Altered Metabolism of Polycyclic Aromatic Hydrocarbons by UDP-Glycosyltransferase 3A2 Missense Variants.

The UDP-glycosyltransferase (UGT) family of enzymes are important in the metabolism of a variety of exogenous substances including polycyclic aromatic hydrocarbons (PAHs), a potent class of environmental carcinogens. As compared to the majority of UGT enzymes, which utilize UDP-glucuronic acid as a cosubstrate, UGT3A2 utilizes alternative cosubstrates (UDP-glucose and UDP-xylose). UGT3A2 is expressed in aerodigestive tract tissues and was highly active against multiple PAHs with both cosubstrates. The goal of the present study was to assess the functional effects of UGT3A2 missense variants (MAF ≥ 0.005) on PAH metabolism and the utilization of cosubstrates. The glycosylation activity (Vmax/Km) of all variants against simple PAHs using both cosubstrates was significantly (P < 0.05) decreased by 42-100% when compared to wild-type UGT3A2. When utilizing UDP-glucose, the variant isoforms exhibited up to a 362-fold decrease in Vmax/Km when compared to wild-type UGT3A2, with a 3.1- to 14-fold decrease for D140N, A344T, and S435Y, a 24- and 43-fold decrease for A436T and R445C, respectively, and a 147- and 362-fold decrease for Y474C and Y74N, respectively. When utilizing UDP-xylose, the variants exhibited up to a 4.0-fold decrease in Vmax/Km when compared to wild-type UGT3A2; Y74N did not exhibit activity, and Y474C did not reach saturation (Km > 4000 µM). Additionally, both wild-type and variant UGT3A2 exhibited a significant (P < 0.05) difference in their utilization of UDP-glucose vs UDP-xylose as cosubstrates using 1-OH-pyrene as substrate. These data suggest that UGT3A2 missense variants decrease the detoxification of PAHs, potentially resulting in altered individual risk for PAH-related cancers.

Structural and Functional Characterization of Dynamic Oligomerization in Burkholderia cenocepacia HMG-CoA Reductase.

The enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (HMGR), in most organisms, catalyzes the four-electron reduction of the thioester (S)-HMG-CoA to the primary alcohol (R)-mevalonate, utilizing NADPH as the hydride donor. In some organisms, including the opportunistic lung pathogen Burkholderia cenocepacia, it catalyzes the reverse reaction, utilizing NAD+ as a hydride acceptor in the oxidation of mevalonate. B. cenocepacia HMGR has been previously shown to exist as an ensemble of multiple non-additive oligomeric states, each with different levels of enzymatic activity, suggesting that the enzyme exhibits characteristics of the morpheein model of allostery. We have characterized a number of factors, including pH, substrate concentration, and enzyme concentration, that modulate the structural transitions that influence the interconversion among the multiple oligomers. We have also determined the crystal structure of B. cenocepacia HMGR in the hexameric state bound to coenzyme A and ADP. This hexameric assembly provides important clues about how the transition among oligomers might occur, and why B. cenocepacia HMGR, unique among characterized HMGRs, exhibits morpheein-like behavior.

Intraoperative brain tumor resection with indocyanine green using augmented microscopy.

Treatment outcomes for brain cancer have seen dismal improvements over the last two decades as evident in available statistical data. Efforts to address this challenge include development of near-infrared contrast agents for improvements in diagnostic and therapeutic modalities. This creates a need for imaging technologies that can support the intraoperative use of such agents. Here, we report implementation of a recently introduced augmented microscope in combination with indocyanine green (ICG), a near-infrared contrast agent, for surgical image guidance of a glioma resection in a rat model. Luc-C6 cells were implanted in rats in the left-frontal lobe and grown for 22 days. Surgical resection was performed by a neurosurgeon using the augmented microscope with ICG contrast. ICG accumulated in the tumor tissue due to enhanced permeation and retention from the compromised blood-brain barrier. Videos and images were acquired to evaluate image quality and resection margins. The augmented microscope highlighted tumor tissue regions via visualization of ICG fluorescence and was capable of guiding the rat glioma resection.

Biomechanical Comparison of Cadaveric and Commercially Available Synthetic Osteoporotic Bone Analogues in a Locked Plate Fracture Model Under Torsional Loading.

OBJECTIVES: Biomechanical studies of osteoporotic bone have used synthetic models rather than cadaveric samples because of decreased variability, increased availability, and overall ease of the use of synthetic models. We compared the torsional mechanical properties of cadaveric osteoporotic bone with those of currently available synthetic osteoporotic bone analogues. METHODS: We tested 12 osteoporotic cadaveric humeri and 6 specimens each of 6 types of synthetic analogues. A 5-mm fracture gap model and posterior plating technique with 4.5-mm narrow 10-hole locking compression plate were used. Torque was applied to a peak of ±10 N·m for 1000 cycles at 0.3 Hz. Data were continuously collected during cyclical and ramped loading with a servohydraulic materials testing system. RESULTS: Cadaveric bone had a 17% failure rate before completing 1000 cycles. Three osteoporotic bone models had 100% failure (P < 0.05), 2 had 17% failure, and 1 had 0% failure before 1000 cycles. Significant differences in the stiffness of the 3 types of synthetic bone models that survived cyclic loading were noted compared with the cadaveric bone model (P < 0.05). Osteoporotic bone analogues had torsional mechanical properties different from those of osteoporotic cadaveric specimens. CONCLUSIONS: The differences between osteoporotic cadaveric humeri and synthetic osteoporotic bone analogues ranged from profound with complete catastrophic failure after a few cycles to subtler differences in stiffness and strain hardening. These findings suggest that different bone analogue models vary substantially in their torsional mechanical properties and might not be appropriate substitutes for cadaveric bone in biomechanical studies of osteoporotic bone.

Columellar Aesthetics in Open Rhinoplasty.

Detailed descriptions of the ideals of columellar aesthetics and nostril shape are conspicuously lacking from the medical literature. Achieving an aesthetic nasal base is critical to an optimal rhinoplasty result. Deviations in the columella and variations in its width and height lead to distortion of nostril shape and frequently compromise function. Six types of columellar disproportion are presented with detailed explanations of how to treat each. By properly recognizing and addressing columellar deformities, the surgeon can optimize both the aesthetic and functional results that are achieved.

Current Concepts in Filler Injection.

When evaluating the face in thirds, the upper face, midface, and lower face, one may assume the lateral the temple, midface, and lateral mandible as the pillars of these subdivisions. Many of our facial aesthetic procedures address these regions, including the lateral brow lift, midface lift, and lateral face lift. As the use of facial fillers has advanced, more emphasis is placed on the correction of the temples, midlateral face, and lateral jaw line. This article is dedicated to these facial aesthetic pillars.

Augmented microscopy: real-time overlay of bright-field and near-infrared fluorescence images.

Intraoperative applications of near-infrared (NIR) fluorescent contrast agents can be aided by instrumentation capable of merging the view of surgical field with that of NIR fluorescence. We demonstrate augmented microscopy, an intraoperative imaging technique in which bright-field (real) and electronically processed NIR fluorescence (synthetic) images are merged within the optical path of a stereomicroscope. Under luminance of 100,000 lx, representing typical illumination of the surgical field, the augmented microscope detects 189 nM concentration of indocyanine green and produces a composite of the real and synthetic images within the eyepiece of the microscope at 20 fps. Augmentation described here can be implemented as an add-on module to visualize NIR contrast agents, laser beams, or various types of electronic data within the surgical microscopes commonly used in neurosurgical, cerebrovascular, otolaryngological, and ophthalmic procedures.

Integration of indocyanine green videoangiography with operative microscope: augmented reality for interactive assessment of vascular structures and blood flow.

BACKGROUND: Preservation of adequate blood flow and exclusion of flow from lesions are key concepts of vascular neurosurgery. Indocyanine green (ICG) fluorescence videoangiography is now widely used for the intraoperative assessment of vessel patency. OBJECTIVE: Here, we present a proof-of-concept investigation of fluorescence angiography with augmented microscopy enhancement: real-time overlay of fluorescence videoangiography within the white light field of view of conventional operative microscopy. METHODS: The femoral artery was exposed in 7 anesthetized rats. The dissection microscope was augmented to integrate real-time electronically processed near-infrared filtered images with conventional white light images seen through the standard oculars. This was accomplished by using an integrated organic light-emitting diode display to yield superimposition of white light and processed near-infrared images. ICG solution was injected into the jugular vein, and fluorescent femoral artery flow was observed. RESULTS: Fluorescence angiography with augmented microscopy enhancement was able to detect ICG fluorescence in a small artery of interest. Fluorescence appeared as a bright-green signal in the ocular overlaid with the anatomic image and limited to the anatomic borders of the femoral artery and its branches. Surrounding anatomic structures were clearly visualized. Observation of ICG within the vessel lumens permitted visualization of the blood flow. Recorded video loops could be reviewed in an offline mode for more detailed assessment of the vasculature. CONCLUSION: The overlay of fluorescence videoangiography within the field of view of the white light operative microscope allows real-time assessment of the blood flow within vessels during simultaneous surgical manipulation. This technique could improve intraoperative decision making during complex neurovascular procedures.

Surface histidine mutations for the metal affinity purification of a ß-carbonic anhydrase.

Metal affinity chromatography using polyhistidine tags is a standard laboratory technique for the general purification of proteins from cellular systems, but there have been no attempts to explore whether the surface character of a protein may be engineered to similar affinity. We present the Arg160His mutation of Haemophilus influenzae carbonic anhydrase (HICA), which mimics the endogenous metal affinity of Escherichia coli carbonic anhydrase (ECCA). The purity and activity of the mutant are reported, and the purification is discussed. This is the first step toward developing a general method to engineer surface metal affinity for use in purification and metal labeling techniques.

Kinetic characterization of an oxidative, cooperative HMG-CoA reductase from Burkholderia cenocepacia.

3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) is a key enzyme in endogenous cholesterol biosynthesis in mammals and isoprenoid biosynthesis via the mevalonate pathway in other eukaryotes, archaea and some eubacteria. In most organisms that express this enzyme, it catalyzes the NAD(P)H-dependent reduction of HMG-CoA to mevalonate. We have cloned and characterized the 6x-His-tagged HMGR from the opportunistic lung pathogen Burkholderia cenocepacia. Kinetic characterization shows that the enzyme prefers NAD(H) over NADP(H) as a cofactor, suggesting an oxidative physiological role for the enzyme. This hypothesis is supported by the fact that the Burkholderia cenocepacia genome lacks the genes for the downstream enzymes of the mevalonate pathway. The enzyme exhibits positive cooperativity toward the substrates of the reductive reaction, but the oxidative reaction exhibits unusual double-saturation kinetics, distinctive among characterized HMG-CoA reductases. The unusual kinetics may arise from the presence of multiple active oligomeric states, each with different Vmax values.

How much vertical displacement of the symphysis indicates instability after pelvic injury?

BACKGROUND: Measures of pubic symphyseal widening are used by at least two classification systems as determinants of injury grade. Recent work has challenged the commonly used parameter of 2.5 cm of pubic symphysis as an accurate marker of pelvic injury grade and has suggested a role of rotation in the flexion-extension plane as a determinant of pelvic stability. We investigated pelvic stability in the flexion-extension plane to determine a threshold of rotational displacement of the hemipelvis above which the potential for instability exists. METHODS: Cadaveric specimens were mounted onto a servohydraulic biaxial testing machine and subjected to a vertically directed flexion moment. Position of hemipelvis was recorded using a three-dimensional motion capture system and video recording. Displacement of the pubic symphysis and changes in length and position of the sacrospinous and sacrotuberous ligaments were recorded. Amount of force applied was measured and recorded. A yield point was determined as the first point at which the force plot exhibited a decrease in force and was correlated to the corresponding displacement. RESULTS: The mean vertical displacement of the pubic symphysis at the yield point was 16 mm (95% confidence interval, 11-22 mm). Mean sacrospinous ligament strain at yield point was 4% (range, 1.0-9.5%). CONCLUSION: Pelves with vertical rotational symphyseal displacement of less than 11 mm can reasonably be expected to have rotational stability in the flexion-extension plane. Those with displacement of greater than 22 mm can be expected to have lost some integrity regarding resistance to pelvic flexion. These values may allow clinicians to infer pelvic stability from amount of vertical symphyseal displacement.

Investigation of multiligamentous knee injury patterns with associated injuries presenting at a level I trauma center.

OBJECTIVES: To characterize multiligamentous knee injury patterns and describe associated morbidities. DESIGN: Retrospective. SETTING: Level I trauma center. PATIENTS: One hundred two patients (106 knees) with multiligamentous knee injuries and/or dislocations from 2000 through 2008. Subgroup of 82 knees with appropriate magnetic resonance images available assessed for ligamentous injury patterns. INTERVENTION: Data obtained from medical records for 106 knees. MAIN OUTCOME MEASUREMENTS: Presence of arterial injuries, nerve injuries, associated fracture patterns, and whole-body morbidities. RESULTS: The most common (43%) injury pattern was a combined disruption of the anterior cruciate ligament, posterior cruciate ligament, and posterolateral corner. Twenty-five percent of knees had associated ipsilateral tibial plateau fractures, and 19% had associated ipsilateral femoral fractures. Peroneal nerve injury occurred in 25% of knees, arterial injury in 21%, and compartment syndrome in 16%. An intra-abdominal injury was present in 13% of patients, a severe closed head injury in 10%, and a symptomatic pulmonary embolism in 5%. CONCLUSIONS: Nearly half the multiligamentous knee injuries involved the anterior cruciate ligament, posterior cruciate ligament, and posterolateral corner; one-fourth had associated ipsilateral tibial plateau fractures. The incidence of peroneal nerve injury (25%) was higher than previously reported (20%), whereas the incidence of arterial injury (21%) was comparable to previous reports (19%). Posterolateral corner injuries were more prevalent than previously reported and were highly associated with peroneal nerve injury. We found a substantial incidence of associated morbidities of the whole body. Multiligamentous knee injuries are a marker of concomitant trauma and should be closely evaluated at presentation and during the hospital course to allow for early intervention for life- or limb-threatening comorbidities.

Predictors of normal electrodiagnostic testing in the evaluation of suspected carpal tunnel syndrome.

Electrodiagnostic studies (electromyography and nerve conduction velocity; EMG/NCV) are used to confirm the diagnosis of carpal tunnel syndrome (CTS) and to evaluate its severity. We investigated the hypothesis that normal electrodiagnostic median nerve testing is predicted by 1) Physician pre-test confidence in the diagnosis of CTS, and 2) Puzzling patient factors. One hundred and forty consecutive patients who underwent electrodiagnostic testing to evaluate for possible CTS were reviewed retrospectively. Both physician confidence in the diagnosis of CTS and puzzling patient factors (heightened illness concern, disproportionate complaints, and vague/nonanatomical/noncharacteristic symptoms) were recorded. Electrodiagnostic testing was used as the reference standard for diagnosis of CTS. Electrodiagnostic testing confirmed CTS in 115 patients and was within normal limits in 25 patients. Low physician confidence in the diagnosis of CTS was highly predictive of a normal electrodiagnostic test (p < 0.001), with high sensitivity (97%), moderate specificity (40%), and high overall accuracy (87%). Puzzling patient factors were moderately predictive of normal electrodiagnostic testing (p < 0.001), with low sensitivity (16%), high specificity (96%), and high overall accuracy (81%). The best multivariable model retained younger age, negative Phalen's test, and low physician confidence as the best predictors of normal electrodiagnostic testing and explained 35% of the variation in test results. A model with low confidence alone explained 19% of the variation in test results. Physician intuition as recorded in the medical record in terms of puzzlement and low confidence are very specific and accurate predictors of normal electrodiagnostic testing in the setting of suspected carpal tunnel syndrome.

Correlation of DASH and QuickDASH with measures of psychological distress.

PURPOSE: In an attempt to shorten the questionnaires given to patients in both clinical and research settings, we studied whether the correlation of commonly used psychological measures was comparable for the standard Disabilities of the Arm, Shoulder, and Hand (DASH) and the shorter QuickDASH questionnaires. METHODS: A cohort of 839 patients with carpal tunnel syndrome, trigger finger, de Quervain's disease, trapeziometacarpal arthrosis, lateral epicondylosis, or a distal radius fracture 2 weeks after surgery, who completed the DASH and 1 or more measures of psychological distress, was created from 10 databases from previously implemented studies. Correlations of the DASH and the QuickDASH with several measures of psychological factors (Center for Epidemiologic Studies Depression Scale [CES-D], Pain Catastrophizing Scale [PCS], and Pain Anxiety Symptoms Scale [PASS-40]) were calculated in both univariate and multivariable analyses. RESULTS: There was a large correlation between the DASH and QuickDASH (r = 0.79; p < .001). QuickDASH scores were significantly higher than DASH scores (p < .001). Correlations of the CES-D, PCS, and PASS-40 with the DASH and QuickDASH ranged from small to medium (range, 0.21-0.31; p < .001). There were no significant differences between correlations of the DASH and the QuickDASH with the psychological factors in the cohort including all patients, nor in subgroups according to diagnosis, gender, and limb dominance. CONCLUSIONS: The correlations of the DASH and QuickDASH with the CES-D, PCS, and PASS-40 were comparable. Our analysis suggests that a shorter and therefore potentially more practical measure of arm-specific disability can be used in studies that evaluate psychosocial aspects of illness behavior. TYPE OF STUDY/LEVEL OF EVIDENCE: Prognostic III.

A survey regarding physician recommendations regarding return to work.

PURPOSE: Returning patients to work may be influenced by subjective factors and physician bias. The purpose of this study was to determine whether factors such as complaints of pain and patient motivation influence physicians' recommendations regarding return to work or activity. METHODS: One hundred twenty-five members of the American Society for Surgery of the Hand completed an online survey describing a 25-year-old patient with surgically treated diaphyseal fractures of the radius and ulna. Physicians were asked whether the patient could be returned to work in 4 distinct scenarios varying with occupation, time since injury, radiographic union, patient motivation, and pain. RESULTS: Logistic regression analysis demonstrated that all 5 predictor variables were highly significant predictors of return to work. Pain and diminished motivation were associated with a significantly lower probability of return to work. CONCLUSIONS: Although in the scenario depicted, objective factors such as radiographic union and job demands are the major determinants of physician clearance to return to work, physicians are also influenced by patient motivation and complaints of pain.

Influence of psychological factors on grip strength.

PURPOSE: Grip strength is widely used to assess upper-extremity function. Although grip strength is a quantitative measure of function, grip strength is a reflection of both physical impairment as well as subjective, psychological factors. We investigated the determinants of grip strength with the hypothesis that psychological factors are associated with diminished grip strength. METHODS: One hundred thirty-four patients with an isolated, discrete upper-extremity condition had grip strength measurements, and, as part of one of several prospective clinical studies, during the same visit they completed 2 or more of the following surveys: the Center for Epidemiologic Studies-Depression (CES-D) scale, Pain Anxiety Symptoms Scale, and Pain Catastrophizing Scale. Univariate and multivariable statistical analysis sought determinants of absolute grip strength and grip strength as a percentage of the opposite, uninvolved limb. RESULTS: Determinants of absolute grip strength included gender and grip strength of the uninvolved limb. The association between CES-D score and absolute grip strength was near significant but very weak. Multivariable regression analysis produced a best-fit model that retained grip strength of the uninvolved limb and CES-D scores. When grip strength of the involved limb was evaluated as a percentage of grip strength of the uninvolved limb, CES-D score was a weak but significant predictor, and patients recovering from a fracture of the distal radius had weaker grip strength than did patients with nontraumatic conditions. CONCLUSIONS: Depression scores were minimally associated with diminished grip strength. Psychological factors appear to affect disability (patient-reported health status) more than they affect performance-based measures of function. TYPE OF STUDY/LEVEL OF EVIDENCE: Prognostic II.

Mass tagging approach for mitochondrial thiol proteins.

A mass tagging approach is described for mitochondrial thiol proteins under nondenaturing conditions. This approach utilizes stable isotope-coded, thiol-reactive (4-iodobutyl)triphenylphosphonium (IBTP) reagents, i.e., the isotopomers IBTP-d(0) and IBTP-d(15). The mass spectrometric properties of IBTP-labeled peptides were evaluated using an ESI-q-TOF and a MALDI-TOF/TOF instrument. High energy collision induced dissociation (CID) in the TOF/TOF instrument caused side-chain fragmentation in the butyltriphenylphosphonium moiety-containing Cys-residue. By contrast, low energy CID in the qTOF instrument yielded sequence tags of IBTP-labeled peptides that were suitable for automated database searching. The IBTP labeling strategy was then applied to the analysis of a protein extract obtained from cardiac mitochondria. The relative abundance measurements for identified IBTP-labeled peptides showed an average variability for peptide quantitation of approximately 10% based on peak area ratios of ion signals for the d(0)/d(15)-tagged peptide pairs. The reactivity of the IBTP reagents was further studied by molecular modeling and visualization. The present study suggests that the IBTP reagent seems to show a bias toward highly surface-exposed protein thiols. Hence, the described mass tagging approach might become potentially useful in redox proteomics studies designed to identify protein thiols that are particularly prone to oxidative modifications.

African-American grandmothers as health educators in the family.

More than 18,000 adolescents die each year in the United States from bicycle, motorcycle, car, and truck accidents. This study sought to understand the role of African-American grandmothers as prevention-oriented health educators in the family. Full Model Fitted Regression Analyses were conducted on a sample of African-American grandmothers (N = 105) with 10- to 19-year-old grandchildren. Findings suggest that grandmothers who adopt a proactive, teaching role with their grandchildren are more committed to doing so, confident about doing so, and in a context to do so. In particular, grandmothers who co-reside with their grandchildren are more likely to teach them about how to prevent transportation accidents than those who do not co-reside. These findings could contribute to innovations in existing grandparent education curricula.

How sequence defines structure: a crystallographic map of DNA structure and conformation.

The fundamental question of how sequence defines conformation is explicitly answered if the structures of all possible sequences of a macromolecule are determined. We present here a crystallographic screen of all permutations of the inverted repeat DNA sequence d(CCnnnN6N7N8GG), where N6, N7, and N8 are any of the four naturally occurring nucleotides. At this point, 63 of the 64 possible permutations have been crystallized from a defined set of solutions. When combined with previous work, we have assembled a data set of 37 single-crystal structures from 29 of the sequences in this motif, representing three structural classes of DNA (B-DNA, A-DNA, and four-stranded Holliday junctions). This data set includes a unique set of amphimorphic sequence, those that crystallize in two different conformations and serve to bridge the three structural phases. We have thus constructed a map of DNA structures that can be walked through in single nucleotide steps. Finally, the resulting data set allows us to dissect in detail the stabilization of and conformational variations within structural classes and identify significant conformational deviations within a particular structural class that result from sequence rather than crystal or crystallization effects.

The vaccinia virus G1L putative metalloproteinase is essential for viral replication in vivo.

The function of the putative metalloproteinase encoded by the vaccinia virus G1L gene is unknown. To address this question, we have generated a vaccinia virus strain in which expression of the G1L gene is dependent on the addition of tetracycline (TET) when infection proceeds in a cell line expressing the tetracycline repressor. The vvtetOG1L virus replicated similarly to wild-type Western Reserve (WR) virus in these cells when TET was present but was arrested at a late stage in viral maturation in the absence of TET. This arrest resulted in the accumulation of 98.5% round immature virus particles compared to 6.9% at a similar time point when TET was present. Likewise, the titer of infectious virus progeny decreased by 98.9% +/- 0.97% when the vvtetOG1L virus was propagated in the absence of TET. Mutant virus replication was partially rescued by plasmid-encoded G1L, but not by G1L containing an HXXEH motif mutated to RXXQR. Modeling of G1L revealed a predicted structural similarity to the alpha-subunit of Saccharomyces cerevisiae mitochondrial processing peptidase (alpha-MPP). The HXXEH motif of G1L perfectly overlaps the HXXDR motif of alpha-MPP in this model. These results demonstrate that G1L is essential for virus maturation and suggest that G1L is a metalloproteinase with structural homology to alpha-MPP. However, no obvious effects on the expression and processing of the vaccinia virus major core proteins were observed in the G1L conditional mutant in the absence of TET compared to results for the TET and wild-type WR controls, suggesting that G1L activity is required after this step in viral morphogenesis.