Qian Wang

Background: The tumor microenvironment (TME) is instrumental in facilitating immune evasion and promoting tumor progression in non-small cell lung cancer (NSCLC). However, the spatial heterogeneity and molecular interactions between immune and non-immune cells are not yet fully understood. This research amalgamated single-cell RNA sequencing (scRNA-seq) and spatial transcriptomic data to examine the cellular composition, ligand-receptor (LR) interactions, and immunosuppressive mechanisms of NSCLC. Methods: ScRNA-seq was performed on NSCLC tumors and paired adjacent normal tissues to analyze immune and stromal cell heterogeneity. Spatial transcriptomics was applied to consecutive tissue sections to map the distribution of major cell types and their interactions. Cell clustering, a differential gene expression analysis, and LR interaction modeling were used to identify key immune regulatory pathways. In vitro co-culture experiments were performed for functional validation. Results: A total of 28,496 high-quality single cells were analyzed, and significant differences in immune cell composition between the tumor and normal tissues were found. The NSCLC tumors exhibited a notable increase in tumor-associated macrophages (TAMs) and fibroblasts, as well as a decrease in the presence of cytotoxic T cells and natural killer (NK) cells. Notably, spatial transcriptomics revealed that the TAMs were predominantly localized in the tumor cores, where they interacted with T cells via immunosuppressive LR pairs. The SPP1-CD44 and NECTIN2-TIGIT axes were identified as major immunosuppressive pathways, with the SPP1 secreted by the TAMs contributing to immune evasion. Additionally, the HLA-E-CD8B interactions were significantly upregulated, suggesting a potential mechanism of antigen presentation modulation. The functional validation showed that SPP1 overexpression (OE) enhanced PDCD1 and CD160 expression, further confirming its role in shaping the immunosuppressive microenvironment. Conclusions: This study established a high-resolution immune atlas of NSCLC, identifying novel macrophage-T cell interactions that drive immune suppression. Our findings suggest that targeting the SPP1-CD44, NECTIN2-TIGIT, and HLA-E-CD8B pathways may improve immune responses in NSCLC. These findings provide novel therapeutic targets and highlight the potential for combination immunotherapies to overcome macrophage-mediated immune evasion.

Intestinal symbionts live in insects’ digestive systems and help with food digestion, nitrogen cycling, and energy use. Numerous symbionts, including bacteria, fungi, and protozoa, colonize the posterior gut of termites. Intestinal microbiota affects the Toll or Imd pathways during viral or entomopathogenic fungus infection in mosquitoes, but the role of intestinal symbionts in individual termite immunity is unclear. In the present research, Odontotermes formosanus and Metarhizium robertsii were selected to investigate the potential mechanism of the intestinal bacteria influencing termite innate immune while encountering an entomopathogen using metagenome sequencing and meta-transcriptome sequencing. We found that termite mortality declined within 48 hours under high spore infections and reached semi-lethal status within 72 hours. We sequenced the control and infestation termite and found that the dominant phyla changed below low-abundance flora during infestation. According to function annotation, the functions of spirochetes are significantly different from those of the rest of the phyla, which are mainly enriched in metabolism and cellular processes. There are 261 upregulated and 459 down-regulated DEGs derived from bacteria after infestation. We identified the glutamate-cysteine ligase gene, gshA, which is significantly upregulated in the infected group, and the GSH content in the regular infected group was higher than the 5% kanamycin-infected group. We speculated on a potential mechanism that one or more bacteria belonging to Spirochetes that upregulated gshA gene expression and increased GSH content in the hindgut to integrate with metarhizium toxins and then excrete out of the intestine.

Background: Polycystic ovary syndrome (PCOS) is characterized by excess androgens, ovulatory dysfunction, and polycystic ovaries. The mechanisms underlying ovulatory and metabolic disorders in PCOS remain elusive, hampering therapeutic development. Enhanced metabolic health correlates with increased microbiota gene content and microbial diversity. We aimed to explore the impact of gut microbiota and serum steroids on PCOS regulation associated with androgen excess.Methods: The fecal samples of patients with hyperandrogenic PCOS (n = 14) and control group with PCOS (n = 14) were analyzed by 16S rRNA gene sequencing. The peripheral venous blood of all subjects was collected to detect serum hormones. The association between gut microbiota and serum hormones was analyzed with the R language.Results: Our findings reveal that the hyperandrogenic PCOS group exhibits lower richness and diversity of gut microbiota compared to the control group. Characteristic genera in PCOS patients with hyperandrogenism include Bifidobacterium, Enterobacteriaceae_unclassified, Streptococcus, Saccharimonadaceae, Enterococcus, and Eubacterium_nodatum_group. Five hormones, including 5β-androsterone, deoxycorticosterone, corticosterone, 11-dehydrocorticosterone, and cortexolone, emerge as potential serum biomarkers for identifying patients with hyperandrogenic-PCOS (HA-PCOS). Furthermore, a lower vitamin D3 level may act as a susceptibility factor, suggesting that vitamin D3 supplementation could serve as a potential intervention for PCOS with hyperandrogenism.Conclusion: Specific fecal microbiota and serum steroids may be used as characteristic markers for clinical diagnosis of hyperandrogenic-PCOS. This research enhances our understanding of the intricate interplay among hormones, gut microbiota, and hyperandrogenemia in patients with PCOS.

Termites are social insects that live in the soil or in decaying wood, where exposure to pathogens should be common. However, these pathogens rarely cause mortality in established colonies. In addition to social immunity, the gut symbionts of termites are expected to assist in protecting their hosts, though the specific contributions are unclear. In this study, we examined this hypothesis in Odontotermes formosanus, a fungus-growing termite in the family Termitidae, by 1) disrupting its gut microbiota with the antibiotic kanamycin, 2) challenging O. formosanus with the entomopathogenic fungus Metarhizium robertsii, and finally 3) sequencing the resultant gut transcriptomes. As a result, 142531 transcripts and 73608 unigenes were obtained, and unigenes were annotated following NR, NT, KO, Swiss-Prot, PFAM, GO, and KOG databases. Among them, a total of 3,814 differentially expressed genes (DEGs) were identified between M. robertsii infected termites with or without antibiotics treatment. Given the lack of annotated genes in O. formosanus transcriptomes, we examined the expression profiles of the top 20 most significantly differentially expressed genes using qRT-PCR. Several of these genes, including APOA2, Calpain-5, and Hsp70, were downregulated in termites exposed to both antibiotics and pathogen but upregulated in those exposed only to the pathogen, suggesting that gut microbiota might buffer/facilitate their hosts against infection by finetuning physiological and biochemical processes, including innate immunity, protein folding, and ATP synthesis. Overall, our combined results imply that stabilization of gut microbiota can assist termites in maintaining physiological and biochemical homeostasis when foreign pathogenic fungi invade.

The crosstalk between gut microbiota and host immunity has emerged as one of the research foci of microbiome studies in recent years. The purpose of this study was to determine how gut microbes respond to fungal infection in termites, given their reliance on gut symbionts for food intake as well as maintaining host health. Here, we used Metarhizium robertsii, an entomopathogenic fungus, to infect Odontotermes formosanus, a fungus-growing termite in the family Termitidae, and documented changes in host gut microbiota via a combination of bacterial 16S rDNA sequencing, metagenomic shotgun sequencing, and transmission electron microscopy. Our analyses found that when challenged with Metarhizium, the termite gut showed reduced microbial diversity within the first 12 h of fungal infection and then recovered and even surpassed pre-infection flora levels. These combined results shed light on the role of gut flora in maintaining homeostasis and immune homeostasis in the host, and the impact of gut flora dysbiosis on host susceptibility to infection.

Plant cell-wall polysaccharides constitute the most abundant but recalcitrant organic carbon source in nature. Microbes residing in the digestive tract of herbivorous bilaterians are particularly efficient at depolymerizing polysaccharides into fermentable sugars and play a significant support role towards their host’s lifestyle. Here, we combine large-scale functional screening of fosmid libraries, shotgun sequencing, and biochemical assays to interrogate the gut microbiota of the wood-feeding “higher” termite Globitermes brachycerastes. A number of putative polysaccharide utilization gene clusters were identified with multiple fibrolytic genes. Our large-scale functional screening of 50,000 fosmid clones resulted in 464 clones demonstrating plant polysaccharide-degrading activities, including 267 endoglucanase-, 24 exoglucanase-, 72 β-glucosidase-, and 101 endoxylanase-positive clones. We sequenced 173 functionally active clones and identified ~219 genes encoding putative carbohydrate-active enzymes (CAZymes) targeting cellulose, hemicellulose and pectin. Further analyses revealed that 68 of 154 contigs encode one or more CAZyme, which includes 35 examples of putative saccharolytic operons, suggesting that clustering of CAZymes is common in termite gut microbial inhabitants. Biochemical characterization of a representative xylanase cluster demonstrated that constituent enzymes exhibited complementary physicochemical properties and saccharolytic capabilities. Furthermore, diverse cellobiose-metabolizing enzymes include β-glucosidases, cellobiose phosphorylases, and phopho-6-β-glucosidases were identified and functionally verified, indicating that the termite gut micro-ecosystem utilizes diverse metabolic pathways to interconnect hydrolysis and central metabolism. Collectively, these results provide an in-depth view of the adaptation and digestive strategies employed by gut microbiota within this tiny-yet-efficient host-associated ecosystem.

Insect prophenoloxidase (PPO) induces melanization around pathogens. Before melanization, PPO is cleaved into phenoloxidase (PO) by serine proteases. Insect PPO can also be activated by exogenous proteases secreted by pathogens as well as by other compounds, such as ethanol and cetylpyridinium chloride (CPC). However, the effect of these activators on the activity of PO is unclear. In this study, the insect endogenous serine protease AMM1, α‐chymotrypsin, and ethanol were used to activate recombinant Drosophila PPO1 (rPPO1), and the PO activity differed depending on the activator applied. The PO‐induced intermediates during melanization also varied markedly in their numbers and abundances. Therefore, this study indicates that the mechanism of PPO activation influences PO activity. It also suggests that PO‐induced different intermediates may affect the antibacterial activity during melanization due to their toxicity.