Structure-activity relationships of pyrazole-4-carbodithioates as antibacterials against methicillin–resistant Staphylococcus aureus

Methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of serious hospital-acquired infections and is responsible for significant morbidity and mortality in residential care facilities. New agents against MRSA are needed to combat rising resistance to current antibiotics. We recently reported 5-hydroxy-3-methyl-1-phenyl-1H-pyrazole-4-carbodithioate (HMPC) as a new bacteriostatic agent against MRSA that appears to act via a novel mechanism. Here, twenty nine analogs of HMPC were synthesized, their anti-MRSA structure-activity relationships evaluated and selectivity versus human HKC-8 cells determined. Minimum inhibitory concentrations (MIC) ranged from 0.5 to 64?μg/mL and up to 16-fold selectivity was achieved. The 4-carbodithioate function was found to be essential for activity but non-specific reactivity was ruled out as a contributor to antibacterial action. The study supports further work aimed at elucidating the molecular targets of this interesting new class of anti-MRSA agents.     

γ-Aminobutyric Acid Type A (GABAA) Receptor Subunits Play a Direct Structural Role in Synaptic Contact Formation via Their N-terminal Extracellular Domains

The establishment of cell-cell contacts between presynaptic GABAergic neurons and their postsynaptic targets initiates the process of GABAergic synapse formation. GABA_A receptors (GABA_ARs), the main postsynaptic receptors for GABA, have been recently demonstrated to act as synaptogenic proteins that can single-handedly induce the formation and functional maturation of inhibitory synapses. To establish how the subunit composition of GABA_ARs influences their ability to induce synaptogenesis, a co-culture model system incorporating GABAergic medium spiny neurons and the HEK293 cells, stably expressing different combinations of receptor subunits, was developed. Analyses of HEK293 cell innervation by medium spiny neuron axons using immunocytochemistry, activity-dependent labeling, and electrophysiology have indicated that the γ2 subunit is required for the formation of active synapses and that its effects are influenced by the type of α/β subunits incorporated into the functional receptor. To further characterize this process, the large N-terminal extracellular domains (ECDs) of α1, α2, β2, and γ2 subunits were purified using the baculovirus/Sf9 cell system. When these proteins were applied to the co-cultures of MSNs and α1/β2/γ2-expressing HEK293 cells, the α1, β2, or γ2 ECD each caused a significant reduction in contact formation, in contrast to the α2 ECD, which had no effect. Together, our experiments indicate that the structural role of GABA_ARs in synaptic contact formation is determined by their subunit composition, with the N-terminal ECDs of each of the subunits directly participating in interactions between the presynaptic and postsynaptic elements, suggesting the these interactions are multivalent and specific.     

Ovčar–Kablar Gorge (SW Serbia) – a new hotspot of Orthoptera diversity

We present a list of Orthoptera from the Ov?ar–Kablar Gorge, situated in western Serbia. During 2015 and 2016 we collected 86 Orthoptera species (50 Ensifera, 36 Caelifera). The majority of the species were registered for the first time for the locality and the presence of Pachytrachis frater (Tettigoniidae) in Serbia is confirmed. This study is intended to provide data for future conservation planning, to expand knowledge about rare species and to point to the high natural value of the Gorge.     

Characterization of the Variability of Epstein-Barr Virus Genes in Nasopharyngeal Biopsies: Potential Predictors for Carcinoma Progression

Epstein-Barr virus (EBV) infection is a significant factor in the pathogenesis of nasopharyngeal carcinoma, especially in the undifferentiated carcinoma of nasopharyngeal type (UCNT, World Health Organization type III), which is the dominant histopathological type in high-risk areas. The major EBV oncogene is latent membrane protein 1 (LMP1). LMP1 gene shows variability with different tumorigenic and immunogenic potentials. EBV nuclear antigen 1 (EBNA1) regulates progression of EBV-related tumors; however, the influence of EBNA1 sequence variability on tumor pathogenesis is controversial. The aims of this study were to characterize polymorphisms of EBV genes in non-endemic nasopharyngeal carcinoma biopsies and to investigate potential sequence patterns that correlate with the clinical presentation of nasopharyngeal carcinoma. In total, 116 tumor biopsies of undifferentiated carcinoma of nasopharyngeal type (UCNT), collected from 2008 to 2014, were evaluated in this study. The genes EBNA2, LMP1, and EBNA1 were amplified using nested-PCR. EBNA2 genotyping was performed by visualization of PCR products using gel electrophoresis. Investigation of LMP1 and EBNA1 included sequence, phylogenetic, and statistical analyses. The presence of EBV DNA was significantly distributed between TNM stages. LMP1 variability showed six variants, with the detection of the first China1 and North Carolina variants in European nasopharyngeal carcinoma biopsies. Newly discovered variants Srb1 and Srb2 were UCNT-specific LMP1 polymorphisms. The B95-8 and North Carolina variants are possible predictors for favorable TNM stages. In contrast, deletions in LMP1 are possible risk factors for the most disfavorable TNM stage, independent of EBNA2 or EBNA1 variability. A newly discovered EBNA1 subvariant, P-thr-sv-5, could be a potential diagnostic marker, as it represented a UCNT-specific EBNA1 subvariant. A particular combination of EBNA2, LMP1, and EBNA1 polymorphisms, type 1/Med/P-thr was identified as a possible risk factor for TNM stage IVB or progression to the N3 stage.     

An approach for the improved immobilization of penicillin G acylase onto macroporous poly(glycidyl methacrylate‐co‐ethylene glycol dimethacrylate) as a potential industrial biocatalyst

The use of penicillin G acylase (PGA) covalently linked to insoluble carrier is expected to produce major advances in pharmaceutical processing industry and the enzyme stability enhancement is still a significant challenge. The objective of this study was to improve catalytic performance of the covalently immobilized PGA on a potential industrial carrier, macroporous poly(glycidyl methacrylate‐co‐ethylene glycol dimethacrylate) [poly(GMA‐co‐EGDMA)], by optimizing the copolymerization process and the enzyme attachment procedure. This synthetic copolymer could be a very promising alternative for the development of low‐cost, easy‐to‐prepare, and stable biocatalyst compared to expensive commercially available epoxy carriers such as Eupergit or Sepabeads. The PGA immobilized on poly(GMA‐co‐EGDMA) in the shape of microbeads obtained by suspension copolymerization appeared to have higher activity yield compared to copolymerization in a cast. Optimal conditions for the immobilization of PGA on poly(GMA‐co‐EGDMA) microbeads were 1 mg/mL of PGA in 0.75 mol/L phosphate buffer pH 6.0 at 25°C for 24 h, leading to the active biocatalyst with the specific activity of 252.7 U/g dry beads. Chemical amination of the immobilized PGA could contribute to the enhanced stability of the biocatalyst by inducing secondary interactions between the enzyme and the carrier, ensuring multipoint attachment. The best balance between the activity yield (51.5%), enzyme loading (25.6 mg/g), and stability (stabilization factor 22.2) was achieved for the partially modified PGA. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 32:43–53, 2016     

The Limitations of Kim’s Reductive Physicalism in Accounting for Living Systems and an Alternative Nonreductionist Ontology

Jaegwon Kim's exclusion argument is a general ontological argument, applicable to any properties deemed supervenient on a microproperty basis, including biological properties. It implies that the causal power of any higher-level property must be reducible to the subset of the causal powers of its lower-level properties. Moreover, as Kim's recent version of the argument indicates, a higher-level property can be causally efficient only to the extent of the efficiency of its micro-basis. In response, I argue that the ontology that aims to capture experimentally based explanations of metabolic control systems and morphogenetic systems must involve causally relevant contextual properties. Such an ontology challenges the exclusiveness of micro-based causal efficiency that grounds Kim's reductionism, since configurations themselves are inherently causally efficient constituents. I anticipate and respond to the reductionist's objection that the nonreductionist ontology's account of causes and inter-level causal relations is incoherent. I also argue that such an ontology is not open to Kim's overdetermination objection.     

Solution structure of Domains IVa and V of the tau subunit of Escherichia coli DNA polymerase III and interaction with the alpha subunit

The solution structure of the C-terminal Domain V of the τ subunit of E. coli DNA polymerase III was determined by nuclear magnetic resonance (NMR) spectroscopy. The fold is unique to τ subunits. Amino acid sequence conservation is pronounced for hydrophobic residues that form the structural core of the protein, indicating that the fold is representative for τ subunits from a wide range of different bacteria. The interaction between the polymerase subunits τ and α was studied by NMR experiments where α was incubated with full-length C-terminal domain (τ_C16), and domains shortened at the C-terminus by 11 and 18 residues, respectively. The only interacting residues were found in the C-terminal 30-residue segment of τ, most of which is structurally disordered in free τ_C16. Since the N- and C-termini of the structured core of τ_C16 are located close to each other, this limits the possible distance between α and the pentameric δτ₂γδ′ clamp–loader complex and, hence, between the two α subunits involved in leading- and lagging-strand DNA synthesis. Analysis of an N-terminally extended construct (τ_C22) showed that τ_C14 presents the only part of Domains IVa and V of τ which comprises a globular fold in the absence of other interaction partners.     

The unstructured C-terminus of the tau subunit of Escherichia coli DNA polymerase III holoenzyme is the site of interaction with the alpha subunit

The τ subunit of Escherichia coli DNA polymerase III holoenzyme interacts with the α subunit through its C-terminal Domain V, τ_C16. We show that the extreme C-terminal region of τ_C16 constitutes the site of interaction with α. The τ_C16 domain, but not a derivative of it with a C-terminal deletion of seven residues (τ_C16Δ7), forms an isolable complex with α. Surface plasmon resonance measurements were used to determine the dissociation constant (K_D) of the α−τ_C16 complex to be ~260pM. Competition with immobilized τ_C16 by τ_C16 derivatives for binding to α gave values of K_D of 7μM for the α−τ_C16Δ7 complex. Low-level expression of the genes encoding τ_C16 and τ_C167, but not τ_C16Δ11, is lethal to E. coli. Suppression of this lethal phenotype enabled selection of mutations in the 3′ end of the τ_C16 gene, that led to defects in α binding. The data suggest that the unstructured C-terminus of τ becomes folded into a helix–loop–helix in its complex with α. An N-terminally extended construct, τ_C24, was found to bind DNA in a salt-sensitive manner while no binding was observed for τ_C16, suggesting that the processivity switch of the replisome functionally involves Domain IV of τ.     

Functional anthology of intrinsic disorder. 3. Ligands, post-translational modifications, and diseases associated with intrinsically disordered proteins

Currently, the understanding of the relationships between function, amino acid sequence, and protein structure continues to represent one of the major challenges of the modern protein science. As many as 50% of eukaryotic proteins are likely to contain functionally important long disordered regions. Many proteins are wholly disordered but still possess numerous biologically important functions. However, the number of experimentally confirmed disordered proteins with known biological functions is substantially smaller than their actual number in nature. Therefore, there is a crucial need for novel bionformatics approaches that allow projection of the current knowledge from a few experimentally verified examples to much larger groups of known and potential proteins. The elaboration of a bioinformatics tool for the analysis of functional diversity of intrinsically disordered proteins and application of this data mining tool to >200 000 proteins from the Swiss-Prot database, each annotated with at least one of the 875 functional keywords, was described in the first paper of this series (Xie, H.; Vucetic, S.; Iakoucheva, L. M.; Oldfield, C. J.; Dunker, A. K.; Obradovic, Z.; Uversky, V.N. Functional anthology of intrinsic disorder. 1. Biological processes and functions of proteins with long disordered regions. J. Proteome Res. 2007, 5, 1882-1898). Using this tool, we have found that out of the 710 Swiss-Prot functional keywords associated with at least 20 proteins, 262 were strongly positively correlated with long intrinsically disordered regions, and 302 were strongly negatively correlated. Illustrative examples of functional disorder or order were found for the vast majority of keywords showing strongest positive or negative correlation with intrinsic disorder, respectively. Some 80 Swiss-Prot keywords associated with disorder- and order-driven biological processes and protein functions were described in the first paper (see above). The second paper of the series was devoted to the presentation of 87 Swiss-Prot keywords attributed to the cellular components, domains, technical terms, developmental processes, and coding sequence diversities possessing strong positive and negative correlation with long disordered regions (Vucetic, S.; Xie, H.; Iakoucheva, L. M.; Oldfield, C. J.; Dunker, A. K.; Obradovic, Z.; Uversky, V. N. Functional anthology of intrinsic disorder. 2. Cellular components, domains, technical terms, developmental processes, and coding sequence diversities correlated with long disordered regions. J. Proteome Res. 2007, 5, 1899-1916). Protein structure and functionality can be modulated by various post-translational modifications or/and as a result of binding of specific ligands. Numerous human diseases are associated with protein misfolding/misassembly/misfunctioning. This work concludes the series of papers dedicated to the functional anthology of intrinsic disorder and describes approximately 80 Swiss-Prot functional keywords that are related to ligands, post-translational modifications, and diseases possessing strong positive or negative correlation with the predicted long disordered regions in proteins.     

Alimentary prion infections: Touchdown in the intestine

Neurodegenerative diseases are caused by proteinaceous aggregates, usually consisting of misfolded proteins which are often typified by a high proportion of β-sheets that accumulate in the central nervous system. These diseases, including Morbus Alzheimer, Parkinson disease and Transmissible Spongiform Encephalopathies (TSEs)—also termed prion disorders—afflict a substantial proportion of the human population and, as such, the etiology and pathogenesis of these diseases has been the focus of mounting research. Although many of these diseases arise from genetic mutations or are sporadic in nature, the possible horizontal transmissibility of neurodegenerative diseases poses a great threat to population health. In this article we discuss recent studies that suggest that the “non-transmissible” status bestowed upon Alzheimer and Parkinson diseases may need to be revised as these diseases have been successfully induced through tissue transplants. Furthermore, we highlight the importance of investigating the “natural” mechanism of prion transmission including peroral and perenteral transmission, proposed routes of gastrointestinal uptake and neuroinvasion of ingested infectious prion proteins. We examine the multitude of factors which may influence oral transmissibility and discuss the zoonotic threats that Chronic Wasting disease (CWD), Bovine Spongiform Encephalopathy (BSE) and Scrapie may pose resulting in vCJD or related disorders. In addition, we suggest that the 37 kDa/67 kDa laminin receptor on the cell surface of enterocytes, a major cell population in the intestine, may play an important role in the intestinal pathophysiology of alimentary prion infections.Key words: prion, 37 kDa/67 kDa laminin receptor, CJD, BSE, CWD, scrapie, Alzheimer disease, Parkinson disease, intestine, enterocytesMany different mechanisms exist which underlie the etiology of the numerous neurodegenerative diseases affecting the human population. Amongst the most prominent are Morbus Alzheimer, prion disorders, Parkinson disease, Chorea Huntington, frontotemporal dementia and amylotrophic lateral sclerosis. The molecular mechanisms underlying these diseases vary; however, all neurodegenerative diseases share a common feature: they are caused by protein aggregation. The only neurodegenerative diseases proven to be transmissible are prion disorders. In contrast to frontotemporal dementia, recent evidence suggests that Alzheimer and Parkinson diseases may also be transmissible. Pre-symptomatic Alzheimer disease (APP23) mice exhibited an increase in the Alzheimer phenotype when brain homogenate of autopsied human Alzheimer disease patients and older, amyloid beta- (Aβ-) laden APP23 mice was injected into their hippocampi.¹ These findings suggest that the Aβ-abundant brain homogenate of Alzheimer disease patients may possess the ability to induce or supplement the overproduction of Aβ, possibly leading to the onset of Alzheimer disease.The pathological feature associated with Parkinson disease is the formation of Lewy bodies in cell bodies and neuronal processes in the brain.² The main component of these protein aggregates is α-synuclein (reviewed in ref. ²). Autopsies of Parkinson disease patients revealed that Lewy bodies had formed on healthy embryonic neurons that had been grafted onto the brain tissue of the patients several years before (prior to said examination).^3–5 It may thus be proposed that α-synuclein transmission is possible from diseased to healthy neurons, suggesting that Parkinson disease may be transmissible from a Parkinson disease patient to a healthy individual. These findings imply that Alzheimer and Parkinson diseases may be transmissible through tissue transplants and the use of contaminated surgical tools.⁶Prion disorders, also termed Transmissible Spongiform Encephalopathies (TSEs), are fatal neurodegenerative diseases that affect the central nervous system (CNS) of multiple animal species. In lieu of the social, economic and political ramifications of such infections, as well as the possible intra- and interspecies transmissibility of such disorders, various routes of experimental transmission have been investigated including intracerebral, intraperitoneal, intraventricular, intraocular, intraspinal and subcutaneous injections (reviewed in ref. ^7–9). However, such routes of transmission are not representative of the “natural” mechanism as the majority of prion disorders are contracted through ingestion of infectious prion (PrP^Sc) containing material. Thus, the peroral and perenteral prion transmission is of greatest consequence with respect to TSE disease establishment. Moreover, the presence of PrP^Sc in the buccal cavity of scrapie-infected sheep¹⁰ (reviewed in ref. ¹¹) and the possible horizontal transfer as a result hereof, as may be similarly proposed for animals suffering from other TSEs, may further contribute to the oral transmissibility of TSEs.A number of model systems have been employed to study TSE transmissibility. Owing to ethical constraints, TSE transmissibility to humans via the oral route may not be directly investigated and as a result hereof, alternative model systems are needed. These may include the use of transgenic mice, cell lines which are permissive to infection¹² and experimental animals such as sheep, calves, goats, minks, ferrets and non-human primates (reviewed in ref. ⁹).Intestinal entry of PrP^Sc has been proposed to occur via two pathways, the membranous (M) cell-dependent and M cell-independent pathways (Fig. 1).^13,14 The former involves endocytic M (microfold)-cells, which cover the intestinal lymphoid follicles (Peyer''s patches)¹⁴ and may take up prions and thereby facilitate the translocation of these proteins across the intestinal epithelium into the lymphoid tissues (reviewed in ref. ⁹) as has been demonstrated in a cellular model.¹³ Following such uptake by the M cells, the prions may subsequently pass to the dendritic cells and follicular dendritic cells (FDCs) (Fig. 1), which allow for prion transport to the mesenteric lymph nodes and replication, respectively.¹⁵ The prion proteins may subsequently gain access to the enteric nervous system (ENS) and ultimately the central nervous system (CNS).¹⁵Open in a separate windowFigure 1Proposed routes of gastrointestinal entry of ingested infectious prions (PrP^Sc) as well as possible pathways of amplification and transport to the central nervous system.However, prion intestinal translocation has been observed in the absence of M cells and has been demonstrated to be as a result of the action of polar, 37 kDa/67 kDa LRP/LR (non-integrin laminin receptor; reviewed in ref. ^16–18) expressing enterocytes. Enterocytes are the major cell population of the intestinal epithelium and due to their ability to endocytose pathogens, nutrients and macromolecules,¹⁹ it has been proposed that these cells may represent a major entry site for alimentary prions (Fig. 1).Since enterocyte prion uptake has been demonstrated to be dependent on the presence of LRP/LR on the apical brush border of the cells,^14,20 the interaction between varying prion protein strains and the receptor^21–23 may be employed as a model system to study possible oral transmissibility of prion disorders across species as well as the intestinal pathophysiology of alimentary prion infections.²⁴ Moreover, the blockage of such interactions through the use of anti-LRP/LR specific antibodies has been reported to reduce PrP^Sc endocytosis¹⁹ and thus these antibodies may serve as potential therapeutics to prevent infectious prion internalization and thereby prevent prion infections. It must be emphasized that the adhesion of prion proteins to cells is not solely dependent on the LRP/LR-PrP^Sc interactions;²⁴ however, this interaction is of importance with regards to internalization and subsequent pathogenesis.We applied the aforementioned cell model to study the possible oral transmission of PrP^BSE, PrP^CWD and ovine PrP^Sc to cervids, cattle, swine and humans.²⁴ The direct transmission of the aforementioned animal prion disorders to humans as a result of dietary exposure and the possible establishment of zoonotic diseases is of great public concern. It must however be emphasized that the study investigated the co-localization of LRP/LR and various prion strains and not the actual internalization process.PrP^BSE was shown to co-localize with LRP/LR on human enterocytes²⁴, thereby suggesting that PrP^BSE is transmissible to humans via the oral route which is widely accepted as the manner by which variant CJD originated. This suspicion was previously investigated using a macaque model, which was successfully perorally infected by BSE-contaminated material and subsequently lead to the development of a prion disorder that resembles vCJD.²⁵ These results, due to the evolutionary relatedness between macaques and humans, allowed researchers to confirm the oral transmissibility of PrP^BSE to humans. PrP^BSE may also potentially lead to prion disorder establishment in swine,²⁴ livestock of great economic and social importance.The prion disorder affecting elk, mule deer and white-tailed deer is termed CWD. Cases of the disease are most prevalent in the US but are also evident in Canada and South Korea.^26,27 As the infectious prion isoform is reported to be present in the blood²⁸ and skeletal muscle,²⁹ hunting, consumption of wild venison and contact with other animal products derived from CWD-infected elk and deer may thereby pose a public health risk. Our studies demonstrate that PrP^CWD co-localizes with LRP/LR on human enterocytes²⁴ thereby suggesting a possible oral transmissibilty of this TSE to humans. This is, however, inconsistent with results obtained during intra-cerebral inoculation of the brains and spinal cords of transgenic mice overexpressing the human cellular prion protein (PrP^c),^26,27 which is essential for TSE disease establishment and progression. Further, discrepancies have also been reported with respect to non-human primates, as squirrel monkeys have been successfully intracerebrally inoculated with mule-deer prion homogenates,³⁰ while cynolmolgus macaques were resistant to infection.³¹ CWD has been transmitted to ferrets, minks and goats³² and as these animals may serve as domestic animals or livestock, secondary transmission from such animals to humans, through direct contact or ingestion of infected material, may be an additional risk factor that merits further scientific investigation.Ovine PrP^Sc co-localization with LRP/LR on human and bovine enterocytes may be indicative of the infectious agents'' ability to effect cross-species infections. The oral transmissibility of Scrapie has been confirmed in hamsters fed with sheep-scrapie-infected material.³³The discrepancies with regards to the transmissibility of certain infectious prion proteins when assessed by different model systems may be due to the experimental transmission route employed. Oral exposure often results in significantly prolonged incubation times when compared to intracerebral inoculation techniques and thus failure of transgenic mice and normal experimental animals to develop disease phenotypes after being fed TSE-contaminated material may not necessarily indicate that the infection process failed.¹⁴ Apart from the route of infection, numerous other factors may influence transmission between species, including dose, PrP polymorphisms and genetic factors, the prion strain employed as well as the efficacy of prion transport to the CNS.³⁴ The degree of homology between the PrP^c protein in the animals serving as the infectious prion source and recipient has also been described as a feature limiting cross-species transmission.³⁴ The negative results, as referred to above, obtained upon prion-protein inoculation of animal models may have resulted due to the slow rate at which the infectious prion induces conformational conversion of the endogenous PrP^c in the animal cells and this in turn results in low levels of infectious prion replication and symptom development.²⁷Furthermore, even in the event that certain prion disorders are not directly transmissible to humans, most are transmissible to at least a single species of domestic animal or livestock. The infectious agents properties may be altered in the secondary host such that it becomes transmissible to humans (reviewed in ref. ³⁵). Thus, interspecies transmission between animals may indirectly influence human health.It is noteworthy to add that although the oral route of PrP^Sc transmission may result in prolonged incubation times, it may broaden the range of susceptible hosts. A common constituent of food is ferritin, a protein that is resistant to digestive enzyme hydrolysis and, due to its homology across species, it may serve as co-transporter of PrP^Sc and facilitate enterocyte internalization of the infectious prion.³⁶ It may thus be proposed that prion internalization may occur via a ferritin-PrP^Sc complex even in the absence of co-localization between the infectious agent and LRP/LR such that many more cross-species infections (provided that the other infection factors are favorable) may be probable.³⁷ In addition, digestive enzymes in the gastrointestinal tract facilitate PrP^Sc binding to the intestinal epithelium and subsequent intestinal uptake³⁶ and thus depending on the individuals'' digestive processes, the susceptibility to infection and the rate of disease development may vary accordingly. As a result hereof, though laboratory experiments in cell-culture and animal models may render a particular prion disorder non-infectious to humans, this may not be true for all individuals.In lieu of the above statements, with particular reference to inconsistencies in reported results and the multiple factors influencing oral transmissibility of TSEs, further transmission studies are required to evaluate the zoonotic threat which CWD, BSE and Scrapie may pose through ingestion.