Mechanisms of precipitate formation during the purification of an Fc-fusion protein.

Protein precipitates that arise during bioprocessing can cause manufacturing challenges, but they can also aid in clearance of host-cell protein (HCP) and DNA impurities. Such precipitates differ from many protein precipitates that have been studied previously in their heterogeneous composition, particularly in the presence of high concentrations of the product protein. Here, we characterize the precipitates that form after neutralization of protein A purified and viral-inactivated material of an Fc-fusion protein produced in Chinese hamster ovary cells. The physical growth of precipitate particles was observed by optical microscopy, transmission electron microscopy, dynamic light scattering, and small-angle and ultra-small-angle X-ray scattering to characterize the precipitate microstructure and growth mechanism. The precipitate microstructure is well-described as a mass fractal with fractal dimension approximately 2. The growth is governed by a diffusion-limited aggregation mechanism as indicated by a power-law dependence on time of the size of the principal precipitate particles. Optical microscopy shows that these primary particles can further aggregate into larger particles in a manner that appears to be promoted by mixing. Absorbance experiments at varying pH and salt concentrations reveal that the growth is largely driven by attractive electrostatic interactions, as growth is hindered by an increase in ionic strength. The solution conditions that resulted in the most significant particle growth are also correlated with the greatest removal of soluble impurities (DNA and HCPs). Proteomic analysis of the precipitates allows identification of O ( 100 ) unique HCP impurities, depending on the buffer species (acetate or citrate) used for the viral inactivation. Most of these proteins have pI values near the precipitation pH, supporting the likely importance of electrostatic interactions in driving precipitate formation.

Local Crystalline Structure in an Amorphous Protein Dense Phase.

Proteins exhibit a variety of dense phases ranging from gels, aggregates, and precipitates to crystalline phases and dense liquids. Although the structure of the crystalline phase is known in atomistic detail, little attention has been paid to noncrystalline protein dense phases, and in many cases the structures of these phases are assumed to be fully amorphous. In this work, we used small-angle neutron scattering, electron microscopy, and electron tomography to measure the structure of ovalbumin precipitate particles salted out with ammonium sulfate. We found that the ovalbumin phase-separates into core-shell particles with a core radius of ∼2 µm and shell thickness of ∼0.5 µm. Within this shell region, nanostructures comprised of crystallites of ovalbumin self-assemble into a well-defined bicontinuous network with branches ∼12 nm thick. These results demonstrate that the protein gel is comprised in part of nanocrystalline protein.

A plasmodesmata-localized protein mediates crosstalk between cell-to-cell communication and innate immunity in Arabidopsis.

Plasmodesmata (PD) are thought to play a fundamental role in almost every aspect of plant life, including normal growth, physiology, and developmental responses. However, how specific signaling pathways integrate PD-mediated cell-to-cell communication is not well understood. Here, we present experimental evidence showing that the Arabidopsis thaliana plasmodesmata-located protein 5 (PDLP5; also known as HOPW1-1-INDUCED GENE1) mediates crosstalk between PD regulation and salicylic acid-dependent defense responses. PDLP5 was found to localize at the central region of PD channels and associate with PD pit fields, acting as an inhibitor to PD trafficking, potentially through its capacity to modulate PD callose deposition. As a regulator of PD, PDLP5 was also essential for conferring enhanced innate immunity against bacterial pathogens in a salicylic acid-dependent manner. Based on these findings, a model is proposed illustrating that the regulation of PD closure mediated by PDLP5 constitutes a crucial part of coordinated control of cell-to-cell communication and defense signaling.

Targeting Borrelia burgdorferi HtpG with a berserker molecule, a strategy for anti-microbial development.

Conventional antimicrobial discovery relies on targeting essential enzymes in pathogenic organisms, contributing to a paucity of new antibiotics to address resistant strains. Here, by targeting a non-essential enzyme, Borrelia burgdorferi HtpG, to deliver lethal payloads, we expand what can be considered druggable within any pathogen. We synthesized HS-291, an HtpG inhibitor tethered to the photoactive toxin verteporfin. Reactive oxygen species, generated by light, enables HS-291 to sterilize Borrelia cultures by causing oxidation of HtpG, and a discrete subset of proteins in proximity to the chaperone. This caused irreversible nucleoid collapse and membrane blebbing. Tethering verteporfin to the HtpG inhibitor was essential, since free verteporfin was not retained by Borrelia in contrast to HS-291. For this reason, we liken HS-291 to a berserker, wreaking havoc upon the pathogen's biology once selectively absorbed and activated. This strategy expands the druggable pathogenic genome and offsets antibiotic resistance by targeting non-essential proteins.

Three-dimensional analysis and computer modeling of the capillary endothelial vesicular system with electron tomography.

OBJECTIVE: We examined the three-dimensional organization of the endothelial vesicular system with TEM tomography of semi-thick sections. MATERIALS AND METHODS: Mouse abdominal muscle capillaries were perfused with terbium to label vesicular compartments open to the luminal surface. The tissue was prepared for TEM and semi-thick (250 nm) sections were cut. Dual axis tilt series, collected from +60° to -60° at 1° increments, were acquired in regions of labeled abluminal caveolae. These tomograms were reconstructed and analyzed to reveal three-dimensional vesicular associations not evident in thin sections. RESULTS: Reconstructed tomograms revealed free vesicles, both labeled and unlabeled, in the endothelial cytoplasm as well as transendothelial channels that spanned the luminal and abluminal membranes. A large membranous compartment connecting the luminal and abluminal surfaces was also present. Computer modeling of tomographic data and video animations provided three-dimensional perspectives to these structures. CONCLUSIONS: Uncertainties associated with other three-dimensional methods to study the capillary wall are remedied by tomographic analysis of semi-thick sections. Transendothelial channels of fused vesicles and free cytoplasmic vesicles give credence to their role as large pores in the transport of solutes across the walls of continuous capillaries.

Expression of the Cameleon calcium biosensor in fungi reveals distinct Ca(2+) signatures associated with polarized growth, development, and pathogenesis.

Calcium is a universal messenger that translates diverse environmental stimuli and developmental cues into specific cellular and developmental responses. While individual fungal species have evolved complex and often unique biochemical and structural mechanisms to exploit specific ecological niches and to adjust growth and development in response to external stimuli, one universal feature to all is that Ca(2+)-mediated signaling is involved. The lack of a robust method for imaging spatial and temporal dynamics of subcellular Ca(2+) (i.e., "Ca(2+) signature"), readily available in the plant and animal systems, has severely limited studies on how this signaling pathway controls fungal growth, development, and pathogenesis. Here, we report the first successful expression of a FRET (Förster Resonance Energy Transfer)-based Ca(2+) biosensor in fungi. Time-lapse imaging of Magnaporthe oryzae, Fusarium oxysporum, and Fusarium graminearum expressing this sensor showed that instead of a continuous gradient, the cytoplasmic Ca(2+) ([Ca(2+)](c)) change occurred in a pulsatile manner with no discernable gradient between pulses, and each species exhibited a distinct Ca(2+) signature. Furthermore, occurrence of pulsatile Ca(2+) signatures was age and development dependent, and major [Ca(2+)](c) transients were observed during hyphal branching, septum formation, differentiation into specialized plant infection structures, cell-cell contact and in planta growth. In combination with the sequenced genomes and ease of targeted gene manipulation of these and many other fungal species, the data, materials and methods developed here will help understand the mechanism underpinning Ca(2+)-mediated control of cellular and developmental changes, its role in polarized growth forms and the evolution of Ca(2+) signaling across eukaryotic kingdoms.

Nanocrystalline protein domains via salting-out.

Protein salting-out is a well established phenomenon that in many cases leads to amorphous structures and protein gels, which are usually not considered to be useful for protein structure determination. Here, microstructural measurements of several different salted-out protein dense phases are reported, including of lysozyme, ribonuclease A and an IgG1, showing that salted-out protein gels unexpectedly contain highly ordered protein nanostructures that assemble hierarchically to create the gel. The nanocrystalline domains are approximately 10-100 nm in size, are shown to have structures commensurate with those of bulk crystals and grow on time scales in the order of an hour to a day. Beyond revealing the rich, hierarchical nanoscale to mesoscale structure of protein gels, the nanocrystals that these phases contain are candidates for structural biology on next-generation X-ray free-electron lasers, which may enable the study of biological macromolecules that are difficult or impossible to crystallize in bulk.

Interspecies Microbial Fusion and Large-Scale Exchange of Cytoplasmic Proteins and RNA in a Syntrophic Clostridium Coculture.

Microbial syntrophy is universal in nature, profoundly affecting the composition and function of microbiomes. We have recently reported data suggesting direct cell-to-cell interactions leading to electron and material exchange between the two microbes in the syntrophy between Clostridium ljungdahlii and C. acetobutylicum Here, transmission electron microscopy and electron tomography demonstrated cell wall and membrane fusions between the two organisms, whereby C. ljungdahlii appears to invade C. acetobutylicum pole to pole. Correlative fluorescence transmission electron microscopy demonstrated large-scale exchange of proteins. Flow cytometry analysis captured the extent and dynamic persistence of these interactions. Dividing hybrid cells were identified containing stained proteins from both organisms, thus demonstrating persistence of cells with exchanged cellular components. Fluorescence microscopy and flow cytometry of one species with stained RNA and the other tagged with a fluorescent protein demonstrated extensive RNA exchange and identified hybrid cells, some of which continued to divide, while some were in an advanced C. acetobutylicum sporulation form. These data demonstrate that cell fusion enables large-scale cellular material exchange between the two organisms. Although unanticipated and never previously reported, these phenomena are likely widely distributed in nature, have profound implications for species evolution and the function of microbial communities, and could find utility in biotechnology. They may shed new light onto little-understood phenomena, such as antibiotic heteroresistance of pathogens, pathogen invasion of human tissues, and the evolutionary trajectory and persistence of unculturable bacteria.IMPORTANCE We report that two different bacterial organisms engage in heterologous cell fusion that leads to massive exchange of cellular material, including proteins and RNA, and the formation of persistent hybrid cells. The interspecies cell fusion observed here involves a syntrophic microbial system, but these heterologous cell fusions were observed even under nonstrict syntrophic conditions, leaving open the possibility that strict syntrophy may not be necessary for interspecies cell fusion and cellular material exchange. Formation of hybrid cells that contain proteins and RNA from both organisms is unexpected and unprecedented. Such fusion events are likely widely distributed in nature, but have gone undetected. The implications are profound and may shed light onto many unexplained phenomena in human health, natural environments, evolutionary biology, and biotechnology.

A comparison of exosome purification methods using serum of Marek's disease virus (MDV)-vaccinated and -tumor-bearing chickens.

Marek's disease (MD) is an alphaherpesvirus (Marek's disease virus, MDV)-induced pathology of chickens associated with paralysis, immunosuppression, neurological signs, and T-cell lymphomas. MD is controlled in poultry production via live attenuated vaccines. The purpose of the current study was to compare methods for precipitating exosomes from vaccinated and protected chicken sera (VEX) and tumor-bearing chicken sera (TEX) for biomarker analysis of vaccine-induced protection and MD lymphomas respectively. A standard polyethylene glycol (PEG, 8%) method was compared to a commercial reagent (total exosome isolation reagent, TEI) for exosome yield and RNA content. Although exosomes purified by PEG or TEI were comparable in size and morphology, TEI-reagent yielded 3-4-fold greater concentration. Relative expression of 8 out of 10 G. gallus- and MDV1-encoded miRNAs examined displayed significant difference depending upon the precipitation method used. Standard PEG yields comparable, albeit lower amounts of exosomes than the TEI-reagent and a distinctive miRNA composition.

Insights Into the Mineralogy and Surface Chemistry of Extracellular Biogenic S0 Globules Produced by Chlorobaculum tepidum.

Elemental sulfur (S0) is produced and degraded by phylogenetically diverse groups of microorganisms. For Chlorobaculum tepidum, an anoxygenic phototroph, sulfide is oxidized to produce extracellular S0 globules, which can be further oxidized to sulfate. While some sulfur-oxidizing bacteria (e.g., Allochromatium vinosum) are also capable of growth on commercial S0 as an electron donor, C. tepidum is not. Even colloidal sulfur sols, which appear indistinguishable from biogenic globules, do not support the growth of C. tepidum. Here, we investigate the properties that make biogenic S0 globules distinct from abiotic forms of S0. We found that S0 globules produced by C. tepidum and abiotic S0 sols are quite similar in terms of mineralogy and material properties, but the two are distinguished primarily by the properties of their surfaces. C. tepidum's globules are enveloped by a layer of organics (protein and polysaccharides), which results in a surface that is fundamentally different from that of abiotic S0 sols. The organic coating on the globules appears to slow the aging and crystallization of amorphous sulfur, perhaps providing an extended window of time for microbes in the environment to access the more labile forms of sulfur as needed. Overall, our results suggest that the surface of biogenic S0 globules may be key to cell-sulfur interactions and the reactivity of biogenic S0 in the environment.

Comparison of exosomes purified via ultracentrifugation (UC) and Total Exosome Isolation (TEI) reagent from the serum of Marek's disease virus (MDV)-vaccinated and tumor-bearing chickens.

Extracellular vesicles (EVs) is a collective term used to refer microparticles, exosomes, and apoptotic bodies produced by a variety of cells and released into interstitial spaces and bodily fluids. Serum exosomes can serve as invaluable biomarkers, containing m/miRNAs, lipids, and proteins, indicative of various conditions. There are currently limited studies on the characterization and mutual consensus of biomarker profiles of serum exosomes purified by different methods. Here we compared the advantages and disadvantages of two commonly used serum exosome purification procedures including ultracentrifugation (UC) and Total Exosome Isolation (TEI) reagent, by analyzing exosome size distribution, concentration, morphology and miRNA expression profiles. Serum was obtained from Marek's disease virus (MDV)-infected chickens that were either vaccinated against Marek's disease (MD), and thus protected, or unvaccinated and bearing MDV-induced tumors. Nanoparticle tracking analysis (NTA) and Transmission Electron Microscopy (TEM) were performed to evaluate particle size, concentration, and morphological integrity, respectively. Our results indicate that the size distribution of particles purified by either procedure is consistent with that of exosomes (30-150 nm). TEI reagent generated higher yields and co-isolated additional EV populations that are slightly larger (∼180 nm). Based on the miRNA expression profiles from a previous high throughput sequencing experiment of exosome small RNAs, we selected six cellular and four MDV1 miRNAs, to validate their expression in UC- and TEI-purified exosomes. miRNA expression profiles displayed relative correlation between the two procedures, but distinctive differences were observed in abundance with TEI-purified exosomes showing higher miRNA expression consistent with higher yield than those purified by UC. TEI-purified exosomes from vaccinated chickens exhibited greater expression of tumor suppressor miRNA, gga-mir-146b and least expression of oncomiR, gga-mir-21 compared to those obtained from tumor-bearing chickens. We propose that gga-mir-146 and -21 can serve as serum exosome biomarkers for vaccine-induced protection and MD tumors respectively.

Stromule extension along microtubules coordinated with actin-mediated anchoring guides perinuclear chloroplast movement during innate immunity.

Dynamic tubular extensions from chloroplasts called stromules have recently been shown to connect with nuclei and function during innate immunity. We demonstrate that stromules extend along microtubules (MTs) and MT organization directly affects stromule dynamics since stabilization of MTs chemically or genetically increases stromule numbers and length. Although actin filaments (AFs) are not required for stromule extension, they provide anchor points for stromules. Interestingly, there is a strong correlation between the direction of stromules from chloroplasts and the direction of chloroplast movement. Stromule-directed chloroplast movement was observed in steady-state conditions without immune induction, suggesting it is a general function of stromules in epidermal cells. Our results show that MTs and AFs may facilitate perinuclear clustering of chloroplasts during an innate immune response. We propose a model in which stromules extend along MTs and connect to AF anchor points surrounding nuclei, facilitating stromule-directed movement of chloroplasts to nuclei during innate immunity.

Effects of the bacterial algicide IRI-160AA on cellular morphology of harmful dinoflagellates.

The algicide, IRI-160AA, induces mortality in dinoflagellates but not other species of algae, suggesting that a shared characteristic or feature renders this class of phytoplankton vulnerable to the algicide. In contrast to other eukaryotic species, the genome of dinoflagellates is stabilized by high concentrations of divalent cations and transition metals and contains large amounts of DNA with unusual base modifications. These distinctions set dinoflagellates apart from other phytoplankton and suggest that the nucleus may be a dinoflagellate-specific target for IRI-160AA. In this study, morphological and ultrastructural changes in three dinoflagellate species, Prorocentrum minimum, Karlodinium veneficum and Gyrodinium instriatum, were evaluated after short-term exposure to IRI-160AA using super resolution structured illumination microscopy (SR-SIM) and transmission electron microscopy (TEM). Exposure to the algicide resulted in cytoplasmic membrane blebbing, differing chloroplast morphologies, nuclear expansion, and chromosome expulsion and/or destabilization. TEM analysis showed that chromosomes of algicide-treated K. veneficum appeared electron dense with fibrous protrusions. In algicide-treated P. minimum and G. instriatum, chromosome decompaction occurred, while for P. minimum, nuclear expulsion was also observed for several cells. Results of this investigation demonstrate that exposure to the algicide destabilizes dinoflagellate chromosomes, although it was not clear if the nucleus was the primary target of the algicide or if the observed effects on chromosomal structure were due to downstream impacts. In all cases, changes in cellular morphology and ultrastructure were observed within two hours, suggesting that the algicide may be an effective and rapid approach to mitigate dinoflagellate blooms.

Localization of fluorescently tagged protein to plasmodesmata by correlative light and electron microscopy.

Plasmodesmata (PD) are intercellular communication channels that form long, membrane-lined cylinders across cellular junctions. A fluorescent-tagging approach is most commonly used for an initial assessment to address whether a protein of interest may localize or associate with PD domain. However, owing to the dimension of PD being at nanoscale, PD-associated fluorescent signals are detected only as small spots scattered at the cell periphery, hence requiring additional confirmatory evidence. Immunogold labeling provides such information, but suitable antibodies are not always available and morphological preservation is often compromised with this approach. Here we describe an alternative approach using a correlative light and electron microscopy (CLEM) technique, which combines fluorescent imaging and transmission electron microscopy. By employing this method, a clear correlation between fluorescent speckles and the presence of individual or clusters of PD is achieved.

The dependences of osteocyte network on bone compartment, age, and disease.

Osteocytes, the most abundant bone cells, form an interconnected network in the lacunar-canalicular pore system (LCS) buried within the mineralized matrix, which allows osteocytes to obtain nutrients from the blood supply, sense external mechanical signals, and communicate among themselves and with other cells on bone surfaces. In this study, we examined key features of the LCS network including the topological parameter and the detailed structure of individual connections and their variations in cortical and cancellous compartments, at different ages, and in two disease conditions with altered mechanosensing (perlecan deficiency and diabetes). LCS network showed both topological stability, in terms of conservation of connectivity among osteocyte lacunae (similar to the "nodes" in a computer network), and considerable variability the pericellular annular fluid gap surrounding lacunae and canaliculi (similar to the "bandwidth" of individual links in a computer network). Age, in the range of our study (15-32 weeks), affected only the pericellular fluid annulus in cortical bone but not in cancellous bone. Diabetes impacted the spacing of the lacunae, while the perlecan deficiency had a profound influence on the pericellular fluid annulus. The LCS network features play important roles in osteocyte signaling and regulation of bone growth and adaptation.

Chloroplast Stromules Function during Innate Immunity.

Inter-organellar communication is vital for successful innate immune responses that confer defense against pathogens. However, little is known about how chloroplasts, which are a major production site of pro-defense molecules, communicate and coordinate with other organelles during defense. Here we show that chloroplasts send out dynamic tubular extensions called stromules during innate immunity or exogenous application of the pro-defense signals, hydrogen peroxide (H2O2) and salicylic acid. Interestingly, numerous stromules surround nuclei during defense response, and these connections correlate with an accumulation of chloroplast-localized NRIP1 defense protein and H2O2 in the nucleus. Furthermore, silencing and knockout of chloroplast unusual positioning 1 (CHUP1) that encodes a chloroplast outer envelope protein constitutively induces stromules in the absence of pathogen infection and enhances programmed cell death. These results support a model in which stromules aid in the amplification and/or transport of pro-defense signals into the nucleus and other subcellular compartments during immunity.

Multi-modal registration for correlative microscopy using image analogies.

Correlative microscopy is a methodology combining the functionality of light microscopy with the high resolution of electron microscopy and other microscopy technologies for the same biological specimen. In this paper, we propose an image registration method for correlative microscopy, which is challenging due to the distinct appearance of biological structures when imaged with different modalities. Our method is based on image analogies and allows to transform images of a given modality into the appearance-space of another modality. Hence, the registration between two different types of microscopy images can be transformed to a mono-modality image registration. We use a sparse representation model to obtain image analogies. The method makes use of corresponding image training patches of two different imaging modalities to learn a dictionary capturing appearance relations. We test our approach on backscattered electron (BSE) scanning electron microscopy (SEM)/confocal and transmission electron microscopy (TEM)/confocal images. We perform rigid, affine, and deformable registration via B-splines and show improvements over direct registration using both mutual information and sum of squared differences similarity measures to account for differences in image appearance.

Robust multimodal dictionary learning.

We propose a robust multimodal dictionary learning method for multimodal images. Joint dictionary learning for both modalities may be impaired by lack of correspondence between image modalities in training data, for example due to areas of low quality in one of the modalities. Dictionaries learned with such non-corresponding data will induce uncertainty about image representation. In this paper, we propose a probabilistic model that accounts for image areas that are poorly corresponding between the image modalities. We cast the problem of learning a dictionary in presence of problematic image patches as a likelihood maximization problem and solve it with a variant of the EM algorithm. Our algorithm iterates identification of poorly corresponding patches and refinements of the dictionary. We tested our method on synthetic and real data. We show improvements in image prediction quality and alignment accuracy when using the method for multimodal image registration.

Atomic force microscopy: a tool for studying biophysical surface properties underpinning fungal interactions with plants and substrates.

One of the primary roles of the cell surface is to provide an effective barrier to various external environmental factors. Specifically, the surface properties of organisms serve as a critical obstacle to pathogen attack. Since its inception, Atomic Force Microscopy (AFM) has enabled nanoscale imaging of cell surfaces in their native state. However AFM has yet to be systematically applied toward resolving surface features and the forces underpinning plant-fungal interactions. In an effort to understand the physical forces involved at the plant-microbe interface, we describe a method for the attachment of fungal spores to AFM tips and the subsequent measurement of unbinding forces between spores with a range of substrates and plant surfaces under physiologically relevant conditions. Investigations of binding events using AFM offer an unexplored, sensitive, and quantitative method for analyzing host-pathogen/microbe-surface interactions.

A cryohistological protocol for preparation of large plant tissue sections for screening intracellular fluorescent protein expression.

In this study, we have developed a robust cryohistological method that allows imaging of virtually any type of plant cell or tissue while preserving fluorescent protein signals and maintaining excellent cellular and subcellular morphology. This method involves modified fixation of plant tissues (i.e., leaves, stems, and petioles), infiltration in a sucrose gradient, freezing, and collection of cryosections directly onto a cryoadhesive tape. Using this method followed by microscopic analysis, we demonstrated a localized accumulation of green fluorescent protein (GFP) in Nicotiana benthamiana plants agroinfiltrated with the movement-incompetent tobacco mosaic virus-based vector and systemic accumulation of GFP in plants infiltrated with the movement-competent vector. Overall, this simple cryohistological procedure reduced sample preparation time and allowed processing of tissue sections for high-resolution imaging of targeted fluorescent proteins in all plant tissues.