Molecular restoration of archived transcriptional profiles by complementary-template reverse-transcription (CT-RT)

Gene expression profiling of formalin-fixed and paraffin-embedded (FFPE) specimens, banked from completed clinical trials and routine clinical care, has the potential to yield valuable information implicating and linking genes with clinical parameters. In order to prepare high-quality cDNA from highly fragmented FFPE-RNA, previously precluded from high-throughput analyses, we have designed a novel strategy based on the nucleic acid restoration of incomplete cDNA sequences prior to T7 in vitro transcription (IVT) amplification. We describe this strategy as complementary-template reverse-transcription (CT-RT) because short single-stranded T7-oligo-dT24-VN-DNA sequences, obtained from FFPE-RNA, are used as primers for the RT of complementary RNA templates contained in a sense-RNA library. We validated our assay by determining the correlation between expression profiles of a matched 10-year-old frozen and FFPE breast cancer sample. We show that T7 IVT-amplification of cDNA transcripts restored by CT-RT is a specific and reliable process that allows recovery of transcriptional features undetectable by direct T7 IVT-amplification of FFPE-RNA. Furthermore, CT-RT restored 35–41% of the transcripts from archived breast and cervical specimens when compared to matched frozen tissue; and profiles included tissue-specific transcripts. Our results indicate that CT-RT allows microarray profiling of severely degraded RNA that could not be analyzed by previous methods

Plectin as a prognostic marker in non-metastatic oral squamous cell carcinoma

Background: Oral squamous cell carcinoma (OSCC) is associated with a poor 5-year survival rate. In general, patients diagnosed with small tumors have a fairly good prognosis, but some small tumors have an aggressive behavior leading to early death. There are at present no reliable prognostic biomarkers for oral cancers. Thus, to optimize treatment for the individual patient, there is a need for biomarkers that can predict tumor behavior. Method: In the present study the potential prognostic value of plectin was evaluated by a tissue microarray (TMA) based immunohistochemical analysis of primary tumor tissue obtained from a North Norwegian cohort of 115 patients diagnosed with OSCC. The expression of plectin was compared with clinicopathological variables and 5 year survival. Results: The statistical analysis revealed that low expression of plectin in the tumor cells predicted a favorable outcome for patients with non-metastatic disease (p = 0.008). Furthermore, the expression of plectin was found to correlate (p = 0.01) with the expression of uPAR, which we have previously found to be a potential prognostic marker for T1N0 tumors. Conclusions: Our results indicate that low expression of plectin predicts a favorable outcome for patients with non-metastatic OSCC and the expression level of plectin may therefore be used in the treatment stratification for patients with early stage disease

Prevention of benzene-induced myelotoxicity by nonsteroidal anti-inflammatory drugs.

Benzene affects hematopoietic progenitor cells leading to bone marrow depression and genotoxic effects such as micronucleus formation. Progenitor cell proliferation and differentiation are inhibited by prostaglandins produced by macrophages. Administration of benzene to DBA/2 or C57BL/6 mice caused a dose-dependent bone marrow depression and a significant increase in marrow prostaglandin E level and both were prevented by the coadministration of indomethacin and other inhibitors of the cyclooxygenase component of prostaglandin H synthase. Levels of benzene that decreased bone marrow cellularity also caused genotoxic effects measured as increased micronucleated polychromatic erythrocytes in peripheral blood, which was also prevented by the coadministration of indomethacin. These results suggest a possible role for prostaglandin synthase in benzene myelotoxicity; a mechanism by which this might occur is presented

Evaluation of chemiluminescence, toluidine blue and histopathology for detection of high risk oral precancerous lesions: A cross-sectional study

<p>Abstract</p> <p>Background</p> <p>Early detection holds the key to an effective control of cancers in general and of oral cancers in particular. However, screening procedures for oral cancer are not straightforward due to procedural requirements as well as feasibility issues, especially in resource-limited countries.</p> <p>Methods</p> <p>We conducted a cross-sectional study to compare the performance of chemiluminescence, toluidine blue and histopathology for detection of high-risk precancerous oral lesions. We evaluated 99 lesions from 55 patients who underwent chemiluminescence and toluidine blue tests along with biopsy and histopathological examination. We studied inter-as well as intra-rater agreement in the histopathological evaluation and then using latent class modeling, we estimated the operating characteristics of these tests in the absence of a reference standard test.</p> <p>Results</p> <p>There was a weak inter-rater agreement (kappa < 0.15) as well as a weak intra-rater reproducibility (Pearson's r = 0.28, intra-class correlation rho = 0.03) in the histopathological evaluation of potentially high-risk precancerous lesions. When compared to histopathology, chemiluminescence and toluidine blue retention had a sensitivity of 1.00 and 0.59, respectively and a specificity of 0.01 and 0.79, respectively. However, latent class analysis indicated a low sensitivity (0.37) and high specificity (0.90) of histopathological evaluation. Toluidine blue had a near perfect high sensitivity and specificity for detection of high-risk lesions.</p> <p>Conclusion</p> <p>In our study, there was variability in the histopathological evaluation of oral precancerous lesions. Our results indicate that toluidine blue retention test may be better suited than chemiluminescence to detect high-risk oral precancerous lesions in a high-prevalence and low-resource setting like India.</p

Spatiotemporal processing of somatosensory stimuli in schizotypy

Unusual interaction behaviors and perceptual aberrations, like those occurring in schizotypy and schizophrenia, may in part originate from impaired remapping of environmental stimuli in the body space. Such remapping is contributed by the integration of tactile and proprioceptive information about current body posture with other exteroceptive spatial information. Surprisingly, no study has investigated whether alterations in such remapping occur in psychosis-prone individuals. Four hundred eleven students were screened with respect to schizotypal traits using the Schizotypal Personality Questionnaire. A subgroup of them, classified as low, moderate, and high schizotypes were to perform a temporal order judgment task of tactile stimuli delivered on their hands, with both uncrossed and crossed arms. Results revealed marked differences in touch remapping in the high schizotypes as compared to low and moderate schizotypes. For the first time here we reveal that the remapping of environmental stimuli in the body space, an essential function to demarcate the boundaries between self and external world, is altered in schizotypy. Results are discussed in relation to recent models of 'self-disorders' as due to perceptual incoherence

Biodegradable Thermosensitive Hydrogel for SAHA and DDP Delivery: Therapeutic Effects on Oral Squamous Cell Carcinoma Xenografts

Background: OSCC is one of the most common malignancies and numerous clinical agents currently applied in combinative chemotherapy. Here we reported a novel therapeutic strategy, SAHA and DDP-loaded PECE (SAHA-DDP/PECE), can improve the therapeutic effects of intratumorally chemotherapy on OSCC cell xenografts. Objective/Purpose: The objective of this study was to evaluate the therapeutic efficacy of the SAHA-DDP/PECE in situ controlled drug delivery system on OSCC cell xenografts. Methods: A biodegradable and thermosensitive hydrogel was successfully developed to load SAHA and DDP. Tumorbeared mice were intratumorally administered with SAHA-DDP/PECE at 50 mg/kg (SAHA) +2 mg/kg (DDP) in 100 ul PECE hydrogel every two weeks, SAHA-DDP at 50 mg/kg(SAHA) +2 mg/kg(DDP) in NS, 2 mg/kg DDP solution, 50 mg/kg SAHA solution, equal volume of PECE hydrogel, or equal volume of NS on the same schedule, respectively. The antineoplastic actions of SAHA and DDP alone and in combination were evaluated using the determination of tumor volume, immunohistochemistry, western blot, and TUNEL analysis. Results: The hydrogel system was a free-flowing sol at 10uC, become gel at body temperature, and could sustain more than 14 days in situ. SAHA-DDP/PECE was subsequently injected into tumor OSCC tumor-beared mice. The results demonstrated that such a strategy as this allows the carrier system to show a sustained release of SAHA and DDP in vivo, and coul

Comparative Proteomic Analysis of Serum from Patients with Systemic Sclerosis and Sclerodermatous GVHD. Evidence of Defective Function of Factor H

BACKGROUND: Systemic sclerosis (SSc) is an autoimmune disease characterized by immunological and vascular abnormalities. Until now, the cause of SSc remains unclear. Sclerodermatous graft-versus-host disease (ScGVHD) is one of the most severe complications following bone marrow transplantation (BMT) for haematological disorders. Since the first cases, the similarity of ScGVHD to SSc has been reported. However, both diseases could have different etiopathogeneses. The objective of this study was to identify new serum biomarkers involved in SSc and ScGVHD. METHODOLOGY: Serum was obtained from patients with SSc and ScGVHD, patients without ScGVHD who received BMT for haematological disorders and healthy controls. Bi-dimensional electrophoresis (2D) was carried out to generate maps of serum proteins from patients and controls. The 2D maps underwent image analysis and differently expressed proteins were identified. Immuno-blot analysis and ELISA assay were used to validate the proteomic data. Hemolytic assay with sheep erythrocytes was performed to evaluate the capacity of Factor H (FH) to control complement activation on the cellular surface. FH binding to endothelial cells (ECs) was also analysed in order to assess possible dysfunctions of this protein. PRINCIPAL FINDINGS: Fourteen differentially expressed proteins were identified. We detected pneumococcal antibody cross-reacting with double stranded DNA in serum of all bone marrow transplanted patients with ScGVHD. We documented higher levels of FH in serum of SSc and ScGVHD patients compared healthy controls and increased sheep erythrocytes lysis after incubation with serum of diffuse SSc patients. In addition, we observed that FH binding to ECs was reduced when we used serum from these patients. CONCLUSIONS: The comparative proteomic analysis of serum from SSc and ScGVHD patients highlighted proteins involved in either promoting or maintaining an inflammatory state. We also found a defective function of Factor H, possibly associated with ECs damage