Discovery Elbow System arthroplasty polyethylene bearing exchange: outcomes and experience.

BACKGROUND: The Discovery Elbow System (DES) utilizes a polyethylene bearing within the ulnar component. An exchange bearing requires preoperative freezing and implantation within 2 minutes of freezer removal to allow insertion. We report our outcomes and experience using this technique. METHODS: This was an analysis of a two-surgeon consecutive series of DES bearing exchange. Inclusion criteria included patients in which exchange was attempted with a minimum 1-year follow-up. Clinical and radiographic review was performed 1, 2, 3, 5, 8 and 10 years postoperative. Outcome measures included range of movement, Oxford Elbow Score (OES), Mayo Elbow Performance Score (MEPS), complications and requirement for revision surgery. RESULTS: Eleven DESs in 10 patients were included. Indications were bearing wear encountered during humeral component revision (n=5); bearing failure (n=4); and infection treated with debridement, antibiotics and implant retention (DAIR; n=2). Bearing exchange was conducted on the first attempt in 10 cases. One case required a second attempt. One patient developed infection postoperatively managed with two-stage revision. Mean follow-up of the bearing exchange DES was 3 years. No further surgery was required, with no infection recurrence in DAIR cases. Mean elbow flexion-extension and pronosupination arcs were 107° (±22°) and 140° (±26°). Mean OES was 36/48 (±12) and MEPS was 83/100 (±19). CONCLUSIONS: Our results support the use of DES bearing exchange in cases of bearing wear with well-fixed stems or acute infection. This series provides surgeons managing DES arthroplasty with management principles, successful and reproducible surgical techniques and expected clinical outcomes in performing DES polyethylene bearing exchange. Level of evidence: IV.

Does long-term follow-up and monitoring of primary shoulder arthroplasty identify failing implants requiring revision?

BACKGROUND: Published scoping review has identified evidence paucity related to long-term follow-up of shoulder arthroplasty. We aim to report effectiveness of elective primary shoulder arthroplasty surveillance in identifying failing implants requiring revision. METHODS: A prospective database recording shoulder arthroplasty and subsequent follow-up surveillance in a shoulder unit was analyzed. Shoulder arthroplasty was performed by 4 fellowship-trained shoulder surgeons for accepted elective indications including the use of anatomic arthroplasty in arthritic shoulders with intact rotator cuff and a reverse prosthesis being used in rotator cuff-deficient shoulders and rotator cuff-competent arthritic shoulders when deemed preferable by the treating surgeon. All shoulder arthroplasty implants used had achieved a minimum 7A Orthopaedic Data Evaluation Panel (ODEP) rating. The included shoulder arthroplasties were performed between May 1, 2004, and December 31, 2021, with minimum 1-year follow-up. Surveillance program involves specialist physiotherapist review at 1, 2, 3, 5, 8, 10, and 15 years postoperatively, including clinical examination, outcome scoring, and radiographs. Patient-initiated review occurred between time points if a patient requested assessment because of symptoms. Outcome measures include ratio of failing implants identified by surveillance and patient-initiated review, with number of surveillance reviews offered and proportion that identified a failing implant requiring revision calculated. RESULTS: A total of 1002 elective primary shoulder arthroplasty with minimum 1-year follow-up were performed (547 reverse total shoulder arthroplasty [rTSA], 234 anatomic total shoulder arthroplasty [aTSA], and 221 hemiarthroplasty [HA]). A total of 238 patients died prior to December 31, 2022, resulting in 4019 surveillance appointments offered. Thirty-eight prostheses required revision ≥1 year postoperatively (6 rTSA, 9 aTSA, and 23 HA), with surveillance identifying requirement in 53% (33% rTSA, 56% aTSA, and 57% HA) and patient-initiated review in 47%. Mean years from implantation to revision was 5.2 (2.7 rTSA, 3.6 aTSA, and 6.6 HA). Revision indications included rotator cuff failure (56% aTSR and 43% HA) and glenoid erosion (57% HA). CONCLUSION: This is the first series reporting effectiveness of shoulder arthroplasty surveillance in identifying implants requiring revision. Surveillance identified more than half of implants requiring revision, although only 0.5% of appointments identified revision requirement. Surveillance enrolment may influence patient-initiated review utilization; therefore, similar studies using only patient-initiated follow-up would help inform recommendations.

Discovery Elbow System arthroplasty polyethylene bearing exchange: outcomes and experience

The Discovery Elbow System (DES) utilizes a polyethylene bearing within the ulnar component. An exchange bearing requires preoperative freezing and implantation within 2 minutes of freezer removal to allow insertion. We report our outcomes and experience using this technique. Methods: This was an analysis of a two-surgeon consecutive series of DES bearing exchange. Inclusion criteria included patients in which exchange was attempted with a minimum 1-year follow-up. Clinical and radiographic review was performed 1, 2, 3, 5, 8 and 10 years postoperative. Outcome measures included range of movement, Oxford Elbow Score (OES), Mayo Elbow Performance Score (MEPS), complications and requirement for revision surgery. Results: Eleven DESs in 10 patients were included. Indications were bearing wear encountered during humeral component revision (n=5); bearing failure (n=4); and infection treated with debridement, antibiotics and implant retention (DAIR; n=2). Bearing exchange was conducted on the first attempt in 10 cases. One case required a second attempt. One patient developed infection postoperatively managed with two-stage revision. Mean follow-up of the bearing exchange DES was 3 years. No further surgery was required, with no infection recurrence in DAIR cases. Mean elbow flexion-extension and pronosupination arcs were 107° (±22°) and 140° (±26°). Mean OES was 36/48 (±12) and MEPS was 83/100 (±19). Conclusions: Our results support the use of DES bearing exchange in cases of bearing wear with well-fixed stems or acute infection. This series provides surgeons managing DES arthroplasty with management principles, successful and reproducible surgical techniques and expected clinical outcomes in performing DES polyethylene bearing exchange. Level of evidence: IV.

Endothelial dysfunction in psoriatic arthritis patients: correlations between insulin resistance and disease activity.

OBJECTIVE: Cardiovascular atherosclerotic comorbidities represent an important cause of morbidity and mortality in patients diagnosed with psoriatic arthritis. In both atherosclerosis and Psoriatic arthritis, inflammation plays a pivotal role. Psoriatic arthritis is considered as an independent risk factor for the development of atherosclerosis with accelerated evolution. Development of atherosclerosis is initiated by the endothelial cell dysfunction along with inflammation and insulin resistance. The main aim of the study was to evaluate the endothelial function in Psoriatic arthritis patients, and to identify if it is related to the insulin resistance and Psoriatic arthritis disease activity. PATIENTS AND METHODS: In this case-control study, a group of 32 age and gender matched healthy controls was formed and compared to the group of 32 Psoriatic arthritis patients. We assessed the following parameters: Disease Activity in Psoriatic Arthritis Score, Homeostatic Model Assessment for Insulin Resistance, serum levels of the tumor necrosis factor alpha (TNFα), and the endothelial dysfunction by means of the flow-mediated dilation at brachial artery. The Student's t-test, the Pearson correlation and the ANOVA test were used to perform the statistical analysis of the data obtained; p-value <0.05 was considered as statistically significant. RESULTS: Compared to the patients in the control group, TNFα and Homeostatic Model Assessment for Insulin Resistance were increased (p-value <0.001), and flow-mediated dilation at brachial artery was decreased (p-value <0.001) in the disease group. In Psoriatic arthritis patients, significant correlations were found between Disease Activity in Psoriatic Arthritis Score and Homeostatic Model Assessment for Insulin Resistance (r=0.8143, p-value <0.001), and between Disease Activity in Psoriatic Arthritis Score and flow-mediated dilation at brachial artery % (r= -0.8376, p-value <0.001). Psoriatic arthritis patients treated with Methotrexate exhibited reduced values of Disease Activity in Psoriatic Arthritis Score and Homeostatic Model Assessment for Insulin Resistance and increased values of flow-mediated dilation at brachial artery, when compared with the untreated patients. CONCLUSIONS: Endothelial dysfunction is present in Psoriatic arthritis patients and has a significant correlation with both, the course of the disease and the insulin resistance.

Minimum five-year outcomes of reverse total shoulder arthroplasty using a trabecular metal glenoid base plate.

AIMS: Reverse total shoulder arthroplasty (RTSA) using trabecular metal (TM)-backed glenoid implants has been introduced with the aim to increase implant survival. Only short-term reports on the outcomes of TM-RTSA have been published to date. We aim to present the seven-year survival of TM-backed glenoid implants along with minimum five-year clinical and radiological outcomes. METHODS: All consecutive elective RTSAs performed at a single centre between November 2008 and October 2014 were reviewed. Patients who had primary TM-RTSA for rotator cuff arthropathy and osteoarthritis with deficient cuff were included. A total of 190 shoulders in 168 patients (41 male, 127 female) were identified for inclusion at a mean of 7.27 years (SD 1.4) from surgery. The primary outcome was survival of the implant with all-cause revision and aseptic glenoid loosening as endpoints. Secondary outcomes were clinical, radiological, and patient-related outcomes with a five-year minimum follow-up. RESULTS: The implant was revised in ten shoulders (5.2%) with a median time to revision of 21.2 months (interquartile range (IQR) 9.9 to 41.8). The Kaplan-Meier survivorship estimate at seven years was 95.9% (95% confidence interval (CI) 91.7 to 98; 35 RTSAs at risk) for aseptic mechanical failure of the glenoid and 94.8% (95% CI 77.5 to 96.3; 35 RTSAs at risk) for all-cause revision. Minimum five-year clinical and radiological outcomes were available for 103 and 98 RTSAs respectively with a median follow-up time of six years (IQR 5.2 to 7.0). Median postoperative Oxford Shoulder Score was 38 (IQR 31 to 45); median Constant and Murley score was 60 (IQR 47.5 to 70); median forward flexion 115° (IQR 100° to 125°); median abduction 95° (IQR 80° to 120°); and external rotation 25° (IQR 15° to 40°) Scapular notching was seen in 62 RTSAs (63.2%). CONCLUSION: We present the largest and longest-term series of TM-backed glenoid implants demonstrating 94.8% all-cause survivorship at seven years. Specifically pertaining to glenoid loosening, survival of the implant increased to 95.9%. In addition, we report satisfactory minimum five-year clinical and radiological outcomes. Cite this article: Bone Joint J 2021;103-B(8):1333-1338.

Screening for acute disseminated histoplasmosis in HIV disease using urinary antigen detection enzyme immunoassay: A pilot study in Cameroon.

Acute disseminated histoplasmosis (ADH) is an AIDS-defining illness and reported in Cameroon, but there are few data about its incidence. Between June and August 2019, we conducted a descriptive cross-sectional study to screen for histoplasmosis in a population of adults with HIV infection, irrespective of their CD4 T-cell counts, using Histoplasma urine antigen detection enzyme immunoassay (EIA) and histoplasmin skin test. Of the 138 participants screened, 36 (26%) had detectable antigen in urine, using an OD cut off of 0.045. Skin lesions were present in two (6%) cases. Of 39 patients tested for histoplasmin skin test positivity, one was positive. Histoplasma antigenuria was associated with a positive history of chest infection (Odds ratio: 3.632, 95% confidence interval: 1.635-8.071, p= 0.001). As 30 (21.7%) of titres were between 0.045 (the current cut off) and 0.25, the cut off may need adjustment in Cameroon, using disease confirmation with alternative, highly sensitive diagnostic approaches such as PCR and bone marrow examination. H. capsulatum infection appears to be common among HIV-infected patients attending outpatient clinics at the Buea Regional Hospital. There is an acute need to improve awareness and management of HIV patients with respect to H. capsulatum infection.

Mid- to long-term survivorship of the cemented, semiconstrained Discovery total elbow arthroplasty.

BACKGROUND: The incidence of total elbow arthroplasty (TEA) is increasing, and an improved understanding of elbow kinematics and biomaterials has driven advances in implant design. In modern practice, cemented, semiconstrained devices are most frequently used. The Discovery TEA has demonstrated promising early results, although there are a paucity of follow-up studies and no dedicated mid- to long-term series. We therefore present the longest, most complete such study to date. METHODS: A prospectively maintained local joint registry was interrogated to yield a consecutive series of Discovery TEAs performed at a single non-design center. The minimum follow-up period was set at 5 years. Revision procedures and TEAs performed for acute trauma were excluded. The primary outcome was survivorship of the implant. The secondary outcomes included clinical, radiographic, and patient-reported outcomes. RESULTS: We identified 67 TEAs in 58 patients for inclusion at a mean of 98.5 ± 20.4 months from surgery. Four cases (6%) were lost to follow-up, and implant survival was censored accordingly. The implant was revised in 14 cases (20.9%). The Kaplan-Meier method showed an implant survivorship rate of 76.8% at 119 months. A significant difference in survival was found between dominant and nondominant elbows (P = .012, Breslow test), with elbow dominance conferring a 4.5-fold increased risk of revision (relative risk, 4.5; 95% confidence interval, 1.1-18.5). Pooled clinical outcomes (70.9% follow-up at minimum of 60 months and median of 77.8 months) were also determined. CONCLUSIONS: We present the longest-term and most complete single-center follow-up study of the Discovery TEA to date. Further long-term survival studies are required to elucidate the performance of this implant compared with more established designs. We have also demonstrated differences in implant survivorship owing to hand dominance for the first time.

First biocompatibility margins for optical stimulation at the eardrum via 532-nm laser pulses in a mouse model.

Hearing impairment affects ∼460 million people worldwide. Conservative therapies, such as hearing aids, bone conduction systems, and middle ear implants, do not always sufficiently compensate for this deficit. The optical stimulation is currently under investigation as an alternative stimulation strategy for the activation of the hearing system. To assess the biocompatibility margins of this emerging technology, we established a method applicable in whole-mount preparations of murine tympanic membranes (TM). We irradiated the TM of anesthetized mice with 532-nm laser pulses at an average power of 50, 89, 99, and 125 mW at two different locations of the TM and monitored the hearing function with auditory brainstem responses. Laser-power-dependent negative side effects to the TM were observed at power levels exceeding 89 mW. Although we did not find any significant negative effects of optical stimulation on the hearing function in these mice, based on the histology results further studies are necessary for optimization of the used parameters.

Frequency-specific activation of the peripheral auditory system using optoacoustic laser stimulation.

Hearing impairment is one of the most common sensory deficits in humans. Hearing aids are helpful to patients but can have poor sound quality or transmission due to insufficient output or acoustic feedback, such as for high frequencies. Implantable devices partially overcome these issues but require surgery with limited locations for device attachment. Here, we investigate a new optoacoustic approach to vibrate the hearing organ with laser stimulation to improve frequency bandwidth, not requiring attachment to specific vibratory structures, and potentially reduce acoustic feedback. We developed a laser pulse modulation strategy and simulated its response at the umbo (1-10 kHz) based on a convolution-based model. We achieved frequency-specific activation in which non-contact laser stimulation of the umbo, as well as within the middle ear at the round window and otic capsule, induced precise shifts in the maximal vibratory response of the umbo and neural activation within the inferior colliculus of guinea pigs, corresponding to the targeted, modelled and then stimulated frequency. There was also no acoustic feedback detected from laser stimulation with our experimental setup. These findings open up the potential for using a convolution-based optoacoustic approach as a new type of laser hearing aid or middle ear implant.

Outcomes for a large metaphyseal volume hemiarthroplasty in complex fractures of the proximal humerus.

BACKGROUND: A large metaphyseal volume shoulder hemiarthroplasty has been in use within our department since 2008; however, no clinical outcome data are available for this prosthesis apart from the designer surgeon series. MATERIALS AND METHODS: During a 5-year period, data were collected for 40 patients (30 women, 10 men) treated consecutively with the Zimmer Anatomical Shoulder Fracture hemiarthroplasty system (Zimmer, Warsaw, IN, USA). RESULTS: The final analysis included 26 patients. The median age was 79 years (range, 58-91 years), and the median follow-up was 3.7 years (range, 2.0-5.8 years). The median Constant Score was 34 points (range, 16-70 points), and the median Oxford Shoulder Score was 27 points (range, 5-46 points). The greater tuberosity healed satisfactorily in 12 patients. Resorption of the greater tuberosity was seen radiologically in 18 patients. The presence of resorption had no significant effect on the Constant Score (P = .264) or the Oxford Shoulder Score (P = .469). Three patients (12%) required revision. CONCLUSIONS: This is the first report from a nondesigner center for outcomes for this prosthesis to date. The results demonstrate reduced functional performance compared with the designer series.

Dissecting human skeletal muscle troponin proteoforms by top-down mass spectrometry.

Skeletal muscles are the most abundant tissues in the human body. They are composed of a heterogeneous collection of muscle fibers that perform various functions. Skeletal muscle troponin (sTn) regulates skeletal muscle contraction and relaxation. sTn consists of 3 subunits, troponin I (TnI), troponin T (TnT), and troponin C (TnC). TnI inhibits the actomyosin Mg(2+)-ATPase, TnC binds Ca(2+), and TnT is the tropomyosin (Tm)-binding subunit. The cardiac and skeletal isoforms of Tn share many similarities but the roles of modifications of Tn in the two muscles may differ. The modifications of cardiac Tn are known to alter muscle contractility and have been well-characterized. However, the modification status of sTn remains unclear. Here, we have employed top-down mass spectrometry (MS) to decipher the modifications of human sTnT and sTnI. We have extensively characterized sTnT and sTnI proteoforms, including alternatively spliced isoforms and post-translationally modified forms, found in human skeletal muscle with high mass accuracy and comprehensive sequence coverage. Moreover, we have localized the phosphorylation site of slow sTnT isoform III to Ser1 by tandem MS with electron capture dissociation. This is the first study to comprehensively characterize human sTn and also the first to identify the basal phosphorylation site for human sTnT by top-down MS.

Augmented phosphorylation of cardiac troponin I in hypertensive heart failure.

An altered cardiac myofilament response to activating Ca(2+) is a hallmark of human heart failure. Phosphorylation of cardiac troponin I (cTnI) is critical in modulating contractility and Ca(2+) sensitivity of cardiac muscle. cTnI can be phosphorylated by protein kinase A (PKA) at Ser(22/23) and protein kinase C (PKC) at Ser(22/23), Ser(42/44), and Thr(143). Whereas the functional significance of Ser(22/23) phosphorylation is well understood, the role of other cTnI phosphorylation sites in the regulation of cardiac contractility remains a topic of intense debate, in part, due to the lack of evidence of in vivo phosphorylation. In this study, we utilized top-down high resolution mass spectrometry (MS) combined with immunoaffinity chromatography to determine quantitatively the cTnI phosphorylation changes in spontaneously hypertensive rat (SHR) model of hypertensive heart disease and failure. Our data indicate that cTnI is hyperphosphorylated in the failing SHR myocardium compared with age-matched normotensive Wistar-Kyoto rats. The top-down electron capture dissociation MS unambiguously localized augmented phosphorylation sites to Ser(22/23) and Ser(42/44) in SHR. Enhanced Ser(22/23) phosphorylation was verified by immunoblotting with phospho-specific antibodies. Immunoblot analysis also revealed up-regulation of PKC-α and -δ, decreased PKCε, but no changes in PKA or PKC-ß levels in the SHR myocardium. This provides direct evidence of in vivo phosphorylation of cTnI-Ser(42/44) (PKC-specific) sites in an animal model of hypertensive heart failure, supporting the hypothesis that PKC phosphorylation of cTnI may be maladaptive and potentially associated with cardiac dysfunction.

Probing the oligomeric state and interaction surfaces of Fukutin-I in dilauroylphosphatidylcholine bilayers.

Fukutin-I is localised to the endoplasmic reticulum or Golgi apparatus within the cell, where it is believed to function as a glycosyltransferase. Its localisation within the cell is thought to to be mediated by the interaction of its N-terminal transmembrane domain with the lipid bilayers surrounding these compartments, each of which possesses a distinctive lipid composition. However, it remains unclear at the molecular level how the interaction between the transmembrane domains of this protein and the surrounding lipid bilayer drives its retention within these compartments. In this work, we employed chemical cross-linking and fluorescence resonance energy transfer measurements in conjunction with multiscale molecular dynamics simulations to determine the oligomeric state of the protein within dilauroylphosphatidylcholine bilayers to identify interactions between the transmembrane domains and to ascertain any role these interactions may play in protein localisation. Our studies reveal that the N-terminal transmembrane domain of Fukutin-I exists as dimer within dilauroylphosphatidylcholine bilayers and that this interaction is driven by interactions between a characteristic TXXSS motif. Furthermore residues close to the N-terminus that have previously been shown to play a key role in the clustering of lipids are shown to also play a major role in anchoring the protein in the membrane.

Interventricular differences in myofilament function in experimental congestive heart failure.

This study was conducted to identify molecular mechanisms which explain interventricular differences in myofilament function in experimental congestive heart failure (CHF). CHF was induced in rats by chronic aortic banding or myocardial infarction for 32-36 weeks. Right and left ventricular (RV, LV) myocytes were mechanically isolated, triton-skinned, and attached to a force transducer and motor arm. Myofilament force-[Ca(2+)] relations assessed maximal Ca(2+)-saturated force (F (max)) and the [Ca(2+)] at 50% of F (max) (EC(50)). Myofilament protein phosphorylation was determined via ProQ diamond phospho-staining. Protein kinase C (PKC)-α expression/activation and site-specific phosphorylation of cardiac troponin I (cTnI) and cardiac troponin T (cTnT) were measured via immunoblotting. Relative to controls, failing RV myocytes displayed a ~45% decrease in F (max) with no change in EC(50), whereas failing LV myocytes displayed a ~45% decrease in F (max) and ~50% increase in EC(50). Failing LV myofilaments were less Ca(2+)-sensitive (37% increase in EC(50)) than failing RV myofilaments. Expression and activation of PKC-α was increased twofold in failing RV myocardium and relative to the RV, PKC-α was twofold higher in the failing LV, while PKC-ß expression was unchanged by CHF. PKC-α-dependent phosphorylation and PP1-mediated dephosphorylation of failing RV myofilaments increased EC(50) and increased F (max), respectively. Phosphorylation of cTnI and cTnT was greater in failing LV myofilaments than in failing RV myofilaments. RV myofilament function is depressed in experimental CHF in association with increased PKC-α signaling and myofilament protein phosphorylation. Furthermore, myofilament dysfunction is greater in the LV compared to the RV due in part to increased PKC-α activation and phosphorylation of cTnI and cTnT.

Redox signaling and cardiac sarcomeres.

Oxidative stress is common in many clinically important cardiac disorders, including ischemia/reperfusion, diabetes, and hypertensive heart disease. Oxidative stress leads to derangements in pump function due to changes in the expression or function of proteins that regulate intracellular Ca(2+) homeostasis. There is growing evidence that the cardiodepressant actions of reactive oxygen species (ROS) also are attributable to ROS-dependent signaling events in the sarcomere. This minireview focuses on myofilament protein post-translational modifications induced by ROS or ROS-activated signaling enzymes that regulate cardiac contractility.

Cardiac troponin T, a sarcomeric AKAP, tethers protein kinase A at the myofilaments.

Efficient and specific phosphorylation of PKA substrates, elicited in response to ß-adrenergic stimulation, require spatially confined pools of PKA anchored in proximity of its substrates. PKA-dependent phosphorylation of cardiac sarcomeric proteins has been the subject of intense investigations. Yet, the identity, composition, and function of PKA complexes at the sarcomeres have remained elusive. Here we report the identification and characterization of a novel sarcomeric AKAP (A-kinase anchoring protein), cardiac troponin T (cTnT). Using yeast two-hybrid technology in screening two adult human heart cDNA libraries, we identified the regulatory subunit of PKA as interacting with human cTnT bait. Immunoprecipitation studies show that cTnT is a dual specificity AKAP, interacting with both PKA-regulatory subunits type I and II. The disruptor peptide Ht31, but not Ht31P (control), abolished cTnT/PKA-R association. Truncations and point mutations identified an amphipathic helix domain in cTnT as the PKA binding site. This was confirmed by a peptide SPOT assay in the presence of Ht31 or Ht31P (control). Gelsolin-dependent removal of thin filament proteins also reduced myofilament-bound PKA-type II. Using a cTn exchange procedure that substitutes the endogenous cTn complex with a recombinant cTn complex we show that PKA-type II is troponin-bound in the myofilament lattice. Displacement of PKA-cTnT complexes correlates with a significant decrease in myofibrillar PKA activity. Taken together, our data propose a novel role for cTnT as a dual-specificity sarcomeric AKAP.

Doxorubicin causes diaphragm weakness in murine models of cancer chemotherapy.

Doxorubicin is a chemotherapeutic agent prescribed for a variety of tumors. While undergoing treatment, patients exhibit frequent symptoms that suggest respiratory muscle weakness. Cancer patients can receive doxorubicin chemotherapy through either intravenous (IV) or intraperitoneal (IP) injections. We hypothesized that respiratory muscle function would be depressed in a murine model of chemotherapy. We tested this hypothesis by treating C57BL/6 mice with a clinical dose of doxorubicin (20 mg/kg) via IV or IP injection. Three days later we measured contractile properties of muscle fiber bundles isolated from the diaphragm. Doxorubicin consistently depressed diaphragm force with both methods of administration (P < 0.01). Doxorubicin IP exaggerated the depression in diaphragm force and stimulated tissue inflammation and muscle fiber injury. These results suggest that clinically relevant doses of doxorubicin cause respiratory muscle weakness and that the loss of function depends, in part, on the route of administration.

Expression and purification of the transmembrane domain of Fukutin-I for biophysical studies.

Fukutin-I is a member of a family of putative O-linked glycosyltransferases linked to the glycosylation of the dystrophin complex. Mutations in this family of proteins have been linked to a number of congenital muscular dystrophies that arise from the hypoglycosylation of alpha-dystroglycan. Critical to the function of Fukutin and other members of this family is their localisation within the cell, which has been shown to depend critically on the interactions between the N-terminal transmembrane domain of these proteins and the lipid bilayer within the ER/Golgi. To investigate how the interactions between the N-terminal transmembrane domain and the lipid bilayer regulate the localisation of Fukutin-I, we have developed an efficient expression and purification protocol in Escherichia coli to allow biophysical studies to be performed. Expressing the N-terminal domain of Fukutin-1 fused to a His(6) tag resulted in the localisation of the protein to the bacterial membrane. A purification strategy has been developed to isolate the highly hydrophobic transmembrane domain of Fukutin-1 from the membrane with yields of approximately 4 mg per litre of minimal media. Preliminary biophysical analyses have confirmed the identity of the peptide and revealed that in hydrophobic solvents mimicking the bilayer, the peptide adopts a well-structured alpha-helix as predicted from the sequence.