Load Adaptation of Lamellipodial Actin Networks.

Actin filaments polymerizing against membranes power endocytosis, vesicular traffic, and cell motility. In vitro reconstitution studies suggest that the structure and the dynamics of actin networks respond to mechanical forces. We demonstrate that lamellipodial actin of migrating cells responds to mechanical load when membrane tension is modulated. In a steady state, migrating cell filaments assume the canonical dendritic geometry, defined by Arp2/3-generated 70° branch points. Increased tension triggers a dense network with a broadened range of angles, whereas decreased tension causes a shift to a sparse configuration dominated by filaments growing perpendicularly to the plasma membrane. We show that these responses emerge from the geometry of branched actin: when load per filament decreases, elongation speed increases and perpendicular filaments gradually outcompete others because they polymerize the shortest distance to the membrane, where they are protected from capping. This network-intrinsic geometrical adaptation mechanism tunes protrusive force in response to mechanical load.

Rescue From Permanent Kidney Injury in Acute Thrombosis of Both Renal Veins, the Inferior Vena Cava, and Both Iliofemoral Veins by Catheter-Based Thrombectomy.

CASE: A 33-year-old man with previously diagnosed lupus membranous nephropathy presented with painful swelling in both legs. Laboratory tests revealed acute kidney injury, and imaging studies by duplex ultrasound and computed tomography scan showed acute thrombosis of both renal veins, the infrahepatic inferior vena cava, and both iliofemoral venous segments. Initially, pharmacomechanical thrombolysis led to an insufficient morphological result. The therapeutic breakthrough was achieved by catheter-based mechanical thrombectomy of the infrarenal vena cava and both renal veins, which successfully cleared all affected venous segments from thrombus, paralleled by improvement of the patient's condition. However, after 1 week, the patient experienced recurrent thrombosis of the right renal vein with hemorrhagic infarction of the right kidney. After further optimization of immunomodulatory and antithrombotic therapy, a repeated catheter-based mechanical thrombectomy resulted in sustained clinical improvement and preservation of renal venous drainage and kidney function. CONCLUSION: Extensive acute thrombosis of both renal veins, the inferior vena cava, and both iliofemoral venous segments is a rare emergency potentially threatening kidney function. Immediate effective thrombus removal is essential to preserve kidney function and can be achieved by catheter-based mechanical thrombectomy embedded in a comprehensive immunomodulatory and antithrombotic therapeutic concept. CLINICAL IMPACT: This case demonstrated the efficacy of a catheter-based therapeutic approach in patients with extensive thrombosis of the venous system. A catheter-based approach must be embedded in a comprehensive medical therapeutic concept, which is essential to achieve a sustainable result.

Suppression PCR-Based Selective Enrichment Sequencing for Pathogen and Antimicrobial Resistance Detection on Cell-Free DNA in Sepsis-A Targeted, Blood Culture-Independent Approach for Rapid Pathogen and Resistance Diagnostics in Septic Patients.

Sepsis is a life-threatening syndrome triggered by infection and accompanied by high mortality, with antimicrobial resistances (AMRs) further escalating clinical challenges. The rapid and reliable detection of causative pathogens and AMRs are key factors for fast and appropriate treatment, in order to improve outcomes in septic patients. However, current sepsis diagnostics based on blood culture is limited by low sensitivity and specificity while current molecular approaches fail to enter clinical routine. Therefore, we developed a suppression PCR-based selective enrichment sequencing approach (SUPSETS), providing a molecular method combining multiplex suppression PCR with Nanopore sequencing to identify most common sepsis-causative pathogens and AMRs using plasma cell-free DNA. Applying only 1 mL of plasma, we targeted eight pathogens across three kingdoms and ten AMRs in a proof-of-concept study. SUPSETS was successfully tested in an experimental research study on the first ten clinical samples and revealed comparable results to clinical metagenomics while clearly outperforming blood culture. Several clinically relevant AMRs could be additionally detected. Furthermore, SUPSETS provided first pathogen and AMR-specific sequencing reads within minutes of starting sequencing, thereby potentially decreasing time-to-results to 11-13 h and suggesting diagnostic potential in sepsis.

Transient localization of the Arp2/3 complex initiates neuronal dendrite branching in vivo.

The formation of neuronal dendrite branches is fundamental for the wiring and function of the nervous system. Indeed, dendrite branching enhances the coverage of the neuron's receptive field and modulates the initial processing of incoming stimuli. Complex dendrite patterns are achieved in vivo through a dynamic process of de novo branch formation, branch extension and retraction. The first step towards branch formation is the generation of a dynamic filopodium-like branchlet. The mechanisms underlying the initiation of dendrite branchlets are therefore crucial to the shaping of dendrites. Through in vivo time-lapse imaging of the subcellular localization of actin during the process of branching of Drosophila larva sensory neurons, combined with genetic analysis and electron tomography, we have identified the Actin-related protein (Arp) 2/3 complex as the major actin nucleator involved in the initiation of dendrite branchlet formation, under the control of the activator WAVE and of the small GTPase Rac1. Transient recruitment of an Arp2/3 component marks the site of branchlet initiation in vivo These data position the activation of Arp2/3 as an early hub for the initiation of branchlet formation.

Smell and taste in idiopathic blepharospasm.

The pathophysiology of blepharospasm is incompletely understood. Current concepts suggest that blepharospasm is a network disorder, involving basal ganglia, thalamus, cortex, and, possibly, the cerebellum. Tracing, imaging, and clinical studies revealed that these structures are also concerned with olfaction and taste. Because of this anatomical overlap, dysfunction of the chemical senses in blepharospasm is expected. Injections of botulinum toxin into the eyelid muscles are the first-line treatment of blepharospasm. Yet, the effects of botulinum toxin on the chemical senses have not been systematically assessed. To contribute to a better understanding of blepharospasm, olfactory and gustatory abilities were assessed in 17 subjects with blepharospasm and 17 age-/sex-matched healthy controls. Sniffin Sticks were used to assess odor threshold, odor discrimination, and odor identification. Results of these three Sniffin Sticks subtests were added to the composite olfactory score. The Taste Strips were applied to assess taste. In an adjacent study, we assessed the sense of smell and taste in eight subjects with blepharospasm before and 4 weeks after botulinum toxin treatment. Subjects with blepharospasm had significantly lower (= worse) scores for odor threshold and for the composite olfactory score than healthy controls, while odor discrimination, odor identification, and the composite taste score were not different between groups. The adjacent study revealed that botulinum toxin did not impact the chemical senses. In this study, subjects with blepharospasm had a lower (= worse) odor threshold than healthy controls. As olfaction is important in daily life, findings justify further research of olfaction in blepharospasm.

Smell and taste in cervical dystonia.

The pathophysiology of cervical dystonia is not completely understood. Current concepts of the pathophysiology propose that it is a network disorder involving the basal ganglia, cerebellum and sensorimotor cortex. These structures are primarily concerned with sensorimotor control but are also involved in non-motor functioning such as the processing of information related to the chemical senses. This overlap lets us hypothesize a link between cervical dystonia and altered sense of smell and taste. To prove this hypothesis and to contribute to the better understanding of cervical dystonia, we assessed olfactory and gustatory functioning in 40 adults with idiopathic cervical dystonia and 40 healthy controls. The Sniffin Sticks were used to assess odor threshold, discrimination and identification. Furthermore, the Taste Strips were applied to assess the combined taste score. Motor and non-motor deficits of cervical dystonia including neuropsychological and psychiatric alterations were assessed as cofactors for regression analyses. We found that cervical dystonia subjects had lower scores than healthy controls for odor threshold (5.8 ± 2.4 versus 8.0 ± 3.2; p = 0.001), odor identification (11.7 ± 2.3 versus 13.1 ± 1.3; p = 0.001) and the combined taste score (9.5 ± 2.2 versus 11.7 ± 2.7; p < 0.001), while no difference was found in odor discrimination (12.0 ± 2.5 versus 12.9 ± 1.8; p = 0.097). Regression analysis suggests that age is the main predictor for olfactory decline in subjects with cervical dystonia. Moreover, performance in the Montreal Cognitive Assessment is a predictor for gustatory decline in cervical dystonia subjects. Findings propose that cervical dystonia is associated with diminished olfactory and gustatory functioning.

Automated MR-based lung volume segmentation in population-based whole-body MR imaging: correlation with clinical characteristics, pulmonary function testing and obstructive lung disease.

OBJECTIVES: Whole-body MR imaging is increasingly utilised; although for lung dedicated sequences are often not included, the chest is typically imaged. Our objective was to determine the clinical utility of lung volumes derived from non-dedicated MRI sequences in the population-based KORA-FF4 cohort study. METHODS: 400 subjects (56.4 ± 9.2 years, 57.6% males) underwent whole-body MRI including a coronal T1-DIXON-VIBE sequence in inspiration breath-hold, originally acquired for fat quantification. Based on MRI, lung volumes were derived using an automated framework and related to common predictors, pulmonary function tests (PFT; spirometry and pulmonary gas exchange, n = 214) and obstructive lung disease. RESULTS: MRI-based lung volume was 4.0 ± 1.1 L, which was 64.8 ± 14.9% of predicted total lung capacity (TLC) and 124.4 ± 27.9% of functional residual capacity. In multivariate analysis, it was positively associated with age, male, current smoking and height. Among PFT indices, MRI-based lung volume correlated best with TLC, alveolar volume and residual volume (RV; r = 0.57 each), while it was negatively correlated to FEV1/FVC (r = 0.36) and transfer factor for carbon monoxide (r = 0.16). Combining the strongest PFT parameters, RV and FEV1/FVC remained independently and incrementally associated with MRI-based lung volume (ß = 0.50, p = 0.04 and ß = - 0.02, p = 0.02, respectively) explaining 32% of the variability. For the identification of subjects with obstructive lung disease, height-indexed MRI-based lung volume yielded an AUC of 0.673-0.654. CONCLUSION: Lung volume derived from non-dedicated whole-body MRI is independently associated with RV and FEV1/FVC. Furthermore, its moderate accuracy for obstructive lung disease indicates that it may be a promising tool to assess pulmonary health in whole-body imaging when PFT is not available. KEY POINTS: • Although whole-body MRI often does not include dedicated lung sequences, lung volume can be automatically derived using dedicated segmentation algorithms • Lung volume derived from whole-body MRI correlates with typical predictors and risk factors of respiratory function including smoking and represents about 65% of total lung capacity and 125% of the functional residual capacity • Lung volume derived from whole-body MRI is independently associated with residual volume and the ratio of forced expiratory volume in 1 s to forced vital capacity and may allow detection of obstructive lung disease.

Performance of Magnetic Resonance Susceptibility-Weighted Imaging for Detection of Calcifications in Patients With Hepatic Echinococcosis.

OBJECTIVE: We evaluated the performance of susceptibility-weighted imaging (SWI) for identification of hepatic calcifications in alveolar echinococcosis and cystic echinococcosis. METHODS: The SWI images of 58 lesions in 40 patients (age, 49 ± 14 y) with alveolar echinococcosis (n = 22) or cystic echinococcosis (n = 18) were reviewed for calcifications. First, calcifications were suggested by visual assessment. Second, ratios of minimum intralesional intensity and mean lumbar muscle intensity were recorded. Computed tomography (CT) served as the criterion standard. RESULTS: Thirty-seven lesions showed calcifications on CT. Susceptibility-weighted imaging provided a sensitivity of 89.2% (95% confidence interval [CI], 50.1-75.7) and a specificity of 57.1% (95% CI, 34.4-77.4) for calcifications detected by visual assessment. Receiver operating characteristic curves demonstrated a sensitivity of 67.6% and a specificity of 85.0% for an intensity ratio of 0.61. A specificity of 100% (95% CI, 80.8-100) and a sensitivity of 84.5% (95% CI, 67.3-93.2) were achieved by SWI for calcifications with a density greater than 184 HU in CT. CONCLUSIONS: Identification of hepatic calcifications is possible with SWI. Susceptibility-weighted imaging offers the potential to reduce the need for of CT imaging for evaluation of echinococcosis.

Electron tomography and simulation of baculovirus actin comet tails support a tethered filament model of pathogen propulsion.

Several pathogens induce propulsive actin comet tails in cells they invade to disseminate their infection. They achieve this by recruiting factors for actin nucleation, the Arp2/3 complex, and polymerization regulators from the host cytoplasm. Owing to limited information on the structural organization of actin comets and in particular the spatial arrangement of filaments engaged in propulsion, the underlying mechanism of pathogen movement is currently speculative and controversial. Using electron tomography we have resolved the three-dimensional architecture of actin comet tails propelling baculovirus, the smallest pathogen yet known to hijack the actin motile machinery. Comet tail geometry was also mimicked in mixtures of virus capsids with purified actin and a minimal inventory of actin regulators. We demonstrate that propulsion is based on the assembly of a fishbone-like array of actin filaments organized in subsets linked by branch junctions, with an average of four filaments pushing the virus at any one time. Using an energy-minimizing function we have simulated the structure of actin comet tails as well as the tracks adopted by baculovirus in infected cells in vivo. The results from the simulations rule out gel squeezing models of propulsion and support those in which actin filaments are continuously tethered during branch nucleation and polymerization. Since Listeria monocytogenes, Shigella flexneri, and Vaccinia virus among other pathogens use the same common toolbox of components as baculovirus to move, we suggest they share the same principles of actin organization and mode of propulsion.

Syringomyelia associated with cervical spondylotic myelopathy causing canal stenosis. A rare association.

BACKGROUND: Although cervical spondylosis is extremely common, only few cases with associated syrinx have been reported. Depending on review of two large data bases, we report this case series. In addition, we evaluated the posterior decompression as the management option in treatment of this rare condition. MATERIALS AND METHODS: Data of all cases with cervical spondylosis and canal stenosis that sought medical advice or needed decompressive laminectomy/laminoplasty between the years 2006 and 2015 were checked in manually. Perioperative data, together with follow up were reviewed. RESULTS: Out of five cases found in the reviewed data; four cases undergone posterior decompression (laminectomy in two cases and laminoplasty in the other). One case refused surgery. Along mean follow up period of 6.25 months; three cases improved markedly, while in one case no improvement occurred. CONCLUSION: Cervical spondylotic myelopathy can rarely cause syringomyelia. Posterior decompression would be the preferable management option with clinical improvement of most of the cases.

A disulfide intercalator toolbox for the site-directed modification of polypeptides.

A disulfide intercalator toolbox was developed for site-specific attachment of a broad variety of functional groups to proteins or peptides under mild, physiological conditions. The peptide hormone somatostatin (SST) served as model compound for intercalation into the available disulfide functionalization schemes starting from the intercalator or the reactive SST precursor before or after bioconjugation. A tetrazole-SST derivative was obtained that undergoes photoinduced cycloaddition in mammalian cells, which was monitored by live-cell imaging.

Tridimensional surface roughness analysis after resin infiltration of (deproteinized) natural subsurface carious lesions.

OBJECTIVES: The objectives of this study were to evaluate ex vivo the effects of resin infiltration on the areal surface roughness of natural non-cavitated proximal subsurface lesions with or without previous deproteinization and to determine differences between E2 and D1 lesions or between premolars and molars. MATERIALS AND METHODS: Forty premolars and 40 molars with proximal carious lesions and macroscopically intact surfaces (International Caries Detection and Assessment System (ICDAS) II; code 2) were radiologically assessed and randomly allocated to four groups (with 20 E2 and 20 D1 lesions, respectively). In each group, 10 lesions were deproteinized (NaOCl; 1%) before etching (HCl; 15%) and resin infiltration (Icon). Areal surface roughness (Sa) at the most demineralized lesion part (DIAGNOdent) was evaluated topometrically before and after deproteinization, after etching, and after infiltration using focus variation 3D scanning microscopy. RESULTS: Pretreatment with NaOCl (n = 40) had no significant effects on Sa (p = 0.208), but resulted in significantly differing Sa values between premolars and molars after etching (p = 0.011). Regarding the effects between etching and baseline, significantly differing Sa values (p = 0.0498) were found for premolars and molars (n = 40/40); Sa after resin infiltration (compared to etching) differed significantly between premolars and molars (p = 0.009). No treatment regimen lead to differences among the radiological grades (E2 vs. D1; p > 0.106). CONCLUSIONS: Resin infiltration showed only minor effects on Sa values of etched subsurface lesions (p < 0.170) and did neither equal nor improve baseline surface roughness (p > 0.401) of the different tooth types. CLINICAL RELEVANCE: Deproteinization should be recommended before etching and infiltration, even if surface roughness of infiltrated advanced (pre-)molar lesions will not be improved.

Monitoring Compliance to Promote Quality Assurance: Development of a Mental Health Clinical Chart Audit Tool in Belize, 2013.

Belize trained psychiatric nurse practitioners (PNPs) in the early 1990s to provide mental health services throughout the country. Despite overwhelming success, the program is limited by lack of monitoring, evaluation, and surveillance. To promote quality assurance, we developed a chart audit tool to monitor mental healthcare delivery compliance for initial psychiatric assessment notes completed by PNPs. After reviewing the Belize Health Information System electronic medical record system, we developed a clinical audit tool to capture 20 essential components for initial assessment clinical notes. The audit tool was then piloted for initial assessment notes completed during July through September of 2013. One hundred and thirty-four initial psychiatric interviews were audited. The average chart score among all PNPs was 9.57, ranging from 3 to 15. Twenty-three charts-or 17.2%-had a score of 14 or higher and met a 70% compliance benchmark goal. Among indicators most frequently omitted included labs ordered and named (15.7%) and psychiatric diagnosis (21.6%). Explicit statement of medications initiated with dose and frequency occurred in 47.0% of charts. Our findings provide direction for training and improvement, such as emphasizing the importance of naming labs ordered, medications and doses prescribed, and psychiatric diagnoses in initial assessment clinical notes. We hope this initial assessment helps enhance mental health delivery compliance by prompting creation of BHIS templates, development of audits tools for revisit follow-up visits, and establishment of corrective actions for low-scoring practitioners. These efforts may serve as a model for implementing quality assurance programming in other low resource settings.

Visible-Light-Induced Click Chemistry.

A rapid and catalyst-free cycloaddition system for visible-light-induced click chemistry is reported. A readily accessible photoreactive 2H-azirine moiety was designed to absorb light at wavelengths above 400 nm. Irradiation with low-energy light sources thus enables efficient small-molecule synthesis with a diverse range of multiple-bond-containing compounds. Moreover, in order to demonstrate the efficiency of the current approach, quantitative ligation of the photoactivatable chromophore with functional polymeric substrates was performed and full conversion with irradiation times of only 1 min at ambient conditions was achieved. The current report thus presents a highly efficient method for applications involving selective cycloaddition to electron-deficient multiple-bond-containing materials.

Efficient Photochemical Approaches for Spatially Resolved Surface Functionalization.

Materials interfaces--with a gas, a liquid, or another solid--are highly important for advanced applications. Besides their topological design, controlling interactions at these interfaces is typically realized by tuning the chemical composition of the materials surface. In areas such as nanoscience or biology, it is, however, highly desirable to impart heterogeneously distributed properties. Photopatterning, more than micro- and nanoprinting methods, is often the method of choice for precise functionalization, especially in terms of versatility. Recently, a range of new or rediscovered photochemistry approaches have been applied to precision surface functionalization, with the common aim of increasing efficiency and resolution while concomitantly lowering the amount of required energy. A survey of such methods is presented in this Review, with a focus on those we have explored.

Actin branching in the initiation and maintenance of lamellipodia.

Using correlated live-cell imaging and electron tomography we found that actin branch junctions in protruding and treadmilling lamellipodia are not concentrated at the front as previously supposed, but link actin filament subsets in which there is a continuum of distances from a junction to the filament plus ends, for up to at least 1 µm. When branch sites were observed closely spaced on the same filament their separation was commonly a multiple of the actin helical repeat of 36 nm. Image averaging of branch junctions in the tomograms yielded a model for the in vivo branch at 2.9 nm resolution, which was comparable with that derived for the in vitro actin-Arp2/3 complex. Lamellipodium initiation was monitored in an intracellular wound-healing model and was found to involve branching from the sides of actin filaments oriented parallel to the plasmalemma. Many filament plus ends, presumably capped, terminated behind the lamellipodium tip and localized on the dorsal and ventral surfaces of the actin network. These findings reveal how branching events initiate and maintain a network of actin filaments of variable length, and provide the first structural model of the branch junction in vivo. A possible role of filament capping in generating the lamellipodium leaflet is discussed and a mathematical model of protrusion is also presented.

Comparative Efficacy and Safety of Neoadjuvant Immunotherapy with Nivolumab vs. Pembrolizumab in Resectable Non-Small Cell Lung Cancer: A Systematic Review.

Non-small cell lung cancer (NSCLC) remains a leading cause of cancer-related mortality worldwide. Immunotherapy has emerged as a promising treatment option due to its favorable toxicity profile. However, selecting the most appropriate immunotherapeutic agent for neoadjuvant use-aimed at curative intent in early-stage NSCLC-based on efficacy and safety remains a critical question. This review aims to compare the efficacy and safety profiles of nivolumab and pembrolizumab when used as neoadjuvant treatments in NSCLC. A systematic review was conducted across PubMed, Scopus, Wiley Online Library, ProQuest Dissertations and Theses Global, and Google Scholar, utilizing the search terms "Nivolumab OR Pembrolizumab AND Neoadjuvant Immunotherapy AND non-small cell lung cancer." Out of 1444 retrieved studies, 4 retrospective studies met the inclusion criteria by providing comparative data on nivolumab and pembrolizumab within the same study cohorts. Despite the critical risk of bias and the evidence quality ranging from moderate to very low across these studies, both nivolumab and pembrolizumab demonstrated efficacy rates exceeding 30% and maintained favorable safety profiles. There is no observed superiority between nivolumab and pembrolizumab in terms of efficacy and safety for the neoadjuvant treatment of early-stage NSCLC.

Targeting the mevalonate or Wnt pathways to overcome CAR T-cell resistance in TP53-mutant AML cells.

TP53-mutant acute myeloid leukemia (AML) and myelodysplastic neoplasms (MDS) are characterized by chemotherapy resistance and represent an unmet clinical need. Chimeric antigen receptor (CAR) T-cells might be a promising therapeutic option for TP53-mutant AML/MDS. However, the impact of TP53 deficiency in AML cells on the efficacy of CAR T-cells is unknown. We here show that CAR T-cells engaging TP53-deficient leukemia cells exhibit a prolonged interaction time, upregulate exhaustion markers, and are inefficient to control AML cell outgrowth in vitro and in vivo compared to TP53 wild-type cells. Transcriptional profiling revealed that the mevalonate pathway is upregulated in TP53-deficient AML cells under CAR T-cell attack, while CAR T-cells engaging TP53-deficient AML cells downregulate the Wnt pathway. In vitro rational targeting of either of these pathways rescues AML cell sensitivity to CAR T-cell-mediated killing. We thus demonstrate that TP53 deficiency confers resistance to CAR T-cell therapy and identify the mevalonate pathway as a therapeutic vulnerability of TP53-deficient AML cells engaged by CAR T-cells, and the Wnt pathway as a promising CAR T-cell therapy-enhancing approach for TP53-deficient AML/MDS.

Cofilin cooperates with fascin to disassemble filopodial actin filaments.

Cells use a large repertoire of proteins to remodel the actin cytoskeleton. Depending on the proteins involved, F-actin is organized in specialized protrusions such as lamellipodia or filopodia, which serve diverse functions in cell migration and sensing. Although factors responsible for directed filament assembly in filopodia have been extensively characterized, the mechanisms of filament disassembly in these structures are mostly unknown. We investigated how the actin-depolymerizing factor cofilin-1 affects the dynamics of fascincrosslinked actin filaments in vitro and in live cells. By multicolor total internal reflection fluorescence microscopy and fluorimetric assays, we found that cofilin-mediated severing is enhanced in fascin-crosslinked bundles compared with isolated filaments, and that fascin and cofilin act synergistically in filament severing. Immunolabeling experiments demonstrated for the first time that besides its known localization in lamellipodia and membrane ruffles, endogenous cofilin can also accumulate in the tips and shafts of filopodia. Live-cell imaging of fluorescently tagged proteins revealed that cofilin is specifically targeted to filopodia upon stalling of protrusion and during their retraction. Subsequent electron tomography established filopodial actin filament and/or bundle fragmentation to precisely correlate with cofilin accumulation. These results identify a new mechanism of filopodium disassembly involving both fascin and cofilin.