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Transient gene expression (TGE) is commonly used to quickly produce protein-based drugs, such as antibodies, that require post-translational modifications. We have previously published a protocol for efficient and inexpensive TGE of multispecific and multivalent antibodies, which we have improved upon and described in the first part of this paper; by replacing the expensive Expi293 expression with BalanCD HEK293 medium, the medium cost was decreased by approximately 90%, and in addition, the harvesting procedure was shortened from 2.5 hours to 15 minutes by mixing the harvested cell media with the mineral compund diatomaceous earth that effectively absorbs and sequester cells and cell debris. The cell media can then be quickly filtered without the need to exchange obstructed filters and without the previously required 1-hour centrifugation step. In the second part of this paper, a small-scale TGE protocol was developed to surmount the cost limitation of testing many culture conditions. The small-scale TGE protocol uses 6-well plates which is the cheapest alternative for scaling down the protein expression, and consumes 83% less material compared to transfections done with the smallest available shaking flasks for cell culture. To test the small-scale TGE protocol we evaluated substances, belonging to a category called longevity molecules, as potential protein expression enhancers. Though the longevity molecules failed to increase protein expression in the conditions tested, our results corroborates the functionality of the small-scale TGE protocol and provides a simple methodology to expediently evaluate factors that can lead to improved protein production protocols in the future.

Background Amyloid-β (Aβ) immunotherapy is a promising therapeutic strategy in the fight against Alzheimer’s disease (AD). A number of monoclonal antibodies have entered clinical trials for AD. Some of them have failed due to the lack of efficacy or side-effects, two antibodies are currently in phase 3, and one has been approved by FDA. The soluble intermediate aggregated species of Aβ, termed oligomers and protofibrils, are believed to be key pathogenic forms, responsible for synaptic and neuronal degeneration in AD. Therefore, antibodies that can strongly and selectively bind to these soluble intermediate aggregates are of great diagnostic and therapeutic interest. Methods We designed and recombinantly produced a hexavalent antibody based on mAb158, an Aβ protofibril-selective antibody. The humanized version of mAb158, lecanemab (BAN2401), is currently in phase 3 clinical trials for the treatment of AD. The new designs involved recombinantly fusing single-chain fragment variables to the N-terminal ends of mAb158 antibody. Real-time interaction analysis with LigandTracer and surface plasmon resonance were used to evaluate the kinetic binding properties of the generated antibodies to Aβ protofibrils. Different ELISA setups were applied to demonstrate the binding strength of the hexavalent antibody to Aβ aggregates of different sizes. Finally, the ability of the antibodies to protect cells from Aβ-induced effects was evaluated by MTT assay. Results Using real-time interaction analysis with LigandTracer, the hexavalent design promoted a 40-times enhanced binding with avidity to protofibrils, and most of the added binding strength was attributed to the reduced rate of dissociation. Furthermore, ELISA experiments demonstrated that the hexavalent design also had strong binding to small oligomers, while retaining weak and intermediate binding to monomers and insoluble fibrils. The hexavalent antibody also reduced cell death induced by a mixture of soluble Aβ aggregates. Conclusion We provide a new antibody design with increased valency to promote binding avidity to an enhanced range of sizes of Aβ aggregates. This approach should be general and work for any aggregated protein or repetitive target.

Alzheimer’s disease is the most common neurodegenerative disorder characterized by the pathological aggregation of amyloid-β (Aβ) peptide. A potential therapeutic intervention in Alzheimer’s disease is to enhance Aβ degradation by increasing the activity of Aβ-degrading enzymes, including neprilysin. The somatostatin (SST) peptide has been identified as an activator of neprilysin. Recently, we demonstrated the ability of a brain-penetrating SST peptide (SST-scFv8D3) to increase neprilysin activity and membrane-bound Aβ42 degradation in the hippocampus of mice overexpressing the Aβ-precursor protein with the Swedish mutation (APPswe). Using LC–MS, we further evaluated the anti-Alzheimer’s disease effects of SST-scFv8D3. Following a triple intravenous injection of SST-scFv8D3, the LC–MS analysis of the brain proteome revealed that the majority of downregulated proteins consisted of mitochondrial proteins regulating fatty acid oxidation, which are otherwise upregulated in APPswe mice compared to wild-type mice. Moreover, treatment with SST-scFv8D3 significantly increased hippocampal levels of synaptic proteins regulating cell membrane trafficking and neuronal development. Finally, hippocampal concentrations of growth-regulated α (KC/GRO) chemokine and degradation of neuropeptide-Y were elevated after SST-scFv8D3 treatment. In summary, our results demonstrate a multifaceted effect profile in regulating mitochondrial function and neurogenesis following treatment with SST-scFv8D3, further suggesting the development of Alzheimer’s disease therapies based on SST peptides.

The presence of the blood brain barrier (BBB) creates a nigh on impenetrable obstacle for large macromolecular therapeutics that need to be delivered to the brain milieu to treat neurological disorders. To overcome this, one of the strategies used is to bypass the barrier with what is referred to as a “Trojan Horse” strategy, where therapeutics are designed to use endogenous receptor-mediated pathways to piggyback their way through the BBB. Even though in vivo methodologies are commonly used to test the efficacy of BBB penetrating biologics, comparable in vitro BBB models are in high demand, as they benefit from being an isolated cellular system devoid of physiological factors that can on occasion mask the processes behind BBB transport via transcytosis. We have developed an in vitro BBB model (In-Cell BBB-Trans assay) based on the murine cEND cells that helps delineate the ability of modified large bivalent IgG antibodies, conjugated to the transferrin-receptor binder scFv8D3, to cross an endothelial monolayer grown on porous cell culture inserts (PCIs). Following the administrating of bivalent antibodies to the endothelial monolayer, a highly sensitive ELISA method is used to determine the concentration in the apical (blood) and basolateral (brain) chambers of the PCI system, allowing for the evaluation of apical recycling and basolateral transcytosis respectively. Our results show that antibodies conjugated to scFv8D3 transcytose at considerably higher levels compared to unconjugated antibodies in the In-Cell BBB-Trans assay. Interestingly, we are able to show that these results mimic in vivo brain uptake studies using identical antibodies. In addition, we are able to transversely section PCI cultured cells, allowing for the identification of biomarkers pertinent to transcytosis of the antibodies. Furthermore, studies using the In-Cell BBB-Trans assay revealed transcytosis of the transferrin-receptor targeting antibodies is dependent on endocytosis. In conclusion, we have designed a simple, reproducible In-Cell BBB-Trans assay based on murine cells that can be used to rapidly determine the BBB penetrating capabilities of transferrin-receptor targeting antibodies. We believe the In-Cell BBB-Trans assay can be used as a powerful, pre-clinical screening platform for therapeutic neurological pathologies.

Amyloid-β (Aβ) oligomers and protofibrils are suggested to be the most neurotoxic Aβ species in Alzheimer’s disease (AD). Hence, antibodies with strong and selective binding to these soluble Aβ aggregates are of therapeutic potential. We have recently introduced HexaRmAb158, a multivalent antibody with additional Aβ-binding sites in the form of single-chain fragment variables (scFv) on the N-terminal ends of Aβ protofibril selective antibody (RmAb158). Due to the additional binding sites and the short distance between them, HexaRmAb158 displayed a slow dissociation from protofibrils and strong binding to oligomers in vitro. In the current study, we aimed at investigating the therapeutic potential of this antibody format in vivo using mouse models of AD. To enhance BBB delivery, the transferrin receptor (TfR) binding moiety (scFv8D3) was added, forming the bispecific-multivalent antibody (HexaRmAb158-scFv8D3). The new antibody displayed a weaker TfR binding compared to the previously developed RmAb158-scFv8D3 and was less efficiently transcytosed in a cell-based BBB model. HexaRmAb158 detected soluble Aβ aggregates derived from brains of tg-ArcSwe and AppNL−G−F mice more efficiently compared to RmAb158. When intravenously injected, HexaRmAb158-scFv8D3 was actively transported over the BBB into the brain in vivo. Brain uptake was marginally lower than that of RmAb158-scFv8D3, but significantly higher than observed for conventional IgG antibodies. Both antibody formats displayed similar brain retention (72 h post injection) and equal capacity in clearing soluble Aβ aggregates in tg-ArcSwe mice. In conclusion, we demonstrate a bispecific-multivalent antibody format capable of passing the BBB and targeting a wide-range of sizes of soluble Aβ aggregates.

The anaerobic bacterium

Background The clinical outcome and health-related quality of life (HRQoL) of living kidney donors is mostly not detrimental, but some donors experience impairment after donation. Gender-specific effects of living kidney donors was evaluated. Methods Clinical outcome was assessed in living kidney donors and HRQoL was obtained by self-reporting validated test systems as the Multidimensional Fatigue Inventory (MFI-20), the Short Form 36 (SF-36), and the Patient Health Questionnaire (PHQ-9). Results Two hundred and eleven (211) living renal donors were evaluated (female 62.2%). Response rate was 80.8%. In both genders, a decrease of renal function of 26% was observed after donation. De novo antihypertensives were introduced in 28.3% of women and 36.5% of men. HRQoL was comparable in female and male donors, except for mental HRQoL, which was lower in 51- to 60-year-old female donors, compared to age-matched male donors and to the female general population. Female donors aged 40–59 years demonstrated more fatigue than the age-matched general population. A low mental HRQoL (MCS; SF-36) was associated with higher values for fatigue (General Fatigue Score; MFI-20) in both genders. Multiple regression analysis detected the General Fatigue score of the MFI-20 questionnaire and depression identified by the PHQ-9 score as independent variables predicting MCS of the SF-36 in both genders. Lower age at time of donation contributed to a lower MCS in female donors. Conclusions Overall, HRQoL in living kidney donors exceeds that of the general population. Inferior mental health status and fatigue seem to be a problem, especially in middle-aged female donors, but not in all female donors. Psychological evaluation pre donation and psychological support post donation are required. Electronic supplementary material The online version of this article (10.1186/s12882-018-1187-8) contains supplementary material, which is available to authorized users.

Clostridioides difficile is the major pathogen causing diarrhea following antibiotic treatment. It is considered to be a strictly anaerobic bacterium, however, previous studies have shown a certain and strain-dependent oxygen tolerance. In this study, the model strain C. difficile 630Δerm was shifted to micro-aerobiosis and was found to stay growing to the same extent as anaerobically growing cells with only few changes in the metabolite pattern. However, an extensive change in gene expression was determined by RNA-Seq. The most striking adaptation strategies involve a change in the reductive fermentation pathways of the amino acids proline, glycine and leucine. But also a far-reaching restructuring in the carbohydrate metabolism was detected with changes in the phosphotransferase system (PTS) facilitated uptake of sugars and a repression of enzymes of glycolysis and butyrate fermentation. Furthermore, a temporary induction in the synthesis of cofactor riboflavin was detected possibly due to an increased demand for flavin mononucleotid (FMN) and flavin adenine dinucleotide (FAD) in redox reactions. However, biosynthesis of the cofactors thiamin pyrophosphate and cobalamin were repressed deducing oxidation-prone enzymes and intermediates in these pathways. Micro-aerobically shocked cells were characterized by an increased demand for cysteine and a thiol redox proteomics approach revealed a dramatic increase in the oxidative state of cysteine in more than 800 peptides after 15 min of micro-aerobic shock. This provides not only a catalogue of oxidation-prone cysteine residues in the C. difficile proteome but also puts the amino acid cysteine into a key position in the oxidative stress response. Our study suggests that tolerance of C. difficile towards O2 is based on a complex and far-reaching adjustment of global gene expression which leads to only a slight change in phenotype.

We have examined the distribution of ribosomes and nucleoids in live Escherichia coli cells under conditions of growth, division, and in quiescence. In exponentially growing cells translating ribosomes are interspersed among and around the nucleoid lobes, appearing as alternative bands under a fluorescence microscope. In contrast, inactive ribosomes either in stationary phase or after treatment with translation inhibitors such as chloramphenicol, tetracycline, and streptomycin gather predominantly at the cell poles and boundaries with concomitant compaction of the nucleoid. However, under all conditions, spatial segregation of the ribosomes and the nucleoids is well maintained. In dividing cells, ribosomes accumulate on both sides of the FtsZ ring at the mid cell. However, the distribution of the ribosomes among the new daughter cells is often unequal. Both the shape of the nucleoid and the pattern of ribosome distribution are also modified when the cells are exposed to rifampicin (transcription inhibitor), nalidixic acid (gyrase inhibitor), or A22 (MreB-cytoskeleton disruptor). Thus we conclude that the intracellular organization of the ribosomes and the nucleoids in bacteria are dynamic and critically dependent on cellular growth processes (replication, transcription, and translation) as well as on the integrity of the MreB cytoskeleton.