An intercellular heme-trafficking protein delivers maternal heme to the embryo during development in C. elegans.

Extracellular free heme can intercalate into membranes and promote damage to cellular macromolecules. Thus it is likely that specific intercellular pathways exist for the directed transport, trafficking, and delivery of heme to cellular destinations, although none have been found to date. Here we show that Caenorhabditis elegans HRG-3 is required for the delivery of maternal heme to developing embryos. HRG-3 binds heme and is exclusively secreted by maternal intestinal cells into the interstitial fluid for transport of heme to extraintestinal cells, including oocytes. HRG-3 deficiency results either in death during embryogenesis or in developmental arrest immediately post-hatching-phenotypes that are fully suppressed by maternal but not zygotic hrg-3 expression. Our results establish a role for HRG-3 as an intercellular heme-trafficking protein.

Lessons from bloodless worms: heme homeostasis in C. elegans.

Heme is an essential cofactor for proteins involved in diverse biological processes such as oxygen transport, electron transport, and microRNA processing. Free heme is hydrophobic and cytotoxic, implying that specific trafficking pathways must exist for the delivery of heme to target hemoproteins which reside in various subcellular locales. Although heme biosynthesis and catabolism have been well characterized, the pathways for trafficking heme within and between cells remain poorly understood. Caenorhabditis elegans serves as a unique animal model for uncovering these pathways because, unlike vertebrates, the worm lacks enzymes to synthesize heme and therefore is crucially dependent on dietary heme for sustenance. Using C. elegans as a genetic animal model, several novel heme trafficking molecules have been identified. Importantly, these proteins have corresponding homologs in vertebrates underscoring the power of using C. elegans, a bloodless worm, in elucidating pathways in heme homeostasis and hematology in humans. Since iron deficiency and anemia are often exacerbated by parasites such as helminths and protozoa which also rely on host heme for survival, C. elegans will be an ideal model to identify anti-parasitic drugs that target heme transport pathways unique to the parasite.

Acute Herpes Simplex Virus Laryngitis Presenting as Airway Obstruction Post Influenza: A Viral Pneumonia.

Herpes simplex virus (HSV) typically presents with mucocutaneous or genital ulcerations but can also manifest with central nervous system involvement and occasionally other visceral or mucosal sites. However, laryngeal involvement almost exclusively presents in infants and children. Very few confirmed adult cases have been reported. Adults present with a broad spectrum of symptoms, usually in the context of significant immunocompromise. Diagnosis is difficult given a wide spectrum of nonspecific presenting symptoms and usually requires tissue biopsy. Frequently, patients have severe laryngeal edema that threatens to compromise the airway and requires tracheostomy. We present a case of HSV laryngitis in a 71-year-old female who presented with septic shock, acute renal failure, and acute hypoxic respiratory failure secondary to Influenza A and bacterial pneumonia for which she required intubation. The hospitalization course included extubation failures due to stridor, a positive cuff leak test resulting in an open tracheostomy, and a laryngeal biopsy confirming HSV infection, which was successfully treated with acyclovir.

A novel heme-responsive element mediates transcriptional regulation in Caenorhabditis elegans.

Hemes are prosthetic groups that participate in diverse biochemical pathways across phylogeny. Although heme can also regulate broad physiological processes by directly modulating gene expression in Metazoa, the regulatory pathways for sensing and responding to heme are not well defined. Caenorhabditis elegans is a heme auxotroph and relies solely on environmental heme for sustenance. Worms respond to heme availability by regulating heme-responsive genes such as hrg-1, an intestinal heme transporter that is up-regulated by >60-fold during heme depletion. To identify the mechanism for the heme-dependent regulation of hrg-1, we interrogated the hrg-1 promoter. Deletion and mutagenesis studies of the hrg-1 promoter revealed a 23-bp heme-responsive element that is both necessary and sufficient for heme-dependent regulation of hrg-1. Furthermore, our studies show that the heme regulation of hrg-1 is mediated by both activation and repression in conjunction with ELT-2 and ELT-4, transcription factors that specify intestinal expression.

The Warburg effect is necessary to promote glycosylation in the blastema during zebrafish tail regeneration.

Throughout their lifetime, fish maintain a high capacity for regenerating complex tissues after injury. We utilized a larval tail regeneration assay in the zebrafish Danio rerio, which serves as an ideal model of appendage regeneration due to its easy manipulation, relatively simple mixture of cell types, and superior imaging properties. Regeneration of the embryonic zebrafish tail requires development of a blastema, a mass of dedifferentiated cells capable of replacing lost tissue, a crucial step in all known examples of appendage regeneration. Using this model, we show that tail amputation triggers an obligate metabolic shift to promote glucose metabolism during early regeneration similar to the Warburg effect observed in tumor forming cells. Inhibition of glucose metabolism did not affect the overall health of the embryo but completely blocked the tail from regenerating after amputation due to the failure to form a functional blastema. We performed a time series of single-cell RNA sequencing on regenerating tails with and without inhibition of glucose metabolism. We demonstrated that metabolic reprogramming is required for sustained TGF-ß signaling and blocking glucose metabolism largely mimicked inhibition of TGF-ß receptors, both resulting in an aberrant blastema. Finally, we showed using genetic ablation of three possible metabolic pathways for glucose, that metabolic reprogramming is required to provide glucose specifically to the hexosamine biosynthetic pathway while neither glycolysis nor the pentose phosphate pathway were necessary for regeneration.

Guided genetic screen to identify genes essential in the regeneration of hair cells and other tissues.

Regenerative medicine holds great promise for both degenerative diseases and traumatic tissue injury which represent significant challenges to the health care system. Hearing loss, which affects hundreds of millions of people worldwide, is caused primarily by a permanent loss of the mechanosensory receptors of the inner ear known as hair cells. This failure to regenerate hair cells after loss is limited to mammals, while all other non-mammalian vertebrates tested were able to completely regenerate these mechanosensory receptors after injury. To understand the mechanism of hair cell regeneration and its association with regeneration of other tissues, we performed a guided mutagenesis screen using zebrafish lateral line hair cells as a screening platform to identify genes that are essential for hair cell regeneration, and further investigated how genes essential for hair cell regeneration were involved in the regeneration of other tissues. We created genetic mutations either by retroviral insertion or CRISPR/Cas9 approaches, and developed a high-throughput screening pipeline for analyzing hair cell development and regeneration. We screened 254 gene mutations and identified 7 genes specifically affecting hair cell regeneration. These hair cell regeneration genes fell into distinct and somewhat surprising functional categories. By examining the regeneration of caudal fin and liver, we found these hair cell regeneration genes often also affected other types of tissue regeneration. Therefore, our results demonstrate guided screening is an effective approach to discover regeneration candidates, and hair cell regeneration is associated with other tissue regeneration.

Comparative approaches to facilitate the discovery of prolongevity interventions: effects of tocopherols on lifespan of three invertebrate species.

Many compounds hold promise for pharmacologic manipulation of aging. However, such claims are difficult to investigate due to time and budget constraints. Here, we took a comparative approach, using short-lived invertebrate species, to directly test the effects of two tocopherols (Vitamin E) on longevity. gamma-Tocopherol represents the most abundant tocopherol in the Western diet, while alpha-tocopherol is selectively enriched in human plasma. Both isoforms demonstrate antioxidant activity and are proposed to have anti-aging activities. We compared the effects of alpha- and gamma-tocopherol supplementation on lifespan in three invertebrate species. gamma-Tocopherol, but not alpha-tocopherol, slightly extended lifespan in nematodes, but neither significantly affected lifespan in two fly species. This study shows that a comparative approach, utilizing multiple invertebrate species, can increase the robustness of invertebrate-based pilot screens for prolongevity interventions.

Inter-organ signalling by HRG-7 promotes systemic haem homeostasis.

Growing evidence in vertebrates predicts that cellular haem levels in animals are maintained not only by a cell's internal capacity for haem synthesis in a cell-autonomous manner, but also by an inter-organ haem trafficking network through cell-non-autonomous regulation. Using Caenorhabditis elegans, a genetically and optically amenable animal model for visualizing haem-dependent signalling, we show that HRG-7, a protein with homology to aspartic proteases, mediates inter-organ signalling between the intestine and extra-intestinal tissues. Intestinal HRG-7 functions as a secreted signalling factor during haem starvation in extra-intestinal tissues and is regulated through a DBL-1, homologous to BMP5, dependent signal from neurons. Given the evidence that vertebrate homologues exist for each of the components of the HRG-7-mediated signalling pathway, it is conceivable that the cell-non-autonomous signalling framework that we uncovered in C. elegans may have functional relevance for inter-organ regulation of iron and haem metabolism in humans.

Loci associated with skin pigmentation identified in African populations.

Despite the wide range of skin pigmentation in humans, little is known about its genetic basis in global populations. Examining ethnically diverse African genomes, we identify variants in or near SLC24A5, MFSD12, DDB1, TMEM138, OCA2, and HERC2 that are significantly associated with skin pigmentation. Genetic evidence indicates that the light pigmentation variant at SLC24A5 was introduced into East Africa by gene flow from non-Africans. At all other loci, variants associated with dark pigmentation in Africans are identical by descent in South Asian and Australo-Melanesian populations. Functional analyses indicate that MFSD12 encodes a lysosomal protein that affects melanogenesis in zebrafish and mice, and that mutations in melanocyte-specific regulatory regions near DDB1/TMEM138 correlate with expression of ultraviolet response genes under selection in Eurasians.

Rapid localized cell trapping on biodegradable polymers using cell surface derivatization and microfluidic networking.

Spatial control over cell attachment is essential for controlling cell behavior and engineering cell-based sensor arrays. Here we report on a patterning procedure that can be utilized on a wide range of adherent and non-adherent cell types without the need to identify the exact peptide sequence or extracellular matrix (ECM) necessary for optimal cell attachment. This is achieved by converting native sialic residues present on the surface of most cells into non-native aldehydes using a mild sodium periodate treatment. The aldehyde groups are then reacted with biotin hydrazide to produce biotinylated cells. Avidin is patterned onto the surface of a biotinylated biodegradable block copolymer, polylactide-poly(ethylene glycol)-biotin (PLA-PEG-biotin) by microfluidic networking using a PDMS stamp. The biotinylated cells then bind specifically to the patterned avidin regions. The PEG that is presented from the PLA-PEG-biotin copolymer in the regions without avidin immobilization minimizes cell binding in the non-patterned regions.

Extracellular HSP60 triggers tissue regeneration and wound healing by regulating inflammation and cell proliferation.

After injury, zebrafish can restore many tissues that do not regenerate well in mammals, making it a useful vertebrate model for studying regenerative biology. We performed a systematic screen to identify genes essential for hair cell regeneration in zebrafish, and found that the heat shock protein Hspd1 (Hsp60) has a critical role in the regeneration of hair cells and amputated caudal fins. We showed HSP60-injected extracellularly promoted cell proliferation and regeneration in both hair cells and caudal fins. We showed that hspd1 mutant was deficient in leukocyte infiltration at the site of injury. Topical application of HSP60 in a diabetic mouse skin wound model dramatically accelerated wound healing compared with controls. Stimulation of human peripheral blood mononuclear cells with HSP60 triggered a specific induction of M2 phase CD163-positive monocytes. Our results demonstrate that the normally intracellular chaperonin HSP60 has an extracellular signalling function in injury inflammation and tissue regeneration, likely through promoting the M2 phase for macrophages.

Culturing Caenorhabditis elegans in axenic liquid media and creation of transgenic worms by microparticle bombardment.

In this protocol, we present the required materials, and the procedure for making modified C. elegans Habituation and Reproduction media (mCeHR). Additionally, the steps for exposing and acclimatizing C. elegans grown on E. coli to axenic liquid media are described. Finally, downstream experiments that utilize axenic C. elegans illustrate the benefits of this procedure. The ability to analyze and determine C. elegans nutrient requirement was illustrated by growing N2 wild type worms in axenic liquid media with varying heme concentrations. This procedure can be replicated with other nutrients to determine the optimal concentration for worm growth and development or, to determine the toxicological effects of drug treatments. The effects of varied heme concentrations on the growth of wild type worms were determined through qualitative microscopic observation and by quantitating the number of worms that grew in each heme concentration. In addition, the effect of varied nutrient concentrations can be assayed by utilizing worms that express fluorescent sensors that respond to changes in the nutrient of interest. Furthermore, a large number of worms were easily produced for the generation of transgenic C. elegans using microparticle bombardment.

It's not what you say, it's how you say it: language use on Facebook impacts employability but not attractiveness.

The expansion and increasing diversity of the Internet has seen a growth in user-generated online content, and an escalation in incorrect and nonstandardized language use (e.g., text speak). This evolution has been exemplified by social networking sites such as Facebook. In our experiment, participants viewed six Facebook profiles whose walls contained status updates that were either spelled correctly, incorrectly, or using text speak, and then rated the profile owners on measures of attractiveness and employability. It was shown that language use had no impact on attractiveness, but users who used correct language were seen as more intelligent, competent, and employable. These results highlight the need to control language in this area of research by demonstrating the variables' seemingly elevated importance to employers compared to peers. The findings also pave the way for further exploration of the Warranting Theory of impression formation online and the role of language in social media-based identity statements and behavioral residue.

Temporal and spatial transcriptional profiles of aging in Drosophila melanogaster.

Temporal and tissue-specific alterations in gene expression have profound effects on aging of multicellular organisms. However, much remains unknown about the patterns of molecular changes in different tissues and how different tissues interact with each other during aging. Previous genomic studies on invertebrate aging mostly utilized the whole body or body parts and limited age-points, and failed to address tissue-specific aging. Here we measured genome-wide expression profiles of aging in Drosophila melanogaster for seven tissues representing nervous, muscular, digestive, renal, reproductive, and storage systems at six adult ages. In each tissue, we identified hundreds of age-related genes exhibiting significant changes of transcript levels with age. The age-related genes showed clear tissue-specific patterns: <10% of them in each tissue were in common with any other tissue; <20% of the biological processes enriched with the age-related genes were in common between any two tissues. A significant portion of the age-related genes were those involved in physiological functions regulated by the corresponding tissue. Nevertheless, we identified some overlaps of the age-related functional groups among tissues, suggesting certain common molecular mechanisms that regulate aging in different tissues. This study is one of the first that defined global, temporal, and spatial changes associated with aging from multiple tissues at multiple ages, showing that different tissues age in different patterns in an organism. The spatial and temporal transcriptome data presented in this study provide a basis and a valuable resource for further genetic and genomic investigation of tissue-specific regulation of aging.