A Qualitative Study on Knowledge and Attitude towards Risk Factors, Early Identification and Intervention of Infant Hearing Loss among Puerperal Mothers- A Short Survey.

INTRODUCTION: Maternal active participation and their support are critical for the success of early hearing loss detection program. Erroneous maternal decisions may have large life long consequences on the infant's life. The mothers' knowledge and their attitudes towards infant hearing loss is the basis for their decisions. AIM: The present study was done to determine the mothers' knowledge and their attitude towards risk factors of infant hearing loss, its early identification and intervention and also awareness of effect of consanguinity on hearing loss. MATERIALS AND METHODS: In this cross-sectional questionnaire survey study, a total of 100 mothers were interviewed using the questionnaire which consisted of three sections namely risk factors, early identification and early intervention of hearing loss. Chi-square test was used to establish relationship between consanguineous and non-consanguineous mother's responses to its effect on hearing loss. A p-value < 0.05 was considered as significant. RESULTS: Mothers' awareness was significantly high for visible causes (ear pain/discharge, head injury and slap to ear) of hearing loss. Positive attitude was seen for importance of screening programs and follow up testing. Moderate level of awareness was found on hazards of consanguinity and benefits of early identification. However, mothers were least aware of neonatal jaundice, NICU admission (>5 days), signs of late-onset and neural hearing loss, management of hearing loss, hearing aid fitting and therapy necessity, which might interfere in early detection and intervention of hearing loss. CONCLUSION: It is crucial to educate mothers on few risk factors and management of hearing loss to reduce its consequences.

Optimization of laccase production from Marasmiellus palmivorus LA1 by Taguchi method of Design of experiments.

BACKGROUND: Fungal laccase has profound applications in different fields of biotechnology due to its broad specificity and high redox potential. Any successful application of the enzyme requires large scale production. As laccase production is highly dependent on medium components and cultural conditions, optimization of the same is essential for efficient product production. RESULTS: Production of laccase by fungal strain Marasmiellus palmivorus LA1 under solid state fermentation was optimized by the Taguchi design of experiments (DOE) methodology. An orthogonal array (L8) was designed using Qualitek-4 software to study the interactions and relative influence of the seven selected factors by one factor at a time approach. The optimum condition formulated was temperature (28 °C), pH (5), galactose (0.8%w/v), cupric sulphate (3 mM), inoculum concentration (number of mycelial agar pieces) (6Nos.) and substrate length (0.05 m). Overall yield increase of 17.6 fold was obtained after optimization. Statistical optimization leads to the elimination of an insignificant medium component ammonium dihydrogen phosphate from the process and contributes to a 1.06 fold increase in enzyme production. A final production of 667.4 ± 13 IU/mL laccase activity paves way for the application of this strain for industrial applications. CONCLUSION: Study optimized lignin degrading laccases from Marasmiellus palmivorus LA1. This laccases can thus be used for further applications in different scales of production after analyzing the properties of the enzyme. Study also confirmed the use of taguchi method for optimizations of product production.

Assessment of nodal involvement and survival analysis in breast cancer patients using image cytometric data: statistical, neural network and fuzzy approaches.

Accurate and reliable decision making in breast cancer prognosis can help in the planning of suitable surgery and therapy and, generally, optimise patient management through the different stages of the disease. In recent years, several prognostic factors have been used as indicators of disease progression in breast cancer. In this paper we investigate a fuzzy method, namely fuzzy k-nearest neighbour technique for breast cancer prognosis, and for determining the significance of prognostic markers and subsets of the markers, which include histology type, tumour grade, DNA ploidy, S-phase fraction, G0G1/G2M ratio, and minimum (start) and maximum (end) nuclear pleomorphism indices. We also compare the method with (a) logistic regression as a statistical method, and (b) multilayer feed forward backpropagation neural networks as an artificial neural network tool, the latter two techniques having been widely used for cancer prognosis. Nodal involvement and survival analyses in breast cancer are carried out for 100 women who were clinically diagnosed with breast disease in the form of carcinoma and benign conditions, and seven prognostic markers collected for each patient. For nodal involvement analysis, node positive and negative patients are predicted whereas survival analysis is carried out for two categories: whether a patient is alive or dead within 5 years of diagnosis. The results obtained show that the fuzzy method yields the highest predictive accuracy of 88% for both nodal involvement and survival analyses obtained from the subsets of [tumour grade, S-phase fraction, minimum (start) nuclear pleomorphism index] and [tumour histology type, DNA ploidy, S-phase fraction, G0G1/G2M ratio], respectively. We believe that this technique has produced more reliable prognostic factor models than those obtained using either the statistical or artificial neural networks-based methods.

DNA ploidy and cell cycle distribution of breast cancer aspirate cells measured by image cytometry and analyzed by artificial neural networks for their prognostic significance.

Chromosomal abnormalities are commonly associated with cancer, and their importance in the pathogenesis of the disease has been well recognized. Also recognized in recent years is the possibility that, together with chromosomal abnormalities, DNA ploidy of breast cancer aspirate cells, measured by image cytometric techniques, may correlate with prognosis of the disease. Here, we have examined the use of an artificial neural network to predict: 1) subclinical metastatic disease in the regional lymph nodes and 2) histological assessment, through the analysis of data obtained by image cytometric techniques of fine needle aspirates of breast tumors. The cellular features considered were: 1) DNA ploidy measured in terms of nuclear DNA content as well as by cell cycle distribution; 2) size of the S-phase fraction; and 3) nuclear pleomorphism. A further objective of the study was to analyze individual markers in terms of impact significance on predicting outcome in both cases. DNA ploidy, indicated by cell cycle distribution, was found markedly to influence the prediction of nodal spread of breast cancer, and nuclear pleomorphism to a lesser degree. Furthermore, a comparison between histological assessment and artificial neural network prediction shows a closer correlation between the neural approach and the development of further metastases as indicated in subsequent follow-up, than does histological assessment. These data demonstrate that artificial neural networks are capable of providing powerful and reliable indicators of possible lymph node metastasis, using measurements of cellular features alone.

S100A4 (MTS1) calcium binding protein in cancer growth, invasion and metastasis.

The S100 family of calcium binding proteins has been shown to be involved in a variety of physiological function, such as cell proliferation, extracellular signal transduction, intercellular adhesion, motility as well as cancer metastasis. The role played by a member of the S100 gene family, viz. S100A4 (also referred to as mtsl, 18A2/mtsl, pEL-98, p9Ka, metastasin) in the control of cell proliferation as well as in cancer invasion and metastasis has now been extensively studied in a number of laboratories. The protein encoded by S100A4 gene is now known to be capable of regulating cell cycle progression, modulating intercellular adhesion and invasive and metastatic properties of cancer cells. The S100A4 protein appears to be able to sequester and disable the p53 suppressor protein which controls G1-S transition of cells as well as the exit of cells from the S phase into mitosis G2-M transition is believed to involve the induction of stathmin (Op18) gene expression. The expression of this gene has been found to parallel that of S100A4, S100A4 also appears to take part in the homeostasis of growth, with apparent involvement also in growth factor signal transduction and apoptotic cell death. There is considerable evidence that S100A4 expression alters the adhesive properties of cells, possibly by remodelling the extracellular matrix and promoting a redeployment of adhesion-mediating macromolecules occurring in the extracellular matrix. Using transfection technology, it has been shown that over-expression of S100A4 enhances lung colonisation by cancer cells. The transfection and expression of antisense constructs, in contrast, inhibit metastatic localisation in the lung. S100 proteins levels in serum and in tumour tissue are increasingly being monitored and have been regarded as good indicators of the state of cancer progression. Valuable evidence has accumulated regarding the expression of S100A4 in human melanomas. In carcinoma of the breast, the level of expression of S100A4 has been found to be closely related to metastatic spread of the cancer to regional lymph nodes. The purpose of this review is to emphasise the need to focus sharply upon the mechanisms by which S100 proteins in general and S100A4 in particular subserve the wide variety of functions currently attributable to them.

Analysis of image cytometry data of fine needle aspirated cells of breast cancer patients: a comparison between logistic regression and artificial neural networks.

Image flow cytometry data of aspirated tumour cells from 102 patients with breast cancer were analysed and used as prognostic markers in an attempt to predict involvement of axillary lymph nodes and histological grade using logistic regression. Prediction was 70% for both nodal status and histological analyses. The outcome of this study is compared to an earlier study using the same cytological information to obtain prediction using a neural approach. Using artificial neural networks, prediction accuracy was 87% and 82% for nodal status and histological assessment, respectively. This study also attempts to identify the impact of individual prognostic factors. The statistical approach identified S-phase fraction and DNA-ploidy as the most important prediction markers for nodal status and histological assessment analyses. A comparison was made between these two quantitative techniques.

Expression of metastasis-associated genes h-mts1 (S100A4) and nm23 in carcinoma of breast is related to disease progression.

The murine 18A2/mts1 and its human homolog h-mts1 (S100A4), encoding a Ca2+-binding protein belonging to the S-100 family, are associated with high invasive and metastatic potentials of murine tumors, human tumor cell lines in vitro, and human tumors growing as xenografts. The nm23 is a putative metastasis-suppressor gene whose expression has been found to correlate inversely with the metastatic potential of some forms of human cancer. The products of both human genes alter cytoskeletal dynamics, with antagonistic effects. In view of the equivocal association of nm23 with the metastatic potential of human cancer, we suspected that the relative expression of h-mts1 and nm23 might reflect tumor progression more accurately than either of them alone. We describe here the expression of these genes in infiltrating ductal carcinomas of the breast and show that high h-mts1 expression is associated with metastatic spread to the regional lymph nodes. The expression of nm23 on its own did not show a statistically significant inverse correlation with nodal spread. However, the expression status of the two genes, taken together, correlated strongly with the occurrence of nodal metastases. Breast cancers with no detectable expression of h-mts1 were found to be estrogen and progesterone receptor positive. Expression of h-mts1 was not related to tumor differentiation. The clinical data, together with the state of expression of steroid receptors and the expression levels of h-mts1 and nm23 genes, were analyzed using artificial neural networks for accuracy in predicting nodal spread of the carcinomas. These analyses support the conclusion that, overall, h-mts1 expression appears to be associated with and indicative of more aggressive disease. Complemented with nm23, h-mts1 could provide a powerful marker of breast cancer prognosis.

Heat shock modulates the expression of the metastasis associated gene MTS1 and proliferation of murine and human cancer cells.

Mts1 is a metastasis-associated gene of the S-100 gene family and codes for a Ca2+-binding protein. It is highly expressed in murine and human cancers of high invasive and metastatic potential. Recent work has shown that the mts1 protein might be involved in cell cycle regulation. An upregulation of its expression drives cells into the S phase, together with an enhanced expression of p53 phosphoprotein, which has led to the suggestion that mtsl protein might be sequestering p53 thereby abrogating the G1-S checkpoint control normally exerted by p53. Preliminary studies showed that expression of mts1 is downregulated by hyperthermia. We present evidence that in murine BL6 melanoma cells and human HUT cells that hyperthermia downregulates the mts1 gene. It is also downregulated in heat-resistant variants of the B16 melanoma and HUT cells. In parallel, there is a decrease in the size of the S phase fraction and an increase in the doubling time of cells. Cell subjected to hyperthermia show an 2- to 3.5-fold increase in the expression of HSP28 which has been shown to possess a proliferation inhibitory action. It is postulated that a complete regulatory loop involving mtsl, p53, and HSP28 might be involved in cell proliferation.

Expression of the transmembrane glycoprotein CD44 and metastasis associated 18A2/MTS1 gene in B16 murine melanoma cells.

CD44 is a transmembrane cell adhesion-mediating protein which occurs as alternatively spliced isoforms in a variety of cell types. CD44 isoforms have been reported to be differentially expressed in cancers and the isoform CD44v6 has often been associated with metastatic potential. The 18A2/mts1 gene, which codes for a Ca(2+)-binding protein of the S-100 family, is a metastasis associated gene and its expression has been shown to be related to cell proliferation, cancer metastasis and invasion. The association of 18A2/mts1 expression with invasion has been attributed to its ability to promote depolymerisation of cytoskeletal elements and it appears to also participate in the remodelling of the extracellular matrix. Here we have examined the expression of CD44v6 in metastatic variants of the B16 melanoma with different levels of 18A2/mts1 expression. We found that metastatic potential was not related to the overall CD44 expression as detected by immunostaining; the high metastasis variant ML8 showed reduced CD44v6 positivity as compared with the low metastasis variant F1. Up-regulation of 18A2/mts1 expression in F1 cells by a-melanocyte stimulating hormone (MSH) and its down-regulation in ML8 cells by RA reduced the numbers of CD44v6 staining cells. In F1 cells the glycoprotein was found to be uniformly associated with the cell membrane. But in F1 cells treated with a-MSH where 18A2/mts1 expression was up-regulated, CD44v6 showed redistribution into a patchy focal pattern. This patchy focal distribution of CD44v6 also occurred in ML8 cells which express 18A2/mts1 at a high level. It is suggested that the patching of CD44v6 molecules is a consequence of the changes in cytoskeletal dynamics brought about by 18A2/mts1 expression, that are conducive to aggregation and patching of these transmembrane glycoproteins. It is postulated that this induction of patching could provide discrete and strong adhesive foci promoting cell adhesion and invasive behaviour.

Induction of 18A2/mts1 gene expression and its effects on metastasis and cell cycle control.

The metastasis associated 18A2/mtsI gene was inserted into the mammalian expression vector pMAMneo placing it under the control of the dexamethasone-inducible MMTV promoter. The construct was transfected into dexamethasone receptor negative F1 and receptor positive F10 cells of the B16 murine melanoma. The transferred gene was switched on in two transfectant clones of F10, by exposure to 10(-6) M dexamethasone, but not in clones of the receptor negative F1 line. One of the F10 transfectant clones (F10-192/10) was characterized further. A 13.5-fold increase in 18A2/mts1 transcripts was found in this clone upon exposure to dexamethasone. There was also a seven-fold increase in lung colonization in an experimental metastasis assay, together with increased expression of depolymerized tubulin and enhanced detection of p53 protein. The number of cells in the S phase increased by 2.5-fold following dexamethasone treatment of the clone. These data suggest a direct involvement of the 18A2/mts1 gene in lung colonization by the tumor cells. The 18A2/mts1 protein promotes tubulin depolymerization, sequesters the p53 phosphoprotein, and induces the cells to enter the S phase, but the relevance of these in the metastatic process remains to be elucidated.

Metastasis-associated mts1 gene expression is down-regulated by heat shock in variant cell lines of the B16 murine melanoma.

Tumour cells are more heat sensitive than corresponding normal cells but the reasons for this are poorly understood. Here we report that induction of heat shock proteins was associated with a down-regulation of the metastasis associated mts1 gene in BL6-B16 murine melanoma cells, and the heat-resistant HTG variant of the BL6 line. Melanocyte stimulating hormone, which does not affect B16 cell proliferation but upregulates mts1 expression, only marginally enhanced heat shock protein expression in F1 cells as determined by immunohistochemical methods. Retinoic acid, which inhibits cell proliferation and down-regulates the mts1 gene, reduced heat shock protein expression in the ML8-B16 variant line. This suggests that the changes in the heat shock protein expression reported here may be cell proliferation related. Heat shock proteins are known to stabilize microtubules, whereas mts1 has been implicated in their depolymerization. Taxol, which stabilizes microtubules and arrests cells at the G1 phase of the cell cycle, down-regulated mts1 gene expression in both F1 and ML8 lines. Taxol also reduced heat shock protein expression in ML8 cells. These data suggest opposing functions of heat shock proteins and the mts1 gene in microtubule polymerization, and may provide a rationale for the use of hyperthermia as a treatment for tumours.

Metastasis-associated mts1 gene expression correlates with increased p53 detection in the B16 murine melanoma.

MTS1 is a metastasis-associated gene highly expressed in high-metastasis tumors. Here we show that the expression of the suppressor gene p53 protein correlates with mts1 expression. In murine melanoma B16-F1 cells, alpha-melanocyte-stimulating hormone up-regulated mts1 and increased p53 positivity in immunohistochemical tests. In B16-ML8 cells, retinoic acid reduced mts1 expression together with a reduction of p53 positivity. The variation of p53 in association with mts1 gene expression suggests that the mts1 product might interact with and stabilize p53. Taxol-induced aneuploidy increased the proportion of G0G1 phase cells, increased p53 positivity, and down-regulated mts1. This suggests that mts1 transcription may have been negatively regulated, possibly on account of the stabilization of microtubules by taxol. We postulate that the control of G1-S transition by p53 could be due to p53 sequestration by mts1, leading to microtubule depolymerization and signaling entry, into the S phase. Thus, a coordinated function of mts1 and p53 may be involved not only in uncontrolled growth but also in cytoskeletal depolymerization that could lead to cancer invasion.

Metastasis associated MTS1 and NM23 genes affect tubulin polymerisation in B16 melanomas: a possible mechanism of their regulation of metastatic behaviour of tumours.

The nm23 and mts1 genes are associated with the expression of the metastatic phenotype. We have shown previously that modulation of metastatic behaviour produces parallel changes in the expression of these genes and that the expression of the two genes is co-regulated. Here we show that modulation of gene expression affects the process of tubulin polymerisation. B16 melanoma cell lines F1 and ML8 were treated with alpha melanocyte stimulating hormone (MSH) and all-trans retinoic acid (RA) respectively. MSH reduced the proportion of nm23+ and increased mts1+ F1 cells, with a 55% decrease in the ratio nm23:mts1. In parallel, MSH increased the expression of depolymerised form of tubulin in these cells. Treatment of ML8 cells with RA decreased mts1 positivity to a greater extent that nm23 positivity and the nm23:mts1 ratio increased by 70% and, in parallel, reduced the expression of depolymerised form of tubulin. These data suggest that nm23 and mts1 gene expression regulates the biological behaviour of the tumour cell and confer on it invasive and metastasizing properties by affecting the state of tubulin polymerisation.

Expression of alpha-melanocyte stimulating hormone and the invasive ability of the B16 murine melanoma.

The expression of alpha melanocyte stimulating hormone (MSH) has been investigated in two variants of the B16 murine melanoma. The presence of MSH was demonstrated by immunohistochemical methods using anti-MSH antibodies. The low metastasis variant B16-F1, which grows as an encapsulated non-invasive tumour, showed no alpha-MSH immunoreactivity. In contrast, the high metastasis variant BL6 was found to be alpha-MSH positive and the immunoreactivity was found predominantly in the peripheral invading zones of the tumour.

Image processing for cell cycle analysis and discrimination in metastatic variant cell lines of the B16 murine melanoma.

Computer-based image analysis offers a wide range of techniques which can be used to make objective and reproducible detection and measurement of subvisual features of microscopic images of cells. We describe here a prototype imaging system for specimen analysis. Using this system, studies have been carried out on the low metastasis variant F1 and high metastasis variant BL6 of the B16 murine melanoma. Cell cycle analyses have been carried out based on the measurement of integrated nuclear density, nuclear area and nucleocytoplasmic ratio of cells stained using standard procedures. It has been possible to discriminate between the two cell populations on the basis of ploidy. The two cell lines had a similar proportion of cells in the S-phase.

Measurement of DNA content and nuclear pleomorphism in metastatic variants of the B16 murine melanoma and hamster lymphoma and its liver metastasis using image analysis techniques.

The DNA content and nuclear pleomorphism (NPM), which are two cellular features consistently employed in the assessment of tumour malignancy, have been measured in B16 murine melanoma metastatic variants and in hamster primary lymphoma and its liver metastasis as tumour models using image analysis techniques. The three melanoma variants studied were the low metastasis variant F1, the BL6 variant selected for high lung metastasis and invasive ability, and ML8, a line isolated from pulmonary metastasis of the BL6 tumour. The cellularity of the melanomas bore no relationship to metastatic ability. The cell cycle distribution of nuclei based on integrated nuclear density (IND) was studied. The ML8 tumour showed higher DNA ploidy. Also, in this tumour the S-phase fraction was approximately 2.0-fold larger than that of the BL6 tumour. Flow cytometry of nuclei isolated from paraffin-embedded tumour tissue showed all three melanomas were aneuploid. In both F1 and BL6, two distinct subpopulations (p2 and p3) of nuclei, based on the degree of their pleomorphism, could be discerned. A significantly higher proportion of the more pleomorphic subpopulation (p2) occurred in BL6 than in F1. In the ML8 alone, a third subpopulation (p1), which was more pleomorphic than p2, was found. The hamster lymphomas (HALY-malignant and N-HALY-non-malignant) were less cellular than the metastatic tumour (HALY-met) in liver. The lymphomas N-HALY and HALY-met had a higher DNA ploidy as compared with its primary tumour HALY. However, the non-malignant lymphoma N-HALY and the moderately malignant hamster fibrosarcoma were also found to be hyperdiploid. The metastatic lymphoma (HALY-met) showed a more pleomorphic nuclear subpopulation as compared with the primary. No differences were found in the size of the S-phase fractions of the hamster tumours. The present work shows that image analysis techniques enable one to make objective measurements of DNA content and nuclear pleomorphism of tumour cells, and suggests that in the tumour models investigated there is increased nuclear pleomorphism and DNA ploidy associated with tumour progression.

Genetic recombination in human melanoma and astrocytoma cell lines involves oncogenes and growth factor genes.

We describe here the chromosomal distribution of sister chromatid exchanges (SCEs) in four human tumor cell lines (two melanomas and two astrocytomas), and have mapped the sister chromatid recombination (SCR) sites. A higher incidence of SCR sites than expected on the basis of chromosome length occurred in chromosomes 2, 4, 5 and 15 in both the RPMI 5966 and MEL57 melanoma cell lines, and in chromosomes 1, 5, 13 and 15 of the IJKt and GUVW astrocytoma cell lines. A majority of the recombination sites occurred close to chromosomal fragile sites. A third of these occurred at the same bands as fragile sites. The recombination sites involved the N-ras and the epidermal growth factor gene in the melanomas. In the astrocytomas, the N-ras, Rb and c-mos genes appeared to be involved in the recombination events. The beta 2-microglobulin gene was involved in both astrocytomas and one melanoma. The erbB2 was involved in SCR only in the RPMI melanoma.

Gene amplification correlates with sister chromatid exchange in B16 murine and human melanoma and human astrocytoma cell lines.

The incidence of double minute chromosomes and sister chromatid exchanges has been examined in four variant lines of the B16 murine melanoma, two human melanoma and two astrocytoma cell lines. The incidence of double minutes and sister chromatid exchanges appears to be strongly associated in these cell lines.

Spontaneous sister chromatid exchange in metastatic variants of the murine B16 melanoma and human astrocytomas in culture.

Three metastatic variants, BL6 (high metastasis), F1 (nonmalignant) and F10 (intermediate malignancy) of the B16 murine melanoma, and a pulmonary metastatic line BL6-ML8 of the BL6 primary tumour have been examined for spontaneous sister chromatid exchange (SCE). Two human astrocytoma cell lines were also examined. SCE was encountered in 29 and 13% of second division metaphases of BL6 and F10. In contrast, only 3% of second division metaphases of the F1 showed SCE. In BL6-ML8, 40% of the metaphases showed SCE. Approximately 2-4% of the human astrocytoma second division cells showed SCE. The variant lines were karyotypically heterogeneous. The pattern of cell distribution according to chromosome number showed an overall similar profile in the melanoma variants. However, the metastatic BL6-ML lines showed a marked shift to a hypertriploid state. SCEs occurred with higher frequency in this hypertriploid subpopulation of BL6 and F10 cells than in F1. SCE incidence in the hypertriploid subpopulation was twofold higher in the metastatic line than in the primary BL6 line. The number of SCEs per chromosome was twice as high in F10, BL6 and BL6-ML8 as in the F1 cells. This hypertriploid subpopulation showed a marked increase of SCEs on exposure to mitomycin C and ethyl methane sulphonate, indicating their mutability. It is suggested that the parallelism between SCE and metastatic potential may be relevant in the context of the generation of the metastatic phenotype.