Apoptosis-inducing factor 1 (AIF1) overexpression is intimately linked to the sensitivity of yeast cells towards hydrogen peroxide or acetic acid. Therefore, studying the mechanism of AIF1 regulation in the cell would provide a significant understanding of the factors guiding yeast apoptosis. In this report, we show the time-dependent induction of AIF1 under hydrogen peroxide stress. Additionally, we find that AIF1 expression in response to hydrogen peroxide is mediated by two transcription factors, Yap5 (DNA binding) and Cdc73 (non-DNA binding). Furthermore, substituting the H3K36 residue with another amino acid significantly abrogates AIF1 expression. However, substituting the lysine (K) in H3K4 or H3K79 with alanine (A) does not affect AIF1 expression level under hydrogen peroxide stress. Altogether, reduced AIF1 expression in cdc73Δ is plausibly due to reduced H3K36me3 levels in the cells.

Enhanced levels of acetic acid reduce the activity of yeast strains employed for industrial fermentation-based applications. Therefore, unraveling the genetic factors underlying the regulation of the tolerance and sensitivity of yeast towards acetic acid is imperative for optimising various industrial processes. In this communication, we have attempted to decipher the acetic acid stress response of the previously reported acetic acid-sensitive histone mutants. Revalidation using spot-test assays and growth curves revealed that five of these mutants, viz., H3K18Q, H3S28A, H3K42Q, H3Q68A, and H3F104A, are most sensitive towards the tested acetic acid concentrations. These mutants demonstrated enhanced acetic acid stress response as evidenced by the increased expression levels of AIF1, reactive oxygen species (ROS) generation, chromatin fragmentation, and aggregated actin cytoskeleton. Additionally, the mutants exhibited active cell wall damage response upon acetic acid treatment, as demonstrated by increased Slt2-phosphorylation and expression of cell wall integrity genes. Interestingly, the mutants demonstrated increased sensitivity to cell wall stress-causing agents. Finally, screening of histone H3 N-terminal tail truncation mutants revealed that the tail truncations exhibit general sensitivity to acetic acid stress. Some of these N-terminal tail truncation mutants viz., H3 [del 1-24], H3 [del 1-28], H3 [del 9-24], and H3 [del 25-36] are also sensitive to cell wall stress agents such as Congo red and caffeine suggesting that their enhanced acetic acid sensitivity may be due to cell wall stress induced by acetic acid.

Invasive candidiasis is the most common and serious fungal disease worldwide, and the development of antifungal drug resistance in Candida spp. is an emerging problem. Miltefosine, approved as an orphan drug for the therapy of invasive candida infections by the US Food and Drug Administration, has broad-spectrum antifungal activity, but its mechanism of action is unclear. This study evaluated the antifungal drug susceptibility of azole-resistant Candida spp. isolates and found that miltefosine showed good activity, with a geometric mean value of 2 µg/mL. Miltefosine was found to increase production of intracellular reactive oxygen species (ROS) and induce apoptosis in Candida albicans. RNA sequencing (RNA-Seq) analysis and iTRAQ-labelling-based quantitative proteomic mass spectrometry analysis were undertaken. Aif1 and the oxidative stress pathway involved in miltefosine-mediated apoptosis were identified using global transcriptomic and proteomic combined screening. Miltefosine increased mRNA and protein expressions of Aif1. The localization of Aif1 was examined using confocal microscopy, and the GFP-Aif1 fusion protein was found to be translocated from the mitochondria to the nucleus when sensing miltefosine. Next, the pex8 Δ/Δ strain was constructed, and the minimum inhibitory concentration of miltefosine was found to decrease four-fold (from 2 to 0.5 µg/mL) and the intracellular ROS increased significantly after knocking out the PEX8 gene. Moreover, miltefosine was found to trigger Hog1 phosphorylation. These findings indicate that Aif1 activation and the Pex8-mediated oxidative stress pathway are the mechanisms of action of miltefosine on C. albicans. The results help to aid understanding of the mechanisms by which miltefosine acts on fungi.

TAR DNA-binding protein 43 (TDP-43) is a nucleic acid-binding protein found in the nucleus that accumulates in the cytoplasm under pathological conditions, leading to proteinopathies, such as frontotemporal dementia and ALS. An emerging area of TDP-43 research is represented by the study of its post-translational modifications, the way they are connected to disease-associated mutations, and what this means for pathological processes. Recently, we described a novel mutation in TDP-43 in an early onset ALS case that was affecting a potential phosphorylation site in position 375 (S375G). A preliminary characterization showed that both the S375G mutation and its phosphomimetic variant, S375E, displayed altered nuclear-cytoplasmic distribution and cellular toxicity. To better investigate these effects, here we established cell lines expressing inducible WT, S375G, and S375E TDP-43 variants. Interestingly, we found that these mutants do not seem to affect well-studied aspects of TDP-43, such as RNA splicing or autoregulation, or protein conformation, dynamics, or aggregation, although they do display dysmorphic nuclear shape and cell cycle alterations. In addition, RNA-Seq analysis of these cell lines showed that although the disease-associated S375G mutation and its phosphomimetic S375E variant regulate distinct sets of genes, they have a common target in mitochondrial apoptotic genes. Taken together, our data strongly support the growing evidence that alterations in TDP-43 post-translational modifications can play a potentially important role in disease pathogenesis and provide a further link between TDP-43 pathology and mitochondrial health.

Hydroxychloroquine (HCQ), which has been proposed as a therapeutic or prophylactic drug for COVID-19, has been administered to thousands of individuals with varying efficacy; however, our understanding of its adverse effects is insufficient. It was reported that HCQ induced psychiatric symptoms in a few patients with autoimmune diseases, but it is still uncertain whether HCQ poses a risk to mental health. Therefore, in this study, we treated healthy mice with two different doses of HCQ that are comparable to clinically administered doses for 7 days. Psychiatric-like behaviors and the expression of related molecules in the brain were evaluated at two time points, i.e., 24 h and 10 days after drug administration. We found that HCQ increased anxiety behavior at both 24 h and 10 days. Furthermore, HCQ decreased the mRNA expression of interleukin-1beta, corticotropin-releasing hormone (Crh), a serotonin transporter (Slc6a4), and a microglia maker (Aif1) in the hippocampus and decreased the mRNA expression of brain-derived neurotrophic factor (Bdnf) in both the hippocampus and amygdala. Lots of these behavioral and molecular changes were sustained beyond 10 days after drug administration, and some of them were dose-dependent. Although this animal study does not prove that HCQ has a similar effect in humans, it indicates that HCQ poses a significant risk to mental health and suggests that further clinical investigation is essential. According to our data, we recommend that HCQ be carefully used as a prophylactic drug in people who are susceptible to mental disorders.

Background: Metastatic breast cancer cells recruit macrophages (metastasis-associated macrophages, or MAMs) to facilitate their seeding, survival and outgrowth. However, a comprehensive understanding of the gene expression program in MAMs and how this program contributes to metastasis remain elusive. Methods: We compared the transcriptomes of MAMs recruited to lung metastases and resident alveolar macrophages (RAMs) and identified a large variety of differentially expressed genes and their associated signaling pathways. Some of the changes were validated using qRT-PCR and immunofluorescence. To probe the functional relevance to metastatic growth, a gene-targeting mouse model of female mice in the C57BL6/J background was used to study allograft inflammatory factor 1 (AIF1, also known as ionized calcium-binding adapter molecule 1 or IBA1). Results: Interferon signaling is one of the most activated pathways in MAMs, with strong upregulation of multiple components of the pathway and a significant enrichment for the gene signatures of interferon-alpha-treated human macrophages. Aif1, an interferon-responsive gene that regulates multiple macrophage activities, was robustly induced in MAMs. Aif1 deficiency in MAMs, however, did not affect development of lung metastases, suggesting that AIF1 indicates MAM activation but is dispensable for regulating metastasis. Conclusions: The drastically different gene expression profile of MAMs as compared to RAMs suggests an important role in promoting metastatic growth. Dissection of the underlying mechanisms and functional validation of potential targets in the profile may provide novel therapeutic strategies for the treatment of metastatic diseases.

Classical swine fever (CSF) is a disease that is characterized by diffuse hemorrhaging, high fever, and high mortality rates. The pro-inflammatory characteristics of allograft inflammatory factor 1 (AIF1) have been well documented; however, insufficient attention has been given to porcine AIF1. In the present study, AIF1 was identified as a key player contributing to CSFV Shimen infection in porcine alveolar macrophage (PAM) 3D4/21 cell line. Our evaluation showed that AIF1 mRNA and protein are expressed at a time-dependent high level in CSFV Shimen-infected PAM 3D4/21 cells. The transcription and translation of IL6 were also significantly upregulated in infected PAM 3D4/21 cells. By utilizing overexpression RNAs approach, we showed that the cellular AIF1 induced an increased IL6 in PAM 3D4/21 cells. Furthermore, silencing of AIF1 suppressed CSFV Shimen-induced IL6 production in PAM 3D4/21 cells and also inhibited CSFV replication, whereas overexpression of recombinant AIF1 was beneficial for the replication of CSFV Shimen and promoting IL6 production in CSFV Shimen-infected PAM 3D4/21 cells. It is suggested CSFV Shimen induced IL6 in PAM 3D4/21 cells via AIF1 activation, which help clarify the AIF1-related inflammatory processes that occur on CSFV Shimen infected macrophages.

Camphor is widely used in pharmacy, the food industry, and cosmetics. In this study, we evaluate inhibitory and cytotoxic effects of camphor in the fission yeast (Schizosaccharomyces pombe), which presents a unicellular model in mechanistic toxicology and cell biology. Low-dose camphor exposure (0.4 mg/mL) activated autophagy, which was shown by GFP-Atg8 dots and transcriptional upregulation of Atg6 (Beclin-1 ortholog). Autophagy was also confirmed by using autophagy-deficient cells, which showed reduction in GFP-Atg8 dot formation. However, high-dose camphor exposure (0.8 mg/mL) caused dramatic cell death ratios, demonstrated by spot and colony-forming assays, even in autophagy-deficient cells. To unravel the underlying mechanism, this time, apoptosis-deficient cells were exposed to low- and high-dose camphor. Apoptosis was also confirmed by acridine orange/ethidium bromide staining. Among yeast apoptosis mediators, Aif1 was found to mediate camphor-induced cell death. In conclusion, differential regulation of autophagy and apoptosis, and switches between them, were found to be dose-dependent. The potential effects of camphor on autophagy and apoptotic cell death and underlying mechanisms were clarified in basic unicellular eukaryotic model, S. pombe.

Glioblastoma (GBM) is the most aggressive malignant primary brain tumor in adults, with a median survival of 14.6 months. Recent efforts have focused on identifying clinically relevant subgroups to improve our understanding of pathogenetic mechanisms and patient stratification. Concurrently, the role of immune cells in the tumor microenvironment has received increasing attention, especially T cells and tumor-associated macrophages (TAM). The latter are a mixed population of activated brain-resident microglia and infiltrating monocytes/monocyte-derived macrophages, both of which express ionized calcium-binding adapter molecule 1 (IBA1). This study investigated differences in immune cell subpopulations among distinct transcriptional subtypes of GBM. Human GBM samples were molecularly characterized and assigned to Proneural, Mesenchymal or Classical subtypes as defined by NanoString nCounter Technology. Subsequently, we performed and analyzed automated immunohistochemical stainings for TAM as well as specific T cell populations. The Mesenchymal subtype of GBM showed the highest presence of TAM, CD8+, CD3+ and FOXP3+ T cells, as compared to Proneural and Classical subtypes. High expression levels of the TAM-related gene AIF1, which encodes the TAM-specific protein IBA1, correlated with a worse prognosis in Proneural GBM, but conferred a survival benefit in Mesenchymal tumors. We used our data to construct a mathematical model that could reliably identify Mesenchymal GBM with high sensitivity using a combination of the aforementioned cell-specific IHC markers. In conclusion, we demonstrated that molecularly distinct GBM subtypes are characterized by profound differences in the composition of their immune microenvironment, which could potentially help to identify tumors amenable to immunotherapy.

UNASSIGNED: Inflammation is a major player in breast cancer (BC) progression. Allograft-inflammatory factor-1 (AIF1) is a crucial mediator in the inflammatory response. AIF1 reportedly plays a role in BC, but the mechanism remains to be elucidated. We identified two AIF1 isoforms, AIF1v1 and AIF1v3, which were differentially expressed between affected and unaffected sisters from families with high risk of BC with no deleterious BRCA1/BRCA2 mutations (BRCAX). We investigated potential functions of AIFv1/v3 in BC of varying severity and breast adipose tissue by evaluating their expression, and association with metabolic and clinical parameters of BC patients.
UNASSIGNED: AIF1v1/v3 expression was determined in BC tissues and cell lines using quantitative real-time PCR. Potential roles and mechanisms were examined in the microenvironment (fibroblasts, adipose tissue, monocytes and macrophages), inflammatory response (cell reaction in BC subgroups), and metabolism [treatment with docosahexaenoic acid (DHA)]. Association of AIF1 transcript expression with clinical factors was determined by Spearman\'s rank correlation. Bioinformatics analyses were performed to characterize transcripts.
UNASSIGNED: AIF1v1/v3 were mostly expressed in the less severe BC samples, and their expression appeared to originate from the tumor microenvironment. AIF1 isoforms had different expression rates and sources in breast adipose tissue; lymphocytes mostly expressed AIF1v1 while activated macrophages mainly expressed AIF1v3. Bioinformatics analysis revealed major structural differences suggesting distinct functions in BC progression. Lymphocytes were the most infiltrating cells in breast tumors and their number correlated with AIF1v1 adipose expression. Furthermore, DHA supplementation significantly lowered the expression of AIF1 isoforms in BRCAX cell lines. Finally, the expression of AIF1 isoforms in BC and breast adipose tissue correlated with clinical parameters of BC patients.
UNASSIGNED: Results strongly suggest that AIF1v1 as much as AIF1v3 play a major role in the crosstalk between BC and infiltrating immune cells mediating tumor progression, implying their high potential as target molecules for BC diagnostic, prognostication and treatment.