Proteomics of larval hemolymph in Bombyx mori reveals various nutrient-storage and immunity-related proteins

The silkworm, Bombyx mori, is an important economic insect for its production of silk. The larvae of many lepidopteran insects are major agricultural pests and often silkworm is explored as a model organism for other lepidopteran pest species. The hemolymph of caterpillars contains a lot of nutrient and immune components. In this study, we applied liquid chromatography–tandem mass spectrometry to gain a better understanding of the larval hemolymph proteomics in B. mori. We identified 752 proteins in hemolymph collected from day-4 fourth instar and day-7 fifth instar. Nearly half the identified proteins (49 %) were predicted to function as binding proteins and 46 % were predicted to have catalytic activities. Apolipophorins, storage proteins, and 30K proteins constituted the most abundant groups of nutrient-storage proteins. Of them, 30K proteins showed large differences between fourth instar larvae and fifth instar larvae. Besides nutrient-storage proteins, protease inhibitors are also expressed very highly in hemolymph. The analysis also revealed lots of immunity-related proteins, including recognition, signaling, effectors and other proteins, comprising multiple immunity pathways in hemolymph. Our data provide an exhaustive research of nutrient-storage proteins and immunity-related proteins in larval hemolymph, and will pave the way for future physiological and pathological studies of caterpillars.     

BnaC.Tic40, a plastid inner membrane translocon originating from Brassica oleracea, is essential for tapetal function and microspore development in Brassica napus

Here, we describe the characteristics of a Brassica napus male sterile mutant 7365A with loss of the BnMs3 gene, which exhibits abnormal enlargement of the tapetal cells during meiosis. Later in development, the absence of the BnMs3 gene in the mutant results in a loss of the secretory function of the tapetum, as suggested by abortive callose dissolution and retarded tapetal degradation. The BnaC.Tic40 gene (equivalent to BnMs3) was isolated by a map-based cloning approach and was confirmed by genetic complementation. Sequence analyses suggested that BnaC.Tic40 originated from BolC.Tic40 on the Brassica oleracea linkage group C9, whereas its allele Bnms3 was derived from BraA.Tic40 on the Brassica rapa linkage group A10. The BnaC.Tic40 gene is highly expressed in the tapetum and encodes a putative plastid inner envelope membrane translocon, Tic40, which is localized into the chloroplast. Transmission electron microscopy (TEM) and lipid staining analyses suggested that BnaC.Tic40 is a key factor in controlling lipid accumulation in the tapetal plastids. These data indicate that BnaC.Tic40 participates in specific protein translocation across the inner envelope membrane in the tapetal plastid, which is required for tapetal development and function.     

Genome-Wide Identification, Classification, and Expression Analysis of Autophagy-Associated Gene Homologues in Rice (Oryza sativa L.)

Autophagy is an intracellular degradation process for recycling macromolecules and organelles. It plays important roles in plant development and in response to nutritional demand, stress, and senescence. Organisms from yeast to plants contain many autophagy-associated genes (ATG). In this study, we found that a total of 33 ATG homologues exist in the rice [Oryza sativa L. (Os)] genome, which were classified into 13 ATG subfamilies. Six of them are alternatively spliced genes. Evolutional analysis showed that expansion of 10 OsATG homologues occurred via segmental duplication events and that the occurrence of these OsATG homologues within each subfamily was asynchronous. The Ka/Ks ratios suggested purifying selection for four duplicated OsATG homologues and positive selection for two. Calculating the dates of the duplication events indicated that all duplication events might have occurred after the origin of the grasses, from 21.43 to 66.77 million years ago. Semi-quantitative RT–PCR analysis and mining the digital expression database of rice showed that all 33 OsATG homologues could be detected in at least one cell type of the various tissues under normal or stress growth conditions, but their expression was tightly regulated. The 10 duplicated genes showed expression divergence. The expression of most OsATG homologues was regulated by at least one treatment, including hormones, abiotic and biotic stresses, and nutrient limitation. The identification of OsATG homologues showing constitutive expression or responses to environmental stimuli provides new insights for in-depth characterization of selected genes of importance in rice.     

Antibacterial Activity,Cytotoxicity and Mechanisms of action of Cathelicidin Peptides against Enteric Pathogens in Weaning Piglets

In the last few decades, long-term and high-dose usage of antibiotics in livestock diets has led to the emergence of antibiotic resistant bacteria, antibiotic residues in animal products and environmental pollution, adversely affecting animal health. Because of these concerns, a study screening cathelicidin peptides from different animal origins (i.e. protegrin-1 [PG-1], PMAP-23, LL-37, indolicidin and cathelicidin-BF [C-BF]) as antibiotic replacements with higher antimicrobial activity and lower cytotoxicity was designed to study their mechanisms towards enteric pathogens in weaning piglets. PG-1 and C-BF proved to be the most effective bacteriocids with the widest spectra of activity, with the MIC values equal to or lower than commonly used antibiotics towards several Escherichia and Salmonella strains, and showed a synergistic effect with aureomycin. Mechanism studies suggested the C-BF killing mechanism is based on membrane permeability, while multiple targets maybe exist for PG-1, including membrane and intracellular biomacromolecules. Cytotoxicity tests showed PMAP-23 and C-BF exhibited the lowest cytotoxic effects, while PG-1, LL-37 and indolicidin displayed cytotoxicity by dose. This study demonstrated that among the peptides tested, C-BF has the capacity to inactivate enteric pathogens with lower cytotoxicity and is potentially a novel anti-bacterial agent. The activity of PG-1 is highly efficient, with the potential to reduce cytotoxicity using molecular design.     

Biological characteristics of foam cell formation in smooth muscle cells derived from bone marrow stem cells

Bone marrow mesenchymal stem cells (BMSC) can differentiate into diverse cell types, including adipogenic, osteogenic, chondrogenic and myogenic lineages. There are lots of BMSC accumulated in atherosclerosis vessels and differentiate into VSMC. However, it is unclear whether VSMC originated from BMSC (BMSC-SMC) could remodel the vessel in new tunica intima or promote the pathogenesis of atherosclerosis. In this study, BMSC were differentiated into VSMC in response to the transforming growth factor β (TGF-β) and shown to express a number of VSMC markers, such as α-smooth muscle actin (α-SMA) and smooth muscle myosin heavy chain1 (SM-MHC1). BMSC-SMC became foam cells after treatment with 80 mg/L ox-LDL for 72 hours. Ox-LDL could upregulate scavenger receptor class A (SR-A) but downregulate the ATP-binding cassette transporter A1 (ABCA1) and caveolin-1 protein expression, suggesting that modulating relative protein activity contributes to smooth muscle foam cell formation in BMSC-SMC. Furthermore, we found that BMSC-SMC have some biological characteristics that are similar to VSMC, such as the ability of proliferation and secretion of extracellular matrix, but, at the same time, retain some biological characteristics of BMSC, such as a high level of migration. These results suggest that BMSC-SMC could be induced to foam cells and be involved in the development of atherosclerosis.     

Digestion-Promoting Effects and Mechanisms of Dashanzha Pill Based on Raw and Charred Crataegi Fructus

Emerging evidence suggests that a high-fat diet (HFD) can influence endoplasmic reticulum (ER) stress and gut microbiota. Crataegi Fructus is a traditional Chinese herb widely used in formulas for dyspepsia, with Dashanzha Pill composed of raw Crataegi Fructus (DR) being a representative drug. Processing products of Crataegi Fructus, however, have a stronger pro-digestive effect, and we hypothesized that Dashanzha Pill composed of charred Crataegi Fructus (DC) is more effective. We found that the contents of glucose 1-phosphate and luteolin in DR and DC were substantially different via ultra-high performance liquid chromatography-hybrid quadrupole-Orbitrap high-resolution mass spectrometry. DC outperformed DR in improving histopathological changes, increasing gastrin and motilin, and decreasing vasoactive intestinal peptides in rats with HFD induced dyspepsia. Fecal microbiota analysis revealed that DC could restore the disturbed intestinal microbiota composition, including that of Bacteroides, Akkermansia, and Intestinimonas to normal levels. Furthermore, DC significantly reduced the mRNA and protein levels of glucose-regulated protein 78, protein kinase R-like ER kinase, and eukaryotic initiation factor 2α. Taken together, DC outperformed DR in relieving dyspepsia by regulating gut microbiota and alleviating ER stress.     

Synchronized oviposition triggered by migratory flight intensifies larval outbreaks of beet webworm

Identifying the reproductive consequences of insect migration is critical to understanding its ecological and evolutionary significance. However, many empirical studies are seemingly contradictory, making recognition of unifying themes elusive and controversial. The beet webworm, Loxostege sticticalis L. is a long-range migratory pest of many crops in the northern temperate zone from 36 °N to 55 °N, with larval populations often exploding in regions receiving immigrants. In laboratory experiments, we examined (i) the reproductive costs of migratory flight by tethered flight, and (ii) the reproductive traits contributing to larval outbreaks of immigrant populations. Our results suggest that the beet webworm does not initiate migratory flight until the 2nd or 3rd night after emergence. Preoviposition period, lifetime fecundity, mating capacity, and egg hatch rate for adults that experienced prolonged flight after the 2nd night did not differ significantly from unflown moths, suggesting these traits are irrelevant to the severity of beet webworm outbreaks after migration. However, the period of first oviposition, a novel parameter developed in this paper measuring synchrony of first egg-laying by cohorts of post-migratory females, for moths flown on d 3 and 5 of adulthood was shorter than that of unflown moths, indicating a tightened time-window for onset of oviposition after migration. The resulting synchrony of egg-laying will serve to increase egg and subsequent larval densities. A dense population offers potential selective advantages to the individual larvae comprising it, whereas the effect from the human standpoint is intensification of damage by an outbreak population. The strategy of synchronized oviposition may be common in other migratory insect pests, such as locust and armyworm species, and warrants further study.     

Microrheological aspects of adhesion of Escherichia coli on glass

The adhesion of both live and fixed bacteria (Escherichia coli) on glass has been studied under well-defined hydrodynamic conditions, created in an impinging jet apparatus. With this technique one can accurately measure the initial deposition rate jo on the surface, the average lifetime of a bacterium on the surface, tau esc, and the surface area blocked per deposited bacterium, normalized by its projected area, gamma. The experimental results are compared to theoretical results for equivalent spheres. It is found that near the stagnation point the deposition rate jo is mainly controlled by convective diffusive transport which, for rod-shaped Eschericia coli, with an axis ratio of about 2, is found to be equal to that for spheres. No differences in jo and tau esc were found between live and fixed bacteria at low flow rates. At high flow rates fixed bacteria adhered to the surface at a slower rate. In both systems jo was found to decrease suddenly at a distance of about 150 microns from the stagnation point, in contrast to systems of spherical particles for which jo is uniform over the surface. Most likely this is due to the rotation of the rod-shaped particles, which vary their distance to the surface periodically with time. The main difference between live and fixed bacteria, besides different deposition rates in strong flows, is that gamma is about 30% larger for fixed bacteria than for live ones, resulting in a much lower final coverage for fixed bacteria. These results imply a larger repulsion between fixed bacteria than between living ones. From detachment experiments we can conclude that not all bacteria stick to the surface with the same bond strength. The variation in the bond strength is due to the aging of the bonds between the bacteria and the surface. The average bond strength corresponds to an energy of about 13-15 kT.     

Intratumoral expression of IL-17 and its prognostic role in gastric adenocarcinoma patients

In this study, we characterized the intratumoral expression of IL-17 and CD8(+) TILs in gastric adenocarcinoma patients after resection and determined the correlation between the survival probability of gastric adenocarcinoma patients and the expression of IL-17 in tumor. Expression of IL-17 and CD8 was assessed by immunohistochemistry, and the prognostic effects of intratumoral IL-17 expression and CD8(+) TILs were evaluated by Cox regression and Kaplan-Meier analysis. Immunohistochemical detection revealed the presence of IL-17 and CD8(+) cells in gastric adenocarcinoma tissue samples (90.6%, 174 out of 192 patients and 96.9%, 186 out of 192 patients, respectively). We have also found that intratumoral IL-17 expression was significantly correlated with age (p=0.004) and that the number of CD8(+)TILs was significantly correlated with UICC staging (p=0.012) and the depth of tumor invasion (p=0.022). The five-year overall survival probability among patients intratumorally expressing higher levels of IL-17 was significantly better than those expressing lower levels of IL-17 (p=0.036). Multivariate Cox proportional hazard analyses revealed that intratumoral IL-17 expression (HR: 0.521; 95% CI: 0.329-0.823; p=0.005) was an independent factor affecting the five-year overall survival probability. We conclude that low levels of intratumoral IL-17 expression may indicate poor prognosis in gastric adenocarcinoma patients.     

Motor neuropathy‐associated mutation impairs Seipin functions in neurotransmission

Gain‐of‐toxic‐function mutations in Seipin (Asparagine 88 to Serine (N88S) and Serine 90 to Leucine (S90L) mutations, both of which disrupt the N‐glycosylation) cause autosomal dominant motor neuron diseases. However, the mechanism of how these missense mutations lead to motor neuropathy is unclear. Here, we analyze the impact of disruption of N‐glycosylation of Seipin on synaptic transmission by over‐expressing mutant Seipin in cultured cortical neurons via lentiviral infection. Immunostaining shows that over‐expressed Seipin is partly colocalized with synaptic vesicle marker synaptophysin. Electrophysiological recordings reveal that the Seipin mutation significantly decreases the frequency, but not the amplitudes of miniature excitatory post‐synaptic currents and miniature inhibitory post‐synaptic currents. The amplitude of both evoked excitatory post‐synaptic currents and inhibitory post‐synaptic current is also compromised by mutant Seipin over‐expression. The readily releasable pool and vesicular release probability of synaptic vesicles are both altered in neurons over‐expressing Seipin‐N88S, whereas neither γ‐amino butyric acid (GABA) nor α‐Amino‐3‐hydroxy‐5‐methyl‐4‐ isoxazolepropionic acid (AMPA) induced whole cell currents are affected. Moreover, electron microscopy analysis reveals decreased number of morphologically docked synaptic vesicles in Seipin‐N88S‐expressing neurons. These data demonstrate that Seipin‐N88S mutation impairs synaptic neurotransmission, possibly by regulating the priming and docking of synaptic vesicles at the synapse.