Detection of circulating tumor cells by nested RT-PCR targeting EGFR/CEA/CK20mRNAs in colorectal carcinoma patients

Background: EGFR is involved in the epidermal growth factors pathway that regulates cellular processes and is associated with the development of many types of cancer including colorectal cancer. Molecular methods with high sensitivity such as nested polymerase chain reaction (PCR) based assays have been used to search for tumor cell specific markers. This study aimed to detect the circulating EGFRmRNA expressing tumor cells and its diagnostic value in colorectal cancer compared with that of known markers of circulating cancer cells CEA and CK20.Subjects and methods: This study included 36 patients diagnosed as having colon cancer of different stages and 18 matched healthy controls. The staging was carried out according to the TNM classification.We used nested RT-PCR-based reverse transcription PCR assay for the detection of circulating cancer cells in the peripheral blood.Results: The blood samples from the colon cancer patients showed detection of EGFR in 15/36 patients (41.7%); CEAmRNA in 22/36 patients (61.1%) and CK20mRNA in 24/36 patients (66.7%). No evidence of EGFR mRNA expression in any of the samples used as controls. 3/18(16.7%) and 4/18 (22.2%) of healthy controls gave a positive result of CEA/CK20mRNAs. There was a statistically significant difference in the prevalence of EGFR/CEA and CK20mRNAs expression between the early disease group (stage I and II) and the advanced disease group (stage IIIand IV) (P < 0.01). Colon cancer patients with a high level of serum CEA exhibited detectable concentrations of EGFR and CEA and CK20mRNAs more often than those with a low serum CEA level, there is significant difference (P < 0.01).Conclusion: EGFR assay might represent a suitable marker for detection of circulating tumor cells in colon cancer patients. CEA and CK20mRNAs are significantly more frequently detected in colon cancer patients than in healthy controls supports the hypothesis that they are promising complementary markers for CRC diagnosis. The assessment of multiple molecular tumor markers improved the sensitivity in detecting circulating tumor cells but due to limited specificity; identification and validation of genes and proteins implicated in metastatic processes need to be furtherinvestigated

Molecular detection of circulating thyroid specific transcripts (TSHR/Tg-mRNAs) in thyroid cancer patients: Their diagnostic significance

Thyroid cancer is the most prevalent endocrine malignancy. The preoperative diagnosis of differentiated thyroid cancer (DTC) that relies solely on fine-needle aspiration (FNAC) biopsy, sometimes possesses conflicting results. New molecular markers for thyroid cancer have been investigated with most of them based on the detection in thyroid nodules or tumor tissue specimens. Recently, it was possible to detect thyroid cancer cells in the circulation by measuring the mRNA of thyroid specific genes. Among these, thyroglobulin and more recently thyroid stimulating hormone receptor mRNAs, TSHR/Tg-mRNAs in peripheral blood might serve as cancer-specific markers. These have become promising new circulating markers for thyroid cancer. The purpose of this study is to assess TSHR/Tg-mRNAs as diagnostic molecular markers for thyroid cancer and if they can be used preoperatively in synergy with FNAC. This study was performed on 60 subjects; 20 healthy volunteers and 40 patients; including 16 patients with benign thyroid diseases, 24 patients with thyroid cancer; 18 patients with newly diagnosed (DTC) and 6 patients with recurrent thyroid cancer. Diagnosis of cancer was based on FNAC and histopathology of surgical specimens. All subjects had TSHR/Tg-mRNAs in peripheral blood measured by reverse transcriptase (RT)-PCR. Based on cytology/pathology; 18 patients had newly diagnosed DTC and 11 had benign thyroid disease. Preoperative FNAC was performed on 29 of 40 patients; FNAC was diagnostic in 11/18 of malignant lesions (61.1%), in 8/11 of benign lesions (72.7%), while 10/29 (34.5%) were indeterminate. TSHR/Tg-mRNAs correctly diagnosed DTC in 20/24 and 19/24 (sensitivity 83.3% and 79.1%) and benign disease in 14/16 and 13/16 (specificity 87.5% and 81.3%), respectively. With indeterminate FNA, TSHR/Tg-mRNAs correctly diagnosed DTC (follicular type) in 5/7 and benign disease in 2/3 (combined sensitivity 71.4%; specificity 66.7%). There was high concordance between RT-PCR results for TSHR-mRNA and Tg-mRNA. Of the controls 19/20 (95%) and 16/20 (80%) were negative for both TSHR- and Tg-mRNAs. With the use of a carefully selected primer pair and qualitative RT-PCR; our results indicate that TSHR/Tg-mRNAs in peripheral blood are both equally sensitive and specific markers for detection of thyroid cancer cells. Combining TSHR/Tg-mRNAs and FNAC and ultrasound enhances the preoperative detection of cancer in patients with thyroid nodules, reducing unnecessary surgeries and correctly classified most follicular cancers and could have spared surgery in patients with benign disease.Keywords: Differentiated thyroid Cancer; TSHR/Tg-mRNAs; Fine-needle aspiration cytology; Thyroid nodules; Indeterminate lesions; Molecular marke

Multiple molecular markers MAGE-1, MAGE-3 and AFP mRNAs expression nested PCR assay for sensitive and specific detection of circulating hepatoma cells: Enhanced detection of hepatocellular carcinoma

Hepatocellular Carcinoma is a multifactorial, multistep and complex process. Its prognosis is poor and early diagnosis and monitoring of metastasis of HCC is of utmost importance. Circulating alpha-fetoprotien mRNA has been proposed as a marker of HCC cells disseminatedinto the circulation but the specificity of this molecular marker and its correlation with the main HCC clinico-pathological parameters remain controversial. In recent years; several different multi-marker assays have been developed for the detection of hepatoma cells in the peripheral bloodof patients with HCC. In this study 58 patients and 15 matched healthy volunteers were included; the patients were divided into three groups; group A: patients with primary HCC (n =32), group B: patients withcirrhosis with no evidence of HCC (n= 12), group C: patients with metastatic cancer in liver (n= 14). Group D: 15 healthy volunteers age and sex matched. The staging of HCC was carried out according to the Tumor/Node/Metastasis (TNM) classification. Peripheral blood samples were obtained from all subjects; MAGE-1 and MAGE-3 and AFP mRNAs were detected by nested RT-PCR. The positive rates of MAGE-1, MAGE-3 and AFP mRNAs were 18/32 (56.3%), 15/32 (46.9%) and 19/32 (59.4%) respectively in the primary HCC patients. In the cirrhotic group only 4/12 (33.3%) patients were positive for AFP mRNA, whereas in the metastatic group 5/14 (35.7%) and 4/14 (28.6%) were positive to MAGE-1 and MAGE-3 mRNAs respectively. MAGE-1 and MAGE-3 mRNAs were correlated with TNM clinical stages; tumor number and tumor size (p&lt;0.05).Our results indicate that a multi-marker nested RT-PCR assay with cancer-specific markers such as MAGE-1 and MAGE-3 in combination with a hepatocyte-specific AFP marker may be a promising diagnostic tool for monitoring hepatocellular carcinoma patients. Nested PCR exhibits highersensitivity, stronger specificity and lower false-positive occurrence as compared to single RT

Fluctuations of radiation from a chaotic laser below threshold

Radiation from a chaotic cavity filled with gain medium is considered. A set of coupled equations describing the photon density and the population of gain medium is proposed and solved. The spectral distribution and fluctuations of the radiation are found. The full noise is a result of a competition between positive correlations of photons with equal frequencies (due to stimulated emission and chaotic scattering) which increase fluctuations, and a suppression due to interaction with a gain medium which leads to negative correlations between photons. The latter effect is responsible for a pronounced suppression of the photonic noise as compared to the linear theory predictions.Comment: 7 pages, 5 figures; expanded version, to appear in Phys. Rev.

Localization of Light: Dual Symmetry between Absorption and Amplification

We study the propagation of radiation through a disordered waveguide with a complex dielectric constant $\epsilon$, and show that dual systems, which differ only in the sign of the imaginary part of $\epsilon$, have the same localization length. Paradoxically, absorption and stimulated emission of radiation suppress the transmittance of the waveguide in the same way.Comment: Added a reference to the paper by Z.Q. Zhang, Phys.Rev.B. 52, 7960 (1995

The Incremental Cooperative Design of Preventive Healthcare Networks

This document is the Accepted Manuscript version of the following article: Soheil Davari, 'The incremental cooperative design of preventive healthcare networks', Annals of Operations Research, first published online 27 June 2017. Under embargo. Embargo end date: 27 June 2018. The final publication is available at Springer via http://dx.doi.org/10.1007/s10479-017-2569-1.In the Preventive Healthcare Network Design Problem (PHNDP), one seeks to locate facilities in a way that the uptake of services is maximised given certain constraints such as congestion considerations. We introduce the incremental and cooperative version of the problem, IC-PHNDP for short, in which facilities are added incrementally to the network (one at a time), contributing to the service levels. We first develop a general non-linear model of this problem and then present a method to make it linear. As the problem is of a combinatorial nature, an efficient Variable Neighbourhood Search (VNS) algorithm is proposed to solve it. In order to gain insight into the problem, the computational studies were performed with randomly generated instances of different settings. Results clearly show that VNS performs well in solving IC-PHNDP with errors not more than 1.54%.Peer reviewe

An otoprotective role for the apoptosis inhibitor protein survivin

Hearing impairment caused by ototoxic insults, such as noise or gentamicin is a worldwide health problem. As the molecular circuitries involved are not yet resolved, current otoprotective therapies are rather empirical than rational. Here, immunohistochemistry and western blotting showed that the cytoprotective protein survivin is expressed in the human and guinea pig cochlea. In the guinea pig model, moderate noise exposure causing only a temporary hearing impairment transiently evoked survivin expression in the spiral ligament, nerve fibers and the organ of Corti. Mechanistically, survivin upregulation may involve nitric oxide (NO)-induced Akt signaling, as enhanced expression of the endothelial NO synthase and phosphorylated Akt were detectable in some surviving-positive cell types. In contrast, intratympanic gentamicin injection inducing cell damage and permanent hearing loss correlated with attenuated survivin levels in the cochlea. Subsequently, the protective activity of the human and the guinea pig survivin orthologs against the ototoxin gentamicin was demonstrated by ectopic overexpression and RNAi-mediated depletion studies in auditory cells in vitro. These data suggest that survivin represents an innate cytoprotective resistor against stress conditions in the auditory system. The pharmacogenetic modulation of survivin may thus provide the conceptual basis for the rational design of novel therapeutic otoprotective strategies

The effects of nail rigidity on fracture healing in rats with osteoporosis

Background and purpose Stress shielding from rigid internal fixation may lead to refracture after removal of the osteosynthesis material. We investigated the effect of a low-rigidity (Ti-24Nb-4Zr-7.9Sn) intramedullary nail regarding stress shielding and bone healing of osteoporotic fractures in the rat

CLIMP-63 is a gentamicin-binding protein that is involved in drug-induced cytotoxicity

Aminoglycoside-induced nephrotoxicity and ototoxicity is a major clinical problem. To understand how aminoglycosides, including gentamicin, induce cytotoxicity in the kidney proximal tubule and the inner ear, we identified gentamicin-binding proteins (GBPs) from mouse kidney cells by pulling down GBPs with gentamicin–agarose conjugates and mass spectrometric analysis. Among several GBPs specific to kidney proximal tubule cells, cytoskeleton-linking membrane protein of 63 kDa (CLIMP-63) was the only protein localized in the endoplasmic reticulum, and was co-localized with gentamicin-Texas Red (GTTR) conjugate after cells were treated with GTTR for 1 h. In western blots, kidney proximal tubule cells and cochlear cells, but not kidney distal tubule cells, exhibited a dithiothreitol (DTT)-resistant dimer band of CLIMP-63. Gentamicin treatment increased the presence of DTT-resistant CLIMP-63 dimers in both kidney proximal (KPT11) and distal (KDT3) tubule cells. Transfection of wild-type and mutant CLIMP-63 into 293T cells showed that the gentamicin-dependent dimerization requires CLIMP-63 palmitoylation. CLIMP-63 siRNA transfection enhanced cellular resistance to gentamicin-induced toxicity, which involves apoptosis, in KPT11 cells. Thus, the dimerization of CLIMP-63 is likely an early step in aminoglycoside-induced cytotoxicity in the kidney and cochlea. Gentamicin also enhanced the binding between CLIMP-63 and 14-3-3 proteins, and we also identified that 14-3-3 proteins are involved in gentamicin-induced cytotoxicity, likely by binding to CLIMP-63

A Phos-Tag-Based Approach Reveals the Extent of Physiological Endoplasmic Reticulum Stress

Cellular response to endoplasmic reticulum (ER) stress or unfolded protein response (UPR) is a key defense mechanism associated with many human diseases. Despite its basic and clinical importance, the extent of ER stress inflicted by physiological and pathophysiological conditions remains difficult to quantitate, posing a huge obstacle that has hindered our further understanding of physiological UPR and its future therapeutic potential. Here we have optimized a Phos-tag-based system to detect the activation status of two proximal UPR sensors at the ER membrane. This method allowed for a quantitative assessment of the level of stress in the ER. Our data revealed quantitatively the extent of tissue-specific basal ER stress as well as ER stress caused by the accumulation of misfolded proteins and the fasting-refeeding cycle. Our study may pave the foundation for future studies on physiological UPR, aid in the diagnosis of ER-associated diseases and improve and facilitate therapeutic strategies targeting UPR in vivo