A combination of genome-wide association study and selection signature analysis dissects the genetic architecture underlying bone traits in chickens.

The bones of chicken play an important role in supporting and protecting the body. The growth and development of bones have a substantial influence on the health and production performance in chickens. However, genetic architecture underlying chicken bone traits is not well understood. The objectives of this study are to dissect the genetic basis of bone traits in chickens and to identify valuable genes and genetic markers for chicken breeding. We performed a combination of genome-wide association study (GWAS) and selection signature analysis (fixation index values and nucleotide diversity ratios) in an F2 crossbred experimental population with different genetic backgrounds (broiler × layer) to identify candidate genes and significant variants related to femur, shank, keel length, chest width, metatarsal claw weight, metatarsal length, and metatarsal circumference. A total of 545 individuals were genotyped based on the whole genome re-sequencing method (26 F0 individuals were re-sequenced at 10 × coverage; 519 F2 individuals were re-sequenced at 3 × coverage). A total of 2 028 112 single-nucleotide polymorphisms (SNPs) remained to carry out analysis after quality control and imputation. The integration of GWAS and selection signature analysis indicated that all significant SNPs responsible for bone traits were mainly localized on chicken chromosomes 1, 4, and 27. Finally, we identified 21 positional candidate genes that might regulate chicken bone growth and development, including LRCH1, RB1, FNDC3A, MLNR, CAB39L, FOXO1, LHFP, TRPC4, POSTN, SMAD9, RBPJ, PPARGC1A, SLIT2, NCAPG, NKX3-2, CPZ, SPOP, NGFR, SOST, ZNF652, and HOXB3. Additionally, an array of uncharacterized genes was identified. The findings provide an in-depth understanding of the genetic architecture of chicken bone traits and offer a molecular basis for applying genomics in practical chicken breeding.

A functional variant in the promoter region of IGF1 gene is associated with chicken abdominal fat deposition.

Insulin-like growth factor 1 (IGF1) plays an important role in the regulation of cell growth, proliferation, differentiation, and apoptosis. Previously several studies revealed that genotypes of chicken IGF1 c.-366A > C were significantly associated with abdominal fat weight and body weight in chickens. But the underlying mechanism is still unknown. To investigate the mechanism underlying the association, herein, we performed IGF1 gene mRNA expression profiling, a dual-luciferase reporter assay and electrophoretic mobility shift assay (EMSA). Quantitative real-time PCR results showed that IGF1 gene was widely expressed in 14 tissues. The mRNA expression levels of IGF1 gene in both abdominal fat and jejunum were significantly higher in fat broilers than in lean broilers. However, the opposite results were observed in the pancreas. The reporter gene assay showed that the promoter luciferase activity of allele A was significantly higher than that of allele C (P < 0.05). In addition, the luciferase activity of allele A promoted by the transcription factor AP1 and OCT1 was higher than that of allele C (P < 0.05). Electrophoretic mobility shift assay result showed that allele A binding to the transcription factor AP1 and OCT1 was stronger than that of allele C. All in all, our data indicated that the IGF1 gene c.-366A > C is a functional SNP responsible for chicken adipose deposition.

[Survival status and influencing factors of HIV/AIDS cases in Liuzhou, 2008-2018].

Objective: To understand the duration of survival and related influencing factors of HIV/AIDS patients in Liuzhou city. Methods: Both life table method and Kaplan-Meier method were used to calculate the average survival time of HIV/AIDS patients aged ≥15 years reported in Liuzhou city from 2008 to 2018. Factors related to the duration of HIV/AIDS patients were analyzed by univariate and multivariate Cox regression models. Results: A total of 14 856 patients with HIV/AIDS were involved in this study and with the average duration of survival time as 98.74 (95%CI: 97.73-99.75) months. The cumulative survival rates of 1, 3, 5 and 10 years were 77.0%, 72.0%, 68.0%, 61.0% respectively. Results from the multivariate Cox proportional risk regression analysis showed that factors as sex, level of education, age when HIV infection was confirmed, occupation, route of transmission, source of samples, results of the first CD(4) test and antiviral treatment were all related to the duration of survival to the HIV/AIDS patients. Conclusions: Strategies involving early detection of HIV infection, improvement of the CD(4) initial detection rate and early antiviral treatment will help to significantly reduce the risk of death in HIV/AIDS population. Focus should be on male, middle-aged and elderly (over 41 years old), junior high school education or below farmers and migrant worker populations.

Residue depletion of ampicillin in eggs.

A residue depletion study of ampicillin (AMP) was performed after oral dosing (60.0 mg/kg and 120.0 mg/kg body weight once a day for 5 days) to laying hens, through the use of reversed-phase high-performance liquid chromatography with fluorescence detection (RP-HPLC-FLD) to achieve detection of ampicillin residue in eggs. Limit of detection was 0.5 ng/g, and limit of quantitation was 1.2 ng/g for ampicillin. Extraction recoveries of ampicillin from samples fortified at 5.0-125.0 ng/g levels ranged from 77.5% to 84.6% in albumen, 77.9% to 87.5% in yolk, and 77.9% to 88.6% in whole egg, with coefficients of variation ≤ 9.3%. The maximum concentrations of ampicillin in albumen, yolk, and whole egg were detected at 1, 2, and 1 day after the last administration of ampicillin, respectively. Ampicillin was not detectable in albumen at day 9 of withdrawal time, at day 10 and 11 in yolk, and day 9 and 11 in whole egg at each of those 2 dose levels. The theoretical withdrawal time of AMP in whole egg was 6.730 and 7.296 days for 60 and 120 mg/kg oral dosage, respectively. This method also proved to be suitable as a rapid and reliable method for the determination of ampicillin in other poultry eggs.

Transcriptomic analysis of Camellia ptilophylla and identification of genes associated with flavonoid and caffeine biosynthesis.

Camellia ptilophylla, or cocoa tea, is naturally decaffeinated and its predominant catechins and purine alkaloids are trans-catechins and theobromine Regular tea [Camellia sinensis (L.) O. Ktze.] is evolutionarily close to cocoa tea and produces cis-catechins and caffeine. Here, the transcriptome of C. ptilophylla was sequenced using the 101-bp paired-end technique. The quality of the raw data was assessed to yield 70,227,953 cleaned reads totaling 7.09 Gbp, which were assembled de novo into 56,695 unique transcripts and then clustered into 44,749 unigenes. In catechin biosynthesis, leucoanthocyanidin reductase (LAR) catalyzes the transition of leucoanthocyanidin to trans-catechins, while anthocyanidin synthase (ANS) and anthocyanidin reductase (ANR) catalyze cis-catechin production. Our data demonstrate that two LAR genes (CpLAR1 and CpLAR2) by C. ptilophylla may be advantageous due to the combined effects of this quantitative trait, permitting increased leucoanthocyanidin consumption for the synthesis of trans-catechins. In contrast, the only ANS gene observed in C. sinensis (CsANS) shared high identity (99.2%) to one homolog from C. ptilophylla (CpANS1), but lower identity (~80%) to another (CpANS2). We hypothesized that the diverged CpANS2 might have lost its ability to synthesize cis-catechins. C. ptilophylla and C. sinensis each contain two copies of ANR, which share high identity and may share the same function. Transcriptomic sequencing captured two N-methyl nucleosidase genes named NMT1 and NMT2. NMT2 was highly identical to three orthologous genes TCS2, PCS2, and ICS2, which did not undergo methylation in vitro; in contrast, NMT1 was less identical to TCS, PCS and ICS, indicating that NMT1 may undergo neofunctionalization.

Charge and charging compensation on oxides and hydroxides in oxygen environmental SEM.

Oxygen environment was applied to the scanning electron microscopy (SEM) analysis of insulating samples. In the high vacuum SEM, a local oxygen pressure was provided, and in the environmental SEM, oxygen atmosphere was used instead of water, the commercial mode. The charging effects in the SEM observation and component characterization of samples such as Al(2)O(3), Al(OH)(3), Mg(OH)(2) and others can be eliminated or significantly reduced. The oxygen environment does not only provide a new approach to releasing the charging difficulty in the analyses using electron beam as a probe, but also provide an insightful hint to the understanding of the charging processes in general.

Combinational synthesis of oligonucleotides and assembly fabrication of oligonucleotide array.

In this paper, a simple, reliable and flexible method, which integrated in situ synthesis with the spotting technique, was reported to fabricate oligonucleotide array. Different oligonucleotide sequences are synthesized on their relative code glass slides through combinational chemistry, thus the slides are broken into smaller pieces, in which the same code pieces have the same probe sequences. An oligonucleotide array is fabricated by arbitrarily assembling these different code pieces onto another solid substrate. In principle experimentation, four different sequences of P16 gene were synthesized and a 5 x 5 array including these four sequences and the control black was fabricated. The analysis results indicated that the hybridization fluorescence intensity of the same sequences locating different sets on the array gave the approximate values, and the fluorescence intensity ratio of matched sequence to one middle location base mismatched, two base mismatched, three middle base mismatched is (1.000+/-0.080):(0.4991+/-0.0671):(0.2360+/-0.0044):(0.0493+/-0.0033). Their relative accuracies were from 6.64 to 10.2%. This result might be used to rapidly screen single-nucleotide polymorphisms (SNPs).

Suppression of post-transcriptional gene silencing by a plant viral protein localized in the nucleus.

Post-transcriptional gene silencing (PTGS) is a homology-dependent RNA degradation process that may target RNA exclusively in the cytoplasm. In plants, PTGS functions as a natural defense mechanism against viruses. We reported previously that the 2b protein encoded by cucumber mosaic cucumovirus (CMV) is a virulence determinant and a suppressor of PTGS initiation in transgenic Nicotiana benthamiana. By fusion with the green fluorescent protein, we now show that the CMV 2b protein localizes to the nuclei of tobacco suspension cells and whole plants via an arginine-rich nuclear localization signal, (22)KRRRRR(27). We further demonstrate that the nuclear targeting of the 2b protein is required for the efficient suppression of PTGS, indicating that PTGS may be blocked in the nucleus. In addition, our data indicate that the PTGS suppressor activity is important, but not sufficient, for virulence determination by the 2b protein.

Strong host resistance targeted against a viral suppressor of the plant gene silencing defence mechanism.

The 2b protein encoded by cucumber mosaic cucumovirus (Cmv2b) acts as an important virulence determinant by suppressing post-transcriptional gene silencing (PTGS), a natural plant defence mechanism against viruses. We report here that the tomato aspermy cucumovirus 2b protein (Tav2b), when expressed from the unrelated tobacco mosaic tobamovirus (TMV) RNA genome, activates strong host resistance responses to TMV in tobacco which are typical of the gene-for-gene disease resistance mechanism. Domain swapping between Cmv2b, which does not elicit these responses, and Tav2b, revealed functional domains in Tav2b critical for triggering virus resistance and hypersensitive cell death. Furthermore, substitution of two amino acids from Tav2b by those found at the same positions in Cmv2b, Lys21-->Val and Arg28-->Ser, abolished the ability to induce hypersensitive cell death and virus resistance. However, in Nicotiana benthamiana, a species related to tobacco, Tav2b functions as a virulence determinant and suppresses PTGS. Thus, a viral suppressor of the host gene silencing defence mechanism is the target of another independent host resistance mechanism. Our results provide new insights into the complex molecular strategies employed by viruses and their hosts for defence, counter-defence and counter counter-defence.

Mitotic stability of infection-induced resistance to plum pox potyvirus associated with transgene silencing and DNA methylation.

Plum pox potyvirus (PPV) infection of transgenic Nicotiana benthamiana plants that expressed the PPV NIb RNA replicase carrying a Gly to Val mutation at the GDD motif (NIbV lines) induced a phenotype of virus resistance and transgene silencing, which was not transmissible to the progeny after self-fertilization (H. S. Guo and J. A. García, Mol. Plant-Microbe Interact. 10:160-170, 1997). Here, we demonstrate that the induced resistance of NIbV plants is mitotically stable after plant propagation by grafting and by in vitro regeneration. Virus replication or residual virus RNA seem not to be required to maintain transgene silencing and virus resistance. Analysis by PCR (polymerase chain reaction) amplification after treatment with methylation-sensitive restriction nucleases indicates that DNA methylation is associated with establishment and maintenance of transgene silencing and virus resistance. Restoration of transgene activity and susceptibility to PPV in sexual progeny correlated with resetting of transgene DNA methylation. On the basis of these and other published results, we present a general model for post-transcriptional gene silencing in which RNA signals, generated either by a silenced nuclear gene or by virus replication, both activate a specific cytoplasmic RNA degradation pathway and induce changes (in particular, DNA methylation) in homologous nuclear genes that switch them from an active to a silenced status.

Susceptibility to recombination rearrangements of a chimeric plum pox potyvirus genome after insertion of a foreign gene.

Infectious RNA transcripts were generated from a chimeric cDNA clone of the plum pox potyvirus (PPV) genome containing the bacterial beta-glucuronidase (GUS) gene inserted between the sequences coding for the P1 and HC proteins. An artificial cleavage site specific for the NIa viral proteinase was engineered between the GUS and HC sequences to produce free GUS and HC proteins. The resulting virus PPVGus/ was stably maintained during the first round of infection, although plants remained symptomless and virus accumulation was delayed with respect to wild-type infection. PPVGus/ deleted variants, missing between 645 and 1779 nt, were detected in a subsequent plant passage. PPVGus/ deletions were confined inside the GUS gene, never affecting the P1 and HC coding regions, in contrast with previous reports of deletions in other potyvirus-based vector, in which deletions frequently reached the HC gene. These results suggest that the N-terminus of the PPV HC protein may be essential for virus viability. Analysis of the deletion endpoints showed short stretches of similarity in donor and acceptor RNAs, as well as oligo A tracts conserved in most junction sites, suggesting that deletions in PPVGus/ might take place by similarity-assisted recombination events.

Plum pox potyvirus resistance associated to transgene silencing that can be stabilized after different number of plant generations.

Nicotiana benthamiana plants were transformed with a fragment of the plum pox potyvirus (PPV) genome that encodes the nuclear inclusion a (NIa) and b (NIb) proteins and the N-terminus of the capsid protein (NIa-NIb-CP). Lines transformed with this PPV genomic fragment harboring mutations in the GDD replicase-motif were also obtained. Plants of NIaDeltaV lines that carry a GDD to VDD mutation in the PPV transgene, were immune to PPV infection. The resistance was highly specific, since it was only partially overcome by a PPV strain different to that from which the transgene was derived, and no resistance was observed after inoculation with a second potyvirus. PPV was not able to replicate in protoplasts isolated from NIaDeltaV transgenic plants, indicating that the resistance was functional at the single cell level. Only a fraction of plants from lines transformed with the NIa-NIb-CP fragment harboring a GDD to ADD mutation (NIaDeltaA lines), were resistant to PPV infection. This same phenotype was observed in plants expressing the wild-type construction (NIaDelta), although the progeny of some non-infected plants seemed to be completely resistant to PPV, independently of the allelic status of the parental plant. In all cases, the resistance phenotype correlated positively with low levels of transgene mRNA accumulation, suggesting that it was mainly due to a gene silencing mechanism. Our results show that, although the transgene was not silenced in all R1 plants from some individual lines, a stable silenced status could be reached in the following generations.

Long sequences in the 5' noncoding region of plum pox virus are not necessary for viral infectivity but contribute to viral competitiveness and pathogenesis.

The 5'-terminal 31 nucleotides of the 146-nucleotides-long 5' noncoding region of plum pox potyvirus (PPV) are highly conserved in all the members of the Potyvirus genus. To map the sequences of the 5' noncoding region that are necessary in vivo for infectivity, we have constructed a nested set of substitution and deletion mutants. While we were not able to infect Nicotiana clevelandii plants with full-length PPV transcripts bearing mutations in the 5'-terminal 35 nucleotides of the viral genome, the deletion of long sequences located between nucleotides 39 and 145 did not alter either the rate of infection or viral accumulation. Nevertheless, these mutants were not able to compete with the wild-type strain in coinoculation experiments. Plants infected with a PPV mutant that lacked nucleotides 127 to 145 showed a very mild symptomathology; the wild-type symptom severity was recovered after spontaneous second-site mutations.

The motif V of plum pox potyvirus CI RNA helicase is involved in NTP hydrolysis and is essential for virus RNA replication.

The plum pox potyvirus (PPV) protein CI is an RNA helicase whose function in the viral life cycle is still unknown. The CI protein contains seven conserved sequence motifs typical of RNA helicases of the superfamily SF2. We have introduced several individual point mutations into the region coding for motif V of the PPV CI protein and expressed these proteins in Escherichia coli as maltose binding protein fusions. Mutations that abolished RNA helicase activity also disturbed NTP hydrolysis. No mutations affected the RNA binding capacity of the CI protein. These mutations were also introduced in the PPV genome making use of a full-length cDNA clone. Mutant viruses carrying CI proteins with reduced RNA helicase activity replicated very poorly in protoplasts and were unable to infect whole plants without rapid pseudoreversion to wild-type. These results indicate that motif V is involved in the NTP hydrolysis step required for potyvirus RNA helicase activity, and that this activity plays an essential role in virus RNA replication inside the infected cell.

Cap-independent leaky scanning as the mechanism of translation initiation of a plant viral genomic RNA.

The genome of plum pox virus contains a single open reading frame that is translated into a large polyprotein. Although the open reading frame starts at nucleotide 36 (36AUG), it is translated from the second, 147AUG, which is in a more favourable context for translation initiation. We have carried out in vitro translation and transient expression analysis in protoplasts of a nested set of substitution and deletion mutants, and the results show that no internal structure in the 5' noncoding region of plum pox virus is necessary for efficient translation initiation. On the other hand, when the cryptic 36AUG was placed in a favourable context, it turned into an efficient initiation codon in vitro. Furthermore, AUGs that were placed in a favourable context, initiating short intraleader open reading frames, repressed translation initiation from the 147AUG in vitro and in vivo. These results point to leaky scanning as the mechanism of translation initiation of plum pox virus RNA. Nevertheless, it is a peculiar leaky scanning where the initiation of translation does not require a cap structure at the 5' end. This fact is congruent with the experimentally predicted absence of a stable secondary structure at the 5' noncoding region.

Modification of atrioventricular conduction by selective AV nodal artery catheterization.

The effects of selective AV nodal artery embolization on AV nodal function was investigated in six closed-chest adult dogs. Programmed atrial stimulation was performed to determine control values for AV nodal effective refractory period (AVN-ERP) and the paced cycle length at which AV nodal Wenckebach conduction occurred (WCL). Using standard percutaneous femoral techniques of coronary artery catheterization, a flexible infusion catheter was positioned selectively in the AV nodal artery. Proper positioning of the catheter was confirmed angiographically and by selective acetylcholine (ACH) infusion into the AV nodal artery, which caused transient complete AV nodal block in three dogs, and for the group, caused lengthening of both AVN-ERP and WCL. Following cessation of ACH infusion and autonomic blockade with atropine 0.04 mg/kg and propranolol 0.2 mg/kg, denervated recontrol values for AVN-ERP and WCL were 192 msec and 243 msec, respectively. The AV nodal artery was then embolized with a suspension of cross-linked collagen fibrils in either normal saline or absolute ethanol. Successful embolization of the AV nodal artery, confirmed angiographically, caused an acute increase in AVN-ERP (243 msec, P less than 0.05 compared to denervated control) and WCL (287 msec, P = 0.058 compared to denervated control). However, at a mean follow-up of 37 days, only one animal exhibited a chronic increase in AVN-ERP and WCL. Selective AV nodal artery catheterization can be performed using standard percutaneous catheterization techniques. Selective administration of agents with direct cidal effects on the AV node using this technique may provide an alternative to conventional methods of catheter ablation of AV conduction in patients with drug-resistant supraventricular arrhythmias.