A PCR primer-specific to Cylindrocarpon heteronema for detection of the pathogen in apple wood

Abstract An oligonucleotide primer (ChInt) was synthesised from the variable internally transcribed spacer (ITS) 1 region of ribosomal DNA (rDNA) of Cylindrocarpon heteronema . PCR with primers ChInt and ITS4 (from a conserved sequence of the rDNA) amplified a 470-bp fragment from several isolates of C. heteronema but not from various apple wood saprophytes. Amplification of this fragment was achieved from 1–2 pg of fungal DNA. These primers amplified a fragment of the same size from DNA extracted from cankered wood but only after impurities were removed from the DNA on a Qiagen tip-5 column. Southern hybridization analysis confirmed the 470-bp fragment from C. heteronema DNA and cankered wood to be identical.     

Anterograde trafficking of Toll-like receptors requires the cargo sorting adaptors TMED-2 and 7

Toll-Like Receptors (TLRs) play a pivotal role in immunity by recognising conserved structural features of pathogens and initiating the innate immune response. TLR signalling is subject to complex regulation that remains poorly understood. Here we show that two small type I transmembrane receptors, TMED2 and 7, that function as cargo sorting adaptors in the early secretory pathway are required for transport of TLRs from the ER to Golgi. Protein interaction studies reveal that TMED7 interacts with TLR2, TLR4 and TLR5 but not with TLR3 and TLR9. On the other hand, TMED2 interacts with TLR2, TLR4 and TLR3. Dominant negative forms of TMED7 suppress the export of cell surface TLRs from the ER to the Golgi. By contrast TMED2 is required for the ER-export of both plasma membrane and endosomal TLRs. Together, these findings suggest that association of TMED2 and TMED7 with TLRs facilitates anterograde transport from the ER to the Golgi.     

Pattern-based clustering of daily weigh-in trajectories using dynamic time warping

“Smart”-scales are a new tool for frequent monitoring of weight change as well as weigh-in behavior. These scales give researchers the opportunity to discover patterns in the frequency that individuals weigh themselves over time, and how these patterns are associated with overall weight loss. Our motivating data come from an 18-month behavioral weight loss study of 55 adults classified as overweight or obese who were instructed to weigh themselves daily. Adherence to daily weigh-in routines produces a binary times series for each subject, indicating whether a participant weighed in on a given day. To characterize weigh-in by time-invariant patterns rather than overall adherence, we propose using hierarchical clustering with dynamic time warping (DTW). We perform an extensive simulation study to evaluate the performance of DTW compared to Euclidean and Jaccard distances to recover underlying patterns in adherence time series. In addition, we compare cluster performance using cluster validation indices (CVIs) under the single, average, complete, and Ward linkages and evaluate how internal and external CVIs compare for clustering binary time series. We apply conclusions from the simulation to cluster our real data and summarize observed weigh-in patterns. Our analysis finds that the adherence trajectory pattern is significantly associated with weight loss.     

Enrichment of adeno-associated virus serotype 5 full capsids by anion exchange chromatography with dual salt elution gradients

Adeno-associated virus-based gene therapies have demonstrated substantial therapeutic benefit for the treatment of genetic disorders. In manufacturing processes, viral capsids are produced with and without the encapsidated gene of interest. Capsids devoid of the gene of interest, or “empty” capsids, represent a product-related impurity. As a result, a robust and scalable method to enrich full capsids is crucial to provide patients with as much potentially active product as possible. Anion exchange chromatography has emerged as a highly utilized method for full capsid enrichment across many serotypes due to its ease of use, robustness, and scalability. However, achieving sufficient resolution between the full and empty capsids is not trivial. In this work, anion exchange chromatography was used to achieve empty and full capsid resolution for adeno-associated virus serotype 5. A salt gradient screen of multiple salts with varied valency and Hofmeister series properties was performed to determine optimal peak resolution and aggregate reduction. Dual salt effects were evaluated on the same product and process attributes to identify any synergies with the use of mixed ion gradients. The modified process provided as high as ≥75% AAV5 full capsids (≥3-fold enrichment based on the percent full in the feed stream) with near baseline separation of empty capsids and achieved an overall vector genome step yield of >65%.     

Bioprocess development to produce a hyperthermostable S-methyl-5′-thioadenosine phosphorylase in Escherichia coli

Nucleoside phosphorylases are important biocatalysts for the chemo-enzymatic synthesis of nucleosides and their analogs which are, among others, used for the treatment of viral infections or cancer. S-methyl-5′-thioadenosine phosphorylases (MTAP) are a group of nucleoside phosphorylases and the thermostable MTAP of Aeropyrum pernix (ApMTAP) was described to accept a wide range of modified nucleosides as substrates. Therefore, it is an interesting biocatalyst for the synthesis of nucleoside analogs for industrial and therapeutic applications. To date, thermostable nucleoside phosphorylases were produced in shake flask cultivations using complex media. The drawback of this approach is low volumetric protein yields which hamper the wide-spread application of the thermostable nucleoside phosphorylases in large scale. High cell density (HCD) cultivations allow the production of recombinant proteins with high volumetric yields, as final optical densities >100 can be achieved. Therefore, in this study, we developed a suitable protocol for HCD cultivations of ApMTAP. Initially, optimum expression conditions were determined in 24-well plates using a fed-batch medium. Subsequently, HCD cultivations were performed using E. coli BL21-Gold cells, by employing a glucose-limited fed-batch strategy. Comparing different growth rates in stirred-tank bioreactors, cultivations revealed that growth at maximum growth rates until induction resulted in the highest yields of ApMTAP. On a 500-mL scale, final cell dry weights of 87.1–90.1 g L⁻¹ were observed together with an overproduction of ApMTAP in a 1.9%–3.8% ratio of total protein. Compared to initially applied shake flask cultivations with terrific broth (TB) medium the volumetric yield increased by a factor of 136. After the purification of ApMTAP via heat treatment and affinity chromatography, a purity of more than 90% was determined. Activity testing revealed specific activities in the range of 0.21 ± 0.11 (low growth rate) to 3.99 ± 1.02 U mg⁻¹ (growth at maximum growth rate). Hence, growth at maximum growth rate led to both an increased expression of the target protein and an increased specific enzyme activity. This study paves the way towards the application of thermostable nucleoside phosphorylases in industrial applications due to an improved heterologous expression in Escherichia coli.     

In situ hybridization of atrial natriuretic peptide mRNA in the endothelial cells of human umbilical vessels

The localization of mRNA encoding preproatrial natriuretic peptide (ANP) was investigated in cultured human umbilical vein endothelial cells (HUVEC) and tissue preparations of umbilical vein and artery. The techniques used were in situ hybridization and in situ hybridization combined with immunocytochemistry, using ³²P-radiolabelled and non-radioactive digoxigenin labelled complementary RNA probes. Human ANP mRNAs are mainly localized in the endothelial cells of the umbilical vein and, to a lesser extent, in the endothelial cells of the umbilical artery. The autoradiographic labelling and the intensity of digoxigenin staining were significantly reduced by treatment with RNase before in situ hybridization. This study provides unequivocal evidence for the expression of the ANP gene in the endothelial cells of human umbilical vessels, confirming that these endothelial cells have the ability to synthesize this peptide. The functional significance of the presence of the ANP mRNA in the endothelial cells of human umbilical vessels is discussed.     

ISOLATION AND PROPERTIES OF A CELL FROM LIVER CHARACTERIZED BY LIPID-RICH PARTICLES

A cell has been isolated from explanted rabbit liver which contains, during all phases of its growth in culture, hundreds of lipid-rich particles with a distinct limiting membrane. The cell grows logarithmically with a generation time of 19 to 20 hours and during mitosis the particles are distributed between the daughter cells. Associated with the particles is the high total lipid content of the rabbit liver cell as compared with a rat liver cell, which contains few, if any, lipid-rich particles. This difference in lipid content between the two cells is due primarily to an increase in the triglyceride fraction, in contradistinction to small differences in the polar lipid and sterol ester fractions. The lipid-rich particles have been isolated and found to contain 90 per cent triglyceride on a dry weight basis. The "genetic" factors responsible for the high concentration of lipid-rich particles and triglycerides in the rabbit liver cell require for their full expression one or more factors which are present in much higher effective concentrations in rabbit serum than in horse serum. The hypothesis is advanced that the lipid-rich particles represent a normal state of the non-structural cell lipid. A procedure is described for the quantitative isolation of the lipid of cultured cells.     

THE FLAGELLAR APPARATUS OF HYMENOMONAS CORONATA (PRYMNESIOPHYTA)1

The ultrastructure of Hymenomonas coronata Mills was reinvestigated to determine the microarchitecture of the flagellar apparatus. Cell morphology and flagellar apparatus structure are very similar to those of Pleurochrysis. Some important variations occur. First, a crystalline root (= compound root) is absent on microtubular root 1. Second, a two-stranded microtubular root emanates at a right angle from microtubular root 2. Third, a fibrous root emanates from the dorsal region between the basal bodies and extends to the cell's right, paralleling microtubular root 3. These similarities and variations in flagellar apparatus characters are discussed in reference to known variations in the Prymnesiophyta.