Cell bank characterization and fermentation optimization for production of recombinant heavy chain C-terminal fragment of botulinum neurotoxin serotype E (rBoNTE(H(c)): antigen E) by Pichia pastoris

A process was developed for production of a candidate vaccine antigen, recombinant C-terminal heavy chain fragment of the botulinum neurotoxin serotype E, rBoNTE(H(c)) in Pichia pastoris. P. pastoris strain GS115 was transformed with the rBoNTE(H(c)) gene inserted into pHILD4 Escherichia coli-P. pastoris shuttle plasmid. The clone was characterized for genetic stability, copy number, and BoNTE(H(c)) sequence. Expression of rBoNTE(H(c)) from the Mut(+) HIS4 clone was confirmed in the shake-flask, prior to developing a fed-batch fermentation process at 5 and 19 L scale. The fermentation process consists of a glycerol growth phase in batch and fed-batch mode using a defined medium followed by a glycerol/methanol transition phase for adaptation to growth on methanol and a methanol induction phase resulting in the production of rBoNTE(H(c)). Specific growth rate, ratio of growth to induction phase, and time of induction were critical for optimal rBoNTE(H(c)) production and minimal proteolytic degradation. A computer-controlled exponential growth model was used for process automation and off-gas analysis was used for process monitoring. The optimized process had an induction time of 9 h on methanol and produced up to 3 mg of rBoNTE(H(c)) per gram wet cell mass as determined by HPLC and Western blot analysis.     

A simple and inexpensive device for collecting urine samples from rats

Many studies require collection of metabolic wastes from laboratory animals, and oftentimes it is important that feces and urine be collected separately. The authors describe an easily assembled and inexpensive device that can be used to collect urine samples from rats without any invasive operations. The device affords reasonable separation of feces and urine.     

Potassium in agriculture – Status and perspectives

In this review we summarize factors determining the plant availability of soil potassium (K), the role of K in crop yield formation and product quality, and the dependence of crop stress resistance on K nutrition. Average soil reserves of K are generally large, but most of it is not plant-available. Therefore, crops need to be supplied with soluble K fertilizers, the demand of which is expected to increase significantly, particularly in developing regions of the world. Recent investigations have shown that organic exudates of some bacteria and plant roots play a key role in releasing otherwise unavailable K from K-bearing minerals. Thus, breeding for genotypes that have improved mechanisms to gain access to this fixed K will contribute toward more sustainable agriculture, particularly in cropping systems that do not have access to fertilizer K. In K-deficient crops, the supply of sink organs with photosynthates is impaired, and sugars accumulate in source leaves. This not only affects yield formation, but also quality parameters, for example in wheat, potato and grape. As K has beneficial effects on human health, its concentration in the harvest product is a quality parameter in itself. Owing to its fundamental roles in turgor generation, primary metabolism, and long-distance transport, K plays a prominent role in crop resistance to drought, salinity, high light, or cold as well as resistance to pests and pathogens. Despite the abundance of vital roles of K in crop production, an improvement of K uptake and use efficiency has not been a major focus of conventional or transgenic breeding in the past. In addition, current soil analysis methods for K are insufficient for some common soils, posing the risk of imbalanced fertilization. A stronger prioritization of these areas of research is needed to counter declines in soil fertility and to improve food security.     

MRI detection of thrombin with aptamer functionalized superparamagnetic iron oxide nanoparticles

Design of smart MRI contrast agent based on superparamagnetic iron oxide nanoparticles and aptamers has been described for the detection of human alpha-thrombin protein. The contrast agent is based on the assembly of the aptamer functionalized nanoparticles in the presence of thrombin. A detectable change in MRI signal is observed with 25 nM thrombin in human serum. Changes were neither observed with control analytes, streptavidin, or bovine serum albumin, nor with inactive aptamer functionalized nanoparticles.     

Proteomics analysis of meclofenamic acid-treated small cell lung carcinoma cells revealed changes in cellular energy metabolism for cancer cell survival

Small cell lung carcinoma (SCLC) is a highly aggressive cancer with low survival rate. Although initial response to chemotherapy in SCLC patients is well-rated, the treatments applied after the disease relapses are not successful. Drug resistance is accepted to be one of the main reasons for this failure. Therefore, there is an urgent need for new treatment strategies for SCLC. Meclofenamic acid, a nonsteroidal anti-inflammatory drug, has been shown to have anticancer effects on various types of cancers via different mechanisms. The aim of this study was to investigate the alterations that meclofenamic acid caused on a SCLC cell line, DMS114 using the tools of proteomics namely two-dimensional gel electrophoresis coupled to MALDI-TOF/TOF and nHPLC coupled to LC-MS/MS. Among the proteins identified by both methods, those showing significantly altered expression levels were evaluated using bioinformatics databases, PANTHER and STRING. The key altered metabolism upon meclofenamic acid treatment appeared to the cellular energy metabolism. Glycolysis was suppressed, whereas mitochondrial activity and oxidative phosphorylation were boosted. The cells underwent metabolic reprogramming to adapt into their new environment for survival. Metabolic reprogramming is known to cause drug resistance in several cancer types including SCLC. The identified differentially regulated proteins in here associated with energy metabolism hold value as the potential targets to overcome drug resistance in SCLC treatment.     

Identification of copy number variants in children and adolescents with autism spectrum disorder: a study from Turkey

Molecular Biology Reports - Copy number variants (CNVs) play a key role in the etiology of autism spectrum disorder (ASD). Therefore, recent guidelines recommend chromosomal microarrays (CMAs) as...     

The role of curcumin on intestinal oxidative stress,cell proliferation and apoptosis after ischemia/reperfusion injury in rats

The aim of this study was to demonstrate the role of curcumin on oxidative stress, cell proliferation and apoptosis in the rat intestinal mucosa after ischemia/reperfusion (I/R). A total of 30 male Wistar albino rats were divided into three groups: sham, I/R and I/R+ curcumin; each group contain 10 animals. Sham group animals underwent laparotomy without I/R injury. After I/R groups animals underwent laparotomy, 1 h of superior mesenteric artery ligation were followed by 1 h of reperfusion. In the curcumin group, 3 days before I/R, curcumin (100 mg/kg) was administered by gastric gavage. All animals were sacrificed at the end of reperfusion and intestinal tissues samples were obtained for biochemical and histopathological investigation in all groups. Curcumin treatment significantly decreased the elevated tissue malondialdehyde levels and increased of reduced superoxide dismutase, and glutathione peroxidase enzyme activities in intestinal tissues samples. I/R caused severe histopathological injury including mucosal erosions and villous congestion and hemorrhage. Curcumin treatment significantly attenuated the severity of intestinal I/R injury, with inhibiting of I/R-induced apoptosis and cell proliferation. These results suggest that curcumin treatment has a protective effect against intestinal damage induced by intestinal I/R. This protective effect is possibly due to its ability to inhibit I/R-induced oxidative stress, apoptosis and cell proliferation.     

Persistence of hyperbaric oxygen-induced oxidative effects after exposure in rat brain cortex tissue

Hyperbaric oxygen (HBO) causes oxidative stress in several organs and tissues. Due to its high rate of blood flow and oxygen consumption, the brain is one of the most sensitive organs to this effect. Many studies have reported oxidative effects of HBO, but there is no comprehensive data about how long this effect persists. The aim of this study was to elucidate the duration of HBO-induced oxidative/antioxidant action. Male Sprague-Dawley rats were divided into 5 groups. Except for the controls, the animals were subjected to 100% oxygen for 2 h at 3 atm and differed from each other by the time to dissection after exposure that began at 30, 60, 90, or 120 min. Thiobarbituric acid-reactive substances (TBARS), as well as superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activity was determined in brain cortex tissue. Additionally, nitrite-nitrate (NO(x)) concentrations were measured. All measured parameters were found to be significantly increased 30 min after exposure. SOD and GSH-Px levels persisted at significantly high levels for 60 min. In conclusion, the oxidative effect of HBO was shown to persist only for 1 h. Further studies should be performed to elucidate the possible molecular interactions during this period.     

Ways of coping as predictors of satisfaction with curriculum and academic success in medical school

The purpose of the present study was to determine the coping strategies of medical students and to investigate the effects of coping strategies on student satisfaction and academic achievement with different instruction methods. A total of 152 medical students was followed throughout the first 2 yr of medical education between 2008 and 2010. Students completed a sociodemographic questionnaire and revised form of the Ways of Coping questionnaire both at the beginning of the first year and at the end of the second year. These forms provided data about the characteristics and main coping strategies (problem focused or emotion focused) of the students and revealed the change over time. At the end of the second year, participants also completed a satisfaction questionnaire asking their satisfaction with lectures, problem-based learning, and practicals. The authors used block, problem-based learning, and practical exam scores of the students attained in the past 2 yr as academic achievement indicators. No sociodemographic variable was related to coping strategy. The majority of students (80.9%) adopted problem-focused coping. A shift occurred in the main coping strategies of some students in both sides. Problem-focused coping scores decreased over time. Problem-focused coping positively correlated with satisfaction with practicals and practical exam scores, whereas emotion-focused coping showed the same correlation negatively. The main coping strategy also predicted satisfaction and exam success in practicals. In conclusion, a main coping strategy may be helpful to predict student satisfaction and academic achievement with some student-centered instruction methods. Determining undesired coping strategies may provide an opportunity for intervention to prevent relevant dissatisfaction and failure.