Collision-Based Electrochemical Detection of Lysozyme Aggregation.

Proteins are utilized across many biomedical and pharmaceutical industries; therefore, methods for rapid and accurate monitoring of protein aggregation are needed to ensure proper product quality. Although these processes have been previously studied, it is difficult to comprehensively evaluate protein folding and aggregation by traditional characterization techniques such as atomic force microscopy (AFM), electron microscopy, or X-ray diffraction, which require sample pre-treatment and do not represent native state proteins in solution. Herein, we report early tracking of lysozyme (Lyz) aggregation states by using single-particle collision electrochemistry (SPCE) of silver nanoparticle (AgNP) redox probes. The method relies on monitoring the rapid interaction of Lyz with AgNPs, which decreases the number of single AgNPs available for collisions and ultimately the frequency of oxidative impacts in the chronoamperometric profile. When Lyz is in a non-aggregated monomeric form, the protein forms a homogeneous coverage onto the surface of AgNPs, stabilizing the particles. When Lyz is aggregated, part of the AgNP surface remains uncoated, promoting the agglomeration of Lyz-AgNP conjugates. The frequency of AgNP impacts decreases with increasing aggregation time, providing a metric to track protein aggregation. Visualizations of integrated oxidation charge-transfer data displayed significant differences between the charge transfer per impact for AgNP samples alone and in the presence of non-aggregated and aggregated Lyz with 99% confidence using parametric ANOVA tests. Electrochemical results revealed meaningful associations with UV-vis, circular dichroism, and AFM, demonstrating that SPCE can be used as an alternative method for studying protein aggregation. This electrochemical technique could serve as a powerful tool to indirectly evaluate protein stability and screen protein samples for formation of aggregates.

G551D mutation impairs PKA-dependent activation of CFTR channel that can be restored by novel GOF mutations.

G551D is a major disease-associated gating mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) protein, an ATP- and phosphorylation-dependent chloride channel. G551D causes severe cystic fibrosis (CF) disease by disrupting ATP-dependent channel opening; however, whether G551D affects phosphorylation-dependent channel activation is unclear. Here, we use macropatch recording and Ussing chamber approaches to demonstrate that G551D impacts on phosphorylation-dependent activation of CFTR, and PKA-mediated phosphorylation regulates the interaction between the x-loop in nucleotide-binding domain 2 (NBD2) and cytosolic loop (CL) 1. We show that G551D not only disrupts ATP-dependent channel opening but also impairs phosphorylation-dependent channel activation by largely reducing PKA sensitivity consistent with the reciprocal relationship between channel opening/gating, ligand binding, and phosphorylation. Furthermore, we identified two novel GOF mutations: D1341R in the x-loop near the ATP-binding cassette signature motif in NBD2 and D173R in CL1, each of which strongly increased PKA sensitivity both in the wild-type (WT) background and when introduced into G551D-CFTR. When D1341R was combined with a second GOF mutation (e.g., K978C in CL3), we find that the double GOF mutation maximally increased G551D channel activity such that VX-770 had no further effect. We further show that a double charge-reversal mutation of D1341R/D173R-CFTR exhibited similar PKA sensitivity when compared with WT-CFTR. Together, our results suggest that charge repulsion between D173 and D1341 of WT-CFTR normally inhibits channel activation at low PKA activity by reducing PKA sensitivity, and negative allostery by the G551D is coupled to reduced PKA sensitivity of CFTR that can be restored by second GOF mutations.

Nanoparticle Characterization Through Nano-Impact Electrochemistry: Tools and Methodology Development.

The field of nanomaterials has been expanding rapidly into many diverse applications within the last 20 years. With this growth, there is a significant need for new method development for the detection and characterization of nanomaterials. Understanding the physical properties of nanoscale entities and their associated reaction kinetics is crucial for monitoring their effect on environmental and human health, and in their use for practical applications. Nano-impact electrochemistry is a novel development in the field of fundamental electrochemistry that provides an ultrasensitive method for analyzing physical and redox properties of nanomaterials and their derivatives. This protocol focuses on the tools required for characterizing silver nanoparticles (AgNPs) by nano-impact electrochemistry, the preparation of microelectrodes and the methodology needed for measurement of the AgNP redox activity. The fabrication of cylindrical carbon fiber as well as gold and platinum microwire electrodes is described in detail. The analysis of nano-impact electrochemistry for the characterization of redox active entities is also outlined with examples of applications.

Slowing ribosome velocity restores folding and function of mutant CFTR.

Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR), with approximately 90% of patients harboring at least one copy of the disease-associated variant F508del. We utilized a yeast phenomic system to identify genetic modifiers of F508del-CFTR biogenesis, from which ribosomal protein L12 (RPL12/uL11) emerged as a molecular target. In the present study, we investigated mechanism(s) by which suppression of RPL12 rescues F508del protein synthesis and activity. Using ribosome profiling, we found that rates of translation initiation and elongation were markedly slowed by RPL12 silencing. However, proteolytic stability and patch-clamp assays revealed RPL12 depletion significantly increased F508del-CFTR steady-state expression, interdomain assembly, and baseline open-channel probability. We next evaluated whether Rpl12-corrected F508del-CFTR could be further enhanced with concomitant pharmacologic repair (e.g., using clinically approved modulators lumacaftor and tezacaftor) and demonstrated additivity of these treatments. Rpl12 knockdown also partially restored maturation of specific CFTR variants in addition to F508del, and WT Cftr biogenesis was enhanced in the pancreas, colon, and ileum of Rpl12 haplosufficient mice. Modulation of ribosome velocity therefore represents a robust method for understanding both CF pathogenesis and therapeutic response.

Easy-to-Use Sensors for Field Monitoring of Copper Contamination in Water and Pesticide-Sprayed Plants.

Copper (Cu2+)-containing pesticides are commonly used in agriculture to control fungal and bacterial diseases, but the release of large quantities of Cu2+ in water and soil can lead to harmful long-term consequences on the environment, organisms, and ecosystem health. Technology available to measure Cu2+ accumulation in the field is too expensive and complicated for general population use. We describe a low-cost sensor with simplified user operation for measuring Cu2+ content in environmental and agricultural samples at sensitivity levels comparable with a laboratory-based atomic absorption spectroscopy (AAS) method. The sensor is based on polyethyeleneimine (PEI), which has a strong chelating ability for Cu2+ ions. The PEI is stabilized on paper by layer-by-layer assembly with the PEI deposited sequentially within electrostatically charged poly(styrenesulfonate) (PSS). The PEI-PSS layers develop a vivid blue complex when interacting with Cu2+, and the resulting color intensity varies with the Cu2+ concentration. Our sensors give a yes or no response with the naked eye down to 10 µM when a preconcentration step was used. A more precise quantitative response can be obtained using a smartphone or scanner and free imaging software within a wide linear range from 10 to 2000 µM with a detection limit of 0.795 µM. The sensors were used for detecting commercial Cu2+-based pesticides in water and pesticide-sprayed plants within 15 min. Considering that these sensors are robust, simple to operate, and extremely stable, they could be ideal for remote monitoring of Cu2+ ion exposure and for the analysis of Cu2+ in environmental water and agricultural fields.

Early vs Delayed Weightbearing After Microfracture of Osteochondral Lesions of the Talus: A Prospective Randomized Trial.

BACKGROUND: Osteochondral lesions of the talus (OLTs) are common injuries in young, active patients. Microfracture is an effective treatment for lesions less than 150 mm2 in size. Most commonly employed postoperative protocols involve delaying weightbearing for 6 to 8 weeks (DWB), though one study suggests that early weightbearing (EWB) may not be detrimental to patient outcomes. The goal of this research is to compare outcomes following EWB and DWB protocols after microfracture for OLTs. METHODS: We performed a prospective, randomized, multicenter clinical trial of subjects with unilateral, primary, unifocal OLTs treated with microfracture. Thirty-eight subjects were randomized into EWB (18 subjects) and DWB (20 subjects) at their first postsurgical visit. The EWB group began unrestricted WB at that time, whereas the DWB group were instructed to remain strictly nonweightbearing for an additional 4 weeks. Primary outcome measures were the American Academy of Orthopaedic Surgery (AAOS) Foot and Ankle score and numeric rating scale (NRS) pain score. RESULTS: The EWB group demonstrated significant improvement in AAOS Foot and Ankle Questionnaire scores at the 6-week follow-up appointment as compared to the DWB group (83.1 ± 13.5 vs 68.7 ± 15.8, P = .017). Following this point, there were no significant differences in AAOS scores between groups. At no point were NRS pain scores significantly different between the groups. CONCLUSIONS: EWB after microfracture for OLTs was associated with improved AAOS scores in the short term. Thereafter and through 2 years' follow-up, no statistically significant differences were seen between EWB and DWB groups. LEVEL OF EVIDENCE: Level II, prospective randomized trial.

Nanomaterial-functionalized Cellulose: Design, Characterization and Analytical Applications.

Cellulose-nanomaterial hybrid systems are promising platforms for the development of portable devices that can be used for fast and inexpensive analysis in the clinical, environmental and food monitoring fields. By combining the chemical and physical properties of the cellulosic network with the unique optical, electrical and catalytic functions of nanomaterials, it is possible to create versatile devices with engineered sensing functions. This review describes the most commonly used types of nanomaterials, their unique properties and assembly in hybrid structures in conjunction with cellulose paper and provides an overview of the most commonly used detection methodologies and their performance for selected applications. Finally, future perspectives and challenges to the implementation of these devices for real world applications are discussed, with focus on method optimization, validation and regulation in order to reach consumers.

Comparison of Calcaneal Exposure Through the Extensile Lateral and Sinus Tarsi Approaches.

The purpose of this study was to compare the exposure of the posterior facet with the extensile lateral (EL) approach compared with the sinus tarsi (ST) approach. We hypothesized that the ST approach will provide a similar exposure of the posterior calcaneal facet. A total of 8 sequential ST then EL approaches were performed on cadavers. Calcaneal landmarks were identified by visualization or palpation. Calibrated digital photographs of the posterior facet and lateral calcaneal body were obtained from standardized positions and used to calculate the exposed surface area. No significant difference was found in the average square area of the posterior facet exposed with the 2 approaches. Significantly more of the lateral calcaneal body was seen with the EL approach. Excluding the posterior facet superomedial quadrant, all the landmarks were visualized in 100% of approaches. The superomedial corner was visualized in significantly more of the cadavers with the EL approach and was palpable in 12.5% of the remaining cadavers in both approaches. Whereas the ST approach exposes less of the lateral wall of the calcaneus, it exposes similar amounts of the posterior facet when compared with the EL approach. LEVELS OF EVIDENCE: Therapeutic, Level V.

The Cystic Fibrosis Transmembrane Conductance Regulator Potentiator Ivacaftor Augments Mucociliary Clearance Abrogating Cystic Fibrosis Transmembrane Conductance Regulator Inhibition by Cigarette Smoke.

Acquired cystic fibrosis transmembrane conductance regulator (CFTR) dysfunction may contribute to chronic obstructive pulmonary disease pathogenesis and is a potential therapeutic target. We sought to determine the acute effects of cigarette smoke on ion transport and the mucociliary transport apparatus, their mechanistic basis, and whether deleterious effects could be reversed with the CFTR potentiator ivacaftor (VX-770). Primary human bronchial epithelial (HBE) cells and human bronchi were exposed to cigarette smoke extract (CSE) and/or ivacaftor. CFTR function and expression were measured in Ussing chambers and by surface biotinylation. CSE-derived acrolein modifications on CFTR were determined by mass spectroscopic analysis of purified protein, and the functional microanatomy of the airway epithelia was measured by 1-µm resolution optical coherence tomography. CSE reduced CFTR-dependent current in HBE cells (P < 0.05) and human bronchi (P < 0.05) within minutes of exposure. The mechanism involved CSE-induced reduction of CFTR gating, decreasing CFTR open-channel probability by approximately 75% immediately after exposure (P < 0.05), whereas surface CFTR expression was partially reduced with chronic exposure, but was stable acutely. CSE treatment of purified CFTR resulted in acrolein modifications on lysine and cysteine residues that likely disrupt CFTR gating. In primary HBE cells, CSE reduced airway surface liquid depth (P < 0.05) and ciliary beat frequency (P < 0.05) within 60 minutes that was restored by coadministration with ivacaftor (P < 0.005). Cigarette smoking transmits acute reductions in CFTR activity, adversely affecting the airway surface. These effects are reversible by a CFTR potentiator in vitro, representing a potential therapeutic strategy in patients with chronic obstructive pulmonary disease with chronic bronchitis.

Analysis of cystic fibrosis-associated P67L CFTR illustrates barriers to personalized therapeutics for orphan diseases.

Emerging knowledge indicates the difficulty in categorizing unusual cystic fibrosis (CF) mutations, with regard to both pathogenic mechanism and theratype. As case in point, we present data concerning P67L mutation of the cystic fibrosis transmembrane conductance regulator (CFTR), a defect carried by a small number of individuals with CF and sometimes attributed to a channel conductance abnormality. Findings from our laboratory and others establish that P67L causes protein misfolding, disrupts maturation, confers gating defects, is thermally stable, and exhibits near normal conductance. These results provide one framework by which rare CF alleles such as P67L can be more comprehensively profiled vis-à-vis molecular pathogenesis. We also demonstrate that emerging CF treatments - ivacaftor and lumacaftor - can mediate pronounced pharmacologic activation of P67L CFTR. Infrequent CF alleles are often improperly characterized, in part, due to the small numbers of patients involved. Moreover, access to new personalized treatments among patients with ultra-orphan genotypes has been limited by difficulty arranging phase III clinical trials, and off-label prescribing has been impaired by high drug cost and difficulty arranging third party reimbursement. Rare CFTR mutations such as P67L are emblematic of the challenges to "precision" medicine, including use of the best available mechanistic knowledge to treat patients with unusual forms of disease.

Comparative Study of Assisted Ambulation and Perceived Exertion With the Wheeled Knee Walker and Axillary Crutches in Healthy Subjects.

BACKGROUND: Functional limitations after lower extremity surgery often require the use of an assistive device for ambulation during rehabilitation and recovery. There are no known objective data evaluating the wheeled knee walker as an assistive device for protected ambulation. The purpose of this study was to compare assisted ambulation and perceived exertion with the wheeled knee walker and the axillary crutches in healthy participants. METHODS: A prospective, randomized crossover study was performed using 24 healthy volunteers. Each participant performed a 6-minute walk test (6MWT) using each assistive device in a crossover manner. Preactivity and postactivity heart rates were recorded. The self-selected walking velocity (SSWV) was calculated and the participant's rating of perceived exertion was recorded using the OMNI Rating of Perceived Exertion (OMNI-RPE). Participant's preference for assistive device was identified. RESULTS: The 6MWT, SSWV, and the Omni-RPE were evaluated using paired t tests and determined to be statistically significant for the wheeled knee walker compared with axillary crutches. Evaluation of the preactivity and postactivity heart rates demonstrated a statistically significant difference for the wheeled knee walker compared with axillary crutches. The wheeled knee walker was preferred by 88% of participants. CONCLUSIONS: The wheeled knee walker provided increased assisted ambulation and had a lower rating of perceived exertion than axillary crutches on level surfaces in healthy participants. LEVEL OF EVIDENCE: Level III, comparative study.

Robust Stimulation of W1282X-CFTR Channel Activity by a Combination of Allosteric Modulators.

W1282X is a common nonsense mutation among cystic fibrosis patients that results in the production of a truncated Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) channel. Here we show that the channel activity of the W1282X-CFTR polypeptide is exceptionally low in excised membrane patches at normally saturating doses of ATP and PKA (single channel open probability (PO) < 0.01). However, W1282X-CFTR channels were stimulated by two CFTR modulators, the FDA-approved VX-770 and the dietary compound curcumin. Each of these compounds is an allosteric modulator of CFTR gating that promotes channel activity in the absence of the native ligand, ATP. Although W1282X-CFTR channels were stimulated by VX-770 in the absence of ATP their activities remained dependent on PKA phosphorylation. Thus, activated W1282X-CFTR channels should remain under physiologic control by cyclic nucleotide signaling pathways in vivo. VX-770 and curcumin exerted additive effects on W1282X-CFTR channel gating (opening/closing) in excised patches such that the Po of the truncated channel approached unity (> 0.9) when treated with both modulators. VX-770 and curcumin also additively stimulated W1282X-CFTR mediated currents in polarized FRT epithelial monolayers. In this setting, however, the stimulated W1282X-CFTR currents were smaller than those mediated by wild type CFTR (3-5%) due presumably to lower expression levels or cell surface targeting of the truncated protein. Combining allosteric modulators of different mechanistic classes is worth considering as a treatment option for W1282X CF patients perhaps when coupled with maneuvers to increase expression of the truncated protein.

Channel Gating Regulation by the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) First Cytosolic Loop.

In this study, we present data indicating a robust and specific domain interaction between the cystic fibrosis transmembrane conductance regulator (CFTR) first cytosolic loop (CL1) and nucleotide binding domain 1 (NBD1) that allows ion transport to proceed in a regulated fashion. We used co-precipitation and ELISA to establish the molecular contact and showed that binding kinetics were not altered by the common clinical mutation F508del. Both intrinsic ATPase activity and CFTR channel gating were inhibited severely by CL1 peptide, suggesting that NBD1/CL1 binding is a crucial requirement for ATP hydrolysis and channel function. In addition to cystic fibrosis, CFTR dysregulation has been implicated in the pathogenesis of prevalent diseases such as chronic obstructive pulmonary disease, acquired rhinosinusitis, pancreatitis, and lethal secretory diarrhea (e.g. cholera). On the basis of clinical relevance of the CFTR as a therapeutic target, a cell-free drug screen was established to identify modulators of NBD1/CL1 channel activity independent of F508del CFTR and pharmacologic rescue. Our findings support a targetable mechanism of CFTR regulation in which conformational changes in the NBDs cause reorientation of transmembrane domains via interactions with CL1 and result in channel gating.

Long-range coupling between the extracellular gates and the intracellular ATP binding domains of multidrug resistance protein pumps and cystic fibrosis transmembrane conductance regulator channels.

The ABCC transporter subfamily includes pumps, the long and short multidrug resistance proteins (MRPs), and an ATP-gated anion channel, the cystic fibrosis transmembrane conductance regulator (CFTR). We show that despite their thermodynamic differences, these ABCC transporter subtypes use broadly similar mechanisms to couple their extracellular gates to the ATP occupancies of their cytosolic nucleotide binding domains. A conserved extracellular phenylalanine at this gate was a prime location for producing gain of function (GOF) mutants of a long MRP in yeast (Ycf1p cadmium transporter), a short yeast MRP (Yor1p oligomycin exporter), and human CFTR channels. Extracellular gate mutations rescued ATP binding mutants of the yeast MRPs and CFTR by increasing ATP sensitivity. Control ATPase-defective MRP mutants could not be rescued by this mechanism. A CFTR double mutant with an extracellular gate mutation plus a cytosolic GOF mutation was highly active (single-channel open probability >0.3) in the absence of ATP and protein kinase A, each normally required for CFTR activity. We conclude that all 3 ABCC transporter subtypes use similar mechanisms to couple their extracellular gates to ATP occupancy, and highly active CFTR channels that bypass defects in ATP binding or phosphorylation can be produced.

Determining optimal ultrasound off time with micropulse longitudinal phacoemulsification.

PURPOSE: To evaluate the optimum off time for the most efficient removal of lens fragments using micropulse ultrasound (US). SETTING: John A. Moran Eye Center Laboratories, University of Utah, Salt Lake City, Utah, USA. DESIGN: Experimental study. METHODS: Porcine lens nuclei were soaked in formalin for 2 hours and then cut into 2.0 mm cubes using the Signature US machine with a bent 0.9 mm phaco tip with a 30-degree bevel. The on time was 7 milliseconds (ms), and the off time was varied from 2 to 20 ms in 2 ms steps. Phacoemulsification efficiency (time for fragment removal) and chatter (number of times the fragment bounced from the tip) were measured. RESULTS: A nonsignificant linear increase in efficiency was observed with 2 to 6 ms of off time (R(2) = .87, P = .24). A significant linear decrease in efficiency was observed with 6 to 20 ms (R(2) = .74, P = .006). CONCLUSIONS: With micropulse longitudinal US, 6 to 7 ms of off time was as efficient as shorter off times; longer off times (8 to 20 ms) showed decreased efficiency. Chatter was minimal and statistically similar throughout. To maximize phacoemulsification US efficiency, an off-time setting of 6 ms is recommended. FINANCIAL DISCLOSURE: No author has a financial or proprietary interest in any material or method mentioned.

An electrostatic interaction at the tetrahelix bundle promotes phosphorylation-dependent cystic fibrosis transmembrane conductance regulator (CFTR) channel opening.

The CFTR channel is an essential mediator of electrolyte transport across epithelial tissues. CFTR opening is promoted by ATP binding and dimerization of its two nucleotide binding domains (NBDs). Phosphorylation of its R domain (e.g. by PKA) is also required for channel activity. The CFTR structure is unsolved but homology models of the CFTR closed and open states have been produced based on the crystal structures of evolutionarily related ABC transporters. These models predict the formation of a tetrahelix bundle of intracellular loops (ICLs) during channel opening. Here we provide evidence that residues E267 in ICL2 and K1060 in ICL4 electrostatically interact at the interface of this predicted bundle to promote CFTR opening. Mutations or a thiol modifier that introduced like charges at these two positions substantially inhibited ATP-dependent channel opening. ATP-dependent activity was rescued by introducing a second site gain of function (GOF) mutation that was previously shown to promote ATP-dependent and ATP-independent opening (K978C). Conversely, the ATP-independent activity of the K978C GOF mutant was inhibited by charge- reversal mutations at positions 267 or 1060 either in the presence or absence of NBD2. The latter result indicates that this electrostatic interaction also promotes unliganded channel opening in the absence of ATP binding and NBD dimerization. Charge-reversal mutations at either position markedly reduced the PKA sensitivity of channel activation implying strong allosteric coupling between bundle formation and R domain phosphorylation. These findings support important roles of the tetrahelix bundle and the E267-K1060 electrostatic interaction in phosphorylation-dependent CFTR gating.

Determining optimal torsional ultrasound power for cataract surgery with automatic longitudinal pulses at maximum vacuum ex vivo.

PURPOSE: To determine the optimal longitudinal power settings for Infiniti OZil Intelligent Phaco (IP) at varying torsional amplitude settings; and to test the hypothesis that increasing longitudinal power is more important at lower torsional amplitudes to achieve efficient phacoemulsification. DESIGN: Laboratory investigation. METHODS: setting: John A. Moran Eye Center, University of Utah, Salt Lake City, Utah. procedure: Individual porcine nuclei were fixed in formalin, then cut into 2.0 mm cubes. Lens cube phacoemulsification was done using OZil IP at 60%, 80%, and 100% torsional amplitude with 0%, 10%, 20%, 30%, 50%, 75%, or 100% longitudinal power. All experiments were done using a 20 gauge 0.9 mm bent reverse bevel phaco tip at constant vacuum (550 mm Hg), aspiration rate (40 mL/min), and bottle height (50 cm). main outcome measure: Complete lens particle phacoemulsification (efficiency). RESULTS: Linear regression analysis showed a significant increase in efficiency with increasing longitudinal power at 60% torsional amplitude (R(2) = 0.7269, P = .01) and 80% torsional amplitude (R(2) = 0.6995, P = .02) but not at 100% amplitude (R(2) = 0.3053, P = .2). Baseline comparison of 60% or 80% vs 100% torsional amplitude without longitudinal power showed increased efficiency at 100% (P = .0004). Increasing longitudinal power to 20% abolished the efficiency difference between 80% vs 100% amplitudes. In contrast, 75% longitudinal power abolished the efficiency difference between 60% vs 100% torsional amplitudes. CONCLUSIONS: Results suggest that longitudinal power becomes more critical at increasing phacoemulsification efficiencies at torsional amplitudes less than 100%. Increasing longitudinal power does not further increase efficiency at maximal torsional amplitudes.

Optimum on-time duty cycle for micropulse technology.

PURPOSE: To evaluate the optimum on time for the most efficient removal of lens fragments using micropulsed ultrasound (US). SETTING: John A. Moran Eye Center Laboratories, University of Utah, Salt Lake City, Utah, USA. DESIGN: Experimental study. METHODS: Twenty porcine lens nuclei were soaked in formalin for 2 hours and then divided into 2.0 mm cubes. Using an US machine with a 0.9 mm bent and a 30-degree bevel tip, the on time was varied every millisecond (ms) from 2 ms to 10 ms with the off time kept constant at 10 ms. Efficiency (time to lens removal) and chatter (number of lens fragment repulsions from the tip) were determined. RESULTS: The most efficient phacoemulsification was achieved with an on time of 6 ms. On times shorter than 6 ms were significantly less efficient (R2=.82, P=.04). Greater on times did not result in a significant difference in efficiency (R2=.03, P=.78) but did appear to have more chatter events when comparing 9 to 10 ms with 2 to 8 ms (P<.0001). CONCLUSIONS: With micropulsed longitudinal US, a 6 ms on time was equally as efficient as longer on times, while shorter on times (2 to 5 ms) had decreased efficiency. At 9 ms and 10 ms on time, significantly more chatter was noted. Therefore, to maximize phacoemulsification, an on-time setting of 6 ms is recommended. FINANCIAL DISCLOSURE: No author has a financial or proprietary interest in any material or method mentioned.

Conserved allosteric hot spots in the transmembrane domains of cystic fibrosis transmembrane conductance regulator (CFTR) channels and multidrug resistance protein (MRP) pumps.

ATP-binding cassette (ABC) transporters are an ancient family of transmembrane proteins that utilize ATPase activity to move substrates across cell membranes. The ABCC subfamily of the ABC transporters includes active drug exporters (the multidrug resistance proteins (MRPs)) and a unique ATP-gated ion channel (cystic fibrosis transmembrane conductance regulator (CFTR)). The CFTR channel shares gating principles with conventional ligand-gated ion channels, but the allosteric network that couples ATP binding at its nucleotide binding domains (NBDs) with conformational changes in its transmembrane helices (TMs) is poorly defined. It is also unclear whether the mechanisms that govern CFTR gating are conserved with the thermodynamically distinct MRPs. Here we report a new class of gain of function (GOF) mutation of a conserved proline at the base of the pore-lining TM6. Multiple substitutions of this proline promoted ATP-free CFTR activity and activation by the weak agonist, 5'-adenylyl-ß,γ-imidodiphosphate (AMP-PNP). TM6 proline mutations exhibited additive GOF effects when combined with a previously reported GOF mutation located in an outer collar of TMs that surrounds the pore-lining TMs. Each TM substitution allosterically rescued the ATP sensitivity of CFTR gating when introduced into an NBD mutant with defective ATP binding. Both classes of GOF mutations also rescued defective drug export by a yeast MRP (Yor1p) with ATP binding defects in its NBDs. We conclude that the conserved TM6 proline helps set the energy barrier to both CFTR channel opening and MRP-mediated drug efflux and that CFTR channels and MRP pumps utilize similar allosteric mechanisms for coupling conformational changes in their translocation pathways to ATP binding at their NBDs.