Biochemical and Structural Characterization of the Atg8/LC3 Lipidation Pathway

Atg8 and its eukaryotic orthologues LC3 and GARBARAP family proteins (referred here to Atg8 family proteins) play crucial roles in autophagy through their covalent ligation to lipids, typically phosphatidylethanolamine (PE), in a process known as lipidation. Lipidation of Atg8 family proteins regulates numerous facets of the autophagy process, including regulating expansion of the phagophore membrane, recruiting selected cargoes for degradation, and providing an autophagosome membrane-bound platform mediating dynamic interactions with other regulatory proteins. Atg8 family proteins are ubiquitin-like proteins (UBLs), and their lipidation involves a divergent UBL conjugation cascade including Atg7, Atg3, and Atg12–Atg5-Atg16 acting as E1, E2, and E3 enzymes, respectively. Atg7 initiates Atg8 conjugation by catalyzing their C-terminal adenylation and conjugation to the catalytic cysteine of Atg3. Ultimately, the Atg12–Atg5-Atg16 complex catalyzes Atg8 ligation to a primary amino group on PE or other acceptor lipids. Molecular mechanisms underlying Atg8 lipidation remain poorly understood despite association of Atg3, the E1 Atg7, and the composite E3 Atg12–Atg5-Atg16 with pathologies including cancers, infections, and neurodegeneration. The first part of this dissertation work describes methods for expressing and purifying human LC3 or GABARAP, ATG7, ATG3, and the ATG12–ATG5-ATG16L1 complex for in vitro studies of LC3/GABARAP lipidation; based on these protocol established, we report that an Atg3 element we term E123IR (E1, E2, and E3-interacting region) is an allosteric switch, by studying yeast enzymes. Nuclear magnetic resonance (NMR), biochemical, crystallographic and genetic data collectively indicate that in the absence of the enzymatic cascade, the Atg3E123IR makes intramolecular interactions restraining Atg3’s catalytic loop, while E1 and E3 enzymes directly remove this brace to conformationally activate Atg3 and elicit Atg8 lipidation in vitro and in vivo. We propose that Atg3’s E123IR protects the E2~UBL thioester bond from wayward reactivity toward errant nucleophiles, while Atg8 lipidation casca

Biomass-derived three-dimensional porous N-doped carbonaceous aerogel for efficient supercapacitor electrodes

Functionalized carbonaceous materials with hierarchical structure and developed porosity are highly desired in energy storage and conversion fields. In this work, a facile and scalable hydrothermal methodology was established to synthesise three-dimensional (3D) N-doped carbonaceous aerogels using biomass-based starting materials and polypyrrole as N-source. The effect of different calcination temperatures on the structural properties, type and content of N-species and electrochemical performance of the 3D N-doped carbonaceous aerogels were uncovered. Thanks to the combinatorial effect of the appropriate N content and porous structure, the obtained samples exhibited excellent electrochemical performance, in particular, an outstanding specific capacitance of 281.0 F g-1 achieved on the sample calcined at 600 °C. This methodology offers a new fabrication strategy to prepare nanoscale carbonaceous materials with desirable morphology and hierarchical architecture of great potentials for the applications in energy fields

Needle δ13C and mobile carbohydrates in Pinus koraiensis in relation to decreased temperature and increased moisture along an elevational gradient in NE China

A tree's crown interacts with atmospheric variables such as CO2, temperature, and humidity. Physioecology of leaves/needles (e.g. δ13C, mobile carbohydrates, and nitrogen) is, therefore, strongly affected by microclimate in and surrounding a tree crown. To understand the physiological responses of leaves to changes in air temperature and moisture, we measured δ13C, soluble sugars, starch, and total nitrogen (N) concentrations in current year and 1-yr-old needles of Pinus koraiensis trees, and compared the growing season air temperature and relative humidity within and outside P. koraiensis crowns along an elevational gradient from 760 to 1,420ma.s.l. on Changbai Mountain, NE China. Our results indicated that needle N and mobile carbohydrates concentrations, as well as needle δ13C values changed continuously with increasing elevation, corresponding to a continuous decrease in air temperature and an increase in relative humidity. Needle carbon and nitrogen status is highly significantly negatively correlated with temperature, but positively correlated with relative humidity. These results indicate that increases in air temperature in combination with decreases in relative humidity may result in lower levels of N and mobile carbohydrates in P. koraiensis trees, suggesting that future climate changes such as global warming and changes in precipitation patterns will directly influence the N and carbon physiology at P. koraiensis individual level, and indirectly affect the competitive ability, species composition, productivity and functioning at the stand and ecosystem level in NE China. Due to the relatively limited range of the transect (760-1,420m) studied, further research is needed to explain whether the present results are applicable to scales across large elevational gradient

Type II taste cells participate in mucosal immune surveillance

The oral microbiome is second only to its intestinal counterpart in diversity and abundance but its effects on taste cells remains largely unexplored. Using single-cell RNASeq, we found that mouse taste cells, in particular, sweet and umami receptor cells that express taste 1 receptor member 3 (Tas1r3), have a gene expression signature reminiscent of Microfold (M) cells, a central player in immune surveillance in the mucosa-associated lymphoid tissue (MALT) such as those in the Peyer’s patch and tonsils. Administration of tumor necrosis factor ligand superfamily member 11 (TNFSF11; also known as RANKL), a growth factor required for differentiation of M cells, dramatically increased M cell proliferation and marker gene expression in the taste papillae and in cultured taste organoids from wild-type (WT) mice. Taste papillae and organoids from knockout mice lacking Spib (SpibKO), a RANKL-regulated transcription factor required for M cell development and regeneration on the other hand, failed to respond to RANKL. Taste papillae from SpibKO mice also showed reduced expression of NF-κB signaling pathway components and proinflammatory cytokines and attracted fewer immune cells. However, lipopolysaccharide-induced expression of cytokines was strongly up-regulated in SpibKO mice compared to their WT counterparts. Like M cells, taste cells from WT but not SpibKO mice readily took up fluorescently labeled microbeads, a proxy for microbial transcytosis. The proportion of taste cell subtypes are unaltered in SpibKO mice; however, they displayed increased attraction to sweet and umami taste stimuli. We propose that taste cells are involved in immune surveillance and may tune their taste responses to microbial signaling and infection

Cross-inhibition to heterologous foot-and-mouth disease virus infection induced by RNA interference targeting the conserved regions of viral genome

AbstractRNA interference (RNAi) is the process by which double-stranded RNA (dsRNA) directs sequence-specific degradation of messenger RNA in animal and plant cells. In mammalian cells, RNAi can be triggered by 21–23 nucleotide duplexes of small interfering RNA (siRNA). Strategies to inhibit RNA virus multiplication based on the use of siRNAs have to consider the high genetic polymorphism exhibited by this group of virus. Here we described a significant cross-inhibition of foot-and-mouth disease (FMD) virus (FMDV) replication in BHK-21 cells by siRNAs targeted to various conserved regions (5′NCR, VP4, VPg, POL, and 3′NCR) of the viral genome. The results showed that siRNAs generated in vitro by human recombinant dicer enzyme gave an inhibition of 10- to 1000-fold in virus yield of both homologous (HKN/2002) and heterologous (CHA/99) isolates of FMDV serotype O at 48 h post-infection (hpi). The inhibition extended to at least 6 days post-infection. For serotype Asia1, the virus yield in YNBS/58-infected cells examined at 12, 24, and 48 hpi decreased by ∼10-fold in cells pretreated with HKN/2002-specific siRNAs, but there was no significant decrease at 60 hpi. The inhibition was specific to FMDV replication, as no reduction was observed in virus yield of pseudorabies virus, an unrelated virus. Moreover, we also demonstrated an enhanced viral suppression could be achieved in BHK-21 cells with siRNA transfection after an infection had been established. These results suggested that siRNAs directed to several conserved regions of the FMDV genome could inhibit FMDV replication in a cross-resistance manner, providing a strategy candidate to treat high genetic variability of FMDV

Non-contact magnetic driving bioinspired Venus flytrap robot based on bistable anti-symmetric CFRP structure

The Venus flytrap takes advantage of its bistability to generate rapid closure motion for capturing its prey. A bioinspired Venus flytrap robot with bistable artificial leaves is presented in this paper. Non-contact electromagnetic driving method is proposed to actuate the Venus flytrap robot's artificial leaves, which are made of anti-symmetric carbon fiber reinforced prepreg (CFRP) cylindrical shells. Magnetic force is generated by using the electromagnet and applied on the shell's curve edge to unbend the shell, and then the bending process transmits from one edge to the whole surface. The required magnetic force for the snap-through process of the bistable CFRP structure is determined from experimental test and compared with the result of finite element simulation. The test of the snap-through process of the Venus flytrap robot show that the Venus flytrap robot can generate a rapid snapping motion by the electromagnet actuation

Prevalence of picky eating behaviour in Chinese school-age children and associations with anthropometric parameters and intelligence quotient. A cross-sectional study

AbstractPrevious studies have demonstrated the importance of eating behaviour regarding dietary variety and nutrient intake of children. However, the association between picky eating and growth of children is still a topic of debate. This study sought to estimate the prevalence of picky eating and to identify possible associations with the growth of school-age children in China. In this survey, 793 healthy children aged 7–12 years were recruited from nine cities and rural areas in China using a multi-stage cluster sampling method. Data collected included socio-demographic information and parents' perceptions of picky eating using a structured questionnaire, nutrient intake using 24-hour dietary recall, weight and height using body measurements, and intelligence using the Wechsler Intelligence Scale for Children. Blood samples were collected and analysed for minerals. The prevalence of picky eating reported by parents was 59.3% in children. Compared with non-picky eaters, picky eaters had a lower dietary intake of energy, protein, carbohydrates, most vitamins and minerals, and lower levels of magnesium, iron, and copper in the blood (p < 0.05), and also had a 0.184 z-score lower in height for age (95% CI: −0.332, 0.036; p = 0.015), a 0.385 z-score lower in weight for age (95% CI: −0.533, −0.237; p < 0.001), a 0.383 z-score lower in BMI for age (95% CI: −0.563, −0.203; p < 0.001), and scored 2.726 points higher on the intelligence test (95% CI: 0.809, 4.643; p = 0.006) when adjusted for children's birth weight and food allergy, mothers' education, and family income. Picky eating behaviour towards meat, eggs and vegetables showed negative associations with growth. Picky eating behaviour is prevalent in school-age children in China and may have a negative effect on growth

High expression of ubiquitin-conjugating enzyme 2C (UBE2C) correlates with nasopharyngeal carcinoma progression

BACKGROUND: Overexpression of ubiquitin-conjugating enzyme 2C (UBE2C) has been detected in many types of human cancers, and is correlated with tumor malignancy. However, the role of UBE2C in human nasopharyngeal carcinoma (NPC) is unclear. In this study, we investigated the role of aberrant UBE2C expression in the progression of human NPC. METHODS: Immunohistochemical analysis was performed to detect UBE2C protein in clinical samples of NPC and benign nasopharyngeal tissues, and the association of UBE2C expression with patient clinicopathological characteristics was analyzed. UBEC2 expression profiles were evaluated in cell lines representing varying differentiated stages of NPC and immortalized nasopharyngeal epithelia NP-69 cells using quantitative RT-PCR, western blotting and fluorescent staining. Furthermore, UBE2C was knocked down using RNA interference in these cell lines and proliferation and cell cycle distribution was investigated. RESULTS: Immunohistochemical analysis revealed that UBE2C protein expression levels were higher in NPC tissues than in benign nasopharyngeal tissues (P<0.001). Moreover, high UBE2C protein expression was positively correlated with tumor size (P=0.017), lymph node metastasis (P=0.016) and distant metastasis (P=0.015) in NPC patients. In vitro experiments demonstrated that UBE2C expression levels were inversely correlated with the degree of differentiation of NPC cell lines, whereas UBE2C displayed low level of expression in NP-69 cells. Knockdown of UBE2C led to significant arrest at the S and G2/M phases of the cell cycle, and decreased cell proliferation was observed in poorly-differentiated CNE2Z NPC cells and undifferentiated C666-1 cells, but not in well-differentiated CNE1 and immortalized NP-69 cells. CONCLUSIONS: Our findings suggest that high expression of UBE2C in human NPC is closely related to tumor malignancy, and may be a potential marker for NPC progression