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Myocilin, a modular multidomain protein, is expressed broadly in the human body but is best known for its presence in the trabecular meshwork extracellular matrix, and myocilin misfolding is associated with glaucoma. Despite progress in comprehending the structure and misfolding of the myocilin olfactomedin domain, the structure and function of full-length myocilin, and contextual changes in glaucoma, remain unknown. Here we expressed and purified milligram-scale quantities of full-length myocilin from suspension mammalian cell culture (Expi293F), enabling molecular characterization in detail not previously accessible. We systematically characterized disulfide-dependent and -independent oligomerization as well as confirmed glycosylation and susceptibility to proteolysis. We identified oligomeric states with glycosylation sites that are inaccessible to enzymatic removal. Low-resolution single particle 2D class averaging from conventional transmission electron microscopy imaging confirms an extended arrangement of tetramers, truncated products consistent with dimers, and a higher-ordered state consistent with octamer. Taken together, our study reveals new myocilin misfolded states and layers of intrinsic heterogeneity, expands our knowledge of olfactomedin-family proteins and lays the foundation for a better molecular understanding of myocilin structure and its still enigmatic biological function., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Recently, two cases of complete remission of classical Hodgkin lymphoma (cHL) and follicular lymphoma (FL) after SARS-CoV-2 infection were reported. However, the precise molecular mechanism of this rare event is yet to be understood. Here, we hypothesize a potential anti-tumor immune response of SARS-CoV-2 and based on a computational approach show that: (i) SARS-CoV-2 Spike-RBD may bind to the extracellular domains of CD15, CD27, CD45, and CD152 receptors of cHL or FL and may directly inhibit cell proliferation. (ii) Alternately, upon internalization after binding to these CD molecules, the SARS-CoV-2 membrane (M) protein and ORF3a may bind to gamma-tubulin complex component 3 (GCP3) at its tubulin gamma-1 chain (TUBG1) binding site. (iii) The M protein may also interact with TUBG1, blocking its binding to GCP3. (iv) Both the M and ORF3a proteins may render the GCP2-GCP3 lateral binding where the M protein possibly interacts with GCP2 at its GCP3 binding site and the ORF3a protein to GCP3 at its GCP2 interacting residues. (v) Interactions of the M and ORF3a proteins with these gamma-tubulin ring complex components potentially block the initial process of microtubule nucleation, leading to cell-cycle arrest and apoptosis. (vi) The Spike-RBD may also interact with and block PD-1 signaling similar to pembrolizumab and nivolumab- like monoclonal antibodies and may induce B-cell apoptosis and remission. (vii) Finally, the TRADD interacting "PVQLSY" motif of Epstein-Barr virus LMP-1, that is responsible for NF-kB mediated oncogenesis, potentially interacts with SARS-CoV-2 M pro , NSP7, NSP10, and spike (S) proteins, and may inhibit the LMP-1 mediated cell proliferation. Taken together, our results suggest a possible therapeutic potential of SARS-CoV-2 in lymphoproliferative disorders.

Ebola virus (EBOV) glycoprotein (GP) can be recognized by neutralizing antibodies (NAbs) and is the main target for vaccine design. Here, we first investigate the contribution of the stalk and heptad repeat 1-C (HR1 C ) regions to GP metastability. Specific stalk and HR1 C modifications in a mucin-deleted form (GPΔmuc) increase trimer yield, whereas alterations of HR1 C exert a more complex effect on thermostability. Crystal structures are determined to validate two rationally designed GPΔmuc trimers in their unliganded state. We then display a modified GPΔmuc trimer on reengineered protein nanoparticles that encapsulate a layer of locking domains (LD) and a cluster of helper T-cell epitopes. In mice and rabbits, GP trimers and nanoparticles elicit cross-ebolavirus NAbs, as well as non-NAbs that enhance pseudovirus infection. Repertoire sequencing reveals quantitative profiles of vaccine-induced B-cell responses. This study demonstrates a promising vaccine strategy for filoviruses, such as EBOV, based on GP stabilization and nanoparticle display.

Royal jelly (RJ) is produced by honeybees (Apis mellifera) as nutrition during larval development. The high viscosity of RJ originates from high concentrations of long lipoprotein filaments that include the glycosylated major royal jelly protein 1 (MRJP1), the small protein apisimin and insect lipids. Using cryo-electron microscopy we reveal the architecture and the composition of RJ filaments, in which the MRJP1 forms the outer shell of the assembly, surrounding stacked apisimin tetramers harbouring tightly packed lipids in the centre. The structural data rationalize the pH-dependent disassembly of RJ filaments in the gut of the larvae.

Hantaviruses are rodent-borne viruses causing serious zoonotic outbreaks worldwide for which no treatment is available. Hantavirus particles are pleomorphic and display a characteristic square surface lattice. The envelope glycoproteins Gn and Gc form heterodimers that further assemble into tetrameric spikes, the lattice building blocks. The glycoproteins, which are the sole targets of neutralizing antibodies, drive virus entry via receptor-mediated endocytosis and endosomal membrane fusion. Here we describe the high-resolution X-ray structures of the heterodimer of Gc and the Gn head and of the homotetrameric Gn base. Docking them into an 11.4-Å-resolution cryoelectron tomography map of the hantavirus surface accounted for the complete extramembrane portion of the viral glycoprotein shell and allowed a detailed description of the surface organization of these pleomorphic virions. Our results, which further revealed a built-in mechanism controlling Gc membrane insertion for fusion, pave the way for immunogen design to protect against pathogenic hantaviruses., Competing Interests: Declaration of Interests E.A.B., N.D.T., F.A.R., and P.G.-C. are coinventors of a pending patent application for development of stabilized immunogens for an Andes virus vaccine. K.C. is a member of the scientific advisory board of Integrum Scientific, LLC. F.A.R. is on the board of Virtexx, SAS., (Copyright © 2020 Elsevier Inc. All rights reserved.)

An elevated concentration of fibrinogen in blood is a significant risk factor during many pathological diseases, as it leads to an increase in red blood cells (RBC) aggregation, resulting in hemorheological disorders. Despite the biomedical importance, the mechanisms of fibrinogen-induced RBC aggregation are still debatable. One of the discussed models is the non-specific adsorption of fibrinogen macromolecules onto the RBC membrane, leading to the cells bridging in aggregates. However, recent works point to the specific character of the interaction between fibrinogen and the RBC membrane. Fibrinogen is the major physiological ligand of glycoproteins receptors IIbIIIa (GPIIbIIIa or αIIββ3 or CD41/CD61). Inhibitors of GPIIbIIIa are widely used in clinics for the treatment of various cardiovascular diseases as antiplatelets agents preventing the platelets' aggregation. However, the effects of GPIIbIIIa inhibition on RBC aggregation are not sufficiently well studied. The objective of the present work was the complex multimodal in vitro study of the interaction between fibrinogen and the RBC membrane, revealing the role of GPIIbIIIa in the specificity of binding of fibrinogen by the RBC membrane and its involvement in the cells' aggregation process. We demonstrate that GPIIbIIIa inhibition leads to a significant decrease in the adsorption of fibrinogen macromolecules onto the membrane, resulting in the reduction of RBC aggregation. We show that the mechanisms underlying these effects are governed by a decrease in the bridging components of RBC aggregation forces.

The envelope glycoprotein I (gI) of herpes simplex virus 1 (HSV-1) is a critical mediator of virus-induced cell-to-cell spread and cell-cell fusion. Here, we report a previously unrecognized property of this molecule. In transfected cells, the HSV-1 gI was discovered to induce rod-shaped structures that were uniform in width but variable in length. Moreover, the gI within these structures was conformationally different from the typical form of gI, as a previously used monoclonal antibody mAb3104 and a newly made peptide antibody to the gI extracellular domain (ECD) (amino acids [aa] 110 to 202) both failed to stain the long rod-shaped structures, suggesting the formation of a higher-order form. Consistent with this observation, we found that gI could self-interact and that the rod-shaped structures failed to recognize glycoprotein E, the well-known binding partner of gI. Further analyses by deletion mutagenesis and construction of chimeric mutants between gI and gD revealed that the gI ECD is the critical determinant, whereas the transmembrane domain served merely as an anchor. The critical amino acids were subsequently mapped to proline residues 184 and 188 within a conserved PXXXP motif. Reverse genetics analyses showed that the ability to induce a rod-shaped structure was not required for viral replication and spread in cell culture but rather correlated positively with the capability of the virus to induce cell fusion in the UL24syn background. Together, this work discovered a novel feature of HSV-1 gI that may have important implications in understanding gI function in viral spread and pathogenesis. IMPORTANCE The HSV-1 gI is required for viral cell-to-cell spread within the host, but the molecular mechanisms of how gI exactly works have remained poorly understood. Here, we report a novel property of this molecule, namely, induction of rod-shaped structures, which appeared to represent a higher-order form of gI. We further mapped the critical residues and showed that the ability of gI to induce rod-shaped structures correlated well with the capability of HSV-1 to induce cell fusion in the UL24syn background, suggesting that the two events may have an intrinsic link. Our results shed light on the biological properties of HSV-1 gI and may have important implications in understanding viral pathogenesis., (Copyright © 2020 American Society for Microbiology.)

Porcine coronavirus SADS-CoV has been identified from suckling piglets with severe diarrhea in southern China in 2017. The SADS-CoV genome shares ~95% identity to that of bat α-coronavirus HKU2, suggesting that SADS-CoV may have emerged from a natural reservoir in bats. Here we report the cryo-EM structures of HKU2 and SADS-CoV spike (S) glycoprotein trimers at 2.38 Å and 2.83 Å resolution, respectively. We systematically compare the domains of HKU2 spike with those of α-, β-, γ-, and δ-coronavirus spikes, showing that the S1 subunit N- and C-terminal domains of HKU2/SADS-CoV are ancestral domains in the evolution of coronavirus spike proteins. The connecting region after the fusion peptide in the S2 subunit of HKU2/SADS-CoV adopts a unique conformation. These results structurally demonstrate a close evolutionary relationship between HKU2/SADS-CoV and β-coronavirus spikes and provide insights into the evolution and cross-species transmission of coronaviruses.

Severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS) coronaviruses (CoVs) are zoonotic pathogens with high fatality rates and pandemic potential. Vaccine development focuses on the principal target of the neutralizing humoral immune response, the spike (S) glycoprotein. Coronavirus S proteins are extensively glycosylated, encoding around 66-87 N-linked glycosylation sites per trimeric spike. Here, we reveal a specific area of high glycan density on MERS S that results in the formation of oligomannose-type glycan clusters, which were absent on SARS and HKU1 CoVs. We provide a comparison of the global glycan density of coronavirus spikes with other viral proteins including HIV-1 envelope, Lassa virus glycoprotein complex, and influenza hemagglutinin, where glycosylation plays a known role in shielding immunogenic epitopes. Overall, our data reveal how organisation of glycosylation across class I viral fusion proteins influence not only individual glycan compositions but also the immunological pressure across the protein surface.

Oviduct-specific glycoprotein (OVGP1) is a high molecular weight chitinase-like protein belonging to GH18 family. It is secreted by non-ciliated epithelial cells of oviduct during estrous cycle providing an essential milieu for fertilization and embryo development. The present study reports the characterization of buffalo OVGP1 through structural modeling, carbohydrate-binding properties and evolutionary analysis. Structural model displayed the typical fold of GH18 family members till the boundary of chitinase-like domain further consisting of a large (β/α)8 TIM barrel sub-domain and a small (α+β) sub-domain. Two critical catalytic residues were found substituted in the catalytic centre (Asp to Phe118, Glu to Leu120) compared with the active chitinase. The carbohydrate-binding groove in TIM barrel was lined with various conserved aromatic residues. Molecular docking with different sugars revealed the involvement of various residues in hydrogen-bonding and non-bonded contacts. Most of the substrate-binding residues were conserved except for a few replacements (Ser13, Lys48, Asp49, Pro50, Asp167, Glu199, Gln272 and Phe275) in comparison with other GH18 members. The residues Trp10, Trp79, Asn80, Gln272, Phe275 and Trp334 were involved in recognition of all six ligands. The α+β sub-domain participated in sugar-binding through Thr270, Gln272, Tyr242 and Phe275. The binding assays revealed significant sugar-binding with purified native and recombinant OVGP1. Phylogenetic analysis revealed that OVGP1 was closely related to AMCases followed by other CLPs and evolution of OVGP1 occurred through several gene duplications. This is the first study describing the structural characteristics of OVGP1 that will further help to understand its interaction with gametes to perform crucial reproductive functions., (© 2019 The Author(s).)

The processes of cell attachment and membrane fusion of Herpes Simplex Virus 1 involve many different envelope glycoproteins. Viral proteins gC and gD bind to cellular receptors. Upon binding, gD activates the gH/gL complex which in turn activates gB to trigger membrane fusion. Thus, these proteins must be located at the point of contact between cellular and viral envelopes to interact and allow fusion. Using super-resolution microscopy, we show that gB, gH/gL and most of gC are distributed evenly round purified virions. In contrast, gD localizes essentially as clusters which are distinct from gB and gH/gL. Upon cell binding, we observe that all glycoproteins, including gD, have a similar ring-like pattern, but the diameter of these rings was significantly smaller than those observed on cell-free viruses. We also observe that contrary to cell-free particles, gD mostly colocalizes with other glycoproteins on cell-bound particles. The differing patterns of localization of gD between cell-free and cell-bound viruses indicates that gD can be reorganized on the viral envelope following either a possible maturation of the viral particle or its adsorption to the cell. This redistribution of glycoproteins upon cell attachment could contribute to initiate the cascade of activations leading to membrane fusion., Competing Interests: The authors have declared that no competing interests exist.

Asymmetrically branched precision glycooligomers are synthesized by solid-phase polymer synthesis for studying multivalent carbohydrate-protein interactions. Through the stepwise assembly of Fmoc-protected oligo(amidoamine) building blocks and Fmoc/Dde-protected lysine, straightforward variation of structural parameters such as the number and length of arms, as well as the number and position of carbohydrate ligands, is achieved. Binding of 1-arm and 3-arm glycooligomers toward lectin receptors langerin and concanavalin A (ConA) was evaluated where the smallest 3-arm glycooligomer shows the highest binding toward langerin, and stepwise elongation of one, two, or all three arms leads to decreased binding. When directly comparing binding toward langerin and ConA, we find that structural variation of the scaffold affects glycomimetic ligand binding differently for the different targets, indicating the potential to tune such ligands not only for their avidity but also for their selectivity toward different lectins.

Proteases are one of attractive therapeutic targets to play key roles in pharmacological action. There are many protease inhibitors in nature, and most of them structurally have cystine knot motifs. Their structures are favorable for recognition of active pockets of proteases, leading to the potent inhibition. However, they also have drawbacks, such as broad cross-reactivity, on the therapeutic application. To create therapeutic proteins derived from a disulfide-rich scaffold, we selected human serine protease inhibitor Kazal type 2 (SPINK2) through a scaffold screening, as a protein scaffold with requirements for therapeutic proteins. We then constructed a diverse library of the engineered SPINK2 by introducing random mutations into its flexible loop region with the designed method. By phage panning against four serine proteases, we isolated potent inhibitors against each target with picomolar K D and sub-nanomolar K i values. Also, they exhibited the desired specificities against target proteases without inhibiting non-target proteases. The crystal structure of kallikrein related peptidase 4 (KLK4)-engineered SPINK2 complex revealed the interface with extensive conformational complementarity. Our study demonstrates that engineered SPINK2 can serve as a scaffold to generate therapeutic molecules against target proteins with groove structures.

Innovative processing technologies, such as ultrasonication, can change the properties of milk, allowing for the improvement or development of dairy foods. Yet taking bench-scale equipment to pilot plant scale has been challenging. Raw milk, standardized to 3% fat and warmed to inlet temperatures of 42 or 54°C, was exposed to continuous, high-intensity, low-frequency ultrasonication (16/20 kHz, 1.36 kW/pass) at flow rates of 0.15, 0.30, and 0.45 L/min that resulted in resident times within the reaction cell of 6, 3, and 2 min per pass, respectively. Multiple passes (3, 5, and 7, respectively) were required to obtain a total exposure time of 14 to 18 min. Evaluation of fat droplet sizes, enzyme coagulation properties, and microstructure of milk and milk gels, as well as determining compositional and lipid properties, were conducted to determine the potential of the ultrasound system to effectively modify milk. Laser scanning particle sizing and confocal microscopy showed that the largest droplets (2.26 ± 0.13 µm) found in raw milk were selectively reduced in size with a concomitant increase in the number of submicron droplets (0.37 ± 0.06 µm), which occurred sooner when exposed to shorter bursts of ultrasonication (0.45 L/min flow rates) and at an inlet temperature of 54°C. Ultrasound processing with milk entering at 42°C resulted in faster gelling times and firmer curds at 30 min; however, extended processing at inlet temperature of 54°C reduced curd firmness and lengthened coagulation time. This showed that ultrasonication altered protein-protein and protein-lipid interactions, thus the strength of the enzyme-set curds. Scanning electron microscopy revealed a denser curd matrix with less continuous and more irregular shaped and clustered strands, whereas transmission electron microscopy showed submicron lipid droplets embedded within the protein strands of the curd matrix. Processing at inlet temperature of 54°C with flow rates of 0.30 and 0.45 L/min also reduced the total aerobic bacterial count by more than 1 log cfu/mL, and the number of psychrophiles below the limit of detection (10 cfu/mL) for this study. Ultrasonication exposures of 14 to 18 min had minimal effect on the milk composition, fatty acid profiles, and lipid heat capacity and enthalpy. The findings show that this continuous ultrasound system, which is conducive to commercial scale-up, modifies the physical and functional properties of milk under the parameters used in this study and has potential use in dairy processing., (Copyright © 2019 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

The subcommissural organ (SCO) is an ancient and conserved brain gland secreting into cerebrospinal fluid (CSF) glycoproteins that form the Reissner fiber (RF). The present investigation was designed to further investigate the dynamic of the biosynthetic process of RF glycoproteins prior and after their release into the CSF, to identify the RF proteome and N-glycome and to clarify the mechanism of assembly of RF glycoproteins. Various methodological approaches were used: biosynthetic labelling injecting 35 S-cysteine and 3 H-galactose into the CSF, injection of antibodies against galectin-1 into the cerebrospinal fluid, light and electron microscopical methods; isolated bovine RF was used for proteome analyses by mass spectrometry and glycome analysis by xCGE-LIF. The biosynthetic labelling study further supported that a small pool of SCO-spondin molecules rapidly enter the secretory pathways after its synthesis, while most of the SCO-spondin molecules are stored in the rough endoplasmic reticulum for hours or days before entering the secretory pathway and being released to assemble into RF. The proteomic analysis of RF revealed clusterin and galectin-1 as partners of SCO-spondin; the in vivo use of anti-galectin-1 showed that this lectin is essential for the assembly of RF. Galectin-1 is not secreted by the SCO but evidence was obtained that it would be secreted by multiciliated ependymal cells lying close to the SCO. Further, a surprising variety and complexity of glycan structures were identified in the RF N-glycome that further expands the potential functions of RF to a level not previously envisaged. A model of the macromolecular organization of Reissner fiber is proposed.

Feeding livestock with n-3 fatty acid (FA) sources (linseed, for example) is a common strategy to improve lipid quality of meat and milk products. However, in monogastric animals, linseed tegument decreases digestibility and alphalinolenic acid (ALA) uptake, while the whole linseed is well used by ruminants. In a context of increasing sustainability of feeding systems, providing monogastric animals and ruminants with linseed products adapted to their digestive systems is an important issue. This research paper addresses the hypotheses: (i) sieved extruded linseed (SEL) specific for ruminants is as or more effective than standard extruded linseed (ii) microalgae DHA Gold® is an interesting source of docosahexaenoic acid (DHA) in feedstuff and (iii) the effects of SEL and microalgae on milk characteristics are complementary and additive. Thirty-two cows were divided into 4 groups with different dietary n-3 fatty acid sources using a continuous design. All the diets were fed as mixed rations based on maize silage, energy concentrate and soybean meal. The first group received a control diet (CTRL) with no additional fat. The 3 other groups received SEL, microalgae DHA Gold® (ALG) and a mixture of microalgae DHA Gold® and SEL (SEL/ALG). Milk was collected from morning milkings after six weeks of dietary treatment. In SEL and SEL/ALG, ALA increased (+0·32 and +0·26% unit, respectively), and DHA increased in ALG and SEL/ALG (+0·43 and +0·15% unit, respectively) compared to CTRL, as a consequence of the initial composition of the n-3 FA sources. In SEL, milk yield, fat and protein contents, milk fat globule size and spontaneous lipolysis (measured to evaluate suitability for milk processing) were not different compared with CTRL. In ALG and SEL/ALG, milk yield decreased (-2·8 and -6·0 kg/d, respectively), fat content was halved, and fat globule size was reduced (-1·46 and -1·31 µm, respectively) compared to CTRL. Spontaneous lipolysis increased in ALG (+0·12 mEq/kg of milk) compared to CTRL. Protected microalgae and the doses of microalgae in the diet need further investigation to prevent FA modification in the rumen and the consequent deleterious effects on milk fat.

Yak milk fat products constitute the base of Qinghai-Tibetan pastoralists' daily food intake. Despite the great importance of fat in processing and pastoralists' health, studies about yak milk fat are scarce. In this study, the lipid composition and the morphological properties of milk fat globule membranes (MFGMs) of yak milk were investigated. The results demonstrated that the yak milk had a higher cholesterol and sphingomyelin content compared to cow milk. In situ structural investigations performed at 25 °C by confocal microscopy showed the presence of lipid domains in yak MFGM, with a larger number and wider size range compared to cow milk. Moreover, the simultaneous localization of glycosylated molecules and polar lipids indicated that glycosylated molecules could be integrated into the lipid domains in yak MFGM. Different characteristics in yak MFGM could be related to the lipid composition and may affect the functions of yak milk lipids during processing and digestion., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Herpes simplex viruses (HSVs) rely on capsid-associated tegument complex (CATC) for long-range axonal transport of their genome-containing capsids between sites of infection and neuronal cell bodies. Here we report cryo-electron microscopy structures of the HSV-1 capsid with CATC up to 3.5-angstrom resolution and atomic models of multiple conformers of capsid proteins VP5, VP19c, VP23, and VP26 and tegument proteins pUL17, pUL25, and pUL36. Crowning every capsid vertex are five copies of heteropentameric CATC, each containing a pUL17 monomer supporting the coiled-coil helix bundle of a pUL25 dimer and a pUL36 dimer, thus positioning their flexible domains for potential involvement in nuclear capsid egress and axonal capsid transport. Notwithstanding newly discovered fold conservation between triplex proteins and bacteriophage λ protein gpD and the previously recognized bacteriophage HK97 gp5-like fold in VP5, HSV-1 capsid proteins exhibit extraordinary diversity in forms of domain insertion and conformational polymorphism, not only for interactions with tegument proteins but also for encapsulation of large genomes., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Niemann-Pick Protein C2 (npc2) is a small soluble protein critical for cholesterol transport within and from the lysosome and the late endosome. Intriguingly, npc2-mediated cholesterol transport has been shown to be modulated by lipids, yet the molecular mechanism of npc2-membrane interactions has remained elusive. Here, based on an extensive set of atomistic simulations and free energy calculations, we clarify the mechanism and energetics of npc2-membrane binding and characterize the roles of physiologically relevant key lipids associated with the binding process. Our results capture in atomistic detail two competitively favorable membrane binding orientations of npc2 with a low interconversion barrier. The first binding mode (Prone) places the cholesterol binding pocket in direct contact with the membrane and is characterized by membrane insertion of a loop (V59-M60-G61-I62-P63-V64-P65). This mode is associated with cholesterol uptake and release. On the other hand, the second mode (Supine) places the cholesterol binding pocket away from the membrane surface, but has overall higher membrane binding affinity. We determined that bis(monoacylglycero)phosphate (bmp) is specifically required for strong membrane binding in Prone mode, and that it cannot be substituted by other anionic lipids. Meanwhile, sphingomyelin counteracts bmp by hindering Prone mode without affecting Supine mode. Our results provide concrete evidence that lipids modulate npc2-mediated cholesterol transport either by favoring or disfavoring Prone mode and that they impose this by modulating the accessibility of bmp for interacting with npc2. Overall, we provide a mechanism by which npc2-mediated cholesterol transport is controlled by the membrane composition and how npc2-lipid interactions can regulate the transport rate.

Recently, aqueous nanoparticles have been used in drug-delivery systems for new type medicines. In particular, milk-casein micelles have been used as drug nanocarriers for targeting cancer cells. Therefore, nanostructure observation of particles and micelles in their native liquid condition is indispensable for analysing their function and mechanisms. However, traditional optical and scanning electron microscopy have difficulty observing the nanostructures of aqueous micelles. Recently, we developed a novel imaging technique called scanning electron-assisted dielectric microscopy (SE-ADM) that enables observation of various biological specimens in water with very little radiation damage and high-contrast imaging without staining or fixation at an 8-nm spatial resolution. In this study, for the first time, we show that the SE-ADM system is capable of high-resolution observation of whole-milk specimens in their natural state. Moreover, we successfully observe the casein micelles and milk-fat globules in an intact liquid condition. Our SE-ADM system can be applied to various biological particles and micelles in a native liquid state., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

The envelope spike (S) proteins of MERS-CoV and SARS-CoV determine the virus host tropism and entry into host cells, and constitute a promising target for the development of prophylactics and therapeutics. Here, we present high-resolution structures of the trimeric MERS-CoV and SARS-CoV S proteins in its pre-fusion conformation by single particle cryo-electron microscopy. The overall structures resemble that from other coronaviruses including HKU1, MHV and NL63 reported recently, with the exception of the receptor binding domain (RBD). We captured two states of the RBD with receptor binding region either buried (lying state) or exposed (standing state), demonstrating an inherently flexible RBD readily recognized by the receptor. Further sequence conservation analysis of six human-infecting coronaviruses revealed that the fusion peptide, HR1 region and the central helix are potential targets for eliciting broadly neutralizing antibodies.

This paper reports a detailed ultrastructural and immunocytochemical investigation of the structure of the milk fat globule membrane (MFGM) in a variety of species. The process follows the same pattern in all mammals so far investigated. The initial (or primary) MFGM immediately on release from the mammary cell is a continuous unit membrane with a thin underlying layer of cytoplasmic origin and a monolayer of phospholipid separating it from the core lipid. This structure changes rapidly as the milk fat globule (MFG) moves into the alveolar lumen. The unit membrane plus the underlying layer of cytoplasm modifies drastically into discontinuous patches and networks. These are superimposed upon a continuous apparently structureless sheet of electron dense material stabilising the MFG and similar to that which bounded the lipid in the cell. The underlying layer of the patches increases in electron density and immunocytochemistry demonstrates localisation of MFGM proteins in this layer. In four species, the dense material shows ordered paracrystalline molecular arrays in section and en face views. All the arrays show the same basic pattern and unit size as determined by optical diffraction. Similar patches, networks and arrays are present on the surface of expressed MFG. Negative staining of lipid-extracted expressed MFGs shows similar patches and networks of membrane. These also occasionally show the crystalline arrays and label with MFGM protein antibodies. Similar networks and strands of plasma membrane on the MFG surface are shown by our CLSM examination of unfixed expressed MFG from mice genetically modified to express a fluorescent molecule as a normal plasma membrane constituent.

The polar lipid assembly and biophysical properties of the biological membrane enveloping the milk fat globules (the MFGM) are yet poorly known, especially in connection with the temperature history that milk can experience after its secretion. However, bioactive mechanisms depend on biological structure, which itself highly depend on temperature. The objectives of this study were to investigate polar lipid packing in hydrated bilayers, models of the MFGM, and to follow at intermolecular level temperature-induced changes in the range 60-6°C, using the combination of differential scanning calorimetry, X-ray diffraction, atomic force microscopy (AFM) imaging and force spectroscopy. MFGM polar lipids, especially sphingomyelin, contain long chain saturated fatty acids with high phase transition temperatures. On cooling, the liquid disordered ld to solid ordered so (gel) phase transition of MFGM polar lipids started at about 40°C, leading to phase separation and formation of so phase domains protruding by about 1nm from the ld phase. Indentation measurements using AFM revealed that the resistance of the so phase domains to rupture was significantly higher than that of the ld phase and that it increased for both the domain and fluid phases with decreasing temperature. However, packing and stability of the bilayers were adversely affected by fast cooling to 6°C or by cooling-rewarming cycle. This study showed that MFGM polar lipid bilayers are dynamic systems. Heterogeneity in the structure and mechanical properties of the membrane was induced by temperature-dependent so/ld phase immiscibility of the lipid components. This could have consequences on the MFGM technological and biological functions (e.g. immunity and milk lipid digestion)., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Foamy viruses (FV) belong to the genus Spumavirus, which forms a distinct lineage in the Retroviridae family. Although the infection in natural hosts and zoonotic transmission to humans is asymptomatic, FVs can replicate well in human cells making it an attractive gene therapy vector candidate. Here we present cryo-electron microscopy and (cryo-)electron tomography ultrastructural data on purified prototype FV (PFV) and PFV infected cells. Mature PFV particles have a distinct morphology with a capsid of constant dimension as well as a less ordered shell of density between the capsid and the membrane likely formed by the Gag N-terminal domain and the cytoplasmic part of the Env leader peptide gp18LP. The viral membrane contains trimeric Env glycoproteins partly arranged in interlocked hexagonal assemblies. In situ 3D reconstruction by subtomogram averaging of wild type Env and of a Env gp48TM- gp80SU cleavage site mutant showed a similar spike architecture as well as stabilization of the hexagonal lattice by clear connections between lower densities of neighboring trimers. Cryo-EM was employed to obtain a 9 Å resolution map of the glycoprotein in its pre-fusion state, which revealed extensive trimer interactions by the receptor binding subunit gp80SU at the top of the spike and three central helices derived from the fusion protein subunit gp48TM. The lower part of Env, presumably composed of interlaced parts of gp48TM, gp80SU and gp18LP anchors the spike at the membrane. We propose that the gp48TM density continues into three central transmembrane helices, which interact with three outer transmembrane helices derived from gp18LP. Our ultrastructural data and 9 Å resolution glycoprotein structure provide important new insights into the molecular architecture of PFV and its distinct evolutionary relationship with other members of the Retroviridae.

Previous efforts to identify cross-neutralizing antibodies to the receptor-binding site (RBS) of ebolavirus glycoproteins have been unsuccessful, largely because the RBS is occluded on the viral surface. We report a monoclonal antibody (FVM04) that targets a uniquely exposed epitope within the RBS; cross-neutralizes Ebola (EBOV), Sudan (SUDV), and, to a lesser extent, Bundibugyo viruses; and shows protection against EBOV and SUDV in mice and guinea pigs. The antibody cocktail ZMapp™ is remarkably effective against EBOV (Zaire) but does not cross-neutralize other ebolaviruses. By replacing one of the ZMapp™ components with FVM04, we retained the anti-EBOV efficacy while extending the breadth of protection to SUDV, thereby generating a cross-protective antibody cocktail. In addition, we report several mutations at the base of the ebolavirus glycoprotein that enhance the binding of FVM04 and other cross-reactive antibodies. These findings have important implications for pan-ebolavirus vaccine development and defining broadly protective antibody cocktails., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Hantaviruses, a geographically diverse group of zoonotic pathogens, initiate cell infection through the concerted action of Gn and Gc viral surface glycoproteins. Here, we describe the high-resolution crystal structure of the antigenic ectodomain of Gn from Puumala hantavirus (PUUV), a causative agent of hemorrhagic fever with renal syndrome. Fitting of PUUV Gn into an electron cryomicroscopy reconstruction of intact Gn-Gc spike complexes from the closely related but non-pathogenic Tula hantavirus localized Gn tetramers to the membrane-distal surface of the virion. The accuracy of the fitting was corroborated by epitope mapping and genetic analysis of available PUUV sequences. Interestingly, Gn exhibits greater non-synonymous sequence diversity than the less accessible Gc, supporting a role of the host humoral immune response in exerting selective pressure on the virus surface. The fold of PUUV Gn is likely to be widely conserved across hantaviruses., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

The microstructures of colostrum and mature bovine milk fat globule membrane (MFGM) were investigated using confocal laser scanning microscopy (CLSM) at different temperatures, and the relationships between microstructure variations and the chemical compositions of the MFGM were also examined. Using a fluorophore-labeled phospholipid probe, we found that non-fluorescent domains on the MFGM were positively correlated with the amount of sphingomyelin at both room (20 °C) and physiological (37 °C) temperatures. However, at the storage temperature (4 °C), there were more non-fluorescent domains on the MFGM. These results indicate that the heterogeneities in the MFGM are most likely to be the result of the lateral segregation of sphingomyelin at the room and physiological temperatures, and at the storage temperature, phospholipids with saturated fatty acids affect the formation of these domains., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Allergens are foreign proteins or glycoproteins that are the target of IgE antibody responses in humans. The relationship between subsequent exposure and the allergic symptoms is often or usually obvious; however, there is increasing evidence that in asthma, atopic dermatitis and some forms of food allergy the induction of symptoms is delayed or chronic. The primary exposure to inhaled allergens is to the particles, which are capable of carrying allergens in the air. Thus, the response reflects not only the properties of the proteins, but also the biological properties of the other constituents of the particle. This is best understood in relation to the mite fecal particles in which the contents include many different immunologically active substances. Allergic disease first became a major problem over 100 years ago, and for many years sensitization to pollens was the dominant form of these diseases. The rise in pediatric asthma correlates best with the move of children indoors, which started in 1960 and was primarily driven by indoor entertainment for children. While the causes of the increase are not simple they include both a major increase in sensitization to indoor allergens and the complex consequences of inactivity. Most recently, there has also been an increase in food allergy. Understanding this has required a reappraisal of the importance of the skin as a route for sensitization. Overall, understanding allergic diseases requires knowing about the sources, the particles and the routes of exposure as well as the properties of the individual allergens., (Copyright © 2015 Japanese Society of Allergology. Production and hosting by Elsevier B.V. All rights reserved.)

The biological membrane that surrounds the milk fat globules exhibits phase separation of polar lipids that is poorly known. The objective of this study was to investigate the role played by cholesterol in the organization of monolayers prepared as models of the milk fat globule membrane (MFGM). Differential scanning calorimetry and X-ray diffraction experiments allowed characterization of the gel to liquid crystalline phase transition temperature of lipids, Tm ~35°C, in vesicles prepared with a MFGM lipid extract. For temperature below Tm, atomic force microscopy revealed phase separation of lipids at 30 mN·m(-1) in Langmuir-Blodgett monolayers of the MFGM lipid extract. The high Tm lipids form liquid condensed (LC) domains that protrude by about 1.5 nm from the continuous liquid expanded (LE) phase. Cholesterol was added to the MFGM extract up to 30% of polar lipids (cholesterol/milk sphingomyelin (MSM) molar ratio of 50/50). Compression isotherms evidenced the condensing effect of the cholesterol onto the MFGM lipid monolayers. Topography of the monolayers showed a decrease in the area of the LC domains and in the height difference H between the LC domains and the continuous LE phase, as the cholesterol content increased in the MFGM lipid monolayers. These results were interpreted in terms of nucleation effects of cholesterol and decrease of the line tension between LC domains and LE phase in the MFGM lipid monolayers. This study revealed the major structural role of cholesterol in the MFGM that could be involved in biological functions of this interface (e.g. mechanisms of milk fat globule digestion)., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Prevention efforts for respiratory syncytial virus (RSV) have been advanced due to the recent isolation and characterization of antibodies that specifically recognize the prefusion conformation of the RSV fusion (F) glycoprotein. These potently neutralizing antibodies are in clinical development for passive prophylaxis and have also aided the design of vaccine antigens that display prefusion-specific epitopes. To date, prefusion-specific antibodies have been shown to target two antigenic sites on RSV F, but both of these sites are also present on monomeric forms of F. Here we present a structural and functional characterization of human antibody AM14, which potently neutralized laboratory strains and clinical isolates of RSV from both A and B subtypes. The crystal structure and location of escape mutations revealed that AM14 recognizes a quaternary epitope that spans two protomers and includes a region that undergoes extensive conformational changes in the pre- to postfusion F transition. Binding assays demonstrated that AM14 is unique in its specific recognition of trimeric furin-cleaved prefusion F, which is the mature form of F on infectious virions. These results demonstrate that the prefusion F trimer contains potent neutralizing epitopes not present on monomers and that AM14 should be particularly useful for characterizing the conformational state of RSV F-based vaccine antigens.

The inhibition of Polyphenol oxidase (PPO) in plants has been widely researched for their important roles in browning reaction. A newly found germin-like protein (GLP) with high PPO activity in Satsuma mandarine was inactivated by low-frequency high-intensity ultrasonic (20 kHz) processing. The effects of ultrasound on PPO activity and structure of GLP were investigated using dynamic light scattering (DLS) analysis, transmission electron microscopy (TEM), circular dichroism (CD) spectral measurement and fluorescence spectral measurement. The lowest PPO activity achieved was 27.4% following ultrasonication for 30 min at 400 W. DLS analysis showed ultrasound caused both aggregation and dissociation of GLP particles. TEM images also demonstrated protein aggregation phenomena. CD spectra exhibited a certain number of loss in α-helix structure content. Fluorescence spectra showed remarkable increase in fluorescence intensity with tiny blue-shift following ultrasonication. In conclusion, ultrasound applied in this study induced structural changes of GLP and eventually inactivated PPO activity., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Lysinibacillus sphaericus produces mosquitocidal binary toxins (Bin toxins) deposited within a balloon-like exosporium during sporulation. Unlike Bacillus cereus group strains, the exosporium of L. sphaericus is usually devoid of the hair-like nap, an external filamentous structure formed by a collagen-like protein, BclA. In this study, a new collagen-like exosporium protein encoded by Bsph_0411 (BclS) from L. sphaericus C3-41 was characterized. Thin-section electron microscopy revealed that deletion of bclS resulted in the loss of the filamentous structures that attach to the exosporium basal layer and spread through the interspace of spores. In vivo visualization of BclS-green fluorescent protein (GFP)/mCherry fusion proteins revealed a dynamic pattern of fluorescence that encased the spore from the mother cell-distal (MCD) pole of the forespore, and the BclS-GFP fusions were found to be located in the interspace of the spore, as confirmed by three-dimensional (3D) superresolution fluorescence microscopy. Further studies demonstrated that the bclS mutant spores were more sensitive to wet-heat treatment and germinated at a lower rate than wild-type spores and that these phenotypes were significantly restored in the bclS-complemented strain. These results suggested novel roles of collagen-like protein in exosporium assembly and spore germination, providing a hint for a further understanding of the genetic basis of the high level of persistence of Bin toxins in nature., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Bone morphogenetic proteins (BMPs) orchestrate key cellular events, such as proliferation and differentiation, in development and homeostasis. Extracellular antagonists, such as chordin, are essential regulators of BMP signaling. Chordin binds to BMPs blocking interaction with receptors, and cleavage by tolloid proteinases is thought to relieve this inhibition. A model has been previously proposed where chordin adopts a horseshoe-like arrangement enabling BMP binding cooperatively by terminal domains (1). Here, we present the nanoscale structure of human chordin using electron microscopy, small angle X-ray scattering, and solution-based biophysical techniques, which together show that chordin indeed has a compact horseshoe-shaped structure. Chordin variants were used to map domain locations within the chordin molecule. The terminal BMP-binding domains protrude as prongs from the main body of the chordin structure, where they are well positioned to interact with the growth factor. The spacing provided by the chordin domains supports the principle of a cooperative BMP-binding arrangement that the original model implied in which growth factors bind to both an N- and C-terminal von Willebrand factor C domain of chordin. Using binding and bioactivity assays, we compared full-length chordin with two truncated chordin variants, such as those produced by partial tolloid cleavage. Cleavage of either terminal domain has little effect on the affinity of chordin for BMP-4 and BMP-7 but C-terminal cleavage increases the efficacy of chordin as a BMP-4 inhibitor. Together these data suggest that partial tolloid cleavage is insufficient to ablate BMP inhibition and the C-terminal chordin domains play an important role in BMP regulation.

Corpora amylacea (CA) are glycoprotein-based depositions that accumulate in the normal aging brain or consecutively to different neuro-degenerative diseases. Although controversies still exists in what it regards their origins and functions, their morphology is described simply as round basophilic entities based on bi-dimensional observations. The aim of the present study was to evaluate the three-dimensional morphology of these bodies in the brain in normal aging. We utilized here brain tissue from six aged patients, and performed an in-depth stereological analysis of CAs based on series of thin serial sections, and 50 μm-thick sections. The thin seriate sections have been counter-stained with Hematoxylin and Eosin, while the thick sections have been immunostained in fluorescence for ubiquitin and GFAP/collagen IV or aquaporin 4. Three-dimensional renderings have been obtained after aligning the serial sections, while high-resolution z-stacks have resulted after deconvolution on the thick sections. More than 70% of all the identified CAs proved to be in fact parts of larger aggregates, where the flattened individual spheroids branched and communicated with other bodies in a complex pattern, and budding of small CAs from larger CAs could be observed. There was a direct correlation between the diameter of the vessels and the number of associated CAs. Astrocyte GFAP and aquaporin 4 signals surrounded CAs, but without any colocalization with the ubiquitin areas, while perivascular CAs were sometimes enclosed in pockets of the basement membranes. In conclusion, as far as we know, this is the first study to describe the three-dimensional branching complexity of corpora amylacea in the brain.

The glycoprotein (GP) molecular fraction structure of the gum exudate of Acacia senegal (gum Arabic) isolated from hydrophobic interaction chromatography was investigated using high-performance size exclusion chromatography-multi angle laser light scattering (HPSEC-MALLS), small angle X-ray scattering (SAXS), synchrotron radiation circular dichroism (SRCD) and transmission electron microscopy (TEM) observations. In solution, GP would be a mixture of spheroidal monomers and more anisotropic oligomers as suggested by the two exponent values found in the Rg vs. Mw relationship and TEM observations. The GP conformation probed by SAXS was ascribed to a thin object with a triaxial ellipsoid morphology, certainly attributed to GP oligomers. A 9 nm diameter particle was also identified by SAXS in agreement with the dimensions identified by TEM on single isolated ring-like structures. The GP oligomerization process, as probed by TEM, would be the result of ring-like subunits self-association. This self-association would lead to more linear or, sometimes, cyclised assembly. At the molecular level, GP fraction was found to have secondary structures mainly made of β-sheets and turns (64%) but also, to a lesser extent, made of polyproline II (PPII) and α-helices (19%). These features were characteristic of hydroxyprolin-rich glycoproteins with arabinosylated and arabinogalactan polysaccharide side chains grafted to the polypeptide backbone. The GP molecular fraction structure from Acacia gum would be an assembly of ring-like glycoproteins modules. These ring-like structures were certainly due to hydroxyproline (Hyp)-arabinogalactan (AG) subunits., (Crown Copyright © 2013. Published by Elsevier Ltd. All rights reserved.)

Closely related peptide epitopes can be recognized by the same T cells and contribute to the immune response against pathogens encoding those epitopes, but sometimes cross-reactive epitopes share little homology. The degree of structural homology required for such disparate ligands to be recognized by cross-reactive TCRs remains unclear. In this study, we examined the mechanistic basis for cross-reactive T cell responses between epitopes from unrelated and pathogenic viruses, lymphocytic choriomeningitis virus (LCMV) and vaccinia virus. Our results show that the LCMV cross-reactive T cell response toward vaccinia virus is dominated by a shared asparagine residue, together with other shared structural elements conserved in the crystal structures of K(b)-VV-A11R and K(b)-LCMV-gp34. Based on analysis of the crystal structures and the specificity determinants for the cross-reactive T cell response, we were able to manipulate the degree of cross-reactivity of the T cell response, and to predict and generate a LCMV cross-reactive response toward a variant of a null OVA-derived peptide. These results indicate that protective heterologous immune responses can occur for disparate epitopes from unrelated viruses.

The impact of washing on the release of proteins from the milk fat globule membrane (MFGM) was examined by applying washing procedures with different degrees of stringency to milk fat globule (MFG) surfaces in simulated milk ultrafiltrate buffer solution. Three washing methods, M1 (3000g, 5 min, three washes), M2 (3750g, 15 min, one wash), and M3 (15000g, 20 min, three washes) were chosen. MFG ζ-potential increased after M3 washing (P < 0.05), suggesting surface damage. For M1, in which the native MFG surface was least damaged, cluster of differentiation 36 (CD 36) and periodic acid schiff 6/7 proteins were more strongly bound to the MFGM compared with other major membrane proteins. For M3, CD 36 together with fatty acid-binding protein was more strongly bound to the MFGM. Washing by centrifugation and redispersal of the fat globules damaged the MFGM, with release into the aqueous phase of some membrane-associated proteins. The current results show the impact of washing processes on retention of functional MFGM proteins.

The first version of a glycoprotein hormone receptor (GPHR) information resource was designed to link functional with structural GPHR information, in order to support sequence-structure-function analysis of the LH, FSH, and TSH receptors (http://ssfa-gphr.de). However, structural information on a binding- and signaling-sensitive extracellular fragment (∼100 residues), the hinge region, had been lacking. A new FSHR crystal structure of the hormone-bound extracellular domain has recently been solved. The structure comprises the leucine-rich repeat domain and most parts of the hinge region. We have not only integrated the new FSHR/FSH structure and the derived homology models of TSHR/TSH, LHCGR/CG, and LHCGR/LH into our web-based information resource, but have additionally provided novel tools to analyze the advanced structural features, with the common characteristics and distinctions between GPHRs, in a more precise manner. The hinge region with its second hormone-binding site allows us to assign functional data to the new structural features between hormone and receptor, such as binding details of a sulfated tyrosine (conserved throughout the GPHRs) extending into a pocket of the hormone. We have also implemented a protein interface analysis tool that enables the identification and visualization of extracellular contact points between interaction partners. This provides a starting point for comparing the binding patterns of GPHRs. Together with the mutagenesis data stored in the database, this will help to decipher the essential residues for ligand recognition and the molecular mechanisms of signal transduction, extending from the extracellular hormone-binding site toward the intracellular G protein-binding sites.

Myocilin is a protein with a molecular weight near 50 kDa. It is expressed in almost all organs and tissues. We showed that the peptide DQLETQTRELETAYSNLLRD corresponding to N-terminal Leucine zipper motif (LZM) of the protein is able to form amyloid-like fibrils. The possible role of this motif in myocilin aggregation is discussed.

The genus Orthobunyavirus within the family Bunyaviridae constitutes an expanding group of emerging viruses, which threaten human and animal health. Despite the medical importance, little is known about orthobunyavirus structure, a prerequisite for understanding virus assembly and entry. Here, using electron cryo-tomography, we report the ultrastructure of Bunyamwera virus, the prototypic member of this genus. Whilst Bunyamwera virions are pleomorphic in shape, they display a locally ordered lattice of glycoprotein spikes. Each spike protrudes 18 nm from the viral membrane and becomes disordered upon introduction to an acidic environment. Using sub-tomogram averaging, we derived a three-dimensional model of the trimeric pre-fusion glycoprotein spike to 3-nm resolution. The glycoprotein spike consists mainly of the putative class-II fusion glycoprotein and exhibits a unique tripod-like arrangement. Protein-protein contacts between neighbouring spikes occur at membrane-proximal regions and intra-spike contacts at membrane-distal regions. This trimeric assembly deviates from previously observed fusion glycoprotein arrangements, suggesting a greater than anticipated repertoire of viral fusion glycoprotein oligomerization. Our study provides evidence of a pH-dependent conformational change that occurs during orthobunyaviral entry into host cells and a blueprint for the structure of this group of emerging pathogens.

The physicochemical properties of human milk fat globules (MFG) at different lactation stages from Danish mothers and the microstructure changes of MFG membrane (MFGM) at varied temperatures were investigated, and the relationship between chemical composition and the microstructure of MFGM was elucidated. The fat content in MFG was found to be significantly increased as lactation progressed, and colostrum MFG had the largest mean diameter of 5.75 ± 0.81 μm and the lowest ζ potential of -5.60 ± 0.12 mV. Chemical composition analyses of MFG revealed the following: (i) Colostrum milk fat constituted higher content in PUFAs (ω-6, and long-chain ω-6 and ω-3) than transitional and mature milk fats, with the corresponding lower content of SFA in its sn-2 position. (ii) The content of polar lipids among total lipids varied during lactation course (maximized at transitional stage); however, in terms of subclasses of polar lipids, no significant change of the relative content of sphingomyelin was observed, while the content of phosphatidycholine in mature milk was higher than that in colostrum and transitional milk. (iii) Inspection of fatty acid composition in phospholipids from different lactation milk revealed no remarkable and regular changes could be generalized; and no obvious difference of the morphologies of MFGM at different lactation stages can be visualized. An investigation of the microstructure change of MFGM vs temperature demonstrated that the segregated domains became larger as temperature decreased to 4 °C, while it became smaller when increased to 37 °C. This phenomenon indicated that, in addition to sphingimyelin and cholesterol, phospholipids might also contribute to increasing the segregated domains at lower temperature, while, at elevated temperature, these domains could be diminished, most likely due to a restructuring or distributing of sphingimyelin and cholesterol.

An in vitro digestion model that simulated gastric and intestinal fasting conditions was used to monitor the physical, chemical, and structural changes of fat globules from raw bovine milk. During in vitro gastric digestion, the fat globules were stable under low-acidic conditions. Some peptides and β-lactoglobulin were resistant to proteolysis by pepsin. Phospholipids, proteins, and peptides stabilized the globules in the stomach model. During in vitro intestinal digestion, most of the β-lactoglobulin and residual peptides were hydrolyzed by trypsin and chymotrypsin, and the lipolytic products, released from the hydrolysis of the triglyceride core of the globules, led to destabilization and coalescence of the globules. By accumulating at the surface of the fat globules, the lipolytic products formed a lamellar phase and their solubilization by bile salts resulted in the formation of disk-shaped micelles. This study brings new interesting insights on the digestion of bovine milk., (Copyright © 2012 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Cartilage oligomeric matrix protein (COMP) is an important non-collagenous cartilage protein that is essential for the structural integrity of the cartilage extracellular matrix. The repeated modular structure of COMP allows it to "bridge" and assemble multiple cartilage extracellular matrix components such as collagens, matrilins, and proteoglycans. With its modular structure, COMP also has the potential to act as a scaffold for growth factors, thereby affecting how and when the growth factors are presented to cell-surface receptors. However, it is not known whether COMP binds growth factors. We studied the binding interaction between COMP and TGF-β1 in vitro and determined the effect of COMP on TGF-β1-induced signal transduction in reporter cell lines and primary cells. Our results demonstrate that mature COMP protein binds to multiple TGF-β1 molecules and that the peak binding occurs at slightly acidic pH. These interactions were confirmed by dual polarization interferometry and visualized by rotary shadow electron microscopy. There is cation-independent binding of TGF-β1 to the C-terminal domain of COMP. In the presence of manganese, an additional TGF-β-binding site is present in the TSP3 repeats of COMP. Finally, we show that COMP-bound TGF-β1 causes increased TGF-β1-dependent transcription. We conclude that TGF-β1 binds to COMP and that TGF-β1 bound to COMP has enhanced bioactivity.

Heterozygous nonsense mutations in the CDSN gene encoding corneodesmosin (CDSN), an adhesive protein expressed in cornified epithelia and hair follicles, cause hypotrichosis simplex of the scalp (HSS), a nonsyndromic form of alopecia. Truncated mutants of CDSN ((mut)CDSN), which bear the N-terminal adhesive Gly/Ser-rich domain (GS domain) of the protein, abnormally accumulate as amorphous deposits at the periphery of hair follicles and in the papillary dermis of the patient skin. Here, we present evidence that the (mut)CDSN deposits display an affinity for amyloidophilic dyes, namely Congo red and thioflavin T. We also detected the serum amyloid protein component in the dermis of HSS patients. We demonstrated that recombinant forms of (mut)CDSN and of the GS domain assemble in vitro into ring-shaped oligomeric structures and fibrils. The amyloid-like nature of the fibrils was demonstrated by dye binding and Fourier transform infrared spectrometry measurements. We showed that the ring-shaped oligomers of (mut)CDSN, but not the fibrillar forms, are toxic to cultured keratinocytes. Finally, online algorithms predicted the GS domain to be a particularly disordered region of CDSN in agreement with circular dichroism measurements. This identifies HSS as a human amyloidosis related to the aggregation of natively unfolded (mut)CDSN polypeptides into amyloid fibrils.

Ultrasound has been used for the last 50 y in different processing applications. Depending on the power and frequency of the sound waves, ultrasound techniques can be classified in different categories. Low-intensity ultrasound uses high frequencies in the range of 100 kHz to 10 MHz and is mostly used for therapeutic purpose (frequencies between 1 and 10 MHz) and to passively monitor the characteristics of materials (frequencies between 100 kHz and 10 MHz). High-intensity ultrasound (HIU), on the other hand, uses lower frequencies in the range of 20 to 100 kHz and it is commonly used for cleaning, disrupting, and restructuring materials. The objective of this study is to evaluate the effect of HIU on functional properties of anhydrous milk fat (AMF), palm kernel oil (PKO), and an all-purpose shortening (Sh). Results from this research shows that HIU induced primary and secondary nucleation in the lipid, generating smaller crystals and as a consequence harder materials. HIU affected hardness more efficiently when applied at higher crystallization temperatures (26 and 28 degrees C) as shown for AMF data, and when the sonication was applied after the first crystals were formed as observed for PKO and Sh systems. In addition to changes in hardness, AMF and Sh networks obtained after sonication were characterized by a steeper and sharper melting profile. This research shows that HIU can be used as an additional processing tool to tailor the functional properties of lipids with the potential to be used in the processing of trans-free shortenings.

Confocal laser scanning microscopy (CLSM) was successfully used to observe the effect of milk processing on the size and the morphology of the milk fat globule in raw milk, raw ultrafiltered milk, and standardized and pasteurized milk prepared for cheese manufacture (cheese-milk) and commercial pasteurized and homogenized milk. Fat globule size distributions for the milk preparations were analyzed using both image analysis and light scattering and both measurements produced similar data trends. Changes to the native milk fat globule membrane (MFGM) were tracked using a MFGM specific fluorescent stain that allowed MFGM proteins and adsorbed proteins to be differentiated on the fat globule surface. Sodium dodecyl sulfate polyacrylamide gel electrophoresis confirmed the identity of native MFGM proteins isolated from the surface of fat globules within raw, UF retentate, and cheese-milk preparations, whereas only casein was detected on the surface of fat globules in homogenized milk. The microstructure, porosity, and gel strength of the rennet induced gel made from raw milk and cheese-milk was also found to be comparable and significantly different to that made from homogenized milk. Our results highlight the potential use of CLSM as a tool to observe the structural details of the fat globule and associated membrane close to its native environment.

The baseplate of bacteriophage T4 is a multicomponent protein complex, which controls phage attachment to the host. It assembles from six wedges and a central hub. During infection the baseplate undergoes a large conformational change from a dome-shaped to a flat, star-shaped structure. We report the crystal structure of the C-terminal half of gene product (gp) 6 and investigate its motion with respect to the other proteins during the baseplate rearrangement. Six gp6 dimers interdigitate, forming a ring that maintains the integrity of the baseplate in both conformations. One baseplate wedge contains an N-terminal dimer of gp6, whereas neighboring wedges are tied together through the C-terminal dimer of gp6. The dimeric interactions are preserved throughout the rearrangement of the baseplate. However, the hinge angle between the N- and C-terminal parts of gp6 changes by approximately 15 degrees , accounting for a 10 A radial increase in the diameter of the gp6 ring.

Rift Valley fever virus (RVFV; Bunyaviridae; Phlebovirus) is an emerging human and veterinary pathogen causing acute hepatitis in ruminants and has the potential to cause hemorrhagic fever in humans. We report a three-dimensional reconstruction of RVFV vaccine strain MP-12 (RVFV MP-12) by cryo-electron microcopy using icosahedral symmetry of individual virions. Although the genomic core of RVFV MP-12 is apparently poorly ordered, the glycoproteins on the virus surface are highly symmetric and arranged on a T=12 icosahedral lattice. Our RVFV MP-12 structure allowed clear identification of inter-capsomer contacts and definition of possible glycoprotein arrangements within capsomers. This structure provides a detailed model for phleboviruses, opens new avenues for high-resolution structural studies of the bunyavirus family, and aids the design of antiviral diagnostics and effective subunit vaccines.