The reliance on model organisms in biotechnology has advanced our understanding of fundamental biology but has failed to capture the complexity of real-world ecosystems, limiting applications in agriculture, biomanufacturing, and environmental remediation. This review critically evaluates the challenges and opportunities of applying CRISPR-Cas genome editing to non-model organisms, structured around a framework that systematically addresses host-specific barriers, enabling technical solutions, and real-world applications. Key obstacles are first delineated, such as restrictive genetic tools, inefficient DNA repair pathways (including NHEJ-dominance, HDR-deficiency, and polyploidy), and delivery limitations. Subsequently, innovative solutions are explored, including the engineering of Cas variants with expanded PAM flexibility and reduced toxicity, the development of host-adapted delivery systems such as phage-based vectors and conjugative plasmids, and the integration of synthetic biology tools and machine learning for optimization. Alternative, DSB-free modalities—comprising base editing, prime editing, CRISPR-associated transposases (CAST), and recombinase-assisted engineering—are further expanded upon, offering enhanced precision and expanded capabilities for complex genetic modifications. Major findings indicate that these approaches can unlock the potential of non-conventional hosts to address global challenges, such as low-energy biomanufacturing, environmental bioremediation, and carbon capture. It is concluded that bridging the gap between foundational CRISPR research and its real-world applications is imperative. Future efforts should focus on democratizing tools via open-source platforms, advancing delivery systems, establishing ethical governance—with detailed considerations for environmental release, horizontal gene transfer, regional regulatory frameworks, and biosafety in extremophile engineering—and fostering sustainable innovation through synthetic biology integration to fully realize the transformative potential of genome editing in organisms beyond model organisms.

Dendrobine, a neuroprotective and anticancer sesquiterpenic alkaloid, is primarily sourced from endangered Dendrobium orchids, posing sustainability challenges to its production. Endophytic fungi, such as Trichoderma longibrachiatum MD33, offer an alternative; however, unresolved biosynthetic pathways and low yields hinder industrial scalability. Enhancing fungal metabolism through nanotechnology could address these limitations; however, nanoparticle-mediated engineering remains unexplored for dendrobine biosynthesis. This study aimed to (1) optimize dendrobine production in T. longibrachiatum MD33 using gold nanoparticles (CH-AuNPs) functionalized with alkaloid precursors and (2) elucidate the biosynthetic pathway to enable targeted metabolic engineering. CH-AuNPs were chemically synthesized, functionalized with L-phenylalanine, L-tyrosine, and tyramine, and applied to fungal cultures at concentrations of 0.5–20.0 mg/L. Multi-omics analyses (transcriptomics, proteomics, and metabolomics) identified pathway enzymes, and oxidative stress markers and dendrobine yields were quantified. Dose-dependent CH-AuNP exposure (10.0 mg/L optimal) elevated dendrobine production by 63.7

Colony-stimulating factor 1 receptor (CSF1R) is a key regulator of macrophage-driven liver inflammation. Here, we report a series of CSF1R inhibitors discovered through a structure-guided optimization strategy for the acute-phase treatment of acetaminophen-induced liver injury. For instance, compound C52 exhibited potent CSF1R inhibition, a kinome-wide selective profile, and low cellular cytotoxicity. C52 rapidly suppressed M-CSF-induced phosphorylation events and downstream signaling in macrophages. In an acute liver injury model, therapeutic administration of C52 during the early inflammatory phase markedly reduced serum transaminase levels, improved hepatic histopathology, and alleviated inflammatory cell infiltration, accompanied by suppression of hepatic p-CSF1R/p-AKT/p-ERK signaling and decreased levels of circulating TNF-α and IL-6. Collectively, these results support pharmacologic CSF1R blockade as a timely strategy to modulate macrophage-mediated inflammation in acute liver injury and warrant further preclinical development of C52.

Background Clinical and preclinical evidence links major depressive disorder (MDD) and Alzheimer’s disease (AD), suggesting MDD treatment could prevent some AD. Dysfunction within the microbiota-gut-brain axis contributes to MDD and AD pathogenesis via dysregulated microbial metabolites. Trilobatin (TLB) functions as a neuroprotective agent modulating gut microbiota. However, its capacity to alleviate depressive-like behavior and cognitive deficits through restoration of gut microbial ecology and metabolite profiles requires clarification. Objective The present research was designed to examine the impact of TLB on depressive-like behavior and cognitive impairments, and the role of the gut microbiota and metabolites. Methods Neuroprotective effects of TLB on MDD and AD were evaluated using an LPS mouse model exhibiting depressive-like behavior and memory impairment. The principal molecular target of TLB was identified through a combination of single-cell sequencing, surface plasmon resonance, and gene knockout approaches. Mechanistic insights into gut microbiota and metabolites were gained through 16S rRNA sequencing and fecal microbiota transplantation (FMT). Results TLB attenuated LPS-induced depressive-like behaviors manifested as lowered sucrose preference, extended immobility, and improved cognitive deficits as reflected by Y-maze and novel object recognition. Mechanistically, TLB directly bound Nrf2, enhanced Nrf2-ARE activity, and suppressed neuroinflammation and oxidative stress. TLB restored gut microbiota homeostasis, elevated Akkermansia muciniphila (AKK) abundance and short-chain fatty acids, and strengthened intestinal tight junction proteins. FMT from TLB-treated mice replicated these benefits in wild-type but not Nrf2-knockout mice. AKK supplementation similarly ameliorated behavioral and cognitive deficits via Nrf2 activation. Conclusion Our findings reveal that TLB mitigates neuropsychiatric deficits by activating Nrf2, remodeling restructuring gut microbiota and fortifying intestinal barrier function. The Nrf2-mediated microbiota-gut-brain axis is suggested as a potential therapeutic target for MDD and AD, positioning TLB as a promising natural Nrf2 activator.

Perfluorooctane sulfonate (PFOS), a persistent environmental pollutant, is associated with cognitive dysfunction through mechanisms involving neuroinflammation, oxidative stress, and metabolic disruption. Icaritin, a bioactive flavonoid with antioxidant and anti-inflammatory properties, exhibits therapeutic potential, though its efficacy against PFOS-induced cognitive impairment remains unexplored. Herein, a mouse model of PFOS-induced cognitive dysfunction was established and treated with oral ICT. Integrated 16S rRNA sequencing and untargeted metabolomics revealed that ICT restored gut microbial homeostasis by enriching beneficial genera (e.g. Akkermansia, Lactobacillus) and reducing ammonia-producing bacteria (e.g. Proteus, Helicobacter, Escherichia), thereby improving gut barrier integrity. Metabolomic profiling identified significant perturbations in ammonia-related pathways, particularly arginine and proline metabolism, underscoring ammonia dysmetabolism as a pivotal mediator of PFOS neurotoxicity. These modifications attenuated systemic and cerebral ammonia accumulation, mitigated neuroinflammation and oxidative stress, and ultimately improved cognitive function. Our findings elucidate ammonia dysmetabolism as a central mechanism in PFOS-induced cognitive decline and highlight the microbiota-gut-brain axis as a promising therapeutic target. This study provides a mechanistic foundation for targeting microbial and metabolic pathways in environmental neurotoxicity.

IntroductionThe endangered orchid Dendrobium nobile is the primary source of dendrobine, a neuroprotective sesquiterpene alkaloid, but unsustainable harvesting necessitates alternative production platforms, such as the endophytic fungus Trichoderma longibrachiatum MD33. However, the fungal dendrobine pathway and its regulatory mechanisms remain uncharacterized, limiting its biotechnological exploitation.MethodsThis study investigated cinnabar (HgS)-induced hormesis to elucidate the stress-mediated metabolic reprogramming of dendrobine biosynthesis through integrated transcriptomic and metabolomic profiling.ResultsSubtoxic HgS concentrations (1.0–4.0 μg/L) triggered ROS signaling, upregulating the mevalonate pathway, terpenoid synthases (TPS1/2), and cytochrome P450 monooxygenases (CYP450s), thereby enhancing dendrobine yields by 24% at 4.0 μg/L. In contrast, cytotoxic doses (6.0 μg/L) suppressed growth (73.9% inhibition) and dendrobine synthesis (73.2% reduction), correlating with metabolic collapse via disrupted CoA biosynthesis and antioxidant depletion. Multi-omics integration revealed biphasic regulation: low HgS activated stress-responsive transcription factors (bZIP, Zn-Cys6) and MAP kinase cascades, redirecting resources toward dendrobine production, whereas high HgS induced apoptotic markers and oxidative damage.ConclusionThese findings establish 4.0 μg/L a hormetic threshold for maximizing dendrobine yields and delineating the genetic and enzymatic architecture of its fungal biosynthesis. This work provides a roadmap for the metabolic engineering of T. longibrachiatum MD33, emphasizing ROS-mediated pathway optimization for sustainable alkaloid production. Future studies should leverage CRISPR-based editing of identified regulatory nodes (e.g., HMGR and FPPS) to enhance stress resilience and dendrobine titers in industrial strains.

Objective:To investigate the in vitro anti-hepatitis B virus (HBV) effects of icariside Ⅱ (ICS Ⅱ) and its impact on mitochondrial fission. Methods:HBV-positive hepatocellular carcinoma HepAD38 cells were used as the cellular model. The cytotoxicity of ICS Ⅱ was assessed via CCK8 assay. The secretion levels of HBV surface antigen (HBsAg) and HBV e antigen (HBeAg), as well as HBV DNA copy numbers, were measured by ELISA and qPCR after treatment with ICS Ⅱ alone or ICS Ⅱ in combination with entecavir (ENT). The effects of ICS Ⅱ on mitochondrial morphology and motility were observed using confocal laser scanning microscopy and transmission electron microscopy (TEM). After ICS Ⅱ treatment, Western blot was performed to analyze the expression levels of key proteins involved in mitochondrial dynamics. Additionally, intracellular reactive oxygen species (ROS) production was evaluated via fluorescence staining. Results:The CCK8 assay results showed that ICS Ⅱ treatment at 25 μmol/L had no significant effect on cell proliferation after 72 h. ICS Ⅱ significantly inhibited the secretion levels of HBsAg and HBeAg, with the respective inhibition rates reaching 54.90% and 39.65% (P < 0.05). Additionally, ICS Ⅱ alone reduced HBV DNA copy numbers by 15.19%, while ENT alone achieved a 34.11% inhibition rate. Notably, ICS Ⅱ in combination with ENT reduced HBV DNA copy numbers by 55.81% (P < 0.05). Furthermore, ICS Ⅱ induced mitochondrial shortening and enhanced mitochondrial motility in HepAD38 cells (P < 0.05). ICS Ⅱ significantly increased the expression levels of mitochondrial motility-related proteins, including Mfn1, Fis1, and phosphorylated Drp1 (ser 616) (P < 0.05), while no significant changes were observed in the expression levels of Mfn2, total Drp1, or Drp1 (ser 637) (P > 0.05). Additionally, ICS Ⅱ significantly suppressed the production of intracellular ROS in HepAD38 cells (P < 0.05). Conclusion:ICS Ⅱ inhibits HBV replication in HepAD38 cells, and the underlying mechanism may be associated with the promotion of mitochondrial fission and suppression of ROS production.

The intricate pathological mechanisms of Alzheimer's disease (AD), along with the restrictive nature of the blood-brain barrier (BBB) that further impedes the drug brain entry, underscore the pressing need for innovative combinational therapy to achieve effective treatment outcomes. Intranasal administration, capable of bypassing BBB by direct transport through olfactory and trigeminal nerves, provides a promising approach for treating neurological disorders. Herein, the guanidinium-modified calix[5]arene (GC5AY) is developed as a single-molecule pleuripotent scaffold, demonstrating small size, positive charge and desirable amphiphilicity, which facilitate its efficient traverse of nasal mucosal barrier. The multifunctionality of GC5AY, including inhibiting amyloid fibrosis, scavenging reactive oxygen species and drug delivery, enables it to serve as a sophisticated platform for constructing multi-target AD therapeutic agents. In light of this, by loading neuroprotective agent Trilobatin (TLB) into the cavity of GC5AY, intranasal administration of the TLB@GC5AY formulation is verified to effectively attenuate the cognitive impairment of AD mice, demonstrating multifaceted pathological improvements, while also possessing good biocompatibility. In response to the growing appeal for combinational therapy of AD, the approach proposed in this study has provided a readily generalizable strategy to fulfill this pursuit.

Amorphophallus konjac is a perennial plant native to Southeast Asia, renowned for its edible corms and rich nutritional value. The bioactive component, konjac glucomannan (KGM), has garnered significant attention due to its broad applications. This review aims to provide a comprehensive overview of the traditional uses, chemical and physical properties, and modern health applications of KGM. It highlights cutting-edge research, discusses challenges and limitations, and identifies future directions for advancing the utility of KGM in health and nutrition. KGM demonstrates remarkable health benefits, including improving metabolic health through weight management, blood glucose stabilization, and lipid profile enhancement. It also plays a vital role in gut health. Emerging evidence highlights its anti-inflammatory and immune-regulatory effects, with applications in managing inflammatory bowel disease, hyperthyroidism, and colorectal cancer (CRC). Recent advancements in multi-omics analyses and high-throughput screening (HTS) approaches have improved KGM extraction, characterization, and evaluation. However, potential side effects such as gastrointestinal discomfort and allergenicity, along with challenges in maintaining purity and molecular consistency, require careful consideration. KGM is a versatile dietary fiber with extensive applications in functional foods, nutraceuticals, and therapeutic interventions. Future research should focus on enhancing KGM's bioavailability, developing targeted delivery systems, and formulating novel applications.

The toxic species formed by the pathological aggregation of α-synuclein (α-Syn) is one of the core pathogenic mechanisms in Parkinson's disease, leading to mitochondrial dysfunction, oxidative stress and ultimately degeneration and loss of dopaminergic neurons. Developing effective inhibitors targeting α-Syn fibrillization critically requires the simultaneous achievement of (1) strong and selective binding of α-Syn for efficient disintegration of fibrils, as well as (2) robust transmembrane capability for efficient cellular uptake. Herein, the co-assembly of guanidinium-modified calixarene (GCA) and cyclodextrin (CD), termed GCA-CD, is screened fully accommodating these conditions. GCA-CD binds tightly and selectively towards α-Syn, thereby effectively inhibiting α-Syn aggregation and disintegrating its fibrils, meanwhile the guanidinium of GCA can additionally improve the transmembrane capability of the co-assembly. In vivo investigations demonstrate that the GCA-CD nanomedicine significantly rescues motor deficits and nigrostriatal degeneration of PD-like rats by decreasing the content of α-Syn as well as restoring mitochondrial dysfunction and suppressing oxidative stress. Astonishingly, transcriptome analysis further reveals the role of GCA-CD in dampening cuproptosis through inhibiting FDX1/LIAS signaling pathway, highlighting the multifaceted therapeutic effects of the co-assembly in PD. The findings in this study underscore the comprehensive exposition on the actual function mechanisms of the therapeutic agents, thereby providing valuable insights for informing material design.

Introduction:To investigate the potential anti-hepatitis B virus (HBV) activity of Icariside Ⅱ (ICS Ⅱ), and elucidate its underlying mitochondrial dynamics mechanisms. Methods:The study employed in vivo and in vitro assays to evaluate anti-HBV effects of ICS Ⅱ. An HBV replicating mouse model was established through hydrodynamic injection of pAAV/HBV1.2, the impact of ICS Ⅱ on HBV replication and liver toxicity was assessed. In vitro cell-based assays used HBV-positive HepG2.2.15 cells. Cytotoxicity was determined with CCK-8 assay, while ELISA and qPCR were employed to measure HBsAg, HBeAg, and HBV DNA levels. The livers of ICS II-treated HBV-infected mice were taken for transcriptome sequencing to screen for different genes, and the results were verified by Western Blot. Mitochondrial morphology and dynamics were visualized using confocal imaging and transmission electron microscopy. Key protein expressions related to mitochondrial fission and fusion were analyzed via WB. Intracellular ROS generation was assessed using fluorescence staining. Results:The study found that ICS Ⅱ exhibited significant anti-HBV effects both in vivo and in vitro. The results of RNA-Seq indicated that ICS Ⅱ modulated the mRNA levels of Fisl, a protein associated with mitochondrial dynamics, during the anti-HBV response. It induced mitochondrial fragmentation and enhanced mitochondrial motility in HBV-positive cells. Notably, key proteins associated with mitochondrial fission and fusion demonstrated alterations favoring fission. Furthermore, ICS Ⅱ effectively reduced ROS production in HBV-positive cells. Conclusion:ICS Ⅱ exhibits significant anti-HBV potential through its regulation of mitochondrial dynamics and ROS production.