Transcriptional control of epithelial to mesenchymal transition by regulatory factors and epigenetic mechanisms

The World Health Organization (WHO) states cancer to be a leading cause of death worldwide accounting for 7.6 million deaths (around 13% of all deaths) and is projected rising to over 11 million in 2030. This is an alarming call to researchers for putting more effort into the analysis of the underlying patho-mechanisms. In a very simplified manner, cancer represents the destruction of healthy tissues and organs by uncontrolled cell proliferation and subsequent formation of a tumor. One key feature of solid tumors that marks the mostly deadly feature of the disease is the acquisition of the potential to invade into the surrounding tissue and form secondary tumors at distant sites, a process called ‘metastasis’. To gain migratory and invasive properties, cancer cells undergo epithelial to mesenchymal transition (EMT) where epithelial cells lose epithelial properties, e.g. their polarized organization and cell-cell junctions, and thus undergo changes in cytoskeleton organization and cell shape and acquire mesenchymal characteristics. Importantly, besides the formation of metastatic lesions, EMT is also involved during development as well as wound healing. To gain insights into the complex process of EMT and to identify new potential markers for ongoing metastasis, we established different in vitro EMT model systems. Global expression profiling during TGF-β-induced EMT revealed genome-wide transcriptome reprogramming during EMT and identified Krupple-like factor 4 (Klf4) and the SRY-Related HMG-Box Gene4 (Sox4) as one of the key transcription factors that were modulated and may possibly contribute to transcriptional changes during EMT. We investigated the role of Klf4 and Sox4 during EMT by employing two different in vitro systems of EMT, using normal murine mammary gland (NMuMG) and Polyoma middle T- breast cancer (Py2T) cells, which undergo a progressive EMT upon transforming growth factor (TGF-β) treatment. We further validated the role of Sox4 in breast cancer carcinogenesis in vivo by orthotropic injection of Sox4-depleted cells into the mammary fat pad of nude mice. In addition, we also investigated whether such TGF-β-induced EMT accompanies epigenetic reprogramming and revealed how Polycomb group (PcG) complex-mediated H3K27me3 modification modulates transcription of key genes underlying this process, thereby regulating EMT. Klf4 is a zinc-finger protein, known to be abnormally expressed in various cancers. It can act as a tumor suppressor or as an oncogene in context dependent manner in different carcinomas. Klf4 is downregulated during TGF-β-induced EMT. Our data reveal a tumor suppressor role for Klf4 in breast carcinogenesis. Klf4 is essential for the maintenance of an epithelial phenotype during EMT, and forced expression of Klf4 leads to blockage of epithelial differentiation. Furthermore, Klf4 is inhibitory to EMT-driven cell migration and also behaves as a survival factor during TGF-β-induced EMT. Genome-wide location analysis by next generation ChIP-seq analysis revealed that Klf4 directly occupies the promoter of many key EMT genes such as N-cadherin, Vimentin, β-catenin and Mapk8. Moreover, one of these Klf4 targets, Mapk8, encoding Jnk1, is upregulated during EMT and a double-knockdown of Klf4 and Jnk1 is able to overcome Klf4 knockdown-induced EMT, migration and apoptosis. These observations underscore a role of Klf4 during EMT by targeting and regulating crucial EMT genes. Sox4 is also known to be deregulated in many cancers. Sox4 is upregulated during TGF-β-induced EMT. We show that Sox4 is required for maintaining mesenchymal identity and depletion of Sox4 prevents TGF-β-induced EMT. Sox4 reduction further impairs the migratory capacity of cells. Moreover, Sox4 provides a survival advantage to cells during breast carcinogenesis. In addition, Sox4 contributes towards TGF-β-induced tumorigenicity and metastatic spread. Gene expression profiling after Sox4 depletion in complementation with Chromatin immunoprecipitation analysis revealed many key EMT genes such as Spred1, Edn1, Palld, Cyr61, Ereg, Areg and Yap1 which are directly targeted by Sox4 for transcriptional regulation. Furthermore, Sox4 also controls many genes which are shown to regulate various other features of EMT as well as cancer development such as angiogenesis, adhesion, migration, morphogenesis, cell cycle and cytoskeleton re-modeling. Ezh2, a catalytic subunit of the Polycomb Repressive Complex 2 (PRC2), has been also found to be transcriptionally regulated by Sox4. To delineate the role of Ezh2 during EMT, a loss of function approach has been used to demonstrate that Ezh2 is required for proper acquisition of EMT and EMT-driven processes such as migration and apoptosis. Taken together, our data provides a role of Sox4 during EMT via transcriptional regulation of key genes, including the Polycomb component, Ezh2. We also studied the role of two prominent epigenetic modifications- DNA methylation and histone 3 lysine 27 tri-methylation (H3K27me3) during TGF-β-induced EMT in a mammary epithelial cell line. Our data revealed no evidence of a reprogramming of DNA methylation during this process. To assess the role of H3K27me3 during EMT, we performed chromatin immunoprecipitation using H3K27me3-specific antibodies followed by next-generation sequencing (ChIP-seq) on 6 different stages of EMT progression. This analysis revealed that many key EMT genes are regulated by H3K27me3 mark including Mcam, Pdgfrb and Itga5 which are repressed by this mark in epithelial cells and loose it during EMT as they get activated conversely, Cdh1, Ocln and Cdx2 gain this mark during EMT and get repressed in mesenchymal cells. We further illustrated that the coordinated activities of Ezh1 and Ezh2 are required for H3K27me3-mediated repression of the gene expression and their co-depletion de-represses target genes and blocks EMT. This study provides novel insights into the important regulatory role of the Polycomb machinery during EMT. In summary, our findings demonstrate how transcription factors, such as Klf4 and Sox4 and the epigenetic machinery, such as PcG proteins, regulate EMT by directly contributing to the transcriptional reprogramming underlying this process

Layout Optimization of a repair facility using discrete event simulation

Technological advancements in the field of simulation have enabled production managers to model and simulate their facilities under various scenarios, in order to optimize system performance. In particular the reconfiguration of factory layouts can be time consuming and expensive; Discrete Event Simulation (DES) can be used to model and assess various scenarios to assist production managers with layout planning. Significant benefits can be achieved through the use of DES for factory layout optimization including: decreased lead times, reduced manufacturing costs, efficient materials handling and increased profit. This paper presents the development of a DES model in WITNESS for the analysis and factory layout optimization of a repair facility. The aim of the model is to allow decision makers to assess various layouts and configurations with a view to optimize production. The model has been built with a link to an Excel spreadsheet to enable data input and the visualization of Key Performance Indicators (KPIs). Specific functions have been built into the simulation model to set and save new layouts within Excel to facilitate layout optimization. The model will be used to optimize the factory configuration

A study on knowledge of breast feeding and effect of antenatal counselling on exclusive breast feeding in primigravida mothers

Background: Exclusive breastfeeding up to 6 months of age is very beneficial not only to baby and mothers, but also to the family, society and the whole nation. In spite of best efforts by various organizations at government and NGO levels, the counselling for breast feeding during ante natal period is almost an unaddressed arena, which if proper attention is given, may result in very high incidence of successful breast feeding.Methods: It is a hospital based prospective study. A total of 150 primiparous mothers (100 study group and 50 control group) were counselled during every antenatal checkup for exclusive breast feeding attending the OPD of Obstetrics & Gynaecology department of Vardhman Institute of Medical sciences, Pawapuri, Nalanda, Bihar from January 2015 to December 2016 and the outcome of counselling was observed during follow up in puerperium for 6 weeks.Results: Similar profile primiparous mothers in both study and control group, the mothers who were counselled on their antenatal visit for exclusive breast feeding, the success rate was much higher in counselled group than study group (p value 0.0001). It was also observed that the prior information about breast feeding were provided by electronic media, papers and family members and in this regard contribution of health system is minimal.Conclusions: Every obstetrician and other care givers must include routine care of breast and counselling on breast feeding during antenatal check up to enhance the confidence of primiparous mothers. A few extra minutes spared in counselling on breast feeding has a long-lasting effect on successful breast feeding during the post-natal period.

A study on effectiveness of lactational amenorrhea as a method of contraception

Background: The lactational amenorrhea method, known as LAM, is part of the World Health Organization’s list of accepted and effective methods of family planning. Studies show LAM to be 98% effective, as effective as the pill and other modern methods, when used according to guidelines. As LAM costs nothing, easy and natural to use, no medication or any devise needed, no interference with natural hormones, no interruption in conjugal life and more over the mother enjoying her motherhood life, it sounds very good as a method of contraception.Methods:This is a prospective non-comparative study. A total of 298 mothers participated in this study. The benefits of exclusive breast feeding with particular emphasis on timely breastfeeding and must feeding during the night hours were explained to the mothers and the physiology of lactational amenorrhea was explained to them. All the mothers were interviewed and followed up on monthly basis. Results:No lactating mothers conceived within the first six months of delivering their baby and only 4% mothers had evidence of pregnancy mainly after the 10th months after delivery.Conclusions:The lactational amenorrhea method is a highly effective method of contraception for 6 months if exclusive breastfeeding practices are followed. In a country like India where use of contraceptive methods are very meager, LAM may prove a boon as it provides dual benefit of exclusive breastfeeding and prevention of pregnancy

JIWHA PARIKSHA- A MIRROR OF HUMAN HEALTH

The tongue is the primary organ of taste and considered as the mirror that reflects the conditions of the body's internal organs, particularly the organs of digestion and metabolism. The tongue also reflects the overall digestive, nutritive and metabolic conditions of the organism. An ideally healthy tongue will look clean and have a pink colour with no white coating. In Ayurveda, Acharya Yogaratnakara has mentioned tongue examination as Jiwha Pariksha under Ashtasthana pariksha. Jiwha (Tongue) tells about the Dosha, Agni and Kostha. The tongue is the window to our digestive system. There is a large amount of information that can be learned from this observation, including the health of various bodily systems, toxins within the system, emotional states, and much more. So, the inspection of the tongue becomes an important starting point in the clinical examination and a careful observation of the state of tongue, its colour, and shape often gives a physician an insight into the current health condition of the patient

Histopathological evaluation of endometrium and its clinical correlation in patients of abnormal uterine bleeding

Background: Abnormal uterine bleeding (AUB) is a very common, challenging gynaecological problem in all age group women. Neoplastic lesions are responsible for very less number of cases but they should be diagnosed early. Endometrial biopsy is necessary for histopathological diagnosis of endometrial lesions and to rule out any malignancy. In present study, we evaluated histopathological pattern of endometrium and different clinical parameters in AUB patients.Methods: A retrospective observational study done in Obstetrics and Gynecology department of SSMC Rewa, Madhya Pradesh in 2 year from 1st October 2017 to 30th September 2019. AUB patients who fulfilled inclusion criteria were included in study. All data were collected from department records in predesigned proforma and statistically analyzed by statistical package for the Social sciences (SPSS) version 20.Results: Total 366 AUB patients were included in study; most frequent clinical presentation was HMB (56%). Mostly patient’s age ranged from 24-84 years, belonged to 41-50 years age group (44%), multiparous (78%), and premenopausal (53%). On histopathological evaluation of endometrium most commonly nonneoplastic lesions (75%) were seen, out of which proliferative phase endometrium was most frequent. Neoplastic lesions were seen in 25% cases. Out of which benign lesions were noted in 7%, premalignant in 17.3% and malignant in 1% (all in >45 year patients).Conclusions: Endometrial biopsy should be done in all AUB patients >45 years and in selected premenopausal in order to get early diagnosis, to rule out malignancy and to help in management. Endometrial biopsy should be considered as first diagnostic modality

Proteome Based Development of Novel Affinity Tail for Immobilized Metal Affinity Chromatography and Hydrophobic Interaction Chromatography

At industrial scale, reducing the step in purification and recovery is desired; this not only decreases the cost but also increases the yield. Hydrophobic Interaction Chromatography (HIC) and Immobilized Metal Affinity Chromatography (IMAC) both are not harsh on biological structure or activity of proteins; also both the techniques are economical and therefore a suitable choice at industrial level. This dissertation comprises of three parts. Purpose for the Part I was to identify and characterize Escherichia coli proteins which display affinity towards both IMAC and Hydrophobic Interaction Chromatography (HIC). Co (II) IMAC was chosen as the primary capture step, followed by HIC employing different concentrations of salt to promote adsorption. Nine out of the ten proteins identified have iso-electric values less than six, and half are considered nonessential. These data indicated that the combination of IMAC and HIC could be developed as a potent method for the purification of recombinant proteins by judicious choice of the salt concentration used to promote HIC, the development of E. coli strain(s) deficient in certain genomic proteins, and the design of an IMAC - HIC affinity tail for recombinant protein isolation based on the very proteins deleted from the genome. Part II comprises of study of E. coli Alpha Galactosidase (MelA) as a candidate for affinity tag for this dual step purification approach. Affinity of MelA towards Co (II) IMAC and HIC was confirmed by fusing with a small peptide. Metal binding motif and various salt binding motifs of MelA were identified and studied. A MelA based affinity tag NT1 was developed by identifying the major salt binding region for HIC. It is desirable to have a tag which doesn\u27t interfere with solubility or biological activity of the fused protein. Solubility has been the biggest issue with the development of affinity tag for HIC. In part III, with successful use of novel proteome, we report development of MelA as a successful affinity tag for HIC especially as it does not affect the expression, solubility or biological activity of the fused protein, with very few co-eluting proteins as contaminants

Fusarium Wilt: A Killer Disease of Lentil

Lentil (Lens culinaris Medikus subsp. culinaris) is an important dietary source of protein and other essential nutrients in South and West Asia, North and East Africa. Lentil crops are vulnerable to a number of diseases caused by fungi, viruses, nematodes, insect pests, parasitic plants and abiotic stresses. Among them, the most significant and serious soil-borne disease is Fusarium wilt (Fusarium oxysporum f.sp. lentis: Fol). Fusarium wilt causes yield loss up to 50% in farmers’ fields. The pathogen showed high levels of phenotypic and genotypic diversity in India, Algeria, Syria and Iran. The disease thrives at 22–25°C temperature and affect lentil either at seedling and vegetative or the reproductive stages of the crop. To minimize yield losses, an integrated management strategy comprising resistant/partial resistant cultivars, adjusting sowing time, bio-control and chemical seed treatments is the best approach to reduce the incidence of the Fusarium wilt of lentil. This review covers past achievements in managing the disease, pathogen diversity and identify gaps in managing Fusarium wilt to improve productivity and production of the crop

Human relationship in the Novels of Nayantara Sahgal

Nayantara Sahgal is an Indian writer in English. She was one of the first female Indian writers in English to receive wide recognition. Her concern for women is based more on humanism rather than that of feminism. Her feminism does not go beyond treating women as an individual. During 1950's period, Nayantara Sahgal emerged as one of the most significant voices among women writers in the contemporary Indian writing in English. Nayantara Sahgal is a prolific writer who has eight novels to her credit, two biographies, two political commentaries and a large number of articles in various newspapers and magazines. She is a recipient of the prestigious `Sahitya Akademi Award' for her novel ‘Rich Like Us’ (1985), the `Sinclair Award' and the 'Common Wealth Award for Eurasia' for her novel ‘Plans For Departure’ (1986). Her fiction is closely interwoven with the fabric of interpersonal relationships not in the political and social milieu of India. Various dimensions of human relationships, man-woman particularly are the main thesis, we do find in all her novels. Man-Woman relationship holds a vital place in these relationships and the novelist deals with this dimension of relationship with full concern and broad perspective