Ex vivo Drug Sensitivity Imaging-based Platform for Primary Acute Lymphoblastic Leukemia Cells.

Resistance of acute lymphoblastic leukemia (ALL) cells to chemotherapy, whether present at diagnosis or acquired during treatment, is a major cause of treatment failure. Primary ALL cells are accessible for drug sensitivity testing at the time of new diagnosis or at relapse, but there are major limitations with current methods for determining drug sensitivity ex vivo. Here, we describe a functional precision medicine method using a fluorescence imaging platform to test drug sensitivity profiles of primary ALL cells. Leukemia cells are co-cultured with mesenchymal stromal cells and tested with a panel of 40 anti-leukemia drugs to determine individual patterns of drug resistance and sensitivity ("pharmacotype"). This imaging-based pharmacotyping assay addresses the limitations of prior ex vivo drug sensitivity methods by automating data analysis to produce high-throughput data while requiring fewer cells and significantly decreasing the labor-intensive time required to conduct the assay. The integration of drug sensitivity data with genomic profiling provides a basis for rational genomics-guided precision medicine. Key features Analysis of primary acute lymphoblastic leukemia (ALL) blasts obtained at diagnosis from bone marrow aspirate or peripheral blood. Experiments are performed ex vivo with mesenchymal stromal cell co-culture and require four days to complete. This fluorescence imaging-based protocol enhances previous ex vivo drug sensitivity assays and improves efficiency by requiring fewer primary cells while increasing the number of drugs tested to 40. It takes approximately 2-3 h for sample preparation and processing and a 1.5-hour imaging time. Graphical overview.

Pharmacotypes across the genomic landscape of pediatric acute lymphoblastic leukemia and impact on treatment response.

Contemporary chemotherapy for childhood acute lymphoblastic leukemia (ALL) is risk-adapted based on clinical features, leukemia genomics and minimal residual disease (MRD); however, the pharmacological basis of these prognostic variables remains unclear. Analyzing samples from 805 children with newly diagnosed ALL from three consecutive clinical trials, we determined the ex vivo sensitivity of primary leukemia cells to 18 therapeutic agents across 23 molecular subtypes defined by leukemia genomics. There was wide variability in drug response, with favorable ALL subtypes exhibiting the greatest sensitivity to L-asparaginase and glucocorticoids. Leukemia sensitivity to these two agents was highly associated with MRD although with distinct patterns and only in B cell ALL. We identified six patient clusters based on ALL pharmacotypes, which were associated with event-free survival, even after adjusting for MRD. Pharmacotyping identified a T cell ALL subset with a poor prognosis that was sensitive to targeted agents, pointing to alternative therapeutic strategies. Our study comprehensively described the pharmacological heterogeneity of ALL, highlighting opportunities for further individualizing therapy for this most common childhood cancer.

Highly-automated, high-throughput replication of yeast-based logic circuit design assessments.

We describe an experimental campaign that replicated the performance assessment of logic gates engineered into cells of Saccharomyces cerevisiae by Gander et al. Our experimental campaign used a novel high-throughput experimentation framework developed under Defense Advanced Research Projects Agency's Synergistic Discovery and Design program: a remote robotic lab at Strateos executed a parameterized experimental protocol. Using this protocol and robotic execution, we generated two orders of magnitude more flow cytometry data than the original experiments. We discuss our results, which largely, but not completely, agree with the original report and make some remarks about lessons learned. Graphical Abstract.

Development of Potent and Selective Janus Kinase 2/3 Directing PG-PROTACs.

Aberrant activation of the JAK-STAT signaling pathway has been implicated in the pathogenesis of a range of hematological malignancies and autoimmune disorders. Here we describe the design, synthesis, and characterization of JAK2/3 PROTACs utilizing a phenyl glutarimide (PG) ligand as the cereblon (CRBN) recruiter. SJ10542 displayed high selectivity over GSPT1 and other members of the JAK family and potency in patient-derived ALL cells containing both JAK2 fusions and CRLF2 rearrangements.

Cognitive Bias and Therapy Choice in Breast Reconstruction Surgery Decision-Making.

BACKGROUND: Understanding how medical experts and their patients process and transfer information is of critical importance for efficient health care provision. Behavioral economics has explored similar credence markets where economic incentives, information asymmetry, and cognitive bias can impact patient and surgeon choice. The aim of the current study is to explore how framing and behavioral bias affect elective restorative surgery decision-making, such as breast reconstruction following cancer treatment. METHODS: The authors' study uses a cross-sectional survey data set of specialist surgeons (n = 53), breast care nurses (n = 101), and former or current breast cancer patients (n = 689). Data collected include participant demographics, medical history, a battery of cognitive bias tests, and a behavioral framing experiment. RESULTS: This study finds statistically significant differences in breast reconstruction surgery preference by patients and nurses when decision options are framed in different ways (i.e., positively versus negatively). The authors' analysis of surgeons, nurses, and patients shows no statistically significant difference across eight common forms of cognitive bias. Rather, the authors find that the behavioral biases are prevalent to the same extent in each group. This may indicate that differences in experience and education seem not to mitigate biases that may affect patient choices and medical professional's recommendations. The authors' multivariate analysis identifies patient age (p < 0.0001), body mass index, and self-perceived health (p < 0.05) as negative correlates for choice of implant-based reconstruction. CONCLUSION: For surgeons, nurses, and patients, the authors find uniform evidence of cognitive bias; more specifically, for patients and nurses, the authors find inconsistency in preference for type of surgical therapy chosen when alternative procedures are framed in different ways (i.e., framing bias).

Assessment of paediatric head shape and management of craniosynostosis.

BACKGROUND: The presentation of a child with an abnormal head shape can be challenging and should be met with an appropriate clinical approach. Craniosynostosis is a common cause of paediatric skull deformity and is best managed by a multispecialty tertiary referral unit with regular follow-up. As craniosynostosis frequently requires time-sensitive surgery, it is important to differentiate between craniosynostosis and common self-limiting conditions such as deformational plagiocephaly. OBJECTIVE: The aim of this article is to outline the clinical approach to paediatric skull deformity in the general practice setting, and to highlight the importance of early referral if there is clinical suspicion of craniosynostosis. DISCUSSION: Parental concern regarding infant head shape is common. General practitioners (GPs) have an important role in assessment, diagnosis and referral for paediatric skull deformities. GPs are well placed to clinically differentiate between deformational plagiocephaly and craniosynostosis and provide timely referrals to optimise patient outcomes.

Sleep and circadian rhythms in Parkinson's disease and preclinical models.

The use of animals as models of human physiology is, and has been for many years, an indispensable tool for understanding the mechanisms of human disease. In Parkinson's disease, various mouse models form the cornerstone of these investigations. Early models were developed to reflect the traditional histological features and motor symptoms of Parkinson's disease. However, it is important that models accurately encompass important facets of the disease to allow for comprehensive mechanistic understanding and translational significance. Circadian rhythm and sleep issues are tightly correlated to Parkinson's disease, and often arise prior to the presentation of typical motor deficits. It is essential that models used to understand Parkinson's disease reflect these dysfunctions in circadian rhythms and sleep, both to facilitate investigations into mechanistic interplay between sleep and disease, and to assist in the development of circadian rhythm-facing therapeutic treatments. This review describes the extent to which various genetically- and neurotoxically-induced murine models of Parkinson's reflect the sleep and circadian abnormalities of Parkinson's disease observed in the clinic.

Exploring Surgeons', Nurses', and Patients' Information Seeking Behavior on Medical Innovations: The Case of 3D Printed Biodegradable Implants in Breast Reconstruction.

Objectives: To explore information seeking behavior on medical innovations. Background: While autologous and alloplastic options for breast reconstruction are well established, it is the advent of the combination of 3D printing technology and the biocompatible nature of a highly porous biodegradable implants that offers new treatment options for the future. While this type of prosthesis is not yet clinically available understanding how patients, surgeons, and nurses take up new medical innovations is of critical importance for efficient healthcare provision. Materials and Methods: Using the largest ever combined sample of breast cancer patients (n = 689), specialist surgeons (n = 53), and breast care nurses (n = 101), we explore participants preference for a new surgical treatment concept rooted in 3D printed and biodegradable implant technologies in the context of breast reconstruction. Results: We find that patients overwhelmingly favor information from a successful patient of the proposed new technology when considering transitioning. Surgeons and nurses instead favor regulatory body advice, peer-reviewed journals, and witnessing the procedure performed (either in person or online). But while 1 in 4 nurses nominated talking to a successful patient as an information source, not a single surgeon chose the same. Our multinomial logit analysis exploring patient preference (controlling for individual differences) showed statistically significant results for both the type of surgical treatment and choice to undergo reconstruction. Women who underwent a type of mastectomy procedure (compared with lumpectomy patients) were more likely to choose a former patient than a surgeon for seeking information relating to a new breast implant technology. Further, women who chose to undergo a reconstruction procedure, compared with those who did not, where more likely to prefer a surgeon for information relating to a new breast implant technology, rather than a successful patient. For medical professionals, we find no statistically significant relationship between medical professionals' preference and their age, nor the number of other medical professionals they work with daily, nor the average number of breast procedures performed in their practice on a weekly basis. Conclusions: As our findings show large variation exists (both within our patient group and compared with medical professionals) in where individuals favor information on new medical innovations, future behavioral research is warranted.

Biomechanical Principles of Breast Implants and Current State of Research in Soft Tissue Engineering for Cosmetic Breast Augmentation.

Currently there are limited implant-based options for cosmetic breast augmentation, and problems associated with those have been increasingly appreciated, most commonly capsular contracture, which occurs due to a chronic foreign body reaction against non-degradable implant materials such as silicone and polyurethane leading to scar tissue formation, pain, and deformity. The underlying biomechanical concepts with implants create a reciprocal stress-strain relationship with local tissue, whilst acting as a deforming force. This means that with time, as the implant continues to have an effect on surrounding tissue the implant and host's biomechanical properties diverge, making malposition, asymmetry, and other complications more likely. Research directed towards development of alternative therapies based on tissue engineering and regenerative medicine seeks to optimize new tissue formation through modulation of tissue progenitors and facilitating tissue regeneration. Scaffolds can guide the process of new tissue formation by providing both an implant surface and a three-dimensional space that promotes the development of a microenvironment that guides attachment, migration, proliferation, and differentiation of connective tissue progenitors. Important to scaffold design are the architecture, surface chemistry, mechanical properties, and biomaterial used. Scaffolds provide a void in which vascularization, new tissue formation, and remodelling can sequentially occur. They provide a conduit for delivery of the different cell types required for tissue regeneration into a graft site, facilitating their retention and distribution. Whilst recent research from a small number of groups is promising, there are still ongoing challenges to achieving clinical translation. This article summarizes the biomechanical principles of breast implants, how these impact outcomes, and progress in scaffold-guided tissue engineering approaches to cosmetic breast augmentation. LEVEL OF EVIDENCE V: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Network-based systems pharmacology reveals heterogeneity in LCK and BCL2 signaling and therapeutic sensitivity of T-cell acute lymphoblastic leukemia.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy, and novel therapeutics are much needed. Profiling patient leukemia' drug sensitivities ex vivo, we discovered that 44.4% of childhood and 16.7% of adult T-ALL cases exquisitely respond to dasatinib. Applying network-based systems pharmacology analyses to examine signal circuitry, we identified preTCR-LCK activation as the driver of dasatinib sensitivity, and T-ALL-specific LCK dependency was confirmed in genome-wide CRISPR-Cas9 screens. Dasatinib-sensitive T-ALLs exhibited high BCL-XL and low BCL2 activity and venetoclax resistance. Discordant sensitivity of T-ALL to dasatinib and venetoclax is strongly correlated with T-cell differentiation, particularly with the dynamic shift in LCK vs. BCL2 activation. Finally, single-cell analysis identified leukemia heterogeneity in LCK and BCL2 signaling and T-cell maturation stage, consistent with dasatinib response. In conclusion, our results indicate that developmental arrest in T-ALL drives differential activation of preTCR-LCK and BCL2 signaling in this leukemia, providing unique opportunities for targeted therapy.

Olfactory flow in the sea catfish, Ariopsis felis (L.): Origin, regulation, and resampling.

The olfactory epithelium of the sea catfish, Ariopsis felis, is found on a pinnate array of lamellae (the olfactory rosette) housed within a nasal chamber. The nasal anatomy of A. felis suggests an ability to capture external water currents. We prepared models from X-ray micro-computed tomography scans of two preserved specimens of A. felis. We then used dye visualisation and computational fluid dynamics to show that an external current induced a flow of water through a) the nasal chamber and b) the sensory channels of the olfactory rosette. The factors responsible for inducing flow through the nasal chamber are common to fishes from two other orders. The dye visualisation experiments, together with observations of sea catfishes in vivo, indicate that flow through the nasal chamber is regulated by a mobile nasal flap. The position of the nasal flap - elevated (significant flow) or depressed (reduced flow) - is controlled by the sea catfish's movements. Flow in the sensory channels of the olfactory rosette can pass through either a single channel or, via multiple pathways, up to four consecutive channels. Flow through consecutive sensory channels (olfactory resampling) is more extensive at lower Reynolds numbers (200 and 300, equivalent to swimming speeds of 0.5-1.0 total lengths s-1), coinciding with the mean swimming speed of the sea catfishes observed in vivo (0.6 total lengths s-1). Olfactory resampling may also occur, via a vortex, within single sensory channels. In conclusion, olfactory flow in the sea catfish is regulated and thoroughly sampled by novel mechanisms.

Use of Onlay Hydroxyapatite Cement for Secondary Cranioplasty.

BACKGROUND: Children who undergo bi-fronto-orbital advancement (BFOA) frequently develop a contour deformity on the temporal and supra-orbital region, with an incidence reported as high as 55% and 75%, respectively. Up to 20% of patients may require correction. Hydroxyapatite cement (HAC) is a good alternative to autogenous tissue. The available literature on its use focusses on the reconstruction of bone defects, but little has been published on its efficacy and safety as an onlay graft over intact cranium. OBJECTIVES: To describe our institution's experience with HAC in the pediatric population. METHODS: Retrospective chart review from 1998 to 2018 on all patients from the Craniofacial Unit at the Sydney Children's Hospital who had either coronal or metopic craniosynostosis and underwent BFOA and later in life required cranioplasty with HAC for contour repair. FINDINGS: We have performed 166 BFOA and nineteen secondary cranioplasties for contour repair using onlay HAC. The mean age at the time of operation was 14 years. Bi-coronal craniosynostosis was most frequently associated with secondary cranioplasty and 37% had an associated syndrome. The mean volume of HAC used was 37 mL. There was only 1 patient who had a complication (5.3%) and required partial removal of allograft. The mean length of admission was 2 days. Mean follow up time of 22.4 months. CONCLUSIONS: HAC represents a safe option when used correctly, with low rates of complication and satisfactory cosmetic outcomes.

The removal of cranial springs used in the treatment of scaphocephaly: A minimal access approach.

The treatment of non-syndromic scaphocephaly with spring-activated cranioplasty offers acceptable outcomes with the potential for reduced surgical morbidity when compared with cranial vault remodelling procedures. A disadvantage of this technique is the need for a second operative intervention to remove the implanted devices. There are many descriptions of the surgical technique for performing spring-activated cranioplasty available in the literature; however, little is documented regarding the procedures used for device removal. The published accounts of spring removal demonstrate a wide range of approaches, from the reopening and dissection of the entire previous surgical field, to attempts to limit the incisions and dissection. In this study we describe our technique for the minimally invasive removal of cranial springs used in the treatment of scaphocephaly. Our technique focuses on minimal soft tissue disruption and uses a Kirschner wire cutter to divide the spring at its mid-point so as to relieve any residual internal forces acting on the footplates.

Quantification of Head Shape and Cranioplasty Outcomes: Six-compartment Volume Method Applied to Sagittal Synostosis.

BACKGROUND: Premature fusion of the sagittal (midline) suture between 2 parietal bones is the most common form of craniosynostosis. Surgical correction is mandated to improve head shape and to decrease the risk of raised intracranial pressure. This study evaluated the utility of 3-dimensional (3D) imaging to quantify the volumetric changes of surgical correction. Currently there is no standardized method used to quantify the outcomes of surgery for craniosynostosis, with the cranial index (width: length ratio) being commonly used. METHODS: A method for quantification of head shape using 3D imaging is described in which the cranium is divided up into 6 compartments and the volumes of 6 compartments are quantified and analyzed. The method is size invariant, meaning that it can be used to assess the long-term postoperative outcomes of patients through growth. The method is applied to a cohort of sagittal synostosis patients and a normal cohort, and is used to follow up a smaller group of synostotic patients 1, 2, and 3 years postoperatively. RESULTS: Statistical analysis of the results shows that the 6-compartment volume quantification method is more accurate in separating normal from synostotic patient head shapes than the cranial index. CONCLUSIONS: Spring-mediated cranioplasty does not return head shape back to normal, but results in significant improvements in the first year following surgery compared with the preoperative sagittal synostosis head shape. 3D imaging can be a valuable tool in assessing the volumetric changes due to surgery and growth in craniosynstosis patients.

Olfactory flow in the sturgeon is externally driven.

Fluid dynamics plays an important part in olfaction. Using the complementary techniques of dye visualisation and computational fluid dynamics (CFD), we investigated the hydrodynamics of the nasal region of the sturgeon Huso dauricus. H. dauricus offers several experimental advantages, including a well-developed, well-supported, radial array (rosette) of visible-by-eye olfactory sensory channels. We represented these features in an anatomically accurate rigid model derived from an X-ray scan of the head of a preserved museum specimen. We validated the results from the CFD simulation by comparing them with data from the dye visualisation experiments. We found that flow through both the nasal chamber and, crucially, the sensory channels could be induced by an external flow (caused by swimming in vivo) at a physiologically relevant Reynolds number. Flow through the nasal chamber arises from the anatomical arrangement of the incurrent and excurrent nostrils, and is assisted by the broad, cartilage-supported, inner wall of the incurrent nostril. Flow through the sensory channels arises when relatively high speed flow passing through the incurrent nostril encounters the circular central support of the olfactory rosette, decelerates, and is dispersed amongst the sensory channels. Vortices within the olfactory flow may assist odorant transport to the sensory surfaces. We conclude that swimming alone is sufficient to drive olfactory flow in H. dauricus, and consider the implications of our results for the three other extant genera of sturgeons (Acipenser, Pseudoscaphirhynchus and Scaphirhynchus), and for other fishes with olfactory rosettes.

Efficient tRNA degradation and quantification in Escherichia coli cell extract using RNase-coated magnetic beads: A key step toward codon emancipation.

Emancipating sense codons toward a minimized genetic code is of significant interest to science and engineering. A key approach toward sense codon emancipation is the targeted in vitro removal of native tRNA. However, challenges remain such as the insufficient depletion of tRNA in lysate-based in vitro systems and the high cost of the purified components system (PURE). Here we used RNase-coated superparamagnetic beads to efficiently degrade E. coli endogenous tRNA. The presented method removes >99% of tRNA in cell lysates, while partially preserving cell-free protein synthesis activity. The resulting tRNA-depleted lysate is compatible with in vitro-transcribed synthetic tRNA for the production of peptides and proteins. Additionally, we directly measured residual tRNA using quantitative real-time PCR. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1401-1407, 2017.

Cell-Free Protein Synthesis Approach to Biosensing hTRß-Specific Endocrine Disruptors.

Here we introduce a Rapid Adaptable Portable In vitro Detection biosensor platform (RAPID) for detecting ligands that interact with nuclear hormone receptors (NHRs). The RAPID platform can be adapted for field use, allowing rapid evaluation of endocrine disrupting chemicals (EDCs) presence or absence in environmental samples, and can also be applied for drug screening. The biosensor is based on an engineered, allosterically activated fusion protein, which contains the ligand binding domain from a target NHR (human thyroid receptor ß in this work). In vitro expression of this protein using cell-free protein synthesis (CFPS) technology in the presence of an EDC leads to activation of a reporter enzyme, reported through a straightforward colorimetric assay output. In this work, we demonstrate the potential of this biosensor platform to be used in a portable "just-add-sample" format for near real-time detection. We also demonstrate the robust nature of the cell-free protein synthesis component in the presence of a variety of environmental and human samples, including sewage, blood, and urine. The presented RAPID biosensor platform is significantly faster and less labor intensive than commonly available technologies, making it a promising tool for detecting environmental EDC contamination and screening potential NHR-targeted pharmaceuticals.

Motion-driven flow in an unusual piscine nasal region.

Fishes have several means of moving water to effect odorant transport to their olfactory epithelium ('olfactory flow'). Here we show that olfactory flow in the adult garpike Belone belone (Belonidae, Teleostei), a fish with an unusual nasal region, can be generated by its motion relative to water (swimming, or an external current, or both). We also show how the unusual features of the garpike's nasal region influence olfactory flow. These features comprise a triangular nasal cavity in which the olfactory epithelium is exposed to the external environment, a papilla situated within the nasal cavity, and an elongated ventral apex. To perform our investigation we first generated life-like plastic models of garpike heads from X-ray scans of preserved specimens. We then suspended these models in a flume and flowed water over them to simulate swimming. By directing filaments of dye at the static models, we were able to visualise flow in the nasal regions at physiologically relevant Reynolds numbers (700-2,000). We found that flow of water over the heads did cause circulation in the nasal cavity. Vortices may assist in this circulation. The pattern of olfactory flow was influenced by morphological variations and the asymmetry of the nasal region. The unusual features of the nasal region may improve odorant sampling in the garpike, by dispersing flow over the olfactory epithelium and by creating favourable conditions for odorant transport (e.g. steep velocity gradients). Unexpectedly, we found that the mouth and the base of the garpike's jaws may assist the sampling process. Thus, despite its apparent simplicity, the garpike's nasal region is likely to act as an effective trap for odorant molecules.