Current state of purification,isolation and analysis of bacteriocins produced by lactic acid bacteria

Applied Microbiology and Biotechnology - The scientific interest for the search of natural means of microbial inhibitors has not faded for several years. A search of natural antibiotics, so-called...     

Using lysozyme amyloid fibrils as a means of scavenging aggregation-inhibiting compounds

The aggregation of amyloidogenic proteins is linked to several amyloidoses, including neurodegenerative disorders, such as Alzheimer's or Parkinson's disease. Currently there are very few effective cures or treatments available, despite countless screenings and clinical trials. One of the most challenging aspects of potential anti-amyloid drug discovery is finding which molecules are the actual inhibitors out of mixtures, which may contain hundreds of distinct compounds. Considering that anti-amyloid compounds would interact with the aggregate, this affinity could be used as a means of separating such compounds from ineffective ones. In this work, we attempt to scavenge potential aggregation-inhibiting molecules out of four, different complexity mixtures, ranging from oxidized gallic acid to tea extract, using lysozyme amyloid fibrils. We show that these compounds bind to aggregates with high affinity and can be later separated from them by different methods.     

Longitudinal monitoring of cell metabolism in biopharmaceutical production using label-free fluorescence lifetime imaging microscopy

Chinese hamster ovary (CHO) cells are routinely used in the biopharmaceutical industry for production of therapeutic monoclonal antibodies (mAbs). Although multiple offline and time-consuming measurements of spent media composition and cell viability assays are used to monitor the status of culture in biopharmaceutical manufacturing, the day-to-day changes in the cellular microenvironment need further in-depth characterization. In this study, two-photon fluorescence lifetime imaging microscopy (2P-FLIM) was used as a tool to directly probe into the health of CHO cells from a bioreactor, exploiting the autofluorescence of intracellular nicotinamide adenine dinucleotide phosphate (NAD(P)H), an enzymatic cofactor that determines the redox state of the cells. A custom-built multimodal microscope with two-photon FLIM capability was utilized to monitor changes in NAD(P)H fluorescence for longitudinal characterization of a changing environment during cell culture processes. Three different cell lines were cultured in 0.5 L shake flasks and 3 L bioreactors. The resulting FLIM data revealed differences in the fluorescence lifetime parameters, which were an indicator of alterations in metabolic activity. In addition, a simple principal component analysis (PCA) of these optical parameters was able to identify differences in metabolic progression of two cell lines cultured in bioreactors. Improved understanding of cell health during antibody production processes can result in better streamlining of process development, thereby improving product titer and verification of scale-up. To our knowledge, this is the first study to use FLIM as a label-free measure of cellular metabolism in a biopharmaceutically relevant and clinically important CHO cell line.     

Lability Landscape and Protease Resistance of Human Insulin Amyloid: A New Insight into Its Molecular Properties

Amyloid formation is a universal behavior of proteins central to many important human pathologies and industrial processes. The extreme stability of amyloids towards chemical and proteolytic degradation is an acquired property compared to the precursor proteins and is a major prerequisite for their accumulation. Here, we report a study on the lability of human insulin amyloid as a function of pH and amyloid ageing. Using a range of methods such as atomic force microscopy, thioflavin T fluorescence, circular dichroism, and gas-phase electrophoretic mobility macromolecule analysis, we probed the propensity of human insulin amyloid to propagate or dissociate in a wide span of pH values and ageing in a low concentration regime. We generated a three-dimensional amyloid lability landscape in coordinates of pH and amyloid ageing, which displays three distinctive features: (i) a maximum propensity to grow near pH 3.8 and an age corresponding to the inflection point of the growth phase, (ii) an abrupt cutoff between growth and disaggregation at pH 8-10, and (iii) isoclines shifted towards older age during the amyloid growth phase at pH 4-9, reflecting the greater stability of aged amyloid. Thus, lability of amyloid strongly depends on the ionization state of insulin and on the structure and maturity of amyloid fibrils. The stability of insulin amyloid towards protease K was assessed by using real-time atomic force microscopy and thioflavin T fluorescence. We estimated that amyloid fibrils can be digested both from the free ends and within the length of the fibril with a rate of ca 4 nm/min. Our results highlight that amyloid structures, depending on solution conditions, can be less stable than commonly perceived. These results have wide implications for understanding the propagation of amyloids via a seeding mechanism as well as for understanding their natural clearance and dissociation under solution conditions unfavorable for amyloid formation in biological systems and industrial applications.     

Biocompatibility evaluation of densified bacterial nanocellulose hydrogel as an implant material for auricular cartilage regeneration

Bacterial nanocellulose (BNC), synthesized by the bacterium Gluconacetobacter xylinus, is composed of highly hydrated fibrils (99 % water) with high mechanical strength. These exceptional material properties make BNC a novel biomaterial for many potential medical and tissue engineering applications. Recently, BNC with cellulose content of 15 % has been proposed as an implant material for auricular cartilage replacement, since it matches the mechanical requirements of human auricular cartilage. This study investigates the biocompatibility of BNC with increased cellulose content (17 %) to evaluate its response in vitro and in vivo. Cylindrical BNC structures (Ø48?×?20 mm) were produced, purified in a built-in house perfusion system, and compressed to increase the cellulose content in BNC hydrogels. The reduction of endotoxicity of the material was quantified by bacterial endotoxin analysis throughout the purification process. Afterward, the biocompatibility of the purified BNC hydrogels with cellulose content of 17 % was assessed in vitro and in vivo, according to standards set forth in ISO 10993. The endotoxin content in non-purified BNC (2,390 endotoxin units (EU)/ml) was reduced to 0.10 EU/ml after the purification process, level well below the endotoxin threshold set for medical devices. Furthermore, the biocompatibility tests demonstrated that densified BNC hydrogels are non-cytotoxic and cause a minimal foreign body response. In support with our previous findings, this study concludes that BNC with increased cellulose content of 17 % is a promising non-resorbable biomaterial for auricular cartilage tissue engineering, due to its similarity with auricular cartilage in terms of mechanical strength and host tissue response.