Cell-based high-throughput proliferation and cytotoxicity assays for screening traditional Chinese herbal medicines

A simple, reliable, high-throughput screening method was developed and used to assess the pharmaceutical effects of extracts of traditional Chinese herbal medicines (TCHMs). This method is based on 3-dimensional (3-D) cultures of mouse embryonic stem (mES) and human colon cancer and breast cancer cells expressing enhanced green fluorescent protein (EGFP) in polyethylene terephthalate (PET) fibrous scaffolds on modified 384-well plates with online monitoring of culture fluorescence for dynamic responses of cells to drugs present in culture media. Cell responses to deoxycholic acid and the extracts of 3 TCHMs (Ganoderma lucidum spores, Ginkgo biloba, and Epimedium brevicornum) at various concentrations were investigated for their effects on proliferation and cytotoxicity. The screening results, i.e., the growth responses of cancer cells to those drugs, were consistent with what have been reported in the literature, confirming the reliability of the new screening approach. Different from previous screening methods for both TCHMs and western medicines that used animal models or 2-D cell-based assays with single cell lines, this 3-D cell-based screening method employs both cancer and normal cells and thereby provides a way for quick, direct evaluation of the anticancer effects of TCHMs. This method also offers assessment on the side effects of TCHMs.     

Rice Performance and Water Use Efficiency under Plastic Mulching with Drip Irrigation

Plastic mulching with drip irrigation is a new water-saving rice cultivation technology, but little is known on its productivity and water-saving capacity. This study aimed to assess the production potential, performance, and water use efficiency (WUE) of rice under plastic mulching with drip irrigation. Field experiments were conducted over 2 years with two rice cultivars under different cultivation systems: conventional flooding (CF), non-flooded irrigation incorporating plastic mulching with furrow irrigation (FIM), non-mulching with furrow irrigation (FIN), and plastic mulching with drip irrigation (DI). Compared with the CF treatment, grain yields were reduced by 31.76–52.19% under the DI treatment, by 57.16–61.02% under the FIM treatment, by 74.40–75.73% under the FIN treatment, which were mainly from source limitation, especially a low dry matter accumulation during post-anthesis, in non-flooded irrigation. WUE was the highest in the DI treatment, being 1.52–2.12 times higher than with the CF treatment, 1.35–1.89 times higher than with the FIM treatment, and 2.37–3.78 times higher than with the FIN treatment. The yield contribution from tillers (YCFTs) was 50.65–62.47% for the CF treatment and 12.07–20.62% for the non-flooded irrigation treatments. These low YCFTs values were attributed to the poor performance in tiller panicles rather than the total tiller number. Under non-flooded irrigation, root length was significantly reduced with more roots distributed in deep soil layers compared with the CF treatment; the DI treatment had more roots in the topsoil layer than the FIM and FIN treatments. The experiment demonstrates that the DI treatment has greater water saving capacity and lower yield and economic benefit gaps than the FIM and FIN treatments compared with the CF treatment, and would therefore be a better water-saving technology in areas of water scarcity.     

Does Serum 25-Hydroxyvitamin D Influence Muscle Development during Puberty in Girls? - A 7-Year Longitudinal Study

Vitamin D is well known for its regulatory role in calcium and phosphate homeostasis, but its role in muscle mass and strength during growth remains inconclusive. We explored the association of serum 25-hydroxyvitamin D (25(OH)D) with muscle development in girls from 11 to 18-years old. Whole body lean tissue mass (LM_WB), appendicular lean mass (aLM), muscle cross-sectional area at the lower leg (mCSA), maximal voluntary contraction of elbow flexors (MVC_elbow) and knee extensors (MVC_knee) were assessed in 217 girls aged 10–13 years (at baseline), 215 in 2-year and 226 in 7.5-year follow-up. Serum concentration of 25(OH)D and intact parathyroid hormone (PTH) were analyzed retrospectively and girls were categorized according to their 25(OH)D levels (consistently insufficient 25(OH)D G_LL <50 nmol/l and consistently sufficient G_HH >50 nmol/l from baseline to 7-year follow-up). We found that 25(OH)D level declined until menarche (p<0.05) while LM_WB, aLM, mCSA, MVC_elbow and MVC_knee continued to increase (p<0.001 for all) post menarche. At pre-menarche, the G_LL (n = 34) had higher LM_WB and aLM than the G_HH (n = 21, p<0.05), while post-menarche the G_HH (n = 15) had a greater catch-up gain in LM_WB (p = 0.004), aLM (p = 0.001) and mCSA (p = 0.027) compared to the G_LL (n = 65) over the first 2-year period. At the age of 18, no differences in muscle mass/strength between the low (n = 151) and high (n = 77) levels of 25(OH)D groups were found. This finding was independent of vitamin D receptor genotype and other confounders. In conclusion, our results showed that levels of 25(OH)D have no significant negative influence on the development of muscle mass and strength during pubertal growth both with longitudinal and cross-sectional comparison. On the contrary, our results suggest that the temporary negative association between 25(OH)D and muscle mass arises as a consequence of fast growth prior to menarche, and this negative association is diminished through catch-up growth after menarche.     

Clinical Characteristics Associated with Borrelia burgdorferi Sensu Lato Skin Culture Results in Patients with Erythema Migrans

Clinical characteristics associated with isolation of Borrelia burgdorferi sensu lato from skin have not been fully evaluated. To gain insight into predictors for a positive EM skin culture, we compared basic demographic, epidemiologic, and clinical data in 608 culture-proven and 501 culture-negative adult patients with solitary EM. A positive Borrelia spp. skin culture was associated with older age, a time interval of >2 days between tick bite and onset of the skin lesion, EM ≥5 cm in diameter, and location of the lesion on the extremities, whereas several other characteristics used as clinical case definition criteria for the diagnosis of EM (such as tick bite at the site of later EM, information on expansion of the skin lesion, central clearing) were not. A patient with a 15-cm EM lesion had almost 3-fold greater odds for a positive skin culture than patients with a 5-cm lesion. Patients with a free time interval between the tick bite and onset of EM had the same probability of a positive skin culture as those who did not recall a tick bite (OR=1.02); however, the two groups had >3-fold greater odds for EM positivity than patients who reported a tick bite with no interval between the bite and onset of the lesion. In conclusion, several yet not all clinical characteristics used in EM case definitions were associated with positive Borrelia spp. skin culture. The findings are limited to European patients with solitary EM caused predominantly by B. afzelii but may not be valid for other situations.     

The Effects of Catalyst Layer Deposition Methodology on Electrode Performance

The catalyst layer of the cathode is arguably the most critical component of low‐temperature fuel cells and carbon dioxide (CO₂) electrolysis cells because their performance is typically limited by slow oxygen (O₂) and CO₂ reduction kinetics. While significant efforts have focused on developing cathode catalysts with improved activity and stability, fewer efforts have focused on engineering the catalyst layer structure to maximize catalyst utilization and overall electrode and system performance. Here, we study the performance of cathodes for O₂ reduction and CO₂ reduction as a function of three common catalyst layer preparation methods: hand‐painting, air‐brushing, and screen‐printing. We employed ex‐situ X‐ray micro‐computed tomography (MicroCT) to visualize the catalyst layer structure and established data processing procedures to quantify catalyst uniformity. By coupling structural analysis with in‐situ electrochemical characterization, we directly correlate variation in catalyst layer morphology to electrode performance. MicroCT and SEM analyses indicate that, as expected, more uniform catalyst distribution and less particle agglomeration, lead to better performance. Most importantly, the analyses reported here allow for the observed differences over a large geometric volume as a function of preparation methods to be quantified and explained for the first time. Depositing catalyst layers via a fully‐automated air‐brushing method led to a 56% improvement in fuel cell performance and a significant reduction in electrode‐to‐electrode variability. Furthermore, air‐brushing catalyst layers for CO₂ reduction led to a 3‐fold increase in partial CO current density and enhanced product selectivity (94% CO) at similar cathode potential but a 10‐fold decrease in catalyst loading as compared to previous reports.     

Entrapment of Ovalbumin into Liposomes—Factors Affecting Entrapment Efficiency,Liposome Size,and Zeta Potential

Various amounts of Ovalbumin (OVA) were encapsulated into positively and negatively charged multilamellar liposomes, with the aim to investigate the entrapment efficiency in different buffers and to study their effects on the liposome size and zeta potential. Results showed that the entrapment efficiency of OVA in anionic liposomes was the same in 10 mM Phosphate Buffer (PB) as in Phosphate-Buffered Saline (PBS; PB?+?0.15 M NaCl). Also, liposome size was approximately 1200 nm for all anionic liposomes incorporating OVA. The entrapment efficiency of OVA in cationic liposomes was highly dependent on ionic strength. The size of cationic liposomes was approximately 1200 nm in PBS, regardless of protein content, but increased with the amount of the incorporated protein in PB. Aggregation of cationic liposomes in PB was observed when the mass of the protein was 2.5 mg or greater. The zeta potential of anionic liposomes was negative and of cationic liposomes positive in the whole range of protein mass tested. These results show how different compositions of lipid and aqueous phases can be used to vary the entrapment efficiency, liposome size, and zeta potential—the factors that are of great importance for the use of liposomes as drug carriers.     

Analysis of B-Class Genes NAP3L3 and NAP3L4 in Narcissus tazetta var. chinensis

Very few flower organ identity genes have been characterized in Chinese narcissus (Narcissus tazetta var. chinensis), which has petaloid sepals. Here, we report the cloning of two full-length B-class genes, namely NAP3L3 and NAP3L4, that are orthologs of the DEFICIENS lineage. Both genes are highly expressed in the second whorl of the perianth and in the stamens. NAP3L4 is also expressed strongly in the ovule. The functions of these two genes were further analyzed using transgenic plants. Ectopic expression of either gene in Arabidopsis gave no obvious floral organ transformation phenotypes. In yeast two-hybrid assays, NAP3L3 and NAP3L4 failed to homodimerize and interacted weakly with each other. The data suggest that these two genes might not be involved in the formation of petaloid sepals. Isolation and functional analysis of other B-class paralogs should be conducted to fully understand petaloid tepal development in Chinese narcissus.     

Aluminum induces low phosphate adaptive responses and modulates primary and lateral root growth by differentially affecting auxin signaling in Arabidopsis seedlings

Aims

The aims of this work were to investigate the aluminum (Al) and phosphate (P) interactions in the regulation of root system architecture of Arabidopsis thaliana seedlings and the contribution of auxin signaling in primary and lateral root growth in response to Al toxicity.

Methods

Detailed analyses of root system architecture and cell division were performed in Arabidopsis WT seedlings and in low phosphorus insensitive mutants lpi1-3 and lpr1-1 lpr2-1 in response to Al. Expression studies of P-deficiency regulated phosphate transporter AtPT2 were also conducted. The role of auxin as a mediator of root morphogenetic changes by Al was evaluated by using the auxin-signaling mutants tir1, tir1 afb2 afb3, and arf7 arf19.

Results

Al inhibited primary root growth by affecting cell cycle progression and causing differentiation of cells in the root meristem. These effects were reduced in low phosphorus insensitive lpi1-3 and low phosphate resistant lpr1-1 lpr2-1 Arabidopsis mutants. Al also activated the expression of the low phosphate-induced P transporter AtPT2 in roots. Lateral root formation by Al decreased in tir1 afb2 afb3 while arf7 arf19 mutants were highly resistant to Al in both primary root inhibition and lateral root induction.

Conclusions

Our results suggest that lateral root formation in response to Al toxicity and P deficiency may involve common signaling mechanisms, while a pathway involving ARF7 and ARF19 is important for primary root growth inhibition by Al.     

Altitudinal variation in growth, photosynthetic capacity and water use efficiency of Abies faxoniana Rehd. et Wils. seedlings as revealed by reciprocal transplantations

Altitudinal variation in morphological, physiological and biochemical characteristics between two provenances of Abies faxoniana Rehd. et Wils. from contrasting elevations (3,500 and 2,850 m) was investigated by reciprocal transplantations in the eastern Qinghai-Tibetan Plateau. From each altitude, 54 seedlings were selected, of which 27 seedlings were simply transplanted at their original altitude, while the other 27 seedlings were reciprocally transplanted to another altitude. The results showed that there is evident altitudinal variation in growth, photosynthetic capacity and water use efficiency among A. faxoniana seedlings. Transplantation of seedlings to a lower altitude decreased the leaf pigment content, leaf N content and water use efficiency, but increased CE, P _n, LMR and PNUE, consequently promoting NAR, the biomass accumulation and branchlet length increment. Besides, the increase in the C/N ratio reduced the risk of pathogen attack. Based on the substantial plasticity in the responses of the seedlings, we infer that the ongoing climate warming might facilitate the growth of A. faxoniana seedlings at high altitudes.