Deficiency Cells Research Articles

Six3 and Six6 jointly control diverse target genes in multiple cell populations over developmental trajectories of mouse embryonic retinal progenitor cells.

How tissue-specific progenitor cells generate adult tissues is a puzzle in organogenesis. Using single-cell RNA sequencing of control and Six3 and Six6 compound-mutant mouse embryonic eyecups, we demonstrated that these two closely related transcription factors jointly control diverse target genes in multiple cell populations over the developmental trajectories of mouse embryonic retinal progenitor cells. In the Uniform Manifold Approximation and Projection for Dimension Reduction (UMAP) graph of control retinas, naïve retinal progenitor cells had two major trajectories leading to ciliary margin cells and retinal neurons, respectively. The ciliary margin trajectory was from naïve retinal progenitor cells in the G1 phase directly to ciliary margin cells, whereas the neuronal trajectory went through an intermediate neurogenic state marked by Atoh7 expression. Neurogenic retinal progenitor cells (Atoh7+) were still proliferative; early retinal neurons branched out from Atoh7+ retina progenitor cells in the G1 phase. Upon Six3 and Six6 dual deficiency, both naïve and neurogenic retinal progenitor cells were defective, ciliary margin differentiation was enhanced, and multi-lineage neuronal differentiation was disrupted. An ectopic neuronal trajectory lacking the Atoh7+ state led to ectopic neurons. Additionally, Wnt signaling was upregulated, whereas FGF signaling was downregulated. Notably, Six3 and Six6 proteins occupied the loci of diverse genes that were differentially expressed in distinct cell populations, and expression of these genes was significantly altered upon Six3 and Six6 dual deficiency. Our findings provide deeper insight into the molecular mechanisms underlying early retinal differentiation in mammals.

Clinical features and predictors of osteoarticular manifestations in common variable immunodeficiency.

This study aimed to assess osteoarticular manifestations in patients with common variable immunodeficiency (CIVD) and to identify the predictive factors. This was a retrospective and prognostic study conducted in the pediatrics: immuno-hematology and stem cell transplantation department, including patients who fit the definition of CVID. A Cox model analysis was used to identify predictive factors. A total of 36 patients were enrolled. Osteoarticular involvement was noted in 15 patients (42%) with a cumulative incidence of 90% after a median follow-up of 25 years. Non-infectious manifestations were reported in 14 patients (39%). The cumulative risk of inflammatory or autoimmune osteoarticular etiology was 74%. Well-characterized rheumatic diseases were retained in six patients and unlabeled autoimmune or inflammatory mechanism in five cases. Bone mineral density revealed osteoporosis in six cases leading to a cumulative risk of degenerative complications of 72%. The cumulative incidence of infectious complications was 17%. In multivariate analysis, predictors of osteoarticular complications were low body weight (HR = 8.67, CI: 1.496-50.278, p = 0.01) and hepatomegaly at diagnosis (HR = 6.2, CI: 1.537-25.075, p = 0.01). Reduced CD4 cells rate < 600 cells/mm3 and hepatomegaly were predictors of autoimmune or inflammatory complications, while chronic diarrhea and iron deficiency were associated with degenerative manifestations. Osteoarticular manifestations have emerged as a real health problem for CVID patients. Risk increases with low body weight, hepatomegaly, chronic diarrhea, iron deficiency, and CD4 cells rate under 600 cell/mm3. Elucidating the mechanisms of these complications in CVID is important for developing preventive strategies. Key Points • This retrospective and prognostic study described the clinical characteristics of osteoarticular manifestations in 36 patients with CVID to ensure better recognition and understanding of this association by clinicians. • Identification of predictive factors of osteoarticular complications according to its etiology is crucial to establish appropriate, optimal and early management of patients at risk.

Tonsillectomy in adults—to do or not to do

Recurrent acute tonsillitis describes the repeated occurrence of acute bacterial infections of palatine tonsils with symptom-free intervals. Acute tonsillitis accounts for around a third of respiratory tract infections in primary care among people of all ages, 1 Petersen I Johnson AM Islam A Duckworth G Livermore DM Hayward AC Protective effect of antibiotics against serious complications of common respiratory tract infections: retrospective cohort study with the UK General Practice Research Database. BMJ. 2007; 335: 982 Crossref PubMed Scopus (210) Google Scholar with an estimated 600 million symptomatic cases of group A streptococcus tonsillitis worldwide annually. 2 Carapetis JR Steer AC Mulholland EK Weber M The global burden of group A streptococcal diseases. Lancet Infect Dis. 2005; 5: 685-694 Summary Full Text Full Text PDF PubMed Scopus (1995) Google Scholar Recurrent episodes occur in 12% of patients with tonsillitis—ie, around 12 000 per 100 000 people. 3 Kvestad E Kværner KJ Røysamb E Tambs K Harris JR Magnus P Heritability of recurrent tonsillitis. Arch Otolaryngol Head Neck Surg. 2005; 131: 383-387 Crossref PubMed Scopus (26) Google Scholar Beyond the relation to group A streptococcus infections, little is known about risk factors for the development of recurrent acute tonsillitis. 4 Roberts AL Connolly KL Kirse DJ et al. Detection of group A Streptococcus in tonsils from pediatric patients reveals high rate of asymptomatic streptococcal carriage. BMC Pediatr. 2012; 12: 3 Crossref PubMed Scopus (97) Google Scholar Recurrent acute tonsillitis could be a genetic immunosusceptibility disease because patients younger than 12 years show some subclinical antibody deficiency and aberrant T-cell function 5 Dan JM Havenar-Daughton C Kendric K et al. Recurrent group A Streptococcus tonsillitis is an immunosusceptibility disease involving antibody deficiency and aberrant TFH cells. Sci Transl Med. 2019; 11eaau3776 Crossref PubMed Scopus (1) Google Scholar and many undergo tonsillectomy. More than 30 000 tonsillectomies are performed annually in the UK. 6 Keltie K Donne A Daniel M et al. Paediatric tonsillectomy in England: a cohort study of clinical practice and outcomes using Hospital Episode Statistics data (2008–2019). Clin Otolaryngol. 2021; 46: 552-561 Crossref PubMed Scopus (16) Google Scholar Tonsillectomy can produce severe postoperative pain, complicated by postoperative bleeding which can result in death in rare cases. Clearly, we need evidence-based policies to guide health-care professionals for the indication of tonsillectomy. 7 Mandavia R Schilder AGM Dimitriadis PA Mossialos E Addressing the challenges in tonsillectomy research to inform health care policy: a review. JAMA Otolaryngol Head Neck Surg. 2017; 143: 943-947 Crossref PubMed Scopus (10) Google Scholar Unfortunately, not much has changed in the past 40 years. National guidelines are still based on the Paradise criteria from 1984, 8 Paradise JL Bluestone CD Bachman RZ et al. Efficacy of tonsillectomy for recurrent throat infection in severely affected children—results of parallel randomized and nonrandomized clinical trials. N Engl J Med. 1984; 310: 674-683 Crossref PubMed Google Scholar stating that a tonsillectomy is indicated when around seven or more treated tonsillitis episodes occur in 1 year. The Paradise criteria were based on paediatrics clinical trials and later extrapolated for adult guidelines without any justifying research in the adult population. Thus, the 2014 Cochrane Review concluded that the effectiveness of tonsillectomy is low. 9 Burton MJ Glasziou PP Chong LY Venekamp RP Tonsillectomy or adenotonsillectomy versus non-surgical treatment for chronic/recurrent acute tonsillitis. Cochrane Database Syst Rev. 2014; 2014CD001802 PubMed Google Scholar Until now, there was no basis to make confident decisions using national guidelines. 7 Mandavia R Schilder AGM Dimitriadis PA Mossialos E Addressing the challenges in tonsillectomy research to inform health care policy: a review. JAMA Otolaryngol Head Neck Surg. 2017; 143: 943-947 Crossref PubMed Scopus (10) Google Scholar Conservative management versus tonsillectomy in adults with recurrent acute tonsillitis in the UK (NATTINA): a multicentre, open-label, randomised controlled trialCompared with conservative management, immediate tonsillectomy is clinically effective and cost-effective in adults with recurrent acute tonsillitis. Full-Text PDF Open Access

KLF14 regulates the growth of hepatocellular carcinoma cells via its modulation of iron homeostasis through the repression of iron-responsive element-binding protein 2

BackgroundHepatocellular carcinoma (HCC) is a multifactor-driven malignant tumor with rapid progression, which causes the difficulty to substantially improve the prognosis of HCC. Limited understanding of the mechanisms in HCC impedes the development of efficacious therapies. Despite Krüpple-Like factors (KLFs) were reported to be participated in HCC pathogenesis, the function of KLF14 in HCC remains largely unexplored.MethodsWe generated KLF14 overexpressed and silenced liver cancer cells, and nude mouse xenograft models for the in vitro and in vivo study. Luciferase reporter assay, ChIP-qPCR, Co-IP, immunofluorescence were performed for mechanism research. The expression of KLF14 in HCC samples was analyzed by quantitative RT-PCR, Western blotting, and immunohistochemistry (IHC) analysis.ResultsKLF14 was significantly downregulated in human HCC tissues, which was highly correlated with poor prognosis. Inhibition of KLF14 promoted liver cancer cells proliferation and overexpression of KLF14 suppressed cells growth. KLF14 exerts its anti-tumor function by inhibiting Iron-responsive element-binding protein 2 (IRP2), which then causes transferrin receptor-1(TfR1) downregulation and ferritin upregulation on the basis of IRP-IREs system. This then leading to cellular iron deficiency and HCC cells growth suppression in vitro and in vivo. Interestingly, KLF14 suppressed the transcription of IRP2 via recruiting SIRT1 to reduce the histone acetylation of the IRP2 promoter, resulting in iron depletion and cell growth suppression. More important, we found fluphenazine is an activator of KLF14, inhibiting HCC cells growth through inducing iron deficiency.ConclusionKLF14 acts as a tumor suppressor which inhibits the proliferation of HCC cells by modulating cellular iron metabolism via the repression of IRP2. We identified Fluphenazine, as an activator of KLF14, could be a potential compound for HCC therapy. Our findings therefore provide an innovative insight into the pathogenesis of HCC and a promising therapeutic target.

SIK2 inhibition enhances PARP inhibitor activity synergistically in ovarian and triple-negative breast cancers.

Poly(ADP-ribose) polymerase inhibitors (PARP inhibitors) have had an increasing role in the treatment of ovarian and breast cancers. PARP inhibitors are selectively active in cells with homologous recombination DNA repair deficiency caused by mutations in BRCA1/2 and other DNA repair pathway genes. Cancers with homologous recombination DNA repair proficiency respond poorly to PARP inhibitors. Cancers that initially respond to PARP inhibitors eventually develop drug resistance. We have identified salt-inducible kinase 2 (SIK2) inhibitors, ARN3236 and ARN3261, which decreased DNA double-strand break (DSB) repair functions and produced synthetic lethality with multiple PARP inhibitors in both homologous recombination DNA repair deficiency and proficiency cancer cells. SIK2 is required for centrosome splitting and PI3K activation and regulates cancer cell proliferation, metastasis, and sensitivity to chemotherapy. Here, we showed that SIK2 inhibitors sensitized ovarian and triple-negative breast cancer (TNBC) cells and xenografts to PARP inhibitors. SIK2 inhibitors decreased PARP enzyme activity and phosphorylation of class-IIa histone deacetylases (HDAC4/5/7). Furthermore, SIK2 inhibitors abolished class-IIa HDAC4/5/7–associated transcriptional activity of myocyte enhancer factor-2D (MEF2D), decreasing MEF2D binding to regulatory regions with high chromatin accessibility in FANCD2, EXO1, and XRCC4 genes, resulting in repression of their functions in the DNA DSB repair pathway. The combination of PARP inhibitors and SIK2 inhibitors provides a therapeutic strategy to enhance PARP inhibitor sensitivity for ovarian cancer and TNBC.

Deficiency of Integrin β4 Results in Increased Lung Tissue Stiffness and Responds to Substrate Stiffness via Modulating RhoA Activity.

The interaction between extracellular matrix (ECM) and epithelial cells plays a key role in lung development. Our studies found that mice with conditional integrin β4 (ITGB4) knockout presented lung dysplasia and increased stiffness of lung tissues. In accordance with our previous studies regarding the functions of ITGB4 in bronchial epithelial cells (BECs), we hypothesize that the decreased ITGB4 expression during embryonic stage leads to abnormal ECM remodeling and increased tissue stiffness, thus impairing BECs motility and compromising lung development. In this study, we examined lung tissue stiffness in normal and ITGB4 deficiency mice using Atomic Force Microscopy (AFM), and demonstrated that ITGB4 deficiency resulted in increased lung tissue stiffness. The examination of ECM components collagen, elastin, and lysyl oxidase (LOX) family showed that the expression of type VI collagen, elastin and LOXL4 were significantly elevated in the ITGB4-deficiency mice, compared with those in normal groups. Airway epithelial cell migration and proliferation capacities on normal and stiff substrates were evaluated through video-microscopy and flow cytometry. The morphology of the cytoskeleton was detected by laser confocal microscopy, and RhoA activities were determined by fluorescence resonance energy transfer (FRET) microscopy. The results showed that migration and proliferation of ITGB4 deficiency cells were noticeably inhibited, along decreased cytoskeleton stabilization, and hampered RhoA activity, especially for cells cultured on the stiff substrate. These results suggest that decreased ITGB4 expression results in increased lung tissue stiffness and impairs the adaptation of bronchial epithelial cells to substrate stiffness, which may be related to the occurrence of broncho pulmonary dysplasia.

Intracellular virus sensor MDA5 mutation develops autoimmune myocarditis and nephritis

Mutations in IFIH1 gene encoding viral RNA sensor MDA5 have been reported responsible for many interferonopathies, including Aicardi-Goutières syndrome (AGS) and monogenic lupus, however, the pathological link between IFIH1 mutations and various autoimmune symptoms remains unclear. Here, we generated transgenic mice expressing human MDA5 R779H mutant (R779H Tg), reported in AGS and monogenic lupus patient. Mice spontaneously developed myocarditis and nephritis with upregulation of type I IFNs in the major organs. R779H Tg Mavs−/− and R779H Tg Ifnar−/− showed no phenotypes, indicating direct MDA5-signaling pathway involvement. Rag-2 deficiency and bone marrow cells transfer from wild type to adult mice did not prevent myocarditis development, while mice with cardiomyocyte-specific expression of hMDA5 R779H showed cardiomegaly and high expression of inflammatory cytokines. Taken together, our study clarifies that type I IFNs production and chemokines from cardiomyocytes starts in neonatal period and is critical for the development of myocarditis. Activated lymphocytes and auto-antibodies exacerbate the pathogenesis but are dispensable for the onset.

Yes-associated protein contributes to magnesium alloy-derivedinflammation in endothelial cells.

Magnesium alloy (Mg alloy) has attracted massive attention in the potential applications of cardiovascular stents because of its good biocompatibility and degradability. However, whether and how the Mg alloy induces inflammation in endothelial cells remains unclear. In the present work, we investigated the activation of Yes-associated protein (YAP) upon Mg alloy stimuli and unveiled the transcriptional function in Mg alloy-induced inflammation. Quantitative RT–PCR, western blotting and immunofluorescence staining showed that Mg alloy inhibited the Hippo pathway to facilitate nuclear shuttling and activation of YAP in human coronary artery endothelial cells (HCAECs). Chromatin immunoprecipitation followed sequencing was carried out to explore the transcriptional function of YAP in Mg alloy-derived inflammation. This led to the observation that nuclear YAP further bonded to the promoter region of inflammation transcription factors and co-transcription factors. This binding event activated their transcription and modified mRNA methylation of inflammation-related genes through regulating the expression of N6-methyladenosine modulators (METTL3, METTL14, FTO and WTAP). This then promoted inflammation-related gene expression and aggravated inflammation in HCAECs. In YAP deficiency cells, Mg alloy-induced inflammation was reduced. Collectively, our data suggest that YAP contributes to the Mg alloy-derived inflammation in HCAECs and may provide a potential therapeutic target that alleviates inflammation after Mg alloy stent implantation.

MiR-181c regulates MCL1 and cell survival in GATA2 deficient cells.

GATA2 is a transcription factor critical for hematopoiesis. Germline mutations in GATA binding protein 2 (GATA2) led to haploinsufficiency, severe cytopenias of multiple cell lineages, susceptibility to infections and strong propensity to develop myelodysplastic syndrome, and acute myeloid leukemia. Mechanisms of progressive cytopenias remain unclear. MicroRNA (miRNA) represents a unique mechanism of post-transcriptional gene regulation. In this study, miRNA profiles were evaluated and eight miRNAs were found to be differentially expressed (≥2-fold, P ≤ 0.05) in patient-derived cell lines (N=13) in comparison to controls (N=10). miR-9, miR-181a-2-3p, miR-181c, miR-181c-3p, miR-486-3p, and miR-582 showed increased expression, whereas miR-223 and miR-424-3p showed decreased expression. Cell death assays indicated that miR-181c potently induces cell death in lymphoid (Ly-8 and SP-53) and myeloid (HL-60) cell lines. miR-181c was predicted to target myeloid cell leukemia (MCL)1, which was confirmed by transfection assays, resulting in significantly reduced MCL1 mRNA and decreased live cell numbers. Bone marrow analysis of 34 GATA2 patients showed significantly decreased cellularity, CD34-positive cells, monocytes, dendritic cells, NK cells, B cells, and B cell precursors in comparison to healthy controls (N=29; P<0.001 for each), which was accompanied by decreased levels of MCL1 (P<0.05). GATA2 expression led to significant repression of miR-181c expression in transfection experiments. Conversely, knockdown of GATA2 led to increased miR-181c expression. These findings indicate that miR-181c expression is increased and MCL1 levels decreased in GATA2 deficiency cells, and that GATA2 represses miR-181c transcription. Increased miR-181c may contribute to elevated cell death and cytopenia in GATA2 deficiency potentially through down-regulation of MCL1.

Abstract 365: Detection of homologous recombination deficiency across cancer types by a real-time activity-based functional biomarker

Abstract Homologous recombination (HR) is an error-free repair pathway to eliminate DNA double-strand breaks and crucial for maintaining genome stability. Pathogenic mutations in genes involved in HR, such as BRCA1 and BRCA2, lead to homologous recombination deficiency (HRD) and sensitive cells to poly(ADP-ribose) polymerase inhibitors (PARPi). As such, the selection of HRD cancer patients becomes an important clinical need. In addition to sequencing HR-related genes, recent studies have developed several approaches to detect HRD including genomic scar score, mutational signatures analysis, and RAD51 foci formation assay. However, these assays are not on a real-time basis and provide an indirect estimation of HR status. To overcome these limitations, we have successfully developed a virus-based functional assay to directly quantify HR activity in cells. Our method detects HR status in a real-time and tumor-only manner. By using this activity-based functional assay, we reveal a universal activity threshold for identifying HRD across cancer types. Here, we present a promising method to accurately detect primary ovarian cancer cells with HRD. Clinical samples that be quantified as HRD show a significant response to PARPi. Therefore, our method can serve as a functional biomarker and companion diagnostic for PARPi. Citation Format: Chih-Ying Lee, Kai-Hang Lei, Shih-Han Huang, Min-Yu Ko, Ko-Yu Chang, Po-Han Lin, Yu-Li Chen, Wen-Fang Cheng, Peter Chi. Detection of homologous recombination deficiency across cancer types by a real-time activity-based functional biomarker [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 365.

T-17, a spirostanol saponin, inhibits p53-independent proliferation and p53-dependent migration of gastric cancer cells

The p53 tumor suppressor gene contributes to a series of life processes of cells. Previously, we have shown that T-17, a spirostanol saponin extracted from Tupistra chinensis induces cell cycle arrest, apoptosis and autophagy in gastric cancer cells. The p53 is essential in the cell cycle arrest induced by T-17, however, the effect of p53 on T-17-induced apoptosis and autophagy is still unclear. Here, our study shows that T-17 has no difference in the sensitivity of gastric cancer cells with different p53 status. By transfecting p53 siRNA into AGS cells (p53 wild type cells) or wild-type p53 into KATO-III cells (p53 deficiency cells), T-17 was found to induce apoptosis and autophagy in gastric cancer cells in a p53-independent manner. Pre-treatment with N-acetylcysteine (NAC, a ROS scavenger) demonstrated that reactive oxygen species (ROS) mediated T-17-induced p53-independent apoptosis. Besides, T-17 induces apoptosis and autophagy in gastric cancer cells by decreasing the expression of HMGB1, also in a p53-independent manner. But when we detected the inhibitory effect of T-17 on gastric cancer cell migration, it was found that p53 is essential. These experimental results showed that T-17 induced apoptosis and autophagy in gastric cancer cells in a p53-independent manner, but inhibited the migration of gastric cancer cells in a p53-dependent manner. Our research indicates that T-17 is a potential candidate for gastric cancer and provides support for better utilization of Tupistra chinensis.

ADORA1 Promotes Hepatocellular Carcinoma Progression via PI3K/AKT Pathway.

ObjectiveHepatocellular carcinoma (HCC) is a common malignancy worldwide. Although the contradictory role of ADORA1 has been explored in certain types of cancers, its clinical significance and function in hepatocellular carcinoma cells are largely unknown.Materials and MethodsThe level of ADORA1 in HCC tissues and cells was evaluated by RT-PCR. The function of ADORA1 overexpression on HCC cell proliferation and invasion was assessed by MTS, transwell analysis, and colony formation assay. In addition, a mouse subcutaneous xenograft model was used to study in vivo effects. The efficacy of knockdown of ADORA1 sensitizes to chemotherapy was assessed by staining with Annexin V/propidium iodide followed with flow cytometry and nuclei fragmentation.ResultsIn this study, ADORA1 was identified to be up-regulated in HCC tissues compared with adjacent normal tissue. High ADORA1 mRNA expression predicted poor survival in hepatocellular carcinoma patients. Ectopic expression of ADORA1 increased hepatocellular carcinoma cell proliferation and invasion. ADORA1 knockdown inhibited HCC cell growth and sensitized to chemotherapy. Furthermore, ADORA1 activated PI3K/AKT oncogenic signaling pathways. Treatment with PI3K inhibitor LY294002 blocked the effects of ADORA1 on tumor growth in either ADORA1-overexpressing or -deficiency cells. Finally, overexpression of ADORA1 stimulates HCC tumor growth in vivo. Treatment of ADORA1 antagonist oppositely suppressed HCC xenograft tumor growth.ConclusionADORA1 serves as an important oncoprotein and a promoter of cell proliferation through PI3K/AKT signaling pathway in hepatocellular carcinoma.

Development of a 3D functional assay and identification of biomarkers, predictive for response of high-grade serous ovarian cancer (HGSOC) patients to poly-ADP ribose polymerase inhibitors (PARPis): targeted therapy

BackgroundPoly(ADP-ribose) polymerase inhibitors (PARPis) specifically target homologous recombination deficiency (HRD) cells and display good therapeutic effect in women with advanced-stage BRCA1/2-mutated breast and epithelial ovarian cancer (EOC). However, about 50% of high grade serous ovarian cancers (HGSOC) present with HRD due to epigenetic BRCA1 inactivation, as well as genetic/epigenetic inactivation(s) of other HR genes, a feature known as “BRCAness”. Therefore, there is a potential for extending the use of PARPis to these patients if HR status can be identified.MethodsWe have developed a 3D (spheroid) functional assay to assess the sensitivity of two PARPis (niraparib and olaparib) in ascites-derived primary cell cultures (AsPCs) from HGSOC patients. A method for AsPCs preparation was established based on a matrix (agarose), allowing for easy isolation and successive propagation of monolayer and 3D AsPCs. Based on this method, we performed cytotoxicity assays on 42 AsPCs grown both as monolayers and spheroids.ResultsThe response to PARPis treatment in monolayer AsPCs, was significantly higher, compared to 3D AsPCs, as 88% and 52% of the monolayer AsPCs displayed sensitivity to niraparib and olaparib respectively, while 66% of the 3D AsPCs were sensitive to niraparib and 38% to olaparib, the latter being more consistent with previous estimates of HRD (40%–60%) in EOC. Moreover, niraparib displayed a significantly stronger cytotoxic effect in both in 3D and monolayer AsPCs, which was confirmed by consecutive analyses of the HR pathway activity (γH2AX foci formation) in PARPis-sensitive and resistant AsPCs. Global gene expression comparison of 6 PARPi-resistant and 6 PARPi-sensitive 3D AsPCs was indicative for the predominant downregulation of numerous genes and networks with previously demonstrated roles in EOC chemoresistance, suggesting that the PARPis-sensitive AsPCs could display enhanced sensitivity to other chemotherapeutic drugs, commonly applied in cancer management. Microarray data validation identified 24 potential gene biomarkers associated with PARPis sensitivity. The differential expression of 7 selected biomarkers was consecutively confirmed by immunohistochemistry in matched EOC tumor samples.ConclusionThe application of this assay and the potential biomarkers with possible predictive significance to PARPis therapy of EOC patients now need testing in the setting of a clinical trial.

Tropisetron attenuates pancreas apoptosis in the STZ-induced diabetic rats: involvement of SIRT1/NF-κB signaling.

Diabetes mellitus (DM) is one of the most common diseases in the worldwide. Type 1 diabetes mellitus (T1DM) is characterized by insulin deficiency and beta cells apoptosis. Tropisetron as a 5-HT3 receptor antagonist has positive effects on the inflammation, apoptosis and glucose lowering. The aim of this study was to investigate the effect of tropisetron on β-cells apoptosis and its possible pathways. Animals were divided into five equal groups: the control, tropisetron, diabetes, tropisetron-DM and glibenclamide-DM (seven in each group). Tropisetron and glibenclamide were administrated for 2weeks after type 1 diabetes induction. Real-time PCR, western blot analysis and TUNEL assay were performed. We found that tropisetron decreased blood glucose and increased insulin secretion. Protein expression of NF-κB was downregulated, while protein expression of SIRT1 upregulated after tropisetron treatment. Moreover, Bax/Bcl2 ratio decreased in tropisetron-DM group and finally, apoptosis improved in pancreas tissue. It seems that tropisetron administration improves STZ-induced apoptosis and diabetes in the animals. This effect might be resulted from involvement in NF-κB/ SIRT1 pathway.

FBXL4 deficiency increases mitochondrial removal by autophagy

Pathogenic variants in FBXL4 cause a severe encephalopathic syndrome associated with mtDNA depletion and deficient oxidative phosphorylation. To gain further insight into the enigmatic pathophysiology caused by FBXL4 deficiency, we generated homozygous Fbxl4 knockout mice and found that they display a predominant perinatal lethality. Surprisingly, the few surviving animals are apparently normal until the age of 8–12 months when they gradually develop signs of mitochondrial dysfunction and weight loss. One‐year‐old Fbxl4 knockouts show a global reduction in a variety of mitochondrial proteins and mtDNA depletion, whereas lysosomal proteins are upregulated. Fibroblasts from patients with FBXL4 deficiency and human FBXL4 knockout cells also have reduced steady‐state levels of mitochondrial proteins that can be attributed to increased mitochondrial turnover. Inhibition of lysosomal function in these cells reverses the mitochondrial phenotype, whereas proteasomal inhibition has no effect. Taken together, the results we present here show that FBXL4 prevents mitochondrial removal via autophagy and that loss of FBXL4 leads to decreased mitochondrial content and mitochondrial disease.

PE_PGRS31-S100A9 Interaction Promotes Mycobacterial Survival in Macrophages Through the Regulation of NF-κB-TNF-α Signaling and Arachidonic Acid Metabolism.

Mycobacterium tuberculosis (M. tb) evades the surveillance of immune responses for survival in macrophages. However, the precise mechanism and toxins/proteins encoded by M. tb involved in the bacterial escape remain elusive. The function of Rv1768 protein (also referred to as PE_PGRS31, belonging to the PE_PGRS family) encoded by the region of deletion 14 (RD-14) in the virulent M. tb H37Rv strain has not, to the best of our knowledge, been reported previously. Here, we found that Rv1768 remarkably promotes bacterial survival in macrophages. Compared to wild type (WT) H37Rv, the Rv1768 deficient strain (H37RvΔ1768) showed significantly decreased colony-forming units in the lungs, spleen, and liver of the murine M. tb infection model. The bacterial burdens of WT H37Rv in WT macrophages and C57BL/6 mice were significantly higher than those in S100A9 deficiency cells and mice, but there were no significant differences for H37RvΔRv1768. Rv1768 binds S100A9 with the proline-glutamic acid domain (PE domain) and blocks the interaction between S100A9 and Toll-like receptor 4 (TLR4), and suppresses TLR4-myeloid differentiation factor 88-nuclear factor-kappa B (NF-κB)-tumor necrosis factor α (TNF-α) signaling in macrophages. Interestingly, Rv1768 binding to S100A9 also disturbs the metabolism of arachidonic acid by activating 5-lipoxygenase, increasing lipotoxin A4, and down-regulating cyclooxygenase-2 and prostaglandin E2 expression, thus, promoting mycobacterial survival. Our results revealed that M. tb Rv1768 promotes mycobacterial survival in macrophages by regulating NF-κB-TNF-α signaling and arachidonic acid metabolism via S100A9. Disturbing the interaction between Rv1768 and S100A9 may be a potential therapeutic target for tuberculosis.

TRNA Queuosine Modification Enzyme Modulates the Growth and Microbiome Recruitment to Breast Tumors.

Background: Transfer RNA (tRNA) queuosine (Q)-modifications occur specifically in 4 cellular tRNAs at the wobble anticodon position. tRNA Q-modification in human cells depends on the gut microbiome because the microbiome product queuine is required for its installation by the enzyme Q tRNA ribosyltransferase catalytic subunit 1 (QTRT1) encoded in the human genome. Queuine is a micronutrient from diet and microbiome. Although tRNA Q-modification has been studied for a long time regarding its properties in decoding and tRNA fragment generation, how QTRT1 affects tumorigenesis and the microbiome is still poorly understood. Results: We generated single clones of QTRT1-knockout breast cancer MCF7 cells using Double Nickase Plasmid. We also established a QTRT1-knockdown breast MDA-MB-231 cell line. The impacts of QTRT1 deletion or reduction on cell proliferation and migration in vitro were evaluated using cell culture, while the regulations on tumor growth in vivo were evaluated using a xenograft BALB/c nude mouse model. We found that QTRT1 deficiency in human breast cancer cells could change the functions of regulation genes, which are critical in cell proliferation, tight junction formation, and migration in human breast cancer cells in vitro and a breast tumor mouse model in vivo. We identified that several core bacteria, such as Lachnospiraceae, Lactobacillus, and Alistipes, were markedly changed in mice post injection with breast cancer cells. The relative abundance of bacteria in tumors induced from wildtype cells was significantly higher than those of QTRT1 deficiency cells. Conclusions: Our results demonstrate that the QTRT1 gene and tRNA Q-modification altered cell proliferation, junctions, and microbiome in tumors and the intestine, thus playing a critical role in breast cancer development.

Dnmt3b ablation impairs fracture repair through upregulation of Notch pathway.

We previously established that DNA methyltransferase 3b (Dnmt3b) is the sole Dnmt responsive to fracture repair and that Dnmt3b expression is induced in progenitor cells during fracture repair. In the current study, we confirmed that Dnmt3b ablation in mesenchymal progenitor cells (MPCs) resulted in impaired endochondral ossification, delayed fracture repair, and reduced mechanical strength of the newly formed bone in Prx1-Cre;Dnmt3bf/f (Dnmt3bPrx1) mice. Mechanistically, deletion of Dnmt3b in MPCs led to reduced chondrogenic and osteogenic differentiation in vitro. We further identified Rbpjκ as a downstream target of Dnmt3b in MPCs. In fact, we located 2 Dnmt3b binding sites in the murine proximal Rbpjκ promoter and gene body and confirmed Dnmt3b interaction with the 2 binding sites by ChIP assays. Luciferase assays showed functional utilization of the Dnmt3b binding sites in murine C3H10T1/2 cells. Importantly, we showed that the MPC differentiation defect observed in Dnmt3b deficiency cells was due to the upregulation of Rbpjκ, evident by restored MPC differentiation upon Rbpjκ inhibition. Consistent with in vitro findings, Rbpjκ blockage via dual antiplatelet therapy reversed the differentiation defect and accelerated fracture repair in Dnmt3bPrx1 mice. Collectively, our data suggest that Dnmt3b suppresses Notch signaling during MPC differentiation and is necessary for normal fracture repair.

MET promotes the proliferation and differentiation of myoblasts

The receptor tyrosine kinase MET plays a vital role in skeletal muscle development and in postnatal muscle regeneration. However, the effect of MET on myogenesis of myoblasts has not yet been fully understood. This study aimed to investigate the effects of MET on myogenesis in vivo and in vitro. Decreased myonuclei and down-regulated expression of myogenesis-related markers were observed in Met p.Y1232C mutant heterozygous mice. To explore the effects of MET on myoblast proliferation and differentiation, Met was overexpressed or interfered in C2C12 myoblast cells through the lentiviral transfection. The Met overexpression cells exhibited promotion in myoblast proliferation, while the Met deficiency cells showed impediment in proliferation. Moreover, myoblast differentiation was enhanced by the stable Met overexpression, but was impaired by Met deficiency. Furthermore, this study demonstrated that SU11274, an inhibitor of MET kinase activity, suppressed myoblast differentiation, suggesting that MET regulated the expression of myogenic regulatory factors (MRFs) and of desmin through the classical tyrosine kinase pathway. On the basis of the above findings, our work confirmed that MET promoted the proliferation and differentiation of myoblasts, deepening our understanding of the molecular mechanisms underlying muscle development.

Lack of mitochondrial NADP(H)-transhydrogenase expression in macrophages exacerbates atherosclerosis in hypercholesterolemic mice.

The atherosclerosis prone LDL receptor knockout mice (Ldlr-/-, C57BL/6J background) carry a deletion of the NADP(H)-transhydrogenase gene (Nnt) encoding the mitochondrial enzyme that catalyzes NADPH synthesis. Here we hypothesize that both increased NADPH consumption (due to increased steroidogenesis) and decreased NADPH generation (due to Nnt deficiency) in Ldlr-/- mice contribute to establish a macrophage oxidative stress and increase atherosclerosis development. Thus, we compared peritoneal macrophages and liver mitochondria from three C57BL/6J mice lines: Ldlr and Nnt double mutant, single Nnt mutant and wild-type. We found increased oxidants production in both mitochondria and macrophages according to a gradient: double mutant > single mutant > wild-type. We also observed a parallel up-regulation of mitochondrial biogenesis (PGC1a, TFAM and respiratory complexes levels) and inflammatory (iNOS, IL6 and IL1b) markers in single and double mutant macrophages. When exposed to modified LDL, the single and double mutant cells exhibited significant increases in lipid accumulation leading to foam cell formation, the hallmark of atherosclerosis. Nnt deficiency cells showed up-regulation of CD36 and down-regulation of ABCA1 transporters what may explain lipid accumulation in macrophages. Finally, Nnt wild-type bone marrow transplantation into LDLr-/- mice resulted in reduced diet-induced atherosclerosis. Therefore, Nnt plays a critical role in the maintenance of macrophage redox, inflammatory and cholesterol homeostasis, which is relevant for delaying the atherogenesis process.