Two new species of Western Australian Abantiades Herrich-Schffer (Lepidoptera: Hepialidae) and a description of the female of Abantiades paradoxa (Tindale).

Two new species, Abantiades concordia sp. nov. and Abantiades malleus sp. nov., are described from Australia. Both species were collected in the Eastern Goldfields subregion of the Coolgardie bioregion in Western Australia. Abantiades concordia sp. nov. is shown to be closely related to A. paradoxa (Tindale, 1932) by sequence similarity of the mtDNA (COI) gene. The female of A. paradoxa is also described here for the first time. Abantiades paradoxa and the new species A. concordia sp. nov. are morphologically similar with respect to the structure of their genitalia, sternite VIII, wing patterning and their antennae with bi-forked rami. Abantiades malleus sp. nov. is quite distinct by sequence similarity of the mtDNA (COI) gene, but related in a clade with A. marcidus Tindale,1932, A. albofasciatus (Swinhoe, 1892), and A. furva (Tindale,1932), the latter species once placed in the synonymised Bordaia Tindale, 1932. Discussion of similar species once grouped under the genus Bordaia and under the genus Trictena Meyrick, 1890 (both junior synonyms of Abantiades Herrich-Schffer, 1855) is also included.

Two new endemic species of emAbantiades/em Herrich-Schäffer (Lepidoptera: Hepialidae) from Kangaroo Island, Australia.

Abantiades penneshawensis Moore Beaver sp. nov. and Abantiades rubrus Moore Beaver sp. nov. are described as new. Both species are endemic to Kangaroo Island, and although both are related to species that occur on the Australian mainland and other islands, they are distinguished from those sister and phenotypically similar species by morphology and mtDNA (COI) barcodes. These two new species raise the number of Abantiades species on Kangaroo Island to six, three being endemic, and 45 species in the genus for the whole of Australia. There are now 13 species of Hepialidae (one undescribed) known from Kangaroo Island and we discuss the potential effects of recent catastrophic fire on some distributions.

Four new species of Splendid Ghost Moths (Lepidoptera: Hepialidae: Aenetus) from Australia and Papua New Guinea.

Four new Aenetus Herrich-Schäffer species are described from northern Australasia; Aenetus simonseni sp. nov. from the top-end of the Northern Territory, Australia, A. maiasinus sp. nov. from the Kimberley region of Western Australia, A. trigonogrammus sp. nov. from south-eastern Queensland, Australia, and A. albadamanteum sp. nov. from eastern Papua New Guinea. Aenetus simonseni sp. nov. and A. maiasinus sp. nov. appear to belong to the tegulatus-group of species (sensu Grehan et al. 2018), A. trigonogrammus sp. nov. is part of the splendens-group of species (sensu Simonsen 2018), while A. albadamanteum sp. nov. shares morphological similarities with A. hampsoni (Joicey Noakes, 1914), A. crameri Viette, 1956, and A. toxopeusi Viette, 1956, from New Guinea, and A. cohici Viette, 1961 from New Caledonia. The four new species are illustrated and compared with superficially similar species in morphology and, for two species, molecular (mtDNA COI gene) sequences.

Description of two new Australian species of Abantiades Herrich-Schäffer (Lepidoptera: Hepialidae) and females of two further species with notes on their biogeography.

Abantiades cephalocorvus sp. nov. and Abantiades tembyi sp. nov. are described, along with the previously undescribed females of A. macropusinsulariae Simonsen, 2018 and A. pallida Simonsen, 2018. All of these species belong to a triforked Abantiades Herrich-Schäffer clade that is loosely centred around the Nullarbor and other arid regions of Australia. We explore DNA barcodes (mtDNA COI gene) from these and other Abantiades and discuss their significance for species recognition. The species distributions are entirely or largely allopatric and we discuss their origins from a widespread common ancestor that was likely distributed over inland and coastal regions in the mid- to late-Mesozoic before the onset of desertification. Notes on new distributional data for all of the known species in this clade are included.

Four new tri-forked species of the Australian genus Abantiades Herrich-Schäffer (Lepidoptera: Hepialidae) from the "dark obscura clade".

A distinct group of Abantiades Herrich-Schäffer species is here confirmed as a valid clade that we refer to as the "dark obscura clade" supported by morphological and mtDNA evidence. The clade is the sister group of A. obscura Simonsen of north-western Australia and comprises four new species: Abantiades centralia sp. nov., A. kayi sp. nov., A. zonatriticum sp. nov., and A. hutchinsoni sp. nov. These species together with A. obscura, are reciprocally allopatric and have a combined distribution spanning much of the western half of Australia and this distribution is consistent with their each differentiating locally from a widespread ancestor. The four new species raise the diversity of Abantiades to 42 species. [Zoobank urn:lsid:zoobank.org:pub:C05458D1-0D34-4432-8EC4-D031ED6B7BEF].

Three new ghost moths of the genus Oxycanus Walker, 1856 from Australia (Lepidoptera: Hepialidae).

Three new species of ghost moth, Oxycanus ephemerous sp. nov., O. flavoplumosus sp. nov., and O. petalous sp. nov. are described from South Australia, New South Wales, and south-west Western Australia, respectively. We illustrate these species and compare morphological and molecular (mtDNA COI gene) characters with similar Oxycanus Walker, 1856 species from Australia. Comparative images of Oxycanus subvaria (Walker, 1856), O. byrsa (Pfitzner, 1933), and O. determinata (Walker, 1856) are figured. The type material of the three new species are held in the Australian National Insect Collection, Canberra, the Western Australian Museum, Perth, and in the South Australian Museum, Adelaide. The type specimens of Oxycanus hildae Tindale, 1964 syn. n. were also examined and the taxon is here considered synonymous with O. subvaria. Concerns are raised about the conservation status of all three new species due to few or localised distribution records.

The complete mitochondrial genome of the brown pansy butterfly, Junonia stygia (Aurivillius, 1894), (Insecta: Lepidoptera: Nymphalidae).

The brown pansy, Junonia stygia (Aurivillius, 1894) (Lepidoptera: Nymphalidae), is a widespread West African forest butterfly. Genome skimming by Illumina sequencing allowed assembly of a complete 15,233 bp circular mitogenome from J. stygia consisting of 79.5% AT nucleotides. Mitochondrial gene order and composition is identical to other butterfly mitogenomes. Junonia stygia COX1 features an atypical CGA start codon, while ATP6, COX1, COX2, ND4, and ND4L exhibit incomplete stop codons. Phylogenetic reconstruction supports a monophyletic Subfamily Nymphalinae, Tribe Junoniini, and genus Junonia. The phylogenetic tree places Junonia iphita and J. stygia as basal mitogenome lineages sister to the remaining Junonia sequences.

RIPK1 is a critical modulator of both tonic and TLR-responsive inflammatory and cell death pathways in human macrophage differentiation.

In this study, we took advantage of human-induced pluripotent stem cells (hiPSC) and CRISPR/Cas9 technology to investigate the potential roles of RIPK1 in regulating hematopoiesis and macrophage differentiation, proinflammatory activation, and cell death pathways. Knock-out of RIPK1 in hiPSCs demonstrated that this protein is not required for erythro-myeloid differentiation. Using a well-established macrophage differentiation protocol, knock-out of RIPK1 did not block the differentiation of iPSC-derived macrophages, which displayed a similar phenotype to WT hiPSC-derived macrophages. However, knock-out of RIPK1 leads to a TNFα-dependent apoptotic death of differentiated hiPSC-derived macrophages (iPS-MΦ) and progressive loss of iPS-MΦ production irrespective of external pro-inflammatory stimuli. Live video analysis demonstrated that TLR3/4 activation of RIPK1 KO hiPSC-derived macrophages triggered TRIF and RIPK3-dependent necroptosis irrespective of caspase-8 activation. In contrast, TLR3/4 activation of WT macrophages-induced necroptosis only when caspases were inhibited, confirming the modulating effect of RIPK1 on RIPK3-mediated necroptosis through the FADD, Caspase-8 pathway. Activation of these inflammatory pathways required RIPK3 kinase activity while RIPK1 was dispensable. However, loss of RIPK1 sensitizes macrophages to activate RIPK3 in response to inflammatory stimuli, thereby exacerbating a potentially pathological inflammatory response. Taken together, these results reveal that RIPK1 has an important role in regulating the potent inflammatory pathways in authentic human macrophages that are poised to respond to external stimuli. Consequently, RIPK1 activity might be a valid target in the development of novel therapies for chronic inflammatory diseases.

The nature and nurture of cell heterogeneity: accounting for macrophage gene-environment interactions with single-cell RNA-Seq.

BACKGROUND: Single-cell RNA-Seq can be a valuable and unbiased tool to dissect cellular heterogeneity, despite the transcriptome's limitations in describing higher functional phenotypes and protein events. Perhaps the most important shortfall with transcriptomic 'snapshots' of cell populations is that they risk being descriptive, only cataloging heterogeneity at one point in time, and without microenvironmental context. Studying the genetic ('nature') and environmental ('nurture') modifiers of heterogeneity, and how cell population dynamics unfold over time in response to these modifiers is key when studying highly plastic cells such as macrophages. RESULTS: We introduce the programmable Polaris™ microfluidic lab-on-chip for single-cell sequencing, which performs live-cell imaging while controlling for the culture microenvironment of each cell. Using gene-edited macrophages we demonstrate how previously unappreciated knockout effects of SAMHD1, such as an altered oxidative stress response, have a large paracrine signaling component. Furthermore, we demonstrate single-cell pathway enrichments for cell cycle arrest and APOBEC3G degradation, both associated with the oxidative stress response and altered proteostasis. Interestingly, SAMHD1 and APOBEC3G are both HIV-1 inhibitors ('restriction factors'), with no known co-regulation. CONCLUSION: As single-cell methods continue to mature, so will the ability to move beyond simple 'snapshots' of cell populations towards studying the determinants of population dynamics. By combining single-cell culture, live-cell imaging, and single-cell sequencing, we have demonstrated the ability to study cell phenotypes and microenvironmental influences. It's these microenvironmental components - ignored by standard single-cell workflows - that likely determine how macrophages, for example, react to inflammation and form treatment resistant HIV reservoirs.

Human Induced Pluripotent Stem Cell-Derived Macrophages Share Ontogeny with MYB-Independent Tissue-Resident Macrophages.

Tissue-resident macrophages, such as microglia, Kupffer cells, and Langerhans cells, derive from Myb-independent yolk sac (YS) progenitors generated before the emergence of hematopoietic stem cells (HSCs). Myb-independent YS-derived resident macrophages self-renew locally, independently of circulating monocytes and HSCs. In contrast, adult blood monocytes, as well as infiltrating, gut, and dermal macrophages, derive from Myb-dependent HSCs. These findings are derived from the mouse, using gene knockouts and lineage tracing, but their applicability to human development has not been formally demonstrated. Here, we use human induced pluripotent stem cells (iPSCs) as a tool to model human hematopoietic development. By using a CRISPR-Cas9 knockout strategy, we show that human iPSC-derived monocytes/macrophages develop in an MYB-independent, RUNX1-, and SPI1 (PU.1)-dependent fashion. This result makes human iPSC-derived macrophages developmentally related to and a good model for MYB-independent tissue-resident macrophages, such as alveolar and kidney macrophages, microglia, Kupffer cells, and Langerhans cells.

MAIT cells are activated during human viral infections.

Mucosal-associated invariant T (MAIT) cells are abundant in humans and recognize bacterial ligands. Here, we demonstrate that MAIT cells are also activated during human viral infections in vivo. MAIT cells activation was observed during infection with dengue virus, hepatitis C virus and influenza virus. This activation-driving cytokine release and Granzyme B upregulation-is TCR-independent but dependent on IL-18 in synergy with IL-12, IL-15 and/or interferon-α/ß. IL-18 levels and MAIT cell activation correlate with disease severity in acute dengue infection. Furthermore, HCV treatment with interferon-α leads to specific MAIT cell activation in vivo in parallel with an enhanced therapeutic response. Moreover, TCR-independent activation of MAIT cells leads to a reduction of HCV replication in vitro mediated by IFN-γ. Together these data demonstrate MAIT cells are activated following viral infections, and suggest a potential role in both host defence and immunopathology.

CRISPR-mediated genotypic and phenotypic correction of a chronic granulomatous disease mutation in human iPS cells.

Chronic granulomatous disease (CGD) is a rare genetic disease characterized by severe and persistent childhood infections. It is caused by the lack of an antipathogen oxidative burst, normally performed by phagocytic cells to contain and clear bacterial and fungal growth. Restoration of immune function can be achieved with heterologous bone marrow transplantation; however, autologous bone marrow transplantation would be a preferable option. Thus, a method is required to recapitulate the function of the diseased gene within the patient's own cells. Gene therapy approaches for CGD have employed randomly integrating viruses with concomitant issues of insertional mutagenesis, inaccurate gene dosage, and gene silencing. Here, we explore the potential of the recently described clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 site-specific nuclease system to encourage repair of the endogenous gene by enhancing the levels of homologous recombination. Using induced pluripotent stem cells derived from a CGD patient containing a single intronic mutation in the CYBB gene, we show that footprintless gene editing is a viable option to correct disease mutations. Gene correction results in restoration of oxidative burst function in iPS-derived phagocytes by reintroduction of a previously skipped exon in the cytochrome b-245 heavy chain (CYBB) protein. This study provides proof-of-principle for a gene therapy approach to CGD treatment using CRISPR-Cas9.

Macrophage infection via selective capture of HIV-1-infected CD4+ T cells.

Macrophages contribute to HIV-1 pathogenesis by forming a viral reservoir and mediating neurological disorders. Cell-free HIV-1 infection of macrophages is inefficient, in part due to low plasma membrane expression of viral entry receptors. We find that macrophages selectively capture and engulf HIV-1-infected CD4+ T cells leading to efficient macrophage infection. Infected T cells, both healthy and dead or dying, were taken up through viral envelope glycoprotein-receptor-independent interactions, implying a mechanism distinct from conventional virological synapse formation. Macrophages infected by this cell-to-cell route were highly permissive for both CCR5-using macrophage-tropic and otherwise weakly macrophage-tropic transmitted/founder viruses but restrictive for nonmacrophage-tropic CXCR4-using virus. These results have implications for establishment of the macrophage reservoir and HIV-1 dissemination in vivo.

Intramolecular oxonium ylide formation-[2,3] sigmatropic rearrangement of diazocarbonyl-substituted cyclic unsaturated acetals: a formal synthesis of hyperolactone C.

Rh(II)-catalyzed oxonium ylide formation-[2,3] sigmatropic rearrangement of α-diazo-ß-ketoesters possessing γ-cyclic unsaturated acetal substitution, followed by acid-catalyzed elimination-lactonization, provides a concise approach to 1,7-dioxaspiro[4.4]non-2-ene-4,6-diones. The process creates adjacent quaternary stereocenters with full control of the relative stereochemistry. An unsymmetrical monomethylated cyclic unsaturated acetal leads to hyperolactone C, where ylide formation-rearrangement proceeds with high selectivity between subtly nonequivalent acetal oxygen atoms.

The productive entry pathway of HIV-1 in macrophages is dependent on endocytosis through lipid rafts containing CD4.

Macrophages constitute an important reservoir of HIV-1 infection, yet HIV-1 entry into these cells is poorly understood due to the difficulty in genetically manipulating primary macrophages. We developed an effective genetic approach to manipulate the sub-cellular distribution of CD4 in macrophages, and investigated how this affects the HIV-1 entry pathway. Pluripotent Stem Cells (PSC) were transduced with lentiviral vectors designed to manipulate CD4 location and were then differentiated into genetically modified macrophages. HIV-1 infection of these cells was assessed by performing assays that measure critical steps of the HIV-1 lifecycle (fusion, reverse transcription, and expression from HIV-1 integrants). Expression of LCK (which tethers CD4 to the surface of T cells, but is not normally expressed in macrophages) in PSC-macrophages effectively tethered CD4 at the cell surface, reducing its normal endocytic recycling route, and increasing surface CD4 expression 3-fold. This led to a significant increase in HIV-1 fusion and reverse transcription, but productive HIV-1 infection efficiency (as determined by reporter expression from DNA integrants) was unaffected. This implies that surface-tethering of CD4 sequesters HIV-1 into a pathway that is unproductive in macrophages. Secondly, to investigate the importance of lipid rafts (as detergent resistant membranes - DRM) in HIV-1 infection, we generated genetically modified PSC-macrophages that express CD4 mutants known to be excluded from DRM. These macrophages were significantly less able to support HIV-1 fusion, reverse-transcription and integration than engineered controls. Overall, these results support a model in which productive infection by HIV-1 in macrophages occurs via a CD4-raft-dependent endocytic uptake pathway.

Informatics calibration of a molecular descriptors database to predict solid dispersion potential of small molecule organic solids.

The use of a novel, in silico method for making an intelligent polymer selection to physically stabilize small molecule organic (SMO) solid compounds formulated as amorphous molecular solid dispersions is reported. 12 compounds (75%, w/w) were individually co-solidified with polyvinyl pyrrolidone:vinyl acetate (PVPva) copolymer by melt-quenching. Co-solidified products were analyzed intact using differential scanning calorimetry (DSC) and the pair distribution function (PDF) transform of powder X-ray diffraction (PXRD) data to assess miscibility. Molecular descriptor indices were calculated for all twelve compounds using their reported crystallographic structures. Logistic regression was used to assess correlation between molecular descriptors and amorphous molecular solid dispersion potential. The final model was challenged with three compounds. Of the 12 compounds, 6 were miscible with PVPva (i.e. successful formation) and 6 were phase separated (i.e. unsuccessful formation). 2 of the 6 unsuccessful compounds exhibited detectable phase-separation using the PDF method, where DSC indicated miscibility. Logistic regression identified 7 molecular descriptors correlated to solid dispersion potential (α=0.001). The atomic mass-weighted third-order R autocorrelation index (R3m) was the only significant descriptor to provide completely accurate predictions of dispersion potential. The three compounds used to challenge the R3m model were also successfully predicted.

Multiple barriers to recombination between divergent HIV-1 variants revealed by a dual-marker recombination assay.

Recombination is a major force for generating human immunodeficiency virus type 1 (HIV-1) diversity and produces numerous recombinants circulating in the human population. We previously established a cell-based system using green fluorescent protein gene (gfp) as a reporter to study the mechanisms of HIV-1 recombination. We now report an improved system capable of detecting recombination using authentic viral sequences. Frameshift mutations were introduced into the gag gene so that parental viruses do not express full-length Gag; however, recombination can generate a progeny virus that expresses a functional Gag. We demonstrate that this Gag reconstitution assay can be used to detect recombination between two group M HIV-1 variants of the same or of different subtypes. Using both gfp and gag assays, we found that, similar to group M viruses, group O viruses also recombine frequently. When recombination between a group M virus and a group O virus was examined, we found three distinct barriers for intergroup recombination. First, similar to recombination within group M viruses, intergroup recombination is affected by the identity of the dimerization initiation signal (DIS); variants with the same DIS recombined at a higher rate than those with different DIS. Second, using the gfp recombination assay, we showed that intergroup recombination occurs much less frequently than intragroup recombination, even though the gfp target sequence is identical in all viruses. Finally, Gag reconstitution between variants from different groups is further reduced compared with green fluorescent protein, indicating that sequence divergence interferes with recombination efficiency in the gag gene. Compared with identical sequences, we estimate that recombination rates are reduced by 3-fold and by 10- to 13-fold when the target regions in gag contain 91% and 72-73% sequence identities, respectively. These results show that there are at least three distinct mechanisms preventing exchange of genetic information between divergent HIV-1 variants from different groups.

Protection of HIV neutralizing aptamers against rectal and vaginal nucleases: implications for RNA-based therapeutics.

RNA-based drugs are an emerging class of therapeutics. They have the potential to regulate proteins, chromatin, as well as bind to specific proteins of interest in the form of aptamers. These aptamers are protected from nuclease attack by chemical modifications that enhance their stability for in vivo usage. However, nucleases are ubiquitous, and as we have yet to characterize the entire human microbiome it is likely that many nucleases are yet to be identified. Any novel, unusual enzymes present in vivo might reduce the efficacy of RNA-based therapeutics, even when they are chemically modified. We have previously identified an RNA-based aptamer capable of neutralizing a broad spectrum of clinical HIV-1 isolates and are developing it as a vaginal and rectal microbicide candidate. As a first step we addressed aptamer stability in the milieu of proteins present in these environments. Here we uncover a number of different nucleases that are able to rapidly degrade 2'-F-modified RNA. We demonstrate that the aptamer can be protected from the nuclease(s) present in the vaginal setting, without affecting its antiviral activity, by replacement of key positions with 2'-O-Me-modified nucleotides. Finally, we show that the aptamer can be protected from all nucleases present in both vaginal and rectal compartments using Zn(2+) cations. In conclusion we have derived a stable, antiviral RNA-based aptamer that could form the basis of a pre-exposure microbicide or be a valuable addition to the current tenofovir-based microbicide candidate undergoing clinical trials.

Structural interpretation in composite systems using powder X-ray diffraction: applications of error propagation to the pair distribution function.

PURPOSE: To develop a method for drawing statistical inferences from differences between multiple experimental pair distribution function (PDF) transforms of powder X-ray diffraction (PXRD) data. METHODS: The appropriate treatment of initial PXRD error estimates using traditional error propagation algorithms was tested using Monte Carlo simulations on amorphous ketoconazole. An amorphous felodipine:polyvinyl pyrrolidone:vinyl acetate (PVPva) physical mixture was prepared to define an error threshold. Co-solidified products of felodipine:PVPva and terfenadine:PVPva were prepared using a melt-quench method and subsequently analyzed using PXRD and PDF. Differential scanning calorimetry (DSC) was used as an additional characterization method. RESULTS: The appropriate manipulation of initial PXRD error estimates through the PDF transform were confirmed using the Monte Carlo simulations for amorphous ketoconazole. The felodipine:PVPva physical mixture PDF analysis determined ±3σ to be an appropriate error threshold. Using the PDF and error propagation principles, the felodipine:PVPva co-solidified product was determined to be completely miscible, and the terfenadine:PVPva co-solidified product, although having appearances of an amorphous molecular solid dispersion by DSC, was determined to be phase-separated. CONCLUSIONS: Statistically based inferences were successfully drawn from PDF transforms of PXRD patterns obtained from composite systems. The principles applied herein may be universally adapted to many different systems and provide a fundamentally sound basis for drawing structural conclusions from PDF studies.