bioRxiv - Pathology最新文献

{"title":"Identification of SPP1-positive macrophages by single-cell spatial analysis in human lung tissues with mycobacterial infection","authors":"Harutaka Katano, Akira Hebisawa, Yuko Sato, Yoshihiko Hoshino","doi":"10.1101/2024.09.12.612778","DOIUrl":"https://doi.org/10.1101/2024.09.12.612778","url":null,"abstract":"Tuberculosis and non-tuberculous mycobacterial (NTM) diseases are infections caused by Mycobacterium tuberculosis and non-tuberculous mycobacteria such as Mycobacterium avium complex, leading to the formation of granulomatous lesions with caseous necrosis in the lungs. Although granulomatous tissues are infiltrated by numerous inflammatory cells, including macrophages, lymphocytes, and neutrophils, the mechanisms underlying granuloma formation by mycobacteria remain unclear. In this study, we performed single-cell spatial analysis on lung tissue samples from patients with tuberculosis and NTM diseases to investigate the infiltrating cell populations. We analyzed seven lung lesions and identified individual cell types infiltrating the granulomatous tissue. Based on gene expression profiles, at least four macrophage subtypes were identified. Notably, SPP1-positive macrophages predominantly infiltrated the granulomatous tissue. Langhans giant cells expressed SPP1, and many SPP1-positive macrophages without giant cell morphology were also present around the granuloma. RNA-seq analysis revealed elevated SPP1 expression in mycobacterium-infected tissues. The SPP1-CD44 signaling pathway was active in SPP1-positive macrophages and their neighboring cells in mycobacterium-infected tissues. SPP1-positive macrophages were also observed around granulomas in other granulomatous diseases, such as granulomatosis with polyangiitis and sarcoidosis. These findings suggest that SPP1-positive macrophages may play a key role in granuloma formation in granulomatous diseases, including mycobacterial infections.","PeriodicalId":501471,"journal":{"name":"bioRxiv - Pathology","volume":"203 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142256922","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Endothelial miR-34a deletion guards against aneurysm development despite endothelial dysfunction","authors":"Anna Grochot-Przeczek, Aleksandra Kopacz, Damian Kloska, Anna Bar, Marta Targosz-Korecka, Dominik Cysewski, Stefan Chlopicki, Alicja Jozkowicz","doi":"10.1101/2024.09.12.612788","DOIUrl":"https://doi.org/10.1101/2024.09.12.612788","url":null,"abstract":"Objective: Our previous study reported a reciprocal link between NRF2, a stress-responsive cytoprotective transcription factor, and aortic and endothelial cell (EC) ageing. We also found that NRF2 transcriptional knockout (tKO) mice are prone to abdominal aortic aneurysm (AAA) development. Given that miRNA-34a is a marker of ageing, in this study we explored its relationship with NRF2 and its role in vascular function and AAA formation.\u0000Approach and Results: The experiments were performed in primary human aortic endothelial cells (HAECs) from young and aged donors and mice devoid of NRF2 transcriptional activity and endothelial miR-34a. The normolipidemic mice were challenged with angiotensin II (Ang II) to develop AAA. We show that premature NRF2-dependent aging of aortic endothelial cells (ECs) depends on miR-34a. Infusion of hypertensive Ang II in mice increases miR-34a in the aortic endothelial layer and serum, especially in mice which develop AAA. Mice deficient in endothelial miR-34a (miR-34a?EC) display severe EC dysfunction. Despite that, such mice are protected from AAA development, also on the NRF2 tKO background. Ang II infusion increases proliferation of intimal ECs in these mice. The protective effect of endothelial miR-34a deletion on AAA formation is reversed by rapamycin that suppresses EC proliferation. MTA2, but not SIRT1, is a direct target of miR-34a abrogating Ang II-induced EC proliferation. Conclusion: These findings reveal that AAA development in NRF2 tKO mice relies on endothelial miR-34a overexpression. Deletion of endothelial miR-34a protects mice from AAA despite inducing endothelial cell dysfunction. The fine-tuning of EC proliferation may play a therapeutic role in the treatment of aneurysm.","PeriodicalId":501471,"journal":{"name":"bioRxiv - Pathology","volume":"31 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142256923","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Machine-learning convergent melanocytic morphology despite noisy archival slides","authors":"Mikio Tada, Garrett Gaskins, Sina Ghandian, Nicholas Mew, Michael J. Keiser, Elizabeth S. Keiser","doi":"10.1101/2024.09.12.612732","DOIUrl":"https://doi.org/10.1101/2024.09.12.612732","url":null,"abstract":"Melanocytic atypia, ranging from benign to malignant, often leads to diagnostic discordance, complicating its prediction by machine learning models. To overcome this, we paired H&E-stained histology images with contiguous or serial sections immunohistochemically (IHC) stained for melanocytic cells via antibodies for MelanA, MelPro, or SOX10. We developed a deep-learning pipeline to identify melanocytic atypia by digitizing a real-world archival dataset of 122 paired whole slide images from 61 confirmed melanoma in situ (MIS) cases at two institutions. Only 37.7% of the cases contained tissue pairs that matched well enough for deep learning. Nonetheless, the MelanA+MelPro models achieved an average area under the receiver-operating characteristic (AUROC) of 0.948 and an average area under the precision-recall curve (AUPRC) of 0.611, while the SOX10 models had an average of 0.867 AUROC and 0.433 AUPRC. Despite learning from biologically different IHC stains, the convolutional neural network (CNN) models independently exhibited an intuitive convergent rationale by explainable AI saliency calculations. Different antibodies, with nuclear versus cytoplasmic staining, provided complementary yet consistent information, which the CNNs integrated effectively. The resulting multi-antibody virtual stains identified morphologic cytologic and small-scale architectural features directly from H&E-stained histology images, which can assist pathologists in assessing cutaneous MIS.","PeriodicalId":501471,"journal":{"name":"bioRxiv - Pathology","volume":"41 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142256924","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Growth Analysis of Trichomonas vaginalis in Different Culture Media: Leveraging Large Language Models (LLMs) to Predict and Optimize In Vitro Growth Conditions","authors":"Shernica L Ferguson, Loria Brown Gordon","doi":"10.1101/2024.09.12.612589","DOIUrl":"https://doi.org/10.1101/2024.09.12.612589","url":null,"abstract":"Trichomonas vagnalis is a tiny protozoan universally known to have one of the highest prevalance rates of any common sexually transmitted disease. Its popularity in HIV transmission and preterm labor highlights its importance in clinical, biological, and epidemiological investigations worldwide. Propagation of T vaginalis in vitro uses modified Diamonds media in commercial and clinical culture environments. Several modifications of this medium exist, but a more recent modification proved the most efficient. Our study aimed to investigate media modifications that could optimize the growth of T. vaginalis in routine laboratory cultivation. RSMT media enriched with quantitated media components was compared to modified Diamond's, Oxoid, and In Pouch culture mediums. Several growth studies were employed to select eight isolates (n=8) of T. vaginalis, including an ATCC reference isolate. These isolates were examined for several days at 37 degrees C in an anaerobic environment. Tests revealed that isolates in RSMT media had over 85% higher concentrations of T vaginalis growth than our testing mediums, with a maximum growth increase of 250%. The composite counts of T. vaginalis in RSMT showed a significant difference (p <0.05) from those of T. vaginalis in the standard Diamonds media Oxoid or In Pouch mediums. This data suggests that more efficient culturing and growth of T. vaginalis requires more vitamins and other growth factors beyond what would conventionally be expended in standard Trichomonas culture mediums.","PeriodicalId":501471,"journal":{"name":"bioRxiv - Pathology","volume":"29 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142256926","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Comparative Analysis of Stain-Free Fourier Ptychographic Microscopy and Traditional Histopathological Light Microscopy in Renal Membranous Nephropathy","authors":"Marika Valentino, Vittorio Bianco, Gioacchino D'Ambrosio, Marco Paulli, Giovanni Smaldone, Valentina Brancato, Lisa Miccio, Marco Salvatore, Marcello Gambacorta, Pietro Ferraro","doi":"10.1101/2024.09.12.612640","DOIUrl":"https://doi.org/10.1101/2024.09.12.612640","url":null,"abstract":"Background: Histology remains a cornerstone in the diagnosis and prognosis of renal diseases, with histopathological analysis of kidney tissue being crucial for understanding renal pathophysiology. The availability of multiple stained sections is essential for conducting a comprehensive histopathological analysis and achieving an accurate diagnosis. Recently, Fourier Ptychographic Microscopy (FPM) earned a spot among the most promising microscopy techniques. The ability to provide high-resolution, quantitative phase-contrast images over a wide area, particularly in a stain-free mode, makes FPM highly appealing to experts in histopathology. Since renal pathologies are characterized by subtle morphological changes encoded in tissue slides, phase maps obtained using FPM are well-suited for providing detailed, high-contrast images of tissue structures. Thus, FPM provides a quantitative imaging tool that can be descriptive of the sample and/or expressive of the disease.\u0000Methods: In this study, we explore FPM capability to image pathological kidney tissue, enabling pathologists to select regions of interest within the intricate architecture of renal tissue and zoom in to observe minute submicron structures, ranging from overall tissue organization and glomeruli distribution to individual cell membranes. Attention is focused on membranous glomerulonephritis since it is a nephropathy highly dependent on histological examination.\u0000Results: The comparative analysis between FPM and traditional light microscopy showed a difference in thickness of glomerular basal membranes between healthy kidney tissues and those affected by membranous glomerulonephritis (MG). Moreover, the results reported in our investigation revealed better glomerular membranes contrast in FPM images with respect to the H&E-stained images.\u0000Conclusions: Our study shows the broad potential of FPM in characterizing hallmarks of MG disease even in stain-free tissue slides.","PeriodicalId":501471,"journal":{"name":"bioRxiv - Pathology","volume":"19 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142256963","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Multifocal cutaneous neoplastic vascular proliferations in a rainbow boa (Epicrates cenchria) collection with boid inclusion body disease","authors":"Anthony Broering Ferreira, Joandes Fonteque, Jessica Withoeft, Renata Assis Casagrande, Ubirajara da Costa, Frank Imkamp, Pauline Goller, Francesca Baggio, Jussi Hepojoki, Udo Hetzel, Anja Kipar","doi":"10.1101/2024.09.12.612643","DOIUrl":"https://doi.org/10.1101/2024.09.12.612643","url":null,"abstract":"Reports on neoplastic processes in snakes are sparse regardless of their location, origin or behavior. Here, we describe the occurrence of multifocal cutaneous neoplastic processes consistent with hemangioma and hemangioendothelioma, with a differential diagnosis of angiomatosis, in a colony of native Brazilian rainbow boas (Epicrates cenchria) which also included animals affected by boid inclusion body disease (BIBD). Thirteen snakes were affected; seven of these had been introduced from other Brazilian sites years earlier, the others had been bred in house but were not offspring of knowingly affected animals. The breeding regime allowed contact between all female and male animals over the years. The cutaneous lesions were first observed over eight years ago, with additional cases detected during the three following years, but no new cases in the last five years. Two affected animals were subjected to a post mortem examination and were found to suffer from peliosis hepatis as one of the additional pathological changes. BIBD was confirmed in five of the eight examined animals, by histology, immunohistology for reptarenavirus nucleoprotein, and multiplex RT-PCR targeting the reptarenavirus S segment. Reptarenavirus infection was also detected in cells in the cutaneous neoplastic processes. PCRs for Bartonella henselae and B. quintana as well as bacterial DNA in general, performed on a pool of six skin lesions, yielded negative results, ruling out ongoing bacterial infection, like bacillary angiomatosis in humans, of the lesions. The results hint towards an association of reptarenavirus infection and BIBD with neoplastic processes which is worth further investigations.","PeriodicalId":501471,"journal":{"name":"bioRxiv - Pathology","volume":"1 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142256925","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"An anatomical investigation of alkaptonuria: Novel insights into ochronosis of cartilage and bone","authors":"Juliette H Hughes, Gemma Charlesworth, Amanda Prior, Claire M Tierney, Paul D Rothwell, Neil P Thomas, Lakshminarayan R Ranganath, James A Gallagher, Alistair P Bond","doi":"10.1101/2024.09.11.612405","DOIUrl":"https://doi.org/10.1101/2024.09.11.612405","url":null,"abstract":"Ochronotic pigmentation of connective tissue is the central pathological process in the rare metabolic disease alkaptonuria (AKU). Tissue pigmentation in AKU occurs due to unmetabolized homogentisic acid (HGA) in the circulation, caused by an enzyme deficiency in the liver. Ochronotic pigmentation, derived from HGA, has previously been reported and described in large joints obtained from arthroplasty surgeries, which typically have advanced disease. Many tissues that are affected by ochronosis are not accessible for study during life, including tissues subjected to early and mid-stage disease. Here, the opportunity arose to anatomically examine a 60-year-old AKU female body donor, allowing the investigation of previously understudied tissue, including those undergoing early-stage pathological changes. Dissection of fresh-frozen tissue was carried out and harvested tissues were fixed and examined histologically using H&E and Schmorls stains to aid identification of ochronotic pigment. This work focusses on osteochondral tissues including extra-skeletal cartilage, viscera and eyes. Gross and histological images demonstrating pigmentation in the cartilage and perichondrium of the ear ossicles, tympanic membrane, and the pubic symphysis fibrocartilaginous disc are described for the first time here. We also show the first examination of the temporomandibular joint, which macroscopically appeared unpigmented, with histological analysis of the fibrocartilaginous disc showing no pigmentation. Pigmentation of non-articular hyaline cartilage was observed in the respiratory tract, in both the hyaline cartilage and perichondrium, confirming previous findings. Within smaller joints, pigmentation of chondrons and the surrounding territorial matrix was observed, but was confined to calcified articular cartilage, and was not generally found in the hyaline articular cartilage. Dark pigmentation of the perichondrium adjacent to the articular surface was observed in numerous small joints, which has not been described before. The calcified bone matrix was not pigmented but ochronosis was identified in a small fraction of trabecular osteocytes in the capitate and radius, with substantially more pigmented osteocytes observed in bone of the ear ossicles. Viscera examined were unpigmented. This anatomical examination of tissues from an AKU individual highlights that most osteochondral tissues are susceptible to HGA-derived pigmentation, including the ear ossicles which are the smallest bones in the body. Within joints, calcified cartilage and perichondrium appear to be the earliest affected tissues, however why this is the case is not understood. Furthermore, why the TMJ disc was unaffected by pigmentation is intriguing. The heterogenous appearance of pigmentation both within and between different tissues indicates that factors other than tissue type (i.e. cartilage, perichondrium) and matrix composition (i.e. collagen-rich, calcified) may affect the process of ochr","PeriodicalId":501471,"journal":{"name":"bioRxiv - Pathology","volume":"17 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142256961","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Partial repair causes permanent defects in papillary structure and function after reversal of urinary obstruction","authors":"Thitinee Vanichapol, Alex Gonzalez, Rachel Delgado, Maya Brewer, Kelly Clouthier, Anna Menshikh, William Snyder, Teebro Raman, Veronika Sander, Alan J Davidson, Mark de Caestecker","doi":"10.1101/2024.09.11.612436","DOIUrl":"https://doi.org/10.1101/2024.09.11.612436","url":null,"abstract":"Urinary obstruction causes injury to the renal papilla and leads to defects in the ability to concentrate urine which predisposes to progressive kidney injury. However, the regenerative capacity of the papilla after reversal of obstruction is poorly understood. To address this, we developed a mouse model of reversible urinary obstruction which is characterized by extensive papillary injury, followed by a robust regeneration response and complete histological recovery over a 3-month period. However, these mice have a pronounced defect in urinary concentrating capacity. We now show that this is due to permanent changes in the composition, organization, and transcriptional signatures of epithelial, endothelial, and interstitial cell lineages in the papilla. There are persistent inflammatory responses that are also seen in patients with renal stone disease but are associated with cell-specific adaptive responses to the increasingly hypoxic environment of the papilla after reversal of obstruction. Taken together, our analysis of a new model of reversible urinary obstruction reveals that partial repair leads to permanent changes in the structure and function of all of the major cellular compartments in the papilla that include both shared and distinct responses to different types of renal papillary injury in humans and mice.","PeriodicalId":501471,"journal":{"name":"bioRxiv - Pathology","volume":"15 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142256960","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Advanced Tissue Technologies of Blood-Brain Barrier Organoids as High Throughput Toxicity Readouts in Drug Development","authors":"Luisa Bell, Claire Simonneau, Chiara Zanini, Elena Kassianidou, Christelle Zundel, Rachel Neff, Bernd Steinhuber, Marco Tecilla, Alex Odermatt, Roberto Villaseñor, Nadine Stokar-Regenscheit","doi":"10.1101/2024.09.09.611987","DOIUrl":"https://doi.org/10.1101/2024.09.09.611987","url":null,"abstract":"Recent advancements in engineering Complex in vitro models (CIVMs) such as Blood-brain barrier (BBB) organoids offer promising platforms for preclinical drug testing. However, their application in drug development, and especially for the regulatory purposes of toxicity assessment, requires robust and reproducible techniques. Here, we developed an adapted set of orthogonal image-based tissue methods including hematoxylin and eosin staining (HE), immunohistochemistry (IHC), multiplex immunofluorescence (mIF), and Matrix Assisted Laser Desorption/Ionization Mass Spectrometry Imaging (MALDI-MSI) to validate CIVMs for drug toxicity assessments. We developed an artificial intelligence (AI) algorithm to increase the throughput and the reliability of histomorphologic evaluations of apoptosis for in vitro toxicity studies. Our data highlight the potential to integrate advanced morphology-based readouts such as histological techniques and digital pathology algorithms for use on CIVMs, as part of a standard preclinical drug development assessment.","PeriodicalId":501471,"journal":{"name":"bioRxiv - Pathology","volume":"5 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142256962","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Coagulation factor XII contributes to renin activation, heart failure progression, and mortality","authors":"Inna P. Gladysheva, Ryan D. Sullivan, Sofiyan Saleem, Francis J. Castellino, Victoria A. Ploplis, Guy L. Reed","doi":"10.1101/2024.09.06.611753","DOIUrl":"https://doi.org/10.1101/2024.09.06.611753","url":null,"abstract":"Symptomatic heart failure (sHF) with cardiac dysfunction, edema, and mortality are driven by overactivation of the renin-angiotensin-aldosterone system (RAAS). Renin is widely recognized as a key initiator of RAAS function, yet the mechanisms that activate renin remain a mystery. We discovered that activated coagulation factor XII generates active renin in the circulation and is directly linked to pathological activation of the systemic RAAS, development of sHF, and increased mortality. These findings suggest a new paradigm for therapeutically modulating the RAAS in sHF and other pathological conditions.","PeriodicalId":501471,"journal":{"name":"bioRxiv - Pathology","volume":"156 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142182949","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}