Association of the SHOX2 and RASSF1A methylation levels with the pathological evolution of early-stage lung adenocarcinoma.

Background The methylation of SHOX2 and RASSF1A shows promise as a potential biomarker for the early screening of lung cancer, offering a solution to remedy the limitations of morphological diagnosis. The aim of this study is to diagnose lung adenocarcinoma by measuring the methylation levels of SHOX2 and RASSF1A, and provide an accurate pathological diagnosis to predict the invasiveness of lung cancer prior to surgery.Material and methods The methylation levels of SHOX2 and RASSF1A were quantified using a LungMe® test kit through methylation-specific PCR (MS-PCR). The diagnostic efficacy of SHOX2 and RASSF1A and the cutoff values were validated using ROC curve analysis. The hazardous factors influencing the invasiveness of lung adenocarcinoma were calculated using multiple regression.Results: The cutoff values of SHOX2 and RASSF1A were 8.3 and 12.0, respectively. The sensitivities of LungMe® in IA, MIA and AIS patients were 71.3% (122/171), 41.7% (15/36), and 16.1% (5/31) under the specificity of 94.1% (32/34) for benign lesions. Additionally, the methylation level of SHOX2, RASSF1A and LungMe® correlated with the high invasiveness of clinicopathological features, such as age, gender, tumor size, TNM stage, pathological type, pleural invasion and STAS. The tumor size, age, CTR values and LungMe® methylation levels were identified as independent hazardous factors influencing the invasiveness of lung adenocarcinoma.Conclusion: SHOX2 and RASSF1A combined methylation can be used as an early detection indicator of lung adenocarcinoma. SHOX2 and RASSF1A combined (LungMe®) methylation is significantly correlated to age, gender, tumor size, TNM stage, pathological type, pleural invasion and STAS. The SHOX2 and RASSF1A methylation levels, tumor size and CTR values could predict the invasiveness of the tumor prior to surgery, thereby providing guidance for the surgical procedure.

Application of ultrasonic-enhanced active seed crystals in the removal of sodium oxalate from alumina refinery waste liquor.

When organic matter, especially sodium oxalate (Na2C2O4), accumulates to a certain extent, it will seriously affect the alumina production process in the refinery and therefore urgently needs to be removed. This work attempts to illuminate the benefits of ultrasonic intensification of the crystallization process of Na2C2O4, taking the alumina refinery waste liquor, i.e., flat plate washing liquor, as a case study. The effects of different operating parameters (seed crystal addition amount, caustic soda concentration, reaction time, ultrasonic power) on the crystallization behavior and yield are discussed, and it is found that ultrasonic can increase the Na2C2O4 removal rate to 70.4%. The addition of ultrasonic promotes the morphological evolution of Na2C2O4 and is of great significance to the optimization of the components of the precipitated Na2C2O4. Specifically, the proportion of Na2C2O4 in the crystallized product reaches 64% with conventional conditions, while it reaches 77% with ultrasonic conditions. Therefore, ultrasonic can greatly reduce the alkali loss caused by the crystallization process of Na2C2O4 in flat plate washing liquor, which has great economic benefits.

Comparative Study on the Effectiveness of Three Inoculation Methods for Valsa sordida in Populus alba var. pyramidalis.

A key step in the study of tree pathology is the identification of an appropriate method for inoculating pathogens of diseases in branches and trunks. Pathogens of diseases in branches and trunks are commonly inoculated through punching, burning, and toothpick inoculation. However, there is a lack of comparative analyses of the inoculation outcomes of these three methods. In this work, six-year-old P. alba var. pyramidalis were inoculated with V. sordida using punching, burning, and toothpick techniques to investigate the differences in the effectiveness of these inoculation methods. Results reveal that the incidence rate was 93.55% in the toothpick inoculation group, significantly higher than the 80.65% in the burning inoculation group (chi-square, n = 90, p = 0.007), while punching inoculation exhibited significant pathological responses in the early stages, with spontaneous healing in the later stage. Additionally, toothpick inoculation was more efficient in inducing Valsa canker when inoculating the pathogen at the bottom of the tree, with lower intra- and inter-row spacing (stand density) providing better outcomes than higher intra- and inter-row spacing. The results of this study demonstrate that toothpick inoculation is an optimal option for studying the artificial inoculation of V. sordida in six-year-old P. alba var. pyramidalis, providing technical support for research on poplar diseases and offering a theoretical basis for the inoculation of other diseases in the branch and trunk.

Combining ozone and ultrasound technology to remove S2- in Bayer liquor.

High content sulfur (S2-) in Bayer liquor can increase alkali consumption, accelerate equipment corrosion, especially seriously affect alumina production. The removal of S2- in Bayer liquor is studied using ultrasonic enhanced ozone method, which significantly improves the removal efficiency. Results indicate that the best removal efficiency of 93.83 % is obtained with reaction duration of 20 min, oxygen flow rate of 80 L/h, ultrasonic power of 60 W and reaction temperature of 60 °C. The comparative analysis shows that the removal efficiency of S2- is 25.34 % higher than that of ozone (O3) system after introducing ultrasound (US), indicating that US accelerates the mass transfer process of O3 and increases the hydroxyl radicals (OH) content. For further explanation of the mechanism of US/O3 system, EPR and XPS spectra are applied to analyze the content of free radical and the form of sulfur in Bayer liquor, indicating that the content of free radical in US/O3 system is more than US and O3 systems, and all sulfur is converted to SO42- after full oxidation.

Immune microenvironment analysis and novel biomarkers of early-stage lung adenocarcinoma evolution.

Background: Lung cancer is the deadliest and most diagnosed type of cancer worldwide. The 5-year survival rate of lung adenocarcinoma (LUAD) dropped significantly when tumor stages advanced. Patients who received surgically resecting at the pre-invasive stage had a 5-year survival rate of nearly 100%. However, the study on the differences in gene expression profiles and immune microenvironment among pre-invasive LUAD patients is still lacking. Methods: In this study, the gene expression profiles of three pre-invasive LUAD stages were compared using the RNA-sequencing data of 10 adenocarcinoma in situ (AIS) samples, 12 minimally invasive adenocarcinoma (MIA) samples, and 10 invasive adenocarcinoma (IAC) samples. Results: The high expression levels of PTGFRN (Hazard Ratio [HR] = 1.45; 95% Confidence Interval [CI]: 1.08-1.94; log-rank P = 0.013) and SPP1 (HR = 1.44; 95% CI: 1.07-1.93; log-rank P = 0.015) were identified to be associated with LUAD prognosis. Moreover, the early LUAD invasion was accompanied by the enhancement of antigen presentation ability, reflected by the increase of myeloid dendritic cells infiltration rate (Cuzick test P < 0.01) and the upregulation of seven important genes participating in the antigen presentation, including HLA-A (Cuzick test P = 0.03), MICA (Cuzick test P = 0.01), MICB (Cuzick test P = 0.01), HLA-DPA1 (Cuzick test P = 0.04), HLA-DQA2 (Cuzick test P < 0.01), HLA-DQB1 (Cuzick test P = 0.03), and HLA-DQB2 (Cuzick test P < 0.01). However, the tumor-killing ability of the immune system was inhibited during this process, as there were no rising cytotoxic T cell activity (Cuzick test P = 0.20) and no increasing expression in genes encoding cytotoxic proteins. Conclusion: In all, our research elucidated the changes in the immune microenvironment during early-stage LUAD evolution and may provide a theoretical basis for developing novel early-stage lung cancer therapeutic targets.

Integrated transcriptome and microRNA sequencing analyses reveal gene responses in poplar leaves infected by the novel pathogen bean common mosaic virus (BCMV).

Recently, a novel poplar mosaic disease caused by bean common mosaic virus (BCMV) was investigated in Populus alba var. pyramidalis in China. Symptom characteristics, physiological performance of the host, histopathology, genome sequences and vectors, and gene regulation at the transcriptional and posttranscriptional levels were analyzed and RT-qPCR (quantitative reverse transcription PCR) validation of expression was performed in our experiments. In this work, the mechanisms by which the BCMV pathogen impacts physiological performance and the molecular mechanisms of the poplar response to viral infection were reported. The results showed that BCMV infection decreased the chlorophyll content, inhibited the net photosynthesis rate (Pn) and stomatal conductance (Gs), and significantly changed chlorophyll fluorescence parameters in diseased leaves. Transcriptome analysis revealed that the expression of the majority of DEGs (differentially expressed genes) involved in the flavonoid biosynthesis pathway was promoted, but the expression of all or almost all DEGs associated with photosynthesis-antenna proteins and the photosynthesis pathway was inhibited in poplar leaves, suggesting that BCMV infection increased the accumulation of flavonoids but decreased photosynthesis in hosts. Gene set enrichment analysis (GSEA) illustrated that viral infection promoted the expression of genes involved in the defense response or plant-pathogen interaction. MicroRNA-seq analysis illustrated that 10 miRNA families were upregulated while 6 families were downregulated in diseased poplar leaves; moreover, miR156, the largest family with the most miRNA members and target genes, was only differentially upregulated in long-period disease (LD) poplar leaves. Integrated transcriptome and miRNA-seq analyses revealed 29 and 145 candidate miRNA-target gene pairs; however, only 17 and 76 pairs, accounting for 2.2% and 3.2% of all DEGs, were authentically negatively regulated in short-period disease (SD) and LD leaves, respectively. Interestingly, 4 miR156/SPL (squamosa promoter-binding-like protein) miRNA-target gene pairs were identified in LD leaves: the miR156 molecules were upregulated, but SPL genes were downregulated. In conclusion, BCMV infection significantly changed transcriptional and posttranscriptional gene expression in poplar leaves, inhibited photosynthesis, increased the accumulation of flavonoids, induced systematic mosaic symptoms, and decreased physiological performance in diseased poplar leaves. This study elucidated the fine-tuned regulation of poplar gene expression by BCMV; moreover, the results also suggested that miR156/SPL modules played important roles in the virus response and development of viral systematic symptoms in plant virus disease.

Stem canker pathogen Botryosphaeria dothidea inhibits poplar leaf photosynthesis in the early stage of inoculation.

Fungal pathogens can induce canker lesions, wilting, and even dieback in many species. Trees can suffer serious physiological effects from stem cankers. In this study, we investigated the effects of Botryosphaeria dothidea (B. dothidea) on Populus bolleana (P. bolleana) leaves photosynthesis and stomatal responses, when stems were inoculated with the pathogen. To provide experimental and theoretical basis for preventing poplar canker early. One-year-old poplar stems were inoculated with B. dothidea using an epidermal scraping method. In the early stage of B. dothidea inoculation (2-14 days post inoculation, dpi), the gas exchange, stomatal dynamics, hormone content, photosynthetic pigments content, chlorophyll fluorescence parameters, and non-structural carbohydrate (NSC) were evaluated to elucidate the pathophysiological mechanism of B. dothidea inhibiting photosynthesis. Compared with the control groups, B. dothidea noteworthily inhibited the net photosynthetic rate (P n), stomatal conductance (G s), intercellular CO2 concentration (C i), transpiration rate (T r), and other photosynthetic parameters of poplar leaves, but stomatal limit value (L s) increased. Consistent with the above results, B. dothidea also reduced stomatal aperture and stomatal opening rate. In addition, B. dothidea not only remarkably reduced the content of photosynthetic pigments, but also decreased the maximum photochemical efficiency (F v/F m), actual photochemical efficiency (Φ PSII), electron transfer efficiency (ETR), and photochemical quenching coefficient (q P). Furthermore, both chlorophyll and Φ PSII were positively correlated with P n. In summary, the main reason for the abated P n under stem canker pathogen was that B. dothidea not merely inhibited the stomatal opening, but hindered the conversion of light energy, electron transfer and light energy utilization of poplar leaves. In general, the lessened CO2 and P n would reduce the synthesis of photosynthetic products. Whereas, sucrose and starch accumulated in poplar leaves, which may be due to the local damage caused by B. dothidea inoculation in phloem, hindering downward transport of these products.

Pathophysiology and transcriptomic analysis of Picea koraiensis inoculated by bark beetle-vectored fungus Ophiostoma bicolor.

Ophiostomatoid fungi exhibit a complex relationship with bark beetles; exhausting of host tree defenses is traditionally regarded as one of the key benefits provided to beetle vectors. Ophiostoma bicolor is one of the dominant species of the mycobiota associated with Ips genus bark beetles which infect the spruce trees across the Eurasian continent. Host spruce trees resist fungal invasion through structural and inducible defenses, but the underlying mechanisms at the molecular level, particularly with respect to the interaction between bark beetle-associated fungi and host trees, remain unclear. The aim of this study was to observe the pathological physiology and molecular changes in Picea koraiensis seedlings after artificial inoculation with O. bicolor strains (TS, BH, QH, MX, and LWQ). This study showed that O. bicolor was a weakly virulent pathogen of spruce, and that the virulent of the five O. bicolor strains showed differentiation. All O. bicolor strains could induce monoterpenoid release. A positive correlation between fungal virulence and release of monoterpenoids was observed. Furthermore, the release rate of monoterpenoids peaked at 4 days post-inoculation (dpi) and then decreased from 4 to 90 dpi. Transcriptomic analysis at 4 dpi showed that many plant-pathogen interaction processes and mitogen-activated protein kinase (MAPK) metabolic processes were activated. The expression of monoterpenoid precursor synthesis genes and diterpenoid synthesis genes was upregulated, indicating that gene expression regulated the release rate of monoterpenoids at 4 dpi. The enriched pathways may reveal the immune response mechanism of spruce to ophiostomatoid fungi. The dominant O. bicolor possibly induces the host defense rather than defense depletion, which is likely the pattern conducted by the pioneers of beetle-associated mycobiota, such as Endoconidiophora spp.. Overall, these results facilitate a better understanding of the interaction mechanism between the dominant association of beetles and the host at the molecular level.

Comparative Genomics Reveals Evolutionary Traits, Mating Strategies, and Pathogenicity-Related Genes Variation of Botryosphaeriaceae.

Botryosphaeriaceae, as a major family of the largest class of kingdom fungi Dothideomycetes, encompasses phytopathogens, saprobes, and endophytes. Many members of this family are opportunistic phytopathogens with a wide host range and worldwide geographical distribution, and can infect many economically important plants, including food crops and raw material plants for biofuel production. To date, however, little is known about the family evolutionary characterization, mating strategies, and pathogenicity-related genes variation from a comparative genome perspective. Here, we conducted a large-scale whole-genome comparison of 271 Dothideomycetes, including 19 species in Botryosphaeriaceae. The comparative genome analysis provided a clear classification of Botryosphaeriaceae in Dothideomycetes and indicated that the evolution of lifestyle within Dothideomycetes underwent four major transitions from non-phytopathogenic to phytopathogenic. Mating strategies analysis demonstrated that at least 3 transitions were found within Botryosphaeriaceae from heterothallism to homothallism. Additionally, pathogenicity-related genes contents in different genera varied greatly, indicative of genus-lineage expansion within Botryosphaeriaceae. These findings shed new light on evolutionary traits, mating strategies and pathogenicity-related genes variation of Botryosphaeriaceae.

Genome Assembly and Annotation of Botryosphaeria dothidea sdau11-99, a Latent Pathogen of Apple Fruit Ring Rot in China.

Botryosphaeria dothidea is a latent and important fungal pathogen on a wide range of woody plants. Fruit ring rot caused by B. dothidea is a major disease in China on apple. This study establishes a high-quality, nearly complete, and well-annotated genome sequence of B. dothidea strain sdau11-99. The findings of this research provide a reference genome resource for further research on the apple fruit ring rot pathogen on apple and other hosts.

Photocatalytic Cleavage of ß-O-4 Ether Bonds in Lignin over Ni/TiO2.

It is of great importance to explore the selective hydrogenolysis of ß-O-4 linkages, which account for 45-60% of all linkages in native lignin, to produce valued-added chemicals and fuels from biomass employing UV light as catalyst. TiO2 exhibited satisfactory catalytic performances in various photochemical reactions, due to its versatile advantages involving high catalytic activity, low cost and non-toxicity. In this work, 20 wt.% Ni/TiO2 and oxidant PCC (Pyridinium chlorochromate) were employed to promote the cleavage of ß-O-4 alcohol to obtain high value chemicals under UV irradiation at room temperature. The Ni/TiO2 photocatalyst can be magnetically recovered and efficiently reused in the following four consecutive recycling tests in the cleavage of ß-O-4 ether bond in lignin. Mechanism studies suggested that the oxidation of ß-O-4 alcohol to ß-O-4 ketone by oxidant PCC first occurred during the reaction, and was followed by the photocatalysis of the obtained ß-O-4 ketone to corresponding acetophenone and phenol derivates. Furthermore, the system was tested on a variety of lignin model substrates containing ß-O-4 linkage for the generation of fragmentation products in good to excellent results.

Fungal canker pathogens trigger carbon starvation by inhibiting carbon metabolism in poplar stems.

Carbon starvation is the current leading hypothesis of plant mortality mechanisms under drought stress; recently, it is also used to explain tree die-off in plant diseases. However, the molecular biology of the carbon starvation pathway is unclear. Here, using a punch inoculation system, we conducted transcriptome and physiological assays to investigate pathogen response in poplar stems at the early stages of Botryosphaeria and Valsa canker diseases. Transcriptome assays showed that the majority of differentially expressed genes (DEGs) in stem phloem and xylem, such as genes involved in carbon metabolism and transportation, aquaporin genes (in xylem) and genes related to the biosynthesis of secondary metabolites and the phenylpropanoid pathway (related to lignin synthesis), were downregulated at 7 days after inoculation (DAI). Results also showed that the expression of the majority of disease-resistance genes upregulated in poplar stems, which may be connected with the downregulation expression of the majority of WRKY family genes. Physiological assays showed that transpiration rate decreased but WUE (water use efficiency) increased the 3 and 7 DAI, while the net photosynthetic rate decreased at 11 DAI in Botryosphaeria infected poplars (ANOVA, P < 0.05). The NSC (non-structural carbohydrates) content assays showed that the soluble sugar content of stem phloem samples increased at 3, 7, and 11 DAI that might due to the impede of pathogen infection. However, soluble sugar content of stem xylem and root samples decreased at 11 DAI; in contrast, the starch content unchanged. Therefore, results revealed a chronological order of carbon related molecular and physiological performance: declination of genes involved in carbon and starch metabolism first (at least at 7 DAI), declination of assimilation and carbon reserve (at 11 DAI) second. Results implied a potential mechanism that affects the host carbon reserve, by directly inhibiting the expression of genes involved in carbon metabolism and transport.

Transcriptome Changes in the Mink Uterus during Blastocyst Dormancy and Reactivation.

Embryo implantation in the mink follows the pattern of many carnivores, in that preimplantation embryo diapause occurs in every gestation. Details of the gene expression and regulatory networks that terminate embryo diapause remain poorly understood. Illumina RNA-Seq was used to analyze global gene expression changes in the mink uterus during embryo diapause and activation leading to implantation. More than 50 million high quality reads were generated, and assembled into 170,984 unigenes. A total of 1684 differential expressed genes (DEGs) in uteri with blastocysts in diapause were compared to the activated embryo group (p < 0.05). Among these transcripts, 1527 were annotated as known genes, including 963 up-regulated and 564 down-regulated genes. The gene ontology terms for the observed DEGs, included cellular communication, phosphatase activity, extracellular matrix and G-protein couple receptor activity. The KEGG pathways, including PI3K-Akt signaling pathway, focal adhesion and extracellular matrix (ECM)-receptor interactions were the most enriched. A protein-protein interaction (PPI) network was constructed, and hub nodes such as VEGFA, EGF, AKT, IGF1, PIK3C and CCND1 with high degrees of connectivity represent gene clusters expected to play an important role in embryo activation. These results provide novel information for understanding the molecular mechanisms of maternal regulation of embryo activation in mink.

Effects of phloem on canopy dieback, tested with manipulations and a canker pathogen in the Corylus avellana/Anisogramma anomala host/pathogen system.

Canker pathogens cause necrosis of the phloem, but in many host/pathogen systems, they also cause canopy dieback, which implicates xylem, not phloem dysfunction. We hypothesize that this dieback distal to the canker is caused by water stress resulting from the lack of a phloem-to-xylem connection, which in a healthy plant would allow delivery of nonstructural carbohydrates (NSCs) and water inward to aid in xylem embolism refilling. We tested several components of this hypothesis in the host/pathogen system Corylus avellana L./Anisogramma anomala (Peck) E. Müll (Eastern filbert blight). Cankers were non-girdling and usually ≥0.1 m long. As expected, healthy controls had higher specific conductivity (Ks) than diseased stems, but unexpectedly, had similar moisture content (m.c.), showing that the lower Ks did not result from more embolisms in the diseased stems. Moreover, manipulations that removed cambium and phloem to simulate a canker, or that shaded stems to lower NSCs, did not result in lower Ks or m.c. than controls. The outer millimeter of xylem adjacent to a canker had infrequent tyloses and/or fungal hyphae in many but not all samples, and dye studies showed little xylem water transport in that region, but the incidence of these blockages was insufficient to cause the observed 19% decrease in Ks. Healthy stems had higher m.c. than diseased stems above the canker (or analogous) location and were longer for the same leaf weight, suggestive of water stress in the upper portion of diseased stems. These results suggest that dieback distal to cankers in this system results from the bottleneck in water transport in the region adjacent to a canker, but did not find evidence to support the requirement of a phloem-to-xylem connection for continued water transport.

Genome-Wide Constitutively Expressed Gene Analysis and New Reference Gene Selection Based on Transcriptome Data: A Case Study from Poplar/Canker Disease Interaction.

A number of transcriptome datasets for differential expression (DE) genes have been widely used for understanding organismal biology, but these datasets also contain untapped information that can be used to develop more precise analytical tools. With the use of transcriptome data generated from poplar/canker disease interaction system, we describe a methodology to identify candidate reference genes from high-throughput sequencing data. This methodology will improve the accuracy of RT-qPCR and will lead to better standards for the normalization of expression data. Expression stability analysis from xylem and phloem of Populus bejingensis inoculated with the fungal canker pathogen Botryosphaeria dothidea revealed that 729 poplar transcripts (1.11%) were stably expressed, at a threshold level of coefficient of variance (CV) of FPKM < 20% and maximum fold change (MFC) of FPKM < 2.0. Expression stability and bioinformatics analysis suggested that commonly used house-keeping (HK) genes were not the most appropriate internal controls: 70 of the 72 commonly used HK genes were not stably expressed, 45 of the 72 produced multiple isoform transcripts, and some of their reported primers produced unspecific amplicons in PCR amplification. RT-qPCR analysis to compare and evaluate the expression stability of 10 commonly used poplar HK genes and 20 of the 729 newly-identified stably expressed transcripts showed that some of the newly-identified genes (such as SSU_S8e, LSU_L5e, and 20S_PSU) had higher stability ranking than most of commonly used HK genes. Based on these results, we recommend a pipeline for deriving reference genes from transcriptome data. An appropriate candidate gene should have a unique transcript, constitutive expression, CV value of expression < 20% (or possibly 30%) and MFC value of expression <2, and an expression level of 50-1,000 units. Lastly, when four of the newly identified HK genes were used in the normalization of expression data for 20 differential expressed genes, expression analysis gave similar values to Cufflinks output. The methods described here provide an alternative pathway for the normalization of transcriptome data, a process that is essential for integrating analyses of transcriptome data across environments, laboratories, sequencing platforms, and species.

Comparative Transcriptome Analysis of Raccoon Dog Skin to Determine Melanin Content in Hair and Melanin Distribution in Skin.

The raccoon dog (Nyctereutes procyonoides) is an important canid fur-bearing animal species worldwide. Chinese raccoon dogs that present a white mutation, especially those with a white coat. Exploring melanin biosynthesis in the hair and skin of raccoon dogs is important for understanding the survival and evolutionary mechanisms of them. In this study, we measured the content of melanin in the hair of two types of raccoon dog and generated stained slices of skin tissue. The results indicated that melanin biosynthesis occurs in the wild-type (W) and white-type (B) raccoon dog skin, although less melanin is produced in B skin. We then sequenced the skin transcriptomes of W and B, compared the similarities and differences in expressed genes. A comparison of the gene expression showed 60 up-regulated genes and 127 down-regulated genes in B skin. We analyzed the unigenes and pathways related to the melanogenesis pathway and found that TYR, TYRP1, MC1R, SLC24a5, SLC45a2 and OCA2 were significantly down-regulated in B skin and these results were verified via qRT-PCR. We surmised that the phenotypic characteristics of the white mutation might be caused by the reduced expression of these genes and this finding provides new insights for future experiments in raccoon dogs.

Prevalence, risk factors and molecular characterization of Enterocytozoon bieneusi in raccoon dogs (Nyctereutes procyonoides) in five provinces of Northern China.

The disease microsporidiosis is found worldwide and is mainly caused by Enterocytozoon bieneusi. E. bieneusi can infect a wide range of hosts; however, information regarding the prevalence and genotyping of E. bieneusi infection in raccoon dogs (Nyctereutes procyonoides) is limited. Therefore, in 2015, we examined 305 faecel samples from 80 farmed raccoon dogs in Jilin Province, from 54 in Hebei Province, from 72 in Liaoning Province, from 29 in Shandong Province, and from 40 in Heilongjiang Province. The overall prevalence of E. bieneusi infection in farmed raccoon dogs was 22.30%. Logistic regression analysis suggests that age, gender and region of raccoon dogs were highly related to the prevalence of E. bieneusi infection. Moreover, six E. bieneusi internal transcribed spacer (ITS) region sequences, including four known genotypes, namely D, CHN-DC1, NCF2, and CHN-F1, and two novel genotypes (NCR1 and NCR2), were identified in the present study. The present study firstly indicated the existence of E. bieneusi genotypes NCF2, NCR1, NCR2and CHN-F1 in infected raccoon dogs in Northern China. Integrated control strategies should be implemented to limit E. bieneusi infection in farmed raccoon dogs, and to prevent transmission of this disease to other animals and humans.

Determination and analysis of complete mitochondrial genome sequence of Peking duck (Anas platyrhychos).

The complete mitochondrial genome (mtDNA) of Peking duck (Anas platyrhychos) was determined. The entire genome was 16,604 bp in length. Similar to the typical mtDNA of vertebrates, it contained 37 genes (13 protein-coding genes, 2 rRNA genes, 22 tRNA genes) and a non-coding region (D-loop). The characteristics of the mitochondrial genome was analyzed and discussed in detail.

Effects of dietary protein level on nutrients digestibility and reproductive performance of female mink (Neovison vison) during gestation.

The objective of this study was to determine whether nutrient digestibility and reproductive performance of pregnant mink (Neovison vison) were affected by different dietary protein levels. One hundred and twenty female mink were randomly assigned to four groups, receiving diets of fresh material with different protein levels. The dietary protein levels, expressed as percentage of dry matter (DM), were 32, 36, 40 and 44% respectively. These values corresponded to average 320, 360, 400 and 440 g protein/kg DM, respectively. Results were as follows. All of crude protein digestibility, nitrogen (N) intake, N retention increased along with dietary protein level increasing. Low protein level (32%) significantly reduced the above indicators (P < 0.05). DM digestibility and ether extract digestibility were not affected by dietary protein level. Results of mated females, barren females, kids per litter, live born kids per mated female, birth survival rate, and birth weight showed that mink achieved optimal reproductive performance when dietary protein level was 36%. In conclusion, dietary protein was anticipated to significantly influence some nutrients' utilization. Adopting the appropriate dietary protein level allow better reproduction performance. The most preferable reproductive performance was achieved when diet contained 275.5 g digestible protein per kg DM for female mink in gestation.