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Abstract Cellular senescence occurs through the accumulation of many kinds of stresses. Senescent cells in tissues also cause various age-related disorders. Therefore, detecting them without labeling is beneficial for medical research and developing diagnostic methods. However, existing biomarkers have limitations of requiring fixation and labeling, or their molecular backgrounds are uncertain. Coherent anti-Stokes Raman scattering (CARS) spectroscopic imaging is a novel option because it can assess and visualize molecular structures based on their molecular fingerprint. Here, we present a new label-free method to visualize cellular senescence using CARS imaging in nucleoli. We found the peak of the nucleolar amide I band shifted to a higher wavenumber in binuclear senescent cells, which reflects changes in the protein secondary structure from predominant α-helices to β-sheets originating from amyloid-like aggregates. Following this, we developed a procedure that can visualize the senescent cells by providing the ratios and subtractions of these two components. We also confirmed that the procedure can visualize nucleolar aggregates due to unfolded/misfolded proteins produced by proteasome inhibition. Finally, we found that this method can help visualize the nucleolar defects in naïve cells even before binucleation. Thus, our method is beneficial to evaluate ongoing cellular senescence through label-free imaging of nucleolar defects.

Abstract Malignant mesothelioma (MMe) is a rare but aggressive malignancy. Although the molecular genetics of MMe is known, including BRCA1-associated protein-1 (BAP1) gene alterations, the prognosis of MMe patients remains poor. Here, we generated BAP1 knockout (BAP1-KO) human mesothelial cell clones to develop molecular-targeted therapeutics based on genetic alterations in MMe. cDNA microarray and quantitative RT-PCR (qRT-PCR) analyses revealed high expression of a calcium/calmodulin-dependent protein kinase type II subunit delta (CAMK2D) gene in the BAP1-KO cells. CAMK2D was highly expressed in 70% of the human MMe tissues (56/80) and correlated with the loss of BAP1 expression, making it a potential diagnostic and therapeutic target for BAP1-deficient MMe. We screened an anticancer drugs library using BAP1-KO cells and successfully identified a CaMKII inhibitor, KN-93, which displayed a more potent and selective antiproliferative effect against BAP1-deficient cells than cisplatin or pemetrexed. KN-93 significantly suppressed the tumor growth in mice xenografted with BAP1-deficient MMe cells. This study is the first to provide a potential molecular-targeted therapeutic approach for BAP1-deficient MMe.

We analyzed the effects of a hypo-osmotic environment on rat Schwann cells, a type of glial cell surrounding neurons, using ultra-broadband multiplex coherent anti-Stokes Raman scattering (CARS) microscopy. After hypo-osmotic treatment, we detected vacuole-like components in the cytoplasm using both bright-field and CARS spectroscopic imaging. An approach integrating both morphological examination and a spectroscopic analysis based on multiple vibrational bands revealed that these structures are predominantly water-filled, and their characteristics closely resembled those of the vacuoles observed in plant cells.

Previous genome-wide association studies have identified risk loci for pancreatic cancer but were centered on individuals of European ancestry. Here the authors identify GP2 gene variants associated with pancreatic cancer susceptibility in populations of East Asian ancestry.

We have developed an ultrabroadband multiplex coherent anti-Stokes Raman scattering (CARS) microspectroscopic system using a supercontinuum (SC) seeded by sub-100-ps (85 ps) laser pulses with a sub-MHz (0.82 MHz) repetition rate. Because of the high peak power and ultrabroadband spectral profile of the SC, we can efficiently generate multiplex CARS signals in the spectral range of 600–3600 cm−1, which covers the entire molecular fingerprint region, as well as the C—H and O—H stretching regions. Due to the high peak power of the new laser source, the exposure time (pixel dwell time) for CARS imaging of polymer beads was reduced to less than 1 ms (0.8 ms), which was limited by the readout time of a CCD camera. Owing to the improvement in CARS spectral quality, clear molecular fingerprinting was achieved for living HeLa cells at different phases in the cell cycle.

Keratins (Ks), the intermediate filament (IF) proteins of epithelia, are coordinately expressed as pairs in a cell-lineage and differentiation manner. Cortical thymic epithelial cells (cTECs) predominantly express the simple epithelium keratin 8/18 (K8/K18) pair, whereas medullary thymic epithelial cells (mTECs) express the stratified epithelium K5/K14 pair, with TECs exhibiting K5 and K8 at the cortico-medullary junction in mature thymus. In the work reported here, we used wild-type (WT) and K8-knockout (K8-null) mice to address the contribution of K8/K18 IFs in the maintenance of the thymic epithelial structure. K8-null thymus maintained the differential cell segregation at the cortex versus the medulla observed in WT thymus, and the distribution of immature thymocytes at the cortex. The K8/K18 loss did not affect thymocyte development. However, it massively perturbed the TEC morphology both at the cortex and the medulla, along with a prominent depletion of cTECs. Such tissue alterations coincided with an increase in apoptosis and a reduced expression of Albatross (Fas-binding factor-1), also known for its capacity to bind K8/18 IFs. In addition, the K8/K18 loss affected the distribution of K5/K14-positive mTECs, but not their differentiation status. Together, the results indicate that K8/K18 IFs constitute key promoters of the thymic epithelium integrity.