Soluble Expression of Human Leukemia Inhibitory Factor with Protein Disulfide Isomerase in Escherichia coli and Its Simple Purification

Human leukemia inhibitory factor (hLIF) is a multifunctional cytokine that is essential for maintaining the pluripotency of embryonic stem cells. hLIF may be also be useful in aiding fertility through its effects on increasing the implantation rate of fertilized eggs. Thus these applications in biomedical research and clinical medicine create a high demand for bioactive hLIF. However, production of active hLIF is problematic since eukaryotic cells demonstrate limited expression and prokaryotic cells produce insoluble protein. Here, we have adopted a hybrid protein disulfide isomerase design to increase the solubility of hLIF in Escherichia coli. Low temperature expression of hLIF fused to the b''a'' domain of protein disulfide isomerase (PDIb''a'') increased the soluble expression in comparison to controls. A simple purification protocol for bioactive hLIF was established that includes removal of the PDIb''a'' domain by cleavage by TEV protease. The resulting hLIF, which contains one extra glycine residue at the N-terminus, was highly pure and demonstrated endotoxin levels below 0.05 EU/μg. The presence of an intramolecular disulfide bond was identified using mass spectroscopy. This purified hLIF effectively maintained the pluripotency of a murine embryonic stem cell line. Thus we have developed an effective method to produce a pure bioactive version of hLIF in E. coli for use in biomedical research.     

DNase I treated DNA-PCR based detection of food pathogens immobilized by metal hydroxides

Bacterial immobilization by metal hydroxides can be used for enrichment of various bacterial strains including Gram (+) and Gram (−). The polymerase chain reaction (PCR)-based bacterial detection without enrichment culture could be implemented by concentrating bacteria from food matrix by metal hydroxides. To distinguish between viable and non-viable cells, it is often required to detect the mRNA, an indicator of viable cells. This technique, although provides accurate and reliable result, is expensive and time-consuming. Here, we report the studies on application of DNase I treatment to eliminate DNA from dead cells and subsequently detect the presence of viable pathogens by conventional PCR. It was found that treatment of immobilized cells with DNase I for 1 h at 37°C prior to DNA extraction could efficiently eliminate false positives due to the presence of non-viable cells. The technique was used to detect the presence of various pathogens in food model. The detection limits for Escherichia coli O157:H7 (384 bp), Listeria monocytogenes (482 bp), and E. coli wild type (580 bp) was 5 × 10¹ cells and that for Salmonella typhimurium (685 bp) was 5 × 10² cells in 10 ml of whole milk. An erratum to this article can be found at     

Surface Renewal: An Advanced Micrometeorological Method for Measuring and Processing Field-Scale Energy Flux Density Data

Advanced micrometeorological methods have become increasingly important in soil, crop, and environmental sciences. For many scientists without formal training in atmospheric science, these techniques are relatively inaccessible. Surface renewal and other flux measurement methods require an understanding of boundary layer meteorology and extensive training in instrumentation and multiple data management programs. To improve accessibility of these techniques, we describe the underlying theory of surface renewal measurements, demonstrate how to set up a field station for surface renewal with eddy covariance calibration, and utilize our open-source turnkey data logger program to perform flux data acquisition and processing. The new turnkey program returns to the user a simple data table with the corrected fluxes and quality control parameters, and eliminates the need for researchers to shuttle between multiple processing programs to obtain the final flux data. An example of data generated from these measurements demonstrates how crop water use is measured with this technique. The output information is useful to growers for making irrigation decisions in a variety of agricultural ecosystems. These stations are currently deployed in numerous field experiments by researchers in our group and the California Department of Water Resources in the following crops: rice, wine and raisin grape vineyards, alfalfa, almond, walnut, peach, lemon, avocado, and corn.     

Deregulated Lipid Sensing by Intestinal CD36 in Diet-Induced Hyperinsulinemic Obese Mouse Model

The metabolic syndrome (MetS) greatly increases risk of cardiovascular disease and diabetes and is generally associated with abnormally elevated postprandial triglyceride levels. We evaluated intestinal synthesis of triglyceride-rich lipoproteins (TRL) in a mouse model of the MetS obtained by feeding a palm oil-rich high fat diet (HFD). By contrast to control mice, MetS mice secreted two populations of TRL. If the smaller size population represented 44% of total particles in the beginning of intestinal lipid absorption in MetS mice, it accounted for only 17% after 4 h due to the secretion of larger size TRL. The MetS mice displayed accentuated postprandial hypertriglyceridemia up to 3 h due to a defective TRL clearance. These alterations reflected a delay in lipid induction of genes for key proteins of TRL formation (MTP, L-FABP) and blood clearance (ApoC2). These abnormalities associated with blunted lipid sensing by CD36, which is normally required to optimize jejunal formation of large TRL. In MetS mice CD36 was not downregulated by lipid in contrast to control mice. Treatment of controls with the proteosomal inhibitor MG132, which prevented CD36 downregulation, resulted in blunted lipid-induction of MTP, L-FABP and ApoC2 gene expression, as in MetS mice. Absence of CD36 sensing was due to the hyperinsulinemia in MetS mice. Acute insulin treatment of controls before lipid administration abolished CD36 downregulation, lipid-induction of TRL genes and reduced postprandial triglycerides (TG), while streptozotocin-treatment of MetS mice restored lipid-induced CD36 degradation and TG secretion. In vitro, insulin treatment abolished CD36-mediated up-regulation of MTP in Caco-2 cells. In conclusion, HFD treatment impairs TRL formation in early stage of lipid absorption via insulin-mediated inhibition of CD36 lipid sensing. This impairment results in production of smaller TRL that are cleared slowly from the circulation, which might contribute to the reported association of CD36 variants with MetS risk.     

The Effects of Electromagnetic Fields on Human Health:Recent Advances and Future

The potential of clectromagnetic ficlds(EMFs)for discase treatment and health enhancement has been actively pursued over the recent decades.This review first provides a general introduction about natural EMFs and related biological effcts.Then the recent progress on the EMF treatment of some common diseases(such as cancer,diabetes,wound healing and neurological diseases,etc.)has been carefully reviewed and summarized.Yet,the blindness on the selection of therapeutic EMF parameters still hinders the broad application ofEMF therapy.Moreover,the unclear mechanism of EMF function and poor reproducibility of experimental rcsults also remain big challenges in the field of bioclectromagnetics.Bionics is a useful methodology that gains inspiration from nature to serve human life and industry.We have discussed the feasibility of applying bionic approach on the selection of the rapeutic EMFs,which is based on thefindings of natural EMFs.Finally,we advocate that the detailed information of EMFs and biological samples should be thoroughly reorded in future research and reported in publications.In addition,the publication of studies with negative results should also be allowed.     

Enhanced biomethanation of kitchen waste by different pre-treatments

Five different pre-treatments were investigated to enhance the solubilisation and anaerobic biodegradability of kitchen waste (KW) in thermophilic batch and continuous tests. In the batch solubilisation tests, the highest and the lowest solubilisation efficiency were achieved with the thermo-acid and the pressure-depressure pre-treatments, respectively. However, in the batch biodegradability tests, the highest cumulative biogas production was obtained with the pressure-depressure method. In the continuous tests, the best performance in terms of an acceptable biogas production efficiency of 60% and stable in-reactor COD_s and VFA concentrations corresponded to the pressure-depressure reactor, followed by freeze-thaw, acid, thermo-acid, thermo and control. The maximum OLR (5 g COD L⁻¹ d⁻¹) applied in the pressure-depressure and freeze-thaw reactors almost doubled the control reactor. From the overall analysis, the freeze-thaw pre-treatment was the most profitable process with a net potential profit of around 11.5 € ton⁻¹ KW.     

Proteasome inhibitor-I enhances tunicamycin-induced chemosensitization of prostate cancer cells through regulation of NF-κB and CHOP expression

Although endoplasmic reticulum (ER) stress induction by some anticancer drugs can lead to apoptotic death of cancer cells, combination therapy with other chemicals would be much more efficient. It has been reported that proteasome inhibitors could induce cancer cell death through ER-stress. Our study, however, showed a differential mechanism of proteasome inhibitor-I (Pro-I)-induced cell death. Pro-I significantly enhanced apoptotic death of PC3 prostate cancer cells pretreated with tunicamycin (TM) while other signaling inhibitors against p38, mitogen activated kinase (MEK) and phosphatidyl-inositol 3-kinase (PI3K) did not, as evidenced by cell proliferation and cell cycle analyses. NF-κB inhibition by Pro-I, without direct effect on ER-stress, was found to be responsible for the TM-induced chemosensitization of PC3 cells. Moreover, TM-induced/enhancer-binding protein (C/EBP) homologous protein (CHOP) expression was enhanced by Pro-I without change in GRP78 expression. CHOP knockdown by siRNA also showed a significant decrease in Pro-I chemosensitization. All these data suggest that although TM could induce both NF-κB activation and CHOP expression through ER-stress, both NF-κB inhibition and increased CHOP level by Pro-I are required for enhanced chemosensitization of PC3 prostate cancer cells. Thus, our study might contribute to the identification of anticancer targets against prostate cancer cells.     

Surveillance for Clostridium difficile infection: ICD-9 coding has poor sensitivity compared to laboratory diagnosis in hospital patients, Singapore

Introduction

Clostridium difficile infection (CDI) is an increasingly recognized nosocomial infection in Singapore. Surveillance methods include laboratory reporting of Clostridium difficile toxin assays (CDTA) or use of International Classification of Diseases, 9^th Revision (ICD-9) discharge code 008.45. Previous US studies showed good correlation between CDTA and ICD-9 codes. However, the use of ICD-9 codes for CDI surveillance has not been validated in other healthcare settings.

Methods

We compared CDI rates based on CDTA to ICD-9 codes for all discharges in 2007 from our hospital to determine sensitivity and specificity of ICD-9 codes. Demographic and hospitalization data were analyzed to determine predictors for missing ICD-9 codes.

Results

During 2007, there were 56,352 discharges. Of these, 268 tested CDTA-positive but only 133 were assigned the CDI ICD-9 code. A total of 141 discharges had the ICD-9 code; 8 were CDTA-negative, the rest were CDTA-positive. Community-acquired CDI accounted for only 3.2% of cases. The sensitivity and specificity of ICD-9 codes compared to CDTA were 49.6% and 100% respectively. Concordance between CDTA and ICD-9 codes was 0.649 (p<.001). Comparing concordant patients (CDTA+/ICD9+) to discordant patients (CDTA+/ICD9−), concordant patients were more likely to be over 50 years of age (OR 3.49, 95% CI 1.66–7.34, p = .001) and have shorter time from admission to testing (OR 0.98, 95% CI 0.97–0.99, p = .009).

Discussion

Unlike previous studies in the US, ICD-9 codes substantially underestimate CDI in Singapore compared to microbiological data. Older patients with shorter time to testing were less likely to have missing ICD-9 codes.