Emergency ABO-incompatible (ABO-I) liver transplantations (LTx) have been performed increasingly to treat severe liver failure. Herein, we report a case of severe hepatic necrosis after ABO-I LTx. A 53-year-old man with blood group O was diagnosed as having severe hepatitis B and acute-on-chronic liver failure, and underwent an emergency liver transplantation implanting a blood-group-B liver from a cardiac-death donor. A routine anti-rejection, anti-infection and anti-virus therapy was given after operation. On post-operative day (POD) 16, the recipient had fever and erythra. Laboratory and radiographic examinations suggested a severe hepatic necrosis of unknown causes. The patient was managed with a 10-d methylprednisolone pulse therapy. He was discharged on POD 35 with stable condition, and no recurrent disease was found during the follow-up.
Liver transplantations; ABO-incompatible; Hepatic necrosis; Graft rejection; Pulse therapy
The DNA methylation inhibitor 5-aza-2′-deoxycytidine (5-aza-CdR) is widely used as an anticancer drug for the treatment of leukemia and solid tumors. Gastric cancer (GC) patients who were positive for caudal type homeobox transcription factor 2 (CDX2) expression showed a higher survival rate compared with those who were CDX2 negative, which suggests that CDX2 performs a tumor suppressor role. However, the molecular mechanisms leading to the inactivation of CDX2 remain unclear. In the present study we demonstrated that the expression levels of CDX2 and DNA methyltransferase enzyme 1 (DNMT1) mRNA were significantly higher in GC compared with distal non-cancerous tissue. The expression of CDX2 mRNA was significantly correlated with Lauren classification, TNM stage and lymph node metastasis. DNMT1 mRNA expression was significantly correlated with TNM stage, pathological differentiation and lymph node metastasis. The expression of CDX2 mRNA was inversely correlated with that of DNMT1 mRNA in GC. Hypermethylation of the CDX2 gene promoter region, extremely low expression levels of CDX2 mRNA and no expression of CDX2 protein were the characteristics observed in MKN-45 and SGC-7901 GC cell lines. Following the treatment of MKN-45 cells with 5-aza-CdR, the hypermethylated CDX2 gene promoter region was demethylated and expression of CDX2 was upregulated, while DNMT1 expression was downregulated. Furthermore, a concentration- and time-dependent growth inhibition as well as increased apoptosis were observed. Caspase-3, −8 and −9 activities increased in a concentration-dependent manner following exposure to different concentrations of 5-aza-CdR. Therefore, our data show that the overexpression of DNMT1 and methylation of the CDX2 gene promoter region is likely to be responsible for CDX2 silencing in GC. 5-Aza-CdR may effectively induce re-expression of the CDX2 gene, inhibit cell proliferation and enhance the caspase-independent apoptosis of MKN-45 cells in vitro.
gastric neoplasms; caudal type homeobox transcription factor 2; DNA methylation; DNA methyltransferase enzyme 1; proliferation; apoptosis
The PTEN tumor suppressor gene is frequently inactivated in human prostate cancer. Using Osr1 (odd skipped related 1)-Cre mice, we generated a novel conditional Pten knockout mouse strain, PtenLoxP:Osr1-Cre. Conditional biallelic and monoallelic Pten knockout mice were viable. Deletion of Pten expression was detected in the prostate of PtenLoxP/LoxP:Osr1-Cre mice as early as 2 weeks of age. Intriguingly, PtenLoxP/LoxP:Osr1-Cre mice develop high-grade prostatic intraepithelial neoplasms (PINs) with high penetrance as early as one-month of age, and locally invasive prostatic tumors after 12-months of age. PtenLoxP/+:Osr1-Cre mice show only mild oncogenic changes after 8-weeks of age. Castration of PtenLoxP/LoxP:Osr1-Cre mice shows no significant regression of prostate tumors, although a shift of androgen receptor (AR) staining from the nuclei to cytoplasm is observed in Pten null tumor cells of castrated mice. Enhanced Akt activity is observed in Pten null tumor cells of castrated PtenLoxP/LoxP:Osr1-Cre. This study provides a novel mouse model that can be used to investigate a primary role of Pten in initiating oncogenic transformation in the prostate and to examine other genetic and epigenetic changes that are required for tumor progression in the mouse prostate.
Clioquinol has been shown to have anticancer activity both in vitro and in vivo. The present study compared the cytotoxicity of clioquinol with six analogues, using human cancer cell lines. Of the analogues tested, 8-hydroxy-5-nitroquinoline (NQ) was the most toxic, with an IC50 that was 5-10 fold lower than other congeners. Its activity was enhanced by copper, but not zinc, and the use of a zinc-sensitive fluorophore showed that unlike clioquinol, NQ is not a zinc ionophore. NQ increased intracellular reactive oxygen species generation, an effect that was significantly enhanced by the addition of copper at levels that approximate those found in human plasma. NQ has been used in humans for the treatment of urinary infections. NQ is an 8-hydroxyquinoline derivative that is more potent than the halogenated 8-hydroxyquinolines, and may be less neurotoxic because it lacks zinc ionophore activity. NQ is another clinical used anti-microbial agent whose properties suggest that it may be useful in treating cancer.
5-amino-8-hydroxyquinoline; clioquinol; Raji; reactive oxygen species; cytotoxicity
This study aimed to review and analyse the computed tomography (CT) imaging results of frequently encountered developmental anomalies of the inferior vena cava (IVC). The underlying clinical significance was evaluated with reference to the relevant literature. CT images of patients who received abdominal or thoracic scanning between July 2009 and September 2011 were reviewed. Developmental anomalies observed in the IVC were identified and categorised. Images of the cases with typical anomalies were presented and their developmental mechanism, as well as clinical significance, was discussed. The most frequently encountered IVC developmental anomalies include the left vena cava, double vena cava, azygos continuation of the IVC, left circumaortic renal vein, left retroaortic renal vein and retrocaval ureter. The embryogenesis of the IVC is a complex process that results in various congenital anomalies. The developmental anomalies of the IVC are distinguished using a CT scan and have significant implications on clinical perspective.
anomaly; inferior vena cava; embryogenesis; cross-sectional imaging; computed tomography; posterior nutcracker phenomenon
Cardiovascular disease is the leading cause of mortality among peritoneal dialysis (PD) patients in Macao. Increased arterial stiffness determined by pulse wave velocity (PWV) has been established as an independent predictor of cardiovascular mortality in end-stage renal disease patients. The present study aims to investigate the relationship between arterial stiffness and its associated risk factors in chronic PD patients.
A total of 96 chronic PD patients (48 males/48 females) were included in the cross-sectional study. Arterial stiffness was assessed by brachial-ankle PWV (baPWV). Patients were divided into two subgroups according to mean baPWV value. On enrollment, clinical characteristics and biochemical parameters were collected.
Compared with low baPWV group patients, high baPWV group patients were significant older (p<0.001) and more likely to have a high proportion of female gender (p=0.004) as well as previous CVD history (p=0.008). Serum albumin, pre-albumin levels and residual renal creatinine clearance (CCr) were significantly lower but the serum ferritin level was significantly higher in high baPWV group patients than in low baPWV group patients (all p<0.01). BaPWV was positively associated with age (r=0.534, p<0.001), Charlson comorbidity index (r=0.350, p<0.001) and serum ferritin level (r=0.340, p=0.001). Meanwhile, baPWV negatively correlated with serum albumin (r=−0.479, p<0.001), pre-albumin levels (r=−0.320, p=0.003) and residual renal CCr (r=−0.177, p=0.048). Age-adjusted partial correlation test found a significant correlation between baPWV and CRP (r=0.462, p<0.001). Multivariate regression analysis showed that baPWV was independently associated with age (p<0.001), serum albumin level (p=0.015), CRP (p=0.019) and residual renal CCr (p=0.045).
Arterial stiffness, assessed by baPWV, had an independent correlation with age, serum albumin level, CRP level and residual renal CCr among PD patients in Macao.
Arterial stiffness; Pulse wave velocity; Cardiovascular disease; Peritoneal dialysis
Rapid growth of the elderly peritoneal dialysis (PD) population is posing a special challenge for renal teams. Peripheral artery disease (PAD) has been reported to be an independent predictor of cardiovascular and all-cause mortality in hemodialysis patients. However, the prevalence and associated risk factors for PAD in elderly PD patients have not yet been fully investigated.
A total of 69 elderly PD patients were included in the present study. PAD was defined as either an ankle-brachial index < 0.9 or a history of intermittent claudication, lower-limb amputation, foot ulcers, or gangrene. On enrollment, clinical and biochemical characteristics were collected.
The overall prevalence of PAD was 31.9%. Compared with non-PAD patients, PAD patients were significantly older and more likely to be female and have longer PD duration and lower diastolic blood pressure (P < 0.001, = 0.002, 0.018, and 0.007, respectively). Serum albumin level (P < 0.001) and residual renal Kt/V value (P < 0.001) were significantly lower, but the serum C-reactive protein level (P = 0.005) was significantly higher, in PAD patients compared with non-PAD patients. Logistic regression analysis showed that serum albumin level (odds ratio = 1.485, P = 0.040) and residual renal Kt/V value (odds ratio = 1.725, P = 0.016) were independently associated with PAD.
A high prevalence of PAD appeared among elderly PD patients in Macao. Serum albumin level and residual renal Kt/V value were independently related to PAD.
ankle-brachial index; atherosclerosis; elderly; peripheral artery disease; peritoneal dialysis
The goal of this study was to investigate the relationship between resting-state functional connectivity and the severity of post-traumatic stress disorder (PTSD) symptoms in 15 people who developed PTSD following recent trauma. Fifteen participants who experienced acute traumatic events underwent a 7.3-min resting functional magnetic resonance imaging scan within 2 days post-event. All the patients were diagnosed with PTSD within 1 to 6 months after trauma. Brain areas in which activity was correlated with that of the posterior cingulate cortex (PCC) were assessed. To assess the relationship between the severity of PTSD symptoms and PCC connectivity, contrast images representing areas positively correlated with the PCC were correlated with the subject’s Clinician-Administered PTSD Scale scores (CAPS) when they were diagnosed. Furthermore, the PCC, medial prefrontal cortex and bilateral amygdala were selected to assess the correlation of the strength of functional connectivity with the CAPS. Resting state connectivity with the PCC was negatively correlated with CAPS scores in the left superior temporal gyrus and right hippocampus/amygdala. Furthermore, the strength of connectivity between the PCC and bilateral amygdala, and even between the bilateral amygdala could predict the severity of PTSD symptoms later. These results suggest that early altered resting-state functional connectivity of the PCC with the left superior temporal gyrus, right hippocampus and amygdala could predict the severity of the disease and may be a major risk factor that predisposes patients to develop PTSD.
Yolk sac tumor (endodermal sinus tumor) is a rare malignant germ cell tumor arising in the testis or ovary. Extragonadal yolk sac tumor is even rarer and has only been described in case reports. Due to the rarity of the tumors, the appropriately optimal treatment remains unclear. We report a case of yolk sac tumor in the seminal vesicle.
A 38-year-old Asian male presented with gross hematuria and hemospermia. Transrectal ultrasound scan showed a solid mass in the left seminal vesicle and the scrotal sonography showed no abnormalities. Bilateral seminal vesicles were resected, and histopathological examination showed a typical pattern of yolk sac tumor (YST). The patient responded poorly to comprehensive treatment of radiotherapy, chemotherapy and surgeries, developed systemic multiple metastases, and died of cachexia one and half years after diagnosis.
Yolk sac tumor; Seminal vesicle; Extragonadal
c-Met, a high-affinity receptor for Hepatocyte Growth Factor (HGF), plays a critical role in cancer growth, invasion and metastasis. Hepatocellular carcinoma (HCC) patients with an active HGF/c-Met signaling pathway have a significantly worse prognosis. Although targeting the HGF/c-Met pathway has been proposed for the treatment of multiple cancers, the effect of c-Met inhibition in HCC remains unclear. Human HCC cell lines, Huh7, Hep3B, MHCC97-L and MHCC97-H, were utilized in this study to investigate the effect of c-Met inhibition using the small molecule, selective c-Met tyrosine kinase inhibitor PHA665752.
MHCC97-L and MHCC97-H cells demonstrate a mesenchymal phenotype with decreased expression of E-cadherin and increased expression of c-Met, Fibronectin and Zeb2 compared to Huh7 and Hep3B cells, which have an epithelial phenotype. PHA665752 treatment blocked phosphorylation of c-Met and downstream PI3K/Akt and MAPK/Erk pathways, inhibited cell proliferation and induced apoptosis in c-Met positive MHCC97-L and MHCC97-H cells. In xenograft models, administration of PHA665752 significantly inhibited c-Met positive MHCC97-L and MHCC97-H tumor growth, and PHA665752 treated tumors demonstrated marked reduction of both c-Met phosphorylation and cell proliferation. c-Met negative Huh7 and Hep3B cells were not affected by c-Met inhibitor treatment in vitro or in vivo. In addition, c-Met positive MHCC97-L and MHCC97-H cells demonstrated cancer stem cell-like characteristics, such as resistance to chemotherapy, tumor sphere-formation, and increased expression of CD44 and ABCG2, and PHA665752 treatment suppressed tumor sphere-formation and inhibited CD44 expression.
c-Met represents a potential target of personalized treatment for HCC with an active HGF/c-Met pathway.
Liver cancer; growth factor signaling; c-Met inhibitor; cancer stem cell; hepatocyte growth factor
Ultrahigh-resolution optical coherence tomography (UR-OCT) has been used for the first time to our knowledge to study single-cell basal cell carcinoma (BCC) in vitro. This noninvasive, in situ, label-free technique with deep imaging depth enables three-dimensional analysis of scattering properties of single cells with cellular spatial resolution. From three-dimensional UR-OCT imaging, live and dead BCC cells can be easily identified based on morphological observation. We developed a novel method to automatically extract characteristic parameters of a single cell from data volume, and quantitative comparison and parametric analysis were performed. The results demonstrate the capability of UR-OCT to detect cell death at the cellular level.
(170.1530) Cell analysis; (170.4500) Optical coherence tomography; (100.2960) Image analysis; (170.1870) Dermatology
km23-1 is a dynein light chain that was identified as a TGFß receptor-interacting protein. To investigate whether km23-1 controls human ovarian carcinoma cell (HOCC) growth, we established a tet-off inducible expression system in SKOV-3 cells in which the expression of km23-1 is induced upon doxycycline removal. We found that forced expression of km23-1 inhibited both anchorage-dependent and anchorage-independent growth of SKOV-3 cells. More importantly, induction of km23-1 expression substantially reduced the tumorigenicity of SKOV-3 cells in a xenograft model in vivo. Fluorescence-activated cell sorting analysis of SKOV-3 and IGROV-1 HOCCs demonstrated that the cells were accumulating at G2/M. Phospho-MEK, phospho-ERK, and cyclin B1 were elevated, as was the mitotic index, suggesting that km23-1 suppresses HOCCs growth by inducing a mitotic delay. Immunofluorescence analyses demonstrated that the cells were accumulating at prometaphase/metaphase with increases in multipolar and multinucleated cells. Further, while the mitotic spindle assembly checkpoint protein BubR1 was present at the prometaphase kinetochore in Dox+/− cells, it was inappropriately retained at the metaphase kinetochore in Dox− cells. Thus, the mechanism by which high levels of km23-1 suppresses ovarian carcinoma growth in vitro and inhibits ovary tumor formation in vivo appears to involve a BubR1-related mitotic delay.
dynein; km23-1; ovarian cancer; mitosis
Lymph node status is a key prognostic factor in penile squamous cell carcinoma. Recently, growing evidence indicates a multimodality approach consisting of neoadjuvant chemotherapy followed by consolidation surgery improves the outcome of locally advanced penile cancer. Thus, accurate estimation of survival probability in node-positive penile cancer is critical for treatment decision making, counseling of patients and follow-up scheduling. This article reviewed evolving developments in assessing the risk for cancer progression based on lymph node related variables, such as the number of metastatic lymph nodes, bilateral lymph node metastases, the ratio of positive lymph nodes, extracapsular extension of metastatic lymph nodes, pelvic lymph node metastases, metastatic deposit in sentinel lymph nodes and N stage in TNM classification. Controversial issues surrounding the prognostic value of these nodal related predictors were also discussed.
Lymph node; Metastasis; Penile cancer; Prognosis
Abnormal activation of the canonical Wnt signaling pathway has been implicated in carcinogenesis. Transcription of Wnt target genes is regulated by nuclear β-catenin, whose over-expression is observed in Hepatocellular Carcinoma (HCC) tissue. Cyr61, a member of the CCN complex family of multifunctional proteins, is also found over-expressed in many types of tumor and plays dramatically different roles in tumorigenesis. In this study, we investigated the relationship between Cyr61 and β-catenin in HCC. We found that while Cyr61 protein was not expressed at a detectable level in the liver tissue of healthy individuals, its expression level was elevated in the HCC and HCC adjacent tissues and was markedly increased in cancer-adjacent hepatic cirrhosis tissue. Over-expression of Cyr61 was positively correlated with increased levels of β-catenin in human HCC samples. Activation of β-catenin signaling elevated the mRNA level of Cyr61 in HepG2 cells, while inhibition of β-catenin signaling reduced both mRNA and protein levels of Cyr61. We identified two TCF4-binding elements in the promoter region of human Cyr61 gene and demonstrated that β-catenin/TCF4 complex specifically bound to the Cyr61 promoter in vivo and directly regulated its promoter activity. Furthermore, we found that over-expression of Cyr61 in HepG2 cells promoted the progression of HCC xenografts in SCID mice. These findings indicate that Cyr61 is a direct target of β-catenin signaling in HCC and may play an important role in the progression of HCC.
Abdominal compartment syndrome (ACS) and intra abdominal hypertension(IAH) are common clinical findings in patients with severe acute pancreatitis(SAP). It is thought that an increased intra abdominal pressure(IAP) is associated with poor prognosis in SAP patients. But the detailed effect of IAH/ACS on different organ system is not clear. The aim of this study was to assess the effect of SAP combined with IAH on hemodynamics, systemic oxygenation, and organ damage in a 12 h lasting porcine model.
Measurements and Methods
Following baseline registrations, a total of 30 animals were divided into 5 groups (6 animals in each group): SAP+IAP30 group, SAP+IAP20 group, SAP group, IAP30 group(sham-operated but without SAP) and sham-operated group. We used a N2 pneumoperitoneum to induce different levels of IAH and retrograde intra-ductal infusion of sodium taurocholate to induce SAP. The investigation period was 12 h. Hemodynamic parameters (CO, HR, MAP, CVP), urine output, oxygenation parameters(e.g., SvO2, PO2, PaCO2), peak inspiratory pressure, as well as serum parameters (e.g., ALT, amylase, lactate, creatinine) were recorded. Histological examination of liver, intestine, pancreas, and lung was performed.
Cardiac output significantly decreased in the SAP+IAH animals compared with other groups. Furthermore, AST, creatinine, SUN and lactate showed similar increasing tendency paralleled with profoundly decrease in SvO2. The histopathological analyses also revealed higher grade injury of liver, intestine, pancreas and lung in the SAP+IAH groups. However, few differences were found between the two SAP+IAH groups with different levels of IAP.
Our newly developed porcine SAP+IAH model demonstrated that there were remarkable effects on global hemodynamics, oxygenation and organ function in response to sustained IAH of 12 h combined with SAP. Moreover, our model should be helpful to study the mechanisms of IAH/ACS-induced exacerbation and to optimize the treatment strategies for counteracting the development of organ dysfunction.
The transcriptional regulator steroidogenic factor 1 (SF-1) and the enzyme cytochrome P450 aromatase (CYP19) play a central role in modulation of a broad range of tissue-specific developmental processes associated with hormone homeostasis that includes differentiation of the central nervous system. SF-1 and CYP19 expression may be targeted by a variety of endocrine disruptive agents prevalent within the environment. In the present study, we cloned and characterized partial sequences for bullfrog (Rana catesbeiana) SF-1 and CYP19 and examined the effects of a 48 h exposure to 1 and 100 μg/L of the herbicide atrazine (ATZ) and its major metabolite desethylatrazine (DEA), as well as 5 ng/L of the estrogenic chemical, 17α-ethynylestradiol (EE2), and 673 ng/L of the thyroid hormone, 3,5, 3′-triiodothyronine (T3), on SF-1 and CYP19 mRNA abundance in the brains of premetamorphic bullfrog tadpoles. Quantitative RT-PCR analysis showed an increase in CYP19 mRNA following a 48 h exposure to EE2 but not T3 while no significant changes in SF-1 transcript levels occurred. We observed a strong positive correlation between CYP19 and SF-1 transcript abundance in the ATZ-exposed animals which was not evident with DEA- or hormone-exposed tadpoles. Our results are intriguing in light of reported behavioral changes in ATZ-exposed frogs and suggest that further research is warranted to examine the relationship and role of CYP19 and SF-1 in amphibian brain development.
atrazine; desethylatrazine; aromatase; steroidogenic factor 1; endocrine disruption; amphibian development; pesticides; gene expression; estrogen; thyroid hormone; low dose
Comparative genomic analysis has revealed that in each genome a large number of open reading frames have no homologues in other species. Such singleton genes have attracted the attention of biochemists and structural biologists as a potential untapped source of new folds. Cthe_2751 is a 15.8 kDa singleton from an anaerobic, hyperthermophile Clostridium thermocellum. To gain insights into the architecture of the protein and obtain clues about its function, we decided to solve the structure of Cthe_2751.
The protein crystallized in 4 different space groups that diffracted X-rays to 2.37 Å (P3121), 2.17 Å (P212121), 3.01 Å (P4122), and 2.03 Å (C2221) resolution, respectively. Crystal packing analysis revealed that the 3-D packing of Cthe_2751 dimers in P4122 and C2221 is similar with only a rotational difference of 2.69° around the C axes. A new method developed to quantify the differences in packing of dimers in crystals from different space groups corroborated the findings of crystal packing analysis. Cthe_2751 is an all α-helical protein with a central hydrophobic core providing thermal stability via π:cation and π: π interactions. A ProFunc analysis retrieved a very low match with a splicing endonuclease, suggesting a role for the protein in the processing of nucleic acids.
Non-Pfam singleton Cthe_2751 folds into a known all α-helical fold. The structure has increased sequence coverage of non-Pfam proteins such that more protein sequences can be amenable to modelling. Our work on crystal packing analysis provides a new method to analyze dimers of the protein crystallized in different space groups. The utility of such an analysis can be expanded to oligomeric structures of other proteins, especially receptors and signaling molecules, many of which are known to function as oligomers.
Radiation-induced skin injury remains a serious concern for radiation therapy. Heme oxygenase-1 (HO-1), the rate-limiting enzyme in heme catabolism, has been reported to have potential antioxidant and anti-apoptotic properties. However, the role of HO-1 in radiation-induced skin damage remains unclear. This study aims to elucidate the effects of HO-1 on radiation-induced skin injury in rats.
A control adenovirus (Ad-EGFP) and a recombinant adenovirus (Ad-HO1-EGFP) were constructed. Rats were irradiated to the buttock skin with a single dose of 45 Gy followed by a subcutaneous injection of PBS, 5 × 109 genomic copies of Ad-EGFP or Ad-HO1-EGFP (n = 8). After treatment, the skin MDA concentration, SOD activity and apoptosis were measured. The expression of antioxidant and pro-apoptotic genes was determined by RT-PCR and real-time PCR. Skin reactions were measured at regular intervals using the semi-quantitative skin injury score.
Subcutaneous injection of Ad-HO1-EGFP infected both epidermal and dermal cells and could spread to the surrounding regions. Radiation exposure upregulated the transcription of the antioxidant enzyme genes, including SOD-1, GPx2 and endogenous HO-1. HO-1 overexpression decreased lipid peroxidation and inhibited the induction of ROS scavenging proteins. Moreover, HO-1 exerted an anti-apoptotic effect by suppressing FAS and FASL expression. Subcutaneous injection of Ad-HO1-EGFP demonstrated significant improvement in radiation-induced skin injury.
The present study provides evidences for the protective role of HO-1 in alleviating radiation-induced skin damage in rats, which is helpful for the development of therapy for radiation-induced skin injury.
Radiation-induced skin injury; HO-1; adenovirus; lipid peroxidation; apoptosis
Background & Aims
The tumor suppressor PTEN inhibits AKT2 signaling; both are aberrantly expressed in liver tumors. We investigated how PTEN and AKT2 regulate liver carcinogenesis. Loss of PTEN leads to spontaneous development of liver tumors from progenitor cells. We investiged how the loss of PTEN activates liver progenitor cells and induces tumorigensis.
We studied mice with liver-specific disruptions in Pten and the combination of Pten and Akt2 to investigate mechanisms of liver carcinogenesis.
PTEN loss leads to hepatic injury and establishes selective pressure for tumor-initiating cells (TICs), which proliferate to form mixed-lineage tumors. The Pten-null mice had increasing levels of hepatic injury before proliferation of hepatic progenitors. Attenuation of hepatic injury by deletion of Akt2 reduced progenitor cell proliferation and delayed tumor development. In Pten/Akt2-null mice given 3,5-diethoxycarbonyl-1,4 dihydrocollidine, the primary effect of AKT2 loss was attenuation of hepatic injury and not inhibition of progenitor-cell proliferation in response to injury.
Liver carcinogenesis in Pten-null mice requires not only the transformation of TICs but selection pressure from hepatic injury and cell death, which activate TIC. Further research is required to elucidate the mechanism for hepatic injury and its relationship with TIC activation.
Liver cancer stem cells; DDC; lipotoxic cell death; mixed-lineage tumors
Theoretical analysis and experimental validation prove that a multi-dataset data-collection strategy produces better diffraction data. The readiness test is a simple and sensitive method for X-ray data-collection system evaluation and a benchmark.
A multi-dataset (MDS) data-collection strategy is proposed and analyzed for macromolecular crystal diffraction data acquisition. The theoretical analysis indicated that the MDS strategy can reduce the standard deviation (background noise) of diffraction data compared with the commonly used single-dataset strategy for a fixed X-ray dose. In order to validate the hypothesis experimentally, a data-quality evaluation process, termed a readiness test of the X-ray data-collection system, was developed. The anomalous signals of sulfur atoms in zinc-free insulin crystals were used as the probe to differentiate the quality of data collected using different data-collection strategies. The data-collection results using home-laboratory-based rotating-anode X-ray and synchrotron X-ray systems indicate that the diffraction data collected with the MDS strategy contain more accurate anomalous signals from sulfur atoms than the data collected with a regular data-collection strategy. In addition, the MDS strategy offered more advantages with respect to radiation-damage-sensitive crystals and better usage of rotating-anode as well as synchrotron X-rays.
multi-dataset data-collection strategy; readiness test
Liver stem cell, or oval cells, proliferate during chronic liver injury, and are proposed to differentiate into both hepatocytes and cholangiocytes. In addition, liver stem cells are hypothesized to be the precursors for a subset of liver cancer, Hepatocellular carcinoma. One of the primary challenges to stem cell work in any solid organ like the liver is the isolation of a rare population of cells for detailed analysis. For example, the vast majority of cells in the liver are hepatocytes (parenchymal fraction), which are significantly larger than non-parenchymal cells. By enriching the specific cellular compartments of the liver (i.e. parenchymal and non-parenchymal fractions), and selecting for CD45 negative cells, we are able to enrich the starting population of stem cells by over 600-fold.The proceduresdetailed in this report allow for a relatively rare population of cells from a solid organ to be sorted efficiently. This process can be utilized to isolateliver stem cells from normal murine liver as well as chronic liver injury models, which demonstrate increased liver stem cell proliferation. This method has clear advantages over standard immunohistochemistry of frozen or formalin fixed liver as functional studies using live cells can be performed after initial co-localization experiments. To accomplish the procedure outlined in this report, a working relationship with a research based flow-cytometry core is strongly encouraged as the details of FACS isolation are highly dependent on specialized instrumentation and a strong working knowledge of basic flow-cytometry procedures. The specific goal of this process is to isolate a population of liver stem cells that can be clonally expanded in vitro.
Tumor initiating stem-like cells (TISCs) are a subset of neoplastic cells that possess distinct survival mechanisms and self-renewal characteristics crucial for tumor maintenance and propagation. The induction of epithelial-mesenchymal-transition (EMT) by TGFβ has been recently linked to the acquisition of TISC characteristics in breast cancer. In HCC, a TISC and EMT phenotype correlates with a worse prognosis. In this work, our aim is to elucidate the underlying mechanism by which cells acquire tumor initiating characteristics after EMT.
Gene and protein expression assays and Nanog-promoter luciferase reporter were utilized in epithelial and mesenchymal phenotype liver cancer cell lines. EMT was analyzed with migration/invasion assays. TISC characteristics were analyzed with tumor-sphere self-renewal and chemotherapy resistance assays. In vivo tumor assay was performed to investigate the role of Snail1 in tumor initiation.
TGFβ induced EMT in epithelial cells through the up-regulation of Snail1 in Smad-dependent signaling. Mesenchymal liver cancer post-EMT demonstrates TISC characteristics such as tumor-sphere formation but are not resistant to cytotoxic therapy. The inhibition of Snail1 in mesenchymal cells results in decreased Nanog promoter luciferase activity and loss of self-renewal characteristics in vitro. These changes confirm the direct role of Snail1 in some TISC traits. In vivo, the down-regulation of Snail1 reduced tumor growth but was not sufficient to eliminate tumor initiation. In summary, TGFβ induces EMT and TISC characteristics through Snail1 and Nanog up-regulation. In mesenchymal cells post-EMT, Snail1 directly regulates Nanog expression, and loss of Snail1 regulates tumor growth without affecting tumor initiation.
Epithelial-to-mesenchymal transition (EMT) is predicted to play a critical role in metastatic disease in hepatocellular carcinoma. In this study, we used a novel murine model of EMT to elucidate a mechanism of tumor progression and metastasis. A total of 2 × 106 liver cells isolated from Ptenloxp/loxp/Alb-Cre+ mice, expanded from a single CD133+CD45− cell clone, passage 0 (P0), were sequentially transplanted to obtain two passages of tumor cells, P1 and P2. Cells were analyzed for gene expression using microarray and real-time polymerase chain reaction. Functional analysis included cell proliferation, migration, and invasion in vitro and orthotopic tumor metastasis assays in vivo. Although P0, P1, and P2 each formed tumors consistent with mixed liver epithelium, within the P2 cells, two distinct cell types were clearly visible: cells with epithelial morphology similar to P0 cells and cells with fibroblastoid morphology. These P2 mesenchymal cells demonstrated increased locomotion on wound healing; increased cell invasion on Matrigel basement membrane; increased EMT-associated gene expression of Snail1, Zeb1, and Zeb2; and down-regulated E-cadherin. P2 mesenchymal cells demonstrated significantly faster tumor growth in vivo compared with P2 epithelial counterparts, with invasion of intestine, pancreas, spleen, and lymph nodes. Furthermore, P2 mesenchymal cells secreted high levels of hepatocyte growth factor (HGF), which we propose acts in a paracrine fashion to drive epithelial cells to undergo EMT. In addition, a second murine liver cancer stem cell line with methionine adenosyltransferase 1a deficiency acquired EMT after sequential transplantations, indicating that EMT was not restricted to Pten-deleted tumors.
EMT is associated with a high rate of liver tumor proliferation, invasion, and metastasis in vivo, which is driven by HGF secreted from mesenchymal tumor cells in a feed-forward mechanism.
It is known that the clade A protein phosphatase 2Cs (PP2Cs), including ABI1 and ABI2 and other PP2C members, are key players that function directly downstream of the PYR/PYL/RCAR abscisic acid (ABA) receptors. Here, identification of a crucial site for function of ABI2 protein phosphatase in ABA signalling is reported. It was observed that a calcium-dependent protein kinase (CDPK) phosphorylation site-like motif (CPL) in the ABI2 molecule is required for the interactions of ABI2 with the two members of the ABA receptors PYL5 and PYL9 and with a downstream protein kinase SnRK2.6, and for the catalytic activity of ABI2 in vitro, as well as for the response of ABI2 to the ABA receptors PYL5/PYL9 in relation to the ABA receptor-induced inhibition of the ABI2 phosphatase activity. Further, genetic evidence was provided to demonstrate that this CPL is required for the function of ABI2 to mediate ABA signalling. These data reveal that this CPL is an important site necessary for both the phosphatase activity of ABI2 and the functional interaction between ABI2 and PYL5/9 ABA receptors, providing new information to understand primary events of ABA signal transduction.
ABA signalling; ABI2; Arabidopsis thaliana; functional site; protein phosphorylation; type 2C protein phosphatase
Little is known about the plasma levels of N-terminal pro-brain natriuretic peptide (NT-proBNP), and the relationship between the severity of coronary heart disease (CHD) with NT-proBNP and multiple biomarkers in diabetic and pre-diabetic patients, compared to individuals with normal glucose levels.
Four hundred and fifteen consecutive Chinese patients of both sexes were assigned to three groups on the basis of the new hemoglobin (Hb) A1c (HbA1c) cut-off points for diagnosis of diabetes and pre-diabetes. The three groups were divided into tertiles according to NT-proBNP, hs-CRP, cystatin C, and troponin T levels. Gensini scores were compared among the three groups and biomarker tertiles. Receiver operating characteristic (ROC) curves were used to obtain the angiographic CHD cut-off points for each biomarker. Stepwise multivariate linear correlation analysis was applied to examine the association between the severity of CHD and biomarker levels.
Gensini scores increased with increasing biomarker tertile levels and HbA1c. Gensini scores were significantly different in the middle and upper NT-proBNP tertiles of the diabetic, pre-diabetic and control groups. NT-proBNP had the highest positive and negative predictive values and area under the curve for CHD. Only NT-proBNP was identified as an independent variable for Gensini score.
Plasma NT-proBNP may be an important biomarker to evaluate the severity of CHD and screen for CHD in diabetic or pre-diabetic patients.