Development of a Machine Learning Algorithm to Correlate Lumbar Disc Height on X-rays with Disc Bulging or Herniation.

Lumbar disc bulging or herniation (LDBH) is one of the major causes of spinal stenosis and related nerve compression, and its severity is the major determinant for spine surgery. MRI of the spine is the most important diagnostic tool for evaluating the need for surgical intervention in patients with LDBH. However, MRI utilization is limited by its low accessibility. Spinal X-rays can rapidly provide information on the bony structure of the patient. Our study aimed to identify the factors associated with LDBH, including disc height, and establish a clinical diagnostic tool to support its diagnosis based on lumbar X-ray findings. In this study, a total of 458 patients were used for analysis and 13 clinical and imaging variables were collected. Five machine-learning (ML) methods, including LASSO regression, MARS, decision tree, random forest, and extreme gradient boosting, were applied and integrated to identify important variables for predicting LDBH from lumbar spine X-rays. The results showed L4-5 posterior disc height, age, and L1-2 anterior disc height to be the top predictors, and a decision tree algorithm was constructed to support clinical decision-making. Our study highlights the potential of ML-based decision tools for surgeons and emphasizes the importance of L1-2 disc height in relation to LDBH. Future research will expand on these findings to develop a more comprehensive decision-supporting model.

Improving Antibody Production in Stably Transfected CHO Cells by CRISPR-Cas9-Mediated Inactivation of Genes Identified in a Large-Scale Screen with Chinese Hamster-Specific siRNAs.

The Chinese hamster ovary (CHO) cell line is commonly used for the production of biotherapeutics. As cell productivity directly affects the cost of production, methods are developed to manipulate the expression of specific genes that are known to be involved in protein synthesis, folding, and secretion to increase productivity. However, there are no large-scale CHO-specific functional screens to identify novel gene targets that impact the production of secreted recombinant proteins. Here, a large-scale, CHO cell-specific small interfering RNA screen is performed to identify genes that consistently enhance antibody production when silenced in a panel of seven CHO cell lines. Four genes, namely, Cyp1a2, Atp5s, Dgki, and P3h2, are identified, and then selected for CRISPR-Cas9 knockout validation in recombinant CHO cell lines. Single knockout of Cyp1a2, Atp5s, or Dgki, but not P3h2, results in a more than 90% increase in specific antibody productivity. Overall, the knockout of Cyp1a2 demonstrates the most significant improvement of antibody production, with a minimal impact on cell growth.

Attenuated glutamine synthetase as a selection marker in CHO cells to efficiently isolate highly productive stable cells for the production of antibodies and other biologics.

Chinese hamster ovary (CHO) cells are the biopharmaceutical industry's primary means of manufacturing therapeutic proteins, including monoclonal antibodies. The major challenge in cell line development for the production of recombinant biopharmaceuticals lies in generating and isolating rare high-producing stable clones, amongst thousands of low-producing or unstable clones, in a short period of time. One approach to accomplish this is to use the glutamine synthetase (GS) selection system, together with the GS inhibitor, methionine sulfoximine (MSX). However, MSX can only increase protein productivity to a limited extent. Often productivity will drop when MSX is removed from the system. We evaluated a congenital GS mutation, R324C, which causes glutamine deficiency in human as an attenuated selection marker for CHO cell line generation. We also created a panel of GS mutants with diminished GS activity. Our results demonstrated that using attenuated GS mutants as selection markers significantly increased antibody production of stably transfected pools. Furthermore, these stably transfected pools sustained high productivity levels for an extended period of time, whereas cells transfected with wild-type GS lost considerable protein productivity over time, particularly after MSX was removed. In summary, the use of attenuated GS as a selection marker in CHO cell line development bypasses the need for MSX, and generates stable clones with significantly higher antibody productivity.Abbreviations: CHO: Chinese hamster ovary; CMV: Cytomegalovirus; DHFR: Dihydrofolate reductase; GFP: Green fluorescent protein; GOI: gene-of-interest; GS: Glutamine synthetase; IRES: internal ribosomal entry site; MSX: Methionine sulfoximine; MTX: Methotrexate; psGS: pseudoGS; RVDs: Repeated variable di-residues; TALENs: transcription activator-like effector nucleases; VCD: Viable cell density; ZFNs: zinc finger nucleases.

The "less-is-more" in therapeutic antibodies: Afucosylated anti-cancer antibodies with enhanced antibody-dependent cellular cytotoxicity.

Therapeutic monoclonal antibodies are the fastest growing class of biological therapeutics for the treatment of various cancers and inflammatory disorders. In cancer immunotherapy, some IgG1 antibodies rely on the Fc-mediated immune effector function, antibody-dependent cellular cytotoxicity (ADCC), as the major mode of action to deplete tumor cells. It is well-known that this effector function is modulated by the N-linked glycosylation in the Fc region of the antibody. In particular, absence of core fucose on the Fc N-glycan has been shown to increase IgG1 Fc binding affinity to the FcγRIIIa present on immune effector cells such as natural killer cells and lead to enhanced ADCC activity. As such, various strategies have focused on producing afucosylated antibodies to improve therapeutic efficacy. This review discusses the relevance of antibody core fucosylation to ADCC, different strategies to produce afucosylated antibodies, and an update of afucosylated antibody drugs currently undergoing clinical trials as well as those that have been approved.

Acute Infarction in the Artery of Percheron Distribution during Cerebral Angiography: A Case Report and Literature Review.

Improvements in techniques, contrast agents, and catheter design have significantly decreased angiography-related neurological deficits and complications. This article reports a case involving an angiographic total obliteration arteriovenous malformation (AVM) in a patient with an acute infarction in the artery of Percheron (AOP) distribution following angiography. Furthermore, imaging of an AOP acute infarction in cerebral angiography is presented.

Comprehensive MDCT evaluation of patients with suspected May-Thurner syndrome.

OBJECTIVE: The purpose of this essay is to introduce the MDCT protocol and interpretation techniques for optimal evaluation of patients with suspected May-Thurner syndrome. CONCLUSION: May-Thurner syndrome is always the working diagnosis when a patient presents with unilateral left lower limb swelling without signs of infection. MDCT is useful for fast, comprehensive evaluation of the vascular system to determine whether May-Thurner syndrome or an alternative condition is present.

Collapsin response mediator proteins (CRMPs) are a new class of microtubule-associated protein (MAP) that selectively interacts with assembled microtubules via a taxol-sensitive binding interaction.

Collapsin response mediator proteins are ubiquitously expressed from multiple genes (CRMPs 1-5) and play important roles in dividing cells and during semaphorin 3A (Sema3A) signaling. Nonetheless, their mode of action remains opaque. Here we carried out in vivo and in vitro assays that demonstrate that CRMPs are a new class of microtubule-associated protein (MAP). In experiments with CRMP1 or CRMP2 and their derivatives, only the C-terminal region (residues 490-572) mediated microtubule binding. The in vivo microtubule association of CRMPs was abolished by taxol or epothilone B, which is highly unusual. CRMP2-depleted cells exhibited destabilized anaphase astral microtubules and altered spindle position. In a cell-based assay, all CRMPs stabilized interphase microtubules against nocodazole-mediated depolymerization, with CRMP1 being the most potent. Remarkably, a 82-residue C-terminal region of CRMP1 or CRMP2, unrelated to other microtubule binding motifs, is sufficient to stabilize microtubules. In cells, we demonstrate that glycogen synthase kinase-3ß (GSK3ß) inhibition potentiates this activity. Thus, CRMPs are a new class of MAP that binds through a unique motif, but in common with others such as Tau, is antagonized by GSK3ß. This regulation is consistent with such kinases being critical for the Sema3A (collapsin) pathway. These findings have implications for cancer and neurodegeneration.

Comprehensive evaluation of patients suspected with deep vein thrombosis using indirect CT venography with multi-detector row technology: from protocol to interpretation.

With the increased availability of multi-detector row CT, indirect CT venography has become an important non-invasive image modality for patients with suspected deep vein thrombosis. Use of indirect CT venography can not only diagnose/exclude deep vein thrombosis, but can also determine if there are other anomalies or diseases which might contribute to the patient's symptoms. In this pictorial essay, we introduce the scanning protocol, post-processing techniques, and interpretation algorithm used in widely available 64 multi-detector row technology. We discuss several cases, including deep vein thrombosis in acute and chronic stages, anatomic variation, vena cava filter, and collateral veins. Lastly, we consider alternative diagnoses including varicose veins, infection, prosthesis failure of arthroplasty, traumatic vessel injury, and other musculoskeletal conditions. Radiologists should be familiar with the comprehensive interpretation of indirect CT venography to facilitate differential diagnosis and further treatment decision.

Arteriovenous fistula and graft evaluation in hemodialysis patients using MDCT: a primer.

OBJECTIVE: Patent arteriovenous fistula (AVF) is related to better prognosis and quality of life for patients on long-term dialysis. When AVF dysfunction is suspected, MDCT is a good noninvasive tool for evaluating the entire AVF structure and determining reversible conditions for treatment. The aim of this article is to introduce the scanning and interpretation techniques and to illustrate the conditions related to early and late fistula failures. CONCLUSION: MDCT is a fast, noninvasive, and accurate technique for diagnosing AVF complications. Radiologists familiar with these techniques can help to improve the prognosis and quality of life for hemodialysis patients.

Comprehensive evaluation of CT pulmonary angiography for patients suspected of having pulmonary embolism.

Recently, CT pulmonary angiography (CTPA), especially performed with multi-detector row CT, has become a key imaging modality for pulmonary embolism. However, CTPA that was performed under clinical suspicion of pulmonary embolism has been shown to lead to high prevalence of alternative diagnosis, up to 25.4%. A comprehensive evaluation of pulmonary and extrapulmonary abnormalities including cardiovascular lesions is critical in proper diagnosis and patient care. Radiologists should be familiar with the comprehensive interpretation of CTPA to facilitate differential diagnosis and further treatment decision.

Comprehensive evaluation of patients with suspected renal hypertension using MDCT: from protocol to interpretation.

OBJECTIVE: The objectives of this article are to, first, describe the reasons for and details of the MDCT protocol for patients with suspected renal hypertension; second, explain the importance of comprehensive evaluation by MDCT in patients with suspected renal hypertension; third, review the image appearances of important conditions that may be encountered in the reader's clinical practice; and, fourth, explain what information should be included in a comprehensive MDCT report for patients with suspected renal hypertension. CONCLUSION: MDCT is widely used for renal artery evaluation in patients with resistant hypertension. Because the regions outside the renal arteries might also have diseases that contribute to the symptoms, a comprehensive interpretation including the renal arteries, renal parenchyma, adrenal glands, and scanned abdomen is very important. The scanning parameters should be adjusted according to the patient's body habitus because some patients with suspected renal hypertension are children or young women. In this article, cases with illustrations showing the process from protocol to interpretation are provided.

MDCT evaluation of congenital mitral-aortic intervalvular fibrosa aneurysm: implications for the aetiology and differential diagnosis.

Mitral-aortic intervalvular fibrosa aneurysm is a rare disease whose aetiology remains a matter of debate. Here we present the youngest reported patient with the disease, a 6-month-old boy, without a history of infection, which supports a congenital origin as initially proposed. Multidetector-row CT (MDCT) surpassed echocardiography in delineating the intracardiac anatomical details with high spatial resolution, confirming the important problem-solving role of MDCT in the diagnosis of congenital heart disease.

Neonatal cardiac multidetector row CT: why and how we do it.

Neonatal congenital heart disease is a most difficult area of diagnostic radiology because of the small patient body size and fast resting heart rate. Recently, the spatial and temporal resolution of multidetector-row CT (MDCT) has evolved so that neonatal congenital heart disease can be precisely diagnosed. We describe the role of MDCT in neonatal congenital heart disease and offer tips for the scanning procedure to familiarize radiologists with this developing field.

Vertebral and carotid artery anomalies in patients with aberrant right subclavian arteries.

BACKGROUND: There is little published evidence regarding the patterns and prevalence of vertebral artery (VA) and common carotid artery (CCA) anomalies in patients with an aberrant right subclavian artery (ARSCA). OBJECTIVE: To study the patterns and prevalence of VA and CCA anomalies in patients with ARSCA. MATERIALS AND METHODS: In a 2-year period we reviewed the children referred with suspected vascular ring who had undergone multidetector-row CT. Patients with ARSCA were reviewed for VA and CCA patterns and their prevalence and relevance were calculated. RESULTS: In total, 102 patients with ARSCA were identified. VA anomalies were present in 16 patients (15.7%), and CCA anomalies (common carotid trunk) in 21 patients (20.6%). In some patients with VA anomalies, the right VA arose from the right CCA and in some the left VA arose from the aortic arch. When the left VA arose from the aortic arch it was situated between the left CCA and the left SCA or between the left SCA and the ARSCA. CONCLUSION: If neurointerventionalists understand these potential anomalies and their prevalence, time and contrast medium could be saved when catheterizing the VA and CCA in patients with ARSCA.

Homogeneous enhancement in pediatric thoracic CT aortography using a novel and reproducible method: contrast-covering time.

OBJECTIVE: This study compares the empiric setting and contrast-covering time (CCT) concept for i.v. contrast injection in pediatric thoracic CT aortography. SUBJECTS AND METHODS: A total of 113 pediatric patients referred for thoracic CT aortography were classified into four groups: group 1 (1-5 years old, CCT), group 2A (6-10 years old, CCT), group 2B (6-10 years old, empiric setting), and group 3 (11-15 years old, CCT). The CT attenuation values from the left common carotid artery to the descending aorta were recorded every 0.5 second. The quantitative bolus geometric analysis of each group included average enhancement, SD within the patient, and slope of enhancement. Groups 2A and 2B were compared to determine whether better bolus geometry could be obtained with the CCT concept than with the traditional empiric setting. Groups 1, 2A, and 3 were compared to determine whether homogeneous bolus geometry could be obtained in different age groups. RESULTS: More homogeneous enhancement was obtained with the CCT concept than the empiric setting with a smaller SD of enhancement (25.5 +/- 8.5 H vs 49.3 +/- 16.2 H, p < 0.001). Furthermore, in the three different age groups (groups 1, 2A, and 3) examined using the CCT concept, there was no significant difference in the average enhancement (415.7 +/- 83.6 H, 422.8 +/- 97.1 H, 392.0 +/- 78.5 H, respectively; all p > 0.05), SD of enhancement (28.5 +/- 9.8 H, 25.5 +/- 8.5 H, 28.5 +/- 14.6 H, respectively; all p > 0.05), or enhancement slopes (-5.6 +/- 18.0 H, -2.7 +/- 10.7 H, -5.4 +/- 12.3 H, respectively; all p > 0.05). CONCLUSION: The CCT concept yields more homogeneous enhancement than the empiric setting. It also can routinely obtain homogeneous bolus geometry in patients in different age groups.

Kinectin-dependent assembly of translation elongation factor-1 complex on endoplasmic reticulum regulates protein synthesis.

Kinectin is an integral membrane protein with many isoforms primarily found on the endoplasmic reticulum. It has been found to bind kinesin, Rho GTPase, and translation elongation factor-1delta. None of the existing models for the quaternary organization of the elongation factor-1 complex in higher eukaryotes involves kinectin. We have investigated here the assembly of the elongation factor-1 complex onto endoplasmic reticulum via kinectin using in vitro and in vivo assays. We established that the entire elongation factor-1 complex can be anchored to endoplasmic reticulum via kinectin, and the interacting partners are as follows. Kinectin binds EF-1delta, which in turn binds EF-1gamma but not EF-1beta; EF-1gamma binds EF-1delta and EF-1beta but not kinectin. In vivo splice blocking of the kinectin exons 36 and 37 produced kinectin lacking the EF-1delta binding domain, which disrupted the membrane localization of EF-1delta, EF-1gamma, and EF-1beta on endoplasmic reticulum, similar to the disruptions seen with the overexpression of kinectin fragments containing the EF-1delta binding domain. The disruptions of the EF-1delta/kinectin interaction inhibited expression of membrane proteins but enhanced synthesis of cytosolic proteins in vivo. These findings suggest that anchoring the elongation factor-1 complex onto endoplasmic reticulum via EF-1delta/kinectin interaction is important for regulating protein synthesis in eukaryotic cells.

An engineered microenvironment for multidimensional microscopy of live cells.

Multidimensional imaging (MD) of live cells is gaining importance in biomedical research as the commercial availability of confocal, nonlinear optical microscopes, environmental chambers, and specific fluorescence probes grows. One crucial aspect of the MD live cell imaging involves the proper immobilization of cells, which refers to the rapid and sufficient immobilization of cells on the microscope stage, neither disrupting the cellular structure and functions nor affecting the optical properties of the cells and the environments. Conventional cell immobilization methods glue the anchoring cells to coated surfaces, but such methods require centrifugation or extended incubation and are not suitable for cells in suspension. Most of the current three-dimensional (3-D) gels either exhibit unsatisfactory optical properties or have adverse effects on cell functions in culture. Recently, an engineered 3-D microcapsule has been developed that involves the complex coacervation of a positively charged collagen and a negatively charged polymer of 2-hydroxyethyl methacrylate--methacrylic acid--methyl methacrylate (HEMA-MMA-MAA). Hence, confocal imaging of live cells in this engineered 3-D microenvironment was investigated for its optical properties and cellular function compatibility. We report here that this microenvironment facilitates efficient cell immobilization, exhibits good optical properties, and can preserve cellular structures and functions, which will be useful in MD imaging of live cells for various applications.

Engineering microenvironment for expansion of sensitive anchorage-dependent mammalian cells.

Tissue engineering involves ex vivo seeding of anchorage-dependent mammalian cells onto scaffolds, or transplanting cells in vivo. The cell expansion currently requires repeated cell detachment from solid substrata by enzymatic, chemical or mechanical means. The report here presents a high yield three-dimensional culture and harvest system circumventing the conventional detachment requirements. Cells mixed with dilute cationic collagen were microencapsulated within an ultra-thin shell of synthetic polymers. The cationic collagen could rapidly form a conformal layer of collagen fibers around cells to support cell proliferation and functions. The collagen could be readily removed from cells with a buffer rinse after harvesting from the fragile microcapsules. The cells harvested from this system demonstrate improved attachment, morphology and functions over conventionally cultured cells, upon binding to ligand-conjugated polymer surfaces. The harvested cells can be re-encapsulated and allowed to proliferate again, or used immediately in applications.

TGF-beta1 regulation in hepatocyte-NIH3T3 co-culture is important for the enhanced hepatocyte function in 3D microenvironment.

Co-culture of hepatocytes or hepatocyte spheroids with the supporting NIH3T3 in a 3D microcapsule formed with a hybrid natural/synthetic matrix has led to enhanced hepatocyte functions. We investigated the mechanism of the functional enhancement in co-culture with respect to the contributions of soluble factors and direct cell-cell interactions. The conditioned media from the co-culture induced higher P450 cytochrome oxidase activity (indicated by EROD assay) in the microencapsulated hepatocytes than the conditioned media from the NIH3T3- or the hepatocytes-alone controls. Conditioned media from physically separated co-culture of hepatocytes-NIH3T3 by a membrane insert reduced the functional enhancement. Among the known stimulators of hepatocyte functions, TGF(beta)1 is primarily responsible for the stimulation of hepatocyte functions in this 3D co-culture since the removal of TGF(beta)1 by antibody depletion eliminated the functional enhancement and the reconstitution of TGF(beta)1 restored the functional enhancement. Activation of latent TGF(beta)1 in an extracellular environment were upregulated in co-culture with no observable increase in the TGF(beta)1 expression at transcriptional and translational levels. Our data led to an improved understanding of how co-culture enhances hepatocyte functions in vitro and pave the way for further innovations in liver tissue engineering, drug metabolism studies, and other applications that require functional hepatocytes cultured in vitro.