Bacillus Calmette-Guérin Packaged for Percutaneous Vaccination Can Be Safely Used for Intravesical Instillation in Patients with Urothelial Carcinoma of the Bladder.

INTRODUCTION: Bacillus Calmette-Guérin production is limited worldwide with stuttering shortages affecting patient access. Our institution received 50 vials of bacillus Calmette-Guérin labeled for percutaneous administration, and upon discussion with our clinical team and approval by the Pharmacy and Therapeutics Committee we used the percutaneous formulation in place of the intravesical formulation. We report our experience. METHODS: Between February and April 2019 patients were treated with a third of a vial dose either with percutaneous or intravesical bacillus Calmette-Guérin. American Urological Association Symptom Score and Quality of Life survey and an additional 6-question survey (querying presence of suprapubic pain, hematuria, fevers, malaise, skin rashes, testicular/groin pain) were administered. Statistical analyses comparing the 2 groups were performed with SPSS version 22 software. RESULTS: A total of 30 patients with 73 intravesical instillations were evaluated with 34 patients receiving intravesical and 39 percutaneous bacillus Calmette-Guérin. We found no significant differences when comparing intravesical vs percutaneous bacillus Calmette-Guérin groups in terms of American Urological Association Symptom Score (6.1 vs 6.9, p=0.177), Quality of Life score (1.3 vs 1.7, p=0.132), fevers (2.9% vs 0%, p=0.300), hematuria (14.7% vs 2.8%, p=0.075), suprapubic pain (10.1% vs 4.3%, p=0.129), skin rashes (1.4% vs 0%, p=0.307) and feeling of general fatigue and malaise (15.7% vs 8.6%, p=0.126). CONCLUSIONS: Intravesical instillation of percutaneous bacillus Calmette-Guérin appears to be a safe alternative to intravesical bacillus Calmette-Guérin during times of shortage. Development of additional strains, use of alternative intravesical therapies and incentivizing bacillus Calmette-Guérin production through policy change and/or alternative funding may also help avoid bacillus Calmette-Guérin supply shortages in the future.

Use of Cannulated Instruments to Localize the Portals in Anterior Ankle Arthroscopy: A Technique Tip.

The use of cannulated instruments under fluoroscopy can improve the localization of the anteromedial and posterolateral portals for use in ankle arthroscopy. This technique is valuable for the less-experienced ankle arthroscopist, in resident education, and for the experienced arthroscopist when surface anatomy palpation and visualization is less than ideal due to soft tissue edema and obesity.

2013 Subtalar Arthroereisis Survey: The Current Practice Patterns of Members of the AOFAS.

BACKGROUND: Subtalar arthroereisis (SA) has been a procedure used for the correction of painful flexible flatfoot deformity in adults and children. Clinical studies of patients who had a SA are sparse and with mixed results and variable indications. The purpose of this study was to determine the current practice among orthopaedic foot and ankle specialists regarding SA. METHODS: Web-based questionnaires were e-mailed to members of the American Orthopaedic Foot and Ankle Society (AOFAS). Requested information included demographics and practice patterns in regard to performing SA surgery. A total of 572 respondents completed the survey (32% response rate). RESULTS: A total of 273 respondents (48%) have performed SA. Of this group, 187 respondents (69%) still perform this procedure (33% of total respondents currently perform SA). Of the respondents, 401 (70%) practice in the United States, 40% have performed SA, and 60% of those still perform this procedure. Of non-US respondents, 66% have performed SA, and 80% of those still perform it. The most common US indications are painful congenital flatfoot, posterior tibial tendon dysfunction, and flatfoot associated with accessory navicular. CONCLUSION: Many doctors have performed SA, and a significant number no longer perform this procedure for various reasons. A greater percentage of non-US practitioners have performed and continue to perform SA than their counterparts in the United States. There is a common list of surgical indications. Most doctors who still perform this procedure have removed the implants, commonly for pain. SA is still being performed in the United States and throughout the world.

Comparison of two fixation methods for arthrodesis of the calcaneocuboid joint: a biomechanical study.

BACKGROUND: The traditional fixation for a calcaneocuboid (CC) arthrodesis in triple arthrodesis is with a 6.5-mm cancellous screw. This procedure can be technically challenging. Fixation with a locking compression plate (LCP) may be easier to perform while achieving compression perpendicular to the fusion site. The purpose of this study was to compare the load to failure and the stiffness for each fixation method. METHODS: Five matched-pair cadaver feet had an arthrodesis of the CC joint. For each matched pair, one was fixed with a screw and the other with an LCP. Surface bead markers were applied. Each specimen was then secured to a material testing machine through the calcaneus. The plantar surface of the cuboid faced the hydraulic ram to simulate weightbearing. A force was applied while the specimen was recorded with a high-resolution camera. The endpoint was maximal force at 2-mm separation between the calcaneus and cuboid measured along a horizontal axis. RESULTS: The average force to failure and the average stiffness in the screw group were significantly less than the LCP group (P < .05). The screw construct failed in pullout from the cuboid; the LCP construct failed by plastic deformation of the plate. CONCLUSION: Calcaneocuboid joint fixation with the LCP withstood a higher load until failure and demonstrated greater stiffness than with a 6.5-mm cancellous lag screw. CLINICAL RELEVANCE: The use of LCP fixation can be considered as an alternative to oblique lag screw fixation for CC arthrodesis in a triple arthrodesis. It remains to be determined if LCP fixation leads to better clinical outcomes.

Amino acids as biomarkers in the SOD1(G93A) mouse model of ALS.

The development of therapies for Amyotrophic Lateral Sclerosis (ALS) has been hindered by the lack of biomarkers for both identifying early disease and for monitoring the effectiveness of drugs. The identification of ALS biomarkers in presymptomatic individuals might also provide clues to the earliest biochemical correlates of the disease. Previous attempts to use plasma metabolites as biomarkers have led to contradictory results, presumably because of heterogeneity in both the underlying genetics and the disease stage in the clinical population. To eliminate these two sources of heterogeneity we have characterized plasma amino acids and other metabolites in the SOD1(G93A) transgenic mouse model for ALS. Presymptomatic SOD1(G93A) mice have significant differences in concentrations of several plasma metabolites compared to wild type animals, most notably in the concentrations of aspartate, cystine/cysteine, and phosphoethanolamine, and in changes indicative of methylation defects. There are significant changes in amino acid compositions between 50 and 70days of age in both the SOD1(G93A) and wild type mice, and several of the age-related and disease-related differences in metabolite concentration were also gender-specific. Many of the SOD1(G93A)-related differences could be altered by treatment of mice with methionine sulfoximine, which extends the lifespan of this mouse, inhibits glutamine synthetase, and modifies brain methylation reactions. These studies show that assaying plasma metabolites can effectively distinguish transgenic mice from wild type, suggesting that one or more plasma metabolites might be useful biomarkers for the disease in humans, especially if genetic and longitudinal analysis is used to reduce population heterogeneity.

Fluoroscopic method for localization of the anteromedial portal for use in ankle arthroscopy: technique tip.

Ankle arthroscopy has become a standard surgical technique for the treatment of many ankle pathologies. Over the past 30 years, the technique has undergone modification and standardization in order to improve surgical performance and outcomes. In contrast to the ankle joint, the hip joint is a deep joint, which makes visualization and palpation of the topographical anatomy quite difficult. The use of fluoroscopy has enabled the surgeon to successfully perform hip arthroscopy for the treatment of selective hip pathologies. Fluoroscopy also can improve the localization of the anteromedial portal for use in ankle arthroscopy. This technique is valuable for the less-experienced ankle arthroscopist, in resident education, and for the experienced arthroscopist when surface anatomy palpation and visualization is less than ideal due to soft tissue edema and obesity.

Effect of sex on lifespan, disease progression, and the response to methionine sulfoximine in the SOD1 G93A mouse model for ALS.

OBJECTIVE: To investigate the role of sex and the role of ammonia and amino acid metabolism, specifically the activity of glutamine synthetase, in survival and disease progression in amyotrophic lateral sclerosis. METHODS: We tested treatment with methionine sulfoximine (MSO) on the lifespan and neuromuscular ability of male and female SOD1 mice as measured by their ability to maintain their grip on an inverted wire grid. We also tested the effects of castration and ovariectomization on those measurements. RESULTS: MSO treatment improves the survival of both male and female mice, but the effects are significantly greater on female mice. Saline-treated (control) female mice have delayed neuromuscular degeneration compared with saline-treated male mice, and MSO further delays disease progression in females, to a greater extent than in males. Ovariectomization or castration completely eliminates the effect of the drug on either survival or neuromuscular deterioration. CONCLUSIONS: Sex is an important factor in disease progression and the response of SOD1 mice to a drug targeting a central enzyme in nitrogen metabolism, with female sex hormones playing a greater role than male sex hormones. Glutamine synthetase, or its reactants and products, therefore plays a role in this disease, and the sex specificity of treatments aimed at this or other metabolic targets may therefore be an important factor in the development of therapies to treat amyotrophic lateral sclerosis.

A glutamine synthetase inhibitor increases survival and decreases cytokine response in a mouse model of acute liver failure.

BACKGROUND: Acute liver failure (ALF) can be induced in mice by administering Escherichia coli lipopolysaccharide (LPS) and D-galactosamine (D-GalN), which induce an inflammatory response involving tumour necrosis factor (TNF)-α production and a hepatocyte-specific transcriptional block. Under these conditions, binding of TNF-α to its cognate receptor on hepatocytes eventually leads to their apoptosis. AIMS: As part of an effort to identify drugs to treat this disease model, we have investigated whether the glutamine synthetase inhibitor methionine sulfoximine (MSO) could play a protective role, given its effectiveness in the inhibition of brain swelling associated with hyperammonaemia. METHODS: Mouse survival, glutamine synthetase activity, hepatocyte apoptosis and induction of inflammatory cytokines were measured in mice treated with MSO before an intraperitoneal injection of LPS/D-GalN. The effect of MSO on viability and on TNF-α release was also assessed on inflammatory and liver cells. RESULTS: We have found that, in mice treated with LPS/D-GalN, MSO (i) drastically increases animal survival; (ii) sharply reduces glutamine synthetase activity, without inhibiting its other target, γ-glutamyl cysteine synthetase; (iii) inhibits death receptor-mediated apoptosis in hepatocytes upstream to cytokine binding; (iv) strongly reduces the overall inflammatory cytokine response, including a significant decrease in TNF-α induction in vivo and ex vivo, and in the interferon-γ level and signalling. CONCLUSIONS: These results demonstrate that the MSO target glutamine synthetase is required for the early steps of the cytokine response to endotoxins, and that its pharmacological inhibition may be exploited to treat inflammation.

Methionine sulfoximine, an inhibitor of glutamine synthetase, lowers brain glutamine and glutamate in a mouse model of ALS.

In an effort to alter the levels of neurochemicals involved in excitotoxicity, we treated mice with methionine sulfoximine (MSO), an inhibitor of glutamine synthetase. Since glutamate toxicity has been proposed as a mechanism for the degeneration of motor neurons in a variety of neurodegenerative diseases, we tested the effects of MSO on the transgenic mouse that overexpresses the mutant human SOD1(G93A) gene, an animal model for the primary inherited form of the human neurodegenerative disease amyotrophic lateral sclerosis (ALS). This treatment in vivo reduced glutamine synthetase activity measured in vitro by 85%. Proton magnetic resonance spectroscopy, with magic angle spinning of intact samples of brain tissue, showed that MSO treatment reduced brain levels of glutamine by 60% and of glutamate by 30% in both the motor cortex and the anterior striatum, while also affecting levels of GABA and glutathione. Kaplan-Meyer survival analysis revealed that MSO treatment significantly extended the lifespan of these mice by 8% (p<0.01). These results show that in the SOD1(G93A) model of neurodegenerative diseases, the concentration of brain glutamate (determined with (1)H-MRS) can be lowered by inhibiting in vivo the synthesis of glutamine with non-toxic doses of MSO.

A surgical approach for flexible flatfeet in adults including a subtalar arthroereisis with the MBA sinus tarsi implant.

BACKGROUND: The purpose of this study was to determine the functional outcomes and radiographic results of adult patients who had an operation for flexible flatfeet without any hindfoot osteotomies or fusions. METHODS: Twenty-eight feet in 23 patients with problems caused by their flexible flatfoot deformities had reconstructive foot and ankle surgery that included a subtalar arthroereisis (the restriction of the range of motion of a joint) with the Maxwell-Brancheau Arthroereisis (MBA) sinus tarsi implant. The American Orthopedic Foot and Ankle Society (AOFAS) Hindfoot Scale and a patient assessment questionnaire were obtained from all patients before surgery and at final follow-up. Preoperative and postoperative standing radiographs were analyzed to determine radiographic correction of the deformities. The average followup was 44 months. The MBA implant was surgically removed in 11 of 28 feet (39%) because of sinus tarsi pain. RESULTS: The average preoperative AOFAS score was 52 and had improved to 87 (p<0.00001) at final followup. The average response to four of five questions in the patient assessment had significantly improved (p<0.05). On a 10-point scale, average patient satisfaction was 8.3 points; 78% said that they would have the surgery again. Correction after surgery was significant (p<0.0001) in each of the three radiographic parameters evaluated for 'correction with MBA' and 'final correction.' With the numbers available, no significant differences could be detected after the MBA was removed. Complications included sinus tarsi pain in 46% (13) of the 28 feet in this study; after implant removal, 73% (8) of 11 feet had less discomfort than before surgery with AOFAS scores 80 or better. CONCLUSIONS: Reconstructive foot and ankle surgery that included a subtalar arthroereisis with the MBA sinus tarsi implant resulted in favorable clinical outcomes and patient satisfaction in 78% (18) of 23 patients. In spite of the high incidence of temporary sinus tarsi pain until the implant was removed, this operative approach compares favorably with other operations for flexible flatfoot deformities in adults.

Activity of the yeast Tat2p tryptophan permease is sensitive to the anti-tumor agent 4-phenylbutyrate.

4-Phenylbutyrate (PB) induces differentiation and is being intensively studied as a treatment for brain, prostate, breast, and hematopoietic cancer. While many different primary targets for PB have been proposed, the mechanism by which it causes cellular differentiation remains unknown. To identify the primary cellular target, we investigated its effects on Saccharomyces cerevisiae and showed that it inhibits tryptophan transport. We show here that PB and sorbic acid induce an ubiquitin-dependent turnover of the tryptophan permease Tat2p. However, the inhibition of transport is not a consequence of the loss of Tat2p, since it also occurs when turnover is prevented by deleting the Tat2p ubiquitination sites. When we tested the effects of PB and other growth inhibitory agents on the growth of amino acid auxotrophs, we found that several auxotrophs are hypersensitive to a number of chemically unrelated agents, including PB and some, but not all, weak acids; and this sensitivity is due to the inhibition of amino acid transport. For the inhibitory weak acids, inhibition is not confined to aromatic amino acid auxotrophs, nor is it a general weak acid stress response, since the degree of inhibition is independent of weak acid hydrophobicity and p Ka. Our results show that diverse agents affect the activity of the Tat2p permease rather than its stability and suggest the hypothesis that the anti-neoplastic action of PB is due to a decrease in the activity of surface receptors or other membrane proteins needed to maintain the transformed state.

Water-mediated hydrogen-bonded network on the cytoplasmic side of the Schiff base of the L photointermediate of bacteriorhodopsin.

The L intermediate in the proton-motive photocycle of bacteriorhodopsin is the starting state for the first proton transfer, from the Schiff base to Asp85, in the formation of the M intermediate. Previous FTIR studies of L have identified unique vibration bands caused by the perturbation of several polar amino acid side chains and several internal water molecules located on the cytoplasmic side of the retinylidene chromophore. In the present FTIR study we describe spectral features of the L intermediate in D(2)O in the frequency region which includes the N-D stretching vibrations of the backbone amides. We show that a broad band in the 2220-2080 cm(-1) region appears in L. By use of appropriate (15)N labeling and mutants, the lower frequency side of this band in L is assigned to the amides of Lys216 and Gly220. These amides are coupled to each other, and interact with Thr46 and Val49 in helix B and Asp96 in helix C via weakly H-bonding water molecules that exhibit O-D stretching vibrations at 2621 and 2605 cm(-1). These water molecules are part of a hydrogen-bonded network characteristic of L which includes other water molecules located closer to the chromophore that exhibit an O-D stretching vibration at 2589 cm(-1). This structure, extending from the Schiff base to the internal proton donor Asp96, stabilizes L and affects the L-to-M transition.

Immunosuppressant-like effects of phenylbutyrate on growth inhibition of Saccharomyces cerevisiae.

Phenylbutyrate (4-phenylbutyric acid; PB) and its metabolite, phenylacetate, are effective anti-neoplastic agents in tissue culture and have shown promise in clinical trials for a variety of neoplasms. PB is a drug of remarkably low toxicity that acts in vitro as a differentiating agent, causing reversion of the transformed phenotype by an unknown mechanism. We attempted to identify the cellular target(s) for PB using Saccharomyces as a model. PB inhibits growth of yeast on rich medium at concentrations of 0.1-1.0 mM, concentrations similar to plasma concentrations observed in human trials. Yeast cells treated with 1 mM PB remain over 90% viable for 24 h. PB inhibits tryptophan uptake, and resistance to PB can be conferred by tryptophan prototrophy, by supplementing tryptophan auxotrophs with the high levels of tryptophan, by overexpression of the aromatic amino acid permeases Tat1p or Tat2p, and by disruption of TAT1. Since tryptophan auxotrophy and transport influences resistance to PB, phytosphingosine, and the immunosuppressant FK506, these drugs might affect the same pathway. We isolated and characterized a mutant resistant to 1 mM PB and identified the mutant as bul1. A chromosomal BUL1 deletion displayed all phenotypes shown by the PB-resistant mutant.

Changes in hydrogen bonding and environment of tryptophan residues on helix F of bacteriorhodopsin during the photocycle: a time-resolved ultraviolet resonance Raman study.

Protein structural changes during the photocycle of bacteriorhodopsin were examined by time-resolved ultraviolet resonance Raman (UVRR) spectroscopy. Most of the 244-nm UVRR difference signals of Trp were assigned to either Trp182 or Trp189 using the Trp182 --> Phe and Trp189 --> Phe mutants. The W17 mode of Trp182 shows a wavenumber downshift in the M(1) --> M(2) transition, indicating an increase in hydrogen bonding strength at the indole nitrogen. On the other hand, Trp189 shows Raman intensity increases of the W16 and W18 modes ascribable to an increased hydrophobic interaction. These observations suggest that the tilt of helix F, which ensures that reprotonation of the Schiff base is from the cytoplasmic side, occurs in the M(1) --> M(2) transition. In the M(2) --> N transition, the environment of Trp189 returns to the initial state, whereas the hydrophobic interaction of Trp182 decreases drastically. The decrease in hydrophobic interaction of Trp182 in the N state suggests an invasion of water molecules that promote the proton transfer from Asp96 to the Schiff base. Structural reorganization of the protein after the tilt of helix F may be important for efficient reprotonation of the Schiff base.

Conformational change of the E-F interhelical loop in the M photointermediate of bacteriorhodopsin.

The conformation of the structured EF interhelical loop of bacteriorhodopsin and its change in the M photointermediate were assessed by measuring the rate of reaction of 16 single engineered cysteine residues along the loop with water-soluble sulfhydryl reagents. The exposure to the bulk in the unilluminated state determined with the cysteine reaction correlated well with the degree of access to water calculated from the crystallographic structure of the loop. The EF-loop should be affected by the well-known outward tilt of helix F in the M and N intermediates of the photocycle. A second mutation in each cysteine mutant, the D96N residue replacement, allowed full conversion to the M state by illumination. The reaction rates measured under these conditions indicated that buried residues tend to become more exposed, and exposed residues become more buried in M. This is to be expected from tilt of helix F. However, the observation of increased exposure of four residues near the middle of the loop, where steric effects are only from other loop residues, indicate that the conformation of the EF-loop itself is changed. Thus, the motion of the loop in M is more complex than expected from simple tilt of helix F, and may include rotation that unwinds its twist.