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PubMed1975

Nitrofurazone-reducing enzymes in E. coli and their role in drug activation in vivo.

Earlier work showed that Escherichia coli contains at least two enzymes which reduce nitrofurazone and other nitrofuran derivatives. One of these enzymes is lacking in some nitrofurazone-resistant mutant strains. We now report that there are three separable nitrofuran reductases in this organism: reductase I (mol. wt. approximately 50 000, insensitive to O2), reductase IIa (mol. wt. approximately 120 000, inhibited by oxygen), reductase IIb (mol. wt. approximately 700 000, inhibited by O2). Unstable metabolites formed during the reduction of nitrofurazone by preparations containing reductases IIa and IIb produce breaks in DNA in vitro. In vivo experiments with nitrofurazone-resistant strains, which lack reductase II but contain reductases IIa and IIb, demonstrated that lethality, mutation, and DNA breakage are all greatly increased when cultures are incubated under anaerobic conditions, i.e., conditions such that reductase II is active. These results provide further evidence for the importance of reductive activation of nitrofurazone.

Aerobiosis↗

PubMed1975

Enzymatic hydrolysis of agar: purification and characterization of neoagarobiose hydrolase and p-nitrophenyl alpha-galactoside hydrolase.

The mixture of polysaccharides in the gelling component of agar (agarose) is hydrolyzed to D-galactose and 3,6-anhydro-L-galactose by a series of hydrolytic enzymes obtained from Pseudomonas atlantica. The final degradative step in the pathway of agarose decomposition is the hydrolysis of the alpha-linkage in the dissaccharide neoagarobiose yielding D-galactose and 3,6-anhydro-L-galactose. Pseudomonas atlantica when grown on agar produces two specific enzymes, p-nitrophenyl alpha-galactose hydrolase and neoagarobiose hydrolase. The purification and partial characterization of both enzymes are presented.

Agar↗

PubMed1975

Regulation and properties of an invertase from Clostridium pasteurianum.

An intracellular invertase was induced in cultures of Clostridium pasteurianum utilizing sucrose as its carbon source for growth. This enzyme synthesis could be repressed by the addition of fructose of a sucrose-growing culture. In contrast, invertase activity was not affected by the addition of glucose to sucrose-growing cells and this enzyme could be induced in a glucose-metabolizing culture by the addition of sucrose. This enzyme was purified 10.5-fold over the induced lese, EC 3.2.1.26) by substrate-specificity studies. Invertase had a pH optimum of 6.5 and an apparent Km of 79.5 mM for sucrose, and required high concentration of potassium phosphate for maximum activity. Invertase was completely inactivated by a 2-min heat treatment at 60 degrees C. This enzyme was strongly inhibited by p-hydroxymercuribenzoate (pCMB) and weakly inhibited by 5,5'-dithiobis(2-nitrobenzoic acid), while cysteine could substantially reverse pCMB) inhibition, suggesting that sulfhydryl group(s) were necessary for invertase activity.

Cell-Free System↗

PubMed2003

Complexes between the LKB1 tumor suppressor, STRAD alpha/beta and MO25 alpha/beta are upstream kinases in the AMP-activated protein kinase cascade.

BACKGROUND: The AMP-activated protein kinase (AMPK) cascade is a sensor of cellular energy charge that acts as a 'metabolic master switch' and inhibits cell proliferation. Activation requires phosphorylation of Thr172 of AMPK within the activation loop by upstream kinases (AMPKKs) that have not been identified. Recently, we identified three related protein kinases acting upstream of the yeast homolog of AMPK. Although they do not have obvious mammalian homologs, they are related to LKB1, a tumor suppressor that is mutated in the human Peutz-Jeghers cancer syndrome. We recently showed that LKB1 exists as a complex with two accessory subunits, STRAD alpha/beta and MO25 alpha/beta. RESULTS: We report the following observations. First, two AMPKK activities purified from rat liver contain LKB1, STRAD alpha and MO25 alpha, and can be immunoprecipitated using anti-LKB1 antibodies. Second, both endogenous and recombinant complexes of LKB1, STRAD alpha/beta and MO25 alpha/beta activate AMPK via phosphorylation of Thr172. Third, catalytically active LKB1, STRAD alpha or STRAD beta and MO25 alpha or MO25 beta are required for full activity. Fourth, the AMPK-activating drugs AICA riboside and phenformin do not activate AMPK in HeLa cells (which lack LKB1), but activation can be restored by stably expressing wild-type, but not catalytically inactive, LKB1. Fifth, AICA riboside and phenformin fail to activate AMPK in immortalized fibroblasts from LKB1-knockout mouse embryos. CONCLUSIONS: These results provide the first description of a physiological substrate for the LKB1 tumor suppressor and suggest that it functions as an upstream regulator of AMPK. Our findings indicate that the tumors in Peutz-Jeghers syndrome could result from deficient activation of AMPK as a consequence of LKB1 inactivation.

AMP-Activated Protein Kinase Kinases↗

PubMed1976

On the nature of the presumed receptor for IgE on mast cells. III. Kinetics of the blocking of the PCA reaction by cell-free particulate preparations from rat peritoneal mast cells and effect of pH and calcium concentration on the reaction.

The 'binding' of IgE to particulate preparations derived from sonicated purified rat mast cells was measured by the blocking of PCA titrations of the supernatant solutions from incubations with such preparations. It was found that the PCA blocking reaction was inhibited by the addition of calcium ion to the incubations. The blocking reaction was strongly dependent on the pH of the incubations, being maximal at pH values lower than 5-0. The blocking reaction proceeded in a linear manner for at least 3 h provided that no more than 70 percent of the amount of IgE initially supplied had been removed by the particulate fraction. Only mast cell-derived preparations were capable of effecting PCA blocking.

Animals↗

PubMed1976

Inhibition of Newcastle disease virus replication by 6-azauridine. I. Inefficacy of purified uridine kinase, effect of adenosine-5'-triphosphate.

As distinct from cell-free extracts prepared from tumour cells, partially purified uridine kinase prepared from the same cells was not effective in 6-azauridine (AzUrd) inhibition of Newcastle disease virus (NDV) replication. This showed that uridine kinase was not the effective component of cell-free extracts. Adenosine-5'-triphosphate (ATP) was found to exert a synergic effect in combination with AzUrd in the inhibition of NDV replication.

Adenosine Triphosphate↗

PubMed2026

A liquid handling platform for standardized quantification of cell-free enzymatic activity encoded by antimicrobial resistance genes.

Antimicrobial resistance (AMR) is a growing global threat to human health, and rapid methods for characterizing emerging antimicrobial resistance genes (ARGs) are needed. Here, we develop a semi-automated workflow using cell-free gene expression systems to measure the activity of two ARGs encoded on plasmid DNA that produce rifampicin-inactivating and gentamicin-inactivating enzymes. We validated the use of a small benchtop Myra liquid handling system compared to manual pipetting, with no statistical differences observed. After optimizing the pre-incubation time of ARGs and dispensing protocol, expression of aac(3)-IIa increased the half-maximal inhibition concentration (IC50) of gentamicin by over 150-fold, whilst arr-3 increased the IC50 of rifampicin by ~20-fold compared to controls. This methodology for rapid, semi-automated ARG characterization offers a strategy to combat AMR by assessing novel ARGs identified through genomic surveillance or profiling activity of new or derivative antibiotics.

aminoglycoside resistance↗

PubMed2025

Detecting known neoepitopes, gene fusions, transposable elements, and circular RNAs in cell-free RNA.

MOTIVATION: Cancer is the second leading cause of death worldwide, and although there have been advances in treatments, including immunotherapies, these often require biopsies which can be costly and invasive to obtain. Due to lack of pre-emptive cancer detection methods, many cases of cancer are detected at a late stage when the definitive symptoms appear. Plasma samples are relatively easy to obtain, and they can be used to monitor the molecular signatures of ongoing processes in the body. Profiling cell-free DNA is a popular method for monitoring cancer, but only a few studies have explored the use of cell-free RNA (cfRNA), which shows the recent footprint of systemic transcription. RESULTS: Here, we developed FastNeo, a computational method for detecting known neoepitopes in human cfRNA. We show that neoepitopes and other biomarkers detected in cfRNA can discern Hepatocellular carcinoma patients from the healthy patients with a sensitivity of 0.84 and a specificity of 0.79. For colorectal cancer we achieve a sensitivity of 0.87 and a specificity of 0.8. An important advantage of our cfRNA based approach is that it also reports putative neoepitopes which are important for therapeutic purposes. AVAILABILITY AND IMPLEMENTATION: The FastNeo package is available at https://github.com/yashumayank/FastNeo and https://zenodo.org/records/11521368. The benchmark pipelines to detect Immune Epitope database and Tumor-Specific Neoantigen database neoepitopes using HaplotypeCaller, bcftools, and Lofreq, and to run FastNeo with STAR instead of Bowtie2 are also available in the above github repository.

Humans↗

PubMed2026

Aging and Reproductive Cancers: An Integrative View on Cell-Free DNA and Transposable Elements.

Aging is one of the strongest risk factors for cancer, and its impact is particularly evident in malignancies of the reproductive system. Ovarian, endometrial, cervical, vulvar, prostate, and penile cancers are mainly diagnosed in older adults and often show different clinical and biological features compared with the same tumors in younger patients. Aging is associated with hormonal changes, immune decline, epigenetic alterations, and accumulation of DNA damage, all of which contribute to cancer development and progression. At the same time, many older patients have frailty and multiple comorbidities, which can limit the use of screening programs and invasive diagnostic procedures. This often leads to delayed diagnosis and worse outcomes. Cell-free DNA (cfDNA) is a minimally invasive biomarker that can be obtained from blood samples and provides molecular information on both tumor and host tissues. Circulating DNA reflects tumor-specific alterations but is also influenced by aging-related changes in DNA release, fragmentation, and methylation. For this reason, aging must be considered when cfDNA-based biomarkers are applied in clinical practice. In this review, we describe how aging influences the biology of reproductive system cancers and how these processes are mirrored in cfDNA profiles. We focus on the clinical use of cfDNA for cancer detection and monitoring in older and fragile patients. Special attention is given to repetitive elements in cfDNA, which are strongly affected by aging and tumor-related epigenetic changes and can be detected with high sensitivity even when the tumor fraction is low. We propose an integrative mechanistic framework in which age-related epigenetic and genomic changes influence both tumor biology and cfDNA composition, with transposable elements acting as a central link between aging and cancer.

Humans↗

PubMed2024

Central conducting lymphatic anomaly: from bench to bedside.

Central conducting lymphatic anomaly (CCLA) is a complex lymphatic anomaly characterized by abnormalities of the central lymphatics and may present with nonimmune fetal hydrops, chylothorax, chylous ascites, or lymphedema. CCLA has historically been difficult to diagnose and treat; however, recent advances in imaging, such as dynamic contrast magnetic resonance lymphangiography, and in genomics, such as deep sequencing and utilization of cell-free DNA, have improved diagnosis and refined both genotype and phenotype. Furthermore, in vitro and in vivo models have confirmed genetic causes of CCLA, defined the underlying pathogenesis, and facilitated personalized medicine to improve outcomes. Basic, translational, and clinical science are essential for a bedside-to-bench and back approach for CCLA.

Cell-Free Nucleic Acids↗

PubMed2026

iMSC-derived extracellular vesicles and their miRNA cargo influence inflammation and oxidative damage in an in vitro osteoarthritis model.

Osteoarthritis is a multifactorial chronic joint disease characterized by progressive cartilage degradation and inflammation. Since there is no effective cure, emerging therapeutic approaches, such as mesenchymal stromal cells (MSCs) transplantation, are currently under investigation. However, the clinical translation of MSC-based therapies is hampered by several limitations, such as donor-dependent variability and heterogeneity related to tissue sources. To address these issues, MSCs derived from induced pluripotent stem cells (iMSCs) have been proposed as a more standardized and scalable alternative. Due to the risks of cell-based therapy, extracellular vesicles (EVs), particularly iMSC-EVs (iEVs), could represent a promising cell-free approach for OA treatment. The present study aimed at characterizing iMSC-derived EVs and evaluating their functional role in modulating inflammatory responses and redox balance in an in vitro OA model. Notably, recent evidence highlights the central role of EV-encapsulated microRNAs (EV-miRNAs) in mediating these effects. EVs isolated from iMSC conditioned media were characterized, and their miRNA content was analyzed at different culture passages. Selected miRNAs were subsequently assessed for their biological activity in an in vitro OA model, with a focus on their impact on inflammatory mediators and oxidative stress parameters. Specifically, six miRNAs such as hsa-miR-17-5p, hsa-miR-20a-5p, hsa-miR-21-5p, hsa-miR-29a-3p, hsa-miR-29b-3p, and hsa-miR-29c-3p differentially reflect the anti-inflammatory and antioxidant effects of iMSCs-EVs treatment, suggesting possible synergistic effects. Their combined effect in the in vitro model confirmed their potential modulation in the expression of pro-inflammatory cytokines. Furthermore, their treatment markedly reduced ROS accumulation and oxidative damage, while restoring antioxidant defense systems. These findings support the therapeutic potential of iMSC-derived EVs as a cell-free strategy for OA treatment. The miRNA cargo encapsulated within iEVs appears to play a pivotal role in modulating inflammation and oxidative stress, emphasizing their promise as a novel, minimally invasive approach for disease modification in OA.

MicroRNAs↗

PubMed2026

Plasma cfDNA hypermethylation at SNCA intron 1 as a potential blood-based epigenetic signal in Parkinson's disease and multiple system atrophy.

BACKGROUND: The accumulation of α-synuclein (SNCA) in the central nervous system is a hallmark of Parkinson's disease (PD) and multiple system atrophy (MSA). SNCA intron 1 methylation is implicated in SNCA transcriptional regulation and may serve as a peripheral epigenetic signal in synucleinopathies. However, studies of SNCA methylation in leukocyte-derived DNA have yielded inconsistent results. We aimed to evaluate whether cell-free DNA (cfDNA)-based SNCA intron 1 methylation differs in PD or MSA compared with normal controls (NC). METHODS: Plasma cfDNA was collected from 105 patients with PD, 50 with MSA, and 114 NC. DNA methylation at CpG sites 10-17 was quantified by bisulfite pyrosequencing. Multivariable linear and logistic regression models, adjusted for age, sex, and education, were used to compare methylation levels and estimate odds ratios (ORs). RESULTS: Patients with PD exhibited hypermethylation at CpG site 14 and higher mean methylation across CpG sites 10-17 compared with NC. Patients with MSA showed hypermethylation at CpG sites 10, 12, 13, and 17 and elevated mean methylation. Elevated mean methylation was also observed in drug-naïve de novo PD and early-stage PD patients. Compared with the lowest tertile, the highest mean methylation tertile was associated with increased odds of PD (OR, 2.49; 95% CI, 1.08-5.92) and MSA (OR, 5.02; 95% CI, 1.58-18.00). CONCLUSION: Plasma cfDNA SNCA intron 1 hypermethylation is associated with PD and MSA and detectable in drug-naïve and early-stage PD. It may represent a peripheral epigenetic alteration and warrants evaluation as an adjunctive signal for early screening.

Humans↗

PubMed2026

Longitudinal genome-wide aneuploidy measurements in circulating cell-free DNA to predict lack of benefit from pembrolizumab in patients with metastatic urothelial cancer.

Accurate prediction of lack of benefit from pembrolizumab in patients with metastatic urothelial cancer (mUC) is an unmet need. We investigated the dynamics of circulating tumor DNA (ctDNA) load, estimated using the modified fast aneuploidy screening test-sequencing system (mFast-SeqS), as a potential biomarker for early on-treatment identification of treatment response. A total of 104 patients with mUC treated with pembrolizumab from two prospective biomarker discovery trials were included and mFast-SeqS was performed on paired blood samples collected at baseline and on-treatment. Patients with a high on-treatment aneuploidy score (≥ 5, n = 26) had a shorter median OS than patients with a low (< 5) score (n = 76) (3 vs 17 months: P-value< 0.001). Patients with an increased (n = 10), stable (n = 66), or decreased (n = 28) on-treatment score relative to their baseline score had a median PFS of 1.5, 4.0, and 8.3 months, respectively. Median OS was 3.0, 11.1, and 18.7 months, respectively. In patients with mUC treated with pembrolizumab, the on-treatment mFast-SeqS-based ctDNA level and its dynamics relative to baseline are independent prognostic markers that can be used to identify patients that are unlikely to benefit from pembrolizumab.

Humans↗

PubMed2025

Cell-free DNA aneuploidy score as a dynamic early response marker in prostate cancer.

Cell-free circulating tumor DNA (ctDNA) has emerged as a promising biomarker for response evaluation in metastatic castration-resistant prostate cancer (mCRPC). The current study evaluated the modified fast aneuploidy screening test-sequencing system (mFast-SeqS), a quick, tumor-agnostic and affordable ctDNA assay that requires a small input of DNA, to generate a genome-wide aneuploidy (GWA) score in mCRPC patients, and correlated this to matched metastatic tumor biopsies. In this prospective multicenter study, GWA scores were evaluated from blood samples of 196 mCRPC patients prior to treatment (baseline) with taxanes (docetaxel and cabazitaxel) and androgen receptor signaling inhibitors (ARSI; abiraterone and enzalutamide), and from 74 mCRPC patients at an early timepoint during treatment (early timepoint; median 21 days). Z-scores per chromosome arm were tested for their association with tumor tissue genomic alterations. We found that a high tumor load in blood (GWAhigh) at baseline was associated with poor response to ARSI [HR: 2.63 (95% CI: 1.86-3.72) P < 0.001] but not to taxanes. Interestingly, GWAhigh score at the early timepoint was associated with poor response to both ARSIs [HR: 6.73 (95% CI: 2.60-17.42) P < 0.001] and taxanes [2.79 (95% CI: 1.34-5.78) P = 0.006]. A significant interaction in Cox proportional hazards analyses was seen when combining GWA status and type of treatment (at baseline P = 0.008; early timepoint P = 0.018). In summary, detection of ctDNA in blood by mFast-SeqS is cheap, fast and feasible, and could be used at different timepoints as a potential predictor for outcome to ARSI and taxane treatment in mCRPC.

Humans↗

PubMed2026

Contactless Co-Culture Assays for Morphometric Studies During Inter-Species Interactions in Fungi.

Cellular behaviour and morphology are usually influenced by various intra- and extracellular factors in a microbial community. Different approaches available to study microbial communication could be tedious and/or require specialised facilities and expertise. Here, two complementary contactless co-culture approaches are described, the membrane insert well plate co-culture assay and the Cell-Free Supernatant (CFS)-based assay, which are based on morphological transition as a measurable response to investigate the role of various factors in a given inter-species interaction. The membrane insert well plate system, while permitting diffusion of extracellular molecules, allows real-time interaction between physically separated species. In comparison, the CFS-based assay provides a simplified, scalable approach for evaluating responses to conditioned media. The protocol presented here includes standardized procedures for culture preparation, generation of conditioned supernatants, assay setup, microscopy, image acquisition, quantitative morphometric analysis using Fiji, and statistical evaluation. This has been demonstrated with an example of fungal morphological response to intrinsic and extrinsic factors. The methods presented here offer accessible and adaptable alternative tools for studying novel microbial interactions in a community, and could be readily extendable to investigate mechanisms underlying multi-species co-existence in a community.

Coculture Techniques↗

PubMed2025

Diffusely metastatic glioblastoma with FGFR3::TACC3 fusion: cell-free DNA fragmentation analyses and molecular characterization of matched primary and metastatic tumor sites.

Extracranial metastasis of IDH-wildtype glioblastoma is very rare and poorly understood at the molecular level. We report a case of FGFR3::TACC3 fusion IDH-wildtype glioblastoma in a 61-year-old male, whose preoperative blood sample showed highly aberrant cfDNA fragmentation patterns, which could be suggestive of early systemic dissemination, undetected by standard-of-care imaging of his body. Eleven months post-resection and adjuvant therapy, he developed widespread extracranial metastases. Comprehensive molecular profiling of matched primary and metastatic tumors revealed broadly conserved genomic, transcriptomic, and copy number landscapes, with the metastasis harboring an additional ERCC6 deletion and enriched expression of receptor tyrosine kinase signaling genes. These findings provide rare insight into the genetic continuity and evolution underlying IDH-wildtype glioblastoma metastasis.

Humans↗

PubMed2026

Discovery and validation of GNA12circle as a first-trimester plasma eccDNA marker for early-onset preeclampsia.

BACKGROUND: Early-onset preeclampsia (EOPE) is a major cause of maternal and perinatal morbidity and is characterized by placental dysfunction, systemic endothelial injury, and hypertensive vascular stress. Because hypertensive disorders of pregnancy may also signal later maternal cardiovascular and cerebrovascular vulnerability, effective biomarkers for first-trimester risk assessment remain clinically important. Extrachromosomal circular DNA (eccDNA), a stable form of circulating cell-free DNA, has emerged as a potential source of disease-associated biomarkers. This study aimed to characterize first-trimester plasma eccDNA alterations associated with subsequent EOPE and to identify and validate a candidate circulating eccDNA marker for early risk assessment. METHODS: A two-stage nested case-control study was conducted within a prospective birth cohort. In the discovery stage, plasma samples collected at 11-13 weeks of gestation from 5 women who subsequently developed EOPE and 5 matched normotensive controls were profiled by Circle-Seq to characterize genome-wide eccDNA alterations. Candidate eccDNAs were prioritized through differential abundance analysis and were further confirmed by outward PCR and Sanger sequencing. In the validation stage, the candidate selected marker was quantified by junction-specific qPCR in an independent cohort of 109 EOPE cases and 109 controls. Its potential predictive value was further evaluated alone and in combination with routine first-trimester clinical variables. RESULTS: In the exploratory discovery analysis, 410 nominally differentially abundant candidate eccDNAs were identified as a hypothesis-generating pool. Among these, GNA12circle (chr7:2876332-2,876,692) was prioritized and experimentally validated at the circular junction. In the independent validation cohort, plasma GNA12circle levels were significantly higher in women who later developed EOPE than in controls. When combined with routine first-trimester variables, GNA12circle improved predictive performance. The RF model showed the best overall cross-validated performance among the evaluated classifiers, with a mean held-out test-fold AUC of 0.843. CONCLUSION: First-trimester plasma eccDNA profiling revealed distinct alterations associated with subsequent EOPE, from which GNA12circle was identified and validated as a candidate circulating marker. These findings support further investigation of circulating eccDNA for early EOPE risk assessment in larger multicenter populations.

Humans↗

PubMed2026

Novel insights into retinoblastoma: From oncogenic circuitry to precision diagnosis and eye-preserving therapies.

Retinoblastoma (RB) represents the most common primary intraocular malignancy in childhood and stands as a paradigm for translating molecular oncology into precision clinical management. This review synthesizes the comprehensive evolution in the understanding and treatment of RB. First, we deconstruct the intricate oncogenic circuitry that extends far beyond Knudson's classic "two-hit" RB1 inactivation model, describing non-classical MYCN-driven pathogenesis, multi-layered epigenetic reprogramming (including chromatin, RNA and histone changes), and distinct histological subtypes with defined clinical correlates, such as the favorable-prognosis cavitary RB. Single-cell genomics has elucidated the cellular origin from cone precursor cells and intratumoral heterogeneity. Risk stratification has been refined through well-defined classification systems, from the therapy-guiding International Intraocular Retinoblastoma Classification (IIRC) to the comprehensive American Joint Committee on Cancer Tumor-Node-metastasis (AJCC TNM) staging. Furthermore, the diagnostic paradigm has advanced from conventional anatomical imaging to liquid biopsies, enabling non-invasive molecular staging and monitoring via tumor-derived cell-free DNA analysis. Concurrently, the therapeutic landscape has undergone a radical shift, moving from enucleation and external-beam radiotherapy to an era dominated by local sight-preserving strategies. We provide a critical synthesis of the evidence for intravenous chemotherapy and the transformative role of super-selective intra-arterial chemotherapy (IAC), and describe essential randomized controlled trials, technical innovations, and optimized drug regimens. Finally, we explore emerging targeted molecular therapies and future directions. By integrating cutting-edge molecular insights with robust, high-level clinical evidence, this review offers the framework for achieving patient and eye survival as well as vision preservation in children with Retinoblastoma.

Intra-arterial chemotherapy↗