Specific, simple and rapid detection of porcine circovirus type 2 using the loop-mediated isothermal amplification method

<p>Abstract</p> <p>Background</p> <p>Porcine circovirus type 2 (PCV2) is the causative agent of postweaning multisystemic wasting syndrome (PMWS), and porcine dermatitis and nephropathy syndrome (PDNS). It has caused heavy losses in global agriculture in recent decades. Rapid detection of PCV2 is very important for the effective prophylaxis and treatment of PMWS.</p> <p>Results</p> <p>A loop-mediated isothermal amplification (LAMP) assay was used to detect PCV2 in this study. Three pairs of primers were specially designed for recognizing eight distinct sequences of the ORF2 gene. This gene lies in the PCV2 virus genome sequence, and encodes the Rep protein that is involved in virus replication. Time and temperature conditions for amplification of PCV2 genes were optimized to be 55 min at 59°C. The analysis of clinical samples indicated that the LAMP method was highly sensitive. The detection limit for PCV2 by the LAMP assay was 10 copies, whereas the limit by conventional PCR was 1000 copies. The assay did not cross-react with PCV1, porcine reproductive and respiratory syndrome virus, porcine epidemic diarrhea virus, transmissible gastroenteritis of pigs virus or rotavirus. When 110 samples were tested using the established LAMP system, 95 were detected as positive.</p> <p>Conclusion</p> <p>The newly developed LAMP detection method for PCV2 was more specific, sensitive, rapid and simple than before. It complements and extends previous methods for PCV2 detection and provides an alternative approach for detection of PCV2.</p

Rapid detection of porcine circovirus type 2 using a TaqMan-based real-time PCR

Porcine circovirus type 2 (PCV2) and the associated disease postweaning multisystemic wasting syndrome (PMWS) have caused heavy losses in global agriculture in recent decades. Rapid detection of PCV2 is very important for the effective prophylaxis and treatment of PMWS. To establish a sensitive, specific assay for the detection and quantitation of PCV2, we designed and synthesized specific primers and a probe in the open reading frame 2. The assay had a wide dynamic range with excellent linearity and reliable reproducibility, and detected between 102 and 1010 copies of the genomic DNA per reaction. The coefficient of variation for Ct values varied from 0.59% to 1.05% in the same assay and from 1.9% to 4.2% in 10 different assays. The assay did not cross-react with porcine circovirus type 1, porcine reproductive and respiratory, porcine epidemic diarrhea, transmissible gastroenteritis of pigs and rotavirus. The limits of detection and quantitation were 10 and 100 copies, respectively. Using the established real-time PCR system, 39 of the 40 samples we tested were detected as positive

Genome Assembly and Annotation of a High-Polymalic Acid (PMLA) Producing Strain Aureobasidium melanogenum CGMCC18996 and Analysis of Its Key Proteins Related to PMLA Synthesis

In this study, we applied PacBio Sequel II and Illumina NovaSeq 6000 sequencing platforms to sequence the genome of a high-polymalic acid (PMLA)-producing strain, Aureobasidium melanogenum CGMCC18996, and used different assemblers to obtain a high-quality genome assembly, which was then annotated using transcriptomic data. The results indicated a total of 6 202 genes were found in the A. melanogenum genome, mainly involved in carbohydrate transport and metabolism, amino acid transport and metabolism, post-translational modification, RNA processing and modification. Meanwhile, functional annotation revealed that most genes in the genome were related to peroxisome in the strain. Transmission electron microscopy (TEM) indicated the existence of a circular peroxisome-like (glyoxysome) structure in the cells, demonstrating the ability to malic acid through the glyoxylate cycle. Finally, we predicted the protein structures of two enzymes related to PMLA biosynthesis, phosphoenolpyruvate carboxykinase (PCKA) and malate synthase (MASY). It was found that the enzymes could have the ability to synthesize malic acid. This study could provide a reference for metabolism regulation in A. melanogenum for improved PMLA production, and the assembled genome has been uploaded to the database, which could provide the basis for the future development and utilization of A. melanogenum CGMCC18996

Self-assembly of activated lipase hybrid nanoflowers with superior activity and enhanced stability

Lipase-inorganic hybrid nanoflowers were prepared using Ca3(PO4)2 as the inorganic component and lipase from Aspergillus oryzae (A. oryzae) as the organic component. The influences of metal ions with different valence, various additives (surfactant), and synthesis conditions on the activity of the lipase hybrid nanoflowers were systematically investigated. Results revealed that the valence state of metal ions played an important role on the shape and activity of lipase hybrid nanoflowers. The synthesized lipase hybrid nanoflowers using bivalence metal ions (Ca2+, Mn2+, and Zn2+) as the inorganic components exhibited relative high activity. However, very low activities were observed in the lipase hybrid nanoflowers using univalent metal ions (Ag+) or trivalent metal ions (Al3+, Fe3+). More importantly, Ca2+ not only induced self-assemble of lipase hybrid nanoflowers, but also activated the enzyme activity by inducing conformational changes in lipase from A. oryzae. As a result, lipase/Ca3(PO4)2 hybrid nanoflowers (hNF-lipase) exhibited the high activity. The hNF-lipase displayed 9, 12, and 61 folds higher activity than lipase/Ag3PO4 hybrid nanoflowers, lipase/AlPO4 hybrid nanoflowers, and lipase/FePO4 nanoflowers, respectively. Compared with free lipase, the hNF-lipase displayed 172 % increase in activities by using 0.15 mM Tween-80 as an activity inducer (activated hNF-lipase). Furthermore, the hNF-lipase and activated hNF-lipase exhibited increased stability against high temperature and denaturant, and had good storage stability and reusability

Impact of opioid-free analgesia on pain severity and patient satisfaction after discharge from surgery: multispecialty, prospective cohort study in 25 countries

Background: Balancing opioid stewardship and the need for adequate analgesia following discharge after surgery is challenging. This study aimed to compare the outcomes for patients discharged with opioid versus opioid-free analgesia after common surgical procedures.Methods: This international, multicentre, prospective cohort study collected data from patients undergoing common acute and elective general surgical, urological, gynaecological, and orthopaedic procedures. The primary outcomes were patient-reported time in severe pain measured on a numerical analogue scale from 0 to 100% and patient-reported satisfaction with pain relief during the first week following discharge. Data were collected by in-hospital chart review and patient telephone interview 1 week after discharge.Results: The study recruited 4273 patients from 144 centres in 25 countries; 1311 patients (30.7%) were prescribed opioid analgesia at discharge. Patients reported being in severe pain for 10 (i.q.r. 1-30)% of the first week after discharge and rated satisfaction with analgesia as 90 (i.q.r. 80-100) of 100. After adjustment for confounders, opioid analgesia on discharge was independently associated with increased pain severity (risk ratio 1.52, 95% c.i. 1.31 to 1.76; P &lt; 0.001) and re-presentation to healthcare providers owing to side-effects of medication (OR 2.38, 95% c.i. 1.36 to 4.17; P = 0.004), but not with satisfaction with analgesia (beta coefficient 0.92, 95% c.i. -1.52 to 3.36; P = 0.468) compared with opioid-free analgesia. Although opioid prescribing varied greatly between high-income and low- and middle-income countries, patient-reported outcomes did not.Conclusion: Opioid analgesia prescription on surgical discharge is associated with a higher risk of re-presentation owing to side-effects of medication and increased patient-reported pain, but not with changes in patient-reported satisfaction. Opioid-free discharge analgesia should be adopted routinely

Prediction and Evaluation of Ecosystem Service Value Based on Land Use of the Yellow River Source Area

Land-use change plays an important role in ecological change; knowing the trends in land-use change can quickly help identify problems in regional ecosystems. In 2000 to 2020, the development of a global economy caused increasing extreme weather events worldwide and lead to exacerbating changes in types of land-use. The Yellow River source area is an important water source and a central part of ecological protection efforts in China. The fragile ecosystems make the area sensitive to environmental changes. Therefore, in protecting the ecological security of the basin, simulating changes in the ecosystem service value under different scenarios is a meaningful procedure. A patch-generating land use simulation model was used to simulate different land use scenarios in 2030, including an ecological protection scenario, a production priority scenario, a carbon neutral scenario and a natural development scenario. The analysis shows that significant progress has been made in water conservation but grassland conservation faces enormous challenges. The rate of development, occupation of farmland and land dedicated to construction has increased. Unused land increased dramatically from 2010&ndash;2020 and has not been mitigated by existing policies. Based on the unit area value equivalent coefficients, the ecosystem service value rankings for the seven land use types were as follows: Grassland &gt; Wetland &gt; Water Area &gt; Forest &gt; Farmland &gt; Unused Land &gt; Construction Land; the four types of ecosystem service value are ranked as follows: regulating services &gt; supporting services &gt; supply services &gt; cultural services; the four scenarios of ecosystem service value are ranked as follows: ecological protection scenario &gt; production priority scenario &gt; carbon neutral scenario &gt; natural development scenario. The ecosystem service value of the Yellow River source area would increase by CNY 1.641 billion in 2030 with ecological protection goals and decrease by CNY 1.421 billion with the current of development. This study provides valuable insights and implications for land use, ecological protection and sustainable development by shedding light on watershed change issues and assessing and predicting the ecological status of the Yellow River source area

Exploring the Relationship between the Eco-Environmental Quality and Urbanization by Utilizing Sentinel and Landsat Data: A Case Study of the Yellow River Basin

Yellow River Basin urban agglomeration (YRBU) is the main carrier of regional socio-economic development in the Yellow River Basin, and its eco-environmental quality, urbanization, and coupling coordination degree are facing higher demands. It is of great significance for the development of YRBU to understand the interactive coupling relationship between the eco-environment and urbanization development from the multi-scale perspective. This research intended to understand the spatio-temporal characteristics of eco-environmental quality, urbanization, and coupling coordination degree in the study area from 2013 to 2021. We proposed an Adjusted Remote Sensing Ecological Index (A-RSEI), integrated Sentinel-2A, Landsat 8, and other remote sensing data to evaluate the eco-environmental quality of the study area, from 2013 to 2021. Coupled coordination degree (CCD) model was used to obtain the CCD between eco-environmental quality and urbanization. In addition, spatio-temporal and multi-scale analysis was carried out from the perspectives of urban agglomeration, municipal, county, and pixel scales. Combined with spatial autocorrelation analysis and Tapio decoupling model, the CCD was further explored. The results show that the proposed A-RSEI model is more suitable for monitoring the eco-environmental quality of the Yellow River Basin. The coupling coordination degree of eco-environment and urbanization in most regions of the study area are rising in a relatively green development trend. The multi-scale analysis among eco-environmental quality, urbanization, and CCD can not only indicate the impact of the central city on its surrounding areas but also help to describe the details of CCD combined with the terrain. The comprehensive discrimination of urban agglomeration and county scale is helpful to express the relationship between urbanization and eco-environmental quality centered on a certain city. The results can provide scientific support for eco-environment protection and high-quality development of the Yellow River Basin

Gas-phase formation of interstellar nucleobases from dehydrogenated formamide and vinyl cyanide

Context. Cytosine, thymine, and uracil are three of the five primary nucleobases that function as the fundamental units of the genetic code in nucleic acids. In searching the extraterrestrial origins of microscopic life, previous studies have reported formation routes of nucleobases in interstellar ice analogs. The present work explores the possibility that nucleobases could form from small molecules through gas-phase reactions in the interstellar medium (ISM). Aims. We aim to search energetically favorable synthetic routes toward the formation of cytosine, thymine, and uracil via gas-phase reactions, using first principles calculations. Based on the computation of a reaction energy barrier and reactant formation energy, we tried to identify the specific interstellar environments favorable to the formation of the nucleobases, with respect to the previously reported detection of relevant reactants in the ISM. Methods. Density functional theory calculations were carried out to investigate the chemical reaction pathways using the M06 functional with 6-31+G(d,p)/6-311++G(d,p) basis sets. An ab initio Møller-Plesset perturbation theory in the second order (MP2) was also used to corroborate the results. Results. We report synthetic routes toward the formation of cytosine, thymine, and uracil through gas-phase reactions between partially dehydrogenated formamide (H2NCHO) and vinyl cyanide (H2CCHCN). The most energetically favorable pathway to the formation of 1H-pyrimidin-2-one (C4H4N2O), a direct precursor of nucleobases, was found in a molecule-radical reaction between HNCHO and H2CCHCN, with an energy barrier of 19.3 kcal mol−1. The energy barriers for the optimal reaction pathways between C4H4N2O and amino, methyl, or hydroxyl to finally produce cytosine, thymine, or uracil are about 11.3, 18.6, or 19.9 kcal mol−1, respectively. Conclusions. The optimal energy barriers of 19.3 and 23.8 kcal mol−1 roughly correspond to a reaction rate coefficient of 10−11 cm3 s−1 at 180 and 220 K, respectively. This indicates that the reaction could be thermally feasible through a gas-phase reaction in hot molecular cores or in the inner part of the protoplanetary disks. In contrast, the energy barriers for the reactions between other dehydrogenated radicals and molecules are relatively high, which corresponds to the extinction energy of far-ultraviolet photons in photo-dissociation regions. Furthermore, the computed pathways suggest that prior H migration in the reactants could be the key rate-determining process for the synthesis of the primary nucleobases

Formation of Interstellar Complex Polycyclic Aromatic Hydrocarbons: Insights from Molecular Dynamics Simulations of Dehydrogenated Benzene

International audienceSmall organic molecules are thought to provide building blocks for the formation of complex interstellar polycyclic aromatic hydrocarbons (PAHs). However, the underlying chemical mechanisms remain unclear, particularly concerning the role of interstellar dust. Using molecular dynamics, we simulate the chemical reaction between dehydro-genated benzene molecules in the gas phase or on the surface of an onion-like carbon nanoparticle (NP). The reaction leads to the formation of PAHs of complex structures. The size of the formed molecules is found to roughly increase at increasing temperature up to 800 K, and to be correlated with the level of dehydrogenation. Morphology analysis features the formation of large rings that contain up to 32 carbon atom at high temperature. Density functional theory (DFT) calculations are performed to search the fundamental energetic reaction pathways. The DFT results quantitatively confirm the correlation between the reactivity and the dehydrogenation level, and the formation of stable C-8 rings. Moreover, the layered nanostructures formed on the NP surface point to a possible layer-by-layer formation mechanism for interstellar fullerene and carbon onions