Search

Injection into the posterior subtalar joint has not been validated for accuracy using radiographic end points. We asked whether needle placement into a normal posterior subtalar joint could be performed accurately and selectively by experienced surgeons without fluoroscopic guidance. Three fellowship-trained orthopaedic foot and ankle surgeons each injected the posterior subtalar joint of 20 cadaveric specimens using an anterolateral approach. Fluoroscopic images were obtained by an independent investigator and blinded. A separate fellowship-trained foot and ankle surgeon interpreted the images. Of 60 injections, 58 were accurate and two were extraarticular based on interpretation by an independent foot and ankle surgeon. Extravasation into the ankle occurred in 14 samples and into the peroneal sheath in two samples. Experienced surgeons can place intraarticular injections into a radiographically normal posterior subtalar joint without fluoroscopy with a high degree of accuracy. However, extravasation into the ankle or peroneal tendon sheath occurred in an unpredictable fashion, suggesting selectivity of injection placement is relatively limited without the use of fluoroscopy. Fluoroscopy may not be necessary for injections used solely for therapeutic purposes. However, if the injection is intended for diagnostic purposes or to assist in surgical decision-making or if the joint is abnormal, we recommend fluoroscopy to ensure the subtalar joint is the only anatomic structure impacted by the injection. [ABSTRACT FROM AUTHOR]

Cystic fibrosis (CF) is caused by defects in the CF transmembrane conductance regulator (CFTR) that functions as a chloride channel in epithelial cells. The most common cause of CF is the abnormal trafficking of CFTR mutants. Therefore, understanding the cellular machineries that transit CFTR from the endoplasmic reticulum to the plasma membrane (PM) is important. The coat protein complex I (COPI) has been implicated in the anterograde and retrograde transport of proteins and lipids between the endoplasmic reticulum and the Golgi. Here, we investigated the role of COPI in CFTR trafficking. Blocking COPI recruitment to membranes by expressing an inactive form of the GBF1 guanine nucleotide exchange factor for ADP-ribosylation factor inhibits CFTR trafficking to the PM. Similarly, inhibiting COPI dissociation from membranes by expressing a constitutively active ADP-ribosylation factor 1 mutant arrests CFTR within disrupted Golgi elements. To definitively explore the relationship between COPI and CFTR in epithelial cells, we depleted β-COP from the human colonic epithelial cell HT-29Cl.19A using small interfering RNA. β-COP depletion did not affect CFTR synthesis but impaired its trafficking to the PM. The arrest occurred pre-Golgi as shown by reduced level of glycosylation. Importantly, decreased trafficking of CFTR had a functional consequence as cells depleted of β-COP showed decreased cAMP-activated chloride currents. To explore the mechanism of COPI action in CFTR traffic we tested whether CFTR was COPI cargo. We discovered that the α-, β-, and γ-subunits of COPI co-immunoprecipitated with CFTR. Our results indicate that the COPI complex plays a critical role in CFTR trafficking to the PM. [ABSTRACT FROM AUTHOR]

Cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels are essential mediators of salt trans- port across epithelia. Channel opening normally requires ATP binding to both nucleotide-binding domains (NBDs), probable dimerization of the two NBDs, and phosphorylation of the R domain. How phosphorylation controls channel gating is unknown. Loss-of-function mutations in the CFTR gene cause cystic fibrosis; thus, there is considerable interest in compounds that improve mutant CFTR function. Here we investigated the mechanism by which CFTR is activated by curcumin, a natural compound found in turmeric. Curcumin opened CFTR channels by a novel mechanism that required neither ATP nor the second nucleotide-binding domain (NBD2). Consequently, this compound potently activated CF mutant channels that are defective for the normal ATP-dependent mode of gating (e.g. G551D and W1282X), including channels that lack NBD2. The stimulation of NBD2 deletion mutants by curcumin was strongly inhibited by ATP binding to NBD1, which implicates NBD1 as a plausible activation site. Curcumin activation became irreversible during prolonged exposure to this compound following which persistently activated channels gated dynamically in the absence of any agonist. Although CFTR activation by curcumin required neither ATP binding nor heterodimerization of the two NBDs, it was strongly dependent on prior channel phosphorylation by protein kinase A. Curcumin is a useful functional probe of CFTR gating that opens mutant channels by circumventing the normal requirements for ATP binding and NBD heterodimerization. The phosphorylation dependence of curcumin activation indicates that the R domain can modulate channel opening without affecting ATP binding to the NBDs or their heterodimerization. [ABSTRACT FROM AUTHOR]

Abstract: The cystic fibrosis transmembrane conductance regulator (CFTR) undergoes rapid turnover at the plasma membrane in various cell types. The ubiquitously expressed N-WASP promotes actin polymerization and regulates endocytic trafficking of other proteins in response to signaling molecules such as Rho-GTPases. In the present study we investigated the effects of wiskostatin, an N-WASP inhibitor, on the surface expression and activity of CFTR. We demonstrate, using surface biotinylation methods, that the steady-state surface CFTR pool in stably transfected BHK cells was dramatically decreased following wiskostatin treatment with a corresponding increase in the amount of intracellular CFTR. Similar effects were observed for latrunculin B, a specific actin-disrupting reagent. Both reagents strongly inhibited macroscopic CFTR-mediated Cl− currents in two cell types including HT29-Cl19A colonic epithelial cells. As previously reported, CFTR internalization from the cell surface was strongly inhibited by a cyclic-AMP cocktail. This effect of cyclic-AMP was only partially blunted in the presence of wiskostatin, which raises the possibility that these two factors modulate different steps in CFTR traffic. In kinetic studies wiskostatin appeared to accelerate the initial rate of CFTR endocytosis as well as inhibit its recycling back to the cell surface over longer time periods. Our studies implicate a role for N-WASP-mediated actin polymerization in regulating CFTR surface expression and channel activity. [Copyright &y& Elsevier]

Efficient endocytosis of the cystic fibrosis transmembrane conductance regulator (CFTR) is mediated by a tyrosine-based internalization signal in the CFTR carboxyl-terminal tail 1424YDSI1427. In the present studies, two naturally occurring cystic fibrosis mutations in the amino terminus of CFTR, R31C, and R31L were examined. To determine the defect that these mutations cause, the Arg-31 mutants were expressed in COS-7 cells and their biogenesis and trafficking to the cell surface tested in metabolic pulse-chase and surface biotinylation assays, respectively. The results indicated that both Arg-31 mutants were processed to band C at ∼50% the efficiency of the wild-type protein. However, once processed and delivered to the cell surface, their half-lives were the same as wild-type protein. Interestingly, indirect immunofluorescence and cell surface biotinylation indicated that the surface pool was much smaller than could be accounted for based on the biogenesis defect alone. Therefore, the Arg-31 mutants were tested in internalization assays and found to be internalized at 2x the rate of the wild-type protein. Patch clamp and 6-methoxy-N-(3-sulfopropyl)quinolinium analysis confirmed reduced amounts of functional Arg-31 channels at the cell surface. Together, the results suggest that both R31C and R31L mutations compromise biogenesis and enhance internalization of CFTR. These two additive effects contribute to the loss of surface expression and the associated defect in chloride conductance that is consistent with a disease phenotype. [ABSTRACT FROM AUTHOR]

Cystic fibrosis (CF) is caused by mutations that disrupt the surface localization and/or gating of the CF transmembrane conductance regulator (CFTR) chloride channel. The most common CF mutant is ΔF508-CFTR, which inefficiently traffics to the surfaces of most cells. The ΔF508 mutation may also disrupt the opening of CFTR channels once they reach the cell surface, but the extent of this gating defect is unclear. Here, we describe potent activators of wild-type and ΔF508-CFTR channels that are structurally related to 5-nitro-2-(3-phenylpropylamino)benzoate (NPPB), a negatively charged pore blocker that we show to have mixed agonistic activity (channel activation plus voltage-dependent pore block). These CFTR agonists include 1) an uncharged NPPB analog that stimulates channel opening at submicromolar concentrations without blocking the pore and 2) curcumin, a dietary compound recently reported to augment ΔF508-CFTR function in mice by an unknown mechanism. The uncharged NPPB analog enhanced the activities of wild-type and ΔF508-CFTR channels both in excised membrane patches and in intact epithelial monolayers. This compound increased the open probabilities of ΔF508-CFTR channels in excised membrane patches by 10-15-fold under conditions in which wild-type channels were already maximally active. Our results support the emerging view that CFTR channel activity is substantially reduced by the ΔF508 mutation and that effective CF therapies may require the use of channel openers to activate mutant CFTR channels at the cell surface. [ABSTRACT FROM AUTHOR]

The cystic fibrosis transmembrane conductance regulator (CFTR) is a phosphorylation- and ATP- dependent chloride channel that modulates salt and water transport across lung and gut epithelia. The relationship between CFTR and oxidized forms of glutathione is of potential interest because reactive glutathione species are produced in inflamed epithelia where they may be modulators or substrates of CFTR. Here we show that CFTR channel activity in excised membrane patches is markedly inhibited by several oxidized forms of glutathione (i.e., GSSG, GSNO, and glutathione treated with diamide, a strong thiol oxidizer). Three lines of evidence indicate that the likely mechanism for this inhibitory effect is glutathionylation of a CFTR cysteine (i.e., formation of a mixed disulfide with glutathione): (a) channels could be protected from inhibition by pretreating the patch with NEM (a thiol alkylating agent) or by lowering the bath pH; (b) inhibited channels could be rescued by reducing agents (e.g., DTT) or by purified glutaredoxins (Grxs; thiol disulfide oxidoreductases) including a mutant Grx that specifically reduces mixed disulfides between glutathione and cysteines within proteins; and (c) reversible glutathionylation of CFTR polypeptides in microsomes could be detected biochemically under the same conditions. At the single channel level, the primary effect of reactive glutathione species was to markedly inhibit the opening rates of individual CFTR channels. CFTR channel inhibition was not obviously dependent on phosphorylation state but was markedly slowed when channels were first "locked open" by a poorly hydrolyzable ATP analogue (AMP-PNP). Consistent with the latter finding, we show that the major site of inhibition is cys-1344, a poorly conserved cysteine that lies proximal to the signature sequence in the second nucleotide binding domain (NBD2) of human CFTR. This region is predicted to participate in ATP-dependent channel opening and to be occluded in the nucleotide-bound state of the channel based on structural comparisons to related ATP binding cassette transporters. Our results demonstrate that human CFTR channels are reversibly inhibited by reactive glutathione species, and support an important role of the region proximal to the NBD2 signature sequence in ATP-dependent channel opening. [ABSTRACT FROM AUTHOR]

Most cases of cystic fibrosis (CF) are caused by mutations that block the biosynthetic maturation of the CF gene product, the CF trans-membrane conductance regulator (CFTR) chloride channel. CFTR- processing mutants fail to escape the endoplasmic reticulum and are rapidly degraded. Current efforts to induce the maturation of CFTR mutants target components of the biosynthetic pathway (e.g., chaperones) rather than CFTR per se. Such methods are inherently nonspecific. Here we show that the most common CF-causing mutant (ΔF508-CFTR) can form mature, functional chloride channels that reach the cell surface when coexpressed with several other CFTR-processing mutants or with amino fragments of the wild-type CFTR protein. This transcomplementation effect required a specific match between the region flanking the disease- causing mutation and the complementing fragment; e.g., amino fragments complemented ΔF508-CFTR but not H1085R (a carboxy-processing mutant), whereas a carboxy fragment complemented H1085R but not ΔF508-CFTR. Transcomplementing fragments did not affect CFTR interactions with Hsc70, a chaperone previously implicated in CFTR biosynthesis. Instead, they may promote CFTR maturation by blocking nonproductive interactions between domains within the same or neighboring CFTR polypeptides that prevent normal processing. These findings indicate that it may be possible to develop CF therapies (e.g., mini-cDNA constructs for gene therapy) that are tailored to specific disease-causing mutants of CFTR. [ABSTRACT FROM AUTHOR]

Studies the role of syntaxin 1A complex in the regulation of cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel. Modulation of channel gating; Interaction between syntaxin 1A and amino terminal tail; Kinetic properties of voltage-gated calcium channels.

Reports on a study which investigated the inhibition of the cystic fibrosis gene (CFTR) chloride channels by syntaxin 1A. In-depth look at the epithelial chloride channel's functional activity; Modulation of the CFTR by components of the vesicle fusion machinery; Methodology used to conduct the study; Results of the study.

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.

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.

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., (© FASEB.)

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., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

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., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Background: Patients with severe lower extremity trauma have significant disability 2 years after injury that worsens by 7 years. Up to 15% seek late amputation. Recently, an energy-storing orthosis demonstrated improved function compared with standard orthoses; however, the effect when integrated with rehabilitation over time is unknown., Questions/purposes: (1) Does an 8-week integrated orthotic and rehabilitation initiative improve physical performance, pain, and outcomes in patients with lower extremity functional deficits or pain? (2) Is the magnitude of recovery different if enrolled more than 2 years after their injury versus earlier? (3) Does participation decrease the number considering late amputation?, Methods: We prospectively evaluated 84 service members (53 less than and 31 > 2 years after injury) who enrolled in the initiative. Fifty-eight sustained fractures, 53 sustained nerve injuries with weakness, and six had arthritis (there was some overlap in the patients with fractures and nerve injuries, which resulted in a total of > 84). They completed 4 weeks of physical therapy without the orthosis followed by 4 weeks with it. Testing was conducted at Weeks 0, 4, and 8. Validated physical performance tests and patient-reported outcome surveys were used as well as questions pertaining to whether patients were considering an amputation., Results: By 8 weeks, patients improved in all physical performance measures and all relevant patient-reported outcomes. Patients less than and greater than 2 years after injury improved similarly. Forty-one of 50 patients initially considering amputation favored limb salvage at the end of 8 weeks., Conclusions: We found this integrated orthotic and rehabilitation initiative improved physical performance, pain, and patient-reported outcomes in patients with severe, traumatic lower extremity deficits and that these improvements were sustained for > 2 years after injury. Efforts are underway to determine whether the Return to Run clinical pathway with the Intrepid Dynamic Exoskeletal Orthosis (IDEO) can be successfully implemented at additional military centers in patients > 2 years from injury while sustaining similar improvements in patient outcomes. The ability to translate this integrated orthotic and rehabilitation program into the civilian setting is unknown and warrants further investigation.

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-β,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., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

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'-(β,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.

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.

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.

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.

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.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a unique ion channel in that its gating is coupled to an intrinsic enzymatic activity (ATP hydrolysis). This enzymatic activity derives from the evolutionary origin of CFTR as an ATP-binding cassette transporter. CFTR gating is distinct from that of a typical ligand-gated channel because its ligand (ATP) is usually consumed during the gating cycle. However, recent findings indicate that CFTR gating exhibits allosteric properties that are common to conventional ligand-gated channels (e.g. unliganded openings and constitutive mutations). Here, we provide a unified view of CFTR gating that combines the allosterism of a ligand-gated channel with its unique enzymatic activity.

Throughout the current conflicts in Afghanistan and Iraq, there have been more than 1100 combat-related major limb amputations, with approximately 80% involving the lower extremity. There is, however, a paucity of data regarding the number of amputations below the level of the ankle. Although not as common, partial foot amputations, in the appropriate setting, offer a way to improve function and decrease energy consumption when compared to proximal amputations. Sound surgical tenets are prerequisite for successful outcomes when performing a distal amputation. Maintaining a robust soft tissue envelope allowing for tension-free wound closure is paramount in determining the feasibility of a partial foot amputation. Careful consideration of tendon balancing is also of utmost importance in avoiding common complications of contracture and deformity. Partial foot amputations present a viable surgical option for successful outcomes and maximization of patient function in the combat injured when certain criteria are met.

CFTR is a cAMP-activated chloride channel responsible for agonist stimulated chloride and fluid transport across epithelial surfaces.(1) Mutations in the CFTR gene lead to cystic fibrosis (CF) which affects the function of secretory organs like the intestine, the pancreas, the airways and the sweat glands. Most of the morbidity and mortality in CF has been linked to a decrease in airway function.(2) The ΔF508 mutation is the most common CF-related mutation in the Caucasian population and represents 90% of CF alleles. Homozygote carriers of this mutation present with a severe CF phenotype.(3) The ΔF508 mutation causes misfolding of the nascent CFTR polypeptide, which leads to inefficient export from the endoplasmic reticulum (ER) and rapid degradation by the proteasome.

Determining whether to perform limb salvage or amputation in the traumatized lower extremity continues to be a difficult problem in the military and civilian sectors. Numerous predictive scores and models have failed to provide definitive criteria for prediction of limb-salvage success. Excellent support is available in the military health care system for soldiers electing to undergo either limb salvage or amputation. Recent experience with soldiers who sustained limb-threatening injuries has shown that delayed amputation after limb-salvage attempts is a viable option for soldiers wounded in combat., (Published by Elsevier Inc.)

Continuous peripheral nerve catheters (CPNCs) have become increasingly popular for postoperative analgesia in orthopedic surgery involving the lower limbs. The CPNC technique has been found to reduce postoperative pain and facilitate earlier discharge and recovery. Until recently, potential infectious complications associated with CPNCs have remained largely unreported. In this report, we present the case of a posterior thigh abscess that developed after placement of a continuous popliteal nerve catheter and required surgical débridement.

Prophylactic and therapeutic treatment of leg compartment syndrome with decompression by double-incision fasciotomy prevents progression of soft-tissue injury in high-energy trauma. This treatment is the standard of care in civilian trauma and combat settings. More controversial is the use of either single- or dual-incision fasciotomy of the foot for prophylactic treatment of foot compartment syndrome. Fasciotomy must be performed in the face of major trauma to the foot with severe swelling and unremitting pain. The role for prophylactic fasciotomy of the foot is unclear and should be considered on a case by case basis. The surgeon must maintain a high degree of vigilance for the development of compartment syndrome in the combat casualty., (Published by Elsevier Inc.)

Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) channel, an ATP binding cassette (ABC) transporter. CFTR gating is linked to ATP binding and dimerization of its two nucleotide binding domains (NBDs). Channel activation also requires phosphorylation of the R domain by poorly understood mechanisms. Unlike conventional ligand-gated channels, CFTR is an ATPase for which ligand (ATP) release typically involves nucleotide hydrolysis. The extent to which CFTR gating conforms to classic allosteric schemes of ligand activation is unclear. Here, we describe point mutations in the CFTR cytosolic loops that markedly increase ATP-independent (constitutive) channel activity. This finding is consistent with an allosteric gating mechanism in which ligand shifts the equilibrium between inactive and active states but is not essential for channel opening. Constitutive mutations mapped to the putative symmetry axis of CFTR based on the crystal structures of related ABC transporters, a common theme for activating mutations in ligand-gated channels. Furthermore, the ATP sensitivity of channel activation was strongly enhanced by these constitutive mutations, as predicted for an allosteric mechanism (reciprocity between protein activation and ligand occupancy). Introducing constitutive mutations into CFTR channels that cannot open in response to ATP (i.e., the G551D CF mutant and an NBD2-deletion mutant) substantially rescued their activities. Importantly, constitutive mutants that opened without ATP or NBD2 still required R domain phosphorylation for optimal activity. Our results confirm that (i) CFTR gating exhibits features of protein allostery that are shared with conventional ligand-gated channels and (ii) the R domain modulates CFTR activity independent of ATP-induced NBD dimerization.

In this study, we compared differences in motion at the bone-prosthesis interface in femora in which a fluted, tapered, or cylindrical distal stem design had been implanted in a revision total hip arthroplasty model. Paired, fresh-frozen, cadaveric femora underwent resection of the proximal femur to simulate the proximal femoral bone loss often present during revision total hip arthroplasty and implantation with either a fluted, tapered stem or a clinically proven cylindrical stem. Specimens were then preloaded and subjected to a synchronous axial and torsional load with continuous monitoring of axial displacement and rotation. For the fluted, tapered stem, mean axial and rotational displacements were 13.33 microm and 9.81 microm, respectively, compared with 18.37 microm and 13.40 microm for the cylindrical stem (both Ps < .05). Therefore, the fluted, tapered stem design that was tested demonstrated superior initial biomechanical stability compared with that of the clinically proven cylindrical design tested. However, both stems demonstrated motion below the threshold necessary for bony ingrowth. Knowledge of the initial biomechanical properties of different stem designs may assist the revision joint surgeon in choosing the optimal prosthesis for implantation.

This is a consecutive study of patients with adolescent idiopathic scoliosis requiring surgical intervention. The purpose was to determine the best radiographic method of evaluating the flexibility of the proximal thoracic (PT) curve in patients with a double thoracic curve pattern. The PT curve averaged 40 degrees (range, 27 degrees-67 degrees) on posteroanterior (PA) radiographs, 30 degrees (range, 12 degrees60 degrees) on supine side-bending radiographs, and 24 degrees (range, 8 degrees -55 degrees) on traction radiographs. The flexibility index (FI) for the PT curve was 0.72 (range, 0.40-0.96) on side-bending and 0.58 (range, 0.29-0.82) on traction. The main thoracic (MT) curve averaged 62 degrees (range, 45 degrees-102 degrees) on PA, 40 degrees (range, 19 degrees-88 degrees) on supine side-bending, and 35 degrees (range, 17 degrees-76 degrees) on traction radiographs. The FI for the MT curve was 0.62 (range, 0.30-0.89) on side bending and 0.56 (range, 0.32-0.81) on traction. There was a statistically significant difference between the flexibility of the PT curve on supine traction as compared with the side-bending films (P<0.0005). Comparison of the MT curve flexibility with the PT curve demonstrated the PT curve to be less flexible in 11 of 15 cases (P = 0.16). The authors conclude that for double thoracic curve patterns, the PT curve is usually the smaller, yet often the stiffer curve. Furthermore, the supine traction radiograph demonstrates greater flexibility of the PT curve than does the supine side-bending radiograph. Clinically, this may assist scoliosis surgeons in assessing the PT curve and achieving a stable, balanced spine.

Background: In an effort to improve the strength and stiffness of anterior cruciate ligament grafts, several authors have advocated alterations of graft structure and orientation, including braiding the tendons in hamstring tendon grafts., Hypothesis: Braiding hamstring tendons does not increase graft strength and stiffness., Study Design: Controlled laboratory study., Methods: Sixteen hamstring tendon and 21 bone-patellar tendon-bone grafts were harvested from 12 cadavers and divided into three groups: 1) braided four-strand hamstring tendon, 2) unbraided four-strand hamstring tendon, and 3) bone-patellar tendon-bone. All grafts were placed under a 50-N preload on a servohydraulic testing device and were tensioned to failure., Results: The strength and stiffness of the tested specimens averaged 427 +/- 36 N and 76 +/- 10 N/mm, respectively, for braided specimens, 532 +/- 44 N and 139 +/- 18 N/mm for unbraided specimens, and 574 +/- 46 N and 158 +/- 15 N/mm for patellar tendon specimens. There was a 20% decrement in hamstring tendon graft tensile strength and a 45% decrease in stiffness after braiding because of the suboptimal multidirectional orientation of individual tendons within the braided grafts., Conclusions: In vitro braided hamstring tendon grafts demonstrated mechanically inferior strength and stiffness characteristics compared with unbraided hamstring tendon grafts and patellar tendon grafts., Clinical Relevance: Braiding of hamstring tendon grafts provides no mechanical advantage in anterior cruciate ligament reconstruction., (Copyright 2002 American Orthopaedic Society for Sports Medicine)