Comparing Genotyping Accuracy Using Buccal Swabs versus Tail Biopsies by PCR in B6;C3-Tg(Prnp-SNCA*A53T)83Vle and B6;C3-Tg(Prnp-SNCA*A53T)83Vle Sncatm1Mjff Mice.

Genotyping is a common and necessary procedure performed on genetically modified animals to distinguish carriers from noncarriers of the variants of interest. Established methods involve collection of tissues such as tips of tails or notches of ears. Noninvasive methods have been described but not widely adopted for reasons including inertia to change, needs to adjust PCR protocols, and the lack of validation; noninvasive genotyping methods are a refinement on animal welfare, but questions remain regarding how they compare with invasive methods in terms of genotyping accuracy rate and reproducibility. To gain answers to these questions, we compared the detection accuracy of the transgene and determination of zygosity in B6;C3-Tg(Prnp-SNCA*A53T)83Vle and B6;C3-Tg(Prnp-SNCA*A53T)83Vle Snca tm1Mjff neonatal mice between tail biopsies and buccal swabs. Moreover, we weighed and observed mice following genotyping to see if any clinical differences can be discerned. Weight data did not support statistically significant differences in mice undergoing different genotyping procedures and control. No statistically significant difference was found between using buccal swabs or tail biopsies for genotyping with PCR or quantitative PCR. None of the pups swabbed was rejected by the dam. Our findings indicate that buccal swabbing is a more humane and feasible alternative to tail biopsies for high-throughput genotyping.

A small molecule exerts selective antiviral activity by targeting the human cytomegalovirus nuclear egress complex.

Human cytomegalovirus (HCMV) is an important pathogen for which new antiviral drugs are needed. HCMV, like other herpesviruses, encodes a nuclear egress complex (NEC) composed of two subunits, UL50 and UL53, whose interaction is crucial for viral replication. To explore whether small molecules can exert selective antiviral activity by inhibiting NEC subunit interactions, we established a homogeneous time-resolved fluorescence (HTRF) assay of these interactions and used it to screen >200,000 compound-containing wells. Two compounds, designated GK1 and GK2, which selectively inhibited this interaction in the HTRF assay with GK1 also active in a co-immunoprecipitation assay, exhibited more potent anti-HCMV activity than cytotoxicity or activity against another herpesvirus. At doses that substantially reduced HCMV plaque formation, GK1 and GK2 had little or no effect on the expression of viral proteins and reduced the co-localization of UL53 with UL50 at the nuclear rim in a subset of cells. GK1 and GK2 contain an acrylamide moiety predicted to covalently interact with cysteines, and an analog without this potential lacked activity. Mass spectrometric analysis showed binding of GK2 to multiple cysteines on UL50 and UL53. Nevertheless, substitution of cysteine 214 of UL53 with serine (C214S) ablated detectable inhibitory activity of GK1 and GK2 in vitro, and the C214S substitution engineered into HCMV conferred resistance to GK1, the more potent of the two inhibitors. Thus, GK1 exerts selective antiviral activity by targeting the NEC. Docking studies suggest that the acrylamide tethers one end of GK1 or GK2 to C214 within a pocket of UL53, permitting the other end of the molecule to sterically hinder UL50 to prevent NEC formation. Our results prove the concept that targeting the NEC with small molecules can selectively block HCMV replication. Such compounds could serve as a foundation for development of anti-HCMV drugs and as chemical tools for studying HCMV.

The CaMKII-dependent phosphorylation of GABAB receptors in the nucleus accumbens was involved in cocaine-induced behavioral sensitization in rats.

BACKGROUND: Previous studies have established that the regulation of prolonged, distal neuronal inhibition by the GABAB heteroreceptor (GABAB R) is determined by its stability, and hence residence time, on the plasma membrane. AIMS: Here, we show that GABAB R in the nucleus accumbens (NAc) of rats affects the development of cocaine-induced behavioral sensitization by mediating its perinucleus internalization and membrane expression. MATERIALS & METHODS: By immunofluorescent labeling, flow cytometry analysis, Co-immunoprecipitation and open field test, we measured the role of Ca2+ /calmodulin-dependent protein kinase II (CaMKII) to the control of GABAB R membrane anchoring and cocaine induced-behavioral sensitization. RESULTS: Repeated cocaine treatment in rats (15 mg/kg) significantly decreases membrane levels of GABAB1 R and GABAB2 R in the NAc after day 3, 5 and 7. The membrane fluorescence and protein levels of GABAB R was also decreased in NAc GAD67 + neurons post cocaine (1 µM) treatment after 5 min. Moreover, the majority of internalized GABAB1 Rs exhibited perinuclear localization, a decrease in GABAB1 R-pHluroin signals was observed in cocaine-treated NAc neurons. By contrast, membrane expression of phosphorylated CaMKII (pCaMKII) post cocaine treatment was significantly increased after day 1, 3, 5 and 7. Baclofen blocked the cocaine induced behavioral sensitization via inhibition of cocaine enhanced-pCaMKII-GABAB1 R interaction. CONCLUSION: These findings reveal a new mechanism by which pCaMKII-GABAB R signaling can promote psychostimulant-induced behavioral sensitization.

Unravelling biological roles and mechanisms of GABABR on addiction and depression through mood and memory disorders.

The metabotropic γ-aminobutyric acid type B receptor (GABABR) remains a hotspot in the recent research area. Being an idiosyncratic G-protein coupled receptor family member, the GABABR manifests adaptively tailored functionality under multifarious modulations by a constellation of agents, pointing to cross-talk between receptors and effectors that converge on the domains of mood and memory. This review systematically summarizes the latest achievements in signal transduction mechanisms of the GABABR-effector-regulator complex and probes how the up-and down-regulation of membrane-delimited GABABRs are associated with manifold intrinsic and extrinsic agents in synaptic strength and plasticity. Neuropsychiatric conditions depression and addiction share the similar pathophysiology of synapse inadaptability underlying negative mood-related processes, memory formations, and impairments. In the attempt to emphasize all convergent discoveries, we hope the insights gained on the GABABR system mechanisms of action are conducive to designing more therapeutic candidates so as to refine the prognosis rate of diseases and minimize side effects.

Coupled Sublattice Melting and Charge-Order Transition in Two Dimensions.

Two-dimensional melting is one of the most fascinating and poorly understood phase transitions in nature. Theoretical investigations often point to a two-step melting scenario involving unbinding of topological defects at two distinct temperatures. Here, we report on a novel melting transition of a charge-ordered K-Sn alloy monolayer on a silicon substrate. Melting starts with short-range positional fluctuations in the K sublattice while maintaining long-range order, followed by longer-range K diffusion over small domains, and ultimately resulting in a molten sublattice. Concomitantly, the charge order of the Sn host lattice collapses in a multistep process with both displacive and order-disorder transition characteristics. Our combined experimental and theoretical analysis provides a rare insight into the atomistic processes of a multistep melting transition of a two-dimensional materials system.

[Analysis of screening strategy of group B streptococcus in the third trimester and its influence on pregnancy outcome].

Objective: To investigate the screening strategy of group B streptococcus (GBS) in the reproductive tract of women in the third trimester and analyze its impact on pregnancy outcome. Methods: A total of 85 461 pregnant women in 35-37 weeks of gestation from Bao'an Maternity and Child Health Hospital, Jinan University from January 2011 to June 2018 were enrolled. They were divided into 3 periods according to different GBS screening strategies, the unscreened period included 31 384 cases (36.72%), 33 267 cases (38.93%) were included in partial screening period, 20 810 cases (24.35%) were included in screening period. All GBS screening positive pregnant women were given intrapartum antibiotic prophylaxis (IAP). The impact on pregnancy outcomes, and the impact of different GBS collection transport and culture methods on the positive rate of GBS screening were analyzed. Results: (1) The incidence of neonatal early onset GBS disease (EOGBSD) in unscreened period was 0.03% (11/31 773), in partial screening period was 0.02%(6/33 887), and in screening period, the incidence of neonatal EOGBSD decreased to 0, the difference was statistically significant (χ(2)=7.86, P=0.02).(2) The incidence of hematogenous infection of GBS in pregnant women was 0.02%(6/33 887) in partial screening period, and there was none in screening period, there was no significant difference (adjusted χ(2)=3.75, P=0.05). (3) In the screening period, the positive rate of GBS was 14.08%(2 719/19 306), which was significantly higher than the positive rate of GBS in the partial screening period (11.48%, 2 058/17 920; χ(2)=56.12, P=0.00). (4) Antibiotic sensitivity tests of 4 777 GBS strains showed that the antibiotics with higher resistance rate were tetracycline (81.52%, 3 896/4 777), erythromycin (66.59%, 3 181/4 777), and clindamycin (64.31%, 3 072/4 777). The combination of erythromycin, clindamycin and tetracycline was the most common resistant pattern, accounting for 48.80% (2 331/4 777). No penicillin, ceftriaxone or vancomycin resistant strains was found. Conclusions: GBS screening strategy in different regions could combine the local neonatal EOGBSD incidence rate, maternal GBS colonization rate, and the socioeconomic factors to determine whether universal GBS screening or screening for high-risk maternal women. GBS screening positive rate is related to the population, scope of the investigation, the sample collection, delivery and culture methods. The multi-drug resistance rate of GBS is high.

The different modes of binding of the dust mite allergens, Der f 7 and Der p 7, on a monoclonal antibody WH9 contribute to the differential reactivity.

BACKGROUND: Der f 7 and Der p 7 are important house dust mite allergens. An IgE-binding inhibition monoclonal antibody WH9 reacts ten folds stronger against Der p 7 than to Der f 7. The purpose of this study is to identify the antigenic determinant(s) and the structural basis of Der f 7 recognize by WH9. METHODS: WH9-reactive determinant(s) on Der f 7 was identified by immunoblot and immunoblot inhibition. The 3-D binary complex structures of WH9 and the group 7 allergens were simulated with homology modeling and docking methods. RESULTS: WH9 reacted with the Der f 7 f9 fragment. Among the five site-directed Der f 7 mutants, WH9 showed reduced immunoblot reactivity against Der f 7 S156A, D159A and P160A mutants. Only the wild-type protein and the Der f 7 I157A and L158A mutants can inhibit significantly the WH9-binding against Der f 7. The structural model of the Der f 7-WH9 complex suggests residues S156 and D159 of Der f 7 can bind to WH9 via potential hydrogen bonds. CONCLUSION: The structure models of Der f 7-WH9 and Der p 7-WH9 complexes revealed that the differential modes of binding of Der p 7 and Der f 7 allergens on WH9 contribute to the differential reactivity of WH9 against the Der f 7 and the Der p 7 mite allergens.

Getting to and through the inner nuclear membrane during herpesvirus nuclear egress.

Herpesviruses, like most DNA viruses, replicate and package their genomes into capsids in the host cell nucleus. Capsids then transit to the cytoplasm in a fascinating process called nuclear egress, which includes several unusual steps: Movement of capsids from the nuclear interior to the periphery, disruption of the nuclear lamina, capsid budding through the inner nuclear membrane, and fusion of enveloped particles with the outer nuclear membrane. Here, we review recent advances and emerging questions relating to herpesvirus nuclear egress, emphasizing controversies regarding mechanisms for capsid trafficking to the nuclear periphery, and implications of recent structures of the two-subunit, viral nuclear egress complex for the process, particularly at the step of budding through the inner nuclear membrane.

Autophagy Modulation by Lanthionine Ketimine Ethyl Ester Improves Long-Term Outcome after Central Fluid Percussion Injury in the Mouse.

Diffuse axonal injury is recognized as a progressive and long-term consequence of traumatic brain injury. Axonal injury can have sustained negative consequences on neuronal functions such as anterograde and retrograde transport and cellular processes such as autophagy that depend on cytoarchitecture and axon integrity. These changes can lead to somatic atrophy and an inability to repair and promote plasticity. Obstruction of the autophagic process has been noted after brain injury, and rapamycin, a drug used to stimulate autophagy, has demonstrated positive effects in brain injury models. The optimization of drugs to promote beneficial autophagy without negative side effects could be used to attenuate traumatic brain injury and promote improved outcome. Lanthionine ketimine ethyl ester, a bioavailable derivative of a natural sulfur amino acid metabolite, has demonstrated effects on autophagy both in vitro and in vivo. Thirty minutes after a moderate central fluid percussion injury and throughout the survival period, lanthionine ketimine ethyl ester was administered, and mice were subsequently evaluated for learning and memory impairments and biochemical and histological changes over a 5-week period. Lanthionine ketimine ethyl ester, which we have shown previously to modulate autophagy markers and alleviate pathology and slow cognitive decline in the 3 × TgAD mouse model, spared cognition and pathology after central fluid percussion injury through a mechanism involving autophagy modulation.

Unexpected features and mechanism of heterodimer formation of a herpesvirus nuclear egress complex.

Herpesvirus nucleocapsids escape from the nucleus in a process orchestrated by a highly conserved, viral nuclear egress complex. In human cytomegalovirus, the complex consists of two proteins, UL50 and UL53. We solved structures of versions of UL53 and the complex by X-ray crystallography. The UL53 structures, determined at 1.93 and 3.0 Å resolution, contained unexpected features including a Bergerat fold resembling that found in certain nucleotide-binding proteins, and a Cys3His zinc finger. Substitutions of zinc-coordinating residues decreased UL50-UL53 co-localization in transfected cells, and, when incorporated into the HCMV genome, ablated viral replication. The structure of the complex, determined at 2.47 Å resolution, revealed a mechanism of heterodimerization in which UL50 clamps onto helices of UL53 like a vise. Substitutions of particular residues on the interaction interface disrupted UL50-UL53 co-localization in transfected cells and abolished virus production. The structures and the identification of contacts can be harnessed toward the rational design of novel and highly specific antiviral drugs and will aid in the detailed understanding of nuclear egress.

Oral TNFα Modulation Alters Neutrophil Infiltration, Improves Cognition and Diminishes Tau and Amyloid Pathology in the 3xTgAD Mouse Model.

Cytokines such as TNFα can polarize microglia/macrophages into different neuroinflammatory types. Skewing of the phenotype towards a cytotoxic state is thought to impair phagocytosis and has been described in Alzheimer's Disease (AD). Neuroinflammation can be perpetuated by a cycle of increasing cytokine production and maintenance of a polarized activation state that contributes to AD progression. In this study, 3xTgAD mice, age 6 months, were treated orally with 3 doses of the TNFα modulating compound isoindolin-1,3 dithione (IDT) for 10 months. We demonstrate that IDT is a TNFα modulating compound both in vitro and in vivo. Following long-term IDT administration, mice were assessed for learning & memory and tissue and serum were collected for analysis. Results demonstrate that IDT is safe for long-term treatment and significantly improves learning and memory in the 3xTgAD mouse model. IDT significantly reduced paired helical filament tau and fibrillar amyloid accumulation. Flow cytometry of brain cell populations revealed that IDT increased the infiltrating neutrophil population while reducing TNFα expression in this population. IDT is a safe and effective TNFα and innate immune system modulator. Thus small molecule, orally bioavailable modulators are promising therapeutics for Alzheimer's disease.

Sickle Cell Hemoglobin with Mutation at αHis-50 Has Improved Solubility.

The unliganded tetrameric Hb S has axial and lateral contacts with neighbors and can polymerize in solution. Novel recombinants of Hb S with single amino acid substitutions at the putative axial (recombinant Hb (rHb) (ßE6V/αH20R) and rHb (ßE6V/αH20Q)) or lateral (rHb (ßE6V/αH50Q)) or double amino acid substitutions at both the putative axial and lateral (rHb (ßE6V/αH20R/αH50Q) and rHb (ßE6V/αH20Q/αH50Q)) contact sites were expressed in Escherichia coli and purified for structural and functional studies. The (1)H NMR spectra of the CO and deoxy forms of these mutants indicate that substitutions at either αHis-20 or αHis-50 do not change the subunit interfaces or the heme pockets of the proteins. The double mutants show only slight structural alteration in the ß-heme pockets. All mutants have similar cooperativity (n50), alkaline Bohr effect, and autoxidation rate as Hb S. The oxygen binding affinity (P50) of the single mutants is comparable with that of Hb S. The double mutants bind oxygen with slightly higher affinity than Hb S under the acidic conditions. In high salt, rHb (ßE6V/αH20R) is the only mutant that has a shorter delay time of polymerization and forms polymers more readily than Hb S with a dextran-Csat value of 1.86 ± 0.20 g/dl. Hb S, rHb (ßE6V/αH20Q), rHb (ßE6V/αH50Q), rHb (ßE6V/αH20R/αH50Q), and rHb (ßE6V/αH20Q/αH50Q) have dextran-Csat values of 2.95 ± 0.10, 3.04 ± 0.17, 11.78 ± 0.59, 7.11 ± 0.66, and 10.89 ± 0.83 g/dl, respectively. rHb (ßE6V/αH20Q/αH50Q) is even more stable than Hb S under elevated temperature (60 °C).

A cell-penetrating ester of the neural metabolite lanthionine ketimine stimulates autophagy through the mTORC1 pathway: Evidence for a mechanism of action with pharmacological implications for neurodegenerative pathologies.

Autophagy is a fundamental cellular recycling process vulnerable to compromise in neurodegeneration. We now report that a cell-penetrating neurotrophic and neuroprotective derivative of the central nervous system (CNS) metabolite, lanthionine ketimine (LK), stimulates autophagy in RG2 glioma and SH-SY5Y neuroblastoma cells at concentrations within or below pharmacological levels reported in previous mouse studies. Autophagy stimulation was evidenced by increased lipidation of microtubule-associated protein 1 light chain 3 (LC3) both in the absence and presence of bafilomycin-A1 which discriminates between effects on autophagic flux versus blockage of autophagy clearance. LKE treatment caused changes in protein level or phosphorylation state of multiple autophagy pathway proteins including mTOR; p70S6 kinase; unc-51-like-kinase-1 (ULK1); beclin-1 and LC3 in a manner essentially identical to effects observed after rapamycin treatment. The LKE site of action was near mTOR because neither LKE nor the mTOR inhibitor rapamycin affected tuberous sclerosis complex (TSC) phosphorylation status upstream from mTOR. Confocal immunofluorescence imaging revealed that LKE specifically decreased mTOR (but not TSC2) colocalization with LAMP2(+) lysosomes in RG2 cells, a necessary event for mTORC1-mediated autophagy suppression, whereas rapamycin had no effect. Suppression of the LK-binding adaptor protein CRMP2 (collapsin response mediator protein-2) by means of shRNA resulted in diminished autophagy flux, suggesting that the LKE action on mTOR localization may occur through a novel mechanism involving CRMP2-mediated intracellular trafficking. These findings clarify the mechanism-of-action for LKE in preclinical models of CNS disease, while suggesting possible roles for natural lanthionine metabolites in regulating CNS autophagy.

Human cytomegalovirus UL97 phosphorylates the viral nuclear egress complex.

UNLABELLED: Herpesvirus nucleocapsids exit the host cell nucleus in an unusual process known as nuclear egress. The human cytomegalovirus (HCMV) UL97 protein kinase is required for efficient nuclear egress, which can be explained by its phosphorylation of the nuclear lamina component lamin A/C, which disrupts the nuclear lamina. We found that a dominant negative lamin A/C mutant complemented the replication defect of a virus lacking UL97 in dividing cells, validating this explanation. However, as complementation was incomplete, we investigated whether the HCMV nuclear egress complex (NEC) subunits UL50 and UL53, which are required for nuclear egress and recruit UL97 to the nuclear rim, are UL97 substrates. Using mass spectrometry, we detected UL97-dependent phosphorylation of UL50 residue S216 (UL50-S216) and UL53-S19 in infected cells. Moreover, UL53-S19 was specifically phosphorylated by UL97 in vitro. Notably, treatment of infected cells with the UL97 inhibitor maribavir or infection with a UL97 mutant led to a punctate rather than a continuous distribution of the NEC at the nuclear rim. Alanine substitutions in both UL50-S216 and UL53-S19 resulted in a punctate distribution of the NEC in infected cells and also decreased virus production and nuclear egress in the absence of maribavir. These results indicate that UL97 phosphorylates the NEC and suggest that this phosphorylation modulates nuclear egress. Thus, the UL97-NEC interaction appears to recruit UL97 to the nuclear rim both for disruption of the nuclear lamina and phosphorylation of the NEC. IMPORTANCE: Human cytomegalovirus (HCMV) causes birth defects and it can cause life-threatening diseases in immunocompromised patients. HCMV assembles in the nucleus and then translocates to the cytoplasm in an unusual process termed nuclear egress, an attractive target for antiviral therapy. A viral enzyme, UL97, is important for nuclear egress. It has been proposed that this is due to its role in disruption of the nuclear lamina, which would otherwise impede nuclear egress. In validating this proposal, we showed that independent disruption of the lamina can overcome a loss of UL97, but only partly, suggesting additional roles for UL97 during nuclear egress. We then found that UL97 phosphorylates the viral nuclear egress complex (NEC), which is essential for nuclear egress, and we obtained evidence that this phosphorylation modulates this process. Our results highlight a new role for UL97, the mutual dependence of the viral NEC and UL97 during nuclear egress, and differences among herpesviruses.

Molecular epidemiology of influenza A(H1N1)pdm09 virus among humans and swine, Sri Lanka.

After multiple discrete introductions of influenza A(H1N1)pdm09 virus into Sri Lanka, the virus was transmitted among humans, then swine. The spread of virus between geographically distant swine farms is consistent with virus dispersal associated with a vehicle used for swine transportation, although this remains unproven.

The transaldolase, a novel allergen of Fusarium proliferatum, demonstrates IgE cross-reactivity with its human analogue.

Fusarium species are among airborne fungi and recognized as causative agents of human atopic disorders. However, Fusarium allergens have not been well characterized and the lack of information limits clinical diagnosis and treatment of fungal allergy. The purpose of this study is to identify and characterize important allergens of F. proliferatum. IgE-reacting F. proliferatum components were identified by immunoblot using serum samples from patients of respiratory atopic diseases. Characterization of allergens and determination of IgE cross-reactivity were performed by cDNA cloning, then homologous expression and immunoblot inhibition studies. We identified nine different F. proliferatum components that can be recognized by IgE antibodies in 17 (28%) of the 60 atopic sera tested. Components with molecular masses of about 43, 37.5 and 36.5 kDa with IgE-binding frequencies of about 88, 47 and 53%, respectively, were considered as important allergens of F. proliferatum. The 37.5 kDa IgE-binding component was putatively considered as a transaldolase protein of F. proliferatum. The full-length cDNA of F. proliferatum transaldolase was subsequently cloned. It encodes an open reading frame of 312 amino acids and has sequence identifies of 73 and 61%, respectively, with Cladosporium and human transaldolases. The purified recombinant F. proliferatum transaldolase can inhibit the IgE-binding against the 37.5 kDa component of F. proliferatum and the transaldolase allergen from Cladosporium cladosporioides. More importantly, the recombinant F. proliferatum transaldolase can inhibit IgE-binding against human transaldolase in a concentration-dependent manner. Thus, a novel and important F. proliferatum transaldolase allergen was identified. In addition to IgE cross-reactivity between the Fusarium and the Cladosporium transaldolase allergens, IgE cross-reactivity between the Fusarium and the human transaldolases also exists and might contribute to atopic manifestations in the absence of exogenous allergen exposure.

A derivative of the brain metabolite lanthionine ketimine improves cognition and diminishes pathology in the 3 × Tg-AD mouse model of Alzheimer disease.

Lanthionine ketimine ([LK] 3,4-dihydro-2H-1,4-thiazine-3,5-dicarboxylic acid) is the archetype for a family of naturally occurring brain sulfur amino acid metabolites, the physiologic function of which is unknown. Lanthionine ketimine and its synthetic derivatives have recently demonstrated neurotrophic, neuroprotective, and antineuroinflammatory properties in vitro through a proposed mechanism involving the microtubule-associated protein collapsin response mediator protein 2. Therefore, studies were undertaken to test the effects of a bioavailable LK ester in the 3 × Tg-AD mouse model of Alzheimer disease. Lanthionine ketimine ester treatment substantially diminished cognitive decline and brain amyloid-ß (Aß) peptide deposition and phospho-Tau accumulation in 3 × Tg-AD mice and also reduced the density of Iba1-positive microglia. Furthermore, LK ester treatment altered collapsin response mediator protein 2 phosphorylation. These findings suggest that LK may not be a metabolic waste but rather a purposeful neurochemical, the synthetic derivatives of which constitute a new class of experimental therapeutics for Alzheimer disease and related entities.

Modeling of material orientation effects on AHSS crush and fracture behavior in axial crush tests.

OBJECTIVE: Due to the rolling manufacturing process, most advanced high-strength steels (AHSS) demonstrate in-plane anisotropic material behavior. This study investigates the effects of material orientation on the axial crush behavior and fracture of AHSS with axial crush tests and computer simulations. METHODS: Crush simulation models considering material anisotropy and damage evolution were developed in LS-DYNA based on the drop-tower crush test results and coupon characterization test data for DP780 steel. The modified Mohr-Coulomb (MMC) isotropic fracture model was employed in the crush simulation models for fracture prediction. RESULTS: The 12-sided components fabricated in the transverse (T) direction of the sheet exhibited slightly higher crush loads and reduced crush distances compared to those in the longitudinal (L) direction. The crush behavior in each direction was generally proportional to ultimate tensile strength. All of the materials investigated in this study showed some cracking in the crush tests for both component orientations, but only DP780 showed significant anisotropy in fracture behavior with more cracking for the T direction compared to the L direction. Overall, the amount of cracking observed in the tests had little or no significant effect on the axial crush performance. The MMC fracture loci in both the L and T directions were determined using a reverse engineering approach, and the stress-strain curves beyond the uniform elongation point were extended using an optimization method. Both material models MAT103 and MAT224 predicted the crush and fracture behavior with reasonably good accuracy. CONCLUSIONS: The predicted fracture mode and force-displacement curves agreed well with the test data for both the L and T directions in axial crush tests of the 12-sided components. The simple isotropic material model MAT224 is adequate for crush simulations to predict material orientation effects on AHSS component crush performance and fracture behavior.

Epitope mapping and in silico characterization of interactions between Der p 7 allergen and MoAb WH9.

Der p 7 is an important house dust mite allergen. However, antigenic determinants of Der p 7 are largely unknown. The purpose of this study is to analyze the determinants of Der p 7 and determine the structural basis of interactions between Der p 7 and WH9, an IgE-binding inhibition mouse monoclonal antibody (MoAb). IgE and WH9-reactive determinant(s) was identified by immunoblot using allergen mutants. A 3-D binary complex structure of Der p 7 and WH9 was simulated with homology modeling and docking methods. Our results obtained showed that among the five Der p 7 mutants (S156A, I157A, L158A, D159A, P160A), serum no. 1045 with IgE-binding against Der p 7 exhibited a reduced IgE immunoblot reactivity against Der p 7 L158A and D159A mutants. WH9 showed reduced immunoblot reactivity against S156A, L158A, D159A and P160A and the observation was confirmed by immunoblot inhibition. The WH9-binding determinant on Der p 7 containing S156, L158, D159 and P160 assumes a loop-like structure. The structural model of the Der p 7-WH9 complex suggests residues S156, I157, L158, D159 and P160 of Der p 7 contribute to WH9 binding via potential hydrogen bonds, electrostatic and hydrophobic interactions. In conclusion, MoAb WH9 interacts with critical residues L158 and D159 of Der p 7 and inhibits IgE-binding to Der p 7. Results obtained advance our understanding on molecular and structural bases of the antigenicity of Der p 7, its interactions with MoAb WH9 and facilitate the design of safer immunotherapy of human atopic disorders.