Between December 2016 and June 2017, 19 Hepatitis A virus (HAV)-positive cases, 17 of which were among men who have sex with men (MSM) were identified in the Tel Aviv area. Seven of the 15 sewage samples collected between January and June 2017 were also HAV-positive. All sequences clustered with two of the three strains identified in the current European HAV outbreak. We demonstrate that despite an efficient vaccination programme, HAV can still be transmitted to an unvaccinated high-risk population. An ongoing hepatitis A outbreak involving 15 European countries with the first case identified in June 2016 in the United Kingdom (UK) [Hepatitis A is a notifiable disease in Israel and only 17 sporadic cases were reported between March 2013 and December 2016 in the Tel Aviv district, when the first case of the current hepatitis A outbreak was identified in a hospitalised man. A case was defined as a report of an individual who had been in the Tel Aviv health district (covering the Tel Aviv metropolitan region) with a clinical presentation of abrupt onset of hepatitis  and laboratory confirmed anti-HAV  serology. By June 2017, 19 cases of HAV infection confirmed by serology were notified to the Tel Aviv district health office . SeventeTo support the outbreak investigation, the presence of HAV sequences was assessed in sewage samples n = 23) collected between August 2016 and June 2017 as part of the routine polio surveillance programme [ collecteAll sewage samples collected between August and the end of December 2016 were HAV- negative. Seven of the fifteen samples , collected between January and June 2017, were found to be HAV-positive.Phylogenetic analysis of clinical and sewage HAV-positive samples showed that all sequences from the current outbreak among MSM in Israel and the seven positive sewage samples, clustered with either RIVM_HAV16–90 (EUROPRIDE) or VRD_521_2016 (UK/SPAIN) isolates identified in the 2016–2017 European HAV outbreak ,5.Three MSM were infected with the VRD_521_2016 (UK/SPAIN) strain and epidemiologically-linked to the same non-household venue. Moreover, sequencing results were available for three of four MSM who reported travelling in the 2 months before symptoms onset. One of them, identified with the VRD_521_2016 (UK/SPAIN) strain in December 2016 (week 52), was most likely infected in Berlin. Another adult MSM, harbouring the RIVM_HAV16–90 (EUROPRIDE) strain and diagnosed in May 2017 (week 18), reported a stay in Poland during the incubation period. The third MSM, diagnosed in late May (week 22), presented with acute hepatitis two weeks after returning from New York and carried the VRD_521_2016 (UK/SPAIN) strain. All other cases had not travelled abroad and were infected in Israel.HAV isolates from sewage also clustered with both HAV strains implicated in the ongoing European outbreak. In two of the sewage samples , both strains could be identified together. HAV sequences from past outbreaks in Tel Aviv formed a separate cluster with HAV 1b sequences , 2.Serum or plasma samples from acute hepatitis A cases positive for anti-HAV IgM were transferred to the national centre of HIV and viral hepatitis in the central virology laboratory of the Ministry of Health. Total nucleic acids were purified from 400 µL of plasma (or from 500 µL of filtered and concentrated sewage samples) and real-time PCR for HAV detection was performed as described previously . PCR andHepatitis A virus infection causes a substantial number of viral hepatitis cases worldwide. It is an acute self-limiting illness, associated with fever, malaise, nausea, anorexia and jaundice, mainly transmitted via the faecal-oral route . A two-dBetween March 2012 and March 2013, there was an outbreak in the Tel Aviv district with 75 cases of acute hepatitis, of which 73% were in non-vaccinated young men. HAV 1b was the predominant subtype identified in clinical as well as in sewage samples collected during the outbreak . In the Anti-HAV vaccine is routinely recommended by the Israeli Ministry of Health to various risk groups, including MSM . FollowiIn the past, molecular and phylogenetic analysis of HAV-positive cases in Israel was only done in specific cases. Following the 2012-13 outbreak, the need for a national diagnostic laboratory to support public health decisions on hepatitis A was emphasised. In June 2017, the viral hepatitis reference laboratory in the central virology laboratory of the Ministry of Health was formally established and an official requirement to send IgM-positive HAV samples for molecular confirmation put in place. Thus future outbreaks are expected to be better assessed. Our findings in the MSM population together with the current reports from Europe and recently also in North America [


Deficits in neuropsychological performance are common in schizophrenia, but their relationship with the fronto-temporal functional abnormalities associated with this condition remains unclear. We explored the relationship between neuropsychological performance as measured using the Brief Assessment of Cognition in Schizophrenia (BACS) and the Social Cognition Screening Questionnaire theory of mind (ToM) subscale and fronto-temporal function in 23 patients with schizophrenia and 23 age- and gender-matched healthy controls (HCs), using 52-channel near-infrared spectroscopy (NIRS). Regional hemodynamic changes were significantly smaller in the schizophrenia group than in the HCs group in the ventro-lateral prefrontal cortex and the anterior part of the temporal cortex (VLPFC/aTC) and dorso-lateral prefrontal cortex and frontopolar cortex (DLPFC/FPC) regions. To dissect the effect of variance in BACS cognitive domains from the relationship between ToM function and fronto-temporal function, we performed additional partial correlation analyses between ToM and NIRS data, using BACS composite score as a control variable. The correlation between ToM and NIRS data remained significant only in the DLPFC/FPC region. This finding is important to models of recovery, as it suggests that intervention programs focusing on enhancing fronto-temporal function may have a greater impact on social and occupational outcomes than traditional rehabilitation programs focusing on neuropsychological performance. Cognitive impairments are recognized as core functional deficits of schizophrenia and are a major factor in the unsuccessful re-entry of patients with schizophrenia into the community after treatment1Neurocognitive and functional disabilities are credible treatment targetsSchizophrenia involves dysfunction of the prefrontal cortex (PFC) via several mechanisms, such as perfusion defects, defective metabolism, and flawed synaptic pruning. The PFC is responsible for higher cognitive function  and motivation78101112In recent years, social cognitive ability has garnered attention in schizophrenia research because of its relationship to functioningExisting research indicates that: 1) both neurocognitive and social cognitive abilities are impaired in schizophrenia and predict the level of functioning182022Although neurocognitive function has been closely associated with social functioning, a recent view has proposed that multiple mediators, including social cognitive ability, beliefs, and motivation, exist between the 2 components2728et al.Our goals in the present study were to test the following hypotheses. (1) Relative to healthy subjects, patients with schizophrenia have detectable abnormalities in working memory-related PFC neural activity, as measured using multi-channel NIRS imaging. (2) This neural activity is related to cognitive ability, in particular ToM (see our previous studyThe participants’ demographic data are shown in The response sensitivity A′ and reaction time (RT) scores in the 2-back task during NIRS measurement were significantly worse in the schizophrenia group than in healthy controls (HCs) .The overall mean oxy-Hb changes waveforms for the 52 channels and the 3 ROIs in each group are shown in In patients with schizophrenia, bivariate Pearson correlations (r) between cognitive function and integral values of oxy-Hb changes are presented in Although neurocognitive and social cognitive abilities segregate as independent dimensions in factor analytical studies, they co-occur in patients with schizophreniaIn this study, we used multi-channel NIRS to identify a relationship between working memory-related hemodynamic responses in fronto-temporal regions and neuropsychological performance as measured using the BACS and the ToM in patients with schizophrenia. At the level of working memory-related hemodynamic responses, we found that activity in the bilateral VLPFC/aTC (left and right region 2 in the present study) were significantly associated with both these cognitive function dimensions, while only the ToM scores  were significantly correlated with the DLPFC/FPC (region 1 the present study). When we controlled for variation in the other dimension, working memory-related hemodynamic responses specifically in region 1 showed significant correlation with ToM scores which was significantly different from that between the BACS composite scores and oxy-Hb changes. None of the partial correlations between the BACS scores and working memory-related hemodynamic responses in fronto-temporal regions  reached statistical significance.Recent studies have assessed the neurocognitive domains together with the social cognitive domains in clinical rating scales, reporting mixed results regarding their link to brain structure and function30313334The DLPFC maintains goal-relevant information during working memory363712394041424345et al.51525354The FPC region plays a key role in coordinating and integrating information from other PFC regions48In addition, the hemodynamic responses correlated positively with the three domains of the BACS  in the VLPFC/aTC regions in patients with schizophrenia. Impairments in working memory and other cognitive functions are cardinal neuropsychological symptoms in schizophrenia, and the PFC is important for mediating and executing these functions56573459Though it seems certain that the PFC contribute to multiple cognitive functions, it remains unclear how these functions should be separated and defined. Deficits in a variety of neurological and neuropsychiatric conditions are entirely explained by loss of fluid intelligence. Partialling out the general effects of fluid intelligence, we propose, may clarify the role of additional, more specific cognitive impairments in conditions such as schizophrenia616263Considering that good social cognitive ability is rare in the presence of poor neurocognitive ability, and that the reverse is far more common, neurocognitive ability may be a “necessary” basis of social cognitive abilityAlthough ToM was broadly associated with working memory-related hemodynamic responses in the DLPFC/FPC regions, these preliminary results should be interpreted within the context of the study limitations. This was a cross-sectional investigation, and because ToM was not observed over time, inferences regarding the causality of the association between ToM and working memory-related hemodynamic responses in the DLPFC/FPC regions cannot be made. Second, this investigation involved a relatively small sample size (N = 23), which may have hampered our ability to detect lesser relationships between ToM and working memory-related hemodynamic responses in the fronto-temporal regions. Further studies with a larger sample size are required to verify our findings. Third, although we did not find any relationships between oxy-Hb signals and the duration of illness or dosages of medication in patients with schizophrenia, most of the patients in this study were chronically ill and were medicated. Thus, to rule out medication effects comprehensively, future studies are warranted using first-episode and/or drug-naïve patients with schizophrenia. Fourth, the sample was obtained from the outpatient population of Tottori University hospital and the number of male and female patients differed markedly, and thus may not be considered to be representative of the general population of patients with schizophrenia. Fifth, we did not administer the SCSQ in normal controls, although in a previous study, we found a reliable discriminatory effect between patients and normal controls using SCSQ total scores and ToM subscale scoresIn conclusion, despite these limitations, our study indicated that impaired ToM function may be related to DLPFC/FPC function in patients with schizophrenia. Our findings extend our knowledge about potential mechanisms underlying ToM function and have implications for the treatment of these patients. In future, these findings should be verified by further studies with a larger sample size.The study was approved by the Ethics Committee of the Faculty of Medicine of Tottori University, Tottori, Japan, and the investigation was carried out in accordance with the latest version of the Declaration of Helsinki. All participants gave informed written consent.The schizophrenia group consisted of 23 patients  who had been diagnosed with schizophrenia based on the Diagnostic and Statistical Manual of Mental Disorders, fourth edition (DSM-IV), using the Mini-International Neuropsychiatric Interview (MINI)Healthy individuals who were appropriately age- and gender-matched to the schizophrenia patients participated as controls in the present study. Inclusion criteria for controls were similar to those for the patient sample, although controls were additionally required to have no previous or current psychiatric illnesses. Twenty-three individuals  meeting these criteria were selected as participants. In the present study, healthy controls participated in the NIRS measurement but not in the cognitive assessment using BACS and SCSQ.All patients were receiving atypical antipsychotic medication. Daily doses of all antipsychotics were converted to the equivalent dose of chlorpromazineBrief Assessment of Cognition in Schizophrenia (BACS).We used the Japanese version of the Brief Assessment of Cognition in Schizophrenia (BACS)7171The Social Cognition Screening Questionnaire (SCSQ).The SCSQ65http://www.jichi.ac.jp/brainlab/virtual_reg.html)A 52-channel NIRS  machine was used to measure relative changes in oxygenated hemoglobin (oxy-Hb) and deoxygenated hemoglobin (deoxy-Hb) at 2 wavelengths (695 and 830 nm) of infrared light, based on the modified Beer–Lambert lawet al.The sampling frequency was 10 Hz. To examine 2-back task-related activation, data were analyzed using the “integral mode” installed on the NIRS machine, in which the pre-task baseline was calculated as the mean over the 10-s period immediately prior to the task period, and the post-task baseline was calculated as the mean over the 5-s period that followed after the 50-s post-task period. Linear fitting was applied to the data recorded between these 2 baselines. A moving-average method, using a 5-s window, was applied to remove any short-term motion artifacts. In addition, we rejected noise related to body-movement artifacts  using the algorithm published by Takizawa We used a 2-back task with a blocked periodic baseline–activation–baseline design to activate brain regions specialized for maintenance components of verbal working memory30A′ = 0.5 + (HR − FAR) (1 + HR − FAR)/4HR (1 − FAR).A high A′ implies high information processing ability. All subjects underwent a brief period of identical training to ensure that they understood the rules of the task prior to measurement.Of our 52 NIRS channels, region 1 was defined as including channels 25–28, 36–38, and 46–49. The right side of region 2 was defined as channels 22–24, 32–35, and 43–45, and the left side of region 2 was defined as channels 29–31, 39–42, and 50–52Statistical analyses were performed using SPSS Statistics 19.0 .Hemodynamic responses during the working memory task in region 1 and left and right region 2 were assessed by the “integral value”80t-tests, while the Mann–Whitney U-test was used for clinical variables that were not normally distributed. Integral value of oxy-Hb changes during the task period were compared between groups using t-tests. When there was a significant between-group difference in the performance level (RT and sensitivity A′), we performed additional analyses of co-variance (ANCOVA) using the performance level (RT and sensitivity A′) as covariates to the integral value of oxy-Hb changes. Bivariate relationships between the 2 cognitive function dimensions (BACS scores and ToM scores) and the integral value of oxy-Hb changes in the region  were assessed using Pearson’s correlation coefficients. Next, we computed partial correlations to assess the relationship between each of the 2 dimensions and the integral value of oxy-Hb changes of each ROI, independently, while controlling for the other dimension.Categorical variables were compared using the chi-square test. In all groups, the clinical variables that had a normal distribution were compared using How to cite this article: Pu, S. et al. Association of fronto-temporal function with cognitive ability in schizophrenia. Sci. Rep.7, 42858; doi: 10.1038/srep42858 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Listeria monocytogenes is a foodborne pathogen that causes abortion, septicemia, gastroenteritis and central nervous system (CNS) infections in ruminants and humans. L. monocytogenes strains mainly belong to two distinct phylogenetic groups, named lineages I and II. In general, clinical cases in humans and animals, in particular CNS infections, are caused by lineage I strains, while most of the environmental and food strains belong to lineage II. Little is known about why lineage I is more virulent than lineage II, even though various molecular factors and mechanisms associated with pathogenesis are known. In this study, we have used a variety of whole genome sequence analyses and comparative genomic tools in order to find characteristics that distinguish lineage I from lineage II strains and CNS infection strains from non-CNS strains. We analyzed 225 strains and identified single nucleotide variants between lineages I and II, as well as differences in the gene content. Using a novel approach based on Reads Per Kilobase per Million Mapped (RPKM), we identified 167 genes predominantly absent in lineage II but present in lineage I. These genes are mostly encoding for membrane-associated proteins. Additionally, we found 77 genes that are largely absent in the non-CNS associated strains, while 39 genes are especially lacking in our defined “non-clinical” group. Based on the RPKM analysis and the metadata linked to the L. monocytogenes strains, we identified 6 genes potentially associated with CNS cases, which include a transcriptional regulator, an ABC transporter and a non-coding RNA. Although there is not a clear separation between pathogenic and non-pathogenic strains based on phylogenetic lineages, the presence of the genes identified in our study reveals potential pathogenesis traits in ruminant L. monocytogenes strains. Ultimately, the differences that we have found in our study will help steer future studies in understanding the virulence mechanisms of the most pathogenic L. monocytogenes strains. Listeria monocytogenes is a rod-shaped Gram-positive bacterium that is an opportunistic food-borne pathogen . This is a 9 kb long region composed of six genes encoding proteins that are required for crucial steps in the intracellular life cycle of L. monocytogenes , phospholipases A and B , zinc metalloproteinase (encoded by mpl), actin assembly-inducing protein (encoded by actA) and the transcriptional activator PrfA (encoded by prfA). PrfA is a 27 kDa site-specific DNA binding protein that regulates the transcription of all LIPI-1 genes  from non-disease related strains, using WGS analyses and comparative genomics tools.In addition to the above mentioned multilocus-based studies, large scale WGS studies recently confirmed the distribution in the four phylogenetic lineages and the clonal population structure of L. monocytogenes, were then transferred to Tryptic Soy Agar (TSA) containing 5% (v/v) sheep blood  and incubated at 37°C for another 24 h. For a few strains, colonies presenting haemolysis on the TSA were then applied to the VITEK Compact 2 phenotypic analysis identification system, using Gram-positive identification cards  for the phenotypic identification of the species Listeria monocytogenes. For the rest of the strains, the species and lineage were defined according to Matrix-Assisted Laser Desorption Ionization-Time Of Flight Mass Spectrometry (MALDI-TOF MS)  sheep blood . Colonies were picked and directly treated with lysozyme . Thereafter, the bacterial cells were lysed in guanidium buffer (60% w/v) , either on MiSeq (300 bp paired-end reads) or HiSeq 2500/3000/4000 (95–150 bp paired-end reads) platforms, according to the manufacturer's protocols. Genome coverage varied from 19 x to more than 1000 x (Table http://www.pacb.com/) in order to combine the high level of accuracy from the short-reads sequencing generated by Illumina technology with the long fragments from PacBio sequencing technology.All strains were sequenced using the Illumina® technology . The total number of genomic variants including short insertions/deletions (INDELs) and single nucleotides variants (SNVs) were identified per strain using SAMtools v0.1.19  were used. Variants with mapping and assertion quality values lower than 30 in Phred-scale and with less than 20 reads supporting the alternate allele were filtered out from the vcf files in individual genomes. A Mann-Whitney-Wilcoxon test (“stats” R-package) was performed to identify differences between the numbers of SNVs per lineage , edited and displayed adding metadata information with CLC Genomics Workbench v.9.5.2 (https://www.qiagenbioinformatics.com/products/clc-genomics-workbench).A phylogenetic tree based on the multiple alignment of the SNVs found (ignoring heterozygous sites) was built using RAxML v8.2.9 . The same approach as detailed above for read mapping and SNVs filtering was repeated with JF5203  as the reference to identify SNVs only present in lineage II strains with respect to lineage I strains.The list of variants present in lineage I with respect to lineage II was filtered by excluding the SNVs with low impact according to SnpEff  were used as input files.Using an Thirty-six published genomes with sufficient information about their lineages (18 belonging to lineage I and 18 to lineage II), along with the genomes of the four internal reference strains underwent pan-genome analyses Table . Using tTo check for the presence of the previously  selected genes present in all lineage I strains of our set but absent in all lineage II strains, we used the information recorded in the bam files  to calculate the Reads Per Kilobase per Million mapped (RPKM) values. RPKM analysis is an established method for RNA sequencing (RNA-seq) data examination  and totalNumReads is the total number of reads mapped to the genome.dnaA and gyrB genes were used as controls. The Mann-Whitney-Wilcoxon test (“stats” R-package) was used to check for differences between the RPKM values for each of the previously identified genes in the two lineages . The post hoc Dunn's test for the Kruskal-Wallis multiple comparison test was performed with “dunn.test” R-package v.1.3.4 to assess the significance among the groups Dinno, .*standard deviation) were retained to obtain a list of genes predominantly absent in lineage II. A matrix was built based on the RPKM values of the selected genes. A heatmap was calculated and plotted in R (“gplots” R-package)  with the ones present in the non-clinical group BC . Genes with a median greater than or equal to 154.01 were kept.After every comparative analysis, the selected genes underwent a Gene Ontology (GO) enrichment step by Blast2GO  of the RPKM values for the 2981 genes in the 225 strains was performed (“stats” R-package). A permutation multivariate analysis of variance (PERMANOVA) test  under the Project number L. monocytogenes lineage I strains from lineage II strains. To this end, we selected two strains belonging to lineage I (JF5203 and JF5861) and two strains of lineage II (JF4839 and LMNC088)  and vB_LmoS_293 (GenBank accession number NC_028929.1), respectively, while contig_3 contained an incomplete prophage. They are bacteriophages of the Siphoviridae family Orthocluster IV which have already been described as L. monocytogenes phages , representing approximately 79% of its length.A 74 kbp plasmid was found in the lineage II strain JF4839, isolated from cheese unrelated to a listeriosis outbreak  and Nucmer . The distribution of the total genomic variants was very similar to the distribution of the SNVs (results not shown).The Illumina sequencing data was used for the detection of variants that can distinguish between lineages I and II. Using the genome sequence of lineage II strain JF4839 as a reference to map all the strains in the study, the average number of SNVs in lineage II was 26,826 while in lineage I this value was 129,632 SNVs. The distribution of all SNVs in each lineage was represented in a kernel density plot Figure , showingA tree based on the number of SNVs at the whole genome level using JF4839 (lineage II) as reference is shown in Figure In lineage I, there are more clinical cases associated with CNS infections (75 compared to 30 cases in lineage II). One septicemia cases is also present in this group. In contrast, in lineage II, environmental strains are more common along with two strains of food origin. Other clinical manifestations such as mastitis, gastroenteritis and abortion are present in both groups. It is also relevant that lineage I has far less SNVs among them than lineage II, showing a closer distribution, while lineage II displays more diversity.The total list of conserved variants in lineage I (present in ≥80% of the strains taking the JF4839 annotated genome of lineage II as a reference) allows the differentiation between the two lineages and all are documented in Supplementary Material Addressing our next aim, the SNVs private to CNS infection cases were analyzed separately in order to find a pattern specific for strains of CNS origin. We looked for genes with high number of SNVs in this fraction of strains. However, we did not identify specific genes correlating to more variations in the neurolisteriosis strains. The maximum percentage of strains having common SNVs was approximately 20%  was done in both directions because both lineages contain elements or genes not present in the other one. Thus, taking the JF5203 annotated genome (lineage I) as a reference, the SNVs were identified for lineage II strains. A list of variants of lineage II present in ≥80% of the strains with respect to lineage I was also created  in lineage II to thymine (T) in lineage I at positions 10 and 13 in the 5′UTR of the prfA gene in all of the 121 lineage I strains analyzed  riboswitch SreA. An Adenine (A) at position 83 in lineage II is substituted by a guanine (G) in lineage I, and a G at position 88 in lineage II is changed to an A in lineage I Table . This SAThese two pairs of variants are given as interesting examples, however there are thousands of other potentially interesting variants to look at.L. monocytogenes genomes (20 of each lineage including our references strains) were analyzed in the MicroScope platform (http://www.genoscope.cns.fr/agc/microscope/home/). The lineage I differential pan-genome comprised a total of 5,730 genes  including the lineage I differential variable-genome of 5,290 genes  and the lineage I differential core-genome of 440 genes. This core-genome corresponds to 22 gene families specific to lineage I  were found between the lineages (Mann-Whitney-Wilcoxon test). All six genes were absent in lineage II and present in all lineage I strains analyzed, with the exception of the LMNC284 strain, where the gene LMOF2365_RS06250 is not present after verification with the Integrative Genomics Viewer (IGV) , the difference between the two lineages is close to zero whereas for the six selected genes, this difference increases to approximately 400, showing similar behavior for all the genes. A Dunn's test revealed significant differences  between the control group and the six selected genes.After RPKM pairwise analysis, box plots of the difference between lineage I and II gene by gene were generated Figure . For theThe RPKM values-based analysis was extended to a whole genome level in order to explore the presence of the 2981 genes from the reference genome JF5203 in the 225 genomes of this study.A heatmap showing the degree of presence/absence of the 167 genes predominantly absent in lineage II was created for the 225 strains Figure . For thep-value < 0.0001; PERMANOVA test). On the other side, two other groups can be distinguished, one with the majority of the clinical strains (either from lineage I or II) and the other one with the majority of the environmental strains. Significant differences were also detected with the PERMANOVA test . Based on these results, the groups were defined as follows: A-lineage I clinical, B-lineage I non-clinical, C-lineage II non-clinical, and D-lineage II clinical. Only one strain (JF5593) was not classified as either clinical or non-clinical, since is located at the middle of the two groups.Figure In order to apply a clinically relevant filter to look for genes that could possibly be related to CNS infections, the RPKM method was applied, but this time to compute the differences of RPKM values between the CNS infection-related and non-related strains. We found that 77 genes are predominantly absent in the non-CNS group of the strains. According to our data, a single gene cannot perfectly separate the two groups and be assigned as CNS-infection causative, but the combination of various genes could be a signature of neurolisteriosis Figure .There are 65 mutual genes from the 167 predominantly absent in the less virulent lineage II and the 77 genes predominantly absent in the non-CNS group. Of these 65 genes, 24 encode for membrane proteins, 5 for transcriptional regulators and the rest have other functions or are hypothetical proteins , LMJF5203_01155 (ncRNA rli38)  and JF4839 (food isolate not related to an outbreak from lineage II). We reported the SNVs in intergenic regions and those in coding regions that have a moderate or high impact, according to SnpEff classification  to higher temperatures (host), by activating the transcription of the downstream virulence genes, faster than lineage II strains.The pathogenicity island LIPI-1 is considered to be the main virulence attribute of The differences between the two lineages are not only due to the single point variants. Differential gene composition is another important criterion. Genes contained in lineage I but missing in lineage II are of particular interest because they represent a list of potential genes or regulatory elements that might facilitate the invasion of the bacteria ultimately producing CNS disorders. Several of these genes encode proteins with particular domains or motifs related to surface location, cell adhesion and internalin-like features.L. monocytogenes. They are known virulence factors involved in the bacterial colonization and cell-to-cell spread in the host .For the comparative genomics study, we successfully used the Microscope platform in a first step with 36 published genomes and our 4 references strains. MicroScope constitutes a user-friendly web-based framework with several integrated tools for analyzing individual or groups of genomes. However, this system requires the assembled genomes to be previously uploaded in the platform. The submission process can take from 4 to 10 weeks per batch and only ten genomes can be compiled per batch , enterotoxigenic E. coli (ETEC), enterohaemorrhagic E. coli (EHEC), enteroaggregative E. coli (EAEC) and enteroinvasive E. coli (EIEC), it has been reported that depending on the pathotype, a group of genes are responsible of the particular pathogenesis (Kaper et al., in vitro and in vivo studies.Since it was not possible to clearly separate the pathogenic form the non-pathogenic strains by lineage classification, two other comparisons were performed: CNS-infection strains against non-CNS infection strains and strains belonging to the clinical group with strains form the non-clinical group Figures , 8. Our L. monocytogenes strains and used them as reference genomes. Subsequently, we sequenced 221 additional strains of both lineages and performed whole genome comparative analyses using a variety of different approaches. We identified a list of private SNVs exclusive to each lineage. While, we could not observe a typical pattern for CNS infection associated strains based on SNVs, we did find two interesting variants in the important 5′UTR of the prfA gene. Future studies should examine if these variants provide an adaptive advantage for the pathogenic strains of lineage I compared to the food and environmental strains of lineage II. Based on a core- pan-genome analysis of published L. monocytogenes genomes together with our reference genomes, we identified a set of putative virulence proteins that are present exclusively in lineage I. Subsequently, a new method based on RPKM-difference values was developed for performing a rapid comparative genomic analysis of our hundreds of strains. After comparing all lineage I strains vs. lineage II, CNS-related strains against non-CNS strains and strains belonging to the clinical group to the ones present in the non-clinical group, a common fraction of 6 genes seems to be relevant for the increased virulence of the CNS disease-related strains. In addition, our study indicates that although there is not a well-defined separation between pathogenic and non-pathogenic strains according to their phylogenetic lineages, the existence of the genes identified suggests a better indication of pathogenesis in the ruminant L. monocytogenes strains analyzed. This work provides an excellent basis for future studies aiming to elucidate L. monocytogenes virulence mechanisms.In this study, we sequenced and fully assembled two lineage I and two lineage II LA-B performed all the bioinformatics and statistical analysis, contributed to the design of the study and wrote the article. PN participated in the design of the study and wrote the manuscript. LR assisted with the development of the scripts. MD contributed to the sample collection and gDNA purification. FO, BG and AO collaborated in the design of the study. JF and LF supervised and conceived the whole study, and wrote the manuscript. All the authors contributed to the article writing.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Previous studies have established a role for premotor cortex in the processing of auditory emotional vocalizations. Inhibitory continuous theta burst transcranial magnetic stimulation (cTBS) applied to right premotor cortex selectively increases the reaction time to a same-different task, implying a causal role for right ventral premotor cortex (PMv) in the processing of emotional sounds. However, little is known about the functional networks to which PMv contribute across the cortical hemispheres. In light of these data, the present study aimed to investigate how and where in the brain cTBS affects activity during the processing of auditory emotional vocalizations. Using functional neuroimaging, we report that inhibitory cTBS applied to the right premotor cortex (compared to vertex control site) results in three distinct response profiles: following stimulation of PMv, widespread frontoparietal cortices, including a site close to the target site, and parahippocampal gyrus displayed an increase in activity, whereas the reverse response profile was apparent in a set of midline structures and right IFG. A third response profile was seen in left supramarginal gyrus in which activity was greater post-stimulation at both stimulation sites. Finally, whilst previous studies have shown a condition specific behavioral effect following cTBS to premotor cortex, we did not find a condition specific neural change in BOLD response. These data demonstrate a complex relationship between cTBS and activity in widespread neural networks and are discussed in relation to both emotional processing and the neural basis of cTBS. Here we assess the effect of theta burst TMS on neural activity in a previously established emotional vocalization perception paradigm.The last two decades have provided increasing evidence that the adult brain is highly susceptible to short-term changes, both in structure and function. Widespread changes in brain organization can occur as a result of both damage or learning and development, over a range of timescales. Studies in humans and animals have demonstrated that neuroplasticity is not restricted to any given developmental phase but remains an ongoing process in the form of learning. In humans, the advent of transcranial magnetic stimulation (TMS) for non-invasive brain stimulation had rendered the human brain accessible for Understanding the emotional states of others is a pivotal aspect of our social interactions. Vocal cues are one of several sources of information that contribute to this ability, with changes in a variety of acoustic cues  providing a rich source of information about the emotional states of others . In receWe recently showed that continuous theta burst stimulation (cTBS) targeted at right PMv (rPMv) disrupts the ability to discriminate between emotional vocalizations, but not speaker identity . ContinuRecent work has shown that repetitive TMS (rTMS) can elicit changes in a number of different ways: Continuous TBS has also been shown to alter activity in functionally connected but distinct sites from the site of stimulation . For exaHere, we sought to utilize and assess the network effects of cTBS based on these previous findings, by combining it with fMRI in order to investigate how the application of cTBS to rPMv influences BOLD responses to emotional sounds (i) across the whole brain, (ii) in regions activated by emotional vocalization perception, (iii) at the target site and finally (iv) at the left hemisphere homolog of the target site. Specifically, we compared BOLD responses during passive listening of different emotional vocalizations as per  before aThe experimental set up required the following conditions: Passive perception of sounds of (1) amusement, (2) triumph, (3) fear, (4) disgust, (5) spectrally rotated versions of a mixture of all four emotional sounds and (6) a silent rest condition (see below for details). Subjects were required to attend two sessions on different days. On each visit subjects underwent two 20-min fMRI experiments which involved the six experimental conditions as listed above. Following the first 20-min fMRI experiment, subjects then underwent cTBS to either the rPMv or to the vertex , before going back into the scanner to repeat the fMRI experiment. On the second visit, subjects underwent the same procedure but had cTBS applied to the other stimulation site. At least a week was left between the active and control site stimulation sessions.Four different types of emotional vocalizations were used, two positive  and two negatively valenced  , 2003. TSixteen healthy right-handed subjects (range 23–49 years) participated in the study. All gave informed consent according to the guidelines approved by UCL Ethics Committee, who provided local ethics approval for this study.2∗-weighted echo-planer images (EPI) were acquired  using BOLD contrast. On each day, subjects were scanned twice, pre- and post- stimulation. On each day of scanning, 250 volumes were acquired in two runs – 125 volumes in the pre-stimulation run and 125 in the post-stimulation run. Subjects were required to come in on two separate days for two separate scanning sessions, during which time they received stimulation to either the vertex or right PMv. In all cases, the first two functional volumes were discarded in order to remove the effect of T1 equilibration. High-resolution T1 anatomical volume images  were also acquired for each subject.Functional imaging data were acquired on a 1.5 Tesla Siemens Avanto system  using a 32-channel birdcage head coil. This type of head coil has been shown to increase signal-to-noise for medical images acquired in the 1.5 Tesla field without increasing the signal drop out associated with higher field strengths . T2∗-weiDuring the main experimental run, subjects lay supine in the scanner in the dark and were asked to close their eyes and listen to the sounds played to them. There was no task involved so as to avoid any form of motor priming that a response, such as a button press, or a cognitive task might entail. Functional data were acquired using a sparse sampling protocol in which four stimuli were presented during the quiet 7 s intervals (±500 ms jitter) between image acquisitions. This approach allowed the presentation of auditory stimuli in the absence of interference from scanner noise . StimuliSounds were presented to the subjects in the scanner via a Denon amplifier  and air conduction headphones  worn by the subjects. During each run, subjects heard stimuli from one of each of the five experimental conditions . There were 20 examples of each condition within each session (pre- and post-TBS) played in a randomized order with ±500 ms onset jitter. The same items were not heard more than once in each run. For the high-level baseline condition, four spectrally rotated versions were played. These were a combination of spectrally rotated versions of all four classes of emotional vocalization. For the silent rest condition subjects heard nothing during the silent period between image acquisitions. The presentation of stimuli was pseudo-randomized to allow a relatively even distribution of the six conditions in the absence of order effects.Between the two 20 min fMRI sessions, subjects were brought out of the scanner in order to administer cTBS. Following application of cTBS subjects were instructed not to speak unless they had to, and were each put back into the scanner immediately. The beginning of the second scanning session was timed such that the functional data acquisition began 5 min after the end of the cTBS.Transcranial magnetic stimulation was delivered via a figure of eight coil with a 70 mm diameter using a Magstim Super Rapid Stimulator  at 40% machine output. This intensity is in accordance with other cTBS studies  and studAn offline cTBS paradigm was used, which consisted of a burst of 3 pulses at 50 Hz repeated at intervals of 200 ms for 20 s, resulting in a total of 300 pulses. Based upon previous findings  the timeLocations for cTBS were identified using Brainsight TMS-magnetic resonance co-registration system . FSL software  was used to transform coordinates for each site to each subject’s individual MRI scan. The coordinates for rPMv  were the averages of neurologically normal participants in an fMRI study of non-verbal auditory emotion processing  found toFunctional data were analyzed using SPM8  running on Matlab 7.4 . All functional images were realigned to the first volume by six-parameter rigid body spatial transformation. Functional images were normalized into standard space using the Montreal Neurological Institute (MNI) template using parameters elicited from a unified segmentation of the T1 anatomical image. Functional images were then smoothed using a Gaussian kernel of full width half medium (FWHM) 8 mm.T-tests were also generated comparing individual conditions pre- and post-stimulation for each site of stimulation. The contrast images generated from these t-tests were entered into group analyses at the second level. Peak activations were localized using the anatomy toolbox available within SPM8  with clear anatomical hypotheses that activity would be observed in superior dorsolateral temporal cortices, statistical maps were thresholded at p < 0.001 with a cluster extent of 30 voxels. In the case of more exploratory comparisons , FDR correction for multiple comparisons was applied .Event-related responses for each condition were modeled as a canonical hemodynamic response function. Event onsets were modeled from the onset of auditory recording with a 4 s duration. For each run (pre- and post- stimulation) each condition was modeled as a separate regressor in a general linear model. Motion parameters (three translations and three rotations) were modeled as six regressors of no interest at the single-subject level. For each subject (first level), contrast images were created to describe the comparisons between each of the experimental conditions compared to silent rest and to each other. hin SPM8 . In the Data were analyzed in a number of different ways:(1)t-tests at the second level.In order to investigate BOLD responses to perception of emotional sounds, the two pre-stimulation sessions were combined into one model, with the 2 days being modeled as separate sessions within one design matrix. Contrasts were then created at the first level of each of the sound categories compared to silent rest and compared to the high-level baseline of rotated sounds. Resulting first level contrasts were entered into (2)[post-stim] > Amusement[pre-stim]). This was performed separately for premotor stimulation and vertex stimulation data. In order to account for within subject variation, these contrasts were then taken up to a second level 2 × 6 fully flexible ANOVA where the two factors were Site (Premotor and Vertex) and Condition . In this way, the contrasts entered into the ANOVA at the second level reflect the difference between pre- and post-stimulation, and so any observed effects of the Site and Condition factors within this model in fact reflect the interaction of these factors with the effect of Session (post- vs. pre-stimulation).In order to investigate the effect of stimulation to either premotor and vertex sites on the neural activity during perception of emotional sounds, contrasts were generated at the first level, comparing perception of each emotional vocalization category post-stimulation with pre-stimulation Region of interest analyses were carried out to investigate mean effect sizes in specific regions across all experimental conditions post-stimulation compared to pre-stimulation using the MarsBar toolbox that is available for use within SPM8 .[post-stim] > Amusement[pre-stim]) [PMv site]/Triumph[post-stim] > Triumph[pre-stim]) [PMv site]/Fear[post-stim] > Fear[pre-stim]) [PMv site]/ Disgust[post-stim] > Disgust[pre-stim]) [PMv site]/Amusement[post-stim] > Amusement[pre-stim]) [Vertex site]/Triumph[post-stim] > Triumph[pre-stim]) [Vertex site]/Fear [post-stim] > Fear[pre-stim]) [Vertex site]/Disgust[post-stim] > Disgust[pre-stim]) [Vertex site] (see Table 1 for anova set up).Amusement > Silent Rest/Amusement > Spectrally rotated/Triumph > Silent Rest/Triumph > Spectrally rotated/Fear > Silent Rest/Fear > Spectrally rotated/Disgust > Silent Rest/Disgust > Spectrally rotated/AmusementFigure 1, red lines). Perception of each emotional vocalization compared to the spectrally rotated sounds  was associated with significant BOLD responses in a more restricted pattern comprising peaks in middle and posterior superior temporal gyri in both hemispheres . In the cases of Fear and Triumph, peaks were also seen in left inferior frontal gyrus . These initial contrasts were thresholded at p < 0.001 with a cluster threshold of 30 voxels.In order to investigate BOLD responses to perception of emotional sounds, the two pre-stimulation sessions were combined into one model. Contrasts were then created at the first level of each of the sound categories compared to silent rest and compared to the high-level baseline of rotated sounds. Perception of emotional sounds pre-stimulation, compared to a silent rest condition was associated with widespread significant activity in the dorsolateral temporal cortices in both hemispheres . We aimed to look at how cTBS modulated BOLD responses (i) across the whole brain, (ii) in regions activated by emotional vocalization perception, (iii) at the target site and finally (iv) at the left hemisphere homolog of the target site (see Table 2 for a list of coordinates).We next addressed whether post- vs. pre-stimulation differences were significantly different between the target sites. In order to look at the different post-stimulation compared to pre-stimulation in the premotor site compared to the vertex site, contrasts such as [Amusement(post>pre)’ . A [Session × Site] interaction was seen in a network of frontal, parietal and midline regions: one bilateral set of clusters lay in inferior frontal gyri with peaks in pars triangularis (BA45) and subpeaks in pars opercularis (BA44), left middle frontal gyrus and right insula cortex and finally in right precentral gyrus (BA6) corresponding to premotor cortex. A second set of clusters lay in bilateral parietal cortex comprising inferior parietal cortex and supramarginal gyrus on the left and postcentral gyrus (BA1) on the right, left supplementary motor area extending into superior frontal gyrus. Finally, a set of midline clusters was seen in right middle cingulate cortex, precuneus and parahippocampal gyrus . Figure 2B displays a t-contrast that reflects the positive difference between post- and pre-stimulation sessions for the two sites ([PMvpost > PMvpre] > [Vertexpost > Vertexpre]). This comprised left inferior frontal gyrus encompassing both pars opercularis and triangularis, left supplementary motor area, cerebellar vermis, right parahippocampal gyrus, bilateral inferior parietal lobe encompassing the angular gyrus, bilateral superior and right middle frontal gyri and right postcentral gyrus . The opposite comparison was associated with activity in left hippocampus, right middle cingulate cortex, right precuneus, left supramarginal gyrus, right supplementary motor area, right inferior frontal gyrus (pars triangularis) and right rolandic operculum .At the first level, contrasts were made comparing perception of each emotional vocalization category post-stimulation with pre-stimulation . This was performed separately for premotor and vertex stimulation data sets. These contrasts were then taken up to a second level 2 × 6 fully factorial ANOVA with two factors ‘Site’ (premotor and vertex) and ‘ConditionFigure 3. Here, a value of zero would indicate no change in activity levels between pre- and post-stimulation. Positive values on the graph indicate that activity was greater post-stimulation than pre-stimulation and vice versa. Similarly, smaller values for contrasts comparing post- to pre-stimulation following premotor stimulation (gray bars) than following vertex stimulation (white bars) indicates that premotor stimulation resulted in a greater reduction in activity. These plots reveal three important factors: first, the effect of stimulation to vertex was considerable and was variable across regions in terms of direction and extent. In roughly half of the regions, activity was greater post vertex stimulation and in the other half activity was less post vertex stimulation. Given that stimulation at the vertex did alter activity in a number of regions, a more meaningful comparison is between the effect post-PMv stimulation to the effect post-vertex stimulation. The second point that is evident from these plots is the presence of three different patterns of activity within this larger network of clusters. A set of regions comprising bilateral IFG and parietal cortices, left SMA, right superior frontal gyrus and parahippocampal gyrus, displayed an increase in activity post-stimulation to PMv but a decrease in activity post vertex stimulation . Secondly, the reverse response profile is apparent in precuneus, middle cingulate cortex and right IFG . Finally a single cluster lying over the left supramarginal gyrus displayed a distinct activity profile whereby activity was greater post-stimulation at both sites, compared to pre-stimulation . Lastly, it is evident from these plots that the effect of cTBS to PMv is affecting activity in these regions across all conditions, including during listening to the high level baseline of rotated sounds and during silent rest trials, not just during the perception of emotional vocalizations. Thus the result that we report is not specific to emotional vocalization perception but is a generalized neural effect of cTBS.In order to look at how BOLD responses were modulated by cTBS at specific cortical sites, region of interest analyses were carried out to investigate mean effect sizes in specific regions across all experimental conditions post-stimulation compared to pre-stimulation. In order to look specifically in regions activated by emotional vocalization perception we plotted mean parameter estimates in regions revealed by the interaction [Session × Site], mean parameter estimates were extracted for each of the clusters and are plotted in Figure 3F] with a subpeak lying at [57 2 34], around 1 cm from the target site [54 -2 44] although this cluster did not encompass precise site of targeting stimulation. In order to investigate BOLD responses within this region further, a 6 mm spherical region of interest was created around the target coordinate and the left homolog [-54 -2 44] and the mean parameter estimates were extracted for each subject. There was no significant difference between activity in this region post-vertex stimulation and post PMv stimulation .Lastly, we wanted to look at BOLD responses at the target site and the left homolog of the target site. The interaction of [Session × Site] revealed a significant cluster of activity in right premotor cortex lying over the post- and precentral gyri in the right hemisphere. The peak of this cluster lay in the postcentral gyrus [66 -10 31, and rotated emotional vocalizations), leading to changes in widespread brain networks involved in the appraisal of affective signals.A distributed network of neural regions contribute to the recognition and appraisal of affect from vocal cues . One comWe report that despite clear evidence to suggest that cTBS inhibits activity in the targeted region, BOLD responses were increased in a region lying close to the target site (compared to post-vertex stimulation) and across wider networks that have been linked to vocalization perception . While TIncreased activity post-stimulation may reflect a rebound in cortical responsiveness following cTBS, as the site of stimulation returns to baseline levels of cortical excitability. While we did not see a significant effect at the target site, we did see a significant increase in activity in a neighboring region lying within right PMv and in a similar region in the left hemisphere. The target site, lying on the ventral bank of the boundary between PMd and PMv , containThe brain may actively compensate for the interference at the site of stimulation and within a wider connected network of regions . Jung anFrontoparietal regions, including rPMv, are known to provide a link between the production and perception of both speech  and non-In addition to the increase of activity in frontoparietal regions, a set of regions displayed the opposite pattern. These were the SMG, right IFG, cingulate cortex and two peaks within the precuneus, all of which have previously been linked with affect processing either through mechanisms of emotion perception or emotion regulation . The antDespite previous findings showing that the effect of cTBS to PMv elicits condition specific behavioral effects, here we did not find evidence of condition specific neural effects. Specifically, that cTBS had a similar effect on BOLD responses for both emotional vocalization perception conditions, but also to spectrally rotated versions of those sounds, suggests a general effect of stimulation on auditory processing. One possibility is that cTBS to PMv serves to prime a general auditory motor network, which behaviorally modulates the processing of biologically relevant sounds, such as emotional vocalizations . Other sGiven the role of each region in affect processing and potential connectivity between the two networks reported here, it is feasible that the regions showing decreased activation may serve to promote the integration and evaluation of emotion stimuli, and that suppressing rPMv results in a reduction of the functional coupling between the frontoparietal network and this set of regions. Previous work has shown that cTBS modulates functional connectivity, many showing a reduction in connectivity, both in humans , and in Whilst it is clear than TMS can elicit neural plasticity and behavioral changes, there are also a range of factors that contribute to high variability in the neural and behavioral responses to stimulation, such as such as age, circadian rhythm, and endogenous brain oscillations . The effPrevious data have shown that cTBS to the rPMv selectively disrupts the ability to perform a same/different task on emotional vocalizations; here we demonstrate that this is due to widespread changes in a network of regions involved in emotion perception and regulation. These data provide the first evidence that cTBS targeted at rPMv modulates activity in distributed cortical regions when processing emotional vocalizations. They add to previous studies e.g.,  by highlThis study was carried out in accordance with the recommendations of UCL Ethics Committee with written informed consent from all subjects in accordance with the Declaration of Helsinki. The protocol was approved by the UCL Ethics Committee.ZA designed and performed the experiments, analyzed the data, and wrote the manuscript. MB designed and performed the experiments, edited the manuscript. CM carried out the experiments and edited the manuscript. VW and SS designed the experiments and edited the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Our observations provide insights on the behavior of chloroplasts, the relationship between stromules and the plastid-body and strongly challenge claims of stromule-specific functions based solely upon protein localization to plastid extensions.Plastids in the viridiplantae sporadically form thin tubules called stromules that increase the interactive surface between the plastid and the surrounding cytoplasm. Several recent publications that report observations of certain proteins localizing to the extensions have then used the observations to suggest stromule-specific functions. The mechanisms by which specific localizations on these transient and sporadically formed extensions might occur remain unclear. Previous studies have yet to address the spatiotemporal relationship between a particular protein localization pattern and its distribution on an extended stromules and/or the plastid body. Here, we have used discrete protein patches found in several transgenic plants as fiducial markers to investigate this relationship. While we consider the inner plastid envelope-membrane localized protein patches of the GLUCOSE 6-PHOSPHATE/PHOSPHATE TRANSLOCATOR1 and the TRIOSE-PHOSPHATE/ PHOSPHATE TRANSLOCATOR 1 as artifacts of fluorescent fusion protein over-expression, stromule formation is not compromised in the respective stable transgenic lines that maintain normal growth and development. Our analysis of chloroplasts in the transgenic lines in the Arabidopsis Columbia background, and in the Observations of the spatiotemporal relationship between plastid envelope localized fluorescent protein fusions of two sugar-phosphate transporters and stromules suggest a stochastic rather than specific localization pattern that questions the idea of independent functions for stromules. Plastids in green plants and algae are bound by a double-membrane envelope and sporadically extend and retract thin tubules  complex , a peroxiredoxin PpPrxQA  leads to 64 gene products with 58 of the proteins directly annotated as stromal. The Arabidopsis Information resource  actually lists 39 records. Amongst these 33 protein records suggest stromule as the place of expression and are traceable to a single publication by Goulas et al. . The chimeric vectors were introduced in Agrobacterium tumefaciens GV3101 through electroporation.The full-length coding sequences for the Arabidopsis GPT1 (At5g54800) TPT1 (At5g46110), were obtained by PCR from a cDNA library and used to create C-terminal fusions with Green Fluorescent Protein , and monomeric EosFP . Experiments aimed at understanding fusion-protein localizations in relation to stromules used stable transgenic lines Arabidopsis thaliana (Columbia ecotype) expressing the plastid stroma-targeted tpFNR-EGFP .All transient arc6 mutant  or the ImageJ / Fiji platform (https://fiji.sc/).All images were captured at a color depth of 24 bit RGB. All images and movies were annotated, cropped and processed for brightness / contrast as complete montages or image stacks using either Adobe Photoshop CS3 .Stromule counts were performed on image z-stacks taken on the hypocotyls of 7-day-old d Hanson , a stromJM conceived and supervised the research. KD, MW, KB, and NG created the different gene fusions and helped with imaging and data collection. NM provided technical assistance. KD and KB analyzed the data. All authors contributed to the writing of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


This review provides an overview of 50 years of basic and clinical research on an oncolytic avian virus, Newcastle Disease Virus (NDV), which has particular anti-neoplastic and immune stimulatory properties. Of special interest is the fact that this biological agent induces immunogenic cell death and systemic anti-tumor immunity. Furthermore, localized oncolytic virotherapy with NDV was shown to overcome systemic tumor resistance to immune checkpoint blockade immunotherapy. Clinical experience attests to low side effects and a high safety profile. This is due among others to the strong virus-induced type I interferon response. Other viral characteristics are lack of interaction with host cell DNA, lack of genetic recombination and independence of virus replication from cell proliferation. In this millennium, new recombinant strains of viruses are being produced with improved therapeutic properties. Clinical applications include single case observations, case series studies and Phase I to III studies. Progress in science and medicine is often driven by new observations or ideas and the development of new concepts and strategies. Sometimes it takes decades before one can say whether such a new idea was right and whether the concept has a lasting effect. This will be exemplified by the concept of oncolytic viruses and post-oncolytic immunity.The first report  of resuloncolytic strain 73 T) as an antineoplastic agent [The 1960s were devoted to oncolytic viruses and later to research on post-oncolytic anti-tumor immunity. All kinds of viruses were found and studied but only few remained of further interest. Among these few was the avian paramyxovirus NDV. By that time, attenuated strains of NDV had been used for almost two decades to prevent Newcastle disease in birds. The inability of this virus to cause serious illness in humans had been established and non-lytic, as well as oncolytic, strains were described . It was ic agent . When, tTwo Phase II clinical studies involving 32 and 51 patients were conducted successively in Stage II metastatic melanoma (AJCC stage III). In these studies, NDV oncolysate was examined as an adjunctive immunotherapeutic agent. A ten-year follow up of these 83 treated patients revealed that over 60% were alive and free of recurrent disease . Older s73 T was used to prepare autologous oncolysates that were given to patients by subcutaneous injection once a week for 8 to 10 weeks, beginning one to three months after radical surgery . Two cytokines, low-dose recombinant interleukin-2 and recombinant interferon-α were added to the oncolysate vaccines. The researchers concluded that the results demonstrated improved disease-free survival (DSF) in comparison with survival data published for similar patients who were treated by surgery alone.A similar Phase II study of NDV oncolysates has been conducted in Germany by Kirchner, Anton and Atzpodien, involving 208 patients with locally advanced renal cell carcinoma . NDV strMTH-68/H. One placebo-controlled Phase II clinical trial [A Hungarian research effort led by Csatary advocated the use of an attenuated oncolytic veterinary vaccine strain, which they termed al trial  includedal trial .MTH-68/H reported on radiographically-documented responses and long survival with improved symptomatology [A selected case series study of four high-grade gliomas treated with matology .HUJ, lentogenic) was administered intravenously to 14 glioblastoma multiforme (GBM) patients using intra-patient dose escalation (1–11 billion infectious units) followed by three cycles of 55 billion infectious units. Toxicity was minimal and a maximal tolerated dose was not achieved. One patient achieved a complete response, although of only transient duration (about three months), while the others developed progressive disease [In Israel, another NDV strain  to syngeneic mice, a local palpable tumor developed within 10 days. To simulate a model system for post-operative immunotherapy, we removed the primary tumor and tried to develop a vaccine against the remaining micrometastatic disease (mainly in the liver).Our concept was to infect tumor cells by a virus to make it more immunogenic and to develop thereof an irradiated virus-infected live cell tumor vaccine. From Lindenmann  we obtained human influenza virus and a mouse strain resistant to this virus. Although we saw occasionally anti-tumor effects in vivo, reproducibility was difficult. Additionally, the virus had to be adapted to the murine tumor cells first and the virus-resistant strain of mice had to be crossed with the strain from which the tumor line arose to be able to perform respective in vivo experiments. There were also safety aspects to be dealt with concerning the laboratory personnel.Ulster) is lentogenic and not oncolytic so that infected tumor cells remain viable and immunogenic, at least within a period of 24–48 h. In contrast to influenza virus, NDV could infect ESb cells without adaptation.All of this changed in 1984, when we got ahold of another virus, namely the avian paramyxovirus NDV. The strain used  who had developed the term “tumor xenogenization”, and had described the higher immunogenicity of live cell vaccines compared to lysates .Ulster). In the control group, which was operated and vaccinated with ESb vaccine without NDV, all animals died within three weeks. In contrast, in one group, which was vaccinated with ESb cells infected by a low dose of virus, more than 50% of the mice survived long-term [In our first post-operative anti-tumor vaccination experiment, we applied i.p. vaccines consisting of 10 million irradiated ESb tumor cells, which had been infected with increasing doses of NDV  the urgent need in the clinic for a new post-operative strategy against metastases and (ii) the development in the USA of an autologous live cell tumor vaccine mixed with Bacillus Calmette Guérin (BCG) by Hanna and colleagues ,26.Of course, there were a number of questions to be solved: Would human tumor cells also be infectable by non-lytic NDV? How could one best isolate tumor cells from a fresh operation sample without losing cell viability? How should one inactivate tumor cells? What dose and where to apply? What type of tumor should be tried first?Like us in Heidelberg, the group of Hanna had based their vaccine on careful pre-clinical post-operative vaccination studies in animals . The terAfter a series of tests of human tumor cell lines or of tumor cells from fresh operation specimens we reported that human tumor cell modification by virus infection is an efficient and safe way to produce cancer vaccine with pleiotropic immune stimulatory properties when using NDV .A Phase I clinical trial in patients with colorectal carcinoma (CRC) helped to establish the optimal number of tumor cells and the optimal amount of virus to use to produce the best possible immune response. Immune responses were monitored by means of a skin delayed-type hypersensitivity (DTH) test using as intra-dermal challenge either irradiated ATV-NDV vaccine or irradiated ATV cells. In one study, 12 of 16 (75%) patients showed increased immune system reactivity against uninfected autologous tumor cells after vaccinations .Post-operative vaccinations were performed three times in two-week intervals. This appeared sufficient to obtain peak anti-tumor DTH reactivity. This means three rounds of expansion and retraction of tumor-reactive T cells involving three waves of memory T cells changing between a status of activated effector memory (EMT) T cells in peripheral tissues and of resting central memory (CMT) T cells in the bone marrow .n = 63), metastatic breast cancer (n = 27) or metastatic ovarian cancer (n = 31) and who had sufficient numbers of recovered tumor cells to allow at least two vaccinations. The ATV-NDV vaccine was classified as either high quality or low quality on the basis of the following parameters: the number of tumor cells and the percentage of live tumor cells. Overall survival (OS), four years after surgery, was estimated to be 96% for the patients with early breast cancer who had received a high quality vaccine (n = 32) compared with an OS of 68% for those who had received a low-quality vaccine (n = 31), a difference that was statistically significant [One clinical study evaluated the effect of vaccine quality on the survival of patients treated with ATV-NDV. In this retrospective study, survival was estimated separately for three groups of patients who had early breast cancer  suggest that the vaccination strategy can stimulate human antitumor immune responses in a manner similar to those found in animal models, and may significantly prolong five-year survival rates in this patient population. The study demonstrated the feasibility and safety of the vaccine regimen, and no major side effects were observed in any of the patients .n = 57). Forty-eight patients were treated with ATV-NDV and nine were treated with autologous tumor cell vaccine (ATV) mixed with BCG bacteria, the adjuvant used by MG Hanna and colleagues in their studies in the USA. Two years after surgery, OS for patients treated by ATV-NDV was 98% compared with 67% for the patients treated with ATV + BCG and to 74% for historical control subjects [A Phase II trial involved patients with locally advanced CRC (subjects .n = 40), whose disease had spread from the kidneys to at least one other organ. After radical nephrectomy the patients were treated by ATV-NDV at three and five weeks after surgery. They were also given subcutaneous injections of low-dose recombinant interleukin-2 and recombinant IFN-α. Five patients had a complete response and six a partial response. After four years of follow-up, OS for these 11 patients was 100%. Among the remaining 29 patients, 12 had stable disease  and 17 had progressive disease  [Another Phase II trial involved renal cell cancer patients ( months) .n = 23) was 40 weeks versus 26 weeks in 87 non-vaccinated control subjects from the same time period and the same clinic. The median OS was 100 weeks . In the vaccinated group, immune monitoring revealed significant increases of skin DTH reactivity, of numbers of tumor-reactive MTCs in the blood and in the number of CD8+ tumor-infiltrating T lymphocytes in frozen tissue slices from GBM recurrences. There was one complete remission of non-resectable remaining brain tumor [A further Phase II study was performed with patients suffering from GBM. They were vaccinated with ATV-NDV vaccine obtained from cell culture. The objective was to assess feasibility, safety and clinical benefit. The median progression-free survival of vaccinated patients  for attachment to ATV-NDV to augment T-cell stimulatory signals and thereby overcome potential T cell anergy .In a phase I clinical study, 14 CRC patients with late-stage disease which could not anymore be subjected to surgery with curative intent were treated with ATV-NDV-bsHN-CD28. No severe adverse events were recorded. While before vaccination none of the patients had detectable levels of tumor-reactive blood circulatory T cells, all patients showed an immunological response of tumor-reactive T cells, at least once during the course of five vaccinations. Additionally, there was a dose-response relationship with the costimulatory molecule attached to the vaccine. A partial response of metastases was documented in four patients. The study suggests that the second-generation three-component vaccine is safe and can reactivate possibly anergic T cells from a chronic disease like advanced-stage cancer.The other strategy was to combine ATV-NDV with dendritic cells (DCs) in order to improve the de novo generation of TAA-specific cells from naïve T cells. A protocol for the generation of viral oncolysate-pulsed DCs (VOL-DCs) has been developed by the Immunological and Oncological Center (IOZK) in Cologne (Germany). In 2015, IOZK received an official permit for an individual application of this product to cancer patients. This permit required, among other factors, the production of high-quality oncolytic NDV under Good Manufacturing Procedure (GMP) guidelines. This was, worldwide, the first time that this had been achieved with NDV.For further information of the strategy which has been developed by IOZK to treat cancer patients, we refer to two recent case reports.One demonstrates long-term remission of prostate cancer with extensive bone metastases. The patient had failed standard therapy, but then achieved complete remission following combined treatment with local hyperthermia (LHT), systemic NDV and vaccination with VOL-DC. The treatment induced a long-lasting antitumor memory T-cell response .The other case report relates to long-term survival of a breast cancer patient with extensive liver metastases. After operation the patient refused further standard therapy. Instead, she was treated with LHT, systemic NDV and VOL-DC. A continuous high quality of life was reported, and the patient survived more than 66 months after the initial diagnosis. No recurrence of further metastases developed under treatment. Following treatment, a long-lasting tumor-reactive memory T-cell responsiveness was documented. This possibly explains the favorable course of disease. Since this combination of therapies is not restricted to a particular tumor type, further exploration is warranted.La Sota) in the treatment of tumors of the digestive tract [In 2003, Liang et al. from China reported results from applications of autologous tumor cell vaccine and NDV vaccine  is a cancer of major significance in China. Bian worked from 2003 to 2006 in my division at the German Cancer Research Center in Heidelberg. Back in China, she focuses on the development of new strategies for fighting this disease. We had developed a bispecific adaptor protein (αHN-IL-2) binding to NDV and to the interleukin-2 receptor IL-2R. The latter could be used as a surrogate tumor-associated target. The aim of her project in Heidelberg was: (i) to specifically target tumor tissue by oncolytic NDV; (ii) to improve the delivery system for systemic application of NDV via the bispecific adapter protein; and (iii) to investigate anti-tumor activity and side effects. Tumor therapy experiments in mice showed that the side-effects of high-dose virotherapy were greatly reduced by the adapter protein, and that the anti-tumor effects were mostly undiminished . This stItalien and generated two recombinant NDVs, carrying a marker gene encoding either enhanced green fluorescent protein (EGFP) or firefly luciferase [rNDV-18HL) with an incorporated antibody gene (cHAb18). This chimeric antibody is directed against tumor-associated antigen CD147 and demonstrated the inhibition of invasion and migration of HCC cells. The new antibody, expressing virus, selectively replicated in orthotopic HCC xenografts leading to antibody expression in situ. It induced tumor necrosis, reduced the intrahepatic metastases, and prolonged the survival in mice [Bian and colleagues managed to construct a reverse genetics system including a minigenome rescue  for NDV ciferase . Recentl in mice .Another finding from this group is of no less importance. Activated hepatic stellate cells (HSCs) are the crucial factor responsible for liver fibrosis and involved in development of HCC by interaction with tumor cells. It was reported in 2009 that NDV can repress the activation of human hepatic stellate cells and can revert the development of hepatic fibrosis in mice .A recent interesting report from China relates to the incorporation into NDV of a suicide gene coding for cytosine deaminase (CD). In animal experiments, the application of 5-FC significantly enhanced cell death of HepG2 tumor cells in vitro and increased survival of H22 tumor-bearing mice .Apart from Bian there were other women scientists from China in my Division who performed excellent and important work with NDV. Bai who, in China, had already done her thesis work on NDV, demonstrated that DCs pulsed with viral oncolysates potently stimulate autologous T cells from cancer patients . Zeng idAdvantages exist in using paramyxoviruses as oncolytic agents versus representatives of other viral families .Lack of gene exchange via recombination. Phylogenetic analyses of the F, HN and M genes from NDV isolates of the last 50 years demonstrate the existence of distinct lines [ct lines ,50. ThesLack of interaction with host cell DNA. The cytoplasmic replication results in a lack of host genome integration and recombination. The cytoplasmic replication makes the virus independent of host cell DNA replication.Virus replication independent of cell proliferation. This is in contrast to chemotherapy (CT) and radiotherapy (RT) which require target cells to be in a proliferating state. Many tumor cells in a resting state, such as tumor stem cells or cells from tumor dormancy, may not be affected by CT or RT, but are likely to become targeted by NDV.Mild side effects in cancer patients. The reported side effects after experience with clinical application for 50 years are grade 1 and 2, with the most common being mild fever.Measles virus (MV), Mumps virus (MuV), Sendai virus (SeV) and NDV. Metastatic cancer cells frequently overexpress on their surface molecules that can serve as virus receptors. For NDV, cell surface receptors are sialylated glycoproteins or glycolipids recognized by the cell adhesion domain of HN. Individual strains of NDV are classified as lytic or nonlytic viruses. Both types of viruses can kill cancer cells, but lytic strains have the potential to do this more quickly because they damage the plasma membrane of infected cells. In addition, lytic strains show multicyclic replication while nonlytic viruses show only monocyclic replication [The list of oncolytic paramyxovirus representatives includes attenuated lication . Nonlytilication , which tParamyxoviruses are capable of inducing syncytium-mediated lysis of cancer cells. Virus release is facilitated by the viral HN-associated neuraminidase and syncytium formation by the viral F protein. This process allows progeny viruses to spread within tumor tissue. In the case of nonlytic strains, progeny virus particles are non-infectious due to an uncleaved F protein, while lytic viruses express a cleaved F protein and release infectious virus progenies.NDV-induced apoptosis is dependent on upregulation of TNF-related apoptosis-inducing ligand (TRAIL) and caspase activation. The virus mediates its oncolytic effect by both intrinsic and extrinsic caspase-dependent pathways of cell death .Identified mechanisms that can explain tumor selectivity of virus replication and oncolysis in non-permissive hosts involve: (i) defects in activation of anti-viral signaling pathways; (ii) defects in type I IFN signaling pathways; (iii) defects in apoptotic pathways; and (iv) activation of Ras signaling and expression of Rac1 protein .In permissive hosts, namely avian cells, NDV has developed a frameshift variant of the viral phosphoprotein P to escape type I interferon mediated anti-viral responses . This V For therapy with oncolytic viruses (OVs), the anti-viral immune response limits OV replication and spread in situ. An initial period of vigorous OV multiplication and lytic activity seems necessary for development of subsequent anti-tumor immunity. Anti-viral T helper cells, however, could also have an interesting effect upon recruitment of DCs to the OV containing vaccination site . The useAn important new paradigm of OV-mediated immunotherapy is the concept of ICD. Classical physiological apoptosis is non-immunogenic. It is characterized by membrane integrity, cell shrinkage, membrane blebbing, release of apoptotic bodies, nuclear condensation and DNA fragmentation. In contrast, immunogenic apoptosis induced by NDV involves immunogenic apoptosis with translocation to the plasma membrane of calreticulin (Ecto-CRT), heat shock proteins (HSPs) and the viral proteins HN and F. This process is then followed by necrosis with the release of cytokines  and chemokines . PAMPs, such as foreign viral RNA in the cytoplasm, recognized by RIG-I receptors , and HN A consequence of ICD is the induction of post-oncolytic anti-tumor immunity. Innate immunity activation involves NK cells, monocytes, macrophages and DCs, including plasmacytoid DCs. This leads to upregulation of TRAIL on NK cells and monocytes, to induction of cytotoxicity by NK cells, to release of nitric oxide (NO) and TNF-α from macrophages, and to the secretion of high amounts of type I IFNs by monocytes, PDCs and DCs .NDV-induced IFN-α serves as a link between innate and adaptive immunity. In normal cells of non-permissive hosts , signaling via RIG-I  and amplification via an IFN induced feedback loop by membrane-expressed type I IFN receptor (IFNRA)  leads toUlster) was shown to lead to upregulation of MHC molecules and of the adhesion molecules ICAM-1 and LFA-3 [Infection of human tumor cells by NDV  or immunology (Schirrmacher) with a different view or vision, and make claims that things could be done in a different, and perhaps better, way, it is clear that this provokes reactions from the oncology establishment. The more the pioneer insists and continues, the more hurdles and roadblocks are put up to make them stop and fail.Thus, the question is not only to have a good idea for future progress in cancer therapy, but a great deal of perseverance is needed to continue, especially when there is lack of industrial and financial support, and of scientific recognition, at times where such topics are not popular. For Cassel, it was the period of the 1960s and 1970s, and for Schirrmacher, the period of the 1980s and 1990s. Progress in NDV research and application could have been much faster if there had been support instead of resistance and prejudice. Considering the positive clinical results summarized in this review, patients could have profited from NDV much earlier.The good news, however, is that the concept of using NDV for the improvements of cancer therapy has survived over five decades. Not only the ideas of viral oncolysis and post-oncolytic immune responses made it survive, but also its high safety profile.E1B gene deleted Adenovirus. In 2015, the FDA approved T-VEC, a GM-CSF gene-modified Herpes Simplex Virus (HSV), for application in advanced melanoma patients. T-VEC improved durable response rates [Two other OVs have been approved for clinical application. H101 was the first oncolytic virus approved for cancer treatment in China, in 2006 . H101 isse rates . The comse rates .When used as a single agent, NDV has been applied in animal models and/or cancer patients by different routes  and used over a large dose range. Specific vaccines, including NDV, were either viral oncolysates, live cell tumor vaccines or DC vaccines pulsed with viral oncolysate.The types of human cancer that were treated are highlighted in this review. They include 10 different types: GBM, Gastrointestinal carcinoma (Ca), CRC, breast Ca, ovarian Ca, HNSCC, renal cell Ca, prostate Ca, melanoma and acute leukemia.Fifty years of study on oncolytic NDV-enhanced immunotherapy of cancer is a long time. In spite of the many different applications of NDV, of different strains, treatment modalities and cancer types, there were no serious adverse events. It can, thus, be concluded that NDV has a very high safety profile for clinical application.It is my hope that this biologic agent, either wildtype or recombinant, will further increase its impact in the coming decades for the benefit of cancer patients.


An effective antitumor remedy is yet to be developed. All previous approaches for a targeted delivery of anticancer medicine have relied on trial and error. The goal of this study was to use structural insights gained from the study of delivery systems and malignant cells to provide for a systematic approach to the development of next-generation drugs. We used doxorubicin (Dox) liposomal formulations. We assayed for cytotoxicity via the electrical current exclusion method. Dialysis of the samples yielded information about their drug release profiles. Information about the surface of the delivery systems was obtained through synchrotron small-angle X-ray scattering (SAXS) measurements. SAXS measurements revealed that Dox-loading yielded an abraded surface of our Dox liposomal formulation containing soybean oil, which also correlated with an effective reduction of the survival of carcinoma cells. Furthermore, a dialysis assay revealed that a higher burst of Dox was released from soybean oil-containing preparations within the first five hours. We conclude from our results that an abraded surface of Dox-loaded drug delivery system increases their efficacy. The apparent match between surface geometry of drug delivery systems and target cells is suggested as a steppingstone for refined development of drug delivery systems. This is the first study to provide a systematic approach to developing next-generation drug carrier systems using structural insights to guide the development of next-generation drug delivery systems with increased efficacy and reduced side effects. Cancerous lesions are predicted to be the leading cause of deaths in the U.S. within the next five years . ApproacReducing the unintended, chemotherapy-associated toxicity profile for cancer patients requires the development of new therapeutic formulations. One approach to address this unmet clinical need is the development of targeted drug delivery using carrier systems with a long shelf life, optimal drug-loading profile, and low inherent toxicity. See ,5 for reDoxorubicin (Dox), either alone ,7,8 or i® prompted the U.S. Food and Drug Administration to approve the usage of a generic version of this liposomal Dox formulation for the treatment of, e.g., advanced or persistent ovarian cancer after prior platinum-derived chemotherapy  in µM against time in hours resulted in the graph shown in In the in vitro tests, we used the differential sensitivity of HeLa and HCT116 cells to chemotherapeutic drugs, such as Dox, ethyl methane-sulfonate, and 5-fluorouracil ,29. We pIn our assays, using a pharmacologically relevant [Dox] of 1 µM  yielded In vitro performance assays using used plain carriers, denoted as S0 and M0, and Dox-loaded carriers (M1: 180 µM and S1: 360 µM) yielded potent effects of STS-containing and Dox-loaded soybean (S) oil-based carrier systems in comparison to their respective Mygliol 812 (M) counterpart (see above). We therefore wished to obtain structural insights into these carrier systems to possibly correlate them with our in vitro performance results. Therefore, SAXS beamline measurements were performed at the Brazilian Synchrotron Light Laboratories as described above. We used plain carriers, denoted as S0 and M0, and Dox-loaded carriers  in our measurements.−1 and q2 = 3.02 nm−1, implying that all the samples presents 3-dimensional cubic structure  of 1 µM when STS is present in these formulations. Structural interrogation of these systems revealed that loading with Dox abraded the initially smooth surface of soybean oil-based carrier formulations, in stark contrast to a diffused surface of Mygliol 812-based formulations under comparable conditions.Our SAXS experimentation revealed support for the notion that the Dox-loaded and STS-containing soybean oil-derived or Mygliol 812 drug delivery systems are indirectly connected to the surface of the system with a dense core regardless of the amounts of Dox loaded. The probability of the formation of hybrid electron density distributions as a result of the scattering event itself allows for estimations of absolute values of the slope α of the intensity of the scattering signal I . There iTherefore, for the STS-containing soybean oil-derived system, the value of the power law scattering exponent |α| demonstrates a fractal surface which explains a better interaction with the fractal surface of HeLa cells . LikewisThe carrier systems surface was abraded after Dox loading of soybean oil carriers. Further studies are necessary to explicate the different surface geometry of the carrier systems studied. The analysis obtained by SAXS consolidates the results observed in vitro using HeLa and HCT116 cells. The SAXS data analysis shows that the interaction of the drug in the rough surface (soybean oil) provides a better delivery when compared to the interaction of the drug in the diffuse surface (Mygliol 812).In conclusion, this is the first study where characteristics of the surface geometry of drug delivery systems, expressed as fractality, is correlated with their antitumor efficacy based on compounds used in food and cosmetic industry, where a substantially reduced risk of HFS can be expected in comparison to the commercially available PEGylated Dox formulation Doxil. Furthermore, this study is providing a new and systematic approach to the development of new anticancer medication with reduced side effects and increased benefits for patients.