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The rapidly expanding catalog of sequenced genomes has revolutionized the pace and scale of microalgal cellular metabolism delineation. However, knowledge of the gene regulation in these genomes is lacking. This is true even for Chlamydomonas reinhardtii , the laboratory model species where transcriptional regulation is best understood, although systematic knowledge of regulatory elements (e.g., promoters) remains elusive. This leads to limitations in microalgae for engineering system designs, which currently rely mainly on characterizing the molecular mechanisms of individual regulatory sequences via low-throughput methods. Here, we take a first step toward multi-promoter dissection and demonstrate systematic dissection of multiple microalgal promoters through quantitative genome-wide comparisons and sequence-based structural annotations. We demonstrate this approach on both well-studied and previously uncharacterized promoters in the oleaginous microalga Nannochloropsis oceanica IMET1. Using in silico design, representative regulatory elements were synthesized and assembled as building blocks for transgene circuits. Assessment of the in vivo activity revealed the maximum transformation efficiency (414 ± 102 transformants/µg DNA) in the plasmid pHN5-2 containing lhp promoter and α-tub terminator. Therefore, the present synthetic approach for establishing engineering systems is superior to the conventional empirical methods. Applying these transforming circuits to the creation and characterization of a gene-indexed loss-of-function mutagenesis library verifies the applicability of this strategy to the discovery and standardization of regulatory parts for microalgae. Moreover, the generality of the approach presented here provides the possibility of quantitative promoter dissection and rational transform system design in N. oceanica and a wide range of other microalgae.

Two-cell-like (2C-like) embryonic stem cells (ESCs) are a small group of ESCs that spontaneously express zygotic genome activation (ZGA) genes and repeats, such as Zscan4 and murine endogenous retrovirus with leucine (MERVL), and are specifically expressed in 2-cell-stage mouse embryos. Although numerous types of treatment and agents elevate the transition of ESCs to 2C-like ESCs, Dux serves as a critical factor in this transition by increasing the expression of Zscan4 and MERVL directly. However, the loss of Dux did not impair the birth of mice, suggesting that Dux may not be the primary transitioning factor in fertilized embryos. It has been reported that for 2-cell embryos derived from somatic cell nuclear transfer (SCNT) and whose expression of ZGA genes and repeats was aberrant, Dux improved the reprogramming efficiency by correcting aberrant H3K9ac modification via its C-terminal domain. We confirmed that the overexpression of full-length Dux mRNA in SCNT embryos improved the efficiency of preimplantation development (62.16% vs. 41.26% with respect to controls) and also increased the expression of Zscan4 and MERVL. Furthermore, we found that the N-terminal double homeodomains of Dux were indispensable for Dux localization and function. The intermediate region was essential for MERVL and Zscan4 activation, and the C-terminal domain was important for elevating level of H3K27ac. Mutant Dux mRNA containing N-terminal double homeodomains with the intermediate region or the C-terminal domain also improved the preimplantation development of SCNT embryos. This is the first report focusing on distinguishing functional domains of Dux in embryos derived from SCNT. Graphical Abstract

The rational dietary ratio of docosahexaenoic acid (DHA) to eicosapentaenoic acid (EPA) can exert neurotrophic and cardiotrophic effects on the human body. The marine microalga Nannochloropsis oceanica produces EPA yet no DHA, and thus, it is considered an ideal EPA-only model to pursue a rational DHA/EPA ratio. In this study, synthetic biological strategy was applied to improve EPA production in N. oceanica. Firstly, to identify promoters and terminators, fifteen genes from N. oceanica were isolated using a transcriptomic approach. Compared to α-tubulin, NO08G03500, NO03G03480 and NO22G01450 exhibited 1.2~1.3-fold increases in transcription levels. Secondly, to identify EPA-synthesizing modules, putative desaturases (NoFADs) and elongases (NoFAEs) were overexpressed by the NO08G03500 and NO03G03480 promoters/terminators in N. oceanica. Compared to the wild type (WT), NoFAD1770 and NoFAE0510 overexpression resulted in 47.7% and 40.6% increases in EPA yields, respectively. Thirdly, to store EPA in triacylglycerol (TAG), NoDGAT2K was overexpressed using the NO22G01450 promoter/terminator, along with NoFAD1770–NoFAE0510 stacking, forming transgenic line XS521. Compared to WT, TAG-EPA content increased by 154.8% in XS521. Finally, to inhibit TAG-EPA degradation, a TAG lipase-encoding gene NoTGL1990 was knocked out in XS521, leading to a 49.2–65.3% increase in TAG-EPA content. Our work expands upon EPA-enhancing approaches through synthetic biology in microalgae and potentially crops.

Cell membrane-derived nanovesicles, particularly those originating from red blood cells (RNVs), have garnered considerable attention as innovative drug delivery vehicles in oncology, owing to their exceptional biocompatibility, immune evasion, and prolonged systemic circulation. Nevertheless, their inherently poor tumor-targeting efficiency and nonspecific biodistribution present major obstacles to their therapeutic translation. This study sought to functionalize RNVs with a diverse array of tumor-targeting ligands - cRGD, transferrin (TRF), folic acid (FA), GE11, and RVG29 - and to systematically compare their tumor-homing efficiency, biodistribution, and biosafety in a breast cancer model. Functionalized RNVs exhibited markedly enhanced tumor affinity relative to unmodified vesicles in both and settings. Among the engineered formulations, RNV@cRGD achieved the most pronounced intratumoral accumulation and cellular uptake, followed sequentially by RNV@GE11, RNV@TRF, RNV@FA, and RNV@RVG29. Fluorescence imaging corroborated the superior tumor selectivity of engineered constructs, all of which also demonstrated robust stability and negligible off-target toxicity in murine models. This work presents systematic comparative evaluation of ligand-engineered RNVs, underscoring cRGD as the most potent targeting moiety for breast cancer. These findings illuminate critical design principles for the rational development of tumor-directed RNV-based drug delivery systems and strengthen the translational promise of biomimetic nanocarriers for clinical oncology.

Hypomecis punctinalis Scopoli, 1763 belongs to the Lepidopteran family Geometridae. We sequenced the complete mitochondrial genome (mitogenome) of H. punctinalis. The mitogenome is 15,648 bp long and contains a typical set of genes (13 protein-coding genes (PCGs), 22 tRNA genes, and two rRNA genes) and a 484 bp AT-rich region. All PCGs start with ATN codons and stop at TAA codon except for cox1 using CGA as initiation codon and nad4 and nad5 using incomplete termination codon T. Within the mitogenome, 17 intergenic spacers and seven overlaps are founded. The intergenic nucleotides are 294 bp in total and two longest intervals locate between trn Gln and nad2 as well as trn Cys and trn Tyr . The overlap nucleotides are 47 bp in total and the maximum overlap lies between cox2 and trn Lys . The AT-rich region of the mitogenome contains an 'ATAGA + polyT' motif, three copies of 30-bp-repeat and a short polyA tail. The phylogenetic tree shows the relationships of four subfamilies of Geometridae are (((Ennominae + Geometrinae)+Larentiinae)+Sterrhinae)) and the relationships within subfamily Ennominae are (((Erannis+Biston)+(Jankowskia+(Hypomecis+(Apocheima+Milionia)))+Ectropis)+Abraxas)+Phthonandria)+Celenna).

In spite of its essential role in culture media, the precise influence of lactate on early mouse embryonic development remains elusive. Previous studies have implicated lactate accumulation in medium affecting histone acetylation. Recent research has underscored lactate-derived histone lactylation as a novel epigenetic modification in diverse cellular processes and diseases. Our investigation demonstrated that the absence of sodium lactate in the medium resulted in a pronounced 2-cell arrest at the late G2 phase in embryos. RNA-seq analysis revealed that the absence of sodium lactate significantly impaired the maternal-to-zygotic transition (MZT), particularly in zygotic gene activation (ZGA). Investigations were conducted employing Cut&Tag assays targeting the well-studied histone acetylation and lactylation sites, H3K18la and H3K27ac, respectively. The findings revealed a noticeable reduction in H3K18la modification under lactate deficiency, and this alteration showed a significant correlation with changes in gene expression. In contrast, H3K27ac exhibited minimal correlation. These results suggest that lactate may preferentially influence early embryonic development through H3K18la rather than H3K27ac modifications.

Image super-resolution technique can improve image quality by increasing image clarity, bringing a better user experience in real production scenarios. However, existing convolutional neural network methods usually have very deep network layers and a large number of parameters, which causes feature information to be lost as the network deepens, and models with a large numbers of parameters are not suitable for deploying on resource-constrained mobile devices. To address the above problems, we propose a novel lightweight image super-resolution network (RepSCN) based on re-parameterization and self-calibration convolution. Specifically, to reduce the computational cost while capturing more high-frequency details, we designed a re-parameterization distillation block (RepDB) and a self-calibrated distillation block (SCDB). They can improve the reconstruction results by aggregating the local distilled feature information under different receptive fields without introducing extra parameters. On the other hand, the positional information of the image is also crucial for super-resolution reconstruction. Nevertheless, existing lightweight SR methods mainly adopt the channel attention mechanism, which ignores the importance of positional information. Therefore, we introduce a lightweight coordinate attention mechanism (CAM) at the end of RepDB and SCDB to enhance the feature representation at both spatial and channel levels. Numerous experiments have shown that our network has better reconstruction performance with reduced parameters than other classical lightweight super-resolution models.

Ribosome biogenesis occurs within the nucleolus, with the initial step being the transcription of ribosomal DNA (rDNA). Although rDNA transcription is limited in somatic cells, it is more active in stem cells. Nevertheless, the mechanisms involved in somatic cell reprogramming remain elusive. Both somatic and stem cell nucleoli exhibit a reticular structure. However, under the electron microscope, we identified an intermediate nucleolar state during reprogramming. This state underwent changes characterised by rDNA hypertranscription, resulting in an enlarged nucleolus, enhanced activity of nucleolus organiser regions (NORs), and a transition from the reticular nucleolar type to an intermediate state of reprogramming, whose three liquid phase boundaries are blurred. Our research revealed that Oct4 was directly targeted to the rDNA enhancer region, promoting its hypertranscription and nucleolar enlargement during reprogramming. Using rDNA transcriptional inhibitors, we proved that nucleolar remodelling and subsequent reprogramming are halted by inhibiting rDNA transcription. But why could rDNA transcriptional activity influence reprogramming? Our findings elucidate that the active nucleoli have the capability to release perinucleolar heterochromatin. By joint analysis of Assay for Transposase‐Accessible Chromatin with high throughput sequencing (ATAC‐seq) and RNA‐seq, we have characterised the perinucleolar chromatin released by the nucleolus in a reprogramming intermediate state. The released chromatin mainly impacted mesenchymal‐to‐epithelial transition (MET)‐related genes. MET is a stage of silencing of mesenchymal genes, accompanied by the activation of epithelial genes. Concurrently, the morphology of mouse embryonic fibroblast cells (MEFs) transitions from elongated spindle‐shaped cells to short roundish forms, exhibiting a propensity to cluster together. MET was considered an early event in reprogramming; our findings suggested that nucleolar remodelling occurred before MET. Active nucleoli have the capability to release perinuclear heterochromatin, which is a novel event of iPSC reprogramming.

Background: Ribosomal DNA (rDNA) transcription by the RNA polymerase I (Pol I) is a rate-limited step for ribosome synthesis, which is critical for cell growth, cell differentiation, and tumorigenesis. Meanwhile rDNA transcription is modulated by DNA methylation and histone epigenetic modification. Though with great progress in epigenetic research recently, it still remains much uncertain about the relationship of histone variant epigenetic modification and rDNA transcription. Results: In this study, epigenetic profiles of silent rDNA in next-generation sequencing datasets were examined. We found that the chaperone of histone variant H3.3, the alpha-thalassemia/mental retardation X-linked syndrome protein (ATRX)/death domain-associated protein (DAXX) complex, and methyltransferase SET domain bifurcated 1 (Setdb1, also known as ESET) help maintain H3.3K9me3 modifications among the promoter and coding regions of silent rDNA. Our experiments further confirmed that DAXX depletion leads to the conversion of silent rDNA into upstream binding factor-bound active rDNA and the release of rDNA transcriptional potency. Support for this model is provided by data from a low-grade glioma in which ATRX is lost and a higher level of ribosomal biosynthesis, nucleolus activity, and proliferation are observed. Conclusions: We demonstrate a model of epigenetic regulation for rDNA with roles for the ATRX/DAXX complex and H3.3/H3K9me3 modifications identified. Thus, loss of ATRX/DAXX may represent a driving force for tumorigenesis due to its contribution to the release of rDNA transcriptional potency.

N-N axially chiral biaryls represent a rarely explored class of atropisomers. Reported herein is construction of diverse classes of diaxially chiral biaryls containing N-N and C-N/C-C diaxes in distal positions in excellent enantioselectivity and diastereoselectivity. The N-N chiral axis in the products provides a handle toward solvent-driven diastereodivergence, as has been realized in the coupling of a large scope of benzamides and sterically hindered alkynes, affording diaxes in complementary diastereoselectivity. The diastereodivergence has been elucidated by computational studies which revealed that the hexafluoroisopropanol (HFIP) solvent molecule participated in an unusual manner as a solvent as well as a ligand and switched the sequence of two competing elementary steps, resulting in switch of the stereoselectivity of the alkyne insertion and inversion of the configuration of the C-C axis. Further cleavage of the N-directing group in the diaxial chiral products transforms the diastereodivergence to enantiodivergence. Molecules with axial chirality are of intense focus to the synthetic organic community, but the axes most commonly explored are carbon–carbon and carbon–heteroatom. Here the authors report the syntheses of diaxially chiral biaryls containing N-N and C-N/C-C axes, achieved via rhodium catalysis.