Body morphometry may predict parastomal hernia following radical cystectomy with ileal conduit.

OBJECTIVE: To investigate whether preoperative body morphometry analysis can identify patients at risk of parastomal hernia (PH), which is a common complication after radical cystectomy (RC). PATIENTS AND METHODS: All patients who underwent RC between 2010 and 2020 with available cross-sectional imaging preoperatively and at 1 and 2 years postoperatively were included. Skeletal muscle mass and total fat mass (FM) were determined from preoperative axial computed tomography images obtained at the level of the L3 vertebral body using Aquarius Intuition software. Sarcopenia and obesity were assigned based on consensus definitions of skeletal muscle index (SMI) and FM index (FMI). PH were graded using both the Moreno-Matias and European Hernia Society criteria. Binary logistic regression and recursive partitioning were used to identify patients at risk of PH. The Kaplan-Meier method with log-rank and Cox proportional hazards models included clinical and image-based parameters to identify predictors of PH-free survival. RESULTS: A total of 367 patients were included in the final analysis, with 159 (43%) developing a PH. When utilising binary logistic regression, high FMI (odds ratio [OR] 1.63, P < 0.001) and low SMI (OR 0.96, P = 0.039) were primary drivers of risk of PH. A simplified model that only relied upon FMI, SMI, and preoperative albumin improved the classification of patients at risk of PH. On Kaplan-Meier analysis, patients who were obese or obese and sarcopenic had significantly worse PH-free survival (P < 0.001). CONCLUSION: Body morphometry analysis identified FMI and SMI to be the most consistent predictors of PH after RC.

Defining Mild Traumatic Brain Injury: From Research Definition to Clinical Practice.

INTRODUCTION: Approximately 75% of traumatic brain injuries (TBIs) qualify as mild. However, there exists no universally agreed upon definition for mild TBI (mTBI). Consequently, treatment guidelines for this group are lacking. The Center for Disease Control (CDC), American College of Rehabilitation Medicine (ACRM), Veterans Affairs and Department of Defense (VA/DoD), Eastern Association for the Surgery of Trauma (EAST), and the University of Arizona's Brain Injury Guidelines (BIG) have each published differing definitions for mTBI. The aim of this study was to compare the ability of these definitions to correctly classify mTBI patients in the acute care setting. METHODS: A single-center, retrospective cohort study comparing the performance of the varying definitions of mTBI was performed at a Level I trauma center from August 2015 to December 2018. Definitions were compared by sensitivity, specificity, positive predictive value, negative predictive value, as well as overtriage and undertriage rates. Finally, a cost-savings analysis was performed. RESULTS: We identified 596 patients suffering blunt TBI with Glasgow Coma Scale 13-15. The CDC/ACRM definitions demonstrated 100% sensitivity but 0% specificity along with the highest rate of undertriage and TBI-related mortality. BIG 1 included nearly twice as many patients than EAST and VA/DoD while achieving a superior positive predictive value and undertriage rate. CONCLUSIONS: The BIG definition identified a larger number of patients compared to the VA/DoD and EAST definitions while having an acceptable and more accurate overtriage and undertriage rate compared to the CDC and ACRM. By eliminating undertriage and minimizing overtriage rates, the BIG maintains patient safety while enhancing the efficiency of healthcare systems. Using the BIG definition, a cost savings of $395,288.95-$401,263.95 per year could be obtained at our level 1 trauma facility without additional mortality.

The Total Mass of Per- and Polyfluoroalkyl Substances (PFASs) in California Cosmetics.

Cosmetics make up one of the consumer product categories most widely known to contain perfluoroalkyl and polyfluoroalkyl substances (PFASs), including precursors to perfluorooctanoic acid (PFOA) and other perfluoroalkyl acids (PFAAs). Because of the way cosmetics are used, most of the PFASs present in these products are likely to reach wastewater treatment plants (WWTPs), which suggests that cosmetics may contribute significantly to the load of PFOA and other PFASs at WWTPs. However, the majority of PFASs present as intentional ingredients in cosmetics cannot be quantified with the available analytical methods. To address this issue, we developed a methodology to estimate the total PFAS mass in cosmetics as well as the corresponding mass of total organic fluorine and of fluorinated side chains associated with PFAA precursors, using various ingredient databases and ingredient concentrations reported by manufacturers. Our results indicate that the cosmetics sold in California during a one-year period cumulatively contain 650-56 000 kg of total PFASs, 370-37 000 kg of organic fluorine, and 330-20 000 kg of fluorinated side chains associated with PFAA precursors. Among the 16 product subcategories considered, >90% of the PFAS mass came from shaving creams and gels, hair care products, facial cleansers, sun care products, and lotions and moisturizers, while the sum of all nine makeup subcategories accounted for <3%. Comparing our estimates to available WWTP influent data from the San Francisco Bay Area suggests that cosmetics may account for at least 4% of the precursor-derived PFAAs measured in wastewater. As the first study ever to estimate the total mass of PFASs contained in cosmetics sold in California, our results shed light on the significance of certain cosmetics as a source of PFASs to WWTPs and can inform effective source reduction efforts.

Unravelling the early photochemical behavior of (8-substituted-7-hydroxyquinolinyl)methyl acetates through electronic structure theory and ultrafast transient absorption spectroscopy.

The photophysical processes and photochemical reactions in the ultrafast time region of (8-bromo-7-hydroxyquinolin-2-yl)methyl acetate (BHQ-OAc) in acetonitrile and neutral aqueous solutions were investigated using quantum chemical calculations and femtosecond transient absorption spectroscopy. After initial excitation into the π,π* excited state, BHQ-OAc undergoes an ultrafast intersystem crossing (ISC) into a π,π* excited triplet state on a timescale of 16 ps. The n,π* and π,π* excited singlet and triplet states involved in the photochemistry were identified by means of their characteristic excited state absorption (ESA) bands and from second order coupled-cluster (CC2) calculations. The high ISC rate of BHQ-OAc and related compounds is traced back to involvement of almost energetically degenerate n,π* excited states that enable efficient ISC that obeys El-Sayed's rules.

Confirmatory factor analytic investigation of variance composition, gender invariance, and validity of the Male Role Norms Inventory-Adolescent-revised (MRNI-A-r).

Confirmatory factor analysis of responses to the Male Role Norms Inventory-Adolescent-revised (MRNI-A-r) from 384 middle school students (163 boys, 221 girls) indicated that the best fit to the data was a bifactor model incorporating the hypothesized 3-factor structure while explicitly modeling an additional, general factor. Specifically, each item-level indicator loaded simultaneously on 2 factors: a general traditional masculinity ideology factor and a specific factor corresponding to 1 of the 3 hypothesized masculine norms for adolescents: Emotionally Detached Dominance, Toughness, and Avoidance of Femininity. Invariance testing across gender supported metric invariance for the general factor only. Although item loadings on the general factor were similar across boys and girls, the specific factor loadings varied substantially, with many becoming nonsignificant in the presence of the general factor for girls. A structural regression analysis predicting latent variables of the Meanings of Adolescent Masculinity Scale (MAMS), the Rosenberg Self-esteem Scale, and the Discipline, School Difficulties, and Positive Behavior Scale (DSDPBS) indicated that the general factor was a strong predictor of MAMS for both genders and DSDPBS for girls. Findings indicate that the MRNI-A-r general factor is a valid and reliable indicator of overall internalization of traditional masculinity ideology in adolescents; however, the specific factors may have different meanings for boys as compared with girls and lack validity in the presence of the general factor. These findings are consistent with a developmental perspective of gender ideology that views adolescence as a time when a differentiated cognitive schema of masculine norms is beginning to develop. (PsycINFO Database Record

The lateral plane delivers higher dose than the frontal plane in biplane cardiac catheterization systems.

The objective of the study is to compare radiation dose between the frontal and lateral planes in a biplane cardiac catheterization laboratory. Tube angulation progressively increases patient and operator radiation dose in single-plane cardiac catheterization laboratories. This retrospective study captured biplane radiation dose in a pediatric cardiac catheterization laboratory between April 2010 and January 2014. Raw and time-indexed fluoroscopic, cineangiographic and total (fluoroscopic + cineangiographic) air kerma (AK, mGy) and kerma area product (PKA, µGym(2)/Kg) for each plane were compared. Data for 716 patients were analyzed: 408 (56.98 %) were male, the median age was 4.86 years, and the median weight was 17.35 kg. Although median beam-on time (minutes) was 4.2 times greater in the frontal plane, there was no difference in raw median total PKA between the two planes. However, when indexed to beam-on time, the lateral plane had a higher median-indexed fluoroscopic (0.75 vs. 1.70), cineangiographic (16.03 vs. 24.92), and total (1.43 vs. 5.15) PKA (p < 0.0001). The median time-indexed total PKA in the lateral plane is 3.6 times the frontal plane. This is the first report showing that the lateral plane delivers a higher dose than the frontal plane per unit time. Operators should consciously reduce the lateral plane beam-on time and incorporate this practice in radiation reduction protocols.

A scoping review of communication outcomes measures in augmentative and alternative communication.

Although outcomes are a critical component of evidence-based practice, measuring augmentative and alternative communication (AAC) outcomes remains problematic. This is, in part, because there is no consensus on how to operationally define AAC communication outcomes. To gain greater insight into AAC communication outcomes, we used the communicative competence framework to determine which areas of AAC intervention have received the greatest attention and how these outcomes are being measured. The following data were charted from the 77 studies that met the inclusion criteria for the scoping review: study design, study participants, study communication target (e.g., language, word learning, etc.), and communication outcome measurements. Across the included studies, researchers used a variety of standardized and non-standardized measures to assess outcomes. Seventy-seven percent of the studies assessed social skills and 62% assessed linguistic skills. A limited number of studies measured operational (14%), strategic (4%), and psychosocial (18%) skills. Using the communicative competence framework enabled us to identify gaps in the research that has been conducted to date.

Unraveling the mechanism of the photodeprotection reaction of 8-bromo- and 8-chloro-7-hydroxyquinoline caged acetates.

Photoremovable protecting groups (PPGs) when conjugated to biological effectors forming "caged compounds" are a powerful means to regulate the action of physiologically active messengers in vivo through 1-photon excitation (1PE) and 2-photon excitation (2PE). Understanding the photodeprotection mechanism is important for their physiological use. We compared the quantum efficiencies and product outcomes in different solvent and pH conditions for the photolysis reactions of (8-chloro-7-hydroxyquinolin-2-yl)methyl acetate (CHQ-OAc) and (8-bromo-7-hydroxyquinolin-2-yl)methyl acetate (BHQ-OAc), representatives of the quinoline class of phototriggers for biological use, and conducted nanosecond time-resolved spectroscopic studies using transient emission (ns-EM), transient absorption (ns-TA), transient resonance Raman (ns-TR(2)), and time-resolved resonance Raman (ns-TR(3)) spectroscopies. The results indicate differences in the photochemical mechanisms and product outcomes, and reveal that the triplet excited state is most likely on the pathway to the product and that dehalogenation competes with release of acetate from BHQ-OAc, but not CHQ-OAc. A high fluorescence quantum yield and a more efficient excited-state proton transfer (ESPT) in CHQ-OAc compared to BHQ-OAc explain the lower quantum efficiency of CHQ-OAc relative to BHQ-OAc.

Resonance Raman characterization of the different forms of ground-state 8-substituted 7-hydroxyquinoline caged acetate compounds in aqueous solutions.

To investigate the substituent effect on the distribution of the forms of the ground-state species of 8-substituted 7-hydroxyquinolines, ultraviolet-absorption and resonance Raman experiments were performed for 8-chloro-7-hydroxyquinoline (CHQ-OAc) and 8-cyano-7-hydroxyquinoline (CyHQ-OAc) in acetonitrile (MeCN), in NaOH-H(2)O/MeCN (60:40, v/v, pH 11-12), and in H(2)O/MeCN (60:40, v/v, pH 6-7) solutions, and these results were compared to those previously reported for the 8-bromo-7-hydroxyquinoline (BHQ-OAc) compound. Swapping a bromine atom in BHQ-OAc for a chlorine atom in CHQ-OAc causes the amount of the tautomeric species to become larger, although the neutral species is still the predominant species for both systems in water-rich solutions. The absorption spectra and the resonance Raman spectra of CyHQ-OAc suggest that, because of the strong electron-withdrawing nature of the cyano substituent, a measurable amount of the anionic species is present and the tautomeric species cannot be easily detected in water-rich solutions. The results reported here reveal large substituent effects on the distribution of the different forms of the XHQ-OAc compounds in largely aqueous solutions. The steric effect of the 8-substituted group and competitive hydrogen bonding between the 8-substituted group and water molecules hinders the formation of a cyclic BHQ-OAc-water complex, and the electron-withdrawing property of the 8-substituted group enhances the deprotonation of the phenol group while disfavoring the formation of the positively charged quinoline nitrogen. We briefly discuss the implications of the substituent effects for using these compounds as phototriggers.

Operon prediction using both genome-specific and general genomic information.

We have carried out a systematic analysis of the contribution of a set of selected features that include three new features to the accuracy of operon prediction. Our analyses have led to a number of new insights about operon prediction, including that (i) different features have different levels of discerning power when used on adjacent gene pairs with different ranges of intergenic distance, (ii) certain features are universally useful for operon prediction while others are more genome-specific and (iii) the prediction reliability of operons is dependent on intergenic distances. Based on these new insights, our newly developed operon-prediction program achieves more accurate operon prediction than the previous ones, and it uses features that are most readily available from genomic sequences. Our prediction results indicate that our (non-linear) decision tree-based classifier can predict operons in a prokaryotic genome very accurately when a substantial number of operons in the genome are already known. For example, the prediction accuracy of our program can reach 90.2 and 93.7% on Bacillus subtilis and Escherichia coli genomes, respectively. When no such information is available, our (linear) logistic function-based classifier can reach the prediction accuracy at 84.6 and 83.3% for E.coli and B.subtilis, respectively.

Bypassing Glutamic Acid Decarboxylase 1 (Gad1) Induced Craniofacial Defects with a Photoactivatable Translation Blocker Morpholino.

γ-Amino butyric acid (GABA) mediated signaling is critical in the central and enteric nervous systems, pancreas, lungs, and other tissues. It is associated with many neurological disorders and craniofacial development. Glutamic acid decarboxylase (GAD) synthesizes GABA from glutamate, and knockdown of the gad1 gene results in craniofacial defects that are lethal in zebrafish. To bypass this and enable observation of the neurological defects resulting from knocking down gad1 expression, a photoactivatable morpholino oligonucleotide (MO) against gad1 was prepared by cyclization with a photocleavable linker rendering the MO inactive. The cyclized MO was stable in the dark and toward degradative enzymes and was completely linearized upon brief exposure to 405 nm light. In the course of investigating the function of the ccMOs in zebrafish, we discovered that zebrafish possess paralogous gad1 genes, gad1a and gad1b. A gad1b MO injected at the 1-4 cell stage caused severe morphological defects in head development, which could be bypassed, enabling the fish to develop normally, if the fish were injected with a photoactivatable, cyclized gad1b MO and grown in the dark. At 1 day post fertilization (dpf), light activation of the gad1b MO followed by observation at 3 and 7 dpf led to increased and abnormal electrophysiological brain activity compared to wild type animals. The photocleavable linker can be used to cyclize and inactivate any MO, and represents a general strategy to parse the function of developmentally important genes in a spatiotemporal manner.

Loss of expression of N-acetylglucosaminyltransferase Va results in altered gene expression of glycosyltransferases and galectins.

We isolated mouse embryo fibroblasts (MEFs) from N-acetylglucosaminyltransferase Va (GnT-Va) knockout mice and studied the effects of loss of expression of GnT-Va on asparagine-linked glycans (N-glycan) synthesis and the gene expression of groups of glycosyltransferases and galectins. Loss of GnT-Va expression caused aberrant expression of several N-glycan structures, including N-linked beta(1,6) branching, poly-N-lactosamine, bisecting N-acetylglucosamine (GlcNAc) and sialic acid. Using quantitative reverse transcriptase-PCR (qRT-PCR), altered gene expression of several groups of glycosyltransferases and galectins was observed in GnT-Va null MEFs, supporting the observed changes in N-glycan structures. These results suggest that genetic disruption of GnT-Va ultimately resulted in altered MEFs gene expression and decreased tumor progression associated with loss of GnT-Va observed may result in part from a combination of effects from these altered gene expressions.

Tibiofemoral Osteoarthritis After Surgical or Nonsurgical Treatment of Anterior Cruciate Ligament Rupture: A Systematic Review.

OBJECTIVE: To determine if surgical or nonsurgical treatment of anterior cruciate ligament rupture affects the prevalence of posttraumatic tibiofemoral osteoarthritis (OA). DATA SOURCES: Studies published between 1983 and April 2012 were identified via EBSCOhost and OVID. Reference lists were then screened in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement. STUDY SELECTION: Studies were included if (a) treatment outcomes focused on a direct comparison of surgical versus nonsurgical treatment of anterior cruciate ligament rupture, (b) the prevalence of tibiofemoral OA was reported, and (c) they were written in English. Studies were excluded if (a) the included patients were treated with cast immobilization after surgery, (b) the mean follow-up was less than 10 years, or (c) the patients underwent anterior cruciate ligament revision surgery. DATA EXTRACTION: Two independent investigators reviewed the included articles using the Newcastle-Ottawa Scale. Frequency of OA, surgical procedure, nonsurgical treatments, and participant characteristics were extracted and summarized. We calculated prevalence (%) and 95% confidence intervals for treatment groups for each individual study and overall. We developed 2 × 2 contingency tables to assess the association between treatment groups (exposed had surgery, referent was nonsurgical treatment) and the prevalence of OA. DATA SYNTHESIS: Four retrospective studies were identified (140 surgical patients, 240 nonsurgical patients). The mean Newcastle-Ottawa Scale score was 5 (range = 4-6 [of 10] points). Average length of follow-up was 11.8 years (range = 10-14 years). The prevalence of OA for surgically treated patients ranged from 32.6% to 51.2% (overall = 41.4%, 95% confidence interval = 35.0%, 48.1%) and for nonsurgical patients ranged from 24.5% to 42.3% (overall = 30.9%, 95% confidence interval = 24.4%, 38.3%). CONCLUSIONS: Although OA prevalence was higher in the surgical treatment group at a mean follow-up of 11.8 years, no definitive evidence supports surgical or nonsurgical treatment after anterior cruciate ligament injury to prevent posttraumatic OA. Current studies have been limited by small sample sizes, low methodologic quality, and a lack of data regarding confounding factors.

Is Participation in Certain Sports Associated With Knee Osteoarthritis? A Systematic Review.

OBJECTIVE: Information regarding the relative risks of developing knee osteoarthritis (OA) as a result of sport participation is critical for shaping public health messages and for informing knee-OA prevention strategies. The purpose of this systematic review was to investigate the association between participation in specific sports and knee OA. DATA SOURCES: We completed a systematic literature search in September 2012 using 6 bibliographic databases (PubMed; Ovid MEDLINE; Journals@Ovid; American College of Physicians Journal Club; Cochrane Central Register of Controlled Trials, Cochrane Database of Systematic Review, Database of Abstracts of Reviews of Effects; and Ovid HealthStar), manual searches (4 journals), and reference lists (56 articles). STUDY SELECTION: Studies were included if they met the following 4 criteria: (1) an aim was to investigate an association between sport participation and knee OA; (2) the outcome measure was radiographic knee OA, clinical knee OA, total knee replacement, self-reported diagnosis of knee OA, or placement on a waiting list for a total knee replacement; (3) the study design was case control or cohort; and (4) the study was written in English. Articles were excluded if the study population had an underlying condition other than knee OA. DATA EXTRACTION: One investigator extracted data (eg, group descriptions, knee OA prevalence, source of nonexposed controls). DATA SYNTHESIS: The overall knee-OA prevalence in sport participants (n = 3759) was 7.7%, compared with 7.3% among nonexposed controls (referent group n = 4730, odds ratio [OR] = 1.1). Specific sports with a significantly higher prevalence of knee OA were soccer (OR = 3.5), elite-level long-distance running (OR = 3.3), competitive weight lifting (OR = 6.9), and wrestling (OR = 3.8). Elite-sport (soccer or orienteering) and nonelite-sport (soccer or American football) participants without a history of knee injury had a greater prevalence of knee OA than nonexposed participants. CONCLUSIONS: Participants in soccer (elite and nonelite), elite-level long-distance running, competitive weight lifting, and wrestling had an increased prevalence of knee OA and should be targeted for risk-reduction strategies.

Five-year clinical outcomes of a randomized trial of anterior cruciate ligament treatment strategies: an evidence-based practice paper.

REFERENCE/CITATION: Frobell RB, Roos HP, Roos EM, Roemer FW, Ranstam J, Lohmander LS. Treatment for acute anterior cruciate ligament tear: five year outcome of randomised trial. BMJ. 2013;346:F232. CLINICAL QUESTION: Does early anterior cruciate ligament (ACL) reconstruction with rehabilitation lead to better patient-reported outcomes and a lower incidence of osteoarthritis at 5 years postinjury compared with delayed ACL reconstruction with rehabilitation? STUDY SELECTION: This randomized controlled trial with extended follow-up at 5 years postrandomization was conducted in 2 Swedish orthopaedic departments. DATA EXTRACTION: The authors studied a total of 121 moderately active adults (age = 18-35 years) with an acute ACL rupture in a knee with no other history of trauma. Excluded were patients with a collateral ligament rupture, full-thickness cartilage defect, or extensive meniscal fixation. One patient assigned to the early ACL-reconstruction group did not attend the 5-year follow-up visit. Patients were randomly assigned to (1) an early ACL reconstruction plus structured rehabilitation group (n = 62, surgery within 10 weeks of injury) or (2) optional-delayed ACL reconstruction plus structured rehabilitation group (n = 59). The primary outcome measure was change in the average of 4 out of 5 subscales of the Knee Injury and Osteoarthritis Outcome Score (KOOS). The authors also assessed crude KOOS (combined 4 subscales), KOOS subscale scores, general physical and mental health (Short-Form 36), activity level (Tegner Activity Scale), mechanical knee stability (Lachman and pivot shift tests), meniscal surgery status, and presence of knee osteoarthritis on radiographs. MAIN RESULTS: Among patients randomized to the optional-delayed ACL-reconstruction group, 30 (51%) opted for an ACL reconstruction. The treatment groups had comparable 5-year patient-reported outcomes and changes in patient-reported outcomes (eg, knee pain, knee symptoms, activities of daily living, sport and recreational levels, knee-related quality of life, general physical health, and general mental health). Patients in the optional-delayed ACL-reconstruction group had greater mechanical knee instability than patients who received early ACL reconstruction; however, this was primarily among the patients opting for conservative management alone. In the overall sample, 61 knees (51%) required meniscal surgery over 5 years, regardless of treatment group. At 5 years, radiographs were available for 113 patients (93%). Overall, 29 patients (26%) had knee osteoarthritis at 5 years. Specifically, 13 patients (12%) developed tibiofemoral radiographic osteoarthritis (9 patients [16%] in the early ACL-reconstruction group, 4 [7%] in the optional-delayed ACL-reconstruction group) and 22 (19%) developed patellofemoral osteoarthritis (14 patients [24%] in the early ACL-reconstruction group, 8 [15%] in the optional-delayed ACL-reconstruction group). Patients with patellar tendon grafts (n = 40) had a greater incidence of ipsilateral patellofemoral osteoarthritis than patients with hamstrings tendon grafts (n = 51), but the 2 groups had similar incidences of ipsilateral tibiofemoral osteoarthritis. Six knees (5%) had both tibiofemoral and patellofemoral osteoarthritis. CONCLUSIONS: Early ACL reconstruction plus rehabilitation did not provide better results at 5 years compared with optional-delayed ACL reconstruction plus rehabilitation. Furthermore, the authors found no radiographic differences among patients with early ACL reconstruction, delayed ACL reconstruction, or no ACL reconstruction (rehabilitation alone).

Rapid generation of stable cell lines expressing high levels of erythropoietin, factor VIII, and an antihuman CD20 antibody using lentiviral vectors.

Lentiviral vectors (LVs) are widely recognized as the most efficient method for the stable delivery of nucleic acid sequences into mammalian cells. Using erythropoietin (EPO), recombinant factor VIII (fVIII), and an anti-CD20 antibody as model proteins, we demonstrate advantages of LV-based gene delivery to achieve high production levels by transduced cells. Highly productive cell clones were able to incorporate up to 100 vector copies per cellular genome, without selection or gene amplification, and were isolated without extensive screening of a large number of clones. The LV transgenes were shown to be distributed throughout the genome, as visualized by fluorescent in situ hybridization. High-expressing clones producing 100-200 pg/cell/day of EPO were isolated and characterized. EPO production was demonstrated for at least 5½ months of continuous culture without selection, during which all the clones displayed high levels of glycosylation despite production levels at 10-20 g/liter. To demonstrate the utility of LV technology for multiple classes of proteins, cell lines producing fVIII and an anti-CD20 antibody were also developed. Cell clones demonstrating high levels of fVIII (100 clot units/ml and anti-CD20 antibody as high as 40-100 pg/cell/day) were isolated and characterized. LV-transduced cells and plasmid-transfected cells were compared for protein production per transgene copy. LV-transduced cells produced significantly higher levels of protein per copy of transgene than plasmid-transfected cells did. This study demonstrates the utility of LV technology for rapid generation of highly productive and stable cell lines over conventional plasmid transfection methods, significantly decreasing the time, cost, and risk of the manufacture of proteins and other complex biological molecules.

Reliability and validity of three quality rating instruments for systematic reviews of observational studies.

To assess the inter-rater reliability, validity, and inter-instrument agreement of the three quality rating instruments for observational studies. Inter-rater reliability, criterion validity, and inter-instrument reliability were assessed for three quality rating scales, the Downs and Black (D&B), Newcastle-Ottawa (NOS), and Scottish Intercollegiate Guidelines Network (SIGN), using a sample of 23 observational studies of musculoskeletal health outcomes. Inter-rater reliability for the D&B (Intraclass correlations [ICC] = 0.73; CI = 0.47-0.88) and NOS (ICC = 0.52; CI = 0.14-0.76) were moderate to good and was poor for the SIGN (κ = 0.09; CI = -0.22-0.40). The NOS was not statistically valid (p = 0.35), although the SIGN was statistically valid (p < 0.05) with medium to large effect sizes (f(2) = 0.29-0.47). Inter-instrument agreement estimates were κ = 0.34, CI = 0.05-0.62 (D&B versus SIGN), κ = 0.26, CI = 0.00-0.52 (SIGN versus NOS), and κ = 0.43, CI = 0.09-0.78 (D&B versus NOS). Reliability and validity are quite variable across quality rating scales used in assessing observational studies in systematic reviews. Copyright © 2011 John Wiley & Sons, Ltd.

Rate, affinity and calcium dependence of nitric oxide synthase isoform binding to the primary physiological regulator calmodulin.

Using interferometry-based biosensors the binding and release of endothelial and neuronal nitric oxide synthase (eNOS and nNOS) from calmodulin (CaM) was measured. In both isoforms, binding to CaM is diffusion limited and within approximately three orders of magnitude of the Smoluchowski limit imposed by orientation-independent collisions. This suggests that the orientation of CaM is facilitated by the charge arrays on the CaM-binding site and the complementary surface on CaM. Protein kinase C phosphorylation of eNOS T495, adjacent to the CaM-binding site, abolishes or greatly slows CaM binding. Kinases which increase the activity of eNOS did not stimulate the binding of CaM, which is already diffusion limited. The coupling of Ca(2+) binding and CaM/NOS binding equilibria links the affinity of CaM for NOS to the Ca(2+) dependence of CaM binding. Hence, changes in the Ca(2+) sensitivity of CaM binding always imply changes in the NOS-CaM affinity. It is possible, however, that in some regimes binding and activation are not synonymous, so that Ca(2+) sensitivity need not be tightly linked to CaM sensitivity of activation. This study is being extended using mutants to probe the roles of individual structural elements in binding and release.

Regulation of glycan structures in animal tissues: transcript profiling of glycan-related genes.

Glycan structures covalently attached to proteins and lipids play numerous roles in mammalian cells, including protein folding, targeting, recognition, and adhesion at the molecular or cellular level. Regulating the abundance of glycan structures on cellular glycoproteins and glycolipids is a complex process that depends on numerous factors. Most models for glycan regulation hypothesize that transcriptional control of the enzymes involved in glycan synthesis, modification, and catabolism determines glycan abundance and diversity. However, few broad-based studies have examined correlations between glycan structures and transcripts encoding the relevant biosynthetic and catabolic enzymes. Low transcript abundance for many glycan-related genes has hampered broad-based transcript profiling for comparison with glycan structural data. In an effort to facilitate comparison with glycan structural data and to identify the molecular basis of alterations in glycan structures, we have developed a medium-throughput quantitative real time reverse transcriptase-PCR platform for the analysis of transcripts encoding glycan-related enzymes and proteins in mouse tissues and cells. The method employs a comprehensive list of >700 genes, including enzymes involved in sugar-nucleotide biosynthesis, transporters, glycan extension, modification, recognition, catabolism, and numerous glycosylated core proteins. Comparison with parallel microarray analyses indicates a significantly greater sensitivity and dynamic range for our quantitative real time reverse transcriptase-PCR approach, particularly for the numerous low abundance glycan-related enzymes. Mapping of the genes and transcript levels to their respective biosynthetic pathway steps allowed a comparison with glycan structural data and provides support for a model where many, but not all, changes in glycan abundance result from alterations in transcript expression of corresponding biosynthetic enzymes.