The safety of high-dose dexmedetomidine after cardiac surgery: a historical cohort study.

PURPOSE: The off-label use of dexmedetomidine beyond the monograph-recommended maximum dose of 0.7 µg·kg-1·hr-1 is common in postoperative cardiac surgical units; however, limited data exist on the association of higher doses and adverse hemodynamic effects. We sought to compare the rate of hypotension or bradycardia in cardiac surgery patients receiving peak infusion doses below and above 0.7 µg·kg-1·hr-1 for any indication or duration. METHODS: In this historical cohort study, we reviewed all patients who received dexmedetomidine infusion after cardiac surgery between June 2013 and July 2017 at a single centre. Regardless of the duration of exposure at the peak infusion dose, patients were categorized into high- or standard-dose groups using 0.7 µg·kg-1·hr-1 as the cutoff value. We compared rates of the primary composite outcome of hypotension or bradycardia, and secondary outcomes (i.e., arrhythmia and hyperglycemia) between groups using the two-proportion z test. Exploratory regression models were fitted to adjust for potential confounders. RESULTS: The median [interquartile range (IQR)] peak infusion dose was 1.0 [1.0-1.4] µg·kg-1·hr-1 in the high-dose group (N = 121) and 0.5 [0.4-0.7] µg·kg-1·hr-1 in the standard-dose group (N = 124). The rates of the primary composite outcome were 73% and 65%, respectively (absolute risk difference, 8%; 95% confidence interval, -3 to 20; P = 0.17). There was no significant difference in primary or secondary outcomes between groups. CONCLUSION: There was a high overall rate of hypotension or bradycardia in patients receiving dexmedetomidine after cardiac surgery; infusion rates below or above 0.7 µg·kg-1·hr-1 had similar rates of adverse hemodynamic events.

RéSUMé: OBJECTIF: L'utilisation non conforme (off-label) de la dexmédétomidine au-delà de la dose maximale recommandée dans la monographie de 0,7 µg·kg−1·h−1 est fréquente dans les unités de chirurgie cardiaque postopératoire; cependant, il n'existe que peu de données sur l'association entre des doses plus élevées et des effets hémodynamiques indésirables. Nous avons cherché à comparer le taux d'hypotension ou de bradycardie chez les patients de chirurgie cardiaque recevant des doses de perfusion maximales inférieures ou supérieures à 0,7 µg·kg−1·h−1 pour toute indication ou durée. MéTHODE: Dans cette étude de cohorte historique, nous avons passé en revue tous les patients qui ont reçu une perfusion de dexmédétomidine après une chirurgie cardiaque entre juin 2013 et juillet 2017 dans un seul centre. Quelle que soit la durée de l'exposition à la dose de perfusion maximale, les patients ont été classés en groupes à dose élevée ou standard selon une valeur seuil de 0,7 µg·kg−1·h−1. Nous avons comparé les taux d'hypotension ou de bradycardie, notre critère d'évaluation principal composite, et les taux des critères d'évaluation secondaires (soit l'arythmie et l'hyperglycémie) entre les groupes à l'aide du test z à deux proportions. Des modèles de régression exploratoire ont été ajustés pour tenir compte des facteurs de confusion potentiels. RéSULTATS: La dose de perfusion maximale médiane [écart interquartile (ÉIQ)] était de 1,0 [1,0­1,4] µg·kg−1·h−1 dans le groupe à forte dose (n = 121) et de 0,5 [0,4­0,7] µg·kg−1·h−1 dans le groupe à dose standard (n = 124). Les taux pour le critère d'évaluation principal composite étaient de 73% et 65%, respectivement (différence de risque absolue, 8%; intervalle de confiance à 95%, -3 à 20; P = 0,17). Aucune différence intergroupe significative n'a été observée dans les critères d'évaluation primaires ou secondaires. CONCLUSION: Nous avons observé un taux global élevé d'hypotension ou de bradycardie chez les patients recevant de la dexmédétomidine après une chirurgie cardiaque; les taux de perfusion inférieurs ou supérieurs à 0,7 µg·kg−1·h−1 ont entraîné des taux similaires d'événements hémodynamiques indésirables.

Movement Patterns During a Jump-Landing Task in Athletes After Sport-Related Concussion and Healthy Control Individuals.

CONTEXT: A relationship between a history of sport-related concussion (SRC) and lower extremity injury has been well established in the literature. OBJECTIVE: To determine if biomechanical differences existed during a double-limb jump landing between athletes who had been released to return to play after SRC and healthy matched control individuals. DESIGN: Cross-sectional study. SETTING: Health system-based outpatient sports medicine center. PATIENTS OR OTHER PARTICIPANTS: A total of 21 participants with SRC (age = 15.38 ± 1.77 years, height = 169.23 ± 8.59 cm, mass = 63.43 ± 7.39 kg, time since release to return to sport after SRC = 16.33 ± 12.7 days) were compared with 21 age-, sex-, and activity-matched healthy participants serving as controls (age = 15.36 ± 1.73 years, height = 169.92 ± 11.1 cm, mass = 65.62 ± 12.08 kg). MAIN OUTCOME MEASURE(S): Biomechanical performance during the double-limb jump landing was assessed using a motion-capture system and force plates. The average of 3 consecutive trials was used to calculate lower extremity joint kinetics and kinematics. The variables of interest were internal knee-extension moment, internal varus moment, and total sagittal-plane knee displacement for the dominant and nondominant limbs. Independent t tests were performed to examine the differences between SRC and control groups for the variables of interest. RESULTS: No differences existed between groups for the descriptive data. The SRC group demonstrated greater internal knee-extension moments in the dominant (-0.028 ± 0.009 Nm/kg, P = .003) and nondominant (-0.018 ± 0.007, P = .02) limbs. The SRC group also exhibited greater internal varus moments in the dominant (0.012 ± 0.004 Nm/kg, P = .005) and nondominant (0.010 ± 0.003, P = .005) limbs. For sagittal-plane knee displacement, the SRC group displayed less knee-flexion displacement in the dominant (-12.56 ± 4.67°, P = .01) but not the nondominant (-8.30 ± 4.91°, P = .10) limb. CONCLUSIONS: Athletes who had been released for return to sport after SRC landed with greater knee valgus than healthy matched control participants.

Protection of the Queuosine Biosynthesis Enzyme QueF from Irreversible Oxidation by a Conserved Intramolecular Disulfide.

QueF enzymes catalyze the nicotinamide adenine dinucleotide phosphate (NADPH)-dependent reduction of the nitrile group of 7-cyano-7-deazaguanine (preQ0) to 7-aminomethyl-7-deazaguanine (preQ1) in the biosynthetic pathway to the tRNA modified nucleoside queuosine. The QueF-catalyzed reaction includes formation of a covalent thioimide intermediate with a conserved active site cysteine that is prone to oxidation in vivo. Here, we report the crystal structure of a mutant of Bacillus subtilis QueF, which reveals an unanticipated intramolecular disulfide formed between the catalytic Cys55 and a conserved Cys99 located near the active site. This structure is more symmetric than the substrate-bound structure and exhibits major rearrangement of the loops responsible for substrate binding. Mutation of Cys99 to Ala/Ser does not compromise enzyme activity, indicating that the disulfide does not play a catalytic role. Peroxide-induced inactivation of the wild-type enzyme is reversible with thioredoxin, while such inactivation of the Cys99Ala/Ser mutants is irreversible, consistent with protection of Cys55 from irreversible oxidation by disulfide formation with Cys99. Conservation of the cysteine pair, and the reported in vivo interaction of QueF with the thioredoxin-like hydroperoxide reductase AhpC in Escherichia coli suggest that regulation by the thioredoxin disulfide-thiol exchange system may constitute a general mechanism for protection of QueF from oxidative stress in vivo.

Sex Differences in Time to Return-to-Play Progression After Sport-Related Concussion.

BACKGROUND: Recently, female sports participation has increased, and there is a tendency for women to experience more symptoms and variable presentation after sport-related concussion (SRC). The purpose of this study was to determine whether sex differences exist in time to begin a return-to-play (RTP) progression after an initial SRC. HYPOTHESIS: After initial SRC, female athletes (11-20 years old) would take longer to begin an RTP progression compared with age-matched male athletes. STUDY DESIGN: Retrospective cohort study. LEVEL OF EVIDENCE: Level 3. METHODS: A total of 579 participants (365 males [mean age, 15.0 ± 1.7 years], 214 females [mean age, 15.2 ± 1.5 years]), including middle school, high school, and collegiate athletes who participated in various sports and experienced an initial SRC were included and underwent retrospective chart review. The following information was collected: sex, age at injury, sport, history of prior concussion, date of injury, and date of initiation of RTP progression. Participants with a history of more than 1 concussion or injury sustained from non-sport-related activity were excluded. RESULTS: Despite American football having the greatest percentage (49.2%) of sport participation, female athletes took significantly longer to start an RTP progression after an initial SRC (29.1 ± 26.3 days) compared with age-matched male athletes (22.7 ± 18.3 days; P = 0.002). CONCLUSION: On average, female athletes took approximately 6 days longer to begin an RTP progression compared with age-matched male athletes. This suggests that sex differences exist between athletes, aged 11 to 20 years, with regard to initiation of an RTP progression after SRC. CLINICAL RELEVANCE: Female athletes may take longer to recover after an SRC, and therefore, may take longer to return to sport. Sex should be considered as part of the clinical decision-making process when determining plan of care for this population.

The Relationship Between Humeral Retrotorsion and Shoulder Range of Motion in Baseball Players With an Ulnar Collateral Ligament Tear.

BACKGROUND: Humeral retrotorsion has been investigated in relation to shoulder range of motion (ROM) in healthy baseball players. Currently, there is limited information on the osseous anatomy and development of ulnar collateral ligament (UCL) tears. PURPOSE: To determine the relationship between humeral retrotorsion and shoulder ROM in baseball players with a UCL tear. STUDY DESIGN: Cross-sectional study; Level of evidence, 3. METHODS: Fifty-four baseball players (mean age, 18.5 ± 2.0 years) with a UCL tear volunteered for this study. Participants were measured bilaterally for shoulder internal (IR) and external rotation (ER) ROM and humeral retrotorsion. Differences between sides (involved to uninvolved) were used to calculate the glenohumeral internal rotation deficit (GIRD), external rotation ROM difference (ERDiff), total rotational motion difference (TRM), and humeral retrotorsion difference (HTDiff). A multivariate regression analysis was performed with GIRD, ERDiff, and TRM regressing on HTDiff. Univariate analysis was performed to further evaluate the effect of the predictors on each outcome separately. To control for the effect of age, weight, duration of symptoms, and years of experience, the variables were included as covariates. An a priori level was set at P < .05. RESULTS: There was a statistically significant relationship between the GIRD, ERDiff, and TRM results compared with HTDiff (P = .003). Independent analysis revealed a statistically significant relationship between GIRD and HTDiff (P = .004) and between ERDiff and HTDiff (P = .003) but no significant relationship between TRM and HTDiff (P = .999). After adjusting for age, weight, duration of symptoms, years of experience, dominant arm, and position, a significant relationship was found between GIRD and HTDiff (P = .05) and between ERDiff and HTDiff (P = .01). No significant relationship was found between TRM and HTDiff (P = .54). Adjusted univariate regression analysis determined that HTDiff explains approximately 16% of the variance in GIRD (r2 = 0.158) and approximately 24% of the variance in ERDiff (r2 = 0.237). CONCLUSION: In baseball players with a UCL tear, approximately 16% of the variance in GIRD and 24% of the variance in ERDiff can be attributed to differences found in humeral retrotorsion between sides. This indicates that humeral retroversion contributes significantly to GIRD and increased ER ROM in baseball players. Recognition of differences in humeral retrotorsion between the dominant and nondominant upper extremities may help explain some but not all of the changes in shoulder ROM commonly seen in baseball players.

Vancouver Transcatheter Aortic Valve Replacement Clinical Pathway: Minimalist Approach, Standardized Care, and Discharge Criteria to Reduce Length of Stay.

We describe the development, implementation, and evaluation of a standardized clinical pathway to facilitate safe discharge home at the earliest time after transfemoral transcatheter aortic valve replacement. Between May 2012 and October 2014, the Heart Team developed a clinical pathway suited to the unique requirements of transfemoral transcatheter aortic valve replacement in contemporary practice. The components included risk-stratified minimalist periprocedure approach, standardized postprocedure care with early mobilization and reconditioning, and criteria-driven discharge home. Our aim was to reduce variation in care, identify a subgroup of patients suitable for early discharge (≤48 hours), and decrease length of stay for all patients. We addressed barriers related to historical practices, complex multidisciplinary stakeholder engagement, and adoption of length of stay as a quality indicator. We retrospectively reviewed the experiences of 393 consecutive patients; 150 (38.2%) were discharged early. At baseline, early discharge patients had experienced less previous balloon aortic valvuloplasty, had higher left ventricular ejection fraction, better cognitive function, and were less frail than the standard discharge group (>48 hours). Early discharge was associated with the use of local anesthesia, implantation of balloon expandable device, avoidance of urinary catheter, and early removal of temporary pacemaker. Median length of stay was 1 day for early discharge and 3 days for other patients; 97.7% were discharged home. There were no differences in 30-day mortality (1.3%), disabling stroke (0.8%), or readmission (10.7%). The implementation of a transcatheter aortic valve replacement clinical pathway shifted the program's approach to combine standardized processes and individual risk stratification. The Vancouver transcatheter aortic valve replacement clinical pathway requires a rigorous assessment to determine its efficacy, safety, and reproducibility.

Benefits of the Nephros Dual Stage Ultrafilter in Chronic Hemodialysis Patients: Evidence for Improved ESA Responsiveness.

Installation of the Nephros Dual Stage Ultrafilter (DSU) added to a conventional hemodialysis unit to achieve ultrapure dialysate was tested in a group of 23 stable outpatients on chronic hemodialysis. Comparing the 6-month period prior to the installation of the filters (as baseline) to the 6-month period after the installation of the filters, we found a significant 40% reduction in the darbepoetin dose needed to maintain a stable hemoglobin level (p < 0.001). In addition, surrogate inflammatory markers, WBC count and serum albumin level, showed small but statistically significant improvements (p = 0.008 and p = 0.042, respectively). In conclusion, the use of the Nephros DSU to further reduce endotoxin exposure in chronic hemodialysis patients can result in improved erythropoiesis-stimulating agent (ESA) responsiveness and a lower ESA dose.

Hospital performance for pharmacologic venous thromboembolism prophylaxis and rate of venous thromboembolism : a cohort study.

IMPORTANCE: Hospitalization for acute medical illness is associated with increased risk of venous thromboembolism (VTE). Although efforts designed to increase use of pharmacologic VTE prophylaxis are intended to reduce hospital-associated VTE, whether higher rates of prophylaxis reduce VTE in medical patients is unknown. OBJECTIVE: To examine the association between pharmacologic VTE prophylaxis rates and hospital-associated VTE. DESIGN, SETTING, AND PARTICIPANTS: Retrospective, multicenter cohort study conducted at 35 Michigan hospitals participating in a statewide quality collaborative from January 1, 2011, through September 13, 2012. Trained medical record abstractors at each hospital collected data from 31 260 general medical patients. Use of VTE prophylaxis on admission, VTE risk factors, and VTE events 90 days after hospital admission were recorded using a combination of medical record review and telephone follow-up. Hospitals were grouped into tertiles of performance based on rate of pharmacologic prophylaxis use on admission for at-risk patients. MAIN OUTCOMES AND MEASURES: Association between hospital performance and time to development of VTE within 90 days of hospital admission. RESULTS: A total of 14 563 of 20 794 patients (70.0%) eligible for pharmacologic prophylaxis received prophylaxis on admission. The rates of pharmacologic prophylaxis use at hospitals in the high-, moderate-, and low-performance tertiles were 85.8%, 72.6%, and 55.5%, respectively. A total of 226 VTE events occurred during 1 765 449 days of patient follow-up. Compared with patients at hospitals in the highest-performance tertile, the hazard of VTE in patients at hospitals in moderate-performance (hazard ratio, 1.10; 95% CI, 0.74-1.62) and low-performance (hazard ratio, 0.96, 95% CI, 0.63-1.45) tertiles did not differ after adjusting for potential confounders. Results remained robust when examining mechanical prophylaxis, prophylaxis use throughout the hospitalization, and subsequent inpatient stays after discharge from the index hospitalization. CONCLUSIONS AND RELEVANCE: The occurrence of 90-day VTE in medical patients after hospitalization is low. Patients who receive care at hospitals that have lower rates of pharmacologic prophylaxis do not have higher adjusted hazards of VTE, even after accounting for individual receipt of pharmacologic prophylaxis. Efforts to increase rates of pharmacologic VTE prophylaxis in hospitalized medical patients may not substantively reduce this adverse outcome.

A regional anesthesia-based "swing" operating room model reduces non-operative time in a mixed orthopedic inpatient/outpatient population.

PURPOSE: We recently reported on the efficacy of a new "swing" room model involving two alternating ORs and regional anesthesia in increasing operating room (OR) throughput in a dedicated ambulatory orthopedic surgery facility. The purpose of this study was to evaluate this model in a main OR suite setting with typical mixed inpatient/outpatient cases. METHODS: We conducted a retrospective matched-pair cohort study of 133 upper extremity surgery patients treated in the swing room model under ultrasound-guided brachial plexus blockade. We compared this cohort with case-matched historical controls treated in the traditional single OR model under general anesthesia. The primary endpoint was non-operative time, defined as the interval between skin closure and incision in the following case. Secondary endpoints included throughput estimated as the median number of cases per eight-hour day, postanesthesia care unit (PACU) bypass rates, and postoperative pain/nausea and vomiting (PONV) intervention rates. RESULTS: Compared with the control group, non-operative times in the swing room group were faster (swing: median 19 min; interquartile range [IQR 8-31] vs control: median 57 min; IQR [49-65]; P < 0.0001). In the swing room model, the estimated daily throughput was 33% greater (swing: median 5.6 cases; IQR [5.0-6.2] vs control: median 4.2 cases; IQR [4.0-4.4]; P < 0.0001), and the PACU bypass rate was higher (swing: 60% vs control: 0%; P < 0.0001). Fewer patients received postoperative opioids (swing: 20% vs control: 82%; P < 0.0001) and treatment for PONV (swing: 2% vs control: 20%; P < 0.0001) in the swing room model. CONCLUSION: The implementation of a "swing" room care model based on ultrasound-guided regional anesthesia in a typical mixed inpatient/outpatient population decreased non-operative times, increased throughput, and improved recovery profiles compared with case-matched historical controls in the traditional model under general anesthesia.

Structural basis of biological nitrile reduction.

The enzyme QueF catalyzes the reduction of the nitrile group of 7-cyano-7-deazaguanine (preQ(0)) to 7-aminomethyl-7-deazaguanine (preQ(1)), the only nitrile reduction reaction known in biology. We describe here two crystal structures of Bacillus subtilis QueF, one of the wild-type enzyme in complex with the substrate preQ(0), trapped as a covalent thioimide, a putative intermediate in the reaction, and the second of the C55A mutant in complex with the substrate preQ(0) bound noncovalently. The QueF enzyme forms an asymmetric tunnel-fold homodecamer of two head-to-head facing pentameric subunits, harboring 10 active sites at the intersubunit interfaces. In both structures, a preQ(0) molecule is bound at eight sites, and in the wild-type enzyme, it forms a thioimide covalent linkage to the catalytic residue Cys-55. Both structural and transient kinetic data show that preQ(0) binding, not thioimide formation, induces a large conformational change in and closure of the active site. Based on these data, we propose a mechanism for the activation of the Cys-55 nucleophile and subsequent hydride transfer.

The use of hospice care in end-stage psychiatric patients.

Hospice care is rarely used in end-stage psychiatric patients, yet situations exist where psychiatric intervention is futile and comfort care is the best option. Delusional disorder is rare, typically begins later in life, and has a chronic course that responds poorly to treatment. The prognosis is affected by factors such as chronicity and insight. A case of a chronic and intractable delusional disorder that affected eating behavior and subsequently caused serious medical complications. Due to the severity of the case and the unique ethical issues it presented, the prognosis was determined to be poor and the patient was discharged home with hospice care. The case presented a rare opportunity to assess hospice care provided to an end-stage psychiatric patient.

Zoospore interspecific signaling promotes plant infection by Phytophthora.

BACKGROUND: Oomycetes attack a huge variety of economically and ecologically important plants. These pathogens release, detect and respond to signal molecules to coordinate their communal behaviors including the infection process. When signal molecules are present at or above threshold level, single zoospores can infect plants. However, at the beginning of a growing season population densities of individual species are likely below those required to reach a quorum and produce threshold levels of signal molecules to trigger infection. It is unclear whether these molecules are shared among related species and what their chemistries are. RESULTS: Zoospore-free fluids (ZFF) from Phytophthora capsici, P. hydropathica, P. nicotianae (ZFFnic), P. sojae (ZFFsoj) and Pythium aphanidermatum were cross tested for stimulating plant infection in three pathosystems. All ZFFs tested significantly increased infection of Catharanthus roseus by P. nicotianae. Similar cross activities were observed in infection of Lupinus polyphyllus and Glycine max by P. sojae. Only ZFFnic and ZFFsoj cross induced zoospore aggregation at a density of 2 × 10³ ml⁻¹. Pure autoinducer-2 (AI-2), a component in ZFF, caused zoospore lysis of P. nicotianae before encystment and did not stimulate plant infection at concentrations from 0.01 to 1000 µM. P. capsici transformants with a transiently silenced AI-2 synthase gene, ribose phosphate isomerase (RPI), infected Capsicum annuum seedlings at the same inoculum concentration as the wild type. Acyl-homoserine lactones (AHLs) were not detected in any ZFFs. After freeze-thaw treatments, ZFF remained active in promoting plant infection but not zoospore aggregation. Heat treatment by boiling for 5 min also did not affect the infection-stimulating property of ZFFnic. CONCLUSION: Oomycetes produce and use different molecules to regulate zoospore aggregation and plant infection. We found that some of these signal molecules could act in an inter-specific manner, though signals for zoospore aggregation were somewhat restricted. This self-interested cooperation among related species gives individual pathogens of the same group a competitive advantage over pathogens and microbes from other groups for limited resources. These findings help to understand why these pathogens often are individually undetectable until severe disease epidemics have developed. The signal molecules for both zoospore aggregation and plant infection are distinct from AI-2 and AHL.

Enzyme-catalyzed transfer of a ketone group from an S-adenosylmethionine analogue: a tool for the functional analysis of methyltransferases.

S-adenosylmethionine (AdoMet or SAM)-dependent methyltransferases belong to a large and diverse family of group-transfer enzymes that perform vital biological functions on a host of substrates. Despite the progress in genomics, structural proteomics, and computational biology, functional annotation of methyltransferases remains a challenge. Herein, we report the synthesis and activity of a new AdoMet analogue functionalized with a ketone group. Using catechol O-methyltransferase (COMT, EC 2.1.1.6) and thiopurine S-methyltransferase (TPMT, EC 2.1.1.67) as model enzymes, this robust and readily accessible analogue displays kinetic parameters that are comparable to AdoMet and exhibits multiple turnovers with enzyme. More importantly, this AdoMet surrogate displays the same substrate specificity as the natural methyl donor. Incorporation of the ketone group allows for subsequent modification via bio-orthogonal labeling strategies and sensitive detection of the tagged ketone products. Hence, this AdoMet analogue expands the toolbox available to interrogate the biochemical functions of methyltransferases.

Zoosporic plant pathogens produce bacterial autoinducer-2 that affects Vibrio harveyi quorum sensing.

The frequent coisolation of bacteria with Phytophthora and Pythium species suggests possible interspecies communication. Zoospore-free fluids (ZFF) from bacteria-free and nutrient-depleted zoospore suspensions were examined to investigate the production of autoinducer-2 (AI-2), a bacterial interspecies signal molecule, by zoosporic oomycetes. ZFF from Phytophthora nicotianae, Phytophthora sojae, and Pythium aphanidermatum triggered luminescence of the Vibrio harve7yi AI-2 reporter, indicating the presence of AI-2 in zoospore extracellular products and the potential of cross-kingdom communication between oomycetes and bacteria. The production of AI-2 by zoospores was confirmed by chemical assays. These results provide a new insight into the physiology and ecology of oomycetes.

Chemical methods for the detection of protein N-homocysteinylation via selective reactions with aldehydes.

Elevated blood levels of homocysteine (Hcy), hyperhomocysteinemia or homocystinuria, have been associated with various diseases and conditions. Homocysteine thiolactone (Hcy TL) is a metabolite of Hcy and reacts with amine groups in proteins to form stable amides, homocystamides, or N-homocysteinylated proteins. It has been proposed that protein N-homocysteinylation contributes to the cytotoxicity of elevated Hcy. Due to its heterogeneity and relatively low abundance, detection of this posttranslational modification remains challenging. On the other hand, the gamma-aminothiol group in homocystamides imparts different chemical reactivities than the native proteins. Under mildly acidic conditions, gamma-aminothiols irreversibly and stoichiometrically react with aldehydes to form stable 1,3-thiazines, whereas the reversible Schiff base formation between aldehydes and amino groups in native proteins is markedly disfavored due to protonation of amines. As such, we have developed highly selective chemical methods to derivatize N-homocysteinylated proteins with various aldehyde tags, thereby facilitating the subsequent analyses. For instance, fluorescent or biotin tagging coupled with gel electrophoresis permits quantification and global profiling of complex biological samples, such as hemoglobin and plasma from rat, mouse and human; affinity enrichment with aldehyde resins drastically reduces sample complexity. In addition, different reactivities of lysine residues in hemoglobin toward Hcy TL were observed.

A naturally occurring brominated furanone covalently modifies and inactivates LuxS.

Halogenated furanones, a group of natural products initially isolated from marine red algae, are known to inhibit bacterial biofilm formation, swarming, and quorum sensing. However, their molecular targets and the precise mode of action remain elusive. Herein, we show that a naturally occurring brominated furanone covalently modifies and inactivates LuxS (S-ribosylhomocysteine lyase, EC 4.4.1.21), the enzyme which produces autoinducer-2 (AI-2).

Mechanistic studies of Bacillus subtilis QueF, the nitrile oxidoreductase involved in queuosine biosynthesis.

The enzyme QueF was recently identified as an enzyme involved in the biosynthesis of queuosine, a 7-deazaguanosine modified nucleoside found in bacterial and eukaryotic tRNA. QueF exhibits sequence homology to the type I GTP cyclohydrolases characterized by FolE, but contrary to the predictions based on sequence analysis the enzyme in fact catalyzes a mechanistically unrelated reaction, the NADPH-dependent reduction of 7-cyano-7-deazaguanine (preQ0) to 7-aminomethyl-7-deazaguanine (preQ1), a late step in the queuosine pathway. The reduction of a nitrile is unprecedented in biology, and we report here characterization and mechanistic studies of the enzyme from Bacillus subtilis. The recombinant enzyme exhibits optimal activity at pH 7.5 and moderate ionic strength, and is not dependent on metal ions for catalytic activity. Steady-state kinetic analysis provided a kcat = 0.66 +/- 0.04 min-1, KM (preQ0) = 0.237 +/- 0.045 microM, and KM (NADPH) = 19.2 +/- 1.1 microM. Based on sequence analysis and homology modeling we predicted previously that Cys55 would be present in the active site and in proximity to the nitrile group of preQ0. Consistent with that prediction we observed that the enzyme was inactivated when preincubated with iodoacetamide, and protected from inactivation when preQ0 was present. Furthermore, titrations of the enzyme with preQ0 in the absence of NADPH were accompanied by the appearance of a new absorption band at 376 nm in the UV-vis spectrum consistent with the formation of an alpha,beta-unsaturated thioimide. Site-directed mutagenesis of Cys55 to Ala or Ser resulted in loss of catalytic activity and no absorption at 376 nm upon addition of preQ0. Based on our data we propose a chemical mechanism for the enzyme-catalyzed reaction, and a chemical rationale for the observation of covalent catalysis.

A riboswitch selective for the queuosine precursor preQ1 contains an unusually small aptamer domain.

A previous bioinformatics-based search for riboswitches yielded several candidate motifs in eubacteria. One of these motifs commonly resides in the 5' untranslated regions of genes involved in the biosynthesis of queuosine (Q), a hypermodified nucleoside occupying the anticodon wobble position of certain transfer RNAs. Here we show that this structured RNA is part of a riboswitch selective for 7-aminomethyl-7-deazaguanine (preQ(1)), an intermediate in queuosine biosynthesis. Compared with other natural metabolite-binding RNAs, the preQ(1) aptamer appears to have a simple structure, consisting of a single stem-loop and a short tail sequence that together are formed from as few as 34 nucleotides. Despite its small size, this aptamer is highly selective for its cognate ligand in vitro and has an affinity for preQ(1) in the low nanomolar range. Relatively compact RNA structures can therefore serve effectively as metabolite receptors to regulate gene expression.

From cyclohydrolase to oxidoreductase: discovery of nitrile reductase activity in a common fold.

The enzyme YkvM from Bacillus subtilis was identified previously along with three other enzymes (YkvJKL) in a bioinformatics search for enzymes involved in the biosynthesis of queuosine, a 7-deazaguanine modified nucleoside found in tRNA(GUN) of Bacteria and Eukarya. Genetic analysis of ykvJKLM mutants in Acinetobacter confirmed that each was essential for queuosine biosynthesis, and the genes were renamed queCDEF. QueF exhibits significant homology to the type I GTP cyclohydrolases characterized by FolE. Given that GTP is the precursor to queuosine and that a cyclohydrolase-like reaction was postulated as the initial step in queuosine biosynthesis, QueF was proposed to be the putative cyclohydrolase-like enzyme responsible for this reaction. We have cloned the queF genes from B. subtilis and Escherichia coli and characterized the recombinant enzymes. Contrary to the predictions based on sequence analysis, we discovered that the enzymes, in fact, catalyze a mechanistically unrelated reaction, the NADPH-dependent reduction of 7-cyano-7-deazaguanineto7-aminomethyl-7-deazaguanine, a late step in the biosynthesis of queuosine. We report here in vitro and in vivo studies that demonstrate this catalytic activity, as well as preliminary biochemical and bioinformatics analysis that provide insight into the structure of this family of enzymes.

Crystallization and preliminary X-ray characterization of the nitrile reductase QueF: a queuosine-biosynthesis enzyme.

QueF (MW = 19.4 kDa) is a recently characterized nitrile oxidoreductase which catalyzes the NADPH-dependent reduction of 7-cyano-7-deazaguanine (preQ0) to 7-aminomethyl-7-deazaguanine, a late step in the biosynthesis of the modified tRNA nucleoside queuosine. Initial crystals of homododecameric Bacillus subtilis QueF diffracted poorly to 8.0 A. A three-dimensional model based on homology with the tunnel-fold enzyme GTP cyclohydrolase I suggested catalysis at intersubunit interfaces and a potential role for substrate binding in quaternary structure stabilization. Guided by this insight, a second crystal form was grown that was strictly dependent on the presence of preQ0. This crystal form diffracted to 2.25 A resolution.