Cysteine-rich intestinal protein 1 enhances the progression of hepatocellular carcinoma via Ras signaling.

The present study aimed to explore the expression and clinical significance of cysteine-rich intestinal protein 1 (CRIP1) mRNA in the serum of patients with hepatocellular carcinoma (HCC). Reverse transcription polymerase chain reaction (RT-PCR) was performed to explore the level of CRIP1 mRNA in the tissues and serum of patients with HCC. Our data showed that the mRNA level of CRIP1 was significantly elevated in the serum and tissues of HCC patients. Moreover, serum CRIP1 mRNA was significantly elevated in HCC patients with larger tumor sizes and higher tumor node metastasis (TNM) stages. Receiver operating characteristic analysis showed that compared with a single marker, the combined detection of alpha-fetoprotein, carcinoembryonic antigen, and CRIP1 had the highest accuracy, sensitivity, and specificity. Further study showed that the overexpression of CRIP1 enhanced the proliferation and migration of HepG2 cells, but the inhibition of CRIP1 decreased the proliferation and migration of HepG2 cells. Microarray assays and KyotoEncyclopedia of Genes and Genomes (KEGG) pathway analysis showed that overexpression of CRIP1 induced the activation of Ras signaling. Co-immunoprecipitation (Co-IP) assays indicated that CRIP1 could interact with Ras. To further evaluate whether CRIP1 interacts with Ras, a specific siRNA targeting Ras was selected. We found that Ras knockdown reduced the activation of Ras/AKT signaling even in HepG2 cells transfected with CRIP1. Moreover, elevated expression of CRIP1 increased the proliferation of HepG2 cells, but such effects could be abolished by silencing Ras. In summary, elevated CRIP1 levels enhanced the progression of CRIP1 via Ras signaling.

Lmo4-resistin signaling contributes to adipose tissue-liver crosstalk upon weight cycling.

Repeated cycles of weight loss and regain, known as weight cycling, is often seen when people try to lose weight. The exact pathophysiological effects and the underlying mechanisms of weight cycling remain largely unclear. Here, we report that weight cycling induced by alternating feeding mice with a low-fat diet or a high-fat diet in a 1-week switch protocol caused further increased epididymal white adipose tissue (eWAT) weight, preadipocyte proliferation, hepatic inflammation, fasting blood glucose level, and glucose intolerance, compared with the continuously HF-fed mice. Combining the secretory protein database with RNA-sequencing and quantitative PCR (qPCR) results in eWAT, the mRNA levels of several adipokines, including Retn (encoding resistin), were found altered by weight cycling. A transcriptional co-factor Lmo4 was found regulated by weight cycling; Lmo4 enhanced preadipocyte proliferation, in vitro adipogenesis, transcription of Retn, and resistin secretion in 3T3-L1 cells. Primary mouse hepatocytes administrated with recombinant mouse resistin (rm-resistin), or exposed to media from Lmo4-overexpressed 3T3-L1 cells, showed increased inflammatory responses and gluconeogenesis. Furthermore, rm-resistin-injected normal chow-fed mice showed upregulated blood glucose level by increasing gluconeogenesis, and upregulated the hepatic inflammatory responses. Together, our results suggest a regulatory role of Lmo4-resistin signaling in weight cycling, indicating a crosstalk between the adipose tissue and liver.

Using the kinetics of C-reactive protein response to improve the differential diagnosis between acute bacterial and viral infections.

PURPOSE: Differential diagnosis between acute viral and bacterial infection is an emerging common challenge for a physician in the emergency department. Serum C-reactive protein (CRP) is used to support diagnosis of bacterial infection, but in patients admitted with low CRP, its ability to discriminate between viral and bacterial infections is limited. We aimed to use two consecutive CRP measurements in order to improve differential diagnosis between bacterial and viral infection. METHODS: A single-center retrospective cohort (n = 1629) study of adult patients admitted to the emergency department with a subsequent microbiological confirmation of either viral or bacterial infection. Trend of CRP was defined as the absolute difference between the first two measurements of CRP divided by the time between them, and we investigated the ability of this parameter to differentiate between viral and bacterial infection. RESULTS: In patients with relatively low initial CRP concentration (< 60 mg/L, n = 634 patients), where the uncertainty regarding the type of infection is the highest, the trend improved diagnosis accuracy (AUC 0.83 compared to 0.57 for the first CRP measurement). Trend values above 3.47 mg/L/h discriminated bacterial from viral infection with 93.8% specificity and 50% sensitivity. CONCLUSIONS: The proposed approach for using the kinetics of CRP in patients whose first CRP measurement is low can assist in differential diagnosis between acute bacterial and viral infection.

Identification of Key Genes Potentially Related to Intervertebral Disk Degeneration by Microarray Analysis.

Aims: This study was designed to investigate differentially expressed genes (DEGs) in the annulus fibrosus (AF), nucleus pulposus (NP), and whole blood (WB) of intervertebral disk degeneration (IDD) patients. Materials and Methods: We retrieved microarray data set GSE70362, which contains the gene expression profiles of 24 AF and 24 NP samples from the Gene Expression Omnibus and identified DEGs in degenerative AF (AF-DEGs) and NP (NP-DEGs) samples compared with nondegenerative samples. We also examined gene expression profiles in WB from patients with IDD and healthy volunteers to identify DEGs in WB (WB-DEGs). We performed functional analyses on the DEGs common to AF-DEGs, NP-DEGs, and WB-DEGs. Expression of the common DEGs was partially validated by quantitative real-time-polymerase chain reaction (QRT-PCR). Results: In total, 846 AF-DEGs, 902 NP-DEGs, and 862 WB-DEGs were identified, and 22 DEGs were common among the three groups. Functional analyses showed that the common DEGs were enriched in 33 biological processes, 16 cellular components, 4 molecular functions, and 9 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways; 13 of the common DEGs were included in the protein-protein interaction (PPI) network and superoxide dismutase 2 (SOD2) was identified as a hub gene in the PPI network. The QRT-PCR results for the expression of the genes protein disulfide isomerase family A member 4, FKBP prolyl isomerase 11, ectonucleotide pyrophosphatase/phosphodiesterase 4, SOD2, and actin binding LIM protein 1, were consistent with the gene chip hybridization results. Conclusions: This study identified key genes for future investigations of the underlying molecular mechanisms of IDD. These genes may provide future targets for the clinical treatment and diagnosis of IDD.

Assessment of alcohol consumption in depression follow-up using self-reports and blood measures including inflammatory biomarkers.

AIMS: Alcohol consumption has been suggested a major role in the pathogenesis and prognosis of depression. However, reliable identification of hazardous drinking continues to be problematic. We compared the accuracy of different biomarkers and self-reports of alcohol consumption in the follow-up study of depression. METHODS: Data from 202 patients with major depressive disorder were obtained through self-reports, AUDIT and AUDIT-C questionnaires and biomarker analyses. The clinical assessments and measurements of biomarkers (GT, CDT, GT-CDT-combination, MCV, ALT, AST, hs-CRP, IL-6) were performed at baseline and after six months of treatment. Based on self-reported alcohol intake at baseline the patients were classified to three subgroups. RESULTS: About 27.2% of patients were categorized to high-risk drinkers, 26.3% low-risk drinkers and 46.5% abstainers. High-risk drinkers showed significantly higher mean values of GT, CDT, GT-CDT-combination and IL-6 than abstainers, diagnostic accuracy being highest with the combined marker of GT-CDT. The accuracy of AUDIT and AUDIT-C to detect high-risk drinking was also significant. During follow-up, the differences observed in the biomarkers at baseline disappeared together with recovery from depression. CONCLUSIONS: Our data suggest the combined use of GT-CDT and AUDIT questionnaires to improve the identification of drinking of patients with depression. This approach could be useful for improving treatment adherence and outcome in depressed patients.

Monocyte Activation Is Associated With Worse Cognitive Performance in HIV-Infected Women With Virologic Suppression.

BACKGROUND: Cognitive impairment persists despite suppression of plasma human immunodeficiency virus (HIV) RNA. Monocyte-related immune activation is a likely mechanism. We examined immune activation and cognition in a cohort of HIV-infected and uninfected women from the Women's Interagency HIV Study (WIHS). METHODS: Blood levels of activation markers, soluble CD163 (sCD163), soluble CD14 (sCD14), CRP, IL-6, and a gut microbial translocation marker (intestinal fatty acid binding protein (I-FABP)) were measured in 253 women (73% HIV-infected). Markers were compared to concurrent (within ± one semiannual visit) neuropsychological testing performance. RESULTS: Higher sCD163 levels were associated with worse overall performance and worse verbal learning, verbal memory, executive function, psychomotor speed, and fine motor skills (P < .05 for all comparisons). Higher sCD14 levels were associated with worse verbal learning, verbal memory, executive function, and psychomotor speed (P < .05 for all comparisons). Among women with virological suppression, sCD163 remained associated with overall performance, verbal memory, psychomotor speed, and fine motor skills, and sCD164 remained associated with executive function (P < .05 for all comparisons). CRP, IL-6, and I-FABP were not associated with worse cognitive performance. CONCLUSIONS: Monocyte activation was associated with worse cognitive performance, and associations persisted despite viral suppression. Persistent inflammatory mechanisms related to monocytes correlate to clinically pertinent brain outcomes.

Autoantibodies against TYMS and PDLIM1 proteins detected as circulatory signatures in Indian breast cancer patients.

PURPOSE: Breast cancer (BC) is the most common invasive cancer in women worldwide. Autoantibodies (AAbs) to tumor-associated antigens (TAAs) have a great potential for the development of diagnostic biomarkers in cancer. This study was performed to identify AAbs and cognate TAAs that may improve detection of this deadly disease. EXPERIMENTAL DESIGN: Serological proteome analysis of plasma samples of BC patients (N = 30) and healthy controls (N = 30) was performed to identify TAAs. Expressions of selected TAAs were also determined in breast tumor tissues (N = 10) by immunohistochemistry. An independent validation cohort (N = 124) was tested to determine diagnostic accuracy of selected AAbs titer by ELISA. RESULTS: Thymidylate synthase (TYMS) and C-terminal LIM domain protein 1 (PDLIM1) were found to react more specifically with plasma samples of BC patients. Both TAAs were also found to be significantly over expressed (p < 0.001) in breast tumor tissues compared to adjacent normal tissues. TYMS AAbs response was positively correlated (r = 0.778, p < 0.008) with TYMS overexpression in BC tissues. TYMS and PDLIM1 AAbs titers discriminated BC from controls with a sensitivity/specificity of 57.81%/95% and 73.44%/58.33%, respectively. CONCLUSION AND CLINICAL RELEVANCE: High titers of both TYMS and PDLIM1 AAbs were significantly more prevalent in BC cases than controls. Our data recommends further investigations for evaluating their potential for BC detection.

Development of autoantibodies against muscle-specific FHL1 in severe inflammatory myopathies.

Mutations of the gene encoding four-and-a-half LIM domain 1 (FHL1) are the causative factor of several X-linked hereditary myopathies that are collectively termed FHL1-related myopathies. These disorders are characterized by severe muscle dysfunction and damage. Here, we have shown that patients with idiopathic inflammatory myopathies (IIMs) develop autoimmunity to FHL1, which is a muscle-specific protein. Anti-FHL1 autoantibodies were detected in 25% of IIM patients, while patients with other autoimmune diseases or muscular dystrophies were largely anti-FHL1 negative. Anti-FHL1 reactivity was predictive for muscle atrophy, dysphagia, pronounced muscle fiber damage, and vasculitis. FHL1 showed an altered expression pattern, with focal accumulation in the muscle fibers of autoantibody-positive patients compared with a homogeneous expression in anti-FHL1-negative patients and healthy controls. We determined that FHL1 is a target of the cytotoxic protease granzyme B, indicating that the generation of FHL1 fragments may initiate FHL1 autoimmunity. Moreover, immunization of myositis-prone mice with FHL1 aggravated muscle weakness and increased mortality, suggesting a direct link between anti-FHL1 responses and muscle damage. Together, our findings provide evidence that FHL1 may be involved in the pathogenesis not only of genetic FHL1-related myopathies but also of autoimmune IIM. Importantly, these results indicate that anti-FHL1 autoantibodies in peripheral blood have promising potential as a biomarker to identify a subset of severe IIM.

Inhibition of G9a methyltransferase stimulates fetal hemoglobin production by facilitating LCR/γ-globin looping.

Induction of fetal hemoglobin (HbF) production in adult erythrocytes can reduce the severity of sickle cell disease and ß-thalassemia. Transcription of ß-globin genes is regulated by the distant locus control region (LCR), which is brought into direct gene contact by the LDB1/GATA-1/TAL1/LMO2-containing complex. Inhibition of G9a H3K9 methyltransferase by the chemical compound UNC0638 activates fetal and represses adult ß-globin gene expression in adult human hematopoietic precursor cells, but the underlying mechanisms are unclear. Here we studied UNC0638 effects on ß-globin gene expression using ex vivo differentiation of CD34(+) erythroid progenitor cells from peripheral blood of healthy adult donors. UNC0638 inhibition of G9a caused dosed accumulation of HbF up to 30% of total hemoglobin in differentiated cells. Elevation of HbF was associated with significant activation of fetal γ-globin and repression of adult ß-globin transcription. Changes in gene expression were associated with widespread loss of H3K9me2 in the locus and gain of LDB1 complex occupancy at the γ-globin promoters as well as de novo formation of LCR/γ-globin contacts. Our findings demonstrate that G9a establishes epigenetic conditions preventing activation of γ-globin genes during differentiation of adult erythroid progenitor cells. In this view, manipulation of G9a represents a promising epigenetic approach for treatment of ß-hemoglobinopathies.

Muscle lim protein isoform negatively regulates striated muscle actin dynamics and differentiation.

Muscle lim protein (MLP) has emerged as a critical regulator of striated muscle physiology and pathophysiology. Mutations in cysteine and glycine-rich protein 3 (CSRP3), the gene encoding MLP, have been directly associated with human cardiomyopathies, whereas aberrant expression patterns are reported in human cardiac and skeletal muscle diseases. Increasing evidence suggests that MLP has an important role in both myogenic differentiation and myocyte cytoarchitecture, although the full spectrum of its intracellular roles has not been delineated. We report the discovery of an alternative splice variant of MLP, designated as MLP-b, showing distinct expression in neuromuscular disease and direct roles in actin dynamics and muscle differentiation. This novel isoform originates by alternative splicing of exons 3 and 4. At the protein level, it contains the N-terminus first half LIM domain of MLP and a unique sequence of 22 amino acids. Physiologically, it is expressed during early differentiation, whereas its overexpression reduces C2C12 differentiation and myotube formation. This may be mediated through its inhibition of MLP/cofilin-2-mediated F-actin dynamics. In differentiated striated muscles, MLP-b localizes to the sarcomeres and binds directly to Z-disc components, including α-actinin, T-cap and MLP. The findings of the present study unveil a novel player in muscle physiology and pathophysiology that is implicated in myogenesis as a negative regulator of myotube formation, as well as in differentiated striated muscles as a contributor to sarcomeric integrity.

Search for peripheral biomarkers in patients affected by acutely psychotic bipolar disorder: a proteomic approach.

Data on neurobiological mechanisms underlying mood disorders are elusive; the aetiology of such states is multifactorial, including genetic predisposition and environmental factors. Diagnosis is currently being made only on an interview-based methodology. Biological markers, which could improve the current classification, and in perspective, stratify patients on a biological basis into more homogeneous clinically distinct subgroups, are highly needed. We describe here a comparative proteomic analysis of peripheral lymphocytes from patients affected by acute psychotic bipolar disorder (PBD) (n = 15), major depressive episode (MDE) with no personal or family history of psychosis (n = 11), and a group of demographically matched healthy controls (HC) (n = 15). All patients were evaluated by means of Structured Clinical Interview for DSM-IV-Patient version (SCID-I-P), Positive and Negative Symptoms Scale (PANSS), Young Mania Rating Scale (YMRS), Hamilton Anxiety Rating Scale (HAM-A) and Hamilton Depression Rating Scale (HAM-D-17) questionnaires. Blood lymphocytes were obtained by gradient separation, and 2-DE was carried out on protein extracts. Significant differences in protein patterns among the three groups were observed. Thirty-six protein spots were found to be differentially expressed in patients compared to controls, which collapsed into 25 different proteins after mass spectrometry identification. Twenty-one of these proteins failed to discriminate between PBD and MDE, suggesting common signatures for these disorders. Nevertheless, after the western blot validation only two of the remaining proteins, namely LIM and SH3 domain protein1, and short-chain specific acyl-CoA dehydrogenase mitochondrial protein, resulted in being significantly upregulated in PBD samples suggesting additional mechanisms that could be associated with the psychotic features of bipolar disorder.

Genomic biomarkers for cardiotoxicity in rats as a sensitive tool in preclinical studies.

The development of safer drugs is a high priority for pharmaceutical companies. Among the various toxicities caused by drugs, cardiotoxicity is an important issue because of its lethality. In addition, cardiovascular toxicity leads to the attrition of many drug candidates in both preclinical and clinical phases. Although histopathological and blood chemistry examinations are the current gold standards for detecting cardiotoxicity in preclinical studies, the large number of withdrawals from clinical studies owing to safety problems indicate that a more sensitive tool is required. We recently identified 32 genes that were candidate genomic biomarkers for cardiotoxicity in rats. Based on their functions, the present study focused on 8 of these 32 genes (Spp1, Fhl1, Timp1, Serpine1, Bcat1, Lmcd1, Rnd1 and Tgfb2). Diagnostic accuracy for the genes was determined by a receiver-operating characteristic (ROC) analysis using more cardiotoxic and non-cardiotoxic compounds. In addition, an optimized support vector machine (SVM) model that was composed of Spp1 and Timp1 was newly constructed. This new multi-gene model exhibited a much higher diagnostic accuracy than that observed for plasma cardiac troponin I (cTnI), which is one of the most useful plasma biomarkers for cardiotoxicity detection. Furthermore, we determined that this multi-gene model could predict potential cardiotoxicity in rats in the absence of any cardiac histopathological lesions or elevations of plasma cTnI. Overall, this multi-gene model exhibited advantages over classic tools commonly used for cardiotoxicity evaluations in rats. Our current results suggest that application of the model could potentially lead to the production of safer drugs.

Release of tissue-specific proteins into coronary perfusate as a model for biomarker discovery in myocardial ischemia/reperfusion injury.

Diagnosis of acute coronary syndromes is based on protein biomarkers, such as the cardiac troponins (cTnI/cTnT) and creatine kinase (CK-MB) that are released into the circulation. Biomarker discovery is focused on identifying very low abundance tissue-derived analytes from within albumin-rich plasma, in which the wide dynamic range of the native protein complement hinders classical proteomic investigations. We employed an ex vivo rabbit model of myocardial ischemia/reperfusion (I/R) injury using Langendorff buffer perfusion. Nonrecirculating perfusate was collected over a temporal profile of 60 min reperfusion following brief, reversible ischemia (15 min; 15I/60R) for comparison with irreversible I/R (60I/60R). Perfusate proteins were separated using two-dimensional gel electrophoresis (2-DE) and identified by mass spectrometry (MS), revealing 26 tissue-specific proteins released during reperfusion post-15I. Proteins released during irreversible I/R (60I/60R) were profiled using gel-based (2-DE and one-dimensional gel electrophoresis coupled to liquid chromatography and tandem mass spectrometry; geLC-MS) and gel-free (LC-MS/MS) methods. A total of 192 tissue-specific proteins were identified during reperfusion post-60I. Identified proteins included those previously associated with I/R (myoglobin, CK-MB, cTnI, and cTnT), in addition to examples currently under investigation in large cohort studies (heart-type fatty acid binding protein; FABPH). The postischemic release profile of a novel cardiac-specific protein, cysteine and glycine-rich protein 3 (Csrp3; cardiac LIM domain protein) was validated by Western blot analysis. We also identified Csrp3 in serum from 6 of 8 patients postreperfusion following acute myocardial infarction. These studies indicate that animal modeling of biomarker release using ex vivo buffer perfused tissue to limit the presence of obfuscating plasma proteins may identify candidates for further study in humans.