Ex Vivo Cytosolic Delivery of Functional Macromolecules to Immune Cells

Intracellular delivery of biomolecules, such as proteins and siRNAs, into primary immune cells, especially resting lymphocytes, is a challenge. Here we describe the design and testing of microfluidic intracellular delivery systems that cause temporary membrane disruption by rapid mechanical deformation of human and mouse immune cells. Dextran, antibody and siRNA delivery performance is measured in multiple immune cell types and the approach’s potential to engineer cell function is demonstrated in HIV infection studies.     

Dynamics of reductive TCE dechlorination in two distinct H<Subscript>2</Subscript> supply scenarios and at various temperatures

Anaerobic microbial dechlorination of trichloroethene (TCE) by a mixed, Dehalococcoides containing culture was investigated at different temperatures (4–60 °C) using propionate and lactate as a slow- and fast-releasing hydrogen (H₂) source, respectively. Distinct temperature-dependent dynamics of substrate fermentation and H₂ levels could explain observed patterns of dechlorination. While varying the temperature caused changes in rate, the overall pattern of dechlorination was characteristic of the supplied electron donor. Feeding cultures with a rapidly fermentable substrate such as lactate generally resulted in high H₂ concentrations and fast and complete dechlorination accompanied by rapid methanogenesis. In contrast, low H₂ release rates resulting from fermentation of propionate were associated with 2 to 3−fold longer time frames necessary for complete dechlorination at intermediate temperatures (15–30 °C). A lag-phase prior to dechlorination of cis-dichloroethene (cDCE), together with a characteristic build-up of H₂ and methane, was consistently observed at slow H₂ supply. At temperatures of 10 °C and lower, the system remained in this lag phase and no dechlorination past cDCE was observed within the experimental time frame. However, when lactate was the substrate, complete dechlorination of TCE occurred within 74 days at 10 °C, accompanied by methane production. The choice of fermentable substrate decisively influenced the rate and degree of dechlorination at an electron donor/TCE ratio as high as 666:1. Temperature-dependent H₂ levels resulting from fermentation of different substrates could be satisfactorily explained through thermodynamic calculations of the Gibbs free energy yield assuming a constant metabolic energy threshold of −20 kJ/(mol reaction).     

Effects of growth phase and nitrogen starvation on expression of fatty acid desaturases and fatty acid composition of Isochrysis aff. galbana (TISO)

Very long-chain polyunsaturated fatty acids (VLC-PUFAs) are important dietary requirements for maintaining human health. Many marine microalgae are naturally high in ω − 3 VLC-PUFAs, however, the molecular mechanisms underpinning fatty acid (FA) desaturation and elongation in algae are poorly understood. An advanced molecular understanding would facilitate improvements of this nascent industry. We aimed to investigate expression responses of four front-end fatty acid desaturase genes and downstream effects on FA profiles to nitrogen limitation and cultivation growth stage in Isochrysis aff. galbana (TISO). Cultures were grown in nitrogen-replete and -deplete medium; samples were harvested during logarithmic, late logarithmic and stationary growth phases to analyse FA content/composition and gene expression of ?⁶-, ?⁸-, ?⁵- and ?⁴-desaturases (d6FAD (putative), d8FAD, d5FAD and d4FAD, respectively). d6FAD (putative) exhibited no differential expression, while d8FAD, d5FAD and d4FAD were significantly upregulated during logarithmic growth of nutrient-replete cultures, coinciding with rapid cell division. In conclusion, it is demonstrated that expression of some FADs in I. aff. galbana varies with culture age and nitrogen status which has downstream consequences on FA desaturation levels. This has implications for the commercial production of VLC-PUFAs where a trade-off between total lipid yield and VLC-PUFAs has to be made.     

Negative regulators of cell death pathways in cancer: perspective on biomarkers and targeted therapies

Cancer is a primary cause of human fatality and conventional cancer therapies, e.g., chemotherapy, are often associated with adverse side-effects, tumor drug-resistance, and recurrence. Molecularly targeted therapy, composed of small-molecule inhibitors and immunotherapy (e.g., monoclonal antibody and cancer vaccines), is a less harmful alternative being more effective against cancer cells whilst preserving healthy tissues. Drug-resistance, however, caused by negative regulation of cell death signaling pathways, is still a challenge. Circumvention of negative regulators of cell death pathways or development of predictive and response biomarkers is, therefore, quintessential. This review critically discusses the current state of knowledge on targeting negative regulators of cell death signaling pathways including apoptosis, ferroptosis, necroptosis, autophagy, and anoikis and evaluates the recent advances in clinical and preclinical research on biomarkers of negative regulators. It aims to provide a comprehensive platform for designing efficacious polytherapies including novel agents for restoring cell death signaling pathways or targeting alternative resistance pathways to improve the chances for antitumor responses. Overall, it is concluded that nonapoptotic cell death pathways are a potential research arena for drug discovery, development of novel biomarkers and targeted therapies.     

Selective mitochondrial accumulation of cytotoxic dinuclear polypyridyl ruthenium(II) complexes

The lipophilic ligand-bridged dinuclear cation Rubb?? is significantly cytotoxic and preferentially accumulates in the mitochondria of the L1210 murine leukemia cancer cell line.     

Expression and regulation of cyclic nucleotide phosphodiesterases in human and rat pancreatic islets

As shown by transgenic mouse models and by using phosphodiesterase 3 (PDE3) inhibitors, PDE3B has an important role in the regulation of insulin secretion in pancreatic β-cells. However, very little is known about the regulation of the enzyme. Here, we show that PDE3B is activated in response to high glucose, insulin and cAMP elevation in rat pancreatic islets and INS-1 (832/13) cells. Activation by glucose was not affected by the presence of diazoxide. PDE3B activation was coupled to an increase as well as a decrease in total phosphorylation of the enzyme. In addition to PDE3B, several other PDEs were detected in human pancreatic islets: PDE1, PDE3, PDE4C, PDE7A, PDE8A and PDE10A. We conclude that PDE3B is activated in response to agents relevant for β-cell function and that activation is linked to increased as well as decreased phosphorylation of the enzyme. Moreover, we conclude that several PDEs are present in human pancreatic islets.     

Characterization of Essential Domains and Plasticity of the Classical Swine Fever Virus Core Protein

Pestiviruses are pathogens of cloven-hoofed animals, belonging to the Flaviviridae. The pestiviral particle consists of a lipid membrane containing the three envelope glycoproteins E^rns, E1, and E2 and a nucleocapsid of unknown symmetry, which is composed of the Core protein and the viral positive-sense RNA genome. The positively charged pestiviral Core protein consists of 86 to 89 amino acids. To analyze the organization of essential domains, N- and C-terminal truncations, as well as internal deletions, were introduced into the Core coding sequence in the context of an infectious cDNA clone of classical swine fever virus strain Alfort. Amino acids 179 to 180, 194 to 198, and 208 to 212 proved to be of special importance for the generation of progeny virus. The results of transcomplementation of a series of C-terminally truncated Core molecules indicate the importance of Ala₂₅₅ at the C terminus. The plasticity of Core protein was examined by the construction of concatemeric arrays of Core coding regions and the insertion of up to three yellow fluorescent protein (YFP) genes between two Core genes. Even a Core fusion protein with more than 10-fold-increased molecular mass was integrated into the viral particle and supported the production of infectious progeny virus. The unexpected plasticity of Core protein brings into question the formation of a regular icosahedric particle and supports the idea of a histone-like protein-RNA interaction. All viruses with a duplicated Core gene were unstable and reverted to the wild-type sequence. Interestingly, a nonviable YFP-Core construct was rescued by a mutation within the C-terminal domain of the nonstructural protein NS3.Several important pathogens of cloven-hoofed animals, such as classical swine fever virus (CSFV) and bovine viral diarrhea virus (BVDV), comprise the genus Pestivirus. The latter, together with the genera Flavivirus and Hepacivirus, belong to the family Flaviviridae. Pestiviruses possess a single-stranded, positive-sense RNA genome of at least 12.3 kb, coding for one polyprotein. It is processed into 12 mature viral proteins by cellular and viral proteases.Pestiviral virions contain four structural proteins, the small, basic Core protein and three envelope glycoproteins, E^rns, E1, and E2. The nucleocapsid consists of Core protein and the viral RNA genome (13, 38, 42). While Flaviviridae typically encode Core protein as the first product of the polyprotein, pestiviruses encode the unique N-terminal protease N^pro at the analogous position (41). N^pro facilitates the degradation of interferon regulatory factor 3 (IRF3) (1) and generates the N terminus of Core by autoproteolytic action. If cleavage is blocked, no generation of infectious particles can be observed and the N^pro-Core protein accumulates in the cytoplasm (39). However, nonviral proteins can be expressed between N^pro and Core if an additional protease cleavage site (2A protease of foot-and-mouth disease virus) is integrated at the Core N terminus (8). The C terminus of Core is created through an intramembrane cleavage by signal peptide peptidase (12). The same proteolytic mechanism is employed in the biosynthesis of Core protein of hepatitis C virus (HCV) (29) but not by members of the genus Flavivirus. Here, the C terminus of the Capsid protein is generated by the viral NS3 protease (4).Recent studies on the structure of the pestiviral Core protein describe it as an intrinsically disordered protein on the basis of far-UV circular dichroism and intrinsic fluorescence spectroscopy analysis (15, 32). Its disordered nature is highlighted in analogy to the Core N terminus of other members of the Flaviviridae, which is often found to be responsible for RNA binding (3, 6, 7, 9, 25). Neither a C-terminal ordered domain, apart from 15 amino acids at the C terminus, nor assembly to an alphahelical, dimeric structure, as described for flaviviruses and HCV, has been reported for the pestiviral Core protein (3, 15, 32). Its interaction with nucleic acids is of low affinity and low specificity, and no specific RNA packaging signals have been identified (32). This unspecific interaction with RNA was further supported by the functional replacement of the RNA binding domain of Sindbis virus Capsid protein by BVDV Core (32). Recently, RNA chaperone activity of BVDV Core protein has been reported, which is responsible for changes in RNA structure without the need of chemical energy provided as ATP (15). RNA chaperone activity relies on a disordered protein stretch that is insensitive to heat.A large internal deletion in Core protein was lethal for recombinant BVDV. However, infectious virus could be recovered by providing Core along with other structural proteins in trans (37). Thus, the importance of pestiviral Core protein for the generation of infectious virus particles is known, but no reports exist on the functional organization of this protein.This study analyzes the domain structure of the pestiviral Core protein by mapping regions important for virus assembly. Truncations and deletions within the Core protein, as well as a dramatic increase of molecular mass, reveal a plasticity that does not fit the strict symmetric requirements that are to be expected for icosahedral nucleocapsids.     

Substrate phosphorylation affects degradation and interaction to endopeptidase 24.15, neurolysin, and angiotensin-converting enzyme

Recent findings from our laboratory suggest that intracellular peptides containing putative post-translational modification sites (i.e., phosphorylation) could regulate specific protein interactions. Here, we extend our previous observations showing that peptide phosphorylation changes the kinetic parameters of structurally related endopeptidase EP24.15 (EC 3.4.24.15), neurolysin (EC 3.4.24.16), and angiotensin-converting enzyme (EC 3.4.15.1). Phosphorylation of peptides that are degraded by these enzymes leads to reduced degradation, whereas phosphorylation of peptides that interacted as competitive inhibitors of these enzymes alters only the K(i)'s. These data suggest that substrate phosphorylation could be one of the mechanisms whereby some intracellular peptides would escape degradation and could be regulating protein interactions within cells.     

Mulch decomposition under agroforestry conditions in a sub-humid tropical savanna processes and influence of perennial plants

For the effective use of mulch materials in tropical agriculture and agroforestry knowledge of the speed of decomposition and nutrient release is of primary importance. The transfer of these informations from one site to another requires comparability of the processes of decomposition and their intensity at the two sites. In a litterbag experiment the decomposition and release of main nutrients from leaves and branches of Cajanus cajan (L.) Millsp. were investigated with regard to the underlying physical and biological processes during an 81 days period. To test the influence of perennial plants on the decomposition process, the study was conducted on an agricultural field in 1.1 m, 6.9 m and 14.9 m distance from a tree and hedge band. During the first 11 days leaching was high, especially for N and P (about 50% lost) and K (75–80% lost). After the 11th day consumption of the mulch material by the soil fauna was the dominating process of decomposition. During this phase the perennial plants significantly retarded the decomposition of Cajanus branches, but not leaves, probably by their influence on termite activity. Ca release was also retarded in leaves. After about 6–7 weeks, more than 90% of all main nutrients except Ca had been released from the samples. To minimize nutrient losses from nutrient-rich mulch materials, they should be applied repeatedly in small quantities according to the nutrient demand of the crop.