Pseudomonas and Bacillus as Potential Sources of Novel Antibiotics

With an increase in antibiotic resistance within the medical field the need for novel antibiotics is evident. Through collaboration with the Tiny Earth Project Initiative, we hope to find novel antibiotics through bacteria found in soil. We were able to successfully isolate two microbes that showed broad spectrum antibiotics against both Staphylococcus epidermis and Bacillus subtilis. Through further exploration and collaboration with the Tiny Earth Project Initiative, we might be able to discover novel antibiotics

Supplementation with fish oil and genistein, individually or in combination, protects bone against the adverse effects of methotrexate chemotherapy in rats

Cancer chemotherapy has been shown to induce long-term skeletal side effects such as osteoporosis and fractures; however, there are no preventative treatments. This study investigated the damaging effects of anti-metabolite methotrexate (MTX) subcutaneous injections (0.75 mg/kg BW) for five days and the potential protective benefits of daily oral gavage of fish oil at 0.5 mL/100 g BW (containing 375 mg of n-3 PUFA/100 g BW), genistein (2 mg/100 g BW), or their combination in young adult rats. MTX treatment alone significantly reduced primary spongiosa height and secondary spongiosa trabecular bone volume. Bone marrow stromal cells from the treated rats showed a significant reduction in osteogenic differentiation but an increase in adipogenesis ex vivo. Consistently, stromal cells had significantly higher mRNA levels of adipogenesis-related proliferator activator activated receptor-γ (PPAR-γ) and fatty acid binding protein (FABP4). MTX significantly increased the numbers of bone-resorbing osteoclasts and marrow osteoclast precursor cell pool while significantly enhancing the mRNA expression of receptor activator for nuclear factor kappa B ligand (RANKL), the RANKL/osteoprotegerin (OPG) ratio, interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) in the bone. Supplementary treatment with fish oil and/or genistein significantly preserved trabecular bone volume and osteogenesis but suppressed MTX-induced adipogenesis and increases in osteoclast numbers and pro-osteoclastogenic cytokine expression. Thus, Fish oil and/or genistein supplementation during MTX treatment enabled not only preservation of osteogenic differentiation, osteoblast number and bone volume, but also prevention of MTX treatment-induced increases in bone marrow adiposity, osteoclastogenic cytokine expression and osteoclast formation, and thus bone loss. © 2013 Raghu Nadhanan et al

Effects of MTX alone or with supplementary treatment with (FO) and/or genistein (Gen) on osteogenic differentiation potential of bone marrow stromal cells isolated from treated rats.

<p>Images of a culture well showing bone marrow stromal cell-derived CFU-f colonies stained positive for alkaline phosphatase (ALP, arrows) of (<b>A</b>) a control rat and (<b>B</b>) a MTX alone treated rat on day 9 post the first MTX injection. (<b>C</b>) Treatment effects on size of osteoprogenitor cell pool in bone marrow. Mineralizing colonies stained positive by Alizarin Red (arrows) of (<b>D</b>) a control rat and (<b>E</b>) a MTX alone treated rat. (<b>F</b>) <i>Ex vivo</i> mineralization assay with bone marrow cells isolated from rats. RT-PCR relative mRNA expression of (<b>G</b>) Runx2 and of (<b>H</b>) bone matrix protein osteocalcin (OCN) assessed in bone marrow stromal cells of treated rats (relative to Cyclophilin-A). Labelled means without a common letter differ (P<0.05).</p

Effects of MTX with or without (FO) and/or genistein (Gen) supplementation on expression of anti-inflammatory cytokines IL-4 and IL-10.

<p>Levels of mRNA expression in metaphysis bones of treated rats as quantitated by real time RT-PCR: (<b>A</b>) IL-4 and (<b>B</b>) IL-10. Protein levels (pg/mL) of circulating (<b>C</b>) IL-4 and (<b>D</b>) IL-10 in plasma samples of treated rats as measured by multiplex cytokine assay. Labelled means without a common letter differ (P<0.05).</p

Effects of MTX with or without (FO) and/or genistein (Gen) supplementation on expression of osteoclastogenesis-regulatory or related genes.

<p>Levels of mRNA expression in metaphysis bones of treated rats as quantitated by real time RT-PCR: (<b>A</b>) RANKL/OPG ratio, (<b>B</b>) TNF-α, and (<b>C</b>) IL-6. (<b>D</b>) Levels (pg/mL) of circulating IL-6 protein in plasma samples of treated rats as measured by multiplex cytokine assay. Labelled means without a common letter differ (P<0.05).</p

Effects of MTX with or without (FO) and/or genistein (Gen) supplementation on osteoclastogenesis potential.

<p>Images of TRAP-stained tibial metaphysis (arrows pointing multinucleated TRAP-positive osteoclasts) of (<b>A</b>) a control rat and (<b>B</b>) a MTX alone treated rat on day 9 post the first MTX injection showing more osteoclasts present. (<b>C</b>) Average osteoclast numbers at tibial primary and secondary spongiosa. Images of TRAP positively-stained cells formed (arrows pointing multinucleated TRAP-positive osteoclast-like cells) in an <i>ex vivo</i> osteoclastogenesis assay of (<b>D</b>) a control rat and (<b>E</b>) a MTX alone treated rat on day 9 post the first MTX injection showing more osteoclasts formed. (<b>F</b>) <i>Ex vivo</i> osteoclast formation from bone marrow cells isolated from treated rats. Labelled means without a common letter differ (P<0.05).</p

Effects of MTX with or without fish oil (FO) and/or genistein (Gen) supplementation on growth plate, primary and secondary spongiosa.

<p>Paraffin sections of the tibial metaphysis region (PS =  Primary spongiosa, SS =  Secondary spongiosa, which are separated by a dashed line) of (<b>A</b>) a normal rat, (<b>B</b>) a MTX+H₂O treated rat showing reduced height of primary spongiosa and. metaphyseal bone volume, (<b>C</b>) a MTX+FO treated rat, and (<b>D</b>) a MTX+Gen treated rat. (<b>E</b>) Growth plate total height (µm). (<b>F</b>) Primary spongiosa height (µm). (<b>G</b>) Secondary spongiosa BV/TV (%). (<b>H</b>) Secondary spongiosa osteoblast/mm² trabecular bone area. Values are means ± SEM; n = 7–8 for all groups. Labelled means without a common letter differ (P<0.05).</p

List of primers used in RT-PCR.

<p>List of primers used in RT-PCR.</p