Pathophysiological Implications of Protein Lactylation in Pancreatic Epithelial Tumors.

Protein lactylation is a post-translational modification associated with glycolysis. Although recent evidence indicates that protein lactylation is involved in epigenetic gene regulation, its pathophysiological significance remains unclear, particularly in neoplasms. Herein, we investigated the potential involvement of protein lactylation in the molecular mechanisms underlying benign and malignant pancreatic epithelial tumors, as well as its role in the response of pancreatic cancer (PC) cells to gemcitabine. Increased lactylation was observed in the nuclei of intraductal papillary mucinous adenoma, non-invasive intraductal papillary mucinous carcinoma, and invasive carcinoma, in parallel to the upregulation of hypoxia-inducible factor-1α. This observation indicated that a hypoxia-associated increase in nuclear protein lactylation could be a biochemical hallmark in pancreatic epithelial tumors. The standard PC chemotherapy drug gemcitabine suppressed histone lactylation in vitro, suggesting that histone lactylation might be relevant to its mechanism of action. Taken together, our findings suggest that protein lactylation may be involved in the development of pancreatic epithelial tumors and could represent a potential therapeutic target for PC.

On-the-fly Raman microscopy guaranteeing the accuracy of discrimination.

Accelerating the measurement for discrimination of samples, such as classification of cell phenotype, is crucial when faced with significant time and cost constraints. Spontaneous Raman microscopy offers label-free, rich chemical information but suffers from long acquisition time due to extremely small scattering cross-sections. One possible approach to accelerate the measurement is by measuring necessary parts with a suitable number of illumination points. However, how to design these points during measurement remains a challenge. To address this, we developed an imaging technique based on a reinforcement learning in machine learning (ML). This ML approach adaptively feeds back "optimal" illumination pattern during the measurement to detect the existence of specific characteristics of interest, allowing faster measurements while guaranteeing discrimination accuracy. Using a set of Raman images of human follicular thyroid and follicular thyroid carcinoma cells, we showed that our technique requires 3,333 to 31,683 times smaller number of illuminations for discriminating the phenotypes than raster scanning. To quantitatively evaluate the number of illuminations depending on the requisite discrimination accuracy, we prepared a set of polymer bead mixture samples to model anomalous and normal tissues. We then applied a home-built programmable-illumination microscope equipped with our algorithm, and confirmed that the system can discriminate the sample conditions with 104 to 4,350 times smaller number of illuminations compared to standard point illumination Raman microscopy. The proposed algorithm can be applied to other types of microscopy that can control measurement condition on the fly, offering an approach for the acceleration of accurate measurements in various applications including medical diagnosis.

Myofibroblasts impair myocardial impulse propagation by heterocellular connexin43 gap-junctional coupling through micropores.

Aim: Composite population of myofibroblasts (MFs) within myocardial tissue is known to alter impulse propagation, leading to arrhythmias. However, it remains unclear whether and how MFs alter their propagation patterns when contacting cardiomyocytes (CMs) without complex structural insertions in the myocardium. We attempted to unveil the effects of the one-sided, heterocellular CM-MF connection on the impulse propagation of CM monolayers without the spatial insertion of MFs as an electrical or mechanical obstacle. Methods and results: We evaluated fluo8-based spatiotemporal patterns in impulse propagation of neonatal rat CM monolayers cultured on the microporous membrane having 8-µm diameter pores with co-culture of MFs or CMs on the reverse membrane side (CM-MF model or CM-CM model, respectively). During consecutive pacing at 1 or 2 Hz, the CM monolayers exhibited forward impulse propagation from the pacing site with a slower conduction velocity (θ) and a larger coefficient of directional θ variation in the CM-MF model than that in the CM-CM model in a frequency-dependent manner (2 Hz >1 Hz). The localized placement of an MF cluster on the reverse side resulted in an abrupt segmental depression of the impulse propagation of the upper CM layer, causing a spatiotemporally non-uniform pattern. Dye transfer of the calcein loaded in the upper CM layer to the lower MF layer was attenuated by the gap-junction inhibitor heptanol. Immunocytochemistry identified definitive connexin 43 (Cx43) between the CMs and MFs in the membrane pores. MF-selective Cx43 knockdown in the MF layer improved both the velocity and uniformity of propagation in the CM monolayer. Conclusion: Heterocellular Cx43 gap junction coupling of CMs with MFs alters the spatiotemporal patterns of myocardial impulse propagation, even in the absence of spatially interjacent and mechanosensitive modulations by MFs. Moreover, MFs can promote pro-arrhythmogenic impulse propagation when in face-to-face contact with the myocardium that arises in the healing infarct border zone.

Correction for Extrinsic Background in Raman Hyperspectral Images.

Raman hyperspectral microscopy is a valuable tool in biological and biomedical imaging. Because Raman scattering is often weak in comparison to other phenomena, prevalent spectral fluctuations and contaminations have brought advancements in analytical and chemometric methods for Raman spectra. These chemometric advances have been key contributors to the applicability of Raman imaging to biological systems. As studies increase in scale, spectral contamination from extrinsic background, intensity from sources such as the optical components that are extrinsic to the sample of interest, has become an emerging issue. Although existing baseline correction schemes often reduce intrinsic background such as autofluorescence originating from the sample of interest, extrinsic background is not explicitly considered, and these methods often fail to reduce its effects. Here, we show that extrinsic background can significantly affect a classification model using Raman images, yielding misleadingly high accuracies in the distinction of benign and malignant samples of follicular thyroid cell lines. To mitigate its effects, we develop extrinsic background correction (EBC) and demonstrate its use in combination with existing methods on Raman hyperspectral images. EBC isolates regions containing the smallest amounts of sample materials that retain extrinsic contributions that are specific to the device or environment. We perform classification both with and without the use of EBC, and we find that EBC retains biological characteristics in the spectra while significantly reducing extrinsic background. As the methodology used in EBC is not specific to Raman spectra, correction of extrinsic effects in other types of hyperspectral and grayscale images is also possible.

Differentiability of cell types enhanced by detrending a non-homogeneous pattern in a line-illumination Raman microscope.

A line illumination Raman microscope extracts the underlying spatial and spectral information of a sample, typically a few hundred times faster than raster scanning. This makes it possible to measure a wide range of biological samples such as cells and tissues - that only allow modest intensity illumination to prevent potential damage - within feasible time frame. However, a non-uniform intensity distribution of laser line illumination may induce some artifacts in the data and lower the accuracy of machine learning models trained to predict sample class membership. Here, using cancerous and normal human thyroid follicular epithelial cell lines, FTC-133 and Nthy-ori 3-1 lines, whose Raman spectral difference is not so large, we show that the standard pre-processing of spectral analyses widely used for raster scanning microscopes introduced some artifacts. To address this issue, we proposed a detrending scheme based on random forest regression, a nonparametric model-free machine learning algorithm, combined with a position-dependent wavenumber calibration scheme along the illumination line. It was shown that the detrending scheme minimizes the artifactual biases arising from non-uniform laser sources and significantly enhances the differentiability of the sample states, i.e., cancerous or normal epithelial cells, compared to the standard pre-processing scheme.

High-throughput line-illumination Raman microscopy with multislit detection.

Raman microscopy is an emerging tool for molecular imaging and analysis of living samples. Use of Raman microscopy in life sciences is, however, still limited because of its slow measurement speed for spectral imaging and analysis. We developed a multiline-illumination Raman microscope to achieve ultrafast Raman spectral imaging. A spectrophotometer equipped with a periodic array of confocal slits detects Raman spectra from a sample irradiated by multiple line illuminations. A comb-like Raman hyperspectral image is formed on a two-dimensional detector in the spectrophotometer, and a hyperspectral Raman image is acquired by scanning the sample with multiline illumination array. By irradiating a sample with 21 simultaneous illumination lines, we achieved high-throughput Raman hyperspectral imaging of mouse brain tissue, acquiring 1108800 spectra in 11.4 min. We also measured mouse kidney and liver tissue as well as conducted label-free live-cell molecular imaging. The ultrafast Raman hyperspectral imaging enabled by the presented technique will expand the possible applications of Raman microscopy in biological and medical fields.

Raman imaging of rat nonalcoholic fatty liver tissues reveals distinct biomolecular states.

An essential challenge in diagnosing states of nonalcoholic fatty liver disease (NAFLD) is the early prediction of progression from nonalcoholic fatty liver (NAFL) to nonalcoholic steatohepatitis (NASH) before the disease progresses. Histological diagnoses of NAFLD rely on the appearance of anomalous tissue morphologies, and it is difficult to segment the biomolecular environment of the tissue through a conventional histopathological approach. Here, we show that hyperspectral Raman imaging provides diagnostic information on NAFLD in rats, as spectral changes among disease states can be detected before histological characteristics emerge. Our results demonstrate that Raman imaging of NAFLD can be a useful tool for histopathologists, offering biomolecular distinctions among tissue states that cannot be observed through standard histopathological means.

Generation of myocyte agonal Ca2+ waves and contraction bands in perfused rat hearts following irreversible membrane permeabilisation.

Although irreversible cardiomyocyte injury provokes intracellular Ca2+ ([Ca2+]i) overload, the underlying dynamics of this response and its effects on cellular morphology remain unknown. We therefore visualised rapid-scanning confocal fluo4-[Ca2+]i dynamics and morphology of cardiomyocytes in Langendorff-perfused rat hearts following saponin-membrane permeabilisation. Our data demonstrate that 0.4% saponin-treated myocytes immediately exhibited high-frequency Ca2+ waves (131.3 waves/min/cell) with asynchronous, oscillatory contractions having a mean propagation velocity of 117.8 µm/s. These waves slowly decreased in frequency, developed a prolonged decay phase, and disappeared in 10 min resulting in high-static, fluo4-fluorescence intensity. The myocytes showing these waves displayed contraction bands, i.e., band-like actin-fibre aggregates with disruption of sarcomeric α-actinin. The contraction bands were not attenuated by the abolition of Ca2+ waves under pretreatment with ryanodine plus thapsigargin, but were partially attenuated by the calpain inhibitor MDL28170, while mechanical arrest of the myocytes by 2,3-butanedione monoxime completely attenuated contraction-band formation. The depletion of adenosine 5'-triphosphate by the mitochondrial electron uncoupler carbonyl cyanide 4-trifluoromethoxy phenylhydrazone also attenuated Ca2+ waves and contraction bands. Overall, saponin-induced myocyte [Ca2+]i overload provokes agonal Ca2+ waves and contraction bands. Contraction bands are not the direct consequence of the waves but are caused by cross-bridge interactions of the myocytes under calpain-mediated proteolysis.

Lipid droplet accumulation and adipophilin expression in follicular thyroid carcinoma.

Follicular neoplasms of the thyroid include follicular thyroid carcinoma (FTC) and follicular thyroid adenoma (FTA). However, the differences in cytological findings between FTC and FTA remain undetermined. Here, we aimed to evaluate the accumulation of lipid droplets (LDs) and the expression of adipophilin (perilipin 2/ADRP/ADFP), a known LD marker, in cultured FTC cells. We also immunohistochemically compared adipophilin expression in the FTC and FTA of resected human thyroid tissues. Cultured FTC (FTC-133 and RO82W-1) possessed increased populations of LDs compared to thyroid follicular epithelial (Nthy-ori 3-1) cells. In vitro treatment with phosphatidylinositol-3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling inhibitors (LY294002, MK2206, and rapamycin) in FTC-133 cells downregulated the PI3K/Akt/mTOR/sterol regulatory element-binding protein 1 (SREBP1) signaling pathway, resulting in a significant reduction in LD accumulation. SREBP1 is a master transcription factor that controls lipid metabolism. Fluorescence immunocytochemistry revealed adipophilin expression in the LDs of FTC-133 cells. Immunohistochemical analysis of surgically resected human thyroid tissues revealed significantly increased expression of adipophilin in FTC compared with FTA and adjacent non-tumorous thyroid epithelia. Taken together, LDs and adipophilin were abundant in cultured FTC; the evaluation of adipophilin expression can help distinguish FTC from FTA in surgical specimens.

Transcatheter Arterial Embolization for Intramammary Hemorrhage Caused by a Seat Belt Injury: Case Series Including Experience with N-butyl-2-cyanoacrylate.

In this study, we report two cases of transcatheter arterial embolization for intramammary hemorrhage caused by seat belt injuries. All patients were female drivers involved in traffic accidents. In each case, we accessed the hemorrhage through the left brachial artery and embolized the perforating branch of the left internal mammary artery with N-butyl-2-cyanoacrylate, obtaining effective hemostasis. Transcatheter arterial embolization is considered effective for breast hemorrhage because of rare but dangerous seat belt injuries.

Label-free Assessment of the Nascent State of Rat Non-alcoholic Fatty Liver Disease Using Spontaneous Raman Microscopy.

Spontaneous Raman microscopy, which can detect molecular vibrations in cells and tissues, could be a useful tool for the label-free assessment of non-alcoholic fatty liver disease (NAFLD). However, it is unclear whether it can be used to evaluate the nascent state of NAFLD. To address this, we analyzed the Raman spectra of rat liver tissues in the nascent state of NAFLD upon excitation at 532 nm. Raman and histochemical analyses were performed of liver tissues from rats fed a high-fat, high-cholesterol diet (HFHCD). Raman microscopic imaging analysis of formalin-fixed thin tissue slices showed hepatic steatosis, as revealed by the Raman band at 2,854 cm-1, whereas lipid droplets were not detectable by hematoxylin-eosin staining of images until 3 days after feeding a HFHCD. Raman signals of retinol at 1,588 cm-1 emitted from hepatic stellate cells were distributed alongside hepatic cords; the retinol content rapidly decreased after feeding a HFHCD, whereas hepatic lipid content increased inversely. Raman microscopic analysis of the surface of fresh ex vivo livers enabled early detection of lipid accumulation after a 1-day feeding a HFHCD. In conclusion, spontaneous Raman microscopy can be applied to the label-free evaluation of the nascent state of NAFLD liver tissues.

Using saturated absorption for superresolution laser scanning transmission microscopy.

We improved the three-dimensional spatial resolution of laser scanning transmission microscopy by exploiting the saturated absorption of dye molecules. The saturated absorption is induced by the high-intensity light irradiation and localises the signal within the centre of the focal spot. Our numerical calculation indicates that the spatial resolution in transmission imaging is significantly improved for both lateral and axial directions using nonlinear transmitted signals induced by saturated absorption. We experimentally demonstrated the improvement of the three-dimensional resolution by observing fine structures of stained rat kidney tissues, which were not able to be visualised by conventional laser scanning transmission microscopy.

Confocal laser scanning microscopy is a powerful technique for three-dimensional imaging to study structures in a specimen. The use of confocal pinhole provides three-dimensional spatial resolution in various types of optical microscopes, such as fluorescence, reflection and scattering. However, in transmission microscopy, the confocal pinhole cannot provide the same effect because the spatial information on the optical axial is not transferred in the imaging system. Therefore, the three-dimensional distribution of light absorbers cannot be observed by laser scanning transmission microscopy. In this paper, we propose the use of saturated absorption to image the three-dimensional distribution of light absorbers in a sample by laser scanning transmission microscopy. The saturated absorption is induced by the high-intensity light irradiation and occurs prominently at the centre of a focal spot. The information of the saturated absorption signal within the focal spot is transferred to the transmitted light, providing the capability of optical sectioning in transmission imaging. In our research, we theoretically and experimentally confirmed that light absorption by dye molecules is saturable at the high illumination intensity, and the saturated absorption signal can be extracted by harmonic demodulation. We obtained the images of a stained rat kidney tissue by selectively detecting the nonlinear transmission signals induced by saturable absorption of the dye molecules. We confirmed the high depth discrimination capability of our technique clearly visualised the fine structures in the specimen that cannot be observed by a conventional laser scanning absorption microscope.

Repression of germline genes by PRC1.6 and SETDB1 in the early embryo precedes DNA methylation-mediated silencing.

Silencing of a subset of germline genes is dependent upon DNA methylation (DNAme) post-implantation. However, these genes are generally hypomethylated in the blastocyst, implicating alternative repressive pathways before implantation. Indeed, in embryonic stem cells (ESCs), an overlapping set of genes, including germline "genome-defence" (GGD) genes, are upregulated following deletion of the H3K9 methyltransferase SETDB1 or subunits of the non-canonical PRC1 complex PRC1.6. Here, we show that in pre-implantation embryos and naïve ESCs (nESCs), hypomethylated promoters of germline genes bound by the PRC1.6 DNA-binding subunits MGA/MAX/E2F6 are enriched for RING1B-dependent H2AK119ub1 and H3K9me3. Accordingly, repression of these genes in nESCs shows a greater dependence on PRC1.6 than DNAme. In contrast, GGD genes are hypermethylated in epiblast-like cells (EpiLCs) and their silencing is dependent upon SETDB1, PRC1.6/RING1B and DNAme, with H3K9me3 and DNAme establishment dependent upon MGA binding. Thus, GGD genes are initially repressed by PRC1.6, with DNAme subsequently engaged in post-implantation embryos.

Super-resolved Raman microscopy using random structured light illumination: Concept and feasibility.

In this article, we report the use of randomly structured light illumination for chemical imaging of molecular distribution based on Raman microscopy with improved image resolution. Random structured basis images generated from temporal and spectral characteristics of the measured Raman signatures were superposed to perform structured illumination microscopy (SIM) with the blind-SIM algorithm. For experimental validation, Raman signatures corresponding to Rhodamine 6G (R6G) in the waveband of 730-760 nm and Raman shift in the range of 1096-1634 cm-1 were extracted and reconstructed to build images of R6G. The results confirm improved image resolution using the concept and hints at super-resolution by almost twice better than the diffraction-limit.

Raman Spectroscopic Assessment of Myocardial Viability in Langendorff-Perfused Ischemic Rat Hearts.

Spontaneous Raman spectroscopy, which senses changes in cellular contents of reduced cytochrome c, could be a powerful tool for label-free evaluation of ischemic hearts. However, undetermined is whether it is applicable to evaluation of myocardial viability in ischemic hearts. To address this issue, we investigated sequential changes in Raman spectra of the subepicardial myocardium in the Langendorff-perfused rat heart before and during ligation of the left coronary artery and its subsequent release and re-ligation. Under 532-nm wavelength excitation, the Raman peak intensity of reduced cytochrome c at 747 cm-1 increased quickly after the coronary ligation, and reached a quasi-steady state within 30 min. Subsequent reperfusion of the heart after a short-term (30-min) ligation that simulates reversible conditions resulted in quick recovery of the peak intensity to the baseline. Further re-ligation resulted in resurgence of the peak intensity to nearly the identical value to the first ischemia value. In contrast, reperfusion after prolonged (120-min) ligation that assumes irreversible states resulted in incomplete recovery of the peak intensity, and re-ligation resulted in inadequate resurgence. Electron microscopic observations confirmed the spectral findings. Together, the Raman spectroscopic measurement for cytochrome c could be applicable to evaluation of viability of the ischemic myocardium without labeling.

Paternal age affects offspring via an epigenetic mechanism involving REST/NRSF.

Advanced paternal age can have deleterious effects on various traits in the next generation. Here, we establish a paternal-aging model in mice to understand the molecular mechanisms of transgenerational epigenetics. Whole-genome target DNA methylome analyses of sperm from aged mice reveal more hypo-methylated genomic regions enriched in REST/NRSF binding motifs. Gene set enrichment analyses also reveal the upregulation of REST/NRSF target genes in the forebrain of embryos from aged fathers. Offspring derived from young mice administrated with a DNA de-methylation drug phenocopy the abnormal vocal communication of pups derived from aged fathers. In conclusion, hypo-methylation of sperm DNA can be a key molecular feature modulating neurodevelopmental programs in offspring by causing fluctuations in the expression of REST/NRSF target genes.

Surface Plasmon Localization-Based Super-resolved Raman Microscopy.

We investigate label-free measurement of molecular distribution by super-resolved Raman microscopy using surface plasmon (SP) localization. Localized SP was formed with plasmonic nanopost arrays (PNAs) for measurement of the molecular distribution in HeLa cells. Compared with conventional Raman microscopy on gold thin films, PNAs induce a localized near-field, which allows for the enhancement of the peak signal-to-noise ratio by as much as 4.5 dB in the Raman shifts. Super-resolved distributions of aromatic amino acids and lipids (C-C stretching and C-H2 twist mode) as targets in HeLa cells were obtained after image reconstruction. Results show almost 4-fold improvement on average in the lateral precision over conventional diffraction-limited Raman microscopy images. Combined with axial imaging in an evanescent field, the results suggest an improvement in optical resolution due to superlocalized light volume by more than an order of magnitude over that of conventional diffraction-limited Raman microscopy.

Comprehensive histochemical profiles of histone modification in male germline cells during meiosis and spermiogenesis: Comparison of young and aged testes in mice.

Human epidemiological studies have shown that paternal aging as one of the risk factors for neurodevelopmental disorders, such as autism, in offspring. A recent study has suggested that factors other than de novo mutations due to aging can influence the biology of offspring. Here, we focused on epigenetic alterations in sperm that can influence developmental programs in offspring. In this study, we qualitatively and semiquantitatively evaluated histone modification patterns in male germline cells throughout spermatogenesis based on immunostaining of testes taken from young (3 months old) and aged (12 months old) mice. Although localization patterns were not obviously changed between young and aged testes, some histone modification showed differences in their intensity. Among histone modifications that repress gene expression, histone H3 lysine 9 trimethylation (H3K9me3) was decreased in the male germline cells of the aged testis, while H3K27me2/3 was increased. The intensity of H3K27 acetylation (ac), an active mark, was lower/higher depending on the stages in the aged testis. Interestingly, H3K27ac was detected on the putative sex chromosomes of round spermatids, while other chromosomes were occupied by a repressive mark, H3K27me3. Among other histone modifications that activate gene expression, H3K4me2 was drastically decreased in the male germline cells of the aged testis. In contrast, H3K79me3 was increased in M-phase spermatocytes, where it accumulates on the sex chromosomes. Therefore, aging induced alterations in the amount of histone modifications and in the differences of patterns for each modification. Moreover, histone modifications on the sex chromosomes and on other chromosomes seems to be differentially regulated by aging. These findings will help elucidate the epigenetic mechanisms underlying the influence of paternal aging on offspring development.

Crack engineering for the construction of arbitrary hierarchical architectures.

Three-dimensional hierarchical morphologies widely exist in natural and biomimetic materials, which impart preferential functions including liquid and mass transport, energy conversion, and signal transmission for various applications. While notable progress has been made in the design and manufacturing of various hierarchical materials, the state-of-the-art approaches suffer from limited materials selection, high costs, as well as low processing throughput. Herein, by harnessing the configurable elastic crack engineering-controlled formation and configuration of cracks in elastic materials-an effect normally avoided in various industrial processes, we report the development of a facile and powerful technique that enables the faithful transfer of arbitrary hierarchical structures with broad material compatibility and structural and functional integrity. Our work paves the way for the cost-effective, large-scale production of a variety of flexible, inexpensive, and transparent 3D hierarchical and biomimetic materials.

High-Resolution Raman Microscopic Detection of Follicular Thyroid Cancer Cells with Unsupervised Machine Learning.

We use Raman microscopic images with high spatial and spectral resolution to investigate differences between human follicular thyroid (Nthy-ori 3-1) and follicular thyroid carcinoma (FTC-133) cells, a well-differentiated thyroid cancer. Through comparison to classification of single-cell Raman spectra, the importance of subcellular information in the Raman images is emphasized. Subcellular information is extracted through a coarse-graining of the spectra at high spatial resolution (∼1.7 µm2), producing a set of characteristic spectral groups representing locations having similar biochemical compositions. We develop a cell classifier based on the frequencies at which the characteristic spectra appear within each of the single cells. Using this classifier, we obtain a more accurate (89.8%) distinction of FTC-133 and Nthy-ori 3-1, in comparison to single-cell spectra (77.6%). We also infer which subcellular components are important to cellular distinction; we find that cancerous FTC-133 cells contain increased populations of lipid-containing components and decreased populations of cytochrome-containing components relative to Nthy-ori 3-1, and that the regions containing these contributions are largely outside the cell nuclei. In addition to increased classification accuracy, this approach provides rich subcellular information about biochemical differences and cellular locations associated with the distinction of the normal and cancerous follicular thyroid cells.