High accuracy of intra-articular needle position during anterior landmark guided glenohumeral injections.

OBJECTIVES: Image-guided ultrasound or fluoroscopic glenohumeral injections have high accuracy rates but require training, equipment, cost, and radiation exposure (fluoroscopy). In contrast, landmark-guided glenohumeral injections do not require additional subspecialist referrals or equipment. An optimal technique would be safe and accurate and have few barriers to implementation. The purpose of this study was to define the accuracy of glenohumeral needle placement via an anterior landmark-guided approach as assessed by direct arthroscopic visualization. METHODS: A consecutive series of adult patients undergoing shoulder arthroscopy in the beach chair position were included in this study. Demographic and procedural data were collected. The time required to perform the injection, the precise location of the needle tip, and factors that affected the accuracy of the injection were also assessed. RESULTS: A standardized anterior landmark-guided glenohumeral joint injection was performed in the operating room prior to surgery, and the location of the needle tip was documented by arthroscopic visualization with a low complication profile and few barriers to implementation. A total of 81 patients were enrolled. Successful intra-articular glenohumeral needle placement by sports medicine and shoulder/elbow fellowship-trained orthopedic surgeons was confirmed in 93.8% (76/81) of patients. The average time to complete the procedure was 24.8 â€‹s. There were no patient-related variables associated with nonintra-articular injections in the cohort. CONCLUSIONS: This study demonstrated that the technique of anterior landmark-guided glenohumeral injection has an accuracy of 93.8% and requires less than 30 â€‹s to perform. This method is safe, yields similar accuracy to image-guided procedures, has improved cost and time efficiency, and requires less radiation exposure. No patient-related factors were associated with inaccurate needle placement. Anterior landmark-guided glenohumeral injections may be utilized with confidence by providers in the clinical setting. LEVEL OF EVIDENCE: Level 5. IRB: Approved under Stanford IRB-56323.

In utero and postnatal ivacaftor/lumacaftor therapy rescues multiorgan disease in CFTR-F508del ferrets.

Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, with F508del being the most prevalent mutation. The combination of CFTR modulators (potentiator and correctors) has provided benefit to CF patients carrying the F508del mutation; however, the safety and effectiveness of in utero combination modulator therapy remains unclear. We created a F508del ferret model to test whether ivacaftor/lumacaftor (VX-770/VX-809) therapy can rescue in utero and postnatal pathologies associated with CF. Using primary intestinal organoids and air-liquid interface cultures of airway epithelia, we demonstrate that the F508del mutation in ferret CFTR results in a severe folding and trafficking defect, which can be partially restored by treatment with CFTR modulators. In utero treatment of pregnant jills with ivacaftor/lumacaftor prevented meconium ileus at birth in F508del kits and sustained postnatal treatment of CF offspring improved survival and partially protected from pancreatic insufficiency. Withdrawal of ivacaftor/lumacaftor treatment from juvenile CF ferrets reestablished pancreatic and lung diseases, with altered pulmonary mechanics. These findings suggest that in utero intervention with a combination of CFTR modulators may provide therapeutic benefits to individuals with F508del. This CFTR-F508del ferret model may be useful for testing therapies using clinically translatable endpoints.

Transgenic ferret models define pulmonary ionocyte diversity and function.

Speciation leads to adaptive changes in organ cellular physiology and creates challenges for studying rare cell-type functions that diverge between humans and mice. Rare cystic fibrosis transmembrane conductance regulator (CFTR)-rich pulmonary ionocytes exist throughout the cartilaginous airways of humans1,2, but limited presence and divergent biology in the proximal trachea of mice has prevented the use of traditional transgenic models to elucidate ionocyte functions in the airway. Here we describe the creation and use of conditional genetic ferret models to dissect pulmonary ionocyte biology and function by enabling ionocyte lineage tracing (FOXI1-CreERT2::ROSA-TG), ionocyte ablation (FOXI1-KO) and ionocyte-specific deletion of CFTR (FOXI1-CreERT2::CFTRL/L). By comparing these models with cystic fibrosis ferrets3,4, we demonstrate that ionocytes control airway surface liquid absorption, secretion, pH and mucus viscosity-leading to reduced airway surface liquid volume and impaired mucociliary clearance in cystic fibrosis, FOXI1-KO and FOXI1-CreERT2::CFTRL/L ferrets. These processes are regulated by CFTR-dependent ionocyte transport of Cl- and HCO3-. Single-cell transcriptomics and in vivo lineage tracing revealed three subtypes of pulmonary ionocytes and a FOXI1-lineage common rare cell progenitor for ionocytes, tuft cells and neuroendocrine cells during airway development. Thus, rare pulmonary ionocytes perform critical CFTR-dependent functions in the proximal airway that are hallmark features of cystic fibrosis airway disease. These studies provide a road map for using conditional genetics in the first non-rodent mammal to address gene function, cell biology and disease processes that have greater evolutionary conservation between humans and ferrets.

Genome Editing in Ferret Airway Epithelia Mediated by CRISPR/Nucleases Delivered with Amphiphilic Shuttle Peptides.

Gene editing strategies are attractive for treating genetic pulmonary diseases such as cystic fibrosis (CF). However, challenges have included the development of safe and effective vector systems for gene editing of airway epithelia and model systems to report their efficiency and durability. The domestic ferret (Mustela putorius furo) has a high degree of conservation in lung cellular anatomy with humans, and has served as an excellent model for many types of lung diseases, including CF. In this study, we evaluated the efficiency of amphiphilic shuttle peptide S10 for protein delivery and gene editing using SpCas9, and AsCas12a (Cpf1) ribonucleoproteins (RNPs). These approaches were evaluated in proliferating ferret airway basal cells, polarized airway epithelia in vitro, and lungs in vivo, by accessing the editing efficiency using reporter ferrets and measuring indels at the ferret CFTR locus. Our results demonstrate that shuttle peptides efficiently enable delivery of reporter proteins/peptides and gene editing SpCas9 or Cpf1 RNP complexes to ferret airway epithelial cells in vitro and in vivo. We measured S10 delivery efficiency of green fluorescent protein (GFP)-nuclear localization signal (NLS) protein or SpCas9 RNP into ferret airway basal cells and fully differentiated ciliated and nonciliated epithelial cells in vitro. In vitro and in vivo gene editing efficiencies were determined by Cas/LoxP-gRNA RNP-mediated conversion of a ROSA-TG Cre recombinase reporter using transgenic primary cells and ferrets. S10/Cas9 RNP was more effective, relative to S10/Cpf1 RNP at gene editing of the ROSA-TG locus. Intratracheal lung delivery of the S10 shuttle combined with GFP-NLS protein or D-Retro-Inverso (DRI)-NLS peptide demonstrated efficiencies of protein delivery that were ∼3-fold or 14-fold greater, respectively, than the efficiency of gene editing at the ROSA-TG locus using S10/Cas9/LoxP-gRNA. Cpf1 RNPs was less effective than SpCas9 at gene editing of LoxP locus. These data demonstrate the feasibility of shuttle peptide delivery of Cas RNPs to the ferret airways and the potential utility for developing ex vivo stem cell-based and in vivo gene editing therapies for genetic pulmonary diseases such as CF.

Hemiarthroplasty for proximal humerus fractures and for fracture sequelae: did not differ in their outcomes.

Background: The role of hemiarthroplasty (HA) in the management of proximal humerus fractures (PHFs) and their sequalae has evolved with the development of contemporary internal fixation techniques and the widespread use of the reverse total shoulder arthroplasty. However, HA may still have a role in certain acute PHFs as well as select fracture sequalae. The aim of this investigation was to evaluate the outcomes of HA when used in acute fractures and fracture sequelae. Methods: Over a 16-year period (2000 - 2016), 122 primary HA performed for either acute PHFs or fracture sequelae were identified. Of these, 70 (57.4%) HA were performed within 4 weeks of the injury, whereas 52 (42.6%) underwent HA for fracture nonunion, malunion, or avascular necrosis. The minimum follow-up period was 2 years. Outcomes included the visual analog scale for pain, range of motion, American Shoulder and Elbow Surgeons (ASES) score, complications, and reoperations inclusive of revision surgery. Cumulative incidence analysis was used to report implant survivorship with death as a competing risk. Results: The mean follow-up time after HA was 4.8 years (range, 2-15 years) with no differences between groups (P = .102). Cohort comparisons demonstrated an older age (67.8 vs. 60.1; P = .004), lower rate of previous procedure (4.3% vs. 51.9%; P < .001), lower bone graft use (28.6% vs. 59.6%; P < .001), and a longer length of stay (5.9 vs. 3.0 days; P < .001) in the acute HA group. Additionally, no differences were observed between the acute and sequalae cohort in pain (2.0 vs. 2.5; P = .523), forward elevation (98° vs. 93°; P = .627), external rotation (30° vs. 23°; P = .215), internal rotation score (4.0 vs. 4.5; P = .589), satisfaction (P = .592), ASES scores (64.4 vs. 57.1; P = .168), complications (27.1% vs. 28.8%; P = .836), or reoperations (11.4% vs. 19.2%; P = .229). When comparing acute fractures and sequalae, the 15-year complication rates were 32.4% and 43.3%, respectively (P = .172), with 15-year reoperation rates of 13.7% and 24%, respectively (P = .098). Conclusions: HA, whether performed acutely for a PHF or in a delayed fashion for fracture sequalae, demonstrated no statistically significant differences in outcomes for all examined parameters. HA in this setting may provide reasonable pain relief. However, limited motion, marginal ASES scores, and elevated rates of complications and reoperations can be expected up to 15 years postoperatively.

Comparing Unicompartmental Knee Arthroplasty and High Tibial Osteotomy for Isolated Medial Compartment Knee Osteoarthritis.

¼: Both unicompartmental knee arthroplasty (UKA) and high tibial osteotomy (HTO) allow for compartment-specific intervention on an arthritic knee joint that preserves bone stock and native soft tissue compared to a total knee arthroplasty (TKA). Both operations give a more natural feeling with native proprioception compared with a TKA. ¼: HTO is better suited in patients who are younger (<55 years-of-age), have a body mass index (BMI) <30 kg/m2, high activity requirements, mechanical malalignment, asymmetric varus, isolated anterior cruciate ligament insufficiency, need for multiplanar correction, and a preference for joint preserving interventions. Recent data suggest that age (>55 years-of-age) should not solely contraindicate a HTO. ¼: UKA may be chosen in patients who are older (>55 years-of-age), low activity requirements, have a BMI <40 kg/m2, severe osteoarthritis with significant joint space narrowing, acceptable coronal alignment, symmetric varus, and patient preference for arthroplasty.

Antibiotic Prophylaxis with Cefazolin Is Associated with Lower Shoulder Periprosthetic Joint Infection Rates Than Non-Cefazolin Alternatives.

BACKGROUND: Although prophylactic antibiotics are considered the standard of care, data with regard to the comparative efficacy of specific antibiotics in the prevention of periprosthetic joint infection (PJI) have remained limited. This study evaluated whether perioperative antibiotic choice affects rates of PJI development in shoulder arthroplasty. METHODS: From 2000 to 2019, all primary shoulder arthroplasty types (hemiarthroplasty, anatomic total shoulder arthroplasty, reverse shoulder arthroplasty) performed for elective and trauma indications with perioperative antibiotic data and a minimum follow-up of 2 years were identified from a single institution. Demographic characteristics, PJI risk factors, and PJI-free survivorship data were retrieved. Multivariable analyses were conducted to determine the association between the antibiotic administered and the development of PJI. RESULTS: Of 7,713 shoulder arthroplasties, cefazolin was administered in 6,879 procedures (89.2%) and non-cefazolin antibiotics consisting of vancomycin (465 procedures [6.0%]), clindamycin (345 procedures [4.5%]), and alternative regimens (24 procedures [0.31%]) were administered in 834 procedures (10.8%). PJIs occurred in 101 shoulder arthroplasties (1.3%), with Cutibacterium acnes as the most common pathogen (44 procedures [43.6%]). PJI-free survivorship was greater in shoulder arthroplasties in which cefazolin was administered compared with those in which non-cefazolin antibiotics were administered, with 0.91% greater survival free of PJI at 1 month, 1.4% at 1 year, and 2.7% at 15 years (p < 0.001). Cefazolin administration, compared with non-cefazolin administration, was associated with a 69% reduction in all-cause PJI risk and a 78% reduction in C. acnes PJI risk (p < 0.001). A higher risk of PJI for both groups was observed with vancomycin; the hazard ratio [HR] was 2.32 (95% confidence interval [CI], 1.22 to 4.40; p = 0.010) for all-cause PJI and 2.94 (95% CI, 1.12 to 7.49; p = 0.028) for C. acnes PJI. A higher risk of PJI was also observed for both groups for clindamycin; the HR was 5.07 (95% CI, 2.83 to 9.05; p < 0.001) for all-cause PJI and 8.01 (95% CI, 3.63 to 17.42; p < 0.001) for C. acnes PJI. CONCLUSIONS: In primary shoulder arthroplasty, cefazolin administration was associated with a significantly lower rate of PJI compared with non-cefazolin alternatives, including both vancomycin and clindamycin. These risk discrepancies were observed across all infectious pathogens and may be considered even greater when C. acnes was the infecting bacterium. LEVEL OF EVIDENCE: Therapeutic Level III. See Instructions for Authors for a complete description of levels of evidence.

Revision Reverse Shoulder Arthroplasty for Anatomical Glenoid Component Loosening Was Not Universally Successful: A Detailed Analysis of 127 Consecutive Shoulders.

BACKGROUND: Glenoid component loosening is a primary cause of failure of anatomical total shoulder arthroplasty (TSA) and is commonly associated with glenoid bone loss. The purpose of the present study was to evaluate the outcome and survival following revision to a reverse total shoulder arthroplasty (RSA) for the treatment of loosening of a polyethylene cemented glenoid component in the setting of failed TSA. METHODS: Between 2010 and 2017, 151 shoulders underwent revision to RSA for the treatment of loosening of an anatomical polyethylene glenoid component. Shoulders with staged reconstruction for the treatment of infection were excluded. One hundred and twenty-seven patients (67 women and 60 men) had a single-stage reconstruction and were available for follow-up. The mean age at the time of surgery was 70 years (range, 41 to 93 years). In all cases, the humeral component was revised and a standard glenoid baseplate was utilized. Bone graft was used at the discretion of the treating surgeon. Medical records and radiographs were reviewed to collect demographic, intraoperative, and postoperative data; to quantify glenoid bone loss; and to determine the radiographic outcome. The mean duration of follow-up was 35 months (range, 24 to 84 months). RESULTS: Revision to RSA resulted in significant improvements in terms of pain and motion. Sixteen shoulders (13%) underwent revision surgery for the treatment of baseplate loosening. Radiographic baseplate loosening was present in 6 additional shoulders (overall rate of baseplate loosening, 17%). Intraoperative fracture or fragmentation of the greater tuberosity occurred in 30 shoulders (24%). Other reoperations included resection for deep infection (3 shoulders), arthroscopic biopsies for unexplained persistent pain (2 shoulders), humeral tray exchange for dislocation (2 shoulders), revision for humeral loosening (1 shoulder), irrigation and debridement for hematoma (1 shoulder), and internal fixation of periprosthetic fracture (1 shoulder) (overall reoperation rate, 20%). Among shoulders with surviving implants at the time of the most recent follow-up, pain was rated as none or mild in 83 shoulders (65.4%) and the average active elevation and external rotation were 132° and 38°, respectively. With the numbers available, no risk factors for failure could be identified. CONCLUSIONS: Revision RSA for the treatment of loosening of an anatomical polyethylene component was associated with a 17% glenoid mechanical failure rate. Although this procedure resulted in improvements in terms of pain and function, it was not universally successful and thus needs further refinement in order to improve outcomes. LEVEL OF EVIDENCE: Therapeutic Level IV. See Instructions for Authors for a complete description of levels of evidence.

Surgical Excision of a Thrombosed Persistent Median Artery Causing Carpal Tunnel-Like Symptoms: A Case Report.

CASE: A 51-year-old man presented with pain and paresthesias in the median nerve distribution and a subjective loss of grip strength. Imaging revealed a thrombosed persistent median artery in the carpal tunnel abutting the median nerve. The thrombosed portion of the artery was surgically excised, and the patient experienced resolution of symptoms. CONCLUSION: Persistent median artery thrombosis is rare and can cause carpal tunnel syndrome. Ultrasound is a useful tool for diagnosis and appropriate surgical planning. Although treatment with systemic anticoagulation is an option, surgical excision resulted in resolution of symptoms and an excellent short-term outcome.

Primary reverse shoulder arthroplasty: how did medialized and glenoid-based lateralized style prostheses compare at 10 years?

BACKGROUND: The purpose of this study was to compare long-term outcomes, complications, and reoperation rates of primary reverse total shoulder arthroplasty (RTSA) performed at a single institution using 2 implant designs: a Grammont medialized prosthesis (medialized [M] group) and a Frankle glenoid-based lateralized prosthesis (glenoid-lateralized [GL] group). METHODS: Between 2004 and 2008, 100 consecutive single-institution primary RTSAs were performed by reconstructive shoulder surgeons who were not design consultants, with the aim of obtaining 10-year follow-up: 56 in the M group and 44 in the GL group. Patients were followed up until death, until revision surgery, or for a minimum of 10 years. RESULTS: Of 100 patients, 87 had more than 2 years' follow-up (mean, 77 months). A subset analysis of 41 patients with an average of 10.2 years' follow-up showed sustained long-term outcomes. RTSA provided clinical improvements without significant differences between the M and GL groups, except for improved active forward elevation in the M group (144° in M group vs. 115° in GL group, P = .002). Reoperation was required in 6 shoulders (10-year cumulative incidence of 3 [5%] in M group vs. 3 [8%] in GL group) for a total of 16 complications (10-year cumulative incidence of 8 [14%] in M group vs. 8 [20%] in GL group). Notching rates were significantly higher in the M group (77% in M group vs. 47% in GL group, P = .013); differences in severe notching (grade 3 or 4) were clinically relevant but did not reach statistical significance (23% in M group vs. 9% in GL group, P = .22). CONCLUSION: Primary RTSA using these first 2 prosthesis designs was associated with good outcomes and low reoperation (5%-8%) and complication (14%-20%) rates at 10 years. The M group had higher rates of notching. These results may provide a benchmark for comparison with newer implants, especially considering that these results include the early RTSA implantation learning curve.

Epidemiology of Hand and Wrist Injuries in NCAA Men's Football: 2009-2010 to 2013-2014.

BACKGROUND: Participation in National Collegiate Athletic Association (NCAA) football is at an all-time high. This population of athletes experiences a substantial injury burden, with many injuries affecting the upper extremities. PURPOSE/HYPOTHESIS: The purpose of this study was to describe the epidemiology of hand and wrist injuries in college football players from the academic years 2009-2010 to 2013-2014. We hypothesized that variables such as event type (practice vs game), mechanism of injury, and player position would have an effect on the injury incidence. STUDY DESIGN: Descriptive epidemiological study. METHODS: An epidemiological study utilizing the NCAA Injury Surveillance Program was performed to investigate rates and patterns of hand and wrist injuries in participating varsity football teams from 2009-2010 to 2013-2014. RESULTS: A total of 725 hand and wrist injuries were captured in 899,225 athlete-exposures. The observed practice injury rate was 0.51 injuries per 1000 athlete-exposures, compared with a game injury rate of 3.60 (P < .01). Player-on-player contact was the most common injury mechanism reported, with blocking being the most common activity at the time of injury. Offensive linemen were most likely to experience an injury. Of all injuries sustained, 71.4% resulted in no time loss from competition, whereas 9.8% of injuries resulted in longer than 7 days of time loss. A fracture resulted in the greatest time loss from competition (mean ± SD, 8.3 ± 24.0 days; median, 0 days [range, 0-148 days] for injuries sustained in a practice setting) (mean ± SD, 7.7 ± 15.8 days; median, 0 days [range, 0-87 days] for injuries sustained in a game setting). CONCLUSION: Hand and wrist injuries were found to be significantly more common in games when compared with practices. This study provides valuable prognostic data regarding expected time loss on a per-injury pattern basis. Further investigation on specific injury subtypes and expected time loss as a result of these injures would provide trainers, players, and coaches with useful information on an expected postinjury recovery and rehabilitation timeline.

Arthroscopy and arthrotomy to address intra-articular pathology during PAO for hip dysplasia demonstrates similar short-term outcomes.

Periacetabular osteotomy (PAO) remains the gold standard procedure for joint preservation in symptomatic developmental dysplasia of the hip (DDH). Hip arthroscopy (HA) and open arthrotomy have been used to correct intra-articular pathology at the time of PAO, but there is limited data regarding differences in outcomes between these techniques when performed at the time of PAO. The aim of this study was to determine if short-term clinical outcomes differed between patients managed with HA versus arthrotomy to evaluate and treat intra-articular pathology at the time of PAO to discern if one technique is associated with better pain and functional results. Data were retrospectively reviewed from two surgeons at one institution managing DDH patients from September 2013 to December 2015. One surgeon treated patients with PAO and arthrotomy (N = 32), while the other performed PAO and HA (N = 39). There were 87% women, median age was 28 years and mean BMI was 25. Seventy-five percent of all patients received an intra-articular intervention. Patients completed 13 PROs at the pre-operative and 1-year post-operative clinical visits. Pre-operatively, there were no differences in any of the 13 PROs between patients treated with HA versus arthrotomy (P ≥ 0.076). Patients treated with PAO and arthrotomy experienced greater mean improvement in two out of the 13 PROs; the other 11 showed no differences. No treatment effect was observed for any of the 13 PROs using multivariable modelling that accounted for severity of dysplasia and degree of arthritis. Few differences were shown in short-term clinical outcomes between HA and arthrotomy at the time of PAO. This work highlights the need for a high quality randomized clinical trial to provide definitive guidance on whether hip preservation surgeons should address intra-articular pathology at the time of PAO for DDH and which technique best serves this purpose.

Patterns of viral load decline with telaprevir-based therapy in patients with genotype 1 chronic HCV infection.

BACKGROUND: Telaprevir-based therapy is associated with rapid decline in HCV RNA, enabling the application of early futility rules. OBJECTIVES: To familiarize physicians with this paradigm, a comprehensive analysis of the most frequent HCV viral load profiles observed during treatment with telaprevir/Peg-IFN/RBV in Phase III trials is provided. DESIGN: HCV RNA profiles were analyzed from 320 HCV genotype 1 treatment-naïve patients enrolled in the ADVANCE study, and 225 prior Peg-IFN/RBV treatment-experienced patients enrolled in the REALIZE study. Patients received 12 weeks of telaprevir with either 24 or 48 weeks of Peg-IFN alfa-2a/RBV. Patients with missing SVR assessments during follow-up, detectable HCV RNA at end of treatment but who did not have viral breakthrough (vBT), or with early vBT who discontinued telaprevir before time of failure were excluded. RESULTS: All analyzed patients experienced a rapid decline in HCV RNA (>2.0 log(10)) by Day 14, irrespective of baseline characteristics and/or prior response to Peg-IFN/RBV (relapse, partial response and null response). Subsequently, HCV RNA continued to decline to undetectable levels in most patients. These patients went on to have one of the following outcomes: sustained virologic response, late vBT (after Week 12, i.e. during the Peg-IFN/RBV phase), or relapse. In the small subset of patients with early vBT or meeting a futility rule before Week 12, HCV RNA usually never became undetectable and/or increased rapidly after reaching the nadir. CONCLUSIONS: HCV RNA profiles with telaprevir/Peg-IFN/RBV are different from those with Peg-IFN/RBV alone. It is important that clinicians understand these HCV RNA profiles and monitor patient viral load in order to apply futility rules correctly.

Characterization of telaprevir treatment outcomes and resistance in patients with prior treatment failure: results from the REALIZE trial.

UNLABELLED: In the Phase 3 REALIZE study, 662 genotype 1 hepatitis C virus (HCV)-infected patients with prior peginterferon/ribavirin treatment failure (including relapsers, partial, and null responders) were randomized to 12 weeks of telaprevir given immediately (T12/PR48) or following 4 weeks of peginterferon/ribavirin (lead-in T12/PR48), or 12 weeks of placebo (PR48), combined with a total of 48 weeks of peginterferon alfa-2a/ribavirin. Sustained virologic response (SVR) rates were 64% (T12/PR48), 66% (lead-in T12/PR48), and 17% (PR48). This analysis aimed to characterize treatment outcomes and viral variants emerging in telaprevir-treated patients not achieving SVR. HCV NS3·4A population sequencing was performed at baseline, during treatment, and follow-up. Telaprevir-resistant variants were classified into lower-level (3- to 25-fold 50% inhibitory concentration [IC(50) ] increase: V36A/M, T54A/S, R155I/K/M/T, and A156S) and higher-level (>25-fold IC(50) increase: V36M+R155K and A156T/V) resistance. Resistant variants were uncommon at baseline. Overall, 18% (52%, 19%, and 1% of prior null and partial responders and relapsers, respectively) of telaprevir-treated patients had on-treatment virologic failure, with no significant difference with or without a lead-in. Virologic failure during the telaprevir-treatment phase was predominantly associated with higher-level resistance; virologic failure during the peginterferon/ribavirin-treatment phase was associated with higher- or lower-level, or wildtype variants, depending on genotype. Relapse occurred in 9% of patients completing assigned treatment and was generally associated with lower-level resistant variants or wildtype. Resistant variants were no longer detectable by study end (median follow-up of 11 months) in 58% of non-SVR patients. CONCLUSION: In REALIZE, variants emerging in non-SVR, telaprevir-treated patients were similar irrespective of the use of a lead-in and were consistent with those previously reported. In most patients, resistant variants became undetectable over time.

Dual targeting of GyrB and ParE by a novel aminobenzimidazole class of antibacterial compounds.

A structure-guided drug design approach was used to optimize a novel series of aminobenzimidazoles that inhibit the essential ATPase activities of bacterial DNA gyrase and topoisomerase IV and that show potent activities against a variety of bacterial pathogens. Two such compounds, VRT-125853 and VRT-752586, were characterized for their target specificities and preferences in bacteria. In metabolite incorporation assays, VRT-125853 inhibited both DNA and RNA synthesis but had little effect on protein synthesis. Both compounds inhibited the maintenance of negative supercoils in plasmid DNA in Escherichia coli at the MIC. Sequencing of DNA corresponding to the GyrB and ParE ATP-binding regions in VRT-125853- and VRT-752586-resistant mutants revealed that their primary target in Staphylococcus aureus and Haemophilus influenzae was GyrB, whereas in Streptococcus pneumoniae it was ParE. In Enterococcus faecalis, the primary target of VRT-125853 was ParE, whereas for VRT-752586 it was GyrB. DNA transformation experiments with H. influenzae and S. aureus proved that the mutations observed in gyrB resulted in decreased susceptibilities to both compounds. Novobiocin resistance-conferring mutations in S. aureus, H. influenzae, and S. pneumoniae were found in gyrB, and these mutants showed little or no cross-resistance to VRT-125853 or VRT-752586 and vice versa. Furthermore, gyrB and parE double mutations increased the MICs of VRT-125853 and VRT-752586 significantly, providing evidence of dual targeting. Spontaneous frequencies of resistance to VRT-752586 were below detectable levels (<5.2x10(-10)) for wild-type E. faecalis but were significantly elevated for strains containing single and double target-based mutations, demonstrating that dual targeting confers low levels of resistance emergence and the maintenance of susceptibility in vitro.

Quorum sensing in Staphylococcus aureus biofilms.

Several serious diseases are caused by biofilm-associated Staphylococcus aureus, infections in which the accessory gene regulator (agr) quorum-sensing system is thought to play an important role. We studied the contribution of agr to biofilm development, and we examined agr-dependent transcription in biofilms. Under some conditions, disruption of agr expression had no discernible influence on biofilm formation, while under others it either inhibited or enhanced biofilm formation. Under those conditions where agr expression enhanced biofilm formation, biofilms of an agr signaling mutant were particularly sensitive to rifampin but not to oxacillin. Time lapse confocal scanning laser microscopy showed that, similar to the expression of an agr-independent fluorescent reporter, biofilm expression of an agr-dependent reporter was in patches within cell clusters and oscillated with time. In some cases, loss of fluorescence appeared to coincide with detachment of cells from the biofilm. Our studies indicate that the role of agr expression in biofilm development and behavior depends on environmental conditions. We also suggest that detachment of cells expressing agr from biofilms may have important clinical implications.

Palmitoylation and plasma membrane localization of Ras2p by a nonclassical trafficking pathway in Saccharomyces cerevisiae.

Subcellular localization of Ras proteins to the plasma membrane is accomplished in part by covalent attachment of a farnesyl moiety to the conserved CaaX box cysteine. Farnesylation targets Ras to the endoplasmic reticulum (ER), where additional processing steps occur, resulting in translocation of Ras to the plasma membrane. The mechanism(s) by which this occurs is not well understood. In this report, we show that plasma membrane localization of Ras2p in Saccharomyces cerevisiae does not require the classical secretory pathway or a functional Golgi apparatus. However, when the classical secretory pathway is disrupted, plasma membrane localization requires Erf2p, a protein that resides in the ER membrane and is required for efficient palmitoylation of Ras2p. Deletion of ERF2 results in a Ras2p steady-state localization defect that is more severe when combined with sec-ts mutants or brefeldin A treatment. The Erf2p-dependent localization of Ras2p correlates with the palmitoylation of Cys-318. An Erf2p-Erf4p complex has recently been shown to be an ER-associated palmitoyltransferase that can palmitoylate Cys-318 of Ras2p (S. Lobo, W. K. Greentree, M. E. Linder, and R. J. Deschenes, J. Biol. Chem. 277:41268-41273, 2002). Erf2-dependent palmitoylation as well as localization of Ras2p requires a region of the hypervariable domain adjacent to the CaaX box. These results provide evidence for the existence of a palmitoylation-dependent, nonclassical endomembrane trafficking system for the plasma membrane localization of Ras proteins.