Non-Coding RNAs: New Biomarkers and Therapeutic Targets for Temporal Lobe Epilepsy

Temporal lobe epilepsy (TLE) is the most common form of focal epilepsy; it is considered a network disorder associated with structural changes. Incomplete knowledge of the pathological changes in TLE complicates a therapeutic approach; indeed, 30 to 50% of patients with TLE are refractory to drug treatment. Non-coding RNAs (ncRNAs), acting as epigenetic factors, participate in the regulation of the pathophysiological processes of epilepsy and are dysregulated during epileptogenesis. Abnormal expression of ncRNA is observed in patients with epilepsy and in animal models of epilepsy. Furthermore, ncRNAs could also be used as biomarkers for the diagnosis and prognosis of treatment response in epilepsy. In summary, ncRNAs can represent important mechanisms and targets for the modulation of brain excitability and can provide information on pathomechanisms, biomarkers and novel therapies for epilepsy. In this review, we summarize the latest research advances concerning mainly molecular mechanisms, regulated by ncRNA, such as synaptic plasticity, inflammation and apoptosis, already associated with the pathogenesis of TLE. Moreover, we discuss the role of ncRNAs, such as microRNAs, long non-coding RNAs and circular RNAs, in the pathophysiology of epilepsy, highlighting their use as potential biomarkers for future therapeutic approaches.     

Chemical Genetic Approaches for the Investigation of Neutral Lipid Metabolism

The coordinated processes of lipid synthesis, degradation, and transport are mediated by enzymes, cofactors, and transport proteins. Accordingly, lipid‐metabolizing enzymes represent logical targets for the treatment of dyslipidemia, a major risk factor for type 2 diabetes, atherosclerosis, and other disorders. Small‐molecule tool compounds, modulating the functions of such proteins, can substantially facilitate the characterization of target proteins. Such molecules complement genetic studies, and allow time‐ and dose‐dependent control of protein activity in biological systems. This can improve our understanding of physiological processes, give insights into the druggability of target proteins, and might finally result in the development of therapeutic compounds. In this review we summarize the current state of available inhibitors targeting key proteins in neutral lipid metabolism, with a focus on metabolic lipases, acyltransferases, and fatty‐acid‐binding proteins.     

High level expression and secretion of Fc-X fusion proteins in mammalian cells

We have developed a general expression system that enhances the production and secretion of proteins in mammalian cells. The protein of interest is expressed as a fusion to a signal peptide and the Fc fragment of immunoglobulin as the N-terminal fusion partner, which can direct the cellular processes into expressing and secreting high levels of many different types of proteins. These include secretory proteins, enzymes and soluble domains of membrane proteins, as well as nuclear and regulatory proteins. Typical expression levels of these proteins from stable cell lines ranged from several to 100 microg/ml in conditioned media. The Fc domain helps to solubilize hydrophobic proteins and provides a handle for easy detection and purification of the fusion proteins; and it can be cleaved off by treatment with protease if desired.      

Chemical Probes and Activity-Based Protein Profiling for Cancer Research

Chemical probes can be used to understand the complex biological nature of diseases. Due to the diversity of cancer types and dynamic regulatory pathways involved in the disease, there is a need to identify signaling pathways and associated proteins or enzymes that are traceable or detectable in tests for cancer diagnosis and treatment. Currently, fluorogenic chemical probes are widely used to detect cancer-associated proteins and their binding partners. These probes are also applicable in photodynamic therapy to determine drug efficacy and monitor regulating factors. In this review, we discuss the synthesis of chemical probes for different cancer types from 2016 to the present time and their application in monitoring the activity of transferases, hydrolases, deacetylases, oxidoreductases, and immune cells. Moreover, we elaborate on their potential roles in photodynamic therapy.     

The Multifaceted Role of Plasminogen in Cancer

Fibrinolytic factors like plasminogen, tissue-type plasminogen activator (tPA), and urokinase plasminogen activator (uPA) dissolve clots. Though mere extracellular-matrix-degrading enzymes, fibrinolytic factors interfere with many processes during primary cancer growth and metastasis. Their many receptors give them access to cellular functions that tumor cells have widely exploited to promote tumor cell survival, growth, and metastatic abilities. They give cancer cells tools to ensure their own survival by interfering with the signaling pathways involved in senescence, anoikis, and autophagy. They can also directly promote primary tumor growth and metastasis, and endow tumor cells with mechanisms to evade myelosuppression, thus acquiring drug resistance. In this review, recent studies on the role fibrinolytic factors play in metastasis and controlling cell-death-associated processes are presented, along with studies that describe how cancer cells have exploited plasminogen receptors to escape myelosuppression.     

Real-Time and Label-Free Measurement of Deubiquitinase Activity with a MspA Nanopore

Covalently attaching ubiquitin (Ub) to cellular proteins as a post-translational modification can result in altered function of modified proteins. Enzymes regulating Ub as a post-translational modification, such as ligases and deubiquitinases, are challenging to characterize in part due to the low throughput of in-vitro assays. Single-molecule nanopore based assays have the advantage of detecting proteins with high specificity and resolution, and in a label-free, real-time fashion. Here we demonstrate the use of a MspA nanopore for discriminating and quantifying Ub proteins. We further applied the MspA pore to measure the Ub-chain disassembly activity of UCH37, a proteasome associated deubiquitinase. The implementation of this MspA system into nanopore arrays could enable high throughput characterizations of unknown deubiquitinases as well as drug screening against disease related enzymes.     

Ubiquitin and Ubiquitin-like Proteins in Cancer,Neurodegenerative Disorders,and Heart Diseases

Post-translational modification (PTM) is an essential mechanism for enhancing the functional diversity of proteins and adjusting their signaling networks. The reversible conjugation of ubiquitin (Ub) and ubiquitin-like proteins (Ubls) to cellular proteins is among the most prevalent PTM, which modulates various cellular and physiological processes by altering the activity, stability, localization, trafficking, or interaction networks of its target molecules. The Ub/Ubl modification is tightly regulated as a multi-step enzymatic process by enzymes specific to this family. There is growing evidence that the dysregulation of Ub/Ubl modifications is associated with various diseases, providing new targets for drug development. In this review, we summarize the recent progress in understanding the roles and therapeutic targets of the Ub and Ubl systems in the onset and progression of human diseases, including cancer, neurodegenerative disorders, and heart diseases.     

Nutritional Interventions for Patients with Mitochondrial POLG-Related Diseases: A Systematic Review on Efficacy and Safety

Ketogenic diet is recommended as a treatment to reduce seizure frequency in patients with intractable epilepsy. The evidence and safety results are sparse for diet interventions in patients with pathogenic polymerase gamma (POLG) variants and intractable epilepsy. The aim of this systematic review is to summarize the efficacy of diet treatment on seizure frequency, clinical symptoms, and potential deleterious effect of liver involvement in patients with mitochondrial diseases caused by pathogenic POLG variants. Literature was searched in PubMed, Embase; and Cochrane in April 2022; no filter restrictions were imposed. The reference lists of retrieved studies were checked for additional literature. Eligibility criteria included verified pathogenic POLG variant and diet treatment. Overall, 880 studies were identified, providing eight case-reports representing nine patients eligible for inclusion. In eight of nine cases, clinical symptoms were improved; six out of nine cases reported improvements in seizure frequency. However, increasing levels of liver enzymes after initiating ketogenic diet were found in four of the nine cases, with one case revealing decreased levels of liver enzymes after initiating long-chain triglyceride restriction. Viewed together, the studies imply that ketogenic diet can have a positive impact on seizure frequency, but may induce progression of liver impairment in patients with pathogenic POLG variants.     

Nutritional qualities of soya protein as affected by processing

The nutritional qualities of soybean proteins are basically determined by amino acid patterns, amino acid availabilities (digestibility) and contents of biologically active components. Of these factors, the last two are most affected by processing conditions, whereas amino acid analysis is least affected, although it too may be modified in those cases in which soybean proteins are fractionated. In the preparation of the large variety of soya products presently available, soybeans are subjected to many different processes, all of which are discussed. Heat treatment appears to be the process which most affects protein nutritional quality; generally, quality first increases with heat treatment due to inactivation of biologically active factors, passes through a maximum and then decreases due to destruction and/or inactivation of essential amino acids such as cystine and lysine. Other processes affect protein nutritional quality to different degrees, inasmuch as they affect amino acid analysis, digestibility and content of biologically active components. When soybean proteins are used to extend animal proteins, supplement other vegetable proteins, or in vegetable protein mixtures, nutritional quality of the combined proteins appears to be affected in the same manner as that of soya proteins alone.     

Immobilization of biologically active species on PA‐6 foils treated by a dielectric barrier discharge

In the field of biomaterials and biomedical devices, surface activation has been focused on creating functional groups capable of preferential adsorption of biologically active species (proteins, enzymes, cells, drugs, etc.). In this way an interface can be created between the synthetic material and the biological medium, with the aim of increasing the compatibility of the implant with the human organism. In our experiments a dielectric barrier discharge (DBD), in helium at atmospheric pressure, was used as the source of energy capable of creating active centers that render the functionalized surface favorable to immobilization of biological molecules. Retention of immunoglobulin (IgG) and heparin biomolecules on polyamide‐6 (PA‐6) surfaces after treatment by the DBD was analyzed by atomic force microscopy, adhesion evaluation, and measurement of the contact angle titration in order to assess this incorporation on the treated surfaces. The marked adsorption of the biomolecules on the active sites created by DBD on the exposed surfaces also was related to a complex set of processes, such as enhanced roughness, increased surface wettability, and modified distribution of cationic and anionic groups on the treated surfaces. All these factors could promote interfacial interactions between the specific groups of the biomolecules existing in the biological medium and the type of cationic and/or anionic groups present on the surface. The efficiency of the DBD treatment showed that the DBD technique is useful for preactivation of the polymer surface for immobilization of other biologically active species (such as drugs and enzymes). © 2003 Wiley Periodicals, Inc. J Appl Polym Sci 90: 1985–1990, 2003     

Recombinant yeast technology at the cutting edge: robust tools for both designed catalysts and new biologicals

Health and safety concerns, enhanced quality criteria, and environmental sustainability, have prompted investigations into production using recombinant yeasts as a feasible alternative for isolation of proteins from natural animal or plant sources, as well as for processes utilising either mammalian cell cultures or bacterial systems. An overview of recent research papers and review articles provides readers with a comprehensive insight into the field of next-generation yeast expression systems. Major breakthroughs in recombinant yeast technology linked to Pichia pastoris are (i) the public availability of tools to generate proteins with tailored and highly homogenous N-glycan structures, similar to the forms assembled in humans, (ii) the recent accomplishment of the annotation of its genome sequence, and finally, (iii) the presence of the first few (non-glycosylated) therapeutic proteins in Pichia on the market. The P. pastoris expression platform is now well developed, as proven by multiple products used in human and veterinary medicine and in industry (e.g., enzymes for chemical synthesis and for the modification/synthesis of pharmaceuticals, drug target proteins used for structural analysis or for high throughput screening, proteins for diagnostics, proteinous biomaterials, vaccines, and therapeutic proteins). Nevertheless, the complexity of protein analysis (monitoring) continues to restrict process development for recombinant products. Drawing on combined expertise in molecular biology and process technology, the Institute of Biotechnology (IBT) at the Zurich University of Applied Science (ZHAW) and its international partners have developed solutions which (i) fully eliminate (or partially reduce) the use of methanol, which is undesirable in high-cell-density and high-productivity processes, (ii) match both strain construction and process design with the target protein characteristics to the benefit of the cells' physiological shape, and (iii) allow multi-gene expressions to be balanced to achieve custom tailored and reproducible protein quality at the level of (engineered) posttranslational modifications. In addition to enabling superior product quality specifications to be achieved with reduced development time, these innovations have helped the industries involved to minimise financial risks and the risk of failure, as well as create an opportunity for (new) drugs with improved functionality at low cost.     

Biofriendly sol-gel processing for the entrapment of soluble and membrane-bound proteins: toward novel solid-phase assays for high-throughput screening

The last decade has seen a revolution in the area of sol-gel-derived biomaterials since the demonstration that these materials can be used to encapsulate biological species such as enzymes, antibodies, and other proteins in a functional state. In particular, recent years have seen tremendous progress in the development of more "protein-friendly" sol-gel processing methods and their use for immobilization of delicate proteins, including key drug targets such as kinases and membrane-bound receptors. The latter example is particularly impressive, given the inherently low stability of membrane receptors and the need to stabilize an amphiphilic bilayer lipid membrane to maintain receptor function. In this Account, we provide an overview of the advances in biofriendly sol-gel processing methods developed in our research group and others and highlight recent accomplishments in the immobilization of both soluble and membrane-bound proteins, with particular emphasis on enzymes and membrane receptors that are drug targets. Emerging applications of sol-gel-entrapped proteins, focusing on the development platforms for high-throughput screening of small molecules, are also described.     

Protein-Metabolite Interactions: Discovery and Significance

Metabolites orchestrate cellular processes as either substrates, co-enzymes, inhibitors, or activators of cellular proteins such as enzymes and receptors. Although traditional biochemical and structural biology-based approaches have been successfully employed for the discovery of protein-metabolite interactions, they often fail to detect transient and low-affinity biomolecular relationships. Another limitation of these approaches is that they are performed under in vitro conditions lacking the physiological context. Recently developed mass spectrometry-based methodologies overcome both these shortcomings, and have resulted in the discovery of global protein-metabolite cellular interaction networks. Herein, we describe traditional and modern approaches for the discovery of protein-metabolite interactions, and discuss the impact of these discoveries on our understanding of cellular physiology and on drug development.     

Elevated Serum Cortisol Levels in Patients with Focal Epilepsy,Depression, and Comorbid Epilepsy and Depression

Background: The hypothalamic-pituitary-adrenal (HPA) axis, inflammatory processes and neurotrophic factor systems are involved in pathogenesis of both epilepsy and depressive disorders. The study aimed to explore these systems in patients with focal epilepsy (PWE, n = 76), epilepsy and comorbid depression (PWCED n = 48), and major depressive disorder (PWMDD, n = 62) compared with healthy controls (HC, n = 78). Methods: Parameters of the HPA axis, neurotrophic factors, and TNF-α were measured in blood serum along with the hemogram. Results: Serum cortisol level was augmented in PWE, PWCED, and PWMDD compared with HC and was higher in PWMDD than in PWE. Serum cortisol negatively correlated with Mini–Mental State Examination (MMSE) score in PWE, and positively with depression inventory–II (BDI-II) score in PWMDD. Only PWMDD demonstrated elevated plasma ACTH. Serum TNF-α, lymphocytes, and eosinophils were augmented in PWMDD; monocytes elevated in PWE and PWCED, while neutrophils were reduced in PWE and PWMDD. Serum BDNF was decreased in PWE and PWCED, CNTF was elevated in all groups of patients. In PWE, none of above indices depended on epilepsy etiology. Conclusions: The results confirm the involvement of HPA axis and inflammatory processes in pathogenesis of epilepsy and depression and provide new insights in mechanisms of epilepsy and depression comorbidity.     

Covalent Inhibition in Drug Discovery

Although covalent inhibitors have been used as therapeutics for more than a century, there has been general resistance in the pharmaceutical industry against their further development due to safety concerns. This inclination has recently been reverted after the development of a wide variety of covalent inhibitors to address human health conditions along with the US Food and Drug Administration (FDA) approval of several covalent therapeutics for use in humans. Along with this exciting resurrection of an old drug discovery concept, this review surveys enzymes that can be targeted by covalent inhibitors for the treatment of human diseases. We focus on protein kinases, RAS proteins, and a few other enzymes that have been studied extensively as targets for covalent inhibition, with the aim to address challenges in designing effective covalent drugs and to provide suggestions in the area that have yet to be explored.     

Dry action of Trichoderma reesei cellulases on cotton fabrics

Following treatment with Trichoderma reesei cellulases, the mechanical properties of cotton fabrics were measured to assess the effect of adsorbed enzymes. The ability of adsorbed cellulases to act as anchors for further wet finishing processes was studied. Dried fabric samples, after 105 days under usual storage conditions, showed neither significant strength loss, nor any changes in the degree of polymerisation. After five months of storage, the enzymes were still found to be active. The adsorption of T reesei cellulases produced an increase in staining levels after dyeing with an acid dye, but washing fastness was poor. Washing with soap under alkaline conditions removed proteins almost completely. Adsorption of T reesei cellulases is reversible and desorption increases from pH 5 to pH 10. Because of the protein desorption under usual alkaline washing conditions, no practical application of adsorbed cellulases for further finishing processes is suggested and more work is required.     

Cancer Cell Response to Anthracyclines Effects: Mysteries of the Hidden Proteins Associated with These Drugs

A comprehensive proteome map of T-lymphoblastic leukemia cells and its alterations after daunorubicin, doxorubicin and mitoxantrone treatments was monitored and evaluated either by paired comparison with relevant untreated control and using multivariate classification of treated and untreated samples. With the main focus on early time intervals when the influence of apoptosis is minimized, we found significantly different levels of proteins, which corresponded to 1%–2% of the total amount of protein spots detected. According to Gene Ontology classification of biological processes, the highest representation of identified proteins for all three drugs belong to metabolic processes of proteins and nucleic acids and cellular processes, mainly cytoskeleton organisation and ubiquitin-proteasome pathway. Importantly, we observed significant proportion of changes in proteins involved in the generation of precursor metabolites and energy typical for daunorubicin, transport proteins participating in response to doxorubicin and a group of proteins of immune system characterising response to mitoxantrone. Both a paired comparison and the multivariate evaluation of quantitative data revealed daunorubicin as a distinct member of the group of anthracycline/anthracenedione drugs. A combination of identified drug specific protein changes, which may help to explain anti-cancer activity, together with the benefit of blocking activation of adaptive cancer pathways, presents important approaches to improving treatment outcomes in cancer.     

Beneficial Effects of Metformin on the Central Nervous System,with a Focus on Epilepsy and Lafora Disease

Metformin is a drug in the family of biguanide compounds that is widely used in the treatment of type 2 diabetes (T2D). Interestingly, the therapeutic potential of metformin expands its prescribed use as an anti-diabetic drug. In this sense, it has been described that metformin administration has beneficial effects on different neurological conditions. In this work, we review the beneficial effects of this drug as a neuroprotective agent in different neurological diseases, with a special focus on epileptic disorders and Lafora disease, a particular type of progressive myoclonus epilepsy. In addition, we review the different proposed mechanisms of action of metformin to understand its function at the neurological level.     

Effects of Lacosamide Treatment on Epileptogenesis,Neuronal Damage and Behavioral Comorbidities in a Rat Model of Temporal Lobe Epilepsy

Clinically, temporal lobe epilepsy (TLE) is the most prevalent type of partial epilepsy and often accompanied by various comorbidities. The present study aimed to evaluate the effects of chronic treatment with the antiepileptic drug (AED) lacosamide (LCM) on spontaneous motor seizures (SMS), behavioral comorbidities, oxidative stress, neuroinflammation, and neuronal damage in a model of TLE. Vehicle/LCM treatment (30 mg/kg, p.o.) was administered 3 h after the pilocarpine-induced status epilepticus (SE) and continued for up to 12 weeks in Wistar rats. Our study showed that LCM attenuated the number of SMS and corrected comorbid to epilepsy impaired motor activity, anxiety, memory, and alleviated depressive-like responses measured in the elevated plus maze, object recognition test, radial arm maze test, and sucrose preference test, respectively. This AED suppressed oxidative stress through increased superoxide dismutase activity and glutathione levels, and alleviated catalase activity and lipid peroxidation in the hippocampus. Lacosamide treatment after SE mitigated the increased levels of IL-1β and TNF-α in the hippocampus and exerted strong neuroprotection both in the dorsal and ventral hippocampus, basolateral amygdala, and partially in the piriform cortex. Our results suggest that the antioxidant, anti-inflammatory, and neuroprotective activity of LCM is an important prerequisite for its anticonvulsant and beneficial effects on SE-induced behavioral comorbidities.     

A Novel Fluorescent Probe for Retaining Galactosyltransferases

Glycosyltransferases (GTs) are a large class of carbohydrate‐active enzymes that are involved, in both pro‐ and eukaryotic organisms, in numerous important biological processes, from cellular adhesion to carcinogenesis. GTs have enormous potential as molecular targets for chemical biology and drug discovery. For the full realisation of this potential, operationally simple and generally applicable GT bioassays, especially for inhibitor screening, are indispensable tools. In order to facilitate the development of GT high‐throughput screening assays for the identification of GT inhibitors, we have developed novel, fluorescent derivatives of UDP‐galactose (UDP‐Gal) that are recognised as donor analogues by several different retaining galactosyltransferases (GalTs). We demonstrate for one of these derivatives that fluorescence emission is quenched upon specific binding to individual GalTs, and that this effect can be used as the read‐out in ligand‐displacement experiments. The novel fluorophore acts as an excellent sensor for several different enzymes and is suitable for the development of a new type of GalT bioassay, whose modular nature and operational simplicity will significantly facilitate inhibitor screening. Importantly, the structural differences between the natural donor UDP‐Gal and the new fluorescent derivatives are minimal, and the general assay principle described herein may therefore also be applicable to other GalTs and/or proteins that use nucleotides or nucleotide conjugates as their cofactor.