Cellular heterogeneity of the developing worker honey bee (Apis mellifera) pupa: a single cell transcriptomics analysis.

It is estimated that animals pollinate 87.5% of flowering plants worldwide and that managed honey bees (Apis mellifera) account for 30-50% of this ecosystem service to agriculture. In addition to their important role as pollinators, honey bees are well-established insect models for studying learning and memory, behavior, caste differentiation, epigenetic mechanisms, olfactory biology, sex determination, and eusociality. Despite their importance to agriculture, knowledge of honey bee biology lags behind many other livestock species. In this study, we have used scRNA-Seq to map cell types to different developmental stages of the worker honey bee (prepupa at day 11 and pupa at day 15) and sought to determine their gene expression signatures. To identify cell-type populations, we examined the cell-to-cell network based on the similarity of the single-cells transcriptomic profiles. Grouping similar cells together we identified 63 different cell clusters of which 17 clusters were identifiable at both stages. To determine genes associated with specific cell populations or with a particular biological process involved in honey bee development, we used gene coexpression analysis. We combined this analysis with literature mining, the honey bee protein atlas, and gene ontology analysis to determine cell cluster identity. Of the cell clusters identified, 17 were related to the nervous system and sensory organs, 7 to the fat body, 19 to the cuticle, 5 to muscle, 4 to compound eye, 2 to midgut, 2 to hemocytes, and 1 to malpighian tubule/pericardial nephrocyte. To our knowledge, this is the first whole single-cell atlas of honey bees at any stage of development and demonstrates the potential for further work to investigate their biology at the cellular level.

Genetics of tibia bone properties of crossbred commercial laying hens in different housing systems.

Osteoporosis and bone fractures are a severe problem for the welfare of laying hens, with genetics and environment, such as housing system, each making substantial contributions to bone strength. In this work, we performed genetic analyses of bone strength, bone mineral density, and bone composition, as well as body weight, in 860 commercial crossbred laying hens from 2 different companies, kept in either furnished cages or floor pens. We compared bone traits between housing systems and crossbreds and performed a genome-wide association study of bone properties and body weight. As expected, the 2 housing systems produced a large difference in bone strength, with layers housed in floor pens having stronger bones. These differences were accompanied by differences in bone geometry, mineralization, and chemical composition. Genome scans either combining or independently analyzing the 2 housing systems revealed no genome-wide significant loci for bone breaking strength. We detected 3 loci for body weight that were shared between the housing systems on chromosomes 4, 6, and 27 (either genome-wide significant or suggestive) and these coincide with associations for bone length. In summary, we found substantial differences in bone strength, content, and composition between hens kept in floor pens and furnished cages that could be attributed to greater physical activity in pen housing. We found little evidence for large-effect loci for bone strength in commercial crossbred hens, consistent with a highly polygenic architecture for bone strength in the production environment. The lack of consistent genetic associations between housing systems in combination with the differences in bone phenotypes could be due to gene-by-environment interactions with housing system or a lack of power to detect shared associations for bone strength.

Effects of Exogenous Transferrin on the Regulation of Iron Metabolism and Erythropoiesis in Iron Deficiency With or Without Anemia.

Erythropoietic response is controlled not only by erythropoietin but also by iron. In addition to its role in iron delivery, transferrin also functions as a signaling molecule, with effects on both iron homeostasis and erythropoiesis. We investigated hematologic parameters, iron status and expression of key proteins, including the hepatic iron regulatory protein hepcidin and the suppressive erythroid factor Erfe, in mice subject to dietary iron deficiency with and without anemia. The acute effect of iron on these parameters was investigated by administration of exogenous iron-loaded transferrin (holoTf) in each of the mouse models. Serum iron in mice with iron deficiency (ID) is modestly lower with hematologic parameters maintained by utilization of iron stores in mice with ID. As expected, erythropoietin expression and concentration, along with marrow Erfe are unaffected in ID mice. Administration of holoTf restores serum iron and Tf saturation levels to those observed in control mice and results in an increase in hepcidin compared to ID mice not treated with holoTf. The expression of the Bmp signaling molecule Bmp6 is not significantly increased following Tf treatment in ID mice. Thus, the expression level of the gene encoding hepcidin, Hamp1, is increased relative to Bmp6 expression in ID mice following treatment with holoTf, leading us to speculate that Tf saturation may influence Bmp sensitivity. In mice with iron deficiency anemia (IDA), decreased hematologic parameters were accompanied by pronounced decreases in serum and tissue iron concentrations, and an increase in serum erythropoietin. In the absence of exogenous holoTf, the greater serum erythropoietin was not reflected by an increase in marrow Erfe expression. HoloTf administration did not acutely change serum Epo in IDA mice. Marrow Erfe expression was, however, markedly increased in IDA mice following holoTf, plausibly accounting for the lack of an increase in Hamp1 following holoTf treatment in the IDA mice. The increase in Erfe despite no change in erythropoietin suggests that Tf acts to increase erythropoietin sensitivity. These observations underscore the importance of Tf in modulating the erythropoietic response in recovery from iron deficiency anemia, with implications for other stress erythropoiesis conditions.

The fluorescent protein iLOV as a reporter for screening of high-yield production of antimicrobial peptides in Pichia pastoris.

The methylotrophic yeast Pichia pastoris is commonly used for the production of recombinant proteins at scale. The identification of an optimally overexpressing strain following transformation can be time and reagent consuming. Fluorescent reporters like GFP have been used to assist identification of superior producers, but their relatively big size, maturation requirements and narrow temperature range restrict their applications. Here, we introduce the use of iLOV, a flavin-based fluorescent protein, as a fluorescent marker to identify P. pastoris high-yielding strains easily and rapidly. The use of this fluorescent protein as a fusion partner is exemplified by the production of the antimicrobial peptide NI01, a difficult target to overexpress in its native form. iLOV fluorescence correlated well with protein expression level and copy number of the chromosomally integrated gene. An easy and simple medium-throughput plate-based screen directly following transformation is demonstrated for low complexity screening, while a high-throughput method using fluorescence-activated cell sorting (FACS) allowed for comprehensive library screening. Both codon optimization of the iLOV_NI01 fusion cassettes and different integration strategies into the P. pastoris genome were tested to produce and isolate a high-yielding strain. Checking the genetic stability, process reproducibility and following the purification of the active native peptide are eased by visualization of and efficient cleavage from the iLOV reporter. We show that this system can be used for expression and screening of several different antimicrobial peptides recombinantly produced in P. pastoris.

The hepcidin regulator erythroferrone is a new member of the erythropoiesis-iron-bone circuitry.

Background: Erythroblast erythroferrone (ERFE) secretion inhibits hepcidin expression by sequestering several bone morphogenetic protein (BMP) family members to increase iron availability for erythropoiesis. Methods: To address whether ERFE functions also in bone and whether the mechanism of ERFE action in bone involves BMPs, we utilize the Erfe-/- mouse model as well as ß-thalassemic (Hbbth3/+) mice with systemic loss of ERFE expression. In additional, we employ comprehensive skeletal phenotyping analyses as well as functional assays in vitro to address mechanistically the function of ERFE in bone. Results: We report that ERFE expression in osteoblasts is higher compared with erythroblasts, is independent of erythropoietin, and functional in suppressing hepatocyte hepcidin expression. Erfe-/- mice display low-bone-mass arising from increased bone resorption despite a concomitant increase in bone formation. Consistently, Erfe-/- osteoblasts exhibit enhanced mineralization, Sost and Rankl expression, and BMP-mediated signaling ex vivo. The ERFE effect on osteoclasts is mediated through increased osteoblastic RANKL and sclerostin expression, increasing osteoclastogenesis in Erfe-/- mice. Importantly, Erfe loss in Hbbth3/+mice, a disease model with increased ERFE expression, triggers profound osteoclastic bone resorption and bone loss. Conclusions: Together, ERFE exerts an osteoprotective effect by modulating BMP signaling in osteoblasts, decreasing RANKL production to limit osteoclastogenesis, and prevents excessive bone loss during expanded erythropoiesis in ß-thalassemia. Funding: YZG acknowledges the support of the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) (R01 DK107670 to YZG and DK095112 to RF, SR, and YZG). MZ acknowledges the support of the National Institute on Aging (U19 AG60917) and NIDDK (R01 DK113627). TY acknowledges the support of the National Institute on Aging (R01 AG71870). SR acknowledges the support of NIDDK (R01 DK090554) and Commonwealth Universal Research Enhancement (CURE) Program Pennsylvania.

Characterisation of 99mTc EDDA/HYNIC-TOC (Tektrotyd) physiological and neuroendocrine tumour uptake using SPECT/CT standardised uptake values: initial experience.

99mTc Ethylene diamine N,N'-diacetic acid hydrazinonicotinamide-conjugated Tyr3-octreotide (99mTc EDDA/HYNIC-TOC) single photon emission tomography/computed tomography (SPECT/CT) imaging of somatostatin receptors is used in the assessment of neuroendocrine tumours (NETs). The objective of this study was to characterise quantitative standardised uptake value (SUV) SPECT/CT of normal physiological uptake and NET disease. Forty-four patients (22 female and 22 male) referred for 99mTc EDDA/HYNIC-TOC SPECT/CT imaging for diagnosis/primary staging (n = 28) or the assessment of residual/recurrent disease (n = 16) were included. SPECT/CT SUVmax values were determined for normal physiological uptake (spleen, kidney, liver and bone) and NET disease (liver metastases, metastatic lymph nodes, bone metastases and intrapulmonary lesions). Statistical testing was performed to compare normal uptake and NET disease uptake in liver and bone (Student's t-test). The highest normal physiological uptake was observed in the spleen (mean SUVmax 29.8, SD 13.7), with lower uptake in the kidneys (16.7, 3.2) and liver (7.3, 2.1). Increased SUVmax values were observed in primary tumour and metastatic disease, greatest in liver metastases (21.8, 13.3), with lower, similar values obtained for metastatic lymph nodes (16.3, 7.5) and intrapulmonary lesions (17.5, 16.8). SUVmax in bone metastases averaged 12.9 (7.0). Significant differences were observed between normal and metastatic SUVmax in the liver and bone (P < 0.01). SPECT/CT SUV quantification is feasible in a manner similar to PET/CT. 99mTc EDDA/HYNIC-TOC SPECT/CT SUVmax has been characterised in NET disease, demonstrating high target to non-target ratios for primary tumours and metastatic lesions.

No evidence that selection for egg production persistency causes loss of bone quality in laying hens.

BACKGROUND: The physiological adaptations that have evolved for egg laying make hens susceptible to bone fractures and keel bone damage. In modern laying hen breeds, longer periods of egg laying could result in a greater risk of poor bone quality, and selection for increased egg production has frequently been stated to be a cause. However, the existing literature does not support this hypothesis. To test the hypothesis that egg production is associated with quality, breaking strength and density of bone, genetic correlations between these traits were estimated in White Leghorn and Rhode Island Red breeds. Genetic correlations of cortical and medullary bone material chemical properties with bone quality were also estimated, in order to identify methods to improve bone quality with appropriately targeted measurement of key traits. RESULTS: Estimates of heritability for bone quality traits were moderate (0.19-0.59) for both White Leghorn and Rhode Island Red breeds, except for the keel bone trait, which had a heritability estimate equal to zero. There was no evidence for genetic or phenotypic relationships between post-peak egg production and bone quality. In the White Leghorn breed, the estimate of the genetic correlation between pre-peak production/age at first egg and bone quality was significant and negative (- 0.7 to - 0.4). Estimates of heritability of thermogravimetric measurements of tibial medullary bone mineralisation were significant (0.18-0.41), as were estimates of their genetic correlations with tibia breaking strength and density (0.6-0.9). CONCLUSIONS: The low genetic correlation of post-peak egg production with bone quality suggests that selection for increased persistency of egg production may not adversely affect bone quality. Onset of puberty and mineralisation of the medullary bone, which is a specialised adaptation for egg laying, were identified as important factors associated with the quality of the skeleton later during egg production. These are traits for which genetic, as well as environmental and management factors can positively impact the overall quality of the skeleton of laying hens.

Hepcidin Status at 2 Months in Infants Fed Breast Milk Compared with Formula.

INTRODUCTION: The basis for the superior absorption of iron from breast milk compared with infant formulas is unclear. The hormone hepcidin downregulates dietary iron absorption. Hepcidin production increases with increased body iron status (reflected in serum ferritin levels). We hypothesized that serum hepcidin levels are suppressed relative to iron status in infants fed breast milk compared with formula. METHODS: Subjects were healthy infants presenting for routine 2-month clinic visit and strictly fed either breast milk or standard infant formula. Urinary hepcidin and ferritin levels (reflective of serum levels) were analyzed and compared across the breast milk- and formula-fed groups. The relationship between urinary hepcidin and ferritin levels within each group was analyzed by linear regression. RESULTS: Twenty-four subjects were enrolled in each group. The median urinary hepcidin level in the group fed breast milk was lower than in formula (130 vs. 359 ng hepcidin/mg creatinine, p < 0.05). However, the median ferritin levels were similar (2.1 vs. 1.9 ng/mL). Within each group, urinary hepcidin correlated with urinary ferritin (r = 0.5, p < 0.05 for each group); however, the slope of the regression line was lower in the group fed breast milk compared with formula (p < 0.005). CONCLUSION: Despite similar urinary ferritin levels, urinary hepcidin levels are lower at 2 months in infants fed breast milk compared with infants fed formula. Hepcidin levels correlate with iron status in each group; however, this relationship is relatively dampened in infants fed breast milk. We speculate that relatively lower infant hepcidin contributes to the superior efficiency of iron absorption from breast milk.

Polarity-Dependent Adsorption of Inverse Micelles.

The adsorption of charged inverse micelles at the electrode-liquid interface has an important effect on field screening and on the voltage drop over diffuse double layers. Recently, we analyzed the behavior of inverse micelles in a nonpolar liquid close to this electrode-liquid interface. For the fluorocarbon/surfactant system under study, we are in the limit of slow adsorption and negligible desorption of inverse micelles on the electrodes. Upon applying a voltage step, this results in a measurable Stern layer buildup in the time range of hours clearly distinguishable from the diffuse double layer buildup, which happens in less than 1 s. This Stern layer buildup manifests itself by a shift in the voltage drop from the diffuse double layer to the Stern layer until the voltage drop over the Stern layers reaches the applied voltage, leaving a zero bulk field without the diffuse double layer. New measurements of the transients of Stern layer buildup show that the buildup of charges in the Stern layer is more complex. We explain the observed transient behavior by introducing an asymmetry in the adsorption rate of charged inverse micelles. We provide an equivalent electrical network, an analytical solution to explain the behavior in more detail, and simulations within the diffuse double layer limit for a range of adsorption rates.

Combined Spatially Resolved Characterization of Antireflection and Antisoiling Coatings for PV Module Glass.

Characterization of photovoltaic (PV) module materials throughout different stages of service life is crucial to understanding and improving the durability of these materials. Currently the large-scale of PV modules (>1 m2) is imbalanced with the small-scale of most materials characterization tools (≤1 cm2). Furthermore, understanding degradation mechanisms often requires a combination of multiple characterization techniques. Here, we present adaptations of three standard materials characterization techniques to enable mapping characterization over moderate sample areas (≥25 cm2). Contact angle, ellipsometry, and UV-vis spectroscopy are each adapted and demonstrated on two representative samples: a commercial multifunctional coating for PV glass and an oxide combinatorial sample library. Best practices are discussed for adapting characterization techniques for large-area mapping and combining mapping information from multiple techniques.

An eQTL in the cystathionine beta synthase gene is linked to osteoporosis in laying hens.

BACKGROUND: Skeletal damage is a challenge for laying hens because the physiological adaptations required for egg laying make them susceptible to osteoporosis. Previously, we showed that genetic factors explain 40% of the variation in end of lay bone quality and we detected a quantitative trait locus (QTL) of large effect on chicken chromosome 1. The aim of this study was to combine data from the commercial founder White Leghorn population and the F2 mapping population to fine-map this QTL and understand its function in terms of gene expression and physiology. RESULTS: Several single nucleotide polymorphisms on chromosome 1 between 104 and 110 Mb (galGal6) had highly significant associations with tibial breaking strength. The alternative genotypes of markers of large effect that flanked the region had tibial breaking strengths of 200.4 vs. 218.1 Newton (P < 0.002) and, in a subsequent founder generation, the higher breaking strength genotype was again associated with higher breaking strength. In a subsequent generation, cortical bone density and volume were increased in individuals with the better bone genotype but with significantly reduced medullary bone quality. The effects on cortical bone density were confirmed in a further generation and was accompanied by increased mineral maturity of the cortical bone as measured by infrared spectrometry and there was evidence of better collagen cross-linking in the cortical bone. Comparing the transcriptome of the tibia from individuals with good or poor bone quality genotypes indicated four differentially-expressed genes at the locus, one gene, cystathionine beta synthase (CBS), having a nine-fold higher expression in the genotype for low bone quality. The mechanism was cis-acting and although there was an amino-acid difference in the CBS protein between the genotypes, there was no difference in the activity of the enzyme. Plasma homocysteine concentration, the substrate of CBS, was higher in the poor bone quality genotype. CONCLUSIONS: Validated markers that predict bone strength have been defined for selective breeding and a gene was identified that may suggest alternative ways to improve bone health in addition to genetic selection. The identification of how genetic variants affect different aspects of bone turnover shows potential for translational medicine.