Reorganization of 3D genome architecture provides insights into pathogenesis of early fatty liver disease in laying hens

Background Fatty liver disease causes huge economic losses in the poultry industry due to its high occurrence and lethality rate.Three-dimensional(3D)chromatin architecture takes part in disease processing by regulating tran-scriptional reprogramming.The study is carried out to investigate the alterations of hepatic 3D genome and H3K27ac profiling in early fatty liver(FLS)and reveal their effect on hepatic transcriptional reprogramming in laying hens.Results Results show that FLS model is constructed with obvious phenotypes including hepatic visible lipid deposi-tion as well as higher total triglyceride and cholesterol in serum.A/B compartment switching,topologically associat-ing domain(TAD)and chromatin loop changes are identified by high-throughput/resolution chromosome conforma-tion capture(HiC)technology.Targeted genes of these alternations in hepatic 3D genome organization significantly enrich pathways related to lipid metabolism and hepatic damage.H3K27ac differential peaks and differential expres-sion genes(DEGs)identified through RNA-seq analysis are also enriched in these pathways.Notably,certain DEGs are found to correspond with changes in 3D chromatin structure and H3K27ac binding in their promoters.DNA motif analysis reveals that candidate transcription factors are implicated in regulating transcriptional reprogram-ming.Furthermore,disturbed folate metabolism is observed,as evidenced by lower folate levels and altered enzyme expression.Conclusion Our findings establish a link between transcriptional reprogramming changes and 3D chromatin struc-ture variations during early FLS formation,which provides candidate transcription factors and folate as targets for FLS prevention or treatment.

Genome-Wide Analysis of the F3′5′H Gene Family in Blueberry(Vaccinium corymbosum L.)Provides Insights into the Regulation of Anthocyanin Biosynthesis

The F3′5′H gene family plays an important role in regulating anthocyanin biosynthesis,abiotic stress,and hormone signaling.In this study,14 F3′5′H genes were identified from the blueberry genome.The chromosomal distribution,physicochemical properties,F3′5′H domain,conserved motifs,cis-acting elements,and intron/exon compositions were analyzed.The functional prediction analysis of these VcF3′5′Hs indicated that their biological functions included light response and other secondary metabolites.The results of qRT-PCR showed that VcF3′5′Hs(especially VcF3′5′H4)were highly expressed at the ripening stage.Subcellular localization revealed that VcF3′5′H4 may be located in the endoplasmic reticulum.Co-expression analysis showed that the VcF3′5′H gene family was related to anthocyanin.This research provides an overview of the blueberry F3′5′H family and helps verify the role of these genes in regulating anthocyanin biosynthesis.

Long noncoding RNA H19 regulates degeneration and regeneration of injured peripheral nerves

Our previous studies have shown that long noncoding RNA(lncRNA)H19 is upregulated in injured rat sciatic nerve during the process of Wallerian degeneration,and that it promotes the migration of Schwann cells and slows down the growth of dorsal root ganglion axons.However,the mechanism by which lncRNA H19 regulates neural repair and regeneration after peripheral nerve injury remains unclear.In this study,we established a Sprague-Dawley rat model of sciatic nerve transection injury.We performed in situ hybridization and found that at 4–7 days after sciatic nerve injury,lncRNA H19 was highly expressed.At 14 days before injury,adeno-associated virus was intrathecally injected into the L4–L5 foramina to disrupt or overexpress lncRNA H19.After overexpression of lncRNA H19,the growth of newly formed axons from the sciatic nerve was inhibited,whereas myelination was enhanced.Then,we performed gait analysis and thermal pain analysis to evaluate rat behavior.We found that lncRNA H19 overexpression delayed the recovery of rat behavior function,whereas interfering with lncRNA H19 expression improved functional recovery.Finally,we examined the expression of lncRNA H19 downstream target SEMA6D,and found that after lncRNA H19 overexpression,the SEMA6D protein level was increased.These findings suggest that lncRNA H19 regulates peripheral nerve degeneration and regeneration through activating SEMA6D in injured nerves.This provides a new clue to understand the role of lncRNA H19 in peripheral nerve degeneration and regeneration.

^(1)H and ^(13)C NMR spectral assignments for low-concentration bile acids in biological samples

Bile acids are the main body of enterohepatic circulation in vivo.They have essential functions such as emulsifying fat,bacteriostasis and regulating multiple metabolic pathways as signal molecules.However,the assignments of NMR signals for some lowconcentration bile acids are still needed.This study combined 1D nuclear magnetic resonance(NMR)and 2D NMR techniques including 1He1H correlation spectroscopy(COSY),1He1H total correlation spectroscopy(TOCSY),1H J-resolved spectroscopy(J-Res),1He13C heteronuclear single quantum coherence spectroscopy(HSQC),and 1He13C heteronuclear multiple bond correlation spectroscopy(HMBC)to assign the 1H and 13C signals of six bile acids in aqueous solution at physiological pH(~7.4)and nine bile acids in methanol.These data are of importance to the NMR-based studies on lipid digestion,absorption,and metabolism.

Prevalence of shiga toxins(stx_1,stx_2),eaeA and hly genes of Escherichia coli O157:H7 strains among children with acute gastroenteritis in southern of Iran

Objective:To survey the prevalence severe diarrhea arising from these bacteria in children under 5 years old in Marvdasht.Methods:In this study faecal sample from 615 children aged <5years old who were hospitalized lor gastroenteritis in Fars hospitals in Iran were collected and then enriched in Escherichia coli(E.coli) broth and modified tryplone soy broth with novobiocin media,fermentation of sorbitol,lactose and β— glucoronidase activity of isolated strains was examined by CT—SMAC,VRBA and chromogenic media respectively.Then isolation of E.coli O157:H7 have been confirmed with the use of specific antisera and with multiplex PCR method presence of virulence genes including:xtx_1.stx_2,eae.A.hly has been analyzed.Results:E.coli O157:H7 was detected in 7(1.14%) stool specimens.A significanl difference was seen between detection rale of isolated bacteria from age groups 18-23 months and other age groups(P=0.004).Out of considered virulence genes.only 1 of the isolated strains(0.16%)he stx,and eaeA genes were seen and also all isolated hacleria had resistance to penicillin,ampicillin and erythromycin antibiotics.Conclusions:We found thai children < 2 years of age were at highest risk of infection with E.coli O157:H7.Regarding severity of E.coli O157:H7 pathogenesis,low infectious dose and lack of routine assay for detection ol these bacleria in clinical laboratory,further and completed studies on diagnosis and genolyping of this E.coli O157:H7 strain has been recommended.

Host markers and correlated mutations in the overlapping genes of influenza viruses: M1, M2;NS1, NS2;and PB1, PB1-F2

The influenza A viruses have three gene segments, M, NS, and PB1, which code for more than one protein. The overlapping genes from the same segment entail their interdependence, which could be reflected in the evolutionary constraints, host distinction, and co-mutations of influenza. Most previous studies of overlapping genes focused on their unique evolutionary constraints, and very little was achieved to assess the potential impact of the overlap on other biological aspects of influenza. In this study, our aim was to explore the mutual dependence in host differentiation and co-mutations in M, NS, and PB1 of avian, human, 2009 H1N1, and swine viruses, with Random Forests, information entropy, and mutual information. The host markers and highly co-mutated individual sites and site pairs (P values < 0.035) in the three gene segments were identified with their relative significance between the overlapping genes calculated. Further, Random Forests predicted that among the three stop codons in the current PB1-F2 gene of 2009 H1N1, the significance of a mutation at these sites for host differentiation was, in order from most to least, that at 12, 58, and 88, i.e., the closer to the start of the gene the more important the mutation was. Finally, our sequence analysis surprisingly revealed that the full-length PB1-F2, if the three stop codons were all mutated, would function more as a swine protein than a human protein, although the PB1 of 2009 H1N1 was derived from human H3N2.

Integrated analysis of human influenza A(H1N1)virus infectionrelated genes to construct a suitable diagnostic model

The genome characteristics and structural functions of coding proteins correlate with the genetic diversity of the H1N1 virus,which aids in the understanding of its underlying pathogenic mechanism.In this study,analyses of the characteristic of the H1N1 virus infection-related genes,their biological functions,and infection-related reversal drugs were performed.Additionally,we used multi-dimensional bioinformatics analysis to identify the key genes and then used these to construct a diagnostic model for the H1N1 virus infection.There was a total of 169 differently expressed genes in the samples between 21 h before infection and 77 h after infection.They were used during the protein-protein interaction(PPI)analysis,and we obtained a total of 1725 interacting genes.Then,we performed a weighted gene co-expression network analysis(WGCNA)on these genes,and we identified three modules that showed significant potential for the diagnosis of the H1N1 virus infection.These modules contained 60 genes,and they were used to construct this diagnostic model,which showed an effective prediction value.Besides,these 60 genes were involved in the biological functions of this infectious virus,like the cellular response to type I interferon and in the negative regulation of the viral life cycle.However,20 genes showed an upregulated expression as the infection progressed.Other 36 upregulated genes were used to examine the relationship between genes,human influenza A virus,and infection-related reversal drugs.This study revealed numerous important reversal drug molecules on the H1N1 virus.They included rimantadine,interferons,and shikimic acid.Our study provided a novel method to analyze the characteristic of different genes and explore their corresponding biological function during the infection caused by the H1N1 virus.This diagnostic model,which comprises 60 genes,shows that a significant predictive value can be the potential biomarker for the diagnosis of the H1N1 virus infection.

Morphofunctional status and the role of mononuclear phagocyte system lung compartment in the pathogenesis of influenza A (H5N1) in mammals

Influenza and other respiratory viral infections account for 80%-90% of infectious pathologies. Influenza A (H5N1) virus has an apparent pneumotropism, and therefore the lung compartment of mononuclear phagocyte system plays an important role in antiviral immunity. Lung macrophages are active phagocytes expressing variety of antiviral factors. The investigation of morphofunctional status of lung macrophages and evaluation of their role in mammal antiviral response in a mouse model were performed within the study. Methods: Light microscopy using standard hematoxylin-eosin, and Van-Gizon’s picrofuchsin staining. Immunohistochemistry using influenza A antigen marker specific primary antibodies, myeloperoxidase, cathepsin D, lysozyme, NO synthase, pro-inflammatory cytokines, cells of CD68 macrophage lineage, PCNA proliferative activity. Morphometric and statistical analysis. Results: Influenza A virus antigen was detected in lung macrophages starting from day 1 to day 14 of infection which corresponds with the beginning of convalescence and may be suggestive of prolonged persistence of virus. On the one hand, the cytopathic effects of the virus lead to lung macrophages death mainly via apoptosis through activation of caspase cascade, including caspase-3 and caspase-9. On the other hand, the observed activation of PCNA proliferation marker, perhaps, allows to support the pool of lung macrophages not only by their recruitment from bone marrow but also by their proliferation in situ. The increase of mononuclear phagocyte system cells expressing antiviral factors depended on the stage of infection. In the early stage, there was an increase of number of cells expressing lysozyme, myeloperoxidase, cathepsin D, endothelial NO synthase (eNOS) followed by the increase of number of macrophages expressing inducible NO synthase (iNOS), pro-inflammatory cytokines and interleukins.

Construction of Recombinant Expression Plasmids Containing H and F Protein Genes of Canine Distemper Virus Isolated from a Mink and Their Expression in Prokaryotic Cells

[Objective] This study aimed to construct the recombinant expression plasmids containing H and F protein genes of Canine distemper virus isolated from a mink and to express these two genes in prekaryotic cells as well as to study the reactogenieity of the expressed products. [ Method ] RT-PCR amplification was used to obtain H and F protein genes; TA cloning and subclonlng techniques were used to construct the cloning plasmids（pMD-18T-H and pMD-18T-F） and recombinant expression plasmids（pET28a-H and pET28a-F） ; SDS-PAGE and Western-blotting were adopted to verify whether the target proteins were successfully expressed. [ Result] The recombinant expression plasmids pET28a-H and pET28a-F containing H and F protein genes of Canine distemper virus isolated from a mink were successfully constructed, and both the expressed H and F proteins with respectively relative molecular mass of 31 400 and 38 200 produced positive reac- tion with the CDV standard positive serum. [ Conclusion] The H and F proteins expressed in prokaryotic cells were the same with the natural ones in terms of reac- togenicity, which can be utilized for diagnosis of a CDV＇s infection or for an epidemiological investigation. Meanwhile, they also provide a basis for developing ge- netically engineered subunit vaccines.

Antimicrobial Effects of Plant Compounds against Virulent <i>Escherichia coli</i>O157:H7 Strains Containing Shiga Toxin Genes in Laboratory Media and on Romaine Lettuce and Spinach

Escherichia coli strains produce Shiga-toxins Stx-1 and Stx-2 that contribute to their virulence. The objective was to evaluate antimicrobial activities of plant essential oils (oregano, cinnamon, lemongrass), their active components (carvacrol, cinnamaldehyde, citral) and plant-extracts (green tea polyphenols, apple skin, black tea, decaffeinated black tea, grapeseed and pomace extracts) against E. coli O157:H7 strains containing Stx-1 and Stx-2 genes, as determined by Multiplex Polymerase Chain Reaction, in vitro and on leafy greens. Antimicrobials at various concentrations in sterile PBS were added to bacterial cultures (~3 - 4 logs CFU/ml), mixed thoroughly, and incubated at 37°C. Surviving bacteria were enumerated at 0, 1, 3, 5 and 24 h. The most effective essential oil (oregano oil;0.5%) and plant extract (green tea;3%) were evaluated against E. coli O157:H7 on romaine lettuce and spinach stored at 4°C for 7 days. Microbial survival was a function of the concentration of antimicrobials and incubation times. All antimicrobials reduced bacterial population to below detection levels in vitro;however, essential oils and active components exhibited greater activity than plant extracts. Oregano oil and green tea reduced E. coli O157:H7 on lettuce and spinach to below detection. Plant-based antimicrobials have the potential to protect foods against E. coli O157:

Construction of HBV-specific ribozyme and its recombinant with HDV and their cleavage activity in vitro

AIM To construct the recombinant of HDVcDNA and HBV-specific ribozyme gene byrecombinant PCR in order to use HDV as atransporting vector carrying HBV-specificribozyme into liver cells for inhibiting thereplication of HBV.METHODS We separately cloned the ribozyme（RZ）gene and recombinant DVRZ（comprisingHDV cDNA and HBV-specific ribozyme gene）intothe downstream of T7 promoter of pTAdv-Tvector and studied the in vitro cleavage activityof their transcripts（rRZ,rDVRZ）on target RNA（rBVCF）from in vitro transcription of HBV Cgene fragment（BVCF）.RESULTS Both the simple（rRZ）and therecombinant ribozyme rDVRZ could efficientlycatalyze the cleavage of target RNA（rBVCF）under different temperatures（37℃,42℃ and55℃）and Mg2+concentrations（10 mmol/L,15 mmol/L and 20 mmol/L）and their catalyticactivity tended to increase as the temperaturewas rising.But the activity of rRZ was evidentlyhigher than that of rDVRZ.CONCLUSION The recombinant of HDV cDNAand ribozyme gene had the potential of beingfurther explored and used in gene therapy of HBVinfection.

Cloning and Sequence of Glycoprotein H Gene of Duck Plague Virus

The glycoprotein H （gH） gene homologue of duck plague virus （DPV） was cloned by degenerate polymerase chain reaction （PCR） and sequenced. It was located immediately downstream from the thymidine kinase gene （TK）. In addition, the 3＇-end of the gene homologue to herpesvirus UL21 was located downstream from the gH gene. DPV gH gene open reading frame （ORF） was 2 505 bp in length and its primary translation product was a polypeptide of 834 amino acids long. It possessed several characteristics of membrane glycoproteins, including an N-terminal hydrophobic signal sequence, an external domain containing eight putative N-linked glycosylation sites, a C-terminal transmembrane domain, and a charged cytoplasmic tail. Comparison with other herpesvirus revealed identities of 20.2, 25.1, 23.0, 23.0, 26.5 and 26.0% with the gH counterparts of the human herpesvirus virus 1 （HSV1）, equine herpesvirus 4 （EHV4）, bovine herpesvirus 1 （BHV1）, pseudorabies virus （PRV）, gallid herpesvirus 2 （GHV2） and gallid herpesvirus 3 （GHV3）, respectively.

Transplantation of Nogo-66 receptor gene-silenced cells in a poly(D,L-lactic-co-glycolic acid) scaffold for the treatment of spinal cord injury

Inhibition of neurite growth, which is in large part mediated by the Nogo-66 receptor, affects neural regeneration following bone marrow mesenchymal stem cell transplantation. The tissue engineering scaffold poly（D,L-lactide-co-glycolic acid） has good histocompatibility and can promote the growth of regenerating nerve fibers. The present study used small interfering RNA to silence Nogo-66 receptor gene expression in bone marrow mesenchymal stem cells and Schwann cells, which were subsequently transplanted with poly（D,L-lactide-co-glycolic acid） into the spinal cord lesion regions in rats. Simultaneously, rats treated with scaffold only were taken as the control group. Hematoxylin-eosin staining and immunohistochemistry revealed that at 4 weeks after transplantation, rats had good motor function of the hind limb after treatment with Nogo-66 receptor gene-silenced ceils prus the poly（O,L-lactide-co-glycolic acid） scaffold compared with rats treated with scaffold only, and the number of bone marrow mesenchymal stem cells and neuron-like cells was also increased. At 8 weeks after transplantation, horseradish peroxidase tracing and transmission electron microscopy showed a large number of unmyelinated and myelinated nerve fibers, as well as intact regenerating axonal myelin sheath following spinal cord hemisection injury. These experimental findings indicate that transplantation of Nogo-66 receptor gene-silenced bone marrow mesenchymal stem cells and Schwann cells plus a poly（D,L-lactide-co-glycolic acid） scaffold can significantly enhance axonal regeneration of spinal cord neurons and improve motor function of the extremities in rats following spinal cord injury.

Comparative Study on Growth Performance of Transgenic(Over-Expressed OsNHX1) and Wild-Type Nipponbare under Different Salinity Regimes

Transgenic Nipponbare which over-expressed a Na＋/H~ antiporter gene OsNHX1 was used to compare its growth performance, water status and photosynthetic efficiency with its wild type under varying salinity regimes. Chlorophyll content, quantum yield and photosynthetic rate were measured to assess the impact of salinity stress on photosynthetic efficiency for transgenic and wild-type Nipponbare. Effects of salinity on water status and gas exchange to both lines were studied by measuring water use efficiency, instantaneous transpiration rate and stomatal conductance. Dry shoot weight and leaf area were determined after three months of growth to assess the impacts of salinity on the growth of those two lines. Our study showed that both lines were affected by salinity stress, however, the transgenic line showed higher photosynthetic efficiency, better utilization of water, and better growth due to low transpiration rate and stomatal conductance. Reduction of photosynthetic efficiency exhibited by the wild-type Nipponbare was correlated to its poor growth under salinity stress.

Alleles contributing to acetyl coenzyme A carboxylase(ACCase)resistance in keng stiffgrass(Pseudosclerochloa kengiana)populations from China

Keng stiffgrass is a grass weed that affects wheat-rice cropping systems in China. The extensive reliance on acetyl coen- zyme A carboxylase （ACCase）-inhibiting herbicides has resulted in keng stiffgrass developing resistance to these herbicides. The objective of this research was to evaluate the resistance level of the putative resistant keng stiffgrass populations to ACCase-inhibiting herbicides and to identify their molecular resistance mechanism. Whole-plant dose-response experi- ments demonstrated that SD-4 （R）, SD-11 （R）, and JS-25 （R） populations were highly resistant to fenoxaprop, clodinafop, and fluazifop, moderately resistant to diclofop, had low resistance to sethoxydim and pinoxaden, but were sensitive to clethodim. Partial chloroplasticACCase sequences showed that there were two copies ofACCase gene in keng stiffgrass, and all homoeologous genes were expressed. The results of sequence analyses of the ACCase CT domain revealed an isoleucine-to-asparagine substitution at position 2041 in SD-4 （R） and SD-11 （R） populations, and a tryptophan-to-cysteine substitution at position 2027 in the JS-25 （R） population. To our knowledge, this is the first report of Ile-2041-Asn and Trp- 2027-Cys mutations in ACCase-resistant keng stiffgrass. In addition, three robust （derived） cleaved amplified polymorphic sequence （（d）CAPS） markers have been developed to rapidly identify these mutations in the ACCase gene of keng stiffgrass.

NQO1 C609T polymorphism correlated to colon cancer risk in farmers from western region of Inner Mongolia

Objective： To investigate the relationship between NAD（P）H：quinone oxidoreductase 1 （NQOI） C609T polymorphism and colon cancer risk in farmers from western region of Inner Mongolia. Methods： Polymerase chain reaction-restriction fragment length polymorphism （PCR-RFLP） was performed to analyze NQO1 C609T polymorphism from 160 healthy controls and 76 colon cancer patients. Results： Among the colon cancer patients, the incidence of NQOI T allele （53.29%） was significantly higher than it in control group （33.75%, P〈0.001）. The individuals with NQO1 T allele had higher risk [2.239 （95% CI： 1.510-3.321） times] to develop colon cancer than individuals with NQO1 C allele. The incidence of NQO1 （TFI-） （34.21%） in colon cancer patients was higher than that in control group （15.62%, P〈0.001）. Odds ratios （OR） analysis suggested that NQO1 （T/F） and NQOI （T/C） genotype carriers had 3.813 （95% CI： 1.836-7.920） times and 2.080 （1.026-4.219） times risk compared with wild-type NQO1 （C/C） gene carriers in developing colon cancer. Individuals with NQO1 （T/I＇） genotype had 2.541 （95% CI： 0.990-6.552） times, 3.713 （95% CI： 1.542-8.935） times, and 3.471 （95% CI： 1.356-8.886） times risk than individuals with NQOI （T/C） or NQOI （C/C） genotype in well- differentiated, moderately-differentiated, and poorly-differentiated colon cancer patients, respectively. Conclusions： NQO1 gene C609T could be one of risk factors of colon cancer in farmers from western region of Inner Mongolia,

Structure,Binding Characteristics,and 3D Model Prediction of a Newly Identified Odorant-Binding Protein from the Cotton Bollworm,Helicoverpa armigera (Hübner)

The full-length sequence of the odorant binding protein 5 gene,HarmOBP5,was obtained from an antennae cDNA library of cotton bollworm,Helicoverpa armigera （Hübner）.The cDNA contains a 444 bp open reading frame,encoding a protein with 147 amino acids,namely HarmOBP5.HarmOBP5 was expressed in Escherichia coli and the recombinant protein was purified by affinity chromatography.SDS-PAGE and Western blot analysis demonstrated that the purified protein can be used for further investigation of its binding characteristics.Competitive binding assays with 113 odorant chemicals indicated that HarmOBP5 has strong affinity to some special plant volatiles,including （E）-β-farnesene,ethyl butyrate,ethyl heptanoate,and acetic acid 2-methylbutyl ester.Based on three-dimensional （3D） model of AaegOBP1 from Aedes aegypti,a 3D model of HarmOBP5 was predicted.The model revealed that some key binding residues in HarmOBP5 may play important roles in odorant perception of H.armigera.This study provides clues for better understanding physiological functions of OBPs in H.armigera and other insects.

Herbivore defense responses and associated herbivore defense mechanism as revealed by comparing a resistant wild soybean with a susceptible cultivar

Plants have evolved sophisticated defense mechanisms against herbivores to help them adapt to the environment. Understanding the defense mechanisms in plants can help us control insects in a more effective manner. In this study, we found that compared with Tianlong 2(a cultivated soybean with insect susceptibility), ED059(a wild soybean line with insect resistance)contains sharper pubescence tips, as well as lower transcript levels of wound-induced protein kinase(WIPK) and salicylic acid-induced protein kinase(SIPK), which are important mitogen-activated protein kinases involved in early defense response to herbivores. The observed lower transcript levels of WIPK and SIPK induced higher levels of jasmonic acid(JA), JA biosynthesis enzymes(AOC3) and some secondary metabolites in ED059. Functional analysis of the KTI1 gene via Agrobacterium-mediated transformation in Arabidopsis thaliana indicated that it plays an important role in herbivore defense in ED059. We further investigated the molecular response of third-instar Helicoverpa armigera(Hübner) larvae to Tianlong 2 and ED059. We found apoptotic cells only in the midguts of larvae that fed on ED059. Compared with larvae reared on the susceptible cultivar Tianlong 2, transcript levels of catalase(CAT) and glutathione S-transferase(GST) were up-regulated, whereas those of CAR, CHSB, and TRY were down-regulated in larvae that fed on the highly resistant variety ED059. We propose that these differences underlie the different herbivore defense responses of ED059 and Tianlong 2.