Owing to increased spending on pharmaceuticals since 2010, discussions about rising costs for the development of new medical technologies have been focused on the pharmaceutical industry. Computational techniques have been developed to reduce costs associated with new drug development. Among these techniques, virtual high-throughput screening (vHTS) can contribute to the drug discovery process by providing tools to search for new drugs with the ability to bind a specific molecular target.Plasmodium falciparum. In this context, Brazilian malaria molecular targets (BraMMT) was generated to execute vHTS experiments on selected molecular targets of P. falciparum were built and evaluated against known antimalarial compounds. In this study, 35 molecular targets of  As a result, it could predict the correct molecular target of market drugs, such as artemisinin. In addition, our findings suggested a new pharmacological mechanism for quinine, which includes inhibition of falcipain-II and a potential new antimalarial candidate, clioquinol.Raccoon software to improve the search for new antimalarial compounds. It can be retrieved from www.drugdiscovery.com.br or download of Supplementary data. The BraMMT is available to perform vHTS experiments using OCTOPUS or  Innovations in health science have resulted in drastic changes in the ability to treat disease, improving the quality of human life. Since the late 1990s, pharmaceutical spending has grown faster than other major components of the health care system. Consequently, discussions on the increased costs to develop new medical technologies have been increasingly focused on the pharmaceutical industry.Among new technologies, bioinformatics tools have been highlighted with the creation of several data banks. These data banks have contributed to the drug discovery process providing tools to search for new drugs able to bind to specific molecular targets. The molecular target is often associated with enzymes, receptors, or other proteins that can be altered by an external stimulus.Many drugs studied in clinical trials fail and are not approved for use in humans. An alternative to overcome this situation is the use of computer-assisted drug design (CADD) for rational development of new drugs.Through computer methods, pharmaceutical companies have introduced new drugs in the market. Among the most successful cases are the development of imunodeficiency virus (HIV) protease inhibitors.,Plasmodium falciparum molecular targets retrieved from the PDB. This data bank permits in silico vHTS experiments against a pool of P. falciparum molecular targets. In this paper, the BraMMT was evaluated through docking tools and a set of known antimalarial compounds.These exemplars show that CADD methods are useful to identify and select molecules able to interact with high affinity and selectivity against a specific molecular target. In summary, two strategies have been used in CADD projects, LBDD and SBDD. As both strategies require an extensive discussion, only SBDD will be highlighted in this manuscript. Among SBDD methods, molecular docking, highlighted by its practice, is fast and has low computation cost. The docking between ligand and the molecular target is the first step in any cell signaling pathway.Selection and preparation of molecular targets - The three-dimensional structures of the receptors were obtained from the PDB database from their respective codesPlasmodium falciparum. For the molecular target with several entries in the PDB, the structure with the lowest values crystallographic resolution was considered, which had the ligand in the binding site. In addition, the molecular targets P. falciparum ATPase calcium pump ortholog (PfATP6) and hexose transporter P. falciparum (PfHT) were constructed by comparative modeling.,,Thereafter, the molecular targets were prepared by removing the replicate residue present at the binding site. Moreover, only water molecules that carried out at least two interactions between the ligand and molecular target were kept.Finally, the druggability of each target was evaluated by TDR platform targets (http://tdrtargets.org). This characteristic predicts whether a protein can bind with high affinity and specificity to small compounds.Evaluation of molecular docking - Re-docking methodology was carried out to evaluate the AutoDock Vina program.,P. falciparum structure following a re-docking process. The crystallographic and re-docking ligands were overlaid for calculation of root mean square deviation (RMSD) using the Discovery Visualizer 4.5 program.50 value were selected from ChEMBL.Additionally, the receiver-operator characteristic (ROC curve) and the area under the ROC curve (AUC) were established for each molecular target to evaluate the ability of the molecular docking methodology to differentiate the active molecules from decoys .,Active compounds and decoys were submitted to the molecular docking calculations in the AutoDock Vina programVirtual screening of antimalarial drugs - The BraMMT data bank was evaluated using 27 antimalarial drugs [see Supplementary data (Table V)] listed by the World Health Organization (WHO) . These molecules were selected from the ChEMBL platform for TBVS through OCTOPUS,Supplementary data (Table I)].Finally, the 27 antimalarial drugs were ranked using the Equation 1, which ∆ values were obtained by the difference between the crystallographic ligand binding energy (obtained from the re-docking step) and antimalarial drugs binding energy (obtained from the vHTS process). Thus, the Δ values greater than 0 demonstrated that the respective compound had a higher binding energy than the crystallographic ligand; therefore, it could be recognised by the molecular target through the intermolecular interaction.∆ = Equation 1. Δ values obtained from the difference between binding energies of the crystallographic ligand and the antimalarial drugs.Molecular dynamics - The molecular dynamics of selected compounds were performed by MolAr in-house software. MolAr was used to refine ligands through MOPAC2016 using the Parametric Method 7 (PM7) and EF routine to search for the structure of local minimum and to perform a molecular dynamic using DOCK 6. In the simulations, ligand flexibility was recorded. Flexible simulations were performed in 3000 steps and 100 energy minimisation cycles.Chemicals - Clioquinol  was purchased from Sigma (CAS Number 130-26-7). Chloroquine diphosphate salt (N4-(7-Chloro-4-quinolinyl)-N1,N1-dimethyl-1,4-pentanediamine diphosphate salt) was purchased from Sigma (CAS Number: 50-63-5).In vitro schizonticidal activity of the clioquinol against P. falciparum - P. falciparum chloroquine-resistant (W2) strain were maintained in continuous culture using human red blood cells in RPMI 1640 medium supplemented with human plasma. Human red blood cells and human plasma were provided by Foundation of Hemotherapy and Hematology of Minas Gerais (Fundação Hemominas). The parasites were synchronised using sorbitol treatment, and the parasitaemias were evaluated microscopically with Giemsa-stained blood smears. The antimalarial activity was determined using an enzyme-linked immunosorbent assay (ELISA) antiHRPII assay. Infected red blood cells were plated in a 96-well plate at 0.05% parasitaemia and 1.5% haematocrit. Different concentrations of the clioquinol were added in triplicate, and twelve drug-free wells were used as controls (six frozen after 24 h as the HRPII background). After the incubation (72 h), the plate was frozen and thawed twice, and an ELISA using antiHRPII antibodies was performed.50) of three assays performed in triplicate, compared with drug-free controls. Curve fitting was performed using OriginPro 8.0 software .The results were expressed as the mean of the half-maximal inhibitory dose (ICIn vitro cytotoxicity test - The noncancerous human lung fibroblast cell line WI-26-VA4 (ATCC CCL-95.1) was used to assess the cell viability after each chemical treatment employing the MTT colorimetric assay. Briefly, 1 × 106 cells were plated in 96-well microplates with RPMI 1640 medium supplemented with foetal bovine serum (FBS). Then, microplates were incubated overnight at 37ºC, 5% CO2, followed by the treatment with each compound solubilised in DMSO 0.1% (v/v). Negative control groups were constituted of cells without treatment. Five serial dilutions (1:10) were made from a stock solution (10 mg·mL-1) using RPMI supplemented with 1% FBS. After 48 h of incubation, cell viability was evaluated by discarding the medium and adding 100 μL of MTT 5%, followed by 3 h of incubation. Then, the supernatant was discarded, and the insoluble formazan product was dissolved in DMSO. The optical density (OD) of each well was measured using a microplate spectrophotometer at 550 nm. The OD in untreated control cells was defined as 100% cell viability. All assays were performed in triplicate. The SI of clioquinol was calculated as: SI = IC50 WI-26-VA4 / IC50P. falciparum.Selection and preparation of molecular targets - The molecular targets were retrieved from PDB by considering the lowest values resolution among similar structures and the presence of a ligand in the binding site. As a result, the BraMMT was constructed using 35 molecular targets. ,The inclusion of water molecules in the binding site was carefully checked by visual inspection. Thus, only water molecules that performed at least two hydrogen bonds between the crystallographic ligand and the respective target remained in the binding site,Mycobacterium leprae and M. tuberculosis; the protozoa Leishmania major, Trypanosoma brucei, T. cruzi, P. falciparum, P. vivax, and Toxoplasma gondii; and the helminths Brugia malayi and Schistosoma mansoni.In addition, the TDR targets platform was used to obtain the druggability of major pathogens of tropical diseases, including Evaluation of molecular docking - The docking methodology was evaluated by re-docking and the ROC curve.11; (ii) the affinity between the ligand and receptor (binding energy) expressed by score functions; (iii) the suitability of the configurations parameters, such as size of the grid box and atomic coordinates, for the respective molecular target; and (iv) the main forces for molecular recognition, as aided by another program, such as LigPlot or Discovery Studio. Supplementary data (Table II)].Supplementary data (Table IV)]. In addition, for the molecular targets 1LYX, 1NHW, 1QNG, 1YWG, 3TLX, and 4C81, it was necessary to select a different organism with the same target. The different targets from other organisms had an identity range of 34-64% [as showed in Supplementary data (Table III)]; therefore, they can use the respective crystallographic ligand found into the binding site.Furthermore, our results were improved by the ROC curve and AUC calculations. ROC curve and AUC are statistical methodologies to discriminate false-positive and true-positive results from a test. The curve ROC is a graph that uses a binary system classification to discriminate active compounds from inactive. This graph shows specificity on the X axis for true positive compounds (1-specificity) and sensitivity on the Y axis for false positive compounds with value 0.In contrast, 26% (nine) of the targets 1LYX, 1TV5, 2AAW, 2ANL, 3TLX, 3PHC, 3T64, 4C81, 4J56, and 4P7S had AUC values of 0.58, 0.59, 0.53, 0.64, 0.51, 0.66, 0.67, 0.58, and 0.61, respectively, being able to discriminate true positives from false negatives at 51-67%. In other words, 26% of the targets had AUCs greater than 0.5 and less than 0.7, showing that methodology is not random . HoweverFinally, targets 1QNG, 2YOG, 4B1B, and PfHT (11%) had values of 0.32, 0.48, 0.44, and 0.44, respectively, lower than 0.5 , showingMoreover, for the 1O5X, 1RL4, 2OK8, 2VN1, 3CLV, 3K7Y, 4N0Z, and PfATP6 (~ 22%) targets, it was not possible to perform the ROC curve analysis as they did not have sufficient active compounds. It is noteworthy that the 1O5X, 1RL4, and 4N0Z targets did not have acceptable RMSD values, suggesting less than 0.5.Virtual screening of antimalarial molecules - The BraMMT data bank was evaluated against antimalarial molecules present on the list of World Health Organization (WHO) drugs. BraMMT, through the OCTOPUS engine, should predict the correct molecular target of these drugs. Owing to the large amount of data, the 3BPF , 1NHW , and PfATP6  molecular targets against the compounds quinine (CHEMBL387326), mefloquine (CHEMBL416956), and artemisinin (CHEMBL269671), respectively, were selected to exemplify the results. 2+ ATPase protein present in the sarcoplastic reticulum, called the P. falciparum Atpase orthological calcium pump (PfATP6). Its inhibition can lead to increased concentration of cytoplasmic Ca2+, compromising the signaling pathways of the parasite proposed byFinally, the mechanism of action of artemisinin and its derivatives is still controversial. A possible mechanism is through inhibition of the CaP. falciparum, present in the digestive vacuole. During the intra-erythrocyte cycle, the parasite internalises and degrades the hemoglobin present in the erythrocyte, using amino acids as an energy source. The hemoglobin digestive process is performed using proteolytic enzymes, such as plasmepsins and falcipain, where the parasite has developed a strategy to promote the polymerisation of the heme group to form hemozoin crystals, also known as malaria pigment.,M. tuberculosisFinally, the mechanism of action of quinine is related to the inhibition of heme polymerisation by Evaluation of molecular dynamics - After to run the molecular dynamics using amber score, a value of -19.41 kcal/mol and -28.07 kcal/mol was obtained for clioquinol and quinine, these results suggest that clioquinol is a potential protease inhibitor, considering that the crystallographic inhibitor presents value of -12.75 kcal/mol.in silico studies, we performed the in vitro antiplasmodial activity of clioquinol.On the basis of our Antiplasmodial activity - Clioquinol was considered active and selective against P. falciparum in vitro, with an IC50 value of 0.56 µM and a high SI of 178.6  SI greater than 10-fold against a human cell line.These criteria include: (i) a preliminary knowledge of the structure-activity relationship; (ii) a cutoff In this work, we carried out 18.150 vHTS simulations, comparable to biological assays, which would have been time consuming and expensive if performed under similar experimental conditions. Thus, the vHTS methodology is a tool to discover the most active molecules inside a bank of millions of compounds and to reduce the number of molecules used in experiments, thus reducing costs by using a rational drug development process.Furthermore, BraMMT was evaluated by traditional methodology metrics, such as RMSD, ROC curve, and AUC. It was possible to conclude that 77% of the targets had RMSD values lower than 2 Å, proving that these targets could be used in inverse virtual screening through the AutoDock Vina program. Approximately 72% of the targets had AUC values greater than 0.5, showing that they were able to discriminate true positives from false positives. Although some targets did not have validation parameters, they could be used together with other methodologies, such as molecular dynamics, to avoid false positive results.P. falciparum in vitro.Moreover, BraMMT was evaluated against known antimalarial compounds, confirming its capacity to predict the correct molecular target. Our experiments suggested new mechanisms for quinine, as a protease inhibitor, which was confirmed by action and structural similarity between the quinolinic ring of quinine and clioquinol drugs. Molecular dynamics showed an amber score value of -19.41 kcal/mol for clioquinol and it was active and selective against Finally, BraMMT is available for vHTS experiments through a new version of the OCTOPUS software. The new version of OCTOPUS can build and evaluate molecular targets, as well as perform docking simulation using Autodock Vina and/or Dock 6. Therefore, it can perform vHTS experiments using an unlimited number of ligands against a database, such as BraMMT.


Loop mediated isothermal amplification of nucleic acid templates is a rapid, sensitive and specific method suitable for molecular diagnostics. However the complexity of primer design and the number of primers involved can lead to false positives from non-specific primer interactions. Standard methods of LAMP detection utilise the increasing concentrations of DNA or inorganic pyrophosphate and therefore lack specificity for identifying the desired LAMP amplification. Molecular beacons used in PCR reactions are target specific and may enhance specificity with LAMP.We present a potential molecular beacon approach to LAMP detection targeting the single stranded region between loops, and test this for LAMP molecular beacons targeting the 35S promoter and NOS terminator sequences commonly used in GM crops. From these studies we show that molecular beacons used in LAMP, despite providing a change in fluorescent intensity with amplification, appear not to anneal to specific target sequences and therefore target specificity is not a benefit of this method. However, molecular beacons demonstrate a change in fluorescence which is indicative of LAMP amplification products. We identify the LAMP loop structure as likely to be responsible for this change in signal.Molecular beacons can be used to detect LAMP amplification but do not provide sequence specificity. The method can be used to determine effectively LAMP amplification from other primer-driven events, but does not discriminate between different LAMP amplicons. It is therefore unsuitable for multiplex LAMP reactions due to non-specific detection of LAMP amplification.The online version of this article (10.1186/s12896-019-0549-z) contains supplementary material, which is available to authorized users. Isothermal nucleic acid amplification methods represent an attractive alternative to PCR technologies which are reliant on thermocycling equipment. There are numerous isothermal methods , 2, but The LAMP reaction utilises 4 or more primers complementary to 6 or more target sequences. At the initiation of the amplification hairpin forming forward and backward inner primers (FIP and BIP) strand invade the double stranded DNA target. These primers anneal and are extended by a polymerase with strong displacement activity. The nascent strands are displaced by further primers (F3 and B3) enabling the subsequent formation of double hairpin structures from which amplification can cycle. Further primers can be added targeting the single stranded DNA hairpin loop structures  to accelThe specificity is inherent from the use of multiple primers but this can also mean that non-specific primer interaction is an issue. The false positives that these interactions can produce are primarily a result of LAMP primer design and the reaction conditions, but also the non-specific nature of some of the detection methods employed. Methods for detection of LAMP amplification have mainly focused on determining the increasing concentrations of DNA or inorganic pyrophosphate for both real time and end point assays and will inherently detect LAMP and potentially also non-specific DNA synthesis. Such methods include SYBR Green , BART biRecent work investigating the use of molecular beacons with LAMP  suggesteThe thermodynamics and kinetics of molecular beacons for PCR have been studied , 14 and In LAMP amplification this process is different due to two factors; namely the single temperature throughout the assay and the displacement polymerase. The recent study  with MB-The mechanism by which increased specificity is conferred from the annealing of the LAMP molecular beacon to the specific target sequence is however unclear. Since assays involve a constant temperature and use a displacement polymerase that would likely displace the molecular beacon. We show here that molecular beacons indeed detect LAMP amplification but this does not require specific annealing to the specific target sequence. However the increase in fluorescence does appear to be linked to increasing concentration of LAMP amplicons and therefore the specificity is greater than methods that report on DNA amplification only since a positive signal is indicative of successful LAMP amplification. The problem of posssible non-specific amplification remains due to the failure of the LAMP molecular beacons to anneal specifically to the target sequence. Nevertheless, the low fluorescence output from the LAMP molecular beacon method enables the quantification of a wider dynamic range of target concentrations to be assayed in comparison to detection with an intercalating fluorescent dye.Agrobacterium tumefaciens, used to express the transgenes. Here we target these transgenic elements in maize event Bt11 in a mass fraction with wild type maize. We have designed molecular beacons for the 35S promoter and NOS terminator sequences to complement LAMP primers previously established for amplification [The detection and quantification of genetically modified materials in food is essential to comply with legislation and two of the most common markers of GMO contamination are the cauliflower mosaic virus 35S promoter and nopaline synthase terminator from fication –18.Our work initially focused on probing the forward loop structure of LAMP amplicons, but this precluded the use of accelerating loop primers . We therAgrobacterium tumefaciens. The genomic DNA was extracted using the Promega Wizard genomic DNA purification kit  and quantified using a Qubit fluorimeter .The plasmid pART7  containiTwo further DNA templates were synthesised by IDT  firstly to create a simplified target for the 35S promoter LAMP primers using a sequence length and structure appropriate to the FIP, BIP, LoopF and LoopB primers only. The second oligonucleotide was designed to match the length of the first primer but without loop forming sequences. The sequences are shown in Table Loop-mediated amplification uses primers with different functions to enable isothermal DNA amplification. The initiation of the process requires the target strand invasion by the forward and backward inner primers (FIP and BIP) with synthesis of the nascent DNA from these primers utilising the displacement activity of Bst polymerase. The initiation event also requires the displacement primers F3 and B3 to displace the products of FIP and BIP extension which can in turn start the cycling between dumbbell LAMP structures (a dynamic structure with single stranded DNA loops at each end). Additional forward and backward loop primers, LoopF and LoopB, anneal and extend from the target loop structures to increase the synthesis of DNA and inorganic pyrophosphate for accelerated detection strategies. This role can also be met by the incorporation of STEM primers that bind between the F1 and B1 primer sequences. The position of the different sets of LAMP primers is shown for the two plasmid targets in Fig. Oligonucleotide primers for LAMP nucleic acid amplification were synthesized and HPLC purified by Sigma Aldrich . Primers were hydrated with molecular grade water to 100 micromolar and stored at minus 20 degrees C. LAMP primers for Moloney Murine Leukaemia Virus LTR were designed with the assistance of PrimerExplorer v4 from Eiken. The primer sequences are shown in Table The design of the 35Sp 5nt LoopF MB for LAMP followed in part the guidelines for molecular beacon design for PCR amplification; arm length 5 to 7 nucleotides, probe sequence 15 to 30 nucleotides, Tm above the annealing temperature of the assay, 5’ fluorophore not attached to a guanine nucleotide with a 3’ non-fluorescent quencher. The length of the probe sequence was restricted by the single stranded elements of the LAMP loop or STEM. The 35Sp LoopF 5nt MB is similar in structure to the first described molecular beacon by Tyagi and Kramer in 1996 , with 5nDNA samples were amplified and detected in real time on a Qiagen  Rotor-Gene thermal cycler acquiring to the green channel. Unless otherwise stated, all reagents are supplied by Sigma Aldrich . The reaction chemistry for LAMP and amplification detection was 1X isothermal buffer , 300 micromolar each dNTP, 0.8 micromolar Betaine, 0.5 micromolar SYTO9 Green, 0.32 units per microlitre Bst polymerase v2.0 warm start (NEB), 0.8 micromolar each LAMP primer, 0.4 micromolar each Loop primer, 0.2 micromolar each displacement primer and molecular grade water for a reaction volume of 20 microlitre. The parameters were set for 60 cycles of 60 seconds at 60 degrees C unless otherwise stated. Melt temperature analysis provided data between 60 and 95 degrees C. LAMP amplification detection with SYTO9 was substituted with LAMP molecular beacons and supplemented with molecular grade water if required.DNA samples were amplified using PCR on a Qiagen Rotor-Gene thermal cycler with detection of SYTO9 on the green channel and detection of the 35Sp STEM 5nt JOE MB on the yellow channel. The primers shown in Table 2) values.Sequences were aligned using CLC sequence viewer 7 and graphically displayed in pdf format. The Basic Local Alignment Search Tool (BLAST) from NCBI was used for sequence specificity and LAMP primers and LAMP molecular beacons were designed with the assistance of Eiken Primer Explorer v4 and Primer3  respectiWe designed a LAMP molecular beacon targeted to the forward loop sequence of a variant of the 35S promoter from Cauliflower mosaic virus Fig. , with a To allow for rapid amplification from the unencumbered use of Loop primers, we therefore designed LAMP molecular beacons to target the region of the LAMP amplicon called the STEM region, lying between the hairpin loops. The probe length of these molecular beacons remained fixed at 10 nucleotides but the length of the self-complementary arms was increased from 4 to 7 nucleotides incrementally. Positive and negative samples were amplified using LAMP reactions containing each of these molecular beacons over a temperature range of 55 to 65 degrees C. All negative template controls showed negative results below the 0.05 normalised fluorescence threshold level set for the assays. The negative results followed a similar line with the exception of assays at 55 degrees C in which the fluorescence from the molecular beacons was reduced Fig. .Fig. 3AThe LAMP molecular beacon results from the positive samples showed stronger signals from the 35Sp 5nt STEM MB  and the LBP LAMP molecular beacon. The fluorescence from the FAM fluorophore was acquired after 60 seconds for a total of 5 min at 60 degrees C. The oligonucleotide designed to form loops, based on one of the 35Sp dumbbell structures, showed an increase in fluorescence when compared to the NTCs with undiluted and diluted template . We conclude that MBs can provide wide dynamic range quantification of LAMP amplification.The LAMP molecular beacon results show clear NTCs throughout the course of the assay and positive results distinguishable from the fluorescence baseline Fig. ; 1A. ForThe quantified linear plasmid stock containing the 35S promoter was diluted to the equivalent of a single DNA copy per partition, and assayed by LAMP amplification using fluorescence detection with either 1 micromolar LAMP molecular beacon or SYTO9. The four LAMP molecular beacons designed to the STEM region were used to compare the sensitivity to the more established technique of intercalating fluorescent dye detection  . We inveThe clearest separation between positive template and negative NTCs at 75 degrees C was for the 35Sp LAMP amplification with detection using the 35Sp LoopF MB Fig. : 1B. PosThe typical structure of a PCR molecular beacon includes 5-7 nucleotide GC rich arms with a 22-30 nucleotide probe sequence  but the The results showed that the real time increase in fluorescence with the LAMP MB was not evident with PCR amplification. With LAMP amplification of the same sample Fig. : 1 and 2The use of a LAMP molecular beacon targeting the 35S promoter sequence and specifically the forward loop structure in the LAMP amplification was successful whether or not the LoopF primer was present, which would be competing for the sequence. Increased fluorescence and faster times were observed when the LoopF primer was included. The faster times can be explained by the accelerated LAMP reaction but the reporting of the faster time with increased fluorescence cannot be explained by the annealing of the probe and the primer to the same site due to the anticipated displacement activity of the polymerase. An alternative and potentially non-specific mechanism for the LAMP molecular beacon may be evident here.We selected the single stranded DNA region between the loops in LAMP amplification to enable us to us to use the full set of primers and hence maximise the reaction kinetics and design MBs for 35S promoter and NOS terminator sequences commonly used in GM crops. Optimisation of these stem LAMP MBs resulted in a 64 degrees C reaction temperature, 1 micromolar probe concentration and 5 nucleotide arms (as predicted for PCR molecular beacons (Marras 2006 )). If itThe increased specificity of LAMP molecular beacons derives from the assumption that a fluorescent signal can only be generated with the target amplification product. More precisely that the molecular beacon can only fluoresce after conformational change brought on by probe:target hybridisation. Non-specific amplification from the interaction between primers should therefore remain undetected. However our investigations showed that the detection of LAMP amplification without a specific molecular beacon binding can occur. This was first shown with 35Sp LAMP amplification detected by a 5 nucleotide arm molecular beacon with a 13 nucleotide probe sequence specific for the stem region of NOS terminator LAMP amplification. This probe sequence is not present in the 35S promoter amplicon. Further analysis on LAMP molecular beacons was conducted on the LAMP amplification of a retroviral LTR sequence of a plasmid DNA template. All the LAMP molecular beacons should have non-specificity to the amplified products however all the 35Sp and NOSt designed MBs produced positive results in the presence of LTR LAMP amplification and negative results with the NTCs. From this data the LAMP molecular beacons are not binding specifically to the LAMP amplicons and therefore the mechanism for the increase in fluorescence is directed towards the increase in DNA Fig. . It is uEnd point LAMP amplification detection with LAMP MBs was previously described   with lowA LAMP molecular beacon in a PCR reaction failed to show increased fluorescence for the positive sample. The shorter probe sequence is likely to be a contributing factor to this. Conversely Liu et al.  showed tIt would be surprising that a molecular beacon could be used successfully in LAMP amplification. Perhaps therefore the increased fluorescence from the molecular beacons is the result of interactions with the LAMP concatemers. Whilst we cannot be sure that this increase in fluorescent signal with LAMP molecular beacons is a general effect applying in all combinations of LAMP amplicons and MBs, it seems clear that whilst they can, at appropriate concentration levels, provide an indication of amplification this does not result from sequence specificity but rather a more general feature of the LAMP amplicon.∘C and achieved sensitivity comparable to the use of SYTO9. The mechanism by which the fluorescence from molecular beacons increases with LAMP amplification is unclear, although it appears that LAMP loop structures are important, but it is clear that there is no gain in specificity from the LAMP molecular beacons used in this study.Molecular beacons for LAMP amplification targeting the stem region between loops are effective but non-specific reporters of LAMP amplification with both 35S promoter and NOS terminator primers. We found the optimum temperature for our assays to be 64Additional file 1Supplementary Information. (DOCX 299 kb)


While electrospinning has been widely employed to spin nanofibers, its low production rate has limited its potential for industrial applications. Comparing with electrospinning, centrifugal spinning technology is a prospective method to fabricate nanofibers with high productivity. In the current study, key parameters of the centrifugal spinning system, including concentration, rotational speed, nozzle diameter and nozzle length, were studied to control fiber diameter. An empirical model was established to determine the final diameters of nanofibers via controlling various parameters of the centrifugal spinning process. The empirical model was validated via fabrication of carboxylated chitosan (CCS) and polyethylene oxide (PEO) composite nanofibers. DSC and TGA illustrated that the thermal properties of CCS/PEO nanofibers were stable, while FTIR-ATR indicated that the chemical structures of CCS and PEO were unchanged during composite fabrication. The empirical model could provide an insight into the fabrication of nanofibers with desired uniform diameters as potential biomedical materials. This study demonstrated that centrifugal spinning could be an alternative method for the fabrication of uniform nanofibers with high yield. Chitosan is a natural polymer that has been widely studied for tissue engineering, due to its excellent biodegradable properties, antibacterial abilities and hemostasis properties ,2,3,4. HSpinning pure or high percentage of continuous bead-free chitosan nanofibers is extremely difficult . PolyethDiameter of fibers plays a significant role in tissue engineering, because the natural collagen nanofibers in tissues are in the range of 50–500 nm . It indiCurrently, electrospinning is the most popular method to produce nanofibers with uniform diameter distribution ,24,25,26Fiber diameter could be controlled by changing the parameters of centrifugal spinning process . HoweverCarboxylated chitosan (low molecular weight) and polyethylene oxide  were purchased from Aladdin Industrial Corporation in Shanghai, China, which were used as received without further modifications. Deionized water was produced in the laboratory.PEO solutions were prepared by dissolving PEO into deionized water. Four different parameters were varied to produce PEO nanofibers via a dedicated designed centrifugal spinning device , respect14 w/v% CCS solution and 7 w/v% PEO solution were prepared by dissolving them into deionized water, respectively. Then, the volume of CCS solution and PEO solution were mixed with ratios of 2:1, 1:1 and 1:2. The ratio of CCS to PEO, AR-2000ex rheometer (TA Instruments) was used to measure the viscosity of PEO solutions (5–7 w/v%), CCS solution (14 w/v%) and CCS/PEO compound solutions with various ratios. A parallel plate (40 mm diameter) was applied to test each sample with a gap in 500 μm. The viscosity of polymer solutions was measured with increasing shear rate from 0.1/s to 1000/s at room temperature . The zerFiber morphologies and diameters were examined by scanning electron microscopy . Before SEM testing, the obtained polymer fibers were coated with Au by sputter coater . The diameters and the alignment of the polymer fibers were measured by ImageJ software . For each of fiber specimen, 10 SEM images were taken and 100 counts of fibers were measured so as to present an objective statistical analysis of fiber morphology and diameter. Fourier transform infrared spectroscopy with a universal attenuated total reflection  was used to test chemical characteristics. All CCS/PEO nanofibers were tested at wavelengths in the range of 4500–600/cm and a resolution of 1/cm with 64 scans. The thermal properties of fibers were tested by differential scanning calorimetric  and thermogravimetric analysis . Around 5 mg polymer fibers were used for each sample. In DSC test, the temperature increased from 30 °C to 250 °C with a heating rate of 10 °C/min. In TGA measurement, experiments were conducted over a temperature range from 10 °C to 800 °C with a heating rate of 10 °C/min. The mechanical test of CCS/PEO nanofibers was performed via an Instron 5943  with a 50 N load cell. The nanofibers were twisted into a yarn with 0.5 mm diameter and cut into 1 cm long, and set the extension rate at 2 mm/min. At least three specimens for each composite nanofibers were tested. Young’s modulus and failure work were calculated from the recorded stress-strain curve.Ca increased with increasing nozzle length. As longer nozzle length led to a slower jet velocity at initial time, both jet stretching speed and solvent evaporation rate decreased, resulting in larger fiber diameter. Besides, the interrelation of these parameters also impacts the final fiber diameter. For instance, when concentration was lower than critical concentration (6 w/v%), no continuous bead-free fibers could be formed, regardless of changing any other parameters, due to polymer chain entanglement being not sufficient against the jet breakup at the jet-extension stage [iW) [Formation of nanofiber is not only influenced by intrinsic material properties, but also effected by operational conditions . Intrinson stage . Therefober (Wi) . These dber (Wi) . As thesGenerally, polymer solution with high viscosities is a non-Newtonian liquid , but it L) are crucial for centrifugal spinning system, because they are explicitly related to the centrifugal force, nozzle velocity and jet velocity at initial time. Therefore, the expected final fiber diameter could be described by a semi-analytical empirical model:The dimensionless parameters had not been used to describe the thinning of filament, until Tripathi et al.  defined n et al.  introduce nozzle . So, theThe relationship between final fiber diameter and elastic processability number was described as 162Pe0.5 . After oDifferent CCS/PEO solutions with different ratios of CCS to PEO were used to test the general relationship between fiber diameter and various parameters. Viscosity was a key parameter for production of continuous bead-free nanofibers . Higher produced . In thisIn order to produce uniform nanofibers from different solutions and validate the empirical model of Equation (3), fiber diameters of three different ratios of CCS/PEO via setting of various parameters were shown in Mechanical properties of nanofibers are strongly influenced by many factors, including chemical structure of polymers and physical properties of fibers  . The mecThermal behaviors of nanofibers with different CCS/PEO ratios were shown in s of CCS ,58. Figus of CCS ,59, a lo−1 was attributed to the C–O stretching on CCS/PEO scaffolds [−1 was due to the vibration of ether group [−1 were attributed to the C–O–C stretching vibrations [−1 were attributed to amide III, amide I and amide II, respectively [−1 was due to the CH2 stretching [−1 was detected due to N–H and O–H stretching of the polysaccharide molecules [Chemical compositions of the three different CCS/PEO nanofiber scaffolds were characterized by FTIR-ATR spectra, as shown in caffolds . The peaer group . The tribrations ,59. The ectively ,61. The retching . A broadolecules . These rIn this study, we investigated various parameters, including nozzle diameter, rotational speed, concentration and nozzle length, for the fabrication of nanofibers. Based on the obtained nanofibers, more parameters were considered into an empirical model, so as to accurately predict the diameter of produced fibers in this centrifugal spinning system. The physical and chemical properties of CCS/PEO nanofibers were characterized via DSC, TG, FTIR-ATR and mechanical test. In this centrifugal spinning system, higher concentration polymers lead to less solvent, comparing with electrospinning. Therefore, less harmful gas will be produced in the air, if the solvent is toxic or contaminative. Continuous bead-free ultra-fine and uniform nanofibers with high content of chitosan was fabricated to improve antibacterial and biocompatible properties of scaffolds for tissue engineering application. Centrifugal spinning could be an alternative method for production of nanofibers with uniform morphology and high efficiency, so as to incorporate the advantages of needle and needleless electrospinning processes. In vitro and vivo experiments will be further studied to assess the advantages and disadvantages of the produced nanofibers.


It is evident that some perfluoroalkyl acids (PFAAs), a group of globally dispersed pollutants, have long biological half-lives in humans and farm animals. However, the effects of PFAAs in domestic animals have not been fully elucidated. The present study investigated how exposure to a single dose of a mixture of 10 PFAAs influenced hepatic and renal gene expression and histopathology, as well as plasma clinical biochemistry, in microminipigs (MMPigs) over 21 days. In animals treated with PFAAs, the mRNA expression of twelve genes related to fatty acid metabolism was upregulated in the kidney, while only few of these genes were induced in the liver. The expression of several kidney injury-associated genes such as, IGFBP1, IGFBP6, GCLC X2, GCLC X3, MSGT1, OLR1 was upregulated in the kidney. Interestingly, the expression of IGFBP-genes was differentially altered in the liver and kidney. Our findings thus identified hepato-renal gene expression changes in MMPigs that were associated with various molecular pathways including peroxisome proliferation, lipid metabolism, kidney injury, and apoptosis. Furthermore, serum HDL levels were significantly decreased following exposure to PFAAs, whereas no significant histopathological changes were detected, as compared to the vehicle group. Taken together, the present study provided the first indication that a single exposure to a mixture of PFAAs can produce changes in MMPig renal gene expression that were observed three weeks post exposure, suggesting that more attention should be paid to the kidney as a primary target organ of PFAAs. For several decades, perfluoroalkyl acids (PFAAs) have been used in a wide range of domestic and industrial applications because of their excellent physical and chemical properties. Consequently, residues of numerous PFAAs have been detected at various concentrations in almost every environmental compartment around the world. For instance, they have been detected in human blood around the globe –7. This PFAAs have been detected in livestock animals ,11. In bMicrominipigs (MMPigs) are the smallest pig species. These animals provide an attractive model for toxicological studies because they are easy to handle and cost-effective. We previously reported the blood elimination half-lives of 10 PFAAs, which are frequently detected in the environment, in MMPigs, in addition to their organ-specific accumulation patterns, following the administration of a single oral dose of mixed PFAAs . However-1 body weight of a nominal mixture of each 10 PFAAs (30 mg kg-1 body weight PFAAs) mixed with sugar powder. These compounds were perfluorobutanoic acid (PFBA), perfluoropentanoic acid (PFPeA), perfluorohexanoic acid (PFHxA), perfluoroheptanoic acid (PFHpA), PFOA, perfluorononanoic acid (PFNA), perfluorononanoic acid (PFDA), perfluorodecanoic acid (PFUnDA), perfluorododecanoic acid (PFDoDA), and PFOS. Blood samples were collected before and after exposure, at 0, 1, 2, 4, 11, 15, and 21 days, and plasma samples were stored at -20°C until use. At 20 days, food was removed from the animals over 24 h before euthanasia under deep anesthesia with sodium pentobarbital, and the animals were necropsied to collect tissue samples. Details of the exposure protocol and the concentrations of PFAAs in blood and tissue samples have been reported previously —GAPDH Ct of the exposure and control groups.The PCR reactions were carried out using primers for cytochrome P4504A21 (CYP4A21), catalase (CAT), glycogen phosphorylase L (PYGL), stearoyl-CoA desaturase (SCD), fatty acid desaturase 1 (FADS1), fatty acid desaturase 2 (FADS2), acyl-CoA dehydrogenase (ACADM), enoyl-CoA hydratase (EHHADH), carnitine palmitoyltransferase 1A (CPT1A), acyl-CoA oxidase 1 (ACOX), peroxisome proliferator activated receptor alpha (PPARα), and ATP binding cassette subfamily D member 3 (ABCD3). The primer sets for kidney injury biomarkers, including insulin like growth factor binding proteins , glutamate-cysteine ligase catalytic subunit (GCLC), transcript variant X2 and X3, microsomal glutathione S-transferase 1 (MGST1), oxidized low density lipoprotein receptor 1 (OLR1), milk fat globule-EGF factor 8 protein (MFGE8), and lipocalin 2 (LCN2), were additionally used. The level of each gene transcript was normalized to the expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and calculated using the 2The biochemical data were presented as the mean ± standard error of the mean, and were analyzed by F-test and either Student’s t-test or Welch’s t-test. The statistical significance threshold was p = 0.05.No exposure-related clinical signs were observed during the 21-day study period [Biochemical analyses of plasma samples revealed significant differences in lipid metabolism. The HDL level was significantly lower in the exposure group, as compared to the control group, at day 21 post exposure  showed the greatest upregulation of the IGFBP family members; IGFBP1 (1.7 ± 0.8-fold) tended to increase slightly, while IGFBP2 tended to decrease (0.4 ± 0.2-fold), as compared to the control group. The expression levels of GCLC X2 (1.9 ± 0.6-fold), GCLC X3 (1.5 ± 0.2-fold), MSGT1 (1.5 ± 0.2-fold), and OLR1 (1.9 ± 0.5-fold) exhibited slight upregulation, as compared to the control group. No clear changes in the expression levels of IGFBP3, 4, 5, MFGE8, or LCN2 were observed in the exposure group, as compared to the control group .In the current study, we investigated changes in plasma clinical biochemistry, histopathology, and the expression of genes related to fat metabolism and kidney injury in MMPigs following the administration of a single dose of a mixture of PFAAs. We employed only 5 animals (control = 2 and treated = 3) to minimize the number of experimental animals. Although hyperplasia, vacuole formation, and atrophy have been reported in rats fed with PFOS , we onlyOur observation of significantly lower plasma HDL concentrations in MMPigs exposed to PFAAs is consistent with previous sub-chronic PFOS exposure studies conducted in monkeys  and chicExposure to PFAAs has been reported to interfere with a large number of PPARα-dependent and -independent genes involved in fatty acid metabolism in rodents –27. For PPARα can be upregulated in the pig liver by peroxisome proliferators, and also by fasting. The expression of genes responsible for PPARα-mediated fatty acid metabolism such as CPT1A, ABCD3, ACOX, ACADM, and EHHADH was significantly or moderately increased in the pig liver in response to both fasting and clofibric acid treatment, while SCD expression was downregulated in fasted pigs, as compared to those receiving a normal diet . In the Epidemiological studies also suggested that kidney function may be associated with the blood levels of PFAAs . As repoThe IGFs and IGFBPs play an important role in cell growth and differentiation . The preS1 Fig†p < 0.1, *p < 0.05 for the comparison between the control group (Cont.) and exposure group (Exp.).(DOCX)Click here for additional data file.


We have reported that a single intra-articular injection of diclofenac etalhyaluronate (SI-613) exerted a potent and long-lasting analgesic effect in experimental arthritis models. In the present study, we investigated the effect of SI-613 on the production of high molecular weight hyaluronic acid (HMW-HA) in synoviocytes from osteoarthritis (OA) patients and compared its efficacy with that of hyaluronic acid (HA).We compared the effect of SI-613, HA, and diclofenac sodium (DF-Na) on high molecular weight HA production by human synoviocytes.SI-613 and exogenous HA induced the production of high molecular weight HA in synoviocytes from OA patients, whereas DF-Na had no effect. The molecular weight of newly produced HA was about 1000 kDa in the HA-treated synoviocytes and much higher than 2400 kDa in the SI-613-treated cells. The effect of the mixture of HA and DF-Na was similar to that of HA alone in that the molecular weight of newly produced HA was around 1000 kDa. SI-613 significantly suppressed hyaluronidase 2 (HYAL2) mRNA expression and significantly enhanced hyaluronan synthase 2 (HAS2) mRNA expression. HA had no effect on the expression levels of HYAL and HAS.The present results clearly demonstrate that SI-613 induces the production of high molecular weight HA in synoviocytes from OA patients, suggesting the long-lasting analgesic and disease modifying effect of SI-613 for OA. Taken together with the anti-inflammatory and analgesic effects we recently reported for the intra-articular administration of SI-613 to experimental animal models, SI-613 holds great promise for the treatment of knee osteoarthritis. N-acetylglucosamine, is a recognized treatment for pain associated with symptomatic knee OA  ([3H]glucosamine) (37 MBq/mL) and Ultima-FloTMM (scintillation solution) were from PerkinElmer Co., Ltd. . OH pak SB-805 HQ and OH pak SB-807 HQ columns were from SHOWA DENKO K.K. . Select-HATM 500 k, Select-HATM 1000 k, and Select-HATM 2500 k were from Hyalose, LLC. .SI-613 was prepared by conjugating high-molecular-weight fermented HA  and DF via a 2-aminoethanol linker extended from the glucuronic acid moieties. The SI-613 active pharmaceutical ingredient was manufactured in accordance with good manufacturing practice (GMP) guidelines, and its solution was prepared in a laminar flow cabinet to maintain its sterility. SI-613 was dissolved in 5 mM phosphate-buffered saline (pH 6.0) at a concentration of 10 mg/mL and diluted appropriately with PBS before use. Although SI-613 has been used at 10 mg/mL in clinical trials, the maximum concentration of SI-613 was set at 1 mg/mL in this study because highly-concentrated SI-613 solution is very viscous and difficult to pipette.2. The medium was changed every 2 days. After confluence, the cells were seeded at a density of 3.0 × 105 cells/2 mL/well in α-MEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin in 6-well plates.Three lots of human fibroblast-like synoviocytes from rheumatoid arthritis (HFLS-RA) patients and three from osteoarthritis (HFLS-OA)  patients were cultured separately in basal medium containing 10% growth supplement and 1% penicillin/streptomycin . Cells were incubated at 37 °C under 5% CO3H (N)] ([3H]glucosamine) and each test material: HA (ARTZ Dispo®), SI-613 (Seikagaku Corporation), diclofenac sodium (DF-Na) , or a mixture of DF-Na and HA (DF-Na + HA). For mRNA expression measurements, [3H] glucosamine was excluded from the medium. The cells were incubated at 37 °C for the indicated periods under 5% CO2.One day after cell seeding, the culture medium was replaced with 2 mL of α-MEM medium containing 10% FBS, 10 ng/mL recombinant human IL-1β/IL-1F2 (IL-1β) , 1% penicillin/streptomycin, 370 kBq/mL glucosamine hydrochloride D- glucosamine and IL-1β for 48 h in the presence of HA or SI-613. Each resulting culture supernatant was then subjected to size exclusion HPLC analysis (OH pak SB-805 HQ column) followed by liquid scintillation counting. The column was calibrated using MW standards of HA. In control experiments, [3H] glucosamine alone was eluted at fractions later than no. 20. The culture supernatant of untreated HFLS-RA cells provided a broad peak with a peak top at fraction no. 10 (MW = 500 kDa) in addition to the [3H] glucosamine peak  of HA-treated culture supernatant disappeared, whereas that of SI-613-treated culture supernatant was only slightly blunted . This result is presumably relevant to the findings of another study  that cho3H]glucosamine-incorporated products in the culture supernatant of HA- and SI-613-treated HFLS-RA cells, the culture supernatant fractions were analyzed by gel filtration HPLC after incubation with or without hyaluronidase. As shown in Fig. 3H]glucosamine-incorporated product in HFLS cells was newly synthesized HMW-HA . For DF-Na, we tested four concentrations (0.014–14 μg/mL), which corresponded to 0.1–100% liberation of DF-Na from a 0.01% SI-613 solution. This study was necessary due to the lack of accurate data for liberated DF-Na in cell cultures, although it was known that 10% of the DF-Na in SI-613 was liberated by incubation at 37 °C for 48 h in cell-free medium (data not shown). We found that the HMW-HA produced by stimulation with HA was similar to that induced by DF-Na + HA  and hyaluronidases . The results revealed that SI-613 significantly suppressed HYAL2 mRNA expression and significantly enhanced HAS2 mRNA expression. On the other hand, HA by itself did not significantly change the mRNA expression levels of HYAL1, HYAL2, HYAL3, HAS1, HAS2, and HAS3.The abundance and molecular size of HA are thought to be controlled by HA synthases and hyaluronidases. Three isoforms  are known as human HA synthases. It has been reported that HAS1 and HAS3 are responsible for producing HA with molecular weights ranging from 200 kDa to 2000 kDa, and HAS2 synthesizes HA larger than 2000 kDa . It has HAS3) mRNA expression and significantly enhanced HAS2 mRNA expression. It had no effect on the expression levels of HAS1 [HAS2 mRNA expression in the present study appears to be in agreement with their findings, although the tested glycosaminoglycans are different.The effect of exogenous HA on high molecular weight sodium hyaluronate production in synoviocytes from OA and RA patients was previously reported by Ikeya et al. However, the molecular weight of newly synthesized HA was not determined. Smith and Ghosh also reported that high molecular weight HA (MW = 4700 kDa) stimulated the HA synthesis, but low molecular weight HA (MW < 500 kDa) showed little or no effect . In our  of HAS1 . Our finIn the present study, we confirmed that exogenous HA induced the production of endogenous HA with molecular weights above 1000 kDa but did not induce the production of HMW-HA > 2400 kDa or change the expression levels of HAS 1, 2, 3 and HYAL 1, 2, 3, in contrast to SI-613. Therefore, HA-induced newly synthesized HA might serve as a substitute substrate for hyaluronidase rather than as functional HA, thus preventing the digestion of high molecular weight endogenous HA.The present results clearly demonstrate that SI-613 induces the production of high molecular weight HA in synoviocytes from OA patients. In conclusion, these findings suggested that SI-613 might exert the long-lasting analgesic and disease modifying effects attributable to HA produced by synoviocytes even after the disappearance of DF. Taken together with the anti-inflammatory and analgesic effects that we recently observed following intra-articular administration of SI-613 to experimental animal models, SI-613 holds great promise for the treatment of knee osteoarthritis.


Candida pathogens are commonly found in women and can cause vulvovaginal candidiasis (VVC), whose infection rate is further increased during pregnancy. We aimed to study the Candida prevalence and strain distribution in pregnant Chinese women with a molecular beacon assay.Candida detection and identification, a unique molecular beacon assay was presented and compared with a traditional phenotypic method. Antifungal susceptibility was tested with the following agents: 5-flucytosine, amphotericin B, fluconazole, itraconazole and voriconazole.From March 2016 to February 2017, a total of 993 pregnant women attending routine antenatal visits at the Beijing Obstetrics and Gynecology Hospital were enrolled. For Candida was found to be 21.8 % when using the molecular method and 15.0 % when using the phenotypic method. The distribution of the Candida spp. was listed in order of decreasing prevalence: Candida albicans (79.8 %), Candida glabrata (13.5 %), Candida parapsilosis (3.7 %), Candida krusei (2.2 %) and Candida tropicalis (1.1 %). We found that 90.7 % of the Candida detection results were consistent between the molecular and the phenotypic methods. In the cases where the sequencing analyses for the Candida isolates resulted in inconsistent identification, the molecular method showed higher sensitivity than the phenotypic method (96.0 vs 64.6 %). C. albicans, C. glabrata and C. parapsilosis were essentially susceptible to all five antifungal agents tested, whereas C. tropicalis and C. krusei were susceptible to voriconazole and amphotericin B.The prevalence of Candida screening platform for pregnant women.By exhibiting good sensitivity and specificity, the molecular assay may offer a fast and accurate  Candida species could be isolated from at least 20 % of asymptomatic healthy women, while the infection rate increased to 30 % during pregnancy [Vulvovaginal candidiasis (VVC) is the second most common cause of vaginitis, affecting 70–75 % of women of reproductive age at least once during their lifetime . About 5regnancy .Candida albicans, accounting for 85–95 % of all cases. Among the remaining non-albicans species, Candida glabrata is the second most common species. C. glabrata, together with other relatively uncommon species, such as Candida parapsilosis, Candida tropicalis and Candida krusei, also causes VVC, with fewer clinical symptoms and more resistance to treatment [C. glabrata infections are more frequently associated with RVVC [The majority of VVC infections result from reatment . Interesith RVVC .Candida spp. to vaginal epithelial cells, resulting in more frequent VVC infection and resistance to treatment [C. albicans and C. glabrata resulted in preterm membrane ruptures or preterm labour [in utero or during delivery. Even in asymptomatic pregnant women, recurrent Candida colonization has been shown to be associated with increased preterm delivery and low birth weight, when compared to women with normal or intermediate flora [Candida colonization occurs in the second trimester [Candida colonization can prevent preterm birth.The primary complaints of VVC-infected patients often include itching, burning, redness, swelling and cottage-cheese-like vaginal discharge. However, these clinical symptoms are non-specific to VVC and their severity varies depending on demographic and behavioural factors during pregnancy, which is a common risk factor for VVC infection . It is freatment . Complicm labour . Congenite flora . These irimester . In a raCandida spp. relies on traditional phenotypic tests such as micromorphology, chromogenic medium (CHROM Agar Candida), germ tube tests, microculture in agar cornmeal and automated biochemical confirmation (VITEK 2) [Candida spp. identification was evaluated in 993 pregnant women at 16 gestational weeks or older. Strain distribution and antifungal susceptibility testing in Candida spp., including C. albicans, C. glabrata, C. parapsilosis, C. tropicalis and C. krusei, were assayed in the recruited patients.The detection of VITEK 2) . These pVITEK 2) . In the From March 2016 to February 2017, a total of 993 pregnant women (16–40 weeks of gestation) attending routine antenatal visits at the Beijing Obstetrics and Gynecology Hospital were recruited to our study. The clinical symptoms in the reproductive tract of recruited patients were not questioned because of privacy concerns. The subjects were excluded if their condition met one of the following exclusion criteria: topical medication in genital tubes within a week, diagnosis of VVC, or diagnosis of gestational diabetes mellitus or any kind of immune deficiency. The research protocol was approved by the Institutional Research Review Board of the Beijing Obstetrics and Gynecology Hospital. All participants enrolled in the study signed consent forms.Two samples of cervical/vaginal mucosa were collected from each patient with sterile cotton-tipped swabs. One swab was used for phenotypic tests and the other swab was used for DNA preparation in the molecular beacon assay.Candida spp., the primers and hybridization probes were designed using Primer Premier 5.0 software (PREMIER Biosoft) . The molecular beacon assay was carried out with the Applied Biosystems 7500 Real-Time PCR (Thermo Fisher). To identify each of the five Candida spp., the species-specific PCR reaction mixtures (40 µl) were set up as follows: 5 ng template DNA, 0.2 mM dNTPs, 0.6 µM primers mix, 0.1 µM probe, 2.5 U Taq polymerase  and 0.2 U uracil-N-glycosylase . Forty amplification cycles were performed (20 s at 94 °C and 45 s at 55 °C) for each reaction tube. The analytical sensitivity of the molecular beacon assay was estimated by serially diluting template genomic DNA prepared from standard cultures of Candida. To confirm the specificity of the primers and probes in differentiating the above five Candida spp., standard strains of C. albicans, C. glabrata, C. parapsilosis, C. tropicalis and C. krusei, together with another 19 fungi or bacteria species  that are common pathogens found in women’s reproductive tracts, were purchased from ATCC or the China General Microbiological Culture Collection Center (CGMCCC) (Table S2). For each standard strain, the genomic DNA was extracted from their cultures, reaching 106 colony-forming units (c.f.u.) ml−1, and this was followed by the molecular beacon assay described previously.DNA was extracted from each sample using the DNA-Free-RY kit  according to the manufacturer’s instructions. Extracted DNA was stored at −20 °C until use, with the concentration being measured with NanoDrop (Thermo Scientific) at 280/260 nm. Based on the distinct sequences of the internal transcribed spacer 2 (ITS2) of Candida Media plates , with incubation at 35 °C for another 24 h. The unresolved isolates from CHROMagar plates were alternatively identified by the Vitek 2 compact system . The antifungal in vitro susceptibility test was performed with the ATB FUNGUS 3  strip in a semi-solid medium under similar conditions to those in the reference method [For the phenotypic identification method, each vaginal/cervical swab was streaked on a chocolate agar plate supplemented with vancomycin for 24–48 h at 35 °C. The suspicious yeast isolates were further inoculated to CHROMagar e method . AccordiWherever necessary, the remaining DNA extracted from the vaginal swabs or colonies in the phenotypic assay was sent out for sequencing in the nuclear ribosomal internal transcribed spacer (ITS) region, which is considered to be a universal DNA barcode marker for fungi . The ITSCandida spp. when determined by molecular and phenotypic methods. The data analyses were performed with IBM SPSS 21.0, with P<0.05 considered to be statistically significant.A chi-square test was carried out to compare the prevalence difference for C. albicans, C. glabrata, C. parapsilosis, C. tropicalis and C. krusei, based on the ITS region of each organism. Each of the five Candida species-specific PCR methods exhibited an analytical sensitivity of 50 copies ml−1 or lower. None of them exhibited cross-reaction with each other or any of the other 19 organisms tested (Table S2), showing the high specificity of the method as a whole.In the present study, the primers and probes in the molecular beacon method were designed specifically to detect and identify Candida pathogens at our hospital, 21.8 % (216/993) tested positive by the molecular method and only 15.0 % (149/993) tested positive by the phenotypic method. The distribution of the five Candida spp. is listed in order of decreasing prevalence : C. albicans (79.8 %), C. glabrata (13.5 %), C. parapsilosis (3.7 %), C. krusei (2.2 %) and C. tropicalis (1.1 %)  . The pre results .Candida spp. screening study is summarized in Candida. For the consistent results, the prevalence and distribution of the each Candida species were similar to those of the total population  and C. glabrata  being the two leading species. With DNA being either aliquoted from the molecular assay or freshly extracted from Candida colonies, ribosomal ITS sequencing was employed as the ‘gold standard’ method to confirm both consistent and inconsistent identification results. Of the subgroup with consistent identification, all four Candida spp., including C. albicans, C. glabrata, C. parapsilosis and C. tropicalis, were positively confirmed by sequencing with randomly selected Candida isolates  and C. krusei (n=1) were susceptible to VRC and AMB . Our molecular identification system contains five independent PCR reactions designed for C. albicans, C. glabrata, C. parapsilosis, C. tropicalis and C. krusei. Relying on species-specific probes that accurately recognize the ITS2 sequence, our assay was highly specific and no cross-reactivity was observed between the Candida spp or with any of the other 19 commonly observed fungal or bacterial pathogens. A similar strategy has been employed successfully in the rapid molecular assay for Candida dubliniensis identification, as reported by Park et al. in 2000 [The mainstream technique for  in 2000 .Candida colonization of 21.8 % in pregnant Chinese women by the molecular method and one of 15.0 % by the phenotypic method. With both symptomatic and asymptomatic participants being recruited, the overall Candida isolation rate reported in the present study was comparable to that observed in pregnancy by Sangare et al. (22.7 %) [This study revealed a prevalence of (22.7 %) , in whicC. albicans and C. glabrata were the two most frequently observed Candida spp. during pregnancy. Their combined prevalence was 93–95 %, similar to that (96 %) revealed by a retrospective study in which 3141 non-pregnant Chinese women were included [C. albicans is the most common Candida species isolated in VVC patients, with a prevalence ranging within 65–97 % in women. Recently, a high genetic heterogeneity of C. albicans in pregnancy was observed between individuals [C. glabrata was more pathogenic in immune-compromised patients than in healthy hosts [Candida spp., including C. parapsilosis, C. tropicalis and C. krusei, was found to be 5.6 % when using the molecular method and 8.1 % when using the phenotypic method, with these values being slightly higher than those in previously published data (2.6 %) from a 10-year study concerning non-pregnant Chinese women [et al., from Burkina Faso, reported that the combined prevalence of C. tropicalis and C. krusei was as high as 26.9 % for local pregnant women, showing the huge influence of geographical and climatic factors.As expected, included . C. albiividuals . This fihy hosts . The comse women . This prCandida detection results were consistent between the molecular and the phenotypic methods. The majority of the inconsistent results  belonged to the ‘molecular positive/phenotypic negative’ results pattern. With limited DNA being left over from the molecular typing assay, 31 ‘molecular positive/phenotypic negative’ isolates were sent for sequencing, with 29 being confirmed positively. Surprisingly, the two isolates identified as C. albicans turned out to be Cladosporium sp. by sequencing. Unfortunately, we found that the amplification primers and probe designed for C. albicans had 73–85 % sequence similarity in the genome of Cladosporium sp. according to the blast search results (https://blast.ncbi.nlm.nih.gov/Blast.cgi). Cladosporium sp. is an anemophilous fungus and is considered to be an exogenous fungal body in humans. Cladosporium sp. has been found to contaminate conventional cervical smears, posing a challenge to cytopathologists [S. cerevisiae isolate was misinterpreted as C. glabrata and one C. lusitaniae isolate was misinterpreted as C. parapsilosis, showing the limitations of the phenotypic method for uncommon and non-Candida species. Indeed, although they are not commonly observed, both S. cerevisiae and C. lusitaniae have been reported in a VVC study [Candida species.As shown in ologists . Common VC study . The thrC. albicans is susceptible to most azoles, and that non-albicans species are less sensitive to azoles [in vitro susceptibility techniques, various observations have been made in these kinds of studies. For example, in the antifungal study by Brandolt et al. [C. albicans displayed >50 % resistance to both FCA and ITR [C. krusei, which was resistant to FCA in our susceptibility test (It is relatively well documented that o azoles . A similt et al. , C. albi and ITR . Another and ITR , which f and ITR . Similar and ITR , VRC andity test .Candida species. Compared to the traditional phenotypic method, the molecular method was more sensitive and involved a shorter turnaround time in the clinical laboratory. Lastly, for the first time, the prevalence and strain distribution of Candida spp. in pregnant Chinese women was reported, along with the in vitro antifungal susceptibility tests. C. albicans and C. glabrata were the two most common species in the present study and were essentially sensitive to all the antifungal drugs tested. However, due to the lack of confirmatory VVC diagnosis in the current study, no conclusion about Candida prevalence and its association with asymptomatic colonization and symptomatic VVC patients could be drawn at this point. Therefore, the ability of the present Candida molecular beacon identification assay to differentiate true infection from simple colonization still needs to be evaluated in future studies.In summary, a highly sensitive and specific molecular method was introduced to detect and identify five


When t-BuP1 was used, the polymerization proceeded in a controlled manner, whereas the obtained homopolymers exhibited the desired molecular weights and narrow polydispersity (Ð < 1.05). In the case of t-BuP2, homopolymers with higher than the theoretical molecular weights and relatively low polydispersity were obtained. On the other hand, in the presence of t-BuP4, the polymerization of styrene was uncontrolled due to the high reactivity of the formed carbanion. The kinetic studies from the polymerization of both monomers showed that the reaction rate follows the order of [t-BuP4]/[sec-BuLi] >>> [t-BuP2]/[sec-BuLi] >> [t-BuP1]/[sec-BuLi] > sec-BuLi. Furthermore, the addition of t-BuP2 and t-BuP1 prior the polymerization of 1,3-butadiene allowed the synthesis of polybutadiene with a high 1,2-microstructure (~45 wt %), due to the delocalization of the negative charge. Finally, the one pot synthesis of well-defined polyester-based copolymers , with predictable molecular weights and a narrow molecular weight distribution (Ð < 1.2), was achieved by sequential copolymerization in the presence of t-BuP2 and t-BuP1.The anionic polymerization of styrene and 1,3-butadiene in the presence of phosphazene bases ( Anionic polymerization has been known for more than sixty years. An early report on anionic polymerization is traced back in the late 1920s by Ziegler and Bähr ,4,5. TheAnionic ring opening polymerization (AROP) proceeds in the same manner but necessitates the use of catalysts in order to form nucleophilic species ,10. The Ka < 43 in acetonitrile) (1 to P4), due to a rise of the delocalization of the charge on the conjugated phosphazenium cation  complex in polar solvent, which served as the initiator for the synthesis of PB-b-PEO and PI-b-PEO [PB: polybutadiene and PI: polyisoprene] block copolymers  were successfully synthesized through a one-pot methodology, combining anionic polymerization, phosphazene bases and ring-opening polymerization (ROP), leading to well-defined block copolymers with controlled molecular characteristics.To the best of our knowledge there are only two works reporting the use of organolithium compounds in combination with d t-BuP4 , while Fpolymers . Thus, itert-butyl-4,4,4-tris(dimethylamino)-2,2-bis[tris(dimethylamino)phosphoranyli-denamino]-2λ5,4λ5-catenadi-(phosphazene) , 1-tert-butyl-2,2,4,4,4-pentakis(dimethylamino)-2λ5,4λ5-catenadi-(phosphazene)  and tert-butylimino-tris(dimethylamino)phosphorane  were used as received. sec-Butyllithium  was diluted to the appropriate concentration in purified benzene, in a specific glass apparatus under high vacuum. Styrene  was purified via consecutive distillations over CaH2  and dibutyl-magnesium  and stored in pre-calibrated ampoules. 1,3-Butadiene  was purified via consecutive distillations over n-BuLi, at −10 °C using ice/salt bath, prior addition to the polymerization reactor. Benzene and toluene  were purified via distillation from CaH2 and stored in glass cylinders, under high vacuum. Ethylene oxide  was used as the terminating agent and was purified by consecutive distillations over CaH2 and n-BuLi at 0 °C, with prior dilution to the appropriate concentration in purified benzene. ε-Caprolactone  was dried over CaH2 twice and distilled under high vacuum. L-Lactide  was recrystallized from ethyl acetate, dried under high vacuum overnight and finally dissolved in purified THF, prior the polymerization. Acetic acid  (terminating agent) was stored under high vacuum and used as received.1-nM) and the polydispersity index (Ð) were determined via size exclusion chromatography (SEC) equipped with an isocratic pump, Styragel HR2 and HR4 columns in series (300 mm × 8 mm), a refractive index detector and THF as the eluent, at a flow rate of 1 mL/min, at 30 °C. The calibration was performed using polystyrene standards . Proton nuclear magnetic resonance (1H-NMR) spectroscopy measurements were carried out using CDCl3  on a Brücker AV-500 spectrometer. The obtained spectra were used to calculate the monomer conversion as well as the microstructure of the synthesized polybutadiene after integration of the corresponding chemical shifts.The number average molecular weight  of t-BuP2 was added and the solution was left under vigorous stirring for 1 h, at 25 °C. Afterwards, 0.82 mL of styrene was added and the reaction left to proceed at room temperature. At time intervals of 30 min, 1 h, 2 h, 4 h, 6 h and 8 h small aliquots were withdrawn from the solution in order to determine the conversion (1H-NMR) as well as the molecular weight and the polydispersity (SEC). Finally, the reaction was quenched by adding acetic acid and the solution precipitated in an excess of methanol. Conversion(PS) = 100% after 2 h, theoretical number-average molecular weight of PS .Sequential copolymerization of sec-BuLi, 0.0302 mmol of t-BuP1 and 0.5 mL of styrene. After 12 h of polymerization at 25 °C, the living carbanion was end-capped with 2–3 monomeric units of ethylene oxide, the benzene was removed under high vacuum and freshly distilled toluene was added. Then, 0.45 g of L-lactide was added and the polymerization left to proceed at room temperature. After 50 h the polymerization quenched by adding acetic acid and the mixture precipitated in an excess of methanol ((LLA) = 75% after 50 h, n,PSM = 16,500 g/mol, n,PLLAM = 11,500 g/mol. The same synthetic protocol was followed for the polymerization of ε-caprolactone.A typical procedure is as follows. The “living” PS with a number-average molecular weight equal to 16,500 g/mol , Entry 4methanol , Entry 7t-BuP4, t-BuP2 and t-BuP1) with respect to the alkyllithium initiator (sec-BuLi). Two additional experiments were designed in the absence of phosphazene base, for comparison reasons. The molecular characteristics of the synthesized homopolymers, as well as the experimental conditions followed during the polymerization, are listed in The polymerization of styrene and 1,3-butadiene was performed in benzene, at a constant temperature of 25 °C, using one or 0.5 equivalent of the corresponding phosphazene superbases  with high polydispersity (Ð > 1.9)  leads to fast but uncontrollable polymerization of styrene (t-BuP4. We assume that during the polymerization either two active populations (aggregated non-active and active initiating species) coexist or the active species partially deactivated by the t-BuP4 and thus the produced polymers exhibit extremely high molecular weight and broad polydispersity. As a consequence, the viscosity of the solution was raised rapidly within 20 min, rendering the monitoring of the polymerization difficult. Further experiments are in progress to explain this complex phenomenon.One experiment , Entry 1Ð > 1.9)  was obtat-BuP2,  was used. The kinetic study revealed that when t-BuP2 was added in a molar ratio 1:1, with respect to sec-BuLi (t-BuP4 (same ratio between the base and the initiator)  and elevated molecular weights. In comparison with t-BuP4, the smaller radius of the phosphazeniun cation {[t-BuP2]Li+} (~3.3 Å) . Even using a lower concentration of phosphazene base with respect to the alkyllithium initiator, the SEC traces  vs. conversion (measured by 1H-NMR) revealed a linear correlation, characteristic of the living character of the polymerization of styrene in the presence of PBs . For entries four and five  prior to the polymerization of a diene  or PB−[(t-BuP1)Li+] complex, respectively. On the contrary, when the polymerization occurred in the absence of a superbase, the synthesized polybutadiene was characterized by 90 wt % 1,4- and 10 wt % 1,2-content  catalyze the ring-opening polymerization of ε-CL and LLA using either various alcoholic initiators or hydroxyl end-functionalized polymers in different solvents   or PS−[(t-BuP2)Li+] (−[(t-BuP1)Li+] or PS−[(t-BuP2)Li+], the living carbanion was end-capped with 2-3 monomeric units of ethylene oxide, to generate PSO−[(t-BuP1)Li+] or PSO−[(t-BuP2)Li+] respectively Li+] as a macroinitiator (b-PLLA) free from unreacted macroinitiator contamination and showed excellent control of molecular weight Li+]} was also used for the ROP of ε-CL Li+] as a macroinitiator was examined in toluene, at room temperature. It has been reported  as the macroinitiator  and DPPLLA = 84, through end-group analysis. The conversion was calculated to be ~75% after 50 h and the polymerization rate was much slower than in the case of PSO−[(t-BuP2)Li+].Furthermore, the polymerization of L-lactide and reported  that usinitiator , Entry 7analysis A,B verifε-CL utilizing PSO−[(t-BuP1)Li+] as the macroinitiator was also performed in toluene at room temperature. This polymerization led to very slow rate and low conversion as indicated from the SEC and 1H-NMR analysis . Kinetic studies showed that the addition of the superbases prior the polymerization of styrene or butadiene increase the reaction rate due to the generation of highly reactive anionic species through the complexation with the lithium cation. The polymerization rate follows the order of [t-BuP4]/[sec-BuLi] >>> [t-BuP2]/[sec-BuLi] >> [t-BuP1]/[sec-BuLi] > sec-BuLi. The high basicity of t-BuP4 generates extremely reactive carbanions, which lead to high molecular weight and broad polydispersity due to the enhanced propagation rate or to the partial deactivation of the active centers by the t-BuP4. NMR studies currently in progress will enlighten the nature of the t-BuP4/sec-BuLi complex. On the other hand, when t-BuP2 was used, a decrease in the propagating site reactivity occurred, resulting in more controlled molecular characteristics. Using the weaker base (t-BuP1), the kinetic study showed that the polymerization rate was slightly increased compared with the conventional use of a metal-based initiating system (sec-BuLi) and that there is absolute control in the molecular weight and in the polydispersity. When polybutadiene was synthesized in the presence of PBs  in non-polar solvent, high vinyl content was obtained  due to the delocalized negative charge. Finally, the one pot synthesis of polyester-based copolymers (PS-b-PCL and PS-b-PLLA) from the previously synthesized “living” homopolymers was feasible, by sequential copolymerization in the presence of t-BuP2 and t-BuP1, leading to a well-defined block of copolymers with desirable molecular characteristics. This study provides useful information towards the designing/synthesis of complex polyester-based polymeric materials.In summary, an investigation of the anionic polymerization of styrene and 1,3-butadiene via high vacuum techniques was performed in the presence of phosphazene bases (


Single‐molecule junctions that are sensitive to compression or elongation are an emerging class of nanoelectromechanical systems (NEMS). Although the molecule–electrode interface can be engineered to impart such functionality, most studies to date rely on poorly defined interactions. We focused on this issue by synthesizing molecular wires designed to have chemically defined hemilabile contacts based on (methylthio)thiophene moieties. We measured their conductance as a function of junction size and observed conductance changes of up to two orders of magnitude as junctions were compressed and stretched. Localised interactions between weakly coordinating thienyl sulfurs and the electrodes are responsible for the observed effect and allow reversible monodentate⇄bidentate contact transitions as the junction is modulated in size. We observed an up to ≈100‐fold sensitivity boost of the (methylthio)thiophene‐terminated molecular wire compared with its non‐hemilabile (methylthio)benzene counterpart and demonstrate a previously unexplored application of hemilabile ligands to molecular electronics. Contact request: Molecular junctions with hemilabile contact moieties show enhanced mechanoresistive behaviour. The molecule–metal contact is forced to transition from a monodentate to a bidentate configuration as the junction is compressed and stretched, with a resulting modulation in conductance of up to two orders of magnitude. Hemilabile ligands are heterobifunctional ligands in which one donor coordinates strongly to a metal and the other coordinates weakly, often reversibly, as a consequence of the chelate effect. They have found wide applications in homogeneous catalysis,G of a molecular electronic device is defined by the Landauer formula,The zero‐bias electrical conductance G0 is the quantum of conductance (2 e2/h≈77.48 μS) and the transmission coefficient T(E) is the probability for an electron of energy E to tunnel through the molecule. The transmission coefficient for a one‐level system with a molecular orbital of energy ϵ coupled to the electrodes can be described by the Breit–Wigner formulaat low temperature, where σ is an energy shift of the orbital due to interactions with the electrodes, and Γ describes the strength of the coupling that causes a broadening of the orbital resonance, as shown in Figure hapto coordination, for instance by using 4‐pyridyl contact groupswhere thioether⋅⋅⋅Sthiophene separation of 2.84 Å. Thiophenes are known to interact with Au electrodes and have been successfully used as molecular termini in several single‐entity electronics studies.We designed molecular wires with hemilabile contacts, where the strong coordination site is placed at the two termini of the molecule, and compression of the junction would allow the interaction of the metal electrode with a weaker coordinating group, promoting a transition to a bidentate configuration and increasing the coupling parameter Γ. We synthesised molecules terminated with chemically‐defined hemilabile (methylthio)thiophene contacts Figure  b. The m1–2 to reduce the conformational‐isomerism issues arising with conventional oligothiophenes, which result in a large spread of conductance values.STM‐BJ) technique.0. The Au–Au junction is then abruptly stretched by 1 nm and a modulation is applied to the piezo signal for 100 ms thiophene moiety, and therefore a mechanically‐induced monodentate⇄bidentate transition is expected.We synthesised the compounds presented in Figure 1. Conductance modulation is reduced when the amplitude of the square wave applied to the piezo actuator is decreased from 3 Å to 2 Å but does not change significantly when the amplitude is increased to 4 Å (See the Supporting Information for density maps). For comparison, the biphenyl‐based compound 3 showed a very small conductance change upon modulation  to the extent of e±0.78/A∘, and similar results were obtained under sinusoidal modulation of the junction size. This absence of a clean coordination change may be of interest for the possible mechanisms of metal‐organic configuration transitions in the fluxional behaviour in solution of thienyl‐basedTo better characterise the phenomena behind the metal–molecule monodentate⇄bidentate contact transitions, we applied more incremental compression/elongation ramps, which would also allow us to assess the evolution of the coupling parameter Γ as the junction is compressed and relaxed to gain insights into the kinetics of hemilabile coordination. Surprisingly, we did not observe a sudden change in conductance as the junction is slowly compressed or stretched, which would mark a clear monodentate⇄bidentate configuration change. The overall results , with no apparent fatigue for up to 1000 modulations. It should be noted that a 10 kHz frequency is at our instrument limit  and it is therefore possible that even higher frequencies can be attained.We also performed high‐frequency modulation experiments  for electrons of energy E passing from the source to the drain electrode via the molecule.4 as an example  to compute the quantum transport properties for all the molecules (see the Supporting Information for details). The quantum‐transport code Gollume Figure  a, we pre Figure  c. As th3 Figure  b,d. It G=G0 T(EF), a value for EF is needed. In Figure T(E) located immediately below and above E−EF=0 respectively. The precise values of the HOMO and LUMO levels relative to EF  depend on environmental conditions and on the unknown shape of the electrodes. However, since the molecules are neither oxidised nor reduced, EF lies within the energy gap between the HOMO and the LUMO, and the qualitative change in the conductance with tip–tip distance is determined by the behaviour of T(E) within the gap. In compound 4, the additional thiophene–Au interactions described earlier result in broadened transport resonances with an accompanying increase of the mid‐gap value of T(E) as the junction is compressed, and therefore the conductance is also expected to increase. In compound 3, which lacks a thiophene moiety, the resonances stay sharp as the junction is compressed and value of T(E) within the gap does not change significantly. Therefore, the conductance is predicted to be almost independent of the electrode separation. The process was repeated for compounds 1 and 2 (details in the Supporting Information) and, in agreement with our experiments, compressing the electrodes leads to strong interactions between the electrodes and the thiophene moieties, causing significant variations of the transmission coefficient over a wide range of energies within the HOMO–LUMO gap. Furthermore, for values of EF lying in the window highlighted as a grey box in Figure 1 having the largest variations in T(E) as the electrode separation is modulated, and compound 3 showing little or no effect. By fitting the Breit–Wigner formula to the LUMO resonance of the T(E) curves for the four compounds presented in this study, we found a pattern of increasing values of Γ for compounds 1, 2, and 4 as the junction is compressed, but Γ for compound 3 does not change significantly over a range of several Å of junction‐size modulation (see the Supporting Information for more details). As further confirmation of the proposed mechanism, the coupling parameters for compound 3 were found to be consistently lower than for compound 1 at similar junction size, in agreement with the reduced conductance modulation observed during junction compression and elongation.To predict the effect on the conductance In conclusion, we have shown how hemilabile ligands, in this case based on the (methylthio)thiophene moiety, can be exploited to impart mechanosensitive behaviour to a single‐molecule junction. The presence of multiple binding sites and the monodentate⇄bidentate contact transition was exploited to modulate the coupling parameter Γ as the junction is compressed and relaxed. The junctions displayed chemically controlled sensitivity, can operate at kHz speed and show no sign of fatigue for up to 1000 cycles. In addition to expanding the molecular‐electronics toolbox with new design ideas for molecular termini and contacts, our results shed light on the mechanism of configuration transitions in thienyl‐based hemilabile ligands, showing that the fabrication of single‐molecule junctions doubles also as a valuable characterisation tool to probe metal–ligand interactions. Furthermore, (methylthio)thiophenes and thiophenethiols are widely used as molecular‐wire termini in molecular electronics,Synthesis of compounds 1–4: Compound 1 was synthesised by methylation of commercial 4H‐cyclopentadithiophene with methyl iodide and potassium hydroxide, followed by selective bromination with NBS, halogen‐lithium exchange with n‐butyllithium and quenching with dimethyl disulfide. Compound 2 was prepared from commercial 2,2′‐bithiophene by tetrabromination with elemental bromine, followed by halogen–lithium exchange with n‐butyllithium and quenching with dimethyl disulfide to yield 3,3′‐dibromo‐5,5′‐bis(thiomethyl)‐2,2′‐bithiophene. Halogen–lithium exchange with n‐butyllithium, followed by low‐temperature quenching with dimethylcarbamoyl chloride gave 2. Compound 3 was prepared by halogen–lithium exchange of 4,4′‐diiodo‐1,1′‐biphenyl with n‐butyllithium, followed by quenching with dimethyl disulfide. Compound 4 was prepared by double direct lithiation of 2,2′:5′,2′′‐terthiophene with n‐butyllithium, followed by quenching with dimethyl disulfide.Scanning‐Tunneling‐Microscope Break Junction: Molecular junctions are formed by repeatedly driving a Au tip into a Au substrate and then pulling it apart in a solution of the desired molecular wire . A constant bias (200 mV) is applied between the tip and the substrate during the measurements, which are performed at room temperature. The tip is retracted in a stepwise manner, creating a nanogap which is held for 100 ms while its size is modulated  for compounds The authors declare no conflict of interest.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.SupplementaryClick here for additional data file.