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Competing Interests: Competing interests: None declared.

BRCA2 is an essential tumor suppressor protein involved in promoting faithful repair of DNA lesions. The activity of BRCA2 needs to be tuned precisely to be active when and where it is needed. Here, we quantified the spatio-temporal dynamics of BRCA2 in living cells using aberration-corrected multifocal microscopy (acMFM). Using multicolor imaging to identify DNA damage sites, we were able to quantify its dynamic motion patterns in the nucleus and at DNA damage sites. While a large fraction of BRCA2 molecules localized near DNA damage sites appear immobile, an additional fraction of molecules exhibits subdiffusive motion, providing a potential mechanism to retain an increased number of molecules at DNA lesions. Super-resolution microscopy revealed inhomogeneous localization of BRCA2 relative to other DNA repair factors at sites of DNA damage. This suggests the presence of multiple nanoscale compartments in the chromatin surrounding the DNA lesion, which could play an important role in the contribution of BRCA2 to the regulation of the repair process., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Accurate calculation of non-covalent interaction energies in nucleotides is crucial for understanding the driving forces governing nucleic acid structure and function, as well as developing advanced molecular mechanics forcefields or machine learning potentials tailored to nucleic acids. Here, we dissect the nucleotides' structure into three main constituents: nucleobases (A, G, C, T, and U), sugar moieties (ribose and deoxyribose), and phosphate group. The interactions among these fragments and between fragments and water were analyzed. Different quantum mechanical methods were compared for their accuracy in capturing the interaction energy. The non-covalent interaction energy was decomposed into electrostatics, exchange-repulsion, dispersion, and induction using two ab initio methods: Symmetry-Adapted Perturbation Theory (SAPT) and Absolutely Localized Molecular Orbitals (ALMO). These calculations provide a benchmark for different QM methods, in addition to providing a valuable understanding of the roles of various intermolecular forces in hydrogen bonding and aromatic stacking. With SAPT, a higher theory level and/or larger basis set did not necessarily give more accuracy. It is hard to know which combination would be best for a given system. In contrast, ALMO EDA2 did not show dependence on theory level or basis set; additionally, it is faster.

Significance: To enable non-destructive longitudinal assessment of drug agents in intact tumor tissue without the use of disruptive probes, we have designed a label-free method to quantify the health of individual tumor cells in excised tumor tissue using multiphoton fluorescence lifetime imaging microscopy (MP-FLIM)., Aim: Using murine tumor fragments which preserve the native tumor microenvironment, we seek to demonstrate signals generated by the intrinsically fluorescent metabolic co-factors nicotinamide adenine dinucleotide phosphate [NAD(P)H] and flavin adenine dinucleotide (FAD) correlate with irreversible cascades leading to cell death., Approach: We use MP-FLIM of NAD(P)H and FAD on tissues and confirm viability using standard apoptosis and live/dead (Caspase 3/7 and propidium iodide, respectively) assays., Results: Through a statistical approach, reproducible shifts in FLIM data, determined through phasor analysis, are shown to correlate with loss of cell viability. With this, we demonstrate that cell death achieved through either apoptosis/necrosis or necroptosis can be discriminated. In addition, specific responses to common chemotherapeutic treatment inducing cell death were detected., Conclusions: These data demonstrate that MP-FLIM can detect and quantify cell viability without the use of potentially toxic dyes, thus enabling longitudinal multi-day studies assessing the effects of therapeutic agents on tumor fragments., (© 2024 The Authors.)

Cell plate formation during cytokinesis entails multiple stages occurring concurrently and requiring orchestrated vesicle delivery, membrane remodelling, and timely deposition of polysaccharides, such as callose. Understanding such a dynamic process requires dissection in time and space; this has been a major hurdle in studying cytokinesis. Using lattice light sheet microscopy (LLSM), we studied cell plate development in four dimensions, through the behavior of yellow fluorescent protein (YFP)-tagged cytokinesis-specific GTPase RABA2a vesicles. We monitored the entire duration of cell plate development, from its first emergence, with the aid of YFP-RABA2a, in both the presence and absence of cytokinetic callose. By developing a robust cytokinetic vesicle volume analysis pipeline, we identified distinct behavioral patterns, allowing the identification of three easily trackable cell plate developmental phases. Notably, the phase transition between phase I and phase II is striking, indicating a switch from membrane accumulation to the recycling of excess membrane material. We interrogated the role of callose using pharmacological inhibition with LLSM and electron microscopy. Loss of callose inhibited the phase transitions, establishing the critical role and timing of the polysaccharide deposition in cell plate expansion and maturation. This study exemplifies the power of combining LLSM with quantitative analysis to decode and untangle such a complex process., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Assessing cell viability is important in many fields of research. Current optical methods to assess cell viability typically involve fluorescent dyes, which are often less reliable and have poor permeability in primary tissues. Dynamic optical coherence microscopy (dOCM) is an emerging tool that provides label-free contrast reflecting changes in cellular metabolism. In this work, we compare the live contrast obtained from dOCM to viability dyes, and for the first time to our knowledge, demonstrate that dOCM can distinguish live cells from dead cells in murine syngeneic tumors. We further demonstrate a strong correlation between dOCM live contrast and optical redox ratio by metabolic imaging in primary mouse liver tissue. The dOCM technique opens a new avenue to apply label-free imaging to assess the effects of immuno-oncology agents, targeted therapies, chemotherapy, and cell therapies using live tumor tissues., Competing Interests: Elephas Biosciences Corporation has filed a provisional patent application 63/459,804 that encompasses aspects of the data described in this paper., (© 2024 Optica Publishing Group.)

Background: Extensor pollicis longus (EPL) tendon rupture is a known complication of distal radius fractures. The Pulvertaft graft technique is currently used for tendon transfer of extensor indicis proprious (EIP) to EPL. This technique can produce unwanted tissue bulkiness and cosmetic concerns as well as hinder tendon gliding. A novel "open book" technique has been proposed, but relevant biomechanical data are limited. We designed a study to examine the biomechanical behaviours of the "open book" versus Pulvertaft techniques. Methods: Twenty matched forearm-wrist-hand samples were harvested from 10 fresh frozen cadavers (2 female, 8 male) with a mean age of 61.7 (±19.25) years. The EIP was transferred to EPL using the Pulvertaft versus "open book" techniques for each matched pair (sides randomly assigned). The repaired tendon segments were mechanically loaded using a Materials Testing System to examine graft biomechanical behaviours. Results: Mann-Whitney U test outcomes demonstrated that there was no significant difference between "open book" versus Pulvertaft techniques for peak load, load at yield, elongation at yield, or repair width. The "open book" technique demonstrated a significantly lower elongation at peak load and repair thickness, as well as significantly higher stiffness when compared with the Pulvertaft technique. Conclusions: Our findings support the use of the "open book" technique, producing comparable biomechanical behaviours compared to the Pulvertaft technique. Incorporating the "open book" technique potentially requires smaller repair volume, producing size and appearance that is more anatomic when compared with the Pulvertaft., Competing Interests: The author(s) declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: Though they are not directly funding and/or influencing this study, the authors would like to disclose the following support for Desirae McKee: Paid teaching and consulting, as well as research support from AxoGen., (© 2021 The Author(s).)

Background: Intraoperative 2-dimensional (2D) fluoroscopy imaging has been commonly adopted for guidance during complex pediatric spinal deformity correction. Despite the benefits, fluoroscopy imaging emits harmful ionizing radiation, which has been well-established to have deleterious effects on the surgeon and operating room staff. This study investigated the difference in intraoperative fluoroscopy time and radiation exposure during pediatric spine surgery between 2D fluoroscopy-based navigation and a novel machine vision navigation system [machine vision image guidance system (MvIGS)]., Methods: This retrospective chart review was conducted at a pediatric hospital with patients who underwent posterior spinal fusion for spinal deformity correction from 2018 to 2021. Patient allocation to the navigation modality was determined by the date of their surgery and the date of implementation of the MvIGS. Both modalities were the standard of care. Intraoperative radiation exposure was collected from the fluoroscopy system reports., Results: A total of 1442 pedicle screws were placed in 77 children: 714 using MvIGS and 728 using 2D fluoroscopy. There were no significant differences in the male-to-female ratio, age range, body mass index, distribution of spinal pathologies, number of levels operated on, types of levels operated on, and the number of pedicle screws implanted. Total intraoperative fluoroscopy time was significantly reduced in cases utilizing MvIGS (18.6 ± 6.3 s) compared with 2D fluoroscopy (58.5 ± 19.0 s) ( P < 0.001). This represents a relative reduction of 68%. Intraoperative radiation dose area product and cumulative air kerma were reduced by 66% (0.69 ± 0.62 vs 2.0 ± 2.1 Gycm 2 , P < 0.001) and 66% (3.4 ± 3.2 vs 9.9 ± 10.5 mGy, P < 0.001) respectively. The length of stay displayed a decreasing trend with MVIGS, and the operative time was significantly reduced in MvIGS compared with 2D fluoroscopy for an average of 63.6 minutes (294.5 ± 15.5 vs 358.1 ± 60.6 min, P < 0.001)., Conclusion: In pediatric spinal deformity correction surgery, MvIGS was able to significantly reduce intraoperative fluoroscopy time, intraoperative radiation exposure, and total surgical time, compared with traditional fluoroscopy methods. MvIGS reduced the operative time by 63.6 minutes and reduced intraoperative radiation exposure by 66%, which may play an important role in reducing the risks to the surgeon and operating room staff associated with radiation in spinal surgery procedures., Level of Evidence: Level III; retrospective comparative study., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2023 The Author(s). Published by Wolters Kluwer Health, Inc.)

A next-generation protocol (Poltype 2) has been developed which automatically generates AMOEBA polarizable force field parameters for small molecules. Both features and computational efficiency have been drastically improved. Notable advances include improved database transferability using SMILES, robust torsion fitting, non-aromatic ring torsion parameterization, coupled torsion-torsion parameterization, Van der Waals parameter refinement using ab initio dimer data and an intelligent fragmentation scheme that produces parameters with dramatically reduced ab initio computational cost. Additional improvements include better local frame assignment for atomic multipoles, automated formal charge assignment, Zwitterion detection, smart memory resource defaults, parallelized fragment job submission, incorporation of Psi4 quantum package, ab initio error handling, ionization state enumeration, hydration free energy prediction and binding free energy prediction. For validation, we have applied Poltype 2 to ~1000 FDA approved drug molecules from DrugBank. The ab initio molecular dipole moments and electrostatic potential values were compared with Poltype 2 derived AMOEBA counterparts. Parameters were further substantiated by calculating hydration free energy (HFE) on 40 small organic molecules and were compared with experimental data, resulting in an RMSE error of 0.59 kcal/mol. The torsion database has expanded to include 3543 fragments derived from FDA approved drugs. Poltype 2 provides a convenient utility for applications including binding free energy prediction for computational drug discovery. Further improvement will focus on automated parameter refinement by experimental liquid properties, expansion of the Van der Waals parameter database and automated parametrization of modified bio-fragments such as amino and nucleic acids., (© 2022 Wiley Periodicals LLC.)

Myosin VI is the only minus-end actin motor and it is coupled to various cellular processes ranging from endocytosis to transcription. This multi-potent nature is achieved through alternative isoform splicing and interactions with a network of binding partners. There is a complex interplay between isoforms and binding partners to regulate myosin VI. Here, we have compared the regulation of two myosin VI splice isoforms by two different binding partners. By combining biochemical and single-molecule approaches, we propose that myosin VI regulation follows a generic mechanism, independently of the spliced isoform and the binding partner involved. We describe how myosin VI adopts an autoinhibited backfolded state which is released by binding partners. This unfolding activates the motor, enhances actin binding and can subsequently trigger dimerization. We have further expanded our study by using single-molecule imaging to investigate the impact of binding partners upon myosin VI molecular organization and dynamics., (© 2022 The Author(s).)

Quantifying molecular dynamics within the context of complex cellular morphologies is essential toward understanding the inner workings and function of cells. Fluorescence recovery after photobleaching (FRAP) is one of the most broadly applied techniques to measure the reaction diffusion dynamics of molecules in living cells. FRAP measurements typically restrict themselves to single-plane image acquisition within a subcellular-sized region of interest due to the limited temporal resolution and undesirable photobleaching induced by 3D fluorescence confocal or widefield microscopy. Here, an experimental and computational pipeline combining lattice light sheet microscopy, FRAP, and numerical simulations, offering rapid and minimally invasive quantification of molecular dynamics with respect to 3D cell morphology is presented. Having the opportunity to accurately measure and interpret the dynamics of molecules in 3D with respect to cell morphology has the potential to reveal unprecedented insights into the function of living cells., (© 2022 The Authors. Small Methods published by Wiley-VCH GmbH.)

During transcription, RNA Polymerase II (RNAPII) is spatially organised within the nucleus into clusters that correlate with transcription activity. While this is a hallmark of genome regulation in mammalian cells, the mechanisms concerning the assembly, organisation and stability remain unknown. Here, we have used combination of single molecule imaging and genomic approaches to explore the role of nuclear myosin VI (MVI) in the nanoscale organisation of RNAPII. We reveal that MVI in the nucleus acts as the molecular anchor that holds RNAPII in high density clusters. Perturbation of MVI leads to the disruption of RNAPII localisation, chromatin organisation and subsequently a decrease in gene expression. Overall, we uncover the fundamental role of MVI in the spatial regulation of gene expression., (© 2022. The Author(s).)

Objectives: Implant failure leading to revision total joint arthroplasty can occur through a variety of different mechanisms which are typically associated with a soft tissue response adjacent to the implant that provide insight into the underlying etiology of implant failure. The objective of this study was to elucidate mechanisms of implant failure as they relate to histological classification and findings of adjacent periprosthetic tissue., Methods: Histological analysis of soft tissue adjacent to the implant was performed in 99 patients with an average age of 64 years old and grouped into four categories based on the study conducted by Morawietz et al.:Type I (N = 47)Wear particle induced typeType II (N = 7)Infectious typeType III (N = 19)Combined type I and IIType IV (N = 26)Indeterminant typeModes of failure were categorized into five groupings based on the study conducted by Callies et al.: Instability (N = 35), Aseptic Loosening (N = 24), Hardware and/or Mechanical Failure (N = 15), Septic (N = 13), and Other failures (N = 12). We calculated odds ratios and conducted regression analysis to assess the relationship between modes of failure and histological findings as well as modes of failure and comorbidities., Results: Hardware/mechanical failure was independently correlated with histological findings of anucleate protein debris, histiocytes, Staphylococcus epidermidis , and synovitis. Furthermore, hardware/mechanical failure was independently correlated with osteosarcoma as a co-morbidity. Septic failure was associated with histological findings of Enterococcus, granulation tissue, and tissue necrosis as well as comorbidities of Crohn's disease, deep venous thrombosis, lung disease, and rheumatoid arthritis. Infection was 5.8 times more likely to be associated with Type II histology. Aseptic loosening was associated with histologic findings of synovitis., Conclusion: Our findings support the existing literature on periprosthetic tissue analysis in revision total joint arthroplasty which may improve surgeon understanding of the patholophysiological mechanisms that contribute to implant failure and revision surgery., Competing Interests: Declaration of conflicting interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article., (© The Author(s) 2021.)

Upon inflammation, leukocytes rapidly transmigrate across the endothelium to enter the inflamed tissue. Evidence accumulates that leukocytes use preferred exit sites, alhough it is not yet clear how these hotspots in the endothelium are defined and how they are recognized by the leukocyte. Using lattice light sheet microscopy, we discovered that leukocytes prefer endothelial membrane protrusions at cell junctions for transmigration. Phenotypically, these junctional membrane protrusions are present in an asymmetric manner, meaning that one endothelial cell shows the protrusion and the adjacent one does not. Consequently, leukocytes cross the junction by migrating underneath the protruding endothelial cell. These protrusions depend on Rac1 activity and by using a photo-activatable Rac1 probe, we could artificially generate local exit-sites for leukocytes. Overall, we have discovered a new mechanism that uses local induced junctional membrane protrusions to facilitate/steer the leukocyte escape/exit from inflamed vessel walls., Competing Interests: JA, EM, MG, LS, IN, EG, Av, ST, BW, Jv, MN, MP, SK, JH, EW, TC, MW, EM, JG, Jv No competing interests declared, (© 2021, Arts et al.)

With age, neural stem cell (NSC) function in the adult ventricular-subventricular zone (V-SVZ) declines, reducing memory and cognitive function in males; however, the impact on females is not well understood. To obtain a global view of how age and sex impact the mouse V-SVZ, we constructed 3D montages after multiplex immunostaining, and used computer-based 3D image analysis to quantify data across the entire niche at 2, 18, and 22 months. We discovered dramatic sex differences in the aging of the V-SVZ niche vasculature, which regulates NSC activity: females showed increased diameter but decreased vessel density with age, while males showed decreased diameter and increased tortuosity and vessel density. Accompanying these vascular changes, males showed significant decline in NSC numbers, progenitor cell proliferation, and more disorganized migrating neuroblast chains with age; however, females did not. By examining the entire 3D niche, we found significant sex differences, with females being relatively spared through very old age., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Filopodia are peripheral F-actin-rich structures that enable cell sensing of the microenvironment. Fascin is an F-actin-bundling protein that plays a key role in stabilizing filopodia to support efficient adhesion and migration. Fascin is also highly up-regulated in human cancers, where it increases invasive cell behavior and correlates with poor patient prognosis. Previous studies have shown that fascin phosphorylation can regulate F-actin bundling, and that this modification can contribute to subcellular fascin localization and function. However, the factors that regulate fascin dynamics within filopodia remain poorly understood. In the current study, we used advanced live-cell imaging techniques and a fascin biosensor to demonstrate that fascin phosphorylation, localization, and binding to F-actin are highly dynamic and dependent on local cytoskeletal architecture in cells in both 2D and 3D environments. Fascin dynamics within filopodia are under the control of formins, and in particular FMNL2, that binds directly to dephosphorylated fascin. Our data provide new insight into control of fascin dynamics at the nanoscale and into the mechanisms governing rapid cytoskeletal adaptation to environmental changes. This filopodia-driven exploration stage may represent an essential regulatory step in the transition from static to migrating cancer cells., (© 2020 Pfisterer et al.)

Summary: Light microscopes can now capture data in five dimensions at very high frame rates producing terabytes of data per experiment. Five-dimensional data has three spatial dimensions (x, y, z), multiple channels (λ) and time (t). Current tools are prohibitively time consuming and do not efficiently utilize available hardware. The hydra image processor (HIP) is a new library providing hardware-accelerated image processing accessible from interpreted languages including MATLAB and Python. HIP automatically distributes data/computation across system and video RAM allowing hardware-accelerated processing of arbitrarily large images. HIP also partitions compute tasks optimally across multiple GPUs. HIP includes a new kernel renormalization reducing boundary effects associated with widely used padding approaches., Availability and Implementation: HIP is free and open source software released under the BSD 3-Clause License. Source code and compiled binary files will be maintained on http://www.hydraimageprocessor.com. A comprehensive description of all MATLAB and Python interfaces and user documents are provided. HIP includes GPU-accelerated support for most common image processing operations in 2-D and 3-D and is easily extensible. HIP uses the NVIDIA CUDA interface to access the GPU. CUDA is well supported on Windows and Linux with macOS support in the future., (© The Author(s) 2019. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The rapid advancement of live-cell imaging technologies has enabled biologists to generate high-dimensional data to follow biological movement at the microscopic level. Yet, the "perceived" ease of use of modern microscopes has led to challenges whereby sub-optimal data are commonly generated that cannot support quantitative tracking and analysis as a result of various ill-advised decisions made during image acquisition. Even optimally acquired images often require further optimization through digital processing before they can be analyzed. In writing this article, we presume our target audience to be biologists with a foundational understanding of digital image acquisition and processing, who are seeking to understand the essential steps for particle/object tracking experiments. It is with this targeted readership in mind that we review the basic principles of image-processing techniques as well as analysis strategies commonly used for tracking experiments. We conclude this technical survey with a discussion of how movement behavior can be mathematically modeled and described. © 2019 by John Wiley & Sons, Inc., (© 2019 John Wiley & Sons, Inc.)

The thenar eminence of the thumb is made up of 4 intrinsic muscles: abductor pollicis brevis, opponens pollicis, flexor pollicis brevis, and the adductor pollicis (ADD). While both heads of the ADD insert on the medial base of the thumb proximal phalanx, the oblique head originates on the capitate and second and third metacarpals, and the transverse head originates on the distal half of the third metacarpal. We present the case of a 36-year-old man who was involved in a motor vehicle collision and sustained a laceration in the first webspace with 1 cm extrusion of the ADD and no neurovascular deficiencies on examination. X-ray imaging noted no bony abnormalities. The only identifiable injury was to the ADD muscle which was avulsed from its origin and was extruded through the open wound in the first webspace. A double-row running locking 3-0 fiberwire suture technique was used to have a solid anchor point with which to secure the muscle to its origin. It was secured to the second metacarpal with Mitek mini suture anchors that was sutured on the opposing side of the double-row fiberwire suture and anchored to the second metacarpal proximally and distally in the metacarpal. We report good clinical outcomes postoperative, with intact range of motion and no surgical complication at the 6-month follow-up visit. The patient has ongoing physical therapy to reduce any residual strength deficits.

One of the most important and error-prone tasks in biological image analysis is the segmentation of touching or overlapping cells. Particularly for optical microscopy, including transmitted light and confocal fluorescence microscopy, there is often no consistent discriminative information to separate cells that touch or overlap. It is desired to partition touching foreground pixels into cells using the binary threshold image information only, and optionally incorporating gradient information. The most common approaches for segmenting touching and overlapping cells in these scenarios are based on the watershed transform. We describe a new approach called pixel replication for the task of segmenting elliptical objects that touch or overlap. Pixel replication uses the image Euclidean distance transform in combination with Gaussian mixture models to better exploit practically effective optimization for delineating objects with elliptical decision boundaries. Pixel replication improves significantly on commonly used methods based on watershed transforms, or based on fitting Gaussian mixtures directly to the thresholded image data. Pixel replication works equivalently on both 2-D and 3-D image data, and naturally combines information from multi-channel images. The accuracy of the proposed technique is measured using both the segmentation accuracy on simulated ellipse data and the tracking accuracy on validated stem cell tracking results extracted from hundreds of live-cell microscopy image sequences. Pixel replication is shown to be significantly more accurate compared with other approaches. Variance relationships are derived, allowing a more practically effective Gaussian mixture model to extract cell boundaries for data generated from the threshold image using the uniform elliptical distribution and from the distance transform image using the triangular elliptical distribution.

The supercritical carbon dioxide diluent is used to control the temperature and to increase the efficiency in oxycombustion fossil fuel energy technology. It may affect the rates of combustion by altering mechanisms of chemical reactions, compared to the ones at low CO 2 concentrations. Here, we investigate potential energy surfaces of the four elementary reactions in the CH 3 + O 2 reactive system in the presence of one CO 2 molecule. In the case of reaction CH 3 + O 2 → CH 2 O + OH (R1 channel), van der Waals (vdW) complex formation stabilizes the transition state and reduces the activation barrier by ∼2.2 kcal/mol. Alternatively, covalently bonded CO 2 may form a six-membered ring transition state and reduce the activation barrier by ∼0.6 kcal/mol. In case of reaction CH 3 + O 2 → CH 3 O + O (R2 channel), covalent participation of CO 2 lowers the barrier for the rate limiting step by 3.9 kcal/mol. This is expected to accelerate the R2 process, important for the branching step of the radical chain reaction mechanism. For the reaction CH 3 + O 2 → CHO + H 2 O (R3 channel) with covalent participation of CO 2 , the activation barrier is lowered by 0.5 kcal/mol. The reaction CH 2 O + OH → CHO + H 2 O (R4 channel) involves hydrogen abstraction from formaldehyde by OH radical. Its barrier is reduced from 7.1 to 0.8 kcal/mol by formation of vdW complex with spectator CO 2 . These new findings are expected to improve the kinetic reaction mechanism describing combustion processes in supercritical CO 2 medium.

The organization of the eukaryotic cell into discrete membrane-bound organelles allows for the separation of incompatible biochemical processes, but the activities of these organelles must be coordinated. For example, lipid metabolism is distributed between the endoplasmic reticulum for lipid synthesis, lipid droplets for storage and transport, mitochondria and peroxisomes for β-oxidation, and lysosomes for lipid hydrolysis and recycling. It is increasingly recognized that organelle contacts have a vital role in diverse cellular functions. However, the spatial and temporal organization of organelles within the cell remains poorly characterized, as fluorescence imaging approaches are limited in the number of different labels that can be distinguished in a single image. Here we present a systems-level analysis of the organelle interactome using a multispectral image acquisition method that overcomes the challenge of spectral overlap in the fluorescent protein palette. We used confocal and lattice light sheet instrumentation and an imaging informatics pipeline of five steps to achieve mapping of organelle numbers, volumes, speeds, positions and dynamic inter-organelle contacts in live cells from a monkey fibroblast cell line. We describe the frequency and locality of two-, three-, four- and five-way interactions among six different membrane-bound organelles (endoplasmic reticulum, Golgi, lysosome, peroxisome, mitochondria and lipid droplet) and show how these relationships change over time. We demonstrate that each organelle has a characteristic distribution and dispersion pattern in three-dimensional space and that there is a reproducible pattern of contacts among the six organelles, that is affected by microtubule and cell nutrient status. These live-cell confocal and lattice light sheet spectral imaging approaches are applicable to any cell system expressing multiple fluorescent probes, whether in normal conditions or when cells are exposed to disturbances such as drugs, pathogens or stress. This methodology thus offers a powerful descriptive tool and can be used to develop hypotheses about cellular organization and dynamics.

Introduction: External rotation and gravity stress radiographs have been described to distinguish stable supination-external rotation-II (SER-II) ankle fractures from unstable SER-IV fractures. It has been previously shown that both external rotation and gravity stress views are equivalent in their ability to diagnose deltoid ligament injury. It has also been shown that the position of the ankle influences the external rotation stress radiograph. However, no data of ankle position exist for gravity stress radiographs., Methods: Eight fresh-frozen cadaveric ankles were dissected and destabilized sequentially according to the SER mechanism of ankle fractures, starting with the anterior inferior tibiofibular ligament, distal fibula osteotomy at the level of the syndesmosis, posterior inferior tibiofibular ligament, superficial deltoid ligament, and finally the deep deltoid ligament. Fluoroscopic radiographs were taken with gravity stress views in both neutral and plantarflexion. Measurements of both dorsal and the medial clear space were taken for each stage. The difference between the dorsal and medial clear space was measured., Results: Positive predictive value (PPV) with a medial clear space cutoff of 5 mm was 80% in plantarflexion and 72.72% in neutral with a negative predictive value (NPV) of 100% in both positions. Using a 6-mm cutoff, the PPV improved to 100% and NPV remained 100% for both neutral and plantarflexion. When the difference measurement is used, a 1.0-mm difference yielded a PPV of 72% and an NPV of 100% in both neutral and plantarflexion. With a 1.5-mm cutoff, the PPV and NPV were 100% for both positions., Conclusions: The position of the ankle during gravity stress radiographs does not influence the clinical effectiveness of the images. Using larger references for stability, the PPV can be improved.

The role of mitochondria in cancer is controversial. Using a genome-wide shRNA screen, we now show that tumours reprogram a network of mitochondrial dynamics operative in neurons, including syntaphilin (SNPH), kinesin KIF5B and GTPase Miro1/2 to localize mitochondria to the cortical cytoskeleton and power the membrane machinery of cell movements. When expressed in tumours, SNPH inhibits the speed and distance travelled by individual mitochondria, suppresses organelle dynamics, and blocks chemotaxis and metastasis, in vivo. Tumour progression in humans is associated with downregulation or loss of SNPH, which correlates with shortened patient survival, increased mitochondrial trafficking to the cortical cytoskeleton, greater membrane dynamics and heightened cell invasion. Therefore, a SNPH network regulates metastatic competence and may provide a therapeutic target in cancer.

The analysis of time-lapse images showing cells dividing to produce clones of related cells is an important application in biological microscopy. Imaging at the temporal resolution required to establish accurate tracking for vertebrate stem or cancer cells often requires the use of transmitted light or phase-contrast microscopy. Processing these images requires automated segmentation, tracking and lineaging algorithms. There is also a need for any errors in the automated processing to be easily identified and quickly corrected. We have developed LEVER, an open source software tool that combines the automated image analysis for phase-contrast microscopy movies with an easy-to-use interface for validating the results and correcting any errors., Availability and Implementation: LEVER is available free and open source, licensed under the GNU GPLv3. Details on obtaining and using LEVER are available at http://n2t.net/ark:/87918/d9rp4t CONTACT: acohen@coe.drexel.edu., (© The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

The supercritical carbon dioxide medium, used to increase efficiency in oxy combustion fossil energy technology, may drastically alter both rates and mechanisms of chemical reactions. Here we investigate potential energy surface of the second most important combustion reaction with quantum chemistry methods. Two types of effects are reported: formation of the covalent intermediates and formation of van der Waals complexes by spectator CO2 molecule. While spectator molecule alter the activation barrier only slightly, the covalent bonding opens a new reaction pathway. The mechanism includes sequential covalent binding of CO2 to OH radical and CO molecule, hydrogen transfer from oxygen to carbon atoms, and CH bond dissociation. This reduces the activation barrier by 11 kcal/mol at the rate-determining step and is expected to accelerate the reaction rate. The finding of predicted catalytic effect is expected to play an important role not only in combustion but also in a broad array of chemical processes taking place in supercritical CO2 medium. It may open a new venue for controlling reaction rates for chemical manufacturing.

Background: Neural stem cells are motile and proliferative cells that undergo mitosis, dividing to produce daughter cells and ultimately generating differentiated neurons and glia. Understanding the mechanisms controlling neural stem cell proliferation and differentiation will play a key role in the emerging fields of regenerative medicine and cancer therapeutics. Stem cell studies in vitro from 2-D image data are well established. Visualizing and analyzing large three dimensional images of intact tissue is a challenging task. It becomes more difficult as the dimensionality of the image data increases to include time and additional fluorescence channels. There is a pressing need for 5-D image analysis and visualization tools to study cellular dynamics in the intact niche and to quantify the role that environmental factors play in determining cell fate., Results: We present an application that integrates visualization and quantitative analysis of 5-D (x,y,z,t,channel) and large montage confocal fluorescence microscopy images. The image sequences show stem cells together with blood vessels, enabling quantification of the dynamic behaviors of stem cells in relation to their vascular niche, with applications in developmental and cancer biology. Our application automatically segments, tracks, and lineages the image sequence data and then allows the user to view and edit the results of automated algorithms in a stereoscopic 3-D window while simultaneously viewing the stem cell lineage tree in a 2-D window. Using the GPU to store and render the image sequence data enables a hybrid computational approach. An inference-based approach utilizing user-provided edits to automatically correct related mistakes executes interactively on the system CPU while the GPU handles 3-D visualization tasks., Conclusions: By exploiting commodity computer gaming hardware, we have developed an application that can be run in the laboratory to facilitate rapid iteration through biological experiments. We combine unsupervised image analysis algorithms with an interactive visualization of the results. Our validation interface allows for each data set to be corrected to 100% accuracy, ensuring that downstream data analysis is accurate and verifiable. Our tool is the first to combine all of these aspects, leveraging the synergies obtained by utilizing validation information from stereo visualization to improve the low level image processing tasks.

Image sequences of live proliferating cells often contain visual ambiguities that are difficult even for human domain experts to resolve. Here we present a new approach to analyzing image sequences that capture the development of clones of hematopoietic stem cells (HSCs) from live cell time lapse microscopy. The HSCs cannot survive long term imaging unless they are cultured together with a secondary cell type, OP9 stromal cells. The HSCs frequently disappear under the OP9 cell layer, making segmentation difficult or impossible from a single image frame, even for a human domain expert. We have developed a new approach to the segmentation of HSCs that captures these occluded cells. Starting with an a priori segmentation that uses a Monte Carlo technique to estimate the number of cells in a clump of touching cells, we proceed to track and lineage the image data. Following user validation of the lineage information, an a posteriori resegmentation step utilizing tracking results delineates the HSCs occluded by the OP9 layer. Resegmentation has been applied to 3031 occluded segmentations from 77 tracks, correctly recovering over 84% of the occluded segmentations.

Parental anxiety is a risk to optimal treatment outcomes for childhood anxiety disorders. The current trial examined whether the addition of a brief parental anxiety management (BPAM) program to family cognitive behavioral therapy (CBT) was more efficacious than family CBT-only in treating childhood anxiety disorders. Two hundred nine children (aged 6-13 years, 104 female, 90% Caucasian) with a principal anxiety disorder were randomly allocated to family CBT with a five-session program of BPAM (n = 109) or family CBT-only (n = 100). Family CBT comprised the Cool Kids program, a structured 12-week program that included both mothers and fathers. Overall, results revealed that the addition of BPAM did not significantly improve outcomes for the child or the parent compared to the CBT-only group at posttreatment or 6-month follow-up. Overall, however, children with nonanxious parents were more likely to be diagnosis free for any anxiety disorder compared to children with anxious parents at posttreatment and 6-month follow-up. BPAM did not produce greater reductions in parental anxiety. The results support previous findings that parent anxiety confers poorer treatment outcomes for childhood anxiety disorders. Nevertheless the addition of BPAM anxiety management for parents in its current format did not lead to additional improvements when used as an adjunct to family CBT in the treatment of the child's anxiety disorder. Future benefits may come from more powerful methods of reducing parents' anxiety.

Biological imaging of live cell and tissue using 3D microscopy is able to capture time-lapse image sequences showing multiple molecular markers labeling different biological structures simultaneously. In order to analyze this complex multi-dimensional image sequence data, there is a need for automated quantitative algorithms, and for methods to visualize and interact with both the data and the analytical results. Traditional computational human input devices such as the keyboard and mouse are no longer adequate for complex tasks such as manipulating and navigating 3+ dimensional volumes. In this paper, we have developed a new interaction system for interfacing with big data sets using the human visual system together with touch, force and audio feedback. This system includes real-time dynamic 3D visualization, haptic interaction via exoskeletal glove, and tonal auditory components that seamlessly create an immersive environment for efficient qualitative analysis.

This protocol and the accompanying software program called LEVER (lineage editing and validation) enable quantitative automated analysis of phase-contrast time-lapse images of cultured neural stem cells. Images are captured at 5-min intervals over a period of 5-15 d as the cells proliferate and differentiate. LEVER automatically segments, tracks and generates lineage trees of the stem cells from the image sequence. In addition to generating lineage trees capturing the population dynamics of clonal development, LEVER extracts quantitative phenotypic measurements of cell location, shape, movement and size. When available, the system can include biomolecular markers imaged using fluorescence. It then displays the results to the user for highly efficient inspection and editing to correct any errors in the segmentation, tracking or lineaging. To enable high-throughput inspection, LEVER incorporates features for rapid identification of errors and for learning from user-supplied corrections to automatically identify and correct related errors.

Objective: Although a potentially useful measure, to date, there has been only one published test of the psychometric properties of the Mini-Social Phobia Inventory (Mini-SPIN). Therefore, the psychometric properties of the Mini-SPIN, a brief 3-item screen for social anxiety disorder, were examined., Method: Participants were 186 patients diagnosed with social anxiety disorder (DSM-IV criteria) attending a specialized anxiety disorders clinic for treatment, and 56 nonclinical participants were recruited to serve as comparisons. Participants were diagnosed using the Anxiety Disorders Interview Schedule for DSM-IV, and they also completed the Mini-SPIN, the Social Interaction Anxiety Scale (SIAS), and the Social Phobia Scale (SPS). Construct validity for the Mini-SPIN was assessed by its correlations with the SIAS and the SPS. Reliability, internal consistency, discriminant validity, and sensitivity to change were also examined, and receiver operating characteristic curve analysis was conducted to determine guidelines regarding cutoff scores for the Mini-SPIN. The study was conducted between April 1999 and December 2001., Results: Supporting findings from a previous study, strong support was found for the Mini-SPIN's ability to discriminate individuals with social anxiety disorder from those without the disorder. Receiver operating characteristic analysis revealed that using a cutoff score of 6 or greater (P < .001), the Mini-SPIN demonstrates excellent sensitivity, specificity, and positive and negative predictive values., Conclusions: Findings suggest that the Mini-SPIN is a reliable and valid instrument for screening social anxiety disorder in adults. Importantly, the use of the Mini-SPIN in primary care may be one way to address the underrecognition of social anxiety disorder in such settings. Due to the ease and brevity of the measure, it also shows potential for use in epidemiology. Given that this study has revealed the ability of the Mini-SPIN to reflect treatment change, the Mini-SPIN may also be considered for use in treatment outcome studies that specifically require minimal assessment.