Optimization of the Lactococcus lactis nisin-controlled gene expression system NICE for industrial applications

BACKGROUND: The nisin-controlled gene expression system NICE of Lactococcus lactis is one of the most widely used expression systems in Gram-positive bacteria. Despite its widespread use, no optimization of the culture conditions and nisin induction has been carried out to obtain maximum yields. As a model system induced production of lysostaphin, an antibacterial protein (mainly against Staphylococcus aureus) produced by S. simulans biovar. Staphylolyticus, was used. Three main areas need optimization for maximum yields: cell density, nisin-controlled induction and protein production, and parameters specific for the target-protein. RESULTS: In a series of pH-controlled fermentations the following parameters were optimized: pH of the culture, use of NaOH or NH(4)OH as neutralizing agent, the addition of zinc and phosphate, the fermentation temperature, the time point of induction (cell density of the culture), the amount of nisin added for induction and the amount of three basic medium components, i.e. yeast extract, peptone and lactose. For each culture growth and lysostaphin production was followed. Lysostaphin production yields depended on all parameters that were varied. In the course of the optimization a three-fold increase in lysostaphin yield was achieved from 100 mg/l to 300 mg/l. CONCLUSION: Protein production with the NICE gene expression system in L. lactis strongly depends on the medium composition, the fermentation parameters and the amount of nisin added for induction. Careful optimization of key parameters lead to a significant increase in the yield of the target protein

MRNA expression profiles of colorectal liver metastases as a novel biomarker for early recurrence after partial hepatectomy

Background: Identification of specific risk groups for recurrence after surgery for isolated colorectal liver metastases (CRLM) remains challenging due to the heterogeneity of the disease. Classical clinicopathologic parameters have limited prognostic value. The aim of this study was to identify a gene expression signature measured in CRLM discriminating early from late recurrence after partial hepatectomy. Methods: CRLM from two patient groups were collected: I) with recurrent disease ≤12 months after surgery (N = 33), and II) without recurrences and disease free for ≥36 months (N = 30). The patients were clinically homogeneous; all had a low clinical risk score (0-2) and did not receive (neo-) adjuvant chemotherapy. Total RNA was hybridised to Illumina arrays, and processed for analysis. A leave-one-out cross validation (LOOCV) analysis was performed to identify a prognostic gene expression signature. Results: LOOCV yielded an 11-gene profile with prognostic value in relation to recurrent disease ≤12 months after partial hepatectomy. This signature had a sensitivity of 81.8%, with a specificity of 66.7% for predicting recurrences (≤12 months) versus no recurrences for at least 36 months after surgery (X2 P < 0.0001). Conclusion: The current study yielded an 11-gene signature at mRNA level in CRLM discriminating early from late or no relapse after partial hepatectomy

Cellular pharmacology of multi-and duplex drugs consisting of ethynylcytidine and 5-fluoro-2′-deoxyuridine

In vivo, ETC-FdUrd and ETC-L-FdURd were orally active. ETC nucleotides accumulated in both tumor and liver tissues. These formulations seem to be effective when a lipophilic linker is used combined with a liposomal formulation

Molecular characteristics of circulating tumor cells resemble the liver metastasis more closely than the primary tumor in metastatic colorectal cancer

Background: CTCs are a promising alternative for metastatic tissue biopsies for use in precision medicine approaches. We investigated to what extent the molecular characteristics of circula

Cellular pharmacology of multi- and duplex drugsconsisting of ethynylcytidine and 5-fluoro-2′-deoxyuridine

Prodrugs can have the advantage over parent drugs in increased activation and cellular uptake. The multidrug ETC-L-FdUrd and the duplex drug ETC-FdUrd are composed of two different monophosphate-nucleosides, 5-fluoro-2′deoxyuridine (FdUrd) and ethynylcytidine (ETC), coupled via a glycerolipid or phosphodiester, respectively. The aim of the study was to determine cytotoxicity levels and mode of drug cleavage. Moreover, we determined whether a liposomal formulation of ETC-L-FdUrd would improve cytotoxic activity and/or cleavage. Drug effects/cleavage were studied with standard radioactivity assays, HPLC and LC-MS/MS in FM3A/0 mammary cancer cells and their FdUrd resistant variants FM3A/TK−. ETC-FdUrd was active (IC50 of 2.2 and 79 nM) in FM3A/0 and TK− cells, respectively. ETC-L-FdUrd was less active (IC50: 7 nM in FM3A/0 vs 4500 nM in FM3A/TK−). Although the liposomal formulation was less active than ETC-L-FdUrd in FM3A/0 cells (IC50:19.3 nM), resistance due to thymidine kinase (TK) deficiency was greatly reduced. The prodrugs inhibited thymidylate synthase (TS) in FM3A/0 cells (80–90%), but to a lower extent in FM3A/TK− (10–50%). FdUMP was hardly detected in FM3A/TK− cells. Inhibition of the transporters and nucleotidases/phosphatases resulted in a reduction of cytotoxicity of ETC-FdUrd, indicating that this drug was cleaved outside the cells to the monophosphates, which was verified by the presence of FdUrd and ETC in the medium. ETC-L-FdUrd and the liposomal formulation were neither affected by transporter nor nucleotidase/phosphatase inhibition, indicating circumvention of active transporters. In vivo, ETC-FdUrd and ETC-L-FdURd were orally active. ETC nucleotides accumulated in both tumor and liver tissues. These formulations seem to be effective when a lipophilic linker is used combined with a liposomal formulation

A reusable simulation model to evaluate the effects of walk-in for diagnostic examinations

Enabling patients to walk in for their diagnostic examination without an appointment has considerable potential in terms of quality of care, patient service, and system efficiency. We present a model to evaluate the effect of implementing a combined walk-in and appointment system, offering appointments to all patients preferring or strictly requiring these, while enabling all other patients to walk in. In a combined system, appointments can be scheduled in periods with low walk-in demand to counterbalance the possible high variability in walk-in arrival rates. We develop a discrete event simulation model, combined with an intelligent algorithmic methodology for appointment schedule optimization, for evaluating the implementation of a combined walk-in and appointment system for diagnostic examinations. Our simulation model is reusable: its component-based structure and generic underlying logic enable it to automatically represent any type of diagnostic facility, for which it can then evaluate the effect of implementing a combined walk-in and appointment system. Applying this approach, we quantitatively investigate the impact of implementing a combined walk-in and appointment system for CT-scans, performing a case study at the Academic Medical Center (AMC) Amsterdam. Inspired by the results, the AMC CT-facility has implemented a combined walk-in and appointment system, thereby shortening patients’ diagnostic trajectories, and decreasing the number of required hospital visits for many patients