Optical Biomarkers of Serous and Mucinous Human Ovarian Tumor Assessed with Nonlinear Optics Microscopies

Background

Nonlinear optical (NLO) microscopy techniques have potential to improve the early detection of epithelial ovarian cancer. In this study we showed that multimodal NLO microscopies, including two-photon excitation fluorescence (TPEF), second-harmonic generation (SHG), third-harmonic generation (THG) and fluorescence lifetime imaging microscopy (FLIM) can detect morphological and metabolic changes associated with ovarian cancer progression.

Methodology/Principal Findings

We obtained strong TPEF + SHG + THG signals from fixed samples stained with Hematoxylin & Eosin (H&E) and robust FLIM signal from fixed unstained samples. Particularly, we imaged 34 ovarian biopsies from different patients (median age, 49 years) including 5 normal ovarian tissue, 18 serous tumors and 11 mucinous tumors with the multimodal NLO platform developed in our laboratory. We have been able to distinguish adenomas, borderline, and adenocarcinomas specimens. Using a complete set of scoring methods we found significant differences in the content, distribution and organization of collagen fibrils in the stroma as well as in the morphology and fluorescence lifetime from epithelial ovarian cells.

Conclusions/Significance

NLO microscopes provide complementary information about tissue microstructure, showing distinctive patterns for serous and mucinous ovarian tumors. The results provide a basis to interpret future NLO images of ovarian tissue and lay the foundation for future in vivo optical evaluation of premature ovarian lesions.     

Evidence of Specialized Tissue in Human Interatrial Septum: Histological,Immunohistochemical and Ultrastructural Findings

Background

There is a paucity of information on structural organization of muscular bundles in the interatrial septum (IAS). The aim was to investigate histologic and ultrastructural organization of muscular bundles in human IAS, including fossa ovalis (FO) and flap valve.

Methods

Macroscopic and light microscopy evaluations of IAS were performed from postmortem studies of 40 patients. Twenty three IAS specimens underwent serial transverse sectioning, and 17 - longitudinal sectioning. The transverse sections from 10 patients were immunolabeled for HCN4, Caveolin3 and Connexin43. IAS specimens from 6 other patients underwent electron microscopy.

Results

In all IAS specimens sections the FO, its rims and the flap valve had muscle fibers consisting of working cardiac myocytes. Besides the typical cardiomyocytes there were unusual cells: tortuous and horseshoe-shaped intertangled myocytes, small and large rounded myocytes with pale cytoplasm. The cells were aggregated in a definite structure in 38 (95%) cases, which was surrounded by fibro-fatty tissue. The height of the structure on transverse sections positively correlated with age (P = 0.03) and AF history (P = 0.045). Immunohistochemistry showed positive staining of the cells for HCN4 and Caveolin3. Electron microscopy identified cells with characteristics similar to electrical conduction cells.

Conclusions

Specialized conduction cells in human IAS have been identified, specifically in the FO and its flap valve. The cells are aggregated in a structure, which is surrounded by fibrous and fatty tissue. Further investigations are warranted to explore electrophysiological characteristics of this structure.     

Quantification of Normal Cell Fraction and Copy Number Neutral LOH in Clinical Lung Cancer Samples Using SNP Array Data

Background

Technologies based on DNA microarrays have the potential to provide detailed information on genomic aberrations in tumor cells. In practice a major obstacle for quantitative detection of aberrations is the heterogeneity of clinical tumor tissue. Since tumor tissue invariably contains genetically normal stromal cells, this may lead to a failure to detect aberrations in the tumor cells.

Principal Finding

Using SNP array data from 44 non-small cell lung cancer samples we have developed a bioinformatic algorithm that accurately models the fractions of normal and tumor cells in clinical tumor samples. The proportion of normal cells in combination with SNP array data can be used to detect and quantify copy number neutral loss-of-heterozygosity (CNNLOH) in the tumor cells both in crude tumor tissue and in samples enriched for tumor cells by laser capture microdissection.

Conclusion

Genome-wide quantitative analysis of CNNLOH using the CNNLOH Quantifier method can help to identify recurrent aberrations contributing to tumor development in clinical tumor samples. In addition, SNP-array based analysis of CNNLOH may become important for detection of aberrations that can be used for diagnostic and prognostic purposes.     

Magnetic Resonance Imaging of Soft Tissue Infection with Iron Oxide Labeled Granulocytes in a Rat Model

Object

We sought to detect an acute soft tissue infection in rats by magnetic resonance imaging (MRI) using granulocytes, previously labeled with superparamagnetic particles of iron oxide (SPIO).

Materials and Methods

Parasternal infection was induced by subcutaneous inoculation of Staphylococcus aureus suspension in rats. Granulocytes isolated from isogenic donor rats were labeled with SPIO. Infected rats were imaged by MRI before, 6 and 12 hours after intravenous injection of SPIO-labeled or unlabeled granulocytes. MR findings were correlated with histological analysis by Prussian blue staining and with re-isolated SPIO-labeled granulocytes from the infectious area by magnetic cell separation.

Results

Susceptibility effects were present in infected sites on post-contrast T2*-weighted MR images in all animals of the experimental group. Regions of decreased signal intensity (SI) in MRI were detected at 6 hours after granulocyte administration and were more pronounced at 12 hours. SPIO-labeled granulocytes were identified by Prussian blue staining in the infected tissue and could be successfully re-isolated from the infected area by magnetic cell separation.

Conclusion

The application of SPIO-labeled granulocytes in MRI offers new perspectives in diagnostic specificity and sensitifity to detect early infectious processes.     

Pathological steps of cancer-related lymphedema: histological changes in the collecting lymphatic vessels after lymphadenectomy

Introduction

To date, an electron microscopy study of the collecting lymphatic vessels has not been conducted to examine the early stages of lymphedema. However, such histological studies could be useful for elucidating the mechanism of lymphedema onset. The aim of this study was to clarify the changes occurring in collecting lymphatic vessels after lymphadenectomy.

Methods

The study was conducted on 114 specimens from 37 patients who developed lymphedema of the lower limbs after receiving surgical treatment for gynecologic cancers and who consulted the University of Tokyo Hospital and affiliated hospitals from April 2009 to March 2011. Lymphatic vessels that were not needed for lymphatico venous anastomosis surgery were trimmed and subsequently examined using electron microscopy and light microscopy.

Results

Based on macroscopic findings, the histochemical changes in the collecting lymphatic vessels were defined as follows: normal, ectasis, contraction, and sclerosis type (NECST). In the ectasis type, an increase in endolymphatic pressure was accompanied by a flattening of the lymphatic vessel endothelial cells. In the contraction type, smooth muscle cells were transformed into synthetic cells and promoted the growth of collagen fibers. In the sclerosis type, fibrous elements accounted for the majority of the components, the lymphatic vessels lost their transport and concentrating abilities, and the lumen was either narrowed or completely obstructed.

Conclusions

The increase in pressure inside the collecting lymphatic vessels after lymphadenectomy was accompanied by histological changes that began before the onset of lymphedema.     

Airway-centered interstitial fibrosis: etiology,clinical findings and prognosis

Background

Airway-centered Interstitial Fibrosis (ACIF) is a common pathologic pattern observed in our practice.

Objectives

The objectives of this study are to describe the causes associated with ACIF in a large sample of patients and its effect on survival.

Methods

A retrospective study in three centers of interstitial lung disease in São Paulo, between January of 1995 and December of 2012. The surgical lung biopsy specimens were reviewed by three pathologists. The clinical, functional and tomographic findings were analyzed by a standardized protocol.

Results

There were 68 cases of ACIF, most of them women. The mean age was 57 ± 12 yr. Dyspnea, cough, restrictive pattern at spirometry and oxygen desaturation at exercise were common. A reticular pattern with peribronchovascular infiltrates was found in 79% of the cases. The etiologies of ACIF were hypersensitivity pneumonitis in 29 (42.6%), gastroesophageal reflux disease in 17 (25.0%), collagen vascular disease in 4 (5.9%), a combination of them in 15 cases and idiopathic in 3 (4.4%). The median survival was 116 months (95% CI = 58.5 – 173.5). Lower values of oxygen saturation at rest, presence of cough and some histological findings - organizing tissue in the airways, fibroblastic foci and microscopic honeycombing - were predictors of worse survival.

Conclusions

ACIF is an interstitial lung disease with a better survival when compared with IPF. The main etiologies are HP and GERD. The oxygen saturation at rest, the presence of cough and some histological findings are predictors of survival.     

Retinal Thickness Measurement Obtained with Spectral Domain Optical Coherence Tomography Assisted Optical Biopsy Accurately Correlates with Ex Vivo Histology

Background

This study determines ‘correlation constants’ between the gold standard histological measurement of retinal thickness and the newer spectral-domain optical coherence tomography (SD-OCT) technology in adult C57BL/6 mice.

Methods

Forty-eight eyes from adult mice underwent SD-OCT imaging and then were histologically prepared for frozen sectioning with H&E staining. Retinal thickness was measured via 10x light microscopy. SD-OCT images and histological sections were standardized to three anatomical sites relative to the optic nerve head (ONH) location. The ratios between SD-OCT to histological thickness for total retinal thickness (TRT) and six sublayers were defined as ‘correlation constants’.

Results

Mean (± SE) TRT for SD-OCT and histological sections was 210.95 µm (±1.09) and 219.58 µm (±2.67), respectively. The mean ‘correlation constant’ for TRT between the SD-OCT and histological sections was 0.96. The retinal thickness for all sublayers measured by SD-OCT vs. histology were also similar, the ‘correlation constant’ values ranged from 0.70 to 1.17. All SD-OCT and histological measurements demonstrated highly significant (p<0.01) strong positive correlations.

Conclusion

This study establishes conversion factors for the translation of ex vivo data into in vivo information; thus enhancing the applicability of SD-OCT in translational research.     

Targeting bone alleviates osteoarthritis in osteopenic mice and modulates cartilage catabolism

Objective

Subchondral bone modifications occur early in the development of osteoarthritis (OA). The level of bone resorption might impact cartilage remodeling. We therefore assessed the in vivo and in vitro effects of targeting bone resorption in OA and cartilage metabolism.

Methods

OA was induced by meniscectomy (MNX) in ovariectomized osteopenic mice (OP) treated with estradiol (E2), pamidronate (PAM), or phosphate buffered saline (PBS) for 6 weeks. We assessed the subchondral bone and cartilage structure and the expression of cartilage matrix proteases. To assess the involvement of bone soluble factors in cartilage metabolism, supernatant of human bone explants pre-treated with E2 or PAM were transferred to cartilage explants to assess proteoglycan release and aggrecan cleavage. OPG/RANKL mRNA expression was assessed in bone explants by real-time quantitative PCR. The role of osteoprotegerin (OPG) in the bone-cartilage crosstalk was tested using an OPG neutralizing antibody.

Results

Bone mineral density of OP mice and osteoclast number were restored by E2 and PAM (p<0.05). In OP mice, E2 and PAM decreased ADAMTS-4 and -5 expression, while only PAM markedly reduced OA compared to PBS (2.0±0.63 vs 5.2±0.95; p<0.05). OPG/RANKL mRNA was increased in human bone explants treated with both drugs (2.2–3.7-fold). Moreover, supernatants from bone explants cultured with E2 or PAM reduced aggrecan cleavage and cartilage proteoglycan release (73±8.0% and 80±22% of control, respectively, p<0.05). This effect was reversed with osteoprotegerin blockade.

Conclusion

The inhibition of bone resorption by pamidronate in osteopenic mice alleviates the histological OA score with a reduction in the expression of aggrecanases. Bone soluble factors, such as osteoprotegerin, impact the cartilage response to catabolic factors. This study further highlights the importance of subchondral bone in the regulation of joint cartilage damage in OA.     

Biochemical Parameters for Longitudinal Monitoring of Liver Function in Rat Models of Partial Hepatectomy Following Liver Injury

Background

While evaluation of liver function in preclinical animal studies is commonly performed at selected time-points by invasive determination of the liver/body weight ratio and histological analyses, the validation of longitudinal measurement tools for monitoring liver function are of major interest.

Aims

To longitudinally evaluate serum cholinesterase (CHE) and total serum bilirubin (TSB) levels as non-invasive markers to determine injury- and partial hepatectomy (PHx)-induced alterations of liver function in rats.

Methods

Male and female Lewis rats were subjected to either methionine/choline deficient (MCD) diet or treatment with FOLFOX chemotherapy prior to PHx. Body weight and CHE/TSB levels are determined weekly. Following PHx and at the study end, histological analyses of liver tissue are performed.

Results

Following MCD diet, but not after FOLFOX chemotherapy treatment, results indicate gender-specific alterations in serum CHE levels and gender-independent alterations in TSB levels. Likewise, histological analyses of resected liver parts indicate significant liver injury following MCD-diet, but not following FOLFOX treatment. While TSB levels rapidly recover following MCD diet/FOLFOX treatment combined with a PHx, serum CHE levels are subject to significant model- and gender-specific differences, despite full histopathological recovery of liver tissue.

Conclusions

Longitudinal measurements of serum CHE levels and TSB levels in rats are highly complementary as non-invasive parameters for evaluation of liver injury and/or recovery.     

Dynamic contrast-enhanced magnetic resonance imaging of metacarpophalangeal joints reflects histological signs of synovitis in rheumatoid arthritis

Introduction

Synovial inflammation and joint destruction in rheumatoid arthritis (RA) may progress despite clinical remission. Dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) is increasingly used to detect synovial inflammation in RA. Although small joints such as metacarpophalangeal (MCP) joints are mainly affected by RA, MRI findings have never been directly compared to histological synovitis in MCP synovial tissue. The objective of the current study was therefore to analyse if DCE-MRI relates to histological signs of synovitis small RA joints.

Methods

In 9 RA patients, DCE-MRI (3 Tesla, dynamic 2D T1 weighted turbo-flash sequence) of the hand was performed prior to arthroscopically-guided synovial biopsies from the second MCP of the imaged hand. Maximum enhancement (ME), rate of early enhancement, and maximum rate of enhancement were assessed in the MCP. Synovial biopsies were stained for determination of sublining CD68 and the Synovitis Score. Correlations between MRI and histological data were calculated according to Spearman.

Results

ME of the MCP significantly correlated to sublining CD68 staining (r = 0.750, P = 0.02), the Synovitis Score (r = 0.743, P = 0.02), and the subscores for lining layer hypertrophy (r = 0.789, P = 0.01) and cellular density (r = 0.842; P = 0.004).

Conclusions

Perfusion imaging of synovial tissue in RA finger joints employing DCE-MRI reflects histological synovial inflammation. According to our study, ME is the most closely associated parameter amongst the measures considered.     

Ultrastructural Features of Ischemic Tissue following Application of a Bio-Membrane Based Progenitor Cardiomyocyte Patch for Myocardial Infarction Repair

Background and Objective

Implantation of cell-sheets into damaged regions of the heart after myocardial infarction (MI) has been shown to improve heart function. However, the tissue morphology following application of induced pluripotent stem cell (iPSC)-derived cardiomyocytes (CM) has not been studied in detail at the level afforded by electron microscopy. We hypothesized that increasing the number of CM derived from iPSC would increase the effectiveness of cell-sheets used to treat ischemic cardiomyopathy. We report here on the ultrastructural features after application of a bio-membrane ‘cell patch’.

Methods

iPSC-derived progenitor cells were transduced using lentivirus vectors with or without NCX1 promoter. iPSC-CM sheets were transplanted over the transmural MI region in a mouse model of regional ischemic cardiomyopathy. Mice were divided into four groups, 1) Sham; 2) MI; 3) MI + iPSC without NCX1 treated cells (MI + iPSC^Null) and 4) MI + iPSC receiving NCX1 promoter treated cells (MI + iPSC^NCX1). Echocardiography was performed 4 weeks after cell patch application, followed by histological and transmission electron microscopy (TEM) analysis.

Results

Large numbers of transplanted CM were observed with significant improvements in left ventricular performance and remodeling in group 4 as compared with group 3. No teratoma formation was detected in any of the treatment groups.

Conclusion

Manipulation of iPSC yields large numbers of iPSC-CM and favorable morphological and ultrastructural tissue changes. These changes have the potential to enhance current methods used for restoration of cardiac function after MI.     

Extraocular Muscle Characteristics Related to Myasthenia Gravis Susceptibility

Background

The pathogenesis of extraocular muscle (EOM) weakness in myasthenia gravis might involve a mechanism specific to the EOM. The aim of this study was to investigate characteristics of the EOM related to its susceptibility to myasthenia gravis.

Methods

Female F344 rats and female Sprague-Dawley rats were assigned to experimental and control groups. The experimental group received injection with Ringer solution containing monoclonal antibody against the acetylcholine receptor (AChR), mAb35 (0.25 mg/kg), to induce experimental autoimmune myasthenia gravis, and the control group received injection with Ringer solution alone. Three muscles were analyzed: EOM, diaphragm, and tibialis anterior. Tissues were examined by light microscopy, fluorescence histochemistry, and transmission electron microscopy. Western blot analysis was used to assess marker expression and ELISA analysis was used to quantify creatine kinase levels. Microarray assay was conducted to detect differentially expressed genes.

Results

In the experimental group, the EOM showed a simpler neuromuscular junction (NMJ) structure compared to the other muscles; the NMJ had fewer synaptic folds, showed a lesser amount of AChR, and the endplate was wider compared to the other muscles. Results of microarray assay showed differential expression of 54 genes in the EOM between the experimental and control groups.

Conclusion

Various EOM characteristics appear to be related to the increased susceptibility of the EOM and the mechanism of EOM weakness in myasthenia gravis.     

Quantitative proteomic analysis of formalin–fixed,paraffin–embedded clear cell renal cell carcinoma tissue using stable isotopic dimethylation of primary amines

Background

Formalin-fixed, paraffin-embedded (FFPE) tissues represent the most abundant resource of archived human specimens in pathology. Such tissue specimens are emerging as a highly valuable resource for translational proteomic studies. In quantitative proteomic analysis, reductive di-methylation of primary amines using stable isotopic formaldehyde variants is increasingly used due to its robustness and cost-effectiveness.

Results

In the present study we show for the first time that isotopic amine dimethylation can be used in a straightforward manner for the quantitative proteomic analysis of FFPE specimens without interference from formalin employed in the FFPE process. Isotopic amine dimethylation of FFPE specimens showed equal labeling efficiency as for cryopreserved specimens. For both FFPE and cryopreserved specimens, differential labeling of identical samples yielded highly similar ratio distributions within the expected range for dimethyl labeling. In an initial application, we profiled proteome changes in clear cell renal cell carcinoma (ccRCC) FFPE tissue specimens compared to adjacent non–malignant renal tissue. Our findings highlight increased levels of glyocolytic enzymes, annexins as well as ribosomal and proteasomal proteins.

Conclusion

Our study establishes isotopic amine dimethylation as a versatile tool for quantitative proteomic analysis of FFPE specimens and underlines proteome alterations in ccRCC.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1768-x) contains supplementary material, which is available to authorized users.     

Quantitative Analysis of Peripheral Tissue Perfusion Using Spatiotemporal Molecular Dynamics

Background

Accurate measurement of peripheral tissue perfusion is challenging but necessary to diagnose peripheral vascular insufficiency. Because near infrared (NIR) radiation can penetrate relatively deep into tissue, significant attention has been given to intravital NIR fluorescence imaging.

Methodology/Principal Findings

We developed a new optical imaging-based strategy for quantitative measurement of peripheral tissue perfusion by time-series analysis of local pharmacokinetics of the NIR fluorophore, indocyanine green (ICG). Time-series NIR fluorescence images were obtained after injecting ICG intravenously in a murine hindlimb ischemia model. Mathematical modeling and computational simulations were used for translating time-series ICG images into quantitative pixel perfusion rates and a perfusion map. We could successfully predict the prognosis of ischemic hindlimbs based on the perfusion profiles obtained immediately after surgery, which were dependent on the preexisting collaterals. This method also reflected increases in perfusion and improvements in prognosis of ischemic hindlimbs induced by treatment with vascular endothelial growth factor and COMP-angiopoietin-1.

Conclusions/Significance

We propose that this novel NIR-imaging-based strategy is a powerful tool for biomedical studies related to the evaluation of therapeutic interventions directed at stimulating angiogenesis.     

Quantification of lung surface area using computed tomography

Objective

To refine the CT prediction of emphysema by comparing histology and CT for specific regions of lung. To incorporate both regional lung density measured by CT and cluster analysis of low attenuation areas for comparison with histological measurement of surface area per unit lung volume.

Methods

The histological surface area per unit lung volume was estimated for 140 samples taken from resected lung specimens of fourteen subjects. The region of the lung sampled for histology was located on the pre-operative CT scan; the regional CT median lung density and emphysematous lesion size were calculated using the X-ray attenuation values and a low attenuation cluster analysis. Linear mixed models were used to examine the relationships between histological surface area per unit lung volume and CT measures.

Results

The median CT lung density, low attenuation cluster analysis, and the combination of both were important predictors of surface area per unit lung volume measured by histology (p < 0.0001). Akaike''s information criterion showed the model incorporating both parameters provided the most accurate prediction of emphysema.

Conclusion

Combining CT measures of lung density and emphysematous lesion size provides a more accurate estimate of lung surface area per unit lung volume than either measure alone.     

Confocal laser scanning microscopy, a new in vivo diagnostic tool for schistosomiasis

Background

The gold standard for the diagnosis of schistosomiasis is the detection of the parasite''s characteristic eggs in urine, stool, or rectal and bladder biopsy specimens. Direct detection of eggs is difficult and not always possible in patients with low egg-shedding rates. Confocal laser scanning microscopy (CLSM) permits non-invasive cell imaging in vivo and is an established way of obtaining high-resolution images and 3-dimensional reconstructions. Recently, CLSM was shown to be a suitable method to visualize Schistosoma mansoni eggs within the mucosa of dissected mouse gut. In this case, we evaluated the suitability of CLSM to detect eggs of Schistosoma haematobium in a patient with urinary schistosomiasis and low egg-shedding rates.

Methodology/Principal Findings

The confocal laser scanning microscope used in this study was based on a scanning laser system for imaging the retina of a living eye, the Heidelberg Retina Tomograph II, in combination with a lens system (image modality). Standard light cystoscopy was performed using a rigid cystoscope under general anaesthesia. The CLSM endoscope was then passed through the working channel of the rigid cystoscope. The mucosal tissue of the bladder was scanned using CLSM. Schistoma haematobium eggs appeared as bright structures, with the characteristic egg shape and typical terminal spine.

Conclusion/Significance

We were able to detect schistosomal eggs in the urothelium of a patient with urinary schistosomiasis. Thus, CLSM may be a suitable tool for the diagnosis of schistosomiasis in humans, especially in cases where standard diagnostic tools are not suitable.     

“Salvage Microbiology”: Detection of Bacteria Directly from Clinical Specimens following Initiation of Antimicrobial Treatment

Background

PCR coupled with electrospray ionization mass spectrometry (ESI-MS) is a diagnostic approach that has demonstrated the capacity to detect pathogenic organisms from culture negative clinical samples after antibiotic treatment has been initiated. [1] We describe the application of PCR/ESI-MS for detection of bacteria in original patient specimens that were obtained after administration of antibiotic treatment in an open investigation analysis.

Methods

We prospectively identified cases of suspected bacterial infection in which cultures were not obtained until after the initiation of antimicrobial treatment. PCR/ESI-MS was performed on 76 clinical specimens that were submitted for conventional microbiology testing from 47 patients receiving antimicrobial treatment.

Findings

In our series, 72% (55/76) of cultures obtained following initiation of antimicrobial treatment were non-diagnostic (45 negative cultures; and 10 respiratory specimens with normal flora (5), yeast (4), or coagulase-negative staphylococcus (1)). PCR/ESR-MS detected organisms in 83% (39/47) of cases and 76% (58/76) of the specimens. Bacterial pathogens were detected by PCR/ESI-MS in 60% (27/45) of the specimens in which cultures were negative. Notably, in two cases of relapse of prosthetic knee infections in patients on chronic suppressive antibiotics, the previous organism was not recovered in tissue cultures taken during extraction of the infected knee prostheses, but was detected by PCR/ESI-MS.

Conclusion

Molecular methods that rely on nucleic acid amplification may offer a unique advantage in the detection of pathogens collected after initiation of antimicrobial treatment and may provide an opportunity to target antimicrobial therapy and “salvage” both individual treatment regimens as well as, in select cases, institutional antimicrobial stewardship efforts.     

Allelic mapping bias in RNA-sequencing is not a major confounder in eQTL studies

Background

RNA sequencing (RNA-seq) is the current gold-standard method to quantify gene expression for expression quantitative trait locus (eQTL) studies. However, a potential caveat in these studies is that RNA-seq reads carrying the non-reference allele of variant loci can have lower probability to map correctly to the reference genome, which could bias gene quantifications and cause false positive eQTL associations. In this study, we analyze the effect of this allelic mapping bias in eQTL discovery.

Results

We simulate RNA-seq read mapping over 9.5 M common SNPs and indels, with 15.6% of variants showing biased mapping rate for reference versus non-reference reads. However, removing potentially biased RNA-seq reads from an eQTL dataset of 185 individuals has a very small effect on gene and exon quantifications and eQTL discovery. We detect only a handful of likely false positive eQTLs, and overall eQTL SNPs show no significant enrichment for high mapping bias.

Conclusion

Our results suggest that RNA-seq quantifications are generally robust against allelic mapping bias, and that this does not have a severe effect on eQTL discovery. Nevertheless, we provide our catalog of putatively biased loci to allow better controlling for mapping bias to obtain more accurate results in future RNA-seq studies.

Electronic supplementary material

The online version of this article (doi:10.1186/s13059-014-0467-2) contains supplementary material, which is available to authorized users.     

Application of Hyperbranched Rolling Circle Amplification for Direct Detection of Mycobacterium Tuberculosis in Clinical Sputum Specimens

Background

Global tuberculosis (TB) control is encumbered by the lack of a rapid and simple detection method for diagnosis, especially in low-resource areas. An isothermal amplification method, hyperbranched rolling circle amplification (HRCA), was optimized to detect Mycobacterium tuberculosis (Mtb) in clinical sputum specimens.

Methods

A clinical validation study was performed to assess the diagnostic accuracy of HRCA. In order to analyze the detection limit of HRCA under optimal conditions, the method was initially used to detect purified H37Rv strain DNA and culture suspensions. Next, three strains of Mycobacterium tuberculosis complex (MTC) and eight strains of non-tuberculosis mycobacterium (NTM) were analyzed in order to evaluate specificity. Sputum specimens from 136 patients with diagnosed pulmonary TB, 38 lung cancer patients, and 34 healthy donors were tested by HRCA to validate the clinical application of HRCA for the rapid detection of Mtb.

Results

The detection limit of HRCA for purified H37Rv DNA and culture suspensions was 740 aM and 200cfu/ml, respectively. The results of all MTC strains were positive in contrast to the NTM specimens which were all negative. The detection sensitivity for the 136 sputum specimens from TB patients was 77.2% (105/136), which was slightly lower than that of quantitative real-time PCR(79.4%, 108/136) and culture (80.9%,110/136). The sensitivity of all three methods was statistically higher than smear microscopy (44.9%, 61/136). The overall specificity of HRCA was 98.6% (71/72) which was similar to that of quantitative real-time PCR (qRT-PCR) and smear/culture methods (100%, 72/72).

Conclusions

Use of the HRCA assay for detection of Mtb within clinical sputum specimens was demonstrated to be highly sensitive and specific. Moreover, the performance of HRCA is simple and cost-effective compared with qRT-PCR and is less time consuming than culture. Therefore, HRCA is a promising TB diagnostic tool that can be used routinely in low-resource clinical settings.