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Time kill assay for <i>E</i>. <i>faecalis</i>.

<p>Time kill assay confirmed growth inhibitory effect of <i>β-</i>lactam compounds (3, 7 and 6a) against <i>E</i>. <i>faecalis</i>. Samples were collected at the indicated times and were evaluated for CFUs. Time kill kinetics of <i>E</i>. <i>faecalis</i> by compound 3 (25 μg/ml) represent dead cells within 1 h. Note: Ut- untreated, Amp- Ampicillin.</p

Synthesis of <i>β-</i>lactam substituted polycyclic fused pyrrolidine/pyrrolizidine cycloadducts.

<p>Synthesis of <i>β-</i>lactam substituted polycyclic fused pyrrolidine/pyrrolizidine cycloadducts.</p

Bacterial live/ dead assay: Tooth samples were infected with <i>E</i>. <i>faecalis</i> (1.5x10⁵) cells for 21 days and were treated with 25 μg/ml of <i>β-</i>lactam compound 3 (MIC).

<p>After incubation, the tooth samples were sliced and stained with fluorescein diacetate/ propidium iodide. A) CLSM image of untreated root canal showed green fluorescence indicating live bacteria B) Marked red fluorescence in the root canal treated with <i>β-</i>lactam compound 3 shows dead bacterial cells C) Root canal treated with ampicillin shows red fluorescence. Note: scale bar: A) 20 μm B and C) 100 μm. D) Graph depicting the percentage of bacterial viability after treatment with various agents. Nonparametric test was done for comparisons of significance for test versus control (<i>p</i>< 0.05). Note: Amp- Ampicillin.</p

Efficacy of <i>β</i>-lactam compounds in <i>ex vivo</i> dentine tubule infection model.

<p>Tooth samples were infected with <i>E</i>. <i>faecalis</i> (1.5x10⁵) cells and were treated with <i>β-</i>lactam compounds (3 and 7) at their MICs. After incubation, dental chips containing resident microbes were harvested at two depths (200 and 400 μm). The mean value of culture O.D was obtained. The percentage of reduction was calculated. Non-parametric test was done for comparisons of significance for test versus control (<i>p</i>< 0.05). Note: Amp- Ampicillin.</p

<i>In vivo</i> antibacterial efficacy of <i>β</i>-lactam compound.

<p>To determine the <i>in vivo</i> efficacy of compound 3, Balb/C mice were inoculated with <i>E</i>. <i>faecalis</i> and compound 3, Ampicillin and saline was administered once daily for three days. After 120 h, mice were killed and organs were isolated and histopathological examinations were conducted. Histopathological observation of <i>E</i>. <i>faecalis</i> infected mice kidney sections shows A) Kidney section from healthy uninfected Balb/C mice stained with haematoxylin- eosin. B) Moderate degree of tubular degeneration in 120 h after infection with <i>E</i>. <i>faecalis</i> C) Inflammatory cells in the interstitum along with the hyaline cast in kidney cells of high dose (50 mg/kg) of <i>β-</i>lactam treated group D) Interstital infiltration with inflammatory cells in ampicillin treated group.</p

Mechanism of action of <i>β</i>-lactam compound 3.

<p>A) FACS based measurement of ROS Production. A(i) Untreated cells showed no fluorescence indicating no ROS production. A(ii) Cells showed fluorescence by compound 3 was monitored by incubation of compound 3 (25 μg/ml) with <i>E</i>. <i>faecalis</i> cells for 1 h. Green fluorescence indicating generation of ROS production after treatment with compound 3. A(iii) Cells showed fluorescence indicating generation of ROS production after treatment with ampicillin. A(iv) Cells showed fluorescence after treatment with hydrogen peroxide (H₂O₂) A(v) Graph indicating the percentage of ROS production after treatment with respective agents. The mean and standard deviations from six triplicates were plotted. B) Flow cytometry analysis to show membrane permeabilization of <i>E</i>. <i>faecalis</i> cells in PI uptake assay. <i>E</i>. <i>faecalis</i> (10⁶ cells) cells were incubated with respective agents for 45 min. B(i) Untreated cells showed no fluorescence indicating no membrane damage B(ii) Cells incubated with <i>β</i>-lactam compound 3 (25 μg/ml) showed increased PI uptake as compared to untreated indicating membrane damage and B(iii) Cells incubated with ampicillin showed increased PI uptake.</p

Docking score of <i>β</i>-lactam derivatives.

<p>Docking score of <i>β</i>-lactam derivatives.</p

Minimum Inhibitory Concentration (μg/ml) of <i>β</i>-lactams against pathogens in root canal infection.

<p>-, no activity, Amp- Ampicillin, RS (1–5)—Resistant strains isolated from Root Canal Treatment failure cases.</p><p>Minimum Inhibitory Concentration (μg/ml) of <i>β</i>-lactams against pathogens in root canal infection.</p