Characterisation of RNA in milk and prediction of microRNA functions

 Milk is considered on of the world’s most ‘complete’ food. To characterise milk composition, Amit investigated RNA present of milk form 8 different species ranging from platypus to human. By applying latest RNA sequencing and bioinformatic techniques, his work led to uncover hundreds of novel milk RNAs.

Programmed function of the tammar mammary gland; the role of milk proteins in gut development

 This thesis aimed to exploit the unique reproductive strategy of marsupials such as the tammar wallaby to prove that milk may regulate postnatal growth and development of organs such as the stomach.

SurR9-C84A mutant nanocarrier and milk protein therapeutics for neurological disorders

 The current project revealed that the novel nanoformulation of SurR9-C84A was able to rescue the neurons following the Alzheimer’s related ß-amyloid toxicity and inflammation. In addition, bovine lactoferrin was found to have potential differentiating effect in the tumor cells and hence is a valuable therapeutic for strengthening the degenerating neurons.

Differential temporal expression of milk miRNA during the lactation cycle of the marsupial tammar wallaby (Macropus eugenii)

Lactation is a key aspect of mammalian evolution for adaptation of various reproductive strategies along different mammalian lineages. Marsupials, such as tammar wallaby, adopted a short gestation and a relatively long lactation cycle, the newborn is immature at birth and significant development occurs postnatally during lactation. Continuous changes of tammar milk composition may contribute to development and immune protection of pouch young. Here, in order to address the putative contribution of newly identified secretory milk miRNA in these processes, high throughput sequencing of miRNAs collected from tammar milk at different time points of lactation was conducted. A comparative analysis was performed to find distribution of miRNA in milk and blood serum of lactating wallaby.

Monotreme lactation protein is highly expressed in monotreme milk and provides antimicrobial protection

Monotremes (platypus and echidna) are the descendants of the oldest ancestor of all extant mammals distinguished from other mammals by mode of reproduction. Monotremes lay eggs following a short gestation period and after an even briefer incubation period, altricial hatchlings are nourished over a long lactation period with milk secreted by nipple-less mammary patches located on the female's abdomen. Milk is the sole source of nutrition and immune protection for the developing young until weaning. Using transcriptome and mass spectrometry analysis of milk cells and milk proteins, respectively, a novel Monotreme Lactation Protein (MLP) was identified as a major secreted protein in milk. We show that platypus and short-beaked echidna MLP genes show significant homology and are unique to monotremes. The MLP transcript was shown to be expressed in a variety of tissues; however, highest expression was observed in milk cells and was expressed constitutively from early to late lactation. Analysis of recombinant MLP showed that it is an N-linked glycosylated protein and biophysical studies predicted that MLP is an amphipathic, α-helical protein, a typical feature of antimicrobial proteins. Functional analysis revealed MLP antibacterial activity against both opportunistic pathogenic Staphylococcus aureus and commensal Enterococcus faecalis bacteria but showed no effect on Escherichia coli, Pseudomonas aeruginosa, Staphylococcus epidermidis, and Salmonella enterica. Our data suggest that MLP is an evolutionarily ancient component of milk-mediated innate immunity absent in other mammals. We propose that MLP evolved specifically in the monotreme lineage supporting the evolution of lactation in these species to provide bacterial protection, at a time when mammals lacked nipples.

Safety and efficacy of antenatal milk expressing for women with diabetes in pregnancy: protocol for a randomised controlled trial

Many maternity providers recommend that women with diabetes in pregnancy express and store breast milk in late pregnancy so breast milk is available after birth, given (1) infants of these women are at increased risk of hypoglycaemia in the first 24 h of life; and (2) the delay in lactogenesis II compared with women without diabetes that increases their infant's risk of receiving infant formula. The Diabetes and Antenatal Milk Expressing (DAME) trial will establish whether advising women with diabetes in pregnancy (pre-existing or gestational) to express breast milk from 36 weeks gestation increases the proportion of infants who require admission to special or neonatal intensive care units (SCN/NICU) compared with infants of women receiving standard care. Secondary outcomes include birth gestation, breastfeeding outcomes and economic impact.

Role of marsupial tammar wallaby milk in lung maturation of pouch young

Background: Marsupials such as the tammar wallaby (M.Eugenii) have a short gestation (29.3 days) and at birth the altricial young resembles a fetus, and the major development occurs postnatally while the young remains in the mother's pouch. The essential functional factors for the maturation of the neonate are provided by the milk which changes in composition progressively throughout lactation (300 days). Morphologically the lungs of tammar pouch young are immature at birth and the majority of their development occurs during the first 100 days of lactation. Results: In this study mouse embryonic lungs (E-12) were cultured in media with tammar skim milk collected at key time points of lactation to identify factors involved in regulating postnatal lung maturation. Remarkably the embryonic lungs showed increased branching morphogenesis and this effect was restricted to milk collected at specific time points between approximately day 40 to 100 lactation. Further analysis to assess lung development showed a significant increase in the expression of marker genes Sp-C, Sp-B, Wnt-7b, BMP4 and Id2 in lung cultures incubated with milk collected at day 60. Similarly, day 60 milk specifically stimulated proliferation and elongation of lung mesenchymal cells that invaded matrigel. In addition, this milk stimulated proliferation of lung epithelium cells on matrigel, and the cells formed 3-dimensional acini with an extended lumen. Conclusions: This study has clearly demonstrated that tammar wallaby milk collected at specific times in early lactation contains bioactives that may have a significant role in lung maturation of pouch young.

Monotreme lactation : characterisation of a novel protein, endocrine regulation and miRNAs in milk

My dissertation is about the fascinating Australian mammals, platypus and echidna milk components function and milk production. I have investigated the platypus and echidna milk proteins and miRNAs that involved in protection or development of the immature monotreme hatchlings, and investigated the hormonal control of monotreme milk production.

The inhibitory effect of stevioside on bacillus cereus growth in milk: validation and its response surface optimization

Stevioside, a glycoside present in the leaves of Stevia rebaudiana Bertoni, offers therapeutic benefits such as anti-hyperglycemic, anti-hypertensive, antiinflammatory, anti-tumor, diuretic and immune influencing properties. In this work antimicrobial activity of stevioside against Bacillus cereus, a major source of milk contamination was investigated. The isolate was confirmed by various biochemical and 16S rRNA gene sequencing. The effect of temperature, incubation time and concentration of stevioside was optimized from a central composite response surface design. The standard plate count (SPC) of pasteurized milk was drastically reduced in comparison to toned and fresh milk. The optimal temperature, incubation time and stevioside concentration were observed to be 60.23°C, 21 h, and 275 μg/ mL respectively. The synergism of stevioside with the external factors (temperature and time) against B. cereus was observed. Our studies showed that addition of stevioside in fresh as well as pasteurised milk would control growth of B. cereus in milk.

Bioactive functions of milk proteins: a comparative genomics approach

The composition of milk includes factors required to provide appropriate nutrition for the growth of the neonate. However, it is now clear that milk has many functions and comprises bioactive molecules that play a central role in regulating developmental processes in the young while providing a protective function for both the suckled young and the mammary gland during the lactation cycle. Identifying these bioactives and their physiological function in eutherians can be difficult and requires extensive screening of milk components that may function to improve well-being and options for prevention and treatment of disease. New animal models with unique reproductive strategies are now becoming increasingly relevant to search for these factors.

Innate immunity in the mammary gland and the role of the milk peptidome

 This research investigated how the mammary gland responds to disease states such as mastitis and how the milk plays a protective role. An in vitro mammary model was developed and shown to replicate the same responses to the breast when challenged with bacterial proteins demonstrating the usefulness of this model for future investigation of therapeutic interventions.

Effect of lactoferrin protein on red blood cells and macrophages: mechanism of parasite-host interaction

BACKGROUND: Lactoferrin is a natural multifunctional protein known to have antitumor, antimicrobial, and anti-inflammatory activity. Apart from its antimicrobial effects, lactoferrin is known to boost the immune response by enhancing antioxidants. Lactoferrin exists in various forms depending on its iron saturation. The present study was done to observe the effect of lactoferrin, isolated from bovine and buffalo colostrum, on red blood cells (RBCs) and macrophages (human monocytic cell line-derived macrophages THP1 cells). METHODS: Lactoferrin obtained from both species and in different iron saturation forms were used in the present study, and treatment of host cells were given with different forms of lactoferrin at different concentrations. These treated host cells were used for various studies, including morphometric analysis, viability by MTT assay, survivin gene expression, production of reactive oxygen species, phagocytic properties, invasion assay, and Toll-like receptor-4, Toll-like receptor-9, and MDR1 expression, to investigate the interaction between lactoferrin and host cells and the possible mechanism of action with regard to parasitic infections. RESULTS: The mechanism of interaction between host cells and lactoferrin have shown various aspects of gene expression and cellular activity depending on the degree of iron saturation of lactoferrin. A significant increase (P<0.05) in production of reactive oxygen species, phagocytic activity, and Toll-like receptor expression was observed in host cells incubated with iron-saturated lactoferrin when compared with an untreated control group. However, there was no significant (P>0.05) change in percentage viability in the different groups of host cells treated, and no downregulation of survivin gene expression was found at 48 hours post-incubation. Upregulation of the Toll-like receptor and downregulation of the P-gp gene confirmed the immunomodulatory potential of lactoferrin protein. CONCLUSION: The present study details the interaction between lactoferrin and parasite host cells, ie, RBCs and macrophages, using various cellular processes and expression studies. The study reveals the possible mechanism of action against various intracellular pathogens such as Toxoplasma, Plasmodium, Leishmania, Trypanosoma, and Mycobacterium. The presence of iron in lactoferrin plays an important role in enhancing the various activities taking place inside these cells. This work provides a lot of information about targeting lactoferrin against many parasitic infections which can rule out the exact pathways for inhibition of diseases caused by intracellular microbes mainly targeting RBCs and macrophages for their survival. Therefore, this initial study can serve as a baseline for further evaluation of the mechanism of action of lactoferrin against parasitic diseases, which is not fully understood to date.