Computational approaches to understanding protein aggregation in neurodegeneration

The generation of toxic non-native protein conformers has emerged as a unifying thread among disorders such as Alzheimer＇s disease, Parkinson＇s disease, and amyotrophic lateral sclerosis. Atomic-level detail regarding dynamical changes that facilitate protein aggre- gation, as well as the structural features of large-scale ordered aggregates and soluble non-native oligomers, would contribute signifi- cantly to current understanding of these complex phenomena and offer potential strategies for inhibiting formation of cytotoxic species. However, experimental limitations often preclude the acquisition of high-resolution structural and mechanistic information for aggregating systems. Computational methods, particularly those combine both aU-atom and coarse-grained simulations to cover a wide range of time and length scales, have thus emerged as crucial tools for investigating protein aggregation. Here we review the current state of computational methodology for the study of protein self-assembly, with a focus on the application of these methods toward understanding of protein aggregates in human neurodegenerative disorders.     

Methoprene application and diet protein supplementation to male melon fly,Bactrocera cucurbitae,modifies female remating behavior

Methoprene （an analogue of juvenile hormone） application and feeding on a protein diet is known to enhance male melon fly, Bactrocera cucurbitae Coquillett （Diptera： Tephritidae）, mating success. In this study, we investigated the effect of these treatments on male B. cucurbitae＇s ability to inhibit female remating. While 14-d-old females were fed on protein diet, 6-d-old males were exposed to one of the following treatments： （i） topical application of methoprene and fed on a protein diet; （ii） no methoprene but fed on a protein diet; （iii） methoprene and sugar-fed only; and （iv） sugar-fed, 14-d-old males acted as controls. Treatments had no effect on a male＇s ability to depress the female remating receptivity in comparison to the control. Females mated with protein-deprived males showed higher remating receptivity than females first mated with protein-fed males. Methoprene and protein diet interaction had a positive effect on male mating success during the first and second mating of females. Significantly more females first mated with sugar-fed males remated with protein-fed males and females first mated with methoprene treated and protein-fed males were more likely to remate with similarly treated males. Females mating latency （time to start mating） was significantly shorter with protein-fed males, and mating duration was significantly longer with protein-fed males compared with protein-deprived males. These results are discussed in the context of methoprene and/or dietary protein as prerelease treatment of sterile males in area-wide control of melon fly integrating the sterile insect technique （SIT）.     

Cloning of heat shock protein genes from the brown planthopper,Nilaparvata lugens,and the small brown planthopper,Laodelphax striatellus,and their expression in relation to thermal stress

Three heat shock protein （HSP） genes （hsp70, hsc70, hsp90） were partially cloned from the brown planthopper Nilaparvata lugens and the small brown planthopper Laodelphax striatellus （Homoptera： Delphacidae）, which are serious pests of the rice plant. Sequence comparisons at the deduced amino acid level showed that the three HSPs of planthoppers were most homologous to corresponding HSPs of dipteran and lepidopteran species. Identities of both heat shock cognate 70 and HSP90 were higher than HSP70 in both species. Identity of the HSP70 between the two planthopper species was only 81%, a value much lower than seen among fly and moth groups. Effects of heat and cold shocks were demonstrated on expression of the three hsp genes in the two planthopper species. Heat shock （40 ℃） upregulated the hsp90 level but did not change the hsc70 level in either the nymph and adult stages of either species. On the other hand, the hsp70 level was only upregulated in L. striatellus. This heat shock response was prompt and lasted only for 1 h after treatment. In contrast, cold shock at 4℃ did not change the expression levels of any hsp in either species.     

Claudin-4 is required for AMPK-modulated paraceliular permeability in submandibular gland ceils

Tight junction plays an important rote in mediating paraceUular permeability in epithelia. We previously found that activation of AMP- activated protein kinase （AMPK） increased saliva secretion by modulating paraceUular permeability in submandibular glands. However, the molecular mechanisms underlying AMPK-modulated paraceUular permeability are unknown. In this study, we found that AICAR, an AMPK agonist, increased saliva secretion in the isolated rat submandibular glands, decreased transepithelial electrical resistance （TER）, and increased 4 kDa FITC-dextran flux in cultured SMG-C6 cells. AICAR also induced redistribution of tight junction protein claudin-4, but not claudin-1, claudin-3, occtudin, or ZO-1, from the cytoplasm to the membrane. Moreover, knockdown of claudin-4 by shRNA suppressed while claudin-4 re-expression restored the TER and 4 kDa FITC-dextran flux responses to AICAR. Additionally, AICAR increased ERK1/2 phosphorylation, and inhibition of ERK1/2 by U0126, an ERK1/2 kinase inhibitor, or by siRNA decreased AICAR-induced TER responses. AICAR induced the serine S199 phosphorylation of claudin-4 and enhanced the inter- action of claudin-4 and occludin. Furthermore, pretreatment with U0126 significantly suppressed AMPK-modulated phosphorytation, redistribution, and interaction with occludin of claudin-4. Taken together, these results indicated that claudin-4 played a crucial role in AMPK-rnodutated paraceUular permeability and ERK1/2 was required in AMPK-modulated tight junction barrier function in subman- dibular gland.     

New combination in Veronica （Scrophulariaceae s.l.） based on morphological characters and the seed storage protein polymorphism

Veronica erciyasdagi （M. A. Fischer） C. Vural comb. ＆ stat. nov., previously regarded as a variety, is proposed as a new combination, based on the morphological characters and seed storage protein polymorphism presented in this study. In addition, information about the ecology and conservation status of Veronica erciyasdagi was reported. This taxon is endemic to central Anatolia, Turkey and is critically endangered.     

Molecular dynamics reveal a novel kinase-substrate interface that regulates protein translation

A key control point in gene expression is the initiation of protein translation, with a universal stress response being constituted by in- hibitory phosphoryiation of the eukaryotic initiation factor 2α （el F2oL）. In humans, four kinases sense diverse physiological stresses to regulate elF2α to control cell differentiation, adaptation, and survival. Here we develop a computational molecular model of elF2α and one of its kinases, the protein kinase R, to simulate the dynamics of their interaction. Predictions generated by coarse-grained dynamics simulations suggest a novel mode of action. Experimentation substantiates these predictions, identifying a previously unrecognized interface in the protein complex, which is constituted by dynamic residues in both elF2α and its kinases that are crucial to regulate protein translation. These findings call for a reinterpretation of the current mechanism of action of the el F2α kinases and demonstrate the value of conducting computational analysis to evaluate protein function.     

Effects of methamidophos and deltamethrin on in vitro protein phosphorylation in Monochamus alternatus

Monochamus alternatus Hope （Coleoptera： Cerambycidae） is not only a serious pest insect to pine trees but also the main vector of pine wood nemadote Bursaphelenchus xylophilus, which causes pine wilt disease. To explore the insecticidal mechanism of insecticides to M. alternatus, we chose methamidophos and deltamethrin as the representatives of two groups of insecticides （organophosphates and pyrethroids）, which are widely used for pest control in China and investigated their effects on phosphorylation of proteins from the insect. Phosphorylation of proteins from the insect fat body and head was determined by in vitro 32P-labelling. In the fat body, deltamethrin obviously reduced basal phosphorylation levels of proteins at 111, 95, 77, and 44 kDa, but enhanced the basal phosphorylation level of a protein at 138 kDa. However, in the presence of calmodulin but not cyclic adenosine monophosphate （cAMP）, deltamethrin increased phosphorylation of the protein at 111 kDa. In the head, deltamethrin inhibited basal phosphorylation levels of proteins at 113, 98, and 51 kDa, but potentiated phosphorylation of a protein at 167 kDa activated by cAMP. Methamidophos inhibited phosphorylation of a protein at 44 kDa in the fat body. Although methamidophos did not impact basal phosphorylation levels of any proteins in the head, it inhibited calcium/calmodulin （Ca^2＋CaM）-stimulated phosphorylation of a protein at 51 kDa. Together, our data indicate that methamidophos and deltamethrin altered phosphorylation levels of various proteins in the head and fat body of the pine insect and these two kinds of insecticides acted on the proteins that can be phosphorylated in the tissues respectively, which is possibly related to their toxicity.     

A suspended cell line from Trichoplusia ni （Lepidoptera）： Characterization and expression of recombinant proteins

A suspended cell line from Trichoplusia ni embryos was established, and its susceptibility to Autographa californica multiple nuclear polyhedrosis virus （AcMNPV） infection was investigated. This cell line had characteristics distinct from the BTI-Tn5B 1- 4 cell line （Tn5B 1-4） from T. ni in growth, and showed approximately the same responses to AcMNPV infection, production of occlusion bodies, and levels of recombinant protein expression. No clumps were observed at maximum cell density at late-log phase in shake-flask or T-flask cultures, and thus the cells represent a useful new contribution for baculovirus research. The cells consist of two major morphological types： approximately 70% spindle-shaped cells and 30% round cells. The cell line was highly susceptible to virus infection and produced around 107 AcMNPV occlusion bodies per cell, on average. Production of β-galactosidase and secreted alkaline phosphatase was high with 3.97 ± 0.13 × 10^4 IU/mL and 3.48 ± 0.40IU/mL, respectively. This cell line may be applicable for studies of scale-up production of viruses or baculovirus-insect cell expression. We also believe the new line can be a source for cell clones with higher production of virus and recombinant proteins compared to the parent or other existing cell lines such as Tn5B 1-4.     

Efficient production of transgenic chickens using self-inactive HIV- based lentiviral vectors

We demonstrated the simple and effective production of transgenic chickens, in which the enhanced green fluorescence protein （EGFP） was expressed by using third-generation self-inactive HIV-based lentiviral vectors. In our experiments, lentiviruses were injected into 204 fertilized eggs, from which 30 （15%） chickens were hatched. The exogenous gene was detected in the genomes of 16 out of 30 （53%） chickens. The green fluorescence signal was observed directly in various body parts, and was particularly significant in the testes. The transgenes were also found in the offspring of these chickens. The results indicate that HIV-based lentiviral vectors can be used to generate transgenic birds economically and effectively [ Current Zoology 55 （5）： 383 - 387,2009].     

Molecular characterization and phylogenetic analysis of three odorant binding protein gene transcripts in Dendrolimus species （Lepidoptera： Lasiocampidae）

Pine caterpillar moths, Dendrolimus spp. （Lepidoptera： Lasiocampidae）, are serious economic pest of pines. Previously, phylogenetic analyses of Dendrolimus using different methods yielded inconsistent results. The chemosensory systems of insects may play fundamental roles in promoting speciation. Odorant-binding proteins （OBPs） participate in the first step of odor detection. Studying the evolution of OBPs in closely related species may help us to identify their role in speciation. We identified three OBPs - one pheromone-binding protein and two general odorant-binding proteins - from male antennae of four Dendrolimus species, D. superans （Butler）, D. punctatus （Walker）, D. kikuchii Matsumura, and D. houi Lajonquiere, the olfactory recognition systems of which had not been previously investigated. We analyzed their molecular characteristics and compared their sequences to those of OBPs in D. tabulaeformis Tsai et Liu. Ka/Ks ratio analyses among the five Dendrolimus species indicate that PBP1 genes experienced more evolutionary pressure than the GOBPs. Phylogenetic relationships of PBP1 and GOBP1 both indicated that D. houi was the basal species, then branched D. kikuchii, while D. tabulaeformis, D. punctatus, and D. superans evolved more recently. These relationships are consistent with the changes in sex pheromone components of these five species. Dendrolimus tabulaeformis and D. punctatus are closely related sister species. However, the distances among GOBP2 sequences in the five Dendrolimus were very short, and the relationships of D. houi and D. la＇kuchii could not be resolved. Integrating our results with those of previous studies, we hypothesized that D. kikuchii, D. punctatus and D. superans evolved from the basal ancestor because of sex pheromone mutations and environmental pressure.     

Effects of different brush border membrane vesicle isolation protocols on proteomic analysis of Cry1Ac binding proteins from the midgut of Helicoverpa armigera

Brush border membrane vesicles （BBMV） isolated from insect midguts have been widely used to study CrylA binding proteins. Sample preparation is important in two- dimensional electrophoresis （2-DE）, so to determine a suitable BBMV preparation method in Helicoverpa armigera for 2-DE, we compared three published BBMV preparation methods mostly used in sodium dodecyl sulfate-polyacrylamide gel electrophoresis （SDS- PAGE）. All methods yielded similar types and numbers of binding proteins, but in different quantities. The Abdul-Rauf and Ellar protocol was the best of the three, but had limitations. Sufficient protein quantity is important for research involving limited numbers of insects, such as studies of insect resistance to Bacillus thuringiensis in the field. Consequently, we integrated the three BBMV isolation methods into a single protocol that yielded high quantities of BBMV proteins from H. armigera larval midguts, which proved suitable for 2- DE analysis.     

Digital imaging approaches for phenotyping whole plant nitrogen and phosphorus response in Bracbypodium distacbyon

This work evaluates the phenotypic response of the model grass （Brachypodium distacbyon （L.） P. Beauv.） to nitrogen and phosphorus nutrition using a combination of imaging techniques and destructive harvest of shoots and roots. Reference line Bd21-3 was grown in pots using 11 phosphorus and 11 nitrogen concentrations to establish a dose-response curve. Shoot biovolume and biomass, root length and biomass, and tissue phosphorus and nitrogen concentrations increased with nutrient concentration. Shoot biovolume, estimated by imaging, was highly correlated with dry weight （R2 〉 0.92） and both biovolume and growth rate responded strongly to nutrient availability. Higher nutrient supply increased nodal root length more than other root types. Photochemical efficiency was strongly reduced by low phosphorus concentrations as early as 1 week after germination, suggesting that this measurement may be suitable for high throughput screening of phosphorus response. In contrast, nitrogen concentration had little effect on photochemical efficiency. Changes in biovolume over time were used to compare growth rates of four accessions in response tonitrogen and phosphorus supply. We demonstrate that a time series image-based approach coupled with mathematical modeling provides higher resolution of genotypic response to nutrient supply than traditional destructive techniques and shows promise for high throughput screening and determina- tion of genomic regions associated with superior nutrient use efficiency.     

Genealogy and phylogeography of Cyprinid fish Labeo rohita （Hamilton, 1822） inferred from ATPase 6 and 8 mitochondrial DNA gene analysis

ATPase 6/8 gene （842 bp） of mitochondrial DNA was sequenced in Labeo rohita samples （n = 253） collected from nine rivers belonging to four river basins; Indus, Ganges, Brahmaputra and Mahanadi. Analysis revealed 44 haplotypes with high haplotype diversity （Hd） 0.694 and low nucleotide diversity （π） 0.001. The within population variation was larger （83.44%） than among population differences （16.56%）. The mean Fsv value （0.166; P 〈 0.05） for overall populations revealed moderate level of genetic structuring in the wild L. rohita populations. The haplotype network presented a single clade for wild L. rohita population, from different rivers. Negative values for Fu＇s index （Fs）, mismatch distribution analysis indicated period of expansion in L. rohita population. The time after recent expansion was estimated for each population, between 0.042 to 0.167 mya. The pattern of Isolation by Distance （IBD） was not significant （r = -0.113, P 〈 0.287）, when all the sampling locations were compared （Mantel test）, however, when an outlier （Indus, Brahmaputra and Mahanadi） was removed from the whole population set, a clear positive correlation between pairwise Fsv and geographic distance （Km） was seen. The analysis of data demonstrated that ATPase6/8 gene polymorphism is a potential marker to understand genetic population structure of wild L. rohita existing in different rivers. The study identified population substructure in wild L. rohita with common ancestral origin [Current Zoology 60 （4）： 460--471, 2014].     

Male size does not correlate with fertilization success in two bufonid toads that show size-assortative mating

We examined sexual size dimorphism （SSD）, mating pattem, fertilization efficiency and female reproductive traits in two bufonid toads （Bufo gargarizans and Duttaphrynus melanostictus） to test the idea that importance of male body size for egg fertilization success depends on the mating pattern. Female-biased SSD was evident only in D. melanostictus. Female B. gar- garizans laid fewer larger eggs nearly three months earlier than did female D. melanostictus. Fertilization efficieneies on average were higher in B. gargarizans （95%） than in D. melanostictus （91%）. Though differing in the degree of SSD, body size, breeding season, clutch size, egg size and fertilization efficiency, the two toads were similar in four aspects： （1） both showed size-assortative mating; （2） females did not tradeoff egg size against egg number; （3） male size, clutch size and clutch dry mass were greater in male-larger than in female-larger pairs after accounting for female snout-vent length （SVL）; and （4） the ratio of male to female SVL did not affect fertilization efficiency. Our data show that： （1） a female preference for large males is likely not important in terms of egg fertilization success; （2） a male preference for large females is likely important because larger females are more fecund; and （3） size-assortative mating arises from a male preference for large females. Our study demonstrates that male size is not always important for egg fertilization success in anurans that show size-assortative mating.     

Antipredator deception in terrestrial vertebrates

Abstract Deceptive antipredator defense mechanisms fall into three categories： depriving predators of knowledge of prey＇s presence, providing cues that deceive predators about prey handling, and dishonest signaling. Deceptive defenses in terrestrial vertebrates include aspects of crypsis such as background matching and countershading, visual and acoustic Batesian mimicry, active defenses that make animals seem more difficult to handle such as increase in apparent size and threats, feigning injury and death, distractive behaviours, and aspects of flight. After reviewing these defenses, I attempt a preliminary evaluation of which aspects of antipredator deception are most widespread in amphibians, reptiles, mammals and birds     

Identification and characterization of genes involved in the jasmonate biosynthetic and signaling pathways in mulberry （Morus notabilis）

Jasmonate （JA） is an important phytohormone regulating growth, development, and environmental response in plants, particularly defense response against herbivorous insects. Recently, completion of the draft genome of the mulberry （Morus notabilis） in conjunction with genome sequencing of silkworm （Bombyx mori） provides an opportuni-ty to study this unique plant-herbivore interaction. Here, we identified genes involved in JA biosynthetic and signaling pathways in the genome of mulberry for the first time, with the majority of samples showing a tissue-biased expression pattern. The analysis of the representative genes 12-oxophy-todienoic acid reductase （OPRs） and jasmonate ZIM-domain （JAZs） was performed and the results indicated that the mulberry genome contains a relatively smal number of JA biosynthetic and signaling pathway genes. A gene encoding an＆amp;nbsp;important repressor, MnNINJA, was identified as an alternative splicing variant lacking an ethylene-responsive element binding factor-associated amphiphilic repression motif. Having this fundamental information wil facilitate future functional study of JA-related genes pertaining to mulberry-silkworm interactions.     

Evolution of deceit by worthless donations in a nuptial gift-giving spider

Males of the nursery web spider Pisaura mirabil usually offer an insect prey wrapped in white silk as a nuptial gift to facilitate copulation. Males exploit female foraging preferences in a sexual context as females feed on the gift during copula- tion. It is possible for males to copulate without a gift, however strong female preference for the gift leads to dramatically higher mating success for gift-giving males. Females are polyandrous, and gift-giving males achieve higher mating success, longer copulations, and increased sperm transfer that confer advantages in sperm competition. Intriguingly, field studies show that ap- proximately one third of males carry a worthless gift consisting of dry and empty insect exoskeletons or plant fragments wrapped in white silk. Silk wrapping disguises gift content and females are able to disclose gift content only after accepting and feeding on the gift, meanwhile males succeed in transferring sperm. The evolution of deceit by worthless gift donation may be favoured by strong intra-sexual competition and costs of gift-construction including prey capture, lost foraging opportunities and investment in silk wrapping. Females that receive empty worthless gifts terminate copulation sooner, which reduces sperm transfer and likely disadvantages males in sperm competition. The gift-giving trait may thus become a target of sexually antagonistic co-evolution, where deceit by worthless gifts leads to female resistance to the trait. We discuss factors such as female mating rate and intensity of sperm competition that may shape the evolution of male deception, and how ecological factors may influence the evolution and maintenance of worthless gifts as an evolutionarily stable alternative mating strategy by frequency dependent selection     

Uropygial gland volatiles may code for olfactory information about sex,individual, and species in Bengalese finches Lonchura striata

Over-shadowed by eye-catching vocal and visual signals, chemical communication has long been overlooked in birds. This study aimed at exploring whether volatile composition of the uropygial gland secretion （UGS） of birds was associated with the information about sex, individual and species. By using dichloromethane extraction and gas chromatography-mass spectrometry （GC-MS）, we analyzed the UGS volatiles of domesticated Bengalese finches （ Lonchura striata, Estrildiea） which is also known as white-rumped munias. We characterized 16 volatile molecules from the UGS, including eight n-alkanols, five diesters, an ester, an aldehyde and a fatty acid, and quantified them in terms of GC peak area percentages （relative abundances） . Among these compounds, hexadecanol and octadecanol were major components in both sexes. The former was richer in males than in females and the latter richer in females than in males, suggesting that they might be male and female pheromone candidates, respectively. The high inter-individual variations, in relative abundance, of the UGS volatiles implied that these compounds might carry information about individuality. The similarity between GC profiles of the UGS and wing feather from same individuals indicates that the birds might preen the secretion to their feathers to transmit chemical cues. Additionally, by comparing with three sympatric passerine species, i. e., zebra finches Taeniopygia guttata, yellow-bowed buntings Emberiza chrysophrys and rooks Corvus frugilegus, we found that the composition of C13 - C18 alkanols in the UGS might code for information about species. Our study also showed that quantitative differences （degree） of same UGS volatiles might be the key for the Bengalese finch to code for information about sex and individuality whereas both the kind and degree of UGS constituents could be utilized to code for information about species [ Current Zoology 55 （5）： 357-365, 2009].