SARS-CoV-2 is a highly pathogenic virus responsible for the COVID-19 disease. It belongs to the Coronaviridae family, characterized by a phospholipid envelope, which is crucial for viral entry and replication in host cells. Hypericin, a lipophilic, naturally occurring photosensitizer, was reported to effectively inactivate enveloped viruses, including SARS-CoV-2, upon light irradiation. In addition to its photodynamic activity, Hyp was found to exert an antiviral action also in the dark. This study explores the mechanical properties of heat-inactivated SARS-CoV-2 viral particles using Atomic Force Microscopy (AFM). Results reveal a flexible structure under external stress, potentially contributing to the virus pathogenicity. Although the fixation protocol causes damage to some particles, correlation with fluorescence demonstrates colocalization of partially degraded virions with their genome. The impact of hypericin on the mechanical properties of the virus was assessed and found particularly relevant in dark conditions. These preliminary results suggest that hypericin can affect the mechanical properties of the viral envelope, an effect that warrants further investigation in the context of antiviral therapies.

In previous work, we reported on iridium(III) (Ir(III)) complex-peptide hybrids as amphiphilic conjugates (IPH-ACs) and triptycene-peptide hybrids as amphiphilic conjugates (TPH-ACs) and found that these hybrid compounds containing three cationic KK(K)GG peptide units through C6-C8 alkyl linkers induce paraptosis II, which is one of the nonapoptotic programmed cell death (PCD) types in Jurkat cells and different from previously reported paraptosis. The details of that study revealed that the paraptosis II induced by IPH-ACs (and TPH-ACs) proceeds via a membrane fusion or tethering of the endoplasmic reticulum (ER) and mitochondria, and Ca2+ transfer from the ER to mitochondria, which results in a loss of mitochondrial membrane potential (ΔΨm) in Jurkat cells. However, the detailed mechanistic studies of paraptosis II have been conducted only in Jurkat cells. In the present work, we decided to conduct mechanistic studies of paraptosis II in HeLa-S3 and A549 cells as well as in Jurkat cells to study the general mechanism of paraptosis II. Simultaneously, we designed and synthesized new TPH-ACs functionalized with peptides that contain cyclohexylalanine, which had been reported to enhance the localization of peptides to mitochondria. We found that TPH-ACs containing cyclohexylalanine promote paraptosis II processes in Jurkat, HeLa-S3 and A549 cells. The results of the experiments using fluorescence Ca2+ probes in mitochondria and cytosol, fluorescence staining agents of mitochondria and the ER, and inhibitors of paraptosis II suggest that TPH-ACs induce Ca2+ increase in mitochondria and the membrane fusion between the ER and mitochondria almost simultaneously, suggesting that our previous hypothesis on the mechanism of paraptosis II should be revised.

The lipopolyplex, a multicomponent nonviral gene carrier, generally demonstrates superior colloidal stability, reduced cytotoxicity, and high transfection efficiency. In this study, a new concept, photochemical reaction-induced transfection, using photosensitizer (PS)-loaded lipopolyplexes was applied, which led to enhanced transfection and cytotoxic effects by photoexcitation of the photosensitizer. Hypericin, a hydrophobic photosensitizer, was encapsulated in the lipid bilayer of liposomes. The preformed nanosized hypericin liposomes enclosed the linear polyethylenimine (lPEI)/pDNA polyplexes, resulting in the formation of hypericin lipopolyplexes (Hy-LPP). The diameters of Hy-LPP containing 50 nM hypericin and 0.25 μg of pDNA were 185.6 ± 7.74 nm and 230.2 ± 4.60 nm, respectively, measured by dynamic light scattering (DLS) and atomic force microscopy (AFM). Gel electrophoresis confirmed the encapsulation of hypericin and pDNA in lipopolyplexes. Furthermore, in vitro irradiation of intracellular Hy-LPP at radiant exposures of 200, 600, and 1000 mJ/cm2 was evaluated. It demonstrated 60- to 75-fold higher in vitro luciferase expression than that in nonirradiated cells. The lactate dehydrogenase (LDH) assay supported that reduced transfection was a consequence of photocytotoxicity. The developed photosensitizer-loaded lipopolyplexes improved the transfection efficiency of an exogenous gene or induced photocytotoxicity; however, the frontier lies in the applied photochemical dose. The light-triggered photoexcitation of intracellular hypericin resulted in the generation of reactive oxygen species (ROS), leading to photoselective transfection in HepG2 cells. It was concluded that the two codelivered therapeutics resulted in enhanced transfection and a photodynamic effect by tuning the applied photochemical dose.

The polycyclic aromatic hydrocarbon content of water (four surface water, six underground water (borehole water), seven sachet water), barbecued food and their fresh equivalents (barbecued beef, fish, plantain, pork, yam, chicken, chevon, potato, corn), oil (three palm oil, nine vegetable oil), and fresh vegetable samples (water leaf, bitter leaf, cabbage, carrot, cucumber, pumpkin, garlic, ginger, green leaf, Gnetum Africana, onion, pepper) were determined by GC-MS analysis. The current study also determined the estimated lifetime cancer risk from ingesting polycyclic aromatic hydrocarbon-contaminated food. The polycyclic aromatic hydrocarbon content of water, oil, vegetable, and food samples were within the United States Environmental Protection Agency/World Health Organization safe limits. The naphthalene, benzo(b)fluoranthene, and benzo(k)fluoranthene levels in surface water were significantly higher than in borehole samples (P = 0.000, 0.047, 0.047). Vegetable oils had higher anthracene and chrysene compared to palm oil (P = 0.023 and 0.032). Significant variations were observed in levels of naphthalene, acenaphthylene, phenanthrene, benzo(b)fluoranthene, benzo(k)fluoranthene, benzo(a)pyrene, and dibenzo(a,h)anthracene among the barbecued and fresh food samples (P <0.05). Barbecued pork, potato, and corn had significantly higher naphthalene compared to their fresh equivalents (P = 0.002, 0.017, and <0.001). Consumption of barbecued food and surface water may be associated with higher exposure risk to polycyclic aromatic hydrocarbons which may predispose to increased cancer health risk. The current work explores in depth the concentration of polycyclic aromatic hydrocarbons in different dietary categories that pose direct risk to humans via direct consumption. These findings add knowledge to support future considerations for human health.

Cardiac fibrosis is a severe outcome of Chagas disease (CD), caused by the protozoan Trypanosoma cruzi. Clinical evidence revealed a correlation between fibrosis levels with impaired cardiac performance in CD patients. Therefore, we sought to analyze the effect of inhibitors of TGF-β (pirfenidone), p38-MAPK (losmapimod) and c-Jun (SP600125) on the modulation of collagen deposition in cardiac fibroblasts (CF) and in vivo models of T. cruzi chronic infection. Sirius Red/Fast Green dye was used to quantify both collagen expression and total protein amount, assessing cytotoxicity. The compounds were also used to treat C57/Bl6 mice chronically infected with T. cruzi, Brazil strain. We identified an anti-fibrotic effect in vitro for pirfenidone (TGF-β inhibitor, IC50 114.3 μM), losmapimod (p38 inhibitor, IC50 17.6 μM) and SP600125 (c-Jun inhibitor, IC50 3.9 μM). This effect was independent of CF proliferation since these compounds do not affect T. cruzi-induced host cell multiplication as measured by BrdU incorporation. Assays of chronic infection of mice with T. cruzi have shown a reduction in heart collagen by pirfenidone. These results propose a novel approach to fibrosis therapy in CD, with the prospect of repurposing pirfenidone to prevent the onset of ECM accumulation in the hearts of the patients.

BACKGROUND: Adult skeletal muscle contains resident muscle stem cells (MuSC) with high myogenic and engraftment potentials, making them suitable for cell therapy and regenerative medicine approaches. However, purification process of MuSC remains a major hurdle to their use in the clinic. Indeed, muscle tissue enzymatic dissociation triggers a massive activation of stress signaling pathways, among which P38 and JNK MAPK, associated with a premature loss of MuSC quiescence. While the role of these pathways in the myogenic progression of MuSC is well established, the extent to which their dissociation-induced activation affects the functionality of these cells remains unexplored.
METHODS: We assessed the effect of P38 and JNK MAPK induction on stemness marker expression and MuSC activation state during isolation by pharmacological approaches. MuSC functionality was evaluated by in vitro assays and in vivo transplantation experiments. We performed a comparative analysis of the transcriptome of human MuSC purified with pharmacological inhibitors of P38 and JNK MAPK (SB202190 and SP600125, respectively) versus available RNAseq resources.
RESULTS: We monitored PAX7 protein levels in murine MuSC during muscle dissociation and demonstrated a two-step decline partly dependent on P38 and JNK MAPK activities. We showed that simultaneous inhibition of these pathways throughout the MuSC isolation process preserves the expression of stemness markers and limits their premature activation, leading to improved survival and amplification in vitro as well as increased engraftment in vivo. Through a comparative RNAseq analysis of freshly isolated human MuSC, we provide evidence that our findings in murine MuSC could be relevant to human MuSC. Based on these findings, we implemented a purification strategy, significantly improving the recovery yields of human MuSC.
CONCLUSIONS: Our study highlights the pharmacological limitation of P38 and JNK MAPK activities as a suitable strategy to qualitatively and quantitatively ameliorate human MuSC purification process, which could be of great interest for cell-based therapies.

BACKGROUND: The search for new bioactive natural compounds with anticancer activity is still of great importance. Even though their potential for diagnostics and treatment of cancer has already been proved, the availability is still limited. Hypericin, a naphthodianthrone isolated essentially from plant source Hypericum perforatum L. along with other related anthraquinones and bisanthraquinones belongs to this group of compounds. Although it has been proven that hypericin is synthesized by the polyketide pathway in plants, none of the candidate genes coding for key enzymes has been experimentally validated yet. Despite the rare occurrence of anthraquinones in plants, their presence in microorganisms, including endophytic fungi, is quite common. Unlike plants, several biosynthetic genes grouped into clusters (BGCs) in fungal endophytes have already been characterized.
RESULTS: The aim of this work was to predict, identify and characterize the anthraquinone BGCs in de novo assembled and functionally annotated genomes of selected endophytic fungal isolates (Fusarium oxysporum, Plectosphaerella cucumerina, Scedosporium apiospermum, Diaporthe eres, Canariomyces subthermophilus) obtained from different tissues of Hypericum spp. The number of predicted type I polyketide synthase (PKS) BGCs in the studied genomes varied. The non-reducing type I PKS lacking thioesterase domain and adjacent discrete gene encoding protein with product release function were identified only in the genomes of C. subthermophilus and D. eres. A candidate bisanthraquinone BGC was predicted in C. subthermophilus genome and comprised genes coding the enzymes that catalyze formation of the basic anthraquinone skeleton (PKS, metallo-beta-lactamase, decarboxylase, anthrone oxygenase), putative dimerization enzyme (cytochrome P450 monooxygenase), other tailoring enzymes (oxidoreductase, dehydrogenase/reductase), and non-catalytic proteins (fungal transcription factor, transporter protein).
CONCLUSIONS: The results provide an insight into genetic background of anthraquinone biosynthesis in Hypericum-borne endophytes. The predicted bisanthraquinone gene cluster represents a basis for functional validation of the candidate biosynthetic genes in a simple eukaryotic system as a prospective biotechnological alternative for production of hypericin and related bioactive anthraquinones.

The persistence and ubiquity of polycyclic aromatic hydrocarbons (PAHs) in the environment necessitate effective remediation strategies. Hence, this study investigated the potential of purified Laccases, TlFLU1L and TpFLU12L, from two indigenous fungi Trichoderma lixii FLU1 (TlFLU1) and Talaromyces pinophilus FLU12 (TpFLU12), respectively for the oxidation and detoxification of anthracene. Anthracene was degraded with vmax values of 3.51 ± 0.06 mg/L/h and 3.44 ± 0.06 mg/L/h, and Km values of 173.2 ± 0.06 mg/L and 73.3 ± 0.07 mg/L by TlFLU1L and TpFLU12L, respectively. The addition of a mediator compound 2,2-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) to the reaction system significantly increased the degradation of anthracene, with up to a 2.9-fold increase in vmax value and up to threefold decrease in Km values of TlFLU1L and TpFLU12L. The GC-MS analysis of the metabolites suggests that anthracene degradation follows one new pathway unique to the ABTS system-hydroxylation and carboxylation of C-1 and C-2 position of anthracene to form 3-hydroxy-2-naphthoic acid, before undergoing dioxygenation and side chain removal to form chromone which was later converted into benzoic acid and CO2. This pathway contrasts with the common dioxygenation route observed in the free Laccase system, which is observed in the second degradation pathways. Furthermore, toxicity tests using V. parahaemolyticus and HT-22 cells, respectively, demonstrated the non-toxic nature of Laccase-ABTS-mediated metabolites. Intriguingly, analysis of the expression level of Alzheimer\'s related genes in HT-22 cells exposed to degradation products revealed no induction of neurotoxicity unlike untreated cells. These findings propose a paradigm shift for bioremediation by highlighting the Laccase-ABTS system as a promising green technology due to its efficiency with the discovery of a potentially less harmful degradation pathway, and the production of non-toxic metabolites.

Glioblastoma multiforme, a highly aggressive and lethal brain tumor, is a substantial clinical challenge and a focus of increasing concern globally. Hematological toxicity and drug resistance of first-line drugs underscore the necessity for new anti-glioma drug development. Here, 43 anthracenyl skeleton compounds as p53 activator XI-011 analogs were designed, synthesized, and evaluated for their cytotoxic effects. Five compounds (13d, 13e, 14a, 14b, and 14n) exhibited good anti-glioma activity against U87 cells, with IC50 values lower than 2 μM. Notably, 13e showed the best anti-glioma activity, with an IC50 value up to 0.53 μM, providing a promising lead compound for new anti-glioma drug development. Mechanistic analyses showed that 13e suppressed the MDM4 protein expression, upregulated the p53 protein level, and induced cell cycle arrest at G2/M phase and apoptosis based on Western blot and flow cytometry assays.

Familial Alzheimer\'s disease (FAD) is a complex and multifactorial neurodegenerative disorder for which no curative therapies are yet available. Indeed, no single medication or intervention has proven fully effective thus far. Therefore, the combination of multitarget agents has been appealing as a potential therapeutic approach against FAD. Here, we investigated the potential of combining tramiprosate (TM), curcumin (CU), and the JNK inhibitor SP600125 (SP) as a treatment for FAD. The study analyzed the individual and combined effects of these two natural agents and this pharmacological inhibitor on the accumulation of intracellular amyloid beta iAβ; hyperphosphorylated protein TAU at Ser202/Thr205; mitochondrial membrane potential (ΔΨm); generation of reactive oxygen species (ROS); oxidized protein DJ-1; proapoptosis proteins p-c-JUN at Ser63/Ser73, TP53, and cleaved caspase 3 (CC3); and deficiency in acetylcholine (ACh)-induced transient Ca2+ influx response in cholinergic-like neurons (ChLNs) bearing the mutation I416T in presenilin 1 (PSEN1 I416T). We found that single doses of TM (50 μM), CU (10 μM), or SP (1 μM) were efficient at reducing some, but not all, pathological markers in PSEN 1 I416T ChLNs, whereas a combination of TM, CU, and SP at a high (50, 10, 1 μM) concentration was efficient in diminishing the iAβ, p-TAU Ser202/Thr205, DJ-1Cys106-SO3, and CC3 markers by -50%, -75%, -86%, and -100%, respectively, in PSEN1 I417T ChLNs. Although combinations at middle (10, 2, 0.2) and low (5, 1, 0.1) concentrations significantly diminished p-TAU Ser202/Thr205, DJ-1Cys106-SO3, and CC3 by -69% and -38%, -100% and -62%, -100% and -62%, respectively, these combinations did not alter the iAβ compared to untreated mutant ChLNs. Moreover, a combination of reagents at H concentration was able to restore the dysfunctional ACh-induced Ca2+ influx response in PSEN 1 I416T. Our data suggest that the use of multitarget agents in combination with anti-amyloid (TM, CU), antioxidant (e.g., CU), and antiapoptotic (TM, CU, SP) actions might be beneficial for reducing iAβ-induced ChLN damage in FAD.