Use of topical methylene blue to image nuclear morphometry with a low-cost scanning darkfield microendoscope.

Significance: Fiber-optic microendoscopy is a promising approach to noninvasively visualize epithelial nuclear morphometry for early cancer and precancer detection. However, the broader clinical application of this approach is limited by a lack of topical contrast agents available for in vivo use. Aim: The aim of this study was to evaluate the ability to image nuclear morphometry in vivo with a novel fiber-optic microendoscope used together with topical application of methylene blue (MB), a dye with FDA approval for use in chromoendoscopy in the gastrointestinal tract. Approach: The low-cost, high-resolution microendoscope implements scanning darkfield imaging without complex optomechanical components by leveraging programmable illumination and the rolling shutter of the image sensor. We validate the integration of our system and MB staining for visualizing epithelial cell nuclei by performing ex vivo imaging on fresh animal specimens and in vivo imaging on healthy volunteers. Results: The results indicate that scanning darkfield imaging significantly reduces specular reflection and resolves epithelial nuclei with enhanced image contrast and spatial resolution compared to non-scanning widefield imaging. The image quality of darkfield images with MB staining is comparable to that of fluorescence images with proflavine staining. Conclusions: Our approach enables real-time microscopic evaluation of nuclear patterns and has the potential to be a powerful noninvasive tool for early cancer detection.

Experimental validation in a neutron exposure frame of the MINAS TIRITH for cell damage simulation.

In the domains of medicine and space exploration, refining risk assessment models for protecting healthy tissue from ionizing radiation is crucial. Understanding radiation-induced effects requires biological experimentations at the cellular population level and the cellular scale modeling using Monte Carlo track structure codes. We present MINAS TIRITH, a tool using Geant4-DNA Monte Carlo-generated databases to study DNA damage distribution at the cell population scale. It introduces a DNA damage location module and proposes a method to convert double-strand breaks (DSB) into DNA Damage Response foci. We evaluate damage location precision and DSB-foci conversion parameters. MINAS TIRITH's accuracy is validated againstγ-H2AX foci distribution from cell population exposed to monoenergetic neutron beams (2.5 or 15.1 MeV) under different configurations, yielding mixed radiation fields. Strong agreement between simulation and experimental results was found demonstrating MINAS TIRITH's predictive precision in radiation-induced DNA damage topology. Additionally, modeling intercellular damage variability within a population subjected to a specific macroscopic dose identifies subpopulations, enhancing realistic fate models. This approach advances our understanding of radiation-induced effects on cellular systems for risk assessment improvement.

A novel cost function for nuclei segmentation and classification in imbalanced histopathology data-sets.

Cancer is a major global health problem, causing millions of deaths yearly. Histopathological analysis plays a crucial role in detecting and diagnosing various types of cancer, enabling an accurate diagnosis to inform targeted treatment planning, allowing for better cancer staging, and ultimately improving prognosis. We aim to detect cancer earlier, which can ultimately help reduce mortality rates and enhance patients' quality of life. However, detecting and classifying rare cells is a key challenge for pathologists and researchers. Many histopathological data-sets contain imbalanced data, with only a few instances of rare cells whose unique morphological structures can impede early diagnosis efforts. Our model, SPNet, a spatially aware convolutional neural network, addresses this problem by employing a spatial data balancing technique, enhancing the classification of rare nuclei by 21.8 %. Since nuclei often cluster and exhibit patterns of the same class, SPNet's novel cost function targets spatial regions, resulting in a 1.9 % increase in the F1 classification of rare class types within the CoNSeP dataset. When integrated with a ResNet50-SE encoder, SPNet increases the mean F1 score for classifying all nuclei in the CoNSeP dataset by 4.3 %, compared to the benchmark set by the state-of-the-art HoVer-Net model. The potential integration of SPNet into existing medical devices could allow us to streamline diagnostic processes and minimise false negatives.

Immunohistochemical Identification and Assessment of the Location of Immunoproteasome Subunits LMP2 and LMP7 in Acquired Cholesteatoma.

Cholesteatoma, accompanied by chronic inflammatory response, is characterized by invasive growth and osteolytic activity. As specific proteasome isoforms, the immunoproteasomes serve as an important modulator of inflammatory responses. The aim of the present study was to determine the biological activity of cholesteatoma through the analysis of the expression and localization of immunoproteasome subunits of low molecule weight protein (LMP) 2 and LMP7. Cholesteatoma specimens were obtained from 15 adults who underwent ear surgery due to acquired attic cholesteatoma. Normal skin specimens were taken from retro-auricular skin incisions from the same patients. The specimens were stained with anti-LMP7 antibody, using immunohistochemistry techniques based on the binding of biotinylated secondary antibody with the enzyme-labeled streptavidin and the Envision FLEX system. In all specimens of cholesteatoma, the immunohistochemical reaction with the antibody against the LMP2 was positive, in both the cytoplasm of the cholesteatoma matrix and the perimatrix. A negative reaction with anti-LMP2 was observed in the cytoplasm and nuclei of control skin cells. A positive nuclear and cytoplasmic immunohistochemical reaction with anti-LMP7 has been demonstrated in numerous cells, in both the matrix and perimatrix of cholesteatoma. We present evidence of the presence of expressions of LMP2 and LMP7 within cholesteatoma tissue. Our results might bring new information concerning immunoproteasome-dependent pathophysiologic mechanisms in cholesteatoma.

Assessment of radiation doses and DNA damage in pediatric patients undergoing interventional procedures for vascular anomalies.

Interventional procedures (IPs) have been widely used to treat vascular anomalies (VA) in recent years. However, patients are exposed to low-dose X-ray ionizing radiation (IR) during these fluoroscopy-guided IPs. We collected clinical information and IR doses during IPs and measured biomarkers including γ-H2AX, chromosome aberrations (CA), and micronuclei (MN), which underpin radiation-induced DNA damage, from 74 pediatric patients before and after IPs. For the 74 children, the range of dose-area product (DAP) values was from 1.2 to 1754.6 Gy∙cm2, with a median value of 27.1 Gy∙cm2. DAP values were significantly higher in children with lesions in the head and neck than in the limbs and trunk; the age and weight of children revealed a strong positive correlation with DAP values. The treated patients as a group demonstrated an increase in all three endpoints relative to baseline following IPs. Children with vascular tumors have a higher risk of dicentric chromosome + centric ring (dic+r) and cytokinesis-block micronucleus (CBMN) after IPs than children with vascular malformations. The younger the patient, the greater the risk of CA after IPs. Moreover, rogue cells (RCs) were found in five children (approximately 10%) after IPs, and the rates of dic+r and CBMN were significantly higher than those of other children (Z = -3.576, p < 0.001). These results suggest that there may be some children with VA who are particularly sensitive to IR, but more data and more in-depth experiments will be needed to verify this in the future.

Interrogating 1000 insect genomes for NUMTs: A risk assessment for estimates of species richness.

The nuclear genomes of most animal species include NUMTs, segments of the mitogenome incorporated into their chromosomes. Although NUMT counts are known to vary greatly among species, there has been no comprehensive study of their frequency/attributes in the most diverse group of terrestrial organisms, insects. This study examines NUMTs derived from a 658 bp 5' segment of the cytochrome c oxidase I (COI) gene, the barcode region for the animal kingdom. This assessment is important because unrecognized NUMTs can elevate estimates of species richness obtained through DNA barcoding and derived approaches (eDNA, metabarcoding). This investigation detected nearly 10,000 COI NUMTs ≥ 100 bp in the genomes of 1,002 insect species (range = 0-443). Variation in nuclear genome size explained 56% of the mitogenome-wide variation in NUMT counts. Although insect orders with the largest genome sizes possessed the highest NUMT counts, there was considerable variation among their component lineages. Two thirds of COI NUMTs possessed an IPSC (indel and/or premature stop codon) allowing their recognition and exclusion from downstream analyses. The remainder can elevate species richness as they showed 10.1% mean divergence from their mitochondrial homologue. The extent of exposure to "ghost species" is strongly impacted by the target amplicon's length. NUMTs can raise apparent species richness by up to 22% when a 658 bp COI amplicon is examined versus a doubling of apparent richness when 150 bp amplicons are targeted. Given these impacts, metabarcoding and eDNA studies should target the longest possible amplicons while also avoiding use of 12S/16S rDNA as they triple NUMT exposure because IPSC screens cannot be employed.

Observation of a Nuclear Recoil Peak at the 100 eV Scale Induced by Neutron Capture.

Coherent elastic neutrino-nucleus scattering and low-mass dark matter detectors rely crucially on the understanding of their response to nuclear recoils. We report the first observation of a nuclear recoil peak at around 112 eV induced by neutron capture. The measurement was performed with a CaWO_{4} cryogenic detector from the NUCLEUS experiment exposed to a ^{252}Cf source placed in a compact moderator. We identify the expected peak structure from the single-γ de-excitation of ^{183}W with 3σ and its origin by neutron capture with 6σ significance. This result demonstrates a new method for precise, in situ, and nonintrusive calibration of low-threshold experiments.

3D quantitative assessment for nuclear morphology in osteocytic spheroid with optical clearing technique.

In recent years, three-dimensional (3D) cell culture has been attracting attention as a cell culture model that mimics an environment closer to that of a living organism. It is known that there is a close relationship between cell nuclear shape and cellular function, which highlights the importance of cell nucleus shape analysis in the 3D culture. On the other hand, it is difficult to observe the cell nuclei inside the 3D culture models because the penetration depth of the laser light under a microscope is limited. In this study, we adopted an aqueous iodixanol solution to the 3D osteocytic spheroids derived from mouse osteoblast precursor cells to make the spheroids transparent for 3D quantitative analysis. With a custom-made image analysis pipeline in Python, we found that the aspect ratio of the cell nuclei near the surface of the spheroid was significantly greater than that at the center, suggesting that the nuclei on the surface were deformed more than those at the center. The results also quantitatively showed that the orientation of nuclei in the center of the spheroid was randomly distributed, whereas those on the surface of the spheroid were oriented parallel to the surface of the spheroid. Our 3D quantitative method with an optical clearing technique will contribute to the 3D culture models including various organoid models to elucidate the nuclear deformation during the development of the organs. Insight box Although 3D cell culture has been a powerful tool in the fields of fundamental biology and tissue engineering, it raises the demand for quantification techniques for cell nuclear morphology in the 3D culture model. In this study, we attempted to optically clear a 3D osteocytic spheroid model using iodixanol solution for the nuclear observation inside the spheroid. Moreover, using a custom-made image analysis pipeline in Python, we successfully quantified the nuclear morphology regarding aspect ratio and orientation. Our quantitative method with the optical clearing technique will contribute to the 3D culture models such as various organoid models to elucidate the nuclear deformation during the development of the organs.

SMILE: Cost-sensitive multi-task learning for nuclear segmentation and classification with imbalanced annotations.

High throughput nuclear segmentation and classification of whole slide images (WSIs) is crucial to biological analysis, clinical diagnosis and precision medicine. With the advances of CNN algorithms and the continuously growing datasets, considerable progress has been made in nuclear segmentation and classification. However, few works consider how to reasonably deal with nuclear heterogeneity in the following two aspects: imbalanced data distribution and diversified morphology characteristics. The minority classes might be dominated by the majority classes due to the imbalanced data distribution and the diversified morphology characteristics may lead to fragile segmentation results. In this study, a cost-Sensitive MultI-task LEarning (SMILE) framework is conducted to tackle the data heterogeneity problem. Based on the most popular multi-task learning backbone in nuclei segmentation and classification, we propose a multi-task correlation attention (MTCA) to perform feature interaction of multiple high relevant tasks to learn better feature representation. A cost-sensitive learning strategy is proposed to solve the imbalanced data distribution by increasing the penalization for the error classification of the minority classes. Furthermore, we propose a novel post-processing step based on the coarse-to-fine marker-controlled watershed scheme to alleviate fragile segmentation when nuclei are with large size and unclear contour. Extensive experiments show that the proposed method achieves state-of-the-art performances on CoNSeP and MoNuSAC 2020 datasets. The code is available at: https://github.com/panxipeng/nuclear_segandcls.

Assessment of Covalently Binding Warhead Compounds in the Validation of the Cytomegalovirus Nuclear Egress Complex as an Antiviral Target.

Herpesviral nuclear egress is a regulated process of viral capsid nucleocytoplasmic release. Due to the large capsid size, a regular transport via the nuclear pores is unfeasible, so that a multistage-regulated export pathway through the nuclear lamina and both leaflets of the nuclear membrane has evolved. This process involves regulatory proteins, which support the local distortion of the nuclear envelope. For human cytomegalovirus (HCMV), the nuclear egress complex (NEC) is determined by the pUL50-pUL53 core that initiates multicomponent assembly with NEC-associated proteins and capsids. The transmembrane NEC protein pUL50 serves as a multi-interacting determinant that recruits regulatory proteins by direct and indirect contacts. The nucleoplasmic core NEC component pUL53 is strictly associated with pUL50 in a structurally defined hook-into-groove complex and is considered as the potential capsid-binding factor. Recently, we validated the concept of blocking the pUL50-pUL53 interaction by small molecules as well as cell-penetrating peptides or an overexpression of hook-like constructs, which can lead to a pronounced degree of antiviral activity. In this study, we extended this strategy by utilizing covalently binding warhead compounds, originally designed as binders of distinct cysteine residues in target proteins, such as regulatory kinases. Here, we addressed the possibility that warheads may likewise target viral NEC proteins, building on our previous crystallization-based structural analyses that revealed distinct cysteine residues in positions exposed from the hook-into-groove binding surface. To this end, the antiviral and NEC-binding properties of a selection of 21 warhead compounds were investigated. The combined findings are as follows: (i) warhead compounds exhibited a pronounced anti-HCMV potential in cell-culture-based infection models; (ii) computational analysis of NEC primary sequences and 3D structures revealed cysteine residues exposed to the hook-into-groove interaction surface; (iii) several of the active hit compounds exhibited NEC-blocking activity, as shown at the single-cell level by confocal imaging; (iv) the clinically approved warhead drug ibrutinib exerted a strong inhibitory impact on the pUL50-pUL53 core NEC interaction, as demonstrated by the NanoBiT assay system; and (v) the generation of recombinant HCMV ∆UL50-ΣUL53, allowing the assessment of viral replication under conditional expression of the viral core NEC proteins, was used for characterizing viral replication and a mechanistic evaluation of ibrutinib antiviral efficacy. Combined, the results point to a rate-limiting importance of the HCMV core NEC for viral replication and to the option of exploiting this determinant by the targeting of covalently NEC-binding warhead compounds.

Modeling of dose and linear energy transfer homogeneity in cell nuclei exposed to alpha particles under various setup conditions.

PURPOSE: Different alpha exposure setups are often used to study the relation between biological responses and LET. This study aimed to estimate the dose heterogeneity and uncertainty in four exposure setups using Geant4 and PARTRAC codes. The importance of the irradiation system characteristics was shown in the context of reporting experimental results, especially in radiobiological studies at the molecular level. MATERIALS AND METHODS: Geant4 was used to estimate the dose distributions in cells grown on a disk exposed to alpha particles penetrating from above and below. The latter setup was simulated without and with a collimator. PARTRAC was used for the validation of Geant4 simulations based on distributions of the number of alpha particles penetrating a round nucleus and the deposited energy. RESULTS: The LET distributions obtained for simulated setups excluding the collimator were wide and non-Gaussian. Using a collimator resulted in a Gaussian LET distribution, but strongly reduced dose rate and dose homogeneity. Comparison between PARTRAC and Geant4 calculations for the cell nucleus exposed to alpha radiation showed an excellent agreement. CONCLUSIONS: The interpretation of results from radiobiological experiments with alpha particles should always cover the characteristics of the experimental setup, which can be done precisely with computational methods.

Leukocyte deep learning classification assessment using Shapley additive explanations algorithm.

INTRODUCTION: A peripheral blood smear is a basic test for hematological disease diagnosis. This test is performed manually in many places worldwide, which requires both time and qualified staff. Large laboratories are equipped with digital morphology analyzers, some of which are based on deep learning methods. However, it is difficult to explain to scientists how they work. In this paper, we proposed to add an explanatory factor to enhance the interpretability of deep learning models in leukocyte classification. METHODS: 10 297 single images of leukocytes obtained from peripheral blood smears were included in this study. Pre-trained and fully trained VGG16 and VGG19 models were used to classify the leukocytes, and Shapley Additive Explanations (SHAP) DeepExplainer was applied to visualize the area of cells that were significant for classification. The output images from the DeepExplainer were compared with cellular elements that are essential to laboratory practice. RESULTS: The accuracy of our fully trained models was 99.81% for VGG16 and 99.79% for VGG19. It achieved slightly better results than the partially trained model, which scored 98.67% for VGG16 and 98.33% for VGG19. Their SHAP explanations indicated the significance of cellular structures in microscopic examination. Explanations in the pre-trained models have proved the cell and nucleus contours to be relevant to classification, while explanations in the fully trained models pointed to the cytoplasm area. CONCLUSION: Despite different SHAP DeepExplainer explanations for fully and partially trained models, this method appears to be helpful for the verification of leukocyte classification in automated peripheral blood smear examination.

A minority of cases of acinic cell carcinoma of the salivary glands are driven by an NR4A2 rearrangement: the diagnostic utility of the assessment of NR4A2 and NR4A3 alterations in salivary gland tumors.

Acinic cell carcinoma (AciCC) is a common salivary gland malignancy, typically composed of neoplastic acinic cells with zymogen granules. The vast majority of cases are driven by a t(4;9)(q13;q31) leading to enhancer hijacking and upregulation of the NR4A3 gene. However, a minority of cases do not display NR4A3 overexpression on immunohistochemical examination and are negative for the rearrangement involving the NR4A3 gene when tested by FISH. Such cases overexpress NR4A2, and the protein product is detectable by immunohistochemistry. In this study, we aimed to assess the utility of NR4A2 and NR4A3 immunohistochemistry in the differential diagnosis of salivary gland tumors. Eighty-five cases of classic low-grade ACiCC, as well as 36 cases with high-grade transformation (HGT) and 7 high-grade AciCC cases were included in the analysis. NR4A3 was at least focally positive in 105/128 (82%) cases. Out of the 23 cases that were immunohistochemically negative for NR4A3, 6 displayed nuclear immunopositivity with the NR4A2 antibody. The NR4A3 rearrangement was confirmed by FISH in 38/52 (73%) cases. In addition, this is the first report of an NR4A2 rearrangement being detected by FISH in 2 AciCC cases that were negative for the NR4A3 rearrangement. Our analysis confirms that the majority of AciCC, including high-grade cases and cases with HGT, are immunopositive for NR4A3, and suggests that NR4A3 immunohistochemistry is a powerful tool in the differential diagnosis of salivary gland tumors. However, its utility is limited in sub-optimally fixed samples which often display weaker and focal positivity. Our study also indicates that in a minority of cases, AciCC might be negative for NR4A3 immunostaining, because the pathogenic genetic event in these cases is instead a rearrangement involving the NR4A2 gene.

Cellular dosimetry of 197Hg, 197mHg and 111In: comparison of dose deposition and identification of the cell and nuclear membrane as important targets.

PURPOSE: To examine the reliability to model cellular S-values for the Auger electron (AE) emitters, 111In, 197Hg and 197mHg with MCNP6 and their relative dose deposition in subcellular targets. METHODS: A model cell was defined as four concentric spheres consisting of the nucleus (N), cytoplasm (Cy), cell and nuclear membranes (CM, NM) in which radionuclides distributed homogeneously. The transport of AE, conversion electrons and photons were simulated by MCNP6 to calculate cellular S values (SN←CM, SN←Cy, SN←NM, SN←N, SCM←CM, SNM←NM). SN←CM, SN←Cy and SN←N were also calculated with MIRDcell. RESULTS: MIRDcell and MCNP6-calculated SN←N were in excellent agreement, but a slight discrepancy on SN←Cy and SN←CM was observed. The ratios of SCM←CM or SNM←NM vs. SN←N were 9.7-51.0 or 10.5-37.4, 7.9-41.8 or 8.4-31.8 and 7.2-36.9 or 8.0-28.1 for 111In, 197Hg, 197mHg, respectively. The mean S(197Hg)/S(111In) and S(197mHg)/S(111In) were 2.5 ± 0.5 and 2.5 ± 0.6, respectively. CONCLUSIONS: Cellular S-values were reliably calculated with MCNP6. 197Hg and 197mHg deposit two-fold more doses than 111In at the subcellular scale. All AE emitters deposit a higher self-dose in the CM and NM than in the N, which warrants studies on the effects of targeting the CM and NM by AE emitters.

Toxicity assessment of potassium bromate and the remedial role of grape seed extract.

In this study, the multiple toxic effects of potassium bromate were investigated in Allium cepa L., an indicator test material. In addition, the toxicity-reducing effects of grape seed extract (GSE) were tested. The toxicity was investigated by some physiological (germination percentage, root length, weight gain, relative injury rate), cytogenetic [mitotic index (MI), micronucleus (MN), and chromosomal abnormalities (CAs)], biochemical [malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), glutathione (GSH) levels] and anatomical parameters. A. cepa bulbs were divided into 6 groups as control and five treatment groups (Group II: 465 mg/L GSE, Group III: 930 mg/L GSE, Group IV: 100 mg/L potassium bromate, Group V: 100 mg/L potassium bromate + 465 mg/L GSE, Group VI: 100 mg /L potassium bromate + 930 mg/L GSE). The bulbs were germinated for 72 h and at the end of the period the bulbs were subjected to routine preparations and made ready for analysis and measurements. As a result, potassium bromate exposure caused statistically significant (p < 0.05) decreases in all physiological parameter values. Potassium bromate application decreased MI by 41.6%, and increased the MN and CAs frequencies. CAs such as fragment, sticky chromosome, and vagrant chromosome, unequal distribution of chromatin, reverse polarization, nuclear bud and disordered mitosis were induced in root meristem cells. The mechanism of potassium bromate genotoxicity has been associated with DNA-potassium bromate interaction supported by spectral shift. Potassium bromate caused a decrease in GSH levels and an increase in MDA, SOD and CAT levels, thereby disrupting the antioxidant/oxidant balance in root tip cells. GSE administration in two different doses together with potassium bromate reduced the toxic effects and caused improvements in all parameters examined. The most significant reduction in toxicity was in group VI, which received 930 mg/L GSE, and there was an improvement about 18% in MI levels and an improvement about 44% in GSH levels in this group. While GSE application increased physiological parameters and GSH levels, it decreased MDA, SOD, CAT levels, MN and CAs frequencies. As a result, it has been determined that potassium bromate causes multi-directional toxicity at high doses and A. cepa is a very reliable indicator in determining this toxicity. In addition, GSE extract has been found to have a strong role in reducing the toxicity induced by potassium bromate.

A novel pre-processing approach based on colour space assessment for digestive neuroendocrine tumour grading in immunohistochemical tissue images.

INTRODUCTION: The complexity of histopathological images remains a challenging issue in cancer diagnosis. A pathologist analyses immunohistochemical images to detect a colour-based stain, which is brown for positive nuclei with different intensities and blue for negative nuclei. Several issues emerge during the eyeballing tissue slide analysis, such as colour variations caused by stain inhomogeneity, non-uniform illumination, irregular cell shapes, and overlapping cell nuclei. To overcome those problems, an automated computer-aided diagnosis system is proposed to segment and quantify digestive neuroendocrine tumours. MATERIAL AND METHODS: We present a novel pre-processing approach based on colour space assessment. A criterion called pertinence degree is introduced to select the appropriate colour channel, followed by contrast enhancement. Subsequently, the adaptive local threshold technique that uses the modified Laplacian filter is applied to minimize the implementation complexity, highlight edges, and emphasize intensity variation between cells across the slide. Finally, the improved watershed algorithm based on the concave vertex graph is applied for cell separation. RESULTS: The performance of the algorithms for nucleus segmentation is evaluated according to both the object-level and pixel-level criteria. Our approach increases segmentation accuracy, with the F1-score equal to 0.986. There is significant agreement between the applied approach and the expert's ground truth segmentation. CONCLUSIONS: The proposed method outperformed the state-of-the-art techniques based on recall, precision, the F1-score, and the Dice coefficient.

Geometrical Properties of the Nucleus and Chromosome Intermingling Are Possible Major Parameters of Chromosome Aberration Formation.

Ionizing radiation causes chromosome aberrations, which are possible biomarkers to assess space radiation cancer risks. Using the Monte Carlo codes Relativistic Ion Tracks (RITRACKS) and Radiation-Induced Tracks, Chromosome Aberrations, Repair and Damage (RITCARD), we investigated how geometrical properties of the cell nucleus, irradiated with ion beams of linear energy transfer (LET) ranging from 0.22 keV/µm to 195 keV/µm, influence the yield of simple and complex exchanges. We focused on the effect of (1) nuclear volume by considering spherical nuclei of varying radii; (2) nuclear shape by considering ellipsoidal nuclei of varying thicknesses; (3) beam orientation; and (4) chromosome intermingling by constraining or not constraining chromosomes in non-overlapping domains. In general, small nuclear volumes yield a higher number of complex exchanges, as compared to larger nuclear volumes, and a higher number of simple exchanges for LET < 40 keV/µm. Nuclear flattening reduces complex exchanges for high-LET beams when irradiated along the flattened axis. The beam orientation also affects yields for ellipsoidal nuclei. Reducing chromosome intermingling decreases both simple and complex exchanges. Our results suggest that the beam orientation, the geometry of the cell nucleus, and the organization of the chromosomes within are important parameters for the formation of aberrations that must be considered to model and translate in vitro results to in vivo risks.

Analysis of a non-Markov transcription model with nuclear RNA export and RNA nuclear retention.

Transcription involves gene activation, nuclear RNA export (NRE) and RNA nuclear retention (RNR). All these processes are multistep and biochemical. A multistep reaction process can create memories between reaction events, leading to non-Markovian kinetics. This raises an unsolved issue: how does molecular memory affect stochastic transcription in the case that NRE and RNR are simultaneously considered? To address this issue, we analyze a non-Markov model, which considers multistep activation, multistep NRE and multistep RNR can interpret many experimental phenomena. In order to solve this model, we introduce an effective transition rate for each reaction. These effective transition rates, which explicitly decode the effect of molecular memory, can transform the original non-Markov issue into an equivalent Markov one. Based on this technique, we derive analytical results, showing that molecular memory can significantly affect the nuclear and cytoplasmic mRNA mean and noise. In addition to the results providing insights into the role of molecular memory in gene expression, our modeling and analysis provide a paradigm for studying more complex stochastic transcription processes.

A combinatorial indexing strategy for low-cost epigenomic profiling of plant single cells.

Understanding how cis-regulatory elements facilitate gene expression is a key question in biology. Recent advances in single-cell genomics have led to the discovery of cell-specific chromatin landscapes that underlie transcription programs in animal models. However, the high equipment and reagent costs of commercial systems limit their applications for many laboratories. In this study, we developed a combinatorial index and dual PCR barcode strategy to profile the Arabidopsis thaliana root single-cell epigenome without any specialized equipment. We generated chromatin accessibility profiles for 13 576 root nuclei with an average of 12 784 unique Tn5 integrations per cell. Integration of the single-cell assay for transposase-accessible chromatin sequencing and RNA sequencing data sets enabled the identification of 24 cell clusters with unique transcription, chromatin, and cis-regulatory signatures. Comparison with single-cell data generated using the commercial microfluidic platform from 10X Genomics revealed that this low-cost combinatorial index method is capable of unbiased identification of cell-type-specific chromatin accessibility. We anticipate that, by removing cost, instrumentation, and other technical obstacles, this method will be a valuable tool for routine investigation of single-cell epigenomes and provide new insights into plant growth and development and plant interactions with the environment.