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Renal ischemia-reperfusion (IR) causes acute kidney injury (AKI) with high mortality and morbidity. The objective of this investigation was to ameliorate kidney IR injury and identify novel biomarkers for kidney injury and repair. Under general anesthesia, left renal ischemia was induced in Wister rats by occluding renal artery for 45 minutes, followed by reperfusion and right nephrectomy. Thirty minutes prior to ischemia, rats (n = 8/group) received Valproic Acid (150 mg/kg; VPA), Dexamethasone (3 mg/kg; Dex) or Vehicle (saline) intraperitoneally. Animals were sacrificed at 3, 24 or 120 h post-IR. Plasma creatinine (mg/dL) at 24 h was reduced (P<0.05) in VPA (2.7±1.8) and Dex (2.3±1.2) compared to Vehicle (3.8±0.5) group. At 3 h, urine albumin (mg/mL) was higher in Vehicle (1.47±0.10), VPA (0.84±0.62) and Dex (1.04±0.73) compared to naïve (uninjured/untreated control) (0.14±0.26) group. At 24 h post-IR urine lipocalin-2 (μg/mL) was higher (P<0.05) in VPA, Dex and Vehicle groups (9.61-11.36) compared to naïve group (0.67±0.29); also, kidney injury molecule-1 (KIM-1; ng/mL) was higher (P<0.05) in VPA, Dex and Vehicle groups (13.7-18.7) compared to naïve group (1.7±1.9). Histopathology demonstrated reduced (P<0.05) ischemic injury in the renal cortex in VPA (Grade 1.6±1.5) compared to Vehicle (Grade 2.9±1.1). Inflammatory cytokines IL1β and IL6 were downregulated and anti-apoptotic molecule BCL2 was upregulated in VPA group. Furthermore, kidney DNA microarray demonstrated reduced injury, stress, and apoptosis related gene expression in the VPA administered rats. VPA appears to ameliorate kidney IR injury via reduced inflammatory cytokine, apoptosis/stress related gene expression, and improved regeneration. KIM-1, lipocalin-2 and albumin appear to be promising early urine biomarkers for the diagnosis of AKI.

Recurrent, clonal somatic mutations in histone H3 are molecular hallmarks that distinguish the genetic mechanisms underlying pediatric and adult high-grade glioma (HGG), define biological subgroups of diffuse glioma, and highlight connections between cancer, development, and epigenetics. These oncogenic mutations in histones, now termed “oncohistones”, were discovered through genome-wide sequencing of pediatric diffuse high-grade glioma. Up to 80% of diffuse midline glioma (DMG), including diffuse intrinsic pontine glioma (DIPG) and diffuse glioma arising in other midline structures including thalamus or spinal cord, contain histone H3 lysine 27 to methionine (K27M) mutations or, rarely, other alterations that result in a depletion of H3K27me3 similar to that induced by H3 K27M. This subgroup of glioma is now defined as diffuse midline glioma, H3K27-altered. In contrast, histone H3 Gly34Arg/Val (G34R/V) mutations are found in approximately 30% of diffuse glioma arising in the cerebral hemispheres of older adolescents and young adults, now classified as diffuse hemispheric glioma, H3G34-mutant. Here, we review how oncohistones modulate the epigenome and discuss the mutational landscape and invasive properties of histone mutant HGGs of childhood. The distinct mechanisms through which oncohistones and other mutations rewrite the epigenetic landscape provide novel insights into development and tumorigenesis and may present unique vulnerabilities for pHGGs. Lessons learned from these rare incurable brain tumors of childhood may have broader implications for cancer, as additional high- and low-frequency oncohistone mutations have been identified in other tumor types.

Macrophages produce genotoxic agents, such as reactive oxygen and nitrogen species, that kill invading pathogens. Here we show that these agents activate the DNA damage response (DDR) kinases ATM and DNA-PKcs through the generation of double stranded breaks (DSBs) in murine macrophage genomic DNA. In contrast to other cell types, initiation of this DDR depends on signaling from the type I interferon receptor. Once activated, ATM and DNA-PKcs regulate a genetic program with diverse immune functions and promote inflammasome activation and the production of IL-1β and IL-18. Indeed, following infection with Listeria monocytogenes, DNA-PKcs-deficient murine macrophages produce reduced levels of IL-18 and are unable to optimally stimulate IFN-γ production by NK cells. Thus, genomic DNA DSBs act as signaling intermediates in murine macrophages, regulating innate immune responses through the initiation of a type I IFN-dependent DDR.

Background Pediatric-type diffuse high-grade glioma (pHGG) is the most frequent malignant brain tumor in children and can be subclassified into multiple entities. Fusion genes activating the MET receptor tyrosine kinase often occur in infant-type hemispheric glioma (IHG) but also in other pHGG and are associated with devastating morbidity and mortality. Methods To identify new treatment options, we established and characterized two novel orthotopic mouse models harboring distinct MET fusions. These included an immunocompetent, murine allograft model and patient-derived orthotopic xenografts (PDOX) from a MET-fusion IHG patient who failed conventional therapy and targeted therapy with cabozantinib. With these models, we analyzed the efficacy and pharmacokinetic properties of three MET inhibitors, capmatinib, crizotinib and cabozantinib, alone or combined with radiotherapy. Results Capmatinib showed superior brain pharmacokinetic properties and greater in vitro and in vivo efficacy than cabozantinib or crizotinib in both models. The PDOX models recapitulated the poor efficacy of cabozantinib experienced by the patient. In contrast, capmatinib extended survival and induced long-term progression-free survival when combined with radiotherapy in two complementary mouse models. Capmatinib treatment increased radiation-induced DNA double-strand breaks and delayed their repair. Conclusions We comprehensively investigated the combination of MET inhibition and radiotherapy as a novel treatment option for MET-driven pHGG. Our seminal preclinical data package includes pharmacokinetic characterization, recapitulation of clinical outcomes, coinciding results from multiple complementing in vivo studies, and insights into molecular mechanism underlying increased efficacy. Taken together, we demonstrate the groundbreaking efficacy of capmatinib and radiation as a highly promising concept for future clinical trials.

Abstract Objectives Lymphocytosis may represent either a lymphoproliferative disorder (LPD) or a reactive process. The absolute lymphocyte count (ALC) threshold for further evaluation of lymphocytosis is not well established. Methods We prospectively performed flow cytometry on blood samples from patients 50 years or older with ALCs of 4.0 × 109 cells/L or greater without a history of an LPD. Results Monoclonal B-cell populations were found in 34 (19.1%) of 178 cases, with incidence increasing with age. In patients younger than 75 years, no monoclonal B-cell population was identified in patients with ALCs less than 4.4 × 109 cells/L, while such clones were found below and above this threshold in patients 75 years and older. Conclusions These findings support a threshold for smear review and flow cytometry no lower than 4.4 × 109 cells/L in patients younger than 75 years and a threshold as low as 4.0 × 109 cells/L in patients 75 years and older.

Background The genus Microbotryum includes plant pathogenic fungi afflicting a wide variety of hosts with anther smut disease. Microbotryum lychnidis-dioicae infects Silene latifolia and replaces host pollen with fungal spores, exhibiting biotrophy and necrosis associated with altering plant development. Results We determined the haploid genome sequence for M. lychnidis-dioicae and analyzed whole transcriptome data from plant infections and other stages of the fungal lifecycle, revealing the inventory and expression level of genes that facilitate pathogenic growth. Compared to related fungi, an expanded number of major facilitator superfamily transporters and secretory lipases were detected; lipase gene expression was found to be altered by exposure to lipid compounds, which signaled a switch to dikaryotic, pathogenic growth. In addition, while enzymes to digest cellulose, xylan, xyloglucan, and highly substituted forms of pectin were absent, along with depletion of peroxidases and superoxide dismutases that protect the fungus from oxidative stress, the repertoire of glycosyltransferases and of enzymes that could manipulate host development has expanded. A total of 14 % of the genome was categorized as repetitive sequences. Transposable elements have accumulated in mating-type chromosomal regions and were also associated across the genome with gene clusters of small secreted proteins, which may mediate host interactions. Conclusions The unique absence of enzyme classes for plant cell wall degradation and maintenance of enzymes that break down components of pollen tubes and flowers provides a striking example of biotrophic host adaptation.

Among combat injured, invasive fungal infections (IFIs) result in significant morbidity. Cultures and histopathology are the primary diagnostic methods for IFIs, but they have limitations. We previously evaluated a panfungal polymerase chain reaction assay, which was 83% sensitive and 99% specific for angioinvasive IFIs. Here, we evaluated 3 less resource-intensive seminested assays targeting clinically relevant fungi in the order Mucorales and genera and . Formalin-fixed paraffin-embedded tissue specimens from a multicenter trauma IFI cohort (2009-2014) were used. Cases were US military personnel injured in Afghanistan with histopathologic IFI evidence. Controls were patients with similar injury patterns and no laboratory IFI evidence (negative culture and histopathology). Seminested assays specific to Mucorales (V4/V5 regions of 18S rDNA), (mitochondrial tRNA), and (internal transcribed spacer [ITS]/28A regions of DNA) were compared with a panfungal assay amplifying the internal transcribed spacer 2 region of rDNA and to histopathology. Specimens from 92 injury sites (62 subjects) were compared with control specimens from 117 injuries (101 subjects). We observed substantial agreement between the seminested and panfungal assays overall, especially for the order Mucorales. Moderate agreement was observed at the genus level for and . When compared with histopathology, sensitivity and specificity of seminested assays were 67.4% and 96.6%, respectively (sensitivity increased to 91.7% when restricted to sites with angioinvasion). Prior studies of seminested molecular diagnostics have focused on culture-negative samples from immunocompromised patients. Our findings underscore the utility of the seminested approach in diagnosing soft-tissue IFIs using formalin-fixed paraffin-embedded tissue samples, especially with angioinvasion.

Objectives: We report a case of hemophagocytosis-related (Asian variant) intravascular large B-cell lymphoma (IVLBCL) in a patient of Western origin initially diagnosed by splenectomy with diffuse large B-cell lymphoma (DLBCL) with a micronodular pattern. The clonal relationship between these two DLBCL subtypes is also investigated. Methods: Hemophagocytosis-related (Asian variant) IVLBCL was identified at autopsy in a 62-year-old Hispanic woman, in North America, following an antemortem diagnosis of massive splenic involvement by DLBCL with a micronodular pattern, a feature not expected of IVLBCL. Results: These two apparently distinct lymphoma types demonstrated similar immunophenotypic profiles and IgH gene rearrangements of identical size suggesting a clonal relationship. The 2008 WHO classification system describes IVLBCL in the spleen as having a sinusoidal pattern. Conclusions: Our observations provide the first molecular genetic support for a seemingly underrecognized micronodular pattern of IVLBCL in the spleen and further support the proposal of a “mixed variant” of IVLBCL with concomitant “intravascular” and “solid” phases of disease.

Dynamic changes to the epigenome are essential regulators of cell differentiation and, when disrupted, can be oncogenic. Pervasive epigenetic alternations are a hallmark of many Non-Hodgkin Lymphomas (NHLs), a heterogeneous group of B and T cell cancers with an incidence of 80,000 new cases and 20,000 deaths in the U.S. per year. My thesis studies have defined the epigenetic landscapes of primary human B and T cell lymphomas and linked them to dynamic changes in the transcriptomes of these cancers.The first of these projects focused on mature B cell lymphoma/leukemia (BCL). To identify common and distinct epigenetic perturbations that promote oncogenesis, we compared three subtypes of BCL with normal B lymphocytes. Purified malignant B cells from 52 patients with follicular or diffuse large B cell lymphoma or chronic lymphocytic leukemia and normal B cell subsets from 28 donor tonsils were subjected to chromatin immunoprecipitation and sequencing (ChIP-seq) for H3K4me1, H3K9/14ac, and H3K27ac; FAIRE-seq for open chromatin; RNA-seq; and genome copy number arrays. We identified 113 novel B cell super enhancers (SE). Functional studies showed that one of these novel SEs is connected to the deregulated expression of two immunoglobulin receptors, which may promote uncontrolled B-cell receptor signaling. Our studies also revealed pervasive downregulation across BCL subtypes of crucial B cell identity transcription factors (TF) and tumor suppressors including PU.1 (SPI1), OCT2 (POU2F2), E2A (TCF3), and RUNX3 in concordance with loss of active chromatin marks in adjacent SEs. Motif analysis and TF ChIP-seq indicate that these TFs and SEs form self-regulatory transcriptional feedback loops whose loss inhibits B cell maturation and promotes proliferation. In sum, this study defined common regulome alterations in the 3 major types of mature BCL and implicates SEs as important hubs of tumor-suppressing transcriptional feedback loops whose disruption is a common mechanism driving mature B cell cancers.T cell lymphomas make up less than 8% of NHL cases, Cutaneous T Cell Lymphoma (CTCL) accounting for nearly half of these. CTCL are often treated with histone deacetylase inhibitors (HDACi), which broadly affect the chromatin landscape and protein acetylation, but only 30% of patients respond to drug treatment. We defined differences in histone acetylation of regulatory elements and target gene expression in HDACi-sensitive versus -resistant CTCL. These include genes in apoptotic (BCL2, BIRC5), cell cycle (CDK1, RRM2), and cell adhesion/migration (CCR6, CXCR4, LAIR2) pathways, many of which were not previously associated with CTCL or HDACi- resistance. Expression of some genes, including LAIR2, was elevated in HDACi-resistant samples before and after therapy, suggesting it may be predictive of resistance. Our findings identified new mechanisms of HDACi-resistance in CTCL and define novel predictive markers as potential targets for therapeutic development.Disease progression of CTCL has not been mapped at the genomic or transcriptomic level and thus factors that contribute to progression or therapy resistance are unknown. To address this, we subjected longitudinal samples from 10 CTCL patients to single-cell RNA-seq (scRNA-seq) and exome + low coverage whole genome sequencing (eWGS). We selected 2-4 specimens per patient, consisting of peripheral blood (PB) samples, skin and lymph node biopsies from distinct clinical timepoints separated by 3 months. scRNA- and T-cell receptor (TCR)-seq were performed on 16 PBMC or CD4+ cells from PB samples from six MF/SS patients, sequencing an average of 7413 cells per sample. For eWGS, DNA was isolated from CD4+ cells (PB), unsorted PBMCs, frozen or FFPE biopsies (skin or lymph node) from 25 samples from the same six patients plus an additional four patients, along with patient-matched benign cells as controls. These data will define clonal evolution of the transcriptome and genome during progression of CTCL. The two complementary T cell lymphoma studies defined mechanisms of HDACi resistance, map the clonal evolution of CTCL, and reveal high-resolution intra and inter-sample differences in malignant cell populations that contribute to the pathogenesis and progression of this challenging disease.Taken together, my dissertation work is a focused ‘omics evaluation of the dynamic changes in gene regulation and epigenome landscape in B and T cell cancers in which I collected, processed, and analyzed hundreds of primary cancer samples. Beyond informatics, I performed molecular and cellular experiments to elucidate the functional impact of a prioritized set of epigenetic perturbations that I identified. In sum, my studies defined novel epigenetic alterations that comprise the malignant underpinnings of these diseases.

We report a case of hemophagocytosis-related (Asian variant) intravascular large B-cell lymphoma (IVLBCL) in a patient of Western origin initially diagnosed by splenectomy with diffuse large B-cell lymphoma (DLBCL) with a micronodular pattern. The clonal relationship between these two DLBCL subtypes is also investigated. Hemophagocytosis-related (Asian variant) IVLBCL was identified at autopsy in a 62-year-old Hispanic woman, in North America, following an antemortem diagnosis of massive splenic involvement by DLBCL with a micronodular pattern, a feature not expected of IVLBCL. These two apparently distinct lymphoma types demonstrated similar immunophenotypic profiles and IgH gene rearrangements of identical size suggesting a clonal relationship. The 2008 WHO classification system describes IVLBCL in the spleen as having a sinusoidal pattern. Our observations provide the first molecular genetic support for a seemingly underrecognized micronodular pattern of IVLBCL in the spleen and further support the proposal of a \"mixed variant\" of IVLBCL with concomitant \"intravascular\" and \"solid\" phases of disease.