EGFR/MEK inhibitor therapy induces a distinct inflammatory hair follicle response that includes a collapse of immune privilege.

AIMS: Inhibitors of epidermal growth factor receptor (EGFRi) or mitogen-activated protein kinase (MEKi) induce a folliculitis in 75-90% of patients, whose pathobiology remains insufficiently understood. OBJECTIVES: (1) Characterize changes in the skin immune status and global transcriptional profile of EGFRi-treated patients (2) Probe whether EGFRi affects the hair follicle's (HF) immune privilege (IP) (3) Identify early pro-inflammatory signals induced by EGFRi/MEKi in human scalp HFs ex vivo. METHODS: Scalp biopsies were taken from long-term EGFRi-treated patients exhibiting folliculitis (Chronic-EGFRi, n=9) vs normal scalp skin (n=9) and patients prior to commencing EGFRi therapy and after two weeks of EGFRi therapy (Acute-EGFRi, n=5). Healthy organ-cultured scalp HFs were exposed to EGFRi (Erlotinib) or MEKi (Cobimetinib) (n=5 patients, each). Samples were assessed by quantatitive immunohistomorphometry, RNAseq and in situ hybridization. RESULTS: The Chronic-EGFRi cohort showed CD8+ T cell infiltration of the bulge alongside a partial collapse of the HF's IP, evidenced by upregulated MHC class I, ß2-microglobulin and MHC class II and decreased TGF-ß1 protein expression. Healthy HFs treated with EGFRi/MEKi ex vivo also showed partial HF IP collapse and increased transcription of HLA-A, HLA-DR, ß2-microglobulin transcripts. RNAseq anlysis showed increased transcription of chemokines (CXCL1, CXCL13, CCL18, CCL3, CCL7) and IL-26 in Chronic-EGFRi biopsies, as well as increased interlukin IL-33 and decreased IL-37 expesssion in both Acute-EGFRi biopsies and organ-cultured HFs. CONCLUSION: These data show that EGFRi/MEKi compromise the physiological IP of human scalp HFs and suggest that future clinical management of EGFRi/MEKi-induced folliculitis requires HF IP protection and inhibition of IL-33.

Myosin-I Synergizes with Arp2/3 Complex to Enhance Pushing Forces of Branched Actin Networks.

Myosin-Is colocalize with Arp2/3 complex-nucleated actin networks at sites of membrane protrusion and invagination, but the mechanisms by which myosin-I motor activity coordinates with branched actin assembly to generate force are unknown. We mimicked the interplay of these proteins using the "comet tail" bead motility assay, where branched actin networks are nucleated by Arp2/3 complex on the surface of beads coated with myosin-I and the WCA domain of N-WASP. We observed that myosin-I increased bead movement efficiency by thinning actin networks without affecting growth rates. Remarkably, myosin-I triggered symmetry breaking and comet-tail formation in dense networks resistant to spontaneous fracturing. Even with arrested actin assembly, myosin-I alone could break the network. Computational modeling recapitulated these observations suggesting myosin-I acts as a repulsive force shaping the network's architecture and boosting its force-generating capacity. We propose that myosin-I leverages its power stroke to amplify the forces generated by Arp2/3 complex-nucleated actin networks.

Force transmission by retrograde actin flow-induced dynamic molecular stretching of Talin.

Force transmission at integrin-based adhesions is important for cell migration and mechanosensing. Talin is an essential focal adhesion (FA) protein that links F-actin to integrins. F-actin constantly moves on FAs, yet how Talin simultaneously maintains the connection to F-actin and transmits forces to integrins remains unclear. Here we show a critical role of dynamic Talin unfolding in force transmission. Using single-molecule speckle microscopy, we found that the majority of Talin are bound only to either F-actin or the substrate, whereas 4.1% of Talin is linked to both structures via elastic transient clutch. By reconstituting Talin knockdown cells with Talin chimeric mutants, in which the Talin rod subdomains are replaced with the stretchable ß-spectrin repeats, we show that the stretchable property is critical for force transmission. Simulations suggest that unfolding of the Talin rod subdomains increases in the linkage duration and work at FAs. This study elucidates a force transmission mechanism, in which stochastic molecular stretching bridges two cellular structures moving at different speeds.

Force transmission by retrograde actin flow-induced dynamic molecular stretching of Talin.

Force transmission at integrin-based adhesions is important for cell migration and mechanosensing. Talin is an essential focal adhesion (FA) protein that links F-actin to integrins. F-actin constantly moves on FAs, yet how Talin simultaneously maintains the connection to F-actin and transmits forces to integrins remains unclear. Here we show a critical role of dynamic Talin unfolding in force transmission. Using single-molecule speckle microscopy, we found that the majority of Talin are bound only to either F-actin or the substrate, whereas 4.1% of Talin is linked to both structures via elastic transient clutch. By reconstituting Talin knockdown cells with Talin chimeric mutants, in which the Talin rod subdomains are replaced with the stretchable ß-spectrin repeats, we show that the stretchable property is critical for force transmission. Simulations suggest that unfolding of the Talin rod subdomains increases in the linkage duration and work at FAs. This study reveals a new mode of force transmission, in which stochastic molecular stretching bridges two cellular structures moving at different speeds.

Cdc42 mobility and membrane flows regulate fission yeast cell shape and survival.

Local Cdc42 GTPase activation promotes polarized exocytosis, resulting in membrane flows that deplete low-mobility membrane-associated proteins from the growth region. To investigate the self-organizing properties of the Cdc42 secretion-polarization system under membrane flow, we developed a reaction-diffusion particle model. The model includes positive feedback activation of Cdc42, hydrolysis by GTPase-activating proteins (GAPs), and flow-induced displacement by exo/endocytosis. Simulations show how polarization relies on flow-induced depletion of low mobility GAPs. To probe the role of Cdc42 mobility in the fission yeast Schizosaccharomyces pombe, we changed its membrane binding properties by replacing its prenylation site with 1, 2 or 3 repeats of the Rit1 C terminal membrane binding domain (ritC), yielding alleles with progressively lower unbinding and diffusion rates. Concordant modelling predictions and experimental observations show that lower Cdc42 mobility results in lower Cdc42 activation level and wider patches. Indeed, while Cdc42-1ritC cells are viable and polarized, Cdc42-2ritC polarize poorly and Cdc42-3ritC is inviable. The model further predicts that GAP depletion increases Cdc42 activity at the expense of loss of polarization. Experiments confirm this prediction, as deletion of Cdc42 GAPs restores viability to Cdc42-3ritC cells. Our combined experimental and modelling studies demonstrate how membrane flows are an integral part of Cdc42-driven pattern formation.

Compressive forces stabilize microtubules in living cells.

Microtubules are cytoskeleton components with unique mechanical and dynamic properties. They are rigid polymers that alternate phases of growth and shrinkage. Nonetheless, the cells can display a subset of stable microtubules, but it is unclear whether microtubule dynamics and mechanical properties are related. Recent in vitro studies suggest that microtubules have mechano-responsive properties, being able to stabilize their lattice by self-repair on physical damage. Here we study how microtubules respond to cycles of compressive forces in living cells and find that microtubules become distorted, less dynamic and more stable. This mechano-stabilization depends on CLASP2, which relocates from the end to the deformed shaft of microtubules. This process seems to be instrumental for cell migration in confined spaces. Overall, these results demonstrate that microtubules in living cells have mechano-responsive properties that allow them to resist and even counteract the forces to which they are subjected, being a central mediator of cellular mechano-responses.

Motion-robust, blood-suppressed, reduced-distortion diffusion MRI of the liver.

PURPOSE: To evaluate feasibility and reproducibility of liver diffusion-weighted (DW) MRI using cardiac-motion-robust, blood-suppressed, reduced-distortion techniques. METHODS: DW-MRI data were acquired at 3T in an anatomically accurate liver phantom including controlled pulsatile motion, in eight healthy volunteers and four patients with known or suspected liver metastases. Standard monopolar and motion-robust (M1-nulled, and M1-optimized) DW gradient waveforms were each acquired with single-shot echo-planar imaging (ssEPI) and multishot EPI (msEPI). In the motion phantom, apparent diffusion coefficient (ADC) was measured in the motion-affected volume. In healthy volunteers, ADC was measured in the left and right liver lobes separately to evaluate ADC reproducibility between the two lobes. Image distortions were quantified using the normalized cross-correlation coefficient, with an undistorted T2-weighted reference. RESULTS: In the motion phantom, ADC mean and SD in motion-affected volumes substantially increased with increasing motion for monopolar waveforms. ADC remained stable in the presence of increasing motion when using motion-robust waveforms. M1-optimized waveforms suppressed slow flow signal present with M1-nulled waveforms. In healthy volunteers, monopolar waveforms generated significantly different ADC measurements between left and right liver lobes ( p = 0 . 0078 $$ p=0.0078 $$ , reproducibility coefficients (RPC) =  470 × 1 0 - 6 $$ 470\times 1{0}^{-6} $$ mm 2 $$ {}^2 $$ /s for monopolar-msEPI), while M1-optimized waveforms showed more reproducible ADC values ( p = 0 . 29 $$ p=0.29 $$ , RPC = 220 × 1 0 - 6 $$ \mathrm{RPC}=220\times 1{0}^{-6} $$ mm 2 $$ {}^2 $$ /s for M1-optimized-msEPI). In phantom and healthy volunteer studies, motion-robust acquisitions with msEPI showed significantly reduced image distortion ( p < 0 . 001 $$ p<0.001 $$ ) compared to ssEPI. Patient scans showed reduction of wormhole artifacts when combining M1-optimized waveforms with msEPI. CONCLUSION: Synergistic effects of combined M1-optimized diffusion waveforms and msEPI acquisitions enable reproducible liver DWI with motion robustness, blood signal suppression, and reduced distortion.

Nivolumab-Induced Lichen Planopilaris: Case Report and Literature Review of Hair Disorders Associated with Targeted Oncological Therapies.

Immune checkpoint inhibitors (ICIs) have revolutionized cancer therapies. Their mechanism promotes a cytotoxic T-cell activation against the tumor cells, but as a consequence, immune-mediated toxicities are increasingly being identified. Cutaneous immune-mediated adverse events (AE) occur in 32% of patients, with possible higher grade AEs seen with anti-programmed cell death protein 1, such as nivolumab. A 67-year-old woman with metastatic melanoma, previously treated for 2 years on dual ICI (ipilimumab and nivolumab), had her treatment interrupted due to grade-3 hepatitis. She was subsequently recommenced on single-agent nivolumab with good response, before discontinuation due to remission. She reported worsening scalp pruritus with associated erythema, scaling, and global hair thinning. On examination, she had significant erythema throughout the scalp with perifollicular scaling and evidence of scarring. She reported severe distress from her symptoms. Her scalp biopsy demonstrated features of scarring alopecia with infundibular and isthmic inflammation and interface change in keeping with lichen planopilaris. Follicular toxicities are rarely reported, possibly due to imprecise AE phenotyping or underreporting. However, growing evidence suggests that patients can develop follicular pigmentary changes and nonscarring alopecia. To our knowledge, this is the first case of scarring alopecia reported with nivolumab. Current treatments for ICI-induced toxicities are limited.

Multistage Testing in Heterogeneous Populations: Some Design and Implementation Considerations.

A central challenge in international large-scale assessments is adequately measuring dozens of highly heterogeneous populations, many of which are low performers. To that end, multistage adaptive testing offers one possibility for better assessing across the achievement continuum. This study examines the way that several multistage test design and implementation choices can impact measurement performance in this setting. To attend to gaps in the knowledge base, we extended previous research to include multiple, linked panels, more appropriate estimates of achievement, and multiple populations of varied proficiency. Including achievement distributions from varied populations and associated item parameters, we design and execute a simulation study that mimics an established international assessment. We compare several routing schemes and varied module lengths in terms of item and person parameter recovery. Our findings suggest that, particularly for low performing populations, multistage testing offers precision advantages. Further, findings indicate that equal module lengths-desirable for controlling position effects-and classical routing methods, which lower the technological burden of implementing such a design, produce good results. Finally, probabilistic misrouting offers advantages over merit routing for controlling bias in item and person parameters. Overall, multistage testing shows promise for extending the scope of international assessments. We discuss the importance of our findings for operational work in the international assessment domain.

Prevalence of wound complications following Mohs micrographic surgery (MMS): a cross-sectional study of 1000 patients undergoing MMS and wound repair in a UK teaching hospital.

BACKGROUND: Mohs micrographic surgery (MMS) for nonmelanoma skin cancer is often quoted as having an excellent safety profile. AIM: To determine the complication rate of patients undergoing MMS in a large UK Mohs unit and subdivide complication rates into mild/intermediate and major, and to identify potential risk factors necessitating a clinical intervention. METHODS: This was a single-centre, cross-sectional study of 1000 consecutive cases of MMS performed with in-house repair. Notes from the postsurgical dressing clinics were reviewed at Visit 1 (Days 7-14) and Visit 2 (approximately Week 6). Based upon the intervention required and effect on cosmetic/functional outcome, complications were classified as minor, intermediate or major. Logistic regression modelling was used to identify risk factors associated with a complication that needed a clinical intervention (i.e. intermediate or major). RESULTS: In total, 1000 Mohs surgeries were performed on 803 patients, resulting in 1067 excisions. Complication rates in our cohort were low (minor 3.6%, intermediate 3.1% and major 0.8%) Potential risk factors for developing a complication included skin graft (unadjusted OR = 4.89, 95% CI 1.93-12.39; fully adjusted OR = 7.13, 95% CI 2.26-22.45) and patients undergoing surgery on the forehead (unadjusted OR = 3.32, 95% CI 0.95-11.58; fully adjusted OR = 5.34, 95% CI 1.40-20.42). Patients whose wounds were allowed to heal by secondary intention healing (6.8%) exhibited no complications. CONCLUSION: We advocate that patients should be informed during the consent procedure that less than 1 in every 100 patients (0.75%) undergoing MMS will have a serious adverse event (major complication) affecting their cosmetic or functional outcome.

Discrete mechanical model of lamellipodial actin network implements molecular clutch mechanism and generates arcs and microspikes.

Mechanical forces, actin filament turnover, and adhesion to the extracellular environment regulate lamellipodial protrusions. Computational and mathematical models at the continuum level have been used to investigate the molecular clutch mechanism, calculating the stress profile through the lamellipodium and around focal adhesions. However, the forces and deformations of individual actin filaments have not been considered while interactions between actin networks and actin bundles is not easily accounted with such methods. We develop a filament-level model of a lamellipodial actin network undergoing retrograde flow using 3D Brownian dynamics. Retrograde flow is promoted in simulations by pushing forces from the leading edge (due to actin polymerization), pulling forces (due to molecular motors), and opposed by viscous drag in cytoplasm and focal adhesions. Simulated networks have densities similar to measurements in prior electron micrographs. Connectivity between individual actin segments is maintained by permanent and dynamic crosslinkers. Remodeling of the network occurs via the addition of single actin filaments near the leading edge and via filament bond severing. We investigated how several parameters affect the stress distribution, network deformation and retrograde flow speed. The model captures the decrease in retrograde flow upon increase of focal adhesion strength. The stress profile changes from compression to extension across the leading edge, with regions of filament bending around focal adhesions. The model reproduces the observed reduction in retrograde flow speed upon exposure to cytochalasin D, which halts actin polymerization. Changes in crosslinker concentration and dynamics, as well as in the orientation pattern of newly added filaments demonstrate the model's ability to generate bundles of filaments perpendicular (actin arcs) or parallel (microspikes) to the protruding direction.

Cell patterning by secretion-induced plasma membrane flows.

Cells self-organize using reaction-diffusion and fluid-flow principles. Whether bulk membrane flows contribute to cell patterning has not been established. Here, using mathematical modeling, optogenetics, and synthetic probes, we show that polarized exocytosis causes lateral membrane flows away from regions of membrane insertion. Plasma membrane­associated proteins with sufficiently low diffusion and/or detachment rates couple to the flows and deplete from areas of exocytosis. In rod-shaped fission yeast cells, zones of Cdc42 GTPase activity driving polarized exocytosis are limited by GTPase activating proteins (GAPs). We show that membrane flows pattern the GAP Rga4 distribution and that coupling of a synthetic GAP to membrane flows is sufficient to establish the rod shape. Thus, membrane flows induced by Cdc42-dependent exocytosis form a negative feedback restricting the zone of Cdc42 activity.

Characterization and correction of cardiovascular motion artifacts in diffusion-weighted imaging of the pancreas.

PURPOSE: To assess the effects of cardiovascular-induced motion on conventional DWI of the pancreas and to evaluate motion-robust DWI methods in a motion phantom and healthy volunteers. METHODS: 3T DWI was acquired using standard monopolar and motion-compensated gradient waveforms, including in an anatomically accurate pancreas phantom with controllable compressive motion and healthy volunteers (n = 8, 10). In volunteers, highly controlled single-slice DWI using breath-holding and cardiac gating and whole-pancreas respiratory-triggered DWI were acquired. For each acquisition, the ADC variability across volunteers, as well as ADC differences across parts of the pancreas were evaluated. RESULTS: In motion phantom scans, conventional DWI led to biased ADC, whereas motion-compensated waveforms produced consistent ADC. In the breath-held, cardiac-triggered study, conventional DWI led to heterogeneous DW signals and highly variable ADC across the pancreas, whereas motion-compensated DWI avoided these artifacts. In the respiratory-triggered study, conventional DWI produced heterogeneous ADC across the pancreas (head: 1756 ± 173 × 10-6 mm2 /s; body: 1530 ± 338 × 10-6 mm2 /s; tail: 1388 ± 267 × 10-6 mm2 /s), with ADCs in the head significantly higher than in the tail (P < .05). Motion-compensated ADC had lower variability across volunteers (head: 1277 ± 102 × 10-6 mm2 /s; body: 1204 ± 169 × 10-6 mm2 /s; tail: 1235 ± 178 × 10-6 mm2 /s), with no significant difference (P ≥ .19) across the pancreas. CONCLUSION: Cardiovascular motion introduces artifacts and ADC bias in pancreas DWI, which are addressed by motion-robust DWI.

Enhancement of cerebrovascular 4D flow MRI velocity fields using machine learning and computational fluid dynamics simulation data.

Blood flow metrics obtained with four-dimensional (4D) flow phase contrast (PC) magnetic resonance imaging (MRI) can be of great value in clinical and experimental cerebrovascular analysis. However, limitations in both quantitative and qualitative analyses can result from errors inherent to PC MRI. One method that excels in creating low-error, physics-based, velocity fields is computational fluid dynamics (CFD). Augmentation of cerebral 4D flow MRI data with CFD-informed neural networks may provide a method to produce highly accurate physiological flow fields. In this preliminary study, the potential utility of such a method was demonstrated by using high resolution patient-specific CFD data to train a convolutional neural network, and then using the trained network to enhance MRI-derived velocity fields in cerebral blood vessel data sets. Through testing on simulated images, phantom data, and cerebrovascular 4D flow data from 20 patients, the trained network successfully de-noised flow images, decreased velocity error, and enhanced near-vessel-wall velocity quantification and visualization. Such image enhancement can improve experimental and clinical qualitative and quantitative cerebrovascular PC MRI analysis.

Assessment of sex differences in ventricular-vascular coupling of left ventricular and aortic flow derived from 4D flow MRI in healthy, young adults.

To gain further insight into male-female differences in cardiovascular conditions it is important to understand sex differences in healthy populations. A previous study from our group of 39 healthy young volunteers (20-35 years) paradoxically found that men had greater left ventricular (LV) kinetic energy (KE) but women had greater LV vorticity. We reanalyzed cardiac four-dimensional flow MRI data from 20 of the original subjects (10 male and 10 female) to quantify aortic flow in addition to LV flow. The combination of LV and aortic flow parameters were then used to calculate ventricular vascular coupling of KE and vorticity. The sex difference found in LV flow were not found in aortic flow and the ventricular-vascular coupling of LV-to-aortic flow was similar between men and women. Dimensional analysis to account for differences in cardiac output and ventricular volume explained the differences found in LV flow. The analysis methods and results of this study may be of further use in understanding ventricular vascular coupling of transported flow variables in healthy sex differences, healthy aging, and various cardiovascular conditions.

Inhibition of Shh Signaling through MAPK Activation Controls Chemotherapy-Induced Alopecia.

Chemotherapy-induced hair loss (alopecia) (CIA) remains a major unsolved problem in clinical oncology. CIA is often considered to be a consequence of the antimitotic and apoptosis-promoting properties of chemotherapy drugs acting on rapidly proliferating hair matrix keratinocytes. Here, we show that in a mouse model of CIA, the downregulation of Shh signaling in the hair matrix is a critical early event. Inhibition of Shh signaling recapitulated key morphological and functional features of CIA, whereas recombinant Shh protein partially rescued hair loss. Phosphoproteomics analysis revealed that activation of the MAPK pathway is a key upstream event, which can be further manipulated to rescue CIA. Finally, in organ-cultured human scalp hair follicles as well as in patients undergoing chemotherapy, reduced expression of SHH gene correlates with chemotherapy-induced hair follicle damage or the degree of CIA, respectively. Our work revealed that Shh signaling is an evolutionarily conserved key target in CIA pathobiology. Specifically targeting the intrafollicular MAPK-Shh axis may provide a promising strategy to manage CIA.

A pilot study of bladder voiding with real-time MRI and computational fluid dynamics.

Lower urinary track symptoms (LUTS) affect many older adults. Multi-channel urodynamic studies provide information about bladder pressure and urinary flow but offer little insight into changes in bladder anatomy and detrusor muscle function. Here we present a novel method for real time MRI during bladder voiding. This was performed in a small cohort of healthy men and men with benign prostatic hyperplasia and lower urinary tract symptoms (BPH/LUTS) to demonstrate proof of principle; The MRI urodynamic protocol was successfully implemented, and bladder wall displacement and urine flow dynamics were calculated. Displacement analysis on healthy controls showed the greatest bladder wall displacement in the dome of the bladder while men with BPH/LUTS exhibited decreased and asymmetric bladder wall motion. Computational fluid dynamics of voiding showed men with BPH/LUTS had larger recirculation regions in the bladder. This study demonstrates the feasibility of performing MRI voiding studies and their potential to provide new insight into lower urinary tract function in health and disease.

Intracranial vascular flow oscillations in Alzheimer's disease from 4D flow MRI.

Recent modeling and experimental evidence suggests clearance of soluble metabolites from the brain can be driven by low frequency flow oscillations (LFOs) through the intramural periarterial drainage (IPAD) pathway. This study investigates the use of 4D flow MRI to derive LFOs from arterial and venous measures of blood flow. 3D radial 4D flow MRI data were acquired on a 3.0 T scanner and reconstructed using a low-rank constraint to produce time resolved measurements of blood flow. Physical phantom experiments were performed to validate the time resolved 4D flow against a standard 2D phase contrast (PC) approach. To evaluate the ability of 4D flow to distinguish physiologic flow changes from noise, healthy volunteers were scanned during a breath-hold (BH) maneuver and compared against 2D PC measures. Finally, flow measures were performed in intracranial arteries and veins of 112 participants including subjects diagnosed with Alzheimer's disease (AD) clinical syndrome (n = 23), and healthy controls (n = 89) on whom apolipoprotein ɛ4 positivity (APOE4+) and parental history of AD dementia (FH+) was known. To assess LFOs, flow range, standard deviation, demeaned temporal flow changes, and power spectral density were quantified from the time series. Group differences were assessed using ANOVA followed by Tukey-Kramer method for pairwise comparison for adjusted means (P < 0.05). Significantly lower LFOs as measured from flow variation range and standard deviations were observed in the arteries of AD subjects when compared to age-matched controls (P = 0.005, P = 0.011). Results suggest altered vascular function in AD subjects. 4D flow based spontaneous LFO measures might hold potential for longitudinal studies aimed at predicting cognitive trajectories in AD and study disease mechanisms.