Interrogation of human microglial phagocytosis by CRISPR genome editing.

Background: Microglia are an integral part of central nervous system, but our understanding of microglial biology is limited due to the challenges in obtaining and culturing primary human microglia. HMC3 is an important cell line for studying human microglia because it is readily accessible and straightforward to maintain in standard laboratories. Although HMC3 is widely used for microglial research, a robust genetic method has not been described. Here, we report a CRISPR genome editing platform, by the electroporation of Cas9 ribonucleoproteins (Cas9 RNP) and synthetic DNA repair templates, to enable rapid and precise genetic modifications of HMC3. For proof-of-concept demonstrations, we targeted the genes implicated in the regulation of amyloid beta (Aß) and glioblastoma phagocytosis in microglia. We showed that CRISPR genome editing could enhance the phagocytic activities of HMC3. Methods: We performed CRISPR gene knockout (KO) in HMC3 by the electroporation of pre-assembled Cas9 RNP. Co-introduction of DNA repair templates allowed site-specific knock-in (KI) of an epitope tag, a synthetic promoter and a fluorescent reporter gene. The editing efficiencies were determined genotypically by DNA sequencing and phenotypically by immunofluorescent staining and flow cytometry. The gene-edited HMC3 cells were examined in vitro by fluorescent Aß and glioblastoma phagocytosis assays. Results: Our platform enabled robust single (>90%) and double (>70%) KO without detectable off-target editing by high throughput DNA sequencing. We also inserted a synthetic SFFV promoter to efficiently upregulate the expression of endogenous CD14 and TREM2 genes associated with microglial phagocytosis. The CRISPR-edited HMC3 showed stable phenotypes and enhanced phagocytosis of fluorescence-labeled Aß1-42 peptides. Confocal microscopy further confirmed the localization of Aß1-42 aggregates in the acidified lysosomes. HMC3 mutants also changed the phagocytic characteristic toward apoptotic glioblastoma cells. Conclusion: CRISPR genome editing by Cas9 RNP electroporation is a robust approach to genetically modify HMC3 for functional studies such as the interrogation of Aß and tumor phagocytosis, and is readily adoptable to investigate other aspects of microglial biology.

Image copy-move forgery detection and localization based on super-BPD segmentation and DCNN.

With the increasing importance of image information, image forgery seriously threatens the security of image content. Copy-move forgery detection (CMFD) is a greater challenge because its abnormality is smaller than other forgeries. To solve the problem that the detection results of the most image CMFD based on convolutional neural networks (CNN) have relatively low accuracy, an image copy-move forgery detection and localization based on super boundary-to-pixel direction (super-BPD) segmentation and deep CNN (DCNN) is proposed: SD-Net. Firstly, the segmentation technology is used to enhance the connection between the same or similar image blocks, improving the detection accuracy. Secondly, DCNN is used to extract image features, replacing conventional hand-crafted features with automatic learning features. The feature pyramid is used to improve the robustness to the scaling attack. Thirdly, the image BPD information is used to optimize the edges of rough detected image and obtain final detected image. The experiments proved that the SD-Net could detect and locate multiple, rotated, and scaling forgery well, especially large-level scaling forgery. Compared with other methods, the SD-Net is more accurately located and robust to various post-processing operations: brightness change, contrast adjustments, color reduction, image blurring, JPEG compression, and noise adding.

The last giants of the Yangtze River: A multidisciplinary picture of what remains of the endemic Chinese sturgeon.

The Chinese sturgeon, an important endemism of the Yangtze River, belongs to 'the most critically endangered group of species' worldwide, with overfishing and habitat destruction being the main drivers towards extinction. Newly obtained microchemical comparisons between animals and water from different river locations revealed a probable shifting of the spawning ground few kilometers downstream compared to the only previously known site, located under the Gezhouba Dam. This offers a glimmer of hope for an adaptive response to habitat perturbation caused by the recently built Three Gorges dam on the Yangtze River. On the other hand, genetic data provide an estimate of about 20 breeders participating in the only significant breeding event of the past 10 years. This warns of a near species extinction forecast if no in situ and ex situ conservation efforts occur promptly. Given these results we propose a list of priority conservation actions that urgently need to be promoted, supported, and put into practice.

Channel estimation based on superimposed pilot and weighted averaging.

Channel estimation based on superimposed pilot (SP) is a challenge in orthogonal frequency division multiplexing (OFDM) systems. To reduce the pilot data interference in the SP and estimate the channel state information accurately, a weighted averaging (WA) channel estimation method based on the superimposed pilot is proposed in this paper. At the transmitter, two signals with data symbols and pilot symbols superimposed at different subcarriers are transmitted. At the receiver, the elimination scheme is proposed to remove the pilot data interference. Based on the temporal correlation of wireless channels, the WA method is used to reduce the interference caused by additive white Gaussian noise in the channel. Simulation results verify that the proposed method can be applied to different channel scenarios. It has better normalized mean square error and bit error rate performance than other existing methods, and the superimposed pilot can improve the throughput of wireless communication systems.

Effectiveness assessment of using riverine water eDNA to simultaneously monitor the riverine and riparian biodiversity information.

Both aquatic and terrestrial biodiversity information can be detected in riverine water environmental DNA (eDNA). However, the effectiveness of using riverine water eDNA to simultaneously monitor the riverine and terrestrial biodiversity information remains unidentified. Here, we proposed that the monitoring effectiveness could be approximated by the transportation effectiveness of land-to-river and upstream-to-downstream biodiversity information flows and described by three new indicators. Subsequently, we conducted a case study in a watershed on the Qinghai-Tibet Plateau. The results demonstrated that there was higher monitoring effectiveness on summer or autumn rainy days than in other seasons and weather conditions. The monitoring of the bacterial biodiversity information was more efficient than the monitoring of the eukaryotic biodiversity information. On summer rainy days, 43-76% of species information in riparian sites could be detected in adjacent riverine water eDNA samples, 92-99% of species information in riverine sites could be detected in a 1-km downstream eDNA sample, and half of dead bioinformation (the bioinformation labeling the biological material that lacked life activity and fertility) could be monitored 4-6 km downstream for eukaryotes and 13-19 km downstream for bacteria. The current study provided reference method and data for future monitoring projects design and for future monitoring results evaluation.

Key Characteristics of Nitrous Oxide-Induced Neurological Disorders and Differences Between Populations.

Background: Nitrous oxide (N2O), commonly known as laughing gas, is inhaled recreationally because it produces the feelings of euphoria and freedom from pain. The risk of neurological dysfunction secondary to N2O abuse and its clinical diagnosis are, however, not yet sufficiently recognized, especially in China. Here, we have summarized the key clinical characteristics of N2O-induced neurological disorders. Materials and Methods: We recruited 20 patients with N2O-induced neurological disorders and analyzed their clinical features, laboratory data, magnetic resonance imaging and electromyography. We also carried out a literature review and compared 99 previously reported patients with our case series to confirm our results. Subgroup analysis was performed to explore the difference in demographical and clinical characteristics of N2O abuse between Asian and non-Asian patients. Results: The most common initial symptoms of N2O-induced neurological disorders were weakness and/or paresthesia. Most patients presented with myelopathy and/or peripheral neuropathy. The most commonly involved segment of the spinal cord was the cervical spinal cord, extending over 4-6 vertebral levels, but more than half of the patients with myelopathy had no sensory change at the corresponding spinal level. Homocysteine was found to be the most sensitive and practical indicator for diagnosis. Subgroup analysis showed that the Asian patients (median: 22.0 years old, Q1-Q3:19.0-26.0 years old) with N2O abuse were younger than non-Asian patients [26.0 (22.3-31.0) years old, P = 2.8 × 10-4]. The incidence of myelopathy combined with peripheral neuropathy was significantly higher in Asian patients than in non-Asian patients, who had myelopathy or peripheral neuropathy (P = 2 × 10-5). Conclusions: Key clinical characteristics of N2O abuse are longitudinally extensive cervical myelopathy and peripheral neuropathy. Recognition of these traits in young people in the age group of 20-30 years will provide important guidance for accurate diagnosis of neurological disease associated with N2O abuse. The clinical manifestations differ in Asian patients and non-Asian patients.

Association Between Air Pollution and the Risk of Uveitis: A Nationwide, Population-Based Cohort Study.

Previous studies have revealed an association between ocular surface disorders and air pollution, few studies have focused on the risk of uveitis. We aimed to investigate whether air pollution increases the risk of uveitis. We used the Taiwan Longitudinal Health Insurance Database (LHID) and the Taiwan Air Quality Monitoring Database (TAQMD) to conduct a retrospective cohort study. Air pollutant concentrations, including those of carbon dioxide (CO2), were grouped into four levels according to quartiles. The outcome was the incidence of uveitis, as defined in the International Classification of Diseases, Ninth Revision. We used univariable and multivariable Cox proportional hazard regression models to calculate the adjusted hazard ratios (aHRs) and determine the potential risk factors of uveitis. Overall, 175,489 subjects were linked to their nearby air quality monitoring stations. We found that for carbon monoxide, the aHRs of uveitis risk for the Q3 and Q4 levels were 1.41 (95% confidence interval (CI) = 1.23-1.61) and 2.19 (95% CI = 1.93-2.47), respectively, in comparison with those for the Q1 level. For nitric oxide, the aHRs for the Q3 and Q4 levels were 1.46 (95% CI = 1.27-1.67) and 2.05 (95% CI = 1.81-2.32), respectively. For nitrogen oxide (NOx), the aHRs for the Q2, Q3, and Q4 levels were 1.27 (95% CI = 1.11-1.44), 1.34 (95% CI = 1.16-1.53), and 1.85 (95% CI = 1.63-2.09), respectively. For total hydrocarbon (THC), the aHRs for the Q2, Q3, and Q4 levels were 1.42 (95% CI = 1.15-1.75), 3.80 (95% CI = 3.16-4.57), and 5.02 (95% CI = 4.19-6.02), respectively. For methane (CH4), the aHRs for the Q3 and Q4 levels were 1.94 (95% CI = 1.60-2.34) and 7.14 (95% CI = 6.01-8.48), respectively. In conclusion, air pollution was significantly associated with incidental uveitis, especially at high THC and CH4 levels. Furthermore, the uveitis risk appeared to increase with increasing NOx and THC levels.

Metformin Use Was Associated With Reduced Risk of Incidental Sjögren's Syndrome in Patients With Type 2 Diabetes: A Population-Based Cohort Study.

Purpose: Previous studies have shown that metformin exhibits an anti-inflammatory effect and may decrease the risk of incidental diabetes. But the effect of metformin on incidental Sjögren's syndrome is unknown. The aim of the study was to examine the association between metformin exposure and Sjögren's syndrome in diabetic patients. Methods: The dataset in this retrospective cohort study was obtained from the National Health Insurance Research Database (2000-2013) in Taiwan. In total, 15,098 type 2 diabetic patients under metformin treatment and an equivalent number without metformin treatment matched for comparison were included. The primary endpoint was the incidence of Sjogren's syndrome. Univariate and multivariate Cox proportional hazards models were used for data analysis. A subgroup analysis and sensitivity test were also performed. Results: The incidence rate of Sjögren's syndrome in non-metformin controls was 40.83 per 100,000 person-years and 16.82 per 100,000 person-years in metformin users. The adjusted hazard ratio (aHR) in diabetic patients under metformin treatment was 0.46 (95% CI, 0.23 to 0.92). In subgroup analysis, men had a lower risk of developing Sjögren's syndrome than women [aHR = 0.15, 95% CI = (0.05, 0.41)]. After prescribing metformin to type 2 diabetic patients aged 60 years or more, those patients had a lower risk of developing Sjögren's syndrome [aHR = 0.34, 95% CI = (0.12, 0.96)]. Conclusion: In this large population-based cohort study, metformin exposure was associated with a reduced risk of developing Sjögren's syndrome in type 2 diabetic patients.

The American Paddlefish Genome Provides Novel Insights into Chromosomal Evolution and Bone Mineralization in Early Vertebrates.

Sturgeons and paddlefishes (Acipenseriformes) occupy the basal position of ray-finned fishes, although they have cartilaginous skeletons as in Chondrichthyes. This evolutionary status and their morphological specializations make them a research focus, but their complex genomes (polyploidy and the presence of microchromosomes) bring obstacles and challenges to molecular studies. Here, we generated the first high-quality genome assembly of the American paddlefish (Polyodon spathula) at a chromosome level. Comparative genomic analyses revealed a recent species-specific whole-genome duplication event, and extensive chromosomal changes, including head-to-head fusions of pairs of intact, large ancestral chromosomes within the paddlefish. We also provide an overview of the paddlefish SCPP (secretory calcium-binding phosphoprotein) repertoire that is responsible for tissue mineralization, demonstrating that the earliest flourishing of SCPP members occurred at least before the split between Acipenseriformes and teleosts. In summary, this genome assembly provides a genetic resource for understanding chromosomal evolution in polyploid nonteleost fishes and bone mineralization in early vertebrates.

Extinction of one of the world's largest freshwater fishes: Lessons for conserving the endangered Yangtze fauna.

The mega river ecosystem of the Yangtze River was once home to diverse aquatic megafauna but is increasingly affected by various anthropogenic stressors that have resulted in continuous loss of biodiversity, such as the probable extinction of Yangtze River Dolphin. The Chinese paddlefish, Psephurus gladius, was one of only two extant members of a relict lineage that was most diverse and widespread 34-75 million years ago. It is also one of the largest freshwater fish species, reaching up to 7 m in length. The Chinese paddlefish was once common in the Yangtze River, with c.25 t being harvested per annum during the 1970s. Populations have, however, declined drastically since the late 1970s as a result of overfishing and habitat fragmentation. Here, a basin-wide capture survey during 2017-2018 found 332 fish species, but did not find a single specimen of Chinese paddlefish. Furthermore, 140 historically reported fish species have not been found and most of them are considered highly endangered. Based on 210 sightings of Chinese paddlefish during the period 1981-2003, we estimated the timing of extinction to be by 2005, and no later than by 2010. In addition, the paddlefish probably became functionally extinct (i.e. it was unable to reproduce) by 1993, before it went extinct. It is likely that the lack of reproduction was among the major causes of extinction. As no individuals exist in captivity, and no living tissues are conserved for potential resurrection, the fish should be considered extinct according to the IUCN Red List criteria. The delayed extinction of Chinese paddlefish resulted from multiple threats, suggesting that optimizing conservation efforts on endangered Yangtze fauna is urgently needed.

Are river protected areas sufficient for fish conservation? Implications from large-scale hydroacoustic surveys in the middle reach of the Yangtze River.

BACKGROUND: The Yangtze River is the third largest river in the world and suffers from extensive anthropogenic impacts. The fishes in the Yangtze River are essential for the sustainable development of freshwater fisheries and the conservation of aquatic biodiversity in China. However, the fishery resources in the Yangtze River Basin have shown rapid decline due to various human activities. In recent years, nature reserves and germplasm resource reserves have become important means to protect fishes in the Yangtze River. However, nature reserves and germplasm resource reserves that regard freshwater fishes as the main object of protection are not common and have been rarely studied in China. In this paper, a hydroacoustic method and systematic conservation planning tool (Marxan) were combined to evaluate the effectiveness of reserves based on the spatial and temporal patterns of mature fishes in the middle reach of the Yangtze River (MRYR) from 2010 to 2017. RESULTS: The hydroacoustic survey results indicated that in the longitudinal direction, low densities of mature fish species were observed in the Jingzhou (S2) and Jianli (S4, S5, S6) sections, whereas high densities of fish were observed in other sections, such as the Yichang (S1), Chenglingji to Huangsangkou (S7-S12), and Hukou (S15) sections. Among the regions preferred by fish, S7, S10 and S12 were non-reserves. No significant difference in mature fish density was observed between the non-reserves and nature reserves, and a similar result was obtained between the non-reserves and germplasm resource reserves. In Marxan, the optimal conservation sites selected for habitat restoration, such as the Chenglingji, Dengjiakou, Zhuankou, Hankou, Yangluo, and Huangsangkou sections, which are located in non-reserves, were identified in the MRYR. CONCLUSIONS: The Chenglingji, Dengjiakou, Zhuankou, Hankou, Yangluo, and Huangsangkou sections, which are located in non-reserves, play equally important roles in the conservation of fish populations in the MRYR. Our results indicated that further optimization is urgently needed for the currently protected areas in this region. These areas should be designated as reserves, and classification protection mechanisms should be adopted to strengthen the effectiveness of fish conservation in the MRYR.

Increasing River Temperature Shifts Impact the Yangtze Ecosystem: Evidence from the Endangered Chinese Sturgeon.

The Yangtze River has the third greatest water flow and is one of the most human-influenced rivers in the world. Since 1950, this river system has experienced drastic human interventions, leading to various environmental changes, including water temperature. In this study, based on observations during the past sixty years, we found that the seasonal temperature regime has been altered, both temporally (1-5 °C variation) and spatially (>626 km distance). Temperature shifts not only delay the timing of fish spawning directly, but also lead to degeneration in gonad development. Temperature regime alterations have delayed the suitable spawning temperature window by approximately 29 days over a decade (2003-2016). It confirmed that a period of lower temperature, higher cumulative temperature, and relatively higher temperature differences promoted the maturation of potential spawners based on the correlation analysis (p < 0.05). Also, thermal alterations were highly correlated with reservoir capacity upstream (R2 = 0.866). On-going cascade dam construction and global warming will lead to further temperature shifts. Currently, rigorous protection measures on the breeding population of the Chinese sturgeon and its critical habitats is urgently needed to prevent the crisis of the species extinction. Increasing river thermal shifts not only threaten the Chinese sturgeon but also affect the entire Yangtze aquatic ecosystem.

Spatiotemporal Distribution and Assemblages of Fishes below the Lowermost Dam in Protected Reach in the Yangtze River Main Stream: Implications for River Management.

Now more and more ecologists concern about the impacts of dam construction on fish. However, studies of fishes downstream Gezhouba Dam were rarely reported except Chinese sturgeon (Acipenser sinensis Gray). In this study, catch investigations and five hydroacoustic detections were completed from 2015 to 2016 to understand the distribution, size, and categories of fishes and their relationship with the environmental factors below Gezhouba Dam in protected reach in the Yangtze River main stream. Results showed significant differences in fish distribution and TS (target strength) between wet and flood seasons. Mean TS in five hydroacoustic detections were -59.98 dB, -54.70 dB, -56.16 dB, -57.90 dB, and -59.17 dB, respectively, and dominant fish species are Coreius guichenoti (Bleeker), Siniperca chuatsi (Basilewsky), and Pelteobagrus vachelli (Richardson). In the longitudinal direction, fish preferred to stay in some specific sections like reaches 2, 4, 7, 8, 11, and 16. Since hydrology factors change greatly in different seasons, environmental characteristics vary along the reaches, and human activities play an important role in the fish behavior, it is concluded that great cross-season changes in hydrology lead to the differences in TS and fish assemblages and that geography characteristics, especially channel geography, together with human activities influence fish longitudinal distribution. This finding provides basic knowledge of spatiotemporal distribution and assemblages of fishes in the extended reaches downstream Gezhouba Dam. In addition, it offers implications for river management. It could also serve as reference of future research on fish habitat.

Hepatocellular carcinoma stem cell-like cells are enriched following low-dose 5-fluorouracil chemotherapy.

It has been proposed that cancer stem cells (CSCs) are involved in tumor resistance to chemotherapy and tumor relapse. The goal of the present study was to determine the effect of low-dose 5-fluorouracil (5-Fu) on enriched hepatocellular CSC-like cells. Increased cell motility and epithelial-mesenchymal transition were observed by migration assay in human hepatoblastoma PLC/RAF/5 cells following 5-Fu treatment, as well as a significant enhancement in their sphere-forming abilities. CSC-like cells were identified by side population cell analysis. The percentage of CSC-like cells in the surviving cells was greatly increased in response to 5-Fu. These findings indicate that low-dose 5-Fu treatment may efficiently enrich the CSC-like cell population in PLC/RAF/5 cells.

[Searching for WDMS Candidates In SDSS-DR10 With Automatic Method].

The Sloan Digital Sky Survey (SDSS) has released the latest data (DR10) which covers the first APOGEE spectra. The massive spectra can be used for large sample research inscluding the structure and evolution of the Galaxy and multi-wave-band identi cation. In addition, the spectra are also ideal for searching for rare and special objects like white dwarf main-sequence star (WDMS). WDMS consist of a white dwarf primary and a low-mass main-sequence (MS) companion which has positive significance to the study of evolution and parameter of close binaries. WDMS is generally discovered by repeated imaging of the same area of sky, measuring light curves for objects or through photometric selection with follow-up observations. These methods require significant manual processing time with low accuracy and the real-time processing requirements can not be satisfied. In this paper, an automatic and efficient method for searching for WDMS candidates is presented. The method Genetic Algorithm (GA) is applied in the newly released SDSS-DR10 spectra. A total number of 4 140 WDMS candidates are selected by the method and 24 of them are new discoveries which prove that our approach of finding special celestial bodies in massive spectra data is feasible. In addition, this method is also applicable to mining other special celestial objects in sky survey telescope data. We report the identfication of 24 new WDMS with spectra. A compendium of positions, mjd, plate and fiberid of these new discoveries is presented which enrich the spectral library and will be useful to the research of binary evolution models.

(-)-Epigallocatechin-3-gallate inhibits VEGF expression induced by IL-6 via Stat3 in gastric cancer.

AIM: To demonstrate that (-)-Epigallocatechin-3-gallate (EGCG) inhibits vascular endothelial growth factor (VEGF) expression and angiogenesis induced by interleukin-6 (IL-6) via suppressing signal transducer and activator of transcription 3 (Stat3) activity in gastric cancer. METHODS: Human gastric cancer (AGS) cells were treated with IL-6 (50 ng/mL) and EGCG at different concentrations. VEGF, total Stat3 and activated Stat3 protein levels in the cell lyses were examined by Western blotting, VEGF protein level in the conditioned medium was measured by enzyme-linked immunosorbent assay, and the level of VEGF mRNA was evaluated by reverse transcription polymerase chain reaction (RT-PCR). Stat3 nuclear translocation was determined by Western blotting with nuclear extract, and Stat3-DNA binding activity was examined with Chromatin immunoprecipitation (ChIP) assay. IL-6 induced endothelial cell proliferation was measured with 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl tetrazoliumbromide assay, in vitro angiogenesis was determined with endothelial cell tube formation assay in Matrigel, and IL-6-induced angiogenesis in vitro was measured with Matrigel plug assay. RESULTS: There was a basal expression and secretion of VEGF in AGS cells. After stimulation with IL-6, VEGF expression was apparently up-regulated and a 2.4-fold increase was observed. VEGF secretion in the conditioned medium was also increased by 2.8 folds. When treated with EGCG, VEGF expression and secretion were dose-dependently decreased. IL-6 also increased VEGF mRNA expression by 3.1 folds. EGCG treatment suppressed VEGF mRNA expression in a dose-dependent manner. EGCG dose-dependently inhibited Stat3 activation induced by IL-6, but did not change the total Stat3 expression. When treated with EGCG or AG490, VEGF expressions were reduced to the level or an even lower level in the tumor cells not stimulated with IL-6. However, PD98059 and LY294002 did not change VEGF expression induced by IL-6. EGCG inhibited Stat3 nucleus translocation, and Stat3-DNA binding activity was also markedly decreased by EGCG. Furthermore, EGCG inhibited IL-6 induced vascular endothelial cell proliferation and tube formation in vitro and angiogenesis in vitro. CONCLUSION: EGCG inhibits IL-6-induced VEGF expression and angiogenesis via suppressing Stat3 activity in gastric cancer, which has provided a novel mechanistic insight into the anti-angiogenic activity of EGCG.

Dissociation between visual line bisection and mental number line bisection in schizophrenia.

Many studies of hemispatial neglect patients have indicated that spatial attention processes operate similarly in visual space and number space. However, some studies have indicated a dissociation of processing between visual line bisection and mental number bisection. A number of investigations have suggested that schizophrenic patients show a mild right pseudo-neglect on visual line bisection tasks. The present study was designed to determine if a functional link exists between performance of visual line and number line bisection in schizophrenic patients. Groups of 40 schizophrenic patients and 40 controls performed each bisection task. In the visual line bisection task, schizophrenic patients showed a significant leftward bias relative to the healthy controls for 9 different line lengths. No significant difference in bias was found between the 2 groups on the mental line bisection task. These results indicated that schizophrenic patients may exhibit attention deficit with respect to visual space but not number space, suggestive of the dissociation of processing between visual line bisection and mental number line bisection. These results provide more insight into the correlation between the visual line and number bisection tasks in schizophrenic patients.