We observed that both EBV- and HCMV-specific T cell responses were significantly lower in SLE patients compared with healthy subjects. We reported decreased EBV- and HCMV-specific T cell responses among medium-high immunosuppressed patients compared to low immunosuppressed patients. Immunosuppressive level could exert a role in the control of herpesviruses reactivation, even if the immunosuppressive condition of SLE remains the driving cause of skewed virus-specific T cell response.Systemic lupus erythematosus (SLE) is an autoimmune disease with a complex etiology. Opportunistic viral pathogens, such as human cytomegalovirus (HCMV) and Epstein-Barr virus (EBV), are particularly relevant. The role of the T cell response in SLE has not been deeply studied; we investigated the role of HCMV- and EBV-specific T cell responses in SLE patients also in relation to their pharmacological immunosuppressive status. PBMCs from 70 SLE patients and 50 healthy controls were stimulated with EBV- and HCMV-specific antigens, and IFN- Herpesviridae family, and they may complicate the disease course [γ) is also suggested to play a crucial role in this context [Systemic lupus erythematosus (SLE) is a complex pathological condition  which mae course –15 or mie course . Furthere course . It has e course , 23, whie course . In this context .γ ELISPOT assay. By using a novel approach we provided a good estimation of both CD4+ and CD8+ antigen-specific T cell responses, avoiding predepletion assay [+ and CD8+ antigen-specific T cell response, avoiding the intracellular cytokine staining approach that is labor intensive and requires a larger number of cells. However, this approach cannot be considered as precise as flow cytometry strategy, but could represent an easier way for the estimation of antigen-specific T cell response. For comparison, T cell response to the nonspecific mitogen (PHA) was also investigated.The aim of this study was to evaluate and characterize T cell responses to HCMV and EBV in SLE patients using IFN-on assay . In thisSeventy patients  38.0-57.8) fulfilling the 1997 ACR classification criteria for SLE  and refeSLE patients had a median age at disease onset of 30 (IQR 23-46) years and a median disease duration of 121.5 (IQR 42.3-228.5) months. In all cases, disease activity was evaluated according to SLEDAI 2k score [For practical purposes, we divided the patients into two groups, according to the degree of pharmacological immunosuppression: patient treatment with hydroxychloroquine and/or with prednisone ≤ 5 mg/day was considered low pharmacological immunosuppression . Patient treatment with mycophenolate mofetil, methotrexate, cyclosporin A, rituximab, belimumab, and/or prednisone > 5 mg/day was considered medium-high pharmacological immunosuppression .μg/ml streptomycin, 25% human albumin , and 10% DMSO ), and stored in liquid nitrogen (10 × 106 cells/ml) until analysis. After thawing, about 50-60% of cells were still viable and could be used in the ELISPOT assay.Peripheral blood was collected into vacutainer tubes (BD) containing heparin. Whole blood was used for viral genome quantification and determination of T cell subsets; plasma was separated for serological analyses. Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation , cryopreserved in freezing medium , IgG anti-VCA, and anti-EBNA were quantified using an ELISA kit , according to the manufacturer's instructions. Healthy subjects and SLE patients were considered EBV-seropositive when IgG anti-VCA and anti-EBNA were positive.IgM anti-HCMV and IgG anti-HCMV were quantified using an ELISA kit , according to the manufacturer's instructions. Healthy subjects and SLE patients were considered HCMV-seropositive when IgG anti-HCMV was positive.DNA was purified using NucliSENS®easyMAG® . EBV DNA and HCMV DNA were quantified using real-time PCR (lower limit detection HCMV and EBV DNA 90 copies/ml), as previously described  were determined by flow cytometry , using Flow-Count Fluorospheres. Gating strategy was set up on CD45+ and side scatter (SSC).Fresh whole blood was stained with anti-CD3-PC5, anti-CD45-FITC, anti-CD4-RD1, and anti-CD8-ECD monoclonal antibodies . After lysis of red blood cells, absolute CD3μg/ml streptomycin with 8% of DMSO. Resuspended peptide pools were used after dilution (1 : 100) in RPMI 1640 supplemented with 2 mM L-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin, and 10% FBS and then used as antigens. A lytic pool, containing peptides spanning the full length of BZLF-1 (59 peptides) and BMRF-1 (99 peptides) EBV proteins; an Epstein-Barr nuclear antigen (EBNA) pool, containing peptides spanning the full length of EBNA 1 (158 peptides), EBNA3a (234 peptides), EBNA 3b (279 peptides), and EBNA 3c (265 peptides) EBV proteins; and a latent membrane protein (LMP) pool, containing peptides spanning the full length of LMP1 (94 peptides) and LMP2 (122 peptides) EBV proteins, were used as EBV-specific antigens  at a final concentration of 0.25 μg/ml for each individual peptide in the corresponding pool. Peptide pools representative of whole HCMV proteins IE-1 (120 peptides), IE-2 (143 peptides), and pp65 (138 peptides) (JPT Peptide Technologies) were used at a final concentration of 0.25 μg/ml for each individual peptide in the corresponding pool.Lyophilized peptide pools, 15 amino acids in length with an 11 amino acid overlap, were resuspended in RPMI 1640 supplemented with 2 mM L-glutamine, 100 U/ml penicillin, and 100 γ ELISPOT kits  and Multiscreen-IP membrane-bottomed 96-well plates  were used as described [γ and stored at 4°C. After washing with PBS, plates were blocked with culture medium  (Euroclone)) for 2 hours at room temperature. Cells were plated in duplicate (1 × 105/100 μl per well) and stimulated with the corresponding antigens or with phytohemagglutinin  or with medium alone (negative control) and incubated at 37°C in a 5% CO2 humidified atmosphere for 24 hours. After washing, plates were incubated overnight at 4°C with biotinylated IFN-γ detection antibody. Plates were washed, streptavidin-alkaline phosphatase conjugate was added, and plates were incubated at 37°C in a 5% CO2 atmosphere for 1 hour. Plates were then washed, and 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (BCIP/NBT) was added for 20 minutes at room temperature. Wells were then washed several times under running water and air-dried overnight. Spots were counted by using an automated AID ELISPOT reader system . The mean number of spots from duplicate wells was adjusted to 1 × 106 PBMCs. The net spots per million PBMCs was calculated by subtracting the number of spots responding to the negative control from the number of spots responding to the corresponding antigenand results were given as net spots/million PBMCs. Furthermore, results were normalized to absolute CD4+ and CD8+ T cell counts, as previously described [Human IFN-escribed –31. Briet-test or Mann-Whitney test was used for quantitative variables in order to perform comparison between groups. Spearman's test was used for the correlation analysis. All tests were two-tailed. A P value < 0.05 was considered statistically significant. Analyses were performed using the GraphPad Prism 5 .Descriptive data were reported or considered as absolute and relative frequencies, mean and standard deviation, median, and interquartile range (IQR) based on the type of the variable distribution. For qualitative variables, Fisher's test was used, while + and CD8+ T cells were significantly lower in SLE patients than in healthy controls (P < 0.0001). In SLE patients, the median CD4+ and CD8+ T cell counts were 434.5 (IQR 270.3-620.3) cells/μl and 287.0 (IQR 196.0-397.3) cells/μl, respectively, while in healthy subjects, the median CD4+ and CD8+ T cell counts were 1054.0 (IQR 760.5-1361.0) cells/μl and 532.0 (IQR 376.8-748.8) cells/μl, respectively. EBV- and HCMV-specific antibodies were analyzed in all enrolled subjects, as well as HCMV and EBV DNAemia. Demographic data are summarized in T cell subsets were analyzed in 70 SLE patients and 50 healthy subjects with homogeneous characteristics. The two groups were not substantially different in terms of age and sex distribution. Both CD4+ and CD8+ T cell responses to all EBNA, LMP, and lytic overlapping 15-mer peptide pools were evaluated in 68 EBV-seropositive SLE patients, 25 EBV-seropositive healthy subjects, and four EBV-seronegative individuals .EBV-specific CD4P = 0.0002)  vs 1.140 (IQR 0.5235-2.6180) EBV-specific CD4+ T cells/μl EBV-specific CD4+ T cells/μl; P < 0.0001) (+ T cell response was significantly different in the two groups of subjects (0.0905 (IQR 0.0215-0.2148) and 0.5620 (IQR 0.2335-1.231) EBV-specific CD8+ T cells/μl, respectively, P < 0.0001) (+ and CD8+ T cell responses with the SLEDAI 2k score in SLE patients (data not shown).Median EBV-specific T cell response was significantly lower in EBV-seropositive SLE patients than in EBV-seropositive healthy subjects (340.5 (IQR 75-738.8) vs. 890 (IQR 360-1983) net spots/million PBMCs;  0.0002) . Normali 0.0001) . Similar 0.0001) . There wn = 25, 36.8%) or mhp-IS . The latter had significantly lower EBV-specific T cell response than the other group (170 (IQR 70-530) and 500 (IQR 237.5-905.0) net spots/million PBMCs, respectively; P = 0.0224) (+ and CD8+ T cells. The EBV-specific CD4+ T cell response was lower in mhp-IS than in lp-IS (0.0620 (IQR 0.0170-0.2680) and 0.2420 (IQR 0.1065-0.4515) EBV-specific CD4+ T cells/μl; P = 0.0124) (+ T cell response in SLE patients receiving mhp-IS was lower than that observed in patients receiving lp-IS (0.1430 (IQR 0.0800-0.3335) and 0.0450 (IQR 0.0180-0.1640) EBV-specific CD8+ T cells/μl; P = 0.0174)  . Results 0.0124) . Similar 0.0174) .+ and CD8+ T cell EBV-specific ELISPOT response in SLE patients with undetectable EBV DNA  and detectable EBV DNA . A trend toward statistical significance was observed when we compared the EBV-specific T cell response measured as net spots/million PBMCs in SLE patients with undetectable (median 387.5 (IQR 119.8-953.8)) and detectable EBV DNA (220 (IQR 56.3-538.8); P = 0.0753) (+ T cells/μl between the two groups (0.202 IQR (0.038-0.450) and 0.072 IQR (0.016-0.236) EBV-specific CD4+ T cells/μl; P = 0.0773)  EBV-specific CD8+ T cells/μl and 0.0710 IQR (0.0157-0.1565) EBV-specific CD8+ T cells/μl; P = 0.1070)  . Similar 0.0773) , althoug 0.1070) .P = 0.0373) (+ T cell response in mhp-IS patients (median 0.040 IQR (0.013-0.160) EBV-specific CD4+ T cells/μl) was compared with lp-IS patients (median 0.223 IQR (0.097-0.398) EBV-specific CD4+ T cells/μl; P = 0.0573) (+ T cell response in mhp-IS (0.034 IQR (0.011-0.120) EBV-specific CD8+T cells/μl and lp-IS patients (0.097 IQR (0.080-0.210) EBV-specific CD8+ T cells/μl; P = 0.0394)  were receiving mhp-IS and nine (30%) lp-IS. In a comparison of the two groups, EBV-specific T cell response measured as net spots/million PBMCs was higher in lp-IS group (median 110 IQR (37.5-460)) than in mhp-IS (median 380 IQR (313-750);  0.0373) . A trend 0.0573) . A signi 0.0394) . No diffP = 0.3679)  vs 0.6065 (IQR 0.1368-1.250) HCMV-specific CD4+ T cells/μl; P = 0.0004) (+ T cell response was significantly different in the two groups of subjects (0.6490 (IQR 0.1480-1.280) vs 2.279 (IQR 0.7108-3.570) HCMV-specific CD8+T cells/μl; P = 0.0059). Both groups of HCMV-seropositive subjects  had HCMV-specific responses higher than HCMV-seronegative controls (P < 0.0001) . Seventeen out of 55 (30.9%) SLE patients were receiving lp-IS, while the remaining 38 (69.1%) were receiving mhp-IS. The HCMV-specific ELISPOT response measured as net spots/million PBMCs was significantly higher in lp-IS patients (median 2695 (IQR 1003-6525)) than in mhp-IS patients (median 1055 (IQR 271-1810)) (P = 0.0282) (+ T cell response was significantly reduced in this mhp-IS patients (0.271 IQR (0.125-1.083) vs 0.927 IQR (0.611-2.315) HCMV-specific CD4+ T cells/μl; P = 0.0179) (+ T cell response between mhp- and lp-IS (0.359 IQR (0.071-0.738) vs 0.910 IQR (0.383-2.090) HCMV-specific CD8+ T cells/μl; P = 0.0111) . HCMV-specific T cell response was lower in HCMV-seropositive SLE patients than in HCMV-seropositive healthy subjects (median 1155 (IQR 415-3925) and 1850 (770-3103) net spots/million PBMCs, respectively), but the difference was not statistically significant ( 0.3679) . Results 0.0004) . Similar 0.0001) . There w 0.0282) . Similar 0.0179) . Finally 0.0111) .We observed that ELISPOT T cell response in the 52 SLE patients with undetectable HCMV DNA was higher than the response observed in the three patients with detectable HCMV DNAemia (data not shown). However, due to the low number of patients in the latter group, the difference was not considered statistically valid.All patients with detectable HCMV DNA were medium-high immunosuppressed. Two of them were also positive for EBV DNA.+ and CD8+ T cells producing IFN-γ in response to PHA, a nonspecific antigen, were significantly lower in 70 SLE patients than in 41 healthy controls. PHA-specific T cell responses measured as net spots/million PBMCs were 4795 (IQR 2956-7193) and 6915 (5449-8165) (P = 0.0140), respectively,  and 6.890 (IQR 4.542-10.89) PHA-specific CD4+ T cells/μl (P < 0.0001), respectively,  and 3.859 (IQR 2.199-5.700) PHA-specific CD8+T cells/μl (P < 0.0001)  . There w+ and CD8+ T cells producing IFN-γ, as previously described [γ ELISPOT is one of the most widespread assays used to evaluate antigen-specific T cell response. It allows the evaluation of total CD4+ and CD8+ antigen-specific T cell response by detection of IFN-γ-producing T cells. As known, IFN-γ is a cytokine mainly produced by activated T helper 1 (Th1) and T cytotoxic cells in response to specific antigens, having a crucial role inactivating lymphocytes to enhance antimicrobial and antitumor effects. The traditional ELISPOT assay does not easily distinguish between CD4+ and CD8+ T cell responses unless separate antigens to detect CD4+ and CD8+ T cell responses or lymphocyte subset depletion are used [+ and CD8+ T cells [+ and CD8+ T cell counts, in order to estimate both CD4+ and CD8+ T cells producing IFN-γ, in response to different viral antigens. Even if it does not allow a precise quantification of CD4+ and CD8+ antigen-specific T cell response, our previous results evidenced a good correlation between intracellular cytokine staining and normalized ELISPOT approach [The aim of this study was to evaluate EBV- and HCMV-specific T cell responses by ELISPOT assay. In this setting, we used the normalization on CD4 and CD8 T cells in order to provide an estimation of CD4escribed . As our are used . EBV- an T cells , 34. Resapproach .Among viral pathogens, EBV infection is the most common in patients with SLE, and more importantly, it has been hypothesized that EBV may play a role in SLE disease induction . In a st+ and CD8+ T cell responses to EBV-specific peptide pools were significantly lower in SLE patients compared with controls. Berner et al. demonstrated that the frequency of EBV-specific CD8+ T cells was similar between SLE and healthy subjects when analyzed by using MHC I tetramers with a lytic cycle EBV antigen peptide. However, CD8+ T cell EBV-specific IFN-γ production was significantly lower in SLE patients than in healthy controls, when assayed by ELISPOT [+ T cell EBV-specific response was provided. In contrast with our results, Kang et al. showed an increased frequency of EBV-specific CD69+ CD4+ T cells producing IFN-γ in SLE patients compared with controls. However, the frequency of EBV-specific CD69+ CD8+ T cells producing IFN-γ tended to be lower in SLE patients [Both CD4 ELISPOT . Neverthpatients . These dγ production upon stimulation with EBNA 1 or EBV early antigen diffuse (EBV-EA/D) in SLE patients, suggesting the decreased control of EBV infection in these subjects [Similarly, Draborg et al. showed a significantly reduced number of activated T cells and IFN-subjects . InteresA few studies –39 have + CD4+ T cells producing IFN-γ and TNF-α was similar in SLE patients and healthy controls, while the frequency of HCMV-specific CD69+ CD8+ T cells producing IFN-γ and TNF-α was lower in SLE patients, although the difference was not statistically significant [+ and CD8+ T cell IFN-γ-producing cells.Larsen et al. did not find immune alterations in HCMV-specific T cell responses in SLE patients compared with controls . Anothernificant . On the Analyzing HCMV-specific T cell response in SLE patients according to immunosuppression level, we do not have the evidence of the difference between lp-IS SLE patients and healthy HCMV-seropositive subjects, while mhp-IS patients showed a markedly reduced HCMV-specific T cell response if compared to lp-IS patients as well as to healthy HCMV-seropositive subjects. This could support the role of immunosuppressive treatment in the control of HCMV reactivation.Interestingly, in our study, a significantly lower T cell response to the specific PHA antigen in SLE patients compared with healthy subjects was demonstrated, while other authors have not shown any differences , 38, sug+ and CD8+ T cell responses in SLE patients treated with medium-high immunosuppression. Similarly, HCMV-specific CD4+ and CD8+ T cells had reduced IFN-γ production in medium-high immunosuppressed SLE patients. On the contrary, there was no difference in terms of PHA-specific T cell response between low and medium-high immunosuppressed SLE patients.In contrast with other studies , 39, we The role of immunosuppressive therapy appears to be crucial mainly to the response to EBV but also to HCMV-specific T cell response. Furthermore, a generally deficient immune response in SLE patients with respect to healthy controls was observed, supporting the hypothesis that SLE disease exerts a general immunosuppressive action regardless of the therapy. To corroborate this hypothesis, virus-specific T cell responses need to be analyzed in a larger number of patients, in order to stratify patients according to immunosuppressive status and iatrogenic risk factors.


The near-complete genome sequence of foot-and-mouth disease virus (FMDV) serotype A potential vaccine strain BAN/CH/Sa-304/2016 is reported here. Its genome revealed antigenic heterogeneity with the current Indian vaccine strain IND40/00, with four amino acid substitutions in antigenically critical sites of the VP1 protein. The near-complete genome sequence of foot-and-mouth disease virus (FMDV) serotype A potential vaccine strain BAN/CH/Sa-304/2016 is reported here. Its genome revealed antigenic heterogeneity with the current Indian vaccine strain IND40/00, with four amino acid substitutions in antigenically critical sites of the VP1 protein. Aphthovirus in the family Picornaviridae. FMDV exists as seven immunologically distinct serotypes  with multiple topotypes, lineages, and sublineages (Foot-and-mouth disease (FMD) is a highly contagious disease of domestic and wild cloven-hoofed animals worldwide . Foot-anlineages . Among alineages . All thelineages , 5.r > 0.3)  . The serr > 0.3) . The ampr > 0.3) .The near-complete genome of the vaccine candidate BAN/CH/Sa-304/2016 is 8,221 nucleotides (nt) in length, consisting of 53.49% GC content, with a 5′ untranslated region (5′ UTR), coding sequence (CDS), and 3′-UTR region of 1,101, 6,999, and 121 nt, respectively. The CDS codes for a polyprotein 2,332 amino acids (aa) long, containing four structural proteins, VP1 to -4, and eight nonstructural proteins , while the 5′-UTR poly(C) tract and 3′-UTR poly(A) tail are 16 nt and 25 nt long, respectively. Phylogenetically, the isolates BAN/CH/Sa-304/2016 and BAN/CH/Sa-302/2016  clustered in a separate clade closely related to other isolates from Bangladesh and India within genotype VII of the Asia topotype .KJ754939) and 91% with Indian vaccine strain IND40/00 (GenBank accession no. HM854025). The VP1, VP2, and VP3 protein sequences of BAN/CH/Sa-304/2016 had 8%, 6%, and 5% variability, with 16, 12, and 9 aa substitutions compared to the respective regions of vaccine strain IND40/00. Only 4 aa substitutions  of VP1 were in antigenically critical sites (59 position of BAN/CH/Sa-304/2016.The homology search in GenBank using BLAST showed that BAN/CH/Sa-304/2016 shares 96% nucleotide similarity with previous serotype A isolate BAN/GA/Sa-197/2013 (GenBank accession no. al sites . One amiWe report here the near-complete genome sequence of FMDV serotype A potential vaccine strain BAN/CH/Sa-304/2016 for FMD control in Bangladesh.MK088171.The near-complete genome sequence of FMDV serotype A strain BAN/CH/Sa-304/2016 has been deposited in the NCBI GenBank database under the accession no.


Trypanosoma equiperdum primarily parasitizes the genital organs and causes dourine in equidae. We isolated a new T. equiperdum strain, T. equiperdum IVM-t1, from the urogenital tract of a horse definitively diagnosed as having dourine in Mongolia. Trypanosoma equiperdum primarily parasitizes the genital organs and causes dourine in equidae. We isolated a new T. equiperdum strain, T. equiperdum IVM-t1, from the urogenital tract of a horse definitively diagnosed as having dourine in Mongolia. Here, we report the whole-genome sequence, the predicted gene models, and their annotations. Trypanosoma equiperdum belongs to the kingdom Protista, phylum Sarcomastigophora, class Zoomastigophorea, order Kinetoplastida, family Trypanosomatidae, genus Trypanosoma, subgenus Trypanozoon together with T. brucei and T. evansi   , EthiopiICT2011) , and Ven in 2005 , 2015 (7T. equiperdum strain IVM-t1 isolate was cultivated using Hirumi's modified Isocove's medium-9 (HMI-9) soft-agarose medium  at 37°C in 5% CO2 (T. equiperdum IVM-t1 was extracted and purified using Tris-EDTA (TE)-saturated phenol (Sigma-Aldrich Japan) and phenol-chloroform isoamyl-alcohol solution (Sigma-Aldrich Japan) , a DNA/polymerase binding kit P6 v2, and a DNA sequencing kit 4.0 v2 for PacBio RS II sequencing . The MiSeq sequencer produced 28,337,050 paired-end reads with an average read length of 300 bp, and the PacBio RS II sequencer produced 7,508 reads with an average read length of 8,523 bp . The low-quality reads were trimmed using FastQC v0.11.5 and FASTX-Toolkit v0.0.13.The h Japan) . PurifieT. equiperdum by Metassembler v1.5 (T. equiperdum IVM-t1 were performed using the published genome of T. brucei brucei (T. b. brucei) TREU927 as a reference via the Companion pipeline (https://companion.sanger.ac.uk/) (Whole-genome assembly was performed with ABySS-2.0.2  with parler v1.5  with the.ac.uk/)  with defN50 value of 859,849 bp and a cumulative length of 26,988,997 bp (≈27 Mbp). The T. equiperdum IVM-t1 draft genome contains 7,718 protein-coding genes, 102 noncoding genes, and 2,473 pseudogenes. Following comparison of the predicted genes of T. equiperdum IVM-t1 with the reference gene sets of T. b. brucei TREU927 using OrthoMCL v2.0.7 (T. equiperdum IVM-t1-specific protein-coding genes were identified. Seven short genes that have less than 25 amino acids were not included in the comparison (https://doi.org/10.6084/m9.figshare.7552262.v1).The integrated draft genome consisted of 45 contigs with an L v2.0.7 , 6,831 pT. equiperdum-specific genes.In conclusion, the whole-genome draft assembly produced in the present study provides a resource for future trypanosome genetic studies and identifies some QSBY00000000. The version described in this paper is the first version, QSBY01000000. Raw sequence reads have been deposited in the NCBI SRA database under the accession no. PRJNA477427.This whole-genome project has been deposited in DDBJ/ENA/GenBank under the accession no.


N-methyl-d-aspartate/glutamate receptor (NMDAR) is one of the major voltage-sensitive ligand-gated cation channel. Several noncompetitive NMDAR antagonists contribute to pathophysiology of schizophrenia and mood disorders; however, the effects of inhibition of NMDAR on several transmitter system have not been well clarified. Thus, this study determined the selective NMDAR antagonist, MK801 (dizocilpine), on thalamocortical, mesothalamic, and mesocortical transmissions associated with l-glutamate, GABA, serotonin, norepinephrine, and dopamine using multiprobe microdialysis. Perfusion with MK801 into the medial prefrontal cortex (mPFC) increased and decreased respective regional releases of monoamine and GABA without affecting l-glutamate. The mPFC MK801-induced monoamine release is generated by the regional GABAergic disinhibition. Perfusion with MK801 into the reticular thalamic nucleus (RTN) decreased GABA release in the mediodorsal thalamic nucleus (MDTN) but increased releases of l-glutamate and catecholamine without affecting serotonin in the mPFC. The RTN MK801-induced l-glutamate release in the mPFC was generated by GABAergic disinhibition in the MDTN, but RTN MK801-induced catecholamine release in the mPFC was generated by activation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate/glutamate receptor (AMPAR) which received l-glutamate release from thalamocortical glutamatergic terminals in the mPFC. Perfusion with MK801 into the dorsal raphe nucleus (DRN) decreased GABA release in the DRN but selectively increased serotonin release in the MDTN and mPFC. These DRN MK801-induced serotonin releases in the both mPFC and MDTN were also generated by GABAergic disinhibition in the DRN. These results indicate that the GABAergic disinhibition induced by NMDAR inhibition plays important roles in the MK801-induced releases of l-glutamate and monoamine in thalamic nuclei and cortex. N-methyl-d-aspartate/glutamate receptor (NMDAR) modulation has been a source of scientific discussion in psychiatry and psychopharmacology, since various clinical studies have demonstrated that a noncompetitive NMDAR antagonist, ketamine contributes to pathophysiology of schizophrenia and mood disorders   = 20.9 (p < 0.05), FTime = 43.7 (p < 0.05), FMK801*Time = 36.4 (p < 0.05)]  = 18.8 (p < 0.05), FTime = 32.0 (p < 0.05), FMK801*Time = 25.9 (p < 0.05)]  = 7.4 (p < 0.05), FTime = 56.2 (p < 0.05), FMK801*Time = 22.2 (p < 0.05)] (l-glutamate level in the mPFC (Perfusions with MK801 (1 and 5 μM) into the mPFC increased regional extracellular levels (mPFC MK801-induced release) of serotonin [F< 0.05)] C, norepi< 0.05)] D and dop< 0.05)] E, but de< 0.05)] B withoutthe mPFC A. Extracthe mPFC C–E; howethe mPFC B. Therefl-glutamate, GABA, serotonin, norepinephrine, or dopamine  = 11.4 (p < 0.05), FTime = 74.3 (p < 0.05), FMK801*Time = 17.0 (p < 0.05)])  = 9.1 (p < 0.05), FTime = 65.2 (p < 0.05), FMK801*Time = 12.1 (p < 0.05)]  = 7.3 (p < 0.05), FTime = 47.1(p < 0.05), FMK801*Time = 5.3(p < 0.05)] ]) C, norepi< 0.05)] D and dop< 0.05)] E without< 0.05)] B. These l-glutamate release in the mPFC    = 16.4 (p < 0.05), FTime = 46.7 (p < 0.05), FMK801*Time  = 18.5 (p < 0.05)]  = 9.0 (p < 0.05), FTime = 49.2 (p < 0.05), FMK801*Time = 21.3 (p <0.05)]  into the RTN increased extracellular levels of < 0.05)] A, norepi< 0.05)] D, and do <0.05)] E without <0.05)] B,C. Extr <0.05)] D,E; howeμM MK801 A. Therefl-glutamate   = 7.2 (p < 0.05), FTime = 21.3 (p < 0.05), FMK801*Time = 7.5 (p < 0.05)]  = 9.0 (p < 0.05), FTime = 73.7 (p < 0.05), FMK801*Time = 12.6 (p < 0.05)]  into the RTN increased extracellular levels of < 0.05)] A and ser< 0.05)] C, and de< 0.05)] B without< 0.05)] D. The exl-glutamate level was increased by 5 μM MK801, but not by 1 μM MK801  and perampanel (AMPAR antagonist) into the MDTN and mPFC on transmitter releases in the mPFC and MDTN induced by 50 μM MK801 perfusion into the RTN .The specific noradrenergic, dopaminergic and serotonergic terminals from respective LC (locus coeruleus), VTA  and DRN  project to deeper layers of frontal cortex ,26,33,37A-R agonist)  and 1 μM perampanel (AMPAR antagonist)  into MDTN reduced 50 μM RTN MK801-induced l-glutamate release in the mPFC  = 7.5 (p < 0.05), FTime = 65.2 (p < 0.05), FPER*Time = 8.5 (p < 0.05)] and mPFC   = 11.6 (p <0.05), FTime = 48.1 (p <0.05), FMus*Time = 17.8 (p <0.05)], but perfusion with 1 μM muscimol into the mPFC did not affect  = 5.0 (p < 0.05), FTime = 107.2 (p < 0.05), FPER*Time = 9.8 (p < 0.05)] and mPFC   = 5.3 (p < 0.05), FTime = 87.2 (p < 0.05), FMus*Time = 22.7 (p < 0.05)], but perfusion with muscimol into the mPFC did not affect (Contrary to < 0.05)] D. Perfust affect D. Simila< 0.05)] E. Perfust affect E.l-glutamate release in the mPFC is generated by the GABAergic disinhibition and relatively activation of AMPAR in the MDTN, but is not modulated by these receptors in the mPFC. Contrary to l-glutamate, RTN MK801-induced catecholamine release in the mPFC is regulated by the GABAergic disinhibition in the MDTN, and activation of AMPAR in the MDTN and mPFC, but is not modulated by GABAA-R in the mPFC. Therefore, RTN MK801-induced catecholamine release in the mPFC is activated by AMPAR in the mPFC via activation of thalamocortical glutamatergic transmission.These results suggest that RTN MK801-induced A-R agonist) into MDTN reduced 50 μM RTN MK801-induced l-glutamate release in the MDTN, whereas perfusion with 1 μM perampanel (AMPAR antagonist) into the MDTN did not affect  was regulated by independent system compared with other catecholamine, since serotonin release in the mPFC is not affected by thalamocortical glutamatergic transmission, but conversely mesothalamic serotonergic transmission possibly affects thalamocortical glutamatergic pathway in the MDTN  . To clarMK801 = 16.8 (p < 0.05), FTime = 42.9 (p < 0.05), FMK801*Time = 16.1 (p < 0.05)]  into the DRN concentration-dependently increased extracellular serotonin level in the mPFC [F< 0.05)] C withoutdopamine A,B,D,E. dopamine C. TherefMK801 = 41.9 (p < 0.05), FTime = 63.8 (p < 0.05), FMK801*Time = 24.0 (p < 0.05)] ] C withoutdopamine A,B,D,E. MK801 = 11.6 (p < 0.05), FTime = 26.0 (p < 0.05), FMK801*Time = 6.3 (p < 0.05)]  into the DRN concentration-dependently increased extracellular serotonin level in the MDTN [F< 0.05)] C withoutdopamine A,B,D. Exdopamine C. Our prMK801 = 21.5 (p < 0.05), FTime = 36.3 (p < 0.05), FMK801*Time = 6.1 (p < 0.05)] ] C. TherefMK801 = 13.6 (p < 0.05), FTime = 53.3 (p < 0.05), FMK801*Time = 15.8 (p < 0.05)]  = 9.9 (p < 0.05), FTime = 42.9 (p < 0.05), FMK801*Time = 12.1 (p < 0.05)]  into the DRN concentration-dependently increased and decreased extracellular levels of serotonin [F< 0.05)] C and GAB< 0.05)] B in the  the DRN A,D,E. Ex the DRN B,C. TherMK801 = 22.0 (p < 0.05), FTime = 42.0 (p < 0.05), FMK801*Time = 6.1 (p < 0.05)] ] C, wherea< 0.05)] B. These The threshold concentration of local administration (perfusion) of MK801 into the mPFC, MDTN, RTN, and DRN on several transmission systems demonstrated by this study and previous reports ,16,17,20l-glutamate, GABA, serotonin, norepinephrine and dopamine in the mPFC, MDTN and DRN. According to the results in this study and published neural circuits [The present study also demonstrated the presence of several regulatory systems in the thalamocortical (RTN-MDTN-mPFC) glutamatergic, mesothalamic (DRN-MDTN), and mesocortical (DRN-mPFC) serotonergic pathways controlling releases of circuits ,38,39,40l-glutamate release [A-R in the mPFC (perfusion with 1 μM muscimol into the mPFC). Therefore, inhibition of NMDAR in the mPFC increases monoamine release induced by presynaptic GABAergic disinhibition in the mPFC. Indeed, the threshold concentrations of local administration of MK801 into the mPFC on GABA release (1 μM) is more sensitive compared with that of monoamine (5 μM)  .l-glutamate, norepinephrine and dopamine in the mPFC without affecting those of GABA or serotonin. The threshold concentration of local administration of MK801 into the RTN on releases of l-glutamate and catecholamine (norepinephrine and dopamine) in the mPFC were 1 μM and 5 μM, respectively. The RTN MK801-induced l-glutamate release in the mPFC was inhibited by the activation of GABAA-R and inhibition of AMPAR in the MDTN, but was not affected by the activation of GABAA-R or inhibition of AMPAR in the mPFC. These results suggest that an activation of glutamatergic neuronal activity induced by GABAergic disinhibition in the MDTN contributes to RTN MK801-induced l-glutamate release in the mPFC.Contrary to intra mPFC regulation system, local administration of MK801 into the RTN increased releases of l-glutamate release in the mPFC is thalamocortical (from MDTN to mPFC) glutamatergic pathway, but generating mechanisms is GABAergic disinhibition from RTN to MDTN through NMDAR inhibition in the RTN.Various thalamic nuclei, which receive GABAergic inhibition from RTN , projectl-glutamate release, the RTN MK801-induced catecholamine release in the mPFC was inhibited by the activation of GABAA-R in the MDTN, inhibition of AMPAR in the MDTN and mPFC, but was not affected by the activation of GABAA-R in the mPFC. Electrophysiological study demonstrated that electrical stimulation of the MDTN increased the releases of glutamate and catecholamine in the mPFC without affecting those of serotonin [Contrary to erotonin . Other lerotonin ,24,40. Terotonin ,24. TakeThis study indicates the several specific regulation systems of serotonergic transmission in the mPFC and MDTN which are independent upon catecholamine release regulation system. The first, intra mPFC regulation of serotonergic transmission associated with NMDAR is resembling to the regulation systems of catecholaminergic transmission, since the selective noradrenergic, dopaminergic and serotonergic terminals in the deeper layers of mPFC receive GABAergic inhibition. However, the serotonergic terminal in the mPFC from DRN does not contact with thalamocortical glutamatergic afferents, whereas mesothalamic serotonergic transmission activates thalamocortical glutamatergic transmission, since an activation of serotonergic neuronal activities in the DRN increases serotonin release in the MDTN. Recent multiprobe microdialysis studies demonstrated that an activation of serotonergic neuronal activity enhances MDTN glutamatergic neurons through activation of excitatory 5-HT7 receptor in the MDTN ,16. In tA-R predominantly, as indicated by decreased regional serotonin release following local administration of muscimol (GABAA-R agonist) into the DRN; however, GABA release is regulated by excitatory 5-HT7 receptor, reflecting decreased regional GABA release following local perfusions with SB269970 (5-HT7R antagonist) into the DRN [A-R and 5-HT7R predominantly inhibit serotonergic neurons and enhance GABAergic neurons, respectively. In the present study, local administration of MK801 into the DRN, decreased and increased releases of GABA and serotonin in the DRN, respectively. The opposite effects of MK801 between releases of GABA and serotonin were inhibited by local administration of muscimol in the DRN. Therefore, NMDAR in the DRN regulates predominantly GABAergic neurons rather than serotonergic neuronal activity in the DRN.In the DRN, serotonin release is regulated by inhibitory GABA the DRN . Previou the DRN . In cont the DRN . Taken wThe present study demonstrated that inhibition of NMDAR directly inhibited GABAergic transmission, but enhanced indirectly glutamatergic and monoaminergic transmissions induced by GABAergic disinhibition. In other words, during resting stage, MK801 selectively inhibits NMDAR on GABAergic neurons, but cannot affect NMDAR on the glutamatergic or monoaminergic neurons. In generally, GABAergic interneuron is more sensitive to NMDAR antagonist compared with other types of neurons, since the resting membrane potential of GABAergic interneurons are more positive (−50 ~−60 mV) rather than those of monoaminergic and glutamatergic neurons ,44. NMDAIn this study, the threshold concentrations of local administrations of MK801 into the mPFC, RTN and DRN on regional GABAergic transmission were almost equal to be 1 μM. Contrary to GABA, the threshold concentrations of local administration of MK801 into the mPFC and RTN on catecholamine release in the mPFC were 5 μM, whereas those into mPFC and DRN on serotonin release in the mPFC were 5 μM and 1 μM, respectively. Therefore, serotonergic transmission is more sensitive to NMDAR antagonist rather than other catecholaminergic transmissions in the mPFC.Subanesthetic doses of ketamine produces transient dissociative and psychotomimetic effects that resemble the positive and negative symptoms of schizophrenia ; howeverR)-ketamine into the infralimbic mPFC and hippocampus also produced antidepressant-like action [R)-ketamine into the prelimbic mPFC nucleus accumbens could not produce [R)-ketamine. Moreover, the binding affinity  of (S)-ketamine and (R)-ketamine to NMDAR are 0.7 and 2.6 μM, respectively [S)-ketamine is potent than that of (R)-ketamine [Neither clinical mechanisms between psychotomimetic and antidepressant effects of ketamine have remained to be clarified. Local administration of ketamine into the infralimbic mPFC reproduced the antidepressant-like actions of systemic ketamine administration . Local ae action , whereas produce . These fectively . Indeed,ketamine . The MK8ketamine . Clinicaketamine ,57. Thesketamine .l-glutamate and secondary catecholamine release in the mPFC, suggests that lower concentration of MK801 affects emotional rather than cognitive disturbances. In the present study, inhibition of AMPAR in the MDTN and mPFC attenuated the MK801-induced glutamatergic and monoaminergic transmissions without affecting GABAergic disinhibition. Several preclinical studies reported the possibility that NMDAR inhibition conversely activates glutamatergic transmission associated with AMPAR through GABAergic disinhibition [The thalamocortical glutamatergic transmission plays important roles in the function of neuro-cognition, including learning, memory, and perceptual integration ,59,60. Thibition ,65. In shibition , whereashibition . The detl-glutamate release in the mPFC activates AMPAR on the regional co-releasing terminals resulting in an increase in releases of norepinephrine and dopamine, but does not affect serotonin release. Inhibition of NMDAR in the DRN enhances serotonin release in the DRN, MDTN and mPFC via GABAergic disinhibition in the DRN. The sensitivities of serotonin release in the mPFC to local administration of MK801 into the DRN is more predominant rather than that of catecholamine release in the mPFC induced by local MK801 administration into the RTN.The present study determined the effects of MK801 on the thalamocortical (RTN-MDTN-mPFC) glutamatergic, mesothalamic (DRN-MDTN), and mesocortical (DRN-mPFC) serotonergic transmissions using multiprobe microdialysis, to clarify the NMDAR associated regulation systems in these three pathways. Inhibition of NMDAR in the RTN activates thalamocortical glutamatergic transmission induced by GABAergic inhibition and secondarily activated AMPAR in the MDTN. The enhanced


Of note, abdominal fat tissue of obese pre-DM patients treated with metformin therapy presented higher SIRT6 expression and lower NF-κB, PPAR-γ, and SREBP-1 expression levels compared to pre-DM control group. Collectively, results show that SIRT6 is involved in the inflammatory pathway of subcutaneous abdominal fat of obese pre-DM patients and its expression responds to metformin therapy.The role of sirtuin 6 (SIRT6) in adipose abdominal tissue of pre-diabetic (pre-DM) patients is poorly known. Here, we evaluated SIRT6 expression in visceral abdominal fat of obese pre-diabetic patients and the potential effects of metformin therapy. Results indicated that obese pre-DM subjects showed low SIRT6 protein expression and high expression of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), peroxisome proliferator-activated receptor gamma (PPAR-γ), and sterol regulatory element-binding transcription factor 1 (SREBP-1). Obese pre-DM patients showed high values of glucose, insulin resistance (HOMA-IR), C reactive protein (CRP), nitrotyrosine, tumor necrosis factor-α (TNF-α) and interleukin 6 (IL-6), and low values of insulin ( Visceral fat and superficial adipose tissue of obese patients express cytokines cross-talking with the cardiovascular system . It has p < 0.05) (p < 0.05), lower values of insulin (p < 0.05), and higher values of HOMA-IR (p < 0.05) than NG obese patients (group 1) (p < 0.05), inflammatory markers, such as CRP (p < 0.05), IL-6 (p < 0.05), TNF-α (p < 0.05), and nitrotyrosine (p < 0.05), than NG obese patients (group 1) (p < 0.05)  . Obese pgroup 1) . Moreovegroup 1) . The sam < 0.05) .p < 0.01). Obese pre-DM not-metformin users presented lower values of SIRT6 (p < 0.01 vs. NG) and higher values of NF-κB, PPAR-γ and SREBP-1 (p < 0.01 vs. NG) (p < 0.05) and lower values of NF-κB, PPAR-γ, and SREBP-1 protein levels (p < 0.05) compared to obese pre-DM not-metformin users  . Moreovein users . p < 0.05). However, a regulatory effect on SIRT6 expression might be the main determinant of oxidative stress and nitrotyrosine reduction in pre-DM receiving an anti-oxidative drug therapy with metformin  buffer, 30 mM Tris-HCl, pH 8.8. Tissues were homogenized with a Precellys-24 system  and centrifuged at 800× p < 0.05 was considered significant. Statistical analysis was performed using the SPSS software package for Windows 17.0 .Data were presented as group mean ± SD. One-way analysis of variance (ANOVA) was used to compare baseline data, followed by Scheffe’s test for pairwise comparisons. Simple and partial correlation were used to evaluate relationships between variables.


Eggerthellaceae within the class Coriobacteriia (phylum Actinobacteria), Adlercreutzia muris WCA-131-CoC-2 (= DSM 29508 = KCTC 15543) and Ellagibacter urolithinifaciens CEBAS 4A (= CCUG 70284 = DSM 104140).Here, we report the annotated draft genome sequences of two type strains belonging to the family  Eggerthellaceae within the class Coriobacteriia (phylum Actinobacteria), Adlercreutzia muris WCA-131-CoC-2 (= DSM 29508 = KCTC 15543) and Ellagibacter urolithinifaciens CEBAS 4A (= CCUG 70284 = DSM 104140).Here, we report the annotated draft genome sequences of two type strains belonging to the family  Eggerthellaceae are typical members of the mammalian gut and have been isolated from, e.g., humans  under anaerobic conditions of N2-CO2 (80:20) in flushed brain heart infusion medium (Merck) supplemented with 0.5% yeast extract, 0.05% l-cysteine monohydrochloride (Roth), 1 mg ml−1 resazurin sodium salt, 2.5 mg liter−1 heme solution, and 2 μg ml−1 vitamin K1 solution (Sigma-Aldrich). Ellagibacter urolithinifaciens DSM 104140T was cultured (37°C) under anaerobic conditions of N2-CO2 (80:20) in flushed anaerobe basal broth (Oxoid). DNA extraction was done using a Qiagen blood and tissue kit. DNA was quantified with a double-stranded DNA (dsDNA) high-sensitivity (HS) assay on a Qubit version 2.0 fluorometer (Thermo Fisher Scientific), according to the manufacturer’s instructions, and adjusted to a concentration of 0.2 ng μl−1.The following type strains were obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ): escribed , 13. AdlA. muris DSM 29508T, while 1,067,866 reads were generated for E. urolithinifaciens DSM 104140T. Data processing was done as previously described  using a BLASTn search . Whole-genome shotgun sequencing was performed on an Illumina MiSeq benchtop sequencer using a 500-cycle v2 kit . In total, 1,469,853 reads were generated for escribed , 13. Seqescribed , 16. The 2.3.3.1 . To elimn search . All conn search . To calcPipeline . The assPipeline  was usedA. muris DSM 29508T and E. urolithinifaciens DSM 104140T, has been deposited at DDBJ/ENA/GenBank under BioProject number PRJNA574580. The versions described in this publication are the first versions and are listed in A. muris DSM 29508T have been deposited under SRA accession number SRX6974936, while the raw reads of E. urolithinifaciens DSM 104140T have been deposited under accession number SRX6974937.This whole-genome shotgun project, including raw reads of


This study explores associations between frailty and oral health in cross-sectional data of 1,202 community-dwelling older people. Two dichotomous outcomes were used: 1. Potential frailty, using routine primary care data; 2. Self-reported frailty, using a questionnaire. Oral health concerned dental record data and self-reported oral problems. Following exploration of univariate associations, age and sex adjusted multivariate logistic regressions were performed. For potential frailty and self-reported frailty associations were found with dental emergency visit (odds ratio (OR)= 2.0, 95% confidence Interval (CI)=1.33;3.02 respectively OR=1.58, 95% CI=1.00;2.49), experiencing oral problems , making dietary adjustments . Additional associations were found for self-reported frailty with wearing dental prosthesis  and missing periodontal information . The cross-sectional data of this study show that in community dwelling older people oral health is associated with frailty.


Scientific Reports 10.1038/s41598-019-38633-4, published online 14 February 2019Correction to: This Article contains an error in the Figure legends of Figures 2 and 3 which have been inadvertently reversed.The correct legend for Figure 2 is:A) Map of specimens CUE JJ_M01-3 showing four-track Minisauripus trackway, and additional isolated fifth track on small unconnected slab. Map based on counterpart cast of track-bearing surface. Red outline shows part of surface preserved as natural impressions. Pterosaur manus tracks, desiccation cracks (stippled areas), raindrop impressions and invertebrate traces also shown. Compare with Fig. 2 and text for details. (B) Shows microstratigraphy of part and counterpart of track-bearing slab. (C) Shows four track-trackway with dashed line to highlight steps and pace angulations. See Table 1 for measurements. Map made by M. G. Lockley and K-S Kim with layout created in Canvas X .”“ Counterpart slab CUE JJ_M01 showing trackway with four consecutive Minisauripus track casts TL1-TR2. (B) Natural impression slab (CUE JJ_M02) showing tracks TL2 and TR2. (C) Isolated track specimen (CUE JJ_M03). Compare with Fig. 3. Photographs by K-S Kim and layout created in Canvas X .”“(


Rhodomyrtus tomentosa, which belongs to the Myrtaceae family. In the current study, we investigated the properties of rhodomyrtone as a potential drug candidate for the treatment of stress-caused depression.Rhodomyrtone is one of the main active compounds derived from We assessed the function of rhodomyrtone in chronic unpredictable mild stress, a well-validated depression model in mice. Depression-like behavior tests, including a sucrose performance test, social interaction test, and forced swimming test, were used to validate the antidepressant effects of rhodomyrtone. The Morris water maze was used to evaluate the mice’s learning and memory ability. Spine density, glycogen synthase kinase-3β, brain-derived neurotrophic factor, postsynaptic density protein 95, and apoptosis-associated protein were detected to reveal the underlying mechanism.Rhodomyrtone was found to prevent source consumption decrease, decreased social behaviors, and increase immobility in the forced swimming test, suggesting a protective effect of rhodomyrtone against depression-like behaviors. Additionally, rhodomyrtone prevented the impairment of spatial memory in mice exposed to chronic unpredictable mild stress. Rhodomyrtone administration also reversed dendritic spine density defects in chronic unpredictable mild stress. Furthermore, rhodomyrtone inhibited the increase of glycogen synthase kinase-3β activity and reversed the decrease of brain-derived neurotrophic factor and postsynaptic density protein 95 in chronic unpredictable mild stress mice. Elevated expression of apoptosis-associated protein Bax and cleaved-caspase 3 was also reversed by rhodomyrtone treatment.These results suggested that the antidepressant effect of rhodomyrtone involves the regulation of neurogenesis, neuronal survival, and synaptic plasticity in the hippocampus. We identified a protection effect of rhodomyrtone against depression-like behaviors in a well-validated depression model in mice. Rhodomyrtone administration can prevent increase of depression-like behaviors, impairment of cognitive abilities, and morphological changes in dendritic spines in CUMS model mice. Rhodomyrtone also prevents CUMS-induced changes in depression-related proteins, including GSK3 activity, BDNF expression, Bax activity, and caspase-3 cleavage. These observations indicate that rhodomyrtone exhibits antidepressant activity involving the promotion of neurogenesis and neuronal survival in the hippocampus.As one of the common mood disorders, depression is correlated with stressful events in life . The cliRhodomyrtus tomentosa, belongs to the acylphloroglucinol derivative family. R. tomentosa has been used in traditional medicine for the treatment of many diseases  and used in the current study. Standard chow and water were supplied ad libitum. Mice were housed in a temperature-controlled (20°C±1°C) room with a normal 12-h-light/-dark cycle, with the lights on at 7:00 Mice in CUMS+vehicle and CUMS+rhodomyrtone groups were exposed to CUMS as described previously . InitialThe SPT wAn adult The mouseSpatial learning and memory were assessed by the Morris water maze (MWM) . The MWMGolgi staining was performed using a GolgiStain kit according to the protocol provided by the manufacturer   on ice. Each sample of lysates (20 μL) was added into SDS-PAGE and electroblotted onto PVDF membranes, blocked by 5% bovine serum albumin, and incubated with primary antibodies overnight at 4°C, including postsynaptic density protein 95 (PSD-95) , cleaved caspase-3 , caspase3 , brain-derived neurotrophic factor (BDNF) , glycogen synthase kinase-3β (GSK3β) , p-GSK3β , Bax (BD), and β-actin (Sigma). Secondary antibodies were purchased from Sigma.Rhodomyrtone  was i.p. injected at 15 mg/kg. The dosages were chosen based on the behavioral results from the 5-mg/kg (low dosage), 15-mg/kg (high dosage 1), and 45-mg/kg (high dosage 2) groups . RhodomyTwo-way ANOVA was used to analyze the effects of exposure to the CUMS protocol and rhodomyrtone treatment, and a posthoc Tukey’s test was conducted. All values were expressed as mean±SEM.F=10.38, CUMS: F=38, 72, Rho: F=17.21, all P<.05; posthoc Tukey’s test CUMS+Veh. vs Control+Veh., P<.01; CUMS+Rho. vs CUMS+Control, P<.01; F=19.72, CUMS: F=14.23, Rho: F=26.02, all P<.05; posthoc Tukey’s test CUMS+Veh. vs Control+Veh., P<.01; CUMS+Rho. vs CUMS+Control, P<.01; F=12.65, P<.01; F=8.41, P<.01; Rho: F=2.66, P=.11; posthoc Tukey’s test CUMS+Veh. vs Control+Veh., P<.01; CUMS+Rho. vs CUMS+Control, P<.01; To investigate the protective effects of rhodomyrtone on CUMS-induced depression, mice were exposed to CUMS protocol for 35 days, and rhodomyrtone or vehicle saline was i.p. injected daily during the last 3 weeks. Meanwhile, mice from vehicle control and rhodomyrtone control group were group-housed with no stress and were i.p. injected with vehicle or rhodomyrtone . DepressF=10.27, P<.01; day 3 interaction: F=15.47, P<.01; day 4 interaction: F=47.60, P<.001; F=0.49, P=.48; F=2.20, P=.15; CUMS: F=13.36, P<.01; rhodomyrtone: F=7.38, P<.05; posthoc Tukey’s test CUMS+Veh. vs Control+Veh., P<.01; CUMS+Rho. vs CUMS+Control, P<.05; 2-way ANOVA; F=8.45, P<.01; CUMS: F=11.25, P<.01; rhodomyrtone: F=7.2, P<.05; posthoc Tukey’s test CUMS+Veh. vs Control+Veh., P<.01; CUMS+Rho. vs CUMS+Control, P<.01; To investigate whether rhodomyrtone could protect against impaired spatial cognition performance in the CUMS group, the MWM task was performed. The escape latency improved in all 4 groups with trial training. However, mice in the CUMS group spent more time to locate the hidden platform during the training. Interestingly, rhodomyrtone treatment significantly improved the latency time of CUMS mice =4.39, P<.05; CUMS: F=12.93, P<.01; rhodomyrtone: F=4.39, P<.05; posthoc Tukey’s test CUMS+Veh. vs Control+Veh., P<.01; CUMS+Rho. vs CUMS+Control, P<.05; Alterations in synaptic and dendritic structure and function are associated with impaired learning and memory in depressive disorders . We deteF=8.01, P<.01; CUMS: F=25.83, P<.01; rhodomyrtone: F=10.59, P<.01; posthoc Tukey’s tests found CUMS+Veh. vs Control+Veh., P<.01; CUMS+Rho. vs CUMS+Control, P<.01; F=2.12, P=.155; CUMS: F=58.38, P<.01; rhodomyrtone: F=7.08, P<.05; posthoc Tukey’s test CUMS+Veh. vs Control+Veh., P<.01; CUMS+Rho. vs CUMS+Control, P<.05; F=0.48, P=.49; CUMS: F=24.26, P<.01; rhodomyrtone: F=9.02, P<.01; posthoc Tukey’s test CUMS+Veh. vs Control+Veh., P<.01; CUMS+Rho. vs CUMS+Control, P<.01; Numerous studies have shown that abnormal activity of GSK3β has multiple effects and is correlated with the severity of depressive symptoms, including neurogenesis defects . Thus, wF=6.21, P<.05; CUMS: F=10.05, P<.01; rhodomyrtone: F=2.23, P>.05; posthoc Tukey’s test CUMS+Veh. vs Control+Veh., P<.01; CUMS+Rho. vs CUMS+Control, P<.05; F=10.75, P<.01; CUMS: F =13.90, P<.01; rhodomyrtone: F=7.59, P<.01; posthoc Tukey’s test CUMS+Veh. vs Control+Veh., P<.01; CUMS+Rho. vs CUMS+Control, P<.01; Volume decrease of the hippocampus in depression is associated with a reduction of neuronal cell body size and increased neural apoptosis in depression patients . AntidepIn the current study, we found that rhodomyrtone exhibited antidepressant activity in a mouse CUMS model. We observed that rhodomyrtone administration protected against CUMS-caused, depression-like behaviors and cognitive defects. Further, CUMS-decreased spine density was reversed by rhodomyrtone treatment. Impairment of neurogenesis and increase of apoptosis are 2 potential mechanisms of depression. We found that rhodomyrtone reversed the CUMS-induced changes in GSK3β activity and BDNF expression level, 2 essential regulators of hippocampal neurogenesis. The activation of apoptosis-related protein Bax and caspase-3 was also reversed by rhodomyrtone treatment. Our findings demonstrated the antidepressant-like effects of rhodomyrtone through regulation of neurogenesis and apoptosis in a classic depressive mouse model induced by CUMS. Studies also showed that rhodomyrtone exhibits antibacterial, anticancer, antiinflammatory, and antioxidant activities . Here, tIt has been reported that defects in emotional memory and spatial learning are key characteristics of depression besides depression-like behaviors . The hipHippocampus shrinkage has been observed in both depressive patients and animal models . InteresAnother important explanation for hippocampus shrinkage is increased apoptosis . ChronicIn summary, for the first time to our knowledge, we showed that rhodomyrtone exhibits antidepressant-like activity in a mouse depression model. Rhodomyrtone can prevent CUMS-induced increase of depression-like behaviors, impairment of cognitive abilities, and morphological changes in dendritic spines. Rhodomyrtone also prevents CUMS-induced changes in GSK3 activation, BDNF expression, Bax activation, and caspase-3 cleavage. These observations indicate that rhodomyrtone has antidepressant effects involving the promotion of neurogenesis and neuronal survival in the hippocampus.None.Supplementary Figure 1Click here for additional data file.Supplementary MaterialClick here for additional data file.


Arabidopsis NBS genes revealed a clade-specific nesting pattern in CNLs, with RNLs nested in the CNL-A clade, and species-specific nesting pattern for TNLs. Surprisingly, we found a moderate bootstrap support (BS = 50%) for CNL-A clade being nested within TNL clade making both the CNL and TNL clades paraphyletic. Arabidopsis and sunflower showed 87 syntenic blocks with 1049 high synteny hits between chromosome 5 of Arabidopsis and chromosome 6 of sunflower. Expression data revealed functional divergence of the NBS genes with basal level tissue-specific expression. This study represents the first genome-wide identification of NBS genes in sunflower paving avenues for functional characterization and potential crop improvement.Nucleotide Binding Site—Leucine-Rich Repeat (NBS-LRR) genes encode disease resistance proteins involved in plants’ defense against their pathogens. Although sunflower is affected by many diseases, only a few molecular details have been uncovered regarding pathogenesis and resistance mechanisms. Recent availability of sunflower whole genome sequences in publicly accessible databases allowed us to accomplish a genome-wide identification of Toll-interleukin-1 receptor-like Nucleotide-binding site Leucine-rich repeat (TNL), Coiled Coil (CC)-NBS-LRR (CNL), Resistance to powdery mildew8 (RPW8)-NBS-LRR (RNL) and NBS-LRR (NL) protein encoding genes. Hidden Markov Model (HMM) profiling of 52,243 putative protein sequences from sunflower resulted in 352 NBS-encoding genes, among which 100 genes belong to CNL group including 64 genes with RX_CC like domain, 77 to TNL, 13 to RNL, and 162 belong to NL group. We also identified signal peptides and nuclear localization signals present in the identified genes and their homologs. We found that NBS genes were located on all chromosomes and formed 75 gene clusters, one-third of which were located on chromosome 13. Phylogenetic analyses between sunflower and  CNL-C (I) clade, sister clade to CNL-C (II) and CNL-D constituted 79 genes. CNL-B clade constituted three genes . The remaining 12 genes did not belong to any clade of Arabidopsis CNL genes. The TNL group formed a species-specific clade, except ten genes that formed a small clade with AT5G36930, named TNL-D clade with strong bootstrap support of 100%. We found a moderate bootstrap support (BS = 50%) for CNL-A clade being nested within TNL clade making both the CNL and TNL clades paraphyletic. Another tree constructed using RNL genes of A. thaliana and H. annuus showed two distinct clades for two lineages: activated disease resistance gene 1 (ADR1) and N-required gene 1 (NRG1)  was used in phylogenetic analyses. Phylogenetic relationships among the sunflower NBS sequences are shown in NL group . The nes1 (NRG1) . The Newhomologs . The synbidopsis .H. annuus clone Ha-NTIR11g CC-NBS-LRR gene (Pl8). HanXRQChr13g0425411, HanXRQChr13g0425361, and HanXRQChr13g0425431 showed more than an 80% identity to the Pl8 gene suggesting the probable homologs to that gene. HanXRQChr04g0123041, belonging to the NL group has shown homology to Lycopersicon esculentum EIX receptor 1 (LeEIX1), a gene that encodes receptor-like proteins (RLPs). Similarly, HanXRQChr17g0552491 showed homology to MLA10, HanXRQChr13g0420141 to N, HanXRQChr17g0552491 to both MLA12 and MLA13 and HanXRQChr17g0552491 to Sr33 protein with greater than 60% identity. Sunflower Genome Database with H. annuus r1 annotations was employed to obtain expression data for predicted NBS genes. We compared accessions of H. annuus r1.2 annotations to H. annuus r1 to obtain the expression data for NBS proteins. Since there were many duplicates for H. annuus r1.2 annotations, we used only the sequences with the unique names. The raw Read Per Kilobase Million) (RPKM) values of gene expression were downloaded separately. The expression values were from bract, corolla, leaves, ligule, ovary, pollen, seed, stamen and stem. Only expression data for 9 CNL type, 33 TNL type, 23 NL type and 6 RNL type genes were retrieved from the database and employed to generate heatmap after deseq normalization of the data using MeV package  [Arabidopsis (~0.43%) [C. sativus (~0.21%) [Carica papaya (~0.21%) [P. vulgaris (~1.19%) [Manihot esculenta (~0.9%) [V. vinifera (~1.3%) [G. max (~0.73%) [Our findings on the NBS-encoding genes in this study is based on recently sequenced sunflower genome . Previoual. 2004  identifial. 2008  identifi(~0.66%) . This nu(~0.43%) , C. sati(~0.21%) , Carica (~0.21%)  and lowe(~1.19%) , Manihot (~0.9%) , V. vini (~1.3%) , and G. (~0.73%) ,24. We p(~0.73%) , Plocik (~0.73%) , and Rad(~0.73%)  showed 7A. thaliana (1:2), A. lyrata (1:2), B. rapa (1:2), Eucalyptus grandis (1:1.25), and Thellungiella salsuginea (1:1.5) as numbers of TNLs were higher than CNLs in these species [M. truncatula, A. thaliana, and B. rapa [A. thaliana and B. rapa [Following the classification of NBS genes by Shao et al. 2016  and Yu e species ,65,66,67 species ,57. The  B. rapa ,27. HanX B. rapa .S. tuberosum [A. thaliana, P. vulgaris, G. max, and P. trichocarpa [P. trichocarpa [B. rapa [A. thaliana, P. trichocarpa, and O. sativa [NBS-encoding genes also called NBS-LRR genes encode proteins having TIR/CC at the N-terminal, NBS domain in the center and LRR at the C-terminal . Among tuberosum . All TIRchocarpa ,24,63,69chocarpa . All of [B. rapa . Kelch m. sativa .Arabidopsis CLAVATA2  [Cladosporium fulvum in tomato [We further compared our pipeline with another pipeline, RGAugury , for theAtRLP10)  is involn tomato .M. esculenta, 143 NBS genes positioned in 39 clusters [C. sativus, 33 NBS genes were located in nine clusters [B. oleracea (3.04), B. rapa (2.7), A. thaliana (2.8) [G. max (4), V. vinifera (6), M. truncatula (5) [Gossypium species such as G. arboretum (3.4), G. raimondii (5.5), G. hirsutum (5.3), and G. barbadense (3.5) [Arabidopsis [aK/sK values of less than one, indicating that these genes are under the influence of purifying selection.A variety of clustering patterns of NBS-encoding genes, frequently observed in almost all plant species, is one of the major reasons for rapid evolution of the NBS genes ,79. The clusters . In C. sclusters , Brassicna (2.8) , Fabaceatula (5) , Gossypise (3.5) . Both sese (3.5) . HoweverC. sativus CNL genes while compared to their respective TNL genes [Arabidopsis, as TNL clades constitute larger numbers of genes than CNL clade [HanXRQChr02g0057361, HanXRQChr02g0057351, and HanXRQChr13g0425771 in the subclade CC (VI) possessed in the range of five to seven. Similarly, subclade TIR (II) possessed gene members with introns in the lowest range (three to five). TIR (I), TIR (III), TIR (IV), TIR (V) and TIR (VI) gene members possessed introns in range of 3 to 17, 2 to 7, 1 to six, 1 to 15, and 1 to 13, respectively. Similar patterns were also observed in the phylogenetic tree of CNL and TNL in C. sativus [Sunflower CNL genes were similar to NL genes . HoweverNL clade . Subclad sativus . The dif sativus . In addi sativus .H. annuus in the analysis as the genome was not available by then. Our results indicate a surprising position of RNLs within TNLs in sunflower making the clades of TNL and CNL potentially paraphyletic. Upon reconstruction of the phylogenetic tree with Arabidopsis NBS genes, RNL genes of sunflower were observed in a CNL-A clade  [ADR1 and NRG1, and two ancient lineages separated before the Angiosperms diversified. The RNL genes, ADR1 and NRG1, have been characterized in Arabidopsis and Nicotiana, respectively. A separate tree, constructed to observe the relationships among sunflower RNLs and Arabidopsis RNLs, formed two clades. The sunflower RNL genes HanXRQChr02g0046611 and HanXRQChr05g0129181 were nested with AT4G3330 (ADR1-L1), AT1G33560 (ADR1) and AT5G04720 (ADR1-L2 or PHX21), with bootstrap support of 90%. On the other hand, HanXRQChr02g0048181, HanXRQChr11g0331571, HanXRQChr03g0067681, HanXRQChr0073241, and HanXRQChr04g0095241 were nested with AT5G66630 (RNL) and AT5G66910 (homologous to NRG1), with bootstrap support of 63%. This suggests that the sunflower RNLs mentioned above are orthologous to the ADR1 and NRG1 homologs of Arabidopsis. ADR1 proteins play a role as helper genes for receiving signals from the R genes in downstream signaling of effector-triggered immunity [A. lyrata (2.5%), A. thaliana (4.2%), B. rapa (4.4%), Capsella rubella (4.7%) and T. salsuginea (5.7%) [a/KsK ratios values for the clade. This supports the hypothesis of high conservation and slow evolutionary rates among the RNL genes [We found that RNLs were nested within the clade of TNLs in sunflower (a member of the Asterids lineage) although RNLs in the families Brassicaceae and Fabaceae (Rosids lineage) were found to be related to CNLs ,30. The s study) . The CNLs study)  suggesteimmunity . Similarimmunity . Since timmunity . Only 5.NL genes .S. tuberosum nematode resistance protein (Gro1.4) [S. tuberosum subsp. andigena RY-1 (conferring resistance to potato virus Y) [N. glutinosa Tobacco Mosaic Virus resistance (N) gene [A. thaliana RPS2 (Resistant to P. syringae 2) [Cucumis melo VAT (resistance to Aphis gossypii) [H. annuus Pl8 [O. sativa Pi36 (conferring resistance to Blast fungus) [H. vulgare subsp. vulgare RPG1 (conferring resistance to stem rust fungus) [http://www.prgdb.org has shown some of them to be the possible homologs of the reference proteins . The Pl8 gene is involved in conferring resistance to P. halstedii, a causative agent to downy mildew [L. esculentum EIX receptor 2 (Eix2), a gene that encodes receptor-like proteins (RLPs) involved in detecting ethylene-inducing xylanase, a fungus elicitor [MLA locus is highly polymorphic and encode allelic CNL type resistance proteins such as MLA1, MLA2, and MLA3 that confer resistance to barley powdery mildew fungus  [Puccinia graminis f. sp. tritici [H. annuus r1.2 annotations compared to H. annuus r1 annotations. From the available expression data, it can be deduced that NBS genes can be expressed at a basal level with tissue specificity in unchallenged conditions [Sunflower NBS proteins identified in this study formed clades with reference proteins such as Pi36, Pl8, Rps2, VAT, RPG1, Gro1.4, RY-1, and N proteins, suggesting their homologous relationships . The sun(Gro1.4) , S. tubevirus Y) , and N. (N) gene . Similaringae 2) , Cucumisossypii) , H. annunuus Pl8 , O. sati fungus) , and H.  fungus) . The BLAproteins . Sunflowy mildew . HanXRQCelicitor . Other iei, Bgh) . Another tritici . We werenditions . In the nditions . Thus, dPl8, LeEIX1, MLA10-13, Sr33 resistance genes. Further characterization of the NBS genes will help us to understand resistance pathways and to develop durable resistance necessary for crop improvement in sunflower, one of the major oilseed crops in the world.We identified 352 NBS genes in sunflower and studied their clustering, phylogenetic relationships, gene homology and functional divergence. These genes formed clusters and showed structural conservation in signature domains and exon/intron architecture in CNL, TNL and RNL types of NBS genes. The RNLs belonged to the CNL-A clade, which in turn was found nested within the TNL clade, making both CNL and TNL clades paraphyletic. This warrants further rigorous analysis. All of the NBS-encoding genes have undergone purifying selection and available expression data have revealed their functional divergence. We confirmed homology of sunflower NBS genes to multiple previously characterized


Histopathological and functional outcomes were evaluated on P10, P18, and P90. Baseline outcome parameters were established in sham-treated and healthy control animals. Gross brain injury did not significantly differ between treatment groups at any time point. On P10, caspase-3 activation and caspase-independent apoptosis were similar between treatment groups; cell proliferation and the number of BrdU-positive vessels did not differ on P18 or P90. Neurobehavioral assessment did not reveal significant differences between treatment groups in accelerod performance, open field behavior, or novel object recognition capacity on P90. Turning behavior was more frequently observed in G-CSF/SCF- and FL-treated animals. No sex-specific differences were detected in any outcome parameter evaluated. In hypoxic-hyperoxic ischemic neonatal brain injury, G-CSF/SCF and FL treatment does not convey neuroprotection. Prior to potential clinical use, meticulous assessment of these hematopoietic growth factors is mandated.Hematopoietic growth factors are considered to bear neuroprotective potential. We have previously shown that delayed treatment with granulocyte colony-stimulating factor (G-CSF)/stem cell factor (SCF) and Fms-related tyrosine kinase 3 ligand (FL) ameliorates excitotoxic neonatal brain injury. The effect of these substances in combined-stressor neonatal brain injury models more closely mimicking clinical conditions has not been investigated. The aim of this study was to assess the short-, mid-, and long-term neuroprotective potential of G-CSF/SCF and FL in a neonatal model of hypoxic-hyperoxic ischemic brain injury. Five-day-old (P5) CD-1 mice were subjected to unilateral common carotid artery ligation and subsequent alternating periods of hypoxia and hyperoxia for 65 minutes. Sixty hours after injury, pups were randomly assigned to intraperitoneal treatment with (i) G-CSF (200  Neonatal brain injury is a problem of great global concern , 2. AdvaOur research group has recently been able to show that systemic treatment with the hematopoietic growth factors/cytokines granulocyte colony-stimulating factor (G-CSF)/stem cell factor (SCF) and Fms-related tyrosine kinase 3 ligand (FL) starting 60 hours after insult protects against N-methyl-D-aspartate receptor-mediated developmental excitotoxic brain damage by reducing injury extent and apoptotic cell death . WhetherFurthermore, any neuromodulatory intervention taking place during potentially critical or sensitive periods of brain development possibly has not only short-, but also long-term impact . The assThus, the aim of the current study was to investigate short-, mid-, and long-term effects of delayed treatment with G-CSF/SCF and FL in a mouse model of combined hypoxic-hyperoxic ischemic neonatal brain injury. From our previous findings we hypothesised that G-CSF/SCF and FL reduce injury extent and apoptotic cell death, stimulate neuroproliferation, and improve neurobehavioral outcome following hypoxia-hyperoxia ischemia.Heparin (Heparin Immuno 1000 IU/ml) was obtained from Ebewe Pharma , and phenobarbital  was obtained from Desitin Arzneimittel GmbH ; murine G-CSF, SCF, and FL were obtained from Peprotech , and 5-bromo-2′-deoxyuridine (BrdU) was obtained from Sigma-Aldrich .μg/kg bw)/SCF (50 μg/kg bw) in vehicle , (ii) FL (100 μg/kg bw) in vehicle, or (iii) vehicle only and received an intraperitoneal (i.p.) injection every 24 hours for three or five consecutive days, depending on timing of endpoint determination. Endpoints were assessed on P10, P18, and P90. Sex was visually determined by assessment of the anogenital distance and confirmed by polymerase chain reaction  or was determined by visual assessment alone  depending on mouse age as described previously ; 1x PBS: 37.1 (11.1), G-CSF/SCF: 37.3 (5.8), FL: 34.6 (6.3); n=11-12 per group, F=0.377, p=0.689, One-Way ANOVA, two degrees of freedom). Female mice were significantly lighter in 1x PBS and G-CSF/SCF treatment groups, whereas body weight did not differ between male and female experimental animals in FL-treated mice (mean bodyweight (SD) [g]; 1x PBS: male 44.6 (9.6), female 28.0 (2.7), n=5-6, t=3.727, p= 0.005, Student's t-test; G-CSF/SCF: male 41.1 (3.8), female 31.9 (2.8), n=5-7, t=4.528, p=0.001, Student's t-test; FL: male 35.5 (7.2), female 33.0 (5.0), n=4-7, t=0.618, p=0.552, Student's t-test; Bonferroni-adjusted level of significance p<0.025).χ2=1.263, p=0.532, Pearson Chi-Square, two degrees of freedom; Turning behavior with counter-clockwise movements and hyperactivity was not observed in healthy control animals, but it occurred in six animals subjected to HHI, indicating severe brain damage (1x PBS: n=1 (9.1%) , G-SCF/SCF: n=2 (16.7%) , FL: n=3 (27.3%) ; With regard to accelerod performance, baseline time until task disruption in healthy control animals was 97.2 ± 46 sec (mean ± SD).In HHI, accelerod performance did not significantly differ between treatment groups (mean time until task disruption (SD) [sec]; 1x PBS: 107.7 (46.7), G-CSF/SCF: 116.3 (35.6), FL: 146.7 (74.4); n=11-12 per group, F=1.624, p=0.213, One-Way ANOVA, two degrees of freedom). No statistically significant sex-specific differences were detected .With regard to open field assessments, baseline anxiety index in sham-treated animals was 80.6%  (median (IQR)), baseline travel distance as an indicator of horizontal activity was 3000.3 cm  (median (IQR)), and baseline time spent rearing as an indicator of vertical activity was 68.1 sec (12.9) (mean (SD)).In HHI, anxiety (median anxiety index (IQR) [%]; 1x PBS: 85.0 , G-CSF/SCF: 78.8 , FL: 85.4 ; n=34, H=2.291, p=0.318, Kruskal-Wallis test, two degrees of freedom), horizontal activity (median travel distance (IQR) [cm]; 1x PBS: 2871.6 ; G-CSF/SCF: 3132.2 , FL: 3420.1 ; n=34, H=4.191, p=0.123, Kruskal-Wallis test, two degrees of freedom), and vertical activity (mean time spent rearing (SD) [sec]; 1x PBS: 71.6 (29.4), G-SCF/SCF: 85.0 (33.4), FL: 68.7 (49.3); n=11-12 per group, F=0.603, p=0.554, One-Way ANOVA, two degrees of freedom) did not significantly differ between treatment groups. No statistically significant sex-specific differences were detected .With regard to novel object recognition, baseline discrimination index (DI) in sham-treated animals was 0.16  (median (IQR)) after one hour, 0.29  (median (IQR)) after three hours, and 0.35  (median (IQR)) after five hours.In HHI, no overall differences in DI between treatment groups were observed after one (median DI (IQR); 1x PBS: 0.41 , G-CSF/SCF: 0.47 , FL: 0.22 ; n=34, H=2.269, p=0.322, Kruskal-Wallis test, two degrees of freedom), three (median DI (IQR); 1x PBS: 0.63 , G-CSF/SCF: 0.61 , FL: 0.46 ; n=34, H=1.942, p=0.379, Kruskal-Wallis test, two degrees of freedom), or five hours (median DI (IQR); 1x PBS: 0.40 , G-CSF/SCF: 0.26 , FL: 0.43 ; n=34, H=1.250, p=0.535, Kruskal-Wallis test, two degrees of freedom). Details can be seen in The identification of novel treatment options for brain injury is one of the biggest challenges encountered in neonatal neuroscience , 40, 41.In the study at hand, we used a combined hypoxic-hyperoxic ischemic brain injury model, thought to more adequately reflect the clinical situation in neonatal intensive care units , 34. AlsOn a histomorphological basis, we did not observe statistically significant differences between treatment groups at any time point, either in gross brain injury, or in caspase-dependent or -independent apoptosis as indicated by AIF-positivity. Another mechanism of action described for G-CSF/SCF is its trophic effect on neurons . An incrThis is in discordance with previous neonatal rodent brain injury studies, reporting reduced damage extent, apoptotic cell death, and increased cell proliferation following both single and repetitive G-CSF treatments , 24, 28.μg/g body weight was chosen, based on previous publications and our own studies, showing a dose response curve [For the present study, a higher dose of G-CSF was selected. The dose of 200 se curve , 30. These curve  The diffse curve  had no ese curve  A directInterestingly, turning behavior as an indicator of severe brain damage was more frequently observed in G-CSF/SCF- and FL-treated animals than in vehicle-treated controls. Even though the finding was not statistically significant, this might still be of relevance as turning behavior has been shown to correlate with histological damage and sensory motor deficit in rodents exposed to cerebral ischemia . CautiouThe importance of evaluating sex-specific differences has been proven by several studies, especially when considering the variable responses of male and female immature rodents to brain injury , 47, 48.To conclude, in this multistressor mouse model of hypoxia-hyperoxia ischemia we cannot confirm the protective effect of G-CSF/SCF and FL on neonatal brain injury previously reported. As adverse effects cannot be ruled out, meticulous assessment of these hematopoietic growth factors in extensive preclinical studies is mandated prior to potential clinical use.


This article has been corrected: Due to a labeling error, the legends for Figures 2 and 3 were accidentally switched. The proper figure legends are given below. The authors declare that these corrections do not change the results or conclusions of this paper.Figure 2: Induction of fibrosis in the mammary gland of NeuT/ATTAC mice following AP21087 treatment for four weeks. Six-week-old NeuT/ATTAC mice were injected i.p. with vehicle (NeuT/ATTAC) or 0.4 mg/kg AP21087 (NeuT/ATTAC+AP) for four weeks. FFPE sections were stained as described in Figure 2. Magnification 400×.Figure 3: Induction of fibrosis in NeuT/ATTAC mice. Mice at 6 weeks-of-age were injected i.p. with vehicle (NeuT/ATTAC) three times weekly for 5.5 months (NeuT/ATTAC) or with 0.4 mg/kg AP21087 (NeuT/ATTAC+AP) for 4 months. FFPE sections were stained with H&E, for collagen (PicroSirius Red) and with antibodies against Ki-67, CD31, α-smooth muscle actin (SMA), F4/80, Cxcl1, Foxp3, CD8 and PD-L1. Magnification 400×.8042-8053. https://doi.org/10.18632/oncotarget.24233Original article: Oncotarget. 2018; 9:


A post-conflict vaccination campaign, Central African Republic. Bull World Health Organ. 2018 Aug 1;96(8):540–47. on page 544,


Scientific Reports 10.1038/s41598-018-34229-6, published online 31 October 2018Correction to: In this Article the Competing Interests section was omitted. This should read:“T.S. received research funding from Sony IP&S Inc.”


This article has been corrected: The correct order of authors' names is given below:1,2, Milind Shivajirao Patole1, Punam Vasudeo Nagvenkar1, Sonali Shankar Kamble2 and Rajesh Nivarti Gacche2,3Bhimashankar Gurushidhappa Utage30304-30323. https://doi.org/10.18632/oncotarget.25717Original article: Oncotarget. 2018; 9:


The correct name is: Ashwani K. Singal. The correct citation is: Axley P, Mudumbi S, Sarker S, Kuo Y-F, Singal AK (2018) Patients with stage 3 compared to stage 4 liver fibrosis have lower frequency of and longer time to liver disease complications. PLoS ONE 13(5): e0197117.