Christophe Lefevre

Hunter Medical Research Institute | HMRI

Assessing the role of lactogenic hormones in human mammary gland development is limited due to issues accessing tissue samples and so development of a human in vitro three-dimensional mammosphere model with functions similar to secretory alveoli in the mammary gland can aid to overcome this shortfall. In this study, a mammosphere model has been cha...

Lactation is a defining feature of mammals. The production of milk by the mammary gland during lactation is generally regarded as a mechanism required to supply nutrition to the neonate before they are able to digest other types of food. Increasing evidence suggests that components of milk have additional functions in addition to nutrition. Monotre...

Milk is a complex secretion that has an important role in mammalian reproduction. It is only recently that sequencing technologies have allowed the identification and quantification of microRNA (miRNA) in milk of a growing number of mammalian species. This provides a novel window on the study of the evolution and functionality of milk through the c...

Significantly preterm and low-birthweight (LBW) babies have diminished lung and gut development, generally fail to thrive, have increased mortality and higher frequency of mature-onset disease. Mothers often cannot breastfeed, and babies receive either formula or pasteurized donor milk, which may further limit the baby's recovery. New approaches ar...

Background: After a short gestation, marsupials give birth to immature neonates with lungs that are not fully developed and in early life the neonate partially relies on gas exchange through the skin. Therefore, significant lung development occurs after birth in marsupials in contrast to eutherian mammals such as humans and mice where lung develop...

The contribution of cows’ milk containing beta-casein protein A1 variant to the development of type 1 diabetes (T1D) has been controversial for decades. Despite epidemiological data demonstrating a relationship between A1 beta-casein consumption and T1D incidence, direct evidence is limited. We demonstrate that early life exposure to A1 beta-casein...

Extracellular matrix (ECM) plays an important role in the normal physiology of tissues and progression to disease. Earlier studies and our external microarray data analysis indicated that mammary matrix from involuting tissue showed upregulation of genes involved in ECM remodeling. The present study examines the fate of mammary and oral cancer cell...

Background The unique lactation strategy of the tammar wallaby (Macropus eugeni) has been invaluable in evaluating the role of lactogenic hormones and the extracellular matrix (ECM) in the local control of mammary gland function. However molecular pathways through which hormones and ECM exert their effect on wallaby mammary gland function remain un...

[This corrects the article DOI: 10.1371/journal.pmed.1002204.].

α-lactalbumin is a protein of dual function found in milk of most mammals. α-lactalbumin binds β-1,4-galactosyltransferase to form the regulatory subunit for lactose synthesis and has also been shown to cause cell death. This study shows, for the first time, that α-lactalbumin isolated in a rare 28kDa dimeric form induces cell death, while 14kDa mo...

Lactation has evolved from an ancient reproductive strategy which appears to have been present long before the evolution of extantmammals. The ability to lactate is a feature only found among mammals and involves a facet of maternal care where mothers secretea nutrient-rich milk which is delivered to the young by the mammary gland. Evolutionary stu...

Background Understanding the cancer genome is seen as a key step in improving outcomes for cancer patients. Genomic assays are emerging as a possible avenue to personalised medicine in breast cancer. However, evolution of the cancer genome during the natural history of breast cancer is largely unknown, as is the profile of disease at death. We soug...

Immunohistochemistry for the protein product of the CDH4 gene, which contains a truncal mutation in TN1. Both primary tumour and liver metastases show evidence of expression. (TIF)

Description of samples. (XLSX)

Validation rates. (XLSX)

Quality control metrics for sequencing. (XLSX)

STROBE Checklist. (DOC)

Primer sequences used for mRNA quantification by RT-PCR.

Table detailing the list of proteins significantly identified from LC-MS/MS analysis and the list of proteins reported in pie chart.

Table detailing the list of proteins analysed through functional categorisation.

Our research is exploiting the marsupial as a model to understand the signals required for lung development. Marsupials have a unique reproductive strategy, the mother gives birth to altricial neonate with an immature lung and the changes in milk composition during lactation in marsupials appears to provide bioactives that can regulate diverse aspe...

It is now clear that milk has multiple functions; it provides the most appropriate nutrition for growth of the newborn, it delivers a range of bioactives with the potential to stimulate development of the young, it has the capacity to remodel the mammary gland (stimulate growth or signal cell death) and finally milk can provide protection from infe...

The molecular processes underlying human milk production and the effects of mastitic infection are largely unknown because of limitations in obtaining tissue samples. Determination of gene expression in normal lactating women would be a significant step toward understanding why some women display poor lactation outcomes. Here, we demonstrate the ut...

The recent upsurge in microbial genome data has revealed that hemoglobin-like (HbL) proteins may be widely distributed among bacteria and that some organisms may carry more than one HbL encoding gene. However, the discovery of HbL proteins has been limited to a small number of bacteria only. This study describes the prediction of HbL proteins and t...

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...

Endocrine regulation of milk protein gene expression in marsupials and eutherians is well studied. However, the evolution of this complex regulation that began with monotremes is unknown. Monotremes represent the oldest lineage of extant mammals and the endocrine regulation of lactation in these mammals has not been investigated. Here we characteri...

Supporting Information.

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...

MicroRNA have been recently discovered in human milk signifying potentially important functions for both the lactating breast and the infant. Whilst human milk microRNA have started to be explored, little data exist on the evaluation of sample processing and analysis to ensure that a full spectrum of microRNA can be obtained. Human milk comprises t...

Cloud and service computing has started to change the way research in science, in particular biology and medicine, is being carried out. Researchers that have taken advantage of this technology (making use of public and private cloud compute resources) can process large amounts of data (big data) and speed up discovery. However, this requires resea...

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...

The rapid advances in technology for both evaluating and understanding the structure of animal genomes and their functional significance have presented opportunities for scientists to more clearly understand the complexity of bovine milk proteins and the control of their expression. Used in conjunction with protein data, we can begin to understand...

Milk has many functions, ranging from the provision of factors crucial to the operation of the mammary gland and the development of the suckled young through to their protection from infection. Lactation evolved about 200 million years ago with the aplacental, egg-laying monotremes, but since that time there has been extensive adaptation to reprodu...

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 chang...

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-...

Recent studies using the mouse showed an inverse correlation between the Caveolin-1 gene expression and lactation, and this was regulated by prolactin. However, current study using mammary explants from pregnant mice showed that while insulin(I), cortisol(F) and prolactin(P) resulted in maximum induction of the β-casein gene, FP and IFP resulted in...

The physicochemical and rheological properties of yoghurt made from unstandardised unhomogenised buffalo milk were investigated during fermentation and 28 days of storage and compared to the properties of yoghurt made from homogenised fortified bovine milk. A number of differences observed in the gel network can be linked to differences in milk com...

Human milk contains both free and encapsulated RNA and microRNA in its cell, lipid and skim milk fractions. Recently, the microRNA content of human milk has attracted much attention due to its high variability and the presence of microRNAs with known immunomodulatory functions. However, optimization of the extraction of RNA and microRNA from differ...

Oral squamous cell carcinoma (OSCC) is a disease with high incidence and mortality in India. There is no clinically proven biomarker for oral cancer and the 5-year survival rate has not improved for the past few years. Better understanding of deregulated process, pathways, and genes involved in the advanced stage tumor (stage III and IV) and furthe...

Plasminogen (Pg), the precursor of the proteolytic and fibrinolytic enzyme of blood, is converted to the active enzyme plasmin (Pm) by different plasminogen activators (tissue plasminogen activators and urokinase), including the bacterial activators streptokinase and staphylokinase, which activate Pg to Pm and thus are used clinically for thromboly...

A method has been developed for predicting blood proteins using the SVM based machine learning approach. In this prediction method a two-step strategy was deployed to predict blood proteins and their subclasses. We have developed models of blood proteins and achieved the maximum accuracies of 90.57% and 91.39% with Matthews correlation coefficient...

Interleukins 2 and 15 (IL-2 and IL-15) are highly differentiated but related cytokines with overlapping, yet also distinct functions, and established benefits for medical drug use. The present study identified a gene for an ancient third IL-2/15 family member in reptiles and mammals, interleukin 15-like (IL-15L), which hitherto was only reported in...

The bone marrow microenvironment maintains a stable balance between self-renewal and differentiation of hematopoietic stem/progenitor cells (HSPCs). This microenvironment, also termed the "hematopoietic niche", is primarily composed of stromal cells and their extracellular matrices (ECM) that jointly regulate HSPC functions. Previously, we have dem...

Breastmilk is a complex secretion of the breast that is the main source of nutrition, protection and developmental programming for the infant. In addition to its biochemical components, breastmilk contains maternal cells and their genetic constituents, including microRNAs. These have been recently discovered in breastmilk, generating numerous quest...

Monotremes are the only oviparous mammals and exhibit a fascinating combination of reptilian and mammalian characters. They represent a component of synapsidal reproduction by laying shelled eggs which are incubated outside the mother's body. This is accompanied by a prototherian lactation process, marking them as representatives of early mammals....

S100 proteins are calcium-binding proteins involved in controlling diverse intracellular and extracellular processes such as cell growth, differentiation, and antimicrobial function. We recently identified a S100-like cDNA from the tammar wallaby (Macropus eugenii) stomach. Phylogentic analysis shows wallaby S100A19 forms a new clade with other mar...

Asynchronous concurrent lactation (ACL) is an extreme lactation strategy in macropod marsupials including the tammar wallaby, that may hold the key to understanding local control of mammary epithelial cell function. Marsupials have a short gestation and a long lactation consisting of three phases; P2A, P2B and P3, representing early, mid and late l...

Fur seals present an unusual lactation, alternating between short periods of feeding the pup on-shore with copious milk production and lengthy foraging trips off-shore away from the pup, where milk supply is suspended. Surprisingly, during foraging, the mammary gland does not progress to involution, which usually rapidly occurs in other mammals at...

The system biology approach has been applied to gene expression data to identify molecular mechanisms of isolated biological systems. Adapting this methodology to incorporate connections between different biological systems and extensive repositories of data can help find novel gene interactions. The devised distributed workflow uses normalized mic...

We have used high throughput sequencing technology to investigate the nucleic acid content of buffalo milk. Buffalo milk contains cells from which messenger RNA can be isolated for transcriptome sequencing and, relatively large amount of extracellular small RNAs including significant amounts of micro-RNA (miRNA). The transcriptome of cells isolated...

##Assembly-Data-START## Sequencing Technology :: Sanger dideoxy sequencing ##Assembly-Data-END##

Micro-RNAs (miRNAs) are small RNA molecules known to participate in important regulatory mechanisms through the targeting of mRNAs by sequence specific interactions, leading to specific inhibition of gene expression. Ongoing studies have revealed the role of miRNAs in the regulation of mammary gland development but a role in lactation is not yet co...

MicroRNAs (miRNAs) are small non-coding RNAs that play a role in post-transcriptional regulation of gene expression in most eukaryotes. They help in fine-tuning gene expression by targeting messenger RNAs (mRNA). The interactions of miRNAs and mRNAs are sequence specific and computational tools have been developed to predict miRNA target sites on m...

The marsupial early lactation protein (ELP) gene is expressed in the mammary gland and the protein is secreted into milk during early lactation (Phase 2A). Mature ELP shares approximately 55.4% similarity with the colostrum-specific bovine colostrum trypsin inhibitor (CTI) protein. Although ELP and CTI both have a single bovine pancreatic trypsin i...

Figure S1 Alignment of the marsupial ELP and eutherian CTI transcripts. Nucleotide sequences of the tammar [GenBank: JN191338], fat-tailed dunnart (FT) [GenBank: JN191339], opossum [GenBank: JN191340], cow (Holstein-Friesian breed) [GenBank: JN191341], dog (Labrador breed) [GenBank: JN191342] and brushtail possum, plus the transcripts predicted fro...

Table S3 Percentage similarity between and within the marsupial ELP and eutherian CTI peptides. Pairwise similarities were determined using MatGAT2.01 software [112]. A. ELP/CTI signal peptide, B. ELP/CTI mature peptide, C. ELP/CTI N-terminus, D. ELP/CTI Kunitz domain motif 2 (51 amino acids), E. ELP/CTI Kunitz domain motif 1 (19 amino acids) and F...

Figure S4 Alignment of the bovine CTI, PTI, STI, TKDP1-5 and SPINT4 precursor proteins. ClustalW2 alignment of the bovine CTI [GenBank: JN191341], PTI [GenBank: P00974], STI [GenBank: NP_991355], TKDP1 [GenBank: NP_991345], TKDP2 [GenBank: AF241777], TKDP3 [GenBank: DAA23071], TKDP4 [GenBank: AAF61250], TKDP5 [GenBank: XP_614808] and SPINT4 [GenBan...

Figure S7 Transposable elements located within the bovine CTIPTISTI and TKDP1-5 genes. Conserved transposable elements within the CTIPTISTITKDP1TKDP2TKDP3TKDP4 and TKDP5 genes (translation start to polyadenylation site, inclusive) were identified using CENSOR [66,108]. The TKDP N-domain-encoding exons located between exon 1 (signal- and pro-peptide...

Table S5 Tammar wallaby mammary gland cDNA microarray data presented in Figure 7. Global normalised Cy3/Cy5 gene expression data for (i) ELP, (ii) LGB and (iii) GAPDH - throughout the lactation cycle derived from custom-made tammar mammary gland cDNA microarrays. Stages of the reproductive cycle investigated included the virgin female mammary gland...

Table S1. Characterisation of the putative functional ELP/CTI gene, transcript and protein.

Table S2 Percentage similarity between and within the marsupial ELP and eutherian CTI transcripts. Pairwise similarities were determined using MatGAT2.01 software [112] based upon alignment of sequence pairs using the BLOSUM62 matrix. A.ELP/CTI transcripts (translation start, ATG, to the polyadenylation signal, AATAAA inclusive), B.ELP/CTI transcri...

Figure S2 Exon 1 and 2 mutations within selected putative eutherian CTI pseudogenes. A (i). ClustalW2 alignment of CTI exon 1 of the sloth, elephant, human and horse compared to dog exon 1 revealed different putative mutations and deletions. For sloth and horse CTI there was a point mutation within the putative translation start site (nt 1–3, methi...

Table S4 Location, identity and orientation of transposable elements within the bovine CTIPTISTI and TKDP1-5 genes. CENSOR [66,108] output tables showing the predicted identity, location and orientation of retroelement fragments within the bovine CTIPTISTITKDP1TKDP2TKDP3TKDP4 and TKDP5 genes.

Figure S3Transposable elements and simple repeats located within the PIGT and ELP/CTI genes and flanking regions. Conserved transposable elements in the region containing the PIGT and ELP/CTI genes of the opossum, tammar, dog, horse, human, elephant and cow were identified using CENSOR [66,108]. The horizontal axis indicates the relative sizes of t...

Figure S5 Relationship between bovine CTI, PTI, STI, TKDP1-5 and SPINT4. The evolutionary history of the protein-coding regions of the bovine CTI, PTI, STI, SPINT4 and TKDP1-5 transcripts was determined by maximum likelihood analysis based upon a molecular clock assumption using PHYLIP. Bovine SLPI was used as an outgroup (data not shown). Numbers...

Figure S6 Genomic arrangement and mVISTA plot of pairwise alignments for the bovine CTI, PTI, STI and TKDP1-5 genes. A. Arrangement and orientation of the bovine chromosome 13 CTI, PTI, STI, TKDP1 TKDP2, TKDP3, TKDP4 and TKDP5 genes. B. (i-viii) Homology between the CTI PTI, STI and TKDP1-5 genes as determined by mVISTA pairwise sequence alignment....

Microarrays and more recently RNA sequencing has led to an increase in available gene expression data. How to manage and store this data is becoming a key issue. In response we have developed EXP-PAC, a web based software package for storage, management and analysis of gene expression and sequence data. Unique to this package is SQL based querying...

Expressed Sequence Tags (ESTs) are short DNA sequences generated by sequencing the transcribed cDNAs coming from a gene expression. They can provide significant functional, structural and evolutionary information and thus are a primary resource for gene discovery. EST annotation basically refers to the analysis of unknown ESTs that can be performed...

WAP four disulfide core domain 2 (WFDC2) is a four disulfide core (4-DSC) protein secreted in the milk of the tammar wallaby. It is comprised of two 4-DSC domains assigned domain III at the NH2-terminal end and domain II at the COOH-terminal end. The WFDC2 gene was expressed only during pregnancy, early lactation, towards the end of lactation and i...

Lactation is a defining feature of mammals. The production of milk by the mammary gland during lactation is generally regarded as a mechanism required to supply nutrition to the neonates before they are able to digest other types of food. Increasing evidence suggests that components of milk have other functions in addition to nutrition. Monotremes,...

We present the genome sequence of the tammar wallaby, Macropus eugenii, which is a member of the kangaroo family and the first representative of the iconic hopping mammals that symbolize Australia to be sequenced. The tammar has many unusual biological characteristics, including the longest period of embryonic diapause of any mammal, extremely sync...

Background We present the genome sequence of the tammar wallaby, Macropus eugenii, which is a member of the kangaroo family and the first representative of the iconic hopping mammals that symbolize Australia to be sequenced. The tammar has many unusual biological characteristics, including the longest period of embryonic diapause of any mammal, ext...

The growth rate of cultured mammalian cells can be influenced by chemical and physical methods such as electromagnetic fields (EMF), light, temperature and plasma. These physical methods have a number of well documented effects on mammalian cells including modification of gene expression, cell cycle, invasion, motility, cell viability, proliferatio...

Supplementary material. Supplementary materials and methods, results and tables [39,42,46,47,58,74,164-192].

Figure S1 - comparison of gene sizes in Monodelphis domestica and Macropus eugenii. One-to-one opossum orthologues of tammar genes located more than 1 kb from the end of a scaffold were downloaded from Ensembl v62. The genomic lengths of the genes are plotted as a scatter plot on the log2 scale. A 1:1 linear relationship between gene sizes is prese...

Figure S2 - analysis of the alignment of transcriptomic reads from different tissues to the tammar genome. (a) Proportion of reads that align to unannotated regions, annotated genes, within 2 kb upstream or downstream of a gene, or fail to align to the tammar genome. (b) Proportion of mapped reads that align to unannotated regions, annotated genes,...

Figure S3 - Comparative analysis of the mammalian casein locus showing the expansion of the casein locus in mammals. Comparison of the casein locus organization in the platypus, tammar, opossum, cattle, mouse and human genomes. Drawn to scale and aligned on the β-casein gene. Genes are represented by a box with a tail arrow pointing in the directio...

We present the genome sequence of the tammar wallaby, Macropus eugenii, which is a member of the kangaroo family and the first representative of the iconic hopping mammals that symbolize Australia to be sequenced. The tammar has many unusual biological characteristics, including the longest period of embryonic diapause of any mammal, extremely sync...

Cathelicidins secreted in milk may be central to autocrine feedback in the mammary gland for optimal development in addition to conferring innate immunity to both the mammary gland and the neonate. This study exploits the unique reproductive strategy of the tammar wallaby (Macropus eugenii) model to analyse differential splicing of cathelicidin gen...

Gene Expression Comparative Analysis allows bio-informatics researchers to discover the conserved or specific functional regulation of genes. This is achieved through comparisons between quantitative gene expression measurements obtained in different species on different platforms to address a particular biological system. Comparisons are made more...

Marsupials provide a suitable alternative model to studying mammary gland involution. They have evolved a different reproductive strategy from eutherians, giving birth to an altricial young and secreting milk that changes in composition during lactation. In this study, we used a marsupial-specific EST microarray to identify 47 up-regulated genes du...

We present the genome sequence of the tammar wallaby, Macropus eugenii, which is a member of the kangaroo family and the first representative of the iconic hopping mammals that symbolize Australia to be sequenced. The tammar has many unusual biological characteristics, including the longest period of embryonic diapause of any mammal, extremely sync...