Characterization of acute-on-chronic liver diseases: A multicenter prospective cohort study.

BACKGROUND: Acute-on-chronic liver disease (AoCLD) accounts for the majority of patients hospitalized in the Department of Hepatology or Infectious Diseases. AIM: To explore the characterization of AoCLD to provide theoretical guidance for the accurate diagnosis and prognosis of AoCLD. METHODS: Patients with AoCLD from the Chinese Acute-on-Chronic Liver Failure (ACLF) study cohort were included in this study. The clinical characteristics and outcomes, and the 90-d survival rate associated with each clinical type of AoCLD were analyzed, using the Kaplan-Meier method and the log-rank test. RESULTS: A total of 3375 patients with AoCLD were enrolled, including 1679 (49.7%) patients with liver cirrhosis acute decompensation (LC-AD), 850 (25.2%) patients with ACLF, 577 (17.1%) patients with chronic hepatitis acute exacerbation (CHAE), and 269 (8.0%) patients with liver cirrhosis active phase (LC-A). The most common cause of chronic liver disease (CLD) was HBV infection (71.4%). The most common precipitants of AoCLD was bacterial infection (22.8%). The 90-d mortality rates of each clinical subtype of AoCLD were 43.4% (232/535) for type-C ACLF, 36.0% (36/100) for type-B ACLF, 27.0% (58/215) for type-A ACLF, 9.0% (151/1679) for LC-AD, 3.0% (8/269) for LC-A, and 1.2% (7/577) for CHAE. CONCLUSION: HBV infection is the main cause of CLD, and bacterial infection is the main precipitant of AoCLD. The most common clinical type of AoCLD is LC-AD. Early diagnosis and timely intervention are needed to reduce the mortality of patients with LC-AD or ACLF.

AcHZP45 is a repressor of chlorophyll biosynthesis and activator of chlorophyll degradation in kiwifruit.

The degradation of chlorophyll during fruit development is essential to reveal a more 'ripe' color that signals readiness to wild dispersers of seeds and the human consumer. Here, comparative biochemical analysis of developing fruit of Actinidia deliciosa cv. Xuxiang ('XX', green-fleshed) and Actinidia chinensis cv. Jinshi No.1 ('JS', yellow-fleshed) indicated that variation in chlorophyll content is the major contributor to differences in flesh color. Four differentially expressed candidate genes were identified: the down-regulated genes AcCRD1 and AcPOR1 involved in chlorophyll biosynthesis, and the up-regulated genes AcSGR1 and AcSGR2 driving chlorophyll degradation. Prochlorophyllide and chlorophyllide, the metabolites produced by AcCRD1 and AcPOR1, progressively reduced in 'JS', but not in 'XX', indicating that chlorophyll biosynthesis was less active in yellow-fleshed fruit. AcSGR1 and AcSGR2 were verified to be involved in chlorophyll degradation, using both transient expression in tobacco and stable overexpression in kiwifruit. Furthermore, a homeobox-leucine zipper (HD-Zip II), AcHZP45, showed significantly increased expression during 'JS' fruit ripening, which led to both repressed expression of AcCRD1 and AcPOR1 and activated expression of AcSGR1 and AcSGR2. Collectively, the present study indicated that different dynamics of chlorophyll biosynthesis and degradation coordinate the changes in chlorophyll content in kiwifruit flesh, which are orchestrated by the key transcription factor AcHZP45.

Nore1 inhibits age-associated myeloid lineage skewing and clonal hematopoiesis but facilitates termination of emergency (stress) granulopoiesis.

Age-associated bone marrow changes include myeloid skewing and mutations that lead to clonal hematopoiesis. Molecular mechanisms for these events are ill defined, but decreased expression of Irf8/Icsbp (interferon regulatory factor 8/interferon consensus sequence binding protein) in aging hematopoietic stem cells may contribute. Irf8 functions as a leukemia suppressor for chronic myeloid leukemia, and young Irf8-/- mice have neutrophilia with progression to acute myeloid leukemia (AML) with aging. Irf8 is also required to terminate emergency granulopoiesis during the innate immune response, suggesting this may be the physiologic counterpart to leukemia suppression by this transcription factor. Identifying Irf8 effectors may define mediators of both events and thus contributors to age-related bone marrow disorders. In this study, we identified RASSF5 (encoding Nore1) as an Irf8 target gene and investigated the role of Nore1 in hematopoiesis. We found Irf8 activates RASSF5 transcription and increases Nore1a expression during emergency granulopoiesis. Similar to Irf8-/- mice, we found that young Rassf5-/- mice had increased neutrophils and progressed to AML with aging. We identified enhanced DNA damage, excess clonal hematopoiesis, and a distinct mutation profile in hematopoietic stem cells from aging Rassf5-/- mice compared with wildtype. We found sustained emergency granulopoiesis in Rassf5-/- mice, with repeated episodes accelerating AML, also similar to Irf8-/- mice. Identifying Nore1a downstream from Irf8 defines a pathway involved in leukemia suppression and the innate immune response and suggests a novel molecular mechanism contributing to age-related clonal myeloid disorders.

A dramatic decline in fruit citrate induced by mutagenesis of a NAC transcription factor, AcNAC1.

Citrate is a common primary metabolite which often characterizes fruit flavour. The key regulators of citrate accumulation in fruit and vegetables are poorly understood. We systematically analysed the dynamic profiles of organic acid components during the development of kiwifruit (Actinidia spp.). Citrate continuously accumulated so that it became the predominate contributor to total acidity at harvest. Based on a co-expression network analysis using different kiwifruit cultivars, an Al-ACTIVATED MALATE TRANSPORTER gene (AcALMT1) was identified as a candidate responsible for citrate accumulation. Electrophysiological assays using expression of this gene in Xenopus oocytes revealed that AcALMT1 functions as a citrate transporter. Additionally, transient overexpression of AcALMT1 in kiwifruit significantly increased citrate content, while tissues showing higher AcALMT1 expression accumulated more citrate. The expression of AcALMT1 was highly correlated with 17 transcription factor candidates. However, dual-luciferase and EMSA assays indicated that only the NAC transcription factor, AcNAC1, activated AcALMT1 expression via direct binding to its promoter. Targeted CRISPR-Cas9-induced mutagenesis of AcNAC1 in kiwifruit resulted in dramatic declines in citrate levels while malate and quinate levels were not substantially affected. Our findings show that transcriptional regulation of a major citrate transporter, by a NAC transcription factor, is responsible for citrate accumulation in kiwifruit, which has broad implications for other fruits and vegetables.

The neutrophil-to-lymphocyte ratio represents a systemic inflammation marker and reflects the relationship with 90-day mortality in non-cirrhotic chronic severe hepatitis.

OBJECTIVES: To investigate the relationship between systemic inflammatory response and short-term mortality in patients with non-cirrhotic chronic severe hepatitis (CSH) by using several indicators of inflammation including neutrophil-to-lymphocyte ratio (NLR), neutrophil (NEU), white blood cell (WBC), platelet-to lymphocyte ratio (PLR), and monocyte-to-lymphocyte ratio (MLR). METHODS: Data were collected from two prospectively enrolled CATCH-LIFE noncirrhotic cohorts. Cox regression analysis was used to investigate the association between systemic inflammatory biomarkers and 90-day liver transplant (LT)-free mortality. A generalized additive model (GAM) was used to illustrate the quantitative curve relationship between NLR and 90-day LT-free mortality. Kaplan-Meier method was used to estimate the 90-year LT-free survival. RESULTS: The prevalence of CSH was 20.5% (226/1103). The 28-day and 90-day LT-free mortality rates were 17.7% and 26.1%, respectively, for patients with non-cirrhotic CSH. Patients with no infection accounted for 75.0% of all CSH patients, and NLR was independently associated with 90-day LT-free mortality. NLR of 2.9 might be related to disease deterioration in CSH patients without infection. CONCLUSIONS: NLR may be an independent risk factor for 90-day LT-free mortality in patients with non-cirrhotic chronic liver disease. A NLR of 2.9 as the cut-off value can be used to predict disease aggravation in CSH patients without infection.

Prevalence and clinical characteristics of autoimmune liver disease in hospitalized patients with cirrhosis and acute decompensation in China.

BACKGROUND: Autoimmune liver disease (AILD) has been considered a relatively uncommon disease in China, epidemiological data for AILD in patients with cirrhosis and acute decompensation (AD) is sparse. AIM: To investigate the prevalence, outcome and risk factors for AILD in cirrhotic patients complicated with AD in China. METHODS: We collected data from patients with cirrhosis and AD from two prospective, multicenter cohorts in hepatitis B virus endemic areas. Patients were regularly followed up at the end of 28-d, 90-d and 365-d, or until death or liver transplantation (LT). The primary outcome in this study was 90-d LT-free mortality. Acute-on-chronic liver failure (ACLF) was assessed on admission and during 28-d hospitalization, according to the diagnostic criteria of the European Association for the Study of the Liver (EASL). Risk factors for death were analyzed with logistic regression model. RESULTS: In patients with cirrhosis and AD, the overall prevalence of AILD was 9.3% (242/2597). Prevalence of ACLF was significantly lower in AILD cases (14%) than those with all etiology groups with cirrhosis and AD (22.8%) (P < 0.001). Among 242 enrolled AILD patients, the prevalence rates of primary biliary cirrhosis (PBC), autoimmune hepatitis (AIH) and PBC-AIH overlap syndrome (PBC/AIH) were 50.8%, 28.5% and 12.0%, respectively. In ACLF patients, the proportions of PBC, AIH and PBC/AIH were 41.2%, 29.4% and 20.6%. 28-d and 90-d mortality were 43.8% and 80.0% in AILD-related ACLF. The etiology of AILD had no significant impact on 28-d, 90-d or 365-d LT-free mortality in patients with cirrhosis and AD in both univariate and multivariate analysis. Total bilirubin (TB), hepatic encephalopathy (HE) and blood urea nitrogen (BUN) were independent risk factors for 90-d LT-free mortality in multivariate analysis. The development of ACLF during hospitalization only independently correlated to TB and international normalized ratio. CONCLUSION: AILD was not rare in hospitalized patients with cirrhosis and AD in China, among which PBC was the most common etiology. 90-d LT-free mortality were independently associated with TB, HE and BUN.

An unusual indole-diterpenoid with C-17 norcassane skeleton from Euphorbia fischeriana induces HEL cell cycle arrest and apoptosis.

Two new polycyclic diterpenoids, euphkanoids H and I (1 and 2), along with 6 known analogues (2-8) were isolated from the roots of Euphorbia fischeriana, a traditional Chinese medicine. Their structures were identified by spectral methods, and the absolute configurations of 1 and 2 were determined by ECD calculation and single crystal X-ray diffraction, respectively. Compound 1 represents the first example of C-17 norcassane indole-diterpenes. All the isolates were screened for antiproliferative activity against a panel of human cancer cell lines using the MTT assay, and 1 showed significant cytotoxicity against HEL cells (IC50 = 3.2 µM). Simple mechanistic study revealed that 1 could induce cell cycle arrest at G0/G1 phase and apoptosis in HEL cells.

The red flesh of kiwifruit is differentially controlled by specific activation-repression systems.

Anthocyanins are visual cues for pollination and seed dispersal. Fruit containing anthocyanins also appeals to consumers due to its appearance and health benefits. In kiwifruit (Actinidia spp.) studies have identified at least two MYB activators of anthocyanin, but their functions in fruit and the mechanisms by which they act are not fully understood. Here, transcriptome and small RNA high-throughput sequencing were used to comprehensively identify contributors to anthocyanin accumulation in kiwifruit. Stable overexpression in vines showed that both 35S::MYB10 and MYB110 can upregulate anthocyanin biosynthesis in Actinidia chinensis fruit, and that MYB10 overexpression resulted in anthocyanin accumulation which was limited to the inner pericarp, suggesting that repressive mechanisms underlie anthocyanin biosynthesis in this species. Furthermore, motifs in the C-terminal region of MYB10/110 were shown to be responsible for the strength of activation of the anthocyanic response. Transient assays showed that both MYB10 and MYB110 were not directly cleaved by miRNAs, but that miR828 and its phased small RNA AcTAS4-D4(-) efficiently targeted MYB110. Other miRNAs were identified, which were differentially expressed between the inner and outer pericarp, and cleavage of SPL13, ARF16, SCL6 and F-box1, all of which are repressors of MYB10, was observed. We conclude that it is the differential expression and subsequent repression of MYB activators that is responsible for variation in anthocyanin accumulation in kiwifruit species.

[Establishment of primary liver cancer model in mice].

Objective: Three modeling methods were used to establish a mouse primary liver cancer model, and compared them to find a more optimal modeling method. Methods: Forty 15-day-old C3H/HeN male mice were randomly divided into groups I-IV, 10 mice in each group. Group Ⅰ were not treated; Group Ⅱ were intraperitoneally injected with 25 mg/kg diethylnitrosamine (DEN) once; Group Ⅲ were intraperitoneally injected with 100 mg/kg DEN once; Group Ⅳ were intraperitoneally injected with 25 mg/kg DEN once and followed by another intraperitoneal injection of 100 mg/kg DEN at 42 days of age. The mortality of mice in each group was analyzed. At the 18th week of modeling, blood was collected from eyeballs after anesthesia, and liver was taken from abdominal cavity after neck was broken. The appearance of liver, the number of cancer nodules and the incidence of liver tumor were observed. The histopathological changes of liver were observed by HE staining. The serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were detected. Results: At the 18th week of modeling, compared with the group I, serum levels of ALT and AST in groups II-IV were increased significantly (P＜0.05); The number of cancer nodules and the incidence of tumors in the surviving mice of groups III and IV were also increased significantly (P＜0.05). At the 18th week of modeling, no mice died in both groups I and II, and the incidence of liver cancer was 0%; The incidence of liver cancer in surviving mice in both groups III and IV was 100%, but the mortality rate of mice in group III was as high as 50%, and that in group IV was only 20%. Conclusion: C3H/HeN male mice can successfully establish a mouse liver cancer model by intraperitoneal injection of 25 mg/kg of DEN once at the age of 15 days and another intraperitoneal injection of 100 mg/kg of DEN once at the age of 42 days with short cycle and low mortality, which is an ideal method to establish a primary liver cancer model.

Plasma Exchange-Based Non-bioartificial Liver Support System Improves the Short-Term Outcomes of Patients With Hepatitis B Virus-Associated Acute-on-Chronic Liver Failure: A Multicenter Prospective Cohort Study.

Background and aims: Hepatitis B virus-associated acute-on-chronic liver failure (HBV-ACLF) is a complicated syndrome with extremely high short-term mortality. Whether plasma exchange (PE) improves HBV-ACLF outcomes remains controversial. Here, PE-based non-bioartificial liver support system (NB-ALSS) effects on short-term HBV-ACLF patient outcomes were investigated. Materials and methods: HBV-ACLF patients from Chinese Acute-on-chronic Liver Failure (CATCH-LIFE) cohort receiving standard medical therapy (SMT) alone or PE-based NB-ALSS in addition to SMT were allocated to SMT and SMT+PE groups, respectively; propensity score matching (PSM) was used to eliminate confounding bias. Short-term (28/90-day and 1-year) survival rates were calculated (Kaplan-Meier). Results: In total, 524 patients with HBV-ACLF were enrolled in this study; 358 received SMT alone (SMT group), and the remaining 166 received PE-based NB-ALSS in addition to SMT (SMT+PE group). PSM generated 166 pairs of cases. In the SMT+PE group, 28-day, 90-day, and 1-year survival rates were 11.90, 8.00, and 10.90%, respectively, higher than those in the SMT group. Subgroup analysis revealed that PE-based NB-ALSS had the best efficacy in patients with ACLF grade 2 or MELD scores of 30-40 (MELD grade 3). In MELD grade 3 patients who received SMT+PE, 28-day, 90-day, and 1-year survival rates were improved by 18.60, 14.20, and 20.10%, respectively. According to multivariate Cox regression analysis, PE-based NB-ALSS was the only independent protective factor for HBV-ACLF patient prognosis at 28 days, 90 days, and 1 year (28 days, HR = 0.516, p = 0.001; 90 days, HR = 0.663, p = 0.010; 1 year, HR = 0.610, p = 0.051). For those who received SMT+PE therapy, PE-based NB-ALSS therapy frequency was the only independent protective factor for short-term prognosis (28-day, HR = 0.597, p = 0.001; 90-day, HR = 0.772, p = 0.018). Conclusions: This multicenter prospective study showed that the addition of PE-based NB-ALSS to SMT improves short-term (28/90 days and 1-year) outcomes in patients with HBV-ACLF, especially in MELD grade 3 patients. Optimization of PE-based NB-ALSS may improve prognosis or even save lives among HBV-ACLF patients.

Symptoms Based on Deficiency Syndrome in Traditional Chinese Medicine Might Be Predictor of Frailty in Elderly Community Dwellers.

BACKGROUND: The most widely used frailty phenotype and frailty indexes are either time-consuming or complicated, thus restricting their generalization in clinical practice; and therefore, an easier and faster screening tool is needed to be developed. OBJECTIVE: To select sensitive symptoms in traditional Chinese medicine (TCM) and study whether they can improve the risk prediction of frailty. METHODS: This is a cross-sectional study enrolling 2249 Chinese elderly community dwellers. Data were collected via face-to-face inquiries, anthropometric measurements, laboratory tests, and community health files. Frailty was the main outcome measure, and it was evaluated by Fried's frailty phenotype (FP). The ordinal logistic regression model was used to identify the factors associated with frailty. The risk assessment plot was used to compare the discriminative ability for frailty among models with and without TCM symptoms. RESULTS: The identified sensitive influential factors for frailty included age, education level, dietary habits, chronic obstructive pulmonary disease, diabetes, cerebral infarction, osteoporosis, cold limbs, lethargy and laziness in speaking and moving, weakness of lower limbs, slow movement, dry mouth and throat, and glazed expression. The risk prediction for "frailty cumulative components ≥1" was not significantly increased, while for "frailty cumulative components ≥2", a new model developed with the above selected TCM symptoms had a higher AUC than the baseline model without it (0.79 VS 0.81, P=0.002). And the NRI and IDI for the new model were 41.4% (P=0.016) and 0.024% (P=0.041), respectively. CONCLUSION: This research might provide an easier and faster way for early identification and risk prediction of frailty in elderly community dwellers.

An ethylene-hypersensitive methionine sulfoxide reductase regulated by NAC transcription factors increases methionine pool size and ethylene production during kiwifruit ripening.

Ethylene plays an important role in regulating fruit ripening by triggering dynamic changes in expression of ripening-associated genes, but the functions of many of these genes are still unknown. Here, a methionine sulfoxide reductase gene (AdMsrB1) was identified by transcriptomics-based analysis as the gene most responsive to ethylene treatment in ripening kiwifruit. The AdMsrB1 protein exhibits a stereospecific activity toward the oxidative stress-induced R enantiomer of methionine sulfoxide (MetSO), reducing it to methionine (Met). Stable overexpression of AdMsrB1 in kiwifruit significantly increased the content of free Met and 1-aminocyclopropane-1-carboxylic acid (ACC), the immediate precursor of ethylene, and increased ethylene production. Dual-luciferase assays indicated that the AdMsrB1 promoter was not directly upregulated by ethylene treatment but was modulated by two ethylene-inducible NAM/ATAF/CUC transcription factors (AdNAC2 and AdNAC72) that bind directly to the AdMsrB1 promoter. Overexpression of AdNAC72 in kiwifruit not only enhanced AdMsrB1 expression, but also increased free Met and ACC content and ethylene production rates. This finding establishes an unexpected regulatory loop that enhances ethylene production and the concentration of its biosynthetic intermediates.

Prevalence and Clinical Significance of Portal Vein Thrombosis in Patients With Cirrhosis and Acute Decompensation.

BACKGROUND & AIMS: Portal vein thrombosis (PVT) is a common and serious complication in patients with cirrhosis. However, little is known about PVT in patients with cirrhosis and acute decompensation (AD). We investigated the prevalence and clinical significance of PVT in nonmalignant patients with cirrhosis and AD. METHODS: We performed a retrospective study of 2 cohorts of patients with acute exacerbation of chronic liver disease who participated in the Chinese AcuTe on CHronic LIver FailurE study, established by the Chinese Chronic Liver Failure Consortium, from January 2015 through December 2016 (n = 2600 patients) and July 2018 through January 2019 (n = 1370 patients). We analyzed data on the prevalence, clinical manifestations, and risk factors of PVT from 2826 patients with cirrhosis, with and without AD. RESULTS: The prevalence of PVT in patients with cirrhosis and AD was 9.36%, which was significantly higher than in patients with cirrhosis without AD (5.24%) (P = .04). Among patients with cirrhosis and AD, 63.37% developed PVT recently (the first detected PVT with no indication of chronic PVT). Compared with patients without PVT, a significantly higher proportion of patients with PVT had variceal bleeding (47.33% vs 19.63%; P < .001) and patients with PVT had a significantly higher median serum level of D-dimer (2.07 vs 1.25; P < .001). Splenectomy and endoscopic sclerotherapy were independent risk factors for PVT in patients with cirrhosis and AD. The 1-year mortality rate did not differ significantly between patients with vs without PVT. CONCLUSIONS: In an analysis of data from 2826 patients with cirrhosis, a significantly higher proportion of those with AD had PVT than those without AD. PVT was associated with increased variceal bleeding, which would increase the risk for AD. Strategies are needed to prevent PVT in patients with cirrhosis, through regular screening, to reduce portal hypertension. ClinicalTrials.gov no: NCT02457637 and NCT03641872.

Methyl Jasmonate Enhances Ethylene Synthesis in Kiwifruit by Inducing NAC Genes That Activate ACS1.

Cross-talk between various hormones is important in regulating many aspects of plant growth, development, and senescence, including fruit ripening. Here, exogenous ethylene (ETH, 100 µL/L, 12 h) rapidly accelerated 'Hayward' kiwifruit (Actinidia deliciosa) softening and ethylene production and was enhanced by supplementing with continuous treatment with methyl jasmonate (MeJA, 100 µM/L, 12 h) (ETH+MeJA). ETH+MeJA enhanced ACC synthase (ACS) activities and 1-aminocyclopropane-1-carboxylic acid (ACC) accumulation but not ACC oxidase (ACO) activity. Increased transcripts of ACS genes AdACS1 and AdACS2, ACS activity, and ethylene production were positively correlated. The abundance of AdACS1 was about 6-fold higher than AdACS2. RNA-seq identified 6 transcription factors among the 87 differentially expressed unigenes induced by ETH+MeJA. Dual-luciferase and electrophoretic mobility shift assays (EMSA) indicated that AdNAC2/3 physically interacted with and trans-activated the AdACS1 promoter 2.2- and 3.5-fold, respectively. Collectively, our results indicate that MeJA accelerates ethylene production in kiwifruit induced by exogenous ethylene, via a preferential activation of AdACS1 and AdACS2.

Pupillary light reaction in preclinical Alzheimer's disease subjects compared with normal ageing controls.

BACKGROUND/AIMS: We wished to determine whether the pupillary light reaction can differentiate preclinical Alzheimer's disease (AD) subjects from normal ageing controls. We performed a prospective study evaluating the pupillary light reaction in a cohort of well-characterised subjects with preclinical AD versus normal ageing controls. METHODS: We recruited 57 subjects from our institution's Memory and Aging Project, part of our Alzheimer's Disease Research Center. All subjects completed PET-PiB imaging, cerebrospinal fluid analysis and at least 1 neuropsychiatric assessment after their baseline assessment. All participants were assigned a clinical dementia rating and underwent a complete neuro-ophthalmic examination. Participants were divided into a dementia biomarker+ (preclinical AD) and biomarker- (normal ageing) group based on preclinical risk for Alzheimer's dementia. Pupillometry measurements were performed by using the NeurOptics PLR-200 Pupillometer. RESULTS: A total of 57 subjects were recruited with 24 dementia biomarker+ and 33 dementia biomarker- individuals. A variety of pupil flash response (PLR) parameters were assessed. Comparisons between groups were analysed using generalised estimating equations. None of the pupillary parameters showed a significant difference between groups. CONCLUSIONS: We found no significant differences in PLR between preclinical AD subjects and normal ageing controls. This suggests that the disease effect on the PLR may be small and difficult to detect at the earliest stages of the disease. Future studies could include larger sample size and chromatic pupillometry.

Inhibition of Fas associated phosphatase 1 (Fap1) facilitates apoptosis of colon cancer stem cells and enhances the effects of oxaliplatin.

Fas associated phosphatase 1 (Fap1) is a ubiquitously expressed protein tyrosine phosphatase. Fap1 substrates include Fas and Gsk3ß, suggesting a role in regulating cell survival. Consistent with this, increased Fap1 expression is associated with resistance to Fas or platinum induced apoptosis in some human colon cancer tumors or cell lines. In the current studies, we found that Fap1 expression was significantly greater in CD133+ colon cancer stem cells compared to CD133- tumor cells. PTPN13 promoter activity (encoding Fap1) was repressed by interferon regulatory factor 2 (irf2), and expression of Fap1 and Irf2 were inversely correlated in CD133+ or CD133- colon cancer cells. We determined that CD133+ cells were relatively resistant to Fas or oxaliplatin induced apoptosis, but this was reversed by Fap1-knockdown or a Fap1-blocking tripeptide (SLV). In a murine xenograft model of colon cancer, we found treatment with SLV peptide significantly decreased tumor growth and relative abundance of CD133+CD44+ cells; associated with increased phosphorylation of Fap1 substrates. SLV peptide also enhanced inhibitory effects of oxaliplatin on tumor growth and Fap1 substrate phosphorylation in this model. Our studies suggest that therapeutically targeting Fap1 may decrease persistence of colon cancer stem cells during treatment with platinum chemotherapy by activating Fap1 substrates. In a murine model of chronic myeloid leukemia, we previously determined that inhibition of Fap1 decreased persistence of leukemia stem cells during tyrosine kinase inhibitor treatment. Therefore, Fap1 may be a tissue agnostic target to increase apoptosis in malignant stem cells.

Four Natural Compounds Separated from Folium Isatidis: Crystal Structures and Antibacterial Activity.

Four natural compounds were obtained by concentrating, separating and purifying from the Folium isatidis. These natural compounds have been characterized by elemental analysis, IR spectrum, NMR and single-crystal X-ray diffraction analysis. The results show that these natural compounds are 4(3H)-quinazolinone (I), 2,4(1H,3H)-quinazolinedione (II), methyl 3,4-dihydro-4-oxoquinazoline-2-carboxylate (III) and ethyl 3,4-dihydro-4-oxoquinazoline-2-carboxylate (IV). The antibacterial activity experiment showed that I and II had better activity against Staphylococcus aureus, Escherichia coli, Bacillus subtilis and Salmonella than III, IV and other multiple components, because III and IV have long branches and steric hindrance effect. Compounds I and II have planar structure, which can more easily combine with these bacteria and kill them. The above results have good guiding significance for studying the antibacterial activity for single components or mixtures from natural origin.

TP53 Haploinsufficiency Rescues Emergency Granulopoiesis in FANCC-/- Mice.

Emergency (stress) granulopoiesis is an episodic process for the production of granulocytes in response to infectious challenge. We previously determined that Fanconi C, a component of the Fanconi DNA-repair pathway, is necessary for successful emergency granulopoiesis. Fanconi anemia results from mutation of any gene in this pathway and is characterized by bone marrow failure (BMF) in childhood and clonal progression in adolescence. Although murine Fanconi anemia models exhibit relatively normal steady-state hematopoiesis, FANCC-/- mice are unable to mount an emergency granulopoiesis response. Instead, these mice develop BMF and die during repeated unsuccessful emergency granulopoiesis attempts. In FANCC-/- mice, BMF is associated with extensive apoptosis of hematopoietic stem and progenitor cells through an undefined mechanism. In this study, we find that TP53 haploinsufficiency completely rescues emergency granulopoiesis in FANCC-/- mice and protects them from BMF during repeated emergency granulopoiesis episodes. Instead, such recurrent challenges accelerated clonal progression in FANCC-/-TP53+/- mice. In FANCC-/- mice, BMF during multiple emergency granulopoiesis attempts was associated with increased ataxia telangiectasia and Rad3-related protein (Atr) and p53 activation with each attempt. In contrast, we found progressive attenuation of expression and activity of Atr, and consequent p53 activation and apoptosis, in the bone marrow of FANCC-/-TP53+/- mice during this process. Therefore, activation of Atr-with consequent Fanconi-mediated DNA repair or p53-dependent apoptosis-is an essential component of emergency granulopoiesis and it protects the bone marrow from genotoxic stress during this process.

Stat3 and CCAAT enhancer-binding protein ß (C/ebpß) activate Fanconi C gene transcription during emergency granulopoiesis.

Interferon consensus sequence-binding protein (Icsbp) is required for terminating emergency granulopoiesis, an episodic event responsible for granulocyte production in response to infections and a key component of the innate immune response. Icsbp inhibits the expression of Stat3 and C/ebpß, transcription factors essential for initiating and sustaining granulopoiesis, and activates transcription of Fanconi C (FANCC), a DNA repair protein. In prior studies, we noted accelerated bone marrow failure in Fancc-/- mice undergoing multiple episodes of emergency granulopoiesis, associated with apoptosis of bone marrow cells with unrepaired DNA damage. Additionally, we found increased expression of Fanconi C and F proteins during emergency granulopoiesis. These findings suggest that Icsbp protects the bone marrow from DNA damage by increasing activity of the Fanconi DNA repair pathway, but the mechanisms for FANCC activation during initiation of emergency granulopoiesis are unclear. In this study, we observed that Stat3 and C/ebpß activate FANCC transcription and contribute to DNA repair. Our findings indicate that FancC expression is increased during Stat3- and C/ebpß-induced initiation of emergency granulopoiesis by these transcription factors and is maintained through termination by Icsbp. Our work reveals that Stat3- and C/ebpß-mediated FancC expression is a critical component for initiating and sustaining key innate immune responses.

The E3 ubiquitin ligase Triad1 influences development of Mll-Ell-induced acute myeloid leukemia.

Chromosomal translocations involving the MLL1 gene characterize a poor prognosis subset of acute myeloid leukemia (AML), referred to as 11q23-AML. Transcription of the HOXA9 and HOXA10 genes is enhanced in hematopoietic stem and progenitor cells in these leukemias. We previously found the ARIH2 gene was repressed by HoxA9 in myeloid progenitors, but activated by HoxA10 during granulopoiesis. ARIH2 encodes the Triad1 protein, an anti-proliferative E3 ubiquitin ligase. In the current study, we investigate the role of Triad1 in leukemogenesis induced by an MLL1 fusion protein (Mll-Ell). We found Mll-Ell increased expression of HoxA9, HoxA10, and Triad1 because HoxA9 represses only one of two ARIH2 cis elements that are activated by HoxA10. Although Triad1 antagonized the generally pro-proliferative effects of the Mll-Ell oncoprotein, we found blocking HoxA9 and HoxA10 phosphorylation shifted the balance to ARIH2 repression in Mll-Ell+ cells. We investigated the significance of these in vitro results in a murine bone marrow transplant model. We found Triad1 knockdown significantly shortened the latency to development of AML in mice transplanted with Mll-Ell-transduced bone marrow. And, Triad1 expression fell during the prolonged AML latency period in mice transplanted with bone marrow expressing Mll-Ell alone. Our studies identify Triad1 as a leukemia suppressor in 11q23-AML. This suggests defining relevant Triad1 substrates may indicate novel therapeutic targets in this disease.