Deconstructing Pancreas Developmental Biology

The relentless nature and increasing prevalence of human pancreatic diseases, in particular, diabetes mellitus and adenocarcinoma, has motivated further understanding of pancreas organogenesis. The pancreas is a multifunctional organ whose epithelial cells govern a diversity of physiologically vital endocrine and exocrine functions. The mechanisms governing the birth, differentiation, morphogenesis, growth, maturation, and maintenance of the endocrine and exocrine components in the pancreas have been discovered recently with increasing tempo. This includes recent studies unveiling mechanisms permitting unexpected flexibility in the developmental potential of immature and mature pancreatic cell subsets, including the ability to interconvert fates. In this article, we describe how classical cell biology, genetic analysis, lineage tracing, and embryological investigations are being complemented by powerful modern methods including epigenetic analysis, time-lapse imaging, and flow cytometry-based cell purification to dissect fundamental processes of pancreas development.     

Quantitative analyses of individual sugars in mixture using FRET‐based biosensors

Molecular biosensors were developed and applied to measure individual sugars in biological mixtures such as bacterial culture broths. As the sensing units, four sugar‐binding proteins (SBPs for allose, arabinose, ribose, and glucose) were selected from the Escherichia coli genome and connected to a cyan fluorescent protein and yellow fluorescent protein via dipeptide linkers (CFP‐L‐SBP‐YFP). The putative sensors were randomized in the linker region (L) and then investigated with regard to the intensity of fluorescence resonance energy transfer on the binding of the respective sugars. As a result, four representatives were selected from each library and examined for their specificity using 16 available sugars. The apparent dissociation constants of the allose, arabinose, ribose, and glucose sensors were estimated to be 0.35, 0.36, 0.17, and 0.18 μM. Finally, the sugar sensors were applied to monitor the consumption rate of individual sugars in an E. coli culture broth. The individual sugar profiles exhibited a good correlation with those obtained using an HPLC method, confirming that the biosensors offer a rapid and easy‐to‐use method for monitoring individual sugars in mixed compositions. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

Synthesis of monoacylglycerol containing pinolenic acid via stepwise esterification using a cold active lipase

High purity monoacylglycerol (MAG) containing pinolenic acid was synthesized via stepwise esterification of glycerol and fatty acids from pine nut oil using a cold active lipase from Penicillium camembertii as a biocatalyst. Effects of temperature, molar ratio, water content, enzyme loading, and vacuum on the synthesis of MAG by lipase‐catalyzed esterification of glycerol and fatty acid from pine nut oil were investigated. Diacylglycerol (DAG) as well as MAG increased significantly when temperature was increased from 20 to 40°C. At a molar ratio of 1:1, MAG content decreased because of the significant increase in DAG content. Water has a profound influence on both MAG and DAG content through the entire course of reaction. The reaction rate increased significantly as enzyme loading increased up to 600 units. Vacuum was an effective method to reduce DAG content. The optimum temperature, molar ratio, water content, enzyme loading, vacuum, and reaction time were 20°C, 1:5 (fatty acid to glycerol), 2%, 600 units, 5 torr, and 24 h, respectively. MAG content further increased via lipase‐catalyzed second step esterification at subzero temperature. P. camembertii lipase exhibited esterification activity up to ?30°C. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

Crucial Role of TSC-22 in Preventing the Proteasomal Degradation of p53 in Cervical Cancer

The p53 tumor suppressor function can be compromised in many tumors by the cellular antagonist HDM2 and human papillomavirus oncogene E6 that induce p53 degradation. Restoration of p53 activity has strong therapeutic potential. Here, we identified TSC-22 as a novel p53-interacting protein and show its novel function as a positive regulator of p53. We found that TSC-22 level was significantly down-regulated in cervical cancer tissues. Moreover, over-expression of TSC-22 was sufficient to inhibit cell proliferation, promote cellular apoptosis in cervical cancer cells and suppress growth of xenograft tumors in mice. Expression of also TSC-22 enhanced the protein level of p53 by protecting it from poly-ubiquitination. When bound to the motif between amino acids 100 and 200 of p53, TSC-22 inhibited the HDM2- and E6-mediated p53 poly-ubiquitination and degradation. Consequently, ectopic over-expression of TSC-22 activated the function of p53, followed by increased expression of p21(Waf1/Cip1) and PUMA in human cervical cancer cell lines. Interestingly, TSC-22 did not affect the interaction between p53 and HDM2. Knock-down of TSC-22 by small interfering RNA clearly enhanced the poly-ubiquitination of p53, leading to the degradation of p53. These results suggest that TSC-22 acts as a tumor suppressor by safeguarding p53 from poly-ubiquitination mediated-degradation.     

Induction of Heme Oxygenase-1 Inhibits Cell Death in Crotonaldehyde-Stimulated HepG2 Cells via the PKC-δ -p38 -Nrf2 Pathway

Background

Crotonaldehyde, an alpha, beta-unsaturated aldehyde present in cigarette smoke, is an environmental pollutant and a product of lipid peroxidation. It also produces adverse effects to humans and is considered as a risk factor for various diseases. Heme oxygenase-1 (HO-1) plays important roles in protecting cells against oxidative stress as a prime cellular defense mechanism. However, HO-1 may be associated with cell proliferation and resistance to apoptosis in cancer cells. The aim of this study was to examine the effects of HO-1 induction on cell survival in crotonaldehyde-stimulated human hepatocellular carcinoma (HepG2) cells.

Methods

To investigate the signaling pathway involved in crotonaldehyde-induced HO-1 expression, we compared levels of inhibition efficiency of specific inhibitors and specific small interfering RNAs (siRNAs) of several kinases. The cell-cycle and cell death was measured by FACS and terminal dUTP nick-end labeling (TUNEL) staining.

Results

Treatment with crotonaldehyde caused a significant increase in nuclear translocation of NF-E2 related factor (Nrf2). Treatment with inhibitors of the protein kinase C-δ (PKC-δ) and p38 pathways resulted in obvious blockage of crotonaldehyde-induced HO-1 expression. Furthermore, treatment with HO-1 siRNA and the specific HO-1 inhibitor zinc-protoporphyrin produced an increase in the G₀/G₁ phase of the cell cycle in crotonaldehyde-stimulated HepG2 cells.

Conclusions

Taken together, the results support an anti-apoptotic role for HO-1 in crotonaldehyde-stimulated human hepatocellular carcinoma cells and provide a mechanism by which induction of HO-1 expression via PKC-δ–p38 MAPK–Nrf2 pathway may promote tumor resistance to oxidative stress.     

Outcomes of Multidrug-Resistant Tuberculosis Treatment with Early Initiation of Antiretroviral Therapy for HIV Co-Infected Patients in Lesotho

Background

Although the importance of concurrent treatment for multidrug-resistant tuberculosis (MDR-TB) and HIV co-infection has been increasingly recognized, there have been few studies reporting outcomes of MDR-TB and HIV co-treatment. We report final outcomes of comprehensive, integrated MDR-TB and HIV treatment in Lesotho and examine factors associated with death or treatment failure.

Methods

We reviewed clinical charts of all adult patients who initiated MDR-TB treatment in Lesotho between January 2008 and September 2009. We calculated hazard ratios (HR) and used multivariable Cox proportional hazards regression to identify predictors of poor outcomes.

Results

Of 134 confirmed MDR-TB patients, 83 (62%) were cured or completed treatment, 46 (34%) died, 3 (2%) transferred, 1 (1%) defaulted, and 1 (1%) failed treatment. Treatment outcomes did not differ significantly by HIV status. Among the 94 (70%) patients with HIV co-infection, 53% were already on antiretroviral therapy (ART) before MDR-TB treatment initiation, and 43% started ART a median of 16 days after the start of the MDR-TB regimen. Among HIV co-infected patients who died, those who had not started ART before MDR-TB treatment had a shorter median time to death (80 days vs. 138 days, p = 0.065). In multivariable analysis, predictors of increased hazard of failure or death were low and severely low body mass index (HR 2.75, 95% confidence interval [CI] 1.27–5.93; HR 5.50, 95% CI 2.38–12.69), and a history of working in South Africa (HR 2.37, 95% CI 1.24–4.52).

Conclusions

Favorable outcomes can be achieved in co-infected patients using a community-based treatment model when both MDR-TB and HIV disease are treated concurrently and treatment is initiated promptly.     

Macrophages homing to metastatic lymph nodes can be monitored with ultrasensitive ferromagnetic iron-oxide nanocubes and a 1.5T clinical MR scanner

Background

Due to the ability of macrophages to specifically home to tumors, their potential use as a delivery vehicle for cancer therapeutics has been suggested. Tracking the delivery and engraftment of macrophages into human tumors with a 1.5T clinical MR scanner requires the development of sensitive contrast agents for cell labeling. Therefore, this study aimed to determine whether intravenously injected macrophages could target a primary tumor as well as metastatic LNs, and whether these cells could be detected in vivo by MRI.

Methodology

Peritoneal macrophages were obtained from BALB/c nude mice. The viability, phagocytotic capacity and migratory activity of the macrophages were assessed. MR imaging was performed using a clinical 1.5 T MR scanner and we estimated the T2* of the labeled macrophages. Metastatic lymph nodes were produced in BALB/c nude mice. We administrated 2×10⁶ macrophages labeled with 50 µg Fe/mL FIONs intravenously into the mice. In the 3D T2* GRE MR images obtained one day after the injection of the labeled macrophages or FION solution, the percentages of pixels in the tumors or LNs below the minimum normalized SI (signal intensity) threshold were summated and reported as the black pixel count (%) for the FION hypointensity. Tumors in the main tumor model as well as the brachial, axillary and inguinal lymph nodes in the metastatic LN models were removed and stained. For all statistical analyses, single-group data were assessed using t test or the Mann-Whitney test. Repeated measurements analysis of variance (ANOVA) with Tukey–Kramer post hoc comparisons were performed for multiple comparisons.

Conclusions

The FION-labeled macrophages, which could be non-invasively monitored using a 1.5 T clinical MR scanner, targeted both the main tumors and LN metastases. Overall, the results of this study suggest that the use of macrophages may have many future applications in the clinic for vectorizing therapeutic agents toward main tumors as well as LN metastases.     

Prospective Validation of ELF Test in Comparison with Fibroscan and FibroTest to Predict Liver Fibrosis in Asian Subjects with Chronic Hepatitis B

Background and Aims

Liver stiffness measurement (LSM) and FibroTest (FT) are frequently used as non-invasive alternatives for fibrosis staging to liver biopsy. However, to date, diagnostic performances of Enhanced Liver Fibrosis (ELF) test, which consists of hyaluronic acid, aminoterminal propeptide of procollagen type-III, and tissue inhibitor of matrix metalloproteinases-1, have not been compared to those of LSM and FT in Asian chronic hepatitis B (CHB) patients.

Methods

Between June 2010 and November 2011, we prospectively enrolled 170 CHB patients who underwent liver biopsies along with LSM, FT, and ELF. The Batts system was used to assess fibrosis stages.

Results

Areas under receiver operating characteristic curves (AUROCs) to predict significant fibrosis (F≥2), advanced fibrosis (F≥3), and cirrhosis (F = 4) were 0.901, 0.860, and 0.862 for ELF, respectively; 0.937, 0.956, and 0.963 for LSM; and 0.896, 0.921, and 0.881 for FT. AUROCs to predict F≥2 were similar between each other, whereas LSM and FT had better AUROCs than ELF for predicting F≥3 (both p<0.05), and LSM predicted F4 more accurately than ELF (p<0.05). Optimized cutoffs of ELF to maximize sum of sensitivity and specificity were 8.5, 9.4, and 10.1 for F≥2, F≥3, and F = 4, respectively. Using suggested ELF, LSM and FT cutoffs to diagnose F1, F2, F3, and F4, 91 (53.5%), 117 (68.8%), and 110 (64.7%) patients, respectively, were correctly classified according to histological results.

Conclusions

ELF demonstrated considerable diagnostic value in fibrosis staging in Asian CHB patients, especially in predicting F≥2. However, LSM consistently provided better performance for predicting F≥3 and F4.     

Induction of neuronal death by microglial AGE-albumin: implications for Alzheimer's disease

Advanced glycation end products (AGEs) have long been considered as potent molecules promoting neuronal cell death and contributing to neurodegenerative disorders such as Alzheimer's disease (AD). In this study, we demonstrate that AGE-albumin, the most abundant AGE product in human AD brains, is synthesized in activated microglial cells and secreted into the extracellular space. The rate of AGE-albumin synthesis in human microglial cells is markedly increased by amyloid-β exposure and oxidative stress. Exogenous AGE-albumin upregulates the receptor protein for AGE (RAGE) and augments calcium influx, leading to apoptosis of human primary neurons. In animal experiments, soluble RAGE (sRAGE), pyridoxamine or ALT-711 prevented Aβ-induced neuronal death in rat brains. Collectively, these results provide evidence for a new mechanism by which microglial cells promote death of neuronal cells through synthesis and secretion of AGE-albumin, thereby likely contributing to neurodegenerative diseases such as AD.     

A Portable Chemotaxis Platform for Short and Long Term Analysis

Flow-based microfluidic systems have been widely utilized for cell migration studies given their ability to generate versatile and precisely defined chemical gradients and to permit direct visualization of migrating cells. Nonetheless, the general need for bulky peripherals such as mechanical pumps and tubing and the complicated setup procedures significantly limit the widespread use of these microfluidic systems for cell migration studies. Here we present a simple method to power microfluidic devices for chemotaxis assays using the commercially available ALZET® osmotic pumps. Specifically, we developed a standalone chemotaxis platform that has the same footprint as a multiwell plate and can generate well-defined, stable chemical gradients continuously for up to 7 days. Using this platform, we validated the short-term (24 hours) and long-term (72 hours) concentration dependent PDGF-BB chemotaxis response of human bone marrow derived mesenchymal stem cells.