Ruling out Color Transparency in Quasielastic

Quasielastic ^{12}C(e,e^{'}p) scattering was measured at spacelike 4-momentum transfer squared Q^{2}=8, 9.4, 11.4, and 14.2 (GeV/c)^{2}, the highest ever achieved to date. Nuclear transparency for this reaction was extracted by comparing the measured yield to that expected from a plane-wave impulse approximation calculation without any final state interactions. The measured transparency was consistent with no Q^{2} dependence, up to proton momenta of 8.5 GeV/c, ruling out the quantum chromodynamics effect of color transparency at the measured Q^{2} scales in exclusive (e,e^{'}p) reactions. These results impose strict constraints on models of color transparency for protons.

Probing the Deuteron at Very Large Internal Momenta.

We measure ^{2}H(e,e^{'}p)n cross sections at 4-momentum transfers of Q^{2}=4.5±0.5 (GeV/c)^{2} over a range of neutron recoil momenta p_{r}, reaching up to ∼1.0 GeV/c. We obtain data at fixed neutron recoil angles θ_{nq}=35°, 45°, and 75° with respect to the 3-momentum transfer q[over →]. The new data agree well with previous data, which reached p_{r}∼500 MeV/c. At θ_{nq}=35° and 45°, final state interactions, meson exchange currents, and isobar currents are suppressed and the plane wave impulse approximation provides the dominant cross section contribution. We compare the new data to recent theoretical calculations, where we observe a significant discrepancy for recoil momenta p_{r}>700 MeV/c.

Influence of protein content and storage temperature on the particle morphology and flowability characteristics of milk protein concentrate powders.

Milk protein concentrate (MPC) powders are widely used as ingredients for food product formulations due to their nutritional profile and sensory attributes. Processing parameters, storage conditions, and composition influences the flow properties of MPC powders. This study investigated the bulk and shear flow properties of 70.3, 81.5, and 88.1% (wt/wt, protein content) MPC after storage for 12 wk at 25 and 40°C. Additionally, the morphological and functional changes of the MPC powders were investigated and correlated with flowability. After 12 wk of storage at 25°C, the basic flow energy values significantly increased from 510 to 930 mJ as the protein content increased from 70 to 90% (wt/wt). Flow rate index was significantly higher for samples with high protein content. Dynamic flow tests indicated that MPC powders with high protein content displayed higher permeability. Shear tests confirmed that the samples stored at 25°C were more flowable than samples stored at 40°C. Likewise, the higher-protein content samples showed poor shear flow behavior. The results indicated that MPC powders stored at 25°C had less cohesiveness and better flow characteristics than MPC powders stored at 40°C. Overall, the MPC powders had markedly different flow properties due to their difference in composition and morphology. This study delivers insights on the particle morphology and flow behavior of MPC powders.

A review on flow characterization methods for cereal grain-based powders.

Flow difficulties during handling, storage, and processing are common in cereal grain-based powder industries. The many studies that focus on the flow properties of powders can be classified as flow indicators, shear properties, and dynamic flow properties. The non-uniformity of physical and chemical characteristics of the individual particles that make up the bulk solid of cereal grain-based powders adds complexity to the characterization of flow behavior. Even so, knowledge of flow behavior is critical to the design of productive and cost-effective equipment for handling and processing of these powders. Because many factors influence flow, a single property/index value may not satisfactorily quantify the flow or no-flow of powders. For powders of biological origin, chemical composition and environmental factors such as temperature and relative humidity complicate flow characterization. This review focuses on the specific flow characteristics that directly affect powder flow during handling, processing, and storage.

Structure of the stationary phase survival protein YuiC from B.subtilis.

BACKGROUND: Stationary phase survival proteins (Sps) were found in Firmicutes as having analogous domain compositions, and in some cases genome context, as the resuscitation promoting factors of Actinobacteria, but with a different putative peptidoglycan cleaving domain. RESULTS: The first structure of a Firmicute Sps protein YuiC from B. subtilis, is found to be a stripped down version of the cell-wall peptidoglycan hydrolase MltA. The YuiC structures are of a domain swapped dimer, although some monomer is also found in solution. The protein crystallised in the presence of pentasaccharide shows a 1,6-anhydrodisaccharide sugar product, indicating that YuiC cleaves the sugar backbone to form an anhydro product at least on lengthy incubation during crystallisation. CONCLUSIONS: The structural simplification of MltA in Sps proteins is analogous to that of the resuscitation promoting factor domains of Actinobacteria, which are stripped down versions of lysozyme and soluble lytic transglycosylase proteins.

Architecturally diverse proteins converge on an analogous mechanism to inactivate Uracil-DNA glycosylase.

Uracil-DNA glycosylase (UDG) compromises the replication strategies of diverse viruses from unrelated lineages. Virally encoded proteins therefore exist to limit, inhibit or target UDG activity for proteolysis. Viral proteins targeting UDG, such as the bacteriophage proteins ugi, and p56, and the HIV-1 protein Vpr, share no sequence similarity, and are not structurally homologous. Such diversity has hindered identification of known or expected UDG-inhibitory activities in other genomes. The structural basis for UDG inhibition by ugi is well characterized; yet, paradoxically, the structure of the unbound p56 protein is enigmatically unrevealing of its mechanism. To resolve this conundrum, we determined the structure of a p56 dimer bound to UDG. A helix from one of the subunits of p56 occupies the UDG DNA-binding cleft, whereas the dimer interface forms a hydrophobic box to trap a mechanistically important UDG residue. Surprisingly, these p56 inhibitory elements are unexpectedly analogous to features used by ugi despite profound architectural disparity. Contacts from B-DNA to UDG are mimicked by residues of the p56 helix, echoing the role of ugi's inhibitory beta strand. Using mutagenesis, we propose that DNA mimicry by p56 is a targeting and specificity mechanism supporting tight inhibition via hydrophobic sequestration.

Molecular architecture and functional analysis of NetB, a pore-forming toxin from Clostridium perfringens.

NetB is a pore-forming toxin produced by Clostridium perfringens and has been reported to play a major role in the pathogenesis of avian necrotic enteritis, a disease that has emerged due to the removal of antibiotics in animal feedstuffs. Here we present the crystal structure of the pore form of NetB solved to 3.9 Å. The heptameric assembly shares structural homology to the staphylococcal α-hemolysin. However, the rim domain, a region that is thought to interact with the target cell membrane, shows sequence and structural divergence leading to the alteration of a phosphocholine binding pocket found in the staphylococcal toxins. Consistent with the structure we show that NetB does not bind phosphocholine efficiently but instead interacts directly with cholesterol leading to enhanced oligomerization and pore formation. Finally we have identified conserved and non-conserved amino acid positions within the rim loops that significantly affect binding and toxicity of NetB. These findings present new insights into the mode of action of these pore-forming toxins, enabling the design of more effective control measures against necrotic enteritis and providing potential new tools to the field of bionanotechnology.

Crystal structures of the human Dysferlin inner DysF domain.

BACKGROUND: Mutations in dysferlin, the first protein linked with the cell membrane repair mechanism, causes a group of muscular dystrophies called dysferlinopathies. Dysferlin is a type two-anchored membrane protein, with a single C terminal trans-membrane helix, and most of the protein lying in cytoplasm. Dysferlin contains several C2 domains and two DysF domains which are nested one inside the other. Many pathogenic point mutations fall in the DysF domain region. RESULTS: We describe the crystal structure of the human dysferlin inner DysF domain with a resolution of 1.9 Ångstroms. Most of the pathogenic mutations are part of aromatic/arginine stacks that hold the domain in a folded conformation. The high resolution of the structure show that these interactions are a mixture of parallel ring/guanadinium stacking, perpendicular H bond stacking and aliphatic chain packing. CONCLUSIONS: The high resolution structure of the Dysferlin DysF domain gives a template on which to interpret in detail the pathogenic mutations that lead to disease.

Hermetic Bags: A Short-Term Solution to Preserve High-Moisture Maize during Grain Drying.

Maintaining maize quality while drying during a rainy season is a major challenge for smallholder farmers in developing countries. We conducted a study to evaluate the impact of temporarily storing wet maize of 18, 21, and 24% moisture content (m.c.) in hermetic Purdue Improved Crop Storage (PICS) and polypropylene (PP) woven (control) bags for 21 days. Oxygen and carbon dioxide concentrations were monitored, and m.c., germination, and visual mold were assessed. In PICS bags, oxygen dropped below 1% within 7, 11.5, and 21 days for maize at 24, 21, and 18% m.c., respectively. After 21 days, the m.c. of maize stored in PICS bags remained constant, but decreased in PP bags. Germination of maize in PICS bags decreased by 0.5, 6.2, and 95.5 percentage points for 18, 21, and 24% m.c., respectively. In PP bags, germination decreased by 17.5, 15.2, and 39.5 percentage points for the respective moisture levels. After 21 days of storage, visible mold was present on maize stored in PP bags at both 21 and 24% m.c. No mold was observed on maize stored in PICS bags, but a fermentation smell was released from maize at 21 and 24% m.c. The results indicate that maize can be effectively stored in PICS bags at 21% m.c. or below for 21 days with minimal germination loss or mold growth. These findings highlight the potential of using hermetic bags for short-term grain quality preservation just before and during drying. This new utility adds to the current use of hermetic bags for protection against pests during long-term storage. Hermetic bags' dual functionality could significantly improve postharvest management on smallholder farms, thereby enhancing food and nutritional security and safety. Field testing is required in order to integrate this approach under smallholder farmers' conditions (e.g., temperature, m.c., drying practices, etc.).

MicroRNA-181a regulates local immune balance by inhibiting proliferation and immunosuppressive properties of mesenchymal stem cells.

Mesenchymal stem cells (MSCs) exhibit extensive self-renewal potential and can modulate immunocyte activation. Our previous study reported that miR-181a expression was significantly increased in placenta from women with severe preeclampsia (PE), but the mechanisms by which miR-181a regulates MSCs are unknown. In this study, we asked if and how miR-181a regulates MSCs' proliferation and immunosuppressive properties. We found that the expression of miR-181a in the MSCs derived from the umbilical cord and decidua of PE patients increased relative to MSCs derived from normal patients. Transfection with miR-181a oligos prevented MSCs proliferation but did not affect MSCs apoptosis. Overexpression of miR-181a blocked activation of the TGF-ß signaling pathway and caused downregulation of target gene (TGFBR1 and TGFBRAP1) mRNA and protein expression. Reporter genes with putative miR-181a binding sites from the TGFBR1 and TGFBRAP1 3'-untranslated regions (3'-UTRs) were downregulated in the presence of miR-181a, suggesting that miR-181a binds to TGFBR1 and TGFBRAP1 3'-UTRs. In contrast, transfection of MSCs with miR-181a oligo enhanced expression of IL-6 and indoleamine 2,3-dioxygenase by activating p38 and JNK signaling pathways, respectively. MSCs transfected with miR-181a also enhanced the proliferation of T cells in a short-term culture. Additionally, treatment with control MSCs, but not miR-181a transfected MSCs, improved dextran sulfate sodium-induced experimental colitis, suggesting that miR-181a attenuates the immunosuppressive properties of MSCs in vivo. Together, our data demonstrate that miR-181a is an important endogenous regulator in the proliferation and immunosuppressive properties of MSCs.

Finite Element Analysis of Extrinsic Parameters on the Onset of Tensile Strength in Powders.

Most food, pharmaceutical, and chemical industries rely heavily on the supply of free-flowing powders that finds their application in raw materials, additives, and manufactured products. Improper storage conditions combined with environmental factors affect the free-flowing ability of powders. An undesirable transformation of these free-flowing powders into a coherent mass that resists flow is called caking. An important metric that can be used to measure the caking propensity of a material is the tensile strength, which is essentially the resistant stress needed to separate two layers of materials using an isostatic tensile strain. Even though several models have quantified the propensity of caking, the complex nature of interactions between the powder and its micro-environment makes the prediction of caking a challenging task. In the present work, the onset of tensile strength in isomalt with changes in temperature, relative humidity, and consolidation pressures using a shear cell was modeled using a finite element approach. The study found that at a consolidation pressure of 3 kPa and relative humidity of 85±0.1%, an increase in temperature by 5˚C increased the tensile strength of isomalt by a factor of 2. On the other hand, at a constant temperature of 25˚C, an increase in relative humidity from 85±0.1% to 86±0.1% registered an increase in tensile strength by 42.7%. This study also found that an increase in consolidation pressure from 3 kPa to 6 and 9 kPa increased the tensile strength by a factor of 1.79 and 2.54, respectively. The model had good agreement with the measurements and had an overall MAPE of 12.13%. This model can be applied to study the influence of extrinsic parameters on the propensity of caking during storage of bulk solids.

Short-Term Hermetic Storage of Wet Maize and Its Effect on Quality.

Maize is a major crop grown in many regions of the world for human consumption, starch production, and animal feed. After harvest, maize is dried to avoid spoilage caused by fungal growth. However, in the humid tropics, drying maize harvested during the rainy season poses challenges. In such instances, temporary storing maize under hermetic conditions may preserve grain quality while waiting for conditions suitable for drying. Wet maize at the moisture contents (m.c.) of 18, 21, and 24% was stored for up to 21 days in both hermetic and non-hermetic jars. The stored maize was assessed, every 7 days, for germination and related parameters, presence of visible mold, and pH. After 21 days of storage at 18, 21, and 24% m.c., maize germination decreased by 28.5, 25.2, and 95.5 percentage points, respectively, in hermetic jars; and by 28.5, 25.2, and 94.5 percentage points in non-hermetic jars (control). There was visible mold on maize stored in non-hermetic jars after 21 days regardless of m.c. Maize at 21 and 24% m.c. stored in hermetic conditions underwent lactic acid fermentation that reduced the pH. The findings suggest that maize at 18 and 21% m.c. can be stored for 14 and 7 days, respectively, under hermetic conditions without significant loss of quality. Further research is needed to thoroughly assess the application of these findings for temporarily storing and subsequently drying maize on farms and along the grain value chain.

Structural analysis of human FANCL, the E3 ligase in the Fanconi anemia pathway.

The Fanconi anemia (FA) pathway is essential for the repair of DNA interstrand cross-links. At the heart of this pathway is the monoubiquitination of the FANCI-FANCD2 (ID) complex by the multiprotein "core complex" containing the E3 ubiquitin ligase FANCL. Vertebrate organisms have the eight-protein core complex, whereas invertebrates apparently do not. We report here the structure of the central domain of human FANCL in comparison with the recently solved Drosophila melanogaster FANCL. Our data represent the first structural detail into the catalytic core of the human system and reveal that the central fold of FANCL is conserved between species. However, there are macromolecular differences between the FANCL proteins that may account for the apparent distinctions in core complex requirements between the vertebrate and invertebrate FA pathways. In addition, we characterize the binding of human FANCL with its partners, Ube2t, FANCD2, and FANCI. Mutational analysis reveals which residues are required for substrate binding, and we also show the domain required for E2 binding.

A conserved peptide in West Nile virus NS4A protein contributes to proteolytic processing and is essential for replication.

The West Nile virus strain Kunjin virus (WNV(KUN)) NS4A protein is a multifunctional protein involved in membrane proliferation, stimulation of cellular pathways, and evasion of host defense and is a major component of the WNV(KUN) RNA replication complex. We identified a highly conserved region ((120)P-E-P-E(123)) upstream of the viral protease dibasic cleavage site and investigated whether this motif was required for WNV(KUN) replication. Single point mutations to alanine and a PEPE deletion mutation were created in a full-length infectious WNV(KUN) molecular clone. All mutations drastically impaired viral replication and virion production, except that of the P122A mutant, which was slightly attenuated. These mutations were subsequently transferred to a WNV(KUN) replicon to specifically assess effects on RNA replication alone. Again, all mutants, except P122A, showed severely reduced negative-sense RNA production as well as decreased viral protein production. Correspondingly, immunofluorescence analyses showed a lack of double-stranded RNA (dsRNA) labeling and a dispersed localization of the WNV(KUN) proteins, suggesting that replication complex formation was additionally impaired. Attempts to rescue replication via conservative mutants largely failed except for substitution of Asp at E121, suggesting that a negative charge at this residue is equally important. Analysis of viral protein processing suggested that cleavage of the 2K peptide from NS4A did not occur with the mutant constructs. These observations imply that the combined effects of proline and negatively charged residues within the PEPE peptide are essential to promote the cleavage of 2K from NS4A, which is a prerequisite for efficient WNV replication.

Recent advances in the study of age-related hearing loss: a mini-review.

Hearing loss is a common age-associated affliction that can result from the loss of hair cells and spiral ganglion neurons (SGNs) in the cochlea. Although hair cells and SGNs are typically lost in the same cochlea, recent analysis suggests that they can occur independently, via unique mechanisms. Research has identified both environmental and genetic factors that contribute to degeneration of cochlear cells. Additionally, molecular analysis has identified multiple cell-signaling mechanisms that likely contribute to pathological changes that result in hearing deficiencies. These analyses should serve as useful primers for future work, including genomic and proteomic analysis, to elucidate the mechanisms driving cell loss in the aging cochlea. Significant progress in this field has occurred in the past decade. As our understanding of aging-induced cochlear changes continues to improve, our ability to offer medical intervention will surely benefit the growing elderly population.

FGF signaling regulates rod photoreceptor cell maintenance and regeneration in zebrafish.

Fgf signaling is required for many biological processes involving the regulation of cell proliferation and maintenance, including embryonic patterning, tissue homeostasis, wound healing, and cancer progression. Although the function of Fgf signaling is suggested in several different regeneration models, including appendage regeneration in amphibians and fin and heart regeneration in zebrafish, it has not yet been studied during zebrafish photoreceptor cell regeneration. Here we demonstrate that intravitreal injections of FGF-2 induced rod precursor cell proliferation and photoreceptor cell neuroprotection during intense light damage. Using the dominant-negative Tg(hsp70:dn-fgfr1) transgenic line, we found that Fgf signaling was required for homeostasis of rod, but not cone, photoreceptors. Even though fgfr1 is expressed in both rod and cone photoreceptors, we found that Fgf signaling differentially affected the regeneration of cone and rod photoreceptors in the light-damaged retina, with the dominant-negative hsp70:dn-fgfr1 transgene significantly repressing rod photoreceptor regeneration without affecting cone photoreceptors. These data suggest that rod photoreceptor homeostasis and regeneration is Fgf-dependent and that rod and cone photoreceptors in adult zebrafish are regulated by different signaling pathways.

The N- or C-terminal domains of DSH-2 can activate the C. elegans Wnt/beta-catenin asymmetry pathway.

Dishevelleds are modular proteins that lie at the crossroads of divergent Wnt signaling pathways. The DIX domain of dishevelleds modulates a beta-catenin destruction complex, and thereby mediates cell fate decisions through differential activation of Tcf transcription factors. The DEP domain of dishevelleds mediates planar polarity of cells within a sheet through regulation of actin modulators. In Caenorhabditis elegans asymmetric cell fate decisions are regulated by asymmetric localization of signaling components in a pathway termed the Wnt/beta-catenin asymmetry pathway. Which domain(s) of Disheveled regulate this pathway is unknown. We show that C. elegans embryos from dsh-2(or302) mutant mothers fail to successfully undergo morphogenesis, but transgenes containing either the DIX or the DEP domain of DSH-2 are sufficient to rescue the mutant phenotype. Embryos lacking zygotic function of SYS-1/beta-catenin, WRM-1/beta-catenin, or POP-1/Tcf show defects similar to dsh-2 mutants, including a loss of asymmetry in some cell fate decisions. Removal of two dishevelleds (dsh-2 and mig-5) leads to a global loss of POP-1 asymmetry, which can be rescued by addition of transgenes containing either the DIX or DEP domain of DSH-2. These results indicate that either the DIX or DEP domain of DSH-2 is capable of activating the Wnt/beta-catenin asymmetry pathway and regulating anterior-posterior fate decisions required for proper morphogenesis.

Comparison of standard moisture loss-on-drying methods for the determination of moisture content of corn distillers dried grains with solubles.

This study quantified the variability among 14 standard moisture loss-on-drying (gravimetric) methods for determination of the moisture content of corn distillers dried grains with solubles (DDGS). The methods were compared with the Karl Fischer (KF) titration method to determine their percent variation from the KF method. Additionally, the thermo-balance method using a halogen moisture analyzer that is routinely used in fuel ethanol plants was included in the methods investigated. Moisture contents by the loss-on-drying methods were significantly different for DDGS samples from three fuel ethanol plants. The percent deviation of the moisture loss-on-drying methods decreased with decrease in drying temperature and, to a lesser extent, drying time. This was attributed to an overestimation of moisture content in DDGS due to the release of volatiles at high temperatures. Our findings indicate that the various methods that have been used for moisture determination by moisture loss-on-drying will not give identical results and therefore, caution should be exercised when selecting a moisture loss-on-drying method for DDGS.

Clostridium perfringens epsilon-toxin shows structural similarity to the pore-forming toxin aerolysin.

Epsilon-toxin from Clostridium perfringens is a lethal toxin. Recent studies suggest that the toxin acts via an unusually potent pore-forming mechanism. Here we report the crystal structure of epsilon-toxin, which reveals structural similarity to aerolysin from Aeromonas hydrophila. Pore-forming toxins can change conformation between soluble and transmembrane states. By comparing the two toxins, we have identified regions important for this transformation.

Developmental expression patterns of protein kinase A catalytic subunits in zebrafish.

Protein kinase A (PKA), also known as cAMP dependent protein kinase, is an essential component of many signaling pathways, many of which regulate key developmental processes. Inactive PKA is a tetrameric holoenzyme, comprised of two catalytic (PRKAC), and two regulatory subunits. Upon cAMP binding, the catalytic subunits are released and thereby activated. There are multiple isoforms of PKA catalytic subunits, but their individual roles are not well understood. In order to begin studying their roles in zebrafish development, it is first necessary to identify the spatial and temporal expression profiles for each prkac subunit. Here we evaluate the expression profiles for the four zebrafish prkacs: prkacαa, αb, ßa, and ßb, at key developmental time points: 24, 48 and 72 h post fertilization. We show that zebrafish prkacs are expressed throughout the developing nervous system, each showing unique expression patterns. This body of work will inform future functional studies into the roles of PKA during development.