timsTOF HT Improves Protein Identification and Quantitative Reproducibility for Deep Unbiased Plasma Protein Biomarker Discovery.

Mass spectrometry (MS) is a valuable tool for plasma proteome profiling and disease biomarker discovery. However, wide-ranging plasma protein concentrations, along with technical and biological variabilities, present significant challenges for deep and reproducible protein quantitation. Here, we evaluated the qualitative and quantitative performance of timsTOF HT and timsTOF Pro 2 mass spectrometers for analysis of neat plasma samples (unfractionated) and plasma samples processed using the Proteograph Product Suite (Proteograph) that enables robust deep proteomics sampling prior to mass spectrometry. Samples were evaluated across a wide range of peptide loading masses and liquid chromatography (LC) gradients. We observed up to a 76% increase in total plasma peptide precursors identified and a >2-fold boost in quantifiable plasma peptide precursors (CV < 20%) with timsTOF HT compared to Pro 2. Additionally, approximately 4.5 fold more plasma peptide precursors were detected by both timsTOF HT and timsTOF Pro 2 in the Proteograph analyzed plasma vs neat plasma. In an exploratory analysis of 20 late-stage lung cancer and 20 control plasma samples with the Proteograph, which were expected to exhibit distinct proteomes, an approximate 50% increase in total and statistically significant plasma peptide precursors (q < 0.05) was observed with timsTOF HT compared to Pro 2. Our data demonstrate the superior performance of timsTOF HT for identifying and quantifying differences between biologically diverse samples, allowing for improved disease biomarker discovery in large cohort studies. Moreover, researchers can leverage data sets from this study to optimize their liquid chromatography-mass spectrometry (LC-MS) workflows for plasma protein profiling and biomarker discovery. (ProteomeXchange identifier: PXD047854 and PXD047839).

Contrasting genetic trajectories of endangered and expanding red fox populations in the western U.S.

As anthropogenic disturbances continue to drive habitat loss and range contractions, the maintenance of evolutionary processes will increasingly require targeting measures to the population level, even for common and widespread species. Doing so requires detailed knowledge of population genetic structure, both to identify populations of conservation need and value, as well as to evaluate suitability of potential donor populations. We conducted a range-wide analysis of the genetic structure of red foxes in the contiguous western U.S., including a federally endangered distinct population segment of the Sierra Nevada subspecies, with the objectives of contextualizing field observations of relative scarcity in the Pacific mountains and increasing abundance in the cold desert basins of the Intermountain West. Using 31 autosomal microsatellites, along with mitochondrial and Y-chromosome markers, we found that populations of the Pacific mountains were isolated from one another and genetically depauperate (e.g., estimated Ne range = 3-9). In contrast, red foxes in the Intermountain regions showed relatively high connectivity and genetic diversity. Although most Intermountain red foxes carried indigenous western matrilines (78%) and patrilines (85%), the presence of nonindigenous haplotypes at lower elevations indicated admixture with fur-farm foxes and possibly expanding midcontinent populations as well. Our findings suggest that some Pacific mountain populations could likely benefit from increased connectivity (i.e., genetic rescue) but that nonnative admixture makes expanding populations in the Intermountain basins a non-ideal source. However, our results also suggest contact between Pacific mountain and Intermountain basin populations is likely to increase regardless, warranting consideration of risks and benefits of proactive measures to mitigate against unwanted effects of Intermountain gene flow.

The use of a novel synthetic resorbable scaffold (TIGR Matrix®) in a clinical quality improvement (CQI) effort for abdominal wall reconstruction (AWR).

PURPOSE: The use of hernia mesh is a common practice in abdominal wall reconstruction (AWR) operations. The high cost of biologic mesh has raised questions about the value of its use in AWR. Resorbable synthetic mesh may have the potential benefits of biologic mesh, minimizing the need for removal when infected, at a lower cost. METHODS: A hernia program has implemented the principles of clinical quality improvement (CQI) to improve patient outcomes. One process improvement attempt was implemented using a newly available resorbable synthetic scaffold. Long-term follow-up was obtained as a part of the CQI process. RESULTS: A total of 91 patients undergoing AWR were included between 8/11 and 9/15 (49 months). There were 58 female (64%) and 33 male (36%) patients. The average age was 57.2 years (28-80). The average BMI was 34.0 (17.6-53.4). There were 52 patients (57%) with recurrent hernias. Mean hernia defect size was 306.6 cm2 (24-720) and mean mesh size was 471.7 cm2 (112-600). Outcomes included a mean length of stay of 7.5 days (0-49), a recurrence rate of 12% (11/91) and a wound complication rate of 27% (25/91). The recurrence rate decreased to 4.5% (3/66) after several improvements, including adopting a transversus abdominus release (TAR) approach, were implemented. There were no mesh-related complications and no mesh removal (partial or total) was required. The mean follow-up length was 42.4 months (0-102). CONCLUSION: In this group of patients, an attempt at process improvement was implemented using a resorbable synthetic scaffold for AWR. With no mesh-related complications and no mesh removals required, there was an improvement in value due to the decrease in mesh cost and improved outcomes over time. Long-term follow-up demonstrated the durability of the repair.

Densely Packed Tethered Polymer Nanoislands: A Simulation Study.

COordinated Responsive Arrays of Surface-Linked polymer islands (CORALS) allow for the creation of molecular surfaces with novel and switchable properties. Critical components of CORALs are the uniformly distributed islands of densely grafted polymer chains (nanoislands) separated by regions of bare surface. The grafting footprint and separation distances of nanoislands are comparable to that of the constituent polymer chains themselves. Herein, we characterize the structural features of the nanoislands and semiflexible polymers within to better understand this critical constituent of CORALs. We observe different characteristics of grafted semiflexible polymers depending on the polymer island's size and distance from the center of the island. Specifically, the characteristics of the chains at the island periphery are similar to isolated tethered polymer chains (full flexible chains), while chains in the center of the island experience the neighbor effect such as chains in the classic polymer brush. Chains close to the edge of the islands exhibit unique structural features between these two regimes. These results can be used in the rational design of CORALs with specific interfacial characteristics and predictable responses to external stimuli. It is hoped that this the discussion of the different morphologies of the polymers as a function of distance from the edge of the polymer will find applications in a wide variety of systems.

Noninvasive Sampling Reveals Short-Term Genetic Rescue in an Insular Red Fox Population.

Genetic factors in the decline of small populations are extremely difficult to study in nature. We leveraged a natural experiment to investigate evidence of inbreeding depression and genetic rescue in a remnant population of subalpine-specialized Sierra Nevada red foxes (Vulpes vulpes necator) using noninvasive genetic monitoring during 2010-2017. Only 7 individuals were detected in the first 2 years. These individuals assigned genetically to the historical population and exhibited genetic hallmarks of inbreeding and no evidence of reproduction. Two years into the study, we detected 2 first-generation immigrant males from a recently expanding population of red foxes in the Great Basin Desert. Through annual resampling of individuals (634 red fox DNA samples, 41 individuals) and molecular reconstruction of pedigrees, we documented 1-3 litters/year for 5 years, all descended directly or indirectly from matings involving immigrant foxes. The observed heterozygosity and allelic richness of the population nearly doubled in 2 years. Abundance increased, indicative of a rapidly expanding population. Throughout the study, adult survival was high. Restoration of gene flow apparently improved the demographic trajectory of this population in the short term. Whether these benefits continue in the longer term could depend on numerous factors, such as maintenance of any locally adapted alleles. This study highlights the value of noninvasive genetic monitoring to assess rapidly shifting conditions in small populations. Uncertainties about the longer-term trajectory of this population underscore the need to continue monitoring and to research potential for both negative and positive aspects of continued genetic infusion.

Transcription-translation coupling: direct interactions of RNA polymerase with ribosomes and ribosomal subunits.

In prokaryotes, RNA polymerase and ribosomes can bind concurrently to the same RNA transcript, leading to the functional coupling of transcription and translation. The interactions between RNA polymerase and ribosomes are crucial for the coordination of transcription with translation. Here, we report that RNA polymerase directly binds ribosomes and isolated large and small ribosomal subunits. RNA polymerase and ribosomes form a one-to-one complex with a micromolar dissociation constant. The formation of the complex is modulated by the conformational and functional states of RNA polymerase and the ribosome. The binding interface on the large ribosomal subunit is buried by the small subunit during protein synthesis, whereas that on the small subunit remains solvent-accessible. The RNA polymerase binding site on the ribosome includes that of the isolated small ribosomal subunit. This direct interaction between RNA polymerase and ribosomes may contribute to the coupling of transcription to translation.

Estrogen Signaling Contributes to Sex Differences in Macrophage Polarization during Asthma.

Allergic asthma is a chronic Th2 inflammation in the lungs that constricts the airways and presents as coughing and wheezing. Asthma mostly affects boys in childhood and women in adulthood, suggesting that shifts in sex hormones alter the course of the disease. Alveolar macrophages have emerged as major mediators of allergic lung inflammation in animal models as well as humans. Whether sex differences exist in macrophage polarization and the molecular mechanism(s) that drive differential responses are not well understood. We found that IL-4-stimulated bone marrow-derived and alveolar macrophages from female mice exhibited greater expression of M2 genes in vitro and after allergen challenge in vivo. Alveolar macrophages from female mice exhibited greater expression of the IL-4Rα and estrogen receptor (ER) α compared with macrophages from male mice following allergen challenge. An ERα-specific agonist enhanced IL-4-induced M2 gene expression in macrophages from both sexes, but more so in macrophages from female mice. Furthermore, IL-4-stimulated macrophages from female mice exhibited more transcriptionally active histone modifications at M2 gene promoters than did macrophages from male mice. We found that supplementation of estrogen into ovariectomized female mice enhanced M2 polarization in vivo upon challenge with allergen and that macrophage-specific deletion of ERα impaired this M2 polarization. The effects of estrogen are long-lasting; bone marrow-derived macrophages from ovariectomized mice implanted with estrogen exhibited enhanced IL-4-induced M2 gene expression compared with macrophages from placebo-implanted littermates. Taken together, our findings suggest that estrogen enhances IL-4-induced M2 gene expression and thereby contributes to sex differences observed in asthma.

Gold-Embedded Hollow Silica Nanogolf Balls for Imaging and Photothermal Therapy.

Hybrid nanocarriers with multifunctional properties have wide therapeutic and diagnostic applications. We have constructed hollow silica nanogolf balls (HGBs) and gold-embedded hollow silica nanogolf balls (Au@SiO2 HGBs) using the layer-by-layer approach on a symmetric polystyrene (PS) Janus template; the template consists of smaller PS spheres attached to an oppositely charged large PS core. ζ Potential measurement supports the electric force-based template-assisted synthesis mechanism. Electron microscopy, UV-vis, and near-infrared (NIR) spectroscopy show that HGBs or Au@SiO2 HGBs are composed of a porous silica shell with an optional dense layer of gold nanoparticles embedded in the silica shell. To visualize their cellular uptake and imaging potential, Au@SiO2 HGBs were loaded with quantum dots (QDs). Confocal fluorescent microscopy and atomic force microscopy imaging show reliable endocytosis of QD-loaded Au@SiO2 HGBs in adherent HeLa cells and circulating red blood cells (RBCs). Surface-enhanced Raman spectroscopy of Au@SiO2 HGBs in RBC cells show enhanced intensity of the Raman signal specific to the RBCs' membrane specific spectral markers. Au@SiO2 HGBs show localized surface plasmon resonance and heat-induced HeLa cell death in the NIR range. These hybrid golf ball nanocarriers would have broad applications in personalized nanomedicine ranging from in vivo imaging to photothermal therapy.

1H NMR Metabolic Profiling of Earthworm (Eisenia fetida) Coelomic Fluid, Coelomocytes, and Tissue: Identification of a New Metabolite-Malylglutamate.

Earthworm metabolism is recognized as a useful tool for monitoring environmental insults and measuring ecotoxicity, yet extensive earthworm metabolic profiling using 1H nuclear magnetic resonance (NMR) spectroscopy has been limited in scope. This study aims to expand the embedded metabolic material in earthworm coelomic fluid, coelomocytes, and tissue to aid systems toxicology research. Fifty-nine metabolites within Eisenia fetida were identified, with 47 detected in coelomic fluid, 41 in coelomocytes, and 54 in whole-worm samples and tissue extracts. The newly detected but known metabolites 2-aminobutyrate, nicotinurate, Nδ,Nδ,Nδ-trimethylornithine, and trigonelline are reported along with a novel compound, malylglutamate, elucidated using 2D NMR and high-resolution MS/MS. We postulate that malylglutamate acts as a glutamate/malate store, chelator, and anionic osmolyte and helps to provide electrolyte balance.

Disaster Preparedness and Response for the Burn Mass Casualty Incident in the Twenty-first Century.

The effective and efficient coordination of emergent patient care at the point of injury followed by the systematic resource-based triage of casualties are the most critical factors that influence patient outcomes after mass casualty incidents (MCIs). The effectiveness and appropriateness of implemented actions are largely determined by the extent and efficacy of the planning and preparation that occur before the MCI. The goal of this work was to define the essential efforts related to planning, preparation, and execution of acute and subacute medical care for disaster burn casualties. This type of MCI is frequently referred to as a burn MCI."

Highly specific SNP detection using 2D graphene electronics and DNA strand displacement.

Single-nucleotide polymorphisms (SNPs) in a gene sequence are markers for a variety of human diseases. Detection of SNPs with high specificity and sensitivity is essential for effective practical implementation of personalized medicine. Current DNA sequencing, including SNP detection, primarily uses enzyme-based methods or fluorophore-labeled assays that are time-consuming, need laboratory-scale settings, and are expensive. Previously reported electrical charge-based SNP detectors have insufficient specificity and accuracy, limiting their effectiveness. Here, we demonstrate the use of a DNA strand displacement-based probe on a graphene field effect transistor (FET) for high-specificity, single-nucleotide mismatch detection. The single mismatch was detected by measuring strand displacement-induced resistance (and hence current) change and Dirac point shift in a graphene FET. SNP detection in large double-helix DNA strands (e.g., 47 nt) minimize false-positive results. Our electrical sensor-based SNP detection technology, without labeling and without apparent cross-hybridization artifacts, would allow fast, sensitive, and portable SNP detection with single-nucleotide resolution. The technology will have a wide range of applications in digital and implantable biosensors and high-throughput DNA genotyping, with transformative implications for personalized medicine.

Dual-Functionalized Theranostic Nanocarriers.

Nanocarriers with the ability to spatially organize chemically distinct multiple bioactive moieties will have wide combinatory therapeutic and diagnostic (theranostic) applications. We have designed dual-functionalized, 100 nm to 1 µm sized scalable nanocarriers comprising a silica golf ball with amine or quaternary ammonium functional groups located in its pits and hydroxyl groups located on its nonpit surface. These functionalized golf balls selectively captured 10-40 nm charged gold nanoparticles (GNPs) into their pits. The selective capture of GNPs in the golf ball pits is visualized by scanning electron microscopy. ζ potential measurements and analytical modeling indicate that the GNP capture involves its proximity to and the electric charge on the surface of the golf balls. Potential applications of these dual-functionalized carriers include distinct attachment of multiple agents for multifunctional theranostic applications, selective scavenging, and clearance of harmful substances.

Magnetically-responsive silica-gold nanobowls for targeted delivery and SERS-based sensing.

Composite colloidal structures with multi-functional properties have wide applications in targeted delivery of therapeutics and imaging contrast molecules and high-throughput molecular bio-sensing. We have constructed a multifunctional composite magnetic nanobowl using the bottom-up approach on an asymmetric silica/polystyrene Janus template consisting of a silica shell around a partially exposed polystyrene core. The nanobowl consists of a silica bowl and a gold exterior shell with iron oxide magnetic nanoparticles sandwiched between the silica and gold shells. The nanobowls were characterized by electron microscopy, atomic force microscopy, magnetometry, vis-NIR and FTIR spectroscopy. Magnetically vectored transport of these nanobowls was ascertained by time-lapsed imaging of their flow in fluid through a porous hydrogel under a defined magnetic field. These magnetically-responsive nanobowls show distinct surface enhanced Raman spectroscopy (SERS) imaging capability. The PEGylated magnetically-responsive nanobowls show size-dependent cellular uptake in vitro.

HESI/FDA workshop on immunomodulators and cancer risk assessment: Building blocks for a weight-of-evidence approach.

Profound immunosuppression (e.g., AIDS, transplant therapy) is epidemiologically associated with an increased cancer risk, and often with oncogenic viruses. It is currently unclear how broadly this association translates to therapeutics that modulate immunity. A workshop co-sponsored by the FDA and HESI examined how perturbing the immune system may contribute to carcinogenesis, and highlighted priorities for improving non-clinical risk assessment of targeted immunomodulatory therapies. Conclusions from the workshop were as follows. 1) While profound altered immunity can promote tumorigenesis, not all components of the immune system are equally important in defense against or promotion of cancer and a similar cancer risk for all immunomodulatory molecules should not be assumed. 2) Rodent carcinogenicity studies have limitations and are generally not reliable predictors of cancer risk associated with immunosuppression. 3) Cancer risk needs to be evaluated based on mechanism-based weight-of-evidence, including data from immune function tests most relevant to tumor immunosurveillance or promotion. 4) Information from nonclinical experiments, clinical epidemiology and immunomodulatory therapeutics show that immunosurveillance involves a complex network of cells and mediators. To support a weight-of-evidence approach, an increased focus on understanding the quantitative relationship between changes in relevant immune function tests and cancer risk is needed.

Cell and Protein Recognition at a Supported Bilayer Interface via In Situ Cavitand-Mediated Functional Polymer Growth.

Water-soluble deep cavitands embedded in a supported lipid bilayer are capable of anchoring ATRP initiator molecules for the in situ synthesis of primary amine-containing polymethacrylate patches at the water:membrane interface. These polymers can be derivatized in situ to incorporate fluorescent reporters, allow selective protein recognition, and can be applied to the immobilization of nonadherent cells at the bilayer interface.

DNA nano-carrier for repeatable capture and release of biomolecules.

DNA can be manipulated to design nano-machines through specific sequence recognition. We report a switchable DNA carrier for repeatable capture and release of a single stranded DNA. The activity of the carrier was regulated by the interactions among a double-stranded actuator, single stranded target, fuel, and anti-fuel DNA strands. Inosine was used to maintain a stable triple-stranded complex when the actuator's conformation was switched between open (capture) and closed (release) configurations. Time lapse fluorescence measurements show repeatable capture and release of target strands. TEM images also show visible capture of target DNA strands when gold nanoparticles were attached to the DNA carrier and the target DNA strand. The carrier activity was controlled by length of toeholds, number of mismatches, and inosine substitutions. Significantly, unlike in previously published work that reported the devices functioned only when there is a perfect match between the interacting DNA strands, the present device works only when there are mismatches in the fuel strand and the best performance is achieved for 1-3 mismatches. The device was used to successfully capture and release gold nanoparticles when linked to the target single-stranded DNA. In general, this type of devices can be used for transport and delivery of theranostic molecules.

Asymmetric Colloidal Janus Particle Formation Is Core-Size-Dependent.

Colloidal particles with asymmetric surface chemistry (Janus particles) have unique bifunctional properties. The size of these particles is an important determinant for their applications in diverse fields from drug delivery to chemical catalysis. The size of Janus particles, with a core surface coated with carboxylate and a partially encapsulating silica shell, depends upon several factors, including the core size and the concentration of carboxylate coating. The role of the carboxylate coating on the Janus particle size is well-understood; however, the role of the core size is not well defined. The role of the carboxylated polystyrene (cPS) core size on the cPS-silica Janus particle morphology (its size and shape) was examined by testing two different silica sizes and five different cPS core sizes. Results from electron microscopy (EM) and dynamic light scattering (DLS) analysis indicate that the composite cPS-silica particle acquires two distinct shapes: (i) when the size of the cPS core is much smaller than the non-cPS silica (b-SiO2) sphere, partially encapsulated Janus particles are formed, and (ii) when the cPS core is larger than or equal to the b-SiO2 sphere, a raspberry-like structure rather than a Janus particle is formed. The cPS-silica Janus particles of ∼100-500 nm size were obtained when the size of the cPS core was much smaller than the non-cPS silica (b-SiO2) sphere. These scalable nanoscale Janus particles will have wide application in a multifunctional delivery platform and catalysis.

Health care: economic impact of caring for geriatric patients.

National health care expenditures constitute a continuously expanding component of the US economy. Health care resources are distributed unequally among the population, and geriatric patients are disproportionately represented. Characterizing this group of individuals that accounts for the largest percentage of US health spending may facilitate the introduction of targeted interventions in key high-impact areas. Changing demographics, an increasing incidence of chronic disease and progressive disability, rapid technological advances, and systemic market failures in the health care sector combine to drive cost. A multidisciplinary approach will become increasingly necessary to balance the delicate relationship between our constrained supply and increasing demand.