Optimization of process conditions for effective ohmic heating of greater lizardfish (saurida tumbil) sausage via response surface methodology.

In current research, the optimization of ohmic heating on greater lizardfish (Saurida tumbil) sausage variables was carried out using response surface methodology-central composite design (RSM-CCD). The effect of process variables including voltage gradient (15-60 V/cm), time (1-15 min), and temperature (60-90°C) on the microbial properties, pH, peroxide value, water holding capacity (WHC), and cooking loss of the sausages was evaluated. The results showed that the characteristics of the sausages were dependent on the ohmic heating conditions and these properties can be modulated. As per the results, the voltage gradient and temperature has a significant effect on the total plate count (p < 0.05). The increase in voltage gradient was the most effective on pH (5.63-6.91). The interaction terms of all items had a significant effect (p < 0.05) on the peroxide value of the fish sausages. Higher amount of temperature and process time were resulted in the more cooking loss. Increasing the voltage gradient was more effective on WHC compared to the temperature. Finally, the process was optimized and the optimized condition was achieved by setting the voltage gradient at 30 V/cm, process time at 4 min, and temperature at 66 °C. Also, it was compared with conventional heating. The results were shown that the differences between the mean values of all responses were statistically significant (p < 0.05) except for pH. Therefore, ohmic method was carried out faster, with lower temperature and obtaining the highest WHC and lowest total plate count, peroxide value, cooking loss, and optimized pH. Generally, this study suggested that the ohmic heating can be used as a rapid and homogeneous cooking method for the preparation of sausages from greater lizardfish as a commercial low-valued fish.

Note: A portable automatic capillary viscometer for transparent and opaque liquids.

A portable automatic capillary viscometer, equipped with an AVR microcontroller, was designed and developed. The viscometer was calibrated with Certified Reference Material (CRM) s200 and utilized for measurement of kinematic viscosity. A quadratic equation was developed for calibration of the instrument at various temperatures. Also, a model was developed for viscosity determination in terms of the viscometer dimensions. Development of the portable viscometer provides for on-site monitoring of engine oil viscosity.

Correlating breakage-fusion-bridge events with the overall chromosomal instability and in vitro karyotype evolution in prostate cancer.

Chromosomal instability (CIN) is thought to underlie the generation of chromosomal changes and genomic heterogeneity during prostatic tumorigenesis. The breakage-fusion-bridge (BFB) cycle is one of the CIN mechanisms responsible for characteristic mitotic abnormalities and the occurrence of specific classes of genomic rearrangements. However, there is little detailed information concerning the role of BFB and CIN in generating genomic diversity in prostate cancer. In this study we have used molecular cytogenetic methods and array comparative genomic hybridization analysis (aCGH) of DU145, PC3, LNCaP, 1532T and 1542T to investigate the in vitro role of BFB as a CIN mechanism in karyotype evolution. Analysis of mitotic structures in all five prostate cancer cell lines showed increased frequency of anaphase bridges and nuclear strings. Structurally rearranged dicentric chromosomes were observed in all of the investigated cell lines, and Spectral Karyotyping (SKY) analysis was used to identify the participating rearranged chromosomes. Multicolor banding (mBAND) and aCGH analysis of some of the more complex chromosomal rearrangements and associated amplicons identified inverted duplications, most frequently involving chromosome 8. Chromosomal breakpoint analysis showed there was a higher frequency of rearrangement at centromeric and pericentromeric genomic regions. The distribution of inverted duplications and ladder-like amplifications was mapped by mBAND and by aCGH. Adjacent spacing of focal amplifications and microdeletions were observed, and focal amplification of centromeric and end sequences was present, particularly in the most unstable line DU145. SKY analysis of this line identified chromosome segments fusing with multiple recipient chromosomes (jumping translocations) identifying potential dicentric sources. Telomere free end analysis indicated loss of DNA sequence. Moreover, the cell lines with the shortest telomeres had the most complex karyotypes, suggesting that despite the expression of telomerase, the reduced telomere length could be driving the observed BFB events and elevated levels of CIN in these lines.

High-resolution cDNA microarray CGH mapping of genomic imbalances in osteosarcoma using formalin-fixed paraffin-embedded tissue.

Formalin-fixed paraffin embedded (FFPE) tumor tissue provides an opportunity to perform retrospective genomic studies of tumors in which chromosomal imbalances are strongly associated with oncogenesis. The application of comparative genomic hybridization (CGH) has led to the rapid accumulation of cytogenetic information on osteosarcoma (OS); however, the limited resolving power of metaphase CGH does not permit precise mapping of imbalances. Array CGH allows quantitative detection and more precise delineation of copy number aberrations in tumors. Unfortunately the high cost and lower density of BACs on available commercial arrays has limited the ability to comprehensively profile copy number changes in tumors such as OS that are recurrently subject to genomic imbalance. In this study a cDNA/EST microarray including 18,980 human cDNAs (which represent all 22 pairs of autosomal chromosomes and chromosome X) was used for CGH analysis of eight OS FFPE. Chromosomes 1, 12, 17, and X harbored the most imbalances. Gain/amplification of X was observed in 4/8 OS, and in keeping with other recent genomic analyses of OS, gain/amplification of 17p11.2 was often accompanied by a distal deletion in the region of the p53 gene. Gain/amplification of the X chromosome was verified using interphase FISH carried out on a subset of OS FFPE sections and OS tissue arrays.

Use of whole genome amplification and comparative genomic hybridisation to detect chromosomal copy number alterations in cell line material and tumour tissue.

We have established that whole genome amplification (WGA), in conjunction with genomic DNA array comparative genomic hybridisation (gaCGH) allows for the identification of genome-wide copy number abnormalities (CNAs) in DNA extracted from both cell line and patient material. To determine the fidelity and reproducibility of WGA to detect copy number imbalances using gaCGH, well characterized cell line genomic DNA was analysed. The gaCGH data obtained from non-amplified DNA and amplified DNA for the neuroblastoma cell line NUB7 and a paediatric medulloblastoma patient was almost identical. In addition, laser capture microdissection (LCM) of prostate tumour cells and subsequent WGA allowed for the detection of a number of CNAs that may not have been identified if DNA had been extracted in bulk from heterogeneous tissue. The results presented here demonstrate the use of WGA for generating sufficient DNA for gaCGH analysis without the introduction of significant sequence representation bias. The combination of amplification and gaCGH using DNA extracted from archival patient material has the potential for permitting the studying of DNA from small cancerous or pre-cancerous foci, which may help to identify potential genomic markers for early diagnosis.

Allelic deletion fingerprinting of urine cell sediments in bladder cancer.

BACKGROUND: Bladder cancer shows frequent nonrandom allelic deletion at various chromosomal regions. Genotypic detection methods could potentially identify patients at risk for recurrent progressive disease. In this study, we examined allelic deletion at specific chromosomal loci in tumor tissue and urine cell sediment samples using a microsatellite-based protocol. Although both allelic deletion and microsatellite instability have been reported in primary bladder cancer, microsatellite instability was not specifically examined in this study. We report a pilot study of 40 patients with bladder cancer in which allelic deletion in tumor tissue and urine cell sediment was compared with conventional urine cytology results. METHODS AND RESULTS: Forty tumors were analyzed using a set of microsatellite primers from chromosomes 3, 4, 8, 11, 14, and 17 to construct allelic deletion fingerprints. Cy5.5-labeled PCR products were analyzed using the OpenGene System and GeneObjects software. Eighty-eight percent of tumors showed allelic deletion. In urine cell sediments, the tumor detection rate was 80% compared with 50% for routine urine cytology. The allelic deletion fingerprinting (ADF) procedure identified 69% of incipient tumors, cases initially classified as normal by routine urine cytology. CONCLUSION: ADF analysis provides a reliable noninvasive method for the detection and monitoring of recurrent cancer in urine cell sediment samples from patients with bladder cancer.

Evidence of chromosomal instability in prostate cancer determined by spectral karyotyping (SKY) and interphase fish analysis.

The way in which cytogenetic aberrations develop in prostate cancer (CaP) is poorly understood. Spectral karyotype (SKY) analysis of CaP cell lines has shown that they have unstable karyotypes and also have features associated with chromosomal instability (CIN). To accurately determine the incidence of de novo structural and numerical aberrations in vitro in CaP, we performed SKY analysis of three independent clones derived from one representative cell line, DU145. The frequent generation of new chromosomal rearrangements and a wide variation in the number of structural aberrations within two to five passages suggested that this cell line exhibited some of the features associated with a CIN phenotype. To study numerical cell-to-cell variation, chromosome 8 aneusomy was assessed in the LNCaP, DU145, and PC-3 cell lines and a patient cohort of 15 CaP primary tumors by interphase fluorescence in situ hybridization (FISH). This analysis showed that a high frequency of numerical alteration affecting chromosome 8 was present in both in vitro and in CaP tissues. In comparison to normal controls, the patient cohort had a statistically significant (P<.05), greater frequency of cells with one and three centromere 8 copies. These data suggest that a CIN-like process may be contributing towards the generation of de novo numerical and structural chromosome abnormalities in CaP.

Genetic characterization of immortalized human prostate epithelial cell cultures. Evidence for structural rearrangements of chromosome 8 and i(8q) chromosome formation in primary tumor-derived cells.

We have utilized a combination of conventional and spectral karyotyping (SKY) techniques and allelotype analysis to assess numerical and structural chromosome alterations in two cell lines derived from normal human prostatic epithelium, and three cell lines derived from human prostate primary tumor epithelium, immortalized with the E6 and E7 transforming genes of human papilloma virus (HPV) 16 or the large T-antigen gene of simian virus 40 (SV40). These studies revealed trisomy for chromosome 20 and rearrangements involving chromosomes 3, 4, 8, 9, 10, 16, 17, 18, 19, 21, or 22. In addition, the four HPV-immortalized cell lines exhibited extensive duplications or translocations involving the 11q chromosomal region. Interestingly, allelotyping data disclosed loss of 8p sequences in two of the three primary tumor-derived cell lines, and SKY data revealed that the loss of 8p sequences was directly due to i(8q) chromosome formation and/or other structural alterations of chromosome 8. This provides intriguing evidence that 8p loss in primary human prostate tumors may, in some cases, result from complex structural rearrangements involving chromosome 8. Moreover, the data reported here provide direct evidence that such complex structural rearrangements sometimes include i(8q) chromosome formation.

Identification of a high frequency of chromosomal rearrangements in the centromeric regions of prostate cancer cell lines by sequential giemsa banding and spectral karyotyping.

BACKGROUND: Currently, prostate cancer (CaP) cytogenetics is not well defined, largely because of technical difficulties in obtaining primary tumor metaphases. METHODS AND RESULTS: We examined three CaP cell lines (LNCaP, DU145, PC-3) using sequential Giemsa banding and spectral karyotyping (SKY) to search for a common structural aberration or translocation breakpoint. No consistent rearrangement common to all three cell lines was detected. A clustering of centromeric translocation breakpoints was detected in chromosomes 4, 5, 6, 8, 11, 12, 14, and 15 in DU145 and PC-3. Both these lines were found to have karyotypes with a greater level of complexity than LNCaP. CONCLUSION: The large number of structural aberrations present in DU145 and PC-3 implicate an underlying chromosomal instability and subsequent accumulation of cytogenetic alterations that confer a selective growth advantage. The high frequency of centromeric rearrangements in these lines indicates a potential role for mitotic irregularities associated with the centromere in CaP tumorigenesis.

Detection of eubacteria in interstitial cystitis by 16S rDNA amplification.

OBJECTIVE: To determine what role non-culturable microorganisms play in the etiology of interstitial cystitis (IC). MATERIALS AND METHODS: Thirty patients fulfilling NIH criteria for the diagnosis of interstitial cystitis and sixteen control patients with culture negative urine gave written informed consent and underwent bladder biopsy. Polymerase chain reaction (PCR) using two sets of universal primers for bacterial 16S rDNA was performed on urine from the cystoscope and on a cold cup bladder biopsy specimen. Of the PCR positive bladder biopsies, three patients with interstitial cystitis and three controls were randomly selected and cloned. Ten clones from each were sequenced and putative taxonomic assignments made. RESULTS: 12/26 (46%) IC and 5/12 (42%) control urine specimens and 16/30 (53%) and 9/15 (60%) bladder biopsies were PCR positive, respectively. The bacterial populations in the two patient groups tested appeared to be different based upon analysis of the 16S rRNA sequences. CONCLUSIONS: Both IC and control patients had non-culturable bacteria in their bladders. A random sampling of the two populations revealed that the bacterial populations are different, suggesting a possible link between one or more bacterial species and IC.