Intact Cell Nuclei Research Articles

Development of mADM-collagen wound dressings for mimicking native skin architecture to enhance skin wound healing

Acellular dermal matrix (ADM) is one of the most promising scaffold materials due to its ability to retain natural extracellular matrix structure. Micronized acellular dermal matrix (mADM) was prepared with no intact cell nuclei and preserved growth factors by High Hydrostatic Pressure (HHP) approach. And mADM-collagen wound dressings were developed with different proportion of type I collagen and recombinant humanized type III collagen. The porous structure of the mADM-collagen wound dressings made them a good candidate for preventing excessive fluid accumulation, while the collagens with gel-like texture combined with mADM powder to form pasty texture wound dressing, which preserving the moisture at the wound site. Moreover, the paste texture of the mADM-collagen wound dressing was easy to reshape to conform any wound shapes and body contours. Furthermore, the resulted mADM-collagen wound dressings showed good biocompatibility by supporting fibroblasts adhesion and proliferation in vitro. Subsequently, a murine model of full-thickness skin wounds was employed to assess its effects on wound healing. Notably, mADM-75% Col-I exhibited superior effects throughout the wound healing process, specifically it promoted neovascularization, skin appendage growth and new skin regeneration. This formulation closely mimicked the collagen ratio found in healthy skin, facilitating the favorable wound repair. These results indicated the superior performance of this mADM-collagen wound dressing providing an optimal environment for wound healing.Graphical

Histone FRET reports the spatial heterogeneity in nanoscale chromatin architecture that is imparted by the epigenetic landscape at the level of single foci in an intact cell nucleus

Genome sequencing has identified hundreds of histone post-translational modifications (PTMs) that define an open or compact chromatin nanostructure at the level of nucleosome proximity, and therefore serve as activators or repressors of gene expression. Direct observation of this epigenetic mode of transcriptional regulation in an intact single nucleus, is however, a complex task. This is because despite the development of fluorescent probes that enable observation of specific histone PTMs and chromatin density, the changes in nucleosome proximity regulating gene expression occur on a spatial scale well below the diffraction limit of optical microscopy. In recent work, to address this research gap, we demonstrated that the phasor approach to fluorescence lifetime imaging microscopy (FLIM) of Förster resonance energy transfer (FRET) between fluorescently labelled histones core to the nucleosome, is a readout of chromatin nanostructure that can be multiplexed with immunofluorescence (IF) against specific histone PTMs. Here from application of this methodology to gold standard gene activators (H3K4Me3 and H3K9Ac) versus repressors (e.g., H3K9Me3 and H3K27Me), we find that while on average these histone marks do impart an open versus compact chromatin nanostructure, at the level of single chromatin foci, there is significant spatial heterogeneity. Collectively this study illustrates the importance of studying the epigenetic landscape as a function of space within intact nuclear architecture and opens the door for the study of chromatin foci sub-populations defined by combinations of histone marks, as is seen in the context of bivalent chromatin.

A comprehensive method to study the DNA's association with lamin and chromatin compaction in intact cell nuclei at super resolution.

Super-resolution fluorescence microscopy has revolutionized multicolor imaging of nuclear structures due to the combination of high labeling specificity and high resolution. Here we expanded the recently developed fBALM (DNA structure fluctuation-assisted binding activated localization microscopy) method by developing a stable methodological sequence that enables dual-color imaging of high-resolution genomic DNA together with an immunofluorescently labeled intranuclear protein. Our measurements of the nuclear periphery, imaging DNA and LaminB1 in biologically relevant samples, show that this novel dual-color imaging method is feasible for further quantitative evaluations. We were able to study the relative spatial signal organization between DNA and LaminB1 by means of highly specific colocalization measurements at nanometer resolution. Measurements were performed with and without the antifade embedding medium ProLong Gold, which proved to be essential for imaging of LaminB1, but not for imaging of SytoxOrange labeled DNA. The localization precision was used to differentiate between localizations with higher and lower amounts of emitting photons. We interpret high intensity localizations to be renatured DNA sections in which a high amount of Sytox Orange molecules were bound. This could give insight into the denaturation kinetics of DNA during fBALM. These results were further complemented by measurements of γH2AX and H3K9me3 signal organization to demonstrate differences within the chromatin landscape, which were quantified with image processing methods such as Voronoi segmentation.

Investigation of Oncogene-Induced Alterations in Chromatin Organization In Vitro by Structured Illumination Microscopy

Oncogenes activation may lead to alterations in chromatin organization at the nanoscale by interfering with fundamental processes such as DNA transcription, DNA replication, and DNA Damage Response [1]. The in vitro observation of these molecular processes is crucial to understand how oncogenes can alter chromatin organization leading to tumor formation.Among all the available techniques to address this issue, optical super-resolution microscopy stands out for its potential in revealing the distribution of molecules within intact cell nuclei. Here we apply structured illumination microscopy (SIM) [2] to investigate a model of oncogene activation in an engineered cell line, U937-PR9, derived from Acute Promyelocytic Leukemia(APL) patients. In APL, 70% of the patients present a mutation that causes the formation of a fusion protein PML-RARalpha. This model allows us to selectively activate the PML-RAR alpha oncogene, simulating cancer behavior. In this work, we exploit multi-color SIM to study the relative spatial distributions of replication and transcription foci in relation to the expression of the oncoprotein. Then we analyze and quantify the distribution of the nuclear sites through object-based method analysis and also through Image Cross-Correlation Spectroscopy (ICCS) [3].[Work supported in part by AIRC MFAG 2018 - ID.21931][1] Negrini et al, Nat. Rev. Mol. Cell. Biol. (2010)[2] Gustafsson et al, Biophys. J. (2008) [3] Oneto et al, Biophys. J. (2019)

Optimized Super-Resolution Imaging of Nuclear Sites in an Engineered Leukemia Cell Line

Stimulated emission depletion (STED) microscopy and Separation of Photons by Lifetime Tuning (SPLIT) provide optical super-resolution imaging, at the nanometer scale, on intact single cell nuclei. The resolution that can be achieved is limited by factors such as photobleaching and/or reduction of signal-to-noise, which depend on the conditions of image acquisition. Here we use image correlation spectroscopy (ICS) to quantify, in an unbiased way, the resolution and signal-to-noise of STED and SPLIT images and determine how they are affected by multiple parameters such as photobleaching, STED power, and number of averages. Imaging conditions optimization is particularly relevant when it comes to analyzing genomic processes, such as DNA replication, occurring within the crowded nuclear environment. As an application, we optimize the imaging conditions on an engineered cell line, U937-PR9, derived from patients suffering from Acute Promyelocytic Leukemia (APL). This cell line allows us to selectively activate the expression of the PML-RARα oncoprotein and to analyze its effects on functional nuclear sites. Carefully optimized super-resolved images will allow the study of nuclear processes at a single-cell level. [Work funded by AIRC MFAG 2018 - ID.21931]

A Multi-Omics Approach to Liver Diseases: Integration of Single Nuclei Transcriptomics with Proteomics and HiCap Bulk Data in Human Liver.

The liver is the largest solid organ and a primary metabolic hub. In recent years, intact cell nuclei were used to perform single-nuclei RNA-seq (snRNA-seq) for tissues difficult to dissociate and for flash-frozen archived tissue samples to discover unknown and rare cell subpopulations. In this study, we performed snRNA-seq of a liver sample to identify subpopulations of cells based on nuclear transcriptomics. In 4282 single nuclei, we detected, on average, 1377 active genes and we identified seven major cell types. We integrated data from 94,286 distal interactions (p < 0.05) for 7682 promoters from a targeted chromosome conformation capture technique (HiCap) and mass spectrometry proteomics for the same liver sample. We observed a reasonable correlation between proteomics and in silico bulk snRNA-seq (r = 0.47) using tissue-independent gene-specific protein abundancy estimation factors. We specifically looked at genes of medical importance. The DPYD gene is involved in the pharmacogenetics of fluoropyrimidine toxicity and some of its variants are analyzed for clinical purposes. We identified a new putative polymorphic regulatory element, which may contribute to variation in toxicity. Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer and we investigated all known risk genes. We identified a complex regulatory landscape for the SLC2A2 gene with 16 candidate enhancers. Three of them harbor somatic motif breaking and other mutations in HCC in the Pan Cancer Analysis of Whole Genomes dataset and are candidates to contribute to malignancy. Our results highlight the potential of a multi-omics approach in the study of human diseases.

An Effective Encoder-Decoder Network for Neural Cell Bodies and Cell Nucleus Segmentation of EM Images.

Neural systems are complicated networks connected by a large number of neurons through gap junctions and synapse. At present, for electron microscopy connectomics research, neuron structure recognition algorithms mostly focus on synapses, dendrites, axons and mitochondria, etc. However, effective methods for automatic recognition of neuronal cell bodies are rare. In this paper, we proposed an effective encoderdecoder network, which extracted segmentation features of neural cell bodies and cell nucleus by the modified residual network and pyramid module. The framework is capable of merging multi-scale contextual information and generating efficient segmentation results by integrating multilevel features. We applied this proposed network on two segmentation tasks for electron microscope (EM) images and compared it with other promising methods as U-Net and deeplab v3+. The results demonstrated that our method achieved the state-of-the-art performance on quality metrics. Finally, we visualized two intact neural cell bodies and cell nucleus to provide a close look into these fine structures.

&lt;italic&gt;In Situ&lt;/italic&gt; Quantification of the Young’s Modulus of Nuclei in Multiple Cellular States Using a Modified Fiber Probe Sensor

In this paper, a modified fiber probe sensor (MFP sensor) with a considerable length–diameter ratio tip was developed to quantitatively characterize the Young’s modulus of the cell nucleus in multiple cellular states. The MFP sensor has a needle tip with an extended length of $30~\mu \text{m}$ and a diameter of 150 nm. The length of the tip exceeds the diameter of most cells so that it can penetrate the cell membrane with minimal damage and reach the nucleus. This allows the measurement of the Young’s modulus of intact cell nuclei under different cellular states without the phenomenon that the cantilever contacts the cell membrane. Moreover, the stiffness of the MFP sensor is reduced by a factor of 25 to better match that of the cell membrane and nucleus, which enables high-precision mechanical measurements. The MFP sensor was successfully used to quantify the Young’s modulus of the intact cell nuclei in the non-adherent, adherent, and early apoptotic states. Experimental results validated the capability and the versatility of the developed method.

Detection and quantification of VEGFR-1 in the nuclei of tumor cells by a new flow cytometry-based method

Flow cytometry using fluorescent antibodies (FC) is the method of choice for the quantitation of proteins expressed at the surface or inside the cell, but, however, does not allow to selectively measure nuclear expression. We therefore sought to develop a method for the extraction of intact cell nuclei, which can be used for their subsequent immunofluorescent analysis by FC. The studied protein was vascular endothelial growth factor-receptor-type 1 (VEGFR-1) which is important in tumor survival and metastasis. Two human cell lines, A431 (epidermoid carcinoma of skin with low invasive and metastatic potential) and BRO (highly aggressive amelanotic melanoma), were used as examples for tumor cells, and normal human fibroblasts PHF served as a control line. The quality of the extracted nuclei was assessed by their intactness and purity from cytoplasm. The high content of the nuclear markers (PCNA = proliferating cell nuclear antigen, lamin A/C) in the extracted nuclei with almost complete absence of the cytoplasmic β-tubulin demonstrated that the protocol can be used to obtain a pure suspension of single intact cell nuclei. The measurement of the nuclear VEGFR-1 content revealed that it was present only in tumor cell nuclei and that in more malignant BRO cells the receptor content was 1.75 times higher than in A431 (p = 0.014). Thus, the developed method of extraction of cell nuclei for subsequent FC analysis is suitable for the quantitative evaluation of protein content in the native nucleus.

Recruitment of 53BP1 Proteins for DNA Repair and Persistence of Repair Clusters Differ for Cell Types as Detected by Single Molecule Localization Microscopy.

DNA double stranded breaks (DSBs) are the most serious type of lesions introduced into chromatin by ionizing radiation. During DSB repair, cells recruit different proteins to the damaged sites in a manner dependent on local chromatin structure, DSB location in the nucleus, and the repair pathway entered. 53BP1 is one of the important players participating in repair pathway decision of the cell. Although many molecular biology details have been investigated, the architecture of 53BP1 repair foci and its development during the post-irradiation time, especially the period of protein recruitment, remains to be elucidated. Super-resolution light microscopy is a powerful new tool to approach such studies in 3D-conserved cell nuclei. Recently, we demonstrated the applicability of single molecule localization microscopy (SMLM) as one of these highly resolving methods for analyses of dynamic repair protein distribution and repair focus internal nano-architecture in intact cell nuclei. In the present study, we focused our investigation on 53BP1 foci in differently radio-resistant cell types, moderately radio-resistant neonatal human dermal fibroblasts (NHDF) and highly radio-resistant U87 glioblastoma cells, exposed to high-LET 15N-ion radiation. At given time points up to 24 h post irradiation with doses of 1.3 Gy and 4.0 Gy, the coordinates and spatial distribution of fluorescently tagged 53BP1 molecules was quantitatively evaluated at the resolution of 10–20 nm. Clusters of these tags were determined as sub-units of repair foci according to SMLM parameters. The formation and relaxation of such clusters was studied. The higher dose generated sufficient numbers of DNA breaks to compare the post-irradiation dynamics of 53BP1 during DSB processing for the cell types studied. A perpendicular (90°) irradiation scheme was used with the 4.0 Gy dose to achieve better separation of a relatively high number of particle tracks typically crossing each nucleus. For analyses along ion-tracks, the dose was reduced to 1.3 Gy and applied in combination with a sharp angle irradiation (10° relative to the cell plane). The results reveal a higher ratio of 53BP1 proteins recruited into SMLM defined clusters in fibroblasts as compared to U87 cells. Moreover, the speed of foci and thus cluster formation and relaxation also differed for the cell types. In both NHDF and U87 cells, a certain number of the detected and functionally relevant clusters remained persistent even 24 h post irradiation; however, the number of these clusters again varied for the cell types. Altogether, our findings indicate that repair cluster formation as determined by SMLM and the relaxation (i.e., the remaining 53BP1 tags no longer fulfill the cluster definition) is cell type dependent and may be functionally explained and correlated to cell specific radio-sensitivity. The present study demonstrates that SMLM is a highly appropriate method for investigations of spatiotemporal protein organization in cell nuclei and how it influences the cell decision for a particular repair pathway at a given DSB site.

Histone Interaction Landscapes Visualized by Crosslinking Mass Spectrometry in Intact Cell Nuclei

Cells organize their actions partly through tightly controlled protein-protein interactions-collectively termed the interactome. Here we use crosslinking mass spectrometry (XL-MS) to chart the protein-protein interactions in intact human nuclei. Overall, we identified ∼8,700 crosslinks, of which 2/3 represent links connecting distinct proteins. From these data, we gain insights on interactions involving histone proteins. We observed that core histones on the nucleosomes expose well-defined interaction hot spots. For several nucleosome-interacting proteins, such as USF3 and Ran GTPase, the data allowed us to build low-resolution models of their binding mode to the nucleosome. For HMGN2, the data guided the construction of a refined model of the interaction with the nucleosome, based on complementary NMR, XL-MS, and modeling. Excitingly, the analysis of crosslinks carrying posttranslational modifications allowed us to extract how specific modifications influence nucleosome interactions. Overall, our data depository will support future structural and functional analysis of cell nuclei, including the nucleoprotein assemblies they harbor.

Decellularization and characterization of a whole intervertebral disk xenograft scaffold.

Intervertebral disk (IVD) degeneration is a multifactor process that results in the physical destruction of the nucleus pulposus (NP) and annulus fibrosus (AF). This compromises IVD function and causes significant disability and economic burden. Strategies to replace the entire composite structure of the IVD are limited and most approaches do not recapitulate the heterogenous biochemical composition, microarchitecture or mechanical properties of the native tissue. Our central hypothesis was that donor IVDs which resemble the size and biochemistry of human lumbar IVDs could be successfully decellularized while retaining the tissue's structure and function with the long-term goal of creating a composite scaffold for tissue engineering the human IVD. Accordingly, we optimized a procedure to decellularize bovine tail IVDs using a combination of detergents, ultrasonication, freeze-thaw cycles, and nucleases. The resultant decellularized whole IVD xenografts retained distinct AF and NP regions which contained no visible intact cell nuclei and minimal residual bovine deoxyribose nucleic acid (DNA; 65.98 ± 4.07 and 47.12 ± 13.22 ng/mg, respectively). Moreover, the NP region of decellularized IVDs contained 313.40 ± 50.67 µg/mg glycosaminoglycan. The presence of collagen type II was confirmed via immunohistochemistry. Additionally, histological analysis of the AF region of decellularized IVDs demonstrated retention of the native angle-ply collagen microarchitecture. Unconfined compression testing demonstrated no significant differences in swelling pressure and toe-region modulus between fresh and decellularized IVDs. However, linear region moduli, peak stress and equilibrium moduli were all significantly reduced. Together, this research demonstrates a successful initial step in developing a biomimetic acellular whole IVD xenograft scaffold for use in IVD tissue engineering. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A:2412-2423, 2018.

Development of a robust immuno-S-FISH protocol using imaging flow cytometry.

Fluorescence in situ hybridization (FISH) is a microscopy technique which uses a fluorescent probe to detect DNA sequences and is generally performed on metaphase spreads or interphase nuclei of intact cells on a slide. In a diagnostic laboratory, cells are hybridized with fluorescent probes and up to 200 cells counted for the number of cells with probe "spots." Recent modifications to standard FISH include immuno-FISH, where chromosomal abnormalities are detected only in cells by their phenotype, and S-FISH where probe hybridization is performed on whole cells in suspension. Here we describe the development of an immuno-S-FISH method that combines immunophenotyping and FISH analysis of cells in suspension followed by analysis on an imaging flow cytometer. This single platform technique couples microscopy with flow cytometry and "spot" detection of bound FISH probe. Automated immuno-S-FISH enables large numbers of analyzed cells to be identified by phenotype and assessed for specific chromosomal determinants by FISH. This novel robust method enables quantitative cell population analysis and "spot" counting for large numbers of cells. We report method optimization of this imaging immuno-S-FISH flow cytometry protocol which has capability for many clinical applications. © 2016 International Society for Advancement of Cytometry.

Shaping mitotic chromosomes: From classical concepts to molecular mechanisms.

How eukaryotic genomes are packaged into compact cylindrical chromosomes in preparation for cell divisions has remained one of the major unsolved questions of cell biology. Novel approaches to study the topology of DNA helices inside the nuclei of intact cells, paired with computational modeling and precise biomechanical measurements of isolated chromosomes, have advanced our understanding of mitotic chromosome architecture. In this Review Essay, we discuss – in light of these recent insights – the role of chromatin architecture and the functions and possible mechanisms of SMC protein complexes and other molecular machines in the formation of mitotic chromosomes. Based on the information available, we propose a stepwise model of mitotic chromosome condensation that envisions the sequential generation of intra-chromosomal linkages by condensin complexes in the context of cohesin-mediated inter-chromosomal linkages, assisted by topoisomerase II. The described scenario results in rod-shaped metaphase chromosomes ready for their segregation to the cell poles.

The Balbiani Ring Story: Synthesis, Assembly, Processing, and Transport of Specific Messenger RNA-Protein Complexes.

Eukaryotic gene expression is the result of the integrated action of multimolecular machineries. These machineries associate with gene transcripts, often already nascent precursor messenger RNAs (pre-mRNAs). They rebuild the transcript and convey properties allowing the processed transcript, the mRNA, to be exported to the cytoplasm, quality controlled, stored, translated, and degraded. To understand these integrated processes, one must understand the temporal and spatial aspects of the fate of the gene transcripts in relation to interacting molecular machineries. Improved methodology is necessary to study gene expression in vivo for endogenous genes. A complementary approach is to study biological systems that provide exceptional experimental possibilities. We describe such a system, the Balbiani ring (BR) genes in polytene cells in the dipteran Chironomus tentans. The BR genes, along with their pre-mRNA-protein complexes (pre-mRNPs) and mRNA-protein complexes (mRNPs), allow the visualization of intact cell nuclei and enable analyses of where and when different molecular machineries associate with and act on the BR pre-mRNAs and mRNAs.

X-linked inhibitor of apoptosis protein (XIAP) lacking RING domain localizes to the nuclear and promotes cancer cell anchorage-independent growth by targeting the E2F1/Cyclin E axis.

The inhibitor of apoptosis protein XIAP (X-linked inhibitor of apoptosis protein) is a well-documented protein that is located in cytoplasm acting as a potent regulator of cell apoptosis. Here, we showed that expressing XIAP with RING (Really Interesting New Gene) domain deletion (XIAP△RING) in cancer cells promoted cancer cell anchorage-independent growth and G1/S phase transition companied with increasing cyclin e transcription activity and protein expression. Further studies revealed that XIAP△RING was mainly localized in nuclear with increased binding with E2F1, whereas XIAP with BIR (Baculoviral IAP Repeat) domains deletion (XIAP△BIRs) was entirely presented in cytoplasma with losing its binding with E2F1, suggesting that RING domain was able to inhibit BIR domains nuclear localization, by which impaired BIRs binding with E2F1 in cellular nucleus in intact cells. These studies identified a new function of XIAP protein in cellular nucleus is to regulate E2F1 transcriptional activity by binding with E2F1 in cancer cells. Our current finding of an effect of XIAP△RING expression on cancer cell anchorage-independent growth suggests that overexpression of this protein may contribute to genetic instability associated with cell cycle and checkpoint perturbations, in addition to its impact on cellular apoptosis.

Imaging feedback for histotripsy by characterizing dynamics of acoustic radiation force impulse (ARFI)-induced shear waves excited in a treated volume.

Our previous study indicated that shear waves decay and propagate at a lower speed as they propagate into a tissue volume mechanically fractionated by histotripsy. In this paper, we hypothesize that the change in the shear dynamics is related to the degree of tissue fractionation, and can be used to predict histotripsy treatment outcomes. To test this hypothesis, lesions with different degrees of tissue fractionation were created in agar-graphite tissue phantoms and ex vivo kidneys with increasing numbers of therapy pulses, from 0 to 2000 pulses per treatment location. The therapy pulses were 3-cycle 750-kHz focused ultrasound delivered at a peak negative/positive pressure of 17/108 MPa and a repetition rate of 50 Hz. The shear waves were excited by acoustic radiation force impulse (ARFI) focused at the center of the lesion. The spatial and temporal behavior of the propagating shear waves was measured with ultrasound plane wave imaging. The temporal displacement profile at a lateral location 10 mm offset to the shear excitation region was detected with M-mode imaging. The decay and delay of the shear waves were quantitatively characterized on the temporal displacement profile. Results showed significant changes in two characteristics on the temporal displacement profile: the peak-to-peak displacement decayed exponentially with increasing numbers of therapy pulses; the relative time-to-peak displacement increased with increasing numbers of therapy pulses, and appeared to saturate at higher numbers of pulses. Correspondingly, the degree of tissues fractionation, as indicated by the percentage of structurally intact cell nuclei, decreased exponentially with increasing numbers of therapy pulses. Strong linear correlations were found between the two characteristics and the degree of tissue fractionation. These results suggest that the characteristics of the shear temporal displacement profile may provide useful feedback information regarding the treatment outcomes.

Nuclear expression of renin-angiotensin system components in NRK-52E renal epithelial cells.

Isolated nuclei of sheep proximal tubules express angiotensin (Ang) receptors as well as angiotensinogen (AGT) and renin. The present study characterized the NRK-52E tubular epithelial cell line for the intracellular expression of renin-angiotensin system (RAS) components. RAS components were visualized by immunofluorescent staining in intact cells and protein expression in isolated nuclei. An antibody to the angiotensin I (Ang I) sequence of AGT (AI-AGT) revealed only cytosolic staining, while an antibody to an internal sequence of AGT (Int-AGT) revealed primarily nuclear staining. Immunoblots of nuclear and cytosolic fractions confirmed the differential cell staining of AGT. Immunostaining for renin was present on nuclei of intact cells. Nuclear renin activity averaged 0.77±0.05 nmol/mg protein/h that was reduced by aliskiren (0.13±0.01 nmol/mg/h, n=3, p<0.01); trypsin activation increased activity three-fold. Peptide staining localized angiotensin II (Ang II) and Ang-(1-7) to the nucleus and peptide content averaged 59±2 and 57±22 fmol/mg (n=4), respectively. Peptide metabolism in isolated nuclei revealed the processing of Ang I to Ang-(1-7) by thimet oligopeptidase. We conclude that the NRK-52E cells express an intracellular RAS localized to the nucleus and may be an appropriate cell model to elucidate the functional relevance of this system.

Characterizing dynamics of shear waves induced with acoustic radiation force impulse in histotripsy lesions for treatment feedback

Histotripsy mechanically fractionates soft tissues into fluid-like homogenates that cannot support shear waves. We hypothesize that dynamics of shear waves excited from a histotripsy lesion using acoustic radiation force impulse (ARFI) change progressively during the fractionation process, and such change is related to the degree of tissue fractionation. To test this hypothesis, lesions with different degrees of fractionation were created in agar-graphite tissue mimicking phantoms and ex vivo kidneys with increasing numbers of histotripsy pulses (3-cycle 750- kHz ultrasound pulses at a peak negative/positive pressure of 17/108 MPa). The shear waves were excited by ARFI focused at the lesion center. The shear-induced temporal displacement profile was measured at a lateral location 10 mm offset to the lesion with M-mode imaging. Results showed significant changes in two characteristics: the peak-to-peak displacement decayed exponentially, and the relative time-to-peak displacement increased and saturated with increasing numbers of histotripsy pulses (N = 6). Correspondingly, the degree of tissues fractionation, as indicated by the percentage of structurally intact cell nuclei, decreased exponentially. Strong linear correlations existed between the two characteristics and the degree of tissue fractionation (R2 &amp;gt; 0.97). These results suggest that characteristics of shear waves induced in a histotripsy lesion may provide useful feedback for treatment outcomes.

Functional implications of genome topology.

Although genomes are defined by their sequence, the linear arrangement of nucleotides is only their most basic feature. A fundamental property of genomes is their topological organization in three-dimensional space in the intact cell nucleus. The application of imaging methods and genome-wide biochemical approaches, combined with functional data, is revealing the precise nature of genome topology and its regulatory functions in gene expression and genome maintenance. The emerging picture is one of extensive self-enforcing feedback between activity and spatial organization of the genome, suggestive of a self-organizing and self-perpetuating system that uses epigenetic dynamics to regulate genome function in response to regulatory cues and to propagate cell-fate memory.