Characterization, biogenesis model, and current bioinformatics of human extrachromosomal circular DNA.

Human extrachromosomal circular DNA, or eccDNA, has been the topic of extensive investigation in the last decade due to its prominent regulatory role in the development of disorders including cancer. With the rapid advancement of experimental, sequencing and computational technology, millions of eccDNA records are now accessible. Unfortunately, the literature and databases only provide snippets of this information, preventing us from fully understanding eccDNAs. Researchers frequently struggle with the process of selecting algorithms and tools to examine eccDNAs of interest. To explain the underlying formation mechanisms of the five basic classes of eccDNAs, we categorized their characteristics and functions and summarized eight biogenesis theories. Most significantly, we created a clear procedure to help in the selection of suitable techniques and tools and thoroughly examined the most recent experimental and bioinformatics methodologies and data resources for identifying, measuring and analyzing eccDNA sequences. In conclusion, we highlighted the current obstacles and prospective paths for eccDNA research, specifically discussing their probable uses in molecular diagnostics and clinical prediction, with an emphasis on the potential contribution of novel computational strategies.

Fluoxetine Successfully Treats Intracranial Enterovirus E18 Infection in a Patient with CD79a Deficiency Arising from Segmental Uniparental Disomy of Chromosome 19.

The pre BCR complex plays a crucial role in B cell production, and its successful expression marks the B cell differentiation from the pro-B to pre-B. The CD79a and CD79b mutations, encoding Igα and Igß respectively, have been identified as the cause of autosomal recessive agammaglobulinemia (ARA). Here, we present a case of a patient with a homozygous CD79a mutation, exhibiting recurrent respiratory infections, diarrhea, growth and development delay, unique facial abnormalities and microcephaly, as well as neurological symptoms including tethered spinal cord, sacral canal cyst, and chronic enteroviral E18 meningitis. Complete blockade of the early B cell development in the bone marrow of the patient results in the absence of peripheral circulating mature B cells. Whole exome sequencing revealed a Loss of Heterozygosity (LOH) of approximately 19.20Mb containing CD79a on chromosome 19 in the patient. This is the first case of a homozygous CD79a mutation caused by segmental uniparental diploid (UPD). Another key outcome of this study is the effective management of long-term chronic enteroviral meningitis using a combination of intravenous immunoglobulin (IVIG) and fluoxetine. This approach offers compelling evidence of fluoxetine's utility in treating enteroviral meningitis, particularly in immunocompromised patients.

Protective effects of the bioactive peptide from maggots against skin flap ischemia‒reperfusion injury in rats.

Ischemia‒reperfusion (I/R) injury is a frequently observed complication after flap surgery, and it affects skin flap survival and patient prognosis. Currently, there are no proven safe and effective treatment options to treat skin flap I/R injury. Herein, the potential efficacies of the bioactive peptide from maggots (BPM), as well as its underlying mechanisms, were explored in a rat model of skin flap I/R injury and LPS- or H2O2-elicited RAW 264.7 cells. We demonstrated that BPM significantly ameliorated the area of flap survival, and histological changes in skin tissue in vivo. Furthermore, BPM could markedly restore or enhance Nrf2 and HO-1 levels, and suppress the expression of pro-inflammatory cytokines, including TLR4, p-IκB, NFκB p65, p-p65, IL-6, and TNF-α in I/R-injured skin flaps. In addition, BPM treatment exhibited excellent biocompatibility with an adequate safety profile, while it exhibited superior ROS-scavenging ability and the upregulation of antioxidant enzymes in vitro. Mechanistically, the above benefits related to BPM involved the activation of Nrf2/HO-1 and suppression of TLR4/NF-κB pathway. Taken together, this study may provide a scientific basis for the potential therapeutic effect of BPM in the prevention of skin flap I/R injury and other related diseases.

The characteristics and prognosis of different disease patterns of multiple primary lung cancers categorized according to the 8th edition lung cancer staging system.

INTRODUCTION: The 8th edition lung cancer staging system was the first to describe the detailed diagnosis and staging of multiple primary lung cancers (MPLC). However, the characteristics and prognosis of MPLC categorized according to the new system have not been evaluated. METHOD: We retrospectively analyzed data from surgically treated MPLC patients in a single center from 2011 to 2013 and explored the characteristics and outcomes of different MPLC disease patterns. RESULTS: In total, 202 surgically treated MPLC patients were identified and classified into different groups according to disease categories and diagnostic time (multifocal ground glass/lepidic (GG/L) nodules: n = 139, second primary lung cancer (SPLC): n = 63, simultaneous MPLC (sMPLC): n = 171, and metachronous MPLC (mMPLC): n = 31). There were significant differences in clinical characteristics between SPLC and GG/L nodule patients and simultaneous and metachronous MPLC patients. The overall 1-, 3-, and 5-year lung cancer-specific survival rates of MPLC were 97.98%, 90.18%, and 82.81%, respectively. Five-year survival was better in patients with multiple GG/L nodules than in those with SPLC (87.94% vs. 71.29%, P < 0.05). Sex was an independent prognostic factor for sMPLC (5-year survival, female vs. male, 88.0% vs. 69.5%, P < 0.05), and in multiple tumors, the highest tumor stage was an independent prognostic factor for all categories of MPLC. CONCLUSIONS: The different disease patterns of MPLC have significantly different characteristics and prognoses. Clinicians should place treatment emphasis on the tumor with the highest stage as it is the main contributor to the prognosis of all categories of MPLC patients.

Application of Chlorophyll Fluorescence Analysis Technique in Studying the Response of Lettuce (Lactuca sativa L.) to Cadmium Stress.

To reveal the impact of cadmium stress on the physiological mechanism of lettuce, simultaneous determination and correlation analyses of chlorophyll content and photosynthetic function were conducted using lettuce seedlings as the research subject. The changes in relative chlorophyll content, rapid chlorophyll fluorescence induction kinetics curve, and related chlorophyll fluorescence parameters of lettuce seedling leaves under cadmium stress were detected and analyzed. Furthermore, a model for estimating relative chlorophyll content was established. The results showed that cadmium stress at 1 mg/kg and 5 mg/kg had a promoting effect on the relative chlorophyll content, while cadmium stress at 10 mg/kg and 20 mg/kg had an inhibitory effect on the relative chlorophyll content. Moreover, with the extension of time, the inhibitory effect became more pronounced. Cadmium stress affects both the donor and acceptor sides of photosystem II in lettuce seedling leaves, damaging the electron transfer chain and reducing energy transfer in the photosynthetic system. It also inhibits water photolysis and decreases electron transfer efficiency, leading to a decline in photosynthesis. However, lettuce seedling leaves can mitigate photosystem II damage caused by cadmium stress through increased thermal dissipation. The model established based on the energy captured by a reaction center for electron transfer can effectively estimate the relative chlorophyll content of leaves. This study demonstrates that chlorophyll fluorescence techniques have great potential in elucidating the physiological mechanism of cadmium stress in lettuce, as well as in achieving synchronized determination and correlation analyses of chlorophyll content and photosynthetic function.

RASFF1A inhibits the epithelial-mesenchymal transition of lens epithelial cells induced by TGFß through regulating HDAC6.

To explore the role of Ras-association domain family 1 A (RASSF1A) in TGFß2-induced changes of lens epithelial cells (LECs) behavior. The human LEC line SRA01/04 cells were treated with TGFß2 in the presence or absence of RASSF1A and histone deacetylase 6 (HDAC6). qRT-PCR and western blot were performed to analysis mRNA and proteins expression. Cell proliferation was evaluated using MTT assay and colony formation assay. Transwell and scratch-wound healing assays were conducted to detected cell migration ability. RASSF1A was downregulated in TGFß2-induced SRA01/04 cells. RASSF1A overexpression inhibited the cell viability, colony formation and migration abilities of SRA01/04 cells induced by TGFß2. Overexpression of RASSF1A suppressed TGFß2-induced EMT of SRA01/04 cells, which was manifested as inhibition of EMT-related proteins α-SMA, Vimentin, Snail and Fn expression. Moreover, RASSF1A down-regulated the expression of HDAC6. Importantly, HDAC6 reversed the effects of RASSF1A on SRA01/04 cells. These findings indicate that RASSF1A prevented TGFß2-induced proliferation, migration, and EMT of LECs by regulating HDAC6 expression, suggesting that RASSF1A holds promise as a potential target for cataracts treatment.

Opportunistic lung cancer screening with low-dose computed tomography in National Cancer Center of China: The first 14 years' experience.

BACKGROUND: In China, over 50% of lung cancer cases occur in nonsmokers. Thus, identifying high-risk individuals for targeted lung cancer screening is crucial. Beyond age and smoking, determining other risk factors for lung cancer in the Asian population has become a focal point of research. Using 30,000 participants in the prospectively enrolled cohort at China's National Cancer Center (NCC) over the past 14 years, we categorized participants by risk, with an emphasis on nonsmoking females. MATERIALS AND METHODS: Between November 2005 and December 2019, 31,431 individuals voluntarily underwent low-dose computed tomography (LDCT) scans for lung cancer screening at the NCC. We recorded details like smoking history, exposure to hazards, and family history of malignant tumors. Using the 2019 NCCN criteria, participants were categorized into high-, moderate-, and low-risk groups. Additionally, we separated non-high-risk groups into female never smokers (aged over 40) exposed to second-hand smoke (SHS) and others. Any positive results from initial scans were monitored per the I-ELCAP protocol (2006), and suspected malignancies were addressed through collaborative decisions between patients and physicians. We analyzed and compared the detection rates of positive results, confirmed lung cancers, and cancer stages across risk, age, and gender groups. RESULTS: Out of 31,431 participants (55.9% male, 44.1% female), 3695 (11.8%) showed positive baseline LDCT scans with 197 (0.6%; 106 females, 91 males) confirmed as lung cancer cases pathologically. Malignancy rate by age was 0.1% among those aged under 40 years, 0.4% among those aged 40-49 years, 0.8% among those aged 50-59 years, and 1.2% among those aged 60 years and older. From the 25,763 participants (56.9% male, 43.1% female) who completed questionnaires, 1877 (7.3%) were categorized as high risk, 6500 (25.2%) as moderate risk, and 17,386 (67.5%) as low risk. Of the 23,886 in the non-high-risk category, 8041 (33.7%) were females over 40 years old exposed to SHS. The high-risk group showed the highest lung cancer detection rate at 1.4%. However, females exposed to SHS had a notably higher detection rate than the rest of the non-high-risk group (1.1% vs. 0.5%; p < 0.0001). In this cohort, 84.8% of the detected lung cancers were at an early stage. CONCLUSIONS: In our study, using LDCT for lung cancer screening proved significant for high-risk individuals. For non-high-risk populations, LDCT screening could be considered for nonsmoking women with exposure to SHS.

Novel Pharmaceutical Cocrystals of Tegafur: Synthesis, Performance, and Theoretical Studies.

PURPOSE: Tegafur (TF) is one of the most important clinical antitumor drugs with poor water solubility, severely reducing its bioavailability. This work develops new cocrystals to improve the solubility of TF and systematically investigates the intermolecular interactions to provide new insights into the formation of cocrystal and changes in physicochemical properties. METHOD: In this paper, two new 1:1 cocrystals of TF with 2,4 dihydroxybenzoic acid (2,4HBA) and p-nitrophenol (PNP) were synthesized. The cocrystal products were identified and characterized by various solid state analysis techniques. And the high performance liquid chromatography (HPLC) was conducted to determine the solubility and dissolution rate of TF and cocrystals. Moreover, the quantum chemistry calculations of crystal structure provided theoretical support for the results. RESULT: Compared with pure TF, the solubility and dissolution rate of TF-2,4HBA is significantly increased in a pH 6.8 buffer at 37°C. Under accelerated storage conditions (40°C, 75% RH), all cocrystal exhibits excellent stability over 8 weeks. Hirshfeld surface (HS) analysis, atoms in molecules (AIM) analysis, interaction region indicator (IRI) analysis, molecular electrostatic potential surface (MEPS) analysis and frontier molecular orbital (HOMO-LUMO) analysis were integrated to understand the hydrogen bonding interaction more comprehensively. The simulation results are in good agreement with the experimental data. The results show that the analysis of physical and chemical properties of TF-PNP cocrystal and TF crystal by quantum chemistry method is reliable at molecular level. CONCLUSION: These results are helpful to provide guiding methods in the cocrystal development and theoretical study of tegafur.

Melatonin decreases GSDME mediated mesothelial cell pyroptosis and prevents peritoneal fibrosis and ultrafiltration failure.

Peritoneal fibrosis together with increased capillaries is the primary cause of peritoneal dialysis failure. Mesothelial cell loss is an initiating event for peritoneal fibrosis. We find that the elevated glucose concentrations in peritoneal dialysate drive mesothelial cell pyroptosis in a manner dependent on caspase-3 and Gasdermin E, driving downstream inflammatory responses, including the activation of macrophages. Moreover, pyroptosis is associated with elevated vascular endothelial growth factor A and C, two key factors in vascular angiogenesis and lymphatic vessel formation. GSDME deficiency mice are protected from high glucose induced peritoneal fibrosis and ultrafiltration failure. Application of melatonin abrogates mesothelial cell pyroptosis through a MT1R-mediated action, and successfully reduces peritoneal fibrosis and angiogenesis in an animal model while preserving dialysis efficacy. Mechanistically, melatonin treatment maintains mitochondrial integrity in mesothelial cells, meanwhile activating mTOR signaling through an increase in the glycolysis product dihydroxyacetone phosphate. These effects together with quenching free radicals by melatonin help mesothelial cells maintain a relatively stable internal environment in the face of high-glucose stress. Thus, Melatonin treatment holds some promise in preserving mesothelium integrity and in decreasing angiogenesis to protect peritoneum function in patients undergoing peritoneal dialysis.

Generation of a human iPSC line (CHCMUi002-A) from peripheral blood mononuclear cells from a chronic granulomatous disease patient.

Chronic granulomatous disease (CGD) is a rare X-linked recessive primary immunodeficiency disease (PID). Herein, a human induced pluripotent stem cell (iPSC) line was generated from the peripheral blood mononuclear cells (PBMCs) of a CGD patient with a mutation (c.785_786delTT) in the CYBB gene. These iPSCs showed the expression of pluripotency markers, the ability to differentiate into three germ layers. They offer a promising technique for studying the pathogenesis and conducting drug screening for CGD patients.

Analysis of Cadmium Contamination in Lettuce (Lactuca sativa L.) Using Visible-Near Infrared Reflectance Spectroscopy.

In order to rapidly and accurately monitor cadmium contamination in lettuce and understand the growth conditions of lettuce under cadmium pollution, lettuce is used as the test material. Under different concentrations of cadmium stress and at different growth stages, relative chlorophyll content of lettuce leaves, the cadmium content in the leaves, and the visible-near infrared reflectance spectra are detected and analyzed. An inversion model of the cadmium content and relative chlorophyll content in the lettuce leaves is established. The results indicate that cadmium concentrations of 1 mg/kg and 5 mg/kg promote relative chlorophyll content, while concentrations of 10 mg/kg and 20 mg/kg inhibit relative chlorophyll content. The cadmium content in the leaves increases with increasing cadmium concentrations. Cadmium stress caused a "blue shift" in the red edge position only during the mature period, while the red valley position underwent a "blue shift" during the seedling and growth periods and a "red shift" during the mature period. The green peak position exhibited a "blue shift". After model validation, it was found that the model constructed using the ratio of red edge area to yellow edge area and the normalized values of red edge area and yellow edge area effectively estimated the cadmium content in lettuce leaves. The model established using the normalized vegetation index of the red edge and the ratio of the peak green value to red shoulder amplitude can effectively estimate the relative chlorophyll content in lettuce leaves. This study demonstrates that the visible-near infrared spectroscopy technique holds great potential for monitoring cadmium contamination and estimating chlorophyll content in lettuce.

Nanocarrier-mediated curcumin delivery: An adjuvant strategy for CNS disease treatment.

Neurological disorders are a major global challenge, which counts for a substantial slice of disease burden around the globe. In these, the challenging landscape of central nervous system (CNS) diseases, including Alzheimer's disease, Parkinson's disease, multiple sclerosis, and neuro-AIDS, demands innovative and novel therapeutic approaches. Curcumin, a versatile natural compound with antioxidant and anti-inflammatory properties, shows great potential as a CNS adjuvant therapy. However, its limited bioavailability and suboptimal permeability to the blood-brain barrier (BBB) hamper the therapeutic efficacy of curcumin. This review explores how nanocarrier facilitates curcumin delivery, which has shown therapeutic efficacy for various non-CNS diseases, for example, cancers, and can also revolutionize the treatment outcomes in patients with CNS diseases. Toward this, intranasal administration of curcumin as a non-invasive CNS drug delivery route can also aid its therapeutic outcomes as an adjuvant therapy for CNS diseases. Intranasal delivery of nanocarriers with curcumin improves the bioavailability of curcumin and its BBB permeability, which is instrumental in promoting its therapeutic potential. Furthermore, curcumin's inhibitory effect on efflux transporters will help to enhance the BBB and cellular permeability of various CNS drugs. The therapeutic potential of curcumin as an adjuvant has the potential to yield synergistic effects with CNS drugs and will help to reduce CNS drug doses and improve their safety profile. Taken together, this approach holds a promise for reshaping CNS disease management by maximizing curcumin's and other drugs' therapeutic benefits.

Curcumin enhances elvitegravir concentration and alleviates oxidative stress and inflammatory response.

In this study, we investigated the potential of using curcumin (CUR) as an adjuvant to enhance the delivery of antiretroviral drug elvitegravir (EVG) across the BBB, and alleviate oxidative stress and inflammatory response, which are the major hallmark of HIV neuropathogenesis. In a mouse model, we compared the biodistribution of EVG alone and in combination with CUR using intraperitoneal (IP) and intranasal (IN) routes. IN administration showed a significantly higher accumulation of EVG in the brain, while both IP and IN routes led to increased EVG levels in the lungs and liver. The addition of CUR further enhanced EVG brain delivery, especially when administered via the IN route. The expression of neural marker proteins, synaptophysin, L1CAM, NeuN, and GFAP was not significantly altered by EVG or CUR alone or their combination, indicating preserved neural homeostasis. After establishing improved brain concentration and safety of CUR-adjuvanted EVG in mice in acute treatment, we studied the effect of this treatment in HIV-infected U1 macrophages. In U1 macrophages, we also observed that the addition of CUR enhanced the intracellular concentration of EVG. The total area under the curve (AUCtot) for EVG was significantly higher in the presence of CUR. We also evaluated the effects of CUR on oxidative stress and antioxidant capacity in EVG-treated U1 macrophages. CUR reduced oxidative stress, as evidenced by decreased reactive oxygen species (ROS) levels and elevated antioxidant enzyme expression. Furthermore, the combination of CUR and EVG exhibited a significant reduction in proinflammatory cytokines (TNFα, IL-1ß, IL-18) and chemokines (RANTES, MCP-1) in U1 macrophages. Additionally, western blot analysis confirmed the decreased expression of IL-1ß and TNF-α in EVG + CUR-treated cells. These findings suggest the potential of CUR to enhance EVG permeability to the brain and subsequent efficacy of EVG, including HIV neuropathogenesis.

Concurrent fabry disease and immunoglobulin a nephropathy: a case report.

BACKGROUND: Fabry disease (FD) is an X-linked, hereditary dysfunction of glycosphingolipid storage caused by mutations in the GLA gene encoding alpha-galactosidase A enzyme. In rare cases, FD may coexist with immunoglobulin A nephropathy (IgAN). We describe a case of concurrent FD, IgAN, and dilated cardiomyopathy-causing mutations in the TTN and BAG3 genes, which has not been reported previously. CASE PRESENTATION: A 60-year-old female patient was admitted with a one-week history of facial and lower-limb edema, two-year history of left ventricular hypertrophy and sinus bradycardia, and recurring numbness and pain in three lateral digits with bilateral thenar muscle atrophy. Renal biopsy revealed concurrent FD (confirmed via an alpha-galactosidase A enzyme assay, Lyso-GL-3 quantification, and GLA gene sequencing) and IgAN. Heterozygous mutations in the TTN (c.30,484 C > A;p.P10162T) and BAG3 (c.88 A > G;p.I30V) genes were observed. The patient reported that two of her brothers had undergone kidney transplantation; one died suddenly at 60 years of age, and the other required a cardiac pacemaker. The 35-year-old son of the patient was screened for the GLA gene mutation and found to be positive for the same mutation as the patient. The patient was administered oral losartan (50 mg/day). Enzyme replacement therapy was refused due to financial reasons. Her renal and cardiac functions were stable yet worth closely monitoring during follow-up. CONCLUSION: The family history of patients with concurrent heart and renal diseases should be assessed in detail. Genetic testing and histological examinations are essential for diagnosing FD with IgAN.

Transcriptome and Lipidomic Analysis Suggests Lipid Metabolism Reprogramming and Upregulating SPHK1 Promotes Stemness in Pancreatic Ductal Adenocarcinoma Stem-like Cells.

Cancer stem cells (CSCs) are considered to play a key role in the development and progression of pancreatic ductal adenocarcinoma (PDAC). However, little is known about lipid metabolism reprogramming in PDAC CSCs. Here, we assigned stemness indices, which were used to describe and quantify CSCs, to every patient from the Cancer Genome Atlas (TCGA-PAAD) database and observed differences in lipid metabolism between patients with high and low stemness indices. Then, tumor-repopulating cells (TRCs) cultured in soft 3D (three-dimensional) fibrin gels were demonstrated to be an available PDAC cancer stem-like cell (CSLCs) model. Comprehensive transcriptome and lipidomic analysis results suggested that fatty acid metabolism, glycerophospholipid metabolism, and, especially, the sphingolipid metabolism pathway were mostly associated with CSLCs properties. SPHK1 (sphingosine kinases 1), one of the genes involved in sphingolipid metabolism and encoding the key enzyme to catalyze sphingosine to generate S1P (sphingosine-1-phosphate), was identified to be the key gene in promoting the stemness of PDAC. In summary, we explored the characteristics of lipid metabolism both in patients with high stemness indices and in novel CSLCs models, and unraveled a molecular mechanism via which sphingolipid metabolism maintained tumor stemness. These findings may contribute to the development of a strategy for targeting lipid metabolism to inhibit CSCs in PDAC treatment.

Ginsenoside Rg1 regulates autophagy and endoplasmic reticulum stress via the AMPK/mTOR and PERK/ATF4/CHOP pathways to alleviate alcohol­induced myocardial injury.

It has been reported that ginsenoside Rg1 (G­Rg1) can alleviate alcoholic liver injury, cardiac hypertrophy and myocardial ischemia, as well as reperfusion injury. Therefore, the present study aimed to investigate the role of G­Rg1 in alcohol­induced myocardial injury, as well as to elucidate its underlying mechanisms of action. For this purpose, H9c2 cells were stimulated with ethanol. Subsequently, H9c2 cell viability and apoptosis were determined using a Cell Counting Kit­8 assay and flow cytometric analysis, respectively. The levels of lactate dehydrogenase and caspase­3 in the H9c2 cell culture supernatant were detected using corresponding assay kits. In addition, the expression of green fluorescent protein (GFP)­light chain 3 (LC3) and that of C/EBP homologous protein (CHOP) were evaluated using GFP­LC3 assay and immunofluorescence staining, respectively. The expression levels of apoptosis­, autophagy­, endoplasmic reticulum stress (ERS)­ and adenosine 5'­monophosphate­activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) pathway­related proteins were detected using western blot analysis. The results revealed that treatment with G­Rg1 enhanced the viability and suppressed the apoptosis of ethanol­stimulated H9c2 cells. G­Rg1 also attenuated autophagy and ERS in ethanol­stimulated H9c2 cells. In addition, the levels of phosphorylated (p)­protein kinase R (PKR)­like ER kinase (PERK), p­eukaryotic translation initiation factor 2a, activating transcription factor 4 (ATF4), CHOP, caspase­12 and p­AMPK were downregulated, while the p­mTOR level was upregulated in ethanol­stimulated H9c2 cells treated with G­Rg1. Furthermore, the co­treatment of G­Rg1­treated ethanol­stimulated H9c2 cells with AICAR, an AMPK agonist, or CCT020312, a PERK agonist, inhibited cell viability and promoted cell apoptosis, autophagy and ERS. Overall, the results of the present study suggest that G­Rg1 suppresses autophagy and ERS via inhibiting the AMPK/mTOR and PERK/ATF4/CHOP pathways to alleviate ethanol­induced H9c2 cell injury.

Gαi1/3 mediate Netrin-1-CD146-activated signaling and angiogenesis.

Netrin-1 binds to the high-affinity receptor CD146 to activate downstream signaling and angiogenesis. Here, we examine the role and underlying mechanisms of G protein subunit alpha i1 (Gαi1) and Gαi3 in Netrin-1-induced signaling and pro-angiogenic activity. In mouse embryonic fibroblasts (MEFs) and endothelial cells, Netrin-1-induced Akt-mTOR (mammalian target of rapamycin) and Erk activation was largely inhibited by silencing or knockout of Gαi1/3, whereas signaling was augmented following Gαi1/3 overexpression. Netrin-1 induced Gαi1/3 association with CD146, required for CD146 internalization, Gab1 (Grb2 associated binding protein 1) recruitment and downstream Akt-mTOR and Erk activation. Netrin-1-induced signaling was inhibited by CD146 silencing, Gab1 knockout, or Gαi1/3 dominant negative mutants. Netrin-1-induced human umbilical vein endothelial cell (HUVEC) proliferation, migration and tube formation were inhibited by Gαi1/3 short hairpin RNA (shRNA), but were potentiated by ectopic Gαi1/3 overexpression. In vivo, intravitreous injection of Netrin-1 shRNA adeno-associated virus (AAV) significantly inhibited Akt-mTOR and Erk activation in murine retinal tissues and reduced retinal angiogenesis. Endothelial knockdown of Gαi1/3 significantly inhibited Netrin1-induced signaling and retinal angiogenesis in mice. Netrin-1 mRNA and protein expression were significantly elevated in retinal tissues of diabetic retinopathy (DR) mice. Importantly, silence of Netrin-1, by intravitreous Netrin-1 shRNA AAV injection, inhibited Akt-Erk activation, pathological retinal angiogenesis and retinal ganglion cells degeneration in DR mice. Lastly, Netrin-1 and CD146 expression is significantly increased in the proliferative retinal tissues of human proliferative diabetic retinopathy patients. Together, Netrin-1 induces CD146-Gαi1/3-Gab1 complex formation to mediate downstream Akt-mTOR and Erk activation, important for angiogenesis in vitro and in vivo.

Screening strategy for 1210 exogenous chemicals in serum by two-dimensional liquid chromatography-mass spectrometry.

Humans are at risk of exogenous exposure to exogenous chemicals. Challenges exist for the comprehensive monitoring of residues with different physical and chemical properties in serum. Here, an on-line two-dimensional liquid chromatography (2D-LC) - high resolution mass spectrometry system (HRMS) was developed, expanding the range of the partition coefficient in octanol/water of the residue analysis from -8 to 12. A high-coverage serum residue screening strategy was further designed by integrating 2D-LC system with HRMS full MS/data independent acquisition and automatic spectral library searching. This strategy enables to simultaneously screen 1210 pesticides, veterinary/human drugs, other chemical pollutants and their metabolites in serum with a single analysis. Method validation showed 92% and 81% of 1022 residues spiked in serum could be detected at 50 ng/mL and 5 ng/mL, respectively. The developed method was applied to the analysis of 24 separately pooled serum samples, 58 suspect residues were found, some of them were detected at high frequencies over than 50%. Among them, 4,6-Dinitro-O-cresol and probable carcinogenic folpet are highly toxic, and cimaterol is banned in China. Collectively, this study developed a 2D-LC-HRMS -based screening strategy for screening pesticides, veterinary/human drugs, and other chemical pollutants in serum, it is helpful for studying the effect of exogenous exposures on human health.

Annotation of Dipeptides and Tripeptides Derivatized via Dansylation Based on Liquid Chromatography-Mass Spectrometry and Iterative Quantitative Structure Retention Relationship.

Small peptides such as dipeptides and tripeptides show various biological activities in organisms. However, methods for identifying dipeptides/tripeptides from complex biological samples are lacking. Here, an annotation strategy involving the derivatization of dipeptides and tripeptides via dansylation was suggested based on liquid chromatography-mass spectrometry (LC-MS) and iterative quantitative structure retention relationship (QSRR) to choose dipeptides/tripeptides by using a small number of standards. First, the LC-autoMS/MS method and initial QSRR model were built based on 25 selected grid-dipeptides and 18 test-dipeptides. To achieve high-coverage detection, dipeptide/tripeptide pools containing abundant dipeptides/tripeptides were then obtained from four dansylated biological samples including serum, tissue, feces, and soybean paste by using the parameter-optimized LC-autoMS/MS method. The QSRR model was further optimized through an iterative train-by-pick strategy. Based on the specific fragments and tR tolerances, 198 dipeptides and 149 tripeptides were annotated. The dipeptides at lower annotation levels were verified by using authentic standards and grid-correlation analysis. Finally, variation in serum dipeptides/tripeptides of three different liver diseases including hepatitis B infection, liver cirrhosis, and hepatocellular carcinoma was characterized. Dipeptides with N-prolinyl, C-proline, N-glutamyl, and N-valinyl generally increased with disease severity. In conclusion, this study provides an efficient strategy for annotating dipeptides/tripeptides from complex samples.