Schizophrenia has been primarily associated with dopamine dysfunction, and treatments have been developed that target the dopamine pathway in the central nervous system. However, accumulating evidence has shown that the core pathophysiology of schizophrenia might involve dysfunction in dopaminergic, glutamatergic, serotonergic, and gamma-aminobutyric acid (GABA) signaling, which may lead to aberrant functioning of interneurons that manifest as cognitive, behavioral, and social dysfunction through altered functioning of a broad range of macro- and microcircuits. The interactions between neurotransmitters can be modeled as nodes and edges by using graph theory, and oxidative balance, immune, and glutamatergic systems may represent multiple nodes interlocking at a central hub; imbalance within any of these nodes might affect the entire system. Therefore, this review attempts to address novel treatment targets beyond the dopamine hypothesis, including glutamate, serotonin, acetylcholine, GABA, and inflammatory cytokines. Furthermore, we outline that these treatment targets can be possibly integrated with novel treatment strategies aimed at different symptoms or phases of the illness. We anticipate that reversing anomalous activity in these novel treatment targets or combinations between these strategies might be beneficial in the treatment of schizophrenia. Since the discovery of chlorpromazine to treat schizophrenia, studies have been focusing on dopamine dysfunction, particularly in the mesolimbic dopamine pathway, which increases dopamine synthesis and release capacity, and can lead to psychosis ,2.Many studies have indicated that schizophrenia is a disorder of the dopamine signal system. Dopamine was initially reported to be related to motor function, but was then found to be associated with reward and motivation in animal studies . CentralThe dopamine hypothesis, which states that the dysregulation of the dopaminergic system is etiologic for schizophrenia, is among the most enduring biological theories in psychiatry. Although variations within genes related to dopaminergic functioning have been associated with schizophrenia, an aggregate test of variations—by using the largest publicly available schizophrenia dataset—has not been conducted. Dopamine dysfunction and its treatment are not sufficient to explain the psychopathology of schizophrenia and its treatment outcomes. In addition to the devastating symptoms of psychosis, many patients with schizophrenia have cognitive impairment. These cognitive symptoms cause substantial dysfunction and can affect their ability to work, to adhere to treatment, and to apply social skills. In addition to the dopamine hypothesis, novel targets have been proposed for schizophrenia treatment. This review intends to address the dopamine hypothesis and the novel targets that have been proposed in recent years .The dopamine hypothesis of schizophrenia was first proposed in the 1960s when the first antipsychotic chlorpromazine was found to successfully treat the positive symptoms of patients with schizophrenia. Since then, the development of newer antipsychotics has generally been following the dopamine hypothesis that patients with schizophrenia have increased dopaminergic activity, which can be normalized using dopamine antagonists, particularly the dopamine D2 receptor antagonist.The dopamine D2 receptor is a G protein-coupled receptor, which is a common target for antipsychotic drugs. The antagonism of dopamine D2 receptor in the mesolimbic pathway is thought to be the main mode of action of antipsychotic medication in treating psychotic symptoms. However, a dopamine receptor antagonist is not clinically effective at treating cortical-related symptoms, such as cognitive deficits, in schizophrenia. Although the exact mechanism underlying these cognitive deficits remains largely unknown, factors such as deficits in cortical dopamine function , dysfuncMolecular imaging studies have supported an association of increased subcortical dopamine transmission with the positive symptoms of schizophrenia, with the limitation that this finding is not pathognomonic, due to the neurochemical heterogeneity of the populations of patients with schizophrenia. Although hyperactivity in subcortical dopaminergic function contributes substantially to aberrant salience (possibly manifesting in positive symptoms), the original dopamine hypothesis must be extended to include contributions of other neurotransmitter systems in the pathophysiology of schizophrenia .N-methyl-d-aspartate receptors (NMDARs) [d-serine, both of which offer additional therapeutic targets. Dysfunction of glutamatergic neurotransmission can be a promising treatment target of schizophrenia for its essential role in the pathophysiology of schizophrenia in terms of negative symptoms and cognitive impairment.Glutamate is one of the excitatory neurotransmitters and the most abundant neurotransmitter in the brain. Glutamate is mediated by (NMDARs) ,15, and (NMDARs) . The ass(NMDARs) . Further(NMDARs) . NMDARs The serotonin  hypothesis of schizophrenia is based on the early studies of interactions between the hallucinogenic drug lysergic acid diethylamide (LSD) and 5-HT . ObservaSerotonin antagonists ameliorate the extrapyramidal effects of antipsychotics. However, direct evidence of serotonergic dysfunction in the pathogenesis of schizophrenia is not yet available; specific serotonin receptors continue to be a focus of interest (particularly 5HT-3 and 5HT-6) in schizophrenia .A significant proportion of patients with schizophrenia are heavy smokers. The high rate and heavy level of smoking observed in this population may be related to the illness or its treatment . StudiesPatients with schizophrenia were found to have a poor inhibition of P50-evoked responses to repeated auditory stimuli, which may reflect impaired sensory gating. However, the effect of smoking on the reversal of diminished auditory sensory gating in schizophrenia may be attenuated due to the desensitization of the nicotine receptor. This observation has been associated with the chromosome 15q14 locus of the α-7 nicotinic receptor gene  and has GABA is the major inhibitory neurotransmitter in the central nervous system. A model has suggested the role of GABA (including GABA–dopamine interactions) in schizophrenia ,30. AlteGABA interneurons are central to brain's rhythm-generating networks, and synchrony of neural oscillations is a fundamental mechanism for the memory, perception and consciousness . GABA abInflammation and oxidative stress are other areas of interest in studies on the pathophysiology of schizophrenia ,37. AlteAn example of an inflammatory model of psychotic disorders is the anti-NMDAR encephalitis syndrome . In thisIn summary, the core pathophysiology of schizophrenia might primarily involve anomalies of the dopaminergic system, and accumulative evidence suggests that glutamatergic, serotonergic, and GABA alterations can lead to aberrant functioning of interneurons, which is manifested as cognitive, behavioral, and social dysfunction through altered functioning of a broad range of macro- and microcircuits. Genetic and early-life environmental risk factors and their interactions can contribute to these abnormalities. Unfortunately, none of the single neurotransmitter systems could explain the full picture of the heterogeneity of schizophrenia symptoms. Intriguingly, the dopaminergic system may interact with other neurotransmitter pathways. For example, postsynaptic density has been suggested to be involved in schizophrenia, and is related to dopaminergic and glutamatergic systems . The intTherefore, identifying the role of novel treatment agents in such complex neurotransmitter networks could be important for understanding their mechanisms of action. Such interactions can be possibly modeled as nodes and edges using graph theory, a novel tool in computational biology for studying complex network interactions. In this approach, dopaminergic, glutamatergic or serotonergic systems may represent multiple nodes connected at a central hub; imbalance within any of these nodes might affect the entire system . ReversaThe relationship between neurobiological findings and clinical symptoms of schizophrenia could lead to the development of novel drug treatments targeting different phases of the illness ,49,50. IA major factor for severe disability in patients with schizophrenia is its persistent and deteriorating negative symptoms. Nevertheless, few current treatments have shown efficacy in this domain . In receCognitive impairment associated with schizophrenia is a significant predictor of social and vocational disability . CurrentPathophysiological processes may vary across different stages of schizophrenia, but current pharmacological treatment does not typically differ during the course of the illness . SeveralAll current antipsychotic therapies have been developed based on the antagonism of dopaminergic receptors or dopamine “stabilizers” including D2/D3 partial agonists, such as the currently available aripiprazole. An exception is the recent introduction of pimavanserin as an antipsychotic in Parkinson’s disease . The devd-serine bind to the NMDAR site as coagonists; thus, an increase in glycine availability by GlyT1 inhibitors is shown to be effective in preclinical studies [Increasing attention has been focused on glutamatergic dysfunction as a pathophysiological mechanism in schizophrenia, and various therapeutic approaches for schizophrenia are being evaluated. Among potential targets related to glutamatergic dysfunction, agents stimulating NMDARs have received the most attention. Because direct NMDAR agonists are neurotoxic, efforts to stimulate NMDARs indirectly by using glycinergic agents  and glycine transport (GlyT1) inhibitors  have been extensively studied. Glycine and  studies . However studies .Metabotropic receptors 1 receptor antagonists have been found to be potential targets for positive symptoms of schizophrenia in animal models ,76, but Second-generation antipsychotics having serotonin–dopamine antagonism provides certain protection against extrapyramidal symptoms, although these agents, except for clozapine, do not provide additional efficacy relative to the first-generation antipsychotics . AlternaGABA is implicated in the pathophysiological mechanisms of schizophrenia. Benzodiazepines  have often been used with antipsychotic medications in patients with schizophrenia. Selective GABA-A agonists and GABA-B antagonists, and more recently, allosteric modulators at GABA-A receptor subtypes are being evaluated in schizophrenia treatment .The cholinergic system has long been a focus of studies on the pathophysiology of schizophrenia, given the clinical observation that nicotine smoking, which most patients with schizophrenia are exposed to, has procognitive effects. Both muscarinic and nicotinic receptors have been targeted in the therapy of schizophrenia ,88,89. ADrugs targeting the neuropeptides associated with dopaminergic and glutamatergic systems have been proposed as a potential treatment strategy for schizophrenia. Cholecystokinin agonists were among the earliest to be evaluated; however, results have been equivocal . CannabiNeurokinin-3 (NK3) receptors have broad modulatory effects on several neurotransmitter systems (including dopamine and GABA); preclinical data with NK3 receptor antagonists have suggested potential efficacy across several symptom dimensions in schizophrenia. Similarly, despite studies indicating the role of neurotensin (NT) in the pathophysiology of schizophrenia , the queMany epidemiological, experimental, and clinical studies have demonstrated significant associations between schizophrenia and inflammatory conditions . A broadd-serine, and one clinical trial has shown positive results with augmenting antipsychotics [DISC1, NOS1, NOS1AP, GRM, Pdxdc1, or ZNF804A, have been implicated in the treatment target for the schizophrenia in preclinical studies. For example, the dopamine D2 receptor has been shown to interact with the DISC1 protein, and disruption of this interaction with a peptide has antipsychotic-like effects in animal models [Pdxdc1 gene is a new antipsychotic treatment target due to its modulatory effect of pre-pulse inhibition [Genome-wide association studies have identified over a hundred single nucleotide polymorphisms (SNPs) associated with schizophrenia risk. Despite the fact that these SNPs account for a modest fraction of genetic predisposition and almost all of them are in the non-coding region, the identified genetic risks of schizophrenia still implicate novel treatment targets via the understanding of neurotransmitter signaling. Certain genes have been already the drug targets for schizophrenia, such as the gene coding for serine racemase (SRR), which synthesizes ychotics . Other gl models . More rehibition , a behavhibition . Additiohibition . It is nIn addition to the aforementioned approaches, epigenetic abnormalities may be a potential target for the treatment of patients with schizophrenia . Recent Schizophrenia treatment based on the dopamine hypothesis has been successful. However, despite many decades of effort by both scientists and drug companies, all currently available clinical treatments still primarily target the dopamine D2 receptor. The reason behind this inconvenient result may be due to the heterogeneity of psychosis. Patients with schizophrenia exhibit marked variations in symptoms, even the biological characterization of symptom domains of schizophrenia remains unclear, and the responses to different therapeutic interventions also vary significantly. The efforts of finding homogenous groups of psychosis using biological markers such as neuroimaging or genetic data may be of help for future pharmacological studies to evaluate novel treatment strategies. Additionally, the lack of reliable animal models of psychosis also contributes to the difficulty in identifying and validating novel treatment agents of schizophrenia. Alternatively, perhaps the dopamine dysfunction is indeed the core psychopathology of schizophrenia, but because of complex interactions between dopamine and other neurotransmitter systems, the development of novel treatment targets cannot be successful without considering these network-like interactions.The difficulty to prospectively predict individual responses to specific treatments leads to a trial-and-error therapeutic strategy. Advances in pharmacogenomics (the study of the genetic determinants of drug response) generate optimism about applying these strategies in treating schizophrenia ,115,116.


However, the physiologically relevant scenario in clinical KS tissue samples is unclear. In the present study, we established a protocol of ChIP-Seq for clinical KS tissue samples and mapped out the epigenetic landscape of KSHV genome in classic KS tissues. We examined AcH3 and H3K27me3 histone modifications on KSHV genome, as well as the genome-wide binding sites of latency associated nuclear antigen (LANA). Our results demonstrated that the enriched AcH3 was mainly restricted at latent locus while H3K27me3 was widespread on KSHV genome in classic KS tissues. The epigenetic landscape at the region of vIRF3 gene confirmed its silenced state in KS tissues. Meanwhile, the abundant enrichment of LANA at the terminal repeat (TR) region was also validated in the classic KS tissues, however, different LANA binding sites were observed on the host genome. Furthermore, we verified the histone modifications by ChIP-qPCR and found the dominant repressive H3K27me3 at the promoter region of replication and transcription activator (RTA) in classic KS tissues. Intriguingly, we found that the TR region in classic KS tissues was lacking in AcH3 histone modifications. These data now established the epigenetic landscape of KSHV genome in classic KS tissues, which provides new insights for understanding KSHV epigenetics and pathogenesis.Kaposi's sarcoma-associated herpesvirus (KSHV) is etiologically related to Kaposi's sarcoma (KS), primary effusion lymphoma (PEL) and multicentric Castleman's disease (MCD). It typically displays two different phases in its life cycle, the default latency and occasional lytic replication. The epigenetic modifications are thought to determine the fate of KSHV infection. Previous studies elegantly depicted epigenetic landscape of latent viral genome in  in vitro cell culture systems was well studied previously. However, the physiologically relevant scenario in clinical KS tissues is unclear. In this study, we performed ChIP-Seq experiments in classic KS tissues and mapped out the AcH3 and H3K27me3 histone modifications on KSHV genome, as well as the genome-wide LANA binding sites. The results revealed a similar H3K27me3 landscape but distinct AcH3 patterns on the KSHV genome compared to the results from in vitro cultured PEL and KSHV infected SLK cells. Intriguingly, there were different LANA binding sites seen on the host genome and a reduced number of AcH3 histone modifications at the TR region of KSHV genome were found. The established epigenetic landscape of KSHV genome in classic KS tissues provides new insights towards our understanding of KSHV epigenetics, which is important for future studies on the mechanism of KSHV infection and pathogenesis.Epigenetic modifications are thought to determine the fate of KSHV infection. The epigenetic landscape of KSHV genome in  Kaposi's sarcoma-associated herpesvirus (KSHV) was first identified in Kaposi's sarcoma (KS) biopsies by Chang and Moore in 1994 and has been proven to be the etiological agent of several human cancers including KS, primary effusion lymphoma (PEL) and multicentric Castleman's disease (MCD) . KSHV isin vitro cell culture systems [in vitro cultured PEL and KSHV-infected endothelial cell lines, the physiologically relevant scenario in clinical KS samples is unclear.Since the epigenetic modifications are thought to determine the fate of KSHV infection, it is important to understand the epigenetic status of viral genome during latency and reactivation –20. Prev systems , 22. It  systems , 22. Whiin vitro culture, thus it is difficult to obtain cell lines reflecting the physiologically relevant scenario in KS tissues [KS is a highly vascular sarcoma on the skin originated from endothelial cells, which is characterized by the infiltrated inflammatory cells and neo-angiogenesis , 23. Acc tissues , 30. TheIn the present study, we established a protocol of ChIP-Seq for clinical KS samples and directly mapped out the epigenetic landscape of KSHV genome in classic KS tissues which are only associated with KSHV infection and derived from Xinjiang area of China. Specifically, we examined AcH3 and H3K27me3 histone modifications on KSHV genome, as well as the genome-wide LANA binding sites. Our results demonstrated that the enriched AcH3 histone modifications were mainly restricted at latent locus while H3K27me3 histone modifications were widespread on KSHV genome in classic KS tissues. The epigenetic landscape at the region of vIRF3 gene confirmed its silenced gene expression in KS tissues. Meanwhile, the abundant enrichment of LANA at the terminal repeat (TR) region was also validated in the classic KS tissues, however, different LANA binding sites were observed on the host genome. Furthermore, the dominant repressive H3K27me3 histone modifications at RTA promoter region were verified by ChIP-qPCR. Intriguingly, we found that the TR region in classic KS tissues was lacking in AcH3 histone modifications with abundant LANA accumulation. Moreover, the established epigenetic landscape in KS tissues was further confirmed in new cases of classic and AIDS-related KS tissues. By analyzing histone modifications and LANA binding sites in classic KS tissues, our study provides new insights for the understanding of KSHV epigentics.in vitro cell culture systems have systemically determined genome-wide distributions of four well-known histone modifications by ChIP-on-ChIP, including AcH3, H3K4me3, H3K27me3 and H3K9me3 [in vitro cell culture systems (BCBL-1 and KSHV infected SLK) were not observed in classic KS tissues while the landscape of H3K27me3 histone modifications in in vitro cell culture systems was much similar to the one in KS tissues. It has been reported that KSHV may have different latency programs in different tissues or cell lines and the expression pattern of viral genes could be affected by the cytokines present in the local cellular milieu [Previous studies on the epigenetic landscape of KSHV genome using  H3K9me3 , 22. The H3K9me3 . The res H3K9me3 , 33. The H3K9me3 . The epi H3K9me3 –37. The  H3K9me3 , 22, we r milieu . The difin vitro cell culture systems were well described in several studies [LANA protein is critical for the maintenance of KSHV episome –41. The  studies –47. Yet  studies –47, the In the meantime, we also analyzed LANA binding sites on the host genome. The identified peaks of LANA binding by MACS were subjected to Peak Annotation and Visualization (PAVIS) analysis . The PAVin vitro cell culture systems described a very similar epigenetic landscape at the RTA promoter region as compared to the results in KS tissues [in vitro studies about RTA regulation could well support and apply to the in vivo scenario.RTA protein encoded by ORF50 is the key switch regulator that controls KSHV reactivation , 14. The tissues , 22, impin vitro cell culture systems [in vitro culture of cells derived from KS tissues was related to the absence of AcH3 histone modifications at TR region. The hyperacetylation of histone H3 can introduce a loosened chromatin structure at TR region, which may facilitate a poised chromatin structure at the long unique region for the topological speculation. However, the hypoacetylation of histone H3 at the TR region in classic KS tissues was shown to correlate a silenced state of viral genome, thus the acetylation of histone at the TR region may not be related to KSHV gene expression according to the results.The TR region of KSHV genome consists of highly repeated sequences of 801 bp with multiple copies which can range over 20 Kb . Previou systems , 22, 49. systems , 50, 51.in vitro cultured cell lines . However, the result in LEC.KSHV was also different from the established results in KS tissues. The strong enrichment of LANA binding and AcH3 histone modifications could be observed at the TR region as the same with other ll lines .To further confirm the established epigenetic landscape in KS tissues, we examined the epigenetic histone modifications of KSHV genome in new cases of KS tissues (two classic and one AIDS-related). As shown in in vitro cell culture systems, which supported the physiological significance of in vitro studies regarding RTA regulation. The distinct AcH3 histone modifications at the TR and vIRF3 coding regions in classic KS tissues provided important clues about the progression of KS disease, which would be a helpful reference for doctors to diagnose clinical patients using epigenetic targeted strategies. We have analyzed the epigenetic landscape of the KSHV genome in classic KS tissues and demonstrated similarities and differences which provide new insights towards understanding KSHV epigenetics, which is important for future studies on the mechanism of KSHV infection and pathogenesis.By analyzing histone modifications and LANA binding sites in classic KS tissues, our study established the epigenetic landscape of KSHV genome in clinical KS samples for the first time. The established epigenetic landscape of KSHV genome in classic KS tissues provided direct evidence to support distinct latent programs in KS tissues. A similar epigenetic landscape was observed at the RTA promoter region in KS tissues as compared to the results from Experiments in the present study were conducted according to the principles in the Declaration of Helsinki. The usage of clinical Kaposi's sarcoma (KS) tissues was reviewed and ethically approved by the Institutional Ethics Committee of the First Teaching Hospital of Xinjiang Medical University . Written informed consent was obtained from all participants, and all samples were anonymized.The clinical KS tissues were collected from four patients who had received a pathological diagnosis of KS, including three classic KS and one AIDS-related KS. The patients were of Uygur and Kazak ethnicities from the local region. All samples were collected from Xinjiang province, northwestern China. Details about the patients/specimen were described in the Body-cavity-based lymphoma (BCBL-1) cell line was derived from KSHV positive primary effusion lymphoma patients . BCBL-1 The collected fresh clinical KS samples (0.1–0.2 g) were stored at -80℃ before usage. The protocol of ChIP-Seq in clinical KS samples is described below:1Pre-treat: Cut tissue into small pieces and transfer tissue into a tube with 1 mL PBS.2g for 5 min to remove the supernatants and wash the pellet with PBS twice.Cross-link: Add formaldehyde to a final concentration of 1.5%. Incubate at room temperature for 20 min. Quench the cross-linking by adding 0.15 mL glycine (1.25 M). Incubate at room temperature for 10 min. Centrifuge at 200 × 3g for 5 min and wash the pellet with PBS.Cell acquisition: Add Collagenase D or P with Dispase (Roche) to a final concentration of 3 mg/mL. Incubate at 37℃ for 1 h with gentle shaking. Grind tissue in douncer on ice. Use 0.7 μm Falcon filter to get the separated cells. Centrifuge at 300 × g for 5 min to remove the supernatants and wash the pellet with PBS twice. Enhanced Cross-link: Add formaldehyde to a final concentration of 1.5%. Incubate at room temperature for 3 min. Quench the cross-linking by adding 0.15 mL glycine (1.25 M). Incubate at room temperature for 10 min. Centrifuge at 200 × 4Normal ChIP procedures: The pellet was lysed with SDS lysis buffer  for 10 min on ice. The lysates were subjected to sonication to obtain 200 bp fragments of DNA , and then centrifuged at 12 000 × g at 4°C for 10 min to obtain the supernatants. The supernatants were diluted 1:10 with RIPA buffer . 10% of the supernatants were kept as input, and the remainder was divided into groups according to the experiment. The aliquots were incubated with pretreated protein A or G beads and corresponding antibody overnight at 4°C. After extensively washing with RIPA buffer, wash buffer  and TE buffer  (four times each), the beads were resuspended in TE buffer. The resuspended beads were subjected to RNase A and proteinase K digestion, and the crosslinking was reversed at 65°C for 8–10 h. DNA was recycled by DNA purification kit (Tiangen).5Library construction and sequencing: The ChIP-seq library was prepared according to the Illumina manual for the preparation of ChIP Sample. The size selection range for the library was 200–400 bp. The established library was validated by Agilent Bioanalyzer and normalized accordingly. The sequencing was performed on the Hiseq 2500 platform.*Note the pretreated beads should not be blocked with sperm DNA.Antibodies in the ChIP-Seq experiments: Anti-Trimethyl-histone H3 (Lys27) (H3K27me3) rabbit polyclonal antibody (07–449) was purchased from Merck Millipore. Anti-acetyl-histone H3 (AcH3) rabbit polyclonal antibody (06–599) was purchased from Merck Millipore. Anti-LANA mouse monoclonal antibody produced by 1B5 hybridoma was made in our laboratory (Antigen source for immunization: LANA 900-1162aa) .−5.. For the analysis of histone modifications with Homer2, the adjusted parameters were set as followed: -style = histone, -size = 100 or 150, -minDist = 300. The default P value cutoff for the peak detection was 10−4. The final result of identified peaks was generated by the combination of MACS and Homer2 analysis. For the analysis of LANA binding sites with MACS, the parameters were set as previously reported:—nomodel,—shiftsize = 50. The Input group was used as control. The P value cutoff for the peak detection was 10−3. Results were visualized by IGV software [y axis of each panel in IGV software was adjusted according to the calculated normalization factors . Reaction mixtures contained 5 μl Master Mix plus Rox, 1 μM each primer, and 4 μl diluted sample. All primers are listed below:ChIP-TR-F: GGGGGACCCCGGGCAGCGAGChIP-TR-R: GGCTCCCCCAAACAGGCTCAChIP-RTAp-F: TCCCCTTCTCCACCGTCAChIP-RTAp-R: TCCGCAATGTCAGGTTCCACChIP-GAPDH-F: TACTAGCGGTTTTACGGGCGChIP-GADPH-R: TCGAACAGGAGGAGCAGAGAGCGAvIRF3-F (ChIP & RT): GTGTGATCTGCGGACTGTCAvIRF3-R (ChIP & RT): ACGAGGGGGACCATATCGAAChIP-vIRF3p (vIRF2)-F: CTCTGGGACTTGGGAGCAAAChIP-vIRF3p (vIRF2)-R: TCTTAACCGCCACCCAATCCChIP-miR-cluster-F: TGTGGCACCAGGTTATGGTCChIP-miR-cluster-R: GCGTCATGACTAAGGGGGAGMORC2-ChIP-F: AAGCCCTGTTTGAGTCCCTGMORC2-ChIP-R: TGCTTTACACTGGGTGCCATRTAp-1.6k-F: TCCACAGGACGGCAAATAGRTAp-1.6k-R: TCCTCATTAGTCGGGACTCGRTAp-0.4k-F: TCCCAGATCAAAGTCATGTCARTAp-0.4k-R: GGCTCAACACCAGACTGAATCThe program set on the 7900HT sequence detection system (Life Technologies) was 95℃ for 5 min, followed by 40 cycles at 95℃ for 15 s, 58℃ for 20 s and 72℃ for 30 s. Melting curve analysis was performed to verify the specificity of the products and each sample was tested in triplicate.The original data have been submitted to SRA (Sequence Read Archive) in NCBI website. The accession number of this project is SRP081036. KSHV genome: HQ404500. TR: NC_009333, 137169–137969.S1 FigBCBL-1 cells and classic KS tissues were collected for RNA extraction, and were reverse transcribed to cDNA. The relative quantity was determined by qPCR. Data were normalized against GAPDH (A). To reduce the influence of heterogeneity in KS tissues, the data were also analyzed by normalizing against LANA (B). Data presented as mean±SD.(TIF)Click here for additional data file.S2 FigN.A. represents no amplification.Results of ChIP-qPCR at different regions of RTA promoter in classic KS tissue (Case2). Samples prepared for ChIP-Seq were divided and a small quantity (1/5) kept for ChIP-qPCR assay before library construction. ChIP–qPCR data were normalized by the percent input method . Data are presented as mean±SD. (TIF)Click here for additional data file.S3 FigN.A. represents no amplification.Results of ChIP-qPCR at the region of TR, RTA promoter, miR-cluster and GAPDH gene (control) in KSHV infected lymphatic endothelial cells (LEC). ChIP–qPCR data were normalized by the percent input method . Data are presented as mean±SD. (TIF)Click here for additional data file.S4 Figy axis represent the relative enrichment of ChIP-Seq signals and has been normalized according to the calculated normalization factors. The epigenetic maps illustrated in the figure contain information from two cases of classic KS tissues. The signals of histone modifications and LANA enrichment were overlaid with the signals of Input (baseline) respectively. Red: AcH3 or H3K27 or LANA. Blue: Input. #: signals at RTA coding region in the LANA group were removed for accidental contamination.Sequence reads for AcH3, H3K27me3, LANA and Input samples were aligned to the KSHV genome (HQ404500+35TR) and visualized in IGV software. Values shown on the (TIF)Click here for additional data file.S1 Supporting Information(ZIP)Click here for additional data file.S1 Dataset(XLS)Click here for additional data file.S1 File(DOCX)Click here for additional data file.S2 File(DOCX)Click here for additional data file.S3 File(DOCX)Click here for additional data file.


Although various computer tools have been elaborately developed to calculate a series of statistics in molecular population genetics for both small- and large-scale DNA data, there is no efficient and easy-to-use toolkit available yet for exclusively focusing on the steps of mathematical calculation. Here, we present PopSc, a bioinformatic toolkit for calculating 45 basic statistics in molecular population genetics, which could be categorized into three classes, including (i) genetic diversity of DNA sequences, (ii) statistical tests for neutral evolution, and (iii) measures of genetic differentiation among populations. In contrast to the existing computer tools, PopSc was designed to directly accept the intermediate metadata, such as allele frequencies, rather than the raw DNA sequences or genotyping results. PopSc is first implemented as the web-based calculator with user-friendly interface, which greatly facilitates the teaching of population genetics in class and also promotes the convenient and straightforward calculation of statistics in research. Additionally, we also provide the Python library and R package of PopSc, which can be flexibly integrated into other advanced bioinformatic packages of population genetics analysis. D value for testing hypothesis of neutral evolution on basis of the observed DNA polymorphisms in 1989 [Theoretical framework of population genetics had been significantly promoted due to the accumulated evidence in molecular biology, which resulted in an interdisciplinary Molecular Population Genetics more than two decades ago . A prima in 1989 , which h in 1989 .Prior to the advent of high-throughput sequencing techniques, the common molecular data available for studies of population genetics always only involve a short DNA fragment directly sequenced with tens of thousands of base pairs in length or several hundreds of polymorphism loci individually genotyped. By aiming at these small-scale and standard molecular data, kinds of computer programs, such as the actively cited DnaSP  and ArleDespite the fact that there are various tools available for population genetics analysis on both small- and large-scale molecular data, we also found two limitations in this field deserving to be addressed. First, it is very inconvenient for using these existing tools, because all of them require strict format of input data, when you intend to compute certain statistics directly starting with the ready-made metadata of variants  instead of raw DNA sequences. So, we believe that the easy-to-use online calculator would be very helpful for conducting some trivial tasks in teaching as well as research of molecular population genetics. Second, there is no toolkit available yet for exclusively performing the calculation step of statistics, which, in expectation, should be able to be directly and flexibly employed and incorporated into an advanced bioinformatic package. Such toolkit, therefore, could facilitate the development of analysis packages for bioinformatists without good background in population genetics, and also for biologists only holding the elementary skills in bioinformatics. In the present study, we specially addressed the two issues.To address the issues as mentioned above, PopSc was designed to exclusively perform the calculation step of statistics of interest in molecular population genetics. Therefore, the initial input data into PopSc are these descriptive metadata, such as the allele or haplotype frequencies, number of segregating sites, counts of different mutation types, and mismatch distribution, etc., which must be generated from upstream raw DNA sequences or genotyping data in advance. After completing the computational duties, PopSc will directly pass the numerical output of statistic on to users. Such philosophy of design will promote the convenient calculation without requiring tedious input of raw DNA sequences and also guarantee the possibility of direct integration of PopSc toolkit into other advanced packages of bioinformatic analysis.Generally guided by the popularity in scientific literatures, we comprehensively collected a total of 45 basic statistics in molecular population genetics into PopSc . All of ab initio input of DNA sequences or other types of raw molecular data. Mathematical formulas and the related documentation for each statistic are also clearly shown on the respective web pages, which would be very helpful for teaching the molecular population genetics in class; and such teaching purpose had also been successfully addressed by the very popular tool of GENALEX, which is an add-in program for Microsoft Excel [Because the web browser is independent of operating system and requires no installation, PopSc was first implemented as online calculator . The webft Excel .http://pypi.python.org and http://cran.r-project.org, respectively) and could be installed by standard commands. The web-based calculator, source codes and reference manuals of PopSc could also be freely available at: http://chenshiyi.com/popsc.html.During the past years, programming languages of Python and R are becoming more and more popular in bioinformatic analyses. Therefore, PopSc was further independently implemented and provided as the Python library and R package, which can be easily integrated into other advanced bioinformatic packages in molecular population genetics. The Python library and R package of PopSc are deposited in official repositories (Of course, PopSc is not an upgrade or even a replacer to the exiting computer tools because it just performs the step of mathematical calculation for each statistic. Therefore, PopSc only accepts the descriptive metadata, which must be independently prepared from raw DNA sequences or genotyping data with the aid of the custom scripts or other computer tools, such as MEGA . PopSc c


Accumulating evidences indicate that non-coding RNAs (ncRNAs) including long non-coding RNAs (lncRNAs) and microRNAs (miRNAs) acting as crucial regulators in osteosarcoma (OS). Previously, we reported that Rho associated coiled-coil containing protein kinase 1 (ROCK1), a metastatic-related gene was negatively regulated by microRNA-335-5p (miR-335-5p) and work as an oncogene in osteosarcoma. Whether any long non-coding RNAs participate in the upstream of miR-335-5p/ROCK1 axial remains unclear.Expression of differentiation antagonizing non-protein coding RNA (DANCR) and miR-335-5p/miR-1972 in osteosarcoma tissues were determined by a qRT-PCR assay and an ISH assay. Osteosarcoma cells’ proliferation and migration/invasion ability changes were measured by a CCK-8/EDU assay and a transwell assay respectively. ROCK1 expression changes were checked by a qRT-PCR assay and a western blot assay. Targeted binding effects between miR-335-5p/miR-1972 and ROCK1 or DANCR were verified by a dual luciferase reporter assay and a RIP assay. In vivo experiments including a nude formation assay as well as a CT scan were applied to detect tumor growth and metastasis changes in animal level.In the present study, an elevated DNACR was found in osteosarcoma tissue specimens and in osteosarcoma cell lines, and the elevated DNACR was closely correlated with poor prognosis in clinical patients. Functional experiments illustrated that a depression of DANCR suppressed ROCK1-mediated proliferation and metastasis in osteosarcoma cells. The results of western blot assays and qRT-PCR assays revealed that DANCR regulated ROCK1 via crosstalk with miR-335-5p and miR-1972. Further cellular behavioral experiments demonstrated that DNACR promoted ROCK1-meidated proliferation and metastasis through decoying both miR-335-5p and miR-1972. Finally, the outcomes of in vivo animal models showed that DANCR promoted tumor growth and lung metastasis of osteosarcoma.LncRNA DANCR work as an oncogene and promoted ROCK1-mediated proliferation and metastasis through acting as a competing endogenous RNA (ceRNA) in osteosarcoma.The online version of this article (10.1186/s12943-018-0837-6) contains supplementary material, which is available to authorized users. Osteosarcoma (OS) is a prevalent malignant bone tumor whose predictive sites are metaphysis of long bones among adolescents . The chaLong non-coding RNAs (lncRNAs), which are a group of transcribed RNA molecules and with length of more than 200 nucleotides, are involved in diverse biological processes in tumor initiation, growth and metastasis through epigenetic, transcriptional and post transcriptional mechanisms –6. lncRNPrevious studies by our group indicated that miR-335 functioned as a tumor suppressor by way of directly targeting to its downstream gene - Rho associated coiled-coil containing protein kinase 1 (ROCK1) , 14. HerNinety-five cases of osteosarcoma tissues and matched para-tumor tissues used in this study were collected under the permissions of patients during tumorectomy in Liaoning Cancer Hospital & Institute from February 2013 to September 2017. None of the patients had received chemotherapy before surgery and all the 95 cases were diagnosed based on a definite pathological diagnosis and the clinical stages of these patients were determined according to the tumor.node.metastasis (TNM) classification of the International Union Against Cancer (UICC). Written informed consent was obtained from all participants. The Institute Research Medical Ethics Committee of Central Hospital affiliated to Shenyang Medical College and Liaoning Cancer Hospital & Institute granted approval for this study.v/v) fetal bovine serum , 100 IU/ml penicillin  and 100 mg/ml streptomycin . All osteosarcoma cell lines were cultured at 37 °C in a humidified atmosphere containing 5% CO2 while human osteoblast cell line hFOB 1.19 was cultured at 34 °C with 5% CO2.Human osteosarcoma cell lines MG-63, U2OS, MNNG/HOS, 143B and human osteoblast cell line hFOB 1.19 were purchased from the Chinese Academy of Sciences Cell Bank  and maintained in DMEM/F12 , DMEM (Gibco), DMEM, MEM, (Gibco) and DMEM, respectively. All mediums were supplemented with 10% . DANCR overexpression plasmids pcDNA-DANCR, wt-DNACR-335, wt-DANCR-1972, mut-DANCR-335 and mut-DANCR-1972 as well as DANCR and ROCK1 knockdown plasmids DANCR shRNA and ROCK1 shRNA with a corresponding negative control shRNA (NC shRNA) were chemically synthesized by GenePharma . The sequence of DANCR shRNA and ROCK1 shRNA were listed in Additional file The procedure was carried out as previously described . Total RTotal proteins were isolated using radio immunoprecipitation assay (RIPA) lysis buffer  and qualified by BCA detecting kit  flollowing to the manufacture’s protocol. Proteins samples were subjected to 10% SDS-PAGE and transferred onto a PVDF membrane, then incubated with ROCK1  and GAPDH antibodies  at 4 °C overnight, respectively. The next day, the membranes were incubated with secondary antibodies  at room temperature for 1 h. Protein bands were detected on X-ray film via enhanced chemiluminescence detection system.3) supplemented with complete growth medium and followed by different transfection 24 h later. At days 1, 2, 3, 4 and 5 after transfection, 10 μl CCK-8 solution was added into each well and incubated for 2 h. The absorbance was measured at an optical density of 450 nm using Microplate reader . Experiments were repeated in triplicate.Osteosarcoma cells’ changes of proliferative capacity were determined by 5-Ethynyl-20-Deoxyuridine (EDU) incorporation assay and cell counting Kit-8 (CCK-8) assay. For EDU incorporation assay, the procedure was carried out according to the manufacturer’s instructions with EDU detection kits . CCK-8 assay was executed as previously reported . In brieThe procedure was carried out as previously described . In shorIn situ hybridization assay was performed on fresh osteosarcoma tissue slices. In brief, slices were washed with1× phosphate buffered saline (PBS)containing 0.5% Triton X-100, afterwards be incubated with appropriate amount of anti-DANCR, anti-miR-335-5p and anti-miR-1972 oligodeoxy-nucleotide probes  with hybridization solution containing 1% blocking solution in humid chamber at 37 °C overnight. The next day, slices were then washed three times for 5 min each at 42 °C with 0.1% Tween-20 in 4× sodium citrate buffer (SSC), once for 5 min in 2× SSC and once for 5 min in 1× SSC in dark. Following, after be rinsed with 1 × PBS for 5 min triply at room temperature and be re-stained by DAPI , fluorescent microscope  was applied to observe and photograph the slices. Images were analyzed using Image - Pro Plus 6.0 software .The procedure of immunohistochemistry was performed as previously described .The procedure was carried out as previously described . Wild anThe procedure was carried out as previously described using Magna RNA-binding protein immunoprecipitation kit  . In brie6 143B cells  with stable overexpression of DANCR (DANCR puro) and with corresponding blank vector (pMSCV puro) were injected subcutaneously or intravenously for the determination of tumor growth and metastasis. Tumor formation and metastasis were monitored by Fluorescent Imager  and Computed Tomography  scan, respectively. The subcutaneous formatted tumor nodes and the lungs from metastatic group were harvested for further detection. This study was carried out in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and was granted by the Institute Research Medical Ethics Committee of Central Hospital affiliated to Shenyang Medical College.Female nude mice (4–5 weeks old) were purchased from Animal Care and Use Committee of Dalian Medical University Ltd., and kept under sterile specific-pathogen-free (SPF) conditions. 1 × 102 test, as appropriate. Survival curves were estimated by the Kaplane-Meier method. The log-rank test was used to determine the statistical differences between survival curves. All statistical analyses were performed using GraphPad Prism V5.0  and SPSS 19.0 . A two-tailed P < 0.05 was considered as statistically significant while P < 0.01 was very significant.The significance of differences between groups was assessed by Student’s t-test, one-way ANOVAs or χThe expression of DANCR in collected 95 cases of osteosarcoma tissues and matched para-tumor tissues were determined by applying of quantitative real-time PCR (qRT-PCR) as normalizing to GAPDH. As the results presented in Fig. Since DANCR was highly expressed in osteosarcoma and was correlated with tumor size and distant metastasis in clinical cases, we wondered about the function DANCR might act on osteosarcoma cells’ proliferation and metastasis. Up-regulation and down-regulation of DANCR by transfection of DANCR-shRNA and pcDNA-DANCR respectively were confirmed by qRT-PCR Fig. -b. Then,Increasing evidences demonstrated that lncRNAs regulated various downstream genes to exert multiple functions. According to our and other previous researches, ROCK1 was closely related to osteosarcoma cells’ proliferation and migration/invasion , 18. HerLately, researches on the competitive endogenous RNA (ceRNA) theory between lncRNAs and miRNAs are the hotspots in the field of noncoding RNAs. Therefore, we specuated whether DANCR might crosstalk with any potential miRNAs in dependence of the same mechanism in osteosarcoma. Firstly, an online lncRNA prediction software (DIANA-LncBase) was applied to predict the potential miRNAs that might interact with DANCR. Among the top 18 miRNAs of which the predictive binding scores were higher than 0.9 Additional file 2:Figure S2. The silenced efficiencies of DANCR and ROCK1 knock down plasmids were qualified by a qRT-PCR assays, and DANCR shRNA-01 as well as ROCK1 shRNA-01 were selected in the following RNAi experiments. (JPG 722 kb)Additional file 3:Figure S1. DIANA TOOLS was applied to predict the potential miRNAs that might interact with DANCR, and the top 18 miRNAs were demonstrated for their higher theoretical binding scores (higher than 0.900). (JPG 3899 kb)


Plants lack the mechanisms needed for adaptive immunity and thus rely entirely on innate immunity to protect themselves from pathogens and pests . Their iComplex and overlapping networks of signal transduction events mediate and integrate the induction of the defense responses that comprise PTI and ETI . SignaliMolecular dissections of successful plant pathogens have revealed that a common strategy for overcoming plant defenses is the production of chemicals and/or effector proteins that can enter inside host cells and interfere with the signaling events responsible for PTI and ETI . A majorPLOS Genetics, Hillmer et al. [Arabidopsis. The study, which integrates mutational analysis, transcript and hormone profiling, and statistical modeling, reveals that the PTI signaling network is highly buffered against interference. In other words, overall transmission of signaling through the network persists even when major components are interrupted, for example by mutations or by the actions of pathogen effectors . Two hormones (SA and JA) and 5,259 genes exhibited responses to flg22 that were statistically significant and also significantly perturbed in at least one genotype. Signal allocation analysis  and 30% were even more deeply buffered, because they remained unaffected by any double mutation.The highly buffered nature of the immune response network provides it with a strong ability to resist interference by pathogen effectors or toxins that may target individual components of the network . Only paAdditionally, the buffering of the network makes it resistant to accurate dissection by conventional genetic approaches that rely on the isolation of single gene mutations. As pointed out by Hillmer et al. , a more A buffered host immune system has important implications for pathogen evolution. If the function provided by the host target of an effector is buffered, then selection for retaining that effector gene in the pathogen genome will be greatly weakened. Similarly, a mutation that enables an effector to acquire a new target will not be selected for if the function of that target is buffered. Thus, in military terms, host plants can confuse pathogens’ weapons development programs by effectively hiding the damage caused by existing and new weapons.The same consideration also affects researchers’ ability to identify key pathogen virulence genes—if the host target of a virulence gene is buffered, then the pathogen gene will appear to be dispensable; this may explain numerous reports of pathogen genes that have obvious virulence-related functions but appear dispensable when individually deleted or silenced ,10. TillArabidopsis defense signaling network, namely the response to flg22. Considering the vast complexity of pathogen molecules exposed to the plant during a natural infection, as well as molecules originating from other members of the plant’s microbiome, much remains to be learned about the way that the immune network integrates a plethora of inputs.Although admirably comprehensive in its design and scope, the study by Hillmer et al.  still ha


Aplysia californica buccal ganglia and compared the water ADCs at cellular and ganglia levels before and after neuronal activation induced by perfusion with a solution containing dopamine. Neural cell swelling, evidenced from optical microscopy imaging, resulted in an intracellular ADC increase and an ADC decrease at ganglia level. Furthermore, the intracellular ADC increase was found to have a significant positive correlation with the increase in cell size. Our results strongly support the hypothesis that the ADC decrease observed with DfMRI upon neuronal activation at tissue level reflects activation-induced neural cell swelling.Diffusion functional magnetic resonance imaging (DfMRI) has been proposed as a method for functional neuroimaging studies, as an alternative to blood oxygenation level dependent (BOLD)-fMRI. DfMRI is thought to more directly reflect neural activation, but its exact mechanism remains unclear. It has been hypothesized that the water apparent diffusion coefficient (ADC) decrease observed upon neural activation results from swelling of neurons or neuron parts. To elucidate the origin of the DfMRI response at cellular level we performed diffusion MR microscopy at 17.2 T in  BOLD-fMRI, which relies on the neurovascular coupling hypothesis and does not reflect neuronal activity directly4, may fail in certain conditions which impair neurovascular coupling5. On the other hand, DfMRI is thought to be more directly linked to neuronal activation, as the diffusion MRI signal is exquisitely sensitive to minute changes occurring in the tissue microstructure upon various physiological or pathological changes6. Studies on rodents have evidenced that while the BOLD fMRI response is abolished by blocking the neurovascular response the DfMRI response is maintained, strongly suggesting that the DfMRI signal is not of vascular origin and that its mechanism differs from that of BOLD7. In addition, the DfMRI response is faster (time to reach the activation peak and time to return to baseline) than the hemodynamically driven BOLD signal response, as revealed by visual stimulation experiments in human subjects8. While these results suggest that DfMRI is a more direct approach for functional imaging than BOLD-fMRI, the exact origin of the DfMRI response remains unclear.Diffusion functional magnetic resonance imaging (DfMRI)12 and that neural swelling is one of the responses associated with neural activation19, it has been hypothesized that the decrease in the water ADC observed during neural evoked responses would originate from the dynamic swelling of neurons or neuron parts, in line with the so-called neuromechanical coupling hypothesis6. To investigate this hypothesis at cellular level, we performed diffusion magnetic resonance (MR) microscopy studies on the Aplysia californica buccal ganglia using an ultra-high magnetic field MRI (17.2 tesla). Aplysia californica is a key animal model in the neuroscience field20. The main advantage of using the buccal ganglia of Aplysia californica is the large size of its neurons which can be resolved and identified by MR microscopy22. Earlier reports showed that the water ADC decreased at the tissue level and increased inside neuron bodies when the cell was exposed to swelling inducers such as hypotonic solution or ouabain (an inhibiter of Na+/K+ pumps)11. In addition, the Aplysia neuronal preparation allows the investigation of neuronal activation in the absence of blood and neurovascular coupling effects. While the DfMRI signal acquired with a low degree of sensitization to diffusion  in the mammalian brains contains a residual BOLD signal23, the absence of blood in the excised Aplysia ganglia guarantees the absence of any BOLD effect in the DfMRI signal acquired in this study. As electrophysiological24 and manganese enhanced MRI (MEMRI) studies22 have previously shown that dopamine promotes neural activation in the Aplysia buccal ganglia, we investigated water ADC changes induced by dopamine application using high resolution DfMRI. The associated neuronal swelling was demonstrated using optical microscopy.Based on earlier reports that the water apparent diffusion coefficient (ADC) decreases in relation to cell swellingAt first, cell swelling was observed by optical microscopy. Representative images of neuron B2 of the buccal ganglia showing cell swelling induced by dopamine stimulation are shown in Fig. −3 mm2/s (ASW) to 0.92 ± 0.02 (dopamine) and from 1.02 ± 0.01 (ASW) to 0.81 ± 0.01 (dopamine), respectively. On the other hand, the ADC averaged over neuron bodies was significantly increased by dopamine stimulation  to fix the ganglia in the same position with respect to the radio frequency (RF) coil during perfusion; 3) to compare the DfMRI signals (water ADC) of the buccal ganglion neurons before and after perfusion with the dopamine neurotransmitter without removing the sample from the magnet bore. Our results show that dopamine stimulation induces an intracellular ADC increase and an ADC decrease in the whole ganglion. These ADC changes were significantly correlated with neuronal volume changes induced by dopamine activation. Our results are consistent with a previous report showing that the water ADC decreased at ganglia tissue level and increased inside neuron bodies after changes in neuronal bodies induced by extra-physiological exposure to hypotonic shock and ouabain11.In this study, we have developed a perfusion system for performing MR microscopy studies using a 17.2 T MRI scanner for the following three purposes: 1) to keep the Aplysia buccal ganglia24. In our current study, optical microscopy measurements demonstrated that the neurons’ cell body diameters volumes were increased by dopamine stimulation, in agreement with earlier reports demonstrating that neural swelling is associated with neuronal activation27. Moreover, as a confirmation of cell viability, we observed that the volume of swollen neuronal bodies decreased back after washing out the dopamine solution.Based on previous MEMRI and electrophysiological studies, dopamine induces neural activation of the 11, the mechanism of the ADC decrease at ganglia or tissue level upon cell swelling remains putative at this stage. Possible explanations include an increased tortuosity within the reduced extracellular space30 or an increase of the neuron membrane surface leading to a volumetric increase in the water molecular layer bound electrostatically to the membranes and adjacent macromolecules31. In our MR microscopy study, the ADC change in subcellular compartments, such as nucleus, cytoplasm, and cellular membrane, could not be assessed due to low signal-to-noise ratio (SNR) and limited spatial resolution. A previous study revealed, however, that ADCs and volumes of nucleus and cytoplasm were increased under hypotonic stress and ouabain exposure11. Much higher spatial resolution studies will be necessary to elucidate whether water diffusion near cell membranes is lower than within the cytoplasm.While the intracellular ADC increase likely reflects a dilution effect in cytoplasm upon cell swellingAplysia neural tissue shares the feature observed in the mammalian brain, namely that neuronal activation is accompanied by cell swelling, however without the confounding effects of blood flow. Our results clearly show that this activation-driven cell swelling leads to a decrease of water diffusion at the neural tissue level, as we have hypothesized earlier33, although water diffusion increases inside swelling neurons, in line with the neuromechanical coupling hypothesis6.Although those results may not be directly extrapolated to the mammalian brain, the Aplysia californica neuronal tissue diffusion MRI microscopy revealed a water diffusion (ADC) increase inside neuron bodies, which correlated positively with cell swelling, and a decrease at ganglia tissue level, in agreement with early findings on the mammalian brain. Our study confirms the hypothesis that the water ADC decrease observed in neural tissue is strongly correlated to activation-induced cell swelling.Upon stimulation with dopamine of Aplysia californica  were used in total. On three samples we performed only MR microscopy while on the other four we also acquired optical images. Aplysia were anesthetized by injection of an isotonic magnesium chloride solution . The animals were dissected on a sylgard-coated petri dish  filled with cold ASW  to isolate the buccal ganglia. All chemicals were purchased from Sigma-Aldrich .Seven juvenile 24.Optical microscopy imaging was performed at room temperature before the MR microscopy study. The isolated buccal ganglia were placed inside a glass chamber  provided with two silicon perfusion tubes attached at each side. The ganglia were fixed inside the chamber using hand-made stainless steel anchors made out of 0.1 mm thick threads placed at 1 mm spacing. The ASW solution was supplied from one side of the chamber at a flow rate of 2.5 ml/h using a syringe pump  and aspirated from the other side at approximately identical flow rate using a peristaltic pump  to keep the same depth of ASW solution. The ASW solution containing dopamine  was prepared by adding 0.05 mM dopamine hydrochloride  and 0.05 mM ascorbic (antioxidant) acid . The time course of optical microscopy experiments is described in Fig. dopamine − XASW)/XASW × 100, where Xdopamine and XASW are the cell axes or volume after dopamine stimulation after and ASW washout, respectively.Measurements of cells’ long and short axes Fig.  were perThe buccal ganglia which were used in the optical microscopy study were transferred from the glass chamber used for optical microscopy imaging to the perfusion system for MR microscopy Fig. . The bil2, Matrix = 80 × 32, Number of slices = 8, Slice thickness = 0.1 mm Resolution = 50 × 50 × 100 μm3, diffusion time δ = 2 ms, ⊿ = 7 ms, 4 b-values = 50, 100, 200, 400 s/mm2, Diffusion gradient encoding direction = , Number of averages = 8, Scan time per b-value = 10 min. A first set of DfMRI images was acquired after 30-min perfusion with dopamine solution. The acquisition lasted for 40 min which coincides with the duration of dopamine-induced neural response24. A second set of DfMRI images was acquired after washout with ASW for 60 min, knowing that the ASW reached the ganglia in approximately 30 min after starting the perfusion. Although ganglia oxygenation was ensured through continuous perfusion, as an extra precaution the DfMRI images under dopamine  + ASW were obtained before those under pure ASW to avoid potentially confounding factors related to hypoxia induced ischemia. Finally, T2 weighted structural images were also acquired with the following parameters: 3D-relaxation enhancement (RARE) sequence; TR = 2000 ms, TE = 16 ms, RARE factor = 4, FOV = 0.4 × 0.16 × 0.3 cm3, Matrix = 160 × 64 × 60, Resolution = 25 × 25 × 50 μm3, Number of averages = 12, Scan time = 6 h 24 min. The total duration of the experiments from the dissection of the Aplysia until the final MR image was approximately 12.5 h. This duration is well within the ganglion expected survival time of 48 h22.MR microscopy was performed at 19 °C on a 17.2 T MRI scanner  equipped with 1 T/m gradients. The radio frequency (RF) transceiver was a home-built surface micro-coil consisting of a single loop (inner diameter of 4.2 mm). The time course of MR microscopy experiments is described in Fig. 22. The MR signals for each b-value were extracted from the DfMRI images and averaged within each ROI for ADC estimation. The ADCs were calculated by fitting S/So(b) = exp(-bADC), where S/So(b) is the signal attenuation at each b value. The ADCs were estimated individually for each neuron body and each bilateral ganglion. The ADC was also calculated in an ROI placed in the ASW region. The significance of the ADC changes induced by dopamine stimulation was quantified using a bilateral paired Student’s t-test, performed on the series of ADC values before and after dopamine stimulation. For neurons B1, B2, B6, and B9, the relative change in ADC was calculated as (ADCdopamine − ADCASW)/ADCASW × 100, where ADCdopamine and ADCASW are the ADC values obtained after dopamine stimulation and after washout, respectively. The relative change in ADC, cell axes, and volume were obtained from 4 neurons  in the ganglia examined by both optical microscopy and MRI. Then, the correlation between the relative increase in ADC and the relative change in cell axes and volume were quantified using the Pearson product-moment correlation coefficient. To provide some indication of the signal level in our DfMRI image data without dopamine solution, signal-noise ratios (SNRs) were estimated by dividing the average signal in whole ganglion region by the standard deviation in the noise region. Averaged SNR values (mean ± sem) were 11.17 ± 0.92 for b50, 10.86 ± 1.20 for b100, 8.98 ± 0.72 for b200 and 7.05 ± 0.84 for b400 (n = 7).All processing was performed using Matlab . For each sample, the structural RARE image and the DfMRI images were co-registered to the same position. For segmentation of cellular and ganglia region, the structural RARE images were displayed as successive slices in Matlab. The neuron bodies  and ganglia region of each side were manually segmented on each slice, based on signal contrast of the RARE image. The positions of these large neurons were easy to identify in the optical and MR microscopy images


The Nobel prize in Chemistry for 2016 was awarded to Jean Pierre Sauvage, Sir James Fraser Stoddart, and Bernard (Ben) Feringa for their contributions to the design and synthesis of molecular machines. oF1 ATPase, and the flagellar motor, function. The ability to “tinker” with separate components of molecular motors allows asking, and answering, specific questions about mechanism, particularly with regard to light driven vs. chemistry driven molecular motors.The Nobel prize in Chemistry for 2016 was awarded to Jean Pierre Sauvage, Sir James Fraser Stoddart, and Bernard (Ben) Feringa for their contributions to the design and synthesis of molecular machines. While this field is still in its infancy, and at present there are no commercial applications, many observers have stressed the tremendous potential of molecular machines to revolutionize technology. However, perhaps the most important result so far accruing from the synthesis of molecular machines is the insight provided into the fundamental mechanisms by which molecular motors, including biological motors such as kinesin, myosin, F The work for which the 2016 Nobel prize in chemistry was awarded has begun to change this state of affairs by presenting inspired synthetic routes to facilely create a host of interlocking structures known as catenanes5 that showed how omnipresent thermal noise can be exploited at the nano-meter scale filtered into the synthetic chemistry community. This insight led to synthesis of molecular machines with the ability to use external energy to power the cyclic motion necessary for creation of true molecular motors. Detailed study of different mechanisms – energy ratchets and information ratchets6 – by which this occurs has led to important gains in understanding how biomolecular machines work. Specifically, the “power-stroke”, presented as the mechanism by which most ATP and ion gradient driven molecular motors work in almost all leading textbooks of biochemistry and cell biology, is shown to be just plain wrong for these chemically driven motors. Instead, the key mechanism for molecular motors driven by a catalysed reaction such as ATP hydrolysis or ion transport is chemical gating in which the specificity for substrate vs. product depends on the state of the molecular motor, a mechanism known as an information ratchet.6At first, many systems operating as simple back and forth molecular switches were proudly proclaimed to be “molecular motors”. Eventually, conceptual advances27 and the other an autonomous rotor driven by catalysis of a chemical reaction from David Leigh's group.8Consider two very different designs for a molecular rotor, one, a light driven rotor from Ben Feringa's group,7 uses optical energy to populate an unstable state by a directional rotation followed by a thermal relaxation to a stable state in a cyclic fashion resulting in completion of a rotation about a carbon–carbon double bond. In cis isomer causes clockwise rotation as the molecule is promoted to an unstable (excited) state (M) trans isomer. This is followed by thermally activated helix inversion to the stable (P) trans isomer. Absorption of a photon by the (P) trans isomer leads to clockwise trans–cis isomerization to the unstable (P) cis isomer followed by thermally activated helix inversion to the starting (M) cis isomer. In the excited state the rotor freely rotates on one side of the plane of the stator but is hindered from helix inversion. In the ground state the barrier for helix inversion is lower than that for rotation about the carbon–carbon double bond, with the net effect being continuous rotation of the molecule when illuminated.The light driven motor described by Feringa and colleaguesG, between unstable and stable states is determined by the intramolecular strain when the methyl group on the rotor is in the equatorial vs. axial positions. It is key that this ΔG be relatively large to ensure directional rotation. The relative likelihood of completing a clockwise vs. counter-clockwise rotation isG is, as are all energies in this paper, expressed in units of the thermal energy.The stability difference, Δ3 were driven by cycles of illumination – heating – cooling – illumination, recent motors have been designed to allow for continuous operation at MHz rotation frequencies at a fixed temperature.7 Similar ideas arise in stochastic conformational pumping mechanisms9a,b where time periodic external driving induces net cycling if some transitions involve a significant ΔG. The mechanical process in which the energy ΔG is dissipated in the environment is very reasonably termed a power stroke. Let us compare the light driven motor that requires a power stroke with a motor driven by catalysis of a chemical reaction.While early light driven motors8 by David Leigh's group using catenanes, a type of the mechanically interlocked molecules held together by mechanical bonds for which Jean Pierre Sauvage1 and Sir Fraser Stoddart2 were awarded the Nobel prize. The rotor described by Wilson et al.8 incorporates the main features of biological molecular motors – chemical catalysis, gating of the binding and release of substrate and product depending on the mechanical state of the motor, and conformational changes involving mechanical motion.Most biomolecular motors use energy from ATP hydrolysis or transport of protons from high to low electrochemical potential across a membrane rather than from light or from external modulations. A longstanding challenge has been to design a synthetic motor that similarly uses chemical energy from a catalyzed reaction to drive directed motion. This goal has recently been achievedThe molecular motor, a [2]catenane, comprises two interlocked rings of different sizes. The small blue ring is free to rotate relative to the larger ring, shuttling between the two recognition sites (shown in aqua) on the larger ring, one of which is labelled by deuterium. There are two catalytic sites (hydroxyl groups) represented by the small red cones, one near each recognition site, that catalyze the conversion of Fmoc-Cl to dibenzofulvene.A kinetic cycle for the mechanism of the motor is shown in kCA > kAC and hence with  driving the transition C → A.It is very natural to imagine that, by analogy with Feringa's light driven motor, clockwise rotation of the blue ring from the perspective of the reader could be enforced by designing the system with a repulsive interaction between the bulky Fmoc protecting group (red sphere) and the dark blue ring, thereby engineering a “power-stroke” in the direct mechanical transitions such that G < 0) or repulsive (ΔG > 0). The directionality arises because the rate of attachment of the protecting group to the catalytic site is faster when the blue ring is at the distal rather than proximal recognition site (αCB > αAB). The rate of cleavage of the Fmoc group as dibenzofulvene, on the other hand, is nearly independent of the position of the ring (βBA ≈ βBC). These two kinetic conditions on the rate constants reflect the fact that installing the protecting group as Fmoc depends on the location of the dark blue ring, but that removing the protecting group as dibenzofulvene does not. The net effect of these two kinetic conditions is to make the transition C → A less likely than the transition A → C, a seemingly slight bias which is nevertheless enough to provide directionality to the rotational motion although the experimentally observed velocity is very small.This intuitive idea however is wrong – the dark blue ring shown in et al.8 and previously described “chemically driven” molecular motors and pumps which were driven by external changes of the chemical environment – redox potential, pH, substrate concentration, etc. Such externally driven motors can, and for the most part do, work by a power stroke mechanism similar to the light driven motor described by Feringa and colleagues7 and the biological pump driven by a fluctuating electric field described by Astumian et al.9aIt is essential at this point to make a very important distinction between the autonomous chemically fuelled motor of Wilson et al.8 on the other hand works under a single reaction condition where substrate (Fmoc-Cl) associates and product (dibenzofulvene) dissociates because the chemical potential of substrate is large and that of product is small. This reproduces a major feature of ATP hydrolysis driven biomolecular motors. Autonomous motors driven by catalysis of a chemical reaction cannot operate by a power stroke mechanism, and instead function as information ratchets6 where the key mechanistic design principle is the kinetic discrimination of the rate of the chemical reaction depending on the mechanical state of the motor by which αCB > αAB.The motor described by Wilson 10 The rate constants must obey the constraint and Keq is the equilibrium constant for the catalyzed reaction. An additional constraintαBA, βBA, αBC, βBC are first order “off” rate constants and the ratio between any two of them is independent of the free energies of the states A, B, and C and can be written as the exponential of the difference between two activation energies. The coefficients αAB, βAB, αCB, βCB on the other hand are pseudo-first order rate constants into which the bulk concentrations [Fmoc-Cl] and [Et3N], for the α's, and [dibenzofulvene], [Et3NHCl], and [CO2], for the β's, have been subsumed. The two relationships eqn (2) and (3) are thermodynamic identities that follow from the principle of microscopic reversibility.11In r > 1 the blue ring rotates clockwise, and when r < 1 the blue ring rotates counter-clockwise, from the perspective of the reader.The directionality of the molecular motor in ad libitum for mathematical convenience rather than for thermodynamic consistency. This approach often leads to plausible but incorrect conclusions. As an example, consider the assignment αAB = αCB = βBA = βBC = γ eμ/4 and αBA = αBC = βAB = βCB = γ eμ/4–, where γ sets the time scale for the chemical transitions relative to the mechanical transition. With these expressions we immediately derive as the ratio of clockwise to counter-clockwise net rate constants the expression r = eG2Δ, seeming to suggest a critical role for the “power-stroke” just as for light driven motors. However, while this assignment for the rate constants is consistent with eqn (2), it is not consistent with eqn (3) and is thus fundamentally incorrect, being tantamount to ignoring both the steric interaction by which αCB > αAB, and any energetic interactions by which kCA ≠ kAC and which must be reflected in the α's and β's according to eqn (3). The negligibility of energetic interactions in the chemical rate constants is absolutely impossible even very far from equilibrium as pointed out first in the context of ion pumps.9aA regrettable tendency in many recent papers is to point out that chemically driven motors “operate far from equilibrium” and then assign rate constants q = αBAβBC/αBCβBA is a ratio of “off” rates and parametrizes the chemical gating. Thus we see that r is independent of ΔG. The term Luc arises from un-coupled cycles12 and setting Luc = 0 represents the optimal directionality of the motor. Not surprisingly, if Δμ = 0 then r = 1. More revelatory is the fact that if q = 1 then r = 1 irrespective of the value of Δμ, i.e., if there is no chemical gating there is no coupled rotation despite continual chemical dissipation. Using the constraints eqn (2) and (3), with αCB > αAB and βBA ≈ βBC we have for the motor in q < 1 and hence that r < 1, irrespective of ΔG.When we carry out the slightly more mathematically complex imposition of the constraints of both eqn (2) and (3) we instead obtain the correct expressionG for the mechanical stroke must be positive in the direction of motion. Howard comments that “If the structural states differ by a distance d, then the power stroke can do work against an external loading force F provided that F·d ≤ ΔG, where ΔG is the decrease in free energy between the two chemical states”.13 and Eisenberg and Hill claim that the maximum efficiency can be no higher than ΔG/Δμ, where Δμ is the free energy released by the driving chemical reaction.14 These assertions regarding the power stroke, based on the incorrect assumption that the “power stroke” must be energetically downhill (exergonic), are just wrong for chemically driven motors. In fact, the thermodynamic properties of a chemically driven motor – efficiency, stoichiometry, stopping force – do not depend on ΔG at all.15 Perhaps having different stabilities for A and C affords a kinetic advantage? This idea, too, is incorrect. While the rate of the mechanical transition may indeed be enhanced when ΔG ≠ 0, the net rate of the chemically driven transitions between A and C through state B is inexorably decreased when the free energies of A and C are different from one another. In fact, the rate of rotation is optimized when the basic free energies of all states are identical, consistent with Jeremy Knowles theory for evolutionary optimization of enzyme catalytic function.16There is a clear presumption in the literature that the Δq, eμ–Δ ≪ 1 so that q eμ–Δ can be neglected in eqn (5). We recognize two distinct regimes of coupled transport.12 When gating is very strong (q ≪ eμ–Δ) the motor operates in the thermodynamic control regime, also known as the tight-coupling regime, in which every conversion from substrate to product is accompanied by a counter-clockwise rotation, and application of torque sufficiently strong as to cause clockwise rotation induces conversion of product to substrate. An example is the F1 ATP synthase where ATP hydrolysis drives rotation in one direction. When an external torque is applied to force rotation in the opposite direction ATP is synthesized.17Now consider the case that μ–Δ ≪ q the motor is regulated by the kinetic gating. In this regime the application of a sufficiently strong torque to cause a motor to move backward results in an increase in the rate of conversion of substrate to product, not in the conversion of product to substrate. This latter behaviour, surprising to many in the field of biological motors, was predicted by Astumian and Bier in 1996  as shown schematically in et al.8 Then an applied oscillating electric field  influences the motion25 and can drive the catalysed reaction away from equilibrium, maintaining a steady-state chemical potential difference26 where  is the dipole moment difference between the states. Such coupled devices could in principle pave the way to new synthetic approaches for high energy  compounds as well as for new sensors.Molecular motors can be designed to interact with a variety of inputs – light, chemical reactions, electric fields, external changes in pH and redox potential, 29 and self-organized assembly30 in soft condensed matter.There are of course approaches other than those based on a synthetic chemistry perspective, including those grounded in surface science431 ‘In the IUPAC definition32 of the principle of microscopic reversibility, “In a reversible reaction, the mechanism in one direction is exactly the reverse of the mechanism in the other direction. This does not apply to reactions that begin with a photochemical excitation”, light driven processes are explicitly excluded from the constraint of microscopic reversibility’.The very different design principle necessary for a motor driven by chemical catalysis – gating – in comparison to that for a light driven or externally driven motor – power stroke – highlights the importance of microscopic reversibility for understanding the mechanism of chemically driven molecular motors.This is because in the absorption of a photon, and its reverse – stimulated emission – there is conversion between energy in one degree of freedom, the photon, and another single degree of freedom, the energy of the photo-chemically active molecule. In contrast, a thermal activation process, including binding of a high free-energy substrate, involves conversion between many degrees of freedom in the bath and only one (or perhaps a few) degrees of freedom in the molecule, irrespective of how far from equilibrium the reaction may be.33 When driven by external changes of the redox potential it is essential that the energy of an intermediate state depend on the oxidation state34 – the pump acts as an energy ratchet. On the other hand, theoretical analysis of a model in which the same process is driven by a catalysed chemical reaction shows that the energy of the intermediate state is irrelevant and the sole determinant of the efficacy of pumping is chemical gating34 – the motor functions as an information ratchet.The importance of microscopic reversibility is also well illustrated in the context of a synthetic molecular pump.e.g. ATP hydrolysis) or osmotic energy  into mechanical motion and the performance of work. The recently synthesized molecular motor driven by chemical catalysis,8 and the concomitant theoretical analysis, shows unequivocally that this mechanism is incorrect. Strain (or other energetic interactions between components) stored in a molecular motor, while of the essence for light driven motors and for motors driven by external modulation of the chemical environment, is irrelevant for molecular motors driven by chemical catalysis, including ion transport.The concept of a power stroke – a viscoelastic mechanical relaxation from a high energy state to a low energy state – is given in almost all textbooks of biochemistry and cell biology to explain how molecular machines such as myosin, kinesin, and the flagellar motor convert chemical energy (In the molecular world, equilibrium is a dynamic state in which all possible motions are explored with equal probability in the forward and reverse directions. The key principle for designing molecular motors that are driven by chemical catalysis is to use the chemical energy to selectively prevent unwanted motion, rather than to cause the desired motion. Any description of the effect of the catalysed reaction in terms of “deposition of chemical energy” or as causing violent kicks or judo throws is wrong and highly misleading.st Solvay Conference on Chemistry held in Brussels in 2007 (see The talk given by Richard Feynman in 1959, “Plenty of room at the bottom”, represents a defining moment in the quest for making molecular motors, as does the 212007 see . At this


Yersinia pestis can lead to pneumonic plague, which without treatment is inevitably fatal. Two novel formulations of liposome-encapsulated ciprofloxacin, ‘ciprofloxacin for inhalation’  and ‘dual release ciprofloxacin for inhalation’  containing CFI and ciprofloxacin solution, are in development. These were evaluated as potential therapies for infection with Y. pestis. In a murine model of pneumonic plague, human-like doses of aerosolized CFI, aerosolized DRCFI or intraperitoneal (i.p.) ciprofloxacin were administered at 24 h (representing prophylaxis) or 42 h (representing treatment) post-challenge. All three therapies provided a high level of protection when administered 24 h post-challenge. A single dose of CFI, but not DRCFI, significantly improved survival compared to a single dose of ciprofloxacin. Furthermore, single doses of CFI and DRCFI reduced bacterial burden in lungs and spleens to below the detectable limit at 60 h post-challenge. When therapy was delayed until 42 h post-challenge, a single dose of CFI or DRCFI offered minimal protection. However, single doses of CFI or DRCFI were able to significantly reduce the bacterial burden in the spleen compared to empty liposomes. A three-day treatment regimen of ciprofloxacin, CFI, or DRCFI resulted in high levels of protection . This study suggests that CFI and DRCFI may be useful therapies for Y. pestis infection, both as prophylaxis and for the treatment of plague.Inhalation of  Yersinia pestis is the causative agent of plague, a disease responsible for the death of an estimated 200 million people through devastating pandemics such as the Black Death . In the Middle Ages, the bodies of dead plague victims were thrown over the walls of a besieged Caffa city by the Tartars , there is the potential for person to person spread and the mortality rate of plague is high . In addition to usefulness for public health indications these inhaled formulations may offer an advantage, compared to an oral or intravenous formulation, in the event of a deliberate release of an aerosolized BW agent. If administered early enough, inhalation of CFI/DRCFI could be used to target the initial site of infection  and prevent establishment of infection. As there is considerable clinical data available, this product could be repurposed as a medical countermeasure for plague under the FDA animal rule following pivotal efficacy studies in animals. Therefore, to determine whether this formulation offers an advantage over therapies currently licensed for plague we have herein sought to compare its efficacy to ciprofloxacin administration in humans .Both formulations have been evaluated in human clinical trials and DRCFI is currently in phase 3 clinical trials for Francisella tularensis Schu S4 infection in a murine model  supplemented with 0.02% haemin or in BAB broth . This strain has a minimum inhibitory concentration of <0.063 mg/L for ciprofloxacin  Act (1986). Female BALB/cAnNCrl (BALB/c) mice, 8–10 weeks old and 20 g (±4 g), were obtained from Charles River Laboratories (UK) where they were implanted with a sub-cutaneous Pico transponder , enabling individual animals to be traced throughout the study. Mice were caged in groups of 5 in polypropylene cages with a stainless steel mesh cover, integral water bottle holder and diet hopper in an ACDP (UK) level 3 rigid wall isolator, complying with British Standard 5726. Corn cob grade 10/14  was used as a nesting material and a range of environmental enrichment was used throughout the study including cellulose dome home and aspen wood wool (IPS). Husbandry conditions consisted of a 12-h light-dark cycle , 19 to 23°C and 45 to 65% relative humidity. Irradiated Labdiet rodent diet 5002 (IPS) and water were freely available during the study. Mice were allowed to acclimatize to their home cage environment for five days before challenge.®, Bayer), liposomal ciprofloxacin for inhalation (CFI)  and dual release ciprofloxacin for inhalation (DRCFI)  were used in this study. Ciproxin, 2 mg/ml solution for infusion, and CFI , 50 mg/ml liposome-encapsulated ciprofloxacin (expressed as hydrochloride) were used as supplied. DRCFI , 35 mg/ml ciprofloxacin hydrochloride, was produced by mixing equal volumes of CFI and ciprofloxacin hydrochloride solution for inhalation (20 mg/ml)  immediately prior to use.Ciprofloxacin (Ciproxinn = 30) or DRCFI (n = 30) aerosols for 20 or 15 min, respectively. Not all of drug in the nebulizer was aerosolized, for example following a 20 min exposure approximately 4 ml of CFI was aerosolized with 2 ml remaining in the nebulizer. The majority of the drug passed through the mouse exposure chamber and was collected in the impinger placed after the exposure chamber (the air is moving through the exposure chamber at 6 L/min whereas mouse respiration is 20 ml/min).Mice were administered either ciprofloxacin, CFI, or DRCFI. Aerosolized CFI and DRCFI were administered using the inhalational therapy system (ITS) , with 6 n = 30) via the intraperitoneal (i.p.) route. The mice (n = 3 /time point/group) were culled at 1, 10, 20, or 30 min and 1, 1.5, 2, 4, 8, 12 h following ciprofloxacin administration and 1, 15, or 30 min and 1, 2, 4, 6, 10, 18, or 24 h following DRCFI or CFI administration. Blood sampling points were chosen based upon the short half-life of ciprofloxacin and longer half-life of CFI  was administered to mice (e of CFI . Blood aThe concentration-time profile of ciprofloxacin in blood and lung homogenate was determined using liquid chromatography mass spectrometry (LC-MS) as previously described . Non-comin vitro measurements of droplet size distribution and emitted dose from the jet nebulizer used clinically  compared to an estimated retained dose of 1.9 × 104 CFU  and the second experiment determined the impact of delaying the initiation of therapy until 42 h post-challenge (the treatment study). In these studies, the pparatus . Mice weculation  using Guculation ). The ae. pestis . Exposin 104 CFU .To determine the bacterial burden of mice administered therapy as prophylaxis or treatment, groups of 5 mice received a single dose of i.p. ciprofloxacin, i.p. PBS, aerosolized empty liposomes, aerosolized CFI or aerosolized DRCFI  at 24 or 42 h post-challenge. A 30 mg/kg dose of i.p. ciprofloxacin administered produces a drug exposure which is similar to the drug exposure observed in humans following a 500 mg dose of oral ciprofloxacin. The empty liposome control was administered using the ITS  with 6 mY. pestis challenge, groups of 10 mice were administered therapy from 24 or 42 h post-challenge. The groups received a single dose of antibiotic or 3 days of therapy; the mice receiving 3 days of therapy were dosed with ciprofloxacin twice daily whereas CFI, DRCFI and empty liposome control were administered once daily to model the human dosing regimen  of approximately 1 (Y. pestis colonization data (transformed to the logarithm of 10) would be around 0.5 , statistical analysis was conducted with SPSS  and power calculations were conducted with SPSS SamplePower (v3.0 IBM). In previous mately 1 . We estiound 0.5 . The higTable 1).The AUC/MIC (area under the curve/minimum inhibitory concentration) measurement has been shown to be the PK parameter most strongly associated with the efficacy of ciprofloxacin in animal models , althougTable 1). In this study, exposure to DRCFI resulted in a lower inhaled ciprofloxacin dose (see Table 1). Therefore, in order to obtain a similar dose of CFI and DRCFI, the length of exposure to DRCFI was increased. Table 1 shows the PK parameters of the shorter DRCFI exposure and the PK of the longer exposure simulated by the one-compartment models.Inhalational drug scaling between humans and animals relies upon attempting to deliver the same amount of drug to the lung per kg of body weight. In Phase II human clinical trials with DRCFI, 6 ml (210 mg) was nebulized, the corresponding dose delivered to the lung is approximately 0.8 mg/kg, assuming a 50-kg patient  which is similar to the dose of CFI delivered to mice  with a greater than 10-fold decrease in the plasma total exposure (as demonstrated by the AUC). Conversely, and as may be expected, inhalational administration of CFI or DRCFI produces a higher Cmax and extended half-life in the lung  resulting in a 20-fold higher overall exposure in the mouse lung compared to ciprofloxacin delivered by the intraperitoneal route.The PK parameters of CFI and DRCFI demonstrate the different distributions of ciprofloxacin following administration of these therapies compared to ciprofloxacin administered by the intraperitoneal route. As previously demonstrated in humans , in the Y. pestis infection, bacteria could be detected (∼2 × 103 CFU/ml) in the lung of all animals at this time, but no bacteria could be detected in the spleen or blood. The control mice (treated with PBS or empty liposomes) succumbed to the infection by day 4 post-challenge whereas a single dose of ciprofloxacin resulted in 60% of mice surviving the lethal challenge. However, a single dose of CFI afforded significantly better protection than ciprofloxacin with all mice surviving until the end of the experiment at day 35 (P = 0.029). There was no significant difference between DRCFI and ciprofloxacin or CFI, with a single dose of DRCFI treatment resulted in 80% survival. When therapy was administered for 3 days, all therapies provided full protection .First, an early initiation of therapy was investigated, with therapy administered from 24 h post-challenge. Using the Balb/c mouse model of aerosolized Figure 3). No bacteria were isolated from the spleen of any antibiotic treated animal and, when compared to controls, this represents significant reductions in spleen bacterial loads (P = 0.023). CFI and DRCFI treatment also reduced the lung bacterial loads to lower than the limit of detection, significantly different to the empty liposome control (P = 0.023). Intraperitoneal ciprofloxacin did reduce the lung bacterial load compared to controls treated with intraperitoneal PBS (P = 0.023) , however, bacteria could still be detected in 2 out of 5 mouse lungs. There were no significant differences in bacterial loads among the different therapies.To further differentiate between CFI, DRCFI, and ciprofloxacin, the bacterial burden in lungs and spleens of infected mice was determined at the onset of clinical signs in the control animals (those treated with PBS or empty liposomes). The bacterial load was high in both lungs and spleens in the control groups whereas all three antibiotics had profound effects on bacterial load  and bacteria could be detected in the blood and spleen of 3 out of 5 mice with an average bacterial burden of ∼3 × 104 CFU/ml and ∼3 × 105 CFU/organ, respectively. All control mice succumbed to infection by day 4 except for 1 mouse dosed with empty liposomes which survived until day 11 post-challenge. A single dose of all three therapies did increase time to death compared to the controls (P < 0.001) but did not offer a high level of protection. A single dose of ciprofloxacin resulted in 60% survival, and a single dose of CFI or DRCFI resulted in 20 and 10% survival, respectively. Here, ciprofloxacin offered significantly better protection than DRCFI (P = 0.016) but not when compared to CFI (P = 0.060) . However, impressively, three days of antibiotic therapy offered high levels of protection with 100, 90, and 90% survival afforded by ciprofloxacin, CFI, and DRCFI, respectively .The efficacy of CFI and DRCFI against systemic Y. pestis was examined when therapy was delayed until 42 h post-challenge. High bacterial loads were present in the lungs and spleens of the control animals. In comparison, bacterial numbers were reduced to levels below a measureable threshold in the lungs and spleen of all the mice treated with CFI and 3 of the 5 mice treated with DRCFI. A comparison of bacterial numbers in the spleen and lung indicated that the bacterial load had been reduced by all three therapies when compared to their relevant control groups (P = 0.023). Furthermore, both CFI and DRCFI treatment significantly reduced bacterial burden in the lung compared to ciprofloxacin treated mice (P = 0.023). CFI treatment also significantly reduced the spleen bacterial burden compared to ciprofloxacin treatment (P = 0.023) . However, there was no significant difference in spleen bacterial burden following DRCFI or ciprofloxacin therapy. In addition, there was no significant difference in the lung or spleen bacterial burden of CFI and DRCFI treated mice.The bacterial burden at the onset of clinical signs in control animals challenged with In the event of a deliberate release or a natural outbreak of pneumonic plague, large numbers of patients and contacts may have to receive antibiotics. Due to the low numbers of natural cases, animal models are needed to evaluate the efficacy of antibiotics against pneumonic plague. The well-characterized Balb/c mouse model of pneumonic plague enables antibiotic evaluation for prophylaxis and, by delaying therapy, for treatment. In reality, those requiring treatment are likely to be in-patients in a hospital, with dedicated medical care and the potential for multiple therapies administered by medical staff. In contrast, those requiring prophylaxis are likely to have different requirements such as a therapy which can be taken without medical supervision and with very few side effects. Those receiving prophylaxis may include doctors or soldiers who need to remain active and able to perform their duties.Delivery of a potent antibiotic directly to the site of the primary infection, the lung, provides the opportunity for much higher antibiotic concentration in proximity of the infection and much lower systemic exposure to avoid systemic side-effects. Indeed, inhaled CFI has been shown to be safe and well tolerated in a number of patient populations . In addiIn this study, when CFI or DRCFI were administered as prophylaxis, at 24 h post challenge, they appear to be as good as, i.p., ciprofloxacin at decreasing the bacterial burden in the lung and spleen and thus reducing the lethality of plague. This may be explained by the PK data which demonstrates that administration of CFI or DRCFI greatly increases the overall exposure to ciprofloxacin in the lung compared to ciprofloxacin administered by intraperitoneal injection. This high concentration of ciprofloxacin could prevent the disease from disseminating from the initial site of infection in the lung.Y. pestis moves from an intracellular to extracellular pathogen, therefore although there are low bacterial numbers in the site of treatment (lung) and the leukocyte rich tissues (spleen) due to CFI treatment there may be other foci of infection enabling the disease to re-emerge. However, increasing the therapy regimen of CFI or DRCFI to three days enables the systemic infection to be successfully treated and offers a high level of protection.Delaying CFI or DRCFI treatment to 42 h post-challenge, a time point at which the infection has become systemic, also provided high levels of protection. This was somewhat unexpected as the systemic exposure to ciprofloxacin (measured by AUC in plasma) is more than 10-fold lower than ciprofloxacin delivered by the intraperitoneal route. Furthermore, CFI and DRCFI were able to reduce bacterial load in the spleen. This may be explained by the hypothesis that liposome-encapsulated antibiotics, like CFI, are taken up by macrophages in a similar manner to the phagocytosis of bacteria  and can Pseudomonas aeruginosa infections in patients with non-cystic fibrosis bronchiectasis, DRCFI appeared more efficacious than CFI in terms of reduction of bacterial burden in the sputum  Act (1986).KH, SH, JB, SA, and HA conceived the concept and designed the experiment. KH, KB, and CD conducted the experiments. KH, SA, and TL performed the data analysis. KH, HA, and JB wrote the manuscript. All the authors reviewed the draft and approved this manuscript for publication.JB is an employee and stockholder of Aradigm Corp. and the other 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.