Search

Apoptosis is programmed cell death and is regulated by a series of related genes. It is of great significance in resistance to pathogen invasion and maintaining homeostasis in the environment. The release of cytochrome c (Cytc) from the mitochondria into the cytoplasm is a key step in the initiation of apoptosis. Increasing evidence from investigation of Cytc in cell apoptosis and immunity shows that it can participate in cell apoptosis induced by virus infection. For example, white spot syndrome virus (WSSV) stimulation can induce Cytc gene expression in Litopenaeus vannamei hepatopancreas and hemocytes, and the apoptosis of Epinephelus akaara hepatocytes induced by red-spotted grouper nervous necrosis virus (RGNNV) is related to the release of Cytc. However, the role of Cytc-mediated apoptosis in Procambarus clarkii WSSV infection has not yet been reported. Therefore, in this study, the full length of the the cytochrome c gene of P. clarkii (PcCytc) was cloned, and the role of PcCytc in P. clarkii was analyzed. Its expression in various tissues of P. clarkii proved that WSSV infection can induce the expression of PcCytc. The mechanism of PcCytc involvement in cell apoptosis during WSSV infection was also explored using RNA interference technology, to gain a deeper understanding of the potential role of apoptosis-related factors in the immune response of P. clarkii.In this study, PcCytc was cloned using RACE technology, with a total length of 897 bp, including the 163 bp 5′-UTR, 419 bp 3′-UTR, and 315 bp open reading frame; it encoded 104 amino acids. The structure prediction showed that PcCytc contained a conserved Cytochrom_C domain, proving that it is related to energy production and tends to be conserved in evolution.The results of the quantitative PCR showed that the PcCytc gene was expressed in all tissues of P. clarkii. The expression was lowest in the stomach and higher in the gills, intestines, and muscles, which showed, respectively, 9.46, 8.65 and 7.88 times greater PcCytc expression than that in the stomach. PcCytc showed relatively high expression in tissues with high energy consumption, such as the intestines and muscles, which is consistent with previous studies in Penaeus vannamei. The highest expression level was observed in the gills of the main immune and respiratory tissues of P. clarkii, indicating that PcCytc may be involved in the related biological processes. Based on the above results, we speculate that PcCytc may play different functions in different tissues.WSSV infection experiments showed that the expression level of PcCytc in the tested hepatopancreas, intestines, and muscle tissues increased after virus infection, and reached the highest value at 24 h (P < 0.01), after which it began to decrease until it returned to a normal level at 96 h; the overall performance was an induced expression pattern. This showed that PcCytc is involved in the process of WSSV infection. In addition, considering that PcCytc can participate in ATP production as a key element in the mitochondrial respiratory chain, the low expression of PcCytc leads to energy deficiency. We speculate that once the virus disrupts the energy metabolism of the host cell, the host may compensate for the loss by upregulating the expression of PcCytc.RNAi technology revealed the role of PcCytc in the process of WSSV infection. At 24 and 48 h after WSSV infection, the WSSV copies of the PcCytc RNAi group were significantly increased compared to the uninterrupted group (P < 0.01), and at 72 h were still significantly increased (P < 0.05). These results indicate that PcCytc plays an important role in inhibiting the replication of WSSV in P. clarkii and delays the infection process. To further confirm whether PcCytc mainly inhibits WSSV infection through the apoptotic pathway, we tested the expression changes of some important apoptosis-related genes (bcl-2, bax, and caspase-3). Among them, caspase-3 is an effector protein that regulates cell apoptosis, and its expression directly reflects the result of cell apoptosis. The ratio of bcl-2/bax is considered to be an indicator of the process of cell apoptosis; an increase in the ratio indicates that apoptosis has been affected. Inhibition (a decrease in the ratio) indicates that apoptosis was promoted. The test results were as follows: compared with the PBS group, the expression of bcl-2, bax, and caspase-3 genes of P. clarkii in the WSSV group was up-regulated to varying degrees, with a very significant difference in values (P < 0.01). This shows that WSSV can cause hemolymph apoptosis in P. clarkii, which is consistent with observations in mud crab and shrimp. In addition, the expression of caspase-3 in the dsCytc injection group was significantly downregulated (P < 0.01), indicating that apoptosis was inhibited after interfering with PcCytc. The value of bcl-2/bax in the dsCytc injection group was significantly increased (P < 0.01), which supported this conclusion.In summary, our results indicate that PcCytc can inhibit WSSV infection by regulating the apoptotic pathway. The results of this study provide new insights into the immune response of P. clarkii to WSSV infection.

This article proposes a novel imaging mode which allows for the acquisition of SAR images with different resolution in a single imaging process, specifically designed for multiple-input–multiple-output synthetic aperture radar (MIMO-SAR). To achieve this, a multimode array system model based on 2-D intrapulse scanning is established, followed by the division of full aperture signals into subaperture signals. Improved azimuth phase coding (APC) technology is then employed to separate multimode echo signals, and spatial filtering technology and range digital beam forming technology are used to remove ambiguity in azimuth and range of the subaperture signals of each mode. Finally, subaperture image coherent fusion algorithm is used to generate high-resolution images corresponding to the full aperture of each mode. Simulation results show that the improved APC can effectively separate multimode echo signals, and the multiresolution characteristics and imaging effect are verified.

Cadmium (Cd2+) and mercury ions (Hg2+) are essential for the quality control of food samples because of their serious toxicity to human health, but the effective and simple strategy for their parallel detection remains challenging. In this paper, a rapid and simple parallel detection method for Cd2+ and Hg2+ was developed using carbon dots (CDs) as fluorescent sensors. A one-step hydrothermal method with a single precursor l-arginine as both the carbon and nitrogen sources was employed to prepare nitrogen-doped CDs (N-CDs). N-CDs exhibited a uniform particle size and excitation-independent fluorescence emission. The maximum emission wavelength of N-CDs was observed at 354 nm with the excitation wavelength at 295 nm. The quantum yield of N-CDs reached as high as 71.6% in water. By using sodium diphosphate and phytic acid as masking agents, the fluorescent sensor can be quenched by Cd2+ and Hg2+ in the linear range of 0–26.8 μM and 0–49.9 μM within 5 min. Other common ions in farm products showed no significant effect on the fluorescence intensity of the sensing system. The results demonstrated that the sensing system had good selectivity and sensitivity for Cd2+ and Hg2+. The detection limits for Cd2+ and Hg2+ were 0.20 and 0.188 μM, respectively. In addition, the fluorescent sensor had been successfully applied for the detection of Cd2+ and Hg2+ in fruits and vegetables, and the recoveries were 86.44–109.40% and 86.62–115.32%, respectively. The proposed fluorescent sensor provides a rapid, simple, and sensitive detection method for Cd2+ and Hg2+ in food samples and thus a novel quantitative detection method for heavy metal ions in foods.

Microsatellites, also known as simple sequence repeats (SSRs), are a type of DNA marker that are highly polymorphic in the genome. However, the microsatellite markers available for the red swamp crayfish Procambarus clarkii are limited. In this study, a sizeable number of microsatellites were identified using the data of the P. clarkii genome survey generated from Illumina Hi-Seq PE sequencing. A total length of 1,625 Mb genomic sequences of P. clarkii were assembled based on 124 Gb raw sequencing data. Based on a Kmer analysis, the P. clarkii genome size was projected as approximately 3.6 Gb. A total of 336,275 SSRs (simple sequence repeats) identified motifs were identified. There were 167,999 the most common type di-nucleotide repeat motifs (49.94%). The second most common repeats were the tri-nucleotide ones (131,405, 39.09%). A total of 312,454 microsatellite loci had flanking sequences. A subset of 56 primer pairs was randomly selected for the PCR experiments to validate the SSRs that had been identified. Forty-six primer pairs (80%) had PCR products that matched the sizes expected, and the products from 40 primer pairs (71%) exhibited polymorphism. Thirty-nine P. clarkii individuals were analyzed for their primer sets, and 22 were identified. The average allelic number in the 22 polymorphic loci was 3.6. The 22 microsatellite loci all had good polymorphisms. Among them, the polymorphism information content of the 12 loci is greater than 0.5, which is highly polymorphic. [ABSTRACT FROM AUTHOR]

Abstract Multiple sclerosis (MS), a chronic neurodegenerative disease driven by damage to the protective myelin sheath, is currently incurable. Today, all clinically available treatments modulate the immune-mediated symptoms of the disease but they fail to stop neurodegeneration in many patients. Remyelination, the regenerative process of myelin repair by oligodendrocytes, which is considered a necessary step to protect demyelinated axons and stop neuronal death, is impaired in MS patients. One of the major obstacles to finding effective remyelinating drugs is the lack of biomimetic drug screening platforms that enable quantification of compounds’ potential to stimulate 3D myelination in the physiologically relevant axon-like environment. To address this need, we built a unique myelination drug discovery platform, by expanding our previously developed technology, artificial axons (AAs), which enables 3D-printing of synthetic axon mimics with the geometry and mechanical properties closely resembling those of biological axons. This platform allows for high-throughput phenotypic myelination assay based on quantification of 3D wrapping of myelin membrane around axons in response to compounds. Here, we demonstrate quantification of 3D myelin wrapping by rat oligodendrocytes around the axon mimics in response to a small library of known pro-myelinating compounds. This assay shows pro-myelinating activity for all tested compounds consistent with the published in vitro and in vivo data, demonstrating predictive power of AA platform. We find that stimulation of myelin wrapping by these compounds is dose-dependent, providing a facile means to quantify the compounds’ potency and efficacy in promoting myelin wrapping. Further, the ranking of relative efficacy among these compounds differs in this 3D axon-like environment as compared to a traditional oligodendrocyte 2D differentiation assay quantifying area of deposited myelin membrane. Together, we demonstrate that the artificial axons platform and associated phenotypic myelin wrapping assay afford direct evaluation of myelin wrapping by oligodendrocytes in response to soluble compounds in an axon-like environment, providing a predictive tool for the discovery of remyelinating therapies.

Summary: Promoting myelination capacity of endogenous oligodendrocyte precursor cells (OPCs) is a promising therapeutic approach for CNS demyelinating disorders such as Multiple Sclerosis (MS). To aid in the discovery of myelination-promoting compounds, we generated a genome-engineered human pluripotent stem cell (hPSC) line that consists of three reporters: identification-and-purification tag, GFP, and secreted-NanoLuc, driven by the endogenous PDGFRA, PLP1, and MBP genes, respectively. Using this cell line, we established a high-throughput drug screening platform and performed a small-molecule screen, which identified at least two myelination-promoting small-molecule (Ro1138452 and SR2211) that target prostacyclin (IP) receptor and retinoic acid receptor-related orphan receptor γ (RORγ), respectively. Single-cell-transcriptomic analysis of differentiating OPCs treated with these molecules further confirmed that they promote oligodendrocyte differentiation and revealed several pathways that are potentially modulated by them. The molecules and their target pathways provide promising targets for the possible development of remyelination-based therapy for MS and other demyelinating disorders.