E-lysis generates empty but structurally intact cell envelopes (BGs) from Gram-negative bacteria which have been suggested as candidate vaccines, immunotherapeutic agents or drug delivery vehicles. E-lysis is a highly dynamic and complex biological process that puts exceptional demands towards process understanding and control. The development of a both economic and robust fed-batch production process for BGs required a toolset capable of dealing with rapidly changing concentrations of viable biomass during the E-lysis phase. This challenge was addressed using a transfer function combining dielectric spectroscopy and soft-sensor based biomass estimation for monitoring the rapid decline of viable biomass during the E-lysis phase. The transfer function was implemented to a feed-controller, which followed the permittivity signal closely and was capable of maintaining a constant specific substrate uptake rate during lysis phase. With the described toolset, we were able to increase the yield of BG production processes by a factor of 8–10 when compared to currently used batch procedures reaching lysis efficiencies >98%. This provides elevated potentials for commercial application of the Bacterial Ghost platform technology.The Bacterial Ghost (BG) platform technology evolved from a microbiological expression system incorporating the  The relative permittivity can be correlated to the membrane-enclosed volume of viable cells, and, therefore, in many cases to the concentration of viable biomass in the suspension as only viable cells with intact, polarizable membranes contribute to the measurement . X0: initial biomass concentration (end of batch phase); V0: initial (batch) volume; S0 substrate (glucose) concentration in the feed; fρ : density of the feed.Equation (4): Calculation of the initial feed rate F(t) in the fed-batch phase (b) in [g/h].Equation (5): Calculation of the feed rate 2 and O2) were quantified using a gas analyzer  using infrared and paramagnetic principle, respectively. Off-gas analysis for the DASGIP quad system was done using a GA4 module  featuring BlueSens sensors for O2 and CO2 .The off-gas concentrations of the Techfors-S fermentations  was applied for in-line measurements of the relative permittivity Δε, which was estimated at frequencies of 10 MHz accounting for non-cellular background and 1.0 MHz attributed to living bacteria.qS during E-lysis phase was implemented using simple calculator devices as outlined in the results section. Screening experiments were monitored and controlled using the software DASGIP Control V.4.5 .The process control system (PCS) Lucullus  was used for monitoring and control of the pilot-scale processes. A tool for estimation of the current broth volume was implemented in Lucullus-integrated interface tool Sim-Fit. A strategy for controlling A first principle rate-based soft-sensor was used for the real-time estimation of viable biomass during the fed-batch phase using a cumulative calculation approach. The approach is based on the real-time calculation of metabolic reaction rates. Generation of an over-determined equation system using the Degree of Reduction (DoR) and the carbon balance allows for estimating unknown rates. The fermentation volume can be calculated through a mass balance. The input term considers mass input through substrate and base inflow as well as oxygen fixation by the microorganisms. The output term considers water stripping, sampling and mass loss through carbon dioxide production. A detailed description of this approach was published .Biomass concentrations as dry cell weight (DCW) were determined off-line for validation of the soft-sensor. The DCW was quantified gravimetrically after drying for a minimum of 72 h at 105 °C. Samples were centrifuged , and the pellet was washed with distilled water.4(3)  for determining cell viability . RH 414 signals were picked up in channel FL2 (orange) defining a gate G1 for exclusion of non-cellular background. Combination of FSC and FL1 (green) signals were used to identify regions for populations of living bacteria (R1), dead but intact bacteria (R2) and Bacterial Ghosts (R3). The method as well as definition of regions R1–R3 have been described in detail [For flow cytometry (FCM), a Cube 6 system  with a 488 nm blue solid state laser was used. Samples were diluted appropriately and stained with two fluorescent dyes: RH 414  for staining cell membranes and DiBACn detail .t after LI can be calculated directly as the ratio of cell counts (CC) in the region R3 (BGs) and total cell counts as given in Equation (6).All given values for E-lysis efficiency (LE) are based on FCM data. LE at any time Equation (6): Calculating the E-lysis efficiency from FCM data.i.e., EcN (pGLysivb), was characterized in prior studies [i.e., glucose) consumption and by-product  formation. During unlimited batch growth on defined medium  EcN shows a maximum specific growth rate of maxµ = 0.75 h−1 and a maximum specific substrate uptake rate S,maxq = 1.67 g/gh. The biomass yield coefficient for such conditions is X/SY = 0.45 g/g while ~1 g/L acetate is produced towards the end of batch phase. In substrate-limited  fed-batch conditions with specific substrate uptake rates distinctly lower than S,maxq (here: Sq < 1.1 g/gh), the strain shows no acetate formation and the biomass yield increases to X/SY = 0.5 g/g.The strain under investigation,  studies  where biTo follow a science-based approach, all critical parameters having potential negative effects on the overall lysis efficiency (LE) had to be identified, both during fed-batch phase (b) as well as during E-lysis phase (c). This was done in a classical risk assessment using a cause-and-effect (Ishikawa) diagram with the focus on LE as a critical quality attribute. All potential critical process parameters are included and rated due to their potential criticality, as shown in The feeding profile was rated the major critical parameter as fed-batch processes are conducted in a state of substrate limitation. For both fed-batch phase (b) and E-lysis phase (c), a potential negative effect of low growth rates on E-lysis performance through the formation of a stationary, non-dividing subpopulation must be prevented.Plasmid stability as second critical parameter for LE was investigated as described above. The E-lysis plasmid (pGLysivb) was retained in 100% of the population when grown on Agar plates without addition of antibiotics.qS(b) = 0.4, 0.7 and 1 g/gh) during fed-batch phase (b) were conducted. Screening-scale experiments were done in parallel, aiming for a biomass concentration of 15 g/L at the end of the fed-batch phase (b). The feed flow rate was set to a fixed value corresponding to a specific substrate rate qS(c) = 1.0 g/gh at the beginning of the E-lysis phase (c) and kept constant all during the lysis phase. LE was determined via FCM at-line measurements as described above. The critical quality attribute LE as a function of the specific substrate uptake rate qS(b) during fed-batch phase (b) is shown in In order to investigate the influence of growth conditions during fed-batch phase (b), screening experiments with different feeding profiles (specific substrate uptake rate qS(b) during fed-batch phase (b) has no impact on the lysis competence of the cells, which gives freedom to operate within this range. Process design was therefore continued with a fixed qS(b) = 0.6 g/gh, after considering economic and process technological aspects such as e.g., process time and oxygen transfer limitations. It can be concluded that, in the investigated range, the specific substrate uptake rate qS(b) = 0.6 g/gh, T = 35 °C). Expression of lysis gene E was induced by shifting the temperature to 42 °C while qS(c) was reduced to 0.1 g/gh (based on the current biomass concentration) and kept on this low level for one hour in order to accumulate protein E but prevent E-lysis. The feed flow rate was then linearly increased within a time frame of two hours. The actual viable biomass concentration was followed by permittivity measurements and a sudden and steep signal drop caused by disruption of the membrane potential [An experimental setup to investigate the conditions for lysis onset was established as follows. For this experimental approach, the strain under investigation was grown at 20 L scale to a biomass concentration of X = 30 g/L in an exponential fed-batch (qS(c) were calculated in retrospect from the feed flow rate based on the assumption that the change in biomass concentration was negligible in the early stage of phase (c). This assumption is supported by the fact that the permittivity signal stagnates after LI before a significant drop indicated the onset of E-lysis at ~14.25 h process time. From that, we concluded that the minimum value for qS(c) in order to see the first indicators for E-lysis is approximately 0.4 g/gh. Since qS(c) is calculated as a function of the feed flow rate and the biomass concentration at the end of the fed-batch phase (b), the values depicted in S(c)q based on the feed-forward approach as described above.We have developed a control strategy aiming to combine the real-time signal for viable biomass estimation provided by a soft-sensor with the permittivity signal that further allows following dynamic process conditions like E-lysis upon membrane depolarization.F(t) proportionally to the decrease of viable biomass, thereby maintaining a constant specific substrate uptake rate qS(c) during E-lysis phase (c).Consequently, the loss of membrane potential can be followed as a drop in the permittivity signal. With the above mentioned transfer function, the PCS is then able to adjust the feed rate X = f (Δε) as mentioned above. R2 = 0.93). At the point of lysis induction (indicated by the arrow in qS(c) could be maintained at a constant average level of 0.92 g/gh over the whole E-lysis phase (c).A fermentation run with EcN (pGLysivb) was conducted to demonstrate the applicability of the capacitance-controlled feeding strategy using the 20 L bioreactor setup as described above. The permittivity signal recorded during fed-batch phase was correlated with the output of the soft-sensor in the fed-batch phase establishing the transfer function qS(b) = 0.6 g/gh. Biomass propagation is followed by dielectric spectroscopy measuring the permittivity, offline biomass sampling and a soft-sensor predicting the viable biomass as described above. Offline biomass samples were taken at thirty-minute intervals to retrospectively validate the biomass estimation by the soft-sensor. Good correlation between online and offline biomass measures as well as linear proportionality between permittivity signal and viable biomass  and E-lysis phase (c), the latter of which can be regarded a threshold for E-lysis onset. Development of a fed-batch production process for BGs therefore involves investigating the effects of the growth conditions on the overall LE by adjusting the specific substrate uptake rate Sq—here considered as a biomass specific measure of total metabolism—during fed-batch (S(b)q) and E-lysis phase (S(c)q). We have shown that the specific substrate rate S(b)q during fed-batch phase in a range of 0.4 to 1.0 does not influence E-lysis performance. For the specific substrate rate during lysis phase, however, we found a threshold value of 0.4 g/gh below which no signs of E-lysis were observed. From these findings, we conclude that at specific substrate uptake rates < 0.4 g/gh, which corresponds to specific growth rates of µ < 0.2 h−1, the vast majority of the bacterial population converges into a state similar to the stationary phase where E-lysis is inhibited. These observations align well with the abovementioned effects of low physiological activity on E-lysis performance.In fed-batch processes, growth conditions are specified by the feeding profile defining a constant substrate uptake rate that is usually chosen at the lower end of the physiologically possible range. However, such low substrate uptake rates translate into low specific growth rates that presumably mimic conditions impeding E-lysis onset. Consequently, one expects that minimum substrate uptake rates i.e., the change in viable biomass predominates the changes in volume. In order to control the specific substrate uptake rate qS(c) during this phase, reliable on-line estimation of the viable cell concentration was crucial. The applied soft-sensor delivered robust and accurate estimations of the viable biomass concentration for standard growth conditions during fed-batch phase (b) but failed to reflect predicting the viable biomass concentration during dynamic, undefined process conditions.The gained process knowledge now demanded for a control strategy that is capable of maintaining the conditions required for E-lysis. Feeding sufficient amounts of substrate during E-lysis phase (c) was equally important to not overfeeding the culture in order to avoid accumulation of substrate. The fed-batch feeding concept as described above (Equations (2) and (3)) comprises a couple of constants as well as the current fermentation volume and viable biomass concentration. The E-lysis phase (c) is a highly dynamic phase where the number of viable cells decreases rapidly and exponentially, F(t) proportionally to the decrease of viable biomass. Using this novel control strategy, we were able to maintain the specific substrate uptake rate qS(c) at an average of 92% of the set-point (1.0 g/gh) over the whole lysis phase (c).Literature reports that E-lysis results in a sudden loss of membrane polarizability . We coulWith the approach we have presented in this contribution, we were able to produce the microbial product Bacterial Ghosts with a lysis efficiency of LE > 98%, maintaining full control over all process phases. The described fed-batch procedure gives 8 to 10-fold higher yields (BG per liter fermentation broth) compared to the previously described batch process .Sq > 0.4 g/gh required for successful E-lysis under substrate-limited conditions.The biological process of E-lysis depends on a certain level of physiological activity; using the specific substrate uptake rate as a biomass specific measure of total metabolism, we were able to determine a threshold value We could show that quantifying the relative permittivity of the culture broth as a measure for membrane polarizability, one can follow the drop of viability in a culture affected by E-lysis.ε) could be aligned with soft-sensor-derived estimation of viable biomass concentrations (X) during bacterial growth.Viability information gained from permittivity measurements (ΔX = f (Δε), we were able to control the specific substrate uptake rate Sq throughout the entire lysis phase.Using a transfer function The described fed-batch process increased the productivity of Bacterial Ghost production by a factor of 8–10 reaching lysis efficiencies of >98%.


Mycobacterium bovis, which infects many domestic animals and is transmitted to humans through consumption of raw milk or from contact with infected animals. The prevalence of BTB in Moroccan cattle is estimated at 18%, and 33% at the individual and the herd level respectively, but the human M. bovis burden needs further clarification. The current control strategy based on test and slaughter should be improved through local context adaptation taking into account a suitable compensation in order to reduce BTB prevalence in Morocco and decrease the disease burden in humans and animals. We established a simple compartmental deterministic mathematical model for BTB transmission in cattle and humans to provide a general understanding of BTB, in particular regarding transmission to humans. Differential equations were used to model the different pathways between the compartments for cattle and humans. Scenarios of test and slaughter were simulated to determine the effects of varying the proportion of tested animals (p) on the time to elimination of BTB  in cattle and humans. The time to freedom from disease ranged from 75 years for p = 20% to 12 years for p = 100%. For p > 60% the time to elimination was less than 20 years. The cumulated cost was largely stable: for p values higher than 40%, cost ranged from 1.47 to 1.60 billion euros with a time frame of 12 to 32 years to reach freedom from disease. The model simulations also suggest that using a 2mm cut off instead of a 4mm cut off in the Single Intradermal Comparative Cervical Tuberculin skin test (SICCT) would result in cheaper and quicker elimination programs. This analysis informs Moroccan bovine tuberculosis control policy regarding time frame, range of cost and levels of intervention. However, further research is needed to clarify the national human-bovine tuberculosis ratio in Morocco.Bovine tuberculosis (BTB) is an endemic zoonosis in Morocco caused by  Tuberculosis is a disease of humans and animals which mainly affects the lungs but can also manifest in other organs. A variety of tuberculosis bacteria cause the disease and are usually transmitted through air, i.e. inhalation of aerosols. Bovine tuberculosis (BTB) occurs predominantly among domestic cattle, although wild animals are an important reservoir for transmission. Humans are usually infected with BTB through contaminated dairy products or close contact with cattle. While BTB has been eliminated in cattle and human populations of most high-income countries, it is still a major health threat in low- and middle-income countries. In Morocco, the disease frequently occurs in cattle and poses a health risk for humans. An effective intervention to reduce BTB among domestic cattle and reduce the risk to humans is slaughter of cattle which test positive for the disease. We simulated BTB in the Moroccan cattle and human populations using a disease transmission model. We assessed effects of test and slaughter in regard to elimination of disease in cattle and humans and estimated the associated costs with the model. The time to elimination of disease depended on the number of cattle tested. Our model suggests that the disease might be eliminated in cattle within 32 years if 40% of Moroccan cattle are tested annually and infected individuals are slaughtered or within 13 years if at least 90% of the cattle population are tested. The estimated total costs for the time periods until elimination ranged from 1.55 billion euros for testing and slaughter at 50% to 1.48 billion euros at 90%. These results can be used as a guide for planning BTB control policy in Morocco with regard to time frames and associated costs. Mycobacterium bovis. It belongs to a group of well-known and newer mycobacteria, together with Mycobacterium tuberculosis, all of which derive from a common ancestor forming the Mycobacterium tuberculosis complex (MTBC)  are tuberculin skin test positive. The initial values of the three compartments IC, EC and SC were calculated such that the pre-intervention endemic equilibrium of the model equals the prevalence of 18% (The most recent estimate (2013) for the cattle population in Morocco is 3,173,000  and infectious cattle (Ic) per year from the herd. The control reproductive number Rc including the test and slaughter intervention as proportion p with a test of sensitivity se was computed as:Although Morocco has a test and slaughter policy for the control of BTB, it is currently not effectively implemented. The BTB transmission model was used to estimate the effect of the proportion of tested and slaughtered tuberculin positive animals on the duration to reach freedom from disease, achieving an individual animal prevalence of less than one in a thousand tested animals (<1/1000) according to the standards of the World Organization for Animal Health (OIE) . The proThe associated costs were estimated in a summaric way, assuming an incremental cost of comparative intradermal or interferon gamma (BOVIGAM) testing of 3 euros per animal. The cost of compensation at 80% of the market value varies from 470 euros for local breeds to 970 euros for improved breeds . For theThe OIE recommended cut-off for SICCT interpretation is 4 mm, however, many studies showed that a severe cut-off of 2mm increased the sensitivity of the test , 68, witc was the most sensitive parameter influencing the dynamics of BTB transmission .0 = 0.6–1.4) and high risk areas (R0 = 1.3–1.9) reported for the United Kingdom [c) was determined to be the most sensitive parameter of the model.To our knowledge this is the first model describing cattle to cattle and cattle to human transmission of BTB in Morocco, although an African buffalo-human model has been published . Time se Kingdom  but is l Kingdom . Using aA key challenge in this model was to distinguish between exposed and infected cattle because the diagnostic test utilized was the intradermal tuberculin test. We used the proportion of cattle with active TB (13.5%) among cattle tested positive by tuberculin skin test, as reported by Ngandolo et al , to calcOur model contains many simplifications. The primary simplification is that of homogeneity: all cattle are not the same. Risk exposure of animals and humans to BTB could change according to sex and age, and we have ignored these differences. Contact between cattle is also not random and is far more likely within herds than between cattle in different herds. Many models have included this heterogeneity in contact patterns within and between herds –54, but M. bovis, which decreased from 5 per 10,000 to 1 per 10,000 after 17 years.BTB prevalence was found to reach less than one per thousand in less than 20 years when the proportion of tested cattle was above 60%. The annual cost for this potential intervention was nearly 77 million euros. Intervention in cattle was found to impact the prevalence of human TB due to The economic assessment presented here is preliminary, and a detailed cost and cost-effectiveness analysis will be published separately. However, our analysis informs Moroccan bovine tuberculosis control policy on the time horizon, range of cost and optimal levels of intervention. An effective control program will depend on the human resources and technical and logistical capacity of the veterinary services to implement testing and slaughtering of animals. If the proportion of cattle subjected to test and slaughter was greater than 60%, freedom from disease would be reached in less than 20 years. The simulation results suggest that switching from a 4mm cut off to a 2mm cut off would be likely to result in significantly shorter durations of elimination programs and much cheaper elimination campaigns.Our model simulates the removal of individual animals rather than whole herds. Past experience in Europe has shown that whole herd removal is critical for effective elimination in low prevalence situations . In addiA recalculation of the intervention cost taking into account stratification by breed, sex and age should be undertaken, as it could lead to a different cost estimation of BTB control strategy.Shortage of human resources should be considered for intervention planning, a maximum of 40% cull rate might be feasible; however, it would be costly in view of current Moroccan economic situation. One may think that test and slaughter implementation would lead to a reduction in cattle population and its by-product. But on the other hand, the increased import of cattle from other countries, with enhanced control measures, could maintain the current population density. In the meantime, as dairy products are provided mostly from highly controlled farms where BTB has a very low prevalence, we could argue that milk production would not be significantly affected.The WHO includes BTB amongst the seven neglected zoonoses which are perceived to be severe threats to public health . FurtherS1 Supporting information(PDF)Click here for additional data file.S2 Supporting information(PDF)Click here for additional data file.S3 Supporting information(XLSX)Click here for additional data file.


CD99 is a cell surface molecule that has emerged as a novel target for Ewing sarcoma (EWS), an aggressive pediatric bone cancer. This report provides the first evidence of methuosis in EWS, a non-apoptotic form of cell death induced by an antibody directed against the CD99 molecule. Upon mAb triggering, CD99 induces an IGF-1R/RAS/Rac1 complex, which is internalized into RAB5-positive endocytic vacuoles. This complex is then dissociated, with the IGF-1R recycling to the cell membrane while CD99 and RAS/Rac1 are sorted into immature LAMP-1-positive vacuoles, whose excessive accumulation provokes methuosis. This process, which is not detected in CD99-expressing normal mesenchymal cells, is inhibited by disruption of the IGF-1R signaling, whereas enhanced by IGF-1 stimulation. Induction of IGF-1R/RAS/Rac1 was also observed in the EWS xenografts that respond to anti-CD99 mAb, further supporting the role of the IGF/RAS/Rac1 axis in the hyperstimulation of macropinocytosis and selective death of EWS cells. Thus, we describe a vulnerability of EWS cells, including those resistant to standard chemotherapy, to a treatment with anti-CD99 mAb, which requires IGF-1R/RAS signaling but bypasses the need for their direct targeting. Overall, we propose CD99 targeting as new opportunity to treat EWS patients resistant to canonical apoptosis-inducing agents. Ewing sarcoma (EWS) is a malignant mesenchymal tumor of children and young adults, with unmet clinical solution and relevant social impact. Despite the use of intensive multidrug treatments combined with surgery and/or radiotherapy, this highly invasive bone tumor forms lung and/or bone metastases in about 30-40% of patients with localized tumor, while approximately 30% of patients have detectable metastasis at diagnosis. Metastatic patients have very poor prognosis and treatment remains a challenge. The characterization of EWS genomes has poorly contributed to the identification of novel therapeutic strategies. EWS shows very low rate of somatic mutations , 2 confiA potential method to kill cancer cells in a caspase-independent mechanism is the hyperstimulation of macropinocytosis, which can induce a form of non-apoptotic cell death, known as methuosis, death by macropinocytosis or catastrophic vacuolation –11. ThisCD99, a cell surface molecule  involvedCD99 promotes cancer cell death when triggered by specific antibodies, such as 0662, O13 murine mAbs or the human single chain fragment variable diabody (dAbd C7) . EngagemHowever, loss of cell viability associated with CD99 engagement is not prevented by the caspase inhibitor z-VAD-fmk and dying cells lack features typical of canonical apoptosis  . ElectroMethuosis relies on the initial formation of vacuoles through clathrin-independent endocytosis and the progressive accumulation and enlargement of vacuoles up to the point of cytoplasmic membrane disruption . Gene exAs EWS cells are not the best model to study endocytosis, having large nuclei, small cytoplasm and growing in suspension, we alternatively used U-2 OS osteosarcoma cells transfected with CD99 . TreatmeCD99 significantly colocalized with caveolin-1, a major component of caveolae, while very limited colocalization was observed between CD99 and clathrin in both LAP-35 and U2/CD99wt57 cells Figure . InhibitCD99-induced macropinocytic vacuoles contain the small GTPase RAB5, a marker of early endosomes, as well as the late endosomal/lysosomal marker LAMP-1 . In factWe previously reported a marked and rapid decrease of MDM2 protein levels following CD99 engagement . MDM2 isActivated forms of RAS and the IGF-1R play an important role in regulating caspase-independent cell death, associated with cytoplasmic vacuolization , 13, 29.Accordingly, triggering of CD99 by 0662mAb in presence of cycloheximide, an inhibitor of protein synthesis, induced partial degradation of CD99 and RAS but not IGF-1R down-regulation (data not shown). Thus, in contrast to CD99 and RAS, which are partially down-regulated upon antibody treatment, the IGF-1R is likely recycled to the cell surface in RAB11-enriched compartments.In TC-71 cells silenced for IGF-1R expression , treatmeOn the contrary, no induction of vacuolization Figure  and a siWe have previously demonstrated that engagement of CD99 does not affect viability of human hematopoietic and mesenchymal stem cells, constitutively expressing high levels of CD99 , 38. Herlow) did not induce RAS up-regulation and cytoplasmic vacuolization, when exposed to the antibody  [MDR1 cDNA and selected in doxorubicin  [® 10 System or POWERPLEX EXS 17 fastsystem, Promega). Bone marrow-derived human mesenchymal (hMSC) stem cells were obtained from healthy donors or patients with benign bone lesions. hMSC cells characterized for mesenchymal markers and differentiative ability were used at early passages.EWS cell lines were grown as previously described , 26. 664w 2009)  were pre, SIGMA) . LAP-35 09  were Transfections were performed using Calcium Phosphate transfection Kit or Lipofectamine 2000 (Life Technologies) according to the manufacturer's protocol. Transient transfection of MDM2 gene was performed with pCMV-MDM2 . For sta® MTT Cell Proliferation Assay kit  accordingly to manufacturer's instructions.Sensitivity to chemotherapeutics was assessed after 72h of treatment with TACS6/ml cells as previously described [The anti-CD99 0662 monoclonal antibody (mAb) was produced in the Unité INSERM 343, Hospital de l'Archet, Nice, France. Treatments with 0662mAb (3μg/ml) were performed in adhesion or at the concentration of 5 × 10escribed , 21. Aftescribed   G4110B 20K arrays. Microarray data are available at GEO database with Series accession number GSE36097 . EnrichmIn order to visualize macropinosomes upon treatments, cells were incubated with 0.75 mg/ml Lucifer Yellow (LY) fluid-phase dye (Thermo Scientific) in IMDM 10% FBS for 15 min at 37°C. Tracer incorporation was evaluated by flow cytometry analysis  or by visual imaging (Nikon ECLIPSE 90i with Plan Apo 60x/NA 1.4 DIC N2). Images were captured under identical conditions using a digital color camera (Nikon DS5MC) and the software NIS-Elements AR 3.10 (Nikon).To track acidic vescicles formation, Acridine Orange  staining was performed as described in t test. Fisher's exact test was used to evaluate the statistical association between two variables.Differences among means were analyzed by Student's


The numerical results show that the NDC behavior of TADHA molecular junction originates from the Stark effect of the applied bias which splits the degeneration of the highest occupied molecular orbital (HOMO) and HOMO-1. The H2O molecule adsorbed on the terminal sulphur atom strongly suppresses the conductance of TADHA molecular device and destroys the NDC behavior in the lower bias regime. Single or separated H2O molecules adsorbed on the backbone of TADHA molecule can depress the energy levels of molecular orbitals, but have little effects on the NDC behavior of the TADHA molecular junction. Aggregate of several H2O molecules adsorbed on one branch of TADHA molecule can dramatically enhance the conductance and NDC behavior of the molecular junction, and result in rectifier behavior.Large negative differential conductance (NDC) at lower bias regime is a very desirable functional property for single molecular device. Due to the non-conjugated segment separating two conjugated branches, the single thiolated arylethynylene molecule with 9,10-dihydroanthracene core (denoted as TADHA) presents excellent NDC behavior in lower bias regime. Based on the  Due to the rapid development of single molecular technologies14, great progresses have been achieved for single-molecule-device fabrications in recent years18. At the meantime, different strategies are designed to control and improve the functional properties of single molecular device22. In order to gain insights into the controlling mechanism of single molecular functional characteristics25, the effects of external ambient28, electrode distance30, molecule-electrode interface35, molecular anchor43, side group47, doping49 and external field55 have been studied intensively. In experimental studies, molecular devices are often fabricated in solution and measured in vacuum or in gas circumstance, thus the effect of the surrounding molecule on the functional properties of molecular device should also be discussed59. Generally, the surrounding molecules play negative effects on the molecular junction, for example, aqueous solution or the water vapor can suppress the electronic transport of molecular junction dramatically58. However, sometimes the surrounding molecules can also have positive effects, our recent research reveals that the small ambient molecules can make molecular junction more stable due to their suppressing the thermal vibrations of the molecular junction14. Therefore, studying the influence of surrounding molecules on molecular device is very helpful to improve the functional characteristics of the molecular device by properly applying surrounding molecules.Utilizing single molecule as functional device in electronic circuit is an ultimate goal of molecular electronics, which has motivated scientists to devote themselves to the investigations of molecular devices for tens of yearset al.60. Although the NDC behavior of single molecular device is often presented in theoretical studies, it is very scarce in experimental findings, and the NDC often shows very small for most cases. Thus, it is significant that Perrin et al. fabricated single TADHA molecular junction with large NDC behavior, especially the NDC behavior was occurred at lower bias regime because high bias voltage may deform the configuration of molecular junction14. Considering that the molecular junction was fabricated in solution, we are very interested in that if the junction is not absolutely dried, i.e., if one or several H2O molecules are left and adsorbed on the functional molecule, whether the NDC behavior can be destroyed or be enhanced? In order to answer this question, according to the density functional theory (DFT), we simulated the adsorptions of H2O molecules on different positions of the TADHA molecule. Then by applying non-equilibrium Green’s function (NEGF) formalism, the electronic transport properties of TADHA molecular junction were studied with the influences of H2O adsorbates. Our study shows that, the H2O molecules adsorbed on the terminal sulphur atoms not only destroy the NDC behavior of TADHA molecular device, but also depress the current of the molecular junction distinctly at lower bias regime. The NDC behavior of TADHA molecular device seems to show some water-immunity character for single or separated H2O molecules adsorbed on the backbone of TADHA molecule, but it is very sensitive to the aggregate of several H2O molecules. Our findings are valuable to the fabrication of TADHA molecular junction in solvent, i.e., one can determine whether there are H2O molecules adsorbed on the terminal S atoms or aggregate of H2O molecules adsorbed on the backbone of functional molecule by the electronic transport properties of molecular junction in experiment.Due to the non-conjugated segment separating two conjugated arms, the single thiolated arylethynylene molecule with 9,10-dihydroanthracene core (denoted as TADHA) shows pronounced negative differential conductance (NDC) behavior, which has recent been detected by Perrin 2O effect, we constructed Au-Molecule-Au systems by sandwiching TADHA molecule between two gold electrodes with one or several H2O molecules adsorbed on different sites of TADHA molecule as shown in Fig. 2O-adsorption samples, based on the geometric optimizations, we found that there are four typical sites for H2O molecule to be adsorbed on, i.e., (1) the aryl ring site on one arm of TADHA molecule near the electrode, denoted as Type I-1; (2) the ethynylene site on one arm of TADHA molecule, denoted as Type I-2; (3) 9,10-dihydroanthracene core site, denoted as Type I-3; (4) the terminal sulphur atom site, denoted as Type I-4 . At the same time, the low-bias current is depressed by about one order in the magnitude, which is similar to the molecular junctions probed by Long et al.28.Figure 2O-adsorbate effects, we presented the transmission spectra for the bias voltages of 0.0 V, ±0.25 V, ±0.5 V and the voltage of peak-current in Fig. 2O adsorbate.In order to understand the NDC behavior of the TADHA molecular junction and the H2O molecule adsorbed on the TADHA molecular junction, due to the strong electronegativity of the O atom, the TADHA molecular orbitals are modulated differently with the H2O molecule adsorbed on different positions, which further induces different changes of the transmission spectra as well as the conductance of the molecular system. In detail, for Type I-1 adsorption, since the H2O molecule is very close to the electrode and the affinity is very weak, the applied bias dominates the split of the energy levels and suppresses the effect of the H2O molecule by the electrodes, hence the transmission spectra show little difference from those of the TADHA molecular junction without H2O adsorbate  eigenstates, which are often used to discuss the electronic transport properties of molecular junctions in literatures23. The MPSH is the self-consistent Hamiltonian of the functional molecule including the H2O adsorbates with the influence of the electrode, which contains the electrode-molecule coupling effects but does not contain the Hamiltonian of the gold electrode, so the energies of the MPSH eigenstates are not perfectly consistent with the positions of the transmission peaks7.In order to gain deep insight into the NDC behavior, we presented spatial distributions of the HOMOs and HOMOs-1 for TADHA molecular systems at 0.0 V, ±0.5 V and at the peak-current voltages in Fig. 2O molecules on the electronic transports more explicitly, we further set two or more H2O molecules adsorbed on the TADHA molecule, which are shown in Fig. 2O molecules being adsorbed on TADHA molecule, Type III is the molecular junction with three H2O adsorbates. After geometric optimizations, we calculated the electronic transport properties for the molecular systems with Type II and Type III configurations. Figure 2O adsorbates as functions of applied bias. From the figure one can see that, TADHA molecular junctions still show low-bias NDC behaviors with the influence of two or three H2O molecules adsorbed on the backbone of TADHA molecule. For the Type II-1 and Type II-2 molecular junctions, the electronic transport properties show similar characteristics to Type I molecular systems. In detail, due to the influences of the H2O molecules adsorbed near the electrodes being suppressed by bias voltage, the electronic transport properties of Type II-1  gap is usually underestimated in standard density functional theory (DFT), which results in overestimate of conductance of single molecular junction. Thus an alternative DFT-based approach65 (e.g. DFT + Σ approach) is needed to give more accurate and physically meaningful understandings of electron-transport properties of TADHA molecular junction in the future. Since the HOMO is much closer to the Fermi level than the LUMO, the electron-transport properties are mainly governed by the HOMO. According to the DFT + Σ method in the literatures63 and the experimental results60, we can roughly estimate that, for the TADHA molecular junction without H2O adsorbate, the HOMO is overestimated by not more than 0.3 eV relative to the Fermi level, which is much smaller than those of weak coupling systems63.As to the accuracy of our work, we found that our calculations successfully reproduced the low-bias NDC behavior of TADHA molecular junction that was investigated experimental by Perrin 2O adsorption effect are investigated by applying the density-functional theory and non-equilibrium Green’s function method. In the lower bias regime, the TADHA molecular junction exhibits excellent NDC behavior, which is attributed to the splitting of the degenerated transmission channels at applied bias voltage. The H2O molecules may depress the energy of the transmission channel, and further influence the electronic transport properties of the molecular junction. The influences of the H2O molecules are closely related to the H2O molecule affinity. Although the single H2O molecule or the separated H2O molecules adsorbed on the backbone of TADHA molecule can lower the energies of the TADHA molecular orbitals and shift the positions of the transmission peaks, they have relatively smaller effects on the current and NDC behavior of the TADHA molecular junction. The aggregate of several H2O molecules adsorbed on one branch of the TADHA molecule can strongly enhance the current and the NDC behavior of the molecular junction. The H2O molecule adsorbed on the terminal S atom strongly depresses the conductive orbitals, which further dramatically suppresses the current of TADHA molecular junction in the lower bias regime and consequently destroys the low-bias NDC behavior of the molecular junction.In summary, the NDC behavior of TADHA molecular junctions and the H2O molecules were optimized using the SIESTA package with a maximum force 0.02 eV/Å67. The Troullier–Martin type norm-conserving pseudopotentials are applied to represent the core electrons68, the generalized gradient approximation (GGA) with Perdew–Burke–Ernzerhof (PBE) formulation is applied as the exchange-correlation functional69. For Au atoms, a single-ζ plus polarization basis set is used, and for other atoms, a double-ζ plus polarization basis set is employed.The geometric structures of TADHA molecular systems without or with H70T is the transmission probability, which depends on the incident energy E of the transmission electrons and the applied bias voltage V. f(E) are the electrochemical potentials of the two electrodes. The transmission probability T is calculated by NEGF method. The differential conductance is defined as −4. A 4 × 4 k-point grid was used for the Brillouin-zone (BZ) sampling in the transverse directions. A 300 K smearing was applied for the electronic Fermi-Dirac distribution.The current through the molecular device with different bias voltage is obtained according to the Landauer–Buttiker formulaSupplementary Information


The compounds exhibit similar conductance profiles, with a low conductance feature being more readily identified by STM‐I(s) methods, and a higher feature by the STM‐BJ method. The lower conductance feature was further characterised by analysis using an unsupervised, automated multi‐parameter vector classification (MPVC) of the conductance traces. The combination of similarly structured HOMOs and non‐resonant tunnelling mechanism accounts for the remarkably similar conductance values across the chemically distinct members of the family 2–5.The compounds and complexes 1,4‐C The development of a range of complementary and relatively facile methods for the measurement of the electrical properties of single molecules has seen a renaissance in the field of molecular electronics.Although the majority of single‐molecule and thin‐film junctions studied to date have been based on organic molecules, such as alkanes,trans‐[Pt(C≡CC6H4SAc)2(PPh3)2] was assembled within a mechanically controlled break‐junction (MCBJ) based molecular junction. From the resulting I/V curves, collected over a bias range of ±5 V, a resistance of 5–50 GΩ  was estimated at the extremes of the bias range, some three orders of magnitude less conductive than similarly contacted organic oligoarylene systems.trans‐[Pt(C≡CC6H4SAc)2(L)2] 3) in crossed‐wire junctions at more modest bias (up to 1 V) revealed a two‐ to threefold higher conductance than 1,4‐(4‐AcSC6H4C≡C)2C6H4, which was ascribed to the shorter sulfur–sulfur distance in the metal complexes.In some earlier studies of organometallic complexes in molecular electronics, the complex trans‐[Ru(C≡CC6H4SAc‐4)2(dppm)2] methane) has been assembled into monolayers and studied within a scanning tunnelling microscope break junction (STM‐BJ), with a comparison made to the oligo(phenyleneethynylene) (OPE) compound 1,4‐(4‐AcSC6H4C≡C)2C6H4 as a benchmark. Extrapolation to single‐molecule conductances gave values of 19±7 nS (ca. 2.5×10−4 G0) for the ruthenium complex and 3.6±2.0 nS (ca. 4.6×10−5 G0) for the OPE. The higher conductance has been attributed to both the shorter molecular length and the extensive Ru(d)−C≡C(π) mixing in the metal complex.trans‐bis motif, with topics of interest including the exploration of surface contacting groups,In seeking to enhance the wire‐like response, significant attention was turned to ruthenium bis complexes, which are generally thought to offer more significant d–π orbital mixing in the occupied frontier molecular orbitals.trans‐bis complexes of platinum and ruthenium, we have turned to such a systematic study here. Here, the 3‐thienyl moiety,I(s)I) as a function of distance (s)) and STM‐BJOne particular advantage of organometallic complexes within the field of molecular electronics lies in the ability to systematically alter the molecular structures of these systems with a fair degree of synthetic ease, which permits a modular approach to molecular designs and a systematic search for structure–property relationships. In seeking to further explore the electrical properties of oligophenyleneethynylene (OPE), and cyclo‐3‐C4H3S, 1) was obtained from the Sonogashira cross‐coupling of 3‐bromothiophene with trimethylsilylacetylene and subsequent deprotection.1 with 1,4‐diiodobenzene gave 1,4‐C6H4(C≡C‐cyclo‐3‐C4H3S)2 2(PEt3)2] (3) was prepared from the CuI‐catalysed reaction of 1 and [PtCl2(PEt3)2] in NEt3,trans‐[Ru(C≡C‐cyclo‐3‐C4H3S)2(dppe)2] ethane) was obtained from the one‐pot reaction of [RuCl(dppe)2]OTf with 1, in the presence of either KOtBu or 1,8‐diazabicyclo[5.4.0]undec‐7‐ene (DBU) and TlBF4.trans‐[Ru(C≡CR)2{P(OEt3)3}4] have been prepared in moderate yield from reaction of trans‐[RuCl2{P(OEt)3}4] with an excess of LiC≡CR.trans‐[Ru(C≡C‐cyclo‐3‐C4H3S)2{P(OEt)3}4] (5) was obtained here simply by allowing the reaction of trans‐[RuCl2{P(OEt)3}4]1, KPF6 and NHiPr2 in ethanol to proceed for 12 days at room temperature, with isolation of the desired compound being achieved by precipitation from methanol. The long reaction time was compensated by the simple reaction conditions and work‐up, compatibility with alkynes substituted with sensitive functional groups and improved yields. The compounds were each characterised by the usual array of 1H, 13C{1H} and, in the case of 2–5, 31P{1H} NMR spectroscopies, mass spectrometry and elemental analysis.The parent alkyne 3‐ethynyl thiophene  or EtOH (5) into CH2Cl2 solutions of the complexes. Plots of the molecules showing the atom labelling schemes are given in Figures Single crystals of 3 is situated on a crystallographic inversion centre. The platinum atom shows the expected square‐planar geometry with trans‐disposed alkynyl and phosphine ligands. The Pt(1)−P(1) (2.3022(5) Å), Pt(1)−C(1) (2.007(2) Å) and C(1)−C(2) (1.185(3) Å) are all within the usual range of values for complexes of this type,In the crystal, trans‐[Ru(C≡CR)2(dppe)2] complexes have been summarised recently,4 offers some points worthy of brief comment. The structure is composed of two independent molecules, both of which are situated on crystallographic inversion centres and which differ in the orientation of the thiophene group. The dihedral angles between the thiophene plane and the plane containing the Ru and C(n1) atoms and the midpoints of the two ligand P atoms are 3.4° for molecule 1, and 96.3 and 77.8° for the two components of the disordered thiophene of molecule 2. Therefore, at least in the crystal, molecules 1 and 2 of 4 are rare examples of such bis(arylacetylide) complexes trans‐[Ru(C≡CR)2(dppe)2] in which the aromatic rings sit close to the “privileged” orientations that allow maximum d–π conjugation along the molecular backbone, and the first in which both conformational isomers are observed for the same chemical compound. The comparable bond lengths within the two molecules are essentially indistinguishable, with the possible exception of the most precisely determined Ru−P bond lengths, which appear to be marginally shorter in molecule 2.The general structural features of 3}4][CF3SO3] has been structurally characterised,5 appears to be the first structurally characterised bis(acetylide) derivative of Ru(C≡CR)2{P(OR)3}4. In the crystal, the tetrakis(triethylphosphite) derivative 5 is situated on a crystallographic 4‾axis so that there is only one unique phosphite group. The dihedral angle between the two thiophene groups  is therefore 90°. The Ru−P (2.3149(3) Å) and Ru−C(1) (2.0592(15) Å) distances in 5 are shorter than in the mixed vinylidene–acetylide derivative (Ru−P 2.341(3)–2.350(2) Å; Ru−C 2.114(8) Å) reflecting the increased electron density at Ru in 5 and increased Ru−P and Ru−C back bonding. Although back‐bonding plays only a modest role in the bonding of metal–alkynyl complexes,5 (1.221(2) Å) and the cation [Ru(C≡CPh){C=C(Me)Ph}{P(OEt)3}4]+ (1.209(11) Å). Overall, with the clear exception of the phosphine and phosphite ligands, there are few if any substantive differences in the structures of 4 and 5.Although the mixed ligand vinylidene–acetylide complex  vs. [Ru(dppe)2] vs. [Ru{P(OEt)3}4]) does not exert a great influence on the conductance of these organometallic complexes within these 3‐thienyl contacted Au|molecule|Au junctions.As shown in Figure up Table . This de2–5 were carried out at the B3LYP level of theory6H4 (2), [Pt(PEt3)2] (3), [Ru(dppe)2] (4), [Ru{P(OEt)3}4] (5)) on the distribution and composition of the frontier molecular orbitals. Plots of the HOMOs are given in Figure In the quest to better understand the conductance behaviour, the electronic properties of the molecules and electrical behaviour of the junctions have been investigated by using DFT‐based methods. Initial studies of the electronic structures of 2 again provides a convenient point to commence discussion and a basis for comparison of the metal complexes 3–5. Unsurprisingly, the lowest energy structure features a co‐planar arrangement of the thienyl and 1,4‐phenylene rings, with the frontier orbitals distributed almost evenly across the molecular backbone, making a linear, π‐type conjugated pathway between the two sulfur atoms. For the platinum complex 3, the lowest energy identified minimum featured the thienyl moieties lying perpendicular to the square plane defining the coordination geometry at the metal centre (perp‐3). However, a second minimum, barely 0.8 kcal mol−1 higher in energy, in which the thienyl moieties lie in the same plane as the metal coordination sphere (planar‐3) was also identified. Again, the HOMOs of these complexes are π‐type and delocalized over the molecular backbone, and feature a small but important metal contribution . The LUMOs are more metal in character  and rather delocalized in the case of planar‐3.The organic compound 4 and 5 offer HOMOs that are similarly structured to those described for 2 and offer only marginally more metal character than planar‐3 . The LUMO of 4 is largely of metal/dppe character, in the case of the phosphite analogue 5 the LUMO is thienyl–π* in character, with the unoccupied metal orbital lying slightly (ca. 0.04 eV) higher in energy.The ruthenium complexes T(E), was calculated by using the GOLLUM code.To provide further insight into the experimentally observed trends, and to better evaluate the properties and behaviour of these molecular junctions, calculations using a combination of DFT (the SIESTA code)EFDFT) is often not reliable,G was computed for a range of Fermi energies EF. The calculated conductances G are plotted as functions of EF−EFDFT in Figure EF was chosen, which gave the closest overall agreement. This yielded a small correction of EF−EFDFT=−0.075 eV, which has been used in all of the theoretical results described below. Thygesen and colleagues have discussed similar situations for C60‐contacted molecular wires, and have shown that critical molecular orbitals can become pinned close to the Fermi level owing to partial charge transfer, leading to good quantitative agreement between calculated and experimentally determined conductance.A key factor governing the conductance of a molecular junction is the position of the Fermi level of a metal electrode with respect to the molecular HOMO and LUMO levels. In turn, this energy alignment is sensitive to not only the chemical nature of the contacting groups that bind the molecule to the electrode, but also the precise configuration of the metal electrode–molecule contact.2–5. This lack of variation occurs because although the HOMO and LUMO transport resonances differ significantly between molecules 2–5, transport in the vicinity of the middle of the HOMO–LUMO gap is similar for all molecules  associated with the molecule, the number calculated on the molecule in the junction (QMG) and hence the number of electrons associated with the thienyl SAu contacts (Γ) (or “bonds”) based on calculated Mulliken charges. Mulliken charges are basis‐set‐dependent mathematical constructions and therefore only approximately coincide with the physical charge on a molecule. However, it is clear from the data in Table Γ, and the nature of the contact, is only weakly dependent on the nature of the backbone and auxiliary ligands in 2–5. Overall, the molecular conductances of these molecules are similar, with minor variations arising through convolution of the strength of the S→Au bond, and the position of the tail of the HOMO resonance relative to the Fermi level of the electrodes 2 (2), trans‐[Pt(C≡C‐cyclo‐3‐C4H3S)2(PEt3)2] (3), trans‐[Ru(C≡C‐cyclo‐3‐C4H3S)2(dppe)2] (4) and trans‐[Ru(C≡C‐cyclo‐3‐C4H3S)2{P(OEt)3}4] (5) have been prepared and studied in metal|molecule|metal junctions by using both STM‐I(s) and STM‐BJ methods. The compounds 2–5 each display two conductance values that differ by a factor of ≈2 within the following range of conductance values: (0.44±0.10–1.03±0.28)×10−4 G0 (low conductance group) and (2.70±0.66–3.18±1.04)×10−4 G0 (high conductance group). The MPVC method has been applied to verify the lowest conductance group in an algorithmically definable fashion. For the 3‐thienyl contact employed here, the conductance values obtained by using MPVC and manual data selection were very similar, although there were some differences between the current–distance data sets assigned by each method. The MPVC method, which allows reproducible and objective analysis of conductance features close to the limit of the current amplifier, is therefore a promising avenue for the further exploration of low conductance features. In addition, with an increase in the number of sub‐clusters the method should also prove useful in the analysis of a wider array of junction configurations or in cases where the junction evolves over time or with distance. Further efforts to develop and exploit the MPVC tool are now underway. A quantum chemical analysis of the electronic structures of the isolated molecules reveals a similarly structured HOMO in each case. Within model junctions, the Fermi level lies slightly towards the HOMO resonance in each case, and a non‐resonant hole tunnelling mechanism mediated by the similarly structured HOMOs is proposed. The positioning of the Fermi level well within the HOMO–LUMO gap is proposed to account for the similar conductance behaviour across the series. Our study demonstrates that although for some systems, platinum complexes may well be less conductive than purely organic analogues or similarly structured complexes of the group 8 metals, this is not a universal situation, and by appropriate use of contacts and ancillary ligands to position key molecular orbitals with respect to the Fermi levels of the electrodes, rather efficient molecular wires may be engineered. For the future, it will be of interest to study thermal transport through such wires, as although they have similar electrical properties, their vibrational properties and phonon thermal conductances are likely to differ significantly. This ability to tune the latter, while preserving electronic conductance is an attractive proposition for the design of thermoelectric thin films.The family of 3‐thienylethynyl contacted compounds 1,4‐C3: C24H36P2PtS2, M=645.69, monoclinic, a=8.6132(1), b=11.1767(2), c=14.1265(2) Å, β=104.958(1)°, U=1313.84(3) Å3, T=180 K, space group P21/n, Z=2, θmax=36.56°, 31 485 reflections measured, 6229 unique (Rint=0.034), R1=0.0230 [I>2σ(I)], wR2=0.0560 , S=1.068, Δρmax,min=1.509, −0.775 e Å−3.4: C64H54P4RuS2⋅CH2Cl2, M=1197.07, triclinic, a=10.3342(2), b=13.2414(3), c=21.4418(5) Å, α=78.892(2), β=84.176(2), γ=71.219(2)°, U=2723.36(10) Å3, T=100 K, space group P1‾, Z=2, θmax=34.38°, 63 208 reflections measured, 21 631 unique (Rint=0.033), R1=0.0464 [I>2σ(I)], wR2=0.1100 , S=1.040, Δρmax,min=1.912, −1.936 e Å−3.The thiophene group on molecule 2 is disordered over two sites with occupancies constrained to 0.5 after trial refinement. Geometries were restrained to ideal values. Both dichloromethane solvent molecules are disordered about crystallographic inversion centres.5: C36H66O12P4RuS2, M=979.96, tetragonal, a=11.7879(1), c=17.5181(3) Å, U=2432.22(5) Å3, T=180 K, space group P4‾21c, Z=2, θmax=37.64, 49 278 reflections measured, 6324 unique (Rint=0.037), R1=0.0325 [I>2σ(I)], wR2=0.0746 . S=1.135, Δρmax,min=0.476, −0.356 e Å−3.The thiophene group was modelled as being disordered about the crystallographic twofold axis. One methyl group of the triethylphosphite ligand was also modelled as being disordered over two sites with occupancies constrained to 0.5 after trial refinement.CCDC 1504230 (3), 1504231 (4), and 1504232 (5) contain the supplementary crystallographic data for this paper. These data can be obtained free of charge from The Cambridge Crystallographic Data Centre.All single‐molecule conductance measurements were recorded at room temperature in mesitylene with an Agilent 5500 SPM microscope. Molecular adlayers were formed on flame‐annealed gold on glass samples, purchased from Arrandee, Germany. These commercially available substrates were rinsed with acetone and flame‐annealed carefully for about 20 s with a butane torch until a slight orange glow was obtained. This flame‐annealing procedure was performed three times and generally resulted in relatively large area flat Au(111) terraces.I(s) methods. The in situ break‐junction method developed by Xu and Tao relies on the formation and cleavage of metallic break junctions between the STM tip and the underlying gold substrate.Single‐molecule electrical measurements were performed by using both the in situ break‐junction (BJ) and I(s) technique, a gold STM tip is brought to a fixed distance, determined by the set point conditions, above the gold surface covered with the target molecule under analysis.I0). The measurement involves first locating the STM tip close to the gold substrate at a given height by setting the I0 and Vbias values. The feedback loop of the STM is then temporary disabled and the STM tip is rapidly retracted (s=distance) while the tunnelling current (I) is continuously recorded. At the initial set‐point conditions, the target molecules can be trapped between the STM tip and the gold substrate as a molecular bridge. In such circumstances, during the retraction of the STM tip, the molecular bridge is then pulled up and stretched in the nanojunction until the molecular junction is cleaved. For both the BJ and the I(s) methods, when the molecular bridge is formed and then cleaved, a characteristic current plateau is typically observed, with a step‐like drop in the current reflecting cleavage of the molecular bridge. On the other hand, if during the tip retraction molecules are not caught in the STM nanogap then the tunnelling current simply decreases exponentially with separation.In the http://datacat.liverpool.ac.uk/187/).Data from the single‐molecule studies are available from the University of Liverpool data catalogue  should be addressed to the authors.SupplementaryClick here for additional data file.


Perhaps unsurprisingly, they find that some important ecologic traits, such as drought response, are coherent within the OTU, but that others vary significantly. Here we discuss their findings in relation to the more general issue of how molecular tools can be effectively used to study microbial ecology. We specifically note the need for investigators to choose the right molecular methods for their biological problem, as nature does not respect the limitations and conventions associated with our methods.Microbial ecology has been transformed by the advent of high-throughput marker gene and metagenomic sequencing methods. These tools provide expansive descriptions of microbial communities, but the descriptions are framed in terms of molecular objects, such as 97% ribosomal operational taxonomic units (OTUs), rather than biological objects, such as species. A recent study by A. B. Chase and colleagues (mBio 8:e01809-17, 2017,  When we try to make sense of microbial communities, we often conceive of a cast of coherent species playing roles determined by their phenotypes and traits. However, the advent of high-throughput DNA sequencing has changed how we observe microbial communities in ways that can distance us from that conceptual framework.Marker gene and metagenomic sequencing (MGS) methods provide expansive descriptions of microbial communities, but the descriptions are framed in terms of molecular measurements that are at best proxies for species and phenotypes. For example, microbial species (to the extent that they exist) are difficult to define and demarcate, while the MGS proxy for a species—the 97% identity ribosomal operational taxonomic unit (OTU)—is clearly defined and can be measured by low-cost marker gene sequencing. Our measurements of molecular objects are shaped by technical limitations, such as sequencing error, and by analytic choices, such as percent identity thresholds, that can begin as technical choices and mature into deeply rooted conventions. The accessibility of MGS methods has led to a growing prominence for molecular objects like OTUs in how we think and talk about microbial communities, thereby abstracting us one step further from biology and making it easy to forget these limitations and conventions. Without care, we can engross ourselves in the ecology of molecules rather than of microbes.mBio article (Curtobacterium. In previous work as part of the Loma Ridge Global Climate Experiment, the authors established Curtobacterium as the most abundant bacterial genus in Southern California leaf litter communities (Curtobacterium isolates for decomposing complex carbohydrates found in leaf litter (In a recent  article , Chase amunities . The higmunities  and the f litter  further Curtobacterium strains contain 16S rRNA genes that are at least 97% similar; thus, Curtobacterium strains have typically been grouped into a single object by previous OTU-based analyses. Chase and colleagues previously found significant genomic diversity within sequenced Curtobacterium isolates (Curtobacterium OTU. This work also contributes to a larger research program pursued by the authors and others investigating the phylogenetic scales over which various microbial phenotypes are conserved and remain coherent (reviewed in reference Known isolates . In the Curtobacterium OTU. A comparison of the genomes of 16 isolates revealed significant variation in nucleotide sequence, protein sequence, and gene content and led the authors to demarcate six distinct Curtobacterium clades. Metagenomic sequencing data collected longitudinally across control, drought, and nitrogen enrichment treatments were mapped onto the isolate genomes, revealing the dynamics of each Curtobacterium clade. The ability of each isolate to degrade cellulose and xylan, the most abundant complex carbohydrates in leaf litter, was directly tested by laboratory growth assays at two different temperatures.In their study, Chase and colleagues  combinedCurtobacterium microbes, but others varied significantly. All clades of Curtobacterium increased their relative abundance in response to drought conditions, and all but one of the strains isolated was able to degrade xylan and cellulose at the hypothesized optimal temperature of 22°C. However, certain clades responded more strongly than others to drought conditions, and major variation between and within clades was observed in xylan and cellulose degradation at the hypothesized maximum viable temperature of 37°C.Some traits were coherent across Vibrionaceae (These findings are consistent with a larger body of work showing that the phylogenetic depths over which ecologically relevant traits are conserved ranges widely among traits, from the deepest evolutionary branches to well below the species level . Toleranionaceae  and viruionaceae .This variation in phylogenetic conservation among traits has clear implications for marker gene and metagenomic studies. We would like to use molecular objects as easily measured proxies for microbial functions within a community, but for this to be successful, those objects must effectively track the relevant functional traits. This will not be the case if our molecular objects group together strains that differ in these traits. Therefore, the phylogenetic resolution of the molecular objects we ultimately analyze must be commensurate with, or finer than, the phylogenetic conservation in the relevant traits.Curtobacterium was coherent across the Curtobacterium OTU, and thus, amplicon sequencing of the 16S rRNA gene could be an effective tool for studying microbial drought response in leaf litter. However, the substantial differences in cellulose and xylan degradation capacity at high temperatures suggest that a different method that can resolve different Curtobacterium strains may be more appropriate if temperature extremes are expected to be an important factor.Because different traits are conserved at different levels, the most appropriate molecular method and analysis will depend on the system being studied and the question being asked . In thisAs genetic data are only ever a proxy for phenotype, the need to match the resolution of molecular methods to the scale of variation in functional traits applies not just to marker gene methods but to all analyses based on DNA sequencing. The analysis of shotgun metagenomic data also relies on explicit or implicit similarity thresholds within which diversity is effectively ignored. When mapping reads to a set of references, for taxonomic assignment or otherwise, the incompleteness of any reference database necessarily leads to some genetically and phenotypically distinct sequences being mapped to the same reference. Similarly, the genomic bins used in the creation of metagenome-assembled genomes can lump together distinct strains that are too similar for the assembly or binning algorithms to distinguish.Molecular approaches that treat the gene, rather than the species, as the fundamental biological object also depend on similarity thresholds that can group together phenotypic diversity. Shotgun metagenomic data are commonly mapped against databases of microbial genes to obtain presence/absence and relative abundance measurements for orthologous genes. Gene-level approaches sidestep the disconnect between functional diversity and organismal phylogeny that can be caused by processes like horizontal gene transfer, but orthologous gene clusters are still aggregates of similar but different genes and thus can group together relevant phenotypic variation. For example, the ability to grow on a particular carbohydrate source might be determined by the presence of an orthologous gene cluster defined at a 10% divergence level, while the growth rate at the prevailing temperature in the environment studied may vary substantially between different orthologs in the cluster.Escherichia coli. However, the problem of matching molecular objects with the scale of the relevant phenotypic variation is not a challenge for amplicon sequencing alone; it is a challenge for all molecular methods used to study patterns and processes driven by function.Too often, molecular and bioinformatic methods are chosen mainly for their ease or popularity. The tremendous variation in the phylogenetic scales over which different phenotypes are conserved requires more attention to matching the molecular objects we use in our analyses with the microbial ecology we wish to understand. This issue is perhaps most widely recognized for amplicon sequencing of the 16S rRNA gene, which is understood to be effective for some problems, such as diagnosing dysbiosis in the vaginal microbiome , but is Curtobacterium OTU revealed that some ecologically relevant traits were coherent across the OTU, while others varied within the OTU. This should not be surprising. There are no 97% OTUs in nature. There is no “microdiversity” in nature. There is only diversity. Nature does not respect the limitations and conventions associated with our methods; therefore, choosing the right tools for a given study may require a break from convention, and careful consideration of the match between potential molecular methods and the functional variation important to our scientific problem.This study by Chase and colleagues  represen


Recent updating of the World Health Organization (WHO) classification of central nervous system (CNS) tumors in 2016 demonstrates the first organized effort to restructure brain tumor classification by incorporating histomorphologic features with recurrent molecular alterations. Revised CNS tumor diagnostic criteria also attempt to reduce interobserver variability of histological interpretation and provide more accurate stratification related to clinical outcome. As an example, diffuse gliomas (WHO grades II–IV) are now molecularly stratified based upon isocitrate dehydrogenase 1 or 2 (IDH) mutational status, with gliomas of WHO grades II and III being substratified according to 1p/19q codeletion status. For now, grading of diffuse gliomas is still dependent upon histological parameters. Independent of WHO classification criteria, multidimensional scaling analysis of molecular signatures for diffuse gliomas from The Cancer Genome Atlas (TCGA) has identified distinct molecular subgroups, and allows for their visualization in 2-dimensional (2D) space. Using the web-based platform Oncoscape as a tool, we applied multidimensional scaling-derived molecular groups to the 2D visualization of the 2016 WHO classification of diffuse gliomas. Here we show that molecular multidimensional scaling of TCGA data provides 2D clustering that represents the 2016 WHO classification of diffuse gliomas. Additionally, we used this platform to successfully identify and define novel copy-number alteration-based molecular subtypes, which are independent of WHO grading, as well as predictive of clinical outcome. The prognostic utility of these molecular subtypes was further validated using an independent data set of the German Glioma Network prospective glioblastoma patient cohort.The online version of this article (doi:10.1186/s40478-017-0443-7) contains supplementary material, which is available to authorized users. ComCDK4 and MDM2. Of note, these alterations were not limited to glioblastomas. The IDH-wildtype diffuse gliomas have frequent chromosome 7 gain, chromosome 10 loss, and 9p loss, among other CNAs. Additional MDS in three dimensions (image available at http://zager.co/glioma/chart.html) demonstrates that the IDH-wildtype cluster can be divided spatially into three subgroups  between subgroups B and C, which are distinguished by gain of chromosome 19  frequencies were analyzed across the molecular clusters defined by MDS to confirm known CNAs as well as identify new cluster-associated CNAs Fig. . The oliutations , is freqCDK4/MDM2 co-amplification for the astrocytic glioma/glioblastoma, IDH-wildtype cluster (W1–W4), as well as CDK4 amplification, CDKN2A deletion, and chromosome 14 gain for the astrocytic glioma/glioblastoma, IDH-mutant cluster (M1–M3) , and vice versa for WHO grade III/IV versus grade II upon adjustment for molecular subgroup  Fig. . For then = 284) of glioblastomas from the German Glioma Network (GGN) was evaluated. Comparing WHO grade IV glioblastomas, median overall survival of the GGN cohort (18.9 months) versus the TCGA cohort (13.5 months) was longer . A caveat to this comparison is that the TCGA gliomas are WHO grade II–IV while the GGN gliomas are all WHO grade IV, which should not be a significant confounder as the molecular subtypes appear to be grade independent  in survival so their combined median overall survival was used for baseline normalization. Normalized median overall survival for each molecular subtype showed similar trends for TCGA and GGN data sets  comparing TCGA and GGN overall survival by molecular subtypes  oligoastrocytoma is discouraged in the new WHO system and how ‘oligoastrocytoma’ easily resolves into either oligodendroglioma or astrocytoma entities , 33, 37.A current area for future clarification and refinement in the WHO classification system is that of grading . While mAlong with reflecting changes in WHO classification of diffuse gliomas, some patterns of genetic alterations become readily apparent by this visualization tool, as was the case for the bimodal age distribution of the IDH-mutant, and 1p/19q-codeleted Oligodendroglioma cluster Fig.  and clusEGFR amplification, and are therefore likely not associated with EGFR-amplified cases in the IDH-wildtype cluster. Epithelioid glioblastomas harbor the BRAF-V600E mutation in about half of all cases [BRAF single nucleotide alterations present exclusively in the IDH-wildtype cluster (not shown), however, they are of various grades  and appear not to group together. Therefore, it will take increased numbers of properly annotated glioblastoma histological subtypes to resolve their spatial distribution, if any. Along these lines, the current datasets for brain tumors in Oncoscape are limited to the diffuse gliomas from the TCGA. Additional efforts are necessary to characterize other CNS tumor types  by multidimensional scaling analysis in order to compare molecular genetic structures and make associations between and amongst histologically disparate brain tumor types.With the TCGA dataset, there are current limitations with regards to placement of some diffuse glioma histologic subtypes on the 2D Oncoscape map. For example, it is not clear where gemistocytic astrocytoma, IDH-mutant  , would bll cases . There aThe ability to visualize brain tumor datasets in 2D space relative to pathologic diagnosis and molecular alterations with tools such as Oncoscape, affords the possibility of using such a tool as a reference point for clinical utility. On the clinical side, molecular information derived from a specific patient’s surgical material can be queried in the reference dataset, and survival and treatment strategies of similar patients can inform prognosis and risk-stratification in real-time. This may have further utility in interdisciplinary settings such as neuro-oncology tumor boards, where management decisions are discussed and planned. This has the potential to ease the decision making process and contribute to the application of precision medicine for individual patients. Given the time and financial constraints that can be associated with whole exome/genome 2D mapping for an individual patient, a more limited or targeted analysis may be more prudent. In this setting, cluster-derived molecular subtypes as the ones described in this study , may be a more appropriate way to risk-stratify in a time-sensitive and cost-effective manner. Beyond brain tumors, Oncoscape can be used to visualize and analyze additional cancer datasets for diagnostic and translational purposes. All 33 TCGA datasets encompassing several organ systems are available for analysis in Oncoscape. Perhaps 2D molecular analysis of this type can help visualize distinct clusters of various other cancers, and help to push the solid tumors from these organ systems to move into integrated diagnoses and risk-stratification, similar to that of neoplasms of the central nervous system.Additional file 1:Supplementary Material. (PDF 190 kb)


Membrane depolarization significantly induced axonal branch-extension in cultured cerebellar granule neurons (CGNs). Local application of nifedipine using a polydimethylsiloxane (PDMS)-based microfluidic device demonstrated that Ca2+ entry from the axonal region via L-type voltage-dependent calcium channels (L-VDCC) is required for branch extension. Furthermore, we developed a method for locally controlling protein levels by combining genetic techniques and use of a microfluidic culture system. A vector for enhanced green fluorescent protein (EGFP) fused to a destabilizing domain derived from E. coli dihydrofolate reductase (ecDHFR) is introduced in neurons by electroporation. By local application of the DHFR ligand, trimethoprim (TMP) using a microfluidic device, we were able to manipulate differentially the level of fusion protein between axons and somatodendrites. The present study revealed the effectiveness of microfluidic devices to address fundamental biological issues at subcellular levels, and the possibility of their development in combination with molecular techniques.The establishment and maintenance of axonal patterning is crucial for neuronal function. To identify the molecular systems that operate locally to control axonal structure, it is important to manipulate molecular functions in restricted subcellular areas for a long period of time. Microfluidic devices can be powerful tools for such purposes. In this study, we demonstrate the application of a microfluidic device to clarify the function of local Ca


It is essential the existence of suitable preclinical models to elucidate the molecular mechanism of a disease and to test potential drugs and therapies. One of the major problems to discover new pharmacological therapies in Parkinson's disease is the lack of suitable preclinical model. This explains why L-dopa is still the most effective drug despite the severe side effects observed after 4-6 years of treatment. There is a long list of clinical studies failed despite being based on robust preclinical results obtained with exogenous neurotoxins as preclinical model. The use of preclinical models based on exogenous neurotoxins  or rotenone) is an important problem to translate successful results to clinical studies and new therapies in Parkinson's disease [Recently, we published that aminochrome can be used as a new preclinical model for Parkinson's disease. The unilateral injection of aminochrome in the striatum induces contralateral behavior without significant loss of dopaminergic neurons accompanied with a significant decrease in dopamine release and a significant increase in GABA levels, generating an imbalance of neurotransmitters in the basal ganglia. Aminochrome induces mitochondrial dysfunction resulting in a significant decrease in ATP level, explaining the significant decrease in the amount of synaptic vesicles in the terminals and a decrease in dopamine release since both axonal transport of vesicles for neurotransmission and dopamine release require ATP .Aminochrome is an o-quinone formed during dopamine oxidation to neuromelanin in dopaminergic neurons containing neuromelanin. It has been demonstrated that aminochrome induces mitochondrial dysfunction, generation of neurotoxic oligomers of alpha-synuclein, protein degradation dysfunction of both proteasomal and lysosomal systems, oxidative and endoplasmic reticulum stress  aminochrome is formed inside of dopaminergic neurons containing neuromelanin lost during the disease; (ii) aminochrome triggers mitochondria dysfunction, formation of alpha-synuclein neurotoxic oligomers, protein degradation dysfunction, oxidative stress and endoplasmic reticulum stress.


In this Research Topic, we have explored the synergistic effects and mechanisms between DNA damage induction and inflammatory response under a variety of cellular stresses like radiations, replication stress, environmental chemicals, bacteria etc. In general, we believe that priority in research must be given to studies following a holistic approach and at the organism level from plants, flies, worms to mouse, and humans. We consider this to be a genuine example of a Systems Biology application in life. Several lines of evidence suggest that DNA-damage especially the one resistant to repair can cause immune reaction and inflammation, and is linked to cancer development and other pathologies  genes and their link with inflammatory proteins, underlying molecular mechanisms justifying the system-level analysis of systemic action, computational and bioinformatic approaches for better description of real biological systems and integration of current empirical or theoretical knowledge.Spanou et al. the role of genetic variability as a regulator of TLR4 and NOD signaling in response to bacterial driven DNA Damage Response (DDR) and Inflammation is discussed with a focus on the gastrointestinal (GI) Tract. The fundamental role of human Toll-like receptors (TLRs) and NOD-like receptors (NLRs), the two most studied pathogen recognition receptors (PRRs), is also explored as one of the protection mechanisms against pathogens and excessive tissue injury. On the same direction, in a mini-review article by Kalisperati et al. the most current picture of the molecular pathways involved in DNA damage and DDR activation during H. pylori infection and gastric tumorigenesis are discussed. As well-known, Helicobacter pylori (H. pylori) is a Gram negative bacterium that colonizes the stomach of almost half of the human population, therefore the interest on the current subject is high.Specifically, in the review article of Eliopoulos et al. uncover the mechanisms by which autophagy and DDR act as partners regulating cellular and organismal homeostasis. In this review, the authors provide novel insights into the pathobiology of conditions associated with accumulation of DNA damage, including cancer and aging, and original concepts for the development of improved therapeutic strategies against these pathologies. Aging is certainly a process associated with DDR and inflammation  progression. Last but certainly not least, Nakad and Schumacher review the very important and recent advances on the understanding of the role(s) of DDR in activating immune signaling. They provide convincing evidence gained into (i) which molecular and cellular pathways of DDR activate immune signaling, (ii) how DNA damage drives chronic inflammation, and (iii) how chronic inflammation causes DNA damage and pathology in humans.Then moving to the DDR-related mechanisms of organism homeostasis, in an analytical review, We are grateful to all contributors of this Research Topic. In total, twenty-five different authors from three (3) different countries contributed with analytical and current review articles. Furthermore, we thank the reviewers which helped us and the authors to create an interesting and high-quality Research Topic.We hope that you enjoy reading this Research Topic as much as we have enjoyed editing it and it will further promote the field under a systems biology perspective.AG wrote the initial draft of the editorial; both authors revised the manuscript and approved the final version of it.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.