Synthetic acyclic receptors, composed of two arms connected with a spacer enabling molecular recognition, have been intensively explored in host-guest chemistry in the past decades. They fall into the categories of molecular tweezers, clefts and clips, depending on the geometry allowing the recognition of various guests. The advances in synthesis and mechanistic studies have pushed them forward to pharmaceutical applications, such as neurodegenerative disorders, infectious diseases, cancer, cardiovascular disease, diabetes, etc. In this review, we provide a summary of the synthetic molecular tweezers, clefts and clips that have been reported for pharmaceutical applications. Their structures, mechanism of action as well as in vitro and in vivo results are described. Such receptors were found to selectively bind biological guests, namely, nucleic acids, sugars, amino acids and proteins enabling their use as biosensors or therapeutics. Particularly interesting are dynamic molecular tweezers which are capable of controlled motion in response to an external stimulus. They proved their utility as imaging agents or in the design of controlled release systems. Despite some issues, such as stability, cytotoxicity or biocompatibility that still need to be addressed, it is obvious that molecular tweezers, clefts and clips are promising candidates for several incurable diseases as therapeutic agents, diagnostic or delivery tools. Host-guest chemistry has experienced tremendous development in the past decades. Initially inspired by Nature, synthetic receptors have been exploited to understand and mimic biological processes, such as molecular recognition, protein interactions  or DNA iThe structures we are interested herein consist in open cavities with one or two binding “arms”, which are able to complex a guest through non-covalent interactions, such as hydrogen bonding, hydrophobic or van der Waals forces, aromatic stacking or metal coordination . Several1, The term “molecular tweezers” was introduced for the first time by Whitlock in 1978 to describe a caffeine-based receptor 1,  [7]. Acc3, Molecular clefts were then described by Rebek in 1985 as rigid receptors exhibiting convergent functional groups directed toward each other and separated by a spacer 3, ,11,12. M4, 2, Klärner, then, discriminated molecular tweezers from molecular clips according to their structure and guest binding mode . In the 5, Finally, the term “molecular switches” has been used by Feringa to describe synthetic structures, including molecular tweezers, where a motion is involved 5,  14]. Af. Af5, 14Several reviews have described the structural diversity of molecular tweezers ,6 or havNature has developed complex systems to recognize selectively biological guests, such as DNA or proteins. Interfering with this process enable a pharmaceutical control over gene expression and regulation. Indeed, many drugs have been developed to interact with DNA, either by intercalation , groove binding , covalent cross-linking , alkylation of bases  or by inducing DNA cleavage  . All the6 n/MBNL1 complexes in a DM1 cell model . Th. Th25]. solvents   and trinucleotide repeats, involved in neurodegenerative diseases. They could be used as diagnostic tools, biosensors or anticancer agents as well as drug carriers. Further applications are expected in the next years since these ligands were found to interfere with DNA hybridation , replicaMolecular tweezers are biomimetic receptors inspired from the lock-and-key model of enzyme-substrate interaction. Therefore, they are excellent candidates for protein interaction, leading to pharmaceutical alteration of biological processes.3, O-methyl ether . Th. Th26, 727, More usually, boronic acids are exploited for sugar recognition, thanks to the covalent bonds they can form with sugar hydroxyl groups. James et al. acknowledge that the spatial disposition of the boronic acid moieties determines which saccharide is bound preferentially . Their sX) thanks to its cleft conformation  thIn addition to their affinity and selectivity, several molecular tweezers exhibit a dynamic behavior controlled by external stimuli such as pH, light, redox, temperature or metal . Such st29, 30, 29,2+ cation brings the recognition arms in close proximity, resulting in a closed U shape, suitable for aromatic guest intercalation. When pyridine-pyrimidyne-pyridine chelating spacer was used instead of terpyridine, 3-state U/S/W conformational metallo-switches were generated. In this second series, the initially uncoordinated tweezers adopt a U-shape . The earliest molecular terpyridine based switches able to reversibly interconvert between a U and a W shape upon metal coordination are derived from Lehn’s work 29, 30,  81]. In. In29, 3hape 30,  favored elements .2+, Zn2+, Pb2+) to the central terpyridine folds the two Pt-salphen arms towards the binding pocket resulting in a U shape -salphen complexes with terpyridine spacer to design dynamic tweezers . Metal chape 31, .2+ or Pb2+, but strongly quenched upon the selective intercalation of a second Hg2+. Such metallo-switches can be applied in the design of luminescent sensors based on phosphorescence enhancement or quenching upon cation recognition. While the closed form was supposed to accommodate a substrate between the two Pt-salphen arms, no complexation was observed upon addition of aromatics. These results were explained by the steric hindrance of the existing tert-butyl groups of the tweezer, preventing π-stacking interactions. Although adding an alkyne spacer to tert-butyl groups and reducing their number did not resolve the problem [32, These tweezers can be reopened by addition of tris(2-aminoethyl)amine as competitive ligand, enabling the reversible process upon demetallation of the central terpyridine. In the open W shape, phosphorescent Pt-Salphen moieties exhibit structured emission with a high quantum yield and long lifetime. The emission properties are slightly modified in response to Zn problem , changinblem 32, . The forblem 32, .33, Another example of Pt-salphen-based luminescent molecular switch for guest binding was reported by Wang et al. . When twLipid nanocarriers such as vesicles, liposomes or nanoparticles are being increasingly used as smart drug delivery systems due to their biocompatibility, non-immunogenicity and high loading capacities. Stimuli-responsive lipid switches have been incorporated into the bilayer where they undergo a conformational switch, in response to the binding of ions, triggering membrane destabilization and cargo release . It has 34, One of the most explored stimuli is pH. pH variations can be found in the gastro-intestinal tract (pH 2–8), in inflamed tissues (pH 6.5–7), in interstitial tumor tissue (pH 6.5–7) or within intracellular compartments, such as endosomes (pH 5–6) or lysosomes (pH 4–5) . Althoug35,a close to 5, suitable for endosomal escape. The corresponding liposomes massively released their content upon acidification, reaching 88% release when pH dropped from 7.4 to 5 in less than 15 min [Further pH-sensitive lipids based on an anisole-pyridine-anisole tricycle were developed in our group 35,. Previoun 15 min . Interesn 15 min . In a ren 15 min .2+ coordination by carboxylic acids induced a spatial reorganization, moving the lipid chains away from each other -dipicolylamine (ZnDPA) 37, . This smStarting from simple synthetic receptors, supramolecular systems have grown in complexity and control leading to molecular motors and nanomachines, recently rewarded by the Nobel Price of Chemistry awarded to Stoddard, Sauvage and Feringa . Such maRecognition between the tweezers/clips/clefts and a substrate requires a complementarity between the receptors and their guests. In the examples described above, it is clear that the geometry or conformation of the substrates, the size of the binding cavity as well as the nature and the number of interactions determine the binding process. For instance, the dynamic metallo-switches described above require metal coordination to switch the host’s conformation to a U shape, favorable for guest inclusion. In the Klarner’s library, the geometry of the receptors and the size of their cavities were tuned by the number and the nature of arenes. Reducing the number of methylene bridges resulted in larger cavities able to surround large guests. The distance between both sidewalls can be tuned using benzene, naphthalene or anthracene naphthalene-spaced clips. Belt-type tweezers 2 that showed a better affinity toward aliphatic guests rather than aromatic ones, were found to undergo a substantial distortion of the receptor geometry upon complexation of benzene rings . FinallyWe truly believe that molecular tweezers can be further explored in pharmaceutical applications, and gain to be known from the pharmaceutical sciences community. Therefore, in this outlook, we summarize the progress and envisage the future applications of tweezers in the following questions:(i)A new drug candidate. In this category, CLR01 is the most advanced compound, showing multiple applications and preclinical data. To progress in the drug development process, the drug-like properties need to be established  and might represent a challenge due to the generally hydrophobic aromatic structure.(ii)A biosensor, for the detection of various molecules or supramolecular assemblies, from cations to DNA mismatches. In this field, the highest challenge is the selectivity and sensitivity of the system in biological medium.(iii)A drug delivery system, usually with responsive properties. The conformational change offers a faster kinetics and a better controlled assembly over other responsive systems, such as hydrolysable polymers. Nevertheless, the biocompatibility and stability of such new excipients with biological media need to be investigated.To summarize the pharmaceutical applications of the molecular tweezers presented in this review, we could say that the tweezers are currently used as:Such concepts have been explored in a large range of diseases, depending on the substrate or the biological process involved, as illustrated on The past years have focused the efforts on optimizing the structure, to gain in selectivity and affinity for their biological targets. Such feature is definitely an asset for drug development, since it ensures a good efficiency and would limit the off-target or side effects. It has to be noted that the synthesis of such structures is already a challenge in itself because the 3-D structure is so precise that it might not suffer a variation of one or few atoms. The next challenges would concern their successful translation in vivo. While Klarner’s molecular tweezer CLR01 proved to be potent in the treatment of various amyloidogenic diseases and showed to be safe in animals , many syAnother—and related—issue would concern the toxicity of such compounds. Although inspired from nature, their polycyclic and/or aromatic composition is far from the natural materials of living organisms . The poor water solubility and the aromatic structure of several tweezers might promote the toxicity of such compounds. Unfortunately, Zimmerman’s tweezers showed poor water solubility and significant toxicity in vivo experiments, making them inappropriate for therapeutic applications . Here agTo overcome biocompatibility issues, synthetic mimics of biomolecules made of nucleic acids, aptamers, nucleotides and peptides have been developed for targeted therapies. For instance, DNA switches are extensively used as sensors  and nuclFinally, another promising development of molecular tweezers is their use as non-covalent linkers in supramolecular structures. Wang and coworkers exploited their host/guest properties to monitor the assembly of chain-like polymers, dendrimers and incorporated them into nanoparticles . As comp


Mycobacterium bovis, remains a major livestock and public health problem in both high and low-income countries. With the current absence of an effective vaccine, control in cattle populations is reliant on regular testing and removal of positive animals. However, surveillance and control are hampered by imperfect diagnostic tests that have poorly described properties in naturally infected populations. Recent research in cattle co-infected with the temperate liver fluke, Fasciola hepatica, has raised concerns about the performance of the intradermal skin test in high fluke incidence areas. Further, recent studies of parasitic co-infections have demonstrated their impact on Th1 and Th2 responses, concurrent disease pathology and susceptibility to mycobacterial infections. Here we report for the first time the association of co-infection with the tropical liver fluke, Fasciola gigantica, with the presence of bTB-like lesions and the IFN-γ response in naturally infected African cattle. After adjusting for age and sex we observed a complex interaction between fluke status and breed. Fulani cattle had a higher risk of having bTB-like lesions than the mixed breed group. The risk of bTB-like lesions increased in the mixed breed group if they had concurrent evidence of fluke pathology but was less clear in the coinfected Fulani breed. Further, we observed a slight decline in the IFN-γ levels in fluke infected animals. Finally we explored factors associated with IFN-γ false negative results compared to the presence of bTB-like lesions. Fulani cattle had a higher risk of having a false negative result compared to the mixed breed group. Further, the mixed breed cattle had an increased risk of being false negative if also co-infected with fluke. Interesting, as with the risk of bTB-like lesions, this association was less clear in the Fulani cattle with weak evidence of a slight decrease in risk of having a false negative test result when fluke pathology positive. This interesting interaction where different breeds appear to have different responses to co-infections is intriguing but further work is needed to confirm and understand more clearly the possible confounding effects of different other co-infections not measured here, breed, management or exposure risks.Bovine tuberculosis (bTB), caused by  Mycobacterium bovis infection has been studied in isolation until relatively recently. Co-infections with Fasiola hepatica have been implicated as a potential reason for poor bTB diagnostic test performance and disagreement between tests (F. hepatica have been shown to down-regulate the Th1 responses (with a resultant dampening of the IFN-γ response), with subsequent predominance of Th2 responses, in order for the parasite to survive and reproduce . In many high-income countries, such as the United Kingdom and New Zealand, compulsory bTB “test and slaughter” programs coupled with compensation have been successful in reducing transmission of M. bovis in livestock populations (M. bovis (Bovine tuberculosis (bTB), caused by the bacterium zoonosis , 10 causzoonosis  amountinulations –14. Diagulations , to remoM. bovis . HoweverM. bovis  resultinF. hepatica co-infection is associated with a reduced Th1 immune response  and a single retropharyngeal lymph node per animal collected for culture as controls.Data were collected at the Ngaoundere municipal abattoir in the Adamawa Region, a major cattle-producing area of Cameroon Figure . The detTissue samples (lesioned lymph nodes) were stored in the vapor phase in liquid nitrogen dry shippers (Taylor-Wharton) and shipped to the Tuberculosis Reference Laboratory (TBRL) Bamenda. Upon arrival at the TBRL the samples were stored at -80°C until processed. Heparinised blood samples were stored in a coolbox at the abattoir (ranging between 10°C to 26°C) and then taken to the lab and kept at room temperature prior to being stimulated in the IFN-γ assay (Bovigam®) described below. Animal data recorded on paper in the abattoir, was transferred to a relational Microsoft Access database where the results could be linked back to individual animals.F. gigantica infection by MINEPIA meat inspectors. The meat inspectors examined the liver systematically to identify gross pathology associated with Fasciola infection by slicing down the common bile duct with an additional 1–2 slices through the liver parenchyma. Once an animal was identified to have gross fasciolosis related pathology, the liver was graded by a member of the research team and scored 0–3  or absence (negative) of F. gigantica pathology for subsequent analysis.All carcases were inspected for evidence of ored 0–3 . A scoreThe tissue samples were prepared and cultured as previously described  followinhttp://www.theincubatorshop.co.uk) run in the field. Following incubation samples were centrifuged at 300g for 10 minutes, the plasma was aliquotted and stored at −20°C in a portable travel freezer (Waeco CF50 12V/240 fridge freezer)). Plasma samples were transported at −20°C to the LEID and the IFN-γ ELISA was conducted as per the published protocol. The acceptable averaged negative bovine OD value was < 0.130 and positive bovine control was >0.700. Animals with a bovine stimulated sample optical density of ≥0.1 above that of the avian PPD sample were classified as test positive and interpreted as the animal being infected with M. bovis.The IFN-γ ELISA (Bovigam®) was conducted as per published protocol , 38. BriFasciola pathology and one of the bTB outcomes .The proportions of cattle with bTB-like lesions, a positive IFN-γ result and liver fluke pathology were calculated and various co-infection definitions were explored using M. bovis culture results. Animal-level explanatory variables  were always included in the models as fixed effects to control for confounding. Model selection was based on the AIC and the best model was selected using the lowest AIC and ΔAIC  models were developed to explore the association between fluke infection and bTB status using a number of different definitions including IFN-γ, bTB-like lesion and and ΔAIC . MLR mod package  with AIC package . PredictSome variables were simplified due to small numbers of observations in some categories. The dentition score (DS) was simplified from number of permanent teeth to a binary age category based on the approximate relationship between age and eruption of permanent incisors in cattle. DS <2 was categorized as “ <3 years old” and DS of ≥2 was categorized as “≥3 years old.” The breed variable was simplified to “mixed”  or “Fulani” (collapsing Red and White Fulani to a single category).During a 4 week period in August 2013, 935 cattle were examined at the Ngaoundere abattoir. The details of bacterial culture results have been presented elsewhere . A total2 test p-value <0.001) but the odds ratios did not change across pathology scores above zero so the effect is captured by collapsing the fluke score into a binary variable  of animals had visible bTB-like lesions observed. The proportion of animals with evidence of liver fluke infection was 49.6% (363/732) and the proportion of animals positive by the IFN-γ assay was 6.6% (48/732). The distribution of the prevalances of bTB-like lesion, liver fluke pathology and IFN-γ based on animal dentition (as a measurable proxy for age) are given in Figure  of animae Figure .F. gigantica pathology status with the probability of observing visible TB-like lesions in cattle in this setting. Fulani cattle had a higher risk of having observable bTB-like lesions than the mixed breed group. However, the risk in the mixed breed group increased if they also had fluke pathology. This association ith fluke pathology was less clear in Fulani cattle. For example, an adult, female, mixed breed animal, that had no fluke pathology had a ~5.2% (95% CI: 2.5–7.8) probability of having a TB-like lesion compared to ~27.7% (95% CI: 16.8–38.4) for a Fulani animal  model of the association between visible lesion and fluke status was developed Table . This sul Figure . The preF. gigantica pathology status was investigated. The raw PPD-B minus PPD-A difference in ELISA OD readings were explored in the subset of animals which were confirmed M. bovis culture positive (n = 53). The raw difference is plotted, stratified by F. gigantica pathology status in Figure p < 0.001, n = 52).The association between IFN-γ result and F. gigantica pathology status, with fluke pathology positive cattle more likely to have a lower OD value difference and to be IFN-γ negative. The association using the binary test results suggest an adult, female, mixed breed cow had a ~5.6% (95%: 3.1–8.1) probability of testing positive which dropped to ~3.5% (95%: 1.7-5.6) if infected with liver fluke compared to a Fulani, adult, female animal which had a ~16.3% (95%: 8.7–24.0) of testing positive which dropped to ~11.1% (95%: 5.9–16.2) if fluke pathology positive  and after accounting for age, sex and breed there was a small mean decrease in the difference of −0.02 (−0.04 to 0.00) in OD value in the fluke pathology positive animals Table . Both the Figure .n = 684) a new variable was generated where a IFN-γ test result was classified as a false negative if there was an observed bTB-like lesion (n = 54) and true negative if there was no lesion observed (n = 630). A multivariable logistic regression analysis to explore the association with fluke pathology was conducted including sex, age and breed as confounders. In addition, for this model the presence or absence of non-tubercular mycobacteria (NTM), based on the Haines typing from cultured samples from lesions, was also included as a known potential confounder for being lesion positive but test negative. The final model is given in Table Using the subset of results where the IFN-γ test result was negative n = 68 a new vaF. hepatica have been shown in the United Kingdom  IFN-γ response in a naturally F. gigantica infected cattle population in Africa.It is well recognized, though still poorly studied, that co-infecting pathogens can have a range of synergistic or antagonist effects. For example,  showed A Kingdom , 3 but lnfection . Here weM. tuberculosis have higher bacterial loads and TB lesion pathology when co-infected with the trematode S. mansoni . Alternatively, the breed association may be due to confounding by other unobserved variables such as differing management between breeds or differences in exposures interacting with different genotypes of M. bovis infections as part of control programs often in combination with the SICCT (F. hepatica co-infection can down-regulate IFN-γ responses to M. bovis infection (The IFN-γ assay is particularly useful to detect early (1-4 weeks post infection) he SICCT , 47. Prenfection , 22. In M. bovis and that a number of these animals with lesions had NTMs based on culture results . Using visible bTB-like lesions as the comparison test, which we know from culture results from these cattle is reasonably specific (sp = 69.7%) and sensitive (95.8%),  or being able to incorporate the likely impact on test performance may improve ante-mortem diagnostic test sensitivity within cattle or wildlife populations.It is clear that co-infections can have complex impacts on test diagnostics and pathogen invasion and this may have important implications for control programmes. As one bacillus is sufficient to establish n a host , leavingF. gigantica on the pathology and detection of M. bovis infections in a natural ecological setting in African cattle. We have shown a complex association between the presence of visible bTB-like lesions in carcasses and the presence of concurrent Fasciola infections which appears to be also affected by breed. Furthermore, we have shown that the IFN-γ response may be slightly dampened down in F. gigantica infected cattle although further data are needed to confirm this. However, it does appear to be sufficient to increase the false negative risk in the mixed breed group at lest in this population. The reduction in sensitivity of the IFN-γ assay by Fasciola spp. co-infection could have profound effects on bTB control and eradication programs as Fasciola spp. are present worldwide  Act, 1986) (ERC No: OS02-13).BB, RK, LN, VT, KM, RN, MS, and NE: conceived and designed the study; RK, SM, NV, NE, and AM: performed the field work; RK, BB, IH, SM, and RC: analyzed the data; RK, BB, IH, SM, MS, and DW: contributed reagents, expertise, materials, analysis tools; RK and BB: wrote the first draft paper; All authors read and contributed to the final draft of the paper.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.


This first report of ultra-high molecular weight (>1000 kDa) linear coordination polymers demonstrates their use in agricultural spray drift control. Mn > 1000 kDa) polymeric drift control adjuvants (DCAs) for agricultural spraying are prone to mechanical degradation and rapidly lose performance. To overcome this, we have designed linear coordination polymers (LCPs) composed of 400 kDa telechelic bis-terpyridine end-functionalised polyacrylamide units, which ‘self-heal’ upon shearing through reformation of coordination bonds. After addition of Fe(ii) to dilute aqueous solutions of the terpyridine telechelics, UHMW LCPs were obtained as demonstrated by UV-vis spectroscopy, MALS GPC and intrinsic viscosity measurements. Importantly, these UHMW LCPs were shown to function as effective DCAs, reducing the formation of fine ‘driftable’ droplets during spray testing at concentrations as low as 100 ppm. Following mechanically-induced coordination bond-scission, the UHMW LCPs were found to recover up to 90% of their performance compared to un-sheared samples, at a rate dependent on the transition metal ion used to form the complex.Ultra-high molecular weight (UHMW,  Mn > 1000 kDa) linear polymers are able to increase solution extensional viscosity at concentrations as low as 100 ppm, with application in mist control,9Ultra-high molecular weight  and association constant (Ka) (eqn (1)),16To overcome this loss of performance for DCAs, we investigated if linear coordination polymers (LCPs) could associate into UHMW species in dilute aqueous solution, and if so, reversibly break and reassociate after shearing. Due to the dynamic property of LCPs, their molecular weight is a function of many variables including concentration, which complicates comparisons across literature. However, in general, molecular weights of LCPs have been limited to the order of 100 kDa at concentrations of 5–20 mg mLUnder dilute conditions the formation of cyclic structures or rings is increasingly favoured, which can act as chain stoppers and reduce LCP molecular weight.et al. investigating the formation of shear stable polymers for reducing jet fuel (organic non-polar solvent) misting. Ultra-high molecular weight ‘megasupramolecules’ were synthesised by end-functionalising large (up to 670 kDa) poly linkers with hydrogen bonding units, resulting in the dilute solution (2 mg mL–1) formation of supramolecular polymers of Mw ≈ 2200 kDa.Limiting cyclic species formation with high molecular weight linker units >100 kDa) has been less explored, and utilises the idea that rings are less likely to form from larger chains due to an increase in the entropic penalty of cyclisation.00 kDa haHerein we report the first UHMW LCP formed in dilute aqueous solution through the use of high molecular weight (∼400 kDa) polyacrylamide linker species end-functionalised with terpyridine ligands. The telechelic polymers were synthesised by RAFT polymerisation of acrylamide (AM) with bis terpyridine-functionalised CTAs, and the resultant LCPs were characterised by GPC, viscometric, screen factor, spray testing and shearing analysis.With the desire to form UHMW LCPs from high molecular weight ∼400 kDa) coordination ligand end-functionalised telechelics, an appropriate means of synthesising such telechelics with high end-group fidelity was needed. When functionalising polymers of high molecular weight, post-polymerisation modification becomes increasingly problematic due to non-quantitative conversion, exacerbated by the high dilution of end-groups.0 kDa coop-methylenephenyl)-2,2′:6′,2′′-terpyridine) trithiocarbonate (tpyTTC) was then synthesised according to a modified literature procedure  monomer in DMSO (where polyacrylamide is soluble at low molecular weights), before chain-extending with AM to approximately 400 kDa (Mn > 1000 kDa) AM polymerisations.45Polyacrylamide is only soluble (particularly at high molecular weights) in water, where both terpyridine RAFT agents are insoluble. To overcome this, macro-CTAs were first synthesised with either AM or  400 kDa . Both ma1H NMR α-proton signal (4.3–5.1 ppm), which is expected to integrate to 2 when each of the terpyridine aromatic proton signals is set to 4   which may in part be due to end-group column interactions  demonstrated the utility of tpyDMAT to successfully form terpyridine end-functionalised polymers by RAFT polymerisation, as determined by GPC and s Fig. S7, furtherii) to these polymers formed the expected purple colouration due to formation of the Fe(ii) bis-terpyridine complex (λmax = 566–568 nm). The Fe(ii) complex was chosen for initial study as it produces a strong visible absorbance clear of other signals and has high stability constants, making it likely to ultimately facilitate successful association into UHMW LCPs. By measuring the peak absorbance value and assuming an end-group fidelity of 100%, an Mn,UV value was calculated for all polymers , in close agreement with Mn,UV (570 kDa).For example, additions of Fe(ii) determined by UV-vis spectroscopic monitored titrations, resulted in the highest molecular weight supramolecular species. Increasing equivalents of Fe(ii) were added to separate 4 mL solutions of T2 (10 mg mL–1), which were then analysed by UV-vis spectroscopy and diluted for relative viscosity, GPC and screen factor (SF) measurements  was observed with increasing equivalents of Fe(ii) until the peak absorbance at 566 nm was recorded. This point corresponds to the experimentally determined stoichiometric equivalence, and further additions of Fe(ii) beyond this point resulted in a reduction in molecular weight. This reduction is expected, as overtitration of Fe(ii) increasingly favours the formation of mono-complexes, reducing the supramolecular DP.Mn,GPC).An increase in all measurements related to molecular weight  400 kDa terpyridine end-functionalised telechelics was observed (ii) complex of T2 resulted in LCPs of similar molecular weight to the Fe(ii) species, while minimal (Co(ii)) to no (Cu(ii), Zn(ii)) increase in molecular weight was observed for the other complexes  and Ni(ii) terpyridine complexes are needed to associate high molecular weight species in dilute solution (≤10 mg mL–1). For simplicity, studies on the formation of UHMW LCPs continued to focus on the Fe(ii) complexes, with the Ni(ii) complex used later during shear recovery experiments.After establishing the utility of UV-vis spectroscopic monitored titrations, other  complexes of T2 were synthesised using the same titration method. Strong qualitative agreement with the trends in observed . The Ni titrations monitored by SF measurements and UV-vis spectroscopy were also performed on T1 and T3. Similar to T2, both T1 and T3 were found to have an increased molecular weight upon Fe(ii) addition, with this maximum occurring at approximately the equivalence point determined by absorbance at 566 nm (ii) complexation. The deviation from an increase in SF with increasing Fe(ii) addition between 0.4 and 0.6 equivalents may be related to inhomogeneities during mixing (and LCP formation) due to the extreme extensional viscosities encountered at 10 mg mL–1 for these species  as the target DP was increased.To assist molecular weight characterisation of the LCPs, control polymers were synthesised by RAFT polymerisation with DMAT  as the CTA C1–C4, . Since tMn,GPC = 350 kDa, Đ = 2.59).In addition to the series of narrow MWD RAFT polymer samples, a commercial  polyacrylamide labelled as 5000–6000 kDa (AM-5-6M) was also used as a benchmark, representative for the type of polyacrylamide currently used in polymeric DCA formulations. This was characterised by the Waters aqueous GPC, highlighting its broad MWD nature (–1 (ii) LCPs (T1 Fe(ii), T2 Fe(ii) and T3 Fe(ii)) are also presented  LCPs are due to the presence of higher molecular weight species than in C4, a minimum supramolecular DP of 3–6 for each LCP can be estimated .The control RAFT polymers were then analysed by SF measurements at 1 mg mL–1 , where l–1 . For refMn > 1000 kDa) species in dilute solution after Fe(ii) complexation. This conclusion was supported by further characterisation on T1 Fe(ii) and T3 Fe(ii) (complexed at 10 mg mL–1).From these SF results it appears that (after careful optimisation of polymerisation conditions) both tpyDMAT and tpyTTC RAFT agents are capable of synthesising ∼400 kDa acrylamide polymers of the required end-group fidelity to associate into UHMW (ii) and the control polymers (η]) is evaluated by extrapolating the reduced viscosity (ηr) of a sample to zero concentration and is related to the polymer molecular weight  by the Mark–Houwink–Sakurada equation. The constants of this equation depend on the polymer, with the values for polyacrylamide presented in eqn (2).47Intrinsic viscosity analysis, as has been previously used to evaluate the molecular weight of terpyridine coordination polymers,polymers . IntrinsMw,visc was in qualitative agreement with Mn,conv. . Importantly, Mw,visc for T1 Fe(ii) was 2180 kDa, indicating the presence of species with supramolecular DP ≥ 4, in agreement with the SF results. The formation of UHMW species for T3 Fe(ii) was supported by absolute molecular weight (MALS GPC) analysis, which recorded a shift from Mn,GPC = 551 kDa (Đ = 1.28) to Mn,GPC = 1056 kDa (Đ = 1.16) after Fe(ii) complexation.It was found that for the control RAFT polymers (C1–C4), Mn,conv. , while fii) and T3 Fe(ii). Many different parameters can be used to describe spray droplet size distributions  was used to quantitate the volume of a spray particularly susceptible to drift. Three different common types of commercial DCAs ) were first tested at standard application concentrations and were found to reduce the V105 from 29%  to 8–12%  was able to significantly reduce V105 at all concentrations (1000 ppm not tested). This data indicates that a minimum molecular weight of approximately 1000 kDa is needed to effectively reduce formation of fine droplets, in general agreement with values reported in the literature for polyethylene oxide based DCAs (minimum of 300–500 kDa).Solutions of the control RAFT polymers (100–1000 ppm) were then analysed under the same conditions , with C1ii) and T3 Fe(ii) were then analysed  and clear reductions in the V105 were observed at all concentrations, with similar performance to that of C4 (V105 of 0.36% was recorded for T1 Fe(ii), however in practice such a high concentration would likely not be used as the simultaneous increase in the formation of large droplets would be detrimental to plant coverage. At 200 ppm (which had a much milder effect on large droplet production), T1 Fe(ii) and T3 Fe(ii) were found to either outperform or match the commercial DCAs with V105 reduced to 6–11%, respectively. This data strongly confirms that these coordination polymer systems are able to form UHMW linear polymer species, which remain present even under extreme dilutions of 100 ppm.The LCPs T1 Fe(at of C4 . At 1000ii), their ability to recover from shear degradation was compared to conventional UHMW covalent polymers. Preliminary shearing experiments identified that shearing AM-5-6M solutions  in an Ultra-Turrax mixer at 3200 RPM for 60 minutes or 5000 RPM for 15 minutes resulted in a 77–91% reduction in SF  terpyridine end-functionalised polymers complexed to Fe and T3 Fe(ii) at 1000 ppm. Solutions were prepared by diluting the un-complexed polymer to 1000 ppm, then adding in the required amount of metal ion. This dilute addition of metal ion was conducted so that the initial un-sheared SF measurements of the LCP would be conducted at equilibrium state, representative of the theoretically ‘fully recovered’ polymer. After an initial SF measurement, the LCP solutions were then divided into three samples; one which would remain un-sheared, one for shearing at 5000 RPM for 15 minutes and one for shearing at 3200 RPM for 60 minutes. Immediately after shearing the samples were analysed by SF again to characterise initial degradation, then further measurements over time were conducted to evaluate the rate of recovery.The effect of these shearing treatments was then examined on both T1 Fe(ii) found that a significant reduction in SF over time occurred for the un-sheared sample, with a reduction from 8.0 to 1.9 over 11 days (ii) is likely, at least in part, due to the presence of a hydrolysable ester linking group between the main-chain and terpyridine end-functionality. To highlight this point, the solution stability of T1 Fe(ii) was compared with T3 Fe(ii), which does not contain an ester linking group (ii) was also observed (4.8 to 3.4 over 10 days), however this was at a significantly reduced rate than for T1 Fe(ii). The cause of SF reduction in the case of T3 Fe(ii) was not determined, however gradual hydrolysis of the central trithiocarbonate group is a likely candidate.Experiments on T1 Fe( 11 days . With su 11 days . This aqng group . Some reii) were then conducted and both shearing treatments resulted in a large initial reduction in SF from 4.5 to 2.1  and 4.5 to 2.3 . This was then followed by a slow recovery over time, which peaked at an SF of approximately 3.0 for both treatments after 5 days, with the majority of this recovery occurring within the first 24 hours , the presence of some telechelic species large enough to undergo covalent bond scission during shearing is possible.Shear recovery experiments for T3 Fe LCPs. Given that increases in SF were observed up to 5 days after mechanical shearing and that only 1 hour is needed to form the Fe(ii) LCP from an un-complexed polymer, recovery through re-association of completely dissociated ligands is unlikely to be the only pathway and may not contribute at all. Therefore, loss in molecular weight after shearing may be due to the formation of cyclic species rather than an increased presence of un-complexed ligand, as was proposed by Sijbesma et al. when investigating smaller LCPs after sonication induced coordination bond scission.ii)–terpyridine complex is relatively long (140 hours at 35.4 °C). By using this insight, the rate of recovery was improved by trialling the Ni(ii) complex, which has a ligand exchange half-life approximately 4.5 times shorter (10.2 hours at 44.8 °C).These results also provide some insight into the mechanism of shear recovery for UHMW terpyridine–Fe(ii) complex to fully form in dilute solution (likely due to its lower formation rate constants) and therefore the shearing was applied 24 hours after Ni(ii) addition to allow for full complex formation (ii) complexed polymer. After shearing, a noticeably accelerated rate of recovery was observed (compared to the Fe(ii) sample), with SF increasing from 2.0 to 3.4 after 24 hours from treatment at 5000 RPM (15 minutes). In the same amount of time, the Fe(ii) sample SF only recovered from 2.1 to 2.6 and eventually reached 3.0 after 5 days. Similar to the Fe(ii) complex, a peak recovery up to 91% of un-sheared SF was observed  complex having a slower association rate, but quicker ligand exchange rate, this data further supports the theory that recovery from mechanical shear for UHMW LCPs is governed by ligand exchange interactions.Preliminary data indicated that 24 hours was needed for the Ni and T3 Ni(ii) before and after (point of greatest recovery) shearing. This demonstrates that (when given enough time) UHMW LCPs can outperform traditional UHMW covalent polymers in terms of minimising SF reduction after shear treatment. On the way to seeing the developed UHMW LCPs applied in spray drift reduction, further investigation into the cause of SF reduction over time in water will be needed, as well as the testing of these polymers in active solutions .To get a picture for how these UHMW LCPs compare to the current covalent polymers after shearing, the data for AM-5-6M before and after shearing at 5000 RPM for 15 minutes (100 ppm) is presented in Mn > 1000 kDa) containing the terpyridine ligand. Telechelic polymers of approximately 400 kDa were synthesised by RAFT polymerisation of AM with bis-terpyridine end-functionalised macro-CTAs. The terpyridine end-functional polymers associated into UHMW LCPs after Fe(ii) complexation, as determined by SF, MALS GPC and intrinsic viscosity measurements. In addition, the UHMW LCPs were found to be effective DCAs, reducing the formation of fine driftable droplets (those <105 μm in diameter) during spray testing at concentrations as low as 100 ppm. These LCPs were also shown to recover from mechanically induced coordination bond scission, with the rate of partial (up to 90% of an un-sheared sample after the same time in solution) SF recovery found to be dependent on the transition metal ion. The Fe(ii) complex recovered over approximately 4–5 days, while the Ni(ii) complex required only 24 hours.This report details the synthesis, testing and optimisation of UHMW LCPs (There are no conflicts of interest to declare.Supplementary informationClick here for additional data file.


Antimicrobial resistance (AMR) is an emerging threat to public health worldwide. A significant evidence has suggested that the knowledge and attitude trends among the community, pharmacists and physicians can play a critical role in managing the ever increasing threat of AMR.A cross-sectional survey was performed using three specific self-administered questionnaires for community members, pharmacists/pharmacy owners and physicians on a randomly selected sample population of 473, 424 and 308 respectively. Bivariate and multivariate logistic regression and Pearson chi-square tests were performed during data analysis.n = 385), 37.7% (n = 160) and 53.9% (n = 166) was achieved for general community, pharmacists/pharmacy owners and physicians respectively. More than half of the community participants  possess poor knowledge of AMR. Furthermore, knowledge and attitude of the community participants were also found to be significantly correlated (r2 = 0.02) with each other. In 90.6% (n = 145) of the pharmacies included in the survey, a qualified pharmacist was not present at the time of the operations. Only 36.9% physicians (n = 60) knew about the environmental route of dissemination of AMR. Majority of the physicians agreed that AMR is a global problem and also recognize the need for initiating AMR stewardship programs.A response rate of 81.2%  contains supplementary material, which is available to authorized users. Antimicrobial resistance (AMR) is one of the most serious and urgent public health concerns which can negatively influence healthcare, veterinary, and agriculture worldwide . The incLike the rest of the world, an upsurge in infections with resistant strains has been reported in Pakistan , 5. The Abuse of antibiotics may arise from a complex interaction between numerous factors, such as patients’ knowledge, beliefs, and attitudes towards antibiotic use, self-medication, patients’ expectations, and patients’ experience with antibiotics . The rolWorld Health Assembly had endorsed a global action plan on AMR in May 2015. Raising public awareness and improving understanding of AMR are the key strategic objectives of this comprehensive plan on AMR. Reports from the world health organization (WHO) have suggested the monitoring and educational interventions aimed at rationalizing the antibiotics prescription, disposal, and consumption to curb AMR [2 and an estimated population of 3,893,672 people [A quantitative and cross-sectional survey was performed in Sialkot, a district in northeastern Punjab with an area of 3016 kmSample size, for the population of 3,893,672, was calculated by using the Raosoft sample size calculator using a confidence interval of 95%. An expected response of 50% was selected as there was utterly no idea about the knowledge and attitude of general public based on the absence of similar studies about the perceptions, knowledge, and attitude of the community members from Sialkot and other cities of Pakistan. The sample size of 385 was selected for the community/consumers. A stratified sampling method was used to approach 476 community members to ensure 385 filled questionnaires. Community members belonging to educational institutes, housewives, businessmen and also from other professions working in the premises of district Sialkot were included in the study. A total of 424 registered pharmacies and 308 physicians currently practicing in Sialkot were also approached to participate in the study.A literature review of similar studies was conducted in order to identify potential questions for the questionnaires used in the study , 17, 18.Similarly, structured questionnaires were also designed for pharmacists/drug store owners and physicians. The questionnaires were first validated and tested for its’ readability by a group of physicians and pharmacists in a pilot study . Both the questionnaires were developed after an extensive literature review of the studies highlighting the role and attitude of pharmacists and healthcare professionals in the context of AMR , 17 and The reliability of the data analyzed in our study was first assessed using Cronbach’s α test . The tesp < 0.05 was accepted in this study. In the bivariate logistic regression, the model was built for all the demographic variables i.e., gender, age, residence, occupation, and monthly income were included whereas in the multivariate logistic regression model only education and age were included. A simple linear regression analysis test between knowledge and attitude of the community members was also performed, and autocorrelation between the two variables was also evaluated by computing Durbin-Watson value. A Pearson Chi-Square test was also applied to test the correlation between the AMR knowledge and availability of the pharmacists.All the questionnaires were manually checked and incomplete questionnaires were not included in the final analysis. All the completed questionnaires were entered into Epi-data version 3.1 and the data was exported to SPSS version 20 for analysis. The results were summarized by mentioning percent distribution in different categories. Association of the sociodemographic characteristics of community respondents with the knowledge of AMR and attitude of antibiotic usage was evaluated by using the bivariate and multivariate logistic regression respectively. A bivariate logistic regression analysis was performed for knowledge because it has been categorized only into two levels (high and low), whereas a multivariate logistic regression model was used for attitude which has been divided into three different categories i.e., bad, average and good. Statistical significance of n = 217) were females, 41.8% (n = 161) had college/ university level education and 33.5% (n = 129) were students (Table n = 214) of the community members under study were possessing low knowledge about AMR whereas 44.4% (n = 171) were categorized in high knowledge possessing group. The participants were also asked about another chronic and life-threatening disease Hepatitis C, and 67.0% (n = 258) people have answered both the relevant questions correctly. The distribution of community respondents according to the bad, average and good attitude was found to be 61.8% (n = 238), 35.1% (n = 135) and 3.1% (n = 12) respectively.A total of 385 community participants were included in the study. Of the 385 participants, 56.4% n = 27 were fen = 80) of the community members in our survey have agreed on disposing of the antibiotics with household waste  = 1.275) was found to be significantly affecting knowledge score of respondents whereas in case of multivariate logistic analysis both age and education as dependent variables gave a perfect model fit (p = 0.169). The results of the full model shows an insignificant effect of the independent variables on the attitude score of the community respondents. However, when the correlation between knowledge and attitude of the respondents was evaluated by simple linear regression, a significant correlation (p = 0.002) was found between the two variables (r2 = 0.02). Moreover, Durbin Watson value (1.71) also confirmed that the data in the two variables was not auto-correlated.About 20% (n = 150) whereas hospital associated pharmacies were only 6.6% (n = 10). Surprisingly in 90.6% (n = 145) of the pharmacies covered in the survey, a qualified pharmacist was not present at the time of operations. 86.2% (n = 125) of the non-qualified pharmacy owners have poor knowledge about AMR and its stewardship programs whereas 73.3% (n = 11) qualified pharmacist not only possess good knowledge of antibiotic resistance but also understands the necessity of the return unwanted medicine (RUM) and AMR stewardship. About three-fourths of the pharmacy owners 74.4% (n = 108) have agreed that patients demand antibiotics from them without the prescription and about 76.6% (n = 111) have also acknowledged the need for antimicrobial stewardship program in Sialkot  was assumed i.e., no relationship exists between the availability of the pharmacist and AMR knowledge. The χ2  value was found to be 30.850  which leads to rejection of null hypothesis i.e., both variables are dependent on each other and significant relationship is present between AMR related knowledge and availability of pharmacists.A total of 424 pharmacies in the vicinity of Sialkot city were approached. Out of which only 160, with 37.7% of response rate, has participated in our study. Independently operated pharmacies included in the study were 93.8% (n = 166) have provided consent and agreed to participate in the study. A majority of physicians i.e., 83.1% (n = 138) recognize that AMR is a worldwide problem while a similar percentage of physicians i.e., 82.6% (n = 137) agreed that patients with common cold demand antibiotics from them. Although only 30% (n = 53) physicians had received any AMR related training in last three years but about three fourths (74.1%) of the total physicians felt the need for the educational/awareness programs for enhancing the knowledge about AMR in the community. When asked about the environmental spread of AMR, surprisingly, only 60 physicians (36.9%) knew about the environmental route of AMR dissemination  of the total community participants had depicted low knowledge about AMR. This percentage was closed to the knowledge percentages reported in similar studies performed in Kuwait and Ethiopia [n = 94; 24.4%) and ‘Antibiotics are an effective therapy against viruses’ . This shows confusion exists among the general community regarding the accurate knowledge about antibiotics efficacy which could have severe implications owing to over the counter access of antibiotics to general public due to the non-regulated drugstores and pharmacies. The significant difference in the knowledge about the AMR and hepatitis C in the general community member could be due to various hepatitis C related awareness campaigns in the region which have educated the general community about the risk, prevention, and treatment of the chronic disease [Primary data about the antibiotic abuse and subsequent resistance is scarcely available in Pakistan. Lack of funding from government and other NGOs in this field is the most probable reason for this data scarcity. Excessive and unnecessary use of antibiotics in hospitals of Pakistan is already well known . The govEthiopia , 18. Com disease . AMR cen disease –25. AddiApart from the consumer, community pharmacists and pharmacy owners also has an important role in ensuring widespread dissemination of knowledge about the proper use and disposal of drugs. To our surprise, a qualified pharmacist was not present in majority of pharmacies at the time of data collection in Sialkot district. Pharmacy owners and non-qualified staff were found to be managing the prescriptions from physicians. This depicts the dearth of regulations in pharmacy stores particularly in Sialkot and generally in Pakistan. Absence of a licensed and qualified pharmacist at an operational pharmacy is also in negation of WHO guidelines . The absLikewise, the role of physicians in controlling the antibiotics’ abuse and AMR spread is already established. In our study, majority of physicians were found to be well aware about the global threat of AMR and also recognizes that AMR guidelines designed specifically at local level would be more beneficial than the international ones. It was also highlighted in our study that physicians’ prescriptions are influenced by the patients demand of antibiotics. This behavior emphasize the need for auditing the antibiotic prescriptions in the health care facilities in general and investigating the consultation behavior. Instead of satisfying the patients by unnecessary prescriptions patients can always be appeased by making them understand their diseases .Environmental dimension of AMR dissemination has recently gained much attention , 34. QueSome limitations which our study possess must be taken into consideration when interpreting the findings and results for the development of broad-scale policy framework. The study was conducted in one of the most developed regions of province Punjab and, also the majority of the population included in the study had an urban background, so it doesn’t allow the generalizability of the results on the provincial and national scale due to demographic constraints. The under representation of the farmers and daily wage laborers and over representation of the students in the present study indicates selection bias. Although maximum efforts have been made to include representative samples but the self-administered questionnaire based study has its own limitations i.e., only persons having ability to read and write can be included in the study. This type of selection bias may affect the external validity in terms of generalization of the results to a diverse community. The cross-sectional nature of the study also highlights that the data is collected at one point in time and, therefore, the current study was not able to track any trends, patterns and/or fluctuations in the knowledge and attitude of respondents towards antibiotic consumption and AMR over time. Despite these limitations, the current findings will provide an update about the knowledge of antibiotic usage and awareness of AMR. Given the global recognition and threat of AMR, a similar study on a national scale in Pakistan among the public, physicians and pharmacists is recommended to provide a more broader and comprehensive picture of the current status of antibiotic usage and awareness status of AMR in Pakistan.Our study has highlighted the gaps in AMR related knowledge and attitude among the community members in district Sialkot, Pakistan. Less knowledge towards AMR in the general public seems to be a serious problem, negatively influencing the antibiotic consumption behavior. The dearth of governance in healthcare systems particularly in pharmacies needs to be addressed by formulating and implementing strict laws and policies ensuring the presence of pharmacists for regulating over the counter access of the potent antibiotics. The study acknowledges the challenge of dissemination of AMR by over the counter and non-regulated availability of antibiotics. The role of physicians is of tantamount importance in the community for controlling AMR spread, so healthy prescription practices should be followed by physicians. Regular monitoring and auditing of prescriptions can help in reducing the appropriate antibiotics consumption. The need for initiating antimicrobial stewardship & RUM programs at local or national levels, with the objective of curbing the dissemination of AMR, has also been realized.Additional file 1:Table S1. AMR related knowledge and attitude of respondent community members in Sialkot. Table S2. AMR related knowledge and attitude of non-qualified pharmacy owners in Sialkot. Table S3. AMR related knowledge and prescription trends of physicians practicing in Sialkot. (DOCX 1591 kb)


We present a proof-of-concept for the adaptive mesh refinement method applied to atmospheric boundary-layer simulations. Such a method may form an attractive alternative to static grids for studies on atmospheric flows that have a high degree of scale separation in space and/or time. Examples include the diurnal cycle and a convective boundary layer capped by a strong inversion. For such cases, large-eddy simulations using regular grids often have to rely on a subgrid-scale closure for the most challenging regions in the spatial and/or temporal domain. Here we analyze a flow configuration that describes the growth and subsequent decay of a convective boundary layer using direct numerical simulation (DNS). We validate the obtained results and benchmark the performance of the adaptive solver against two runs using fixed regular grids. It appears that the adaptive-mesh algorithm is able to coarsen and refine the grid dynamically whilst maintaining an accurate solution. In particular, during the initial growth of the convective boundary layer a high resolution is required compared to the subsequent stage of decaying turbulence. More specifically, the number of grid cells varies by two orders of magnitude over the course of the simulation. For this specific DNS case, the adaptive solver was not yet more efficient than the more traditional solver that is dedicated to these types of flows. However, the overall analysis shows that the method has a clear potential for numerical investigations of the most challenging atmospheric cases. The aim of the present study is to introduce adaptive mesh refinement (AMR) as an efficient tool for numerical investigations of the atmospheric boundary layer (ABL) using turbulence resolving methods. This refers typically to models that rely on direct numerical simulation (DNS) or large-eddy simulation (LES) techniques. In general, AMR solvers aim to distribute the available computational resources efficiently over a domain by dynamically refining and coarsening the computational grid in space and time. AMR techniques have successfully been employed in studies concerning flows with a high degree of scale separation throughout the spatial and/or temporal domain. Such studies concern a wide range of topics, e.g. cosmological hydrodynamics Teyssier , electroSeveral methods that meet a varying resolution requirement throughout the spatial domain have already been successfully applied in studies on ABL turbulence. For example, stretching and squeezing of grids  is typically much shallower than the CBL and, furthermore, the length scales of the turbulent structures that account for the mixing of heat and momentum within this layer are only a fraction of the size of those associated with daytime convective turbulence  for the adaptive-grid runs and do not assess alternatives.We realize that it is impractical to address all questions regarding the AMR technique in relation to ABL simulations. For example, here we focus on a single case whereas we will argue that the performance of an AMR solver varies depending on the particular case specifications  discretized with an even number (n) of elements i such that ith entry of D) that approximates n / 2 elements,U) that samples f. Note that the described method is formally linked to wavelet thresholding that has already been employed for fluid dynamical simulations . As the grid structure may change during a simulation run, an important issue is load-balancing; the decomposition of the domain between processors must then be modified as the grid is locally refined or coarsened. This is achieved in the Basilisk solver using the natural decomposition of a Z-ordering space-filling curve applied to the quad/octree structure (Griebel and Zumbusch http://turbulence.pha.jhu.edu/). We analyze a 2D slice of the data  that show a significant deviation in E(k) from the original appear to decrease. We point out that in order to evaluate the spectrum we have linearly interpolated the data from the non-uniform grids to an equidistant grid with When grid cells with a small but finite The example presented in Fig. As indicated in the Introduction, we ran a DNS case from the referenced literature to validate, benchmark and exemplify the adaptive-grid approach. The cases from virtually all atmospheric-turbulence-resolving studies prescribe the periodicity of the solution in the horizontal directions. Unfortunately, at the time of writing, the Basilisk solver cannot yet handle an adaptive grid in combination with periodic boundaries. To circumvent this limitation, we limit ourselves to a case where there is no mean horizontal forcing such that we can apply a no-penetration boundary condition for the normal velocity component at the lateral boundaries. This is supplemented with a Neumann-boundary condition for the tangential velocity components, pressure and buoyancy fields. We realize that this choice might affect the solution and therefore its impact is assessed by re-running the case using a fixed and regular grid with both sets of lateral boundary conditions (not shown). It appears that for the chosen set-up of the case, the simulation results are insensitive to the choice of the horizontal boundary conditions. Note that in future work, we will update the adaptive solver such that periodic boundary conditions can be combined with the AMR technique.b) is used as the thermodynamic variable. The buoyancy is related to the potential temperature  and buoyancy (b), the Navier–Stokes equations for an incompressible fluid are solved under the Boussinesq approximation, according to,For the evolution of the three velocity components  is fixed throughout the simulations. Therefore, the relative overhead of MPI-domain communication routines compared to actual calculations increases as the number of grid cells decreases.For coarse grids, the physical timestep taken per integration timestep increases (Courant–Friedrichs–Lewy criterion). Diagnostic analysis of the solution is performed with a regular interval in the physical time, i.e. In Fig. As discussed in the introduction, for highly dynamic flow configurations such as a diurnal cycle, model performance may benefit from the AMR approach. Although the present case of decaying convection is less dynamic than a full diurnal cycle, it is tempting to compare the simulation performance of the AMR-based run to its counterparts using a fixed and regular grid. Thereupon, several performance characteristics are presented in Fig. Fornberg . If we cIn Fig. We have based our test and performance benchmark on an idealized flow configuration of a CBL using DNS, providing a ground truth for our intercomparison. In the future, we plan to study more practically-oriented cases by using an LES formulation. For many atmospheric cases, LES is preferred over DNS, because it provides an efficient tool for studying high-Reynolds-number flows. Therefore, the next step is to test the AMR approach in combination with an LES formulation. In this section, we briefly discuss some preliminary results on this topic. Because this is part of ongoing research, we do not perform a quantitative discussion of the test runs, the results and performance characteristics. The presented results aim to exemplify the AMR method for a different case and show the flexibility of the AMR approach. The example is based on the LES intercomparison study case by Bretherton et al. , in whicFor this case, the AMR algorithm dynamically adapts to the flow by redirecting the grid refinement to those regions of the spatial domain where it is required. Hence in this case, adaptation is predominantly spatially focussed, whereas in the DNS case the refinement was most prominent in the temporal domain see Fig. a. As sucsurface boundary conditions also becomes important and provides an additional cause of scale separation that may call for adaptive grid refinement. For example, refinement may be preferred at sharp transitions between different types of land use, such as the land–sea interface.Finally, we note the following; the present cases where restricted to spatially homogeneous set-ups, where ‘scale separation’ naturally occurs through the internal variability of turbulence, originating from the non-linearity of the governing equations. In reality, heterogeneity in the We have introduced and tested an adaptive mesh refinement (AMR) method for studies of the atmospheric boundary layer (ABL). This work is motivated by a desire to numerically study highly dynamic cases. Such cases are characterized by a high degree of scale separation throughout the spatial and temporal domain. This work should be viewed as the first step in our AMR-based research that assesses the usage of an AMR method for studies of the ABL. We have based our adaptive-grid simulations on the flow solver implemented in the Basilisk code.The method is tested using DNS based on a case introduced by Heerwaarden and Mellado , describG) is expected to scale asD is the effective  dimension of the physical process  outperformed all other runs in terms of computational efficiency, indicating that there is an overhead associated with the use of the adaptive solver. In general, the exact impact of this overhead depends on the details of the studied case. The most challenging ABL systems typically owe their complexity to the dynamical interplay between various processes at different length and time scales. Hence, the AMR technique is likely to be more favourable as complexity increases. More specifically, as discussed in Popinet , the cos


Positive emotions refer to emotions accompanied by pleasant feelings, such as happiness, interest, satisfaction, pride, and love, which are generated by individuals in response to internal and external stimuli and events. Positive emotions are conscious processes that involve a variety of components, such as a pleasant experience, facial/body expressions, evaluations, and especially behavioural plans and activation states. People's cognitive process is often accompanied by emotions, and the influence of positive emotions on cognitive activities has gradually become the focus of research in recent years. This study constructs a regulation model and an assessment model of positive emotions, and analyses the neural mechanisms of the effects of dopamine substances on human positive emotions. According to a large number of previous studies, the cognitive promotion effect of positive emotions can be summarized as broadening the cognitive scope and promoting cognitive flexibility.Positive emotion refers to the emotion accompanied by pleasure feelings generated by an individual due to internal and external stimuli and events to meet individual needs, including happiness, interest, satisfaction, pride, love, etc. Positive emotion is a conscious process that includes pleasure experience, facial/body expression, evaluation, especially behavioral planning and activation state. People’s cognitive process is often accompanied by emotions, and the influence of positive emotions on cognitive activities has gradually become a research hotspot in recent years.1.1In the early stage of human cognition, people put their consciousness to the outside world and thought about what the origin of the world was. After modern times, the object of philosophical study turns to the subject itself and studies how to understand the possible problem, which is modern epistemological philosophy. After the middle of the 20th century, philosophy began to turn its eyes to the language of the intermediate link between the subject and the object, which is the contemporary western philosophy of language. In the study of linguistics and philosophy of language, linguists and philosophers of language have found that language is a reflection of the mind, and the mind is a function of the brain. Many of them have moved from the study of language to the study of mind and cognition.” Language is the mirror of the soul,” Chomsky said. “Language is a fundamental function of the human mind,” said sale. After the mid-1970s, the object of philosophy naturally turned to the human mind itself. According to sale, “the most important development in cognitive science is the shift of cognitive scientists from computational models of cognitive science to models of cognitive neuroscience. This shows that the brain as the basis of cognition has replaced the digital computer as the basis of cognition. We see the neurobiological brain as the basis of human cognition, and this is a very important shift. He added: “the most promising area of research is cognitive neuroscience, not just brain micro nanotechnology.. The structure of the previously internationally recognized discipline of cognitive science is shown in Cognition is the process and activity in which the brain and nervous system produce the minds. Generally speaking, animals with brain and nervous system have a certain degree of mind. Cognitive science is a science that takes cognitive process and rules as research object, and cognition involves learning, memory, thinking, understanding, and other behaviours that occur in the cognitive process During the development of cognitive science, 6 new development directions are produced within the original 6 supporting disciplines, including mental philosophy, cognitive psychology, cognitive linguistics (or language and cognition), cognitive anthropology , artificial intelligence, and cognitive neuroscience. These 6 emerging disciplines are the six major branches of cognitive science. These 6 supporting disciplines intersect with each other to produce 11emerging interdisciplinary subjects: (1) cybernetics; (2) neurolinguistics; (3) neuropsychology; (4) cognitive process simulation; (5) computational linguistics; (6) psycholinguistics; (7) psychological philosophy; (8) language philosophy; (9) anthropological linguistics; (10) cognitive anthropology; and (11) brain evolution.. These developments in brain science have prepared conditions for us to reveal the secrets of the mind.The important progress of cognitive science has benefited from the development of brain science, which in turn has benefited from the great progress of brain imaging technology. Computer tomography (CT), magnetic resonance imaging (MRl), functional magnetic resonance imaging (fMRl) and positron emission tomography (PET) are widely used in the research of brain and neuroscience, which promotes the development of brain and neuroscience 1.2. According to this model, people preliminarily define the positive emotion and the negative emotion.The “positive” in positive emotions is derived in contrast with “negative” in negative emotions. According to Russell’s circumplex model , emotions are considered to contain two dimensions, valence and arousal. The valence dimension is divided into pleasantness-unpleasantness, and the arousal degree is divided into high-low  that the cognitive flexibility view does not. Gable and harmon-jones recently proposed a motivational dimension model, suggesting that the reason for the narrowing/broadening of attention and cognition by emotion is motivational intensity rather than emotional valence. Only positive emotions that approach low motivational intensity can broaden the cognitive process and flexibility, while positive emotions that approach high motivational intensity narrow the cognitive process and flexibility. Past studies have concluded that positive emotions promote cognition because the motivation intensity of induced positive emotions is relatively low, which can broaden the cognitive process. The method of the trigger positive emotions to participants unexpected gifts , look at the pictures lead to positive emotions or comedy movies, imagine happy event, induced by positive emotional words and music, report success in the task, and so on, is usually caused by happy, satisfaction, pleasure, etc., all these emotions are “goals” or “irrelevant” low motivation intensity of positive emotions., which act on different emotion generation stages, as shown in .Based on the process model of emotion generation in the previous section, emotion regulation can act on any stage of emotion generation. Therefore, the emotional adjustment process can be divided into five categories: situation selection, situation modification, attentional deployment, cognitive change and response modulation 3.2.The process model of emotion regulation mainly focuses on the execution process of emotion regulation, that is, the process in which the individual adopts a specific strategy to regulate emotion. However, some recent studies have suggested that emotion regulation actually involves multiple stages of psychological processes. For example, after contain emotion is generated; the individual shall determine whether the emotion needs to be regulated, choose the appropriate emotion regulation strategy, and then adopt the specific strategy for regulation. The basic idea of an extended model of emotion regulation is that, like other types of emotions, emotions involve the process of assessment. Specifically, there are four components in the assessment system: world, perception, assessment, and action . The cycle of the appraisal system stops only when the difference between the individual’s target state and the changed world is below a certain threshold.Through the comparison of the assessment system with the previous process model of emotion regulation, it is not difficult to find that there is a one-to-one relationship between them: situation-world, attention-perception, appraisal- assessment and response - action. The most important part of the assessment system is its dynamics. First, the behaviour produced by each appraisal cycle is targeted at one or more aspects of the “world” in the appraisal cycle. When the “world” part of the cycle changes, the perception of the “world” cycle by the subsequent appraisal cycle will change, resulting in a change in the subsequent appraisal and action see  [9]. The44.1. Similarly, the role of positive emotions in the human body is also directly related to dopamine, whose structural model is shown in Dopamine is a neurotransmitter that helps cells to transmit pulsed chemicals. This brain secretion is associated with human eroticism and feelings, which conveys messages of excitement and happiness. It is a precursor of NA, and a key neurotransmitter in the hypothalamus and pituitary gland. The concentration of dopamine in the nervous system is influenced by mental factors, there is axon connection and interaction between GnRH of the nerve endings and dopamine, and dopamine can inhibit the secretion of GnRH. The neuronal substance dopamine in the midbrain directly affects people’s emotions. In theory, the addition of this substance can make people excited, but it can be addictive, too. Dopamine appears in the forebrain and basal ganglia, of which the latter responsible for dealing with fear. However, because of dopamine, the feeling of fear is replaced, thus there are many people who are addicted to dopamine Ashby et al. put forward the neuropsychological theory that positive emotion promotes cognition, believing that many effects of positive emotion on behaviour and cognition  are mediated by the dopamine system. The more important mesolimbic system is made up of dopamine-producing cells projected into the VTA, which is associated with reward and motivation. Many of the important dopamine projections may be the neural mechanisms responsible for the cognitive effects of positive emotions. For example, studies have shown that improvements in creative problem solving are due to positive emotional conditions associated with increased dopamine levels in the frontal cortex. The theory has two hypotheses about VTA projection: first, positive emotions may alter the processing of any structure that receives a direct VTA projection. Second, behavioural changes resulting from positive emotions are unlikely to be mediated by structures that do not receive VTA direct projections.Experiments have shown that decreased dopamine levels are associated with anhedonia  rather than negative emotions. Stressful or anxiety-inducing events that cause people to experience negative emotional states actually seem to increase dopamine levels in specific brain regions. So do high and low dopamine levels explain the motivational dimensional model to some extent? Ashby speculated that the correlation between dopamine levels and task performance might be inverted u-shaped, so any increase or decrease in the optimal level of dopamine would impair performance. Additional research has shown that very high levels of dopamine are associated with three characteristics of borderline personality disorder - mood disorders, impulsive behaviour, and cognitive-perceptual deficits. It can be seen from the above evidence that dopamine may have an optimal level when mediating the effect of positive emotion on cognition, and according to the motivational dimension model, the promotion effect of positive emotion on cognition may also present an inverted u-shaped distribution in the intensity of motivation approach. So whether there is a relationship between dopamine levels and the motivational approach intensity of positive emotions is not studied in the available literature.In fact, when comparing the moderately positive emotion and neutral emotion induced, not only the increase of dopamine level was found in various brain regions, but also the peripheral blood circulation system, such as the dopamine concentration in serum, showed a significant increase. In addition, they found increased activity in NK cells, a type of immune cell associated with positive emotions. Be aware of the effects of neurotransmitters and neuromodulators on positive emotions in addition to dopamine.Many of the effects of positive emotions on behaviour and cognition  are mediated by the dopamine system. The more important mesolimbic system consists of dopamine-producing cells projecting into the marginal and ventral tegmental area of the cortex, which are associated with reward and motivation.4.2.Many important projections of dopamine see  are the Experiments have shown that a drop in dopamine levels is associated with lack of pleasure (apathy or lack of pleasure) rather than with negative emotions. Stressful events that cause people to develop negative emotional states actually seem to increase the level of dopamine in certain regions of the brain. The correlation between the levels of dopamine and task performance is inverted U-shaped, so an increase or decrease in dopamine from the optimal level can impair performance. In addition, studies have shown that high levels of dopamine are associated with three characteristics of borderline personality disorder, such as emotional disorders, impulsive behaviour, and cognitive-perceptual defects, that’s, high dopamine levels have a detrimental effect on cognition. It can be seen from the above evidence that dopamine may have an optimal level in mediating the effect of positive emotions on cognition. However, according to the motivation dimension model, the positive effect of positive emotions on cognition may also show an inverted U-shaped distribution in the approach- motivation intensity.In fact, the comparison of induced moderate positive emotions and neutral emotions shows that an increase in dopamine levels is not only found in the brain regions, but also in the peripheral blood circulation system. For instance, the concentration of dopamine in the serum shows a significant increase. In addition, there is an increase in the activity of immune cells - NK cells  under positive emotions. The effects of other neurotransmitters and neuromodulators on positive emotions shall be noted in addition to dopamine.5At present, there are still some problems in the research on the effect of positive emotions on cognitive enhancement. In the process of inducing positive emotions, the positive emotions induced by different methods are not only not quantified or even qualitatively unclear. As Gable et al. pointed out, most of the inducing methods in past studies produced positive emotions with low motivation, which is because the motivation dimension of positive emotions has not been taken into account in the past.Based on cognitive science, this study briefly describes the basic characteristics and functions of positive emotions, constructs the regulation process model and assessment model of positive emotions, and probes into the role of dopamine in the process of emotion generation, intervention and development. The study on the effects of positive emotions on cognition and its neural mechanisms can help to understand normal and abnormal neuropsychological functions. For example, a decrease in positive emotions may make it difficult for people to perform tasks that rely on executive function, which is a common phenomenon in depressed individuals. This study has a great significance on the education cause. Teachers shall treat each student as an individual with creative potential, and provide an environment that helps them to exert their creativity, one of which is the emotional state of pleasure.


To deeply understand its molecular mechanism, miRNA-216a-5p was identified as a target of DANCR by bioinformatic analysis. Experiments demonstrated that miRNA-216a-5p interacted with DANCR and its inhibitor could weaken the influences induced by DANCR knockdown for cancer cells, including cell proliferation and invasion, and the expression of Nanog, SOX2, and OCT4. Therefore, DANCR might act as a tumor promoter by targetting miRNA-216a-5p, which might provide a potential therapy target for breast cancer treatment.Breast cancer, the most frequently occurring malignant tumor, has high mortality rate, especially triple-negative breast cancer (TNBC). LncRNA-differentiation antagonizing non-protein coding RNA (lncRNA DANCR) has been found that its aberrant expression was associated with tumor progression and it was promising to be a potential target for cancer therapy. The goal of the present study was to explore the biological effects and underlying mechanism of DANCR in breast cancer. Our results showed that DANCR was up-regulated in TNBC tissues and breast cancer cells compared with normal breast tissues and cells, and higher DANCR level suggested poorer prognosis, implying that it was promising to be a novel biomarker used for TNBC diagnosis and prognosis. To better research the functions and mechanism of DANCR on breast cancer cells, we selected two cell lines used for next study: one TNBC cell line–MDA-MB-231 and one ER-positive breast cancer cell line–MCF-7. Further study indicated that DANCR overexpression significantly promoted cell proliferation and invasion  Globally, breast cancer is the most frequently occurring malignant tumor for females, with about 1.7 million new diagnosed patients and 522,000 breast cancer-caused deaths every year . Based oLncRNAs, one subtype of ncRNAs, are characterized with length more than 200 nts and have limited or no protein coding ability . Plenty in vitro. Xenograft animal study was used to further confirm DANCR effects on tumor progression. Moreover, we explored its possible molecular mechanism in breast cancer.LncRNA-differentiation antagonizing non-protein coding RNA (DANCR), a novel identified lncRNA from human cancers, is located on chromosome 4. Recently, some studies have reported that lncRNA DANCR is up-regulated in various human cancers and its aberrant expression could promote tumor progression by improving cancer cell proliferation and invasion, such as glioma, non-small cell lung cancer, and prostate cancer . Based oThe present study was approved by the Ethics Committees of The Third Affiliated Hospital of Harbin Medical University, and all patients have signed consent form. The research has been carried out in accordance with the World Medical Association Declaration of Helsinki. A total of 57 pairs of breast cancer tissues and paired adjacent non-tumor tissue were collected from patients with TNBC in The Third Affiliated Hospital of Harbin Medical University from August 2012 to December 2014. Tissue samples were immediately put into liquid nitrogen and stored at −80°C.2.Human breast cancer cell lines  and normal human breast epithelial cell (MCF-10A) were purchased from ATCC . All cells were cultured in RPMI-1640 medium  supplemented with 10% fetal bovine serum  in an incubator at 37°C with 5% COIt was well known that RNA transcripts can have multiple exons aligning to different non-contiguous regions of a chromosome. Alignment searches were performed with NCBI’s BLAST suite. For alignment to RNA sequences, the top search result with a value <0.01 was reported. By analysis, we chose miRNA-216a-5p (miR-216a) used for further experiments because it was highly combined with DANCR.C−ΔΔT method. GADPH and U6 were respectively as loading control of DANCR and miR-216a.Total RNA was extracted from tissues and cells using a miRNeasy Mini Kit  in accordance with the manufacturer’s instructions, immediately the concentration and quality of RNA were detected by NanoDrop 2000 . Then, the obtained RNA was used to synthesize the first-strand complementary DNA (cDNA), using TransScript first-strand cDNA synthesis SuperMix  as per the manufaturer’s protocol. RT-PCR experiment was performed with SYBR green qPCR SuperMix  in ABI prism 7500 sequence detection system . Experimental conditions were as below: 55°C for 10 min, 40 cycles of 95°C for 30 s, 55–59°C for 30 s, and 72°C for 42 s. Cycle threshold (Ct) values were detected and used to calculate the relative expression levels of DANCR and miR-216a according to 2Oligonucleotides sequences were chemically synthesized by GenePharma , including siRNA against DANCR (si-DANCR), miR-216a inhibitor (216a-inhibitor), miR-216a mimics (216a-mimics), and their negative controls (NCs). Oligonucleotide transfection was performed using Lipofectamine 2000  according to the manufacturer’s protocol. The cells without transfection were as control group. After transfection, the expression level of DANCR or miR-216a was measured by qRT-PCR.First, oligonucleotide sequences containing DANCR cDNA fragment (including miRNA binding sites) were amplified and cloned into the pmirGLO plasmids . Then, pmirGLO-DANCR-Mutant was generated by site-directed mutagenesis PCR with platinum pfx DNA polymerase as per the product manual, which acted as NC. Luciferase reporter plasmids and miR-216a-5p mimics/miR-NC mimics (miR-NC mimics) were cotransfected into DANCR-mutant cells (DANCR-MUT) and DANCR-wild type cells (DANCR-WT) by Lipofectamine 2000. After transfection 48 h, relative luciferase activity of cells was examined via a luminometer by Dual-Luciferase Reporter Assay System .2. After transfection 24, 48, 72, and 96 h, 10 μl of CCK-8 reagent  was added to each well and continued incubating for 2 h at 37°C. Finally, the absorbance values of all wells at 450 nm were measured by an enzyme immunoassay analyzer .The cell proliferation assay was performed using Cell Counting Kit-8 (CCK-8) method. MCF-7 and MDA-MB-231 cells were first transfected with siRNA-negative control (si-NC), si-DANCR, 216a-inhibitor, or si-DANCR+ 216a-inhibitor. Then the cells were seeded into 96-well plates and cultured in an incubator at 37°C with 5% COThe migration and invasion of breast cancer cells were measured using wound-healing assay and transwell assay. Each kind of breast cancer cells was divided into four groups based on transfection: si-NC, si-DANCR, 216a-inhibitor, and si-DANCR+ 216a-inhibitor. For wound-healing assay, breast cancer cells were seeded in six-well plates and incubated until they grew to 90% confluence. Then, the media were removed and the monolayer cells were manually scraped using sterile pipette tip. At incubation 0 and 24 h, the width of the wounding scratches was observed and measured under a microscope . For transwell assay, cell-culture insert  was first plated with Matrigel gelatin  according to the manufacturer’s protocol. Then, breast cancer cells were resuspended in serum-free medium and planted in the top chamber. The normal medium containing 10% fetal bovine serums was added to the lower chamber. After incubation 24 h, the cells invaded into the lower chamber were fixed in 100% cold methanol and stained by 0.05% crystal violet. The number of invading cells in 5 randomly selected views was recorded.g, and the protein concentration was measured by BCA test. Then, equal amounts of protein was separated via SDS-PAGE and transferred to PVDF membrane . PVDF membrane was blocked with 5% skim milk and then incubated with primary antibodies at 4°C. The primary antibodies were obtained from Abcam  and used with 1:1000 dilutions, including anti-E-cadherin, anti-Nanog, anti-SOX2, anti-OCT4, and anti-GADPH. Amongst these, GADPH acted as a loading control. Subsequently, the membrane was washed by PBS-T buffer (Sigma) and incubated with conjugated goat antirabbit IgG (Abcam) at room temperature for 2 h. Finally, the signals were measured using ECL detection kit .Breast cancer cells were lysed using RIPA buffer  containing protease inhibitors cocktail . The total protein was obtained by centrifuging with 12,000 7 cells per mouse) to obtain tumor growth model. At 8 weeks post injection, the mice were killed and the tumors were put out. The relative expression levels of DANCR and miR-216a in tumors were detected by qRT-PCR.First, MDA-MB-231 cells were transfected with lentiviral vectors (LV). shRNA plasmid directly targetting DANCR (sh-DANCR) and empty shRNA plasmid (sh-control) were constructed by Shanghai Genechem (China) and introduced into pFU-GW-RNAi vector that carried green fluorescent protein reporter gene driven by the U6 promoter. Then cancer cells were planted in six-well plates and infected with LV-sh-DANCR or LV-sh-control. Xenograft animal study was approved by the Institutional Animal Care and Use Committee of The Third Affiliated Hospital of Harbin Medical University and carried out based on the experimental protocols. Briefly, lentiviral-transfected cancer cells were injected subcutaneously into the left posterior flank of 6-week-old BALB/c mice  and autoclaved for 90 s at 121°C. After washing with PBS, sections were blocked with goat serum  for 30 min at room temperature and then incubated with Ki67 antibody  overnight at 4°C. Next, sections were washed by PBS and incubated using Polink-1 HRP DAB Detection System One-step polymer detection system  for 20 min at room temperature. Finally, tissue sections were counterstained via hematoxylin.t-test or one-way ANOVA followed by the LSD post hoc test. If P value < 0.05, the result was considered to be statistically significant.Each experiment was repeated at last three times, and all data were presented as the means ± S.D. Expression correlation assays were performed using Pearson’s coefficient correlation. The comparisons between groups were analyzed by two-tailed Student’s To explore whether DANCR disorder occurred in TNBC patients, qRT-PCR was performed to detect its expression level in breast cancer tissues and paired adjacent non-tumor tissues (n = 57). To study the influence of DANCR on breast cancer cells, cell transfection assay was performed. Before transfection, DANCR was also highly expressed in two breast cancer cells (MCF-7 and MDA-MB-231) A. After Bioinformatic analysis showed that in theory, DANCR had a binding site of miRNA-216a-5p A. To conTo further research whether the effects of DANCR on breast cancer cells were related with miRNA-216a-5p, MCF-7, and MDA-MB-231 cells were transfected with si-NC, si-DANCR, 216a-inhibitor, or si-DANCR+ 216a-inhibitor. The result of CCK-8 exhibited that the cells, co-transfected with si-DANCR+ 216a-inhibitor, had higher optical density values than those transfected by only si-DANCR, which implied that 216a-inhibitor could inhibit the effect of si-DANCR on cell proliferation A. In addin vivo. LV-sh-DANCR-transfected MDA-MB-231 cells were subcutaneously injected into nude mice for 8 weeks. As shown as in vivo. Moreover, the results of qRT-PCR also displayed that LV-sh-DANCR was able to decrease DANCR level and increase miRNA-216a-5p level in tumors  hypothesis that lncRNAs could communicate with other protein-coding RNA transcripts to modulate the level or functions of miRNAs via shared miRNA binding sites , 28. SurIn order to better research the functions and mechanism of DANCR on breast cancer, one ER-positive breast cancer cell line–MCF-7 was also selected to verify whether it also had promoting functions on other subtype of breast cancer. Regrettably, the results displayed that DANCR played same effects on MCF-7 cells by targetting miR-216a-5p, which suggested that it probably played same roles on some subtypes of breast cancer, including TNBC and ER-positive breast cancer. Surely, there were some limitations in our study. We did not select another TNBC cell line to verify our results, so the results might be not extrapolated to all TNBC.In conclusion, our work found that DANCR had promoting functions on cell proliferation, invasion, and migration in breast cancer through working as ceRNA to target miR-216a-5p, which indicated that the new axis of DANCR/miR-216a-5p might provide a potential therapy target for breast cancer treatments, not only TNBC but also ER-positive breast cancer. Moreover, these results also indicated DANCR was promising to be a novel biomarker used for breast cancer diagnosis and prognosis.The analyzed datasets generated during the study are available from the corresponding author on reasonable request.