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Ethosomes are widely applied as the carriers for the transdermal delivery of hydrophobic and hydrophilic drugs. Herein, curcumin-loaded ethosomes (CE) with different phospholipid composition were formulated and thoroughly compared. A significant interaction between the unsaturated phosphatidylcholine (PC) and saturated hydrogenated phosphatidylcholine (HPC) was found by molecular simulation and differential scanning calorimetry (DSC), which led to the reduction of PC peroxidation with the presence of HPC. Subsequently, the composite phospholipid ethosomes containing curcumin were prepared for the first time to evaluate their properties in comparison with the conventional ethosomes composed of PC (CE-P) or HPC (CE-H). CE with PC/HPC ratio of 1:1 (CE-P1H1) with the best vesicle stability and flexibility significantly decreased the uptake by HaCaT cells compared to CE-H and free curcumin, indicating reduced skin cell toxicity. Compared with free curcumin, CE-P1H1 had the highest transdermal efficiency (p 0.001), followed by CE-P (p 0.05), partly due to the fact that CE-P1H1 could disturb lipid domain of stratum corneum (SC). Moreover, CE-P1H1 was found to promote curcumin for deep penetration of the skin via the hair follicles route. Our study has shown that using composite phospholipid ethosomes as lipid vesicular carriers could enhance transdermal penetration of drugs and increase in the vesicle stability.

Polyploidy is a prominent route to speciation in plants; however, this entails resolving the challenges of meiotic instability facing abrupt doubling of chromosome complement. This issue remains poorly understood. We subjected progenies of a synthetic hexaploid wheat, analogous to natural common wheat, but exhibiting extensive meiotic chromosome instability, to heat or salt stress. We selected stress-tolerant cohorts and generated their progenies under normal condition. We conducted fluorescent in situ hybridization/genomic in situ hybridization-based meiotic/mitotic analysis, RNA-Seq and virus-induced gene silencing (VIGS)-mediated assay of meiosis candidate genes. We show that heritability of stress tolerance concurred with increased euploidy frequency due to enhanced meiosis stability. We identified a set of candidate meiosis genes with altered expression in the stress-tolerant plants vs control, but the expression was similar to that of common wheat (cv Chinese Spring, CS). We demonstrate VIGS-mediated downregulation of individual candidate meiosis genes in CS is sufficient to confer an unstable meiosis phenotype mimicking the synthetic wheat. Our results suggest that heritable regulatory changes of preexisting meiosis genes may be hitchhiked as a spandrel of stress tolerance, which significantly improves meiosis stability in the synthetic wheat. Our findings implicate a plausible scenario that the meiosis machinery in hexaploid wheat may have already started to evolve at its onset stage.

The non-random spatial packing of chromosomes in the nucleus plays a critical role in orchestrating gene expression and genome function. Here, we present a Hi-C analysis of the chromatin interaction patterns in rice (Oryza sativa L.) at hierarchical architectural levels. We confirm that rice chromosomes occupy their own territories with certain preferential inter-chromosomal associations. Moderate compartment delimitation and extensive TADs (Topologically Associated Domains) were determined to be associated with heterogeneous genomic compositions and epigenetic marks in the rice genome. We found subtle features including chromatin loops, gene loops, and off-/near-diagonal intensive interaction regions. Gene chromatin loops associated with H3K27me3 could be positively involved in gene expression. In addition to insulated enhancing effects for neighbor gene expression, the identified rice gene loops could bi-directionally (+/-) affect the expression of looped genes themselves. Finally, web-interleaved off-diagonal IHIs/KEEs (Interactive Heterochromatic Islands or KNOT ENGAGED ELEMENTs) could trap transposable elements (TEs) via the enrichment of silencing epigenetic marks. In parallel, the near-diagonal FIREs (Frequently Interacting Regions) could positively affect the expression of involved genes. Our results suggest that the chromatin packing pattern in rice is generally similar to that in Arabidopsis thaliana but with clear differences at specific structural levels. We conclude that genomic composition, epigenetic modification, and transcriptional activity could act in combination to shape global and local chromatin packing in rice. Our results confirm recent observations in rice and A. thaliana but also provide additional insights into the patterns and features of chromatin organization in higher plants.

Although a distinct karyotype with defined chromosome number and structure characterizes each biological species, it is intrinsically labile. Polyploidy or whole-genome duplication has played a pervasive and ongoing role in the evolution of all eukaryotes, and is the most dramatic force known to cause rapid karyotypic reconfiguration, especially at the initial stage. However, issues concerning transgenerational propagation of karyotypic heterogeneity and its translation to phenotypic diversity in nascent allopolyploidy, at the population level, have yet to be studied in detail. Here, we report a large-scale examination of transgenerationally propagated karyotypic heterogeneity and its phenotypic manifestation in an artificially constructed allotetraploid with a genome composition of AADD, that is, involving two of the three progenitor genomes of polyploid wheat. Specifically, we show that 1) massive organismal karyotypic heterogeneity is precipitated after 12 consecutive generations of selfing from a single euploid founder individual, 2) there exist dramatic differences in aptitudes between subgenomes and among chromosomes for whole-chromosome gain and/or loss and structural variations, 3) majority of the numerical and structural chromosomal variations are concurrent due to mutual contingency and possible functional constraint, 4) purposed and continuous selection and propagation for euploidy over generations did not result in enhanced karyotype stabilization, and 5) extent of karyotypic variation correlates with variability of phenotypic manifestation. Together, our results document that allopolyploidization catalyzes rampant and transgenerationally heritable organismal karyotypic heterogeneity that drives population-level phenotypic diversification, which lends fresh empirical support to the still contentious notion that whole-genome duplication enhances organismal evolvability.

Summary Many ectotherms hibernate in face of the harsh winter conditions to improve their survival rate. However, the molecular mechanism underlying this process remains unclear. Here, we explored the hibernation mechanism of Chinese alligator using integrative multi-omics analysis. We revealed that (1) the thyroid hormone biosynthesis, nutrition absorption and metabolism, muscle contraction, urinary excretion and immunity function pathways are overall downregulated during hibernation; (2) the fat catabolism is completely suppressed, contrasting with the upregulation of hepatic fatty-acid-transporter CPT1A, suggesting a unique energy-saving strategy that differs from that in hibernating mammals; (3) the hibernation-related genes are not only directly regulated by DNA methylation but also controlled by methylation-dependent transcription networks. In addition, we identified and compared tissue-specific, species-specific, and conserved season-biased miRNAs, demonstrating complex post-transcriptional regulation during hibernation. Our study revealed the genetic and epigenetic mechanisms underlying hibernation in the Chinese alligator and provided molecular insights into the evolution of hibernation regulation., Graphical Abstract, Highlights • Metabolic and physiological pathways are overall suppressed during hibernation • Suppressed fat catabolism with active CPT1A suggests a unique energy-saving strategy • Hibernation-related genes are controlled by methylation-dependent transcription network • miRNAs play complex post-transcriptional regulation roles during hibernation, Biological Sciences; Evolutionary Ecology; Transcriptomics

A number of nonclassical MHC Ib molecules recognizing distinct microbial antigens have been implicated in the immune response to Mycobacterium tuberculosis (Mtb). HLA-E has been identified to present numerous Mtb peptides to CD8+ T cells, with multiple HLA-E-restricted cytotoxic T lymphocyte (CTL) and regulatory T cell lines isolated from patients with active and latent tuberculosis (TB). In other disease models, HLA-E and its mouse homolog Qa-1 can act as antigen presenting molecules as well as regulators of the immune response. However, it is unclear what precise role(s) HLA-E/Qa-1 play in the immune response to Mtb. In this study, we found that murine Qa-1 can bind and present Mtb peptide antigens to CD8+ T effector cells during aerosol Mtb infection. Further, mice lacking Qa-1 (Qa-1-/-) were more susceptible to high-dose Mtb infection compared to wild-type controls, with higher bacterial burdens and increased mortality. The increased susceptibility of Qa-1-/- mice was associated with dysregulated T cells that were more activated and produced higher levels of pro-inflammatory cytokines. T cells from Qa-1-/- mice also had increased expression of inhibitory and apoptosis-associated cell surface markers such as CD94/NKG2A, KLRG1, PD-1, Fas-L, and CTLA-4. As such, they were more prone to cell death and had decreased capacity in promoting the killing of Mtb in infected macrophages. Lastly, comparing the immune responses of Qa-1 mutant knock-in mice deficient in either Qa-1-restricted CD8+ Tregs (Qa-1 D227K) or the inhibitory Qa-1-CD94/NKG2A interaction (Qa-1 R72A) with Qa-1-/- and wild-type controls indicated that both of these Qa-1-mediated mechanisms were involved in suppression of the immune response in Mtb infection. Our findings reveal that Qa-1 participates in the immune response to Mtb infection by presenting peptide antigens as well as regulating immune responses, resulting in more effective anti-Mtb immunity., Author summary The disease tuberculosis (TB) is caused by the microbe Mycobacterium tuberculosis (Mtb), and remains a major public health concern. More research is needed to understand the diverse immune responses against Mtb to develop better vaccines. Mouse Qa-1 and its human counterpart HLA-E are nonclassical MHC I molecules that can activate or inhibit immune responses in a variety of diseases. However, their role during the immune response to Mtb remains unknown. We found that Qa-1 can present Mtb peptides to activate CD8+ T effector cells during aerosol Mtb infection. Further, Mtb-infected mice that lacked Qa-1 (Qa-1-/-) had higher numbers of bacteria and died more often than infected mice that expressed Qa-1 (Qa-1+/+). The lack of Qa-1 results in over-activation of the immune response upon infection, which is less efficient in controlling Mtb. Using mice expressing different mutant forms of Qa-1, we showed that Qa-1 can regulate immune responses against Mtb through the interaction with inhibitory CD94/NKG2A receptors as well as the activation of regulatory CD8+ T cells. We believe our study sheds light on the diverse mechanisms at play in generating protective immune responses against Mtb and will inform future mouse and human studies.

MHC Ib-restricted CD8+ T cells have been implicated in host defense against Mycobacterium tuberculosis (Mtb) infection. However, the relative contribution of various MHC Ib-restricted T cell populations to anti-mycobacterial immunity remains elusive. In this study, we used mice that lack MHC Ia (Kb-/-Db-/-), MHC Ia/H2-M3 (Kb-/-Db-/-M3-/-), or β2m (β2m-/-) to study the role of M3-restricted and other MHC Ib-restricted T cells in immunity against Mtb. Unlike their dominant role in Listeria infection, we found that M3-restricted CD8+ T cells only represented a small proportion of the CD8+ T cells responding to Mtb infection. Non-M3, MHC Ib-restricted CD8+ T cells expanded preferentially in the lungs of Mtb-infected Kb-/-Db-/-M3-/- mice, exhibited polyfunctional capacities and conferred protection against Mtb. These MHC Ib-restricted CD8+ T cells recognized several Mtb-derived protein antigens at a higher frequency than MHC Ia-restricted CD8+ T cells. The presentation of Mtb antigens to MHC Ib-restricted CD8+ T cells was mostly β2m-dependent but TAP-independent. Interestingly, a large proportion of Mtb-specific MHC Ib-restricted CD8+ T cells in Kb-/-Db-/-M3-/- mice were Qa-2-restricted while no considerable numbers of MR1 or CD1-restricted Mtb-specific CD8+ T cells were detected. Our findings indicate that nonclassical CD8+ T cells other than the known M3, CD1, and MR1-restricted CD8+ T cells contribute to host immune responses against Mtb infection. Targeting these MHC Ib-restricted CD8+ T cells would facilitate the design of better Mtb vaccines with broader coverage across MHC haplotypes due to the limited polymorphism of MHC class Ib molecules., Author Summary Tuberculosis, the disease caused by Mycobacterium tuberculosis (Mtb), remains a major global health burden. As T cells are crucial to the control of Mtb infection, it is imperative to decipher the role of different T cell subsets in anti-Mtb immunity for the development of more effective vaccines. While the contribution of conventional T cells to protective immunity against Mtb is well established, the involvement of unconventional T cells is less clear. In this study, we used mutant mice that lack distinct MHC I molecules to characterize immune responses mediated by unconventional T cells during Mtb infection. We found that unconventional CD8+ T cells preferentially expanded in the lung after Mtb infection. These CD8+ T cells responded to numerous mycobacterial antigens, produced multiple cytokines, and contributed to protection against Mtb. A large proportion of unconventional T cells induced by Mtb infection are Qa-2 restricted CD8+ T cells, suggesting this group of T cells may play a greater role in anti-mycobacterial immunity than other unconventional T cell populations that have been characterized previously. Targeting these unconventional T cells may facilitate the design of TB vaccines that are universally effective in ethnically diverse populations due to the non-polymorphic nature of MHC Ib molecules.