The evolution and diversification of oakleaf butterflies.

Oakleaf butterflies in the genus Kallima have a polymorphic wing phenotype, enabling these insects to masquerade as dead leaves. This iconic example of protective resemblance provides an interesting evolutionary paradigm that can be employed to study biodiversity. We integrated multi-omic data analyses and functional validation to infer the evolutionary history of Kallima species and investigate the genetic basis of their variable leaf wing patterns. We find that Kallima butterflies diversified in the eastern Himalayas and dispersed to East and Southeast Asia. Moreover, we find that leaf wing polymorphism is controlled by the wing patterning gene cortex, which has been maintained in Kallima by long-term balancing selection. Our results provide macroevolutionary and microevolutionary insights into a model species originating from a mountain ecosystem.

Directed evolution of adeno-associated virus for efficient gene delivery to microglia.

As the resident immune cells in the central nervous system (CNS), microglia orchestrate immune responses and dynamically sculpt neural circuits in the CNS. Microglial dysfunction and mutations of microglia-specific genes have been implicated in many diseases of the CNS. Developing effective and safe vehicles for transgene delivery into microglia will facilitate the studies of microglia biology and microglia-associated disease mechanisms. Here, we report the discovery of adeno-associated virus (AAV) variants that mediate efficient in vitro and in vivo microglial transduction via directed evolution of the AAV capsid protein. These AAV-cMG and AAV-MG variants are capable of delivering various genetic payloads into microglia with high efficiency, and enable sufficient transgene expression to support fluorescent labeling, Ca2+ and neurotransmitter imaging and genome editing in microglia in vivo. Furthermore, single-cell RNA sequencing shows that the AAV-MG variants mediate in vivo transgene delivery without inducing microglia immune activation. These AAV variants should facilitate the use of various genetically encoded sensors and effectors in the study of microglia-related biology.

The effects of methylphenidate and atomoxetine on Drosophila brain at single-cell resolution and potential drug repurposing for ADHD treatment.

The stimulant methylphenidate (MPH) and the non-stimulant atomoxetine (ATX) are frequently used for the treatment of attention-deficit/hyperactivity disorder (ADHD); however, the function of these drugs in different types of brain cells and their effects on related genes remain largely unknown. To address these questions, we built a pipeline for the simultaneous examination of the activity behavior and transcriptional responses of Drosophila melanogaster at single-cell resolution following drug treatment. We selected the Drosophila with significantly increased locomotor activities (hyperactivity-like behavior) following the administration of each drug in comparison with the control (same food as the drug-treated groups with 5% sucrose, yeast, and blue food dye solution) using EasyFlyTracker. Subsequently, single cell RNA sequencing (scRNASEQ) was used to capture the transcriptome of 82,917 cells, unsupervised clustering analysis of which yielded 28 primary cell clusters representing the major cell types in adult Drosophila brain. Indeed, both neuronal and glial cells responded to MPH and ATX. Further analysis of differentially expressed genes (DEGs) revealed distinct transcriptional changes associated with these two drugs, such as two well-studied dopamine receptor genes (Dop2R and DopEcR) were responsive to MPH but not to ATX at their optimal doses, in addition to genes involved in dopamine metabolism pathways such as Syt1, Sytalpha, Syt7, and Ih in different cell types. More importantly, MPH also suppressed the expression of genes encoding other neurotransmitter receptors and synaptic signaling molecules in many cell types, especially those for Glu and GABA, while the responsive effects of ATX were much weaker. In addition to monoaminergic neuronal transmitters, other neurotransmitters have also shown a similar pattern with respect to a stronger effect associated with MPH than with ATX. Moreover, we identified four distinct glial cell subtypes responsive to the two drugs and detected a greater number of differentially expressed genes associated with ensheathing and astrocyte-like glia. Furthermore, our study provides a rich resource of candidate target genes, supported by drug set enrichment analysis (P = 2.10E-4; hypergeometric test), for the further exploration of drug repurposing. The whole list of candidates can be found at ADHDrug ( http://adhdrug.cibr.ac.cn/ ). In conclusion, we propose a fast and cost-efficient pipeline to explore the underlying molecular mechanisms of ADHD drug treatment in Drosophila brain at single-cell resolution, which may further facilitate drug repurposing applications.

Purification and characterization of recombinant human superoxide dismutase integrated with resilin-like polypeptide.

Human superoxide dismutase (hSOD1) plays an important role in the aerobic metabolism and free radical eliminating process in the body. However, the production of existing SOD faces problems such as complex purification methods, high costs, and poor product stability. This experiment achieved low-cost, rapid, and simple purification of hSOD1 through ammonium sulfate precipitation method and heat resistance of recombinant protein. We constructed a recombinant protein hSOD1-LR containing a resilin-like polypeptide tag and expressed it. The interest protein was purified by ammonium sulfate precipitation method, and the results showed that the purification effect of 1.5 M (NH4)2SO4 was the best, with an enzyme activity recovery rate of 80 % after purification. Then, based on its thermal stability, further purification of the interest protein at 60 °C revealed a purification fold of up to 24 folds, and the purification effect was similar to that of hSOD1-6xHis purified by nickel column affinity chromatography. The stability of hSOD1-LR showed that the recombinant protein hSOD1-LR has better stability than hSOD-6xHis. hSOD1-LR can maintain 76.57 % activity even after 150 min of reaction at 70 °C. At same time, hSOD1-LR had activity close to 80 % at pH < 5, indicating good acid resistance. In addition, after 28 days of storage at 4 °C and 40 °C, hSOD1-LR retained 92 % and 87 % activity, respectively. Therefore, the method of purifying hSOD1-LR through salt precipitation may have positive implications for the study of SOD purification.

Tobacco Transcription Factor NtWRKY70b Facilitates Leaf Senescence via Inducing ROS Accumulation and Impairing Hydrogen Sulfide Biosynthesis.

Leaf senescence is the terminal stage of leaf development, and its initiation and progression are closely controlled by the integration of a myriad of endogenous signals and environmental stimuli. It has been documented that WRKY transcription factors (TFs) play essential roles in regulating leaf senescence, yet the molecular mechanism of WRKY-mediated leaf senescence still lacks detailed elucidation in crop plants. In this study, we cloned and identified a tobacco WRKY TF gene, designated NtWRKY70b, acting as a positive regulator of natural leaf senescence. The expression profile analysis showed that NtWRKY70b transcript levels were induced by aging and hydrogen peroxide (H2O2) and downregulated upon hydrogen sulfide (H2S) treatment. The physiological and biochemical assays revealed that overexpression of NtWRKY70b (OE) clearly promoted leaf senescence, triggering increased levels of reactive oxygen species (ROS) and decreased H2S content, while disruption of NtWRKY70b by chimeric repressor silencing technology (SRDX) significantly delayed the onset of leaf senescence, leading to a decreased accumulation of ROS and elevated concentration of H2S. The quantitative real-time PCR analysis showed that the expression levels of various senescence-associated genes and ROS biosynthesis-related genes (NtRbohD and NtRbohE) were upregulated in OE lines, while the expression of H2S biosynthesis-related genes (NtDCD and NtCYSC1) were inhibited in OE lines. Furthermore, the Yeast one-hybrid analysis (Y1H) and dual luciferase assays showed that NtWRKY70b could directly upregulate the expression of an ROS biosynthesis-related gene (NtRbohD) and a chlorophyll degradation-related gene (NtPPH) by binding to their promoter sequences. Accordingly, these results indicated that NtWYKY70b directly activated the transcript levels of NtRbohD and NtPPH and repressed the expression of NtDCD and NtCYCS1, thereby promoting ROS accumulation and impairing the endogenous H2S production, and subsequently accelerating leaf aging. These observations improve our knowledge of the regulatory mechanisms of WRKY TFs controlling leaf senescence and provide a novel method for ensuring high agricultural crop productivity via genetic manipulation of leaf senescence in crops.

The subcommissural organ regulates brain development via secreted peptides.

The subcommissural organ (SCO) is a gland located at the entrance of the aqueduct of Sylvius in the brain. It exists in species as distantly related as amphioxus and humans, but its function is largely unknown. To explore its function, we compared transcriptomes of SCO and non-SCO brain regions and found three genes, Sspo, Car3, and Spdef, that are highly expressed in the SCO. Mouse strains expressing Cre recombinase from endogenous promoter/enhancer elements of these genes were used to genetically ablate SCO cells during embryonic development, resulting in severe hydrocephalus and defects in neuronal migration and development of neuronal axons and dendrites. Unbiased peptidomic analysis revealed enrichment of three SCO-derived peptides, namely thymosin beta 4, thymosin beta 10, and NP24, and their reintroduction into SCO-ablated brain ventricles substantially rescued developmental defects. Together, these data identify a critical role for the SCO in brain development.

The subcommissural organ regulates brain development via secreted peptides.

The subcommissural organ (SCO) is a gland located at the entrance of the aqueduct of Sylvius in the brain. It exists in species as distantly related as amphioxus and humans, but its function is largely unknown. Here, to explore its function, we compared transcriptomes of SCO and non-SCO brain regions and found three genes, Sspo, Car3 and Spdef, that are highly expressed in the SCO. Mouse strains expressing Cre recombinase from endogenous promoter/enhancer elements of these genes were used to genetically ablate SCO cells during embryonic development, resulting in severe hydrocephalus and defects in neuronal migration and development of neuronal axons and dendrites. Unbiased peptidomic analysis revealed enrichment of three SCO-derived peptides, namely, thymosin beta 4, thymosin beta 10 and NP24, and their reintroduction into SCO-ablated brain ventricles substantially rescued developmental defects. Together, these data identify a critical role for the SCO in brain development.

Long-read genome assemblies reveal a cis-regulatory landscape associated with phenotypic divergence in two sister Siniperca fish species.

Due to the difficulty in accurately identifying structural variants (SVs) across genomes, their impact on cis-regulatory divergence of closely related species, especially fish, remains to be explored. Recently identified broad H3K4me3 domains are essential for the regulation of genes involved in several biological processes. However, the role of broad H3K4me3 domains in phenotypic divergence remains poorly understood. Siniperca chuatsi and S. scherzeri are closely related but divergent in several phenotypic traits, making them an ideal model to study cis-regulatory evolution in sister species. Here, we generated chromosome-level genomes of S. chuatsi and S. scherzeri, with assembled genome sizes of 716.35 and 740.54 Mb, respectively. The evolutionary histories of S. chuatsi and S. scherzeri were studied by inferring dynamic changes in ancestral population sizes. To explore the genetic basis of adaptation in S. chuatsi and S. scherzeri, we performed gene family expansion and contraction analysis and identified positively selected genes (PSGs). To investigate the role of SVs in cis-regulatory divergence of closely related fish species, we identified high-quality SVs as well as divergent H3K27ac and H3K4me3 domains in the genomes of S. chuatsi and S. scherzeri. Integrated analysis revealed that cis-regulatory divergence caused by SVs played an essential role in phenotypic divergence between S. chuatsi and S. scherzeri. Additionally, divergent broad H3K4me3 domains were mostly associated with cancer-related genes in S. chuatsi and S. scherzeri and contributed to their phenotypic divergence.

Role and modulation of the secondary structure of antimicrobial peptides to improve selectivity.

Since the development of bacterial resistance, the decreasing effectiveness of antibiotics is becoming one of the most critical problems worldwide. Novel antibacterial agents are urgently needed to prevent humanity from falling back into the "post-antibiotic era". As an important part of the innate immune system, antimicrobial peptides (AMPs) are one of the most promising antibacterial agents showing broad-spectrum activity against bacteria and low propensity for drug resistance. However, the shortcomings of AMPs, such as high toxicity and easy digestion by proteases, limit their clinical application. This review mainly focuses on the effect of the secondary structure on the antimicrobial activity and cytotoxicity of AMPs and the strategies of designing conformationally transitionable AMPs with improved selectivity towards bacteria.