Oxidative Folding of Proteins: The “Smoking Gun” of Glutathione

Glutathione has long been suspected to be the primary low molecular weight compound present in all cells promoting the oxidative protein folding, but twenty years ago it was found “not guilty”. Now, new surprising evidence repeats its request to be the “smoking gun” which reopens the criminal trial revealing the crucial involvement of this tripeptide.     

Dynamic “Molecular Portraits” of Biomembranes Drawn by Their Lateral Nanoscale Inhomogeneities

To date, it has been reliably shown that the lipid bilayer/water interface can be thoroughly characterized by a sophisticated so-called “dynamic molecular portrait”. The latter reflects a combination of time-dependent surface distributions of various physicochemical properties, inherent in both model lipid bilayers and natural multi-component cell membranes. One of the most important features of biomembranes is their mosaicity, which is expressed in the constant presence of lateral inhomogeneities, the sizes and lifetimes of which vary in a wide range—from 1 to 10³ nm and from 0.1 ns to milliseconds. In addition to the relatively well-studied macroscopic domains (so-called “rafts”), the analysis of micro- and nanoclusters (or domains) that form an instantaneous picture of the distribution of structural, dynamic, hydrophobic, electrical, etc., properties at the membrane-water interface is attracting increasing interest. This is because such nanodomains (NDs) have been proven to be crucial for the proper membrane functioning in cells. Therefore, an understanding with atomistic details the phenomena associated with NDs is required. The present mini-review describes the recent results of experimental and in silico studies of spontaneously formed NDs in lipid membranes. The main attention is paid to the methods of ND detection, characterization of their spatiotemporal parameters, the elucidation of the molecular mechanisms of their formation. Biological role of NDs in cell membranes is briefly discussed. Understanding such effects creates the basis for rational design of new prospective drugs, therapeutic approaches, and artificial membrane materials with specified properties.     

Slaying (Yet Again) the Brain-Eating Zombie Called the “Isochore Theory”: A Segmentation Algorithm Used to “Confirm” the Existence of Isochores Creates “Isochores” Where None Exist

The isochore theory, which was proposed more than 40 years ago, depicts the mammalian genome as a mosaic of long, homogeneous regions that are characterized by their guanine and cytosine (GC) content. The human genome, for instance, was claimed to consist of five compositionally distinct isochore families. The isochore theory, in all its reincarnations, has been repeatedly falsified in the literature, yet isochore proponents have persistently resurrected it by either redefining isochores or by proposing alternative means of testing the theory. Here, I deal with the latest attempt to salvage this seemingly immortal zombie—a sequence segmentation method called isoSegmenter, which was claimed to “identify” isochores while at the same time disregarding the main characteristic attribute of isochores—compositional homogeneity. I used a series of controlled, randomly generated simulated sequences as a benchmark to study the performance of isoSegmenter. The main advantage of using simulated sequences is that, unlike real data, the exact start and stop point of any isochore or homogeneous compositional domain is known. Based on three key performance metrics—sensitivity, precision, and Jaccard similarity index—isoSegmenter was found to be vastly inferior to isoPlotter, a segmentation algorithm with no user input. Moreover, isoSegmenter identified isochores where none exist and failed to identify compositionally homogeneous sequences that were shorter than 100−200 kb. Will this zillionth refutation of “isochores” ensure a final and permanent entombment of the isochore theory? This author is not holding his breath.     

From Molecular Recognition to the “Vehicles” of Evolutionary Complexity: An Informational Approach

Countless informational proposals and models have explored the singular characteristics of biological systems: from the initial choice of information terms in the early days of molecular biology to the current bioinformatic avalanche in this “omic” era. However, this was conducted, most often, within partial, specialized scopes or just metaphorically. In this paper, we attempt a consistent informational discourse, initially based on the molecular recognition paradigm, which addresses the main stages of biological organization in a new way. It considers the interconnection between signaling systems and information flows, between informational architectures and biomolecular codes, between controlled cell cycles and multicellular complexity. It also addresses, in a new way, a central issue: how new evolutionary paths are opened by the cumulated action of multiple variation engines or mutational ‘vehicles’ evolved for the genomic exploration of DNA sequence space. Rather than discussing the possible replacement, extension, or maintenance of traditional neo-Darwinian tenets, a genuine informational approach to evolutionary phenomena is advocated, in which systemic variation in the informational architectures may induce differential survival (self-construction, self-maintenance, and reproduction) of biological agents within their open ended environment.     

The Recruitment-Secretory Block (“R-SB”) Phenomenon and Endoplasmic Reticulum Storage Diseases

In this article, we review the biological and clinical implication of the Recruitment-Secretory Block (“R-SB”) phenomenon. The phenomenon refers to the reaction of the liver with regard to protein secretion in conditions of clinical stimulation. Our basic knowledge of the process is due to the experimental work in animal models. Under basal conditions, the protein synthesis is mainly carried out by periportal (zone 1) hepatocytes that are considered the “professional” synthesizing protein cells. Under stimulation, midlobular and centrolobular (zones 2 and 3) hepatocytes, are progressively recruited according to lobular gradients and contribute to the increase of synthesis and secretion. The block of secretion, operated by exogenous agents, causes intracellular retention of all secretory proteins. The Pi MZ phenotype of Alpha-1-antitrypsin deficiency (AATD) has turned out to be the key for in vivo studies of the reaction of the liver, as synthesis and block of secretion are concomitant. Indeed, the M fraction of AAT is stimulated for synthesis and regularly exported while the Z fraction is mostly retained within the cell. For that reason, the phenomenon has been designated “Recruitment-Secretory Block” (“R-SB”). The “R-SB” phenomenon explains why: (a) the MZ individuals can correct the serum deficiency; (b) the resulting immonohistochemical and electron microscopic (EM) patterns are very peculiar and specific for the diagnosis of the Z mutation in tissue sections in the absence of genotyping; (c) the term carrier is no longer applicable for the heterozygous condition as all Pi MZ individuals undergo storage and the storage predisposes to liver damage. The storage represents the true elementary lesion and consequently reflects the phenotype-genotype correlation; (d) the site and function of the extrahepatic AAT and the relationship between intra and extracellular AAT; (e) last but not least, the concept of Endoplasmic Reticulum Storage Disease (ERSD) and of a new disease, hereditary hypofibrinogenemia with hepatic storage (HHHS). In the light of the emerging phenomenon, described in vitro, namely that M and Z AAT can form heteropolymers within hepatocytes as well as in circulation, we have reviewed the whole clinical and experimental material collected during forty years, in order to evaluate to what extent the polymerization phenomenon occurs in vivo. The paper summarizes similarities and differences between AAT and Fibrinogen as well as between the related diseases, AATD and HHHS. Indeed, fibrinogen gamma chain mutations undergo an aggregation process within the RER of hepatocytes similar to AATD. In addition, this work has clarified the intriguing phenomenon underlying a new syndrome, hereditary hypofibrinogenemia and hypo-APO-B-lipoproteinemia with hepatic storage of fibrinogen and APO-B lipoproteins. It is hoped that these studies could contribute to future research and select strategies aimed to simultaneously correct the hepatocytic storage, thus preventing the liver damage and the plasma deficiency of the two proteins.     

The FMN “140s Loop” of Cytochrome P450 Reductase Controls Electron Transfer to Cytochrome P450

Cytochrome P450 reductase (CYPOR) provides electrons to all human microsomal cytochrome P450s (cyt P450s). The length and sequence of the “140s” FMN binding loop of CYPOR has been shown to be a key determinant of its redox potential and activity with cyt P450s. Shortening the “140s loop” by deleting glycine-141(ΔGly141) and by engineering a second mutant that mimics flavo-cytochrome P450 BM3 (ΔGly141/Glu142Asn) resulted in mutants that formed an unstable anionic semiquinone. In an attempt to understand the molecular basis of the inability of these mutants to support activity with cyt P450, we expressed, purified, and determined their ability to reduce ferric P450. Our results showed that the ΔGly141 mutant with a very mobile loop only reduced ~7% of cyt P450 with a rate similar to that of the wild type. On the other hand, the more stable loop in the ΔGly141/Glu142Asn mutant allowed for ~55% of the cyt P450 to be reduced ~60% faster than the wild type. Our results reveal that the poor activity of the ΔGly141 mutant is primarily accounted for by its markedly diminished ability to reduce ferric cyt P450. In contrast, the poor activity of the ΔGly141/Glu142Asn mutant is presumably a consequence of the altered structure and mobility of the “140s loop”.     

The “Angiogenic Switch” and Functional Resources in Cyclic Sports Athletes

Regular physical activity in cyclic sports can influence the so-called “angiogenic switch”, which is considered as an imbalance between proangiogenic and anti-angiogenic molecules. Disruption of the synthesis of angiogenic molecules can be caused by local changes in tissues under the influence of excessive physical exertion and its consequences, such as chronic oxidative stress and associated hypoxia, metabolic acidosis, sports injuries, etc. A review of publications on signaling pathways that activate and inhibit angiogenesis in skeletal muscles, myocardium, lung, and nervous tissue under the influence of intense physical activity in cyclic sports. Materials: We searched PubMed, SCOPUS, Web of Science, Google Scholar, Clinical keys, and e-LIBRARY databases for full-text articles published from 2000 to 2020, using keywords and their combinations. Results: An important aspect of adaptation to training loads in cyclic sports is an increase in the number of capillaries in muscle fibers, which improves the metabolism of skeletal muscles and myocardium, as well as nervous and lung tissue. Recent studies have shown that myocardial endothelial cells not only respond to hemodynamic forces and paracrine signals from neighboring cells, but also take an active part in heart remodeling processes, stimulating the growth and contractility of cardiomyocytes or the production of extracellular matrix proteins in myofibroblasts. As myocardial vascularization plays a central role in the transition from adaptive heart hypertrophy to heart failure, further study of the signaling mechanisms involved in the regulation of angiogenesis in the myocardium is important in sports practice. The study of the “angiogenic switch” problem in the cerebrovascular and cardiovascular systems allows us to claim that the formation of new vessels is mediated by a complex interaction of all growth factors. Although the lungs are one of the limiting systems of the body in cyclic sports, their response to high-intensity loads and other environmental stresses is often overlooked. Airway epithelial cells are the predominant source of several growth factors throughout lung organogenesis and appear to be critical for normal alveolarization, rapid alveolar proliferation, and normal vascular development. There are many controversial questions about the role of growth factors in the physiology and pathology of the lungs. The presented review has demonstrated that when doing sports, it is necessary to give a careful consideration to the possible positive and negative effects of growth factors on muscles, myocardium, lung tissue, and brain. Primarily, the “angiogenic switch” is important in aerobic sports (long distance running). Conclusions: Angiogenesis is a physiological process of the formation of new blood capillaries, which play an important role in the functioning of skeletal muscles, myocardium, lung, and nervous tissue in athletes. Violation of the “angiogenic switch” as a balance between proangiogenic and anti-angiogenic molecules can lead to a decrease in the functional resources of the nervous, musculoskeletal, cardiovascular, and respiratory systems in athletes and, as a consequence, to a decrease in sports performance.     

Swift and Reliable “Easy Lab” Methods for the Sensitive Molecular Detection of African Swine Fever Virus

African swine fever (ASF) is a contagious viral hemorrhagic disease of domestic pigs and wild boars. The disease is notifiable to the World Organisation for Animal Health (OIE) and is responsible for high mortality and serious economic losses. PCR and real-time PCR (qPCR) are the OIE-recommended standard methods for the direct detection of African swine fever virus (ASFV) DNA. The aim of our work was the simplification and standardization of the molecular diagnostic workflow in the lab. For validation of this “easy lab” workflow, different sample materials from animal trials were collected and analyzed (EDTA blood, serum, oral swabs, chewing ropes, and tissue samples) to identify the optimal sample material for diagnostics in live animals. Based on our data, the EDTA blood samples or bloody tissue samples represent the best specimens for ASFV detection in the early and late phases of infection. The application of prefilled ready-to-use reagents for nucleic acid extraction or the use of a Tissue Lysis Reagent (TLR) delivers simple and reliable alternatives for the release of the ASFV nucleic acids. For the qPCR detection of ASFV, different published and commercial kits were compared. Here, a lyophilized commercial kit shows the best results mainly based on the increased template input. The good results of the “easy lab” strategy could be confirmed by the ASFV detection in field samples from wild boars collected from the 2020 ASFV outbreak in Germany. Appropriate internal control systems for extraction and PCR are key features of the “easy lab” concept and reduce the risk of false-negative and false-positive results. In addition, the use of easy-to-handle machines and software reduces training efforts and the misinterpretation of results. The PCR diagnostics based on the “easy lab” strategy can realize a high sensitivity and specificity comparable to the standard PCR methods and should be especially usable for labs with limited experiences and resources.     

The Anti-Inflammatory Role of Bilirubin on “Two-Hit” Sepsis Animal Model

Introduction: Bilirubin is a product of the heme catabolism pathway, and it is excreted in bile and removed from the body through the urine. Bilirubin has potent antioxidant properties but also plays a role in anti-inflammation by protecting the body against endotoxin-induced lung inflammation, down-regulating the expression of adhesion molecules, and inhibiting the infiltration of inflammatory cells. Thus, bilirubin is a promising agent that could use in inflammation disease treatment. The application of bilirubin on the “two-hit” sepsis animal model has been, to date, unknown. Methods: we used lipopolysaccharide to induce initial insults in C57BL/6 mice. After 24 h, mice underwent cecal ligation and puncture to induce the “two-hit” sepsis model. Next, mice were administered 30 mg/kg bilirubin and we observed an improvement. Results: We observed that bilirubin inhibited the expression of pro-inflammatory cytokines, while the levels of anti-inflammatory cytokines were significantly augmented in the lung. Bilirubin improved the survival rate in the sepsis model. Furthermore, we suggest that bilirubin can modulate the accumulation of T-regulatory cells and myeloid-derived suppressor cells. Notably, bilirubin suppressed the activation and functions of T-cells. Conclusions: These results clarified that bilirubin might improve tissue injury in sepsis through anti-inflammatory mechanisms.     

Selective Antimicrobial Therapies for Periodontitis: Win the “Battle and the War”

Traditional antimicrobial therapies for periodontitis (PD) have long focused on non-selective and direct approaches. Professional cleaning of the subgingival biofilm by instrumentation of dental root surfaces, known as scaling and root planning (SRP), is the mainstay of periodontal therapy and is indisputably effective. Non-physical approaches used as adjuncts to SRP, such as chemical and biological agents, will be the focus of this review. In this regard, traditional agents such as oral antiseptics and antibiotics, delivered either locally or systemically, were briefly reviewed as a backdrop. While generally effective in winning the “battle” against PD in the short term, by reducing its signs and symptoms, patients receiving such therapies are more susceptible to recurrence of PD. Moreover, the long-term consequences of such therapies are still in question. In particular, concern about chronic use of systemic antibiotics and their influence on the oral and gut microbiota is warranted, considering antibiotic resistance plasmids, and potential transfer between oral and non-oral microbes. In the interest of winning the “battle and the war”, new more selective and targeted antimicrobials and biologics for PD are being studied. These are principally indirect, blocking pathways involved in bacterial colonization, nutrient acquisition, inflammation or cellular invasion without directly killing the pathogens. This review will focus on current and prospective antimicrobial therapies for PD, emphasizing therapies that act indirectly on the microbiota, with clearly defined cellular and molecular targets.     

The Impact of the “Osteo” Component of Osteosarcopenia on Fragility Fractures in Post-Menopausal Women

Osteosarcopenia, the coexistence of bone and muscle loss, is common in older adults, but its definition lacks international consensus. This cross-sectional study (n = 1199 post-menopausal women) aimed to determine the association between osteosarcopenia and fragility fractures and to investigate the impact of the definition of the “osteo” component. Bone mineral density and bone microarchitecture were measured by dual-energy X-ray absorptiometry and the trabecular bone score (TBS), respectively. The “osteo” component of osteosarcopenia was classified as osteoporosis (T-score ≤ −2.5 SD), osteopenia/osteoporosis (T-score < −1 SD), and high-fracture-risk osteopenia (−2.5 SD < T-score < −1 SD)/osteoporosis (T-score ≤ −2.5 SD). The Fracture Risk Assessment Tool was used to identify high-fracture-risk osteopenia. Altogether, 30.3%, 32.2%, 14.4%, and 23.1% of participants had osteosarcopenia, osteoporosis alone, sarcopenia alone, and neither condition, respectively. The odds ratios between osteosarcopenia and fragility fractures were 3.70 (95% CI: 1.94–7.04) for osteosarcopenia, 2.48 (95% CI: 1.30–4.71) for osteoporosis alone, and 1.87 (95% CI: 0.84–4.14) for sarcopenia alone. Women with osteosarcopenia also had lower TBS, indicating worse bone microarchitecture. In conclusion, women with osteosarcopenia were more likely to have previously sustained a fracture compared to those without osteosarcopenia, with sarcopenia alone, and with osteoporosis alone. The relationship between osteosarcopenia and fracture risk may be best identified when considering high-fracture-risk osteopenia and osteoporosis.     

Design and Engineering of “Green” Nanoemulsions for Enhanced Topical Delivery of Bakuchiol Achieved in a Sustainable Manner: A Novel Eco-Friendly Approach to Bioretinol

In the present work, we establish novel “environmentally-friendly” oil-in-water nanoemulsions to enhance the transdermal delivery of bakuchiol, the so-called “bioretinol” obtained from powdered Psoralea corylifolia seeds via a sustainable process, i.e., using a supercritical fluid extraction approach with pure carbon dioxide (SC-CO₂). According to Green Chemistry principles, five novel formulations were stabilized by “green” hybrid ionic surfactants such as coco-betaine—surfactin molecules obtained from coconut and fermented rapeseed meal. Preliminary optimization studies involving three dispersion stability tests, i.e., centrifugation, heating, and cooling cycles, indicated the most promising candidates for further physicochemical analysis. Finally, nanoemulsion colloidal characterization provided by scattering (dynamic and electrophoretic light scattering as well as backscattering), microscopic (transmission electron and confocal laser scanning microscopy), and spectroscopic (UV–Vis spectroscopy) methods revealed the most stable nanocarrier for transdermal biological investigation. In vitro, topical experiments provided on human skin cell line HaCaT keratinocytes and normal dermal NHDF fibroblasts indicated high cell viability upon treatment of the tested formulation with a final 0.02–0.2 mg/mL bakuchiol concentration. This excellent biocompatibility was confirmed by ex vivo and in vivo tests on animal and human skin tissue. The improved permeability and antiaging potential of the bakuchiol-encapsulated rich extract were observed, indicating that the obtained ecological nanoemulsions are competitive with commercial retinol formulations.     

Ribosome Protection Proteins—“New” Players in the Global Arms Race with Antibiotic-Resistant Pathogens

Bacteria have evolved an array of mechanisms enabling them to resist the inhibitory effect of antibiotics, a significant proportion of which target the ribosome. Indeed, resistance mechanisms have been identified for nearly every antibiotic that is currently used in clinical practice. With the ever-increasing list of multi-drug-resistant pathogens and very few novel antibiotics in the pharmaceutical pipeline, treatable infections are likely to become life-threatening once again. Most of the prevalent resistance mechanisms are well understood and their clinical significance is recognized. In contrast, ribosome protection protein-mediated resistance has flown under the radar for a long time and has been considered a minor factor in the clinical setting. Not until the recent discovery of the ATP-binding cassette family F protein-mediated resistance in an extensive list of human pathogens has the significance of ribosome protection proteins been truly appreciated. Understanding the underlying resistance mechanism has the potential to guide the development of novel therapeutic approaches to evade or overcome the resistance. In this review, we discuss the latest developments regarding ribosome protection proteins focusing on the current antimicrobial arsenal and pharmaceutical pipeline as well as potential implications for the future of fighting bacterial infections in the time of “superbugs.”     

A Regulator Based “Semi-Targeted” Approach to Activate Silent Biosynthetic Gene Clusters

By culturing microorganisms under standard laboratory conditions, most biosynthetic gene clusters (BGCs) are not expressed, and thus, the products are not produced. To explore this biosynthetic potential, we developed a novel “semi-targeted” approach focusing on activating “silent” BGCs by concurrently introducing a group of regulator genes into streptomycetes of the Tübingen strain collection. We constructed integrative plasmids containing two classes of regulatory genes under the control of the constitutive promoter ermE*p (cluster situated regulators (CSR) and Streptomyces antibiotic regulatory proteins (SARPs)). These plasmids were introduced into Streptomyces sp. TÜ17, Streptomyces sp. TÜ10 and Streptomyces sp. TÜ102. Introduction of the CSRs-plasmid into strain S. sp. TÜ17 activated the production of mayamycin A. By using the individual regulator genes, we proved that Aur1P, was responsible for the activation. In strain S. sp. TÜ102, the introduction of the SARP-plasmid triggered the production of a chartreusin-like compound. Insertion of the CSRs-plasmid into strain S. sp. TÜ10 resulted in activating the warkmycin-BGC. In both recombinants, activation of the BGCs was only possible through the simultaneous expression of aur1PR3 and griR in S. sp. TÜ102 and aur1P and pntR in of S. sp. TÜ10.     

Propellanes as Rigid Scaffolds for the Stereodefined Attachment of σ-Pharmacophoric Structural Elements to Achieve σ Affinity

Following the concept of conformationally restriction of ligands to achieve high receptor affinity, we exploited the propellane system as rigid scaffold allowing the stereodefined attachment of various substituents. Three types of ligands were designed, synthesized and pharmacologically evaluated as σ₁ receptor ligands. Propellanes with (1) a 2-methoxy-5-methylphenylcarbamate group at the “left” five-membered ring and various amino groups on the “right” side; (2) benzylamino or analogous amino moieties on the “right” side and various substituents at the left five-membered ring and (3) various urea derivatives at one five-membered ring were investigated. The benzylamino substituted carbamate syn,syn-4a showed the highest σ₁ affinity within the group of four stereoisomers emphasizing the importance of the stereochemistry. The cyclohexylmethylamine 18 without further substituents at the propellane scaffold revealed unexpectedly high σ₁ affinity (K_i = 34 nM) confirming the relevance of the bioisosteric replacement of the benzylamino moiety by the cyclohexylmethylamino moiety. Reduction of the distance between the basic amino moiety and the “left” hydrophobic region by incorporation of the amino moiety into the propellane scaffold resulted in azapropellanes with particular high σ₁ affinity. As shown for the propellanamine 18, removal of the carbamate moiety increased the σ₁ affinity of 9a (K_i = 17 nM) considerably. Replacement of the basic amino moiety by H-bond forming urea did not lead to potent σ ligands. According to molecular dynamics simulations, both azapropellanes anti-5 and 9a as well as propellane 18 adopt binding poses at the σ₁ receptor, which result in energetic values correlating well with their different σ₁ affinities. The affinity of the ligands is enthalpy driven. The additional interactions of the carbamate moiety of anti-5 with the σ₁ receptor protein cannot compensate the suboptimal orientations of the rigid propellane and its N-benzyl moiety within the σ₁ receptor-binding pocket, which explains the higher σ₁ affinity of the unsubstituted azapropellane 9a.