Plasmids are used extensively in basic and applied biology. However, design construction of plasmids, specifically the ones carrying complex genetic information, remains one most time-consuming, labor-intensive, rate-limiting steps performing sophisticated biological experiments. Here, we report development a versatile, robust, automated end-to-end platform named PlasmidMaker that allows error-free plasmids with virtually any sequences high throughput manner. This consists versatile DNA...

Pleckstrin homology (PH) domains are presumed to bind phosphoinositides (PIPs), but specific interaction with and regulation by PIPs for most PH domain-containing proteins unclear. Here we employ a single-molecule pulldown assay study interactions of lipid vesicles full-length in mammalian whole cell lysates. Of 67 human initially examined, 36 (54%) found have affinity various specificity, the majority which not been reported before. Further investigation ARHGEF3 reveals distinct structural...

Significance Cell migration is central in physiological and pathological conditions such as immune response cancer metastasis. The excitable network hypothesis can account for recent observations of propagating waves signal transduction cytoskeleton events well behaviors migrating cells. However, the molecular feedback loops involved these networks that bring about excitability are poorly understood. Here, we provide evidence a positive-feedback loop based on mutual inhibitory interaction...

Skeletal muscle regeneration after injury is essential for maintaining function throughout aging. ARHGEF3, a RhoA/B-specific GEF, negatively regulates myoblast differentiation through Akt signaling independently of its GEF activity in vitro. Here, we report ARHGEF3's role skeletal revealed by ARHGEF3-KO mice. These mice exhibit indiscernible phenotype under basal conditions. Upon acute injury, however, ARHGEF3 deficiency enhances the mass/fiber size and regenerating muscles both young...

The CRISPR-Cas12a system is more advantageous than the widely used CRISPR-Cas9 in terms of specificity and multiplexibility. However, its on-target editing efficiency typically much lower that system. Here we improved by simply incorporating 2-aminoadenine (base Z, which alters canonical Watson-Crick base pairing) into crRNA to increase binding affinity between complementary DNA target. resulting (named zCRISPR-Cas12a thereafter) shows an comparable but with off-target effects mammalian...

Proteins are the molecular machines of life with numerous applications in energy, health, and sustainability. However, engineering proteins desired functions for practical remains slow, expensive, specialist-dependent. Here we report a generally applicable platform autonomous protein that integrates machine learning large language models biofoundry automation to eliminate need human intervention, judgement, domain expertise. Requiring only an input sequence quantifiable way measure fitness,...

Skeletal muscle in adults retains a robust ability to regenerate after injury, which progressively declines with age. Many of the regulators skeletal myogenesis are unknown or incompletely understood. Intriguingly, cells secrete wide variety factors, such as cytokines, can influence development and regeneration an autocrine paracrine manner. Here we describe chemokine (C-X-C motif) ligand 14 (Cxcl14) novel negative regulator myogenesis. We found that Cxcl14 expression myoblasts prevented...

Chemically modified mRNAs hold great potential for therapeutic applications in vivo. Currently, the base modification scheme largely preserves canonical Watson-Crick pairing, thus missing one mode of mRNA modulation by altering its secondary structure. Here we report incorporation Z (2-aminoadenine) into to create Z-mRNA with improved translational capacity, decreased cytotoxicity, and drastically reduced immunogenicity compared unmodified mammalian cells. In particular, A-to-Z substitution...

Mitochondria play a key role in energy production and cellular metabolism, making them promising target for metabolic engineering disease treatment. However, despite the known influence of passenger proteins on localization efficiency, only few protein-localization tags have been characterized mitochondrial targeting. To address this limitation, we exploited Variational Autoencoder (VAE), an unsupervised deep learning framework, to design novel targeting sequences (MTSs). In silico analysis...

Abstract Natural products (NPs) produced by bacteria, fungi and plants are a major source of drug leads. Streptomyces species particularly important in this regard as they produce numerous natural with prominent bioactivities. Here we report fully utomated, s calable high-throughput platform for discovery bioactive n atural p roducts S treptomyces (FAST-NPS). This comprises computational prediction prioritization target biosynthetic gene clusters (BGCs) guided self-resistance genes, highly...

ABSTRACT Pleckstrin homology (PH) domains are presumed to bind phosphoinositides (PIPs), but specific interaction with and regulation by PIPs for most PH domain-containing proteins unclear. Here we employed a single-molecule pulldown assay study interactions of lipid vesicles full-length in mammalian whole cell lysates. Of 67 human domaincontaining examined, 36 (54%) were found have affinity various specificity, the majority which had not been reported before. Further investigation ARHGEF3...

Regenerative myogenesis after injury is a unique capacity of adult skeletal muscle to maintain normal tissue structure. This ability declines with aging or diseases. Muscle regeneration consists precisely organized processes such as cell cycle withdrawal followed by myocyte fusion. However, regulatory mechanisms remain incompletely understood. Interestingly, cells express variety cytokines, which may play roles in an autocrine paracrine manner. Previously, we performed RNAi screen that...

Summary Skeletal muscle regeneration is essential for restoring function upon injury and the maintenance of health with aging. ARHGEF3, a Rho-specific GEF, negatively regulates myoblast differentiation via mammalian target rapamycin complex 2 (mTORC2)-Akt signaling in GEF-independent manner vitro . Here, we investigated ARHGEF3’s role skeletal by creating ARHGEF3 KO mice. These mice exhibited no discernible phenotype under normal conditions. Upon injury, however, deficiency enhanced mass,...

Protein kinase B (PKB)/AKT is a serine/threonine with wide range of essential functions in cell proliferation, survival, differentiation, and metabolism. Growth factor stimulation phosphoinositide‐3 (PI3K) results generation phosphatidylinositol‐3,4,5‐trisphosphate [PI(3,4,5)P 3 ] or PI(3,4)P 2 at the plasma membrane, which recruits AKT. At membrane AKT phosphorylated activated, subsequently dissociates from translocates to cytoplasm nucleus order phosphorylate its targets. Hence, it...