pp 535-544 September 2017 RESEARCH ARTICLE
WNT-ß-catenin-TCF pathway is involved in carcinogenesis and foetal development. As a member of the WNT gene family, Wnt8A encodes secreted signalling proteins and responds to many biological processes.However, similar research on the effects of genetic variations of Wnt8A gene on growth traits is lacking. Therefore, in this study, polymorphisms of Wnt8A were detectedin 396 animals from Chinese Qinchuan cattle using DNA pool sequencing and PCR-RFLP methods. Four novel single-nucleotide polymorphisms (SNPs) of Wnt8A gene were identified, including three mutations in introns (g.T-445C, g.G244C and g.G910A) and one in exon (g.T4922C). Additionally, we examined the associations of four SNPs with growth traits. The results revealed thatSNP2 (g.G244C) was significantly associated with shoulder height, hip height, body length, hip width, and body weight (P <0.05). SNP3 (g.G910A) also displayed notable effects on hip width (P < 0.05). Meanwhile, the haplotype combination CC-GC-GA-CC was strongly associated with heavier, taller and longer animals (P < 0.05). These results show that the Wnt8A gene may be apotential candidate gene, and the SNPs could be used as molecular markers in early marker-assisted selection in beef cattle breeding programmes.
pp 545-550 September 2017 RESEARCH ARTICLE
Cytosine methylation is an effectiveway to modulate gene transcription.However, very little is knownabout the epigenetic changes in the host that is infected with Salmonella enterica. In this study, we usedmethylatedDNA immunoprecipitation sequencing to analyse the genomewide DNA methylation changes in domestic chickens after infected with Salmonella. The level of DNA methylation was slightly higher in the genomic regions around the transcription start termination sites in a Salmonella-infected group compared to the controls. Overall, 879 peaks were differentially methylated between Salmonella-infected and control groups, amongwhich 135 were located in the gene promoter regions. Genes including MHC class IV antigen, GABARAPL1, MR1 and KDM1B were shown to be methylated more heavily after infected with Salmonella, whereas DYNLRB2, SEC14L3 and ANKIB1 tended to have fewer methylated cytosine residues in the promoter regions.Gene interaction network analysis of differentiallymethylated genesin the promoter regions revealed extensive connections with immune-related genes, indicating the possible impact of infection with Salmonella on the epigenetic status of the host.
pp 551-561 September 2017 RESEARCH ARTICLE
Pigeon pea (Cajanus cajan), an important legume crop is predominantly cultivated in tropical and subtropical regions of Asia and Africa. It is normally considered to have a low degree of genetic diversity, an impediment in undertaking crop improvement programmes.We have analysed genetic polymorphism of domesticated pigeon pea germplasm (47 accessions) across the world using earlier characterized panzee retrotransposon-based molecularmarkers. Itwas conjectured that since retrotransposons are interspersed throughout the genome, retroelements-based markers would be able to uncover polymorphism possibly inherent in the diversity of retroelement sequences. Two PCR-based techniques, sequence-specific amplified polymorphism (SSAP) and retrotransposon microsatellite amplified polymorphism (REMAP) were utilized for the analyses.We show that a considerable degree of polymorphism could be detected using these techniques. Three primer combinations in SSAP generated 297 amplified products across 47 accessionswith an average of 99 amplicons per assay. Degree of polymorphism varied from 84–95%. In the REMAP assays, the number of amplicons was much less but up to 73% polymorphism could be detected. On the basis of similarity coefficients, dendrograms were constructed. The results demonstrate that the retrotransposon-based markers could serve as a better alternative for the assessment of genetic diversity in crops with apparent low genetic base.
pp 563-570 September 2017 RESEARCH ARTICLE
The high molecular weight glutenin subunits (HMW-GS) in bread wheat are major determinants of the viscoelastic properties of dough and the end-use quality of wheat flour. Two novel HMW-GSs, 1Cx1.1 and 1Cy9.1, from the diploid speciesAegilops markgrafii (CC) were identified in the present study. The corresponding open-reading frames of the genes of 1Cx1.1 and 1Cy9.1 were isolated and sequenced using allele-specific polymerase chain reaction. Sequence comparison demonstrated that the HMW-GSs from Ae. markgrafii possess a similar primary structure to the homologous proteins in wheat and related species. A tandem tripeptide exists in the central repetitive domain of 1Cx1.1, and this unique structure is very rare in the HMW-GSs of other genomes. To confirm the authenticity of these isolated endogenous HMW-GS, the heterologous proteins produced by removing the signal peptides expressed by E. coli exhibited the same electrophoretic mobility as the native proteins. Subsequently, the singleprotein was purified at a sufficient scale for incorporation into flour to performsodium dodecyl sulphate (SDS) sedimentation testing. Notably, the SDS sedimentation volume was less with the addition of 1Cx1.1 than it was with 1Cy9.1.
pp 571-582 September 2017 RESEARCH ARTICLE
Salinity is the second most important abiotic stress after drought that hampers rice production, especially in south and Southeast Asia. Breeding approach supplemented with molecular markers-assisted selection is the most promising approach in terms of efficiency to increase the productivity under salt-affected soils. Thirty-day-old rice seedlings of 300 F5:6 recombinant-inbred lines derived from a cross between the salt sensitive, IR29 (indica), and a salt tolerant, Hasawi (aus), were used to identify quantitative trait loci (QTLs) linked to salinity tolerance. One hundred and ninety four polymorphic SNP markers were used to construct a genetic linkage map involving 142 selected RILs that covered 1441.96 cM genome with an average distance of 7.88 cM between loci. Twentynew QTLs (LOD > 3) were identified on chromosomes 1, 2, 4, 6, 8, 9 and 12 using composite interval mapping with R2 as high as >20% with LODvalue of 7.21. Many earlier studies reported big qSaltol for seedling stage salinity tolerance in rice is on short arm of chromosome 1 but none of the QTL in our study was on qSaltol or nearby position, therefore, Hasawi conferred salinity tolerance in RILs due to novel QTLs. It is suggested to fine map the novel QTLs so that the level of salinity tolerance could be further enhanced by pyramiding of the different QTLs in one genetic background through marker-assisted selection.
pp 583-589 September 2017 RESEARCH ARTICLE
Gaucher disease (GD), the most frequent lysosomal storage disease, is caused by heterogeneous mutations in the locus coding for glucocerebrosidase (GBA). It is an autosomal recessive disorder with different phenotypes of which the most frequent is the nonneuronopathic or type 1, prevalent worldwide. To date, more than 430 mutations have been described, but their frequency distribution varies in different populations with four, N370S, L444P, IVS2 + 1G > A and 84insG, being the most frequent ones. In Venezuela, 20 unrelated index cases with GD type I were assessed for GBA mutation detection and for their in-phase haplotypeidentification, to gather genetic epidemiological data on the disease in the country and of its eventual ethnic origin. Ten missense mutations and two complex alleles were identified. The most frequent were N370S (42.5%), L444P (20%), IVS2+1G > A (10%) and R48W (5%); mutations R120W, P245H, H311R, R496H, W36X and R433G which were carried by a single chromosome each one.Three geographical foci were identified, displaying mutation heterogeneity. N370S had multiple genetic origins, different from the Ashkenazi’s; a single common remote ancestor for this mutation in the country was dismissed, according to the haplotype analysis. All mutations have a likely European Caucasoid descent.
pp 591-597 September 2017 RESEARCH ARTICLE
The essential role of the acetyl-CoA carboxylase (ACACA) enzyme in milk fatty acid (FA) synthesis suggests that it may be responsible for the phenotypic variability observed inmilk.Before attempting association analyses between this gene and/or enzyme and phenotypic traits, a study on the genetic variability within this locus is required. The aim of this work was to sequence the entirecoding region of ACACA gene in Valle del Belice sheep breed to identify polymorphic sites. A total of 51 coding exons of ACACA gene were sequenced in 32 individuals of Valle del Belice sheep breed. Sequencing analysis and alignment of obtained sequences showed the presence of 23 polymorphic sites. The most polymorphic was exon 53 which showed presence of 12 single-nucleotide polymorphisms (SNPs), ofwhich eight were missense mutations, caused amino acid changes and therefore may affect protein functionor stability causing variation in phenotype. The identified polymorphisms showed high variability of the ACACA gene. Sequences analysis allowed to find six new SNPs in exon 53 (6832C>T; 6835C>A; 6840G>A; 6847G>T; 6852C>T and 6860G>C). A total of 31 haplotypes were inferred. Although this study could not provide association study with production traits, it shows finding of novelSNPs that might be important in future studies and laid the basis for further association analyses needed to evaluate the potential use of these SNPs as genetic markers for fat content and FAs composition in milk of Valle del Belice sheep breed.
pp 599-612 September 2017 RESEARCH ARTICLE
While the acquisition of drug resistance is often accompanied by fitness costs, Mycobacterium tuberculosis has developed mechanisms to overcome these costs in the form of compensatory mutations. In an attempt to dissect strain-specific differences in biological fitness, 10 M. tuberculosis genomes, representing F15/LAM4/KZN, Beijing, F11 and F28 genotypes were sequenced on the Illumina MiSeq platform. Drug-susceptible F15/LAM4/KZN strains differed by 43 SNPs, demonstrating that heterogeneity exists even among closely-related strains. We found unique, nonsynonymous single-nucleotide polymorphisms (SNPs) in the sigA and grcC1 genes of multidrug resistant (MDR) and XDR F15/LAM4/KZN strains, respectively. The F28 MDR strain harboured a novel ubiA mutation in combination with its embB M306I mutation, which may be related to ethambutol resistance. In addition, it possessed a low-frequency rpoC mutation, suggesting that this strain was in the process of developing compensation. In contrast, nocompensatory mutations were identified in Beijing and F11 MDR strains, corroborating its low in vitro fitness. Clinical strains also harboured unique SNPs in a number of important genes associated with virulence, highlighting the need for future studies which examine the correlation of genetic variations with phenotypic diversity. In summary, whole-genome sequencing revealed the presence of fitness-compensatory mutations in F15/LAM4/KZN and F28 genotypes which predominate in MDR and/or extensively drug resistant (XDR) forms in KwaZulu-Natal, South Africa.
pp 613-623 September 2017 RESEARCH ARTICLE
Smac/DIABLO gene is essential for the apoptosis mechanism in mammals. This study is the first report of the Megalobrama amblycephala (ma) diablo gene, and the first report of the tertiary structure of a Diablo polypeptide in fish. Madiablois 1540-bp long with an open reading frame of 792 bp, encoding a putative protein of 263 amino acids with a molecular weight of 29.2 kDa. Phylogenetic analysis indicates that it is closely related to the zebrafish Diablo-a homologue. It also indicates the existence of two diablo copies (a and b) in teleosts; apart fromthe Percomorpha group,where diablo-b has been lost, but diablo-a had undergone an independent duplication. Madiablo protein contains a long Smac_DIABLO super family domain (Leu32–Asp263) and alpha heliceswere prevalent in the secondary structure. Homology model of madiablo protein was constructed using the comparative modelling method. Expression of madiablo mRNA transcript was investigated using qPCR: (i) in five tissues from a healthy blunt snout bream, indicating the highest constitutive expression level in liver. (ii) During the embryo and juvenile development, indicating a spike inexpression during hatching and in later phases of the juvenile development. (iii) In response to Aeromonas hydrophila infection, indicating the downregulation in liver, spleen and kidney during the first 12 h postinfection and upregulation in spleen and kidney after 24 h postinfection (hpi). The results imply that madiablo is homologous to Diablo orthologues in other species, both structurallyand functionally, and that, it probably plays a role in the immune system of M. amblycephala.
pp 625-631 September 2017 RESEARCH ARTICLE
Esterases are known to play essential role inmetabolism, reproductive physiology and behaviour of Drosophila. Esterases are highly polymorphic enzymes in Drosophila, but the polymorphism of these enzymes is not well studied in Drosophila ananassae. Recent studies on esterase polymorphism in D. ananassae revealed that Est-4 locus comprises Est-4 active and Est-4 null allelesdepending on enzymatic activity. For the in vivo functional characterization of this locus, homozygous lines of genotypes Est-4 active and Est-4 null were derived from the flies collected from Gangtok, Sikkim, in 2006. Mating propensity, mating pattern, fecundity, fertility and productivity of female, life span and triglycerides level were investigated in the flies bearing either Est-4 active or Est-4 null genotypes. Results showed that mating occurred randomly with nonsignificant difference in mating propensity between Est-4 active and Est-4 null flies. However, a significant difference in fecundity and strong dependency between genotypes and the rate of fertility was found. The median values of progeny produced per female were 24 and 20 for Est-4 active and Est-4 null genotypes,respectively. The life span assay showed a significant difference in the survivorship between the two genotypes. Triglycerides level was higher in Esterase-4 active larval haemolymph as well as in mature flies’ homogenate than that of Esterase-4 null. Thus, Esterase-4 locus of D. ananassae has its role in fecundity, fertility and productivity of female, life span control and lipid metabolism.
pp 633-639 September 2017 RESEARCH ARTICLE
The detoxifying activity of glutathione S-transferases (GST) enzymes not only protect cells from the adverse effects of xenobiotics, but also alters the effectiveness of drugs in cancer cells, resulting in toxicity or drug resistance. In this study, we aimed to evaluate the association of GSTM1, GSTT1 and GSTP1 Ile105Val polymorphisms with treatment response among Malaysian chronic myeloid leukaemia (CML) patients who everyday undergo 400 mg of imatinib mesylate (IM) therapy. Multiplex polymerase chain reaction (multiplex-PCR) was performed to detect GSTM1 and GSTT1 polymorphisms simultaneously and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis was conducted to detect the GSTP1 Ile195Val polymorphism. On evaluating the association of the variant genotype with treatment outcome, heterozygous variant (AG) and homozygous variant (GG) of GSTP1 Ile105Val showed significantly a higher risk for the development of resistance to IM withOR: 1.951 (95% CI: 1.186–3.209, P = 0.009) and OR: 3.540 (95% CI: 1.305–9.606, P = 0.013), respectively. Likewise, GSTT1 null genotype was also associated with a significantly higher risk for the development of resistance to IM with OR = 1.664 (95% CI: 1.011–2.739, P = 0.045). Our results indicate the potential usefulness of GST polymorphism genotyping in predicting the IMtreatment response among CML patients.
pp 641-646 September 2017 RESEARCH ARTICLE
Paratuberculosis is one of the chronic granulomatous enteritis that predominantly affects ruminantsworldwide, caused by Mycobacterium avium ssp. paratuberculosis (MAP). In ruminants, microsatellite polymorphisms of the 3' untranslated region (3'UTR) of the solute carrier family 11 member A1 (SLC11A1) gene were associated with resistance to intracellular pathogen infections. Thisresearch was carried out to detect the polymorphisms in A and B regions of the 3'UTR of SLC11A1 gene and to evaluate the potential association between these polymorphisms and MAP infection in goats. MAP-specific antibodies were detected by ELISA and MAP infection was confirmed by IS900 PCR in 150 adult goats from different regions of Kerala, India. The polymorphism ofmicrosatellite regions A and B at 3'UTR of the SLC11A1 gene was analysed in goats by an automated technique, fragment analysis, using fluorescent-tagged forward primers. Eight alleles with sizes ranging from 221 to 239 bp were found in region A. Region B revealed two alleles, 117 bp (B7) and 119 bp (B8). Animals with B8 alleles were found to have higher incidence of paratuberculosis than animals with B7alleles (P < 0.01). There was no statistically significant association found between region A genotypes and paratuberculosis incidence. These results suggest that caprine SLC11A1 gene has significant role in paratuberculosis resistance ingoats and further studies might help in development of a PCR-based genotyping test for paratuberculosis resistance and selection of superior animals for future goat breeding programmes.
pp 647-652 September 2017 RESEARCH ARTICLE
Hand-foot-genital syndrome (HFGS) is a rare autosomal dominant inherited syndrome characterized by limb malformations and urogenital defects. HFGS is caused by mutations in the HOXA13 gene. The aim of this study was to identifycausative mutations in individuals and to explore the molecular pathogenesis in a Chinese family with HFGS. We performed Sanger sequencing and identified a recurrent missense mutation in the homeodomain (c.1123G>T, p.V375F) of HOXA13, molecular modelling predicted the mutation would affect DNA binding, and a luciferase reporter assay indicated that it impaired the ability ofHOXA13 to activate transcription of the human EPHA7 promoter. This is the first report of the molecular basis for HFGS caused by missense mutations of HOXA13.
pp 653-663 September 2017 RESEARCH ARTICLE
The apical membrane antigen-1 (AMA-1) of Plasmodium spp. is a merozoite surface antigen that is essential for the recognition and invasion of erythrocytes. Polymorphisms occurring in this surface antigen will cause major obstacles in developing effective malaria vaccines based on AMA-1. The objective of this study was to characterize ama1 gene in Plasmodium knowlesi isolates from Sabah. DNA was extracted from blood samples collected from Keningau, Kota Kinabalu and Kudat. The Pkama1 gene was amplified using nested PCR and subjected to bidirectional sequencing. Analysis of DNA sequence revealed that most of the nucleotide polymorphisms were synonymous and concentrated in domain I of PkAMA-1. Forteen haplotypes were identified based on amino acid variations and haplotype K5 was the most common haplotype. dN/dS ratios implied that purifying selection was prevalent in Pkama1 gene. Fu and Li’s D and F values further provided evidence of negative selection acting on domain II ofPkama1. Lownucleotide diversitywas also detected for the Pkama1 sequences,which is similar to reports on Pkama1 from Peninsular Malaysia and Sarawak. The presence of purifying selection and low nucleotide diversity indicated that domain II of Pkama1 can be used as a target for vaccine development.
pp 665-671 September 2017 RESEARCH ARTICLE
Genus Astyanax is well distributed in Neotropical freshwater environments and its taxonomic position is uncertain, as is the case with other Characidae genera allocated in the group incertae sedis. This study aimed to analyse the karyotype of different populations of Astyanax fasciatus (Corumbataí River basin) using Giemsa staining, C-band technique, and fluorescence in situ hybridization for the H3 histone and 5S rRNA genes, in addition we describe for the first time the chromosomal organization of H3 histone and 5S rRNAgenes in A. marionae (ParaguayRiver basin). Chromosomes of three A. fasciatus populations were analysed (two with 2n = 50 and one with 2n = 48) and the heterochromatin was organized in two forms (blocks with blurred boundaries and distinct blocks). H3 histone and 5S rRNA genes were observed in all the three populations of A. fasciatus on two chromosome pairs (one metacentric chromosome showing H3 histone and 5S rRNA gene clusters). In A. marionae (2n = 48), H3 histone and5S rRNA genes were observed in one acrocentric chromosome pair (different pairs). Further, differences between karyotypes and heterochromatin, as well as the chromosomal organization of H3 histone and 5S rRNA genes in Astyanax species, focussing on chromosome evolution in the group are discussed.
pp 673-679 September 2017 RESEARCH ARTICLE
The objective of this study was to investigate the effect of the polymorphism in the Myf5 gene on meat quality traits in the Ira and Tianfu Black rabbit breeds using polymerase chain reaction and DNA sequencing. A total of six SNPs and four haplotypes were found in Ira rabbits and only two SNPs were found in Tianfu Black rabbits. The two rabbit breeds had intermediate levels of genetic diversity according to their polymorphic information content values. The SNP association analysis in Ira indicated that SNP1-6 had a significant association with redness, yellowness and intramuscular fat values in the biceps femoris muscle, and alsoa significantly effect on redness in the longissimus dorsi muscle. The haplotype association analysis indicated that some haplotypes could be selected to get higher or lower meat redness, yellowness and intramuscular fat content in longissimus dorsi in Ira rabbits. Several SNPs and haplotypes of Myf5 identified here could be considered as molecular markers to improve the meat quality of Iraand Tianfu Black rabbits.
pp 681-685 September 2017 RESEARCH NOTE
Epilepsy is one of the most common neurological disorders with about 500 genes thought to be involved across the phenotypic spectrum (Busch et al. 2014; Ran et al. 2014), which includes monogenic, multigenic, epistatic and pleiotropic phenotype manifestations (Busch et al. 2014; Thomas et al. 2014), driving the need for a comprehensive diagnostic test. Next-generation sequencing (NGS) allows for the simultaneous investigation of a large number of genes, making it a very attractive option for a condition as diverse as epilepsy at a low cost compared to traditional Sanger sequencing (Lemke et al. 2012; Németh et al. 2013). Our 377 gene epilepsy NGS test was developed to include genes known to cause or have published association with epilepsy and seizure-related disorders. Given the scale of information that is generated, the efficacy of an NGS panel depends on a number of factors, including the genes present on the panel, prebioinformatic and postbioinformatic analysis protocols, as well as reporting criteria, prompting the current study, a retrospective analysis of 305 cases tested for the epilepsy panel.
pp 687-693 September 2017 RESEARCH NOTE
YIRUI WANG YIMIN SUN YONGQING HUANG YONGCHU PAN BING SHI JIAN MA LAN MA FEIFEI LAN YUXI ZHOU JIAYU SHI JINFANG ZHU HONGBING JIANG LEI ZHANG XUE XIAO MIN JIANG AIHUA YIN LILI YU LIN WANG JING CHENG YINXUE YANG
Nonsyndromic cleft lip with or without cleft palate (NSCL/P) is a common birth defect due to abnormal orofacial development. Previous studies report abnormal sonic hedgehog (SHH) signalling activity during NSCL/P pathogenesis and propose several genes in the SHH pathway as candidate risk genes. As such, we focussed on GLI3, a downstream modulator of the SHH pathway. In the present study,we genotyped 34 tag SNPs covering GLI3 and performed association analysis with NSCL/P in 504 cases and 455 healthy controls. Our preliminary results identified risk variants of GLI3 that are associated with NSCL/P susceptibility in a Chinese population. In particular, rs3801161 and its haplotypes rs3801161–rs7785287 displayed significant association with NSCL/Pand survived Bonferroni correction for multiple comparisons. The robustness of the association between GLI3 and NSCL/P is worth further examination in the future across different populations.
pp 695-700 September 2017 RESEARCH NOTE
Themutations of androgen receptor (AR) gene are the most common cause for complete androgen insensitivity syndrome (CAIS). We aimed to characterize the six cases enrolled in our hospital (the First People’s Hospital of Yunnan, China) and explore the molecular mechanism of CAIS. Between 2010 and 2013, six female cases were enrolled in our hospital for the agenesis of secondary sexual characteristics. The clinical examinations such as sex hormone test and B ultrasound were performed and the genetic characterization of patients were evaluated by karyotype analysis, polymerase chain reaction and DNA sequencing. The six cases with 46, XY karyotype were diagnosed with CAIS and four novel AR mutations were discovered, which were responsible forChinese CAIS. The molecular study of the AR gene facilitated the understanding of the mechanism of CAIS and provided the genetic counselling clinically.
pp 701-706 September 2017 RESEARCH NOTE
A rapid decline in temperature poses a major challenge for poikilothermic fish, as their entire metabolism depends on ambient temperature. The gene expression of rainbow trout Oncorhynchus mykiss having undergone such a cold shock (0◦C) was compared to a control (5◦C) in a microarray and quantitative real-time PCR based study. The tissues of gill, kidney and liver were examined. The most differently expressed genes were found in liver, many of them contributing to the network ‘cellular compromise, cellular growth and proliferation’.However, the number of genes found to be regulated at 0◦Cwas surprisingly low. Instead of classical genes involved in temperature shock, the three genes encoding fibroblast growth factor 1 (fgf1), growth arrest and DNA-damageinducible,alpha (gadd45a) and sclerostin domain-containing protein 1 (sostdc1) were upregulated in the liver upon cold shock in two different rainbow trout strains, suggesting that these genes may be considered as general biomarkers for cold shock in rainbow trout.
pp 707-715 September 2017 RESEARCH NOTE
Aggression is a heritable trait and genetically related to neurotransmitter-related genes. Behavioural characteristics of some pig breeds are different. To compare the genetic differences between breeds, backtest and aggressive behaviour assessments, and genotyped using Sequenom iPLEX platform were performed in 50 Chinese indigenous Mi pigs and 100 landrace-large white (LLW) cross pigs with 32 SNPs localized in 11 neurotransmitter-related genes. The genetic polymorphisms of 26 SNPs had notable differences (P < 0.05) between Mi and LLW. The most frequent haplotypes were different in DBH, HTR2A, GAD1, HTR2B,MAOA and MAOB genes between Mi and LLW. The mean of backtest scores was significantly lower (P < 0.001) for Mi than LLW pigs. Skin lesion scores were greater (P < 0.01) in LLW pigs than Mi pigs. In this study, we have confirmed that Chinese Mi pigs are less active and less aggressive than European LLW pigs, and the genetic polymorphisms of neurotransmitter-related genes, which have been proved previously associated with aggressive behaviour, have considerable differences between Mi and LLW pigs.
Volume 99, 2020
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