Felix A Habermann, Herbert Kaltner, Alonso M Higuero, Gabriel García Caballero, Anna-Kristin Ludwig, Joachim C Manning, José Abad-Rodríguez, Hans-Joachim Gabius
{"title":"What Cyto- and Histochemistry Can Do to Crack the Sugar Code.","authors":"Felix A Habermann, Herbert Kaltner, Alonso M Higuero, Gabriel García Caballero, Anna-Kristin Ludwig, Joachim C Manning, José Abad-Rodríguez, Hans-Joachim Gabius","doi":"10.1267/ahc.21-00017","DOIUrl":"10.1267/ahc.21-00017","url":null,"abstract":"<p><p>As letters form the vocabulary of a language, biochemical 'symbols' (the building blocks of oligo- and polymers) make writing molecular messages possible. Compared to nucleotides and amino acids, sugars have chemical properties that facilitate to reach an unsurpassed level of oligomer diversity. These glycans are a part of the ubiquitous cellular glycoconjugates. Cyto- and histochemically, the glycans' structural complexity is mapped by glycophenotyping of cells and tissues using receptors ('readers', thus called lectins), hereby revealing its dynamic spatiotemporal regulation: these data support the concept of a sugar code. When proceeding from work with plant (haem)agglutinins as such tools to the discovery of endogenous (tissue) lectins, it became clear that a broad panel of biological meanings can indeed be derived from the sugar-based vocabulary (the natural glycome incl. post-synthetic modifications) by glycan-lectin recognition <i>in situ</i>. As consequence, the immunocyto- and histochemical analysis of lectin expression is building a solid basis for the steps toward tracking down functional correlations, for example in processes leading to cell adhesion, apoptosis, autophagy or growth regulation as well as targeted delivery of glycoproteins. Introduction of labeled tissue lectins to glycan profiling assists this endeavor by detecting counterreceptor(s) <i>in situ</i>. Combining these tools and their applications strategically will help to take the trip toward the following long-range aim: to compile a dictionary for the glycan vocabulary that translates each message (oligosaccharide) into its bioresponse(s), that is to crack the sugar code.</p>","PeriodicalId":6888,"journal":{"name":"Acta Histochemica Et Cytochemica","volume":null,"pages":null},"PeriodicalIF":2.4,"publicationDate":"2021-04-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/90/23/ahc-054-31.PMC8116616.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38999690","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"Raman Spectroscopic Assessment of Myocardial Viability in Langendorff-Perfused Ischemic Rat Hearts.","authors":"Koki Ikemoto, Kosuke Hashimoto, Yoshinori Harada, Yasuaki Kumamoto, Michiyo Hayakawa, Kentaro Mochizuki, Kazuhiko Matsuo, Kenta Yashiro, Hitoshi Yaku, Tetsuro Takamatsu, Hideo Tanaka","doi":"10.1267/ahc.21-00016","DOIUrl":"https://doi.org/10.1267/ahc.21-00016","url":null,"abstract":"<p><p>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<sup>-1</sup> 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.</p>","PeriodicalId":6888,"journal":{"name":"Acta Histochemica Et Cytochemica","volume":null,"pages":null},"PeriodicalIF":2.4,"publicationDate":"2021-04-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/14/f7/ahc-054-65.PMC8116620.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38999692","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"Development of Pathological Diagnosis Support System Using Micro-computed Tomography.","authors":"Tomonari Hayakawa, Atsushi Teramoto, Yuka Kiriyama, Tetsuya Tsukamoto, Ayumi Yamada, Kuniaki Saito, Hiroshi Fujita","doi":"10.1267/ahc.20-00033","DOIUrl":"https://doi.org/10.1267/ahc.20-00033","url":null,"abstract":"<p><p>In pathological diagnosis, the cutting position of pathological materials is subjectively determined by pathologists. This leads to a low cutting accuracy, which in turn may lead to incorrect diagnoses. In this study, we developed a system that supports the determination of the cutting position by visualizing and analyzing the internal structure of pathological material using micro-computed tomography (CT) before cutting. This system consists of a dedicated micro-CT and cutting support software. The micro-CT system has a fixture for fixing the target, enabling the scanning of easily deformable pathological materials. In the cutting support software, a function that interactively selects the extraction plane while displaying the volume rendering image and outputs a pseudo-histological image was implemented. Our results confirmed that the pseudo-histological image showed the fine structure inside the organ and that the latter image was highly consistent with the pathological image.</p>","PeriodicalId":6888,"journal":{"name":"Acta Histochemica Et Cytochemica","volume":null,"pages":null},"PeriodicalIF":2.4,"publicationDate":"2021-04-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/70/4c/ahc-054-49.PMC8116619.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38999689","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"Altered Immunohistochemical Expression Patterns of HLA Class I during the Clinical Course of Cervical Intraepithelial Neoplasia.","authors":"Okuto Koguchi, Haruna Nishimaki, Yoko Nakanishi, Hiroko Kobayashi, Sumie Ohni, Xiaoyan Tang, Yoshiaki Kusumi, Shinobu Masuda","doi":"10.1267/ahc.21-00010","DOIUrl":"https://doi.org/10.1267/ahc.21-00010","url":null,"abstract":"<p><p>It is unclear how immunohistochemical expression patterns of HLA class I in the pre-malignant phase of cervical intraepithelial neoplasia (CIN) alter during the clinical follow-up period. The present study aimed to demonstrate the correlation between the immunohistochemical expression pattern of HLA class I and the CIN grade through repeated examinations during the clinical course. Expression patterns of HLA class I, p16INK4a, and PD-L1 were immunohistochemically examined using formalin-fixed paraffin-embedded (FFPE) sections of biopsy or conization samples that were obtained from 20 patients diagnosed with CIN. The mRNA expression levels of HLA class I were analyzed by real-time reverse transcription polymerase chain reaction using FFPE sections of 14 patients, who were examined metachronously during the follow-up period. HLA class I expression was limited to the lower part of the epithelial thickness (M1 pattern) in more than half of CIN1 cases, and was present throughout the epithelial thickness (M2 pattern) in one fourth of CIN1 and CIN2 cases approximately. Heterogeneous expression (H pattern) was detected in half of CIN2 and CIN3 cases and in the all of squamous cell carcinoma cases. Metachronous examinations revealed that these immunohistochemical patterns altered more frequently than the CIN grade. The rate of change of HLA class I mRNA expression level was higher in cases with a progressed immunohistochemical pattern compared to those with regressed immunohistochemical pattern. In conclusion, the immunohistochemical pattern of HLA class I expression is associated with the CIN grade, and it is alterable during the clinical course, especially in CIN2.</p>","PeriodicalId":6888,"journal":{"name":"Acta Histochemica Et Cytochemica","volume":null,"pages":null},"PeriodicalIF":2.4,"publicationDate":"2021-04-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/55/40/ahc-054-57.PMC8116618.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38999691","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Junji Itou, Akihiro Nakamura, Hideki Hokazono, Masakazu Toi
{"title":"Supplementation with Fermented Barley Extract Prevents Mammary Epithelial Cell Invasion in an Early Breast Cancer Model.","authors":"Junji Itou, Akihiro Nakamura, Hideki Hokazono, Masakazu Toi","doi":"10.1267/ahc.20-00029","DOIUrl":"https://doi.org/10.1267/ahc.20-00029","url":null,"abstract":"<p><p>Diet-based prevention of malignant transformation contributes to the maintenance of quality of life by avoiding a battle against cancer. Invasion is one of the features of malignant breast cancer, and the prevention of invasion may reduce breast cancer malignancy. A recently established early breast cancer model system showed mammary ductal dysplasia with invasion in mice. This study utilized the model system and investigated the effect of fermented barley extract (FBE), a food material. The elastic fiber layer is the outermost layer of the mammary duct. A reduction in the elastic fiber layer was observed in the mammary glands of the model system, whereas supplementation with 8% FBE containing water prevented this reduction. Moreover, we found that FBE supplementation prevented mammary epithelial cell invasion. Based on our findings, FBE might be a candidate material for a diet-based prevention of early breast cancer invasion.</p>","PeriodicalId":6888,"journal":{"name":"Acta Histochemica Et Cytochemica","volume":null,"pages":null},"PeriodicalIF":2.4,"publicationDate":"2021-04-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/94/b7/ahc-054-73.PMC8116617.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"39000168","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"The Periodontium Damage Induces Neuronal Cell Death in the Trigeminal Mesencephalic Nucleus and Neurodegeneration in the Trigeminal Motor Nucleus in C57BL/6J Mice.","authors":"Ashis Dhar, Eriko Kuramoto, Makoto Fukushima, Haruki Iwai, Atsushi Yamanaka, Tetsuya Goto","doi":"10.1267/ahc.20-00036","DOIUrl":"https://doi.org/10.1267/ahc.20-00036","url":null,"abstract":"Proprioception from masticatory apparatus and periodontal ligaments comes through the trigeminal mesencephalic nucleus (Vmes). We evaluated the effects of tooth loss on neurodegeneration of the Vmes and trigeminal motor nucleus (Vmo). Bilateral maxillary molars of 2-month-old C57BL/6J mice were extracted under anesthesia. Neural projections of the Vmes to the periodontium were confirmed by injecting Fluoro-Gold (FG) retrogradely into the extraction sockets, and for the anterograde labeling adeno-associated virus encoding green fluorescent protein (AAV-GFP) was applied. For immunohistochemistry, Piezo2, ATF3, Caspase 3, ChAT and TDP-43 antibodies were used. At 1 month after tooth extraction, the number of Piezo2-immunoreactive (IR) Vmes neurons were decreased significantly. ATF3-IR neurons were detected on day 5 after tooth extraction. Dead cleaved caspase-3-IR neurons were found among Vmes neurons on days 7 and 12. In the Vmo, neuronal cytoplasmic inclusions (NCIs) formation type of TDP-43 increased at 1 and 2 months after extraction. These indicate the existence of neural projections from the Vmes to the periodontium in mice and that tooth loss induces the death of Vmes neurons followed by TDP-43 pathology in the Vmo. Therefore, tooth loss induces Vmes neuronal cell death, causing Vmo neurodegeneration and presumably affecting masticatory function.","PeriodicalId":6888,"journal":{"name":"Acta Histochemica Et Cytochemica","volume":null,"pages":null},"PeriodicalIF":2.4,"publicationDate":"2021-02-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/2e/0e/ahc-054-11.PMC7947638.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"25488573","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"Matrix Metalloproteinase-2 Activated by Ultraviolet-B Degrades Human Ciliary Zonules <i>In Vitro</i>.","authors":"Yuki Shiroto, Ryo Saga, Hironori Yoshino, Yoichiro Hosokawa, Keitaro Isokawa, Eichi Tsuruga","doi":"10.1267/ahc.20-00021","DOIUrl":"https://doi.org/10.1267/ahc.20-00021","url":null,"abstract":"<p><p>The ciliary zonules, also known as the zonules of Zinn, help to control the thickness of the lens during focusing. The ciliary zonules are composed of oxytalan fibers, which are synthesized by human nonpigmented ciliary epithelial cells (HNPCEC). The ciliary zonules are exposed to ultraviolet (UV), especially UV-A and UV-B, throughout life. We previously demonstrated that UV-B, but not UV-A, degrades fibrillin-1- and fibrillin-2-positive oxytalan fibers. However, the mechanism by which UV-B degrades oxytalan fibers remains unknown. In this study, we investigate the involvement of matrix metalloproteinase-2 (MMP-2) in the UV-B-induced degradation of fibrillin-1- and fibrillin-2-positive oxytalan fibers in cultured HNPCECs. Enzyme-linked immunosorbent assay revealed that UV-B irradiation at levels of 100 and 150 mJ/cm<sup>2</sup> significantly increased the level of active MMP-2. Notably, MMP-2 inhibitors completely suppressed the degradation of fibrillin-1- and fibrillin-2-positive oxytalan fibers. In addition, we show that UV-B activates MMP-2 via stress-responsive kinase p38. Taken together, the results suggest that UV-B activates a production of active type of MMP-2 via the p38 pathway, and subsequently, an active-type MMP-2 degrades the fibrillin-1- and fibrillin-2-positive oxytalan fibers in cultured HNPCECs.</p>","PeriodicalId":6888,"journal":{"name":"Acta Histochemica Et Cytochemica","volume":null,"pages":null},"PeriodicalIF":2.4,"publicationDate":"2021-02-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/3d/68/ahc-054-1.PMC7947639.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"25488572","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"Comparative Analysis of Immunohistochemical Staining Intensity Determined by Light Microscopy, ImageJ and QuPath in Placental Hofbauer Cells.","authors":"Katerina Cizkova, Tereza Foltynkova, Mariam Gachechiladze, Zdenek Tauber","doi":"10.1267/ahc.20-00032","DOIUrl":"https://doi.org/10.1267/ahc.20-00032","url":null,"abstract":"<p><p>Software based analyses of immunohistochemical staining are designed for obtaining quantitative, reproducible, and objective data. However, often times only a certain type of positive cells or structures need to be quantified thus whole image analysis cannot be performed. Such an example is Hofbauer placental cells, which show positivity of some antigens together with trophoblast, but only Hofbauer cells represent the regions of interest (ROIs). Two independent observers evaluated the immunohistochemical staining intensity of Hofbauer cells in placenta samples stained for cytoplasmic antigens by ImageJ, QuPath and light microscopy. Thus, the precise manual determination of ROIs, i.e. Hofbauer cells, was necessary. We detected low inter-observer variability in staining intensity. Almost perfect agreement between observers was reached for ImageJ and QuPath whilst substantial agreement was reached for light microscopy evaluation. As for the comparison of ImageJ, QuPath and light microscopy, the agreement of all three methods (identical immunohistochemical intensity) was achieved for 38.1% samples. The almost perfect agreement of staining intensities was reached between ImageJ and QuPath, and moderate agreement for comparison of the light microscopy to both software. Software analyses are much more time-consuming, thus their utilization is at least questionable to evaluate ROIs with selection.</p>","PeriodicalId":6888,"journal":{"name":"Acta Histochemica Et Cytochemica","volume":null,"pages":null},"PeriodicalIF":2.4,"publicationDate":"2021-02-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/17/df/ahc-054-21.PMC7947637.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"25488574","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"Minocycline Alleviates Cluster Formation of Activated Microglia and Age-dependent Dopaminergic Cell Death in the Substantia Nigra of Zitter Mutant Rat.","authors":"Daisuke Taguchi, Ayuka Ehara, Taro Kadowaki, Shin-Ichi Sakakibara, Kazuhiko Nakadate, Koichi Hirata, Shuichi Ueda","doi":"10.1267/ahc.20-00022","DOIUrl":"https://doi.org/10.1267/ahc.20-00022","url":null,"abstract":"<p><p>Microglial activation is a component of neurodegenerative pathology. Here, we examine whether activated microglia participate in age-related dopaminergic (DA) cell death in the substantia nigra pars compacta (SNc) of the zitter (<i>zi/zi</i>) rat, a mutant characterized by deletion of the attractin gene. Confocal microscopy with double-immunohistochemical staining revealed activated microglia-formed cell-clusters surrounding DA neurons in the SNc from 2 weeks after birth. An immunoelectron microscopic study showed that the cytoplasm of activated microglia usually contains phagosome-like vacuoles and lamellar inclusions. Expression levels of the pro-inflammatory cytokines <i>interleukin-1β</i> (<i>IL-1β</i>), <i>tumor necrosis factor-α</i> (<i>TNF-α</i>) and <i>inducible nitric oxide synthase</i> (<i>iNOS</i>) were increased in the midbrain of 2-month-old <i>zi/zi</i> rats. Chronic treatment with the anti-inflammatory agent minocycline altered the morphology of the microglia, reduced cluster formation by the microglia, and attenuated DA cell death in the SNc, and reduced the expression of <i>IL-1β</i> in the midbrain. These results indicate that activated microglia, at least in part and especially at the initial phase, contribute to DA cell death in the SNc of the <i>zi/zi</i> rat.</p>","PeriodicalId":6888,"journal":{"name":"Acta Histochemica Et Cytochemica","volume":null,"pages":null},"PeriodicalIF":2.4,"publicationDate":"2020-12-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7785462/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38812593","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"Mercury and Selenium Localization in the Cerebrum, Cerebellum, Liver, and Kidney of a Minamata Disease Case.","authors":"Masumi Marumoto, Mineshi Sakamoto, Kohji Marumoto, Shozo Tsuruta, Yoshihiro Komohara","doi":"10.1267/ahc.20-00009","DOIUrl":"https://doi.org/10.1267/ahc.20-00009","url":null,"abstract":"<p><p>Minamata disease is a methylmercury poisoning caused by consumption of marine food contaminated by man-made methylmercury environmental pollution, and its most prominent feature is marked pathological changes in the central nervous system. Morphological alterations are less pronounced in the liver and the kidney, although their mercury levels are higher than those of the brain. In marine mammals, methylmercury is known to be easily converted to inorganic mercury and it combines with selenium forming mercury selenide, which may counteract the toxicity of mercury. However, little is known about the formation of mercury and selenium complex in human organs. In the present study, we examined the cerebrum, cerebellum, liver, and kidney of a Minamata disease case to study the mercury and selenium localization using electron probe microanalysis. Our results indicated the mercury and selenium localization in the specified tissue of the brain, liver, and kidney such as glial cells, Kupffer cells, and renal tubules.</p>","PeriodicalId":6888,"journal":{"name":"Acta Histochemica Et Cytochemica","volume":null,"pages":null},"PeriodicalIF":2.4,"publicationDate":"2020-12-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7785461/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38812594","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}