Molecular and transcriptional basis of bidirectional CD4+ T cell exhaustion in oropharyngeal squamous cell carcinoma.

Tumor-infiltrating CD4+ T cells orchestrate the adaptive immune response through remarkable plasticity, and the expression patterns of exhaustion-related inhibitory receptors in these cells differ significantly from those of CD8+ T cells. Thus, a better understanding of the molecular basis of CD4+ T cell exhaustion and their responses to immune checkpoint blockade (ICB) is required. Here, we integrated multiomics approaches to define the phenotypic and molecular profiles of exhausted CD4+ T cells in oropharyngeal squamous cell carcinoma (OPSCC). Two distinct immune-promoting (Module 1) and immunosuppressive (Module 2) functional modules in tumor-infiltrating CD4+ T cells were identified, and both the immune-promoting function of Module 1 cells and immunosuppressive function of Module 2 cells were positively associated with their corresponding exhaustion states. Furthermore, the application of ICBs targeting effector CD4+ T cells in Module 1 (αPD-1) and Treg cells in Module 2 (αCTLA-4) in mouse models could help reinvigorate the effector function of Module 1-exhausted CD4+ T cells and reduce the immunosuppressive function of Module 2-exhausted CD4+ T cells, ultimately promoting OPSCC tumor regression. Taken together, our study provides a crucial cellular basis for the selection of optimal ICB in treating OPSCC.

Proliferative exhausted CD8+ T cells exacerbate long-lasting anti-tumor effects in human papillomavirus-positive head and neck squamous cell carcinoma.

The survival prognosis of human papillomavirus (HPV)-positive and HPV-negative head and neck squamous cell carcinoma (HNSCC) is largely different, and little is known about the anti-tumor mechanism of tumor-infiltrated exhausted CD8+ T cells (Tex) in HNSCC. We performed cell-level multi-omics sequencing on human HNSCC samples to decipher the multi-dimensional characteristics of Tex cells. A proliferative exhausted CD8+ T cell cluster (P-Tex) which was beneficial to survival outcomes of patients with HPV-positive HNSCC was identified. Interestingly, P-Tex cells expressed CDK4 genes as high as cancer cells, which could be simultaneously inhibited by CDK4 inhibitors and might be a potential reason for the ineffectiveness of CDK4 inhibitors in treating HPV-positive HNSCC. P-Tex cells could aggregate in the antigen-presenting cell niches and activate certain signaling pathways. Together, our findings suggest a promising role for P-Tex cells in the prognosis of patients with HPV-positive HNSCC by providing modest but persistent anti-tumor effects.

Current Therapies in Patients With Posterior Semicircular Canal BPPV, a Systematic Review and Network Meta-analysis.

OBJECTIVE: To compare the efficacy of different treatments for posterior semicircular canal benign paroxysmal positional vertigo (PC-BPPV) by using direct and indirect evidence from existing randomized data. METHODS: Randomized case-control studies that compared the efficacy of various nonsurgical treatments in PC-BPPV patients at 1 week and 1 month of follow-up were comprehensively screened. Bayesian network meta-analysis was performed to evaluate direct and indirect treatment comparisons. We further conducted subgroup pairwise meta-analysis to explore the inconsistency between comparisons of the Epley versus a sham maneuver and the Epley versus the Semont maneuver. RESULTS: A total of 41 parallel, randomized controlled studies were included. The Epley with vestibular rehabilitation (EVR), Epley, Semont and Hybrid maneuvers were effective in eliminating nystagmus during a Dix-Hallpike test at 1 week of follow-up (odds ratios [ORs]: 11.41-23.8, 95% credible interval [CrI]: excluding null), among which EVR showed the best efficacy (the surface area under the cumulative ranking curves [SUCRA] = 77.5%). However, at 1 month of follow-up, only the Semont (rank first, SUCRA = 76.1%) and Epley maneuvers (rank second, SUCRA = 65.3%) were effective in eliminating nystagmus during a Dix-Hallpike test. In the pairwise subgroup meta-analysis, for patients younger than 55 years of age, the efficacy of the Epley maneuver was comparable to that of the Semont maneuver [rate ratio (RR): 0.99, 95% confidence interval (CI): 0.93-1.05]; for patients with a longer duration before treatment, the effect of the Epley maneuver was equivalent to that of a sham maneuver (RR: 1.07, 95% CI: 0.90-1.29). CONCLUSION: Among the 12 types of PC-BPPV treatments, the Epley, Semont, EVR, and Hybrid maneuvers were effective in eliminating nystagmus during a Dix-Hallpike test for PC-BPPV at 1 week of follow-up, whereas only the Epley and Semont maneuvers were effective at 1 month of follow-up. The duration before treatments and the age of patients might contribute to the efficacy of treatments.

Single-Cell Transcriptome Profiling Identifies Phagocytosis-Related Dual-Feature Cells in A Model of Acute Otitis Media in Rats.

Background: The molecular mechanisms of acute otitis media (AOM) development, and the intercellular crosstalk within the multicellular ecosystem of AOM, are not clear. Methods: We established a model of AOM in rats (with normal rats as controls) and undertook single-cell RNA sequencing (scRNA-seq) for the middle-ear mucosa (MEM). Cell clustering and trajectory analyses were undertaken using Seurat and Monocle 2 packages in R software. Pathway analyses were done by gene set enrichment analysis (GSEA). Cell-cell interactions were inferred by CellChat. Cell scores were calculated to identify cells with dual-feature. Results: A total of 7023 cells from three samples of inflamed MEM and 5258 cells from three samples of healthy MEM underwent scRNA-seq, which identified 20 cell clusters belonging to eight major cell types. After exposure to lipopolysaccharide, the MEM underwent significant conversion of cell types characterized by rapid infiltration of macrophages and neutrophils. M2 macrophages seemed to play a key part in inflammatory intercellular crosstalk, which facilitated the maintenance and proliferation of macrophages, cell chemotaxis, and regulation of the proinflammatory activities of cytokines. Three rare cell clusters with phagocytosis-related dual-feature were also identified. They coexisted with professional phagocytes in the MEM, and displayed distinct immunoregulatory functions by maintaining a normal immune microenvironment or influencing inflammation progression. Conclusions: Macrophages might be the "master" initiators and regulators of the inflammatory response of the MEM to external stimuli. And their functions are fulfilled by a specific polarization status (M2) and sophisticated intercellular crosstalk via certain signaling pathways. Besides, the coexistence of professional phagocytes and non-professional phagocytes as well as their interplay in the MEM provides new clues for deciphering the underlying pathogenic mechanisms of AOM.

The PIFs Redundantly Control Plant Defense Response against Botrytis cinerea in Arabidopsis.

Endogenous and exogenous signals are perceived and integrated by plants to precisely control defense responses. As a crucial environmental cue, light reportedly plays vital roles in plant defenses against necrotrophic pathogens. Phytochrome-interacting factor (PIF) is one of the important transcription factors which plays essential roles in photoreceptor-mediated light response. In this study, we revealed that PIFs negatively regulate plant defenses against Botrytis cinerea. Gene expression analyses showed that the expression level of a subset of defense-response genes was higher in pifq (pif1/3/4/5) mutants than in the wild-type control, but was lower in PIF-overexpressing plants. Chromatin immunoprecipitation assays proved that PIF4/5 binds directly to the ETHYLENE RESPONSE FACTOR1 (ERF1) promoter. Moreover, genetic analyses indicated that the overexpression of ERF1 dramatically rescues the susceptibility of PIF4-HA and PIF5-GFP transgenic plants, and that PIF controls the resistance to B. cinerea in a COI1- and EIN2-dependent manner. Our results provide compelling evidence that PIF, together with the jasmonate/ethylene pathway, is important for plant resistance to B. cinerea.

Arabidopsis Class II TCP Transcription Factors Integrate with the FT-FD Module to Control Flowering.

The appropriate timing of flowering is critical for plant reproductive success. Although the FLOWERING LOCUS T (FT)-FD module plays crucial roles in the photoperiodic flowering pathway, the underlying mechanisms and signaling pathways involved still remain elusive. Here, we demonstrate that class II TCP transcription factors (TFs) integrate into the FT-FD complex to control floral initiation in Arabidopsis (Arabidopsis thaliana). Class II CINCINNATA (CIN) TCP TFs function as transcriptional activators by directly binding to the promoters of downstream floral meristem identity genes, such as APETALA1 (AP1). In addition, these TCPs directly interact with FD, a basic Leu zipper TF that plays a critical role in photoperiodic flowering, which further activates AP1 expression. Genetic analyses indicated that class II CIN TCP TFs function synergistically with FT and FD, to positively regulate flowering in an AP1-dependent manner. Thus, our results provide compelling evidence that class II CIN TCP TFs act directly at the AP1 promoter to enhance its transcription, thus further elucidating the molecular mechanisms underlying the regulation of photoperiodic flowering in Arabidopsis.

Microcystin-LR increases genotoxicity induced by aflatoxin B1 through oxidative stress and DNA base excision repair genes in human hepatic cell lines.

Aflatoxin B1 (AFB1) and microcystin-LR (MC-LR) simultaneously exist in polluted food and water in humid and warm areas, and each has been reported to be genotoxic to liver and associated with hepatocellular carcinoma (HCC). However, the genotoxic effects of the two biotoxins in combination and potential mechanism remain unknown. We treated the human hepatic cell line HL7702 with AFB1 and MC-LR together at different ratios, examined their genotoxic effects using micronuclei and comet assays, and evaluated the possible mechanism by measuring oxidative stress markers and DNA base excision repair (BER) genes. Our data show that co-exposure to AFB1 and MC-LR significantly increased DNA damage compared with AFB1 or MC-LR alone as measured by the levels of both micronuclei and tail DNA. Meanwhile, AFB1 and MC-LR co-exposure showed biphasic effects on ROS production, and a gradual trend towards increased Glutathione (GSH) levels and activity of Catalase (CAT) and Superoxide Dismutase (SOD). Furthermore, MC-LR, with or without AFB1, significantly down-regulated the expression of the base excision repair (BER) genes 8-oxoguanine glycosylase-1 (OGG1) and X-ray repair cross complementing group 1 (XRCC1). AFB1 and MC-LR in combination upregulated the expression of the BER gene apurinic/apyrimidinic endonuclease 1 (APE1), whereas either agent alone had no effect. In conclusion, our studies show that MC-LR exacerbates AFB1-induced genotoxicity and we report for the first time that this occurs through effects on oxidative stress and the deregulation of DNA base excision repair genes.

Arabidopsis WRKY45 Interacts with the DELLA Protein RGL1 to Positively Regulate Age-Triggered Leaf Senescence.

Leaf senescence can be triggered and promoted by various environmental stressors, developmental cues, and endogenous hormone signals. Several lines of evidence have suggested the involvement of WRKY transcription factors in regulating leaf senescence, but the underlying mechanisms and signaling pathways involved remain elusive. In this study, we identified Arabidopsis thaliana WRKY DNA-binding protein 45 (WRKY45) as a positive regulator of age-triggered leaf senescence. Loss of WRKY45 function resulted in increased leaf longevity in age-triggered senescence, whereas overexpression of WRKY45 significantly accelerated age-triggered leaf senescence. Consistently, expression of SENESCENCE-ASSOCIATED GENEs (SAGs) was significantly reduced in wrky45 mutants but markedly enhanced in transgenic plants overexpressing WRKY45. Chromatin immunoprecipitation assays revealed that WRKY45 directly binds the promoters of several SAGs such as SAG12, SAG13, SAG113, and SEN4. Both in vivo and in vitro biochemical analyses demonstrated that WRKY45 interacts with the DELLA protein RGA-LIKE1 (RGL1), a repressor of the gibberellin (GA) signaling pathway. We found that RGL1 repressed the transcription activation function of WRKY45, thereby attenuating the expression of its regulon. Consistent with this finding, overexpression of RGL1 resulted in significantly increased leaf longevity in age-triggered senescence. Taken together, our results provide compelling evidence that WRKY45 functions as a critical component of the GA-mediated signaling pathway to positively regulate age-triggered leaf senescence.

A dynamic smoke generation and nose-only inhalation exposure system for rats: preliminary results from studies of selected transportation materials.

CONTEXT: Smoke inhalation injury is the main cause of fatalities for fire victims. Understanding in the pathophysiology of the injury has not been fully explored in recent years. To further explore the pathophysiological mechanism, a dynamic and controllable animal model is necessary. OBJECTIVE: To develop a rat model of smoke inhalation injury to simulate human victims in air-restricted vehicle cabin fires. MATERIALS AND METHODS: Smoke concentration, including CO, O2, VOCs and smoke temperature under different combustion conditions, were detected. Levels of COHb, respiratory function, lung wet-to-dry weight ratio and protein concentration in BALF and blood were measured. Pathological evaluations of lung in tissues were conducted at 1, 6, 24 and 48 h post-exposure. RESULTS: Smoke concentration rose with the increase of combustion temperature and decrease of oxygen flow. Further, 215 kinds of VOCs in the smoke were detected, and the concentrations of benzene, methylbenzene, ethylbenzene, dimethylbenzene, phenylethylene and trimethylbenzene was 32.93, 402.06, 764.03, 113.73, 1006.61 and 89.28 mg/m(3), respectively. Significant hypoxemia and CO poisoning occurred in rats. The FCOHb after exposure for 14 min immediately rose to (44.2 ± 12.3) % and then gradually decrease to a normal level at 300 min post-exposure. At 24 h post-exposure, Penh increased significantly (p < 0.05), and high pulmonary vascular permeability and significant lung edema (p < 0.05) were observed in the smoke inhalation group. DISCUSSION AND CONCLUSION: In summary, the novel rat model of smoke inhalation injury system used in the study is dynamic and controllable, and appropriate for use in smoke inhalation injury studies of air-restricted cabins in vehicles.

WRKY8 transcription factor functions in the TMV-cg defense response by mediating both abscisic acid and ethylene signaling in Arabidopsis.

WRKY transcription factors are key players in the plant immune response, but less is known about their involvement in antiviral defense than about their roles in defense against bacterial or fungi pathogens. Here, we report that Arabidopsis thaliana WRKY DNA-binding protein 8 (WRKY8) has a role in mediating the long-distance movement of crucifer-infecting tobacco mosaic virus (TMV-cg). The expression of WRKY8 was inhibited by TMV-cg infection, and mutation of WRKY8 accelerated the accumulation of TMV-cg in systemically infected leaves. Quantitative RT-PCR analysis showed that the expression of ABA insensitive 4 (ABI4) was reduced and the expression of 1-aminocyclopropane-1-carboxylic acid synthase 6 (ACS6) and ethylene response factor 104 (ERF104) was enhanced in the systemically infected leaves of wrky8. Immunoprecipitation assays demonstrated that WRKY8 could bind selectively to putative W-boxes of the ABI4, ACS6, and ERF104 promoters. Furthermore, TMV-cg infection enhanced WRKY8 binding to the ABI4 promoter but reduced the binding of WRKY8 to the ACS6 and ERF104 promoters, indicating that regulation of ABI4, ACS6, and ERF104 by WRKY8 is at least partially dependent on TMV-cg. Exogenous applications of abscisic acid (ABA) reduced the systemic accumulation of TMV-cg. Mutations in ABA deficient 1, ABA deficient 2, ABA deficient 3, or abi4 accelerated systemic TMV-cg accumulation. In contrast, exogenous application of aminocyclopropane-1-carboxylic acid enhanced the systemic accumulation of TMV-cg, but mutations in acs6, erf104, or an octuple acs mutant inhibited systemic TMV-cg accumulation. Our results demonstrate that WRKY8 is involved in the defense response against TMV-cg through the direct regulation of the expression of ABI4, ACS6, and ERF104 and may mediate the crosstalk between ABA and ethylene signaling during the TMV-cg-Arabidopsis interaction.

[Aggressive fibromatosis of the head and neck (A report of two cases and literature review)].

OBJECTIVE: To evaluate the clinic manifestation, pathologic behavior, therapy and prognosis of rare aggressive fibromatosis in the head and neck. METHOD: Two cases of aggressive fibromatosis were analyzed and relevant literatures were reviewed. RESULT: Aggressive fibromatosis was characterized as infiltrative, locally aggressive and tended to recur after surgical resection. Pathology showed fibroblastic monoclonal proliferation. Fibromatosis was composed of well-differentiated fibroblasts and myofibroblasts, lacking cytological features of malignancy and scanty or absent mitotic activity. Complete surgical excision of aggressive fibromatosis was considered to be the only effective method of cure by most authorities. Chemotherapy and radiotherapy can be used together with surgery in recurrence or unsatisfactory surgical margin. In our study, one patient recurred after the first operation, and after another operation, the patient did not recur after 6 months follow up, and the other one did not recur after 6 months follow up. CONCLUSION: The diagnosis of aggressive fibromatosis depended on pathological examination. Radical removal was an important way to reduce recurrence rate. Radiation therapy and chemotherapy can be used as adjuvant therapy in patients with recurrent or unresectable or inoperable disease.

Chromosomal damage and polymorphisms of metabolic genes among 1, 3-butadiene-exposed workers in a matched study in China.

1, 3-Butadiene (BD) is a high-efficiency carcinogen in rodents and was classified as a human carcinogen in 2008 by the International Agency for Research on Cancer. However, its ability to induce genetic damage and the influence of metabolic polymorphisms to such damage in humans are both controversial claims. This study was conducted to investigate the relationships between exposure to BD, the polymorphisms of metabolic genes and the chromosomal damage in 45 pairs of occupationally exposed workers in a BD product workshop and matched control workers in an administrative office and circulatory water workshop in China. Exposure to BD was evaluated by personal sampling and stationary sampling. Different chromosomal damage endpoints in peripheral blood lymphocytes were determined using the cytokinesis-blocked micronucleus (CBMN) cytome assay; polymorphisms of metabolic genes [cytochrome P450 2E1 (CYP2E1), glutathione S-transferases (GST) and microsomal epoxide hydrolase (mEH)] in BD-exposed group were detected by polymerase chain reaction (PCR) or PCR-restriction fragment length polymorphism analysis. The results show that the average BD measurements of the exposed group were significantly higher than those for the control group (a personal sampling and stationary sampling, respectively). The BD-exposed workers exhibited increased frequencies of micronuclei (MNi) (8.00 ± 3.78‰ versus 5.62 ± 2.41‰) and nucleoplasmic bridges (NPBs) (2.58 ± 2.79‰ versus 1.13 ± 1.34‰) and a decreased nuclear division index (2.20 ± 0.14 versus 2.35 ± 0.27) when compared subjects in the control group. Meanwhile, BD-exposed workers carrying CYP2E1 c1c2/c2c2 or mEH intermediate (I)/high (H) group had a significantly higher NPB frequency than those carrying CYP2E1 c1c1 [frequency ratio (FR) = 2.60, 95% confidence interval (CI) 1.72-3.93; P < 0.0001) or the mEH low(S) group (FR = 2.06, 95% CI% 1.17-3.62; P < 0.05), respectively. Our study suggests that MNi and NPB frequency in CBMN cytome assay could be potential genotoxic biomarkers for BD exposure in humans. The polymorphism of CYP2E1 and mEH could also affect the chromosomal instability of BD workers.