On‐line yeast propagation process monitoring and control using an intelligent automatic control system

The monitoring and control of bioprocesses is a challenging task. This applies particularly if the actions to the process have to be carried out in real‐time. This work presents a system for on‐line monitoring and control of batch yeast propagation under limiting conditions based on a virtual plant operator, which uses the concept of intelligent control algorithms by means of fuzzy logic theory. Process information is provided on‐line using a sensor array comprising the measurement of OD, operating temperature, pressure, density, dissolved oxygen, and pH value. In this context practical problems arising through on‐line sensing and signal processing are addressed. The preprocessed sensor data are fed to a neural network for on‐line biomass estimation. The root mean squared error of prediction is 4 × 10⁶ cells/mL. The proposed system then triggers temperature and aeration by usage of a temperature dependent metabolic growth model and sensor data. The deviation of the predicted biomass from that of the reference trajectory as modeled by the metabolic growth model and its temporal derivative are used as inputs for the fuzzy temperature controller. The inputs used by the fuzzy aeration controller are the deviation of measured extract from that of the reference trajectory, the predicted cell count, and the dissolved oxygen concentration. The fuzzy‐based expert system allows to provide the desired yeast cell concentration of 100–120 × 10⁶ cells/mL at a minimum residual extract limit of 6.0 g/100 g at the required point of time. Thus, a dynamic adjustment of the propagation process to the overall production schedule is possible in order to produce the required amount of biomass at the right time.     

IL22/IL-22R Pathway Induces Cell Survival in Human Glioblastoma Cells

Interleukin-22 (IL-22) is a member of the IL-10 cytokine family that binds to a heterodimeric receptor consisting of IL-22 receptor 1 (IL-22R1) and IL-10R2. IL-22R expression was initially characterized on epithelial cells, and plays an essential role in a number of inflammatory diseases. Recently, a functional receptor was detected on cancer cells such as hepatocarcinoma and lung carcinoma, but its presence was not reported in glioblastoma (GBM). Two GBM cell lines and 10 primary cell lines established from patients undergoing surgery for malignant GBM were used to investigate the expression of IL-22 and IL-22R by using quantitative RT-PCR, western blotting and confocal microscopy studies. The role of IL-22 in proliferation and survival of GBM cell lines was investigated in vitro by BrdU and ELISA cell death assays. We report herein that the two subunits of the IL-22R complex are expressed on human GBM cells. Their activation, depending on exogenous IL-22, induced antiapoptotic effect and cell proliferation. IL-22 treatment of GBM cells resulted in increased levels of phosphorylated Akt, STAT3 signaling protein and its downstream antiapoptotic protein Bcl-xL and decreased level of phosphorylated ERK1/2. In addition, IL-22R subunits were expressed in all the 10 tested primary cell lines established from GBM tumors. Our results showed that IL-22R is expressed on GBM established and primary cell lines. Depending on STAT3, ERK1/2 and PI3K/Akt pathways, IL-22 induced GBM cell survival. These data are consistent with a potential role of IL-22R in tumorigenesis of GBM. Since endogenous IL-22 was not detected in all studied GBM cells, we hypothesize that IL-22R could be activated by immune microenvironmental IL-22 producing cells.     

Within-host Competition Does Not Select for Virulence in Malaria Parasites; Studies with Plasmodium yoelii

In endemic areas with high transmission intensities, malaria infections are very often composed of multiple genetically distinct strains of malaria parasites. It has been hypothesised that this leads to intra-host competition, in which parasite strains compete for resources such as space and nutrients. This competition may have repercussions for the host, the parasite, and the vector in terms of disease severity, vector fitness, and parasite transmission potential and fitness. It has also been argued that within-host competition could lead to selection for more virulent parasites. Here we use the rodent malaria parasite Plasmodium yoelii to assess the consequences of mixed strain infections on disease severity and parasite fitness. Three isogenic strains with dramatically different growth rates (and hence virulence) were maintained in mice in single infections or in mixed strain infections with a genetically distinct strain. We compared the virulence (defined as harm to the mammalian host) of mixed strain infections with that of single infections, and assessed whether competition impacted on parasite fitness, assessed by transmission potential. We found that mixed infections were associated with a higher degree of disease severity and a prolonged infection time. In the mixed infections, the strain with the slower growth rate was often responsible for the competitive exclusion of the faster growing strain, presumably through host immune-mediated mechanisms. Importantly, and in contrast to previous work conducted with Plasmodium chabaudi, we found no correlation between parasite virulence and transmission potential to mosquitoes, suggesting that within-host competition would not drive the evolution of parasite virulence in P. yoelii.     

Evaluation and identification of hepatitis B virus entry inhibitors using HepG2 cells overexpressing a membrane transporter NTCP

Hepatitis B virus (HBV) entry has been analyzed using infection-susceptible cells, including primary human hepatocytes, primary tupaia hepatocytes, and HepaRG cells. Recently, the sodium taurocholate cotransporting polypeptide (NTCP) membrane transporter was reported as an HBV entry receptor. In this study, we established a strain of HepG2 cells engineered to overexpress the human NTCP gene (HepG2-hNTCP-C4 cells). HepG2-hNTCP-C4 cells were shown to be susceptible to infection by blood–borne and cell culture-derived HBV. HBV infection was facilitated by pretreating cells with 3% dimethyl sulfoxide permitting nearly 50% of the cells to be infected with HBV. Knockdown analysis suggested that HBV infection of HepG2-hNTCP-C4 cells was mediated by NTCP. HBV infection was blocked by an anti-HBV surface protein neutralizing antibody, by compounds known to inhibit NTCP transporter activity, and by cyclosporin A and its derivatives. The infection assay suggested that cyclosporin B was a more potent inhibitor of HBV entry than was cyclosporin A. Further chemical screening identified oxysterols, oxidized derivatives of cholesterol, as inhibitors of HBV infection. Thus, the HepG2-hNTCP-C4 cell line established in this study is a useful tool for the identification of inhibitors of HBV infection as well as for the analysis of the molecular mechanisms of HBV infection.     

How does a hopping kangaroo breathe?

We developed a model to demonstrate how a hopping kangaroo breathes. Interestingly, a kangaroo uses less energy to breathe while hopping than while standing still. This occurs, in part, because rather than using muscle power to move air into and out of the lungs, air is pulled into (inspiration) and pushed out of (expiration) the lungs as the abdominal organs "flop" within the kangaroo's body. Specifically, as the kangaroo hops upward, the abdominal organs lag behind, and the insertion of the diaphragm is pulled toward its origin, flattening the dome and increasing the vertical dimension of the thoracic cavity (the thoracic cavity and lungs enlarge). Increasing the volume of the thoracic cavity reduces alveolar pressure below atmospheric pressure (barometric pressure), and air moves into the alveoli by bulk flow. In contrast, the impact of the organs against the diaphragm at each landing causes expiration. Specifically, upon landing, the abdominal organs flop into the diaphragm, causing it to return to its dome shape and decreasing the vertical dimension of the thoracic cavity. This compresses the alveolar gas volume and elevates alveolar pressure above barometric pressure, so air is expelled. To demonstrate this phenomenon, the plunger of a syringe model of the respiratory system was inserted through a compression spring. Holding the syringe and pressing the plunger firmly against a hard surface expels air from the lungs (the balloon within the syringe deflates) and compresses the spring. This models the kangaroo landing after a hop forward. Subsequently, the compression spring provides the energy for the "kangaroo" to "hop" forward upon the release of the syringe, and air enters the lungs (the balloon within the syringe inflates). The model accurately reflects how a hopping kangaroo breathes. A model was chosen to demonstrate this phenomenon because models engage and inspire students as well as significantly enhance student understanding.     

A fast algal bioassay for assessment of copper toxicity in water using <Emphasis Type="Italic">Euglena gracilis</Emphasis>

A rapid, sensitive algal bioassay was investigated to monitor copper toxicity using photosynthetic activity and motility parameters as end points in the photosynthetic flagellate Euglena gracilis. Effects on motility parameters were determined using the automatic bioassay ECOTOX. Photosynthetic efficiency was measured using chlorophyll fluorescence by means of a fast, noninvasive pulse amplitude modulated fluorometer. These parameters were assessed regarding their effectiveness as end points for short- (0–24 h) and long-term (1–5 days) toxicity tests. The model organism showed significant responses to the tested concentrations at both motility and photosynthesis examined levels. EC₅₀ values for movement parameters immediately after the cells were mixed with copper are 19.09 (300.4 μM), 20.4 (312 μM) and 23.09 (363.35 μM) mg L⁻¹ for motility, r-value and velocity, respectively. The current study proofs that Euglena is a convenient biotest organism where motility parameters show rapid sensitive responses to copper (direct exposure to copper). Also, the photosynthetic parameters appear to be appropriate for acute and chronic toxicity tests for higher and lower copper concentrations, respectively.     

The effect of diode laser and light emitting diode on the bacterial contamination of semen medium for artificial insemination.

Animal artificial insemination (AI) suffers from bacterial contamination of semen media which results in decreased success of the process of artificial insemination. It is difficult to treat the semen extender medium to reduce the bacterial growth in the presence of semen using the conventional techniques of bacterial inhibition. In the present work, a new optical method developed for bacterial growth inhibition in semen containing extender medium using diode laser (DL) and commercial cheap light emitting diode (LED) is presented. Certain wavelengths and exposure times suitable for the process of artificial insemination are found to be optimum at reducing bacterial growth with a minimum significant effect on the semen motility and viability.     

Adhesion of Yersinia enterocolitica to non-cultured epithelial cells from pig and rabbit ilea

A simple and reliable method was developed to isolate intact epithelial cells from pig and rabbit ilea and these were used to investigate the adhesion of Yersinia enterocolitica. Hydrophobic interaction was eliminated by treating the bacterial culture with 0.8 M tetramethyl urea (TMU). Virulent strains of Y. enterocolitica had significantly greater attachment than avirulent strains but both attached in a linear dose-dependant fashion. Epithelial cells prepared from pig ilea were attached to more readily than those prepared from rabbit ilea.     

Phenology, distribution, and host specificity of Solenopsis invicta virus-1

Studies were conducted to examine the phenology, geographic distribution, and host specificity of the Solenopsis invicta virus-1 (SINV-1). Two genotypes examined, SINV-1 and -1A, exhibited similar seasonal prevalence patterns. Infection rates among colonies of S. invicta in Gainesville, Florida, were lowest from early winter (December) to early spring (April) increasing rapidly in late spring (May) and remaining high through August before declining again in the fall (September/October). Correlation analysis revealed a significant relationship between mean monthly temperature and SINV-1 (p<0.0005, r=0.82) and SINV-1A (p<0.0001, r=0.86) infection rates in S. invicta colonies. SINV-1 was widely distributed among S. invicta populations. The virus was detected in S. invicta from Argentina and from all U.S. states examined, with the exception of New Mexico. SINV-1 and -1A were also detected in other Solenopsis species. SINV-1 was detected in Solenopsis richteri and the S. invicta/richteri hybrid collected from northern Alabama and Solenopsis geminata from Florida. SINV-1A was detected in S. geminata and Solenopsis carolinensis in Florida and the S. invicta/richteri hybrid in Alabama. Of the 1989 arthropods collected from 6 pitfall trap experiments from Gainesville and Williston, Florida, none except S. invicta tested positive for SINV-1 or SINV-1A. SINV-1 did not appear to infect or replicate within Sf9 or Dm-2 cells in vitro. The number of SINV-1 genome copies did not significantly increase over the course of the experiment, nor were any cytopathic effects observed. Phylogenetic analyses of SINV-1/-1A nucleotide sequences indicated significant divergence between viruses collected from Argentina and the U.S.