Comparative analysis of three studies measuring fluorescence from engineered bacterial genetic constructs.

Reproducibility is a key challenge of synthetic biology, but the foundation of reproducibility is only as solid as the reference materials it is built upon. Here we focus on the reproducibility of fluorescence measurements from bacteria transformed with engineered genetic constructs. This comparative analysis comprises three large interlaboratory studies using flow cytometry and plate readers, identical genetic constructs, and compatible unit calibration protocols. Across all three studies, we find similarly high precision in the calibrants used for plate readers. We also find that fluorescence measurements agree closely across the flow cytometry results and two years of plate reader results, with an average standard deviation of 1.52-fold, while the third year of plate reader results are consistently shifted by more than an order of magnitude, with an average shift of 28.9-fold. Analyzing possible sources of error indicates this shift is due to incorrect preparation of the fluorescein calibrant. These findings suggest that measuring fluorescence from engineered constructs is highly reproducible, but also that there is a critical need for access to quality controlled fluorescent calibrants for plate readers.

Author Correction: Robust estimation of bacterial cell count from optical density.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Robust estimation of bacterial cell count from optical density.

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals  <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data.

Radiographic Study of Humeral Stem in Shoulder Arthroplasty After Lesser Tuberosity Osteotomy or Subscapularis Tenotomy.

Lesser tuberosity osteotomy (LTO) and subscapularis tenotomy (ST) are used for takedown of the subscapularis during shoulder arthroplasty. LTO offers the theoretical but unproven benefit of improved healing and function of the subscapularis. However, humeral stem subsidence and loosening may be greater when osteotomy is performed, which may compromise functional outcomes. Our hypothesis is that no difference in proximal collar press-fit humeral stem subsidence or loosening exists, with no impairment of functional outcomes using the LTO technique. Radiographs of 39 shoulders from 35 patients who underwent shoulder arthroplasty with a minimum of 1 year of radiographic follow-up were included in the study cohort. All patients received the same press-fit implant (Bigliani-Flatow; Zimmer Biomet). We collected data including demographic information; radiographic measurements, including humeral-acromial distance (HAD); subsidence; subluxation index; the presence of lucent lines >2 mm; and functional outcome scores using the Western Ontario Osteoarthritis of the Shoulder Index, the Disabilities of the Arm, Shoulder and Hand (DASH) questionnaire, and the Constant score. Subsidence was 2.8 ± 3.1 mm for LTO vs 2.5 ± 3.1 mm for ST (P = .72). HAD did not differ between the LTO and ST groups preoperatively (9.5 ± 2.4 mm vs 10.9 ± 2.7 mm, P = .11). The first postoperative and final follow-up films for HAD for the LTO and ST groups showed a statistically significant difference (first postoperative film, 11.9 ± 3.7 mm vs 15.9 ± 4.5 mm, P = .005; final follow-up film, 11.8 ± 3.2 mm vs 14.5 ± 3.9 mm, P = .03). We identified no differences in subsidence, lucent lines >2 mm, posterior subluxation, and Constant, and DASH functional outcome scores for patients undergoing total shoulder arthroplasty via the LTO vs ST techniques with the same proximal collar press-fit humeral stem at short-term follow-up.

Validity of QuickDASH at day of surgery versus day of initial consultation: Does informed consent make a difference?

INTRODUCTION: The trend toward requiring explicit consent from patients participating in observational research increases time and resources required to perform such research. Informed consent introduces the potential for "consent bias"-either through selection bias or through the "Hawthorne effect," where patients may alter responses based upon the awareness of participation in a study, thus potentially limiting its applicability to a generalized orthopedic practice. We hypothesized that administering Quick Disabilities of the Arm, Shoulder, and Hand Questionnaire (QuickDASH) to patients on the day of surgery with informed consent would alter responses in a statistically and clinically meaningful way compared to patients who complete QuickDASH as a quality control measure. METHODS: We previously instituted the QuickDASH questionnaire as the standard new patient intake and postoperative questionnaire for quality assurance purposes. We retrospectively reviewed data on a cohort of patients who underwent isolated carpal tunnel release (CTR) who had completed preoperative and postoperative QuickDASH forms without providing consent for study participation. Next, a cohort of patients scheduled to undergo isolated CTR who completed the intake questionnaire was approached on the day of surgery for consent to participate in the study. After obtaining consent but prior to surgery, these patients completed a second questionnaire and then completed a postoperative questionnaire on follow-up at a mean of 8 weeks postoperatively. RESULTS: Thirty-nine patients and 35 patients were included in the retrospective and prospective cohorts, respectively. No significant differences were observed in age, gender, symptom duration, nerve conduction study/electromyography results, or disease severity between the two groups. We identified no statistically significant difference in preoperative or postoperative QuickDASH score between the retrospective and prospective cohorts (39.8 ± 22.7 vs. 39.7 ± 19.1 preoperatively; 27.3 ± 24.7 vs. 18.7 ± 13.3 postoperatively) or within the prospective cohort before and after obtaining informed consent. CONCLUSION: Informed consent did not significantly alter patient responses to the QuickDASH questionnaire. These results suggest that both "opt-in" and "opt-out" approaches to observational research in hand surgery provide results that may be applicable to a generalized orthopedic practice. CLINICAL RELEVANCE: This study provides evidence that will inform the interpretation of observational research findings in hand surgery.

Variables Prognostic for Delayed Union and Nonunion Following Ulnar Shortening Fixed With a Dedicated Osteotomy Plate.

PURPOSE: To examine potential risk factors for the development of delayed or nonunion following elective ulnar shortening osteotomy using a dedicated osteotomy plating system. METHODS: We performed a retrospective review of all patients who underwent elective ulnar shortening using the TriMed single osteotomy dynamic compression plating system by 1 of 2 fellowship-trained hand surgeons over a 5-year period. Demographic data and medical, surgical, and social histories were reviewed. Time to bony union was determined radiographically by a blinded reviewer. Bivariate statistical analysis was performed to examine the effect of explanatory variables on the time to union and the incidence of delayed or nonunion. Those variables associated with the development of delayed or nonunion were used in a multivariate logistic regression model. Complications, including the need for additional surgery, were also recorded. RESULTS: Seventy-two ulnar shortening osteotomy procedures were performed in 69 patients. Delayed union, defined as ≥ 6 months to union, occurred in 8 of 72 cases (11%). Of 72 surgeries, 4 (6%) resulted in nonunions, all of which required additional surgery. Hardware removal was performed in 13 of 72 (18%) of the cases. Time to union was significantly increased in smokers (6 ± 3 months) versus nonsmokers (3 ± 1 months). On multivariable analysis, diabetics and active smokers demonstrated a significantly higher risk of developing delayed union or nonunion. Patient age, sex, body mass index, thyroid disease, worker's compensation status, alcohol use, and amount smoked daily did not have an effect on the time to union or the incidence of delayed or nonunion. CONCLUSIONS: Despite the use of an osteotomy-specific plating system, smokers and diabetics were at significantly higher risk for both delayed union and nonunion following elective ulnar shortening osteotomy. Other known risk factors for suboptimal bony healing were not found to have a deleterious effect.

Inhibition of beta-catenin signaling by Pb leads to incomplete fracture healing.

There is strong evidence in the clinical literature to suggest that elevated lead (Pb) exposure impairs fracture healing. Since Pb has been demonstrated to inhibit bone formation, and Wnt signaling is an important anabolic pathway in chondrocyte maturation and endochondral ossification, we investigated the impact of Wnt therapy on Pb-exposed mice undergoing bone repair in a mouse tibial fracture model. We established that tibial fracture calluses from Pb-treated mice were smaller and contained less mineralized tissue than vehicle controls. This resulted in the persistence of immature cartilage in the callus and decreased ß-catenin levels. Reduction of ß-catenin protein was concurrent with systemic elevation of LRP5/6 antagonists DKK1 and sclerostin in Pb-exposed mice throughout fracture healing. ß-catenin stimulation by the GSK3 inhibitor BIO reversed these molecular changes and restored the amount of mineralized callus. Overall, Pb is identified as a potent inhibitor of endochondral ossification in vivo with correlated effects on bone healing with noted deficits in ß-catenin signaling, suggesting the Wnt/ß-catenin as a pivotal pathway in the influence of Pb on fracture repair.

Analysis of subscapularis integrity and function after lesser tuberosity osteotomy versus subscapularis tenotomy in total shoulder arthroplasty using ultrasound and validated clinical outcome measures.

BACKGROUND: The optimal method-subscapularis peel (SP) or lesser tuberosity osteotomy (LTO)-for takedown of the subscapularis during total shoulder arthroplasty (TSA) is controversial. This study compares the functional outcomes in a 2-surgeon cohort using the 2 techniques. METHODS: Patients who underwent TSA with a minimum 1 year of follow-up were evaluated. Physical and ultrasound examinations of the operative shoulder were performed. Radiographs were evaluated for osteotomy healing. Patients completed the Western Ontario Osteoarthritis of the Shoulder (WOOS) index, Disability of the Arm, Shoulder, and Hand (DASH), and Constant Scores. RESULTS: Subscapularis tenotomy (n = 32) and LTO (n = 28) patients were similar in age, hand dominance, and sex. Follow-up duration for subscapularis tenotomy and LTO patients differed (31.7 vs 22.1 months, P = .003). SP patients demonstrated increased external rotation (69° ± 12° vs 60° ± 11°). Belly press and bear hug resistance were not significantly different. WOOS (P = .13), DASH (P = .71), and Constant Scores (P = .80) were not significantly different. After controlling for follow-up imbalance, the WOOS score difference was statistically significant (91.5 ± 10.2 for LTO vs 82.1 ± 18.9 for SP, P = .05) but not clinically significant. By ultrasonography assessment, 4 subscapularis tendons were abnormal in the SP group (3 attenuated, 1 ruptured), and all tendons were normal in the LTO group. Patients with an abnormal ultrasound result had significantly inferior WOOS (88 ± 15 vs 65 ± 18) and DASH (11.5 ± 11.4 vs 25.9 ± 11.2) scores. Belly press resistance was significantly decreased, bear hug resistance trended lower, and external rotation was increased in the abnormal ultrasound group. CONCLUSIONS: Abnormal subscapularis tendons identified by ultrasonography only in the SP group correlate with clinically significant inferior functional outcome scores.

The effect of informed consent on results of a standard upper extremity intake questionnaire.

PURPOSE: The trend toward requiring explicit consent from patients participating in observational research introduces the potential for consent bias, either through selection bias or through the Hawthorne effect. In the Hawthorne effect, patients may alter responses based on awareness of participation in a study, thus potentially limiting its applicability to a generalized orthopedic practice. We hypothesized that study subjects' awareness of participation in an observational study by informed consent would alter responses to a standard upper extremity questionnaire in a statistically and clinically meaningful way compared with patients who filled out the questionnaire as a quality control measure. METHODS: We retrospectively reviewed data on 39 patients who underwent isolated carpal tunnel release, who had completed preoperative and postoperative Quick Disabilities of the Arm, Shoulder, and Hand (Quick DASH) forms without providing consent for study participation. Next, we approached 35 patients scheduled to undergo isolated carpal tunnel release who completed the intake questionnaire on the day of surgery, for consent to participate in the study. After obtaining consent but before surgery, these patients completed a second questionnaire and then completed a postoperative questionnaire at a mean of 8 weeks postoperatively. RESULTS: There were no significant differences in age, sex, insurance status, symptom duration, nerve conduction study and electromyography results, or disease severity between groups. We identified no statistically significant difference in preoperative or postoperative Quick DASH score between the retrospective and prospective cohorts (40 ± 23 vs 40 ± 19 preoperatively; 27 ± 25 vs 19 ± 13 postoperatively) or within the prospective cohort before and after obtaining informed consent. CONCLUSIONS: Informed consent did not significantly alter patients' responses to the Quick DASH questionnaire. These results suggest that both opt-in and opt-out approaches to observational research in hand surgery provide results that may be applicable to a generalized orthopedic practice. CLINICAL RELEVANCE: This study provides evidence that will inform the interpretation of observational research findings in hand surgery.

Design and selection of Toca 511 for clinical use: modified retroviral replicating vector with improved stability and gene expression.

Retroviral replicating vectors (RRVs) are a nonlytic alternative to oncolytic replicating viruses as anticancer agents, being selective both for dividing cells and for cells that have defects in innate immunity and interferon responsiveness. Tumor cells fit both these descriptions. Previous publications have described a prototype based on an amphotropic murine leukemia virus (MLV), encoding yeast cytosine deaminase (CD) that converts the prodrug 5-fluorocytosine (5-FC) to the potent anticancer drug, 5-fluorouracil (5-FU) in an infected tumor. We report here the selection of one lead clinical candidate based on a general design goal to optimize the genetic stability of the virus and the CD activity produced by the delivered transgene. Vectors were tested for titer, genetic stability, CD protein and enzyme activity, ability to confer susceptibility to 5-FC, and preliminary in vivo antitumor activity and stability. One vector, Toca 511, (aka T5.0002) encoding an optimized CD, shows a threefold increased specific activity in infected cells over infection with the prototype RRV and shows markedly higher genetic stability. Animal testing demonstrated that Toca 511 replicates stably in human tumor xenografts and, after 5-FC administration, causes complete regression of such xenografts. Toca 511 (vocimagene amiretrorepvec) has been taken forward to preclinical and clinical trials.

Brain tumor eradication and prolonged survival from intratumoral conversion of 5-fluorocytosine to 5-fluorouracil using a nonlytic retroviral replicating vector.

Patients with the most common and aggressive form of high-grade glioma, glioblastoma multiforme, have poor prognosis and few treatment options. In 2 immunocompetent mouse brain tumor models (CT26-BALB/c and Tu-2449-B6C3F1), we showed that a nonlytic retroviral replicating vector (Toca 511) stably delivers an optimized cytosine deaminase prodrug activating gene to the tumor lesion and leads to long-term survival after treatment with 5-fluorocytosine (5-FC). Survival benefit is dose dependent for both vector and 5-FC, and as few as 4 cycles of 5-FC dosing after Toca 511 therapy provides significant survival advantage. In the virally permissive CT26-BALB/c model, spread of Toca 511 to other tissues, particularly lymphoid tissues, is detectable by polymerase chain reaction (PCR) over a wide range of levels. In the Tu-2449-B6C3F1 model, Toca 511 PCR signal in nontumor tissues is much lower, spread is not always observed, and when observed, is mainly detected in lymphoid tissues at low levels. The difference in vector genome spread correlates with a more effective antiviral restriction element, APOBEC3, present in the B6C3F1 mice. Despite these differences, neither strain showed signs of treatment-related toxicity. These data support the concept that, in immunocompetent animals, a replicating retroviral vector carrying a prodrug activating gene (Toca 511) can spread through a tumor mass, leading to selective elimination of the tumor after prodrug administration, without local or systemic pathology. This concept is under investigation in an ongoing phase I/II clinical trial of Toca 511 in combination with 5-FC in patients with recurrent high-grade glioma (www.clinicaltrials.gov NCT01156584).

Lead induces chondrogenesis and alters transforming growth factor-beta and bone morphogenetic protein signaling in mesenchymal cell populations.

BACKGROUND: It has been established that skeletal growth is stunted in lead-exposed children. Because chondrogenesis is a seminal step during skeletal development, elucidating the impact of Pb on this process is the first step toward understanding the mechanism of Pb toxicity in the skeleton. OBJECTIVES: The aim of this study was to test the hypothesis that Pb alters chondrogenic commitment of mesenchymal cells and to assess the effects of Pb on various signaling pathways. METHODS: We assessed the influence of Pb on chondrogenesis in murine limb bud mesenchymal cells (MSCs) using nodule formation assays and gene analyses. The effects of Pb on transforming growth factor-beta (TGF-beta) and bone morphogenetic protein (BMP) signaling was studied using luciferase-based reporters and Western analyses, and luciferase-based assays were used to study cyclic adenosine monophosphate response element binding protein (CREB), beta-catenin, AP-1, and nuclear factor-kappa B (NF-kappaB) signaling. We also used an ectopic bone formation assay to determine how Pb affects chondrogenesis in vivo. RESULTS: Pb-exposed MSCs showed enhanced basal and TGF-beta/BMP induction of chondrogenesis, evidenced by enhanced nodule formation and up-regulation of Sox-9, type 2 collagen, and aggrecan, all key markers of chondrogenesis. We observed enhanced chondrogenesis during ectopic bone formation in mice preexposed to Pb via drinking water. In MSCs, Pb enhanced TGF-beta but inhibited BMP-2 signaling, as measured by luciferase reporter assays and Western analyses of Smad phosphorylation. Although Pb had no effect on basal CREB or Wnt/beta-catenin pathway activity, it induced NFkappaB signaling and inhibited AP-1 signaling. CONCLUSIONS: The in vitro and in vivo induction of chondrogenesis by Pb likely involves modulation and integration of multiple signaling pathways including TGF-beta, BMP, AP-1, and NFkappaB.