S417 in the CC3 region of STIM1 is critical for STIM1-Orai1 binding and CRAC channel activation.

Store-operated Ca2+ entry (SOCE) is a universal Ca2+ influx pathway that is important for the function of many cell types. SOCE is controlled by the interaction of the ER Ca2+ sensor STIM1 with the plasma membrane Ca2+ channel Orai1. S417 is located in the third coiled-coil (CC3) domain of the C-terminus of STIM1. We found that single-point mutation of this residue (S417G) abolished STIM1 C-terminus interactions with Orai1. Mutation of S417 also abolished CAD-Orai1 binding and Orai1 channel activation, eliminated STIM1 puncta formation, and co-localization with Orai1 and SOCE. 2-APB was found to restore the binding of the STIM1 C-terminus mutant (S417G) to Orai1 and dose-dependently activate Orai1 channel. Both CBD and NBD of Orai1 are required for 2-APB-induced coupling between the Orai1 and STIM1 C-terminus mutant (S417G) and CRAC channel activation. We also demonstrated that 2-APB led to delayed activation of Orai1-K85E channel, although Orai1-K85E obviously impairs 2-APB-induced STIM1 C-terminus mutant (S417G)-Orai1 coupling. Our results suggest S417 in the CC3 domain of STIM1 is essential for STIM1-Orai1 binding and CRAC channel activation.

Multi-extraction system with identical supported semi-liquid membrane: Enhanced stability for coextraction of acidic and basic drugs.

Coextraction of different groups of analytes is vital for saving sample volumes and simplifying analytical procedures in bioanalysis. Conventionally, coextraction was achieved by using multi-extraction systems with different supported liquid membranes (SLMs). However, the different membrane solvents tended to diffuse into the aqueous solutions and the other SLM to reach distribution equilibrium during extraction process, causing the system instability. In this work, a stable multi-extraction system (integration of liquid-phase microextraction and electromembrane extraction, LPME/EME) based on the identical supported semi-liquid membrane (SsLM) was developed. Principally, the state of distribution equilibrium of the membrane solvent (polypropylene glycol with molecular weight 4000) in SsLM could be reached at the beginning of extraction, which enhanced the coextraction stability. With this multi-extraction system, acidic and basic analytes were simultaneously extracted from practical biological samples. The extraction recoveries of the six model drugs in undiluted urine samples were over 70%. Followed by LC-MS/MS, the limits of quantification (LOQs) were in the range of 5-10 ng mL-1. The multi-extraction system using the identical SsLM in this study shows promising potential in construction of other stable multi-extraction systems (e.g., LPME/LPME and EME/EME) in the future, which will greatly benefit the group separation of analytes in complicated biological samples.

Successive liquid-phase microextraction of acidic and basic analytes.

Practical biological and environmental samples always contain both acidic and basic substances, and the samples are always precious. Thus, separation of analytes with different nature from the same sample was of great significance. Successive liquid phase microextraction (sLPME) of acidic and basic analytes under optimal extraction conditions was therefore proposed for the first time. The concept of sLPME was proved by using three acidic analytes (naproxen, flurbiprofen and diclofenac) and three basic analytes (haloperidol, fluoxetine and sertraline) as model analytes, and using polypropylene glycol with an average molecular weight of 4000 (PPG4000) as SLM. The recoveries of all target analytes by sLPME were similar to that by individual LPME due to good affinity of PPG4000 to both acidic and basic analytes. Under optimal extraction conditions, the recoveries for all analytes by sLPME from urine samples were in the range of 62%-95%. Moreover, combined with LC-MS/MS, such sLPME approach was also evaluated with urine samples. The matrix effect of sLPME-LC-MS/MS at different levels for all analytes ranged from -14.1%-13.2%. The linear ranges with R2 > 0.996 were 5-1000 ng mL-1 for basic analytes, and 20-1000 ng mL-1 for acidic analytes except diclofenac (1-1000 ng mL-1). The repeatability and accuracy at four levels were in the range of 3%-10% and 86%-120%, respectively. The limit of detection (LOD, S/N = 3) and limit of quantification (LOQ, S/N = 10) were found to be 0.07-0.49 ng mL-1 and 0.25-1.63 ng mL-1, respectively. Finally, the strategy for constructing a sLPME system was further confirmed with urine, plasma and saliva using another two versatile SLM solvents possessing high affinity to both acidic and basic analytes. Successive LPME enabled separation of acidic and basic analytes from the same sample under optimum extraction conditions for all target analytes. Thus, we believe that the sLPME system will become a potent platform for forensic toxicology analysis, food science, environmental analysis and epidemiology study.

Versatile Integration of Liquid-Phase Microextraction and Fluorescent Aptamer Beacons: A Synergistic Effect for Bioanalysis.

Fluorescent aptamer beacons (FABs) are a major category of biosensors widely used in environmental analysis. However, due to their low compatibility, it is difficult to use the common FABs for biological samples. To overcome this challenge, construction of FABs with complex structures to adapt the nature of biological samples is currently in progress in this field. Unlike previous works, we moved our range of vision from the FAB itself to the biological sample. Inspired by this idea, in this work, flat membrane-based liquid-phase microextraction (FM-LPME) with sufficient sample cleanup and preconcentration capacities was integrated with FABs. With the merits of both FM-LPME and FABs, the integrated LPME-FAB system displayed a clear synergistic enhancement for target analysis. Specifically, LPME in the LPME-FAB system provided purified and enriched Hg2+ for the FAB recognition, while the FAB recognition event promoted the extraction efficiency of LPME. Due to superior performances, the LPME-FAB system achieved highly sensitive analysis of Hg2+ in urine samples with a detection limit of 27 nM and accuracies in the range of 98-113%. To the best of our knowledge, this is the first time that an integrated LPME-FAB system was constructed for target analysis in biological samples. We believe that this study will provide a new insight into the next generation of biosensors, where the integration of sample preparation with detection probes is as important as the design of complex probes in the field of bioanalysis.

Characterization of the Poplar R2R3-MYB Gene Family and Over-Expression of PsnMYB108 Confers Salt Tolerance in Transgenic Tobacco.

The MYB, one of the largest transcription factor families in plants, is related to various biological processes. For an example, the R2R3-MYB family plays an important role in regulation of primary and secondary metabolism, plant growth and development, and responses to hormones and stresses. However, functional studies on the poplar R2R3-MYB genes are limited. In this study, we identified 207 poplar R2R3-MYB genes that are unevenly distributed on the 19 chromosomes of poplar, followed by characterization of their conserved domains. On the basis of phylogenetic analysis, these genes can be divided into 23 groups. Evidence from synteny analyses indicated that the poplar R2R3-MYB gene family is featured by tandem and segmental duplication events. On the basis of RNA-Seq data, we investigated salt responsive genes and explored their expression patterns. Furthermore, we cloned the PsnMYB108 gene from poplar, which is significantly up-regulated in roots and leaves in response to salt stress. To validate its function, we developed transgenic tobacco plants that over-express the PsnMYB108 gene. It appears that the transgenic lines are more tolerant to salt stress than the wild type does. Evidence from physiological analyses demonstrated that over-expression of PsnMYB108 may improve tobacco salt stress tolerance by increasing the reactive oxygen species scavenging ability and the accumulation of proline. These results laid the foundation for future analysis and functional studies of poplar R2R3-MYB family members, and revealed that PsnMYB108 plays an important role in improving plant salt stress tolerance.

Blood Group Antigen Shielding Facilitated by Selective Cell Surface Engineering.

Production of red blood cells (RBCs) without immunogenicity of blood group antigens is of special interest in blood transfusion therapy in clinical chemistry. In this study, a selective cell surface engineering method was developed for the preparation of antigen-shielded RBCs based on molecular imprinting. Using an epitope imprinting method, biocompatible molecularly imprinted nanogels (MIgels) were prepared with a high affinity to the blood group antigens of RBCs. The antigen-shielded RBCs could avoid the agglutination caused by blood group mismatch, resulting in the antigen-shielded RBCs in efficiently substituting RBCs in case of a shortage of blood supply. Moreover, the antigen-shielded RBCs could maintain the normal physiological structure and functions of the original RBCs. We believe that the selective cell surface engineering presented in this work may offer significant benefits in specific cell protection for biomedical application.

Hybrid breath figure method: A new insight in Petri dishes for cell culture.

The traditional Petri dish, which has not been changed in almost 60 years, has clear limitations when it is applied to cell cultures in a modern biological laboratory. In this work, by integrating the advantages of both the semidirect breath figure (sDBF) method and the traditional breath figure (BF) method, we proposed for the first time a novel hybrid BF method for the fabrication of honeycomb-patterned Petri dishes with mPEG self-assembly in the pores. Due to the amphiphilic structure of mPEG, the active OH groups of mPEG were located inside the pores of the dishes, which could covalently couple with other functional materials. For instance, in this work, an antibacterial agent was immobilized onto the dish surface via a typical coupling reaction. Because of the size difference between the bacteria and cells, the prepared dishes had selective antibacterial activity but noncytotoxicity against mammalian cells. The present hybrid BF method provides a new insight for endowing commercial PS Petri dishes or other membranes with special topographical structures and functions, which could solve the long-term challenges of cell cultures in the future.

SOCE induced calcium overload regulates autophagy in acute pancreatitis via calcineurin activation.

Acute pancreatitis (AP) is an acute inflammatory process of the pancreas that is characterized by inflammation, edema, vacuolization and necrosis, which has significant morbidity and lethality. The pathogenesis of AP has not been established completely. An early and critical feature of AP is the aberrant signaling of Calcium (Ca2+) within the pancreatic acinar cell, termed Ca2+ overload. Store-operated Ca2+ (SOC) channels are the principal Ca2+ influx channels that contribute to Ca2+ overload in pancreatic acinar cells. Store-operated Ca2+ entry (SOCE) has been proved to be a key pathogenic step in AP development that leads to trypsin activation, inflammation and vacuolization. However, the molecular mechanisms are still poorly understood. By establishing Ca2+ overload model and mouse AP model using caerulein, we found that caerulein triggered SOCE via inducing interaction between STIM1 and Orai1, which activated calcineurin (CaN); CaN activated the nuclear factor of activated T cells (NFAT) and transcription factor EB (TFEB), thus promoting the transcriptional activation of multiple chemokines genes and autophagy-associated genes respectively. To the best of our knowledge, this is the first evidence showing that SOCE activates TFEB via CaN activation, which may have noticeable longer-term effects on autophagy and vacuolization in AP development. Our findings reveal the role for SOCE/CaN in AP development and provide potential targets for AP treatment.

Anaerobic Ammonium-Oxidizing Bacteria in Cow Manure Composting.

Composting is widely used to transform waste into valuable agricultural organic fertilizer. Anaerobic ammonium-oxidizing (anammox) bacteria play an important role in the global nitrogen cycle, but their role in composting remains poorly understood. In the present study, the community structure, diversity, and abundance of anammox bacteria were analyzed using cloning and sequencing methods by targeting the 16S rRNA gene and the hydrazine oxidase gene (hzo) in samples isolated from compost produced from cow manure and rice straw. A total of 25 operational taxonomic units were classified based on 16S rRNA gene clone libraries, and 14 operational taxonomic units were classified based on hzo gene clone libraries. The phylogenetic tree analysis of the 16S rRNA gene and deduced HZO protein sequences from the corresponding encoding genes indicated that the majority of the obtained clones were related to the known anammox bacteria Candidatus "Brocadia," Candidatus "Kuenenia," and Candidatus "Scalindua." The abundances of anammox bacteria were determined by quantitative PCR, and between 2.13 × 105 and 1.15 × 106 16S rRNA gene copies per gram of compost were found. This study provides the first demonstration of the existence of anammox bacteria with limited diversity in cow manure composting.

Impacts of drought stress on soluble carbohydrates and respiratory enzymes in fruit body of Auricularia auricula.

In order to study the survival mechanisms to drought stress for fruit body of Auricularia auricula, soluble carbohydrates and respiratory enzymes were investigated. Fruit bodies were exposed to sunlight and were naturally dehydrated. Samples were taken at different levels of water loss (0%, 10%, 30%, 50% and 70%) to measure the content of soluble sugars and polysaccharides. The activities of phosphoglucose isomerase (PGI), combined glucose-6-phosphate dehydrogenase (G-6-PDH) and 6-phosphogluconate dehydrogenase (6-PGDH), and malate dehydrogenase (MDH), were also determined. The results showed that with the increase in water loss, soluble sugars and MDH activity declined, whereas the activities of G-6-PDH and 6-PGDH increased. Soluble polysaccharides content and PGI activity decreased with water loss up to 30% and increased afterwards. These results suggested that the pentose phosphate pathway (PPP), as demonstrated by activities of G-6-PDH and 6-PGDH, could be one of the mechanisms for survival during drought stress in the fruit body of A. auricula. Moreover, soluble polysaccharides may play a part in protecting the fruit body in further drought stress.

Development of SSR Markers and Genetic Diversity in White Birch (Betula platyphylla).

In order to study genetic diversity of white birch (Betula platyphylla), 544 primer pairs were designed based on the genome-wide Solexa sequences. Among them, 215 primer pairs showed polymorphism between five genotypes and 111 primer pairs that presented clear visible bands in genotyping 41 white birch plants that were collected from 6 different geographical regions. A total of 717 alleles were obtained at 111 loci with a range of 2 to 12 alleles per locus. The results of statistic analysis showed that polymorphic frequency of the alleles ranged from 17% to 100% with a mean of 55.85%; polymorphism information content (PIC) of the loci was from 0.09 to 0.58 with a mean of 0.30; and gene diversity between the tested genotypes was from 0.01 to 0.66 with a mean of 0.36. The results also indicated that major allele frequency ranged from 0.39 to 1.00 with an mean of 0.75; expected heterozygosity from 0.22 to 0.54 with a mean of 0.46; observed heterozygosity from 0.02 to 0.95 with a mean of 0.26; Nei's index from 0.21 to 0.54 with a mean of 0.46; and Shannon's Information from 0.26 to 0.87 with a mean of 0.66. The 41 white birch genotypes at the 111 selected SSR loci showed low to moderate similarity (0.025-0.610), indicating complicated genetic diversity among the white birch collections. The UPGMA-based clustering analysis of the allelic constitution of 41 white birch genotypes at 111 SSR loci suggested that the six different geographical regions can be further separated into four clusters at a similarity coefficient of 0.22. Genotypes from Huanren and Liangshui provenances were grouped into Cluster I, genotypes from Xiaobeihu and Qingyuan provenances into Cluster II, genotypes from Finland provenance into Cluster III, and genotypes from Maoershan into Cluster IV. The information provided in this study could help for genetic improvement and germplasm conservation, evaluation and utilization in white birch tree breeding program.

Structure and immunobiological activity of a new polysaccharide from Bletilla striata.

A new polysaccharide (designated BSPF2), with a molecular weight of 2.35 × 10(5)Da, was isolated from the tubers of Bletilla striata. It contained mannose, glucose, and galactose in a molar ratio of 9.4:2.6:1.0. The acetyl content of BSPF2 was estimated to be 2.9%, and acetyl groups were located in positions 3 and 6 of mannosyl residues. The structural features were elucidated by a combination of monosaccharide composition analysis, periodate oxidation, partial acid hydrolysis, acetolysis, and methylation analysis. The results indicated that the backbone of BSPF2 consisted of (1→4)-linked mannosyl residues and (1→4)-linked glucosyl residues in a molar ratio of 2:1. About three fifths of glucosyl residues in the backbone were branched at O-6 position, and the terminal sugar residues were composed of mannosyl residues. Immunological assay results demonstrated that BSPF2 significantly induced the spleen cell proliferation in a dose-dependent manner.

Lycium ruthenicum polysaccharide attenuates inflammation through inhibiting TLR4/NF-κB signaling pathway.

Polysaccharide has been reported to possess diverse biological activities, however, the inflammatory activity of polysaccharide isolated from Lycium ruthenicum remains unknown so far. In the present study, we investigated the effects of L. ruthenicum polysaccharide (LRGP3) on inflammatory reaction induced by lipopolysaccharide (LPS) in mouse macrophage RAW264.7 cells and some potential underlying mechanisms. Our results showed that LRGP3 treatment significantly inhibited the LPS-induced NO production and the mRNA expression of iNOS, as well as the level of Toll-like receptor 4 (TLR4). Furthermore, LRGP3 treatment prevented the IκBα degradation and reduced phospho-NF-κB p65 protein expression in LPS-stimulated RAW264.7 cells. Meanwhile, the levels of pro-inflammatory cytokines, such as interleukin (IL)-α, IL-6, tumor necrosis factor (TNF)-α were suppressed by LRGP3 in LPS-stimulated RAW264.7 cells. Taken together, our results suggested that LRGP3 attenuated LPS-induced inflammation via inhibiting TLR4/NF-κB signaling pathway.