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Supersonic gas-solid injection technology finds extensive use in drug particle delivery systems. However, the combined impact of particle diameter and mass flow rate on the delivery efficiency remain insufficiently explored. Within the Euler-Lagrange framework, this study utilizes the discrete phase method (DPM) for the numerical simulation of supersonic gas-particle flow in a needle-free injector. After validating the model’s accuracy with experiment results, further investigations were conducted into the influences of particle size and mass flow rate on particle behavior and flow field properties. The results indicate that the impact of larger particles on the compressible structure is stronger, while higher mass flow rate absorbs greater energy from the gas phase, reducing the gas expansion capacity, which results in lower velocity, Mach number, and higher temperature. The jet core zone is approximately x/X  = 0.3 in length. Outside core zone, the gas velocity rapidly decays and temperature rises sharply. Within the jet core zone, drug particles are accelerated and cooled, while beyond core zone, they decelerate and heat up. The strongest inter-phase interactions occur primarily in the nozzle expansion area and the jet core zone. Smaller particles reach maximum velocity upstream. This implies that in designing needle-free injectors, the nozzle-to-skin distance must match the drug particle diameter to achieve maximum penetration effectiveness. Furthermore, the particle temperature decreases with smaller sizes. As the particle diameter rises from 10 μm to 100 μm, the minimum temperatures of the particles are 145 K and 264 K, respectively, indicating the need to match the particle diameter with the minimum temperature at which the drug particles remain effective. Additionally, higher mass flow rate doses reduce injection velocity and penetration ability, necessitating the rational control of the administered dose range. These results offer significant theoretical guidance for the design and improvement of needle-free injection.

Background Egg-laying mammals (monotremes) evolved multiple sex chromosomes independently of therian mammals and lack the sex-determining gene SRY . The Y-localized anti-Müllerian hormone gene ( AMHY ) is the candidate sex-determination gene in monotremes. Here, we describe the evolution of monotreme AMHX and AMHY gametologues and for the first time, investigate their expression during gonad sexual differentiation in a monotreme. Results Monotreme AMHX and AMHY have significant sequence divergence at the promoter, gene, and protein level, likely following an original allele inversion in the early stages of monotreme sex chromosome differentiation but retaining the conserved features of TGF-β molecules. We show that the expression of sexual differentiation genes in the echidna fetal gonad, including DMRT1 and SOX9 , is significantly different from that of therian mammals. Importantly, AMHY is expressed exclusively in the male gonad during sexual differentiation consistent with a role as the primary sex-determination gene whereas AMHX is expressed in both sexes. Experimental ectopic expression of platypus AMHX or AMHY in the chicken embryo did not masculinize the female urogenital system, as does chicken AMH, a possible result of mammalian-specific changes to AMH proteins preventing function in the chicken. Conclusions Our results provide insight into the early steps of monotreme sex chromosome evolution and sex determination with developmental expression data strongly supporting AMHY as the primary male sex-determination gene of platypus and echidna.

Bursaphelenchus xylophilus is a notorious invasive species, causing extensive losses to pine ecosystems globally. Previous studies had shown that the development of B. xylophilus was seriously suppressed by light. However, the mechanism involved in the inhibition is unknown. Here, it is the first report that Bxy-madd-4 is a light-regulated gene, plays a potential role in B. xylophilus in responding to the blue light. Transcriptome sequencing revealed that the expression level of Bxy-madd-4 declined by 86.39% under blue light. The reverse transcription quantitative real-time PCR results were in accord with the transcriptome sequencing, confirming the expression level of Bxy-madd-4 was suppressed by blue light. Bxy-madd-4 promoter::mCherry reporter constructed in Caenorhabditis elegans were utilized to mimic the spatiotemporal expression patterns of Bxy-madd-4 . Bxy-madd-4A promoter activity had a strong continuity throughout all development stages in C. elegans . Further RNA interference indicated that only 36.8% of the Bxy-madd-4 dsRNA treated embryos were hatched. Moreover, 71.6% of the hatched nematodes were abnormal, such as particles on the body surface and concave tissues. Our findings contribute towards a better understanding of the mechanism of light against the destructive invasive nematode, providing a promising hint for control of the destructive invasive nematode.

Janus kinase (JAK) inhibitors, such as tofacitinib (Xeljanz) and filgotinib (Jyseleca), have been approved for treatment of ulcerative colitis with several other JAK inhibitors in late-stage clinical trials for inflammatory bowel disease (IBD). Despite their impressive efficacy, the risk of adverse effects accompanying the use of JAK inhibitors has brought the entire class under scrutiny, leading to them receiving an FDA black box warning. In this study we investigated whether ileocolonic-targeted delivery of a pan-JAK inhibitor, tofacitinib, can lead to increased tissue exposure and reduced systemic exposure compared to untargeted formulations. The stability of tofacitinib in the presence of rat colonic microbiota was first confirmed. Next, in vivo computed tomography imaging was performed in rats to determine the transit time and disintegration site of ileocolonic-targeted capsules compared to gastric release capsules. Pharmacokinetic studies demonstrated that systemic drug exposure was significantly decreased, and colonic tissue exposure increased at 10 mg/kg tofacitinib dosed in ileocolonic-targeted capsules compared to gastric release capsules and an oral solution. Finally, in a rat model of LPS-induced colonic inflammation, targeted tofacitinib capsules significantly reduced concentrations of proinflammatory interleukin 6 in colonic tissue compared to a vehicle-treated control (p = 0.0408), unlike gastric release tofacitinib capsules and orally administered dexamethasone. Overall, these results support further development of ileocolonic-targeted tofacitinib, and potentially other specific JAK inhibitors in pre-clinical and clinical development, for the treatment of IBD.

Background The HOX gene clusters are thought to be highly conserved amongst mammals and other vertebrates, but the long non-coding RNAs have only been studied in detail in human and mouse. The sequencing of the kangaroo genome provides an opportunity to use comparative analyses to compare the HOX clusters of a mammal with a distinct body plan to those of other mammals. Results Here we report a comparative analysis of HOX gene clusters between an Australian marsupial of the kangaroo family and the eutherians. There was a strikingly high level of conservation of HOX gene sequence and structure and non-protein coding genes including the microRNAs miR-196a , miR-196b , miR-10a and miR-10b and the long non-coding RNAs HOTAIR , HOTAIRM1 and HOX A11AS that play critical roles in regulating gene expression and controlling development. By microRNA deep sequencing and comparative genomic analyses, two conserved microRNAs ( miR-10a and miR-10b ) were identified and one new candidate microRNA with typical hairpin precursor structure that is expressed in both fibroblasts and testes was found. The prediction of microRNA target analysis showed that several known microRNA targets, such as miR-10 , miR-414 and miR-464 , were found in the tammar HOX clusters. In addition, several novel and putative miRNAs were identified that originated from elsewhere in the tammar genome and that target the tammar HOXB and HOXD clusters. Conclusions This study confirms that the emergence of known long non-coding RNAs in the HOX clusters clearly predate the marsupial-eutherian divergence 160 Ma ago. It also identified a new potentially functional microRNA as well as conserved miRNAs. These non-coding RNAs may participate in the regulation of HOX genes to influence the body plan of this marsupial.

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.

Background Kangaroos and wallabies have specialised limbs that allow for their hopping mode of locomotion. The hindlimbs differentiate much later in development but become much larger than the forelimbs. The hindlimb autopod has only four digits, the fourth of which is greatly elongated, while digits two and three are syndactylous. We investigated the expression of two genes, HOXA13 and HOXD13 , that are crucial for digit patterning in mice during formation of the limbs of the tammar wallaby. Results We describe the development of the tammar limbs at key stages before birth. There was marked heterochrony and the hindlimb developed more slowly than the forelimb. Both tammar HOXA13 and HOXD13 have two exons as in humans, mice and chickens. HOXA13 had an early and distal mRNA distribution in the tammar limb bud as in the mouse, but forelimb expression preceded that in the hindlimb. HOXD13 mRNA was expressed earlier in the forelimb than the hindlimb and was predominantly detected in the interdigital tissues of the forelimb. In contrast, the hindlimb had a more restricted expression pattern that appeared to be expressed at discrete points at both posterior and anterior margins of the limb bud, and was unlike expression seen in the mouse and the chicken. Conclusions This is the first examination of HOXA and HOXD gene expression in a marsupial. The gene structure and predicted proteins were highly conserved with their eutherian orthologues. Interestingly, despite the morphological differences in hindlimb patterning, there were no modifications to the polyalanine tract of either HOXA13 or HOXD13 when compared to those of the mouse and bat but there was a marked difference between the tammar and the other mammals in the region of the first polyserine tract of HOXD13 . There were also altered expression domains for both genes in the developing tammar limbs compared to the chicken and mouse. Together these findings suggest that the timing of HOX gene expression may contribute to the heterochrony of the forelimb and hindlimb and that alteration to HOX domains may influence phenotypic differences that lead to the development of marsupial syndactylous digits.

Background WNT4 is a critical signalling molecule in embryogenesis and homeostasis, but the elements that control its transcriptional regulation are largely unknown. This study uses comparative cross species sequence and functional analyses between humans and a marsupial (the tammar wallaby, Macropus eugenii ) to refine the mammalian Wnt4 promoter. Results We have defined a highly conserved 89 bp minimal promoter region in human WNT4 by comparative analysis with the tammar wallaby. There are many conserved transcription factor binding sites in the proximal promoter region, including SP1, MyoD, NFκB and AP2, as well as highly conserved CpG islands within the human, mouse and marsupial promoters, suggesting that DNA methylation may play an important role in WNT4 transcriptional regulation. Conclusion Using a marsupial model, we have been able to provide new information on the transcriptional regulators in the promoter of this essential mammalian developmental gene, WNT4 . These transcription factor binding sites and CpG islands are highly conserved in two disparate mammals, and are likely key controlling elements in the regulation of this essential developmental gene.

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.

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 down-regulated 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 hormonesmakes these genes potential targets of EEDs in the etiology of abnormal phallus development including hypospadias. Summary Sentence SHH and WNT genes are tightly regulated by hormones during phallus differentiation and are critical for its development, making these genes potential targets of endocrine disruption, leading to malformations of the external genitalia.