A pan-cancer single-cell panorama of human natural killer cells.

Natural killer (NK) cells play indispensable roles in innate immune responses against tumor progression. To depict their phenotypic and functional diversities in the tumor microenvironment, we perform integrative single-cell RNA sequencing analyses on NK cells from 716 patients with cancer, covering 24 cancer types. We observed heterogeneity in NK cell composition in a tumor-type-specific manner. Notably, we have identified a group of tumor-associated NK cells that are enriched in tumors, show impaired anti-tumor functions, and are associated with unfavorable prognosis and resistance to immunotherapy. Specific myeloid cell subpopulations, in particular LAMP3+ dendritic cells, appear to mediate the regulation of NK cell anti-tumor immunity. Our study provides insights into NK-cell-based cancer immunity and highlights potential clinical utilities of NK cell subsets as therapeutic targets.

Parallel single-cell and bulk transcriptome analyses reveal key features of the gastric tumor microenvironment.

BACKGROUND: The tumor microenvironment (TME) has been shown to strongly influence treatment outcome for cancer patients in various indications and to influence the overall survival. However, the cells forming the TME in gastric cancer have not been extensively characterized. RESULTS: We combine bulk and single-cell RNA sequencing from tumors and matched normal tissue of 24 treatment-naïve GC patients to better understand which cell types and transcriptional programs are associated with malignant transformation of the stomach. Clustering 96,623 cells of non-epithelial origin reveals 81 well-defined TME cell types. We find that activated fibroblasts and endothelial cells are most prominently overrepresented in tumors. Intercellular network reconstruction and survival analysis of an independent cohort imply the importance of these cell types together with immunosuppressive myeloid cell subsets and regulatory T cells in establishing an immunosuppressive microenvironment that correlates with worsened prognosis and lack of response in anti-PD1-treated patients. In contrast, we find a subset of IFNγ activated T cells and HLA-II expressing macrophages that are linked to treatment response and increased overall survival. CONCLUSIONS: Our gastric cancer single-cell TME compendium together with the matched bulk transcriptome data provides a unique resource for the identification of new potential biomarkers for patient stratification. This study helps further to elucidate the mechanism of gastric cancer and provides insights for therapy.

Genomic and microbial factors affect the prognosis of anti-pd-1 immunotherapy in nasopharyngeal carcinoma.

Antibodies targeting the programmed cell death protein-1 (PD-1) molecule have been reported to hold promising antitumor activities in patients with nasopharyngeal carcinoma (NPC). However, only a small subset of NPC patients benefits from the anti-PD-1 monotherapy and factors that affect the treatment response need further investigation. This study aimed to examine the impact of multiple genetic and environmental factors on outcome of anti-PD-1 immunotherapy by identifying tumor size, tumor mutation burden (TMB) based on whole exon sequencing, human leukocyte antigen class I (HLA-I) homo-/heterozygosity and supertypes, blood Epstein-Barr virus (EBV) DNA load, T cell proportions, and interferon-γ(IFN-γ) levels in a cohort of 57 NPC patients that received Nivolumab or Camrelizumab treatment. Moreover, we profiled the longitudinal changes in gut microbiota composition using shotgun metagenomics sequencing. We observed that high TMB combined with HLA-I heterozygosity was associated with improved clinical outcomes. In agreement with previous studies, we found that patients with higher plasma EBV DNA load showed worse progression-free survival. We found no evidence for an effect of gut bacterial diversity on the treatment response, but identified a higher abundance of seven specific gut bacteria at baseline of non-responders, including Blautia wexlera and Blautia obeum, as well as four other bacteria belonging to the Clostridiales order, and one Erysipelatoclostridium. Combined, this study provides insight into the influence of several genetic and environmental factors on anti-PD-1 immunotherapy responses in NPC patients.

A genetic model for in vivo proximity labelling of the mammalian secretome.

Organ functions are highly specialized and interdependent. Secreted factors regulate organ development and mediate homeostasis through serum trafficking and inter-organ communication. Enzyme-catalysed proximity labelling enables the identification of proteins within a specific cellular compartment. Here, we report a BirA*G3 mouse strain that enables CRE-dependent promiscuous biotinylation of proteins trafficking through the endoplasmic reticulum. When broadly activated throughout the mouse, widespread labelling of proteins was observed within the secretory pathway. Streptavidin affinity purification and peptide mapping by quantitative mass spectrometry (MS) proteomics revealed organ-specific secretory profiles and serum trafficking. As expected, secretory proteomes were highly enriched for signal peptide-containing proteins, highlighting both conventional and non-conventional secretory processes, and ectodomain shedding. Lower-abundance proteins with hormone-like properties were recovered and validated using orthogonal approaches. Hepatocyte-specific activation of BirA*G3 highlighted liver-specific biotinylated secretome profiles. The BirA*G3 mouse model demonstrates enhanced labelling efficiency and tissue specificity over viral transduction approaches and will facilitate a deeper understanding of secretory protein interplay in development, and in healthy and diseased adult states.

Distinct Functional Metagenomic Markers Predict the Responsiveness to Anti-PD-1 Therapy in Chinese Non-Small Cell Lung Cancer Patients.

Background: Programmed death 1 (PD-1) and the ligand of PD-1 (PD-L1) are central targets for immune-checkpoint therapy (ICT) blocking immune evasion-related pathways elicited by tumor cells. A number of PD-1 inhibitors have been developed, but the efficacy of these inhibitors varies considerably and is typically below 50%. The efficacy of ICT has been shown to be dependent on the gut microbiota, and experiments using mouse models have even demonstrated that modulation of the gut microbiota may improve efficacy of ICT. Methods: We followed a Han Chinese cohort of 85 advanced non-small cell lung cancer (NSCLC) patients, who received anti-PD-1 antibodies. Tumor biopsies were collected before treatment initiation for whole exon sequencing and variant detection. Fecal samples collected biweekly during the period of anti-PD-1 antibody administration were used for metagenomic sequencing. We established gut microbiome abundance profiles for identification of significant associations between specific microbial taxa, potential functionality, and treatment responses. A prediction model based on random forest was trained using selected markers discriminating between the different response groups. Results: NSCLC patients treated with antibiotics exhibited the shortest survival time. Low level of tumor-mutation burden and high expression level of HLA-E significantly reduced progression-free survival. We identified metagenomic species and functional pathways that differed in abundance in relation to responses to ICT. Data on differential enrichment of taxa and predicted microbial functions in NSCLC patients responding or non-responding to ICT allowed the establishment of random forest algorithm-adopted models robustly predicting the probability of whether or not a given patient would benefit from ICT. Conclusions: Overall, our results identified links between gut microbial composition and immunotherapy efficacy in Chinese NSCLC patients indicating the potential for such analyses to predict outcome prior to ICT.

An Asia-specific variant of human IgG1 represses colorectal tumorigenesis by shaping the tumor microenvironment.

Emerging studies have focused on ways to treat cancers by modulating T cell activation. However, whether B cell receptor signaling in the tumor microenvironment (TME) can be harnessed for immunotherapy is unclear. Here, we report that an Asia-specific variant of human IgG1 containing a Gly396 to Arg396 substitution (hIgG1-G396R) conferred improved survival of patients with colorectal cancer (CRC). Mice with knockin of the murine functional homolog mIgG2c-G400R recapitulated the alleviated tumorigenesis and progression in murine colon carcinoma models. Immune profiling of the TME revealed broad mobilizations of IgG1+ plasma cells, CD8+ T cells, CD103+ DCs, and active tertiary lymphoid structure formation, suggesting an effective antitumor microenvironment in hIgG1-G396R CRC patients. Mechanistically, this variant potentiated tumor-associated antigen-specific (TAA-specific) plasma cell differentiation and thus antibody production. These elevated TAA-specific IgG2c antibodies in turn efficiently boosted the antibody-dependent tumor cell phagocytosis and TAA presentation to effector CD8+ T cells. Notably, adoptive transfer of TAA-specific class-switched memory B cells harboring this variant exhibited therapeutic efficacy in murine tumor models, indicating their clinical potential. All these results prompted a prospective investigation of hIgG1-G396R in patients with CRC as a biomarker for clinical prognosis and demonstrated that manipulating the functionality of IgG1+ memory B cells in tumors could improve immunotherapy outcomes.

Pan-cancer single-cell landscape of tumor-infiltrating T cells.

T cells play a central role in cancer immunotherapy, but we lack systematic comparison of the heterogeneity and dynamics of tumor-infiltrating T cells across cancer types. We built a single-cell RNA-sequencing pan-cancer atlas of T cells for 316 donors across 21 cancer types and revealed distinct T cell composition patterns. We found multiple state-transition paths in the exhaustion of CD8+ T cells and the preference of those paths among different tumor types. Certain T cell populations showed specific correlation with patient properties such as mutation burden, shedding light on the possible determinants of the tumor microenvironment. T cell compositions within tumors alone could classify cancer patients into groups with clinical trait specificity, providing new insights into T cell immunity and precision immunotherapy targeting T cells.

A pan-cancer single-cell transcriptional atlas of tumor infiltrating myeloid cells.

Tumor-infiltrating myeloid cells (TIMs) are key regulators in tumor progression, but the similarity and distinction of their fundamental properties across different tumors remain elusive. Here, by performing a pan-cancer analysis of single myeloid cells from 210 patients across 15 human cancer types, we identified distinct features of TIMs across cancer types. Mast cells in nasopharyngeal cancer were found to be associated with better prognosis and exhibited an anti-tumor phenotype with a high ratio of TNF+/VEGFA+ cells. Systematic comparison between cDC1- and cDC2-derived LAMP3+ cDCs revealed their differences in transcription factors and external stimulus. Additionally, pro-angiogenic tumor-associated macrophages (TAMs) were characterized with diverse markers across different cancer types, and the composition of TIMs appeared to be associated with certain features of somatic mutations and gene expressions. Our results provide a systematic view of the highly heterogeneous TIMs and suggest future avenues for rational, targeted immunotherapies.

Resolving the genetic paradox of invasions: Preadapted genomes and postintroduction hybridization of bigheaded carps in the Mississippi River Basin.

The genetic paradox of biological invasions is complex and multifaceted. In particular, the relative role of disparate propagule sources and genetic adaptation through postintroduction hybridization has remained largely unexplored. To add resolution to this paradox, we investigate the genetic architecture responsible for the invasion of two invasive Asian carp species, bighead carp (Hypophthalmichthys nobilis) and silver carp (H. molitrix) (bigheaded carps) that experience extensive hybridization in the Mississippi River Basin (MRB). We sequenced the genomes of bighead and silver carps (~1.08G bp and ~1.15G bp, respectively) and their hybrids collected from the MRB. We found moderate-to-high heterozygosity in bighead (0.0021) and silver (0.0036) carps, detected significantly higher dN/dS ratios of single-copy orthologous genes in bigheaded carps versus 10 other species of fish, and identified genes in both species potentially associated with environmental adaptation and other invasion-related traits. Additionally, we observed a high genomic similarity (96.3% in all syntenic blocks) between bighead and silver carps and over 90% embryonic viability in their experimentally induced hybrids. Our results suggest intrinsic genomic features of bigheaded carps, likely associated with life history traits that presumably evolved within their native ranges, might have facilitated their initial establishment of invasion, whereas ex-situ interspecific hybridization between the carps might have promoted their range expansion. This study reveals an alternative mechanism that could resolve one of the genetic paradoxes in biological invasions and provides invaluable genomic resources for applied research involving bigheaded carps.

Comparative analysis of sequencing technologies for single-cell transcriptomics.

Single-cell RNA-seq technologies require library preparation prior to sequencing. Here, we present the first report to compare the cheaper BGISEQ-500 platform to the Illumina HiSeq platform for scRNA-seq. We generate a resource of 468 single cells and 1297 matched single cDNA samples, performing SMARTer and Smart-seq2 protocols on two cell lines with RNA spike-ins. We sequence these libraries on both platforms using single- and paired-end reads. The platforms have comparable sensitivity and accuracy in terms of quantification of gene expression, and low technical variability. Our study provides a standardized scRNA-seq resource to benchmark new scRNA-seq library preparation protocols and sequencing platforms.

Endangered Père David's deer genome provides insights into population recovering.

The Milu (Père David's deer, Elaphurus davidianus) were once widely distributed in the swamps (coastal areas to inland areas) of East Asia. The dramatic recovery of the Milu population is now deemed a classic example of how highly endangered animal species can be rescued. However, the molecular mechanisms that underpinned this population recovery remain largely unknown. Here, different approaches (genome sequencing, resequencing, and salinity analysis) were utilized to elucidate the aforementioned molecular mechanisms. The comparative genomic analyses revealed that the largest recovered Milu population carries extensive genetic diversity despite an extreme population bottleneck. And the protracted inbreeding history might have facilitated the purging of deleterious recessive alleles. Seventeen genes that are putatively related to reproduction, embryonic (fatal) development, and immune response were under high selective pressure. Besides, SCNN1A, a gene involved in controlling reabsorption of sodium in the body, was positively selected. An additional 29 genes were also observed to be positively selected, which are involved in blood pressure regulation, cardiovascular development, cholesterol regulation, glycemic control, and thyroid hormone synthesis. It is possible that these genetic adaptations were required to buffer the negative effects commonly associated with a high-salt diet. The associated genetic adaptions are likely to have enabled increased breeding success and fetal survival. The future success of Milu population management might depend on the successful reintroduction of the animal to historically important distribution regions.

Purification and characterization of a novel intracellular α-amylase with a wide variety of substrates hydrolysis and transglycosylation activity from Paenibacillus sp. SSG-1.

Intracellular α-amylase was a special glycoside hydrolase in the cytoplasm. We cloned and expressed an intracellular α-amylase, Amy, from Paenibacillus sp. SSG-1. The recombinant enzyme was purified by metal-affinity chromatography, exhibited a molecular mass of 71.7 kDa. Amy exhibited unexpectedly sequence similarity and evolutionary relationships with alpha-glucanotransferase. The docked results of Amy with maltose showed it had similar catalytic residues with α-amylase and glucanotransferase. The substrate specificity experiment showed that Amy could hydrolyze typical substrates into glucose and maltose. It was noteworthy that Amy showed the catalytic capacity of cyclomaltodextrinase and pullulanase. Meanwhile, Amy could transfer sugar molecules and form maltotetraose upon the hydrolysis of substrates. These results indicated that Amy was a novel intracellular α-amylase with distinct catalytic ability characteristics of hydrolyzing glycogen/cyclodextrin/pullulan and transglycosylation. We deduced that Amy may play an important role in utilizing maltooligosaccharides that released from extracellular α-glucan or storage α-glucan (glycogen) in Paenibacillus sp. SSG-1.

Comparative genomic analysis of Paenibacillus sp. SSG-1 and its closely related strains reveals the effect of glycometabolism on environmental adaptation.

The extensive environmental adaptability of the genus Paenibacillus is related to the enormous diversity of its gene repertoires. Paenibacillus sp. SSG-1 has previously been reported, and its agar-degradation trait has attracted our attention. Here, the genome sequence of Paenibacillus sp. SSG-1, together with 76 previously sequenced strains, was comparatively studied. The results show that the pan-genome of Paenibacillus is open and indicate that the current taxonomy of this genus is incorrect. The incessant flux of gene repertoires resulting from the processes of gain and loss largely contributed to the difference in genomic content and genome size in Paenibacillus. Furthermore, a large number of genes gained are associated with carbohydrate transport and metabolism. It indicates that the evolution of glycometabolism is a key factor for the environmental adaptability of Paenibacillus species. Interestingly, through horizontal gene transfer, Paenibacillus sp. SSG-1 acquired an approximately 150 kb DNA fragment and shows an agar-degrading characteristic distinct from most other non-marine bacteria. This region may be transported in bacteria as a complete unit responsible for agar degradation. Taken together, these results provide insights into the evolutionary pattern of Paenibacillus and have implications for studies on the taxonomy and functional genomics of this genus.

Complete Chloroplast Genome Sequence of Coptis chinensis Franch. and Its Evolutionary History.

The Coptis chinensis Franch. is an important medicinal plant from the Ranunculales. We used next generation sequencing technology to determine the complete chloroplast genome of C. chinensis. This genome is 155,484 bp long with 38.17% GC content. Two 26,758 bp long inverted repeats separated the genome into a typical quadripartite structure. The C. chinensis chloroplast genome consists of 128 gene loci, including eight rRNA gene loci, 28 tRNA gene loci, and 92 protein-coding gene loci. Most of the SSRs in C. chinensis are poly-A/T. The numbers of mononucleotide SSRs in C. chinensis and other Ranunculaceae species are fewer than those in Berberidaceae species, while the number of dinucleotide SSRs is greater than that in the Berberidaceae. C. chinensis diverged from other Ranunculaceae species an estimated 81 million years ago (Mya). The divergence between Ranunculaceae and Berberidaceae was ~111 Mya, while the Ranunculales and Magnoliaceae shared a common ancestor during the Jurassic, ~153 Mya. Position 104 of the C. chinensis ndhG protein was identified as a positively selected site, indicating possible selection for the photosystem-chlororespiration system in C. chinensis. In summary, the complete sequencing and annotation of the C. chinensis chloroplast genome will facilitate future studies on this important medicinal species.

Horizontal Transfer of a Novel Soil Agarase Gene from Marine Bacteria to Soil Bacteria via Human Microbiota.

Seaweed is receiving an increasing amount of attention as a "sea vegetable". The microbiota of coastal populations may acquire seaweed associated enzymes through marine food. Several agarases have been found in non-marine environments; however, their origin is unknown. In this study, a hypothetical protein, Aga1, was identified as an agarase from an inland soil agar-degrading bacterium, Paenibacillus sp. SSG-1.Having low similarity to known glycoside hydrolases, Aga1 may be a distant member of the glycoside hydrolase family 86. Aga1 has good pH stability (pH 3-11) and is stable in the presence of various metal ions. Aga1 is an exo-type ß-agarase that produces NA 4 (neoagarotetraose) and NA 6 (neoagarohexaose) as its main products. In addition, Aga1 may be a cell-surface-binding protein. The bioinformatic analysis showed aga1 may have been transfered together with its surrounding genes, from marine bacteria to soil bacteria via human microbiota. The use of seaweed as food and the disposal of human faeces or saliva were the most likely reasons for this gene transfer pathway. Notably, the results also indicated that microbes from inland humans may degrade agar and that these microbes may have acquired seaweed associated genes because of increased seaweed in diets.