Di(2-ethylhexyl)phthalate (DEHP), a common environmental endocrine disruptor and agricultural organic pollutant, has drawn wide concern for its hepatotoxicity. However, the molecular mechanisms underlying DEHP-induced hepatic fibrosis remain unclear. Through network toxicology analysis, lipid metabolism and autophagy pathways are involved in DEHP-related hepatic fibrosis. Subchronic DEHP exposure (50-500 mg/kg) induced dose-dependent hepatic fibrosis in mice, while its metabolite MEHP synergized with TGF-β1 to promote LX2 cell activation in vitro. Crucially, MEHP activates lipophagy by targeting Beclin1, thereby mobilizing energy for the activation of hepatic stellate cells (HSCs). Attenuating autophagy with 3-Methyladenine (3-MA) or Chloroquine (CQ), or via Beclin1 knockdown, all rescued the activation phenotype induced by MEHP. These results suggest that DEHP acts as a trigger for fibrosis, promoting this process by inducing the activation of HSCs. This work identifies the lipophagy pathway as a crucial mechanistic link between environmental toxin exposure and HSC-mediated hepatic fibrosis progression.

Porcine reproductive and respiratory syndrome virus (PRRSV) induces excessive autophagy and aberrant nuclear factor kappa-light-chain-enhancer of activated B cell (NF-κB) hyperactivation, triggering severe respiratory and reproductive disorders in pigs and causing massive economic losses to the global swine industry. Hydroxychloroquine sulfate (HCQ), a classic autophagy inhibitor, restricts PRRSV proliferation in vitro; however, its in vivo protective efficacy and dual-targeted molecular mechanisms remain poorly characterized. In this study, intramuscular HCQ administration conferred robust protection against lethal PRRSV infection in piglets: it completely eliminated virus-induced mortality, relieved fever and interstitial pulmonary lesions, and restored normal growth performance. In vitro assays confirmed that HCQ with negligible cytotoxicity within the concentration range of 1-100 μM interferes with multiple stages of PRRSV infection. Combined network pharmacology and cellular functional experiments revealed a dual regulatory mechanism of HCQ against PRRSV: HCQ blocks autophagosome-lysosome fusion to disrupt complete autophagic flux, and suppresses the IκBα-p65 signaling cascade to reduce the secretion of proinflammatory mediators. Dose-response analyses demonstrated that HCQ exerts potent antiviral activity against divergent field PRRSV isolates, porcine epidemic diarrhea virus (PEDV) and swine acute diarrhea syndrome coronavirus (SADS-CoV) within a wide safe therapeutic window, with all broad-spectrum in vitro antiviral activity against diverse swine RNA viruses validated in cell culture models. Collectively, HCQ suppresses PRRSV replication by simultaneously regulating autophagic flux and NF-κB-dependent inflammatory signaling, supporting its repurposing as a promising therapeutic candidate for porcine reproductive and respiratory syndrome (PRRS) control. Although HCQ exhibits broad-spectrum inhibitory activity against divergent PRRSV strains, PEDV, and SADS-CoV in cell culture, all anti-PEDV and anti-SADS-CoV effects are only validated in vitro without in vivo animal verification.

Porcine astrovirus (PAstV) infection predominantly results in diarrhea and encephalitis in suckling piglets, yet the underlying pathogenic mechanisms remain poorly understood. In this study, the PAstV1-GX1 strain, previously isolated, was utilized to infect PK-15 cells. The results demonstrated that PAstV infection induced pronounced endoplasmic reticulum (ER) stress, as evidenced by the upregulation of HSPA5/GRP78 expression, and macroautophagy/autophagy, indicated by the increased conversion of MAP1LC3/LC3 from LC3-I to LC3-II and decreased expression of SQSTM1/p62. Pharmacological mitigation of ER stress effectively suppressed autophagy, suggesting that PAstV facilitates autophagy via ER stress. Further mechanistic investigations identified that PAstV infection selectively activated the EIF2AK3/PERK-EIF2S1/eIF2α-ATF4 pathway within the unfolded protein response, establishing this pathway as the pivotal upstream signal mediating autophagy. Through the screening of viral proteins, the capsid protein was identified as the primary viral component responsible for inducing ER stress and autophagy. This protein was concurrently localized to both mitochondria and the ER, where it interacted with the MAVS (mitochondrial antiviral signaling protein). The capsid protein facilitated the degradation of MAVS via the autophagy pathway and an apoptosis-related pathway in a non-classical, caspase-3-independent manner, thereby suppressing the production of type I interferons. Modulating ER stress, autophagy, or apoptosis greatly impacted the capsid protein's suppression of type I interferons. This suggested that the capsid protein hindered type I interferon production by triggering ER stress, which activated autophagy and possibly apoptosis, resulting in MAVS degradation. These insights lay the groundwork for understanding PAstV's pathogenic mechanisms and aid in developing new antiviral treatments.Abbreviations: 3-MA: 3-methyladenine; 4-PBA: 4-phenylbutyric acid; ACTB/β-actin: actin beta; ATF4: activating transcription factor 4; ATF6: activating transcription factor 6; CCK-8: Cell Counting Kit-8; Co-IP: co-immunoprecipitation; CQ: chloroquine; DAPI: 4,6-diamidino-2-phenylindole; DDIT3/CHOP: DNA damage inducible transcript 3; EGFP: enhanced green fluorescent protein; EIF2AK3/PERK: eukaryotic translation initiation factor 2 alpha kinase 3; EIF2S1/eIF2α: eukaryotic translation initiation factor 2 subunit alpha; ER: endoplasmic reticulum; ERN1/IRE1: endoplasmic reticulum to nucleus signaling 1; hpi: hours post-infection; HSPA5/GRP78: heat shock protein family A (Hsp70) member 5; IFIT1/ISG56: interferon induced protein with tetratricopeptide repeats 1; IFN: interferon; IFNB1/IFN-β: interferon beta 1; ISG15: ISG15 ubiquitin like modifier; mAb: monoclonal antibody; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MAVS: mitochondrial antiviral signaling protein; MOI: multiplicity of infection; PBS: phosphate-buffered saline; PAstV: porcine astrovirus; phospho-EIF2S1: phosphorylated EIF2S1; PPP1R15A/GADD34: protein phosphatase 1 regulatory subunit 15A; RAPA: rapamycin; RT-PCR: reverse transcription polymerase chain reaction; RT-qPCR: reverse transcription quantitative polymerase chain reaction; SeV: Sendai virus; siRNA: small interfering RNA; SQSTM1/p62: sequestosome 1; TG: thapsigargin; TU: tunicamycin; UPR: unfolded protein response; UV: ultraviolet; VSV-GFP: vesicular stomatitis virus expressing green fluorescent protein; WCL: whole-cell lysate; XBP1: X-box binding protein 1.

Antimalarial resistance is one of the greatest threats to malaria elimination. Following an outbreak of Plasmodium falciparum infection in Sabah Malaysia in 2019, 98 samples were sequenced to search for adaptations driving the outbreak. The genomic data revealed evidence of clonal expansion of a strain carrying multiple copies of the chloroquine resistance transporter gene (pfcrt) in 86 cases. A TaqMan qPCR assay was developed to quantify pfcrt copy number, confirming the in silico evidence of duplication. Whilst point mutations in pfcrt have been associated with resistance to chloroquine and other drugs, copy number amplification has not been widely explored as a resistance mechanism. We investigated the genetic architecture of the duplication, revealing a wildtype haplotype (3D7 reference-type with 76K variant) and a novel mutant (with 76T mutation). Application of the TaqMan assay in 65 P. falciparum cases from neighbouring Palawan Island, the Philippines, identified a further two cases with pfcrt duplication. Assessment of the global genomic data in the MalariaGEN Pf8 repository identified a further 86 cases, including 73 (85%) from West Africa with evidence of pfcrt duplication. Amongst 47 monoclonal MalariaGEN cases, majority (95%) comprised wild type and mutant variants at codon 76. Our study reveals a potential previously unconsidered antimalarial resistance mechanism for P. falciparum and provides an assay for surveillance in other populations.

Malaria continues to be a profound global health problem, with escalating drug resistance in Plasmodium falciparum paving way for the necessity of affordable synthetic antimalarial agents. A virtual library of 300 tetraoxane derivatives was designed and subjected to in silico ADMET and molecular docking against falcipain-2 (PDB: 3BPF). Seven classified drug candidates (KD-1 to KD-7) were synthesised via two-step acid-catalyzed cyclic condensation of gem-dihydroperoxides. These compounds were evaluated in vitro against chloroquine-resistant (PfRKL-9) and chloroquine-sensitive (Pf3D7) strains. All derivatives exhibited inhibition dose-dependently, with KD-7 demonstrating the highest potency, followed by KD-1 and KD-2. Microscopic observation indicated effective inhibition of parasite development at the schizont stage. Although the synthesised scaffolds currently demonstrate promising foundational activity, this work unlocks an opportunity to scrutinise structural flexibility with synthetic approaches for further optimisation to combat the escalating trend of antimalarial resistance.

This study aimed to compare demographic, clinical, laboratory, and autoantibody profiles between anti-dense fine speckled 70 (DFS70)-positive and -negative patients with undifferentiated connective tissue disease (UCTD). We retrospectively analyzed 284 UCTD patients followed at a tertiary rheumatology centre between January 2017 and December 2024. Patients fulfilling established classification criteria for a defined connective tissue disease were excluded. Patients were stratified according to anti-DFS70 status. Group comparisons were performed, and the Benjamini-Hochberg procedure was applied to control the false discovery rate. A multivariable logistic regression model was additionally used to evaluate the potential confounding effect of hydroxychloroquine use. Of 284 patients, 75 (26.4%) were anti-DFS70 positive and 209 (73.6%) were negative. After false discovery rate correction, arthritis/arthralgia was more frequent in the anti-DFS70-negative group than in the positive group (81.8% vs. 61.3%, adjusted p = 0.034), as was homogeneous ANA pattern (33.5% vs. 12.0%, adjusted p = 0.034). In multivariable analysis, homogeneous ANA pattern remained associated with anti-DFS70 negativity after adjustment for hydroxychloroquine use (OR 2.71, 95% CI 1.16-6.30, p = 0.021), whereas arthritis/arthralgia did not remain statistically significant (OR 1.92, 95% CI 0.95-3.90, p = 0.071). Anti-DFS70 negativity in UCTD was associated with homogeneous ANA pattern. Clinical associations, particularly arthritis/arthralgia, should be interpreted cautiously because of potential confounding by treatment exposure. Further prospective studies are warranted to clarify the clinical significance of anti-DFS70 status in UCTD.

Stimulator of interferon genes (STING) is critical for antiviral and intracellular antibacterial immune defense and is involved in pulmonary fibrotic diseases. However, the role of STING in regulating autophagy and airway remodeling in asthma remains unclear. An ovalbumin (OVA)-induced mouse model of asthma airway remodeling was established. Expression levels of STING and autophagy-related gene 5 (ATG5) in lung tissues were detected. The effects of STING inhibitor C-176 on autophagy and airway remodeling were evaluated in vivo. In human bronchial epithelial BEAS-2B cells stimulated with interleukin (IL)-13, the relationships among STING, autophagy, and cellular fibrosis were investigated in vitro. In OVA-challenged mice, C-176, chloroquine (CQ), and dexamethasone (DX) reduced airway hyperresponsiveness and eosinophil counts. C-176 and CQ alleviated inflammatory cell infiltration, mucus secretion, and collagen deposition, and suppressed autophagy and fibrosis in airway epithelium. In IL-13-stimulated BEAS-2B cells, C-176, ATG5 inhibition, or PERK pathway inhibition downregulated autophagy and fibrosis‑related markers. STING upregulation in airway epithelial cells promotes autophagy and subsequent airway remodeling in asthma. Inhibition of STING or autophagy attenuates these pathological changes, indicating a potential therapeutic target for asthma.

Spring viremia of carp virus (SVCV) severely threatens cyprinid aquaculture, yet the role of autophagy in SVCV infection remains unclear. Here, SVCV infection was associated with enhanced autophagic activity and autophagic flux in common carp and EPC cells. Pharmacological perturbation of autophagy revealed that rapamycin treatment increased SVCV gene expression and aggravated SVCV-induced cytopathic effects, whereas chloroquine blocked autophagic flux, suppressed viral gene expression, and mitigated virus-induced cellular damage. Transcriptomic analysis identified 1,868 differentially expressed genes after chloroquine treatment, with major changes in lysosome/autophagy-related pathways, glycan degradation, ion transport, cell cycle, DNA replication, and DNA repair. Metabolomic and integrated multi-omics analyses revealed changes in amino sugar and nucleotide sugar metabolism, steroid biosynthesis, riboflavin metabolism, and amino acid metabolism. Functional validation showed that diflorasone and all-trans-retinal reduced SVCV-G expression, alleviated cytopathic effects, and improved survival after SVCV infection. These findings suggest that SVCV infection is associated with enhanced autophagic flux, whereas chloroquine treatment blocks autophagic flux and is accompanied by reduced SVCV gene expression.

Malaria remains a major parasitic disease caused mainly by Plasmodium falciparum and Plasmodium vivax, and drug-resistant parasites continue to challenge disease control. Drug repurposing may help identify compounds with antimalarial potential. Here, we evaluated gallopamil, an L-type calcium channel blocker, using in vitro, ex vivo, in vivo and in silico approaches. Gallopamil inhibited the chloroquine-resistant P. falciparum W2 strain in vitro (IC50 = 3.65 µM) and showed ex vivo activity against clinical isolates of P. falciparum and P. vivax from CEPEM/RO, with median IC50 values of 6.4 and 9.87 µM, respectively. Selectivity indices calculated using HepG2 cytotoxicity data ranged from >51 to >137, indicating in vitro selectivity relative to HepG2 cells. In a preliminary murine P. berghei suppressive assay, gallopamil at 5 mg/kg reduced parasitemia by more than 40% at the day-5 endpoint, as measured by light microscopy and flow cytometry. Stage-specific assays showed that gallopamil inhibited parasite development when treatment began at the ring stage (p < 0.000001), whereas no significant inhibition was observed when treatment started with predominantly schizont-stage parasites (p = 0.30). Molecular docking predicted interactions between gallopamil and residues of Plasmodium hexose transporters, including LYS-51, GLN-167, ASN-309 and ASN-339, with binding energies of -6.1 kcal/mol for PvHT1 and -7.8 kcal/mol for PfHT1. These findings support gallopamil as an early antiplasmodial hit compound and provide a transporter-related mechanistic hypothesis for future functional validation.

The biofilms of Mycobacterium Tuberculosis (Mtb) use extracellular DNA (eDNA) as a protective structural scaffold that shields resident bacteria from host immunity and antibiotics, promoting persistent infection. An FDA-approved DNase can partially hydrolyze eDNA but has limited affinity and nucleolytic activity against Z-form DNA (Z-DNA). After confirming the abundance of Z-DNA in Mtb biofilms, we develop Nano-NucleOTAC, a nano-nucleolysis targeting chimera that combines an eDNA-binding ligand (lactoferrin), a DNA intercalator (chloroquine) and DNase. Nano-NucleOTAC anchors to Z-DNA in Mtb H37Ra biofilms, enabling effective biofilm degradation via Z-DNA transformation and enzymatic cleavage. Following treatment, dispersed Mtb H37Ra clusters induce neutrophil extracellular trap (NET) formation and cytokine secretion (TNF, IL-1β, and IL-10) to recruit macrophages, while NET remodeling and nucleolysis enhance macrophage bactericidal effect vs. DNase alone. In mouse lung infection models, Nano-NucleOTAC improves lung drug retention, reduces pathology, and reactivates host immune responses. It also demonstrates high efficacy against Mtb strains of varying virulence (H37Ra, H37Rv) and non-tuberculous mycobacteria (Mycobacterium smegmatis). In clinical sputum samples, Nano-NucleOTAC potentiates rifampicin efficacy, and enhances neutrophil and macrophage antibacterial immunity in bronchoalveolar lavage fluid (BALF) and pus. This safe, versatile nucleic-acid-targeting strategy modulates bacterial biophysical barriers and host immunity to address biofilm-associated multidrug-resistant infections.