Molecular mechanisms and energetics of lipid droplet formation and directional budding.

The formation and budding of lipid droplets (LDs) are known to be governed by the LD size and by membrane tensions in the endoplasmic reticulum (ER) bilayer and LD-monolayers. Using coarse-grained simulations of an LD model, we first show that ER-embedded LDs of different sizes can form through a continuous transition from wide LD lenses to spherical LDs at a fixed LD size. The ER tendency to relax its bilayer modulates the transition via a subtle interplay between the ER and LD lipid densities. By calculating the energetic landscape of the LD transition, we demonstrate that this size-independent transition is regulated by the mechanical force balance of ER and LD-tensions, independent from membrane bending and line tension whose energetic contributions are negligible according to our calculations. Our findings explain experimental observation of stable LDs of various shapes. We then propose a novel mechanism for directional LD budding where the required membrane asymmetry is provided by the exchange of lipids between the LD-monolayers. Remarkably, we demonstrate that this budding process is energetically neutral. Consequently, LD budding can proceed by a modest energy input from proteins or other driving agents. We obtain equal lipid densities and membrane tensions in LD-monolayers throughout budding. Our findings indicate that unlike LD formation, LD budding by inter-monolayer lipid exchange is a tension-independent process.

Single-Point Fixation for Noncomminuted Zygomaticomaxillary Complex Fractures-A 20-Year Experience.

PURPOSE: Zygomaticomaxillary complex (ZMC) fractures occur often. However, no clinical consensus has been reached regarding the number of fixation points required when performing open reduction and internal fixation (ORIF). The objective of the present study was to explore the utility of single-point fixation in the management of noncomminuted ZMC fractures. PATIENTS AND METHODS: We analyzed the data from a retrospective case series of 211 patients treated during a 20-year period. RESULTS: The mean length of follow-up was 3.4 months. Of the 211 patients, 162 with noncomminuted ZMC fractures had been treated with single-point fixation of the zygomaticomaxillary buttress. During the follow-up period, 1 patient experienced tooth loss because of a root present in the fracture line, 7 experienced intraoral plate exposure, with 2 subsequently undergoing plate exchange, and 8 developed a wound infection. No patients required orthognathic surgery or cheek implants for malar asymmetry. No patient developed hypoglobus or enophthalmos, and none required revision ORIF of their ZMC fracture. CONCLUSIONS: To the best of our knowledge, the present study represents the largest series in the literature reporting the surgical results and outcomes of patients with noncomminuted ZMC fractures treated with single-point fixation. In experienced hands, we believe this is a viable surgical option if appropriate surgical considerations are made.

I-PINE web server: an integrative probabilistic NMR assignment system for proteins.

Various methods for understanding the structural and dynamic properties of proteins rely on the analysis of their NMR chemical shifts. These methods require the initial assignment of NMR signals to particular atoms in the sequence of the protein, a step that can be very time-consuming. The probabilistic interaction network of evidence (PINE) algorithm for automated assignment of backbone and side chain chemical shifts utilizes a Bayesian probabilistic network model that analyzes sequence data and peak lists from multiple NMR experiments. PINE, which is one of the most popular and reliable automated chemical shift assignment algorithms, has been available to the protein NMR community for longer than a decade. We announce here a new web server version of PINE, called Integrative PINE (I-PINE), which supports more types of NMR experiments than PINE (including three-dimensional nuclear Overhauser enhancement and four-dimensional J-coupling experiments) along with more comprehensive visualization of chemical shift based analysis of protein structure and dynamics. The I-PINE server is freely accessible at http://i-pine.nmrfam.wisc.edu . Help pages and tutorial including browser capability are available at: http://i-pine.nmrfam.wisc.edu/instruction.html . Sample data that can be used for testing the web server are available at: http://i-pine.nmrfam.wisc.edu/examples.html .

Vesicle constriction by rings of Janus nanoparticles and aggregates of curved proteins.

Membrane constriction and associated scission by proteins and nano structures are crucial to many processes in cellular and synthetic biology. We report mechanical constriction of vesicles by rings of adsorbed Janus nanoparticles that represent synthetic nano structures and mimic contractile proteins, and by aggregates of curved crescents that mimic scaffold proteins. Membrane energetics from Monte Carlo simulations and simulated annealing of the elastic membrane model confirms spontaneous vesicle constriction by aggregates of sufficiently-curved crescents of various lengths and by rings of Janus nanoparticles with a variety of ring lengths, particle sizes, and particle area fractions. We show that shorter rings of smaller particles with higher area fractions reinforce the constriction by increasing the energetic drive towards the constricted vesicle with smaller constriction radius. We demonstrate that vesicle constriction by crescent aggregates strongly depends on the crescent curvature. In contrast to aggregates of sufficiently-curved crescents that are capable of inducing full vesicle constriction, those of near flat crescents with negligible curvature leave the vesicle unconstricted. Our results offer promising perspectives for designing membrane-constricting nano structures such as nanoparticle aggregates and clusters of synthetic curved proteins such as DNA origami scaffolds with applications in synthetic biology. Our findings reveal the significant contribution of highly-curved F-BAR domains to cell division and explain how contractile protein rings such as dynamin GTPase, actomyosin rings, and endosomal sorting complexes required for transport constrict the membrane.

Preoperative radiation and complication rates after double free flap reconstruction of head and neck cancer.

INTRODUCTION: In this study, we explore whether preoperative external beam radiation affects complication rates in patients that have undergone double simultaneous free tissue transfer for head and neck defects. STUDY DESIGN, SETTING, SUBJECTS AND METHODS: Approval was obtained from the JPS Institutional Review Board. We performed a retrospective analysis of patients who underwent double free flap reconstruction of head and neck defects between August 1997 and April 2017. Minimum follow up was 6 months. Patients were grouped according to preoperative radiation status (XRT vs non-XRT). The chi-squared test was used for all comparisons. P-values and 95% confidence intervals (CI) were reported as (P, 95% CI). RESULTS: 90 flaps were performed on 45 patients. The most common flap combination utilized was fibula plus radial forearm free flap (RFF) in 17 out of 45 patients. There were no statistically significant differences in frequency of flap failure (0.35, -15.9-20.1), wound infection (0.75, -22.1-19.3), hematoma (0.16, -5.3-36.7), or fistula formation (0.69, -22.5-24.6). There were also no statistically significant differences in cardiac complications (0.57, -10.3-28.2) and DVT (0.22, -12.4-25.3). CONCLUSION: Our findings suggest that double free flap patients who had preoperative radiation are not more likely to have complications compared to non- radiated patients. Simultaneous double free flaps should be reserved for the most complex cases. Extensive discussion should be had with the patient about possible morbidity and mortality.

A Pediatric Patient With an Orbital Respiratory Epithelial Cyst.

Respiratory epithelial cysts are rare orbital cysts that can arise secondary to choristomatous rests of respiratory epithelium. Approximately 15 congenital cases have been described in the literature, making it a rare disease entity. We present a case of a 14-month-old Middle Eastern male with a right infraorbital respiratory epithelial cyst. Magnetic resonance imaging of the brain and orbits revealed a right infraorbital cyst hyperintense on T1-weighted images and followed fluid density on T2-weighted images. This cyst was noted to displace the globe superiorly and inferior rectus muscle laterally. This cyst was excised using a transconjunctival approach. Histologically, the cyst wall was lined by ciliated columnar cells with interspersed mucus-containing cells and ciliated transitional epithelium was present, establishing the diagnosis of respiratory epithelial cyst. To our knowledge, this is the youngest patient with a respiratory epithelial cyst of the orbit reported in the literature.

ADAPT-NMR Enhancer: complete package for reduced dimensionality in protein NMR spectroscopy.

SUMMARY: ADAPT-nuclear magnetic resonance (ADAPT-NMR) offers an automated approach to the concurrent acquisition and processing of protein NMR data with the goal of complete backbone and side chain assignments. What the approach lacks is a useful graphical interface for reviewing results and for searching for missing peaks that may have prevented assignments or led to incorrect assignments. Because most of the data ADAPT-NMR collects are 2D tilted planes used to find peaks in 3D spectra, it would be helpful to have a tool that reconstructs the 3D spectra. The software package reported here, ADAPT-NMR Enhancer, supports the visualization of both 2D tilted planes and reconstructed 3D peaks on each tilted plane. ADAPT-NMR Enhancer can be used interactively with ADAPT-NMR to automatically assign selected peaks, or it can be used to produce PINE-SPARKY-like graphical dialogs that support atom-by-atom and peak-by-peak assignment strategies. Results can be exported in various formats, including XEASY proton file (.prot), PINE pre-assignment file (.str), PINE probabilistic output file, SPARKY peak list file (.list) and TALOS+ input file (.tab). As an example, we show how ADAPT-NMR Enhancer was used to extend the automated data collection and assignment results for the protein Aedes aegypti sterol carrier protein 2. AVAILABILITY: The program, in the form of binary code along with tutorials and reference manuals, is available at http://pine.nmrfam.wisc.edu/adapt-nmr-enhancer.

FIM: A fatigued-injured muscle model based on the sliding filament theory.

Skeletal muscle modeling has a vital role in movement studies and the development of therapeutic approaches. In the current study, a Huxley-based model for skeletal muscle is proposed, which demonstrates the impact of impairments in muscle characteristics. This model focuses on three identified ions: H+, inorganic phosphate Pi, and Ca2+. Modifications are made to actin-myosin attachment and detachment rates to study the effects of H+ and Pi. Additionally, an activation coefficient is included to represent the role of calcium ions interacting with troponin, highlighting the importance of Ca2+. It is found that maximum isometric muscle force decreases by 9.5% due to a reduction in pH from 7.4 to 6.5 and by 47.5% in case of the combination of a reduction in pH and an increase of Pi concentration up to 30 mM, respectively. Then the force decline caused by a fall in the active calcium ions is studied. When only 15% of the total calcium in the myofibrillar space is able to interact with troponin, up to 80% force drop is anticipated by the model. The proposed fatigued-injured muscle model is useful to study the effect of various shortening velocities and initial muscle-tendon lengths on muscle force; in addition, the benefits of the model go beyond predicting the force in different conditions as it can also predict muscle stiffness and power. The power and stiffness decrease by 40% and 6.5%, respectively, due to the pH reduction, and the simultaneous accumulation of H+ and Pi leads to a 50% and 18% drop in power and stiffness.

RNA-PAIRS: RNA probabilistic assignment of imino resonance shifts.

The significant biological role of RNA has further highlighted the need for improving the accuracy, efficiency and the reach of methods for investigating RNA structure and function. Nuclear magnetic resonance (NMR) spectroscopy is vital to furthering the goals of RNA structural biology because of its distinctive capabilities. However, the dispersion pattern in the NMR spectra of RNA makes automated resonance assignment, a key step in NMR investigation of biomolecules, remarkably challenging. Herein we present RNA Probabilistic Assignment of Imino Resonance Shifts (RNA-PAIRS), a method for the automated assignment of RNA imino resonances with synchronized verification and correction of predicted secondary structure. RNA-PAIRS represents an advance in modeling the assignment paradigm because it seeds the probabilistic network for assignment with experimental NMR data, and predicted RNA secondary structure, simultaneously and from the start. Subsequently, RNA-PAIRS sets in motion a dynamic network that reverberates between predictions and experimental evidence in order to reconcile and rectify resonance assignments and secondary structure information. The procedure is halted when assignments and base-parings are deemed to be most consistent with observed crosspeaks. The current implementation of RNA-PAIRS uses an initial peak list derived from proton-nitrogen heteronuclear multiple quantum correlation ((1)H-(15)N 2D HMQC) and proton-proton nuclear Overhauser enhancement spectroscopy ((1)H-(1)H 2D NOESY) experiments. We have evaluated the performance of RNA-PAIRS by using it to analyze NMR datasets from 26 previously studied RNAs, including a 111-nucleotide complex. For moderately sized RNA molecules, and over a range of comparatively complex structural motifs, the average assignment accuracy exceeds 90%, while the average base pair prediction accuracy exceeded 93%. RNA-PAIRS yielded accurate assignments and base pairings consistent with imino resonances for a majority of the NMR resonances, even when the initial predictions are only modestly accurate. RNA-PAIRS is available as a public web-server at http://pine.nmrfam.wisc.edu/RNA/.

Compartmentalizing and sculpting nanovesicles by phase-separated aqueous nanodroplets.

Phase-separated liquid droplets inside giant vesicles have been intensely studied as biomimetic model systems to understand cellular microcompartmentation and molecular crowding and sorting. On the nanoscale, however, how aqueous nanodroplets interact with and shape nanovesicles is poorly understood. We perform coarse-grained molecular simulations to explore the architecture of compartmentalized nanovesicles by phase-separated aqueous nanodroplets, and their morphological evolution under osmotic deflation. We show that phase separation of a biphasic liquid mixture can form both stable two-compartment and meta-stable multi-compartment nanovesicles. We identify morphological transitions of stable two-compartment nanovesicles between tube, sheet and cup morphologies, characterized by membrane asymmetry and phase-separation propensity between the aqueous phases. We demonstrate that the formation of local sheets and in turn cup-shaped nanovesicles is promoted by negative line tensions resulting from large separation propensities, an exclusive nanoscale phenomenon which is not expected for larger vesicles where energetic contributions of the line tensions are dominated by those of the membrane tensions. Despite their instability, we observe long-lived multi-compartment nanovesicles, such as nanotubules and branched tubules, whose prolonged lifetime is attributed to interfacial tensions and membrane asymmetry. Aqueous nanodroplets can thus form novel membrane nanostructures, crucial for cellular processes and forming cellular organelles on the nanoscale.

High-resolution compositional mapping of surfaces in non-contact atomic force microscopy by a new multi-frequency excitation.

In this paper, a new multi-frequency excitation method based on combination resonance is introduced to enhance the non-contact atomic force microscopy performance. In combination resonance, excitation frequencies are selected so that summation/subtraction of excitation frequencies is close to the natural frequencies of the microcantilever. Due to the nonlinear nature of this method, the probe response to excitation is very sensitive to change in tip-sample forces. This could be used to generate high-resolution compositional mapping and topographical images of the surface. The present study reveals that both amplitude and phase shift of the combination resonance are sensitive to change in parameters such as Hamaker constant, damping coefficient, Young's modulus and tip-sample initial distance. It is observed that because of high sensitivity to Hamaker constant a small change in the surface material leads to considerable variations in amplitude and phase shift. This sensitivity is employed to improve compositional mapping of the surface materials. It is also found out that the response amplitude in the combination resonance is very sensitive to change in the tip-sample initial distance. This sensitivity may be used to reduce the vertical noise and increase image resolution, especially in environments with low quality factors. Overall, using this technique the image contrast increases significantly and high resolution compositional mapping of surfaces is achieved.

Biodegradable carboxymethyl cellulose-polyvinyl alcohol composite incorporated with Glycyrrhiza Glabra L. essential oil: Physicochemical and antibacterial features.

Glycyrrhiza glabra L. root essential oil (GGEO) has well-known antimicrobial and therapeutic features. In this study, a new antimicrobial carboxymethyl cellulose-polyvinyl alcohol (CMC-PVA) binary film was developed using GGEO as an active compound. The effects of various concentrations of GGEO (0.25%, 0.50%, and 0.75%) were scrutinized on the physicochemical and antibacterial properties of composites. It was discovered that GGEO significantly reduced the composite ultimate tensile strength from 17.01 to 3.86 MPa. Further, by increasing the concentration of GGEO to 0.75%, the water vapor permeability and moisture content increased to 13.61 × 10-9 g/m s-1 Pa-1 and 41.06%, respectively. The results indicated that the active films possessed good inhibitory effects against the gram-positive bacteria (L. monocytogenes and Staphylococcus aureus) and were less powerful against gram-negative bacteria (Escherichia coli and S. typhimurium). Finally, the results highlighted that GGEO can act as an excellent antimicrobial agent in combination with CMC-PVA composite.

PINE-SPARKY: graphical interface for evaluating automated probabilistic peak assignments in protein NMR spectroscopy.

SUMMARY: PINE-SPARKY supports the rapid, user-friendly and efficient visualization of probabilistic assignments of NMR chemical shifts to specific atoms in the covalent structure of a protein in the context of experimental NMR spectra. PINE-SPARKY is based on the very popular SPARKY package for visualizing multidimensional NMR spectra (T. D. Goddard and D. G. Kneller, SPARKY 3, University of California, San Francisco). PINE-SPARKY consists of a converter (PINE2SPARKY), which takes the output from an automated PINE-NMR analysis and transforms it into SPARKY input, plus a number of SPARKY extensions. Assignments and their probabilities obtained in the PINE-NMR step are visualized as labels in SPARKY's spectrum view. Three SPARKY extensions (PINE Assigner, PINE Graph Assigner, and Assign the Best by PINE) serve to manipulate the labels that signify the assignments and their probabilities. PINE Assigner lists all possible assignments for a peak selected in the dialog box and enables the user to choose among these. A window in PINE Graph Assigner shows all atoms in a selected residue along with all atoms in its adjacent residues; in addition, it displays a ranked list of PINE-derived connectivity assignments to any selected atom. Assign the Best-by-PINE allows the user to choose a probability threshold and to automatically accept as "fixed" all assignments above that threshold; following this operation, only the less certain assignments need to be examined visually. Once assignments are fixed, the output files generated by PINE-SPARKY can be used as input to PINE-NMR for further refinements. AVAILABILITY: The program, in the form of source code and binary code along with tutorials and reference manuals, is available at http://pine.nmrfam.wisc.edu/PINE-SPARKY.

Probabilistic interaction network of evidence algorithm and its application to complete labeling of peak lists from protein NMR spectroscopy.

The process of assigning a finite set of tags or labels to a collection of observations, subject to side conditions, is notable for its computational complexity. This labeling paradigm is of theoretical and practical relevance to a wide range of biological applications, including the analysis of data from DNA microarrays, metabolomics experiments, and biomolecular nuclear magnetic resonance (NMR) spectroscopy. We present a novel algorithm, called Probabilistic Interaction Network of Evidence (PINE), that achieves robust, unsupervised probabilistic labeling of data. The computational core of PINE uses estimates of evidence derived from empirical distributions of previously observed data, along with consistency measures, to drive a fictitious system M with Hamiltonian H to a quasi-stationary state that produces probabilistic label assignments for relevant subsets of the data. We demonstrate the successful application of PINE to a key task in protein NMR spectroscopy: that of converting peak lists extracted from various NMR experiments into assignments associated with probabilities for their correctness. This application, called PINE-NMR, is available from a freely accessible computer server (http://pine.nmrfam.wisc.edu). The PINE-NMR server accepts as input the sequence of the protein plus user-specified combinations of data corresponding to an extensive list of NMR experiments; it provides as output a probabilistic assignment of NMR signals (chemical shifts) to sequence-specific backbone and aliphatic side chain atoms plus a probabilistic determination of the protein secondary structure. PINE-NMR can accommodate prior information about assignments or stable isotope labeling schemes. As part of the analysis, PINE-NMR identifies, verifies, and rectifies problems related to chemical shift referencing or erroneous input data. PINE-NMR achieves robust and consistent results that have been shown to be effective in subsequent steps of NMR structure determination.

Progressive Scalp Thinning Over Mesh Cranioplasty and the Role of Lipotransfer.

OBJECTIVES: To evaluate the role of lipotransfer in progressive scalp thinning following titanium mesh cranioplasty. METHODS: Retrospective review of single surgeon, single tertiary referral experience of all patients who underwent mesh cranioplasty. Patient demographics, prior radiotherapy, frequency and timing of scalp thinning, and treatment course data were obtained. RESULTS: A total of 144 patients were included, 77 male and 67 female with mean ages 58.2 and 54.8, respectively. One hundred four patients (72%) developed mesh exposure or impending exposure requiring reconstruction. Fifty-six patients (54%) with scalp thinning were treated with lipotransfer, 40 of which were salvaged and the remainder of these patients definitively managed with cranioplasty and reconstruction. Prior radiotherapy was found to be associated with higher rates of mesh exposure (P = .0028), but not predictive of response to lipotransfer. CONCLUSION: Lipotransfer is a useful technique in managing moderate scalp thinning following mesh cranioplasty. Mesh exposure or severe thinning require definitive cranioplasty and reconstruction. LEVEL OF EVIDENCE: IV Laryngoscope, 130: 1926-1931, 2020.

Pharyngeal Reconstruction with Microvascular Free Tissue Transfer.

Reconstruction of pharyngoesophageal defects after total laryngectomies and extirpation of hypopharyngeal and upper esophageal carcinomas presents a challenging task. Goals of reconstruction include adequate voice rehabilitation and restoration of normal swallowing. The reconstructive armamentarium contains many options for reconstruction and creation of a new upper digestive tract. This review article focuses on the most commonly used free tissue transfer options for the reconstruction of these defects, with an assessment of their advantages and disadvantages.

Physico-mechanical and antimicrobial properties of tragacanth/hydroxypropyl methylcellulose/beeswax edible films reinforced with silver nanoparticles.

The novel trinary bio-composite film based on Tragacanth/Hydroxypropyl methylcellulose/Beeswax reinforced silver nanoparticles (AgNPs) was developed. This study investigated the effect of AgNPs (2, 4 and 8%) on some physico-mechanical and antimicrobial properties of bio-composite film. It was discovered that AgNPs reduced the composite tensile strength from 33.64 to 16.12 MPa. However, water vapor permeability was improved by addition of the nanoparticles (4.57-2.16 × 10-13 g m/m2 s Pa). The use of AgNPs influenced the apparent color of bio-composite film. The microscopic surface structure and topography of the films were also examined by scanning electron microscopy and Fourier transform infrared, respectively. Dynamic mechanical thermal analysis results showed that the thermal stability of film was slightly decreased through incorporation with AgNPs. Finally, nano-composite films demonstrated strong antibacterial activity against tested pathogen bacteria in the contact surface zone. The antimicrobial results suggest that new nanocomposite film may be used as food active packaging.

Cranioplasty Using a Mixture of Biologic and Nonbiologic Agents.

IMPORTANCE: A surgeon faces challenges with cranioplasty techniques to achieve a successful result with relatively few complications. OBJECTIVE: To describe a unique technique for incorporating both biologic autologous bone and nonbiologic allograft materials for defect coverage in cranioplasty with favorable outcomes and low occurrence of complications. DESIGN, SETTING, AND PARTICIPANTS: A retrospective medical records review of all 26 patients who underwent primary cranioplasty procedure with a modified technique between January 2011 and December 2015 at a high-volume head and neck oncologic reconstructive practice was conducted; data analysis was also performed during that period. After several years of experience with traditional cranioplasty maneuvers, the modified technique has evolved to incorporate both autologous bone grafts and alloplastic materials in the formation of a shapeable on-lay material. Data were collected on demographics, need for cranioplasty, materials used, outcomes, and risk factors. MAIN OUTCOMES AND MEASURES: Rates of infection, hematoma, flap loss or resorption, cerebrospinal fluid leak, hardware exposure or malfunction, and repeated reconstruction. RESULTS: Of the 26 patients, 21 (81%) were men; mean (SD) age was 65.8 (14.3) years. Eight (31%) patients had a history of diabetes, 4 (15%) patients were receiving immunosuppressive drugs, and 5 (19%) patients were active smokers at the time of surgery. Neoplasia was the most common cause of the calvarial defect seen, responsible for 20 of 28 (71%) operative defects and necessitated procedures. All but 1 patient achieved successful mineralization following primary cranioplasty with the modified technique; this success was verified based on physical examination and follow-up imaging. Complications were rare and involved only 3 patients who developed postoperative infection; 1 (4%) of these patients lost the integrity of the cranioplasty. Thus, the rate of infection was 11% and loss rate was 4%. Preoperative and postoperative radiotherapy appeared to have no bearing on graft survival. CONCLUSIONS AND RELEVANCE: The results using a unique technique for incorporating both biologic autologous bone and nonbiologic allograft materials for defect coverage in cranioplasty are favorable, with satisfactory aesthetic outcomes and limited postoperative complications. LEVEL OF EVIDENCE: 4.