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PubMed2002

Systematic learning of gene functional classes from DNA array expression data by using multilayer perceptrons.

Recent advances in microarray technology have opened new ways for functional annotation of previously uncharacterised genes on a genomic scale. This has been demonstrated by unsupervised clustering of co-expressed genes and, more importantly, by supervised learning algorithms. Using prior knowledge, these algorithms can assign functional annotations based on more complex expression signatures found in existing functional classes. Previously, support vector machines (SVMs) and other machine-learning methods have been applied to a limited number of functional classes for this purpose. Here we present, for the first time, the comprehensive application of supervised neural networks (SNNs) for functional annotation. Our study is novel in that we report systematic results for ~100 classes in the Munich Information Center for Protein Sequences (MIPS) functional catalog. We found that only ~10% of these are learnable (based on the rate of false negatives). A closer analysis reveals that false positives (and negatives) in a machine-learning context are not necessarily "false" in a biological sense. We show that the high degree of interconnections among functional classes confounds the signatures that ought to be learned for a unique class. We term this the "Borges effect" and introduce two new numerical indices for its quantification. Our analysis indicates that classification systems with a lower Borges effect are better suitable for machine learning. Furthermore, we introduce a learning procedure for combining false positives with the original class. We show that in a few iterations this process converges to a gene set that is learnable with considerably low rates of false positives and negatives and contains genes that are biologically related to the original class, allowing for a coarse reconstruction of the interactions between associated biological pathways. We exemplify this methodology using the well-studied tricarboxylic acid cycle.

Algorithms↗

PubMed2002

Molecular imaging: new applications for biochemistry.

Molecular imaging can reveal in vivo analysis and quantification of biochemical reactions. To enable cell-surface imaging of receptors, novel ligands have been developed which can be radiolabeled or imaged by bioluminescence. Specific examples include somatostatin receptors, estrogen and progesterone receptors, receptors involved in adhesion and externalization of phosphatidyl serine as an indicator of apoptosis. Central nervous system imaging can be carried out using ligands for receptors including dopamine, serotonin and Gamma amino butyric acid (GABA). In addition, tumor and metabolic imaging can be carried out with the Na-K ATPase pump using the tracer thallium-201 for SPECT or F-18 FDG for PET imaging. Finally, novel receptors or endogenous metabolic pathways can be analyzed combining cell-gene therapy to create specific tracer targets in cells that can be studied by molecular imaging. The challenge of molecular imaging is to first identify key pathways that are unique for a specific disease processes, such as atherosclerosis, cancer, CNS disorders, immunologic and arthritis disorders and next to devise a high-affinity specific small molecular ligand that can be adapted to be a radiolabeled tracer to study this pathway. Advances in genomics and proteomics combine with new peptide-chemistry approaches should provide a large number of targets and tracers in the near future to achieve these imaging objectives.

Animals↗

PubMed2026

ClearDepthIAS enables automated high-throughput quantification of roots in soil-grown taproot crops.

Understanding root system architecture is critical for improving crop productivity and resilience, yet phenotyping root traits such as root growth angle and rooting depth remains technically challenging, especially at high throughput. Here, we present ClearDepthIAS, a high-throughput imaging and analysis platform that enables nondestructive, automated quantification of root architecture traits in taproot system crops. By capturing and stitching 360° images of roots growing along the transparent walls of pots and applying deep learning-based segmentation (ClearDepth-WRT), we measured wall root shallowness (WRS)-a proxy for root growth angle-with high precision. We demonstrated for the tap root systems of soybean and canola that the system accurately detects root tips, quantifies their vertical distribution, and extracts biologically meaningful traits such as root area, distribution indices, and growth angles. Validation experiments in canola and soybean demonstrated that WRS can correlate with root crown architecture in mature plants, both in greenhouse and field settings. Furthermore, WRS and root distribution indices derived from ClearDepthIAS are predictors of early root architecture and can be correlated with root biomass distribution across soil depths under field conditions; however, environmental interactions may influence these relationships and weaken or even negate such correlations, as observed when comparing field to field variation in root system architecture. Our system enables efficient phenotyping of genetically diverse populations, with medium to high trait heritability, supporting its utility for genome-wide association studies and breeding. ClearDepthIAS accelerates the development of root ideotypes for improved resource acquisition and carbon sequestration, offering a scalable tool for supporting climate-resilient agriculture.

Plant Roots↗

PubMed2025

Fluorescence Loss After Photoactivation (FLAPh): A Pulse-Chase Cellular Assay for Understanding Kinetics and Dynamics of Viral Inclusions.

Influenza A virus (IAV) relies on host cellular machinery for replication. Upon infection, the eight genomic segments, independently packed as viral ribonucleoproteins (vRNPs), are released into the cytosol before nuclear import for viral replication. After nucleocytoplasmic transport, the resulting progeny vRNPs reach the cytosol, accumulating in highly mobile and dynamic viral inclusions that display liquid properties. Being sites postulated to support IAV genome assembly, the biophysical properties of IAV inclusions may be critical for function. In agreement, imposing liquid-to-solid transitions was demonstrated to impact viral replication negatively. Therefore, screening for host factors or compounds able to alter the material properties may provide the molecular basis for how influenza genomic complex forms as well as identify novel antivirals. Conventional techniques employed to investigate biomolecular condensates' material properties include fluorescence correlation spectroscopy, raster image correlation spectroscopy, single molecule or microrheology particle tracking, and Fluorescence Recovery After Photobleaching (FRAP). These approaches allow measuring molecular dynamics in systems that do not move very much. However, the analysis of highly mobile intracellular condensates, such as IAV inclusions, poses significant challenges as these structures not only constantly move within the cell but also exchange material, fusing, and dividing, rendering the quantitation of internal rearrangements and diffusion coefficients of molecules within condensates inaccurate. As an alternative, we opted for measuring the kinetics and the exchange of material between IAV inclusions using the Fluorescence Loss After Photoactivation (FLAPh) technique. It involves pulse photoactivation of individual or pools of viral inclusions in the cell, and chasing over time in photoactivated and non-photoactivated regions. This approach is suitable for quantifying the movement and spatial distribution of components within inclusions over time, enabling the determination of both the distance and speed from a specific cellular location. As a result, this method allows the quantification of decay profiles, half-lives, decay constant rate, and mobile and immobile fractions in viral inclusions. It, therefore, enables high throughput screenings for compounds or host factors that affect this dynamism and indirectly allows assessing the material properties of IAV inclusions.

Humans↗

PubMed2026

Calibrated Prediction Intervals for Polygenic Scores: Updated Comparisons, Contextual Calibration, and Data Normalization.

Calibrated prediction intervals for polygenic scores (PGS) are essential for communicating individual-level uncertainty in genomic medicine. We present updated comparisons of two methods for constructing such intervals: CalPred, a parametric approach, and PredInterval, a non-parametric approach. Our results show that both methods can achieve calibrated coverage, although CalPred additionally requires a sufficiently large calibration set. The two methods also exhibit complementary trade-offs with respect to dataset size and risk identification. We further show that contextual calibration, as introduced in Hou et al. and followed in Shi et al., is most naturally achieved through appropriate phenotype normalization and data preprocessing. Apparent miscalibration can arise from inadequate normalization or from providing contextual information to some methods but not others. In UK Biobank, standard GWAS phenotype normalization procedures are sufficient to achieve contextual calibration for traits analyzed. In the extreme simulations of Hou et al. and Shi et al., supplying contextual covariates to PredInterval restores contextual calibration without normalization, and appropriate normalization can achieve contextual calibration without supplying covariates, while also substantially improving upstream tasks including association power and PGS accuracy. Together, these results underscore the central role of phenotype normalization and data preprocessing in GWAS analyses, including reliable uncertainty quantification for PGS.

Journal Article↗

PubMed2001

Assessment of Helicobacter pylori gene expression within mouse and human gastric mucosae by real-time reverse transcriptase PCR.

Despite increasing knowledge on the biology of Helicobacter pylori, little is known about the expression pattern of its genome during infection. While mouse models of infection have been widely used for the screening of protective antigens, the reliability of the mouse model for gene expression analysis has not been assessed. In an attempt to address this question, we have developed a quantitative reverse transcriptase PCR (RT-PCR) that allowed the detection of minute amounts of mRNA within the gastric mucosa. The expression of four genes, 16S rRNA, ureA (encoding urease A subunit), katA (catalase), and alpA (an adhesin), was monitored during the course of a 6-month infection of mice and in biopsy samples from of 15 infected humans. We found that the selected genes were all expressed within both mouse and human infected mucosae. Moreover, the relative abundance of transcripts was the same (16S rRNA > ureA > katA > alpA), in the two models. Finally, results obtained with the mouse model suggest a negative effect of bacterial burden on the number of transcripts of each gene expressed per CFU (P < 0.05 for 16S rRNA, alpA, and katA). Overall, this study demonstrates that real-time RT-PCR is a powerful tool for the detection and quantification of H. pylori gene expression within the gastric mucosa and strongly indicates that mice experimentally infected with H. pylori provide a valuable model for the analysis of bacterial gene expression during infection.

Animals↗

PubMed2000

Use of single nucleotide polymorphisms (SNP) and real-time polymerase chain reaction for bone marrow engraftment analysis.

Allogeneic bone marrow transplant engraftment assays use polymorphisms in the human genome to determine the relative percentages of donor and recipient cells present in the recipient. We describe a novel posttransplant assay approach using single nucleotide polymorphisms (SNPs), the most common type of polymorphism in humans. Using samples of defined genotype, we used real-time polymerase chain reaction (PCR) and allele-specific fluorescent TaqMan probes to assay a SNP of the cytochrome P450 CYP2C9 gene. Standard curves of chimeric mixes showed a linear relationship between the ratio of two alleles and the ratio of their respective fluorophore emission, except for mixes with a low percentage (< 5%) of the less common allele. We validated the SNP real-time PCR assay by comparing it to Southern hybridization analysis, analyzing DNA mixes in a blinded fashion with both methods. The correlation between the two methods was high. We have produced a statistical model that varies allele frequency to predict how many SNPs would be required to produce a functional SNP panel. Additional development will be necessary to produce such a panel of highly informative SNPs for clinical use. A real-time PCR SNP assay may ultimately provide more accurate quantification and shortened turnaround time compared to current post-engraftment assays.

Alleles↗

PubMed1988

Fluorescence in situ hybridization to interphase cell nuclei in suspension allows flow cytometric analysis of chromosome content and microscopic analysis of nuclear organization.

Fluorescence hybridization to interphase nuclei in liquid suspension allows quantification of chromosome-specific DNA sequences using flow cytometry and the analysis of the three-dimensional positions of these sequences in the nucleus using fluorescence microscopy. The three-dimensional structure of nuclei is substantially intact after fluorescence hybridization in suspension, permitting the study of nuclear organization by optical sectioning. Images of the distribution of probe and total DNA fluorescence within a nucleus are collected at several focal planes by quantitative fluorescence microscopy and image processing. These images can be used to reconstruct the three-dimensional organization of the target sequences in the nucleus. We demonstrate here the simultaneous localization of two human chromosomes in an interphase nucleus using two probe labeling schemes (AAF and biotin). Alternatively, dual-beam flow cytometry is used to quantify the amount of bound probe and total DNA content. We demonstrate that the intensity of probe-linked fluorescence following hybridization is proportional to the amount of target DNA over a 100-fold range in target content. This was shown using four human/hamster somatic cell hybrids carrying different numbers of human chromosomes and diploid and tetraploid human cell lines hybridized with human genomic DNA. We also show that populations of male, female, and XYY nuclei can be discriminated by measuring their fluorescence intensity following hybridization with a Y-chromosome-specific repetitive probe. The delay in the increase in Y-specific fluorescence until the end of S-phase in consistent with the results recorded in previous studies indicating that these sequences are among the last to replicate in the genome. A chromosome-17-specific repetitive probe is used to demonstrate that target sequences as small as one megabase (Mb) can be detected using fluorescence hybridization and flow cytometry.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell Line↗

PubMed2003

Evaluation of PCR amplification bias by terminal restriction fragment length polymorphism analysis of small-subunit rRNA and mcrA genes by using defined template mixtures of methanogenic pure cultures and soil DNA extracts.

Terminal restriction fragment length polymorphism (T-RFLP) analysis is a widely used method for profiling microbial community structure in different habitats by targeting small-subunit (SSU) rRNA and also functional marker genes. It is not known, however, whether relative gene frequencies of individual community members are adequately represented in post-PCR amplicon frequencies as shown by T-RFLP. In this study, precisely defined artificial template mixtures containing genomic DNA of four different methanogens in various ratios were prepared for subsequent T-RFLP analysis. PCR amplicons were generated from defined mixtures targeting not only the SSU rRNA but also the methyl-coenzyme M reductase (mcrA/mrtA) genes of methanogens. Relative amplicon frequencies of microorganisms were quantified by comparing fluorescence intensities of characteristic terminal restriction fragments. SSU ribosomal DNA (rDNA) template ratios in defined template mixtures of the four-membered community were recovered absolutely by PCR-T-RFLP analysis, which demonstrates that the T-RFLP analysis evaluated can give a quantitative view of the template pool. SSU rDNA-targeted T-RFLP analysis of a natural community was found to be highly reproducible, independent of PCR annealing temperature, and unaffected by increasing PCR cycle numbers. Ratios of mcrA-targeted T-RFLP analysis were biased, most likely by PCR selection due to the degeneracy of the primers used. Consequently, for microbial community analyses, each primer system used should be evaluated carefully for possible PCR bias. In fact, such bias can be detected by using T-RFLP analysis as a tool for the precise quantification of the PCR product pool.

Bias↗

PubMed2002

Multicenter study of the LCx HIV RNA quantitative assay--a new competitive reverse transcriptase-PCR which targets pol genomic region of HIV-1 for the measurement of type B, non-type B and group O HIV-1 RNA.

Performance characteristics of the Abbott LCx HIV RNA Quantitative Assay (LCx HIV) were established in a multicenter study comparing it with the manual (Amplicor v1.5) and automated (Cobas) ultra-sensitive Roche Amplicor HIV-1 Monitor v1.5, the Bayer Quantiplex HIV RNA 3.0 (bDNA v3.0), and the Organon NucliSens HIV QT 2.0 (NucliSens). Within-run precision of LCx HIV assessed in clinical specimens was SD log10 0.210 at approximately 50 copies/ml, and log10 0.133 at approximately 400 copies/ml. Total precision in a reconstituted type B HIV-1 RNA panel was SD log10 0.380 at 100 copies/ml, and SD log10 0.180 at 1000 copies/ml. Type B HIV-1 RNA sensitivity (1 ml input) assessed at a 50%, 75% and 95% detection rate ranged from 29 to 41, 54 to 75 and 94 to 176 copies/ml, respectively. Overall specificity in HIV seronegative individuals was 99.78%. Linear regression indicated close assay correlations and agreements for measurement of type B HIV-1 RNA. Pearson's correlations and (Log10LCx=aLog10x + b) linear regressions were 0.91 (y=0.892 Log10Amplicor + 0.595), 0.93 (y=0.827 Log10Cobas + 0.969), 0.93 (y=0.951 Log10bDNA + 0.550), and 0.79 (y=0.834 Log10NucliSens + 0.911). LCx HIV was least affected by the genetic variability of HIV-1. LCx HIV detected 99% of non-type B HIV-1 group M samples (subtypes A-G), Amplicor v1.5 detected 96%, and bDNA v3.0 detected 99%. The assays detected 10/11, 1/11 and 8/11, respectively of the HIV-1 group O samples. LCx HIV vs. Amplicor/bDNA Spearman's rank correlations for quantification of non-type B HIV-1 RNA were 0.76/0.84 (A), 0.93/0.93 (C), 0.73/0.99 (D), 0.86/0.98 (E), and 0.40/0.83 (group O). LCx HIV assays consistently detect and quantify type B, non-type B and group O HIV-1 RNA.

Acquired Immunodeficiency Syndrome↗

PubMed1999

Immunohistochemical localization and biochemical characterization of two novel decapeptides derived from POMC-A in the trout hypothalamus.

Several vertebrate species which underwent duplication of their genome, such as trout, salmon and Xenopus, possess two proopiomelanocortin (POMC) genes. In the trout, one of the POMC molecules, called POMC-A, exhibits a unique C-terminal extension of 25 amino acids which has no equivalent in other POMCs characterized so far. This C-terminal peptide contains three pairs of basic residues, suggesting that it may be the source of novel regulatory peptides. The aim of the present study was to investigate the occurrence of these peptides in the brain of the trout Oncorhynchus mykiss by using specific antibodies raised against two epitopes derived from the C-terminal extension of POMC-A, i.e., EQWGREEGEE and YHFQ-NH2. Immunohistochemical labeling of brain sections revealed the presence of EQWGREEGEE- and YHFQ-NH2-immunoreactive cell bodies in the anterior part of the nucleus lateralis tuberis of the hypothalamus. Immunoreactive fibers were observed in the dorsal hypothalamus, the thalamus, the telencephalon, the optic tectum and the medulla oblongata. In contrast, no labeling was detected using antibodies against the non-amidated peptide YHFQG. Biochemical characterization was performed by combining high-performance liquid chromatography (HPLC) analysis with radioimmunoassay (RIA) quantification. Two peptides exhibiting the same retention time as synthetic EQWGREEGEE and ALGERKYHFQ-NH2 were resolved. However, no peptide co-eluting with YHFQ-NH2 or YHFQG could be detected. These results demonstrate that, in the trout brain, post-translational processing of POMC-A generates the two decapeptides EQWGREEGEE and ALGERKYHFQ-NH2. The wide distribution of immunoreactive fibers in the diencephalon, telencephalon, optic tectum and medulla oblongata suggests that these peptides may exert neurotransmitter and/or neuromodulator activities.

Animals↗

PubMed2026

Monitoring kinetic changes and restriction of influenza A virus RNA species during infection using a Flu-Stranded CRISPR platform.

UNLABELLED: Influenza A virus (IAV) generates three closely related RNA species: viral RNA (vRNA), complementary RNA (cRNA), and messenger RNA (mRNA), whose strand-specific quantification remains limited by sensitivity and quantitative dynamic range, particularly at low RNA abundance. Here, we developed Flu-Stranded CRISPR-Cas12a, a strand-specific detection platform integrating tagged reverse transcription, segment-specific PCR, and Cas12a collateral cleavage to support quantitative analysis of all three RNA species across a broad dynamic range. The assay enables reliable detection down to 102 copies per reaction, extending the lower quantitative boundary relative to both SYBR Green and TaqMan reverse transcription quantitative PCR (RT-qPCR) under matched conditions. Validated in infected cell lines, murine lung tissues, and clinical nasopharyngeal specimens, the platform enabled subtype-discriminating, strand-resolved detection, including samples near or below the quantitative range of SYBR Green RT-qPCR. Using finely resolved infection time-course analyses in NP and NA segments, we identified a reproducible early vRNA decline within the early post-infection phase. This decline was partially attenuated in RIG-I knockout A549 cells, while subsequent vRNA accumulation was enhanced, consistent with a modulatory rather than essential role for RIG-I in early viral RNA dynamics. Subcellular fractionation localized this decline to cytoplasmic incoming genomes. In contrast, importazole-mediated inhibition of nuclear import abolished vRNA recovery without affecting the early decline, indicating that nuclear entry functionally separates early genome reduction from subsequent productive replication. These findings establish Flu-Stranded CRISPR-Cas12a as a strand-resolved framework for monitoring IAV RNA dynamics and reveal an early window of genome vulnerability during cytoplasmic transit that shapes infection outcome. IMPORTANCE: The early fate of incoming influenza virus genomes remains unclear, limiting our understanding of how infection is established or aborted in host cells. We developed Flu-Stranded CRISPR-Cas12a, a strand-specific platform for sensitive and quantitative analysis of influenza viral RNA (vRNA), complementary RNA (cRNA), and messenger RNA (mRNA) across experimental and clinical samples. Using high-resolution time-course analysis, we identified a reproducible early decline in vRNA during the post-entry phase. Our data suggest that this early genome loss arises from multiple processes, with RIG-I acting as a modulatory factor rather than a primary driver. Subcellular fractionation localized this effect to cytoplasmic incoming genomes, whereas importin-β-mediated nuclear entry was required for subsequent vRNA recovery. These findings support a model of an early cytoplasmic phase of genome attrition that is distinct from replication and provide a framework for understanding early influenza RNA kinetics and for guiding strand-resolved diagnostics and antiviral evaluation.

CRISPR-Cas12a↗

PubMed2004

Enteroviral hepatitis in children.

BACKGROUND: The pathogen causing enteroviral hepatitis is often not found despite careful examination. METHODS: This study investigates the enterovirus genome in serum and liver tissue obtained from patients who showed abnormal liver function without negative data of usual studies and cytomegalovirus (CMV) serologically positive cases.' RESULTS: Nine out of 21 serum samples were positive by using reverse transcriptase-polymerase chain reaction (RT-PCR) for enterovirus. The 21 samples had CMV-IgM antibodies in five cases. These CMV serologically positive cases were all negative for enterovirus using RT-PCR. Therefore, nine out of 16 (60%) were of unknown etiology. Some cases showed liver dysfunction over a period of more than 6 months. The liver function revealed that all cases finally improved. The sequences coincided with those of Coxsackie B5 or B6 with the highest score by gene homology search. The liver pathology revealed that two of three subjected cases had mild fibrosis and small cell infiltration. RT-PCR of liver tissue for enterovirus were positive in all three cases comparing the house keeping gene. The viral load was high in acute phase and low in convalescent phase. CONCLUSIONS: In more than half of children with illnesses of unknown etiology, the pathogen was found to be enteroviruses, and RT-PCR and quantification of serum is an easy method to identify these diseases.

Child, Preschool↗

PubMedDate not supplied

Quantitative complete tooth variation among east Asians and Native Americans: developmental biology as a tool for the assessment of human divergence.

The quantification of total tooth structure derived from X-rays of Vietnamese, Southern Chinese, Mongolians, Western Eskimos, and Peruvian pre-Inca (Huari Empire) populations was used to examine dental divergence and the morphogenetics of change. Multivariate derived distances between the samples helped identify a quasicontinuous web of ethnic groups with two binary clusters ensconced within the web. One cluster was composed of Mongolians, Western Eskimos, and pre-Inca, and the other group consisted of the Southern Chinese and Vietnamese. Mongolians entered the quasicontinuum from a divergent angle (externally influenced) from that of the Southeast Asians. The Chinese and pre-Inca formed the polar samples of the distance superstructure. The pre-Inca sample was the most isolated, its closest neighbor being the Western Eskimos. Univariate and multivariate analyses suggested that the pre-Inca, whose ancestors arrived in America perhaps approximately 30,000 years ago, was the least derived sample. Clearly, microevolutionary change occurred among the samples, but the dental phenotype was resistant to environmental developmental perturbations. An assessment of dental divergence and developmental biology suggested that the overall dental phenotype is a complex multigenic morphological character, and that the observed variation evolved through total genomic drift. The quantified dental phenotype is greater than its highly multigenic algorithm and its development homeostasis is tightly controlled, or canalized, by the deterministic organization of a complex nonlinear epigenetic milieu. The overall dental phenotype quantified here was selectively neutral and a good character to help reconstruct the sequence of human evolution, but if the outlying homeostatic threshold was or will be exceeded in antecedents and descendants, respectively, evolutionary saltation occurs.

Analysis of Variance↗

PubMed2004

A diffusion-reaction model for DNA microarray assays.

DNA microarrays are extensively used for the quantification of the degree of differential mRNA expression. The assay involves hybridization of mobile DNA strands with immobilized complementary DNA strands to form duplexes. The overall duplex formation rate depends on the rate of transport of strands in solution to the corresponding spot on the surface, and the rate of the hybridization reaction. We present a theoretical model that incorporates both kinetics of the reversible hybridization reaction and diffusional transport of the labeled strands, and analyze DNA microarray hybridization using this model. Simulations are carried out in a geometrically realistic domain for labeled DNA concentrations corresponding to rare and abundant transcripts for typical assay conditions. The rate of strand diffusion in solution is shown to strongly affect the overall hybridization rate. We compute the minimum inter-spot spacing for replicate spots to enhance sensitivity. We also determine the hybridization time for which reliable estimates of the relative mRNA abundance of two species can be obtained using total fluorescence intensities. An analytical solution for the concentration distribution of mobile strands at intermediate hybridization times provides a convenient tool to calculate the mobile strand concentration profiles. This model provides a framework for the process analysis of all microarray assays currently used for genomic transcriptional analysis.

Computer-Aided Design↗

PubMed1999

Indications for an active process underlying spontaneous and radiation-induced micronucleation in L929 cells.

PURPOSE: To investigate the mechanism of micronucleus formation in irradiated L929 cells. MATERIALS AND METHODS: Radiation-induced micronuclei (MN) of L929 cells isolated at 48 and 72 h after irradiation were processed for detection of DNA-laddering and higher-order chromatin fragments using conventional gel electrophoresis and pulse-field gel electrophoresis. Quantification of double-strand breaks in micronuclei and nuclei was performed with the TdT assay and quantified using image analysis. The number of binucleated cells containing micronuclei (cytochalasin B method) was counted after application of three unspecific endonuclease inhibitors (aurin, ATA, spermine), a topoisomerase II inhibitor (VM-26), administration of two PKC inhibitors (H-7, Gö6983) and after addition of N-acetylsphingosine (C2-ceramide). PKC activity was determined by measuring the incorporation of [gamma-32P]ATP into a suitable specific substrate. Proliferation was measured by detection of PCNA, RFP-A and BrdU (30-min pulse labelling) using both conventional immunofluorescence and laser scanning microscopy. RESULTS: (1) Higher chromatin fragments accumulated in MN with a size as they occur during early stages of apoptosis; (2) the frequency of MN was influenced by drugs known to play an important role in signalling and execution of apoptosis (endonucleases, topoisomerase II, protein kinases, ceramide); (3) MN are characterized by a reduced transcription ability (PCNA, RFP-A). CONCLUSIONS: A proportion of L929 MN may be formed by an active process comparable with the early stages of apoptosis; it may play a role in the re-organization of the damaged genome.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed2002

Physical and biological parameters affecting DNA double strand break misrejoining in mammalian cells.

In an attempt to investigate the effect of radiation quality, dose and specific repair pathways on correct and erroneous rejoining of DNA double strand breaks (DSBs), an assay was applied that allows the identification and quantification of incorrectly rejoined DSB ends produced by ionising radiation. While substantial misrejoining occurs in mammalian cells after high acute irradiation doses, decreasing misrejoining frequencies were observed in dose fractionation experiments with X rays. In line with this finding, continuous irradiation with gamma rays at low dose rate leads to no detectable misrejoining. This indicates that the probability for a DSB to be misrejoined decreases drastically when DSBs are separated in time and space. The same dose fractionation approach was applied to determine DSB misrejoining after alpha particle exposure. In contrast to the results with X rays, there was no significant decrease in DSB misrejoining with increasing fractionation. This suggests that DSB misrejoining after alpha irradiation is not significantly affected by a separation of particle tracks. To identify the enzymatic pathways that are involved in DSB misrejoining, cell lines deficient in non-homologous end-joining (NHEJ) were examined. After high X ray doses, DSB misrejoining is considerably reduced in NHEJ mutants. Low dose rate experiments show elevated DSB misrejoining in NHEJ mutants compared with wild-type cells. The authors propose that NHEJ serves as an efficient pathway for rejoining correct break ends in situations of separated breaks but generates genomic rearrangements if DSBs are close in time and space.

Alpha Particles↗

PubMed2026

Quantifying uncertainty of predictions from cancer progression models.

MOTIVATION: Cancer progresses through the accumulation of genomic events. Cancer progression models such as Mutual Hazard Networks (MHNs) describe this dynamic, enabling prediction of temporal event positions and patient-specific risks of acquiring mutations. However, current MHN analyses rely on single most likely models and do not quantify the uncertainty inherent to parameter estimation. Assessing forecast stability is essential before using them to anticipate treatment-relevant mutations, adapt targeted therapies, or prioritize monitoring of patients at elevated progression risk. RESULTS: We address a key prerequisite for the responsible clinical use of cancer progression models by making MHN-derived predictions uncertainty-aware. We present a Bayesian framework for MHN that uses Markov Chain Monte Carlo to sample from the posterior distributions of model parameters and derived predictions. For practical use we implemented the Random-Walk Metropolis, Metropolis-Adjusted Langevin Algorithm (MALA), and simplified manifold MALA samplers as part of the existing mhn Python package. Only MALA and smMALA were successful in sampling from MHN posteriors, with MALA performing best. While most MHN parameters and predictions showed low posterior variance, a small subset displayed greater variability across the posterior distribution. This differentiation cannot be obtained from a single most likely model, emphasizing the need for uncertainty quantification, especially in clinical contexts. As an illustrative example, posterior sampling identified a subgroup of STK11$-$, KRAS$+$ lung adenocarcinoma patients with a high predicted short-term risk-with low variance across posterior samples-to develop an STK11 mutation. This subgroup exhibited poorer survival under immunotherapy, resembling patterns observed in STK11+ patients. AVAILABILITY AND IMPLEMENTATION: Our implementation is part of version 1.2.0 of the mhn package (https://github.com/spang-lab/LearnMHN). All analyses including the code to produce all figures in this article can be found under https://github.com/huy29433/MCMC-sampling-for-MHN (https://doi.org/10.5281/zenodo.21160219).

Humans↗