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SAF-A is conserved throughout vertebrates and has emerged as an important factor regulating a multitude of nuclear functions, including lncRNA localization, gene expression, and splicing. SAF-A has several functional domains, including an N-terminal SAP domain that binds directly to DNA. Phosphorylation of SAP domain serines S14 and S26 are important for SAF-A localization and function during mitosis, however whether these serines are involved in interphase functions of SAF-A is not known. In this study we tested for the role of the SAP domain, and SAP domain serines S14 and S26 in X chromosome inactivation, protein dynamics, gene expression, splicing, and cell proliferation. Here we show that the SAP domain serines S14 and S26 are required to maintain XIST RNA localization and polycomb-dependent histone modifications on the inactive X chromosome in female cells. In addition, we present evidence that an Xi localization signal resides in the SAP domain. We found that that the SAP domain is not required to maintain gene expression and plays only a minor role in mRNA splicing. In contrast, the SAF-A SAP domain, in particular serines S14 and S26, are required for normal protein dynamics, and to maintain normal cell proliferation. We propose a model whereby dynamic phosphorylation of SAF-A serines S14 and S26 mediates rapid turnover of SAF-A interactions with DNA during interphase.

Mutations in BRAF are present in 4% of non-small cell lung cancer (NSCLC), of which half are well-characterized activating variants affecting codon 600 (classified as class I). These mutations, most commonly BRAF V600E, have been associated with response to BRAF/MEK-directed small molecule kinase inhibitors. NSCLC with kinase-activating BRAF mutations occurring at other codons (class II variants) represent a substantial portion of BRAF-mutated NSCLC, but use of targeted therapy in these tumors is still under investigation. Class II mutations have been described in other tumor types and have been associated with response to BRAF/MEK-targeted agents, although optimal treatment strategies for these patients are lacking. This report presents a case of a woman with metastatic NSCLC harboring a class II BRAF p.N486_P490del variant who had a sustained clinical response to combination therapy with dabrafenib and trametinib. This first report of the use of BRAF/MEK-targeted therapy for this variant in NSCLC supports consideration of such treatment for tumors with class II BRAF variants.

Staphylococcus aureus is a significant human pathogen with a formidable propensity for antibiotic resistance. Worldwide, it is the leading cause of skin and soft tissue infections (SSTI), septic arthritis, osteomyelitis, and infective endocarditis originating from both community- and healthcare-associated settings. Although often grouped by methicillin resistance, both methicillin-resistant (MRSA) and methicillin-sensitive (MSSA) strains are known to cause significant pathologies and injuries. Virulence factors and growing resistance to antibiotics play major roles in the pathogenicity of community-associated strains. In our study, we examined the genetic variability and acquired antibiograms of 122 S. aureus clinical isolates from SSTI, blood, and urinary tract infections originating from pediatric patients within the southeast region of Virginia, USA. We identified a suite of clinically relevant virulence factors and evaluated their prevalence within these isolates. Five genes ( clfA , spA , sbi , scpA , and vwb ) with immune-evasive functions were identified in all isolates. MRSA isolates had a greater propensity to be resistant to more antibiotics as well as significantly more likely to carry several virulence factors compared to MSSA strains. Further, the carriage of various genes was found to vary significantly based on the infection type (SSTI, blood, urine).

Mitosis results in a dramatic reorganization of chromatin structure to promote chromosome compaction and segregation to daughter cells. Consequently, mitotic entry is accompanied by transcriptional silencing and removal of most chromatin-bound RNA from chromosomes. As cells exit mitosis, chromatin rapidly decondenses and transcription restarts as waves of differential gene expression. However, little is known about the fate of chromatin-bound RNAs following cell division. Here we explored whether nuclear RNA from the previous cell cycle is present in G1 nuclei following mitosis. We found that half of all nuclear RNA is inherited in a transcription-independent manner following mitosis. Interestingly, the snRNA U2 is efficiently inherited by G1 nuclei, while the lncRNAs NEAT1 and MALAT1 show no inheritance following mitosis. We found that the nuclear protein SAF-A, which is hypothesized to tether RNA to DNA, did not play a prominent role in nuclear RNA inheritance, indicating that the mechanism for RNA inheritance may not involve RNA chaperones that have chromatin-binding activity. Instead, we observe that the timing of RNA inheritance indicates that a select group of nuclear RNAs are reimported into the nucleus after the nuclear envelope has reassembled. Our work demonstrates that there is a fraction of nuclear RNA from the previous cell cycle that is reimported following mitosis and suggests that mitosis may serve as a time to reset the interaction of lncRNAs with chromatin.

In this work, we: (i) review likelihood-based inference for parameter estimation and the construction of confidence regions; and (ii) explore the use of techniques from information geometry, including geodesic curves and Riemann scalar curvature, to supplement typical techniques for uncertainty quantification, such as Bayesian methods, profile likelihood, asymptotic analysis and bootstrapping. These techniques from information geometry provide data-independent insights into uncertainty and identifiability, and can be used to inform data collection decisions. All code used in this work to implement the inference and information geometry techniques is available on GitHub.

Staphylococcus aureus employs a multitude of immune-evasive tactics to circumvent host defenses including the complement system, a component of innate immunity central to controlling bacterial infections. With antibiotic resistance becoming increasingly common, there is a dire need for novel therapies. Previously, we have shown that S. aureus binds the complement regulator factor H (FH) via surface protein SdrE to inhibit complement. To address the need for novel therapeutics and take advantage of the FH:SdrE interaction, we examined the effect of a fusion protein comprised of the SdrE-interacting domain of FH coupled with IgG Fc on complement-mediated opsonophagocytosis and bacterial killing of community associated methicillin-resistant S. aureus. S. aureus bound significantly more FH-Fc compared to Fc-control proteins and FH-Fc competed with serum FH for S. aureus binding. FH-Fc treatment increased C3-fragment opsonization of S. aureus for both C3b and iC3b, and boosted generation of the anaphylatoxin C5a. In 5 and 10% serum, FH-Fc treatment significantly increased S. aureus killing by polymorphonuclear cells. This anti-staphylococcal effect was evident in 75% (3/4) of clinical isolates tested. This study demonstrates that FH-Fc fusion proteins have the potential to mitigate the protective effects of bound serum FH rendering S. aureus more vulnerable to the host immune system. Thus, we report the promise of virulence-factor-targeted fusion-proteins as an avenue for prospective anti-staphylococcal therapeutic development., Competing Interests: The authors have declared that no competing interests exist.

Tumour spheroids are common in vitro experimental models of avascular tumour growth. Compared with traditional two-dimensional culture, tumour spheroids more closely mimic the avascular tumour microenvironment where spatial differences in nutrient availability strongly influence growth. We show that spheroids initiated using significantly different numbers of cells grow to similar limiting sizes, suggesting that avascular tumours have a limiting structure; in agreement with untested predictions of classical mathematical models of tumour spheroids. We develop a novel mathematical and statistical framework to study the structure of tumour spheroids seeded from cells transduced with fluorescent cell cycle indicators, enabling us to discriminate between arrested and cycling cells and identify an arrested region. Our analysis shows that transient spheroid structure is independent of initial spheroid size, and the limiting structure can be independent of seeding density. Standard experimental protocols compare spheroid size as a function of time; however, our analysis suggests that comparing spheroid structure as a function of overall size produces results that are relatively insensitive to variability in spheroid size. Our experimental observations are made using two melanoma cell lines, but our modelling framework applies across a wide range of spheroid culture conditions and cell lines., Competing Interests: AB, JS, RM, GG, BL, KB, NH, MS No competing interests declared, (© 2021, Browning et al.)

Optimal control theory provides insight into complex resource allocation decisions. The forward-backward sweep method (FBSM) is an iterative technique commonly implemented to solve two-point boundary value problems arising from the application of Pontryagin's maximum principle (PMP) in optimal control. The FBSM is popular in systems biology as it scales well with system size and is straightforward to implement. In this review, we discuss the PMP approach to optimal control and the implementation of the FBSM. By conceptualizing the FBSM as a fixed point iteration process, we leverage and adapt existing acceleration techniques to improve its rate of convergence. We show that convergence improvement is attainable without prohibitively costly tuning of the acceleration techniques. Furthermore, we demonstrate that these methods can induce convergence where the underlying FBSM fails to converge. All code used in this work to implement the FBSM and acceleration techniques is available on GitHub at https://github.com/Jesse-Sharp/Sharp2021.

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 characterised using human mammary epithelial cells grown on either mouse extracellular matrix or agarose and showed insulin is essential for formation of mammospheres. Insulin was shown to up-regulate extracellular matrix genes. Microarray analysis of these mammospheres revealed an up-regulation of differentiation, cell-cell junctions, and cytoskeleton organisation functions, suggesting mammosphere formation may be regulated through ILK signalling. Comparison of insulin and IGF-1 effects on mammosphere signalling showed that although IGF-1 could induce spherical structures, the cells did not polarise correctly as shown by the absence of up-regulation of polarisation genes and did not induce the expression of milk protein genes. This study demonstrated a major role for insulin in mammary acinar development for secretory differentiation and function indicating the potential for reduced lactational efficiency in women with obesity and gestational diabetes.

A series of Zn-Al-Li alloys with potential application in bioresorbable implants were cast, thermomechanically processed and tested. The formation of secondary phases, such as LiZn 4 , LiZn 3 Al and Al 3 Li, contributed to both dynamic recrystallization and grain refinement of the matrix (η-phase) during the hot-extrusion process, leading to grain sizes as small as 1.75 μm for Zn-4Al-0.6Li alloy (wt%). This alloy exhibited an ultimate tensile strength (UTS) of 451 MPa, a total elongation of 46% and a corrosion rate of 60 μm/year in simulated body fluid. The grain refinement played a major role in increasing the strength, but it also weakened the basal texture and promoted non-basal slip and grain boundary sliding, thus contributing to the increased plastic deformation of the alloy. The corrosion rate was affected by a layer of zinc oxide and phosphate formed in the early stages of the immersion tests. The corrosion products protected the substrate and tended to reduce the corrosion rate over time. The developed Zn-4Al-0.6Li and Zn-6Al-0.4Li alloys which showed promising mechanical and corrosion properties appeared to be cytocompatible in the mouse fibroblast cell line and human umbilical mesenchymal stem cells making them promising candidates for bioresorbable stent and implant applications., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Bacteria invest in a slow-growing subpopulation, called persisters, to ensure survival in the face of uncertainty. This hedging strategy is remarkably similar to financial hedging, where diversifying an investment portfolio protects against economic uncertainty. We provide a new, to our knowledge, theoretical foundation for understanding cellular hedging by unifying the study of biological population dynamics and the mathematics of financial risk management through optimal control theory. Motivated by the widely accepted role of volatility in the emergence of persistence, we consider several models of environmental volatility described by continuous-time stochastic processes. This allows us to study an emergent cellular hedging strategy that maximizes the expected per capita growth rate of the population. Analytical and simulation results probe the optimal persister strategy, revealing results that are consistent with experimental observations and suggest new opportunities for experimental investigation and design. Overall, we provide a new, to our knowledge, way of conceptualizing and modeling cellular decision making in volatile environments by explicitly unifying theory from mathematical biology and finance., (Copyright © 2020 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

During mitosis, the genome is transformed from a decondensed, transcriptionally active state to a highly condensed, transcriptionally inactive state. Mitotic chromosome reorganization is marked by the general attenuation of transcription on chromosome arms, yet how the cell regulates nuclear and chromatin-associated RNAs after chromosome condensation and nuclear envelope breakdown is unknown. SAF-A/hnRNPU is an abundant nuclear protein with RNA-to-DNA tethering activity, coordinated by two spatially distinct nucleic acid-binding domains. Here we show that RNA is evicted from prophase chromosomes through Aurora-B-dependent phosphorylation of the SAF-A DNA-binding domain; failure to execute this pathway leads to accumulation of SAF-A-RNA complexes on mitotic chromosomes, defects in metaphase chromosome alignment, and elevated rates of chromosome missegregation in anaphase. This work reveals a role for Aurora-B in removing chromatin-associated RNAs during prophase and demonstrates that Aurora-B-dependent relocalization of SAF-A during cell division contributes to the fidelity of chromosome segregation., (© 2020 Sharp et al.)

Chronic kidney disease (CKD) with type 2 diabetes (T2D) is a major public health problem, resulting in significant cardiovascular and kidney adverse outcomes worldwide. Despite the widespread use of standard-of-care therapies for CKD with T2D over the past few decades, rates of progression to end-stage kidney disease remain high with no beneficial impact on its accompanying burden of cardiovascular disease. The advent of the newer classes of antihyperglycemic agents, including SGLT2 (sodium glucose cotransporter 2) inhibitors and GLP-1 (glucagon-like peptide-1) receptor agonists, has changed the landscape of therapeutic options for patients with CKD with T2D, with demonstration of significant reductions in cardiovascular adverse events and progression to end-stage kidney disease. Several potential mechanisms exist through which these beneficial effects are achieved in both drug classes, which may be independent of their antihyperglycemic effects. This scientific statement summarizes the current literature on the cardiorenal protective effects with SGLT2 inhibitors and GLP-1 receptor agonists in patients with CKD and T2D. It reviews potential mechanistic pathways that may drive these benefits and summarizes the literature on adverse effects in patients with CKD and T2D at risk for or with established cardiovascular disease. Last, it provides practical guidance on a proposed collaborative care model bridging cardiologists, nephrologists, endocrinologists, and primary care physicians to facilitate the prompt and appropriate integration of these therapeutic classes in the management of patients with T2D and CKD.

Strategic management of populations of interacting biological species routinely requires interventions combining multiple treatments or therapies. This is important in key research areas such as ecology, epidemiology, wound healing and oncology. Despite the well developed theory and techniques for determining single optimal controls, there is limited practical guidance supporting implementation of combination therapies. In this work we use optimal control theory to calculate optimal strategies for applying combination therapies to a model of acute myeloid leukaemia. We present a versatile framework to systematically explore the trade-offs that arise in designing combination therapy protocols using optimal control. We consider various combinations of continuous and bang-bang (discrete) controls, and we investigate how the control dynamics interact and respond to changes in the weighting and form of the pay-off characterising optimality. We demonstrate that the optimal controls respond non-linearly to treatment strength and control parameters, due to the interactions between species. We discuss challenges in appropriately characterising optimality in a multiple control setting and provide practical guidance for applying multiple optimal controls. Code used in this work to implement multiple optimal controls is available on GitHub., (Crown Copyright © 2020. Published by Elsevier Ltd. All rights reserved.)

Stress reactivity and glucocorticoid signaling alterations are reported in mouse models of autism spectrum disorder (ASD). BALB/c mice display decreased locomotor activity in the presence of stimulus mice and spend less time exploring enclosed stimulus mice; this mouse strain has been validated as an ASD model. VU0410120, a glycine type 1 transporter (GlyT1) inhibitor, improved sociability in BALB/c mice, consistent with data that NMDA Receptor (NMDAR) activation regulates sociability, and the endogenous tone of NMDAR-mediated neurotransmission is altered in this strain. Effects of a prosocial dose of VU0410120 on conspecific-provoked immobility, and relationships between conspecific-provoked immobility and corticosterone response were explored. VU0410120-treated BALB/c mice showed reduced immobility in the presence of conspecifics and increased the conspecific-provoked corticosterone response. However, the intensity of conspecific-provoked immobility in VU0410120-treated BALB/c mice did not differ as a function of corticosterone response. Expression profiles of 88 glucocorticoid signaling associated genes within frontal cortex and hippocampus were examined. BALB/c mice resistant to prosocial effects of VU0410120 had increased mRNA expression of Ddit4, a negative regulator of mTOR signaling. Dysregulated mTOR signaling activity is a convergent finding in several monogenic syndromic forms of ASD. Prosocial effects of VU0410120 in the BALB/c strain may be related to regulatory influences of NMDAR-activation on mTOR signaling activity. Because corticosterone response is a marker of social stress, the current data suggest that the stressfulness of a social encounter alone may not be the sole determinant of increased immobility in BALB/c mice; this strain may also display an element of social disinterest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

EchAMP, the tenth most abundant transcript expressed in the mammary gland of echidna, has in vitro broad-spectrum antibacterial effects. However, the effects of EchAMP on mastitis, a condition where inflammation is triggered following mammary gland infection, has not been investigated. To investigate the impact of EchAMP against mastitis, EchAMP transgenic mice were generated. In antibacterial assays, the whey fractions of milk from transgenic mice significantly reduced growth of Staphylococcus aureus, Bacillus subtilis, Escherichia coli and Pseudomonas aeruginosa compared with whey fractions from wildtype mice. Furthermore, a mastitis model created by infecting mammary gland with these four bacterial strains displayed a significant reduction in bacterial load in transgenic mice injected with S. aureus and B. subtilis. On further confirmation, histomorphologic analysis showed absence of necrosis and cell infiltration in the mammary glands of transgenic mice. To understand the role of EchAMP against inflammation, we employed an LPS-injected mastitis mouse model. LPS is known to induce phopshorylation of NF-κB and MAPK pathways, which in turn activate downstream proinflammatory signaling mediators, to promote inflammation. In LPS-treated EchAMP transgenic mice, phosphorylation levels of NF-κB, p38 and ERK1/2 were significantly downregulated. Furthermore, in mammary gland of transgenic mice, there was a significant downregulation of mRNA levels of proinflammatory cytokines, namely TNF-α, IL-6 and IL-1β. Taken together, these data suggest that EchAMP has an antiinflammatory response and is effective against S. aureus and B. subtilis. We suggest that EchAMP may be a potential prophylactic protein against mastitis in dairy animals by expressing this gene in their mammary gland.

Blending synthetic biology and synthetic chemistry represents a powerful approach to diversity complex molecules. To further enable this, compatible synthetic tools are needed. We report the first Buchwald Hartwig amination reactions with unprotected halotryptophans under aqueous conditions and demonstrate this methodology is applicable also to the modification of unprotected tripeptides and the natural product barettin.

Acute myeloid leukaemia (AML) is a blood cancer affecting haematopoietic stem cells. AML is routinely treated with chemotherapy, and so it is of great interest to develop optimal chemotherapy treatment strategies. In this work, we incorporate an immune response into a stem cell model of AML, since we find that previous models lacking an immune response are inappropriate for deriving optimal control strategies. Using optimal control theory, we produce continuous controls and bang-bang controls, corresponding to a range of objectives and parameter choices. Through example calculations, we provide a practical approach to applying optimal control using Pontryagin's Maximum Principle. In particular, we describe and explore factors that have a profound influence on numerical convergence. We find that the convergence behaviour is sensitive to the method of control updating, the nature of the control, and to the relative weighting of terms in the objective function. All codes we use to implement optimal control are made available., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Background: Antibiotic resistance is a problem that necessitates the identification of new antimicrobial molecules. Milk is known to have molecules with antimicrobial properties (AMPs). Echidna Antimicrobial Protein (EchAMP) is one such lactation specific AMP exclusively found in the milk of Echidna, an egg-laying mammal geographically restricted to Australia and New Guinea. Previous studies established that EchAMP exhibits substantial bacteriostatic activity against multiple bacterial genera. However, the subsequent structural and functional studies were hindered due to the unavailability of pure protein., Results: In this study, we expressed EchAMP protein using a heterologous expression system and successfully purified it to >95% homogeneity. The purified recombinant protein exhibits bacteriolytic activity against both Gram-positive and Gram-negative bacteria as confirmed by live-dead staining and scanning electron microscopy. Structurally, this AMP belongs to the family of intrinsically disordered proteins (IDPs) as deciphered by the circular-dichroism, tryptophan fluorescence, and NMR spectroscopy. Nonetheless, EchAMP has the propensity to acquire structure with amphipathic molecules, or membrane mimics like SDS, lipopolysaccharides, and liposomes as again observed through multiple spectroscopic techniques., Conclusions: Recombinant EchAMP exhibits broad-spectrum bacteriolytic activity by compromising the bacterial cell membrane integrity. Hence, we propose that this intrinsically disordered antimicrobial protein interact with the bacterial cell membrane and undergoes conformational changes to form channels in the membrane resulting in cell lysis., General Significance: EchAMP, the evolutionarily conserved, lactation specific AMP from an oviparous mammal may find application as a broad-spectrum antimicrobial against pathogens that affect mammary gland or otherwise cause routine infections in humans and livestock., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Objective Beta-casein is a major protein in breast milk and an important source for several bioactive peptides that are encrypted within the sequence. Beta-casomorphins (BCMs) are short-chain proteolytic peptides that are derived from the beta-casein protein and have opioid effects in newborns. Human milk is known to contain naturally occurring milk-protein-derived bioactive peptides but the identification of naturally occurring beta-casein-derived BCMs in human breast milk has been limited due to difficulties in the detection of BCM peptides, which are small and circulate in low concentrations. Methods The present study aimed to identify the naturally occurring BCM peptides from beta-casein in human breast milk using liquid chromatography-tandem mass spectrometry. The BCM peptides identified in the breast milk were analysed to predict the milk proteases responsible for the cleavage patterns using a computational tool EnzymePredictor. Results In-depth peptidomics analysis of breast milk samples that were collected at different lactation stages during human lactation revealed the presence of BCMs including BCM-8, -9, -10, and -11 as well as precursors and truncated forms of the original peptide, which suggests that milk protease activity in the mammary gland generates biologically relevant BCMs. Conclusions To our knowledge, this is the first report to describe the presence of naturally occurring human BCM-10 and -11 in breast milk. Our study provides evidence of beta-casein-derived BCM peptides in human milk before infant digestion. Proteases that are present in milk are likely specific in their proteolysis of beta-casein. The identified bioactive BCM-8, -9, -10, and -11 as well as the precursor peptides meet the structural requirements to elicit opioid, immunomodulatory, antioxidative, and satiety functions in newborns., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

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 are required to manage the early stages of neonatal development. The tammar wallaby, an Australian marsupial, has a short gestation and a simple placenta, and gives birth to an altricial young equivalent to a final trimester human embryo. The neonate remains in the pouch and attached to the teat for 100 days postpartum. The mother slows growth of the young and progressively changes the composition of the milk to deliver signals for organ development, including the lung and gut. This closely resembles the relationship between the human fetus and delivery of placental and uterine bioactives. Datasets comprised of differentially expressed genes coding for secreted proteins in early lactation in the tammar mammary gland have been compared to databases produced from human placenta, amniotic fluid, colostrum and milk to identify human homologues for the putative signaling molecules for organ development. These data will be used to develop milk fortifiers for treatment of preterm and LBW babies in both the developed and the developing world., (© 2019 Nestlé Nutrition Institute, Switzerland/S. Karger AG, Basel.)

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 development occurs predominantly in the embryo. To explore the mechanisms of marsupial lung development in comparison to eutherians, morphological and gene expression analysis were conducted in the gray short-tailed opossum (Monodelphis domestica)., Results: Postnatal lung development of Monodelphis involves three key stages of development: (i) transition from late canalicular to early saccular stages, (ii) saccular and (iii) alveolar stages, similar to developmental stages overlapping the embryonic and perinatal period in eutherians. Differentially expressed genes were identified and correlated with developmental stages. Functional categories included growth factors, extracellular matrix protein (ECMs), transcriptional factors and signalling pathways related to branching morphogenesis, alveologenesis and vascularisation. Comparison with published data on mice highlighted the conserved importance of extracellular matrix remodelling and signalling pathways such as Wnt, Notch, IGF, TGFβ, retinoic acid and angiopoietin. The comparison also revealed changes in the mammalian gene expression program associated with the initiation of alveologenesis and birth, pointing to subtle differences between the non-functional embryonic lung of the eutherian mouse and the partially functional developing lung of the marsupial Monodelphis neonates. The data also highlighted a subset of contractile proteins specifically expressed in Monodelphis during and after alveologenesis., Conclusion: The results provide insights into marsupial lung development and support the potential of the marsupial model of postnatal development towards better understanding of the evolution of the mammalian bronchioalveolar lung.

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 cells grown in the ECM from lactating mammary gland. Our findings show that non-tumorigenic cells, MCF10A and DOK cells did not proliferate but the tumorigenic and metastatic cells, SCC25 and MDA-MB-231, underwent apoptosis when grown on mammary ECM isolated from lactating mice. In addition, the cytokinesis marker, CEP55, was repressed in the oral and breast cancer cells. In contrast, these cells proliferated normally on mammary ECM isolated from mice undergoing involution. External microarray data analysis of mammary tissue further revealed over expression (~16 fold) of QSOX1 gene, which promotes cellular quiescence, in lactating mammary gland. A recent study has indicated that QSOX1 overexpression in breast cancer cells led to reduced proliferation and tumorigenic properties. This extracellular protein in mammary ECM may be responsible for reduced cellular proliferation. The present study has shown that ECM from lactating mammary gland can regulate signals to oral and breast cancer cells to halt cell division. This preliminary observation provided insights into the potential role of ECM factors present in lactating mammary gland as therapeutic targets to control cancer cell division. This preliminary study is an attempt to understand not only the requirement of ECM remodeling factors essential for the growth and survival of cancer cells but also the factors present in the lactation matrix that simultaneously halts cell division and selectively inhibits the growth of cancer cells., (Creative Commons Attribution License)

Monotreme lactation protein (MLP) is a recently identified protein with antimicrobial activity. It is present in the milk of monotremes and is unique to this lineage. To characterize MLP and to gain insight into the potential role of this protein in the evolution of lactation, the crystal structure of duck-billed platypus (Ornithorhynchus anatinus) MLP was determined at 1.82 Å resolution. This is the first structure to be reported for this novel, mammalian antibacterial protein. MLP was expressed as a FLAG epitope-tagged protein in mammalian cells and crystallized readily, with at least three space groups being observed (P1, C2 and P2 1 ). A 1.82 Å resolution native data set was collected from a crystal in space group P1, with unit-cell parameters a = 51.2, b = 59.7, c = 63.1 Å, α = 80.15, β = 82.98, γ = 89.27°. The structure was solved by SAD phasing using a protein crystal derivatized with mercury in space group C2, with unit-cell parameters a = 92.7, b = 73.2, c = 56.5 Å, β = 90.28°. MLP comprises a monomer of 12 helices and two short β-strands, with much of the N-terminus composed of loop regions. The crystal structure of MLP reveals no three-dimensional similarity to any known structures and reveals a heretofore unseen fold, supporting the idea that monotremes may be a rich source for the identification of novel proteins. It is hypothesized that MLP in monotreme milk has evolved to specifically support the unusual lactation strategy of this lineage and may have played a central role in the evolution of these mammals.

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 monomeric α-lactalbumin is inactive. In contrast to the casein derived and chemically induced α-lactalbumin variants, MAL and HAMLET/BAMLET, the effects of 28kDa α-lactalbumin are calcium independent and, unlike MAL and HAMLET, 28kDa α-lactalbumin dimer causes cell death of primary mammary cells and a variety of immortalised cell lines, which are committed to cell death pathways within 1-4h of exposure. Microarray analysis confirmed that cell death was the result of an apoptotic response. Functional assays determined that the mechanism by which 28kDa α-lactalbumin kills cells involved inhibition of histone deacetylase activity mediated by NF-kB. We also show that 28kDa α-lactalbumin occurs naturally in the milk of cows, goats and sheep, is low in concentration during mid-lactation, but accumulates during milk stasis, consistent with a role in involution., (Copyright © 2017 Elsevier Inc. All rights reserved.)

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 infection and inflammation when the mammary gland is susceptible to these challenges. There is increasing evidence to support studies using an Australian marsupial, the tammar wallaby (Macropus eugenii), as an interesting and unique model to study milk bioactives. Reproduction in the tammar wallaby is characterized by a short gestation, birth of immature young and a long lactation. All the major milk constituents change substantially and progressively during lactation and these changes have been shown to regulate growth and development of the tammar pouch young and to have roles in mammary gland biology. This review will focus on recent reports examining the control of lactation in the tammar wallaby and the timed delivery of milk bioactivity., (Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.)

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 characterised the proximal promoter and hormonal regulation of two platypus milk protein genes, Beta-lactoglobulin (BLG), a whey protein and monotreme lactation protein (MLP), a monotreme specific milk protein, using in vitro reporter assays and a bovine mammary epithelial cell line (BME-UV1). Insulin and dexamethasone alone provided partial induction of MLP, while the combination of insulin, dexamethasone and prolactin was required for maximal induction. Partial induction of BLG was achieved by insulin, dexamethasone and prolactin alone, with maximal induction using all three hormones. Platypus MLP and BLG core promoter regions comprised transcription factor binding sites (e.g. STAT5, NF-1 and C/EBPα) that were conserved in marsupial and eutherian lineages that regulate caseins and whey protein gene expression. Our analysis suggests that insulin, dexamethasone and/or prolactin alone can regulate the platypus MLP and BLG gene expression, unlike those of therian lineage. The induction of platypus milk protein genes by lactogenic hormones suggests they originated before the divergence of marsupial and eutherians., (Copyright © 2015 Elsevier Inc. All rights reserved.)

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 aspects of lung development, including branching morphogenesis, cell proliferation and cell differentiation. These effects are seen with milk collected between 25 and 100days postpartum. To better understand the temporal effects of milk composition on postnatal lung development we used a cross-fostering technique to restrict the tammar pouch young to milk composition not extending beyond day 25 for 45days of its early postnatal life. These particular time points were selected as our previous study showed that milk protein collected prior to ~day 25 had no developmental effect on mouse embryonic lungs in culture. The comparative analysis of the foster group and control young at day 45 postpartum demonstrated that foster pouch young had significantly reduced lung size. The lungs in fostered young were comprised of large intermediate tissue, had a reduced size of airway lumen and a higher percentage of parenchymal tissue. In addition, expression of marker genes for lung development (BMP4, WNT11, AQP-4, HOPX and SPB) were significantly reduced in lungs from fostered young. Further, to identify the potential bioactive expressed by mammary gland that may have developmental effect on pouch young lungs, we performed proteomics analysis on tammar milk through mass-spectrometry and listed the potential bioactives (PDGF, IGFBP5, IGFBPL1 and EGFL6) secreted in milk that may be involved in regulating pouch young lung development. The data suggest that postnatal lung development in the tammar young is most likely regulated by maternal signalling factors supplied through milk., (Copyright © 2016 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.)

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 utility of RNA obtained directly from human milk cells to detect mammary epithelial cell (MEC)-specific gene expression. Milk cell RNA was collected from five time points (24 h prepartum during the colostrum period, midlactation, two involutions, and during a bout of mastitis) in addition to an involution series comprising three time points. Gene expression profiles were determined by use of human Affymetrix arrays. Milk cells collected during milk production showed that the most highly expressed genes were involved in milk synthesis (e.g., CEL, OLAH, FOLR1, BTN1A1, and ARG2), while milk cells collected during involution showed a significant downregulation of milk synthesis genes and activation of involution associated genes (e.g., STAT3, NF-kB, IRF5, and IRF7). Milk cells collected during mastitic infection revealed regulation of a unique set of genes specific to this disease state, while maintaining regulation of milk synthesis genes. Use of conventional epithelial cell markers was used to determine the population of MECs within each sample. This paper is the first to describe the milk cell transcriptome across the human lactation cycle and during mastitic infection, providing valuable insight into gene expression of the human mammary gland.

Embryonic development involves diffusion and proliferation of cells, as well as diffusion and reaction of molecules, within growing tissues. Mathematical models of these processes often involve reaction-diffusion equations on growing domains that have been primarily studied using approximate numerical solutions. Recently, we have shown how to obtain an exact solution to a single, uncoupled, linear reaction-diffusion equation on a growing domain, 0 < x < L(t), where L(t) is the domain length. The present work is an extension of our previous study, and we illustrate how to solve a system of coupled reaction-diffusion equations on a growing domain. This system of equations can be used to study the spatial and temporal distributions of different generations of cells within a population that diffuses and proliferates within a growing tissue. The exact solution is obtained by applying an uncoupling transformation, and the uncoupled equations are solved separately before applying the inverse uncoupling transformation to give the coupled solution. We present several example calculations to illustrate different types of behaviour. The first example calculation corresponds to a situation where the initially-confined population diffuses sufficiently slowly that it is unable to reach the moving boundary at x = L(t). In contrast, the second example calculation corresponds to a situation where the initially-confined population is able to overcome the domain growth and reach the moving boundary at x = L(t). In its basic format, the uncoupling transformation at first appears to be restricted to deal only with the case where each generation of cells has a distinct proliferation rate. However, we also demonstrate how the uncoupling transformation can be used when each generation has the same proliferation rate by evaluating the exact solutions as an appropriate limit.

The complement system has been increasingly recognized to play a pivotal role in a variety of inflammatory and autoimmune diseases. Consequently, therapeutic modulators of the classical, lectin and alternative pathways of the complement system are currently in pre-clinical and clinical development. Our laboratory has identified a peptide that specifically inhibits the classical and lectin pathways of complement and is referred to as Peptide Inhibitor of Complement C1 (PIC1). In this study, we determined that the lead PIC1 variant demonstrates a salt-dependent binding to C1q, the initiator molecule of the classical pathway. Additionally, this peptide bound to the lectin pathway initiator molecule MBL as well as the ficolins H, M and L, suggesting a common mechanism of PIC1 inhibitory activity occurs via binding to the collagen-like tails of these collectin molecules. We further analyzed the effect of arginine and glutamic acid residue substitution on the complement inhibitory activity of our lead derivative in a hemolytic assay and found that the original sequence demonstrated superior inhibitory activity. To improve upon the solubility of the lead derivative, a pegylated, water soluble variant was developed, structurally characterized and demonstrated to inhibit complement activation in mouse plasma, as well as rat, non-human primate and human serum in vitro. After intravenous injection in rats, the pegylated derivative inhibited complement activation in the blood by 90% after 30 seconds, demonstrating extremely rapid function. Additionally, no adverse toxicological effects were observed in limited testing. Together these results show that PIC1 rapidly inhibits classical complement activation in vitro and in vivo and is functional for a variety of animal species, suggesting its utility in animal models of classical complement-mediated diseases.

We consider the motion of a diffusive population on a growing domain, 0

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.

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.

Background: 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., Results: Results showed that high levels of miRNA secreted in milk and allowed the identification of differentially expressed milk miRNAs during the lactation cycle as putative markers of mammary gland activity and functional candidate signals to assist growth and timed development of the young. Comparative analysis of miRNA distribution in milk and blood serum suggests that milk miRNAs are primarily expressed from mammary gland rather than transferred from maternal circulating blood, likely through a new putative exosomal secretory pathway. In contrast, highly expressed milk miRNAs could be detected at significantly higher levels in neonate blood serum in comparison to adult blood, suggesting milk miRNAs may be absorbed through the gut of the young., Conclusion: The function of miRNA in mammary gland development and secretory activity has been proposed, but results from the current study also support a differential role of milk miRNA in regulation of development in the pouch young, revealing a new potential molecular communication between mother and young during mammalian lactation.

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 downregulation of Caveolin 1. Additionally, IF, FP and IFP resulted in the downregulation of Caveolin 2. Immunohistochemistry confirmed localisation of Caveolin 1 specific to myoepithelial cells and adipocytes. Comparative studies with the tammar wallaby showed Caveolin 1 and 2 had 70-80% homology with the mouse proteins. However, in contrast to the mouse, Caveolin 1 and 2 genes showed a significantly increased level of expression in the mammary gland during lactation. The regulation of tammar Caveolin 1 and 2 gene expression was examined in mammary explants from pregnant tammars, and no significant difference was observed either in the absence or in the presence of IFP., (Crown Copyright © 2014. Published by Elsevier B.V. All rights reserved.)

Background: Prevention of acute hemolytic transfusion reactions is a worldwide concern. The objective of this study was to develop a simple rat model of complement-mediated acute intravascular hemolysis., Study Design and Methods: Human AB red blood cells (RBCs) were incubated with complement-sufficient or complement-deficient Wistar rat serum (WRS) in the presence and absence of human RBC antibody in vitro to elucidate the mechanism of hemolysis. To study the role of complement in acute intravascular hemolysis in vivo, Wistar rats were treated either with or without cobra venom factor (CVF) to deplete complement activity. Human AB RBCs were then injected into both groups of rats, followed by serial blood draws up to 2 hours. Venous blood clearance and lysis of transfused RBCs at each time point were measured by flow cytometry and spectrophotometry. RBC sequestration was determined in the liver, spleen, and kidney by immunohistochemistry., Results: In vitro incubation of human RBCs with WRS demonstrated that RBC lysis was mediated via the classical complement pathway and that hemolysis was antibody dependent. Transfusion of human RBCs into rats showed significantly less hemolysis in the CVF group versus untreated group. RBC sequestration in the spleen and liver 2 hours posttransfusion were not quantitatively different between the two groups., Conclusions: Given the much higher degree of similarity for rat and human complement compared to mice, this simple rat model is ideal for testing novel inhibitors of classical pathway activation for the prevention and treatment of acute intravascular hemolysis., (© 2014 AABB.)

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., (© The Author(s) 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

The classical pathway of complement plays multiple physiological roles including modulating immunological effectors initiated by adaptive immune responses and an essential homeostatic role in the clearance of damaged self-antigens. However, dysregulated classical pathway activation is associated with antibody-initiated, inflammatory diseases processes like cold agglutinin disease, acute intravascular hemolytic transfusion reaction (AIHTR), and acute/hyperacute transplantation rejection. To date, only one putative classical pathway inhibitor, C1 esterase inhibitor (C1-INH), is currently commercially available and its only approved indication is for replacement treatment in hereditary angioedema, which is predominantly a kinin pathway disease. Given the variety of disease conditions in which the classical pathway is implicated, development of therapeutics that specifically inhibits complement initiation represents a major unmet medical need. Our laboratory has identified a peptide that specifically inhibits the classical and lectin pathways of complement. In vitro studies have demonstrated that these peptide inhibitors of complement C1 (PIC1) bind to the collagen-like region of the initiator molecule of the classical pathway, C1q. PIC1 binding to C1q blocks activation of the associated serine proteases (C1s-C1r-C1r-C1s) and subsequent downstream complement activation. Rational design optimization of PIC1 has resulted in the generation of a highly potent derivative of 15 amino acids. PIC1 inhibits classical pathway mediated complement activation in ABO incompatibility in vitro and inhibiting classical pathway activation in vivo in rats. This review will focus on the pre-clinical development of PIC1 and discuss its potential as a therapeutic in antibody-mediated classical pathway disease, specifically AIHTR.

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 marsupial and monotreme S100A19, while this group shows similarity to eutherian S100A7 and S100A15 genes. This is also supported by amino acid and domain comparisons. We show S100A19 is developmentally-regulated in the tammar wallaby gut by demonstrating the gene is expressed in the forestomach of young animals at a time when the diet consists of only milk, but is absent in older animals when the diet is supplemented with herbage. During this transition the forestomach phenotype changes from a gastric stomach into a fermentation sac and intestinal flora changes with diet. We also show that S100A19 is expressed in the mammary gland of the tammar wallaby only during specific stages of lactation; the gene is up-regulated during pregnancy and involution and not expressed during the milk production phase of lactation. Comparison of the tammar wallaby S100A19 protein sequence with S100 protein sequences from eutherian, monotreme and other marsupial species suggest the marsupial S100A19 has two functional EF hand domains, and an extended His tail. An evolutionary analysis of S100 family proteins was carried out to gain a better understanding of the relationship between the S100 family member functions. We propose that S100A19 gene/protein is the ancestor of the eutherian S100A7 gene/protein, which has subsequently modified its original function in eutherians. This modified function may have arisen due to differentiation of evolutionary pressures placed on gut and mammary gland developmental during mammal evolution. The highly regulated differential expression patterns of S100A19 in the tammar wallaby suggests that S100A19 may play a role in gut development, which differs between metatherians and eutherians, and/or include a potential antibacterial role in order to establish the correct flora and protect against spiral bacteria in the immature forestomach. In the mammary gland it may protect the tissue from infection at times of vulnerability during the lactation cycle., (Crown Copyright © 2013. Published by Elsevier Inc. All rights reserved.)

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 lactation respectively and characterised by profound changes in milk composition. A lactating tammar is able to concurrently produce phase 2A and 3 milk from adjacent glands in order to feed a young newborn and an older sibling at heel. Physiological effectors of ACL remain unknown and in this study the extracellular matrix (ECM) is investigated for its role in switching mammary phenotypes between phases of tammar wallaby lactation. Using the level of expression of the genes for the phase specific markers tELP, tWAP, and tLLP-B representing phases 2A, 2B and 3 respectively we show for the first time that tammar wallaby mammary epithelial cells (WallMECs) extracted from P2B acquire P3 phenotype when cultured on P3 ECM. Similarly P2A cells acquire P2B phenotype when cultured on P2B ECM. We further demonstrate that changes in phase phenotype correlate with phase-specific changes in ECM composition. This study shows that progressive changes in ECM composition in individual mammary glands provide a local regulatory mechanism for milk protein gene expression thereby enabling the mammary glands to lactate independently., (Copyright © 2013. Published by Elsevier B.V.)

We have previously shown that the gene for MaeuCath1, a cathelicidin secreted in wallaby milk is alternately spliced into two variants, MaeuCath1a and MaeuCath1b which are temporally regulated in order to provide antimicrobial protection to the newborn and stimulate mammary growth, respectively. The current study investigated the extracellular matrix (ECM) for its regulatory role in MaeuCath1 gene expression. Reverse transcription qPCR using RNA isolated from mammary epithelial cells (WallMEC) cultured on ECM showed that ECM regulates MaeuCath1a gene expression in a lactation phase-dependent manner. Luciferase reporter-based assays and in silico analysis of deletion fragments of the 2245bp sequence upstream of the translation start site identified ECM-dependent positive regulatory activity in the -709 to -15 region and repressor activity in the -919 to -710 region. Electrophoretic Gel Mobility Shift Assays (EMSA) using nuclear extract from ECM-treated WallMEC showed differential band shift in the -839 to -710 region., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Many organisms localize mRNAs to specific subcellular destinations to spatially and temporally control gene expression. Recent studies have demonstrated that the majority of the transcriptome is localized to a nonrandom position in cells and embryos. One approach to identify localized mRNAs is to biochemically purify a cellular structure of interest and to identify all associated transcripts. Using recently developed high-throughput sequencing technologies it is now straightforward to identify all RNAs associated with a subcellular structure. To facilitate transcript identification it is necessary to work with an organism with a fully sequenced genome. One attractive system for the biochemical purification of subcellular structures are egg extracts produced from the frog Xenopus laevis. However, X. laevis currently does not have a fully sequenced genome, which hampers transcript identification. In this article we describe a method to produce egg extracts from a related frog, X. tropicalis, that has a fully sequenced genome. We provide details for microtubule polymerization, purification and transcript isolation. While this article describes a specific method for identification of microtubule-associated transcripts, we believe that it will be easily applied to other subcellular structures and will provide a powerful method for identification of localized RNAs.

Purpose: To evaluate the safety of an intensive cataract surgery training programme., Methods: An intensive cataract surgery training programme was implemented in August 2010 for year 3 ophthalmology trainees in the East Midlands Deanery North Rotation (United Kingdom). Trainees participated in extra-ocular surgery and 50 h of virtual reality cataract surgery simulator training over a 2-year period. Their third year comprised 6 months of intensive phacoemulsification training in a tertiary centre followed by a 6-month period of consolidation in a district general hospital. The complication rates and case numbers were evaluated after the first 2 years of implementation., Results: At 2 years, three trainees had completed a full year of intensive training. In the first 6 months of training, Trainee 1 completed 156 cases, Trainee 2 completed 194 cases, and Trainee 3 completed 151 full cases as primary surgeons with an average rate of posterior capsule rupture (PCR) of 1%. At 12 months, Trainee 1 completed 291, Trainee 2 completed 318, and Trainee 3 completed 294 cases, with an average PCR rate of 0.66%. The trainees required 84 lists on average to complete 150 full cataract procedures., Conclusion: The combination of simulation and the new intensive training programme is safer than the traditional programme for cataract surgery training.

Previous experiments from our laboratories have identified peptides derived from the human astrovirus coat protein (CP) that bind C1q and mannose binding lectin (MBL) inhibiting activation of the classical and lectin pathways of complement, respectively. The purpose of this study was to evaluate the function of these coat protein peptides (CPPs) in an in vitro model of complement-mediated disease (ABO incompatibility), preliminarily assess their in vivo complement suppression profile and develop more highly potent derivatives of these molecules. E23A, a 30 amino acid CPP derivative previously demonstrated to inhibit classical pathway activation was able to dose-dependently inhibit lysis of AB erythrocytes treated with mismatched human O serum. Additionally, when injected into rats, E23A inhibited the animals' serum from lysing antibody-sensitized erythrocytes, providing preliminary in vivo functional evidence that this CPP can cross the species barrier to inhibit serum complement activity in rodents. A rational drug design approach was implemented to identify more potent CPP derivatives, resulting in the identification and characterization of a 15 residue peptide (polar assortant (PA)), which demonstrated both superior inhibition of classical complement pathway activation and robust binding to C1q collagen-like tails. PA also inhibited ABO incompatibility in vitro and demonstrated in vivo complement suppression up to 24h post-injection. CPP's ability to inhibit ABO incompatibility in vitro, proof of concept in vivo inhibitory activity in rats and the development of the highly potent PA derivative set the stage for preclinical testing of this molecule in small animal models of complement-mediated disease., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

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. The only extant monotremes are the platypus, and the short- and long- beaked echidnas, and their distributions are limited to Australia and New Guinea. Apart for a short weaning period, milk is the sole source of nutrition and protection for the hatchlings which are altricial and immunologically naive. The duration of lactation in these mammals is prolonged relative to the gestational length and period of incubation of eggs. Much of the development of monotreme young occurs in the non-sterile ex-utero environment. Therefore the role of milk in the growth, development and disease protection of the young is of significant interest. By sequencing the cDNA of cells harvested from monotreme milk, we have identified a novel monotreme- specific transcript, and the corresponding gene was designated as the EchAMP. The expression profile of this gene in various tissues revealed that it is highly expressed in milk cells. The peptides corresponding to the EchAMP protein have been identified in a sample of echidna milk In silico analysis indicated putative antimicrobial potential for the cognate protein of EchAMP. This was further confirmed by in vitro assays using a host of bacteria. Interestingly, EchAMP did not display any activity against a commensal gut floral species. These results support the hypothesis of enhancement of survival of the young by antimicrobial bioactives of mammary gland origin and thus emphasize the protective, non- nutritional role of milk in mammals.

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 involution. The WFDC2 protein showed antibacterial activity against Staphylococcus aureus, Salmonella enterica and Pseudomonas aeruginosa and this activity resided with domain II. There was no antibacterial activity detected against Enterococcus faecalis. The observed expression pattern of tammar WFDC2 and its antibacterial activity suggests a role to either reduce mastitis in the mammary gland caused by S. aureus or to protect the gut of the young at a time when it is not immune-competent. The latter effect could be achieved without disturbing the balance of commensal gut flora such as E. faecalis., (Crown Copyright © 2011. Published by Elsevier Ltd. All rights reserved.)

Similar to other highly successful invasive bacterial pathogens, Staphylococcus aureus recruits the complement regulatory protein factor H (fH) to its surface to inhibit the alternative pathway of complement. Here, we report the identification of the surface-associated protein SdrE as a fH-binding protein using purified fH overlay of S. aureus fractionated cell wall proteins and fH cross-linking to S. aureus followed by mass spectrometry. Studies using recombinant SdrE revealed that rSdrE bound significant fH whether from serum or as a purified form, in both a time- and dose-dependent manner. Furthermore, rSdrE-bound fH exhibited cofactor functionality for factor I (fI)-mediated cleavage of C3b to iC3b which correlated positively with increasing amounts of fH. Expression of SdrE on the surface of the surrogate bacterium Lactococcus lactis enhanced recruitment of fH which resulted in increased iC3b generation. Moreover, surface expression of SdrE led to a reduction in C3-fragment deposition, less C5a generation, and reduced killing by polymorphonuclear cells. Thus, we report the first identification of a S. aureus protein associated with the staphylococcal surface that binds factor H as an immune evasion mechanism.