Journal: BMC Microbiology

Article Title: Role of Mycobacterium tuberculosis pknD in the Pathogenesis of central nervous system tuberculosis

doi: 10.1186/1471-2180-12-7

Figure Lengend Snippet: Invasion and survival of M. tuberculosis pknD mutant in host-derived cells . A . BALB/c mice were infected with M. tuberculosis CDC1551 or pknD mutant, and sacrificed at days 1 and 49 after infection. The mutant for M. tuberculosis pknD was significantly attenuated (P = 0.004) in mouse brain, but not lung tissue, 49 days after infection. No defect was observed in the lungs at either time point. Bacterial burden is represented as log 10 CFU/organ for all animal experiments. B . Invasion of host-cell monolayers by wild-type CDC1551, wild-type intergenic transposon control, pknD transposon mutant (pknD:Tn), and pknD genetic complement (pknD:Comp) was examined and normalized to the wild-type control. Invasion assays were performed in brain microvascular endothelial cells (HBMEC), epithelial A549 cells, and umbilical vein endothelia (HUVEC). No difference in invasion was observed in A549 cells (P = 0.31) or HUVEC (P = 0.41). A significant reduction in invasive capacity, however, was observed in the CNS-derived HBMEC (P = 0.02). This defect was restored by genetic complementation with the native pknD/pstS2 operon. N.S. = not significantly different. C . Intracellular survival of each of the above M. tuberculosis strains was examined in HBMEC at days 1, 3, 5, and 7 after infection. The pknD:Tn mutant demonstrated an invasion and intracellular survival defect in HBMEC relative to wild-type over the course of the seven day infection. D . Survival was also examined by infection of activated J774 macrophages. No corresponding survival defect for the pknD:Tn mutant was observed in these cells during the seven day infection. A mutant for the gene Rv0442c , known to be attenuated in the macrophage model, is included as a control. All CFU counts are represented as mean ± standard deviation.

Article Snippet: Primary human brain microvascular endothelial cells and HUVEC were kind gifts from Dr. Kwang Sik Kim, Department of Pediatrics, Johns Hopkins University School of Medicine.

Techniques: Mutagenesis, Derivative Assay, Infection, Control, Standard Deviation

Journal: Pathogens

Article Title: Impact of Neospora caninum Infection on the Bioenergetics and Transcriptome of Cerebrovascular Endothelial Cells

doi: 10.3390/pathogens9090710

Figure Lengend Snippet: Top differentially expressed genes (DEGs) detected in human and rat endothelial cells after 24 h of N. caninum infection.

Article Snippet: Rat Brain Microvascular Endothelial Cell line (rBMVEC), GPNT strain, was obtained from the European Collection of Authenticated Cell Cultures (ECACC, Health Protection Agency Culture Collections (HPACC), Salisbury, UK).

Techniques: Infection, Ubiquitin Proteomics, Membrane, Binding Assay, Transduction

Journal: Pathogens

Article Title: Impact of Neospora caninum Infection on the Bioenergetics and Transcriptome of Cerebrovascular Endothelial Cells

doi: 10.3390/pathogens9090710

Figure Lengend Snippet: The effects of Neospora caninum on the expression levels of the genes of hBMECs and rBMVECs determined by microarray analysis at 24 hpi. PCA scatter plots of gene expression profiles of ( a ) hBMECs and ( d ) rBMVECs showing a clear infection-dependent effect in both cell lines. The plots demonstrate a clear separation in the expression profile of infected and uninfected (control) cells. ( b , e ) Volcano plots showing individual genes as dots. The red and blue dots represent genes with increased and decreased expression, respectively. Genes whose expression levels were not significantly changed are shown in grey. X -axis shows Fold Change (FC) and y -axis shows p -value. ( n = 3/group; p -value < 0.05; FC ± 1). ( c , f ) Heat map showing hierarchical clustering of DEGs between infected and uninfected hBMECs and rBMVECs at 24 hpi. Each group had three replicates. Red and blue colors indicate genes with increased and decreased expression, respectively. Gene expression in infected cells was compared to uninfected cells and the criterion for differential expression was ≥ 1FC with a p -value of < 0.05.

Article Snippet: Rat Brain Microvascular Endothelial Cell line (rBMVEC), GPNT strain, was obtained from the European Collection of Authenticated Cell Cultures (ECACC, Health Protection Agency Culture Collections (HPACC), Salisbury, UK).

Techniques: Expressing, Microarray, Gene Expression, Infection, Control, Quantitative Proteomics

Journal: Cell Communication and Signaling : CCS

Article Title: TGFβ1-induced hedgehog signaling suppresses the immune response of brain microvascular endothelial cells elicited by meningitic Escherichia coli

doi: 10.1186/s12964-023-01383-y

Figure Lengend Snippet: Analysis of miR-155 binding sites on kras 3’UTR and Gli1/2 binding sites on mir155hg promotor by dual-luciferase reporter assays. A Conservation of the miR-155 sequence among different species (upper panel), and conservation of the miR-155 target sequence in KRAS among different species (lower panel). Human, Homo sapiens ; Mouse, Mus musculus ; Rat, Rattus norvegicus . B The miRNA response elements (MREs) of miR-155 were shown on the sequence of kras 3′-UTR, and mutations were introduced on these MREs. Both wild-type and mutated sequences were cloned into the psiCHECK-2 plasmid. C dual-luciferase reporter assay testing miR-155 binding with kras 3’UTR. HEK-293 T cells were co-transfected with has-miR-155 mimics or mimics NC as control oligonucleotide (final concentration at 50 nM) together with the wild-type (KRAS-WT-3’UTR) or mutated (KRAS-Mut-3’UTR) kras 3′-UTR luciferase reporter plasmids. The renilla luciferase activity was measured and normalized to firefly luciferase activity after 36 h. ** p < 0.01. The assays were performed in triplicates, and results are presented as mean ± SEM. D Schematic of the three predicted Gli1/2 binding sites on mir155 hg promotor (upper panel) and their binding sequences accordingly (lower panel). The binding sites were located at − 1088 to − 1077 (site 1), − 461 to − 447 (site 2), and − 407 to − 393 (site 3) of the mir155hg promotor. The gli2 luciferase activities were tested by applying a series of truncations ( E ) as well as site-targeted mutations ( F ) on the mir155hg promoter, along with pcDNA3.1-Gli1 and pcDNA3.1-Gli2 and pRL-TK plasmids. The specific constructs used in the truncation assays ( E ) included pGL3-basic vector, pGL3-mir155hg-promo-WT (containing promotor region from − 1440 to + 226), pGL3-mir155hg-promo-truncation1 (from − 913 to + 226) and pGL3-mir155hg-promo-truncation2 (from − 339 to + 226). The specific constructs used in the site-mutation assays ( F ) included pGL3-basic vector, pGL3- mir155hg-promo-WT (containing all 3 sites), pGL3-mir155hg-promo-mut1 (lack of site 1), pGL3-mir155hg-promo-mut2 (lack of site 2) and pGL3-mir155hg-promo-mut3 (lack of site 3). The luciferase activities were determined and presented as the ratio of firefly and renilla luciferase activity. The assays were performed with three replicates and data are presented as mean ± SEM. ** p < 0.01. G Schematic of the Gli1 and Gli2 binding to the mir155hg promotor at around − 1088 in hBMECs

Article Snippet: The human BMECs (hBMECs) were kindly gifted from Prof. Kwang Sik Kim at Johns Hopkins University School of Medicine, and the Gli-KO hBMECs cell lines were constructed and tested in a previous study [ ].

Techniques: Binding Assay, Luciferase, Sequencing, Clone Assay, Plasmid Preparation, Reporter Assay, Transfection, Control, Concentration Assay, Activity Assay, Construct, Mutagenesis

Journal: Cell Communication and Signaling : CCS

Article Title: TGFβ1-induced hedgehog signaling suppresses the immune response of brain microvascular endothelial cells elicited by meningitic Escherichia coli

doi: 10.1186/s12964-023-01383-y

Figure Lengend Snippet: rTGFβ1 inhibited ERK1/2 signaling and immune reaction of RS218 infected hBMECs relying on inducted noncanonical HH signaling. A Detecting expression alterations of IL-6, MIP-2, and E-selectin in hBMECs with qPCR or Western blot. The cells were infected with RS218 with or without rTGFβ1 pre-treatment (at 50 ng/mL). ** p < 0.01. The qPCR assays were performed in triplicates, and results are presented as mean ± SEM. B qPCR detecting IL-6, MIP-2, and E-selectin transcription upon RS218 infection in wild-type hBMECs or Gli-KO hBMECs. The wild-type hBMECs were pretreated with rTGFβ1 (at 50 ng/mL) or together with GANT61 (at 10 μM). The Gli-KO hBMECs were pretreated with rTGFβ1 (at 50 ng/mL). ** p < 0.01. The qPCR assays were performed in triplicates, and results are presented as mean ± SEM. C Western blot detecting phosphorylation of p65, JNK, ERK1/2, and p38 in RS218 infected hBMECs. The cells were pretreated with or without rTGFβ1 at 50 ng/mL. D Western blot detecting expression of E-selectin and ERK1/2 phosphorylation in RS218 infected hBMECs. The cells were pretreated with rTGFβ1 (at 50 ng/mL) or GANT61 (at 10 μM). E Detecting expression alterations of IL-6, MIP-2, and E-selectin in hBMECs with qPCR or Western blot. The cells were infected with RS218 with or without U0126 treatment (at 5 μM). ** p < 0.01. The qPCR assays were performed in triplicates, and results are presented as mean ± SEM

Article Snippet: The human BMECs (hBMECs) were kindly gifted from Prof. Kwang Sik Kim at Johns Hopkins University School of Medicine, and the Gli-KO hBMECs cell lines were constructed and tested in a previous study [ ].

Techniques: Infection, Expressing, Western Blot, Phospho-proteomics

Journal: Cell Communication and Signaling : CCS

Article Title: TGFβ1-induced hedgehog signaling suppresses the immune response of brain microvascular endothelial cells elicited by meningitic Escherichia coli

doi: 10.1186/s12964-023-01383-y

Figure Lengend Snippet: TGF-HH axis suppressed ERK1/2 signaling in RS218 infected hBMECs via modulating miR155 and KRAS. A Western blot and qPCR detecting expression of KRAS in RS218 infected hBMECs. The cells were pretreated with rTGFβ1 (at 50 ng/mL) or GANT61 (at 10 μM). ** p < 0.01. The qPCR assays were performed in triplicates, and results are presented as mean ± SEM. B IF assays showing the KRAS expression in BMECs of mice challenged by RS218 with or without rTGFβ1 pre-treatment (at 1 μg/kg). The KRAS was stained in red. CD31 was specifically applied for labeling the micro-vessels in green. The cell nucleus was stained in blue with DAPI. Scale bar = 50 μm. C qPCR detecting MIR155HG and miR-155 expression upon RS218 infection in hBMECs. The cells were pretreated with rTGFβ1 (at 50 ng/mL) or GANT61 (at 10 μM). ** p < 0.01. The qPCR assays were performed in triplicates, and results are presented as mean ± SEM. D Western blot detecting expression of E-selectin, KRAS and ERK1/2 phosphorylation in RS218 infected hBMECs with or without rTGFβ1 pre-treatment (at 50 ng/mL). The cells were transfected with has-miR-155 mimics, mimics NC, has-miR-155 inhibitors, or inhibitors NC at 50 nM as indicated. E qPCR detecting expression alterations of IL-6, MIP-2, and E-selectin in RS218 infected hBMECs with or without rTGFβ1 pre-treatment (at 50 ng/mL). The cells were transfected with has-miR-155 mimics, mimics NC, has-miR-155 inhibitors, or inhibitors NC at 50 nM as indicated. ** p < 0.01. The qPCR assays were performed in triplicates, and results are presented as mean ± SEM

Article Snippet: The human BMECs (hBMECs) were kindly gifted from Prof. Kwang Sik Kim at Johns Hopkins University School of Medicine, and the Gli-KO hBMECs cell lines were constructed and tested in a previous study [ ].

Techniques: Infection, Western Blot, Expressing, Staining, Labeling, Phospho-proteomics, Transfection

Journal: Cell Communication and Signaling : CCS

Article Title: TGFβ1-induced hedgehog signaling suppresses the immune response of brain microvascular endothelial cells elicited by meningitic Escherichia coli

doi: 10.1186/s12964-023-01383-y

Figure Lengend Snippet: SAG repressed BMECs immune reaction and neuroinflammation of infected mice in vivo and in vitro. A Western blot detecting expression of E-selectin, KRAS and ERK1/2 phosphorylation in RS218 infected hBMECs with or without SAG pre-treatment (at 10 μM). B qPCR detecting expression alterations of IL-6, MIP-2, and E-selectin in RS218 infected hBMECs with or without SAG pre-treatment (at 10 μM). ** p < 0.01. The qPCR assays were performed in triplicates, and results are presented as mean ± SEM. C qPCR detecting MIR155HG, miR-155, and KRAS expression upon RS218 infection in hBMECs. The cells were pretreated with or without SAG pre-treatment (at 10 μM). * p < 0.05, ** p < 0.01. The qPCR assays were performed in triplicates, and results are presented as mean ± SEM. D Effects of the SAG pre-treatment at 10 mg/kg (for 12 h) on the survival of the mice after the challenge of RS218 ( n = 10). ** p < 0.01. E ELISA analysis of IL-6 and MIP-2 in brain lysates from RS218 challenged mice with or without SAG pre-treatment at 10 mg/kg. Data are presented as mean ± SEM from five individual mice in each group. F IF assays showing the E-selectin and KRAS expression in brains of mice challenged by RS218 with or without SAG pre-treatment (at 10 mg/kg). The E-selection and KRAS were stained in red. CD31 was specifically applied for labeling the micro-vessels in green. The cell nucleus was stained in blue with DAPI. Scale bar = 50 μm

Article Snippet: The human BMECs (hBMECs) were kindly gifted from Prof. Kwang Sik Kim at Johns Hopkins University School of Medicine, and the Gli-KO hBMECs cell lines were constructed and tested in a previous study [ ].

Techniques: Infection, In Vivo, In Vitro, Western Blot, Expressing, Phospho-proteomics, Enzyme-linked Immunosorbent Assay, Selection, Staining, Labeling

Journal: Cell Communication and Signaling : CCS

Article Title: TGFβ1-induced hedgehog signaling suppresses the immune response of brain microvascular endothelial cells elicited by meningitic Escherichia coli

doi: 10.1186/s12964-023-01383-y

Figure Lengend Snippet: Schematic representation of the TGFβ1 or SAG immunosuppression effects on BMECs through upregulating miR-155 and negative regulating KRAS as well as downstream ERK1/2 signaling. The exogenous TGFβ1 or SAG triggered the HH signaling in E. coli -infected BMECs and upregulated intracellular miR-155. Further, the promoted miR-155 suppressed the ERK1/2 activation by negatively regulating KRAS, thus decreasing IL-6, MIP-2, and E-selectin expression

Article Snippet: The human BMECs (hBMECs) were kindly gifted from Prof. Kwang Sik Kim at Johns Hopkins University School of Medicine, and the Gli-KO hBMECs cell lines were constructed and tested in a previous study [ ].

Techniques: Infection, Activation Assay, Expressing