Co-circulation of alpha- and beta-coronaviruses in Pteropus vampyrus flying foxes from Indonesia.

Bats are important reservoirs for alpha- and beta-coronaviruses. Coronaviruses (CoV) have been detected in pteropodid bats from several Southeast Asian countries, but little is known about coronaviruses in the Indonesian archipelago in proportion to its mammalian biodiversity. In this study, we screened pooled faecal samples from the Indonesian colonies of Pteropus vampyrus with unbiased next-generation sequencing. Bat CoVs related to Rousettus leschenaultii CoV HKU9 and Eidolon helvum CoV were detected. The 121 faecal samples were further screened using a conventional hemi-nested pan-coronavirus PCR assay. Three positive samples were successfully sequenced, and phylogenetic reconstruction revealed the presence of alpha- and beta-coronaviruses. CoVs belonging to the subgenera Nobecovirus, Decacovirus and Pedacovirus were detected in a single P. vampyrus roost. This study expands current knowledge of coronavirus diversity in Indonesian flying foxes, highlighting the need for longitudinal surveillance of colonies as continuing urbanization and deforestation heighten the risk of spillover events.

LncRNA PVT-1 promotes osteosarcoma cancer stem-like properties through direct interaction with TRIM28 and TSC2 ubiquitination.

Osteosarcoma, the most common pediatric bone tumor, is an aggressive heterogeneous malignancy defined by complex chromosomal aberrations. Overall survival rates remain at ~70%, but patients with chemoresistant or metastatic disease have extremely poor outcomes of <30%. A subgroup of tumors harbor amplification of chromosome 8q24.2 and increased expression of the oncogenic long noncoding RNA (lncRNA) Plasmacytoma Variant Translocation-1 (PVT-1), which is associated with an extremely poor clinical prognosis. This study demonstrates that PVT-1 is critical for osteosarcoma tumor-initiation potential. Chromatin Hybridization by RNA Purification analysis identified Tripartite-Motif Containing Family 28 (TRIM28) as a novel PVT-1 binding partner. Mechanistically, co-immunoprecipitation studies showed the PVT-1/TRIM28 complex binds and increases SUMOylation of phosphatidylinositol 3-kinase catalytic subunit type 3 (Vps34), which leads to enhanced ubiquitination and degradation of tumor suppressor complex 2 (TSC2), thus contributing to increased self-renewal and stem cell phenotypes. Furthermore, we identified that osteosarcoma cells with increased PVT-1 have enhanced sensitivity to the SUMOylation inhibitor, TAK-981. Altogether, this study elucidated a role for PVT-1 in the enhancement of cancer stem-like behaviors, including migration and invasion, in osteosarcoma, and identified the novel PVT-1/TRIM28 axis signaling cascade as a potential therapeutic target for osteosarcoma treatment.

Detection of Tioman Virus in Pteropus vampyrus Near Flores, Indonesia.

Diverse paramyxoviruses have coevolved with their bat hosts, including fruit bats such as flying foxes (Chiroptera: Pteropodidae). Several of these viruses are zoonotic, but the diversity and distribution of Paramyxoviridae are poorly understood. We screened pooled feces samples from three Pteropus vampyrus colonies and assayed tissues, rectal swabs, and oral swabs from 95 individuals of 23 pteropodid species sampled at 17 sites across the Indonesian archipelago with a conventional paramyxovirus PCR; all tested negative. Samples from 43 individuals were screened with next generation sequencing (NGS), and a single Pteropus vampyrus collected near Flores had Tioman virus sequencing reads. Tioman virus is a bat-borne virus in the genus Pararubulavirus with prior evidence of spillover to humans. This work expands the known range of Tioman virus, and it is likely that this isolated colony likely has sustained intergenerational transmission over a long period.

Interrogating Phylogenetic Discordance Resolves Deep Splits in the Rapid Radiation of Old World Fruit Bats (Chiroptera: Pteropodidae).

The family Pteropodidae (Old World fruit bats) comprises $>$200 species distributed across the Old World tropics and subtropics. Most pteropodids feed on fruit, suggesting an early origin of frugivory, although several lineages have shifted to nectar-based diets. Pteropodids are of exceptional conservation concern with $>$50% of species considered threatened, yet the systematics of this group has long been debated, with uncertainty surrounding early splits attributed to an ancient rapid diversification. Resolving the relationships among the main pteropodid lineages is essential if we are to fully understand their evolutionary distinctiveness, and the extent to which these bats have transitioned to nectar-feeding. Here we generated orthologous sequences for $>$1400 nuclear protein-coding genes (2.8 million base pairs) across 114 species from 43 genera of Old World fruit bats (57% and 96% of extant species- and genus-level diversity, respectively), and combined phylogenomic inference with filtering by information content to resolve systematic relationships among the major lineages. Concatenation and coalescent-based methods recovered three distinct backbone topologies that were not able to be reconciled by filtering via phylogenetic information content. Concordance analysis and gene genealogy interrogation show that one topology is consistently the best supported, and that observed phylogenetic conflicts arise from both gene tree error and deep incomplete lineage sorting. In addition to resolving long-standing inconsistencies in the reported relationships among major lineages, we show that Old World fruit bats have likely undergone at least seven independent dietary transitions from frugivory to nectarivory. Finally, we use this phylogeny to identify and describe one new genus. [Chiroptera; coalescence; concordance; incomplete lineage sorting; nectar feeder; species tree; target enrichment.].

Targeting PAK4 Inhibits Ras-Mediated Signaling and Multiple Oncogenic Pathways in High-Risk Rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is the most prevalent pediatric soft-tissue sarcoma. Multimodal treatment, including surgery and traditional chemotherapy with radiotherapy, has contributed to improvements in overall survival rates. However, patients with recurrent or metastatic disease have 5-year survival rates of less than 30%. One reason for the lack of therapeutic advancement is identification and targeting of critical signaling nodes. p21-activated kinases (PAK) are a family of serine/threonine kinases downstream of multiple critical tumorigenic receptor tyrosine kinase receptors and oncogenic regulators, including IGFR and RAS signaling, that significantly contribute to aggressive malignant phenotypes. Here, we report that RMS cell lines and tumors exhibit enhanced PAK4 expression levels and activity, which are further activated by growth factors involved in RMS development. Molecular perturbation of PAK4 in multiple RMS models in vitro and in vivo resulted in inhibition of RMS development and progression. Fusion-positive and -negative RMS models were sensitive to two PAK4 small-molecule inhibitors, PF-3758309 and KPT-9274, which elicited significant antitumor and antimetastatic potential in several primary and metastatic in vivo models, including a relapsed RMS patient-derived xenograft model. Transcriptomic analysis of PAK4-targeted tumors revealed inhibition of the RAS-GTPase, Hedgehog, and Notch pathways, along with evidence of activation of antitumor immune response signatures. This PAK4-targeting gene signature showed prognostic significance for patients with sarcoma. Overall, our results show for the first time that PAK4 is a novel and viable therapeutic target for the treatment of high-risk RMS. SIGNIFICANCE: These data demonstrate a novel oncogenic role for PAK4 in rhabdomyosarcoma and show that targeting PAK4 activity is a promising viable therapeutic option for advanced rhabdomyosarcoma.

Dispersal out of Wallacea spurs diversification of Pteropus flying foxes, the world's largest bats (Mammalia: Chiroptera).

AIM: Islands provide opportunities for isolation and speciation. Many landmasses in the Indo-Australian Archipelago (IAA) are oceanic islands, and founder-event speciation is expected to be the predominant form of speciation of volant taxa on these islands. We studied the biogeographic history of flying foxes, a group with many endemic species and a predilection for islands, to test this hypothesis and infer the biogeographic origin of the group. LOCATION: Australasia, Indo-Australian Archipelago, Madagascar, Pacific Islands. TAXON: Pteropus (Pteropodidae). METHODS: To infer the biogeographic history of Pteropus, we sequenced up to 6169 bp of genetic data from 10 markers and reconstructed a multilocus species tree of 34 currently recognized Pteropus species and subspecies with 3 Acerodon outgroups using BEAST and subsequently estimated ancestral areas using models implemented in BioGeoBEARS. RESULTS: Species-level resolution was occasionally low because of slow rates of molecular evolution and/or recent divergences. Older divergences, however, were more strongly supported and allow the evolutionary history of the group to be inferred. The genus diverged in Wallacea from its common ancestor with Acerodon; founder-event speciation out of Wallacea was a common inference. Pteropus species in Micronesia and the western Indian Ocean were also inferred to result from founder-event speciation. MAIN CONCLUSIONS: Dispersal between regions of the IAA and the islands found therein fostered diversification of Pteropus throughout the IAA and beyond. Dispersal in Pteropus is far higher than in most other volant taxa studied to date, highlighting the importance of inter-island movement in the biogeographic history of this large clade of large bats.

Long non-coding RNAs regulation of therapeutic resistance.

Non-protein coding RNAs have emerged as a regulator of cell signaling and cancer progression through regulation of cell proliferation, metastatic burden, and cancer stem cell capacity. A subtype of non-protein coding RNA is long non-protein coding RNA (lncRNA). Besides their aforementioned roles in cancer cell biology, dysregulation of lncRNAs contribute to resistance to therapeutic treatments. A couple of important therapeutic classes are chemotherapy and targeted/hormone therapies. This review highlights the variety of malignancies affected by lncRNA dysregulation and the underlying mechanism causing therapeutic resistance.

Quantifying the bat bushmeat trade in North Sulawesi, Indonesia, with suggestions for conservation action.

The intense consumption of flying foxes in North Sulawesi, Indonesia has raised hunting pressure and extirpation is expected to spread into other regions. To assess local cultural attitudes towards bats for formulating a targeted conservation campaign, we conducted a survey of consumption practices of bats in 2013 at the eight major markets near Manado. Locals eat flying foxes at least once a month, but the frequency increases tenfold around Christian holidays. Approximately 500 metric tons of bats are imported from other provinces, with South Sulawesi as the main provider at 38%. No action has been taken to conserve the bats, as continued abundance in the market masks the effects of the bushmeat trade on wild populations. We suggest: (1) engaging churches as conduits for environmental education and quota enforcement; (2) legal regulation of interprovincial trade; (3) substituting bats with a sustainable option; (4) involving local students as campaigners to ensure higher receptiveness from local communities. Grassroots conservation initiatives combined with enforcement of existing laws aim to affect change on a local level, which has been successful in other conservation programs. These efforts would not only progress bat conservation, but conservation of other rare, endemic mammals common to the bushmeat trade.

The senescence-associated secretory phenotype promotes benign prostatic hyperplasia.

Benign prostatic hyperplasia (BPH) is characterized by increased tissue mass in the transition zone of the prostate, which leads to obstruction of urine outflow and considerable morbidity in a majority of older men. Senescent cells accumulate in human tissues, including the prostate, with increasing age. Expression of proinflammatory cytokines is increased in these senescent cells, a manifestation of the senescence-associated secretory phenotype. Multiplex analysis revealed that multiple cytokines are increased in BPH, including GM-CSF, IL-1α, and IL-4, and that these are also increased in senescent prostatic epithelial cells in vitro. Tissue levels of these cytokines were correlated with a marker of senescence (cathepsin D), which was also strongly correlated with prostate weight. IHC analysis revealed the multifocal epithelial expression of cathepsin D and coexpression with IL-1α in BPH tissues. In tissue recombination studies in nude mice with immortalized prostatic epithelial cells expressing IL-1α and prostatic stromal cells, both epithelial and stromal cells exhibited increased growth. Expression of IL-1α in prostatic epithelial cells in a transgenic mouse model resulted in increased prostate size and bladder obstruction. In summary, both correlative and functional evidence support the hypothesis that the senescence-associated secretory phenotype can promote the development of BPH, which is the single most common age-related pathology in older men.

FGFR1-WNT-TGF-ß signaling in prostate cancer mouse models recapitulates human reactive stroma.

The reactive stroma surrounding tumor lesions performs critical roles ranging from supporting tumor cell proliferation to inducing tumorigenesis and metastasis. Therefore, it is critical to understand the cellular components and signaling control mechanisms that underlie the etiology of reactive stroma. Previous studies have individually implicated fibroblast growth factor receptor 1 (FGFR1) and canonical WNT/ß-catenin signaling in prostate cancer progression and the initiation and maintenance of a reactive stroma; however, both pathways are frequently found to be coactivated in cancer tissue. Using autochthonous transgenic mouse models for inducible FGFR1 (JOCK1) and prostate-specific and ubiquitously expressed inducible ß-catenin (Pro-Cat and Ubi-Cat, respectively) and bigenic crosses between these lines (Pro-Cat × JOCK1 and Ubi-Cat × JOCK1), we describe WNT-induced synergistic acceleration of FGFR1-driven adenocarcinoma, associated with a pronounced fibroblastic reactive stroma activation surrounding prostatic intraepithelial neoplasia (mPIN) lesions found both in in situ and reconstitution assays. Both mouse and human reactive stroma exhibited increased transforming growth factor-ß (TGF-ß) signaling adjacent to pathologic lesions likely contributing to invasion. Furthermore, elevated stromal TGF-ß signaling was associated with higher Gleason scores in archived human biopsies, mirroring murine patterns. Our findings establish the importance of the FGFR1-WNT-TGF-ß signaling axes as driving forces behind reactive stroma in aggressive prostate adenocarcinomas, deepening their relevance as therapeutic targets.

Highly specific targeting of the TMPRSS2/ERG fusion gene using liposomal nanovectors.

PURPOSE: The TMPRSS2/ERG (T/E) fusion gene is present in half of all prostate cancer tumors. Fusion of the oncogenic ERG gene with the androgen-regulated TMPRSS2 gene promoter results in expression of fusion mRNAs in prostate cancer cells. The junction of theTMPRSS2- and ERG-derived portions of the fusion mRNA constitutes a cancer-specific target in cells containing the T/E fusion gene. Targeting the most common alternatively spliced fusion gene mRNA junctional isoforms in vivo using siRNAs in liposomal nanovectors may potentially be a novel, low-toxicity treatment for prostate cancer. EXPERIMENTAL DESIGN: We designed and optimized siRNAs targeting the two most common T/E fusion gene mRNA junctional isoforms (type III or type VI). Specificity of siRNAs was assessed by transient co-transfection in vitro. To test their ability to inhibit growth of prostate cancer cells expressing these fusion gene isoforms in vivo, specific siRNAs in liposomal nanovectors were used to treat mice bearing orthotopic or subcutaneous xenograft tumors expressing the targeted fusion isoforms. RESULTS: The targeting siRNAs were both potent and highly specific in vitro. In vivo they significantly inhibited tumor growth. The degree of growth inhibition was variable and was correlated with the extent of fusion gene knockdown. The growth inhibition was associated with marked inhibition of angiogenesis and, to a lesser degree, proliferation and a marked increase in apoptosis of tumor cells. No toxicity was observed. CONCLUSIONS: Targeting the T/E fusion junction in vivo with specific siRNAs delivered via liposomal nanovectors is a promising therapy for men with prostate cancer.

Sexual selection, multiple male ornaments, and age- and condition-dependent signaling in the common yellowthroat.

In many animals, sexual selection has resulted in complex signaling systems in which males advertise aspects of their phenotypic or genetic quality through elaborate ornamentation and display behaviors. Different ornaments might convey different information or be directed at different receivers, but they might also be redundant signals of quality that function reliably at different times (ages) or in different contexts. We explored sexual selection and age- and condition-dependent signaling in the common yellowthroat (Geothlypis trichas), a sexually dichromatic warbler with two prominent plumage ornaments--a melanin-based, black facial "mask" and carotenoid-based, UV-yellow "bib." In a three-year study, variance among males in the number of social (M(w)) and extra-pair (M(e)) mates generated strong sexual selection on mask and bib attributes. Some traits (mask size, bib yellow brightness) were correlated with male age and did not experience selection beyond age-related increases in M(w) and M(e). Other traits showed age-specific (bib size) or age-reversed (ultraviolet brightness) patterns of selection that paralleled changes in the information-content of each ornament. The components of male fitness generating selection in young versus old males were distinct, reflecting different sources of variation in male fertilization success. Age- and context-dependent changes in the strength, direction, and target of selection may help explain the maintenance of multiple ornaments in this and other species.

Profile changes in orthodontic patients treated with mandibular advancement surgery.

INTRODUCTION: The potential to improve facial esthetics is often the deciding factor in treatment planning of borderline orthodontic patients who can be treated with either orthognathic surgery or dental camouflage. The purpose of this study was to determine the degree of skeletal and soft-tissue Class II disharmony necessary before a significant esthetic benefit is derived from mandibular advancement surgery. METHODS: Twenty laypeople, 20 orthodontists, and 20 oral surgeons rated the attractiveness of before and after treatment profiles of 20 mandibular advancement patients using a 5-point Likert scale. The Spearman rank correlation tested for relationships between amount of profile change and varying pretreatment ANB and profile angles. Plots of the distribution of profile changes with varying ANB and profile angles were then examined. RESULTS: There was a tendency for inverse correlations between profile change and profile angle, and for positive correlations between profile change and ANB angles, but only the relationship between profile change and ANB angles judged by the orthodontists was statistically significant (P <0.05). Orthodontists, oral surgeons, and laypeople found that profiles consistently improved when profile angles were < or = 159 degrees, < or = 158 degrees, and < or = 157 degrees, respectively. Orthodontists and oral surgeons found profiles consistently improved when ANB angles were > or = 5.5 degrees and > or = 6.5 degrees, respectively, whereas laypeople showed no trend between ANB angle and profile change. The incidence of having less desirable profiles after treatment was 2.6 to 5.0 times higher when the pretreatment profile angles were larger than the threshold profile angles, and 4.5 to 7.9 times higher when the pretreatment ANB angles were less than threshold ANB angles. CONCLUSIONS: Pretreatment profile angles < 160 degrees and ANB angles > 6 degrees are necessary for profiles to be consistently perceived as improved after surgery and to minimize the incidence of the profile worsening after treatment.

Microleakage of Class II posterior composite restorations with gingival margins placed entirely within dentin.

PURPOSE: Composite restorations are more frequently being placed with margins apical to the cementoenamel junction. However, margins within dentin are prone to microleakage. The purpose of this in vitro study was to evaluate various restorative procedures in terms of their ability to reduce microleakage in posterior composite restorations with gingival margins within dentin. We also examined the effect of staining time on microleakage. MATERIALS AND METHODS: Mesio-occlusal and disto-occlusal preparations were made in 50 extracted molars. Teeth were randomly assigned to receive 1 of 5 treatments followed by restoration with Z100 composite resin: acid etch (control); Clearfil SE Bond; Prompt-L-Pop; Vitrebond/Scotchbond Multipurpose Plus (closed-sandwich technique); or Geristore/Tenure (open-sandwich technique). After 48 hours of water storage followed by sectioning buccolingually, 1 restoration from each tooth was randomly assigned to either 2- or 4-hour immersion in 50% by weight silver nitrate solution. Restorations were removed and gingival floors analyzed to determine the percentage of surface area stained in each of 3 0.5-mm wide zones. RESULTS: Repeated measures ANOVA did not reveal statistically significant differences in staining for 2 and 4 hours. Compared with the control group, Clearfil SE bond produced statistically significant reductions in leakage in all 3 zones. Prompt-L-Pop did not reduce leakage significantly except in zone 3 (closest to the pulp). Vitrebond and Geristore both reduced microleakage in zones 2 and 3, but the reduction was greater with the use of Vitrebond. CONCLUSION: Both Clearfil SE Bond and Vitrebond in a closed-sandwich technique were effective methods for reducing microleakage within dentin.