ARID1A suppresses R-loop-mediated STING-type I interferon pathway activation of anti-tumor immunity.

Clinical trials have identified ARID1A mutations as enriched among patients who respond favorably to immune checkpoint blockade (ICB) in several solid tumor types independent of microsatellite instability. We show that ARID1A loss in murine models is sufficient to induce anti-tumor immune phenotypes observed in ARID1A mutant human cancers, including increased CD8+ T cell infiltration and cytolytic activity. ARID1A-deficient cancers upregulated an interferon (IFN) gene expression signature, the ARID1A-IFN signature, associated with increased R-loops and cytosolic single-stranded DNA (ssDNA). Overexpression of the R-loop resolving enzyme, RNASEH2B, or cytosolic DNase, TREX1, in ARID1A-deficient cells prevented cytosolic ssDNA accumulation and ARID1A-IFN gene upregulation. Further, the ARID1A-IFN signature and anti-tumor immunity were driven by STING-dependent type I IFN signaling, which was required for improved responsiveness of ARID1A mutant tumors to ICB treatment. These findings define a molecular mechanism underlying anti-tumor immunity in ARID1A mutant cancers.

A quantitative gibberellin signaling biosensor reveals a role for gibberellins in internode specification at the shoot apical meristem.

Growth at the shoot apical meristem (SAM) is essential for shoot architecture construction. The phytohormones gibberellins (GA) play a pivotal role in coordinating plant growth, but their role in the SAM remains mostly unknown. Here, we developed a ratiometric GA signaling biosensor by engineering one of the DELLA proteins, to suppress its master regulatory function in GA transcriptional responses while preserving its degradation upon GA sensing. We demonstrate that this degradation-based biosensor accurately reports on cellular changes in GA levels and perception during development. We used this biosensor to map GA signaling activity in the SAM. We show that high GA signaling is found primarily in cells located between organ primordia that are the precursors of internodes. By gain- and loss-of-function approaches, we further demonstrate that GAs regulate cell division plane orientation to establish the typical cellular organization of internodes, thus contributing to internode specification in the SAM.

Masseter muscle thickness is predictive of cancer cachexia in patients with head and neck cancer.

BACKGROUND: Cancer cachexia is prevalent in head and neck cancer patients. The L3 skeletal muscle index (SMI) is often used to assess sarcopenia and cachexia but is infrequently able to be measured in this population. Masseter muscle thickness (MT) may serve as an alternative predictor of cachexia. METHODS: SMI and MT were calculated from 20 trauma (CTRL) and 40 cachectic (CA-CX) and non-cachectic (CA-NCX) head and neck cancer patients. Area Under the Curve of the Receiver Operating Characteristics (AUC-ROC) analysis was performed for SMI and MT. RESULTS: Both SMI and MT were significantly decreased in CA-CX patients (vs. CA-NCX mean difference -19.5 cm2/m2 and -2.06 mm, respectively) and significant predictors of CA-CX (AUC = 0.985 and 0.805, respectively). When analyzed by sex, the same findings were observed for MT in males and trended toward significance in females. CONCLUSIONS: Compared with SMI, MT is a good alternative prognostic biomarker to determine CA-CX status in HNC patients.

Spider mite herbivory induces an abscisic acid-driven stomatal defense.

Arthropod herbivory poses a serious threat to crop yield, prompting plants to employ intricate defense mechanisms against pest feeding. The generalist pest two-spotted spider mite (Tetranychus urticae) inflicts rapid damage and remains challenging due to its broad target range. In this study, we explored the Arabidopsis (Arabidopsis thaliana) response to T. urticae infestation, revealing the induction of abscisic acid (ABA), a hormone typically associated with abiotic stress adaptation, and stomatal closure during water stress. Leveraging a FRET-based ABA biosensor (nlsABACUS2-400n), we observed elevated ABA levels in various leaf cell types post-mite feeding. While ABA's role in pest resistance or susceptibility has been debated, an ABA-deficient mutant exhibited increased mite infestation alongside intact canonical biotic stress signaling, indicating an independent function of ABA in mite defense. We established that ABA-triggered stomatal closure effectively hinders mite feeding and minimizes leaf cell damage through genetic and pharmacological interventions targeting ABA levels, ABA signaling, stomatal aperture, and density. This study underscores the critical interplay between biotic and abiotic stresses in plants, highlighting how the vulnerability to mite infestation arising from open stomata, crucial for transpiration and photosynthesis, reinforces the intricate relationship between these stress types.

How an intrinsically disordered regulatory subunit assembles a PP1:eIF2 complex.

Fatalska et al.1 use an interdisciplinary strategy to elucidate how an intrinsically disordered regulatory subunit of protein phosphatase 1 binds trimeric eIF2 and positions the phosphatase-substrate complex for dephosphorylation. As validation, they show that a disease mutation abolishes the interaction.

Post-translational modification-centric base editor screens to assess phosphorylation site functionality in high throughput.

Signaling pathways that drive gene expression are typically depicted as having a dozen or so landmark phosphorylation and transcriptional events. In reality, thousands of dynamic post-translational modifications (PTMs) orchestrate nearly every cellular function, and we lack technologies to find causal links between these vast biochemical pathways and genetic circuits at scale. Here we describe the high-throughput, functional assessment of phosphorylation sites through the development of PTM-centric base editing coupled to phenotypic screens, directed by temporally resolved phosphoproteomics. Using T cell activation as a model, we observe hundreds of unstudied phosphorylation sites that modulate NFAT transcriptional activity. We identify the phosphorylation-mediated nuclear localization of PHLPP1, which promotes NFAT but inhibits NFκB activity. We also find that specific phosphosite mutants can alter gene expression in subtle yet distinct patterns, demonstrating the potential for fine-tuning transcriptional responses. Overall, base editor screening of PTM sites provides a powerful platform to dissect PTM function within signaling pathways.

Auxin co-receptor IAA17/AXR3 controls cell elongation in Arabidopsis thaliana root solely by modulation of nuclear auxin pathway.

The nuclear TIR1/AFB-Aux/IAA auxin pathway plays a crucial role in regulating plant growth and development. Specifically, the IAA17/AXR3 protein participates in Arabidopsis thaliana root development, response to auxin and gravitropism. However, the mechanism by which AXR3 regulates cell elongation is not fully understood. We combined genetical and cell biological tools with transcriptomics and determination of auxin levels and employed live cell imaging and image analysis to address how the auxin response pathways influence the dynamics of root growth. We revealed that manipulations of the TIR1/AFB-Aux/IAA pathway rapidly modulate root cell elongation. While inducible overexpression of the AXR3-1 transcriptional inhibitor accelerated growth, overexpression of the dominant activator form of ARF5/MONOPTEROS inhibited growth. In parallel, AXR3-1 expression caused loss of auxin sensitivity, leading to transcriptional reprogramming, phytohormone signaling imbalance and increased levels of auxin. Furthermore, we demonstrated that AXR3-1 specifically perturbs nuclear auxin signaling, while the rapid auxin response remains functional. Our results shed light on the interplay between the nuclear and cytoplasmic auxin pathways in roots, revealing their partial independence but also the dominant role of the nuclear auxin pathway during the gravitropic response of Arabidopsis thaliana roots.

Elective nodal dissection for cN0 intermediate-grade parotid mucoepidermoid carcinoma: A NCDB study.

PURPOSE: To determine the occult nodal disease rate and whether elective regional lymph node dissection (RLND) confers any 10-year overall survival (OS) in cN0 intermediate-grade mucoepidermoid carcinoma (MEC) of the parotid gland. MATERIALS & METHODS: The National Cancer Database was reviewed from 2004 to 2016 on adults with cT1-4aN0M0 intermediate-grade parotid MEC undergoing resection with/without RLND. Comparisons between patients with and without RLND were made. Occult nodal rate and 10-year overall survival (OS) were determined. RESULTS: Out of 898 included patients with cN0 intermediate grade parotid MEC undergoing elective RLND, the occult nodal rate was 7.6%. This was significantly different from low-grade (3.9%) and high-grade (25.7%) cN0 disease. When stratified by pT-classification, marginal differences were identified between low-grade and intermediate-grade tumors, whereas high-grade tumors demonstrated increased occult nodal disease with low T-stage (pT1-pT2, 20.4% vs. 5.1%) and high T-stage (pT3-pT4a, 32.1% vs. 17.6%). Patients undergoing elective RLND were more often treated at an academic facility (53.8% vs. 41.2%), had higher pT3-pT4 tumors (19.2% vs. 10.4%), and more frequently underwent total/radical parotidectomy (46.0% vs. 29.9%) with adjuvant radiation therapy (53.8% vs. 41.0%) Cox-proportional hazard modeling did not identify RLND, regardless if stratified by nodal yield or pT-classification, nor nodal positivity as significant predictors of 10-year OS. CONCLUSIONS: The occult nodal disease in intermediate-grade parotid MEC is low and similar to low-grade. Elective RLND may have a limited impact on OS, though its effect on locoregional control remains unknown. LEVEL OF EVIDENCE: III.

Whole genome sequencing of resistance and virulence genes in multi-drug resistant Pseudomonas aeruginosa.

BACKGROUND: Pseudomonas aeruginosa is an opportunistic bacterium that causes serious hospital-acquired infections. To assess the risk of clinically isolated P. aeruginosa to human health, we analyzed the resistance and virulence mechanisms of a collection of clinical isolates. METHODS: This was a retrospective study in which P. aeruginosa isolates collected from January 1, 2018 to August 31, 2019 were analyzed using phenotypic and whole-genome sequencing (WGS) methods. The analysis included 48 clinical samples. Median patient age was 54.0 (29.5) years, and 58.3% of patients were women. Data from the microbiology laboratory database were reviewed to identify P. aeruginosa isolates. All unique isolates available for further testing were included, and related clinical data were collected. Infections were defined as hospital acquired if the index culture was obtained at least 48 h after hospitalization. RESULTS: High-risk P. aeruginosa clones, including sequence types (STs) ST235 and ST111, were identified, in addition to 12 new STs. The isolates showed varying degrees of biofilm formation ability when evaluated at room temperature, along with reduced metabolic activity, as measured by metabolic staining, suggesting their ability to evade antimicrobial therapy. Most isolates (77.1%) were multidrug resistant (MDR), with the highest resistance and susceptibility rates to beta-lactams and colistimethate sodium, respectively. CONCLUSIONS: The MDR phenotypes of the examined isolates can be explained by the high prevalence of efflux-mediated resistance- and hydrolytic enzyme-encoding genes. These isolates had high cytotoxic potential, as indicated by the detection of toxin production-related genes.

Bridging Neuroscience and Robotics: Spiking Neural Networks in Action.

Robots are becoming increasingly sophisticated in the execution of complex tasks. However, an area that requires development is the ability to act in dynamically changing environments. To advance this, developments have turned towards understanding the human brain and applying this to improve robotics. The present study used electroencephalogram (EEG) data recorded from 54 human participants whilst they performed a two-choice task. A build-up of motor activity starting around 400 ms before response onset, also known as the lateralized readiness potential (LRP), was observed. This indicates that actions are not simply binary processes but rather, response-preparation is gradual and occurs in a temporal window that can interact with the environment. In parallel, a robot arm executing a pick-and-place task was developed. The understanding from the EEG data and the robot arm were integrated into the final system, which included cell assemblies (CAs)-a simulated spiking neural network-to inform the robot to place the object left or right. Results showed that the neural data from the robot simulation were largely consistent with the human data. This neurorobotics study provides an example of how to integrate human brain recordings with simulated neural networks in order to drive a robot.

Proteome-wide base editor screens to assess phosphorylation site functionality in high-throughput.

Signaling pathways that drive gene expression are typically depicted as having a dozen or so landmark phosphorylation and transcriptional events. In reality, thousands of dynamic post-translational modifications (PTMs) orchestrate nearly every cellular function, and we lack technologies to find causal links between these vast biochemical pathways and genetic circuits at scale. Here, we describe "signaling-to-transcription network" mapping through the development of PTM-centric base editing coupled to phenotypic screens, directed by temporally-resolved phosphoproteomics. Using T cell activation as a model, we observe hundreds of unstudied phosphorylation sites that modulate NFAT transcriptional activity. We identify the phosphorylation-mediated nuclear localization of the phosphatase PHLPP1 which promotes NFAT but inhibits NFκB activity. We also find that specific phosphosite mutants can alter gene expression in subtle yet distinct patterns, demonstrating the potential for fine-tuning transcriptional responses. Overall, base editor screening of PTM sites provides a powerful platform to dissect PTM function within signaling pathways.

Validity of the UCEEM in use: How Does it Triangulate with Qualitative Data in Measuring the Effect of an Educational Intervention?

Objectives: Improving medical student placements relies on being able to reliably evaluate how students experience clinical learning environments. The Undergraduate Clinical Education Environment Measure (UCEEM) is an increasingly used validated tool designed to allow such evaluations. This study aims to further characterize how the UCEEM relates to qualitative evaluation. methods: Students on placement at one hospital were invited to complete the UCEEM before and after the implementation of an innovative new placement structure. Additionally, focus groups were employed to collect qualitative data on their experiences. a novel protocol to triangulate the output of the UCEEM with the qualitative data was developed. Results: The UCEEM showed good internal consistency (Cronbach's Alpha 0.79-0.91) and internal correlation. Implementation of the intervention created significant improvements in the overall UCEEM scores (P = .008) and in the "Learning in and through work and quality of supervision" (P = .048), "Preparedness for student entry" (P = .033) and "Workplace interaction patterns and student inclusion" (P = .039) domains. The triangulation of qualitative data with UCEEM output showed that the UCEEM allowed evaluation of some perceptions not reached through open questioning. However, mixed interpretations of UCEEM items by students led to the conflation of themes and challenges in deriving the meaning behind the score. This appeared to be the case for 14 of the 24 UCEEM items. Conclusion: This investigation adds to the literature supporting the UCEEM as a validated tool. It also elucidates the limitations and relationships to qualitative data that investigators need to be aware of in its use.

Null effects of temporal prediction on recognition memory but evidence for differential neural activity at encoding. A registered report.

Previous research has demonstrated that rhythmic presentation of stimuli during encoding boosts subsequent recognition and is associated with distinct neural activity compared with when stimuli are presented in an arrhythmic manner. However, it is unclear whether the effect is driven by automatic entrainment to rhythm or non-rhythmic temporal prediction. This registered report presents an Electroencephalographic (EEG) study aimed at establishing the cognitive and neural mechanisms of the effect of temporal prediction on recognition. In a blocked design, stimulus onset during encoding was systematically manipulated in four conditions prior to recognition testing: rhythmic fixed (RF), rhythmic variable (RV), arrhythmic fixed (AF), and arrhythmic variable (AV). By orthogonally varying rhythm and temporal position we were able to assess their independent contributions to recognition enhancement. Our behavioural results did not replicate previous findings that show a difference in recognition memory based on temporal predictability at encoding. However, event-related potential (ERP) component analysis did show an early (N1) interaction effect of temporal position and rhythm, and later (N2 and Dm) effects driven by temporal position only. Taken together, we observed effects of temporal prediction at encoding, but these differences did not translate to later effects of memory, suggesting that effects of temporal prediction on recognition are less robust than previously thought.

Highlighter: An optogenetic system for high-resolution gene expression control in plants.

Optogenetic actuators have revolutionized the resolution at which biological processes can be controlled. In plants, deployment of optogenetics is challenging due to the need for these light-responsive systems to function in the context of horticultural light environments. Furthermore, many available optogenetic actuators are based on plant photoreceptors that might crosstalk with endogenous signaling processes, while others depend on exogenously supplied cofactors. To overcome such challenges, we have developed Highlighter, a synthetic, light-gated gene expression system tailored for in planta function. Highlighter is based on the photoswitchable CcaS-CcaR system from cyanobacteria and is repurposed for plants as a fully genetically encoded system. Analysis of a re-engineered CcaS in Escherichia coli demonstrated green/red photoswitching with phytochromobilin, a chromophore endogenous to plants, but also revealed a blue light response likely derived from a flavin-binding LOV-like domain. We deployed Highlighter in transiently transformed Nicotiana benthamiana for optogenetic control of fluorescent protein expression. Using light to guide differential fluorescent protein expression in nuclei of neighboring cells, we demonstrate unprecedented spatiotemporal control of target gene expression. We implemented the system to demonstrate optogenetic control over plant immunity and pigment production through modulation of the spectral composition of broadband visible (white) light. Highlighter is a step forward for optogenetics in plants and a technology for high-resolution gene induction that will advance fundamental plant biology and provide new opportunities for crop improvement.

Single-residue mutation in protein kinase C toggles between cancer and neurodegeneration.

Conventional protein kinase C (cPKC) isozymes tune the signaling output of cells, with loss-of-function somatic mutations associated with cancer and gain-of-function germline mutations identified in neurodegeneration. PKC with impaired autoinhibition is removed from the cell by quality-control mechanisms to prevent the accumulation of aberrantly active enzyme. Here, we examine how a highly conserved residue in the C1A domain of cPKC isozymes permits quality-control degradation when mutated to histidine in cancer (PKCß-R42H) and blocks down-regulation when mutated to proline in the neurodegenerative disease spinocerebellar ataxia (PKCγ-R41P). Using FRET-based biosensors, we determined that mutation of R42 to any residue, including lysine, resulted in reduced autoinhibition as indicated by higher basal activity and faster agonist-induced plasma membrane translocation. R42 is predicted to form a stabilizing salt bridge with E655 in the C-tail and mutation of E655, but not neighboring E657, also reduced autoinhibition. Western blot analysis revealed that whereas R42H had reduced stability, the R42P mutant was stable and insensitive to activator-induced ubiquitination and down-regulation, an effect previously observed by deletion of the entire C1A domain. Molecular dynamics (MD) simulations and analysis of stable regions of the domain using local spatial pattern (LSP) alignment suggested that P42 interacts with Q66 to impair mobility and conformation of one of the ligand-binding loops. Additional mutation of Q66 to the smaller asparagine (R42P/Q66N), to remove conformational constraints, restored degradation sensitivity. Our results unveil how disease-associated mutations of the same residue in the C1A domain can toggle between gain- or loss-of-function of PKC.

Frontal Sinus Anatomy Variations in Race and Sex Using the International Frontal Sinus Anatomy Classification.

Objective: To identify frontal sinus anatomical variations grouped by patient sex, race, and presence of chronic rhinosinusitis with frontal sinus involvement (CRFS) using the International Frontal Sinus Anatomy Classification (IFAC) system. Methods: A retrospective review from 2015 to 2020 was performed of consecutive adult patients with computed tomography sinus imaging. Prevalence of frontal sinus cells using the IFAC system was recorded. Comparisons were made between patient race, sex, and CRFS groups. Results: A total of 184 patients (368 sides) were included, 90 (48.9%) of which had CRFS. The racial distribution was 50 white (27.2%), 50 black (27.2%), 45 Hispanic/Latino (24.5%), and 39 Asian (21.2%) patients. The supra agger cell was most prevalent in the white population (P = 0.009), and supraorbital ethmoid cells were more prevalent in the Asian population (P = 0.017). Patients with frontal sinus disease were more likely to have a supraorbital ethmoid cell (P = 0.024). Overall, CRFS was more prevalent in the Asian population (P = 0.013). Conclusion: Significant differences in frontal sinus anatomy and disease exist between patient race and sex. Supraorbital ethmoid cells are associated with the development of CRFS. These patterns in frontal sinus anatomy should be noted prior to frontal sinus surgery to improve surgical awareness and outcomes.

Whole-Body Magnetic Resonance Imaging Assessment of the Contributions of Adipose and Nonadipose Tissues to Cardiovascular Remodeling in Adolescents.

Background Greater body mass index is associated with cardiovascular remodeling in adolescents. However, body mass index cannot differentiate between adipose and nonadipose tissues. We examined how visceral and subcutaneous adipose tissue are linked with markers of early cardiovascular remodeling, independently from nonadipose tissue. Methods and Results Whole-body magnetic resonance imaging was done in 82 adolescents (39 overweight/obese; 36 female; median age, 16.3 [interquartile range, 14.4-18.1] years) to measure body composition and cardiovascular remodeling markers. Left ventricular diastolic function was assessed by echocardiography. Waist, waist:height ratio, and body mass index z scores were calculated. Residualized nonadipose tissue, subcutaneous adipose tissue, and visceral adipose tissue variables, uncorrelated with each other, were constructed using partial regression modeling to allow comparison of their individual contributions in a 3-compartment body composition model. Cardiovascular variables mostly related to nonadipose rather than adipose tissue. Nonadipose tissue was correlated positively with left ventricular mass (r=0.81), end-diastolic volume (r=0.70), stroke volume (r=0.64), left ventricular mass:end-diastolic volume (r=0.37), and systolic blood pressure (r=0.35), and negatively with heart rate (r=-0.33) (all P<0.01). Subcutaneous adipose tissue was associated with worse left ventricular diastolic function (r=-0.42 to -0.48, P=0.0007-0.02) and higher heart rates (r=0.34, P=0.007) but linked with better systemic vascular resistance (r=-0.35, P=0.006). There were no significant relationships with visceral adipose tissue and no associations of any compartment with pulse wave velocity. Conclusions Simple anthropometry does not reflect independent effects of nonadipose tissue and subcutaneous adipose tissue on the adolescent cardiovascular system. This could result in normal cardiovascular adaptations to growth being misinterpreted as pathological sequelae of excess adiposity in studies reliant on such measures.

Next-generation ABACUS biosensors reveal cellular ABA dynamics driving root growth at low aerial humidity.

The plant hormone abscisic acid (ABA) accumulates under abiotic stress to recast water relations and development. To overcome a lack of high-resolution sensitive reporters, we developed ABACUS2s-next-generation Förster resonance energy transfer (FRET) biosensors for ABA with high affinity, signal-to-noise ratio and orthogonality-that reveal endogenous ABA patterns in Arabidopsis thaliana. We mapped stress-induced ABA dynamics in high resolution to reveal the cellular basis for local and systemic ABA functions. At reduced foliar humidity, root cells accumulated ABA in the elongation zone, the site of phloem-transported ABA unloading. Phloem ABA and root ABA signalling were both essential to maintain root growth at low humidity. ABA coordinates a root response to foliar stresses, enabling plants to maintain foraging of deeper soil for water uptake.

Enabling Deoxygenative C(sp2)-C(sp3) Cross-Coupling for Parallel Medicinal Chemistry.

Herein we report the development of an automated deoxygenative C(sp2)-C(sp3) coupling of aryl bromide with alcohols to enable parallel medicinal chemistry. Alcohols are among the most diverse and abundant building blocks, but their usage as alkyl precursors has been limited. Although metallaphotoredox deoxygenative coupling is becoming a promising strategy to form C(sp2)-C(sp3) bond, the reaction setup limits its widespread application in library synthesis. To achieve high throughput and consistency, an automated workflow involving solid-dosing and liquid-handling robots has been developed. We have successfully demonstrated this high-throughput protocol is robust and consistent across three automation platforms. Furthermore, guided by cheminformatic analysis, we examined alcohols with comprehensive chemical space coverage and established a meaningful scope for medicinal chemistry applications. By accessing the rich diversity of alcohols, this automated protocol has the potential to substantially increase the impact of C(sp2)-C(sp3) cross-coupling in drug discovery.

STrain Analysis and Mapping of the Plantar Surface (STAMPS): A novel technique of plantar load analysis during gait.

Diabetic foot ulceration is driven by peripheral neuropathy, resulting in abnormal foot biomechanics and elevated plantar load. Plantar load comprises normal pressure and tangential shear stress. Currently, there are no in-shoe devices measuring both components of plantar load. The STAMPS (STrain Analysis and Mapping of the Plantar Surface) system was developed to address this and utilises digital image correlation (DIC) to determine the strain sustained by a plastically deformable insole, providing an assessment of plantar load at the foot-surface interface during gait. STAMPS was developed as a multi-layered insole, comprising a deformable mid-layer, onto which a stochastic speckle pattern film is applied. A custom-built imaging platform is used to obtain high resolution pre- and post-walking images. Images are imported into commercially available DIC software (GOM Correlate, 2020) to obtain pointwise strain data. The strain and displacement data are exported and post-processed with custom analysis routines (MATLAB, Mathworks Inc.), to obtain the resultant global and regional peak strain (SMAG), antero-posterior strain (SAP) and medio-lateral strain (SML). To validate the core technique an experimental test process used a Universal Mechanical Tester (UMT) system (UMT TriboLab, Bruker) to apply controlled vertical and tangential load regimes to the proposed multi-layer insole. A pilot study was then conducted to assess the efficacy of using the STAMPS system to measure in-shoe plantar strain in three healthy participants. Each participant walked 10 steps on the STAMPS insole using a standardised shoe. They also walked 10 m in the same shoe using a plantar pressure measurement insole (Novel Pedar®) to record peak plantar pressure (PPP) as a gold-standard comparator. The results of the experimental validation tests show that with increased normal force, at a constant shear distance, SMAG increased in a linear fashion. Furthermore, they showed that with increased shear distance, at a constant force, SMAG increased. The results of the pilot study found participant 1 demonstrated greatest SMAG in the region toes 3-5 (15.31%). The highest mean SMAG for participant 2 was at the hallux (29.31%). Participant 3 exhibited highest strain in the regions of the first and second metatarsal heads (58.85% and 41.62% respectively). Increased PPP was strongly associated with increased SMAG with a Spearman's correlation coefficient 0.673 (p < 0.0001). This study has demonstrated the efficacy of a novel method to assess plantar load across the plantar surface of the foot. Experimental testing validated the sensitivity of the method to both normal pressure and tangential shear stress. This technique was successfully incorporated into the STAMPS insole to reliably measure and quantify the cumulative degree of strain sustained by a plastically deformable insole during a period of gait, which can be used to infer plantar loading patterns. Future work will explore how these measures relate to different pathologies, such as regions at risk of diabetic foot ulceration.