The optical properties of dye molecules in confined spaces can differ from the solution phase due to confinement effects. Pre-organized mesoscale channels of metal-organic frameworks (MOFs) are very suited for hosting various dyes, and the robust frameworks often render the encapsulated dyes with certain preferential geometries, which are different from those found in solution. Furthermore, pre-organized open channels can efficiently guide the uniform and unique spatial distribution of dye molecules in a controlled manner, which are otherwise difficult to achieve. Thus, sufficiently large dye molecules can avoid the formation of complex aggregates when captured inside open channels. In contrast, small dye molecules can form well-defined dimers or aggregates. The resulting dye-encapsulated MOFs can display unusual photophysical properties of the captured dyes. An anionic framework of In-BTB with mesoscale 3D channels is utilized for the efficient encapsulation of various cationic dyes through cation-exchange processes. Six different cationic dyes are encapsulated in the anionic framework of In-BTB, and their crystal structures are completely solved. Novel photophysical properties of these spatially distributed dye molecules in dye@In-BTBs are investigated. Additionally, MOFs usually possess high surfaces and large pore volumes and thereby are suitable for various advanced applications6. Selective CO2 capture7 at moderate conditions and high storage ability of energy-related gases, such as H2 and CH4, have accelerated the research for new types of crystalline hybrid porous materials8. Additionally, MOFs are good heterogeneous catalysts as well as superior catalyst supports for a number of organic transformations and electrocatalysis9.The unique physicochemical properties of metal-organic frameworks (MOFs) primarily stem from either their metal-based nodes or polytopic organic bridging ligands with a wide range of functionalities10. Drug delivery systems based on MOFs encapsulating drugs have increasingly attracted attention13. The robust and tailorable open channels of MOFs can effectively host various biologically active drug molecules for biomedical purposes due to their large voids. Likewise, various dye molecules have also been successfully encapsulated inside MOF channels to visibly demonstrate the large potential voids of MOFs. For instance, high-surface-area MOF-177, [Zn4O(BTB)2] where BTB = 1,3,5-benzenetribenzoate, can encapsulate 16 molecules of Astrazon Orange R (AOR), 2 molecules of Nile red (NR), and 1 molecule of Reichardt’s dye (RD) per unit cell14. The physical dimensions of these dyes increase in the order of AOR < NR < RD. Notably, dye@MOF-177 exhibited the characteristic color of each dye because MOF-177 is colorless. There are numerous examples of reported dye encapsulation by MOFs21. In most cases, however, it is not clear where the guest molecules are placed inside the void spaces of MOFs due to lack of crystallographic information of the dye-encapsulated MOFs (dye@MOFs). In addition, the mechanistic information of the encapsulation process is not well investigated. Although the colors of MOFs encapsulating specific dye molecules display the characteristic colors of the dyes, there is not much information about the penetration depth of dye molecules into the channels of MOFs. Obviously, once the MOF crystals are placed into the solution of the guest molecules, these molecules start to diffuse into the available channels of the MOF crystals from the outermost parts of the single crystal. Detailed information of the penetration depths for dyes will shed light on the understanding of mechanisms for molecular sieving effects, catalysis and drug delivery.Meanwhile, MOFs can also be utilized for encapsulating various functional guest molecules or ions with high efficacy2NH2]3[In3(BTB)4], through a cation-exchange process22. The anionic framework of In-BTB is an especially good host for RD cations despite its large molecular dimensions compared to small neutral acridine orange (AO). The encapsulation amount of AO by In-BTB is much smaller than RD under the same encapsulation conditions. Furthermore, In-BTB containing RD, RD@In-BTB, exhibited distinct visible absorption properties compared to non-encapsulated free RD based on solid-state UV/Vis absorption spectroscopy. To elucidate these different optical properties, the crystal structure of RD@In-BTB was successfully refined for the first time22. The structural comparison of the encapsulated RD and the free RD crystal grown in solution showed large distortion of the main molecular axis of RD in RD@In-BTB. This severe distortion is mainly responsible for the weak light absorption in the visible region due to less effective intramolecular charge transfer. This example implies that the photoluminescence (PL) properties of luminescent dyes captured inside pre-organized MOF channels can also be effectively altered due to isolation, confinement, and unique spatial ordering of dye molecules inside mesoscale channels.In this regard, we have been investigating the crystal structures of dye@MOFs. The optical properties of dyes in solution strongly depend on their environment, such as the solvent. In contrast, the lack of detailed crystallographic information of dyes encapsulated in MOFs often hampers the systematic correlation between the structures of dyes and their optical properties. Thus, the spatial positioning of dye molecules inside pre-organized channels of MOFs is an ideal method to investigate the structure-dependent optical properties of dyes. For example, we recently demonstrated that large RD was effectively encapsulated in mesoscale channels of anionic In-BTB, [Et4]− motifs as nodes of infinite 3D networks possess anionic frameworks with periodically arranged counter-cations located near the anionic InIII centers27. The resulting anionic nodes of In-MOFs would be beneficial for encapsulating a range of functional cationic guests within the frameworks through a simple cation-exchange process because of favorable host-guest electrostatic interactions. Notwithstanding, the guest encapsulation abilities of In-MOFs in solution have been rarely studied in detail compared to their gas sorption properties27. In order to explore the encapsulation of guests by an In-MOF in solution, we carefully chose six different cationic dyes for encapsulation, as shown in Fig. Motivated from these interesting findings, we attempted to further elucidate the relationship between the positional parameters and/or geometry of a series of cationic dyes with varying dimensions and functionalities encapsulated in the anionic framework of In-BTB and the corresponding PL properties of the captured dyes. In general, In-MOFs containing mononuclear pseudotetrahedral 3[In3(BTB)4]·10DEF·14H2O. The overall structure is best described as a 2-fold interpenetrated 3D network  that were not successfully refined. Six representative colored guest molecules  were chosen based on their physical dimensions and charges to systematically investigate the encapsulation properties of the micron-sized as-prepared In-BTB crystals. The large void of In-BTB may be sufficient to capture large cationic dyes, as the center-to-center distances on each BTB3− linker are as large as 15.486(1) Å and 27.034(1) Å alternatively. The dimensions of the free dye molecules are approximately 10.49 × 7.02 Å2 for Rh6G, 11.84 × 4.69 Å2 for NBA, 11.68 × 4.88 Å2 for AOG, 12.25 × 10.77 Å2 for CV, 13.19 × 5.70 Å2 for DEOCy, and 11.47 × 3.79 Å2 for DMMP. The as-prepared In-BTB (10 mg) was immersed into an ethanol solution (10 mL) of each dye with a concentration of 2 mM for 7 d, except for CV and AOG. Saturated ethanol solutions of CV and AOG were used instead because of their low solubility.Single crystals of In-BTB were prepared by the reaction between In with C3 symmetry along the c-axis 0.529 and (Rh6G)2[CdCl4]·(EtOH)·(H2O)30 with tilting angles of 77.5° and 79.75°, respectively. The carbonyl group is located near the xanthene ring, and it is the most likely configuration, as determined from previous theory31.To investigate the interactions between In-BTB and the dye cations, we analyzed the single-crystal structures of each dye@In-BTB. The occupancies of all the dye cation atoms were fixed to obtain the best fit with the largest residual peaks Table , but theBTB Fig. . Partialxis Fig. . Rh6G laB Tables . ConsideC3 symmetry alternatively along the c-axis  of the cation- and solvent-free In-BTB based on PLATON analysis . These indicate that the dyes were encapsulated by 25.8% for Rh6G, 13.5% for NBA, 13.8% for AOG, 15.6% for CV, 12.8% for DEOCy, and 21.5% for DMMP of the original void volume (71.0%) of In-BTB.2NH2]+ counter-cations with Rh6G cations mostly occurs at the outermost parts of the crystal. This can be accounted for by the fact that the initial cation exchange near the crystal surfaces gradually blocks the available channels to reduce the channel dimensions because the large Rh6G cations can be relatively strongly held near the anionic InIII nodes through electrostatic interaction. Therefore, additional Rh6G cations cannot be easily encapsulated further after a certain period of time. It can also be inferred from the series of Z-stack CLSM images in Fig. Fluorescence confocal laser scanning microscopy (CLSM) of a single crystal of Rh6G@In-BTB was performed to check the distribution and penetration depth of Rh6G cations. The morphologies of single crystals of In-BTB were analyzed by scanning electron microscopy (SEM) to acquire crystal morphology information before performing the CLSM investigation. The crystals have two seemingly different morphologies. Relatively large crystals exhibited more complex shapes with pentagonal crystal facets, while small crystals showed truncated cubic shapes Fig. . However34. Therefore, its PL properties are of significance in many optical applications; however, Rh6G has a problem of forming molecular aggregates. Rh6G encapsulated in a solid matrix, such as silica gel, usually displays uncontrollable aggregation, which often strongly affects its emission properties35. De Camargo and coworkers studied means to reduce dye aggregation. The dye aggregation is believed to occur through π-π interactions of the aromatic rings. To prevent aggregation, they used mesoporous sol-gel hosts. De Camargo et al. mentioned that to minimize aggregation effects, the most favorable host matrices are those in which the anionic binding sites are diluted and highly dispersed34. Thus, MOFs with anionic frameworks are good candidates to satisfy these two conditions to prevent dye aggregation.Rh6G is a well-known cationic xanthene dye used in dye lasers due to its high quantum yield (QY)36. The Rh6G dipoles are represented as arrows to display how Rh6G is placed inside the In-BTB framework, as shown in Fig. In our system, Rh6G@In-BTB showed a very uniform spatial distribution of Rh6G with unprecedented regularity inside the mesoscale channels of In-BTB, as shown in Fig. −6 M), as shown in Fig. ex = 534 nm) for Rh6G in ethanol. In contrast, the spectrum for Rh6G@In-BTB (λex = 520 nm) showed two bands centered at 577 and 637 nm at room temperature, which can be further deconvoluted into five well-defined emission bands centered at 578, 604, 633, 655, and 675 nm, as depicted in Fig. −2 M at 77 K37. The former and latter emission bands are attributed to monomer emission and aggregate emission, respectively. The relative intensity ratio of IMonomer/IAggregate was reported to be 1:0.9. The ratio varied upon changing the measurement temperature. When the temperature was increased to 100 K, the ratio became 0.67:1. Further increasing the temperature led to the two bands merging at 140 K. At 184 K (the melting point of 2-propanol), only a single emission band at 563 nm corresponding to monomer emission was observed. This phenomenon was mainly attributed to the fact that the energy transfer from monomeric Rh6G to the aggregates became less efficient in solution despite the high concentration.The PL properties of Rh6G@In-BTB were investigated using solid-state fluorescence measurements. The crystalline sample was used without grinding to preserve the original state of Rh6G in In-BTB crystals, whose structure was obtained from X-ray crystallography. The solid-state excitation and fluorescence emission spectra of Rh6G@In-BTB are depicted in Fig. 3(bpdc)3(DMF)]·5DMF·18H2O , with large hexagonally shaped 1D channels with a dimension of 27.9 × 24.5 Å38. Unlike Rh6G@In-BTB, the emission spectrum of Rh6G@JUC-48 only showed a broad band at 563 nm (λex = 541 nm) at room temperature. Decreasing the temperature to 77 K for Rh6G@JUC-48 only resulted in a slightly redshifted emission at 570 nm. No other redshifted emission bands with emission maxima greater than 600 nm were observed for Rh6G@JUC-48. Although it is difficult to analyze the positions of the Rh6G cations in Rh6G@JUC-48 due to a lack of crystallographic information, the single emission band can be conclusively assigned to diluted monomeric Rh6G distributed in the JUC-48 channels. Therefore, the four well-defined emission bands at 604, 633, 655, and 675 nm for Rh6G@In-BTB at room temperature can be attributed to new emission modes due to the unprecedented spatial ordering of the Rh6G cations in the pre-organized mesoscale channels of In-BTB. It is notable that these emissions are fundamentally different from the commonly observed aggregate emission in both the solution phase and solid state because Rh6G in Rh6G@In-BTB cannot form such aggregates. Moreover, the redshifts for the new emissions are prominent for Rh6G@In-BTB, owing to either efficient reabsorption of fluorescence by Rh6G in the channels and/or new energy transfer pathways. The control emission spectrum of bulk crystalline Rh6G (Sigma-Aldrich) excited at the same wavelength of 520 nm was also obtained for comparison. Bulk Rh6G only showed a broad single band deconvoluted into two emission bands at 618 and 645 nm, as shown in Fig. 39. Therefore, the emission properties of Rh6G@In-BTB are quite different from those of bulk crystalline Rh6G.Zhu and coworkers also reported Rh6G@JUC-48, where JUC-48 is styryl)−1-methylpyridinium (DMMP). The dyes are thought to be encapsulated through simple cation-exchange processes. The crystal structures of dye@In-BTBs were completely solved, and the PL spectra of three of these species were investigated. The successful refinements of the crystal structures of dye@In-BTBs can be attributed to the ideal dimensions of the cationic dyes and mesoscale channels of the anionic framework of In-BTB. Particularly, Rh6G@In-BTB exhibited distinct PL properties due to the unprecedented spatial ordering and isolation of the Rh6G cations in the mesoscale channels compared to free Rh6G in ethanol and bulk crystalline Rh6G. Both monomer emission and new emissions at longer wavelengths were identified at room temperature. The corresponding QY was higher than that of the control bulk crystalline Rh6G. NBA cations captured as a dimeric form did not give strong emission due to self-quenching. These results suggest that the photophysical properties of dyes can be tuned in a controllable manner by simply encapsulating into suitable channels of MOFs. There are numerous high surface MOFs having different charges in the frameworks3BTB) was prepared according to previous literature47. In(NO3)3 hydrate, rhodamine 6G (Rh6G), nile blue A (NBA), Astrazon Orange G (AOG), crystal violet (CV), 3,3′-diethyloxacarbocyaine iodide (DEOCy), 2-[4-dimethylamino]styryl)-1-methylpyridinium (DMMP), deuterium chloride solution (35 wt% in D2O), and DMSO-d6 were purchased from Sigma-Aldrich and used as received. N,N-Diethylformamide (DEF) was purchased from TCI and used without further purification. All other reagent grade solvents were used as received.1,3,5-Benzenetribenzoic acid  was added to a 2.0 mM solution of Rh6G dissolved in ethanol (10 mL) in a vial and gently shaken at room temperature. The vial was covered with aluminum foil. Aliquots of the solution were periodically withdrawn and diluted with ethanol to give a 250-fold diluted solution for quantification by UV-Vis spectroscopy. The measured molar extinction coefficient of 1.0 mM Rh6G in ethanol was 106463 M−1 cm−1 at λmax = 629.0 nm. The crystallographic data of NBA@In-BTB were collected using the retrieved crystals from the mixture after 7 d.The mixture containing as-prepared In-BTB (10 mg) and a 2.0 mM ethanol solution of NBA (10 mL) was gently shaken at room temperature in dark condition. Aliquots of the solution were periodically withdrawn and 200-fold diluted with ethanol for quantification by UV-Vis spectroscopy. The measured molar extinction coefficient of 1.0 mM NBA in ethanol was 72769 MThe as-prepared In-BTB (10 mg) was added to a saturated ethanol solution of AOG (10 mL) and gently shaken at room temperature in dark condition. The crystallographic data of AOG@In-BTB were collected using the retrieved crystals from the mixture after 7 d.The as-prepared In-BTB (10 mg) was added to a saturated ethanol solution of CV (10 mL) and gently shaken at room temperature in dark condition. The crystallographic data of CV@In-BTB were collected using the retrieved crystals from the mixture after 7 d.−1 cm−1 at λmax = 481.1 nm. The crystallographic data of DEOCy@In-BTB were collected using the retrieved crystals from the mixture after 7 d.The as-prepared In-BTB (10 mg) was added to a 2.0 mM ethanol solution of DEOCy (10 mL) and gently shaken at room temperature in dark condition. Aliquots of the solution were periodically withdrawn and 300-fold diluted with ethanol for quantification by UV-Vis spectroscopy. The measured molar extinction coefficient of 2.0 mM NBA in ethanol was 145242 M−1 cm−1 at λmax = 465.2 nm. The crystallographic data of DMMP@In-BTB were collected using the retrieved crystals from the mixture after 7 d.The as-prepared In-BTB (10 mg) was added to a 2.0 mM ethanol solution of DMMP (10 mL) and gently shaken at room temperature in dark condition. Aliquots of the solution were periodically withdrawn and 100-fold diluted with ethanol for quantification by UV-Vis spectroscopy. The measured molar extinction coefficient of 2.0 mM DMMP in ethanol was 42947 M = 1.54178 Å). The X-ray diffraction data for other dye@In-BTB MOFs were collected by a Bruker APEX-II diffractometer equipped with a monochromator and a Mo Kα X-ray source (λ = 0.71073 Å). Rh6G@In-BTB and NBA@In-BTB crystals were mounted on the loop for X-ray data collections at 223 K. The other dye@In-BTB crystals were inserted into a Lindemann capillary. The capillary was then filled with the mother liquor and sealed off by epoxy glue for data collection at room temperature. Bruker-SAINT software package was used for the integration and scaling of the collected CCD data. The crystal structures were solved and refined using SHELXL 201448. All hydrogen atoms were placed in their calculated positions. SQUEEZE/PLATON function was employed in structural refinement. The crystallographic data are listed in Table The X-ray intensity data of Rh6G@In-BTB were measured with a Bruker AXS X8 Prospector system equipped with a multilayer monochromator and a Cu IμSTM microfocus sealed-tube X-ray source λ = .54178 Å.1H) spectrometer. The proton chemical shifts of samples were calibrated with respect to the reference proton resonance signals occurring from the protic residues of the deuterated solvents. Elemental compositions of MOF samples were analyzed at the Organic Chemistry Research Center . PXRD spectra were recorded on a Bruker D8 Focus diffractometer . SEM images were recorded on a Coxem CX-100S . UV/Vis spectra were collected by a Scinco S-3100 spectrophotometer. Diffuse reflectance UV/Vis spectra were collected by a Jasco V-550 spectrophotometer. FT-IR spectra were obtained with Jasco FT/IR-4100 spectrometer using the KBr pellet method. Solid-state fluorescence spectra were collected using a Hitachi F-7000 fluorescence spectrophotometer. Absolute quantum yields were measured using a Quanta-φ integrating sphere system connected to a Fluorolog-3 fluorescence spectrophotometer (Horiba).NMR spectra were recorded using a Bruker Ascend 400  data acquisition route. The acquisition time was 2 ms for each pixel. Exponential fittings for the obtained PL decays were performed using the Symphotime-64 software.Time-resolved photoluminescence (TRPL) imaging was performed using an inverted-type scanning confocal microscope  with a 60× (water) objective. The measurements were performed at the Korea Basic Science Institute (KBSI), Daegu Center. A single-mode pulsed diode laser (470 nm with ~30 ps pulse width and an average power of < 1 μW) was used as an excitation source. A dichroic mirror , a long-pass filter , and a single-photon avalanche diode  were used to collect the emission from each sample. A time-correlated single-photon counting (TCSPC) technique was used to count emitting photons. TRPL images with a dimension of 80 × 80 μmSupplementary Information


Brain microvascular endothelial cell (BMEC) survival and angiogenesis after ischemic stroke has great significance for improving the prognosis of stroke. Abnormal variants of lncRNAs are closely associated with stroke. In this study, we examined the effects and molecular mechanisms of differentiation antagonizing non-protein coding RNA (DANCR) on apoptosis, migration, and angiogenesis of oxygen-glucose deprivation (OGD)-treated BMECs. We found that DANCR expression significantly increased at 2, 4, 6, 8, and 10 h after OGD. DANCR overexpression promoted cell viability, migration, and angiogenesis in OGD-treated BMECs. Additionally, we found that X-box binding protein l splicing (XBP1s) expression was positively correlated with DANCR expression. DANCR overexpression promoted XBP1s expression in OGD-treated BMECs. Silenced XBP1s reversed the effect of DANCR in OGD-treated BMECs. Furthermore, we found that microRNA (miR)-33a-5p bound to DANCR and the 3'-UTR of XBP1. miR-33a-5p overexpression inhibited proliferation, migration, angiogenesis, and XBP1s expression in OGD-treated DANCR-overexpressing BMECs, reversing the protective effect of DANCR. Finally, we found that XBP1s expression promoted proliferation, migration, and angiogenesis, reversing the damaging effect of miR-33a-5p. In conclusion, DANCR enhanced survival and angiogenesis in OGD-treated BMECs through the miR-33a-5p/XBP1s axis. Stroke causes neurological dysfunction and long-term disability, has a high incidence, and imposes an extremely heavy burden worldwide including in China , 2. The Non-coding RNAs (ncRNAs), including microRNAs (miRNAs) and long ncRNAs (lncRNAs), modulate gene expression and function through different mechanisms , 6. lncRVarious studies have shown that neuro-protectants are effective in animal models of stroke, although they have shown less effectiveness in the clinical research of stroke, suggesting that focusing only on neuro-protection is inadequate , 15. HenDANCR expression was measured using qRT-PCR. DANCR expression levels were the highest at 4 h and the lowest at 10 h after OGD treatment . AdditioAfter the optimization of the OGD-treated BMEC model, we explored the effect of DANCR on cell proliferation. BMECs were first transfected with DANCR-pcDNA3.1 and si-DANCR at 48 h, and we evaluated DANCR mRNA expression using qRT-PCR after the 4-h OGD treatment. DANCR expression was significantly inhibited in cells transfected with si-DANCR-2 and, particularly, si-DANCR-3, compared with si-NC-transfected cells . Thus, sIn our previous study, we found that XBP1s promotes BMEC survival and induces angiogenesis, thereby attenuating ischemia-induced BMEC injury . In the First, a bioinformatics prediction analysis was performed using StarBase V2.0 and Targetscan V7.2 to analyze miRNAs that can bind DANCR and XBP1. We found that DANCR and the 3′-UTR of XBP1 could competitively combine with miR-33a-5p, miR-33b-5p, miR-1251-5p, and miR-4731-5p . PreviouTo further study the influence of the interaction between miR-33a-5p and DANCR on OGD-treated BMECs, BMECs were transfected with both miR-33a-5p and DANCR. From the PCR results, we found that miR-33a-5p expression in the DANCR +miR-33a-5p mimic group was significantly higher than that in the DANCR+NC mimic group . CompareBased on the previous results, we decided to investigate whether XBP1s may promote cell viability and angiogenesis in OGD-treated BMECs even under the interference of miR-33a-5p overexpression. Hence, miR-33a-5p mimic and XBP1s-pcDNA3.1 were co- transfected into BMECs and confirmed using qRT-PCR and western blot analysis. From the results, it can be seen that owing to the XBP1s-pcDNA3.1 transfection, XBP1s expression remained higher in the co-transfected miR-33a-5p mimic and XBP1s-pcDNA3.1 BMECs after the 4-h OGD treatment . ConversA previous study found that DANCR activates Wnt/β-catenin signaling to promote glioma proliferation . In the Abnormal variants of lncRNAs are closely associated with stroke. In this study, we found that DANCR expression was upregulated in OGD-induced BMECs. Additionally, DANCR promoted BMEC proliferation and angiogenesis through the regulation of miR-33a-5p/XBP1s in OGD-induced BMECs, which may be important for ischemic stroke therapies.Ischemic stroke induces BMEC death and BBB disruption and aggravates neurological injury . BMEC anWe further investigated the mechanism of the DANCR effect. DANCR has been found to act as a ceRNA to sponge miRNAs. Previous studies have also identified that DNACR can sponge miR-33a-5p to regulate cell growth and metastasis in glioma and osteosarcoma , 23. In Silencing of the Wnt/β-catenin pathway contributes to the development of ischemic stroke . Activatin vivo.There are several limitations to this study. First, we only explored the effect of miR-33a-5p on DANCR-regulated XBP1s expression. In fact, miR-33b-5p, miR-1251-5p, and miR-4731-5p may also play important roles. Additionally, miR-33a-5p may have more target genes other than XBP1s. Finally, the function of the DANCR/miR-33a-5p/XBP1s axis needs to be verified In conclusion, we found that DANCR enhanced the survival and angiogenesis in OGD-treated BMECs through the miR-33a-5p/XBP1s axis, which provides a better understanding of the roles of DANCR and miR-33a-5p in ischemic stroke. Our findings indicate that DANCR overexpression and miR-33a-5p knockdown exhibit a protective effect in ischemic stroke. Thus, DANCR and miR-33a-5p may be a therapeutic target for the treatment of stroke.2 and 95% air into a 75-cm2 flask. Three passages of BMECs were cultured in D-Hank’s medium, instead of normal culture medium, and incubated under hypoxic conditions . BMECs were exposed to OGD for 0, 2, 4, 6, 8, and 10 h.This study was approved by the Animal Care Committee of Xiangya Hospital, Central South University, and all efforts were made to minimize the discomfort to the animals. Sprague-Dawley (SD) rats (aged 3–5 weeks) were fed as previously described . Rat BME-ΔΔCq method. All reactions were performed in triplicate. The primer sequences used were as follows: DANCR-F: 5′-GAAAGTGCACCAAAGGGTAT-3′ DANCR-R: 5′-AGAATTGCCACCATTGTCTC-3′ XBP1s-F: 5′-AAGAAGAGAACCAGAAACTCC-3′ XBP1s-R: 5′-TGAGACCTCTTCAGTAACCA-3′ β-actin-F: 5′-AGGGAAATCGTGCGTGACAT-3′ β-actin-R: 5′-GAACCGCTCATTGCCGATAG-3′ miR-33a-5p-F: 5′-GTGCATTGTAGTTGCATTGCA-3′ miR-33a-5p-R: 5'-GTGCAGGGTCCGAGGT-3′ U6-F: 5′-GCTTCGGCAGCACATATACTAAAAT-3′ U6-R: 5′-CGCTTCACGAATTTGCGTGTCAT-3′.Quantitative real-time polymerase chain reaction (qRT-PCR) was used to evaluate the expression levels of DANCR, miR-33a-5p, and XBP1s. Total RNA was extracted from cultured cells using Trizol reagent . For gene expression analysis, 1 μg of total RNA was reverse transcribed in a final volume of 20 μL to synthesize first-strand cDNA using the ImProm-II reverse transcription system . qRT-PCR was performed using the SYBR® Premix ExTaq™ II kit  on a 7500 Real-Time PCR System . β-actin and U6 were used as endogenous controls to normalize mRNA/lncRNA and miRNA expressions, respectively. The relative expression levels of mRNA/lncRNA and miRNA were calculated using the 2Collected cells or tissues were homogenized in a radioimmunoprecipitation assay buffer. After, the homogenized solutions were centrifuged at 12 000 rpm for 15 min, and the supernatants were collected. Protein concentration was determined using the bicinchoninic acid assay. An equal amount of proteins was electrophoresed in 12% SDS-PAGE and then transferred to a polyvinylidene difluoride membrane and blocked with 5% non-fat milk in Tris-Buffered Saline Tween-20 (TBST) buffer for 1 h. Subsequently, the membranes were incubated with anti-XBP1s  antibodies at 1:500 dilution in 5% non-fat milk overnight at 4°C, followed by incubation with anti-rabbit antibodies conjugated with horseradish peroxidase  at 1:2000 dilution for 1 h at 25°C, followed by three times washing with TBST. The protein bands were visualized on a gel-imaging system using ECL luminol reagent. β-actin was used as an internal reference to confirm the comparable amount of proteins in each lane.5 cells/well) were transfected with 1 μg/μL ov-DANCR without and with 50 nM miR-33a-5p mimic using 1 μL Lipofectamine® 2000 , according to the manufacturer’s instructions. Additionally, three siRNAs for DANCR  and a negative control (si-NC) were synthesized by GenePharma, with the following sequences, si-NC: 5′-GAACUGGGGUGCGUGUGAUdTdT-3′; si-DANCR-1: 5′-CTGAATACTCTGCAGCTGCdTdT-3′; si-DANCR-2: 5′-GATTACACCCCCTTGTAAAdTdT-3′; si-DANCR-3: 5′-GGTGGAGAAGCCTGGCAGGdTdT-3'. Cells were transfected using lipofectamine RNAiMAX (Invitrogen). At 48 h after transfection, BMECs were exposed to OGD for 4 h.miR-33a-5p mimic (5′-GUGCAUUGUAGUUGCA UUGCA-3′) and miRNA mimic negative control  were purchased from GenePharma . DANCR sequence, which included Nhe1 and Xho1 restriction enzyme cutting sites, was chemically synthesized by GENEWIZ  and then cloned into pcDNA 3.1 (ov-DANCR). The empty pcDNA 3.1 plasmid served as a negative control (ov-NC). BMECs , and Cell cycle detection kit  and Annexin V-FITC apoptosis detection kit (Keygen) were used to detect the cell cycle and apoptosis rates, respectively. Migration assay and tube formation assay were analyzed according to the protocols detailed in our previous study  .p < 0.05 (*); p < 0.01(**), or p < 0.001(***) were considered statistically significant.All experiments were performed in triplicate and data were expressed as mean ± standard deviation. Differences between the control and experimental groups were analyzed using the two-tailed Student’s t-test. An analysis of variance (ANOVA) was used for the overall comparison of the measurement indices between the groups, followed by post-hoc tests of the least significant difference (LSD). Differences of


A rapid review of current evidence examining psychological issues regarding the use of antibiotics and antimicrobials and resistance to these in both human and animal populations was conducted. Specific areas of interest were studies examining psychological determinants of AMR and interventions which attempt to change behavior with regard to AMR in the general population; animals; and fish, in particular. Although there is some evidence of the effectiveness of behavior change in general human populations, there is limited evidence in farmed animals, with a particular dearth in fish farming. We conclude there is an urgent need for more psychological research to identify major barriers and facilitators to change and evaluate the effectiveness of theory-based interventions aimed at reducing AM use in food production animals, including the promotion of alternatives to AMs, such as vaccination. Antimicrobial resistance (AMR) is an escalating risk to global health in both animals and humans, and progress to tackle its potential threat to life appears to be slow. Many bacteria have developed means of resistance to antimicrobials (AMs), which can spread to other bacteria, reducing the effectiveness of subsequent AM treatments in both humans and animals. Antibiotic (AB) and AM use in animals has important implications for AMR because of the potential of inter-species transmission of resistance. AM use in animals has been increasing at a concerning rate and so poses a considerable threat to AMR in both animals and humans. Accordingly, infectious disease experts have called for “strategies to minimise the risk of spreading antibiotic resistance among all populations”   (p. 47).Psychological factors play an important role in determining behavior. This literature review will add to existing evidence on associations between psychological factors and issues surrounding AM usage and AMR development, in members of the public as well as animal producers and owners, a little-studied area. Few studies have examined the role of psychological factors as drivers for AMR in humans, and even fewer have examined their consequence in AMR development within the animal food production sector . VeterinThe concept of AB or AM stewardship is concerned with the judicious use of AB/AMs in order to ensure their continued efficacy in both humans and animals. It is all-encompassing: contexts in which ABs are used including hospitals, general practitioner surgeries, dentists, the livestock industry (including fish), veterinary surgeries, and other groups such as prisons, nursing homes, and even racehorses and domestic pets. In food production not only veterinarians, but farmers themselves, are responsible for administering antibiotics; in humans, patients’ behavior  may place undue influence on health professionals’ prescribing behavior. It is therefore important to understand the role of non-health professionals in determining AB usage, and this forms the focus of this review.The aim was to provide a rapid review of the research evidence examining psychological issues surrounding the understanding of the use (appropriate or otherwise) of ABs/AMs and attitudes to AMR in the general public and animal farmers/owners. A rapid review was chosen as it provides a knowledge synthesis in a timely manner by simplifying the systematic review process . ApproprSpecific areas of interest were studies examining psychological determinants of AMR and interventions which attempt to change behavior with regard to AMR in the general population and all animals, including farmed fish. In particular we searched for theoretically-based studies, as these provide better scientific rigor, and these have been reported separately. Theory-based studies are important because they offer the potential to determine not only if behavior changes but also the mechanism of change, e.g., via potential mediating constructs such as perceived necessity, concerns, attitudes, intention, and cost. This can then inform interventions that can be targeted at modifying key constructs, e.g., reducing AB necessity and increasing concerns beliefs. A particular focus of this review is to inform our Vaccines against AMR in Aquaculture project (VAAC), which aims to deliver novel aquatic vaccines and test innovative machine vaccination to reduce antibiotic use in the Vietnamese catfish sector. As part of this project, Vietnamese catfish farmers’ attitudes towards antibiotic use, AMR, and vaccination will be examined. Knowledge, attitudes, and beliefs of farmers are likely to differ between Western and developing cultures, as government legislation will vary; therefore, it was important for us to pay particular attention to studies conducted in developing countries and in aquaculture.Search terms for this rapid review included ‘antibiotic’, ‘antibiotic stewardship’, ‘antibiotic resistance’, ‘antimicrobial’, ‘antimicrobial stewardship’, ‘antimicrobial resistance’, ‘AMR’, ‘human’, ‘animal’, ‘farming’, ‘fish’, ‘aquaculture’, ‘psychology’, ‘psychological’, ‘psychosocial’, ‘attitudes’, ‘knowledge’, ‘beliefs’, ‘intervention’. Reference lists of relevant articles were also searched for additional research papers not identified during the main searches. All articles reviewed have been published since 2007.Studies included in this rapid review which involve the use of ABs/AMs in humans are summarized in There have been a number of recent systematic reviews and meta-analyses examining knowledge, attitude, or behavior towards antibiotic (AB) usage and/or AMR in the general population. Gualano et al.  conducteMcCullough et al.  conducteTwo systematic reviews examined parents’ attitudes, knowledge, and behavior towards AB use in their children in qualitative and quantitative research in the UK, US, Europe, Asia, the Middle East, and South America ,7. Most A recent qualitative study examined the drivers of AMR in pet owners (n = 23) and veterinarians (n = 16) across the UK . They foGovernment policies regarding AB and AM usage differ between countries, and this may contribute towards inappropriate AB usage on a global scale. This may be a particular issue in developing countries: with lower overall levels of education amongst the general public, and potentially less access to medical care, it might be expected that poorer AB/AM awareness and more inappropriate use of ABs would be more evident in such countries. However, there is less research conducted in these countries on which to base evidence. A few of the studies included in the above reviews with the general public were conducted in Asian countries, including Malaysia, China, Indonesia, and South Korea ,5. FindiThe TPB  is an esAlthough there are a few studies examining the TPB in healthcare practitioners , with inThere have been a number of reviews of educational interventions to improve knowledge, attitudes, and beliefs, as well as behavior towards AB usage and AMR. A recent Public Health England report examined current behavior change research and its application to reducing AB usage . AlthougA National Institute of Health and Care Excellence (NICE) review found that educational interventions led to increased knowledge about the appropriate use of AMs  but not to better knowledge of AMR   but also governed by emotional responses . The authors of the Price et al.  review awww.antibioticguardian.com) and choose a pledge. Pledges were based on an Implementation Intentions (II) approach which aims to get people to decide, in advance when, where and how they will act in a particular situation using an If-then approach. For example one of the quoted public Antibiotic Guardian pledges states: ‘For infections that our bodies are good at fighting off on their own, like coughs, colds, sore throats and flu, I pledge to talk to my pharmacist about how to treat the symptoms first rather than going to the GP’. The authors found that both self-reported knowledge and a sense of personal responsibility increased and they concluded that online pledge systems could be effective in changing behaviors with regard to AMR. However, the sample was biased towards people with previous AMR awareness, 70% of respondents were females aged 44–54 and response to the survey required online access, so it’s not clear how generalizable it would be to other groups. Pinder et al. [IIs provide a framework for developing interventions which address habitual behaviors and so can be effective at changing usual responses, such as always seeking a GP prescription for ABs for colds. IIs involve a person planning, in advance, when, where and how they will implement a particular behavior . Chaintar et al.  also recRönnerstrand and Andersson Sundell  examinedWhilst many of the studies which have examined for knowledge, attitudes and behavior towards AM usage in the general public have recommended better information campaigns, there is mixed evidence regarding the effectiveness of mass media interventions . Direct Nonetheless, Pinder et al.  identifiStudies included in this rapid review which involve the use of ABs/AMs in food-producing animals are summarized in There is currently a dearth of studies examining psychological determinants of behavior in relation to AMR in animals . A rapidMost of the studies which touch on psychological issues, including attitudes, barriers, and motivations regarding AM use, have been conducted in Western cultures and on cattle, sheep, and pig farms. Coyne et al.  conducteIn a later mixed-methods study with UK pig farmers , Coyne eAnother recent UK qualitative study, conducted in the cattle, sheep and pig sectors , assesseIn the US, Friedman et al.  conducteAnother recent study examined associations between Dutch farmers’ beliefs and actual AM usage . The autA few studies have examined the TPB or social norms in relation to AM usage. Jones et al.  examinedMcIntosh and colleagues have conducted a number of studies in the US cattle feedlot industry, which examine TPB concepts including social norms and beliefs . OveralAnother study examined social factors which influence farmers’ decisions (n = 38) about how long to administer antibiotics in the treatment of mastitis in dairy cows in the Netherlands and in Germany . They foA few recent studies have considered attitudes towards AM usage in countries in Southeastern Asia. In many Asian countries, antibiotics can be used in animals without needing to be prescribed by veterinarians. In contrast to Western farmers, knowledge of AMR risk in Asian farmers appears poor. A questionnaire study of Malaysian ruminant farmers (n = 84) found little awareness of AMR and its impact on animals and public health   who produce for the domestic market, concluded that farmers had poor knowledge of the purpose of using antibiotics on their farms . Almost No interventions aimed at changing farmers’ attitudes to antibiotic use in fish were found anywhere.Awareness of appropriate AB/AM usage (including ineffectiveness against viruses) and the causes of ABR/AMR is often poor in the general population, and this is more evident in lower-income and less educated populationsAwareness of AMR in farmers is also often poor, especially in developing countries; however, good knowledge does not translate into reduced AM usageThe general public’s expectations of being prescribed ABs for common viruses persistFarmers are more influenced by social norms, including other farmers’ opinions, than advice from veterinarians when deciding whether to use AMs in animals. Practical considerations like economic factors and the animal’s health are also more determinant of farmers’ usage of ABs than consideration of ABRPeople attribute the rise in ABR/AMR to other people’s behavior (including clinicians) and do not see it as a problem for themselves or their children; even those with good knowledge of ABR believe that overuse in individuals raises the personal risk for those individuals but do not believe it is a risk for other people who restrict their own AB usageFarmers do not associate over-usage of AMs in animals with AMR in humans, including farmworkers; vaccination is seen as a viable alternative by someThere is inconclusive evidence that mass media campaigns aimed at the general public are effective; educational interventions aimed at parents and/or children may have merit in changing AB behaviorsThere is a dearth of evidence on changing psychological determinants of behavior with regard to AMR in farm animalsTo date, there has been limited research conducted with regard to psychological issues surrounding appropriate AM usage or AM stewardship. There is some published research regarding awareness, concern, and behavior around AMR usage in the general public, but much less examining changing behavior regarding the consumption of AMs in food-producing animals, suggesting there is a relative neglect of changing the risk posed to human health via AMR-induced by farming practices. A key contribution of the current research is to draw together two distinct bodies of literature that each contribute to our understanding of psychological determinants of AB use, and hence, pathways to AMR. By doing so, we hope to highlight blind spots in current policy and practice  and to help identify how research aimed at changing the behavior of food-producing farmers is needed to prevent AB misuse.Regarding AB usage in both humans and animals, there is a clear need for better education to improve awareness as well as interventions aimed at changing behavior. These may be better achieved via using techniques such as IIs (if-then planning) to change usual behaviors, such as requesting ABs for colds or flu or using AMs in animal feed, and/or developing a new social norm that ABs should be used as a last resort.This review examined the psychological determinants of AMR in the general public and animal producers and did not specifically look at studies or interventions which might consider attitudes towards alternatives to antibiotics. Nonetheless, many of the reported reviews concluded that inappropriate or excessive antibiotic use tended to increase when people did not believe there were effective alternatives. This has been shown to be an issue in lower socioeconomic countries where, for example, hygiene was problematic and there was an increased risk of bacterial infection, prompting more or less preventive use of second-line antibiotics . Effective vaccination can also reduce the need for antibiotics. A recent review by Brewer et al.  providedThe review highlights that there is a particular dearth of evidence to guide interventions to try and change food farmers’ attitudes, beliefs, and behaviors to AM use. We urgently need psychologically-informed studies to: (a) identify the major barriers and facilitators to change, and (b) evaluate the effectiveness of theory-based interventions aimed at reducing AM use in animal and fish farming, in particular, including the promotion of alternatives to AMs such as vaccination.


Nanoscale lubrication measurements around nanodefects point to local molecular ordering as the main cause for friction. The lubrication properties of nanoconfined liquids underpin countless natural and industrial processes. However, our current understanding of lubricated friction is still limited, especially for nonideal interfaces exhibiting nanoscale chemical and topographical defects. Here, we use atomic force microscopy to explore the equilibrium and dynamical behavior of a model lubricant, squalane, confined between a diamond tip and graphite in the vicinity of an atomic step. We combine high-resolution imaging of the interface with highly localized shear measurements at different velocities and temperatures to derive a quantitative picture of the lubricated friction around surface defects. We show that defects tend to promote local molecular order and increase friction forces by reducing the number of stable molecular configurations in their immediate vicinity. The effect is general, can propagate over hundreds of nanometers, and can be quantitatively described by a semiempirical model that bridges the molecular details and mesoscale observations. Friction plays a crucial role in many phenomena, ranging from the molecular diffusion of solutes within cells  computer simulations to investigate the molecular organization and dynamics of nanoconfined squalane near atomic steps of a graphite substrate. Squalane is a branched alkane with six methyl sidegroups  . A value of φS = 0° indicates a purely elastic behavior of the sheared squalane layer, whereas 90° corresponds to a purely viscous behavior. With a confinement area of ∼20 nm2 , FS can be expressed also as a function of the confining pressure  and the nanopositioning system of the AFM to place the tip, it is possible to conduct highly localized shear measurements at any desired location visible in the high-resolution image –coated AFM probes to ensure a system as symmetrical as possible with the model lubricant confined between two hydrophobic surfaces , but the MD results clearly confirm the fact that squalane molecules move more freely away from the step. The threefold increase in the squalane diffusion coefficient matches remarkably well the magnitude of the reduction in FS measured with AFM. The simulations also show an increase in the lateral layering of the squalane molecules close to the step edge, confirming the AFM observation that surface singularities promote molecular order . Furthermore, the simulations show that the average orientation of squalane molecules tends to be aligned to the step edge when in close proximity but progressively lose this alignment at larger distances with no clear ordering for dSE > 6 nm (fig. S6A). This step-induced molecular orientation is consistent with the row-like features observed in AFM image  substrate reveal a comparable behavior (fig. S7). Similarly, mesoscale experiments taken over larger areas of the graphite’s surface show a clear correlation between the apparent surface roughness and the resulting lubrication force despite the tip shearing over a variety of singularities (fig. S7). However, the nanoscale AFM results cannot fully address the link between molecular-level effects and macroscopic friction, and complementary results also involving other techniques , but a viscoelastic response is always present, confirming that the AFM tip probes the shear response of the confined lubricant and not of the surface itself. These results confirm the importance of the vicinity regions to modulate friction, with the equilibrium molecular arrangement alone able to decrease FS by more than 50% . However, ηeff converges exponentially toward a maximum value for pressures larger than ~0.5 GPa, consistent with the existence of solid-like ordered molecular domains of lubricant molecules. Piezo-viscous effects are also more pronounced for tip-surface gaps smaller than 4 nm (see fig. S9).The relaxation dynamics of the lubricant molecules has a marked impact on the lubricant’s effective viscosity ηFL ~10 to 30 nN; d, which can be understood as plastic viscosity in the modified Bingham plastic model. Second, it bypasses the difficulties associated with modeling the yield stress part of the curve (FL ≲ 10 nN) where the shear curves tend to present irregular features and shapes. A poor reproducibility at lower loads is inevitable given the stochastic nature of intermolecular bond rupture and possible variations in the local nanoscale details of the squalane/tip system during the initial stages of the shearing. Using the proposed linear fitting, Y0  is simply the ordinate at originTogether, these shearing experiments confirm the dominating role played by the lubricant’s molecular relaxation and provide clues to modeling its behavior. Building on this information, we systematically explored the impact of temperature and shear velocity on the resulting lubricated friction force, probing 14 different temperatures (between 298 and 368 K) and at least five different shear velocities at each temperature. For each measurement, at least 20 individual curves were acquired and subsequently averaged to ensure reliability of the results. The goal is to exploit this comprehensive set of measurements to develop a semiempirical model able to quantitatively describe the dynamical response of the sheared lubricant in all our experiments. To simplify as much as possible the modeling and avoid any overfitting, we fitted the linear region of the shear force curves  over all the temperatures and velocities measured . The negative sign is a consequence of Ea and explore neighboring configurations as in the Prandtl-Tomlinson model (Ea ≅ kBT)  at 378 K  mounted on a stainless steel disk using silver paint . High-performance liquid chromatography grade squalane with a purity of ≥99% was purchased from Sigma-Aldrich and used without any further purification .kf, = 2.8 (1.2 to 5.5) N m−1 and typical tip radii of 38 ± 4 nm (see fig. S4). Each cantilever was calibrated using its thermal spectrum , equipped with temperature control. All the experiments were conducted with a DLC-coated cantilevers  with nominal flexural spring constant, kt, returned a value for kt = 92 ± 2 N m −1   the associated phase, φS  and Python. Before and after changing location for force spectroscopy measurements, high-resolution imaging ensured that no shifts of the sample had taken place affecting the area under investigation.3 at 298.15 K, in agreement with the experimental value of 804 kg/m3 (z direction. During the simulations, the HOPG atoms were not allowed to move. All simulations were performed with a 0.002-ps time step. Before use, the squalane box was equilibrated for 20 ns. After combining with the stepped HOPG, the system was equillibrated for 17 ns before collecting the data over a further 12 ns. The diffusion constant was calculated using the Einstein relation by least squares fitting of the mean square displacement of the squalane molecules (The simulations were performed using the MD package GROMACS version 2016 (


Recent advancements in molecular robotics have been greatly contributed by the progress in various fields of science and technology, particularly in supramolecular chemistry, bio- and nanotechnology, and informatics. Yet one of the biggest challenges in molecular robotics has been controlling a large number of robots at a time and employing the robots for any specific task as flocks in order to harness emergent functions. Swarming of molecular robots has emerged as a new paradigm with potentials to overcome this hurdle in molecular robotics. In this review article, we comprehensively discuss the latest developments in swarm molecular robotics, particularly emphasizing the effective utilization of bio- and nanotechnology in swarming of molecular robots. Importance of tuning the mutual interaction among the molecular robots in regulation of their swarming is introduced. Successful utilization of DNA, photoresponsive molecules, and natural molecular machines in swarming of molecular robots to provide them with processing, sensing, and actuating ability is highlighted. The potentials of molecular swarm robots for practical applications by means of their ability to participate in logical operations and molecular computations are also discussed. Prospects of the molecular swarm robots in utilizing the emergent functions through swarming are also emphasized together with their future perspectives.  The Living organisms perform tasks which are essential for life by using the molecular machines already available in nature. Some of the examples include moving large cargoes in cells using biomolecular motors , convertIn these developments very small size of the molecular robots has been a big advantage since it enables creating a large number of robots and managing them to perform tasks coherently. Allowing small individuals to interact with each other and produce a more complex, collective task, has been motivated by collective behavior of living organisms ,39. Loca2.1.A molecular robot can be best described as an integrated system formed through the combination of different molecular parts or devices that may work as processor or logic gates, actuator, and sensors  Figure . A molec2.2.Three basic components are essential for constructing molecular robots: actuator, processor, and sensor . Actuato2.3.Biomolecular motor, the power source of a molecular robot, is the smallest natural molecular machine that can perform various tasks in cells by consuming chemical energy . By virt2.4.Microtubule/kinesin, a cytoskeletal motor protein system, was used as the biomolecular motor for fabricating molecular robot. The basic unit of the molecular robot was prepared by using a chemical technique to conjugate microtubules with single-strand DNA . Further2.5.As already discussed, local interaction plays an important role in group formation by self-propelled objects. Swarming of molecular robots was realized by utilizing the molecular recognition ability of DNA in controlling local interactions between molecular robots . Using a2.6.−24 Nm2 [The morphology of the swarms of molecular robots was also varied not only by local interactions introduced via DNA but also by tuning the length and rigidity of microtubules that were the basic units of molecular robots. For example, a molecular robot synthesized from microtubules having relatively high bending stiffness of 62 × 10−24 Nm2  formed a−24 Nm2 . Changes−24 Nm2 .2.7.Over the last decade, extensive attempts have been undertaken in pursuit of more powerful computers. Looking beyond the currently used silicon chip, a promising alternative has been DNA which is able to perform more complex computing . An abil2.8.Orthogonality has been one of the biggest challenges in the operation of molecular robots, particularly with respect to their swarming. We define orthogonality in the context of swarm robotics as a feature that enables swarming or solitary activity of different groups/types of robots without interacting or interfering with each other. Molecular robots making up an orthogonal system are strongly connected to the robots of their own group but still lacks the ability to interact with robots from other groups. Orthogonality is useful in providing groups of robots with enhanced precision in accomplishing multiple tasks simultaneously. The high molecular recognition ability of DNA forms the basis of orthogonal swarming. Information carried by a DNA sequence can be transmitted only to the target molecules without interfering with any other input signals. By making use of such orthogonality, it has been possible to independently control the group formation of molecular robots and swarming even in a complex assembly of different types of robots. For example, two types of DNA input signals were designed, one for a rigid molecular robot and the other one for a flexible molecular robot prepared from microtubules. Mechanical properties of microtubules were modulated by manipulating their polymerization conditions . The rig2.9.Light was successfully used to drive a nanocar, although coordinated motion of many cars was not accomplished . Photo-r3.By overcoming the hurdles related to the size and number of individual molecular robots prepared from biomolecular motors, DNA, and photosensitive molecules, swarming of molecular robots have been executed as an emergent function. The size of robots has been scaled down from centimeters to nanometers, and the number of robots participating in swarming has been successfully increased from one thousand to millions. Further optimization of the molecular robots is necessary for their applications to process, store, and transmit information which are subject to future work . Molecul


Recently, quantum contextuality has been proved to be the source of quantum computation’s power. That, together with multiple recent contextual experiments, prompts improving the methods of generation of contextual sets and finding their features. The most elaborated contextual sets, which offer blueprints for contextual experiments and computational gates, are the Kochen–Specker (KS) sets. In this paper, we show a method of vector generation that supersedes previous methods. It is implemented by means of algorithms and programs that generate hypergraphs embodying the Kochen–Specker property and that are designed to be carried out on supercomputers. We show that vector component generation of KS hypergraphs exhausts all possible vectors that can be constructed from chosen vector components, in contrast to previous studies that used incomplete lists of vectors and therefore missed a majority of hypergraphs. Consequently, this unified method is far more efficient for generations of KS sets and their implementation in quantum computation and quantum communication. Several new KS classes and their features have been found and are elaborated on in the paper. Greechie diagrams are discussed. Recently, it has been discovered that quantum contextuality might have a significant place in a development quantum communication ,2, quantExperimental contextual tests involve subtle issues, such as the possibility of noncontextual hidden variable models that can reproduce quantum mechanical predictions up to arbitrary precision . These mQuantum contextual configurations that have been elaborated on the most in the literature are the KS sets, and, in this paper, we consider just them. In order to obtain KS sets, so far, various methods of exploiting correlations, symmetries, geometry, qubit states, Pauli states, Lie algebras, etc., have been found and used for generating master sets i.e., big sets which contain all smaller contextual sets ,35,36,37n-tuples of orthogonalities from which a master set can be read off or finding a structure, e.g., a polytope, from which again a list of vectors and orthogonalities can be read off as well as a master set they build. In the present paper, we take the simplest possible vector components within an n-dimensional Hilbert space, e.g., n-tuples and then filter out KS sets from the sets in which the vectors are organized. For a particular choice of components, the chances of getting KS sets are very high. We generate KS sets for even-dimensional spaces, up to 32, that properly contain all previously obtained and known KS sets, present their features and distributions, give examples of previously unknown sets, and present a blueprint for implementation of a simple set with a complex coordinatization.All of these methods boil down either to finding a list of vectors and their n-dimensional (nD) Hilbert space (n-tuples (in our approach represented by MMP hypergraph edges) of orthogonal vectors (MMP hypergraph vertices) which exhaust themselves in forming configurations/sets of vectors (MMP hypergraphs). Already in  _ ‘ { | } ~ [n-dimensional KS set with k vectors and m n-tuples is represented by an MMP hypergraph with k vertices and m edges which we denote as a k-m set. In its graphical representation, vertices are depicted as dots and edges as straight or curved lines connecting m orthogonal vertices. We handle MMP hypergraphs by means of algorithms in the programs SHORTD, MMPSTRIP, MMPSUBGRAPH, VECFIND, STATES01, and others [m edges are successively given, separated by commas, and followed by assignments of coordinatization to k vertices  as defined via lists and tables of vectors used to build the KS master sets that one can find in the literature. These master sets serve us to obtain billions of non-isomorphic smaller KS sets  which define k-m classes. In doing so , we keep to minimal, critical, KS subhypergraphs in the sense that a removal of any of their edges turns them into non-KS sets. Critical KS hypergraphs are all we need for an experimental implementation: additional orthogonalities that bigger KS sets  might possess do not add any new property to the ones that the minimal critical core already has. The smallest hypergraphs we give in the table are therefore the smallest criticals. Many more of them, as well as their distributions, the reader can find in the cited references. Some coordinatizations are over-complicated in the original literature. For example (as shown in [In shown in ), for thSome of the smallest KS hypergraphs in the table have ASCII characters assigned and some do not. This is to stress that we can assign them in an arbitrary and random way to any hypergraph and then the program VECFIND will provide them with a coordinatization in a fraction of a second. For instance,18-9: 1234,4567,789A,ABCD,DEFG,GHI1,I29B,35CE,68FH.{1={0,0,0,1},2={0,0,1,0},3={1,1,0,0},4={1,-1,0,0},5={0,0,1,1},6={1,1,1,-1},7={1,1,-1,1}, 8={1,-1,1,1},9={1,0,0,-1},A={0,1,1,0},B={1,0,0,1},C={1,-1,1,-1},D={1,1,-1,-1},E={1,-1,-1,1},F={0,1,0,1},G={1,0,1,0},H={1,0,-1,0},I={0,1,0,0}}.However, a real finding is that we can go the other way round and determine the KS sets from nothing but vector components -master, the program builds an internal list of all possible non-zero vectors containing these components. From this list, it finds all possible edges of the hypergraph, which it then generates. MMPSTRIP via its option -U separates unconnected MMP subgraphs. We pipe the obtained hypergraphs through the program STATES01 to keep those that possess the KS property. We can use other programs of ours, MMPSTRIP, MMPSHUFFLE, SHORTD, STATES01, LOOP, etc., to obtain smaller KS subsets and analyze their features.We put simplest possible vector components, which might build vectors and therefore provide a coordinatization to MMP hypergraphs, into our program VECFIND. Via its option i)(the input set of components for generating two-qubit KS hypergraphs (4D) should contain number pairs of opposite signs, e.g., ii) means that, no matter how many different numbers we use as our input components, we will not get a KS master if at least to one of the numbers, the same number with the opposite sign is not added. Thus, e.g., Feature (As for the features (ii) and (iii) above, components In , the reaThe 60-75 list-master contains criticals with up to 41 edges and 60 vertices, while the 2316-3052 component-master generated from the same vector components contains criticals with up to close to 200 edges and 300 vertices;The 60-105 list-master contains criticals with up to 40 edges and 60 vertices, while the 156-249 component-master generated from the same vector components contains criticals with up to at least 58 edges and 88 vertices;Components In , we geneWe did not generate 16D and 32D masters because that would take too many CPU days and we already generated a huge number of criticals from submasters which are also defined by means of the same vector components in . See alsWe obtained the following particular results which show the extent to which component-masters give a more populated distribution of KS criticals than list-masters. We also closed several open questions:http://goo.gl/xbx8U2. They are developed in [Our methods for obtaining quantum contextual sets boil down to algorithms and programs within the MMP language we developed to generate and handle KS MMP hypergraphs as the most elaborated and implemented kind of these sets. The programs we make use of, VECFIND, STATES01, MMPSTRIP, MMPSHUFFLE, SUBGRAPH, LOOP, SHORTD, etc., are freely available from our repository loped in ,40,47,48The main result we obtain is that our vector component generation of KS hypergraphs (sets) exhaustively use all possible vectors that can be constructed from chosen vector components. This is in contrast to previous studies, which made use of serendipitously obtained lists of vectors curtailed in number due to various methods applied to obtain them. Hence, we obtain a thorough and maximally dense distribution of KS classes in all dimensions whose critical sets can therefore be much more effectively used for possible implementation in quantum computation and communication. A comparison of In vertices . It stem vertex) , on the  vertex) ), on theOur future engagement would be to tackle odd dimensional KS hypergraphs. Notice that, in a 3D Hilbert space, it is possible to explore similarities between Greechie diagrams and MMP hypergraphs because then neither of them can have edges/blocks which share more than one vertex/atom (via their respective definitions) and loops in both of them are of the order five or higher ,39.


We considered unwanted loneliness, ascertained using the UCLA Loneliness Scale, as primary measure of social defeat. We found no statistically significant difference in striatal dopamine synthesis capacity between ASD and controls . In ASD, striatal dopamine synthesis capacity was not significantly associated with loneliness . Secondary analyses showed comparable results when examining the associative, limbic, and sensorimotor sub-regions of the striatum . Results were similar before and after adjusting for age, sex, smoking-status, and PET/CT-scanner-type. In conclusion, in unmedicated, non-psychotic adults with ASD, striatal dopamine synthesis capacity is not increased and not associated with social defeat.Alterations in dopamine signalling have been implied in autism spectrum disorder (ASD), and these could be associated with the risk of developing a psychotic disorder in ASD adults. Negative social experiences and feelings of social defeat might result in an increase in dopamine functioning. However, few studies examined dopamine functioning in vivo in ASD. Here we examine whether striatal dopamine synthesis capacity is increased in ASD and associated with social defeat. Forty-four unmedicated, non-psychotic adults diagnosed with ASD and 22 matched controls, aged 18–30 years, completed a dynamic 3,4-dihydroxy-6-[ However, risk factors and neurobiological alterations associated with this risk remain poorly understood2.Adults with autism spectrum disorder (ASD) are at an increased risk of developing a psychotic disorder3. This increase in presynaptic dopamine functioning has also been reported in individuals at-risk of psychotic disorder5 and in individuals with a non-psychotic disorder when psychotic symptoms are present6.A well-replicated finding of neurotransmitter functioning in psychosis has been that of increased presynaptic dopamine functioning. In vivo molecular imaging studies using positron emission tomography (PET) or single-photon emission computed tomography (SPECT) have consistently shown that psychosis is strongly related to increased presynaptic dopamine synthesis capacity and endogenous dopamine release in the striatum7. For instance, this has been implied in theoretical work8, gene studies9, and studies examining dopamine-modulating medication11. However, molecular imaging studies, in particular of presynaptic dopamine, are lacking12. No studies examined striatal dopamine release, and only two studies used 3,4-dihydroxy-6-[18F]-fluoro-L-phenylalanine ([18F]-FDOPA) PET to measure dopamine synthesis capacity. The first13 found no significant increase in striatal dopamine synthesis capacity in autistic children (n = 14) relative to non-autistic peers (n = 10), but most (n = 11) autistic children were sedated using propofol which can influence dopamine concentrations14. The second15 obtained evidence of increased striatal dopamine synthesis capacity in adults with Asperger syndrome (n = 8) relative to controls (n = 5), but the number of participants was small and the influence of possible risk factors was not examined.Several researchers have suggested that alterations in dopamine functioning exist in ASD16. According to the social defeat hypothesis of schizophrenia, the long-term subjective experience of outsider status or subordinate position leads to increased baseline activity and/or sensitization of the brain dopamine system19. In line with this hypothesis, increased presynaptic dopamine functioning has been reported in non-psychotic groups presumed to be more socially defeated, such as immigrants, individuals with severe hearing impairment, and individuals with a history of childhood trauma23. Whether social defeat also increases presynaptic dopamine functioning in individuals with ASD, who are widely exposed to defeating experiences such as loneliness24, bullying victimization26, and discrimination27, remains unknown.Evidence indicates that psychosocial stress can influence dopamine functioning18F]-FDOPA PET/computed tomography (PET/CT), in non-psychotic adults with ASD and controls aged 18–30 years. We expect (hypothesis 1a) that striatal dopamine synthesis capacity is increased in ASD relative to controls. Furthermore, we expect (hypothesis 1b) that in ASD, striatal dopamine synthesis capacity is positively associated with loneliness, as an approximate measure of social defeat. As secondary outcomes, we expect (hypothesis 2a) that adults with ASD report more social defeat than controls, and (hypothesis 2b) that striatal dopamine synthesis capacity is positively related to other measures of social defeat (besides loneliness) and to subclinical psychotic symptoms. Finally, we expect (hypothesis 3) the strongest associations with dopamine synthesis capacity in the associative striatum, since the largest striatal dopaminergic disruptions in psychosis have been reported in this sub-region3 (with reference to hypotheses 1a-b and 2b).Here we examine striatal presynaptic dopamine synthesis capacity, measured with -FDOPA PET/CT-scan. All participants signed informed consent prior to the start of the study. The study was approved by the medical ethics committee of Leiden University Medical Center (reference NL54244.058.15), and pre-registered within the Netherlands Trial Register .Participants were assessed on three separate testing days, during which they were screened for in- and exclusion criteria, underwent a magnetic resonance imaging (MRI) scan (see below), and completed measures of social defeat and an -FDOPA metabolites49. Immediately prior to the PET acquisition, a low-dose CT-scan of the brain  was acquired for attenuation correction purposes. Subsequently, 150 MBq [18F]-FDOPA was administered intravenously as a bolus, which was followed by a 90-min dynamic PET acquisition. Head movement was minimalised with a headrest and head strap, and participants were monitored throughout the PET acquisition so that any discomfort could be observed and addressed. Data were collected in list mode and histogrammed into 25 timeframes ). PET data were reconstructed iteratively with resolution modelling and 2-mm full-width-at-half-maximum Gaussian smoothing filter with a voxel size of 2 x 2 x 2 mm3.A structural T1-weighted MRI (3D fast field echo sequence) was obtained for each participant on a 3T Ingenia MR scanner . Due to a necessary replacement of the PET/CT-scanner at the hospital in which this study was conducted, PET/CT data were collected on two different PET/CT systems, i.e. a Biograph Horizon with TrueV option  or Vereos . Both systems received EARL PET/CT accreditation. Reconstruction settings were harmonized using acquisitions of a Hoffman 3D brain phantom51, based on mutual information52, to spatially align the images and compensate for minor head motion. Head movement was quantified by taking the square root of the sum of movement in millimetres squared in x, y, and z directions from the last PET frame relative to the reference frame. Prior to pharmacokinetic analysis, volumes of interest (cerebellum and striatum) were generated automatically using PVElab 54 based on a maximum probability atlas55. In addition, SPM12  was used to segment the MRI image into grey (GM) and white matter (WM). Subsequently, the GM cerebellum was used as a reference region to calculate the influx constant kicer  as a measure of dopamine synthesis capacity using reference Patlak graphical analysis56 as implemented in PPET 57. PET frames acquired between minutes 25 and 90 were used for linear fit, resulting in a whole-brain parametric image. From this parametric image, kicer for the GM striatum was extracted.Image processing was conducted by researchers who were blind to ASD status. The MRI- and PET-images (except for the first two frames) were rigidly co-registered to a single PET-frame acquired 7 min post-injection using Vinci 58, and a standard Montreal Neurological Imaging (MNI) brain template were extracted from FSLeyes v0.3 in FSL 60. Using Vinci, the MNI-template was warped to match the participant MRI using a non-linear affine transformation. The resulting transformation matrix was applied to the striatal sub-regions, thereby warping them from standard- to subject-space. From the parametric image, we extracted GM kicer values for voxels with at least 60% probability of belonging to the functional striatal sub-regions61.The functional striatal sub-regions , defined in the Oxford-GSK-Imanova brain atlaskicer value obtained from the three sub-regions weighted by their respective volumes and compared this to the kicer value for the striatum obtained from the maximum probability atlas. Second, we repeated our main analyses with kicer values extracted from the combined GM and WM tissue, since previous studies examining dopamine synthesis capacity did not always separate these tissues. Third, in the analyses with the striatal sub-regions, we additionally investigated a stricter threshold of at least 90% probability. Fourth, we compared the mean standardized uptake values (SUV) in GM cerebellum in ASD and controls, to examine whether non-specific uptake of [18F]-FDOPA differed between groups. Finally, we repeated the main analyses for the two PET/CT-scanners separately.We conducted several additional analyses to ensure the validity of the methods that we used . Since imputation of these missing data did not change the outcomes of the study, we report the results without these data imputed.kicer between ASD and controls using ANCOVA. We examined the relationship between striatal kicer and loneliness within ASD by conducting linear regression analysis. Analyses were adjusted for age, sex, smoking-status, and scanner-type   and striatal kicer. Finally, we conducted the previously-described analyses using the kicer in the three striatal sub-regions (rather than whole striatum) as dependent variables.In the secondary analyses, group differences in social defeat were examined using independent-samples n = 1 ASD, n = 2 controls). Thus, the final sample included 44 adults with ASD and 22 controls. Table kicer values of these participants fell within the range of values of the other participants. Moreover, excluding them did not alter the results.Forty-five adults with ASD and 24 controls completed the study. For technical reasons, we were unable to use the data of three participants  and controls  in the striatum after adjusting for covariates . Moreover, as shown in Fig. M = 0.0155, SD = 0.0024; controls: M = 0.0155, SD = 0.0027; F1,60 = 0.003, p = 0.96), limbic , or sensorimotor  striatal sub-regions after adjusting for covariates.Figure β = 0.01, p = 0.96) , limbic , or sensorimotor  striatal sub-regions after adjusting for covariates.In contrast to hypothesis 1b, among adults with ASD there was no statistically significant association between loneliness and dopamine synthesis capacity in the striatum after adjusting for covariates . However, this association was largely driven by one outlier  and became non-significant after adjusting the p-value for multiple testing.The relations between the secondary measures of social defeat and striatal dopamine synthesis capacity in adults with ASD are shown in Supplement Notably, contrary to our expectations, exploratory post-hoc analyses in controls showed a significant negative association between childhood trauma and striatal dopamine synthesis capacity  reported more psychotic symptoms on the PQ-16 than controls . However, these symptoms were not significantly associated with dopamine synthesis capacity in the striatum in ASD  or in controls  after adjusting for covariates. Results were also non-significant for the striatal sub-regions chronically defeated rodents67. In contrast, in line with post-hoc analyses with reference to childhood trauma in our control group, two recent studies observed negative associations between different types of social adversity and dopamine synthesis capacity68 or amphetamine-induced striatal dopamine release69. One possibility is that social defeat only increases dopamine functioning in certain individuals and that, for example, individuals with ASD respond neurochemically different to defeat than other risk groups. Nevertheless, given the high rates of social defeat in adults with ASD and the absence of a relationship with presynaptic dopamine, one can conclude from this study that social defeat per se does not necessarily upregulate dopamine synthesis capacity.The social defeat hypothesis of schizophrenia, which predicts that non-psychotic adults with ASD show an increase in dopamine synthesis capacity and that this capacity is associated with measures of social defeat70. To the best of our knowledge, presynaptic dopamine release has not yet been examined in ASD and it might be worthwhile to do so. It should be performed with great care, however, since inducing dopamine release might precipitate psychotic symptoms.Second, dopamine sensitization might have been demonstrated if presynaptic dopamine release had been examined, for instance after a challenge with amphetamine. Since knowledge about the relationship between presynaptic dopamine synthesis capacity and amphetamine-induced dopamine release is limited, it is difficult to predict the outcome of this challengeThis study has several strengths. First, this is by far the largest PET/CT-study to examine dopamine synthesis capacity in ASD and the first to examine an association with possible risk factors. Given the (for PET/CT studies) large, a priori determined sample size, it is highly unlikely that the negative findings are due to a lack of statistical power. Second, we had strict inclusion measures, which might limit the generalizability of the findings to the entire ASD population but does reduce the influence of external factors such as (dopaminergic) medication use. Third, in addition to our main analysis with loneliness, we used multiple measures to assess social defeat, which consistently showed an absence of relation with striatal dopamine functioning.The study also has several limitations. First, the study was cross-sectional. To understand how dopaminergic abnormalities arise, longitudinal studies are necessary. Second, we relied on self-report measures of social defeat, which may be prone to several types of bias. Third, PET/CT-scans were acquired on two different PET/CT systems. However, reconstruction parameters for the two scanners were harmonized as much as possible and scanner-type was added as a covariate to the analyses. Moreover, conclusions did not change when we conducted the main analyses for the two PET/CT-scanners separately.In conclusion, non-psychotic, unmedicated adults with ASD do not show a significant increase in striatal dopamine synthesis capacity, and this capacity is not associated with measures of social defeat.Supplementary information