A Vivens Ex Vivo Study on the Synergistic Effect of Electrolysis and Freezing on the Cell Nucleus

Freezing—cryosurgery, and electrolysis—electrochemical therapy (EChT), are two important minimally invasive surgery tissue ablation technologies. Despite major advantages they also have some disadvantages. Cryosurgery cannot induce cell death at high subzero freezing temperatures and requires multiple freeze thaw cycles, while EChT requires high concentrations of electrolytic products—which makes it a lengthy procedure. Based on the observation that freezing increases the concentration of solutes (including products of electrolysis) in the frozen region and permeabilizes the cell membrane to these products, this study examines the hypothesis that there could be a synergistic effect between freezing and electrolysis in their use together for tissue ablation. Using an animal model we refer to as vivens ex vivo, which may be of value in reducing the use of animals for experiments, combined with a Hematoxylin stain of the nucleus, we show that there are clinically relevant protocols in which the cell nucleus appears intact when electrolysis and freezing are used separately but is affected by certain combinations of electrolysis and freezing.     

Analysis of DNA Methylation of Multiple Genes in Microdissected Cells From Formalin-fixed and Paraffin-embedded Tissues

A procedure for simultaneous quantification of DNA methylation of several genes in minute amounts of sample material was developed and applied to microdissected formalin-fixed and paraffin-embedded breast tissues. The procedure is comprised of an optimized bisulfite treatment protocol suitable for samples containing only few cells, a multiplex preamplification and subsequent locus specific reamplification, and a novel quantitative bisulfite sequencing method based on the incorporation of a normalization domain into the PCR product. A real-time PCR assay amplifying repetitive elements was established to quantify low amounts of bisulfite-treated DNA. Ten prognostic and diagnostic epigenetic breast cancer biomarkers (PITX2, RASSF1A, PLAU, LHX3, PITX3, LIMK1, SLITRK1, SLIT2, HS3ST2, and TFF1) were analyzed in tissue samples obtained from two patients with invasive ductal carcinoma of the breast. The microdissected samples were obtained from several areas within the tumor tissue, including intraductal and invasive carcinoma, adenosis, and normal ductal epithelia of adjacent normal tissue, as well as stroma, tumor infiltrating lymphocytes, and adipose tissue. Overall, reliable quantification was possible for all genes. For most genes, increased DNA methylation in invasive and intraductal carcinoma cells compared with other tissue components was observed. For TFF1, decreased methylation levels were observed in tumor cells. (J Histochem Cytochem 57:477–489, 2009)     

Oxygen-stabilized enzyme electrode for d-glucose analysis in fermentation broths

A new principle for the construction of oxygen-dependent enzyme electrodes is presented. The enzyme electrode is based on a galvanic oxygen electrode which is furnished with an electrolysis anode covered by immobilized enzyme and placed close to the oxygen-sensing surface. An ordinary oxygen electrode is used as a reference electrode. The enzymatic consumption of oxygen in the enzyme electrode generates a potential difference between the electrodes which is utilized to control electrolytic generation of oxygen from water in such a way that zero differential potential is maintained. Thus, the enzyme electrode operates under ambient oxygen tension and does not suffer from oxygen limitation. The electrolytic current in this system gives a measure of the concentration of substrate surrounding the enzyme electrode. The electrode has been applied for continuous d-glucose analysis in situ during batch cultures of Candida utilis.     

GALVANIC STIMULATION OF LUMINESCENCE IN PELAGIA NOCTILUCA

1. Pelagia noctiluca responds to galvanic stimulation by a luminescent glow at the anode. If placed near the cathode a secondary glow occurs also on the cathodal side. 2. The luminescent slime of Pelagia when subjected to the galvanic current glows around the cathode. This is referred partly to the movement of hydrogen bubbles, but in the main to the alkali formed at the cathode. 3. The cause of galvanic stimulation in Pelagia is ionic. (1) Anodal stimulation is referred to the blocking of positive ions by the tissue on that side. (2) Cathodal stimulation, when the animal lies near the cathode, is due to the diffusion of alkali outward from a region of high concentration (the cathode). 4. Only the margin of the bell is excited to luminescence by the galvanic current. It is therefore concluded that nervous elements are the seat of excitation. 5. Luminescence is not a result of muscular contraction, since K ion causes relaxation of musculature but a continuous luminescent glow in Pelagia. The galvanic current causes pulsations of the bell (contraction and relaxation of the musculature) but a continuous glow.     

Psychosocial correlates of pregnant women's attitudes toward prenatal maternal serum screening and invasive diagnostic testing: beyond traditional risk status

This study examined whether psychosocial variables predict pregnant women's attitudes toward maternal serum screening and invasive diagnostic testing, beyond the influence of traditional obstetric risk status (based on advanced maternal age, history of genetic disorders, etc.). In a sample of 612 pregnant women (66.5% high risk, 33.5% low risk) we assessed responses to hypothetical scenarios of invasive testing following normal or abnormal maternal serum screening. We also assessed psychosocial variables stemming from the theory of planned behavior (e.g., knowledge, concern for fetus, attitudes toward termination, health locus of control). Overall, two thirds of the women would want serum screening. Follow-up invasive diagnostic testing would be sought by 37.2% of the women after a negative screening, and by 75.0% after a positive screening. As expected, traditional risk status predicted desire for screening and also invasive testing following either a negative or positive screen. Yet, controlling for risk status, many psychosocial variables predicted a women's interest in screening and in invasive testing: more knowledge about prenatal testing, concern about fetal health, willingness to terminate a pregnancy, and an internal or medical profession health locus of control. We conclude that psychosocial variables influence women's desire for screening or invasive testing beyond traditional risk status.     

Screening of Potential Biomarkers for Gastric Cancer with Diagnostic Value Using Label-free Global Proteome Analysis

Gastric cancer (GC) is known as a top malignant type of tumors worldwide. Despite the recent decrease in mortality rates, the prognosis remains poor. Therefore, it is necessary to find novel biomarkers with early diagnostic value for GC. In this study, we present a large-scale proteomic analysis of 30 GC tissues and 30 matched healthy tissues using label-free global proteome profiling. Our results identified 537 differentially expressed proteins, including 280 upregulated and 257 downregulated proteins. The ingenuity pathway analysis (IPA) results indicated that the sirtuin signaling pathway was the most activated pathway in GC tissues whereas oxidative phosphorylation was the most inhibited. Moreover, the most activated molecular function was cellular movement, including tissue invasion by tumor cell lines. Based on IPA results, 15 hub proteins were screened. Using the receiver operating characteristic curve, most of hub proteins showed a high diagnostic power in distinguishing between tumors and healthy controls. A four-protein (ATP5B-ATP5O-NDUFB4-NDUFB8) diagnostic signature was built using a random forest model. The area under the curve (AUC) values of this model were 0.996 and 0.886 for the training and testing sets, respectively, suggesting that the four-protein signature has a high diagnostic power. This signature was further tested with independent datasets using plasma enzyme-linked immune sorbent assays, resulting in an AUC value of 0.778 for distinguishing GC tissues from healthy controls, and using immunohistochemical tissue microarray analysis, resulting in an AUC value of 0.805. In conclusion, this study identifies potential biomarkers and improves our understanding of the pathogenesis, providing novel therapeutic targets for GC.     

Methylation status and protein expression of RASSF1A in breast cancer patients

Recently genetics and epigenetics alterations have been found to be characteristic of malignancy and hence can be used as targets for detection of neoplasia. RAS association domain family protein 1A (RASSF1A) gene hypermethylation has been a subject of interest in recent researches on cancer breast patients. The aim of the present study was to evaluate whether RASSF1A methylation status and RASSF1A protein expression are associated with the major clinico-pathological parameters. One hundred and twenty breast cancer Egyptian patients and 100-control subjects diagnosed with benign lesions of the breast were enrolled in this study. We evaluated RASSF1A methylation status in tissue and serum samples using Methyl specific PCR together with RASSF1A protein expression in tissues by immunohistochemistry. Results were studied in relation to known prognostic clinicopathological features in breast cancer. Frequency of RASSF1A methylation in tissues and serum were 70 and 63.3 % respectively and RASSF1A protein expression showed frequency of 46.7 %. There was an association between RASSF1A methylation in tissues, serum and loss of protein expression in tissues with invasive carcinoma, advanced stage breast cancer, L.N. metastasis, ER/PR and HER2 negativity. RASSF1A methylation in serum showed high degree of concordance with methylation in tissues (Kappa = 0.851, P < 0.001). RASSF1A hypermethylation in tissues and serum and its protein expression may be a valid, reliable and sensitive tool for detection and follow up of breast cancer patients.     

The Microbiological and Clinical Characteristics of Invasive Salmonella in Gallbladders from Cholecystectomy Patients in Kathmandu,Nepal

Gallbladder carriage of invasive Salmonella is considered fundamental in sustaining typhoid fever transmission. Bile and tissue was obtained from 1,377 individuals undergoing cholecystectomy in Kathmandu to investigate the prevalence, characteristics and relevance of invasive Salmonella in the gallbladder in an endemic area. Twenty percent of bile samples contained a Gram-negative organism, with Salmonella Typhi and Salmonella Paratyphi A isolated from 24 and 22 individuals, respectively. Gallbladders that contained Salmonella were more likely to show evidence of acute inflammation with extensive neutrophil infiltrate than those without Salmonella, corresponding with higher neutrophil and lower lymphocyte counts in the blood of Salmonella positive individuals. Antimicrobial resistance in the invasive Salmonella isolates was limited, indicating that gallbladder colonization is unlikely to be driven by antimicrobial resistance. The overall role of invasive Salmonella carriage in the gallbladder is not understood; here we show that 3.5% of individuals undergoing cholecystectomy in this setting have a high concentration of antimicrobial sensitive, invasive Salmonella in their bile. We predict that such individuals will become increasingly important if current transmission mechanisms are disturbed; prospectively identifying these individuals is, therefore, paramount for rapid local and regional elimination.     

Performance of a rapid immuno-chromatographic test (Schistosoma ICT IgG-IgM) for detecting Schistosoma-specific antibodies in sera of endemic and non-endemic populations

BackgroundSchistosomiasis, an acute and chronic parasitic disease caused by human pathogenic Schistosoma species, is a neglected tropical disease affecting more than 220 million people worldwide.For diagnosis of schistosomiasis, stool and urine microscopy for egg detection is still the recommended method, however sensitivity of these methods is limited. Therefore, other methods like molecular detection of DNA in stool, detection of circulating cathodic antigen in urine or circulating anodic antigen in urine and serum, as well as serological tests have gained more attention. This study examines the sensitivity and specificity of a rapid diagnostic test based on immunochromatography (Schistosoma ICT IgG-IgM, LD Bio, Lyon, France) for simultaneous detection of specific IgG and IgM antibodies in serum, against Schistosoma spp. in endemic and non-endemic populations.Methodology/Principal findingsFrozen banked serum samples from patients with confirmed schistosomiasis, patients with other helminth infections, patients with seropositive rheumatoid arthritis and healthy blood donors were used to assess the sensitivity and the specificity of the Schistosoma ICT IgG-IgM rapid diagnostic test.The test showed a sensitivity of 100% in patients with parasitologically confirmed schistosomiasis, irrespective of the species (S. mansoni, S. haematobium, S. japonicum, S. mekongi). In healthy blood donors and patients with rheumatoid factor positive rheumatoid arthritis from Europe, specificity was 100%. However, in serum samples of patients with other tissue invasive helminth infections, the test showed some cross-reactivity, resulting in a specificity of 85%.Conclusion/SignificanceWith its high sensitivity, the Schistosoma ICT IgG-IgM rapid diagnostic test is a suitable screening test for detection of Schistosoma specific antibodies, including S. mekongi. However, in populations with a high prevalence of co-infection with other tissue invasive helminths, positive results should be confirmed with other diagnostic assays due to the test’s imperfect specificity.     

A lymphoma-like neoplasm arising from hematopoietic tissue in the white shrimp, Penaeus vannamei Boone (Crustacea: Decapoda)

A pond-reared adult female Pacific white shrimp (Penaeus vannamei Boone, Crustacea: Decapoda) from an experimental shrimp culture facility in Hawaii was found to possess a lymphatic neoplasm. Present in this animal were bilateral hypertrophied hematopoietic nodules present ventral and lateral to the ventral nerve cord, and smaller multiple ectopic foci of similar appearing lymphoid cells in the gills, the subcutis, and other tissues. The lesions contained numerous anaplastic and hypertrophied lymphoid cells, many of which displayed bizarre mitotic figures, including polypolar metaphase figures. Numerous multinuclear giant cells present in the lesion were presumed to have originated from cells with polypolar mitotic figures. The characteristics of the cells composing these lesions, the expansive and invasive nature of the lesions, and the presence of ectopic foci of neoplastic cells support classification of this lesion as a hematopoietic sarcoma. Focal lesions of the type that are diagnostic of infections by the penaeid shrimp virus IHHN were present in the neoplastic hematopoietic tissue and other tissues of this shrimp, suggesting the possible role of viral infection in the development of neoplastic lesions in this animal.     

Spatially and temporally regulated alpha6 integrin cleavage during Xenopus laevis development

The α6 integrin is essential for early nervous system development in Xenopus laevis. We have previously reported a uPA cleaved form of integrin α6 (α6p), in invasive human prostate cancer tissue, whose presence correlates with increased migration and invasive capacity. We now report that α6 is cleaved during the normal development of Xenopus in a spatially and temporally controlled manner. In addition, unlike normal mammalian tissues, which lack α6p, the major form of the α6 integrin present in adult Xenopus is α6p. The protease responsible for the cleavage in mammals, uPA, is not involved in the cleavage of Xenopus α6. Finally, overexpression of a mammalian α6 mutant which cannot be cleaved leads to developmental abnormalities suggesting a potential role for the cleavage in development.     

Mass spectrometric analysis of metabolite excretion in five Japanese patients with the late-onset form of glutaric aciduria type II.

The variability of clinical and biochemical features in five Japanese patients with the late-onset form of glutaric aciduria type II (GAII) was studied using mass spectrometric procedures. The age at onset ranged from 5 months to five years, presenting acute episodes such as lethargy, hypotonia, hyperammonaemia, hypoglycaemia or Reye's syndrome-like illness, while one of the five cases was asymptomatic at 1 year of age. Organic acid analysis as oxime-trimethylsilyl derivatives by gas chromatography/mass spectrometry revealed the presence of several abnormalities characteristic of GAII in clinically asymptomatic conditions of three patients but not of the two others. Quantitative acylglycine analysis using a stable isotope dilution method and qualitative acylcarnitine analysis by fast atom bombardment mass spectrometry provided diagnostic information in all five patients, regardless of their clinical conditions. However, significant differences in the respective metabolite profiles as well as in their clinical pictures were noted. Although an increased excretion of both isovalerylglycine and isovalerylcarnitine was found in four patients, the fifth showed normal isovalerylglycine excretion during both the acute stage and in remission, despite the increased amount of isovalerylcarnitine in urine. From these results, it was suggested that the variations in clinical severity and metabolite excretion among GAII patients may be attributed not only to the residual enzyme activity at the defective site but also to differences in the capability to conjugate accumulated acyl-coenzyme A.     

Proline-Rich Tyrosine Kinase 2 (Pyk2) Regulates IGF-I-Induced Cell Motility and Invasion of Urothelial Carcinoma Cells

The insulin-like growth factor receptor I (IGF-IR) plays an essential role in transformation by promoting cell growth and protecting cancer cells from apoptosis. We have recently demonstrated that the IGF-IR is overexpressed in invasive bladder cancer tissues and promotes motility and invasion of urothelial carcinoma cells. These effects require IGF-I-induced Akt- and MAPK-dependent activation of paxillin. The latter co-localizes with focal adhesion kinases (FAK) at dynamic focal adhesions and is critical for promoting motility of urothelial cancer cells. FAK and its homolog Proline-rich tyrosine kinase 2 (Pyk2) modulate paxillin activation; however, their role in regulating IGF-IR-dependent signaling and motility in bladder cancer has not been established. In this study we demonstrate that FAK was not required for IGF-IR-dependent signaling and motility of invasive urothelial carcinoma cells. On the contrary, Pyk2, which was strongly activated by IGF-I, was critical for IGF-IR-dependent motility and invasion and regulated IGF-I-dependent activation of the Akt and MAPK pathways. Using immunofluorescence and AQUA analysis we further discovered that Pyk2 was overexpressed in bladder cancer tissues as compared to normal tissue controls. Significantly, in urothelial carcinoma tissues there was increased Pyk2 localization in the nuclei as compared to normal tissue controls. These results provide the first evidence of a specific Pyk2 activity in regulating IGF-IR-dependent motility and invasion of bladder cancer cells suggesting that Pyk2 and the IGF-IR may play a critical role in the invasive phenotype in urothelial neoplasia. In addition, Pyk2 and the IGF-IR may serve as novel biomarkers with diagnostic and prognostic significance in bladder cancer.     

Adipose-derived stem cells: Implications in tissue regeneration

Adipose-derived stem cells (ASCs) are mesenchymal stem cells (MSCs) that are obtained from abundant adipose tissue, adherent on plastic culture flasks, can be expanded in vitro, and have the capacity to differentiate into multiple cell lineages. Unlike bone marrow-derived MSCs, ASCs can be obtained from abundant adipose tissue by a minimally invasive procedure, which results in a high number of cells. Therefore, ASCs are promising for regenerating tissues and organs damaged by injury and diseases. This article reviews the implications of ASCs in tissue regeneration.     

DNA methylome profiling identifies novel methylated genes in African American patients with colorectal neoplasia

The identification of genes that are differentially methylated in colorectal cancer (CRC) has potential value for both diagnostic and therapeutic interventions specifically in high-risk populations such as African Americans (AAs). However, DNA methylation patterns in CRC, especially in AAs, have not been systematically explored and remain poorly understood. Here, we performed DNA methylome profiling to identify the methylation status of CpG islands within candidate genes involved in critical pathways important in the initiation and development of CRC. We used reduced representation bisulfite sequencing (RRBS) in colorectal cancer and adenoma tissues that were compared with DNA methylome from a healthy AA subject’s colon tissue and peripheral blood DNA. The identified methylation markers were validated in fresh frozen CRC tissues and corresponding normal tissues from AA patients diagnosed with CRC at Howard University Hospital. We identified and validated the methylation status of 355 CpG sites located within 16 gene promoter regions associated with CpG islands. Fifty CpG sites located within CpG islands—in genes ATXN7L1 (2), BMP3 (7), EID3 (15), GAS7 (1), GPR75 (24), and TNFAIP2 (1)—were significantly hypermethylated in tumor vs. normal tissues (P < 0.05). The methylation status of BMP3, EID3, GAS7, and GPR75 was confirmed in an independent, validation cohort. Ingenuity pathway analysis mapped three of these markers (GAS7, BMP3 and GPR) in the insulin and TGF-β1 network—the two key pathways in CRC. In addition to hypermethylated genes, our analysis also revealed that LINE-1 repeat elements were progressively hypomethylated in the normal-adenoma-cancer sequence. We conclude that DNA methylome profiling based on RRBS is an effective method for screening aberrantly methylated genes in CRC. While previous studies focused on the limited identification of hypermethylated genes, ours is the first study to systematically and comprehensively identify novel hypermethylated genes, as well as hypomethylated LINE-1 sequences, which may serve as potential biomarkers for CRC in African Americans. Our discovered biomarkers were intimately linked to the insulin/TGF-B1 pathway, further strengthening the association of diabetic disorders with colon oncogenic transformation.     

Electrical Impedance Tomography of Electrolysis

The primary goal of this study is to explore the hypothesis that changes in pH during electrolysis can be detected with Electrical Impedance Tomography (EIT). The study has relevance to real time control of minimally invasive surgery with electrolytic ablation. To investigate the hypothesis, we compare EIT reconstructed images to optical images acquired using pH-sensitive dyes embedded in a physiological saline agar gel phantom treated with electrolysis. We further demonstrate the biological relevance of our work using a bacterial E.Coli model, grown on the phantom. The results demonstrate the ability of EIT to image pH changes in a physiological saline phantom and show that these changes correlate with cell death in the E.coli model. The results are promising, and invite further experimental explorations.