Prenatal Exposure to Dexamethasone in the Mouse Alters Cardiac Growth Patterns and Increases Pulse Pressure in Aged Male Offspring

Exposure to synthetic glucocorticoids during development can result in later cardiovascular and renal disease in sheep and rats. Although prenatal glucocorticoid exposure is associated with impaired renal development, less is known about effects on the developing heart. This study aimed to examine the effects of a short-term exposure to dexamethasone (60 hours from embryonic day 12.5) on the developing mouse heart, and cardiovascular function in adult male offspring. Dexamethasone (DEX) exposed fetuses were growth restricted compared to saline treated controls (SAL) at E14.5, but there was no difference between groups at E17.5. Heart weights of the DEX fetuses also tended to be smaller at E14.5, but not different at E17.5. Cardiac AT_1aR, Bax, and IGF-1 mRNA expression was significantly increased by DEX compared to SAL at E17.5. In 12-month-old offspring DEX exposure caused an increase in basal blood pressure of ∼3 mmHg. In addition, DEX exposed mice had a widened pulse pressure compared to SAL. DEX exposed males at 12 months had an approximate 25% reduction in nephron number compared to SAL, but no difference in cardiomyocyte number. Exposure to DEX in utero appears to adversely impact on nephrogenesis and heart growth but is not associated with a cardiomyocyte deficit in male mice in adulthood, possibly due to compensatory growth of the myocardium following the initial insult. However, the widened pulse pressure may be indicative of altered vascular compliance.     

Donor milk intake and infant growth in a South African neonatal unit: a cohort study

Background

Implications of donor milk feedings on infant growth in resource limited settings remain uncertain. This knowledge gap includes the impact of donor milk availability on infant intake of mother’s own milk. Therefore, this investigation aimed to measure intake and growth in infants receiving donor milk when born to women from resource limited backgrounds with high rates of human immunodeficiency virus (HIV).

Methods

A retrospective cohort study enrolled eligible infants admitted to a South African combined neonatal intensive and secondary high care unit, within a one year admission period during 2015, with signed consent for donor milk feedings. A certified milk bank provided donor milk. Daily nutritional intake during the first month was recorded. Details included proportional intake of donor milk, mother’s own milk and infant formula. The primary outcome of infant growth velocity from day back to birth weight to discharge was calculated when length of stay was ≥14 days. Analyses primarily used T-tests; mixed effects models compared weekly calorie intake.

Results

One hundred five infants with donor milk consent were born at 30.9?±?3.6 weeks of gestation, weighing 1389?±?708 g. Forty percent of mothers had HIV. Infant growth velocity did not differ based on percent of feedings as donor milk (≥ 50%: 11.8?±?4.9 g/kg/d; <?50%: 13.5?±?5.3 g/kg/d; p?=?0.3). Percent of feedings from donor milk was similar based on maternal HIV status (positive: 31?±?25%; negative: 36?±?29%; p?=?0.4), as was percent of feedings as mother’s milk (positive: 53?±?35%; negative: 58?±?30%; p?=?0.4). Calorie intake increased markedly during the first two weeks and then plateaued (p?<?0.0001).

Conclusions

Donor milk feedings in higher proportions did not further impair growth of infants managed in a South African combined neonatal intensive and secondary high care unit with growth rates already below reference ranges. The provision of donor milk contributed to feedings being composed of primarily human milk during the first month. Increasing early calorie intake may improve infant growth in this center.

     

Citizen science in schools: Engaging students in research on urban habitat for pollinators

Citizen science can play an important role in school science education. Citizen science is particularly relevant to addressing current societal environmental sustainability challenges, as it engages the students directly with environmental science and gives students an understanding of the scientific process. In addition, it allows students to observe local representations of global challenges. Here, we report a citizen science programme designed to engage school‐age children in real‐world scientific research. The programme used standardized methods deployed across multiple schools through scientist–school partnerships to engage students with an important conservation problem: habitat for pollinator insects in urban environments. Citizen science programmes such as the programme presented here can be used to enhance scientific literacy and skills. Provided key challenges to maintain data quality are met, this approach is a powerful way to contribute valuable citizen science data for understudied, but ecologically important study systems, particularly in urban environments across broad geographical areas.     

A genetic screen in rodent malaria parasites identifies five new apicoplast putative membrane transporters,one of which is essential in human malaria parasites

The malaria‐causing parasite, Plasmodium, contains a unique non‐photosynthetic plastid known as the apicoplast. The apicoplast is an essential organelle bound by four membranes. Although membrane transporters are attractive drug targets, only two transporters have been characterised in the malaria parasite apicoplast membranes. We selected 27 candidate apicoplast membrane proteins, 20 of which are annotated as putative membrane transporters, and performed a genetic screen in Plasmodium berghei to determine blood stage essentiality and subcellular localisation. Eight apparently essential blood stage genes were identified, three of which were apicoplast‐localised: PbANKA_0614600 (DMT2), PbANKA_0401200 (ABCB4), and PbANKA_0505500. Nineteen candidates could be deleted at the blood stage, four of which were apicoplast‐localised. Interestingly, three apicoplast‐localised candidates lack a canonical apicoplast targeting signal but do contain conserved N‐terminal tyrosines with likely roles in targeting. An inducible knockdown of an essential apicoplast putative membrane transporter, PfDMT2, was only viable when supplemented with isopentenyl diphosphate. Knockdown of PfDMT2 resulted in loss of the apicoplast, identifying PfDMT2 as a crucial apicoplast putative membrane transporter and a candidate for therapeutic intervention.     

Phage display and structural studies reveal plasticity in substrate specificity of caspase‐3a from zebrafish

The regulation of caspase‐3 enzyme activity is a vital process in cell fate decisions leading to cell differentiation and tissue development or to apoptosis. The zebrafish, Danio rerio, has become an increasingly popular animal model to study several human diseases because of their transparent embryos, short reproductive cycles, and ease of drug administration. While apoptosis is an evolutionarily conserved process in metazoans, little is known about caspases from zebrafish, particularly regarding substrate specificity and allosteric regulation compared to the human caspases. We cloned zebrafish caspase‐3a (casp3a) and examined substrate specificity of the recombinant protein, Casp3a, compared to human caspase‐3 (CASP3) by utilizing M13 bacteriophage substrate libraries that incorporated either random amino acids at P5‐P1′ or aspartate fixed at P1. The results show a preference for the tetrapeptide sequence DNLD for both enzymes, but the P4 position of zebrafish Casp3a also accommodates valine equally well. We determined the structure of zebrafish Casp3a to 2.28Å resolution by X‐ray crystallography, and when combined with molecular dynamics simulations, the results suggest that a limited number of amino acid substitutions near the active site result in plasticity of the S4 sub‐site by increasing flexibility of one active site loop and by affecting hydrogen‐bonding with substrate. The data show that zebrafish Casp3a exhibits a broader substrate portfolio, suggesting overlap with the functions of caspase‐6 in zebrafish development.     

Proteomic analysis reveals novel proteins associated with the Plasmodium protein exporter PTEX and a loss of complex stability upon truncation of the core PTEX component,PTEX150

The Plasmodium translocon for exported proteins (PTEX) has been established as the machinery responsible for the translocation of all classes of exported proteins beyond the parasitophorous vacuolar membrane of the intraerythrocytic malaria parasite. Protein export, particularly in the asexual blood stage, is crucial for parasite survival as exported proteins are involved in remodelling the host cell, an essential process for nutrient uptake, waste removal and immune evasion. Here, we have truncated the conserved C‐terminus of one of the essential PTEX components, PTEX150, in Plasmodium falciparum in an attempt to create mutants of reduced functionality. Parasites tolerated C‐terminal truncations of up to 125 amino acids with no reduction in growth, protein export or the establishment of new permeability pathways. Quantitative proteomic approaches however revealed a decrease in other PTEX subunits associating with PTEX150 in truncation mutants, suggesting a role for the C‐terminus of PTEX150 in regulating PTEX stability. Our analyses also reveal three previously unreported PTEX‐associated proteins, namely PV1, Pf113 and Hsp70‐x (respective PlasmoDB numbers; PF3D7_1129100, PF3D7_1420700 and PF3D7_0831700) and demonstrate that core PTEX proteins exist in various distinct multimeric forms outside the major complex.     

Identifying Patient-Specific Epstein-Barr Nuclear Antigen-1 Genetic Variation and Potential Autoreactive Targets Relevant to Multiple Sclerosis Pathogenesis

Background

Epstein-Barr virus (EBV) infection represents a major environmental risk factor for multiple sclerosis (MS), with evidence of selective expansion of Epstein-Barr Nuclear Antigen-1 (EBNA1)-specific CD4+ T cells that cross-recognize MS-associated myelin antigens in MS patients. HLA-DRB1*15-restricted antigen presentation also appears to determine susceptibility given its role as a dominant risk allele. In this study, we have utilised standard and next-generation sequencing techniques to investigate EBNA-1 sequence variation and its relationship to HLA-DR15 binding affinity, as well as examining potential cross-reactive immune targets within the central nervous system proteome.

Methods

Sanger sequencing was performed on DNA isolated from peripheral blood samples from 73 Western Australian MS cases, without requirement for primary culture, with additional FLX 454 Roche sequencing in 23 samples to identify low-frequency variants. Patient-derived viral sequences were used to predict HLA-DRB1*1501 epitopes (NetMHCII, NetMHCIIpan) and candidates were evaluated for cross recognition with human brain proteins.

Results

EBNA-1 sequence variation was limited, with no evidence of multiple viral strains and only low levels of variation identified by FLX technology (8.3% nucleotide positions at a 1% cut-off). In silico epitope mapping revealed two known HLA-DRB1*1501-restricted epitopes (‘AEG’: aa 481–496 and ‘MVF’: aa 562–577), and two putative epitopes between positions 502–543. We identified potential cross-reactive targets involving a number of major myelin antigens including experimentally confirmed HLA-DRB1*15-restricted epitopes as well as novel candidate antigens within myelin and paranodal assembly proteins that may be relevant to MS pathogenesis.

Conclusions

This study demonstrates the feasibility of obtaining autologous EBNA-1 sequences directly from buffy coat samples, and confirms divergence of these sequences from standard laboratory strains. This approach has identified a number of immunogenic regions of EBNA-1 as well as known and novel targets for autoreactive HLA-DRB1*15-restricted T cells within the central nervous system that could arise as a result of cross-reactivity with EBNA-1-specific immune responses.     

Glycosylation of a Fasciclin-Like Arabinogalactan-Protein (SOS5) Mediates Root Growth and Seed Mucilage Adherence via a Cell Wall Receptor-Like Kinase (FEI1/FEI2) Pathway in Arabidopsis

Fundamental processes that underpin plant growth and development depend crucially on the action and assembly of the cell wall, a dynamic structure that changes in response to both developmental and environmental cues. While much is known about cell wall structure and biosynthesis, much less is known about the functions of the individual wall components, particularly with respect to their potential roles in cellular signaling. Loss-of-function mutants of two arabinogalactan-protein (AGP)-specific galactosyltransferases namely, GALT2 and GALT5, confer pleiotropic growth and development phenotypes indicating the important contributions of carbohydrate moieties towards AGP function. Notably, galt2galt5 double mutants displayed impaired root growth and root tip swelling in response to salt, likely as a result of decreased cellulose synthesis. These mutants phenocopy a salt-overly sensitive mutant called sos5, which lacks a fasciclin-like AGP (SOS5/FLA4) as well as a fei1fei2 double mutant, which lacks two cell wall-associated leucine-rich repeat receptor-like kinases. Additionally, galt2gal5 as well as sos5 and fei2 showed reduced seed mucilage adherence. Quintuple galt2galt5sos5fei1fei2 mutants were produced and provided evidence that these genes act in a single, linear genetic pathway. Further genetic and biochemical analysis of the quintuple mutant demonstrated involvement of these genes with the interplay between cellulose biosynthesis and two plant growth regulators, ethylene and ABA, in modulating root cell wall integrity.     

The effect of tetrathiomolybdate on the metabolism of copper by hepatocytes and fibroblasts

Tetrathiomolybdate (TTM) has been examined for its effect on copper metabolism in mouse hepatocytes in primary culture and human fibroblasts. It decreased the amount of copper inside hepatocytes, decreased the rate of copper uptake by hepatocytes in a concentration dependent manner, and increased the copper efflux from the cells. TTM appeared to remove copper preferentially from the labile pool, but with a lower affinity than cage chelators. In fibroblasts, TTM only had a marginal effect on copper levels below a concentration of 100 microM and had no clear effect on the rate of copper uptake. TTM was not toxic to human fibroblasts, but in some preparations, a concentration of more than 50 microM was toxic to hepatocytes.