Key Motifs Research Articles

The Significance of Victorian England for the Cottagecore Aesthetic

This article contemplates the idyllic imagery of Elizabeth Gaskell's rural texts in relation to the key visual motifs of the Internet aesthetic "cottagecore." Meanwhile, the paper also strives to highlight the importance of both the Victorian era, particularly its literature and art, with regard to this popular Internet aesthetic. With some brief references to influential figures of the age, the cultural timeframe surrounding Gaskell's rural fiction is shown to offer significant historical relevance to the romanticisation of the English country-cottage life. The literary and pictorial texts serve as examples of this cultural process. Considering the author's mostly ornamental use of cottages in Wives and Daughters as well as her employment of floral characterisation, the paper also highlights the visual aesthetics of the cottage art of Helen Allingham and Myles Birket Foster as well as rural depictions made by illustrators of Gaskell's provincial works that display the visual after-life of Gaskell's rural texts.

“I CONCEIVED THIS THING IN A HARD TIME”: ON THE QUESTION OF THE SIGNIFICANCE OF THE STORY ENOUGH IN THE WORKS OF I.S. TURGENEV

The article deals with I. Turgenev’s story Enough in terms of its role in the writer’s work, which has not yet been determined, despite of the increased interest in texts created after Fathers and Sons in recent decades. Enough traditionally attracts researchers as an example of Turgenev’s philosophical narrative, saturated with numerous allusions, and as an experience of a new literary form, which, however, is not always seen as successful and relevant to the tasks set by the writer himself. In general, scientific analytics is dominated by the history of the perception of this work by Turgenev’s contemporaries, who, with rare exceptions, reacted to Enough negatively. The article offers a brief overview of the responses to Turgenev’s story. The main attention of the author of the article was attracted by the judgments of L. Tolstoy. His assessments of Turgenev’s Enough varied throughout his life. At first, Tolstoy took the general position of denying this text, although the author of the article saw in some of the writer’s ‘Schopenhauer’ statements in War and Peace an indifferent polemic with Turgenev’s judgments from Enough. But already in the 1880s Tolstoy develops a view that Enough is Turgenev’s key text, which most capaciously and accurately expresses one of the most important “phases” (in total Tolstoy counted three of them) in the life and work of the writer. Tolstoy was attentive to Turgenev’s “phase” of “doubt in everything”, appreciated his sincerity and honesty in this matter and saw a close connection between Turgenev’s extreme doubt and faith and new religiosity. The author of the article also proposes an analysis of the poetics of Enough in the light of the key topoi and motifs of the writer’s prose of the 1850s, traces the correlation of the novel with the characteristic trends in the development of Turgenev’s lyrical prose miniature (a combination of lyrical subjectivity and generalization), which confirms the correctness of Tolstoy.

Image of “Fallen Woman” in Novels of G. Fallada’s “Wolf Among Wolves” and F. M. Dostoevsky’s “Crime and Punishment”: Comparative Analysis

The image of the “fallen woman” in the novels of G. Fallada’s “Wolf Among Wolves” and F. M. Dostoevsky’s “Crime and Punishment” is examined. Traces of the literary influence of the Russian classic on the poetics of the German writer’s novel at the thematic and character levels are identified. It is established that the key motif in both works is the theme of moral resurrection of the fallen individual. It is shown that, in addressing the problem of the “humiliated and insulted”, both authors incorporate the image of the “fallen woman” into the system of characters in their novels. It is noted that the writers pay special attention to the reasons for her social downfall. Through the use of comparative-historical analysis, parallels in the images of “fallen” women presented in these works — Petra Ledig and Sonya Marmeladova — are identified. The author of the article concludes that the heroines share a number of common features: Christian semantics of their names, moral purity despite physical vice, a tendency towards selfsacrifice, great inner strength despite outward timidity, a desire to show mercy, the ability to motivate their loved ones towards spiritual rebirth and thereby “rebirth” themselves. The results of the study allow to demonstrate the significance of F. M. Dostoevsky’s humanistic ideas for the aesthetic principles and worldview position of G. Fallada in the novel “Wolf Among Wolves”.

Design, synthesis, crystal structure and DFT analysis of (E)-N-(tert-butyl)-11H-benzo[4,5]imidazo[2,1-a]isoindol-11-imines with dual-state emission property

Benzoimidazoisoindoles are key structural motifs, widely found in pharmaceutical drugs and functional materials. However, very few strategies have been developed for the synthesis of benzoimidazoisoindoles, which greatly limited their applications. Herein we report an efficient approach for synthesizing benzoimidazoisoindole derivatives via palladium-catalyzed tandem cyclization of 2-(2-bromophenyl)-1H-benzo[d]imidazoles and isocyanides. Various benzoimidazoisoindoles were prepared in 63–86 % yields under air atmosphere at 100°C. The benzoimidazoisoindoles were characterized by UV–vis, photo-fluorescence, 1H and 13C NMR and HMRS. Furthermore, the single crystal structure of compound 3aa was determined to be of monoclinic system, space group I2/a, with α = 17.0080(4), b = 5.89820(10), c = 29.3286(7) Å, β = 102.948(2)°, V = 2867.34(11) Å3, Dc = 1.276 g/cm3, Z = 8, F(000) = 1168.0, μ(Cu Kα) = 0.599 mm−1, R = 0.0373 and wR = 0.0961. Photophysical property tests demonstrated that the designed benzoimidazoisoindoles exhibited typical dual-state emission (DSE) phenomenon. Density functional theory (DFT) calculation was also conducted to help understand the relationship between electronic structures and photophysical properties. Modified skeleton, the fused system includes benzoimidazoisoindole ring based on the structure–property relationship helps to design new, more effective types of dual-state emission materials.

Wotan’s Biopunk: The Grim(m) German God and His English Bloodsport in Sarban’s The Sound of His Horn

Abstract The essay analyzes Sarban’s 1952 novel The Sound of His Horn, one of the first alternate histories to depict a victorious Third Reich. The depiction of the latter is a strange mixture. On the one hand, the novel is a product of its day by presenting a regressive, resolutely anti-modern Nazi Germany headed back into a barbarian past. On the other hand, it anticipates later depictions (both in the alternate history genre as well as in historiography proper) by highlighting the constitutive role of technology and the regime’s inner divisions. The latter results in narrowing the gap between the (British) observer and his (German) environment. I argue that this narrowing can be traced by analyzing both the chief villain Hackelnberg, a figure borrowed from German folklore that Jacob Grimm associated with the Germanic god Wotan, and the key motif of hunting. Second, the narrowing is structurally embedded in the novel by virtue of the fact that the counterfactual Nazi domain is confined to a nested narrative. It may be a mere projection, in which case Hackelnberg’s deadly hunts and English bloodsports are not that far apart.

Adaptive organization in tourism promotion in the city of Makassar

Within the framework of Makassar, Indonesia, this qualitative study investigates adaptive organizational techniques in tourism promotion. Based on document analysis, interviews, and on-site observations, the study indicates a discernible shift in stakeholder perspectives away from conventional promotional strategies. Participants stress the necessity for adaptability and the careful balancing act needed to maintain Makassar's cultural identity while utilizing modern improvements. Stakeholders see community participation as a key motif, acknowledging the community as active participants in the promotional story. Visitors respond favorably to the promotional materials' integration of technology, yet the presence of traditional brochures presents a subtle hybrid model. Various visitor demographics emphasize the value of customized approaches, and the study's conclusions imply that Makassar's future prosperity depends on a robust, inclusive, Within the framework of Makassar, Indonesia, this qualitative study investigates adaptive organizational techniques in tourism promotion. Based on document analysis, interviews, and on-site observations, the study indicates a discernible shift in stakeholder perspectives away from conventional promotional strategies. Participants stress the necessity for adaptability and the careful balancing act needed to maintain Makassar's cultural identity while utilizing modern improvements. Stakeholders see community participation as a key motif, acknowledging the community as active participants in the promotional story. There is a clear and genuine approach to promoting tourism while incorporating technology into promotional materials. The results offer significant perspectives for Makassar and other locations attempting to manage the intricacies of the changing tourism terrain.

Novel Small Molecule VLA-4 Inhibitors Optimized for Extended Hematopoietic and Progenitor Cell Mobilization

Background: Hematopoietic stem and progenitor cell (HSPC) transplant is an essential treatment for a variety of blood disorders and malignancies, and more recently, for gene editing therapies such as sickle cell anemia. A key step in this procedure is the effective mobilization of a large quantity of primitive donor HSPCs to ensure a durable and long lasting engraftment capable of developing into all blood cell lineages. The most commonly used regimen for donor mobilization is a 5-day course of Granulocyte Colony Stimulating Factor (G-CSF). However, G-CSF does not effectively mobilize a sufficient number or quality of stem cells in all donors, can have unwanted side effects, and is contraindicated in sickle cell disease. Therefore, it is desirable to identify an alternative treatment that is safe, rapid and cost effective. The cell surface integrin receptor VLA-4 (a4b1) and the chemokine receptor CXCR4 are essential for HSPC homing and retention in the bone marrow. Inhibition of these receptors rapidly mobilizes HSPCs within an hour, as opposed to the multi-day process using G-CSF. Small molecule inhibitors of VLA-4 have been reported previously, but most have poor pharmacokinetic properties and/or weak inhibition of VLA4. These inhibitors rapidly mobilize HSPCs into the peripheral blood of mice after a single s.c. injection, but their effect is short lived, yielding an inadequate number of HSPCs necessary for stem cell transplantation or for gene therapy. Therefore, VLA4 inhibitors with improved pharmacokinetic properties imparting extended and greater HSPC mobilization after a single dose is desirable. Methods: We designed and synthesized multiple novel VLA-4 inhibitor molecules and tested their potency using soluble VCAM-1 binding assays. Inhibitors determined to be most potent with improved aqueous solubility were then tested in vivo in DBA mice for extended HSPC mobilization beyond 4 hours after a single SC injection, alone and in combination with the CXCR4 inhibitors. Results: We developed two main core molecules that each incorporate key VLA4 receptor binding motifs critical for potent inhibition of VLA4 binding to VCAM-1. Our first generation of inhibitors were quite potent and rapidly mobilized HSPCs, but their effect was short lived and yielded insufficient numbers of HSPCs for gene editing and engraftment studies. Most had poor solubility and very short plasma half-lives after s.c. injection in mice. We designed later generation analogues that strategically exploited a region of each optimized core, allowing for covalent addition of polyethylene glycol (PEG) chains of increasing length without decreasing potency. We determined that a minimum PEG length of 24 PEG units was required for extended HSPC mobilization in mice. WU-106, one of our VLA-4 inhibitors containing 24 PEG units, exhibited excellent aqueous solubility, longer plasma half-life after s.c. dosing, and mobilized 2-fold more murine HSPCs for a longer time than previously published best-in-class VLA-4 inhibitors or our first generation VLA4 inhibitor leads. Importantly, a synergistic effect on HSPC mobilization was achieved when WU-106 was co-administered with CXCR4 inhibitors (Plerixafor or BL-8040). Further optimization by the addition of longer PEG chains, even up to 40KD, retained sub nM potency and extended mobilization beyond 24 hours after a single injection. Our 20kD PEG analogues attached to our two different core molecules (WU-125 and WU-133) allowed for dosing of the drug in the evening, with maximum mobilization still present the next morning, thereby enabling HSPC collection to initiate soon (2-4 hours) after CXCR4 inhibitor dosing in the morning. The quantity of HSPCs obtained using this staggered dosing regimen was greater than simultaneous administration of the VLA-4 and CXCR4 inhibitors ( Fig. 1). Furthermore, our novel, long-acting PEG analogues demonstrated superior HSPC mobilization compared to an alpha 4-targeted antibody (anti-CD49d) in mice ( Fig.2). Conclusion: We have developed novel, potent PEGylated small molecule VLA-4 inhibitors that have excellent aqueous solubility and pharmacokinetics. The kinetics and magnitude of HSPC mobilization by our PEGylated inhibitors are superior to other previously described VLA-4 inhibitors and an antibody to alpha-4(anti-CD49d).

Exploring complexity of class-A Beta-lactamase family using physiochemical-based multiplex networks

The Beta-lactamase protein family is vital in countering Beta-lactam antibiotics, a widely used antimicrobial. To enhance our understanding of this family, we adopted a novel approach employing a multiplex network representation of its multiple sequence alignment. Each network layer, derived from the physiochemical properties of amino acids, unveils distinct insights into the intricate interactions among nodes, thereby enabling the identification of key motifs. Nodes with identical property signs tend to aggregate, providing evidence of the presence of consequential functional and evolutionary constraints shaping the Beta-lactamase family. We further investigate the distribution of evolutionary links across various layers. We observe that polarity manifests the highest number of unique links at lower thresholds, followed by hydrophobicity and polarizability, wherein hydrophobicity exerts dominance at higher thresholds. Further, the combinations of polarizability and volume, exhibit multiple simultaneous connections at all thresholds. The combination of hydrophobicity, polarizability, and volume uncovers shared links exclusive to these layers, implying substantial evolutionary impacts that may have functional or structural implications. By assessing the multi-degree of nodes, we unveil the hierarchical influence of properties at each position, identifying crucial properties responsible for the protein’s functionality and providing valuable insights into potential targets for modulating enzymatic activity.

Engineering minimal tissue inhibitors of metalloproteinase targeting MMPs via gene shuffling and yeast surface display.

Overexpression of specific matrix metalloproteinases (MMPs) has a key role in development of several diseases, such as cancer, neurological disorders, and cardiovascular diseases due to their critical role in degradation and remodeling of the extracellular matrix (ECM). Tissue inhibitors of metalloproteinases (TIMPs), a family of four in humans, are endogenous inhibitors of MMPs. TIMPs have a high level of sequence and structure homology, with a broad range of binding and inhibition to the family of MMPs. It is important to identify the key motifs of TIMPs responsible for inhibition of MMPs to developefficient therapeutics targeting specific MMPs. We used DNA shuffling between the human TIMP family to generate a minimal TIMP hybrid library in yeast to identify the dominant minimal MMP inhibitory regions. The minimal TIMP variants screened toward MMP-3 and MMP-9 using fluorescent-activated cell sorting (FACS). Interestingly, several minimal TIMP variants selected after screening toward MMP-3cd or MMP-9cd, with lengths as short as 20 amino acids, maintained or improved binding to MMP-3 and MMP-9. The TIMP-MMP binding dissociation constant (KD ), in the nM range, and MMP inhibition constants (Ki ), in the pM range, of these minimal TIMP variants were similar to the N-terminal domain of TIMP-1 on the yeast surface and in solution indicating the potency of these minimal variants as MMP inhibitors. We further used molecular modeling simulation, and molecular docking of the minimal TIMP variants in complex with MMP-3cd to understand the binding and inhibition mechanism of these variants.

Deep Learning-Assisted Design of Novel Promoters in Escherichia coli.

Deep learning (DL) approaches have the ability to accurately recognize promoter regions and predict their strength. Here, the potential for controllably designing active Escherichia coli promoter is explored by combining multiple deep learning models. First, "DRSAdesign," which relies on a diffusion model to generate different types of novel promoters is created, followed by predicting whether they are real or fake and strength. Experimental validation showed that 45 out of 50 generated promoters are active with high diversity, but most promoters have relatively low activity. Next, "Ndesign," which relies on generating random sequences carrying functional -35 and -10 motifs of the sigma70 promoter is introduced, and their strength is predicted using the designed DL model. The DL model is trained and validated using 200 and 50 generated promoters, and displays Pearson correlation coefficients of 0.49 and 0.43, respectively. Taking advantage of the DL models developed in this work, possible 6-mers are predicted as key functional motifs of the sigma70 promoter, suggesting that promoter recognition and strength prediction mainly rely on the accommodation of functional motifs. This work provides DL tools to design promoters and assess their functions, paving the way for DL-assisted metabolic engineering.

Discovery and preclinical development of LY3473329, a potent small molecule inhibitor of lipoprotein(a) formation

Abstract Background Lipoprotein(a) [Lp(a)] is a lipoprotein particle consisting of a low-density lipoprotein (LDL) particle and apolipoprotein(a) [apo(a)]. Lp(a) is a causal risk factor for cardiovascular disease. The formation of Lp(a) requires an initial interaction of apo(a) Kringle IV Domains 7 and 8 (KIV7, KIV8) with apoB-100 on LDL, followed by formation of a disulfide bond between apo(a) and apoB-100. Blocking the initial apo(a)-apoB interaction is a viable therapeutic approach to reduce circulating levels of Lp(a). The apo(a) protein is encoded by the LPA gene that arose from the plasminogen gene through an evolutionary gene duplication event. Because of sequence conservation between apo(a) and plasminogen, compound selectivity for apo(a) is imperative for clinical development. Purpose We report the discovery and preclinical characterization of LY3473329, a novel selective small molecule inhibitor of Lp(a) formation. Methods Computational and synthetic chemistry approaches coupled with multiple biochemical and biophysical assays were used to create apo(a) KIV7,8 binders. Chemical optimization led to molecules with potent inhibition of in vitro Lp(a) formation. Both apo(a) and plasminogen binding assays were conducted to evaluate compound selectivity. The potential for LY3473329 to lower steady state Lp(a) levels was assessed in human Lp(a) transgenic mice and in cynomolgus monkeys, and its effect on plasminogen activity was assessed in rat and monkey. Results Computational modeling, synthetic chemistry and small molecule library screens identified compounds that bind apo(a) KIV7 and KIV8 domains, and subsequent chemical optimization yielded compounds with sub-micromolar Lp(a) inhibition potency. Exploration of compound multivalency created inhibitors with sub-nanomolar potency, including LY3473329. LY3473329 engages multiple apo(a) KIV motifs, allowing it to potently inhibit Lp(a) formation in vitro. Oral doses of LY3473329 given to human Lp(a) transgenic mice and cynomolgus monkeys caused dose-dependent decreases in plasma Lp(a). The key binding motif for LY3473329 is shared by two KIV domains in rat and monkey plasminogen, but only one KIV domain in human plasminogen. LY3473329 was non-selective for rat and monkey plasminogen, but showed ∼ 50-fold selectivity for human plasminogen in in vitro binding assays. In cynomolgus monkeys and rats, LY3473329 significantly reduced plasminogen activity levels without adverse effects. Given its selectivity against human plasminogen, LY3473329 is expected to lower Lp(a) in human without affecting plasminogen. LY3473329 has been evaluated in a human Phase 1 study and is currently in Phase 2 clinical development. Conclusions LY3473329 is a potent and selective small molecule inhibitor of Lp(a) formation and is the only orally administered Lp(a) investigational therapeutic in clinical development.

Napoli fürs Sofa

Abstract Using a famous commercial shown on German-language television from the 1980s, this article analyses the media staging, reactivation and transformation of audio-visual images of Italy at the turn of the 20th and 21st centuries. The case study operates at the intersection of music history, the advertising and insurance industries, mass media and global tourism. It interweaves musical, technological, socio-cultural and discursive perspectives. In doing so, it demonstrates how key acoustic motifs become nationally connoted carriers of meaning in the context of the cross-media production and dissemination of clichés relating to Italy in international everyday culture.

Difluoromethoxylated Ketones as Building Blocks for the Synthesis of Challenging OCF2H‐Bearing N‐Heterocycles

AbstractInvited for this issue's Front Cover is the group of Frédéric. R. Leroux. The cover picture illustrates the potential of difluoromethoxylated ketones as building blocks for accessing challenging structures bearing the key OCF2H motif. These are represented here by a rocket, a highly technical achievement. Obviously, a parallel can be drawn between the building block strategy and a famous construction game. On the righthand side of the cover we can see the rocket heading towards a cell, highlighting the importance of fluorinated molecules in life sciences. Cover design by A. Loison. Read the full text of the article at 10.1002/ejoc.202300695.

Molecular design and systematic optimization of a halogen-bonding system between the asthma interleukin-5 receptor and its cyclic peptide ligand.

Human interleukin-5 (IL-5) functions as an important pro-inflammatory factor by binding to its specific receptor, IL-5Rα, which has been implicated in the pathogenesis of asthma. Previously, a disulfide-bonded cyclic peptide AF17121 obtained from random library screening and sequence variation was found to competitively disrupt the cognate IL-5Rα/IL-5 interaction with moderate potency. In this study, the crystal complex of IL-5Rα with AF17121 was investigated at structural and energetic levels. It is revealed that the side-chain indole moiety of the AF17121 Trp5 residue is a potential site for a stem putative halogen bond (X-bond) with IL-5Rα, which is just located within the key 3 EXXR6 motif region recognized specifically by IL-5Rα. We systematically examined four halogen substitution types at five positions of the indole moiety; QM/MM calculations theoretically unraveled that only halogenations at 5 and 6 positions can form effective X-bonds with the side-chain hydroxyl oxygen of the IL-5Rα Thr21 residue and the backbone carbonyl oxygen of Ala66 residue, respectively. Binding assays observed that I-substitution at the 5 position and Br-substitution at the 6 position can result in two potent halogenated peptides, [5I]AF17121 and [6Br]AF17121, which are improved by 1.6-fold and 3.5-fold relative to the native AF17121, respectively. 5I/6Br-double substitution, resulting in [5I/6Br]AF17121, can further enhance the peptide affinity by 7.5-fold. Structural analysis revealed that the X-bond stemming from 6Br-substitution is also involved in an orthogonal interaction system with a H-bond; they share a common backbone carbonyl oxygen acceptor of IL-5Rα Ala66 residue and exhibit a significant synergistic effect between them.

Color tunable benzothiadiazole-based small molecules for lightening applications

Abstract 2,1,3-Benzothiadiazole (BTD) is a key motif for the chemistry of photoluminescence molecules and its application in lightening technology as well as cell imagining. Evaluating the characteristics and reaction mechanisms of the BTD derivatives in molecular is critical for molecular designing. Research has been turned to BTD-extended derivatives owing to their possible usage in organic photovoltaics, organic light-emitting diodes, liquid crystal devices, dye-sensitized solar cells, and many other applications. Herein, three small molecules with a BTD core, 4,7-di-m-tolylbenzo[c][1,2,5]thiadiazole (NK1), 4,7-bis(3,4,5-trifluorophenyl)benzo[c][1,2,5]thiadiazole (NK2), and 4,7-bis(5-(m-tolyl)thiophen-2-yl)benzo[c][1,2,5]thiadiazole (NK3), were designed and synthesized and their molecular structures were elucidated. Additionally, their photophysical and electrochemical behaviors as well as the aggregation-induced enhanced emission properties were examined to investigate their possible usage in cell imaging and organic electronic applications. The HOMO energy level of molecules has been found as −5.96, −5.58, and −5.60 eV, respectively. In the emission spectra, it was seen that molecules showed green, blue, and red emission, respectively.

Decolonizing Ourselves

AbstractThis article outlines some key steps and motifs for developing a decolonial relational ethics that will be vital not just to the work of decolonization but also to the practice of the ecumenical movement if it is to both add its weight to the calls for decolonization and itself be decolonized. The author interrogates key dimensions of her own field of study in Christian ethics and engagement in the ecumenical movement. She highlights some of the key spaces and often marginalized communities and persons who can shape the ecumenical movement's further engagement in decolonization. It offers a critique and a vision rooted in the author's lived experience and the scholarship of decolonization while seeking application in methodologies for colonial systems, relationships and mindsets to be challenged and reset.

Ascorbate Peroxidase 2 (APX2) of Chlamydomonas Binds Copper and Modulates the Copper Insertion into Plastocyanin.

Recent phylogenetic studies have unveiled a novel class of ascorbate peroxidases called "ascorbate peroxidase-related" (APX-R). These enzymes, found in green photosynthetic eukaryotes, lack the amino acids necessary for ascorbate binding. This study focuses on the sole APX-R from Chlamydomonas reinhardtii referred to as ascorbate peroxidase 2 (APX2). We used immunoblotting to locate APX2 within the chloroplasts and in silico analysis to identify key structural motifs, such as the twin-arginine transport (TAT) motif for lumen translocation and the metal-binding MxxM motif. We also successfully expressed recombinant APX2 in Escherichia coli. Our in vitro results showed that the peroxidase activity of APX2 was detected with guaiacol but not with ascorbate as an electron donor. Furthermore, APX2 can bind both copper and heme, as evidenced by spectroscopic, and fluorescence experiments. These findings suggest a potential interaction between APX2 and plastocyanin, the primary copper-containing enzyme within the thylakoid lumen of the chloroplasts. Predictions from structural models and evidence from 1H-NMR experiments suggest a potential interaction between APX2 and plastocyanin, emphasizing the influence of APX2 on the copper-binding abilities of plastocyanin. In summary, our results propose a significant role for APX2 as a regulator in copper transfer to plastocyanin. This study sheds light on the unique properties of APX-R enzymes and their potential contributions to the complex processes of photosynthesis in green algae.

Discovery and Anti-Inflammatory Activity of a Cyanobacterial Fatty Acid Targeting the Keap1/Nrf2 Pathway.

The monounsaturated fatty acid 7(E)-9-keto-hexadec-7-enoic acid (1) and three structurally related analogues with different oxidation states and degrees of unsaturation (2-4) were discovered from a marine benthic cyanobacterial mat collected from Delta Shoal, Florida Keys. Their structures were elucidated using NMR spectroscopy and mass spectrometry. The structure of 1 contained an α,β-unsaturated carbonyl system, a key motif required for the activation of the Keap1/Nrf2-ARE pathway that is involved in the activation of antioxidant and phase II detoxification enzymes. Compounds 1-4 were screened in ARE-luciferase reporter gene assay using stably transfected HEK293 cells, and only 1 significantly induced Nrf2 activity at 32 and 10 µM, whereas 2-4 were inactive. As there is crosstalk between inflammation and oxidative stress, subsequent biological studies were focused on 1 to investigate its anti-inflammatory potential. Compound 1 induced Nqo1, a well-known target gene of Nrf2, and suppressed iNos transcript levels, which translated into reduced levels of nitric oxide in LPS-activated mouse macrophage RAW264.7 cells, a more relevant model for inflammation. RNA sequencing was performed to capture the effects of 1 on a global level and identified additional canonical pathways and upstream regulators involved in inflammation and immune response, particularly those related to multiple sclerosis. A targeted survey of marine cyanobacterial samples from other geographic locations, including Guam, suggested the widespread occurrence of 1. Furthermore, the previous isolation of 1 from marine diatoms and green algae implied a potentially important ecological role across marine algal eukaryotes and prokaryotes. The previous isolation from sea lettuce raises the possibility of dietary intervention to attenuate inflammation and related disease progression.

CXCL17 binds efficaciously to glycosaminoglycans with the potential to modulate chemokine signaling.

CXCL17 is a mucosally secreted protein, and the most recently identified human chemokine, an assignment based on protein fold prediction and chemotactic activity for leukocytes. However, these credentials have been the subject of much recent discussion and no experimental evidence has been presented regarding the definitive structure of CXCL17. In this study, we evaluated the structural and chemoattractant credentials of CXCL17 to better characterize this molecule, and gain deeper insights into its functional role as a glycosaminoglycan (GAG) binding protein. In the absence of structural information, in silico modeling techniques assessed the likelihood of CXCL17 adopting a chemokine fold. Recombinant CXCL17 was synthesized in mammalian and prokaryotic systems. Modified Boyden chamber and real-time chemotaxis assays assessed the ability of CXCL17 to promote chemotaxis of murine splenocytes, human neutrophils, and CXCR1 transfectants. The efficacy of CXCL17 binding to GAGs was quantified with solid-phase assays and bio-layer interferometry techniques. All modeling efforts failed to support classification of CXCL17 as a chemokine based on its predicted conformation. Recombinant CXCL17 was observed to dimerize as a function of concentration, a characteristic of several chemokines. Contrary to a previous report, CXCL17 was not chemotactic for murine splenocytes, although it was a low-potency chemoattractant for human neutrophils at micromolar concentrations, several orders of magnitude higher than those required for CXCL8. As anticipated owing to its highly basic nature, CXCL17 bound to GAGs robustly, with key C-terminal motifs implicated in this process. While inactive via CXCR1, CXCL17 was found to inhibit CXCR1-mediated chemotaxis of transfectants to CXCL8 in a dose-dependent manner. In summary, despite finding little evidence for chemokine-like structure and function, CXCL17 readily bound GAGs, and could modulate chemotactic responses to another chemokine in vitro. We postulate that such modulation is a consequence of superior GAG binding, and that C-terminal fragments of CXCL17 may serve as prototypic inhibitors of chemokine function.

Insights into the missing apiosylation step in flavonoid apiosides biosynthesis of Leguminosae plants

Apiose is a natural pentose containing an unusual branched-chain structure. Apiosides are bioactive natural products widely present in the plant kingdom. However, little is known on the key apiosylation reaction in the biosynthetic pathways of apiosides. In this work, we discover an apiosyltransferase GuApiGT from Glycyrrhiza uralensis. GuApiGT could efficiently catalyze 2″-O-apiosylation of flavonoid glycosides, and exhibits strict selectivity towards UDP-apiose. We further solve the crystal structure of GuApiGT, determine a key sugar-binding motif (RLGSDH) through structural analysis and theoretical calculations, and obtain mutants with altered sugar selectivity through protein engineering. Moreover, we discover 121 candidate apiosyltransferase genes from Leguminosae plants, and identify the functions of 4 enzymes. Finally, we introduce GuApiGT and its upstream genes into Nicotiana benthamiana, and complete de novo biosynthesis of a series of flavonoid apiosides. This work reports an efficient phenolic apiosyltransferase, and reveals mechanisms for its sugar donor selectivity.