Fluorescent reporter systems for protein expression enable real-time, dynamic, and single-cell visualization of gene expression in living cells, but lack sufficient sensitivity for detecting low-abundance proteins. Herein, we developed a signal-amplified split superfolder green fluorescent protein (sfGFP) reporter system: sfGFP was split into the GFP1-10 fragment driven by a constitutively strong promoter and a 20× tandem GFP11 tag controlled by weak promoters, with the incorporation of coiled-coil dimerization domains to facilitate fragment complementation. This design enhanced fluorescent signals by approximately 10-fold, significantly improving detection sensitivity and enabling clear visualization of green fluorescence even at extremely low mRNA levels. Application of this system to evaluate Tet-On inducible systems revealed substantial basal leakage in Tet-On 2G. Moreover, real-time monitoring of endogenous weakly promoters (e.g., VIM, HPRT1, GUSB) demonstrated that the fluorescent intensity was approximately 10-fold higher than that of conventional reporter systems. This platform provides a highly sensitive and versatile tool for weak promoter activity analysis, dynamic tracking of low-abundance genes, and investigation of cell types and states.

C18H22O5, orthorhombic, P212121 (no. 19), a = 8.62400(10) & Aring;, b = 20.6110(2) & Aring;, c = 27.4020(2) & Aring;, V = 4870.69(8) & Aring;3, Z = 12, R gt (F) = 0.0398, wR ref (F2) = 0.1047, T = 149.99(10) K.

C16H11NO2, monoclinic, P21/n (no. 14), a = 13.4514(3) & Aring;, b = 4.5021(1) & Aring;, c = 19.8068(4) & Aring;, beta = 96.185(2)degrees, V = 1,192.51(4) & Aring;3, Z = 4, Rgt(F) = 0.0420, wRref(F2) = 0.1251, T = 298.35(10) K.

The FKBP-based chemically induced dimerization (CID) technology is a fundamental tool for spatiotemporal precise modulation of protein functions in living cells, widely used in gene editing, protein function regulation, disease therapy, and drug development. However, its widespread application is limited by the inherent drawbacks including issues with immunosuppressive activity, stability, reversibility, and in vivo delivery. Recent years have seen remarkable progress in addressing these challenges: orthogonalization strategies eliminate immunosuppressive effects, fast-dissociating ligands and optogenetic systems enable reversible regulation, and protein engineering optimizes the FKBP/FRB domains to enhance stability and reduce immunogenicity. Moreover, integration with novel delivery technologies broadens its application scope greatly. This review summarizes the key optimization strategies and innovative applications of this technology in the cutting-edge biological research, aiming to reference the development of next-generation chemogenetic tools with higher precision, better safety, and greater application potential.

Parkinson’s disease (PD) has been associated with the microbiota-gut-brain axis, and probiotics have been shown to alleviate PD symptoms via various mechanisms. Akkermansia muciniphila has demonstrated therapeutic effects in some neurological disorders; however, its role in PD treatment remains debated. This study revealed that orally administered hypoactive Akkermansia muciniphila inhibited dopaminergic neuron loss in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD mouse model. Its ingestion also reduced astrocyte activation and mitigated inflammatory responses in both the brain and colon. Additionally, hypoactive Akkermansia muciniphila modulated intestinal microbiota composition in PD model mice. These findings suggest that hypoactive Akkermansia muciniphila reduces neurotoxicity in an MPTP-induced PD model and may offer potential for inhibiting PD progression.IMPORTANCERecent research suggests a connection between Parkinson’s disease (PD) and the microbiota-gut-brain axis, with evidence indicating that probiotics may alleviate PD symptoms. Although Akkermansia muciniphila has demonstrated potential benefits for certain neurological disorders, its efficacy in the treatment of PD is still a subject of ongoing debate. Here, we demonstrated that hypoactive Akkermansia muciniphila ameliorated dopaminergic neuronal death, correlating to the reduction of glial hyperactivation and neuroinflammation. Hypoactive Akkermansia muciniphila also induced microbiota fluctuation, which may perform a sophisticated effect on PD progression. Our study may provide an innovative strategy for using hypoactive Akkermansia muciniphila as a therapy or adjuvant therapy for PD.

Messenger RNA (mRNA), emerging as a revolutionary therapeutic tool, has shown remarkable potential in diverse fields such as vaccine development, tumor immunotherapy, gene therapy, and protein replacement therapy, attributed to its high programmability and safety. However, traditional engineered mRNA therapeutics encounter obstacles like short half-lives and limited protein expression, which impede their extensive application in the biomedical domain. In recent years, with the progress of RNA modification technologies and the advent of novel RNA techniques, including mRNA, self-amplifying RNA (saRNA), circular RNA (circRNA), and branched RNA (brRNA), researchers have achieved substantial breakthroughs in enhancing RNA stability, prolonging protein expression, and reducing immunogenicity. This article comprehensively reviews the structure, function, optimization strategies, and biomedical applications of these protein-coding RNAs.

The regenerative capacity of the central nervous system is extremely limited, posing significant challenges for repairing brain injuries. Human induced pluripotent stem cells (hiPSCs) offer a promising cell source for neural regeneration, but their clinical application is hindered by inefficient neuronal differentiation, and poor post-transplant survival. To overcome these challenges, we prepared electrospun fibrous membranes composed of acetylated glucomannan, gelatin, polycaprolactone, and carbon nanotubes. These scaffolds exhibited anti-inflammatory properties in vitro and in vivo. Their excellent adhesion and support properties mimic the extracellular matrix, facilitating the 3D culture of our previous engineered NILB-hiPSCs in vitro. After loading with doxycycline (differentiation inducer) and cyclosporin A (immunosuppressant), the scaffolds achieved sustained release of these agents, ensuring timely neuronal differentiation and maintaining localized immunosuppression, thereby circumventing the need for high systemic doses. Moreover, the composites improved the survival and differentiation of NILB-hiPSCs, promoting the neural repairment in controlled cortex injury mice. Our study highlights the importance of customized multifunctional biomaterials in stem cell transplantation therapy.

C 17 H 12 ClN 3 O 3 , monoclinic, P 2 1 / c (no. 14), a = 13.8689(2) Å, b = 7.19130(10) Å, c = 15.9230(2) Å, β = 107.584(1)°, V = 1513.88 Å 3 , Z = 4, R gt ( F ) = 0.0338, w R ref ( F 2 ) = 0.0937, T = 200(10) K.

Stem cell-based therapy holds great potential for substituting degenerated motor neurons (MNs) in amyotrophic lateral sclerosis (ALS). Missing protocols for advanced differentiation of transplanted cells into MNs, immune rejection, and the lack of suitable ALS models for preclinical trials have slowed the development of effective therapies. Here, we employed multiplex genetic-editing to generate a novel human pluripotent stem cell line containing doxycycline (Dox)-inducible MNs-specific transcription factors and comprehensively modified immunomodulatory genes. We transplanted these cells into the spinal cord of ALS large animal models (SOD1G93A pigs and TIA1P362L rabbits), which faithfully recapitulate pathologies and symptoms observed in ALS patients. The transplanted cells could efficiently differentiate into functional MNs upon Dox treatment in vivo, distribute throughout the spinal cord and motor cortex via extensive migration, survive long-term without the need for immunosuppression. Notably, these MNs integrated into host neural circuits, as evidenced by their long projection of peripheral axons to target muscle and reformation of neuromuscular junctions. As result, pathologies and motor deficits were substantially ameliorated in both animal models. One Sentence Summary Hypoimmunogenic human motor neurons induced from iPSCs in vivo reform neuromuscular junctions and ameliorate ALS disease in pig and rabbit models. ### Competing Interest Statement The authors have declared no competing interest.

The occurrence and development of pre-eclampsia (PE) is closely related to genetics. However, multi-omics analysis does not provide sufficient evidence to define significant genes. Therefore, we aimed to identify significant genes and pathways using summary statistics from genome-wide association studies (GWAS). Based on the summary statistics, we used linkage disequilibrium score regression (LDSC) to discover genetic correlation between PE and complex traits. Leveraging summary statistics of tissue-specific expression quantitative trait loci (eQTL), we used FUSION to define significant genes, Bayesian colocalization analysis to identify pleiotropic genes, and Multi-marker Analysis of GenoMic Annotation (MAGMA) to determine the associated pathways. Specifically, considering the potential relationship between PE and tissues, we included 11 tissues, such as kidney cortex. Our integrative analysis revealed that the observed heritability of PE was 0.0179 (standard error [SE] = 0.0021, P -value < 0.001). Also, based on the Bonferroni correction, we defined 238 traits genetically correlated to PE, such as the other cardiovascular diseases (r = −0.55) and furosemide (r = 0.79). Integrating eQTL summary statistics across eleven tissues, we identified 30 significant genes, such as EIF2S1 in the uterus (TWAS. Z = 4.44, TWAS. P = 8.95 × 10 −6 ), and PAWRP2 in ovary (TWAS. Z = 4.34, TWAS. P = 1.45 × 10 −5 ). Based on colocalization, we identified 26 pleiotropic genes. We found that three genes, including RPS26 , SULT1A2 , OBSCN-AS1 , and SUOX , were simultaneously defined by FUSION and colocalization. Moreover, we found that the significant enrichment was in the FOXG1_TARGET_GENES pathway regulated by the transcription factor FOXG1 (P FDR = 0.049). The findings of post-GWAS analysis for PE indicate that there are 30 significant genes and 26 pleiotropic genes. Future studies are required to investigate the efficacy of targeting pleiotropic genes to reduce the risk of PE.

Long-term use of naproxen can lead to serious side effects. Inspired by the biological activity of cinnamic acid, a series of cinnamic acid derivatives containing naproxen were designed and synthesized, and their anti-inflammatory activities and mechanisms were explored in vitro. Our results indicated that all of naproxen derivatives showed more significant inhibition against lipopolysaccharide (LPS)-induced nitric oxide (NO) production and had a lower degree of cytotoxicity than that of naproxen. The present studies revealed that compound 23 (IC50 = 5.66 ± 1.66 µM) markedly inhibited the LPS-induced NO production and the over-expression of pro-inflammatory cytokines, including interleukin (IL)-1β, inducible NO synthase (iNOS), and cyclooxygenase-2 (COX-2). Furthermore, it blocked the activation of NF-κB signaling pathway and pyrin domain-containing protein 3 (NLRP-3) inflammasome in a concentration-dependent manner. Additionally, docking studies confirmed that compound 23 exhibited a well-fitting into the NLRP3 active site. Considering these results, compound 23 might be a novel NLRP3 inhibitor to treat inflammatory diseases.

INTRODUCTION:Electroacupuncture (EA) has been demonstrated as an effective therapeutic intervention for cerebral ischemia-reperfusion injury (CIRI); however, the fundamental processes underlying EA therapy remain largely elusive, which hinders the optimization and broader clinical application of EA. It has been reported that during CIRI, cellular metabolism undergoes a shift from oxidative phosphorylation to glycolysis, giving rise to an accumulation of lactate, but whether and how lactate is involved in CIRI and EA therapy is not fully understood. OBJECTIVES:To explore the role of lactate in EA therapy against CIRI and the underlying mechanisms. METHODS:Neurological outcome evaluations and TTC staining were performed to assess CIRI in mice. Western blotting and immunofluorescence were used to detect histone lactylation, and genes regulated by histone lactylation were identified by CUT&Tag, ATAC-seq and RNA-seq, followed by investigation of the role(s) of an iron transporter encoding gene Zip14 in EA therapy. Arterial lactate levels in stroke patients were assessed 30 min after recanalization using arterial blood gas analysis. RESULTS:Lactate markedly enhances histone H4 lysine 12 lactylation (H4K12la) in neurons, concomitantly upregulating PKM2 expression-a pivotal regulator of lactate production. EA protects neurons in the ischemic penumbra and improves neurological outcomes following CIRI by suppressing PKM2-mediated H4K12la. Furthermore, CIRI elevates H4K12la enrichment at Zip14, which facilitates chromatin accessibility, activates Zip14 transcription, and ultimately triggers ferroptosis. EA treatment attenuates ferroptosis and mitigates CIRI by decreasing ZIP14 expression. Clinically, EA significantly reduces arterial lactate levels after recanalization in patients with ischemic stroke. CONCLUSIONS:This study uncovered a previously unrecognized mechanism by which lactate is involved in EA therapy against CIRI, highlighting H4K12la-dependent ZIP14 expression as a potential therapeutic target for CIRI management and EA optimization.

Essential oils (EOs), derived from medicinal plants, has been reported to possess various bioactivities to treat inflammatory diseases. Therefore, it is urgent to investigate the effect and the mechanism of EOs essential oils as naturally anti-inflammatory agents. The present study indicated thirty-one EOs had different inhibition on lipopolysaccharide (LPS)-induced nitric oxide (NO) production. Moreover, the most potent EOs against NO production in BV2 cells were chuan-xiong, schizonepeta tenuifolia, lemongrass and zedoary turmeric with IC50 values of 12.59, 23.12, 22.76 and 21.88 μg/mL, respectively. The further studies revealed that ligustilide, tributyl citrate and longifolene, the main constituents of chuan-xiong EO, schizonepeta tenuifolia EO and zedoary turmeric EO, were responsible for their activities against NO production with IC50 values of 8.75, 6.05 and 20.32 μg/mL. Above all, theses EOs were confirmed to be promising anti-inflammatory agents.

Gene-switch techniques hold promising applications in contemporary genetics research, particularly in disease treatment and genetic engineering. Here, we developed a compact drug-induced splicing system that maintains low background using a human ubiquitin C (hUBC) promoter and optimized drug (LMI070) binding sequences based on the Xon switch system. To ensure precise subcellular localization of the protein of interest (POI), we inserted a 2A self-cleaving peptide between the extra N-terminal peptide and POI. This streamlined and optimized switch system, named miniXon2G, effectively regulated POIs in different subcellular localizations both in vitro and in vivo. Furthermore, miniXon2G could be integrated into endogenous gene loci, resulting in precise, reversible regulation of target genes by both endogenous regulators and drugs. Overall, these findings highlight the performance of miniXon2G in controlling protein expression with great potential for general applicability to diverse biological scenarios requiring precise and delicate regulation.

Glycosylase base editor (GBE) can induce C-to-G transversion in mammalian cells, showing great promise for the treatment of human genetic disorders. However, the limited efficiency of transversion and the possibility of off-target effects caused by Cas9 restrict its potential clinical applications. In our recent study, we have successfully developed TaC9-CBE and TaC9-ABE by separating nCas9 and deaminase, which eliminates the Cas9-dependent DNA off-target effects without compromising editing efficiency. We developed a novel GBE called TaC9-GBE(YE1), which utilizes the deaminase and UNG-nCas9 guided by TALE and sgRNA, respectively. TaC9-GBEYE1 showed comparable levels of on-target editing efficiency to traditional GBE at 19 target sites, without any off-target effects caused by Cas9 or TALE. The TaC9-GBE(YE1) is a safe tool for gene therapy.

Spinal muscular atrophy (SMA) is a devastating neuromuscular disease caused by mutations in the survival motor neuron 1 ( SMN1) gene. Gene editing technology repairs the conversion of the 6th base T to C in exon 7 of the paralogous SMN2 gene, compensating for the SMN protein expression and promoting the survival and function of motor neurons. However, low editing efficiency and unintended off-target effects limit the application of this technology. Here, we optimized a TaC9-adenine base editor (ABE) system by combining Cas9 nickase with the transcription activator-like effector (TALE)-adenosine deaminase fusion protein to effectively and precisely edit SMN2 without detectable Cas9 dependent off-target effects in human cell lines. We also generated human SMA-induced pluripotent stem cells (SMA-iPSCs) through the mutation of the splice acceptor or deletion of the exon 7 of SMN1. TaC9-R10 induced 45% SMN2 T6>C conversion in the SMA-iPSCs. The SMN2 T6>C splice-corrected SMA-iPSCs were directionally differentiated into motor neurons, exhibiting SMN protein recovery and anti-apoptosis ability. Therefore, the TaC9-ABE system with dual guides from the combination of Cas9 with TALE could be a potential therapeutic strategy for SMA with high efficacy and safety.

Naproxen, one type of non-steroidal anti-inflammatory drugs, has excellent anti-inflammatory activity. However, long-term use of it causes serious adverse effects. Inspired by the biological diversification of cinnamic acid, novel naproxen derivatives containing cinnamic acid are synthesized and used to improve their antiinflammatory activity and safety. Our results indicated that thirty naproxen derivatives have different inhibitory effects on RAW264.7 macrophage cells. It is showed that most of the target naproxen derivatives possess the lower degree of cytotoxicity than that of naproxen. Further studies indicated that compound 22 (IC50 = 8.74 +/- 2.13 mu M) concentration-dependently inhibits the over-expression of iNOS, COX-2 and IL-113 by blocking the activation of nuclear factor kappa-B (NF-KB) signaling pathway and NLRP-3 inflammasome, respectively. Docking studies showed that the binding of compound 22 to NLRP3 using a well-fitting mode. It is found that compound 22 will be a novel anti-inflammatory agent to treat inflammatory diseases with an improved safety profile.

Current methodologies for hepatocyte induction from human induced pluripotent stem cells (hiPSCs) have limited efficacy due to lack of a functional hepatocyte reporter. To address this, we developed an endogenous albumin (ALB)-sfGFP reporter system in hiPSCs using homologous directed recombination (HDR)-mediated knock-in. The hiPSCs maintained the characteristic morphology, pluripotency, and normal karyotype while demonstrating successful differentiation into all three germ layers both in vitro and in vivo. Co-expression of EGFP and ALB was observed in the derived hepatocyte-like cells (HLCs). This reporter system holds promise for functional hepatocyte induction.

Naproxen, widely used to treat anti-inflammatory diseases, would cause serious of side effects. Based on the biological activities of cinnamic acid, naproxen derivatives containing cinnamic acid were designed, synthesized and used to enhance their anti-inflammatory activities and safeties. The results investigated that thirty novel naproxen derivatives had inhibitory effects on the nitric oxide (NO) release in RAW264.7 macrophage cells. A majority of naproxen derivatives showed the lower degree of cytotoxicity than that of naproxen. In vitro studies revealed that A22 (IC50 = 7.38 +/- 1.96 mu M) blocked the activation of nuclear transcription factor kappa B (NF-kappa B) signaling pathway and pyrin domain containing protein 3 (NLRP-3) inflammasome in a concentration dependent manner, thereby down-regulating the expression of pro-inflammatory cytokines, such as interleukin (IL)-1 beta, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). Docking studies confirmed that A22 exhibited a well-fitting into the NLRP3 active site. Accordingly, A22 might be a novel NLRP3 inhibitor to treat inflammatory diseases.

Gemcitabine (GEM) is a standard chemotherapeutic agent for patients with pancreatic cancer; however, GEM-based chemotherapy has a high rate of toxicity. A combination of GEM and active constituents from natural products may enhance its therapeutic efficacy and reduce its toxicity. This study investigated the synergistic effects of the combination of liriopesides B (LirB) from Liriope spicata var. prolifera and GEM on human pancreatic cancer cells. The results of our study showed that the combination of LirB and GEM synergistically decreased the viability of pancreatic cancer cells. The combination also caused a strong increase in apoptosis and a strong decrease in cell migration and invasion. Furthermore, LirB combined with GEM had potent inhibitory effects on pancreatic cancer stem cells (CSCs). Studies on the mechanisms of action showed that the combination more potently inhibited protein kinase B (Akt) and nuclear factor kappa B (NF-κB), as well as the downstream antiapoptotic molecules B-cell lymphoma 2 (Bcl-2) and survivin than either agent used alone. The results of this study suggest that the combination of LirB with GEM may improve the efficacy of GEM for the treatment of pancreatic cancer.