Gene Expression in the Microfilariae of Brugia pahangi

Filarial nematodes have a complex developmental life cycle involving mammalian and mosquito hosts. The microfilariae (mf) which circulate in the blood stream are developmentally blocked prior to transfer to the mosquito. The switch between the different environments must be accompanied by changes in gene expression to allow development within the new host. The aim of this study was to isolate mf genes from the mf of B. pahangi that are differentially expressed within the two hosts. To this end a mammalian-derived mf cDNA library was constructed and differentially screened using mammalian and mosquito-derived mf cDNA probes. Due to the difficulty of obtaining in vivo parasites from the mosquito, the cDNA probes were produced by RT-PCR from mf culture in vitro under either mammalian or mosquito like conditions. Nine independent cDNA clones were isolated, of which five hybridised more strongly to the mammalian-derived mf probe and four to the vector-derived mf probe. Analysis of the cDNAs nucleotide sequence revealed that five of the clones were homologous to ribosomal protein mRNAs previously characterised from other species, one cDNA corresponded to the B. pahangi heat shock protein 90 mRNA and three cDNAs represented novel genes of unknown function. A more detailed molecular analysis was conducted on two of the cDNAs of unknown function, Bp-vmc-2 (B. pahangi-vector-derived mf cDNA-2) and Bp-mmc- 1 (B. pahangi-mammalian-derived mf cDNA-1). Analysis of the expression of the mRNAs by semi-quantitative RT-PCR showed that Bp-vmc-2 was expressed at varying levels throughout the life-cycle of B. pahangi. The mRNA abundance of Bp- vmc-2 increased as the parasite matured in the mosquito host and reached peak expression in infective L3 parasites. In contrast Bp-mmc-1 was exclusive to the mf stage and was not expressed by mf developing in utero. An antiserum raised to the recombinant protein localised MMC-1 throughout the body of the mf, but it was not present on the surface of the sheath or cuticle of the parasite. Western blotting using MMC-1 antiserum, reacted specifically with mf extracts, confirming the specificity of temporal expression. Analysis of immune responses to the recombinant protein showed that significant amounts of IL-5 were produced by T-cells in response to stimulation with MMC-1. Human serum from patients infected with B. malayi recognised MMC-1 and the predominant reactive immunoglobulin subclasses were IgGl and IgG3, which have been associated with disease pathology in other studies

The Human Postsynaptic Density Shares Conserved Elements with Proteomes of Unicellular Eukaryotes and Prokaryotes

The animal nervous system processes information from the environment and mediates learning and memory using molecular signaling pathways in the postsynaptic terminal of synapses. Postsynaptic neurotransmitter receptors assemble to form multiprotein complexes that drive signal transduction pathways to downstream cell biological processes. Studies of mouse and Drosophila postsynaptic proteins have identified key roles in synaptic physiology and behavior for a wide range of proteins including receptors, scaffolds, enzymes, structural, translational, and transcriptional regulators. Comparative proteomic and genomic studies identified components of the postsynaptic proteome conserved in eukaryotes and early metazoans. We extend these studies, and examine the conservation of genes and domains found in the human postsynaptic density with those across the three superkingdoms, archaeal, bacteria, and eukaryota. A conserved set of proteins essential for basic cellular functions were conserved across the three superkingdoms, whereas synaptic structural and many signaling molecules were specific to the eukaryote lineage. Genes involved with metabolism and environmental signaling in Escherichia coli including the chemotactic and ArcAB Two-Component signal transduction systems shared homologous genes in the mammalian postsynaptic proteome. These data suggest conservation between prokaryotes and mammalian synapses of signaling mechanisms from receptors to transcriptional responses, a process essential to learning and memory in vertebrates. A number of human postsynaptic proteins with homologs in prokaryotes are mutated in human genetic diseases with nervous system pathology. These data also indicate that structural and signaling proteins characteristic of postsynaptic complexes arose in the eukaryotic lineage and rapidly expanded following the emergence of the metazoa, and provide an insight into the early evolution of synaptic mechanisms and conserved mechanisms of learning and memory

Method for RNA extraction and transcriptomic analysis of single fungal spores

Transcriptomic analysis of single cells has been increasingly in demand in recent years, thanks to technological and methodological advances as well as growing recognition of the importance of individuals in biological systems. However, the majority of these studies have been performed in mammalian cells, due to their ease of lysis and high RNA content. No single cell transcriptomic analysis has yet been applied to microbial spores, even though it is known that heterogeneity at the phenotype level exists among individual spores. Transcriptomic analysis of single spores is challenging, in part due to the physically robust nature of the spore wall. This precludes the use of methods commonly used for mammalian cells. Here, we describe a simple method for extraction and amplification of transcripts from single fungal conidia (asexual spores), and its application in single-cell transcriptomics studies. The method can also be used for studies of small numbers of fungal conidia, which may be necessary in the case of limited sample availability, low-abundance transcripts or interest in small subpopulations of conidia.• The method allows detection of transcripts from single conidia of Aspergillus niger• The method allows detection of genomic DNA from single conidia of Aspergillus nige

Novel insights into viral infection and oncogenesis from Koala Retrovirus (KoRV) infection of HEK293T cells

Koala retrovirus is thought to be an underlying cause of high levels of neoplasia and immunosuppression in koalas. While epidemiology studies suggest a strong link between KoRV and disease it has been difficult to prove causality because of the complex nature of the virus, which exists in both endogenous and exogenous forms. It has been difficult to identify koalas completely free of KoRV, and infection studies in koalas or koala cells are fraught with ethical and technical difficulties, respectively. This study uses KoRV infection of the susceptible human cell line HEK293T and RNAseq to demonstrate gene networks differentially regulated upon KoRV infection. Many of the pathways identified are those associated with viral infection, such as cytokine receptor interactions and interferon signalling pathways, as well as viral oncogenesis pathways. This study provides strong evidence that KoRV does indeed behave similarly to infectious retroviruses in stimulating antiviral and oncogenic cellular responses. In addition, it provides novel insights into KoRV oncogenesis with the identification of a group of histone family genes that are part of several oncogenic pathways as upregulated in KoRV infection

Koala retrovirus viral load and disease burden in distinct northern and southern koala populations

Koala retrovirus (KoRV) displays features of both an endogenous and exogenous virus and is linked to neoplasia and immunosuppression in koalas. This study explores the apparent differences in the nature and impact of KoRV infection between geographically and genetically separated "northern" and "southern" koala populations, by investigating the disease status, completeness of the KoRV genome and the proviral (DNA) and viral (RNA) loads of 71 northern and 97 southern koalas. All northern animals were positive for all KoRV genes (gag, pro-pol and env) in both DNA and RNA forms, whereas many southern animals were missing one or more KoRV genes. There was a significant relationship between the completeness of the KoRV genome and clinical status in this population. The proviral and viral loads of the northern population were significantly higher than those of the southern population (P

Investigative power of Genomic Informational Field Theory (GIFT) relative to GWAS for genotype-phenotype mapping

Identifying associations between phenotype and genotype is the fundamental basis of genetic analyses. Inspired by frequentist probability and the work of R.A. Fisher, genome-wide association studies (GWAS) extract information using averages and variances from genotype-phenotype datasets. Averages and variances are legitimated upon creating distribution density functions obtained through the grouping of data into categories. However, as data from within a given category cannot be differentiated, the investigative power of such methodologies is limited. Genomic Informational Field Theory (GIFT) is a method specifically designed to circumvent this issue. The way GIFT proceeds is opposite to that of GWAS. Whilst GWAS determines the extent to which genes are involved in phenotype formation (bottom-up approach), GIFT determines the degree to which the phenotype can select microstates (genes) for its subsistence (top-down approach). Doing so requires dealing with new genetic concepts, a.k.a. genetic paths, upon which significance levels for genotype-phenotype associations can be determined. By using different datasets obtained in ovis aries related to bone growth (Dataset-1) and to a series of linked metabolic and epigenetic pathways (Dataset-2), we demonstrate that removing the informational barrier linked to categories enhances the investigative and discriminative powers of GIFT, namely that GIFT extracts more information than GWAS. We conclude by suggesting that GIFT is an adequate tool to study how phenotypic plasticity and genetic assimilation are linked.</p

Unbiased analysis of the impact of micropatterned biomaterials on macrophage behaviour provides insights beyond pre-defined polarisation states

Macrophages are master regulators of immune responses towards implanted biomaterials. The activation state adopted by macrophages in response to biomaterials determines their own phenotype and function as well as those of other resident and infiltrating immune and non-immune cells in the area. A wide spectrum of macrophage activation states exists, with M1 (pro-inflammatory) and M2 (anti-inflammatory) representing either ends of the spectrum. In biomaterials research, cellinstructive surfaces that favour or induce M2 macrophages have been considered as beneficial due to the anti-inflammatory and pro-regenerative properties of these cells. In this study, we used a gelatin methacryloyl (GelMA) hydrogel platform to determine whether micropatterned surfaces can modulate the phenotype and function of human macrophages. The effect of microgrooves/ridges and micropillars on macrophage phenotype, function, and gene expression profile were assessed using conventional methods (morphology, cytokine profile, surface marker expression, phagocytosis) and gene microarrays. Our results demonstrated that micropatterns did induce distinct gene expression profiles in human macrophages cultured on microgrooves/ridges and micropillars. Significant changes were observed in genes related to primary metabolic processes such as transcription, translation, protein trafficking, DNA repair and cell survival. However, interestingly conventional phenotyping methods, relying on surface marker expression and cytokine profile, were not able to distinguish between the different conditions, and indicated no clear shift in cell activation towards an M1 or M2 phenotypes. This highlights the limitations of studying the effect of different physicochemical conditions on macrophages by solely relying on conventional markers that are primarily developed to differentiate between cytokine polarised M1 and M2 macrophages. We therefore, propose the adoption of unbiased screening methods in determining macrophage responses to biomaterials. Our data clearly shows that the exclusive use of conventional markers and methods for determining macrophage activation status could lead to missed opportunities for understanding and exploiting macrophage responses to biomaterials

Genetic diversity of Koala retrovirus (KoRV) env gene subtypes: Insights into northern and southern koala populations

Koala retrovirus (KoRV) is a recently endogenised retrovirus associated with neoplasia and immunosuppression in koala populations. The virus is known to display sequence variability and to be present at varying prevalence in different populations, with animals in southern Australia displaying lower prevalence and viral loads than northern animals. This study used a PCR and next generation sequencing strategy to examine the diversity of the KoRV env gene in both proviral DNA and viral RNA forms in two distinct populations representative of the “northern” and “southern” koala genotypes. The current study demonstrated that the full range of KoRV subtypes is present across both populations, and in both healthy and sick animals. KoRV-A was the predominant proviral subtype in both populations, but there was marked diversity of DNA and RNA subtypes within individuals. Many of the northern animals displayed a higher RNA viral diversity than evident in their proviral DNA, indicating relatively higher replication efficiency of non-KoRV-A subtypes. The southern animals displayed a lower absolute copy number of KoRV than the northern animals as reported previously and a higher preponderance of KoRV-A in individual animals. These discrepancies in viral replication and diversity remain unexplained but may indicate relative protection of the southern population from KoRV replication due to either viral or host factors and may represent an important protective effect for the host in KoRV’s ongoing entry into the koala genome

Author Correction:The Beta-adrenergic agonist, Ractopamine, increases skeletal muscle expression of Asparagine Synthetase as part of an integrated stress response gene program

© 2019, The Author(s). In the Supplementary Information file originally published with this Article, Table 2 was omitted. This error has been corrected in the Supplementary Information that now accompanies the Article