Recent studies highlighted the clinicopathologic importance of the tumor microenvironment (TME) in delineating molecular attributes and therapeutic potentials. However, the overall TME cell infiltration landscape in nonsquamous non–small cell lung cancer (NSCLC) has not been comprehensively characterized. In this study, we used consensus non-negative matrix factorization molecular subtyping to determine TME cell infiltration patterns and identified 3 TME clusters (TME-C1, -C2, -C3) characterized by distinct clinicopathologic features, infiltrating cells, and biological processes. Proteomics analyses revealed that cyclic GMP-AMP–stimulator of interferon genes immune signaling–mediated protein and phosphorylation levels were significantly upregulated in inflammation-related TME-C2 clusters. The score extracted from the TME-related signature (TMEsig-score) divided patients with NSCLC into high- and low-score subgroups, where a high score was associated with favorable prognosis and immune infiltration. The genomic landscape revealed that patients with low TMEsig-score harbored more somatic copy number alterations and higher mutation frequency of driver genes involving STK11 , KEAP1 , SMARCA4 , and others. Drug sensitivity analyses suggested that tumors with high TMEsig-score were responsible for favorable clinical response to immune checkpoint inhibitor treatment. In summary, this study highlights that comprehensive recognizing of the TME cell infiltration landscape will contribute to enhancing our understanding of TME immune regulation and promote effectiveness of precision biotherapy strategies.

Background: Pulmonary infections caused by non-diphtheriae corynebacteria are increasing. However, rapid identification of Corynebacterium species poses a challenge due to the low genetic variation within the genus. Methods: Three reference strains and 99 clinical isolates were used in this study. A qPCR followed by high-resolution melting (HRM) targeting ssrA was performed to simultaneously identify C. striatum, C. propinquum and C. simulans. To further evaluate this assay’s performance, 88 clinical sputum samples were tested by HRM and the detection results were compared with those of the traditional culture method and multiple cross-displacement amplification (MCDA) assay. Results: The melting curve produced by a pair of universal primers generated species-specific HRM curve profiles and could distinguish the three target species from other related bacteria. The limit of detection of HRM assay for DNA from the three purified Corynebacterium species was 100 fg. Compared with the culture method, HRM detected 22 additional positive specimens, representing a 23.9% relative increase in detection rate. The HRM assay had 98.4% (95% confidence interval [CI], 90.5–99.9%) sensitivity and 100% (95% CI, 82.8–100%) specificity. Additionally, 95.5% concordance between HRM and MCDA (κ = 0.89 [95% CI, 0.79–0.99]) was noted. Conclusions: The HRM assay was a simple, rapid, sensitive, and specific diagnostic tool for detecting C. striatum, C. propinquum, and C. simulans, with the potential to contribute to early diagnosis, epidemiological surveillance, and rapid response to outbreak.

Background: Nocardia is a facultative intracellular pathogen that infects the lungs and brains of immunocompromised patients with consequences that can be fatal. The incidence of such infections is rising, immunocompetent individuals are also being infected, and there is a need to learn more about this neglected bacterial pathogen and the interaction with its human host. Results: We have applied dual RNA-seq to assess the global transcriptome changes that occur simultaneously in Nocardia farcinica (N. farcinica) and infected human epithelial alveolar host cells, and have tested a series of mutants in this in vitro system to identify candidate determinants of virulence. Using a mouse model, we revealed the profiles of inflammation-related factors in the lung after intranasal infection and confirmed that nbtB and nbtS are key virulence genes for Nocardia infection in vivo. Regarding the host response to infection, we found that the expression of many histones was dysregulated during the infection of lung cells, indicating that epigenetic modification might play a crucial role in the host during Nocardia infection. In our mouse model, Nocardia infection led to neurological symptoms and we found that 15 of 22 Nocardia clinical strains tested could cause obvious PD-like symptoms. Further experiments indicated that Nocardia infection could activate microglia and drive M1 microglial polarization, promote iNOS and CXCL-10 production, and cause neuroinflammation in the substantia nigra, all of which may be involved in causing PD-like symptoms. Importantly, the deletion of nbtS in N. farcinica completely attenuated the neurological symptoms. Conclusions: Our data contribute to an in-depth understanding of the characteristics of both the host and Nocardia during infection and provide valuable clues for future studies of this neglected human pathogen, especially those addressing the underlying causes of infection-related neurological symptoms.

Nocardia is emerging as a serious and easily neglected pathogen in clinical practice with multidrug resistance that extends the treatment period for months or even years. This has led to the investigation of a vaccine approach to prevent Nocardia infections. However, studies on the protective proteins of Nocardia have not yet been carried out. In the present work, over 500 proteins in the supernatant of N. farcinica IFM10152 were identified by LC–MS/MS. In silico analysis of these proteins with a high content (score > 2000) predicted that NFA49590 was one of the conserved proteins in N. farcinica strains with potential antigenicity. After the rNFA49590 protein was cloned and expressed in E. coli (DE3) and purified using a Ni-NTA column, its good antigenicity was confirmed with sera from mice immunized with different Nocardia species by Western blot. Then we confirmed its ability to activate innate immunity by examining the phosphorylation status of ERK1/2, JNK, p38, and p65 and the cytokine levels of IL-6, TNF-α, and IL-10. Finally, we evaluated its immunoprotective effect in BALB/c mice, and we found that mice immunized with rNFA49590 protein exhibited high antibody titers, enhanced bacterial clearance ability, and generated robust protective effects from the N. farcinica challenge. These results offer strong support for the use of NFA49590 protein as a vaccine candidate and open the possibilities for the exploration of a large array of immunoprotective proteins.