Background: Epilepsy remains challenging to treat still no etiologic treatment has been identified, however, some antiepileptic drugs (AEDs) are able to modify the pathogenesis of the disease. Lacosamide (LCM) has been shown to possess complex anticonvulsant and neuroprotective actions, being an enhancer of the slow inactivation of voltage-gated sodium channels, and it has the potential to prevent epileptogenesis. Recent evidence has shown that LCM indirectly improves the function of GABAA receptors. Receptors at most GABAergic synapses involve the gamma-2 subunit, which contributes to both phasic and tonic inhibition, and its presence assures benzodiazepine sensitivity. Moreover, mutant gamma-2 subunits were associated with generalized epilepsy syndromes. In animal models, the expression of the gamma-2 subunit of the gamma-aminobutyric acid A receptor (GABAAg2) was shown to be increased in pentylenetetrazole (PTZ)-induced chemical kindling in Wistar rats. Objective: This study hypothesized that LCM might affect the kindling process by influencing the expression of GABAA receptors in the hippocampus. Methods: The gene and protein expression levels of the GABAAg2 were studied using RT-qPCR and immunofluorescent staining. Results: It was found that LCM treatment (10 mg/kg i.p. daily for 57 days) reduced the maximal intensity of the PTZ-induced seizures but did not prevent kindling. On the other hand, LCM treatment reverted the increase of mRNA expression of GABAAg2 in the hippocampus and prevented the decrease of GABAAg2 protein in the hippocampal CA1 region. Conclusion: LCM could exhibit modulatory effects on the GABAergic system of the hippocampus that may be independent of the anticonvulsant action.

Succinic acid production through biological fermentation led to new pathways in the integration of renewable feedstock from different industries into biosynthesis. In this article, we investigate the population growth dynamics and succinic acid production potential of the recently isolated natural succinic acid producer, Basfia succiniciproducens, using in silico constraint-based metabolic models as well as in vitro experiments. Our work focuses on the influence of different renewable substrates and added yeast extract on fermentation dynamics, and the produced metabolites of the strain cultured in mineral (minimal) medium. According to our experiments, which were carried out as small-scale fermentations and in bioreactor conditions, glucose is the preferred carbon source, while the addition of 1% yeast extract has a significant positive effect on biomass formation. In the case of B. succiniciproducens cultured in minimal salt medium, a production potential as high as 47.09 mM succinic acid was obtained in these conditions. Industrial applications related to this bacterial strain could contribute to new possibilities for the re-use of byproducts by using fermentation processes, leading to high added-value compounds.

A continuous effort is needed to develop sustainable production methods for industrial platform chemicals. B. succiniciproducens, a natural succinic acid-producer, can metabolize five and six carbon atoms containing sugars in pure form as well as from agro-industrial wastes. In our work, we investigate the conversion of industrial by-products, apple pomace from apple juice production, and whey waste from milk processing to succinic acid and other organic acids (lactic, formic, and acetic acid). We obtained a succinic acid yield of 0.224 g/g total consumed fermentable sugars, lactic acid yield was 0.087 g/g, in turn, formic acid was produced at a 0.034 g/g yield, and acetic acid was obtained at 0.010 g/g total consumed fermentable sugars, using a thermal pretreated apple pomace-based medium. In the case of pretreated whey-based medium formulation, we obtained a succinic acid yield of 0.236 g/g consumed lactose, while formic acid and acetic acid were produced as well (0.09 g/g and 0.101 g/g, respectively). We demonstrate that lactose is a promising carbon source for organic acid production by B. succiniciproducens, while our study is the first to propose the use of a similarly available agro-industrial by-product, apple pomace, for the fermentative production of succinic acid by B. succiniciproducens.

Background/Objectives: Immune checkpoint blockade, particularly targeting the programmed cell death 1 (PD-1) receptor, is a promising strategy in cancer immunotherapy. The interaction between PD-1 and its ligands, PD-L1 and PD-L2, is crucial in immune evasion by tumors. Blocking this interaction with monoclonal antibodies like Nivolumab can restore anti-tumor immunity. This study aims to develop a stable expression system for Nivolumab-based anti-PD-1 in the Chinese Hamster Ovary (CHO) DG44 cell line using two different expression vector systems with various signal sequences. Methods: The heavy chain (HC) and light chain (LC) of Nivolumab were cloned into two expression vectors, pOptiVEC and pcDNA3.3. Each vector was engineered with two distinct signal sequences, resulting in the creation of eight recombinant plasmids. These plasmids were co-transfected into CHO DG44 cells in different combinations, allowing for the assessment of stable antibody production. Results: Both pOptiVEC and pcDNA3.3 vectors were successful in stably integrating and expressing the Nivolumab-based anti-PD-1 antibody in CHO DG44 cells. This study found that the choice of signal sequence significantly influenced the quantity of antibodies produced. The optimization of production conditions further enhanced antibody yield, indicating the potential for large-scale production. Conclusions: This study demonstrates that both pOptiVEC and pcDNA3.3 expression systems are effective for the stable production of Nivolumab-based anti-PD-1 in CHO DG44 cells. Signal sequences play a critical role in determining the expression levels, and optimizing production conditions can further increase antibody yield, supporting future applications in cancer immunotherapy.

New proteins as molecular targets in development of therapies are discovered every day. However, study of their interactions with other proteins or binding partners in complex cellular environments has its limits. Therefore, high-yield production of these proteins in heterologous systems is a valid necessity, while obtaining these proteins linked to suitable fluorescent markers represents a step ahead in protein-protein interaction studies and cellular or subcellular localization. In this study, we present production of human SMAC/Diablo recombinant protein with EGFP as a fusion partner. High-yield expression of the fusion protein was carried out in E. coli Rosetta™(DE3)pLysS strain, and an acceptable purity of the protein was obtained after affinity chromatography purification and gel filtration. The obtained protein can be further used in protein-protein interaction studies, whereas our method represents a cost-effective and efficient production method for EGFP-fused proteins, applicable for a number of therapeutically important polypeptides.

Human proteins expressed in prokaryotic systems tend to form inclusion bodies. Proteins in inclusion bodies are inactive and the refolding of these densely packed protein molecules is affected by several factors depending on the applied refolding technique. To obtain the active form of protein the most common technique is denaturation of the protein aggregates followed by refolding of inclusion proteins. Conventional denaturants for solubilization are urea, guanidine hydrochloride and sodium dodecyl sulphate (SDS), while refolding can be achieved by several techniques found in the literature. In our study, the recombinant GST-tagged XIAP (X-linked Inhibitor of Apoptosis protein) construct was expressed as inclusion bodies. The protein was solubilized with high efficiency using N-Lauroylsarcosine (ionic detergent). A chromatography-based method using different ratios of detergents was investigated for the refolding process. Batch mode affinity purification was successfully executed using Glutathione Sepharose 4B resin and TritonX-100, n-octyl β-D-thio-glucopyranoside (OTG) and 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate hydrate (CHAPS) detergents in the appropriate ratio. Finally, the refolded protein was purified by size-exclusion chromatography and investigated by western blot analysis.