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.

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.

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.

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.

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.

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.

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.

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.

Comparison of functional outcomes following bridge synostosis with non-bone-bridging transtibial combat-related amputations.

BACKGROUND: The prevalence of penetrating wartime trauma to the extremities has increased in recent military conflicts. Substantial controversy remains in the orthopaedic and prosthetic literature regarding which surgical technique should be performed to obtain the most functional transtibial amputation. We compared self-reported functional outcomes associated with two surgical techniques for transtibial amputation: bridge synostosis (modified Ertl) and non-bone-bridging (modified Burgess). METHODS: A review of the prospective military amputee database was performed to identify patients who had undergone transtibial amputation between June 2003 and December 2010 at three military institutions receiving the majority of casualties from the most recent military conflicts; two of those institutions, Walter Reed Army Medical Center and National Naval Medical Center, have since been consolidated. Short Form-36, Prosthesis Evaluation Questionnaire, and functional data questions were completed by twenty-seven modified Ertl and thirty-eight modified Burgess isolated transtibial amputees. RESULTS: The average duration of follow-up after amputation (and standard deviation) was 32 ± 22.7 months, which was similar between groups. Residual limb length was significantly longer in the modified Ertl cohort by 2.5 cm (p < 0.005), and significantly more modified Ertl patients had delayed amputations (p < 0.005). There were no significant differences between groups with regard to any of the Short Form-36 domains or Prosthesis Evaluation Questionnaire subsections. CONCLUSIONS: The modified Ertl and Burgess techniques offer similar functional outcomes in the young, active-duty military population managed with transtibial amputation.

Converting nonhydrolyzable nucleotides to strong cystic fibrosis transmembrane conductance regulator (CFTR) agonists by gain of function (GOF) mutations.

Cystic fibrosis transmembrane conductance regulator (CFTR) is the only ligand-gated ion channel that hydrolyzes its agonist, ATP. CFTR gating has been argued to be tightly coupled to its enzymatic activity, but channels do open occasionally in the absence of ATP and are reversibly activated (albeit weakly) by nonhydrolyzable nucleotides. Why the latter only weakly activates CFTR is not understood. Here we show that CFTR activation by adenosine 5'-O-(thiotriphosphate) (ATPγS), adenosine 5'-(ß,γ-imino)triphosphate (AMP-PNP), and guanosine 5'-3-O-(thio)triphosphate (GTPγS) is enhanced substantially by gain of function (GOF) mutations in the cytosolic loops that increase unliganded activity. This enhancement correlated with the base-line nucleotide-independent activity for several GOF mutations. AMP-PNP or ATPγS activation required both nucleotide binding domains (NBDs) and was disrupted by a cystic fibrosis mutation in NBD1 (G551D). GOF mutant channels deactivated very slowly upon AMP-PNP or ATPγS removal (τdeac ∼ 100 s) implying tight binding between the two NBDs. Despite this apparently tight binding, neither AMP-PNP nor ATPγS activated even the strongest GOF mutant as strongly as ATP. ATPγS-activated wild type channels deactivated more rapidly, indicating that GOF mutations in the cytosolic loops reciprocally/allosterically affect nucleotide occupancy of the NBDs. A GOF mutation substantially rescued defective ATP-dependent gating of G1349D-CFTR, a cystic fibrosis NBD2 signature sequence mutant. Interestingly, the G1349D mutation strongly disrupted activation by AMP-PNP but not by ATPγS, indicating that these analogs interact differently with the NBDs. We conclude that poorly hydrolyzable nucleotides are less effective than ATP at opening CFTR channels even when they bind tightly to the NBDs but are converted to stronger agonists by GOF mutations.

Treatment of open proximal femoral fractures sustained in combat.

BACKGROUND: Open proximal femoral fractures are rare injuries that often result from wartime high-energy causes. Limited data exist regarding the treatment and complications of these injuries. METHODS: We retrospectively reviewed the records of combat casualties treated at two institutions between March 2003 and March 2008. The casualty patient databases, medical records, radiographs, and laboratory data were reviewed to determine time to union, complication rates, and patient outcomes. RESULTS: Forty-one patients (thirty-nine men and two women) with a mean age of 25.7 years were identified as receiving treatment for open proximal femoral fractures. The mechanisms of injury for these forty-one patients were blast (twenty-nine patients [71%]), gunshot wound (eight patients [20%]), motor vehicle crash (three patients [7%]), and helicopter crash (one patient [2%]). There were thirty Type-IIIA, six Type-IIIB, and five Type-IIIC open fractures. The predominant method of definitive fixation was a cephalomedullary or reconstruction nail in thirty-four patients (83%). Thirty-nine patients had at least two years of follow-up data available for assessment of complications and radiographic union. The mean time to union was 5.1 months (range, 2.8 to 16.0 months). Complications requiring reoperation occurred in twenty-two (56%) of thirty-nine patients. Wound infection (twelve patients [31%]) and symptomatic heterotopic ossification (ten patients [26%]) were the most common complications. CONCLUSIONS: Cephalomedullary nail fixation of open Type-III wartime subtrochanteric and pertrochanteric femoral fractures can be reliably used to effect fracture union in a timely manner. The most frequent complications of treatment are wound infection and symptomatic heterotopic ossification.

The CFTR ion channel: gating, regulation, and anion permeation.

Cystic fibrosis transmembrane conductance regulator (CFTR) is an ATP-gated anion channel with two remarkable distinctions. First, it is the only ATP-binding cassette (ABC) transporter that is known to be an ion channel--almost all others function as transport ATPases. Second, CFTR is the only ligand-gated channel that consumes its ligand (ATP) during the gating cycle--a consequence of its enzymatic activity as an ABC transporter. We discuss these special properties of CFTR in the context of its evolutionary history as an ABC transporter. Other topics include the mechanisms by which CFTR gating is regulated by phosphorylation of its unique regulatory domain and our current view of the CFTR permeation pathway (or pore). Understanding these basic operating principles of the CFTR channel is central to defining the mechanisms of action of prospective cystic fibrosis drugs and to the development of new, rational treatment strategies.

Quantification of posterior ankle exposure through an achilles tendon-splitting versus posterolateral approach.

BACKGROUND: The optimal surgical exposure to the posterior ankle for trauma and reconstruction is a source of debate. We hypothesized that the Achilles tendon-splitting approach would provide greater exposure to the posterior ankle than the posterolateral approach. METHODS: Forty surgical approaches were performed from twenty fresh-frozen cadavers. Achilles tendon-splitting and posterolateral approaches were performed using a randomized crossover design for surgical sequence. Six landmarks (medial malleolus, ankle joint, subtalar joint, incisura fibularis, lateral malleolus and medial gutter) were identified by direct visualization or palpation. A calibrated digital photograph was taken and Image J (http://rsb.info.nih.gov/ij/) was used to calculate the surface area of the distal tibia and talus exposed in neutral and dorsiflexion. RESULTS: Using a posterolateral approach, the average distal tibia exposed was 11.3cm(2) in neutral and 10.2 cm(2) in dorsiflexion. The average talus exposed was 2.0 cm(2) in neutral and 2.4 cm(2) in dorsiflexion. Using an Achilles tendon-splitting approach, the average exposed distal tibia was 33% more (15.0 cm(2)) in neutral and 43% more (14.6 cm(2)) in dorsiflexion. The average talus exposed was 47% more (3.0 cm(2)) in neutral and 76% more (4.2 cm(2)) in dorsiflexion. All increases in exposure were statistically significant. The medial malleolus was visualized in 19 tendon-splitting and six posterolateral approaches. The medial gutter was visualized in 20 tendon-splitting and 13 posterolateral approaches. These differences were statistically significant. All other landmarks could be visualized through both approaches. CONCLUSION: The Achilles tendon-splitting approach provided significantly greater exposure of the posterior distal tibia and talus compared to the posterolateral approach. CLINICAL RELEVANCE: Prospective studies will help determine if the tendon-splitting approach is a safe and clinically useful approach for surgeries in which direct access to the entire posterior ankle and subtalar joint are required.

Deployment after limb salvage for high-energy lower-extremity trauma.

BACKGROUND: Many wounded warriors experienced high-energy lower-extremity trauma (HELET) that may be limb threatening. Volumetric muscle loss, posttraumatic osteoarthritis, nerve injuries, and pain may severely limit physical function. Several wounded warriors express a strong desire to return to their units and be deployed in their original military occupational specialty. We began the return-to-run (RTR) clinical pathway at our institution 2 years ago to facilitate high-performance goals such as these. It involves an energy storing ankle foot orthosis, the intrepid dynamic exoskeletal orthosis in combination with high-intensity, progression-oriented rehabilitation. We sought to determine the rate of deployment or predeployment training after participation in this noninvasive intervention. METHODS: A retrospective analysis of the RTR database was performed to determine the rate of deployment or predeployment training among those service members who began participation in the RTR between November of 2009 and March of 2011. Medical records were reviewed for demographics, injury, surgical data, and major complications. Requests for delayed amputation were recorded, and charts were reviewed to determine if patients eventually elected to proceed with amputation or if they chose to continue with limb salvage. RESULTS: Between November 2009 and March 2011, 87 service members completed the RTR. Of these, 17 (19.5%) have been deployed to combat or are in predeployment training. Sixteen serve in combat arms (nine Special Forces, four infantry/ranger, two combat engineers, and one gunner), and one is a member of the military intelligence community. Fifteen patients sustained their injuries as a result of HELET (four gunshot, five motor vehicle collisions, four explosions, one parachute injury, and one fall from height), one had idiopathic avascular necrosis of the talus, and one had an iatrogenic nerve injury after pelvic surgery. Six of the patients underwent circular external fixation, five received joint fusions (three ankle, two subtalar joint), and nine had major nerve injuries. Four initially desired amputation of their injured limb but have subsequently countermanded their request. CONCLUSION: Returning to high-level physical function after HELET is challenging. After implementation of the RTR clinical pathway with the intrepid dynamic exoskeletal orthosis, 19.5% of wounded warriors treated with the RTR have been deployed or will be deployed in the coming year.

A comparison of limb-socket kinematics of bone-bridging and non-bone-bridging wartime transtibial amputations.

BACKGROUND: While there are proponents of both bone-bridging and non-bone-bridging transtibial amputation techniques, there is a lack of evidence describing functional differences between these two techniques. The goal of the present investigation was to objectively compare the techniques of bone-bridging and non-bone-bridging with respect to limb socket displacement during physiologic loading. METHODS: Fifteen male subjects with an average age of twenty-seven years (range, twenty-two to thirty-two years) who had undergone a unilateral transtibial amputation secondary to a traumatic wartime injury were prospectively evaluated. Seven patients had undergone a bone-bridging amputation, and eight had undergone a non-bone-bridging amputation. Digital fluoroscopic video was used to measure the vertical displacement of the limb within a total-surface-bearing socket with weight-bearing from 0% to 100% of body weight. RESULTS: There was no difference in limb-socket displacement between amputation techniques with initial loading (12.78 mm for the bone-bridging group, compared with 12.43 mm for the non-bone-bridging group; p = 0.88) or with total loading (p = 0.98). Similarly, there was no difference between suspension mechanisms in limb-socket displacement with initial loading (12.15 mm for patients with pin lock suspension, compared with 12.98 mm for those with suction sleeve suspension; p = 0.72) or with total loading (18.24 mm for patients with pin lock suspension, compared with 21.42 mm for those with suction sleeve suspension, p = 0.21). CONCLUSIONS: The current study demonstrated no difference between surgical techniques with respect to bone-socket displacement. These data provide no evidence to support statements that bone-bridging contributes to a more efficient platform in the total-surface-bearing socket.

Comparative effect of orthosis design on functional performance.

BACKGROUND: High-energy extremity trauma is common in combat. Orthotic options for patients whose lower extremities have been salvaged are limited. A custom energy-storing ankle-foot orthosis, the Intrepid Dynamic Exoskeletal Orthosis (IDEO), was created and used with high-intensity rehabilitation as part of the Return to Run clinical pathway. We hypothesized that the IDEO would improve functional performance compared with a non-custom carbon fiber orthosis (BlueRocker), a posterior leaf spring orthosis, and no brace. METHODS: Eighteen subjects with unilateral dorsiflexion and/or plantar flexion weakness were evaluated with six functional tests while they were wearing the IDEO, BlueRocker, posterior leaf spring, or no brace. The brace order was randomized, and five trials were completed for each of the functional measures, which included a four-square step test, a sit-to-stand five times test, tests of self-selected walking velocity over level and rocky terrain, and a timed stair ascent. They also completed one trial of a forty-yard (37-m) dash, filled out a satisfaction questionnaire, and indicated whether they had ever considered an amputation and, if so, whether they still intended to proceed with it. RESULTS: Performance was significantly better with the IDEO with respect to all functional measures compared with all other bracing conditions (p < 0.004), with the exception of the sit-to-stand five times test, in which there was a significant improvement only as compared with the BlueRocker (p = 0.014). The forty-yard dash improved by approximately 35% over the values for the posterior leaf spring and no-brace conditions, and by 28% over the BlueRocker. The BlueRocker demonstrated a significant improvement in the forty-yard dash compared with no brace (p = 0.033), and a significant improvement in self-selected walking velocity on level terrain compared with no brace and the posterior leaf spring orthosis (p < 0.028). However, no significant difference was found among the posterior leaf spring, BlueRocker, and no-brace conditions with respect to any other functional measure. Thirteen patients initially considered amputation, but after completion of the clinical pathway, eight desired limb salvage, two were undecided, and three still desired amputation. CONCLUSIONS: Use of the IDEO significantly improves performance on validated tests of agility, power, and speed. The majority of subjects initially considering amputation favored limb salvage after this noninvasive intervention.

Thermally unstable gating of the most common cystic fibrosis mutant channel (ΔF508): "rescue" by suppressor mutations in nucleotide binding domain 1 and by constitutive mutations in the cytosolic loops.

Most cystic fibrosis (CF) cases are caused by the ΔF508 mutation in the CF transmembrane conductance regulator (CFTR), which disrupts both the processing and gating of this chloride channel. The cell surface expression of ΔF508-CFTR can be "rescued" by culturing cells at 26-28 °C and treating cells with small molecule correctors or intragenic suppressor mutations. Here, we determined whether these various rescue protocols induce a ΔF508-CFTR conformation that is thermally stable in excised membrane patches. We also tested the impact of constitutive cytosolic loop mutations that increase ATP-independent channel activity (K978C and K190C/K978C) on ΔF508-CFTR function. Low temperature-rescued ΔF508-CFTR channels irreversibly inactivated with a time constant of 5-6 min when excised patches were warmed from 22 °C to 36.5 °C. A panel of CFTR correctors and potentiators that increased ΔF508-CFTR maturation or channel activity failed to prevent this inactivation. Conversely, three suppressor mutations in the first nucleotide binding domain rescued ΔF508-CFTR maturation and stabilized channel activity at 36.5 °C. The constitutive loop mutations increased ATP-independent activity of low temperature-rescued ΔF508-CFTR but did not enhance protein maturation. Importantly, the ATP-independent activities of these ΔF508-CFTR constructs were stable at 36.5 °C, whereas their ATP-dependent activities were not. Single channel recordings of this thermally stable ATP-independent activity revealed dynamic gating and unitary currents of normal amplitudes. We conclude that: (i) ΔF508-CFTR gating is highly unstable at physiologic temperature; (ii) most rescue protocols do not prevent this thermal instability; and (iii) ATP-independent gating and the pore are spared from ΔF508-induced thermal instability, a finding that may inform alternative treatment strategies.