In this report, we designed and synthesized polyacrylonitrile/silver (PAN/AgNPs) nanofibers via an in-situ method to obtain a washable with high-dispersed silver nanoparticles membrane to form the hierarchically organized antibacterial mask to prevent the two-way effect of bacteria from person to environment and environment to person. For this objective, the electrospun PAN nanofibers were stabilized via the heating method. Different amounts of AgNPs were loaded into the PAN nanofibers by using silver nitrate and sodium hydroxide solutions. The basic results showed that AgNPs was homogenously loaded in PAN nanofiber matrixes. Furthermore, the release profile based on two-stage release theory showed that when the negligible amount of AgNPs was loaded into the nanofibers, the release significantly decreased, whereas antibacterial activity increased. The greatest potential antibacterial activity of the lowest amount of AgNPs showed controllable AgNPs release from PAN nanofibers that has a direct relationship with the washability and could promote the application of the produced product. Over the last 50 years, antibiotic resistance has been a major health concern in the world since bacteria progressively adapted to antibiotics . ConsequHowever, many antibacterial additives such as phenols, inorganic salts, organometallics , organicHowever, the researchers did not focus on the stabilized AgNPs in the nanofiber structures to produce an antibacterial mask by hierarchically organizing the nanofiber membrane to control the AgNPs release by chemical bonding. Nanofibers showed the ability to be made into an ultrafine solid with continuous fibers and a controlled surface and molecular structures for antibacterial properties . In addiTherefore, we attempted to fabricate a polyacrylonitrile/silver (PAN/AgNPs) nanofibers composite membrane for a washable antibacterial mask by the hierarchical arranging of layers. PAN/AgNPs nanofibers composite membrane, due to its two-way protection against bacteria and dust, was chosen as the main component of this research. The morphology of nanofiber and nanoparticles has been studied by scanning electron microscope and transmission electron microscope (TEM). Attenuated total reflection (ATR) and X-ray photoelectron spectroscopy (XPS) have been employed for chemical characterization and confirming the presence of AgNPs in the nanofibers. Inductively coupled plasma mass spectrometry (ICP-MS) was used for direct measurement of the ppm amount of AgNPs upon release and loaded amount in nanofibers. Agar disc diffusion method was chosen for antibacterial activity test.N,N-Dimethylformamide (DMF) (99.59%). Silver nitrate , sodium hydroxide , and hydrochloric acid  were purchased from Wako Pure Chemical Industries, Ltd.  and deionized water was used.Polyacrylonitrile (PAN) with  dimethyl sulfoxide (99.9%) (DMSO) was purchased from Sigma Aldrich Corporation USA, w/w PAN solution was prepared in DMF with stirring for 5 h at room temperature. The spinning solution was loaded in a plastic syringe with a tip diameter of 0.6 mm and a copper wire was chosen as the positive electrode. The distance from the capillary tip to the collector was kept at 13 cm and then supplied with a voltage of 12 kV. The nanofibers were formed without beads at room temperature and in 45% humidity. The resultant nanofibers mat was stabilized via heating at 250 °C for 2 h in an air atmosphere. The PAN/AgNPs was prepared by dipping the nanofibers mat in a silver nitrate solution, followed by dipping in an alkaline solution. For this purpose, 1 gL silver nitrate solution was prepared and the PAN nanofibers mat was supplied directly to 40 mL of silver nitrate solution for 12 h. Then, PAN nanofibers containing the silver ions were rinsed with deionized water and subjected to 40 mL sodium hydroxide solution of 1 g/L for 12 h. Nanofibers were placed in deionized water for 15 min and shaken to wash out the extra sodium hydroxide and silver ions. This cycle was repeated for three times to prepare the different amounts of silver nanoparticles through the PAN nanofibers. Finally, the obtained PAN/AgNPs was dried at 30 °C and used for characterization. The mechanism of chemical reaction of AgNPs with PAN nanofibers [The 10% nofibers  is shownThe morphology of the nanofibers was observed by using scanning electron microscopy  with accelerating voltage of 12 kV. The dispersion of nanoparticles on nanofibers was studied by transmission electron microscopy  with an accelerating voltage of 200 kV that was used to analyze the dispersion of AgNPs on the nanofibers. The average diameter and size distributions of the nanofibers and AgNPs were determined from SEM and TEM micrographs by image analysis software . −1 with a resolution of 4 cm−1 and the addition of 128 scans at room temperature. The samples in solid and dry form were directly supplied to ATR.Attenuated total reflectance (ATR) spectroscopy  of the nanofibers was recorded between the wavelengths of 450 and 4000 cmX-ray photoelectron spectroscopy (XPS) was conducted on a Shimadzu-Kratos AXIS-ULTRA HAS SV  by using an Al X-ray source set at 10 kV and 15 mA to confirm the presence of AgNPs in the nanofibers. Mt is the amount of AgNPs released after 72 h, Mi is the total amount of AgNPs loaded in PAN nanofibers, and all calculations are done based on using 1 g nanofibers.The amount of loaded and released AgNPs from nanofibers was directly measured by inductively coupled plasma mass spectrometry (ICP-MS) . The release profile over a period of 72 h was studied by dipping 0.1 g of nanofibers in a plate containing 20 mL of deionized water by shaking at 28 °C. During the release, 2 mL of solution was removed and exchanged by deionized water at precise times . The solution containing AgNPs, which was removed from the source each time, was subjected to ICP-MS spectrometry for measurement of AgNPs released into the deionized water. The amount of silver loaded into nanofibers was measured by dissolving the 0.01 g nanofibers in 5 mL DMSO and diluting the 100 µL of the obtained clear solution with 900 µL deionized water and supplied to ICP-MS spectrometry. The release profile was presented as a percentage based on the below formula:Staphylococcus and Pseudomonas) with the disc diffusion method. In this method, Pseudomonas and Staphylococcus were used as model bacteria. In this method, the bacteria are cultured in tryptic soy broth liquid medium and incubated overnight at 35 °C. Bacteria from the prepared source were seeded by dispersing TSB containing the bacteria on the surface of nutrient agar plates and nanofibers placed on the cultured diffusion plates for each strain and incubated overnight at 35 °C. To measure the quantified antibacterial properties of samples, colony formation unit method used base on ISO 22196. Briefly, the cell concentration of 105 per mL was prepared by using each strain bacteria in PBS 1X after seeding in tryptic soy broth. The samples were placed in each solution under shaking at 35 °C for 24 h. The series of dilutions were prepared using PBS 1X and 20 µL of each diluted solution containing bacteria placed on agar plates and incubated for almost 24 h at 35 °C. The percentage of viable bacteria was calculated using the formula below:S shows the colony formation unit for samples and CFUC is assigned to colony formation unit for control. The antibacterial properties of PAN nanofibers containing different amounts of silver nanoparticles were examined with two bacterial strains  to grade 4 (severe toxic) evaluation, trypan blue was added after 24 h in each plate and observed by microscope.The mechanical properties of the final composite nanofiber with one cycle loaded AgNPS before and after wash during 72 h were assessed using a universal testing machine  under a cross-head speed of 1 mm/min at room temperature. In accordance with ASTM D-638, the nonwoven nanofibers were prepared in the shape of a dumbbell to test their tensile strength. Stress and strain were calculated by Formulas (3) and (4) as mentioned below:In order to investigate the morphology of PAN/AgNPs nanofibers, SEM was employed to observe the composite nanofibers as shown in In order to investigate the AgNPs morphology, TEM was used to analyze the dispersion of nanoparticles into the nanofibers as shown in −1 and C≡N band was around 2241 cm−1 stretching, and a strong signal at 1666 cm−1 appeared due to the C=O band. After stabilization, obvious peaks were observed about C≡N and C=O changed and an extensive band at 3344 cm−1 was related to symmetric stretching of the NH group formed, and in comparison to Zhang et al., it seems that this peak appeared due to stabilization of PAN nanofibers [2 vibration appeared in 1634, 1452, and 1351 cm−1. However, the presence of a strong peak at 1031 cm−1 was attributed to the C-N functions group of PAN nanofibers [In order to investigate the chemical structure of the functional groups of a pure PAN nanofiber mat, a stabilized pure PAN nanofiber mat and a PAN nanofiber mat containing the different amounts of AgNPs were studied by using ATR-FTIR spectroscopy as shown in nofibers . −1 and 1031 cm−1 decreased significantly. It seems that this phenomenon appeared due to the reaction between the Ag ions with NH and the C-N group through the nanofibers [−1, 2243 cm−1, and 1452 cm−1, respectively, due to the presence of CH2 stretching, C≡N, and CH2 vibrations, appeared without any changes in the various amounts of AgNPs and it seems that the presence of AgNPs did not have any effect on changing the chemical structure of PAN nanofibers. The chemical reaction between the AgNPs with PAN nanofibers occurred flowing by covalence bonding with NH and C-N groups as shown in Table 1On the other hand, with the in situ synthesis of the AgNPs on the nanofibers, the intensity of strong peaks at 3344 cmnofibers . However5/2, Ag3d3/2, and the slitting between core levels calculated by 6.2 eV that indicate a normal state of AgNPs. However, the peaks related to AgNPs have been compared with literature that focused on synthesizing the AgNPs with the immersed polymer matrix [In order to confirm the presence of silver nanoparticles on the nanofibers, the XPS spectra were studied and the resultant graph is shown in r matrix . The resThe amount of loaded AgNPs in 1 g PAN nanofibers was calculated as 2447, 4898, and 11,262 ppm for the cycles 1, 2, and 3, respectively, as shown in Pseudomonas and Staphylococcus bacteria were used as model bacteria. Mishra S. K. et al. in 2016 showed that the antibacterial properties of silver nanoclusters which synthesized the AgNCs by in situ method had toxicity around 56% at the maximum amount of AgNCs [co-glycolide)/AgNPs. Herein, they concluded that 3 wt % AgNPs incorporated with poly (l-lactide-co-glycolide) has good biocompatibility despite this concentration showing weak antibacterial results [The antibacterial efficiency of the PAN/AgNPs nanofibers was examined by agar diffusion method. In this method, of AgNCs . However results .Pseudomonas and Staphylococcus bacteria but it also shows that AgNPs presence in the nanofiber matrix has more effect against Pseudomonas than against Staphylococcus. When the numbers of immersing cycles were increased, the antibacterial properties were also increased as shown in The results of antibacterial efficiency are shown in The results as displayed in In order to investigate the mechanical strength behavior of the PAN nanofibers, PAN/AgNPs nanofibers and washed PAN/AgNPs nanofibers with one cycle loaded AgNPs were studied as shown in Herein, we successfully designed a washable, hierarchically organized, antibacterial mask by in situ synthesis of metallic nanoparticles on PAN/AgNPs nanofibers. On the basis of characterizations, it was concluded that PAN/AgNPs nanofibers, which were fabricated with one cycle loading AgNPs (2247 ppm/gr nanofiber) made ideal membranes for antibacterial properties despite their lowest antibacterial properties in comparison to cycles two and three. It seems that the ability of one cycle loading AgNPs is enough for the preparation of a washable antibacterial mask. The release profile of AgNPs in the 1 gr of PAN nanofibers has been shown to control the release ratio during 120 h that emphasized the ability of this method for preparing washable antibacterial products. Therefore, PAN/AgNPs nanofibers fabricated with one cycle of loading AgNPs had the optimal potential for antibacterial properties and biocompatibility. Results showed this type of membrane is the most suitable use of nanofibers to form the hierarchically organized antibacterial breath mask with appropriate biocompatibility (less than grade 1) to prevent the two-way effect of bacteria from person to environment and environment to person. The tensile strength results showed loading silver nanoparticles increased the mechanical properties of PAN nanofibers even after 120 h washing with deionized water. Hence, this proposed antibacterial breath mask would fulfill the stated and implied needs of customers.


Journal of Clinical Medicine was specifically dedicated to research evaluating the effects of exercise on cognitive function. This Special Issue published 23 articles dedicated to this topic (Cognitive function is associated with longevity and is of critical importance for optimal daily functioning . As suchis topic . These sSeveral interesting findings were observed from this collective body of work. As an example, Chen et al.  evaluate


Infection by Kaposi’s sarcoma-associated herpesvirus (KSHV) is necessary for the development of Kaposi’s sarcoma (KS), which most often develops in HIV-infected individuals. KS frequently has oral manifestations and KSHV DNA can be detected in oral cells. Numerous types of cancer are associated with the alteration of microbiome including bacteria and virus. We hypothesize that oral bacterial microbiota affects or is affected by oral KS and the presence of oral cell-associated KSHV DNA. In this study, oral and blood specimens were collected from a cohort of HIV/KSHV-coinfected individuals all previously diagnosed with KS, and were classified as having oral KS with any oral cell-associated KSHV DNA status , no oral KS but with oral cell-associated KSHV DNA , or with neither oral KS nor oral cell-associated KSHV DNA . We sequenced the hypervariable V1-V2 region of the 16S rRNA gene present in oral cell-associated DNA by next generation sequencing. The diversity, richness, relative abundance of operational taxonomic units (OTUs) and taxonomic composition of oral microbiota were analyzed and compared across the 3 studied groups. We found impoverishment of oral microbial diversity and enrichment of specific microbiota in O-KS individuals compared to O-KSHV or No KSHV individuals. These results suggest that HIV/KSHV coinfection and oral microbiota might impact one another and influence the development of oral KS. Kaposi’s sarcoma (KS) is the most common cancer occurring in HIV-infected individuals worldwide, and often involves the mouth. While infection by Kaposi’s sarcoma-associated herpesvirus (KSHV) is necessary for the development of KS, other cofactors remain unclear. In this study, we evaluated the impact of oral bacterial microbiota on the development of oral KS and the presence of oral cell-associated KSHV DNA by studying a cohort of HIV/KSHV-coinfected individuals all previously diagnosed with KS, classified as having oral KS with any oral cell-associated KSHV DNA status (O-KS), no oral KS but with oral cell-associated KSHV DNA (O-KSHV), or with neither oral KS nor oral cell-associated KSHV DNA (No KSHV). We observed impoverishment of oral microbial diversity and enrichment of specific types of microbes in O-KS individuals compared to O-KSHV or No KSHV individuals. Hence, HIV/KSHV coinfection and oral microbiota might impact one another and influence the development of oral KS. Infection by Kaposi’s sarcoma-associated virus (KSHV), also called human herpesvirus-8 (HHV-8), is associated with several human malignancies or hyperinflammatory conditions including Kaposi’s sarcoma (KS), primary effusion lymphoma (PEL), multicentric Castleman’s disease (MCD) and KSHV inflammatory cytokine syndrome (KICS), 2. KSHVKS is the most common KSHV-associated malignancy worldwide. It is characterized by the proliferation of vascular spindle tumors cells, and extensive inflammatory infiltration and angiogenesis. AIDS-asHelicobacter pylori and Fusobacterium nucleatum, respectively[Propionibacterium acnes and the development of prostate cancer[Escherichia coli (E. coli) strains are associated with tumorigenesis in mouse models of colorectal cancers[E. coli C25 strain could promote KSHV-induced tumorigenesis in a KS-like mouse model[The development of next generation sequencing (NGS) has enabled the identification and quantification of the microbiome including bacteria, viruses and fungi in healthy individuals, or in a context of disease. Indeed,pectively, 17. Othte cancer. Some enl cancers. We haveuse model.E. coli infection or bacterial ligands such as LPS can promote KSHV-induced tumorigenesis in mice through the activation of TLR4 and pro-inflammatory pathways[Numerous pathogenic mechanisms, including the production of genotoxic inducers, the activation of TLRs and pro-inflammatory pathways, the inhibition of the immune response, and the increase of the cellular turnover have been proposed to explain the oncogenic properties of some types of bacteria. Bacteri pathways.In this study, we examined the oral bacterial microbiota in a cohort consisting of HIV/KSHV-coinfected individuals all previously diagnosed with KS, with or without oral KS whose status of oral cell-associated KSHV DNA was assessed. We showed a diminution of the oral microbial diversity and enrichment of specific bacteria in HIV/KSHV-coinfected individuals with oral KS, which was independent of the presence of oral cell-associated KSHV DNA. Hence, the composition of the oral microbiota may contribute to the development of oral KS.Table 1). While all individuals had KS when admitted into the cohort, 5 of these subjects no longer had detectable KS at the time of sampling but had either KSHV-MCD or PEL (Table 1). Intermittent oral cell-associated KSHV DNA was defined based on the detection of KSHV DNA in oral cells in longitudinal follow-ups in at least 3 visits. Oral microbiota, oral cell-associated KSHV DNA, KSHV blood DNA detected in peripheral blood mononuclear cells (PBMC), and HIV load were examined at the time of sampling while CD4+ T cell count and CD8+ T cell count were obtained from medical records. Of note, while all individuals were infected with HIV when they were admitted into the cohort, only nine had detectable (>50 copies/mL) HIV RNA at the time of sampling, two of whom had >400 copies/ml. Two individuals in the O-KSHV group were negative for oral cell-associated KSHV DNA at the time of sampling; however, they had a significant history of detectable oral cell-associated KSHV DNA with one individual positive for 17 of 20 time points (85%) tested over a 43 months period and the second individual positive for 8 of 15 time points (53%) tested over a 55 months period. All individuals were men and had a median age of 45 years.We recruited 29 HIV/KSHV-coinfected individuals with a history of pathology-confirmed KS by physician physical examination, including 9 individuals who had oral KS involvement with any oral cell-associated KSHV DNA status (O-KS), 10 individuals who had detectable intermittent oral cell-associated KSHV DNA without oral KS (O-KSHV), and 10 individuals who had neither oral cell-associated KSHV DNA nor oral KS (No KSHV) . Likewise, no significant difference in HIV load  was noted between groups. There were 5 of 9 and 8 of 10 individuals who had detectable oral cell-associated KSHV DNA in the O-KS and O-KSHV groups, respectively, but no significant difference was observed between the O-KSHV and O-KS groups at the time of collection , and 8 of 9 individuals in the O-KS group had advanced immunodeficiency with a CD4+ T cell count <200 cells/μL. However, no difference of CD8+ T cell count was observed between the O-KS and either of the other two groups . There was a significant positive correlation between oral cell-associated KSHV DNA and CD4+ T cell count  as previously described[Fig 1G). There was a weak positive correlation between HIV load and PBMC KSHV DNA  as previously described[Fig 1I).The CD4+ T cells was significantly lower in the O-KS group than in the other two groups . Using Venn diagram, we observed that all 3 groups shared 1,242 OTUs, whereas 62, 129 and 97 OTUs were specific to the O-KS, O-KSHV and No KSHV groups, respectively .We sequenced the hypervariable V1-V2 region of 16S rRNA gene from oral specimens of the 29 individuals using an Illumina MiSeq system. A total of 15,261,199 raw sequences were generated. After quality control and filtering, 13,098,094 high-quality sequences with an average length of 263 bp were recovered for further analysis, with an average of 451,658 reads per sample . After alignment with QIIME database, unique representative sequences were classified into 1,886 operational taxonomic units (OTUs), from which 16 phyla, 28 classes, 41 orders, 79 families, 125 genera and 148 species were identified. Shannon diversity index was used to evaluate the sequencing depth. All the rarefaction curves reached plateau indicating that there was sufficient sequencing coverage depth  and the α-diversity of the observed species (using the Shannon index measuring how evenly OTUs are distributed in a sample) among all groups . We observed a strong diminution of the α-diversity and richness in individuals in the O-KS group compared to the other two groups. Using Wilcoxon signed-rank test, we tested the statistical difference between the richness and α-diversity of each pair of groups . We observed a significant dissimilarity between the O-KS and O-KSHV groups , and between the O-KS and No KSHV groups , whereas the O-KSHV and No KSHV groups were similar . Hence, individuals of the O-KS group tend to cluster together and were more distant from individuals of the O-KSHV or No KSHV groups, suggesting an association between the oral microbiota and the presence of oral KS in HIV/KSHV-positive individuals.We calculated the species richness (using the Ace and Chao1 nonparametric methods for estimating the number of species in a community) , which were consistent with previous reports[Streptococcus, Prevotella, Lactobacillus, Dispar and Selenomonas were the top 5 most abundant species for all 3 groups . To investigate the taxonomic composition of the bacterial populations in all 29 subjects, heatmaps were constructed by clustering each individual OTU compositions , as well as each mean of relative OTU abundances for the 3 studied groups . The heatmaps constructed at phylum and species levels demonstrated that the O-KSHV and No KSHV groups clustered together, whereas the O-KS group was phylogenetically more distant from other groups , indicating that distinct oral microbiome might influence the development of oral KS in HIV/KSHV-coinfected individuals or that the oral microbiome is perturbed by the development of oral KS.The relative abundances of OTUs, as well as the taxonomic compositions of bacterial populations in individuals of the 3 studied groups were analyzed at different taxonomic levels. The top 5 most abundant identifiable phyla in the oral microbiota were s reports. At the Pasteurellales and Burkholderiales at order level in the O-KS group compared to the O-KSHV and No KSHV groups . At family level, Bacillaceae was enriched in the O-KS group, whereas the abundance of Burkholderiaceae decreased . At genus level, the abundances of Aggregibacter and Lautropia were decreased in the O-KS group, whereas those of Corynebacterium and Shuttleworthia were increased . At the species level, the abundances of Dialister and Satelles were increased whereas those of Lautropia and Porphyromonas were decreased in the O-KS group . No significant differences were observed among all 3 groups at phylum and class levels. The increase in OTUs in the O-KS group clustered in the Firmicutes and Actinobacteria phylum, whereas the decrease in OTUs in the O-KS group centered in the Bacteroides and Proteobacteria phylum .We further examined the differences of bacterial distributions across all 3 studied groups at all phylogenetic levels. We observed a significant diminution of Bacillales order increased in both O-KSHV and O-KS groups , whereas those of Gemellaceae family and the Gemella species decreased .When individual bacterial orders, families, or species groups were examined, there were some significant differences between individuals with oral cell-associated KSHV DNA (O-KSHV) as compared to those without oral cell-associated KSHV DNA (No KSHV). Some of these differences were independent of oral KS status as they were also observed between the O-KS group and the No KSHV group, most likely due to the fact that 5 of 9 individuals in the O-KS group had detectable oral cell-associated KSHV DNA. For examples, the abundance of Altogether, these results indicated that alterations of the oral microbiota might influence the detection of oral cell-associated KSHV DNA and development of oral KS in HIV/KSHV-coinfected individuals, or that the presence of oral cell-associated KSHV DNA and development of oral KS may influence the composition of the oral microbiome.E. coli strain K12 or C25 promoted KSHV-induced cellular transformation and tumorigenesis in a KS-like animal model[Altered oral microbiota has been observed in several diseases including diabetes, bacteremia, endocarditis, cancer and autoimmune disease, and in some cases can influence disease progression or tumormal model. Therefomal model, 30, andFig 1D). The impoverishment in oral microbiota may have implications for the immune reconstitution during cART in HIV-infected patients developing oral KS. In melanoma patients undergoing anti-PD-1 immunotherapy, significant differences were observed in the diversity and composition of the gut microbiome of responders versus non-responders. Higher gut microbiome diversity was associated with improved response to anti-PD-1 immunotherapy in patients with metastatic melanoma[We observed a strong diminution of microbial α-diversity and richness in individuals developing oral KS compared to the other two groups without oral KS. Imbalance in microbial flora composition is correlated with impaired immune cell activity and the decrease of oral microbial diversity could affect immune responses. We inde melanoma. NeverthFirmicutes, Proteobacteria, Bacteroidetes, Actinobacteria, and Fusobacteria, with a predominance in the genus Streptococcus followed by Prevotella, Veillonella, Neisseria, and Haemophilus[Fig 3A). At species level, because of the high variability within each individual, no strict consensus has been identified so far regarding the bacterial relative abundance in the oral cavity in healthy individuals[Streptococcus, Prevotella, Lactobacillus, Dispar sp. and Selenomonas as the top 5 species in all 3 groups of HIV/KSHV-coinfected individuals . Although there was no difference in the distribution of the top 5 phyla or species across all 3 groups , the cluster analysis showed that the phylum and species in the O-KS group were distinct from the O-KSHV and No KSHV groups , highlighting the modification of the microbial pattern in individuals developing cancer as previously reported[In healthy individuals, the oral microbiota is usually composed of the phyla emophilus. As obseemophilus, the topdividuals, 30. How reported.Capnocytophaga gingivalis and Prevotella melaninogenica belonging to the Bacteroidetes phylum, and Streptococcus mitis belonging to the Firmicutes phylum, as well as decreases of Citrobacter and Neisseraceae belonging to the Proteobacteria phylum compared to the cancer-free control groups[Burkholderiales and Pasteurellales order belonging to the Proteobacteria phylum, and a decrease in species of Porphyromonas belonging to the Bacteroidetes phylum in individuals who developed oral KS compared to control groups without oral KS. In parallel, there were increases in OTUs belonging to the Firmicutes phylum such as Satelles species, Dialister order and Bacillaceae family in the O-KS group . Hence, the bacterial signature of oral KS is different from that of OSCC despite both types of cancer having an increase in OTUs of Firmicutes phylum and a decrease in OTUs of Proteobacteria phylum. However, the diminution of OTUs belonging to the Bacteroidetes phylum seems to be specific to individuals who developed oral KS and has not been described in other types of oral cancer. Moreover, as observed in the O-KS group, an increase of Firmicutes and decreases of Bacteroidetes and Proteobacteria phylotypes have previously been reported in other types of malignancies such as colorectal cancer (CRC)[Oral squamous cell cancer (OSCC) is the oral cancer with the highest incidence, and ranked the 15th place in frequency among all types of cancer in 2012. OSCC haol groups–35. In ocer (CRC).Porphyromonas sp. and Corynebacterium order in HIV-infected individuals compared to HIV-negative individuals[Various factors such as diet, oral hygiene, tobacco and alcohol consumption, stress, hormonal imbalance, diabetes, and gingival inflammation can perturb the oral bacterial community. Studiesdividuals, 42.Fig 5). These results suggested that these differences were independent of KS status despite 5 of 9 individuals in the O-KS group had detectable oral cell-associated KSHV DNA  and those with oral KS (O-KS) or detectable oral cell-associated KSHV DNA (O-KSHV) , which allowed the evaluation of oral cell-associated KSHV DNA. Individuals were selected from cohorts of AIDS-KS patients who were participating in clinical studies. Participants with at least 3 oral cell-associated KSHV DNA measurements were included. KS diagnosis was initially made by the attending physicians and confirmed by pathological examination by NCI pathologists with a biopsy by immunohistochemistry and sometimes by PCR for the presence of KSHV DNA. The KS stage of all patients was determined using the AIDS Clinical Trials Group Network (ACTG) criteria. PresencoC prior to extraction. PBMC materials were matched with oral cells (same draw date) in every incidence except one intermittent shedder who had PBMC draw next day and for two never shedders who had PBMC materials within a month of the collection of oral cells. PBMC were isolated from blood collected in acid citrate dextrose (ACD) tubes by Ficoll  centrifugation with Leucosep tubes (Greiner bio-one). Red blood cells were lysed with ammonium-chloride-potassium (ACK) buffer following manufacturer’s protocol (Thermo Scientific). The purified PBMC were counted using a Nexcellom Cellometer Vision instrument. Pellets of approximately 1–2 million PBMC were obtained for each individual and stored at -80°C prior to DNA extraction.Approximately 5 ml of Scope mouthwash was used to collect oral fluids and cellular materials. Samples were centrifuged at 8,000 g for 5 min to pellet cellular materials. The supernatant was transferred to another tube without disturbing the pellet. Pellets were stored at -80 oC.Genomic DNA was extracted from oral cells and PBMC pellets using QIAamp Blood Mini Kits according to the manufacturer’s instructions (Qiagen). The quality and quantity of the DNA extracted were measured using a UV spectrophotometer at 260 and 280 nm (Nanodrop 2000). All DNA samples were stored at -80 6 cells. The assays are Clinical Laboratory Improvement Amendments (CLIA) certified and conducted using stringent procedures to prevent contamination.KSHV DNA in oral cell pellet and PBMC collected at the time of oral sampling were measured as previously described. BrieflyKSHV serological status was determined using recombinant protein ELISA for ORF73 and K8.1. Participants were considered KSHV-seropositive if they had antibodies to either antigen.oC, 25 cycles of 30 sec denaturation at 95 oC, 30 sec elongation at 72 oC, and a 5 min one final extension at 72 oC. Illumina sequencing adapters and indexes were attached using Nextera XT Index kit . The amplicon library was built and sequenced with the Illumina MiSeq V3 600 Cycle Kit according to the manufacturer’s instructions (Illumina) using the universal 16S primers 27F (5'-AGAGTTTGATCMTGGCTCAG-3’) and 355R (5'-GCTGCCTCCCGTAGGAGT-3’)[The first PCR amplification of the bacterial 16S rRNA gene hypervariable V1-V2 region was performed with DNA isolated from oral cell pellet using the forward primer 5’-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-3’ and the reverse primer 5’-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-3’, both of which contain overhang adapters. The PCR reaction was carried out by 2 min initial denaturation at 95 GGAGT-3’).Raw pyrosequencing results from the hypervariable region V1-V2 of the 16S rRNA gene were filtered and unique reads were assigned to the corresponding samples after mapping with barcode and primer sequences. FastQC was used to evaluate the sample reads for sequencing quality. SequencFor all statistic tests, P-value ≤0.05 (*), ≤0.005 (**) or ≤0.0005 (***) were considered significant.All patients were enrolled on National Institutes of Health Clinical Center Protocol 01-C-0038 , which was approved by the National Cancer Institute Institutional Review Board, and the samples were obtained and studied as part of this protocol. All individuals gave written informed consent to clinical examination, sample acquisition, and testing on clinical samples. However, all the data were analyzed anonymously.S1 Fig(A) Relative OTU abundances at phylum level clustered by each individual subject. (B) Relative OTU abundances of the 30 most abundant species clustered by each individual subject.(TIF)Click here for additional data file.


Graphite is used in conjunction with SiOx to overcome the limited energy density of graphite, and to lessen the adverse effects of volume expansion of Si. However, electrodes based on graphite/SiOx composites can be made with only 3–5 wt % SiOx because of the increased failure of electrodes with higher SiOx contents. Here, we developed a new polymer binder, by combining dopamine-grafted heparin with the commercial binder carboxymethyl cellulose (CMC)/styrene butadiene rubber (SBR), in order to more effectively hold the SiOx particles together and prevent disintegration of the electrode during charging and discharging. The crosslinking using acid-base interactions between heparin and CMC and the ion-conducting sulfonate group in heparin, together with the strong adhesion properties of dopamine, yielded better physical properties for the dopamine-heparin-containing CMC/SBR-based electrodes than for the commercial CMC/SBR-based electrodes, and hence yielded excellent cell performance with a retention of 73.5% of the original capacity, a Coulombic efficiency of 99.7% at 150 cycles, and a high capacity of 200 mAh g−1 even at 20 C. Furthermore, a full cell test using the proposed electrode material showed stable cell performance with 89% retention at the 150th cycle.Graphite is used commercially as the active material in lithium ion batteries, frequently as part of a graphite/SiO However, currently available lithium-ion secondary batteries use graphite as the anode material, and as a result do not have sufficient energy density to efficiently power such high-energy applications.Lithium-ion secondary batteries are being intensively studied due to their potential uses as large-scale energy storage devices in applications such as electric vehicles as well as energy storage systems9. For example, the silicon electrode has a high theoretical capacity, more than ten times greater than that of the currently used anode material for lithium-ion batteries , which has a theoretical capacity of only about 372 mAh g−1. However, silicon has inherent problems due to volume expansion and shrinkage of about 400% during the charge-discharge process of Si + 4.4Li ⇆ Li4.4Si6. When such changes in volume are repeated, mechanical stresses build up that damage the anode, induce its components  to separate from one another, and cause loss of electrical pathways by isolating Si particles having insulation properties, leading to rapid deterioration of the battery system12. Thus, many researchers have examined modified forms of silicon, for example, nano-sized particles, in order to reduce the stress caused by the volume expansion of Si14. The preparation of these nano-architectured silicon anode materials, however, inevitably increases the production costs.Much effort is hence being devoted to increasing the energy density levels of lithium-ion batteries. To achieve this, materials based on Li alloys, such as lithium-tin and lithium-silicon, have attracted much recent attention as alternate anode materials due to their high capacitiesx), on the other hand, are relatively inexpensive to produce and have been commonly used as composite materials with Si to lessen the adverse effects of the volume expansion of Si. In these composites, either crystalline or amorphous Si cores are uniformly dispersed in a SiO2 matrix, and the stable lithium-silicates formed after lithiation of this material have been shown to reduce the magnitude of the changes in the volume of Si during cycling17. Indeed, in currently used and commercialized active materials based on graphite, replacing some of the graphite with SiOx has been found to be the most practical way of overcoming the limited energy density of the graphite. The SiOx content of the active material, however, has been limited to at most 3–5 wt.% due to the rapid increase in electrode failure with increasing SiOx content as a result of the above-mentioned side reactions of Si.Silicon oxides /styrene butadiene rubber (SBR), the binder currently used in commercial graphite anodes. In addition, cross-linkages via covalent bonds26 or even noncovalent ones such as hydrogen bonds31 have been exploited to further improve the physical properties of these polymer binders, yielding markedly improved cell performance.Aside from the problems directly related to the silicon itself, developing a strong polymer binder that can endure large volume changes of Si and prevent electrodes from rupturing has also been widely pursued recently. Polymers such as poly(acrylic acid) (PAA) that consist of many carboxylic acid functional groupsx/graphite composite-based anodes. Heparin is a highly sulfonated, biocompatible, water-soluble, and naturally derived polysaccharide. It has various functional groups, which have contributed to its effectiveness and hence wide use as an anticoagulant and inhibitor of both angiogenesis and tumor growth32. Although heparin is difficult to apply directly as a binder to Si anodes due to its poor physical properties when used alone33, the many functional groups of heparin allow it to interact with CMC. These functional groups include amines, which can reversibly interact with the acid groups in CMC through hydrogen bonding, and sulfonates, which can further contribute to Li+ conduction. In addition, dopamine, inspired by its role as an adhesive in mussel foot protein, was further incorporated onto heparin due to dopamine’s well-known excellent adhesion to various surfaces including Si anodes34. Therefore, we produced dopamine-functionalized heparin and added it to the CMC/SBR binder in order to (1) mechanically strengthen this binder by crosslinking, (2) improve the adhesion property of the corresponding binder to Si and hence suppress the volume expansion of Si, and (3) endow the crosslinked binder system with the ability to conduct ions.We report herein a new polymer binder based on dopamine-functionalized heparin/CMC/SBR, in which dopamine-grafted heparin was used as an additive in the commercially available CMC/SBR binder, and show that it improves the performance of SiOThe addition of dopamine to heparin was accomplished via the N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) coupling reaction, as shown in Fig. 1) was carried out by performing UV-Vis and FT-IR spectroscopy investigations. The UV-Vis spectrum of 1 . A dramatic increase in the binding affinity was further obtained for the electrode with the CMC/SBR binder containing dopamine-heparin (dopamine-heparin/CMC/SBR), with a measured value of 1.287 N. These results suggested that the addition of heparin to CMC/SBR mechanically strengthened the electrode due to the formation of physical interactions between CMC and heparin, and that the durability of the electrode in the presence of mechanical stress was further increased by the strong adhesion resulting from the inclusion of dopamine in the dopamine-grafted heparin 34.The binding affinities for the three electrodes  were then investigated by carrying out 180-degree peel tests Fig. . In the 28. The tapes pulled off the pristine CMC/SBR-based electrode were stained with a lot of slurry and transmitted little light, indicating that much of the Si material in these cases became detached from the electrode and transferred to the tape. In contrast, the tapes pulled off the heparin/CMC/SBR-based electrode were stained with only a very small amount of slurry, and were observed to transmit a considerable amount of light, indicative of the better ability of heparin/CMC/SBR than of pristine CMC/SBR to prevent material from being detached from the electrode and consistent with the stronger adhesion force of the heparin/CMC/SBR binder based on the peel test. And this trend continued with the inclusion of dopamine: tapes pulled off the dopamine-heparin/CMC/SBR-based electrode, showed the least amount of slurry and transmitted the most light, consistent with the dopamine-heparin/CMC/SBR binder having displayed an adhesion force greater than those of the other binders.We further used an optical microscope to examine the surface of the tape detached from the electrode after the peel test to determine whether the addition of dopamine-heparin (to form a dopamine-heparin/CMC/SBR binder) reduced the amount of Si exfoliated from the electrode. As shown in Fig. 3a−1), and compared the results with the cell performance of the pristine CMC/SBR-based electrodes  did not cause any particular side reactions. In addition, the voltage profiles of both the heparin- and dopamine-heparin-based electrodes showed lower resistance during lithiation than did the electrode prepared from CMC/SBR. The enhanced physical properties of the heparin and dopamine-heparin binder systems have been suggested to result in less polarization due to the reduction of the contact resistance of the electrode36. Having the highest mechanical strength, the dopamine-heparin-based electrode indeed showed the lowest resistance.The voltage profiles of the prepared cells during the 1der Fig. , suggest−1 and a retention of 62.3% of the original specific capacity. After about 100 cycles, this electrode showed particularly poor cycling stability. This poor stability was attributed to the CMC/SBR binder, with its poor adhesion properties, no longer being able to accommodate the repeated changes in the volume of the silicon electrode. The cell performances of the electrode made of the heparin/CMC/SBR binder and that made with the dopamine-heparin/CMC/SBR binder, however, were much better than the cell performance of the electrode made with the pristine CMC/SBR binder. The superior performance of the former two cells was attributed to their enhanced physical properties. A specific capacity of 380 mAh g−1, representing a retention of 81.3% of the original capacity, was observed at 100 cycles, and a specific capacity of 268 mAh g−1 and retention of 57.3%, were observed at 150 cycles for the electrode including heparin/CMC/SBR. The dopamine-heparin/CMC/SBR-based binder system showed the best cell performance. Although the specific capacity and retention values of the dopamine-heparin/CMC/SBR-based binder system at 100 cycles, with values of 378 mAh g−1 and 81.3%, respectively, were similar to those of the heparin/CMC/SBR-based system, a much greater reversible capacity of 343 mAh g−1 and retention of 73.5% were obtained at 150 cycles for the dopamine-heparin/CMC/SBR-based system than for the heparin/CMC/SBR-based system. Since the three cells were subjected to the same conditions, the superior specific capacity and percent retention of original specific capacity up to the 150 cycles for the cell having the dopamine-heparin/CMC/SBR binder were therefore attributed mainly to the superior adhesion properties of this binder material.In addition, the specific discharge capacities of the cells prepared using the three binders were also determined in order to assess the effect of the binder on cyclability Fig. . The speth cycle, and very high value of 99.7% at the 150th cycle. These values were higher than those of the CMC/SBR- and heparin/CMC/SBR-based electrodes.The dopamine-heparin-CMC/SBR electrode also displayed excellent Coulombic efficiency Fig. . It disp−1 even at 20 C. We attributed the enhanced rate properties of the heparin and dopamine-heparin electrodes to the improved physical properties of these binders, which helped to prevent the conducting agents and active materials from detaching from the electrodes even at high C rates. In addition, the functional groups in the heparin structure were thought to further provide a lithium transfer pathway capable of conducting Li+ ions41.We also performed rate capability tests for the three types of electrode Fig. . Their 0Electrochemical impedance spectra (EIS) were also acquired for the electrodes with and without the dopamine-heparin, and the overall resistance on the electrode surface was found to be smaller when the dopamine-heparin was included Fig. . Since t0.6Co0.2Mn0.2O2 positive electrode and the composite negative electrode containing the dopamine-heparin/CMC/SBR binder, was tested to evaluate the binder in a practical battery application , as shown in Fig. x and the polymer binder, and of silicon atoms, which originated from SiOx) showed that the materials comprising the electrodes were well dispersed for both electrodes irrespective of the addition of the dopamine-heparin to the CMC/SBR  analyses of the prepared electrodes  produce physical crosslinking using acid-base interactions between heparin and CMC, (2) increase the ion conductivity using the sulfonate groups of heparin, and (3) strengthen the adhesion of the binder to the electrode due to the inclusion of dopamine. While the addition of heparin to the CMC/SBR binder  improved the physical properties of the electrodes and hence the overall performance of the corresponding cells, even better physical properties were achieved when dopamine-heparin was added (to produce dopamine-heparin/CMC/SBR electrodes). Also, while previously described electrodes could be made with only 3–5 wt.% SiOx because of the increased failure of these electrodes with increasing SiOx content due to side reactions of Si, we were able to achieve excellent cell performance for the dopamine-heparin/CMC/SBR-based electrode with SiOx/graphite composite active material containing up to 7 wt.% SiOx. Specifically, the resulting dopamine-heparin/CMC/SBR-based electrode displayed a specific capacity of 343 mAh g−1 and retention of 73.5% of the original capacity, and Coulombic efficiency of 99.7% after 150 cycles, together with a high capacity of 200 mAh g−1 even at 20 C. Furthermore, a stable cell performance with 89% retention at the 150th cycle was achieved in a full cell test using this electrode. Overall, the inclusion of dopamine-grafted heparin not only enhanced the physical properties of the corresponding dopamine-heparin/CMC/SBR-based electrode but also its ionic conductivity, and hence demonstrated its potential for use in practical Si-based lithium-ion batteries.We developed a new polymer binder system based on dopamine-grafted heparin (dopamine-heparin) as an additive to the CMC/SBR binder for SiO6 in ethylene carbonate/ethylmethyl carbonate (EC:EMC = 3:7 v/v) with 10 wt.% fluoroethylene carbonate (FEC)) were purchased from PanaxEtec. Silicon monoxide (SiOx) was obtained from Osaka Titanium Technologies. Carboxymethyl cellulose (CMC) was purchased from Nippon Paper Chemicals. Styrene-butadiene rubber (SBR) was purchased from ZEON.Heparin sodium salt, N-hydroxysuccinimide (NHS), and dopamine hydrochloride were purchased from Sigma-Aldrich. N-(3-Dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (EDC), sodium dihydrogen phosphate and sodium hydrogen phosphate were purchased from Alfa-Aesar. Dimethyl carbonate and electrolyte (1 M LiPFHeparin (1.0 g) was added to an aqueous phosphate buffer solution  in a round-bottom flask, and this mixture was stirred until a homogeneous solution was obtained. Thereafter, EDC  and NHS , dissolved in a PBS solution (pH = 6.0), were subsequently added to the heparin solution. Finally, to this mixture was added an aqueous solution of dopamine  in PBS (pH = 6.0). The resulting mixture was stirred for 9 h at r.t. After completion of the reaction, the dopamine-grafted heparin was purified from the reaction mixture by subjecting this mixture to dialysis twice using a cellulose membrane  in distilled water for 6 h, and the product was collected by freeze-drying.1 was calculated by performing quantitative analysis using UV-Vis spectroscopy :CMC:SBR. To make a SiO−1.FT-IR spectra were recorded on a PerkinElmer Spectrum Two ATR spectrometer. UV-vis spectra were recorded on a PerkinElmer Lambda 365 spectrometer. Scanning Electron Microscopy (SEM) was performed using a JEOL JSM-7000F instrument. To see the surface morphology, the cycled Si electrodes were washed with dimethyl carbonate before being imaged. For evaluating the mechanical properties of the electrodes with various polymer binders, the prepared electrodes were each cut into rectangular shapes (1.2 cm × 3.0 cm) and attached to 12-mm-wide 3 M tape. The peel strength of each tested electrode specimen was then recorded with a universal testing machine  by pulling the tape at a constant displacement rate of 30 mm min6 in ethylene carbonate/ethylmethyl carbonate (EC:EMC = 3:7 v/v) with 10 wt.% fluoroethylene carbonate (FEC) (PanaxEtec) as an electrolyte. Galvanostatic discharge−charge cycling was then performed using a CPS-Lab battery cycler (Basytec) at 25 °C in a temperature-controlled chamber. All of the cells were repeatedly discharged to 0.005 V vs Li/Li+ and charged to 2.0 V vs Li/Li+ at a constant C-rate of C/20 lithiation (discharge)-C/10 delithiation (charge) in the first cycle and C/10 lithiation-C/10 delithiation in the next two cycles for pre-cycling, and then at C/5 lithiation- C/2 in the following cycles. Rate capability tests were carried out by repeating discharging to 0.005 V vs Li/Li+ and charging to 2.0 V vs Li/Li+. The pre-cycling was performed before the cyclability test, and the current for lithiation was fixed at C/20, and the currents for delithiation were varied from 1/2 to 20 C . For postmortem analyses, the cycled cells were disassembled in an Ar-filled glove box and recovered electrodes were washed with dimethyl carbonate to eliminate residual electrolyte. Then, the surface morphologies of cycled electrodes were investigated by performing scanning electron microscopy . The EIS was measured at a frequency range of 10 mHz–100 kHz with an AC amplitude of 10 mV at 0.01 V.To evaluate the electrochemical performances of the prepared electrodes, 2032-type coin cells were assembled in an Ar-filled glove box, using multi-layered porous PE/PP/PE as the separator, a lithium disc as the counter electrode, and 1 M LiPFx/Graphite composite electrode was used for the negative electrode and fabricated 94:3:3 ratio of active materials:super-P:binder. The composite cathode electrode being fabricated with a 90:5:5 ratio of LiNi0.6Co0.2Mn0.2O2:super-P:PVdF were used. The full cells were repeatedly charged to 4.1 V and discharged to 3.0 V at a constant C-rate of C/10 for both the 1st charge and discharge as a pre-cycling step and at C/5 of lithiation and C/2 of delithiation in the subsequent cycles. And 1 C was determined to be 170 mA per gram of the positive electrode material.The full-cell was assembled using an N/P ratio of 1.1. The same SiOSupplementary Information


Vibrio cholerae, closely related with Vibrio vulnificus, has been reported in plants and vegetables. Here, we investigated the possibility of Vibrio vulnificus 96-11-17M, an opportunistic human pathogen, to infect and colonize Arabidopsis thaliana plants, resulting in typical disease symptoms at 5 and 7 days post-inoculation in vitro and in planta under artificial and favorable conditions, respectively. RNA-Seq analysis revealed 5,360, 4,204, 4,916 and 3,741 differentially expressed genes (DEGs) at 12, 24, 48 and 72 h post-inoculation, respectively, compared with the 0 h time point. Gene Ontology analysis revealed that these DEGs act in pathways responsive to chemical and hormone stimuli and plant defense. The expression of genes involved in salicylic acid (SA)-, jasmonic acid (JA)- and ethylene (ET)-dependent pathways was altered following V. vulnificus inoculation. Genetic analyses of Arabidopsis mutant lines verified that common pathogen-associated molecular pattern (PAMP) receptors perceive the V. vulnificus infection, thus activating JA and ET signaling pathways. Our data indicate that the human bacterial pathogen V. vulnificus 96-11-17M modulates defense-related genes and host defense machinery in Arabidopsis thaliana under favorable conditions.Because of the recent increase in the demand for fresh produce, contamination of raw food products has become an issue. Foodborne diseases are frequently caused by the infection of leguminous plants by human bacterial pathogens. Moreover, contamination by  Salmonella [Escherichia coli O157 contaminate fresh fruits and vegetables [Salmonella spp. and norovirus were reported in the US, and Salmonella has been ranked as the major bacterial pathogen responsible for the hospitalization and death of humans [Over the past few decades, the availability of fresh foods for optimum human health has received considerable attention. Although suppliers have put tremendous effort into reducing the contamination of fresh foods, the number of cases related to contaminated food products has been increasing ,2. Contalmonella  and Eschgetables . Betweenf humans .Plants may be contaminated at any stage of growth or during post-harvest processing. Human pathogen population increases both before the harvest of plants ,8 as welV. vulnificus is a Gram-negative halophilic bacterium and a lactose-positive opportunistic human pathogen [V. vulnificus grows in aquatic environments worldwide, especially in warm waters in tropical and subtropical regions [V. vulnificus has been classified into three distinct biotypes, based on its lipopolysaccharide (LPS) antigens, expression of capsule and host range [V. vulnificus occurs via two main routes: 1) septicaemia resulting from the consumption of V. vulnificus-contaminated raw seafood; and 2) wound infection [V. vulnificus infection has been reported to cause gastroenteritis [To date, pathogen . V. vuln regions ,13. V. vst range ,15. Biotst range . Biotypest range . Biotypenfection . Recentlnteritis .V. cholerae shares many similarities with V. vulnificus [V. cholerae has been reported in diverse plants and vegetables for a long time [V. vulnificus has not yet been reported to cause any plant disease. This notion prompts us to test the possibility of V. vulnificus causes disease in plants and how it modulates the plant defense responses under artificial and favorable conditions. To investigate further experiments, we first identified the strains of V. vulnificus that clearly contribute to the development of pathogenicity in Arabidopsis in vitro. Among the tested strains, V. vulnificus 96-11-17M was selected for further analyses. Next, we examined the disease severity and pathogen population density in Arabidopsis plants inoculated with this strain in planta. To obtain molecular evidence, we performed RNA-sequencing (RNA-Seq) analysis of Arabidopsis plants infiltrated with V. vulnificus 96-11-17M in planta. The results showed that V. vulnificus 96-11-17M infection altered the transcript levels of genes involved in salicylic acid (SA)-, jasmonic acid (JA)- and ethylene (ET)-dependent pathways at the tested time points relative to time zero (control). Furthermore, several differentially expressed genes (DEGs) identified in Arabidopsis plants inoculated with V. vulnificus 96-11-17M significantly overlapped with those identified in Arabidopsis plants inoculated with Pseudomonas syringae pv. maculicola ES4326, a common plant bacterial pathogen. Taken together, our results provide that V. vulnificus modulates defense-related genes and host defense machinery in Arabidopsis thaliana under favorable conditions used in this study.It is known that lnificus . Moreoveong time ,21,22,23Arabidopsis ecotype Col-0 were surface-sterilized with 6% sodium hypochlorite for 5 min and then washed five times with sterile distilled water. The sterilized seeds were plated on half-strength Murashige and Skoog (0.5× MS) medium supplemented with 3% sucrose and 0.6% plant agar. Seeds were germinated and incubated in a growth chamber at 24 ± 2°C under a 12 h light/12 h dark photoperiod and fluorescent light  for 3 days. The germinated seeds were transferred to fresh 0.5× MS plates and incubated for 2 weeks under the same conditions for further experiments.Seeds of fls2, efr1, fls2/efr1, NahG, sid2, npr1, jar1 and etr1) plants were planted in soil-less potting medium . Plants were grown at 24 ± 2°C under a 8 h light/16 h dark photoperiod and fluorescent light  for 3 weeks.Seeds of wild-type (Col-0) and mutant  of 2.0 and with Pseudomonas syringae pv. tomato DC3000 . Briefly, bacterial strains were picked from each glycerol stock at -70°C, streaked on LB agar medium containing 2% NaCl  and 5 μg/ml rifampicin  and incubated at 30°C for 2 days. P. syringae pv. tomato DC3000 was also steaked on LB agar medium containing 5 μg/ml rifampicin and incubated at 30°C for 2 days. After incubation, colonies were scripted by plastic bar using sterilized 10% PBS . The 10% PBS buffer contained NaCl (13.7 mM), KCl (0.27 mM), Na2HPO4 (1 mM) and KH2PO4 (0.18 mM) at pH 7.4. The inoculated plants were incubated in a growth chamber for 5 days and then scored for disease severity. Plants inoculated with P. syringae pv. tomato DC3000 re-suspended in 10% PBS were scored on a scale from 0 to 5, where 0 indicated no symptoms, 1 indicated the development of yellowish color, 2 indicated chlorosis only, 3 indicated necrosis and chlorosis, 4 indicated partial necrosis of the inoculated area and 5 indicated complete necrosis of the inoculated area [To perform ted area . The 10%in vitro assays, leaves at the same stage and age were collected at 5 days after drop-inoculation of V. vulnificus 96-11-17M and P. syringae pv. tomato DC3000, homogenized, spread on LB agar medium containing 2% NaCl and 5 μg/ml rifampicin and LB agar medium containing 5 μg/ml rifampicin, respectively, and finally incubated at 30°C for 2 days. P. syringae pv. tomato DC3000 was also steaked on and incubated at 30°C for 2 days. The number of V. vulnificus 96-11-17M and P. syringae pv. tomato DC3000 colonies was counted at 2 days after incubation.For population quantification V. vulnificus 96-11-17M was prepared as described in above part. To determine the population density of V. vulnificus 96-11-17M in planta, leaves of 3-week-old wild-type (Col-0) seedlings only  and collected at 3, 4 or 7 days post-infiltration. The collected leaves were homogenized and resuspended in 10% PBS. Subsequently, the resuspended samples were spread on LB agar medium containing 2% NaCl and 5 μg/ml rifampicin and incubated at 30°C. The number of V. vulnificus 96-11-17M colonies was counted at 2 days after incubation. Disease symptom was observed by V. vulnificus 96-11-17M infiltration in planta and photograph was taken at 7 days after infiltration.The suspensions of ngs only  or Col-0ls2/efr1  or Col-0eedlings , were inV. vulnificus 96-11-17M (OD600 = 1 in 10% PBS) were harvested at 0, 12, 24, 48 and 72 h post-infiltration and immediately frozen in liquid nitrogen. Subsequently, the leaf tissues were ground to a fine powder using a mortar and pestle. Total RNA was isolated from 100 mg ground tissue using RNeasy® Plus Mini kit, according to the manufacturer’s protocol . First-strand cDNA was synthesized using 2 μg total RNA, oligo-dT primer, dNTPs and Moloney murine leukaemia virus reverse transcriptase . To validate the RNA-Seq data, qRT-PCR was performed using a Chromo4 Real-time PCR System . Each reaction mixture contained 2× Brilliant SYBR Green Supermix , cDNA template and 0.5 μM gene-specific primers. Sequences were amplified using the following conditions: initial denaturation at 95°C for 10 min, followed by 44 cycles of denaturation at 95°C for 30 sec, annealing at 60°C for 30 sec and extension at 72°C for 42 sec. Gene expression levels were normalized relative to the expression of AtACT2 (GenBank accession no. Q96292). Primer sets used in this study are listed in Leaves of 3-week-old wild-type plants infiltrated with Arabidopsis seedlings were infiltrated with V. vulnificus 96-11-17M (OD600 = 1 in 10% PBS) and collected at 0, 12, 24, 48 and 72 h post-infiltration; two independent replicates were performed in planta for this experiment. Intact total RNA was extracted from the infiltrated leaves, as described above. The quality and integrity of RNA were confirmed by agarose gel electrophoresis, and unwanted contamination with proteins and solvents was assessed using a Nanodrop spectrophotometer . An RNA-Seq library was constructed using TruSeq RNA Sample Preparation kit v2 , according to the manufacturer’s guidelines. Briefly, mRNA was purified from total RNA using magnetic beads attached to specific poly oligo-dT primers. The purified mRNA was then fragmented and used as template for cDNA synthesis. The cDNA was ligated to adapters, and the fragments were amplified by PCR. The RNA-Seq library was sequenced to generate paired-end (2 × 100 bp) reads using the Illumina Hiseq-2000 system  at the Human Derived Material Center of KRIBB, Daejeon, South Korea.Leaves of 3-week-old wild-type A. thaliana was first obtained from The Arabidopsis Information Resource . The bowtie2-build component of Bowtie2 (ver. 2.0) and SAMtools (ver. 0.1.18) were used for reference genome indexing. Reads from V. vulnificus-infiltrated samples at different time points were mapped onto the reference genome (ver. 2.0) using the Tophat2 reference genome mapper. The RNA-Seq data generated in this study were deposited at the NCBI Gene Expression Omnibus (GEO) public database under the accession number GSE61418 and data were publicly available.To analyze the RNA-Seq data, the reference genome sequence of To analyze the transcriptome, a hierarchical clustering algorithm that uses a centered correlation coefficient as the measure of similarity and complete linkage clustering was applied, as described previously . When peP = 0.05). When a significant F-test was obtained, separation of means was accomplished using Fisher’s protected least significant difference test at P = 0.05.Analysis of variance (ANOVA) of experimental data sets was performed using JMP software version 5.0 . Significant effects of treatment were determined based on the magnitude of the F-value  DC3000 (positive control), while no disease symptoms were observed in Arabidopsis leaves inoculated with 10% phosphate buffer saline (PBS) (negative control) . We drop-inoculated with this bacterial suspension onto 2-week-old-Arabidopsis leaves and confirmed the same disease symptom at 5 days after inoculation. To confirm that V. vulnificus colonized Arabidopsis plants, we monitored the total bacterial population density after drop-inoculation with V. vulnificus 96-11-17M and Pto DC3000 in the same age and stage leaves. The population density of V. vulnificus 96-11-17M and Pto DC3000 was approximately 10-fold higher on day 5 than on day 0, whereas the negative control showed no V. vulnificus colonies (in vitro). For consistent results, we infiltrated with V. vulnificus 96-11-17M and Pto DC3000 in Arabidopsis plants grown in soil; this was accompanied by a 15-fold increase in the total population density of V. vulnificus 96-11-17M and Pto DC3000 (in planta). These results indicate that V. vulnificus causes disease in A. thaliana.To assess whether the human pathogenic marine bacterium control) . After sV. vulnificus modulates Arabidopsis plants at the molecular level, we investigated gene expression levels in Arabidopsis leaves infiltrated with V. vulnificus 96-11-17M at 0, 12, 24, 48 and 72 h post-inoculation in planta analysis. The correlation coefficients of all samples were highly similar, suggesting that none of the samples exhibited abnormal or artificial variation in gene expression levels . A total of 5,360, 4,204, 4,916 and 3,741 DEGs were identified at 12, 24, 48 and 72 h post-inoculation with  control . Express control . This reSeq data .V. vulnificus affects the plant defense mechanism for its propagation in planta, we monitored the population density of V. vulnificus in wild-type (Col-0) and mutant plants of Arabidopsis; these knockout mutants lacked genes that encode pattern recognition receptors (PRRs), such as Flagellin Sensing2 (FLS2) and Elongation Factor 2 Related 1 (EFR1), that recognize pathogen/microbe-associated molecular patterns (PAMPs/MAMPs). Higher V. vulnificus population densities were observed in the knockout mutant lines than in wild-type plants at 3 days post-infiltration  and Nonexpressor of Pathogenesis-Related gene 1 mutant (npr1) lines compared with the Col-0 (control). By contrast, the bacterial population density was at least 10-fold higher in the JA response mutant (jar1) and Ethylene Receptor 1 mutant (etr1) than in the Col-0 (control)  .V. vulnificus modulated Arabidopsis defense-related genes, we compared the transcriptome data obtained from Arabidopsis plants inoculated with V. vulnificus with transcriptome data obtained from Arabidopsis plants inoculated with P. syringae pv. maculicola ES4326 (GSE 5685), a well known bacterial plant pathogen. This comparison revealed 3,486 and 3,108 DEGs in Arabidopsis at 24 and 48 h post-inoculation with P. syringae pv. maculicola ES4326, respectively , FLG22-INDUCED RECEPTOR-LIKE KINASE 1 (FRK1), NITRATE TRANSPORTER 2.6 (NRT2.6), SENESCENCE-ASSOCIATED GENE 13 (SAG13) and ELICITOR PEPTIDE 3 PRECURSOR (PROPEP3) were top-5 inducible genes in Arabidopsis at 24 h and PATHOGENESIS-RELATED 1 (PR1), CYTOCHROME P450 FAMILY 71 SUBFAMILY A POLYPEPTIDE 12 (CYP71A12), CINNAMOYL COA REDUCTASE (CCR2), EARLY FLOWERING 4 (EFL4) and FLG22-INDUCED RECEPTOR-LIKE KINASE 1 (FRK1) genes were listed in Arabidopsis at 48 h. The results of GO analysis showed that, under the biological process category, genes overlapping between the two transcriptome data sets were classified under response to JA, response to ET, response to stimulus, immune system process, flavonoid biosynthesis, flavonoid metabolism, phenylpropanoid metabolism, developmental process, and wax metabolism and biosynthesis, regardless of the sampling time point -rich environment [V. vulnificus uptakes nutrients and converts amino acids to amines and CO2 under acidic conditions [2 levels of the plant apoplast were appropriate for facilitating the infection of Arabidopsis plants by V. vulnificus.To complete its life cycle under unfavorable conditions, man body . The plaironment . V. vulnnditions ,30. The nditions . IntriguV. vulnificus. The notion that V. vulnificus acts as a plant pathogen is based on the hypothesis that plants have evolved an elaborate immune system that activates defense pathways in response to V. vulnificus infection. The first line of plant defense is the activation of PRRs localized in the plasma membrane upon the recognition of PAMPs/MAMPs [Arabidopsis are involved in the perception of flagellin and elongation factor, respectively, and thus in the activation of the plant defense response, we quantified the population density of V. vulnificus in wild-type and PAMP receptor mutant plants of Arabidopsis. The bacterial population density was 10-fold higher in the mutants than in wild-type plants Click here for additional data file.S2 FigArabidopsis at 12, 24, 48 and 72 h after V. vulnificus 96-11-17M infiltration compared to the control (0 h) using DAVID software.GO categories were assigned for DEGs from (TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.S6 Table(DOCX)Click here for additional data file.S7 Table(DOCX)Click here for additional data file.


Correction to: Molecular Cancer (2014) 13:176.http://www.molecular-cancer.com/content/13/1/176After the publication of this work , the aut


We suggest a switching mechanism that relies on resonant tunnelling via the superatom molecular orbitals (SAMOs) of the fullerene cage as a means of Li activation, thereby bypassing the typical vibronic excitation of the carbon cage that is known to cause molecular decomposition.The field of molecular electronics aims at advancing the miniaturization of electronic devices, by exploiting single molecules to perform the function of individual components. A molecular switch is defined as a molecule that displays stability in two or more states  and upon application of a controlled external perturbation, electric or otherwise, undergoes a reversible change such that the molecule is altered. Previous work has shown multi-state molecular switches with up to four and six distinct states. Using low temperature scanning tunnelling microscopy and spectroscopy, we report on a multi-state single molecule switch using the endohedral fullerene Li@C 60 endofullerene can be switched between 14 molecular states—distinguished by the location of the Li atom inside the fullerene cage—by resonant tunnelling through the superatom molecular orbitals.Molecular switches that can access more than just a few states are difficult to realize. Here, the authors use scanning tunnelling microscopy and spectroscopy to show that a surface-bound Li@C This radical new concept has subsequently led to a vast array of novel single-molecule devices developed to mimic the electronic functionalities of their macroscopic counterparts, such as wires, and transistors5. Amongst the various nanoscale components reported, molecular switches figure predominantly, not least because of their potential to drastically increase data storage density12. Nowadays, silicon technology requires of the order of one million atoms to store one bit (i.e. 0 or 1). In contrast, a single magnetic atom was recently demonstrated to act as a reliable memory bit, capable of being toggled between two magnetic states13. This represents the ultimate miniaturisation of an electronic component.Molecular electronics was born from the desire to continue technological progression beyond the physical limits of the semiconducting materials and technology currently employed. In 1974, Aviram and Ratner designed a theoretical rectifier made from just one molecule6. But the need for complex molecules often results in large species with intricate chemistry. One family of molecules that has garnered considerable attention is that of the endohedral fullerenes due to their stable carbon shell that acts to shield the encapsulated species16. Fullerenes have the advantage that, if chosen appropriately, the internal species can potentially be manipulated without compromising molecular integrity, making such species appealing for incorporating into nano-architectures. The fullerene studied in the following work, Li@C60, is particularly interesting because of its near-spherical shape and the loss of symmetry due to the off-centre position of the Li. A range of values have been theoretically predicted for the off-centre displacement of Li for gas-phase molecules (1.2–1.5 Å), with an energetically favourable coordination towards a C6 hexagonal face26. This is supported by second harmonic generation studies on films containing Li@C6021, third-order susceptibility studies on Li@C60 solutions27, and x-ray diffraction studies on [Li@C60]+PF6− crystals28. Migration of the Li between the faces of the isolated molecule has been studied theoretically24 implying that the Li can easily move between local minima at elevated temperatures. Experimental evidence for the motion of Li within the cage for temperatures above 100–150 K has been obtained in THz spectroscopy30, and time-dependent dielectric measurements on [Li@C60]+PF6− crystals31.One strategy currently being explored relies on the use of more complex molecules with the aim of gaining access to more states than just two (0 and 1), thereby allowing storage density to increase even further. Molecular switches with multiple states, like that observed by Auwärter et al. (switching occurs amongst four distinct molecular states) match these criteria32, the prototypical endofullerene Li@C60 could hence potentially reveal multi-state switching capabilities by exploiting the relocation of the off-centred Li atom within the carbon cage. The following work demonstrates a 14-state switching behaviour of individual Li@C60 molecules condensed on a Au(111) surface using low temperature, ultrahigh-vacuum scanning tunnelling microscopy and spectroscopy. Furthermore, careful analysis of variations in the superatom molecular orbital energies (SAMO34) allows the potential energy landscape inside an endofullerene to be explored.As first predicted 25 years ago6 hexagonal faces, hence potentially enabling Li@C60 to act as a 20-state molecular switch. With all measurements being performed near liquid-helium temperatures, we rule out migration of the Li within the cage16 unless purposefully induced by our STM experiments. In the gas phase, the 20 Li-cage coordinations are degenerate. This will not be the case when adsorbed on a metal surface. The presence of the Au(111) support is expected to lift the degeneracy for C6 faces situated at different heights from the Au surface. Our recent STM studies35 show that the Li@C60 is always oriented with a C6 face sitting on the Au. It follows that six different Li-cage coordination Z-levels can be expected as illustrated in Fig. 6 face, furthest and closest to the Au(111) surface, respectively. Levels 2 (green) and 5 (orange) each refer to three C6 faces arranged in a three-fold symmetric distribution around the cage. These two levels are mirror-symmetric. Finally, levels 3 (blue) and 4 (cyan) are also mirror-symmetric, and refer each to six C6 faces arranged near the equator in three pairs of two adjacent faces (i.e. sharing one C = C double bond). Therefore, the distribution of C6 faces from levels 1 to 6 follows the sequence 1–3–6–6–3–1. Figure 26. Our experimental data for a Au(111) substrate are consistent with this prediction36.The premises of our interpretation rely on the fact that the host molecule possesses 20 C60/Li@C60 which adopts the well-known commensurate (2√3 × 2√3)R30° arrangement38. The variation in apparent height within the island, also observed by other groups for pure C6039, is due to different rotational orientations of C60, and not due to the presence of encapsulated Li atoms, as discussed by Stefanou et al. 35. The Li@C60 molecules in the midst of the island are revealed by imaging the same area with a variety of different tunnelling biases  as exhibited in Fig. 60 rotational orientations previously reported in the literature39. However, upon imaging the same area at +2.5 V, ~13% of the fullerenes show a remarkable apparent height increase and appear circular as opposed to all other topographic signatures . Amongst such C6 fullerenes, there are two possible orientations that are 180° rotations of each other. These are referred to as the majority species  and the minority species . The M-C6 and m-C6 adsorption registries are rendered energetically inequivalent due to second-layer effects of the substrate. Figure 60, although the topographical data alone is an insufficient proof of Li content. For this, we compile in Fig. I/dV spectroscopy data acquired on the four species. The three grey-coloured dI/dV traces show the C60 data to be almost independent of rotational alignment (peak assignments follow those reported in our recent PES-STM-DFT study35). These observations are in line with Schull and co-workers39. The red dI/dV trace corresponding to Li@C60 exhibits a completely different resonance structure, proving that this molecule is electronically inequivalent to C60. Careful inspection of Fig. 6 adsorption orientation  measurements43. An example is reported in Fig. I(t) spectrum to identify the change in conductance involved  surface in the M-C6 orientation prior to any induced switch. The excitation event is monitored by the conductance trace in the inset of Fig. I/dV spectrum in Fig. z-SAMO resonances35. The STM image of the filled states . The results are summarised in Fig. The measurements in Fig. I/dV spectra acquired before and after excitation results in the identification of 14 distinct species. Several of these exhibit identical but rotated topographic signatures, yielding the five topographic groups reported in Fig. 60 in the M-C6 orientation prior to any induced switch  separation distance. Rather, they indicate that the measured 3–6–3–1 = 13 Li-cage coordinations (14 minus the native one) are equally probable after excitation, for which we expect 23 ± 3%, 46 ± 3%, 23 ± 3%, and 8 ± 2%, respectively (errors are standard deviations associated with 270 random draws of four outcomes with weighted probabilities of 3–6–3–1).42 and Supplementary Note d-states of the metal substrate plays a major role in determining the energetically preferred adsorption coordination of Li@C60 on the Au(111) substrate (native state identified as level 1 with the Li located furthest from the surface). The Pz-SAMO resonance also shows a significant change but it is difficult to quantify due to its overlap with the LUMO+1. The Px,y-SAMO and D-SAMO resonances, all located above 3 eV, shift in energy only marginally with Li position, indicating a significantly weaker coupling to the metal substrate. Supplementary Note As mentioned earlier, since the SAMO resonances are cage-centred (see refs 60 switching measurements presented so far derive from tunnelling excitations of +5.0 V and 2.0 μA. These severe activation conditions raise the question of which switching mechanism is at play. Our observations can be summarised as follows: (1) only positive polarity (tunnelling into empty electronic states of Li@C60/Au(111)) leads to switching. At negative polarity, increasing the current and/or the bias leads to the collapse of the tunnelling junction (tip crash and/or molecule decomposition) before any switching event can be recorded. (2) At positive polarity, the switch rate increases both with current and/or voltage. 2.0 μA of current are needed at 5.0 V to observe switch rates of about 1 Hz. Further increasing the current or the voltage leads to increased rate but also more frequent tunnel junction collapse and/or molecular decomposition . Further decreasing the current or the voltage leads to the switch rate to decrease. The lowest energy at which we have observed switching is +3.0 V, with very low rates and with a current in excess of 3 μA. At energies below 3.0 V, increasing the current leads to cage decomposition with no prior observable switching.All Li@C43. The fact that the polarity plays such a major role suggests an EF activation. However, we rule out this mechanism on the basis of Fig. 3N molecules in C80 cages on the Cu(110)–O(2 × 1) surface is feasible with energies as low as about ±80 meV, corresponding to the antisymmetric stretch vibration of the Sc3N molecule2. If so, we would anticipate an activation of 40–110 meV in line with the theoretically predicted energy barriers involved in the diffusion of the Li atom within a C60 cage25. Our experimental conditions far exceed these energies and are polarity dependent.The two most commonly invoked non-thermal activation mechanisms in single-molecule systems are inelastic electron tunnelling (IET), and electric field (EF)60 molecules on various metal surfaces, including Au(111), utilising an STM setup45. They demonstrate that resonant tunnelling through molecular states of an individual C60 can generate the necessary vibrational excitation (temperature) to decompose the molecule (via inelastic electron scattering with molecular vibrations). The authors limit their experimental data to excitations below 3.25 eV, i.e. encompassing only the LUMO, LUMO+1, and LUMO+2/3 resonances of C60, but not the SAMO resonances  system, it is reasonable to assume that the decomposition behaviour at lower energies is similar to that of C60/Au(111) with the exception that the tunnelling currents required for decomposition are overall larger for Li@C60 due to a stronger coupling with the substrate45. Molecular heating of the carbon cage, and ultimately decomposition, is achieved by resonant tunnelling mainly through the LUMO+X resonances below 3 V, which exhibit an efficient coupling to the molecular vibrational modes. The LUMO+X resonances differ from the SAMO resonances in that these are carbon-atom-centred orbitals, whereas the latter are Rydberg-like, diffuse orbitals centred on the fullerene cage. Whilst the former have their density spread over the cage (hence the efficient coupling to C60 vibrations), the latter have their density extending beyond as well as within the cage . Resonant tunnelling through LUMOs or SAMOs is therefore expected to lead to different mechanisms and rates of molecular excitation, with the LUMOs likely to be more conducive to cage heating than the SAMOs. In particular for Li@C60, it is conceivable that the SAMOs facilitate access to the inner Li atom since their density also penetrates the fullerene cage. We hence propose a mechanism whereby resonant tunnelling through SAMOs is key to Li activation. The Pz-SAMO and S-SAMO of Li@C60, both located below 3 V, exhibit a strong coupling to the empty states of Au(111)  support .Schulze and co-workers have studied in detail the electron-mediated heating, heat dissipation and decomposition of C45. More interestingly, in a handful of excitation events (<1%), we were able to witness irreversible changes to Li@C60 that can only be ascribed to the ejection of the Li atom without any subsequent damage to the carbon cage. The STM images in Fig. 60 M-C6 final state). The most compelling evidence for Li ejection stems from the constant-current dI/dV spectra recorded before and after excitation, as shown in Fig. 60 molecule and an empty C60 molecule, respectively. The conductance change accompanying the Li ejection was also recorded in I(t) data. Two such spectra are reported in Supplementary Note 6 hexagon and requires at least 5 eV to succeed. This is consistent with the energetic threshold for implanting Li+ into a C60 cage48 and is also supported by ab initio molecular dynamics simulations49. The Li is likely ejected to the gas phase (as a consequence of the strong positive EF applied by the STM tip), since our STM images do not reveal tell-tale signs of the Li fate, e.g. the formation of an exofullerene.A non-negligible fraction of excitation attempts at 5 V and ~2 μA  lead to irreversible damage to the target molecule and its surroundings. Such events were discarded from the analysis reported in this paper, but their occurrence is not surprising following the work of Schulze et al. 60/Li@C60 on a Au(111) surface. By applying EFs in excess of 3 V and very high current densities (in the μA range) to selected Li@C60 molecules, 14 distinct Li-cage coordinations are statistically populated and unambiguously identified. To the best of our knowledge, this represents by far the largest number of accessible, reversible, and stable states observed for a surface-bound single molecule switch. However, we recognise the current lack of final-state control compared to the degree of hierarchical control at low bias voltage previously reported for the rotation of Sc3N inside C807. We propose that Li activation is a result of resonant tunnelling via SAMO resonances, as initially suggested in a theoretical study by Jorn et al. 22. Further exploration of the isomerisation mechanism could be applied to isolated Li@C60 molecules. We suspect, however, that if the manipulation parameters did not merely result in molecular diffusion, we could expect 20 distinct cage coordinations due to the lack of neighbouring fullerenes affecting the internal surface potential of the Li@C60.To conclude, we have shown through a combination of scanning tunnelling microscopy and spectroscopy that it is possible to purposefully induce reversible Li-cage coordination changes in Li endofullerenes condensed in a close-packed island of C60]+(PF6)– salt  was placed in a molecular evaporator consisting of a small quartz-glass capillary tube containing the molecular mixture and surrounded by a heating element. The material was heated at a range of temperatures up to 590 K for >100 h to remove the ligands and any lingering impurities. In this conditioning phase prior to deposition, some Li content is lost through a slow thermal decay process35. The temperature was then raised to 665 K for molecular sublimation, and the Au(111) surface  was held at about 80 K and exposed to the effusive molecular beam for 4 min at a partial pressure of 5 × 10–8 mbar. The sample then underwent a post-adsorption anneal to 570 K for 30 s in order to desorb additional unwanted pollutants and induce molecular diffusion to form large, hexagonally close-packed islands of molecules. The sublimation temperature used here is compatible with what is reported for the sublimation of unfilled C60 molecules36 and was also the temperature used to prepare gas-phase targets of Li@C60 from the same starting material35.Commercial, purified [Li@C−11 mbar) at sample temperatures of ~5 K. Constant-current STM images and constant-current dI/dV spectra (measured by lock-in technique with a superimposed modulation amplitude of 50 mV peak-to-peak and frequency of 1470 Hz) are used to identify the fullerenes.All measurements were conducted with a commercially available CreaTec ultrahigh vacuum low-temperature scanning tunnelling microscope (UHV LT-STM) using an electrochemically etched tungsten tip under ultra-high vacuum (<1 × 10Supplementary InformationPeer Review


In this study, we explore the functional role of striatal cholinergic interneurons, hereinafter referred to as tonically active neurons (TANs), via computational modeling; specifically, we investigate the mechanistic relationship between TAN activity and dopamine variations and how changes in this relationship affect reinforcement learning in the striatum. TANs pause their tonic firing activity after excitatory stimuli from thalamic and cortical neurons in response to a sensory event or reward information. During the pause striatal dopamine concentration excursions are observed. However, functional interactions between the TAN pause and striatal dopamine release are poorly understood. Here we propose a TAN activity-dopamine relationship model and demonstrate that the TAN pause is likely a time window to gate phasic dopamine release and dopamine variations reciprocally modulate the TAN pause duration. Furthermore, this model is integrated into our previously published model of reward-based motor adaptation to demonstrate how phasic dopamine release is gated by the TAN pause to deliver reward information for reinforcement learning in a timely manner. We also show how TAN-dopamine interactions are affected by striatal dopamine deficiency to produce poor performance of motor adaptation. It is widely accepted that the basal ganglia play an important role in action selection, the process by which contextually appropriate actions are chosen in response to presented stimuli. To determine the appropriateness of an action, in the basal ganglia perform reinforcement learning occurs to establish action-stimulus associations. This learning process is facilitated by dopaminergic activity in the striatum, where a reward prediction error is encoded by the dopamine concentration excursion from its baseline level. When a subject performs context-appropriate actions, there is a phasic increase in striatal dopamine if the received reward is above the expectation, which means a positive reward prediction error is computed. Over time, the synapses that correspond to appropriate stimulus-action association in the striatal network are strengthened by long-term potentiation, and inappropriate actions are suppressed by long-term depression Frank, . AlthougThe neural populations within the striatum consist of GABAergic medium spiny neurons (MSNs), cholinergic interneurons, and GABAergic interneurons Kita, . Many prTonically active neurons (TANs) are so-called because they exhibit tonic firing activity (5~10 Hz) . The sAHP lasts several seconds and induces a pause in tonic firing , is involved in quick recovery from sAHP. Deng et al. showed that partially blocking Ih resulted in a prolonged TAN pause duration, and that Ih was modulated by dopamine primarily via D2 inhibitory receptors  that is mainly controlled by apamin-sensitive calcium dependent potassium current  current. The sAHP current is activated by TAN depolarization—represented in the model as TAN activity in excess of a specified threshold. The kinetics of this current are defined on a timescale of hundreds of milliseconds. This mechanism—intrinsic to the TAN population—is responsible for generating the pause in TAN activity, following a stimulus from the cortex/thalamus.h-current. This inward current activates when TANs are hyperpolarized, and the timescale of its kinetics is similar to the sAHP current. The h-current thus contributes to the recovery of TANs from the pause in activity. In the model, the h-current deactivates in response to an increase in the concentration of dopamine—an implementation of D2-receptor agonism, which serves as a dopamine-based modulation of TAN activity .We also assume that the deviation of the firing rate of dopaminergic neurons from its baseline encodes the difference between the expected and received reward—the reward prediction error , which in turn shortens the pause duration. Then—because cocaine is a dopamine transporter antagonist, which results in an increase in extracellular dopamine—we simulated the cocaine condition by increasing the tonic dopamine concentration in the model until the TAN pause duration matched the experimental results.Sulpiride is a selective D2 receptor antagonist; thus, in the model administration of sulpiride corresponds to maximal activation of h-current . Thus, both tonic (baseline) and phasic dopamine levels are decreased by the same factor; Using the model, we investigated the mechanisms of levodopa-based treatments for dopamine deficiency. Levodopa (L-DOPA) is a common medication for Parkinson's Disease patients to increase overall dopamine concentration in the brain Brooks, . LevodopIn the model, the concentration of levodopa is represented as a constant added to the baseline dopamine concentration. In addition to our analysis of the local effects of dopamine deficiency on the striatal dopamine concentration, we also simulated the effects of dopamine deficiency on motor adaptation by incorporating the current model of TAN-dopamine interactions into our previously published model of reward-based motor adaptation . Eight sessions per subject were performed and averaged. Subjects in the control group gradually overcame the visual perturbation and reduced the distance error, but Parkinson's Disease subjects showed poor learning performance  activation on dopaminergic axon terminals  current, and hence more time is required for its subsequent deactivation. This prediction is consistent with the To illustrate this relationship, we performed simulations, varying the duration of thalamic stimulation (from 100 to 400 ms) as shown in Furthermore, the TAN pause duration is dependent on any change in the extracellular dopamine concentration—not just the RPE-determined phasic dopamine release. Therefore, we also produced simulations demonstrating the effects of dopamine deficiency as well as the effect of levodopa administration on the TAN pause duration. Importantly, dopamine deficiency has almost no effect on the TAN pause duration when the reward prediction error is at a minimum  to the cholinergic interneurons in nucleus accumbens . The following differential equation governs its dynamics:Our model describes the collective dynamics of a population of striatal tonically active neurons (TANs). The model represents the aggregate firing rate (activity) of the population treated as a smooth function of time TAN is a time constant, σ (x) = Θ (x)·tanh(x) is a sigmoid function, Θ (x) is Heaviside's function, and ITAN(t) is a term representing an aggregate input composed of intrinsic current inputs and synaptic inputs to the TAN population:where τVThal(t) is a thalamic stimulus equal to 1 during stimulation and 0 otherwise, WThal is a synaptic weight of the thalamic input, DrvTAN is a constant drive that defines the baseline firing rate, IsAHP(t) is a slow after-hyperpolarization current input, and IH(t) is an h-current input.Here IsAHP(t) is a hyperpolarizing current activated when the TAN activity exceeds certain threshold; the dynamics of this current are defined asThe slow after-hyperpolarization current sAHP is a time constant, gsAHP is the activation gain, and θsAHP is the threshold for activation.where τIsAHP, the depolarizing h-current IH(t) is activated when the TAN activity is below certain threshold, and its activation is modulated by the dopamine concentration. Its dynamics is defined by the following equation.In contrast to H is a time constant, gH is the activation gain, WDA is the dopamine weight coefficient, [DA] is the concentration of striatal dopamine, and θH is the h-current activation threshold.where τThe temporal dynamics of striatal dopamine are defined byDA is the time constant, RPE is the reward prediction error, θDA is the nicotinic receptor threshold, [DA]0 is the baseline dopamine concentration.where τTo calibrate the model, we replicated experimental data published by Ding et al.  who recoWDA = 0 as sulpiride is a selective antagonist of dopamine D2 receptors. To simulate the effect of suppressed dopamine reuptake by cocaine , and feed back to the cortex through the thalamus. Action preference is facilitated by increased excitatory projections from sensory cortical neurons representing the stimulus to direct pathway striatal neurons (D1 MSNs). Suppression of unwanted competing actions is assumed to occur because of lateral inhibition among the loops at some level of the network in a winner-takes-all manner.In the model, novel cue-action associations are formed based on reinforcement learning in the striatum. Eventually, the preferable behavior is reliably selected due to potentiated projections from the neurons in prefrontal cortex (PFC), activated by the provided stimulus, to D1 MSNs, corresponding to the preferred behavior. In technical terms, the output of basal ganglia model is the activation levels of thalamocortical relay neurons in response to the input from PFC neurons activated by visual cues. Each cure represents one of the possible reaching targets. These levels are used as coefficients of the linear combination of all possible actions which represents the motor program selected for execution. The resulting motor program is used to calculate the endpoint of the movement using neuro-mechanical arm model  excurses from the baseline ([DA]0) during a short pause in TAN activity only, while the degradation process occurs continuously on a lot longer timescale, we can approximately rewrite these equations in a difference form by integrating over the pause duration:Considering that dopamine concentration ; TK, RC, KH, WB, DT, EL, SM, IR, and YM: writing (review and editing); TK, RC, and SM: visualization; IR, and YM: supervision, project administration, and funding acquisition.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.


In the biomedical field, human organ repair and regeneration represent a very important and challenging tasks  control of intrinsical multifunctional properties for enhanced bioactivity and drug/gene delivery; (2) Control of bioactivity and biodegradation; and (3) understanding molecular mechanism of nanomaterials controlling tissue regeneration. This Research Topic has attracted a series of papers that show the recent advances in the synthesis and biomedical application of bioactive nanomaterials especially bioactive glass nanoparticles, and provide new insights on designing bioactive nanomaterials. In this Research Topic, The editors hope that the Research Topic “Multifunctional Bioactive Nanomaterials for Tissue Regeneration” will contribute to the progress of research and development activities in the field of novel bioactive nanomaterials for regenerative medicine, inspiring future work leading to the expansion of the biomedical applications of such bioactive nanomaterials.BL proposed the Research Topic and editorial and in charge of 2 manuscript for review process. AB revised the topic and editorial. XC was in charge of 1 manuscript for review process. All authors listed have made a substantial, direct and intellectual contribution to the work.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.