Development of a novel and fast XRF instrument for large area heavy metal detection integrated with UAV.

The disadvantages of the current chemical and instrumental analysis methods for soil heavy metal pollution are that they have a high detection cost, long cycle times, and may cause secondary pollution. The aims of this study were to improve the rapid detection of soil heavy metal pollution over large areas. This study combined aircraft technology, embedded development, computer software, electronic information, and other technical methods to create a novel solution to the problem, i.e., an integrated unmanned aerial vehicle (UAV) based soil heavy metal pollution rapid detection system (UAV-SHMPRDS) was built. The key technologies required for a rapid detection system were developed, including the development of a hardware system based on a UAV and an X-ray fluorescence spectrum (XRF) analyzer, the design and implementation of a control system software system, and the implementation of a data inversion processing algorithm. Finally, a prototype UAV-SHMPRDS was constructed. Testing showed that the system improved regionalized soil heavy metal pollution detection efficiency. This study provides new solutions for the current problems encountered in the actual rapid detection of soil heavy metal pollution.

Mitochondrial genome characteristics of Somena scintillans (Lepidoptera: Erebidae) and comparation with other Noctuoidea insects.

In this study, mitogenome of Somena scintillans (Lepidoptera: Erebidae) were sequenced and compared with other Noctuoidea species. The mitogenome is 15,410 base pairs in length. All 13 protein-coding genes (PCGs) are initiated by ATN codons except cox1 with CGA and all of PCGs terminate with TAA except nad4 with TAG. The codons ACG and CGC are absent. All the tRNA genes could be folded into the typical cloverleaf secondary structure except the trnS1 which not only loses dihydrouridine (DHU) arm but also mutates its anticodon into TCT. In the AT-rich region of the mitogenome the motif 'ATAGA' mutates to 'ATATA' and two copies of 161 bp-tandem repeats and two 'TA' short tandem repeats are founded. Phylogenetic analyses showed that S. scintillans is clustered into subfamily Lymatriinae. The phylogenetic relationships within Noctuoidea is (((Nolidae + (Euteliidae + Noctuidae)) + Erebidae) + Notodontidae).

Comparative Evaluation of Small Molecular Additives and Their Effects on Peptide/Protein Identification.

Detergents and salts are widely used in lysis buffers to enhance protein extraction from biological samples, facilitating in-depth proteomic analysis. However, these detergents and salt additives must be efficiently removed from the digested samples prior to LC-MS/MS analysis to obtain high-quality mass spectra. Although filter-aided sample preparation (FASP), acetone precipitation (AP), followed by in-solution digestion, and strong cation exchange-based centrifugal proteomic reactors (CPRs) are commonly used for proteomic sample processing, little is known about their efficiencies at removing detergents and salt additives. In this study, we (i) developed an integrative workflow for the quantification of small molecular additives in proteomic samples, developing a multiple reaction monitoring (MRM)-based LC-MS approach for the quantification of six additives (i.e., Tris, urea, CHAPS, SDS, SDC, and Triton X-100) and (ii) systematically evaluated the relationships between the level of additive remaining in samples following sample processing and the number of peptides/proteins identified by mass spectrometry. Although FASP outperformed the other two methods, the results were complementary in terms of peptide/protein identification, as well as the GRAVY index and amino acid distributions. This is the first systematic and quantitative study of the effect of detergents and salt additives on protein identification. This MRM-based approach can be used for an unbiased evaluation of the performance of new sample preparation methods. Data are available via ProteomeXchange under identifier PXD005405.

Development of cell-active N6-methyladenosine RNA demethylase FTO inhibitor.

The direct nucleic acid repair dioxygenase FTO is an enzyme that demethylates N(6)-methyladenosine (m(6)A) residues in mRNA in vitro and inside cells. FTO is the first RNA demethylase discovered that also serves a major regulatory function in mammals. Together with structure-based virtual screening and biochemical analyses, we report the first identification of several small-molecule inhibitors of human FTO demethylase. The most potent compound, the natural product rhein, which is neither a structural mimic of 2-oxoglutarate nor a chelator of metal ion, competitively binds to the FTO active site in vitro. Rhein also exhibits good inhibitory activity on m(6)A demethylation inside cells. These studies shed light on the development of powerful probes and new therapies for use in RNA biology and drug discovery.

DPP10-AS1-Mediated Downregulation of MicroRNA-324-3p Is Conducive to the Malignancy of Pancreatic Cancer by Enhancing CLDN3 Expression.

OBJECTIVES: Network of long noncoding RNA-microRNA (miRNA)-mRNA is becoming increasingly pivotal roles in carcinogenesis mechanism. Herein, we aim to delineate the mechanistic understanding of dipeptidyl peptidase like 10-antisense RNA 1 (DPP10-AS1)/miRNA-324-3p/claudin 3 (CLDN3) axis in the malignancy of pancreatic cancer (PC). METHODS: Microarray profiling and other bioinformatics methods were adopted to predict differentially expressed long noncoding RNA-miRNA-mRNA in PC, followed by verification of expression of DPP10-AS1, microRNA-324-3p (miR-324-3p), and CLDN3 in PC cells. The relationship among DPP10-AS1, miR-324-3p, and CLDN3 were further assessed. The PC cell invasion and migration were evaluated by scratch test and transwell assay. Tumor formation and lymph node metastasis were assessed in nude mice. RESULTS: Highly expressed DPP10-AS1 and CLDN3 and poorly expressed miR-324-3p were identified in PC cells. The competitively binding between DPP10-AS1 and miR-324-3p was identified, and CLDN3 was targeted and downregulated by miR-324-3p. In addition, DPP10-AS1 was found to sequester miR-324-3p to release CLDN3 expression. DPP10-AS1 knockdown or miR-324-3p restoration diminished migration, invasion, tumor formation, microvessel density, and lymph node metastasis of PC cells, which was associated with CLDN3 downregulation. CONCLUSIONS: Taken together, the study identified the regulatory role of DPP10-AS1/miR-324-3p/CLDN3 axis in PC, offering a mechanistic basis suggesting DPP10-AS1 ablation as a therapeutic target against PC.

The complete mitochondrial genome of Huainan partridge chicken (Gallus gallus).

Huainan Partridge chicken is one of the indigenous chicken breeds in China. In this study, the first complete mitochondrial DNA (mtDNA) sequence of Huainan Partridge chicken had been obtained using PCR amplification, sequencing and assembling. The mitogenome of Huainan Partridge chicken is 16785 bp in length, including a control region (D-loop), 13 protein-coding genes, 22 transfer genes and 2 ribosomal genes. The base composition of the complete mtDNA sequence is 30.27% for A, 23.73% for T, 13.50%for G, 32.50% for C. This study will provide reference for the phylogenetic analysis of Huainan Partridge chicken.

Rapid structural determination of alkaloids in a crude extract of Stemona saxorum by high-performance liquid chromatography/electrospray ionization coupled with tandem mass spectrometry.

The electrospray ionization (ESI) mass spectrometric behavior of five Stemona alkaloids, stemokerrin, oxystemokerrin, oxystemokerrilactone, oxystemokerrin N-oxide and stemokerrin N-oxide, was studied using an ESI tandem mass technique (MS(n)). These compounds, isolated from Stemona saxorum endemic in Vietnam, represent a class of alkaloids containing a pyrido[1,2-a]azepine A,B-ring core with a 1-hydroxypropyl side chain attached to C-4. Their fragmentation pathways were elucidated by ESI-MS(n) results and the elemental composition of the major product ions was confirmed by accurate mass measurement. In order to rationalize some fragmentation pathways, the relative Gibbs free energies of some product ions were estimated using the B3LYP/6-31+G(d) method. Based on the ESI-MS(n) results of five reference compounds, a reversed-phase high-performance liquid chromatography with tandem mass spectrometry (RP-HPLC/MS(n)) method was developed for the characterization of Stemona alkaloids with a pyrido[1,2-a]azepine A,B-ring core from the extract of S. saxorum. A total of 41 components were rapidly identified or tentatively characterized, of which 12 compounds were identified as Stemona alkaloids with a pyrido[1,2-a]azepine A,B-ring core, including four new compounds. This method is convenient and sensitive, especially for minor components in complex natural product extracts.

Two new Daphniphyllum alkaloids from the stems and leaves of Daphniphyllum calycinum.

Two new Daphniphyllum alkaloids, 9,10-epoxycalycinine A (1) and homodaphniphyllate (2), together with eight known related alkaloids, were isolated from the stems and leaves of Daphniphyllum calycinum. The structures of the new compounds 1 and 2 were elucidated on the basis of in-depth spectroscopic-data analysis, by chemical derivatization, and by comparison of their spectroscopic data with those of known compounds.

Further Daphniphyllum alkaloids with insecticidal activity from the bark of Daphniphyllum macropodum M(IQ).

Two new Daphniphyllum alkaloids, macropodumines J and K (1 and 2, resp.), together with six known structurally related alkaloids, 3-8, were isolated from the bark of Daphniphyllum macropodum M(IQ). The structures of the new compounds 1 and 2 were elucidated on the basis of a comprehensive analysis of their spectroscopic and chemical data. Macropodumine J (1) contains a CN group which is relatively rare in naturally occurring alkaloids. All isolated compounds were tested for their insecticidal activities against a number of insect species. Daphtenidine C (5) is the most active compound against Plutella xylostella. This is the first report of insecticidal properties of Daphniphyllum alkaloids.

Complete mitogenome of Parum colligata (Lepidoptera: Sphingidae) and its phylogenetic position within the Sphingidae.

In this study, the complete mitochondrial DNA sequence of Parum colligata (Lepidoptera: Sphingidae: Smerinthinae) was sequenced firstly. The mitogenome is 15,288 bp in size, containing 13 protein-coding genes (PCGs), 22 transfer RNA genes (tRNAs), two ribosomal RNA genes (rRNAs), and an A+T-rich region. In the mitogenome, Ile, Leu2, and Phe are the most frequently used codon families, while codons GCG, TGC, GGC, CTG, AGG, and ACG are absent. The A+T-rich region is 358 bp in length including a motif 'ATAGA', an 18 bp poly-T stretch, three copies of a 12 bp 'TATATATATATA', and a short poly-A element. The nucleotides sequence of A+T-rich region is closer to Sphinginae than Macroglossinae. Phylogenetic analyses, based on the PCGs by using Maximum Likelihood (ML) and Bayesian Inference (BI) methods, generated consistent results that Smerinthinae was clustered together with Sphinginae to be the sister groups rather than Macroglossinae.

Complete mitochondrial genome of the Partridge Shank chicken (Galliformes: Phasianidae).

Partridge Shank chicken is a valuable broiler breed in China. The first complete mitochondrial DNA (mtDNA) sequence of Partridge Shank chicken had been obtained using PCR amplification, sequencing and assembling. The complete mitochondrial genome was 16,788 bp in length, with the base composition of 30.2% for A, 23.7% for T, 32.5% for C and 13.5% for G. It exhibited the typical mitochondrial structure, including 2 ribosomal RNA genes, 13 protein-coding genes, 22 transfer RNA genes, and a non-coding control region (D-loop region). The phylogenetic tree construced with maximum-likelihood (ML) method based on the complete chicken mitochondrial genomes showed that the 26 chicken breeds could be divided into two groups.

A new exploration of health risk assessment quantification from sources of soil heavy metals under different land use.

Heavy metals in the topsoil affected adversely human health through inhalation, ingestion and dermal contact. The health risk assessment, which are quantified from soil heavy metals sources under different land use, can provide an important reference basis for preventing and controlling the soil heavy metals pollution from the source. In this study, simple statistical analysis and Positive Matrix Factorization (PMF) were used to quantify sources of soil heavy metals; then a health risk assessment (HRA) model combined with PMF was proposed to assess quantificationally the human health risk (including non-cancer risk and cancer risk) from sources under residential-land, forest-land and farm land. Xiang River New District (XRNQ) was chosen as the example and four significant sources were quantitatively analyzed in the study. For cancer risk, industrial discharge was the largest source and accounted for about 69.6%, 69.7%, 56.5% for adults under residential-land, forest-land and farm-land, respectively. For non-cancer risk, industrial discharge was still the largest significant source under residential-land and forest-land and accounted for about 41.7%, 39.2% for adult, respectively; while agricultural activities accounted for about 51.8% for adult under farm-land. The risk trend of children, including cancer risk and non-cancer risk, was similar with adults. However, the non-cancer risk areas of adults affected by industrial discharge was higher than that of children, while the cancer risk areas of adults were on the contrary. The new exploration was useful to assess health risk quantification from sources under different land use, thus providing certain reference in preventing and controlling the pollution from the source for local authorities effectively.

Immune response induced by spike protein from transmissible gastroenteritis coronavirus expressed in mouse mammary cells.

The present study is undertaken to investigate the immune response that was induced by the recombinant spike (S) protein from swine-transmissible gastroenteritis virus (TGEV) expressed in mouse mammary cells. A mammary-specific expression vector pEBS containing the full-length cDNA of S gene was constructed and expressed in the mouse mammary cells (EMT6). The recombinant S protein from culture supernatant of transgenic EMT6 was harvested and immunized BALB/c mice. The results demonstrated recombinant S protein was expressed at high levels in mammary cells by Western blotting and enzyme-linked immunosorbent assay (ELISA) detection. The antibody titer in BALB/c mice following immunization with recombinant S protein was detectable after the first immunization. Maximum titers of antibody (8.86+/-0.19 ng/ml of serum) were attained after the second immunization. In conclusion, the recombinant S protein expressed in mammary cells was able to elicit substantial immunological response against TGEV. This lays the basis for using mammary gland bioreactor generating edible vaccine.

Macropodumines D and E, two new alkaloids with unusual skeletons from Daphniphyllum macropodum Miq.

[structure: see text] Two new complex polycyclic alkaloids, macropodumines D (1) and E (2), both possessing unprecedented skeletons, along with four known related alkaloids, were isolated from the leaves and barks of Daphniphyllum macropodum Miq. The structures including the relative stereochemistry of new compounds 1 and 2 were elucidated on the basis of detailed spectroscopic data analysis.

Immortalization of bovine germ line stem cells by c-myc and hTERT.

The limited life span of bovine germ line stem cells in vitro is one of the obstacles to spermatogenesis analysis, genetic manipulation and generating transgenic animal. The aim of this study is to establish immortalized bovine germ line stem cells by c-myc or hTERT. We constructed pEMY and pETE expression vectors and transfected germ line cells from 5-month-old bovine. After G418 screening, four types of positive clones were obtained. The results showed that they expressed exogenous genes c-myc or hTERT at mRNA and protein level by RT-PCR and Western blotting detection. Presumable cell lines GM7, GT3, GMT5 all expressed germ-line-stem-cell-specific makers by immunocytochemical analysis, such as c-kit, Oct-4 and GFRalpha-1. The putative cell lines also had higher capacity of proliferation than freshly isolated bovine spermatogonial stem cells. So we can conclude that exogenous genes c-myc or hTERT have integrated into the genome of bovine germ cells and upregulated the expression of telomerase.

Complete mitochondrial genome of Cnidocampa flavescens (Lepidoptera: Limacodidae).

Cnidocampa flavescens, lives in Nepal, Bhutan, China, Far East of Russia, Korea, and Japan, belongs to the Lepidoptera family Limacodidae. In this study, we describe the genomic features of the mitogenome sequences of the insects. The mitogenome of C. flavescens is 15,406 bp long consisting a typical set of genes (13 protein-coding genes, 22 tRNA genes, and 2 rRNA genes) and one major 415 bp non-coding A + T-rich region. All PCGs of C. flavescens start with ATN codons and end with TAA codons. The gene arrangement of C. flavescens mitogenome is same to Monema flavescens while the intergenic spacers and overlaps are different. The 415 bp A + T-rich region contains a conserved ATAGA motif followed a poly-T stretch.

Complete mitochondrial genome of Histia rhodope Cramer (Lepidoptera: Zygaenidae).

Histia rhodope Cramer, found in India, Chinese, Burma, Indonesia and other Southeast Asian regions, belongs to Lepidoptera family Zygaenidae. In this study, we describe the genomic features of the mitogenome sequences of these insects. The mitogenome of Histia rhodope Cramer is 15,205 bp long consisting a typical set of genes (13 protein-coding genes, 22 tRNA genes and two rRNA genes) and one major 376 bp non-coding A + T-rich region. All PCGs of Histia rhodope Cramer start with ATN codons except cox1 which start with CGA codon and all PCGs stop at TAA codons. Phylogenetic analysis demonstrates that Histia rhodope Cramer and Rhodopsona rbiginosa are clustered together into a monophyletic group Zygaenidae. Zygaenidae is phylogenetically closer to Limacodiae than Tortricidea.

In vitro biochemical and thermodynamic characterization of nucleocapsid protein of SARS.

The major biochemical and thermodynamic features of nucelocapsid protein of SARS coronavirus (SARS_NP) were characterized by use of non-denatured gel electrophoresis, size-exclusion chromatographic and surface plasmon resonance (SPR) techniques. The results showed that SARS_NP existed in vitro as oligomer, more probably dimer, as the basic functional unit. This protein shows its maximum conformational stability near pH 9.0, and it seems that its oligomer dissociation and protein unfolding occur simultaneously. Thermal-induced unfolding for SARS_NP was totally irreversible. Both the thermal and chemical denaturant-induced denaturation analyses showed that oligomeric SARS_NP unfolds and refolds through a two-state model, and the electrostatic interactions among the charge groups of SARS_NP made a significant contribution to its conformational stability.

Characteristic ion clusters as determinants for the identification of pyrrolizidine alkaloid N-oxides in pyrrolizidine alkaloid-containing natural products using HPLC-MS analysis.

Pyrrolizidine alkaloid (PA)-containing plants are widely distributed in the world. PAs are hepatotoxic, affecting livestock and humans. PA N-oxides are often present together with PAs in plants and also exhibit hepatotoxicity but with less potency. HPLC-MS is generally used to analyze PA-containing herbs, although PA references are unavailable in most cases. However, to date, without reference standards, HPLC-MS methodology cannot distinguish PA N-oxides from PAs because they both produce the same characteristic ions in mass spectra. In the present study, the mass spectra of 10 PA N-oxides and the corresponding PAs were systemically investigated using HPLC-MS to define the characteristic mass fragment ions specific to PAs and PA N-oxides. Mass spectra of toxic retronecine-type PA N-oxides exhibited two characteristic ion clusters at m/z 118-120 and 136-138. These ion clusters were produced by three unique fragmentation pathways of PA N-oxides and were not found in their corresponding PAs. Similarly, the nontoxic platynecine-type PA N-oxides also fragmented via three similar pathways to form two characteristic ion clusters at m/z 120-122 and 138-140. Further application of using these characteristic ion clusters allowed successful and rapid identification of PAs and PA N-oxides in two PA-containing herbal plants. Our results demonstrated, for the first time, that these characteristic ion clusters are unique determinants to discriminate PA N-oxides from PAs even without the availability of reference samples. Our findings provide a novel and specific method to differentiate PA N-oxides from PAs in PA-containing natural products, which is crucial for the assessment of their intoxication.

Extensive crosstalk between O-GlcNAcylation and phosphorylation regulates Akt signaling.

O-linked N-acetylglucosamine glycosylations (O-GlcNAc) and O-linked phosphorylations (O-phosphate), as two important types of post-translational modifications, often occur on the same protein and bear a reciprocal relationship. In addition to the well documented phosphorylations that control Akt activity, Akt also undergoes O-GlcNAcylation, but the interplay between these two modifications and the biological significance remain unclear, largely due to the technique challenges. Here, we applied a two-step analytic approach composed of the O-GlcNAc immunoenrichment and subsequent O-phosphate immunodetection. Such an easy method enabled us to visualize endogenous glycosylated and phosphorylated Akt subpopulations in parallel and observed the inhibitory effect of Akt O-GlcNAcylations on its phosphorylation. Further studies utilizing mass spectrometry and mutagenesis approaches showed that O-GlcNAcylations at Thr 305 and Thr 312 inhibited Akt phosphorylation at Thr 308 via disrupting the interaction between Akt and PDK1. The impaired Akt activation in turn resulted in the compromised biological functions of Akt, as evidenced by suppressed cell proliferation and migration capabilities. Together, this study revealed an extensive crosstalk between O-GlcNAcylations and phosphorylations of Akt and demonstrated O-GlcNAcylation as a new regulatory modification for Akt signaling.