H3K27me3 timely dictates uterine epithelial transcriptome remodeling and thus transformation essential for normal embryo implantation.

Uterine luminal epithelia (LE), the first layer contacting with the blastocyst, acquire receptivity for normal embryo implantation. Besides the well-accepted transcriptional regulation dominated by ovarian estrogen and progesterone for receptivity establishment, the involvement of epigenetic mechanisms remains elusive. This study systematically profiles the transcriptome and genome-wide H3K27me3 distribution in the LE throughout the preimplantation. Combining genetic and pharmacological approaches targeting the PRC2 core enzyme Ezh1/2, we demonstrate that the defective remodeling of H3K27me3 in the preimplantation stage disrupts the differentiation of LE, and derails uterine receptivity, resulting in implantation failure. Specifically, crucial epithelial genes, Pgr, Gata2, and Sgk1, are transcriptionally silenced through de novo deposition of H3K27me3 for LE transformation, and their sustained expression in the absence of H3K27me3 synergistically confines the nuclear translocation of FOXO1. Further functional studies identify several actin-associated genes, including Arpin, Tmod1, and Pdlim2, as novel direct targets of H3K27me3. Their aberrantly elevated expression impedes the morphological remodeling of LE, a hindrance alleviated by treatment with cytochalasin D which depolymerizes F-actin. Collectively, this study uncovers a previously unappreciated epigenetic regulatory mechanism for the transcriptional silencing of key LE genes via H3K27me3, essential for LE differentiation and thus embryo implantation.

OsPRMT6a-mediated arginine methylation of OsJAZ1 regulates jasmonate signaling and spikelet development in rice.

Although both protein arginine methylation (PRMT) and jasmonate (JA) signaling are crucial for regulating plant development, the relationship between these processes in the control of spikelet development remains unclear. In this study, we used the CRISPR/Cas9 technology to generate two OsPRMT6a loss-of-function mutants that exhibit various abnormal spikelet structures. Interestingly, we found that OsPRMT6a can methylate arginine residues in JA signal repressors OsJAZ1 and OsJAZ7. We showed that arginine methylation of OsJAZ1 enhances the binding affinity of OsJAZ1 with the JA receptors OsCOI1a and OsCOI1b in the presence of JAs, thereby promoting the ubiquitination of OsJAZ1 by the SCFOsCOI1a/OsCOI1b complex and degradation via the 26S proteasome. This process ultimately releases OsMYC2, a core transcriptional regulator in the JA signaling pathway, to activate or repress JA-responsive genes, thereby maintaining normal plant (spikelet) development. However, in the osprmt6a-1 mutant, reduced arginine methylation of OsJAZ1 impaires the interaction between OsJAZ1 and OsCOI1a/OsCOI1b in the presence of JAs. As a result, OsJAZ1 proteins become more stable, repressing JA responses, thus causing the formation of abnormal spikelet structures. Moreover, we discovered that JA signaling reduces the OsPRMT6a mRNA level in an OsMYC2-dependent manner, thereby establishing a negative feedback loop to balance JA signaling. We further found that OsPRMT6a-mediated arginine methylation of OsJAZ1 likely serves as a switch to tune JA signaling to maintain normal spikelet development under harsh environmental conditions such as high temperatures. Collectively, our study establishes a direct molecular link between arginine methylation and JA signaling in rice.

Terminally Chlorinated and Thiophene-linked Acceptor-Donor-Acceptor Structured 3D Acceptors with Versatile Processability for High-efficiency Organic Solar Cells.

To exploit the potential of our newly developed three-dimensional (3D) dimerized acceptors, a series of chlorinated 3D acceptors (namely CH8-3/4/5) were reported by precisely tuning the position of chlorine (Cl) atom. The introduction of Cl atom in central unit affects the molecular conformation. Whereas, by replacing fluorinated terminal groups (CH8-3) with chlorinated terminal groups (CH8-4 and CH8-5), the red-shift absorption and enhanced crystallization are achieved. Benefiting from these, all devices received promising power conversion efficiencies (PCEs) over 16 % as well as decent thermal/photo-stabilities. Among them, PM6:CH8-4 based device yielded a best PCE of 17.58 %. Besides, the 3D merits with multi alkyl chains enable their versatile processability during the device preparation. Impressive PCEs of 17.27 % and 16.23 % could be achieved for non-halogen solvent processable devices prepared in glovebox and ambient, respectively. 2.88 cm2 modules also obtained PCEs over 13 % via spin-coating and blade-coating methods, respectively. These results are among the best performance of dimerized acceptors. The decent performance of CH8-4 on small-area devices, modules and non-halogen solvent-processed devices highlights the versatile processing capability of our 3D acceptors, as well as their potential applications in the future.

Dingkun pill alleviates metabolic abnormalities in polycystic ovary syndrome through brown adipose tissue activation.

BACKGROUND: Traditional Chinese medicine has been used for a long time to treat a variety of gynecological diseases. Among various traditional Chinese medicine, Dingkun Pill (DK) has been used for the treatment of female gynecological diseases. However, DK therapeutic effect on PCOS and the target tissue for its potential effect need to be explored. This study aims to explore the therapeutic effect of DK for PCOS in mice from three aspects: metabolism, endocrine and fertility, and determine whether the brown adipose tissue is the target organ to alleviate the PCOS phenotype. METHODS: PCOS mouse model was constructed by subcutaneous injection of DHEA. The estrous cycle, ovulation, and pregnancy outcome was examined in mice. The level of hormone including the LH, FSH, estrogen and testosterone in the serum were measured by ELISA. Both the glucose sensitivity and insulin sensitivity were determined in mice with different treatment. The histomorphology and lipid contents in the brown adipose tissue were analyzed. RNA-Seq was conducted for the brown adipose tissue and different expression of critical metabolism marker genes was confirmed by real-time PCR. RESULTS: The data showed that the fertility in PCOS mice with DK treatment was significantly increased, and the metabolic disorder was partially restored. Both the whiten of brown adipose tissue and reduced UCP1 expression induced by DHEA was rescued by the DK. The RNA-Seq data further demonstrated both the DHEA induced downregulation of lipolysis genes and oxidative phosphorylation genes were at least partially rescued by DK in the brown adipose tissue. CONCLUSIONS: DK has therapeutic effect on PCOS in DHEA treated mice and the brown adipose tissue is at least one critical target organ to alleviate the PCOS. This is achieved by not only regulating the lipid mobilization of brown adipose, but also restoring its thermogenic function.

A synthetic peptide from Sipunculus nudus promotes bone formation via Estrogen/MAPK signal pathway based on network pharmacology.

The tripeptide Leu-Pro-Lys (LPK), derived from the Sipunculus nudus protein, was synthesized and studied to investigate its potential protective effect on bone formation. The effect and mechanism of LPK were analyzed through network pharmacology, bioinformatics, and experimental pharmacology. The study found that LPK at concentrations of 25 µg/mL and 50 µg/mL significantly increased ALP activity and mineralization in C3H10 cells. LPK also increased the expression of COL1A1 and promoted bone formation in zebrafish larvae. Network pharmacology predicted 148 interaction targets between LPK and bone development, and analysis of the protein-protein interaction network identified 13 hub genes, including ESR1, MAPK8, and EGFR, involved in bone development. Through KEGG enrichment pathways analysis, it was determined that LPK promotes bone development by regulating endocrine resistance, the relaxin signaling pathway, and the estrogen signaling pathway. Molecular docking results showed direct interactions between LPK and ESR1, MAPK8, and MAPK14. Additional verification experiments using western blot assay revealed that LPK significantly upregulated the expression of genes related to bone formation, including COL1A1, OPG, RUNX2, ESR1, phosphorylated MAPK14, and phosphorylated MAPK8 in C3H10 cells. These results suggest that LPK promotes bone formation by activating the estrogen/MAPK signaling pathway.

Lycorine improves peripheral nerve function by promoting Schwann cell autophagy via AMPK pathway activation and MMP9 downregulation in diabetic peripheral neuropathy.

Diabetic peripheral neuropathy (DPN) is the most common complication of diabetes mellitus and no effective therapy is approved. Here, lycorine, a natural alkaloid, was identified as a potential drug for DPN by the bioinformatics analysis of GEO datasets and Connectivity Map database. Lycorine administration improved peripheral nerve function and autophagy-associated proteins of diabetic mice. Again, in vitro high glucose-cultured rat Schwann cells (RSC96) showed enhanced autophagosome marker LC3-II with the treatment of lycorine. Additionally, beclin-1 and Atg3 were decreased in high glucose-stimulated RSC96 cells, which were reversed by lycorine treatment. Furthermore, DPN-associated differentially expressed genes (DEGs) from GEO datasets and lycorine-drug targets from PubChem and PharmMapper were visually analyzed and revealed that MMP9 was both DPN-associated DEGs and lycorine-drug target. Functional enrichment analysis of MMP9-relevant genes showed that cell energy metabolism was involved. Moreover, lycorine reduced high glucose-enhanced MMP9 expression in RSC96 cells. Overexpression of MMP9 attenuated lycorine-induced the expression of beclin-1, Atg3 and LC3-II in high glucose-cultured RSC96 cells. In addition, AMPK pathway activation was confirmed in lycorine-treated high glucose-cultured RSC96 cells. Then AMPK pathway inhibition attenuated lycorine-reduced MMP9 expression in high glucose-treated RSC96 cells. Molecular docking analysis revealed that lycorine bound the domain of AMPK containing Thr 172 site, which affected AMPK (Thr 172) phosphorylation. Finally, AMPK pathway activation and MMP9 downregulation were also revealed in the sciatic nerves of diabetic mice administrated with lycorine. Taken together, lycorine was advised to promote Schwann cell autophagy via AMPK pathway activation and MMP9 downregulation-induced LC3-II transformation in diabetic peripheral neuropathy.

Screening and identification of key genes in imatinib-resistant chronic myelogenous leukemia cells: a bioinformatics study.

BACKGROUND: Chronic myelogenous leukemia (CML) is one of the most common cancers in the world. Imatinib is one of the most effective therapeutic strategies to inhibit the BCR-ABL tyrosine Kinase in patients with CML, but resistance is increasingly encountered. MATERIAL AND METHODS: Microarray data GSE7114, GSE92624 and GSE97562 were downloaded and analyzed from Gene Expression Omnibus (GEO) to identify the candidate genes in the imatinib-resistant CML cells. The differentially expressed genes (DEGs) were appraised, and the protein-protein interaction (PPI) network was created by using STRING and Cytoscape. RESULTS: We screened a total of 217 DEGs, including 151 upregulated genes and 66 downregulated genes. The enriched functions and pathways of genes include insulin-like growth factor I binding, cysteine-type endopeptidase inhibitor activity involved in apoptotic process, cell adhesion, positive regulation of nitric oxide biosynthetic process and hematopoietic cell lineage. Nine hub genes were appraised and Gene Ontology enrichment analysis revealed that these genes are mainly enriched in cell cycle, peptidase inhibitor activity and cell division. Several genes such as BIRC5, CCNE2 and MCM4 were identified in survival analysis and these genes alteration are significantly associated with worse overall survival and disease-free survival. CONCLUSIONS: These genes have the potential to become surrogate markers for a clinical evaluation of imatinib-resistant CML patients. Our results provide potential target genes for diagnosis and treatment of imatinib-resistant CML patients.

Spray-dried assembly of 3D N,P-Co-doped graphene microspheres embedded with core-shell CoP/MoP@C nanoparticles for enhanced lithium-ion storage.

The advancement of novel synthetic approaches for micro/nanostructural manipulation of transition metal phosphide (TMP) materials with precisely controlled engineering is crucial to realize their practical use in batteries. Here, we develop a novel spray-drying strategy to construct three-dimensional (3D) N,P co-doped graphene (G-NP) microspheres embedded with core-shell CoP@C and MoP@C nanoparticles (CoP@C⊂G-NP, MoP@⊂G-NP). This intentional design shows a close correlation between the microstructural G-NP and chemistry of the core-shell CoP@C/MoP@C nanoparticle system that contributes towards their anode performance in lithium-ion batteries (LIBs). The obtained structure features a conformal porous G-NP framework prepared via the co-doping of heteroatoms (N,P) that features a 3D conductive highway that allows rapid ion and electron passage and maintains the overall structural integrity of the material. The interior carbon shell can efficiently restrain volume evolution and prevent CoP/MoP nanoparticle aggregation, providing excellent mechanical stability. As a result, the CoP@C⊂G-NP and MoP@⊂G-NP composites deliver high specific capacities of 823.6 and 602.9 mA h g-1 at a current density of 0.1 A g-1 and exhibit excellent cycling stabilities of 438 and 301 mA h g-1 after 500 and 800 cycles at 1 A g-1. The present work details a novel approach to fabricate core-shell TMPs@C⊂G-NP-based electrode materials for use in next-generation LIBs and can be expanded to other potential energy storage applications.

InDel marker development and QTL analysis of agronomic traits in mung bean [Vigna radiate (L.) Wilczek].

The stem color of young mung bean is a very useful tool in germplasm identification. Flowering time and plant height (PH) are known to be strongly correlated with crop adaption and yield. However, few studies have focused on elucidating the genetic mechanisms that regulate these five particular traits: young stem color (YSC), days to first flowering (DFF), days to maturity (DM), PH, and nodes on the main stem (NMS). In this study, a genetic linkage map for the F2 population was constructed using 129 InDel markers that were developed based on the sequence variations between parents. A total of 14 QTLs related to YSC, DFF, DM, PH, and NMS were detected. These QTLs were distributed on six chromosomes (1, 3, 4, 6, 7, and 10), which individually accounted for 1.32 to 90.07% of the total phenotypic variation. Using a short and high-density linkage map for the F3 population, six of the seven QTLs which clustered at two intervals on chromosomes 3 and 10 were detected again. Further analysis found that four QTLs between InDel markers R3-15 and R3-19 controlled DFF, DM, PH, and NMS, and each QTL accounted for a large percent of the total phenotypic variation. Analysis of two separated F2:3 lines also found that the phenotype was highly corresponded to its genotype which was between R3-15 and R3-19. Phenotype and genotype analysis for 30 mung bean accessions showed that the major effect QTL qDFF3 was a key regulator for DFF. Using a map-based cloning method, the major effect QTL qYSC4 for YSC was mapped in a 347 Kb interval on chromosome 4. Candidate gene analysis showed that sequence variations and expression level differences existed in the predicted candidate gene between the parents. These results provide a theoretical basis for cloning these QTLs and marker-assisted selection. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-021-01233-0.

The chaperone Hsp70 is a BH3 receptor activated by the pro-apoptotic Bim to stabilize anti-apoptotic clients.

The chaperone heat shock protein 70 (Hsp70) is crucial for avoiding protein misfolding under stress, but is also up-regulated in many kinds of cancers, where its ability to buffer cellular stress prevents apoptosis. Previous research has suggested Hsp70 interacts with pro-apoptotic Bcl-2 family proteins, including Bim and Bax. However, a definitive demonstration of this interaction awaits, and insights into the structural basis and molecular mechanism remain unclear. Earlier studies have identified a Bcl-2 homology 3 (BH3) domain present in Bcl-2 family members that engages receptors to stimulate apoptosis. We now show that Hsp70 physically interacts with pro-apoptotic multidomain and BH3-only proteins via a BH3 domain, thereby serving as a novel BH3 receptor, using in vitro fluorescent polarization (FP), isothermal titration calorimetry (ITC), and cell-based co-immunoprecipitation (co-IP) experiments, 1H-15N-transverse relaxation optimized spectroscopy (TROSY-HSQC), trypsin proteolysis, ATPase activity, and denatured rhodanese aggregation measurements further demonstrated that BimBH3 binds to a novel allosteric site in the nucleotide-binding domain (NBD) of Hsp70, by which Bim acts as a positive co-chaperone to promote the ATPase activity and chaperone functions. A dual role of Hsp70's anti-apoptotic function was revealed that when it keeps Bim in check to inhibit apoptosis, it simultaneously stabilizes oncogenic clients including AKT and Raf-1 with the aid of Bim. Two faces of Bim in cell fate regulation were revealed that in opposite to its well-established pro-apoptotic activator role, Bim could help the folding of oncogenic proteins.

Identification of the Apoptosis and Autophagy Bi-functional Proteins.

Although autophagy and apoptosis, two main kinds of programmed cell death, constitute distinct cellular processes with often opposing outcomes, there have been shown a complex interplay between them in recent years. This interplay is surely critical to the overall fate of the cell. However, a full-scale identification of those bi-functional proteins involved in both functions is currently beyond reach by existing databases or traditional biological experiments. And that makes the interplay impossible to be well understood. Here we built a large, comprehensiveness apoptosis and autophagy related PPI (protein-protein interaction) network and then used topology clustering to undergo a network analysis workflow. Finally, we concluded a list of 151 apoptosis and autophagy bi-functional proteins from this network. By this way we showed a global view on these bi-functional proteins about their unique characteristics and provided clues of new functions of some proteins which are not focused in present researches.

Earlier Degraded Tapetum1 (EDT1) Encodes an ATP-Citrate Lyase Required for Tapetum Programmed Cell Death.

In flowering plants, the tapetum cells in anthers undergo programmed cell death (PCD) at the late meiotic stage, providing nutrients for further development of microspores, including the formation of the pollen wall. However, the molecular basis of tapetum PCD remains elusive. Here we report a tapetum PCD-related mutant in rice (Oryza sativa), earlier degraded tapetum 1 (edt1), that shows complete pollen abortion associated with earlier-than-programmed tapetum cell death. EDT1 encodes a subunit of ATP-citrate lyase (ACL), and is specifically expressed in the tapetum of anthers. EDT1 localized in both the nucleus and the cytoplasm as observed in rice protoplast transient assays. We demonstrated that the A and B subunits of ACL interacted with each other and might function as a heteromultimer in the cytoplasm. EDT1 catalyzes the critical steps in cytosolic acetyl-CoA synthesis. Our data indicated a decrease in ATP level, energy charge, and fatty acid content in mutant edt1 anthers. In addition, the genes encoding secretory proteases or lipid transporters, and the transcription factors known to regulate PCD, were downregulated. Our results demonstrate that the timing of tapetum PCD must be tightly regulated for successful pollen development, and that EDT1 is involved in the tapetum PCD process. This study furthers our understanding of the molecular basis of pollen fertility and fecundity in rice and may also be relevant to other flowering plants.

Rice albino 1, encoding a glycyl-tRNA synthetase, is involved in chloroplast development and establishment of the plastidic ribosome system in rice.

The chloroplast is an important organelle that performs photosynthesis as well as biosynthesis and storage of many metabolites. Aminoacyl-tRNA synthetases (aaRSs) are key enzymes in protein synthesis. However, the relationship between chloroplast development and aaRSs still remains unclear. In this study, we isolated a rice albino 1 (ra1) mutant through methane sulfonate (EMS) mutagenesis of rice japonica cultivar Ningjing 4 (Oryza sativa L.), which displayed albinic leaves in seedling stage due to abnormal chloroplast development. Compared with wild type (WT), ra1 showed significantly decreased levels of chlorophylls (Chl) and carotenoids (Car) in 2-week-old seedlings, which also showed obvious plastidic structural defects including abnormal thylakoid membrane structures and more osmiophilic particles. These defects caused albino phenotypes in seedlings. Map-based cloning revealed that RA1 gene encodes a glycyl-tRNA synthetase (GlyRS), which was confirmed by genetic complementation and knockout by Crispr/Cas9 technology. Sequence analysis showed that a single base mutation (T to A) occurred in the sixth exon of RA1 and resulted in a change from Isoleucine (Ile) to Lysine (Lys). Real-time PCR analyses showed that RA1 expression levels were constitutive in most tissues, but most abundant in the leaves and stems. By transient expression in Nicotiana benthamiana, we found that RA1 protein was localized in the chloroplast. Expression levels of chlorophyll biosynthesis and plastid development related genes were disordered in the ra1 mutant. RNA analysis revealed biogenesis of chloroplast rRNAs was abnormal in ra1. Meanwhile, western blotting showed that synthesis of proteins associated with plastid development was significantly repressed. These results suggest that RA1 is involved in early chloroplast development and establishment of the plastidic ribosome system in rice.

Systems analysis of phosphorylation-regulated Bcl-2 interactions establishes a model to reconcile the controversy over the significance of Bcl-2 phosphorylation.

BACKGROUND AND PURPOSE: The biological significance of the multi-site phosphorylation of Bcl-2 at its loop region (T69, S70 and S87) has remained controversial for decades. This is a major obstacle for understanding apoptosis and anti-tumour drug development. EXPERIMENTAL APPROACH: We established a mathematical model into which a phosphorylation and de-phosphorylation process of Bcl-2 was integrated. Paclitaxel-treated breast cancer cells were used as experimental models. Changes in the kinetics of binding with its critical partners, induced by phosphorylation of Bcl-2 were experimentally obtained by surface plasmon resonance, using a phosphorylation-mimicking mutant EEE-Bcl-2 (T69E, S70E and S87E). KEY RESULTS: Mathematical simulations combined with experimental validation showed that phosphorylation regulates Bcl-2 with different dynamics depending on the extent of Bcl-2 phosphorylation and the phosphorylated Bcl-2-induced changes in binding kinetics. In response to Bcl-2 homology 3 (BH3)-only protein Bmf stress, Bcl-2 phosphorylation switched from diminishing to enhancing the Bcl-2 anti-apoptotic ability with increased phosphorylation of Bcl-2, and the turning point was 50% Bcl-2 phosphorylation induced by 0.2 µM paclitaxel treatment. In contrast, Bcl-2 phosphorylation enhanced the anti-apoptotic ability of Bcl-2 towards other BH3-only proteins Bim, Bad and Puma, throughout the entire phosphorylation procedure. CONCLUSIONS AND IMPLICATIONS: The model could accurately predict the effects of anti-tumour drugs that involve the Bcl-2 family pathway, as shown with ABT-199 or etoposide.

Proteome-Wide Identification of On- and Off-Targets of Bcl-2 Inhibitors in Native Biological Systems by Using Affinity-Based Probes (AfBPs).

Selective inhibition of proteins of the Bcl-2 family by small-molecule inhibitors is a promising new approach in drug discovery. However, information about how these molecules interact with their cellular targets (on- and off-) is highly limited. We have designed and synthesized photoreactive and "clickable" affinity-based probes (AfBPs)-Nap-2 and Nap-5-by introducing photo-crosslinkers onto Nap-1, a fluorescent derivative of small-molecule Bcl-2 inhibitor S1-6. The resulting trifunctional probes Nap-2 and Nap-5 can enrich, visualize, and enable the identification of cellular on- and off-targets of Bcl-2 inhibitors both in vitro and in situ. Tubulin was validated as an off-target of Bcl-2 inhibitors (Nap-1 and S1-6) by large-scale cell-based proteome profiling and pull-down/western blotting (PD/WB) with Nap-2 and Nap-5. It was preliminarily illustrated to be a BH3-containing protein because some well-known Bcl-2 inhibitors can block the labeling of tubulin by Nap-2.

A selfish genetic element confers non-Mendelian inheritance in rice.

Selfish genetic elements are pervasive in eukaryote genomes, but their role remains controversial. We show that qHMS7, a major quantitative genetic locus for hybrid male sterility between wild rice (Oryza meridionalis) and Asian cultivated rice (O. sativa), contains two tightly linked genes [Open Reading Frame 2 (ORF2) and ORF3]. ORF2 encodes a toxic genetic element that aborts pollen in a sporophytic manner, whereas ORF3 encodes an antidote that protects pollen in a gametophytic manner. Pollens lacking ORF3 are selectively eliminated, leading to segregation distortion in the progeny. Analysis of the genetic sequence suggests that ORF3 arose first, followed by gradual functionalization of ORF2 Furthermore, this toxin-antidote system may have promoted the differentiation and/or maintained the genome stability of wild and cultivated rice.

Evaluation of water quality in surface water and shallow groundwater: a case study of a rare earth mining area in southern Jiangxi Province, China.

This study was conducted to evaluate the quality of surface water and shallow groundwater near a rare earth mining area in southern Jiangxi Province, China. Water samples from paddy fields, ponds, streams, wells, and springs were collected and analyzed. The results showed that water bodies were characterized by low pH and high concentrations of total nitrogen (total N), ammonium nitrogen (NH4 (+)-N), manganese (Mn), and rare earth elements (REEs), which was likely due to residual chemicals in the soil after mining activity. A comparison with the surface water standard (State Environmental Protection Administration & General Administration of Quality Supervision, Inspection and Quarantine of China GB3838, 2002) and drinking water sanitary standard (Ministry of Health & National Standardization Management Committee of China GB5749, 2006) of China revealed that 88 % of pond and stream water samples investigated were unsuitable for agricultural use and aquaculture water supply, and 50 % of well and spring water samples were unsuitable for drinking water. Moreover, significant cerium (Ce) negative and heavy REEs enrichment was observed after the data were normalized to the Post-Archean Australian Shales (PAAS). Principal component analysis indicated that the mining activity had a more significant impact on local water quality than terrace field farming and poultry breeding activities. Moreover, greater risk of water pollution and adverse effects on local residents' health was observed with closer proximity to mining sites. Overall, these findings indicate that effective measures to prevent contamination of surrounding water bodies from the effects of mining activity are needed.

Stabilization of collagen nanofibers with L-lysine improves the ability of carbodiimide cross-linked amniotic membranes to preserve limbal epithelial progenitor cells.

To overcome the drawbacks associated with limited cross-linking efficiency of carbodiimide modified amniotic membrane, this study investigated the use of L-lysine as an additional amino acid bridge to enhance the stability of a nanofibrous tissue matrix for a limbal epithelial cell culture platform. Results of ninhydrin assays and zeta potential measurements showed that the amount of positively charged amino acid residues incorporated into the tissue collagen chains is highly correlated with the L-lysine-pretreated concentration. The cross-linked structure and hydrophilicity of amniotic membrane scaffolding materials affected by the lysine molecular bridging effects were determined. With an increase in the L-lysine-pretreated concentration from 1 to 30 mM, the cross-linking density was significantly increased and water content was markedly decreased. The variations in resistance to thermal denaturation and enzymatic degradation were in accordance with the number of cross-links per unit mass of amniotic membrane, indicating L-lysine-modulated stabilization of collagen molecules. It was also noteworthy that the carbodiimide cross-linked tissue samples prepared using a relatively high L-lysine-pretreated concentration (ie, 30 mM) appeared to have decreased light transmittance and biocompatibility, probably due to the influence of a large nanofiber size and a high charge density. The rise in stemness gene and protein expression levels was dependent on improved cross-link formation, suggesting the crucial role of amino acid bridges in constructing suitable scaffolds to preserve limbal progenitor cells. It is concluded that mild to moderate pretreatment conditions (ie, 3-10 mM L-lysine) can provide a useful strategy to assist in the development of carbodiimide cross-linked amniotic membrane as a stable stem cell niche for corneal epithelial tissue engineering.

Immunogenicity and protective effects of inactivated Singapore grouper iridovirus (SGIV) vaccines in orange-spotted grouper, Epinephelus coioides.

Vaccination is one of the best methods against viral diseases. In this study, experimental inactivated Singapore grouper iridovirus (SGIV) vaccines were prepared, and immunogenicity and protection against virus infection of the vaccines were investigated in orange-spotted grouper, Epinephelus coioides. Two kinds of vaccines, including ß-propiolactone (BPL) inactivated virus at 4°C for 12 h and formalin inactivated virus at 4°C for 12 d, was highly protective against the challenge at 30-day post-vaccination and produced relative percent of survival rates of 91.7% and 100%, respectively. These effective vaccinations induced potent innate immune responses mediated by pro-inflammatory cytokines and type I interferon (IFN)-stimulated genes (ISGs). It is noteworthy that ISGs, such as Mx and ISG15, were up-regulated only in the effective vaccine groups, which suggested that type I IFN system may be the functional basis of early anti-viral immunity. Moreover, effective vaccination also significantly up-regulated of the expression of MHC class I gene and produced substantial amount of specific serum antibody at 4 weeks post-vaccination. Taken together, our results clearly demonstrated that effective vaccination in grouper induced an early, nonspecific antiviral immunity, and later, a specific immune response involving both humoral and cell-mediated immunity.

Selection and identification of Singapore grouper iridovirus vaccine candidate antigens using bioinformatics and DNA vaccination.

In this study, we described a rapid and efficient method which integrated the bioinformatic prediction and DNA vaccine technology to identify vaccine candidates against Singapore grouper iridovirus (SGIV). The 162 previously defined open reading frames (ORFs) of SGIV were subjected to extensive sequence similarity searches, as well as motif, cellular location, and domain prediction. Based on our analysis, 13 genes were chosen and cloned into the eukaryotic expression vector pcDNA 3.1. In vitro and in vivo expression of these DNA vaccine constructs was examined in Epinephelus akaara spleen cells (EAGS) and immunized fish by Western blot and RT-PCR analysis, respectively. Three weeks after the second booster, immunized fish were challenged with SGIV and the level of protection and survival was assessed. Fish vaccinated with plasmid DNA encoding viral ORF072, ORF039 and ORF036 (designated as pcDNA-72, pcDNA-39 and pcDNA-36, respectively) exhibited 66.7%, 66.7% and 58.3% relative percent survival rates, respectively, in comparison with the control fish. These three DNA vaccines induced innate immune responses, raising significantly high level of Mx expression relative to the fish vaccinated with the empty plasmid at 3 days post-vaccination. Furthermore, recombinant protein from ORF072 was also used to immunize another set of fish and similar protective effect was obtained. Taken together, our results validated the applicability of bioinformatics in genome mining, resulting in the identification of three protective antigens. The promising results obtained in the present study have prompted further testing to improve the immunogenicity of these potential DNA vaccines.