Stochastic Tunneling of Two Mutations in a Population of Cancer Cells

Cancer initiation, progression, and the emergence of drug resistance are driven by specific genetic and/or epigenetic alterations such as point mutations, structural alterations, DNA methylation and histone modification changes. These alterations may confer advantageous, deleterious or neutral effects to mutated cells. Previous studies showed that cells harboring two particular alterations may arise in a fixed-size population even in the absence of an intermediate state in which cells harboring only the first alteration take over the population; this phenomenon is called stochastic tunneling. Here, we investigated a stochastic Moran model in which two alterations emerge in a cell population of fixed size. We developed a novel approach to comprehensively describe the evolutionary dynamics of stochastic tunneling of two mutations. We considered the scenarios of large mutation rates and various fitness values and validated the accuracy of the mathematical predictions with exact stochastic computer simulations. Our theory is applicable to situations in which two alterations are accumulated in a fixed-size population of binary dividing cells.     

Intrarenal Renin Angiotensin System Revisited: ROLE OF MEGALIN-DEPENDENT ENDOCYTOSIS ALONG THE PROXIMAL NEPHRON

The existence of a local renin angiotensin system (RAS) of the kidney has been established. Angiotensinogen (AGT), renin, angiotensin-converting enzyme (ACE), angiotensin receptors, and high concentrations of luminal angiotensin II have been found in the proximal tubule. Although functional data have documented the relevance of a local RAS, the dualism between biosynthesis and endocytotic uptake of its components and their cellular processing has been incompletely understood. To resolve this, we have selectively analyzed their distribution, endocytosis, transcytosis, and biosynthesis in the proximal tubule. The presence of immunoreactive AGT, restricted to the early proximal tubule, was due to its retrieval from the ultrafiltrate and storage in endosomal and lysosomal compartments. Cellular uptake was demonstrated by autoradiography of radiolabeled AGT and depended on intact endocytosis. AGT was identified as a ligand of the multiple ligand-binding repeats of megalin. AGT biosynthesis was restricted to the proximal straight tubule, revealing substantial AGT mRNA expression. Transgenic AGT overexpression under the control of an endogenous promoter was also restricted to the late proximal tubule. Proximal handling of renin largely followed the patterns of AGT, whereas its local biosynthesis was not significant. Transcytotic transport of AGT in a proximal cell line revealed a 5% recovery rate after 1 h. ACE was expressed along late proximal brush-border membrane, whereas ACE2 was present along the entire segment. Surface expression of ACE and ACE2 differed as a function of endocytosis. Our data on the localization and cellular processing of RAS components provide new aspects of the functional concept of a “self-contained” renal RAS.     

Development of Adenoviral Delivery Systems to Target Hepatic Stellate Cells In Vivo

Hepatic stellate cells (HSCs) are known as initiator cells that induce liver fibrosis upon intoxication or other noxes. Deactivation of this ongoing remodeling process of liver parenchyma into fibrotic tissue induced by HSCs is an interesting goal to be achieved by targeted genetic modification of HSCs. The most widely applied approach in gene therapy is the utilization of specifically targeted vectors based on Adenovirus (Ad) serotype 5. To narrow down the otherwise ubiquitous tropism of parental Ad, two modifications are required: a) ablating the native tropism and b) redirecting the vector particles towards a specific entity solely present on the cells of interest. Therefore, we designed a peptide of the nerve growth factor (NGF_p) with specific affinity for the p75 neurotrophin receptor (p75NTR) present on HSCs. Coupling of this NGF_p to vector particles was done either via chemical conjugation using bifunctional polyethylene glycol (PEG) or, alternatively, by molecular bridging with a fusion protein specific for viral fiber knob and p75NTR. Both Ad vectors transmit the gene for the green fluorescent protein (GFP). GFP expression was monitored in vitro on primary murine HSCs as well as after systemic administration in mice with healthy and fibrotic livers using intravital fluorescence microscopy. Coupling of NGF_p to Ad via S11 and/or PEGylation resulted in markedly reduced liver tropism and an enhanced adenoviral-mediated gene transfer to HSCs. Transduction efficiency of both specific Ads was uniformly higher in fibrotic livers, whereas Ad.GFP-S11-NGF_p transduce activated HSCs better than Ad.GFP-PEG-NGF_p. These experiments contribute to the development of a targeted gene transfer system to specifically deliver antifibrotic compounds into activated HSCs by systemically applied adenoviral vector modified with NGF_p.     

Establishing a Fed‐Batch Process for Protease Expression with Bacillus licheniformis in Polymer‐Based Controlled‐Release Microtiter Plates

Introducing fed‐batch mode in early stages of development projects is crucial for establishing comparable conditions to industrial fed‐batch fermentation processes. Therefore, cost efficient and easy to use small‐scale fed‐batch systems that can be integrated into existing laboratory equipment and workflows are required. Recently, a novel polymer‐based controlled‐release fed‐batch microtiter plate is described. In this work, the polymer‐based controlled‐release fed‐batch microtiter plate is used to investigate fed‐batch cultivations of a protease producing Bacillus licheniformis culture. Therefore, the oxygen transfer rate (OTR) is online‐monitored within each well of the polymer‐based controlled‐release fed‐batch microtiter plate using a µRAMOS device. Cultivations in five individual polymer‐based controlled‐release fed‐batch microtiter plates of two production lots show good reproducibility with a mean coefficient of variation of 9.2%. Decreasing initial biomass concentrations prolongs batch phase while simultaneously postponing the fed‐batch phase. The initial liquid filling volume affects the volumetric release rate, which is directly translated in different OTR levels of the fed‐batch phase. An increasing initial osmotic pressure within the mineral medium decreases both glucose release and protease yield. With the volumetric glucose release rate as scale‐up criterion, microtiter plate‐ and shake flask‐based fed‐batch cultivations are highly comparable. On basis of the small‐scale fed‐batch cultivations, a mechanistic model is established and validated. Model‐based simulations coincide well with the experimentally acquired data.     

Lumi-ergometrine – structural identification and occurrence in sclerotia

The fungus Claviceps purpurea grows on grasses and cereal grains and produces six predominant ergot alkaloids. These toxic substances undergo different transformation reactions during storage and cereal processing. One of these reactions is the addition of water to a double bond in the ergoline skeleton. Since light is required for this process, the substances formed were named lumi-ergot alkaloids. From these, a new asymmetric carbon and consequently two epimers with different polarities are formed. For investigations of lumi-ergot alkaloids, ergometrine was used exemplarily as it represents one of the six ergot alkaloids predominantly formed by Claviceps purpurea. The main reaction product, the less polar compound of the two lumi-ergometrine epimers, was separated by HPLC and unambiguously identified as 10-(S)-lumi-ergometrine using X-ray structural analysis. A HPLC-MS/MS method was developed for the detection of this substance in sclerotia extracts. Using this method, the existence of both epimeric forms of lumi-ergometrine could be proved in the sclerotia. This is the first time that the existence of a lumi-transformation product of ergot alkaloids was proved in naturally grown samples.     

Viscoelastic Properties of Differentiating Blood Cells Are Fate- and Function-Dependent

Although cellular mechanical properties are known to alter during stem cell differentiation, understanding of the functional relevance of such alterations is incomplete. Here, we show that during the course of differentiation of human myeloid precursor cells into three different lineages, the cells alter their viscoelastic properties, measured using an optical stretcher, to suit their ultimate fate and function. Myeloid cells circulating in blood have to be advected through constrictions in blood vessels, engendering the need for compliance at short time-scales (<seconds). Intriguingly, only the two circulating myeloid cell types have increased short time scale compliance and flow better through microfluidic constrictions. Moreover, all three differentiated cell types reduce their steady-state viscosity by more than 50% and show over 140% relative increase in their ability to migrate through tissue-like pores at long time-scales (>minutes), compared to undifferentiated cells. These findings suggest that reduction in steady-state viscosity is a physiological adaptation for enhanced migration through tissues. Our results indicate that the material properties of cells define their function, can be used as a cell differentiation marker and could serve as target for novel therapies.     

A quantitative analysis of spontaneous isoaspartate formation from N-terminal asparaginyl and aspartyl residues

The formation of isoaspartate (isoAsp) from asparaginyl or aspartyl residues is a spontaneous post-translational modification of peptides and proteins. Due to isopeptide bond formation, the structure and possibly function of peptides and proteins is altered. IsoAsp modifications within the peptide chain have been reported for many cytosolic proteins. Amyloid peptides (Aβ) deposited in Alzheimer’s disease may carry an N-terminal isoAsp-modification. Here, we describe a quantitative investigation of isoAsp-formation from N-terminal Asn and Asp using model peptides similar to the Aβ N-terminus. The study is based on a newly developed separation of peptides using capillary electrophoresis (CE). ¹H NMR was employed to validate the basic finding of N-terminal isoAsp-formation from Asp and Asn. Thereby, the isomerization of Asn at neutral pH (0.6 day^?1, peptide NGEF) is approximately six times faster than that within the peptide chain (AANGEF). The difference in velocity between Asn and Asp isomerization is approximately 50-fold. In contrast to N-terminal Asn, Asp isomerization is significantly accelerated at acidic pH. The kinetic solvent isotope (k _D2O/k _H2O) effect of 2.46 suggests a rate-limiting proton transfer in isoAsp-formation. The proton inventory is consistent with transfer of one proton in the transition state, supporting the previous notion of rate-limiting deprotonation of the peptide backbone amide during succinimide-intermediate formation. The study provides evidence for a spontaneous N-terminal isoAsp-formation within peptides and might explain the accumulation of N-terminal isoAsp in amyloid deposits.