Chinese hamster ovary (CHO) cells are the most widely used mammalian hosts for recombinant protein production. However, by conventional random integration strategy, development of a high-expressing and stable recombinant CHO cell line has always been a difficult task due to the heterogenic insertion and its caused requirement of multiple rounds of selection. Site-specific integration of transgenes into CHO hot spots is an ideal strategy to overcome these challenges since it can generate isogenic cell lines with consistent productivity and stability. In this study, we investigated three sites with potential high transcriptional activities: C12orf35, HPRT, and GRIK1, to determine the possible transcriptional hot spots in CHO cells, and further construct a reliable site-specific integration strategy to develop recombinant cell lines efficiently. Genes encoding representative proteins mCherry and anti-PD1 monoclonal antibody were targeted into these three loci respectively through CRISPR/Cas9 technology. Stable cell lines were generated successfully after a single round of selection. In comparison with a random integration control, all the targeted integration cell lines showed higher productivity, among which C12orf35 locus was the most advantageous in both productivity and cell line stability. Binding affinity and N-glycan analysis of the antibody revealed that all batches of product were of similar quality independent on integrated sites. Deep sequencing demonstrated that there was low level of off-target mutations caused by CRISPR/Cas9, but none of them contributed to the development process of transgene cell lines. Our results demonstrated the feasibility of C12orf35 as the target site for exogenous gene integration, and strongly suggested that C12orf35 targeted integration mediated by CRISPR/Cas9 is a reliable strategy for the rapid development of recombinant CHO cell lines.

The type VI secretion system (T6SS) is a double‐tubular nanomachine widely found in gram‐negative bacteria. Its spear‐like Hcp tube is capable of penetrating a neighboring cell for cytosol‐to‐cytosol protein delivery. However, gram‐positive bacteria have been considered impenetrable to such T6SS action. Here we report that the T6SS of a plant pathogen, Acidovorax citrulli (AC), could deliver an Rhs‐family nuclease effector RhsB to kill not only gram‐negative but also gram‐positive bacteria. Using bioinformatic, biochemical, and genetic assays, we systematically identified T6SS‐secreted effectors and determined that RhsB is a crucial antibacterial effector. RhsB contains an N‐terminal PAAR domain, a middle Rhs domain, and an unknown C‐terminal domain. RhsB is subject to self‐cleavage at both its N‐ and C‐terminal domains and its secretion requires the upstream‐encoded chaperone EagT2 and VgrG3. The toxic C‐terminus of RhsB exhibits DNase activities and such toxicity is neutralized by either of the two downstream immunity proteins, RimB1 and RimB2. Deletion of rhsB significantly impairs the ability of killing Bacillus subtilis while ectopic expression of immunity proteins RimB1 or RimB2 confers protection. We demonstrate that the AC T6SS not only can effectively outcompete Escherichia coli and B. subtilis in planta but also is highly potent in killing other bacterial and fungal species. Collectively, these findings highlight the greatly expanded capabilities of T6SS in modulating microbiome compositions in complex environments.

The type VI secretion system (T6SS) is a spear-like nanomachine found in gram-negative pathogens for delivery of toxic effectors to neighboring bacterial and host cells. Its assembly requires a tip spike complex consisting of a VgrG-trimer, a PAAR protein, and the interacting effectors. However, how the spike controls T6SS assembly remains elusive. Here we investigated the role of three VgrG-effector pairs in Aeromonas dhakensis strain SSU, a clinical isolate with a constitutively active T6SS. By swapping VgrG tail sequences, we demonstrate that the C-terminal ~30 amino-acid tail dictates effector specificity. Double deletion of vgrG1&2 genes (VgrG3+) abolished T6SS secretion, which can be rescued by ectopically expressing chimeric VgrG3 with a VgrG1/2-tail but not the wild type VgrG3. In addition, deletion of effector-specific chaperones also severely impaired T6SS secretion, despite the presence of intact VgrG and effector proteins, in both SSU and Vibrio cholerae V52. We further show that SSU could deliver a V. cholerae effector VasX when expressing a plasmid-borne chimeric VgrG with VasX-specific VgrG tail and chaperone sequences. Pull-down analyses show that two SSU effectors, TseP and TseC, could interact with their cognate VgrGs, the baseplate protein TssK, and the key assembly chaperone TssA. Effectors TseL and VasX could interact with TssF, TssK and TssA in V. cholerae. Collectively, we demonstrate that chimeric VgrG-effector pairs could bypass the requirement of heterologous VgrG complex and propose that effector-stuffing inside the baseplate complex, facilitated by chaperones and the interaction with structural proteins, serves as a crucial structural determinant for T6SS assembly.

When public health emergencies occur, relevant information containing different topics, sentiments, and emotions spread rapidly on social media. From the cognitive and emotional dimensions, this paper explores the relationship between information attributes and information dissemination behavior. At the same time, the moderating role of the media factor (user influence) and the time factor (life cycle) in information attributes and information transmission is also discussed. The results confirm differences in the spread of posts under different topic types, sentiment types, and emotion types on social media. At the same time, the study also found that posts published by users with a high number of followers and users of a media type are more likely to spread on social media. In addition, the study also found that posts with different information attributes are easier to spread on social media during the outbreak and recurrence periods. The driving effect of life cycles is more obvious, especially for topics of prayer and fact, negative sentiment, emotions of fear, and anger. Relevant findings have specific contributions to the information governance of public opinion, the development of social media theory, and the maintenance of network order, which can further weaken the negative impact of information epidemic in the occurrence of public health emergencies, maintain normal social order, and thus create favorable conditions for the further promotion of global recovery.

This paper investigates the resource allocation problem in the downlink of OFDMA cooperative relaying networks with one source (base station) and multiple relay and destination nodes. Assuming that the base station knows all the instantaneous channel gains of all links, we propose a dynamic joint subchannel and power allocation (SPA) scheme whose objective is to maximize the worst user's data rate with the constraint of total transmission power. Since the optimal solution to this combinatorial problem is extremely computationally complex, we propose a low-complexity suboptimal algorithm that allocates subchannel and power separately. In the proposed algorithm, subchannels are firstly paired by relay nodes according to their equivalent channel gains and then assigned to a specific user. Subsequently, an optimal power distribution algorithm is designed for the proposed subchannel allocation (SA) scheme. The simulation results show that the performance of the proposed algorithm approaches asymptotically to that of the optimal one, and maximal fairness is guaranteed for all users.