Integrated proteomic profiling identifies amino acids selectively cytotoxic to pancreatic cancer cells.

Pancreatic adenocarcinoma (PDAC) is one of the most deadly cancers, characterized by extremely limited therapeutic options and a poor prognosis, as it is often diagnosed during late disease stages. Innovative and selective treatments are urgently needed, since current therapies have limited efficacy and significant side effects. Through proteomics analysis of extracellular vesicles, we discovered an imbalanced distribution of amino acids secreted by PDAC tumor cells. Our findings revealed that PDAC cells preferentially excrete proteins with certain preferential amino acids, including isoleucine and histidine, via extracellular vesicles. These amino acids are associated with disease progression and can be targeted to elicit selective toxicity to PDAC tumor cells. Both in vitro and in vivo experiments demonstrated that supplementation with these specific amino acids effectively eradicated PDAC cells. Mechanistically, we also identified XRN1 as a potential target for these amino acids. The high selectivity of this treatment method allows for specific targeting of tumor metabolism with very low toxicity to normal tissues. Furthermore, we found this treatment approach is easy-to-administer and with sustained tumor-killing effects. Together, our findings reveal that exocytosed amino acids may serve as therapeutic targets for designing treatments of intractable PDAC and potentially offer alternative treatments for other types of cancers.

EvIPqPCR, Target Circulating Tumorous Extracellular Vesicles for Detection of Pancreatic Cancer.

Pancreatic cancer patients predominantly present with advanced disease at diagnosis, contributing to its high mortality. A noninvasive, fast screening method to detect this disease is an unmet need. Tumor-derived extracellular vesicles (tdEVs) bearing information from parental cells have emerged as a promising cancer diagnostic biomarker. However, most tdEV-based assays have impractical sample volumes and time-consuming, complex, and costly techniques. To overcome these limitations, we developed a novel diagnostic method for pancreatic cancer screening. Our approach utilizes the mitochondrial DNA to nuclear DNA ratio of EVs as a collective cell-specific characteristic. We introduce EvIPqPCR, a fast method that combines immunoprecipitation (IP) and qPCR quantification to detect tumor-derived EVs directly from serum. Importantly, our method employs DNA isolation-free and duplexing probes for qPCR, saving at least 3 h. This technique has the potential to serve as a translational assay for cancer screening with a weak correlation to prognosis biomarkers and sufficient discriminatory power among healthy controls, pancreatitis, and pancreatic cancer cases.

Cytokine receptor gp130 promotes postnatal proliferation of cardiomyocytes required for the normal functional development of the heart.

Mammalian ventricular cardiomyocytes are premature at birth and exhibit substantial phenotypic changes before weaning. Mouse ventricular myocytes undergo cell division several times after birth; however, the regulatory mechanisms and roles of cardiomyocyte division in postnatal heart development remain unclear. Here, we investigated the physiological role of glycoprotein 130 (gp130), the main subunit of multifunctional receptors for the IL-6 family of cytokines, in postnatal cardiomyocyte proliferation. Pharmacological inhibition of gp130 within the first month after birth induced significant systolic dysfunction of the left ventricle in mice. Consistently, mice with postnatal cardiomyocyte-specific gp130 depletion exhibited impaired left ventricular contractility compared with control mice. In these mice, cardiomyocytes exhibited a moderately decreased size and dramatically inhibited proliferation in the left ventricle but not in the right ventricle. Stereological analysis revealed that this change significantly decreased the number of cardiomyocytes in the left ventricle. Furthermore, IL-6 was mainly responsible for promoting ventricular cardiomyocyte proliferation by activating the JAK/STAT3 pathway. Taken together, the IL-6/gp130/JAK/STAT3 axis plays a crucial role in the physiological postnatal proliferation and hypertrophy of left ventricular cardiomyocytes to ensure normal cardiac functional development.NEW & NOTEWORTHY Although cardiomyocytes undergo proliferation in the early postnatal period, the regulatory mechanisms and physiological importance of this process have not been clarified. We found that the pharmacological and genetic depletion of gp130 in preweaning mice resulted in significant impairment of cardiomyocyte proliferation, thinning of the myocardium, and systolic dysfunction of the left but not right ventricle by perturbing JAK/STAT3 signaling. Thus, the IL-6/gp130/JAK/STAT3 axis is crucial for the postnatal functional development of the left ventricle.

Delineating regions of interest for mass spectrometry imaging by multimodally corroborated spatial segmentation.

Mass spectrometry imaging (MSI), which localizes molecules in a tag-free, spatially resolved manner, is a powerful tool for the understanding of underlying biochemical mechanisms of biological phenomena. When analyzing MSI data, it is essential to delineate regions of interest (ROIs) that correspond to tissue areas of different anatomical or pathological labels. Spatial segmentation, obtained by clustering MSI pixels according to their mass spectral similarities, is a popular approach to automate ROI definition. However, how to select the number of clusters (#Clusters), which determines the granularity of segmentation, remains to be resolved, and an inappropriate #Clusters may lead to ROIs not biologically real. Here we report a multimodal fusion strategy to enable an objective and trustworthy selection of #Clusters by utilizing additional information from corresponding histology images. A deep learning-based algorithm is proposed to extract "histomorphological feature spectra" across an entire hematoxylin and eosin image. Clustering is then similarly performed to produce histology segmentation. Since ROIs originating from instrumental noise or artifacts would not be reproduced cross-modally, the consistency between histology and MSI segmentation becomes an effective measure of the biological validity of the results. So, #Clusters that maximize the consistency is deemed as most probable. We validated our strategy on mouse kidney and renal tumor specimens by producing multimodally corroborated ROIs that agreed excellently with ground truths. Downstream analysis based on the said ROIs revealed lipid molecules highly specific to tissue anatomy or pathology. Our work will greatly facilitate MSI-mediated spatial lipidomics, metabolomics, and proteomics research by providing intelligent software to automatically and reliably generate ROIs.

ß-Sheet Richness of the Circulating Tumor-Derived Extracellular Vesicles for Noninvasive Pancreatic Cancer Screening.

Tumor-derived extracellular vesicles (EVs) are under intensive study for their potential as noninvasive diagnosis biomarkers. Most EV-based cancer diagnostic assays trace supernumerary of a single cancer-associated marker or marker signatures. These types of biomarker assays are either subtype-specific or vulnerable to be masked by high background signals. In this study, we introduce using the ß-sheet richness (BR) of the tumor-derived EVs as an effective way to discriminate EVs originating from malignant and nonmalignant cells, where EV contents are evaluated as a collective attribute rather than single factors. Circular dichroism, Fourier transform infrared spectroscopy, fluorescence staining assays, and a de novo workflow combining proteomics, bioinformatics, and protein folding simulations were employed to validate the collective attribute at both cellular and EV levels. Based on the BR of the tumorous EVs, we integrated immunoprecipitation and fluorescence labeling targeting the circulating tumor-derived EVs in serum and developed the process into a clinical assay, named EvIPThT. The assay can distinguish patients with and without malignant disease in a pilot cohort, with weak correlations to prognosis biomarkers, suggesting the potential for a cancer screening panel with existing prognostic biomarkers to improve overall performance.

Sequence determinants of human junctophilin-2 protein nuclear localization and phase separation.

Junctophilin-2 (JPH2) was conventionally considered as a structural membrane binding protein. Recently, it was shown that proteolytically truncated mouse JPH2 variants are imported into nucleus to exert alternative functions. However, the intranuclear behaviors of human JPH2 (hJPH2) and underlying molecular determinants have not been explored. Here, we demonstrate that full-length hJPH2 is imported into nucleus in human cells by two nuclear localization signals (NLSs), including a newly discovered one at the C-terminus. Importantly, unlike the JPH2 N-terminal truncation which diffuses throughout the nucleus, full-length hJPH2 forms nuclear bodies behaving like liquid-liquid phase separated droplets that are separated from chromatin. The C-terminal transmembrane domain is required for the formation of hJPH2 droplets. Oxidation mimicking substitution of residues C678 and M679 augments the formation of hJPH2 nuclear droplets, suggesting nuclear hJPH2 liquid-liquid phase separation could be modulated by oxidative stress. Mutation A405D, which introduces a negatively charged residue into an intrinsic disordered region (IDR) of hJPH2, turns liquid-like droplets into amyloid-like aggregates. Depletion of an Alanine Rich Region in the IDR recapitulates the liquid-amyloid phase transition. The MORN repeat regions of hJPH2 encodes intrinsic tendency to form amyloid-like structure. Together, these data revealed the novel intrinsic properties of hJPH2 to form nuclear liquid droplets, and identified critical functional domains encoding these properties. We propose that hJPH2 droplets could function as membrane-less organelles participating in nuclear regulatory processes.

Deinococcus detaillensis sp. nov., isolated from humus soil in Antarctica.

A Gram-staining-positive, non-motile, coccus or short-rod-shaped bacterium, designated H1T, was isolated from a humus soil sample in the Detaille Island of Antarctica. The 16S rRNA gene sequence result indicated that strain H1T shared the highest 16S rRNA gene sequence identity with the type strain of Deinococcus alpinitundrae (96.2%). Growth of strain H1T occurred at 4-25 °C, pH 6.0-8.0 and in the presence of 0-1.0% NaCl (w/v). The respiratory quinone was MK-8. The major fatty acids were C16:0, C17:0 cyclo and summed feature 3 (C16:1 ω7c/C16:1 ω6c). The polar lipids were aminoglycophospholipid, aminophospholipid, glycolipid and glycophospholipid. The cell wall peptidoglycan type was A3ß. The genomic DNA G + C content was 61.3 mol%. The average nucleotide identity (ANI) between strain H1T and the closely related Deinococcus members was below the cut-off level (95-96%) for species identification. Based on the above results, strain H1T represents a novel species of the genus Deinococcus, for which the name Deinococcus detaillensis sp. nov. is proposed. Type strain is H1T (= CGMCC 1.13938T = JCM 33291T).

Analysis of Cardiac Myocyte Maturation Using CASAAV, a Platform for Rapid Dissection of Cardiac Myocyte Gene Function In Vivo.

RATIONALE: Loss-of-function studies in cardiac myocytes (CMs) are currently limited by the need for appropriate conditional knockout alleles. The factors that regulate CM maturation are poorly understood. Previous studies on CM maturation have been confounded by heart dysfunction caused by whole organ gene inactivation. OBJECTIVE: To develop a new technical platform to rapidly characterize cell-autonomous gene function in postnatal murine CMs and apply it to identify genes that regulate transverse tubules (T-tubules), a hallmark of mature CMs. METHODS AND RESULTS: We developed CRISPR/Cas9/AAV9-based somatic mutagenesis, a platform in which AAV9 delivers tandem guide RNAs targeting a gene of interest and cardiac troponin-T promoter-driven Cre to RosaCas9GFP/Cas9GFP neonatal mice. When directed against junctophilin-2 (Jph2), a gene previously implicated in T-tubule maturation, we achieved efficient, rapid, and CM-specific JPH2 depletion. High-dose AAV9 ablated JPH2 in 64% CMs and caused lethal heart failure, whereas low-dose AAV9 ablated JPH2 in 22% CMs and preserved normal heart function. In the context of preserved heart function, CMs lacking JPH2 developed T-tubules that were nearly morphologically normal, indicating that JPH2 does not have a major, cell-autonomous role in T-tubule maturation. However, in hearts with severe dysfunction, both adeno-associated virus-transduced and nontransduced CMs exhibited T-tubule disruption, which was more severe in the transduced subset. These data indicate that cardiac dysfunction disrupts T-tubule structure and that JPH2 protects T-tubules in this context. We then used CRISPR/Cas9/AAV9-based somatic mutagenesis to screen 8 additional genes for required, cell-autonomous roles in T-tubule formation. We identified RYR2 (Ryanodine Receptor-2) as a novel, cell-autonomously required T-tubule maturation factor. CONCLUSIONS: CRISPR/Cas9/AAV9-based somatic mutagenesis is a powerful tool to study cell-autonomous gene functions. Genetic mosaics are invaluable to accurately define cell-autonomous gene function. JPH2 has a minor role in normal T-tubule maturation but is required to stabilize T-tubules in the failing heart. RYR2 is a novel T-tubule maturation factor.

Sildenafil ameliorates left ventricular T-tubule remodeling in a pressure overload-induced murine heart failure model.

AIM: Sildenafil, a phosphodiesterase 5 (PDE5) inhibitor, has been shown to exert beneficial effects in heart failure. The purpose of this study was to test whether sildenafil suppressed transverse-tubule (T-tubule) remodeling in left ventricular (LV) failure and thereby providing the therapeutic benefits. METHODS: A pressure overload-induced murine heart failure model was established in mice by thoracic aortic banding (TAB). One day after TAB, the mice received sildenafil (100 mg·kg(-1)·d(-1), sc) or saline for 5 weeks. At the end of treatment, echocardiography was used to examine LV function. Then the intact hearts were dissected out and placed in Langendorff-perfusion chamber for in situ confocal imaging of T-tubule ultrastructure from epicardial myocytes. RESULTS: TAB surgery resulted in heart failure accompanied by remarkable T-tubule remodeling. Sildenafil treatment significantly attenuated TAB-induced cardiac hypertrophy and congestive heart failure, improved LV contractile function, and preserved T-tubule integrity in LV cardiomyocytes. But sildenafil treatment did not significantly affect the chamber dilation. The integrity of LV T-tubule structure was correlated with cardiac hypertrophy (R(2)=0.74, P<0.01) and global LV function (R(2)=0.47, P<0.01). CONCLUSION: Sildenafil effectively ameliorates LV T-tubule remodeling in TAB mice, revealing a novel mechanism underlying the therapeutic benefits of sildenafil in heart failure.

Phospholamban knockout breaks arrhythmogenic Ca²âº waves and suppresses catecholaminergic polymorphic ventricular tachycardia in mice.

RATIONALE: Phospholamban (PLN) is an inhibitor of cardiac sarco(endo)plasmic reticulum Ca²âº ATPase. PLN knockout (PLN-KO) enhances sarcoplasmic reticulum Ca²âº load and Ca²âº leak. Conversely, PLN-KO accelerates Ca²âº sequestration and aborts arrhythmogenic spontaneous Ca²âº waves (SCWs). An important question is whether these seemingly paradoxical effects of PLN-KO exacerbate or protect against Ca²âº-triggered arrhythmias. OBJECTIVE: We investigate the impact of PLN-KO on SCWs, triggered activities, and stress-induced ventricular tachyarrhythmias (VTs) in a mouse model of cardiac ryanodine-receptor (RyR2)-linked catecholaminergic polymorphic VT. METHODS AND RESULTS: We generated a PLN-deficient, RyR2-mutant mouse model (PLN-/-/RyR2-R4496C+/-) by crossbreeding PLN-KO mice with catecholaminergic polymorphic VT-associated RyR2-R4496C mutant mice. Ca²âº imaging and patch-clamp recording revealed cell-wide propagating SCWs and triggered activities in RyR2-R4496C+/- ventricular myocytes during sarcoplasmic reticulum Ca²âº overload. PLN-KO fragmented these cell-wide SCWs into mini-waves and Ca²âº sparks and suppressed the triggered activities evoked by sarcoplasmic reticulum Ca²âº overload. Importantly, these effects of PLN-KO were reverted by partially inhibiting sarco(endo)plasmic reticulum Ca²âº ATPase with 2,5-di-tert-butylhydroquinone. However, Bay K, caffeine, or Li⁺ failed to convert mini-waves to cell-wide SCWs in PLN-/-/RyR2-R4496C+/- ventricular myocytes. Furthermore, ECG analysis showed that PLN-KO mice are not susceptible to stress-induced VTs. On the contrary, PLN-KO protected RyR2-R4496C mutant mice from stress-induced VTs. CONCLUSIONS: Our results demonstrate that despite severe sarcoplasmic reticulum Ca²âº leak, PLN-KO suppresses triggered activities and stress-induced VTs in a mouse model of catecholaminergic polymorphic VT. These data suggest that breaking up cell-wide propagating SCWs by enhancing Ca²âº sequestration represents an effective approach for suppressing Ca²âº-triggered arrhythmias.

Calsequestrin accumulation in rough endoplasmic reticulum promotes perinuclear Ca2+ release.

Molecular mechanisms underlying Ca(2+) regulation by perinuclear endoplasmic/sarcoplasmic reticulum (ER/SR) cisternae in cardiomyocytes remain obscure. To investigate the mechanisms of changes in cardiac calsequestrin (CSQ2) trafficking on perinuclear Ca(2+) signaling, we manipulated the subcellular distribution of CSQ2 by overexpression of CSQ2-DsRed, which specifically accumulates in the perinuclear rough ER. Adult ventricular myocytes were infected with adenoviruses expressing CSQ2-DsRed, CSQ2-WT, or empty vector. We found that perinuclear enriched CSQ2-DsRed, but not normally distributed CSQ2-WT, enhanced nuclear Ca(2+) transients more potently than cytosolic Ca(2+) transients. Overexpression of CSQ2-DsRed produced more actively propagating Ca(2+) waves from perinuclear regions than did CSQ2-WT. Activities of the SR/ER Ca(2+)-ATPase and ryanodine receptor type 2, but not inositol 1,4,5-trisphosphate receptor type 2, were required for the generation of these perinuclear initiated Ca(2+) waves. In addition, CSQ2-DsRed was more potent than CSQ2-WT in inducing cellular hypertrophy in cultured neonatal cardiomyocytes. Our data demonstrate for the first time that CSQ2 retention in the rough ER/perinuclear region promotes perinuclear Ca(2+) signaling and predisposes to ryanodine receptor type 2-mediated Ca(2+) waves from CSQ2-enriched perinuclear compartments and myocyte hypotrophy. These findings provide new insights into the mechanism of CSQ2 in Ca(2+) homeostasis, suggesting that rough ER-localized Ca(2+) stores can operate independently in raising levels of cytosolic/nucleoplasmic Ca(2+) as a source of Ca(2+) for Ca(2+)-dependent signaling in health and disease.