Association of a missense mutation in the bovine leptin gene with carcass fat content and leptin mRNA levels

Previously, we have shown that alleles of the BM1500 microsatellite, located 3.6 kb downstream of the leptin gene in cattle, were associated with carcass fat measures in a population of 154 unrelated beef bulls. Subsequently, a cytosine (C) to thymine (T) transition that encoded an amino acid change of an arginine to a cysteine was identified in exon 2 of the leptin gene. A PCR-RFLP was designed and allele frequencies in four beef breeds were correlated with levels of carcass fat. The T allele was associated with fatter carcasses and the C allele with leaner carcasses. The frequencies of the SNP alleles among breeds indicated that British breeds have a higher frequency of the T allele whereas the continental breeds have a higher occurrence of the C allele. A ribonuclease protection assay was developed to quantify leptin mRNA in a separate group of animals selected by genotype. Animals homozygous for thymine expressed higher levels of leptin mRNA. This may suggest that the T allele, which adds an extra cysteine to the protein, imparts a partial loss of biological function and hence could be the causative mutation.     

Genomic evidence of contemporary hybridization between Schistosoma species

Hybridization between different species of parasites is increasingly being recognised as a major public and veterinary health concern at the interface of infectious diseases biology, evolution, epidemiology and ultimately control. Recent research has revealed that viable hybrids and introgressed lineages between Schistosoma spp. are prevalent across Africa and beyond, including those with zoonotic potential. However, it remains unclear whether these hybrid lineages represent recent hybridization events, suggesting hybridization is ongoing, and/or whether they represent introgressed lineages derived from ancient hybridization events. In human schistosomiasis, investigation is hampered by the inaccessibility of adult-stage worms due to their intravascular location, an issue which can be circumvented by post-mortem of livestock at abattoirs for Schistosoma spp. of known zoonotic potential. To characterise the composition of naturally-occurring schistosome hybrids, we performed whole-genome sequencing of 21 natural livestock infective schistosome isolates. To facilitate this, we also assembled a de novo chromosomal-scale draft assembly of Schistosoma curassoni. Genomic analyses identified isolates of S. bovis, S. curassoni and hybrids between the two species, all of which were early generation hybrids with multiple generations found within the same host. These results show that hybridization is an ongoing process within natural populations with the potential to further challenge elimination efforts against schistosomiasis.     

The birds and the bees: pollinator behaviour and variation in the mating system of the rare shrub Grevillea macleayana

Background and Aims

In Australia, honey-bees have invaded systems that evolved without social insect pollinators, where many plants are adapted to vertebrate pollination. Behavioural differences between pollinators are likely to influence mating patterns, but few studies have examined this empirically in long-lived, woody, perennials. It was shown previously that outcrossing rates in Grevillea macleayana vary among populations. Here tests were conducted to determine whether the behaviour of birds and honey-bees differed between a population previously found to be highly outcrossed and two inbreeding populations.

Methods

Visit frequencies and movement patterns of the visitors to inflorescences at three sites over two seasons were compared. A caging experiment was used to test the effects of excluding birds on pollen removal from newly opened flowers and on pollen deposition on stigmas that had been washed clean.

Key Results

Honey-bees were the most frequent visitors overall, but honeyeaters were more frequent visitors in the population previously found to have a high outcrossing rate than they were in either of the other populations. More visits by honeyeaters were from distant plants. Pollen removal did not vary greatly among sites, and was not affected by bird exclusion; however, more pollen was deposited on the stigmas of cleaned pollen presenters in the population previously observed to be highly outcrossing than in the other two. This high level of pollen deposition was reduced by experimental bird exclusion.

Conclusions

Honey-bees were the most frequent visitors, by an order of magnitude, and excluding vertebrates revealed that bees were removing most of the pollen but deposited fewer pollen grains on stigmas. Birds were more frequent visitors at the site previously found to be outcrossing than the other two sites, and they moved further between plants and visited fewer inflorescences on a plant during a foraging bout than bees did. These characteristics of bird visits to G. macleayana would be sufficient to produce significant variation in outcrossing rates among sites.Key words: Grevillea macleayana, Apis mellifera, honey-bees, honeyeaters, pollinator behaviour, pollen removal, pollen deposition, outcrossing rate     

Human cerebral organoids reveal progenitor pathology in EML1‐linked cortical malformation

Malformations of human cortical development (MCD) can cause severe disabilities. The lack of human‐specific models hampers our understanding of the molecular underpinnings of the intricate processes leading to MCD. Here, we use cerebral organoids derived from patients and genome edited‐induced pluripotent stem cells to address pathophysiological changes associated with a complex MCD caused by mutations in the echinoderm microtubule‐associated protein‐like 1 (EML1) gene. EML1‐deficient organoids display ectopic neural rosettes at the basal side of the ventricular zone areas and clusters of heterotopic neurons. Single‐cell RNA sequencing shows an upregulation of basal radial glial (RG) markers and human‐specific extracellular matrix components in the ectopic cell population. Gene ontology and molecular analyses suggest that ectopic progenitor cells originate from perturbed apical RG cell behavior and yes‐associated protein 1 (YAP1)‐triggered expansion. Our data highlight a progenitor origin of EML1 mutation‐induced MCD and provide new mechanistic insight into the human disease pathology.     

Genetic and biological characterisation of three cryptic Eimeria operational taxonomic units that infect chickens (Gallus gallus domesticus)

More than 68 billion chickens were produced globally in 2018, emphasising their major contribution to the production of protein for human consumption and the importance of their pathogens. Protozoan Eimeria spp. are the most economically significant parasites of chickens, incurring global costs of more than UK £10.4 billion per annum. Seven Eimeria spp. have long been recognised to infect chickens, with three additional cryptic operational taxonomic units (OTUs) first described more than 10 years ago. As the world’s farmers attempt to reduce reliance on routine use of antimicrobials in livestock production, replacing drugs that target a wide range of microbes with precise species- and sometimes strain-specific vaccines, the breakthrough of cryptic genetic types can pose serious problems. Consideration of biological characteristics including oocyst morphology, pathology caused during infection and pre-patent periods, combined with gene-coding sequences predicted from draft genome sequence assemblies, suggest that all three of these cryptic Eimeria OTUs possess sufficient genetic and biological diversity to be considered as new and distinct species. The ability of these OTUs to compromise chicken bodyweight gain and escape immunity induced by current commercially available anticoccidial vaccines indicates that they could pose a notable threat to chicken health, welfare, and productivity. We suggest the names Eimeria lata n. sp., Eimeria nagambie n. sp. and Eimeria zaria n. sp. for OTUs x, y and z, respectively, reflecting their appearance (x) or the origins of the first isolates of these novel species (y, z).     

Induction of Na+/K+/2Cl- cotransporter expression mediates chronic potentiation of intestinal epithelial Cl- secretion by EGF

Alterations in EGF receptor (EGFR) signaling occur in intestinal disorders associated with dysregulated epithelial transport. In the present study, we investigated a role for the EGFR in the chronic regulation of intestinal epithelial secretory function. Epithelial Cl(-) secretion was measured as changes in short-circuit current (Isc) across voltage-clamped monolayers of T84 cells in Ussing chambers. Acute treatment of T84 cells with EGF (100 ng/ml, 15 min) chronically enhanced Isc responses to a broad range of secretagogues. This effect was apparent within 3 h, maximal by 6 h, and sustained for 24 h after treatment with EGF. The Na+/K+/2Cl(-) cotransporter (NKCC1) inhibitor bumetanide (100 microM) abolished the effect of EGF, indicating increased responses are due to potentiated Cl(-) secretion. Neither basal nor agonist-stimulated levels of intracellular Ca2+ or PKA activity were altered by EGF, implying that the effects of the growth factor are not due to chronic alterations in levels of second messengers. EGF increased the expression of NKCC1 with a time course similar to that of its effects on Cl(-) secretion. This effect of EGF was maximal after 6 h, at which time NKCC1 expression in EGF-treated cells was 199.9 +/- 21.9% of that in control cells (n = 21, P < 0.005). EGF-induced NKCC1 expression was abolished by actinomycin D, and RT-PCR analysis demonstrated EGF increased expression of NKCC1 mRNA. These data increase our understanding of mechanisms regulating intestinal fluid and electrolyte transport and reveal a novel role for the EGFR in the chronic regulation of epithelial secretory capacity through upregulation of NKCC1 expression.     

Development aggravates the severity of skeletal muscle catabolism induced by endotoxemia in neonatal pigs

Accretion rates of muscle protein are elevated in normal neonates, but this anabolic drive decreases with maturation. As this change occurs, it is not known whether development also influences muscle protein catabolism induced by sepsis. We hypothesize that protein degradation in skeletal muscle induced by endotoxemia becomes more severe as the neonate develops. Fasted 7- and 26-day-old pigs were infused for 8 h with LPS (0 and 10 μg·kg(-1)·h(-1)), while plasma amino acids (AA), 3-methylhistidine (3-MH), and α-actin concentrations and muscle protein degradation signal activation were determined (n = 5-7/group/age). Plasma full-length α-actin was greater in 7- than 26-day-old pigs, suggesting a higher baseline protein turnover in neonatal pigs. LPS increased plasma total AA, 3-MH, and full-length and cleaved α-actin in 26- than in 7-day-old pigs. In muscle of both age groups, LPS increased AMPK and NF-κB phosphorylation, the abundances of activated caspase 3 and E-3 ligases MuRF1 and atrogin1, as well as the abundance of cleaved α-actin, suggesting activation of muscle proteolysis by endotoxin in muscle. LPS decreased Forkhead box 01 (Fox01) and Fox04 phosphorylation and increased procaspase 3 abundance in muscle of 26-day-old pigs despite the lack of effect of LPS on PKB phosphorylation. The results suggest that skeletal muscle in healthy neonatal pigs maintains high baseline degradation signal activation that cannot be enhanced by endotoxin, but as maturation advances, the effect of LPS on muscle protein catabolism manifests its severity.     

A tumor-associated beta 1 integrin mutation that abrogates epithelial differentiation control

SCC4 human keratinocytes are derived from a squamous cell carcinoma of the tongue and undergo very little spontaneous differentiation. Introduction of a wild-type beta 1 integrin subunit into SCC4 cells stimulates differentiation, suggesting either that the cells have a defect in the integrin signaling pathways that control differentiation or that the beta1 subunit itself is defective. Here we describe a heterozygous mutation in the SCC4 beta 1 subunit. The mutation, T188I, maps to the I-like domain. It results in constitutive activation of ligand binding, irrespective of the partner alpha subunit, in solid phase assays with recombinant protein and in living cells. The mutation promotes cell spreading, but not proliferation, motility, or invasiveness. It results in sustained activation of Erk MAPK independent of cell spreading. When introduced into SCC4 keratinocytes, the wild-type beta1 integrin stimulates differentiation, whereas the mutant is inactive. Activation of beta 1 integrins in normal keratinocytes also suppresses differentiation. These results establish, for the first time, mutation as a mechanism by which integrins can contribute to neoplasia, because the degree of differentiation in epithelial cancers is inversely correlated with prognosis. They also provide new insights into how integrins regulate keratinocyte differentiation.     

The Physical Gene Hsp70 Map on Polytene Chromosomes of Anopheles darlingi from the Brazilian Amazon

In situ hybridization was used to determine the physical location of the Hsp70 genes in salivary polytene chromosomes of Anopheles darlingi from Manaus and Macapá, Brazil, and to assess the usefulness of the Hsp70 locus as a genetic marker in A. darlingi populations. In both populations, the double markings corresponding to the Hsp70-12A and Hsp70-14A genes were located on the right arm of chromosome 2. The Hsp70 locus was considered to be an excellent marker for studying chromosomal evolution and relationships among A. darlingi populations.