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At least 1,063 records · Page 59

Mass spectrometry structural analysis of intrinsically disordered phosphoproteins

Phosphorylation is a ubiquitous protein modification that is known to play important roles in many biological phenomena including cell signaling, the opening and closing of membrane protein channels, and even triggering of amyloid protein aggregation. Despite the effects phosphorylation has on protein function, the impact phosphorylation has on the structure of proteins is not well understood. Here, to determine how phosphorylation affects the structure of proteins, top-down mass spectrometry (TD-MS) and ion mobility-mass spectrometry (IM-MS) were performed on various phosphorylated proteins and their dephosphorylated proteoforms. TD-MS with collision- and electron-based fragmentation techniques was utilized to locate phosphorylation sites on the intrinsically disordered amyloid proteins β-casein and α-synuclein. TD-MS also provided evidence that alkaline phosphatase dephosphorylates β-casein from the N-terminus to the C-terminus. Furthermore, IM-MS of common phosphorylated proteins such as β-casein, α-casein, ovalbumin, and phosvitin indicates that phosphorylation promotes compaction of protein structure in denaturing as well as native conditions. Increases in abundance of more compact conformers are also observed when the disease related amyloid protein α-synuclein is phosphorylated at serine 129. We interpret the increased abundance of more compact conformers when proteins are phosphorylated as evidence that salt bridges form between negatively charged phosphates and positively charged residues, which alters protein structure. Salt bridge formation due to phosphorylation could be a mechanism for regulating protein function and be responsible for many of the phenomena observed in nature.

Amyloid proteins↗

A Viable New Strategy for the Discovery of Peptide Proteolytic Cleavage Products in Plant-Microbe Interactions

Small peptides that are proteolytic cleavage products (PCPs) of less than 100 amino acids are emerging as key signaling molecules that mediate cell-to-cell communication and biological processes that occur between and within plants, fungi, and bacteria. Yet, the discovery and characterization of these molecules is largely overlooked. Today, selective enrichment and subsequent characterization by mass spectrometry–based sequencing offers the greatest potential for their comprehensive characterization, however qualitative and quantitative performance metrics are rarely captured. Herein, we addressed this need by benchmarking the performance of an enrichment strategy, optimized specifically for small PCPs, using state-of-the-art de novo–assisted peptide sequencing. As a case study, we implemented this approach to identify PCPs from different root and foliar tissues of the hybrid poplar Populus × canescens 717-1B4 in interaction with the ectomycorrhizal basidiomycete Laccaria bicolor . In total, we identified 1,660 and 2,870 Populus and L. bicolor unique PCPs, respectively. Qualitative results supported the identification of well-known PCPs, like the mature form of the photosystem II complex 5-kDa protein (approximately 3 kDa). A total of 157 PCPs were determined to be significantly more abundant in root tips with established ectomycorrhiza when compared with root tips without established ectomycorrhiza and extramatrical mycelium of L. bicolor . These PCPs mapped to 64 Populus proteins and 69 L. bicolor proteins in our database, with several of them previously implicated in biologically relevant associations between plant and fungus.

59 BASIC BIOLOGICAL SCIENCES↗

The histone code of the fungal genus Aspergillus uncovered by evolutionary and proteomic analyses

Chemical modifications of DNA and histone proteins impact the organization of chromatin within the nucleus. Changes in these modifications, catalysed by different chromatin-modifying enzymes, influence chromatin organization, which in turn is thought to impact the spatial and temporal regulation of gene expression. While combinations of different histone modifications, the histone code, have been studied in several model species, we know very little about histone modifications in the fungal genus Aspergillus, whose members are generally well studied due to their importance as models in cell and molecular biology as well as their medical and biotechnological relevance. Here, we used phylogenetic analyses in 94 Aspergilli as well as other fungi to uncover the occurrence and evolutionary trajectories of enzymes and protein complexes with roles in chromatin modifications or regulation. We found that these enzymes and complexes are highly conserved in Aspergilli, pointing towards a complex repertoire of chromatin modifications. Nevertheless, we also observed few recent gene duplications or losses, highlighting Aspergillus species to further study the roles of specific chromatin modifications. SET7 (KMT6) and other components of PRC2 (Polycomb Repressive Complex 2), which is responsible for methylation on histone H3 at lysine 27 in many eukaryotes including fungi, are absent in Aspergilli as well as in closely related Penicillium species, suggesting that these lost the capacity for this histone modification. We corroborated our computational predictions by performing untargeted MS analysis of histone post-translational modifications in Aspergillus nidulans. This systematic analysis will pave the way for future research into the complexity of the histone code and its functional implications on genome architecture and gene regulation in fungi.

59 BASIC BIOLOGICAL SCIENCES↗

In situ , in vivo , and in operando imaging and spectroscopy of liquids using microfluidics in vacuum

This review offers a succinct overview of the development of a vacuum-compatible microfluidic reactor system for analysis at the liquid vacuum interface (SALVI), and its diverse applications in in situ, in vivo, and in operando imaging of liquid surfaces as well as the air-liquid (a-l), liquid-liquid (l-l), and solid-liquid (s-l) interfaces in the past decade. SALVI is one of the first microfluidics-based reactors that has enabled direct analysis of volatile liquids in vacuum surface tools such as scanning electron microscopy (SEM) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). Its integration into ambient and vacuum spectroscopy and microscopy is illustrated. Several applications are highlighted including (1) imaging nanoparticles in liquid using in situ SEM; (2) mapping the evolving l-l interface using in situ x-ray absorption spectroscopy and ToF-SIMS; (3) following complex a-l interfacial oxidation reaction products using in situ ToF-SIMS; (4) capturing biological interfaces of cells and microbes via in vivo multimodal and correlative imaging; and (5) monitoring the dynamic solid electrode and liquid electrolyte interface using in operando molecular imaging. Finally, outlook and recommendations are presented. Besides showing the holistic information volume obtained by real-time multiplexed imaging, this review intends to convey the importance of tool development in revolutionizing surface and interface analysis using vacuum platforms previously limited to solid surfaces. Microfluidics is manifested to be not limited to ambient conditions in many examples in this review. Moreover, fundamental interfacial phenomena underpinning mass and charge transfer can now be pursued in real time via innovated chemical imaging and spectroscopy.

42 ENGINEERING↗

Accessing pluripotent materials through tempering of dynamic covalent polymer networks

Pluripotency, which is defined as a system not fixed as to its developmental potentialities, is typically associated with biology and stem cells. Here, inspired by this concept, we report synthetic polymers that act as a single “pluripotent” feedstock and can be differentiated into a range of materials that exhibit different mechanical properties, from hard and brittle to soft and extensible. To achieve this, we have exploited dynamic covalent networks that contain labile, dynamic thia-Michael bonds, whose extent of bonding can be thermally modulated and retained through tempering, akin to the process used in metallurgy. In addition, we show that the shape memory behavior of these materials can be tailored through tempering and that these materials can be patterned to spatially control mechanical properties.

36 MATERIALS SCIENCE↗

Green Factor

Los Alamos is designing a screening platform coupled to our biosensor to customize production processes in cell-free environments that offer biomanufacturers higher titers and yields, better resource utilization and cost-effective bioprocess. Our technology aims at enabling cell-free biosynthetic systems by providing enzyme variants generated by coupling with our biosensor technology, that can utilize cheap raw material cofactor analogues. This will enable bioprocesses based on cell free synthetic biology for a sustainable bioeconomy.

42 ENGINEERING↗

Decoding FGF/FGFR Signaling: Insights into Biological Functions and Disease Relevance

Fibroblast Growth Factors (FGFs) and their cognate receptors, FGFRs, play pivotal roles in a plethora of biological processes, including cell proliferation, differentiation, tissue repair, and metabolic homeostasis. This review provides a comprehensive overview of FGF-FGFR signaling pathways while highlighting their complex regulatory mechanisms and interconnections with other signaling networks. Further, we briefly discuss the FGFs involvement in developmental, metabolic, and housekeeping functions. By complementing current knowledge and emerging research, this review aims to enhance the understanding of FGF-FGFR-mediated signaling and its implications for health and disease, which will be crucial for therapeutic development against FGF-related pathological conditions.

Edirisinghe, Oshadi↗

The effect of fluid mechanical stress on cellular arachidonic acid metabolism

The effect of sublytic levels of mechanical perturations of cells on cell metabolism were investigated by analyzing the products of arachidonic acid (used as a marker metabolite) in blood platelets, polymorphonuclear leucocytes, and cultured umbilical-vein endothelial cells after the suspensions of these cells were subjected to a shear stress in a modified viscometer. It is shown that the sublytic levels of mechanical stress stimulated the arachidonic acid metabolism in all these cell types. Possible biological implications of this stress-metabolism coupling are discussed.

Mcintire, L. V.↗

The centrifuge facility - A life sciences research laboratory for Space Station Freedom

The paper describes the centrifugal facility that is presently being developed by NASA for studies aboard the Space Station Freedom on the role of gravity, or its absence, at varying intensities for varying periods of time and with multiple model systems. Special attention is given to the design of the centrifuge system, the habitats designed to hold plants and animals, the glovebox system designed for experimental manipulations of the specimens, and the service unit. Studies planned for the facility will include experiments in the following disciplines: cell and developmental biology, plant biology, regulatory physiology, musculoskeletal physiology, behavior and performance, neurosciences, cardiopulmonary physiology, and environmental health and radiation.

Fuller, Charles A.↗

STS-70 Space Shuttle Mission Report - September 1995

The STS-70 Space Shuttle Program Mission Report summarizes the Payload activities as well as the Orbiter, External Tank (ET), Solid Rocket Booster (SRB), Reusable Solid Rocket Motor (RSRM), and the Space Shuttle main engine (SSME) systems performance during the seventieth flight of the Space Shuttle Program, the forty-fifth flight since the return-to-flight, and the twenty-first flight of the Orbiter Discovery (OV-103). In addition to the Orbiter, the flight vehicle consisted of an ET that was designated ET-71; three SSMEs that were designated as serial numbers 2036, 2019, and 2017 in positions 1, 2, and 3, respectively; and two SRBs that were designated 81-073. The RSRMs, designated RSRM-44, were installed in each SRB and were designated as 36OL044A for the left SRB, and 36OL044B for the right SRB. The primary objective of this flight was to deploy the Tracking and Data Relay Satellite-G/Inertial Upper Stage (TDRS-G/IUS). The secondary objectives were to fulfill the requirements of the Physiological and Anatomical Rodent Experiment/National Institutes of Health-Rodents (PARE/NIH-R); Bioreactor Demonstration System (BDS); Commercial Protein Crystal Growth (CPCG) experiment; Space Tissue Loss/National Institutes of Health - Cells (STL/NIH-C) experiment; Biological Research in Canisters (BRIC) experiment; Shuttle Amateur Radio Experiment-2 (SAREX-2); Visual Function Tester-4 (VFT-4); Hand-Held, Earth-Oriented, Real-Time, Cooperative, User-Friendly Location-Targeting and Environmental System (HERCULES); Microencapsulation in Space-B (MIS-B) experiment; Window Experiment (WINDEX); Radiation Monitoring Equipment-3 (RME-3); and the Military Applications of Ship Tracks (MAST) payload.

Fricke, Robert W., Jr.↗

Genomic instability, bystander effect, cytoplasmic irradiation and other phenomena that may achieve fame without fortune

The possible risk of induced malignancies in astronauts, as a consequence of the radiation environment in space, is a factor of concern for long term missions. Cancer risk estimates for high doses of low LET radiation are available from the epidemiological studies of the A-bomb survivors. Cancer risks at lower doses cannot be detected in epidemiological studies and must be inferred by extrapolation from the high dose risks. The standard setting bodies, such as the ICRP recommend a linear, no-threshold extrapolation of risks from high to low doses, but this is controversial. A study of mechanisms of carcinogenesis may shed some light on the validity of a linear extrapolation. The multi-step nature of carcinogenesis suggests that the role of radiation may be to induce a mutation leading to a mutator phenotype. High energy Fe ions, such as those encountered in space are highly effective in inducing genomic instability. Experiments involving the single particle microbeam have demonstrated a "bystander effect", ie a biological effect in cells not themselves hit, but in close proximity to those that are, as well as the induction of mutations in cells where only the cytoplasm, and not the nucleus, have been traversed by a charged particle. These recent experiments cast doubt on the validity of a simple linear extrapolation, but the data are so far fragmentary and conflicting. More studies are necessary. While mechanistic studies cannot replace epidemiology as a source of quantitative risk estimates, they may shed some light on the shape of the dose response relationship and therefore on the limitations of a linear extrapolation to low doses.

NASA Discipline Radiation Health↗

The development of spaceflight experiments with Arabidopsis as a model system in gravitropism studies

Experiments with Arabidopsis have been developed for spaceflight studies in the European Space Agency's Biorack module. The Biorack is a multiuser facility that is flown on the United States Space Shuttle and serves as a small laboratory for studying cell and developmental biology in unicells, plants, and small invertebrates. The purpose of our spaceflight research was to investigate the starch-statolith model for gravity perception by studying wild-type (WT) and three starch-deficient mutants of Arabidopsis. Since spaceflight opportunities for biological experimentation are scarce, the extensive ground-based testing described in this paper is needed to ensure the success of a flight project. Therefore, the specific aims of our ground-based research were: (1) to modify the internal configuration of the flight hardware, which originally was designed for large lentil seeds, to accommodate small Arabidopsis seeds; (2) to maximize seed germination in the hardware; and (3) to develop favorable conditions in flight hardware for the growth and gravitropism of seedlings. The hardware has been modified, and growth conditions for Arabidopsis have been optimized. These experiments were successfully flown on two Space Shuttle missions in 1997.

STS-84 Shuttle Project↗

New Method Developed to Measure Contact Angles of a Sessile Drop

The spreading of an evaporating liquid on a solid surface occurs in many practical processes and is of importance in a number of practical situations such as painting, textile dyeing, coating, gluing, and thermal engineering. Typical processes involving heat transfer where the contact angle plays an important role are film cooling, boiling, and the heat transfer through heat pipes. The biological phenomenon of cell spreading also is analogous to a drop spreading (ref. 1). In the study of spreading, the dynamic contact angle describes the interfacial properties on solid substrates and, therefore, has been studied by physicists and fluid mechanics investigators. The dynamic contact angle of a spreading nonvolatile liquid drop provides a simple tool in the study of the free-boundary problem, but the study of the spreading of a volatile liquid drop is of more practical interest because the evaporation of common liquids is inevitable in practical processes. The most common method to measure the contact angle, the contact radius, and the height of a sessile drop on a solid surface is to view the drop from its edge through an optical microscope. However, this method gives only local information in the view direction. Zhang and Yang (ref. 2) developed a laser shadowgraphy method to investigate the evaporation of sessile drop on a glass plate. As described here, Zhang and Chao (refs. 3 and 4) improved the method and suggested a new optical arrangement to measure the dynamic contact angle and the instant evaporation rate of a sessile drop with much higher accuracy (less than 1 percent). With this method, any fluid motion in the evaporating drop can be visualized through shadowgraphy without using a tracer, which often affects the field under investigation.

Chao, David F.↗

Plant Science in Reduced Gravity: Lessons Learned

The effect of gravity on the growth and development of plants has been the subject of scientific investigation for over a century. The results obtained in space to test specific hypotheses on gravitropism, gene expression, seed formation, or growth rate are affected by both the primary effect of the microgravity and secondary effects of the spaceflight environment. The secondary effects of the spaceflight environment include physical effects arising from physical changes, such as the absence of buoyancy driven convective mixing, altered behavior of liquids and gases, and the environmental conditions in the spacecraft atmosphere. Thus, the design of biological experiments (e.g. cells, plants, animals, etc.) conducted in microgravity must account for changes in the physical forces, as well as the environmental conditions, imposed by the specific spaceflight vehicle and experimental hardware. In addition, researchers must become familiar with other aspects of spaceflight experiments: payload integration with hardware developers, safety documentation and crew procedures, and the logistics of conducting flight and ground controls. This report reviews the physical and environmental factors that directly and indirectly affect the results of plant science experiments in microgravity and is intended to serve as a guide in the design and implementation plant experiments in space.

Stutte, Gary W.↗