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Terpene synthesis stands at the forefront of modern synthetic chemistry and represents the state-of-the-art in the chemist's toolbox. Notwithstanding, these endeavors are inherently tied to the current availability of natural cyclic building blocks. Addressing this limitation, the stereocontrolled cyclization of abundant unbiased linear terpenes emerges as a valuable tool, which is still difficult to achieve with chemical catalysts. In this study, we showcase the remarkable capabilities of squalene-hopene cyclases (SHCs) in the chemoenzymatic synthesis of head-to-tail-fused terpenes. By combining engineered SHCs and a practical reaction setup, we generate ten chiral scaffolds with >99% ee and de, at up to decagram scale. Our mechanistic insights suggest how cyclodextrin encapsulation of terpenes may influence the performance of the membrane-bound enzyme. Moreover, we transform the chiral templates to valuable (mero)-terpenes using interdisciplinary synthetic methods, including a catalytic ring-contraction of enol-ethers facilitated by cooperative iodine/lipase catalysis., (© 2024. The Author(s).)

Membrane forces shift the equilibria of mechanosensitive channels enabling them to convert mechanical cues into electrical signals. Molecular tools to stabilize and methods to capture their highly dynamic states are lacking. Cyclodextrins can mimic tension through the sequestering of lipids from membranes. Here we probe the conformational ensemble of MscS by EPR spectroscopy, the lipid environment with NMR, and function with electrophysiology under cyclodextrin-induced tension. We show the extent of MscS activation depends on the cyclodextrin-to-lipid ratio, and that lipids are depleted slower when MscS is present. This has implications in MscS' activation kinetics when distinct membrane scaffolds such as nanodiscs or liposomes are used. We find MscS transits from closed to sub-conducting state(s) before it desensitizes, due to the lack of lipid availability in its vicinity required for closure. Our approach allows for monitoring tension-sensitive states in membrane proteins and screening molecules capable of inducing molecular tension in bilayers., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Cyclodextrins (CDs) are important starch derivatives and commonly comprise α-, β-, and γ-CDs. Their hydrophilic surface and hydrophobic inner cavity enable regulation of enzyme catalysis through direct or indirect interactions. Clarifying interactions between CDs and enzyme is of great value for enzyme screening, mechanism exploration, regulation of catalysis, and applications. We summarize the interactions between CDs and glycoside hydrolases (GHs) according to two aspects: 1) CD as products, substrates, inhibitors and activators of enzymes, directly affecting the reaction process; 2) CDs indirectly affecting the enzymatic reaction by solubilizing substrates, relieving substrate/product inhibition, increasing recombinant enzyme production and storage stability, isolating and purifying enzymes, and serving as ligands in crystal structure to identify functional amino acid residues. Additionally, CD enzyme mimetics are developed and used as catalysts in traditional artificial enzymes as well as nanozymes, making the application of CDs no longer limited to GHs. This review concerns the regulation of GHs catalysis by CDs, and gives insights into research on interactions between enzymes and ligands., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The influenza BM2 transmembrane domain ( BM2 TM ), an acid-activated proton channel, is an attractive antiviral target due to its essential roles during influenza virus replication, whereas no effective inhibitors have been reported for BM2. In this study, we draw inspiration from the properties of cyclodextrins (CDs) and hypothesize that CDs of appropriate sizes may possess the potential to act as inhibitors of the BM2TM proton channel. To explore this possibility, molecular dynamics simulations were employed to assess their inhibitory capabilities. Our findings reveal that CD4, CD5, and CD6 are capable of binding to the BM2TM proton channel, resulting in disrupted water networks and reduced hydrogen bond occupancy between H19 and the solvent within the BM2TM channel necessary for proton conduction. Notably, CD4 completely obstructs the BM2TM water channel. Based on these observations, we propose that CD4, CD5, and CD6 individually contribute to diminishing the proton transfer efficiency of the BM2 protein, and CD4 demonstrates promising potential as an inhibitor for the BM2 proton channel.

Cyclodextrinases are carbohydrate-active enzymes involved in the linearization of circular amylose oligosaccharides. Primarily thought to function as part of starch metabolism, there have been previous reports of bacterial cyclodextrinases also having additional enzymatic activities on linear malto-oligosaccharides. This substrate class also includes environmentally rare α-diglucosides such as kojibiose (α-1,2), nigerose (α-1,3), and isomaltose (α-1,6), all of which have valuable properties as prebiotics or low-glycemic index sweeteners. Previous genome sequencing of three Cellvibrio japonicus strains adapted to utilize these α-diglucosides identified multiple, but uncharacterized, mutations in each strain. One of the mutations identified was in the amy13E gene, which was annotated to encode a neopullulanase. In this report, we functionally characterized this gene and determined that it in fact encodes a cyclodextrinase with additional activities on α-diglucosides. Deletion analysis of amy13E found that this gene was essential for kojibiose and isomaltose metabolism in C. japonicus . Interestingly, a Δ amy13E mutant was not deficient for cyclodextrin or pullulan utilization in C. japonicus ; however, heterologous expression of the gene in E. coli was sufficient for cyclodextrin-dependent growth. Biochemical analyses found that Cj Amy13E cleaved multiple substrates but preferred cyclodextrins and maltose, but had no activity on pullulan. Our characterization of the Cj Amy13E cyclodextrinase is useful for refining functional enzyme predictions in related bacteria and for engineering enzymes for biotechnology or biomedical applications.IMPORTANCEUnderstanding the bacterial metabolism of cyclodextrins and rare α-diglucosides is increasingly important, as these sugars are becoming prevalent in the foods, supplements, and medicines humans consume that subsequently feed the human gut microbiome. Our analysis of a cyclomaltodextrinase with an expanded substrate range is significant because it broadens the potential applications of the GH13 family of carbohydrate active enzymes (CAZymes) in biotechnology and biomedicine. Specifically, this study provides a workflow for the discovery and characterization of novel activities in bacteria that possess a high number of CAZymes that otherwise would be missed due to complications with functional redundancy. Furthermore, this study provides a model from which predictions can be made why certain bacteria in crowded niches are able to robustly utilize rare carbon sources, possibly to gain a competitive growth advantage., Competing Interests: The authors declare no conflict of interest.

9α-Hydroxyandroster-4-ene-3,17-dione (9-OH-AD) is a representative steroid drug intermediate that can be prepared by phytosterols (PS) biotransformation with mycobacteria in a resting cell-cyclodextrin system. In this study, over-expression of 17β-hydroxysteroid dehydrogenase (Hsd4A) was testified to enhance the side-chain degradation of PS and to reduce the incomplete degradation by-products. Meanwhile, the complete degradation product 4-androstene-3,17-dione (AD) was increased due to the lack of 3-Ketosteroid 9α-Hydroxylase (KshA1) activities. To increase the production and purity of 9-OH-AD, the metabolic pathway of the side-chain degradation of PS and 9-position hydroxylation was modulated by balancing the over-expression of Hsd4A and KshA1 in mycobacteria and reducing the bioconversion rate via lowering the ratio of PS and cyclodextrin. The production and purity of 9-OH-AD in broth were improved from 22.18 g L -1 and 77.13% to 28.27 g L -1 and 87.84%, with a molar yield of 78.32%., (© 2023 Wiley-VCH GmbH.)

Cyclodextrin glucanotransferase (CGTase) is an extracellular enzyme of the GH13 α-amylase family that catalyzes a unique intramolecular reaction known as cyclization to transform α-1, 4-glucans and similar starches into cyclodextrins. They also catalyze intermolecular transglycosylation reactions namely coupling, disproportionation, and some hydrolyzing effects on starch. The monomeric structures of the CGTase exhibit five domains (A, B, C, D, and E domains) with different molecular weights and amino acid sequences depending on the source. Among bacteria, Bacillus genus covers approximately 90% of the CGTase producers, while other genera like Klebsiella, Paenibacillus, and Thermoanaerobacter also shown decent contributions in recent studies. CGTase production is highly supported by alkaliphilic bacteria under submerged fermentation rather than solid-state fermentation. The bacterial sources, biochemical properties, production conditions, and structure of CGTases are compiled in this review. Cyclodextrins have the unique property of making inclusion complexes with various compounds, hence widely used in the food, pharmaceutical, cosmetics, laundry, and chemical sectors. This review presents a comprehensive view of CGTase produced by Bacillus spp., and other bacterial genera like Klebsiella, Paenibacillus, and Microbacterium. It also gives insight of the properties and recent biotechnological applications of cyclodextrins. KEY POINTS: • Transglycosylation reactions catalyzed by CGTase and their structural properties. • Comparative data of CGTase production by various genera and Bacillus spp. • Structures, properties, and applications of different cyclodextrins., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Adipocytes store a significant amount of cholesterol and triglycerides. However, whether cholesterol modulates adipocyte function remains largely unknown. We modulated the cholesterol level in adipocytes to examine its effect on the secretion of adiponectin, an important hormone specifically secreted by adipocytes. Treating differentiated 3T3-L1 adipocytes with 4 mM methyl-β-cyclodextrin (MβCD), a molecule with a high affinity for cholesterol, rapidly depleted cholesterol in adipocytes. Interestingly, MβCD treatment increased adiponectin in the medium without affecting its intracellular level, suggesting a modulation of secretion. By contrast, cholesterol addition did not affect adiponectin secretion, suggesting that cholesterol-depletion-induced intracellular cholesterol trafficking, but not reduced cholesterol level, accounted for MβCD-induced adiponectin secretion. MβCD-induced adiponectin secretion was reduced after 10 μg/mL U18666A treatment that suppressed cholesterol transport out of late endosomes/lysosomes. Depleting Niemann-Pick type C1 (NPC1) or NPC2 proteins, which mediate endosomal/lysosomal cholesterol export, consistently reduced MβCD-induced adiponectin secretion. Furthermore, treatment with 1 μM bafilomycin A1, which neutralized acidic endosomes/lysosomes, also attenuated MβCD-induced adiponectin secretion. Finally, MβCD treatment redistributed cellular adiponectin to lower-density fractions in sucrose gradient fractionation. Our results show that MβCD-mediated cholesterol depletion elevates the secretion of adiponectin, highlighting the involvement of endosomes and lysosomes in adiponectin secretion in adipocytes.

The endogenous peptides from peanut hairy root culture were induced upon elicitor treatment with chitosan (CHT), methyl jasmonate (MeJA), and cyclodextrin (CD): CHT+MeJA+CD. The peptides secreted into the liquid culture medium play an important role in plant signaling and stress responses. By performing gene ontology (GO) analysis, a number of plant proteins involved in biotic and abiotic defense responses were identified, such as endochitinase, defensin, antifungal protein, cationic peroxidase and Bowman-Birk type protease inhibitor A-II. The bioactivity of 14 peptides synthesized from secretome analysis was determined. Peptide BBP1-4, derived from the diverse region of Bowman-Birk type protease inhibitor, displayed high antioxidant activity and mimicked the property of chitinase and β-1,3-glucanase enzymes. The antimicrobial activity against S. aureus, S. typhimurium, and E. coli was evidenced with different peptide concentrations. Additionally, peptide BBP1-4 has the potential to be a useful candidate for an immune response property, as it was found to increase the expression of some pathogenesis-related (PR) proteins and stilbene biosynthesis genes in peanut hairy root tissues. The findings indicate that secreted peptides may play a role in plant responses to both abiotic and biotic stresses. These peptides, which possess bioactive properties, could be considered as potential candidates for use in the pharmaceutical, agricultural, and food industries., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Cyclodextrin (CD), a cyclic oligosaccharide, is a pharmaceutical additive that improves the solubility of hydrophobic compounds. Recent research has focused on the potential active pharmaceutical abilities of CD. Lysosomal storage diseases are inherited metabolic diseases characterized by lysosomal dysfunction and abnormal lipid storage. Niemann-Pick disease type C (NPC) is caused by mutations in cholesterol transporter genes (NPC1, NPC2) and is characterized by cholesterol accumulation in lysosomes. A biocompatible cholesterol solubilizer 2-hydroxypropyl-β-cyclodextrin (HP-β-CD) was recently used in NPC patients for compassionate use and in clinical trials. HP-β-CD is an attractive drug candidate for NPC; however, its adverse effects, such as ototoxicity, should be solved. In this review, we discuss the current use of HP-β-CD in basic and clinical research and discuss alternative CD derivatives that may outperform HP-β-CD, which should be considered for clinical use. The potential of CD therapy for the treatment of other lysosomal storage diseases is also discussed., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Purpose: Tyrosine kinase inhibitors inhibit VEGF receptors. If delivered to the retina, they might inhibit oedema and neovascularization such as in age-related macular degeneration and diabetic retinopathy. The aim of this study was to formulate cediranib maleate, a potent VEGF inhibitor, as γ-cyclodextrin nanoparticle eye drops and measure the retinal delivery and overall ocular pharmacokinetics after a single-dose administration in rabbits., Methods: A novel formulation technology with 3% cediranib maleate as γ-cyclodextrin micro-suspension was prepared by autoclaving method. Suitable stabilizers were tested for heat-stable eye drops. The ophthalmic formulation was topically applied to one eye in rabbits. The pharmacokinetics in ocular tissues, tear film and blood samples were studied at 1, 3 and 6 hr after administration., Results: γ-cyclodextrin formed complex with cediranib maleate. The formation of γ-cyclodextrin nanoparticles occurred in concentrated complexing media. Combined stabilizers prevented the degradation of drug during the autoclaving process. Three hours after administration of the eye drops, treated eyes showed cediranib levels of 737 ± 460 nM (mean ± SD) in the retina and 10 ± 6 nM in the vitreous humour., Conclusions: Cediranib maleate in γ-cyclodextrin nanoparticles were stable to heat in presence of stabilizers. The drug as eye drops reached the retina in concentrations that are more than 100 times higher than the 0.4 nM IC 50 value reported for the VEGF type-II receptor and thus, presumably, above therapeutic level. These results suggest that γ-cyclodextrin-based cediranib maleate eye drops deliver effective drug concentrations to the retina in rabbits after a single-dose administration., (© 2022 Acta Ophthalmologica Scandinavica Foundation. Published by John Wiley & Sons Ltd.)

Nanotechnology has been developed to deliver cargos effectively to the vascular system. Nanomedicine is a novel and effective approach for targeted vascular disease treatment including atherosclerosis, coronary artery disease, strokes, peripheral arterial disease, and cancer. It has been well known for some time that vascular disease patients have a higher cancer risk than the general population. During atherogenesis, the endothelial cells are activated to increase the expression of adhesion molecules such as Intercellular Adhesion Molecule 1 (ICAM-1), Vascular cell adhesion protein 1 (VCAM-1), E-selectin, and P-selectin. This biological activation of endothelial cells gives a targetability clue for nanoparticle strategies. Nanoparticle formation has a passive targeting pathway due to the increased adhesion molecule expression on the cell surface as well as increased cell activation. In addition, the VCAM-1-targeting peptide has been widely used to target the inflamed endothelial cells. Biomimetic nanoparticles using platelet and leukocyte membrane fragment strategies have been promising techniques for targeted vascular disease treatment. Cyclodextrin, a natural oligosaccharide with a hydrophobic cavity, increase the solubility of cholesterol crystals at the atherosclerotic plaque site and has been used to deliver the hydrophobic drug statin as a therapeutic in a targeted manner. In summary, nanoparticles decorated with various targeting molecules will be an effective and promising strategy for targeted vascular disease treatment.

Owing to the similarity of hydrogels to cartilage extracellular matrix, they have been extensively utilized in the chondral lesions. Moreover, their tunable administration properties are desirable for reducing injuries in lesion sites. Generally, injectable hydrogels are mechanically weak, requiring some modifications for being used as a cell carrier in place of articular cartilage. In this study, a combination of β -cyclodextrin-grafted alginate (Alg- β -CD) and pluronic-amine with multiple physical crosslinking was used for the first time. Supramolecular interactions, including electrostatic forces, host-guest interaction, and hydrophobic interaction with increasing temperature maintain injectability of hydrogels while these interactions boost mechanical properties to the extent that shear modulus surpassed 40 kPa. Vacant β -CD cavities in conjunction with gel network were exploited for kartogenin (KGN) loading. All groups had gel time of less than one minute and gel temperature was 28 °C. No toxic effect of hydrogels on encapsulated cells was observed. While the optimum combination of polymers provided a sustainable release for KGN, it also extended the in vitro degradation time of hydrogels from six days to two weeks. KGN facilitated encapsulated mesenchymal stem cells differentiation towards chondrocytes. Taken together, the synthesized hydrogel proved to be a promising candidate for being utilized in cartilage regeneration., (© 2022 IOP Publishing Ltd.)

The γ-cyclodextrin glycosyltransferase (γ-CGTase) from Evansella caseinilytica was expressed on the cell surface of E. coli using pAIDA-I autotransporter and was further utilized in the conversion of starch to γ-cyclodextrins (CDs). Maximum cyclization activity of 2.28 ± 0.46 U/g biomass was achieved after 3 h of induction using 0.1 mM IPTG at 37 ºC. Surface expression of γ-CGTase was confirmed using flow cytometry employing a FITC-conjugated anti-HIS antibody. Biochemical characterization of surface-displayed γ-CGTase revealed optima at pH 10.0 and 40 ºC along with a t 1/2 of 24.75 min at 50ºC. The K m and V max values on soluble potato starch were 10.94 mg/ml and 4.33 µmoles min -1 g -1 DCW respectively, and the activation energy was calculated to be 89.8 kJ/mol. The surface displayed γ-CGTase was further utilized for CD production and specifically, γ-CD conversion was obtained. The maximum conversion was achieved at 50 ºC, pH 9.0 using soluble potato starch (2.5%; w/v) taking a final enzyme concentration of 0.6 U/g starch. The surface-displayed γ-CGTase was able to convert soluble potato starch (2.5%) into γ-CDs with a 72.7% specific yield and no other peaks corresponding to α- and β-CDs were observed on HPLC. The enzyme was found to be ~100% operationally stable for up to 2 consecutive cycles of 24 h, with > 75% storage stability at - 20 ºC even after 7 days., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Gangliosides are glycosphingolipids of the plasma membrane and are highly enriched in the nervous system where they play a vital role in normal cell functions. Furthermore, several studies suggest their potential involvement in the pathogenesis of neurological conditions. Since cyclodextrins (CDs) can form inclusion complexes with various lipids, methylated beta-CDs are widely used in biomedical research to extract cholesterol from the membrane and study its cellular role. Despite CDs being known to interact with other membrane lipid components, their effect on gangliosides is poorly characterized. The aim of this research was to investigate the effect of dimethyl-beta-cyclodextrin (DIMEB), hydroxypropyl-beta-cyclodextrin (HPBCD), randomly methylated-alpha-cyclodextrin (RAMEA), and hydroxypropyl-alpha-cyclodextrin (HPACD) on ganglioside and cholesterol levels in rat brain synaptosomes. Their effect on membrane integrity and viability was also assessed. We examined the role of lipid depletion by CDs on the release of the major excitatory neurotransmitter, glutamate. Selective concentration range for cholesterol depletion was only found with HPBCD, but not with DIMEB. Selective depletion of gangliosides was achieved by both RAMEA and HPACD. The inhibition of stimulated glutamate release upon ganglioside depletion was found, suggesting their potential role in neurotransmission. Our study highlights the importance of the characterization of the lipid depleting capability of different CDs.

Background: Approximately 10% of infants are born preterm. Preterm birth leads to short and long-term changes in cardiac shape and function. By using a rat model of neonatal high-oxygen (80%O 2 ) exposure, mimicking the premature hyperoxic transition to the extrauterine environment, we revealed a major role of the renin-angiotensin system peptide Angio II (angiotensin II) and its receptor AT1 (angiotensin receptor type 1) on neonatal O 2 -induced cardiomyopathy. Here, we tested whether treatment with either orally active compounds of the peptides Angio-(1-7) or alamandine included in cyclodextrin could prevent postnatal cardiac remodeling and the programming of cardiomyopathy induced by neonatal high-O 2 exposure., Methods: Sprague-Dawley pups were exposed to room air or 80% O 2 from postnatal day 3 (P3) to P10. Neonatal rats were treated orally from P3 to P10 and assessed at P10 and P28. Left ventricular (LV) shapes were characterized by tridimensional computational atlases of ultrasound images in addition to histomorphometry., Results: At P10, high O 2 -exposed rats presented a smaller, globular and hypertrophied LV shape versus controls. Treatment with cyclodextrin-Angio-(1-7) significantly improved LV function in the O 2 -exposed neonatal rats and slightly changed LV shape. Cyclodextrin-alamandine and cyclodextrin-Angio-(1-7) treatments similarly reduced hypertrophy at P10 as well as LV remodeling and dysfunction at P28. Both treatments upregulated cardiac angiotensin-converting enzyme 2 in O 2 -exposed rats at P10 and P28., Conclusions: Our findings demonstrate LV remodeling changes induced by O 2 -stress and the potential benefits of treatments targeting the cardioprotective renin-angiotensin system axis, supporting the neonatal period as an important window for interventions aiming at preventing cardiomyopathy in people born preterm.

Main Conclusion: Peanut cultivars are known to produce stilbene compounds. Transcriptional control plays a key role in the early stages of the stress response mechanism, involving both PR-proteins and stilbene compounds. In this study, the production of stilbenoid compounds, especially prenylated, was investigated in two cultivars of peanut hairy root lines, designated as K2-K599 and T9-K599 elicited with a combination of chitosan (CHT), methyl jasmonate (MeJA), and cyclodextrin (CD): CHT + MeJA + CD. The antioxidant activities and stilbenoid content of both K2-K599 and T9-K599 hairy root lines increased significantly during the elicitation period. The T9-K599 hairy root line expressed higher ABTS and FRAP antioxidant activities than the K2-K599 line while the latter exhibited greater total phenolic content than the former at all-time points. Additionally, the K2-K599 line exhibited more stilbene compounds, including trans-resveratrol, trans-arachidin-1, and trans-arachidin-3 than the T9-K599 line, which showed statistically significant differences at all-time points. Gene expression of the enzyme involved in the stilbene biosynthesis pathway (PAL, RS, RS3) was observed, responding early to elicitor treatment and the metabolic production of a high level of stilbenoid compounds at a later stage. The antioxidant enzyme (CuZn-SOD, APX, GPX) and pathogenesis-related protein (PR; PR4A, PR5, PR10, chitinase) genes were strongly expressed after elicitor treatment at 24 h and decreased with an increasing elicitation time. Investigation of the response mechanism illustrates that the elicitor treatment can affect various plant responses, including plant cell wall structure and integrity, antioxidant system, PR-proteins, and secondary plant metabolites at different time points after facing external environmental stimuli., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Panose is a type of functional sugar with diverse bioactivities. The enzymatic conversion bioprocess to produce high purity panose with high efficiency has become increasingly important. Here, a new neopullulanase (NPase), Amy117 from B. pseudofirmus 703, was identified and characterized. Amy117 presented the optimal activity at pH 7.0 and 30 °C, its activity is over 40% at 10 °C and over 80% at 20 °C, which is cold-active. The enzyme cleaved α-1, 4-glycosidic linkages of pullulan to generate panose as the only hydrolysis product, and degraded cyclodextrins (CDs) and starch to glucose and maltose, with an apparent preference for CDs. Furthermore, Amy117 can produce 72.7 mg/mL panose with a conversion yield of 91% ( w / w ) based on 80 mg/mL pullulan. The sequence and structure analysis showed that the low proportion of Arg, high proportion of Asn and Gln, and high α-helix levels in Amy117 may contribute to its cold-active properties. Root mean square deviation (RMSD) analysis also showed that Amy117 is more flexible than two mesophilic homologues. Hence, we discovered a new high-efficiency panose-producing NPase, which so far achieves the highest panose production and would be an ideal candidate in the food industry.

Cyclodextrins (CDs) are yielded by cyclodextrin glycosyltransferase (CGTase) using starches or their derivatives as substrates. However, how starch fine structure affects it production is still ambiguous. This study aimed to decipher the relevance between the external chain length (ECL) and CD production. The waxy maize starch was hydrolyzed by β-amylase to prepare starchy substrates with regular gradient ECL for CGTase. The HPAEC results reflected that β-amylolysis lower the starch chain fractions with DP range of 13-24 by 30%, while the fractions with DP < 6 promoted by 30%. The molecular weight distribution and iodine binding results reflected that this process had limited impact on overall starch molecular size parameters. After CGTase conversion, it is interesting to find that the ECL was positively correlated with CD contents, which also has a high consistency among α-CD, β-CD, and γ-CD. This research would provide a favorable perspective for CD production improvement., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

The glucose oligosaccharide-derived cyclodextrins (CDs) are used for improving bioactive compound production in plant cell cultures because, in addition to their elicitation activity, CDs promote product removal from cells. However, despite these advantages, the industrial application of CDs is hampered by their high market price. A strategy to overcome this constraint was recently tested, in which reusable CD polymers coated with magnetic Fe 3 O 4 nanoparticles were harnessed in Vitis vinifera cell cultures to produce t-resveratrol (t-R). In this study, we applied hydroxypropyl-β-CDs (HPCD) and HPCDs coated with magnetic nanoparticles (HPCD-EPI-MN) in methyl jasmonate (MJ)-treated transgenic Silybum marianum cultures ectopically expressing either a stilbene synthase gene (STS) or a chalcone synthase gene (CHS), and compared their effects on the yields of t-R and naringenin (Ng), respectively. HPCD-EPI-MN at 15 g/L stimulated the accumulation of metabolites in the culture medium of the corresponding transgenic cell lines, with up to 4 mg/L of t-R and 3 mg/L of Ng released after 3 days. Similar amounts were produced in cultures treated with HPCD. Concentrations higher than 15 g/L of HPCD-EPI-MN and prolonged incubation periods negatively affected cell growth and viability in both transgenic cell lines. Reutilization of HPCD-EPI-MN was possible in three elicitation cycles (72 h each), after which the polymer retained 25-30% of its initial efficiency, indicating good stability and reusability. Due to their capacity to adsorb metabolites and their recyclability, the application of magnetic CD polymers may reduce the costs of establishing efficient secondary metabolite production systems on a commercial scale. KEY POINTS: • Long-term transgenic S. marianum suspensions stably produce transgene products • t-R and Ng accumulated extracellularly in cultures elicited with HPCD and HPCD-EPI-MN • The recyclability of HPCD-EPI-MN for metabolite production was proven., (© 2022. The Author(s).)

Dynamic systems of cyclodextrins (CDs) enabled by a native cyclodextrin glucanotransferase (CGTase) can incorporate unnatural glucopyranose-derived building blocks, expanding the applicability of enzyme-mediated dynamic combinatorial chemistry by using synthetically modified substrates. Starting dynamic combinatorial libraries from CDs with a single 6-modified glucopyranose results in a dynamic mixture of CDs containing several modified glucopyranoses. The relative concentrations of modified α, β or γ-CDs can be controlled by the addition of templates, providing a novel way to access modified CDs.

Carbohydrate metabolism via cyclodextrins (CM-CD) is an uncommon starch-converting pathway that thoroughly depends on extracellular cyclomaltodextrin glucanotransferases (CGTases) to transform the surrounding starch substrate to α-(1,4)-linked oligosaccharides and cyclodextrins (CDs). The CM-CD pathway has emerged as a convenient microbial adaptation to thrive under extreme temperatures, as CDs are functional amphipathic toroids with higher heat-resistant values than linear dextrins. Nevertheless, although the CM-CD pathway has been described in a few mesophilic bacteria and archaea, it remains obscure in extremely thermophilic prokaryotes (T opt  ≥ 70 °C). Here, a new monophyletic group of CGTases with an exceptional three-domain ABC architecture was detected by (meta)genome mining of extremely thermophilic Thermoanaerobacterales living in a wide variety of hot starch-poor environments on Earth. Functional studies of a representative member, CldA, showed a maximum activity in a thermoacidophilic range (pH 4.0 and 80 °C) with remarkable product diversification that yielded a mixture of α:β:γ-CDs (34:62:4) from soluble starch, as well as G3-G7 linear dextrins and fermentable sugars as the primary products. Together, comparative genomics and predictive functional analysis, combined with data of the functionally characterized key proteins of the gene clusters encoding CGTases, revealed the CM-CD pathway in Thermoanaerobacterales and showed that it is involved in the synthesis, transportation, degradation, and metabolic assimilation of CDs., (© 2022. The Author(s).)

Around 5% of the population of the world is affected with the disease called diabetes mellitus. The main medication of the diabetes is the insulin; the active form is the insulin monomer, which is an instable molecule, because the long storage time, or the high temperature, can cause the monomer insulin to adapt an alternative fold, rich in β-sheets, which is pharmaceutically inactive. The aim of this study is to form different insulin complexes with all the cyclodextrin used for pharmaceutical excipients (native cyclodextrin, methyl, hydroxyethyl, hydroxypropyl and sulfobutylether substituted β-cyclodextrin), in silico condition, with the AutoDock molecular modeling program, to determine the best type of cyclodextrin or cyclodextrin derivate to form a complex with an insulin monomer, to predict the molar ratio, the conformation of the complex, and the intermolecular hydrogen bonds formed between the cyclodextrin and the insulin. From the results calculated by the AutoDock program it can be predicted that insulin can make a stable complex with 5-7 molecules of hydroxypropyl-β-cyclodextrin or sulfobutylether-β-cyclodextrin, and by forming a complex potentially can prevent or delay the amyloid fibrillation of the insulin and increase the stability of the molecule.

Highly reliable separation and determination of various biologically active compounds were achieved using capillary electrophoresis (CE) based on β-cyclodextrin-functionalized graphene quantum dots (βcd-GQDs) as the background electrolyte additive. βcd-GQDs improve the separation efficiency between peaks of all analytes. No addition of surfactants or organic solvents was needed in the running buffer containing βcd-GQDs. Up to eight consecutive runs were acquired with high precision for the separation of resveratrol, pyridoxine, riboflavin, catechin, ascorbic acid, quercetin, curcumin, and even of several of their structural analogs. Baseline separation was achieved within just 13 min as a result of the effective mobility of the analytes along the capillary owing to the differential interaction with the additive. The proposed analytical method displayed a good resolution of peaks for all species selecting two absorption wavelengths in the diode array detector. Detection limits lower than 0.28 µg mL -1 were found for all compounds and precision values were in the range of 2.1-4.0% in terms of the peak area of the analytes. The usefulness of the GQD-assisted selectivity-enhanced CE method was verified by the analysis of food and dietary supplements. The applicability to such complex matrices and the easy and low-cost GQD preparation open the door for routine analyses of food and natural products. The concept of using such a dual approach (macromolecules and nanotechnology) has been explored to tackle the separation of various bioactive compounds in nutritional supplements and food. Schematic illustration of the electrophoretic separation of the bioactive molecules in the capillary which is filled with the running solution without (top) and with βcd-GQDs (bottom). The fused silica capillary with negatively ionizable silanol groups at the wall. The voltage is applied at positive polarity at the outlet. R, riboflavin; r, resveratrol; P, pyridoxine; C, catechin; c, curcumin; A, ascorbic acid; Q, quercetin., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

The bacterial mechanosensitive channel of small conductance (MscS) has been extensively studied to understand how mechanical forces are converted into the conformational changes that underlie mechanosensitive (MS) channel gating. We showed that lipid removal by β-cyclodextrin can mimic membrane tension. Here, we show that all cyclodextrins (CDs) can activate reconstituted Escherichia coli MscS, that MscS activation by CDs depends on CD-mediated lipid removal, and that the CD amount required to gate MscS scales with the channel's sensitivity to membrane tension. Importantly, cholesterol-loaded CDs do not activate MscS. CD-mediated lipid removal ultimately causes MscS desensitization, which we show is affected by the lipid environment. While many MS channels respond to membrane forces, generalized by the "force-from-lipids" principle, their different molecular architectures suggest that they use unique ways to convert mechanical forces into conformational changes. To test whether CDs can also be used to activate other MS channels, we chose to investigate the mechanosensitive channel of large conductance (MscL) and demonstrate that CDs can also activate this structurally unrelated channel. Since CDs can open the least tension-sensitive MS channel, MscL, they should be able to open any MS channel that responds to membrane tension. Thus, CDs emerge as a universal tool for the structural and functional characterization of unrelated MS channels., Competing Interests: The authors declare no competing interest.

The transport of membrane impermeable compounds into cells is a prerequisite for the efficient cellular delivery of hydrophilic and amphiphilic compounds and drugs. Transport into the cell's cytosolic compartment should ideally be controllable and it should involve biologically compatible and degradable vehicles. Addressing these challenges, nanocontainers based on cyclodextrin amphiphiles that are stabilized by a biodegradable peptide shell are developed and their potential to deliver fluorescently labeled cargo into human cells is analyzed. Host-guest mediated self-assembly of a thiol-containing short peptide or a cystamine-cross-linked polypeptide shell on cyclodextrin vesicles produce short peptide-shelled (SPSV ss ) or polypeptide-shelled vesicles (PPSV ss ), respectively, with redox-responsive and biodegradable features. Whereas SPSV ss are permeable and less stable, PPSV ss effectively encapsulate cargo and show a strictly regulated release of membrane impermeable cargo triggered by either reducing conditions or peptidase treatment. Live cell experiments reveal that the novel PPSV SS are readily internalized by primary human endothelial cells (human umbilical vein endothelial cells) and cervical cancer cells and that the reductive microenvironment of the cells' endosomes trigger release of the hydrophilic cargo into the cytosol. Thus, PPSV SS represent a highly efficient, biodegradable, and tunable system for overcoming the plasma membrane as a natural barrier for membrane-impermeable cargo., (© 2021 The Authors. Advanced Science published by Wiley-VCH GmbH.)

Cyclodextrinase (CDase) and cyclodextrin glucosyltransferase (CGTase) were synergistically used to provide a novel enzymatic method in lowing in vitro digestibility of waxy maize starch. The molecular structure, malto-oligosaccharide composition, and digestibility properties of the generated products were investigated. The molecular weight was reduced to 0.3 × 10 5  g/mol and 0.2 × 10 5  g/mol by simultaneous and sequential treatment with CDase and CGTase, while the highest proportion of chains with degree of polymerization (DP) < 13 was obtained by simultaneous treatment. The resistant starch contents were increased to 27.5% and 36.9% by simultaneous and sequential treatments respectively. Dual-enzyme treatment significantly promoted the content of malto-oligosaccharides (MOSs) by hydrolyzing cyclodextrins from CGTase with CDase. However, the replacement of cyclodextrins by MOSs did not obviously influence the digestibility of the products. The starch digestion kinetics further revealed the hydrolysis pattern of these two enzymes on the starch hydrolysate. It was proved that the starch digestibility could be lowered by modulating the molecular structure and beneficial MOSs content by this dual-enzyme treatment., Competing Interests: Declaration of competing interest No conflicts of interest are declared for any of the authors., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Cyclodextrin glucanotransferase (CGTase; EC 2.4.1.19) produces cycloamyloses (CAs), which are large cyclic glucans, and subsequently transforms them to α-, β-, and γ-cyclodextrins. We developed a novel encapsulation process based on the cyclization activity of CGTase and applied it to the formation of CA inclusion complexes with resveratrol (RVT), which has limited bioavailability due to its low water solubility. The encapsulated RVT (CA-RVT) was purified using preparative high-performance liquid chromatography. The water solubility of CA-RVT was 6,000-fold higher than that of RVT. CA-RVT in water demonstrated 98% stability for 1 week at 4 °C. According to radical scavenging activity and anti-inflammatory assays, CA-RVT in aqueous solution exhibited similar activities as an equal amount of RVT in dimethyl sulfoxide, suggesting the limited solubility of RVT can be overcome through CA encapsulation by CGTase, thus enhancing its nutraceutical value as a functional ingredient in the food industry., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Hydroxylated polycyclic aromatic hydrocarbons (OH-PAHs) can bind with functional biomacromolecules and thus cause toxic effects in vivo. Four types of cyclodextrins (CDs) were selected to explore their potential ability to regulate the bindings between 1-hydroxypyrene (1-OHPyr) and bovine serum albumin (BSA) using multi-spectroscopic methods combined with molecular docking. The results showed that the four CDs caused varied modulating effects on the binding of BSA with 1-OHPyr, and the effects of γ-CD and (2-hydroxypropyl)-β-CD (HPCD) are the most significant. Specifically, γ-CD and HPCD could significantly reduce the binding affinity between 1-OHPyr and BSA, inhibit the micro-environmental changes of tryptophan residues, and slightly recover the helicity of BSA. The interactions and inclusion behavior of CDs with 1-OHPyr was the main reason why CDs could affect the binding of 1-OHPyr to BSA. The results indicated that γ-CD and HPCD might have potential application value in regulating the toxic effects of OH-PAHs., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Cyclodextrins (CDs) are doughnut-shaped cyclic oligosaccharides having a cavity and two rims. Inclusion binding in the cavity has long served as a classic model of molecular recognition, and rim binding has been neglected. We found that CDs recognize guests by size-sensitive binding using the two rims in addition to the cavity, using single-molecule electron microscopy and a library of graphitic cones as a solid-state substrate for complexation. For example, with its cavity and rim binding ability combined, γ-CD can recognize a guest of radius between 4 and 9 Å with a size-recognition precision of better than 1 Å, as shown by structural analysis of thousands of individual specimens and statistical analysis of the data thereof. A 2.5 ms resolution electron microscopic video provided direct evidence of the process of size recognition. The data suggest the occurrence of the rim binding mode for guests larger than the size of the CD cavity and illustrate a unique application of dynamic molecular electron microscopy for deciphering the spatiotemporal details of supramolecular events.

The efficiency of enzymatic cyclodextrin production using cyclodextrin glycosyltransferases (CGTases) is limited by product inhibition. In this study, maltose binding site 2 (MBS2) of the β-CGTase from Bacillus circulans STB01 was modified to decrease product inhibition. First, two point mutants were prepared at position 599 (A599V and A599N). Then, two double mutants incorporating alanine at position 633 (A599N/Y633A and A599V/Y633A) were prepared. Finally, the entire MBS2 region was replaced by that of the α-CGTase from Paenibacillus macerans JFB05-01 to form multipoint mutant MBS2 β → α. All five mutants exhibited mixed-type product inhibition, although both the competitive and uncompetitive components of this inhibition were decreased. The total cyclization activities of A599N, A599V and A599V/Y633A were 15.6%, 76.8% and 70.9% lower than that of the wild-type, respectively, while that of A599N/Y633A was 22.4% higher. Among the mutants, only MBS2 β → α showed catalytic efficiency (k cat /K m ) comparable with that of the wild-type. Moreover, A599N, A599N/Y633A and MBS2 β → α produced cyclodextrin yields 13.1%, 15.8% and 19.7% greater than that of the wild-type, respectively. These results suggest that A599N, A599N/Y633A and MBS2 β → α may be more suitable than the wild-type for cyclodextrin production., Competing Interests: Declaration of competing interest The authors declare no competing financial interest. Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by USDA. USDA is an equal opportunity provider and employer., (Copyright © 2021. Published by Elsevier B.V.)

Cyclodextrins (CDs) have been widely used as pharmaceutical excipients for formulation purposes for different delivery systems. Recent studies have shown that CDs are able to form complexes with a variety of biomolecules, such as cholesterol. This has subsequently paved the way for the possibility of using CDs as drugs in certain retinal diseases, such as Stargardt disease and retinal artery occlusion, where CDs could absorb cholesterol lumps. However, studies on the retinal toxicity of CDs are limited. The purpose of this study was to examine the retinal toxicity of different beta-(β)CD derivatives and their localization within retinal tissues. To this end, we performed cytotoxicity studies with two different CDs-2-hydroxypropyl-βCD (HPβCD) and randomly methylated β-cyclodextrin (RMβCD)-using wild-type mouse retinal explants, the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, and fluorescence microscopy. RMβCD was found to be more toxic to retinal explants when compared to HPβCD, which the retina can safely tolerate at levels as high as 10 mM. Additionally, studies conducted with fluorescent forms of the same CDs showed that both CDs can penetrate deep into the inner nuclear layer of the retina, with some uptake by Müller cells. These results suggest that HPβCD is a safer option than RMβCD for retinal drug delivery and may advance the use of CDs in the development of drugs designed for intravitreal administration.

Per(6-O-tert-butyldimethylsilyl)-α-, β- and γ-cyclodextrin derivatives are well-known as synthetic intermediates that enable the selective mono-, partial, or perfunctionalization of the secondary face of the macrocycles. Although silylation of the primary rim is readily achieved by treatment with tert-butyldimethylsilyl chloride in the presence of pyridine (either alone or mixed with a co-solvent), the reaction typically results in a mixture containing both under- and oversilylated byproducts that are difficult to remove. To address this challenge in preparing a pure product in high yield, we describe an approach that centers on the addition of a controlled excess of silylating agent to avoid the presence of undersilylated species, followed by the removal of oversilylated species by column chromatography elution with carefully designed solvent mixtures. This methodology works well for 6-, 7-, and 8-member rings (α-, β-, and γ-cyclodextrins, respectively) and has enabled us to repeatedly prepare up to ⁓35 g of ≥98% pure product (as determined by HPLC) in 3 d. We also provide procedures for lower-scale reactions, as well as an example of how the β-cyclodextrin derivative can be used for functionalization of the secondary face of the molecule.

Cyclodextrins (CDs) are cyclic oligomers broadly used in food manufacturing as food additives for different purposes, e.g., to improve sensorial qualities, shelf life, and sequestration of components. In this review, the latest advancements of their applications along with the characteristics of the uses of the different CDs (α, β, γ and their derivatives) were reviewed. Their beneficial effects can be achieved by mixing small amounts of CDs with the target material to be stabilized. Essentially, they have the capacity to form stable inclusion complexes with sensitive lipophilic nutrients and constituents of flavor and taste. Their toxicity has been also studied, showing that CDs are innocuous in oral administration. A review of the current legislation was also carried out, showing a general trend towards a wider acceptance of CDs as food additives. Suitable and cost-effective procedures for the manufacture of CDs have progressed, and nowadays it is possible to obtain realistic prices and used them in foods. Therefore, CDs have a promising future due to consumer demand for healthy and functional products.

A newly cloned 4-α-glucanotransferase (αGT) from Deinococcus geothermalis and two typical bacterial αGTs from Thermus scotoductus and Escherichia coli (MalQ) were investigated. Among 4 types of catalysis, the cyclization activity of αGTs that produces cycloamylose (CA), a valuable carbohydrate making inclusion complexes, was intensively studied. The new αGT, DgαGT, showed close protein sequence to the αGT from T. scotoductus (TsαGT). MalQ was clearly separated from the other two αGTs in the phylogenetic and the conserved regions analyses. The reaction velocities of disproportionation, cyclization, coupling, and hydrolysis of three αGTs were determined. Intriguingly, MalQ exhibited more than 100-fold lower cyclization activity than the others. To lesser extent, the disproportionation activity of MalQ was relatively low. DgαGT and TsαGT showed similar kinetics results, but TsαGT had nearly 10-fold lower hydrolysis activity than DgαGT. Due to the very low cyclizing activity of MalQ, DgαGT and TsαGT were selected for further analyses. When amylose was treated with DgαGT or TsαGT, CA with a broad DP range was generated immediately. The DP distribution of CA had a bimodal shape (DP 7 and 27 as peaks) for the both enzymes, but larger DPs of CA quickly decreased in the DgαGT. Cyclomaltopentaose, a rare cyclic sugar, was produced at early reaction stage and accumulated as the reactions went on in the both enzymes, but the increase was more profound in the TsαGT. Taken together, we clearly demonstrated the catalytic differences between αGT groups from thermophilic and pathogenic bacteria that and showed that αGTs play different roles depending on their lifestyle.

In the current work we report on our participation in the SAMPL7 challenge calculating absolute free energies of the host-guest systems, where 2 guest molecules were probed against 9 hosts-cyclodextrin and its derivatives. Our submission was based on the non-equilibrium free energy calculation protocol utilizing an averaged consensus result from two force fields (GAFF and CGenFF). The submitted prediction achieved accuracy of [Formula: see text] in terms of the unsigned error averaged over the whole dataset. Subsequently, we further report on the underlying reasons for discrepancies between our calculations and another submission to the SAMPL7 challenge which employed a similar methodology, but disparate ligand and water force fields. As a result we have uncovered a number of issues in the dihedral parameter definition of the GAFF 2 force field. In addition, we identified particular cases in the molecular topologies where different software packages had a different interpretation of the same force field. This latter observation might be of particular relevance for systematic comparisons of molecular simulation software packages. The aforementioned factors have an influence on the final free energy estimates and need to be considered when performing alchemical calculations.

We investigate the binding of native β-cyclodextrin (β-CD) and eight novel β-CD derivatives with two different guest compounds, using isothermal calorimetry and 2D NOESY NMR. In all cases, the stoichiometry is 1:1 and binding is exothermic. Overall, modifications at the 3' position of β-CD, which is at the secondary face, weaken binding by several kJ/mol relative to native β-CD, while modifications at the 6' position (primary face) maintain or somewhat reduce the binding affinity. The variations in binding enthalpy are larger than the variations in binding free energy, so entropy-enthalpy compensation is observed. Characterization of the bound conformations with NOESY NMR shows that the polar groups of the guests may be situated at either face, depending on the host molecule, and, in some cases, both orientations are populated. The present results were used in the SAMPL7 blinded prediction challenge whose results are detailed in the same special issue of JCAMD.

Membrane protein pores have demonstrated applications in nanopore technology. Previous studies have mostly focused on β-barrel protein pores, whereas α-helix-based transmembrane protein pores are rarely explored in nanopore applications. Here, we developed a synthetic transmembrane peptide pore built entirely from short synthetic α-helical peptides. We examined the formation of a stable uniform ion-selective pore in single-channel electrical recordings. Furthermore, we show that cyclodextrins (CDs) block the peptide pores and determine the kinetics of CD binding and translocation. We suggest that such designed synthetic transmembrane pores will be useful for several applications in biotechnology, including stochastic sensing.

Background Venous neointimal hyperplasia and venous stenosis (VS) formation can result in a decrease in arteriovenous fistula (AVF) patency in patients with end-stage renal disease. There are limited therapies that prevent VNH/VS. Systemic delivery of simvastatin has been shown to reduce VNH/VS but local delivery may help decrease the side effects associated with statin use. We determined if microparticles (MP) composed of cyclodextrins loaded with simvastatin (MP-SV) could reduce VS/VNH using a murine arteriovenous fistula model with chronic kidney disease. Methods and Results Male C57BL/6J mice underwent nephrectomy to induce chronic kidney disease. Four weeks later, an arteriovenous fistula was placed and animals were randomized to 3 groups: 20 μL of PBS or 20 μL of PBS with 16.6 mg/mL of either MP or MP-SV. Animals were euthanized 3 days later and the outflow veins were harvested for quantitative reverse transcriptase-polymerase chain reaction analysis and 28 days later for immunohistochemistical staining with morphometric analysis. Doppler ultrasound was performed weekly. Gene expression of vascular endothelial growth factor-A ( Vegf-A ), matrix metalloproteinase-9 ( Mmp-9 ), transforming growth factor beta 1 ( Tgf-β1 ), and monocyte chemoattractant protein-1 ( Mcp-1 ) were significantly decreased in MP-SV treated vessels compared with controls. There was a significant decrease in the neointimal area, cell proliferation, inflammation, and fibrosis, with an increase in apoptosis and peak velocity in MP-SV treated outflow veins. MP-SV treated fibroblasts when exposed to hypoxic injury had decreased gene expression of Vegf-A and Mmp-9 . Conclusions In experimental arteriovenous fistulas, periadventitial delivery of MP-SV decreased gene expression of Vegf-A , Mmp-9 , Tgf-β1 and Mcp-1, VNH/VS, inflammation, and fibrosis.

Cyclic α-maltosyl-(1→6)-maltose (CMM) is a cyclic glucotetrasaccharide with alternating α-1,4 and α-1,6 linkages. Here, we report functional and structural analyses on CMM-binding protein (CMMBP), which is a substrate-binding protein (SBP) of an ABC importer system of the bacteria Arthrobacter globiformis. Isothermal titration calorimetry analysis revealed that CMMBP specifically bound to CMM with a Kd value of 9.6 nM. The crystal structure of CMMBP was determined at a resolution of 1.47 Å, and a panose molecule was bound in a cleft between two domains. To delineate its structural features, the crystal structure of CMMBP was compared with other SBPs specific for carbohydrates, such as cyclic α-nigerosyl-(1→6)-nigerose and cyclodextrins. These results indicate that A. globiformis has a unique metabolic pathway specialized for CMM., Competing Interests: The authors of this manuscript have the following competing interests: Three authors (MK, TM,TN) are paid employees of Hayashibara Co, Ltd. This commercial affiliation does not alter our adherence to PLOS ONE policies on sharing data and materials. There are no patents, products in development or marketed products associated with this research to declare.

The plasma membrane of eukaryotic cells is asymmetric with respect to its phospholipid composition. Analysis of the lipid composition of the outer leaflet is important for understanding cell membrane biology in health and disease. Here, a method based on cyclodextrin-mediated lipid exchange to characterize the phospholipids in the outer leaflet of red blood cells (RBCs) is reported. Methyl-α-cyclodextrin, loaded with exogenous lipids, was used to extract phospholipids from the membrane outer leaflet, while delivering lipids to the cell to maintain cell membrane integrity. Thin layer chromatography and lipidomics demonstrated that the extracted lipids were from the membrane outer leaflet. Phosphatidylcholines (PC) and sphingomyelins (SM) were the most abundant phospholipids in the RBCs outer leaflet with PC 34:1 and SM 34:1 being the most abundant species. Fluorescence quenching confirmed the delivery of exogenous lipids to the cell outer leaflet. The developed lipid exchange method was then used to remove phosphatidylserine, a phagocyte recognition marker, from the outer leaflet of senescent RBCs. Senescent RBCs with reconstituted membranes were phagocytosed in significantly lower amounts compared to control cells, demonstrating the efficiency of the lipid exchange process and its application in modifying cell-cell interactions.

As the intertissue delivery of hydrophobic temoporfin (mTHPC) remains inefficient, we propose the use of cyclodextrin-based nanosponges as a smart, advanced system for improved mTHPC delivery. Recently, we demonstrated that cyclodextrins (CDs) allow mTHPC to penetrate into tumor spheroids via a nanoshuttle mechanism. However, the CD complexes were very sensitive to the dilution, thus limiting their translation invivo. Hypercrosslinked CD monomers in a three-dimensional network (namely, CD nanosponges), however, may form both inclusion and non-inclusion complexes with drug molecules, providing controlled release and prolonged exposure to the drug. In the present work, we demonstrate that epichlorohydrin-crosslinked CD nanosponges based on β-CD (βCDp) and carboxymethyl-β-CD (CMβCDp) monomers efficiently encapsulated mTHPC. We calculated the apparent binding constants between mTHPC and CD polymers (K=(6.3-8.8) × 10 6 M -1 and K=(1.2-1.7) × 10 6 M -1 for βCDp and CMβCDp, respectively) using fluorescence titration curve fitting. The encapsulation of mTHPC in a CD polymer matrix had slower photosensitizer (PS) release compared to monomer CD units, providing deep penetration of mTHPC in 3D tumor spheroids in a concentration-dependent manner. However, the improvement of mTHPC penetration in 3D human pharynx squamous cell carcinoma (FaDu) spheroids using CD polymers was strongly accompanied by the inhibition of PS cellular uptake, demonstrating the delicate balance between the accumulation and the penetration of PS in FaDu spheroids. In summary, mTHPC-loaded CD nanosponges are a strong candidate for further invivo study in preclinical models, which could be considered as an advanced smart system for mTHPC delivery., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Enzyme-mediated dynamic combinatorial chemistry combines the concept of thermodynamically controlled covalent self-assembly with the inherent biological relevance of enzymatic transformations. A system of interconverting cyclodextrins has been explored, in which the glycosidic linkage is rendered dynamic by the action of cyclodextrin glucanotransferase (CGTase). External factors, such as pH, temperature, solvent, and salinity are reported to modulate the composition of the dynamic cyclodextrin library. Dynamic libraries of cyclodextrins (CDs) could be obtained in wide ranges of pH (5.0-9.0), temperature (5-37 °C), and salinity (up to 7.5 m NaNO 3 ), and with high organic solvent content (50 % by volume of ethanol), showing that enzyme-mediated dynamic systems can be robust and not limited to physiological conditions. Furthermore, it is demonstrated how strategic choice of reaction conditions can enhance template effects, in this case, to achieve highly selective production of α-CD, an otherwise challenging target due to competition from the structurally similar β-CD., (© 2020 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

This work investigated the interaction between cyclodextrins and pullulanase to provide insight into the production and application of cyclodextrins. Enzyme activity and kinetic assays showed that α-cyclodextrin (α-CD), β-cyclodextrin (β-CD) and γ-cyclodextrin (γ-CD) inhibited pullulanase in a competitive manner. Circular dichroism spectra and fluorescence spectroscopy suggested the formation of cyclodextrin and pullulanase complexes. According to ITC assays and molecular docking results, compared with α-CD and γ-CD, β-CD had the strongest affinity for pullulanase because of its appropriate cavity geometric dimensions. In addition, cyclodextrins interacted with pullulanase through hydrogen bonds, van der Waals force and hydrophobic interactions, the latter of which were verified as the major driving force. Phenylalanine 476 was the key amino acid residue in pullulanase for cyclodextrin recognition and binding., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Cyclodextrin-based nanosponges (CD-NS) are considered as safe and biocompatible systems for removing toxic molecules from the body. Rapid removal of toxic molecules that are formed in the body from certain food constituents, is relevant especially for patients affected by chronic kidney disease. Within the scope of this study, innovative cyclodextrin polymers were synthesized to form nanosponges able to remove indole, before it could form the toxic indoxyl sulfate in the body. Furthermore, in vivo studies were carried out using the two optimal CD-NS formulations by assessing physicochemical properties, stability, indole adsorption capacity and in vitro cytotoxicity. NS prepared from β-cyclodextrin cross-linked with toluene diisocyanate was found to be the most effective NS with an in vitro indole adsorption capacity of over 90%. In addition, this derivative was more stable in gastrointestinal media. Animal studies further revealed that oral CD-NSs did not tend to accumulate and damage gastrointestinal tissues and are excreted from the GI tract with minimal absorption. In conclusion, this study suggests that CD-NS formulations are effective and safe in removing toxic molecules from the body. Their potential use in veterinary or human medicine could reduce dialysis frequency and avoid hepatic and cardiac toxicity avoiding the indole formation., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Complex formation between cyclodextrins and ionizable guest molecules depends on pH. In general, the neutral species of an ionizable guest molecule has the highest affinity for the cyclodextrin cavity, but ionized species will also be able to form complexes with cyclodextrins. This work presents a theoretical expression for the relationship between the stability constant and pH for interaction between neutral cyclodextrins and ionizable guest molecules with multiple pK a values. Input parameters for the theoretical expression are pK a values of the guest molecule and stability constants for the complex at specific pH values. The pH profile of the stability constant for a complex depends on the acid-base properties of the guest and the closeness of the pK a values, and examples of pH profiles for polyprotic acids, bases and amphoteric guests are shown. Empirical data sets from the literature were used to confirm the accuracy of the theoretical expression, and Monte Carlo simulations were used to validate that the theoretical expression yield a good fit to empirical data. Lastly, an experimental protocol was suggested, and a freely available graphical user interface was presented to facilitate easy use of the theoretical expression., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The graphene road in nanomedicine still seems very long and winding because the current knowledge about graphene/cell interactions and the safety issues are not yet sufficiently clarified. Specifically, the impact of graphene exposure on gene expression is a largely unexplored concern. Herein, we investigated the intracellular fate of graphene (G) decorated with cyclodextrins (CD) and loaded with doxorubicin (DOX) and the modulation of genes involved in cancer-associated canonical pathways. Intracellular fate of GCD@DOX, tracked by FLIM, Raman mapping and fluorescence microscopy, evidenced the efficient cellular uptake of GCD@DOX and the presence of DOX in the nucleus, without graphene carrier. The NanoString nCounter™ platform provided evidence for 34 (out of 700) differentially expressed cancer-related genes in HEp-2 cells treated with GCD@DOX (25 µg/mL) compared with untreated cells. Cells treated with GCD alone (25 µg/mL) showed modification for 16 genes. Overall, 14 common genes were differentially expressed in both GCD and GCD@DOX treated cells and 4 of these genes with an opposite trend. The modification of cancer related genes also at sub-cytotoxic G concentration should be taken in consideration for the rational design of safe and effective G-based drug/gene delivery systems. The reliable advantages provided by NanoString ® technology, such as sensibility and the direct RNA measurements, could be the cornerstone in this field.

Rutin is one of the most important flavonoids with poor bioavailability. This work aimed at addressing the issue of poor biopharmaceutical performance of rutin by applying a combination of complexation with secondary processing into tablets. Mechanical activation was the most suitable method of rutin complex formation with (2-hydroxypropyl)-β-cyclodextrin (HP-β-CD), while the β-cyclodextrin (β-CD) complex successfully formed by kneading with an ethanol/water mixture. Complexation was confirmed by thermal analysis, powder X-ray diffraction and vibrational spectroscopy. Dynamic vapour sorption showed that stability of powders at high humidity conditions was satisfactory, however, the β-CD complex retained around 8% of moisture. The complexes were compacted with or without tricalcium phosphate (TRI-CAFOS) filler at a range of compression pressures (19-113 MPa). The best tabletability was determined for rutin/HP-β-CD, compressibility for the TRI-CAFOS blends with complexes and compactibility for the rutin/HP-β-CD + TRI-CAFOS mix. Dissolution studies showed quicker and more complete dissolution (pH 1.2) of rutin/HP-β-CD tablets, however the compacts comprising the filler were superior than pure complexes. The tablets manufactured in this study appear to be promising delivery systems of rutin and it is recommended to combine rutin/HP-β-CD with TRI-CAFOS and compact at 38-76 MPa., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Understanding lipid oxidation mechanisms in low moisture foods is necessary to develop antioxidant strategies to increase shelf life and/or to improve nutritional quality by increasing polyunsaturated fatty acid concentrations. In this study, we examined the influence of water activity (a w ), sugars (glucose, maltose, maltodextrin, and cyclodextrin), and proteins (casein and gluten) on the lipid hydroperoxide and hexanal lag phases of model crackers. Oxidative stability of crackers was in an order: a w 0.7 > a w 0.4 > a w 0.2 > a w 0.05. Higher water activities resulted in bigger differences between hydroperoxide lag phases and hexanal lag phases. Compared to non-reducing cyclodextrin and no added sugar controls, reducing sugars including glucose, maltose, and maltodextrin at the same dextrose equivalence increased both hydroperoxide and hexanal lag phases. At the same dextrose equivalence, oxidative stability was in the order of maltose > maltodextrin > glucose > control (no sugar added). The antioxidant effectiveness of maltose, a low sweetness profile sugar, increased with increasing concentrations from 1.1 to 13.8%. Increasing a w increased the antioxidant activity of maltose. For example, 1.1% maltose increased both hydroperoxides and hexanal lag phases by 9 days at an a w of 0.2, but increased hydroperoxide lag phase by 24 days and hexanal lag phase by 15 days at an a w of 0.7. Gluten was able to inhibit lipid oxidation with activity increasing with increasing a w while casein showed minimal antioxidant impact. Antioxidant activity of gluten decreased when its sulfhydryl groups were blocked by N-ethylmaleimide suggesting that cysteine was an important antioxidant component of gluten. Adjusting water activity and addition of reducing sugars and gluten could be strategies to increase oxidative stability of low moisture crackers., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Sesquiterpenoids are one of the most diverse groups in natural compounds with various chemical structures and bioactivities. In our previous work, we developed the chemoenzymatic oxygenation method based on the combination of Fe(II)-EDTA and ferric-chelate reductase that could synthesize (-)-rotundone, a key aroma sesquiterpenoid of black pepper. Fe(II)-EDTA catalyzed the oxygenation of sesquiterpene to sesquiterpenoid, and ferric-chelate reductase catalyzed the supply and regeneration of Fe(II)-EDTA in this system. We then investigated the effect of various Fe 2+ -chelates on the catalytic oxygenation of sesquiterpene and applied this system to the synthesis of odor sesquiterpenoids. We determined Fe(II)-NTA to be an efficient oxygenation catalyst by the screening approach focusing on ligand structures and coordination atoms of Fe 2+ -chelates. Valuable odor sesquiterpenoids such as (+)-nootkatone, (-)-isolongifolenone, and (-)-β-caryophyllene oxide were oxygenatively synthesized from each precursor sesquiterpene by 66%, 82%, and 67% of the molar conversion rate, respectively. Abbreviations: EDTA: ethylenediaminetetraacetate; NTA: nitrilotriacetate; DTPA: diethylenetriaminepentaacetate; phen: o -phenanthroline; cyclam: 1,4,8,11-tetraazacyclotetradecane; TPA: tris(2-pyridylmethyl)amine; GlcDH: glucose dehydrogenase; HP-β-CD: hydroxypropyl-β-cyclodextrin.