Biosynthesis of natural and halogenated plant monoterpene indole alkaloids in yeast.

Monoterpenoid indole alkaloids (MIAs) represent a large class of plant natural products with marketed pharmaceutical activities against a wide range of indications, including cancer, malaria and hypertension. Halogenated MIAs have shown improved pharmaceutical properties; however, synthesis of new-to-nature halogenated MIAs remains a challenge. Here we demonstrate a platform for de novo biosynthesis of two MIAs, serpentine and alstonine, in baker's yeast Saccharomyces cerevisiae and deploy it to systematically explore the biocatalytic potential of refactored MIA pathways for the production of halogenated MIAs. From this, we demonstrate conversion of individual haloindole derivatives to a total of 19 different new-to-nature haloserpentine and haloalstonine analogs. Furthermore, by process optimization and heterologous expression of a modified halogenase in the microbial MIA platform, we document de novo halogenation and biosynthesis of chloroalstonine. Together, this study highlights a microbial platform for enzymatic exploration and production of complex natural and new-to-nature MIAs with therapeutic potential.

Antimullerian Hormone, a Marker of Ovarian Reserve, Is Protective Against Presence and Severity of NASH in Premenopausal Women.

BACKGROUND & AIMS: Antimüllerian hormone (AMH) is a marker of ovarian reserve with emerging data linking lower levels to some metabolic and inflammatory diseases in women. Whether AMH levels influence nonalcoholic fatty liver disease (NAFLD) is unknown. METHODS: Leveraging the NASH Clinical Research Network we determined the association of AMH levels within 6 months of liver biopsy with presence and severity of histologic measures of NAFLD in premenopausal women. Outcomes included presence of nonalcoholic steatohepatitis (NASH), presence and severity of fibrosis, and NAFLD Activity Score and its components. Logistic and ordinal logistic regression models were adjusted for age, race/ethnicity, homeostatic model assessment for insulin resistance, body mass index, dyslipidemia, polycystic ovary syndrome, estrogen-progestin use, and menstrual cyclicity. RESULTS: Median cohort age was 35 years; 73% were white and 24% Hispanic. Thirty-three percent had diabetes, 81% had obesity, and 95% had dyslipidemia. On biopsy 71% had NASH, 68% had any fibrosis, and 15% had advanced fibrosis. On adjusted analysis (n = 205), higher AMH quartiles were inversely associated with NAFLD histology including prevalent NASH (adjusted odds ratio [AOR], 0.64; 95% confidence interval [CI], 0.41-1.00), NAFLD Activity Score ≥5 (AOR, 0.52; 95% CI, 0.35-0.77), Mallory hyaline (AOR, 0.54; 95% CI, 0.35-0.82), and higher fibrosis stage (AOR, 0.70; 95% CI, 0.51-0.98). The protective effects of AMH were more pronounced among women without polycystic ovary syndrome (n = 164), including lower odds of NASH (AOR, 0.53; 95% CI, 0.32-0.90) and any NASH fibrosis (AOR, 0.54; 95% CI, 0.32-0.93). CONCLUSIONS: AMH may reflect a unique biomarker of NASH in premenopausal women and findings suggest a novel link between reproductive aging and histologic severity of NAFLD in women.

Per-patient Negative Predictive Value of the CT and MRI Liver Imaging Reporting and Data System Version 2018 Treatment Response Algorithm for Hepatocellular Carcinoma.

Background The Liver Imaging Reporting and Data System version 2018 (LI-RADS) treatment response algorithm (TRA) is a high-specificity, lower-sensitivity grading system to diagnose hepatocellular carcinoma (HCC) and recurrence after local-regional therapy. However, the emphasis on specificity can result in disease understaging, potentially leading to poorer posttransplant outcomes. Purpose To determine the negative predictive value (NPV) of pretransplant CT and MRI assessment for viable HCC on a per-patient basis using the LI-RADS TRA, considering explant pathology as the reference standard. Materials and Methods Patient records from 218 consecutive adult patients from a single institution with HCC who underwent liver transplant from January 2011 to November 2017 were retrospectively reviewed. Two readers blinded to the original report reviewed immediate (within 90 days) pretransplant imaging and characterized observations according to the LI-RADS TRA. Based on this, patients with LR-4, LR-5, or LR-TR (treatment response) viable tumors were designated as viable tumor; patients with solely LR-3 or LR-TR equivocal tumors were designated as equivocal; and patients with only LR-TR nonviable lesions were designated as no viable disease. Patients were designated as within or outside the Milan criteria. These per-patient designations were compared with the presence of viable disease at explant pathology. Fisher exact test was used to compare the differences between CT and MRI. Weighted κ values were used to calculate interreader reliability. Results Final study sample consisted of 206 patients (median age, 61 years [IQR, 57-65 years]; 157 male patients and 49 female patients). Per-patient LI-RADS TRA assessment of pretransplant imaging had an NPV of 32% (95% CI: 27, 38) and 26% (95% CI: 20, 33) (readers 1 and 2, respectively) for predicting viable disease. Seventy-five percent (reader 1) and 77% (reader 2) of patients deemed equivocal had residual tumors at explant pathology. Weighted interreader reliability was substantial (κ = 0.62). Conclusion Patient-based stratification of viable, equivocal, and nonviable disease at pretransplant CT or MRI, based on LI-RADS TRA, demonstrated low negative predictive value in excluding HCC at explant pathology. © RSNA, 2023 See also the editorial by Tamir and Tau in this issue.

Integrated Genomic and Clinicopathologic Approach Distinguishes Pancreatic Grade 3 Neuroendocrine Tumor From Neuroendocrine Carcinoma and Identifies a Subset With Molecular Overlap.

Distinguishing grade 3 pancreatic neuroendocrine tumor (G3 PanNET) from neuroendocrine carcinoma (PanNEC) is a known diagnostic challenge, and accurate classification is critical because clinical behavior and therapies differ. Although current recommendations suggest that immunohistochemistry for p53, Rb, ATRX, and DAXX can distinguish most cases, some cases remain difficult to classify using this approach. In this study, we reviewed 47 high-grade neoplasms originally diagnosed as pancreatic neuroendocrine neoplasms. In addition to the currently recommended stains, we performed capture-based sequencing of approximately 500 cancer genes and immunohistochemistry for p16 and trypsin or chymotrypsin. Using an integrated molecular and clinicopathologic approach, 42 (89%) of 47 cases had a clear final diagnosis of either G3 PanNET (n = 17), PanNEC (n = 17), or mixed acinar-NEC (n = 8). The 17 G3 PanNETs demonstrated frequent alterations in MEN1 (71%), DAXX (47%), ATRX (24%), TSC2 (35%), SETD2 (42%), and CDKN2A (41%). Contrary to prior reports, TP53 alterations were also common in G3 PanNETs (35%) but were always mutually exclusive with CDKN2A alterations in this group. The 17 PanNECs demonstrated frequent alterations in TP53 (88%), cell cycle genes RB1 (47%), CCNE1/CCND1 (12%), CDKN2A (29%), and in KRAS (53%) and SMAD4 (41%); TP53 was coaltered with a cell cycle gene in 76% of PanNECs. Diffuse strong p16 staining was observed in 69% of PanNECs in contrast to 0% of G3 PanNETs. The 8 acinar-NECs had recurrent alterations in ATM (25%), APC (25%), and STK11 (25%). Five cases remained difficult to classify, 3 of which exhibited overlapping molecular features with alterations in MEN1 with or without ATRX, and RB1 with or without TP53, making it unclear whether to classify as PanNET or PanNEC. Our data demonstrate that molecular profiling and immunohistochemistry for p16 greatly improve the diagnostic accuracy of high-grade pancreatic neuroendocrine neoplasms and identify a subset of rare cases with overlapping features of both PanNET and PanNEC.

Understanding toxicity associated with boron nitride nanotubes: Review of toxicity studies, exposure assessment at manufacturing facilities, and read-across.

Boron nitride nanotubes (BNNT) are produced by many different methods leading to variances in physicochemical characteristics and impurities in the final product. These differences can alter the toxicity profile. The importance of understanding the potential pathological implications of this high aspect ratio nanomaterial is increasing as new approaches to synthesize and purify in large scale are being developed. In this review, we discuss the various factors of BNNT production that can influence its toxicity followed by summarizing the toxicity findings from in vitro and in vivo studies conducted to date, including a review of particle clearance observed with various exposure routes. To understand the risk to workers and interpret relevance of toxicological findings, exposure assessment at manufacturing facilities was discussed. Workplace exposure assessment of BNNT from two manufacturing facilities measured boron concentrations in personal breathing zones from non-detectable to 0.95 µg/m3 and TEM structure counts of 0.0123 ± 0.0094 structures/cm3, concentrations well below what was found with other engineered high aspect ratio nanomaterials like carbon nanotubes and nanofibers. Finally, using a purified BNNT, a "read-across" toxicity assessment was performed to demonstrate how known hazard data and physicochemical characteristics can be utilized to evaluate potential inhalation toxicity concerns.

Low Testosterone Is Associated With Nonalcoholic Steatohepatitis and Fibrosis Severity in Men.

With rising prevalence of obesity and diabetes, nonalcoholic fatty liver disease (NAFLD) is now a leading cause of chronic liver disease. One-third of obese or diabetic men have subnormal free and bioavailable testosterone concentrations.1 Several studies have further shown low testosterone to be associated with imaging-confirmed NAFLD in men,2 although it is unknown whether low testosterone confers increased risk of more clinically relevant manifestations of NAFLD, including nonalcoholic steatohepatitis (NASH) and NASH fibrosis. We therefore aimed to evaluate the association of testosterone with histologic features of NAFLD among a representative cohort of men from the multicenter NASH Clinical Research Network (NASH CRN).

Testosterone is Associated With Nonalcoholic Steatohepatitis and Fibrosis in Premenopausal Women With NAFLD.

BACKGROUND & AIMS: Higher testosterone contributes to imaging-confirmed nonalcoholic fatty liver disease (NAFLD) in women, but whether testosterone influences their disease severity is unknown. METHODS: The association of free testosterone (free T) with nonalcoholic steatohepatitis (NASH) was determined in pre-menopausal women with biopsy-confirmed NAFLD (n = 207). Interaction testing was performed for age and free T given decline in testosterone with age, and association of aging with NASH. Regression models adjusted for abdominal adiposity, diabetes, and dyslipidemia. RESULTS: Median age was 35 yrs (interquartile range, 29-41); 73% were white, 25% Hispanic; 32% had diabetes, 93% abdominal adiposity, and 95% dyslipidemia. 69% had NASH, 67% any fibrosis, and 15% advanced fibrosis. Higher free T levels were associated with NAFLD severity in younger women (interaction P value <.02). In the youngest age quartile, free T was independently associated with NASH (odds ratio [OR], 2.3; 95% CI, 1.2-4.4), NASH fibrosis (OR, 2.1; 95% CI, 1.1-3.8), and higher fibrosis stage (OR, 1.9; 95% CI, 1.1-3.4), P value .02. In these women, the proportion with NASH steadily rose from 27% to 88%, and with NASH fibrosis rose from 27% to 81%, with higher free T quartiles (P < .01). Free T was additionally associated with abdominal adiposity among all pre-menopausal women (OR, 2.2; 95% CI, 1.2-4.1: P = .02). CONCLUSIONS: In young women with NAFLD, higher testosterone levels conferred a 2-fold higher risk of NASH and NASH fibrosis, and increased risk of abdominal adiposity, supporting a potential mechanistic link of abdominal fat on testosterone-associated liver injury. Testosterone may represent an early risk factor for NASH progression in young women, prior to their onset of more dominant, age-related metabolic risk factors.

CRISPR/Cas9 recombineering-mediated deep mutational scanning of essential genes in Escherichia coli.

Deep mutational scanning can provide significant insights into the function of essential genes in bacteria. Here, we developed a high-throughput method for mutating essential genes of Escherichia coli in their native genetic context. We used Cas9-mediated recombineering to introduce a library of mutations, created by error-prone PCR, within a gene fragment on the genome using a single gRNA pre-validated for high efficiency. Tracking mutation frequency through deep sequencing revealed biases in the position and the number of the introduced mutations. We overcame these biases by increasing the homology arm length and blocking mismatch repair to achieve a mutation efficiency of 85% for non-essential genes and 55% for essential genes. These experiments also improved our understanding of poorly characterized recombineering process using dsDNA donors with single nucleotide changes. Finally, we applied our technology to target rpoB, the beta subunit of RNA polymerase, to study resistance against rifampicin. In a single experiment, we validate multiple biochemical and clinical observations made in the previous decades and provide insights into resistance compensation with the study of double mutants.

Diagnostic challenges of focal nodular hyperplasia: interobserver variability, accuracy, and the utility of glutamine synthetase immunohistochemistry.

AIMS: The diagnosis of focal nodular hyperplasia (FNH) and the interpretation of glutamine synthetase (GS) staining can be challenging on biopsies. We aimed to evaluate the reproducibility of needle biopsy diagnosis of FNH, the effect of GS immunohistochemistry on FNH diagnosis, and which histological features are most useful for the diagnosis of FNH. METHODS AND RESULTS: The study included virtual needle biopsies generated from 75 resection specimens (30 FNHs, 15 hepatocellular adenomas, 15 hepatocellular carcinomas, and 15 non-lesional liver specimens). Pathologists were reasonably accurate (83.1%) in the diagnosis of FNH with haematoxylin and eosin alone. Ductular reaction and nodularity had the highest sensitivity for a diagnosis of FNH (88.1% and 82.2%, respectively), whereas central scar was the most specific feature (90.6%). The presence of two or more of the classic histological features had 89.6% sensitivity and 86.2% specificity for a diagnosis of FNH. Diagnostic accuracy was significantly higher with the addition of a GS stain. A map-like GS staining pattern was highly specific (99.3%) for FNH. However, GS staining was interpreted as non-map-like in 14.4% of reviews of true FNH cases, and overall interobserver agreement for interpretation of the GS staining pattern was only moderate (kappa = 0.42). CONCLUSIONS: Pathologists are reasonably accurate in the diagnosis of FNH on virtual biopsies, and GS staining improves accuracy. However, a subset of FNH cases remain challenging. Steatosis and a pseudo-map-like GS staining pattern were associated with increased difficulty. Therefore, although a map-like GS staining pattern is useful for confirmation of a diagnosis, the lack of a map-like GS staining pattern on needle biopsy does not necessarily exclude a diagnosis of FNH.

Glycogenosis is common in nonalcoholic fatty liver disease and is independently associated with ballooning, but lower steatosis and lower fibrosis.

BACKGROUND/AIMS: Glycogen synthesis and storage are normal hepatocyte functions. However, glycogenosis, defined as excess hepatocyte glycogen visible by routine H&E light microscopy, has not been well characterized in nonalcoholic fatty liver disease (NAFLD). METHODS: Glycogenosis in NAFLD liver biopsies was graded as "none", "focal" (in <50% of hepatocytes), or "diffuse" (in ≥50% of hepatocytes). Clinical and pathological variables associated with glycogenosis were assessed. 2047 liver biopsies were prospectively analysed. RESULTS: In adults and children, any glycogenosis was present in 54% of cases; diffuse glycogenosis was noted in approximately 1/3 of cases. On multiple logistic regression analysis, adults with glycogenosis tended to be older (P = .003), female (P = .04), have higher serum glucose (P = .01), and use insulin (P = .02). Adults tended to have lower steatosis scores (P = .006) and lower fibrosis stages (P = .005); however, unexpectedly, they also tended to have more hepatocyte injury including ballooning (P = .003). On multiple logistic regression analysis, paediatric patients with glycogenosis were more likely to be Hispanic (P = .03), have lower body weight (P = .002), elevated triglycerides (P = .001), and a higher fasting glucose (P = .007). Paediatric patients with glycogenosis also had less steatosis (P < .001) than those without. CONCLUSIONS: Glycogenosis is common in adult and paediatric NAFLD, and is associated with clinical features of insulin resistance. Glycogenosis is important to recognize histologically because it may be misinterpreted as ballooning, and when diffuse, confusion with glycogen storage disorders or glycogenic hepatopathy must be avoided. The newly observed dichotomous relationship between glycogenosis and increased liver cell injury but decreased steatosis and fibrosis requires further study.

A versatile platform strain for high-fidelity multiplex genome editing.

Precision genome editing accelerates the discovery of the genetic determinants of phenotype and the engineering of novel behaviors in organisms. Advances in DNA synthesis and recombineering have enabled high-throughput engineering of genetic circuits and biosynthetic pathways via directed mutagenesis of bacterial chromosomes. However, the highest recombination efficiencies have to date been reported in persistent mutator strains, which suffer from reduced genomic fidelity. The absence of inducible transcriptional regulators in these strains also prevents concurrent control of genome engineering tools and engineered functions. Here, we introduce a new recombineering platform strain, BioDesignER, which incorporates (i) a refactored λ-Red recombination system that reduces toxicity and accelerates multi-cycle recombination, (ii) genetic modifications that boost recombination efficiency, and (iii) four independent inducible regulators to control engineered functions. These modifications resulted in single-cycle recombineering efficiencies of up to 25% with a 7-fold increase in recombineering fidelity compared to the widely used recombineering strain EcNR2. To facilitate genome engineering in BioDesignER, we have curated eight context--neutral genomic loci, termed Safe Sites, for stable gene expression and consistent recombination efficiency. BioDesignER is a platform to develop and optimize engineered cellular functions and can serve as a model to implement comparable recombination and regulatory systems in other bacteria.

Genome engineering of E. coli for improved styrene production.

Microbial production of exogenous organic compounds is challenging as biosynthetic pathways are often complex and produce metabolites that are toxic to the hosts. Biogenic styrene is an example of this problem, which if addressed could result in a more sustainable supply of this important component of the plastics industry. In this study, we engineered Escherichia coli for the production of styrene. We systematically optimized the production capability by first screening different pathway expression levels in E. coli strains. We then further designed and constructed a transcription regulator library targeting 54 genes with 85,420 mutations, and tested this library for increased styrene resistance and production. A series of tolerant mutants not only exhibited improved styrene tolerance but also produced higher styrene concentrations compared to the parent strain. The best producing mutant, ST05 LexA_E45I, produced a 3.45-fold increase in styrene compared to the parent strain. The produced styrene was extracted via gas stripping into dodecane and used in a direct free radical synthesis of polystyrene.

Clinicopathological features and outcomes of parenchymal rejection in liver transplant biopsies.

AIMS: The aim of this study was to perform a comprehensive retrospective analysis of liver transplant biopsies with parenchymal rejection (PR) at our institution, including histological features, laboratory values and follow-up biopsies, and to compare PR with portal-based acute cellular rejection (ACR). METHODS AND RESULTS: Biopsies from 173 patients were evaluated (retrospective database search 1990-2017), including 49 isolated PR, 35 PR with portal ACR (PR/ACR), 34 mild ACR and 52 moderate ACR cases. The rise and fall of serum liver enzymes was calculated as a measure of acute liver injury and response to immunotherapy, respectively. Isolated PR was associated with delayed-onset acute rejection (P < 0.001), as well as younger age (P = 0.004), and showed a similar rise in liver enzymes to mild ACR. PR/ACR and moderate ACR showed the highest elevations in transaminases (P < 0.05). Isolated PR on an initial biopsy was associated with recurrent episodes of PR (P = 0.01), chronic ductopaenic rejection (P = 0.002) and chronic vascular rejection (P = 0.017). Immunohistochemistry for C4d was performed, and strong C4d staining of venules was only detected in one severe isolated PR case (one of three, 33%) and one moderate ACR case (one of 20, 5%). CONCLUSIONS: Isolated PR represents a form of late acute rejection with distinct clinical and histological features. There is value in reporting PR in liver transplant biopsies to identify patients at higher risk of developing recurrent PR and chronic rejection. Standardisation of terminology and histological criteria of PR can help in uniform reporting and ensure appropriate management.

Polycystic ovary syndrome (PCOS) is associated with NASH severity and advanced fibrosis.

BACKGROUND: Polycystic ovary syndrome (PCOS) affects 10% of reproductive-aged women, and is marked by irregular menses and high androgens. PCOS is a known risk factor for imaging-confirmed steatosis, and we now aim to evaluate whether PCOS influences histologic severity of non-alcoholic fatty liver disease (NAFLD). METHODS: Retrospective study of women ages 18-45 years with biopsy-confirmed NAFLD between 2008 and 2019. Metabolic comorbidities were captured within 6 months of biopsy. Histologic features of non-alcoholic steatohepatitis (NASH) were independently evaluated by two pathologists blinded to PCOS status. RESULTS: Among 102 women meeting study criteria, 36% (n = 37) had PCOS; median age was 35 years; 27% were white, 6% black, 19% Asian and 47% reported Hispanic ethnicity. Women with PCOS had higher LDL (122 vs 102 mg/dL, P = .05) and body mass index(BMI) (38 vs 33 kg/cm2 , P < .01). NASH was present in 76% of women with PCOS vs 66% without PCOS (P = .3), and a higher proportion with PCOS had severe ballooning (32% vs 13%, P = .02), presence of any fibrosis (84% vs 66%, P = .06) and advanced fibrosis (16% vs 6%, P = .10). Adjusted for age and BMI, PCOS remained associated with severe hepatocyte ballooning (OR 3.4, 95% CI 1.1-10.6, P = .03) and advanced fibrosis (OR 7.1, 95% CI 1.3-39, P = .02). Among women with advanced fibrosis, median age was 5 years younger in those with as compared to those without PCOS (40 vs 45 years, P = .02). CONCLUSION: Polycystic ovary syndrome is independently associated with more severe NASH, including advanced fibrosis. Hepatologists should routinely inquire about PCOS in reproductive-aged women with NAFLD, and evaluate for more severe liver disease in this population.

Complexities in the diagnosis of large B-cell lymphomas, classic Hodgkin lymphomas and overlapping peripheral T-cell lymphomas simplified: An evidence-based guide.

The diagnosis of a large B-cell lymphoma and classic Hodgkin lymphoma (CHL) is often straightforward. However, in select circumstances, these simple diagnoses can be quite complex. In part, diagnostic difficulty may be due to uncertainty in the evaluation of morphologic and immunophenotypic features along a biologic continuum, or alternatively arise from uncertainty in predicting the behavior and outcomes of patients. Here, we systematically discuss and review areas of diagnostic difficulty in the diagnosis of large B-cell lymphomas (LBCL), classic Hodgkin lymphomas (CHL) and peripheral T-cell lymphomas (PTCL). We provide careful data-driven analyses and evidence-based approaches to help guide pathologists and clinicians. We discuss: 1) marginal zone lymphomas with increased large cells versus diffuse large B-cell lymphoma (DLBCL), 2) chronic lymphocytic leukemia with expanded proliferation centers versus diffuse large B-cell lymphoma (DLBCL), 3) chronic lymphocytic leukemia with Hodgkin/Reed-Sternberg-like cells versus CHL arising from chronic lymphocytic leukemia, 4) complex cases of follicular lymphoma versus DLBCL, 5) PTCL with large B-cell proliferations versus PTCL with LBCL, 6) PTCL with Hodgkin/Reed-Sternberg-like cells versus CHL, and finally 7) blastoid/pleomorphic mantle cell lymphoma versus DLBCL. Our evidence and data driven approach may serve as a useful diagnostic guide.

Multiplex navigation of global regulatory networks (MINR) in yeast for improved ethanol tolerance and production.

Multiplex navigation of global regulatory networks (MINR) is an approach for combinatorially reprogramming gene expression to manipulate complex phenotypes. We designed, constructed, and mapped MINR libraries containing 43,020 specific mutations in 25 regulatory genes expected to perturb the yeast regulatory network. We selected growth competition experiments for library mutants conferring increased ethanol and/or glucose tolerance. We identified specific mutants that not only possessed improved ethanol and/or glucose tolerance but also produced ethanol at concentrations up to 2-fold higher than those produced by the wild-type strain. We further determined that mutations increasing ethanol tolerance were transferable to a diploid industrial yeast strain. The facile construction and mapping of 43,020 designer regulatory mutations provide a roadmap for how to access and engineer complex phenotypes in future synthetic biology and broader efforts.

Deep scanning lysine metabolism in Escherichia coli.

Our limited ability to predict genotype-phenotype relationships has called for strategies that allow testing of thousands of hypotheses in parallel. Deep scanning mutagenesis has been successfully implemented to map genotype-phenotype relationships at a single-protein scale, allowing scientists to elucidate properties that are difficult to predict. However, most phenotypes are dictated by several proteins that are interconnected through complex and robust regulatory and metabolic networks. These sophisticated networks hinder our understanding of the phenotype of interest and limit our capabilities to rewire cellular functions. Here, we leveraged CRISPR-EnAbled Trackable genome Engineering to attempt a parallel and high-resolution interrogation of complex networks, deep scanning multiple proteins associated with lysine metabolism in Escherichia coli We designed over 16,000 mutations to perturb this pathway and mapped their contribution toward resistance to an amino acid analog. By doing so, we identified different routes that can alter pathway function and flux, uncovering mechanisms that would be difficult to rationally design. This approach sets a framework for forward investigation of complex multigenic phenotypes.

DNA flow cytometric and interobserver study of crypt cell atypia in inflammatory bowel disease.

AIMS: The pathological features and diagnostic reliability of crypt cell atypia (CCA) arising in inflammatory bowel disease (IBD) and its clinical significance are unknown. METHODS AND RESULTS: DNA flow cytometry (FCM) was performed on 14 colon biopsies of CCA from seven IBD patients (male-to-female ratio, 5:2; mean age, 53 years; mean IBD duration, 15 years) using paraffin-embedded tissue. Seven gastrointestinal pathologists were asked to diagnose each biopsy as negative for dysplasia (NEG), indefinite for dysplasia (IND), low-grade dysplasia (LGD) or high-grade dysplasia (HGD) by morphology alone, then again with knowledge of FCM results. Aneuploidy was detected in all 14 biopsies, and five of eight biopsies (63%) also showed strong and diffuse nuclear staining for p53 in the areas of CCA. Six (86%) patients developed HGD (n = 5) or adenocarcinoma (n = 1) in the same colonic segment where CCA had been diagnosed within a mean follow-up time of 27 months. No follow-up information was available in the remaining one patient. When diagnoses were grouped as NEG or 'atypical' (including IND, LGD or HGD), the overall agreement rate of 76% (kappa = 0.51) based on morphology alone improved to 90% (kappa = 0.81) with knowledge of FCM results. Even when categorised as NEG or dysplasia (LGD or HGD) with each of the IND diagnoses reclassified into three categories (NEG, LGD or HGD) based on the degree of suspicion for dysplasia, the overall agreement rate of 63% (kappa = 0.25) based on morphology alone improved to 73% (kappa = 0.46) with knowledge of FCM results. However, when grouped as NEG, LGD or HGD, the overall agreement rate was less than 40% (kappa < 0.09) regardless of knowledge of FCM results. CONCLUSIONS: The presence of aneuploidy, p53 positivity and development of HGD or adenocarcinoma on follow-up indicate that CCA likely represents a dysplastic lesion (at least LGD) and is a histological marker of neoplastic progression. Although the grading of CCA, largely based on cytological abnormalities, is subject to significant interobserver variability, CCA can be histologically identified and should lead to a recommendation of increased endoscopic surveillance, especially if aneuploidy is detected.

Histology of portal vascular changes associated with idiopathic non-cirrhotic portal hypertension: nomenclature and definition.

Idiopathic non-cirrhotic portal hypertension (INCPH) is a rare vascular liver disease that has attracted new interest in recent years. It is characterised by clinical signs of portal hypertension in the absence of cirrhosis or severe fibrosis and any known cause of portal hypertension. As much uncertainty exists about INCPH pathophysiology, and no definite diagnostic tests are available, liver biopsy is an essential tool for achieving a definite diagnosis. Unfortunately, the histological diagnosis of INCPH is not always straightforward, as the characteristic lesions are unevenly distributed, vary greatly in their severity, are often very subtle, and are not all necessarily present in a single case. Furthermore, specifically for the characteristic portal vessel changes observed in INCPH, the terminology and definition are ambiguous, which adds complexity to the already complex clinicopathological scenario. An international study group of liver pathologists and hepatologists pursued a consensus on nomenclature for the portal vascular lesions of INCPH. Such standardisation may assist pathologists in the recognition of such lesions, and will possibly facilitate further advancement in this field.

Computational Discovery of Niclosamide Ethanolamine, a Repurposed Drug Candidate That Reduces Growth of Hepatocellular Carcinoma Cells In Vitro and in Mice by Inhibiting Cell Division Cycle 37 Signaling.

BACKGROUND & AIMS: Drug repositioning offers a shorter approval process than new drug development. We therefore searched large public datasets of drug-induced gene expression signatures to identify agents that might be effective against hepatocellular carcinoma (HCC). METHODS: We searched public databases of messenger RNA expression patterns reported from HCC specimens from patients, HCC cell lines, and cells exposed to various drugs. We identified drugs that might specifically increase expression of genes that are down-regulated in HCCs and reduce expression of genes up-regulated in HCCs using a nonparametric, rank-based pattern-matching strategy based on the Kolmogorov-Smirnov statistic. We evaluated the anti-tumor activity of niclosamide and its ethanolamine salt (NEN) in HCC cell lines (HepG2, Huh7, Hep3B, Hep40, and PLC/PRF/5), primary human hepatocytes, and 2 mouse models of HCC. In one model of HCC, liver tumor development was induced by hydrodynamic delivery of a sleeping beauty transposon expressing an activated form of Ras (v12) and truncated ß-catenin (N90). In another mouse model, patient-derived xenografts were established by implanting HCC cells from patients into livers of immunocompromised mice. Tumor growth was monitored by bioluminescence imaging. Tumor-bearing mice were fed a regular chow diet or a chow diet containing niclosamide or NEN. In a separate experiment using patient-derived xenografts, tumor-bearing mice were given sorafenib (the standard of care for patients with advanced HCC), NEN, or niclosamide alone; a combination of sorafenib and NEN; or a combination sorafenib and niclosamide in their drinking water, or regular water (control), and tumor growth was monitored. RESULTS: Based on gene expression signatures, we identified 3 anthelmintics that significantly altered the expression of genes that are up- or down-regulated in HCCs. Niclosamide and NEN specifically reduced the viability of HCC cells: the agents were at least 7-fold more cytotoxic to HCCs than primary hepatocytes. Oral administration of NEN to mice significantly slowed growth of genetically induced liver tumors and patient-derived xenografts, whereas niclosamide did not, coinciding with the observed greater bioavailability of NEN compared with niclosamide. The combination of NEN and sorafenib was more effective at slowing growth of patient-derived xenografts than either agent alone. In HepG2 cells and in patient-derived xenografts, administration of niclosamide or NEN increased expression of 20 genes down-regulated in HCC and reduced expression of 29 genes up-regulated in the 274-gene HCC signature. Administration of NEN to mice with patient-derived xenografts reduced expression of proteins in the Wnt-ß-catenin, signal transducer and activator of transcription 3, AKT-mechanistic target of rapamycin, epidermal growth factor receptor-Ras-Raf signaling pathways. Using immunoprecipitation assays, we found NEN to bind cell division cycle 37 protein and disrupt its interaction with heat shock protein 90. CONCLUSIONS: In a bioinformatics search for agents that alter the HCC-specific gene expression pattern, we identified the anthelmintic niclosamide as a potential anti-tumor agent. Its ethanolamine salt, with greater bioavailability, was more effective than niclosamide at slowing the growth of genetically induced liver tumors and patient-derived xenografts in mice. Both agents disrupted interaction between cell division cycle 37 and heat shock protein 90 in HCC cells, with concomitant inhibition of their downstream signaling pathways. NEN might be effective for treatment of patients with HCC.