Self-assembly in situ, where synthetic molecules are programmed to organize in a specific and complex environment i.e., within living cells, can be a unique strategy to influence cellular functions. Here we present a small series of rationally designed oligothiophene analogues that specifically target, locate and dynamically self-report their supramolecular behavior within the confinement of a cell. Through the recognition of the terminal alkyl substituent and the amphiphilic pyridine motif, we show that the cell provides different complementary pathways for self-assembly that can be traced easily with fluorescence microscopy as their molecular organization emits in distinct fluorescent bands. Importantly, the control and induction of both forms are achieved by time, temperature and the use of the intracellular transport inhibitor, bafilomycin A1. We showcase the importance of both intrinsic (cell) and extrinsic (stimulus) factors for self-organization and the potential of such a platform toward developing synthetic functional components within living cells. Self-assembly of synthetic molecules in living cells can influence cell function, but is extremely challenging due to the complex environment of cells. Here the authors report the self-assembly of small organic molecules that locate, target and self-report their supramolecular behavior in living cells. Displayed coherently in the body, the dynamics of nanostructured biocomponents  drives cellular processes that are regulated in a highly specific way to ensure survival and proliferation. Although much efforts have been directed to understanding the influence of small molecules on cellular functions, the impact of materials at the nanometer scale assembled by seemingly individual synthetic molecules inside living cells have only very recently received increasing attention3 or the transient formation of microtubules that dictates essential intracellular transport4. As such, there is a need to look beyond molecular functions when it is necessary to change or modify a biological condition, especially since biological superstructures are always dynamically involved in most pathways. In this aspect, a new perspective toward developing highly specific therapeutics or to engineer artificial cells5 can be drawn if there is a possibility to control processes by integrating synthetic nanostructures within the cellular body7. Nonetheless, nanostructure assemblies of synthetic molecules within a living cell are extremely challenging to achieve due to the presence of a plethora of biological components in the environment. To circumvent this from a molecular design perspective, conjugated π-systems offer a very convenient scaffold as its orthogonality toward an environment predominated by hydrogen bonds provides a natural impetus for self-organization. Conventionally, π-conjugated heteroaromatic compounds have been extensively studied on superstructure formation in thin films especially for charge transport applications  and these devices are well understood. However, considering their self-assembly in aqueous biological medium, these molecules are rarely investigated, due to solubility, even though they possess attractive multifold functions .In fact, whether it is a malignancy or a normal cellular process, it is crucial to appreciate that many of these systems exert their biological functions at the superstructure level, such as the assembly of peptide fragments into amyloid plaques that devastate neurological functions10. Naturally, this conformation dependent emission profile becomes an exceptional tool especially in real-time monitoring of self-assembly processes within a complex environment11. This feature is even more appreciable because other well-known assembly systems in biology i.e., peptides, lipids, DNA, or proteins are incapable of providing an optical readout beyond the autofluorescence of the cell in the native state. In this aspect, Barbarella’s group has shown in a seminal study the co-assembly of cell-permeable dithienothiophene-S,S-dioxide with type-I collagen to form a series of conducting microfibers within HeLa cells12. The complementary properties of the eventual hybrid material are astounding considering that nature plays an unexpected but active role in its formation. This leads toward the notion that the integration of biological processes into the construction of intracellular assemblies can be evolved as an elegant strategy14. In developing such strategies into applications, Rao’s group has taken advantage of the enrichment effect of intracellular nano-assemblies to amplify both the diagnostic signal and therapeutic profile within cancer cells15. On a similar note, Maruyama’s group recently investigated that cancer cells can be killed purely by mechanical stress imposed by the programmed self-assembly of a peptide gelator within the cell16. Based on these studies, a strong potential toward developing an intracellular self-assembly platform as a forthcoming strategy for altering biological functions can be foreseen.Among different conjugated π-systems, substituted oligothiophenes have been shown in preliminary studies to be able to bind, in a structure dependent fashion, to specific peptides and proteins often accompanied by an associated fluorescence changeN-heteroaromatic head groups of varying polarities to show that intracellular self-assembly and self-reporting systems can be rationally designed and monitored in a facile manner  with pyridine (1P3T) and subsequent N-methylation (Me1P3T), we would sequentially assess the biological impact of (1) increasing polar interactions and electron acceptor character of the head group (Ar) as well as their impact on the terthiophene π-system, (2) how small structural changes to the oligothiophene scaffold can notably direct intracellular self-assembly. On the other hand, the carbosilane motif that incorporates long, branched alkyl chains was designed to further enhance self-organization behavior through van der Waals interactions19, as well as to support important lipid-mediated intracellular translocation20. In particular, this substituent promotes binding toward human serum albumin (HSA), an ubiquitous protein in the bloodstream that has shown broad binding capabilities toward lipophilic molecules21, allowing their solubilization in water, as well as transport to cells. The result, therefore, is a biocompatible hybrid complex that facilitates membrane interactions and transport, with both amphiphilic quaterthiophene analogues individually displaying highly exclusive fluorescent features that provide real-time reporting of self-assembly processes inside living cells.Herein, we present carbosilane substituted terthiophenes, which are functionalized by ner Fig. . The oli22, the reaction conditions have been substantially modified and improved. Having accomplished the substituent R, the monosubstituted thiophene 1 was lithiated in the remaining α-position, followed by stannylation or borylation. Resulting 2 or 3 were coupled with 5-bromo-2,2′-bithiophene affording the corresponding terthiophene 4. The Suzuki pathway was performed under milder reaction conditions with a more efficient palladium catalyst system compared to our previous protocol22 via Stille route leading to an improvement in yield from 65 to 87%. A further advantage of the Suzuki cross-coupling reaction is particularly the non-toxic nature of the boronic ester precursors. In order to introduce the last coupling step, the monosubstituted terthiophene 4 was again borylated  was exclusively carried out by a Suzuki-type cross-coupling reaction of monosubstituted terthienylboronic pinacol ester 5 with 4-bromopyridine hydrochloride. The Stille pathway was not pursued, because the prior mentioned coupling to 4 worked much better with the boron-containing species. Additionally, the required basic reaction conditions for Suzuki coupling reactions were utilized to neutralize, in situ, the building block from its hydrochloride salt into the free base of 4-bromopyridine. In the last step of the synthetic route, the basic functionality of 1P3T was modified via N-methylation reaction using methyl trifluoromethanesulfonate to afford 7 (Me1P3T). These two analogues and the quaterthiophene 4T form the basis of scaffold design by which the biological workflow is built upon , a co-solvent system with THF in water or buffer is necessary to prevent precipitation. The maximum amount of water content tolerated by 4T and 1P3T (20 µM) without precipitation is 80% v/v H2O/THF and 95% v/v H2O/THF for Me1P3T. Comparing the absorbance spectra, both the quaterthiophene 4T and the pyridine-functionalized terthiophene 1P3T exhibit a maximum at 400 nm while the quaternization of the pyridine by N-methylation resulted in a bathochromic shift of the absorbance (λmax = 439 nm) , 4T exhibits a maximum at 552 nm and a fine structure that is characteristic of oligothiophenes having multiple vibrational modes due to different molecular orientation and/or assembly  and the large energy difference between the emission peaks suggest the formation of supramolecular structures in solution.As these oligothiophenes alone are insoluble in polar solvents  to ascertain the perceived supramolecular behavior (Supplementary Methods). In THF/H nm Fig. . As a co nm Fig.  while th 2O Fig.  can be a2O where aggregates of 1P3T were formed, Me1P3T remains, however, strictly in a molecular state (< 0.5 nm) . On the contrary, in pure THF, Me1P3T showed an emission at 636 nm  Fig. . The cor nm Fig. , which s nm Fig. . To demo23. HSA has a highly dynamic structure and has been known to temporarily stabilize and shuttle molecules i.e., drugs24, fatty acids25, and even carbon nanotubes26 in water. The synthesis of the protein complex can be achieved by mixing a stock solution (20% THF/H2O) of the oligothiophenes into a buffered solution of human serum albumin (HSA) for 16 h. THF is removed completely by freeze drying and the mixture is purified by size exclusion chromatography. The oligothiophene-HSA complexes are well soluble in water and were subsequently characterized by fluorescence spectroscopy and atomic force microscopy to understand the spectral changes upon interaction with the protein.In order to apply these oligothiophene amphiphiles in cellular studies, they need to be processible under entirely aqueous conditions. In cells, very lipophilic or amphiphilic molecules are often transported by proteins. Inspired by such natural transport opportunities, we have selected alkyl chains in our design as they are very well known to interact and bind to serum albumin Fig. , a prote27. As such, oligothiophenes can exist within the protein as a single molecule or as a small group of aggregated molecules. As the extinction of the molecules cannot be accurately determined when the oligothiophenes are complexed to the protein, a very rough estimate of ratio between protein and oligothiophene  can be seen. A higher protein to oligothiophene ratio is plausible due to the natural presence of HSA multimers in solution28. Height topographic images of the oligothiophene-HSA complexes were analyzed in fluid operation AFM demonstrating a homogenous distribution of particles of ~2 nm  that report the molecular and aggregated forms of the amphiphilic quaterthiophene analogues.λem = 577 nm). In comparison, the spectra of the oligothiophene analogues in THF resembles that in phosphatidylcholine, suggesting that the membrane-type microenvironments provide a similar environment to that by THF. Taken together, the use of THF as a co-solvent to study the respective aggregation behavior remains largely relevant. Nonetheless, the main differences comparing the two sets of spectra include the appearance of vibronic fine structures of 1P3T with phosphatidylcholine and a strong hypsochromic shift (~60 nm) for Me1P3T. The shift of the emission of Me1P3T toward higher energy is a consequence of the electron withdrawing capability of the pyridinium cation being partially compensated by the negative charge on phosphatidylcholine. The affinity of Me1P3T toward the cellular membrane is not unique and is supported by the host of cationic surfactants in the literature30. Hence, in terms of toxicity (on both cellular and organism level), the mechanism by which the integrity of the cellular membrane is compromised by the hydrophobic cation is very well known32. In contrast, the treatment of 4T and 1P3T does not seem to influence the cell morphology  were observed within A549 cells after 24 h treatment Fig. . Systemaion Fig. . The simion Fig. .Fig. 6In34. This concept was commonly known to be exploited in the albumin-paclitaxel formulation (Abraxane) for anti-cancer therapy35. On the other hand, intracellular transport of HSA only happens via cell signaling or in selective cell types 36. Therefore, the significantly reduced toxicity of Me1P3T-HSA is due to HSA preventing larger aggregates of the oligothiophenes and regulating the affinity of Me1P3T toward the cellular membrane. In this way, there is no sudden onset of oligothiophenes  bound onto the cellular surface to cause the membrane collapse and consequently cell death.Based on these results, we investigate in detail the intracellular processes to elucidate the role of HSA using pathway inhibitors and subcellular stains. To probe the transport function of HSA, the protein was labeled with ATTO 647 that can be excited independently from the oligothiophenes. It was clearly found that the HSA protein does not internalize into the cells at the tested concentrations  or mitochondria (Me1P3T) was observed while red fluorescence of aggregates were clearly absent Fig. . At 24 hThese previous studies give an insight into the relationship between small variations in chemical functionalities and targeted intracellular trafficking. This knowledge is fundamental for the design of smart materials within the cell where we showed that these intracellular supramolecular assemblies can be manipulated. As most supramolecular systems are largely influenced also by temperature changes, we utilize a combination of methods to specifically initiate self-assembly within targeted intracellular compartments. From the aforementioned experiments, we established that Me1P3T transports first to the mitochondria before trafficking across to the perinuclear region. By locking the translocation pathway using bafilomycin A1, the mitochondria localized Me1P3T was subjected to 4 °C treatment for 2 h Fig. . CompariThe synthesis of oligothiophene cellular reporters has been achieved by performing critical and concise chemical modifications toward the conjugated π-electron system. The resulting oligothiophene derivatives were able to respond, target and self-assemble specifically within mammalian cells. Integration of the three investigated oligothiophenes  into HSA was essential to provide full water solubility at high concentrations up to 100 µM) of these otherwise insoluble molecules, resulting in the formation of non-cytotoxic complexes. Their fluorescent properties directly represent, to various degrees, nanostructural changes in solution allowing their dynamic behavior within intracellular environments to be evaluated. By interpreting these fluorescence changes, HSA is shown to be integral in mediating the cell/molecule interface where it delivers the oligothiophenes as molecules, which are subsequently recognized by the cells and sorted into different downstream pathways . This unique feature facilitates the capability to initiate self-assembly and localize aggregates, which would not be possible under standard cell culture conditions. Within these studies, we highlight the importance of structural design toward assembly in living cells where very stringent  and dynamic intracellular processes  are involved.Collectively, we have presented the ability to trigger supramolecular assemblies within a complex cellular system, which provides a unique perspective toward constructing dynamic nanostructures to possibly reprogram cellular functions on a supramolecular level. Development in this area would potentially create a new platform for therapeutics where higher ordered architectures within the cells are targeted instead of a specific protein/enzyme. Furthermore, the ability to create an artificial intracellular architecture remains a powerful capability of synthetic supramolecular chemistry that may very well represent an entirely new perspective of biomedical science.1H NMR and 13C NMR spectra were recorded at room temperature on a Bruker AMX400. High temperature NMR spectroscopy was conducted on a Bruker AMX500. All NMR measurements were done in CDCl3, C2D2Cl4, or CD3OD/CDCl3 mixture, respectively, with the solvent residual peak as an internal reference . Mass spectrometry (MALDI-TOF) was performed on Bruker Reflex III. The elemental composition was determined with an Elementar Vario EL system. The synthesis of the precursors can be found in Supplementary Information.19.4T was prepared according to the literature2O (6 mL) was added. The solution was degassed for 1 h. Then 4-bromopyridine hydrochloride , Pd2(dba)3.CHCl3 , HP(t-Bu)3BF4  and K2CO3  were added and the reaction mixture was stirred at 81 °C for 19 h. The organic phase was separated and the aqueous phase was extracted with chloroform. The combined organic phases were dried over MgSO4 and the solvent was evaporated. The crude product was purified by column chromatography  to give 6 as a yellow solid . M.p.: 72 °C. 1H-NMR , δ [p.p.m.]: 8.57  = 4.6, 1.6 Hz, 2H, 2-HP, 6-HP), 7.43  = 4.6, 1.6 Hz, 2H, 3-HP, 5-HP), 7.40  = 3.9 Hz, 1H, 4″-HT), 7.14  = 3.8 Hz, 1H, 3″-HT), 7.10  = 3.8 Hz, 1H, 4′-HT), 7.00  = 3.9 Hz, 1H, 3′-HT), 6.99  = 3.6 Hz, 1H, 3-HT), 6.68  = 3.6 Hz, 1H, 4-HT), 2.78  = 7.3 Hz, 2H, α-CH2), 1.68–1.61 , 1.29-1.23 , 0.85  = 6.8 Hz, 9H, CH3), 0.58-0.54 , 0.49–0.45 . 13C-NMR , δ [p.p.m.]: 150.3, 145.9, 141.0, 139.2, 137.9, 134.6, 134.2, 126.2, 125.1, 125.0, 124.4, 123.7, 123.6, 119.4, 34.2, 33.9, 31.9, 29.70, 29.65, 29.39, 29.34, 26.4, 23.9, 22.7, 14.1, 12.4, 12.2. MS (MALDI-TOF), m/z: 817.5 [M]+. Elemental analysis: Calcd. for C50H79NS3Si (818.45): C 73.38, H 9.73, N 1.71; found: C 73.35, H 9.79, N 1.66.Terthienylboronic pinacol ester 5  was dissolved in THF (20 mL) and H2/dichloromethane: methanol = 5: 1) to give 7 as a red solid . M.p.: 96 °C. 1H-NMR , δ [p.p.m.]: 8.65  = 6.1 Hz, 2H, 2-HP, 6-HP), 8.12  = 6.2 Hz, 2H, 3-HP, 5-HP), 8.02  = 4.0 Hz, 1H, 4″-HT), 7.38  = 4.0 Hz, 1H, 3″-HT), 7.35  = 3.9 Hz, 1H, 4′-HT), 7.11  = 3.8 Hz, 1H, 3′-HT), 7.09  = 3.5 Hz, 1H, 3-HT), 6.73  = 3.5 Hz, 1H, 4-HT), 4.26 , 2.82  = 7.0 Hz, 2H, α-CH2), 1.71–1.63 , 1.32-1.24 , 0.85  = 6.9 Hz, 9H, CH3), 0.61–0.57 , 0.52–0.48 . 1H-NMR , δ [p.p.m.]: 8.62 , 7.96 , 7.84 , 7.33 , 7.12 , 6.78 , 4.40 , 2.88  = 7.3 Hz, 2H, α-CH2), 1.81–1.74 , 1.42–1.34 , 0.95  = 6.8 Hz, 9H, CH3), 0.71–0.68 , 0.61–0.58 . MS (MALDI-TOF), m/z: 832.5 [M]+. Elemental analysis: Calcd. for C52H82F3NO3S4Si (982.55): C 63.57, H 8.41, N 1.43; found: C 63.31, H 8.52, N 1.54.To a solution of 6  in dry dichloromethane (10 mL) methyl trifluoromethanesulfonate  was added at 0 °C. After complete addition the reaction mixture was stirred at the same temperature for 0.5 h. Then the mixture was allowed to warm to room temperature and was stirred for 3 h. The crude product was purified by column chromatography (SiO2. CellTiter-Glo Cell Viability Assay was purchased from Promega and used according to the given protocol. Luminescence intensities are measured from Glomax Multi 96-well plate reader (Promega). Confocal laser scanning microscopy was performed using Zeiss LSM 710, Observer.Z1 and processed using Zen Blue/Black software. Separate studies were also independently performed using Leica TCS SP5.Cell culture of A549 cells was performed in Dulbecco’s Modified Eagle’s Medium  supplemented with 10% FBS, 1% penicillin/streptomycin and 1× MEM non-essential amino acid with incubation conditions set at 37 °C, 5% COOligothiophenes  were pre-dissolved in THF at a concentration of 1 mg/mL. Human serum albumin  was dissolved in 1.2 mL of phosphate buffer  and added to the respective oligothiophenes (52.5 nmol) in 300 µL THF. The resulting mixture was put onto an orbital shaker for 16 h at room temperature and subsequently freeze dried to remove all solvents.2O and the solution was filtered through 0.2 µm syringe filter. The solution was purified using Sephadex G25M  size exclusion chromatography using MilliQ water as the mobile phase. The desalted solution was subsequently freeze dried to afford the oligothiophene-albumin complex  in ≈80% yield.The crude solid was re-dissolved in 1 mL HAnalysis was accomplished via Superdex 200 FPLC  using phosphate buffered saline .N-hydroxysuccinimide ester  predissolved in anhydrous DMF. The reaction was stirred at room temperature for 16 h and purified using Sephadex G25M  size exclusion chromatography using MilliQ water as the mobile phase. The desalted solution was subsequently freeze dried to obtain ATTO 647-HSA in 92% yield.Human serum albumin  was dissolved in 2 mL phosphate buffer  and added ATTO 647 Atomic force microscopy was measured with a Bruker Dimension FastScan Bio AFM equipped with the ScanAsyst mode. The respective 1P3T-HSA and Me1P3T-HSA solution  was deposited onto freshly cleaved mica surface and left for 5 min at room temperature to allow the protein complexes to adsorb. After an addition of additional 20 µL of MilliQ Water into the sample, the sample was scanned at the rate between 1 and 3 Hz. Several images in different areas were taken to ensure reproducibility of the results. All images were analyzed using the NanoScope Analysis 1.50 and Gwyddion 2.38 software.2 prior to each experiment.A549/MCF-7/HeLa cells were cultured in standard T-75 flasks using high glucose DMEM fortified with 10% fetal bovine serum, 1% penicillin/streptomycin and 1% MEM non-essential amino acids. The cells were split at near confluency and incubated at 37 °C, 5% CO2. The respective oligothiophenes  and/or oligothiophene-HSA complexes  were added into each well at concentrations (15 µM) and incubated for 5 and 24 h separately at 37 °C, 5% CO2. The treated cells were washed twice with DMEM to remove non-specific adsorption and imaged using Zeiss LSM 710. For fluorescence visualization, a 405 nm excitation laser was used in conjunction with emission filters at 450–520 nm and 550–750 nm to monitor the molecular and assembled species, respectively.Pre-cultured A549 cells were seeded at a density of 15,000 cells per well in an 8-well confocal microscopy chamber (Ibidi) and left to adhere overnight at 37 °C, 5% CO2 and imaged as described above.To exclusively monitor intracellular localization, the medium containing the oligothiophene-HSA complex was exchanged with pure DMEM after 24 h incubation. The well was aspirated and pure DMEM (300 µL) was added. The cells were incubated for a further 24 h at 37 °C, 5% CO2. Bafilomycin A1 or Nocodazole (100 nM) was introduced separately into different wells and incubated for 30 min at 37 °C, 5% CO2. The complexes  were prepared at concentrations (15 µM) were subsequently co-incubated with the inhibitor for an additional 24 h at 37 °C, 5% CO2. Following which, the cells were washed twice with DMEM prior to imaging using Zeiss LSM 710 and with parameters as stated above.Pre-cultured A549 cells were seeded at a density of 15,000 cells per well in an 8-well confocal microscopy chamber (Ibidi) and left to adhere overnight at 37 °C, 5% CO2 and were washed twice with DMEM prior to imaging. The procedures for ERTracker Red and NuclearMask Deep Red were performed using manufacturer’s protocol.MitoTracker Deep Red (Molecular Probes) was dissolved in DMSO  to produce a 1 mM stock solution. The staining medium was prepared by performing a 50,000× dilution (1 µL stock into 50 mL DMEM) to a final concentration of 20 nM. The medium of the respective cells, incubated with the different oligothiophene-albumin complexes, were removed and added 300 µL of the diluted staining medium. The cells were stained for 15 min at 37 °C, 5% CO2. Bafilomycin A1 (100 nM) was introduced separately into different wells and incubated for 30 min at 37 °C, 5% CO2. Me1P3T-HSA was added into each well and co-incubated with the inhibitor for 24 h at 37 °C, 5% CO2. Subsequently, one set of the microscopy chamber was transferred into a sterile 4 °C refridgerator and incubated for a further 2 h. These cells were washed with cold DMEM and left on ice prior to imaging. The other set of cells were kept at 37 °C and was treated similar to previous sections as a control experiment. Both set of cells were imaged using Zeiss LSM 710.Pre-cultured A549 cells were seeded at a density of 15,000 cells per well in 2 separate 8-well confocal microscopy chamber (Ibidi) and left to adhere overnight at 37 °C, 5% CO2. The oligothiophenes and the respective HSA-complexes were introduced at concentrations (0.1–100 µM) and incubated for 24 h at 37 °C, 5% CO2. CellTiter-Glo (Promega) was employed following manufacturer’s protocol and the resultant luminescence read out was detected using Glomax Multi 96-well plate reader (Promega).Pre-cultured A549 cells were seeded at a density of 6500 cells per well in a white 96-well plate  and allowed to adhere overnight at 37 °C, 5% COThe data supplementing the effect of pH and hydrocarbon solvents on 1P3T are found in Supplementary Information (Supplementary Figs. Supplementary Information


The regression equations were obtained with good linearity (r2 > 0.99) and limit of quantifications was higher than 0.1 µg/mL. The precision  and accuracy (higher than 81.7% and lower than 107.6%) of the methods were adequate. Barbatusin (1) was detected in P. grandis (15.432 ± 2.28 mg/g) and P. barbatus (5.198 ± 3.45 mg/g) extracts, while compound 2 was detected in P. grandis (4.068 ± 3.34 mg/g), P. barbatus (0.654 ± 5.86 mg/g), P. amboinicus (0.160 ± 7.25 mg/g), and P. ornatus (0.763 ± 5.10 mg/g). The evaluated validation parameters were satisfactorily achieved, and the developed methodology represents a suitable tool for application in the quantification of barbatusin (1) and 3β-hydroxy-3-deoxybarbatusin (2) in Plectranthus species. The concentration of diterpenes barbatusin ( Plectranthus genus (Labiatae) is represented by ca. 300 species, which occur essentially in Africa, Asia, Australia, and some Pacific islands. Several species of this genus are cultivated for their edible tubes or as essential oil crops. In addition, some species are used for medicinal purposes such as treating vomiting, nausea, and ear infections; relieving toothache, headache, sores, and burns; or as antiseptic [ Plectranthus show biosynthetic ability to produce bioactive diterpenes like the labdane diterpenoid forskolin, from P. forskohlii, that shows significant blood pressure lowering properties [ Staphylococcus aureus and vancomycin-resistant Enterococcus faecalis [ Plectranthus species are popularly known as “boldo” and widely used as medicinal plants. P. grandis is used in the treatment of gastric and hepatic disturbs, and the gastric hyposecretory activity of this plant was already proven [ P. grandis in the treatment of digestive problems. The abietane diterpenes barbatusin (1) and 3β-hydroxy-deoxybarbatusin (2)   were isobarbatus , 8 and sφ mm 0,063–0,200) purchased from Vetec®. Analytical TLC was performed on precoated 0.25 mm thick plates of silica gel 60 F254 from Macherey-Nagel®, and the spots were visualized under UV lamp (254 nm) and by spraying with a solution of perchloric acid-vanillin in EtOH, followed by heating.Acetonitrile and methanol (HPLC grade) were purchased from Tedia®. Deionized water was obtained from a Millipore Milli-Q system. All other solvents and reagents were purchased from Synth®. Chromatography columns were developed on silica ( P. grandis (#28377), P. barbatus (#24408), P. ornatus (#31929), and P. amboinicus (#28601), grown under environmental conditions at the Horto de Plantas Medicinais Francisco José de Abreu Matos , were collected in January 2013, and their voucher specimens are deposited in Prisco Bezerra Herbarium-UFC.Leaves ofµm) analytical column in combination with a Phenomenex guard column.A Shimadzu UFLC chromatographic system (model LC-20A), with DAD detector (Diode Assay Detector) SPD-M20A, and LC solutions software (version 1.25) were used for data processing. The analyses were conducted on a Phenomenex® Luna 5u C18 (2) 100A  and 45% acetonitrile in water for compound 2 (method 2). Both methods were carried out at flow rate of 0.8 mL/min. Temperature of the column was 40°C, and detection wavelength was 254 nm. Analyses were carried out in triplicate.The methodology was based on the analysis of diterpenes in plants by HPLC-DAD . Isocrat P. grandis were extracted with ethanol (5 × 8 L) and yielded 295.0 g of extract after solvent evaporation under reduced pressure. The crude extract was dissolved in 3 L of water/methanol (4 : 6 v/v) and submitted to liquid-liquid extraction with dichloromethane (6 × 100 mL) to yield 60.0 g of dichloromethane fraction. Successive chromatographic treatments in column, using SiO2 as stationary phase and dichloromethane as mobile phase, gave 3.4 g of 1 and 140.0 mg of 2. The structures of both diterpenes were confirmed by high-resolution mass spectra, 1H-NMR , 13C-NMR , and comparison with literature data [The dried leaves (3.5 Kg) ofhane 6 × 0 mL to y Plectranthus species  were dried at 40°C, pulverized to powder, and stored in a desiccator. An accurately weighted amount of 1.0 g of each sample was extracted with 100 mL of ethanol in ultrasonic bath during 1 hour at 25°C. Extracts were filtered and the solvent was completely removed by vacuum distillation at 40°C. Each plant extract was dissolved in methanol (HPLC grade) and transferred to a 25 mL volumetric flask for a final concentration 0.5 mg/mL to be analyzed by HPLC-DAD.Leaves from four1) and 3β-3-hydroxy-3-deoxybarbatusin (2) isolated from leaves of P. grandis were used as standards for the analytical curves and method validation. Each diterpene was separately dissolved in methanol (HPLC grade), and the final concentration was adjusted to 1.0 mg/mL. The purities of standard compounds 1 and 2 were determined as ≥99% by HPLC-DAD analyses.Barbatusin  and general standard curve equation were obtained from linear least-square regression analysis (Graphpad Prism® 5.03 software). The method selectivity was determined through the analyses of the standard compounds 1 and 2 and extracts of the four Plectranthus species. In all extracts, the peaks corresponding to 1 and 2 were identified by comparison of their retention times with those from the standard compounds.Solutions of the standard compounds 1 and 2 were 0.015 and 0.22 µg/mL, and the limits of quantification  were 0.1 and 0.6 µg/mL, respectively.The limits of detection  of the two diterpenesThe precision of the method was evaluated in two levels, intraday and interdays. Intraday precision was evaluated by nine determinations (triplicate) in three different concentrations. Interdays precision was evaluated using the same three concentrations, during two consecutive days, and by changing operators. The analyses were performed separately for each standard compound. The relative standard deviations (RSD) were calculated for each determination and taken as a measure of precision and repeatability. 1  and 2 , in quintuplicate, were analyzed, besides the extracts with no addition of standard compounds. The recoveries of both standard compounds were calculated from the corresponding calibration curve according to the following equation: The accuracy was determined by recovery tests, which is an estimate of the accuracy of the methods. Extracts containing known amounts of 1 (method 1) and compound 2 (method 2). The linearity of the concentrations versus peak area plot for both methods was determined, and good correlation coefficients were achieved (r2 = 0.9931 and r2 = 0.9966).  1 and 2 and P. grandis extract are shown in Two analytical curves were obtained, one for each standard compound 1 and 2, which were accurate with recovery rates ranging from 81.7 to 104.0% for 1 and from 102.1 to 107.6% for 2. The found recovery rates  was identified only in the extracts from P. grandis and P. barbatus, while compound 2 was present in all Plectranthus species. The highest amount of both compounds 1 and 2 was found in P. grandis. This is the first report on the identification of 3β-hydroxy-3-deoxybarbatusin (2) in the extracts from P. amboinicus and P. ornatus.In summary, the analytical methods were adequate for quantification of diterpenes


The heat shock protein HSP27 has been correlated in ovarian cancer (OC) patients with aggressiveness and chemoresistance and, therefore, represents a promising potential biomarker for OC diagnosis, prognosis, and treatment response. Notably, secretion of soluble HSP27 has been described by a few cell types and may take place as well in OC cells. Therefore, we studied HSP27 secretion mechanisms under diverse cellular conditions in an OC cell model system. Secretion of HSP27 was characterized after overexpression of HSP27 by transfected plasmids and after heat shock. Intra- and extracellular HSP27 amounts were assessed by Western blotting and ELISA. Protein secretion was blocked by brefeldin A and the impact of the HSP27 phosphorylation status was analyzed overexpressing HSP27 phosphomutants. The present study demonstrated that HSP27 secretion by OVCAR-3 and SK-OV-3 cells depends on intracellular HSP27 concentrations. Moreover, HSP27 secretion is independent of the endoplasmic reticulum secretory pathway and HSP27 phosphorylation. Notably, analysis of OC cell-born exosomes not only confirmed the concentration-dependent correlation of HSP27 expression and secretion but also demonstrated a concentration-dependent incorporation of HSP27 protein into exosomes. Thus, secreted HSP27 may become more important as an extracellular factor which controls the tumor microenvironment and might be a noninvasive biomarker. Ovarian cancer (OC) therapy is restricted to a great extent by a limited understanding of the driving force of OC breakout and progression. OC represents the eighth most common female malignancy and first most common cause of death amongst gynaecological malignancies in the Western hemisphere . HoweverHeat shock proteins (HSP) are cellular factors whose expression levels are frequently upregulated in carcinoma tissue with respect to the nonmalignant tissue counterpart. Recently, accumulating evidence has emphasized the pivotal role of HSP in initiation and progression of cancer. Cytoprotective and thus frequently prooncogenic activities of HSP are particularly in demand when cellular proteins are disordered. Moreover, HSP are mostly stress-inducible cell survival factors that are upregulated by chemical and physical stresses  as well as radio- and chemotherapy. HSP functions are closely linked to bind specifically to client proteins and subsequently to control turnover and activity of these proteins , 3. WhilElevated levels of HSP27 are associated with treatment resistance and poor prognosis in numerous types of malignancies including breast, prostate, liver, and gastric carcinoma –9. HSP27Notably, a few studies have demonstrated that HSP27 is liberated by a so far unknown cellular machinery , 15 and The objective of the present study was to examine HSP27 secretion by OC cells against the background of future applications in gynecooncological diagnosis and prognosis. Thus, analysis of intra- and extracellular HSP27 levels was performed in an OC cell culture model.2.The human OC cell lines OVCAR-3 and SK-OV-3 were both received from Cell Lines Service  and propagated in RPMI 1640 medium  containing 10% foetal bovine serum (Biochrom), 0.125% gentamicin , and 0.1% insulin  and DMEM/F12  supplemented with 5% foetal bovine serum and 0.125% gentamicin, respectively. Cells were passaged twice a week and maintained in a humidified atmosphere at 37°C and 5% COµl of cell suspension was analyzed in triplicate. Measurement was performed using a capillary of 150 µm in diameter and cell line specific gate settings were used to discriminate between living cells, dead cells, and cellular debris .The proliferation of OC cells was evaluated utilizing a CASY Cell Counter and Analyzer Model TT . Briefly, adherent cells were detached with trypsin at different time points, suspended at 1 : 100 dilution into CASYton buffer (Roche Applied Science) and 400 HSP27 concentration in cell culture supernatant was determined by use of the DuoSet ELISA kit human HSP27  with Substrate Reagent Pack containing stabilized hydrogen peroxide  and stabilized tetramethylbenzidine  according to the manufacturer's instructions. Duplicates of each sample were measured. Cells and cellular debris were sedimented , the supernatant was incubated with capture antibodies , and soluble HSP27 was determined using a BMG FLUOstar OPTIMA Microplate Reader with OPTIMA software 2.10 .β-mercaptoethanol). Equal amounts of cell lysate were separated by SDS-PAGE using 10% polyacrylamide  and blotted onto a polyvinylidene fluoride membrane. Subsequently, the membrane was blocked  and incubated with primary antibodies directed against HSP27 , β-actin , and glyceraldehyde-3-phosphate dehydrogenase  overnight at 4°C. After washing, blot membranes were incubated with dye-conjugated mouse- and rabbit-specific secondary antibodies  for 1 h. Protein signals were visualized by enhanced chemiluminescence with a LI-COR Odyssey Infrared Imager System (LI-COR Biotechnology) according to the manufacturer's instructions. Protein levels of HSP27, β-actin, and GAPDH were quantified using the Image Studio Lite Version 4.0 software (LI-COR Biotechnology). β-Actin signals served as loading control for lysates; GAPDH was the loading control in exosome preparations. HSP27 expression levels were normalized to controls (control = 1.0).Intracellular proteins were extracted from the OC cell lines by lysing the samples in Laemmli buffer  glycerol, 0.01% bromophenol blue, and 5.0%  5 cells/well into a 6-well cell culture plate containing the appropriate medium without foetal bovine serum and gentamicin. 3.0 μg of plasmid DNA/well  or cells were uniquely exposed to 39°C for 4 h. Control cells were treated at 37°C for 72 h and 4 h, respectively.For heat shock experiments SK-OV-3 cells were plated at a density of 1.5 × 105 cells/well into a 6-well cell culture plate containing the appropriate medium. After 24 h brefeldin A was supplemented at a final concentration of 80 nM using ethanol vehicle as control. At indicated time points, cells as well as cell culture supernatant were harvested and intracellular and extracellular HSP27 concentrations were determined by Western blotting and by ELISA analysis.Cells were plated at a density of 1.5 × 10Preparation of exosomes was followed as previously described . Brieflyt-test with P ≤ 0.05 (∗), P ≤ 0.01 (∗∗), and P ≤ 0.001 (∗∗∗) given as significant.The statistical analysis was performed using SPSS 13.0 software . Data were evaluated using the graphics and statistics software program Graph Pad Prism 5.01 . Statistical comparisons were performed using Student's β-actin: 2.71 ± 0.80) was only slightly enhanced compared to OVCAR-3 cells . Notably, the subtle distinction between intracellular HSP27 expressions in both cell lines was linked with significantly elevated levels of secreted HSP27 protein in OVCAR-3 cells (P = 0.0197) has been exhibited.Secretion of the low molecular weight protein HSP27 from OC tissue has been described previously , 20. How-3 cells . In OVCAP = 0.0131; 48 h: 3.50 ± 2.90, P = 0.0203; 72 h: 1.68 ± 0.48, P = 0.0041) compared to pcDNA3.1 transfected controls. Parallel determination of HSP27 protein in the cell culture supernatant by ELISA demonstrated elevated amounts of secreted HSP27 compared to control transfections   .P = 0.0037; 4 h: 1.71 ± 0.61, P = 0.0315) compared to 37°C control incubations. As shown in P = 0.0001). After 4 h heat shock, however, no changes in secreted HSP27 concentrations were detectable .To verify these data, a comparable approach was used in which the endogenous HSP27 expression in SK-OV-3 cells was activated by heat shock induction. A 37°C/39°C/37°C heat shock cycle for 24 h each temperature (cycle) and a unique 39°C heat shock for 4 h (4 h) were performed and analyzed by Western blotting Figures . In bothP = 0.0497; 24 h: 0.73 ± 0.16, P = 0.0056; 48 h: 0.76 ± 0.18, P = 0.0086) (P < 0.0001) followed by significantly increased levels of secreted IL-6 after 24 h and 48 h of incubation   . In SK-O 0.0065) .P = 0.0048; 24 h: 0.97 ± 0.21, P = 0.7389; 48 h: 1.00 ± 0.19, P = 0.9567)      nor in S 0.8327) . ELISA a 0.9013)  and SK-O 0.1040)  comparedP = 0.0093; 48 h: 1.99 ± 0.46, P = 0.0014; 72 h: 2.41 ± 0.86, P = 0.0064), the phosphomutants HSP27-3A , and HSP27-3D   . Again,  0.0085) ; howeverP = 0.0135) induction of HSP27 expression in cell lysates and a 10.25 ± 4.47-fold (P = 0.0061) increase in HSP27 incorporation into extracellular exosomes  for oligomerization and a partially conserved region with so far unknown function. HSP27's C-terminus is defined by the proteins . A typicThe block of IL-6 liberation in the presence of brefeldin A served as positive control and demonstrated an unexpected increase in IL-6 secretion after 24 h restricted to SK-OV-3 cells . As brefUnder physiological conditions, HSP27 is predominantly localized to the cytoplasm. Nevertheless, Wong et al. have shown that the phosphorylation of HSP27 may cause HSP27's translocation into the nucleus , suggestWhile IL-6 secretion was significantly blocked in the presence of the endoplasmic reticulum secretory pathway inhibitor brefeldin A, HSP27 expression and secretion were not affected. Surprisingly, for periods of brefeldin A incubation longer than 5 h, IL-6 levels in SK-OV-3 cell culture supernatants increased. This effect may reflect differences in OVCAR-3 and SK-OV-3 cell response to the cytotoxic agent brefeldin A. HSP27 secretion, however, could not be inhibited by brefeldin A, pointing to a differing secretion pathway for HSP27 release.Protein secretion may also occur by direct translocation through the cytoplasmic membrane or by uptake into different types of export vesicles including exosomes . In prosThe tumor progression factor HSP27 is liberated from OC cells by exosomes. Protein's secretion appears independently of the phosphostatus as well as independently of the classical endoplasmic secretion pathway. Notably, HSP27 liberation is correlated with the intracellular HSP27 levels. This should be taken into consideration when anticancer treatment may induce HSP27  and subsRegardless of the so far unknown functionality of extracellular HSP27 in OC cells and adjacent tumor tissues, secreted HSP27 may become a promising biomarker for OC. Zhao and coworkers compared 24 blood samples from healthy women to blood samples from 48 patients with epithelial OC. An association of elevated blood levels of secreted HSP27 with metastatic OC was found , but bec


Non-covalent binding of water reduction catalysts and photosensitizers to hydrophobic silica particles represents a general approach for heterogenizing molecular water splitting components. 18-alkyl chains and immobilized by adsorption on hydrophobic fumed silica. The resulting loaded support was suspended in water with anionic or cationic surfactants and subjected to heterogeneous photocatalytic H2 production with ascorbate as sacrificial electron donor (SED). No leaching was observed under catalytic conditions, thus catalysis was truly heterogeneous. The catalytic performance of immobilized PS and WRC clearly exceeded that of homogeneous catalysis at low concentrations. At high concentration, diffusion and light limitation lead to lower reaction rates, but the same stability as for homogeneous reactions was still achieved. WRC concentration variations indicated a relatively high stability (up to 1300 H2/Co) and mobility of amphiphilic catalysts on the hydrophobic silica surface. Comparison of fumed silica with porous and non-porous silica showed, that a high BET surface area along with a good accessibility from the reaction media are crucial for catalytic performance. Mechanistic investigations by transient absorption spectroscopy displayed reductive quenching of excited PS by ascorbate followed by on particle electron transfer to WRC as reaction pathway. Particles with additional cationic surfactants exhibited a significantly higher catalytic performance as compared to anionic surfactants. Non-covalent anchoring of correspondingly derivatized WRCs or PSs to reversed-phase silica offers a rapid and versatile transition from homogeneous to heterogeneous molecular proton reduction.Heterogeneous photocatalytic hydrogen production with a non-covalently immobilized molecular ruthenium based photosensitizer (PS) and a cobalt polypyridyl based water reducing catalyst (WRC) is reported. PS and WRC were derivatized with C They observed “through particle” electron transfer (ET) for TiO2 and “on particle” ET for ZrO2.2 particles for photo- and electrocatalytically active electrodes.Photocatalytic water splitting into HSilica particles, coated with hydrophobic, long alkyl chains, represent a combination between oxidic nanoparticles and membranes. These so-called reversed-phase materials as used in HPLC and preparative column chromatography are chemically and physically inert and interact strongly with molecules comprising pendent lipophilic groups. These strong, non-covalent interactions suppress leaching while keeping mobility on the alkylated surface intact. With minimal derivatization, essentially any catalyst or photosensitizer (or both) can be anchored on these materials. Hydrophobic interactions22IIBr(appy)]Br 1 together with Re or Ru based PSs exhibited excellent proton reducing properties in homogeneous aqueous solution (via hydrophobic adsorption is straightforward and displacement of the components in aqueous media by protonation or competing (ionic or polar) species are greatly diminished.Acyclic cobalt complexes with poly-pyridyl ligands are a focus of recent research.solution .52,54 A 1 and [Ru(bpy)3]Cl2, both derivatized with long alkyl chains, on hydrophobic fumed silica particles, a cheap, robust and commercially available high BET surface area material. As fumed silica is non-porous with high accessibility, good catalytic performance was expected from these composites compared to other silica based particles. For comparison, the catalysts were also anchored on porous silica with very high BET surface area but low accessibility and on non-porous, spherical silica with high accessibility but low BET surface area. These nanocomposites are applied to heterogeneous, photocatalytic proton reduction and efficiency is compared to catalysis in homogeneous solution under equal conditions. The concept of anchoring catalysts on the hydrophobic surface of nano- or micromaterials is an excellent way of assessing the long-term performance of molecular systems.We present in this study a flexible method to immobilize WRC 18-appy)]Br (3) was synthesized in two steps; first, the hydroxy group of the basic polypyridyl ligand framework 2 (appy) was alkylated with octadecyliodide, then WRC 3 was obtained by coordination to CoII in 79% overall yield. The alkylated bpy ligand 4-methyl-4′-nonadecyl-2,2′-bipyridine  was obtained by deprotonation of 4,4′-dimethyl-2,2′-bipyridyl with Li[N(isopropyl)2] (LDA) followed by reaction with 1-bromooctadecane. The PS [Ru(bpy)2(C19-bpy)]2+ (5) finally was prepared from cis-[Ru(bpy)2Cl2] with 4 and subsequent counter-ion exchange with NH4PF6 (WRC Cl2 to the separated solution and continued irradiation 3]Cl2 and 1 in solution, assuming that the alkyl chains in 5 and 3 would not lead to significant activity differences with the latter PS and WRC. 2 at different (WRC and PS) concentrations. For the heterogeneous reactions, the amount of f-SiO2-C18 was reduced in order to keep the loading densities of 5 and 3 constant. At low catalysts concentrations (<20 μM PS and 1 μM WRC), immobilized complexes 5 and 3 exceed the performance of [Ru(bpy)3]Cl2 and 1 by far 3]Cl2 and 1. Apart from these limitations, immobilization does not adversely affect the catalyst performance since TONs for both systems are about identical  phosphine (TCEP).3, while keeping PS 5 and all other parameters constant. If mobility's of 3 and 5 on f-SiO2-C18 were low, WRC “dilution” will entail a linear decrease in rates with concentrations. Two distinct domains can be seen in 2 evolution rate, accompanied by a decrease in TONCo between 5 and 20 μM WRC and a linear decrease in rate, accompanied by constant TONCo below [WRC] = 5 μM.Complex 5) cycle becomes rate limiting . Although [Ru(bpy)3]2+ is known to undergo rapid electron self-exchange reactions (PS + PS– ↔ PS– + PS) at an estimated rate of ∼108 M–1 s–1,–2) is too large for efficient electron hopping to the WRC, if catalysts were not mobile. We thus conclude that 3 and 5 remain mobile on hydrophobic silica which enables rapid dynamic reorganization. The surface can be considered as a two dimensional liquid in interfacial contact with an aqueous solution. König and co-workers observed a similar mobility for complexes embedded into membranes.2 obtained in the first domain . As the stability of 3 is expected to be similar to 1 (>30 000 H2/Co (3 (–5 + 3 → 5 + –3) becomes too slow at low [WRC]. Thus only reduced PS within a maximal distance of a WRC molecule can deliver its reduction equivalents, whereas those that are too far away will decompose.In the second domain, below 5 μM [WRC], we assume that the WRC cycle limits the rate. In contrast to the homogeneous reaction, TON00 H2/Co ) we hypo3 and 5 (5 resp. 100 μM) on varying amounts of f-SiO2-C18 (0.02–0.27 molecules per nm2) gave a decrease of catalysis rate at low loading, whereas constant rates were observed above  and 7 (cationic) are shown in 2 evolution rates and total H2 amounts increased distinctly with decreasing concentrations of anionic surfactant 6 and otherwise identical conditions. Cationic surfactant 7, on the other hand, gave higher catalytic activity and stability in the concentration range accessible for catalysis. Experiments using surfactants 6 and 7 in a homogenous system gave no significant effect on the rate of photocatalysis  becomes slower which entails a decreased reaction rate. In parallel, electron back transfer to the continuously formed, neutral DHA is reduced which results in more H2 with the cationic surfactant 7. Cationic surfactants are therefore superior to anionic counterparts in heterogeneous photocatalytic H2 production.Maximal HTable SI2. Electro3 and PS 5 immobilized on three silica-based supports with different BET surface areas and accessibilities, but with similar particle diameters. Commercially available C18-silica (d = 40–70 μm) as used in reverse phase column chromatography is highly porous  and has a very large BET surface area (480 m2 g–1). Catalysts adsorbed in the pores might show altered kinetics , and catalytic activity should therefore be low. Calcination of porous silica (d = 10 μm) at 1100 °C followed by C18-grafting, gives spherical, non-porous hydrophobic silica . These two silica based supports were compared to f-SiO2-C18, which is non-porous but consists of small silica chains and branches, aggregated to larger particles . Thus, fumed silica combines both advantages of porous and non-porous silica; a high BET surface area and good accessibility. As expected, porous silica showed an even distribution of PS through the whole particle – similarly to f-SiO2-C18 aggregates – whereas for non-porous silica PS was only observed on the particle surface . Yet, 3 and 5 exhibited a 2–3 times higher maximal H2 evolution rate on non-porous compared to porous silica albeit H2 evolution ceased quickly due to low amounts of catalysts 3]Cl2 and 1. There are two possible pathways; excited PS* is reductively quenched by Hasc– followed by electron transfer to 1 and subsequent proton reduction (1 followed by reduction of oxidized [Ru(bpy)3]2+ (RuIII) with Hasc–. Although less likely, the second pathway cannot be excluded “a priori” since on the particle, 3 and 5 are in intimate vicinity. Transient absorption spectroscopy in water with [CoBr(appy)]Br (1) and [Ru(bpy)3]Cl2 did, however, not show any detectable formation of RuIII or CoI species, but non-productive quenching, most likely by energy transfer from PS* to 1 with kq = 2.2 ± 0.1 × 108 M–1 s–1  was expected.–PS to 1 is indeed close to diffusion control with kET = 1.6 ± 0.1 × 109 M–1 s–1  over anionic surfactants (6). According to our detailed and recent study with a similar tetra-pyridyl based Co WRC, we propose the final formation of H2 to occur by protonation of CoI → CoIII–H, reduction of CoIII–H → CoII–H and subsequent protonation and H2 release.69For obtaining a closer insight into the mechanism of Heduction ; 1.–4. oFig. SI10. In cont3 and PS 5. No leaching was observed, in agreement with strong hydrophobic interactions. At low concentrations, catalytic activity was still observed while the homogenous systems ceased to produce H2. At high concentrations, the homogenous system exceeds the immobilised one in terms of rate, but stability is retained. A proper choice of surfactants improved performance without altering the basic catalytic system. A careful analysis of the concentration dependency showed that immobilized catalysts remained mobile on their supports, and that PS stability limits turnover of catalysis, in line with results from recycling experiments. In summary, highly active molecular catalysts were immobilized by a straightforward and flexible method on cheap and robust supports, which is an important step towards heterogenization and thus physical separation of HER and potentially OER in a molecular water splitting architecture.A convenient method to immobilize molecular PSs and WRCs on hydrophobic supports by non-covalent interactions is presented. This photocatalytic architecture can be applied to various molecular PSs and WRCs since derivatizations with long alkyl chains are synthetically versatile, and no active functionalities are present, potentially interfering with the basic catalyst framework. The approach allows moving from homogeneous to heterogeneous catalysis, while mechanistic insights can still be obtained from studies in solution. We exemplify this concept with WRC 2 (appy), [Ru(bpy)2Cl2] and [Ru(bpy)3]Cl2 were prepared according to reported procedures.Poly-pyridine ligand 2  and NaH  were stirred in dry DMF (20 mL) for 45 min at rt. Iodo-octadecane  was added and stirring continued for 15 h at rt. CH2Cl2 and aq. NaOH (ca. 2 M) were added and the phases separated. The aqueous phase was washed three times with CH2Cl2, the combined organic phases dried over MgSO4, filtered and concentrated. The resulting yellowish oil was dissolved in MeOH/Et2O  and Co(ClO4)2 hexahydrate  added. The resulting brown solution was stirred for 3 h at rt followed by precipitation with excess Et2O. The solid was filtered off, washed 3× with Et2O and dried in vacuo to give 3 as light brown solid .ESI-MS (MeOH): m/z = 364 [M – 2 ClO4]2+ (100%), [M – ClO4]+ (50%), elemental analysis: calcd. for [Co(C18H37-appy)(H2O)](ClO4)2 (%): C, 55.88; H, 6.07; N, 7.40. Found: C, 56.05; H, 5.98; N, 7.29; UV/vis absorption (MeOH): λmax = 248 nm (ε = 1800 M–1 cm–1); 299 nm (ε = 2400 M–1 cm–1); 455 nm .Ligand 4 as colourless solid . 1H NMR : δ = 8.55 , 8.23 , 7.14 , 2.70 , 2.45 , 1.72–1.64 , 1.26 , 0.89 . 13C[1H] NMR : δ = 152.9, 148.9, 148.1, 128.4, 127.0, 125.6, 124.6, 123.9, 122.0, 121.3, 43.8, 35.8, 35.6, 31.9, 31.1, 30.5, 29.7, 29.54, 29.49, 29.44, 29.38, 29.2, 23.9, 22.7, 21.2, 14.1. ESI-MS (MeOH): m/z = 437.7 [M + H]+ (100%). Elemental analysis: calcd for C30H48N2 (%): C, 82.51; H, 11.08; N, 6.41. Found: C, 82.55; H, 11.04; N, 6.38.4,4′-Dimethyl-2,2′-bipyridyl  was suspended in dry THF (3 mL) and cooled to –45 °C in an MeOH/dry ice bath. LDA  was slowly added over 30 minutes and the mixture stirred for 2 h at –45 °C. Afterwards, a solution of 1-bromooctadecane  in dry THF (1 mL) was added drop wise to the reddish/brown solution, and the reaction mixture allowed to warm up to room temperature and further stirred for 15 h. The reaction mixture was quenched with MeOH (3 mL) and stirred for another 30 min at room temperature followed by concentration. The crude product was purified by flash chromatography  to give 2Cl2]·2H2O  and 4  were suspended in H2O/EtOH  and heated up to 100 °C for 24 h. The hot reaction mixture was filtered through celite, cooled down to rt and concentrated. The residual solid was washed 3× with 10 mL of hexane and dissolved in water. A threefold excess of NH4PF6 as concentrated aqueous solution was added dropwise to precipitate the complex as PF6–salt. Filtration and washing with water followed by drying gave 5 as red solid . UV/vis absorption (H2O): λmax = 453 nm (ε = 13 300 M–1 cm–1). Luminescence (H2O): λem = 623 nm . 1H-NMR  δ = 9.04–8.99 , 8.56 , 8.49 , 8.17–8.10 , 7.81–7.73 , 7.63–7.48 , 7.36–7.29 , 2.86 , 2.64 , 1.73 , 1.26 , 0.88 . ESI-MS (MeOH): m/z = 425.4 [M]2+ (100%), elemental analysis: calcd for C50H64N6P2F6Ru (%): C, 52.67; H, 5.66; N, 7.37. Found: C, 52.42; H, 5.60; N, 7.31.Cl2 and WRC 1 added. Finally NaOTf  was added as solid and the solution diluted up to 10 mL.The ascorbate buffer solution was transferred into the reaction vessel and corresponding amount of [Ru(bpy)–7 mol s–1). H2 evolution was measured as described in a previous publication.52The reaction mixtures were connected to an automated sampling system linked to a GC-TCD, degassed with Ar followed by illumination with 453 nm LED (85 ± 2 mW; photon flux: 3 × 10Supplementary informationClick here for additional data file.


RECORD-4 assessed everolimus in patients with metastatic renal cell carcinoma (mRCC) who progressed after 1 prior anti-vascular endothelial growth factor (VEGF) or cytokine and reinforced the clinical benefit of second-line everolimus. Because of the high percentage of patients from China enrolled in RECORD-4 (41%) and some reported differences in responses to certain targeted agents between Chinese and Western patients, this subanalysis evaluated outcomes in Asian versus non-Asian patients.RECORD-4 enrolled patients with clear cell mRCC into 3 cohorts based on prior first-line therapy: sunitinib, other anti-VEGF , or cytokines. Patients received everolimus 10 mg/d until progression of disease  or intolerance. Primary end point was progression-free survival per investigator review. Data cutoff was Sept 1, 2014.n = 55) versus non-Asian (n = 79) patients, 98% versus 84% had good/intermediate MSKCC prognosis; 73% versus 65% were men, and 85% versus 73% were < 65 years of age. All (100%) Asian patients were of Chinese ethnicity. Median duration of exposure was 5.5 mo for Asian and 6.0 mo for non-Asian patients. Among Asian versus non-Asian patients, median progression-free survival (months) was 7.4 versus 7.8 overall, 7.4 versus 4.0 with prior sunitinib, and 5.7 versus 9.2 with prior other anti-VEGFs. Clinical benefit rate was similar between populations: 74.5% (95% CI 61.0–85.3) for Asian patients and 74.7% (95% CI 63.6–83.8) for non-Asian patients. Most patients achieved stable disease as best overall response . Overall rate of grade 3/4 adverse events appeared similar for Asian (58%) and non-Asian patients (54%).Among Asian ; ClinicalTrials.gov identifier: The online version of this article (10.1186/s12885-018-4091-5) contains supplementary material, which is available to authorized users. Everolimus, a mammalian target of rapamycin inhibitor, is a second-line treatment option for patients with metastatic renal cell carcinoma (mRCC) refractory to vascular endothelial growth factor (VEGF) receptor inhibitors , 2. ApprThe RECORD-4 primary analysis has been published [n = 55) and non-Asian (n = 79) populations, respectively, 98% and 84% had good/intermediate Memorial Sloan Kettering Cancer Center (MSKCC) prognosis, 73% and 65% were men, and 85% and 73% were < 65 years of age . Median duration of exposure was 5.5 and 6.0 months for Asian and non-Asian patients, respectively.In the Asian (n = 55) and 7.8 months (95% CI 5.3–12.9) for non-Asian patients (n = 79)  and 4.0 months (95% CI 2.5–12.9) in the non-Asian population (n = 29)  and 9.2 months (95% CI 5.5–18.0) in the non-Asian population (n = 41)  and 12.9 months (95% CI 2.6–NE) in the non-Asian population (n = 9)  and non-Asian patients (n = 55), respectively . Clinical benefit rate  was 75% (95% CI 61.0–85.3) for Asian patients (n = 41) and 75% (95% CI 63.6–83.8) for non-Asian patients (n = 51).Median PFS was 7.4 months (95% CI 5.5–11.0) for Asian patients (79) Fig. . In the 29) Fig. . In the 41) Fig. . In the  9) Fig. . Best ovTable S3). The most common grade 3 and 4 AEs were anemia (7%), decreased hemoglobin level (6%), hypertriglyceridemia (6%), mouth ulceration (6%), proteinuria (6%), respiratory failure (6%), and stomatitis (5%) among Asian patients and anemia (17%), hyperglycemia (5%), and stomatitis (5%) among non-Asian patients. In the Asian and non-Asian populations, respectively, 22 (40%) and 35 (45%) patients required dose adjustment or study drug interruption to manage AEs. Eleven patients (20%) in the Asian population and 13 patients (17%) in the non-Asian population discontinued treatment because of AEs. Causes of on-treatment deaths in the Asian population were disease progression (n = 3), respiratory failure (n = 2), and multiorgan failure (n = 1) and in the non-Asian population were multiorgan failure (n = 2), cardiopulmonary failure (n = 1), disease progression (n = 1), sepsis (n = 1), sudden death (n = 1), and unknown cause (n = 1).The overall rate of grade 3 and 4 AEs (irrespective of relationship to study drug) was 58% and 54% for Asian and non-Asian patients, respectively  are currently approved for the treatment of advanced RCC in China, and everolimus and axitinib carry the highest level of evidence for second-line treatment after failure of first-line TKI in Chinese and Asia consensus treatment guidelines –9. Thus,In conclusion, second-line everolimus had comparable efficacy and safety in Asian and non-Asian patients with mRCC.Additional files 1:Table S1 Baseline demographics and disease characteristics of Asian and non-Asian patients in the overall population and in the first-line therapy cohorts. (DOCX 15 kb)Additional files 2:Table S2 Tumor responses (RECIST v1.0) of Asian and non-Asian patients in the overall population and in the first-line therapy cohorts. (DOCX 13 kb)Additional files 3:Table S3 Grade 3 and 4 adverse events reported by Asian and non-Asian patients in the overall population and in the first-line therapy cohorts. (DOCX 13 kb)


Their structures were established by MS and NMR data. Compounds 1–6 showed no activity to five human cancer cell lines.Two new seco-sativene sesquiterpenoids, bipolenins D (The online version of this article (doi:10.1007/s13659-016-0116-4) contains supplementary material, which is available to authorized users. Cochliobolus, Drechslera, and Bipolaris, et al., while some of them displayed bioactive diversity such as antiplasmodial activity and cytotoxicity + , corresponding to four degrees of unsaturation. The IR absorption bands at 1631, 1454, and 1368 cm−1 revealed the existence of double bond. In the 1H NMR spectrum of compound 1 , 0.94 , and 1.19  were ascribable for three methyl groups, while signals at δH 4.82 and 4.72 should be assigned to a terminal double bond. The 13C NMR and DEPT spectra showed 16 carbon resonances which were ascribable for four methyl (including one methoxy), four methylene, six methine and two quaternary carbons (Table 1 was suggested to possess a seco-sativene backbone with similar structure to that of prehelminthosporol (4) + . The 13C NMR spectrum displayed seventeen carbon resonances. Of them, signals at δC 21.0 (q) and 171.2 (s) were ascribed to one acetoxyl group, while other fifteen signals were similar to those of secolongifolene diol D20 +28.8 . IR (KBr) νmax cm−1: 3429, 2952, 2848, 1657, 1453, 1386, 1119, 981, 882; 1H NMR  and 13C NMR  data, see Table m/z: 477.3340 [M + Na]+ .Colorless oil. [α]D17 +1.6 . 1H NMR  and 13C NMR  data, see Table m/z: 280.2025 [M]+ .White powder; [α]Supplementary material 1 (PDF 1499 kb)Below is the link to the electronic supplementary material.


Edwardsiella hoshinae is a Gram-negative facultative anaerobe that has primarily been isolated from avians and reptiles. We report here the complete and annotated genome sequence of an isolate from a monitor lizard (Varanus sp.), which contains a chromosome of 3,811,650 bp and no plasmids. Edwardsiella genus was first recognized in the 1960s based on the new species Edwardsiella tarda, constituting a group of enteric Gram-negative bacteria  technology to a depth of 25× genome coverage 96.5 Mb). Reads ≥8,000 bp were error corrected with shorter PacBio reads and assembled using Canu version 1.0 . Illumin6.5 Mb. Rhttp://enve-omics.ce.gatech.edu/ani/; dDDH, http://ggdc.dsmz.de/distcalc2.php).The circularized and completed genome sequence was submitted to the National Center for Biotechnology Information (NCBI) Prokaryotic Genome Annotation Pipeline (PGAP) for annotation and submission to GenBank. The genome sequence was also submitted for Rapid Annotations using Subsystems Technology (RAST) analysis , 12 emplE. hoshinae genome consists of one circular chromosome with 3,811,650 bp (56.9% G+C content). PGAP annotation predicted 3,401 genes encoding 3,204 proteins and 101 tRNAs. RNAmmer (E. tarda isolate FL95-01 (E. anguillarum isolates LADL05-105 (E. piscicida isolates S11-285 (E. ictaluri isolate 93-146 (Edwardsiella hoshinae will be useful for the proper determination of taxonomic affiliation of future Edwardsiella species genomes, as well as investigations into host-pathogen interactions.This  RNAmmer  predicte FL95-01 ; 83.1% (DL05-105 , EA18101DL05-105 , 20; 83. S11-285  and C07- S11-285 ; and 82.e 93-146 . We deteEdwardsiella hoshinae isolate ATCC 35051 has been deposited in GenBank under accession no. CP016043.The complete genome sequence for


UBE3A gene. Ube3A protein is an E3 ubiquitin ligase that ubiquitinates proteins and targets them for degradation. The immediate-early gene Arc, a master regulator of synaptic plasticity, was identified as a putative substrate of Ube3A, but there have been conflicting reports on whether Arc is a bona fide E3 ligase substrate. Using multiple approaches, we found no evidence for a physical interaction between Arc and Ube3A in vivo. Nonetheless, activity-induced subcellular distribution of Arc is altered in brains from Ube3am−/p+ mice, with abnormal concentration of Arc at synapses. Furthermore, although activation of Arc transcription is normal, the stability of Arc protein is enhanced in dendrites of hippocampal neurons cultured from Ube3am−/p+ mice. Finally, homeostatic synaptic scaling of surface AMPA receptors does not occur in Ube3am−/p+ hippocampal neurons, reminiscent of neurons that lack Arc protein. Although Ube3A does not seem to bind Arc in a canonical E3 ligase-substrate interaction, Arc-dependent synaptic plasticity is still altered in Ube3am−/p+ mice, which may underlie the cognitive deficits observed in AS.Angelman syndrome (AS) is a neurodevelopmental disorder that results from deletions or mutations in chromosome 15, which usually includes the  UBE3A on the 15q11.2-13 chromosome  is a neurodevelopmental disorder affecting about one in 15,000 children, and presents with intellectual disability, ataxia, inability to acquire language and seizures  were then bred to male WT C57BL/6J mice  to create litters containing Ube3am−/p+ and WT (Ube3am+/p+) mice on the C57 background. Both male and female Ube3am−/p+ and WT mice were used. Arc KO mice were described previously  were a generous gift from Yong-Hui Jiang ; DAPI ; mouse anti-GluA1-NT  or synaptoneurosome buffer  or in immunoprecipitation buffer for immunoprecipitation (600 μL). The other aliquot was filtered through a 5-μm filter  at 12,000× g to isolate the synaptoneurosome pellet  or hippocampal tissue was homogenized in synaptoneurosome buffer with a glass dounce homogenizer , then frozen overnight at −20°C to assist in obtaining clean nuclear fractions . Total protein in each lane was detected using either Ponceau stain for immunoprecipitations, or the Pierce reversible protein stain kit (Thermo Fisher Scientific) for synaptoneurosome Western blots, then imaged using an Azure c300 gel dock . Membranes were blocked in 5% milk + 1× tris-buffered saline  for 30 min at RT, then incubated in primary antibody in 1× TBS for either 1 h at RT or overnight at 4°C. Membranes were washed 3 × 10 min in 1× TBS, then incubated in an HRP-conjugated secondary antibody in block for 1 h at RT. After 3 × 10 min in 1× TBS, a chemiluminescent kit  was used to detect the protein bands, and the membranes were imaged on a c300 gel dock.Immunoblotted membranes were analyzed using the gel analysis plugin in ImageJ . Values for bands for Ube3A and Arc were normalized to the total protein, determined by the Pierce reversible protein stain kit, in each lane. The efficacy of the synaptoneurosome preparation was determined by the reversible protein stain, and if no total protein was observed, then that sample was removed from subsequent analysis  and papain , then triturated with a fire-polished glass pipette to obtain a single-cell suspension. Cells were pelleted at 1000× g for 4 min, the supernatant removed, and cells resuspended and counted with a TC-20 cell counter (Bio-Rad). Neurons were plated on glass coverslips  coated with poly-L-lysine  in 12-well plates  at 100,000 cells/mL. Neurons were initially plated in Neurobasal media containing 5% horse serum, 2% GlutaMAX, 2% B-27, and 1% penicillin/streptomycin (Thermo Fisher Scientific) in a 37°C incubator with 5% CO2. On DIV4, neurons were fed via half media exchange with astrocyte-conditioned Neurobasal media containing 1% horse serum, GlutaMAX and penicillin/streptomycin, 2% B-27, and 5 μM cytosine β-D-arabinofuranoside . Neurons were fed with astrocyte-conditioned media every 3 days thereafter.The neuron culture protocol was based on Shepherd et al. . HippocaAt DIV18–20, neuron cultures were treated with 1 μM tetrodotoxin  or 10 μM bicuculline , then fixed for 15 min with 4% sucrose/4% formaldehyde (Thermo Fisher Scientific) in 1× PBS. Neurons were washed 3 × 5 min with 1× PBS, permeabilized for 10 min with 0.2% Triton X-100  in 1× PBS, and blocked for 30 min in 5% normal donkey serum (Jackson ImmunoResearch) in 1× PBS. Neurons were then incubated in primary antibody diluted in block for 1 h at RT, washed 3 × 5 min in 1× PBS, and incubated in secondary antibody diluted in block for 1 h at RT. Neurons on coverslips were mounted on glass slides in Fluoromount (Thermo Fisher Scientific) and dried overnight at RT. For live-labeling of surface GluA1 receptors . Healthy neurons were chosen based on MAP2 immunostaining. Arc and GluA1 immunostaining were analyzed using ImageJ software. The most intense immunostaining in each condition was used to set an arbitrary pixel intensity threshold, which was applied to every image in the experiment. Thresholds were set to ensure that pixel values were in the linear range for all treatment groups with both WT and t-tests or Tukey HSD tests were used to make post hoc comparisons.One- or two-way analysis of variances (ANOVAs) were performed for each experiment as described in the text using JMP Pro statistical software . Student’s in vivo  for 24 h, then exposed them to an enriched environment  for 2 h before euthanasia . Mice were either sacrificed in the animal colony , or dark-housed for 24 h to normalize activity, then exposed to light and novel objects (“enriched environment” condition) for 2 h before sacrifice. Hippocampi were dissected out, homogenized and subcellular fractionation performed to isolate the nucleus and synaptoneurosome. Lysates were immunoblotted for Arc and Ube3A  = 0.005, p = 0.95; Ube3am−/p+ syn/in: 0.34 ± 0.06, WT syn/in: 0.38 ± 0.04; one-way ANOVA, F = 0.43, p = 0.55). However, after exposure to an enriched environment  = 51.43, p = 0.002; Ube3am−/p+ syn/in: 1.31 ± 0.27, WT syn/in: 0.18 ± 0.03; one-way ANOVA, F = 18.11, p = 0.013). This suggests that activity-dependent Arc subcellular localization is abnormal in Ube3am−/p+ mice. Interestingly, we found that expression of Ube3A also changes dramatically after activity  = 137.35, p < 0.0001). We therefore performed immunoprecipitation from both whole hippocampal lysate as well as fractionated hippocampal lysate to ensure that we were not diluting out a potential Arc-Ube3A interaction  = 15.74, p = 0.0002), treatment  = 8.35, p = 0.0005), and a significant genotype × treatment interaction  = 5.67, p = 0.005; Figure Ube3am−/p+ neurons expressed more Arc than WT neurons . Arc levels were not significantly different from baseline in either genotype at 4 h . By 10 h, Arc levels had declined significantly in WT neurons , but remained unchanged from baseline in Ube3am−/p+ neurons . Furthermore, Arc levels in Ube3am−/p+ neurons were higher than WT at 10 h . When examining cytoplasmic levels of Arc in isolation from the nucleus , there was a significant main effect of genotype  = 7.08, p = 0.009), treatment  = 11.81, p < 0.0001), and a genotype × treatment interaction  = 4.99, p = 0.009). Arc levels were lower at 10 h than 4 h regardless of genotype , although Arc expression was significantly higher in Ube3am−/p+ neurons than WT at baseline . Nuclear Arc  levels did not exhibit any significant genotype differences. A two-way ANOVA showed that there was a significant main effect of treatment  = 6.64, p = 0.002), but no effect of genotype  = 0.32, p = 0.57) and no interaction  = 0.63, p = 0.54). Arc expression at 10 h was significantly lower than at 4 h  and baseline (p = 0.003). These data suggest that the stability of Arc protein is selectively altered in the dendritic compartment.Despite a lack of a physical interaction with Ube3A, we set out to determine whether the stability of Arc protein is altered in Ube3am−/p+ dendrites could either be due to altered Arc protein stability, as the results above suggest, or due to enhanced activity-dependent induction (transcription and/or translation) of Arc expression. To determine whether enhanced activity-dependent Arc induction occurs in Ube3am−/p+ neurons, we treated DIV19–21 cultured hippocampal neurons with TTX for 24 h to normalize basal neuronal activity and Arc levels. We then replaced the culture media with fresh conditioned media containing 10 μM BIC for either 4 or 10 h to induce Arc. Neurons were then fixed, permeabilized, and immunostained for Arc, and two 30-μm segments of dendrite/neuron were analyzed  = 13.09, p = 0.0005), treatment  = 39.09, p < 0.0001), and an interaction  = 17.65, p < 0.0001). Post hoc Tukey HSD tests showed that while Arc levels were not significantly different between WT and Ube3am−/p+ neurons at 24 h TTX  or at 4 h BIC , at 10 h BIC, Ube3am−/p+ neurons expressed significantly more Arc than WT . At 10 h BIC, WT neurons expressed less Arc than at 4 h (p = 0.04). Interestingly, and mirroring the results from the TTX experiment above, the difference in Arc expression between genotypes was only observed in dendrites. In the cytoplasm . In the nucleus  = 8.79, p = 0.0003). BIC treatment increased Arc expression over the 24 h TTX baseline . In this experiment, we did not observe a significant difference in basal dendritic Arc levels in Ube3am−/p+ neurons . The cumulative frequency of the integrated density of the GluA1 puncta was graphed and statistically analyzed using the Kolmogorov-Smirnov test. At baseline, there was no difference between the cumulative frequency distribution in WT and Ube3am−/p+ neurons (p = 0.5). In WT, as expected, 24 h TTX increased  and 24 h BIC decreased  the cumulative frequency of surface GluA1 puncta integrated density . Strikingly, TTX and BIC were unable to induce homeostatic scaling in Ube3am−/p+ neurons, with no change in the distribution of surface GluA1 puncta integrated density . One explanation for this result is that Arc expression is uncoupled from neuronal activity in Ube3am−/p+ neurons. To test this hypothesis, we measured Arc levels in the same neurons that had undergone homeostatic scaling to determine whether Arc expression correlated with the lack of BIC-induced GluA1 scaling observed in Ube3am−/p+ neurons  = 7.87, p = 0.006), treatment  = 3.22, p = 0.043), and an interaction  = 4.96, p = 0.008). We did not observe a significant difference between baseline and BIC-induced Arc levels in WT neurons, although there was a trend towards higher levels. This may be due to already high basal neuronal activity levels. However, BIC treatment did significantly induce Arc expression over TTX-treated WT neurons . However, in Ube3am−/p+ neurons, BIC-induced Arc levels were not significantly different from TTX-induced , and WT neurons expressed more Arc after BIC treatment than Ube3am−/p+ neurons . These results show that Arc protein expression is uncoupled from neuronal activity in Ube3am−/p+ neurons, which may lead to deficits in homeostatic scaling.s Figure . Neuronss Figure . Neuronsin vivo. Despite a lack of interaction, we show for the first time that activity-dependent subcellular distribution of Arc is altered in Ube3am−/p+ neurons, in vivo. Moreover, stability of Arc protein in dendrites is altered, which leads to deficits in homeostatic synaptic scaling of AMPARs, revealing an unknown synaptic deficit that may underlie the cognitive dysfunction in AS. These results suggest that Arc is not a direct E3 ligase substrate of Ube3A, and thus Ube3A controls Arc expression either through another direct substrate or a non-canonical E3 ligase function of Ube3A.Here we show that, under multiple conditions, Arc and Ube3A do not physically interact in vitro binding methods. However, these two subsequent studies were performed under very disparate experimental paradigms: either using non-neuronal cells, or cultured cortical neurons that had been silenced for 24 h. Mabb et al.  signaling (Huber et al., EDP performed experiments and analyzed and interpreted the data; JDS and EDP designed experiments, interpreted the data, and wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.