Search Results (100)

In acute myeloid leukemia (AML) it is important to elucidate the biological events that lead from remission to relapse, which have a high probability of leading to an adverse disease outcome. The cancer stem cell marker aldehyde dehydrogenase 1 (ALDH1A1) is underexpressed in AML cells when compared to healthy cells, both at the RNA level and at the protein level, and at least in the former, both in the bone marrow and in peripheral blood. Nonetheless, ALDH1A1/ALDH1A2 activity increases in AML cells during disease relapse and is higher in adverse prognosis AML in comparison with favorable prognosis AML. Furthermore, especially in relapsed AML and in unfavorable AML, AML cells rich in ALDH1A1 can contain high levels of reactive oxygen species (ROS), in parallel with high ALDH1A1/2 activity. This metabolic feature is clearly incompatible with normal stem cells. The term “stem-like” therefore is useful to coin malignant cells with a variety of genetic makeups, metabolic programming and biomarkers that converge in the function of survival of clones sufficient to sustain, spread and re-establish neoplastic disease. Therefore, AML “stem-like” cells survive cancer treatment that eradicates other malignant cell clones. This fact differentiates AML “stem-like” cells from normal stem and progenitor cells that function in tissue regeneration as part of a distinct hierarchical order of cell phenotypes. The ODYSSEY clinical trial is a Phase I/II study designed to evaluate the safety, tolerability, pharmacokinetics, and pharmacodynamics of ABD-3001, a novel therapeutic agent, in patients with AML who have relapsed or are refractory to standard treatments. In this context, ABD-3001 is used as an inhibitor of cytosolic ALDH1 enzymes, such as ALDH1A1 and ALDH1A2. Full article

CK2α is a kinase important for essential cellular and biological processes. CK2α is ubiquitously expressed, albeit at different tissue levels, and its transcript levels are dysregulated in disease. However, there is limited knowledge on the regulation of CK2α gene expression. The best one studied, the human CSNK2A1 (CK2α) gene promoter, contains uncharacterized binding motifs for NF-κB. Our goal was to investigate the role of NF-κB in Csnk2a1 promoter regulation. We cloned the mouse Csnk2a1 promoter which had significant sequence homology with the human CSNK2A1 promoter. Using promoter deletions, we identified a minimal promoter region containing transcription factor motifs (NF-κB, Ets-1, Sp1) consistent with those published for the CSNK2A1 promoter. Electrophoretic mobility shift assays demonstrated specific NF-κB subunit binding to the minimal promoter. NF-κB subunit transfection and extracellular NF-κB stimulation in non-tumor cell lines led to increased transactivation of the mouse minimal promoter. These data, together with data on the regulation of NF-κB by CK2 kinase activity, suggest a positive-feedback loop between CK2α and NF-κB. Non-tumor cell line re-plating and increased percent confluence upregulated Csnk2a1 transcript levels which differed from tumor cell line published data. In summary, Csnk2a1 promoter is regulated by NF-κB signaling and during cellular proliferation. Full article

Objective: We developed a targeted integration-based CHO cell platform for simultaneous antibody display and secretion, enabling a streamlined transition from antibody library screening to production without requiring the re-cloning of antibody genes. Methods: The platform consists of a CHO master cell line with a single-copy landing pad, a helper vector expressing FLPe recombinase, and bi-functional targeting vectors. Recombinase-mediated cassette exchange was utilized to integrate targeting vectors into the landing pad. Bi-functional vectors were designed by incorporating a minimal furin cleavage sequence (mFCS), RRKR, and various 2A peptides between the heavy chain (HC) and a membrane anchor. Results: Incomplete cleavage at the mFCS and 2A sites facilitated the expression of both membrane-bound and secreted antibodies, while mutations in the 2A peptide produced a range of display-to-secretion ratios. However, a fraction of secreted antibodies retained 2A residues attached to the HC polypeptides. Further analysis demonstrated that modifying the first five amino acids of the 2A peptide significantly influenced furin cleavage efficiency, resulting in different display-to-secretion ratios for targeting vectors containing mFCS-2A variant combinations. To overcome this, we designed nine-amino-acid FCS variants that, when placed between the HC and membrane anchor, provided a range of display-to-secretion ratios and eliminated the issue of attached 2A residues in the secreted antibodies. Vectors with lower display levels proved more effective at distinguishing cells expressing high-affinity antibodies with closely matched binding affinities. The platform also demonstrated high sensitivity in isolating high-affinity antibody-expressing cells and supported robust antibody production. Conclusion: This targeted integration-based CHO platform enables efficient, in-format screening and production of antibodies with tunable display-to-secretion profiles. It provides a powerful and scalable tool for accelerating the development of functional, manufacturable therapeutic antibodies. Full article

Background/Objectives: The interleukin 17 (IL-17) family, known for its proinflammatory properties, is important in immune responses against bacterial and fungal infections. To exert its immune function, the IL-17 family typically binds to IL-17 receptor (IL-17R) to facilitate signal transduction. Methods: This study identified, cloned and analyzed seven IL-17 and nine IL-17R family members in snakeheads. Results: A duplication event occurred in snakehead IL-17s and IL-17Rs, but bioinformatics analyses indicated that these genes were conserved in both protein domains and evolutionary processes. Tissue distribution analysis revealed that IL-17s/IL-17Rs were widely distributed in the detected tissues, with relatively high expression levels in immune tissues. Upon Nocardia seriolae stimulation, most members were expressed, particularly IL-17C2, IL-17D, IL-17N, IL-17RA1, IL-17RA2, IL-17RC1, and IL-17RE1, which were significantly upregulated in gill and intestine. Conclusions: These results suggested that IL-17s and IL-17Rs played a crucial role in mucosal immunity against bacterial infection, providing insights into immunoprophylactic strategies for bacterial diseases in aquaculture. Full article

Chikungunya virus (CHIKV) is a mosquito-transmitted alphavirus that, since its re-emergence in 2004, has become recognised as a major public health concern throughout many tropical and sub-tropical regions of the world. Amongst the insights gained from studies on other alphaviruses, several key determinants of virulence have been identified, including one present at the P3 position in the nsP1/nsP2 cleavage domain of the S.A.AR86 Sindbis (SINV) strain. This strain is associated with neurovirulence in adult mice; however, when a threonine-to-isoleucine substitution is engineered at this P3 position, an attenuated phenotype results. A reverse genetics system was developed to evaluate the phenotype that resulted from the substitution of alanine, present at the P3 position in the wild-type CHIKV clone, with valine. The A533V-mutant CHIKV induced milder disease symptoms in the C57BL/6 mouse model than the wild-type virus, in terms of severity of inflammation, length of viraemic period, and histological changes. Furthermore, the induction of type I IFN occurred more rapidly in both CHIKV-infected cell cultures and the mouse model with the mutant CHIKV. Full article

Microcystis aeruginosa is an important species causing cyanobacterial blooms, which can be effectively infected and lysed by cyanophages. Several strategies have been developed by M. aeruginosa to resist cyanophage infections, including the CRISPR-Cas systems. However, detailed information on the CRISPR-Cas systems in M. aeruginosa is rare. In the present study, the CRISPR-Cas systems of M. aeruginosa FACHB-524 were analyzed by genome re-sequencing, which showed that there are two type I (Cluster 1, I-B1; Cluster 2, I-D) and three type III-B (Cluster 3/4/5) CRISPR-Cas systems in the cyanobacteria. Further comparison revealed that spacer sequences of two type III-B systems targeted several genes of the cyanophage MaMV (M. aeruginosa myovirus) strains. One of the type III systems (Cluster 4) was then cloned and expressed in Escherichia coli BL21 (DE3). Protein purification and mass spectrometry identification revealed that a Cmr-crRNA effector complex formed in the E. coli. Subsequently, T4 phage (T4) was used to infect the E. coli, expressing the Cmr-crRNA complex with or without accessory proteins. The results showed that the Cmr-crRNA effector complex exhibited anti-phage activity and the accessory protein Csx1 enhanced the immune activity of the complex. Collectively, our results comprehensively demonstrate the CRISPR systems encoded by a strain of M. aeruginosa, and for the first time, one of the CRISPR systems was constructed into E. coli, providing a foundation for further in-depth analysis of cyanobacterial CRISPR systems. Full article

Background: The passion fruit (Passiflora edulis Sims) is a diploid plant (2n = 2x = 18) and is a perennial scrambling vine in Southern China. However, the occurrence and spread of stem rot in passion fruit severely impact its yield and quality. Methods: In this study, we re-sequenced a BC₁F₁ population consisting of 158 individuals using whole-genome resequencing. We constructed a high-density genetic linkage map and identified the quantitative trait locus (QTL), and analyzed candidate genes associated with stem rot resistance in passion fruit. Results: Based on the passion fruit reference genome (MER), a high-density genetic linkage map was constructed with 1,180,406 single nucleotide polymorphisms (SNPs). The map contains nine linkage groups, covering a total genetic distance of 1559.03 cM, with an average genetic distance of 311.81 cM. The average genetic distance between 4206 bins was 0.404 cM, and the average gap length was 10.565 cM. The collinearity correlation coefficient between the genetic map and the passion fruit genome was 0.9994. Fusarium solani was used to infect the BC₁F₁ population, and the resistance to stem rot showed a continuous distribution. A QTL, qPSR5, was mapped to the 113,377,860 bp–114,811,870 bp genomic region on chromosome 5. We performed RNA sequencing (RNA-seq) and real-time quantitative polymerase chain reaction (RT-qPCR) to analyze the expression levels of predicted genes in the candidate region and identified ZX.05G0020740 and ZX.05G0020810 as ideal candidate genes for stem rot resistance in passion fruit. Conclusions: The findings in this study not only lay the foundation for cloning the qPSR5 responsible for stem rot resistance but also provide genetic resources for the genetic improvement of passion fruit. Full article

Background: Whole genome sequencing of clinical bacterial isolates holds promise in predicting their susceptibility to antibiotic therapy, based on a detailed understanding of the phenotypic manifestation of genotypic variation. The aac(6′) aminoglycoside acetyltransferase gene family is the most abundant aminoglycoside resistance determinant encountered in clinical practice. A variety of AAC(6′) isozymes have been described, suggesting a phenotypic distinction between subtype I, conferring resistance to amikacin (AMK), and subtype II, conferring resistance to gentamicin (GEN) instead. However, the epidemiology and thus clinical relevance of the various and diverse isozymes and their phenotypic distinction demand systematic and contemporary re-assessment to reliably predict bacterial susceptibility to aminoglycoside antibiotics. Methods: We analyzed the resistance gene annotations of 657,603 clinical bacterial isolates to assess the prevalence and diversity of aac(6′) genes. Seventeen unique aac(6′) amino acid sequences were cloned and expressed under defined promoter control in otherwise isogenic E. coli cells for phenotypic analysis with twenty distinct aminoglycoside antibiotics. A panel of clinical isolates was analyzed for the genotype–phenotype correlation of aac(6′). Results: An aac(6′) resistance gene annotation was found in 139,236 (21.2%) of the clinical isolates analyzed. AMK resistance-conferring aac(6′)-I genes dominated in Enterobacterales (28.5%). In Pseudomonas aeruginosa and Acinetobacter baumannii, a gene conferring the aac(6′)-II phenotype but annotated as aac(6′)-Ib₄ was the most prevalent. None of the aac(6′) genes were annotated as subtype III, but gene aac(6′)-Ii identified in Gram-positive isolates displayed a subtype III phenotype. Genes that were annotated as aac(6′)-Ib₁₁ in Enterobacterales conferred resistance to both AMK and GEN, which we propose constitutes a novel subtype IV when applying established nomenclature. A phenotypic assessment facilitated structural re-assessment of the substrate promiscuity of AAC(6′) enzymes. Conclusions: Our study provides the most comprehensive analysis of clinically relevant aac(6′) gene sequence variations to date, providing new insights into a differentiated substrate promiscuity across the genotypic spectrum of this gene family, thus translating into a critical contribution towards the development of amino acid sequence-based in silico antimicrobial susceptibility testing (AST). Full article

Myogenic regulator factors (MRFs) are essential for skeletal muscle development in vertebrates, including fish. This study aimed to characterize the role of myogenic regulatory factor 4 (MRF4) in muscle development in Nile tilapia by cloning NT-MRF4 from muscle tissues. To explore the function of NT-MRF4, CRISPR/Cas9 gene editing was employed. The NT-MRF4 cDNA was 1146 bp long and had encoded 225 amino acids, featuring a myogenic basic domain, a helix-loop-helix domain, and a nuclear localization signal. NT-MRF4 mRNA was exclusively expressed in adult muscle tissues, with expression also observed during embryonic and larval stages. Food-deprived Nile tilapia exhibited significantly lower NT-MRF4 mRNA levels than the controls while re-feeding markedly increased expression. The CRISPR/Cas9 gene editing of NT-MRF4 successfully generated two types of gene disruption, leading to a frame-shift mutation in the NT-MRF4 protein. Expression analysis of MRF and MEF2 genes in gene-edited (GE) Nile tilapia revealed that MyoG expressions nearly doubled compared to wild-type (WT) fish, suggesting that MyoG compensates for the loss of MRF4 function. Additionally, MEF2b, MEF2d, and MEF2a expressions significantly increased in GE Nile tilapia, supporting continued muscle development. Overall, these findings suggest that NT-MRF4 regulates muscle development, while MyoG may compensate for its inactivation to sustain normal muscle growth. Full article

Some Corynebacterium strains produce toxins that are similar to those produced by Corynebacterium diphtheriae, leading to human infections that are often transmitted through zoonotic diseases. A novel species, which is formerly classified as Corynebacterium ulcerans lineage II, was recently re-evaluated and renamed “Corynebacterium ramonii sp. nov.”. We isolated C. ramonii from a human skin ulcer in Japan in 2023 (KCU0303-001) and identified it as ST344 using a genomic analysis. In addition, C. ramonii KPHES-18084 (ST344) and six strains of C. ulcerans (ST337/ST1011) were isolated from the oral cavities of 7/208 rescued cats (3.4%). The human ulcer strain KCU0303-001 and the rescued cat strain KPHES-18084 were found to be ST344 and closely related clones by core-genome and pan-genome analyses, suggesting that ST344 may be endemic to both clinical and companion animals in Japan. In support of this finding, another clinical isolate of ST344 (TSU-28 strain) was reported in Japan in 2019. Although ST337 is the most common C. ulcerans infection, the second most recent clinical isolate of C. ramonii, ST344, might be increasing; therefore, further genomic surveillance is required to monitor C. ramonii and C. ulcerans infections. Full article

Thiosemicarbazones and their metal complexes have been studied for their biological activities against bacteria, cancer cells and protozoa. Short-term in vitro treatment with one gold (III) complex (C3) and its salicyl-thiosemicarbazone ligand (C4) selectively inhibited proliferation of T. gondii. Transmission Electron Microscopy (TEM) detected transient structural alterations in the parasitophorous vacuole membrane and the tachyzoite cytoplasm, but the mitochondrial membrane potential appeared unaffected by these compounds. Proteins potentially interacting with C3 and C4 were identified using differential affinity chromatography coupled with mass spectrometry (DAC-MS). Moreover, long-term in vitro treatment was performed to investigate parasitostatic or parasiticidal activity of the compounds. DAC-MS identified 50 ribosomal proteins binding both compounds, and continuous drug treatments for up to 6 days caused the loss of efficacy. Parasite tolerance to both compounds was, however, rapidly lost in their absence and regained shortly after re-exposure. Proteome analyses of six T. gondii ME49 clones adapted to C3 and C4 compared to the non-adapted wildtype revealed overexpression of ribosomal proteins, of two transmembrane proteins involved in exocytosis and of an alpha/beta hydrolase fold domain-containing protein. Results suggest that C3 and C4 may interfere with protein biosynthesis and that adaptation may be associated with the upregulated expression of tachyzoite transmembrane proteins and transporters, suggesting that the in vitro drug tolerance in T. gondii might be due to reversible, non-drug specific stress-responses mediated by phenotypic plasticity. Full article

Castanopsis hystrix, a precious tree species in Southeast Asia, has the advantages of rapid growth and high-quality wood materials. However, there are problems such as its long breeding cycle and low efficiency, and being time-consuming, which greatly restricts the industrial development of C. hystrix. Performing genome selection (GS) for growth and wood traits for the early selection of superior progeny has great significance for the rapid breeding of new superior varieties of C. hystrix. We used 226 clones in the main distribution and 479 progenies within 23 half-sib families as experimental materials in this study. Genotyping datasets were obtained by high-throughput re-sequencing technology, and GS studies were conducted on the growth (tree height (H), diameter at breast height (DBH)) and wood (wood density (WD), fiber length (FL), and fiber length–width ratio (LWR)) traits. The coefficient of variation (CV) of five phenotypic traits ranged from 10.1% to 22.73%, the average CV of growth traits was 19.93%, and the average CV of wood traits was 9.72%. The Pearson correlation coefficients between the five traits were almost all significantly positive. Based on the Genomic Best Linear Unbiased Prediction (GBLUP) model, the broad-sense heritabilities of growth traits were higher than those of wood quality traits, and the different number of SNPs had little effect on the heritability estimation. GS prediction accuracy first increased and then reached a plateau at around 3K SNPs for all five traits. The broad-sense heritability of these five traits was significantly positively correlated with their GS predictive ability (r = 0.564, p < 0.001). Bayes models had better GS prediction accuracy than the GBLUP model. The 15 excellent progeny individuals were selected, and their genetic gain ranged from 0.319% to 2.671%. These 15 superior offspring individuals were 4388, 4438, 4407, 4468, 4044, 4335, 4410, 4160, 4212, 4461, 4052, 4014, 4332, 4389, and 4007, mainly from three families F5, F6, and F11. Our research lays out the technical and material foundation for the rapid breeding of new superior varieties of C. hystrix in southern China. Full article

Fasciolosis, a globally re-emerging zoonotic disease, is mostly caused by the parasitic infection with Fasciola hepatica, often known as the liver fluke. This disease has a considerable impact on livestock productivity. This study aimed to evaluate the fluke burdens and faecal egg counts in goats that were administered phage clones of cathepsin L mimotopes and then infected with F. hepatica metacercariae. Additionally, the impact of vaccination on the histology of the reproductive system, specifically related to egg generation in adult parasites, was examined. A total of twenty-four goats, which were raised in sheds, were divided into four groups consisting of six animals each. These groups were randomly assigned. The goats were then subjected to two rounds of vaccination. Each vaccination involved the administration of 1 × 10¹³ phage particles containing specific mimotopes for cathepsin L2 (group 1: PPIRNGK), cathepsin L1 (group 2: DPWWLKQ), and cathepsin L1 (group 3: SGTFLFS). The immunisations were carried out on weeks 0 and 4, and the Quil A adjuvant was used in combination with the mimotopes. The control group was administered phosphate-buffered saline (PBS) (group 4). At week 6, all groups were orally infected with 200 metacercariae of F. hepatica. At week 22 following the initial immunisation, the subjects were euthanised, and adult F. hepatica specimens were retrieved from the bile ducts and liver tissue, and subsequently quantified. The specimens underwent whole-mount histology for the examination of the reproductive system, including the testis, ovary, vitellaria, Mehlis’ gland, and uterus. The mean fluke burdens following the challenge were seen to decrease by 50.4%, 62.2%, and 75.3% (p < 0.05) in goats that received vaccinations containing cathepsin L2 PPIRNGK, cathepsin L1 DPWWLKQ, and cathepsin L1 SGTFLFS, respectively. Animals that received vaccination exhibited a significant reduction in the production of parasite eggs. The levels of IgG1 and IgG2 isotypes in vaccinated goats were significantly higher than in the control group, indicating that protection is associated with the induction of a mixed Th1/Th2 immune response. The administration of cathepsin L to goats exhibits a modest level of efficacy in inducing histological impairment in the reproductive organs of liver flukes, resulting in a reduction in egg output. Full article

Ginseng (Panax ginseng C. A. Meyer) is an ancient and valuable Chinese herbal medicine, and ginsenoside, as the main active ingredient of ginseng, has received wide attention because of its various pharmacological active effects. Cytochrome P450 is the largest family of enzymes in plant metabolism and is involved in the biosynthesis of terpenoids, alkaloids, lipids, and other primary and secondary plant metabolites. It is significant to explore more PgCYP450 genes with unknown functions and reveal their roles in ginsenoside synthesis. In this study, based on the five PgCYP450 genes screened in the pre-laboratory, through the correlation analysis with the content of ginsenosides and the analysis of the interactions network of the key enzyme genes for ginsenoside synthesis, we screened out those highly correlated with ginsenosides, PgCYP309, as the target gene from among the five PgCYP450 genes. Methyl jasmonate-induced treatment of ginseng adventitious roots showed that the PgCYP309 gene responded to methyl jasmonate induction and was involved in the synthesis of ginsenosides. The PgCYP309 gene was cloned and the overexpression vector pBI121-PgCYP309 and the interference vector pART27-PgCYP309 were constructed. Transformation of ginseng adventitious roots by the Agrobacterium fermentum-mediated method and successful induction of transgenic ginseng hairy roots were achieved. The transformation rate of ginseng hairy roots with overexpression of the PgCYP309 gene was 22.7%, and the transformation rate of ginseng hairy roots with interference of the PgCYP309 gene was 40%. Analysis of ginseng saponin content and relative gene expression levels in positive ginseng hairy root asexual lines revealed a significant increase in PPD, PPT, and PPT-type monomeric saponins Re and Rg2. The relative expression levels of PgCYP309 and PgCYP716A53v2 genes were also significantly increased. PgCYP309 gene promotes the synthesis of ginsenosides, and it was preliminarily verified that PgCYP309 gene can promote the synthesis of dammarane-type ginsenosides. Full article

The first member of the arrestin family, visual arrestin-1, was discovered in the late 1970s. Later, the other three mammalian subtypes were identified and cloned. The first described function was regulation of G protein-coupled receptor (GPCR) signaling: arrestins bind active phosphorylated GPCRs, blocking their coupling to G proteins. It was later discovered that receptor-bound and free arrestins interact with numerous proteins, regulating GPCR trafficking and various signaling pathways, including those that determine cell fate. Arrestins have no enzymatic activity; they function by organizing multi-protein complexes and localizing their interaction partners to particular cellular compartments. Today we understand the molecular mechanism of arrestin interactions with GPCRs better than the mechanisms underlying other functions. However, even limited knowledge enabled the construction of signaling-biased arrestin mutants and extraction of biologically active monofunctional peptides from these multifunctional proteins. Manipulation of cellular signaling with arrestin-based tools has research and likely therapeutic potential: re-engineered proteins and their parts can produce effects that conventional small-molecule drugs cannot. Full article