Highlights d High rate of NF1 loss in the R0 compared to neoadjuvant chemotherapy (NACT) group d Lower chromothripsis-like pattern and higher neoantigens in the R0 versus NACT group d Increased number of infiltrated T cells and decreased macrophages in the R0 group d Significant transcriptomic and proteomic variations between HGSC subgroups

Ifosfamide and other oxazaphosphorines can result in hemorrhagic cystitis, a constellation of complications caused by acrolein metabolites. We previously showed that a single dose of IPSE (Interleukin-4-inducing principle from Schistosoma eggs), a schistosome-derived host modulatory protein, can ameliorate ifosfamide-related cystitis; however, the mechanisms underlying this urotoxicity and its prevention are not fully understood. To provide insights into IPSE’s protective mechanism, we undertook transcriptional profiling of bladders from ifosfamide-treated mice, with or without pretreatment with IPSE or IPSE-NLS (a mutant of IPSE lacking nuclear localization sequence). Ifosfamide treatment upregulated a range of proinflammatory genes. The IL-1β-TNFα-IL-6 proinflammatory cascade via NFκB and STAT3 pathways was identified as the key driver of inflammation. The NRF2-mediated oxidative stress response pathway, which regulates heme homoeostasis and expression of antioxidant enzymes, was highly activated. Anti-inflammatory cascades, namely Wnt, Hedgehog and PPAR pathways, were downregulated. IPSE drove significant downregulation of major proinflammatory pathways including the IL-1β-TNFα-IL-6 pathways, interferon signaling, and reduction in oxidative stress. IPSE-NLS reduced inflammation but not oxidative stress. Taken together, we have identified signatures of acute-phase inflammation and oxidative stress in ifosfamide-injured bladder, which are reversed by pretreatment with IPSE. This work revealed several pathways that could be therapeutically targeted to prevent ifosfamide-induced hemorrhagic cystitis.

34Ifosfamide and other oxazaphosphorines can result in hemorrhagic cystitis, a constellation of complications 35 caused by acrolein metabolites. We previously showed that a single dose of IPSE, a schistosome-derived host 36 modulatory protein, can ameliorate ifosfamide-related cystitis; however, the exact mechanisms underlying this 37 urotoxic effect and its prevention are not fully understood. To provide insights into IPSE's protective 38 mechanism, we undertook transcriptional profiling of bladders from ifosfamide-treated mice, with or without 39 IPSE pretreatment. Following ifosfamide challenge, there was upregulation of a range of pro-inflammatory 40 genes. The pro-inflammatory pathway involving the IL-1ߚ, TNFߙ and IL-6 triad via NFߢB and STAT3 41 signaling pathways was identified as the key driver of inflammation. The NRF2-mediated oxidative stress 42 response pathway, which regulates both Hmox1-mediated heme homoeostasis and expression of antioxidant 43 enzymes, was highly activated. Anti-inflammatory and cellular proliferation cascades implicated in tissue 44 repair, namely Wnt, Hedgehog and PPAR pathways, were downregulated. IPSE administration before 45 ifosfamide injection resulted in significant downregulation of major proinflammatory pathways including the 46 triad of IL-1ߚ, TNFߙ and IL-6 pathways, the interferon signaling pathway, and less apparent reduction in 47 oxidative stress responses. Taken together, we have identified signatures of acute phase inflammation and 48 oxidative stress responses in the ifosfamide-injured bladder, which are reversed by pretreatment with IPSE, a 49 parasite derived anti-inflammatory molecule. In addition to providing new insights into the underlying 50 mechanism of IPSE's therapeutic effects, this work has revealed several pathways that could be therapeutically 51 targeted to prevent and treat ifosfamide-induced hemorrhagic cystitis. 52 53 Introduction 54

Introduction: Recurrent urinary tract infections have been linked to increased risk of bladder cancer, suggesting a potential role of the urinary microbiome in bladder cancer pathogenesis.Objective: Compare the urinary microbiomes in mice with and without bladder. Methods:Longitudinal study of mice exposed to a dilute bladder-specific carcinogen (0.05% nbutyl-n-(4-hydroxybutyl) nitrosamine, BBN mice, n=10), and control mice (n=10). Urine was sampled monthly from individual mice for 4 months. Microbial DNA was extracted from the urine, and the V4 region of the 16S rRNA gene sequenced. Animals were sacrificed and their bladders harvested for histopathology. Bladder sections were graded by a blinded pathologist. The composition and diversity of the urinary microbiome were compared between the BBN and control mice. Metabolic pathway analysis was completed using PICRUST.Results: Bladder histology in the BBN group showed normal tissue with inflammation (BBNnormal, n=5), precancerous pathologies, (BBN-precancerous, n=3), and invasive cancer (BBNcancer, n=2). Alpha diversity did not differ between the mice exposed to BBN and the control mice at any timepoint. There were no differences in the urinary microbiomes between the BBN and control mice at baseline. At month 4, mice exposed to BBN had higher proportion of both Gardnerella and Bifidobacterium compared to control mice. There were no differences in proportions of specific bacteria between either the BBN-precancer or BBN-cancer and controls at month 4. However, the BBN-normal mice had higher proportions of Gardnerella, Haemophilus, Bifidobacterium, and Ureaplasma Actinobaculum, and lower proportions of Actinomyces, compared to control mice at month 4. Functional pathway analysis demonstrated increases in genes related to purine metabolism, phosphotransferase systems, peptidases, protein folding, and bacterial toxins in the BBN-mice compared to control mice at month 4.Conclusion: Mice exposed to 4 months of BBN, a bladder-specific carcinogen, have distinct urine microbial profiles compared to control mice.

Summary Aims Mouse bladder wall injection with Schistosoma haematobium eggs has been used to overcome limitations in animal models of urogenital schistosomiasis. However, the effect of the absence of cercarial infection on immune responses to eggs in this model is unknown. We hypothesized that cercarial infection would alter local bladder and systemic immune responses to eggs in this model. Methods and results Mice were infected or not infected with S haematobium cercariae, and then, their bladder walls injected with S haematobium eggs or vehicle 5 weeks following cercarial infection. Three weeks later, mice were bled, sacrificed, perfused and their bladders harvested. Parasitological parameters and gross bladder pathology were not changed in egg‐injected bladders by cercarial exposure. Figure shows no changes in either granulomas or fibrosis. The only bladder cytokine upregulated in egg‐injected bladders by cercarial exposure (vs no exposure) was leptin. Cercarial exposure, compared to no exposure, resulted in increased serum, IL‐1α, IL‐13 and TGF‐β in bladder egg‐injected mice. Conclusion Cercarial exposure altered systemic responses of several cytokines in bladder egg‐injected mice, but surprisingly, only modified leptin expression in bladder tissue. This suggests that depending on the specific application, cercarial exposure may not be strictly necessary to model local immune responses in the bladder wall egg injection mouse model of urogenital schistosomiasis.

Analyses of whole organs from parasite-infected animals can reveal the entirety of the host tissue transcriptome, but conventional approaches make it difficult to dissect out the contributions of individual cellular subsets to observed gene expression. Computational deconvolution of gene expression data may be one solution to this problem. We tested this potential solution by deconvoluting whole bladder gene expression microarray data derived from a model of experimental urogenital schistosomiasis. A supervised technique was used to group B-cell and T-cell related genes based on their cell types, with a semi-supervised technique to calculate the proportions of urothelial cells. We demonstrate that the deconvolution technique was able to group genes into their correct cell types with good accuracy. A clustering-based methodology was also used to improve prediction. However, incorrectly predicted genes could not be discriminated using this methodology. The incorrect predictions were primarily IgH- and IgK-related genes. To our knowledge, this is the first application of computational deconvolution to complex, parasite-infected whole tissues. Other computational techniques such as neural networks may need to be used to improve prediction.

The mammalian transcriptome comprises a vast family of long noncoding (lnc)RNAs implicated in physiologic processes such as myogenesis, through which muscle forms during embryonic development and regenerates in the adult. However, the specific molecular mechanisms by which lncRNAs regulate human myogenesis are poorly understood. Here, we identified a novel muscle-specific lncRNA, lncFAM71E1-2:2 (lncFAM), which increased robustly during early human myogenesis. Overexpression of lncFAM promoted differentiation of human myoblasts into myotubes, while silencing lncFAM suppressed this process. As lncFAM resides in the nucleus, chromatin isolation by RNA purification followed by mass spectrometry (ChIRP-MS) analysis was employed to identify the molecular mechanisms whereby it might promote myogenesis. Analysis of lncFAM-interacting proteins revealed that lncFAM recruited the RNA-binding protein HNRNPL to the promoter of MYBPC2, in turn increasing MYBPC2 mRNA transcription and enhancing production of the myogenic protein MYBPC2. These results highlight a mechanism whereby a novel ribonucleoprotein complex, lncFAM-HNRNPL, elevates MYBPC2 expression transcriptionally to promote myogenesis.

Changes in the transcriptomes of human tissues with advancing age are poorly cataloged. Here, we sought to identify the coding and long noncoding RNAs present in cultured primary skin fibroblasts collected from 82 healthy individuals across a wide age spectrum (22–89 years old) who participated in the GESTALT (Genetic and Epigenetic Signatures of Translational Aging Laboratory Testing) study of the National Institute on Aging, NIH. Using high‐throughput RNA sequencing and a linear regression model, we identified 1437 coding RNAs (mRNAs) and 1177 linear and circular long noncoding (lncRNAs) that were differentially abundant as a function of age. Gene set enrichment analysis (GSEA) revealed select transcription factors implicated in coordinating the transcription of subsets of differentially abundant mRNAs, while long noncoding RNA enrichment analysis (LncSEA) identified RNA‐binding proteins predicted to participate in the age‐associated lncRNA profiles. In summary, we report age‐associated changes in the global transcriptome, coding and noncoding, from healthy human skin fibroblasts and propose that these transcripts may serve as biomarkers and therapeutic targets in aging skin.