Through the looking-glass: Observing HSCs through the lens of infection reveals unexpected insights into HSC function

Comment on: Hall CJ, et al. Cell Stem Cell 2012; 10:198-209.     

Feline immunodeficiency virus in puma: Estimation of force of infection reveals insights into transmission

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Inflammatory modulation of HSCs: viewing the HSC as a foundation for the immune response

Cells of the innate and adaptive immune systems are the progeny of a variety of haematopoietic precursors, the most primitive of which is the haematopoietic stem cell. Haematopoietic stem cells have been thought of generally as dormant cells that are only called upon to divide under extreme conditions, such as bone marrow ablation through radiation or chemotherapy. However, recent studies suggest that haematopoietic stem cells respond directly and immediately to infections and inflammatory signals. In this Review, we summarize the current literature regarding the effects of infection on haematopoietic stem cell function and how these effects may have a pivotal role in directing the immune response from the bone marrow.     

Voltage-dependant anion channels: Novel insights into isoform function through genetic models

Voltage-dependant Anion Channels, also known as mitochondrial porins, are pore-forming proteins located in the mitochondrial outer membrane (MOM) that, in addition to forming complexes with other proteins that localize to the MOM, also function as the main conduit for transporting metabolites between the cytoplasm and mitochondria. VDACs are encoded by a multi-member gene family, and the number of isoforms and specific functions of VDACs varies between species. Translating the well-described in vitro characteristics of the VDAC isoforms into in vivo functions has been a challenge, with the generation of animal models of VDAC deficiency providing much of the available information about isoform-specific roles in biology. Here, we review the approaches used to create these insect and mammalian animal models, and the conclusions reached by studying the consequences of loss of function mutations on the genetic, physiologic, and biochemical properties of the resulting models. This article is part of a Special Issue entitled: VDAC structure, function, and regulation of mitochondrial metabolism.     

Production of human type I collagen in yeast reveals unexpected new insights into the molecular assembly of collagen trimers

Substantial evidence supports the role of the procollagen C-propeptide in the initial association of procollagen polypeptides and for triple helix formation. To evaluate the role of the propeptide domains on triple helix formation, human recombinant type I procollagen, pN-collagen (procollagen without the C-propeptides), pC-collagen (procollagen without the N-propeptides), and collagen (minus both propeptide domains) heterotrimers were expressed in Saccharomyces cerevisiae. Deletion of the N- or C-propeptide, or both propeptide domains, from both proalpha-chains resulted in correctly aligned triple helical type I collagen. Protease digestion assays demonstrated folding of the triple helix in the absence of the N- and C-propeptides from both proalpha-chains. This result suggests that sequences required for folding of the triple helix are located in the helical/telopeptide domains of the collagen molecule. Using a strain that does not contain prolyl hydroxylase, the same folding mechanism was shown to be operative in the absence of prolyl hydroxylase. Normal collagen fibrils were generated showing the characteristic banding pattern using this recombinant collagen. This system offers new opportunities for the study of collagen expression and maturation.     

Interrogating the substrate specificity landscape of UvrC reveals novel insights into its non-canonical function

Although it is relatively unexplored, accumulating data highlight the importance of tripartite crosstalk between nucleotide excision repair (NER), DNA replication, and recombination in the maintenance of genome stability; however, elucidating the underlying mechanisms remains challenging. While Escherichia coli uvrA and uvrB can fully complement polAΔ cells in DNA replication, uvrC attenuates this alternative DNA replication pathway, but the exact mechanism by which uvrC suppresses DNA replication is unknown. Furthermore, the identity of bona fide canonical and non-canonical substrates for UvrCs are undefined. Here, we reveal that Mycobacterium tuberculosis UvrC (MtUvrC) strongly binds to, and robustly cleaves, key intermediates of DNA replication/recombination as compared with the model NER substrates. Notably, inactivation of MtUvrC ATPase activity significantly attenuated its endonuclease activity, thus suggesting a causal link between these two functions. We built an in silico model of the interaction of MtUvrC with the Holliday junction (HJ), using a combination of homology modeling, molecular docking, and molecular dynamic simulations. The model predicted residues that were potentially involved in HJ binding. Six of these residues were mutated either singly or in pairs, and the resulting MtUvrC variants were purified and characterized. Among them, residues Glu595 and Arg597 in the helix-hairpin-helix motif were found to be crucial for the interaction between MtUvrC and HJ; consequently, mutations in these residues, or inhibition of ATP hydrolysis, strongly abrogated its DNA-binding and endonuclease activities. Viewed together, these findings expand the substrate specificity landscape of UvrCs and provide crucial mechanistic insights into the interplay between NER and DNA replication/recombination.     

Systematic whole-genome sequencing reveals an unexpected diversity among actinomycetoma pathogens and provides insights into their antibacterial susceptibilities

Mycetoma is a neglected tropical chronic granulomatous inflammatory disease of the skin and subcutaneous tissues. More than 70 species with a broad taxonomic diversity have been implicated as agents of mycetoma. Understanding the full range of causative organisms and their antibiotic sensitivity profiles are essential for the appropriate treatment of infections. The present study focuses on the analysis of full genome sequences and antibiotic inhibitory concentration profiles of actinomycetoma strains from patients seen at the Mycetoma Research Centre in Sudan with a view to developing rapid diagnostic tests. Seventeen pathogenic isolates obtained by surgical biopsies were sequenced using MinION and Illumina methods, and their antibiotic inhibitory concentration profiles determined. The results highlight an unexpected diversity of actinomycetoma causing pathogens, including three Streptomyces isolates assigned to species not previously associated with human actinomycetoma and one new Streptomyces species. Thus, current approaches for clinical and histopathological classification of mycetoma may need to be updated. The standard treatment for actinomycetoma is a combination of sulfamethoxazole/trimethoprim and amoxicillin/clavulanic acid. Most tested isolates had a high IC (inhibitory concentration) to sulfamethoxazole/trimethoprim or to amoxicillin alone. However, the addition of the β-lactamase inhibitor clavulanic acid to amoxicillin increased susceptibility, particularly for Streptomyces somaliensis and Streptomyces sudanensis. Actinomadura madurae isolates appear to have a particularly high IC under laboratory conditions, suggesting that alternative agents, such as amikacin, could be considered for more effective treatment. The results obtained will inform future diagnostic methods for the identification of actinomycetoma and treatment.     

Apolipoprotein E structure: insights into function

Human apolipoprotein E (apoE) is a member of the family of soluble apolipoproteins. Through its interaction with members of the low-density lipoprotein receptor family, apoE has a key role in lipid transport both in the plasma and in the central nervous system. Its three common structural isoforms differentially affect the risk of developing atherosclerosis and neurodegenerative disorders, including Alzheimer's disease. Because the function of apoE is dictated by its structure, understanding the structural properties of apoE and its isoforms is required both to determine its role in disease and for the development of therapeutic strategies.     

Novel insulin/GIP co-producing cell lines provide unexpected insights into Gut K-cell function in vivo

Enteroendocrine (EE) cells represent complex, rare, and diffusely-distributed intestinal epithelial cells making them difficult to study in vivo. A specific sub-population of EE cells called Gut K-cells produces and secretes glucose-dependent insulinotropic peptide (GIP), a hormone important for glucose homeostasis. The factors that regulate hormone production and secretion, as well as the timing of peptide release, are remarkably similar for K-cells and islet beta-cells suggesting engineering insulin production by K-cells is a potential gene therapeutic strategy to treat diabetes. K-cell lines could be used to study the feasibility of this potential therapy and to understand Gut K-cell physiology in general. Heterogeneous STC-1 cells were transfected with a plasmid (pGIP/Neo) encoding neomycin phosphotransferase, driven by the GIP promoter-only cells in which the GIP promoter was active survived genetic selection. Additional clones expressing pGIP/Neo plus a GIP promoter/insulin transgene were isolated-only doubly transfected cells produced preproinsulin mRNA. Bioactive insulin was stored and then released following stimulation with arginine, peptones, and bombesin-physiological GIP secretagogues. Like K-cells in vivo, the GIP/insulin-producing cells express the critical glucose sensing enzyme, glucokinase. However, glucose did not regulate insulin or GIP secretion or mRNA levels. Conversely, glyceraldehyde and methyl-pyruvate were secretagogues, indicating cells depolarized in response to changes in intracellular metabolite levels. Potassium channel opening drugs and sulphonylureas had little effect on insulin secretion by K-cells. The K-cell lines also express relatively low levels of Kir 6.1, Kir 6.2, SUR1, and SUR2 suggesting secretion is independent of K(ATP) channels. These results provided unexpected insights into K-cell physiology and our experimental strategy could be easily modified to isolate/characterize additional EE cell populations.     

A degron-based strategy reveals new insights into Aurora B function in C. elegans

The widely conserved kinase Aurora B regulates important events during cell division. Surprisingly, recent work has uncovered a few functions of Aurora-family kinases that do not require kinase activity. Thus, understanding this important class of cell cycle regulators will require strategies to distinguish kinase-dependent from independent functions. Here, we address this need in C. elegans by combining germline-specific, auxin-induced Aurora B (AIR-2) degradation with the transgenic expression of kinase-inactive AIR-2. Through this approach, we find that kinase activity is essential for AIR-2’s major meiotic functions and also for mitotic chromosome segregation. Moreover, our analysis revealed insight into the assembly of the ring complex (RC), a structure that is essential for chromosome congression in C. elegans oocytes. AIR-2 localizes to chromosomes and recruits other components to form the RC. However, we found that while kinase-dead AIR-2 could load onto chromosomes, other components were not recruited. This failure in RC assembly appeared to be due to a loss of RC SUMOylation, suggesting that there is crosstalk between SUMOylation and phosphorylation in building the RC and implicating AIR-2 in regulating the SUMO pathway in oocytes. Similar conditional depletion approaches may reveal new insights into other cell cycle regulators.     

Neurobiology through the looking-glass: D-serine as a new glial-derived transmitter

D-Amino acids have been known to be present in bacteria for more than 50 years, but only recently they were identified in mammals. The occurrence of D-amino acids in mammals challenge classic concepts in biology in which only L-amino acids would be present or thought to play important roles. Recent discoveries uncovered a role of endogenous D-serine as a putative glial-derived transmitter that regulates glutamatergic neurotransmission in mammalian brain. Free D-serine levels in the brain are about one third of L-serine values and its extracellular concentration is higher than many common L-amino acids. D-Serine occurs in protoplasmic astrocytes, a class of glial cells that ensheath the synapses and modulate neuronal activity. Biochemical and electrophysiological studies suggest that endogenous D-serine is a physiological modulator at the co-agonist site of NMDA-type of glutamate receptors. We previously showed that D-serine is synthesized by a glial serine racemase, a novel enzyme converting L- to D-serine in mammalian brain. The enzyme requires pyridoxal 5'-phosphate and it was the first racemase to be cloned from eucaryotes. Inhibitors of serine racemase have therapeutic implications for pathological processes in which over-stimulation of NMDA receptors takes place, such as stroke and neurodegenerative diseases. Here, we review the role of endogenous D-serine in modulating NMDA neurotransmission, its biosynthetic apparatus and the potential usefulness of serine racemase inhibitors as a novel neuroprotective strategy to decrease glutamate/NMDA excitotoxicity.     

First insights into the function of the sawshark rostrum through examination of rostral tooth microwear

Potential roles of the rostrum of sawsharks (Pristiophoridae), including predation and self‐defence, were assessed through a variety of inferential methods. Comparison of microwear on the surface of the rostral teeth of sawsharks and sawfishes (Pristidae) show that microwear patterns are alike and suggest that the elongate rostra in these two elasmobranch families are used for a similar purpose (predation). Raman spectroscopy indicates that the rostral teeth of both sawsharks and sawfishes are composed of hydroxyapatite, but differ in their collagen content. Sawfishes possess collagen throughout their rostral teeth whereas collagen is present only in the centre of the rostral teeth of sawsharks, which may relate to differences in ecological use. The ratio of rostrum length to total length in the common sawshark Pristiophorus cirratus was found to be similar to the largetooth sawfish Pristis pristis but not the knifetooth sawfish Anoxypristis cuspidata. Analysis of the stomach contents of P. cirratus indicates that the diet consists of demersal fishes and crustaceans, with shrimp from the family Pandalidae being the most important dietary component. No prey item showed evidence of wounds inflicted by the rostral teeth. In light of the similarities in microwear patterns, rostral tooth chemistry and diet with sawfishes, it is hypothesised that sawsharks use their rostrum in a similar manner for predation (sensing and capturing prey) and possibly for self‐defence.     

Glycosphingolipids in engineered mice: insights into function

Recent success in the cloning of glycosyl-transferase genes involved in the synthesis of GSLs has enabled us to modulate the expression profiles of GSLs in cultured cells and experimental animals, and allowed novel approaches to obtain clear elucidation of individual enzyme products by observing the resulting phenotypic changes in the mutant animals and transfected cells. In this review, recent progress in the study of glycosyltransferases involved in the synthesis and modification of GSLs has been summarized with special emphasis on their function.     

Common dolphins form unexpected strong social bonds: insights into social plasticity of delphinids

When a social species inhabits disparate environments with different requirements, it presents an ideal study framework for investigating plasticity in social structure. Common dolphins (Delphinus delphis) are wide-ranging offshore delphinids that generally form societies with fission-fusion dynamics within large schools and exhibit weak social bonds. In Port Phillip, southeastern Australia, common dolphins of the same species are, against expectations, resident to an embayment. Residency in this species provides a unique opportunity to investigate whether their social structure resembles that of their offshore conspecifics with weak social bonds, or whether bay living leads to stronger social bonds. We investigated the social structure of 12 resident adult common dolphins, between 2007 and 2014, in Port Phillip. Network analyses revealed nonrandom associations and several strong bonds, a social structure unusual for this species. The study shows that the social structure of a wide-ranging gregarious species in Port Phillip reflects the requirements of a confined environment with limited but predictable resources. Their social structure in the bay resembles that of inshore delphinids, rather than of its own species. Our study highlights the extreme plasticity in social structure that common dolphins are capable of and the importance of the environment for social bonds.