BioCanRx Summit for Cancer Immunotherapy 2022 Proceedings.

From 19 to 21 November 2022, BioCanRx held its first post-pandemic in-person Summit for Cancer Immunotherapy in Montreal, Canada. The meeting was well attended by patients, trainees, researchers, clinicians, and industry professionals, who came together to discuss the current state and future of biotherapeutics for cancer in Canada and beyond. Three plenaries, three keynote speakers, a lively debate, and panel discussions, together with poster sessions and a social event, made the event memorable and productive. The current state of cellular therapies, cellular engineering, clinical trials, and the role of the cancer microbiome were discussed in plenary session, and the patient voice was welcomed and present throughout the meeting, in large part due to the Learning Institute, a BioCanRx initiative to include patient partners in research. In this meeting review, we highlight the platform presentations, keynote speakers, debate combatants, panellists, and the patient perspective on the annual meeting.

A Visual, In-Expensive, and Wireless Capillary Rheometer for Characterizing Wholly-Cellular Bioinks.

Rheological measurements with in situ visualization can elucidate the microstructural origin of complex flow behaviors of an ink. However, existing commercial rheometers suffer from high costs, the need for dedicated facilities for microfabrication, a lack of design flexibility, and cabling that complicates operation in sterile or enclosed environments. To address these limitations, a low-cost ($300) visual, in-expensive and wireless rheometer (VIEWR) using 3D-printed and off-the-shelf components is presented. VIEWR measurements are validated by steady-state and transient flow responses for different complex fluids, and microstructural flow profiles and evolution of yield-planes are revealed via particle image velocimetry. Using the VIEWR, a wholly-cellular bioink system comprised of compacted cell aggregates is characterized, and complex yield-stress and viscoelastic responses are captured via concomitantly visualizing the spatiotemporal evolution of aggregate morphology. A symmetric hyperbolic extensional-flow geometry is further constructed inside a capillary tube using digital light processing. Such geometries allow for measuring the extensional viscosity at varying deformation rates and further visualizing the alignment and stretching of aggregates under external flow. Synchronized but asymmetric evolution of aggregate orientation and strain through the neck is visualized. Using varying geometries, the jamming and viscoelastic deformation of aggregates are shown to contribute to the extensional viscosity of the wholly-cellular bioinks.

2022 Optical Interference Coatings Conference: Manufacturing Problem Contest [Invited].

Participants in the 2022 Manufacturing Problem Contest were challenged to fabricate an optical filter with a specified stepped transmittance spanning three orders of magnitude from 400 to 1100 nm. The problem required that contestants be versed in the design, deposition, and measurement of optical filters to achieve good results. Nine samples from five institutions were submitted with total thicknesses between 5.9 and 53.5 µm with between 68 and 1743 layers. The filter spectra were measured by three independent laboratories. The results were presented in June 2022 at the Optical Interference Coatings Conference in Whistler, B.C., Canada.

Biophysical principles of choanoflagellate self-organization.

Inspired by the patterns of multicellularity in choanoflagellates, the closest living relatives of animals, we quantify the biophysical processes underlying the morphogenesis of rosette colonies in the choanoflagellate Salpingoeca rosetta We find that rosettes reproducibly transition from an early stage of 2-dimensional (2D) growth to a later stage of 3D growth, despite the underlying variability of the cell lineages. Our perturbative experiments demonstrate the fundamental importance of a basally secreted extracellular matrix (ECM) for rosette morphogenesis and show that the interaction of the ECM with cells in the colony physically constrains the packing of proliferating cells and, thus, controls colony shape. Simulations of a biophysically inspired model that accounts for the size and shape of the individual cells, the fraction of ECM, and its stiffness relative to that of the cells suffices to explain our observations and yields a morphospace consistent with observations across a range of multicellular choanoflagellate colonies. Overall, our biophysical perspective on rosette development complements previous genetic perspectives and, thus, helps illuminate the interplay between cell biology and physics in regulating morphogenesis.

A mode of cell adhesion and migration facilitated by CD44-dependent microtentacles.

The structure and mechanics of many connective tissues are dictated by a collagen-rich extracellular matrix (ECM), where collagen fibers provide topological cues that direct cell migration. However, comparatively little is known about how cells navigate the hyaluronic acid (HA)-rich, nanoporous ECM of the brain, a problem with fundamental implications for development, inflammation, and tumor invasion. Here, we demonstrate that glioblastoma cells adhere to and invade HA-rich matrix using microtentacles (McTNs), which extend tens of micrometers from the cell body and are distinct from filopodia. We observe these structures in continuous culture models and primary patient-derived tumor cells, as well as in synthetic HA matrix and organotypic brain slices. High-magnification and superresolution imaging reveals McTNs are dynamic, CD44-coated tubular protrusions containing microtubules and actin filaments, which respectively drive McTN extension and retraction. Molecular mechanistic studies reveal that McTNs are stabilized by an interplay between microtubule-driven protrusion, actomyosin-driven retraction, and CD44-mediated adhesion, where adhesive and cytoskeletal components are mechanistically coupled by an IQGAP1-CLIP170 complex. McTNs represent a previously unappreciated mechanism through which cells engage nanoporous HA matrix and may represent an important molecular target in physiology and disease.

Cofilin is required for polarization of tension in stress fiber networks during migration.

Cell migration is associated with the establishment of defined leading and trailing edges, which in turn requires polarization of contractile forces. While the actomyosin stress fiber (SF) network plays a critical role in enforcing this polarity, precisely how this asymmetry is established remains unclear. Here, we provide evidence for a model in which the actin-severing protein cofilin (specifically cofilin-1) participates in symmetry breakage by removing low-tension actomyosin filaments during transverse arc assembly. Cofilin knockdown (KD) produces a non-polarized SF architecture that cannot be rescued with chemokines or asymmetric matrix patterns. Whereas cofilin KD increases whole-cell prestress, it decreases prestress within single SFs, implying an accumulation of low-tension SFs. This notion is supported by time-lapse imaging, which reveals weakly contractile and incompletely fused transverse arcs. Confocal and super-resolution imaging further associate this failed fusion with the presence of crosslinker-rich, tropomyosin-devoid nodes at the junctions of multiple transverse arc fragments and dorsal SFs. These results support a model in which cofilin facilitates the formation of high-tension transverse arcs, thereby promoting mechanical asymmetry.

Malpractice Litigation in Plastic Surgery: Can We Identify Patterns?

BACKGROUND: Malpractice litigation has a significant impact on healthcare costs and important professional implications for healthcare providers. OBJECTIVES: The authors sought to comprehensively characterize the litigation landscape in plastic surgery across its different subspecialties. METHODS: The authors utilized the Westlaw legal database to conduct a comprehensive search of malpractice cases in the United States in the following categories: cosmetic, reconstructive, hand, craniofacial, and gender affirmation surgery. They conducted both a Boolean and a natural language search to identify cases in which a plastic surgeon was the defendant. Data were analyzed employing descriptive statistics, logistic regression, and relative risk calculations. RESULTS: In total, 165 cases were included. Most surgeons accused of malpractice worked in a private setting (148 [90%]). Among the 22 (13%) cases that contained information on board certification status, most surgeons were board certified (17 [77%]). Resident involvement was mentioned in only 5 (3%) cases. The majority of cases were successfully defended by surgeons (98 [60%] vs 65 [40%]), particularly in craniofacial surgery (risk ratio: 1.54; P = 0.03; 95% CI: 1.03-2.3). Surgeons who successfully defended a case were more likely to benefit from summary judgment (P = 0.005). CONCLUSIONS: Malpractice litigation is commonplace in medical practice, and no specialty is spared. Legal outcomes were in favor of plastic surgeons in the majority of cases, particularly those that proceeded to summary judgment. Surgeons can avoid litigation by maintaining detailed office and surgical notes, always obtaining informed consent, adequately following and monitoring patients after surgery, and ensuring compliance by communicating frequently and effectively.

Vaccinia virus proteins A36 and F12/E2 show strong preferences for different kinesin light chain isoforms.

Vaccinia virus (VACV) utilizes microtubule-mediated trafficking at several stages of its life cycle, of which virus egress is the most intensely studied. During egress VACV proteins A36, F12 and E2 are involved in kinesin-1 interactions; however, the roles of these proteins remain poorly understood. A36 forms a direct link between virions and kinesin-1, yet in its absence VACV egress still occurs on microtubules. During a co-immunoprecipitation screen to seek an alternative link between virions and kinesin, A36 was found to bind isoform KLC1 rather than KLC2. The F12/E2 complex associates preferentially with the C-terminal tail of KLC2, to a region that overlaps the binding site of cellular 14-3-3 proteins. F12/E2 displaces 14-3-3 from KLC and, unlike 14-3-3, does not require phosphorylation of KLC for its binding. The region determining the KLC1 specificity of A36 was mapped to the KLC N-terminal heptad repeat region that is responsible for its association with kinesin heavy chain. Despite these differing binding properties F12/E2 can co-operatively enhance A36 association with KLC, particularly when using a KLC1-KLC2 chimaera that resembles several KLC1 spliceforms and can bind A36 and F12/E2 efficiently. This is the first example of a pathogen encoding multiple proteins that co-operatively associate with kinesin-1.

The North Carolina Experience with Mucopolysaccharidosis Type I Newborn Screening.

OBJECTIVE: To evaluate the performance of a 2-tiered newborn screening method for mucopolysaccharidosis type I (MPS I) in North Carolina. STUDY DESIGN: The screening algorithm included a flow injection analysis-tandem mass spectrometry assay as a first-tier screening method to measure α-L-iduronidase (IDUA) enzyme activity and Sanger sequencing of the IDUA gene on dried blood spots as a second-tier assay. The screening algorithm was revised to incorporate the Collaborative Laboratory Integrated Reports, an analytical interpretive tool, to reduce the false-positive rate. A medical history, physical examination, IDUA activity, and urinary glycosaminoglycan (GAG) analysis were obtained on all screen-positive infants. RESULTS: A total of 62 734 specimens were screened with 54 screen-positive samples using a cut-off of 15% of daily mean IDUA activity. The implementation of Collaborative Laboratory Integrated Reports reduced the number of specimens that screened positive to 19 infants. Of the infants identified as screen-positive, 1 had elevated urinary GAGs and a homozygous pathogenic variant associated with the severe form of MPS I. All other screen-positive infants had normal urinary GAG analysis; 13 newborns had pseudodeficiency alleles, 3 newborns had variants of unknown significance, and 2 had heterozygous pathogenic variants. CONCLUSIONS: An infant with severe MPS I was identified and referred for a hematopoietic stem cell transplant. Newborn IDUA enzyme deficiency is common in North Carolina, but most are due to pseudodeficiency alleles in infants with normal urinary GAG analysis and no evidence of disease. The pilot study confirmed the need for second-tier testing to reduce the follow-up burden.

Early Check: translational science at the intersection of public health and newborn screening.

BACKGROUND: Newborn screening (NBS) occupies a unique space at the intersection of translational science and public health. As the only truly population-based public health program in the United States, NBS offers the promise of making the successes of translational medicine available to every infant with a rare disorder that is difficult to diagnose clinically, but for which strong evidence indicates that presymptomatic treatment will substantially improve outcomes. Realistic NBS policy requires data, but rare disorders face a special challenge: Screening cannot be done without supportive data, but adequate data cannot be collected in the absence of large-scale screening. The magnitude and scale of research to provide this expanse of data require working with public health programs, but most do not have the resources or mandate to conduct research. METHODS: To address this gap, we have established Early Check, a research program in partnership with a state NBS program. Early Check provides the infrastructure needed to identify conditions for which there have been significant advances in treatment potential, but require a large-scale, population-based study to test benefits and risks, demonstrate feasibility, and inform NBS policy. DISCUSSION: Our goal is to prove the benefits of a program that can, when compared with current models, accelerate understanding of diseases and treatments, reduce the time needed to consider inclusion of appropriate conditions in the standard NBS panel, and accelerate future research on new NBS conditions, including clinical trials for investigational interventions. TRIAL REGISTRATION: Clinicaltrials.gov registration # NCT03655223 . Registered on August 31, 2018.

Lessons Learned from Newborn Screening in Pilot Studies.

This commentary discusses the importance of conducting newborn screening pilot studies in North Carolina and the lessons learned from performing three pilots for severe combined immunodeficiency (SCID), mucopolysaccharidosis type I (MPS I), and X-linked adrenoleukodystrophy (X-ALD).

RUTHENIUM-106 BRACHYTHERAPY IN THE TREATMENT OF CIRCUMSCRIBED CHOROIDAL HEMANGIOMA.

PURPOSE: To examine the efficacy and safety of Ruthenium-106 plaque radiotherapy in the treatment of circumscribed choroidal hemangioma. METHODS: Twenty-one eyes of 21 patients diagnosed with symptomatic circumscribed choroidal hemangioma who underwent Ruthenium-106 plaque radiotherapy were included in the study. Clinical response, ancillary tests finding improvement, and major side effects were evaluated. RESULTS: From the initial to the 1-year follow-up visits, vision improved in 12 eyes (57%), was stable in 7 eyes (33%), and became worse in 2 eyes (10%). Based on fluorescein angiography and optical coherence tomography, subretinal fluid and cystoid macular edema resolved in all patients. Changes in logarithm of minimum angle of resolution visual acuity (P = 0.038); tumor thickness (P = 0.0001) and largest diameter (P = 0.007) on ultrasonography; and subfoveal thickness on optical coherence tomography (P < 0.0001), were statistically significant between the initial and the 1-year follow-up visits. Side effects as observed during the follow-up period included: radiation-related retinopathy in 5 (24%) eyes, radiation-related papillopathy in 1 eye (5%), and subretinal fibrosis in 2 eyes (10%). Subretinal fibrosis was the only permanent radiation-related side effect. CONCLUSION: Ruthenium-106 plaque radiotherapy is an effective and safe method of treatment for symptomatic circumscribed choroidal hemangiomas. The incidence of permanent visual loss is low with prompt treatment of complications.

Tagging of the vaccinia virus protein F13 with mCherry causes aberrant virion morphogenesis.

Vaccinia virus produces two distinct infectious virions; the single-enveloped intracellular mature virus (IMV), which remains in the cell until cell lysis, and the double-enveloped extracellular enveloped virus (EEV), which mediates virus spread. The latter is derived from a triple-enveloped intracellular enveloped virus (IEV) precursor, which is transported to the cell periphery by the kinesin-1 motor complex. This transport involves the viral protein A36 as well as F12 and E2. A36 is an integral membrane protein associated with the outer virus envelope and is the only known direct link between virion and kinesin-1 complex. Yet in the absence of A36 virion egress still occurs on microtubules, albeit at reduced efficiency. In this paper double-fluorescent labelling of the capsid protein A5 and outer-envelope protein F13 was exploited to visualize IEV transport by live-cell imaging in the absence of either A36 or F12. During the generation of recombinant viruses expressing both A5-GFP and F13-mCherry a plaque size defect was identified that was particularly severe in viruses lacking A36. Electron microscopy showed that this phenotype was caused by abnormal wrapping of IMV to form IEV, and this resulted in reduced virus egress to the cell surface. The aberrant wrapping phenotype suggests that the fluorescent fusion protein interferes with an interaction of F13 with the IMV surface that is required for tight association between IMVs and wrapping membranes. The severity of this defect suggests that these viruses are imperfect tools for characterizing virus egress.

Cultivated Sub-Populations of Soil Microbiomes Retain Early Flowering Plant Trait.

The collection of microorganisms found in the root zone of soil, termed the rhizosphere microbiome, has been shown to impact plant growth and development. Here, we tease apart the function of the cultivable portion of the microbiome from the whole microbiome in retaining plant traits modified through artificial selection on flowering time. Specifically, the whole microbiome associated with earlier flowering time of Arabidopsis thaliana was cultivated on four types of solid media to create cultivated fractions of the microbiome. These cultivated microbiomes were subsequently preserved in glycerol, frozen, and revived to yield a portion of the cultivable fraction to compare (1) whole microbiome, (2) cultivable microbiome, and (3) revived, cultivable microbiome controls on early flowering time. Plants grown in soils inoculated with bacteria grown on 25 % Luria broth and 10 % tryptic soy agar retained the early flowering trait. An increase in leaf biomass with two of the cultivated microbiomes (49.4 and 38.5 %) contrasted the lowered biomass effect of the whole microbiome. Inoculation with the cultivated microbiomes that were cryopreserved in glycerol showed no effect on flowering time or leaf biomass. The results indicate that the cultivable portion of a plant's microbiome retains the early flowering effect in A. thaliana, but cryopreservation of the cultivated microbiomes disrupts the microbial effects on flowering time. Furthermore, the contrasting effects on leaf biomass (an indirect response from selection on early flowering time), seen with the whole microbiome versus the cultivable portion, suggests versatility in using cultivation methods to modify multiple traits of plants.

Ocular morbidities of juvenile idiopathic arthritis-associated uveitis in adulthood: results from a tertiary center study.

PURPOSE: To describe the clinical and visual outcomes of juvenile idiopathic arthritis (JIA)-associated uveitis in adults and to examine risk factors for ongoing inflammation in adulthood. METHODS: Medical records were reviewed for patients with JIA-associated uveitis who were >16 years old at the final visit (the last visit prior to data collection). RESULTS: In total, 135 eyes of 77 patients (70 female, 7 male) were included. The mean age of patients at the final visit was 29.72 ± 11.27 years. The number of eyes with visual acuity of ≤20/50 and ≤20/200 at the final visit was 37 (28 %) and 20 (15 %), respectively; at least one ocular complication was present in 72 % of eyes. Band keratopathy was the most frequent complication (42 %), followed by cataract (25 %), posterior synechiae (22 %), maculopathy (22 %), ocular hypertension (13 %), and hypotony (5 %). At the final visit, patients who were >16 years of age at presentation to the Massachusetts Eye Research and Surgery Institution had more ocular complications and a greater degree of vision loss than patients who were ≤16 years of age. Ongoing inflammation at the final visit was noted in 40 patients (52 %). The presence of posterior synechiae, hypotony, cataract at presentation, and a history of cataract surgery prior to presentation were predictive of ongoing inflammation in adulthood in univariate analysis. The presence of hypotony and posterior synechiae at the initial visit were predictive factors in multivariate analysis. CONCLUSIONS: JIA-associated uveitis may be associated with ongoing inflammation, ocular complications, and severe visual impairment in adulthood. The presence of posterior synechiae and hypotony at the initial visit is predictive of ongoing inflammation.

Designing lipids for selective partitioning into liquid ordered membrane domains.

Self-organization of lipid molecules into specific membrane phases is key to the development of hierarchical molecular assemblies that mimic cellular structures. While the packing interaction of the lipid tails should provide the major driving force to direct lipid partitioning to ordered or disordered membrane domains, numerous examples show that the headgroup and spacer play important but undefined roles. We report here the development of several new biotinylated lipids that examine the role of spacer chemistry and structure on membrane phase partitioning. The new lipids were prepared with varying lengths of low molecular weight polyethylene glycol (EGn) spacers to examine how spacer hydrophilicity and length influence their partitioning behavior following binding with FITC-labeled streptavidin in liquid ordered (Lo) and liquid disordered (Ld) phase coexisting membranes. Partitioning coefficients (Kp Lo/Ld) of the biotinylated lipids were determined using fluorescence measurements in studies with giant unilamellar vesicles (GUVs). Compared against DPPE-biotin, DPPE-cap-biotin, and DSPE-PEG2000-biotin lipids, the new dipalmityl-EGn-biotin lipids exhibited markedly enhanced partitioning into liquid ordered domains, achieving Kp of up to 7.3 with a decaethylene glycol spacer (DP-EG10-biotin). We further demonstrated biological relevance of the lipids with selective partitioning to lipid raft-like domains observed in giant plasma membrane vesicles (GPMVs) derived from mammalian cells. Our results found that the spacer group not only plays a pivotal role for designing lipids with phase selectivity but may also influence the structural order of the domain assemblies.

Characterization of dental anatomy and gingival biotype in Asian populations.

Gingival and dental characteristics are risk factors for periodontal problems. With short or fused roots, a decreased periodontium results in some attachment loss, compromising periodontal stability. Similarly, with an increased incidence of thin gingival biotype, inflammatory and traumatic insults may result in gingival recession. Anecdotally, Asian dentitions have been described as having short roots with "thin gingiva". This cross-sectional study will utilize clinical data and radiographic interpretation to ascertain whether this clinical impression is valid.

Expression of EMMPRIN modulates mediators of tumor invasion in oral squamous cell carcinoma.

Squamous cell carcinoma (SCC) accounts for 96 percent of all intraoral malignancies. The five-year survival rate is 50 percent and has not improved in 60 years. During SCC progression, subsets of SCC cells undergo an epithelial-to-mesenchymal transition (EMT) to become highly invasive. The extracellular matrix metalloproteinase inducer (EMMPRIN) contributes to EMT by activating local matrix metalloproteinases (MMPs). In this study, we found that EMMPRIN modulates the invasive phenotype and may be a potential therapeutic target.

A Case of Retinal Atherosclerotic Oxalosis due to Hyperoxaluria in a Patient with Short Gut Syndrome.

PURPOSE: To describe a severe case of crystalline retinopathy secondary to hyperoxaluria from short gut syndrome. METHODS: Case report. RESULTS: A 62-year-old Caucasian female with short gut syndrome and end-stage renal disease from renal oxalosis presented with chronic bilateral vision loss. She had previously been treated for presumed occlusive vasculitis. Visual acuity on initial exam was 20/400 OD and 20/100 OS with an afferent pupillary defect of the right eye.Exam revealed attenuated retinal vasculature and diffuse crystalline infiltration of retinal arterial lumens and throughout the retinas bilaterally. Optical coherence tomography revealed inner retinal atrophy with crystalline deposition in the inner retinal layers. Fluorescein angiography demonstrated delayed vascular filling and dropout consistent with severe ischemic vasculopathy. It was concluded that the short-gut syndrome led to over-absorption of oxalate with subsequent hyperoxaluria leading to retinal atherosclerotic oxalosis. CONCLUSION: Retinal calcium oxalate deposits due to hyperoxaluria have been previously noted; however, this degree of severe retinal vascular infiltration has not been described. Our patient was receiving hemodialysis, which is associated with high rebound increases in systemic oxalate concentrations. It is important to keep hyperoxaluria in mind as a potential cause of retinopathy in patients with end-stage renal disease presenting with vision loss.

Killer instincts: natural killer cells as multifactorial cancer immunotherapy.

Natural killer (NK) cells integrate heterogeneous signals for activation and inhibition using germline-encoded receptors. These receptors are stochastically co-expressed, and their concurrent engagement and signaling can adjust the sensitivity of individual cells to putative targets. Against cancers, which mutate and evolve under therapeutic and immunologic pressure, the diversity for recognition provided by NK cells may be key to comprehensive cancer control. NK cells are already being trialled as adoptive cell therapy and targets for immunotherapeutic agents. However, strategies to leverage their naturally occurring diversity and agility have not yet been developed. In this review, we discuss the receptors and signaling pathways through which signals for activation or inhibition are generated in NK cells, focusing on their roles in cancer and potential as targets for immunotherapies. Finally, we consider the impacts of receptor co-expression and the potential to engage multiple pathways of NK cell reactivity to maximize the scope and strength of antitumor activities.