Circulating cell-free DNA as a biomarker for diagnosis of Schistosomiasis japonica.

BACKGROUND: Schistosomiasis, a neglected tropical disease, remains an important public health problem. Although there are various methods for diagnosing schistosomiasis, many limitations still exist. Early diagnosis and treatment of schistosomiasis can significantly improve survival and prognosis of patients. METHODOLOGY: Circulating cell-free (cf)DNA has been widely used in the diagnosis of various diseases. In our study, we evaluated the diagnostic value of circulating cfDNA for schistosomiasis caused by Schistosoma japonicum. We focused on the tandem sequences and mitochondrial genes of S. japonicum to identify highly sensitive and specific targets for diagnosis of Schistosomiasis japonica. RESULTS: Through data screening and analysis, we ultimately identified four specific tandem sequences (TD-1, TD-2, TD-3. and TD-4) and six mitochondrial genes (COX1(1), COX1(2), CYTB, ATP6, COX3, and ND5). We designed specific primers to detect the amount of circulating cfDNA in S. japonicum-infected mouse and chronic schistosomiasis patients. Our results showed that the number of tandem sequences was significantly higher than that of the mitochondrial genes. A S. japonicum infection model in mice suggested that infection of S. japonicum can be diagnosed by detecting circulating cfDNA as early as the first week. We measured the expression levels of circulating cfDNA (TD-1, TD-2, and TD-3) at different time points and found that TD-3 expression was significantly higher than that of TD-1 or TD-2. We also infected mice with different quantities of cercariae (20 s and 80 s). The level of cfDNA (TD-3) in the 80 s infection group was significantly higher than in the 20 s infection group. Additionally, cfDNA (TD-3) levels increased after egg deposition. Meanwhile, we tested 42 patients with chronic Schistosomiasis japonica and circulating cfDNA (TD-3) was detected in nine patients. CONCLUSIONS: We have screened highly sensitive targets for the diagnosis of Schistosomiasis japonica, and the detection of circulating cfDNA is a rapid and effective method for the diagnosis of Schistosomiasis japonica. The levels of cfDNA is correlated with cercariae infection severity. Early detection and diagnosis of schistosomiasis is crucial for patient treatment and improving prognosis.

Inflammation and comorbidities of chronic obstructive pulmonary disease: The cytokines put on a mask!

OBJECTIVE: Chronic obstructive pulmonary disease (COPD) is a well-known complex multicomponent disease characterized by systemic inflammation that frequently coexists with other conditions. We investigated the relationship between some inflammatory markers and complications in COPD patients to explore the possible roles of inflammation in these comorbidities. METHODS: This study used cross-sectional and case-control methods. We included 336 hospitalized COPD patients, 64 healthy controls, and 42 major depression patients and evaluated all participants using the Hamilton Rating Scale. C-reactive protein (CRP), red blood cell distribution width (RDW), neutrophil/lymphocyte ratio (NLR), platelet/lymphocyte ratio (PLR), monocyte/lymphocyte ratio (MLR), interleukin-1ß (IL-1ß) and interleukin-6 (IL-6) were collected and measured in the study population. Statistical methods were used to analyze the association of inflammatory markers with COPD comorbidities. RESULTS: Cor pulmonale and psychological comorbidities (depression and anxiety) were more common in this study on COPD patients. We found that MLR (OR = 2.054, 95 % CI 1.129-3.735, p = 0.018) and RDW (OR = 1.367, 95 % CI 1.178-1.586, p = 0.000) were related to COPD patients complicated with cor pulmonale, while IL-6 (OR = 1.026, 95 % CI 1.001-1.053, p = 0.045) and RDW (OR = 1.280, 95 % CI 1.055-1.552, p = 0.012) were related to depression symptoms. CONCLUSION: MLR, RDW and IL-6 were closely related to cor pulmonale and depression in COPD patients. IL-1 ß and IL-6 are closely related to depression in humans.

Multiple Factor Analysis of Depression and/or Anxiety in Patients with Acute Exacerbation Chronic Obstructive Pulmonary Disease.

Objective: To reveal the risk factors, the symptom distribution characteristics, the clinical values of white blood cell counts (WBC counts), red blood cell distribution width (RDW), neutrophil-to-lymphocyte ratio (NLR), platelet-to-lymphocyte ratio (PLR) and monocyte-to-lymphocyte ratio (MLR) in hospitalized patients with acute exacerbation of chronic obstructive pulmonary disease (AECOPD) combined with depression and/or anxiety. Methods: The study included prospective cross-sectional and case-control studies, and was executed in the Affiliated Hospital of Zunyi Medical University, Guizhou, China. Previously diagnosed chronic obstructive pulmonary disease (COPD) patients who admitted to the hospital with AECOPD, patients with depression and/or anxiety, and healthy people were enrolled in the study. The Hamilton Rating Scales were used to assess all subjects, and the complete blood counts (CBC) were collected. Baseline data and clinical measurement data [spirometry, arterial blood gas analysis, and COPD evaluation test (the CAT scale)] from patients with AECOPD were collected. Results: Of the 307 patients with AECOPD included, 63.5% (N=195) had depressive and/or anxiety symptoms, and 36.5% (N=112) had no symptoms. Sex, respiratory failure, number of comorbidities, number of acute exacerbations in the previous year and the CAT score were closely related to AECOPD combined with depression and/or anxiety (p<0.05). The CAT scale score were the independent risk factor (OR=6.576, 95% CI 3.812-11.342) and significant predictor of AECOPD with depression and/or anxiety (AUC=0.790,95% CI 0.740-0.834); the patients with depression and/or anxiety were more severe and characteristic than the patients with AECOPD combined with depression and/or anxiety; RDW was associated with AECOPD with depression and/or anxiety (p=0.020, OR1.212,95% CI1.03-1.426), and had certain clinical diagnostic value (AUC=0.570,95% CI 0.531-0.626). Conclusion: Depression and anxiety should not be ignored in patients with AECOPD. The severity and quality of life of COPD were closely related to the occurrence of depression and/or anxiety symptoms. In most cases, perhaps depression and anxiety in AECOPD are only symptoms and not to the extents of the diseases. RDW had clinical diagnostic value in AECOPD combined with depression and/or anxiety. NLR, PLR, MLR, and RDW may become the novel indicators for evaluating the degree of inflammation of AECOPD and deserve further research.

Deubiquitinase DUB3 Regulates Cell Cycle Progression via Stabilizing Cyclin A for Proliferation of Non-Small Cell Lung Cancer Cells.

The deubiquitinase DUB3 is frequently overexpressed in non-small cell lung cancer (NSCLC) and contributes to its malignant phenotype. However, the underlying molecular mechanism of DUB3 in NSCLC is largely unknown. In this study, we report that DUB3 regulates cell cycle progression by deubiquitinating cyclin A that links to proliferation of NSCLC cells. We found that knockdown of DUB3 decreases cyclin A levels, whereas overexpression of DUB3 strongly increases cyclin A levels. Mechanistically, DUB3 interacts with cyclin A, which removes the polyubiquitin chains conjugated onto cyclin A and stabilizes the cyclin A protein. Furthermore, we demonstrate that DUB3 regulates cell cycle progression by stabilizing cyclin A, because ablation of DUB3 arrests cell cycle from G0/G1 to S phase and the resulting effect can be rescued by introducing cyclin A into NSCLC cells. Functionally, we found that the effect of DUB3 on cyclin A mediates proliferation of NSCLC cells. Moreover, a significant correlation between DUB3 abundance and cyclin A expression levels were also found in NSCLC samples. Taken together, these results reveal that DUB3 functions as a novel cyclin A regulator through maintaining cyclin A stability, and that the DUB3-cyclin A signaling axis plays a critical role in cell cycle progression for proliferation of NSCLC.

Engineering of a near-infrared fluorescent probe for real-time simultaneous visualization of intracellular hypoxia and induced mitophagy.

Mitophagy induced by hypoxia plays an important role in regulating cellular homeostasis via the removal of dysfunctional mitochondria in the lysosomal degradation pathway, which results in physiological changes in the mitochondria, such as the pH, polarity and viscosity. However, the lack of an effective method for imaging of both the hypoxic microenvironment and the resulting variable mitochondria limits the visualization of hypoxia-induced mitophagy. Based on the specific mitochondrial pH changes during the hypoxia-induced mitophagy process, we have reported a near-infrared fluorescent probe (NIR-HMA) for real-time simultaneous visualization of the hypoxic microenvironment and the subsequent mitophagy process in live cells. NIR-HMA selectively accumulated in the hypoxic mitochondria in the NIR-MAO form, emitting at 710 nm, and then transformed into NIR-MAOH, emitting at 675 nm, in the acidified mitochondria-containing autolysosomes. Importantly, by smartly tethering the hypoxia-responsive group to the hydroxyl group of the NIR-fluorochrome, which shows ratiometric pH changes, NIR-HMA can differentiate between different levels of the hypoxic microenvironment and mitophagy. Furthermore, using NIR-HMA, we could track the complete mitophagy process from the mitochondria to the autolysosomes and visualize mitophagy caused only by hypoxia both in cancer cells and normal cells. Finally, NIR-HMA was applied to investigate the role that mitophagy plays in the hypoxic microenvironment via the cycling hypoxia-reoxygenation model. We observed a decreased fluorescence ratio after reoxygenation and a further increased mitophagy level after hypoxia was induced again, suggesting that mitophagy might be a self-protective process that allows cells to adapt to hypoxia. Our work may provide an attractive way for real-time visualization of relevant physiological processes in hypoxic microenvironments.

WDR79 mediates the proliferation of non-small cell lung cancer cells by regulating the stability of UHRF1.

WD repeat protein 79 (WDR79) is a member of the WD-repeat protein family characterized by the presence of a series of WD-repeat domains and is a scaffold protein that participates in telomerase assembly, Cajal body formation and DNA double strand break repair. Although previous studies have revealed that WDR79 is frequently overexpressed in non-small cell lung cancer (NSCLC) and promotes the proliferation of NSCLC cells, the underlying mechanism responsible for WDR79-mediated NSCLC proliferation is not fully understood. In this study, we report a novel molecular function of WDR79 that mediates NSCLC cell proliferation by controlling the stability of UHRF1. In the nucleus, WDR79 colocalized and interacted with UHRF1. As a result, overexpression of WDR79 stabilized UHRF1, whereas ablation of WDR79 decreased the level of UHRF1. Meanwhile, we showed that WDR79 can protect UHRF1 from poly-ubiquitination-mediated proteolysis, which facilitated the stabilization of UHRF1. We further demonstrated that WDR79 exerts a proliferation effect on NSCLC cells by stabilizing UHRF1. These findings reveal that WDR79 is a novel UHRF1 regulator by maintaining UHRF1 stability, and they also provide a clue as to how to explore WDR79 for potential therapeutic application in NSCLC.

WDR79 promotes the proliferation of non-small cell lung cancer cells via USP7-mediated regulation of the Mdm2-p53 pathway.

WD repeat protein 79 (WDR79) is a member of the WD-repeat protein family and functions as a scaffold protein during telomerase assembly, Cajal body formation and DNA double strand break repair. We have previously shown that WDR79 is frequently overexpressed in cell lines and tissues derived from non-small cell lung cancer (NSCLC) and it accelerates cell proliferation in NSCLC. However, the detailed mechanism underlying the role of WDR79 in the proliferation of NSCLC cells remains unclear. Here, we report the discovery of a molecular interaction between WDR79 and USP7 and show its functional significance in linking the Mdm2-p53 pathway to the proliferation of NSCLC cells. We found that WDR79 colocalized and interacted with USP7 in the nucleus of NSCLC cells. This event, in turn, reduced the ubiquitination of Mdm2 and p53, thereby increasing the stability and extending the half-life of the two proteins. We further found that the functional effects of WDR79 depended upon USP7, because the knockdown of USP7 resulted in their attenuation. Finally, we demonstrated that WDR79 promoted the proliferation of NSCLC cells via USP7. Taken together, our findings reveal a novel molecular function of WDR79 and may lead to broadly applicable and innovative therapeutic avenues for NSCLC.

Crystal structure of a soluble form of human monoglyceride lipase in complex with an inhibitor at 1.35 Å resolution.

A high-resolution structure of a ligand-bound, soluble form of human monoglyceride lipase (MGL) is presented. The structure highlights a novel conformation of the regulatory lid-domain present in the lipase family as well as the binding mode of a pharmaceutically relevant reversible inhibitor. Analysis of the structure lacking the inhibitor indicates that the closed conformation can accommodate the native substrate 2-arachidonoyl glycerol. A model is proposed in which MGL undergoes conformational and electrostatic changes during the catalytic cycle ultimately resulting in its dissociation from the membrane upon completion of the cycle. In addition, the study outlines a successful approach to transform membrane associated proteins, which tend to aggregate upon purification, into a monomeric and soluble form.

Crystal structure of the tyrosine kinase domain of colony-stimulating factor-1 receptor (cFMS) in complex with two inhibitors.

The cFMS proto-oncogene encodes for the colony-stimulating factor-1 receptor, a receptor-tyrosine kinase responsible for the differentiation and maturation of certain macrophages. Upon binding its ligand colony-stimulating factor-1 cFMS autophosphorylates, dimerizes, and induces phosphorylation of downstream targets. We report the novel crystal structure of unphosphorylated cFMS in complex with two members of different classes of drug-like protein kinase inhibitors. cFMS exhibits a typical bi-lobal kinase fold, and its activation loop and DFG motif are found to be in the canonical inactive conformation. Both ATP competitive inhibitors are bound in the active site and demonstrate a binding mode similar to that of STI-571 bound to cABL. The DFG motif is prevented from switching into the catalytically competent conformation through interactions with the inhibitors. Activation of cFMS is also inhibited by the juxtamembrane domain, which interacts with residues of the active site and prevents formation of the activated kinase. Together the structures of cFMS provide further insight into the autoinhibition of receptor-tyrosine kinases via their respective juxtamembrane domains; additionally the binding mode of two novel classes of kinase inhibitors will guide the design of novel molecules targeting macrophage-related diseases.

Protein engineering of the colony-stimulating factor-1 receptor kinase domain for structural studies.

A parallel approach to designing crystallization constructs for the c-FMS kinase domain was implemented, resulting in proteins suitable for structural studies. Sequence alignment and limited proteolysis were used to identify and eliminate unstructured and surface-exposed domains. A small library of chimeras was prepared in which the kinase insert domain of FMS was replaced with the kinase insert domain of previously crystallized receptor-tyrosine kinases. Characterization of the newly generated FMS constructs by enzymology and thermoshift assays demonstrated similar activities and compound binding to the FMS full-length cytoplasmic domain. Two chimeras were evaluated for crystallization in the presence and absence of a variety of ligands resulting in crystal structures, and leading to a successful structure-based drug design project for this important inflammation target.

Effect of construct design on MAPKAP kinase-2 activity, thermodynamic stability and ligand-binding affinity.

MAPK-activated protein kinase-2 (MAPKAPK2) regulates the synthesis of tumor necrosis factor and other cytokines and is a potential drug target for inflammatory diseases. Five protein constructs were produced in 4-10mg quantities per liter of culture media using baculovirus-infected insect cells and characterized for kinase activity, thermal stability, and ligand-binding affinity. Compared to construct 1-370, removal of the C-terminal autoinhibitory peptide in 1-338 resulted in a destabilized but partially active nonphosphorylated enzyme; phosphorylation of 1-338 by p38alpha further increased activity 12-fold. A putative constitutively active mutant, 1-370/T222E/T334E, was 6.3-fold less active than phosphorylated 1-370. ThermoFluor, an equilibrium ligand-binding assay, was used to measure nucleotide analogue affinity for various constructs. Binding of phosphorylated nucleotides was Mg(2+)-dependent. Residues 1-40 were required for high-affinity binding of ADP, ATPgammaS, staurosporine, and K252a. A mutation M138A rendered 1-370 susceptible to p38-inhibitors SB-203580 and SB-202190 with IC50 values of 17.4 and 14.1 microM, respectively. Taken together, these studies provide information on the mechanism of ligand-binding to MAPKAPK2 that can be used in the search for selective small-molecule inhibitors.