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PubMed2006

In vivo biocombinatorial synthesis of lipopeptides by COM domain-mediated reprogramming of the surfactin biosynthetic complex.

The intermolecular communication within NRPS complexes relies on the coordinated interplay of donor and acceptor communication-mediating (COM) domains. In this study, the potential of COM domains was exploited in vivo by establishing a system for the true biocombinatorial synthesis of lipopeptides via directed reprogramming of a natural NRP biosynthetic assembly line (i.e., surfactin). By means of COM domain swapping, these experiments verified the decisive role of COM domains for the control of protein-protein interactions between NRPSs, demonstrated the functionality of COM domain pairs even in the context of a heterologous host and NRPS system, and allowed for the intended skipping of a biosynthetic enzyme within a multienzymatic biosynthetic complex. Ultimately, abrogation of the selectivity barrier provided by COM domains afforded the successful simultaneous, biocombinatorial synthesis of distinct lipopeptide products.

Bacillus subtilis↗
PubMed2005

Reprogramming autologous skeletal myoblasts to express cardiomyogenic function. Challenges and possible approaches.

Cell transplantation therapy is emerging as a promising mode of treatment following myocardial infarction. Of the various cell types that can potentially be used for transplantation, autologous skeletal myoblasts appear particularly attractive, because this would avoid issues of immunogenicity, tumorigenesis, ethics and donor availability. Additionally, skeletal myoblasts display much higher levels of ischemic tolerance and graft survival compared to other cell types. There is some evidence for improvement in heart function with skeletal myoblast transplantation. However, histological analysis revealed that transplanted myoblasts do not transdifferentiate into functional cardiomyocytes in situ. This is evident by the lack of expression of cardiac-specific antigens, and the absence of intercalated disc formation. Instead, there is differentiation into myotubes that are not electromechanically coupled to neighboring cardiomyocytes. This could in turn limit the clinical efficacy of treatment. This review would therefore examine the various challenges faced in attempting to reprogram autologous skeletal myoblast to express cardiomyogenic function, together with the various possible strategies that could be employed to achieve this objective.

Cell Differentiation↗
PubMed2006

Reprogramming of committed T cell progenitors to macrophages and dendritic cells by C/EBP alpha and PU.1 transcription factors.

The differentiation potential of T lineage cells becomes restricted soon after entry of multipotent precursors into the thymus and is accompanied by a downregulation of the transcription factors C/EBP alpha and PU.1. To investigate this restriction point, we have expressed C/EBP alpha and PU.1 in fully committed pre-T cells and found that C/EBP alpha (and C/EBP beta) induced the formation of functional macrophages. In contrast, PU.1 converted them into myeloid dendritic cells under identical culture conditions. C/EBP alpha-induced reprogramming is complex because upregulation of some but not all myelomonocytic markers required endogenous PU.1. Notch signaling partially inhibited C/EBP alpha-induced macrophage formation and completely blocked PU.1-induced dendritic cell formation. Likewise, expression of intracellular Notch or the transcription factor GATA-3 inhibited C/EBP alpha-induced lineage conversion. Our data show that committed T cell progenitors remain susceptible to the lineage instructive effects of myeloid transcription factors and suggest that Notch signaling induces T lineage restriction by downregulating C/EBP alpha and PU.1 in multilineage precursors.

Animals↗
PubMed2026

In vivo CAR-T therapy: The shift from ex vivo culturing to direct in situ immune reprogramming.

CAR T-cell therapy using chimeric antigen receptors (CARs) has provided a radical shift in the treatment of several hematological malignancies, producing high response rates and durable remissions. However, conventional ex vivo manufacturing is limited by complex processing steps, high costs, variability in product quality, and clinically relevant delays that restrict patient eligibility. In vivo manufacturing has emerged as a next-generation approach in which immune cells are reprogrammed directly within the patient, eliminating the need for exogenous handling and culture. This strategy uses viral and non-viral delivery platforms, including lentiviral vectors, adeno-associated viruses, lipid nanoparticles, and targeted polymer systems, together with DNA, mRNA, and genome editing tools such as CRISPR-based technologies. Early feasibility data are supported mainly by preclinical models and translational studies, while safety remains a central concern due to potential immunotoxicity, off-target transduction, and regulatory challenges. This review highlights key engineering strategies enabling in vivo CAR T-cell generation, summarizes emerging clinical research and development, and discusses future opportunities for expanding in vivo CAR T-cell therapies as scalable immunotherapy platforms.

Humans↗
PubMed2026

Siglec-7 orchestrates mitochondrial dynamics and metabolic reprogramming to restrain human NK cell cytotoxic function.

Natural killer (NK) cells are innate lymphocytes that directly eliminate tumor and virus-infected cells by integrating signals from activating and inhibitory receptors, and their effector functions are tightly coupled to cellular metabolism. Given that the inhibitory receptor PD-1 reprograms T cell metabolism to shape functional fate, the bioenergetic consequences of inhibitory receptor engagement on human NK cells remain largely unexplored, particularly for sialic acid-binding immunoglobulin-like lectin (Siglec-7), a glyco-immune checkpoint receptor. Here, we investigated metabolic programs and effector functions associated with Siglec-7 expression and antibody-mediated Siglec-7 ligation in primary NK cells and NK-92MI cells. Siglec-7POS NK cells exhibited selectively impaired CD107a degranulation under glycolytic and oxidative phosphorylation inhibition, whereas Siglec-7NEG cells remained relatively resistant, indicating distinct energetic wiring between these subsets. Engagement of Siglec-7 by an agonistic antibody induced mitochondrial fission with altered Drp1 phosphorylation, transient mitochondrial depolarization, and broadly suppressed mitochondrial respiration, while concurrently enhancing glycolytic capacity, consistent with a dual metabolic shift upon Siglec-7 ligation. In contrast, sustained Siglec-7 expression in NK-92MI-S cells was associated with globally enhanced mitochondrial respiratory capacity, indicating that sustained Siglec-7 expression and short-term treatment with an agonistic anti-Siglec-7 antibody were associated with distinct metabolic profiles in NK cells. Furthermore, Siglec-7POS NK cells showed increased accumulation of autophagic vacuole, reduced proliferation, and heightened apoptotic susceptibility compared with Siglec-7NEG counterparts. Collectively, these findings support an association between Siglec-7 status, mitochondrial homeostasis, and metabolic fitness in NK cells, with Siglec-7NEG cells retaining a metabolically robust, cytotoxic phenotype.

Journal Article↗
PubMed2026

Reprogrammed Komagataella phaffii for enhanced secretory expression of human lactoferrin.

Human lactoferrin (hLF) is a multifunctional glycoprotein of the transferrin family derived from milk and mucosal secretions, which exhibits antibacterial, anti-tumor, and immunomodulatory functions, and is an important component of infant formula. Conventional methods for lactoferrin expression are often inefficient, primarily due to inadequate protein synthesis capabilities and poor stability within microbial hosts. Herein, a Komagataella phaffii yeast strain capable of high-level secretory expression of hLF was constructed by reprogramming the endoplasmic reticulum (ER) and vacuole using CRISPR/Cas9 technology. A dual-expression cassette containing the AOX1 promoter, an α-secretion signal peptide, the hLF gene, and a terminator was integrated into three different sites of the K. phaffii genome. The stepwise strategy combining expansion of the ER membrane involved in protein synthesis with knockout of vacuolar proteases further enhanced hLF production. Subsequently, 0.1 g/L FeCl₃ was added to the medium to reduce the toxicity of hLF and improve its stability. After high-density cultivation of K. phaffii through optimization of cultivation conditions in shake flasks and a 5 L bioreactor, the secretory intact hLF titer reached 2214 mg/L, representing a 76.3-fold increase achieved through these engineering strategies. In addition, antibacterial experiments demonstrated that this secretory hLF had a significant inhibitory effect on Escherichia coli, Staphylococcus aureus, and yeast. Overall, the developed K. phaffii protein expression platform enabled efficient production of lactoferrin, demonstrating its potential for expressing other lactoproteins.

Lactoferrin↗
PubMed2006

Competitive signal discrimination, methylation reprogramming and genomic imprinting.

Genomic imprinting (parent-of-origin-dependent gene regulation) is associated with intra-genomic evolutionary conflict over the optimal pattern of gene expression. Most theoretical models of imprinting focus on the conflict between the maternally and paternally derived alleles at an imprinted locus. Recently, however, more attention has been focused on multi-directional conflicts involving not only the imprinted gene itself, but also the genes that encode the regulatory machinery responsible for establishing and maintaining imprinted gene expression. In this paper, I examine the conflict involved in epigenetic reprogramming of imprinted genes in early mammalian embryonic development. In the earliest phase of development, maternal-store proteins are responsible for most regulatory activity in the embryo. These proteins are under selection to maximize the mother's inclusive fitness, which is not identical to that of either of the sets of genes present in the embryo. Both the maternally and paternally derived genomes in the embryo favor maintenance of the epigenetic modifications established in the female and male germlines, respectively. Maternal-store proteins favor maintenance of some of these modifications, but erasure of others. Here I consider the logical structure of the machinery responsible for these two activities. Methylation maintenance is most effectively performed by AND-linked architectures, which may explain the unusual trafficking behavior of the oocyte-specific DNA methyltransferase, Dnmt1o. By contrast, demethylation is better supported by OR-linked architectures, which may explain the difficulty in identifying the factor(s) responsible for the active demethylation of the paternal genome following fertilization.

Alleles↗
PubMed2026

Transmitochondrial pigs reveal causal effects of mitochondrial DNA on backfat thickness via nuclear epigenetic reprogramming.

Mitochondrial DNA (mtDNA) polymorphisms have been associated with production traits in farm animals, including backfat thickness in pigs, yet direct in vivo evidence establishing a causal link between specific mtDNA haplotypes and fat deposition remains limited. In this study, we generated transmitochondrial pigs (mitopigs) by combining the Dapulian nuclear genome with Wuzhishan mtDNA via somatic cell nuclear transfer, introducing 23 mtDNA mutations relative to controls. Mitopigs exhibited significantly increased backfat thickness at 5 months, a difference that persisted in their offspring, without significant differences in body weight, body size, or litter size. Fibroblasts derived from mitopigs exhibited reduced mtDNA copy numbers, decreased expression of mitochondrial biogenesis genes (PPARA, PPARGC1A, RRM2B, and LRPPRC), impaired mitochondrial respiration, elevated reactive oxygen species (ROS), and upregulated adipogenic transcription factors (CEBPA, CEBPB, and PPARG). Consistent with these fibroblast findings, backfat tissue of mitopigs showed corresponding upregulation of adipogenic transcription factors and downregulation of mitochondrial biogenesis genes. Integrated transcriptomic and whole-genome bisulfite sequencing (WGBS) analyses revealed nuclear transcriptional reprogramming that was closely associated with differential DNA methylation, predominantly affecting mitochondrial function and lipid metabolism pathways. Mitopig fibroblasts also showed a pro-inflammatory response to lipopolysaccharide stimulation, with elevated expression of IL-12, NOS2, RELA, and TNF-α. Our findings provide direct in vivo evidence that mtDNA variants regulate adiposity in pigs through mitochondrial dysfunction, oxidative stress, and nuclear epigenetic modulation, highlighting the potential for incorporating mtDNA haplotype information into pig breeding programs as a complementary strategy to nuclear genomic selection.

Adipogenesis↗
PubMed2026

Targeting microbial bile salt hydrolase reprograms bile acid metabolism and ameliorates metabolic dysfunction-associated steatohepatitis in mice.

Microbial bile salt hydrolase (BSH) plays a central role in shaping bile acid composition and gut-liver metabolic signaling, yet its therapeutic potential in metabolic dysfunction-associated steatohepatitis (MASH) remains incompletely defined. Here, we evaluated the efficacy of the non-absorbable BSH inhibitor GR-7 in a diet-induced mouse model of steatohepatitis using early and late intervention strategies with different dosing regimens. GR-7 reduced food intake and exerted stage- and dose-dependent therapeutic effects, with early intervention robustly suppressing hepatic fibrosis even at a low dose, whereas late-stage administration of high-dose GR-7 markedly reduced hepatic steatosis and inflammation, as evidenced by decreased liver weight, hepatic triglyceride and cholesterol levels, and plasma ALT. Although late intervention did not result in statistically significant histological reversal of fibrosis, a trend toward improvement was observed, together with suppression of fibrogenic gene expression, suggesting that prolonged treatment may further enhance antifibrotic efficacy. Mechanistically, GR-7 effectively inhibited microbial BSH activity in vivo, leading to reduced cecal unconjugated primary and secondary bile acids-including deoxycholic acid and lithocholic acid, which was associated with improved gut barrier integrity and reduced hepatic inflammation. In parallel, BSH inhibition reprogrammed hepatic bile acid metabolism toward activation of the alternative CYP27A1-mediated synthesis pathway, accompanied by reduced food intake, thereby contributing to reduced hepatic lipid accumulation. Furthermore, late-stage high-dose treatment selectively remodeled the hepatic immune landscape rather than fully restoring homeostasis, highlighting immune recalibration as a key component of therapeutic response. Together, these findings identify microbial BSH inhibition as a promising microbiome-targeted therapeutic strategy for MASH.

Animals↗
PubMed2026

Chronic nitric oxide mediates dual-layer gene regulation through mRNA m6A positional remodeling and parallel transcriptional reprogramming.

Nitric oxide (NO) is a pleiotropic free radical that functions as a master regulator of gene expression, and its sustained production within the tumor microenvironment reshapes the epitranscriptomic state of cancer cells. We previously demonstrated that NO inhibits the m6A mRNA demethylases FTO and ALKBH5 through dinitrosyliron complex formation while leaving the methyltransferase METTL3 intact, a demethylase-specific perturbation that increases global m6A on mRNA. Here, integrating m6A-RIP-seq and RNA-seq from triple-negative breast cancer cells, we show that chronic NO does not produce the uniform hypermethylation anticipated from demethylase inhibition. Instead, it redistributes m6A on mRNA, enriching the 5'UTR and coding sequence while depleting the 3'UTR and departing from the canonical stop-codon and 3'UTR topology. We found that the position of m6A, rather than its intensity or mere presence, shapes the outcome, in part by determining which reader protein is predicted to recognize it. In parallel, NO drives a canonical NF-κB and inflammatory transcriptional program. The transcriptional program is independent of the m6A methylome in both which genes respond and how strongly they respond, ruling out a linear methylome-to-transcriptome cascade; even so, m6A position remains associated with the direction of change among responding transcripts. The 3'UTR is the primary site of m6A loss and shows a suggestive computational link to miRNA-mediated regulation. Sense-antisense coordination reinforces the transcriptional response without bridging the two programs. These findings demonstrate that NO not only increases m6A abundance, but it also rewrites the m6A positional code, establishing spatial reprogramming of the epitranscriptome as a previously unrecognized mode of gene regulation.

RNA Methylation↗
PubMed2005

Cardiac intracrine renin angiotensin system. Part of genetic reprogramming?

The hypothesis that intracrine renin-angiotensin system activated during heart failure is part of the tendency of the heart to return to embryological conditions when organogenesis is possible is presented and discussed. The hypothesis proposes that the change in genetic makeup, which is known to occur during heart failure, includes a drastic change of intercellular chemical and electrical communication such as second messengers and other signal molecules which are involved in cell proliferation and growth. The role of angiotensin II, which is a growth factor, reduces cell coupling in the failing heart through the activation of AT1 receptors and intracellular pathways, such as PKC, MAPK family and increment of intracellular calcium, might play a key role in the genetic reprogramming of the failing heart.

Animals↗
PubMed2004

Stepwise reprogramming of B cells into macrophages.

Starting with multipotent progenitors, hematopoietic lineages are specified by lineage-restricted transcription factors. The transcription factors that determine the decision between lymphoid and myeloid cell fates, and the underlying mechanisms, remain largely unknown. Here, we report that enforced expression of C/EBPalpha and C/EBPbeta in differentiated B cells leads to their rapid and efficient reprogramming into macrophages. C/EBPs induce these changes by inhibiting the B cell commitment transcription factor Pax5, leading to the downregulation of its target CD19, and synergizing with endogenous PU.1, an ETS family factor, leading to the upregulation of its target Mac-1 and other myeloid markers. The two processes can be uncoupled, since, in PU.1-deficient pre-B cells, C/EBPs induce CD19 downregulation but not Mac-1 activation. Our observations indicate that C/EBPalpha and beta remodel the transcription network of B cells into that of macrophages through a series of parallel and sequential changes that require endogenous PU.1.

Animals↗
PubMed2000

Isolation of pluripotent embryonic stem cells from reprogrammed adult mouse somatic cell nuclei.

Pluripotent human stem cells isolated from early embryos represent a potentially unlimited source of many different cell types for cell-based gene and tissue therapies [1-3]. Nevertheless, if the full potential of cell lines derived from donor embryos is to be realised, the problem of donor-recipient tissue matching needs to be overcome. One approach, which avoids the problem of transplant rejection, would be to establish stem cell lines from the patient's own cells through therapeutic cloning [3,4]. Recent studies have shown that it is possible to transfer the nucleus from an adult somatic cell to an unfertilised oocyte that is devoid of maternal chromosomes, and achieve embryonic development under the control of the transferred nucleus [5-7]. Stem cells isolated from such a cloned embryo would be genetically identical to the patient and pose no risk of immune rejection. Here, we report the isolation of pluripotent murine stem cells from reprogrammed adult somatic cell nuclei. Embryos were generated by direct injection of mechanically isolated cumulus cell nuclei into mature oocytes. Embryonic stem (ES) cells isolated from cumulus-cell-derived blastocysts displayed the characteristic morphology and marker expression of conventional ES cells and underwent extensive differentiation into all three embryonic germ layers (endoderm, mesoderm and ectoderm) in tumours and in chimaeric foetuses and pups. The ES cells were also shown to differentiate readily into neurons and muscle in culture. This study shows that pluripotent stem cells can be derived from nuclei of terminally differentiated adult somatic cells and offers a model system for the development of therapies that rely on autologous, human pluripotent stem cells.

Animals↗
PubMed2001

Nuclear reprogramming of somatic cells by in vitro hybridization with ES cells.

The resetting of a somatic epigenotype to a totipotential state has been demonstrated by successful animal cloning, via transplantation of somatic nuclei into enucleated oocytes. We have established an experimental system, which reproduces the nuclear reprogramming of somatic cells in vitro by fusing adult thymocytes with embryonic stem (ES) cells. Analysis of the lymphoid-cell-specific V-(D)-J DNA rearrangement of the T cell receptor and immunoglobin genes shows that the ES cells have hybridized with differentiated cells. In these ES cell hybrids, the inactivated X chromosome derived from a female thymocyte adopts some characteristics of an active X chromosome, including early replication timing and unstable Xist transcription. We also found that an Oct4-GFP transgene, which is normally repressed in thymocytes, is reactivated 48 hr after cell fusion. The pluripotency of the ES-thymocyte hybrid cells is shown in vivo, since they contribute to all three primary germ layers of chimeric embryos. The somatic DNA methylation pattern of the imprinted H19 and Igf2r genes is maintained in these hybrids, unlike hybrids between ES and EG (embryonic germ) cells in which the differential methylation is erased. Thus, ES cells have the capacity to reset certain aspects of the epigenotype of somatic cells to those of ES cells.

Animals↗
PubMed2001

Delayed and incomplete reprogramming of chromosome methylation patterns in bovine cloned embryos.

Full-term development has now been achieved in several mammalian species by transfer of somatic nuclei into enucleated oocytes [1, 2]. Although a high proportion of such reconstructed embryos can evolve until the blastocyst stage, only a few percent develop into live offspring, which often exhibit developmental abnormalities [3, 4]. Regulatory epigenetic markers such as DNA methylation are imposed on embryonic cells as normal development proceeds, creating differentiated cell states. Cloned embryos require the erasure of their somatic epigenetic markers so as to regain a totipotent state [5]. Here we report on differences in the dynamics of chromosome methylation between cloned and normal bovine embryos before implantation. We show that cloned embryos fail to reproduce distinguishable parental-chromosome methylation patterns after fusion and maintain their somatic pattern during subsequent stages, mainly by a highly reduced efficiency of the passive demethylation process. Surprisingly, chromosomes appear constantly undermethylated on euchromatin in morulae and blastocysts, while centromeric heterochromatin remains more methylated than that of normal embryos. We propose that the abnormal time-dependent methylation events spanning the preimplantation development of clones may significantly interfere with the epigenetic reprogramming, contributing to the high incidence of physiological anomalies occurring later during pregnancy or after clone birth.

Animals↗
PubMed2004

Reprogrammed genetic decoding in cellular gene expression.

Reprogrammed genetic decoding signals in mRNAs productively overwrite the normal decoding rules of translation. These "recoding" signals are associated with sites of programmed ribosomal frameshifting, hopping, termination codon suppression, and the incorporation of the unusual amino acids selenocysteine and pyrrolysine. This review summarizes current knowledge of the structure and function of recoding signals in cellular genes, the biological importance of recoding in gene regulation, and ways to identify new recoded genes.

Animals↗
PubMed2006

Reprogramming of human somatic cells by embryonic stem cell cytoplast.

Somatic cell nuclear transfer (SCNT) provides the basis for the development of patient-specific stem cell lines. Recent progress in SCNT suggested the presence of reprogramming factors in human embryonic stem (hES) cells, although no method is currently available for replacement of nuclei of hES cells by somatic cell nuclei. An original technique has been developed, involving the fusion of different types of somatic cells with hES cells, which allowed a complete replacement of the nuclei of hES cells by nuclei of somatic cells. The resulting 'cybrids' were demonstrated to have the genotype of the donor somatic cells and 'stemness' of the recipient hES cells. However, the colonies isolated from the resulting fusion contained a mixture of these cybrid cells with the cells with the recipient nuclei, as well as hybrid cells containing both donor and recipient nuclei, so future purification will be necessary before the technique can be considered for future practical application.

Cell Fusion↗
PubMed2001

Rational reprogramming of the R2 subunit of Escherichia coli ribonucleotide reductase into a self-hydroxylating monooxygenase.

The outcome of O2 activation at the diiron(II) cluster in the R2 subunit of Escherichia coli (class I) ribonucleotide reductase has been rationally altered from the normal tyrosyl radical (Y122*) production to self-hydroxylation of a phenylalanine side-chain by two amino acid substitutions that leave intact the (histidine)2-(carboxylate)4 ligand set characteristic of the diiron-carboxylate family. Iron ligand Asp (D) 84 was replaced with Glu (E), the amino acid found in the cognate position of the structurally similar diiron-carboxylate protein, methane monooxygenase hydroxylase (MMOH). We previously showed that this substitution allows accumulation of a mu-1,2-peroxodiiron(III) intermediate, which does not accumulate in the wild-type (wt) protein and is probably a structural homologue of intermediate P (H(peroxo)) in O2 activation by MMOH. In addition, the near-surface residue Trp (W) 48 was replaced with Phe (F), blocking transfer of the "extra" electron that occurs in wt R2 during formation of the formally Fe(III)Fe(IV) cluster X. Decay of the mu-1,2-peroxodiiron(III) complex in R2-W48F/D84E gives an initial brown product, which contains very little Y122* and which converts very slowly (t1/2 approximately 7 h) upon incubation at 0 degrees C to an intensely purple final product. X-ray crystallographic analysis of the purple product indicates that F208 has undergone epsilon-hydroxylation and the resulting phenol has shifted significantly to become a ligand to Fe2 of the diiron cluster. Resonance Raman (RR) spectra of the purple product generated with 16O2 or 18O2 show appropriate isotopic sensitivity in bands assigned to O-phenyl and Fe-O-phenyl vibrational modes, confirming that the oxygen of the Fe(III)-phenolate species is derived from O2. Chemical analysis, experiments involving interception of the hydroxylating intermediate with exogenous reductant, and Mössbauer and EXAFS characterization of the brown and purple species establish that F208 hydroxylation occurs during decay of the peroxo complex and formation of the initial brown product. The slow transition to the purple Fe(III)-phenolate species is ascribed to a ligand rearrangement in which mu-O2- is lost and the F208-derived phenolate coordinates. The reprogramming to F208 monooxygenase requires both amino acid substitutions, as very little epsilon-hydroxyphenylalanine is formed and pathways leading to Y122* formation predominate in both R2-D84E and R2-W48F.

Amino Acid Substitution↗