Retuning Rieske-type oxygenases to expand substrate range

Rieske-type oxygenases are promising biocatalysts for the destruction of persistent pollutants or for the synthesis of fine chemicals. In this work, we explored pathways through which Rieske-type oxygenases evolve to expand their substrate range. BphAE(p4), a variant biphenyl dioxygenase generated from Burkholderia xenovorans LB400 BphAE(LB400) by the double substitution T335A/F336M, and BphAE(RR41), obtained by changing Asn(338), Ile(341), and Leu(409) of BphAE(p4) to Gln(338), Val(341), and Phe(409), metabolize dibenzofuran two and three times faster than BphAE(LB400), respectively. Steady-state kinetic measurements of single- and multiple-substitution mutants of BphAE(LB400) showed that the single T335A and the double N338Q/L409F substitutions contribute significantly to enhanced catalytic activity toward dibenzofuran. Analysis of crystal structures showed that the T335A substitution relieves constraints on a segment lining the catalytic cavity, allowing a significant displacement in response to dibenzofuran binding. The combined N338Q/L409F substitutions alter substrate-induced conformational changes of protein groups involved in subunit assembly and in the chemical steps of the reaction. This suggests a responsive induced fit mechanism that retunes the alignment of protein atoms involved in the chemical steps of the reaction. These enzymes can thus expand their substrate range through mutations that alter the constraints or plasticity of the catalytic cavity to accommodate new substrates or that alter the induced fit mechanism required to achieve proper alignment of reaction-critical atoms or groups.     

Bacteriological follow-up of pulmonary tuberculosis treatment: a study with a simple colorimetric assay

The viability of Mycobacterium tuberculosis (MTB) in serial sputum specimens from persistently smear positive patients was evaluated. The assay was based on oxidation-reduction of Alamar Blue and Malachite Green dyes that change their color in response to MTB growth. A total of 280 sputum specimens from 40 persistently smear positive TB patients and 40 sputa from non-tuberculosis patients were digested, decontaminated and examined microscopically. To check the MTB viability, the sediments from decontaminated samples were inoculated into three culture media: Lowenstein-Jensen (LJ) slants, Alamar Blue and Malachite Green culture tubes. We found that out of 280 smear positive specimens, the LJ culture was positive in 124 (44%). The numbers of correctly identified S+/C+ cases by Alamar Blue and Malachite Green were 118 (95%) and 116 (93%), respectively. The mean time required for reporting the positive signal in Alamar Blue culture tubes was 9 versus 11 days by Malachite Green culture tubes. In the standard LJ culture media the average detection time was 27 days (P < 0.05). The sensitivity of LJ was 99%, Alamar Blue 95% and Malachite Green 93%. The specificity was 100%, 92% and 93%, respectively. The oxidation-reduction method is rapid, sensitive and inexpensive in monitoring the treatment response of patients with pulmonary TB. Thus, using this method can be of paramount importance, particularly in resource-constrained areas.     

Taxonomic implications of pollen exine morphology in infrageneric classification of Scutellaria (Lamiaceae)

Scutellaria L. in Lamiaceae subfamily Scutellarioideae is a subcosmopolitan genus with about 360 species. According to the latest subgeneric classification, two subgenera are recognized: S. subgen. Scutellaria and S. subgen. Apeltanthus. We studied pollen grains from 29 species of Scutellaria belonging to both subgenera and from both the Old and the New World to evaluate their taxonomic importance for the infrageneric classification of the genus. Pollen grains were acetolysed and studied by scanning electron microscope (SEM) and light microscope (LM). All examined pollen grains are isopolar, tricolpate, suboblate, oblate spheroidal, prolate spheroidal to subprolate. The exine is mainly bireticulate perforate, but with variations in lumina shape and size as well as perforation size and uniformity. Based on the exine sculpturing, two subtypes are recognized within the bireticulate pollen type. In subtype I the primary reticulum of the exine consists of angular muri; the lumen has an irregular polygonal shape and the perforations of the secondary reticulum are more or less uniform and of the same size. This pollen subtype was observed in species of subgenus Scutellaria, sectt. Scutellaria p.p., Anaspis, Perilomia and Salazaria. In subtype II the primary reticulum of the exine consists of a more or less curved murus; the lumen is rounded or obtusely angular and the secondary reticulum consists of one or more large central perforations surrounded by some smaller ones. This pollen subtype was observed in species of subgenus Scutellaria sectt. Scutellaria p.p., Salviifoliae and in all members of subgenus Apeltanthus. The results of this study reveal that the exine ornamentation is a diagnostic character useful for the infrageneric classification of Scutellaria.     

Effects of foliar application of 6-benzylaminopurine on yield and oil content in two spring safflower (Carthamus tinctorius L.) cultivars

To study the effect of an exogenous cytokinin application on safflower yield, an experiment was conducted in 2012–2013. Two cultivars of safflower (Goldasht and Zendehrood) and five concentrations of 6-benzylaminopurine (BAP) (0, 25, 50, 75, and 100 μM) were applied at the flowering stage. Results indicated that the application of 75 μM of BAP showed increased seed and oil yield by 17.54 and 18.29 % over the control, respectively. The increase in seed yield by application of BAP was attributed to the increase in characters like number of heads per plant, number of seeds per head, and 1,000 seed weight. Applying of BAP increased oil content compared with the control. To determine the concentration of cytokinin which has the highest performance for increasing seed yield, regression analysis were estimated showing that in the Zendehrood cultivar, the application of 43 μM of BAP produced the highest seed yield, and in the Goldasht cultivar the application of 73 μM of BAP during flowering produced the highest seed yield.     

Herbivore-induced indirect defense across bean cultivars is independent of their degree of direct resistance

We tested the extent to which resistance of common bean (Phaseolus vulgaris) cultivars to the spider mite Tetranychus urticae parallels the extent to which these plants display indirect defenses via the induced attraction of the predatory mite Phytoseiulus persimilis. First, via field and greenhouse trials on 19 commercial bean cultivars, we selected two spider mite-resistant (Naz and Ks41128) and two susceptible (Akthar and G11867) cultivars and measured the spider mite-induced volatiles and the subsequently induced attraction of predatory mites via olfactory choice assays. The two major volatiles, 4,8,12-trimethyltrideca-1,3,7,11-tetraene (TMTT) and (Z)-3-hexenyl-acetate, were induced in the resistant but not in the susceptible cultivars. However, uninfested susceptible cultivars emitted these volatiles at levels similar to those of mite-infested resistant cultivars. Significant induction of several minor components was observed for all four cultivars except for the infested-susceptible cultivar G11867. Both, the spider mite-resistant cultivar Naz and the susceptible cultivar G11867, attracted more predatory mites when they were infested. In contrast, spider mites induced increased emission of two major and five minor volatiles in Ks41128, but predatory mites did not discriminate between infested and uninfested plants. Overall, the attraction of predatory mites appeared to correlate positively with the presence of TMTT and (Z)-3-hexenyl acetate and negatively with β-caryophyllene and α-pinene in the bean headspace. Taken together, our data suggest that resistance and attraction of natural enemies via induced volatiles are independent traits. We argue that it should be possible to cross predator-attraction promoting traits into resistant cultivars that lack sufficiently inducible indirect defenses.     

Rip3 knockdown rescues photoreceptor cell death in blind pde6c zebrafish

Achromatopsia is a progressive autosomal recessive retinal disease characterized by early loss of cone photoreceptors and later rod photoreceptor loss. In most cases, mutations have been identified in CNGA3, CNGB3, GNAT2, PDE6C or PDE6H genes. Owing to this genetic heterogeneity, mutation-independent therapeutic schemes aimed at preventing cone cell death are very attractive treatment strategies. In pde6c^w59 mutant zebrafish, cone photoreceptors expressed high levels of receptor-interacting protein kinase 1 (RIP1) and receptor-interacting protein kinase 3 (RIP3) kinases, key regulators of necroptotic cell death. In contrast, rod photoreceptor cells were alternatively immunopositive for caspase-3 indicating activation of caspase-dependent apoptosis in these cells. Morpholino gene knockdown of rip3 in pde6c^w59 embryos rescued the dying cone photoreceptors by inhibiting the formation of reactive oxygen species and by inhibiting second-order neuron remodelling in the inner retina. In rip3 morphant larvae, visual function was restored in the cones by upregulation of the rod phosphodiesterase genes (pde6a and pde6b), compensating for the lack of cone pde6c suggesting that cones are able to adapt to their local environment. Furthermore, we demonstrated through pharmacological inhibition of RIP1 and RIP3 activity that cone cell death was also delayed. Collectively, these results demonstrate that the underlying mechanism of cone cell death in the pde6c^w59 mutant retina is through necroptosis, whereas rod photoreceptor bystander death occurs through a caspase-dependent mechanism. This suggests that targeting the RIP kinase signalling pathway could be an effective therapeutic intervention in retinal degeneration patients. As bystander cell death is an important feature of many retinal diseases, combinatorial approaches targeting different cell death pathways may evolve as an important general principle in treatment.     

Recurrent pregnancy loss in a subject with heterozygote factor V Leiden mutation; a case report

Recurrent pregnancy loss is usually defined as the loss of two or more consecutive pregnancies before 20 weeks of gestation, which occurs in approximately 5% of reproductive-aged women. It has been suggested that women with thrombophilia have an increased risk of pregnancy loss and other adverse pregnancy outcomes. Thrombophilia is an important predisposition to blood clot formation and is considered as a significant risk factor for recurrent pregnancy loss. The inherited predisposition to thrombophilia is most often associated with factor V Leiden mutation, prothrombin G20210A mutation, and methylenetetrahydrofolate reductase C677T and A1298C gene variants. The net effect is an increased cleavage of prothrombin to thrombin and excessive blood coagulation. Key Words: Recurrent pregnancy loss, Hereditary thrombophilia, Factor V Leiden mutation     

Endophytic Bacteria Induce Growth Promotion and Wilt Disease Suppression in Oilseed Rape and Tomato

To determine whether bacteria isolated from within plant tissue can have plant growth-promotion potential and provide biological control against soilborne diseases, seeds and young plants of oilseed rape (Brassica napus L. cv. Casino) and tomato (Lycopersicon lycopersicum L. cv. Dansk export) were inoculated with individual bacterial isolates or mixtures of bacteria that originated from symptomless oilseed rape, wild and cultivated. They were isolated after surface sterilization of living roots and stems. The effects of these isolates on plant growth and soilborne diseases for oilseed rape and tomato were evaluated in greenhouse experiments. We found isolates that not only significantly improved seed germination, seedling length, and plant growth of oilseed rape and tomato but also, when used for seed treatment, significantly reduced disease symptoms caused by their vascular wilt pathogens Verticillium dahliae Kleb and Fusarium oxysporum f. sp. lycopersici (Sacc.), respectively.