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.

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.

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.

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.

High-Throughput Cell Imaging and Classification by Narrowband and Low-Spectral-Resolution Raman Microscopy.

We investigated the use of narrowband Raman spectra for rapid label-free molecular imaging aimed at cell classification using principal component regression and linear discriminant analysis. In the classification of breast nontumorigenic epithelial and cancer cell lines, the classification accuracies using a spectral range of 100 cm-1 were equivalent to or better than that with using the fingerprint and high-wavenumber regions. Narrowing the Raman spectral range for analysis allows reduction of the charge-coupled device (CCD) pixels required for spectrum detection, resulting in the improvement of image acquisition speed with adequate classification accuracy. Our measurements revealed that the wavenumber region at 1397-1501 cm-1 can provide molecular information sufficient for cell classification without causing notable errors in the baseline-correction. A spectral resolution of ∼9 cm-1 was found to be sufficient to provide high accuracy in cell classification, which allowed us to apply pixel binning at the CCD readout for further acceleration of the imaging speed. As a result, the acquisition time for a 1200 × 1500 pixels Raman hyperspectral image at 1397-1501 cm-1 was reduced to 21 min. Under this condition, different cell lines were classified at accuracies higher than 90%. The presented approach will improve throughput of cell and tissue analysis and classification using Raman spectroscopy and extend practical uses of Raman imaging in biology and medicine.

Dnd1-mediated epigenetic control of teratoma formation in mouse.

Spontaneous testicular teratoma develops from primordial germ cells (PGCs) in embryos; however, the molecular mechanisms underlying teratoma formation are not fully understood. Mutation of the dead-end 1 (Dnd1) gene, which encodes an RNA-binding protein, drastically enhances teratoma formation in the 129/Sv mouse strain. To elucidate the mechanism of Dnd1 mutation-induced teratoma formation, we focused on histone H3 lysine 27 (H3K27) trimethylation (me3), and found that the levels of H3K27me3 and its responsible methyltransferase, enhancer of zeste homolog 2 (Ezh2), were decreased in the teratoma-forming cells of Dnd1 mutant embryos. We also showed that Dnd1 suppressed miR-26a-mediated inhibition of Ezh2 expression, and that Dnd1 deficiency resulted in decreased H3K27me3 of a cell-cycle regulator gene, Ccnd1 In addition, Ezh2 expression or Ccnd1 deficiency repressed the reprogramming of PGCs into pluripotent stem cells, which mimicked the conversion of embryonic germ cells into teratoma-forming cells. These results revealed an epigenetic molecular linkage between Dnd1 and the suppression of testicular teratoma formation.

Sclerosing mesenteritis mimicking metachronous peritoneal metastases from descending colon adenocarcinoma.

BACKGROUND: Sclerosing mesenteritis is a non-neoplastic inflammatory disease that occurs in the bowel mesentery. Distinguishing sclerosing mesenteritis from neoplasms may be difficult because of the clinical and radiographic similarities between the two disease entities. CASE PRESENTATION: We report a case of sclerosing mesenteritis mimicking peritoneal metastases of colorectal carcinoma. A 73-year-old man with stage II descending colon adenocarcinoma with poor prognostic features was found to have developed left lower abdominal quadrant masses on computed tomography (CT) 9 months after undergoing radical surgery. These masses were diagnosed as peritoneal metastases because they grew in size and displayed fluorodeoxyglucose (FDG) uptake 3 months later; thus, a laparotomy was performed. The masses, which were localized in the jejunal mesentery, were excised completely via segmental jejunal resection. Histopathological analysis confirmed that the masses were sclerosing mesenteritis. The patient showed no signs of sclerosing mesenteritis or colorectal carcinoma recurrence during follow-up. CONCLUSIONS: In patients suspected of having localized peritoneal metastasis from malignancies, any masses must be sampled by surgical excisional biopsy and subsequently examined to rule out alternative diagnoses, such as sclerosing mesenteritis.

DNA methylation of the Fthl17 5'-upstream region regulates differential Fthl17 expression in lung cancer cells and germline stem cells.

The Ferritin heavy polypeptide-like 17 (Fthl17) gene is a member of the cancer/testis antigen gene family, and is preferentially expressed in cancer cells and in testis. Although DNA methylation has been linked to the regulation of human FTHL17 gene expression, detailed epigenetic regulation of its expression has not been investigated. To address this, we assessed the epigenetic regulation of murine Fthl17 gene expression in cancer cells and germ cells. Fthl17 was more highly expressed in testis, a murine lung cancer cell line, KLN205, and in germline stem cells (GSCs) than in normal lung tissues. Furthermore, the Fthl17 expression level in GSCs was significantly higher than in KLN205 cells. We performed bisulfite-sequencing and luciferase (luc) reporter assays to examine the role of DNA methylation of the Fthl17 promoter in the regulation of Fthl17 expression. In KLN205 cells, testis, and GSCs, the Fthl17 5'-upstream region was hypo-methylated compared with normal lung tissues. Luc reporter assays indicated that hypo-methylation of the -0.6 kb to 0 kb region upstream from the transcription start site (TSS) was involved in the up-regulation of Fthl17 expression in KLN205 cells and GSCs. Because the -0.6 kb to -0.3 kb or the -0.3 kb to 0 kb region were relatively more hypo-methylated in KLN205 cells and in GSCs, respectively, compared with other regions between -0.6 kb to 0 kb, those regions may contribute to Fthl17 up-regulation in each cell type. Following treatment with 5-Azacytidine, the -0.3 kb to 0 kb region became hypo-methylated, and Fthl17 expression was up-regulated in KLN205 cells to a level comparable to that in GSCs. Together, the results suggest that hypo-methylation of different but adjacent regions immediately upstream of the Fthl17 gene contribute to differential expression levels in lung cancer cells and GSCs, and hypo-methylation of the TSS-proximal region may be critical for high level expression.

Usefulness of selective renal artery embolization for urinary fistula following partial nephrectomy: Two case reports.

The present study reported two cases in which selective artery embolization were identified to assist in resolving urinary fistulae following partial nephrectomies. The first case involved a 51-year-old male who received a mini-incision partial nephrectomy with renorrhaphy. Following the operation, urine continued to discharge from the retroperitoneal drain. Selective renal artery embolization of the upper calyx at post-operation day 20 was highly effective and urine output from the drain stopped immediately. Case 2 involved a 66-year-old male, who also suffered from a urinary fistula following a partial nephrectomy. Selective renal artery embolization performed at post-operation day 21 was again effective. In each case, the upper calyx was separated from the renal pelvis. These cases demonstrated that suturing of the collecting system and renal parenchyma may result in the separation of the urine pathway, and that selective renal artery embolization appears to be a highly effective treatment in such cases.

Induction of neurite outgrowth in PC12 cells treated with temperature-controlled repeated thermal stimulation.

To promote the functional restoration of the nervous system following injury, it is necessary to provide optimal extracellular signals that can induce neuronal regenerative activities, particularly neurite formation. This study aimed to examine the regulation of neuritogenesis by temperature-controlled repeated thermal stimulation (TRTS) in rat PC12 pheochromocytoma cells, which can be induced by neurotrophic factors to differentiate into neuron-like cells with elongated neurites. A heating plate was used to apply thermal stimulation, and the correlation of culture medium temperature with varying surface temperature of the heating plate was monitored. Plated PC12 cells were exposed to TRTS at two different temperatures via heating plate (preset surface temperature of the heating plate, 39.5°C or 42°C) in growth or differentiating medium for up to 18 h per day. We then measured the extent of growth, neuritogenesis, or acetylcholine esterase (AChE) activity (a neuronal marker). To analyze the mechanisms underlying the effects of TRTS on these cells, we examined changes in intracellular signaling using the following: tropomyosin-related kinase A inhibitor GW441756; p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580; and MAPK/extracellular signal-regulated kinase (ERK) kinase (MEK) inhibitor U0126 with its inactive analog, U0124, as a control. While a TRTS of 39.5°C did not decrease the growth rate of cells in the cell growth assay, it did increase the number of neurite-bearing PC12 cells and AChE activity without the addition of other neuritogenesis inducers. Furthermore, U0126, and SB203580, but not U0124 and GW441756, considerably inhibited TRTS-induced neuritogenesis. These results suggest that TRTS can induce neuritogenesis and that participation of both the ERK1/2 and p38 MAPK signaling pathways is required for TRTS-dependent neuritogenesis in PC12 cells. Thus, TRTS may be an effective technique for regenerative neuromedicine.

Poorly enhanced areas of pancreatic adenocarcinomas on late-phase dynamic computed tomography: comparison with pathological findings.

OBJECTIVES: The aim of the study was to retrospectively compare image findings of poorly enhanced areas (PEAs) of pancreatic adenocarcinomas that show almost no enhancement or obviously hypoattenuating area relative to the surrounding carcinoma on late-phase dynamic computed tomography (CT) with pathological findings. METHODS: Thirty-nine patients with pancreatic adenocarcinoma underwent dynamic CT and surgery. Poorly enhanced areas were classified according to their size, attenuation value, position, and border on CT imaging and signal intensity on magnetic resonance imaging. RESULTS: Of the 33 PEAs, 12 showed neoplastic duct-like structure that contained both large tumor gland and dilated pancreatic duct with atypia, 11 showed necrosis, 4 showed retention cyst, 2 showed dilated pancreatic duct without atypia or with limited invasion, 1 showed mucin, and 3 showed no remarkable differences in characteristics compared with surrounding tissue. Neoplastic duct-like structures tended to be well defined (P < 0.01). Necrotic portions tended to show a high attenuation value (P < 0.01) and central position (P < 0.01) and were ill defined (P < 0.01). Retention cysts tended to show a peripheral position (P < 0.01). CONCLUSIONS: Poorly enhanced areas corresponded to cystic, necrotic, and mucinous components. Image findings demonstrated these characteristics. Necrotic component can be visualized and distinguished with other components and can be a prognostic factor.

MDCT findings of extrapancreatic nerve plexus invasion by pancreas head carcinoma: correlation with en bloc pathological specimens and diagnostic accuracy.

OBJECTIVE: To elucidate the multi-detector row computed tomography (MDCT) findings of extrapancreatic nerve plexus (PLX) invasion by pancreas head carcinoma (PhC) by "point-by-point" correlation with en bloc pathological specimens and to assess their diagnostic accuracy. METHODS: Each pathological section of PhC and adjusted double oblique multiplanar reconstruction MDCT images were correlated in 554 sections from 37 patients. The diagnostic accuracy of the MDCT patterns derived was assessed by blind reading. RESULTS: PLX invasion with fibrosis showed mass or strand shape (85.6%) or coarse reticula (13.3%). The CT findings were divided into fine reticular and linear, coarse reticular, mass and strand, and nodular patterns. PLX invasion was revealed pathologically in 92% of the regions of investigation showing the mass and strand pattern and 63% of the coarse reticular pattern (all continuous with PhC), and they were highly suggestive of PLX invasion by PhC on MDCT images (p < 0.001). Sensitivity, specificity, accuracy, and positive and negative predictive values of these MDCT findings in the diagnosis of PLX invasion were 100% (25/25), 83.3% (10/12), 94.6% (35/37), 92.6% (25/27) and 100% (10/10), respectively. CONCLUSION: The mass and strand pattern and the coarse reticular pattern continuous with PhC on MDCT images were highly suggestive of PLX invasion by PhC.

Enhancement patterns of pancreatic adenocarcinoma on conventional dynamic multi-detector row CT: correlation with angiogenesis and fibrosis.

AIM: To evaluate retrospectively the correlation between enhancement patterns on dynamic computed tomography (CT) and angiogenesis and fibrosis in pancreatic adenocarcinoma. METHODS: Twenty-three patients with pancreatic adenocarcinoma underwent dynamic CT and tumor resection. In addition to the absolute and relative enhanced value that was calculated by subtracting the attenuation value on pre-contrast from those on contrast-enhanced CT in each phase, we defined one parameter, "tumor-aorta enhancement ratio", which was calculated by dividing enhancement of pancreatic cancer by enhancement of abdominal aorta in each phase. These enhancement patterns were correlated with the level of vascular endothelial growth factor (VEGF), microvessel density (MVD), and extent of fibrosis. RESULTS: The absolute enhanced value in the arterial phase correlated with the level of VEGF and MVD (P = 0.047, P = 0.001). The relative enhanced value in arterial phase and tumor-aorta enhancement ratio (arterial) correlated with MVD (P = 0.003, P = 0.022). Tumor-aorta enhancement ratio (arterial) correlated negatively with the extent of fibrosis (P = 0.004). The tumors with greater MVD and higher expression of VEGF tended to show high enhancement in the arterial dominant phase. On the other hand, the tumors with a larger amount of fibrosis showed a negative correlation with the grade of enhancement during the arterial phase. CONCLUSION: Enhancement patterns on dynamic CT correlated with angiogenesis and may be modified by the extent of fibrosis.

[Imaging of gastrointestinal stromal tumor (GIST): relation between CT findings and grade of malignancy].

PURPOSE: To report the relation between CT findings and the grade of malignancy in gastrointestinal stromal tumor (GIST), especially the uncommitted type of GIST. MATERIALS AND METHODS: A total of 14 patients with histologically proven GIST (uncommitted type) underwent CT. Tumors were divided into three grades. HISTOLOGICALLY: Benign (mitotic index [MI] < 2/10 high-power fields [HPF]), borderline (2/10 HPF < or = MI < or = 5/10 HPF), and malignant (5/10 HPF < MI). We evaluated tumor size, cystic component, margin, and early enhancement. RESULTS: All benign tumors were smaller than 5 cm, and most malignant tumors reached 5 cm. The size of borderline tumors was between the sizes of benign and malignant tumors. No benign tumors had cystic components, whereas all borderline and malignant tumors except for one case had cystic components. Only two huge malignant tumors had unclear margins. The relation between early enhancement and the grade of malignancy showed no tendency, but all duodenal tumors showed marked early enhancement irrespective of grade. CONCLUSION: The grade of malignancy of GIST (uncommitted type) and size, presence of cystic components, and margin were highly correlated. That is, 1) tumors smaller than 5 cm with no cystic components can be diagnosed as benign, whereas 2) tumors that have cystic components are borderline or malignant. 3) Tumors that have cystic components and unclear margin can be diagnosed as actively malignant.