Publications by authors named "Kai Peng"

57 Publications

Long-Term Efficacy of Ultrasound-Guided Percutaneous Laser Ablation for Low-Risk Papillary Thyroid Microcarcinoma: A 5-Year Follow-Up Study.

Biomed Res Int 2021 23;2021:6616826. Epub 2021 Jul 23.

Department of Ultrasound, The Third Xiangya Hospital, Central South University, Changsha, Hunan, China 410013.

Objective: To evaluate the long-term efficacy and safety of ultrasound-guided percutaneous laser ablation (PLA) for the treatment of low-risk papillary thyroid microcarcinoma (PTMC).

Methods: From June 2012 to May 2015, 105 patients with solitary, pathologically confirmed PTMC lesions were treated with ultrasound-guided PLA. Nodule location, nodule volume, thyroid function, and clinical symptoms were evaluated before ablation. Contrast-enhanced ultrasound (CEUS) was performed 1 h after treatment to evaluate whether the ablation was complete. Ultrasound examination was performed at 1, 3, 6, and 12 months after ablation and every 6 months thereafter to determine the size of the ablation area and search for recurrence in the thyroid parenchyma and lymph node metastasis. Thyroid function was examined before and 1 month after ablation. Fine needle aspiration biopsy was performed for any suspicious metastatic lymph nodes and recurrent lesions in the thyroid.

Results: All 105 lesions were completely inactivated after one ablation, making the success rate for single ablation 100%. The average ablation time was 2.78 ± 1.05 min, and the average ablation energy was 505 ± 185 J. All patients could tolerate and complete the ablation. No serious complications occurred during the treatment; only minor side effects such as pain and local discomfort were reported. The volume reduction rates were -781.14 ± 653.29% at 1 h posttreatment and -268.65 ± 179.57%, -98.39 ± 76.58%, 36.78 ± 30.32%, 75.55 ± 21.81%, 96.79 ± 10.57%, and 100% at 1, 3, 6, 12, 18, and 24 months after ablation, respectively. This rate remained 100% at the later follow-up times. Overall, 28 (26.67%), 74 (70.48%), 96 (91.43%), and 103 (100%) were completely absorbed by 6, 12, 18, and 24 months after PLA. One patient developed another lesion 12 months after ablation, and two patients had central cervical lymph node metastasis 24 months after ablation.

Conclusion: PLA is a safe and effective alternative clinical treatment for low-risk PTMC.
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http://dx.doi.org/10.1155/2021/6616826DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8324339PMC
July 2021

[Spatio-temporal Distribution Characteristics and Driving Factors of Zooplankton in Hongze Lake].

Huan Jing Ke Xue 2021 Aug;42(8):3753-3762

School of Ecology and Environment, Anhui Normal University, Wuhu 241002, China.

Hongze Lake is the fourth largest freshwater lake in China and is an important source of water for surrounding industrial and agricultural processes and fishery resources. Analyzing the changes in the zooplankton community structure in Hongze Lake can provide scientific support for the scientific management of its ecology and environment. A one-year monthly monitoring study was conducted from March 2017 to February 2018 to analyze the temporal and spatial changes in species composition, density, and biomass of zooplankton in Hongze Lake, as well as the seasonal changes in community diversity and dominant species. Canonical correspondence analysis was employed to explore the relationships between the temporal and spatial changes in zooplankton and the environmental factors of Hongze Lake. The results showed that the average annual density of zooplankton in Hongze Lake was 383.87 ind ·L, and the average annual biomass was 1.36 mg ·L. The community structure of zooplankton in Hongze Lake varied greatly across time and space. Community structure varied greatly in summer, and zooplankton density and biomass reached a maximum in autumn. The community structure of the zooplankton was the simplest in winter. Chengzi Bay and Lihewa Bay exhibited an abundance of many different zooplankton species with limited spatial differentiation, whereas the zooplankton in the overflow area comprised fewer species but exhibited greater spatial variation. In summer, water level and temperature are the dominant factors, whereas in autumn and winter, the dominant factors are water temperature, nutrients, and chlorophyll. Canonical correspondence analysis showed that the temporal and spatial changes in zooplankton community structure in Hongze Lake were mainly determined by water level, total phosphorus, water temperature, and total nitrogen content. Water level fluctuation has the greatest direct impact on zooplankton community structure, and water quality regulation has indirect impact.
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http://dx.doi.org/10.13227/j.hjkx.202010195DOI Listing
August 2021

Yeast Synthetic Minimal Biosensors for Evaluating Protein Production.

ACS Synth Biol 2021 07 14;10(7):1640-1650. Epub 2021 Jun 14.

ARC Centre of Excellence in Synthetic Biology, Department of Molecular Sciences, Macquarie University, Sydney, NSW 2109, Australia.

The unfolded protein response (UPR) is a highly conserved cellular response in eukaryotic cells to counteract endoplasmic reticulum (ER) stress, typically triggered by unfolded protein accumulation. In addition to its relevance to human diseases like cancer, the induction of the UPR has a significant impact on the recombinant protein production in eukaryotic cell factories, including the industrial workhorse. Being able to accurately detect and measure this ER stress response in single cells, enables the rapid optimization of protein production conditions and high-throughput strain selection strategies. Current methodologies to monitor the UPR in are often temporally and spatially removed from the cultivation stage or lack updated systematic evaluation. To this end, we constructed and systematically evaluated a series of high-throughput UPR sensors by different designs, incorporating either yeast native UPR promoters or novel synthetic minimal UPR promoters. The native promoters of and were identified to have suitable UPR biosensor properties and served as an expression level guide for orthogonal sensor benchmarking. Our best synthetic minimal sensor is only 98 bp in length, has minimal homology to other native yeast sequences and displayed superior sensor characteristics. The synthetic minimal UPR sensor was able to accurately distinguish between cells expressing different heterologous proteins and between the different secretion levels of the same protein. This work demonstrated the potential of synthetic UPR biosensors as high-throughput tools to predict the protein production capacity of strains, interrogate protein properties hampering their secretion, and guide rational engineering strategies for optimal heterologous protein production.
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http://dx.doi.org/10.1021/acssynbio.0c00633DOI Listing
July 2021

Characterization of novel ISAba1-bounded tet(X15)-bearing composite transposon Tn6866 in Acinetobacter variabilis.

J Antimicrob Chemother 2021 Aug;76(9):2481-2483

Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, College of Veterinary Medicine, Yangzhou University, Yangzhou, China.

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http://dx.doi.org/10.1093/jac/dkab182DOI Listing
August 2021

Identification of a Novel Plasmid-Mediated Carbapenemase-Encoding Gene, , in Vibrio diabolicus.

Antimicrob Agents Chemother 2021 07 16;65(8):e0020621. Epub 2021 Jul 16.

Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, College of Veterinary Medicine, Yangzhou University, Yangzhou, Jiangsu Province, People's Republic of China.

Here, we characterized a carbapenem-resistant Vibrio diabolicus strain of shrimp origin with various experiments and bioinformatics analysis. A novel metallo-β-lactamase (MBL) gene, , that confers resistance to β-lactams, including meropenem and cephalosporins, was identified on a plasmid-borne composite transposon, IS-IS---IS, which is capable of generating a -bearing circular intermediate. IS was found to be disseminated in multidrug-resistant (MDR) pathogens, arousing the concern of further transmission of a -bearing circular intermediate to clinical Enterobacterales strains via such insertion sequences, which warrants further investigations.
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http://dx.doi.org/10.1128/AAC.00206-21DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8284434PMC
July 2021

CRKL is a critical target of Hh-GLI2 pathway in lung adenocarcinoma.

J Cell Mol Med 2021 Jun 2. Epub 2021 Jun 2.

Department of Respiratory and Critical Care, The First Affiliated Hospital of Nanchang University, Nanchang, China.

Lung adenocarcinoma (LUAD) is one of the important components of non-small-cell lung cancer (NSCLC) and leads to many deaths every year. During the initiation and progression of the LUAD, the Hh-GLI2 pathway plays critical roles. Several components of this pathway have been shown to be amplified or overexpressed in LUAD, providing this pathway as an attractive target for therapeutics. However, a gap in our understanding of the Hh-GLI2 pathway is the identity of transcriptional targets of GLI2 that drive LUAD tumorigenesis. Here, we show that the oncogene CRKL is a direct target of GLI2. GLI2 turns on CRKL transcription through binding its second intron. Furthermore, CRKL is an essential mediator for GLI2-driven proliferation and migration of LUAD cells. Depletion of CRKL blunts Hh-GLI2 pathway-mediated cell proliferation and invasion. Lastly, we find that CRKL knockout cells are more sensitive to EGFR-TKI and chemotherapeutics. Taken together, our work here identifies a specific target for Hh-related malignancies and provides CRKL as a promising therapeutic target for LUAD.
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http://dx.doi.org/10.1111/jcmm.16592DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8256351PMC
June 2021

Chromosome-level genome of Himalayan yew provides insights into the origin and evolution of the paclitaxel biosynthetic pathway.

Mol Plant 2021 Jul 2;14(7):1199-1209. Epub 2021 May 2.

Key Laboratory of Systems Microbial Biotechnology, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China; National Center of Technology Innovation for Synthetic Biology, Tianjin 300308, China.

Taxus, commonly known as yew, is a well-known gymnosperm with great ornamental and medicinal value. In this study, by assembling a chromosome-level genome of the Himalayan yew (Taxus wallichiana) with 10.9 Gb in 12 chromosomes, we revealed that tandem duplication acts as the driving force of gene family evolution in the yew genome, resulting in the main genes for paclitaxel biosynthesis, i.e. those encoding the taxadiene synthase, P450s, and transferases, being clustered on the same chromosome. The tandem duplication may also provide genetic resources for the nature to sculpt the core structure of taxoids at different positions and subsequently establish the complex pathway of paclitaxel by neofunctionalization. Furthermore, we confirmed that there are two genes in the cluster encoding isoenzymes of a known enzyme in the paclitaxel biosynthetic pathway. The reference genome of the Himalayan yew will serve as a platform for decoding the complete biosynthetic pathway of paclitaxel and understanding the chemodiversity of taxoids in gymnosperms.
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http://dx.doi.org/10.1016/j.molp.2021.04.015DOI Listing
July 2021

Pt-based (Zn, Cu) nanodendrites with enhanced catalytic efficiency and durability toward methanol electro-oxidation via trace Ir-doping engineering.

J Colloid Interface Sci 2021 Sep 9;598:126-135. Epub 2021 Apr 9.

Institute for Energy Research, Jiangsu University, 301 Xuefu Road, Zhenjiang 212013, PR China. Electronic address:

Pt-based alloy nanomaterials with nanodendrites (NDs) structures are efficient electrocatalysts for methanol oxidation reaction (MOR), however their durability is greatly limited by the issue of transition metals dissolution. In this work, a facile trace Ir-doping strategy was proposed to fabricate Ir-PtZn and Ir-PtCu alloy NDs catalysts in aqueous medium, which significantly improved the electrocatalytic activity and durability for MOR. The as-prepared Ir-PtZn/Cu NDs catalysts showed distinct dendrites structures with the averaged diameter of 4.1 nm, and trace Ir doping subsequently improved the utilization of Pt atoms and promoted the oxidation efficiency of methanol. The electrochemical characterizations further demonstrated that the obtained Ir-PtZn/Cu NDs possessed enhanced mass activities of nearly 1.23 and 1.28-fold higher than those of undoped PtZn and PtCu, and approximately 2.35 and 2.67-fold higher than that of Pt/C in acid medium. More excitingly, after long-term durability test, the proposed Ir-PtZn and Ir-PtCu NDs still retained about 88.9% and 91.6% of its initial mass activities, which further highlights the key role of Ir-doping in determining catalyst performance. This work suggests that trace Ir-doping engineering could be a promising way to develop advanced electrocatalysts toward MOR for direct methanol fuel cell (DMFC) applications.
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http://dx.doi.org/10.1016/j.jcis.2021.04.014DOI Listing
September 2021

Characterization of TMexCD3-TOprJ3, an RND-Type Efflux System Conferring Resistance to Tigecycline in Proteus mirabilis, and Its Associated Integrative Conjugative Element.

Antimicrob Agents Chemother 2021 06 17;65(7):e0271220. Epub 2021 Jun 17.

Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, College of Veterinary Medicine, Yangzhou University, Yangzhou, Jiangsu Province, People's Republic of China.

The emergence and transmission of novel antimicrobial resistance genes pose a great threat to public health globally. Recently, the plasmid-encoding RND efflux pump TMexCD1-TOprJ1 in Klebsiella pneumoniae was reported to reduce the sensitivity of multiple antimicrobials. Here, we identified a pandrug-resistant Proteus mirabilis isolate that harbored the novel gene cluster located on SXT/R391 ICE. This study expands current knowledge of the transfer mechanisms of like gene clusters among P. mirabilis isolates and warrants further genomic epidemiology investigations.
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http://dx.doi.org/10.1128/AAC.02712-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8218640PMC
June 2021

HDAC1 and 2 regulate endothelial VCAM-1 expression and atherogenesis by suppressing methylation of the promoter.

Theranostics 2021 20;11(11):5605-5619. Epub 2021 Mar 20.

Key Laboratory of Targeted Intervention of Cardiovascular Disease, Collaborative Innovation Center for Cardiovascular Disease Translational Medicine, Nanjing Medical University, Nanjing, China.

Increased expression of vascular cell adhesion molecule (VCAM)-1 on the activated arterial endothelial cell (EC) surface critically contributes to atherosclerosis which may in part be regulated by epigenetic mechanisms. This study investigated whether and how the clinically available histone deacetylases 1 and 2 (HDAC1/2) inhibitor drug Romidepsin epigenetically modulates VCAM-1 expression to suppress atherosclerosis. VCAM-1 expression was analyzed in primary human aortic EC (HAEC) treated with Romidepsin or transfected with HDAC1/2-targeting siRNA. Methylation of GATA6 promoter region was examined with methylation-specific PCR assay. Enrichment of STAT3 to GATA6 promoter was detected with chromatin immunoprecipitation. Lys685Arg mutation was constructed to block STAT3 acetylation. The potential therapeutic effect of Romidepsin on atherosclerosis was evaluated in mice fed with a high-fat diet. Romidepsin significantly attenuated TNFα-induced VCAM-1 expression on HAEC surface and monocyte adhesion through simultaneous inhibition of HDAC1/2. This downregulation of VCAM-1 was attributable to reduced expression of transcription factor GATA6. Romidepsin enhanced STAT3 acetylation and its binding to DNA methyltransferase 1 (DNMT1), leading to hypermethylation of the promoter CpG-rich region at +140/+255. Blocking STAT3 acetylation at Lys685 disrupted DNMT1-STAT3 interaction, decreased promoter methylation, and reversed the suppressive effects of HDAC1/2 inhibition on GATA6 and VCAM-1 expression. Finally, intraperitoneal administration of Romidepsin reduced diet-induced atherosclerotic lesion development in mice, accompanied by a reduction in GATA6/VCAM-1 expression in the aorta. HDAC1/2 contributes to VCAM-1 expression and atherosclerosis by suppressing STAT3 acetylation-dependent promoter methylation. These findings may provide a rationale for HDAC1/2-targeting therapy in atherosclerotic heart disease.
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http://dx.doi.org/10.7150/thno.55878DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8039941PMC
July 2021

Identification of Two Plasmids Coharboring Carbapenemase Genes and in Clinical Klebsiella pneumoniae ST2667.

Antimicrob Agents Chemother 2021 05 18;65(6). Epub 2021 May 18.

Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, College of Veterinary Medicine, Yangzhou University, Yangzhou, Jiangsu, China

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http://dx.doi.org/10.1128/AAC.00625-21DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8316132PMC
May 2021

Emergence of a multidrug resistance efflux pump with carbapenem resistance gene blaVIM-2 in a Pseudomonas putida megaplasmid of migratory bird origin.

J Antimicrob Chemother 2021 05;76(6):1455-1458

College of Veterinary Medicine, Yangzhou University, Yangzhou, Jiangsu Province, P. R. China.

Background: Tigecycline and carbapenems are regarded as vital antimicrobials to treat serious bacterial infections. Co-occurrence of resistance genes conferring resistance to both tigecycline and carbapenems in Pseudomonas spp. was not investigated.

Objectives: To characterize a megaplasmid co-harbouring tmexCD1-toprJ1 and blaVIM-2 in Pseudomonas putida of migratory bird origin.

Methods: One tigecycline- and carbapenem-resistant strain was isolated and characterized by antimicrobial susceptibility testing, conjugation assay, WGS and bioinformatics analysis.

Results: The strain P. putida ZXPA-20 resistant to meropenem and tigecycline was positive for blaVIM-2 and tmexCD1-toprJ1 genes. The gene blaVIM-2 was inserted into the backbone of the megaplasmid pZXPA-20 within a Tn5090-like structure. The genetic context of tmexCD1-toprJ1 in the megaplasmid was identical to many chromosomal tmexCD1-toprJ1 of Pseudomonas species. Plasmid-mediated tmexCD1-toprJ1 gene cluster in Pseudomonas spp. was more common than that in Klebsiella pneumoniae. To the best of our knowledge, this is the first report of co-occurrence of blaVIM-2 and tmexCD1-toprJ1 in one plasmid.

Conclusions: Emergence of plasmid-mediated carbapenem and tigecycline resistance genes in P. putida from migratory birds highlighted the importance of surveillance of novel mobile resistance genes in migratory birds, which may play a vital role in global transmission of novel resistance genes.
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http://dx.doi.org/10.1093/jac/dkab044DOI Listing
May 2021

Effects of added calcium-based additives on swine manure derived biochar characteristics and heavy metals immobilization.

Waste Manag 2021 Mar 8;123:69-79. Epub 2021 Feb 8.

School of Chemistry and Chemical Engineering, Huaiyin Normal University, Huai'an 223300, China. Electronic address:

Although pyrolysis is a promising way for treating animal manure, the application is restricted with some limitations of biochar. To improve the quality of biochar derived from swine manure and enhance the immobilization of heavy metals (Cu and Zn) in it, swine manure was mixed with four types of Ca-based additives (CaO, CaCO, Ca(OH), and Ca(HPO)) prior to pyrolysis at 300-700 °C. The thermogravimetric characteristics of swine manure were obviously influenced The addition of CaO, CaCO, and Ca(OH) during the whole decomposition process. Furthermore, with the addition of CaO and Ca(OH), the emission of CO and CO was substantially decreased at 200-500 °C, whereas the formation of CO, H, CO, and CH was drastically increased at 600-800 °C. The biochar produced with CaO addition had the highest pH, surface area and carbon content. Moreover, by addition of Ca-based additives, except for Ca(HPO), the transformation of labile Cu and Zn to the stable fraction was promoted, and the leachability and environmental risk of them were simultaneously reduced. In contrast, CaO and Ca(OH) were more favorable for the immobilization of Cu and Zn than CaCO. Our study indicated that the catalytic pyrolysis using CaO was an effective and valuable method of animal manure treatment.
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http://dx.doi.org/10.1016/j.wasman.2021.01.020DOI Listing
March 2021

The complete mitochondrial genome of (Diptera, Chironomidae).

Mitochondrial DNA B Resour 2019 Nov 12;4(2):3983-3984. Epub 2019 Nov 12.

State key laboratory of Lake Science and Environment, Nanjing Institute of Geography and Limnology, Chinese Academy of Science, Nanjing, People's Republic of China.

is one of the most important genera of Chironomidae. However, the systemically classification and taxonomic studies have so far been limited. In this study, we report the complete mitochondrial genome sequence of . . The mitogenome has 15,632 base pairs (77.58% A + T content) and made up of total of 37 genes (13 protein-coding, 22 transfer RNAs, and 2 ribosomal RNAs). This study will provide useful genetic information for future phylogenetic and taxonomic classification of Chironomidae.
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http://dx.doi.org/10.1080/23802359.2019.1688703DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7707779PMC
November 2019

Synthesis of Ligustrazine from Acetaldehyde by a Combined Biological-Chemical Approach.

ACS Synth Biol 2020 11 6;9(11):2902-2908. Epub 2020 Nov 6.

Key Laboratory of Systems Microbial Biotechnology, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, China.

Ligustrazine is an important active alkaloid in medicine and in the food industry. Here, we developed a combined biological-chemical approach to produce ligustrazine from acetaldehyde. First, we constructed a whole-cell biocatalytic system to produce the precursor acetoin from acetaldehyde by overexpressing formolase (FLS). Second, a two-step strategy was developed to enhance protein expression of FLS by codon usage optimization at the first 14 codons and the introduction of an overlapping gene before the start codon. Through expression optimization and directed evolution of FLS, we improved the titer of acetoin about 40 fold when the concentration of acetaldehyde was 1.5 M. Finally, after reaction conditions optimization, the titer of acetoin and ligustrazine reached 222 g L and 94 g L, with a 86.5% and 48% conversion rate from acetaldehyde, respectively. The developed one-pot synthesis for acetoin and ligustrazine is expected to be applied to industrial production in the future with the advantages of a green process, high efficiency, and low cost.
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http://dx.doi.org/10.1021/acssynbio.0c00113DOI Listing
November 2020

Conserving purple prairie clover (Dalea purpurea Vent.) as hay and silage had little effect on the efficacy of condensed tannins in modulating ruminal fermentation in vitro.

J Sci Food Agric 2021 Feb 20;101(3):1247-1254. Epub 2020 Nov 20.

Agriculture and Agri-Food Canada, Lethbridge Research and Development Centre, Lethbridge, Alberta, Canada.

Background: The objective of this study was to determine the effects of forage conservation method and condensed tannins (CT) in conserved forage on rumen fermentation. Purple prairie clover (PPC; Dalea purpurea Vent.) containing 84.5 g kg dry matter (DM) of CT was harvested at full flower and preserved as freeze-dried green chop (F), hay (H) or silage (S). Batch culture incubations were conducted using conserved forages as a substrate and treatments were arranged as 3 × 2 factorial design of forage type (F, H and S) with or without polyethylene glycol (PEG). PEG was used to isolate the effects of CT on fermentation. Incubation was repeated twice with quadruplicate vials for each treatment in each incubation. N-labelled ammonium sulfate was used as microbial N marker and headspace gas was sampled to determine methane production.

Results: Concentrations of neutral detergent fiber (NDF) and acid detergent fiber were lower (P < 0.01) in F than in H or S. Ensiling decreased (P < 0.001) total phenolics and extractable CT, but had no effect on total CT, whereas none of these phenolic fractions were altered in H. Hay and silage had lower (P < 0.01) true DM disappearance (TDMD) and NDF disappearance (NDFD) than F. Inclusion of PEG did not affect TDMD or NDFD after 8, 24 or 72 h of incubation. Productions of total gas, methane and total volatile fatty acid and the rate of gas production (c) were also similar, but ammonia was higher (P < 0.05) and microbial N was lower (P < 0.05) with than without PEG after 72 h of incubation. After 72 h, a lower (P < 0.001) proportion of acetate but higher (P < 0.05) proportion of propionate was noted with S, resulting in a lower (P < 0.001) acetate:propionate ratio as compared to F or H after 8 h of incubation.

Conclusion: Condensed tannins in PPC decreased protein degradation in vitro, but had minimal effects on overall rumen fermentation, and conservation of PPC as hay or silage had little effect on the efficacy of CT in modulating rumen fermentation. © 2020 Her Majesty the Queen in Right of Canada. Journal of The Science of Food and Agriculture © 2020 Society of Chemical Industry.
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http://dx.doi.org/10.1002/jsfa.10913DOI Listing
February 2021

Microdroplet enabled cultivation of single yeast cells correlates with bulk growth and reveals subpopulation phenomena.

Biotechnol Bioeng 2021 02 21;118(2):647-658. Epub 2020 Oct 21.

School of Engineering, Macquarie University, Sydney, NSW, Australia.

Yeast has been engineered for cost-effective organic acid production through metabolic engineering and synthetic biology techniques. However, cell growth assays in these processes were performed in bulk at the population level, thus obscuring the dynamics of rare single cells exhibiting beneficial traits. Here, we introduce the use of monodisperse picolitre droplets as bioreactors to cultivate yeast at the single-cell level. We investigated the effect of acid stress on growth and the effect of potassium ions on propionic acid tolerance for single yeast cells of different species, genotypes, and phenotypes. The results showed that the average growth of single yeast cells in microdroplets experiences the same trend to those of yeast populations grown in bulk, and microdroplet compartments do not significantly affect cell viability. This approach offers the prospect of detecting cell-to-cell variations in growth and physiology and is expected to be applied for the engineering of yeast to produce value-added bioproducts.
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http://dx.doi.org/10.1002/bit.27591DOI Listing
February 2021

Emergence of Carbapenem- and Tigecycline-Resistant of Animal Origin.

Front Microbiol 2020 14;11:1940. Epub 2020 Aug 14.

College of Veterinary Medicine, Yangzhou University, Yangzhou, China.

The emergence of (X) and carbapenemase genes in Enterobacterales pose significant challenges to the treatment of infectious diseases. Convergence of these two categories of genes in an individual pathogen would deteriorate the antimicrobial resistance (AMR) crisis furthermore. Here, tigecycline-resistant Enterobacterales strains were isolated and detected with carbapenemase genes, characterized by antimicrobial susceptibility testing, PCR, conjugation assay, whole genome sequencing, and bioinformatics analysis. Three tigecycline-resistant isolates consisting of one plasmid-mediated (X4)-bearing and two chromosomal (X6)-bearing were recovered from chicken feces. The (X4) was located on a conjugative IncX1 plasmid pHNCF11W-tetX4 encoding the identical structure as reported (X4)-bearing IncX1 plasmids in . Among two strains, (X6) was located on two similar chromosomal MDR regions with genetic contexts IS---IS--(X6)---IS---IS-- and IS---IS--(X6)---IS--. Apart from (X6), HNCF44W harbored a novel transposon Tn positive for on a conjugative plasmid. This study probed the genomic basis of three (X)-bearing, tigecycline-resistant strains, one of which coharbored and (X6), and identified as the important reservoir of (X6) variants. Emergence of encoding both and (X6) reveals a potential public health risk.
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http://dx.doi.org/10.3389/fmicb.2020.01940DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7457074PMC
August 2020

Quantifying the effects of climate change on hydrological regime and stream biota in a groundwater-dominated catchment: A modelling approach combining SWAT-MODFLOW with flow-biota empirical models.

Sci Total Environ 2020 Nov 17;745:140933. Epub 2020 Jul 17.

Department of Bioscience, Aarhus University, Silkeborg, Denmark; Sino-Danish Centre for Education and Research, Beijing, China.

Climate change may affect stream ecosystems through flow regime alterations, which can be particularly complex in streams with a significant groundwater contribution. To quantify the impacts of climate change on hydrological regime and subsequently the stream biota, we linked SWAT-MODFLOW (A model coupling the Soil and Water Assessment Tool and the Modular Finite-difference Flow Model) with flow-biota empirical models that included indices for three key biological taxonomic identities (fish, macroinvertebrates and macrophytes) and applied the model-complex to a groundwater-dominated catchment in Denmark. Effects of predicted climate change towards the end of this century relative to the reference period (1996-2005) were tested with two contrasting climate change scenarios of different greenhouse gas emissions (Representative Concentration Pathway 2.6 (RCP 2.6) and RCP 8.5) and analysed for all subbasins grouped into streams of three size classes. The total water yield in the catchment did not change significantly (-1 ± 4 (SD) mm yr) from the baseline in the RCP2.6 scenario, while it increased by 9 ± 11 mm yr in the RCP8.5 scenario. The three stream size classes underwent different alterations in flow regime and also demonstrated different biotic responses to climate change. All large and some small streams were impacted most heavily by the climate change, where fish and macrophyte indices decreased up to 14.4% and 11.2%, respectively, whereas these indices increased by up to 14.4% and 6.0%, respectively, in the medium and some small streams. The climate change effects were, as expected, larger in the RCP8.5 scenario than in the RCP2.6 scenario. Our study is the first to quantify the impacts of streamflow alterations induced by climate change on stream biota beyond specific species.
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http://dx.doi.org/10.1016/j.scitotenv.2020.140933DOI Listing
November 2020

[Succession Pattern of Phytoplankton and Its Drivers in Lake Luoma, Jiangsu Province].

Huan Jing Ke Xue 2020 Apr;41(4):1648-1656

State Key Laboratory of Lake Science and Environment, Nanjing Institute of Geography and Limnology, Chinese Academy of Sciences, Nanjing 210008, China.

As a water storage lake for the South-to-North Water Diversion Project, it is crucial to examine changes in aquatic ecosystem structures in Lake Luoma, Jiangsu province. Field sampling was carried out in Lake Luoma monthly from 2014 to 2018 to study the relationship between the phytoplankton community structure and environmental factors. During the studied period, total nitrogen, permanganate index, and electrical conductivity in water column gradually increased, whereas fluoride content declined. The pattern of total phosphorus and dissolved oxygen was not distinct. A total of 71 genera of phytoplankton were identified from 2014 to 2018, and the average monthly biomass variation ranged from 0.16 to 5.51 mg·L. Bacillariophyta and Chlorophyta were the dominant phyla in the four years, followed by Pyrrophyta and Cryptophyta. The dominant genera were sp., spp spp., sp., spp. spp., sp. sp. and spp. The results showed that the phytoplankton community structure significantly changed in the four years, which was mainly ascribed to the redistribution of biomass. Specifically, in addition to the dominance of Bacillariophyta and Chlorophyta, the dominance of Pyrrophyta and Cyanophyta increased during the last two years. Non-metric multidimensional scaling analysis showed that variation of the phytoplankton community in Lake Luoma was mainly related to total nitrogen, fluoride, water temperature, total phosphorus, dissolved oxygen, pH, conductivity, and permanganate index, among which the total nitrogen, water temperature, and fluoride concentration dominated the phytoplankton community change after the generalized additive model test. Water temperature is the driving factor affecting seasonal changes of the phytoplankton community. Total nitrogen and fluoride concentrations are the driving factors affecting the interannual variation in the phytoplankton community. Our study indicated that in recent years, the implementation of the ban on sand mining and demolition of the enclosed aquaculture in Lake Luoma has affected the water environment, resulting in a significant succession of the phytoplankton community.
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http://dx.doi.org/10.13227/j.hjkx.201909146DOI Listing
April 2020

Characterization of a Novel -Bearing Plasmid in ST395 of Chicken Origin.

Infect Drug Resist 2020 16;13:1781-1784. Epub 2020 Jun 16.

Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, College of Veterinary Medicine, Yangzhou University, Yangzhou, People's Republic of China.

The emergence of mobile colistin resistance genes undermines the efficacy of colistin as the last-resort drug for multi-drug resistance infections and constitutes a great public health concern. Plasmids play a critical role in the transmission of genes among bacteria. One colistin-resistant strain of chicken origin was collected and analyzed by antimicrobial susceptibility testing, PCR, conjugation assay and S1-PFGE. Whole-genome sequencing (WGS) approach combining Illumina and MinION platforms was utilized to decipher the underlying colistin resistance mechanism and genetic context. A novel -bearing plasmid p2019036D-mcr8-345kb with 345 655 bp in size encoding various resistance genes including , , (D), and was identified responsible for the colistin resistance phenotype. Plasmid comparison has shown that the -bearing plasmid differed from other reported plasmids positive for but shared the same core -bearing conserved region. This study demonstrates the emergence of -bearing of animal origin is a potential risk to humans.
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http://dx.doi.org/10.2147/IDR.S256544DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7305932PMC
June 2020

Transthoracic closure of ventricular septal defects guided by transesophageal echocardiography.

Turk Gogus Kalp Damar Cerrahisi Derg 2019 Jun 22;28(2):250-256. Epub 2020 Apr 22.

Department of Cardiovascular Surgery, Union Hospital, Fujian Medical University, Fuzhou, China.

Background: This study aims to investigate the effectiveness and safety of transthoracic closure of ventricular septal defects totally guided by transesophageal echocardiography.

Methods: A total of 119 patients (62 males, 57 females; mean age 2.1±5.2 years; range, 11 months to 50 years) who underwent transthoracic closure of ventricular septal defects in our center between April 2017 and November 2018 were included. All patients were evaluated in terms of the diameter and morphological features of ventricular septal defects via transesophageal echocardiography. During the procedure, transthoracic echocardiography was used as the only guiding tool for occluder implantation.

Results: Of the patients, 116 underwent successful transthoracic device closure procedure. Two patients were switched to surgical repair due to new-onset aortic regurgitation in one patient and severe arrhythmias after device release in the other patient. One patient underwent a second operation for occluder migration during the hospital stay. In the first attempt, 106 ventricular septal defect occluders were correctly positioned. Transthoracic echocardiography revealed the insecure position or significant residual shunting in 10 patients. The original device was replaced with an asymmetric device or a more extensive occluder, and satisfactory results were finally obtained. No complications such as new-onset aortic regurgitation, residual shunt, complete heart block, or device dislodgement occurred during follow-up.

Conclusion: Transthoracic closure of ventricular septal defects under the total guidance of transthoracic echocardiography is a safe and effective method.
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http://dx.doi.org/10.5606/tgkdc.dergisi.2020.18745DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7298365PMC
June 2019

Exploring tet(X)-bearing tigecycline-resistant bacteria of swine farming environments.

Sci Total Environ 2020 Sep 11;733:139306. Epub 2020 May 11.

College of Veterinary Medicine, Yangzhou University, Yangzhou, Jiangsu Province, PR China; Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, Jiangsu Province, PR China. Electronic address:

Emergence of high-level tigecycline resistance tet(X) variants in animal and human Enterobacterales is posing a public health concern. Recently, novel tet(X) variants including tet(X3), tet(X4), tet(X5) and tet(X6) were detected in Enterobacterales and Acinetobacter baumannii. Here, we comprehensively investigated the prevalence of tet(X) variants among different bacterial species in swine farm environment with nanopore sequencing. The tet(X6) gene was found located on both plasmids and ICEs in Proteus, but tet(X4) was in plasmids in E. coli. To our knowledge, this is the first report of the emergence of IncA/C2-type plasmid-mediated tet(X6). The bacterial host adaptation of different tet(X) variants implies they evolved in microbiota separately, but ISCR2 should be the key element facilitating horizontal transfer of various tet(X) variants through circular intermediates. Our findings further expand the knowledge about reservoirs of mobile tigecycline resistance genes and the epidemic characteristics of tet(X) variants in animals and related environments.
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http://dx.doi.org/10.1016/j.scitotenv.2020.139306DOI Listing
September 2020

Cracking Behavior of René 104 Nickel-Based Superalloy Prepared by Selective Laser Melting Using Different Scanning Strategies.

Materials (Basel) 2020 May 6;13(9). Epub 2020 May 6.

Engineering Research Center of Tribology and Materials Protection, Ministry of Education, Henan University of Science and Technology, Luoyang 71003, China.

Eliminating cracks is a big challenge for the selective laser melting (SLM) process of low-weldable Nickel-based superalloy. In this work, three scanning strategies of the snake, stripe partition, and chessboard partition were utilized to prepare René 104 Ni-based superalloy, of which the cracking behavior and the residual stress were investigated. The results showed that the scanning strategies had significant effects on the cracking, residual stress, and relative density of the SLMed René 104 superalloy. The scanning strategies with more partitions boosted the emergence of cracks, as high-density cracks occurred in these samples. The overlapping zone (OZ) of the scanning partition was also susceptible to cracking, which increased the size, number, and density of the cracks. The cracking performance was relatively moderate in the snake-scanned samples, while that in the chessboard-partition-scanned samples was the most severe. It is concluded that the partition scanning strategies induced more cracks in the SLMed René 104 superalloy, of which the residual stress was apparently reduced. Therefore, it is necessary to design scanning strategies with optimized scanning partitions and overlaps to avoid cracking and acquire a high-quality, near fully dense, low-weldable Nickel-based superalloy using SLM.
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http://dx.doi.org/10.3390/ma13092149DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7254403PMC
May 2020

Deciphering the Structural Diversity and Classification of the Mobile Tigecycline Resistance Gene (X)-Bearing Plasmidome among Bacteria.

mSystems 2020 Apr 28;5(2). Epub 2020 Apr 28.

College of Veterinary Medicine, Yangzhou University, Yangzhou, Jiangsu, People's Republic of China

The emergence of novel plasmid-mediated resistance genes constitutes a great public concern. Recently, mobile (X) variants were reported in diverse pathogens from different sources. However, the diversity of (X)-bearing plasmids remains largely unknown. In this study, the phenotypes and genotypes of all the (X)-positive tigecycline-resistant strains isolated from a slaughterhouse in China were characterized by antimicrobial susceptibility testing, conjugation, pulsed-field gel electrophoresis with S1 nuclease (S1-PFGE), and PCR. The diversity and polymorphism of (X)-harboring strains and plasmidomes were investigated by whole-genome sequencing (WGS) and single-plasmid-molecule analysis. Seventy-four (X4)-harboring strains and one (X6)-bearing strain were identified. The (X4)-bearing elements in 27 strains could be transferred to the recipient strain via plasmids. All (X4)-bearing plasmids isolated in this study and 15 (X4)-bearing plasmids reported online were analyzed. (X4)-bearing plasmids ranged from 9 to 294 kb and were categorized as ColE2-like, IncQ, IncX1, IncA/C2, IncFII, IncFIB, and hybrid plasmids with different replicons. The core (X4)-bearing genetic contexts were divided into four major groups: IS-(X4)-, △IS--(X4)-IS, IS-(X4)-IS--, and -(X4)-IS----IS-- Tandem repeats of (X4) were universally mediated by IS Different (X)-bearing strains existed in the same microbiota. Reorganization of (X4)-bearing multidrug resistance plasmids was found to be mediated by IS and other homologous regions. Finally, single-plasmid-molecule analysis captured the heterogenous state of (X4)-bearing plasmids. These findings significantly expand our knowledge of the (X)-bearing plasmidome among microbiotas, which establishes a baseline for investigating the structure and diversity of human, animal, and environmental tigecycline resistomes. Characterization of (X) genes among different microbiotas should be performed systematically to understand the evolution and ecology. Tigecycline is an expanded-spectrum tetracycline used as a last-resort antimicrobial for treating infections caused by superbugs such as carbapenemase-producing or colistin-resistant pathogens. Emergence of the plasmid-mediated mobile tigecycline resistance gene (X4) created a great public health concern. However, the diversity of (X4)-bearing plasmids and bacteria remains largely uninvestigated. To cover this knowledge gap, we comprehensively identified and characterized the (X)-bearing plasmidome in different sources using advanced sequencing technologies for the first time. The huge diversity of (X4)-bearing mobile elements demonstrates the high level of transmissibility of the (X4) gene among bacteria. It is crucial to enhance stringent surveillance of (X) genes in animal and human pathogens globally.
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http://dx.doi.org/10.1128/mSystems.00134-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7190383PMC
April 2020

Characterization of a porcine Proteus cibarius strain co-harbouring tet(X6) and cfr.

J Antimicrob Chemother 2020 06;75(6):1652-1654

Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, College of Veterinary Medicine, Yangzhou University, Yangzhou, P. R. China.

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http://dx.doi.org/10.1093/jac/dkaa047DOI Listing
June 2020

Water column nutrient concentrations are related to excretion by benthic invertebrates in Lake Taihu, China.

Environ Pollut 2020 Jun 10;261:114161. Epub 2020 Feb 10.

Department of Bioscience, Aarhus University, Vejlsøvej 25, 8600, Silkeborg, Denmark; Sino-Danish Centre for Education and Research (SDC), University of Chinese Academy of Sciences, 100049, Beijing, China; Limnology Laboratory and EKOSAM, Department of Biological Sciences, Middle East Technical University, Ankara, 06800, Turkey.

Internal release of nutrients is an important contributor to the nutrient dynamics in shallow eutrophic lakes. Zoobenthic organisms may contribute to this release by excreting nutrients to the overlaying water. Based on experiments and using results from previous experimental studies as well as field monitoring density data from 2007 to 2017, we calculated the annual and seasonal nutrient excretions of the two most common macroinvertebrates (Corbicula fluminea and Limnodrilus hoffmeisteri) in Lake Taihu, China. We compared these rates with the concentrations of NH-N, total nitrogen (TN), PO-P and total phosphorus (TP) in the lake water as well as with previous results of release rates from undisturbed sediments collected in the lake. The spatial distribution of nutrient excretion by the two invertebrate species varied markedly among sites and years. Regression analyses revealed significant relationships between total nutrient excretions by these two species and the concentrations of NH-N, TN, PO-P and TP in the lake, but with seasonal differences. The relationship was overall strongest in winter, followed by spring, and weakest in summer and autumn. The flux of NH-N and PO-P released by the two macroinvertebrate species were equivalent to as much as 50% and 66%, respectively, of the sediment release recorded in lab experiments under undisturbed conditions; however, the percentages would be somewhat lower under field conditions where the sediment is subjected to frequent wind-induced resuspension and fish disturbance, enhancing the release rates. The release declined during the study period due to a reduction in the density of macroinvertebrates, perhaps indicating increasing stocking of fish since 2007. Our results indicate that benthic invertebrates are important contributor to the internal loading in shallow eutrophic lakes.
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http://dx.doi.org/10.1016/j.envpol.2020.114161DOI Listing
June 2020

IRF-1 mediates the suppressive effects of mTOR inhibition on arterial endothelium.

J Mol Cell Cardiol 2020 03 19;140:30-41. Epub 2020 Feb 19.

Key Laboratory of Targeted Intervention of Cardiovascular Disease, Collaborative Innovation Center for Cardiovascular Disease Translational Medicine, Nanjing Medical University, Nanjing, China; Key laboratory of Human Functional Genomics of Jiang Province, Nanjing, China. Electronic address:

Aims: Mammalian target of rapamycin (mTOR) inhibitors used in drug-eluting stents (DES) to control restenosis have been found to delay endothelialization and increase incidence of late-stent thrombosis through mechanisms not completely understood. We revealed that mTOR inhibition (mTORi) upregulated the expression of cell growth suppressor IRF-1 in primary human arterial endothelial cells (HAEC). This study aimed to examine how mTOR-regulated IRF-1 expression contributes to the suppressive effect of mTORi on arterial endothelial proliferation.

Methods And Results: Western blotting, quantitative PCR, and a dual-luciferase reporter assay indicated that mTOR inhibitors rapamycin and torin 1 upregulated IRF-1 expression and increased its transcriptional activity. IRF-1 in turn contributed to the suppressive effect of mTORi by mediating HAEC apoptosis and cell cycle arrest in part through upregulation of caspase 1 and downregulation of cyclin D3, as revealed by CCK-8 assay, Annexin V binding assay, measurement of activated caspase 3, BrdU incorporation assay, and matrigel tube formation assay. In a mouse model of femoral artery wire injury, administration of rapamycin inhibited EC recovery, an effect alleviated by EC deficiency of IRF-1. Chromatin immunoprecipitation assay with HAEC and rescue expression of wild type or dominant-negative IRF-1 in EC isolated from Irf1 mice confirmed transcriptional regulation of IRF-1 on the expression of CASP1 and CCND3. Furthermore, mTORi activated multiple PKC members, among which PKCζ was responsible for the growth-inhibitory effect on HAEC. Activated PKCζ increased IRF1 transcription through JAK/STAT-1 and NF-κB signaling. Finally, overexpression of wild type or mutant raptor incapable of binding mTOR indicated that mTOR-free raptor contributed to PKCζ activation in mTOR-inhibited HAEC.

Conclusions: The study reveals an IRF-1-mediated mechanism that contributes to the suppressive effects of mTORi on HAEC proliferation. Further study may facilitate the development of effective strategies to reduce the side effects of DES used in coronary interventions.
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http://dx.doi.org/10.1016/j.yjmcc.2020.02.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7138750PMC
March 2020

Effects of inclusion of purple prairie clover ( Vent.) with native cool-season grasses on fermentation and digestibility of mixed forages.

J Anim Sci Biotechnol 2020 14;11:23. Epub 2020 Feb 14.

2Agriculture and Agri-Food Canada, Lethbridge Research and Development Centre, Lethbridge, AB T1J 4B1 Canada.

Background: Incorporation of legume species into native North American pastures is considered an effective method to increase native pasture productivity and improve the nutritive value of forage. This study evaluated the effects of inclusion of purple prairie clover (PPC, Vent.), a native legume forage, with native cool-season grasses on the fermentation and digestibility of mixed forages.

Methods: Whole plant PPC and mixtures of cool-season grasses were harvested when the PPC reached the vegetative (VEG), full flower (FL) and seedpod (SP) stages, and were combined in ratios (DM basis) of 0:100, 25:75, 50:50, 75:25 and 100:0 at each maturity. ruminal incubations using these mixtures were conducted for 48 h to determine gas production (GP), DM disappearance (IVDMD), total volatile fatty acids (VFA) and ammonia-N production. Mixtures of forages harvested when the PPC reached the FL stage and 50:50 mixture of forages harvested at VEG, FL and SP stages were incubated in the rumen of three heifers for 0, 2, 6, 12, 24, 48, 72 and 96 h to determine degradabilities of DM, neutral detergent fibre (aNDF) and crude protein (CP).

Results: Contents of aNDF and ADF increased ( < 0.01), while CP decreased ( < 0.001) as PPC matured. Concentrations of extractable condensed tannins in PPC ranked as FL > VEG > SP ( < 0.05). Regardless of PPC proportions in the mixture, GP decreased ( < 0.05) with increasing PPC maturity. Increasing PPC proportions linearly increased ( < 0.001) GP, IVDMD and total VFA at VEG, but linearly decreased ( < 0.001) them at SP. Irrespective of PPC maturity, ammonia-N production linearly increased ( < 0.01) with increasing proportions of PPC and the concentration was higher ( < 0.05) at VEG than at FL and SP stages. Increasing proportion of PPC at either maturity linearly increased ( < 0.001) molar percentage of acetate (A) and branched-chain VFA, but linearly decreased ( < 0.001) molar percentage of propionate (P), resulting in a linearly increase ( < 0.001) in the A:P ratio. Increasing FL PPC in the mixture linearly and quadratically ( < 0.01) increased (soluble fraction), but linearly and quadratically decreased ( < 0.01) (potentially degradable fraction) for DM and aNDF, resulting in linear ( < 0.05) and quadratic ( < 0.01) increases in DM and aNDF maximum potential degradabilities ( + ). Effective degradabilities of DM and aNDF were also linearly and quadratically increased ( < 0.05), and CP was quadratically increased ( < 0.05) with increasing FL PPC, with the greatest effective degradability being observed with ratios between 50:50 and 75:25. Ruminal maximum potential degradabilities of DM and aNDF decreased ( < 0.001) as the forage matured. Effective degradability of DM ranked as VEG > FL > SP ( < 0.001), whereas the effective degradability of aNDF was similar between VEG and FL and both were greater ( < 0.01) than SP.

Conclusions: Inclusion of vegetative PPC in a mixed forage diet resulted in the greatest digestibility and incorporation of PPC before seedpod stage with native grasses had a positive effect on ruminal fermentation. Effects of PPC on ruminal digestion depend on both the stage of maturity and its proportion in mixed legume-grass pastures. Pastures containing 50% of PPC in full flower stage would likely provide the greatest quality diet to grazing ruminants subject to potential animal selectivity.
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http://dx.doi.org/10.1186/s40104-019-0418-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7020374PMC
February 2020

Reorganization of mcr-1-bearing large MDR plasmids resolved by nanopore sequencing.

J Antimicrob Chemother 2020 06;75(6):1645-1647

College of Veterinary Medicine, Yangzhou University, Yangzhou, Jiangsu Province, P. R. China.

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http://dx.doi.org/10.1093/jac/dkaa046DOI Listing
June 2020
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