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Rationale and objectives This study seeks to generate a model based on two linear measurements, anteroposterior (AP) diameter and interpedicular (IPD) distance, to approximate the cervical central canal (CCC) area in a non-pathologic patient population by employing area calculations of shapes such as ellipse, rectangle, and triangle. Secondarily, this study aims to generate second-order approximations (SOAs), using the aforementioned shape approximations, to increase the utility of this linear measurement-based model. Methods The authors reviewed medical and radiographic records of patients aged 18-35 who received computed tomography (CT) imaging of the cervical spine to collect AP diameter, IPD distance, and area of the CCC from C2-3 to C6-7. Subsequently, shape approximations were calculated for each patient at all cervical spine levels. Lastly, SOAs were generated by combining optimal ratios of shape approximations to improve the statistical reliability of approximations. Results The ellipse shows the closest approximation to manual measurements of the individual shape approximations. Percent error analysis demonstrated the superiority of the ellipse, followed by the rectangle, and lastly the triangular approximation. The highest correlation of approximations was observed at C6-7. All individual shape approximations demonstrated statistical differences from manual measurements. SOAs combining ellipse and rectangle measurements demonstrated superior accuracy and were not statistically different from manual measurements. Conclusion Individual shape approximations based on AP diameter and IPD distance show some value as a model for the assessment of the CCC area. SOAs demonstrated greater utility than individual shape approximations and show promise as a linear measurement-based tool to assess the CCC area.
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Introduction: Distal fibula osteoporotic comminuted fractures are challenging to treat and are often treated with periarticular locking plates. This study examined the biomechanical difference between locked plating and dual non-locked one-third tubular plating. Methods: Using an osteoporotic Sawbones fibula model, simulated fracture were fixated with one-third tubular dual plating and locked periarticular plating. The samples were then torqued to failure and peak torque, stiffness, and displacement were recorded. Results: The peak torque of the dual plating group was found to be statistically higher than the periarticular locked plating group (0.841 Nm and 0.740 Nm respectively; p = 0.024). However overall stiffness calculated at each 10° increment of displacement was noted to have no significant difference between the two constructs. Conclusion: Dual non-locked plating of distal fibula osteoporotic comminuted fractures is biomechanically equivalent to locked periarticular plating.
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Purpose: The purpose of this study is to determine if machine learning is an effective method to identify features of patients who may need a longer postoperative stay following a patellar tendon repair. Methods: The American College of Surgeons National Quality Improvement Program (ACS-NSQIP) was used to collect 1173 patients who underwent patellar tendon repair. Machine learning (ML) was then applied to determine features of importance in this patient population. Several algorithms were used: Random Forest, Artificial Neural Network, Gradient Boosting, and Support Vector Machine. These were then compared to the American Society of Anesthesiologists (ASA) classification system based logistic regression as a control. Results: Random Forest (RF) was determined to be the best performing algorithm, with an AUC of 0.72, accuracy of 77.66 %, and precision of 0.79, and recall of 0.96. All other algorithms performed similarly to the control. RF gave the highest permutation feature importance to age (PFI 0.25), BMI (PFI 0.19), ASA classification (PFI 0.14), hematocrit (PFI 0.12), and height (PFI 0.11). Conclusions: This study shows that machine learning can be used as a tool to identify features of importance for length of postoperative stay in patients undergoing patellar tendon repair. RF was found to be a better performing model than logistic regression at determining patients predisposed to longer length of stay as determined by AUC. This supported the study's hypothesis that ML can provide an effective method for identifying features of importance in patients requiring a longer postoperative stay after patellar tendon repair.
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BACKGROUND: This study focused on using deep learning neural networks to classify the severity of osteoarthritis in the knee. A continuous regression score of osteoarthritis severity has yet to be explored using artificial intelligence machine learning, which could offer a more nuanced assessment of osteoarthritis. MATERIALS AND METHODS: This study used 8260 radiographic images from The Osteoarthritis Initiative to develop and assess four neural network models (VGG16, EfficientNetV2 small, ResNet34, and DenseNet196). Each model generated a regressor score of the osteoarthritis severity based on Kellgren-Lawrence grading scale criteria. Primary performance outcomes assessed were area under the curve (AUC), accuracy, and mean absolute error (MAE) for each model. Secondary outcomes evaluated were precision, recall, and F-1 score. RESULTS: The EfficientNet model architecture yielded the strongest AUC (0.83), accuracy (71%), and MAE (0.42) compared with VGG16 (AUC: 0.74; accuracy: 57%; MAE: 0.54), ResNet34 (AUC: 0.76; accuracy: 60%; MAE: 0.53), and DenseNet196 (AUC: 0.78; accuracy: 62%; MAE: 0.49). CONCLUSION: Convolutional neural networks offer an automated and accurate way to quickly assess and diagnose knee radiographs for osteoarthritis. The regression score models evaluated in this study demonstrated superior AUC, accuracy, and MAE compared with standard convolutional neural network models. The EfficientNet model exhibited the best overall performance, including the highest AUC (0.83) noted in the literature. The artificial intelligence-generated regressor exhibits a finer progression of knee osteoarthritis by quantifying severity of various hallmark features. Potential applications for this technology include its use as a screening tool in determining patient suitability for orthopedic referral. [Orthopedics. 202x;4x(x):xx-xx.].
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Introduction Orthopedics has a reputation for being competitive but not diverse. Leaders of the orthopedics community have been making efforts to increase the participation of women and minorities in the field by raising awareness and strengthening pipeline programs. We aim to explore the trends in the risk of not matching by comparing the proportions of women and underrepresented populations in the applicant pools versus proportions in residency programs. Simultaneously, we aim to evaluate if women or underrepresented population applicants exhibit a lower likelihood of applying to orthopedics compared to male and White applicants. Methods The study received an IRB exemption. The authors collected Accreditation Council for Graduate Medical Education (ACGME) data books for the years 2015-2016 to 2022-2023 to obtain demographic information on orthopedic residents in training during each of those academic years. The pool of corresponding applicants (for example: residents in training during the 2021-2022 academic year would consist of five classes, made up of applicants from 2016-2017 to 2020-2021) was then tabulated from Electronic Residency Applications Service (ERAS) statistics, which are publicly available on the Association of American Medical Colleges (AAMC) website. The race and gender composition of the applicant pool was compared to that of corresponding enrolled residents to calculate the relative risk (RR) of women not matching compared to men and underrepresented population applicants (Blacks, Hispanics, Asians, and Native Americans) compared to White applicants. ERAS data was subsequently used to calculate the percentages of each demographic applying to all residency programs and orthopedic programs. Results For female applicants into orthopedic residency, they had a similar RR of going unmatched when compared to their male counterparts. In the academic year 2020-2021, there was an exception to this as women had a slightly higher RR of going unmatched. All underrepresented populations had a higher risk of not matching compared to White applicants for all cycles, peaking for residents in training in 2020-2021. The trendline improved for residents in the following year. Throughout the study, women accounted for 46.61% of applicants applying for any residency; however, they only accounted for 16.98% of applicants applying for orthopedic residency. A similar discrepancy is noted among Asian applicants but not Black or Hispanic applicants. Conclusions Underrepresented populations were increasingly less likely to match into orthopedics relative to White applicants until 2021. In the academic year of 2021-2022, there was an improvement in this trend for all studied underrepresented populations. Although the exact explanation for this is unclear, it is associated with the transition to virtual applicant interactions. The female gender did not appear to be a consistent advantage or disadvantage in the match. Women and Asian applicants were less likely to apply to orthopedics than other specialties.
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Background: Machine learning algorithms are finding increasing use in prediction of surgical outcomes in orthopedics. Random forest is one of such algorithms popular for its relative ease of application and high predictability. In the process of sample classification, algorithms also generate a list of variables most crucial in the sorting process. Total shoulder arthroplasty (TSA) is a common orthopedic procedure after which most patients are discharged home. The authors hypothesized that random forest algorithm would be able to determine most important variables in prediction of nonhome discharge. Methods: Authors filtered the National Surgical Quality iImprovement Program database for patients undergoing elective TSA (Current Procedural Terminology 23472) between 2008 and 2018. Applied exclusion criteria included avascular necrosis, trauma, rheumatoid arthritis, and other inflammatory arthropathies to only include surgeries performed for primary osteoarthritis. Using Python and the scikit-learn package, various machine learning algorithms including random forest were trained based on the sample patients to predict patients who had nonhome discharge (to facility, nursing home, etc.). List of applied variables were then organized in order of feature importance. The algorithms were evaluated based on area under the curve of the receiver operating characteristic, accuracy, recall, and the F-1 score. Results: Application of inclusion and exclusion criteria yielded 18,883 patients undergoing elective TSA, of whom 1813 patients had nonhome discharge. Random forest outperformed other machine learning algorithms and logistic regression based on American Society of Anesthesiologists (ASA) classification. Random forest ranked age, sex, ASA classification, and functional status as the most important variables with feature importance of 0.340, 0.130, 0.126, and 0.120, respectively. Average age of patients going to facility was 76 years, while average age of patients going home was 68 years. 78.1% of patients going to facility were women, while 52.7% of patients going home were. Among patients with nonhome discharge, 80.3% had ASA scores of 3 or 4, while patients going home had 54% of patients with ASA scores 3 or 4. 10.5% of patients going to facility were considered of partially/totally dependent functional status, whereas 1.3% of patients going home were considered partially or totally dependent (P value < .05 for all). Conclusion: Of various algorithms, random forest best predicted discharge destination following TSA. When using random forest to predict nonhome discharge after TSA, age, gender, ASA scores, and functional status were the most important variables. Two patient groups (home discharge, nonhome discharge) were significantly different when it came to age, gender distribution, ASA scores, and functional status.
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BACKGROUND: Spin is a reporting bias that misrepresents research. Ultimately it can affect surgeon decision making and patient care. Anterior cruciate ligament (ACL) reconstruction is common, but debate continues over optimal treatment modalities. PURPOSE: To identify the prevalence of spin in meta-analysis and systematic review abstracts regarding the treatment of ACL injuries with quadriceps tendon graft. STUDY DESIGN: Cross-sectional study. METHODS: Electronic libraries (MEDLINE, Embase, Web of Science, Google Scholar) were searched for meta-analyses and systematic reviews regarding the treatment of ACL tears with quadriceps tendon graft. The 9 most severe types of spin commonly found in abstracts were used as an evaluation tool to assess the articles. Two reviewers each performed a blinded assessment of each article for spin. A third reviewer helped after review was done to address any discrepancies between the original reviewers. Further evaluation included year of publication, number of citations, journal impact factor, and A Measurement Tool to Assess Systematic Reviews 2 (AMSTAR-2) score. RESULTS: The electronic database search resulted in 986 articles, of which 13 met our inclusion criteria. After review, we found that 53.8% (7/13) of the included articles contained 1 of the 9 most severe forms of spin. Of the 13 articles, 15.4% (n = 2) contained 2 types of spin, and 38.5% (n = 5) contained 1 type of spin. No studies contained ≥3 types of spin. Of the types of spin evaluated, the most prevalent (n = 4; 30.8%) was type 3 ("selective reporting of or overemphasis on efficacy outcomes or analysis favoring the beneficial effect of the experimental intervention"). All studies, regardless of the presence of spin, were found to be low or critically low quality according to the AMSTAR-2 assessment. CONCLUSION: This study demonstrated the presence of spin in 53.8% of meta-analysis and systematic review abstracts pertaining to quadriceps tendon graft for ACL reconstruction. Orthopaedic surgeons should learn to recognize spin as they review articles when deciding the treatment course for ACL injuries. Additionally, strict criteria should be considered to reduce the prevalence of spin in orthopaedic literature.
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Background and Objective: Intracranial hemorrhage following spinal surgery is an infrequent but severe complication. Due to its rarity, the etiology, clinical characteristics, and treatment have not yet been fully elucidated. This literature review analyzed the incidence, clinical manifestations, hemorrhage location, current therapeutic strategies, location of operation, and interval time between surgery and bleeding. The objectives of the article were to provide insights for clinicians to promptly identify and prevent potential cases of intracranial hemorrhage. Methods: The authors queried PubMed and Web of Science databases using predefined keywords and included published literature reporting on intracranial hemorrhage after spinal surgery. Relevant case reports, case series, and reviews describing the mechanism of intracranial hemorrhage after spinal surgery and meeting diagnostic criteria for intracranial hemorrhage related to spinal surgery were included. Clinico-demographc data, presentations symptoms, location, index surgery type, and neurological outcomes after brain hemorrhage. Oxford Centre Level of Evidence guidelines was used to evaluate the quality of included studies. Descriptive statistics were used to synthesize the results. Key Content and Findings: A total of 80 publications of level of evidence IV involving 108 patients with median age at diagnosis was 58.5 years (inter-quartile range: 6-85) were analyzed. The incidence of intracranial hemorrhage was 0.08-0.37% among patients who underwent spinal surgery, and this complication occurred predominantly within 48 hours postoperatively. The initial presentation included headache, reduced level of consciousness, dysarthria, nausea, vomiting, hearing loss, blurred vision, neck rigidity, and delayed recovery from anesthesia. More than half (58.3%) of patients improved, while 23.1% still experienced neurological dysfunctions, and 7.4% died. Conclusions: The present study is limited by the levels of evidence of the included studies. There is heterogeneity among cases with respect to patient demographics and medical history. Angiography is critical in assessing the presence and extent of underlying vascular diseases. Intracranial hemorrages may be caused by intraoperative or postoperative cerebrospinal fluid leakage that will lead to intracranial pressure change and induced by intracranial venous or arterial bleeding. The treatment strategies include conservative medical management and surgical treatment. Individualized treatment should be emphasized.
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OBJECTIVES: Spin is a reporting bias that presents the beneficial effect of an experimental treatment as greater than what is found in the results of the study. This bias can result in patient care recommendations that are more subjective than objective. The purpose of this study is to identify the prevalence of spin in meta-analysis and systematic review abstracts regarding treatment of midshaft clavicle fractures. METHODS: Electronic libraries (MEDLINE, Embase, Web of Science, Google Scholar) were systematically searched. Meta-analyses and systematic reviews regarding treatment of midshaft clavicular fractures were analyzed. The nine most severe types of spin commonly found in abstracts were used as an evaluation tool to assess the articles. Other variables analyzed include year of publication, journal impact factor, number of citations, and methodologic quality according to A Measurement Tool to Assess Systematic Reviews (AMSTAR-2). RESULTS: The database search resulted in 401 articles, of which 53 met inclusion criteria. After review, it was found that 52.8% (28/53) of the included articles contained spin within the abstract. Of the nine most severe types of spin found in abstracts, type 3 spin ("selective reporting of or overemphasis on efficacy outcomes or analysis favoring the beneficial effect of the experimental intervention") was found to be the most prevalent 28.3% (15/53). CONCLUSION: This study demonstrated the presence of spin in the majority of meta-analyses and systematic review abstracts pertaining to midshaft clavicular fractures. Orthopedic surgeons should be aware and recognize spin as they review articles when deciding the treatment course for such injuries. LEVEL OF EVIDENCE: Level 3. See Instructions for Authors for a complete description of levels of evidence.
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Background: Determine effects of pre-operative opiate use on anterior cervical discectomy and fusion (ACDF) surgery outcomes. Methods: The study design was a single center retrospective cohort study. Patient records were reviewed from 2013 and 2018 for elective 1 to 2 level ACDF surgeries. Patients were classified as: opiate naive (ON: no history of opiate) use, acute opiate (AO: <6 months preoperatively) use, and chronic opiate (CO: 6-12 months preoperatively) use based on prescription history before surgery. Opiate use was quantified by milligram morphine equivalents (MME) at 6-12 months preop, 0-6 months preop, 0-6 months postop, and 6-12 months postop. Charts were reviewed for American Society of Anesthesiologists (ASA) physical status classification and smoking history. Results: Readmission rates were 9.8% for ON, 9.1% for AO, and 30% for CO (P value <0.05). Average opiate use measured in MME 6-12 months post-surgery was 5.76 for ON, 18.44 for AO, and 39.92 for CO (P value <0.05). Readmission rate between nonsmokers, former smokers, and active smokers was 4.4%, 0%, and 10.8% (P value <0.05) at 30-90 days post-surgery, and 1.1%, 14.5%, and 2.5% (P value <0.05) in the 91 days to 1-year post-surgery. Conclusions: There is statistically significant relationship between CO and higher readmission rates after ACDF. Preoperative opiate use is also associated with increased opiate use 6-12 months after surgery. Smoking history is also associated with increased readmission rates.
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Patients with Adolescent Idiopathic Scoliosis (AIS) have increased prevalence of lower bone mineral density (BMD) compared to healthy adolescents. Our goal was to compare bone density in patients with AIS and a nonaffected control group using an MRI-based vertebral bone quality (VBQ) score. We reviewed 50 consecutive lumbar MRIs of patients with AIS and of 50 nonaffected controls that were matched for age and sex. The VBQ score was calculated and compared between the groups. The VBQ score for the AIS group was 2.5 (SD 0.4) and for the control group 2.1 (SD 0.3) (p < 0.001). Multiple linear regression analyses were performed for control of confounding from body weight, body height, corrected BMI, and Cobb angle. Besides the diagnosis of AIS, there was no variable that was independently associated with the VBQ score. This is the first study to evaluate bone density in pediatric patients with AIS. We found a significant difference in the VBQ-score between patients with AIS and nonaffected controls matched for sex, age, and race. MRI can be used to assess the bone quality in patients with adolescent idiopathic scoliosis.
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Cifose , Escoliose , Adolescente , Densidade Óssea , Criança , Humanos , Imageamento por Ressonância Magnética , Coluna VertebralRESUMO
PURPOSE: To assess the intra- and inter-observer reliability of the novel vertebral bone quality (VBQ) scoring system. METHODS: Four orthopedic surgery residents at various levels of training (PGY1-4) evaluated 100 noncontrast, T1-weighted MRIs of the lumbar spine. VBQ was calculated as quotient of the median of L1-L4 average signal intensity (SI) and the L3 cerebral spinal fluid (CSF) SI, as described by Ehresman et al. All measurements were repeated 2 weeks later. We performed a stratified analysis based on patient history of instrumentation, pathology, and MRI manufacturer/magnet strength to determine their effect on VBQ reliability. Spinal pathologies included compression fracture, burst fracture, vertebral osteomyelitis, epidural abscess, or neoplasm. The interclass correlation coefficient (ICC) two-way mixed model on absolute agreement was used to analyze inter-rater and intra-rater reliability. ICC less than 0.40 was considered poor, 0.40-0.59 as fair, 0.60-0.74 as good, and greater than 0.75 as excellent. RESULTS: Intra-observer reliability was excellent (≥ 0.75) for all four observers. When stratified by history of spinal instrumentation or spinal pathology, all raters showed excellent intra-observer reliability except one (0.71 and 0.69, respectively). When stratified by MRI manufacturer, intra-observer reliability was ≥ 0.75 for all raters. Inter-observer reliability was excellent (0.91) and remained excellent (≥ 0.75) when stratified by history of spinal instrumentation, spinal pathology, or MRI-manufacturer. CONCLUSIONS: VBQ scores from spine lumbar MRIs demonstrate excellent intra-observer and inter-observer reliability. These scores are reliably reproduced in patients regardless of previous instrumentation, spinal pathology, or MRI manufacturer/magnetic field strength.
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Fraturas por Compressão , Vértebras Lombares , Humanos , Vértebras Lombares/diagnóstico por imagem , Região Lombossacral , Imageamento por Ressonância Magnética , Variações Dependentes do Observador , Reprodutibilidade dos TestesRESUMO
BACKGROUND: Renal transplantation improves long-term outcomes in patients with end-stage renal disease (ESRD); however, patients with impaired left ventricular ejection fraction (LVEF) are less likely to be selected for renal transplantation. We sought to evaluate the effect of renal transplantation in this population. METHODS: We retrospectively evaluated 181 patients who underwent renal transplantation between 2011 and 2016. For patients with pretransplant LVEF <50% (cohort 1) and ≥50% (cohort 2), we evaluated the effect of renal transplantation on LVEF, graft failure, and mortality. RESULTS: Cohort 1 comprised 24 patients (mean age, 47 years; pretransplant LVEF 38%). Cohort 2 comprised 157 patients (mean age, 53 years; pretransplant LVEF 64%). Forty-six percent of cohort 1 experienced significant improvement in LVEF posttransplant, with mean LVEF improvement from 38% to 66%. There was no significant association between pretransplant LVEF and graft failure (hazard ratio [HR] = 2.7; 95% confidence interval [CI], 0.6-11.4; P = .1) or mortality (HR = 1.02; 95% CI, 0.3-3.6; P = .9). Coronary artery disease predicted mortality (HR = 3.12; 95% CI, 1.2-8.4; P = .02). Older age trended toward higher mortality (HR = 1.04; 95% CI, 1.0-1.1; P = .05). Younger age predicted graft failure (HR = 0.96; 95% CI, 0.8-0.9; P = .02). CONCLUSIONS: In patients with ESRD undergoing renal transplantation, there was no significant association between pretransplant LVEF and mortality or graft failure, suggesting that patients with ESRD with impaired LVEF can experience positive posttransplant outcomes.
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Transplante de Rim , Disfunção Ventricular Esquerda , Função Ventricular Esquerda , Doença da Artéria Coronariana , Insuficiência Cardíaca , Humanos , Transplante de Rim/efeitos adversos , Pessoa de Meia-Idade , Estudos Retrospectivos , Volume SistólicoRESUMO
Undergraduate students participating in the UCLA Undergraduate Research Consortium for Functional Genomics (URCFG) have conducted a two-phased screen using RNA interference (RNAi) in combination with fluorescent reporter proteins to identify genes important for hematopoiesis in Drosophila. This screen disrupted the function of approximately 3500 genes and identified 137 candidate genes for which loss of function leads to observable changes in the hematopoietic development. Targeting RNAi to maturing, progenitor, and regulatory cell types identified key subsets that either limit or promote blood cell maturation. Bioinformatic analysis reveals gene enrichment in several previously uncharacterized areas, including RNA processing and export and vesicular trafficking. Lastly, the participation of students in this course-based undergraduate research experience (CURE) correlated with increased learning gains across several areas, as well as increased STEM retention, indicating that authentic, student-driven research in the form of a CURE represents an impactful and enriching pedagogical approach.
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Drosophila , Genômica/educação , Universidades , Animais , Células Sanguíneas , Drosophila/genética , Humanos , EstudantesRESUMO
The performance of three research-use-only, dual HIV and syphilis rapid diagnostic tests (RDTs) was evaluated for 150 patient serum samples and compared to reference HIV and Treponema pallidum antibody detection methods. The RDTs performed comparably, with sensitivities of 93 to 99% and specificities of 97 to 100%. The kappa statistic between the RDTs was 0.95.
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Anticorpos Antibacterianos/sangue , Testes Diagnósticos de Rotina/métodos , Anticorpos Anti-HIV/sangue , Infecções por HIV/diagnóstico , Sífilis/diagnóstico , Adulto , Feminino , HIV/imunologia , Infecções por HIV/virologia , Humanos , Imunoensaio/métodos , Masculino , Sensibilidade e Especificidade , Sífilis/microbiologia , Treponema pallidum/imunologiaRESUMO
Polycrystalline Er3+/Yb3+ co-doped CaMoO4 (CaMoO4:Er3+/Yb3+) film was successfully fabricated by a spray coating method. Crystal structure, surface morphology and upconversion (UC) luminescent properties were investigated. Under 980-nm excitation, CaMoO4:Er3+/Yb3+ film exhibited strong green UC emissions at 530 and 550 nm (2H,11/2 --> 4S3/2 - 4I15/2) visible to the naked eye with a weak red emission near 660 nm (4F9/2 --> 4I15/2) corresponding to the intra 4f transitions of Er3+. A possible UC mechanism related to the pump-power dependence is discussed in detail.
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Cálcio/química , Érbio/química , Medições Luminescentes , Molibdênio/química , Oxigênio/química , Itérbio/química , Propriedades de SuperfícieRESUMO
A green lighting upconversion (UC) system was successfully achieved from Er3+/Yb3+ co-doped BaMoO4 synthesized by the complex citrate-gel method. Under 980 nm laser excitation, the Er3+/Yb3+ co-doped BaMoO4 emitted strong green luminescence around 530 and 550 nm and weak red luminescence near 660 nm, which corresponded to the intra 4f-4f transitions in Er3+. Optimal doping concentrations of Er3+/Yb3+ into the BaMoO4 matrix were investigated. Moreover, based on excitation power dependence, the UC luminescent mechanism in the Er3+/Yb3+ co-doped BaMoO4 was presented in detail.
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Botulinum neurotoxin serotype B (BoNT/B)-specific Fab was expressed in a cell-free protein synthesis system derived from an E. coli extract. The cell-free synthesized antibody fragment was found to be effective in neutralizing the toxicity of BoNT/B in animal studies. Expression of functional Fab required an appropriately controlled and stably maintained redox potential. Under an optimized redox condition, the cell extract, whose disulfide reducing activity had been exhausted, could generate bio-functional Fab molecules. Use of a cell extract enriched with molecular chaperones (GroEL/ES) and disulfide bond isomerases were effective in obtaining larger quantities of functional Fab. Under the optimized reaction conditions, approximately 30 microg of functional Fab was obtained after purification from 1 mL reaction mixture.
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Anticorpos Monoclonais/biossíntese , Fragmentos Fab das Imunoglobulinas/biossíntese , Proteínas Recombinantes/biossíntese , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Toxinas Botulínicas/imunologia , Toxinas Botulínicas Tipo A , Sistema Livre de Células , Escherichia coli/metabolismo , Fragmentos Fab das Imunoglobulinas/genética , Fragmentos Fab das Imunoglobulinas/imunologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologiaRESUMO
Major vault protein (MVP) represents the main component of vaults and has been linked to multi-drug resistance (MDR) in cancer cells. We previously reported that MVP plays an important role in the resistance of senescent human diploid fibroblasts (HDFs) to apoptosis and also that MVP expression is markedly reduced in young HDFs but not in senescent HDFs. In this study, designed to elucidate the regulation of MVP in young and senescent HDFs, we examined the levels of transcriptional factors for the MVP gene, which revealed that among the putative transcriptional factors, p53 decreased only in young HDFs, but not in senescent HDFs in response to H(2)O(2) treatment in the same mode as the expression of MVP. Moreover, the phosphorylation status of p53 increased only in senescent HDFs but not in young HDFs in response to H(2)O(2) treatment. Therefore, we tested the possibility of MVP regulation by p53 status. MVP is upregulated in p53 over-expressing young HDFs, while MVP is downregulated in p53-specific small interfering RNA (siRNA)-transfected senescent HDFs, which suggests that the expression of MVP would be p53 dependent. Furthermore, using chromatin immunoprecipitation (ChIP) assay, we observed that p53 binds directly to the MVP promoter. Taken together, these results suggest that p53 would be a major transcriptional factor for MVP gene expression.
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Senescência Celular , Proteína Supressora de Tumor p53/metabolismo , Partículas de Ribonucleoproteínas em Forma de Abóbada/metabolismo , Apoptose , Diploide , Fibroblastos/metabolismo , Humanos , Peróxido de Hidrogênio/farmacologia , RNA Interferente Pequeno/metabolismo , Transcrição Gênica , Proteína Supressora de Tumor p53/genética , Partículas de Ribonucleoproteínas em Forma de Abóbada/genéticaRESUMO
Although the apoptosis of chondrocytes plays an important role in endochondral ossification, its mechanism has not been elucidated. In this study, we show that guanosine induces chondrocyte apoptosis based on the results of acridine orange/ethidium bromide staining, caspase-3 activation, and sub-G1 fraction analysis. The potent inhibitory effect of dipyridamole, a nucleoside transporter blocker, indicates that extracellular guanosine must enter the chondrocytes to induce apoptosis. We found that guanosine promotes Fas-Fas ligand interaction which, in turn, leads to chondrocyte apoptosis. These findings indicate a novel mechanism for endochondral ossification via metabolic regulation.