Early changes in gene expression in two models of Batten disease

Infantile and juvenile neuronal ceroid lipofuscinosis (NCLs) are progressive neurodegenerative disorders of childhood with distinct ages of clinical onset, but with a similar pathological outcome. Infantile and juvenile NCL are inherited in an autosomal recessive manner due to mutations in the CLN1 and CLN3 genes, respectively. Recently developed Cln1- and Cln3-knockout mouse models share similarities in pathology with the respective human disease. Using oligonucleotide arrays we identified reproducible changes in gene expression in the brains of both 10-week-old Cln1- and Cln3-knockout mice as compared to wild-type controls, and confirmed changes in levels of several of the cognate proteins by immunoblotting. Despite the similarities in pathology, the two mutations affect the expression of different, non-overlapping sets of genes. The possible significance of these changes and the pathological mechanisms underlying NCL diseases are discussed.     

Elimination of ergoline alkaloids following treatment of<Emphasis Type="Italic"> Ipomoea asarifolia</Emphasis> (Convolvulaceae) with fungicides

Ergoline alkaloids are constituents of Clavicipitaceous fungi living on Poaceae plants. Ergoline alkaloids as well as volatile oil are also present in Ipomoea asarifolia Roem. & Schult (Convolvulaceae). Treatment of this plant with two fungicides (Folicur, Pronto Plus) eliminates the ergoline alkaloids but not the volatile oil. Elimination of ergoline alkaloids occurs concomitantly with loss of fungal hyphae associated with secretory glands on the upper leaf surface of the Ipomoea plant. Our observations suggest that accumulation of ergoline alkaloids in the Convolvulaceae may depend on the presence of a plant-associated fungus.Dedicated to Wolfgang Steglich, München, on the occasion of his 70th birthday     

Determination of endogenous extracellular signal-regulated protein kinase by microchip capillary electrophoresis

The application of microchip capillary electrophoresis (CE) to the assay of extracellular signal-regulated protein kinase (ERK) is presented. In this assay, ERK catalyzes the transfer of gamma-phosphate from adenosine 5(')-triphosphate to the threonine residue of a fluorescently labeled nonapeptide (APRTPGGRR), and the phosphorylated and nonphosphorylated peptides were detected by fluorescence. The phosphorylated and nonphosphorylated peptides and the internal standard were separated within 20s, and the increase in magnitude of the phosphorylated peptide peak was monitored to assess ERK activity. ERK reactions were prepared off-chip and analyzed on a single-lane glass microchip fabricated by standard methods. It was demonstrated that microchip CE could be used to measure endogenous amounts of ERK by spiking known concentrations of recombinant ERK2 into the lysates of serum-starved human umbilical vein endothelial cells (HUVEC) and recovering between 90 and 100% for all samples. Endogenous ERK activity was determined by microchip where HUVEC were stimulated with 500pM vascular endothelial growth factor (VEGF) at different times before cell lysis. The results showed a transient VEGF-mediated ERK activation that peaked at 10min, which was consistent with previous reports using conventional techniques. The microchip assay provided a rapid, accurate, and precise alternative to conventional methods of determining endogenous ERK activity.     

Lipase production from a novel thermophilic Bacillus sp.: application of Plackett-Burman design for evaluating culture conditions affecting enzyme formation

A novel thermophilic Bacillus sp. capable of producing lipase was locally isolated. Phylogenetic analysis based on 16SrDNA sequence revealed its close relationship to Bacillus thermoleovorans. Plackett-Burman experimental design was used to evaluate cultural conditions affecting lipase production process. Fifteen variables and four dummy variables were examined in the experimental design. Tween 80, temperature, olive oil, aeration, beef extract and inoculum age were found to be the highest positive significant variables affecting lipase activity, whereas pH and calcium chloride were the highest negative significant variables. Moreover, Tween 80, temperature and olive oil positively affected lipase specific activity. On the other hand, gum arabic, inoculum size and calcium chloride had the highest negative effect on lipase specific activity. This study would improve the further optimization steps on the bioprocess development track.     

Decision making using hybrid rough sets and neural networks

A methodology for using rough sets theory for preference modeling in decision problem is presented in this paper. We will introduce a new method where neural network systems and rough sets theory are completely integrated into a hybrid system and are used cooperatively for decision and classification support. At the first glance, the two methods we discuss have not much in common. But, in spite of the differences between them, it is interesting to try to incorporate both into one combined system, and apply it in the building of a decision support system.     

Investigation of a failed DNA assay leads to identification of an unexpected contaminant in a commonly used chromatography buffer

For mammalian cell-derived recombinant biotherapeutics, controlling host cell DNA levels below a threshold is a regulatory requirement to ensure patient safety. DNA removal during drug substance manufacture is accomplished by a series of chromatography-based purification steps and a qPCR-based analytical method is most used to measure DNA content in the purified drug substance to enable material disposition. While the qPCR approach is mature and its application to DNA measurement is widespread in the industry, it is susceptible to trace levels of process-related contaminants that are carried forward. In this study, we observed failures in spike recovery studies that are an integral component of the qPCR-based DNA testing, suggesting the presence of an inhibitory compound in the sample matrix. We generated hypotheses around the origin of the inhibitory compound and generated multiple sample matrices and deployed a suite of analytical techniques including Raman and NMR spectroscopy to determine the origin and identity of the inhibitory compound. The caustic wash step and depth filter extractables were ruled out as root causes after extensive experimentation and DNA testing. Subsequently, 2-(N-morpholino)ethanesulfonic acid (MES), a buffer used in the chromatography unit operations, was identified as the source of the contaminant. A 500-fold concentration followed by Raman and NMR spectroscopy analysis revealed the identity of the inhibitory compound as polyvinyl sulfone (PVS), an impurity that originates in the MES manufacturing process. We have implemented PVS concentration controls for incoming MES raw material, and our work highlights the need for rigor in raw material qualification and control.     

Recent advances in mRNA-based vaccine for cancer therapy; bench to bedside

The messenger RNA (mRNA) vaccines have progressed from a theoretical concept to a clinical reality over the last few decades. Compared to conventional vaccination methods, these vaccines have a number of benefits, such as substantial potency, rapid growth, inexpensive production, and safe administration. Nevertheless, their usefulness was restricted up to now due to worries about the erratic and ineffective circulation of mRNA in vivo. Thankfully, these worries have largely been allayed by recent technological developments, which have led to the creation of multiple mRNA vaccination platforms for cancer and viral infections. The mRNA vaccines have been demonstrated as a powerful alternative to traditional conventional vaccines because of their high potency, safety and efficacy, capacity for rapid clinical development, and potential for rapid, low-cost manufacturing. The paper will examine the present status of mRNA vaccine technology and suggest future paths for the advancement and application of this exciting vaccine platform as a common therapeutic choice.     

Synthesis and characterization of high-sensitivity Dy,Eu co-doped CaSO4 thermoluminescent phosphor using coprecipitation technique

In this work, (99 − x)CaSO₄-Dy₂O₃–xEu₂O₃, (where x = 0, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5) thermoluminescence phosphors were prepared using a coprecipitation method. The thermoluminescence (TL) dosimetry (TLD) characteristics such as TL sensitivity, dose–response, minimum detectable dose, thermal fading, and the effect of sunlight on the prepared phosphors were investigated. The obtained results indicated that the most sensitive phosphor was obtained at x = 0.05. Large thermal fading of 6% after 1 h and 26% after 24 h from irradiation followed by 71% after 1 month with no additional fading was observed within a time frame exceeding 2 months throughout the remaining duration of the investigation, which also spanned over 2 months. Despite the phosphor's high fading rate, the relative sensitivity of the prepared samples was ~90% compared with TLD-100. The marked effect of day sunlight was also determined. High dose–response within the low-dose range from 0.01 to 5 Gy was observed. The obtained results suggested that the synthesized phosphor is well suited for applications involving radiation biology and radiotherapy dosimetry.     

Determination the population trends of cereal aphids and associated parasitoids by yellow sticky traps with reference to aphid management on wheat

Population trends of cereal aphids and their associated parasitoids inhabiting wheat plantations were monitored by yellow sticky traps. The identified aphids exhibited one seasonal peak for each and were found to be active during the first half of March. Data revealed that, Rhopalosiphum padi L peak occurred one week later than both of Schizaphis graminum (Rondani) and Rhopalosiphum maidis (Fitch). The numbers of aphid complex was recorded more at 90 than at 30 and 60 cm height. Hymenopterous parasitoids activity is synchronised with aphid species, whereas they appeared in small numbers at early and late wheat season and showed their peak on March, with a positive correlation coefficient with aphid populations. The tested compounds (Karate, Biscaya, Match 5% EC, Tracer 24% SC and Neem Azal T/S) showed 100% reduction in aphid numbers after 24 h post application. However, the general reduction percentages indicated 99.31 > 98.32 > 97.97 > 97.06 > 95.66%, by using the abovementioned compounds, respectively.     

Interaction of FXYD10 (PLMS) with Na,K-ATPase from shark rectal glands. Close proximity of Cys74 of FXYD10 to Cys254 in the a domain of the alpha-subunit revealed by intermolecular thiol cross-linking

FXYD domain-containing proteins are tissue-specific regulators of the Na,K-ATPase that have been shown to have significant physiological implications. Information about the sites of interaction between some FXYD proteins and subunits of the Na,K-ATPase is beginning to emerge. We previously identified an FXYD protein in plasma membranes from shark rectal gland cells and demonstrated that this protein (FXYD10) modulates shark Na,K-ATPase activity. The present study was undertaken to identify the location of the C-terminal domain of FXYD10 on the alpha-subunit of Na,K-ATPase, using covalent cross-linking combined with proteolytic cleavage. Treatment of Na,K-ATPase-enriched membranes with the homobifunctional thiol cross-linker 1,4-bismaleimidyl-2,3-dihydroxybutane resulted in cross-linking of FXYD10 to the alpha-subunit. Cross-linking was not affected by preincubation with sodium or potassium but was significantly reduced after pre-incubation with the non-hydrolyzable ATP analog beta,gamma-methyleneadenosine 5'-triphosphate (AMP-PCP). A peptic assay was developed, in which pepsin treatment of Na,K-ATPase at low pH resulted in extensive cleavage of the alpha-subunit while FXYD10 was left intact. Proteolytic fragments of control and cross-linked preparations were isolated by immunoprecipitation and analyzed by gel electrophoresis. A proteolytic fragment containing FXYD10 cross-linked to a fragment from the alpha-subunit could be localized on SDS gels. Sequencing of this fragment showed the presence of FXYD10 as well as a fragment within the A domain of the alpha-subunit comprising 33 amino acids, including a single Cys residue, Cys254. Thus, regulation of Na,K-ATPase by FXYD10 occurs in part via cytoplasmic interaction of FXYD10 with the A domain of the shark alpha-subunit.