Impaired ESX-3 Induces Bedaquiline Persistence in Mycobacterium abscessus Growing Under Iron-Limited Conditions.

ESX-3 is a secretion pathway which is essential for mycobactin-mediated iron acquisition under iron-limited conditions. Although present in all Mycobacterium sp., ESX-3 remains to be elucidated in Mycobacterium abscessus. In the study reported here, impaired ESX-3 seriously restricts the growth of M. abscesses under iron-limited conditions; growth is salvaged by functional ESX-3 or iron supplementation. Notably, impaired ESX-3 does not kill M. abscesses when environmental iron is insufficient but induces persistence to bedaquiline, a diarylquinoline class antibiotic used to treat multidrug-resistant mycobacteria. One potential mechanism contributing to persistence is the iron deficiency due to impaired ESX-3 suppressing succinate dehydrogenase activity, which dysregulates the tricarboxylic acid cycle and inactivates bedaquiline. Experiments conducted here also demonstrate that the regulator, MtrA, can bind ESX-3 and promote the survival of M. abscessus. As such, this study suggests that a novel pathway involving MtrA, ESX-3, iron metabolism, and the TCA cycle contributes to bedaquiline persistence in M. abscesses growing under iron-limited conditions.

YY1 lactylation in microglia promotes angiogenesis through transcription activation-mediated upregulation of FGF2.

BACKGROUND: Ocular neovascularization is a leading cause of blindness. Retinal microglia have been implicated in hypoxia-induced angiogenesis and vasculopathy, but the underlying mechanisms are not entirely clear. Lactylation is a novel lactate-derived posttranslational modification that plays key roles in multiple cellular processes. Since hypoxia in ischemic retinopathy is a precipitating factor for retinal neovascularization, lactylation is very likely to be involved in this process. The present study aimed to explore the role of lactylation in retinal neovascularization and identify new therapeutic targets for retinal neovascular diseases. RESULTS: Microglial depletion by the colony-stimulating factor 1 receptor (CSF1R) inhibitor PLX3397 suppresses retinal neovascularization in oxygen-induced retinopathy. Hypoxia increased lactylation in microglia and accelerates FGF2 expression, promoting retinal neovascularization. We identify 77 sites of 67 proteins with increased lactylation in the context of increased lactate under hypoxia. Our results show that the nonhistone protein Yin Yang-1 (YY1), a transcription factor, is lactylated at lysine 183 (K183), which is regulated by p300. Hyperlactylated YY1 directly enhances FGF2 transcription and promotes angiogenesis. YY1 mutation at K183 eliminates these effects. Overexpression of p300 increases YY1 lactylation and enhances angiogenesis in vitro and administration of the p300 inhibitor A485 greatly suppresses vascularization in vivo and in vitro. CONCLUSIONS: Our results suggest that YY1 lactylation in microglia plays an important role in retinal neovascularization by upregulating FGF2 expression. Targeting the lactate/p300/YY1 lactylation/FGF2 axis may provide new therapeutic targets for proliferative retinopathies.

iPSC-based model of Vogt-Koyanagi-Harada disease for phenotype recapitulation and drug screening.

Vogt-Koyanagi-Harada (VKH) disease, a major blinding eye disease, is characterized by an autoimmune response against melanocytes in multiple organs throughout the body. Currently, the aetiology and pathogenesis of VKH disease are unclear, and the treatment strategy needs to be further optimized. The retinal pigment epithelium (RPE), a monolayer of pigmented cells of the fundus, is essential for maintaining normal visual function and is involved in both the acute and chronic stages of VKH disease. Therefore, the functions of the RPE may play a critical role in the aetiology and treatment of VKH disease. Herein, we established a human induced pluripotent stem cell (hiPSC) RPE model of VKH disease by reprogramming peripheral blood mononuclear cells (PBMCs) into iPSCs and then differentiating them into RPE cells. Patient-derived RPE cells exhibited barrier disruption, impaired phagocytosis, and depigmentation compared with those from normal controls, which was consistent with the features of VKH disease. Furthermore, a small molecular compound targeting EGR2 was found to rescue the barrier and phagocytic functions of the hiPSC-RPE cells through high-throughput virtual screening and functional studies, suggesting a promising strategy for the treatment of VKH disease.

Ginsenoside Rg1 protects against d-galactose induced fatty liver disease in a mouse model via FOXO1 transcriptional factor.

AIMS: Rg1 is the most active component of traditional Chinese medicine ginseng, having anti-aging and anti-oxidative stress features in multiple organs. Cellular senescence of hepatocytes is involved in the progression of a wide spectrum of chronic liver diseases. In this study, we investigated the potential benefits and mechanism of action of Rg1 on aging-driven chronic liver diseases. MATERIALS AND METHODS: A total of 40 male C57BL/6 mice were randomly divided into four groups: control group; Rg1 group; Rg1+d-gal group; and d-gal group. Blood and liver tissue samples were collected for determination of liver function, biochemical and molecular markers, as well as histopathological investigation. KEY FINDINGS: Rg1 played an anti-aging role in reversing d-galactose induced increase in senescence-associated SA-ß-gal staining and p53, p21 protein in hepatocytes of mice and sustained mitochondria homeostasis. Meanwhile, Rg1 protected livers from d-galactose caused abnormal elevation of ALT and AST in serum, hepatic steatosis, reduction in hepatic glucose production, hydrogenic degeneration, inflammatory phenomena including senescence-associated secretory phenotype (SASP) IL-1ß, IL-6, MCP-1 elevation and lymphocyte infiltration. Furthermore, Rg1 suppressed drastic elevation in FOXO1 phosphorylation resulting in maintaining FOXO1 protein level in the liver after d-galactose treatment, followed by FOXO1 targeted antioxidase SOD and CAT significant up-regulation concurrent with marked decrease in lipid peroxidation marker MDA. SIGNIFICANCE: Rg1 exerts pharmaceutic effects of maintaining FOXO1 activity in liver, which enhances anti-oxidation potential of Rg1 to ameliorate SASP and to inhibit inflammation, also promotes metabolic homeostasis, and thus protects livers from senescence induced fatty liver disease. The study provides a potential therapeutic strategy for alleviating chronic liver pathology.

The development of an herbal material quality control strategy considering the effects of manufacturing processes.

BACKGROUND: Quality by design (QbD) is an advanced drug quality control concept that has been gradually implemented in the optimization of manufacturing processes of Chinese medicines. However, the variation of Chinese medicinal material quality has rarely been considered in published works. Because manufacturing processes may lower the variation introduced through different batches of materials, a material quality control strategy should be developed considering the influences of manufacturing processes. METHODS: In this work, the processes of extraction, concentration, water precipitation, and chromatography for notoginseng total saponin (NTS) production were investigated while considering Panax notoginseng quality variation as a sample. Ten process parameters were studied simultaneously using a definitive screening design. After the process critical quality attributes (CQAs) were determined, critical process parameters (CPPs) and critical material attributes (CMAs) were identified simultaneously. Then, models utilizing the CMAs, CPPs, and process CQAs were developed. The design space was then calculated using a Monte Carlo simulation method with an acceptable probability of 0.90. A material quality control strategy considering the influences of manufacturing processes was proposed. RESULTS: The ginsenoside Rd purity and total saponin purity in the eluate were identified as process CQAs. The ethanol solution concentration used for extraction, the ethanol solution concentration used for elution, and elution time were identified as CPPs. The extractable dry matter content of Panax notoginseng was one of the CMAs. The extractable contents of notoginsenoside R1, ginsenoside Rg1, ginsenoside Rb1, and ginsenoside Rd were the other CMAs. The inequalities implemented to discriminate the high quality and low quality of Panax notoginseng were developed according to the NTS standard of the Xuesaitong injection. Low quality Panax notoginseng should not be released for NTS production. High quality Panax notoginseng can be treated with feasible manufacturing processing parameters. Verification experiments were carried out successfully for 2 batches of high quality Panax notoginseng. CONCLUSIONS: In this work, a quality control strategy for herbal materials was developed considering the matching of process characteristics and material quality attributes. This strategy is promising for application to other Chinese medicines.

Cortical network underlying audiovisual semantic integration and modulation of attention: An fMRI and graph-based study.

Many neuroimaging and electrophysiology studies have suggested that semantic integration as a high-level cognitive process involves various cortical regions and is modulated by attention. However, the cortical network specific to semantic integration and the modulatory mechanism of attention remain unclear. Here, we designed an fMRI experiment using "bimodal stimulus" to extract information regarding the cortical activation related to the effects of semantic integration with and without attention, and then analyzed the characteristics of the cortical network and the modulating effect of attention on semantic integration. To further investigate the related cortical regions, we constructed a functional brain network for processing attended AV stimuli to evaluate the nodal properties using a graph-based method. The results of the fMRI and graph-based analyses showed that the semantic integration with attention activated the anterior temporal lobe (ATL), temporoparietal junction (TPJ), and frontoparietal cortex, with the ATL showing the highest nodal degree and efficiency; in contrast, semantic integration without attention involved a relatively small cortical network, including the posterior superior temporal gyrus (STG), Heschl's gyrus (HG), and precentral gyrus. These results indicated that semantic integration is a complex cognitive process that occurs not only in the attended condition but also in the unattended condition, and that attention could modulate the distribution of cortical networks related to semantic integration. We suggest that semantic integration with attention is a conscious process and needs a wide cortical network working together, in which the ATL plays the role of a central hub; in contrast, semantic integration without attention is a pre-attentive process and involves a relatively smaller cortical network, in which the HG may play an important role. Our study will provide valuable insights into semantic integration and will be useful for investigations on multisensory integration and attention mechanism at multiple processing stages and levels within the cortical hierarchy.

Structural and developmental expression of Ss-riok-2, an RIO protein kinase encoding gene of Strongyloides stercoralis.

RIO kinases are essential atypical protein kinases in diverse prokaryotic and eukaryotic organisms, playing significant roles in yeast and humans. However, little is known about their functions in parasitic nematodes. In the present study, we have isolated and characterized the full-length cDNA, gDNA and a putative promoter of a RIOK-2 protein kinase (Ss-RIOK-2) encoding gene (Ss-riok-2) from Strongyloides stercoralis, a medically important parasitic nematode (Order Rhabditida). A three-dimensional structure (3D) model of Ss-RIOK-2 was generated using the Chaetomium thermophilum RIOK-2 protein kinase (Ct-RIOK-2) crystal structure 4GYG as a template. A docking study revealed some critical sites for ATP binding and metal binding. The putative promoter of Ss-riok-2 contains a number of conserved elements. RNAseq analysis revealed the highest levels of the Ss-riok-2 transcript in free-living females and parasitic females. To identify anatomical patterns of Ss-riok-2 expression in S. stercoralis, we observed expression patterns of a transgene construct encoding green fluorescent protein under the Ss-riok-2 promoter in post free-living S. stercoralis. Expression driven by this promoter predominated in intestinal cells. This study demonstrates significant advancement in molecular and cellular biological study of S. stercoralis and of parasitic nematodes generally, and provides a foundation for further functional genomic studies.