On the choice of the optimal periodic operation for a continuous fermentation process

In this contribution we investigate the impact of the forcing waveform on the productivity of a continuous bioreactor governed by an unstructured, nonlinear kinetic model. The (periodic) forcing is applied on the substrate concentration in the feed. To this end, some alternative waveforms commonly encountered in practice are evaluated and their performance is compared. An analytical/numerical approach is used. The preliminary analytical step is based on the π‐criterion that gives useful information for small amplitudes. The extension to larger amplitudes, when significant improvements are expected, is then performed through a continuation‐optimization procedure. It is found that the choice of the specific waveform has an impact on the performance of the process and there is no unique best forcing for any process condition, but its choice depends on the operating parameters and the forcing amplitude and frequency values. Further, the influence of the waveform functions on the wash‐out conditions are extensively examined. The analysis shows that all the waveforms examined in this work may lead to significant enlargement of the nontrivial regime with respect to a steady state operation. In particular, square‐wave forcing leads in practice to the extinction of the wash‐out conditions for any feed substrate concentration and for a well defined choice of the forcing parameters. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Multiomic Metabolic Enrichment Network Analysis Reveals Metabolite–Protein Physical Interaction Subnetworks Altered in Cancer

Metabolism is recognized as an important driver of cancer progression and other complex diseases, but global metabolite profiling remains a challenge. Protein expression profiling is often a poor proxy since existing pathway enrichment models provide an incomplete mapping between the proteome and metabolism. To overcome these gaps, we introduce multiomic metabolic enrichment network analysis (MOMENTA), an integrative multiomic data analysis framework for more accurately deducing metabolic pathway changes from proteomics data alone in a gene set analysis context by leveraging protein interaction networks to extend annotated metabolic models. We apply MOMENTA to proteomic data from diverse cancer cell lines and human tumors to demonstrate its utility at revealing variation in metabolic pathway activity across cancer types, which we verify using independent metabolomics measurements. The novel metabolic networks we uncover in breast cancer and other tumors are linked to clinical outcomes, underscoring the pathophysiological relevance of the findings.     

The effects of monocyte-conditioned medium and interleukin 1 on the synthesis of collagenous and non-collagenous proteins by mouse bone and human bone cells in vitro

Cultural adherent human mononuclear cells produce factor(s) which stimulate the release of calcium from new-born mouse calvaria in organ culture. This stimulation of bone resorption is accompanied by an inhibition of the incorporation of [³H]proline into collagen which is independent of increased prostaglandin production by the bone. When human osteoblast-like cells are treated with conditioned medium from human mononuclear cells, collagen accounts for a decreased proportion of the protein synthesised. This effect on matrix synthesis is not accompanied by an inhibitory action of the monocyte-conditioned medium preparations on net cell proliferation. In human osteoblast-like cell cultures, partially purified human interleukin 1 also inhibits the production of the bone-specific protein osteocalcin in a dose-dependent fashion. These observations are consistent with the hypothesis that products of human monocytes similar to, or identical with, human interleukin 1 may be important regulators of bone metabolism and may contribute to the bone loss seen in diseases such as chronic rheumatoid arthritis.     

MicroID2: A Novel Biotin Ligase Enables Rapid Proximity-Dependent Proteomics

Identifying protein–protein and other proximal interactions is central to dissecting signaling and regulatory processes in cells. BioID is a proximity-dependent biotinylation method that uses an “abortive” biotin ligase to detect proximal interactions in cells in a highly reproducible manner. Recent advancements in proximity-dependent biotinylation tools have improved efficiency and timing of labeling, allowing for measurement of interactions on a cellular timescale. However, issues of size, stability, and background labeling of these constructs persist. Here we modified the structure of BioID2, derived from Aquifex aeolicus BirA, to create a smaller, highly active, biotin ligase that we named MicroID2. Truncation of the C terrminus of BioID2 and addition of mutations to alleviate blockage of biotin/ATP binding at the active site of BioID2 resulted in a smaller and highly active construct with lower background labeling. Several additional point mutations improved the function of our modified MicroID2 construct compared with BioID2 and other biotin ligases, including TurboID and miniTurbo. MicroID2 is the smallest biotin ligase reported so far (180 amino acids [AAs] for MicroID2 versus 257 AAs for miniTurbo and 338 AAs for TurboID), yet it demonstrates only slightly less labeling activity than TurboID and outperforms miniTurbo. MicroID2 also had lower background labeling than TurboID. For experiments where precise temporal control of labeling is essential, we in addition developed a MicroID2 mutant, termed lbMicroID2 (low background MicroID2), that has lower labeling efficiency but significantly reduced biotin scavenging compared with BioID2. Finally, we demonstrate utility of MicroID2 in mass spectrometry experiments by localizing MicroID2 constructs to subcellular organelles and measuring proximal interactions.     

Induction of unscheduled DNA synthesis by the carcinogen 7-bromomethylbenz(a)anthracene and its removal from the DNA of normal and xeroderma pigmentosum lymphocytes

The carcinogen 7-bromomethylbenz(a)anthracene (BBA), which can bind strongly to DNA, induces unscheduled DNA synthesis (DNA repair) in normal lymphocytes but almost none in lymphocytes from patients with Xeroderma pigmentosum (XP), and inherited disease known to be defective in excision repair of ultraviolet-damaged DNA. We studied [³H]BBA's ability to bind to DNA of normal and XP lymphocytes, its influence on unscheduled DNA synthesis, and its removal from the DNA of both cell types. We found that 20–30% of the BBA is bound to macromolecules other than DNA and that its binding to DNA is essentially complete after 30 min. The induction of unscheduled DNA synthesis by the carcinogen in XP lymphocytes was approximately 10% of that induced in normal lymphocytes. While 15–20% of the BBA was removed from the DNA of normal cells 6 h after treatment, only 1–2% was removed from the DNA of XP cells. Thus, XP cells not only are defective in repairing ultraviolet-damaged DNA and excising thymine dimers but also fail to repair DNA damaged by certain carcinogens, and, most importantly, fail to remove the DNA-bound carcinogen, BBA.     

Operation of a Benchtop Bioreactor

Fermentation systems are used to provide an optimal growth environment for many different types of cell cultures. The ability afforded by fermentors to carefully control temperature, pH, and dissolved oxygen concentrations in particular makes them essential to efficient large scale growth and expression of fermentation products. This video will briefly describe the advantages of the fermentor over the shake flask. It will also identify key components of a typical benchtop fermentation system and give basic instruction on setup of the vessel and calibration of its probes. The viewer will be familiarized with the sterilization process and shown how to inoculate the growth medium in the vessel with culture. Basic concepts of operation, sampling, and harvesting will also be demonstrated. Simple data analysis and system cleanup will also be discussed.     

Decision Support through Mass and Energy Flow Management in the Vehicle-Refinishing Sector

In the past few decades, major advances in environmental protection within the coating application industry have been made. In spite of this technological progress, approximately 50% of industrial-solvent emissions still come from the paint-application sector. The advances made in reducing emissions for plants requiring licensing have unfortunately had no influence on the environmental efforts of smaller companies. Solvent-reduced painting systems, such as high-solid paints, water-based coating, and powder coating have not been able to achieve acceptance, nor have innovative application technologies. The principal arguments against a conversion to these ecologically more favorable alternatives were related to cost and quality.
Recently, the EU Solvent Directive (1999/13/EC) went into effect, aiming to significantly reduce industrial-solvent emissions. Up until this point, however, instruments enabling smaller companies to determine their solvent emissions and to simultaneously develop process-improvement potentials while keeping costs in mind have been missing.
Using the mass and energy flow-management approach, cost structures and environmental benefits can be made transparent to the entrepreneur. The primary result of the research projects presented here is the computer-based mass and energy flow model called the individual computer-aided mass and energy flow model for the vehicle-refinishing sector (IMPROVE). It can be used as a detailed business-consultancy tool. Based upon this, practical guidelines were developed for easy orientation and activity planning. They can be used by companies to help them fulfill the requirements of environmental legislation and to display the benefits that can be achieved by various emission-reduction measures.     

Optimizing the expression of chimeric genes in plant cells

Summary The Serratia marcescens chiA gene encodes a secreted chitinase activity which contributes to the fungal growth inhibition exhibited by this bacterium. The coding region from the chiA gene was fused to the promoter and 3 polyadenylation region of the Agrobacterium nopaline synthase gene. Site-directed mutagenesis of specific nucleotides surrounding the initiating AUG of the coding sequence of this chimeric gene resulted in up to an eight-fold increase in the amount of chitinase protein detected in transformed plant tissue. Analysis of the chiA mRNA indicated that these nucleotides also affected mRNA levels. At least 50% of the chitinase protein produced in transformed tobacco cells was the same molecular weight as the S. marcescen secreted protein.