Growth and development of the Mahogany Glider (Petaurus gracilis).

The growth and development of the endangered Mahogany Glider (Petaurus gracilis) was monitored in a captive population at Burleigh Heads, Queensland, Australia. Video surveillance confirmed that the gestation period for this species was 16 days. Morphometric data and developmental milestones were recorded from 10 Mahogany Gliders from birth to weaning. Growth curves were developed for head length, ulna length, tail length, and body weight. Weekly inspections of female pouches revealed the young's eyelid margins were visible by Day 21, the first hair erupted on the bridge of the nose at Day 30, pigmentation of the body developed at Day 63, and they started detaching from the teat intermittently, and the body was covered in short fur by Day 70. The young were left in the nest alone from Days 84 to 87, their eyes opened between Days 84 and 94, and there was a rapid increase in length and density of fur from Day 98 onwards. At Days 101 to 105 of age the young left the nest box with its mother as back young. Weaning occurred from 184 to 187 days. Typically, the reproductive rate was two young per annum per pair, but one pair produced five young in 19 months. Females produced young from 12 months to 7 years of age, males up to 9.4 years of age. The average longevity of Mahogany Gliders in the studbook in 2018 was 11.6 years. This study provides data on the reproductive biology of the Mahogany Glider that will assist in its captive breeding, management, and conservation.

Gene expression analyses support fallopian tube epithelium as the cell of origin of epithelial ovarian cancer.

Folate receptor alpha (FOLR1/FRA) is reported to be overexpressed in epithelial ovarian cancers (EOC), especially the serous histotype. Further, while dysregulation of the folate-dependent 1-carbon cycle has been implicated in tumorogenesis, little is known relative to the potential mechanism of action of FOLR1 expression in these processes. We therefore investigated the expression of FOLR1, other folate receptors, and genes within the 1-carbon cycle in samples of EOC, normal ovary and fallopian tube on a custom TaqMan Low Density Array. Also included on this array were known markers of EOC such as MSLN, MUC16 and HE4. While few differences were observed in the expression profiles of genes in the 1-carbon cycle, genes previously considered to be overexpressed in EOC (e.g., FOLR1, MSLN, MUC16 and HE4) showed significantly increased expression when comparing EOC to normal ovary. However, when the comparator was changed to normal fallopian tube, these differences were abolished, supporting the hypothesis that EOC derives from fallopian fimbriae and, further, that markers previously considered to be upregulated or overexpressed in EOC are most likely not of ovarian origin, but fallopian in derivation. Our findings therefore support the hypothesis that the cell of origin of EOC is tubal epithelium.

Key developmental transitions in human germinal center B cells are revealed by differential CD45RB expression.

We previously reported that RO(+) expression correlated with increased mutation, activation, and selection among human germinal center (GC) B cells. Here, we subdivided human tonsillar B cells, including IgD(-)CD38(+) GC B cells, into different fractions based on RB expression. Although each subset contained RB(+) cells, when used as an intrasubset marker, differential RB expression effectively discriminated between phenotypically distinct cells. For example, RB(+) GC B cells were enriched for activated cells with lower AID expression. RB inversely correlated with mutation frequency, demonstrating a key difference between RB- and RO-expressing GC B cells. Reduced RB expression during the transition from pre-GC (IgM(+)IgD(+)CD38(+)CD27(-)) to GCB cells was followed by a dramatic increase during the GC-to-plasmablast (IgD(-)CD38(++)CD27(+)) and memory (IgD(-)CD38(-)CD27(+)) transition. Interestingly, RB(+) GC B cells showed increased signs of terminal differentiation toward CD27(+) post-GC early plasmablast (increased CD38 and RO) or early memory (decreased CD38 and RO) B cells. We propose that as in T cells, differential RB expression directly correlates with development- and function-based transitions in tonsillar B cells. Application of this RB:RO system should advance our understanding of normal B-cell development and facilitate the isolation of more discrete B-cell populations with potentially different propensities in disease pathogenesis.

Phylogenetic and morphological significance of an overlooked flying squirrel (Pteromyini, Rodentia) from the eastern Himalayas with the description of a new genus.

The flying squirrels (Pteromyini, Rodentia) are the most diverse and widely distributed group of gliding mammals. Taxonomic boundaries and relationships within flying squirrels remain an area of active research in mammalogy. The discovery of new specimens of Pteromys ( Hylopetes) leonardi Thomas, 1921 previously considered a synonym of Hylopetes alboniger, in Yunnan Province, China allowed a morphological and genetic reassessment of the status of this taxon. Phylogenetic reconstruction was implemented using sequences of two mitochondrial (12S ribosomal DNA and 16S ribosomal DNA) and one nuclear (interphotoreceptor retinoid-binding protein) gene fragments. Morphological assessments involved examinations of features preserved on skins, skulls, and penises of museum specimens, supplemented with principal component analysis of craniometric data. Together these assessments revealed that this taxon should be recognized not only as a distinct species, and should also be placed within a new genus, described here as Priapomys.

Discovery and description of a mysterious Asian flying squirrel (Rodentia, Sciuridae, Biswamoyopterus) from Mount Gaoligong, southwest China.

The flying squirrels of the tribe Pteromyini (Family Sciuridae) currently include 15 genera of which the genus Biswamoyopterus comprises two recognized species, B.biswasi Saha, 1981 and B.laoensis Sanamxay et al., 2013. These two species were each described from only one specimen that are separated from each other by 1,250 kilometres in southern Asia, where they occur in northeast India and central Lao PDR respectively. In 2017 and 2018, two specimens of Biswamoyopterus were discovered from Mount Gaoligong, west Yunnan province, southwest China (between the type locality of the two recognized species). This study aimed to evaluate the taxonomic status of these two newly acquired specimens of Biswamoyopterus by comparing their morphology with the two described species of the genus. The results of this study showed that the specimens from Yunnan province (China) differed from both B.laoensis and B.biswasi in both pelage colour and craniology, and should be recognised as a distinct species, B.gaoligongensis sp. nov., which is formally described here. This study contributes to the understanding of the flying squirrels of southern Asia and identifies an additional species that appears to be endemic to southwest China; however, more research is required to provide details of its ecology, distribution, and conservation status.

The Dogma of Dingoes-Taxonomic status of the dingo: A reply to Smith et al.

Adopting the name Canis dingo for the Dingo to explicitly denote a species-level taxon separate from other canids was suggested by Crowther et al.  (2014) as a means to eliminate taxonomic instability and contention. However, Jackson et al.  (2017), using standard taxonomic and nomenclatural approaches and principles, called instead for continued use of the nomen C. familiaris for all domestic dogs and their derivatives, including the Dingo. (This name, C. familiaris, is applied to all dogs that derive from the domesticated version of the Gray Wolf, Canis lupus, based on nomenclatural convention.) The primary reasons for this call by Jackson et al.  (2017) were: (1) a lack of evidence to show that recognizing multiple species amongst the dog, including the Dingo and New Guinea Singing Dog, was necessary taxonomically, and (2) the principle of nomenclatural priority (the name familiaris Linnaeus, 1758, antedates dingo Meyer, 1793). Overwhelming current evidence from archaeology and genomics indicates that the Dingo is of recent origin in Australia and shares immediate ancestry with other domestic dogs as evidenced by patterns of genetic and morphological variation. Accordingly, for Smith et al.  (2019) to recognise Canis dingo as a distinct species, the onus was on them to overturn current interpretations of available archaeological, genomic, and morphological datasets and instead show that Dingoes have a deeply divergent evolutionary history that distinguishes them from other named forms of Canis (including C. lupus and its domesticated version, C. familiaris). A recent paper by Koepfli et al.  (2015) demonstrates exactly how this can be done in a compelling way within the genus Canis-by demonstrating deep evolutionary divergence between taxa, on the order of hundreds of thousands of years, using data from multiple genetic systems. Smith et al.  (2019) have not done this; instead they have misrepresented the content and conclusions of Jackson et al.  (2017), and contributed extraneous arguments that are not relevant to taxonomic decisions. Here we dissect Smith et al.  (2019), identifying misrepresentations, to show that ecological, behavioural and morphological evidence is insufficient to recognise Dingoes as a separate species from other domestic dogs. We reiterate: the correct binomial name for the taxon derived from Gray Wolves (C. lupus) by passive and active domestication, including Dingoes and other domestic dogs, is Canis familiaris. We are strongly sympathetic to arguments about the historical, ecological, cultural, or other significance of the Dingo, but these are issues that will have to be considered outside of the more narrow scope of taxonomy and nomenclature.

Human immunoglobulin selection associated with class switch and possible tolerogenic origins for C delta class-switched B cells.

Current paradigms of peripheral B cell selection suggest that autoreactive B cells are controlled by clonal deletion, anergy, and developmental arrest. We report that changes to the human antibody repertoire likely resulting from these mechanisms both for a well-characterized autoreactivity from antibodies encoded by the V(H)4-34 gene and for other hallmarks of an autoreactive repertoire are apparent mainly for class-switched B cells and not for IgM germinal center, IgM memory, or IgM plasma cells. Other possible indicators of autoreactivity found selected with immunoglobulin class include J(H)6 gene segment usage, increased frequency of B cells with long third hypervariable regions, and distal J(kappa) gene segment bias. Of particular interest is the finding that B cells with these same characteristics are selected into the lineage of B cells that have undergone the unusual class switch from constant region C mu to C delta (C delta-CS). The C delta-CS population also displays an increased frequency of charged amino acids localized to the complementarity-determining regions, further suggesting autoreactivity, and evidence is presented that these B cells had undergone extensive receptor editing. Thus, the C delta-CS lineage may be a "sink" for B cells harboring autoreactive specificities in normal humans. A model for a new tolerizing mechanism that could account for the C delta-CS lineage is presented.

The bovine antibody repertoire.

Cattle are able to produce a full range of Ig classes including the long-elusive IgD through rearrangement of their germline genes. Several IgL groupings have been reported but as in several other livestock species (e.g. sheep, rabbits, chickens), rearrangement per se fails to generate significant IgH diversity. This is largely because of the modest number of bovine VH segments that participate in rearrangement and their conserved sequences. Perhaps in compensation, bovine Ig heavy chains carry CDR3 sequences of exceptional length. Processes that operate post-rearrangement to generate diversity remain ill defined as are the location, timing and triggers to these events. Reagents are needed to understand better the maturation of B lymphocytes, their responses to antigens and cytokines, and to provide standards for the quantitation of Ig responses in cattle; recombinant methods may help meet this need as Ab engineering technologies become more widely used.

Novel antibody probes for the characterization of endosialin/TEM-1.

Endosialin (Tumor Endothelial Marker-1 (TEM-1), CD248) is primarily expressed on pericytes of tumor-associated microvasculature, tumor-associated stromal cells and directly on tumors of mesenchymal origin, including sarcoma and melanoma. While the function of endosialin/TEM-1 is incompletely understood, studies have suggested a role in supporting tumor growth and invasion thus making it an attractive therapeutic target. In an effort to further understand its role in cancer, we previously developed a humanized anti-endosialin/TEM-1 monoclonal antibody (mAb), called ontuxizumab (MORAb-004) for testing in preclinical and clinical studies. We herein report on the generation of an extensive panel of recombinant endosialin/TEM-1 protein extracellular domain (ECD) fragments and novel mAbs against ECD motifs. The domain-specific epitopes were mapped against ECD sub-domains to identify those that can detect distinct structural motifs and can be potentially formatted as probes suitable for diagnostic and functional studies. A number of mAbS were shown to cross-react with the murine and human protein, potentially allowing their use in human animal models and corresponding clinical trials. In addition, pairing of several mAbs supported their use in immunoassays that can detect soluble endosialin/TEM-1 (sEND) in the serum of healthy subjects and cancer patients.

A SCA7 CAG/CTG repeat expansion is stable in Drosophila melanogaster despite modulation of genomic context and gene dosage.

CAG and CTG repeat expansions are the cause of at least a dozen inherited neurological disorders. In these so-called "dynamic mutation" diseases, the expanded repeats display dramatic genetic instability, changing in size when transmitted through the germline and within somatic tissues. As the molecular basis of the repeat instability process remains poorly understood, modeling of repeat instability in model organisms has provided some insights into potentially involved factors, implicating especially replication and repair pathways. Studies in mice have also shown that the genomic context of the repeat sequence is required for CAG/CTG repeat instability in the case of spinocerebellar ataxia type 7 (SCA7), one of the most unstable of all CAG/CTG repeat disease loci. While most studies of repeat instability have taken a candidate gene approach, unbiased screens for factors involved in trinucleotide repeat instability have been lacking. We therefore attempted to use Drosophila melanogaster to model expanded CAG repeat instability by creating transgenic flies carrying trinucleotide repeat expansions, deriving flies with SCA7 CAG90 repeats in cDNA and genomic context. We found that SCA7 CAG90 repeats are stable in Drosophila, regardless of context. To screen for genes whose reduced function might destabilize expanded CAG repeat tracts in Drosophila, we crossed the SCA7 CAG90 repeat flies with various deficiency stocks, including lines lacking genes encoding the orthologues of flap endonuclease-1, PCNA, and MutS. In all cases, perfect repeat stability was preserved, suggesting that Drosophila may not be a suitable system for determining the molecular basis of SCA7 CAG repeat instability.

A Drosophila homolog of the polyglutamine disease gene SCA2 is a dosage-sensitive regulator of actin filament formation.

Spinocerebellar ataxia type 2 (SCA2) is a neurodegenerative disorder caused by the expansion of a CAG repeat encoding a polyglutamine tract in ataxin-2, the SCA2 gene product. The normal cellular function of ataxin-2 and the mechanism by which polyglutamine expansion of ataxin-2 causes neurodegeneration remain unknown. In this study we have used genetic and molecular approaches to investigate the function of a Drosophila homolog of the SCA2 gene (Datx2). Like human ataxin-2, Datx2 is found throughout development in a variety of tissue types and localizes to the cytoplasm. Mutations that reduce Datx2 activity or transgenic overexpression of Datx2 result in female sterility, aberrant sensory bristle morphology, loss or degeneration of tissues, and lethality. These phenotypes appear to result from actin filament formation defects occurring downstream of actin synthesis. Further studies demonstrate that Datx2 does not assemble with actin filaments, suggesting that the role of Datx2 in actin filament formation is indirect. These results indicate that Datx2 is a dosage-sensitive regulator of actin filament formation. Given that loss of cytoskeleton-dependent dendritic structure defines an early event in SCA2 pathogenesis, our findings suggest the possibility that dysregulation of actin cytoskeletal structure resulting from altered ataxin-2 activity is responsible for neurodegeneration in SCA2.

Mitochondrial dysfunction in NnaD mutant flies and Purkinje cell degeneration mice reveals a role for Nna proteins in neuronal bioenergetics.

The Purkinje cell degeneration (pcd) mouse is a recessive model of neurodegeneration, involving cerebellum and retina. Purkinje cell death in pcd is dramatic, as >99% of Purkinje neurons are lost in 3 weeks. Loss of function of Nna1 causes pcd, and Nna1 is a highly conserved zinc carboxypeptidase. To determine the basis of pcd, we implemented a two-pronged approach, combining characterization of loss-of-function phenotypes of the Drosophila Nna1 ortholog (NnaD) with proteomics analysis of pcd mice. Reduced NnaD function yielded larval lethality, with survivors displaying phenotypes that mirror disease in pcd. Quantitative proteomics revealed expression alterations for glycolytic and oxidative phosphorylation enzymes. Nna proteins localize to mitochondria, loss of NnaD/Nna1 produces mitochondrial abnormalities, and pcd mice display altered proteolytic processing of Nna1 interacting proteins. Our studies indicate that Nna1 loss of function results in altered bioenergetics and mitochondrial dysfunction.

CD45RO enriches for activated, highly mutated human germinal center B cells.

To date, there is no consensus regarding the influence of different CD45 isoforms during peripheral B-cell development. Examining correlations between surface CD45RO expression and various physiologic processes ongoing during the germinal center (GC) reaction, we hypothesized that GC B cells, like T cells, that up-regulate surface RO should progressively acquire phenotypes commonly associated with activated, differentiating lymphocytes. GC B cells (IgD(-)CD38(+)) were subdivided into 3 surface CD45RO fractions: RO(-), RO(+/-), and RO(+). We show here that the average number of mutations per IgV(H) transcript increased in direct correlation with surface RO levels. Conjunctional use of RO and CD69 further delineated low/moderately and highly mutated fractions. Activation-induced cytidine deaminase (AID) mRNA was slightly reduced among RO(+) GC B cells, suggesting that higher mutation averages are unlikely due to elevated somatic mutation activity. Instead, RO(+) GC B cells were negative for Annexin V, comprised mostly (93%) of CD77(-) centrocytes, and were enriched for CD69(+) cells. Collectively, RO(+) GC B cells occupy what seems to be a specialized niche comprised mostly of centrocytes that may be in transition between activation states. These findings are among the first to sort GC B cells into populations enriched for live mutated cells solely using a single extracellular marker.

IgH V-region sequence does not predict the survival fate of human germinal center B cells.

Germinal center (GC) B cell survival fate is governed in part by the outcome of successful/failed BCR-mediated interactions with accessory cells. However, the extent to which the BCR primary sequence influences such interactions is not fully understood. Over 1000 IgV(H)4 family cDNAs were sequenced from living (annexin V(-)) and apoptotic (annexin V(+) or from within tingible body macrophages) GC B cell fractions from seven tonsils. Results surprisingly demonstrate that living and dying GC B cells do not significantly differ in IgV(H), D, or J(H) gene segment use; HCDR3 length or positive charge; or mutation frequency. Additionally, equivalent IgH cDNA sequences were identified in both fractions, suggesting that BCR sequence alone is an unreliable predictor of GC B cell survival.

An A-kinase anchoring protein is required for protein kinase A regulatory subunit localization and morphology of actin structures during oogenesis in Drosophila.

Protein kinase A (PKA) holoenzyme is anchored to specific subcellular regions by interactions between regulatory subunits (Pka-R) and A-kinase anchoring proteins (AKAPs). We examine the functional importance of PKA anchoring during Drosophila oogenesis by analyzing membrane integrity and actin structures in mutants with disruptions in Akap200, an AKAP. In wild-type ovaries, Pka-RII and Akap200 localized to membranes and to the outer rim of ring canals, actin-rich structures that connect germline cells. In Akap200 mutant ovaries, Pka-RII membrane localization decreased, leading to a destabilization of membrane structures and the formation of binucleate nurse cells. Defects in membrane integrity could be mimicked by expressing a constitutively active PKA catalytic subunit (Pka-C) throughout germline cells. Unexpectedly, nurse cells in Akap200 mutant ovaries also had enlarged, thin ring canals. In contrast, overexpressing Akap200 in the germline resulted in thicker, smaller ring canals. To investigate the role of Akap200 in regulating ring canal growth, we examined genetic interactions with other genes that are known to regulate ring canal morphology. Akap200 mutations suppressed the small ring canal phenotype produced by Src64B mutants, linking Akap200 with the non-receptor tyrosine kinase pathway. Together, these results provide the first evidence that PKA localization is required for morphogenesis of actin structures in an intact organism.