Multiple Novel Functions of Henipavirus O-glycans: The First O-glycan Functions Identified in the Paramyxovirus Family

O-linked glycosylation is a ubiquitous protein modification in organisms belonging to several kingdoms. Both microbial and host protein glycans are used by many pathogens for host invasion and immune evasion, yet little is known about the roles of O-glycans in viral pathogenesis. Reportedly, there is no single function attributed to O-glycans for the significant paramyxovirus family. The paramyxovirus family includes many important pathogens, such as measles, mumps, parainfluenza, metapneumo- and the deadly Henipaviruses Nipah (NiV) and Hendra (HeV) viruses. Paramyxoviral cell entry requires the coordinated actions of two viral membrane glycoproteins: the attachment (HN/H/G) and fusion (F) glycoproteins. O-glycan sites in HeV G were recently identified, facilitating use of the attachment protein of this deadly paramyxovirus as a model to study O-glycan functions. We mutated the identified HeV G O-glycosylation sites and found mutants with altered cell-cell fusion, G conformation, G/F association, viral entry in a pseudotyped viral system, and, quite unexpectedly, pseudotyped viral F protein incorporation and processing phenotypes. These are all important functions of viral glycoproteins. These phenotypes were broadly conserved for equivalent NiV mutants. Thus our results identify multiple novel and pathologically important functions of paramyxoviral O-glycans, paving the way to study O-glycan functions in other paramyxoviruses and enveloped viruses.     

ORM Expression Alters Sphingolipid Homeostasis and Differentially Affects Ceramide Synthase Activity

Sphingolipid synthesis is tightly regulated in eukaryotes. This regulation in plants ensures sufficient sphingolipids to support growth while limiting the accumulation of sphingolipid metabolites that induce programmed cell death. Serine palmitoyltransferase (SPT) catalyzes the first step in sphingolipid biosynthesis and is considered the primary sphingolipid homeostatic regulatory point. In this report, Arabidopsis (Arabidopsis thaliana) putative SPT regulatory proteins, orosomucoid-like proteins AtORM1 and AtORM2, were found to interact physically with Arabidopsis SPT and to suppress SPT activity when coexpressed with Arabidopsis SPT subunits long-chain base1 (LCB1) and LCB2 and the small subunit of SPT in a yeast (Saccharomyces cerevisiae) SPT-deficient mutant. Consistent with a role in SPT suppression, AtORM1 and AtORM2 overexpression lines displayed increased resistance to the programmed cell death-inducing mycotoxin fumonisin B₁, with an accompanying reduced accumulation of LCBs and C16 fatty acid-containing ceramides relative to wild-type plants. Conversely, RNA interference (RNAi) suppression lines of AtORM1 and AtORM2 displayed increased sensitivity to fumonisin B₁ and an accompanying strong increase in LCBs and C16 fatty acid-containing ceramides relative to wild-type plants. Overexpression lines also were found to have reduced activity of the class I ceramide synthase that uses C16 fatty acid acyl-coenzyme A and dihydroxy LCB substrates but increased activity of class II ceramide synthases that use very-long-chain fatty acyl-coenzyme A and trihydroxy LCB substrates. RNAi suppression lines, in contrast, displayed increased class I ceramide synthase activity but reduced class II ceramide synthase activity. These findings indicate that ORM mediation of SPT activity differentially regulates functionally distinct ceramide synthase activities as part of a broader sphingolipid homeostatic regulatory network.Sphingolipids play critical roles in plant growth and development as essential components of endomembranes, including the plasma membrane, where they constitute more than 40% of the total lipid (Sperling et al., 2005; Cacas et al., 2016). Sphingolipids also are highly enriched in detergent-insoluble membrane fractions of the plasma membrane that form microdomains for proteins with important cell surface activities, including cell wall biosynthesis and hormone transport (Cacas et al., 2012, 2016; Perraki et al., 2012; Bayer et al., 2014). In addition, sphingolipids, particularly those with very-long-chain fatty acids (VLCFAs), are integrally associated with Golgi-mediated protein trafficking that underlies processes related to the growth of plant cells (Bach et al., 2008, 2011; Markham et al., 2011; Melser et al., 2011). Furthermore, sphingolipids function through their bioactive long-chain base (LCB) and ceramide metabolites to initiate programmed cell death (PCD), important for mediating plant pathogen resistance through the hypersensitive response (Greenberg et al., 2000; Liang et al., 2003; Shi et al., 2007; Bi et al., 2014; Simanshu et al., 2014).Sphingolipid biosynthesis is highly regulated in all eukaryotes. In plants, the maintenance of sphingolipid homeostasis is vital to ensure sufficient sphingolipids for growth (Chen et al., 2006; Kimberlin et al., 2013) while restricting the accumulation of PCD-inducing ceramides and LCBs until required for processes such as the pathogen-triggered hypersensitive response. Serine palmitoyltransferase (SPT), which catalyzes the first step in LCB synthesis, is generally believed to be the primary control point for sphingolipid homeostasis (Hanada, 2003). SPT synthesizes LCBs, unique components of sphingolipids, by catalyzing a pyridoxal phosphate-dependent condensation of Ser and palmitoyl (16:0)-CoA in plants (Markham et al., 2013). Similar to other eukaryotes, the Arabidopsis (Arabidopsis thaliana) SPT is a heterodimer consisting of LCB1 and LCB2 subunits (Chen et al., 2006; Dietrich et al., 2008; Teng et al., 2008). Research to date has shown that SPT is regulated primarily by posttranslational mechanisms involving physical interactions with noncatalytic, membrane-associated proteins that confer positive and negative regulation of SPT activity (Han et al., 2009, 2010; Breslow et al., 2010). These proteins include a 56-amino acid small subunit of SPT (ssSPT) in Arabidopsis, which was recently shown to stimulate SPT activity and to be essential for generating sufficient amounts of sphingolipids for pollen and sporophytic cell viability (Kimberlin et al., 2013).Evidence from yeast and mammalian research points to a more critical role for proteins termed ORMs (for orosomucoid-like proteins) in sphingolipid homeostatic regulation (Breslow et al., 2010; Han et al., 2010). The Saccharomyces cerevisiae Orm1p and Orm2p negatively regulate SPT through reversible phosphorylation of these polypeptides in response to intracellular sphingolipid levels (Breslow et al., 2010; Han et al., 2010; Roelants et al., 2011; Gururaj et al., 2013; Muir et al., 2014). Phosphorylation/dephosphorylation of ORMs in S. cerevisiae presumably affects the higher order assembly of SPT to mediate flux through this enzyme for LCB synthesis (Breslow, 2013). In this sphingolipid homeostatic regulatory mechanism, the S. cerevisiae Orm1p and Orm2p are phosphorylated at their N termini by Ypk1, a TORC2-dependent protein kinase (Han et al., 2010; Roelants et al., 2011). The absence of this phosphorylation domain in mammalian and plant ORM homologs brings into question the nature of SPT reversible regulation by ORMs in other eukaryotic systems (Hjelmqvist et al., 2002).Sphingolipid synthesis also is mediated by the N-acylation of LCBs by ceramide synthases to form ceramides, the hydrophobic backbone of the major plant glycosphingolipids, glucosylceramide (GlcCer) and glycosyl inositolphosphoceramide (GIPC). Two functionally distinct classes of ceramide synthases occur in Arabidopsis, designated class I and class II (Chen et al., 2008). Class I ceramide synthase activity resulting from the Longevity Assurance Gene One Homolog2 (LOH2)-encoded ceramide synthase acylates, almost exclusively, LCBs containing two hydroxyl groups (dihydroxy LCBs) with 16:0-CoA to form C16 ceramides, which are used primarily for GlcCer synthesis (Markham et al., 2011; Ternes et al., 2011; Luttgeharm et al., 2016). Class II ceramide synthase activities resulting from the LOH1- and LOH3-encoded ceramide synthases are most active in the acylation of LCBs containing three hydroxyl groups (trihydroxy LCBs) with VLCFA-CoAs, including primarily C₂₄ and C₂₆ acyl-CoAs (Markham et al., 2011; Ternes et al., 2011; Luttgeharm et al., 2016). Class II (LOH1 and LOH3) ceramide synthase activity is essential for producing VLCFA-containing glycosphingolipids to support the growth of plant cells, whereas class I (LOH2) ceramide synthase activity is nonessential under normal growth conditions (Markham et al., 2011; Luttgeharm et al., 2015b). It was speculated recently that LOH2 ceramide synthase functions, in part, as a safety valve to acylate excess LCBs for glycosylation, resulting in a less cytotoxic form (Luttgeharm et al., 2015b; Msanne et al., 2015). Recent studies have shown that the Lag1/Lac1 components of the S. cerevisiae ceramide synthase are phosphorylated by Ypk1, and this phosphorylation stimulates ceramide synthase activity in response to heat and reduced intracellular sphingolipid levels (Muir et al., 2014). This finding points to possible coordinated regulation of ORM-mediated SPT and ceramide synthase activities to regulate sphingolipid homeostasis, which is likely more complicated in plants and mammals due to the occurrence of functionally distinct ceramide synthases in these systems (Stiban et al., 2010; Markham et al., 2011; Ternes et al., 2011; Luttgeharm et al., 2016).RNA interference (RNAi) suppression of ORM genes in rice (Oryza sativa) has been shown to affect pollen viability (Chueasiri et al., 2014), but no mechanistic characterization of ORM proteins in plants has yet to be reported. Here, we describe two Arabidopsis ORMs, AtORM1 and AtORM2, that suppress SPT activity through direct interaction with the LCB1/LCB2 heterodimer. We also show that strong up-regulation of AtORM expression impairs growth. In addition, up- or down-regulation of ORMs is shown to differentially affect the sensitivity of Arabidopsis to the PCD-inducing mycotoxin fumonisin B₁ (FB₁), a ceramide synthase inhibitor, and to differentially affect the activities of class I and II ceramide synthases as a possible additional mechanism for regulating sphingolipid homeostasis.     

The Protective Association between Pet Ownership and Depression among Street-involved Youth: A Cross-sectional Study

Street-involved youth represent a particularly vulnerable subsection of the homeless population and are at increased risk of health problems, substance abuse, and depression. Qualitative research has demon- strated that animal companions help homeless youth cope with loneliness, are motivators for positive change, such as decreasing drug or alcohol use, provide unconditional love without judgement, and improve youths’ sense of health. To quantitatively investigate the association between depression and pet ownership among street-involved youth, a cross-sectional study was per- formed with a convenience sample of 189 street-involved youths who were surveyed in four cities in Ontario, Canada, 89 of whom were pet owners and 100 of whom were not. Logistic regression modelling found pet ownership to be negatively associated with depression in the study population (controlling for gender, regular use of drugs, and time since youth left home), with the odds of being depressed three times greater for youths who did not own pets. While pet ownership among street-involved youth has many liabilities, includ- ing impairing youths’ ability to access shelter, services, and housing and employment opportunities, companion animals may offer both physical and psychosocial benefits that youth have difficult attaining. This finding highlights the importance of increased awareness among youth service providers of the potential impacts of pet ownership for street-involved youth.     

Plant–pollinator interactions in a Mexican Acacia community

Competition for pollination is thought to be an important factor structuring flowering in many plant communities, particularly among plant taxa with morphologically similar and easily accessible flowers. We examined the potential for heterospecific pollen transfer (HPT) in a community of four Acacia species in a highly seasonal tropical habitat in Mexico. Partitioning of pollen flow among sympatric species appears to be achieved, in part, through segregation of flowering in seasonal time, and interspecific differences in pollinator guilds. However, two coflowering species (Acacia macracantha and Acacia angustissima) shared multiple flower visitors, raising the possibility of HPT. Each of these coflowering species showed high intraspecific daily synchrony in pollen release, but dehisce at different times of day. Pollinators rapidly harvested available pollen from one species before abandoning it to visit the flowers of the second later in the day. The activity of shared pollinators, predominantly bees, is thus structured throughout the day, and potential for HPT reduced. Suggestive evidence in favour of a resource partitioning explanation for this pattern is provided by the fact that A. macracantha showed significantly greater intraspecific synchrony when coflowering with a potential competitor (A. angustissima) than when flowering alone. We discuss our results in light of previous work on coflowering acacia assemblages in Tanzania and Australia. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.

Distributed representations accelerate evolution of adaptive behaviours

Animals with rudimentary innate abilities require substantial learning to transform those abilities into useful skills, where a skill can be considered as a set of sensory–motor associations. Using linear neural network models, it is proved that if skills are stored as distributed representations, then within-lifetime learning of part of a skill can induce automatic learning of the remaining parts of that skill. More importantly, it is shown that this “free-lunch” learning (FLL) is responsible for accelerated evolution of skills, when compared with networks which either 1) cannot benefit from FLL or 2) cannot learn. Specifically, it is shown that FLL accelerates the appearance of adaptive behaviour, both in its innate form and as FLL-induced behaviour, and that FLL can accelerate the rate at which learned behaviours become innate.     

Basis for the reduced affinity of beta T- and gamma T-thrombin for hirudin

Partial proteolysis of human alpha-thrombin by trypsin results in the formation of beta T-thrombin and gamma T-thrombin which have a reduced affinity for the inhibitor hirudin and the cell-surface cofactor thrombomodulin as well as reduced activity with fibrinogen. The basis of the reduction in affinity of these thrombin derivatives for hirudin has been investigated by examining their kinetics of interaction with a number of hirudin mutants differing in their C-terminal charge properties as well as with a truncated form of hirudin. The results indicate that the reduced affinity of beta T-thrombin for hirudin is most likely due to a decrease in the strength of nonionic interactions between thrombin and the C-terminal region of hirudin. No decrease in the strength of ionic interactions was observed with beta T-thrombin. In contrast, the reduced affinity of gamma T-thrombin was due to a decrease in the strength of both ionic and nonionic interactions. The N-terminal core region of hirudin, which interacts predominantly with the active-site cleft of thrombin, exhibited similar affinities for alpha-, beta T-, and gamma T-thrombin, indicating that thrombin-hirudin interactions within the active site are largely preserved in beta T- and gamma T-thrombin.     

Intercalation of the (1R,2S,3R,4S)-N6-[1-(1,2,3,4-tetrahydro-2,3,4-trihydroxybenz[a]anthracenyl)]-2'-deoxyadenosyl adduct in the N-ras codon 61 sequence: DNA sequence effects

The structure of the bay region (1R,2S,3R,4S)-N6-[1-(1,2,3,4-tetrahydro-2,3,4-trihydroxybenz[a]anthracenyl)]-2'-deoxyadenosyl adduct at X(7) of 5'-d(CGGACAXGAAG)-3'.5'-d(CTTCTTGTCCG)-3', incorporating codons 60, 61 (underlined), and 62 of the human N-ras protooncogene, was determined by NMR. This was the bay region benz[a]anthracene RSRS (61,3) adduct. The BA moiety intercalated above the 5'-face of the modified base pair. NOE connectivities between imino protons were disrupted at T16 and T17. Large chemical shifts at the lesion site were consistent with ring current shielding arising from the BA moiety. A large chemical shift dispersion was observed for the BA aromatic protons. An increased rise of 8.17 A was observed between base pairs A6 x T17 and X7 x T(16). The PAH moiety stacked with the purine ring of A6, the 5'-neighbor nucleotide. This resulted in buckling of the 5'-neighbor A6 x T17 base pair, evidenced by exchange broadening for the T17 imino resonance. It also interrupted sequential NOE connectivities between nucleotides C5 and A6. The A6 deoxyribose ring showed an increased percentage of the C3'-endo conformation. This differed from the bay region BA RSRS (61,2) adduct, in which the lesion was located at position X6 [Li, Z., Mao, H., Kim, H.-Y., Tamura, P. J., Harris, C. M., Harris, T. M., and Stone, M. P. (1999) Biochemistry 38, 2969-2981], but was similar to the benzo[a]pyrene BP SRSR (61,3) adduct [Zegar I. S., Chary, P., Jabil, R. J., Tamura, P. J., Johansen, T. N., Lloyd, R. S., Harris, C. M., Harris, T. M., and Stone, M. P. (1998) Biochemistry 37, 16516-16528]. The altered sugar pseudorotation at A6 appears to be common to both bay region BA RSRS (61,3) and BP SRSR (61,3) adducts. It could not be discerned if the C3'-endo conformation at A6 in the BA RSRS (61,3) adduct altered base pairing geometry at X7 x T16, as compared to the C2'-endo conformation. The structural studies suggest that the mutational spectrum of this adduct may be more complex than that of the BA RSRS (61,2) adduct.