Perhaps the most controversy is regarding the function of RNAs found in introns of annotated transcripts, where most of the reads that map outside of exons are usually found. However, it has been reported that the levels of RNA in introns are minor relative to those of the corresponding exons, and that changes in the levels of intronic RNAs correlate tightly with that of adjacent exons. This would suggest that RNAs produced from the vast expanse of intronic space are just pieces of pre-mRNAs or excised introns We present data that challenges the notion that intronic RNAs are mere by-standers in the cell. By performing a highly quantitative RNAseq analysis of transcriptome changes during an inflammation time course, we show that intronic RNAs have a number of features that would be expected from functional, standalone RNA species. We show that there are thousands of introns in the mouse genome that generate RNAs whose overall abundance, which changes throughout the inflammation timecourse, and other properties suggest that they function in yet unknown ways.So far, the focus of non-coding RNA discovery has shied away from intronic regions as those were believed to simply encode parts of pre-mRNAs. Results presented here suggest a very different situation – the sequences encoded in the introns appear to harbor a yet unexplored reservoir of novel, functional RNAs. As such, they should not be ignored in surveys of functional transcripts or other genomic studies. Mammalian cells require molecular machineries with sufficient complexity and diversity to acquire, process, and distribute vast amounts of information. The unique features of non-coding RNAs could facilitate key steps in the information processing activities of hundreds of regulatory pathways, suggesting a role for them as the major informational “currency” of the cell,2. The lHowever, the prevalent belief is that these RNAs simply represent pre-mRNAs en route to splicing or spliced-out introns en route to degradation. Moreover, it has been suggested that the levels of RNAs in the intronic regions are consistently lower than those in exons and that the levels of intronic and exonic RNAs highly correlate – all coTo gain insight into the functional significance of non-exonic RNA, we asked whether introns could indeed harbor functional transcripts in the cell whose features and physiological behavior differed from that of pre-mRNAs or spliced out introns. In this report, we present the results of a global investigation of intronic transcription during the mammalian inflammation cascade. The cascade sets in motion a series of intricate responses to the perturbation of invading pathogens, using many interlocking feedback loops to determine the precise nature and extent of the challenge, and carefully regulating the corresponding pathways to resolution. Thus, it is expected to require a significant intracellular information exchange, where we and others expect non-coding RNAs to function-15 and aMice were treated with lipo-polysaccharide (LPS) by inhalation, followed by isolation of RNA from lung at 3, 6, 12, 24 and 48 hours post-treatment. In total 42 animals were studied: 7 animals at each time-point and a group of control, un-treated animals. We have chosen the single-molecule sequencing (SMS) platform for RNA analysis due to its superior performance and reproducibility for quantification of RNA expression, in part due to the fact that it does not depend on PCR amplification and ligation for the library preparation. Total RTotal RNA from each sample was treated with DNAse, subjected to a RiboMinus procedure to deplete ribosomal RNAs and sequenced by SMS. The basic statistics reflecting the distribution of mapped reads in annotated exons, introns and intergenic regions are shown in TableConsistent with our previously published-results, 63.6–64It is worth noting that in our analysis intronic RNAs refer to any RNAs encoded by genomic regions annotated as introns, including excised introns, alternative isoforms of exons, as well as standalone sense or antisense transcripts transcribed from their own promoters that overlap intronic regions. The first clue that intronic RNAs should not be disregarded came from the observation that the signal from intronic RNA was not evenly spread among genes: rather, annotated genes differed significantly in the amounts of intronic RNAs Figure. On one As the next step, we investigated whether levels of intronic RNAs in general mirrored those of the exons of their associated transcripts, or whether the two were relatively independent from each other. Some controversy in this regard exists in the literature. The report by van Bakel et. al. found a We then calculated Spearman rank correlation between RNAseq densities of each intron and the corresponding exons throughout the time course by comparing 42 exon-intron densities for each animal in each time point for each intron-exon pair (Methods). The histogram of the resulting correlation coefficients for 220,645 exon-intron pairs from this analysis is shown in FigureWe then asked how well different introns of the same transcript correlate with each other. We used two metrics – correlation of different introns of the same transcript with each other throughout the time course, and the range of differences of the intronic RNAseq signal within the same transcript. We expected that if an intron indeed harbored independently regulated transcripts, these transcripts would not correlate with the levels of either exons or other introns from the same locus. The correlations between an intron N and all other introns in a given locus were split into bins according to the correlation that this intron had with the corresponding exons as shown in FigureIndeed, introns that correlated well with their corresponding exons also had higher correlations with the other introns, as evidenced by the upward trend of the minimal correlation box plots in the right portion of the FigureWe then asked how introns of the same transcript might differ in terms of abundance with their corresponding RNAs. For each transcript with 2 or more introns analyzed in FigureThe median ratio of intronic densities to their corresponding exonic densities was found to be 11.95% across all samples in the “Total Intron-Exon Pair-Wise Dataset” (Methods). However, as mentioned above we also observed heterogeneity in the level of intronic RNA signal, even among different introns of the same gene. Therefore, we asked how many individual introns have at least comparable or higher read densities than that of their corresponding exons in at least one animal at any time point. We calculated the maximum ratio of intron/exon densities for each intron that could be found in any of the 42 animals at any timepoint. The distribution of these ratios is shown in FigureWe were intrigued by the introns whose densities are higher than that of exons in this analysis and investigated this further. The introns were stratified based on the maximum ratio of intron/exon density. In the >10 fold category, represented by 96 introns, most (~90%) contained annotated non-coding RNAs, with half of the introns containing snoRNAs found only in the Ensembl database Table. While tAlso as expected, once we lowered the maximum intron/exon density ratio to include the introns with still significant, but lower ratio of 2–10 and 1–2 fold, the situation reversed. More than 90% of introns in these much more numerous categories did not have any annotated RNA . The remaining 7 seem to represent novel RNAs with no BLAST hits to any sequence of known function and 4 examples are shown in FigureThese two examples shown in FigureAccurate and precise regulation of a biological molecule separates functional behavior from noise. Therefore, we asked whether the steady-state levels of intronic RNAs could be as tightly regulated as those of exons. We reasoned that the level of control exerted over the steady-state level of a transcript would manifest itself in the variation of its levels measured among different genetically-identical individuals maintained in the same conditions. Levels of tightly regulated RNAs would vary less than those of RNAs under little regulatory control. Our system is well-suited for measurement of such variation – we have profiled 7 animals of the same gender and similar age per time point from a genetically-homogeneous strain of mice using SMS, which generates highly reproducible results, a key tTo accomplish this, we calculated the coefficient of variation (CV) of each individual intron and the corresponding exons among 7 animals for each time point, resulting in 1,231,535 intron-exon combinations (Methods). While the much lower intronic density precludes a direct comparison between the CV’s of the introns and exons (Methods), we asked how many introns had the same or lower CV than the corresponding exons at any time point. Surprisingly, 40,948 out of 98,190 introns used in this analysis satisfied this criterion, despite the fact that the RNAseq median densities within these introns were 20.78% of that of the corresponding exons. In other words, the levels of 41.7% of introns have the same or lower animal-to-animal variation within the same time point of LPS treatment (or control) as the corresponding exons, even though their relative levels are on average ~5 fold lower, suggesting an extraordinary level of biological control of steady state expression of intronic RNA.As a next step, we interrogated the entire genome  for the presence of RNAs that change during different stages of inflammation irrespective of the presence of annotation. Determining un-annotated RNAs presents a unique challenge because their sequences are not known and the read length is shorter than the sequence of the entire RNA molecule. Therefore, we have chosen an unbiased approach to identify such regions, based on splitting the genome into a series of non-overlapping bins of different sizes, and then counting the number of SMS reads in each bin, in each sample, and then comparing to the control. We have previously reported this approach with theAn example of a known non-coding RNA detected with this method, the primary transcript for miRNA mir-21, whose transcription increases during inflammation, is showTableOverall, thousands of such examples exist: just at the 3 hr time point, we found 3,890 annotated transcripts corresponding to 1,556 loci that had at least one intronic down-regulated bin and 6,165/2,472 transcripts/loci that had at least one intronic up-regulated bin with no accompanying changes in exons of these genes  RNA regions were first identified based on profiling of histone modifications associated with elongating RNA Pol II in mouse and thenWe thus investigated the distribution of our informative reads and DE bins among the mouse linc RNA regions published by Guttman et al. In total, we could remap 1,666 out of the original 1,673 regions to mm9. We realized that 530 of those regions originally found in the intergenic space now map to or overlap UCSC Genes as exemplified by FigureIt has been reported that cells depleted for the components of RNA degradation machinery, such as the exosome, have higher levels of some non-coding RNAs,38. It hThe discovery of pervasive transcription of the mammalian genome,32,41-43Here, we challenge this general notion and provide evidence that many intronic RNAs can display features consistent with function: their levels of expression and their biological variation during a physiological time course, or among different individuals of the same strain, can often occur with a magnitude similar to that of exons. Furthermore, levels of intronic RNAs often do not depend on levels of their exonic counterparts. Based on these criteria, many introns produce RNA molecules whose fates in the cell are different from that of exonic RNAs, in response to the important biological stimulus of LPS induced inflammation. The combination of these and other parameters as shown in TableWhile these arguments do not tell us how intronic RNAs function, they suggest that they should not be ignored or automatically assigned to the category of annotated pre-mRNAs as suggested by van Bakel et al.,9. On thThis knowledge has an immediate application in molecular diagnostics as intronic RNAs can apparently provide additional information about a biological state that cannot be obtained just by the analysis of exons alone. As a matter of fact, microarray-based analysis of specific biological systems have indeed shown that non-exonic RNAs, including intronic RNAs, not only can be used as diagnostic markers,46, theyThis begs the question of what potential functions could be carried out by intronic RNAs. We think that there are at least two likely possibilities: precursors to smaller functional RNAs, and RNA scaffolds. The general theme of production of smaller RNAs from larger precursors is now recognized in the field. For exaWhile the fact of pervasive transcription is firmly established now as mentioned above, as history shows, it can fade somewhat with time, with focus instead shifting to separately developed lists of long non-coding RNAs detected in specific experiments or filtered by certain properties that hint at functionality,34,52-55This study presents a highly quantitative, comprehensive and unbiased RNAseq dataset showing that most of the RNAs that change during inflammation correspond to the non-coding, predominately intronic, regions of the genome. These non-coding RNAs would not have been detected if only the exons of protein-coding mRNAs or relatively few non-coding RNAs from existing lists were considered as most of the genomic regions they are derived from are not annotated. In general, large numbers of intronic RNAs have properties comparable to those of stand-alone functional RNA species, suggesting that they are more than just discardable parts of pre-mRNAs. In summary, these results argue for a global change in thinking away from one where intronic RNAs are automatically relegated into the pre-mRNA category. Rather, the community should analyze RNAs from these regions with the same interest and rigor as it does mRNAs or linc RNAs.Female Balb/c mice, approx. 6–8 weeks old  were used in this study as a well-established model of LPS-induced inflammation. All animals were weighed and randomized prior to their first challenge: in consideration of their weight they were distributed evenly to groups of eight animals each. For the induction of respiratory inflammation, all animals were exposed to a defined LPS aerosol, except the negative control group which was exposed to clean air only. The aerosols were generated using an Aeroneb nebulizer. The LPS inhalation was done using aerosolized lipopolysaccharide (deposited dose approx. 20 ng LPS) with 0.021% LPS solution for an inhalation period of 10 min on three consecutive days. After the final challenge a necropsy was performed at the following time points: 0 , 3, 6, 12, 24 and 48 hours.Animals were treated using the vehicle 7 days before first inhalative challenge and daily 1 hour before challenge to LPS or clean air (the control group). Animals from all groups were sacrificed painlessly with an overdose of pentobarbital sodium (Narcoren®) 0 (control) and 3, 6, 12, 24 and 48 hours after LPS-challenge. Seven animals were assessed per time point. The inflammatory status in lungs was analysed including the numbers of macrophages/monocytes, neutrophils, eosinophils and lymphocytes by counting a total number of 300 cells per cytospot.Whole lungs were excised, cut into small pieces (max 0.5 cm diameter) and transferred in a sufficient volume of RNAlater (Ambion) and frozen in liquid nitrogen. The tissue samples were stored at −70°C until RNA isolation.Total RNA from tissue samples and cell lines was extracted using TRIzol® reagent  using manufacturer’s protocol. The sample was homogenized while suspended within an appropriate volume (10x sample volume) of reagent. After a brief incubation at room temperature, the samples were centrifuged to remove insoluble material and the supernatant transferred to a fresh tube. Choloroform (0.2x TRIzol volume) was then added to the contents of the new tube and vigorously vortexed. After a few minutes of incubation at room temperature samples were then centrifuged for 15 minutes at 12000 x g at 4°C. This results in the formation of three distinct phases. The topmost aqueous phase, which contains RNA, was then carefully transferred to a new tube. The addition of isopropyl alcohol (0.5X TRIzol volume) to the samples followed by a 10 minute room temperature incubation and a subsequent 10 minute centrifugation at 12000 x g at 4°C precipitated the RNA into a gel-like pellet at the bottom of the tube. After the removal of the supernatant, the pellet was washed twice in 1 ml of ice-cold 75% ethanol. The resultant pellet of RNA was then allowed to dry for about 5–10 minutes and finally resuspended in DEPC-treated water. The quality and the quantity of the resulting RNA was then measured using spectrophotometric techniques on a NanoDrop instrument (Thermo Scientific).Total RNA was first DNase treated to remove any residual DNA. Approximately 40 μg of total RNA , 2 μl Rnase-out  in a total volume of 200ul) is incubated for 30 minutes at 37°C. Samples are then cleaned using the RNeasy MinElute cleanup kit  following manufacturer’s protocol. In brief, 700 μl Buffer RLT and 500 μl 100% Ethanol are added to the sample which is then added to a MinElute spin column. The columns are washed with 500ul Buffer RPE followed by 500 μl 80% Ethanol. After an additional 2 minute centrifugation to remove any residual ethanol the sample is eluted in 14ul DEPC treated water. Quality of RNA was then assessed using an Agilent 2100 Bioanalyzer and their RNA 6000 Nano Kit  using manufacturer's protocol and the sample was quantified using a Nanodrop as per above. Samples were depleted for rRNA through the use of the RiboMinus Eukaryote Kit for RNA-Seq  following the manufacturer's protocol. The RNA was then converted into cDNA, and subjected to SMS as previously described[All genomic coordinates listed throughout this work correspond to the mm9 version of the mouse genome.We extracted intronic intervals from the knownGene.txt file downloaded from the UCSC genome browser site, based on the mm9 version of the mouse genome and last modified 30-May-2011 00:13. We then removed parts of introns that overlapped exons annotated in the same knownGene.txt file, also see Additional fileTo calculate intronic densities, we counted number of reads that fall within each intronic interval  to generate the “Total Intron-Exon Per Time-Point Dataset”. One complication that we faced was that the CV of a transcript level among animals depends on the expression level of a transcript, with more abundant transcripts having less variation – as would be expected, since more abundant transcripts could be measured more reliably than the less abundant ones. Indeed, the Spearman rank correlation between average RNA signal density and the corresponding CV was −0.58, indicating that transcripts with lower abundance have higher CV. Since introns had a tendency to have lower abundance than exons, their variation would be higher due to the intrinsically higher variation of detection of lower-abundant RNAs. To avoid intronic transcripts with low levels of expression in this experiment, we sorted the “Total Intron-Exon Per Time-Point Dataset” by average intronic density and selected the top half of data points for analysis where average exonic density was positive . The median CV for introns and exons in the resulting dataset were 35.37% and 18.97% respectively and the median density of an intron being 18.16% of the corresponding exons.t-test was used to estimate the fold change significance across 7 animals and p-value < 0.001 cutoff was applied. If one group consists only of zero values, then artificially 0.5 read is added to one animal and 0.5 read is subtracted from another animal of the group before expression density and Student score calculation.Optimal detection of regions of transcription requires the use of nested bins of different sizes. For example, exons of protein-coding mRNAs that have a median size on the order of 100 bp in the mouse genome would be expected to be optimally detected with a smaller size bin on the order of 100 bp. On the other hand, relatively low abundance longer transcripts would be detected best with a bin of longer size that has a chance to capture more SMS reads representing such transcripts. Therefore, genome sequence was split into non-overlapping bins of variable size 100, 200, 500 and 1000 bp and expression density of each bin for each animal for each time point was calculated. Up-regulated and down-regulated bins  on each time point comparing to control (which is zero time point) were identified. One-tailed paired Student’s Bins of different sizes were then merged together. Whenever a bin of smaller size overlapped a bin of a larger size, a bin of the smaller size was chosen as it is more likely to represent the accurate bounds of the detected transcribed element. The power of a more precise bin approach is illustrated by detection of a specific isoform of Adora 3 consisting of only two exons out of a total of 9 known for this locus and upregulated at the 3 hour time point  of annotated UCSC Genes were selected from list of up-regulated and down-regulated on 3 hrs after LPS bins described above. Up-regulated or down-regulated bins where the corresponding gene had respectively at least one up-regulated or down-regulated exon at 3 hrs after LPS  were filtered out from the list.1,383 snoRNAs annotated by the Ensembl and not UCSC Genes database were selected to assess enrichment of functional non-coding RNAs presented in TablewhereN - number of unique Introns in the tested dataset,M - number of unique Introns from tested dataset passing the threshold,n - number of unique Introns from N overlapping snoRNAs,m - number of unique Introns from M overlapping snoRNAs.First strand synthesis was performed using Superscript III  following the manufacturer’s protocols with 200 ng Total DNase-free RNA (see above) as a template. See the Additional fileAuthors declare no competing interests.DS, TE and PK performed the statistical and bioinformatics analyses; MT and ZY performed sample preparation and SMS sequencing; CW and SUI provided theoretical insights; GstL, BS and TAM designed and performed the inflammation time course study; PK and GstL provided the theoretical framework and guidance for the intronic RNA analysis and wrote the manuscript. All authors read and approved the final manuscript.Figure S1. A scheme of the strategy to partition intronic coordinates in the cases of overlapping transcripts. Boxes –exons, lines – introns. Regions 1–4 were used to calculate the average read density of the corresponding introns.Click here for fileTable S1. Correlation of specified introns of Prkca and Slc24a3 with their corresponding exons.Click here for fileTable S2. Properties of individual mouse introns.Click here for fileTable S3. Details of RT-PCR analysis in selected mouse introns.Click here for fileFigure S2. An example of DE bins specifically detecting a specific up-regulated isoform of Adora3 locus.Click here for fileFigure S3. Examples of DE bins detecting regions around annotated miRNAs, found both in an intron  and an interegenic region .Click here for file


In vivo knockdown of GAP-43 in olivary neurons, leads to the atrophy of their CFs and a reduction in the ability to sprout toward surrounding denervated PCs. These findings demonstrate that GAP-43 is essential for promoting denervation-induced sprouting and maintaining normal CF architecture.Structural plasticity occurs physiologically or after brain damage to adapt or re-establish proper synaptic connections. This capacity depends on several intrinsic and extrinsic determinants that differ between neuron types. We reviewed the significant endogenous regenerative potential of the neurons of the inferior olive (IO) in the adult rodent brain and the structural remodeling of the terminal arbor of their axons, the climbing fiber (CF), under various experimental conditions, focusing on the growth-associated protein GAP-43. CFs undergo remarkable collateral sprouting in the presence of denervated Purkinje cells (PCs) that are available for new innervation. In addition, severed olivo-cerebellar axons regenerate across the white matter through a graft of embryonic Schwann cells. In contrast, CFs undergo a regressive modification when their target is deleted.  Structural plasticity is limited in the central nervous system (CNS) of adult mammals, constituting a significant impediment to recovery from injuries such as those caused by trauma, stroke, and neurodegenerative and demyelinating diseases Duffau, . NeverthThe cerebellar climbing fiber (CF), the terminal arbor of the olivo-cerebellar axons, has provided the first example, in the mammalian CNS, of individually observed fibers undergoing sprouting after brain injury . CFs undergo lesion-induced sprouting, activity-dependent remodeling, expansion of their area of innervation in response to an enlarged target territory, and regressive modifications after elimination of their target  respond dramatically to axonal injury. The resection of olivo-cerebellar axons leads to the regression of the remaining stump and the death of many axotomized neurons in the IO during the first few weeks after injury  are linked to agenesis of the corpus callosum and severe mental retardation , a model of human muscular dystrophy, in which degeneration-regeneration events in muscle fibers are accompanied by remodeling of intramuscular terminal nerve fibers , mossy fibers grow ectopically to their normal target layer, innervating the distal L1CAM and NCAM are expressed at constitutively high levels in the IO (Horinouchi et al., In addition to maintaining of CF structure, GAP-43 might also govern the organization of the presynaptic terminal and, consequently, neurotransmitter release. When GAP-43 is silenced, CF varicosities undergo alteration in morphology, becoming rounder and larger compared with control varicosities (Grasselli et al., Several studies have also established the involvement of GAP-43 in neurotransmitter release and synaptic plasticity (Dekker et al., Thus, increasing evidence suggest that GAP-43 has a double role in mature CFs in sustaining both injury-induced sprouting and the maintaining their structure under normal conditions, possibly by mediating cytoskeletal reorganization that is triggered by cell adhesion molecules and CFs interactions with their target. In addition GAP-43 appears to regulate the organization of CF presynaptic terminal and neurotransmitter release.Emerging technologies, such as 2-photon microscopy and laser axotomy, will allow us to monitor cells during injury and repair in live mammalian brains and induce microscopic lesions, enabling us to determine the sequence of structural remodeling events that occur in single fibers after axotomy (Holtmaat and Svoboda, 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.


Previous studies suggest that the responsiveness of TrkB receptor to BDNF is developmentally regulated in rats. Antidepressant drugs (AD) have been shown to increase TrkB signalling in the adult rodent brain, and recent findings implicate a BDNF-independent mechanism behind this phenomenon. When administered during early postnatal life, ADs produce long-lasting biochemical and behavioural alterations that are observed in adult animals.We have here examined the responsiveness of brain TrkB receptors to BDNF and ADs during early postnatal life of mouse, measured as autophosphorylation of TrkB (pTrkB).ex vivo. Basal p-TrkB status in the brain of BDNF deficient mice was significantly reduced only during early postnatal period. Enhancing cAMP (cyclic adenosine monophosphate) signalling failed to facilitate TrkB responsiveness to BDNF. Reduced responsiveness of TrkB to BDNF was not produced by the developmental increase in the expression of dominant-negative truncated TrkB.T1 because this reduction was similarly observed in the brain microslices of trkB.T1−/− mice. Moreover, postnatal AD administration produced long-lasting behavioural alterations observable in adult mice, but the responses were different when mice were treated during the time when ADs did not (P4-9) or did (P16-21) activate TrkB.We found that ADs fail to induce TrkB signalling before postnatal day 12 (P12) after which an adult response of TrkB to ADs was observed. Interestingly, there was a temporally inverse correlation between the appearance of the responsiveness of TrkB to systemic ADs and the marked developmental reduction of BDNF-induced TrkB in brain microslices We have found that ADs induce the activation of TrkB only in mice older than 2 weeks and that responsiveness of brain microslices to BDNF is reduced during the same time period. Exposure to ADs before and after the age when ADs activate TrkB produces differential long-term behavioural responses in adult mice. Brain-derived neurotrophic factor (BDNF) and its receptor TrkB are critical regulators of neuronal survival, differentiation, axonal and dendritic growth, and synapse formation BDNF and TrkB play a central role in the mechanism of action of antidepressant drugs (AD) Previous finding suggests that the responsiveness of TrkB to BDNF is regulated during development; BDNF readily activates TrkB in early postnatal rat brain microslices, but this effect of BDNF is strongly blunted after the second week postnatally bdnf and trkB mRNA expression in mice Exposure to ADs during early life has been shown to produce long-lasting behavioural changes in rodents that are evident even in adulthood. Specifically, depressive and anxiety-like, motor and sexual behaviours are modified in adulthood ex vivo BDNF at different stages of mouse postnatal development. Our results suggest the intriguing hypothesis that during the postnatal development, there is a shift in TrkB responsiveness from a receptor that is readily activated by BDNF but refractory to ADs to a TrkB clearly phosphorylated by ADs but only weakly activated by BDNF. This shift may play a role in the development of certain adult emotional phenotypes.We have here studied the phosphorylation response of TrkB to systemic ADs and We have previously shown that a single intraperitoneal (i.p.) injection of AD induces phosphorylation of TrkB within an hour in the prefrontal cortex (PFC) and hippocampus (HC) of adult rodent brain Our results showed that, along postnatal period, TrkB response to ADs is age-dependently regulated. In the PFC, acute IMI treatment of mice between P5 and P11 failed to increase TrkB phosphorylation at the PLCγ1 site (pY816) . Furthermore, as already reported in adult brain data not shown).We have previously shown that ADs specifically induce phosphorylation of TrkB at the PLCγ1 site (Y816) and the phosphorylation of CREB (cAMP response element binding protein), the downstream target of this pathway, whereas no increase in phosphorylation levels is observed at the Shc site (pY515) or its downstream target Akt et alFigure S2).Knüsel et aldata not shown).The studies by Knüsel The developmentally reduced ability of BDNF to induce TrkB phosphorylation in the mouse brain microslices is probably not due to reduced penetrance of BDNF into mature brain tissue since a highly related neurotrophin, NGF (nerve growth factor), readily induced TrkA phosphorylation (Y674/5) in hippocampal microslices obtained from P24 mice  Bdnf+/− mice  and/or serotonin (5-HT) that subsequently can activate Gs –linked postsynaptic receptors. Consequently, we next tested whether acute treatment with IMI might restore the responsiveness of TrkB to BDNF ex vivo in adult hippocampus. As expected, IMI treatment increased the pTrkB levels in the hippocampus but no further phosphorylation was observed when BDNF was applied .Cyclic adenosine monophosphate (cAMP) signalling has been shown to facilitate or “gate” BDNF-induced TrkB signalling in mature hippocampal neurons ex vivo assay in brain microslices obtained from adult mice pre-treated with fluoxetine (FLX) via drinking water (0.08 mg/ml) for 21 days Long-term systemic treatment with antidepressant fluoxetine has been shown to reopen developmental-like plasticity in the adult rodent brain in vitro cell-free kinase assay to examine whether there are any structural modifications in the TrkB receptor protein itself that might prevent BDNF from binding to and activating TrkB in the mature brain tissue. When the adult PFC and HC homogenates were subjected to the kinase assay in the presence of adenosine triphosphate , a clear increase in TrkB phosphorylation in response to BDNF was observed (ex vivo assay (.We used an data not shown), suggesting that long-lasting alterations of the levels of BDNF or TrkB do not explain the behavioural phenotype observed in adult animals.Exposure of rodents to ADs during postnatal life has been shown to elicit alterations in emotional behaviour that are evident in adulthood, long after the cessation of the treatment Table S1 and post hoc test showed that mice exposed to L-PS CLO show a significantly increased Time in the light .When we tested behaviour of adult animals at 3 months of age, we found that animals exposed to CLO during E-PS or L-PS, long before the behavioural testing, showed differential alterations in exploratory locomotion and anxiety-related behaviour that correlated with the age of AD treatment. Specifically, exposure to CLO treatment produced strong long-term behavioural effects in the Light-Dark test when the drug was given during the L-PS, but not when it was administered during the E-PS (Table S1 and post hoc test showed a reduced latency to feed in mice treated with CLO in the E-PS when compared to control mice at the same age (p<0.05) (Table S1 and Table S1), but the post hoc test did not show any difference among controls and CLO treated animals. No differences were found between the groups for weight loss before the test for postnatal age and treatment (Table S1).In the novelty-suppressed feeding (NSF) test, only the administration of CLO during the E-PS significantly influenced adult behaviour. In this paradigm, food-deprived mice are presented a food pellet in a novel environment and the latency to approach the food is measured ex vivo BDNF stimulation in late postnatal stage and adulthood. AD exposure before or after this developmental switch produces differential long-lasting effects on motor and anxiety-like behaviours in adult animals. Although an inverse temporal correlation between the appearance of the responsiveness of TrkB to systemic ADs and the robust reduction of BDNF-induced TrkB phosphorylation in ex vivo microslices cannot be taken as any evidence of causality, it is tempting to speculate that the same maturation processes that bring about the AD responsiveness, lead to developmental changes that restrict the effects of BDNF on TrkB.In the present study, we observed an age-dependent switch from a TrkB receptor readily activated by BDNF but not by ADs during early postnatal stage to a receptor activated by ADs but less sensitive to direct Pharmacologically, ADs such as the drugs used in the present study bind to and inhibit the monoamine reuptake pumps which lead to enhanced monoamine receptor signalling. While monoaminergic neurons are among the first neurons to be generated during early embryonic development, full maturation of these cells extends beyond postnatal life in rodents Ser133 is induced by a variety of signalling pathways, including PKA (protein kinase A), Ca2+-calmodulin kinase II and Mitogen-activated protein kinase pathways AD-induced TrkB phosphorylation during late postnatal period takes place specifically at the Y816 (binding site of PLCγ1), but not at the Y515 (binding site of Shc), which is consistent to our previous findings in adult brain et alWe have here confirmed and extended the unexpected observations previously reported by Knüsel trkB.T1−/− mice. Another possible explanation could be that the ability of BDNF to induce TrkB activation in the mature brain depends on additional gating mechanisms such as cAMP or adenosine i signalling, might facilitate or restore the ability of BDNF to activate TrkB, but this did not turn out to be the case. Moreover, direct up-regulation of [cAMP]i signalling in the ex vivo assay failed to facilitate BDNF-induced TrkB phosphorylation in mature microslices. It is possible that functional maturation of neuronal networks and closure of sensitive periods may modify the responsiveness of TrkB to BDNF (and ADs). Chronic treatment with AD fluoxetine has been shown to reopen developmental-like plasticity in the adult rodent cortex ex vivo BDNF in the adult mouse brain. However, the cell-free TrkB kinase assay suggests that there does not appear to be any structural modifications in the TrkB protein itself that would prevent the receptor from binding to and being activated by BDNF.It was previously suggested that the developmentally regulated increase in the truncated TrkB.T1 receptor that can act as dominant-negative partner for full-length TrkB, might explain the specific reduction of the responsiveness of TrkB to BDNF. We ruled out this possibility, since hippocampal TrkB receptors responded similarly to BDNF and ADs in the tissue samples prepared from WT and ex vivo assay after early postnatal life remains unclear. Although recent evidence suggest that lack of BDNF has only minor effects on the survival and structure of cortical neurons in the CNS in vivo infusion of BDNF into the adult brain increases TrkB phosphorylation, albeit only at relatively high concentrations via TrkB bdnf+/− mice and in mice over-expressing an inhibitory form of trkB (TrkB.T1) in brain The reason for and significance of the apparently complete loss of TrkB responsiveness to BDNF , 2, the brains quickly removed and bilateral hippocampus and prefrontal cortex were dissected out on a dish cooled on dry-ice. Samples were homogenized in a NP++ buffer  and 2 mM Na3VO4). After incubation on ice for 15 min, samples were centrifuged  and the supernatant collected for further analysis.Dams with their litters were housed individually. For postnatal acute antidepressant treatments, age-matched litters (P5-21) were randomly assigned to receive i.p. injection of saline (SAL) , imipramine (IMI) , or clomipramine  and following indicated lag-time (30–60 min) hippocampus and medial prefrontal cortex were collected as described in Figure S4).Dams with their litters were housed individually. The litters were randomly assigned to 4 groups: saline injected (SAL) , and clomipramine injected (CLO)  Mice were weaned on P22 and males were housed together (4–6 mice/cage) until the behavioural experiments. For biochemical analyses the animals were killed after 2 weeks of the last behavioural test and their tissues were collected and processed as described before.For the acute AD treatments, adult (∼P90) mice received a single i.p. injection of imipramine  or SAL and were killed 30 minutes after. For chronic AD treatment, adult (∼P90) mice had free access to either tap water or to 0.08 mg/ml solution of fluoxetine  for 21 days ex vivo BDNF stimulation assay was performed according to Knüsel et alThe et al2, 0.05% BSA, 2% DMSO with or without 100 µM ATP and/or 50 ng/mL BDNF. The reaction was initiated adding 40 µg of NP++ lyzed protein extract to the mix and the incubation was allowed to proceed for 10 min at 37°C. For a subset of samples, the reaction was quenched by adding an equal volume of 4× Laemmli sample buffer and proteins were separated by SDS-PAGE and TrkB phosphorylation status analyzed with western blotting as described below. Rest of the samples we immediately transferred to Trk antibody containing ELISA (enzyme-linked immunosorbent assay) plates, 3% BSA/PBS-T (+2 mM Na3VO4) added ad 200 µl, and phospho-TrkB ELISA proceeded as previously described Assays were performed according to the procedures described by Angeles Y816 , anti-p-TrkA/BY490/Y515 , MA, USA), anti-p-TrkA/BY674-5/Y705-6 , anti-TrkBout , anti-p-CREBS133 , anti-CREB , CA, USA), anti-p-AktThr308 , anti-AKT , anti-p-PLCγ1Y783  or anti-Trk (#sc-11 (rabbit), 1∶1000, SCB). Further the membranes were washed with TBS/0.01% Tween® (TBST) and incubated with horseradish peroxidase conjugated secondary antibodies . After subsequent TBST washes, secondary antibodies were visualized using electrochemiluminescence kits (Amersham Biosciences) followed by an exposure to Fuji LAS-3000 Camera  for ECL detection.Sample protein concentrations were measured using the Lowry Method (Bio-Rad DC protein assay). Lectin precipitation was carried out essentially as described Battery of behavioural tests Testing was performed for 10 min in an acrylic cage (28.5×28.5×20 cm)  divided into two equal sized compartments: one part with transparent walls, open topped and brightly illuminated (∼450 lx by a 40 W light bulb fixed 55 cm above the floor), the other part made from black plastic (passing infrared light) and covered by a lid. The two compartments were separated by a partition containing an opening (7×5 cm) in its centre at floor level. The animal was placed in the centre of the light compartment facing away from the opening, and the latency to enter dark area, time spent in the compartments, total distance travelled, immobility time and the number of entries to dark were measured over 10 min. In addition, rearing time was also calculated. Testing apparatus was thoroughly cleaned before each animal using 70% ethanol.ad libitum. On testing day each animal was removed from its home cage and placed in a holding cage for 30 min before the test and then put in one corner of the arena. The latency to beginning of a feeding episode was measured (maximum over a period of 5 min). Immediately, after starting of feeding the mouse was removed from the arena and placed with the food pellet in its home cage and allowed to feed ad libitum over a period of 5 min. The amount of food consumed was quantified by weighting the pellet.Novelty-Suppressed Feeding: The Novelty-Suppressed Feeding (NSF) was performed as previously described post hoc test. The criterion for significance was set to p<0.05.Immunoblot bands were quantified using NIH ImageJ. All the data are expressed as mean ± SEM (Standard Error of Mean) and as percentage of control. Statistical analyses were performed using GraphPad Prism 4.0 for Windows . For comparison between two groups Two-way Student's t-test was used. Two-way ANOVA or two-way ANOVA for repeated measures was used to reveal main effect and interaction between the factors followed by Bonferroni Figure S1Effect of imipramine on the phosphorylations of TrkB Shc binding site and Akt during development. (a) Phosphorylation of TrkB Shc binding site (Y515) after acute imipramine treatment  in prefrontal cortex (PFC) and hippocampus (HC). (b) Phosphorylation of Akt (Thr308) after acute imipramine treatment  in prefrontal cortex (PFC) and hippocampus (HC). Phospho-protein values are normalized against corresponding total protein levels. (TIF)Click here for additional data file.Figure S2Acute BDNF stimulations does not regulate total TrkB protein levels in brain microslices. Representative blots showing the levels of full-length TrkB after control or BDNF stimulation in cortical (PFC) or hippocampal (HC) microslices prepare from P5-P60 old mice.(TIF)Click here for additional data file.Figure S3Imipramine and fluoxetine does not directly induce TrkB phosphorylation in hippocampal microslices ex vivo. P20 hippocampal microslices were incubate with vehicle or different concentrations of imipramine  (a) or fluoxetine  (b) for 15 min at 37°C and TrkB phosphorylation (Y705/6) analyzed with western blotting. n = 3 per group.(TIF)Click here for additional data file.Figure S4The effect of postnatal clomipramine on weight gain of mouse. A daily dose of clomipramine  or saline during early  or late  postnatal period produced no changes on weight gain during (P4-21) (a) or after the treatments (P28-120) (b). n = 10–15 per group.(TIF)Click here for additional data file.Table S1Postnatal treatments with clomipramine lead to long-lasting and distinct behaviours depending on early exposure period. The effect of early  and late  postnatal clomipramine treatment  on behaviour in light-dark box test and novelty suppressed feeding test in adult (P90) animals. Two-Way ANOVA followed with Bonferroni post hoc test was performed for statistical analysis; *P<0.05, ** P<0.01. n = 10–15 per group.(DOCX)Click here for additional data file.


New paradigms have been recently proposed in the pathogenesis of both chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF), evidencing surprising similarities between these deadly diseases, despite their obvious clinical, radiological and pathologic differences. There is growing evidence supporting a "double hit" pathogenic model where in both COPD and IPF the cumulative action of an accelerated senescence of pulmonary parenchyma (determined by either telomere dysfunction and/or a variety of genetic predisposing factors), and the noxious activity of cigarette smoke-induced oxidative damage are able to severely compromise the regenerative potential of two pulmonary precursor cell compartments . The consequent divergent derangement of signalling pathways involved in lung tissue renewal (mainly Wnt and Notch), can eventually lead to the distinct abnormal tissue remodelling and functional impairment that characterise the alveolar parenchyma in these diseases . Chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF) are two severe multifactorial pulmonary disorders characterised by quite distinct clinical and pathological features. COPD is characterised by a poorly reversible and progressive airflow limitation that is determined by the concurrence of airways inflammation and emphysema (from now on both included in the acronym COPD),2, whereNevertheless, a number of similarities can be recognised between the two disorders. Firstly, both COPD and IPF are chronic and progressive diseases of elderly people , that severely affect the lung function, and both are related to long term inhalation of external noxious agents (mainly tobacco smoking),4,6,7. SDespite the great deal of research, effective treatments are lacking for both COPD and IPF. This can be a consequence, at least in part, of the limited understanding of their pathogenesis, despite the overwhelming plethora of studies and theories proposed so far. Interestingly, for both diseases a gradual shift from "inflammatory-based" pathogenic theories to more complex approaches occurred in recent years ,13. In tWAF1 and p16INK4a )[The most striking new information linking the pathogenesis of IPF and COPD relates to their proposed inclusion within the category of diseases with alveolar senescence and lung "premature aging" -33. The 16INK4a ),25,34,3516INK4a ),30. In a16INK4a ).Although abnormalities directly affecting telomerase genes have not been demonstrated in COPD, telomere decreased length has been demonstrated in either lung cells or peripheral leukocytes in COPD patients, compared with control subjects ,24-27. TThe pathogenic role of genetic abnormalities in IPF is much more evident than in COPD, and familial IPF has been recently included within the category of genetic diseases with "telomere dysfunction" together with acquired aplastic anaemia and dyskeratosis congenita -44. ThesFamilial IPF cases have been linked not only to mutations of telomerase genes, but also to mutations in surfactant proteins coding genes (surfactant protein-C and A2)-54. ChroThus different mechanisms can be responsible of the progressive loss of pneumocyte precursors in IPF, including accelerated senescence, surfactant abnormalities and endoplasmic reticulum stress, all potentially causing precursor cell exhaustion and abnormal alveolar re-epithelialisation -59. The In COPD a large amount of data suggest that parenchymal remodelling and progressive dilation of alveolar spaces is related to decreased and/or deregulated production of extracellular matrix proteins, in particular elastin, with the eventual impaired capability to sustain connective and epithelial tissue repair. Connective tissue insufficiency can be either caused by genetic defects as observed in alpha-1 anti-trypsin deficiency , or it may be ascribed to ill defined causes inducing mesenchymal precursor cell senescence and progressive decrease of matrix protein production, as suggested in this review. Accordingly, senescence related markers in COPD are mainly demonstrable in mesenchymal cells  , and a vAll this taken into account, it is possible to hypothesise that in the pathogenesis of both COPD and IPF the main driving abnormality is the precocious senescence of pulmonary parenchyma.But how can be reconciled this proposed similarity between the basic pathogenic mechanisms underlying COPD and IPF with the obvious diversity of their clinical and pathological presentations? A possible explanation can be searched in the diversity of the genetic alterations, including genetic/epigenetic inheritance or predisposing gene polymorphisms, compromising the renewal capacity of different target cells -71. As pHuman and experimental studies provide strong evidence that molecular networks regulating parenchymal lung tissue renewal are perturbed in both COPD and IPF, although in different ways. Among these networks, particular relevance has been focused on the interplay between Notch and Wnt, two signalling pathways playing critical roles in epithelial and mesenchymal precursor cell maintenance and differentiation -78.In IPF the senescent phenotype seems to mainly affect the epithelial precursors of alveolar tissue (type-II pneumocytes), thus preventing a correct epithelial renewal at anatomical sites where mechanical stress and alveolar damage are expected to be maximal  ,79. Pneuwaf1 and p53 [But how can senescent pneumocytes abnormally trigger these pathways in IPF? Signals provided by the milieu of damaged alveolar cells can trigger a variety of reparative mechanisms, including the recruitment and stimulation of endogenous and exogenous progenitors , and it  and p53 ,97. Smal and p53 , express and p53 ,99. Bron and p53 ,56,99. I and p53 . Further and p53 ,100. Int and p53 .Interestingly, the same signalling pathways involved in IPF (Wnt- and Notch), seem to have a relevant role also in COPD, but in the opposite way. In fact, both Wnt- and Notch- appear as significantly inhibited in COPD, rather than activated as observed in IPF ,102,103,The observed inhibition of Wnt-signalling in COPD may be ascribed to a variety of concurrent causes, including the smoking-related up-regulation of extracellular Wnt antagonists (e.g. secreted frizzled-related proteins) . InteresAll these data taken together, it is possible to hypothesise that in COPD the abnormal senescence of mesenchymal precursors can cause both an impaired production of extracellular matrix proteins (e.g. elastin), as well as a derangement of the interplay between signalling pathways that regulate alveologenesis (in particular Wnt and Notch). These abnormalities are likely sufficient to cause the progressive weakening of the scaffold interstitial structures sustaining the pulmonary parenchyma, with eventual alveolar dilatation and emphysema.Interestingly, the here proposed pathogenic scheme, centred on alveolar senescence and mesenchymal precursor cell insufficiency, can in part reconcile some controversial issues regarding the significance and role of inflammation and autoimmunity in the development of airway disease occurring in COPD ,109-115.In summary, in both COPD and IPF a common pathogenic scheme can be traced where an accelerated cellular senescence determined by the "two hits" paradigm (genetic predisposition to cell senescence with the concurrence of tobacco smoke), determines an impaired regeneration of the lung parenchyma after damage. The divergence in two horns in this model is provided by the affected precursor cells , the relevance of genetic background, as well as by the basic signalling pathways involved in the development of either emphysema or fibrosis Figure . Both meThe complexity of this network is difficult to be completely deciphered in both IPF and COPD, since beyond precursor cell senescence, as here described, other genetic predisposing factors and molecular mechanisms are likely involved in both diseases, including micro-RNA regulation -133. IntThese evolving concepts open new options to better understand the pathogenesis of both IPF and COPD, as far as the involvement of both parenchymal and small airway components are concerned ,143, andThe authors declare that they have no competing interests.All Authors participated in the design of the review and helped to draft the manuscript. All authors read and approved the final manuscript.


Irradiation dynamics of a single graphene sheet bombarded by hydrogen atoms is studied in the incident energy range of 0.1 to 200 eV. Results for reflection, transmission, and adsorption probabilities, as well as effects of a single adsorbed atom to the electronic properties of graphene, are obtained by the quantum-classical Monte Carlo molecular dynamics within a self-consistent-charge-density functional tight binding formalism We compare these results with those, distinctly different, obtained by the classical molecular dynamics.PACS: 61.80.Az, 61.48.Gh, 61.80.Jh, 34.50.Dy. Graphen storage ,6. These storage ,8. The r storage ,9. For eIn this paper, we study the perpendicular impact of hydrogen on a single graphene sheet over more than three decades of impact energies (0.1 to 200 eV) using methods of quantum-classical Monte Carlo molecular dynamics. Our approach is described in detail in the second section entitled 'Methods'. The irradiated target was an infinite graphene sheet obtained by applying 2-D periodic boundary conditions to a graphene cell of size 29.12 × 28.53 Å (336 C atoms). The graphene was prepared at a temperature of 300 K by a Nose-Hoover thermostat and left free during each collision event, which lasted 200 to 500 fs, depending on the impact energy. The irradiation was performed by more than 1,000 independent trajectories for each impact energy, with randomly chosen position of emission of an atom above the surface of the graphene cell. In this method, the total electronic energy of the system is solved quantum-mechanically at the beginning of each time step (on the order of a femtosecond), maintaining fixed atom positions; after incorporating the nucleus-nucleus interaction into the total electronic energy, forces on each atom are updated, and the atoms are moved classically within the time step. The electronic structure is solved here by the self-consistent-charge-density functional tight binding (SCC-DFTB) method -12. To al-hE, in the difference, l-hE, of the (discrete) lowest unoccupied molecular orbital and highest occupied molecular orbital energies in response to the hydrogen adsorption. These changes are indicative of possible changes in the graphene sheet conductance. They are, surprisingly, on the order of 1 eV and depend on the vibrational energy of the adsorbed hydrogen. Adsorption occurs only for the low-energy impacts (< 1 eV). This confirms some predictions in literature on the extreme sensitivity of the highest occupied molecular orbital (HOMO)-lowest unoccupied molecular orbital (LUMO) gap and transport properties of graphene and SWCNT to the adsorption of hydrogen and other atoms and molecules [Results for reflection and transmission probabilities, angular distributions, and adsorption probabilities at low energies (0.1 to 1 eV) are shown and analyzed in the first part of the 'Results and discussion' section, entitled 'Irradiation dynamics and effects on electronic structure'. Additionally, changes in the molecular orbital levels close to the Fermi energy, which influence the non-equilibrium ballistic electron transport properties  of the system, are calculated and characterized by the changes, ∆olecules -19.In the second part of the 'Results and discussion' section entitled 'Comparison with classical molecular dynamics', we perform classical molecular dynamics (CMD) calculations with two state-of-the-art bond order hydrocarbon potentials, reactive empirical bond-order (REBO)  and adapTel = 1,000 K, which has a similar effect to averaging over many electronic states near the Fermi level.To simulate effects of irradiation on graphene, one can apply direct molecular dynamics methods in which electronic structure is treated explicitly using quantum mechanics, while the motion of the nuclei is described by the means of the classical dynamics. This allows one to accurately describe bond breaking and formation as well as the interatomic potentials. Such an approach is, however, computationally very expensive, which greatly limits the system sizes, timescales, and choice of quantum mechanics-based methods. To mimic the dynamics observed by experiment, we apply a Monte Carlo approach to the trajectories, i.e., using a large number of trajectories, randomly varying 'impact parameters' to obtain acceptable statistics of the collision events. Even using this approach, we must use a less expensive and more approximate quantum-mechanical approach. Here, we use the SCC-DFTB method, an approximate density functional theory (DFT) method in which only valence electron interactions are considered. Although a full DFT treatment would be ideal, this is currently too expensive computationally, even for a handful of trajectories. In SCC-DFTB, the total electronic densities and energies are expressed by solution of the Schrodinger equation in the Kohn-Sham form, using predetermined Hamiltonian and overlap integrals as well as repulsive splines fit to reference systems . The tight binding methods applied to the large (solid-state) systems have a long history. Here, we use a self-consistent charge version developed by Bremen Group  -12. SCC To safely allow for high-energy bombardment simulations (in our case 200 eV), we use a refitted version of the original DFTB PBC-0-3  parametez-position above the graphene/coronene plane. The coronene potentials show bonding that is roughly 1 eV weaker and a potential barrier at the hexagon center that is 1 eV higher, reflecting the changes in electronic structure between hydrogen-terminated and periodic sp2 carbon. Despite these differences, the forms of the H-graphene and H-coronene interactions are very similar. Thus, the agreement of SCC-DFTB with DFT calculations of the coronene molecule in Figure Figure Notably, there are two bonding regions in the H-graphene potential. For incidence directly upon a lattice carbon, the potential minimum occurs at approximately 1.1 Å, while incidence upon a C-C bond center shows a shallower potential with minimum close to 1.0 Å. Indeed, there are many potential wells in the 3-D multibody potential that are responsible for capturing impinging hydrogen atoms; these will later be shown to have an effect on the electronic structure of resultant H-graphene surfaces. There are repulsive barriers at the bond center and hexagon center of heights 17 and 2.5 eV, respectively. Notably, hydrogen encounters no barrier before entering the potential well when incident directly on a lattice carbon.z = 0 plane and periodically extended in the xy coordinate plane. To simulate the bombardment in a real-world environment, the sample is thermostated (via Nose-Hoover scheme) to 300 K before bombardment and left to evolve freely during approximately 0.1 to 1 ps (depending on incident energy) simulation time. The impinging hydrogen atom is released from a random  position in the z = 10 Å plane, with velocity perpendicular to the graphene sheet.About ten per decade incident kinetic energies ranging from 0.1 to 200 eV are considered for the impinging hydrogen atom. While cumulative bombardment is not investigated, 1,008 single impact simulations are performed for each incident energy; this is achieved using 1,008 processors, one for each trajectory, on the Kraken Cray XT5 supercomputer . The target graphene surface described in the 'Background' is situated in the Three outcomes of the bombardment are observed: reflection, transmission, and adsorption of the incident hydrogen atom; no sputtering of any type was observed in our quantum-classical approach. Figure Reflection of the incident hydrogen can occur at all points in the graphene lattice. As can be seen in Figure x-y positions for adsorption. Adsorbed atoms are clustered around the carbon atoms, often showing some lateral vibration.By examining the position within the hexagon where incident atoms are reflected, transmitted, or adsorbed, one can infer the form of the many-body potential at nonsymmetrical parts of the lattice. Figure Reflection is distributed evenly around the perimeter of the hexagon, indicating that incident atoms are deflected away from the hexagon center due to the relatively low force experienced here. Also due to the weak interaction at the hexagon center, it is the most probable location for transmission to occur. Thus, atoms incident upon or deflected toward this position are both able to penetrate. These results agree with those from a previous study , which fThe scattering of incident noble gas atoms has been investigated at high energies (keV), where transmitted particles were found to have very little angular deflection while leaving the graphene relatively unaffected . Here, sNmax normalizes the distribution, and the differential solid angle dΩ becomes 2π sin θ dθ due to the azimultal symmetry of the problem. N(θ ± Δθ/2) is the number of atoms scattered into a bin of width Δθ centered at polar angle θ.Here, 1/x- or y-components of an atom's linear momentum are much more visible for low incident energies, where these changes can be comparable to the initial momentum. In the SCC-DFTB simulations, atoms with such low incident energy tend to reflect when not adsorbed, and the reflected angular distribution shows much more scattering. Transmitting hydrogen atoms in these simulations tend to have higher incident energies, so the small x- or y-forces don't produce a significant angular displacement of their momenta. While atoms incident at 5 and 10 eV have a wider distribution than at the higher energies, they tend to penetrate only near the center, where the H-C interactions are weakest.Small changes in the l-h Equantity of the affected graphene. However, roughly a third of the incident atoms are found to bond to the surface after initially being reflected at a large angle relative to their initial momenta. These 'wandering' hydrogen atoms, primarily seen at 0.5 eV incidence, generally drift above the graphene surface at a distance of about 3 Å for 2 to 5 fs before falling toward a lattice carbon and adsorbing. Roughly 10% of these 'wanderers' do not bond to a carbon within the simulation time. Therefore, while they are counted as adsorbed in Figure The dominance of adsorption in SCC-DFTB simulations at impact energies below 1 eV provides enough statistical weight for an investigation of the effects of H-adsorption on the l-h Eby subtracting the energy of the highest occupied orbital from that of the lowest unoccupied orbital. The 1,000-K electronic temperature used creates a 'smearing' of the orbital occupations near the Fermi level. We use occupations of 1.8 for h  and 0.2 for l . This allows us to accomplish significant statistics while accounting for the different sites of adsorption and variety of vibrational states in which atom is adsorbed. The system is a 336-atom supercell, equivalent to an 18 × 18 × 1 k-point grid.The graphene band gap is often computed using a band structure or density of states calculation. However, the graphene system studied here is subject to thermal motion as well as bombardment, and the impinging particle should not be included in Brillouin zone integration. As discussed earlier, we simply define a quantity l-h Eover a number of time steps at the end of the simulation. When doing this time-averaging, it is important to avoid time steps at which some of the hydrogen atoms have not yet bound to the graphene surface. Figure z-position distribution averaged over 1, 4, 12, and 24 fs of simulation time. In all cases, the mean value is within a single standard deviation of 1.2 Å.Figure z-positions. As shown in Figure l-h Eon average.One can see that the standard deviation, representative of the distribution's width, is higher for low averaging times. Additionally, atoms incident with higher kinetic energy are adsorbed with greater vibrational energy, so they display a wider distribution of l-h Eis 171.5 meV for 0.1 eV incidence, 165.1 meV for 0.2 eV incidence, and 157.7 meV for 0.5 eV incidence  and high-energy (0.5 eV incidence) oscillations are 0.1 and 0.3 Å, respectively. The change in l-h Edecreases with increasing adsorbate distance, and the flattening observed below 1 Å causes the minimum position in oscillation to affect the gap less than the maximum position. Thus, larger oscillation amplitudes produce, on average, a smaller change in the l-h Equantity. These averages are shown as thick dashed lines and have a difference of 30 meV, which is on the order of the 14 meV difference between average changes induced by 0.1 and 0.5 eV bombardments.Figure In the classical molecular dynamics approach, the physical accuracy of the simulation is determined mainly by the quality of the interatomic potentials. Like its predecessor, the REBO potential, AIREBO is a member of the classical bond-order family of potentials ,21 of thcluster, i.e., no periodic boundary conditions. All three potentials display a well near 1.2 Å for incidence upon a lattice carbon, and the subsequent repulsive barriers agree well as they are all fit to ZBL [z = 0 for AIREBO and REBO, only 2 eV higher than the corresponding SCC-DFTB curve. REBO is consistently 2 to 5 eV more repulsive than DFTB, but qualitatively very similar. The most distinctive difference between these three potentials is their treatment of the graphene π-orbitals. Clearly, the AIREBO Lennard-Jones interactions coming from the six adjacent carbons produce a potential barrier at the graphene hexagon center that is more than 60 eV (525%) higher than the potential in its predecessor, which is in turn roughly 10 eV (380%) higher than DFTB. The REBO potentials clearly agree much more with the DFTB calculations than those of AIREBO, which indicates that the Lennard-Jones interactions which produce the observed potential barriers likely overestimate the hydrogen-graphene interaction.Figure t to ZBL . HoweverIto et al. ,30, Nakad Eis a carbon atom displacement energy from the rapheme, then the kinetic energy of the impact atom in the head-on binary collision is Another result of the increased repulsiveness of AIREBO is the occurrence of physical carbon sputtering upon impact of hydrogen. Figure x- or y-momentum, resulting in a cos(θ-θo) distribution with maximum at roughly 37°. Transmitting atoms also interact with the potential barriers on the opposite side of the surface, which are responsible for deflecting these atoms and producing the much wider distribution than observed in the SCC-DFTB calculations.Lastly, the CMD calculations result in larger angular scattering effects than SCC-DFTB, as can be seen in Figure l-h Equantity, analogous to the HOMO-LUMO gap in clusters, were investigated, predicting a notable change of the graphene electrical conductivity for even one H-atom chemisorbed. Adsorption was found to be the dominant process below 1 eV, with transmission dominating above 20 eV and reflection dominating at the intermediate energies. Reflection was found to have a more significant scattering effect than transmission.Understanding the effects of irradiation is paramount in developing graphene-based nanosensors and nanoelectronics. Thus, in this work, simulations of single-layer graphene bombarded by hydrogen atoms for a wide range of incident energies were carried out using quantum-classical molecular dynamics based on the self-consistent-charge-density functional tight binding method for treatment of the electron dynamics, combined with classical dynamics of the nuclei. The effects of this bombardment on the graphene sheet and the scattered particle distributions were analyzed in terms of reflection, transmission, and adsorption probabilities and angular distributions. Particularly significant effects of adsorption on the graphene A comparison between results of the SCC-DFTB simulations and classical MD simulations employing the AIREBO potential was made, showing a significant difference in the calculated probabilities and chemistry, mainly caused by differences in the multibody potentials. The AIREBO H-graphene potential overestimates (in comparison to SCC-DFTB) the interaction at the hexagon center  and C-C bond center  lattice positions. A comparison of REBO and AIREBO showed that the overestimate is a result of the Lennard-Jones terms in AIREBO. The effect of this added repulsiveness permeated all of the dynamics, producing wider scattering, a much smaller adsorption probability, and nonzero sputtering yields. Refitting of these terms may significantly improve the accuracy of AIREBO.l-h Equantity, qualitatively associated to the H-L gap and electric conductance of graphene, were found to depend on incident atom energy. Using an averaging time of 32 fs, in addition to averaging over all adsorbed trajectories, the adsorption effect on the l-h Ediffered by roughly 10 meV between incident energies. By virtue of higher vibrational energy, larger incident kinetic energies are found to have a smaller effect on the band gap, as shown in Figure l-h Echanges and/or adsorbed vibrational modes could support the application of graphene in a hypersensitive slow single-particle detector in agreement with the sensitivity to a single biomolecule being coupled to a graphene sheet [l-h Equantity to hydrogen adsorption indicates that the functionality of graphene-based nanoelectronics could be adversely affected by the irradiation by light, chemically reactive species.Changes in the graphene ne sheet . This hyThe authors declare that they have no competing interests.RCE carried out computations, and analyzed results with PSK and JD. RCE and PSK prepared the manuscript, which was finalized together with JD, JJ, and PRCK. JJ provided help in using DFTB. All authors read and approved the final manuscript.


The kink turn is a widespread RNA motif that introduces an acute kink into the axis of duplex RNA, typically comprising a bulge followed by a G⋅A and A⋅G pairs. The kinked conformation is stabilized by metal ions, or the binding of proteins including L7Ae. We now demonstrate a third mechanism for the stabilization of k-turn structure, involving tertiary interactions within a larger RNA structure. The SAM-I riboswitch contains an essential standard k-turn sequence that kinks a helix so that its terminal loop can make a long-range interaction. We find that some sequence variations in the k-turn within the riboswitch do not prevent SAM binding, despite preventing the folding of the k-turn in isolation. Furthermore, two crystal structures show that the sequence-variant k-turns are conventionally folded within the riboswitch. This study shows that the folded structure of the k-turn can be stabilized by tertiary interactions within a larger RNA structure. 2+ ions in isolation ► Nucleotide substitution in the G•A pairs of the k-turn prevent folding in isolation, but some can be stabilized in the context of the riboswitch, allowing binding of SAM ligand ► The crystal structure of a SAM-I riboswitch with a variant k-turn provides the first example of a k-turn with an A•A pair at the 2b•2n position► The folded conformation of a kink turn is stabilized by long-range tertiary interactions in RNA ► The SAM-I riboswitch contains a k-turn, that folds in the presence of Mg Both subunits of the ribosome contain multiple k-turns , and k-tg/kturn/ .trans sugar edge-Hoogsteen edge base pairs that direct the minor groove edges of the two adenine bases toward the minor groove of the C helix. Intimate A-minor interactions and a number of specific hydrogen bonds , and on its 5′ side by regular base pairing (the C helix) A and 1B.en bonds  stabiliz2+ ions induces folding into the structure observed in the crystal in a noncooperative two-state process with a midpoint of the order of 100 μM  have beIn principle, a third process might lead to stabilization of the kinked conformation of the k-turn. The sharp change in helical trajectory at the turn is frequently used to create long-range architecture in large RNA molecules. Conversely, tertiary interactions in such species might stabilize the kinked geometry of the k-turn, so that in a sense the RNA structure forces the k-turn to adopt its folded conformation. We therefore set out to investigate this possibility, and for this purpose we chose the SAM riboswitch as a model system.cis-acting elements occurring in mRNA of principally prokaryotic organisms, that bind small molecules in a manner that leads to metabolic autoregulation      are supee: 3cc2) , with ane: 3npb)  and T. t2+-induced folding of the k-turn of the SAM-I riboswitch as an isolated species. For this purpose, we constructed an RNA duplex of 25 bp with a central sequence corresponding to the riboswitch k-turn. In order to study this by fluorescence resonance energy transfer (FRET), fluorescein (donor) and Cy3 (acceptor) fluorophores were attached to the 5′ termini (We have studied the possible Mg termini A. FoldinEFRET) was measured by steady-state fluorimetry, using the acceptor normalization method  resulted in no detectable evolution of heat on addition SAM B. EvidenKd = 0.31 ± 0.06 μM is similar to that of the natural riboswitch. The conclusion that emerges is that the G2nA substitution that prevented the folding of the isolated k-turn is tolerated in the context of the riboswitch, indicating that the riboswitch structure has stabilized the variant k-turn that was not capable of folding in isolation. The riboswitch carrying a G3nU substitution in its k-turn also binds SAM exothermically . The same modification was introduced into the complete riboswitch, which was titrated with SAM C. In conrmically D, with trmically . Thus thG° = −33 ± 0.4 kJ mol-1 , with an rmsd of 0.53 Å B. Thus, n region C shows te k-turn  are pres network . The for network .We have also solved the crystal structure for the SAM-I riboswitch in which the k-turn contains the double G2nU, G3nU substitution. As with the G2nA, this intrinsically unstable k-turn is stabilized in the context of the riboswitch. The crystals diffracted to 2.9 Å, but due to low completeness between 2.9 and 3.0 Å the data set is regarded as 3 Å resolution. The overall fold and structure of the riboswitch is again maintained A, with a2+ ions in free solution, but folding is prevented by sequence substitutions that disrupt the G⋅A pairs at the 1b⋅1n and 2b⋅2n positions. In all respects, the behavior of the SAM-I riboswitch k-turn is closely similar to that of a standard k-turn such as H. marismortui Kt-7.The SAM-I riboswitch contains a near-standard k-turn that plays an important role in the architecture of the functional riboswitch. The axial kink of the k-turn generates the correct trajectory of helix P2b that permits its terminal hairpin loop to make a loop-loop interaction with an acceptor in helix P4. Inserted into a simple helix, the k-turn folds normally on addition of MgWe observed that the substitutions G2nA  and G2nU, G3nU (disrupting both the 2b⋅2n and 3b⋅3n pairs) prevented significant folding into the kinked structure in the isolated helical RNA. Yet, those same substitutions within the SAM-I riboswitch did not prevent SAM binding. This suggested that in the context of the more complex RNA structure, the k-turn could fold despite the disruption of the 2b⋅2n and 3b⋅3n pairs. This was confirmed more directly by solving the X-ray crystal structures of the two variant riboswitches, revealing that the modified k-turn adopts a standard k-turn fold in both cases. Thus sequence substitutions that prevent metal ion-induced folding of the isolated RNA do not prevent folding within the larger structure of the riboswitch. Evidently, the tertiary interactions in the riboswitch “force” the impaired k-turns to fold. There is likely to be some overall stabilization resulting from binding the SAM ligand to the riboswitch. But since this occurs at a site that is remote from the k-turn or loop-loop interaction the stabilization of the k-turn must be mediated through the tertiary structure of the RNA. The overall free energy of riboswitch folding is sufficient to stabilize some compromised k-turn structures. By contrast, the A1nC- or A1nU-substituted riboswitches did not bind SAM, indicating that these variants could not be induced to fold by the overall RNA structure. Disruption of the 1b⋅1n is clearly more destabilizing, such that the free energy of riboswitch folding is insufficient to overcome the destabilization of the k-turn in this case. Further support for the stabilization of the k-turn in the context of the riboswitch is provided by NAIM experiments of H. marismortui Kt-7. Unfortunately the resolution of the G2nU, G3nU riboswitch is insufficient to be confident about the interactions stabilizing the core of that structure. However, the positions of all the nucleobases of the G2nA riboswitch are very well defined in that structure, and so the hydrogen bonding can be identified with good confidence. All the standard, well-conserved hydrogen bonds are present, and the adenine nucleobases at the 1n and 2b positions are placed correctly so that A-minor interactions between the minor groove edges of the C and NC helices are formed. The variant sequence forms an A⋅A pair at the 2b⋅2n position, held in place by a number of hydrogen bonds, and this study provides the first crystallographic structure of a standard k-turn with an A⋅A base pair at this position. Thus the A⋅A pair is evidently not sufficient to permit the stable formation of this k-turn in isolation, yet the k-turn can fold when stabilized by the tertiary interactions. Interestingly, an A⋅A pair occurs naturally at the 2b⋅2n position of the sequence of T. solenopsae Kt-23 in the small ribosomal subunit, and can be induced to fold by the binding of L7Ae protein. At present there is no structure available for this k-turn, but a crystallographic study of this RNA is underway in this laboratory, and it will be interesting to see if the structure formed in Kt-23 is the same as that observed here. In principle, an A⋅U pair could form at the 2b⋅2n position of the G2nU, G3nU variant, but we cannot be sure that it does so on the basis of the present structure for that riboswitch. In some natural k-turn sequences, the usual G at the 2n position is replaced by U, exemplified by Kt-23 of the T. thermophilus 16S rRNA. In that case, U2n forms a reverse Hoogsteen A⋅U pair with the A2b  were measured using the acceptor normalization method  at 4°C using an SLM-Aminco 8100 fluorimeter. Values of FRET efficiency  as described by 3gx5  and BM-14 (G2nA) at the European Synchrotron Radiation Facility in Grenoble, France. The structure of G2nU, G3nU was solved by performing a rigid body refinement using REFMAC5  with the3gx5  as a preFuller experimental details are presented in the


One of the variables which interfere in the maintenance of sterility in the materials is the storage. There are many official and not-official recommendations for temperature and air humidity control in the storage area, however without any theoretical or experimental basis. Some hospitals do not have a system that makes the control of temperature and humidity possible, and, in most cases, the sterilizing chambers are next to the storage area, constantly releasing heat and water vapour. Considering that one of the functions of the wraps is to maintain the sterility of the content, even under adverse conditions, the recommendations to have an environmental control in the storage area, a priori, would have a secondary importance. For these reason the doubt about the real importance of the temperature and humidity in the contamination of the materials stored after the autoclave sterilization arise.Serratia marcescens 106and stored in an environment with temperature around 35°C and air humidity around 75%. This group was compared with another group, the negative control, stored in temperature around 20 °C and air humidity around 60%. After a period of 30 days of storage, the carriers were removed from the boxes and incubated.Therefore, an experiment was developed in which boxes with surgical instruments and cylinders carriers were packed in cotton sheets, crepe paper, SMS and surgical-grade paper, sterilized, and intentionally contaminated externally with No bacterial growth was detected in any of the samples.The experiment results allowed concluding that the high temperature and high air humidity do not interfere in the barrier efficiency of the packs.None declared.


Graphene grown on crystalline metal surfaces is a good candidate to act as a buffer layer between the metal and organic molecules that are deposited on top, because it offers the possibility to control the interaction between the substrate and the molecules. High-resolution angular-resolved ultraviolet photo electron spectroscopy (ARPES) is used to determine the interaction states of iron phthalocyanine molecules that are adsorbed onto graphene on Ni(111). The iron phthalocyanine deposition induces a quenching of the Ni d surface minority band and the appearance of an interface state on graphene/Ni(111). The results have been compared to the deposition of iron phthalocyanine on graphene/Ir(111), for which a higher decoupling of the organic molecule from the underlying metal is exerted by the graphene buffer layer. The interest in the preparation of ordered layers of organic molecules is rapidly growing, because of the possibility to realize advanced electronic- and spin-based devices –3. TransThe magnetic and electronic properties of the adsorbed molecules may be strongly influenced by the interface and can be potentially tuned by using an appropriate buffer layer. Graphene (Gr), thanks to its unique electronic properties  and to t−10 mbar range at the LOTUS laboratory of the Università La Sapienza (Roma). The Ni(111) single crystal was cleaned by several sputtering–annealing cycles . Graphene was obtained by exposing the sample, which was kept slightly below 600 °C to 6000 L of ethylene (1 L = 10−6 torr·s). The formation of graphene on the Ni(111) surface is complicated by the segregation of carbon from the bulk, because of the high solubility of carbon in Ni [+ for 30 min, 1200 °C for 60 s). The preparation of graphene was done by several 120-seconds long exposures to ethylene while flash-heating the sample up to 1100 °C.Experiments were performed in situ in ultra-high-vacuum (UHV) chambers with base pressures in the low 10on in Ni –20. The The deposition of FePc was carried out by using a custom-made quartz crucible and the molecular deposition was controlled by using a quartz microbalance. One single-layer (SL) is defined as the molecular density of flat molecules fully covering the graphene layer, and it corresponds to a nominal thickness of about 3.4 Å.α (21.218 eV) and HeIIα (40.814 eV), was provided by a SCIENTA VUV-5050 monochromatic source.Low energy electron diffraction (LEED) was used to check the symmetry of both clean and Gr-covered surfaces. LEED was performed in the energy range of the primary beam of 90–140 eV. High-resolution angular-resolved photoelectron spectroscopy data was carried out by using a SCIENTA SES-200 analyzer with an angular acceptance of ±8° and a resolution of 16 meV. All spectra have been taken along the ΓK direction. The UV radiation, HeIThe ARPES band structure of the Gr/Ni(111) and Gr/Ir(111) systems along the ΓK direction of the two-dimensional (2D) Brillouin Zone (BZ) is presented in * states with the Ni d bands [The presence of a distinct single π band for both Gr sheets (on Ni and on Ir) reveals the single-layer nature of graphene. For Gr/Ir111), the Dirac point is localized on a projected bulk band gap and the graphene π band looks very similar to the band of free-standing graphene, the linear dispersion of the π band is preserved close to the K point with only a slight p-doping, which is in agreement with the literature –13. The 1, the Di d bands .The photoemission data in the low binding energy region for the iron phthalocyanine molecules deposited on the Gr/Ni surface, and on Gr/Ir for comparison, as a function of the thickness of the FePc layer is shown in In order to better understand the nature of the interaction, we also analyze data at the K point of the BZ, as shown in When used as a buffer layer between an organic molecule and a metal surface, graphene plays a different role in the molecule–metal interaction that depends on the interaction of graphene with the metal substrate. Graphene on Ni(111) reveals a strong interaction with the substrate and strong alteration of the ideal graphene π band. After deposition of small amounts of FePc molecules, by means of high-resolution UV photoemission we give direct experimental evidence of an interaction of the molecule with Ni through graphene, as shown by the emerging of an interface state at about 0.3 eV binding energy in normal emission and by the quenching of the Gr–Ni π−d hybrid state at the K point of the BZ.


Use of Hirshfeld surfaces calculated from crystal structure determinations on various transition metal ion complexes of three terpyridine ligands carrying trimethoxyphenyl substituents has enabled an assessment of the contribution made by the ligand components to the interactions determining the lattice structures, interactions expected also to be present in metallomesogens derived from similar ligands. The form of the link joining the trimethoxyphenyl substituent to the 4′ position of 2,2′;6′,2″-terpyridine is of some importance. In the case of the Co(II) complexes of two of the ligands, their spin-crossover characteristics can be rationalised in terms of the different interactions seen in their lattices. Thus, there is again minimal evidence for a significant role of aromatic–aromatic, specifically phenyl–pyridyl, interactions in defining the lattice of these complexes, although the formation of bridges via π-type interactions between acetonitrile pairs and pyridyl rings is a feature of all three structures . Examination of the three structures using CrystalExplorer shows that the role of the trimethoxyphenyl groups in lattice construction is largely associated with the methoxy groups, principally through O…π interactions and H-bond acceptance by the oxygen atoms and H-bond donation, including CH…π, by the methyl groups, some examples of these interactions being shown for [Fe(L1)2](ClO4)2·CH3CN in Given that the metal ions are encased in a large ligand sheath and that perchlorate and tetrafluoroborate are tetrahedral anions of similar size, it is unsurprising that [Fe(L2 produces a lattice of [Co(L2)2](BF4)2·H2O which is very similar to those just described for L1 complexes. The space group is again P-1 and the shortest Co…Co separation of 9.177(3) Å, significantly shorter than that of [Co(L1)2](BF4)2·CH3CN·0.5H2O, is again found in pairs (isolated in this case) linked by aromatic-CH…FBF…HC-aromatic bridges, here coupled with aromatic-CH…O(water)…FBF…HC-aromatic bridges, with no “terpyridine embrace” evident. Viewed down a, the lattice has a very similar appearance to that of [Co(L1)2](BF4)2·CH3CN·0.5H2O in that sheets can be seen edge-on, sheets in which cation chains lie side by side, very much as seen in various complexes of terpyridine with simple polyphenyl substituents [L2)2](BF4)2·H2O, these sheets lie parallel to the (0 1 2) plane and the chains  within these sheets (L2)2](BF4)2·H2O, where heating and cooling cycles produce continuous changes (L1)2](BF4)2·CH3CN·0.5H2O (ClO4)2·CH3CN·Et2O has a somewhat different composition to its Co(II) analog, it also crystallises in space group P-1 and the lattice is very similar to that just described, except that the cation sheets lie parallel to the (0 1 −1) plane. The shortest Cd…Cd separation is 9.73(2) Å, but there are other pairs with a separation of 9.90(2) Å and in fact all the cation pairs here can be considered part of a chain with alternating links of the slightly different distances resulting from bridging by differently oriented perchlorate anions. In the sheets parallel to (0 1 −1), it is again possible to discern chains of cations with overlapping aromatic units, here with Cd…Cd alternating between 15.51(3) and 15.56(3) Å, but what is striking is that there is now no indication of stacking interactions involving the triple-bond carbon atoms (their separations being ~0.15 Å greater than in the Co(II) complex). There are various contacts indicative of π-type interactions but only some of these involve the facing aromatics (While the complex [Cd(romatics  and onceL3)2](ClO4)2·CH3CN, once again belonging to space group P-1, can be considered as composed of slightly undulating sheets of cations lying parallel to the bc plane and separated by layers containing the acetonitrile molecules and perchlorate anions. 2](ClO4)2·CH3CN and [Ni(L1)2](ClO4)2·CH3CN, with no indication of any of the π-type interactions seen in those cases (and in the lattice of [Co(L1)2](BF4)2·CH3CN·0.5H2O), confirming thus their secondary importance. A possibly significant feature of the hydroxymethylene link between the aromatic units of L3 is that it appears to allow not only bending of the mean plane of the ligand, as seen for one ligand of the two on a metal ion in all the present cases (an asymmetry noted in various related systems [L3)2](ClO4)2·CH3CN that this additional flexibility may explain is the retention of a vestigial “embrace” array (The lattice of [Cu( anions.  Viewed dL3)2]ClO2·CH3CN,  systems ) but alse” array , althougL3)2](BF4)2 was determined at two temperatures (100(2) and 294(2) K) corresponding, on the basis of magnetic susceptibility measurements (see ahead), to low- and high-spin species, respectively. The same space group, P-1, was retained at both temperatures, so the spin state change is not complicated by phase changes. The change in size of the Co(II) centre due to the change in spin state is accommodated in bond length changes (L3)2](BF4)2 all major cation–anion interactions via aromatic-CH…F bonding are retained and it is essentially all π-type interactions (including O…π) which are lost or weakened. Thus, the only C…C interaction apparent is one still involving C43 but now with C14′ (C43…C14′ 3.32(6) Å) for cations where Co…Co is 10.22(3) Å. The rings involved are not parallel.The crystal structure of [Co2](BF4)2 proved to be rather different to those of [Co(L1)2](BF4)2·CH3CN·0.5H2O, with a spin state change occurring quite abruptly, though without any hysteresis (L3)2](BF4)2 may, as we have suggested for various related systems, [L3 compared to L1 noted above and also the lack of the rather unusual specific interactions involving lattice acetonitrile seen in [Co(L1)2](BF4)2·CH3CN·0.5H2O. The fact that there are numerous points of interaction possible for cation and anion and thus that stabilisation can be achieved in multiple arrangements may be a general factor influencing spin state changes in all the Co(II) complexes presently considered.Somewhat unexpectedly, the magnetic properties of terpyridine-4′-ol , 5-(bromomethyl)-1,2,3-trimethoxybenzene , and K2CO3  in dry DMF (50 mL) was heated at 100 °C for 12 h. After cooling to room temperature, the precipitate was filtered out and the solvent was removed under reduced pressure. The residue was subjected to chromatography on silica using ethylacetate/hexane  eluent to give the desired product as a pale yellow powder. Yield, 0.32 g, 37%. 1H NMR : δ = 8.74 , 8.68 , 8.18 , 7.92 , 7.40 , 7.37 , 7.06 , 5.36 , 3.95 , 3.93 .Ligand L1)2](BF4)2·CH3CN·0.5H2O [L1 only.All metal ion complexes were prepared and crystallised for structure determinations by methods essentially identical to those used for [Co(L1)2](ClO4)2·CH3CN: L1  was dissolved in hot CH3CN (10 mL) and Fe(ClO4)2·H2O  in CH3CN (5 mL) slowly added. The mixture was stirred for 2 h at 60 °C, then evaporated to dryness under reduced pressure. The purple residue was washed with cold MeOH and dried in air. Yield: 0.11 g. Vapour diffusion of diethyl ether into an acetonitrile solution of the product gave purple crystals suitable for an X-ray structure determination. 1H NMR : δ = 9.18 , 8.71 , 7.97 , 7.58 , 7.23 , 7.14 , 7.12 , 4.15 , 3.95 . Anal. Calcd. for C50H45Cl2FeN7O14: C, 54.86; H, 4.14; N, 8.96. Found: C, 54.80; H, 4.16; N, 9.01%.[Fe(L1)2](ClO4)2·CH3CN: Substitution of Ni(ClO4)2.6H2O  for Fe(ClO4)2·H2O in the above procedure gave a pale yellow powder. Yield: 0.10 g. Anal. Calcd. for C50H45Cl2NiN7O14: C, 54.72; H, 4.13; N, 8.93. Found: C, 54.76; H, 4.12; N, 8.90%.[Ni(L2)2](BF4)2·H2O: Use of L2  and Co(BF4)2.6H2O  gave a deep orange powder, Yield: 0.12 g. Anal. Calcd. for C52H44B2CoF8N6O7: C, 56.91; H, 4.04; N, 7.66. Found: C, 56.95; H, 4.01; N, 7.60%.[Co(L2)2](ClO4)2·CH3CN·Et2O: Use of L2  and Cd(ClO4)2.H2O  gave a pale yellow powder. Yield: 0.09 g. 1H NMR : δ = 8.82 , 8.65 , 8.27 , 8.13 , 7.55 , 7.53 , 7.08 , 3.94 , 3.83 . Anal. Calcd. for C58H55CdCl2N7O15: C, 54.71; H, 4.35; N, 7.70. Found: C, 54.80; H, 4.26; N, 7.75%.[Cd(L3)2](BF4)2: Use of L3  and Co(BF4)2·6H2O  gave an orange powder. Yield: 0.11 g. Anal. Calcd. for C52H48B2CoF8N4O8: C, 57.32; H, 4.44; N, 5.14. Found: C, 56.98; H, 4.41; N, 5.10%.[Co(L3)2](ClO4)2·CH3CN: Use of L3  and Cu(ClO4)2·6H2O  gave a green powder. Yield: 0.12 g. Anal. Calcd. for C54H51Cl2CuN5O16: C, 55.89; H, 4.43; N, 6.03. Found: C, 56.01; H, 4.39; N, 6.11%.[Cu(3.3.L1)2](ClO4)2·CH3CN, crystals were coated with Paratone-N oil and the diffraction data measured at temperatures between 100 and 294 K depending on the particular species with synchrotron radiation  on an ADSC Quantum-210 detector at 2D SMC with a silicon (111) double crystal monochromator (DCM) at the Pohang Accelerator Laboratory, Korea. The ADSC Q210 ADX program [L1)2](ClO4)2·CH3CN, data were collected at 173(2) K using graphite-monochromated MoKα radiation with a CCD area detector on a Bruker APEX2 diffractometer [F2 using SHELXL-97 [viawww.ccdc.cam.ac.uk/data_request/cif by citing CCDC 955464–955470.For all complexes other than [Fe( program  was used program  was usedctometer . The strctometer  and refiHELXL-97 . The pos4.viz., interactions of the aromatic–CH…base type, should generally be considered more important influences upon the lattice structures than those arising from any face-to-face contacts of aromatic groups within separate cations. This is consistent with known theoretical calculations for systems even where there are such contacts giving rise to the terpyridine embrace [The use of Hirshfeld surfaces calculated from single-crystal X-ray structure determinations is a convenient means of identifying labile interactions in metal complexes of functionalised terpyridine ligands. It shows that peripheral interactions of aromatic groups in cationic complexes with their counteranions,  embrace ,9. It is