Comparative Mitogenomic Analyses of Darkling Beetles (Coleoptera: Tenebrionidae) Provide Evolutionary Insights into tRNA-like Sequences.

Tenebrionidae is widely recognized owing to its species diversity and economic importance. Here, we determined the mitochondrial genomes (mitogenomes) of three Tenebrionidae species (Melanesthes exilidentata, Anatolica potanini, and Myladina unguiculina) and performed a comparative mitogenomic analysis to characterize the evolutionary characteristics of the family. The tenebrionid mitogenomes were highly conserved with respect to genome size, gene arrangement, base composition, and codon usage. All protein-coding genes evolved under purifying selection. The largest non-coding region (i.e., control region) showed several unusual features, including several conserved repetitive fragments (e.g., A+T-rich regions, G+C-rich regions, Poly-T tracts, TATA repeat units, and longer repetitive fragments) and tRNA-like structures. These tRNA-like structures can bind to the appropriate anticodon to form a cloverleaf structure, although base-pairing is not complete. We summarized the quantity, types, and conservation of tRNA-like sequences and performed functional and evolutionary analyses of tRNA-like sequences with various anticodons. Phylogenetic analyses based on three mitogenomic datasets and two tree inference methods largely supported the monophyly of each of the three subfamilies (Stenochiinae, Pimeliinae, and Lagriinae), whereas both Tenebrioninae and Diaperinae were consistently recovered as polyphyletic. We obtained a tenebrionid mitogenomic phylogeny: (Lagriinae, (Pimeliinae, ((Tenebrioninae + Diaperinae), Stenochiinae))). Our results provide insights into the evolution and function of tRNA-like sequences in tenebrionid mitogenomes and contribute to our general understanding of the evolution of Tenebrionidae.

Taxonomic status and phylogenetic relationship of Anomura (Crustacea: Decapoda) based on mitochondrial sequences and gene order rearrangements.

The complete mitochondrial genome (mitogenome) contributed crucial information, which could improve the better understanding of molecular phylogenetic analysis, evolution, and gene rearrangements. However, only a few Coenobitidae and Albuneidae mitogenomes have been described, and controversies about the Anomuran phylogeny remain. Here, we determined three Coenobitidae species (Coenobita perlatus, Coenobita rugosus, Coenobita clypeatus) and one Albuneidae species (Emerita talpoida). As the representative species of genera or families, and even the first species of the genus, supplementing these four species is essential to study the phylogenetic relationships within Anomura. Most of the four novel mitogenomes have the typical 37 genes, except for E. talpoida, which lacked trnF. Through combining these new data with 31 Anomuran mitogenomes from Genbank, different gene rearrangement patterns and internal phylogenetic relationships of Anomura were investigated. In our phylogeny analysis, all the Anomuran species were clustered into one group, and the polyphyly of Paguroidea was well supported. Moreover, various peculiar mitochondrial gene orders (MGOs) were summarized among Anomura and preliminary determined their rearrangement mechanisms through CREx. Twenty different MGOs were suggested in our research, and we were focused seven MGO patterns (Pattern A-J) in the process of discussing the gene rearrangement mechanism. Currently making full use of the large taxon sampling, our results provide valuable information on the evolutionary status of Anomuran species and are available for systematic rearrangement and phylogenetic analyses.

The mitochondrial genomes of Crispatotrochus rubescens and Crispatotrochus rugosus (Hexacorallia; Scleractinia): new insights on the phylogeny of the family Caryophylliidae.

BACKGROUND: Caryophylliidae is one of the most diverse scleractinian families, however it was recovered as polyphyletic in multiple molecular studies. Recently, the mitochondrial gene order was proposed as a character for a taxonomic revision of the family. Here we describe the first mitogenome of the caryophylliid genus Crispatotrochus, whose phylogenetic position remains uncertain. METHODS AND RESULTS: The complete mitochondrial genomes of Crispatotrochus rubescens and Crispatotrochus rugosus were sequenced, assembled, and annotated. The two mitogenomes are identical and circular, have a length of 16,536 bp, a GC content of 35.9%, and contain 13 protein-coding genes, 2 ribosomal RNAs and 2 transfer RNAs. Both species have a transposition of a three gene block - cob, nad2, and nad6 - similarly to a group of caryophylliid genera that were recovered as monophyletic, including the type genus (Caryophyllia) of the family. The phylogenetic analyses recovered Crispatotrochus within the clade that presents the gene rearrangement and specifically as sister taxa of the genus Caryophyllia, a result consistent with previous studies and the similar gross morphology of the two genera. CONCLUSIONS: We determined the mitochondrial genomes of the genus Crispatotrochus to investigate their relations within Scleractinia. Results from this study provide insights on the phylogenetic position of the genus and corroborate that the mitochondrial gene order could be used as taxonomic character for the family Caryophylliidae.

Projection in genomic analysis: A theoretical basis to rationalize tensor decomposition and principal component analysis as feature selection tools.

Identifying differentially expressed genes is difficult because of the small number of available samples compared with the large number of genes. Conventional gene selection methods employing statistical tests have the critical problem of heavy dependence of P-values on sample size. Although the recently proposed principal component analysis (PCA) and tensor decomposition (TD)-based unsupervised feature extraction (FE) has often outperformed these statistical test-based methods, the reason why they worked so well is unclear. In this study, we aim to understand this reason in the context of projection pursuit (PP) that was proposed a long time ago to solve the problem of dimensions; we can relate the space spanned by singular value vectors with that spanned by the optimal cluster centroids obtained from K-means. Thus, the success of PCA- and TD-based unsupervised FE can be understood by this equivalence. In addition to this, empirical threshold adjusted P-values of 0.01 assuming the null hypothesis that singular value vectors attributed to genes obey the Gaussian distribution empirically corresponds to threshold-adjusted P-values of 0.1 when the null distribution is generated by gene order shuffling. For this purpose, we newly applied PP to the three data sets to which PCA and TD based unsupervised FE were previously applied; these data sets treated two topics, biomarker identification for kidney cancers (the first two) and the drug discovery for COVID-19 (the thrid one). Then we found the coincidence between PP and PCA or TD based unsupervised FE is pretty well. Shuffling procedures described above are also successfully applied to these three data sets. These findings thus rationalize the success of PCA- and TD-based unsupervised FE for the first time.

Caryophylliids (Anthozoa, Scleractinia) and mitochondrial gene order: Insights from mitochondrial and nuclear phylogenomics.

Molecularly, the family Caryophylliidae is polyphyletic and different sets of genetic data converge towards a consensus that a taxonomic review of this family is necessary. Overall, the order of genes in the mitochondrial genome (mitogenome) together with DNA sequences have been used to successfully untangle evolutionary relationships in several groups of organisms. Published mitogenomes of two caryophylliid genera (Desmophyllum and Solenosmilia) present a transposition of the gene block containing cob, nad2, and nad6, which is located between nad5 5' exon and trnW, while that of Polycyathus chaishanensis presents the same gene order as the majority of scleractinian corals. In molecular-based evolutionary reconstructions, caryophylliids that have the mitochondrial gene rearrangement were recovered as a monophyletic lineage ("true" caryophylliids), while members of the genus Polycyathus were placed in a different position. In this study, additional mitogenomes of this family were assembled and included in evolutionary reconstructions of Scleractinia in order to improve our understanding on whether the mitogenome gene rearrangement is limited to and, therefore, could be a synapomorphy of the actual members of Caryophylliidae. Specimens of Caryophyllia scobinosa, Premocyathus sp., Heterocyathus sulcatus, and Trochocyathus caryophylloides, as well as Desmophyllum pertusum and Solenosmilia variabilis from the Southwest Atlantic were sequenced using Illumina platforms. Then, mitochondrial genomes were assembled and annotated, and nuclear datasets were recovered in-silico from assembled contigs using a previously published set of baits. Evolutionary reconstructions were performed using mitochondrial and nuclear datasets and based on Maximum Likelihood and Bayesian Inference. Obtained mitogenomes are circular and range between 15,816 and 18,225 bp in size and from 30.76% to 36.63% in GC content. The gene rearrangement is only seen in C. scobinosa, D. pertusum, Premocyathus sp., and S. variabilis, which were recovered as a monophyletic clade in both mitochondrial and nuclear phylogenies. On the other hand, the "caryophylliids" with the canonical mitogenome gene order were not recovered within this clade. Differences in features of the skeleton of "true" caryophylliids in comparison to traditional members of the family were observed and offer further support that the gene rearrangement might be seen as a synapomorphy of family Caryophylliidae.

Fragmented mitochondrial genomes of seal lice (family Echinophthiriidae) and gorilla louse (family Pthiridae): frequent minichromosomal splits and a host switch of lice between seals.

BACKGROUND: The mitochondrial (mt) genomes of 15 species of sucking lice from seven families have been studied to date. These louse species have highly dynamic, fragmented mt genomes that differ in the number of minichromosomes, the gene content, and gene order in a minichromosome between families and even between species of the same genus. RESULTS: In the present study, we analyzed the publicly available data to understand mt genome fragmentation in seal lice (family Echinophthiriidae) and gorilla louse, Pthirus gorillae (family Pthiridae), in particular the role of minichromosome split and minichromosome merger in the evolution of fragmented mt genomes. We show that 1) at least three ancestral mt minichromosomes of sucking lice have split in the lineage leading to seal lice, 2) one minichromosome ancestral to primate lice has split in the lineage to the gorilla louse, and 3) two ancestral minichromosomes of seal lice have merged in the lineage to the northern fur seal louse. Minichromosome split occurred 15-16 times in total in the lineages leading to species in six families of sucking lice investigated. In contrast, minichromosome merger occurred only four times in the lineages leading to species in three families of sucking lice. Further, three ancestral mt minichromosomes of sucking lice have split multiple times independently in different lineages of sucking lice. Our analyses of mt karyotypes and gene sequences also indicate the possibility of a host switch of crabeater seal louse to Weddell seals. CONCLUSIONS: We conclude that: 1) minichromosome split contributes more than minichromosome merger in mt genome fragmentation of sucking lice, and 2) mt karyotype comparison helps understand the phylogenetic relationships between sucking louse species.

Phylogenetic position of Bopyroides hippolytes, with comments on the rearrangement of the mitochondrial genome in isopods (Isopoda: Epicaridea: Bopyridae).

BACKGROUND: Classification of parasitic bopyrids has traditionally been based on morphological characteristics, but phylogenetic relationships have remained elusive due to limited information provided by morphological data and tendency for loss of morphological features as a result of parasitic lifestyle. Subfamily Argeiinae was separated from Bopyrinae based on morphological evidence, although the assignment of all genera has not been phylogenetically evaluated. Bopyroides hippolytes has been traditionally classified in Bopyrinae, but divergent morphological characters make this assignment questionable. To investigate the relationship of bopyrines, we sequenced the complete mitochondrial genome of B. hippolytes and four mitochondrial genes of two other Bopyrinae. RESULTS: The phylogenetic trees based on separate and combined cox1and 18S sequence data recovered Bopyridae as robustly monophyletic, but Bopyrinae as polyphyletic. Bopyroides hippolytes was a close sister to Argeia pugettensis, type species to Argeiinae. Mitochondrial phylogenomics also suggested that B. hippolytes was close to Argeiinae. We also found a novel gene order in B. hippolytes compared to other isopods. CONCLUSIONS: Bopyroides hippolytes should be excluded from the Bopyrinae and has a close affinity with Argeia pugettensis based on molecular and morphological data. The conserved syntenic blocks of mitochondrial gene order have distinctive characteristics at a subordinal level and may be helpful for understanding the higher taxonomic level relationships of Isopoda.

Components of a HIV-1 vaccine mediate virus-like particle (VLP)-formation and display of envelope proteins exposing broadly neutralizing epitopes.

The sequence diversity of HIV-1 is the biggest hurdle for the design of a prophylactic vaccine. Mosaic (Mos) antigens consisting of synthetically shuffled epitopes from various HIV-1 strains are currently tested in the clinical vaccine trial Mosaico (NCT03964415). Besides adenovirus vectors encoding variants of Mos.Gag-Pol and soluble Mos.Env proteins, the Mosaico vaccine entails vectors mediating gene transfer and expression of the membrane-anchored Env-variant Mos2S.Env. We thus examined whether the expression of mosaic Gag mediates the formation of virus-like particles (VLPs). Mos1.Gag- and Mos2.Gag-VLP-formation was readily detected using Western blot- and electron microscopic-analysis. Upon co-expression of both mosaic Gag variants with Mos2S.Env, incorporation of Env into Gag-formed VLPs was observed. The display of the respective neutralization-sensitive target epitopes on Mos2S.Env-decorated VLPs was demonstrated employing a panel of broadly neutralizing antibodies (bNAbs) in a VLP-capture assay. This opens new perspectives for future HIV vaccine designs.

Mitogenome sequence comparison in the endangered congeneric Pinna nobilis and Pinna rudis bivalves.

BACKGROUND: The pen shells Pinna nobilis and Pinna rudis are large wedge-shaped bivalve molluscs. Both species are threatened by different anthropogenic pressures. In the last few years, P. nobilis populations have significantly reduced due to massive mortality events. The complete mitochondrial DNA sequences of these congeneric species have been determined and compared for the first time. RESULTS: The mitogenome sequences of P. nobilis and P. rudis were 18,919 bp and 18,264 bp in length, respectively. Each mitogenome is composed of 12 protein-coding genes, 2 ribosomal RNA, 22 transfer RNA (tRNAs) genes and non-coding regions. A putative Adenosine Triphosphate synthase subunit 8 gene could only be proposed for P. nobilis. Both newly sequenced mitogenomes present a conserved gene order between them, comparable to the closely related Atrina pectinata, but global arrangement greatly differs from other available bivalve mitochondrial sequences. Multiple copies of tRNA-Cys were identified, located in different positions probably due to mechanisms of mitochondrial genome rearrangements, and detected 2 and 3 times in P. rudis and in P. nobilis, respectively. CONCLUSION: A close relationship was shown between Pinna species and Atrina pectinata and a consistent clustering showing a monophyletic origin of Pinnidae family sequences was evidenced. The mitochondrial genomes will provide a valuable genetic resource for further studies on population genetics and species identification.

Characterization of recombinant ß subunit of human MUC4 mucin (rMUC4ß).

MUC4 is a transmembrane mucin expressed on various epithelial surfaces, including respiratory and gastrointestinal tracts, and helps in their lubrication and protection. MUC4 is also aberrantly overexpressed in various epithelial malignancies and functionally contributes to cancer development and progression. MUC4 is putatively cleaved at the GDPH site into a mucin-like α-subunit and a membrane-tethered growth factor-like ß-subunit. Due to the presence of several functional domains, the characterization of MUC4ß is critical for understanding MUC4 biology. We developed a method to produce and purify multi-milligram amounts of recombinant MUC4ß (rMUC4ß). Purified rMUC4ß was characterized by Far-UV CD and I-TASSER-based protein structure prediction analyses, and its ability to interact with cellular proteins was determined by the affinity pull-down assay. Two of the three EGF-like domains exhibited typical ß-fold, while the third EGF-like domain and vWD domain were predominantly random coils. We observed that rMUC4ß physically interacts with Ezrin and EGFR family members. Overall, this study describes an efficient and simple strategy for the purification of biologically-active rMUC4ß that can serve as a valuable reagent for a variety of biochemical and functional studies to elucidate MUC4 function and generating domain-specific antibodies and vaccines for cancer immunotherapy.

APC-Targeted DNA Vaccination Against Reticulocyte-Binding Protein Homolog 5 Induces Plasmodium falciparum-Specific Neutralizing Antibodies and T Cell Responses.

Targeted delivery of antigen to antigen presenting cells (APCs) is an efficient way to induce robust antigen-specific immune responses. Here, we present a novel DNA vaccine that targets the Plasmodium falciparum reticulocyte-binding protein homolog 5 (PfRH5), a leading blood-stage antigen of the human malaria pathogen, to APCs. The vaccine is designed as bivalent homodimers where each chain is composed of an amino-terminal single chain fragment variable (scFv) targeting unit specific for major histocompatibility complex class II (MHCII) expressed on APCs, and a carboxyl-terminal antigenic unit genetically linked by the dimerization unit. This vaccine format, named "Vaccibody", has previously been successfully applied for antigens from other infectious diseases including influenza and HIV, as well as for tumor antigens. Recently, the crystal structure and key functional antibody epitopes for the truncated version of PfRH5 (PfRH5ΔNL) were characterized, suggesting PfRH5ΔNL to be a promising candidate for next-generation PfRH5 vaccine design. In this study, we explored the APC-targeting strategy for a PfRH5ΔNL-containing DNA vaccine. BALB/c mice immunized with the targeted vaccine induced higher PfRH5-specific IgG1 antibody responses than those vaccinated with a non-targeted vaccine or antigen alone. The APC-targeted vaccine also efficiently induced rapid IFN-γ and IL-4 T cell responses. Furthermore, the vaccine-induced PfRH5-specific IgG showed inhibition of growth of the P. falciparum 3D7 clone parasite in vitro. Finally, sera obtained after vaccination with this targeted vaccine competed for the same epitopes as PfRH5-specific mAbs from vaccinated humans. Robust humoral responses were also induced by a similar P. vivax Duffy-binding protein (PvDBP)-containing targeted DNA vaccine. Our data highlight a novel targeted vaccine platform for the development of vaccines against blood-stage malaria.

Fusion of apoptosis-related protein Cytochrome c with anti-HER-2 single-chain antibody targets the suppression of HER-2+ breast cancer.

Cancer treatment has gradually developed from toxic chemotherapy to targeted therapy with fewer side effects. Approximately 30% of breast cancer patients overexpress human epidermal growth factor receptor 2 (HER-2). Previous studies have successfully produced single-chain antibodies (scFv) targeting HER-2+ breast cancer; however, scFv have poor stability, easy aggregation and a shorter half-life, which have no significant effect on targeting therapy. Moreover, scFv has been considered as a drug delivery platform that can kill target cells by effector molecules. However, the functional killing domains of immunotoxins are mainly derived from plant or bacterial toxins, which have a large molecular weight, low tissue permeability and severe side effects. To address these concerns, we designed several apoptotic immune molecules to replace exogenous toxins using endogenous apoptosis-related protein DNA fragmentation factor 40 (DFF40) and tandem-repeat Cytochrome c base on caspase-3 responsive peptide (DEVD). Our results suggest that DFF40 or Cytc fusion scFv specifically targets HER-2 overexpressing breast cancer cells (SK-BR-3 and BT-474) rather than HER-2 negative cells (MDA-MB-231 and MCF-7). Following cellular internalization, apoptosis-related proteins inhibited tumour activity by initiating endogenous apoptosis pathways, which significantly reduced immunogenicity and toxic side effects. Therefore, we suggest that immunoapoptotic molecules may become potential drugs for targeted immunotherapy of breast cancer.

The ß2Tubulin, Rad50-ATPase and enolase cis-regulatory regions mediate male germline expression in Tribolium castaneum.

Genetics-based pest management processes, including the sterile insect technique, are an effective method for the control of some pest insects. However, current SIT methods are not directly transferable to many important pest insect species due to the lack of genetic sexing strains. Genome editing is revolutionizing the way we conduct genetics in insects, including in Tribolium castaneum, an important genetic model and agricultural pest. We identified orthologues of ß2Tubulin, Rad50-ATPase and enolase in T. castaneum. Using RT-PCR, we confirmed that these genes are predominantly expressed in the testis. PiggyBac-based transformation of T. castaneum cis-regulatory regions derived from Tc-ß2t, Tc-rad50 or Tc-eno resulted in EGFP expression specifically in the T. castaneum testis. Additionally, we determined that each of these regulatory regions regulates EGFP expression in different cell types of the male gonad. Cis-regulatory regions from Tc-ß2t produced EGFP expression throughout spermatogenesis and also in mature sperms; Tc-rad50 resulted in expression only in the haploid spermatid, while Tc-eno expressed EGFP in late spermatogenesis. In summary, the regulatory cis-regions characterized in this study are not only suited to study male gonadal function but could be used for development of transgenic sexing strains that produce one sex in pest control strategies.

Full-length dystrophin restoration via targeted exon integration by AAV-CRISPR in a humanized mouse model of Duchenne muscular dystrophy.

Targeted gene-editing strategies have emerged as promising therapeutic approaches for the permanent treatment of inherited genetic diseases. However, precise gene correction and insertion approaches using homology-directed repair are still limited by low efficiencies. Consequently, many gene-editing strategies have focused on removal or disruption, rather than repair, of genomic DNA. In contrast, homology-independent targeted integration (HITI) has been reported to effectively insert DNA sequences at targeted genomic loci. This approach could be particularly useful for restoring full-length sequences of genes affected by a spectrum of mutations that are also too large to deliver by conventional adeno-associated virus (AAV) vectors. Here, we utilize an AAV-based, HITI-mediated approach for correction of full-length dystrophin expression in a humanized mouse model of Duchenne muscular dystrophy (DMD). We co-deliver CRISPR-Cas9 and a donor DNA sequence to insert the missing human exon 52 into its corresponding position within the DMD gene and achieve full-length dystrophin correction in skeletal and cardiac muscle. Additionally, as a proof-of-concept strategy to correct genetic mutations characterized by diverse patient mutations, we deliver a superexon donor encoding the last 28 exons of the DMD gene as a therapeutic strategy to restore full-length dystrophin in >20% of the DMD patient population. This work highlights the potential of HITI-mediated gene correction for diverse DMD mutations and advances genome editing toward realizing the promise of full-length gene restoration to treat genetic disease.

Action-at-a-distance mutations at 5'-GpA-3' sites induced by oxidised guanine in WRN-knockdown cells.

G:C sites distant from 8-oxo-7,8-dihydroguanine (GO, 8-hydroxyguanine) are frequently mutated when the lesion-bearing plasmid DNA is replicated in human cells with reduced Werner syndrome (WRN) protein. To detect the untargeted mutations preferentially, the oxidised guanine base was placed downstream of the reporter supF gene and the plasmid DNA was introduced into WRN-knockdown cells. The total mutant frequency seemed higher in the WRN-knockdown cells as compared to the control cells. Mutation analyses revealed that substitution mutations occurred at the G:C pairs of 5'-GpA-3'/5'-TpC-3' sites, the preferred sequence for the apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3 (APOBEC3)-family cytosine deaminases, in the supF gene in both control and knockdown cells. These mutations were observed more frequently at G sites than C sites on the DNA strand where the GO base was originally located. This tendency was promoted by the knockdown of the WRN protein. The present results imply the possible involvement of APOBEC3-family cytosine deaminases in the action-at-a-distance (untargeted) mutations at G:C (or G) sites induced by GO and in cancer initiation by oxidative stress.

Gene Position Index Mutation Detection Algorithm Based on Feedback Fast Learning Neural Network.

In the detection of genome variation, the research on the internal correlation of reference genome is deepening; the detection of variation in genome sequence has become the focus of research, and it has also become an effective path to find new genes and new functional proteins. The targeted sequencing sequence is used to sequence the exon region of a specific gene in cancer gene detection, and the sequencing depth is relatively large. Traditional alignment algorithms will lose some sequences, which will lead to inaccurate mutation detection. This paper proposes a mutation detection algorithm based on feedback fast learning neural network position index. By establishing a position index relationship for ACGT in the DNA sequence, the subsequence is decomposed into the position relationship of different subsequences corresponding to the main sequence. The positional relationship of the subsequence in the main sequence is determined by the positional relationship. Analyzing SNP and InDel mutations, even structural mutations, through the position correlation of sequences has the advantages of high precision and easy implementation by personal computers. The feedback fast learning neural network is used to verify whether there is a linear relationship between two or more positions. Experimental results show that the mutation points detected by position index are more than those detected by Bcftools, Freebye, Vanscan2, and Gatk.

Innovative dual system approach for selective eradication of cancer cells using viral-based delivery of natural bacterial toxin-antitoxin system.

The inactivation of p53, a tumor suppressor, and the activation of the RAS oncogene are the most frequent genetic alterations in cancer. We have shown that a unique E. coli MazF-MazE toxin-antitoxin (TA) system can be used for selective and effective eradication of RAS-mutated cancer cells. This out of the box strategy holds great promise for effective cancer treatment and management. We provide proof of concept for a novel platform to selectively eradicate cancer cells using an adenoviral delivery system based on the adjusted natural bacterial system. We generated adenoviral vectors carrying the mazF toxin (pAdEasy-Py4-SV40mP-mCherry-MazF) and the antitoxin mazE (pAdEasy-RGC-SV40mP-MazE-IRES-GFP) under the regulation of RAS and p53, resp. The control vector carries the toxin without the RAS-responsive element (pAdEasy-ΔPy4-SV40mP-mCherry-MazF). In vitro, the mazF-mazE TA system (Py4-SV40mP-mCherry-MazF+RGC-SV40mP-MazE-IRES-GFP) induced massive, dose-dependent cell death, at 69% compared to 19% for the control vector, in a co-infected HCT116 cell line. In vivo, the system caused significant tumor growth inhibition of HCT116 (KRASmut/p53mut) tumors at 73 and 65% compared to PBS and ΔPY4 control groups, resp. In addition, we demonstrate 65% tumor growth inhibition in HCT116 (KRASmut/p53wt) cells, compared to the other two control groups, indicating a contribution of the antitoxin in blocking system leakage in WT RAS cells. These data provide evidence of the feasibility of using mutations in the p53 and RAS pathway to efficiently kill cancer cells. The platform, through its combination of the antitoxin (mazE) with the toxin (mazF), provides effective protection of normal cells from basal low activity or leakage of mazF.

CRISPR/Cas9 Guided Mutagenesis of Grain Size 3 Confers Increased Rice (Oryza sativa L.) Grain Length by Regulating Cysteine Proteinase Inhibitor and Ubiquitin-Related Proteins.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein (Cas9)-mediated genome editing has become an important way for molecular breeding in crop plants. To promote rice breeding, we edited the Grain Size 3 (GS3) gene for obtaining valuable and stable long-grain rice mutants. Furthermore, isobaric tags for the relative and absolute quantitation (iTRAQ)-based proteomic method were applied to determine the proteome-wide changes in the GS3 mutants compared with wild type (WT). Two target sites were designed to construct the vector, and the Agrobacterium-mediated method was used for rice transformation. Specific mutations were successfully introduced, and the grain length (GL) and 1000-grain weight (GWT) of the mutants were increased by 31.39% and 27.15%, respectively, compared with WT. The iTRAQ-based proteomic analysis revealed that a total of 31 proteins were differentially expressed in the GS3 mutants, including 20 up-regulated and 11 down-regulated proteins. Results showed that differentially expressed proteins (DEPs) were mainly related to cysteine synthase, cysteine proteinase inhibitor, vacuolar protein sorting-associated, ubiquitin, and DNA ligase. Furthermore, functional analysis revealed that DEPs were mostly enriched in cellular process, metabolic process, binding, transmembrane, structural, and catalytic activities. Pathway enrichment analysis revealed that DEPs were mainly involved in lipid metabolism and oxylipin biosynthesis. The protein-to-protein interaction (PPI) network found that proteins related to DNA damage-binding, ubiquitin-40S ribosomal, and cysteine proteinase inhibitor showed a higher degree of interaction. The homozygous mutant lines featured by stable inheritance and long-grain phenotype were obtained using the CRISPR/Cas9 system. This study provides a convenient and effective way of improving grain yield, which could significantly accelerate the breeding process of long-grain japonica parents and promote the development of high-yielding rice.

RNA silencing suppressor-influenced performance of a virus vector delivering both guide RNA and Cas9 for CRISPR gene editing.

We report on further development of the agroinfiltratable Tobacco mosaic virus (TMV)-based overexpression (TRBO) vector to deliver CRISPR/Cas9 components into plants. First, production of a Cas9 (HcoCas9) protein from a binary plasmid increased when co-expressed in presence of suppressors of gene silencing, such as the TMV 126-kDa replicase or the Tomato bushy stunt virus P19 protein. Such suppressor-generated elevated levels of Cas9 expression translated to efficient gene editing mediated by TRBO-G-3'gGFP expressing GFP and also a single guide RNA targeting the mgfp5 gene in the Nicotiana benthamiana GFP-expressing line 16c. Furthermore, HcoCas9 encoding RNA, a large cargo insert of 4.2 kb, was expressed from TRBO-HcoCas9 to yield Cas9 protein again at higher levels upon co-expression with P19. Likewise, co-delivery of TRBO-HcoCas9 and TRBO-G-3'gGFP in the presence of P19 also resulted in elevated levels percentages of indels (insertions and deletions). These data also revealed an age-related phenomenon in plants whereby the RNA suppressor P19 had more of an effect in older plants. Lastly, we used a single TRBO vector to express both Cas9 and a sgRNA. Taken together, we suggest that viral RNA suppressors could be used for further optimization of single viral vector delivery of CRISPR gene editing parts.

The BCMA-Targeted Fourth-Generation CAR-T Cells Secreting IL-7 and CCL19 for Therapy of Refractory/Recurrent Multiple Myeloma.

Chimeric antigen receptor (CAR) technology has revolutionized cancer treatment, particularly in malignant hematological tumors. Currently, the BCMA-targeted second-generation CAR-T cells have showed impressive efficacy in the treatment of refractory/relapsed multiple myeloma (R/R MM), but up to 50% relapse remains to be addressed urgently. Here we constructed the BCMA-targeted fourth-generation CAR-T cells expressing IL-7 and CCL19 (i.e., BCMA-7 × 19 CAR-T cells), and demonstrated that BCMA-7 × 19 CAR-T cells exhibited superior expansion, differentiation, migration and cytotoxicity. Furthermore, we have been carrying out the first-in-human clinical trial for therapy of R/R MM by use of BCMA-7 × 19 CAR-T cells (ClinicalTrials.gov Identifier: NCT03778346), which preliminarily showed promising safety and efficacy in first two enrolled patients. The two patients achieved a CR and VGPR with Grade 1 cytokine release syndrome only 1 month after one dose of CAR-T cell infusion, and the responses lasted more than 12-month. Taken together, BCMA-7 × 19 CAR-T cells were safe and effective against refractory/relapsed multiple myeloma and thus warranted further clinical study.