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In this work, volume of fluid method coupled with discrete element method are established to explore the multi-scale flow hydrodynamics in the gas-liquid-solid system with multi-inlet configuration. The numerical method is well validated by experimental measurements. The results reveal that: (i) the squeezing effect of the densely packed particles largely determines the multi-channel flow pattern by affecting the initial size, shape and moving direction of the bubbles newly detached from the dense bed; (ii) in the two-channel system, the unbalanced radial squeezing effect of particles leads to parenthesis-shape bubble ascending path; (iii) due to bubble collision and breakup, the volume, velocity, chord length and aspect ratio of bubbles all have a wide distribution and exhibit a characteristic of chaotic and nonlinear. The findings obtained provide meaningful insights for the understanding of multi-channel flow behaviors in gas-solid-liquid reactors.

Bursaphelenchus xylophilus, causal agent of pine wilt disease, causes extensive damage worldwide. Strategies are needed to inhibit population growth or block the spread of the invasive nematode to control pine wilt disease. The gene daf-8 plays crucial roles in larval development and dauer formation in Caenorhabditis elegans, but little is known about its orthologue in B. xylophilus. In the present molecular characterization and functional analysis of daf-8 in B. xylophilus (Bx-daf-8), RT-qPCR showed that the expression of Bx-daf-8 gradually increased during the embryonic stage, peaked in the second-stage juvenile (J2), then dramatically dropped in the J3, and remained at that low level for the rest of its life. Bx-daf-8-transgenic C. elegans was employed to mimic the spatiotemporal expression of Bx-daf-8, which was expressed close to the pharynx during early embryogenesis and weakly throughout the whole body during late embryogenesis. It was observed in head neurons and tail ganglions throughout all postembryonic stages. B. xylophilus embryos were severely abnormal, and hatching rate decreased sharply after Bx-daf-8 knockdown. daf-16-1 and da-f16-2, downstream genes in the IIS pathway, also dropped sharply after Bx-daf-8 knockdown. We propose that TGFβ may crosstalk with the IIS pathway upstream of Bx-daf-16 and that daf-8 may act as a master regulator of daf-16 in B. xylophilus. However, knockdown of Bx-daf-8 did not lead to constitutive developmental arrest at the dauer larval stage, indicating that dauer entry in B. xylophilus might be controlled by several genes and is more complicated than in C. elegans. Bx-daf-8 alone did not control the dauer entry in B. xylophilus, but it was indispensable for embryogenesis, providing a potential target for suppressing population growth of B. xylophilus.

Bursaphelenchus xylophilus is an invasive plant-parasitic nematode causing the notorious pine wilt disease (PWD) worldwide, which results in huge economic losses. G protein-coupled receptors (GPCRs) play an essential role in mating and reproduction behavior of animals. As a unique biogenic amine in invertebrates, octopamine (OA) can regulate a variety of physiological and behavioral responses by binding specific GPCRs. These specific GPCRs are also called octopamine receptors (OARs), and octr-1 is one of them. However, Bxy-octr-1 is unknown in B. xylophilus. Therefore, we investigated the expression pattern and biological function of Bxy-octr-1. Bioinformatics analysis indicated that Bxy-octr-1 was evolutionarily conserved. The real-time quantitative PCR data revealed that Bxy-octr-1 expression was required throughout the entire life of B. xylophilus. mRNA in situ hybridization showed that Bxy-octr-1 was mainly located in the cephalopharynx, body wall muscle, intestine, and gonadal organs of B. xylophilus. RNA interference (RNAi) showed that embryo hatching rates and locomotion speeds were both dramatically decreased. Obvious abnormal phenotypes were observed in the second-stage of juveniles after RNAi treated. Furthermore, its ontogenesis was stunting. Lack of Bxy-octr-1 reduced fecundity of females, of which 31.25% of them could not successfully ovulate. In addition, the error positioning ratio of the nematode was significantly increased. Our study suggests that Bxy-octr-1 is indispensable for locomotion, early ontogenesis and mating behavior in B. xylophilus.

At present, the energy loss (EL) mechanism of intracranial aneurysm (IA) rupture is explored based on the global EL calculated by Bernoulli equation, but the details of EL are still unclear. This study aimed to explore the temporal and spatial characteristics of EL of IAs and reveal its mechanism. A novel method for calculating the EL of IAs based on dissipation function (DF) was proposed. DF was derived from the differential form of the energy equation and reflected the irreversible conversion from mechanical energy to internal energy caused by the friction between the fluid micelles. Eight sidewall IAs located at the posterior communicating segment of the internal carotid artery were collected; the three-dimensional (3D) geometric models of IAs were established employing image segmentation and 3D reconstruction. Computational fluid dynamics was applied to obtain hemodynamic parameters of IAs. The temporal and spatial characteristics of EL of IAs were achieved utilizing our proposed method. The simulation results indicated that EL occurred mainly in the boundary layer and the region adjacent to high-velocity inflow jet, EL increased rapidly during cardiac systole and reached its maximum at end-systolic phase and then decreased gradually during diastole until the end of cardiac cycle. The proposed method achieved some improvements over the traditional Bernoulli equation–based method by acquiring the temporal and spatial characteristics of EL, and it could provide insights into the EL of IAs and contribute to further rupture mechanism investigation.

Background: A highly potent pan-Janus kinase (JAK) inhibitor with excellent kinome selectivity was developed for topical delivery to treat severe asthma. This poorly soluble drug discovery candidate, iJAK-001, is expected to exhibit long duration of JAK/STAT pathway inhibition at low doses in asthmatics because of depot effect after dry powder inhalation. Human dose projection for inhaled molecules with low aqueous solubility remains to be a daunting challenge because of several limitations: (1) bioanalytical measurement of dissolved fraction after inhalation of solid particles is uncertain; (2) distribution of these particles is not homogenous in the lung; (3) in vitro solubility measurements to estimate fraction dissolved may not be a reflection of local surface lung concentration; (4) lack of a surrogate biomarker of lung target engagement, and (5) invasive procedure needed to sample human lung tissue in the clinic. Methods: We leveraged in silico, in vitro, and in vivo tools preclinically and found significant differences in lung to plasma partition ratio when iJAK-001 was given intravenously (IV) or intratracheally in a solution-based formulation versus that in suspension, as well as pharmacodynamic response in preclinical asthma models when delivered systemically via IV infusion versus inhaled. Results and Conclusion: The combined results from above suggest that caution must be exercised using either lung or plasma exposure for human dose projection. Instead, using the local inhibitor concentration estimate based on delivery efficiency, dose, fraction absorbed, and rate of absorption normalized by lung (cardiac) blood flow may be more appropriate for dose projection.

BACKGROUND The pine wood nematode (PWN) Bursaphelenchus xylophilus is the causal agent of pine wilt disease (PWD). This disease is a serious threat to pine forests globally. The fuca gene encodes α-L-fucosidase, which plays crucial roles in numerous biological and pathological processes in bacteria, fungi, plants and animals. To find promising control strategies against PWD, we investigated the expression and functions of Bxy-fuca in B. xylophilus. RESULTS Bxy-fuca encoding α-L-fucosidase is highly conserved within the deduced functional domains and key residues. It is expressed continuously across all developmental stages of B. xylophilus. mRNA in situ hybridization demonstrated that Bxy-fuca was mainly localized in the body wall muscles and intestine. RNA interference indicated that the zygotic expression of Bxy-fuca was indispensable for embryogenesis. The rate of B. xylophilus egg hatch was significantly decreased after Bxy-fuca was interfered. Postembryonic silence of Bxy-fuca resulted in a dramatic decrease in the longevity of and the total number of eggs produced by B. xylophilus. In addition, the motility of the nematode was greatly hampered with a significant drop in head thrashing frequency. CONCLUSION Bxy-fuca plays crucial roles in development, lifespan and reproduction of B. xylophilus. Our results provide promising hints for control of PWD by blocking embryogenesis and ontogenesis, decreasing nematode fecundity, and disrupting the connection between B. xylophilus and its vector beetle by preventing nematode movement into the tracheal system. © 2019 Society of Chemical Industry.

PI3Kδ catalytic activity is required for immune cell activation, and has been implicated in inflammatory diseases as well as hematological malignancies in which the AKT pathway is overactive. A purine PI3Kδ inhibitor bearing a benzimidazolone-piperidine motif was found to be poorly tolerated in dog, which was attributed to diffuse vascular injury. Several strategies were implemented to mitigate this finding, including reconstruction of the benzimidazolone-piperidine selectivity motif. Structure-based design led to the identification of O- and N-linked heterocycloalkyls, with pyrrolidines being particularly ligand efficient and kinome selective, and having an improved safety pharmacology profile. A representative was advanced into a dog tolerability study where it was found to be well tolerated, with no histopathological evidence of vascular injury.

We leveraged a clinical pharmacokinetic (PK)/pharmacodynamics (PD)/efficacy relationship established with an oral phosphatidylinositol 3-kinase (PI3K)δ inhibitor (Idelalisib) in a nasal allergen challenge study to determine whether a comparable PK/PD/efficacy relationship with PI3Kδ inhibitors was observed in preclinical respiratory models of type 2 T helper cell (TH2) and type 1 T helper cell (TH1) inflammation. Results from an in vitro rat blood basophil (CD63) activation assay were used as a PD biomarker. IC50 values for PI3Kδ inhibitors, MSD-496486311, MSD-126796721, Idelalisib, and Duvelisib, were 1.2, 4.8, 0.8, and 0.5 μM. In the ovalbumin Brown Norway TH2 pulmonary inflammation model, all PI3Kδ inhibitors produced a dose-dependent inhibition of bronchoalveolar lavage eosinophils (maximum effect between 80% and 99%). In a follow-up experiment designed to investigate PK attributes [maximum (or peak) plasma concentration (Cmax), area under the curve (AUC), time on target (ToT)] that govern PI3Kδ efficacy, MSD-496486311 [3 mg/kg every day (QD) and 100 mg/kg QD] produced 16% and 93% inhibition of eosinophils, whereas doses (20 mg/kg QD, 10 mg/kg twice per day, and 3 mg/kg three times per day) produced 54% to 66% inhibition. Our profiling suggests that impact of PI3Kδ inhibitors on eosinophils is supported by a PK target with a ToT over the course of treatment close to the PD IC50 rather than strictly driven by AUC, Cmax, or Cmin (minimum blood plasma concentration) coverage. Additional studies in an Altenaria alternata rat model, a sheep Ascaris-sensitive sheep model, and a TH1-driven rat ozone exposure model did not challenge our hypothesis, suggesting that an IC50 level of TE (target engagement) sustained for 24 hours is required to produce efficacy in these traditional models. We conclude that the PK/PD observations in our animal models appear to align with clinical results associated with a TH2 airway disease.

MAPKs affect gonadal differentiation in mice and humans, but whether this applies to all mammals is as yet unknown. Thus, we investigated MAPK expression during gonadal differentiation and after treatment with oestrogen in a distantly related mammal, the marsupial tammar wallaby, using our model of oestrogen-induced gonadal sex reversal. High-throughput RNA-sequencing was carried out on gonads collected from developing tammar 2 days before birth to 8 days after birth to characterise MAPK and key sexual differentiation markers. Day 25 foetal testes were cultured for 120 h in control medium or medium supplemented with exogenous oestrogen and processed for RNA-seq to identify changes in gene expression in response to oestrogen. MAPK pathway genes in the tammar were highly conserved at the sequence and amino acid level with those of mice and humans. Marsupial MAP3K1 and MAP3K4 clustered together in a separate branch from eutherian mammals. There was a marked decrease in the expression of male-determining genes SOX9 and AMH and increase in the female marker FOXL2 in oestrogen-treated male gonads. Only MAP3K1 expression increased in male gonads in response to oestrogen while other MAPK genes remained unaffected. This study suggests that MAP3K1 can be influenced by exogenous oestrogens during gonadal differentiation in this marsupial.

The approvals of idelalisib and duvelisib have validated PI3Kδ inhibitors for the treatment for hematological malignancies driven by the PI3K/AKT pathway. Our program led to the identification of structurally distinct heterocycloalkyl purine inhibitors with excellent isoform and kinome selectivity; however, they had high projected human doses. Improved ligand contacts gave potency enhancements, while replacement of metabolic liabilities led to extended half-lives in preclinical species, affording PI3Kδ inhibitors with low once-daily predicted human doses. Treatment of C57BL/6-Foxp3-GDL reporter mice with 30 and 100 mg/kg/day of 3c (MSD-496486311) led to a 70% reduction in Foxp3-expressing regulatory T cells as observed through bioluminescence imaging with luciferin, consistent with the role of PI3K/AKT signaling in Treg cell proliferation. As a model for allergic rhinitis and asthma, treatment of ovalbumin-challenged Brown Norway rats with 0.3 to 30 mg/kg/day of 3c gave a dose-dependent reduction in pulmonary bronchoalveolar lavage inflammation eosinophil cell count.

Prostaglandin E2 (PGE2), an arachidonic acid pathway metabolite produced by cyclooxygenase (COX)-1/2, has been shown to impair anti-tumor immunity through engagement with one or more E-type prostanoid receptors (EP1-4). Specific targeting of EP receptors, as opposed to COX-1/2 inhibition, has been proposed to achieve preferential antagonism of PGE2–mediated immune suppression. Here we describe the anti-tumor activity of MF-766, a potent and highly selective small-molecule inhibitor of the EP4 receptor. EP4 inhibition by MF-766 synergistically improved the efficacy of anti-programmed cell death protein 1 (PD-1) therapy in CT26 and EMT6 syngeneic tumor mouse models. Multiparameter flow cytometry analysis revealed that treatment with MF-766 promoted the infiltration of CD8+ T cells, natural killer (NK) cells and conventional dendritic cells (cDCs), induced M1-like macrophage reprogramming, and reduced granulocytic myeloid-derived suppressor cells (MDSC) in the tumor microenvironment (TME). In vitro experiments demonstrated that MF-766 restored PGE2-mediated inhibition of lipopolysaccharide (LPS)-induced tumor necrosis factor (TNF)-α production in THP-1 cells and human blood, and PGE2-mediated inhibition of interleukin (IL)-2-induced interferon (IFN)-γ production in human NK cells. MF-766 reversed the inhibition of IFN-γ in CD8+ T-cells by PGE2 and impaired suppression of CD8+ T-cells induced by myeloid-derived suppressor cells (MDSC)/PGE2. In translational studies using primary human tumors, MF-766 enhanced anti-CD3-stimulated IFN-γ, IL-2, and TNF-α production in primary histoculture and synergized with pembrolizumab in a PGE2 high TME. Our studies demonstrate that the combination of EP4 blockade with anti-PD-1 therapy enhances antitumor activity by differentially modulating myeloid cell, NK cell, cDC and T-cell infiltration profiles.

Prostaglandin E2 (PGE2), an arachidonic acid pathway metabolite produced by cyclooxygenase (COX)-1/2, has been shown to impair anti-tumor immunity through engagement with one or more E-type prostanoid receptors (EP1-4). Specific targeting of EP receptors, as opposed to COX-1/2 inhibition, has been proposed to achieve preferential antagonism of PGE2–mediated immune suppression. Here we describe the anti-tumor activity of MF-766, a potent and highly selective small-molecule inhibitor of the EP4 receptor. EP4 inhibition by MF-766 synergistically improved the efficacy of anti-programmed cell death protein 1 (PD-1) therapy in CT26 and EMT6 syngeneic tumor mouse models. Multiparameter flow cytometry analysis revealed that treatment with MF-766 promoted the infiltration of CD8+ T cells, natural killer (NK) cells and conventional dendritic cells (cDCs), induced M1-like macrophage reprogramming, and reduced granulocytic myeloid-derived suppressor cells (MDSC) in the tumor microenvironment (TME). In vitro experiments demonstrated that MF-766 restored PGE2-mediated inhibition of lipopolysaccharide (LPS)-induced tumor necrosis factor (TNF)-α production in THP-1 cells and human blood, and PGE2-mediated inhibition of interleukin (IL)-2-induced interferon (IFN)-γ production in human NK cells. MF-766 reversed the inhibition of IFN-γ in CD8+ T-cells by PGE2 and impaired suppression of CD8+ T-cells induced by myeloid-derived suppressor cells (MDSC)/PGE2. In translational studies using primary human tumors, MF-766 enhanced anti-CD3-stimulated IFN-γ, IL-2, and TNF-α production in primary histoculture and synergized with pembrolizumab in a PGE2 high TME. Our studies demonstrate that the combination of EP4 blockade with anti-PD-1 therapy enhances antitumor activity by differentially modulating myeloid cell, NK cell, cDC and T-cell infiltration profiles.

Background Bursaphelenchus xylophilus is one of the most destructive invasive species, causing extensive economic losses worldwide. The sex ratio of female to male of B. xylophilus plays an important role in the nematode infestation. However, little is known about the processes of its sex determination. The double sex/mab-3-related family of transcription factors are highly conserved in animals, playing crucial roles in sex determination, spermatogenesis and ontogenesis. We therefore investigated its orthologue, Bxy-mab-3, in B. xylophilus. Results Bxy-mab-3 has two typical conserved DM domains. It was observed in J2 (the second-stage of juveniles), J3, J4 and male adults (specifically on the spicules), but not in eggs or female adults via mRNA in situ hybridization. RNA-seq indicated significantly higher expression in males. RNAi showed that the body size and sperm size of male adults were markedly smaller than those of the controls. Meanwhile, almost all the RNAi treated males failed to mate with the normal females, even 26.34% of interfered males did not produce sperm. However, RNAi of Bxy-mab-3 had no effect on the sex ratio of B. xylophilus. Conclusion Bxy-mab-3 is indispensable for spermatogenesis, ontogenesis and mating behavior. It is a typical sex-determination gene with the differential expression in males and females. However, knocking down Bxy-mab-3 expression could not alter the sex ratio as seen in other species. Our findings contribute towards a better understanding of the molecular events of Bxy-mab-3 in B. xylophilus, which provides promising hints for control of pine wilt disease by blocking ontogenesis and decreasing nematode fecundity. This article is protected by copyright. All rights reserved.

MAPKs affect gonadal differentiation in mice and humans, but whether this applies to all mammals is as yet unknown. Thus, we investigated MAPK expression during gonadal differentiation and after treatment with oestrogen in a distantly related mammal, the marsupial tammar wallaby, using our model of oestrogen-induced gonadal sex reversal. High-throughput RNA-sequencing was carried out on gonads collected from developing tammar 2 days before birth to 8 days after birth to characterise MAPK and key sexual differentiation markers. Day 25 foetal testes were cultured for 120 h in control medium or medium supplemented with exogenous oestrogen and processed for RNA-seq to identify changes in gene expression in response to oestrogen. MAPK pathway genes in the tammar were highly conserved at the sequence and amino acid level with those of mice and humans. Marsupial MAP3K1 and MAP3K4 clustered together in a separate branch from eutherian mammals. There was a marked decrease in the expression of male-determining genes SOX9 and AMH and increase in the female marker FOXL2 in oestrogen-treated male gonads. Only MAP3K1 expression increased in male gonads in response to oestrogen while other MAPK genes remained unaffected. This study suggests that MAP3K1 can be influenced by exogenous oestrogens during gonadal differentiation in this marsupial.

Summary The pine wood nematode (PWN) Bursaphelenchus xylophilus is the causal agent of pine wilt disease (PWD). To understand the light influence on PWN, we investigated its phototactic behaviour. Our data indicated the mixed population of propagative PWN had a positive response to red, orange, yellow, green, blue and white lights, but a weak negative response to violet. For age-synchronised propagative nematodes, however, phototactic behavioural features changed with development. Interestingly, the dispersal fourth-stage juveniles (JIV) showed negative response to all tested lights, which was almost completely the reverse of the propagative fourth-stage juveniles (J4). Further bioassays proved that green, blue and white lights suppressed the host transmission of dispersal JIV from vector beetle to healthy pine branches. Our results revealed that night could be the peak of host transmission. With the results of previous studies, we speculate volatiles from the host tree and light may play pull-and-push roles to accelerate the host transmission of B. xylophilus.

The discovery of a potent selective low dose Janus kinase 1 (JAK1) inhibitor suitable for clinical evaluation is described. As part of an overall goal to minimize dose, we pursued a medicinal chemistry strategy focused on optimization of key parameters that influence dose size, including lowering human Clint and increasing intrinsic potency, bioavailability, and solubility. To impact these multiple parameters simultaneously, we used lipophilic ligand efficiency as a key metric to track changes in the physicochemical properties of our analogs, which led to improvements in overall compound quality. In parallel, structural information guided advancements in JAK1 selectivity by informing on new vector space, which enabled the discovery of a unique key amino acid difference between JAK1 (Glu966) and JAK2 (Asp939). This difference was exploited to consistently produce analogs with the best balance of JAK1 selectivity, efficacy, and projected human dose, ultimately culminating in the discovery of compound 28.

Sex determination and sexual differentiation pathways are highly conserved between marsupials and eutherians. There are 2 different pathways of prostaglandin D2 (PGD2) synthesis: prostaglandin D synthase (PTGDS) and haematopoietic prostaglandin D synthase (HPGDS). PGD2 regulates the subcellular localization of SOX9 during gonadal sexual differentiation. To investigate the function of PGD2 in the tammar gonad, we cultured undifferentiated male gonads in the presence of the HPGDS inhibitor HQL-79 and female gonads with exogenous PGD2 to mimic activation of the PTGDS-PGD2 pathway. Tammar PTGDS and HPGDS have only 50% similarity with mouse and human orthologues, but functional domains are conserved. The expression of SOX9 was unchanged by the treatments in cultured gonads, but its subcellular localization was markedly affected. SOX9 remained cytoplasmic in the Sertoli cells of testes treated with HQL-79. Treated testes developed a thickened ovary-like surface epithelium. In contrast, SOX9 became nuclear in the granulosa cells of developing ovaries treated with PGD2 and the surface epithelium was thin, as in testes. These results demonstrate that PGD2 regulates the subcellular localization of SOX9 and subsequent gonadal development in the developing marsupial gonads, as it does in mice, and that it must have been an ancestral mechanism.

There is increasing evidence that long non-coding RNAs (lncRNAs) are important for normal reproductive development, yet very few lncRNAs have been identified in phalluses so far. Unlike eutherians, phallus development in the marsupial tammar wallaby occurs post-natally, enabling manipulation not possible in eutherians in which differentiation occurs in utero. We treated with sex steroids to determine the effects of androgen and oestrogen on lncRNA expression during phallus development. Hormonal manipulations altered the coding and non-coding gene expression profile of phalluses. We identified several predicted co-regulatory lncRNAs that appear to be co-expressed with the hormone-responsive candidate genes regulating urethral closure and phallus growth, namely IGF1, AR and ESR1. Interestingly, more than 50% of AR-associated coding genes and lncRNAs were also associated with ESR1. In addition, we identified and validated three novel co-regulatory and hormone-responsive lncRNAs: lnc-BMP5, lnc-ZBTB16 and lncRSPO4. Lnc-BMP5 was detected in the urethral epithelium of male phalluses and was downregulated by oestrogen in males. Lnc-ZBTB16 was downregulated by oestrogen treatment in male phalluses at day 50 post-partum (pp). LncRSPO4 was downregulated by adiol treatment in female phalluses but increased in male phalluses after castration. Thus, the expression pattern and hormone responsiveness of these lncRNAs suggests a physiological role in the development of the phallus.

The kinase-activating mutation G2019S in leucine-rich repeat kinase 2 (LRRK2) is one of the most common genetic causes of Parkinson's disease (PD) and has spurred development of LRRK2 inhibitors. Preclinical studies have raised concerns about the safety of LRRK2 inhibitors due to histopathological changes in the lungs of nonhuman primates treated with two of these compounds. Here, we investigated whether these lung effects represented on-target pharmacology and whether they were reversible after drug withdrawal in macaques. We also examined whether treatment was associated with pulmonary function deficits. We conducted a 2-week repeat-dose toxicology study in macaques comparing three different LRRK2 inhibitors: GNE-7915 (30 mg/kg, twice daily as a positive control), MLi-2 (15 and 50 mg/kg, once daily), and PFE-360 (3 and 6 mg/kg, once daily). Subsets of animals dosed with GNE-7915 or MLi-2 were evaluated 2 weeks after drug withdrawal for lung function. All compounds induced mild cytoplasmic vacuolation of type II lung pneumocytes without signs of lung degeneration, implicating on-target pharmacology. At low doses of PFE-360 or MLi-2, there was ~50 or 100% LRRK2 inhibition in brain tissue, respectively, but histopathological lung changes were either absent or minimal. The lung effect was reversible after dosing ceased. Lung function tests demonstrated that the histological changes in lung tissue induced by MLi-2 and GNE-7915 did not result in pulmonary deficits. Our results suggest that the observed lung effects in nonhuman primates in response to LRRK2 inhibitors should not preclude clinical testing of these compounds for PD.

The development of the mammalian phallus involves hormone-dependent mesenchymal–epithelial signalling mechanisms that contribute to urethral closure and regulation of phallus elongation and growth. In marsupials, most differentiation and growth of the phallus occurs post-natally, making them amenable to direct hormone treatment. Expression of IGFs, FGFs, EFNB2, MAFB, DLX5 and AP-1 mRNAs in the phallus at day 50 post-partum (pp) were altered after treatment of tammar wallaby young from day 20 to 40 pp with androgen, oestrogen or after castration at day 25 pp. However, the most interesting changes occurred in the IGF pathway genes. Androgen treatment upregulated IGF1 in female phalluses and oestrogen treatment upregulated IGF1 in male phalluses, but it was downregulated by castration. IGFBP3 was higher in female phalluses and downregulated by androgen. IGF1 expression was higher in all untreated male than in female phalluses from day 50 to 150 pp, but IGFBP3 had the reverse pattern. At day 90 pp, when urethral closure in males is progressing and male phallus growth is accelerating. IGF1 and PCNA protein were only detected in the male urorectal septum, suggesting for the first time that closure and elongation may involve IGF1 activation of cell proliferation specifically in male phalluses. These effects of sex steroids on gene expression and on the IGF1 signalling pathway in particular, suggest that the developing phallus may be especially susceptible to perturbation by exogenous hormones.

We leveraged a clinical pharmacokinetic (PK)/pharmacodynamics (PD)/efficacy relationship established with an oral phosphatidylinositol 3-kinase (PI3K)

Environmental endocrine disruptors (EEDs) that affect androgen or estrogen activity may disrupt gene regulation during phallus development to cause hypospadias or a masculinized clitoris. We treated developing male tammar wallabies with estrogen and females with androgen from day 20-40 postpartum (pp) during the androgen imprinting window of sensitivity. Estrogen inhibited phallus elongation but had no effect on urethral closure and did not significantly depress testicular androgen synthesis. Androgen treatment in females did not promote phallus elongation but initiated urethral closure. Phalluses were collected for transcriptome sequencing at day 50 pp when they first become sexually dimorphic to examine changes in two signaling pathways, sonic hedgehog (SHH) and wingless-type MMTV integration site family (WNT)/β-catenin. SHH mRNA and β-catenin were predominantly expressed in the urethral epithelium in the tammar phallus, as in eutherian mammals. Estrogen treatment and castration of males induced an upregulation of SHH, while androgen treatment downregulated SHH. These effects appear to be direct since we detected putative estrogen receptor α (ERα) and androgen receptor (AR) binding sites near SHH. WNT5A, like SHH, was downregulated by androgen, while WNT4 was upregulated in female phalluses after androgen treatment. After estrogen treatment, WIF1 and WNT7A were both downregulated in male phalluses. After castration, WNT9A was upregulated. These results suggest that SHH and WNT pathways are regulated by both estrogen and androgen to direct the proliferation and elongation of the phallus during differentiation. Their response to exogenous hormones makes these genes potential targets of EEDs in the etiology of abnormal phallus development including hypospadias.

Sex determination and sexual differentiation pathways are highly conserved between marsupials and eutherians. There are 2 different pathways of prostaglandin D2 (PGD2) synthesis: prostaglandin D synthase (PTGDS) and haematopoietic prostaglandin D synthase (HPGDS). PGD2 regulates the subcellular localization of SOX9 during gonadal sexual differentiation. To investigate the function of PGD2 in the tammar gonad, we cultured undifferentiated male gonads in the presence of the HPGDS inhibitor HQL-79 and female gonads with exogenous PGD2 to mimic activation of the PTGDS-PGD2 pathway. Tammar PTGDS and HPGDS have only 50% similarity with mouse and human orthologues, but functional domains are conserved. The expression of SOX9 was unchanged by the treatments in cultured gonads, but its subcellular localization was markedly affected. SOX9 remained cytoplasmic in the Sertoli cells of testes treated with HQL-79. Treated testes developed a thickened ovary-like surface epithelium. In contrast, SOX9 became nuclear in the granulosa cells of developing ovaries treated with PGD2 and the surface epithelium was thin, as in testes. These results demonstrate that PGD2 regulates the subcellular localization of SOX9 and subsequent gonadal development in the developing marsupial gonads, as it does in mice, and that it must have been an ancestral mechanism.

The first sign of mammalian germ cell sexual differentiation is the initiation of meiosis in females and of mitotic arrest in males. In the mouse, retinoic acid induces ovarian Stra8 expression and entry of germ cells into meiosis. In developing mouse testes, cytochrome P450 family 26, subfamily b, polypeptide 1 (CYP26B1) produced by the Sertoli cells degrades retinoic acid, preventing Stimulated by Retinoic Acid Gene 8 (Stra8), expression and inhibiting meiosis. However, in developing humans, there is no evidence that CYP26B1 acts a meiosis-inhibiting factor. We therefore examined aspects of the retinoic acid/STRA8/CYP26B1 pathway during gonadal development in the tammar wallaby, a marsupial, to understand whether retinoic acid stimulation of STRA8 and CYP26B1 degradation of retinoic acid was conserved between widely divergent mammals. In tammar ovaries, as in human ovaries and unlike the pattern in mice, CYP26B1 expression was not downregulated before the onset of meiosis. Exposure of pre-meiotic tammar ovaries to exogenous retinoic acid in vitro upregulated STRA8 expression compared to controls. We conclude that retinoic acid and STRA8 are conserved factors that control the initiation of meiosis amongst mammals but the role of CYP26B1 as a meiosis-inhibiting factor may be specific to rodents. The identity of the marsupial meiosis-inhibiting factor remains unknown.

A method for observing and quantifying the mating behavior of the pinewood nematode, Bursaphelenchus xylophilus, was established under a stereomicroscope. To improve the mating efficiency of B. Xylophilus and to increase the chances of mating observation, virgin adults were cultured and used for the investigation. Eggs were obtained by keeping the nematodes in water and allowing the females to lay eggs for 10 min. The second-stage juveniles (J2) were synchronized by incubating the eggs for 24 h at 25 °C in the dark, and the early J4 were obtained by culturing the J2 with grey mold, Botrytis cinerea, for another 52 h. At this time point, most J4 nematodes could be clearly distinguished as being male or female using their genital morphology. The male and female J4 were collected and cultured separately in two different Petri dishes for 24 h to get virgin adult nematodes. A virgin male and a virgin female were paired in a drop of water in the well of a concave slide. The mating behavior was filmed with a video recorder under a stereomicroscope. The whole period of the mating process was 82.8 ±3.91 min (mean ±SE) and could be divided into 4 different phases: searching, contacting, copulating, and lingering. The mean minutes of duration were 21.8 ± 2.0, 28.0 ± 1.9, 25.8 ± 0.7 and 7.2 ± 0.5, respectively. Eleven sub-behaviors were described: cruising, approaching, encountering, touching, hooping, locating, attaching, ejaculating, separating, quiescence, and roaming. Interestingly, obvious intra-sexual competition was observed when one female was grouped with 3 males or one male with 3 females. This protocol is useful and valuable, not only in investigating the mating behavior of B. xylophilus, but also in acting as a reference for ethological studies of other nematodes.

Spleen tyrosine kinase (SYK) is a key activator of signaling pathways downstream of multiple surface receptors implicated in asthma. SYK function has been extensively studied in mast cells downstream of the high-affinity IgE receptor, FcεR1. Preclinical studies have demonstrated a role for SYK in models of allergic inflammation, but a role in airway constriction has not been demonstrated. Here, we have used a potent and selective pharmacological inhibitor of SYK to determine the role of SYK in allergen-mediated inflammation and airway constriction in preclinical models. Attenuation of allergic airway responses was evaluated in a rat passive anaphylaxis model and rat and sheep inhaled allergen challenge models, as well as an ex vivo model of allergen-mediated airway constriction in rats and cynomolgus monkeys. Pharmacological inhibition of SYK dose-dependently blocked IgE-mediated tracheal plasma extravasation in rats. In a rat ovalbumin-sensitized airway challenge model, oral dosing with an SYK inhibitor led to a dose-dependent reduction in lung inflammatory cells. Ex vivo analysis of allergen-induced airway constriction in ovalbumin-sensitized brown Norway rats showed a complete attenuation with treatment of a SYK inhibitor, as well as a complete block of allergen-induced serotonin release. Similarly, allergen-mediated airway constriction was attenuated in ex vivo studies from nonhuman primate lungs. Intravenous administration of an SYK inhibitor attenuated both early- and late-phase allergen-induced increases in airway resistance in an Ascaris-sensitive sheep allergen challenge model. These data support a key role for SYK signaling in mediating allergic airway responses.

Background: At birth, marsupial neonates have precociously developed forelimbs. The development of the tammar wallaby (Macropus eugenii) hindlimbs lags significantly behind that of the forelimbs. This differs from the grey short-tailed opossum, Monodelphis domestica, which has relatively similar fore- and hindlimbs at birth. This study examines the expression of the key patterning genes TBX4, TBX5, PITX1, FGF8, and SHH in developing limb buds in the tammar wallaby. Results: All genes examined were highly conserved with orthologues from opossum and mouse. TBX4 expression appeared earlier in development than in the mouse, but later than in the opossum. SHH expression is restricted to the zone of polarising activity, while TBX5 (forelimb) and PITX1 (hindlimb) showed diffuse mRNA expression. FGF8 is specifically localised to the apical ectodermal ridge, which is more prominent than in the opossum. Conclusions: The most marked divergence in limb size in marsupials occurs in the kangaroos and wallabies. The faster development of the fore limb compared to that of the hind limb correlates with the early timing of the expression of the key patterning genes in these limbs. Developmental Dynamics 243:324–338, 2014. © 2013 Wiley Periodicals, Inc.

Carbapenems are intravenous lifesaving hospital antibiotics. Once patients leave the hospital, they are sent home with antibiotics other than carbapenems since they cannot be administered orally due to lack of oral absorption primarily because of very highly polarity. A prodrug approach is a bona fide strategy to improve oral absorption of compounds. Design and synthesis, in vitro and in vivo evaluation of diversified prodrugs of ertapenem, one of the only once daily dosed carbapenems is described. Many of the prodrugs prepared for evaluation are rapidly hydrolyzed in rat plasma. Only bis-(5-methyl-2-oxo-1,3-dioxol-4-yl)methyl (medoxomil) ester prodrug was rapidly hydrolyzed in most of the plasmas including rat, human, dog, and monkey. Although the rate of conversion of ertapenem diethyl ester prodrug (6) was slow in in vitro plasma hydrolysis, it showed the best in vivo pharmacokinetic profile in dog by an intraduodenal dosing giving >31% total oral absorption.

In transonic centrifugal compressors, the losses and blockage are increased dominantly by the shock, shock/tip leakage vortex (TLV) interaction and shock/boundary layers interaction on the blade suction side. Therefore, a detailed investigation of shock effects is seriously important to improve the aerodynamic performance and extend the stable operating range of a centrifugal compressor. This paper firstly analyzes the shock structures in the transonic impeller at the design and off-design conditions. It is found that the shock structure is three-dimensional and swept along the span, with the shock becoming normal to the shroud near the blade tip. As the mass flow rate decreases, the swept shock moves upstream, and the shock/TLV interaction becomes stronger, inducing the vortex breakdown at the near stall condition. Then a comparative study is conducted on the flow structures in the transonic centrifugal impeller without the tip clearance (OTC impeller). Compared with the impeller with a tip clearance (WTC impeller), the splitter shock strength in the passage between the main blade and splitter is enhanced and the separation caused by the splitter shock /boundary layers interaction is induced, due to the lack of the blockage in the inducer induced by the shock/TLV of main blade interaction in the WTC impeller. Thus, at a large mass flow rate, the efficiency of the OTC impeller is lower than that of the WTC impeller because the stronger splitter shock induces larger flow separation in the passages between the main blade and splitter. However, at a small mass flow rate, the WTC impeller has a lower efficiency because the losses in the inducer induced by the interaction between the shock and the TLV of main blade are dominant.

Kallmann syndrome is characterized by hypogonadotrophic hypogonadism and anosmia. The syndrome can be caused by mutations in several genes, but the X-linked form is caused by mutation in the Kallmann syndrome 1 (KAL1). KAL1 plays a critical role in gonadotropin-releasing hormone (GnRH) neuronal migration that is essential for the normal development of the hypothalamic-pituitary-gonadal axis. Interestingly, KAL1 appears to be missing from the rodent X, and no orthologue has been detected as yet. We investigated KAL1 during development and in adults of an Australian marsupial, the tammar wallaby, Macropus eugenii. Marsupial KAL1 maps to an autosome within a group of genes that was added as a block to the X chromosome in eutherian evolution. KAL1 expression was widespread in embryonic and adult tissues. In the adult testis, tammar KAL1 mRNA and protein were detected in the germ cells at specific stages of differentiation. In the adult testis, the protein encoded by KAL1, anosmin-1, was restricted to the round spermatids and elongated spermatids. In the adult ovary, anosmin-1 was not only detected in the oocytes but was also localized in the granulosa cells throughout folliculogenesis. This is the first examination of KAL1 mRNA and protein localization in adult mammalian gonads. The protein localization suggests that KAL1 participates in gametogenesis not only through the development of the hypothalamic-pituitary-gonadal axis by activation of GnRH neuronal migration, but also directly within the gonads themselves. Because KAL1 is autosomal in marsupials but is X-linked in eutherians, its conserved involvement in gametogenesis supports the hypothesis that reproduction-related genes were actively recruited to the eutherian X chromosome.

Marsupials are especially valuable for comparative genomic studies of mammals. Two distantly related model marsupials have been sequenced: the South American opossum (Monodelphis domestica) and the tammar wallaby (Macropus eugenii), which last shared a common ancestor about 70 Mya. The six-fold opossum genome sequence has been assembled and assigned to chromosomes with the help of a cytogenetic map. A good cytogenetic map will be even more essential for assembly and anchoring of the two-fold wallaby genome. As a start to generating a physical map of gene locations on wallaby chromosomes, we focused on two chromosomes sharing homology with the human X, wallaby chromosomes X and 5. We devised an efficient strategy for mapping large conserved synteny blocks in non-model mammals, and applied this to generate dense maps of the X and 'neo-X' regions and to determine the arrangement of large conserved synteny blocks on chromosome 5. Comparisons between the wallaby and opossum chromosome maps revealed many rearrangements, highlighting the need for comparative gene mapping between South American and Australian marsupials. Frequent rearrangement of the X, along with the absence of a marsupial XIST gene, suggests that inactivation of the marsupial X chromosome does not depend on a whole-chromosome repression by a control locus.

To understand the efficacy of emamectin benzoate, avermectin, milbemectin, and thiacloprid on the reproduction and development of Bursaphelenchus xylophilus, seven parameters, namely population growth, fecundity, egg hatchability, larval lethality, percent larval development, body size, and sexual ratio, were investigated using sublethal (LC20) doses of these compounds in the laboratory. Emamectin benzoate treatment led to a significant suppression in population size, brood size, and percent larval development with 411, 3.50, and 49.63%, respectively, compared to 20850, 24.33, and 61.43% for the negative control. The embryonic and larval lethality increased obviously from 12.47% and 13.70% to 51.37% and 75.30%, respectively. In addition, the body length was also significantly reduced for both males and females in the emamectin benzoate treatment. Avermectin and milbemectin were also effective in suppressing population growth by increasing larval lethality and reducing larval development, although they did not affect either brood size or embryonic lethality. Body length for both male and female worms was increased by avermectin. Thiacloprid caused no adverse reproductive effects, although it suppressed larval development. Sexual ratio was not affected by any of these four nematicides. Our results indicate that emamectin benzoate, milbemectin, and avermectin are effective against the reproduction of B. xylophilus. We think these three nematicides can be useful for the control of pine wilt disease.