Journal: MycoKeys

Article Title: Segregation of the genus Parahypoxylon ( Hypoxylaceae , Xylariales ) from Hypoxylon by a polyphasic taxonomic approach

doi: 10.3897/mycokeys.95.98125

Figure Lengend Snippet: Characteristic stromatal pigments and other secondary metabolites of Hypoxylon species. (+)-mitorubrin ( 1 ); (+)-6˝-hydroxymitorubrinol acetate ( 2 ); (+)-mitorubrinol acetate ( 3 ); (+)-6˝-hydroxymitorubrinol ( 4 ); (+)-mitorubrinol ( 5 ); sporothriolide ( 6 ); dihydroisosporothric acid ( 7 ); cohaerin E ( 8 ); 8-methoxy naphthol ( 9 ); 1,8-naphthol ( 10 ); hypoxylone ( 11 ).

Article Snippet: Hypoxylon hinnuleum , ATCC 36255, MUCL 3621 , MK287537 , MK287549 , MK287562 , MK287575 , USA (T) , .

Techniques:

Journal: MycoKeys

Article Title: Segregation of the genus Parahypoxylon ( Hypoxylaceae , Xylariales ) from Hypoxylon by a polyphasic taxonomic approach

doi: 10.3897/mycokeys.95.98125

Figure Lengend Snippet: Inferred molecular phylogenetic maximum Likelihood (lLn = -122825.7921) tree of selected Hypoxylaceae , Graphostromataceae and Xylariaceae sequences. The analysis was calculated by using IQ-Tree with posterior probability support calculated from Bayesian inference methodology and support values generated from 1000 bootstrap replicates using a multigene alignment (ITS, LSU, tub 2 and rpb 2). The tree was rooted with Xylaria hypoxylon CBS 122620, X. arbuscula CBS 126415 ( Xylariaceae ) and Graphostroma platystomum CBS 27087 ( Graphostromataceae ). Type material is highlighted in bold letters. Bayesian posterior probability scores ≥ 0.95 / Bootstrap support values ≥ 70 are indicated along branches.

Article Snippet: Hypoxylon hinnuleum , ATCC 36255, MUCL 3621 , MK287537 , MK287549 , MK287562 , MK287575 , USA (T) , .

Techniques: Generated

Journal: Scientific Reports

Article Title: Inflammatory Eicosanoids Increase Amyloid Precursor Protein Expression via Activation of Multiple Neuronal Receptors

doi: 10.1038/srep18286

Figure Lengend Snippet: hTP-hAPP cells were transfected with 50 nM of control siRNA or siRNA directed to Gα q , Gα 12 , or Gα 13 and cultured for 72 h, with IBOP (10 nM) added over the last 48 h. ( A ) qRT-PCR analysis revealed a reduction in IBOP-induced APP mRNA levels only in cells treated with Gα q siRNA [R.Q. (relative quantification) values of APP/GAPDH from qPCR are plotted relative to non-IBOP-treated cells without siRNA addition]. ( B ) Only cells treated with Gα q siRNA showed reduced APP protein expression, as determined by immunoblot analysis with 5685 antibody [values are relative to non-IBOP-treated cells without siRNA addition, with normalization to α-tubulin]. Statistical analyses consisted of a mixed-effects model, with values representing estimates from the least squares means fit of the mixed procedure from 2–6 independent studies with 1–3 replicates for each treatment/study. Error bars represent SEM; **p < 0.01; ***p < 0.001.

Article Snippet: The following siRNAs were used: Cntl, Gαq and PKCβ siRNA (Santa Cruz Biotechnologies, Inc.; Dallas, TX); Gα and Gα 13 (Thermo Scientific; Waltham, MA); and PKCε and PKCα (Qiagen; Hilden, Germany).

Techniques: Transfection, Control, Cell Culture, Quantitative RT-PCR, Quantitative Proteomics, Expressing, Western Blot

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Persistence of Mycobacterium tuberculosis in response to infection burden and host-induced stressors

doi: 10.3389/fcimb.2022.981827

Figure Lengend Snippet: Strains and plasmids utilized in this study.

Article Snippet: pST5552 , Bacterial Expression vector, Kan R GFP under control of the theophylline inducible riboswitch , Addgene, USA (Plasmid #36255); .

Techniques: Construct, Control, Expressing, Plasmid Preparation

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Persistence of Mycobacterium tuberculosis in response to infection burden and host-induced stressors

doi: 10.3389/fcimb.2022.981827

Figure Lengend Snippet: Flow cytometry gating strategy. (A) A primary gate was applied to the bacterial population according to forward scatter (FSC) and side scatter (SSC) properties. For cell enumeration, a secondary gate was applied to the non-fluorescent bead population. (B) M. tuberculosis Δ leuD Δ panCD ::pTiGc was cultured in the presence of 4 mM theophylline to allow induction of the riboswitch-based promoter for expression of TurboFP635. Constitutive expression of GFP is observed, whilst fluorescence of TurboFP635 is observed following induction with theophylline. (C) Selecting on the bacterial population, a rectangle gate was created to select for live cells, according to their GFP positivity. Single colour controls ensured optimal voltage settings for positive fluorescence of GFP (pST5552) and TurboFP635 (pSTCHARGE), above that of the autofluorescence of unstained cells. (D) The fluorescence dilution technique allows monitoring of bacterial replication for 5 generations. To improve detection of the fluorescent signal to allow measurement over 5 days, theophylline, was retained in culture for 24 hours. Bacterial replication could thus effectively be monitored from day 2 onwards since the fluorescent signal remained stable in vitro and intracellularly between day 0 and day 1. (E) Following harvesting of intracellular bacteria, cells were stained with CV-AM for analyses of metabolic esterase activity. CV-AM is a non-polar, cell permeable fluorogenic probe that is rapidly hydrolysed to a polar, fluorescent compound by intracellular esterases of live cells. Dead cells no longer possess esterase activity, and will thus not convert to the fluorescent calcein, whilst calcein is stably retained in live cells. (F) Selecting on the live cells population, dilution of the TurboFP635 fluorescent signal provided an indication of bacterial replication following removal of the inducer, theophylline (Theo). The high red gate was created based on maximum TurboFP635 fluorescence observed at D0 using the range tool and used to detect mycobacteria that retain their TurboFP635 fluorescence from later time points (D3 and D5), representing slow or non-growing bacteria. The low TurboFP635 gate was created to distinguish replicating intracellular bacteria, as visually assessed when overlayed with in vitro day 3 or day 5 bacteria. (G) Selecting on the high and low TurboFP635 subpopulations, the esterase activity of intracellular bacteria was assessed by overlaying on a histogram plot. (H) Fluorescence dilution of the TurboFP635 signal over time. The geometric median fluorescent intensity (MFI) of TurboFP635 enabled determination of the number of bacterial generations during infection. CV-AM, calcein violet AM.

Article Snippet: pST5552 , Bacterial Expression vector, Kan R GFP under control of the theophylline inducible riboswitch , Addgene, USA (Plasmid #36255); .

Techniques: Flow Cytometry, Cell Culture, Expressing, Fluorescence, In Vitro, Bacteria, Staining, Activity Assay, Stable Transfection, Infection

Journal: The Journal of Biological Chemistry

Article Title: The store-operated Ca 2+ channel Orai1α is required for agonist-evoked NF-κB activation by a mechanism dependent on PKCβ2

doi: 10.1016/j.jbc.2023.102882

Figure Lengend Snippet: Cartoon summarizing Orai1α-dependent, agonist-stimulated, NF-κB transcriptional activity. G protein–coupled receptor occupation leads to the activation of phospholipase C (PLC) that generates inositol 1,4,5-trisphosphate (IP 3 ), which, in turn, induces Ca 2+ release from the endoplasmic reticulum (ER). Ca 2+ store depletion leads to Ca 2+ influx via Orai1α, which enhances the Ca 2+ concentration nearby. Orai1α and the Ca 2+ -regulated conventional PKC isoform, PKCβ2, are maintained in close apposition by the scaffold protein AKAP79. Therefore, the rise in cytosolic Ca 2+ concentration nearby the channel results in activation of PKCβ2, which has been reported to lead to the activation of NF-κB via the classical IκB kinase (IKK)–IκB signaling pathway, thus, leading to IκB serine phosphorylation and proteasomal degradation, which releases active, dimeric, NF-κB that translocates to the nucleus and initiates transcription of NF-κB-responsive genes.

Article Snippet: PKCβ shRNA (catalog number: sc-29450-SH; from Santa Cruz Biotechnology).

Techniques: Activity Assay, Activation Assay, Concentration Assay, Phospho-proteomics