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Imbalanced joint load distribution across the tibiofemoral surface is a risk factor for osteoarthritic changes to this joint. Lateral wedge insoles, with and without arch support, are a form of biomechanical intervention that can redistribute tibiofemoral joint load, as estimated by external measures of knee load. The objective of this study was to examine the effect of these insoles on the internal joint contact characteristics of osteoarthritic knees during weightbearing. Fifteen adults with tibiofemoral osteoarthritis underwent magnetic resonance imaging of the affected knee, while standing under 3 insole conditions: flat control, lateral wedge alone, and lateral wedge with arch support. Images were processed, and the surface area and centroid location of joint contact were quantified separately for the medial and lateral tibiofemoral compartments. Medial contact surface area was increased with the 2 lateral wedge conditions compared with the control (P ≤.012). A more anterior contact centroid was observed in the medial compartment in the lateral wedge with arch support compared with the lateral wedge alone (P =.009). Significant changes in lateral compartment joint contact outcomes were not observed. These findings represent early insights into how loading at the tibiofemoral interface may be altered by lateral wedge insoles as a potential intervention for knee osteoarthritis. [ABSTRACT FROM AUTHOR]

Due to the potential applications in biomedical engineering, it becomes more and more important to understand the process of engulfment and internalization of nanoparticles (NPs) by cell membranes. Despite the fact that the interaction between cell membranes and passive particles has been widely studied, the interaction between cell membranes and self-propelled nonspherical NPs remains to be elucidated. Here, we present a theoretical model to systematically investigate the influence of the active force, aspect ratio of NPs, particle size, and membrane properties (adhesion energy density and membrane tension) on the cellular uptake of a nonspherical nanoparticle. It is found that the active force generated by an NP can trigger a type of first-order wrapping transition from a small partial wrapping state to a large one. In addition, the phase diagram in the force-aspect ratio (particle size, adhesion energy density, and membrane tension) space displays more complex behaviors compared with that for the passive wrapping mediated merely by adhesion. These results may provide useful guidance to the study of activity-driven cellular entry of active particles into cells. [ABSTRACT FROM AUTHOR]

Cone beam computed tomography (CBCT) is a widely available modality, but its clinical utility has been limited by low detail conspicuity and quantitative accuracy. Convenient post-reconstruction denoising is subject to back projected patterned residual, but joint denoise-reconstruction is typically computationally expensive and complex. In this study, we develop and evaluate a novel Metric-learning guided wavelet transform reconstruction (MEGATRON) approach to enhance image domain quality with projection-domain processing. Projection domain based processing has the benefit of being simple, efficient, and compatible with various reconstruction toolkit and vendor platforms. However, they also typically show inferior performance in the final reconstructed image, because the denoising goals in projection and image domains do not necessarily align. Motivated by these observations, this work aims to translate the demand for quality enhancement from the quantitative image domain to the more easily operable projection domain. Specifically, the proposed paradigm consists of a metric learning module and a denoising network module. Via metric learning, enhancement objectives on the wavelet encoded sinogram domain data are defined to reflect post-reconstruction image discrepancy. The denoising network maps measured cone-beam projection to its enhanced version, driven by the learnt objective. In doing so, the denoiser operates in the convenient sinogram to sinogram fashion but reflects improvement in reconstructed image as the final goal. Implementation-wise, metric learning was formalized as optimizing the weighted fitting of wavelet subbands, and a res-Unet, which is a Unet structure with residual blocks, was used for denoising. To access quantitative reference, cone-beam projections were simulated using the X-ray based Cancer Imaging Simulation Toolkit (XCIST). In both learning modules, a data set of 123 human thoraxes, which was from Open-Source Imaging Consortium (OSIC) Pulmonary Fibrosis Progression challenge, was used. Reconstructed CBCT thoracic images were compared against ground truth FB and performance was assessed in root mean square error (RMSE), peak signal-to-noise ratio (PSNR), and structural similarity index (SSIM). MEGATRON achieved RMSE in HU value, PSNR, and SSIM were 30.97 ± 4.25, 37.45 ± 1.78, and 93.23 ± 1.62, respectively. These values are on par with reported results from sophisticated physics-driven CBCT enhancement, demonstrating promise and utility of the proposed MEGATRON method. We have demonstrated that incorporating the proposed metric learning into sinogram denoising introduces awareness of reconstruction goal and improves final quantitative performance. The proposed approach is compatible with a wide range of denoiser network structures and reconstruction modules, to suit customized need or further improve performance.

Circulating tumor cells (CTCs) and tumor-derived extracellular vesicles (tEVs) are two crucial methodologies of liquid biopsy. Given their distinct size differences and release dynamics, CTCs and tEVs potentially offer synergistic capabilities in the non-invasive detection of differentiated thyroid cancer (DTC), a typically indolent tumor. We present the Combined DTC CTC/tEV Assay, integrating dual liquid biopsy processes: i) DTC CTC enrichment by Click Chips, followed by analysis of seven DTC-specific genes, and ii) DTC tEV enrichment by Click Beads, succeeded by mRNA cargo quantification in DTC tEVs. This method utilizes click chemistry, leveraging a pair of biorthogonal and highly reactive functional motifs (tetrazine, Tz, and trans-cyclooctene, TCO), to overcome the challenges encountered in the conventional immunoaffinity-based enrichment of CTCs and tEVs. The Combined DTC CTC/tEV Assay synergistically combines the diagnostic precision of CTCs with the sensitivity of tEVs, demonstrating superior diagnostic accuracy in DTC detection and boasting an AUROC of 0.99. This outperforms the individual diagnostic performance of using either DTC CTC or DTC tEV alone. This integration enables full utilization of a patient's blood sample, and marks a significant evolution in the development of nanomaterial-based liquid biopsy technologies to address challenging unmet clinical needs in cancer care.

Temporal development of neural electrophysiology follows genetic programming, similar to cellular maturation and organization during development. The emergent properties of this electrophysiological development, namely neural oscillations, can be used to characterize brain development. Recently, we utilized the innate programming encoded in the human genome to generate functionally mature cortical organoids. In brief, stem cells are suspended in culture via continuous shaking and naturally aggregate into embryoid bodies before being exposed to media formulations for neural induction, differentiation and maturation. The specific culture format, media composition and duration of exposure to these media distinguish organoid protocols and determine whether a protocol is guided or unguided toward specific neural fate. The 'semi-guided' protocol presented here has shorter induction and differentiation steps with less-specific patterning molecules than most guided protocols but maintains the use of neurotrophic factors such as brain-derived growth factor and neurotrophin-3, unlike unguided approaches. This approach yields the cell type diversity of unguided approaches while maintaining reproducibility for disease modeling. Importantly, we characterized the electrophysiology of these organoids and found that they recapitulate the maturation of neural oscillations observed in the developing human brain, a feature not shown with other approaches. This protocol represents the potential first steps toward bridging molecular and cellular biology to human cognition, and it has already been used to discover underlying features of human brain development, evolution and neurological conditions. Experienced cell culture technicians can expect the protocol to take 1 month, with extended maturation, electrophysiology recording, and adeno-associated virus transduction procedure options.

When a new molecular entity is predicted to exhibit high clearance in humans, pharmaceutical sponsors almost universally search for similar acting back-up compounds that will demonstrate low clearance. Here we show that, except for oral dosing, there can be marked advantages to developing and commercializing controlled release formulations of high clearance drugs, the expertise of readers of this journal. Our recent publications demonstrate that the universally held pharmacokinetic principle that drug delivery rate has no effect on measured drug clearance is not correct. Rather, we show that if clearance from the drug delivery site is markedly less than the iv bolus clearance of a drug, the in vivo drug clearance can be the drug delivery clearance controlled by the designed dosage form. This approach will be especially advantageous for high hepatic clearance drugs. These advantages include not being concerned with: a) saturable nonlinear kinetics, b) significant pharmacogenomic differences, c) drug-drug induction mechanisms, and d) in many cases drug-drug inhibition interactions. This is due to the ability of a drug sponsor to design clearance, independent of the pharmacokinetic characteristics for high clearance compounds, where clearance from the dosage form becomes the drug clearance from the patient. Recognition of this principle, as described here, results from our development of the use of Kirchhoff's Laws from physics to derive rate-defining clearance and rate constant elimination processes independent of differential equation derivations. The key message for readers of this journal is that high clearance drugs are potentially drugable through formulation design and should not be outright disregarded, since for such drugs the dose-corrected area under the curve can be increased if the release rate from the injection site is controlled and slow resulting in drug clearance from the body controlled by clearance from the dosage form. The concepts presented here describe previously unrecognized advantages of controlled release formulations.

It remains a challenge to produce soft robots that can mimic the responsive adaptability of living organisms. Rather than fabricating soft robots from bulk hydrogels,hydrogels are integrated into the interfacial assembly of aqueous two-phase systems to generate ultra-soft and elastic all-aqueous aquabots that exhibit responsive adaptability, that can shrink on demand and have electrically conductive functions. The adaptive functions of the aquabots provide a new platform to develop minimally invasive surgical devices, targeted drug delivery systems, and flexible electronic sensors and actuators.

Background: Autologous tissues such as buccal mucosa (BM) are widely used for reconstruction of urethral strictures; however, limitations such as donor site morbidity and scarce tissue supply require the development of alternative biomaterials for urethral repair. The goals of this study were to determine the safety and efficacy of bi-layer silk fibroin (BLSF) matrices for urethral stricture repair and compare histological and functional outcomes to the standard approach, BM urethroplasty under good laboratory practices.Material and methods: A total of 13 rabbits exhibiting urethral stricture formation following electrocoagulation injury were treated with onlay urethroplasty with either acellular BLSF (N = 7) or autologous BM (N = 6) grafts for 3 months. Uninjured control rabbits were maintained in parallel (N = 4).Results and conclusion: Animals receiving BLSF implants were demonstrated to be functionally equivalent to BM grafts in their ability to restored strictured calibers, support micturition and promote tissue regeneration with minimal inflammation.

Effective transport of biological systems as cargo during space travel is a critical requirement to use synthetic biology and biomanufacturing in outer space. Bioproduction using microbes will drive the extent to which many human needs can be met in environments with limited resources. Vast repositories of biological parts and strains are available to meet this need, but their on-site availability requires effective transport. Here, we explore an approach that allows DNA plasmids, ubiquitous synthetic biology parts, to be safely transported to the International Space Station and back to the Kennedy Space Center without low-temperature or cryogenic stowage. Our approach relied on the cyanobacterium Nostoc punctiforme PC73102, which is naturally tolerant to prolonged desiccation. Desiccated N. punctiforme was able to carry the non-native pSCR119 plasmid as intracellular cargo safely to space and back. Upon return to the laboratory, the extracted plasmid showed no DNA damage or additional mutations and could be used as intended to transform the model synbio host Escherichia coli to bestow kanamycin resistance. This proof-of-concept study provides the foundation for a ruggedized transport host for DNA to environments where there is a need to reduce equipment and infrastructure for biological parts stowage and storage.

Fish oils rank among the world's most popular nutritional supplements and are purported to have numerous health benefits. Previous work suggested that fish oils increase collagen production; however, the effect of fish oils on musculoskeletal health is poorly understood. Further, the divergent effects of omega-3 (Ω3FA) and saturated fatty acids (SFA) remains poorly understood. We tested the effects of Ω3FA and SFAs on in vitro-engineered human ligament (EHL) function. EHLs were treated with bovine serum albumin (BSA)-conjugated eicosapentaenoic acid (EPA, 20:5(n-3)), palmitic acid (PA, 16:0), or a BSA control for 6 days. EPA did not significantly alter, whereas PA significantly decreased EHL function and collagen content. To determine whether this was an in vitro artifact, mice were fed a control or high-lard diet for 14 weeks and musculoskeletal mass, insulin sensitivity, and the collagen content, and mechanics of tendon and bone were determined. Body weight was 40 % higher on a HFD, but muscle, tendon, and bone mass did not keep up with body weight resulting in relative losses in muscle mass, tendon, and bone collagen, as well as mechanical properties. Importantly, we show that PA acutely decreases collagen synthesis in vitro to a similar extent as the decrease in collagen content with chronic treatment. These data suggest that Ω3FAs have a limited effect on EHLs, whereas SFA exert a negative effect on collagen synthesis resulting in smaller and weaker musculoskeletal tissues both in vitro and in vivo.

PurposePhoton counting CT (PCCT) provides spectral measurements for material decomposition. However, the image noise (at a fixed dose) depends on the source spectrum. Our study investigates the potential benefits from spectral optimization using fast kV switching and filtration to reduce noise in material decomposition.ApproachThe effect of the input spectra on noise performance in both two-basis material decomposition and three-basis material decomposition was compared using Cramer-Rao lower bound analysis in the projection domain and in a digital phantom study in the image domain. The fluences of different spectra were normalized using the CT dose index to maintain constant dose levels. Four detector response models based on Si or CdTe were included in the analysis.ResultsFor single kV scans, kV selection can be optimized based on the imaging task and object size. Furthermore, our results suggest that noise in material decomposition can be substantially reduced with fast kV switching. For two-material decomposition, fast kV switching reduces the standard deviation (SD) by ∼10% . For three-material decomposition, greater noise reduction in material images was found with fast kV switching (26.2% for calcium and 25.8% for iodine, in terms of SD), which suggests that challenging tasks benefit more from the richer spectral information provided by fast kV switching.ConclusionsThe performance of PCCT in material decomposition can be improved by optimizing source spectrum settings. Task-specific tube voltages can be selected for single kV scans. Also, our results demonstrate that utilizing fast kV switching can substantially reduce the noise in material decomposition for both two- and three-material decompositions, and a fixed Gd filter can further enhance such improvements for two-material decomposition.

Human serum albumin (HSA) is currently used as a plasma expander (PE) to increase blood volume during hypovolemic conditions, such as blood loss. However, its effectiveness is suboptimal in septic shock and burn patients due to their enhanced endothelial permeability, resulting in HSA extravasation into the tissue space leading to edema, and deposition of toxic HSA-bound metabolites. Hence, to expand HSA's applicability toward treating patients with compromised endothelial permeability, HSA has been previously polymerized to increase its molecular size thus compartmentalizing the polymerized HSA (PolyHSA) molecules in the vascular space. Previous studies bracketed PolyHSA between 100 kDa and 0.2 μm. In this research, PolyHSA was synthesized at two cross-link densities 43:1 and 60:1 (i.e., molar ratios of glutaraldehyde to HSA) and subsequently fractionated via tangential flow filtration (TFF) into two narrower brackets: bracket A (500 kDa and 0.2 μm) and bracket B (50-500 kDa). PolyHSA within the same size bracket at different cross-link densities exhibited similar solution viscosity, zeta potential, and osmolality but differed in hydrodynamic diameter. At the same cross-link density, the PolyHSA A bracket showed higher viscosity, lowered zeta potential, and a larger hydrodynamic diameter compared with the PolyHSA B bracket while maintaining osmolality. Interestingly, PolyHSA 43:1 B, PolyHSA 60:1 A, and PolyHSA 60:1 B brackets exhibited colloid osmotic pressure similar to HSA, indicating their potential to serve as PEs.

Acute limb ischaemia (ALI) is an emergent clinical condition that strains pre-hospital resources and impacts healthcare costs and patient quality of life. Hypothermia has long been used in clinical and research settings to mitigate ischaemic damage in tissues, but prompt reperfusion is needed to prevent loss of limb or function from ALI. To address the unmet need for pre-hospital intervention of threatened limbs awaiting definitive specialty care, we have focused on controlled application of hypothermia. Over years of animal experiments, phantom limb creation, and materials selection, we conceptualised and created a portable limb-cooling device that can be used alone or combined with a traditional tourniquet or resuscitative endovascular balloon occlusion of the aorta. Here, we describe our process of building and testing the device, from computer simulation through animal-limb metabolic studies, to prototype testing.

Diabetic foot ulcers (DFU) are chronic, refractory wounds caused by diabetic neuropathy, vascular disease, and bacterial infection, and have become one of the most serious and persistent complications of diabetes mellitus because of their high incidence and difficulty in healing. Its malignancy results from a complex microenvironment that includes a series of unfriendly physiological states secondary to hyperglycemia, such as recurrent infections, excessive oxidative stress, persistent inflammation, and ischemia and hypoxia. However, current common clinical treatments, such as antibiotic therapy, insulin therapy, surgical debridement, and conventional wound dressings all have drawbacks, and suboptimal outcomes exacerbate the financial and physical burdens of diabetic patients. Therefore, development of new, effective and affordable treatments for DFU represents a top priority to improve the quality of life of diabetic patients. In recent years, nanozymes-based diabetic wound therapy systems have been attracting extensive interest by integrating the unique advantages of nanomaterials and natural enzymes. Compared with natural enzymes, nanozymes possess more stable catalytic activity, lower production cost and greater maneuverability. Remarkably, many nanozymes possess multienzyme activities that can cascade multiple enzyme-catalyzed reactions simultaneously throughout the recovery process of DFU. Additionally, their favorable photothermal-acoustic properties can be exploited for further enhancement of the therapeutic effects. In this review we first describe the characteristic pathological microenvironment of DFU, then discuss the therapeutic mechanisms and applications of nanozymes in DFU healing, and finally, highlight the challenges and perspectives of nanozyme development for DFU treatment.

Virulent infectious agents such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and methicillin-resistant Staphylococcus aureus (MRSA) induce tissue damage that recruits neutrophils, monocyte, and macrophages, leading to T cell exhaustion, fibrosis, vascular leak, epithelial cell depletion, and fatal organ damage. Neutrophils, monocytes, and macrophages recruited to pathogen-infected lungs, including SARS-CoV-2-infected lungs, express phosphatidylinositol 3-kinase gamma (PI3Kγ), a signaling protein that coordinates both granulocyte and monocyte trafficking to diseased tissues and immune-suppressive, profibrotic transcription in myeloid cells. PI3Kγ deletion and inhibition with the clinical PI3Kγ inhibitor eganelisib promoted survival in models of infectious diseases, including SARS-CoV-2 and MRSA, by suppressing inflammation, vascular leak, organ damage, and cytokine storm. These results demonstrate essential roles for PI3Kγ in inflammatory lung disease and support the potential use of PI3Kγ inhibitors to suppress inflammation in severe infectious diseases.

Algae biotechnology holds immense promise for revolutionizing the bioeconomy through the sustainable and scalable production of various bioproducts. However, their development has been hindered by the lack of advanced genetic tools. This study introduces a synthetic biology approach to develop such tools, focusing on the construction and testing of synthetic promoters. By analyzing conserved DNA motifs within the promoter regions of highly expressed genes across six different algal species, we identified cis-regulatory elements (CREs) associated with high transcriptional activity. Combining the algorithms POWRS, STREME, and PhyloGibbs, we predicted 1511 CREs and inserted them into a minimal synthetic promoter sequence in 1, 2, or 3 copies, resulting in 4533 distinct synthetic promoters. These promoters were evaluated in vivo for their capacity to drive the expression of a transgene in a high-throughput manner through next-generation sequencing post antibiotic selection and fluorescence-activated cell sorting. To validate our approach, we sequenced hundreds of transgenic lines showing high levels of GFP expression. Further, we individually tested 14 identified promoters, revealing substantial increases in GFP expression─up to nine times higher than the baseline synthetic promoter, with five matching or even surpassing the performance of the native AR1 promoter. As a result of this study, we identified a catalog of CREs that can now be used to build superior synthetic algal promoters. More importantly, here we present a validated pipeline to generate building blocks for innovative synthetic genetic tools applicable to any algal species with a sequenced genome and transcriptome data set.

Three-dimensional variation in structural components or fiber alignments results in complex mechanical property distribution in tissues and biomaterials. In this paper, we use a physics-informed UNet-based neural network model (El-UNet) to discover the three-dimensional (3D) internal composition and space-dependent material properties of heterogeneous isotropic and transversely isotropic materials without a priori knowledge of the composition. We then show the capabilities of El-UNet by validating against data obtained from finite-element simulations of two soft tissues, namely, brain tissue and articular cartilage, under various loading conditions. We first simulated compressive loading of 3D brain tissue comprising of distinct white matter and gray matter mechanical properties undergoing small strains with isotropic linear elastic behavior, where El-UNet reached mean absolute relative errors under 1.5 % for elastic modulus and Poisson's ratio estimations across the 3D volume. We showed that the 3D solution achieved by El-UNet was superior to relative stiffness mapping by inverse of axial strain and two-dimensional plane stress/plane strain approximations. Additionally, we simulated a transversely isotropic articular cartilage with known fiber orientations undergoing compressive loading, and accurately estimated the spatial distribution of all five material parameters, with mean absolute relative errors under 5 %. Our work demonstrates the application of the computationally efficient physics-informed El-UNet in 3D elasticity imaging and provides methods for translation to experimental 3D characterization of soft tissues and other materials. The proposed El-UNet offers a powerful tool for both in vitro and ex vivo tissue analysis, with potential extensions to in vivo diagnostics. STATEMENT OF SIGNIFICANCE: Elasticity imaging is a technique that reconstructs mechanical properties of tissue using deformation and force measurements. Given the complexity of this reconstruction, most existing methods have mostly focused on 2D problems. Our work is the first implementation of physics-informed UNets to reconstruct three-dimensional material parameter distributions for isotropic and transversely isotropic linear elastic materials by having deformation and force measurements. We comprehensively validate our model using synthetic data generated using finite element models of biological tissues with high bio-fidelity-the brain and articular cartilage. Our method can be implemented in elasticity imaging scenarios for in vitro and ex vivo mechanical characterization of biomaterials and biological tissues, with potential extensions to in vivo diagnostics.

PurposeHigh quality scan prescription that optimally covers the area of interest with scan planes aligned to relevant anatomical structures is crucial for error-free radiologic interpretation. The goal of this project was to develop a machine learning pipeline for oblique scan prescription that could be trained on localizer images and metadata from previously acquired MR exams.MethodsA novel Multislice Rotational Region-based Convolutional Neural Network (MS-R2CNN) architecture was developed. Based on this architecture, models for automated prescription sagittal lumbar spine acquisitions from axial, sagittal, and coronal localizer slices were trained. The automated prescription pipeline was integrated with the scanner console software and evaluated in experiments with healthy volunteers (N = 3) and patients with lower-back pain (N = 20).ResultsExperiments in healthy volunteers demonstrated high accuracy of automated prescription in all subjects. There was good agreement between alignment and coverage of manual and automated prescriptions, as well as consistent views of the lumbar spine at different positions of the subjects within the scanner bore. In patients with lower-back pain, the generated prescription was applied in 18 cases (90% of the total number). None of the cases required major adjustment, while in 11 cases (55%) there were minor manual adjustments to the generated prescription.ConclusionsThis study demonstrates the ability of oriented object detection-based models to be trained to prescribe oblique lumbar spine MRI acquisitions without the need of manual annotation or feature engineering and the feasibility of using machine learning-based pipelines on the scanner for automated prescription of MRI acquisitions.

BackgroundApplying ultra-high dose rates to radiation therapy, otherwise known as FLASH, has been shown to be just as effective while sparing more normal tissue compared to conventional radiation therapy. However, there is a need for a dosimeter that is able to detect such high instantaneous dose, particularly in vivo. To fulfill this need, protoacoustics is introduced, which is an in vivo range verification method with submillimeter accuracy.PurposeThe purpose of this work is to demonstrate the feasibility of using protoacoustics as a method of in vivo real-time monitoring during FLASH proton therapy and investigating the resulting protoacoustic signal when dose per pulse and pulsewidth are varied through multiple simulation studies.MethodsThe dose distribution of a proton pencil beam was calculated through a Monte Carlo toolbox, TOPAS. Next, the k-Wave toolbox in MATLAB was used for performing protoacoustic simulations, where the initial proton dose deposition was inputted to model acoustic propagations, which were also used for reconstructions. Simulations involving the manipulation of the dose per pulse and pulsewidth were performed, and the temporal and spatial resolution for protoacoustic reconstructions were investigated as well. A 3D reconstruction was performed with a multiple beam spot profile to investigate the spatial resolution as well as determine the feasibility of 3D imaging with protoacoustics.ResultsOur results showed consistent linearity in the increasing dose-per-pulse, even up to rates considered for FLASH. The simulations and reconstructions were performed for a range of pulsewidths from 0.1 to 10 μs. The results show the characteristics of the proton beam after convolving the protoacoustic signal with the varying pulsewidths. 3D reconstruction was successfully performed with each beam being distinguishable using an 8 cm × 8 cm planar array. These simulation results show that measurements using protoacoustics has the potential for in vivo dosimetry in FLASH therapy during patient treatments in real time.ConclusionThrough this simulation study, the use of protoacoustics in FLASH therapy was verified and explored through observations of varying parameters, such as the dose per pulse and pulsewidth. 2D and 3D reconstructions were also completed. This study shows the significance of using protoacoustics and provides necessary information, which can further be explored in clinical settings.

Vitamin B12 is critical for hematopoiesis and myelination. Deficiency can cause neurologic deficits including loss of coordination and cognitive decline. However, diagnosis relies on measurement of vitamin B12 in the blood, which may not accurately reflect the concentration in the brain. Using programmable phage display, we identified an autoantibody targeting the transcobalamin receptor (CD320) in a patient with progressive tremor, ataxia, and scanning speech. Anti-CD320 impaired cellular uptake of cobalamin (B12) in vitro by depleting its target from the cell surface. Despite a normal serum concentration, B12 was nearly undetectable in her cerebrospinal fluid (CSF). Immunosuppressive treatment and high-dose systemic B12 supplementation were associated with increased B12 in the CSF and clinical improvement. Optofluidic screening enabled isolation of a patient-derived monoclonal antibody that impaired B12 transport across an in vitro model of the blood-brain barrier (BBB). Autoantibodies targeting the same epitope of CD320 were identified in seven other patients with neurologic deficits of unknown etiology, 6% of healthy controls, and 21.4% of a cohort of patients with neuropsychiatric lupus. In 132 paired serum and CSF samples, detection of anti-CD320 in the blood predicted B12 deficiency in the brain. However, these individuals did not display any hematologic signs of B12 deficiency despite systemic CD320 impairment. Using a genome-wide CRISPR screen, we found that the low-density lipoprotein receptor serves as an alternative B12 uptake pathway in hematopoietic cells. These findings dissect the tissue specificity of B12 transport and elucidate an autoimmune neurologic condition that may be amenable to immunomodulatory treatment and nutritional supplementation.

Glycosylation is a ubiquitous modification present across all of biology, affecting many things such as physicochemical properties, cellular recognition, subcellular localization, and immunogenicity. Nucleotide sugars are important precursors needed to study glycosylation and produce glycosylated products. Saccharomyces cerevisiae is a potentially powerful platform for producing glycosylated biomolecules, but it lacks nucleotide sugar diversity. Nucleotide sugar metabolism is complex, and understanding how to engineer it will be necessary to both access and study heterologous glycosylations found across biology. This review overviews the potential challenges with engineering nucleotide sugar metabolism in yeast from the salvage pathways that convert free sugars to their associated UDP-sugars to de novo synthesis where nucleotide sugars are interconverted through a complex metabolic network with governing feedback mechanisms. Finally, recent examples of engineering complex glycosylation of small molecules in S. cerevisiae are explored and assessed.

Synthetic biology constitutes a scientific domain focused on intentional redesign of organisms to confer novel functionalities or create new products through strategic engineering of their genetic makeup. Leveraging the inherent capabilities of nature, one may address challenges across diverse sectors including medicine. Inspired by this concept, we have developed an innovative bioengineering platform, enabling high-yield and large-scale production of biological small interfering RNA (BioRNA/siRNA) agents via bacterial fermentation. Herein, we show that with the use of a new tRNA fused pre-miRNA carrier, we can produce various forms of BioRNA/siRNA agents within living host cells. We report a high-level overexpression of nine target BioRNA/siRNA molecules at 100% success rate, yielding 3-10 mg of BioRNA/siRNA per 0.25 L of bacterial culture with high purity (>98%) and low endotoxin (

PurposeTo report on cases of unilateral perimacular atrophy after treatment with voretigene neparvovec-rzyl, in the setting of previous contralateral eye treatment with a different viral vector.DesignSingle-center, retrospective chart review.MethodsIn this case series, four patients between the ages of six and 11 years old with RPE65-related retinopathy were treated unilaterally with rAAV2-CB-hRPE65 as part of a gene augmentation clinical trial (NCT00749957). Six to 10 years later the contralateral eyes were treated with the Food and Drug Administration-approved drug, voretigene neparvovec-rzyl. Best-corrected visual acuity (BCVA), fundus photos, ocular coherence tomography, two-color dark-adapted perimetry, full field stimulus threshold testing (FST), and location of subretinal bleb and chorioretinal atrophy were evaluated.ResultsThree out of four patients showed unilateral perimacular atrophy after treatment with voretigene, ranging from five to 22 months after treatment. Areas of robust visual field improvement were followed by areas of chorioretinal atrophy. Despite perimacular changes, BCVA, FST, and subjective improvements in vision and nyctalopia were maintained. Perimacular atrophy was not observed in the first eye treated with the previous viral vector.ConclusionsWe observed areas of robust visual field improvement followed by perimacular atrophy in voretigene treated eyes, as compared to the initially treated contralateral eyes.Translational relevanceCaution is advised when using two different viral vectors between eyes in gene therapy. This may become an important issue in the future with increasing gene therapy clinical trials for inherited retinal dystrophies.

Some gene polymorphisms can lead to monogenic diseases, whereas other polymorphisms may confer beneficial traits. A well-characterized example is congenital erythrocytosis-the non-pathogenic hyper-production of red blood cells-that is caused by a truncated erythropoietin receptor. Here we show that Cas9-mediated genome editing in CD34+ human haematopoietic stem and progenitor cells (HSPCs) can recreate the truncated form of the erythropoietin receptor, leading to substantial increases in erythropoietic output. We also show that combining the expression of the cDNA of a truncated erythropoietin receptor with a previously reported genome-editing strategy to fully replace the HBA1 gene with an HBB transgene in HSPCs (to restore normal haemoglobin production in cells with a β-thalassaemia phenotype) gives the edited HSPCs and the healthy red blood cell phenotype a proliferative advantage. Combining knowledge of human genetics with precise genome editing to insert natural human variants into therapeutic cells may facilitate safer and more effective genome-editing therapies for patients with genetic diseases.

Evaluation of the 3D bone morphology of the glenohumeral joint is necessary for pre-surgical planning. Zero echo time (ZTE) magnetic resonance imaging (MRI) provides excellent bone contrast and can potentially be used in the place of computed tomography. Segmentation of the shoulder anatomy, particularly the humeral head and the acetabulum, is needed for the detailed assessment of each anatomy and for pre-surgical preparation. In this study, we compared the performance of two popular deep learning models based on Google’s DeepLab and U-Net to perform automated segmentation on ZTE MRI of human shoulders. Axial ZTE images of normal shoulders (n = 31) acquired at 3-Tesla were annotated for training with DeepLab and 2D U-Net, and the trained model was validated with testing data (n = 13). While both models showed visually satisfactory results for segmenting the humeral bone, U-Net slightly over-estimated while DeepLab under-estimated the segmented area compared to the ground truth. Testing accuracy quantified by Dice score was significantly higher (p < 0.05) for U-Net (88%) than DeepLab (81%) for the humeral segmentation. We have also implemented the U-Net model onto an MRI console for push-button DL segmentation processing. Although this is an early work with limitations, our approach has the potential to improve shoulder MR evaluation hindered by manual post-processing and may provide clinical benefit for quickly visualizing bones of the glenohumeral joint.

Objective. Prompt gamma timing (PGT) uses the detection time of prompt gammas emitted along the range of protons in proton radiotherapy to verify the position of the Bragg peak (BP). Cherenkov detectors offer the possibility of enhanced signal-to-noise ratio (SNR) due to the inherent physics of Cherenkov emission which enhances detection of high energy prompt gamma rays relative to other induced uncorrelated signals. In this work, the PGT technique was applied to 3 semiconductor material slabs that emit only Cherenkov light for use in a full scale system: a 3 × 3 × 20 mm3TlBr, a 12 × 12 × 12 mm3TlBr, and a 5 × 5 × 5 mm3TlCl.Approach. A polymethyl methacrylate (PMMA) target was exposed to a 67.5 MeV, 0.5 nA proton beam and shifted in 3 mm increments at the Crocker nuclear laboratory (CNL) in Davis, CA, USA. A fast plastic scintillator coupled to a photomultiplier tube (PMT) provided the start reference for the proton time of flight. Time of flight (TOF) distributions were generated using this reference and the gamma-ray timestamp in the Cherenkov detector.Main results. The SNR of the proton correlated peaks relative to the background was 20, 29, and 30 for each of the three samples, respectively. The upper limit of the position resolutions with the TlCl sample were 2 mm, 3 mm, and 5 mm for 30k, 10k, and 5k detected events, respectively. The time distribution of events with respect to the reference reproduced with clarity the periodicity of the beam, implying a very high SNR of the Cherenkov crystals to detect prompt gammas. Background presence from the neutron-induced continuum, prompt gammas from deuterium, or positron activation were not observed. Material choice and crystal dimensions did not seem to affect significantly the outcome of the results.Significance. These results show the high SNR of the pure Cherenkov emitters TlBr and TlCl for the detection of prompt gammas in a proton beam with current of clinical significance and their potential for verifying the proton range. The accuracy in determining shifts of the BP was highly dependent on the number of events acquired, therefore, the performance of these detectors are expected to vary with different beam conditions such as current, pulse repetition, and proton bunch width.

4D MRI with high spatiotemporal resolution is desired for image-guided liver radiotherapy. Acquiring densely sampled k-space data is time-consuming. Accelerated acquisition with sparse sampling is desirable but often causes degraded image quality or long reconstruction time. We propose the Reconstruct Paired Conditional Generative Adversarial Network (Re-Con-GAN) for shortening the 4D MRI reconstruction time while maintaining the reconstruction quality. Methods: Patients underwent free-breathing liver 4D MRI were included in the study. Fully- and retrospectively under-sampled data at 3, 6 and 10 times (3x, 6x and 10x) were first reconstructed using the nuFFT algorithm. Re-Con-GAN then trained input and output in pairs. Three types of networks, ResNet9, UNet and reconstruction swin transformer, were explored as generators. PatchGAN was selected as the discriminator. Re-Con-GAN processed the data (3D+t) as temporal slices (2D+t). A total of 48 patients with 12332 temporal slices were split into training (37 patients with 10721 slices) and test (11 patients with 1611 slices). Compressed sensing (CS) reconstruction with spatiotemporal sparsity constraint was used as a benchmark. Reconstructed image quality was further evaluated with a liver gross tumor volume (GTV) localization task using Mask-RCNN trained from a separate 3D static liver MRI dataset (70 patients; 103 GTV contours).Results: Re-Con-GAN consistently achieved comparable/better PSNR, SSIM, and RMSE scores compared to CS/UNet models. The inference time of Re-Con-GAN, UNet and CS are 0.15s, 0.16s, and 120s. The GTV detection task showed that Re-Con-GAN and CS, compared to UNet, better improved the dice score (3x Re-Con-GAN 80.98%; 3x CS 80.74%; 3x UNet 79.88%) of unprocessed under-sampled images (3x 69.61%). Conclusion: A generative network with adversarial training is proposed with promising and efficient reconstruction results demonstrated on an in-house dataset. The rapid and qualitative reconstruction of 4D liver MR has the potential to facilitate online adaptive MR-guided radiotherapy for liver cancer.

We herein present a multifunctional self-priming hairpin probe-based isothermal amplification, termed MSH, enabling one-pot detection of target nucleic acids. The sophisticatedly designed multifunctional self-priming hairpin (MSH) probe recognizes the target and rearranges to prime itself, triggering the amplification reaction powered by the continuously repeated extension, nicking, and target recycling. As a consequence, a large number of double-stranded DNA (dsDNA) amplicons are produced that could be monitored in real-time using a dsDNA-intercalating dye. Based on this unique design approach, the nucleocapsid (N) and the open reading frame 1 ab (ORF1ab) genes of SARS-CoV-2 were successfully detected down to 1.664 fM and 0.770 fM, respectively. The practical applicability of our method was validated by accurately diagnosing 60 clinical samples with 93.33% sensitivity and 96.67% specificity. This isothermal one-pot MSH technique holds great promise as a point-of-care testing protocol for the reliable detection of a wide spectrum of pathogens, particularly in resource-limited settings.

Rapid and precise detection of hydrogen peroxide (H2O2) holds great significance since it is linked to numerous physiological and inorganic catalytic processes. We herein developed a label-free and washing-free strategy to detect H2O2 by employing a hand-held personal glucose meter (PGM) as a signal readout device. By focusing on the fact that the reduced redox mediator ([Fe(CN)6]4-) itself is responsible for the final PGM signal, we developed a new PGM-based strategy to detect H2O2 by utilizing the target H2O2-mediated oxidation of [Fe(CN)6]4- to [Fe(CN)6]3- in the presence of horseradish peroxidase (HRP) and monitoring the reduced PGM signal in response to the target amount. Based on this straightforward and facile design principle, H2O2 was successfully determined down to 3.63 μM with high specificity against various non-target molecules. We further demonstrated that this strategy could be expanded to identify another model target choline by detecting H2O2 produced through its oxidation promoted by choline oxidase. Moreover, we verified its practical applicability by reliably determining extracellular H2O2 released from the breast cancer cell line, MDA-MB-231. This work could evolve into versatile PGM-based platform technology to identify various non-glucose target molecules by employing their corresponding oxidase enzymes, greatly advancing the portable biosensing technologies.

The intricate interplay between biochemical and physical cues dictates pluripotent stem cell (PSC) differentiation to form various tissues. While biochemical modulation has been extensively studied, the role of biophysical microenvironments in early lineage commitment remains elusive. Here, we introduce a novel 3D cell culture system combining electrospun nanofibers with microfabricated polydimethylsiloxane (PDMS) patterns. This system enables the controlled formation of semispherical human induced pluripotent stem cell (hiPSC) colonies, facilitating the investigation of local mechanical stem cell niches on mechano-responsive signaling and lineage specification. Our system unveiled spatially organized RhoA activity coupled with actin-myosin cable formation, suggesting mechano-dependent hiPSC behaviors. Nodal network analysis of RNA-seq data revealed RhoA downstream regulation of YAP signaling, DNA histone modifications, and patterned germ layer specification. Notably, altering colony morphology through controlled PDMS microwell shaping effectively modulated the spatial distribution of mechano-sensitive mediators and subsequent differentiation. This study provides a cell culture platform to decipher the role of biophysical cues in early embryogenesis, offering valuable insights for material design in tissue engineering and regenerative medicine applications.

Dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) is a routine method to noninvasively quantify perfusion dynamics in tissues. The standard practice for analyzing DCE-MRI data is to fit an ordinary differential equation to each voxel. Recent advances in data science provide an opportunity to move beyond existing methods to obtain more accurate measurements of fluid properties. Here, we developed a localized convolutional function regression that enables simultaneous measurement of interstitial fluid velocity, diffusion, and perfusion in 3D. We validated the method computationally and experimentally, demonstrating accurate measurement of fluid dynamics in situ and in vivo. Applying the method to human MRIs, we observed tissue-specific differences in fluid dynamics, with an increased fluid velocity in breast cancer as compared to brain cancer. Overall, our method represents an improved strategy for studying interstitial flows and interstitial transport in tumors and patients. We expect that our method will contribute to the better understanding of cancer progression and therapeutic response.

BackgroundAutologous tissues derived from bowel, buccal mucosa and skin are primarily used to repair or replace diseased vaginal segments as well as create neovaginas for male-to-female transgenders. These grafts are often limited by scarce tissue supply, donor site morbidity and post-operative complications. Bi-layer silk fibroin (BLSF) biomaterials represent potential alternatives for vaginoplasty given their structural strength and elasticity, low immunogenicity, and processing flexibility. The goals of the current study were to assess the potential of acellular BLSF scaffolds for vaginal tissue regeneration in respect to conventional small intestinal submucosal (SIS) matrices in a rat model of vaginoplasty.MethodsInlay vaginoplasty was performed with BLSF and SIS scaffolds (N = 21 per graft) in adult female rats for up to 2 months of implantation. Nonsurgical controls (N = 4) were investigated in parallel. Outcome analyses included histologic, immunohistochemical and histomorphometric evaluations of wound healing patterns; µ-computed tomography (CT) of vaginal continuity; and breeding assessments.ResultsAnimals in both scaffold cohorts exhibited 100% survival rates with no severe post-operative complications. At 2 months post-op, µ-CT analysis revealed normal vaginal anatomy and continuity in both graft groups similar to controls. In parallel, BLSF and SIS grafts also induced comparable constructive remodeling patterns and were histologically equivalent in their ability to support formation of vascularized vaginal neotissues with native tissue architecture, however with significantly less smooth muscle content. Vaginal tissues reconstructed with both implants were capable of supporting copulation, pregnancy and similar amounts of live births.ConclusionsBLSF biomaterials represent potential "off-the-shelf" candidates for vaginoplasty.

SignificanceIt has long been hypothesized that naturally occurring electric fields (EFs) aid wound healing by guiding cell migration. Consequently, the application of EFs has significant potential for promoting wound healing. However, the mechanisms underlying the cellular response to EFs remain unclear. Recent Advances： Although the directed migration of isolated single cells under EFs has been studied for decades, only recently has experimental evidence demonstrated the distinct collective migration of large sheets of keratinocytes and corneal epithelial cells in response to applied EFs. Accumulating evidence suggests that the emergent properties of cell groups in response to EF guidance offer new opportunities for EF-assisted directional migration.Critical issuesIn this review, we provide an overview of the field of collective electrotaxis, highlighting key advances made in recent years. We also discuss advanced engineering strategies utilized to manipulate collective electrotaxis.Future directionsWe outline a series of unanswered questions in this field and propose potential applications of collective electrotaxis in developing electrical stimulation technologies for wound healing.

Tissue engineering can provide in vitro models for drug testing, disease modeling, and perhaps someday, tissue/organ replacements. For building 3D heart tissue, the alignment of cardiac cells or cardiomyocytes (CMs) is important in generating a synchronously contracting tissue. To that end, researchers have generated several fabrication methods for building heart tissue, but direct comparisons of pros and cons using the same cell source is lacking. Here, we derived cardiomyocytes (CMs) from human induced pluripotent stem cells (hiPSCs) and compare the assembly of these cells using three fabrication methods: cardiospheres, muscle rings, and muscle strips. All three protocols successfully generated compacted tissue comprised of hiPSC-derived CMs stable for at least 2 weeks. The percentage of aligned cells was greatest in the muscle strip (55%) and the muscle ring (50%) compared with the relatively unaligned cardiospheres (35%). The iPSC-derived CMs within the muscle strip also exhibited the greatest elongation, with elongation factor at 2.0 compared with 1.5 for the muscle ring and 1.2 for the cardiospheres. This is the first direct comparison of various fabrication techniques using the same cell source.

ObjectiveFast and accurate deformable image registration (DIR) that include DIR uncertainty estimation are essential for safe and reliable clinical deployment. While recent deep learning models have shown promise in predicting DIR with its uncertainty, challenges persist in proper uncertainty evaluation and hyperparameter optimization for these methods. This work aims to develop and evaluate a model that can perform fast DIR and predict its uncertainty in seconds.ApproachIn this study, we introduce a novel probabilistic multi-resolution image registration model utilizing convolutional neural networks (CNNs) to estimate a multivariate normal distributed dense displacement field (DDF) in a multimodal image registration problem. To assess the quality of the DDF distribution predicted by the model, we propose a new metric based on the Kullback-Leibler divergence. The performance of our approach was evaluated against three other DIR algorithms (VoxelMorph, Monte Carlo Drop-Out, and Monte Carlo B-splines) capable of predicting uncertainty. The evaluation of the models included not only the quality of the deformation but also the reliability of the estimated uncertainty. Our application investigated registration of a treatment planning computed tomography (CT) to follow-up cone beam CT for daily adaptive radiotherapy.Main resultsThe hyperparameter tuning of the models showed that there is a trade-off between the reliability of the estimated uncertainty and the accuracy of the deformation. In the optimal trade-off our model excelled in contour propagation and uncertainty estimation (p < 0.01) compared to existing uncertainty estimation models. We obtained an average dice similarity coefficient of 0.89 and a KL-divergence of 0.15.SignificanceBy addressing challenges in DIR uncertainty estimation and evaluation, our work showed that both the DIR and its uncertainty can be reliably predicted paving the way for safe deployment in clinical environment..

Osteoarthritis is characterized by enzymatic breakdown of the articular cartilage via the disruption of chondrocyte homeostasis, ultimately resulting in the destruction of the articular surface. Decades of research have highlighted the importance of inflammation in osteoarthritis progression, with inflammatory cytokines shifting resident chondrocytes into a pro-catabolic state. Inflammation can result in poor outcomes for cells implanted for cartilage regeneration. Therefore, a method to promote the growth of new cartilage and protect the implanted cells from the pro-inflammatory cytokines found in the joint space is required. In this study, we fabricate two gel types: polymer network hydrogels composed of chondroitin sulfate and hyaluronic acid, glycosaminoglycans (GAGs) known for their anti-inflammatory and prochondrogenic activity, and interpenetrating networks of GAGs and collagen I. Compared to a collagen-only hydrogel, which does not provide an anti-inflammatory stimulus, chondrocytes in GAG hydrogels result in reduced production of pro-inflammatory cytokines and enzymes as well as preservation of collagen II and aggrecan expression. Overall, GAG-based hydrogels have the potential to promote cartilage regeneration under pro-inflammatory conditions. Further, the data have implications for the use of GAGs to generally support tissue engineering in pro-inflammatory environments.

The magnetoelectric behavior of epitaxial Fe-Ga microstructures on top of a (001)-oriented PMN-PT piezoelectric substrate is imaged with magnetic X-ray microscopy. Additionally, the micron-scale strain distribution in PMN-PT is characterized by X-ray microdiffraction and examined with respect to the results of the Fe-Ga magnetoelectric switching. The magnetic reorientation of Fe-Ga is found to be strongly correlated with size, shape, and crystallographic orientation of the microstructures. In the case of square-shaped structures, size dictates the influence of the strain distribution on both the initialization of the ground state and on the magnetic reorientation during application of voltage. On the other hand, elliptical microstructures demonstrate completely different magnetic responses depending on the relative orientation of their long axis with respect to the crystallographic directions of the PMN-PT. This study demonstrates that engineering the behavior of highly magnetostrictive epitaxial microdevices is possible. It further elucidates that voltage-induced actuation can be largely tuned to achieve the desired type of magnetic switching ranging from vortex circulation reversal, domain wall motion, to a large rotation of magnetization. Because of the outstanding properties of the investigated material system, the reported findings are expected to be of great interest for the realization of next-generation energy-efficient magnetic memory and logic devices.

Surface gel layers on commercially available contact lenses have been shown to reduce frictional shear stresses and mitigate damage during sliding contact with fragile epithelial cell layers in vitro. Spencer and co-workers recently demonstrated that surface gel layers could arise from oxygen-inhibited free-radical polymerization. In this study, polyacrylamide hydrogel shell probes (7.5 wt % acrylamide, 0.3 wt % N,N'-methylenebisacrylamide) were polymerized in three hemispherical molds listed in order of decreasing surface energy and increasing oxygen permeability: borosilicate glass, polyether ether ketone (PEEK), and polytetrafluoroethylene (PTFE). Hydrogel probes polymerized in PEEK and PTFE molds exhibited 100× lower elastic moduli at the surface (EPEEK* = 80 ± 31 and EPTFE* = 106 ± 26 Pa, respectively) than those polymerized in glass molds (Eglass* = 31,560 ± 1,570 Pa), in agreement with previous investigations by Spencer and co-workers. Biotribological experiments revealed that hydrogel probes with surface gel layers reduced frictional shear stresses against cells (τPEEK = 35 ± 15 and τPTFE = 22 ± 16 Pa) more than those without (τglass = 68 ± 15 Pa) and offered greater protection against cell damage when sliding against human telomerase-immortalized corneal epithelial (hTCEpi) cell monolayers. Our work demonstrates that the "mold effect" resulting in oxygen-inhibition polymerization creates hydrogels with surface gel layers that reduce shear stresses in sliding contact with cell monolayers, similar to the protection offered by gradient mucin gel networks across epithelial cell layers.

Six decades after its conception, proton computed tomography (pCT) and proton radiography have yet to be used in medical clinics. However, good progress has been made on relevant detector technologies in the past two decades, and a few prototype pCT systems now exist that approach the performance needed for a clinical device.The tracking and energy-measurement technologies in common use are described, as are the few pCT scanners that are in routine operation at this time.Most of these devices still look like detector R\&D efforts as opposed to medical devices, are difficult to use, are at least factor of five slower than desired for clinical use, and are too small to image many parts of the human body.Recommendations are made for what to consider when engineering a pre-clinical pCT scanner that is designed to meet clinical needs in terms of performance, cost, and ease of use.

Pancreatic ductal adenocarcinoma (PDAC) accounts for about 90% of all pancreatic cancer cases. Five-year survival rates have remained below 12% since the 1970s, in part due to the difficulty in detection prior to metastasis (migration and invasion into neighboring organs and glands). Mechanical memory is a concept that has emerged over the past decade that may provide a path toward understanding how invading PDAC cells "remember" the mechanical properties of their diseased ("stiff", elastic modulus, E ≈ 10 kPa) microenvironment even while invading a healthy ("soft", E ≈ 1 kPa) microenvironment. Here, we investigated the role of mechanical priming by culturing a dilute suspension of PDAC (FG) cells within a 3D, rheologically tunable microgel platform from hydrogels with tunable mechanical properties. We conducted a suite of acute (short-term) priming studies where we cultured PDAC cells in either a soft (E ≈ 1 kPa) or stiff (E ≈ 10 kPa) environment for 6 h, then removed and placed them into a new soft or stiff 3D environment for another 18 h. Following these steps, we conducted RNA-seq analyses to quantify gene expression. Initial priming in the 3D culture showed persistent gene expression for the duration of the study, regardless of the subsequent environments (stiff or soft). Stiff 3D culture was associated with the downregulation of tumor suppressors (LATS1, BCAR3, CDKN2C), as well as the upregulation of cancer-associated genes (RAC3). Immunofluorescence staining (BCAR3, RAC3) further supported the persistence of this cellular response, with BCAR3 upregulated in soft culture and RAC3 upregulated in stiff-primed culture. Stiff-primed genes were stratified against patient data found in The Cancer Genome Atlas (TCGA). Upregulated genes in stiff-primed 3D culture were associated with decreased survival in patient data, suggesting a link between patient survival and mechanical priming.

Macrophages are innate immune cells that interact with complex extracellular matrix environments, which have varied stiffness, composition, and structure, and such interactions can lead to the modulation of cellular activity. Collagen is often used in the culture of immune cells, but the effects of substrate functionalization conditions are not typically considered. Here, we show that the solvent system used to attach collagen onto a hydrogel surface affects its surface distribution and organization, and this can modulate the responses of macrophages subsequently cultured on these surfaces in terms of their inflammatory activation and expression of adhesion and mechanosensitive molecules. Collagen was solubilized in either acetic acid (Col-AA) or N-(2-hydroxyethyl)piperazine-N'-ethanesulfonic acid (HEPES) (Col-HEP) solutions and conjugated onto soft and stiff polyacrylamide (PA) hydrogel surfaces. Bone marrow-derived macrophages cultured under standard conditions (pH 7.4) on the Col-HEP-derived surfaces exhibited stiffness-dependent inflammatory activation; in contrast, the macrophages cultured on Col-AA-derived surfaces expressed high levels of inflammatory cytokines and genes, irrespective of the hydrogel stiffness. Among the collagen receptors that were examined, leukocyte-associated immunoglobulin-like receptor-1 (LAIR-1) was the most highly expressed, and knockdown of the Lair-1 gene enhanced the secretion of inflammatory cytokines. We found that the collagen distribution was more homogeneous on Col-AA surfaces but formed aggregates on Col-HEP surfaces. The macrophages cultured on Col-AA PA hydrogels were more evenly spread, expressed higher levels of vinculin, and exerted higher traction forces compared to those of cells on Col-HEP. These macrophages on Col-AA also had higher nuclear-to-cytoplasmic ratios of yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ), key molecules that control inflammation and sense substrate stiffness. Our results highlight that seemingly slight variations in substrate deposition for immunobiology studies can alter critical immune responses, and this is important to elucidate in the broader context of immunomodulatory biomaterial design.

The current paradigm of medicine is mostly designed to block or prevent pathological events. Once the disease-led tissue damage occurs, the limited endogenous regeneration may lead to depletion or loss of function for cells in the tissues. Cell therapy is rapidly evolving and influencing the field of medicine, where in some instances attempts to address cell loss in the body. Due to their biological function, engineerability, and their responsiveness to stimuli, cells are ideal candidates for therapeutic applications in many cases. Such promise is yet to be fully obtained as delivery of cells that functionally integrate with the desired tissues upon transplantation is still a topic of scientific research and development. Main known impediments for cell therapy include mechanical insults, cell viability, host's immune response, and lack of required nutrients for the transplanted cells. These challenges could be divided into three different steps: 1) Prior to, 2) during the and 3) after the transplantation procedure. In this review, we attempt to briefly summarize published approaches employing biomaterials to mitigate the above technical challenges. Biomaterials are offering an engineerable platform that could be tuned for different classes of cell transplantation to potentially enhance and lengthen the pharmacodynamics of cell therapies.

Cardiovascular organ-on-a-chip (OoC) devices are composed of engineered or native functional tissues that are cultured under controlled microenvironments inside microchips. These systems employ microfabrication and tissue engineering techniques to recapitulate human physiology. This review focuses on human OoC systems to model cardiovascular diseases, to perform drug screening, and to advance personalized medicine. We also address the challenges in the generation of organ chips that can revolutionize the large-scale application of these systems for drug development and personalized therapy.

Blood flow index (BFI) is an optically accessible parameter, with unit distance-squared-over-time, that is widely used as a proxy for tissue perfusion. BFI is defined as the dynamic scattering probability (i.e. the ratio of dynamic to overall reduced scattering coefficients) times an effective Brownian diffusion coefficient that describes red blood cell (RBC) motion. Here, using a wavelength division multiplexed, time-of-flight- (TOF) - resolved iNIRS system, we obtain TOF-resolved field autocorrelations at 773 nm and 855 nm via the same source and collector. We measure the human forearm, comprising biological tissues with mixed static and dynamic scattering, as well as a purely dynamic scattering phantom. Our primary finding is that forearm BFI increases from 773 nm to 855 nm, though the magnitude of this increase varies across subjects (23% ± 19% for N = 3). However, BFI is wavelength-independent in the purely dynamic scattering phantom. From these data, we infer that the wavelength-dependence of BFI arises from the wavelength-dependence of the dynamic scattering probability. This inference is further supported by RBC scattering literature. Our secondary finding is that the higher-order cumulant terms of the mean squared displacement (MSD) of RBCs are significant, but decrease with wavelength. Thus, laser speckle and related modalities should exercise caution when interpreting field autocorrelations.

Background: Asynchronous telepsychiatry (ATP) consultations are a novel form of psychiatric consultation. Studies comparing patient and provider satisfaction for ATP with that for synchronous telepsychiatry (STP) do not exist. Methods: This mixed-methods study is a secondary analysis of patients' and primary care providers' (PCPs) satisfaction from a randomized clinical trial of ATP compared with STP. Patients and their PCPs completed satisfaction surveys, and provided unstructured feedback about their experiences with either ATP or STP. Differences in patient satisfaction were assessed using mixed-effects logistic regression models, and the qualitative data were analyzed using thematic analysis with an inductive coding framework. Results: Patient satisfaction overall was high with 84% and 97% of respondents at 6 months reported being somewhat or completely satisfied with ATP and STP, respectively. Patients in the STP group were more likely to report being completely satisfied, to recommend the program to a friend, and to report being comfortable with their care compared with ATP (all p < 0.05). However, there was no difference between the patients in ATP and STP in perceived change in clinical outcomes (p = 0.51). The PCP quantitative data were small, and thus only summarized descriptively. Conclusions: Patients expressed their overall satisfaction with both STP and ATP. Patients in ATP reported more concerns about the process, likely because feedback after ATP was slower than that after STP consultations. PCPs had no apparent preference for STP or ATP, and reported implementing the psychiatrists' recommendations for both groups when such recommendations were made, which supports our previous findings. Trial Registration: ClinicalTrials.gov NCT02084979; https://clinicaltrials.gov/ct2/show/NCT02084979.