Seed Polyphenolic Profile,Antioxidative Activity,and Fatty Acids Composition of Wild and Cultivated Carthamus Species

Total flavonoid content (TFC) and cyanidin‐3‐glucoside (Cyd‐3‐glu) of seed and seed coat extract of 16 genotypes from five species of Carthamus were studied, and their major polyphenolic compounds and antioxidant activity of the seed coat extracts were determined using HPLC analysis and DPPH assay, respectively. Additionally, fatty acids composition of the seed oil was analyzed by GC. In general, TFC and Cyd‐3‐glu content of seed coat extracts were higher than those of seed extracts. A novel breeding line with black seed coat (named A82) depicted lower TFC (3.79 mg QUE/g DW) but higher Cyd‐3‐glu (24.64 mg/g DW) compared to the white and other seed‐pigmented genotypes. DPPH radical scavenging activity showed a strong association with Cyd‐3‐glu content (r = 0.84), but no correlation with TFC (r = ?0.32). HPLC analysis of seed coat extracts revealed that four compounds were dominant constituents including rutin (7.23 – 117.95 mg/100 g DW), apigenin (4.37 – 64.88 mg/100 g DW), quercetin (3.09 – 14.10 mg/100 g DW), and ferulic acid (4.49 – 30.41 mg/100 g DW). Interestingly, the genotype A82 with an appropriate polyunsaturated/saturated fatty acids index (5.46%) and a moderate linoleic fatty acid content (64.70%) had higher nutritional and pharmaceutical value than all the other genotypes.     

First efficient CRISPR‐Cas9‐mediated genome editing in Leishmania parasites

Protozoan pathogens that cause leishmaniasis in humans are relatively refractory to genetic manipulation. In this work, we implemented the CRISPR‐Cas9 system in Leishmania parasites and demonstrated its efficient use for genome editing. The Cas9 endonuclease was expressed under the control of the Dihydrofolate Reductase‐Thymidylate Synthase (DHFR‐TS) promoter and the single guide RNA was produced under the control of the U6snRNA promoter and terminator. As a proof of concept, we chose to knockout a tandemly repeated gene family, the paraflagellar rod‐2 locus. We were able to obtain null mutants in a single round of transfection. In addition, we confirmed the absence of off‐target editions by whole genome sequencing of two independent clones. Our work demonstrates that CRISPR‐Cas9‐mediated gene knockout represents a major improvement in comparison with existing methods. Beyond gene knockout, this genome editing tool opens avenues for a multitude of functional studies to speed up research on leishmaniasis.     

Head Movements Evoked in Alert Rhesus Monkey by Vestibular Prosthesis Stimulation: Implications for Postural and Gaze Stabilization

The vestibular system detects motion of the head in space and in turn generates reflexes that are vital for our daily activities. The eye movements produced by the vestibulo-ocular reflex (VOR) play an essential role in stabilizing the visual axis (gaze), while vestibulo-spinal reflexes ensure the maintenance of head and body posture. The neuronal pathways from the vestibular periphery to the cervical spinal cord potentially serve a dual role, since they function to stabilize the head relative to inertial space and could thus contribute to gaze (eye-in-head + head-in-space) and posture stabilization. To date, however, the functional significance of vestibular-neck pathways in alert primates remains a matter of debate. Here we used a vestibular prosthesis to 1) quantify vestibularly-driven head movements in primates, and 2) assess whether these evoked head movements make a significant contribution to gaze as well as postural stabilization. We stimulated electrodes implanted in the horizontal semicircular canal of alert rhesus monkeys, and measured the head and eye movements evoked during a 100ms time period for which the contribution of longer latency voluntary inputs to the neck would be minimal. Our results show that prosthetic stimulation evoked significant head movements with latencies consistent with known vestibulo-spinal pathways. Furthermore, while the evoked head movements were substantially smaller than the coincidently evoked eye movements, they made a significant contribution to gaze stabilization, complementing the VOR to ensure that the appropriate gaze response is achieved. We speculate that analogous compensatory head movements will be evoked when implanted prosthetic devices are transitioned to human patients.     

Association between TPO gene polymorphisms and Anti-TPO level in Tehranian population: TLGS

Introduction

Thyroid peroxidase (TPO) gene variations are one cause of thyroid autoimmune diseases. The aim of this study was to examine the association between the T1936C, T2229C and A2257C polymorphisms of the TPO gene and Anti-TPO level.

Materials and methods

In this case–control study, 188 individuals (86 males and 102 females), aged 20–80 years, were randomly selected from among the Tehran Lipid and Glucose Study (TLGS) population. A2257C and T2229C SNPs were detected with RFLP by use of BsrI and Eco57I as the restriction enzymes respectively, while the T1936C SNP was determined with ARMS-PCR.

Results

In the presence of the C allele of T1936C, Anti-TPO level was significantly increased (CC: 238 ± 43.3, CT: 47.7 ± 15.9, TT: 74.1 ± 11.3 IU/L p = 0.002); however, this association was attenuated after adjustment for sex and age (p = 0.059). No significant difference, before and after adjustment, was found in Anti-TPO level in the presence of T2229C SNP (CC: 129.1 ± 24.5, CT: 43.5 ± 12.6, TT: 126.5 ± 13.8 IU/L p = 0.196). The association between A2257C and Anti-TPO level was only significant after adjustment for potential confounders (p = 0.007). The association between ATC and CTT haplotypes and Anti-TPO level was significant (p = 0.023, 0.021 respectively), the association between CTT and Anti-TPO concentration was also significant after adjustment for sex (p = 0.014).

Conclusion

The results of the present study confirmed the association between TPO gene polymorphisms and Anti-TPO level in the Tehranian population.     

Dehydroquinate synthase: the role of divalent metal cations and of nicotinamide adenine dinucleotide in catalysis

The cofactor requirements of dehydroquinate synthase from Escherichia coli have been characterized. The homogeneous enzyme, purified from the overproducing strain RB791 (pJB14), is a monomeric metalloenzyme of Mr = 39,000 that contains 1 mol of tightly bound Co(II) according to atomic absorption analysis. The holoenzyme rapidly loses activity upon incubation with EDTA, giving rise to a stable but catalytically inactive apoenzyme. Activity is fully restored by reconstitution with Co(II) and partially restored with other divalent cations. Reconstitution of the apoenzyme with Zn(II) (which is probably the functioning metal in vivo) restores activity to 53% of the level observed with the Co(II)-holoenzyme. The presence of the substrate 3-deoxy-D-arabino-heptulosonate 7-phosphate (1) blocks the inactivation by EDTA. Dehydroquinate synthase also binds 1 mol of NAD+, the presence of which is essential for catalytic activity. The rate constant for the dissociation of NAD+ from the Co(II)-holoenzyme was found to be 0.024 min-1. Under turnover conditions with saturating levels of substrate, the dissociation rate of NAD+ increases by a factor of 40, to 1 min-1. Under these conditions (pH 7.5, 20 degrees C), the Km for NAD+ was determined to be 80 nM.     

Suppressive effect of exogenous miR-16 and miR-34a on tumorigenesis of breast cancer cells

Recent investigations have shown tumor-suppressive roles for miR-16 and miR-34a. They also share some features in regard to targeting cancer cell signaling pathways which they control. Therefore, in this study, we aimed to further scrutinize whether exogenous induction of mature miR-34a and miR-16 can collaborate in breast tumor suppression. MDA-MB-231 and SK-BR-3 human breast cancer cell lines were cultured and transfected twice with hsa-miR-16-5p and hsa-miR-34a-5p mimics individually or in combination. The cells were analyzed for apoptosis rate and cell cycle indices by flow cytometry. Also, the expression of several invasion and the epithelial-mesenchymal transition markers was evaluated at gene and protein levels by quantitative real-time polymerase chain reaction and western blot analysis, respectively. Assessment of invasiveness and migratory potential of the transfected cells was performed using three-dimensional spheroid formation and wound-healing assay, respectively. In both cell lines, miR-16 and miR-34a induced apoptosis and cell-cycle arrest and also suppressed invasion and migration. Some of these effects, like cell-cycle arrest and induction of apoptosis, were significantly higher when using both microRNAs than when using them individually for transfection of the cells. Our results are indicating that miR-16 and miR-34a can collaborate in breast tumor suppression.     

Human Calprotectin： Effect of Calcium and Zinc on its Secondary and Tertiary Structures, and Role of pH in its Thermal Stability

Calprotectin, a heterodimeric complex belonging to the S 100 protein family, has been found predominantly in the cytosolic fraction of neutrophils. In the present study, human calprotectin was purified from neutrophils using two-step ion exchange chromatography. The purified protein was used for circular dichroism study and fluorescence analysis in the presence of calcium and zinc at physiological concentrations, as well as for assessment of its inhibitory activity on the K562 leukemia cell line. The thermal stability of the protein at pH 7.0 （physiological pH） and 8.0 （similar to intestinal pH） was also compared. The results of cell proliferation analysis revealed that human calprotectin initiated growth inhibition of the tumor cells in a dose- dependent manner. The intrinsic fluorescence emission spectra of human calprotectin （50 ktg/ml） in the presence of calcium and zinc ions show a reduction in fluorescence intensity, reflecting a conformational change within the protein with exposure of aromatic residues to the protein surface that is important for the biological function of calprotectin. The far ultraviolet-circular dichroism spectra of human calprotectin in the presence of calcium and zinc ions at physiological concentrations show a decrease in the m-helical content of the protein and an increase in [3- and other structures. Our results also show that increasing the pH level from 7.0 to 8.0 leads to a marked elevation in the thermal stability of human calprotectin, indicating a significant role for pH in the stability of calprotectin in the gut.