Transgenic rice endosperm as a bioreactor for molecular pharming

Plants provide a promising expression platform for producing recombinant proteins with several advantages in terms of high expression level, lower production cost, scalability, and safety and environment-friendly. Molecular pharming has been recognized as an emerging industry with strategic importance that could play an important role in economic development and healthcare in China. Here, this review represents the significant advances using transgenic rice endosperm as bioreactor to produce various therapeutic recombinant proteins in transgenic rice endosperm and large-scale production of OsrHSA, and discusses the challenges to develop molecular pharming as an emerging industry with strategic importance in China.     

Rapid detection of Down's syndrome using quantitative real-time PCR (qPCR) targeting segmental duplications on chromosomes 21 and 11

Objective

Development of a qPCR test for the detection of trisomy 21 using segmental duplications.

Methods

Segmental duplications in the TTC3 gene on chromosome 21 and the KDM2A gene on chromosome 11 were selected as molecular markers for the diagnostic qPCR assay. A set of consensus primers selected from the conserved regions of these segmental duplications were used to amplify internal diverse sequences that were detected and quantified with different probes labeled with distinct fluorescence. The copy numbers of these two fragments were determined based on the ΔCq values of qPCR. The results of qPCR for prenatal and neonatal screening of Down's syndrome were compared with the conventional karyotype analysis by testing 82 normal individuals and 50 subjects with Down's syndrome.

Results

The ΔCq values of segmental duplications on chr21 and 11 ranged between 0.33 and 0.75 in normal individuals, and between 0.91 and 1.18 in subjects with Down's syndrome. The ΔCq values of these two segmental duplications clearly discriminated Down's syndrome from normal individuals (P < 0.001). Furthermore, the qPCR results were consistent with karyotype analysis.

Conclusion

Our qPCR can be used for rapid prenatal and neonatal screening of Down's syndrome.     

Asymmetric Addition of Triethylaluminium to Aromatic Aldehydes Catalyzed by Titanium‐(5,5’‐Biquinoline‐6,6’‐Diol) Complexes

A novel convenient procedure for the resolution of 5,5’‐biquinoline‐6,6’‐diol (BIQOL) was achieved by separating the corresponding diastereomeric mixture of (S)‐(+)‐camphorsulfonates on a semiprepared XDB‐C8 column followed by hydrolysis. The efficient asymmetric addition of triethylaluminium to aromatic aldehydes catalyzed by Ti‐(+)/(–)BIQOL complexes under mild conditions is described. The reactions led to the formation of 1‐arylpropan‐1‐ol in up to 87.5% ee. Chirality 26:268‐271, 2014. © 2014 Wiley Periodicals, Inc.     

Molecular screening of Chinese medicinal plants for progestogenic and anti-progestogenic activity

Estrogen and progestins have adverse effects, and many of these adverse effects are caused by progestins. Due to this, many women choose to use botanical alternatives for hormone replacement therapy, which does not trigger steroidogenic properties. Therefore, it is necessary to screen these herbs for progestogenic and anti-progestogenic properties. Extract of 13 Chinese medicinal plants were analysed for progestogenic and anti-progestogenic activities by using progesterone response element-driven luciferase reporter gene bioassay. MTT assay was carried out to investigate the cytotoxic effect of herb extract on PAE cells. Among the 13 herbs, Dipsacus asperoides extract exhibited progestogenic activity, and 10 species – Cortex eucommiae, Folium artemisiae argyi, Glycyrrhiza uralensis, Angelica sinensis, Atractylodes macrocephala koidz, Scutellaria baicalensis, Cuscuta chinensis, Euscaphis japonica, Ailanthus altissima, and Dioscorea opposita – were recognized to have anti-progestogenic like activities. Extract of Dipsacus asperoides demonstrated dose-dependent progestogenic activity, and the progestogenic activity of 100 μg/mL extracts was equivalent to 31.45 ng/mL progesterone activity. Herbs extracts that exhibited anti-progestogenic-like activity also inhibited the 314.46 ng/mL progesterone activity in a dose-response manner. None of the herb extracts shown significant toxic effect on PAE cells at 40–100 μg/mL compared to control. This discovery will aid selection of suitable herbs for hormone replacement therapy.     

Knockdown of VEGFR2 Inhibits Proliferation and Induces Apoptosis in Hemangioma-Derived Endothelial Cells

Angiogenesis is a process of development and growth of new capillary blood vessels from pre-existing vessels. Angiogenic growth factors play important roles in the development and maintenance of some malignancies, of which vascular endothelial growth factor (VEGF)/VEGFR2 interactions are involved in proliferation, migration, and survival of many cancer cells. The aim of this study was to investigate the function of VEGFR2 in human hemangiomas (HAs). Using immunohistochemistry assay, we examined the expression levels of VEGF, VEGFR2, Ki-67, glucose transporter-1 (Glut-1), phosphorylated protein kinase B (p-AKT) and p-ERK in different phases of human HAs. Positive expression of VEGF, VEGFR2, Ki-67, Glut-1, p-AKT and p-ERK was significantly increased in proliferating phase HAs, while decreased in involuting phase HAs (P=0.001; P=0.003). In contrast, cell apoptotic indexes were decreased in proliferating phase HAs, but increased in involuting phase HAs (P<0.01). Furthermore, we used small hairpin RNA (shRNA)-mediated VEGFR2 knockdown in primary HA-derived endothelial cells (HemECs) to understand the role of VEGF/VEGFR2 signaling. Knockdown of VEGFR2 by Lv-shVEGFR2 inhibited cell viability and induced apoptosis in primary HemECs companied with decreased expression of p-AKT, p-ERK, p-p38MAPK and Ki-67 and increased expression of caspase-3 (CAS-3); Overexpression of VEGFR2 promoted cell viability and blocked apoptosis in Lv-VEGFR2-transfected HemECs. Taken together, our findings demonstrate that, increased expression of VEGFR2 is involved in the development of primary HemECs possibly through regulation of the AKT and ERK pathways, suggesting that VEGFR2 may be a potential therapeutic target for HAs.Key words: vascular endothelial growth factor receptor 2, hemangioma, proliferation, apoptosis     

The dual role of phytoene synthase genes in carotenogenesis in carrot roots and leaves

Carrot (Daucus carota L.) is an important food crop and is useful for studying carotenogenesis due to the quantity and diversity of carotenoids in its roots. Phytoene synthase catalyzes the first committed step in the carotenoid biosynthesis pathway, and its overexpression is the main driving force in the orange phenotype. At present, we lack fundamental knowledge of the role of these genes and their effects on carotenoid accumulation in leaves. In the present study, three backcross inbred lines (BC2S4) with different colored roots derived from a cross between the orange inbred line (Af) and related wild species were used to investigate the role of the duplicated DcPSY genes in root carotenogenesis. Promoter analysis showed that DcPSY genes have diverged substantially in their regulatory sequences after gene duplication. Expression levels of DcPSY1 and DcPSY2 were generally positively correlated with carotenoid content during root development. In mature leaves, total carotenoid content was higher than that in the roots, DcPSY1 expression increased extremely higher than DcPSY2 expression compared with roots, and DcPSY1 was more sensitive than DcPSY2 during leaf de-etiolation under sunlight. These results suggest that DcPSY1 seems to make an important contribution to carotenoid accumulation in the leaves and is important for photosynthesis and photoprotection, but they are not the determining factors of root color. This expands our understanding of the regulation of carotenoid biosynthesis in carrot.     

Ursolic Acid Inhibits Leucine-Stimulated mTORC1 Signaling by Suppressing mTOR Localization to Lysosome

Ursolic acid (UA), a pentacyclic triterpenoid widely found in medicinal herbs and fruits, has been reported to possess a wide range of beneficial properties including anti-hyperglycemia, anti-obesity, and anti-cancer. However, the molecular mechanisms underlying the action of UA remain largely unknown. Here we show that UA inhibits leucine-induced activation of the mechanistic target of rapamycin complex 1 (mTORC1) signaling pathway in C2C12 myotubes. The UA-mediated inhibition of mTORC1 is independent of Akt, tuberous sclerosis complex 1/2 (TSC1/2), and Ras homolog enriched in brain (Rheb), suggesting that UA negatively regulates mTORC1 signaling by targeting at a site downstream of these mTOR regulators. UA treatment had no effect on the interaction between mTOR and its activator Raptor or inhibitor Deptor, but suppressed the binding of RagB to Raptor and inhibited leucine-induced mTOR lysosomal localization. Taken together, our study identifies UA as a direct negative regulator of the mTORC1 signaling pathway and suggests a novel mechanism by which UA exerts its beneficial function.     

Pallidal Index as Biomarker of Manganese Brain Accumulation and Associated with Manganese Levels in Blood: A Meta-Analysis

Objective

The current study was designed to evaluate the sensitivity, feasibility, and effectiveness of the pallidal index (PI) serving as a biomarker of brain manganese (Mn) accumulation, which would be used as an early diagnosis criteria for Mn neurotoxicity.

Methods

The weighted mean difference (WMD) of the PI between control and Mn-exposed groups was estimated by using a random-effects or fixed-effects meta-analysis with 95% confidence interval (CI) performed by STATA software version 12.1. Moreover, the R package “metacor” was used to estimate correlation coefficients between PI and blood Mn (MnB).

Results

A total of eight studies with 281 occupationally Mn-exposed workers met the inclusion criteria. Results were pooled and performed with the Meta-analysis. Our data indicated that the PI of the exposed group was significantly higher than that of the control (WMD: 7.76; 95% CI: 4.86, 10.65; I² = 85.7%, p<0.0001). A random effects model was used to perform meta-analysis. These findings were remarkably robust in the sensitivity analysis, and publication bias was shown in the included studies. Seven out of the eight studies reported the Pearson correlation (r) values. Significantly positive correlation between PI and MnB was observed (r = 0.42; 95% CI, 0.31, 0.52).

Conclusions

PI can be considered as a sensitive, feasible, effective and semi-quantitative index in evaluating brain Mn accumulation. MnB can also augment the evaluation of brain Mn accumulation levels in the near future. However, the results should be interpreted with caution.     

Differences in TCR-Vβ Repertoire and Effector Phenotype between Tumor Infiltrating Lymphocytes and Peripheral Blood Lymphocytes Increase with Age

Tumor infiltrating lymphocytes (TIL) reflect the host''s anti-tumor immune response, and can be a valuable predictor of prognosis. However, many properties of TIL are not fully understood. In the present study, TCR-Vβ repertoires of cancer patients were primarily analyzed by flow cytometry. Abnormally expressed TCR-Vβ subfamilies were generally found in both TIL and peripheral blood lymphocytes (PBL) of each patient. Of note, increased patient age was associated with increasingly biased TCR-Vβ repertoire in TIL but not in PBL, and the dispersion degree of the differences of TCR-Vβ subfamilies between TIL and PBL correlated positively with age (P = 0.007). Utilizing immunoscope analysis, we identified the age-related reduction in TCR-Vβ diversity, but polyclonal pattern was predominant in significantly expanded TCR-Vβ subfamilies. In addition, we found that older patients possessed a decreased ratio of CD8⁺CD62L⁺ non-effector cells in TIL compared to PBL, implying age-related increase of CD8⁺CD62L⁻ effector cells in TIL. The colocalization analysis of CD8 and CD3, however, suggested the suppressed activity of these effector cells in tumor microenvironment. These findings further elucidate the properties of TIL, showing an increasing difference between TIL and PBL with age, which may provide insight for the development of effective immunotherapies for cancer patients of different ages.