Endolysosomal Trafficking Controls Yolk Granule Biogenesis in Vitellogenic Drosophila Oocytes

Endocytosis and endolysosomal trafficking are essential for almost all aspects of physiological functions of eukaryotic cells. As our understanding on these membrane trafficking events are mostly from studies in yeast and cultured mammalian cells, one challenge is to systematically evaluate the findings from these cell-based studies in multicellular organisms under physiological settings. One potentially valuable in vivo system to address this challenge is the vitellogenic oocyte in Drosophila, which undergoes extensive endocytosis by Yolkless (Yl), a low-density lipoprotein receptor (LDLR), to uptake extracellular lipoproteins into oocytes and package them into a specialized lysosome, the yolk granule, for storage and usage during later development. However, by now there is still a lack of sufficient understanding on the molecular and cellular processes that control yolk granule biogenesis. Here, by creating genome-tagging lines for Yl receptor and analyzing its distribution in vitellogenic oocytes, we observed a close association of different endosomal structures with distinct phosphoinositides and actin cytoskeleton dynamics. We further showed that Rab5 and Rab11, but surprisingly not Rab4 and Rab7, are essential for yolk granules biogenesis. Instead, we uncovered evidence for a potential role of Rab7 in actin regulation and observed a notable overlap of Rab4 and Rab7, two Rab GTPases that have long been proposed to have distinct spatial distribution and functional roles during endolysosomal trafficking. Through a small-scale RNA interference (RNAi) screen on a set of reported Rab5 effectors, we showed that yolk granule biogenesis largely follows the canonical endolysosomal trafficking and maturation processes. Further, the data suggest that the RAVE/V-ATPase complexes function upstream of or in parallel with Rab7, and are involved in earlier stages of endosomal trafficking events. Together, our study provides s novel insights into endolysosomal pathways and establishes vitellogenic oocyte in Drosophila as an excellent in vivo model for dissecting the highly complex membrane trafficking events in metazoan

Gene Expression from the ORF50/K8 Region of Kaposi's Sarcoma-Associated Herpesvirus

The ORF50 gene of Kaposi's sarcoma (KS)-associated herpesvirus, or human herpesvirus 8 (KSHV), activates viral replication and is weakly homologous to the herpesvirus family of R transactivators; therefore, the transcription and translation events from this region of KSHV are key events in viral reactivation. We demonstrate that ORF50 is expressed in a bicistronic message after induction of the viral lytic cycle. ORF50 migrated as a series of polypeptides: the major ones as 119 and 101 kDa, respectively. Using 3' rapid amplification of cDNA ends, RT-PCR, and cDNA library screening, we demonstrate that the major ORF50 transcript also encodes K8. The ORF50/K8 transcript was resistant to cyclohexamide, whereas the K8 transcript was only partially resistant to cyclohexamide at early timepoints. Both transcripts showed partial resistance after 12 h of phorbol ester induction. Using a GAL4-ORF50 fusion protein expression vector, we demonstrate that the transactivation domain of ORF50 resides within a 160-amino-acid region of the carboxyl portion of the ORF. Upstream regions of both ORF50 and K8 have basal promoter activity in KSHV-infected cells. K8, which had sequence homology to Bzip proteins, did not activate either promoter. However, both promoters were activated after cotransfection of ORF50 in BCBL-1 cells

Sealing of oil-gas reservoir caprock: Destruction of shale caprock by micro-fractures

The sealing ability of caprock is affected by many factors, such as cap thickness, displacement pressure, fracture development, and lithology of caprock. Shale is one of the ideal materials for oil and gas sealing cap formation due to its low porosity and permeability. Microfractures can destroy the sealing property of shale caprock. When buried deep enough, shale will change from toughness to brittleness. In general, the greater the brittleness of shale, the more developed the fractures will be. In areas with high tectonic stress, such as the anticline axis, syncline axis and stratum dip end, the strata stress is high and concentrated, and it is easier to generate fractures. When the stress state of the caprock changes, new micro-cracks are formed or previously closed cracks are re-opened, reducing the displacement pressure of the caprock. These micro-fractures are interconnected to form microleakage spaces, which reduces the sealing capacity of the caprock

Revealing mechanism of pore defect formation in laser directed energy deposition of aluminum alloy via in-situ synchrotron X-ray imaging

Laser metal additive manufacturing technology is capable of producing components with complex geometries and compositions that cannot be realized by conventional manufacturing methods. However, a large number of pores generated during the additive manufacturing process greatly affect the mechanical properties of the additively manufactured parts, and the mechanism of such pore generation has not been revealed by direct observation clearly. Here, we report the mechanism of pore generation in the laser direct energy deposition process as revealed by {\it in-situ} high-speed high-resolution synchrotron X-ray imaging. We found that dissolution and re-precipitation of external gases and precipitation of metal vapors are the two main mechanisms of pore formation. We further explored the effects of different process parameters on the generation of pores and optimized the process to suppress pore generation. This work provides important insights into the formation of porosity defects during laser metal additive manufacturing, and can provide guidance for related process optimization.Comment: 7 figure

iTRAQ-Based Quantitative Proteomics Analysis of the Protective Effect of Yinchenwuling Powder on Hyperlipidemic Rats

Yinchenwuling powder (YCL) is an effective traditional Chinese medicine formula to modulate lipid levels. In this study, we established hyperlipidemic rat models and treated them with YCL. The serum concentrations of lipid, malondialdehyde (MDA), endothelin-1 (ET-1), and calcitonin gene-related peptide (CGRP) were measured. Adventitia-free vascular proteins between hyperlipidemic rats and YCL-treated rats were identified using iTRAQ-based quantitative proteomics research approach. Proteins with 1.3-fold difference were analyzed through bioinformatics, and proteomic results were verified by Western blot. The results showed that the serum levels of TC, TG, LDL-C, ET-1, and MDA were significantly decreased, whereas the HDL-C and CGRP levels were significantly increased in the YCL-treated group. Proteomics technology identified 4,382 proteins, and 15 proteins were selected on the basis of their expression levels and bioinformatics. Of these proteins, 2 (Adipoq and Gsta1) were upregulated and 13 (C3, C4, C6, Cfh, Cfp, C8g, C8b, Lgals1, Fndc1, Fgb, Fgg, Kng1, and ApoH) were downregulated in the YCL-treated rats. Their functions were related to immunity, inflammation, coagulation and hemostasis, oxidation and antioxidation, and lipid metabolism and transport. The validated results of ApoH were consistent with the proteomics results. This study enhanced our understanding on the therapeutic effects and mechanism of YCL on hyperlipidemia

Copper cocatalyst modulated radical generation for selective heterogeneous photosynthesis of α‑haloketones

The α-haloketones are important precursors for synthetic chemistry and pharmaceutical applications; however, their production relies heavily on traditional synthetic methods via halogenation of ketones that are toxic and environmentally risky. Here, we report a heterogeneous photosynthetic strategy of α-haloketone production from aromatic olefins using copper-modified graphitic carbon nitride (Cu–C3N4) under mild reaction conditions. By employing NiX2 (X = Cl, Br) as the halogen source, a series of α-haloketones can be synthesized using atmospheric air as the oxidant under visible-light irradiation. In comparison with pristine carbon nitride, the addition of Cu as a cocatalyst provides a moderate generation rate of halogen radicals and selective reduction of molecular oxygen into •OOH radicals, thus leading to a high selectivity to α-haloketones. The Cu–C3N4 also exhibits high stability and versatility, rendering it a promising candidate for solar-driven synthetic applications

E1A-engineered human umbilical cord mesenchymal stem cells as carriers and amplifiers for adenovirus suppress hepatocarcinoma in mice

Gene therapy is an attractive approach for hepatocellular carcinoma (HCC) patients. Nevertheless, efficient transgene delivery remains a challenge. In this study, we explored a new targeted system based on human umbilical cord-derived mesenchymal stem cells (HUMSCs), which were engineered to deliver adenovirus to tumor sites, and to replicate and assemble into new adenovirus against HCC. Our results showed that HUMSCs infected by Ad-hTERTp-IL24 followed by LentiR.E1A infection could specifically migrate to HepG2 tumor cells and support adenoviral replication in vitro and in vivo 36 h after LentiR.E1A infection. Ad-hTERTp-IL24 specifically inhibited HepG2 cells growth, and this inhibitory effect was enhanced by low doses of 5-fluorouracil (5-Fu), because the expression levels of coxsackie adenovirus receptor (CAR) and integrin ανβ3 on tumor cells were significantly increased, causing higher viral uptake. Compared with the no treatment groups, Ad-hTERTp-IL24 and LentiR.E1A co-loaded HUMSCs exhibited significant anti-tumor activity in vivo, particularly in combination with low doses of 5-Fu. In summary, this study provides a promising targeted gene therapeutic strategy dependent on the tumor tropism of HUMSCs, to improve the outcome of virotherapy for tumor patients especially those with metastatic diseases