CRISPR-Mediated Library Screening of Gene-Knockout Cell Lines for Investigating Antiviral Innate Immunity.

The application of CRISPR-mediated library screening has fundamentally transformed functional genomics by revealing the complexity of virus-host interactions. This protocol describes the use of CRISPR-mediated library screening to identify key functional genes regulating the innate immune response to PEDV infection. We detail a step-by-step process, starting from the design and construction of a customized CRISPR knockout library targeting genes involved in innate immunity to the effective delivery of these constructs into cells using lentiviral vectors. Subsequently, we outline the process of identifying functional genes postviral attack, including the use of next-generation sequencing (NGS), to analyze and identify knockout cells that exhibit altered responses to infection. This integrated approach provides researchers in immunology and virology with a resource and a robust framework for uncovering the genetic basis of host-pathogen interactions and the arsenal of the innate immune system against viral invasions.

"All in one" lipid-polymer nanodelivery system for gene therapy of ischemic diseases.

Gene therapy offers a promising avenue for treating ischemic diseases, yet its clinical efficacy is hindered by the limitations of single gene therapy and the high oxidative stress microenvironment characteristic of such conditions. Lipid-polymer hybrid vectors represent a novel approach to enhance the effectiveness of gene therapy by harnessing the combined advantages of lipids and polymers. In this study, we engineered lipid-polymer hybrid nanocarriers with tailored structural modifications to create a versatile membrane fusion lipid-nuclear targeted polymer nanodelivery system (FLNPs) optimized for gene delivery. Our results demonstrate that FLNPs facilitate efficient cellular uptake and gene transfection via membrane fusion, lysosome avoidance, and nuclear targeting mechanisms. Upon encapsulating Hepatocyte Growth Factor plasmid (pHGF) and Catalase plasmid (pCAT), HGF/CAT-FLNPs were prepared, which significantly enhanced the resistance of C2C12 cells to H2O2-induced injury in vitro. In vivo studies further revealed that HGF/CAT-FLNPs effectively alleviated hindlimb ischemia-induced gangrene, restored motor function, and promoted blood perfusion recovery in mice. Metabolomics analysis indicated that FLNPs didn't induce metabolic disturbances during gene transfection. In conclusion, FLNPs represent a versatile platform for multi-dimensional assisted gene delivery, significantly improving the efficiency of gene delivery and holding promise for effective synergistic treatment of lower limb ischemia using pHGF and pCAT.

Perilla frutescens leaf extracts alleviate acute lung injury in mice by inhibiting KAT2A.

ETHNOPHARMACOLOGICAL RELEVANCE: Acute lung injury (ALI) can lead to respiratory failure and even death. KAT2A is a key target to suppress the development of inflammation. A herb, perilla frutescens, is an effective treatment for pulmonary inflammatory diseases with anti-inflammatory effects; however, its mechanism of action remains unclear. AIM OF THE STUDY: The purpose of this study was to investigate the therapeutic effect and underlying mechanism of perilla frutescens leaf extracts (PLE), in the treatment of ALI by focusing on its ability to treat inflammation. MATERIALS AND METHODS: In vivo and in vitro models of ALI induced by LPS. Respiratory function, histopathological changes of lung, and BEAS-2B cells damage were assessed upon PLE. This effect is also tested under conditions of KAT2A over expression and KAT2A silencing. RESULTS: PLE significantly attenuated LPS-induced histopathological changes in the lungs, improved respiratory function, and increased survival rate from LPS stimuation background in mice. PLE remarkably suppressed the phosphorylation of STAT3, AKT, ERK (1/2) and the release of cytokines (IL-6, TNF-α, and IL-1ß) induced by LPS via inhibiting the expression of KAT2A. CONCLUSIONS: PLE has a dose-dependent anti-inflammatory effect by inhibiting KAT2A expression to suppress LPS-induced ALI n mice. Our study expands the clinical indications of the traditional medicine PLE and provide a theoretical basis for clinical use of acute lung injury.

Sea buckthorn extract mitigates chronic obstructive pulmonary disease by suppression of ferroptosis via scavenging ROS and blocking p53/MAPK pathways.

ETHNOPHARMACOLOGICAL RELEVANCE: Sea buckthorn (Hippophae rhamnoides), a traditional Tibetan medicinal herb, exhibits protective effects against cardiovascular and respiratory diseases. Although Sea buckthorn extract (SBE) has been confirmed to alleviate airway inflammation in mice, its therapeutic effect and underlying mechanism on chronic obstructive pulmonary disease (COPD) requires further clarification. AIM OF THE STUDY: To elucidate the alleviative effect and molecular mechanism of SBE on lipopolysaccharides (LPS)/porcine pancreatic elastase (PPE)-induced COPD by blocking ferroptosis. METHODS: The anti-ferroptotic effects of SBE were evaluated in human BEAS-2B bronchial epithelial cells using CCK8, RT-qPCR, western blotting, and transmission electron microscopy. Transwell was employed to detect chemotaxis of neutrophils. COPD model was induced by intranasally administration of LPS/PPE in mice and measured by alterations of histopathology, inflammation, and ferroptosis. RNA-sequencing, western blotting, antioxidant examination, flow cytometry, DARTS, CETSA, and molecular docking were then used to investigate its anti-ferroptotic mechanisms. RESULTS: In vitro, SBE not only suppressed erastin- or RSL3-induced ferroptosis by suppressing lipid peroxides (LPOs) production and glutathione (GSH) depletion, but also suppressed ferroptosis-induced chemotactic migration of neutrophils via reducing mRNA expression of chemokines. In vivo, SBE ameliorated LPS/PPE-induced COPD phenotypes, and inhibited the generation of LPOs, cytokines, and chemokines. RNA-sequencing showed that p53 pathway and mitogen-activated protein kinases (MAPK) pathway were implicated in SBE-mediated anti-ferroptotic action. SBE repressed erastin- or LPS/PPE-induced overactivation of p53 and MAPK pathway, thereby decreasing expression of diamine acetyltransferase 1 (SAT1) and arachidonate 15-lipoxygenase (ALOX15), and increasing expression of glutathione peroxidase 4 (GPX4) and solute carrier family 7 member 11 (SLC7A11). Mechanistically, erastin-induced elevation of reactive oxygen species (ROS) was reduced by SBE through directly scavenging free radicals, thereby contributing to its inhibition of p53 and MAPK pathways. CETSA, DARTS, and molecular docking further showed that ROS-generating enzyme nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 4 (NOX4) may be the target of SBE. Overexpression of NOX4 partially impaired the anti-ferroptotic activity of SBE. CONCLUSION: Our results demonstrated that SBE mitigated COPD by suppressing p53 and MAPK pro-ferroptosis pathways via directly scavenging ROS and blocking NOX4. These findings also supported the clinical application of Sea buckthorn in COPD therapy.

A novel glycopeptide from mountain-cultivated ginseng residue protects type 2 diabetic symptoms-induced heart failure.

ETHNOPHARMACOLOGICAL RELEVANCE: Mountain-cultivated Panax ginseng C.A.Mey. (MCG) with high market price and various properties was valuable special local product in Northeast of Asia. MCG has been historically used to mitigate heart failure (HF) for thousand years, HF is a clinical manifestation of deficiency of "heart-qi" in traditional Chinese medicine. However, there was little report focus on the activities of extracted residue of MCG. AIM OF THE STUDY: A novel glycopeptide (APMCG-1) was isolated from step ethanol precipitations of alkaline protease-assisted extract from MCG residue. MATERIALS AND METHODS: The molecular weight and subunit structure of APMCG-1 were determined by FT-IR, HPLC and GPC technologies, as well as the H9c2 cells, Tg (kdrl:EGFP) zebrafish were performed to evaluated the protective effect of APMCG-1. RESULTS: APMCG-1 was identified as a glycopeptide containing seven monosaccharides and seven amino acids via O-lined bonds. Further, in vitro, APMCG-1 significantly decreased reactive oxygen species production and lactate dehydrogenase contents in palmitic acid (PA)-induced H9c2 cells. APMCG-1 also attenuated endoplasmic reticulum stress and mitochondria-mediated apoptosis in H9c2 cells via the PI3K/AKT signaling pathway. More importantly, APMCG-1 reduced the blood glucose, lipid contents, the levels of heart injury, oxidative stress and inflammation of 5 days post fertilization Tg (kdrl:EGFP) zebrafish with type 2 diabetic symptoms in vivo. CONCLUSIONS: APMCG-1 protects PA-induced H9c2 cells while reducing cardiac dysfunction in zebrafish with type 2 diabetic symptoms. The present study provides a new insight into the development of natural glycopeptides as heart-related drug therapies.

Dachengqi decoction dispensing granule ameliorates LPS-induced acute lung injury by inhibiting PANoptosis in vivo and in vitro.

ETHNOPHARMACOLOGICAL RELEVANCE: Acute lung injury (ALI) is a serious health-threatening syndrome of intense inflammatory response in the lungs, with progression leading to acute respiratory distress syndrome (ARDS). Dachengqi decoction dispensing granule (DDG) has a pulmonary protective role, but its potential modulatory mechanism to alleviate ALI needs further excavation. AIM OF THE STUDY: This study aims to investigate the effect and potential mechanism of DDG on lipopolysaccharide (LPS)-induced ALI models in vivo and in vitro. MATERIALS AND METHODS: LPS-treated Balb/c mice and BEAS-2B cells were used to construct in vivo and in vitro ALI models, respectively. Hematoxylin-eosin (HE), Wet weight/Dry weight (W/D) calculation of lung tissue, and total protein and Lactic dehydrogenase (LDH) assays in BALF were performed to assess the extent of lung tissue injury and pulmonary edema. Enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of tumor necrosis factor-alpha (TNF-α), interleukin-1ß (IL-1ß), and interleukin-18 (IL-18) in BALF, serum, and cell supernatant. The qRT-PCR was used to detect inflammatory factors, Z-DNA binding protein 1 (ZBP1), and receptor-interacting protein kinase 1 (RIPK1) expression in lung tissues and BEAS-2B cells. Double immunofluorescence staining and co-immunoprecipitation were used to detect the relative expression and co-localization of ZBP1 and RIPK1. The effects of LPS and DDG on BEAS-2B cell activity were detected by Cell Counting Kit-8 (CCK-8). Western blot (WB) was performed to analyze the expression of PANoptosis-related proteins in lung tissues and BEAS-2B cells. RESULTS: In vivo, DDG pretreatment could dose-dependently improve the pathological changes of lung tissue in ALI mice, and reduce the W/D ratio of lung, total protein concentration, and LDH content in BALF. In vitro, DDG reversed the inhibitory effect of LPS on BEAS-2B cell viability. Meanwhile, DDG significantly reduced the levels of inflammatory factors in vitro and in vivo. In addition, DDG could inhibit the expression levels of PANoptosis-related proteins, especially the upstream key regulatory molecules ZBP1 and RIPK1. CONCLUSION: DDG could inhibit excessive inflammation and PANoptosis to alleviate LPS-induced ALI, thus possessing good anti-inflammatory and lung-protective effects. This study establishes a theoretical basis for the further development of DDG and provides a new prospect for ALI treatment by targeting PANoptosis.

In vivo and in vitro study on the regulatory mechanism of XiaoChaiHu decoction on PANoptosis in sepsis-induced cardiomyopathy.

ETHNOPHARMACOLOGICAL RELEVANCE: In accordance with the tenets of traditional Chinese medicine, sepsis is categorized into three distinct syndromes: heat syndrome, blood stasis syndrome, and deficiency syndrome. Xiaochaihu decoction (XCHD) has many functions, including the capacity to protect the liver, cholagogue, antipyretic, anti-inflammatory, and anti-pathogenic microorganisms. XCHD exerts the effect of clearing heat and reconciling Shaoyang. The XCHD contains many efficacious active ingredients, yet the mechanism of sepsis-induced cardiomyopathy (SIC) remains elusive. AIM OF THE STUDY: To investigate the molecular mechanisms underlying the protective effects of XCHD against SIC using an integrated approach combining network pharmacology and molecular biology techniques. MATERIALS AND METHODS: Network pharmacology methods identified the active ingredients, target proteins, and pathways affected by XCHD in the context of SIC. We conducted in vivo experiments using mice with lipopolysaccharide-induced SIC, evaluating cardiac function through echocardiography and histology. XCHD-containing serum was analyzed to determine its principal active components using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The effects of XCHD-containing serum on SIC were further tested in vitro in LPS-treated H9c2 cardiac cells. Protein expression levels were quantified via Western blotting and enzyme-linked immunosorbent assay (ELISA). Additionally, molecular docking was performed between the active components and ZBP1, a potential target protein. Overexpression of ZBP1 in H9c2 cells allowed for a deeper exploration of its role in modulating SIC-associated gene expression. RESULTS: UPLC-MS/MS identified 31 shared XCHD and XCHD-containing serum components. These included organic acids, terpenoids, and flavonoids, which have been identified as the active components of XCHD. Our findings revealed that XCHD alleviated LPS-induced myocardial injury, improved cardiac function, and preserved cardiomyocyte morphology in mice. In vitro studies, we demonstrated that XCHD-containing serum significantly suppressed the expression of inflammatory cytokines (IL-6, IL-1ß, and TNF-α) in LPS-induced H9c2 cells. Mechanistic investigations showed that XCHD downregulated genes associated with PANoptosis, a novel cell death pathway, suggesting its protective role in sepsis-damaged hearts. Conversely, overexpression of ZBP1 abolished the protective effects of XCHD and amplified PANoptosis-related gene expression. CONCLUSIONS: Our study provides the first evidence supporting the protective effects of XCHD against SIC, both in vitro and in vivo. The underlying mechanism involves the inhibition of ZBP1-initiated PANoptosis, offering new insights into treating SIC using XCHD.

Integration of network pharmacology and experimental verification to reveal the active components and molecular mechanism of modified Danzhi Xiaoyao San in the treatment of depression.

ETHNOPHARMACOLOGICAL RELEVANCE: Modified Danzhi Xiaoyao San (MDXS) is an effective clinical prescription for depression in China, which was deprived of Danzhi Xiaoyao San in the Ming Dynasty. MDSX has significant implications for the development of new antidepressants, but its pharmacological mechanism has been rarely studied. AIM OF THE STUDY: To reveal the active components and molecular mechanism of MDXS in treating depression through network pharmacology and experimental verification in vivo and in vitro. MATERIALS AND METHODS: UPLC-Q-TOF-MS/MS was used to identify the chemical components in the MDXS freeze-dried powder, drug-containing serum, and cerebrospinal fluid (CSF). Based on the analysis of prototype components in the CSF, the major constituents, potential therapeutic targets and possible pharmacological mechanisms of MDXS in treating depression were investigated using network pharmacological and molecular docking. Then corticosterone (CORT)-induced mice model of depression was established to investigate the antidepressant effects of MDXS. HT22 cells were cultured to verify the neuroprotective effects and core targets of the active components. RESULTS: There were 81 compounds in MDXS freeze-dried powder, 36 prototype components in serum, and 13 prototype components in CSF were identified, respectively. Network pharmacology analysis showed that these 13 prototype components in the CSF shared 190 common targets with depression, which were mainly enriched in MAPK and PI3K/AKT signaling pathways. PPI analysis suggested that AKT1 and MAPK1 (ERK1/2) were the core targets. Molecular docking revealed that azelaic acid (AA), senkyunolide A (SA), atractylenolide III (ATIII), and tokinolide B (TB) had the highest binding energy with AKT1 and MAPK1. Animal experiments verified that MDXS could reverse CORT-induced depression-like behaviors, improve synaptic plasticity, alleviate neuronal injury in hippocampal CA3 regions, and up-regulate the protein expression of p-ERK1/2 and p-AKT. In HT22 cells, azelaic acid, senkyunolide A, and atractylenolide III significantly protected the cell injury caused by CORT, and up-regulated the protein levels of p-ERK1/2 and p-AKT. CONCLUSIONS: These results suggested that MDXS may exert antidepressant effects partially through azelaic acid, senkyunolide A, and atractylenolide III targeting ERK1/2 and AKT.

Exploring the wound healing potential of Lobostemon fruticosus using in vitro and in vivo bioassays.

ETHNOPHARMACOLOGICAL RELEVANCE: Lobostemon fruticosus (L.) H.Buek is a perennial and woody shrub of the Boraginaceae family, found in the Cape region of South Africa. The leaves and twigs are used to treat dermatological conditions such as wounds, burns, ringworm, erysipelas and eczema. Anti-inflammatory, antibacterial, antiviral and anti-proliferative activities of L. fruticosus have been reported. However, there is a void in research which reports on the wound healing properties of this plant. AIM OF THE STUDY: Aligned with the traditional use of L. fruticosus, our study aimed to use in vitro and in vivo bioassays to confirm the wound healing potential of the plant. MATERIALS AND METHODS: An aqueous methanol extract (80% v/v) of L. fruticosus was prepared using a sample collected from the Western Cape Province of South Africa and chromatographically profiled by ultra-performance liquid chromatography coupled to mass spectrometry (UPLC-MS). The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cytotoxicity assay was performed to determine the non-toxic concentrations of the extract for subsequent use in the in vitro scratch assay. Both the human keratinocyte (HaCaT) and fibroblast (BJ-5ta) cell lines were employed in the in vitro scratch assay. The in vivo caudal fin amputation assay was used to assess the wound healing potential of L. fruticosus, by monitoring fin regeneration in zebrafish larvae treated with the plant extract at various concentrations. RESULTS: Six major compounds were tentatively identified in the L. fruticosus extract namely; globoidnan A, globoidnan B, rutin, rabdosiin, sagerinic acid and rosmarinic acid. The potentially toxic pyrrolizidine alkaloids were also identified and quantitatively confirmed to be present at a low concentration of 119.58 ppm (m/m). Treatment of HaCaT and BJ-5ta cells with the plant extract in the scratch assay resulted in an increase in cell migration, which translates to accelerated wound closure. After 24 hr treatment with 100 µg/mL of extract, wound closure was recorded to be 91.1 ± 5.7% and 94.1 ± 1.3% for the HaCaT and BJ-5ta cells, respectively, while the untreated (medium) controls showed 72.3 ± 3.3% and 73.0 ± 4.3% for the two cell lines, respectively. Complete wound closure was observed between 24 and 36 hr, while the untreated control group did not achieve 100% wound closure by the end of the observation period (48 hr). In vivo, the crude extract at 100 µg/mL accelerated zebrafish caudal fin regeneration achieving 100.5 ± 3.8% regeneration compared to 68.3 ± 6.6% in the untreated control at two days post amputation. CONCLUSIONS: The study affirms the wound healing properties, as well as low toxicity of L. fruticosus using both in vitro and in vivo assays, which supports the traditional medicinal use. Other in vitro assays that target different mechanisms involved in wound healing should be investigated to support the current findings.

Exosomal let-7b-5p deriving from parietal epithelial cells attenuate renal fibrosis through suppression of TGFßR1 and ARID3a in obstructive kidney disease.

As renal progenitor cells, parietal epithelial cells (PECs) have demonstrated multilineage differentiation potential in response to kidney injury. However, the function of exosomes derived from PECs has not been extensively explored. Immunofluorescent staining of Claudin-1 was used to identify primary PECs isolated from mouse glomeruli. Transmission electron microscopy, nanoparticle tracking analysis, and western blotting were used to characterize the properties of PECs-derived exosomes (PEC-Exo). The therapeutic role of PEC-Exo in tubulointerstitial fibrosis was investigated in the unilateral ureteral obstruction (UUO) mouse model and TGF-ß1-stimulated HK-2 cells. High-throughput miRNA sequencing was employed to profile PEC-Exo miRNAs. One of the most enriched miRNAs in PEC-Exo was knocked down by transfecting miRNA inhibitor, and then we investigated whether this candidate miRNA was involved in PEC-Exo-mediated tubular repair. The primary PECs expressed Claudin-1, PEC-Exo was homing to obstructed kidney, and TGF-ß1 induced HK-2 cells. PEC-Exo significantly alleviated renal inflammation and ameliorated tubular fibrosis both in vivo and in vitro. Mechanistically, let-7b-5p, highly enriched in PEC-Exo, downregulated the protein levels of transforming growth factor beta receptor 1(TGFßR1) and AT-Rich Interaction Domain 3A(ARID3a) in tubular epithelial cells (TECs), leading to the inhibition of p21 and p27 to restoring cell cycle. Furthermore, administration of let-7b-5p agomir mitigated renal fibrosis in vivo. Our findings demonstrated that PEC-derived exosomes significantly repressed the expression of TGFßR1 and ARID3a by delivering let-7b-5p, thereby alleviating renal fibrosis. This study provides novel insights into the role of PEC-Exo in the repair of kidney injury and new ideas for renal fibrosis intervention.

Comparative evaluation of cigarette smoke and a heated tobacco product on microglial toxicity, oxidative stress and inflammatory response.

BACKGROUND: Tobacco smoking is the leading cause of preventable death and disease worldwide, with over 8 million annual deaths attributed to cigarette smoking. This study investigates the impact of cigarette smoke and heated tobacco products (HTPs) on microglial function, focusing on toxicological profiles, inflammatory responses, and oxidative stress using ISO standard and clinically relevant conditions of exposure. METHODS: We assessed cell viability, reactive oxygen species (ROS) production, lipid peroxidation, mitochondrial function, unfolded protein response, and inflammation in human microglial cells (HMC3) exposed to cigarette smoke, HTP aerosol or nicotine. RESULTS: Our findings show that cigarette smoke significantly reduces microglial viability, increases ROS formation, induces lipid peroxidation, and reduces intracellular glutathione levels. Cigarette smoke also alters the expression of genes involved in mitochondrial dynamics and biogenesis, leading to mitochondrial dysfunction. Additionally, cigarette smoke impairs the unfolded protein response, activates the NF-κB pathway, and induces a pro-inflammatory state characterized by increased TNF and IL-18 expression. Furthermore, cigarette smoke causes DNA damage and decreases the expression of the aging marker Klotho ß. In contrast, HTP, exhibited a lesser degree of microglial toxicity, with reduced ROS production, lipid peroxidation, and mitochondrial dysfunction compared to conventional cigarettes. CONCLUSION: These results highlight the differential toxicological profile of cigarette smoke and HTP on microglial cells, suggesting a potential harm reduction strategy for neurodegenerative disease for smokers unwilling or unable to quit.

Intracellular delivery of antiviral shRNA using penetratin-based complexes effectively inhibits respiratory syncytial virus replication and host cell apoptosis.

BACKGROUND: Cell-penetrating peptides (CPPs) are effective for delivering therapeutic molecules with minimal toxicity. This study focuses on the use of penetratin, a well-characterized CPP, to deliver a DNA vector encoding short hairpin RNA (shRNA) targeting the respiratory syncytial virus (RSV) F gene into infected cells. RSV is known to cause severe lower respiratory infections in infants and poses significant risks to immunocompromised individuals and the elderly. We evaluated the antiviral efficacy of the penetratin-shRNA complex by comparing its ability to inhibit RSV replication and induce apoptosis with ribavirin treatment. METHODS: Penetratin-shRNA complexes were prepared at different ratios and analyzed using gel retardation assays, dynamic light scattering, and zeta potential measurements. The complexes were tested in HEp-2 and A549 cells for transfection efficiency, cytotoxicity, viral load, and apoptosis using plaque assays, real-time reverse transcription-polymerase chain reaction (RT-PCR), DNA fragmentation, propidium iodide staining, and caspase 3/7 activation assays. RESULTS: The gel shift assay determined that a 20:1 CPP-to-shRNA ratio was optimal for effective complexation, resulting in particles with a size of 164 nm and a zeta potential of 8.7 mV. Transfection efficiency in HEp-2 cells was highest at this ratio, reaching up to 93%. The penetratin-shRNA complex effectively silenced the RSV F gene, reduced viral titers, and decreased DNA fragmentation and apoptosis in infected cells. CONCLUSION: Penetratin effectively delivers shRNA targeting the RSV F gene, significantly reducing viral load and preventing apoptosis without toxicity. This approach surpasses Lipofectamine and shows potential for future therapeutic interventions, especially when combined with ribavirin, against RSV infection.

Recombinant lactate-assimilating cyanobacteria reduce high-concentration culture-associated cytotoxicity in mammalian cells.

In the fields of cultured meat, biopharmaceuticals, cell therapy, and tissue engineering, large numbers of mammalian cells are required; thus, highly-concentrated cell cultures are widely adopted. In general, such cultures can lead to cell damage caused by waste product accumulation and nutritional inadequacy. In this study, a novel co-culture system where the recombinant lactate-assimilating cyanobacterial strain, KC0110, derived from euryhaline Picosynechococcus sp. PCC 7002, and mammalian muscle cells cultured across porous membranes been developed. By using the KC0110 strain, the amount of ammonium and lactate excreted from C2C12 mouse muscle cells into the culture significantly decreased. Importantly, pyruvate and some amino acids, including pyruvate-derived amino acids, also increased significantly compared to those in monoculture of C2C12 cells. It is believed that the organic acids secreted by the KC0110 strain enhance the growth of mammalian cells, leading to a reduction in high-concentration culture-induced mammalian cell damage [lactate dehydrogenase (LDH) release] through cyanobacterial co-culture. These results show that, through co-cultivation with cyanobacteria, it is possible to culture mammalian cells, alleviating cell damage, even in highly-concentrated cultures. This study demonstrated an in vitro "symbiotic circular system" that can interchange metabolites produced by phototrophs and mammalian cells.

Replication kinetics of pathogenic Eurasian orthohantaviruses in human mesangial cells.

BACKGROUND: Eurasian pathogenic orthohantaviruses cause hemorrhagic fever with renal syndrome (HFRS) characterized by acute kidney injury (AKI). The virulence of orthohantaviruses varies enormously and direct infection of different renal cell types contribute to pathogenesis. Glomerular mesangial cells play an essential role in the interplay between kidney cells and proper kidney function. Therefore, we analyzed the replication competence of different orthohantavirus species in primary mesangial cells and a mesangial cell line. METHODS: We tested the suitability of the mesangial cell line CIHGM-1 (conditionally immortalized human glomerular mesangial cells) as cell culture model for orthohantavirus kidney infection by comparison with primary human renal mesangial cells (HRMCs). We analyzed infection with high pathogenic Hantaan virus (HTNV), moderate pathogenic Puumala virus (PUUV) and non-/low-pathogenic Tula virus (TULV). RESULTS: Effective viral spread was observed for PUUV only, whereas infection with HTNV and TULV was abortive. However, in contrast to TULV, HTNV exhibits an initially high infection rate and declines afterwards. This replication pattern was observed in HRMCs and CIHGM-1 cells. Viability or adhesion was neither impaired for PUUV-infected CIHGM-1 nor HRMCs. A loss of migration capacity was observed in PUUV-infected CIHGM-1 cells, but not in HRMCs. CONCLUSIONS: The identification of differences in the replication competence of pathogenic orthohantavirus strains in renal mesangial cells is of special interest and may provide useful insights in the virus-specific mechanisms of orthohantavirus induced AKI. The use of CIHGM-1 cells will facilitate the research in a relevant cell culture system.

SULF1 expression is increased and promotes fibrosis through the TGF-ß1/SMAD pathway in idiopathic pulmonary fibrosis.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive interstitial lung disease of unknown etiology. Despite the increasing global incidence and poor prognosis, the exact pathogenic mechanisms remain elusive. Currently, effective therapeutic targets and treatment methods for this disease are still lacking. This study tried to explore the pathogenic mechanisms of IPF. We found elevated expression of SULF1 in lung tissues of IPF patients compared to normal control lung tissues. SULF1 is an enzyme that modifies heparan sulfate chains of heparan sulfate proteoglycans, playing a critical role in biological regulation. However, the effect of SULF1 in pulmonary fibrosis remains incompletely understood. Our study aimed to investigate the impact and mechanisms of SULF1 in fibrosis. METHODS: We collected lung specimens from IPF patients for transcriptome sequencing. Validation of SULF1 expression in IPF patients was performed using Western blotting and RT-qPCR on lung tissues. ELISA experiments were employed to detect SULF1 concentrations in IPF patient plasma and TGF-ß1 levels in cell culture supernatants. We used lentiviral delivery of SULF1 shRNA to knock down SULF1 in HFL1 cells, evaluating its effects on fibroblast secretion, activation, proliferation, migration, and invasion capabilities. Furthermore, we employed Co-Immunoprecipitation (Co-IP) to investigate the regulatory mechanisms involved. RESULTS: Through bioinformatic analysis of IPF transcriptomic sequencing data (HTIPF) and datasets GSE24206, and GSE53845, we identified SULF1 may potentially play a crucial role in IPF. Subsequently, we verified that SULF1 was upregulated in IPF and predominantly increased in fibroblasts. Furthermore, SULF1 expression was induced in HFL1 cells following exposure to TGF-ß1. Knockdown of SULF1 suppressed fibroblast secretion, activation, proliferation, migration, and invasion under both TGF-ß1-driven and non-TGF-ß1-driven conditions. We found that SULF1 catalyzes the release of TGF-ß1 bound to TGFßRIII, thereby activating the TGF-ß1/SMAD pathway to promote fibrosis. Additionally, TGF-ß1 induces SULF1 expression through the TGF-ß1/SMAD pathway, suggesting a potential positive feedback loop between SULF1 and the TGF-ß1/SMAD pathway. CONCLUSIONS: Our findings reveal that SULF1 promotes fibrosis through the TGF-ß1/SMAD pathway in pulmonary fibrosis. Targeting SULF1 may offer a promising therapeutic strategy against IPF.

Deletion of exons 45 to 55 in the DMD gene: from the therapeutic perspective to the in vitro model.

BACKGROUND: Gene editing therapies in development for correcting out-of-frame DMD mutations in Duchenne muscular dystrophy aim to replicate benign spontaneous deletions. Deletion of 45-55 DMD exons (del45-55) was described in asymptomatic subjects, but recently serious skeletal and cardiac complications have been reported. Uncovering why a single mutation like del45-55 is able to induce diverse phenotypes and grades of severity may impact the strategies of emerging therapies. Cellular models are essential for this purpose, but their availability is compromised by scarce muscle biopsies. METHODS: We introduced, as a proof-of-concept, using CRISPR-Cas9 edition, a del45-55 mimicking the intronic breakpoints harboured by a subset of patients of this form of dystrophinopathy (designing specific gRNAs), into a Duchenne patient's cell line. The edited cell line was characterized evaluating the dystrophin expression and the myogenic status. RESULTS: Dystrophin expression was restored, and the myogenic defects were ameliorated in the edited myoblasts harbouring a specific del45-55. Besides confirming the potential of CRISPR-Cas9 to create tailored mutations (despite the low cleavage efficiency of our gRNAs) as a useful approach to generate in vitro models, we also generated an immortalized myoblast line derived from a patient with a specific del45-55. CONCLUSIONS: Overall, we provide helpful resources to deepen into unknown factors responsible for DMD-pathophysiology.

Role of canonical and non-canonical cAMP sources in CRHR2α-dependent signaling.

Hippocampal neurons exhibit activation of both the conventional transmembrane adenylyl cyclases (tmACs) and the non-canonical soluble adenylyl cyclase (sAC) as sources of cyclic AMP (cAMP). These two cAMP sources play crucial roles in mediating signaling pathways downstream of CRHR1 in neuronal and neuroendocrine contexts. In this study, we investigate the involvement of both cAMP sources in the molecular mechanisms triggered by CRHR2α. Here we provide evidence demonstrating that UCN1 and UCN3 exert a neuritogenic effect on HT22-CRHR2α cells, which is solely dependent on the cAMP pool generated by sAC and PKA activity but independent of ERK1/2 activation. Through the characterization of the effectors implicated in neurite elongation, we found that CREB phosphorylation and c-Fos induction rely on PKA activity and ERK1/2 phosphorylation, underscoring the critical role of signaling pathway regulation. These findings strengthen the concept that localized cAMP microdomains actively participate in the regulation of these signaling processes.

Oxycodone alleviates LPS-induced neuroinflammation by regulating the CREB/miR-181c/PDCD4 axis.

BACKGROUND: Neuroinflammation plays a critical role in various neurological disorders. Oxycodone has anti-inflammatory properties. The purpose of this work was to look into the effect of oxycodone in controlling lipopolysaccharide (LPS)-induced neuroinflammation in microglia. METHODS: LPS-induced HMC3 cells were subjected to oxycodone (2.5, 5, 10 and 20 µg/mL). The mRNA and protein expressions were examined by qRT-PCR and western blotting. TNF-α, IL-1ß, IL-6, and IL-8 levels were assessed by ELISA. MTT assay was adopted to measure cell viability. The interactions between CREB, miR-181c and PDCD4 were analyzed by dual-luciferase reporter assay, ChIP and/or RIP assays. RESULTS: Oxycodone treatment alleviated LPS-induced inflammation in HMC3 cells and increased p-CREB level, but reduced PDCD4 and iNOS levels in LPS-treated cells. Mechanistically, oxycodone mitigated LPS-induced neuroinflammation by upregulating miR-181c. In addition, CREB promoted miR-181c expression by directly binding to the MIR181C promoter, and miR-181c inhibited PDCD4 expression by directly binding to PDCD4 3'UTR. As expected, oxycodone alleviated LPS-induced neuroinflammation by regulating the CREB/miR-181c/PDCD4 axis. CONCLUSION: Oxycodone attenuated LPS-induced neuroinflammation in microglia by regulating the CREB/miR-181c/PDCD4 axis. These findings proved that oxycodone is a potential drug for treating neuroinflammation and elucidate the mechanisms involved.

SNX27:Retromer:ESCPE-1-mediated early endosomal tubulation impacts cytomegalovirus replication.

Introduction: Cytomegaloviruses (CMVs) extensively reorganize the membrane system of the cell and establish a new structure as large as the cell nucleus called the assembly compartment (AC). Our previous studies on murine CMV (MCMV)-infected fibroblasts indicated that the inner part of the AC contains rearranged early endosomes, recycling endosomes, endosomal recycling compartments and trans-Golgi membrane structures that are extensively tubulated, including the expansion and retention of tubular Rab10 elements. An essential process that initiates Rab10-associated tubulation is cargo sorting and retrieval mediated by SNX27, Retromer, and ESCPE-1 (endosomal SNX-BAR sorting complex for promoting exit 1) complexes. Objective: The aim of this study was to investigate the role of SNX27:Retromer:ESCPE-1 complexes in the biogenesis of pre-AC in MCMV-infected cells and subsequently their role in secondary envelopment and release of infectious virions. Results: Here we show that SNX27:Retromer:ESCPE1-mediated tubulation is essential for the establishment of a Rab10-decorated subset of membranes within the pre-AC, a function that requires an intact F3 subdomain of the SNX27 FERM domain. Suppression of SNX27-mediated functions resulted in an almost tenfold decrease in the release of infectious virions. However, these effects cannot be directly linked to the contribution of SNX27:Retromer:ESCPE-1-dependent tubulation to the secondary envelopment, as suppression of these components, including the F3-FERM domain, led to a decrease in MCMV protein expression and inhibited the progression of the replication cycle. Conclusion: This study demonstrates a novel and important function of membrane tubulation within the pre-AC associated with the control of viral protein expression.