Investigating the inoculum dynamics of Cladosporium on the surface of raspberry fruits and in the air

Raspberry production is under threat from the emerging fungal pathogenic genus Cladosporium. We used amplicon-sequencing, coupled with qPCR, to investigate how fruit age, fruit location within a polytunnel, polytunnel location and sampling date affected the fruit epiphytic microbiome. Fruit age was the most important factor impacting the fungal microbiome, followed by sampling date and polytunnel location. In contrast, polytunnel location and fruit age were important factors impacting the bacterial microbiome composition, followed by the sampling date. The within-tunnel location had a small significant effect on the fungal microbiome and no effect on the bacterial microbiome. As fruit ripened, fungal diversity increased and the bacterial diversity decreased. Cladosporium was the most abundant fungus of the fruit epiphytic microbiome, accounting for nearly 44% of all fungal sequences. Rotorod air samplers were used to study how the concentration of airborne Cladosporium inoculum (quantified by qPCR) varied between location (inside and outside the polytunnel) and time (daytime vs. nighttime). Quantified Cladosporium DNA was significantly higher during the day than the night and inside the polytunnel than the outside. This study demonstrated the dynamic nature of epiphytic raspberry fruit microbiomes and airborne Cladosporium inoculum within polytunnels, which will impact disease risks on raspberry fruit.     

Process-wide control and automation of an integrated continuous manufacturing platform for antibodies

Integrated continuous manufacturing is entering the biopharmaceutical industry. The main drivers range from improved economics, manufacturing flexibility, and more consistent product quality. However, studies on fully integrated production platforms have been limited due to the higher degree of system complexity, limited process information, disturbance, and drift sensitivity, as well as difficulties in digital process integration. In this study, we present an automated end-to-end integrated process consisting of a perfusion bioreactor, CaptureSMB, virus inactivation (VI), and two polishing steps to produce an antibody from an instable cell line. A supervisory control and data acquisition (SCADA) system was developed, which digitally integrates unit operations and analyzers, collects and centrally stores all process data, and allows process-wide monitoring and control. The integrated system consisting of bioreactor and capture step was operated initially for 4 days, after which the full end-to-end integrated run with no interruption lasted for 10 days. In response to decreasing cell-specific productivity, the supervisory control adjusted the loading duration of the capture step to obtain high capacity utilization without yield loss and constant antibody quantity for subsequent operations. Moreover, the SCADA system coordinated VI neutralization and discharge to enable constant loading conditions on the polishing unit. Lastly, the polishing was sufficiently robust to cope with significantly increased aggregate levels induced on purpose during virus inactivation. It is demonstrated that despite significant process disturbances and drifts, a robust process design and the supervisory control enabled constant (optimum) process performance and consistent product quality.     

Prostaglandin EP4 receptor enhances BCR-induced apoptosis of immature B cells

Prostaglandin E2 (PGE2) is emerging as an important co-modulator of B cell responses. Using a pharmacological approach, we aimed to delineate the role of PGE2 in B cell receptor (BCR) induced apoptosis of immature B cells. Gene and protein expression analyses showed that, of the four PGE2 receptors subtypes, only EP4 receptor is upregulated upon BCR cross-linking, leading to sensitization of WEHI 231 cells towards PGE2 mediated inhibitory effects. EP4 receptor antagonist ONO-AE3-208, was able to completely revert the observed effects of PGE2. The engagement of EP4 receptor promotes BCR-induced G0/G1 arrest of WEHI 231 cells, resulting in enhanced caspase mediated, BCR-induced apoptosis. We addressed, mechanistically, the interplay between BCR and EP4 receptor signaling components. Prostaglandin1-alcohol (Pge1-OH), a selective EP4 receptor agonist inhibits BCR-induced activation of NF-κB by suppression of BCR-induced IκBα phosphorylation. Disruption of prosurvival pathways is a possible mechanism by which PGE2 enhances BCR-induced apoptosis in immature B lymphocytes.     

Globular domain of the prion protein needs to be unlocked by domain swapping to support prion protein conversion

Prion diseases are fatal transmissible neurodegenerative diseases affecting many mammalian species. The normal prion protein (PrP) converts into a pathological aggregated form, PrPSc, which is enriched in the β-sheet structure. Although the high resolution structure of the normal PrP was determined, the structure of the converted form of PrP remains inaccessible to high resolution techniques. To map the PrP conversion process we introduced disulfide bridges into different positions within the globular domain of PrP, tethering selected secondary structure elements. The majority of tethered PrP mutants exhibited increased thermodynamic stability, nevertheless, they converted efficiently. Only the disulfides that tether subdomain B1-H1-B2 to subdomain H2-H3 prevented PrP conversion in vitro and in prion-infected cell cultures. Reduction of disulfides recovered the ability of these mutants to convert, demonstrating that the separation of subdomains is an essential step in conversion. Formation of disulfide-linked proteinase K-resistant dimers in fibrils composed of a pair of single cysteine mutants supports the model based on domain-swapped dimers as the building blocks of prion fibrils. In contrast to previously proposed structural models of PrPSc suggesting conversion of large secondary structural segments, we provide evidence for the conservation of secondary structural elements of the globular domain upon PrP conversion. Previous studies already showed that dimerization is the rate-limiting step in PrP conversion. We show that separation and swapping of subdomains of the globular domain is necessary for conversion. Therefore, we propose that the domain-swapped dimer of PrP precedes amyloid formation and represents a potential target for therapeutic intervention.     

Selective Cytotoxicity of Amidinopiperidine Based Compounds Towards Burkitt's Lymphoma Cells Involves Proteasome Inhibition

Serine proteases have proven to be promising pharmacological targets in contemporary drug discovery for cancer treatment. Since azaphenylalanine-based compounds manifest cytotoxic activity, we have selected serine protease inhibitors designed and synthesized in-house with large hydrophobic naphthalene moiety for screening. The cytotoxic potential of screened molecules was correlated to modifications of R(1) residues. The most cytotoxic were compounds with greater basicity; amidinopiperidines, piperidines and benzamidines. Amidinopiperidine-based compounds exert cytotoxicity in low μM range, with IC(50) 18 μM and 22 μM for inhibitors 15 and 16 respectively. These compounds exhibited selective cytotoxicity towards the Burkitt's lymphoma cells Ramos and Daudi, and proved nontoxic to PMBC, Jurkat and U937. They induce caspase-dependent apoptotic cell death, as demonstrated by the use of a pan-caspase inihibitor, zVADfmk, which was able to rescue Ramos cells from compound(s)-induced apoptosis. We confirm a disruption of the pro-survival pathway in Burkitt's lymphoma through NFκB inhibition. The accumulation of phosphorylated precursor (p105) and inhibitory (IκB) molecules with no subsequent release of active NFκB implicated the involvement of proteasome. Indeed, we show that the amidinopiperidine-based compounds inhibit all three proteolytical activities of the human 20S proteasome, with the most prominent effect being on the trypsin-like activity. Consistently, treatment of Ramos cells with these compounds led to an increase in ubiquitinated proteins. The amidinopiperidine-based serine protease inhibitors presented are, as selective inducers of apoptosis in Burkitt's lymphoma cells, promising leads for the development of novel chemotherapeutics.     

Iron enriched yeast biomass--a promising mineral feed supplement

Yeast biomass enriched with iron could represent a new and safer solution for prevention from anaemia development. Such an iron source is less toxic and has better absorbability in organisms. The purpose of our research was the determination of the most suitable iron source in the cultivation medium for the yeast Saccharomyces cerevisiae, regarding good growth and iron accumulation in cells. Iron(III) citrate, iron(III) chloride, iron(III) nitrate and Fe-EDTA complex were used. The uptake of the chosen iron compound, Fe(III) citrate, by the yeasts Candida intermedia and Kluyveromyces marxianus was also investigated. Different growth behaviour of the three yeast strains in the presence of Fe(III) citrate was observed. The highest amounts of accumulated iron in S. cerevisiae, C. intermedia and K. marxianus biomass were about 13, 20 and 34mgFeg(-1)dry wt., respectively. To optimise the accumulation of iron in K. marxianus and to characterise iron enriched yeast biomass, further experiments are needed.     

Biogenic volatile organic compounds as potential carbon sources for microbial communities in soil from the rhizosphere of Populus tremula

Catabolism of a (14)C-labelled volatile monoterpene compound (geraniol) to (14)CO(2) was investigated in soils taken from the rhizosphere at distances up to 200 cm from the trunks of three small Populus tremula trees growing at different sites in Slovenia. Emissions of limonene of up to 18 microg m(-2) h(-1) were detected from the soil surface at each site. Evolution of (14)C-labelled CO(2) was measured as a product of catabolism of (14)C-labelled geraniol introduced into the soil samples. Indigenous soil microorganisms degraded the geraniol rapidly. There was a significant difference in relative lag times and rates of catabolism along the gradient from the tree trunks, with relatively longer lag times and lower rates occurring in soil samples from the farthest point from the tree.     

Nucleus pulposus repair with cultured autologous elastic cartilage derived chondrocytes

Low back pain is one of the most common medical conditions in the Western world. Disc degeneration, an inevitable process of ageing, is one of the major causes of low back pain. Autologous chondrocyte transplantation (ACT) is an increasingly popular method of addressing pathological disorders of cartilage. The purpose of our study was to determine whether autologous chondrocytes from elastic cartilage could survive and synthesise a cartilage specific matrix in the intervertebral disc of rabbits. Sixteen lumbar intervertebral discs (IVD) of New Zealand White rabbits were analysed. In 6 IVD, the nucleus pulposus was evacuated and replaced with tissue engineered autologous chondrocytes from auricular cartilage. In the second group, only the nucleus pulposus was evacuated from 6 IVD, with no chondrocytes implantation. Four non-operated IVD were used as a control. Six months after the operation, the animals were euthanized and the IVD were analysed histologically. Autologous cartilage implants were well tolerated by the host for up to six months in vivo. There was only hyaline-like cartilage in the place of the nucleus pulposus. We could not detect any elastic fibres in the new cartilage matrix. In IVD from which only the nucleus pulposus was evacuated and no chondrocytes were implanted, just fibrous tissue was found instead of nucleus pulposus. The overall histological analysis of new cartilage produced after implantation in our study confirmed the hypothesis that ACT from auricular cartilage can be implanted into the IVD instead of the nucleus pulposus and that a significant percentage of implanted chondrocytes survive and produce hyaline-like cartilage.