Taxonomic reevaluation of <i>Eremias strauchi strauchi</i> Kessler, 1878 and <i>Eremias strauchi kopetdaghica</i> Szczerbak, 1972, based on nuclear and mitochondrial DNA sequences (Reptilia: Lacertidae)

<div><p>Strauch's Racerunner, <i>Eremias strauchi</i>, is represented by two subspecies, <i>Eremias strauchi strauchi</i> Kessler, 1878 and <i>Eremias strauchi kopetdaghica</i> Szczerbak, 1972, occurring in opposite margins on the northern Iranian Plateau. We sequenced 3926 base pairs of nuclear and mitochondrial DNA from 16 samples of <i>Eremias strauchi</i>, <i>Eremias lalezharica</i> and <i>Eremias velox</i> collected from northeastern, northwestern and southern Iran. Phylogenetic analyses revealed that <i>Eremias lalezharica</i> is sister to <i>Eremias strauchi kopetdaghica</i> and caused the currently recognised species <i>Eremias strauchi</i> to be paraphyletic. According to the estimated genetic distances in the mitochondrial fragments among the lineages, <i>E. s. strauchi</i> diverged from <i>E. s. kopetdaghica</i> and <i>E. lalezharica</i> with a mean genetic distance of 14.0% and 13.9% respectively. Our data indicate enough molecular divergence between the two currently recognised subspecies of <i>E. strauchi</i> and justify upgrading them to full species level as <i>Eremias strauchi</i> (for the north-western clade) and <i>Eremias kopetdaghica</i> (for the north-eastern clade).</p></div

UHPLC-MS characterisation of principal triterpene glycosides and biological activities of different solvent extracts of <i>Allochrusa gypsophiloides</i> (Caryophyllaceae)

A crude methanol extract of the roots of Allochrusa gypsophiloides (syn. Acanthophyllum gypsophiloides) (collected from the Tashkent region of Uzbekistan) was chemically characterised by UHPLC-ESI-QTOF-MS/MS analysis. The results indicate the presence of six major bisdesmosidic saponins derived from gypsogenin, gypsogenic and quillaic acids, including five compounds reported for the first time for this species. The chloroform, methanol and water extracts of A. gypsophiloides showed weak antioxidant and anthelmintic activities. Among the tested extracts, the water extract exhibited the highest level of cytotoxicity in CCRF-CEM and CEM/ADR5000 cell lines with IC50 values of 23.6 and 31.9 µg/mL, respectively.</p

Genomic DNA and transcript analysis of hairy root cultures.

<p>(A) Southern blot analysis was performed using the genomic DNA of transformed hairy root clones. (B) RT-PCR analysis of <i>EPO</i> mRNA in hairy root cultures ^pK7cal/rh<i>EPO</i>^HR and ^pK7rh<i>EPO</i>^HR. The presence of mRNA for tobacco <i>β actin</i> gene is shown as an internal control for transcript analysis.</p

Supplemental Files.zip

Data of the manuscript 'Cucurbitacins: Elucidation of their interactions with the cytoskeleton'<br

HPLC profile of rhEPO in transformed and control hairy root cultures.

<p>(A) Positive control rhEPO^CHO standard recombinant human erythropoietin from CHO cells. (B) Hairy root culture ^pK7cal/rh<i>EPO</i>^HR. (C) Negative control hairy root culture Nt/ATCC^HR. The arrow indicates the presence of peaks.</p

Regeneration of T₀ tobacco plantlets.

<p>(A) The 2 week old transgenic or control calli were cultured in regeneration medium. Scale bars are 5.8 cm. (B) The 4 week old transgenic or control regenerated T₀ plantlets (^pK7cal/rh<i>EPO</i>^RP or Nt/ATCC^RP respectively) were grown in MS medium. Scale bars are 5.9 cm.</p

GFP expression in hairy root cultures and in leaves of regenerated plant.

<p>(A) Confocal images obtained from 15 d old hairy root cultures. (B) Confocal images obtained from 4 week old leaf tissues of regenerated T₀ transgenic plantlets. Images are representative for three experiments and the scale bars are 50 μm.</p

<i>Agrobacterium rhizogenes</i> mediated transformation and growth kinetics of the hairy root cultures.

<p>(A) Hairy root culture with cal signal sequence ^pK7cal/rh<i>EPO</i>^HR, hairy root culture without cal signal sequence ^pK7rh<i>EPO</i>^HR and control hairy root cultures Nt/ATCC^HR were shown. Scale bars are 1 cm. (B) Establishment of the initiated hairy roots (1–2 cm) in 50 mL of liquid WPM. Scale bars are 5 cm. (C) Graphical representation of growth kinetics for the hairy root cultures. All samples were measured in triplicates and each data represents the average ± SD.</p

Western blot and ELISA analysis for effect of PVP treatment on the expression or yield of rhEPO.

<p>(A) Western blot analysis of intracellular and extracellular total protein was performed for 20 d old hairy root cultures following the addition of PVP to the growth medium. For size comparison, recombinant human erythropoietin rhEPO^CHO of molecular size 37 kDa from CHO cells (Sigma) was used. (B) ELISA analysis of extracellular rhEPO^HR concentration in hairy root culture ^pK7cal/rh<i>EPO</i>^HR is shown as ng ml^-1 for three different intervals 10, 20 and 35 d. (C) ELISA analysis of intracellular rhEPO^HR concentration for 20 d old hairy root culture ^pK7cal/rh<i>EPO</i>^HR is shown as ng/μg of total soluble protein is shown as ng μg^-1 of total soluble protein. (D) ELISA analysis of intracellular rhEPO^RP concentration in 4 w old leaf tissues of transgenic plants ^pK7cal/rh<i>EPO</i>^RP regenerated from hairy root cultures ^pK7cal/rh<i>EPO</i>^HR is shown as ng μg^-1 of total soluble protein. Each bar represents the average ± SD in panels B–D. Student’s t-test was performed to analyze significance, N = 3, *** and ⁺⁺⁺ p<0.001, ** and ⁺⁺ p<0.01, and n.s. indicates no significance in panels B–D.</p

Bio-assay of rhEPO in a retinal pigmented epithelial (ARPE) cells.

<p>(A) Impact of rhEPO^HR on viability in ARPE cell lines was measured with MTT assay. (B) Impact of rhEPO^HR on proliferation of ARPE cells was analysed with BrdU cell proliferation assay. The results are presented as percentage of cell viability in Panel A and percentage of cell proliferation in panel B. Student’s t-test was performed for Panels A and B to analyze significance, N = 3, *** p<0.001 and ** p<0.01 as compared to the corresponding data of untreated group (grey bars).</p