The coiled-coil domain protein EPG-8 plays an essential role in the autophagy pathway in C. elegans

Macroautophagy (hereafter referred to as autophagy) involves the formation of a closed, double membrane structure, called the autophagosome. Most of the Atg proteins that are essential for autophagosome formation are evolutionarily conserved between yeast and higher eukaryotes. The functions of some Atg proteins, however, are mediated by highly divergent proteins in mammalian cells. In this study, we identified a novel coiled-coil domain protein, EPG-8, that plays an essential role in the autophagy pathway in C. elegans. Mutations in epg-8 cause defects in degradation of various autophagy substrates and also compromise survival of animals under nutrient-depletion conditions. In epg-8 mutants, lipidated LGG-1 (the C. elegans Atg8 homolog) accumulates but does not form distinct punctate structures. EPG-8 directly interacts with the C. elegans Beclin 1 homolog, BEC-1. Our study demonstrates that epg-8 may function as a highly divergent homolog of the yeast autophagy gene Atg14.     

Genomic structure,expression and association study of the porcine <Emphasis Type="Italic">FSD2</Emphasis>

The fibronectin type III and SPRY domain containing 2 (FSD2) on porcine chromosome 7 is considered a candidate gene for pork quality, since its two domains, which were present in fibronectin and ryanodine receptor. The fibronectin type III and SPRY domains were first identified in fibronectin and ryanodine receptor, respectively, which are candidate genes for meat quality. The aim of this study was to elucidate the genomic structure of FSD2 and functions of single nucleotide polymorphisms (SNPs) within FSD2 that are related to meat quality in pigs. Using a bacterial artificial chromosome clone sequence, we revealed that porcine FSD2 consisted of 13 exons encoding 750 amino acids. In addition, FSD2 was expressed in heart, longissimus dorsi muscle, psoas muscle, and tendon among 23 kinds of porcine tissues tested. A total of ten SNPs, including four missense mutations, were identified in the exonic region of FSD2, and two major haplotypes were obtained based on the SNP genotypes of 633 Berkshire pigs. Both haplotypes were associated significantly with intramuscular fat content (IMF, P < 0.020) and moisture percentage (MP, P < 0.002). Moreover, haplotype 2 was associated with meat color, affecting yellowness (P = 0.002). These haplotype effects were further supported by the alteration of putative protein structures with amino acid substitutions. Taken together, our results suggest that FSD2 haplotypes are involved in regulating meat quality including IMF, MP, and meat color in pigs, and may be used as meaningful molecular makers to identify pigs with preferable pork quality.     

Expression,purification, and breast cancer cell inhibiting effect of recombinant human lactoferrin C-lobe

Lactoferrin (LTF), a multifunctional glycoprotein of the transferrin family mainly found in exotic secretions in mammals, is an important defense molecule against not only microbial invasion but also tumors. It folds into two globular domains (N- and C-lobes) each containing an iron-binding site. The cationic antimicrobial peptide in N-lobe is known to exert anti-tumor effect via a non-receptor-mediated pathway. However, whether LTF C-lobe also contributes to its anti-tumor activity remains to be investigated. In this study, a human LTF fragment (amino acid residues 343–682) covering the C-lobe was expressed with a histidine tag in E. coli and the purified polypeptide refolded through a series of buffer changing procedure. The resultant recombinant protein caused significant growth arrest of breast carcinoma cells MDA-MB-231 in a dose- and time-dependent manner, evidently via induction of apoptosis of the cell. Our data suggest a positive role for the C-lobe of human LTF in controlling tumors in vitro.     

Immobilization of glucose oxidase on graphene oxide for highly sensitive biosensors

Glucose oxidase (GOx) was immobilized onto graphene oxide (GRO) via three different preparation methods: enzyme adsorption (EA), enzyme adsorption and crosslinking (EAC), and enzyme adsorption, precipitation and crosslinking (EAPC). EAPC formulations, prepared via enzyme precipitation with 60% ammonium sulfate, showed 1,980 and 1,630 times higher activity per weight of GRO than those of EA and EAC formulations, respectively. After 59 days at room temperature, EAPC maintained 88% of initial activity, while EA and EAC retained 42 and 45% of their initial activities, respectively. These results indicate that the steps of precipitation and crosslinking in the EAPC formulation are critical to achieve high enzyme loading and stability of EAPC. EA, EAC and EAPC were used to prepare enzyme electrodes for use as glucose biosensors. Optimized EAPC electrode showed 93- and 25-fold higher sensitivity than EA and EAC, respectively. To further increase the sensitivity of EAPC electrode, multi-walled carbon nanotubes (MWCNTs) were mixed with EAPC for the preparation of enzyme electrode. Surprisingly, the EAPC electrode with additional 99.5 wt% MWCNTs showed 7,800-fold higher sensitivity than the EAPC electrode without MWCNT addition. Immobilization and stabilization of enzymes on GRO via the EAPC approach can be used for the development of highly sensitive biosensors as well as to achieve high enzyme loading and stability.     

Leaf meristems: an easily ignored component of the response to human disturbance in alpine grasslands

Grazing and fencing are two important factors that influence productivity and biomass allocation in alpine grasslands. The relationship between root (R) and shoot (S) biomass and the root:shoot ratio (R/S) are critical parameters for estimating the terrestrial carbon stocks and biomass allocation mechanism responses to human activities. Previous studies have often used the belowground:aboveground biomass ratio (M_b/M_a) to replace the R/S in alpine ecosystems. However, these studies may have neglected the leaf meristem biomass, which belongs to the shoot but occurs below the soil surface, leading to a significant overestimation of the R/S ratio. We conducted a comparative study to explore the differences between the R/S and M_b/M_a at both the species (Stipa purpurea, Carex moorcroftii, and Artemisia nanschanica) and community levels on a Tibetan alpine grassland with grazing and fencing management blocks. The results revealed that the use of the M_b/M_a to express the R/S appeared to overestimate the actual value of the R/S, both at species and community levels. For S. purpurea, the M_b/M_a was three times higher than the R/S. The M_b/M_a was approximately two times higher than the R/S for the species of C. moorcroftii and A. nanschanica and at the community level. The relationships between the R‐S and M_b‐M_a exhibited different slopes for the alpine plants across all the management practices. Compared to the fenced grasslands, the plants in the grazing blocks not only allocated more biomass to the roots but also to the leaf meristems. The present study highlights the contribution of leaf meristems to the accurate assessment of shoot and belowground biomasses. The R/S and M_b/M_a should be cautiously used in combination in the future research. The understanding of the distinction between the R‐S and M_b‐M_a may help to improve the biomass allocation mechanism response to human disturbances in an alpine area.     

Ca2+ Signaling and Cell Death Induced by Protriptyline in HepG2 Human Hepatoma Cells

The effect of protriptyline on Ca²⁺ physiology in human hepatoma is unclear. This study explored the effect of protriptyline on [Ca²⁺]_i and cytotoxicity in HepG2 human hepatoma cells. Protriptyline (50–150 μM) evoked [Ca²⁺]_i rises. The Ca²⁺ entry was inhibited by removal of Ca²⁺. Protriptyline‐induced Ca²⁺ entry was confirmed by Mn²⁺‐induced quench of fura‐2 fluorescence. Except nifedipine, econazole, SKF96365, GF109203X, and phorbol 12‐myristate 13 acetate did not inhibit Ca²⁺ entry. Treatment with the endoplasmic reticulum Ca²⁺ pump inhibitor 2,5‐di‐tert‐butylhydroquinone (BHQ) inhibited 40% of protriptyline‐induced response. Treatment with protriptyline abolished BHQ‐induced response. Inhibition of phospholipase C (PLC) suppressed protriptyline‐evoked response by 70%. At 20–40 μM, protriptyline killed cells which was not reversed by the Ca²⁺ chelator 1,2‐bis(2‐aminophenoxy)ethane‐N,N,N′,N′‐tetraacetic acid‐acetoxymethyl ester (BAPTA/AM). Together, in HepG2 cells, protriptyline induced [Ca²⁺]_i rises that involved Ca²⁺ entry through nifedipine‐sensitive Ca²⁺ channels and PLC‐dependent Ca²⁺ release from endoplasmic reticulum. Protriptyline induced Ca²⁺‐independent cell death.     

Possible target‐related proteins and signal network of bufalin in A549 cells suggested by both iTRAQ‐based and label‐free proteomic analysis

Bufalin (BF) exhibited antiproliferation and antimigration effects on human A549 lung cancer cells. To search its target‐related proteins, protein expression profiles of BF‐treated and control cells were compared using two quantitative proteomic methods, iTRAQ‐based and label‐free proteomic analysis. A total of 5428 proteins were identified in iTRAQ‐based analysis while 6632 proteins were identified in label‐free analysis. The number of common identified proteins of both methods was 4799 proteins. By application of 1.20‐fold for upregulated and 0.83‐fold for downregulated cutoff values, 273 and 802 differentially expressed proteins were found in iTRAQ‐based and label‐free analysis, respectively. The number of common differentially expressed proteins of both methods was 45 proteins. Results of bioinformational analysis using Metacore^TM showed that the two proteomic methods were complementary and both suggested the involvement of oxidative stress and regulation of gene expression in the effects of BF, and fibronectin‐related pathway was suggested to be an important pathway affected by BF. Western blotting assay results confirmed BF‐induced change in levels of fibronectin and other related proteins. Overexpression of fibronectin by plasmid transfection ameliorated antimigration effects of BF. Results of the present study provided information about possible target‐related proteins and signal network of BF.     

Comparison of three transgenic Bt rice lines for insecticidal protein expression and resistance against a target pest,Chilo suppressalis (Lepidoptera: Crambidae)

Two transgenic rice lines (T2A‐1 and T1C‐19b) expressing cry2A and cry1C genes, respectively, were developed in China, targeting lepidopteran pests including Chilo suppressalis (Walker) (Lepidoptera: Crambidae). The seasonal expression of Cry proteins in different tissues of the rice lines and their resistance to C. suppressalis were assessed in comparison to a Bt rice line expressing a cry1Ab/Ac fusion gene, Huahui 1, which has been granted a biosafety certificate. In general, levels of Cry proteins were T2A‐1 > Huahui 1 > T1C‐19b among rice lines, and leaf > stem > root among rice tissues. The expression patterns of Cry protein in the rice line plants were similar: higher level at early stages than at later stages with an exception that high Cry1C level in T1C‐19b stems at the maturing stage. The bioassay results revealed that the three transgenic rice lines exhibited significantly high resistance against C. suppressalis larvae throughout the rice growing season. According to Cry protein levels in rice tissues, the raw and corrected mortalities of C. suppressalis caused by each Bt rice line were the highest in the seedling and declined through the jointing stage with an exception for T1C‐19b providing an excellent performance at the maturing stage. By comparison, T1C‐19b exhibited more stable and greater resistance to C. suppressalis larvae than T2A‐1, being close to Huahui 1. The results suggest cry1C is an ideal Bt gene for plant transformation for lepidopteran pest control, and T1C‐19b is a promising Bt rice line for commercial use for tolerating lepidopteran rice pests.     

Overcoming Electrode‐Induced Losses in Organic Solar Cells by Tailoring a Quasi‐Ohmic Contact to Fullerenes via Solution‐Processed Alkali Hydroxide Layers

It is shown that the performance of inverted organic solar cells can be significantly improved by facilitating the formation of a quasi‐ohmic contact via solution‐processed alkali hydroxide (AOH) interlayers on top of n‐type metal oxide (aluminum zinc oxide, AZO, and zinc oxide, ZnO) layers. AOHs significantly reduce the work function of metal oxides, and are further proven to effectively passivate defect states in these metal oxides. The interfacial energetics of these electron collecting contacts with a prototypical electron acceptor (C₆₀) are investigated to reveal the presence of a large interface dipole and a new interface state between the Fermi energy and the C₆₀ highest occupied molecular orbital for AOH‐modified AZO contacts. These novel interfacial gap states are a result of ground‐state electron transfer from the metal hydroxide‐functionalized AZO contact to the adsorbed molecules, which are hypothesized to be electronically hybridized with the contact. These interface states tail all the way to the Fermi energy, providing for a highly n‐doped (metal‐like) interfacial molecular layer. Furthermore, the strong “light‐soaking” effect is no longer observed in devices with a AOH interface.     

Exposure of RNA templates and encapsidation of spliced viral RNA are influenced by the arginine-rich domain of human hepatitis B virus core antigen (HBcAg 165-173)

Previously, human hepatitis B virus (HBV) mutant 164, which has a truncation at the C terminus of the HBV core antigen (HBcAg), was speculated to secrete immature genomes. For this study, we further characterized mutant 164 by different approaches. In addition to the 3.5-kb pregenomic RNA (pgRNA), the mutant preferentially encapsidated the 2.2-kb or shorter species of spliced RNA, which can be reverse transcribed into double-stranded DNA before virion secretion. We observed that mutant 164 produced less 2.2-kb spliced RNA than the wild type. Furthermore, it appeared to produce at least two different populations of capsids: one encapsidated a nuclease-sensitive 3.5-kb pgRNA while the other encapsidated a nuclease-resistant 2.2-kb spliced RNA. In contrast, the wild-type core-associated RNA appeared to be resistant to nuclease. When arginines and serines were systematically restored at the truncated C terminus, the core-associated DNA and nuclease-resistant RNA gradually increased in both size and signal intensity. Full protection of encapsidated pgRNA from nuclease was observed for HBcAg 1-171. A full-length positive-strand DNA phenotype requires positive charges at amino acids 172 and 173. Phosphorylation at serine 170 is required for optimal RNA encapsidation and a full-length positive-strand DNA phenotype. RNAs encapsidated in Escherichia coli by capsids of HBcAg 154, 164, and 167, but not HBcAg 183, exhibited nuclease sensitivity; however, capsid instability after nuclease treatment was observed only for HBcAg 164 and 167. A new hypothesis is proposed here to highlight the importance of a balanced charge density for capsid stability and intracapsid anchoring of RNA templates.