Sci Rep Research Articles

First Report of Colletotrichum gloeosporioides Causing Anthracnose on Pepper in Shaanxi Province, China.

Pepper (Capsicum annuum L.) is an important solanaceous vegetable crop, with high nutritional and economic value. However, it is susceptible to Colletotrichum spp. infection during its growth and development, which seriously affects production yield and quality. Chili anthracnose, caused by Colletotrichum spp., is one of the most destructive diseases of pepper. In August 2020, chili anthracnose was observed with widespread distribution in the horticulture field of Northwest A&F University (34.16° N, 108.04° E) in Shaanxi Province, China. Approximately 60% of the pepper plants had disease symptoms typical of anthracnose. Lesions on pepper fruits were dark, circular, sunken, and necrotic, with the presence of orange to pink conidial masses (Figure S1A). To perform fungal isolation, the tissue at the lesion margin was cut from eight symptomatic fruits, surface disinfested with 75% ethanol for 30 s, and 2% NaClO for 1 min, then rinsed three times with sterile distilled water and dried on sterile filter paper. The tissues were placed on potato dextrose agar (PDA) and incubated at 28 ºC in the dark. After 3 days, hyphae growing from tissue of each lesion were recultured on PDA (Liu et al. 2016). A representative single-spore isolate (NWAFU2) was used for morphological characterization, molecular analysis, phylogenetic analysis, and pathogenicity tests. NWAFU2 colonies had gray-white aerial mycelium, and the reverse side of the colonies was dark gray to light yellow after 10-days growth on PDA (Figure S1B-C). Conidia were cylindrical, aseptate, with obtuse to slightly rounded ends, and measured 10.1 to 16.9 (length) × 4.7 to 7.0 (width) μm (n=50) (Figure S1D). Based on morphological features, the isolate was consistent with the description of C. gloeosporioides species complex (Weir et al. 2012). For molecular identification, genomic DNA was extracted using a CTAB method and the internal transcribed spacer (ITS) region, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and partial sequences of actin (ACT) genes were amplified and sequenced using primers ITS1F/ITS4, GDF1/GDR1 and ACT-512F/ACT-783R, respectively (Dowling et al. 2020). Using the BLAST, ITS, ACT, GAPDH gene sequences (GenBank accession nos. MW258690, MW258691 and MW258692, respectively) were 100%, 100% and 98.19% identical to ZJL-4 of C. gloeosporioides (GenBank accession nos. MN075757, MN058142 and MN075666, respectively). Phylogenetic analysis was conducted using MEGA-X (Version 10.0) based on the concatenated sequences of published ITS, ACT and GAPDH for Colletotrichum species using Neighbor-Joining algorithm. The identified isolate (NWAFU2) was closely related to C. gloeosporioides (Figure S2). To confirm the pathogenicity, ten healthy pepper fruits were surface-sterilized and 2 μL of conidial suspension (1×106 conidia/mL) was injected the surface of pepper. Five fruits were inoculated with 2μL sterile distilled water as controls. After inoculation, the fruits were kept in a moist chamber at 28°C in the dark. The experiment was repeated three times. Anthracnose symptoms similar to those observed in the field, were observed 7 days after inoculation (Figure S1F) and control fruits remained healthy. A similarly inoculated detached leaf assay resulted in water-soaked lesions 3 days after inoculation. C. gloeosporioides was reisolated from the infected pepper fruits, fulfilling Koch's postulates. C. gloeosporioides has been reported to cause chili anthracnose in Sichuan Province, China (de Silva et al. 2019; Liu et al. 2016). However, Shaanxi is one of the main pepper producing areas in china and it is geographically distinct from Sichuan; its climate and environmental conditions are different from Sichuan. Knowledge that C. gloeosporioides causes chili anthracnose of pepper in Shaanxi province, China may aid in the selection of appropriate management tactics for this disease. Reference: de Silva, D. D., Groenewald, J. Z., Crous, P. W., Ades, P. K., Nasruddin, A., Mongkolporn, O., and Taylor, P. W. J. 2019. Identification, prevalence and pathogenicity of Colletotrichum species causing anthracnose of Capsicum annuum in Asia. IMA Fungus 10:8. Dowling, M., Peres, N., Villani, S., and Schnabel, G. 2020. Managing Colletotrichum on Fruit Crops: A "Complex" Challenge. Plant Dis 104:2301-2316. Liu, F. L., Tang, G. T., Zheng, X. J., Li, Y., Sun, X. F., Qi, X. B., Zhou, Y., Xu, J., Chen, H. B., Chang, X. L., Zhang, S. R., and Gong, G. S. 2016. Molecular and phenotypic characterization of Colletotrichum species associated with anthracnose disease in peppers from Sichuan Province, China. Sci Rep 6. Weir, B. S., Johnston, P. R., and Damm, U. 2012. The Colletotrichum gloeosporioides species complex. Stud Mycol 73:115-180.

Understanding the thermodynamic properties of insect swarms

Sinhuber et al. (Sci Rep 11:3773, 2021) formulated an equation of state for laboratory swarms of the non-biting midge Chironomus riparius that holds true when the swarms are driven through thermodynamic cycles by the application external perturbations. The findings are significant because they demonstrate the surprising efficacy of classical equilibrium thermodynamics for quantitatively characterizing and predicting collective behaviour in biology. Nonetheless, the equation of state obtained by Sinhuber et al. (2021) is anomalous, lacking a physical analogue, making its’ interpretation problematic. Moreover, the dynamical processes underlying the thermodynamic cycling were not identified. Here I show that insect swarms are equally well represented as van der Waals gases and I attribute the possibility of thermodynamic cycling to insect swarms consisting of several overlapping sublayers. This brings about a profound change in the understanding of laboratory swarms which until now have been regarded as consisting of non-interacting individuals and lacking any internal structure. I show how the effective interactions can be attributed to the swarms’ internal structure, the external perturbations and to the presence of intrinsic noise. I thereby show that intrinsic noise which is known to be crucial for the emergence of the macroscopic mechanical properties of insect swarms is also crucial for the emergence of their thermodynamic properties as encapsulated by their equation of state.

Can we talk about myoblast fusion?

Can we talk about myoblast fusion?

Murine models of Duchenne muscular dystrophy: is there a best model?

Murine models of Duchenne muscular dystrophy: is there a best model?

Polygenic scores for handedness and their association with asymmetries in brain structure

Handedness is the most widely investigated motor preference in humans. The genetics of handedness and especially the link between genetic variation, brain structure, and right-left preference have not been investigated in detail. Recently, several well-powered genome-wide association studies (GWAS) on handedness have been published, significantly advancing the understanding of the genetic determinants of left and right-handedness. In the present study, we estimated polygenic scores (PGS) of handedness-based on the GWAS by de Kovel and Francks (Sci Rep 9: 5986, 2019) in an independent validation cohort (n = 296). PGS reflect the sum effect of trait-associated alleles across many genetic loci. For the first time, we could show that these GWAS-based PGS are significantly associated with individual handedness lateralization quotients in an independent validation cohort. Additionally, we investigated whether handedness-derived polygenic scores are associated with asymmetries in gray matter macrostructure across the whole brain determined using magnetic resonance imaging. None of these associations reached significance after correction for multiple comparisons. Our results implicate that PGS obtained from large-scale handedness GWAS are significantly associated with individual handedness in smaller validation samples with more detailed phenotypic assessment.

Experimental demonstration of the novel \u201cvan-Hove integral method (vHI)\u201d for measuring diffusive dynamics by elastic neutron scattering

Quasi-elastic neutron scattering (QENS)—based on the seminal work of Nobel Laureate Brockhouse—has been one of the major methods for studying pico-second to nano-second diffusive dynamics over the past 70 years. This is regarded as an “inelastic” method for dynamics. In contrast, we recently proposed a new neutron-scattering method for dynamics, which uses the elastic line of the scattering to access system dynamics directly in the time domain (Benedetto and Kearley in Sci Rep 9:11284, 2019). This new method has been denoted “vHI” that stands for “van Hove Integral”. The reason is that, under certain conditions, the measured elastic intensity corresponds to the running-time integral of the intermediate scattering function, Ileft( {Q,t} right), up to a time that is inversely proportional to the energy band-width incident on the sample. As a result, Ileft( {Q,t} right) is accessed from the time derivative of the measured vHI profile. vHI has been supported by numerical and Monte-Carlo simulations, but has been difficult to validate experimentally due to the lack of a suitable instrument. Here we show that vHI works in practice, which we achieved by using a simple modification to the standard QENS backscattering spectrometer methodology. Basically, we varied the neutron-energy band-widths incident at the sample via a step-wise variation of the frequency of the monochromator Doppler-drive. This provides a measurement of the vHI profile at the detectors. The same instrument and sample were also used in standard QENS mode for comparison. The intermediate scattering functions, Ileft( {Q,t} right), obtained by the two methods—vHI and QENS—are strikingly similar providing a direct experimental validation of the vHI method. Perhaps surprisingly, the counting statistics of the two methods are comparable even though the instrument used was expressly designed for QENS. This shows that the methodology modification adopted here can be used in practice to access vHI profiles at many of the backscattering spectrometers worldwide. We also show that partial integrations of the measured QENS spectrum cannot provide the vHI profile, which clarifies a common misconception. At the same time, we show a novel approach which does access Ileft( {Q,t} right) from QENS spectra.

Abstract 2336: Eradicating melanoma cells using combination of nutritional stress and checkpoint inhibitors

Abstract Arginine (Arg) deprivation can inhibit tumors which do not express argininosuccinate synthetase (ASS), a key enzyme for synthesis of Arg. Arg deiminase (ADIPEG20) has shown antitumor activity in such tumors, including melanoma and mesothelioma. Resistance to this treatment occurs due to re-expression of ASS. We have shown that this is mediated via cMyc (positive regulator) and HIF1α ( negative regulator ). Arg starvation rapidly induces chromatin remodeling complex P300-HDAC2-Sin3A which epigenetically deacetylates H3K14ac and H3K27ac at the ASS promoter. Following the PHD2-derived HIF1α-degrading system, the promoter-bound HIF1α is degraded (Sci Rep 7:10814). This allows cMyc (an E-Box binder), to turn on ASS. Arg starvation rapidly triggers externalization of Gas6 to interact with its receptor tyrosine kinase (RTK) Axl. This activates the downstream Ras-PI3K/AKT/GSK3β pathway, resulting in stabilization of cMyc. However, this activation also turns on PDL1 gene expression. This is not surprising since cMyc has been shown to bind to PDL1 promoter and increase its expression. Thus, re-expression of ASS also leads to PDL1 expression. We studied these findings in two other cell lines, Mel114 and Skmel2, and obtained similar results, Interestingly, in three cell lines with high levels of antiapoptotic protein Bcl-2, Arg starvation only leads to growth inhibition, with only less than 2 fold increase in ASS and no PDL1 expression. These results suggest that increased Bcl-2 expression negates the necessity to increase PDL1 and ASS to prevent cell death from nutritional stress. In contrast, transfection of ASS in A2058, A375, Mel1220 and SkMel2 will only confer resistance to Arg starvation, and does not affect PDL1, Bcl2 expression or other alterations in PI3K/AKT pathway. Furthermore, in samples from patients who failed ADIPEG20 due to increased ASS expression, the primary cultures or re-biopsy tumor also exhibited high levels of PDL1 expression, which most likely will make them susceptible to anti PDL1 treatment. On the other hand, six primary cultures derived from patients who failed BRAF/MEK inhibitor (BMR) also showed low levels of ASS and increase in PDL1 expression (1.5-4 fold increase both in mRNA and proteins level compared to their parental cells). These results also applied to 5 pairs of BMR cell lines (A375/BMR, A2058/BMR, Skmel2/BMR, UACC62/BMR, MelRR/BMR). These cells also are very susceptible to combination of ADIPEG20 and PD-L1 treatment. Overall, our data suggest that combination of Arg deprivation and check point inhibitors is effective in patients whose tumors do not express ASS and as salvage therapy for BRAF/MEK inhibitor resistant patients. Supported by the VA Merit Review Award(1BX003328) Citation Format: Lynn G. Feun, Chunjing Wu, Ying-Ying Li, Min You, Medhi Wangpaichitr, Miguel Suarez, Niramol G. Savaraj. Eradicating melanoma cells using combination of nutritional stress and checkpoint inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2336.

Abstract 3156: Over-expression of CEACAM6 negatively modulates the tumor microenvironment in pancreatic cancer

Abstract Background: CEACAM6 is a cell adhesion receptor of the Ig-family over-expressed exclusively in primates and human pancreatic ductal adenocarcinoma (PDA). It is present in all subtypes and is enriched classical activated stroma subtype. CEACAM6 has multifaceted roles in KRAS mutant PDA and is a poor prognostic maker (Pandey et al. Sci Rep 2019). We report here in PDA, high CEACAM6 modulates the tumor microenvironment with an immunosuppressive landscape. Methods: RNA-Seq data of PDA patient samples from TCGA and GEO were obtained, normalized and log transformed for analysis. Tumor samples were stratified into cohorts of high and low CEACAM6. Pearson correlation coefficient is utilized to identify CEACAM6 correlated genes and enrichment of signaling pathways. The two cohorts were scored by ESTIMATE for stromal and immune population. Abundance of different cell populations were calculated utilizing MCPcounter and ConsensusTME methods. All analysis was performed using R (v 4.3). Results: High CEACAM6 PDA samples had both stromal and immune components. Half of the high expressing samples had a higher immune score vs stromal score. The other half had both high or both low, immune and stromal score. Investigating the immune genes that are utilized for scoring, we found several immune genes differentially expressed in high CEACAM6 (up-regulated genes - ADAM8, IL2RG, LYZ, SELPLG involved in innate immunity) compared to low CEACAM6 samples. Genes under expressed in high CEACAM6 samples were CD27, NKG7, IL18RAP, LILRB1, KLKRK1 involved in promoting immune response and cell protection. Overall TCGA PDA samples have lower enrichment of immune cells compared to higher enrichment of fibroblasts, endothelial cells, macrophages, monocytes and dendritic cells. The NK cells are overall low in PDA but enriched in some lower CEACAM6 samples. Plasma cells, B-cells, CD8 cells, NK cells, Cytotoxic T-cells, Tγδ-cells have very poor representation in higher CEACAM6 samples but present in some low CEACAM6 expressing samples. Some of the TCGA PDA samples have T-reg cells but high CEACAM6 do not enrich of T-reg cells. High CEACAM6 samples are enriched for metabolic pathways. Conclusions: CEACAM6 over-expressed in PDA have pathologic functions which include reshaping the stroma, suppressing host immunity and inhibiting apoptosis. An anti-CEACAM6 Mab increases apoptosis, necrosis and anti-tumor activity in PDA. Citation Format: Ritu Pandey, Yuliang Chen, Mihir Patil, Patrick J. Conway, Aisha Al-Khinji, Daruka Mahadevan. Over-expression of CEACAM6 negatively modulates the tumor microenvironment in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3156.

Reconstruction enhancement via projection screening in holographic tomography

This paper presents an algorithm for automatic detection of erroneous amplitude and phase components of a sample’s optical field, acquired by a holographic tomograph with a limited angle of projection. By applying image processing methods and statistical analysis to find and remove unfit projections, the quality of tomographic reconstruction of a 3D refractive index distribution of an object is greatly improved. The proposed methods can find their application in preprocessing of data in holographic tomography. Full Text: PDF ReferencesA. Kuś, W. Krauze, P. L. Makowski, and M. Kujawińska, "Holographic tomography: hardware and software solutions for 3D quantitative biomedical imaging (Invited paper)", ETRI Journal, 41, 1 (2019). CrossRef V. Balasubramani et al., "Phase unwrapping in ICF target interferometric measurement via deep learning", Appl. Opt., 60, 10 (2021). CrossRef Y. Park, C. Depeursinge, and G. Popescu, "Quantitative phase imaging in biomedicine", Nature Photonics, 12, 10 (2018). CrossRef W. Krauze, P. Makowski, M. Kujawińska, and A. Kuś, "Generalized total variation iterative constraint strategy in limited angle optical diffraction tomography", Opt. Express, 24, 5 (2016). CrossRef D. Ryu et al., "A non-calorimetric approach for investigating the moisture-induced ageing of a pyrotechnic delay material using spectroscopies", Sci Rep, 9, 1 (2019). CrossRef B. S. Lipkin, Picture Processing and Psychopictorics. (Saint Louis, Elsevier Science 2014). DirectLink A. M. Taddese, N. Verrier, M. Debailleul, J.-B. Courbot, and O. Haeberlé, "Optimizing sample illumination scanning in transmission tomographic diffractive microscopy", Appl. Opt., 60, 6 (2021). CrossRef

Highly Sensitive Viral RNA-Seq Is a Powerful Tool to Analyze the Viral Transcripts in HTLV-1-Infected Individuals

IntroductionHuman T-cell leukemia virus type 1 (HTLV-1) is a retrovirus which causes two major pathologies in infected individuals, Adult T-cell leukemia-lymphoma (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). There are tens of thousands of different infected clones in asymptomatic HTLV-1 carriers (AC) and each clone can be identified by the viral integration site. Monoclonal integration of HTLV-1 provirus is observed in the host genomic DNA in ATL cells, suggesting that these originate from a single virus-infected clone. It is well known that Tax and HBZ, encoded in the sense and antisense strands of HTLV-1 provirus, respectively, have the ability to induce oncogenesis. The question is how a certain infected clone, among various different ones, becomes a malignant clone. One hypothesis is that a malignant clone may express viral gene transcripts, associated with oncogenesis, more abundantly than a non-malignant clone. Another hypothesis is that HTLV-1 integration may induce an abnormal transcript in the host cell, such as the aberrant expression of the host gene nearby the integration site or the generation of a chimeric transcript between the virus and the host. To analyze HTLV-1 transcript or explore the presence of virus-host chimeric transcripts efficiently, we aimed to establish a method with higher resolution than the conventional RNA-seq by using DNA probe targeting for the entire HTLV-1 sequence. And then, we applied the protocol to characterize the pattern of transcription from HTLV-1 provirus and analyzed virus-host chimeric transcripts in patients' samples.MethodsTo check the efficiency and possible bias of the highly sensitive viral RNA-seq analysis, we analyzed viral transcript of an HTLV-1-infected clone, TBX-4B. Subsequently, mRNA-seq was performed with PBMCs from 6 AC, 4 HAM/TSP, and 5 ATL patients. After cDNA libraries synthesis, HTLV-1 sequences were enriched by the probe-based capture method as we reported previously for DNA-seq (Miyazato et al, Sci Rep 2016). The enriched libraries were sequenced in an Illumina NextSeq device.ResultsWe analyzed HTLV-1 transcript of a virus-infected T-cell clone both before and after the enrichment by viral-specific DNA probes. The proportion of viral sequences within the total number of reads was significantly increased from 0.05% to 80.62% (1600-fold) by the enrichment method. We found that most of the viral regions were significantly and similarly enriched by the additional enrichment step, suggesting the DNA probe-based enrichment of HTLV-1 sequences is applicable to RNA-seq. Next, we applied the protocol to analyze PBMC samples from HTLV-1-infected individuals. The results showed that the level of anti-sense HTLV-1 transcription was significantly higher than that of the sense transcription, not only in ATL patients, which is consistent with previous reports by other groups, but also in AC and HAM/TSP patients. Average proportion of sense and antisense signals were 3.42% and 96.46%, respectively. Even after the enrichment step, tax expression, a sense viral transcript, was undetectable in most of the samples except for one AC and one ATL patient, while an anti-sense transcript HBZ was detectable in all infected individuals. There was no significant difference in the expression level of HBZ between different clinical states. We identified more abundant virus-host chimeric transcripts in ATL samples than in HAM/TSP patients or AC. In ATL cases, HTLV-1 generated chimeric transcripts with the following host genes, HSD17B11, COLEC12, AHSA2, EEF2, TAOK1, KIAA0100, and KIF1B . The presence of abundant chimeric transcripts was validated by RT-PCR.DiscussionWe have established a highly sensitive viral RNA-seq for HTLV-1 transcript analysis. That significantly increased the sensitivity of detection of proviral and chimeric transcripts between virus and the host genome. The result was not consistent with the hypothesis that a malignant clone may express viral gene transcripts more abundantly than a non-malignant clone. Thus, the presence of chimeric transcripts could play a role in cell proliferation and help us reveal the mechanism of clonal selection for HTLV-1-infected cells. We are now going to analyze virus-host chimeric transcripts detected by the high sensitive RNA-seq in more patients' samples to clarify their association with oncogenesis. [Display omitted] DisclosuresYamano:Daiichi Sankyo: Research Funding; Ono Pharma: Research Funding. Utsunomiya:Celgene: Honoraria; Novartis Pharma: Honoraria; Roche Diagnostics: Honoraria; Japan Blood Products Organization: Honoraria; Daiichi Sankyo: Honoraria; Kyowa Hakko Kirin: Honoraria, Research Funding; Astellas Pharma: Honoraria; Chugai Pharma: Honoraria; Bristol-Myers Squibb: Honoraria; Siemens: Honoraria; Nippon Shinyaku: Honoraria; HUYA Bioscience International: Honoraria; Pfizer: Honoraria.

Landscape of Immune Reconstitution after Allogeneic Hematopoietic Cell Transplantation Is Superdominated By Distinct T Cell Populations Bearing T-Cell Receptor Clonotypes Shared Among Different Individuals

Background: Enormous diversity of T-cell receptor (TCR) repertoire is generated by somatic rearrangement of gene segments that encode TCRα and TCRβ subunits. Next-generation TCR sequencing is emerging as a powerful tool to elucidate TCR repertoire at very high resolution. We recently found that a small fraction of TCRs shared among different individuals confers immunologic predominance on T-cell reactivities against some viral antigens (Miyama, Sci Rep 2017). To investigate the role of T cells bearing such “shared” TCRs during immune reconstitution after allogeneic hematopoietic cell transplantation (allo-HCT), we herein performed semi-quantitative NGS analysis of TCR repertoire in peripheral blood (PB) of post-transplant patients.Methods: This study was conducted according to a protocol approved by the ethics committee of Hiroshima University. We collected PB mononuclear cells from 15 patients who received allo-HCT at our center for hematologic malignancies (acute leukemia=9; myelodysplastic syndrome=3; lymphomas=3) prior to and at different time points (1, 2, 3, 4, 6, 8 weeks and 3, 6, and 12 months) after transplantation. 10 patients received HLA-8/8 or 7/8-matched unrelated-donor bone marrow and 5 received HLA-mismatched single-cord blood unit. Using these samples, unbiased NGS analysis of TRB V-D-J gene segments was performed. We assessed the TCR repertoire diversity of each sample by calculating the Simpson's Reciprocal Index (SRI, 1/D) using NGS data. We defined TCRβ clonotypes found in two or more patients using the same TRBV/TRBJ gene segments and CDR3 amino acid sequences as a “shared” clonotype. To determine whether the read numbers of a shared TCR clonotype in a given sample occupy the higher or lower rank compared with those of unshared clonotypes, we performed the nonparametric Mann-Whitney test. P values less than 0.05 were considered statistically significant.Results: All patients achieved neutrophil engraftment at a median of 19 days with complete donor-type chimerism. A total of 12 patients developed grade II-IV acute GVHD at a median of 23.5 (range 6-60) days and 10 patients experienced cytomegalovirus reactivation at a median onset of 36 (range 15-56) days after transplantation. Among 36,281,897 TRB sequence reads obtained from the collected samples, 2,782,129 were identified as a unique read that defines a single TCRβ clonotype. Among these unique clonotypes, 42,436 (1.5%) were shared among different patients. Although SRIs in the samples obtained from 2 to 4 weeks post-transplant were nearly equivalent to those in pre-transplant samples, the diversity indices from 6 weeks to 12 months were significantly decreased before transplant (median 1/D; pre-transplant=137, 6w=29.9, 8w=40.5, 3m=35.8, 6m=33.6, 12m=28.3, P<0.05), indicating the persistent reduction of TCR repertoire diversity. Intriguingly, although the proportions of “shared” clonotypes among total TCRβ clonotypes later than 1 week post-transplant were relatively small (median 5.2%, range 0.55-16.9%), the ratio of unique reads encoding shared clonotypes among total TRB reads ranged from 3.6% to 93.3% (median 50.7%), which was significantly higher than that of unshared unique reads (P<0.05). Such predominance of shared TCRβ clonotypes was observed in both CD4+ and CD8+ T cell populations.Conclusions: T-cell repertoire reconstitution after allo-HCT is characterized by extreme predominance of T cells bearing TCRβ clonotypes shared among different individuals. Our results suggest that a distinct population of T cells expressing “shared” TCRs strongly affects the homeostasis of T cell repertoire after allo-HCT. Functional assays of these superdominant TCRs are strongly warranted in the context of their reactivities against microbial or allogenic antigens. DisclosuresKitaura:Repertoire Genesis Incorporation: Employment. Shin-I:BITS Incorporation: Equity Ownership. Suzuki:Repertoire Genesis Incorporation: Equity Ownership.

186-OR: Risk Prediction of Early Decline in Renal Function in Diabetic Kidney Disease with Algorithm Including Fractional Excretion of Glycated Amino Acids

Diabetic kidney disease occurs in ca. 40% patients with diabetes. Approximately 1 in 5 patients with type 1 diabetes mellitus (T1DM) develop early decline in renal function (EDRF), requiring renal dialysis after 5 - 20 years. Currently, it is uncertain which patients are at high risk of EDRF. With Joslin Kidney Study investigators, we found patients with T1DM with and without microalbuminuria who later develop EDRF (Decliners) have higher fractional excretion (FE) of 6 glycated amino acids - fructosyl-lysine and 5 advanced glycation endproducts (AGEs), compared to patients with stable renal function (Non-decliners) (Perkins et al., Sci Rep 10, 12709, 2020). No individual FE could classify patients. In this study, we applied artificial intelligence machine learning to develop diagnostic algorithms to classify Decliners and Non-decliners, exploring A1C, log(ACR), 14 glycated and oxidized amino acids in plasma, urine, related FEs and other features. Data from patients with T1DM with stable renal function (n = 52) and EDRF (n = 33) were available. Algorithms were trained and tested on 90%/10% data split, repeated 1000 times, using Extreme Gradient Boosting. Optimum classification required measurement of 3 AGEs: Nꞷ-carboxymethylarginine (CMA) and glyoxal-derived hydroimidazolone (G-H1) in plasma and urine, and N-carboxymethyl-lysine (CML) in plasma; measured in 15 min by liquid chromatography-tandem mass spectrometry. Optimum algorithm features were: A1C, log[ACR], FECMA, FEG-H1 and plasma CML free adduct. The diagnostic performance for risk prediction of future EDRF was (mean ± SD): sensitivity 0.74 ± 0.09, specificity 0.91 ± 0.04 and accuracy 87 ± 4%. The positive likelihood ratio LR+ of 11.0 indicates that this method gives strong, often conclusive evidence of future EDRF in patients with T1DM. With further validation, including in patients with type 2 diabetes mellitus, this method may markedly improve risk prediction of EDRF. Disclosure P. Thornalley: None. A. De la fuente: None. N. Rabbani: None. Funding Qatar Foundation; Qatar University

MSphere of Influence: the Key Role of Neutrophils in Tuberculosis and Type 2 Diabetes Comorbidity.

ABSTRACTAnnie Mayer Bridwell works in the field of tuberculosis pathogenesis from the host perspective. She is fascinated by comorbidities, and in this paper, she reflects on three publications that shaped her model of neutrophil-centric pathology in tuberculosis and type 2 diabetes comorbidity. She explains that “Systems immunology of diabetes-tuberculosis comorbidity reveals signatures of disease complications” (C. A. Prada-Medina, K. F. Fukutani, N. Pavan Kumar, L. Gil-Santana, et al., Sci Rep 7:1999, 2017, https://doi.org/10.1038/s41598-017-01767-4) led her to consider neutrophils as a central immunological player in comorbid patients. “Type I IFN exacerbates disease in tuberculosis-susceptible mice by inducing neutrophil-mediated lung inflammation and NETosis” (L. Moreira-Teixeira, P. J. Stimpson, E. Stavropoulos, S. Hadebe, et al., Nat Commun 11:5566, 2020, https://doi.org/10.1038/s41467-020-19412-6) and “Diabetes primes neutrophils to undergo NETosis, which impairs wound healing” (S. L. Wong, M. Demers, K. Martinod, M. Gallant, et al., Nat Med 21:815–819, 2015, https://doi.org/10.1038/nm.3887) then shed light on neutrophil extracellular trap (NET) formation as a common pathological feature of dysregulated neutrophils in tuberculosis and diabetes, respectively. Together, these works laid the foundation for Dr. Mayer Bridwell's interest in metabolic regulation of NETosis during TB infection and diabetes comorbidity.

Direct Numerical Simulation of Pore-Scale Trapping Events During Capillary-Dominated Two-Phase Flow in Porous Media

This study focuses on direct numerical simulation of imbibition, displacement of the non-wetting phase by the wetting phase, through water-wet carbonate rocks. We simulate multiphase flow in a limestone and compare our results with high-resolution synchrotron X-ray images of displacement previously published in the literature by Singh et al. (Sci Rep 7:5192, 2017). We use the results to interpret the observed displacement events that cannot be described using conventional metrics such as pore-to-throat aspect ratio. We show that the complex geometry of porous media can dictate a curvature balance that prevents snap-off from happening in spite of favourable large aspect ratios. We also show that pinned fluid-fluid-solid contact lines can lead to snap-off of small ganglia on pore walls; we propose that this pinning is caused by sub-resolution roughness on scales of less than a micron. Our numerical results show that even in water-wet porous media, we need to allow pinned contacts in place to reproduce experimental results.

Surgical skill assessment using machine learning algorithms

Abstract Objective Surgical skill is correlated with clinical outcomes. Therefore, the assessment of surgical skill is of major importance to improve clinical outcomes and increase patient safety. However, surgical skill assessment often lacks objectivity and reproducibility. Furthermore, it is time-consuming and expensive. Therefore, we developed an automated surgical skill assessment using machine learning algorithms. Methods Surgical skills were assessed in videos of laparoscopic cholecystectomy using a three-step machine learning algorithm. First, a three-dimensional convolutional neural network was trained to localize and classify the instruments within the videos. Second, movement patterns of the instruments were recorded over time and extracted. Third, the movement patterns were correlated with human surgical skill ratings using a linear regression model to predict surgical skill ratings automatically. Machine ratings were compared against human ratings of four board certified surgeons using a score ranging from 1 (poor skills) to 5 (excellent skills). Results Human raters and machine learning algorithms assessed surgical skills in 242 videos. Inter-rater reliability for human raters was excellent (79%, 95%CI 72-85%). Instrument detection showed an average precision of 78% and average recall of 82%. Machine learning algorithms showed an 87% accuracy in predicting good or poor surgical skills, when compared to human raters. Conclusion Machine learning algorithms can be trained to distinguish good and poor surgical skills with high accuracy. This work was published in Sci Rep 11, 5197 (2021). https://doi.org/10.1038/s41598-021-84295-6

Pathological Downstaging and Survival Outcomes Associated with Neoadjuvant Chemotherapy for Variant Histology Muscle Invasive Bladder Cancer.

Patients with muscle invasive bladder cancer (MIBC) of variant histology have a poor prognosis. It is unclear if neoadjuvant chemotherapy prior to radical cystectomy is associated with pathological downstaging or improved overall survival (OS) for patients with variant histology. Our objective was to assess for associations between receipt of neoadjuvant chemotherapy, pathological downstaging and OS for patients with variant histology MIBC. Patients were identified in the National Cancer Database from 2004 to 2017 with MIBC, without metastases, who underwent radical cystectomy. Patients were stratified by histological subgroup, and receipt or nonreceipt of neoadjuvant chemotherapy. Pathological downstaging was defined as pT0N0 or pT ≤1N0, and OS from the time of diagnosis to date of death or censoring at last followup. Multivariable logistic regression analysis determined associations between neoadjuvant chemotherapy and pathological downstaging. Multivariable Cox regression analysis determined associations between neoadjuvant chemotherapy and OS. A total of 31,218 patients were included in the final study population (urothelial carcinoma [UC]: 27,779; sarcomatoid UC: 501; micropapillary UC: 418; squamous cell carcinoma: 1,141; neuroendocrine carcinoma: 629; adenocarcinoma: 750). Neoadjuvant chemotherapy was associated with pathological downstaging to pT0N0 in all histological subgroups (UC: OR 5.1 [4.6-5.6]; sarcomatoid UC: OR 13.8 [5.5-39.0]; micropapillary UC: OR 9.7 [2.8-46.8]; squamous cell carcinoma: OR 7.4 [2.1-24.5]; neuroendocrine: OR 4.7 [2.6-9.2]; adenocarcinoma: OR 23.3 [8.0-74.2]). Neoadjuvant chemotherapy was associated with improved OS for UC (HR 0.8 [0.77-0.84]), sarcomatoid UC (HR 0.64 [0.44-0.91]) and neuroendocrine carcinoma (HR 0.55 [0.43-0.70]). Neoadjuvant chemotherapy was associated with pathological downstaging for all MIBC histological variants, with improved OS for patients with UC, sarcomatoid variant UC and neuroendocrine carcinoma.

Fentanyl and Morphine Cause Dose‐Dependent Sleep Disruption in C57BL/6J (B6) Mice

Opioids are important for pain management but have the unwanted side effect of sleep disruption in humans (Sleep Med Clin 13:271, 2018), rats (Sci Rep 9:14122, 2019), and mice (Neurosci Abstr Online 502.02, 2019). Dose-response data are needed for efforts to develop countermeasures to the undesired effects of opioids. Therefore, this study is testing the hypothesis that the sleep-disrupting effects of opioids are dose dependent. Experiments were conducted in accordance with the FASEB Statement of Principles for the use of Animals in Research and Education. Adult male B6 mice (n=8) were housed in a temperature- and humidity-controlled environment and 12:12h light:dark cycle. Mice were implanted with telemeters (DSI HD-X02) to record cortical electroencephalogram (EEG) and neck muscle electromyogram. These measures made it possible to objectively quantify the effects of opioids on states of wakefulness, rapid eye movement (REM) sleep, and non-REM (NREM) sleep. One week after recovery from surgery, each mouse began receiving subcutaneous injections (0.3 mL) within 60 min of light onset. States of sleep and wakefulness were recorded for 4 h after each injection. Repeated injections into the same mouse were separated by 3 or 4 days. Saline (vehicle control) was followed by increasing, half log doses (mg/kg) of morphine (0.01 to 30) or fentanyl (0.001 to 3). Every 10 s of each 4-h recording was scored by two individuals, one of whom was blinded to the treatment condition. Sleep scoring concordance between the scorers was >90%. Data were analyzed using repeated measures ANOVA and Tukey's multiple comparisons test (GraphPad Prism V9). Morphine (F(8, 56) = 104.4, p <0.0001) and fentanyl (F(8, 56) = 94.65, p < 0.0001) dose-dependently increased wakefulness, decreased NREM sleep (F(8, 56) = 110.2, p <0.0001; F(8, 56) = 96.30, p < 0.0001) and decreased REM sleep (F(8, 56) = 34.00, p <0.0001; F(8, 56)= 51.80, p <0.0001). The lowest dose of morphine that significantly increased wakefulness and decreased NREM sleep was 1 mg/kg (p = 0.04, p = 0.03); and 3 mg/kg for decreasing REM sleep (p = 0.0004). For fentanyl, the lowest dose that significantly increased wakefulness and decreased NREM and REM sleep was 0.3 mg/kg (p = 0.001; p = 0.0016; p = 0.0007). Higher doses of morphine and fentanyl eliminated NREM and REM sleep. Both opioids also significantly and differentially altered EEG power during wakefulness (see companion abstract by O’Brien et al.). Nonlinear regression was used to calculate the half-maximal dose (mg/kg) for increasing wakefulness (ED50) and inhibiting sleep (ID50). ED50 and ID50 data were used to compare drug potency. Morphine ED50 was 2.2 to increase wakefulness, ID50 2.3 to decrease NREM sleep, and 1.3 to decrease REM sleep. Fentanyl ED50 was 0.2 to increase wakefulness, ID50 0.2 to decrease NREM sleep, and 0.1 to decrease REM sleep. These data demonstrate that opioid-induced sleep disruption is dose-dependent, and that fentanyl is more potent than morphine to disrupt sleep.

Fentanyl, but not Morphine, Increases Cortical Electroencephalographic (EEG) Delta Power during Wakefulness in C57BL/6J (B6) Mice

Cortical EEG signals comprise differing oscillations that represent lower-level traits characteristic of higher-level behavioral phenotypes such as sleep and wakefulness (Physiology 32:60, 2017). Opioid-induced changes in neurobehavioral function also are reflected in altered EEG frequency and amplitude. These EEG features have the potential to provide insights regarding the desired actions and undesired side effects of opioids. In rat, morphine (Anesthesiology 103:779, 2005) and fentanyl (Sci Rep 9:14122, 2019) increase EEG slow waves. Studies of mice provide the additional opportunity to use mutated or transgenic animals with features potentially relevant to respiratory depression or addiction. This study is testing the hypothesis that subcutaneous injection of fentanyl and morphine causes dose-dependent EEG changes in wild-type mice. Experiments were conducted in accordance with the FASEB Statement of Principles for the use of Animals in Research and Education. Adult male B6 mice (n = 4) were implanted with telemeters (DSI HD-X02) for wireless recording of cortical EEG and neck muscle electromyogram. The recordings were used to quantify the effects of opioids on sleep and wakefulness. These studies demonstrated dose-dependent sleep disruption by both opioids (see companion abstract by Zebadua Unzaga et al.). EEG data were digitized by DSI Ponemah software and analyzed using public domain software for creating multi-tapered EEG spectrograms (J Vis Exp 153:e60333, 2019). Analyses were based on EEG data from the first 30 min after opioid injection, when all mice were awake. EEG power was expressed as percent change relative to saline (vehicle control) for six frequency bands: delta (0.5 to 4 Hz), theta (4 to 8 Hz), alpha (8 to 13 Hz), sigma (12 to 15 Hz), beta (13 to 30 Hz), and gamma (30 to 60 Hz). Increasing half log doses (mg/kg) ranged from 0.001 to 3 for fentanyl and 0.01 to 30 for morphine. Repeated measures ANOVA with the Geisser-Greenhouse correction showed a significant dose main-effect of fentanyl on EEG delta power (F(1.857, 5.571) = 7.454, p = 0.0276) and alpha power (F(1.705, 5.116) = 6.316, p = 0.0439). For fentanyl (3 mg/kg) compared to saline, Cohen's d = 2.9 for delta power. By contrast, for morphine there was a significant main effect of dose only for EEG sigma power (F(2.162, 6.486) = 6.557, p = 0.0265). Comparing morphine (30 mg/kg) to saline, the Cohen's d = 1.1 for sigma power. Although both fentanyl and morphine are full mu-opioid receptor agonists, they significantly altered EEG power at different frequency bands. Fentanyl, but not morphine, blocks alpha-1 adrenergic receptor subtypes and vesicular reuptake of several monoamine transmitters (J Pharmacol Exp Ther 343:376, 2020). These results support the interpretation that the differential effects of fentanyl and morphine on EEG power may be due, in part, to non-mu-opioid receptor of fentanyl.

The Impact of Hypercholesterolemia on Tendon Injury Repair

Background Hypercholesterolemia is a condition characterized by high blood cholesterol levels and is associated with tendon xanthomas (cholesterol deposits found on superficial tendons). Lipid accumulation in the tendon's extracellular spaces1 may disrupt the parallel organization of collagen substructures within the tendon, and negatively affect its mechano-sensing and mechanical properties, which may lead to injury. We hypothesized that tendon strength and metabolism would be inferior in a high cholesterol environment and attenuate the tendon's ability to repair after injury. Methods 50 Sprague-Dawley (SD, control) and 50 apolipoprotein E knock-out rats (ApoE, Envigo, IL, USA) rats bred in-house were subject to a unilateral patellar tendon (PT) injury at 12 weeks of age; the uninjured limb served as a control. Animals were euthanased at 3, 14 or 42 days post-injury, time-points chosen to represent tendon inflammatory, reparative and remodelling repair phases, respectively. Animals were randomly assigned for investigating gene expression (SD/ApoE per timepoint n=4/4), tissue histology (n=6/6), biomechanical testing (n=10/10; 14 and 42 days only). Equal male/female ratios were used where possible (total SD M23/F27, ApoE M23/F26). Results ApoE total cholesterol was over double that of SD rats (mean 2.12 vs 0.99 mg/ml, p<0.001). Despite this, there was little histological evidence of lipid content (Oil Red-O staining) in tendons of either group, and no apparent differences in tendon mechanical or material properties (Fig. 1). However, there were differences in gene expression (Fig. 2). ApoE rats demonstrated significantly greater COX2 expression than SD; other inflammatory markers (TGFβ, IL-1, IL-6) did not differ between groups. COL1A1, SCX, LOXα were significantly upregulated 14 days post injury in SD compared to ApoE. A greater COL3A1 response was also observed in ApoE compared to SD rats. Discussion We found differences in injury repair at the cellular level, using an ApoE high cholesterol model. There was little evidence of lipid accumulation at the tissue level, therefore the PT may have been a poor choice for an injury study in this instance and it is not surprising that biomechanical differences were not found. Moreover, ApoE rats were recently found to demonstrate only modest early atherosclerotic characteristics2, thus the young age of our cohort may be a primary factor in the lack of physical evidence of lipid accumulation. Nonetheless, our results indicate that even with a mild phenotype, high cholesterol significantly modulates tendon inflammatory and healing cellular activity, as indicated by altered mRNA levels, which may contribute to the known consequences of tendon cholesterol on tendons in humans. References 1Grewal et al. 2014. Accumulation of oxidized LDL in the tendon tissues of C57BL/6 or apolipoprotein E knock-out mice that consume a high fat diet: potential impact on tendon health. PLoS One 9(12): e114214. 2Rune et al., 2018. Long-term Western diet fed apolipoprotein E-deficient rats exhibit only modest early atherosclerotic characteristics. Sci Rep 8(1): 5416.

Development of a Novel Sparse Labeling Method by Machine Learning‐Guided Engineering of Cre‐ lox Recombination

Background Neural networks generate complex behaviors. The relationships between neural networks and behaviors aren't understood well. Optogenetics is a powerful technique that enables on-demand photo-regulation of neural activity and exploration of the mechanism of neural computation. Conventional optogenetics presents three difficult challenges: 1. a hypothesis is necessary to conduct experiments; 2. optogenetics is low-throughput because different transformants are required to test each hypothesis: and 3. single-cell analysis is difficult due to the lack of single-cell-specific promoters. To overcome these difficulties, we have developed a new methodology named “functional cellomics,” which can annotate the function of neural networks in a hypothesis-free, high-throughput, and single-cell resolution manner. The key factor is the stochastic labeling of opsin by applying the Cre-lox system. We designed a genetic circuit in which two sets of lox variants, lox2272 and loxP sequences, are inserted alternately. A transcription factor, QF2w, is interposed between loxP sequences. Induced Cre excises DNA exclusively either between loxP sequences or between lox2272 sequences in every single cell. When Cre is allowed to act on lox2272 sequences, QF2w is expressed. Then, QF2w induces expression of opsin. We succeeded in the stochastic labeling of opsin dependent on the induction of Cre. However, the labeling rate of opsin was about 30% [1]. If opsin is labeled in many neurons, it becomes difficult to distinguish the function of individual neurons. In this study, we developed a new methodology to enable precise control of the labeling rate of opsin to conduct an in-depth analysis of the function of the nervous system. Methods In our genetic circuit, the opsin is expressed in neurons in which the Cre-lox recombination event occurs between the lox2272 sequences. Thus, we hypothesized that the labeling rate of opsin reduces by using lox2272 variants which is difficult to be excised by Cre. We generated a library of randomized lox sequences by PCR and introduced them in S. cerevisiae. Then, we induced Cre production in the S. cerevisiae library and evaluated the excision rates of lox2272 variants by using NGS. To develop a machine learning model to predict the cleavage rate of lox2272 variants, we trained a Gaussian process (GP) model using data containing lox2272 variant sequences and their cleavage rates by Cre. We randomly chose 1000 lox2272 variants as training data and 100 variants to test prediction accuracy. Results and Discussion We quantified the excision rates of more than 2000 lox2272 variants by NGS and successfully found variants that have excision efficiencies ranging from 0.05% to 100%. We confirmed the results of NGS analysis by an in vivo assay using S. cerevisiae and qPCR. Then, we build a GP model and observed a high correlation between the actual cleavage rates and predicted cleavage rates of the test data (R = 0.93). We used the GP model to predict the efficiency of unevaluated lox2272 sequences and succeeded in the identification of lox2272 variants that had various cleavage rates. These results indicate the feasibility of precise control of the labeling rate of opsin in functional cellomics [2]. 1. Aoki et al, Sci Rep, 8, 10380, 2018. 2. Yamauchi et al, submitted.