NAD+ supplementation improves mitochondrial functions and normalizes glaucomatous trabecular meshwork features.

Glaucoma is characterized by pathological elevation of intraocular pressure (IOP) due to dysfunctional trabecular meshwork (TM), which is the primary cause of irreversible vision loss. There are currently no effective treatment strategies for glaucoma. Mitochondrial function plays a crucial role in regulating IOP within the TM. In this study, primary TM cells treated with dexamethasone were used to simulate glaucomatous changes, showing abnormal cellular cytoskeleton, increased expression of extracellular matrix, and disrupted mitochondrial fusion and fission dynamics. Furthermore, glaucomatous TM cell line GTM3 exhibited impaired mitochondrial membrane potential and phagocytic function, accompanied by decreased oxidative respiratory levels as compared to normal TM cells iHTM. Mechanistically, lower NAD + levels in GTM3, possibly associated with increased expression of key enzymes CD38 and PARP1 related to NAD + consumption, were observed. Supplementation of NAD + restored mitochondrial function and cellular viability in GTM3 cells. Therefore, we propose that the aberrant mitochondrial function in glaucomatous TM cells may be attributed to increased NAD + consumption dependent on CD38 and PARP1, and NAD + supplementation could effectively ameliorate mitochondrial function and improve TM function, providing a novel alternative approach for glaucoma treatment.

NGS panel enhance precise diagnosis of myeloid neoplasms under WHO-HAEM5 and International Consensus Classification: An observational study.

This study aimed to assess hematological diseases next-generation sequencing (NGS) panel enhances the diagnosis and classification of myeloid neoplasms (MN) using the 5th edition of the WHO Classification of Hematolymphoid Tumors (WHO-HAEM5) and the International Consensus Classification (ICC) of Myeloid Tumors. A cohort of 112 patients diagnosed with MN according to the revised fourth edition of the WHO classification (WHO-HAEM4R) underwent testing with a 141-gene NGS panel for hematological diseases. Ancillary studies were also conducted, including bone marrow cytomorphology and routine cytogenetics. The cases were then reclassified according to WHO-HAEM5 and ICC to assess the practical impact of these 2 classifications. The mutation detection rates were 93% for acute myeloid leukemia (AML), 89% for myelodysplastic syndrome (MDS), 94% for myeloproliferative neoplasm (MPN), and 100% for myelodysplasia/myeloproliferative neoplasm (MDS/MPN) (WHO-HAEM4R). NGS provided subclassified information for 26 and 29 patients with WHO-HAEM5 and ICC, respectively. In MPN, NGS confirmed diagnoses in 16 cases by detecting JAK2, MPL, or CALR mutations, whereas 13 "triple-negative" MPN cases revealed at least 1 mutation. NGS panel testing for hematological diseases improves the diagnosis and classification of MN. When diagnosed with ICC, NGS produces more classification subtype information than WHO-HAEM5.

VEGF-C and 5-Fluorouracil Improve Bleb Survival in a Rabbit Glaucoma Surgery Trabeculectomy Model.

Purpose: To evaluate VEGF-C-induced lymphoproliferation in conjunction with 5-fluorouracil (5-FU) antimetabolite treatment in a rabbit glaucoma filtration surgery (GFS) model. Methods: Thirty-two rabbits underwent GFS and were assigned to four groups (n = 8 each) defined by subconjunctival drug treatment: (a) VEGF-C combined with 5-FU, (b) 5-FU, (c) VEGF-C, (d) and control. Bleb survival, bleb measurements, and IOP were evaluated over 30 days. At the end, histology and anterior segment OCT were performed on some eyes. mRNA was isolated from the remaining eyes for RT-PCR evaluation of vessel-specific markers (lymphatics, podoplanin and LYVE-1; and blood vessels, CD31). Results: Qualitatively and quantitatively, VEGF-C combined with 5-FU resulted in blebs which were posteriorly longer and wider than the other conditions: vs. 5-FU (P = 0.043 for longer, P = 0.046 for wider), vs. VEGF-C (P < 0.001, P < 0.001) and vs. control (P < 0.001, P < 0.001). After 30 days, the VEGF-C combined with 5-FU condition resulted in longer bleb survival compared with 5-FU (P = 0.025), VEGF-C (P < 0.001), and control (P < 0.001). Only the VEGF-C combined with 5-FU condition showed a negative correlation between IOP and time that was statistically significant (r = -0.533; P = 0.034). Anterior segment OCT and histology demonstrated larger blebs for the VEGF-C combined with 5-FU condition. Only conditions including VEGF-C led to increased expression of lymphatic markers (LYVE-1, P < 0.001-0.008 and podoplanin, P = 0.002-0.011). Expression of CD31 was not different between the groups (P = 0.978). Conclusions: Adding VEGF-C lymphoproliferation to standard antimetabolite treatment improved rabbit GFS success and may suggest a future strategy to improve human GFSs.

Discovery of novel and selective farnesoid X receptor antagonists through structure-based virtual screening, preliminary structure-activity relationship study, and biological evaluation.

Farnesoid X receptor (FXR) is a bile acids receptor and plays a crucial role in regulating bile acids, lipids, and glucose metabolism. Previous research suggests that inhibiting FXR activation can be beneficial in reducing cholesterol and low-density lipoprotein cholesterol (LDL-C) levels, offering potential treatment options for metabolic syndrome with lipid disorders. Herein, we report p-acetylaminobenzene sulfonate derivatives as a novel scaffold of FXR antagonists by multistage screening. Among these derivatives, compound F44-A13 exhibited a half-maximal inhibitory concentration of 1.1 µM. Furthermore, compound F44-A13 demonstrated effective inhibition of FXR activation in cellular assays and exhibited high selectivity over eleven other nuclear receptors. Besides, compound F44-A13 significantly suppressed the regulation of FXR target genes Shp, Besp, and Cyp7a1, while reducing cholesterol levels in human hepatoma HepG2 cells. Pharmacological studies conducted on C57BL/6 mice further confirmed that compound F44-A13 had beneficial effects in reducing cholesterol, triglycerides, and LDL-C levels. These findings highlight that F44-A13 is a highly selective FXR antagonist that might serve as a useful molecule for further FXR studies as well as the development of FXR antagonists for the potential treatment of metabolic diseases with lipid disorders.

Single-cell detection of DMSO promoted HL-60 differentiation toward granulocyte based on DC-iDEP for medicine screening.

The most common form of leukemia in adults is acute leukemia. Drug differentiation control is an extremely critical treatment for acute leukemia. Unfortunately, current techniques detecting differentiation control experience long time and complex steps of verification hindering the pace of medicine discovery: flow cytometry and RT-PCR are highly accurate and efficient at a cost of inconvenient fluorescent labeling or a high risk of contamination; conventional staining leads to cell death unavailable for further pharmacological tests. There is a great interest in developing simple, fast, and non-invasive techniques to screen medicine. DC-iDEP is an emerging label-free identification technique taking advantage of the whole cell native biophysical property for sorting cell populations. Here, HL-60 cell line has been used as a model to study the differentiation process toward granulocytes and medicine efficacy. The results showed that DEP succeeded in detecting the DMSO promoted HL-60 differentiation degree by the weighted average characterization factor. This factor is related to the single cell biophysical property, which accumulates to generate differences in each population with distinct constitutions. Furthermore, cichoric acid was investigated to be capable of promoting DMSO-induced differentiation efficiently. Using the change induced by cichoric acid, the HL-60 medicine screening application has been first attempted based on DEP. A rapid, label-free medicine screening method has been established to monitor HL-60 differentiation toward granulocyte and has great potential for medicine screening.

Role of m6A modification in immune microenvironment of digestive system tumors.

Digestive system tumors are huge health problem worldwide, largely attributable to poor dietary choices. The role of RNA modifications in cancer development is an emerging field of research. RNA modifications are associated with the growth and development of various immune cells, which, in turn, regulate the immune response. The majority of RNA modifications are methylation modifications, and the most common type is the N6-methyladenosine (m6A) modification. Here, we reviewed the molecular mechanism of m6A in the immune cells and the role of m6A in the digestive system tumors. However, further studies are required to better understand the role of RNA methylation in human cancers for designing diagnostic and treatment strategies and predicting the prognosis of patients.

Detection of LPS-induced macrophage activation with single-cell resolution through DC insulator-based electrokinetic devices.

Macrophages are considered critical in the initiation, maintenance, and resolution of inflammation. Lipopolysaccharide (LPS)-induced inflammation is often used as a model to understand the cell inflammation responses. Current techniques identifying the LPS-induced inflammation are experiencing cell destruction or cell labeling or are based on the whole cell population information with low identification degree. This limits the detection process with time-consuming cytokine selection, low resolution of population heterogeneity, and unavailability for their next use. Direct current insulator-based electrokinetics (DC-iEK) is introduced to achieve an easier and noninvasive identification of inflamed cells with high resolution. A biophysical scale is also established first time for screening medicine in the treatment of inflammation. The new microfluidic design concentrates cells with applied voltages forming streamline providing more stable cell capture conditions and unique biophysical factors at different capture positions. The average electric field of the cell capture positions is recorded to characterize each cell population. The characterization value of macrophage decreases from to 1.61 × 104  V/m when treated with 0.1 mM LPS and to 1.42 × 104  V/m when treated with 1 mM LPS. By treating the inflamed macrophages with representative effective medicines, healing signals could also be detected by a newly established inflammation scale. The cells showed proliferation and functional activity after extraction. DC-iEK has provided an easy and noninvasive approach to identify inflammation for further fundamental and clinical precision medicine use.

Label-free microfluidic device reveals single cell phagocytic activity and screens plant medicine rapidly.

Phagocytic activity is an extremely important indicator that evaluates medicinal effects related to the immune system and functions to investigate the mechanism of how a drug works under conditions such as immunological regulation, immune tolerance, inflammation, cancer, etc. Current techniques based on flow cytometry, fluorescence imaging or numbering CFUs after cell lysis for detecting phagocytosis suffer from long terms of bacteria culturing and complex preparation steps for fluorescent labeling or require a large amount of cell samples to be tested. This study aims at developing a simple and fast method for testing the phagocytic activity of unlabeled and native cells, taking advantage of very high-resolution direct current insulator-based dielectrophoresis (DC-iDEP). The properties of cells are characterized by native whole cell biophysical properties. This strategy not only eliminates the time-consuming bacterial culture work after cell lysis, but also lowers the expenses of bacteria labeling. The introduction of microfluidics reduces the sample volume or reagent needed. The analysis of the biophysical property distributions of native cells and medicine treated cells may lead to a less expensive and rapid tool for evaluating medicinal effects. Furthermore, berberine was investigated for decreasing the phagocytic activity of macrophages and used for comparison of activities. This study works on establishing a label-free, unbiased, and non-destructive method to determine cell phagocytic activity and investigate its use in evaluating medicinal effects on phagocytosis in a single step within a short time.

iTRAQ-based proteomic analysis of 17ß-Estradiol-induced anti-proliferation and apoptosis in mouse thymic epithelial cells by disturbing ribosomal biogenesis.

Many evidences have suggested that estrogen was associated with thymic atrophy and suppressed thymocyte functions. Thymic epithelial cells (TECs), as a crucial constituent of thymic stroma support a unique microenvironment for thymocyte maturation, but the effects of estrogen on TECs were poorly understood. In our study, we found that 17ß-Estradiol (17ß-E2), one of the primary estrogens, could significantly inhibit cell proliferation, and cause cell cycle arrest in G2/M phase and apoptosis in mouse thymic epithelial cell line 1 (MTEC1 cells) with time- and dose- dependent. Above all, we provided the systemic and sufficient proteomic profiling of 17ß-E2 (50 nmol/L) acting on MTEC1 cells through isobaric tags for relative and absolute quantitation and LC-MS/MS (Liquid Chromatography Mass Spectrometry/Mass Spectrometry). A total of 71 differentially expressed proteins were identified, of which 61 were up-regulated and 10 were down-regulated. Particularly, the differential expression of abundant ribosomal proteins (RPs) was drawing our attention, including RPL3, RPL4, RPS11, RPL17, RPL5, RPS9, RPL13, RPL23A, RPLP2, RPS15A, and RPL29. Most of these proteins have been widely reported exerting extra-ribosomal function associated with the proliferation and apoptosis of distinct cell types, but not yet observed in TECs. Moreover, bioinformatics analysis revealed that disturbance of ribosomal biogenesis was closely related to the anti-proliferation and apoptosis in MTEC1 cells upon 17ß-E2. These data highlighted the possible mechanisms of 17ß-E2 on MTEC1 cells through showing adequate differential protein expression profiles. We inferred that 17ß-E2 induced anti-proliferation and apoptosis in MTEC1 cells in response to alterations of ribosome biogenesis and RPs expression, which will contribute to gaining insight into the internal mechanism of thymic degeneration and exploiting to treat autoimmune diseases in the future.

Discovery of Betulinic Acid Derivatives as Potent Intestinal Farnesoid X Receptor Antagonists to Ameliorate Nonalcoholic Steatohepatitis.

Farnesoid X receptor (FXR) has emerged as a promising therapeutic target for nonalcoholic steatohepatitis (NASH) because of its tightly interwoven relationship with bile acid homeostasis, inflammation, fibrosis, and glucose and lipid metabolism. Evidence showed that intestinal FXR antagonism exhibited remarkable metabolic improvements in mice. Herein, we developed a series of betulinic acid derivatives as potent intestinal FXR antagonists, and F6 was identified as the most potent one with an IC50 at 2.1 µM. F6 selectively inhibited intestinal FXR signaling and ameliorated the hepatic steatosis, inflammation, and fibrosis in Gubra-amylin NASH (GAN) and high-fat with methionine and choline deficiency (HFMCD) diet-induced NASH models. The beneficial effects were achieved by direct antagonism of intestinal FXR and feedback activation of hepatic FXR, thereby decreasing ceramides and repressing inflammasome activation in the liver. Collectively, our work substantially supports F6 as a promising drug candidate against NASH and demonstrates that antagonism of intestinal FXR signaling is a practical strategy for treating metabolic diseases.

Discovery of novel ataxia telangiectasia mutated (ATM) kinase modulators: Computational simulation, biological evaluation and cancer combinational chemotherapy study.

Ataxia-telangiectasia mutated (ATM) kinase is a serine/threonine protein kinase and plays a key role in DNA double-strand breaks repair. Thus, ATM is considered a promising target for radiotherapy and chemotherapy sensitizing. Herein, we report the discovery of ATM agonist A22 and inhibitor A41 by computational methods and further biological evaluation. Among them, A22 exhibited low cytotoxicity in vitro and might serve as a useful tool for ATM research. Moreover, we firstly proved that ATM inhibitors could sensitize Irinotecan and Etoposide in a time-dependent manner on MCF-7 and SW480 cells, antagonism in a short period treatment while synergy at a long-term treatment and ATM agonist worked in an opposite way of ATM inhibitors. Further mechanism study demonstrated that the antagonism effect of ATM inhibitors with chemotherapeutic agents in a short period was resulting from inhibiting the p53/p21 axis to accelerate G1/S phase cell-cycle transition and promote cell survival. Additionally, A41 displayed antitumor effects combined with a chemotherapeutic drug in the SW480 xenograft model, indicating that A41 is a promising ATM inhibitor, which could increase the antitumor effect of chemotherapeutic drugs in vivo. All in all, these findings will guide the combination of ATM inhibitors with chemotherapeutic agents in further preclinical and clinical studies.

Hyperglycemia induces corneal endothelial dysfunction through attenuating mitophagy.

Hyperglycemia increases the risk of corneal endothelial dysfunction, resulting in damage to corneal endothelial structure and function. However, the effect and mechanism of hyperglycemia-induced corneal endothelial damage remain elusive. In this study, we demonstrated that hyperglycemia reduced the expression of pump-related protein Na+/K+ ATPase and barrier-related protein ZO-1. Moreover, we found hyperglycemia caused abnormal changes of morphological mitochondria and dynamics in vitro. In addition, the decreased levels of mitophagy were further confirmed Western blotting and LC3B-Mitotracker Immunofluorescence. Exogenous application of mitophagy agonist carbonyl cyanide m-chlorophenyl hydrazine (CCCP) increases the expression of Na+/K+ ATPase and ZO-1 in corneal endothelial cells through up-regulated mitophagy in vitro. In addition, CCCP effectively reverses the phenomenon of corneal opacity and increased corneal thickness in diabetic mice. Therefore, our demonstrated the novel function of mitophagy in the pathogenesis of diabetic cornea endothelial dysfunction, and provide potential approach for treating diabetic corneal endothelial dysfunction.

Growth differentiation factor 11 attenuates cardiac ischemia reperfusion injury via enhancing mitochondrial biogenesis and telomerase activity.

It has been reported that growth differentiation factor 11 (GDF11) protects against myocardial ischemia/reperfusion (IR) injury, but the underlying mechanisms have not been fully clarified. Considering that GDF11 plays a role in the aging/rejuvenation process and that aging is associated with telomere shortening and cardiac dysfunction, we hypothesized that GDF11 might protect against IR injury by activating telomerase. Human plasma GDF11 levels were significantly lower in acute coronary syndrome patients than in chronic coronary syndrome patients. IR mice with myocardial overexpression GDF11 (oe-GDF11) exhibited a significantly smaller myocardial infarct size, less cardiac remodeling and dysfunction, fewer apoptotic cardiomyocytes, higher telomerase activity, longer telomeres, and higher ATP generation than IR mice treated with an adenovirus carrying a negative control plasmid. Furthermore, mitochondrial biogenesis-related proteins and some antiapoptotic proteins were significantly upregulated by oe-GDF11. These cardioprotective effects of oe-GDF11 were significantly antagonized by BIBR1532, a specific telomerase inhibitor. Similar effects of oe-GDF11 on apoptosis and mitochondrial energy biogenesis were observed in cultured neonatal rat cardiomyocytes, whereas GDF11 silencing elicited the opposite effects to oe-GDF11 in mice. We concluded that telomerase activation by GDF11 contributes to the alleviation of myocardial IR injury through enhancing mitochondrial biogenesis and suppressing cardiomyocyte apoptosis.

High expression of SRY-box transcription factor 30 associates with well differentiation, absent lymph node metastasis and predicts longer survival in nonsmall-cell lung cancer patients.

The present study aimed to investigate SRY-box transcription factor 30 (SOX30) expression in nonsmall-cell lung cancer (NSCLC) tumor tissues and adjacent noncancerous tissues, and further explore the correlation of tumor SOX30 expression with clinical characteristics and survival profiles in patients with NSCLC.Totally, 365 patients with NSCLC who underwent resection were screened, and SOX30 expression was detected in their tumor tissues and adjacent noncancerous tissues via immunohistochemistry (IHC) assay, which was assessed by a semiquantitative method considering the multiplying staining intensity score and staining density score. According to the tumor SOX30 expression, patients were categorized as tumor SOX30 low (IHC score ≤3) and high (IHC score 4-12) patients, the latter were further divided into tumor SOX30 high+ (IHC score 4-6), high++ (IHC score 7-9), and high+++ (IHC score 10-12) patients.SOX30 was downregulated in NSCLC tumor tissues compared with adjacent noncancerous tissues. Meanwhile, tumor SOX30 high expression associated with well differentiation, absent lymph node metastasis, decreased TNM stage, but did not associated with age, gender, history of smoke and drink, hypertension, hyperlipidemia, diabetes, tumor size, or carcinoembryonic antigen level. Both accumulating disease-free survival and overall survival were the longest in tumor SOX30 high+++ patients, followed by tumor SOX30 high++ patients, and tumor SOX30 high+ patients, and the shortest in tumor SOX30 low patients. Besides, tumor SOX30 high expression was an independent predictor for longer disease-free survival and overall survival.Tumor SOX30 exhibits the potential to be a novel biomarker for survival prediction of patients with NSCLC.

Suppressed epithelial-mesenchymal transition and cancer stem cell properties mediate the anti-cancer effects of ethyl pyruvate via regulation of the AKT/nuclear factor-κB pathway in prostate cancer cells.

Castration-resistant prostate cancer (CRPC) is a leading cause of mortality among cases of prostate cancer (PCa). Current treatment options for CRPC are limited. Ethyl pyruvate (EP), a lipophilic derivative of pyruvic acid, has been reported to have antitumor activities. In the present study, the efficacy of EP against PCa was investigated using two human PCa cell lines and a mouse xenograft tumor model. PC3 and CWR22RV1 cells were treated with EP, and cytotoxicity was evaluated via Cell Counting Kit-8 and colony formation assays, while cell cycle distribution was assessed by flow cytometry. Changes in cell migration and invasion caused by EP treatment were also evaluated with Transwell and wound healing assays, and changes in the expression of intracellular signaling pathway components were detected by western blotting. EP treatment reduced cell viability, induced G1 arrest, and activated the intrinsic apoptosis pathway. Additionally, the in vivo experiments revealed that EP administration markedly inhibited tumor growth. EP also reversed epithelial-mesenchymal transition and suppressed cancer stem cell properties in part through negative regulation of AKT/nuclear factor-κB signaling. These results indicate that EP has anticancer activity in vitro and in vivo, and is therefore a promising therapeutic agent for the treatment of PCa.

Integrated microRNA and mRNA sequencing analysis of age-related changes to mouse thymic epithelial cells.

MicroRNAs (miRNAs) play key roles in the regulation of gene expression during multiple physiological processes, including early development, differentiation, and ageing. However, their involvement in age-related thymic involution is not clear. In this study, we profiled the global transcriptome and miRNAome of thymic epithelial cells in 1- and 3-month-old male and female mice, and predicted the possible transcription factors and target genes of the four most significantly differentially expressed miRNAs (DEMs) (miR-183-5p, miR-199b-5p, miR-205-5p, and miR-200b-3p) by performing bioinformatics analyses. We also evaluated the relationships between the significantly DEMs and mRNAs. We performed quantitative polymerase chain reaction to confirm the changes in the expression of the miRNAs and their predicted target genes. We found that miR-183-5p, miR-199b-5p, miR-205-5p, and miR-200b-3p can be used as a biomarker group for mouse thymus development and involution. In addition, the predicted target genes (Ptpn4, Slc2a9, Pkib, Pecam1, and Prkdc), which were identified by mRNA sequencing analysis, were mainly involved in growth, development, and accelerated senescence. In conclusion, miRNAs and their predicted target genes likely play important roles in thymus development and involution. To the best of our knowledge, this is the first study to systematically analyze the relevance of miRNAs and their targets by mRNA sequencing in mouse thymic epithelial cells. © 2018 IUBMB Life, 70(7):678-690, 2018.

Structure, chain conformation, and immunomodulatory activity of the polysaccharide purified from Bacillus Calmette Guerin formulation.

A polysaccharide, coded as BDP, purified from the injection powder of Bacillus Calmette Guerin (BCG) polysaccharide and nucleic acid, was composed mainly of α-D-(1→4)-linked glucan with (1→6)-linked branches and trace amounts of fucose and mannose from the results of FT-IR, HPAEC-PAD and NMR spectrum. The Mw, Mn, Mz, and [Formula: see text] were determined to be 1.320×10(5)g/mol, 1.012×10(5)g/mol, 2.139×10(5)g/mol, and 21.8±3.2%nm by using HPSEC-MALLS, respectively. The ν value from [Formula: see text] was calculated to be 0.52±0.01, which firstly clarified that BDP existed as random coils in 0.9% NaCl aqueous solution. AFM and SEM combined with Congo-red test also revealed that the polysaccharide was irregular globular like or curly structure. Furthermore, in vitro tests on RAW264.7 murine macrophages cells revealed that BDP exhibited significant immunomodulatory activity.