PET imaging for the early evaluation of ocular inflammation in diabetic rats by using [18F]-DPA-714.

Ocular complications of diabetes mellitus (DM) are the leading cause of vision loss. Ocular inflammation often occurs in the early stage of DM; however, there are no proven quantitative methods to evaluate the inflammatory status of eyes in DM. The 18 kDa translocator protein (TSPO) is an evolutionarily conserved cholesterol binding protein localized in the outer mitochondrial membrane. It is a biomarker of activated microglia/macrophages; however, its role in ocular inflammation is unclear. In this study, fluorine-18-DPA-714 ([18F]-DPA-714) was evaluated as a specific TSPO probe by cell uptake, cell binding assays and micro positron emission tomography (microPET) imaging in both in vitro and in vivo models. Primary microglia/macrophages (PMs) extracted from the cornea, retina, choroid or sclera of neonatal rats with or without high glucose (50 mM) treatment were used as the in vitro model. Sprague-Dawley (SD) rats that received an intraperitoneal administration of streptozotocin (STZ, 60 mg/kg once) were used as the in vivo model. Increased cell uptake and high binding affinity of [18F]-DPA-714 were observed in primary PMs under hyperglycemic stress. These findings were consistent with cellular morphological changes, cell activation, and TSPO up-regulation. [18F]-DPA-714 PET imaging and biodistribution in the eyes of DM rats revealed that inflammation initiates in microglia/macrophages in the early stages (3 weeks and 6 weeks), corresponding with up-regulated TSPO levels. Thus, [18F]-DPA-714 microPET imaging may be an effective approach for the early evaluation of ocular inflammation in DM.

The JMJD3 histone demethylase inhibitor GSK-J1 ameliorates lipopolysaccharide-induced inflammation in a mastitis model.

Jumonji domain-containing 3 (JMJD3/KDM6B) is a histone demethylase that plays an important role in regulating development, differentiation, immunity, and tumorigenesis. However, the mechanisms responsible for the epigenetic regulation of inflammation during mastitis remain incompletely understood. Here, we aimed to investigate the role of JMJD3 in the lipopolysaccharide (LPS)-induced mastitis model. GSK-J1, a small molecule inhibitor of JMJD3, was applied to treat LPS-induced mastitis in mice and in mouse mammary epithelial cells in vivo and in vitro. Breast tissues were then collected for histopathology and protein/gene expression examination, and mouse mammary epithelial cells were used to investigate the mechanism of regulation of the inflammatory response. We found that the JMJD3 gene and protein expression were upregulated in injured mammary glands during mastitis. Unexpectedly, we also found JMJD3 inhibition by GSK-J1 significantly alleviated the severity of inflammation in LPS-induced mastitis. These results are in agreement with the finding that GSK-J1 treatment led to the recruitment of histone 3 lysine 27 trimethylation (H3K27me3), an inhibitory chromatin mark, in vitro. Furthermore, mechanistic investigation suggested that GSK-J1 treatment directly interfered with the transcription of inflammatory-related genes by H3K27me3 modification of their promoters. Meanwhile, we also demonstrated that JMJD3 depletion or inhibition by GSK-J1 decreased the expression of toll-like receptor 4 and negated downstream NF-κB proinflammatory signaling and subsequently reduced LPS-stimulated upregulation of Tnfa, Il1b, and Il6. Together, we propose that targeting JMJD3 has therapeutic potential for the treatment of inflammatory diseases.

Phosphodiesterase 4A confers resistance to PGE2-mediated suppression in CD25+ /CD54+ NK cells.

Inadequate persistence of tumor-infiltrating natural killer (NK) cells is associated with poor prognosis in cancer patients. The solid tumor microenvironment is characterized by the presence of immunosuppressive factors, including prostaglandin E2 (PGE2), that limit NK cell persistence. Here, we investigate if the modulation of the cytokine environment in lung cancer with IL-2 or IL-15 renders NK cells resistant to suppression by PGE2. Analyzing Cancer Genome Atlas (TCGA) data, we found that high NK cell gene signatures correlate with significantly improved overall survival in patients with high levels of the prostaglandin E synthase (PTGES). In vitro, IL-15, in contrast to IL-2, enriches for CD25+ /CD54+ NK cells with superior mTOR activity and increased expression of the cAMP hydrolyzing enzyme phosphodiesterase 4A (PDE4A). Consequently, this distinct population of NK cells maintains their function in the presence of PGE2 and shows an increased ability to infiltrate lung adenocarcinoma tumors in vitro and in vivo. Thus, strategies to enrich CD25+ /CD54+ NK cells for adoptive cell therapy should be considered.

The causal association between peripheral blood eosinophils and nasal polyps: a Mendelian randomization study.

OBJECTIVE: Observational studies suggested that peripheral blood eosinophils were associated with the risk of nasal polyps. However, these studies did not confirm the causality. This study aims to apply Mendelian randomization (MR) method to comprehensively assess the potential causal association between peripheral blood eosinophils and nasal polyps. METHODS: Genetic instrumental variables were extracted from the largest available genome-wide association study (GWAS) of European participants, which were used to investigate the relationship between peripheral blood eosinophils and nasal polyps. The inverse variance weighted method, the MR Egger method, and the weighted median method were applied for this analysis. MR-Egger intercept tests, leave-one-out analyses, and funnel plots were performed for the sensitivity analysis. RESULTS: With the inverse variance weighted method, the MR analysis suggested that there was a significant difference between peripheral blood eosinophils and the risk of nasal polyps (ukb-a-97, OR 1.004, 95% CI 1.003-1.005, p < 0.001; ukb-a-541, OR 1.005, 95% CI 1.004-1.006, p < 0.001; ukb-b-7211, OR 1.004, 95% CI 1.003-1.005, p < 0.001; ukb-b-8425, OR 1.004, 95% CI 1.003-1.005, p < 0.001; finn-b-J10_NASALPOLYP, OR 3.089, 95% CI 2.537-3.761, p < 0.001). Consistent results were also proved by using the weighted median method and the MR Egger method. CONCLUSIONS: Our findings reveal the causal effect of peripheral blood eosinophils on the increased risk of nasal polyps.

Genes, Structural, and Biochemical Characterization of Four Chlorophyllases from Solanum lycopersicum.

Recent studies have confirmed that chlorophyllase (CLH), a long-found chlorophyll (Chl) dephytylation enzyme for initiating Chl catabolism, has no function in leaf senescence-related Chl breakdown. Yet, CLH is considered to be involved in fruit degreening and responds to external and hormonal stimuli. The purpose of this work was to elucidate in detail the biochemical, structural properties, and gene expression of four CLHs from the Solanum lycopersicum genome so as to understand the roles of Solanum lycopersicum chlorophyllases (SlCLHs). SlCLH1/4 were the predominantly expressed CLH genes during leaf and fruit development/ripening stages, and SlCLH1 in mature green fruit was modulated by light. SlCLH1/2/3/4 contained a highly conserved GHSXG lipase motif and a Ser-Asp-His catalytic triad. We identified Ser159, Asp226, and His258 as the essential catalytic triad by site-directed mutagenesis in recombinant SlCLH1. Kinetic analysis of the recombinant enzymes revealed that SlCLH1 had high hydrolysis activities against Chl a, Chl b, and pheophytin a (Phein a), but preferred Chl a and Chl b over Phein a; SlCLH2/3 only showed very low activity to Chl a and Chl b, while SlCLH4 showed no Chl dephytylation activity. The recombinant SlCLH1/2/3 had different pH stability and temperature optimum. Removal of the predicted N-terminal processing peptide caused a partial loss of activity in recombinant SlCLH1/2 but did not compromise SlCLH3 activity. These different characteristics among SlCLHs imply that they may have different physiological functions in tomato.

Cell Lysate Microarray for Mapping the Network of Genetic Regulators for Histone Marks.

Proteins, as the major executer for cell progresses and functions, its abundance and the level of post-translational modifications, are tightly monitored by regulators. Genetic perturbation could help us to understand the relationships between genes and protein functions. Herein, to explore the impact of the genome-wide interruption on certain protein, we developed a cell lysate microarray on kilo-conditions (CLICK) with 4837 knockout (YKO) and 322 temperature-sensitive (ts) mutant strains of yeast (Saccharomyces cerevisiae). Taking histone marks as examples, a general workflow was established for the global identification of upstream regulators. Through a single CLICK array test, we obtained a series of regulators for H3K4me3, which covers most of the known regulators in S. cerevisiae We also noted that several group of proteins are involved in negatively regulation of H3K4me3. Further, we discovered that Cab4p and Cab5p, two key enzymes of CoA biosynthesis, play central roles in histone acylation. Because of its general applicability, CLICK array could be easily adopted to rapid and global identification of upstream protein/enzyme(s) that regulate/modify the level of a protein or the posttranslational modification of a non-histone protein.

Current applications of antibody microarrays.

The concept of antibody microarrays is one of the most versatile approaches within multiplexed immunoassay technologies. These types of arrays have increasingly become an attractive tool for the exploratory detection and study of protein abundance, function, pathways, and potential drug targets. Due to the properties of the antibody microarrays and their potential use in basic research and clinical analytics, various types of antibody microarrays have already been developed. In spite of the growing number of studies utilizing this technique, few reviews about antibody microarray technology have been presented to reflect the quality and future uses of the generated data. In this review, we provide a summary of the recent applications of antibody microarray techniques in basic biology and clinical studies, providing insights into the current trends and future of protein analysis.

Construction of a metabolomics profile of arsenic trioxide effect in gastric carcinoma cell line SGC7901.

Arsenic trioxide (ATO) is highly effective for treating acute promyelocytic leukemia. It also holds the promise for treating solid tumors, including gastric carcinoma. However, the molecular mechanism of the effectiveness of ATO to solid tumor is still poorly understood. In this study, we chosed gastric carcinoma as an example and tried to reveal the antitumor mechanism through metabolomics. Gastric carcinoma cell line SGC7901 was treated with ATO for 6, 12, and 24 h. The global metabolite profiles were monitored by metabolomics analysis using gas chromatography (GC)/mass spectrometry (MS) and liquid chromatography/MS/MS. A total of 281 certified metabolites were reliably detected. Bioinformatics analysis showed that glycerophospholipid synthesis, one-carbon synthesis, and glutathione synthesis were affected dramatically. Other cellular functions/pathways that had been affected included inflammatory response, nicotinamide adenine dinucleotide (NAD(+)), and polyamine biosynthesis pathway. The metabolomics data from this study, in combination with previous transcriptomics and proteomics data, could serve as valuable resources for the understanding of the specific antitumor mechanism of ATO treatment.

Benefit-finding profiles and comparison of caregiving ability among informal caregivers of patients with lung cancer: A latent profile analysis.

Objective: This study aimed to explore the benefit finding (BF) profiles among informal caregivers of patients with lung cancer, identify demographic and disease characteristics, and analyze differences in caregiving ability between profiles. Methods: This cross-sectional study utilized convenience sampling to select 272 informal caregivers of patients with lung cancer from a tertiary care hospital in Guangzhou, China. The research instruments used included the Demographic and Disease Characteristics Questionnaire, the revised version of the BF Scale, and the Chinese version of the Family Caregiver Task Inventory. Data analysis was performed using latent profile analysis, chi-square test, Fisher's exact probability test, Kruskal-Wallis test, and multivariate logistic regression. Results: (1) BF can be divided into three profiles: "high benefit-family and personal growth" (Profile 1, 7.7%), "moderate benefit-unclear perception" (Profile 2, 44.9%), and "low benefit-coping ability deficient" (Profile 3, 47.4%). (2) Having a cocaregiver and a disease duration of 6-12 months were more likely to belong to Profile 1; caregivers of patients aged 40-60 years tended to belong to Profile 2; caregivers of older patients with disease duration > 12 months and clinical stage II or III were more likely to belong to Profile 3. (3) There were significant differences in the total score of caregiving ability and the scores of each dimension among the different BF profiles (P < 0.001), and the caregiving abilities of Profile 1 and Profile 2 were higher than those of Profile 3. Conclusions: There was heterogeneity in BF among informal caregivers of patients with lung cancer. Healthcare professionals can identify the key profiles of lung-cancer caregivers based on characteristics such as age, clinical stage, disease duration, and cocaregiver status and enhance their caregiving ability through targeted nursing guidance.

Immunosuppressive role of BDNF in therapy-induced neuroendocrine prostate cancer.

Prostate stromal cells play a crucial role in the promotion of tumor growth and immune evasion in the tumor microenvironment (TME) through intricate molecular alterations in their interaction with prostate cancer (PCa) cells. While the impact of these cells on establishing an immunosuppressive response and influencing PCa aggressiveness remains incompletely understood. Our study shows that the activation of the leukemia inhibitory factor (LIF)/LIF receptor (LIFR) pathway in both prostate tumor and stromal cells, following androgen deprivation therapy (ADT), leads to the development of an immunosuppressive TME. Activation of LIF/LIFR signaling in PCa cells induces neuroendocrine differentiation (NED) and upregulates immune checkpoint expression. Inhibition of LIF/LIFR attenuates these effects, underscoring the crucial role of LIF/LIFR in linking NED to immunosuppression. Prostate stromal cells expressing LIFR contribute to NED and immunosuppressive marker abundance in PCa cells, while LIFR knockdown in prostate stromal cells reverses these effects. ADT-driven LIF/LIFR signaling induces brain-derived neurotrophic factor (BDNF) expression, which, in turn, promotes NED, aggressiveness, and immune evasion in PCa cells. Clinical analyses demonstrate elevated BDNF levels in metastatic castration-resistant PCa (CRPC) and a positive correlation with programmed death-ligand 1 (PDL1) and immunosuppressive signatures. This study shows that the crosstalk between PCa cells and prostate stromal cells enhances LIF/LIFR signaling, contributing to an immunosuppressive TME and NED in PCa cells through the upregulation of BDNF.

MCTP1 increases the malignancy of androgen-deprived prostate cancer cells by inducing neuroendocrine differentiation and EMT.

Neuroendocrine prostate cancer (PCa) (NEPC), an aggressive subtype that is associated with poor prognosis, may arise after androgen deprivation therapy (ADT). We investigated the molecular mechanisms by which ADT induces neuroendocrine differentiation in advanced PCa. We found that transmembrane protein 1 (MCTP1), which has putative Ca2+ sensing function and multiple Ca2+-binding C2 domains, was abundant in samples from patients with advanced PCa. MCTP1 was associated with the expression of the EMT-associated transcription factors ZBTB46, FOXA2, and HIF1A. The increased abundance of MCTP1 promoted PC3 prostate cancer cell migration and neuroendocrine differentiation and was associated with SNAI1-dependent EMT in C4-2 PCa cells after ADT. ZBTB46 interacted with FOXA2 and HIF1A and increased the abundance of MCTP1 in a hypoxia-dependent manner. MCTP1 stimulated Ca2+ signaling and AKT activation to promote EMT and neuroendocrine differentiation by increasing the SNAI1-dependent expression of EMT and neuroendocrine markers, effects that were blocked by knockdown of MCTP1. These data suggest an oncogenic role for MCTP1 in the maintenance of a rare and aggressive prostate cancer subtype through its response to Ca2+ and suggest its potential as a therapeutic target.

Natural killer cells drive 4-1BBL positive uveal melanoma towards EMT and metastatic disease.

BACKGROUND: Inflammation in the eye is often associated with aggravated ocular diseases such as uveal melanoma (UM). Poor prognosis of UM is generally associated with high potential of metastatic liver dissemination. A strong driver of metastatic dissemination is the activation of the epithelial-mesenchymal transition (EMT) regulating transcription factor ZEB1, and high expression of ZEB1 is associated with aggressiveness of UM. While ZEB1 expression can be also associated with immune tolerance, the underlying drivers of ZEB1 activation remain unclear. METHODS: Transcriptomic, in vitro, ex vivo, and in vivo analyses were used to investigate the impact on clinical prognosis of immune infiltration in the ocular tumor microenvironment. A metastatic liver dissemination model of was developed to address the role of natural killer (NK) cells in driving the migration of UM. RESULTS: In a pan-cancer TCGA analysis, natural killer (NK) cells were associated with worse overall survival in uveal melanoma and more abundant in high-risk monosomy 3 tumors. Furthermore, uveal melanoma expressed high levels of the tumor necrosis factor superfamily member 4-1BB ligand, particularly in tumors with monosomy 3 and BAP1 mutations. Tumors expressing 4-1BB ligand induced CD73 expression on NK cells accompanied with the ability to promote tumor dissemination. Through ligation of 4-1BB, NK cells induced the expression of the ZEB1 transcription factor, leading to the formation of liver metastasis of uveal melanoma. CONCLUSIONS: Taken together, the present study demonstrates a role of NK cells in the aggravation of uveal melanoma towards metastatic disease.

Tumor-associated NK cells drive MDSC-mediated tumor immune tolerance through the IL-6/STAT3 axis.

Apart from their killer identity, natural killer (NK) cells have integral roles in shaping the tumor microenvironment. Through immune gene deconvolution, the present study revealed an interplay between NK cells and myeloid-derived suppressor cells (MDSCs) in nonresponders of immune checkpoint therapy. Given that the mechanisms governing the outcome of NK cell-to-myeloid cell interactions remain largely unknown, we sought to investigate the cross-talk between NK cells and suppressive myeloid cells. Upon contact with tumor-experienced NK cells, monocytes and neutrophils displayed increased expression of MDSC-related suppressive factors along with increased capacities to suppress T cells. These changes were accompanied by impaired antigen presentation by monocytes and increased ER stress response by neutrophils. In a cohort of patients with sarcoma and breast cancer, the production of interleukin-6 (IL-6) by tumor-infiltrating NK cells correlated with S100A8/9 and arginase-1 expression by MDSCs. At the same time, NK cell-derived IL-6 was associated with tumors with higher major histocompatibility complex class I expression, which we further validated with b2m-knockout (KO) tumor mice models. Similarly in syngeneic wild-type and IL-6 KO mouse models, we then demonstrated that the accumulation of MDSCs was influenced by the presence of such regulatory NK cells. Inhibition of the IL-6/signal transducer and activator of transcription 3 (STAT3) axis alleviated suppression of T cell responses, resulting in reduced tumor growth and metastatic dissemination. Together, these results characterize a critical NK cell-mediated mechanism that drives the development of MDSCs during tumor immune escape.

Production of L-Malic Acid by Metabolically Engineered Aspergillus nidulans Based on Efficient CRISPR-Cas9 and Cre-loxP Systems.

Aspergillus nidulans has been more extensively characterized than other Aspergillus species considering its morphology, physiology, metabolic pathways, and genetic regulation. As it has a rapid growth rate accompanied by simple nutritional requirements and a high tolerance to extreme cultural conditions, A. nidulans is a promising microbial cell factory to biosynthesize various products in industry. However, it remains unclear for whether it is also a suitable host for synthesizing abundant L-malic acid. In this study, we developed a convenient and efficient double-gene-editing system in A. nidulans strain TN02A7 based on the CRISPR-Cas9 and Cre-loxP systems. Using this gene-editing system, we made a L-malic acid-producing strain, ZQ07, derived from TN02A7, by deleting or overexpressing five genes (encoding Pyc, pyruvate carboxylase; OahA, oxaloacetate acetylhydrolase; MdhC, malate dehydrogenase; DctA, C4-dicarboxylic acid transporter; and CexA, citric acid transporter). The L-malic acid yield in ZQ07 increased to approximately 9.6 times higher (up to 30.7 g/L titer) than that of the original unedited strain TN02A7, in which the production of L-malic acid was originally very low. The findings in this study not only demonstrate that A. nidulans could be used as a potential host for biosynthesizing organic acids, but also provide a highly efficient gene-editing strategy in filamentous fungi.

A tractable microscopy- and flow cytometry-based method to measure natural killer cell-mediated killing and infiltration of tumor spheroids.

Understanding the anti-tumor activity of immune cells and testing cancer immunotherapies requires conditions that are as life-like as possible. The tumor microenvironment (TME) describes a complex sum of cellular and acellular actors that influence both immune cells and tumor cells as well as their interplay. Yet in development phases of new immunotherapies, the screening of drugs and adoptive cell products benefits from reproducible and controlled conditions. Two-dimensional (2D) cell cultures cannot simultaneously meet these two challenges therefore lacking considerably predictive power owing to their artificial nature. Various 3D tumor models have therefore been implemented to mimic the architecture and intrinsic heterogeneity of a microtumor. This protocol provides an easy-to-follow, time-efficient, material-limited method for live cell killing and infiltration of single tumor spheroids. It uses multicellular tumor spheroids grown scaffold-free and allows co-culture with immune cells. This protocol is optimized for natural killer (NK) cell functionality assays. However, it can be transferred to other immune cells, in particular cytotoxic T cells. This assay can be analysed using life cell imaging (here with the IncuCyte S3 system) and/or flow cytometry.

CD25bright NK cells display superior function and metabolic activity under regulatory T cell-mediated suppression.

Infusion of natural killer (NK) cells is an attractive therapeutic modality in patients with cancer. However, the activity of NK cells is regulated by several mechanisms operating within solid tumors. Regulatory T (Treg) cells suppress NK cell activity through various mechanisms including deprivation of IL-2 via the IL-2 receptor alpha (CD25). Here, we investigate CD25 expression on NK cells to confer persistence in Treg cells containing solid tumor models of renal cell carcinoma (RCC). Compared with IL-2, stimulation with IL-15 increases the expression of CD25 resulting in enhanced response to IL-2 as evidenced by increased phosphorylation of STAT5. Compared with CD25dim NK cells, CD25bright NK cells isolated from IL-15 primed NK cells display increased proliferative and metabolic activity as well as increased ability to persist in Treg cells containing RCC tumor spheroids. These results support strategies to enrich for or selectively expand CD25bright NK cells for adoptive cellular therapy of NK cells.

Targeting PKLR/MYCN/ROMO1 signaling suppresses neuroendocrine differentiation of castration-resistant prostate cancer.

Conventional treatment of prostate cancer (PCa) uses androgen-deprivation therapy (ADT) to inhibit androgen receptor (AR) signaling-driven tumor progression. ADT-induced PCa recurrence may progress to an AR-negative phenotype with neuroendocrine (NE) histologic features, which are associated with metabolic disturbances and poor prognoses. However, the metabolic pathways that regulate NE differentiation (NED) in PCa remain unclear. Herein, we show a regulatory mechanism in NED-associated metabolism dysfunction induced by ADT, whereby overexpression of pyruvate kinase L/R (PKLR) mediates oxidative stress through upregulation of reactive oxygen species modulator 1 (ROMO1), thereby promoting NED and aggressiveness. ADT mediates the nuclear translocation of PKLR, which binds to the MYCN/MAX complex to upregulate ROMO1 and NE-related genes, leading to altered mitochondrial function and NED of PCa. Targeting nuclear PKLR/MYCN using bromodomain and extra-terminal motif (BET) inhibitors has the potential to reduce PKLR/MYCN-driven NED. Abundant ROMO1 in serum samples may provide prognostic information in patients with ADT. Our results suggest that ADT resistance leads to upregulation of PKLR/MYCN/ROMO1 signaling, which may drive metabolic reprogramming and NED in PCa. We further show that increased abundance of serum ROMO1 may be associated with the development of NE-like PCa.

Specific pupylation as IDEntity reporter (SPIDER) for the identification of protein-biomolecule interactions.

Protein-biomolecule interactions play pivotal roles in almost all biological processes. For a biomolecule of interest, the identification of the interacting protein(s) is essential. For this need, although many assays are available, highly robust and reliable methods are always desired. By combining a substrate-based proximity labeling activity from the pupylation pathway of Mycobacterium tuberculosis and the streptavidin (SA)-biotin system, we developed the Specific Pupylation as IDEntity Reporter (SPIDER) method for identifying protein-biomolecule interactions. Using SPIDER, we validated the interactions between the known binding proteins of protein, DNA, RNA, and small molecule. We successfully applied SPIDER to construct the global protein interactome for m6A and mRNA, identified a variety of uncharacterized m6A binding proteins, and validated SRSF7 as a potential m6A reader. We globally identified the binding proteins for lenalidomide and CobB. Moreover, we identified SARS-CoV-2-specific receptors on the cell membrane. Overall, SPIDER is powerful and highly accessible for the study of protein-biomolecule interactions.

Tumor MHC class I expression alters cancer-associated myelopoiesis driven by host NK cells.

Downregulation of MHC class I (MHCI) molecules on tumor cells is recognized as a resistance mechanism of cancer immunotherapy. Given that MHCI molecules are potent regulators of immune responses, we postulated that the expression of MHCI by tumor cells influences systemic immune responses. Accordingly, mice-bearing MHCI-deficient tumor cells showed reduced tumor-associated extramedullary myelopoiesis in the spleen. Depletion of natural killer (NK) cells abrogated these differences, suggesting an integral role of immune-regulatory NK cells during tumor progression. Cytokine-profiling revealed an upregulation of TNF-α by NK cells in tumors and spleen in mice-bearing MHCI expressing tumors, and inhibition of TNF-α enhanced host myelopoiesis in mice receiving adoptive transfer of tumor-experienced NK cells. Our study highlights a critical role of NK cells beyond its identity as a killer lymphocyte and more importantly, the potential host responses to a localized tumor as determined by its MHCI expression.

The Heterotrimeric Transcription Factor CCAAT-Binding Complex and Ca2+-CrzA Signaling Reversely Regulate the Transition between Fungal Hyphal Growth and Asexual Reproduction.

The life cycle of filamentous fungi generally comprises hyphal growth and asexual reproduction. Both growth and propagation processes are critical for invasion growth, spore dissemination, and virulence in fungal pathogens and for the production of secondary metabolites or for biomass accumulation in industrial filamentous fungi. The CCAAT-binding complex (CBC) is a heterotrimeric transcription factor comprising three subunits, HapB, HapC, and HapE, and is highly conserved in fungi. Previous studies revealed that CBC regulates sterol metabolism by repressing several genes in the ergosterol biosynthetic pathway in the human fungal pathogen Aspergillus fumigatus. In the present study, we found dysfunction of CBC caused the abnormal asexual reproduction (conidiation) in submerged liquid culture. CBC suppresses the activation of the brlA gene in the central regulatory pathway for conidiation combined with its upstream regulators fluG, flbD, and flbC by binding to the 5'-CCAAT-3' motif within conidiation gene promoters, and lack of CBC member HapB results in the upregulation of these genes. Furthermore, when the expression of brlA or flbC is repressed, the submerged conidiation does not happen in the hapB mutant. Interestingly, deletion of HapB leads to enhanced transient cytosolic Ca2+ levels and activates conidiation-positive inducer Ca2+-CrzA modules to enhance submerged conidiation, demonstrating that CrzA works with CBC as a reverse regulator of fungal conidiation. To the best of our knowledge, the finding of this study is the first report for the molecular switch mechanism between vegetative hyphal growth and asexual development regulated by CBC, in concert with Ca2+-CrzA signaling in A. fumigatus. IMPORTANCE A precisely timed switch between vegetative hyphal growth and asexual development is a crucial process for the filamentous fungal long-term survival, dissemination, biomass production, and virulence. However, under the submerged culture condition, filamentous fungi would undergo constant vegetative growth whereas asexual conidiation rarely occurs. Knowledge about possible regulators is scarce, and how they could inhibit conidiation in liquid culture is poorly understood. Here, we demonstrated that the transcription factor heterotrimeric CBC dominantly maintains vegetative growth in liquid-submerged cultures by directly suppressing the conidiation-inductive signal. In contrast, calcium and the transcription factor CrzA, are positive inducers of conidiation. Our new insights into the CBC and Ca2+-CrzA regulatory system for transition control in the submerged conidiation of A. fumigatus may have broad repercussions for all filamentous fungi. Moreover, our elucidation of the molecular mechanism for submerged conidiation may support new strategies to precisely control vegetative growth and asexual conidiation in aspergilli used in industry.