Development of a robotic cluster for automated and scalable cell therapy manufacturing.

BACKGROUND AIMS: The production of commercial autologous cell therapies such as chimeric antigen receptor T cells requires complex manual manufacturing processes. Skilled labor costs and challenges in manufacturing scale-out have contributed to high prices for these products. METHODS: We present a robotic system that uses industry-standard cell therapy manufacturing equipment to automate the steps involved in cell therapy manufacturing. The robotic cluster consists of a robotic arm and customized modules, allowing the robot to manipulate a variety of standard cell therapy instruments and materials such as incubators, bioreactors, and reagent bags. This system enables existing manual manufacturing processes to be rapidly adapted to robotic manufacturing, without having to adopt a completely new technology platform. Proof-of-concept for the robotic cluster's expansion module was demonstrated by expanding human CD8+ T cells. RESULTS: The robotic cultures showed comparable cell yields, viability, and identity to those manually performed. In addition, the robotic system was able to maintain culture sterility. CONCLUSIONS: Such modular robotic solutions may support scale-up and scale-out of cell therapies that are developed using classical manual methods in academic laboratories and biotechnology companies. This approach offers a pathway for overcoming manufacturing challenges associated with manual processes, ultimately contributing to the broader accessibility and affordability for personalized immunotherapies.

ScMiles2: A Script to Conduct and Analyze Milestoning Trajectories for Long Time Dynamics.

Milestoning is a theory and an algorithm that computes kinetics and thermodynamics at long time scales. It is based on partitioning the (phase) space into cells and running a large number of short trajectories between the boundaries of the cells. The termination points of the trajectories are analyzed with the Milestoning theory to obtain kinetic and thermodynamic information. Managing the tens to hundreds of thousands of Milestoning trajectories is a challenge, which we handle with a python script, ScMiles. Here, we introduce a new version of the python script ScMiles2 to conduct Milestoning simulations. Major enhancements are: (i) post analysis of Milestoning trajectories to obtain the free energy, mean first passage time, the committor function, and exit times; (ii) similar to (i) but the post analysis is for a single long trajectory; (iii) we support the use of the GROMACS software in addition to NAMD; (iv) a restart option; (v) the automated finding, sampling, and launching trajectories from new milestones that are found on the fly; and (vi) support Milestoning calculations with several coarse variables and for complex reaction coordinates. We also evaluate the simulation parameters and suggest new algorithmic features to enhance the rate of convergence of observables. We propose the use of an iteration-averaged kinetic matrix for a rapid approach to asymptotic values. Illustrations are provided for small systems and one large example.

Effect of a Nurse-Led Protocol on Time to Treatment of Partial Occlusions in Central Venous Access Devices in Pediatric Oncology.

Central venous access devices (CVADs) are the standard of care in pediatric oncology. Occlusion is a common complication that can lead to delays in therapy, readmission, and CVAD removal and reinsertion. Early treatment of partial occlusions using a standardized protocol may restore patency and increase life span of CVADs. The objective of this study was to develop and evaluate a nurse-led protocol to manage partial CVAD occlusions in pediatric oncology and autologous bone marrow transplant patients. The protocol enabled nurses to manage partially occluded CVADs by administering thrombolytic therapy following an algorithm and patient-specific standing order. The primary outcome was time from recognition of the partial occlusion to instillation of a thrombolytic. Secondary outcomes were thrombolytic dwell time, number of complete occlusions, and CVAD life span. We used a quasi-experimental, after-only, nonequivalent control group design to compare patients not exposed (retrospective cohort, n = 137) and patients exposed (prospective cohort, n = 101) to the nurse-led protocol. Mann-Whitney U tests were used to compare time to treatment, dwell time, and CVAD life span between cohorts, and χ2 was used to compare the proportion of occlusions classified as complete. Time to treatment was significantly lower in the prospective cohort (M = 99.9 minutes) versus the retrospective cohort (M = 483.7 minutes), U = 1366.50, p < .01, as was thrombolytic dwell time, U = 282.50, p < .01. Proportion of complete occlusions and CVAD life span did not differ between cohorts. The nurse-led protocol was effective to manage partial CVAD occlusions in pediatric oncology patients.

Postoperative aspirin use and its effect on bone healing in the treatment of ankle fractures.

BACKGROUND: There is hesitancy to administer nonsteroidal anti-inflammatories (NSAIDs) within the postoperative period following fracture care due to concern for delayed union or nonunion. However, aspirin (ASA) is routinely used for chemoprophylaxis of deep vein thrombosis (DVT) and is gaining popularity for use after treatment of ankle fractures. The current study examines the incidence of nonunion of operative ankle fractures and risk of DVT in patients who did and did not receive postoperative ASA. METHODS: A retrospective chart review was performed on all patients treated between 2008 and 2018 for ankle fractures requiring operative fixation by three Foot and Ankle fellowship trained orthopaedic surgeons at a single institution. Demographics, preoperative comorbidities, and postoperative medical and surgical complications were compared between patients who did and did not receive ASA postoperatively. For both groups, union was evaluated by clinical exam as well as by radiograph, for those with 6-week, 12-week, or 24-week follow-up. RESULTS: Five-hundred and six patients met inclusion criteria: 152 who received ASA and 354 who did not. Radiographic healing at six weeks was demonstrated in 95.9% (94/98) and 98.6% (207/210) respectively (p-value .2134). There was no significant difference in time to radiographic union between groups. The risk of postoperative DVTs in those with and without ASA was not significantly different (0.7% (1/137) vs 1.2% (4/323), respectively; p-value .6305). CONCLUSION: Postoperative use of ASA does not delay radiographic union of operative ankle fractures or affect the rate of postoperative DVT. This is the first and largest study to examine the effect of ASA on time to union of ankle fractures. LEVEL OF EVIDENCE: III.

A Novel External Fixation Technique for Injuries of the Proximal Interphalangeal Joint.

Digital external fixation is often used for the management of complex injuries involving the proximal interphalangeal joint, including pilon fractures of the middle phalanx base and unstable fracture-dislocations. Several dynamic "homemade" constructs have been described which utilize only K-wires and rubber bands and allow early range of motion within the construct. Although these constructs are inexpensive and their application is fairly straightforward, their designs pose a few potential problems when the construct is stressed during rehabilitation efforts. These designs utilize a blocking K-wire which relies on pin-to-pin contact to maintain reduction and creates unnecessary friction that can impede motion and result in pin loosening in bone. Furthermore, rubber band rupture can occur and destabilizes the construct. Here we present a novel technique which utilizes only K-wires and K-wire caps, provides adequate joint distraction and stabilization throughout the arc of motion, and avoids the aforementioned pitfalls of existing designs.

Extraskeletal Osteochondroma of the Great Toe in a Teenager.

Osteochondromas are common, benign surface tumors of bone, composed of the cartilage-capped bone confluent with the medullary canal of the metaphyseal bone. Extraskeletal osteochondromas have the same gross appearance and histologic characteristics as a typical osteochondroma but do not have any boney attachment to the surrounding osseous structures. They are rare and most frequently reported in the middle-age and older adults. We present the first case of an extraskeletal osteochondroma of the foot reported in a teenager. Our patient was a 17-year-old male complaining of a slow-growing mass along the medial border of the great toe that he first noted at the age of 14 years. The increasing size of the mass and frequency of complaints with shoe wear prompted medical attention. Imaging studies showed an ossified 1-cm boney mass with trabecular detail, located on the medial aspect of the great toe at the level of the interphalangeal joint, without any connection to the surrounding structures. An excisional biopsy revealed a well-circumscribed, easily removable mass, which proved to be an extraskeletal osteochondroma both clinically and histologically.

Purpose in adolescents diagnosed with an autism spectrum disorder.

INTRODUCTION: Drawing from positive youth development theory, the research team examined purpose in life among adolescents diagnosed with an autism spectrum disorder (ASD). METHODS: Members of the research team conducted paired interviews about purpose in life with adolescents diagnosed with an ASD and one of each adolescent's parents. Data collection took place in the south-central region of the US. The eight adolescent participants were in middle school, high school, and early college. The research team open-coded interview transcripts and condensed these codes into meta-codes to aid in determining the form of purpose for each participant. RESULTS: Similar to what has been found in studies of neurotypical youth, participants distributed diversely across the forms of purpose, with all but one participant demonstrating some aspect of purpose. CONCLUSIONS: The authors recommend practitioners consider the variety of supports they could provide to adolescents diagnosed with an ASD and consider encouraging these youth when their creative interests are sparked. Additionally, the research team invites the scholarly community to further investigate specific contextual supports and to develop ways of measuring purpose that do not rely on advanced language and social skills.

A case example and literature review of catastrophic wear before catastrophic failure: identification of trunnionosis and metallosis in metal-on-polyethylene hip arthroplasty prior to frank failure or fracture.

Though rare, there are documented failures of femoral prosthesis due to corrosion of the head-neck interface in total hip arthroplasty (THA), a phenomenon known as trunnionosis. This wear can result in metallosis, whereby metal debris scatters the surrounding soft tissues. We present on a 58-year-old female who presented with increase in hip and back pain 10 years following right THA using a metal-on-polyethylene construct with a large femoral head (44 mm). Aspiration withdrew metallic fluid, and intraoperative findings showed corrosion of the head-neck taper with surrounding metallosis and pseudocapsule formation. Despite advances in THA design, corrosion and wear between components still exists and may be cause for failure. We present on both the subtle clinical findings and the recommended workup when suspicion is high for trunnionosis, metallosis, or wear, ideally with identification prior to catastrophic failure such as component dislocation or fracture as previously reported.

Periarticular Injection After Total Knee Arthroplasty Using Liposomal Bupivacaine vs a Modified Ranawat Suspension: A Prospective, Randomized Study.

BACKGROUND: The purpose of this study is to compare liposomal bupivacaine to a modified (Ranawat) local injection for total knee arthroplasty (TKA). METHODS: This is a prospective, randomized study of 105 consecutive patients undergoing primary TKA. Group A patients received a periarticular injection with liposomal bupivacaine and group B with a mixture of ropivacaine, epinephrine, ketorolac, and clonidine. There were 54 patients in the group A (liposomal bupivacaine) and 51 in group B. RESULTS: There were no differences in the groups with respect to age, sex, and preoperative knee scores. There were no differences with respect to postoperative narcotic usage and knee range of motion. CONCLUSION: Liposomal bupivacaine as a periarticular injection after TKA demonstrated similar pain levels, narcotic usage, and range of motion compared to a modified Ranawat suspension but improved walking distance.

Posterior tibial tendon entrapment within an intact ankle mortise: a case report.

The present case report demonstrates a rare finding associated with irreducible ankle fracture dislocations. To our knowledge, posterior tibial tendon entrapment with an intact ankle mortise has not yet been documented in published studies. In the case of our patient, a high-energy, 12-ft fall resulted in a comminuted intra-articular fracture of the medial malleolus, confirmed by the initial radiographs. Preoperative magnetic resonance imaging showed the Achilles tendon to be ruptured and the posterior tibial tendon to be both displaced and entrapped between the medial malleolar fracture fragments, preventing initial closed reduction. At operative repair for the ruptured Achilles tendon and the medial malleolus fracture, the posterior tibial tendon was removed from the fracture site and was found to be intact with no evidence of laceration or rupture. The tendon was returned back to its anatomic position, and the tendon sheath was reapproximated. Although uncommon, it is important that entrapment of the posterior tibial tendon be considered in cases of irreducible ankle fracture. This injury type can be addressed during open reduction internal fixation to achieve reduction.

The effectiveness of a pre-procedural mouthrinse in reducing bacteria on radiographic phosphor plates.

PURPOSE: This study assessed the effectiveness of three antimicrobial mouthrinses in reducing microbial growth on photostimulable phosphor (PSP) plates. MATERIALS AND METHODS: Prior to performing a full-mouth radiographic survey (FMX), subjects were asked to rinse with one of the three test rinses (Listerine®, Decapinol®, or chlorhexidine oral rinse 0.12%) or to refrain from rinsing. Four PSP plates were sampled from each FMX through collection into sterile containers upon exiting the scanner. Flame-sterilized forceps were used to transfer the PSP plates onto blood agar plates (5% sheep blood agar). The blood agar plates were incubated at 37℃ for up to 72 h. An environmental control blood agar plate was incubated with each batch. Additionally, for control, 25 gas-sterilized PSP plates were plated onto blood agar and analyzed. RESULTS: The mean number of bacterial colonies per plate was the lowest in the chlorhexidine group, followed by the Decapinol, Listerine, and the no rinse negative control groups. Only the chlorhexidine and Listerine groups were significantly different (p=0.005). No growth was observed for the 25 gas-sterilized control plates or the environmental control blood agar plates. CONCLUSION: The mean number of bacterial colonies was the lowest in the chlorhexidine group, followed by the Decapinol, Listerine, and the no rinse groups. Nonetheless, a statistically significant difference was found only in the case of Listerine. Additional research is needed to test whether a higher concentration (0.2%) or longer exposure period (two consecutive 30 s rinse periods) would be helpful in reducing PSP plate contamination further with chlorhexidine.

Impact of an acute care surgery service on timeliness of care and surgeon satisfaction at a Canadian academic hospital: a retrospective study.

INTRODUCTION: In January 2012 an acute care surgery (ACS) model was introduced at St. Paul's Hospital, Saskatoon, Saskatchewan. The goal of implementing an ACS service was to improve the delivery of care for emergent, non-trauma surgical patients. We examined whether the ACS model improved wait time to surgery, decreased the proportion of surgeries performed after hours, and shortened post-surgical length of stay. We also assessed whether the surgeons working in an ACS system had higher on-call satisfaction than surgeons working in a non- ACS system. METHODS: A retrospective pre-post analysis was performed using data from the Discharge Abstract Database and the Organizing Medical Networked Information database. Surgeon satisfaction was evaluated using a questionnaire that was mailed to all general surgeons in Saskatoon. RESULTS: An ACS service significantly reduced wait time to surgery for patients with all acute general surgery diagnoses from 221 minutes to 192 minutes (ρ = 0.015; CI = 5.8-52.2). Post-surgery length of stay for patients operated on for acute appendicitis, or acute cholecystitis was not reduced. On average, patients with bowel obstruction had increased length of stay following ACS service implementation. Most surgeries in our study were performed between 16:00 hours and 08:00 hours but the introduction of an ACS significantly reduced the number of afterhours surgeries (60.0% vs. 72.6%) (ρ < 0.0001). Our survey had a response rate of 75%. Overall, surgeons on an ACS service had greater satisfaction with the organization of their call schedule than surgeons not on an ACS service. CONCLUSION: Introduction of an ACS service in Saskatoon has decreased wait time to surgery and reduced the proportion of afterhours emergency surgeries, with no reduction in the length of post-surgery hospital stay. Satisfaction may be higher for surgeons in an ACS service.

Diagnostic imaging for temporomandibular disorders and orofacial pain.

The focus of this article is diagnostic imaging used for the evaluation of temporomandibular disorders and orofacial pain patients. Imaging modalities discussed include conventional panoramic radiography, panoramic temporomandibular joint imaging mode, cone beam computed tomography, and magnetic resonance imaging. The imaging findings associated with common diseases of the temporomandibular joint are presented and indications for brain imaging are discussed. Advantages and disadvantages of each imaging modality are presented as well as illustrations of the various imaging techniques.

Robustness of Helicobacter pylori infection conferred by context-variable redundancy among cysteine-rich paralogs.

Deletion of single genes from expanded gene families in bacterial genomes often does not elicit a phenotype thus implying redundancy or functional non-essentiality of paralogous genes. The molecular mechanisms that facilitate evolutionary maintenance of such paralogs despite selective pressures against redundancy remain mostly unexplored. Here, we investigate the evolutionary, genetic, and functional interaction between the Helicobacter pylori cysteine-rich paralogs hcpG and hcpC in the context of H. pylori infection of cultured mammalian cells. We find that in natural H. pylori populations both hcpG and hcpC are maintained by positive selection in a dual genetic relationship that switches from complete redundancy during early infection, whereby ΔhcpC or ΔhcpG mutants themselves show no growth defect but a significant growth defect is seen in the ΔhcpC,ΔhcpG double mutant, to quantitative redundancy during late infection wherein the growth defect of the ΔhcpC mutant is exacerbated in the ΔhcpC,ΔhcpG double mutant although the ΔhcpG mutant itself shows no defect. Moreover, during early infection both hcpG and hcpC are essential for optimal translocation of the H. pylori HspB/GroEL chaperone, but during middle-to-late infection hcpC alone is necessary and sufficient for HspB/GroEL translocation thereby revealing the lack of functional compensation among paralogs. We propose that evolution of context-dependent differences in the nature of genetic redundancy, and function, between hcpG and hcpC may facilitate their maintenance in H. pylori genomes, and confer robustness to H. pylori growth during infection of cultured mammalian cells.

A phase 2 trial of once-weekly hypofractionated breast irradiation: first report of acute toxicity, feasibility, and patient satisfaction.

PURPOSE: To report on early results of a single-institution phase 2 trial of a 5-fraction, once-weekly radiation therapy regimen for patients undergoing breast-conserving surgery (BCS). METHODS AND MATERIALS: Patients who underwent BCS for American Joint Committee on Cancer stage 0, I, or II breast cancer with negative surgical margins were eligible to receive whole breast radiation therapy to a dose of 30 Gy in 5 weekly fractions of 6 Gy with or without an additional boost. Elective nodal irradiation was not permitted. There were no restrictions on breast size or the use of cytotoxic chemotherapy for otherwise eligible patients. Patients were assessed at baseline, treatment completion, and at first posttreatment follow-up to assess acute toxicity (Common Terminology Criteria for Adverse Events, version 3.0) and quality of life (European Organization for Research and Treatment of Cancer QLQ-BR23). RESULTS: Between January and September 2011, 42 eligible patients underwent weekly hypofractionated breast irradiation immediately following BCS (69.0%) or at the conclusion of cytotoxic chemotherapy (31.0%). The rates of grade ≥2 radiation-induced dermatitis, pain, fatigue, and breast edema were 19.0%, 11.9%, 9.5%, and 2.4%, respectively. Only 1 grade 3 toxicity-pain requiring a course of narcotic analgesics-was observed. One patient developed a superficial cellulitis (grade 2), which resolved with the use of oral antibiotics. Patient-reported moderate-to-major breast symptoms (pain, swelling, and skin problems), all decreased from baseline through 1 month, whereas breast sensitivity remained stable over the study period. CONCLUSIONS: The tolerance of weekly hypofractionated breast irradiation compares well with recent reports of daily hypofractionated whole-breast irradiation schedules. The regimen appears feasible and cost-effective. Additional follow-up with continued accrual is needed to assess late toxicity, cosmesis, and disease-specific outcomes.

Evaluation of an ameloblastic fibro-odontoma with cone beam computed tomography.

BACKGROUND: The clinical presentation, radiographic features, and histopathological findings of a case of ameloblastic fibro-odontoma are described. The clinical and radiographic presentations of this case are typical of this benign odontogenic tumor. The patient was young and asymptomatic with the exception of the clinical absence of the permanent mandibular left first molar. Treatment consisted of simple enucleation with removal of the primary mandibular left second molar. METHODS: A panoramic image and cone beam computed tomography (CBCT) scan were acquired in order to determine the reason for clinical absence of the permanent mandibular left first molar. The CBCT image proved to be a valuable supplement to the conventional radiographic examination as it provided information not apparent on the 2-dimensional panoramic image. Both the extent of the lesion and its effects on adjacent structures were better delineated with CBCT. Additionally, the presence of calcifications, an important radiographic characteristic of this particular lesion, were visible on CBCT versus the panoramic image. CONCLUSIONS: This case report demonstrates that the CBCT scan provided important management and treatment planning information that was not apparent in the panoramic image.

The inhibitors of apoptosis (IAPs) as cancer targets.

Apoptosis has been accepted as a fundamental component in the pathogenesis of cancer, in addition to other human diseases including neurodegeneration, coronary disease and diabetes. The origin of cancer involves deregulated cellular proliferation and the suppression of apoptotic processes, ultimately leading to tumor establishment and growth. Several lines of evidence point toward the IAP family of proteins playing a role in oncogenesis, via their effective suppression of apoptosis. The central mechanisms of IAP apoptotic suppression appear to be through direct caspase and pro-caspase inhibition (primarily caspase 3 and 7) and modulation of, and by, the transcription factor NF-kappaB. Thus, when the IAPs are over-expressed or over-active, as is the case in many cancers, cells are no longer able to die in a physiologically programmed fashion and become increasingly resistant to standard chemo- and radiation therapies. To date several approaches have been taken to target and eliminate IAP function in an attempt to re-establish sensitivity, reduce toxicity, and improve efficacy of cancer treatment. In this review, we address IAP proteins as therapeutic targets for the treatment of cancer and emphasize the importance of novel therapeutic approaches for cancer therapy. Novel targets of IAP function are being identified and include gene therapy strategies and small molecule inhibitors that are based on endogenous IAP antagonists. As well, molecular mechanistic approaches, such as RNAi to deplete IAP expression, are in development.

Granzyme B-induced apoptosis requires both direct caspase activation and relief of caspase inhibition.

Cytotoxic lymphocytes employ Granzyme B as a potent initiator of apoptosis to cleave and activate effector caspases. Unexpectedly, cells transfected with Bcl-2 were resistant to granzyme B-induced killing, suggesting that a mitochondrial pathway was critical. Utilizing cells expressing a dominant-negative caspase 9, the current study demonstrated that caspase activation via the apoptosome was not required. Indeed, cleavage of caspase 3 to p20 still occurred in Bcl-2-transfectants but processing to p17 was blocked. This blockade was recapitulated by the Inhibitor-of-Apoptosis-Protein XIAP and relieved by Smac/DIABLO. Thus granzyme B mediates direct cleavage of caspase 3 and also activates mitochondrial disruption, resulting in the release of proapoptotic proteins that suppress caspase inhibition. Engagement of both pathways is critical for granzyme-induced killing.

A novel ubiquitin fusion system bypasses the mitochondria and generates biologically active Smac/DIABLO.

Smac/DIABLO is a mitochondrial protein that is proteolytically processed and released during apoptosis along with cytochrome c and other proapoptotic factors. Once in the cytosol, Smac protein binds to inhibitors of apoptosis (IAP) proteins and disrupts the ability of the IAPs to inhibit caspases 3, 7, and 9. The requirement for mitochondrial processing and release has complicated efforts to delineate the effect of Smac overexpression and IAP inhibition on cell death processes. In this report, we document a novel expression system using ubiquitin fusions to express mature, biologically active Smac in the cytosol of transfected cells. Processing of the ubiquitin-Smac fusions is rapid and complete and generates mature Smac protein initiating correctly with the Ala-Val-Pro-Ile tetrapeptide sequence that is required for proper function. The biological activity of this exogenous protein was demonstrated by its interaction with X-linked IAP, one of the most potent of the IAPs. The presence of mature Smac was not sufficient to trigger apoptosis of healthy cells. However, cells with excess Smac protein were greatly sensitized to apoptotic triggers such as etoposide exposure. Cancer cells typically display deregulated apoptotic pathways, including Bcl2 overexpression, thereby suppressing the release of cytochrome c and Smac. The ability to circumvent the requirement for mitochondrial processing and release is critical to developing Smac as a possible gene therapy payload in cancer chemosensitization.