and of return to flying. This study supports early use of CMR in occupational assessment for high-hazard occupations.

Development and Assessment of a Microcomputed Tomography Compatible Five Degrees-of-Freedom Knee Joint Motion Simulator.

Currently available knee joint kinematic tracking systems fail to nondestructively capture the subtle variation in joint and soft tissue kinematics that occur in native, injured, and reconstructed joint states. Microcomputed tomography (CT) imaging has the potential as a noninvasive, high-resolution kinematic tracking system, but no dynamic simulators exist to take advantage of this. The purpose of this work was to develop and assess a novel micro-CT compatible knee joint simulator to quantify the knee joint's kinematic and kinetic response to clinically (e.g., pivot shift test) and functionally (e.g., gait) relevant loading. The simulator applies closed-loop, load control over four degrees-of-freedom (DOF) (internal/external rotation, varus/valgus rotation, anterior/posterior translation, and compression/distraction), and static control over a fifth degree-of-freedom (flexion/extension). Simulator accuracy (e.g., load error) and repeatability (e.g., coefficient of variation) were assessed with a cylindrical rubber tubing structure and a human cadaveric knee joint by applying clinically and functionally relevant loads along all active axes. Micro-CT images acquired of the joint at a loaded state were then used to calculate joint kinematics. The simulator loaded both the rubber tubing and the cadaveric specimen to within 0.1% of the load target, with an intertrial coefficient of variation below 0.1% for all clinically relevant loading protocols. The resultant kinematics calculated from the acquired images agreed with previously published values, and produced errors of 1.66 mm, 0.90 mm, 4.41 deg, and 1.60 deg with respect to anterior translation, compression, internal rotation, and valgus rotation, respectively. All images were free of artifacts and showed knee joint displacements in response to clinically and functionally loading with isotropic CT image voxel spacing of 0.15 mm. The results of this study demonstrate that the joint-motion simulator is capable of applying accurate, clinically and functionally relevant loads to cadaveric knee joints, concurrent with micro-CT imaging. Nondestructive tracking of bony landmarks allows for the precise calculation of joint kinematics with less error than traditional optical tracking systems.

Polymer Assembly Encapsulation of Lanthanide Nanoparticles as Contrast Agents for In Vivo Micro-CT.

Despite recent technological advancements in microcomputed tomography (micro-CT) and contrast agent development, preclinical contrast agents are still predominantly iodine-based. Higher contrast can be achieved when using elements with higher atomic numbers, such as lanthanides; lanthanides also have X-ray attenuation properties that are ideal for spectral CT. However, the formulation of lanthanide-based contrast agents at the high concentrations required for vascular imaging presents a significant challenge. In this work, we developed an erbium-based contrast agent that meets micro-CT imaging requirements, which include colloidal stability upon redispersion at high concentrations, evasion of rapid renal clearance, and circulation times of tens of minutes in small animals. Through systematic studies with poly(ethylene glycol) (PEG)-poly(propylene glycol), PEG-polycaprolactone, and PEG-poly(l-lactide) (PLA) block copolymers, the amphiphilic block copolymer PEG114-PLA53 was identified to be ideal for encapsulating oleate-coated lanthanide-based nanoparticles for in vivo intravenous administration. We were able to synthesize a contrast agent containing 100 mg/mL of erbium that could be redispersed into colloidally stable particles in saline after lyophilization. Contrast enhancement of over 250 HU was achieved in the blood pool for up to an hour, thereby meeting the requirements of live animal micro-CT.

Smartphone-Enabled Heart Rate Variability and Acute Mountain Sickness.

INTRODUCTION: The autonomic system and sympathetic activation appears integral in the pathogenesis of acute mountain sickness (AMS) at high altitude (HA), yet a link between heart rate variability (HRV) and AMS has not been convincingly shown. In this study we investigated the utility of the smartphone-derived HRV score to predict and diagnose AMS at HA. METHODS: Twenty-one healthy adults were investigated at baseline at 1400 m and over 10 days during a trek to 5140 m. HRV was recorded using the ithlete HRV device. RESULTS: Acute mountain sickness occurred in 11 subjects (52.4%) at >2650 m. HRV inversely correlated with AMS Scores (r = -0.26; 95% CI, -0.38 to -0.13: P < 0.001). HRV significantly fell at 3700, 4100, and 5140 m versus low altitude. HRV scores were lower in those with both mild (69.7 ± 14.0) and severe AMS (67.1 ± 13.1) versus those without AMS (77.5 ± 13.1; effect size n = 0.043: P = 0.007). The HRV score was weakly predictive of severe AMS (AUC 0.74; 95% CI, 0.58-0.89: P = 0.006). The change (delta) in the HRV Score (compared with baseline at 1400 m) was a moderate diagnostic marker of severe AMS (AUC 0.80; 95% CI, 0.70-0.90; P = 0.0004). A fall in the HRV score of >5 had a sensitivity of 83% and specificity of 60% to identify severe AMS (likelihood ratio 1.9). Baseline HRV at 1400 m was not predictive of either AMS at higher altitudes. CONCLUSIONS: The ithlete HRV score can be used to help in the identification of severe AMS; however, a baseline score is not predictive of future AMS development at HA.

Varus tibial alignment is associated with greater tibial baseplate migration at 10 years following total knee arthroplasty.

PURPOSE: To examine implant migration and articular behavior of primary total knee arthroplasty (TKA) at 10 years after index surgery and correlate to implant alignment. METHODS: Thirty-five patients underwent a cemented posterior stabilized total knee arthroplasty with a surgical objective of neutral alignment and were enrolled in a long-term radiostereometric analysis (RSA) study. At 10 years after surgery, patients were analyzed for implant migration using RSA as well as radiographic assessment of articular behavior at four positions of knee flexion. Implant position and alignment was measured on full-length radiographs. Patient demographics and reported outcomes were also collected. RESULTS: No difference between patient demographics or patient-reported outcomes were found. When categorized into neutral and varus groupings, no difference in migration was present. If alignment was considered as a continuous variable, there was no correlation between overall leg alignment and migration, however, migration increased with an increasing varus tibial alignment. Although contact location did not differ between neutral and varus groups through a range of motion, condylar liftoff was much more common in the varus group, of which all were lateral liftoff. CONCLUSIONS: Increased tibial varus results in increased implant migration. Overall varus limb alignment is correlated with isolated lateral compartment liftoff, and liftoff occurs more commonly than in neutral aligned knees. The increased migration and liftoff raise concerns about the longevity of malaligned total knee replacements. If a goal of overall varus limb alignment is desired for TKA, the tibia should remain neutral. LEVEL OF EVIDENCE: Level III.

In-vitro comparison of different palatal sites for orthodontic miniscrew insertion: Effect of bone quality and quantity on primary stability.

INTRODUCTION: This experiment was undertaken to assess the primary stability of orthodontic miniscrews inserted at different sites in human cadaveric palatal bone for temporary skeletal anchorage, and to determine the effect of bone quality and quantity on their primary stability using microcomputed tomography imaging. METHODS: A total of 10 cadaveric maxillary hard palates were used for insertion of 130 orthodontic miniscrews (VectorTAS; Ormco, Orange, Calif; length, 6 mm; diameter, 1.4 mm). Upon insertion, maximal insertion torque (IT) was recorded. Imaging (microcomputed tomography) was performed before and after insertion for assessment of bone quality and quantity parameters (bone mineral density [BMD], bone thickness [BT], and length of screw engagement [LSE]). Differences in each parameter were assessed at the various insertion sites. Correlations between IT and measurements of BMD, BT, and LSE were evaluated. RESULTS: Significant differences (P < 0.001) were found among insertion sites for IT, BT, and LSE, but not for BMD (P = 0.004). Correlations were found between IT and BMD (rs = 0.42; P < 0.001), IT and BT (rs = 0.58; P < 0.001), and IT and LSE (rs = 0.58; P < 0.001). Most perforations of miniscrews into the nasal cavity occurred posterior to the permanent second premolars. CONCLUSIONS: The primary stability of orthodontic miniscrews in the palate is affected by bone quality and quantity, with higher primary stability obtained anterior to the second premolars and parasagittally at the level of the permanent first molars.

Whole-body vibration of mice induces progressive degeneration of intervertebral discs associated with increased expression of Il-1ß and multiple matrix degrading enzymes.

OBJECTIVE: Whole-body vibration (WBV) is a popular fitness trend based on claims of increased muscle mass, weight loss and reduced joint pain. Following its original implementation as a treatment to increase bone mass in patients with osteoporosis, WBV has been incorporated into clinical practice for musculoskeletal disorders, including back pain. However, our recent studies revealed damaging effects of WBV on joint health in a murine model. In this report, we examined potential mechanisms underlying disc degeneration following exposure of mice to WBV. METHODS: Ten-week-old male mice were exposed to WBV (45 Hz, 0.3 g peak acceleration, 30 min/day, 5 days/week) for 4 weeks, 8 weeks, or 4 weeks WBV followed by 4 weeks recovery. Micro-computed tomography (micro-CT), histological, and gene expression analyses were used to assess the effects of WBV on spinal tissues. RESULTS: Exposure of mice to 4 or 8 weeks of WBV did not alter total body composition or induce significant changes in vertebral bone density. On the other hand, WBV-induced intervertebral disc (IVD) degeneration, associated with decreased disc height and degenerative changes in the annulus fibrosus (AF) that did not recover within 4 weeks after cessation of WBV. Gene expression analysis showed that WBV for 8 weeks induced expression of Mmp3, Mmp13, and Adamts5 in IVD tissues, changes preceded by increased expression of Il-1ß. CONCLUSIONS: Progressive IVD degeneration induced by WBV was associated with increased expression of Il-1ß within the IVD that preceded Mmp and Adamts gene induction. Moreover, WBV-induced IVD degeneration is not reversed following cessation of vibration.

Changes in appetite, energy intake, body composition, and circulating ghrelin constituents during an incremental trekking ascent to high altitude.

PURPOSE: Circulating acylated ghrelin concentrations are associated with altitude-induced anorexia in laboratory environments, but have never been measured at terrestrial altitude. This study examined time course changes in appetite, energy intake, body composition, and ghrelin constituents during a high-altitude trek. METHODS: Twelve participants [age: 28(4) years, BMI 23.0(2.1) kg m-2] completed a 14-day trek in the Himalayas. Energy intake, appetite perceptions, body composition, and circulating acylated, des-acylated, and total ghrelin concentrations were assessed at baseline (113 m, 12 days prior to departure) and at three fixed research camps during the trek (3619 m, day 7; 4600 m, day 10; 5140 m, day 12). RESULTS: Relative to baseline, energy intake was lower at 3619 m (P = 0.038) and 5140 m (P = 0.016) and tended to be lower at 4600 m (P = 0.056). Appetite perceptions were lower at 5140 m (P = 0.027) compared with baseline. Acylated ghrelin concentrations were lower at 3619 m (P = 0.046) and 4600 m (P = 0.038), and tended to be lower at 5140 m (P = 0.070), compared with baseline. Des-acylated ghrelin concentrations did not significantly change during the trek (P = 0.177). Total ghrelin concentrations decreased from baseline to 4600 m (P = 0.045). Skinfold thickness was lower at all points during the trek compared with baseline (P ≤ 0.001) and calf girth decreased incrementally during the trek (P = 0.010). CONCLUSIONS: Changes in plasma acylated and total ghrelin concentrations may contribute to the suppression of appetite and energy intake at altitude, but differences in the time course of these responses suggest that additional factors are also involved. Interventions are required to maintain appetite and energy balance during trekking at terrestrial altitudes.

Markers of physiological stress during exercise under conditions of normoxia, normobaric hypoxia, hypobaric hypoxia, and genuine high altitude.

PURPOSE: To investigate whether there is a differential response at rest and following exercise to conditions of genuine high altitude (GHA), normobaric hypoxia (NH), hypobaric hypoxia (HH), and normobaric normoxia (NN). METHOD: Markers of sympathoadrenal and adrenocortical function [plasma normetanephrine (PNORMET), metanephrine (PMET), cortisol], myocardial injury [highly sensitive cardiac troponin T (hscTnT)], and function [N-terminal brain natriuretic peptide (NT-proBNP)] were evaluated at rest and with exercise under NN, at 3375 m in the Alps (GHA) and at equivalent simulated altitude under NH and HH. Participants cycled for 2 h [15-min warm-up, 105 min at 55% Wmax (maximal workload)] with venous blood samples taken prior (T0), immediately following (T120) and 2-h post-exercise (T240). RESULTS: Exercise in the three hypoxic environments produced a similar pattern of response with the only difference between environments being in relation to PNORMET. Exercise in NN only induced a rise in PNORMET and PMET. CONCLUSION: Biochemical markers that reflect sympathoadrenal, adrenocortical, and myocardial responses to physiological stress demonstrate significant differences in the response to exercise under conditions of normoxia versus hypoxia, while NH and HH appear to induce broadly similar responses to GHA and may, therefore, be reasonable surrogates.

Practical fabrication of microfluidic platforms for live-cell microscopy.

We describe a simple fabrication technique - targeted towards non-specialists - that allows for the production of leak-proof polydimethylsiloxane (PDMS) microfluidic devices that are compatible with live-cell microscopy. Thin PDMS base membranes were spin-coated onto a glass-bottom cell culture dish and then partially cured via microwave irradiation. PDMS chips were generated using a replica molding technique, and then sealed to the PDMS base membrane by microwave irradiation. Once a mold was generated, devices could be rapidly fabricated within hours. Fibronectin pre-treatment of the PDMS improved cell attachment. Coupling the device to programmable pumps allowed application of precise fluid flow rates through the channels. The transparency and minimal thickness of the device enabled compatibility with inverted light microscopy techniques (e.g. phase-contrast, fluorescence imaging, etc.). The key benefits of this technique are the use of standard laboratory equipment during fabrication and ease of implementation, helping to extend applications in live-cell microfluidics for scientists outside the engineering and core microdevice communities.

A bony connection signals laryngeal echolocation in bats.

Echolocation is an active form of orientation in which animals emit sounds and then listen to reflected echoes of those sounds to form images of their surroundings in their brains. Although echolocation is usually associated with bats, it is not characteristic of all bats. Most echolocating bats produce signals in the larynx, but within one family of mainly non-echolocating species (Pteropodidae), a few species use echolocation sounds produced by tongue clicks. Here we demonstrate, using data obtained from micro-computed tomography scans of 26 species (n = 35 fluid-preserved bats), that proximal articulation of the stylohyal bone (part of the mammalian hyoid apparatus) with the tympanic bone always distinguishes laryngeally echolocating bats from all other bats (that is, non-echolocating pteropodids and those that echolocate with tongue clicks). In laryngeally echolocating bats, the proximal end of the stylohyal bone directly articulates with the tympanic bone and is often fused with it. Previous research on the morphology of the stylohyal bone in the oldest known fossil bat (Onychonycteris finneyi) suggested that it did not echolocate, but our findings suggest that O. finneyi may have used laryngeal echolocation because its stylohyal bones may have articulated with its tympanic bones. The present findings reopen basic questions about the timing and the origin of flight and echolocation in the early evolution of bats. Our data also provide an independent anatomical character by which to distinguish laryngeally echolocating bats from other bats.