Impaired cerebral microvascular endothelial cells integrity due to elevated dopamine in myasthenic model.

Myasthenia gravis is an autoimmune disease characterized by pathogenic antibodies that target structures of the neuromuscular junction. However, some patients also experience autonomic dysfunction, anxiety, depression, and other neurological symptoms, suggesting the complex nature of the neurological manifestations. With the aim of explaining the symptoms related to the central nervous system, we utilized a rat model to investigate the impact of dopamine signaling in the central nervous and peripheral circulation. We adopted several screening methods, including western blot, quantitative PCR, mass spectrum technique, immunohistochemistry, immunofluorescence staining, and flow cytometry. In this study, we observed increased and activated dopamine signaling in both the central nervous system and peripheral circulation of myasthenia gravis rats. Furthermore, changes in the expression of two key molecules, Claudin5 and CD31, in endothelial cells of the blood-brain barrier were also examined in these rats. We also confirmed that dopamine incubation reduced the expression of ZO1, Claudin5, and CD31 in endothelial cells by inhibiting the Wnt/ß-catenin signaling pathway. Overall, this study provides novel evidence suggesting that pathologically elevated dopamine in both the central nervous and peripheral circulation of myasthenia gravis rats impair brain-blood barrier integrity by inhibiting junction protein expression in brain microvascular endothelial cells through the Wnt/ß-catenin pathway.

Effect of ST36 electroacupuncture on the switch of skeletal muscle fibres in mice with sciatic nerve dissociation.

Skeletal muscle is striated muscle that moves autonomously and is innervated by peripheral nerves. Peripheral nerve injury is very common in clinical treatment. However, the commonly used treatment methods often focus on the regeneration of the injured nerve but overlook the pathological changes in the injured skeletal muscle. Acupuncture, as the main treatment for denervated skeletal muscle atrophy, is used extensively in clinical practice. In the present study, a mouse model of lower limb sciatic nerve detachment was constructed and treated with electroacupuncture Stomach 36 to observe the atrophy of lower limb skeletal muscle and changes in skeletal muscle fibre types before and after electroacupuncture Stomach 36 treatment. Mice with skeletal muscle denervation showed a decrease in the proportion of IIa muscle fibres and an increase in the proportion of IIb muscle fibres, after electroacupuncture Stomach 36. The changes were reversed by specific activators of p38 MAPK, which increased IIa myofibre ratio. The results suggest that electroacupuncture Stomach 36 can reverse the change of muscle fibre type from IIb to IIa after denervation of skeletal muscle by inhibiting p38 MAPK. The results provide an important theoretical basis for the treatment of clinical peripheral nerve injury diseases with electroacupuncture, in addition to novel insights that could facilitate the study of pathological changes of denervated skeletal muscle.

A combinatorial therapeutic approach to enhance FLT3-ITD AML treatment.

Internal tandem duplication mutations of the FMS-like tyrosine kinase-3 (FLT3-ITDs) occur in 25%-30% of patients with acute myeloid leukemia (AML) and are associated with dismal prognosis. Although FLT3 inhibitors have demonstrated initial clinical efficacy, the overall outcome of patients with FLT3-ITD AML remains poor, highlighting the urgency to develop more effective treatment strategies. In this study, we reveal that FLT3 inhibitors reduced protein stability of the anti-cancer protein p53, resulting in drug resistance. Blocking p53 degradation with proteasome inhibitors restores intracellular p53 protein levels and, in combination with FLT3-ITD inhibitors, shows superior therapeutic effects against FLT3-ITD AML in cells, mouse models, and patients. These data suggest that this combinatorial therapeutic approach may represent a promising strategy to target FLT3-ITD AML.

Expression patterns and influence of the two-component system in Vibrio parahaemolyticus of different genotypes.

Vibrio parahaemolyticus is a foodborne pathogen that threatens global food security and human health. The two-component system (TCS) is a primary method for bacteria self-regulate and adapt to the environment. Previous studies have shown that V. parahaemolyticus has four hemolytic genotypes with diverse biological phenotypes and environmental adaptability, but the mechanism is unclear. In this study, we investigated TCS expression patterns in V. parahaemolyticus with different genotypes for the first time and explored the differences in TCS between strains. The results showed similarities in the TCS expression pattern between VPC17 (tdh+/trh-) and VPC44 (tdh-/trh-), while VPC85(tdh-/trh+) had the least similar TCS expression pattern to the other three strains. Analysis of biological information revealed that different regulations of C4 dicarboxylate transport, tetrathionate uptake, antibiotic resistance, and flagellar synthesis involved in the TCS might influence strains' growth, antibiotic resistance, biofilm, and virulence. The different TCS regulatory abilities of strains might be one of the reasons for diverse biological characteristics and different environmental adaptations. This work provides a theoretical basis and a new research direction for the strain variability of V. parahaemolyticus.

Metformin inhibits the pathogenic functions of AChR-specific B and Th17 cells by targeting miR-146a.

Myasthenia gravis (MG) is characterized by fatigable skeletal muscle weakness with a fluctuating and unpredictable disease course and is caused by circulating autoantibodies and pathological T helper cells. Regulation of B-cell function and the T-cell network may be a potential therapeutic strategy for MG. MicroRNAs (miRNAs) have emerged as potential biomarkers in immune disorders due to their critical roles in various immune cells and multiple inflammatory diseases. Aberrant miR-146a signal activation has been reported in autoimmune diseases, but a detailed exploration of the relationship between miR-146a and MG is still necessary. Using an experimental autoimmune myasthenia gravis (EAMG) rat model, we observed that miR-146a was highly expressed in the spleen but expressed at low levels in the thymus and lymph nodes in EAMG rats. Additionally, miR-146a expression in T and B cells was also quite different. EAMG-specific Th17 and Treg cells had lower miR-146a levels, while EAMG-specific B cells had higher miR-146a levels, indicating that targeted intervention against miR-146a might have diametrically opposite effects. Metformin, a drug that was recently demonstrated to alleviate EAMG, may rescue the functions of both Th17 cells and B cells by reversing the expression of miR-146a. We also investigated the downstream target genes of miR-146a in both T and B cells using bioinformatics screening and qPCR. Taken together, our study identifies a complex role of miR-146a in the EAMG rat model, suggesting that more caution should be paid in targeting miR-146a for the treatment of MG.

SIRT7 is a Prognostic Biomarker in Kidney Renal Clear Cell Carcinoma That is Correlated with Immune Cell Infiltration.

Background: SIRT7 has been shown to be expressed in many cancer types, including clear cell renal cell carcinoma (KIRC), but its functional role in this oncogenic context remains to be firmly defined. This study was designed to explore correlations between SIRT7 and KIRC characteristics using the TCGA database. Methods: Relationships between SIRT7 expression and KIRC patient clinicopathological characteristics were assessed through Kruskal-Wallis tests, Wilcoxon signed-rank tests, and logistic regression analyses. Area under the ROC curve (AUC) values were used to assess the prognostic value of SIRT7 as a means of classifying clear cell renal cell carcinoma patients. The functional role of SIRT7 in this cancer type was assessed through GO/KEGG enrichment analyses and immune cell infiltration analyses. Results: In KIRC patients, higher levels of SIRT7 expression were associated with Race, M stage, T stage (all P < 0.05). SIRT7 offered significant diagnostic value in ROC curve analyses (AUC = 0.912), and elevated SIRT7 levels were linked to worse patient overall survival (OS; P < 0.001). The expression of SIRT7 was independently related with KIRC patient OS (HR: 1.827; 95% CI: 1.346-2.481; P<0.001). In GO/KEGG analyses, SIRT7 was found to be associated with ubiquitin-mediated proteolysis and nucleotide excision repair. Higher SIRT7 expression was related to the enhanced infiltration of certain immune cells. Conclusion: Increased SIRT7 expression was associated with a worse KIRC patient prognosis, and immune infiltrates, suggesting it may offer value as a prognostic biomarker for this cancer type.

Silencing uridine diphosphate N-acetylglucosamine pyrophosphorylase gene impairs larval development in Henosepilachna vigintioctopunctata.

BACKGROUND: Uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc) diphosphorylase (UAP) catalyzes the formation of UDP-GlcNAc, the precursor for the production of chitin in ectodermally derived epidermal cells and midgut, for GlcNAcylation of proteins and for generation of glycosyl-phosphatidyl-inositol anchors in all tissues in Drosophila melanogaster. RESULTS: Here, we identified a putative HvUAP gene in Henosepilachna vigintioctopunctata. Knockdown of HvUAP at the second-, third- and fourth-instar stages impaired larval development. Most resultant HvUAP hypomorphs showed arrested development at the third-, fourth-instar larval or prepupal stages, and became paralyzed, depending on the age when treated. Some HvUAP-silenced larvae had weak and soft scoli. A portion of HvUAP-depleted beetles formed misshapen pupae. No HvUAP RNA interference pupae successfully emerged as adults. Dissection and microscopic observation revealed that knockdown of HvUAP affected gut growth and food ingestion, reduced cuticle thickness, and negatively affected the formation of newly generated cuticle layers during ecdysis. Furthermore, HvUAP deficiency inhibited development of the tracheal respiratory system and thinned tracheal taenidia. CONCLUSION: The phenotypical defects in HvUAP hypomorphs suggest that HvUAP is involved in the production of chitin. Moreover, our findings will enable the development of a double-stranded RNA-based pesticide to control H. vigintioctopunctata. © 2021 Society of Chemical Industry.

The Oncogenic Role and Immune Infiltration for CARM1 Identified by Pancancer Analysis.

Chromatin-modifying enzymes, especially protein arginine methyltransferases (PRMTs), have been identified as candidate targets for cancer. Cellular or animal-based evidence has suggested an association between coactivator-linked arginine methyltransferase 1 (CARM1) and cancer progression. However, the relationship between CARM1 and patient prognosis and immune infiltration in pancancer patients is unknown. On the basis of the GEO and TCGA databases, we first investigated the possible oncogenic functions of CARM1 in thirty-three tumor types. CARM1 expression was elevated in many types of tumors. In addition, there was a significant association between CARM1 expression and the survival rate of tumor patients. Uterine corpus endometrial carcinoma (UCES) samples had the highest CARM1 mutation frequency of all cancer types. In head and neck squamous cell carcinoma (HNSC) and lung squamous cell carcinoma (LUSC), CARM1 expression was associated with the level of CD8+ T cell infiltration, and cancer-associated fibroblast infiltration was also observed in other tumors including kidney renal papillary cell carcinoma (KIRC) and prostate adenocarcinoma (PRAD). CARM1 was involved in immune modulation and played an important role in the tumor microenvironment (TME). Furthermore, activities associated with RNA transport and its metabolism were included in the possible mechanisms of CARM1. Herein, our first pancancer research explores the oncogenic role of CARM1 in various tumors. CARM1 is associated with immune infiltrates and can be employed as a predictive biomarker in pancancer.

RNAi for chitin synthase 1 rather than 2 causes growth delay and molting defect in Henosepilachna vigintioctopunctata.

Chitin synthase (CHS) plays a critical role in chitin synthesis and excretion. In most insects, CHSs have been segregated into 1 and 2 classes. CHS1 is responsible for chitin production in the ectodermally-derived epidermal cells. CHS2 is dedicated to chitin biosynthesis in the midgut peritrophic matrix (PM). Henosepilachna vigintioctopunctata is a serious pest of Solanaceae and Cucurbitaceae plants. In this study, we identified HvCHS1 and HvCHS2. We found that HvCHS1 was abundantly transcribed in the larval tracheae and epidermis, whereas HvCHS2 was mainly expressed in the guts. Escherichia coli HT115 expressed double stranded RNAs targeting HvCHS1 and HvCHS2 (dsCHS1 and dsCHS2) were used to immerse potato foliage and the treated leaves were provided to the newly-molted fourth- and third-instar larvae. Ingestion of dsCHS1 by the fourth-instar larvae significantly diminished the target mRNA level and had slight influence on the expression of HvCHS2. In contrast, consumption of dsCHS2 significantly lowered the target mRNA level but triggered the transcription of HvCHS1. Knockdown of HvCHS1, rather than HvCHS2, arrested larval development and impaired larva-pupa-adult transition. A large proportion of HvCHS1 hypomorphs became stunting prepupae, deformed pupae or misshapen adults. Moreover, knockdown of HvCHS1 damaged gut integrity, decreased cuticle thickness, and delayed the formation of newly-generated cuticle layer during ecdysis. Furthermore, depletion of HvCHS1 inhibited the development of trachea system and thinned tracheal taenidia. Ingestion of dsCHS1 at the third-instar stage caused similar but severe negative effects. Our results demonstrated that HvCHS1 is responsible for chitin biosynthesis during ecdysis. Moreover, HvCHS1 is a potential amenable target gene and young larvae are more susceptible to dsRNA.

RNAi of vacuolar-type H+-ATPase genes causes growth delay and molting defect in Henosepilachna vigintioctopunctata.

Henosepilachna vigintioctopunctata is one of the most serious insect pests to a large number of nightshades and cucurbits. RNA interference (RNAi) triggered by double-stranded RNA (dsRNA) offers a reduced risk approach to control the beetle. Identification of amenable target genes and determination of appropriate life stage for dsRNA treatment are two critical steps in order to improve RNAi efficiency. In the present paper, we identified three vATPase genes, namely HvvATPaseC, HvvATPaseE and HvvATPaseH. We found that the three transcripts were widely expressed in the eggs, first- to fourth-instar larvae, prepupae, pupae and adults. They were abundantly transcribed in the hindgut and Malpighian tubules, in contrast to the epidermis and fat body. Three days' ingestion of dsvATPaseC, dsvATPaseE and dsvATPaseH by the fourth-instar larvae significantly decreased corresponding transcript level by 90.1, 88.9 and 97.2%, greatly reduced larval fresh weight by 28.0, 29.9 and 28.0%, and caused 66.7, 100 and 78.7% larval lethality respectively. Comparably, 3 days' exposure of the third-instar larvae to dsvATPaseC significantly reduced HvvATPaseC mRNA level by 89.5%, decreased approximately 80% of the larval fresh weight, and killed 100% of the treated larvae. Therefore, the three vATPase genes, especially HvvATPaseE, are potential amenable target genes and young larvae are more susceptible to dsRNA. Our findings will enable the development of the dsRNA-based pesticide to control H. vigintioctopunctata.

Involvement of microRNA-155 in the mechanism of electroacupuncture treatment effects on experimental autoimmune encephalomyelitis.

Multiple sclerosis (MS) is a neurodegenerative and demyelinating autoimmune disease mediated by autoreactive T cells that affects the central nervous system (CNS). Electroacupuncture (EA) has emerged as an alternative or supplemental treatment for MS, but the mechanism by which EA may alleviate MS symptoms is unresolved. Here, we examined the effects of EA at the Zusanli (ST36) acupoint on mice with experimental autoimmune encephalomyelitis (EAE), the predominant animal model of MS. The effects of EA on EAE emergence, inflammatory cell levels, proinflammatory cytokines, and spinal cord pathology were examined. EA treatment attenuated the EAE clinical score and associated spinal cord demyelination, while reducing the presence of proinflammatory cytokines in mononuclear cells (MNCs), downregulating microRNA (miR)-155, and upregulating the opioid peptide precursor proopiomelanocortin (POMC) in the CNS. Experiments in which cultured neurons were transfected with a miR-155 mimic or a miR-155 inhibitor further showed that the direct modulation of miR-155 levels could regulate POMC levels in neurons. In conclusion, the alleviation of EAE by EA is characterized by reduced proportions of Th1/Th17 cells and increased proportions of Th2 cells, POMC upregulation, and miR-155 downregulation, while miR-155 itself can suppress POMC expression. These results, support the hypothesis that the effects of EA on EAE may involve the downregulation of miR-155.

RNA interference targeting ecdysone receptor blocks the larval-pupal transition in Henosepilachna vigintioctopunctata.

Henosepilachna vigintioctopunctata is a serious insect pest which attacks a large number of nightshades and cucurbits in Asian countries, Brazil and Australia. Prolonged application of traditional pesticides has caused environmental pollution and exerted deleterious effects on human health. Finding new approaches with high target specificity and low environmental contamination has become an urgent task. RNA interference (RNAi) induced by double-stranded RNA (dsRNA) is expected to be applicable to managing this pest. Here we evaluated the effects of Escherichia coli-expressed dsRNAs targeting ecdysone receptor (EcR) gene via dietary delivery in laboratory and foliar spraying in a greenhouse. The target transcript was successfully knocked down when the 4th-instar larvae had fed on potato foliage dipped with dsEcR in a laboratory bioassay. Around 85% of the HvEcR RNAi larvae remained as prepupae or became abnormal pupae, and failed to emerge into adults. Ingestion of dsEcR-immersed foliage by the 3rd-instar larvae effectuated a comparable RNAi response and brought about more severe defects: all the resultant larvae arrested development, remained as prepupae and finally died. For assay in the greenhouse, a dsEcR-contained E. coli suspension was directly sprayed to the foliage of greenhouse-growing potato plants and the 3rd- and 4th-instar larvae were transferred to the leaves. High RNAi efficacy was obtained and identical RNAi phenotypes were observed in treated larvae. In addition, spraying dsEcR reduced leaf damage. Our results indicate a possibility of practical application of dsEcR as an environmentally friendly RNA pesticide to control H. vigintioctopunctata larvae.

Combining miR-23b exposure with mesenchymal stem cell transplantation enhances therapeutic effects on EAE.

Bone marrow mesenchymal stem cells (BMSCs) have the immuno-modulatory capacity to ameliorate autoimmune diseases, such as multiple schlerosis (MS), systemic lupus erythematosus and rheumatoid arthritis. However, BMSC-mediated immunosuppression can be challenging to achieve. The efficacy of BMSC transplantation may be augmented by an adjuvant therapy. Here, we demonstrated that treatment of mice with experimental autoimmune encephalomyelitis (EAE), a model of MS, with BMSCs over-expressing microRNA (miR)-23b provided better synergistic and longer-term therapeutic effects than treatment with traditional BMSCs. Over-expression of miR-23b enhanced the ability of BMSCs to inhibit differentiation of Th17 cells and reduced IL-17 secretion. Compared to traditional BMSCs, the miR-23b over-expressing BMSCs (miR23b-BMSCs) exhibited enhanced secretion of tumor growth factor beta 1 (TGF-ß1), a cytokine that promotes the differentiation of regulatory T (Treg) cells. Pathologically, miR23b-BMSC transplantation delayed EAE progression, apparently by reducing the Th17/Treg cell ratio and inhibiting inflammatory cell infiltration across the blood-brain barrier, and thus slowing spinal cord demyelination. These results may lead to better utility of BMSCs as a treatment for autoimmune diseases.

Inference of Subpathway Activity Profiles Reveals Metabolism Abnormal Subpathway Regions in Glioblastoma Multiforme.

Glioblastoma, also known as glioblastoma multiforme (GBM), is the most malignant form of glioma and represents 81% of malignant brain and central nervous system (CNS) tumors. Like most cancers, GBM causes metabolic recombination to promote cell survival, proliferation, and invasion of cancer cells. In this study, we propose a method for constructing the metabolic subpathway activity score matrix to accurately identify abnormal targets of GBM metabolism. By integrating gene expression data from different sequencing methods, our method identified 25 metabolic subpathways that were significantly abnormal in the GBM patient population, and most of these subpathways have been reported to have an effect on GBM. Through the analysis of 25 GBM-related metabolic subpathways, we found that (S)-2,3-Epoxysqualene, which was at the central region of the sterol biosynthesis subpathway, may have a greater impact on the entire pathway, suggesting a potential high association with GBM. Analysis of CCK8 cell activity indicated that (S)-2,3-Epoxysqualene can indeed inhibit the activity of U87-MG cells. By flow cytometry, we demonstrated that (S)-2,3-Epoxysqualene not only arrested the U87-MG cell cycle in the G0/G1 phase but also induced cell apoptosis. These results confirm the reliability of our proposed metabolic subpathway identification method and suggest that (S)-2,3-Epoxysqualene has potential therapeutic value for GBM. In order to make the method more broadly applicable, we have developed an R system package crmSubpathway to perform disease-related metabolic subpathway identification and it is freely available on the GitHub (https://github.com/hanjunwei-lab/crmSubpathway).

IFN-γ regulates the transformation of microglia into dendritic-like cells via the ERK/c-myc signaling pathway during cerebral ischemia/reperfusion in mice.

Cerebral ischemia-reperfusion injury induces a secondary immune inflammatory reaction that exacerbates brain injury and clinical prognosis. Dendritic cells (DCs) and microglia are both important regulators of neuroinflammation. Studies have confirmed that a large number of cells express the DC surface marker CD11c in the ischemic area, and some of these cells also express microglial markers. However, the specific mechanism of transformation between microglia and DCs and their roles in the process of cerebral ischemia-reperfusion injury are still not clear. In this study, we established a mouse model and flow cytometry was used to detect the expression of mature DC surface molecules in activated microglia. IFN-γ knockout mice were used to determine the regulatory effect of IFN-γ on microglial transformation. We found that CD11c+ cells were derived from microglia after ischemia-reperfusion injury, and this group of cells highly expressed MHC-II molecules and other costimulatory molecules, such as CD80 and CD86, which were regulated by IFN-γ and its downstream signaling molecules ERK/c-myc. In summary, our results showed in cerebral ischemia-reperfusion injury, IFN-γ regulates the transformation of microglia to DC-like cells. Microglial-derived DC-like cells possess the ability to present antigens and activate naïve T cells which is regulated by the ERK/c-myc signaling pathway.

MicroRNA-182 exacerbates blood-brain barrier (BBB) disruption by downregulating the mTOR/FOXO1 pathway in cerebral ischemia.

Cerebral ischemia causes damage to the structure and function of the blood-brain barrier (BBB) and alleviating BBB destruction will be of great significance for the treatment and prognosis of ischemic stroke. Recently, microRNAs have been shown to play a critical role in BBB integrity. However, the potential mechanism by which microRNA-182 (miR-182) affects the BBB in ischemic stroke remains unclear. We demonstrated for the first time that cerebral ischemia leads to a significant progressive increase in miR-182 after pMCAO, and bEnd.3 cells are the primary target cells of miR-182. In miR-182 KD transgenic mice, infarct volume, and BBB permeability were attenuated, and tight junction (TJ) proteins increased. Inhibition of miR-182 with an antagomir reduced OGD-induced apoptosis of bEnd.3 cells and the loss of ZO-1 and Occludin. To further explore the mechanism by which miR-182 regulates BBB integrity, we detected the apoptotic proteins Bcl-2/Bax and demonstrated that mTOR and FOXO1 were the targets of miR-182. Inhibition of mTOR/FOXO1 by rapamycin/AS1842856 decreased the ratio of Bcl-2/Bax and exacerbated TJ protein loss. Taken together, inhibition of miR-182 protects BBB integrity by reducing endothelial cell apoptosis through the mTOR/FOXO1 pathway. Thus, miR-182 may be a potential target for the treatment of BBB disruption during cerebral ischemia.

Neural-specific distribution of transmembrane protein TMEM240 and formation of TMEM240-Body.

Mutation in TMEM240 is suggested to cause SCA21, but the specific mechanism has not been clarified. The subcellular localization, specific biological function, and corresponding mechanism of action of TMEM240 have also not been delineated. In this study, the mRNA and protein expression of TMEM240 were assessed using qPCR and western blotting, respectively. Live cell imaging was used to establish the sub-cellular location of TMEM240, and electron microscopy was used to determine the morphology and distribution of TMEM240 in the cell. TMEM240 was specifically expressed in the neurons. Exogenous TMEM240 formed a multilayered cell structure, which we refer to as TMEM240-Body (T240-Body). T240-Body was separated and purified by centrifugation and filtration. An anchor protein His-tagged-GFP-BP on Ni-NTA agarose was used to pull down T240-GFP binding proteins. Both the N-terminal and the C-terminal of TMEM240 were confirmed to be inside the T240-Body. Co-localization experiments suggested that peroxisomes might contribute to T240-Body formation, and the two transmembrane regions of TMEM240 appear to be essential for formation of the T240-Body. Emerin protein contributed to formation of T240-Body when combined with TMEM240. Overall, this study provides new insights into TMEM240, which inform future research to further our understanding of its biological function.

Involvement of Leptinotarsa hormone receptor 38 in the larval-pupal transition.

In insects, nuclear receptors (NRs) including EcR (NR1H1), USP (NR2B4), E75 (NR1D3), HR3 (NR1F), HR4 (NR6) and FTZ-F1 (NR5A3) mediate the 20-hydroxyecdysone (20E) signaling cascade to play a critical role during larval metamorphosis. In this present paper, we focused on hormone receptor 38 (HR38) in Leptinotarsa decemlineata, the only insect homolog of the NR4A subclass. RNA interference (RNAi) of LdHR38 in the penultimate (third) instar larvae reduced the expression of an ecdysteroidogenesis gene and declined the titer of 20E. Knockdown of LdHR38 intensified the expression of LdUSP, LdE75, LdE74, LdE93, LdBroad and LdHR3, whereas repressed the transcription of LdFTZ-F1. Disruption of 20E signaling inhibited chitin biosynthesis in the larval cuticle. Approximately 25% of the LdHR38 RNAi larvae died, around 40% of the resultant larvae remained as prepupae or become deformed pupae. The body surface of the HR38 depleted abnormal prepupae and pupae looked wet, just like the cuticle being covered with a layer of liquid. Moreover, the increase of larval mortality, and the impairment of pupation and emergence exhibited dose-dependent manners. Furthermore, silencing LdHR38 at the final (fourth) instar caused similar but less severe impairment of pupation. Dietary supplement with 20E for the third instar larvae did not rescue the high larval death and only slightly alleviated the low pupation rate in the LdHR38 RNAi hypomorphs. Accordingly, we propose that HR38 is necessary for tune of ecdysteroidogenesis and for mediation of 20E signaling during metamorphosis in L. decemlineata.

FLT3 inhibition upregulates HDAC8 via FOXO to inactivate p53 and promote maintenance of FLT3-ITD+ acute myeloid leukemia.

Internal tandem duplication (ITD) mutations within the FMS-like receptor tyrosine kinase-3 (FLT3) can be found in up to 25% to 30% of acute myeloid leukemia (AML) patients and confer a poor prognosis. Although FLT3 tyrosine kinase inhibitors (TKIs) have shown clinical responses, they cannot eliminate primitive FLT3-ITD+ AML cells, which are potential sources of relapse. Therefore, elucidating the mechanisms underlying FLT3-ITD+ AML maintenance and drug resistance is essential to develop novel effective treatment strategies. Here, we demonstrate that FLT3 inhibition induces histone deacetylase 8 (HDAC8) upregulation through FOXO1- and FOXO3-mediated transactivation in FLT3-ITD+ AML cells. Upregulated HDAC8 deacetylates and inactivates p53, leading to leukemia maintenance and drug resistance upon TKI treatment. Genetic or pharmacological inhibition of HDAC8 reactivates p53, abrogates leukemia maintenance, and significantly enhances TKI-mediated elimination of FLT3-ITD+ AML cells. Importantly, in FLT3-ITD+ AML patient-derived xenograft models, the combination of FLT3 TKI (AC220) and an HDAC8 inhibitor (22d) significantly inhibits leukemia progression and effectively reduces primitive FLT3-ITD+ AML cells. Moreover, we extend these findings to an AML subtype harboring another tyrosine kinase-activating mutation. In conclusion, our study demonstrates that HDAC8 upregulation is an important mechanism to resist TKIs and promote leukemia maintenance and suggests that combining HDAC8 inhibition with TKI treatment could be a promising strategy to treat FLT3-ITD+ AML and other tyrosine kinase mutation-harboring leukemias.