INTRODUCTION ( Around 200 million years ago (mya), prototherians

INTRODUCTION Mammals are divided in to three subclasses into three subclasses namely allotheria, prototheria and theria. The allotherian mammals are extinct and consisted of multituberculates ( Around 200 million years ago (mya), prototherians split into a separate subclass giving rise to monotremes as illustrated in figure-1 (1). Monotremes are primitive mammals possessing mammalian and reptilian features. They have mammary glands that lack nipples, they possess hair, single jaw bone, are endothermic and have a three bone middle ear structure which are the classic features of mammals. Like reptiles, they lay egg covered with leathery egg shells and have a cloaca instead of vagina indicating non-placental growth of the young one ( Echidnas and platypus are the only members of the Order of monotremes as given in figure-1. They are abundantly found in New Guinea and Australia (1). Milk is the major source of nourishment and protection for the young one (puggle) (1).  Figure 1: Classification of mammals. The extant subclasses of class mammals include Prototheria and Theria. While Monotremes are the only order under the subclass Prototheria, Theria is further divided into infraclass Metatheria and Eutheria which consists of several orders, families and species. Echidna falls under the order Monotremes.Previous study from our lab has found an antimicrobial protein EchAMP (Echidna antimicrobial protein) from echidna (Tachyglossus aculeatus). It also showed that EchAMP is highly expressed in milk cells (during late lactation) and lower expression of the EchAMP protein was recorded in intestine, liver, testis and penis. EchAMP shows a broad spectrum anti-microbial activity against both gram positive and gram negative bacteria that includes Escherischia coli, Salmonella enterica, Pseudomonas aeruginosa, Staphylococcus epidermidis and Staphylococcus aureus but does not affect the commensal gut species of Enterococcus faecalis (PMID: 23326486).Previous study from our laboratory produced transgenic mice expressing the EchAMP gene in its milk/mammary gland to explore the role of EchAMP as an antimicrobial protein. EchAMP gene knock in, confirmed by southern blotting and quantitative PCR (Polymerase Chain Reaction) techniques have indicated the integration and transcription of the gene into RNA (Unpublished data). But the expression of the protein in the transgenic mice has not been confirmed yet. Since there are no commercial antibodies available to study the expression of EchAMP protein in transgenic mice milk, the first aim of the present study focuses on developing antibodies against the EchAMP protein in rabbits. Several studies on milk proteins have revealed their important role in anti-fungal, anti-bacterial, anti-parasitic and anti-viral activities (doi: 10.1111/1541-4337.12126, PMID: 12769734, PMID: 27579006, PMID: 10877382). Milk contains immunologically active proteins which serves as the first vaccine for the neonate. The immunoglobulins (Igs) forms the first line of defence in the new born and milk serves as the richest source of Igs. Other milk proteins such as lactoferrin, lactoglobulin, casein and lactalbumin are also known for their excellent activity as anti-microbial proteins (AMPs) (PMID: 12769734). Studies show that these antimicrobial milk proteins are equipped with anti-inflammatory activities also. Human and bovine milk have proven to protect infants from gut inflammatory disorders resulting from imbalance in gut inflammatory mediators and bio actives secreted from gut microbioal flora. This is usually found in growth restricted or premature born infants. Milk AMPs activate toll-like receptors, complementary system and anti-inflammatory cytokines to combat this (PMID: 23660296). On the other hand, it’s been shown that gut microbiome imbalance also leads to cancer in the presence of microorganisms that alter the macrophages and other immune components (PMID: 24906974, PMID: 28481406). Parallel studies in our lab also indicates that EchAMP has anti-inflammatory activities against S. aureus, in EchAMP knocked in transgenic mice. Previous studies also show that EchAMP is harmful only against pathogenic microbiome and does not affect the commensal gut flora.Cancer is the second largest cause of death due to non-communicable diseases according to the World Health Organisation (WHO) ( The current therapies on cancer are not completely accurate and effective. Hence an alternative new therapy is the need of the hour. Mammary glands, milk and colostrum are excellent home of host defence peptides (PMID: 12769734). Recent studies demonstrate that AMPs have a tremendous potential as an anti-cancer drug. Human host defence peptide LL-37, a cathelicidin, is an excellent example of AMPs role in tumour control in mammals. LL-37 has proven to be operative against melanoma, colon cancer and ovarian cancer. It is currently under phase 2 clinical trial against melanoma ( Lactoferrin is a milk protein whose role as AMP and ACP has been extensively studied. They are present in several exocrine secretions of mammals including milk (PMID: 28626840). This iron binding glycoprotein has been found to play a role of primary defence protein against tumorigenesis and was found to be effective against melanoma in both in vitro and in vivo (mice) studies (PMID: 8162571). Based on all these findings, the second part of the study focuses on exploring the preliminary in vitro anti-tumorigenic property of EchAMP protein.  The fact that EchAMP is also equipped with anti-inflammatory properties in mice and does not affect intestinal gut microbiome, alteration of which leads to cancer, supports the hypothesis. REVIEW OF LITERATUREEVOLUTIONARY AND MAMMALIAN TRAITS OF PROTOTHERIANS AND THERIANSMammals are present in all habitats including sea (whales), land and air (bat). They are not a diverse group with their diversity being approximately half of the birds. They have prominent physical traits which distinguish them from other classes. The presence of mammary gland is an obvious characteristic of the species in this class. Other defining characters include three bone structure of the middle ear (stapes, incus and malleus), presence of one single jaw bone, endothermic that is internal regulation of body temperature against reptiles which are ectothermic and allows the environment to regulate the body temperature and cellular and structural differences ( The existing subclasses of mammalian class includes Theria and Prototheria (1). The order of monotremes come under the subclass prototherians while Theria consists of Metatherians and Eutherians (  While the Metatherians and Eutherians give birth to young ones, Monotremes are egg laying mammals, hence their other name ovoviviparous. They egg shell however is leathery in nature. The egg is carried in the pouch and hatches in about ten days from which raises an premature young ones. The Metatherians give birth to an under developed young one due to the lack of placenta and incapability to nourish the young one in the worm. They possess an egg shell like membrane inside their uterus. This also renders it impossible to transfer necessary bioactives to the young one. The young ones of both prototherians and Metatherians have strong forearm structure facilitating them to climb from the pouch to mother’s teat for milk consumption. . In both the subclasses milk is the major source of nourishment. Unlike the Eutherians these species are born underdeveloped and do not have placental nourishment from the mother. Hence the milk of these species is enriched with high amounts of bio actives ( Both the subclasses possess the important mammalian characteristics. Monotremes have only 5 surviving species, four of Echidna and one Platypus. Our lab and its collaborative project was interested in Echidna specie Tachyglossus aculeatus and proceeded to research upon it. Echidna are shy animals which dwells in an unclean environment (as shown in Figure 2) and feed on ants and termites. Like other monotremes, Echidna milk is a rich source of nutrient and protective bio actives and the young one feeds on mother’s milk (as shown in Figure 2) for a minimum period of three months (1).  Figure 2: Puggle suckling echidna. The Echidna suckles her puggle until three months. The hostile and unclean environment that the puggle has to encounter necessitates the need for immune boosting bio-actives in the milk. IMPORTANCE OF MILK IN COMBATING PATHOGENSStudies have shown that colostrum and milk are a rich source of anti-microbial proteins. Milk is a natural technique for nourishing the neonate for better immunity (PMID: 26115887).  Mother’s milk is an impeccable source of immunogenicity and anti-inflammatory proteins for neonates. Experiments have shown that kids who did not go through breast feeding are more prone to severe infections and diseases such as asthma, type-2 diabetes mellitus and leukaemia (Bookshelf ID: NBK52687). Analysis on several milk proteins like lactoferrin, ?-casein, lactalbumin, lactoperoxidase, lysozyme and immunoglobulins (IgA) demonstrate that they have exceptional and specific antimicrobial activity (PMID: 12769734). Hence, these proteins are also termed as AMPs. Our lab has found out a novel monotreme antimicrobial protein EchAMP. The expression of EchAMP was very high in milk especially during the late lactation period when the young one is ready to leave the mother’s pouch and low in intestine, liver, testis and penis. Since milk proteins have antimicrobial activity and monotremes immunize and nourish the young one through milk, previous studies in our lab explored EchAMP for its antimicrobial activity and found that it is a broad spectrum antimicrobial protein. EchAMP is effective against both gram positive and gram negative bacteria but not Enterococcus faecalis,   a commensal gut specie (PMID: 23326486).ADVANTAGES OF ANTIMICROBIAL PROTEINS OVER CONVENTIONAL TREATMENTSAMPs have gained their momentum in recent times. Scientists are constantly searching for novel drugs against harmful microorganisms, as the latter have been developing resistance against the presently available antibiotics. In addition to that, these antibiotics are known to have a detrimental effect on gut microbiome, which may lead to aggression of several diseases (PMID: 27028893). Recent study has shown that antibiotics treated infants were more prone to Salmonellosis resulting in severe diarrhoea and stomach infections (PMID: 24995874). Hence, a search for an alternative source of treatment is in need. AMPs overcome multidrug resistance of microorganisms and does not alter the gut microbiome, thus making them a good alternative (PMID: 25421473).MECHANISM OF AMPSInitially AMP’s were thought to be solely cationic in nature destroying the microbes with anionic surface membranes through membrane lysis. But recent researches has surfaced the fact that anionic AMPs also exists (PMID: 19751192). While magainins, defensins, cecropinA, lactoferrin are examples of cationic AMPs, kappacins and chromacins belong to the category of anionic AMPs (PMID: 22725698, PMID: 19751192). AMPs mainly work by three major mechanisms, namely membrane depolarization, lysis through cell penetration and activation of immune components. The first mechanism is mostly adopted by cationic AMPs as they depolarize the negatively charged microbial membrane through electrostatic interaction (PMID: 22725698). The amphipathic nature of AMPs enhance the binding with lipid bilayer for the execution of this mechanism. This leads to an osmotic dysregulation and eventually leads to cell lysis. The second mechanism involves the entry of AMPs into the cell either by disrupting the lipid bilayer of the membrane or translocation into the cell with the aid of divalent cations. While the former is mostly implemented by cationic AMPs, anionic AMPs work with the aid of divalent cations such as Zn2+, Ca2+ and Mg2+ (PMID: 22725698, PMID: 19751192). Disruption of lipid bilayer occurs through three methods, namely barrel stave model, toroidal pore model and carpet model.  In barrel stave model, the proteins from AMP interact with membrane molecules and form ion-protein channel. The peptide interaction with the membrane leads to the recruitment of additional peptides which later lines the membrane to form a transmembrane pore. The hydrophobic side of the peptides interacts with the lipid layer and the hydrophilic layer faces inside (Figure 2a). In toroidal pore model the phospholipids are forced to bend away making a way for the peptides to enter the cell (Figure 2b). In carpet model, disruption of membrane takes place. This happens as the hydrophilic side of the peptides faces the hydrophobic head group due to which the polarity of the bilayer reverses and disrupts (Figure 2c). Subsequently, as they enter into the cell they can disrupt DNA, RNA or essential proteins important for the survival of the cell (PMID: 22725698). The human host defence protein LL-37 disrupts the membrane using toroidal pore model and damages the DNA by chromatin condensation (PMID: 12767238). The third mechanism is highly specific to higher order species. The AMPs actuates the immune system of the organism. It either recruits immune components such as macrophages, dendritic cells and anti-inflammatory proteins to lyse the microorganism or activate the complement system to destroy the foreign particles (PMID: 19751192).  Figure 2: The mechanism of membrane disruption by AMPs. a) The barrel stave model in which, peptides (red cylinder) form a transmembrane pore with the hydrophobic side of the peptides interacting with the hydrophobic side of the bilayer and vice versa eg: alamethicin. b) In toroidal pore model, peptides (red rods) force their way through the lipid bilayer while some of the peptides line the mouth of the pore stabilizing the pore eg: mellitins magainins. c) In carpet model, peptides (red rods) align themselves on the surface of membrane and depolarize them by inverting the bilayer eg: LL-37, cecropinOTHER FUNCTIONAL ROLES OF AMPSThe function of AMPs is not limited to antimicrobial activity. Several experiments have proven that AMPs function as anti-inflammatory proteins, antioxidants and anti-tumorigenic proteins as they also modulate the immune reactions (doi: 10.1111/1541-4337.12126). Recent findings suggest that milk proteins also have anti-inflammatory activity and protect the gut of a new born from necrotizing enter colitis a disease which is responsible for high infant mortality rate. This is prevented by administering milk to the infants. Milk proteins activates toll like receptors, anti-inflammatory cytokines, matrix metalloproteinase, leukotrienes and complement factors and supresses inflammatory metabolites to reduce the inflammation (PMID: 23660296). As host defence peptides they not only defend against external threats but guard the system against a defective internal organisation as well. There are numerous studies on how an AMP functions as anti-carcinogenic protein (ACP) due to the similarities between a microbial and a cancer cell. Both microbial and cancer cells have high negative surface charge which isolates them from the normal cells. The high negative charge in cancer cells is due to the expression of excessive amounts of phosphatidylserine (PS) and phosphatidylethanolamine (PE) on the outer leaflet of the lipid bilayer unlike the expression in the inner leaflet in the normal cells. Other sources of negative charge includes the linking of sialic acid to glycoproteins like mucin and proteoglycans conjugated to heparin sulphate or chondroitin which carry a high negative charge. The AMPs are specific to certain type of cancers as they are not solely attracted to negative charge but also tumour specific receptors present on the surface of the tumour cells (PMID: 22725698). For example, LL-37 is effective against melanoma and ovarian cancer but not lung cancer (PMID: 23812430, PMID: 19068548). The mechanisms adopted by ACPs does not vary much from AMPs. Similar to AMPs, they recruit immune components to the cancer sites, for e.g. site of cancer like lactoferrin which activates NK cells and macrophages, have membrane lysis properties on the cell as in the case of magainins or translocate into the cell to attack DNA, RNA or proteins. ACPs follow an additional protocol of apoptosis which is the preferred mechanism over necrosis (PMID: 22725698). Apoptosis occurs either by intrinsic (mitochondrial pathway) or extrinsic (death receptor pathway) caspase dependent mechanism (both converge in the same execution pathway involving caspase-3) or by caspase independent mechanism (DNA damage through chromatin condensation) as observed in the case of AMPs like LL-37 (PMID: 23100468). In cancer, there is an imbalance between pro-apoptotic and anti-apoptotic proteins. Restoring the balance between these two can induce the apoptosis of cancer cells. Bax-related pore-forming proteins and BH3 proteins follow this mechanism. They bind to pro-apoptotic proteins and inhibit their interaction with anti-apoptotic proteins thus inducing apoptosis (PMID: 22725698). Several AMPs found in the milk, namely lactoferrin, ?-lacto albumin from bovine milk, ?-lacto albumin from human milk, ?-caseins etcetera, are found to have competent ACP activity as given in table 1. The table 1 describes the mechanisms by which several milk proteins function as an AMP and an ACP. Table 1: The mechanism of milk proteins against microorganisms and tumour cellsMilk protein Mechanism of action against tumour cells Mechanism of action against bacterial cellsLactoferrin It is a cationic protein which induces apoptosis and inhibition of cell proliferation in melanoma cells, colon carcinoma and mammalian leukaemia cells (DOI: 10.1111/1541-4337.12126) It binds to the cell and effects an opsonin like activity. It activates NK cells, polymorphomuclear leukocytes and macrophages. Increases the number of T-cells and B-cells (PMID: 12769734)HAMLET (Human Alpha-lactalbumin Made Lethal to Tumour cells It’s an anionic protein from human; interacts with cell membrane, trans locates into the cell networking with cell organelles and activating several signal pathways in cell nuclei. The tumour activity requires removal of Ca2+ and incorporation of C18:1 fatty acid chain. It induces caspase independent chromatin condensation active against human skin papillomas and bladder cancer (PMID: 20186341). It attains a bactericidal conformation which includes elimination of Ca2+ and addition of C18:1 fatty acid. It alterates the substrate specificity of enzymes from N-acetyl glucosamine to Glucose thus altering the role of lactose synthase complex (PMID: 12769734)BAMLET (Bovine Alpha-lactalbumin Made Lethal to Tumour cells) It is a bovine anionic protein. It inhibits mTORC1 and induces autophagy. It alsoCauses caspase independent chromatin condensation; it activates lysosomal cell death pathway. The tumour activity requires removal of Ca2+ and incorporation of C18:1 fatty acid chain. It is effective against human prostate adenocarcinoma, bladder cell papilloma and cervical adenocarcinoma (PMID: 20053771) The antimicrobial mechanism is the same as HAMLET (PMID: 12769734)Casein It is a cationic protein. It inhibits proliferation of tumour cells by hindering the BCL-2 pathway and inducing apoptosis in ovarian cancer cells, melanoma and breast cancer cells. Stimulates phagocytosis and enhances the activity of macrophages by increasing the expression of Major Histocompatibility Complex class II antigen on the bacterial cells (PMID: 12769734). RATIONALE:1. EchAMP gene was knocked in mice to give raise to transgenic mice producing EchAMP in its milk with the purpose of finding out its activity against mastitis causing microorganisms. Though the expression of EchAMP was found in DNA and RNA levels, there are no commercial antibodies to study the expression of EchAMP in protein. Hence raising antibodies will help in identification of protein expression.2. EchAMP is an antimicrobial protein and parallel studies in our lab indicates that it’s equipped with anti-inflammatory properties in mice. It is also an anionic milk protein with amphiphilic structure. Studies indicate that anionic AMPs with amphiphilic structure can be exceptional anti-cancerous protein. Hence, it’s hypothesized that EchAMP may have anti-carcinogenic activity.  AIMS AND OBJECTIVES:1) The first aim of this study is directed towards generating an antibody against EchAMP by following methods: • To clone and transform EchAMP gene in E.coli (BL21 ? DE3 strain)• To optimize, produce and purify EchAMP protein• To administer EchAMP protein in rabbits for raising antibody • To identify the presence of antibody in rabbit sera 2. The second aim of this study is to find out if EchAMP has in vitro anti-cancerous property with following objectives: • To study the effect of EChAMP protein on cancer cells in-vitro • To understand the mechanism by which it destroys the cancer cells