Low-Temperature Plasma Probe Mass Spectrometry for Analytes Separated on Thin-Layer Chromatography Plates: Direct vs Laser Assisted Desorption.

Thin-layer chromatography (TLC) is a widespread technique because it allows fast, simple, and inexpensive analyte separations. In addition, direct analysis of the compounds separated on TLC plates via mass spectrometry (MS) has been shown to provide high sensitivity and selectivity while avoiding time-consuming sample extraction protocols. Here, direct desorption low-temperature plasma-mass spectrometry (LTP-MS) as well as diode laser assisted desorption (LD) LTP-MS are studied for direct spatially resolved analysis of compounds from TLC plates. Qualitative and quantitative characterization of amino acids, pharmaceuticals, and structural isomers were performed. The nature of the TLC plate stationary phase was found to have a significant influence, together with the analyte's characteristics, on the desorption efficiency. Tandem MS is shown to greatly improve the limits of detection (LODs). Direct desorption LTP-MS, without external thermal assisted desorption, demonstrates its best performance with cellulose TLC plates (LODs, 0.01 ng/mm2 to 2.55 ng/mm2) and restricted performance with normal-phase (NP) TLC plates (several analytes without observable signal). LD LTP-MS, with systematic optimization of irradiance and focal point diameter, is shown to overcome the direct-desorption limitations and reach significantly improved LODs with NP TLC plates (up to ×1000 better). In addition, a wide-ranging characterization of amino acid analytical figures of merit with LD LTP-MS shows that LODs from 84 pg/mm2 down to 0.3 pg/mm2 are achieved on NP TLC plates.

Regulation of the expression of key signalling molecules in mTOR pathway of skeletal muscle satellite cells in neonatal chicks: Effects of leucine and glycine-leucine peptide.

This study was conducted to analyse the effects of leucine (Leu) and glycine (Gly)-Leu peptide on expressions of key signalling molecules in mTOR pathway of skeletal muscle satellite cells in neonatal chicks. The 4-day-old male AA broilers with similar weight were selected to obtain the broiler skeletal muscle satellite cells with the two-step method of collagenase-I and trypsin digestion. The satellite cells were subjected to primary culture in vitro, and they were cultured in DMEM medium with the Leu concentration of 0.2 mM and 2 mM as well as with the Gly-Leu peptide concentration of 0.2 mM and 2 mM. The experiment lasted for 5 days. The results showed that TOR, S6K1 and 4E-BP1 mRNA expressions in the medium with Leu concentration of 2 mM were significantly higher than that in 0.2 mM group (p < 0.05). There was no difference between the medium with Gly-Leu concentration of 2 mM and 0.2 mM on the TOR, S6K1 and 4E-BP1 mRNA expressions (p > 0.05). In conclusion, Leu significantly increases TOR, S6K1 and 4E-BP1 mRNA expressions of skeletal muscle satellite cells, but Gly-Leu peptide has no effect on them.

Genetic map construction and QTL analysis of leaf-related traits in soybean under monoculture and relay intercropping.

Soybean (Glycine max L.) is an important food and oil crop widely planted by intercropping in southwest China. The shade caused by intercropping changes plant growth traits, such as soybean leaf and dry mass, thereby reducing yields. To improve the yield and elucidate the genetic mechanism of the leaf-related traits in intercropped soybeans, we measured the F6:7-8 recombinant inbred lines (RILs) derived from the cross of 'Nandou 12' and 'Jiuyuehuang' for six leaf-related traits under monoculture and relay intercropping in 2015 and 2016. We found 6366 single-nucleotide polymorphisms (SNPs) markers that covered the whole genome of soybean distributed in 20 linkage groups, which spanned 2818.67 cM with an average interval of 0.44 cM between adjacent markers. Nineteen quantitative trait loci (QTLs) were detected in two environments in 2 years. Three candidate genes associated to leaf-related traits were found according to gene expression and GO enrichment analyses. These results revealed the susceptibility of leaf phenotype to shading and helped elucidate the mechanisms that control leaf-related traits.

Effects of antibiotic on microflora in ileum and cecum for broilers by 16S rRNA sequence analysis.

An experiment was conducted to analyze and compare the microbial composition, abundance, dynamic distribution, and functions without and with antibiotic fed to broilers. A 16S rRNA-sequencing approach was used to evaluate the bacterial composition of the gut of male broilers under different groups. A total of 240 1-day old AA male broilers were randomly assigned to two groups, with 120 broilers per group. The treatment group was administered an antibiotic with their feed, while the control group was not administered antibiotic (control group). A total of 10 replicates were assessed per treatment. The control group was fed a basal diet containing corn, soybean meal, and cottonseed meal and met the nutritional requirement. The antibiotic group was fed 100 mg/kg aureomycin (based on the basal diet). The trial lasted 42 days. Operational taxonomic unit partition and classification, alpha diversity, taxonomic composition, beta diversity, and microflora comparative analyses along with key species screening were performed for all of the treatment groups. Our data indicate that aureomycin treatment in broilers is directly correlated with variations of the gut content of specific bacterial taxa, and herein provide insights into the impact of antibiotic on microbial communities in cecum and ileum of broiler chickens.

WITHDRAWN:Microbiota transplantation modifies the microfloras of the ileum and the cecum in broiler chickens: a study based on 16S rRNA sequence analysis.

Ahead of Print article withdrawn by publisher.

Effects of increasing concentrations of an Escherichia coli phytase on the apparent ileal digestibility of amino acids and the apparent total tract digestibility of energy and nutrients in corn-soybean meal diets fed to growing pigs.

An experiment was conducted to test the hypothesis that inclusion of increasing concentrations of an Escherichia coli phytase to a corn-soybean meal (SBM) diet results in improved digestibility of DM, GE, CP, NDF, ADF, macrominerals, microminerals, and AA. Twenty-four growing barrows (initial BW: 37.0 ± 1.4 kg) were equipped with a T-cannula in the distal ileum and placed individually in metabolism crates, and allotted to a 2-period switch-back design with 6 diets and 4 replicate pigs per diet in each period. The positive control diet was a corn-SBM diet that contained limestone and dicalcium phosphate to meet the requirement for standardized total tract digestible (STTD) P and Ca (0.31% STTD P and 0.70% Ca). A negative control diet that was similar to the positive control diet, with the exception that no dicalcium phosphate was used, was also formulated, and this diet contained 0.16% STTD P and 0.43% Ca. Four additional diets were formulated by adding 500, 1,000, 2,000, or 4,000 units of microbial phytase (FTU) to the negative control diet. Each period lasted 14 d. Fecal and urine samples were collected from the feed provided from days 6 to 11 of each period following 5 d of adaptation to the diets. Ileal digesta were collected for 8 h on days 13 and 14. Results indicated that addition of the E. coli phytase to the negative control diet tended to quadratically improve the apparent ileal digestibility of Phe (P = 0.086) and Asp (P = 0.054), and linearly increased (P < 0.05) the apparent total tract digestibility (ATTD) of ADF, K, and Fe. Microbial phytase also quadratically increased (P < 0.05) the ATTD of NDF and Mg, and linearly and quadratically increased (P < 0.05) the ATTD and retention of Ca and P. However, no effects of the phytase on ATTD of GE or the concentration of DE were observed. In conclusion, the increased absorption of several minerals including Ca, P, K, Mg, and Fe that was observed as increasing concentrations of an E. coli phytase was added to a corn-SBM meal diet indicates that the dietary provision of these minerals may be reduced if phytase is fed.

Additivity of values for phosphorus digestibility in corn, soybean meal, and canola meal in diets fed to growing pigs.

OBJECTIVE: This study was conducted to determine the apparent and standardized total tract digestibility (ATTD and STTD) of phosphorus (P) in corn, soybean meal (SBM), and canola meal (CM), and additivity of values for ATTD and STTD of P in corn, SBM, and CM in diets fed to growing pigs. METHODS: Thirty-six growing barrows (initial body weight of 21.6±1.7 kg) were placed in metabolism crates and allotted to a completely randomized design with 6 diets and 6 pigs per diet. Six diets were formulated using corn, SBM or CM as the sole source of P, or corn and SBM, or corn and CM, or corn, SBM, and CM as the P source in each diet, respectively. Fecal samples were collected for 5 d following a 7 d adaptation period to the diets. RESULTS: Values for ATTD and STTD of P in corn, SBM, and CM in growing pigs were 33.12% and 37.76%, 50.19% and 56.62%, 34.93% and 39.45%, respectively. The ATTD and STTD of P in SBM were greater (p<0.05) than those in corn and CM. However, there were no differences in the ATTD or STTD of P between corn and CM. The determined STTD of P in the mixture of corn and SBM, corn and CM, and corn, SBM, and CM is not different from the calculated STTD values. CONCLUSION: Values for STTD of P in corn, SBM, and CM are additive in their mixture fed to growing pigs.

Cytoprotective effect of deferiprone against aluminum chloride-induced oxidative stress and apoptosis in lymphocytes.

Aluminum (Al) is a toxic metal, and excessive Al accumulation causes immunosuppression. Deferiprone (DFP) is a well-known chelator and used in dialysis patients for removing Al from tissues. The present study aimed to investigate whether DFP treatment can attenuate immunotoxicity induced by aluminum chloride (AlCl3) in cultured lymphocytes. Lymphocytes were treated with 0 and 0.6 mmol/L AlCl3∙6H2O (pH 7.2) and/or 1.8 mmol/L DFP, respectively. Immune function of lymphocytes was assessed by T and B lymphocytes proliferation rates, T lymphocyte subpopulations and IL-2, IL-6 and TNF-α contents. In addition, lymphocyte damage was assessed by LDH activity, NO and MDA contents, NOS, SOD and GSH-Px activities, lymphocyte apoptosis index. These results showed that AlCl3 exposure reduced T and B lymphocyte proliferation rates, CD3+ and CD4+ T lymphocyte subpopulations, CD4+/CD8+ ratio, IL-2, IL-6 and TNF-α contents, SOD and GSH-Px activities, early and later lymphocyte apoptosis indexes while enhanced CD8+ T lymphocyte subpopulation, NO and MDA contents, LDH activity. DFP treatment attenuated the immunotoxicity of lymphocytes and reduced oxidative stress and lymphocyte apoptosis induced by AlCl3, indicating that DFP could protect lymphocytes against immunosuppression induced by AlCl3 through attenuating oxidative stress and apoptosis.

Effects of proteinate complex zinc on growth performance, hepatic and splenic trace elements concentrations, antioxidative function and immune functions in weaned piglets.

OBJECTIVE: To assess the effects of proteinate complex zinc (PC-Zn) on growth performance, antioxidative function, trace element concentrations and immune function in weaned piglets. METHODS: Three hundred newly weaned barrows (Duroc×Landrace×Yorkshire), 28 days of age, were randomly allotted to 3 dietary groups of 5 replicate pens per group for 4 weeks of feeding. Experimental diets were: i) zinc deficient diet (ZnD, 24 mg/kg Zn supplementation from ZnSO4), ii) inorganic Zn diet supplemented with 120 mg/kg of Zn from Zn sulfate (ZnSO4), and iii) organic Zn diet supplemented with 120 mg/kg of Zn from PC-Zn. The body weight of pigs were recorded at the beginning, at the middle and at the end of the experiment, and the amount of feed supplied each day was recorded. Five barrows from each dietary treatment group were selected to be anesthetized and euthanized at the end of the trial to determine the Zn, Cu, Fe, and Mn concentrations, the hepatic metallothionein content, the levels of methane dicarboxylic aldehyde (MDA), Mn, and Cu/Zn superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px) in the spleen, the levels of interleukin (IL)-2, IL-4, IL-10, interferon (IFN)-γ, CD3+, CD4+, and CD8+ T lymphocyte. RESULTS: The accumulation of Zn in the spleen, levels of SOD, GSH-Px, IL-4, IL-10, the proportions of CD3+ and CD4+ T lymphocyte, and the ratio of CD4+/CD8+ T lymphocyte were increased by organic Zn supplementation compared to ZnD, while the levels of MDA, IFN-γ, and proportion of CD8+ T lymphocyte were lowered. CONCLUSION: These findings indicate that Zn can improve the antioxidant potential and immune functions of weaned piglets.

Methodological aspects of determining phosphorus digestibility in swine: A review.

The role of phosphorus (P) in swine nutrition has been taken on new significance in recent years. Methods to determine the available phosphorus (AP) content of swine feeds include relative bioavailability (RBV), apparent total tract digestibility (ATTD), standardized total tract digestibility (STTD), and true total tract digestibility (TTTD). The RBV of P is determined by measuring bone ash or bone P, whereas the ATTD of P is determined by calculating the difference between P intake and P excretion in feces. Recent research has shown that the use of ATTD of P underestimates the AP due to the existence of endogenous P in feces and digesta. The STTD can be calculated from ATTD by taking basal endogenous phosphorus losses (EPL) into consideration. The basal EPL in pigs can be measured by feeding a P-free diet. Values for STTD of P are believed to be additive in mixed diets but not for ATTD of P. The regression method is a common approach to determine total EPL and TTTD of P, which measures the linear relationship between fecal P excretion and the dietary intake of total P. In addition, in vitro methods such as the bionic enzymatic method are being increasingly utilized because they can be done quickly and simply. Several dietary factors such as P and Ca concentrations, phytate, Ca to P ratio and vitamin D may affect AP. This review summarizes the evolution of methods to measure AP and factors that can affect AP, which may provide information to formulate swine diet more accurately. Moreover, the knowledge about AP may help to reduce the P waste in swine production and thus decrease its impact on the environment.

[Study on influence factors of seed germination and seeding growth of Lonicera macranthoides].

In order to improve reproductive efficiency and quality standard, the influence factors of seed germination and seeding growth of Lonicera macranthoides werew studied. The fruit and seed morphological characteristics of L. macranthoides were observed, the seed water absorbing capacity was determined, and different wet sand stratification time, temperature and germination bed treatment were set up. The effects of the parameters on seed germination and seedling growth were analysed. There was no obstacles of water absorption on L. macranthoides seed, quantity for 22 h water absorption was close to saturation. In the first 80 d, with the increase of the stratification time, seed initial germination time was shortened, germination rate and germination potential was improved. Stratification for 100 d, germination rate decreased. At 15 ℃, seed germination and seedling growth indicators were the best. The seedling cotyledon width in light was significantly higher than that in dark. Seeds on the top of paper and top of sand germination rate, germination potential, and germination index was significantly higher than that of other germination bed and mildew rate is low. The optimal conditions of seeds germination test was stratified in 4 ℃ wet sand for 80 d, 15 ℃ illuminate culture on the top of paper or top of sand. The first seeding counting time was the 4th day after beginning the test, the final time was the 23th day. The germination potential statistical time was the 13th day after beginning the test.

[Study on seed quality test and quality standard of Lonicera macranthoides].

Referring to the rules for agricultural seed testing (GB/T 3543-1995) issued by China, the test of sampling, purity, thousand seed weight, moisture, viability, relative conductivity and germination rate had been studied for seed quality test methods of Lonicera macranthoides. The seed quality from 38 different collection areas was measured to establish quality classification standard by K-means clustering. The results showed that at least 7.5 g seeds should be sampled, and passed 20-mesh sieve for purity analysis.The 500-seed method used to measure thousand seed weight. The moisture was determined by crushed seeds dried in high temperature (130±2) ℃ for 3 h.The viability determined by 25 ℃ 0.1% TTC stained 5h in dark. 1.0 g seeds soaked in 50 ml ultra pure water in 25 ℃ for 12 hours to determine the relative conductivity. The seed by 4 ℃stratification for 80 days were cultured on paper at 15 ℃. Quality of the seeds from different areas was divided into three grades. The primary seed quality classification standard was established.The I grade and II grade were recommend use in production.

The Effect of Inclusion Level of Soybean Oil and Palm Oil on Their Digestible and Metabolizable Energy Content Determined with the Difference and Regression Method When Fed to Growing Pigs.

This experiment was conducted to determine the effects of inclusion level of soybean oil (SO) and palm oil (PO) on their digestible and metabolism energy (DE and ME) contents when fed to growing pigs by difference and regression method. Sixty-six crossbred growing barrows (Duroc×Landrace×Yorkshire and weighing 38.1±2.4 kg) were randomly allotted to a 2×5 factorial arrangement involving 2 lipid sources (SO and PO), and 5 levels of lipid (2%, 4%, 6%, 8%, and 10%) as well as a basal diet composed of corn and soybean meal. The barrows were housed in individual metabolism crates to facilitate separate collection of feces and urine, and were fed the assigned test diets at 4% of initial body weight per day. A 5-d total collection of feces and urine followed a 7-d diet adaptation period. The results showed that the DE and ME contents of SO and PO determined by the difference method were not affected by inclusion level. The DE and ME determined by the regression method for SO were greater compared with the corresponding respective values for PO (DE: 37.07, ME: 36.79 MJ/kg for SO; DE: 34.11, ME: 33.84 MJ/kg for PO, respectively). These values were close to the DE and ME values determined by the difference method at the 10% inclusion level (DE: 37.31, ME: 36.83 MJ/kg for SO; DE: 34.62, ME: 33.47 MJ/kg for PO, respectively). A similar response for the apparent total tract digestibility of acid-hydrolyzed ether extract (AEE) in lipids was observed. The true total tract digestibility of AEE in SO was significantly (p<0.05) greater than that for PO (97.5% and 91.1%, respectively). In conclusion, the DE and ME contents of lipid was not affected by its inclusion level. The difference method can substitute the regression method to determine the DE and ME contents in lipids when the inclusion level is 10%.

Isolation and characterization of a crude oil degrading bacteria from formation water: comparative genomic analysis of environmental Ochrobactrum intermedium isolate versus clinical strains.

In this study, we isolated an environmental clone of Ochrobactrum intermedium, strain 2745-2, from the formation water of Changqing oilfield in Shanxi, China, which can degrade crude oil. Strain 2745-2 is aerobic and rod-shaped with optimum growth at 42 °C and pH 5.5. We sequenced the genome and found a single chromosome of 4 800 175 bp, with a G+C content of 57.63%. Sixty RNAs and 4737 protein-coding genes were identified: many of the genes are responsible for the degradation, emulsification, and metabolizing of crude oil. A comparative genomic analysis with related clinical strains (M86, 229E, and LMG3301(T)) showed that genes involved in virulence, disease, defense, phages, prophages, transposable elements, plasmids, and antibiotic resistance are also present in strain 2745-2.

Aluminum Trichloride Disorders Bile Acid Secretion and Induces Hepatocyte Apoptosis in Rats.

The aim of this study was to examine effects of aluminum trichloride (AlCl3) on the bile acid secretion and hepatocyte apoptosis in rats. Forty male Wistar rats (5 weeks old) weighed 110-120 g were orally exposed to AlCl3 at doses of 0, 64.18 (1/20LD50), 128.36 (1/10LD50), and 256.72 (1/5LD50) mg/kg body weight in drinking water for 120 days. Each group had ten rats. The serum total bile acid (TBA) concentration, the early and total hepatocyte apoptosis indexes, and protein and mRNA expression of Bcl-2 and Bax were determined. The results showed that the serum TBA concentration, the early and total hepatocyte apoptosis indexes, and protein and mRNA expression of Bax increased, while protein and mRNA expression of Bcl-2 decreased in AlCl3-treated rats. The results indicate that AlCl3 disorders bile acid secretion and induces hepatocyte apoptosis in rats.

Interaction between abscisic acid receptor PYL3 and protein phosphatase type 2C in response to ABA signaling in maize.

Abscisic acid (ABA) is a ubiquitous hormone that regulates plant growth, development and responses to environmental stresses. In recent researches, pyrabactin resistance 1-like protein (PYL) and protein phosphatase type 2C (PP2C) were identified as the direct receptor and the second component of ABA signaling pathway, respectively. However, a lot of PYL and PP2C members were found in Arabidopsis and several other plants. Some of them were found not to be involved in ABA signaling. Because of the complex diversity of the genome, few documents have been available on the molecular details of the ABA signal perception system in maize. In the present study, we conducted bioinformatics analysis to find out the candidates (ZmPYL3 and ZmPP2C16) of the PYL and PP2C members most probably involved in ABA signaling in maize, cloned their encoding genes (ZmPYL3 and ZmPP2C16), verified the interaction between these two proteins in response to exogenous ABA induction by yeast two-hybrid assay and bimolecular fluorescence complementation, and investigated the expression patterns of these two genes under the induction of exogenous ABA by real-time fluorescence quantitative PCR. The results indicated that the ZmPYL3 and ZmPP2C16 proteins interacted in vitro and in vivo in response to the induction of exogenous ABA. The downregulated expression of the ZmPYL3 gene and the upregulated expression of the ZmPP2C16 gene are responsive to the induction of exogenous ABA. The ZmPYL3 and ZmPP2C16 proteins are the most probable members of the receptors and the second components of ABA signaling pathway, respectively.

Expression profile of maize microRNAs corresponding to their target genes under drought stress.

Microarray assay of four inbred lines was used to identify 303 microRNAs differentially expressed under drought stress. The microRNAs were used for bioinformatics prediction of their target genes. The majority of the differentially expressed microRNA families showed different expression profiles at different time points of the stress process among the four inbred lines. Digital gene expression profiling revealed 54 genes targeted by 128 of the microRNAs differentially expressed under the same stress conditions. The differential expression of miR159 and miR168 was further validated by locked nucleic acid northern hybridization. These results indicated that miR159 and miR168, as well as numerous other microRNAs, play critical roles in signaling pathways of maize response to drought stress. However, the level of the post-transcriptional regulation mediated by microRNAs had different responses among genotypes, and the gene expression related to signaling pathways under drought stress is also regulated, possibly by multiple mechanisms.

Heterologous expression of betaine aldehyde dehydrogenase gene from Ammopiptanthus nanus confers high salt and heat tolerance to Escherichia coli.

Betaine aldehyde dehydrogenase (BADH) catalyzes the synthesis of glycine betaine, a regulator of osmosis, and therefore BADH is considered to play a significant role in response of plants to abiotic stresses. Here, based on the conserved residues of the deduced amino acid sequences of the homologous BADH genes, we cloned the AnBADH gene from the xerophytic leguminous plant Ammopiptanthus nanus by using reverse transcription PCR and rapid amplification of cDNA ends. The full-length cDNA is 1,868 bp long without intron, and contains an open reading frame of 1512 bp, and 3'- and 5'-untranslated regions of 294 and 62 bp. It encodes a 54.71 kDa protein of 503 amino acids. The deduced amino acid sequence shares high homology, conserved amino acid residues and sequence motifs crucial for the function with the BADHs in other leguminous species. The sequence of the open reading frame was used to construct a prokaryotic expression vector pET32a-AnBADH, and transform Escherichia coli. The transformants expressed the heterologous AnBADH gene under the induction of isopropyl ß-D-thiogalactopyranoside, and demonstrated significant enhancement of salt and heat tolerance under the stress conditions of 700 mmol L(-1) NaCl and 55°C high temperature. This result suggests that the AnBADH gene might play a crucial role in adaption of A. nanus to the abiotic stresses, and have the potential to be applied to transgenic operations of commercially important crops for improvement of abiotic tolerance.

Genomovar assignment of Pseudomonas stutzeri populations inhabiting produced oil reservoirs.

Oil reservoirs are specific habitats for the survival and growth of microorganisms in general. Pseudomonas stutzeri which is believed to be an exogenous organism inoculated into oil reservoirs during the process of oil production was detected frequently in samples from oil reservoirs. Very little is known, however, about the distribution and genetic structure of P. stutzeri in the special environment of oil reservoirs. In this study, we collected 59 P. stutzeri 16S rRNA gene sequences that were identified in 42 samples from 25 different oil reservoirs and we isolated 11 cultured strains from two representative oil reservoirs aiming to analyze the diversity and genomovar assignment of the species in oil reservoirs. High diversity of P. stutzeri was observed, which was exemplified in the detection of sequences assigned to four known genomovars 1, 2, 3, 20 and eight unknown genomic groups of P. stutzeri. The frequent detection and predominance of strains belonging to genomovar 1 in most of the oil reservoirs under study indicated an association of genomovars of P. stutzeri with the oil field environments.