Sertoli cells play a pivotal role in spermatogenesis by providing structural support and delivering essential nutrients and signaling molecules to developing germ cells. A key function of these cells is the regulation of androgen-binding protein (ABP), which maintains the high intratesticular testosterone levels necessary for proper spermatogenic progression. Despite the critical nature of ABP, the mechanisms underlying its degradation within Sertoli cells remain poorly understood. This study investigates the role of the valosin-containing protein (p97/VCP) in the autophagic degradation of ABP, with a particular focus on molecular pathways that may contribute to male infertility and testicular dysfunction. Sertoli 15P-1 cells were treated with testosterone, chloroquine, and rapamycin, and subjected to single and double immunofluorescence staining, Western blotting, and siRNA-mediated knockdown experiments to explore the interaction between p97/VCP and ABP. Western blot analysis demonstrated that p62 expression was highest in the chloroquine + testosterone (CQ + T) group compared to control siRNA (Csi), although not statistically significant (p > 0.05). LC3 expression peaked in the chloroquine (CQ) group (p > 0.05). As expected, the lowest expression of p97/VCP was detected in the VCPsi group following siRNA transfection. Immunofluorescence analyses revealed significant differences in protein colocalization patterns across all treatment groups, except for p62 in rapamycin-treated samples. These findings provide the first evidence in the literature of a direct relationship between p97/VCP and ABP within Sertoli cells, underscoring the relevance of autophagic pathways in the regulation of male reproductive hormones. The data suggest a novel regulatory mechanism by which p97/VCP may influence spermatogenesis and male fertility through ABP degradation. Furthermore, this work lays a foundational framework for future investigations in other ABP-expressing tissues such as the brain, liver, and placenta, potentially linking p97/VCP expression to ABP-associated endocrine and pathophysiological disorders beyond the testis.

Sirtuin-1 (SIRT1) is an NAD[+]-dependent deacetylase implicated in autophagosome formation; however, whether SIRT1 also regulates autophagosome clearance during late-stage autophagy remains unclear. Here, we investigated the role of SIRT1 in autophagosome clearance during autophagy and mitophagy in cardiomyocytes. Mitochondrial stress induced by carbonyl cyanide m-chlorophenyl hydrazone (CCCP) decreased mitochondrial protein levels and increased phosphorylation of ubiquitin, a PINK1 target, in H9c2 cardiomyocytes. These CCCP-induced decreases in mitochondrial proteins were prevented by co-treatment with chloroquine, an inhibitor of lysosomal degradation, supporting the induction of CCCP-triggered mitophagy. SIRT1 knockdown similarly prevented the CCCP-induced reduction in mitochondrial proteins and led to the accumulation of autophagosomes containing fragmented mitochondria without attenuating ubiquitin phosphorylation, suggesting that SIRT1 acts downstream of mitochondrial tagging. Tandem GFP-RFP LC3 assay and LC3-LAMP1 colocalization analysis demonstrated impaired autophagosome-lysosome fusion following SIRT1 knockdown. In vivo, cardiomyocyte-specific SIRT1 knockout mice exhibited elevated basal LC3-II levels and a blunted LC3-II response to chloroquine, consistent with impaired autophagic flux. In a doxorubicin (DOX)-treated model, SIRT1 deficiency attenuated autophagosome degradation during the early period after DOX administration. Mechanistically, SIRT1 interacted with Rab7, a key regulator of autophagosome-lysosome fusion, raising the possibility that SIRT1 might regulate fusion through post-translational modification of Rab7 or related components. Collectively, these findings identify SIRT1 as a regulator of autophagosome-lysosome fusion that promotes autophagosome degradation during autophagy and mitophagy in cardiomyocytes.

An expedited synthesis of 2-sulfonamido-substituted 4H-chromene derivatives via reaction between para-quinone methides and ynamides in the presence of triflic acid at room temperature is described. Most of the synthesized compounds displayed potent antiplasmodial activity against both the chloroquine-sensitive and -resistant strains of Plasmodium falciparum with a good selectivity index.

Motivated by the antimalarial potential demonstrated by benzamide derivatives of 4-alkylamino-7-chloroquinolines against chloroquine-resistant Plasmodium falciparum, we designed and synthesized a rational series of N-2-(7-chloroquinolin-4-ylamino)ethyl amides to investigate the effects of electron-withdrawing and donating substituents on antiplasmodial and cytotoxicity properties, from prediction to in vitro assays. From an initial screening of 22 chloroquine (CQ) analogs, 16 compounds with two distal amino groups in the alkyl linker showed IC50 values below 1000 nM against the CQ-sensitive 3D7 strain. Next, their activity against the CQ-resistant K1 strain, in silico toxicity, predicted using the pkCSM program, and in vitro cytotoxicity on HepG2 cells were evaluated. Benzamides containing electron-withdrawing chlorine and nitro groups showed the most promising profiles, with IC50 values against the CQ-resistant K1 strain (IC50 [K1], 55-72 nM) and resistance index (RI, 0.7-1.9) values outperforming CQ (IC50 [K1] = 141 nM, RI = 7.1). Although predicted to be hepatotoxic, these compounds were not cytotoxic to HepG2 cells at the tested concentrations, showing CC50 values above 12,500 nM and selectivity indices greater than 115. Compared with CQ, a 3D qualitative SAR model indicated that increased lipophilicity around the distal benzamide moiety enhances antiplasmodial activity against the K1 strain, contributing to the observed potency.

Head and neck squamous cell carcinoma (HNSCC) exhibits profound resistance to chemoradiotherapy and immune checkpoint inhibition, which is closely associated with therapy-induced cellular senescence. However, the regulatory mechanism of the senescence-independent senescence-associated secretory phenotype (SASP) driven by double-stranded RNA (dsRNA)-mediated innate immune activation after chemoradiotherapy remains largely unknown. The dsRNA mimic polyinosinic-polycytidylic acid [poly(I:C)] was used to stimulate CAL27 cells, with chloroquine (CQ) intervention to inhibit autophagy. Integrated transcriptomic analysis, in vitro functional assays and clinical specimen validation were performed to explore the regulation, transcellular cascade and prognostic value of senescence-independent SASP. dsRNA-mediated innate immune stimulation triggered NF-κB-dependent senescence-independent SASP in an autophagy-dependent manner without inducing proliferative arrest or classic senescence marker expression. CXCL1 was identified as a candidate key mediator of this SASP subtype. Furthermore, senescence-independent SASP initiates an IL-6-mediated transcellular inflammatory cascade to activate SASP in tumor-associated macrophages, remodeling the immunosuppressive tumor microenvironment. Autophagy inhibition effectively suppressed this SASP, reduced IL-6 secretion and disrupted the tumor-macrophage inflammatory loop. Clinically, the TLR3-autophagy-NF-κB-IL-6 signature was validated as a reliable prognostic biomarker for oral squamous cell carcinoma (HNSCC). This study reveals a novel autophagy-NF-κB-IL-6 axis governing senescence-independent SASP and the downstream inflammatory cascade in HNSCC, representing a promising therapeutic strategy to improve chemoradiotherapy plus immunotherapy efficacy.

Neutrophil extracellular traps (NETs) perpetuate chronic inflammation in ulcerative colitis (UC), yet current neutrophil-targeting therapies focus on acute intervention rather than sustained daily control. Here, we propose an oral microsupplement for long-term UC management that concurrently scavenges reactive oxygen species (ROS) and suppresses NET formation. The microsupplement integrates a catalase-mimetic CoAl-layered double hydroxide (LDH) nanozyme intercalated with the autophagy inhibitor chloroquine (CQ), together with dopamine (DOPA). The LDH nanozyme catalytically converts pathological ROS into oxygen, driving in situ polymerization of DOPA into polydopamine (PDA), a mucoadhesive polymer that prolongs colonic retention of CQ@LDH and promotes mucosal healing. RNA sequencing reveals that internalized CQ@LDH inhibits autophagic flux in neutrophils, thereby upregulating catalase expression and peroxisomal function to prevent NET formation. Consequently, NET-driven M1 macrophage polarization is reduced from 29.5% to 17.9%, and Th17 differentiation is markedly suppressed, accompanied by a decrease in colonic Th17/Treg ratio from 8.68 to 1.31. In a murine UC model, this microsupplement restores colonic mucosal integrity, rebalances the gut microbiota, and outperforms the clinical drug 5-aminosalicylic acid in both immune regulation and epithelial restitution. This neutrophil-regulating, colon-adhesive platform offers a promising strategy for routine daily UC management.

Peptide nucleic acids (PNAs) are a promising platform targeting disease-associated microRNAs due to their high binding affinity, excellent sequence specificity and resistance to enzymatic degradation. However, their therapeutic application is often limited by inefficient cellular uptake. In this study, we present the design, synthesis and physicochemical characterisation of cell-penetrating peptide (CPP)-PNA conjugates that target the oncogenic microRNA miRNA-221-5p, as well as their preliminary biological evaluation. Anti-miRNA-221-5p PNAs functionalised for conjugation with CPPs via Cu(I)-catalysed azide-alkyne cycloaddition (CuAAC) or disulfide-mediated coupling were synthesised using solid-phase peptide synthesis. Circular dichroism and UV-melting analyses confirmed the formation of a stable, sequence-specific PNA-RNA heteroduplex with miRNA-221-5p. This demonstrated strong hybridisation and high thermodynamic stability compared to scrambled control sequences. Cell proliferation assays indicated modest but reproducible antiproliferative effects for the CPP-PNA conjugates co-incubated with chloroquine, with no evidence of acute cytotoxicity. These findings emphasise the potential of CPP-PNA systems for the intracellular delivery of antisense agents and highlight the importance of the linker nature in determining their biological performance.

Xanthostemon chrysanthus (F.Muell.) Benth. is a species from the Myrtaceae family with limited phytochemical and pharmacological studies. In this study, ten compounds (1-10) were isolated and purified from X. chrysanthus, among which compound 2, identified as des-O-methyllasiodiplodin (DML), exhibited the most potent anti-inflammatory activity. This is the first report of DML being isolated from this species. DML significantly inhibited lipopolysaccharide (LPS)-induced inflammation in RAW264.7 macrophages by reducing the expression of TNF-α, IL-1β, and IL-6, as well as nitric oxide (NO) and reactive oxygen species (ROS) production. Moreover, DML promoted autophagy by increasing LC3 II/LC3 I conversion and decreasing p62 expression, accompanied by suppression of the PI3K/AKT/mTOR signaling pathway. The autophagy inhibitor chloroquine (CQ) reversed these effects, confirming that DML exerts its anti-inflammatory action through autophagy activation. In a mouse ear edema model, DML effectively reduced inflammation, showing even greater efficacy than indomethacin. Collectively, this study provides the first evidence that DML isolated from X. chrysanthus exerts strong anti-inflammatory effects by promoting autophagy and modulation of the PI3K/AKT/mTOR pathway. These findings enrich the chemical and pharmacological understanding of X. chrysanthus and highlight DML as a promising natural candidate for the development of novel anti-inflammatory therapeutics.

Behavioral scoring is central to preclinical pain research but is typically performed manually, which is inherently low-throughput and subject to variability between observers. Automated alternatives exist, but cost and programming expertise limit adoption beyond the laboratories that build them. Here we describe PixelPaws, an open-source platform combining a near-infrared imaging enclosure built from consumer parts for under $300, multi-camera acquisition software, a bundled pose-estimation network, and eight classifiers in a graphical user interface. We evaluated the platform across standard assays of pain, itch, and opioid withdrawal. Licking, scratching, and jumping classifiers were validated against trained human observers by leave-one-session-out evaluation. In the formalin assay, classifier-scored licking followed the expected biphasic time course and was reduced dose-dependently by oxycodone, with a phase 2 potency estimate comparable to blinded manual scoring. Chloroquine roughly tripled scratching, and naloxone precipitated a burst of jumping in oxycodone-treated mice. From the same recordings we derived a set of paw contour metrics. Contour intensity fell after formalin and tracked the oxycodone dose series, and contour area shifted toward symmetry after oxycodone in nerve-injured mice. Behavioral sequencing revealed distinct changes in the transi.

As a ubiquitous organic pollutant in industrial settings, 1,2-dichloroethane (1,2-DCE) has been documented to induce cognitive impairment; however, the underlying molecular mechanisms remain poorly understood. In our animal experiments, mice were exposed to 0, 0.225, 0.45, and 0.9 g/m[3] of 1,2-DCE via static whole-body inhalation for 3.5 h daily over 56 days. Our results demonstrate that 56-day repeated 1,2-DCE exposure induces oxidative damage, activates autophagy, and promotes apoptosis in mouse hippocampal neural cells. We also observed impaired lysosomal function following 1,2-DCE treatment. On this basis, we hypothesized that 1,2-DCE disrupted autophagic flux, which was verified by blocked p62 degradation and an elevated LC3-II/I ratio, ultimately leading to neuronal apoptosis. These findings were further validated in vitro using the autophagy agonist rapamycin and the inhibitor chloroquine. In addition, 2-chloroethanol (2-CE), the primary metabolite of 1,2-DCE in vivo, increased p62 and LC3-II/I levels in PC12 cells in both dose- and time-dependent manners. Importantly, in vivo studies revealed that the ROS-JNK/c-Fos/c-Jun/FoxO1/FoxO3a axis acts as a critical upstream regulator, modulating transcription of core autophagy-related genes to initiate and enhance autophagy. These results were confirmed in vitro via pharmacological inhibition of ROS and JNK. Furthermore, activated JNK may promote autophagy through Bcl-2 phosphorylation, thereby facilitating subsequent apoptosis. Collectively, our study demonstrates for the first time that 1,2-DCE exposure triggers ROS-mediated and JNK-dependent autophagy, thereby inducing autophagic flux blockade and subsequent neuronal apoptosis.

The discovery of novel anti-malarial drugs has become urgent due to increasing drug resistance. Our previous work identified ONX-0914, which targets parasite proteolytic systems, as promising against Plasmodium falciparum. This study aims to further evaluate the antiplasmodial activity of ONX-0914 and related proteasome inhibitors (PIs) against various Plasmodium strains. The in vitro antimalarial activity of the inhibitors was evaluated against the Plasmodium knowlesi A1H1 strain and compared with that against the chloroquine (CQ)-sensitive P. falciparum 3D7 strain. Potent synergistic interactions between compounds and artemisinin (ART) were analyzed against PkA1H1 and ART-resistant PfDd2R539T(+) using an isobologram. To explore the mode of action, the disruption of the ubiquitin-proteasome system (UPS) in PkA1H1 and Pf3D7 was investigated. In vivo efficacy of ONX-0914 was further validated against CQ-resistant Plasmodium yoelii. PIs significantly inhibited PkA1H1 proliferation with low-nanomolar IC50 values; ONX-0914 and LU-005i were more potent against PkA1H1 than Pf3D7, and effectively blocked multiple blood stages. Inhibition of parasite growth correlated with potent disruption of UPS at nanomolar concentrations (IC50 < 50 nM). ONX-0914 demonstrated strong synergistic activity with ART and other PIs (∑FIC50 < 0.8). In vivo, ONX-0914 significantly suppressed P. yoelii infection, improving lifespan, with ⁓84% efficacy at an intraperitoneal (IP) dose of 20 mg/kg, and ⁓90% in combination with artemisinin at a dose of 10 mg/kg (IP). Further optimization of the structure and dosing regimen is able to enhance the therapeutic potential of PIs as potential antimalarial drug candidates.

We report a case of bilateral vortex keratopathy associated with long-term ribociclib therapy in a 67-year-old woman with metastatic breast cancer. The corneal deposits were identified during routine ophthalmic examination after 4 years of continuous treatment. Slit-lamp biomicroscopy revealed bilateral whorl-like epithelial deposits with a subtle morphological appearance compared with classic amiodarone-associated cornea verticillata. The patient had no history of tamoxifen, amiodarone, or chloroquine use, and there were no clinical features suggestive of Fabry disease. The combined severity of the bilateral cortical cataracts and macular changes associated with dry age-related macular degeneration was considered sufficient to explain the reduced visual acuity, whereas the subtle corneal deposits were not considered visually significant. The patient remained asymptomatic, and systemic therapy was continued without modification. This case suggests that ribociclib-associated vortex keratopathy may be detected after prolonged exposure and remain clinically silent at presentation, highlighting the importance of careful clinical assessment to avoid unnecessary interruption of essential oncologic treatment.