Pairwise variation analysis between NFn and NFn+1 to determine the optimal number of genes for reliable normalization.

<p>Values below the 0.15 threshold mean that <i>n</i> genes might be sufficient.</p

Specificity of primers used for RT-qPCR analysis in <i>B</i>. <i>poitrasii</i>.

<p><b>A)</b> Agarose gel electrophoresis and analysis of amplified products obtained from RT-qPCR. The electrophoresis was done on 2% agarose gel. The names of candidate reference genes are mentioned on top. The figures on left indicate the size of PCR amplicons in base pairs. B) Melting curves indicating single peaks for three representative genes. Arrow indicates the melting curve for the RT-qPCR reaction without template.</p

Distribution overview of expression of each candidate reference gene in absolute Ct values for RNA samples of different morphological forms in <i>B</i>. <i>poitrasii</i>.

<p>Samples 1, yeast cells grown in YPG (1%) medium for 12 h; Sample 2, yeast cells grown in YPG medium for 24h; Sample 3, yeast cells grown in YPG (0.1%) medium for 12 h; Sample 4, hyphae grown in YP medium for 12 h; Sample 5, hyphae grown in YP medium for 24 h; Sample 6, hyphae grown in YPG(0.1%) for 12 h.; Sample 7, 5 d old sporangiospores; Sample 8, 10 d old sporangiospores; Sample 9, 15 d old sporangiospores; Sample 10, 7 d old zygospores; Sample 11, 15 d old zygospores; Sample 12, 30 d old zygospores.</p

Selection of reference genes for quantitative real-time RT-PCR assays in different morphological forms of dimorphic zygomycetous fungus <i>Benjaminiella poitrasii</i>

<div><p><i>Benjaminiella poitrasii</i>, a dimorphic non-pathogenic zygomycetous fungus, exhibits a morphological yeast (Y) to hypha (H) reversible transition in the vegetative phase, sporangiospores (S) in the asexual phase and zygospores (Z) in the sexual phase. To study the gene expression across these diverse morphological forms, suitable reference genes are required. In the present study, 13 genes <i>viz</i>. <i>ACT</i>, <i>18</i>S r<i>RNA</i>, <i>eEF1α</i>, <i>eEF-Tu</i>,<i>eIF-1A</i>, <i>Tub-α</i>, <i>Tub-b</i>, <i>Ubc</i>, <i>GAPDH</i>, <i>Try</i>, <i>WS-21</i>, <i>NADGDH</i> and <i>NADPGDH</i> were evaluated for their potential as a reference, particularly for studying gene expression during the Y-H reversible transition and also for other asexual and sexual life stages of <i>B</i>. <i>poitrasii</i>. Analysis of RT-qPCR data using geNorm, normFinder and BestKeeper software revealed that genes such as <i>Ubc</i>, <i>18S rRNA</i> and <i>WS-21</i> were expressed at constant levels in each given subset of RNA samples from all the morphological phases of <i>B</i>. <i>poitrasii</i>. Therefore, these reference genes can be used to elucidate the role of morpho-genes in <i>B</i>. <i>poitrasii</i>. Further, use of the two most stably expressed genes (<i>Ubc</i> and <i>WS-21</i>) to normalize the expression of the ornithine decarboxylase gene (Bp<i>odc</i>) in different morphological forms of <i>B</i>. <i>poitrasii</i>, generated more reliable results, indicating that our selection of reference genes was appropriate.</p></div

List of the candidate reference genes evaluated in <i>B</i>. <i>poitrasii</i> and sequences of primers along with optimized annealing temperatures, size of the fragments they amplified and PCR efficiency.

<p>List of the candidate reference genes evaluated in <i>B</i>. <i>poitrasii</i> and sequences of primers along with optimized annealing temperatures, size of the fragments they amplified and PCR efficiency.</p

Protein, cAMP, and NAD(P)H contents of different morphological forms of <i>Benjaminiella poitrasii</i>.

<p>Protein, cAMP, and NAD(P)H contents of different morphological forms of <i>Benjaminiella poitrasii</i>.</p

Silicon Incorporated Morpholine Antifungals: Design, Synthesis, and Biological Evaluation

Known morpholine class antifungals (fenpropimorph, fenpropidin, and amorolfine) were synthetically modified through silicon incorporation to have 15 sila-analogues. Twelve sila-analogues exhibited potent antifungal activity against different human fungal pathogens such as <i>Candida albicans</i>, <i>Candida glabrata</i>, <i>Candida tropicalis</i>, <i>Cryptococcus neoformans</i>, and <i>Aspergillus niger</i>. Sila-analogue <b>24</b> (fenpropimorph analogue) was the best in our hands, which showed superior fungicidal potential than fenpropidin, fenpropimorph, and amorolfine. The mode of action of sila-analogues was similar to morpholines, i.e., inhibition of sterol reductase and sterol isomerase enzymes of ergosterol synthesis pathway