Computational and Empirical Studies Predict Mycobacterium tuberculosis-Specific T Cells as a Biomarker for Infection Outcome

Identifying biomarkers for tuberculosis (TB) is an ongoing challenge in developing immunological correlates of infection outcome and protection. Biomarker discovery is also necessary for aiding design and testing of new treatments and vaccines. To effectively predict biomarkers for infection progression in any disease, including TB, large amounts of experimental data are required to reach statistical power and make accurate predictions. We took a two-pronged approach using both experimental and computational modeling to address this problem. We first collected 200 blood samples over a 2- year period from 28 non-human primates (NHP) infected with a low dose of Mycobacterium tuberculosis. We identified T cells and the cytokines that they were producing (single and multiple) from each sample along with monkey status and infection progression data. Machine learning techniques were used to interrogate the experimental NHP datasets without identifying any potential TB biomarker. In parallel, we used our extensive novel NHP datasets to build and calibrate a multi-organ computational model that combines what is occurring at the site of infection (e.g., lung) at a single granuloma scale with blood level readouts that can be tracked in monkeys and humans. We then generated a large in silico repository of in silico granulomas coupled to lymph node and blood dynamics and developed an in silico tool to scale granuloma level results to a full host scale to identify what best predicts Mycobacterium tuberculosis (Mtb) infection outcomes. The analysis of in silico blood measures identifies Mtb-specific frequencies of effector T cell phenotypes at various time points post infection as promising indicators of infection outcome. We emphasize that pairing wetlab and computational approaches holds great promise to accelerate TB biomarker discovery.     

MiR-422a as a Potential Cellular MicroRNA Biomarker for Postmenopausal Osteoporosis

Background

MicroRNAs (miRNAs) are a class of short non-coding RNA molecules that regulate gene expression by targeting mRNAs. Recently, miRNAs have been shown to play important roles in the etiology of various diseases. However, little is known about their roles in the development of osteoporosis. Circulating monocytes are osteoclast precursors that also produce various factors important for osteoclastogenesis. Previously, we have identified a potential biomarker miR-133a in circulating monocytes for postmenopausal osteoporosis. In this study, we aimed to further identify significant miRNA biomarkers in human circulating monocytes underlying postmenopausal osteoporosis.

Methodology/Principal Findings

We used ABI TaqMan miRNA array followed by qRT-PCR validation in human circulating monocytes from 10 high BMD and 10 low BMD postmenopausal Caucasian women to identify miRNA biomarkers. MiR-422a was up-regulated with marginal significance (P = 0.065) in the low compared with the high BMD group in the array analysis. However, a significant up-regulation of miR-422a was identified in the low BMD group by qRT-PCR analysis (P = 0.029). We also performed bioinformatic target gene analysis and found several potential target genes of miR-422a which are involved in osteoclastogenesis. Further qRT-PCR analyses of the target genes in the same study subjects showed that the expression of five of these genes (CBL, CD226, IGF1, PAG1, and TOB2) correlated negatively with miR-422a expression.

Conclusions/Significance

Our study suggests that miR-422a in human circulating monocytes (osteoclast precursors) is a potential miRNA biomarker underlying postmenopausal osteoporosis.     

Identification of a Plasma Four-microRNA Panel as Potential Noninvasive Biomarker for Osteosarcoma

Background

Circulating microRNAs (miRNAs) are emerging as promising biomarkers for human cancer. Osteosarcoma is the most common human primary malignant bone tumor in children and young adults. The objective of this study was to investigate whether circulating miRNAs in plasma could be a useful biomarker for detecting osteosarcoma and monitoring tumor removal dynamics.

Methods

Plasma samples were obtained from 90 patients before surgery, 50 patients after one month of surgery, and 90 healthy individuals. The study was divided into three steps: First, initial screening of the profiles of circulating miRNAs in pooled plasma samples from healthy controls and pre-operative osteosarcoma patients using a TaqMan low density array (TLDA). Second, evaluation of miRNA concentration in individual plasma samples from 90 pre-operative osteosarcoma patients and 90 healthy controls by a quantitative real time PCR (qRT-PCR) assay. Third, evaluation of miRNA concentration in paired plasma samples from 50 pre- and post-operative osteosarcoma patients by qRT-PCR assay.

Results

Four plasma miRNAs including miR-195-5p, miR-199a-3p, miR-320a, and miR-374a-5p were significantly increased in the osteosarcoma patients. Receiver operating characteristics curve analysis of the combined populations demonstrated that the four-miRNA signature could discriminate cases from controls with an area under the curve of 0.9608 (95% CI 0.9307-0.9912). These 4 miRNAs were markedly decreased in the plasma after operation. In addition, circulating miR-195-5p and miR-199a-3p were correlated with metastasis status, while miR-199a-3p and miR-320a were correlated with histological subtype.

Conclusions

Our data suggest that altered levels of circulating miRNAs might have great potential to serve as novel, non-invasive biomarkers for osteosarcoma.     

Gelsolin in Cerebrospinal Fluid as a Potential Biomarker of Epilepsy

Gelsolin is an actin regulatory protein that generally distributed in a wide variety of body tissues, especially the brain tissues and cerebrospinal fluid. In this study we found that lumbar CSF-gelsolin concentrations markedly decreased in epileptic patients by enzyme linked immunosorbent assay. In order to help judge the result, we determined gelsolin expression in temporal lobe tissues of patients with temporal lobe epilepsy using double-label immunofluorescence to location and using western blot to quantitation. Then we observed that gelsolin was co-expressed with microtubule-associated protein-2 in axons and cytoplasms of neurons and gelsolin protein level was also down-regulated in temporal lobe tissues of epileptic patients. Our findings suggested that CSF-gelsolin level might reflect the alteration of gelsolin in brain tissue of epileptic patients and CSF-gelsolin seems to be a potential biomarker for epilepsy.     

Blood Plasma Pseudocholinesterase as a Potential Biomarker of Autoimmune Diseases

We studied the blood plasma butyrylcholinesterase (BChE, pseudocholinesterase) activity as a potential marker for differentiating such autoimmune pathologies as type 1 diabetes mellitus (DM1), autoimmune thyroiditis (AIT) and rheumatoid arthritis (RA) from diseases having similar symptoms but different etiology. The blood plasma pseudocholinesterase activity was analyzed for patients with different types of diabetes mellitus. We showed its fivefold increase in DM1 compared to the control group, while in the second type of diabetes, which is metabolic pathology, it decreases two times. It was demonstrated that the activity of blood plasma BChE in patients with AIT and RA also increased fivefold compared to the group of patients without autoimmune disorders. Thus, the butyrylcholinesterase activity increases in diseases of an autoimmune nature, whereas in metabolic pathology it has the tendency to decrease; it indicates the adequacy of the selected criterion for the further development of a screening test-system that would allow differentiation of autoimmune diseases from the diseases of another origin, but with similar symptoms.     

Cell Stiffness Is a Biomarker of the Metastatic Potential of Ovarian Cancer Cells

The metastatic potential of cells is an important parameter in the design of optimal strategies for the personalized treatment of cancer. Using atomic force microscopy (AFM), we show, consistent with previous studies conducted in other types of epithelial cancer, that ovarian cancer cells are generally softer and display lower intrinsic variability in cell stiffness than non-malignant ovarian epithelial cells. A detailed examination of highly invasive ovarian cancer cells (HEY A8) relative to their less invasive parental cells (HEY), demonstrates that deformability is also an accurate biomarker of metastatic potential. Comparative gene expression analyses indicate that the reduced stiffness of highly metastatic HEY A8 cells is associated with actin cytoskeleton remodeling and microscopic examination of actin fiber structure in these cell lines is consistent with this prediction. Our results indicate that cell stiffness may be a useful biomarker to evaluate the relative metastatic potential of ovarian and perhaps other types of cancer cells.     

Liposomes Containing Sphingomyelin and Cholesterol: Detergent Solubilisation and Infrared Spectroscopic Studies

Abstract

The solubilisation by Triton X-100 of large unilamellar vesicles consisting of pure egg sphingomyelin (T_m 39°) or sphingomyelinxholesterol mixtures has been tested at various temperatures. For pure sphingomyelin, solubilisation occurs most readily at temperatures just below T_m. In general, egg sphingomyelin is solubilised by Triton X-100 more easily than egg phosphatidylcholine. Mixtures of sphingomyelin and cholesterol are detergent-insoluble under most conditions. Infrared spectroscopy has been applied to explore the interactions of cholesterol and sphingomyelin at the level of the lipid-water interface. Moreover, various cholesterol analogues (cholestane, cholestanone, androstenol) have been used in parallel solubilisation experiments and IR observations. The results show that cholesterol modifies the conformation (or H-bonding properties, or both) of the sphingomyelin polar head-group, both above and below T_m. Moreover, both the hydroxyl group at C3 and the hydrocarbon chain at C17 of the steroid nucleus appear to be required for insolubility to be detected. Perturbation of the polar environment of the sphingomyelin: cholesterol bilayers by 3M urea makes them soluble in Triton X-100. These results may be related to the observed insolubility of cell membrane 'rafts' in cold detergent.     

Network Analysis Identifies SOD2 mRNA as a Potential Biomarker for Parkinson's Disease

Increasing evidence indicates that Parkinson''s disease (PD) and type 2 diabetes (T2DM) share dysregulated molecular networks. We identified 84 genes shared between PD and T2DM from curated disease-gene databases. Nitric oxide biosynthesis, lipid and carbohydrate metabolism, insulin secretion and inflammation were identified as common dysregulated pathways. A network prioritization approach was implemented to rank genes according to their distance to seed genes and their involvement in common biological pathways. Quantitative polymerase chain reaction assays revealed that a highly ranked gene, superoxide dismutase 2 (SOD2), is upregulated in PD patients compared to healthy controls in 192 whole blood samples from two independent clinical trials, the Harvard Biomarker Study (HBS) and the Diagnostic and Prognostic Biomarkers in Parkinson''s disease (PROBE). The results from this study reinforce the idea that shared molecular networks between PD and T2DM provides an additional source of biologically meaningful biomarkers. Evaluation of this biomarker in de novo PD patients and in a larger prospective longitudinal study is warranted.     

Bioremediation Potential of Chlorella: Spectroscopic,Kinetics, and Sem Studies

A dead dried alga, Chlorella sp., was used for the uptake of Cr⁺³, Cr₂O₇ ^?2, Cu⁺², and Ni⁺² from the aqueous solutions of these metal ions. The equilibrium data were fitted using the Langmuir and Freundlich isotherm model and the maximum uptakes for Cr⁺³, Cr₂O₇ ^?2, Ni⁺², and Cu⁺² were 98, 104, 108, and 183 mg/g, respectively. The Freundlich model, in comparison to the Langmuir model, better represented the sorption process. The kinetics of metal ions uptake by Chlorella sp. was best described by a pseudo-second order rate equation. Infrared spectroscopic data were employed to identify the site(s) of bonding in Chlorella sp. A scanning electron microscopic (SEM) study of pure dead Chlorella sp. and the species treated with different metal ions provided an idea of the extent of metal uptake by this species. The dead Chlorella sp took up maximum Cu(II). The size of the cell of the metal-treated Chlorella sp. obtained from SEM data is in agreement with the extent of metal uptake.     

Antibody Phage Display Assisted Identification of Junction Plakoglobin as a Potential Biomarker for Atherosclerosis

To date, no plaque-derived blood biomarker is available to allow diagnosis, prognosis or monitoring of atherosclerotic vascular diseases. In this study, specimens of thrombendarterectomy material from carotid and iliac arteries were incubated in protein-free medium to obtain plaque and control secretomes for subsequent subtractive phage display. The selection of nine plaque secretome-specific antibodies and the analysis of their immunopurified antigens by mass spectrometry led to the identification of 22 proteins. One of them, junction plakoglobin (JUP-81) and its smaller isoforms (referred to as JUP-63, JUP-55 and JUP-30 by molecular weight) were confirmed by immunohistochemistry and immunoblotting with independent antibodies to be present in atherosclerotic plaques and their secretomes, coronary thrombi of patients with acute coronary syndrome (ACS) and macrophages differentiated from peripheral blood monocytes as well as macrophage-like cells differentiated from THP1 cells. Plasma of patients with stable coronary artery disease (CAD) (n = 15) and ACS (n = 11) contained JUP-81 at more than 2- and 14-fold higher median concentrations, respectively, than plasma of CAD-free individuals (n = 13). In conclusion, this proof of principle study identified and verified JUP isoforms as potential plasma biomarkers for atherosclerosis. Clinical validation studies are needed to determine its diagnostic efficacy and clinical utility as a biomarker for diagnosis, prognosis or monitoring of atherosclerotic vascular diseases.     

Egg-derived tyrosine phosphatase as a Potential Biomarker for Muscle Ageing and Degeneration in Drosophila melanogaster

Ageing is associated with declined activity of behaviors, physiology and metabolic processes （Arking, 2006）. In- vestigations in model organisms have indicated the exis- tence of ＂functional senescence＂, the progressive decline of biological functions with age and the decline in the activity may vary from tissue to tissue. Consequently, studies per- taining to the key organs/tissues whose functions deterio- rate/fail with age have led to the development of tissue specific ageing biomarkers （Grotewiel et al., 2005; Demontis et al., 2013）.     

Imaging the Perivascular Space as a Potential Biomarker of Neurovascular and Neurodegenerative Diseases

Although the brain lacks conventional lymphatic vessels found in peripheral tissue, evidence suggests that the space surrounding the vasculature serves a similar role in the clearance of fluid and metabolic waste from the brain. With aging, neurodegeneration, and cerebrovascular disease, these microscopic perivascular spaces can become enlarged, allowing for visualization and quantification on structural MRI. The purpose of this review is to: (i) describe some of the recent pre-clinical findings from basic science that shed light on the potential neurophysiological mechanisms driving glymphatic and perivascular waste clearance, (ii) review some of the pathobiological etiologies that may lead to MRI-visible enlarged perivascular spaces (ePVS), (iii) describe the possible clinical implications of ePVS, (iv) evaluate existing qualitative and quantitative techniques used for measuring ePVS burden, and (v) propose future avenues of research that may improve our understanding of this potential clinical neuroimaging biomarker for fluid and metabolic waste clearance dysfunction in neurodegenerative and neurovascular diseases.     

Heat Shock Protein 70 Acts as a Potential Biomarker for Early Diagnosis of Heart Failure

Early identification for heart failure (HF) may be useful for disease modifying treatment in order to reduce heart disease progression or even to reverse it. In our previous studies, we have revealed a group of heat shock proteins (HSPs) which might be related to neonatal rat cardiomyocyte hypertrophy by proteomic approach. Here, we confirm that HSPs, including HSP27 and HSP70, altered in the early stage of cardiac remodeling in vivo animal model. Furthermore, plasma concentrations of those HSPs and their potential screening value were evaluated at different stages in 222 patient subjects. Plasma HSP27, HSP70 and HSP90 were measured using enzyme-linked immunosorbent assay. Results indicate that HSP70 was positively correlated to the severity (progression) of HF (r = 0.456, p<0.001). The area under the rate of change (ROC) curve was 0.601 (p = 0.017) in patients with stage B HF and 0.835 (p<0.001) in those with stage C HF. However, HSP27 and HSP90 did not display significant changes in any stage of HF in this study. Taken together, plasma concentrations of HSP70 elevated with the progression of HF and might act as a potential screening biomarker for early diagnosis of HF.     

Synthesis and Studies of Modified Oligonucleotides- Directed Triple Helix Formation at the Purine-Pyrimidine Interrupted Site

Abstract

Triple helix formation is still restricted to oligopurine-oligopyrimidine double stranded DNA target. Herein we focus on our progress achieved in nucleobase and oligonucleotide modifications area to address the chemical challenge to circumvent the recognition of a purine-pyrimidine base pair interruption in an oligopyrimidine-oligopurine DNA sequence.     

Role of a Neural Cell Adhesion Molecule Found in Cerebrospinal Fluid as a Potential Biomarker for Epilepsy

The neural cell adhesion molecule (NCAM-1) plays an important role in cell adhesion and synaptic plasticity. We designed this study to evaluate NCAM-1 as a potential biomarker for epilepsy. We performed a quantitative evaluation of the levels of NCAM-1 in cerebrospinal fluid (CSF) and serum and noted differences in patients with epilepsy compared to control subjects. We used sandwich enzyme-linked immunosorbent assays to measure NCAM-1 concentrations in CSF and serum samples of 76 epileptic patients (subdivided into the following subgroups: drug-refractory epilepsy, DRE; first-diagnosis epilepsy, FDE; and drug-effective epilepsy, DEE) and 44 control subjects. Our results show that cerebrospinal fluid–NCAM-1 (CSF–NCAM-1) concentrations and NCAM-1 Indices in the epileptic group were lower than in the control group. Both the CSF–NCAM-1 concentration and the NCAM-1 Indices in the drug-refractory epilepsy group were lower than in the drug-effective epilepsy group. These differences were statistically significant (P < 0.05). However, serum–NCAM-1 levels were not statistically different when comparing the epilepsy group to the control group (P > 0.05). Our results indicate that CSF–NCAM-1 is a potential biomarker for drug-effective epilepsy and drug-refractory epilepsy.     

早期胃癌血清特异标志物过氧化物酶4核酸适配体的筛选、鉴定与应用

胃癌是世界上死亡率第三的重大疾病,而在早期阶段却有着良好的治愈率和生存率。因此,找到针对早期胃癌的特异性标志物从而提高早期胃癌的检出率,是目前亟待解决的问题。在本实验室的早期研究中,发现过氧化物酶4（peroxiredoxin-4,PRDX4）有极大的潜能作为早期胃癌的特异性标志物,并且由于蛋白质结构的特殊性,能够分泌至血清中,为早期胃癌的无创化诊断提供了可能。本文为寻找血清中PRDX4蛋白的特异性适配体,通过消减-SELEX方法找到9种适配体。经特异性和亲和力分析后,证实其中Ap-EGACS-11在9种适配体中具有最高的特异性和亲和力。随后在适配体的验证研究中证实,相对于进展期胃癌病人血清、结直肠癌病人血清和正常人血清,Ap-EGACS-11对早期胃癌病人血清的检出率最高。该结果表明,PRDX4具有早期胃癌特异性血清标志物的潜能,且Ap-EGACS-11可直接作为早期胃癌的检测试剂。     

Validation of LRG1 as a Potential Biomarker for Detection of Epithelial Ovarian Cancer by a Blinded Study

Background

Leucine-rich alpha-2-glycoprotein (LRG1) was found to be differentially expressed in sera from patients with Epithelial Ovarian Cancer (EOC). The aim of this study is to investigate the performance of LRG1 for detection of EOC, including early stage EOC, and to evaluate if LRG1 can complement CA125 in order to improve EOC detection using two independent blinded sample sets.

Methods and Results

Serum LRG1 and CA125 were measured by immunoassays. All assays were performed blinded to clinical data. Using the two independent sample sets (156 participants for sample set 1, and 233 for sample set 2), LRG1 was differentially expressed in EOC cases as compared to healthy, surgical, and benign controls, and its performance was not affected by the conditions of blood collection. The areas under the ROC curve (AUC) for LRG1 in differentiating EOC cases from non-cases were 0.797 and 0.786 for sample set 1 and 2. For differentiating EOC cases from healthy controls, the AUC values for LRG1 were 0.792 and 0.794. At a fixed specificity of 95%, LRG1 detects 52%, and 53.5% of EOC cases from healthy controls for sample set 1 and 2. When combining LRG1 and CA125, the AUC value increased to 0.927, which was improved compared to CA125 (AUC=0.916) (p=0.008) alone in distinguishing EOC cases from non-cases. More importantly, LRG1 also showed potential performance in differentiating early stage EOC from non-cases with an AUC of 0.715 for sample set 1, and 0.690 for sample set 2. The combination of LRG1 and CA125 resulted in an AUC of 0.838, which outperforms CA125 (AUC=0.785) (p=0.018) in detecting early stage EOC cases from non-cases using the larger sample set.

Conclusions

LRG1 could be a useful biomarker alone or in combination with CA125 for the diagnosis of ovarian cancer.