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Molecular studies of resected intestinal tissue of control and Crohn disease (CD) patients reveal different host immune responses that confirm the existence of two different forms of CD namely perforating and nonperforating. This result confirms the previous epidemiological evidences on the existence of two CD forms. Increased interleukin (IL)-1p, IL-1ra mRNA expressions lead to the nonperforating CD which is the more benign form. Manifestation of a specific form of CD in an individual depends on host immune response.

Background: Some studies suggest that mesalazine (5-ASA) can attenuate the rate of progression of ulcerative colitis (UC) to colorectal cancer (CRC). Little is known about where in the colitis-dysplasiacarcinoma sequence [...]

The objective of this study was to determine sTNF-R, type I (p55) and type II (p75) in sera of HIV-infected male homosexuals and correlate them to T lymphocyte subpopulations and course of HIV infection. Serum samples were obtained from 39 HIV-1+ asymptomatic male homosexuals, 10 symptomatic (ARC and AIDS) male homosexuals and 44 HIV- non-homosexual healthy controls. sTNF-R levels were determined by ELISA with specific MoAbs and polyclonal antibodies to the sTNF-R proteins. sTNF-RI and II levels were significantly elevated in 72% and 74% respectively of HIV+ asymptomatic male homosexuals and in all of the symptomatic male homosexuals. In sequential studies a highly significant positive correlation was found between sTNF-RI and sTNF-RII (r = 0.8, P < 0.001) and between both sTNF-R and CD8+ lymphocyte counts (r= 0.6 and 0.92, respectively, P < 0.001-0.001) during the asymptomatic stage of the infection. All these correlations were lost, however, during the symptomatic phase of the disease. These results suggest that: (i) HIV infection is associated with elevation of sTNF-R serum levels: (ii) sTNF-R levels are strongly correlated to CD8+ lymphocytes during the asymptomatic stage of HIV infection. [ABSTRACT FROM AUTHOR]

Abstract Background The p53 tumor suppressor gene is commonly mutated in colorectal cancer. While the effect of p53 mutations on colorectal cancer prognosis has been heavily studied, less is known about how epidemiologic risk factors relate to p53 status, particularly in early colorectal neoplasia prior to clinically invasive colorectal cancer (including adenomas, carcinoma in situ (CIS), and intramucosal carcinoma). Methods We examined p53 status, as measured by protein overexpression, in 157 cases with early colorectal neoplasia selected from three New York City colonoscopy clinics. After collecting paraffin-embedded tissue blocks, immunohistochemistry was performed using an anti-p53 monoclonal mouse IgG2a [BP53-12-1] antibody. We analyzed whether p53 status was different for risk factors for colorectal neoplasia relative to a polyp-free control group (n = 508). Results p53 overexpression was found in 10.3%, 21.7%, and 34.9%, of adenomatous polyps, CIS, and intramucosal cases, respectively. Over 90% of the tumors with p53 overexpression were located in the distal colon and rectum. Heavy cigarette smoking (30+ years) was associated with cases not overexpressing p53 (OR = 1.8, 95% CI = 1.1–2.9) but not with those cases overexpressing p53 (OR = 1.0, 95% CI = 0.4–2.6). Heavy beer consumption (8+ bottles per week) was associated with cases overexpressing p53 (OR = 4.0, 95% CI = 1.3–12.0) but not with cases without p53 overexpression (OR = 1.6, 95% CI = 0.7–3.7). Conclusion Our findings that p53 overexpression in early colorectal neoplasia may be positively associated with alcohol intake and inversely associated with cigarette smoking are consistent with those of several studies of p53 expression and invasive cancer, and suggest that there may be relationships of smoking and alcohol with p53 early in the adenoma to carcinoma sequence.

Background & Aims: Histologic evaluation of mucosal healing in Crohn's disease is an evolving treatment target. We evaluated histologic outcomes for mirikizumab efficacy and associations with endoscopic and 1-year outcomes., Methods: Biopsy specimens from 1 ileal and 4 colonic segments were evaluated at weeks 0, 12, and 52 from each of the 170 SERENITY participants. Criteria for the weeks 12 and 52 histologic response were no epithelial neutrophils or epithelial damage, or >50% decrease in either the Robarts Histopathology Index or the active Global Histologic Disease Activity Score, and remission (no mucosal neutrophils and no epithelial damage) had to be met in all biopsy specimens. Agreement was evaluated between histologic and endoscopic end points. Associations between 1-year outcomes and week 12 histologic and endoscopic response were evaluated., Results: At week 12, 1000 mg mirikizumab resulted in greater rates of histologic response (66% vs 27%; P < .001) and remission (26% vs 6%; P < .01) than placebo. Rates were numerically similar at 1 year (mirikizumab pooled response, 46%-69%; remission, 13%-31%). Agreement between week 12 histologic and endoscopic response was 69% (Cohen's kappa coefficient [κ] = 0.40) and remission was 83% (κ = 0.38) in all pooled arms, including placebo. At 1 year, the percentage of participants who received any dose of mirikizumab and achieved endoscopic remission differed by their week 12 response: histologic (20%), endoscopic (25%), combined histology-endoscopy (45%), or neither (4%) (P = .003)., Conclusions: In a post hoc analysis of phase 2 data, mirikizumab induced and sustained histologic response and remission in Crohn's disease over 52 weeks. Early combined histologic-endoscopic response was associated with endoscopic remission after 1 year of treatment with mirikizumab (ClinicalTrials.gov NCT02891226)., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Sustained chronic inflammation of the large intestine leads to tissue damage and repair, which is associated with an increased incidence of colitis-associated colorectal cancer (CAC). The genetic makeup of CAC is somewhat similar to sporadic colorectal carcinoma (sCRC), but there are differences in the sequence and timing of alterations in the carcinogenesis process. Several models have been developed to explain the development of CAC, particularly the "field cancerization" model, which proposes that chronic inflammation accelerates mutagenesis and selects for the clonal expansion of phenotypically normal, pro-tumorigenic cells. In contrast, the "Big Bang" model posits that tumorigenic clones with multiple driver gene mutations emerge spontaneously. The details of CAC tumorigenesis-and how they differ from sCRC-are not yet fully understood. In this Review, we discuss recent genetic, epigenetic, and environmental findings related to CAC pathogenesis in the past five years, with a focus on unbiased, high-resolution genetic profiling of non-dysplastic field cancerization in the context of inflammatory bowel disease (IBD)., (© 2023. The Author(s).)

The histone H3 variant, H3.3, is localized at specific regions in the genome, especially promoters and active enhancers, and has been shown to play important roles in development. A lysine to methionine substitution in position 27 (H3.3K27M) is a main cause of Diffuse Intrinsic Pontine Glioma (specifically Diffuse Midline Glioma, K27M-mutant), a lethal type of pediatric cancer. H3.3K27M has a dominant-negative effect by inhibiting the Polycomb Repressor Complex 2 (PRC2) activity. Here, we studied the immediate, genome-wide, consequences of the H3.3K27M mutation independent of PRC2 activity. We developed Doxycycline (Dox)-inducible mouse embryonic stem cells (ESCs) carrying a single extra copy of WT-H3.3, H3.3K27M and H3.3K27L, all fused to HA. We performed RNA-Seq and ChIP-Seq at different times following Dox induction in undifferentiated and differentiated ESCs. We find increased binding of H3.3 around transcription start sites in cells expressing both H3.3K27M and H3.3K27L compared with WT, but not in cells treated with PRC2 inhibitors. Differentiated cells carrying either H3.3K27M or H3.3K27L retain expression of ESC-active genes, in expense of expression of genes related to neuronal differentiation. Taken together, our data suggest that a modifiable H3.3K27 is required for proper histone incorporation and cellular maturation, independent of PRC2 activity., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Breast cancer, the most frequent cancer in women, is generally classified into several distinct histological and molecular subtypes. However, single-cell technologies have revealed remarkable cellular and functional heterogeneity across subtypes and even within individual breast tumors. Much of this heterogeneity is attributable to dynamic alterations in the epigenetic landscape of the cancer cells, which promote phenotypic plasticity. Such plasticity, including transition from luminal to basal-like cell identity, can promote disease aggressiveness. We now report that the tumor suppressor LATS1, whose expression is often downregulated in human breast cancer, helps maintain luminal breast cancer cell identity by reducing the chromatin accessibility of genes that are characteristic of a "basal-like" state, preventing their spurious activation. This is achieved via interaction of LATS1 with the NCOR1 nuclear corepressor and recruitment of HDAC1, driving histone H3K27 deacetylation near NCOR1-repressed "basal-like" genes. Consequently, decreased expression of LATS1 elevates the expression of such genes and facilitates slippage towards a more basal-like phenotypic identity. We propose that by enforcing rigorous silencing of repressed genes, the LATS1-NCOR1 axis maintains luminal cell identity and restricts breast cancer progression., (© 2022. The Author(s).)

Cancer cells are highly heterogeneous at the transcriptional level and epigenetic state. Methods to study epigenetic heterogeneity are limited in throughput and information obtained per cell. Here, we adapted cytometry by time-of-flight (CyTOF) to analyze a wide panel of histone modifications in primary tumor-derived lines of diffused intrinsic pontine glioma (DIPG). DIPG is a lethal glioma, driven by a histone H3 lysine 27 mutation (H3-K27M). We identified two epigenetically distinct subpopulations in DIPG, reflecting inherent heterogeneity in expression of the mutant histone. These two subpopulations are robust across tumor lines derived from different patients and show differential proliferation capacity and expression of stem cell and differentiation markers. Moreover, we demonstrate the use of these high-dimensional data to elucidate potential interactions between histone modifications and epigenetic alterations during the cell cycle. Our work establishes new concepts for the analysis of epigenetic heterogeneity in cancer that could be applied to diverse biological systems., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Background & Aims: Strictures in Crohn's disease (CD) are classically attributed to fibromuscular hypertrophy of the intestinal wall. We have identified and characterized CD-related ileal strictures that result instead from mural constriction (ie, reduced external circumference)., Methods: Twenty-four strictures and internal controls from 17 adults with obstructive CD were analyzed by cross-sectional morphometry., Results: The stricture-to-control circumference ratios (CRs) ranged from 0.53 to 1.7. Six strictures with CR ≥1.0, designated hypertrophic, had concentrically thickened walls, mean 3-fold increases in cross-sectional area and stainable fibromucular tissue, and high transmural inflammation scores. In contrast, 18 strictures with CR <1.0, designated constrictive, had thin, pliant walls, cross-sectional areas and stainable fibromuscular tissue comparable with control values, and low transmural inflammation scores. Eight mildly constrictive strictures also showed mild fibromuscular mural expansion that fell short of statistical significance. Twelve of 18 constrictive strictures (67%) occurred multiply (2-4 strictures per specimen) in contrast with hypertrophic strictures, all of which occurred singly (P = .01). Constriction correlated quantitatively with circumferential serosal fat wrapping (P = .003) and was associated with myenteric lymphocytic plexitis (P = .02). Disease duration was shortest among subjects with constrictive strictures and correlated with increasing circumference (CR ≤0.8, 6.3 ± 6.2 years; CR >0.8, 8.7 ± 6.4 years; and CR ≥1.00, 13.7 ± 5.0 years, respectively; P = .03)., Conclusions: Constrictive ileal strictures in CD differ pathologically and clinically from hypertrophic strictures, featuring little or no fibromuscular mural expansion, frequent multiplicity, and earlier onset. Mesenteric fat wrapping and myenteric plexitis may contribute to their pathogenesis. Pathologic manifestations of constriction and hypertrophy can coexist, suggesting that stricture heterogeneity may be shaped in part by the dynamics of constrictive and hypertrophic processes., (Copyright © 2022 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Background: With the emergence of whole slide imaging (WSI) and widespread access to high-speed Internet, pathology labs are now poised to implement digital pathology as a way to access diagnostic pathology expertise. This paper describes a collaborative partnership between a high-volume reference diagnostic laboratory (Labcorp) and an academic pathology department (Mount Sinai Hospital) in the transition from a traditional glass slide service to a digital platform. Using the standard framework of implementation science, we evaluate the consistency and quality of the Philips IntelliSite Pathology Solution (PIPS) in delivering save and efficient diagnostic services., Materials and Methods: Digital and glass slide diagnoses of all consult cases were documented over a 12-month period. The Proctor guideline was used to quantitatively and qualitatively measure (e.g., focus group studies, field notes, and administrative data) implementation success. Lean techniques (e.g., value stream mapping) were applied to measure changes in efficiency with the transition to a digital platform., Results: Our study supports the acceptability, high adoption, appropriateness, feasibility, fidelity, and sustainability of the digital pathology platform. The digital portal also improved the quality of patient care by increasing efficiency, effectiveness, safety, and timeliness. The intraobserver concordance rate was 100%. The digital transition resulted in a reduction in turnaround time from 86 h to an average 35 min and a 20-fold increase in efficiency of the consultation process., Conclusion: As the pathology community contemplates digital pathology as a transformational tool in providing broad access to diagnostic expertise across time and space, our study provides an implementation strategy along with evidence that the digital platform is safe, effective, and efficient., Competing Interests: There are no conflicts of interest., (Copyright: © 2021 Journal of Pathology Informatics.)

Background: Neoadjuvant chemotherapy (NAC) is frequently used in gastrointestinal cancers (GIC), and pathological, radiological, and tumor marker responses are assessed during and after NAC., Aim: To evaluate the relationship between pathologic, radiologic, tumor marker responses and recurrence-free survival (RFS), overall survival (OS), adjuvant chemotherapy (AC) decisions, and the impact of changing to a different AC regimen after poor response to NAC., Methods and Results: Medical records of GIC patients treated with NAC at Mount Sinai between 1/2012 and 12/2018 were reviewed. One hundred fifty-six patients (58.3% male, mean age 63 years) were identified. Primary tumor sites were: 43 (27.7%) pancreas, 62 (39.7%) gastroesophageal, and 51 (32.7%) colorectal. After NAC, 31 (19.9%) patients had favorable pathologic response (FPR; defined as College of American Pathologists [CAP] score 0-1). Of 107 patients with radiological data, 59 (55.1%) had an objective response, and of 113 patients with tumor marker data, 61 (54.0%) had a ≥50% reduction post NAC. FPR, but not radiographic or serological responses, was associated with improved RFS (HR 0.28; 95% CI 0.11-0.72) and OS (HR 0.13; 95% CI 0.2-0.94). Changing to a different AC regimen from initial NAC, among all patients and specifically among those with unfavorable pathological response (UPR; defined as CAP score 2-3) after NAC, was not associated with improved RFS or OS., Conclusions: GIC patients with FPR after NAC experienced significant improvements in RFS and OS. Patients with UPR did not benefit from changing AC. Prospective studies to better understand the role of pathological response in AC decisions and outcomes in GIC patients are needed., (© 2021 The Authors. Cancer Reports published by Wiley Periodicals LLC.)

Background and Aims: Disease extent varies in ulcerative colitis (UC) from proctitis to left-sided colitis to pancolitis and is a major prognostic factor. When the extent of UC is limited there is often a sharp demarcation between macroscopically involved and uninvolved areas and what defines this or subsequent extension is unknown. We characterized the demarcation site molecularly and determined genes associated with subsequent disease extension., Methods: We performed RNA sequence analysis of biopsy specimens from UC patients with endoscopically and histologically confirmed limited disease, of which a subset later extended. Biopsy specimens were obtained from the endoscopically inflamed upper (proximal) limit of disease, immediately adjacent to the uninvolved colon, as well as at more proximal, endoscopically uninflamed colonic segments., Results: Differentially expressed genes were identified in the endoscopically inflamed biopsy specimens taken at each patient's most proximal diseased site relative to healthy controls. Expression of these genes in the more proximal biopsy specimens transitioned back to control levels abruptly or gradually, the latter pattern supporting the concept that disease exists beyond the endoscopic disease demarcation site. The gradually transitioning genes were associated with inflammation, angiogenesis, glucuronidation, and homeodomain pathways. A subset of these genes in inflamed biopsy specimens was found to predict disease extension better than clinical features and were responsive to biologic therapies. Network analysis revealed critical roles for interferon signaling in UC inflammation and poly(ADP-ribose) polymerase 14 (PARP14) was a predicted key driver gene of extension. Higher PARP14 protein levels were found in inflamed biopsy specimens of patients with limited UC that subsequently extended., Conclusion: Molecular predictors of disease extension reveal novel strategies for disease prognostication and potential therapeutic targeting., (Copyright © 2021 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its associated clinical syndrome COVID-19 are causing overwhelming morbidity and mortality around the globe and disproportionately affected New York City between March and May 2020. Here, we report on the first 100 COVID-19-positive autopsies performed at the Mount Sinai Hospital in New York City. Autopsies revealed large pulmonary emboli in six cases. Diffuse alveolar damage was present in over 90% of cases. We also report microthrombi in multiple organ systems including the brain, as well as hemophagocytosis. We additionally provide electron microscopic evidence of the presence of the virus in our samples. Laboratory results of our COVID-19 cohort disclose elevated inflammatory markers, abnormal coagulation values, and elevated cytokines IL-6, IL-8, and TNFα. Our autopsy series of COVID-19-positive patients reveals that this disease, often conceptualized as a primarily respiratory viral illness, has widespread effects in the body including hypercoagulability, a hyperinflammatory state, and endothelial dysfunction. Targeting of these multisystemic pathways could lead to new treatment avenues as well as combination therapies against SARS-CoV-2 infection., (© 2021. The Author(s), under exclusive licence to United States & Canadian Academy of Pathology.)

Background & Aims: Histopathology is an emerging treatment target in ulcerative colitis (UC) clinical trials. Our aim was to provide guidance on standardizing biopsy collection protocols, identifying optimal evaluative indices, and defining thresholds for histologic response and remission after treatment., Methods: An international, interdisciplinary expert panel of 19 gastroenterologists and gastrointestinal pathologists was assembled. A modified RAND/University of California, Los Angeles appropriateness methodology was used to address relevant issues. A total of 138 statements were derived from a systematic review of the literature and expert opinion. Each statement was anonymously rated as appropriate, uncertain, or inappropriate using a 9-point scale. Survey results were reviewed and discussed before a second round of voting., Results: Histologic measurements collected using a uniform biopsy strategy are important for assessing disease activity and determining therapeutic efficacy in UC clinical trials. Multiple biopsy strategies were deemed acceptable, including segmental biopsies collected according to the endoscopic appearance. Biopsies should be scored for architectural change, lamina propria chronic inflammation, basal plasmacytosis, lamina propria and epithelial neutrophils, epithelial damage, and erosions/ulcerations. The Geboes score, Robarts Histopathology Index, and Nancy Index were considered appropriate for assessing histologic activity; use of the modified Riley score and Harpaz Index were uncertain. Histologic activity at baseline should be required for enrollment, recognizing this carries operational implications. Achievement of histologic improvement or remission was considered an appropriate and realistic therapeutic target. Current histologic indices require validation for pediatric populations., Conclusions: These recommendations provide a framework for standardized implementation of histopathology in UC trials. Additional work is required to address operational considerations and areas of uncertainty., (Copyright © 2021 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Background & Aims: Given that gastrointestinal (GI) symptoms are a prominent extrapulmonary manifestation of COVID-19, we investigated intestinal infection with SARS-CoV-2, its effect on pathogenesis, and clinical significance., Methods: Human intestinal biopsy tissues were obtained from patients with COVID-19 (n = 19) and uninfected control individuals (n = 10) for microscopic examination, cytometry by time of flight analyses, and RNA sequencing. Additionally, disease severity and mortality were examined in patients with and without GI symptoms in 2 large, independent cohorts of hospitalized patients in the United States (N = 634) and Europe (N = 287) using multivariate logistic regressions., Results: COVID-19 case patients and control individuals in the biopsy cohort were comparable for age, sex, rates of hospitalization, and relevant comorbid conditions. SARS-CoV-2 was detected in small intestinal epithelial cells by immunofluorescence staining or electron microscopy in 15 of 17 patients studied. High-dimensional analyses of GI tissues showed low levels of inflammation, including down-regulation of key inflammatory genes including IFNG, CXCL8, CXCL2, and IL1B and reduced frequencies of proinflammatory dendritic cells compared with control individuals. Consistent with these findings, we found a significant reduction in disease severity and mortality in patients presenting with GI symptoms that was independent of sex, age, and comorbid illnesses and despite similar nasopharyngeal SARS-CoV-2 viral loads. Furthermore, there was reduced levels of key inflammatory proteins in circulation in patients with GI symptoms., Conclusions: These data highlight the absence of a proinflammatory response in the GI tract despite detection of SARS-CoV-2. In parallel, reduced mortality in patients with COVID-19 presenting with GI symptoms was observed. A potential role of the GI tract in attenuating SARS-CoV-2-associated inflammation needs to be further examined., (Copyright © 2021 AGA Institute. All rights reserved.)

Background and Aims: The presence of gastrointestinal symptoms and high levels of viral RNA in the stool suggest active severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replication within enterocytes., Methods: Here, in multiple, large cohorts of patients with inflammatory bowel disease (IBD), we have studied the intersections between Coronavirus Disease 2019 (COVID-19), intestinal inflammation, and IBD treatment., Results: A striking expression of ACE2 on the small bowel enterocyte brush border supports intestinal infectivity by SARS-CoV-2. Commonly used IBD medications, both biologic and nonbiologic, do not significantly impact ACE2 and TMPRSS2 receptor expression in the uninflamed intestines. In addition, we have defined molecular responses to COVID-19 infection that are also enriched in IBD, pointing to shared molecular networks between COVID-19 and IBD., Conclusions: These data generate a novel appreciation of the confluence of COVID-19- and IBD-associated inflammation and provide mechanistic insights supporting further investigation of specific IBD drugs in the treatment of COVID-19. Preprint doi: https://doi.org/10.1101/2020.05.21.109124., (Copyright © 2021 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Eukaryotic cells have evolved organelles that allow the compartmentalization and regulation of metabolic processes. Knowledge of molecular mechanisms that allow temporal and spatial organization of enzymes within organelles is therefore crucial for understanding eukaryotic metabolism. Here, we show that the yeast malate dehydrogenase 2 (Mdh2) is dually localized to the cytosol and to peroxisomes and is targeted to peroxisomes via association with Mdh3 and a Pex5-dependent piggybacking mechanism. This dual localization of Mdh2 contributes to our understanding of the glyoxylate cycle and provides a new perspective on compartmentalization of cellular metabolism, which is critical for the perception of metabolic disorders and aging., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

Cellular function requires molecular motors to transport cargoes to their correct intracellular locations. The regulated assembly and disassembly of motor-adaptor complexes ensures that cargoes are loaded at their origin and unloaded at their destination. In Saccharomyces cerevisiae, early in the cell cycle, a portion of the vacuole is transported into the emerging bud. This transport requires a myosin V motor, Myo2, which attaches to the vacuole via Vac17, the vacuole-specific adaptor protein. Vac17 also binds to Vac8, a vacuolar membrane protein. Once the vacuole is brought to the bud cortex via the Myo2-Vac17-Vac8 complex, Vac17 is degraded and the vacuole is released from Myo2. However, mechanisms governing dissociation of the Myo2-Vac17-Vac8 complex are not well understood. Ubiquitylation of the Vac17 adaptor at the bud cortex provides spatial regulation of vacuole release. Here, we report that ubiquitylation alone is not sufficient for cargo release. We find that a parallel pathway, which initiates on the vacuole, converges with ubiquitylation to release the vacuole from Myo2. Specifically, we show that Yck3 and Vps41, independent of their known roles in homotypic fusion and protein sorting (HOPS)-mediated vesicle tethering, are required for the phosphorylation of Vac17 in its Myo2 binding domain. These phosphorylation events allow ubiquitylated Vac17 to be released from Myo2 and Vac8. Our data suggest that Vps41 is regulating the phosphorylation of Vac17 via Yck3, a casein kinase I, and likely another unknown kinase. That parallel pathways are required to release the vacuole from Myo2 suggests that multiple signals are integrated to terminate organelle inheritance., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)