High-Throughput Host-Microbe Single-Cell RNA Sequencing Reveals Ferroptosis-Associated Heterogeneity during Acinetobacter baumannii Infection.

Interactions between host and bacterial cells are integral to human physiology. The complexity of host-microbe interactions extends to different cell types, spatial aspects, and phenotypic heterogeneity, requiring high-resolution approaches to capture their full complexity. The latest breakthroughs in single-cell RNA sequencing (scRNA-seq) have opened up a new era of studies in host-pathogen interactions. Here, we first report a high-throughput cross-species dual scRNA-seq technology by using random primers to simultaneously capture both eukaryotic and bacterial RNAs (scRandom-seq). Using reference cells, scRandom-seq can detect individual eukaryotic and bacterial cells with high throughput and high specificity. Acinetobacter baumannii (A.b) is a highly opportunistic and nosocomial pathogen that displays resistance to many antibiotics, posing a significant threat to human health, calling for discoveries and treatment. In the A.b infection model, scRandom-seq witnessed polarization of THP-1 derived-macrophages and the intracellular A.b-induced ferroptosis-stress in host cells. The inhibition of ferroptosis by Ferrostatin-1 (Fer-1) resulted in the improvement of cell vitality and resistance to A.b infection, indicating the potential to resist related infections. scRandom-seq provides a high-throughput cross-species dual single-cell RNA profiling tool that will facilitate future discoveries in unraveling the complex interactions of host-microbe interactions in infection systems and tumor micro-environments.

Hepatic HDAC3 Regulates Systemic Iron Homeostasis and Ferroptosis via the Hippo Signaling Pathway.

Histone deacetylases (HDACs) are epigenetic regulators that play an important role in determining cell fate and maintaining cellular homeostasis. However, whether and how HDACs regulate iron metabolism and ferroptosis (an iron-dependent form of cell death) remain unclear. Here, the putative role of hepatic HDACs in regulating iron metabolism and ferroptosis was investigated using genetic mouse models. Mice lacking Hdac3 expression in the liver (Hdac3-LKO mice) have significantly reduced hepatic Hamp mRNA (encoding the peptide hormone hepcidin) and altered iron homeostasis. Transcription profiling of Hdac3-LKO mice suggests that the Hippo signaling pathway may be downstream of Hdac3. Moreover, using a Hippo pathway inhibitor and overexpressing the transcriptional regulator Yap (Yes-associated protein) significantly reduced Hamp mRNA levels. Using a promoter reporter assay, we then identified 2 Yap-binding repressor sites within the human HAMP promoter region. We also found that inhibiting Hdac3 led to increased translocation of Yap to the nucleus, suggesting activation of Yap. Notably, knock-in mice expressing a constitutively active form of Yap (Yap K342M) phenocopied the altered hepcidin levels observed in Hdac3-LKO mice. Mechanistically, we show that iron-overload-induced ferroptosis underlies the liver injury that develops in Hdac3-LKO mice, and knocking down Yap expression in Hdac3-LKO mice reduces both iron-overload- and ferroptosis-induced liver injury. These results provide compelling evidence supporting the notion that HDAC3 regulates iron homeostasis via the Hippo/Yap pathway and may serve as a target for reducing ferroptosis in iron-overload-related diseases.

Causal Effects of Circulating Lipid Traits on Epithelial Ovarian Cancer: A Two-Sample Mendelian Randomization Study.

Ovarian cancer (OC), and particularly epithelial OC (EOC), is an increasing challenge for women. Circulating lipids play different roles in the occurrence and development of OC, but no causal relationship has been confirmed. We used two-sample Mendelian randomization (MR) to evaluate the genetic effects of circulating Apolipoprotein A1 (APOA1), Apolipoprotein B (APOB), high-density lipoprotein (HDL) cholesterol, low-density lipoprotein (LDL) cholesterol, and triglyc-erides (TG) on EOC risks based on summary data obtained from the UK Biobank and the Ovarian Cancer Association Consortium. We used the inverse-variance weight as the main statistical method and the MR-Egger, weighted median, and MR-PRESSO for sensitivity analysis. A 1-SD increment in HDL gave odds ratios (OR) and 95% confidence intervals (CI) of OR = 0.80 (95% CI: 0.69-0.93), OR = 0.77 (95% CI: 0.66-0.90), and OR = 0.76 (95% CI: 0.63-0.90) for low malignant potential OC (LMPOC), low-grade low malignant OC (LGLMSOC), and low malignant serous OC (LMSOC), respectively. Genetic liability due to TG was associated with an increased risk of LGLMSOC and LGSOC and a suggestive association with an increased risk of LMSOC (p = 0.001, p = 0.007, and p = 0.027, respectively). Circulating HDL was negatively associated with the risk of LMPOC, LGLMSOC, and LMSOC, while elevated circulating TG levels genetically predicted an increased risk of LGLMSOC and LGSOC. Further research is needed to investigate the causal effects of lipids on EOC and potential intervention and therapeutic targets.

Causal Associations of Circulating Lipids with Osteoarthritis: A Bidirectional Mendelian Randomization Study.

Osteoarthritis (OA) imposes an increasing social burden due to global activity limitations, especially among the aged. Links between circulating lipids and OA have been reported; however, confounding data from observational studies have hindered causal conclusions. We used Mendelian randomization (MR) approach to evaluate the genetic causal effects of circulating apolipoproteins and lipoprotein lipids on OA risk. Genetic instruments at the genome-wide significance level (p < 5 × 10−8) were selected from genome-wide association studies (n = 393,193−441,016 individuals). Summary-level OA data were obtained from the UK Biobank (39,427 cases, 378,169 controls). Bidirectional two-sample Mendelian randomization (MR) analyses used MR-Egger, weighted median, and MR-PRESSO for sensitivity analysis. Genetic predisposition to 1-SD increments of Apolipoprotein B (APOB), and low-density lipoprotein (LDL) was associated with a decreased risk of knee or hip OA (KHOA) (odds ratio (OR) = 0.925, 95% confidence interval (95% CI): 0.881−0.972, p = 0.002; OR = 0.898, 95% CI: 0.843−0.957, p = 0.001) and hip OA (HOA) (OR = 0.894; 95% CI: 0.832−0.961, p = 0.002; OR = 0.870 95% CI: 0.797−0.949, p = 0.002). Genetically predicted APOB showed an association with knee OA (KOA) (OR per SD increase, 0.930, 95% CI: 0.876−0.987, p = 0.016). The OR of KOA was 0.899 (95% CI: 0.835−0.968, p = 0.005) for a 1-SD increase in LDL. Apolipoprotein A1, high-density lipoprotein, and triglycerides showed no association. Inverse MR showed no causal effect of KOA, HOA, or KHOA on these serum lipids. Distinct protective genetic-influence patterns were observed for APOB and LDL on OA, offering new insights into relationships between lipids and OA risk and a better understanding of OA etiology.

ACSL4 contributes to ferroptosis-mediated rhabdomyolysis in exertional heat stroke.

BACKGROUND: Rhabdomyolysis (RM) is a common complication of exertional heat stroke (EHS) and constitutes a direct cause of death. However, the mechanism underlying RM following EHS remains unclear. METHODS: The murine EHS model was prepared by our previous protocol. RNA sequencing is applied to identify the pathological pathways that contribute to RM following EHS. Inhibition of the acyl-CoA synthetase long-chain family member 4 (ACSL4) was achieved by RNA silencing in vitro prior to ionomycin plus heat stress exposure or pharmacological inhibitors in vivo prior to heat and exertion exposure. The histological changes, the iron accumulation, oxidized phosphatidylethanolamines species, as well as histological evaluation and levels of lipid metabolites in skeletal muscle tissues were measured. RESULTS: We demonstrated that ferroptosis contributes to RM development following EHS. Ferroptosis inhibitor ferrostatin-1 administration once EHS onset significantly ameliorated the survival rate of EHS mice from 35.357% to 52.288% within 24 h after EHS (P = 0.0028 compared with control) and markedly inhibited RM development induced by EHS. By comparing gene expression of between sham heat rest (SHR) (n = 3) and EHS (n = 3) mice in the gastrocnemius (Gas) muscle tissue, we identified that Acsl4 mRNA expression is elevated in Gas muscle tissue of EHS mice (P = 0.0038 compared with SHR), so as to its protein levels (P = 0.0001 compared with SHR). Followed by increase in creatine kinase (CK) and myoglobin (MB) levels, the labile iron accumulation, decrease in glutathione peroxidase 4 (GPX4) expression, and elevation of lipid peroxidation products. From in vivo and in vitro experiments, inhibition of Acsl4 significantly improves muscle cell death caused by EHS, thereby ameliorating RM development, followed by reduction in CK and MB levels by 30-40% (P < 0.0001; n = 8-10) and 40% (P < 0.0001; n = 8-10), restoration of GPX4 expression, and decrease in lipid peroxidation products. Mechanistically, ACSL4-mediated RM seems to be Yes-associated protein (YAP) dependent via TEA domain transcription factor1/TEA domain transcription factor4. CONCLUSIONS: These findings demonstrate an important role of ACSL4 in mediating ferroptosis activation in the development of RM following EHS and suggest that targeting ACSL4 may represent a novel therapeutic strategy to limit the skeletal muscle cell death and prevent RM after EHS.

[Progress on epigenetic regulation of iron homeostasis].

Iron homeostasis plays an important role for the maintenance of human health. It is known that iron metabolism is tightly regulated by several key genes, including divalent metal transport-1(DMT1), transferrin receptor 1(TFR1), transferrin receptor 2(TFR2), ferroportin(FPN), hepcidin(HAMP), hemojuvelin(HJV) and Ferritin H. Recently, it is reported that DNA methylation, histone acetylation, and microRNA (miRNA) epigenetically regulated iron homeostasis. Among these epigenetic regulators, DNA hypermethylation of the promoter region of FPN, TFR2, HAMP, HJV and bone morphogenetic protein 6 (BMP6) genes result in inhibitory effect on the expression of these iron-related gene. In addition, histone deacetylase (HADC) suppresses HAMP gene expression. On the contrary, HADC inhibitor upregulates HAMP gene expression. Additional reports showed that miRNA can also modulate iron absorption, transport, storage and utilization via downregulation of DMT1, FPN, TFR1, TFR2, Ferritin H and other genes. It is noteworthy that some key epigenetic regulatory enzymes, such as DNA demethylase TET2 and histone lysine demethylase JmjC KDMs, require iron for the enzymatic activities. In this review, we summarize the recent progress of DNA methylation, histone acetylation and miRNA in regulating iron metabolism and also discuss the future research directions.

Cloning and characterization of metallothionein gene (HcMT) from Halostachys caspica and its expression in E. coli.

Halostachys caspica is a short shrub distributed in the semi-arid and saline-alkali area, which evolved various mechanisms for modulating salt and metal level. In the present study, a Type 2 metallothionein (HcMT) gene was cloned from the salt induced suppression subtractive hybridization (SSH) cDNA library of H.caspica. Quantitative real time PCR (qRT-PCR) analysis indicated that HcMT gene was up-regulated under the stress of Cu(2+), Zn(2+) and Cd(2+), and the tolerance of E. coli strain harboring with the recombinant HcMT (pET-32a-HcMT) to Cu(2+), Zn(2+) and Cd(2+) was enhanced compared to strain with control vector (pET-32a). Moreover, the purified TrxA-HcMT fusion protein from E. coli cells grown in the presence of 0.3mM CuSO4, 0.3mM ZnSO4, or 0.1mM CdCl2 could bind more metal ions than TrxA alone. The predicted 3D structure showed that HcMT could form a single metal-thiolate cluster, which confers the ability to bind five divalent metal ions through fourteen cysteine residues. These data indicate that HcMT may be involved in processes of metal tolerance in H. caspica and could be employed as a potential candidate for heavy metal phytoremediation.