Flow cytometric resolution of yeast is affected by enzymatic treatment and culture media.

Saccharomyces cerevisiae is an important model organism and a typical fungal representative for studies of eukaryotes. The cell cycle of yeast can be analyzed by flow cytometry, and refining the cytometric resolution of the cell cycle with this technique is important. Such refinement is potentially influenced by multiple factors, including enzymatic treatment and variations in the culture media used, although this has been subject to only limited investigation. Here, we examined the effect of different enzymatic pre-treatments and various media on cytometric resolution. We show that cytometric resolution is significantly altered by both enzymatic conditions and the media used. Culture media with different amino nitrogen concentrations potentially impact the protein content in the yeast cell wall, which may affect the permeability of the cell wall and alter cytometric resolution. The present study provides beneficial technical information about the influence of media and enzymes on the cytometric resolution of the yeast cell cycle and most likely other fungi, which should be considered in future research.

Autocrine Canonical Wnt Signaling Primes Noncanonical Signaling through ROR1 in Metastatic Castration-Resistant Prostate Cancer.

Wnt signaling driven by genomic alterations in genes including APC and CTNNB, which encodes ß-catenin, have been implicated in prostate cancer development and progression to metastatic castration-resistant prostate cancer (mCRPC). However, nongenomic drivers and downstream effectors of Wnt signaling in prostate cancer and the therapeutic potential of targeting this pathway in prostate cancer have not been fully established. Here we analyzed Wnt/ß-catenin signaling in prostate cancer and identified effectors distinct from those found in other tissues, including aryl hydrocarbon receptor and RUNX1, which are linked to stem cell maintenance, and ROR1, a noncanonical Wnt5a coreceptor. Wnt/ß-catenin signaling-mediated increases in ROR1 enhanced noncanonical responses to Wnt5a. Regarding upstream drivers, APC genomic loss, but not its epigenetic downregulation commonly observed in prostate cancer, was strongly associated with Wnt/ß-catenin pathway activation in clinical samples. Tumor cell upregulation of the Wnt transporter Wntless (WLS) was strongly associated with Wnt/ß-catenin pathway activity in primary prostate cancer but also associated with both canonical and noncanonical Wnt signaling in mCRPC. IHC confirmed tumor cell WLS expression in primary prostate cancer and mCRPC, and patient-derived prostate cancer xenografts expressing WLS were responsive to treatment with Wnt synthesis inhibitor ETC-1922159. These findings reveal that Wnt/ß-catenin signaling in prostate cancer drives stem cell maintenance and invasion and primes for noncanonical Wnt signaling through ROR1. They further show that autocrine Wnt production is a nongenomic driver of canonical and noncanonical Wnt signaling in prostate cancer, which can be targeted with Wnt synthesis inhibitors to suppress tumor growth. SIGNIFICANCE: This work provides fundamental insights into Wnt signaling and prostate cancer cell biology and indicates that a subset of prostate cancer driven by autocrine Wnt signaling is sensitive to Wnt synthesis inhibitors.

An improved staining method of cell cycle analysis with Sybr Green I for fungi: Cryptococcus neoformans and Saccharomyces cerevisiae.

Cryptococcus neoformans is a pathogenic fungus which causes millions of deaths and infections, especially threatening immunocompromised individuals. During the development of new drugs, the ubiquitination has been found to play an important role in the regulation of the virulence and cell cycle of this fungus. Based on this mechanism, ubiquitination-related mutant strains exhibiting cell cycle arrest have been established for drug development for the fungus. However, flow cytometry detection of the cell cycle in fungi is generally difficult because the thick cell wall and capsule of fungi generally contribute to a nonspecific signal of cytometry. In this study, an improved method, derived from Saccharomyces cerevisiae assays, is developed to specifically stain C. neoformans, in whose cell cycle the G1 and G2 peaks are separated enough to be allowed for cell cycle analysis. As a result, the improved method facilitates the detection of the alterations in the cell cycle of C. neoformans with a mutation that results in cell cycle arrest, which distinctly delays the cell division of C. neoformans. Thus, the improved method reported here provides detailed technical information regarding assays on C. neoformans and, more importantly, offers a solution for assessing the cell cycle in other fungi in the future. Abbreviation: PI: propidium iodide.

Corrigendum: Mechanism of Growth Regulation of Yeast Involving Hydrogen Sulfide From S-Propargyl-Cysteine Catalyzed by Cystathionine-γ-Lyase.

[This corrects the article DOI: 10.3389/fmicb.2021.679563.].

Mechanism of Growth Regulation of Yeast Involving Hydrogen Sulfide From S-Propargyl-Cysteine Catalyzed by Cystathionine-γ-Lyase.

Pathogenic fungi are recognized as a progressive threat to humans, particularly those with the immunocompromised condition. The growth of fungi is controlled by several factors, one of which is signaling molecules, such as hydrogen sulfide (H2S), which was traditionally regarded as a toxic gas without physiological function. However, recent studies have revealed that H2S is produced enzymatically and endogenously in several species, where it serves as a gaseous signaling molecule performing a variety of critical biological functions. However, the influence of this endogenous H2S on the biological activities occurring within the pathogenic fungi, such as transcriptomic and phenotypic alternations, has not been elucidated so far. Therefore, the present study was aimed to decipher this concern by utilizing S-propargyl-cysteine (SPRC) as a novel and stable donor of H2S and Saccharomyces cerevisiae as a fungal model. The results revealed that the yeast could produce H2S by catabolizing SPRC, which facilitated the growth of the yeast cells. This implies that the additional intracellularly generated H2S is generated primarily from the enhanced sulfur-amino-acid-biosynthesis pathways and serves to increase the growth rate of the yeast, and presumably the growth of the other fungi as well. In addition, by deciphering the implicated pathways and analyzing the in vitro enzymatic activities, cystathionine-γ-lyase (CYS3) was identified as the enzyme responsible for catabolizing SPRC into H2S in the yeast, which suggested that cystathionine-γ-lyase might play a significant role in the regulation of H2S-related transcriptomic and phenotypic alterations occurring in yeast. These findings provide important information regarding the mechanism underlying the influence of the gaseous signaling molecules such as H2S on fungal growth. In addition, the findings provide a better insight to the in vivo metabolism of H2S-related drugs, which would be useful for the future development of anti-fungal drugs.

Quantitative acetylome analysis reveals histone modifications that may predict prognosis in hepatitis B-related hepatocellular carcinoma.

Lysine acetylation (Kac) as an important posttranslational modification of histones is essential for the regulation of gene expression in hepatocellular carcinoma (HCC). However, the atlas of whole acetylated proteins in HCC tissues and the difference in protein acetylation between normal human tissues and HCC tissues are unknown. In this report, we characterized the proteome and acetyl proteome (acetylome) profile of normal, paracancerous, and HCC liver tissues in human clinical samples by quantitative proteomics techniques. We identified 6781 acetylation sites of 2582 proteins and quantified 2492 acetylation sites of 1190 proteins in normal, paracancerous, and HCC liver tissues. Among them, 15 proteins were multiacetylated with more than 10 lysine residues. The histone acetyltransferases p300 and CBP were found to be hyperacetylated in hepatitis B virus pathway. Moreover, we found that 250 Kac sites of 214 proteins were upregulated and 662 Kac sites of 451 proteins were downregulated in HCC compared with normal liver tissues. Additionally, the acetylation levels of lysine 120 in histone H2B (H2BK120ac), lysine 18 in histone H3.3 (H3.3K18ac), and lysine 77 in histone H4 (H4K77ac) were increased in HCC. Interestingly, the higher levels of H2BK120ac, H3.3K18ac, and H4K77ac were significantly associated with worse prognosis, such as poorer survival and higher recurrence in an independent clinical cohort of HCC patients. Overall, this study lays a foundation for understanding the functions of acetylation in HCC and provides potential prognostic factors for the diagnosis and therapy of HCC.

Mesenchymal stromal cell therapies: immunomodulatory properties and clinical progress.

Mesenchymal stromal cells (MSCs) are a subset of heterogeneous non-hematopoietic fibroblast-like cells that can differentiate into cells of multiple lineages, such as chondrocytes, osteoblasts, adipocytes, myoblasts, and others. These multipotent MSCs can be found in nearly all tissues but mostly located in perivascular niches, playing a significant role in tissue repair and regeneration. Additionally, MSCs interact with immune cells both in innate and adaptive immune systems, modulating immune responses and enabling immunosuppression and tolerance induction. Understanding the biology of MSCs and their roles in clinical treatment is crucial for developing MSC-based cellular therapy for a variety of pathological conditions. Here, we review the progress in the study on the mechanisms underlying the immunomodulatory and regenerative effects of MSCs; update the medical translation of MSCs, focusing on the registration trials leading to regulatory approvals; and discuss how to improve therapeutic efficacy and safety of MSC applications for future.

Ribosomal Protein L40e Fused With a Ubiquitin Moiety Is Essential for the Vegetative Growth, Morphological Homeostasis, Cell Cycle Progression, and Pathogenicity of Cryptococcus neoformans.

Ubiquitin is a highly conserved protein required for various fundamental cellular processes in eukaryotes. Herein, we first report the contribution of the ubiquitin fusion protein Ubi1 (a ubiquitin monomer fused with the ribosome protein L40e, Rpl40e) in the growth and pathogenicity of Cryptococcus neoformans. UBI1 deletion resulted in severe growth restriction of C. neoformans, whose growth rate was positively correlated with UBI1 expression level. The growth defect of the ubi1Δ strain could be closely associated with its morphological abnormalities, such as its reduced ribosome particles. In addition, the ubi1Δ mutant also displayed increased cell ploidy, cell cycle arrest, and decreased intracellular survival inside macrophages. All these phenotypes were reversed by the reconstitution of the full-length UBI1 gene or RPL40a domain. Mouse survival and fungal burden assays further revealed a severely attenuated pathogenicity for the ubi1Δ mutant, which is probably associated with its reduced stress tolerance and the induction of T-helper 1-type immune response. Taken together, Ubi1 is required for maintaining the vegetative growth, morphological homeostasis, cell cycle progression, and pathogenicity in vivo of C. neoformans. The pleiotropic roles of Ubi1 are dependent on the presence of Rpl40e and associated with its regulation of cryptococcal ribosome biogenesis.

Correction to: DNMT3A reads and connects histone H3K36me2 to DNA methylation.

The author would like to add the below information in this correction. A similar study from Chao Lu group was published online on 5 September 2019 in Nature, entitled "The histone mark H3K36me2 recruits DNMT3A and shapes the intergenic DNA methylation landscape" (Weinberg et al., 2019). Although both the studies reported the preferential recognition of H3K36me2 by DNMT3A PWWP, ours in addition uncovered a stimulation function by such interaction on the activity of DNMT3A. On the disease connections, we used a NSD2 gain-of-function model which led to the discovery of potential therapeutic implication of DNA inhibitors in the related cancers, while the other study only used NSD1 and DNMT3A loss-of-function models.

Application of PD-1 Blockade in Cancer Immunotherapy.

The programmed cell death protein 1 (PD-1) pathway has received considerable attention due to its role in eliciting the immune checkpoint response of T cells, resulting in tumor cells capable of evading immune surveillance and being highly refractory to conventional chemotherapy. Application of anti-PD-1/PD-L1 antibodies as checkpoint inhibitors is rapidly becoming a promising therapeutic approach in treating tumors, and some of them have successfully been commercialized in the past few years. However, not all patients show complete responses and adverse events have been noted, suggesting a better understanding of PD-1 pathway mediated immunosuppression is needed to predict patient response and improve treatment efficacy. Here, we review the progresses on the studies of the mechanistic role of PD-1 pathway in the tumor immune evasion, recent clinical development and commercialization of PD-1 pathway inhibitors, the toxicities associated with PD-1 blockade observed in clinical trials as well as how to improve therapeutic efficacy and safety of cancer immunotherapy.

New method for quantification of gasotransmitter hydrogen sulfide in biological matrices by LC-MS/MS.

Hydrogen sulfide exists widely in mammalian tissues and plays a vital role in physiological and pathophysiological processes. However, striking differences with orders of magnitude were observed for the detected hydrogen sulfide concentrations in biological matrices among different measurements in literature, which lead to the uncertainty for examination the biological relevance of hydrogen sulfide. Here, we developed and validated a liquid chromatography- mass spectrometry (LC-MS/MS) method for the determination of hydrogen sulfide in various biological matrices by determination of a derivative of hydrogen sulfide and monobromobimane named sulfide dibimane (SDB). 36S-labeled SDB was synthesized and validated for using as an internal standard. This method has been successfully used to measure hydrogen sulfide levels in a broad range of biological matrices, such as blood, plasma, tissues, cells, and enzymes, across different species. Moreover, a novel mode that hydrogen sulfide could loosely and non-covalently bind to human serum protein (HSA) and hemoglobin (HB) was revealed by using the developed method.

The Natural Product Resveratrol Inhibits Yeast Cell Separation by Extensively Modulating the Transcriptional Landscape and Reprogramming the Intracellular Metabolome.

An increasing number of studies have shown that the promising compound resveratrol treats multiple diseases, such as cancer and aging; however, the resveratrol mode-of-action (MoA) remains largely unknown. Here, by virtue of multiple omics approaches, we adopted fission yeast as a model system with the goal of dissecting the common MoA of the anti-proliferative activity of resveratrol. We found that the anti-proliferative activity of resveratrol is mainly due to its unique role of inhibiting the separation of sister cells, similar phenotype with the C2H2 zinc finger transcription factor Ace2 knock-out strain. Microarray analysis shown that resveratrol has extensive impact on the fission yeast transcription levels. Among the changed gene's list, 40% of up-regulated genes are Core Environmental Stress Responses genes, and 57% of the down-regulated genes are periodically expressed. Moreover, resveratrol leverages the metabolome, which unbalances the intracellular pool sizes of several classes of amino acids, nucleosides, sugars and lipids, thus reflecting the remodulated metabolic networks. The complexity of the resveratrol MoA displayed in previous reports and our work demonstrates that multiple omics approaches must be applied together to obtain a complete picture of resveratrol's anti-proliferative function.

D-2-hydroxyglutarate is essential for maintaining oncogenic property of mutant IDH-containing cancer cells but dispensable for cell growth.

Cancer-associated isocitrate dehydrogenase (IDH) 1 and 2 mutations gain a new activity of reducing α-KG to produce D-2-hydroxyglutarate (D-2-HG), which is proposed to function as an oncometabolite by inhibiting α-KG dependent dioxygenases. We investigated the function of D-2-HG in tumorigenesis using IDH1 and IDH2 mutant cancer cell lines. Inhibition of D-2-HG production either by specific deletion of the mutant IDH1-R132C allele or overexpression of D-2-hydroxyglutarate dehydrogenase (D2HGDH) increases α-KG and related metabolites, restores the activity of some α-KG-dependent dioxygenases, and selectively alters gene expression. Ablation of D-2-HG production has no significant effect on cell proliferation and migration, but strongly inhibits anchorage independent growth in vitro and tumor growth in xenografted mouse models. Our study identifies a new activity of oncometabolite D-2-HG in promoting tumorigenesis.