Nanoneedles are emerging as a safe and scalable strategy for the genetic modification of primary human cells. However, a limited understanding of how interactions at the biointerface lead to functional gene expression continues to hinder clinical translation. While direct membrane penetration, permeabilization, and endocytosis have been proposed as intracellular delivery avenues, the mechanistic connection between delivery and successful transfection remains unclear. Here, we identify caveolae-mediated endocytosis, dependent on Caveolin-1, as a key mechanism enabling nanoneedle transfection. By selectively modulating Caveolin-1 expression in primary human regulatory T cells and MG63 cells and investigating endolysosomal processing, we show that although nucleic acids can be efficiently delivered in the absence of Caveolin-1, gene expression occurs only when caveolar endocytosis is present. These findings reveal a mechanistic basis and establish a broader design principle for nanoneedle transfection: interfacing must be accompanied by the engagement of permissive cellular trafficking pathways to achieve gene expression.

The projected 2.7-fold increase in population in sub-Saharan Africa by the end of the century demands consideration as to how agricultural output can keep pace. Augmenting nitrogen inputs is a practical necessity, but this must be accomplished in such a way that avoids the environmental costs of past advances and also places the resource in the hands of those who will be the most affected. Biological nitrogen fixation might play an important role. The realization that certain algae are able to provide for their own nitrogen needs by fixing atmospheric N2 raises the possibility that an endosymbiont responsible for the nitrogen might be transferred to crop plants. For this to take place, it is necessary that the endosymbionts be (or be made to be) sufficiently independent of their hosts so that they may establish themselves in crop plants appropriate to African agriculture. Genomes from six endosymbionts from diatoms within the family Rhopalodiaceae were analyzed. They were compared to genomes from free-living cyanobacteria and to those of the nitroplast UCYN-A and chromatophore from Paulinella, to which they are related. Unlike the latter two endosymbionts, the six from Rhopalodia encode all the enzymes considered that underlie metabolic processes and provide the energy to power N-fixation. Some of the endosymbionts also appear able to synthesize cofactors essential for central metabolism. The analysis points to possible carbon sources the endosymbionts might take up from their hosts, including glycerol and chitobiose. Possible routes of nitrogen export to the host were also examined. Within the limits of genome analysis, some of the Rhopalodian endosymbionts appear to be metabolically independent of their hosts, except for requiring a carbon source. However, the choice of carbon source and the likely means of nitrogen export are not compatible with crop plants. Genetic modification would surely be necessary for any prospect of propagation of an endosymbiont in a plant of agricultural importance, and significant questions must first be answered in the laboratory. To this end, the endosymbiont of Epithemia clementina may be best suited for such investigations, eventually after transfer to the model diatom Phaeodactyllum tricornutum.

Combining metabolic engineering and fermentation optimization to achieve cost-effective oil production by Cutaneotrichosporon oleaginosus.

Gene manipulation is essential for understanding biological mechanisms, yet genetic modification in the social amoebas (Dictyostelia) has been largely limited to Dictyostelium discoideum. Here, we aimed to establish a CRISPR/Cas9-based genome-editing system applicable across the phylogenetic breadth of Dictyostelia, spanning Groups 1-4. Using an extrachromosomal CRISPR/Cas9 vector from D. discoideum, we disrupted stlA and pkaC in Polysphondylium violaceum and pkaC in two early-branching species, Heterostelium pallidum and Cavenderia fasciculata. In D. discoideum, co-introduction of donor oligos with the CRISPR vector enabled selection-free knockout generation of pkaC with 28.6% efficiency. In H. pallidum, where genome editing is typically inefficient, co-electroporation of donor oligos with the CRISPR/Cas9 vector followed by 4 days of drug selection increased the frequency of pkaC disruption from 0.9% to 8.3%. These results demonstrated that the D. discoideum CRISPR/Cas9 system can be extended across Dictyostelia, providing a versatile platform for comparative genetic and evolutionary developmental studies.

Pharmaceutical protein production by transgenic chickens: several viewpoints towards the next stage.

Regulatory T cells, or Tregs, are designed to limit unnecessary inflammation and serve as a safeguard mechanism to prevent tissue damage caused by heightened inflammatory responses from activated macrophages or effector T cells. Impaired Treg function has implications in autoimmunity and neuroinflammation. Neuroinflammation triggered by amyloid proteins and protein aggregates accelerates neurodegeneration due to increased cytokines and chemokines in the brains of individuals with Alzheimer’s Disease and Parkinson’s Disease. A simple approach involves preventing inflammation by suppressing T-effector cell activity in affected brains through boosting Tregs’ function. Super-Tregs, with enhanced anti-inflammatory properties, can be engineered in vitro to combat inflammation in various tissues and, after homotropic transfer to the target tissue, prevent damage caused by inflammation. The development of Super-Tregs can be achieved through specific genetic and epigenetic modifications. Efforts to generate Super-Tregs utilizing miRNAs and miRNA-containing extracellular vesicles hold promise in treating neuroinflammation with miRNA-engineered Super-Tregs. In this review, we discuss the potential, progress, challenges, and limitations of Super-Treg development and their application in the treatment of neurodegeneration.

Plant branching, encompassing both vegetative and reproductive forms, is a complex and crucial process that shapes overall architecture and determines crop yield and biomass. MicroRNAs (miRNAs) have emerged as master regulators in fine-tuning the intricate genetic and hormonal networks that govern plant branching. This review systematically synthesises recent advances in understanding how miRNA-target gene modules regulate essential pathways to orchestrate the branching patterns. We highlight a central insight that specific miRNA families form hierarchical, stage-specific networks that facilitate the independent optimisation of vegetative and reproductive branching. Furthermore, we explore the potential applications of miRNA manipulation in optimising branching architecture to improve crop yield. By critically evaluating strategies such as artificial miRNAs, target mimics and CRISPR/Cas9 genome editing, we discuss how modulating miRNA networks can engineer ideal plant architecture. Finally, we provide a forward-looking perspective on overcoming challenges in miRNA-based crop improvement, emphasising the integration of single-cell omics and epigenetic insights to achieve precise genetic modifications. This review underscores the transformative potential of miRNAs in designing future crops for enhanced productivity.

Vitamin A deficiency (VAD), a major global health concern, has driven efforts to develop staple crops with enhanced pro-vitamin A (pVA) content. Delivering meaningful nutritional benefits, however, requires technologies that maintain elevated carotenoid levels under field conditions. Previous proof-of-concept work demonstrated that pVA content can be substantially increased in Cavendish bananas through genetic modification, providing a platform for transferring the technology into East African Highland banana (EAHB) cultivars relevant to reducing VAD in Uganda. To evaluate performance under agronomic conditions, we conducted multi-generational field assessments of 27 transgenic Cavendish lines generated from seven constructs enabling constitutive or fruit-preferred expression of three carotenoid biosynthesis genes: ZmPsy1, MtPsy2a and PaCrtI. Constitutive expression was driven by the maize Ubi promoter, while fruit expression was regulated by Exp1 or ACO promoters. Agronomic performance and fruit carotenoid levels were analysed across three generations to explore factors influencing pVA enhancement. All transgenic lines exhibited increased fruit pVA, with the highest accumulation observed in lines constitutively expressing MtPsy2a. Promoter-transgene combinations significantly affected carotenoid accumulation and the stability of the trait in the field. PVA accumulation was the highest in the initial sucker crop and declined in subsequent ratoons, reflecting sensitivity to seasonal conditions. While ACO- and Ubi-driven lines were less affected by seasonal temperature changes, these variations significantly constrained pVA accumulation in wild-type and Exp1-regulated lines. This comprehensive assessment helps elucidate the complex interplay of promoter, isoform, and environmental factors that are essential for the long-term viability of nutritional interventions aimed at combating VAD in the region.

Direct visualization of postsynaptic scaffolds in living neurons is essential for dissecting synaptic dynamics and plasticity. Existing methods for live synapse visualization have major constraints, relying on genetic engineering or multistep application of live-cell incompatible antibodies or nanobodies. Available affinity probes and delivery strategies lack the required contrast due to incomplete or excess delivery. Here, we introduce Sylives, a set of compact, synthetic fluorescent peptides that enable high-contrast live imaging of inhibitory (gephyrin) and excitatory (PSD-95) postsynaptic scaffolds in native neurons. Critically, by pre-purification of the redox-cleavable CPP-probe conjugate we overcome side-product formation of in-situ coupling strategies, achieving reliable cytosolic delivery and restored scaffold binding after intracellular reduction. The Sylive design addresses the need for nanomolar probe levels versus micromolar CPP for clean labelling and efficient delivery by decoupling targeting and uptake. Through quantitative evaluation of uptake and off-target binding, we defined a transferrable parameter space for effective intracellular delivery. Near traceless Sylive uptake and target specificity are validated by direct comparison to transiently expressed proteins and immunolabeling in fixed neurons. The reduction-sensitive Sylive conjugates enable high-contrast, specificity-restored labelling of endogenous postsynaptic sites without genetic modification and offer a modular platform for targeting alternative intracellular proteins in living primary neurons.

Efficacy and safety of autologous CD5-KO anti-CD5 CAR-T cells in relapsed/refractory CD5+ hematological malignancies.

Aphids are one of the most damaging piercing-sucking pests in rice production, whose population outbreaks cause crop losses amounting to hundreds of millions of dollars annually worldwide. To address this challenge, it is highly imperative to develop aphid-resistant rice varieties through techniques such as genetic modification. In this study, we successfully targeted the (E)-β-farnesene synthase (EβFS) to the chloroplast using the promoter of the rice Rubisco small subunit gene (rbcS) and its chloroplast transit peptide (ctp), which significantly enhanced the production of (E)-β-farnesene (EβF) in transgenic rice. At the seedling stage, the EβFS protein content was 17.6-fold higher in transgenic rice compared with that in the rice previously generated using the cauliflower mosaic virus 35S promoter (35S::EβFS). At the tillering stage, the EβF release rate reached 12.7 μg per plant per day in the transgenic rice. Olfactory behavioral assays demonstrated that the transgenic rice line generated in this study had significantly enhanced ability to repel bird cherry-oat aphids (Rhopalosiphum padi) ('Push' effect) and attract its natural enemy multicolored Asian lady beetles (Harmonia axyridis) ('Pull' effect) compared with the 35S::EβFS lines. In addition, the rbcS promoter can achieve specific expression of the EβFS gene in photosynthetic organs, avoiding unnecessary consumption of energy and resources associated with constitutive expression. This Push-Pull strategy could protect rice plants from aphid infestation by controlling the aphid populations, providing an ecologically friendly and sustainable approach for integrated pest management in rice fields. © 2025 Society of Chemical Industry.

Mesenchymal stem cells (MSCs) are adult stem cells with extensive differentiation potential, sourced from bone marrow, adipose tissue, umbilical cord blood, and other tissues. MSCs from different origins exhibit distinct functional characteristics. These cells have demonstrated therapeutic efficacy in various neurological disorders, primarily by modulating immune responses, promoting neovascularization, and aiding neural circuit reconstruction. Notably, the strong proangiogenic properties of MSCs play a crucial role in disease treatment and regression. This review focuses on the application of MSCs and their derivatives in neurological disorders, primarily exploring strategies to enhance their angiogenic effects, including pharmacological interventions, genetic modification, modulation of the culture environment, and the application of novel materials. Furthermore, the article prospects the potential application of MSC-mediated angiogenesis in the treatment of neurological disorders, specifically in the surgical management of ischemic cerebrovascular diseases.

Immunotherapies, including cell therapies, are effective anti-cancer agents. However, cellular product persistence can be limiting with short functional duration of activity contributing to disease relapse. A variety of manufacturing protocols are used to generate therapeutic engineered T cells; these differ in techniques used for T-cell isolation, activation, genetic modification, and other methodology. We sought to determine how pre-selection affected the phenotype of T cells engineered to secrete a CD123xCD3 bispecific engager (ENG-T). These cells were designed to treat acute myeloid leukemia (AML). We evaluated the effect of T-cell selection on transduction efficiency, T-cell activation, short- and long-term anti-AML cytotoxicity, and gene transcription. Unselected, CD4, CD8, and CD4/CD8 pre-selected ENG-T cells have minor differences in T-cell subset components, equivalent activation, and equal cytotoxicity in short-term assays. While unselected and CD4/ CD8-selected ENG-T cells have identical CD4:CD8 composition prior to target cell exposure, serial stimulation in vitro showed CD4/CD8 pre-selection supports ENG-T-cell survival and long-term activity. Likewise, CD4 and CD4/CD8 pre-selected ENG-T cells display superior anti-tumor efficacy and prolong murine survival in AML xenografts. Unselected ENG-T cells are exposed to cytokines during early manufacture that imprint upregulation of intracellular inflammatory pathways. This early activation likely underpins long-term observed functional differences. Pre-selection of T cells from banked patient biospecimens decreased blast contamination, exposure to inflammatory cytokines, and may improve T-cell expansion during manufacture. Pre-selection of T-cell products should continue to be performed to enhance the quality of clinical cellular therapeutics.

Biotechnology of isopentenols production from microbial cell factories.

Leveraging advances in RNAi and CRISPR for improved biological pest control.

After many decades of experimental studies and the advent of genetic engineering for pig organs, xenotransplantation has finally reached clinical application. Clinical trials are beginning, and a review of the recent preclinical and clinical studies is paramount. More specifically, reviewing the pathologic findings in biopsies and terminal samples of transplanted pig organs are integral to assessing xenograft acceptance and clinical success. This review aims to summarize the current literature in xenotransplantation and enumerate both typical and novel pathological findings in xenografts. This holds an opportunity to identify both diagnostic clues and gaps that are critical to treatment protocol modifications and to improving xenograft survival. While thrombotic microangiopathy and antibody mediated rejection are known pathologic entities in xenograft pathology, we re-examine their occurrence in the context of other diagnostic parameters, and we revisit the importance of integrating clinical findings and molecular techniques to aid pathological diagnosis. We also describe the novel findings in xenografts from both nonhuman primate and human recipients. With the recently observed pathological features, a structured approach to xenograft pathology diagnosis can be proposed for use in clinical practice and is an effort that can impact therapeutic and genetic modification strategies.

We investigated unauthorized glyphosate-tolerant rice plants from fields where no genetically modified herbicide-resistant varieties have regulatory approval. The unusual herbicide tolerance phenotype suggested potential unauthorized genetic modification, necessitating comprehensive molecular characterization. We employed integrated analytical approaches: PCR screening for transgenic elements, quantitative polymerase chain reaction for copy number determination, Illumina whole-genome sequencing, and PacBio long-read sequencing. Bioinformatic analysis identified integration sites and insertion structures. Gene-specific and event-specific detection assays were developed following international regulatory standards. We identified six independent cisgenic rice events, each containing 4-9 tandem copies of a mutated rice OsEPSPS gene conferring glyphosate tolerance. Each event exhibited unique chromosomal integration sites, distinct flanking sequences, and complex tandem repeat structures. The mutations were absent from natural rice germplasm databases (3K RG), confirming intentional genetic modification. Detection assays achieved 0.05-0.1% limits of detection, meeting international performance standards. This study reveals critical gaps in current genetically modified organism monitoring systems that fail to detect cisgenic products. Our findings demonstrate that unauthorized cisgenic crops can evade conventional regulatory oversight, challenging biosafety management and international trade under process-based frameworks. This work underscores the urgent need to transition from element-based detection to comprehensive genomic approaches, providing essential methodologies for detecting new breeding technique products and maintaining regulatory oversight.

Tumors often exhibit an extracellular matrix with elevated stiffness due to excessive accumulation and cross-linking of proteins, particularly collagen. This elevated stiffness acts as a physical barrier, impeding the infiltration of immune cells and the effective delivery of various immunotherapeutic agents, such as lipid nanoparticle-based RNA therapeutics. Here, we investigate the ability of ultrasound-activated nanobubbles (US-NBs) to increase the permeability and immunogenicity of tumors. Our results show that US-NBs physically remodel the tumor tissue by decreasing its stiffness by 60% 5 days after a single treatment. US-NB-treated tumors display randomly oriented collagen with a 5.47-fold lower deposition compared to untreated tumors. This leads to the effective delivery and widespread distribution of lipid nanoparticles (LNPs) in the tumor. Importantly, when assisted by US-NB, LNPs exhibit superior gene-transfection efficiency across pan-immune cells and achieve efficient genetic modification of T cells directly in vivo. This combined approach engages both innate and adaptive immunity, enhancing tumor immunogenicity and boosting cytotoxic cell infiltration by 4-fold compared to LNPs alone. These results indicate that gentle mechanical stimulation of the tumor using US-NB offers a promising strategy to augment the delivery and efficacy of existing immunotherapies.

Oral wound healing is a robust process; however, complications from surgery, systemic diseases, and aging can impair healing. While some treatments exist, regenerative therapies to promote mucosal wound healing remain limited. In recent years, there has been a significant rise in FDA-approved cell-based therapies; however, extracellular vesicles represent an emerging cell-free alternative that may mitigate risks associated with cellular therapies, including tumorigenesis and immunogenicity. These lipid-encapsulated nanovesicles can deliver therapeutic cargo, such as proteins, lipids, nucleic acids, or drugs, to the wound site. Extracellular vesicles can be derived from mesenchymal stromal cells, immune cells, bodily fluids, or bacteria, and engineered through genetic modification, preconditioning, or direct cargo loading to enhance therapeutic potency. Furthermore, advanced delivery platforms, including hydrogels, microneedles, and aerosols, allow for sustained and localized EV delivery to the oral wound site. This review examines differences between cutaneous and oral wound healing; factors that impair oral repair; extracellular vesicle sources and engineering strategies; and delivery strategies for developing EV-based therapeutics for oral wound healing.

The epidermis is uniquely exposed to the effects of environmental factors, such as ultraviolet radiation, which induce progressive genetic and epigenetic modifications contributing to aging and the onset of keratinocyte carcinomas. DNA methylation is the best-characterized epigenetic modification and a valuable biomarker for assessing epidermal health. However, broad screening approaches have been hindered by the limited quantity and quality of the genomic DNA obtained from the epidermis and the resulting need for invasive sampling methods. Here we describe an integrated method that enables the non-invasive sampling of epidermal DNA for subsequent analysis by DNA methylation microarrays. This procedure combines a gel-based adhesive tape for keratinocyte collection, a robust gDNA extraction protocol and a curated selection of microarray probes optimized for low-input DNA conditions. Analysis of >100 corresponding methylomes demonstrates that our approach can be used for both the training of epigenetic clocks capable of predicting epidermal age with high accuracy, as well as the investigation of various DNA methylation-based biomarkers relevant to keratinocyte cancer development. These findings underscore the potential of our method for broad and non-invasive skin health assessment and cancer prevention strategies.