Cost-Effective Whole Transcriptome Sequencing Landscape and Diagnostic Potential Biomarkers in Active Tuberculosis.

Tuberculosis (TB) is a prevalent and severe infectious disease that poses a significant threat to human health. However, it is frequently disregarded as there are not enough quick and accurate ways to diagnose tuberculosis. Here, we develop a strategy for tuberculosis detection to address the challenges, including an experimental strategy, namely, Double Adapter Directional Capture sequencing (DADCSeq), an easily operated and low-cost whole transcriptome sequencing method, and a computational method to identify hub differentially expressed genes as well as the diagnosis of TB based on whole transcriptome data using DADCSeq on peripheral blood mononuclear cells (PBMCs) from active TB and latent TB or healthy control. Applying our approach to create a robust and stable TB multi-mRNA risk probability model (TBMMRP) that can accurately distinguish active and latent TB patients, including active TB and healthy controls in clinical cohorts, this diagnostic biomarker was successfully validated by several independent cross-platform cohorts with favorable performance in differentiating active TB from latent TB or active TB from healthy controls and further demonstrated superior or similar diagnostic accuracy compared to previous diagnostic markers. Overall, we develop a low-cost and effective strategy for tuberculosis diagnosis; as the clinical cohort increases, we can expand to different disease kinds and learn new features through our disease diagnosis strategy.

Engineering of a compact, high-fidelity EbCas12a variant that can be packaged with its crRNA into an all-in-one AAV vector delivery system.

The CRISPR-associated endonuclease Cas12a has become a powerful genome-editing tool in biomedical research due to its ease of use and low off-targeting. However, the size of Cas12a severely limits clinical applications such as adeno-associated virus (AAV)-based gene therapy. Here, we characterized a novel compact Cas12a ortholog, termed EbCas12a, from the metagenome-assembled genome of a currently unclassified Erysipelotrichia. It has the PAM sequence of 5'-TTTV-3' (V = A, G, C) and the smallest size of approximately 3.47 kb among the Cas12a orthologs reported so far. In addition, enhanced EbCas12a (enEbCas12a) was also designed to have comparable editing efficiency with higher specificity to AsCas12a and LbCas12a in mammalian cells at multiple target sites. Based on the compact enEbCas12a, an all-in-one AAV delivery system with crRNA for Cas12a was developed for both in vitro and in vivo applications. Overall, the novel smallest high-fidelity enEbCas12a, this first case of the all-in-one AAV delivery for Cas12a could greatly boost future gene therapy and scientific research.

Engineering of Cas12a nuclease variants with enhanced genome-editing specificity.

The clustered regularly interspaced short palindromic repeat (CRISPR)-Cas12a system is a powerful tool in gene editing; however, crRNA-DNA mismatches might induce unwanted cleavage events, especially at the distal end of the PAM. To minimize this limitation, we engineered a hyper fidelity AsCas12a variant carrying the mutations S186A/R301A/T315A/Q1014A/K414A (termed HyperFi-As) by modifying amino acid residues interacting with the target DNA and crRNA strand. HyperFi-As retains on-target activities comparable to wild-type AsCas12a (AsCas12aWT) in human cells. We demonstrated that HyperFi-As has dramatically reduced off-target effects in human cells, and HyperFi-As possessed notably a lower tolerance to mismatch at the position of the PAM-distal region compared with the wild type. Further, a modified single-molecule DNA unzipping assay at proper constant force was applied to evaluate the stability and transient stages of the CRISPR/Cas ribonucleoprotein (RNP) complex. Multiple states were sensitively detected during the disassembly of the DNA-Cas12a-crRNA complexes. On off-target DNA substrates, the HyperFi-As-crRNA was harder to maintain the R-loop complex state compared to the AsCas12aWT, which could explain exactly why the HyperFi-As has low off-targeting effects in human cells. Our findings provide a novel version of AsCas12a variant with low off-target effects, especially capable of dealing with the high off-targeting in the distal region from the PAM. An insight into how the AsCas12a variant behaves at off-target sites was also revealed at the single-molecule level and the unzipping assay to evaluate multiple states of CRISPR/Cas RNP complexes might be greatly helpful for a deep understanding of how CRISPR/Cas behaves and how to engineer it in future.

Decoding the transcriptional heterogeneity, differentiation lineage, clinical significance in tissue-resident memory CD8 T cell of the small intestine by single-cell analysis.

Resident memory T (Trm) cells which are specifically located in non-lymphoid tissues showed distinct phenotypes and functions compared to circulating memory T cells and were vital for the initiation of robust immune response within tissues. However, the heterogeneity in the transcriptional features, development pathways, and cancer response of Trm cells in the small intestine was not demonstrated. Here, we integrated scRNA-seq and scTCR-seq data pan-tissue T cells to explore the heterogeneity of Trm cells and their development pathways. Trm were enriched in tissue-specific immune response and those in the DUO specially interacted with B cells via TNF and MHC-I signatures. T cell lineage analyses demonstrated that Trm might be derived from the T_CD4/CD8 subset within the same organ or migrated from spleen and mesenteric lymph nodes. We compared the immune repertoire of Trm among organs and implied that clonotypes in both DUO and ILE were less expanded and hydrophilic TRB CDR3s were enriched in the DUO. We further demonstrated that Trm in the intestine infiltrated the colorectal cancer and several effector molecules were highly expressed. Finally, the TCGA dataset of colorectal cancer implied that the infiltration of Trm from the DUO and the ILE was beneficial for overall survival and the response to immune checkpoint blockade.

Cell volume controlled by LRRC8A-formed volume-regulated anion channels fine-tunes T cell activation and function.

Biosynthesis drives the cell volume increase during T cell activation. However, the contribution of cell volume regulation in TCR signaling during T lymphoblast formation and its underlying mechanisms remain unclear. Here we show that cell volume regulation is required for optimal T cell activation. Inhibition of VRACs (volume-regulated anion channels) and deletion of leucine-rich repeat-containing protein 8A (LRRC8A) channel components impair T cell activation and function, particularly under weak TCR stimulation. Additionally, LRRC8A has distinct influences on mRNA transcriptional profiles, indicating the prominent effects of cell volume regulation for T cell functions. Moreover, cell volume regulation via LRRC8A controls T cell-mediated antiviral immunity and shapes the TCR repertoire in the thymus. Mechanistically, LRRC8A governs stringent cell volume increase via regulated volume decrease (RVD) during T cell blast formation to keep the TCR signaling molecules at an adequate density. Together, our results show a further layer of T cell activation regulation that LRRC8A functions as a cell volume controlling "valve" to facilitate T cell activation.

Aspirin induces immunogenic cell death and enhances cancer immunotherapy in colorectal cancer.

The use of aspirin is associated with reduced incidence of colorectal cancer (CRC). However, the detailed mechanism remains unclear. In this study, we reported that colon cancer cells treated with aspirin showed the hallmarks of immunogenic cell death (ICD), including surface expression of calreticulin (CRT) and heat shock protein 70 (HSP70). Mechanistically, aspirin induced endoplasmic reticulum (ER) stress in colon cancer cells. In addition, aspirin decreased the expression of the glucose transporters, GLUT3, and reduced the key enzyme of glycolysis, including HK2, PFKM, PKM2 and LDHA. The changes of tumor glycolysis after aspirin treatment were associated with c-MYC downregulation. Moreover, aspirin potentiated the antitumor efficacy of anti-PD-1 antibody and anti-CTLA-4 antibody in CT26 tumors. However, this antitumor activity of aspirin in combination with anti-PD-1 antibody was abolished by the depletion of CD8+ T cells. Vaccination with tumor antigens is one of the strategies for activating T-cell response against tumors. Here, we demonstrated that aspirin-treated tumor cells in combination with tumor antigens (AH1 peptide) or protective substituted peptide (A5 peptide) could be served as a potent vaccine to eradicate tumors. Overall, our data indicated that aspirin can be used as an inducer of ICD for CRC therapy.

SpBNIP3 regulates apoptosis and autophagy in mud crab (Scylla paramamosain) during white spot syndrome virus infection.

BNIP3 (BCL2 and adenovirus E1B 19-kDa-interacting protein 3), which is a pro-apoptotic protein in the BCL-2 family involves a variety of cell signaling pathways, including mitochondrial dysfunction, mitochondrial autophagy, and apoptosis in vertebrates. However, the role of BNIP3 in the regulation of apoptosis and/or autophagy in crustaceans suffering virus infection is still limited. In this study, the mud crab (Scylla paramamosain) BNIP3 (SpBNIP3) was identified and studied to elucidate its association with the white spot syndrome virus (WSSV) infection. SpBNIP3 was widely expressed in all tested tissues and significantly down-regulated in the hemocytes of mud crab after WSSV infection. Knockdown of SpBNIP3 using RNA interference increased the apoptosis rate and Caspase 3 activity but decreased the mitochondrial membrane potential and autophagy levels, as well as viral copy number in mud crabs infected with WSSV. Additionally, the relationship between the viral infection and the autophagy of hemocytes was observed. The level of autophagy was reduced upon WSSV infection, and the activation of autophagy enriched the viral copy number. Taken together, the results of this study provide a new finding on the mechanism that SpBNIP3 may participate in the WSSV infection through the regulation of apoptosis and autophagy processes in mud crabs.

Cas12a variants designed for lower genome-wide off-target effect through stringent PAM recognition.

Cas12a is an RNA-guided endonuclease that has been widely used for convenient multiplex gene editing with low off-target effects. To minimize off-targeting in gene editing, we engineered a variant of LbCas12a (termed Lb-K538R) with more stringent PAM recognition, lower off-targeting capability, and similar editing efficiency in vivo compared with LbCas12a. We also demonstrated that Lb2Cas12a from Lachnospiraceae bacterium MA2020 has extensive gene-editing activities in mammalian cells. Similar to Lb-K538R, the designed Lb2Cas12a variant (termed Lb2-K518R) not only had a more stringent PAM sequence change from YYN to TYN (Y is T or C, N is A, T, C, or G), but also displayed lower off-target effects, thereby enabling more potential target site selections with low off-targeting than the common TTTV (V is A, G, or C) PAM. To determine whether this type of mutation at the homologous position had similar effects in other Cas12a, As-K548R was evaluated. Based on the results of the genome-wide off-target test, As-K548R displayed lower off-target effects. Collectively, our findings indicate that the Cas proteins could be designed to be stringent in PAM recognition to reduce their off-target effects, which suggests a promising and practical approach for minimizing off-targets effects in genome editing.

Molting Alters the Microbiome, Immune Response, and Digestive Enzyme Activity in Mud Crab (Scylla paramamosain).

Molting is a crucial lifelong process in the growth, development, and reproduction of crustaceans. In mud crab (Scylla paramamosain), new exoskeleton, gills, and appendages are formed after a molting, which contributes to a 40 to 90% increase in body weight. However, little is currently known about the associations between molting and the dynamic changes of microbiota and physiological characteristics in mud crabs. In this study, the effects of molting on changes of the microbiome, immune response, and digestive enzyme activities in mud crabs were investigated. The results showed dynamic changes in the abundances and community compositions of crab-associated microbiota harboring the gills, subcuticular epidermis, hepatopancreas, midgut, and hemolymph during molting. Renewed microbiota was observed in the gills and midgut of crabs at the postmolt stages, which seems to be related to the formation of a new exoskeleton after the molting. A significant positive correlation between the expression of two antimicrobial peptide (AMP) genes (SpALF5 and SpCrustin) and the relative abundance of two predominant microorganisms (Halomonas and Shewanella) in hemolymph was observed in the whole molt cycle, suggesting that AMPs play a role in modulating hemolymph microbiota. Furthermore, digestive enzymes might play a vital role in the changes of microbiota harboring the hepatopancreas and midgut, which provide suitable conditions for restoring and reconstructing host-microbiome homeostasis during molting. In conclusion, this study confirms that molting affects host-associated microbiota and further sheds light on the effects on the immune response and the digestive systems as well. IMPORTANCE Molting is crucial for crustaceans. In mud crab, its exoskeleton is renewed periodically during molting, and this process is an ideal model to study the effects of host development on its microbiota. Here, multiple approaches were used to investigate the changes in microbial taxa, immune response, and digestive enzyme activity with respect to molting in mud crab. The results found that a renewed microbiota was generated in the gills and midgut of crab after a molt. A significant positive correlation between changes in the relative abundances of microbes (such as Halomonas and Shewanella) and the expression of AMP genes (SpALF5 and SpCrustin) was observed in the hemolymph of crabs during the whole molt cycle, suggesting the modulation of hemolymph microbes by AMPs. Furthermore, the digestive enzymes were found to participate in the regulation of microbiota in hepatopancreas and midgut, consequently providing a suitable condition for the restoration and reconstruction of host-microbiome homeostasis during the molting. This study confirms that molting affects the microbial communities and concomitantly influences the immune and digestive systems in mud crabs. This is also the first time the homeostasis of the host and microbiome, and the associations between molting and physiological characteristics in crustaceans, have been revealed.

SpATF2 participates in maintaining the homeostasis of hemolymph microbiota by regulating dual oxidase expression in mud crab.

Activating transcription factors 2 (ATF2) is a transcription factor of the members of ATF/CREB family that is phosphorylated and activated by the mitogen-activated protein kinase (MAPK) in responding to the stimulation of stimuli. In present study, SpATF2 from mud crab (Scylla paramamosain) was identified and studied. The open reading frame of SpATF2 with 2136 bp in length encodes a protein with 711 amino acids. The SpATF2 protein includes the putative zinc finger domain in the N-terminus and bZIP type DNA-binding domain in the C-terminal. Tissue distribution of SpATF2 transcripts showed that SpATF2 was ubiquitously expressed in all examined tissues of the untreated mud crabs, with the highest expression levels in muscle and hepatopancreas. The transcriptional level of SpATF2 was up-regulated in the hemocytes after Vibrio parahemolyticus or WSSV infection. Reporter gene assays indicated that SpATF2 could activate the expression of dual oxidase (SpDuox1) in S. paramamosain. The RNA interference (RNAi) of SpATF2 significantly decreased the expression of SpDuox1, and consequently reduced reactive oxygen species production thereby significantly increased the bacterial load in the hemolymph of mud crabs. Similarly, significant reduction in bacterial clearance of hemolymph was observed after the V. parahemolyticus infection in SpATF2 knockdown mud crabs. This study showed that SpATF2 played a vital role in maintaining homeostasis of the hemolymph microbiota through regulating the expression of dual oxidase of mud crab.

Comparison of the complete mitochondrial genome of Phyllophorus liuwutiensis (Echinodermata: Holothuroidea: Phyllophoridae) to that of other sea cucumbers.

Sea cucumber species are abundant (>1400 species) and widely distributed globally. mtDNA sequencing is frequently used to identify the phylogenetic and evolutionary relationships among species. However, there are no reports on the mitochondrial genome of Phyllophorus liuwutiensis. Here, we performed mtDNA sequencing of P. liuwutiensis to examine its phylogenetic relationships with other echinoderms. Its mitochondrial genome (15 969 bp) contains 37 coding genes, including 13 protein-coding genes, 22 tRNA genes and 2 rRNA genes. Except for one protein-coding gene (nad6) and five tRNA genes encoded on the negative strand, all other genes were encoded on the positive strand. The mitochondrial bases of P. liuwutiensis were composed of 29.55% T, 22.16% C, 35.64% A and 12.64% G. The putative control region was 703 bp in length. Seven overlapping regions (1-10 bp) were found. The noncoding region between the genes ranged from 1 to 130 bp in length. One putative control region has been found in the P. liuwutiensis mitogenome. All of the tRNA genes were predicted to fold into a cloverleaf structure. In addition, we compared the gene arrangements of six echinoderms, revealing that the gene order of P. liuwutiensis was a new arrangement.

Effects of prebiotic mixtures on growth performance, intestinal microbiota and immune response in juvenile chu's croaker, Nibea coibor.

Prebiotics has been known to be growth promoter and immunostimulant in aquatic animals. In this study, we investigated the effects of prebiotics on growth performance, intestinal microbiota, short-chain fatty acids (SCFAs) production and immune response of the marine fish, juvenile chu's croaker (Nibea coibor). The fish were fed IG (including 0.5% inulin and 0.5% GOS), GS (0.5% GOS and 0.5% D-sorbitol), IGS (0.33% inulin, 0.33% GOS and 0.33% D-sorbitol) or control diets for 8 weeks. The results showed that the growth performance of the fish was promoted by IG and GS, but not by IGS. The intestinal microbiota in NDC (non-digestible carbohydrates, NDC)-supplemented groups was clearly separated from that of the control, and the highest Shannon and Simpson diversity indices were observed in the IGS group. In the intestine of the croaker, Proteobacteria, Firmicutes, and Bacteroidetes were dominant; among them, 24 taxa revealed a significant difference among groups. Most of these bacteria are able to produce SCFAs, which were significantly increased in all NDC-supplemented groups. Moreover, NDCs were found to activate the immune system of the fish by modulating the serum complements, cytokine levels, lysozyme activities and antioxidant capacity. Furthermore, the results of this study revealed correlations among intestinal microbiota, SCFAs production, innate immunity, antioxidant capacity and digestive enzymes in the croaker fed NDCs. Taken together, our results demonstrated that NDC mixtures might promote growth performance, antioxidant capacity and immune responses of the croaker through modulating the composition of intestinal microbiota and the subsequent SCFAs production, which suggest that NDCs were efficient feed additives for marine fish.

Spp38 MAPK participates in maintaining the homeostasis of hemolymph microbiota in Scylla paramamosain.

The p38 mitogen-activated protein kinases (MAPKs) are evolutionally conserved from yeasts to mammals, and are involved in the regulation of cells response to various extracellular stimuli. In this study, the p38 MAPK gene (designated as Spp38) of mud crab (Scylla paramamosain) was identified and studied. Spp38 contained the conserved Thr-Gly-Tyr (TGY) motif and a Ala-Thr-Arg-Trp (ATRW) substrate-binding site. Spp38 transcript was ubiquitously expressed in all tissues examined, with the highest expression found in muscle and hepatopancras. Quantitative real-time PCR revealed that Spp38 was upregulated in hemocytes and hepatopancras after infection with Vibrio parahemolyticus and Lipopolysaccharides (LPS). Reporter gene assays indicated that Spp38 activated the expression of anti-lipopolysaccharides (SpALF1 - SpALF6) in S. paramamosian. RNA interference (RNAi)-mediated knockdown of Spp38 or inhibition of Spp38 by SB203580 decreased the expression levels of SpALF1-6 and dual oxidase (SpDuox1 and SpDuox2) in S. paramamosian, which consequently reduced reactive oxygen species (ROS) production thereby significantly increasing the bacterial count in the hemolymph of mud crabs. Similarly, there was a significant reduction in bacterial clearance ability of hemolymph after Spp38 knockdown followed by V. parahemolyticus infection. Taken together, the current data indicated that Spp38 could play a vital role in maintaining the homeostasis of hemolymph microbiota in S. paramamosain.

Complete mitochondrial genome of Holothuria leucospilata (Holothuroidea, Holothuriidae) and phylogenetic analysis.

The complete Holothuria leucospilata mitochondrial genome was determined and analyzed in this work. It had a circular mapping molecular with a total length of 15,904 bp and contained 13 protein-coding genes, 2 rRNA genes, 22 tRNA genes, and 1 putative control region. Phylogenetic analysis showed that H. leucospilata clustered together with Holothuria scabra and Holothuria forskali. The complete mitochondrial genome provided in this work would be used for elucidation of Holothuroidea conservation genetics and evolutionary relationships.

Dual oxidases participate in the regulation of hemolymph microbiota homeostasis in mud crab Scylla paramamosain.

Dual oxidases (DUOXs) were originally identified as NADPH oxidases (NOXs), found to be associated with the reactive oxygen species (ROS) hydrogen peroxide (H2O2) production at the plasma membrane and crucial in host biological processes. In this study, SpDUOX1 and SpDUOX2 of mud crab (Scylla paramamosain) were identified and studied. Both SpDUOX1 and SpDUOX2 are transmembrane proteins, including an N-signal peptide region and a peroxidase homology domain in the extracellular region, transmembrane regions, and three EF (calcium-binding region) domains, a FAD-binding domain, and a NAD binding domain in the intracellular region. The SpDUOXs were expressed in all tissues examined, but mainly in hepatopancreas, heart, and mid-intestine. The expression of the SpDUOXs in the hemolymph of mud crabs was up-regulated after challenge with Vibrio parahemolyticus or LPS. RNA interference (RNAi) of the SpDUOXs resulted in reduced ROS production in hemolymph. The bacterial count increased in the hemolymph of mud crabs injected with SpDUOX1 or SpDUOX2-RNAi, while the bacterial clearance ability of hemolymph significantly reduced. At the phylum level, the phyla Bacteroidetes and Actinobacteria were significantly increased, while Proteobacteria were significantly reduced following SpDUOX2 knockdown. There was a significant increase in the relative abundance of the genera Marinomonas, Pseudoalteromonas, Shewanella, and Hydrogenoph in SpDUOX2 depleted mud crabs compared with the controls. Our current findings therefore indicated that SpDUOXs might play important roles in maintaining the homeostasis in the hemolymph microbiota of mud crab.

Hemolymph Microbiomes of Three Aquatic Invertebrates as Revealed by a New Cell Extraction Method.

Symbiotic microorganisms have been found in the hemolymph (blood) of many aquatic invertebrates, such as crabs, shrimp, and oysters. Hemolymph is a critical site in the host immune response. Currently, studies on hemolymph microorganisms are mostly performed with culture-dependent strategies using selective media (e.g., thiosulfate-citrate-bile salts-sucrose [TCBS], 2216E, and LB) for enumerating and isolating microbial cells. However, doubts remain about the "true" representation of the microbial abundance and diversity of symbiotic microorganisms in hemolymph, particularly for uncultivable microorganisms, which are believed to be more abundant than the cultured microorganisms. To explore this, we developed a culture-independent cell extraction method for separating microbial cells from the hemolymph of three aquatic invertebrates (Scylla paramamosain [mud crab], Litopenaeus vannamei [whiteleg shrimp], and Crassostrea angulata [Portuguese oysters]) involving filtration through a 5-µm-pore-size mesh filter membrane (the filtration method). A combination of the filtration method with fluorescence microscopy and high-throughput sequencing technique provides insight into the abundances and diversity of the total microbiota in the hemolymph of these three invertebrates. More than 2.6 × 104 cells/ml of microbial cells dominated by Escherichia-Shigella and Halomonas, Photobacterium and Escherichia-Shigella, and Pseudoalteromonas and Arcobacter were detected in the hemolymph of Scylla paramamosain, Litopenaeus vannamei, and Crassostrea angulata, respectively. A parallel study for investigating the hemolymph microbiomes by comparing the filtration method and a culture-dependent method (the plate count method) showed significantly higher microbial abundances (between 26- and 369-fold difference; P < 0.05) and less biased community structures of the filtration method than those of the plate count method. Furthermore, hemolymph of the three invertebrates harbored many potential pathogens, including Photobacterium, Arcobacter, and Vibrio species. Finally, the filtration method provides a solution that improves the understanding of the metabolic functions of uncultivable hemolymph microorganisms (e.g., metagenomics) devoid of host hemocyte contamination.IMPORTANCE Microorganisms are found in the hemolymph of invertebrates, a critical site in the host immune response. Currently, studies on hemolymph microorganisms are mostly performed with culture-dependent strategies. However, doubts remain about the "true" representation of the hemolymph microbiome. This study developed a culture-independent cell extraction method that could separate microbial cells from the hemolymph of three aquatic invertebrates (S. paramamosain, L. vannamei, and C. angulata) based on filtration through a 5-µm-pore-size mesh filter membrane (the filtration method). A combination of the filtration method with fluorescence microscopy and a high-throughput sequencing technique provides insight into the abundances and diversity of the total microbiota in the hemolymph of these three invertebrates. Our results demonstrate that the hemolymph of aquatic invertebrates harbors a much higher microbial abundance and more distinct microbial community composition than previously estimated. Furthermore, this work provides a less biased solution for studying the metabolic functions of uncultivable hemolymph microbiota devoid of host hemocyte contamination.