Geographical identification of strawberries based on stable isotope ratio and multi-elemental analysis coupled with multivariate statistical analysis: A Slovenian case study.

The geographical classification and authentication of strawberries were attempted using discriminant and class-modelling methods applied to stable isotopes of light elements and elemental composition. The work involved creating a database of 92 authentic Slovenian strawberry samples and 32 imported samples. All samples were harvested between 2018 and 2020. A good geographical classification of Slovenian and non-Slovenian strawberries was obtained despite different production years using discriminant approaches. However, for verifying compliance with a given specification (geographical indications), a class-modelling approach was used to build an unbiased verification model. Class models generated by data-driven soft independent modelling of class analogy (DD-SIMCA) had high sensitivity (96% to 97%) and good specificity (81% to 91%) on a yearly basis, while a more generalised model combining total yearly data gave a lower specificity (63%). Of the 33 commercially available samples (test samples) with declared Slovenian origin, 39% were from outside of Slovenia.

Dynamic multiscale metabolic network modeling of Chinese hamster ovary cell metabolism integrating N-linked glycosylation in industrial biopharmaceutical manufacturing.

Experimental and modeling work, described in this article, is focused on the metabolic pathway of Chinese hamster ovary (CHO) cells, which are the preferred expression system for monoclonal antibody protein production. CHO cells are one of the primary hosts for monoclonal antibodies production, which have extensive applications in multiple fields like biochemistry, biology and medicine. Here, an approach to explain cellular metabolism with in silico modeling of a microkinetic reaction network is presented and validated with unique experimental results. Experimental data of 25 different fed-batch bioprocesses included the variation of multiple process parameters, such as pH, agitation speed, oxygen and CO2 content, and dissolved oxygen. A total of 151 metabolites were involved in our proposed metabolic network, which consisted of 132 chemical reactions that describe the reaction pathways, and include 25 reactions describing N-glycosylation and additional reactions for the accumulation of the produced glycoforms. Additional eight reactions are considered for accumulation of the N-glycosylation products in the extracellular environment and one reaction to correlate cell degradation. The following pathways were considered: glycolysis, pentose phosphate pathway, nucleotide synthesis, tricarboxylic acid cycle, lipid synthesis, protein synthesis, biomass production, anaplerotic reactions, and membrane transport. With the applied modeling procedure, different operational scenarios and fed-batch techniques can be tested.

Host Cell Protein Profiling in Biopharmaceutical Harvests.

Biopharmaceuticals contain residual host cell protein (HCP) impurities, a complex mixture of endogenous proteins from production cell lines such as Chinese hamster ovary (CHO) cells. The composition of HCP impurities at harvest hinges on multiple factors, e.g., identity of cell line, cell density and viability at harvest, or other process parameters. Two-dimensional differential gel electrophoresis (2-D DIGE) was used to compare HCP in 15 null cell culture harvest supernatants, which are representative for a wide range of manufacturing processes of therapeutic antibodies, using five different CHO cell lines. Numerical metrics were developed to quantitatively compare HCP composition, which may be used to assess the suitability of a platform HCP assay standard for a new product or to assess the impact of process changes. A very similar HCP composition was found for the 15 analyzed CHO null cell culture harvests, demonstrating that even the wide range of applied manufacturing processes did not have a strong influence on the HCP impurities.

Biosimilar structural comparability assessment by NMR: from small proteins to monoclonal antibodies.

Biosimilar drug products must have a demonstrated similarity with respect to the reference product's molecules in order to ensure both the effectiveness of the drug and the patients' safety. In this paper the fusion framework of a highly sensitive NMR fingerprinting approach for conformational changes and mathematically-based biosimilarity metrics is introduced. The final goal is to translate the complex spectral information into biosimilarity scores, which are then used to estimate the degree of similarity between the biosimilar and the reference product. The proposed method was successfully applied to a small protein, i.e., filgrastim (neutropenia treatment), which is the first biosimilar approved in the United States, and a relatively large protein, i.e., monoclonal antibody rituximab (lymphoma treatment). This innovative approach introduces a new level of sensitivity to structural changes that are induced by, e.g., a small pH shift or other changes in the protein formulation.

N-acylated peptides derived from human lactoferricin perturb organization of cardiolipin and phosphatidylethanolamine in cell membranes and induce defects in Escherichia coli cell division.

Two types of recently described antibacterial peptides derived from human lactoferricin, either nonacylated or N-acylated, were studied for their different interaction with membranes of Escherichia coli in vivo and in model systems. Electron microscopy revealed striking effects on the bacterial membrane as both peptide types induced formation of large membrane blebs. Electron and fluorescence microscopy, however demonstrated that only the N-acylated peptides partially induced the generation of oversized cells, which might reflect defects in cell-division. Further a different distribution of cardiolipin domains on the E. coli membrane was shown only in the presence of the N-acylated peptides. The lipid was distributed over the whole bacterial cell surface, whereas cardiolipin in untreated and nonacylated peptide-treated cells was mainly located at the septum and poles. Studies with bacterial membrane mimics, such as cardiolipin or phosphatidylethanolamine revealed that both types of peptides interacted with the negatively charged lipid cardiolipin. The nonacylated peptides however induced segregation of cardiolipin into peptide-enriched and peptide-poor lipid domains, while the N-acylated peptides promoted formation of many small heterogeneous domains. Only N-acylated peptides caused additional severe effects on the main phase transition of liposomes composed of pure phosphatidylethanolamine, while both peptide types inhibited the lamellar to hexagonal phase transition. Lipid mixtures of phosphatidylethanolamine and cardiolipin revealed anionic clustering by all peptide types. However additional strong perturbation of the neutral lipids was only seen with the N-acylated peptides. Nuclear magnetic resonance demonstrated different conformational arrangement of the N-acylated peptide in anionic and zwitterionic micelles revealing possible mechanistic differences in their action on different membrane lipids. We hypothesized that both peptides kill bacteria by interacting with bacterial membrane lipids but only N-acylated peptides interact with both charged cardiolipin and zwitterionic phosphatidylethanolamine resulting in remodeling of the natural phospholipid domains in the E. coli membrane that leads to defects in cell division.

MARCKS as a negative regulator of lipopolysaccharide signaling.

Myristoylated alanine-rich C kinase substrate (MARCKS) is an intrinsically unfolded protein with a conserved cationic effector domain, which mediates the cross-talk between several signal transduction pathways. Transcription of MARCKS is increased by stimulation with bacterial LPS. We determined that MARCKS and MARCKS-related protein specifically bind to LPS and that the addition of the MARCKS effector peptide inhibited LPS-induced production of TNF-α in mononuclear cells. The LPS binding site within the effector domain of MARCKS was narrowed down to a heptapeptide that binds to LPS in an extended conformation as determined by nuclear magnetic resonance spectroscopy. After LPS stimulation, MARCKS moved from the plasma membrane to FYVE-positive endosomes, where it colocalized with LPS. MARCKS-deficient mouse embryonic fibroblasts (MEFs) responded to LPS with increased IL-6 production compared with the matched wild-type MEFs. Similarly, small interfering RNA knockdown of MARCKS also increased LPS signaling, whereas overexpression of MARCKS inhibited LPS signaling. TLR4 signaling was enhanced by the ablation of MARCKS, which had no effect on stimulation by TLR2, TLR3, and TLR5 agonists. These findings demonstrate that MARCKS contributes to the negative regulation of the cellular response to LPS.

Structural features governing the activity of lactoferricin-derived peptides that act in synergy with antibiotics against Pseudomonas aeruginosa in vitro and in vivo.

Pseudomonas aeruginosa is naturally resistant to many antibiotics, and infections caused by this organism are a serious threat, especially to hospitalized patients. The intrinsic low permeability of P. aeruginosa to antibiotics results from the coordinated action of several mechanisms, such as the presence of restrictive porins and the expression of multidrug efflux pump systems. Our goal was to develop antimicrobial peptides with an improved bacterial membrane-permeabilizing ability, so that they enhance the antibacterial activity of antibiotics. We carried out a structure activity relationship analysis to investigate the parameters that govern the permeabilizing activity of short (8- to 12-amino-acid) lactoferricin-derived peptides. We used a new class of constitutional and sequence-dependent descriptors called PEDES (peptide descriptors from sequence) that allowed us to predict (Spearman's ρ = 0.74; P < 0.001) the permeabilizing activity of a new peptide generation. To study if peptide-mediated permeabilization could neutralize antibiotic resistance mechanisms, the most potent peptides were combined with antibiotics, and the antimicrobial activities of the combinations were determined on P. aeruginosa strains whose mechanisms of resistance to those antibiotics had been previously characterized. A subinhibitory concentration of compound P2-15 or P2-27 sensitized P. aeruginosa to most classes of antibiotics tested and counteracted several mechanisms of antibiotic resistance, including loss of the OprD porin and overexpression of several multidrug efflux pump systems. Using a mouse model of lethal infection, we demonstrated that whereas P2-15 and erythromycin were unable to protect mice when administered separately, concomitant administration of the compounds afforded long-lasting protection to one-third of the animals.

Expression, purification and structural studies of a short antimicrobial peptide.

We have produced a small antimicrobial peptide PFWRIRIRR in bacteria utilizing production in the form of insoluble fusion protein with ketosteroid isomerase. The recombinant peptide was rapidly and efficiently isolated by acidic cleavage of the fusion protein based on the acid labile Asp-Pro bond at the N-terminus of the peptide. The peptide has antibacterial activity and neutralizes macrophage activation by LPS. The selectivity of the peptide against bacteria correlates with preferential binding to acidic phospholipid vesicles. Solution structure of the peptide in SDS and DPC micelles was determined by NMR. The peptide adopts a well-defined structure, comprising a short helical segment. Cationic and hydrophobic clusters are segregated along the molecular axis of the short helix, which is positioned perpendicular to the membrane plane. The position of the helix is shifted in two micellar types and more nonpolar surface is exposed in anionic micelles. Overall structure explains the advantageous role of the N-terminal proline residue, which forms an integral part of the hydrophobic cluster.

Structural origin of endotoxin neutralization and antimicrobial activity of a lactoferrin-based peptide.

Treatment of Gram-negative bacterial infections with antimicrobial agents can cause release of the endotoxin lipopolysaccharide (LPS), the potent initiator of sepsis, which is the major cause of mortality in intensive care units worldwide. Structural information on peptides bound to LPS can lead to the development of more effective endotoxin neutralizers. Short linear antimicrobial and endotoxin-neutralizing peptide LF11, based on the human lactoferrin, binds to LPS, inducing a peptide fold with a "T-shaped" arrangement of a hydrophobic core and two clusters of basic residues that match the distance between the two phosphate groups of LPS. Side chain arrangement of LF11 bound to LPS extends the previously proposed LPS binding pattern, emphasizing the importance of both electrostatic and hydrophobic interactions in a defined geometric arrangement. In anionic micelles, the LF11 forms amphipathic conformation with a smaller hydrophobic core than in LPS, whereas in zwitterionic micelles, the structure is even less defined. Protection of tryptophan fluorescence quenching in the order SDS>LPS>DPC and hydrogen exchange protection indicates the decreasing extent of insertion of the N terminus and potential role of peptide plasticity in differentiation between bacterial and eukaryotic membranes.

Comparison of backbone dynamics of monomeric and domain-swapped stefin A.

Three-dimensional domain swapping has been observed in increasing number of proteins and has been implicated in the initial stages of protein aggregation, including that of the cystatins. Stefin A folds as a monomer under native conditions, while under some denaturing conditions domain-swapped dimer is formed. We have determined the backbone dynamics of the monomeric and domain-swapped dimeric forms of stefin A by (15)N relaxation using a model-free approach. The overall correlation times of the molecules were determined to be 4.6 +/- 0.1 ns and 9.2 +/- 0.2 ns for the monomer and the dimer, respectively. In the monomer, decreased order parameters indicate an increased mobility for the N-terminal trunk, the first and the second binding loops. At the opposite side of the molecule, the loop connecting the alpha-helix with strand B, the beginning of strand B and the loop connecting strands C and D show increased localized mobility. In the domain-swapped dimer, a distinctive feature of the structure is the concatenation of strands B and C into a single long beta-strand. The newly formed linker region between strands B and C, which substitutes for the first binding loop in the monomer, has order parameters typical for the remainder of the beta-strands. Thus, the interaction between subunits that occurs on domain-swapping has consequences for the dynamics of the protein at long-range from the site of conformational change, where an increased rigidity in the newly formed linker region is accompanied by an increased mobility of loops remote from that site.