We previously reported the Marimo cell line, which was established from the bone marrow cells of a patient with essential thrombocythemia (ET) at the last stage after transformation to acute myeloid leukemia (AML). This cell line is widely used for the biological analysis of ET because it harbors CALR mutation. However, genetic processes during disease progression in the original patient were not analyzed. We sequentially analyzed the genetic status in the original patient samples during disease progression. The ET clone had already acquired CALR and MPL mutations, and TP53 and NRAS mutations affected the disease progression from ET to AML in this patient. Particularly, the variant allele frequency of the NRAS mutation increased along with the disease progression after transformation, and the NRAS-mutated clone selectively proliferated in vitro, resulting in the establishment of the Marimo cell line. Although CALR and MPL mutations co-existed, MPL was not expressed in Marimo cells or any clinical samples. Furthermore, mitogen-activated protein kinase (MAPK) but not the JAK2-STAT pathway was activated. These results collectively indicate that MAPK activation is mainly associated with the proliferation ability of Marimo cells.

Serum laminin-γ2 monomer (Lm-γ2m) is a potent predictive biomarker for hepatocellular carcinoma (HCC) onset in patients with hepatitis C infection who achieve a sustained virologic response with liver cirrhosis (LC) and for the onset of extrahepatic metastases in early-stage HCC. Although Lm-γ2m involvement in late-stage cancer progression has been well investigated, its precise roles in HCC onset remain to be systematically investigated. Therefore, we analyzed an HCC model, human hepatocytes and cholangiocytes, and surgically resected liver tissues from patients with HCC to understand the roles of Lm-γ2m in HCC onset. Ck-19- and EpCAM-positive hepatic progenitor cells (HPCs) in the liver of pdgf-c transgenic HCC mouse model with ductular reaction showed ectopic expression of Lm-γ2m. Forced expression of Lm-γ2m in hepatocytes adjacent to HPCs resulted in enhanced tumorigenicity, cell proliferation, and migration in immortalized hepatocytes, but not in cholangiocytes in vitro. Further, pharmacological inhibition of epidermal growth factor receptor (EGFR) and c-Jun activator JNK suppressed Lm-γ2m-induced hepatocyte transformation, suggesting the involvement of EGFR/c-Jun signaling in the transformation, leading to HCC development. Finally, immunohistochemical staining of HCC tissues revealed a high level of Lm-γ2 expression in the HPCs of the liver with ductular reaction in normal liver adjacent to HCC tissues. Overall, HPC-derived Lm-γ2m in normal liver with ductular reaction acts as a paracrine growth factor on surrounding hepatocytes and promotes their cellular transformation through the EGFR/c-Jun signaling pathway. Furthermore, this is the first report on Lm-γ2m expression detected in the normal liver with ductular reaction, a human precancerous lesion of HCC.

Visible light-driven intimately coupled photocatalysis and biodegradation (VDICPB) is an efficient technology for removing recalcitrant contaminants, but the degradation pathway on 17β-estradiol 3-Sulfate (E2-3S) is still not clear. In this study, VDICPB based on N-doped TiO2 as a photocatalyst was established to investigate the removal and transformation of E2-3S in synthetic wastewater. VDICPB showed a satisfactory removal efficiency of 97.8 ± 0.4 %, which was much higher than that of independent photocatalysis (84.0 ± 2.2 %) or biodegradation system (71.4 ± 1.8 %). Steroid C/D-rings of E2-3S was broken in VDICPB since the transformation process reached terminal central pathway. Primary metabolites did not accumulate in VDICPB, resulting in a low expression of functional genes. E2-3S was mainly removed by cooperative interaction of photocatalysis and co-metabolism of biofilm. Photocatalysis led to deconjugation and microbes acted to mineralization. This study provides technical reference and theoretical support for the removal of new pollutants.

The bacterial world offers diverse strains for understanding medical and environmental processes and for engineering synthetic biological chassis. However, genetically manipulating these strains has faced a long-standing bottleneck: how to efficiently transform DNA. Here, we report imitating methylation patterns rapidly in TXTL (IMPRINT), a generalized, rapid, and scalable approach based on cell-free transcription-translation (TXTL) to overcome DNA restriction, a prominent barrier to transformation. IMPRINT utilizes TXTL to express DNA methyltransferases from a bacterium\'s restriction-modification systems. The expressed methyltransferases then methylate DNA in vitro to match the bacterium\'s DNA methylation pattern, circumventing restriction and enhancing transformation. With IMPRINT, we efficiently multiplex methylation by diverse DNA methyltransferases and enhance plasmid transformation in gram-negative and gram-positive bacteria. We also develop a high-throughput pipeline that identifies the most consequential methyltransferases, and we apply IMPRINT to screen a ribosome-binding site library in a hard-to-transform Bifidobacterium. Overall, IMPRINT can enhance DNA transformation, enabling the use of sophisticated genetic manipulation tools across the bacterial world.

Aeromonas trota is sensitive to most antibiotics and the sole species of this genus susceptible to ampicillin. This susceptibility profile could be related to its inability to acquire exogenous DNA. In this study, A. trota isolates were analyzed to establish their capacity to incorporate foreign DNA. Fourteen strains were identified as A. trota by multilocus phylogenetic analysis (MLPA). Minimal inhibitory concentrations of antibiotics (MIC) were assessed, confirming the susceptibility to most antibiotics tested. To explore their capacity to be transformed, A. trota strains were used as recipients in different horizontal transfer assays. Results showed that around fifty percent of A. trota strains were able to incorporate pBAMD1-2 and pBBR1MCS-3 plasmids after conjugal transfer. In all instances, conjugation frequencies were very low. Interestingly, several isoforms of plasmid pBBR1MCS-3 were observed in transconjugants. Strains could not receive pAr-32, a native plasmid from A. salmonicida. A. trota strains were unable to receive DNA by means of electroporation, natural transformation or vesiduction. These results confirm that A. trota species are extremely refractory to horizontal gene transfer, which could be associated to plasmid instability resulting from oligomerization or to the presence of defense systems against exogenous genetic material in their genomes. To explain the poor results of horizontal gene transfer (HGT), selected genomes were sequenced and analyzed, revealing the presence of defense systems, which could prevent the stable incorporation of exogenous DNA in A. trota.

Maize (Zea mays L.) is an important crop that has been widely studied for its agronomic and industrial applications and is one of the main classical model organisms for genetic research. Agrobacterium-mediated transformation of immature maize embryos is a commonly used method to introduce transgenes, but a low transformation frequency remains a bottleneck for many gene-editing applications. Previous approaches to enhance transformation included the improvement of tissue culture media and the use of morphogenic regulators such as BABY BOOM and WUSCHEL2. Here, we show that the frequency can be increased using a pVS1-VIR2 virulence helper plasmid to improve T-DNA delivery, and/or expressing a fusion protein between a GROWTH-REGULATING FACTOR (GRF) and GRF-INTERACTING FACTOR (GIF) protein to improve regeneration. Using hygromycin as a selection agent to avoid escapes, the transformation frequency in the maize inbred line B104 significantly improved from 2.3 to 8.1% when using the pVS1-VIR2 helper vector with no effect on event quality regarding T-DNA copy number. Combined with a novel fusion protein between ZmGRF1 and ZmGIF1, transformation frequencies further improved another 3.5- to 6.5-fold with no obvious impact on plant growth, while simultaneously allowing efficient CRISPR-/Cas9-mediated gene editing. Our results demonstrate how a GRF-GIF chimera in conjunction with a ternary vector system has the potential to further improve the efficiency of gene-editing applications and molecular biology studies in maize.

Mesenchymal stem cell (MSCs) therapy, as a rapidly developing area of medicine, holds great promise for the treatment of a variety of medical conditions. MSCs are multipotent stem cells that can be isolated from various tissues and could self-renew and differentiate. They secrete cytokines and trophic factors that create a regenerative microenvironment and have immunomodulatory properties. Although clinical trials have been conducted with MSCs in various diseases, concerns regarding the possibility of malignant transformation of these cells have been raised. The studies showed a higher rate of hematological malignancy and carcinogenesis in experimental models after MSC transplantation. The mechanisms underlying malignant transformation of MSCs are complex and not fully understood, but they are believed to involve the presence of special signaling molecules and alterations in cell behavior regulation pathways. Possible pathways that lead to MSCs\' oncogenic transformation occur through two mechanisms: spontaneous and stimulated malignant transformation, including cell fusion, fusion proteins, and the tumor microenvironment. MSC-based therapies have the potential to revolutionize medicine, and addressing the issue of malignancy is crucial to ensure their safety and efficacy. Therefore, the purpose of the present review is to summarize the potential mechanisms of the malignant transformation of MSCs. [Figure: see text].

Genotype-to-phenotype mapping is an essential problem in the current genomic era. While qualitative case-control predictions have received significant attention, less emphasis has been placed on predicting quantitative phenotypes. This emerging field holds great promise in revealing intricate connections between microbial communities and host health. However, the presence of heterogeneity in microbiome datasets poses a substantial challenge to the accuracy of predictions and undermines the reproducibility of models. To tackle this challenge, we investigated 22 normalization methods that aimed at removing heterogeneity across multiple datasets, conducted a comprehensive review of them, and evaluated their effectiveness in predicting quantitative phenotypes in three simulation scenarios and 31 real datasets. The results indicate that none of these methods demonstrate significant superiority in predicting quantitative phenotypes or attain a noteworthy reduction in Root Mean Squared Error (RMSE) of the predictions. Given the frequent occurrence of batch effects and the satisfactory performance of batch correction methods in predicting datasets affected by these effects, we strongly recommend utilizing batch correction methods as the initial step in predicting quantitative phenotypes. In summary, the performance of normalization methods in predicting metagenomic data remains a dynamic and ongoing research area. Our study contributes to this field by undertaking a comprehensive evaluation of diverse methods and offering valuable insights into their effectiveness in predicting quantitative phenotypes.

Nano-scale particles (NPs) have gained increased interest as non-viral vectors for nucleic acid delivery due to their ability to penetrate through unabraded cell membranes. The previous studies performed have evaluated the nanomaterials for their microbial transformation proficiency but have not compared the relative efficacy. The present study aims to identify the most proficient nano-delivery vehicle among the chemically synthesized/functionalized non-metal oxide, metal/metal oxide, and carbon-based (carbon nanotube (CNT), graphene oxide (GO)) nanomaterial(s) (NMs) for the transformation of two gram-negative bacteria, i.e., Escherichia coli and Agrobacterium tumefaciens. The microscopy and spectroscopy studies helped to identify the interaction, adhesion patterns, transformation efficiencies, better delivery, and expression of the target gfp gene by use of NMs. Loading of pgfp on all NMs imparted protection to DNAse I attack except ZnO NPs with maximum by chitosan, layered double hydroxide (LDH), and GO NM-plasmid DNA conjugates. The CNTs and GO significantly enhanced the extra- and intra-cellular protein content, respectively, in both bacteria. However, GO and CNT significantly decreased the cell viability in a time-dependent manner while AuNPs exhibited negligible cell toxicity. Therefore, this study identified the comparative efficiency of metal/metal oxide, non-metal oxide, and carbon nanomaterials with AuNPs as the most biosafe while LDH and chitosan NPs being the most proficient alternative tools for the genetic transformation of gram-negative bacteria by simple incubation method.

Little is known about the effect of surface coatings on the fate and toxicity of CeO2 nanoparticles (NPs) to aquatic plants. In this study, we modified nCeO2 with chitosan (Cs) and alginate (Al) to obtain positively charged nCeO2@Cs and negatively charged nCeO2@Al, respectively, and exposed them to a representative aquatic plant, duckweed (Lemna minor L.). Uncoated nCeO2 could significantly inhibit the growth of duckweed, induce oxidative damage and lead to cell death, whereas nCeO2@Cs and nCeO2@Al exhibited lower toxicity to duckweed. ICP-MS analysis revealed that the Ce content in duckweed from the nCeO2 group was 1.74 and 2.85 times higher than that in the nCeO2@Cs and nCeO2@Al groups, respectively. Microscopic observations indicated that the positively charged nCeO2@Cs was more readily adsorbed on the root surface of duckweed than the negatively charged nCeO2@Al. The results of XANES and LCF demonstrated that a certain percentage of Ce(Ⅳ) was reduced to Ce(Ⅲ) after the interaction of the three NPs with duckweed, but the degree of biotransformation differed among the treatments. Specifically, the absolute contents of Ce(III) produced of nCeO2@Cs and nCeO2@Al through biotransformation were reduced by 55.5% and 83.5%, respectively, compared with that of the nCeO2 group, which might be the key factor for the diminished phytotoxicity of the coated nCeO2 to the duckweed. These findings were valuable for understanding the toxicity of metal-based NPs to aquatic plants and for the synthesis of environmentally friendly nanomaterials.