Cellular Neonatal Fc Receptor Recycling Efficiencies can Differentiate Target-Independent Clearance Mechanisms of Monoclonal Antibodies.

In vivo clearance mechanisms of therapeutic monoclonal antibodies (mAbs) encompass both target-mediated and target-independent processes. Two distinct determinants of overall mAb clearance largely separate of target-mediated influences are non-specific cellular endocytosis and subsequent pH-dependent mAb recycling mediated by the neonatal Fc receptor (FcRn), where inter-mAb variability in the efficiency of both processes is observed. Here, we implemented a functional cell-based FcRn recycling assay via Madin-Darby canine kidney type II cells stably co-transfected with human FcRn and its light chain β2-microglobulin.

Pharmacokinetics of Levetiracetam Seizure Prophylaxis in Severe Traumatic Brain Injury.

Background: Drug pharmacokinetics (PK) are altered in neurocritically ill patients, and optimal levetiracetam dosing for seizure prophylaxis is unknown.

Objective: This study evaluates levetiracetam PK in critically ill patients with severe traumatic brain injury (sTBI) receiving intravenous levetiracetam 1000 mg every 8 (LEV8) to 12 (LEV12) hours for seizure prophylaxis.

Methods: This prospective, open-label study was conducted at a level 1 trauma, academic, quaternary care center.

Using quantitative single molecule localization microscopy to optimize multivalent HER2-targeting ligands.

Introduction: The progression-free survival of patients with HER2-positive metastatic breast cancer is significantly extended by a combination of two monoclonal antibodies, trastuzumab and pertuzumab, which target independent epitopes of the extracellular domain of HER2. The improved efficacy of the combination over individual antibody therapies targeting HER2 is still being investigated, and several molecular mechanisms may be in play: the combination downregulates HER2, improves antibody-dependent cell mediated cytotoxicity, and/or affects the organization of surface-expressed antigens, which may attenuate downstream signaling.

Methods: By combining protein engineering and quantitative single molecule localization microscopy (qSMLM), here we both assessed and optimized clustering of HER2 in cultured breast cancer cells.

Engineering protein glycosylation in CHO cells to be highly similar to murine host cells.

Since 2015 more than 34 biosimilars have been approved by the FDA. This new era of biosimilar competition has stimulated renewed technology development focused on therapeutic protein or biologic manufacturing. One challenge in biosimilar development is the genetic differences in the host cell lines used to manufacture the biologics.

Titin-truncating variants in hiPSC cardiomyocytes induce pathogenic proteinopathy and sarcomere defects with preserved core contractile machinery.

Titin-truncating variants (TTNtv) are the single largest genetic cause of dilated cardiomyopathy (DCM). In this study we modeled disease phenotypes of A-band TTNtv-induced DCM in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) using genome editing and tissue engineering technologies. Transcriptomic, cellular, and micro-tissue studies revealed that A-band TTNtv hiPSC-CMs exhibit pathogenic proteinopathy, sarcomere defects, aberrant Na channel activities, and contractile dysfunction.

Molecular Imaging of HER2 in Patient Tissues with Touch Prep-Quantitative Single Molecule Localization Microscopy.

Biomolecules can be investigated at the nanoscale with quantitative single molecule localization microscopy (qSMLM). This technique, which achieves single molecule sensitivity, can probe how membrane receptors are organized under both normal and pathological conditions. While a number of receptors have been extensively studied in cultured cells, technical challenges have largely impeded their robust quantification in tissue samples.

Characterizing Binding Interactions That Are Essential for Selective Transport through the Nuclear Pore Complex.

Specific macromolecules are rapidly transported across the nuclear envelope via the nuclear pore complex (NPC). The selective transport process is facilitated when nuclear transport receptors (NTRs) weakly and transiently bind to intrinsically disordered constituents of the NPC, FG Nups. These two types of proteins help maintain the selective NPC barrier.

Endocutter Staple Height Auto-Adjusts to Tissue Thickness.

Background: Surgeon choice of the appropriate staple height has been cited as a factor in the mechanical integrity of a staple line. However, tissue measured at the industry standard 8 g/mm is usually thicker than the formed staple height of the staples that hold it together. This means that the pressure that the staples apply must be greater than 8g/mm.

Impact of antithrombin III and enoxaparin dosage adjustment on prophylactic anti-Xa concentrations in trauma patients at high risk for venous thromboembolism: a randomized pilot trial.

The impact of antithrombin III activity (AT-III) on prophylactic enoxaparin anti-factor Xa concentration (anti-Xa) is unknown in high-risk trauma patients. So too is the optimal anti-Xa-adjusted enoxaparin dosage. This prospective, randomized, pilot study sought to explore the association between AT-III and anti-Xa goal attainment and to preliminarily evaluate two enoxaparin dosage adjustment strategies in patients with subprophylactic anti-Xa.

Efficacy of Noninvasive Technologies in Triaging Traumatic Brain Injury and Correlating With Intracranial Pressure: A Prospective Study.

Background: There is interest in methods of measuring noninvasive intracranial pressure (ICP), including pupillometry, ultrasonographic transcranial Doppler (TCD), and optic nerve sheath diameter (ONSD), for diagnosing traumatic brain injury (TBI) in limited resource environments. Whether these technologies have diagnostic agreement is unknown. We hypothesized that ONSD, pupillometry, and TCD could both distinguish severe TBI and correlate with ICP.

Assessment of a novel stapler performance for laparoscopic sleeve gastrectomy.

Background: Optimal stapler selection during laparoscopic sleeve gastrectomy requires careful balance between tissue compression, hemostasis, and mechanical integrity. Junctions along a staple line can further increase the risks of technical or mechanical staple line failures. The aim of this study was to compare two commonly utilized laparoscopic linear gastrointestinal staplers (Ethicon, Medtronic) with a novel linear stapler (Titan) designed to perform a sleeve gastrectomy with a single stapler firing.

Single molecule characterization of individual extracellular vesicles from pancreatic cancer.

Biofluid-accessible extracellular vesicles (EVs) may represent a new means to improve the sensitivity and specificity of detecting disease. However, current methods to isolate EVs encounter challenges when they are used to select specific populations. Moreover, it has been difficult to comprehensively characterize heterogeneous EV populations at the single vesicle level.

Ethanol and Naltrexone Have Distinct Effects on the Lateral Nano-organization of Mu and Kappa Opioid Receptors in the Plasma Membrane.

The complex spatiotemporal organization of proteins and lipids in the plasma membrane is an important determinant of receptor function. Certain substances, such as ethanol, can penetrate into the hydrophobic regions of the plasma membrane. By altering protein-lipid and protein-protein interactions, these substances can modify the dynamic lateral organization and the function of plasma membrane receptors.

Single molecule localization microscopy coupled with touch preparation for the quantification of trastuzumab-bound HER2.

All breast cancers are assessed for levels of human epidermal growth factor receptor 2 (HER2). Fluorescence in situ hybridization (FISH) and immunohistochemistry are currently used to determine if a patient is eligible for anti-HER2 therapy. Limitations of both tests include variability and relatively long processing times.

Dynamic lateral organization of opioid receptors (kappa, mu and mu ) in the plasma membrane at the nanoscale level.

Opioid receptors are important pharmacological targets for the management of numerous medical conditions (eg, severe pain), but they are also the gateway to the development of deleterious side effects (eg, opiate addiction). Opioid receptor signaling cascades are well characterized. However, quantitative information regarding their lateral dynamics and nanoscale organization in the plasma membrane remains limited.

The FcεRI Signaling Cascade and Integrin Trafficking Converge at Patterned Ligand Surfaces.

We examined the spatial targeting of early and downstream signaling mediated by the IgE receptor (FcεRI) in RBL mast cells utilizing surface-patterned 2,4 dinitrophenyl (DNP) ligands. Micron-sized features of DNP are presented as densely immobilized conjugates of bovine serum albumin (DNP-BSA) or mobile in a supported lipid bilayer (DNP-SLB). Although soluble anti-DNP IgE binds uniformly across features for both pattern types, IgE bound to FcεRI on cells shows distinctive distributions: uniform for DNP-SLB and edge-concentrated for DNP-BSA.

Graphene Oxide Nanosheets Stimulate Ruffling and Shedding of Mammalian Cell Plasma Membranes.

Graphene oxide (GO) has attracted intense interest for use in living systems and environmental applications. GO's compatibility with mammalian cells is sometimes inferred from its low cytotoxicity, but such conclusions ignore non-lethal effects that will influence GO's utility. Here we demonstrate, with rat basophilic leukemia (RBL) cells, profound plasma membrane (PM) ruffling and shedding induced by GO using confocal and live cell fluorescence microscopy, as well as scanning electron microscopy.

Molecular signatures of mu opioid receptor and somatostatin receptor 2 in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC), a particularly aggressive malignancy, has been linked to atypical levels, certain mutations, and aberrant signaling of G-protein-coupled receptors (GPCRs). GPCRs have been challenging to target in cancer because they organize into complex networks in tumor cells. To dissect such networks with nanometer-scale precision, here we combine traditional biochemical approaches with superresolution microscopy methods.

Inhomogeneity Based Characterization of Distribution Patterns on the Plasma Membrane.

Cell surface protein and lipid molecules are organized in various patterns: randomly, along gradients, or clustered when segregated into discrete micro- and nano-domains. Their distribution is tightly coupled to events such as polarization, endocytosis, and intracellular signaling, but challenging to quantify using traditional techniques. Here we present a novel approach to quantify the distribution of plasma membrane proteins and lipids.

Spatially defined EGF receptor activation reveals an F-actin-dependent phospho-Erk signaling complex.

We investigated the association of signaling proteins with epidermal growth factor (EGF) receptors (EGFR) using biotinylated EGF bound to streptavidin that is covalently coupled in an ordered array of micron-sized features on silicon surfaces. Using NIH-3T3 cells stably expressing EGFR, we observe concentration of fluorescently labeled receptors and stimulated tyrosine phosphorylation that are spatially confined to the regions of immobilized EGF and quantified by cross-correlation analysis. We observe recruitment of phosphorylated paxillin to activated EGFR at these patterned features, as well as β1-containing integrins that preferentially localize to more peripheral EGF features, as quantified by radial fluorescence analysis.

Near-field fluorescence cross-correlation spectroscopy on planar membranes.

The organization and dynamics of plasma membrane components at the nanometer scale are essential for biological functions such as transmembrane signaling and endocytosis. Planarized nanoscale apertures in a metallic film are demonstrated as a means of confining the excitation light for multicolor fluorescence spectroscopy to a 55 ± 10 nm beam waist. This technique provides simultaneous two-color, subdiffraction-limited fluorescence correlation spectroscopy and fluorescence cross-correlation spectroscopy on planar membranes.