Hibernation is a biological status during which hibernating animals acclimatize themselves to reduced energy consumption through extreme but governed decline in self-metabolism. The role of mitochondria (Mt) in metabolic suppression during hibernation has already been elaborated in different organs and species. Nonetheless, the concretely changing process of mitochondrial architecture and the mechanism underlying this transformation during hibernation remains unclear. Herein, the present study was aimed at clarifying the detailed alteration of mitochondrial morphology and its potential role in the Chinese soft-shelled turtle (Pelodiscus sinensis) during different stages of hibernation. Compared with the nonhibernation period, the mitochondrial architecture was changing from round to crescent, and lipid droplet (LD)/Mt interaction was enhanced during hibernation, as observed by transmission electron microscopy (TEM). Further ultrastructural analysis uncovered that mitochondrial fusion was promptly accelerated in the early stage of hibernation, followed by mitochondrial fission in the middle stage, and mitophagy was boosted in the late stage. Moreover, gene and protein expression related to mitochondrial fusion, fission, and mitophagy accorded closely with the mitochondrial ultrastructural changes in different stages of hibernation. Taken together, our results clarified that the transformation of mitochondrial architecture and mitochondrial dynamics are of vital importance in maintaining internal environment homeostasis of Pelodiscus sinensis.

The blood-brain barrier (BBB) is an important internal barrier. Herein, the electron microscope examination of duck BBB was performed during the brain development. Meanwhile, the genes/proteins of tight junctions (TJs) including zonula occludens-1, occludin, and claudin-5 in the duck brain were detected by Q-PCR and immunohistochemistry. The results showed the density of capillaries in the brain gradually increased during the embryonic period. The generation of the BBB and the specialization of its components occurred mainly in the embryonic stage. During this period, the endothelial cells (ECs) became thinner and pinocytic vesicles decreased; the TJs between EC membranes became longer and more electron-dense; the basement membrane surrounding ECs and pericytes gradually thickened; and the astrocyte foot processes appeared to wrap around the vessels. By the day of hatching (P1), the whole set of duck BBB structures was completely assembled and gradually improved in the subsequent growth process. Interestingly, compared with the cerebrum and cerebellum, the maturity level of the midbrain BBB was earlier seen during the embryonic stage. The expression of TJs increased during the embryonic period and remained stable by post-hatching. The study systematically investigated the histochemical and ultrastructural features of duck BBB during development and explored the corresponding relationship between structure and function.

Post-testicular maturation of spermatozoa is crucial for attaining the morphological and functional capabilities needed for successful fertilization. Epididymal epithelia offer a favorable environment for spermatozoa that are stored long term in the turtle epididymis; however, sperm–epithelial interactions during storage, which are enormously important for sperm functional and morphological maturation, are still largely unknown in turtles. The present study examined the epididymis during the sperm-storage period (November–April) in the Chinese soft-shelled turtle (Pelodiscus sinensis). Light and transmission electron microscopy were used to determine the cellular features of each epididymal segment (caput, corpus, and cauda) and their epithelial interactions with the spermatozoa. Spermatozoa were mainly located in the lumena of caput, corpus, and cauda epididymides. Numerous spermatozoa were bound to apical surfaces of the epithelia, and several were even embedded in the epithelial cytoplasm of the caput and corpus epididymides. No embedded spermatozoa were found in the cauda epididymis. In all epididymal segments, principal and clear cells showed the synthetic activity, evidenced by a well-developed endoplasmic reticulum network and high and low electron-dense secretory materials, respectively. Principal and clear cells in the caput and corpus segments showed embedded spermatozoa in electron-dense secretions and in the lipid droplets within the cytoplasm. No lysosomes were observed around the embedded spermatozoa. The lumena of the caput and corpus segments showed few apocrine and low electron density secretions. In the lumen of the cauda epididymidis, different secretions, such as holocrine with low and high electron density and their fragmentation, apocrine, and dictyosome, were found and are summarized. Altogether, sperm physical interactions with secretions either in the cytoplasm of epithelium or in the lumen may support the viability, morphological maintenance, and transfer of various proteins involved in long-term sperm storage in the turtle. This interaction could help us to understand the mechanisms of long-term sperm storage and provide more insights into the reproductive strategies of turtle sperm preservation.

The seminiferous tubule (ST) is the location of spermatogenesis, where mature spermatozoa are produced with the assistance of Sertoli cells. The role of extracellular vesicles in the direct communication between Sertoli-germ cells in the ST is still not fully understood. In this study, we reported multivesicular bodies (MVBs) and their source of CD63-enriched exosomes by light and ultrastructure microscopy during the reproductive phases of turtles. Strong CD63 immunopositivity was detected at the basal region in the early and luminal regions of the ST during late spermatogenesis by immunohistochemistry (IHC), immunofluorescence (IF), and western blot (WB) analysis. Labeling of CD63 was detected in the Sertoli cell cytoplasmic processes that surround the developing germ cells during early spermatogenesis and in the lumen of the ST with elongated spermatids during late spermatogenesis. Furthermore, ultrastructure analysis confirmed the existence of numerous MVBs in the Sertoli cell prolongations that surround the round and primary spermatogonia during acrosome biogenesis and with the embedded heads of spermatids in the cytoplasm of Sertoli cells. Additionally, in spermatids, Chrysanthemum flower centers (CFCs) generated isolated membranes involved in MVBs and autophagosome formation, and their fusion to form amphiosomes was also observed. Additionally, autophagy inhibition by 3-methyladenine (after 24 h) increased CD63 protein signals during late spermatogenesis, as detected by IF and WB. Collectively, our study found MVBs and CD63 rich exosomes within the Sertoli cells and their response to autophagy inhibition in the ST during the spermatogenesis in the turtle.

The present study was designed to investigate the in vivo biological processes of multivesicular bodies (MVBs) and exosomes in mitochondria-rich cells (MRCs), goblet cells (GCs), and absorptive cells (ACs) in turtle intestines during hibernation. The exosome markers, cluster of differentiation 63 (CD63) and tumor susceptibility gene 101 (TSG101), were positively expressed in intestinal villi during turtle hibernation. The distribution and formation processes of MVBs and exosomes in turtle MRCs, GCs, and ACs were further confirmed by transmission electron microscopy. During hibernation, abundantly secreted early endosomes (ees) were localized in the luminal and basal cytoplasm of the MRCs and ACs, and late endosomes (les) were dispersed with the supranuclear parts of the MRCs and ACs. Many “heterogeneous” MVBs were identified throughout the cytoplasm of the MRCs and ACs. Interestingly, the ees, les, and MVBs were detected in the cytoplasm of the GCs during hibernation; however, they were absent during nonhibernation. Furthermore, the exocytosis pathways of exosomes and autophagic vacuoles were observed in the MRCs, GCs, and ACs during hibernation. In addition, the number of different MVBs with intraluminal vesicles (ILVs) and heterogeneous endosome–MVB–exosome complexes was significantly increased in the MRCs, GCs, and ACs during hibernation. All these findings indicate that intestinal epithelial cells potentially perform a role in the secretion of MVBs and exosomes, which are essential for mucosal immunity, during hibernation.

The third generation of the BeiDou navigation satellite system (BDS-3) is a global navigation system, and is expected to be in full operation by 2020. High-precision orbits are a precondition for BDS-3 to provide a highly accurate service, which needs a global tracking and monitoring capability for the operational satellites. However, it is difficult for BDS to construct global ground monitoring stations. Fortunately, Ka-band Inter-Satellite Link (ISL) antennae fitted to the BDS-3 satellites can be used to extend the visible arc of the Medium Earth Orbit (MEO) satellites and to enhance the ground stations for orbit determination. This paper analyses the ISL-enhanced orbit determination for eight BDS-3 satellites, using the data from ten Chinese domestic stations and 13 international Global Navigation Satellite System (GNSS) Monitoring and Assessment System (iGMAS) overseas stations. The results show that the Three-Dimensional (3D) position Root Mean Square (RMS) error of the Overlapping Orbit Differences (OODs) is approximately 1 m when only ten regional stations are used. When the ISL measurements are added, the 3D position RMS error is decreased to 0·5 m, and the accuracy of the 24-hour orbit prediction can also be improved from 2 m to 0·7 m, which is even better than that of the orbits determined using globally distributed stations. It can be expected that with the subsequent launch of BDS-3 satellites and the increasing number of ISLs, the advantage of the ISL enhanced orbit determination will become more significant.

Camellia oleifera is an important woody plant producing healthy edible oils. People need a large number of molecular markers, especially microsatellite, in breeding of C. oleifera. In this study, we sequenced the root transcriptomes of C. oleifera, and then designed a novel set of microsatellite markers based on the root-expressed genes. We assembled a total of 57,121 unigenes with a length of 42.63 Mb, which harboured 15,902 microsatellites. Among these microsatellites, di-nucleotide repeat motifs were the most abundant group (56.45%), then followed by tri- (25.20%), mono- (12.12%), hexa- (3.21%), penta- (2.18%) and quad-nucleotide ones (0.84%). In total, 6738 primer pairs were designed successfully to amplify the microsatellite loci. To test these microsatellite markers, 48 primer pairs were randomly selected and synthesized and validated in C. oleifera and its eight relatives. Up to 75% of the primer pairs amplified in C. oleifera and its relatives, and 62.5% displayed polymorphism. The transferability and diverse alleles across its eight relatives were detected for each polymorphic primer pair. The novel set of microsatellites derived from the root transcriptomes here provided a useful resource for future molecular genetics improvement of C. oleifera and its relatives.

This paper describes CMP challenges in development of copper-low k process technology. As copper/oxide or copper/FSG backend schemes are being implemented successfully in early manufacturing, development focus has shifted to Cu/OSG (organo-silicate glass) integration development. Cu-OSG presents unique challenges with CMP integration, as these films tend to have much lower hardness than silicon dioxide. Significant process challenges have to be overcome prior to successfully implementing CMP process which does not mechanically damage the softer films and at the same time can achieve planarization requirements expected from CMP process. In addition, the OSG films tend to be hydrophobic leading to a need for developing improved cleaning processes/consumables. It was determined that Applied Materials ElectraPolishTM barrier slurry is extendable to OSG films. Good removal rate and removal profile can be achieved with ElectraPolishTM slurry. A proprietary cleaning solution reduced defect counts by 2 orders of magnitude as detected by SurfScan SS6200 on blanket OSG wafers. The same cleaning solution can be applied to copper/low-k patterned damascene wafers to clean both copper and dielectric surface. Polished OSG films have RMS roughness less than 2 angstroms and copper surface roughness about 5 angstroms with good surface finish. Blanket and patterned wafer results are presented to demonstrate final capability. Future directions for process enhancement are suggested.