A novel approach to sequence validating protein expression clones with automated decision making

<p>Abstract</p> <p>Background</p> <p>Whereas the molecular assembly of protein expression clones is readily automated and routinely accomplished in high throughput, sequence verification of these clones is still largely performed manually, an arduous and time consuming process. The ultimate goal of validation is to determine if a given plasmid clone matches its reference sequence sufficiently to be "acceptable" for use in protein expression experiments. Given the accelerating increase in availability of tens of thousands of unverified clones, there is a strong demand for rapid, efficient and accurate software that automates clone validation.</p> <p>Results</p> <p>We have developed an Automated Clone Evaluation (ACE) system – the first comprehensive, multi-platform, web-based plasmid sequence verification software package. ACE automates the clone verification process by defining each clone sequence as a list of multidimensional discrepancy objects, each describing a difference between the clone and its expected sequence including the resulting polypeptide consequences. To evaluate clones automatically, this list can be compared against user acceptance criteria that specify the allowable number of discrepancies of each type. This strategy allows users to re-evaluate the same set of clones against different acceptance criteria as needed for use in other experiments. ACE manages the entire sequence validation process including contig management, identifying and annotating discrepancies, determining if discrepancies correspond to polymorphisms and clone finishing. Designed to manage thousands of clones simultaneously, ACE maintains a relational database to store information about clones at various completion stages, project processing parameters and acceptance criteria. In a direct comparison, the automated analysis by ACE took less time and was more accurate than a manual analysis of a 93 gene clone set.</p> <p>Conclusion</p> <p>ACE was designed to facilitate high throughput clone sequence verification projects. The software has been used successfully to evaluate more than 55,000 clones at the Harvard Institute of Proteomics. The software dramatically reduced the amount of time and labor required to evaluate clone sequences and decreased the number of missed sequence discrepancies, which commonly occur during manual evaluation. In addition, ACE helped to reduce the number of sequencing reads needed to achieve adequate coverage for making decisions on clones.</p

Dementia and delirium, the outcomes in elderly hip fracture patients

Background: Delirium in hip fractured patients is a frequent complication. Dementia is an important risk factor for delirium and is common in frail elderly. This study aimed to extend the previous knowledge on risk factors for delirium and the consequences. Special attention was given to patients with dementia and delirium. Methods: This is a retrospective cohort study performed in the Amphia Hospital, Breda, the Netherlands. A full electronic patient file system (Hyperspace Version IU4: Epic, Inc., Verona, WI, USA) was used to assess data between January 2014 and September 2015. All patients presented were aged ≥70 years with a hip fracture, who underwent surgery with osteosynthesis or arthroplasty. Patients were excluded in case of a pathological or a periprosthetic hip fracture, multiple traumatic injuries, and high-energy trauma. Patient and surgical characteristics were documented. Postoperative outcomes were noted. Delirium was screened using Delirium Observation Screening Scale and dementia was assessed from medical notes. Results: Of a total of 566 included patients, 75% were females. The median age was 84 years (interquartile range: 9). Delirium was observed in 35%. Significant risk factors for delirium were a high American Society of Anesthesiology score, delirium in medical history, functional dependency, preoperative institutionalization, low hemoglobin level, and high amount of blood transfusion. Delirium was correlated with a longer hospital stay (P=0.001), increased association with comp

Formation and Shaping of the Antirrhinum Flower through Modulation of the CUP Boundary Gene

Boundary domain genes, expressed within or around organ primordia, play a key role in the formation, shaping, and subdivision of planar plant organs, such as leaves. However, the role of boundary genes in formation of more elaborate 3D structures, which also derive from organ primordia, remains unclear. Here we analyze the role of the boundary domain gene CUPULIFORMIS (CUP) in formation of the ornate Antirrhinum flower shape. We show that CUP expression becomes cleared from boundary subdomains between petal primordia, most likely contributing to formation of congenitally fused petals (sympetally) and modulation of growth at sinuses. At later stages, CUP is activated by dorsoventral genes in an intermediary region of the corolla. In contrast to its role at organ boundaries, intermediary CUP activity leads to growth promotion rather than repression and formation of the palate, lip, and characteristic folds of the closed Antirrhinum flower. Intermediary expression of CUP homologs is also observed in related sympetalous species, Linaria and Mimulus, suggesting that changes in boundary gene activity have played a key role in the development and evolution of diverse 3D plant shapes

Magnetic resonance imaging findings in 46 elbows with a radial head fracture

Radial head fractures are common, and may be associated with other injuries of clinical importance. We present the results of a standard additional MRI scan for patients with a radial head fracture. PATIENTS AND METhods: 44 patients (mean age 47 years) with 46 radial head fractures underwent MRI. 17 elbows had a Mason type-I fracture, 23 a Mason type-II fracture, and 6 elbows had a Mason type-III fracture. Associated injuries were found in 35 elbows: 28 elbows had a lateral collateral ligament lesion, 18 had capitellar injury, 1 had a coronoid fracture, and 1 elbow had medial collateral ligament injury. The incidence of associated injuries with radial head fractures found with MRI was high. The clinical relevance should be investigate

A randomized controlled trial of nonoperative treatment versus open reduction and internal fixation for stable, displaced, partial articular fractures of the radial head: The RAMBO trial

Background: The choice between operative or nonoperative treatment is questioned for partial articular fractures of the radial head that have at least 2 millimeters of articular step-off on at least one radiograph (defined as displaced), but less than 2 millimeter of gap between the fragments (defined as stable) and that are not associated with an elbow dislocation, interosseous ligament injury, or other fractures. These kinds of fractures are often classified as Mason type-2 fractures. Retrospective comparative studies suggest that operative treatment might be better than nonoperative treatment, but the long-term results of nonoperative treatment are very good. Most experts agree that problems like reduced range of motion, painful crepitation, nonunion or bony ankylosis are infrequent with both nonoperative and operative treatment of an isolated displaced partial articular fracture of the radial head, but determining which patients will have problems is difficult. A prospective, randomized comparison would help minimize bias and determine the balance between operative and nonoperative risks and benefits. Methods/Design. The RAMBO trial (Radial Head - Amsterdam - Amphia - Boston - Others) is an international prospective, randomized, multicenter trial. The primary objective of this study is to compare patient related outcome defined by the \u27Disabilities of Arm, Shoulder and Hand (DASH) score\u27 twelve months after injury between operative and nonoperative treated patients. Adult patients with partial articular fractures of the radial head that comprise at least 1/3rd of the articular surface, have ≥ 2 millimeters of articular step-off but less than 2 millimeter of gap between the fragments will be enrolled. Secondary outcome measures will be the Mayo Elbow Performance Index (MEPI), the Oxford Elbow Score (OES), pain intensity through the \u27Numeric Rating Scale\u27, range of motion (flexion arc and rotational arc), radiographic appearance of the fracture (heterotopic ossification, radiocapitellar and ulnohumeral arthrosis, fracture healing, and signs of implant loosening or breakage) and adverse events (infection, nerve injury, secondary interventions) after one year. Discussion. The successful completion of this trial will provide evidence on the best treatment for stable, displaced, partial articular fractures of the radial head. Trial registration. The trial is registered at the Dutch Trial Register: NTR3413. © 2014Bruinsma et al.; licensee BioMed Central Ltd

A randomized controlled trial of nonoperative treatment versus open reduction and internal fixation for stable, displaced, partial articular fractures of the radial head: The RAMBO trial

Background: The choice between operative or nonoperative treatment is questioned for partial articular fractures of the radial head that have at least 2 millimeters of articular step-off on at least one radiograph (defined as displaced), but less than 2 millimeter of gap between the fragments (defined as stable) and that are not associated with an elbow dislocation, interosseous ligament injury, or other fractures. These kinds of fractures are often classified as Mason type-2 fractures. Retrospective comparative studies suggest that operative treatment might be better than nonoperative treatment, but the long-term results of nonoperative treatment are very good. Most experts agree that problems like reduced range of motion, painful crepitation, nonunion or bony ankylosis are infrequent with both nonoperative and operative treatment of an isolated displaced partial articular fracture of the radial head, but determining which patients will have problems is difficult. A prospective, randomized comparison would help minimize bias and determine the balance between operative and nonoperative risks and benefits. Methods/Design. The RAMBO trial (Radial Head - Amsterdam - Amphia - Boston - Others) is an international prospective, randomized, multicenter trial. The primary objective of this study is to compare patient related outcome defined by the 'Disabilities of Arm, Shoulder and Hand (DASH) score' twelve months after injury between operative and nonoperative treated patients. Adult patients with partial articular fractures of the radial head that comprise at least 1/3rd of the articular surface, have ≥ 2 millimeters of articular step-off but less than 2 millimeter of gap between the fragments will be enrolled. Secondary outcome measures will be the Mayo Elbow Performance Index (MEPI), the Oxford Elbow Score (OES), pain intensity through the 'Numeric Rating Scale', range of motion (flexion arc and rotational arc), radiographic appearance of the fracture (heterotopic ossification, radiocapitellar and ulnohumeral arthrosis, fracture healing, and signs of implant loosening or breakage) and adverse events (infection, nerve injury, secondary interventions) after one year. Discussion. The successful completion of this trial will provide evidence on the best treatment for stable, displaced, partial articular fractures of the radial head. Trial registration. The trial is registered at the Dutch Trial Register: NTR3413

A hinged external fixator for complex elbow dislocations: A multicenter prospective cohort study

Background: Elbow dislocations can be classified as simple or complex. Simple dislocations are characterized by the absence of fractures, while complex dislocations are associated with fractures of the radial head, olecranon, or coronoid process. The majority of patients with these complex dislocations are treated with open reduction and internal fixation (ORIF), or arthroplasty in case of a non-reconstructable radial head fracture. If the elbow joint remains unstable after fracture fixation, a hinged elbow fixator can be applied. The fixator provides stability to the elbow joint, and allows for early mobilization. The latter may be important for preventing stiffness of the joint. The aim of this study is to determine the effect of early mobilization with a hinged external elbow fixator on clinical outcome in patients with complex elbow dislocations with residual instability following fracture fixation. Methods/Design. The design of the study will be a multicenter prospective cohort study of 30 patients who have sustained a complex elbow dislocation and are treated with a hinged elbow fixator following fracture fixation because of residual instability. Early active motion exercises within the limits of pain will be started immediately after surgery under supervision of a physical therapist. Outcome will be evaluated at regular intervals over the subsequent 12 months. The primary outcome is the Quick Disabilities of the Arm, Shoulder, and Hand score. The secondary outcome measures are the Mayo Elbow Performance Index, Oxford Elbow Score, pain level at both sides, range of motion of the elbow joint at both sides, radiographic healing of the fractures and formation of periarticular ossifications, rate of secondary interventions and complications, and health-related quality of life (Short-Form 36). Discussion. The outcome of this study will yield quantitative data on the functional outcome in patients with a complex elbow dislocation and who are treated with ORIF and additional stabilization with a hinged elbow fixator. Trial Registration. The trial is registered at the Netherlands Trial Register (NTR1996)

Derivation of High Purity Neuronal Progenitors from Human Embryonic Stem Cells

The availability of human neuronal progenitors (hNPs) in high purity would greatly facilitate neuronal drug discovery and developmental studies, as well as cell replacement strategies for neurodegenerative diseases and conditions, such as spinal cord injury, stroke, Parkinson's disease, Alzheimer's disease, and Huntington's disease. Here we describe for the first time a method for producing hNPs in large quantity and high purity from human embryonic stem cells (hESCs) in feeder-free conditions, without the use of exogenous noggin, sonic hedgehog or analogs, rendering the process clinically compliant. The resulting population displays characteristic neuronal-specific markers. When allowed to spontaneously differentiate into neuronal subtypes in vitro, cholinergic, serotonergic, dopaminergic and/or noradrenergic, and medium spiny striatal neurons were observed. When transplanted into the injured spinal cord the hNPs survived, integrated into host tissue, and matured into a variety of neuronal subtypes. Our method of deriving neuronal progenitors from hESCs renders the process amenable to therapeutic and commercial use