Journal: Clinical Orthopaedics and Related Research

Article Title: Phosphatidylcholine Coatings Deliver Local Antimicrobials and Reduce Infection in a Murine Model: A Preliminary Study

doi: 10.1007/s11999-016-5211-7

Figure Lengend Snippet: Eluate turbidity testing against Staphylococcus aureus and Pseudomonas aeruginosa

Article Snippet: The presence of C2DA in the eluate lowered the amount of amikacin needed to inhibit UAMS-1 growth; an eluate with no C2DA and 156 μg/mL amikacin was unable to inhibit UAMS-1 growth, but a sample with 19 μg/mL amikacin and 88 μg/mL C2DA was inhibitory (Table ). table ft1 table-wrap mode="anchored" t5 Table 1 caption a7 Amikacin loading C2DA loading Measured amikacin (μg/mL) Measured C2DA (μg/mL) Inhibited UAMS-1 Inhibited PA-ATCC 27317 Low None 156 0 N Y Low Low 61 47 N Y Low Medium 94 129 N Y Low High 62 99 N Y Medium None 65 0 N Y Medium Low 19 88 Y Y Medium Medium 232 283 Y Y Medium High 254 150 Y Y High None 237 0 Y Y High Low 248 94 Y Y High Medium 158 291 Y Y High High 154 685 Y Y Open in a separate window Eluates were diluted 1:10 during the assay; C2DA = cis-2-decenoic acid; N = no; Y = yes.

Techniques:

Journal: eLife

Article Title: Protein kinase C is a calcium sensor for presynaptic short-term plasticity

doi: 10.7554/eLife.03011

Figure Lengend Snippet: Synaptic plasticity was examined at the calyx of Held following tetanic stimulation ( B and F ) or bath application of the phorbol ester PDBu ( C and G ) for wild-type (wt, black ), PKCαβ dko animals ( purple ), and PKCαβ dko animals expressing PKCβ WT -YFP ( green ). ( A ) Domain arrangement of PKC Ca . DAG and PDBu bind to the C1 domain and Ca 2+ binds to the C2 domain. ( B , C , F , G ) Left, example EPSCs recorded prior to ( bold traces ) and after ( light traces ) synaptic enhancement for each experimental condition. Right, EPSCs are plotted as a function of time (mean ± SEM). For ( B ), wild-type: 62 ± 12%; αβ dko: 2.4 ± 1.8%. Also see and accompanying legend for PTP induced under elevated-temperature conditions. Similar to PTP induced at room temperature, PTP at near-physiological temperature requires PKC Ca . For ( C ), at steady state: wild-type: 97 ± 12%; αβ dko: 3.2 ± 3.4%; for ( F ), PKCβ WT -YFP: 61 ± 7%; for ( G ), 84 ± 11%. In F and G , the αβ dko group data from B and C respectively are re-plotted for comparison. Also see and . ( D ) In this schematic of the auditory brainstem, the ventral cochlear nucleus (VCn) and medial nuclei of the trapezoid body (MNTB) are labeled. An AAV expressing PKCβ WT -YFP was injected in the VCn at postnatal day 4. ( E ) Confocal images of a brain section labeled with an antibody against vGlut1 ( red ) are shown for a calyx of Held expressing PKCβ WT -YFP ( green ) in a PKCαβ dko animal at postnatal day 18. Scale bar: 10 µm. ( H and I ) The synaptic mechanism through which PKCβ rescues PTP was examined under conditions that relieve AMPA receptor desensitization and saturation. ( H ) Left, overlay of EPSCs (10 ms inter-stimulus interval) delivered prior to ( bold traces ) and 10 s after ( light traces ) PTP-inducing tetanus. Middle, traces are normalized to the first EPSC to allow comparison of PPR. Right, PPR POST (after tetanus) over PPR PRE (before tetanus) (mean ± SEM, see ). Wild-type: p=0.49; αβ dko expressing PKCβ WT -YFP: p=0.68. ( I ) Summary of the readily releasable pool (RRP) and release probability ( p ) contributions to PTP (mean ± SEM, also see and ). RRP WT : 37 ± 9%; RRP PKCβWT-YFP : 39 ± 12%; p=0.88. Scale bars in B , C , F , and G : 2 nA, 1 ms. Scale bars in H : 2 nA, 5 ms. DOI: http://dx.doi.org/10.7554/eLife.03011.003 10.7554/eLife.03011.004 Figure 1—source data 1. Summary and statistical analyses of synaptic properties during PTP and PDBu-induced potentiation. DOI: http://dx.doi.org/10.7554/eLife.03011.004 10.7554/eLife.03011.005 Figure 1—source data 2. Summary and statistical analyses of basal synaptic properties. DOI: http://dx.doi.org/10.7554/eLife.03011.005

Article Snippet: To generate bacterial expression plasmids of the PKCβ C2 domain, the sequences for C2 WT or C2 D/A (a.a. 157–294) ( ) (see also ) were inserted into a pGEX-KG vector (Addgene database #2890) using XbaI and NcoI and the following primers: 5′-TCCGGTGGTGGTGGTGGAATTCTAGAAGAACGCCGTGGCCGCATC-3′ and 3’-AAGCTTGAGCTCGAGTCGACCCATGGTCATCCTTCCGGCGGCACCG-5′.

Techniques: Expressing, Comparison, Labeling, Injection

Journal: eLife

Article Title: Protein kinase C is a calcium sensor for presynaptic short-term plasticity

doi: 10.7554/eLife.03011

Figure Lengend Snippet: ( A ) A partial sequence of the PKCβ C2 domain is shown with Ca 2+ -coordinating aspartates in green. These aspartates were mutated to alanines ( blue ) in the C2 D/A construct. Also see . ( B ) Coomassie blue-stained gel of recombinant wild-type (C2 WT ) and mutant (C2 D/A ) PKCβ C2 domains. ( C ) Averaged intrinsic tryptophan fluorescence is shown for C2 WT and C2 D/A . Fluorescence emission spectra were recorded in 0 mM Ca 2+ ( bold traces ) and 1 mM Ca 2+ ( light traces ). Peak fluorescence intensity change: C2 WT :17 ± 1.3%; C2 D/A : −1.3 ± 2.0%. ( D ) Translocation of PKCβ WT -YFP (left) and PKCβ D/A -YFP (right) in HEK293T cells was monitored in response to the Ca 2+ ionophore ionomycin and in response to PDBu. Ca 2+ increases caused PKCβ WT -YFP to translocate, but not PKCβ D/A -YFP. Both PKCβ WT -YFP and PKCβ D/A -YFP translocated in response to PDBu. Scale bar: 10 µm. DOI: http://dx.doi.org/10.7554/eLife.03011.012

Article Snippet: To generate bacterial expression plasmids of the PKCβ C2 domain, the sequences for C2 WT or C2 D/A (a.a. 157–294) ( ) (see also ) were inserted into a pGEX-KG vector (Addgene database #2890) using XbaI and NcoI and the following primers: 5′-TCCGGTGGTGGTGGTGGAATTCTAGAAGAACGCCGTGGCCGCATC-3′ and 3’-AAGCTTGAGCTCGAGTCGACCCATGGTCATCCTTCCGGCGGCACCG-5′.

Techniques: Sequencing, Construct, Staining, Recombinant, Mutagenesis, Fluorescence, Translocation Assay