The Fujian oyster (Crassostrea angulate) is an important marine bivalve mollusk with high economic value. Gene function research and gene editing techniques have broad application prospects in oyster. The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system has been widely used for genome engineering in many species. CRISPR-mediated gene editing has also been used successfully in the Pacific oyster through direct delivery of the CRISPR/Cas9 components into oyster embryos by microinjection. However, the low throughput and operational difficulties associated with microinjection is one of the factors limiting the widespread application of CRISPR/Cas9 in oysters. In this study, we attempted to deliver the CRISPR/Cas9-system into the embryos of C. angulate by electroporation. An all-in-one CRISPR/Cas9 vector plasmid was used as CRISPR/Cas9 system in this study. Electroporation was carried out using both eggs and blastula larvae. A large number of larvae became malformed or die after electroporation. A single base substitution mutation was detected in the D-larvae developed from electroporated eggs. Our results demonstrate that the CRISPR/Cas9 system can be delivered into embryos of C. angulate for gene editing by electroporation, which provides a reference and will further contribute to the future application of electroporation in mollusks.

Clarification of postmortem metabolite changes can help characterize the process of biological degradation and facilitate investigations of forensic casework, especially in the estimation of postmortem interval (PMI). Metabolomics can provide information on the molecular profiles of tissues, which can aid in investigating postmortem metabolite changes. In this study, liquid chromatography-mass spectrometric (LC-MS) analysis was performed to identify the metabolic profiles of rat femoral muscle at ten periods of time after death within 168 h. The results obtained by reversed-phase liquid chromatography (RPLC)- and hydrophilic interaction liquid chromatography (HILIC)- electrospray ionization (ESI±) have revealed more than 16,000 features from all four datasets. Furthermore, 915 of these features were identified using an in-house database. Principal component analysis (PCA) demonstrated the time-specific features of molecular profiling at each period of time after death. Moreover, results from partial least squares projection to latent structures-discriminant analysis (PLS-DA) disclosed a strong association of metabolic alterations of at least 59 metabolites with the time since death, especially within 48 h after death, which expounds these metabolites as potential indicators in PMI estimation. Altogether, our results illustrate the potentiality of metabolic profiling in the evaluation of PMI and provide candidate metabolite markers with strong correlation with time since death for forensic purpose.

Deep Neural Networks are well known to be vulnerable to adversarial attacks and backdoor attacks, where minor modifications on the input can mislead the models to give wrong results. Although defenses against adversarial attacks have been widely studied, research on mitigating backdoor attacks is still at an early stage. It is unknown whether there are any connections and common characteristics between the defenses against these two attacks.In this paper, we present a unified framework for detecting malicious examples and protecting the inference results of Deep Learning models. This framework is based on our observation that both adversarial examples and backdoor examples have anomalies during the inference process, highly distinguishable from benign samples. As a result, we repurpose and revise four existing adversarial defense methods for detecting backdoor examples. Extensive evaluations indicate these approaches provide reliable protection against backdoor attacks, with a higher accuracy than detecting adversarial examples. These solutions also reveal the relations of adversarial examples, backdoor examples and normal samples in model sensitivity, activation space and feature space. This can enhance our understanding about the inherent features of these two attacks, as well as the defense opportunities.

Retinoic acid (RA) signaling pathways mediated by RA receptors (RARs) are essential for many physiological processes such as organ development, regeneration, and differentiation in animals. Recent studies reveal that RARs identified in several mollusks, including Pacific oyster Crassostrea gigas, have a different function mechanism compared with that in chordates. In this report, we identified the molecular characteristics of CgRAR to further explore the mechanism of RAR in mollusks. RT-qPCR analysis shows that CgRAR has a higher expression level in the hemocytes and gonads, indicating that CgRAR may play roles in the processes of development and metabolism. The mRNA expression level of both CgRAR and CgRXR was analyzed by RT-qPCR after injection with RA. The elevated expression of CgRAR and CgRXR was detected upon all-trans-RA (ATRA) exposure. Finally, according to the results of Yeast Two-Hybrid assay and co-immunoprecipitation analysis, CgRAR and CgRXR can interact with each other through the C-terminal region. Taken together, our results suggest that CgRAR shows a higher expression level in gonads and hemocytes. ATRA exposure up-regulates the expression of CgRAR and CgRXR. Besides, CgRAR can interact with CgRXR to form a heterodimer complex.

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