This study develops low-fat microwaved peanut snacks (LMPS) using partially defatted peanuts (PDP) with different defatting ratios, catering to people’s pursuit of healthy, low-fat cuisine.  The effects of defatting treatment on the structural characteristics, texture, color, and nutrient composition of LMPS were comprehensively explored.  The structural characteristics of LMPS were characterized using X-ray micro-computed tomography (Micro-CT) and scanning electron microscope (SEM).  The results demonstrated that the porosity, pore number, pore volume, brightness, brittleness, protein content, and total sugar content of LMPS all significantly increased (P<0.05) with the increase in the defatting ratio.  At the micro level, porous structure, cell wall rupture, and loss of intracellular material could be observed in LMPS after defatting treatments.  LMPS made from PDP with a defatting ratio of 64.44% had the highest internal pore structural parameters (porosity 59%, pore number 85.3×105, pore volume 68.23 mm³), the brightest color (L* 78.39±0.39), the best brittleness (3.64±0.21) mm^–1), and the best nutrition (high protein content, (34.02±0.38)%; high total sugar content, (17.45±0.59)%; low-fat content, (27.58±0.85)%).  The study provides a theoretical basis for the quality improvement of LMPS.

The Chinese crested duck is a unique duck breed having a bulbous feather shape on its duck head.  However, the mechanisms involved in its formation and development are unclear.  In the present study, RNA sequencing analysis was performed on the crested tissues of 6 Chinese crested ducks and the scalp tissues of 6 cherry valley ducks (CVs) from 2 developmental stages.  This study identified 261 differentially expressed genes (DEGs), 122 upregulated and 139 downregulated, in the E28 stage and 361 DEGs, 154 upregulated and 207 downregulated in the D42 stage between CC and CV ducks.  The subsequent results of weighted gene co-expression network analysis (WGCNA) revealed that the turquoise and cyan modules were associated with the crest trait in the D42 stage, meanwhile, the green, brown, and pink modules were associated with the crest trait in the E28 stage.  Venn analysis of the DEGs and WGCNA showed that 145 and 45 genes are associated between the D42 and E28 stages, respectively.  The expression of WNT16, BMP2, SLC35F2, SLC6A15, APOBEC2, ABHD6, TNNC2, MYL1, and TNNI2 were verified by real-time quantitative PCR.  This study provides an approach to reveal the molecular mechanisms underlying the crested trait development.

Cotton is an important natural fiber crop worldwide which plays a vital role in our daily life.  High yield is a constant goal of cotton breeding, and lint percentage (LP) is one of the important components of cotton fiber yield.  A stable QTL controlling LP, qLP_A01.1, was identified on chromosome A01 from Gossypium hirsutum introgressed lines with G. tomentosum chromosome segments in a previous study.  To fine-map qLP_A01.1, an F₂ population with 986 individuals was established by crossing G. hirsutum cultivar CCRI35 with the chromosome segment substitution line HT_390.  A high-resolution genetic map including 47 loci and spanning 56.98 cM was constructed in the QTL region, and qLP_A01.1 was ultimately mapped into an interval corresponding to an ~80 kb genome region of chromosome A01 in the reference genome, which contained six annotated genes.  Transcriptome data and sequence analysis revealed that S-acyltransferase protein 24 (GoPAT24) might be the target gene of qLP_A01.1.  This result provides the basis for cotton fiber yield improvement via marker-assisted selection (MAS) and further studies on the mechanism of cotton fiber development.

Although African swine fever (ASF) has been prevalent for more than a century, it remains the number one swine disease that seriously endangers the global pig industry, and there is no effective means of prevention and treatment (Wang et al. 2023). Due to its enormous economic and social impact, it is listed as a notifiable animal disease by the World Organization for Animal Health (Costard et al. 2013). ASF has been present in sub-Saharan Africa since its first discovery in Kenya. During the 1950s and 1970s, it spread to Western Europe and Latin America, but most were quickly eradicated. In 2007, it spread to Russia and its neighboring Eastern European countries (Pejsak et al. 2014; Martinez-Lopez et al. 2015), and in 2018, it was first transmitted to China and quickly spread to other Asian countries and regions, posing a serious threat to global pork production (Dixon et al. 2020).

African swine fever virus (ASFV), the causative agent of ASF, is an icosahedral nucleocytoplasmic large DNA virus belonging to the genus Asfivirus in the family Asfarviridae (Alonso et al. 2018). To date, more than 150 proteins encoded by ASFV have been identified; however, the functions of most of them remain unknown. The structure of ASFV virion is extremely complex, consisting of five layers, namely outer envelope, capsid, inner envelope, core shell, and nucleoid (Liu et al. 2019). Among them, the core shell is mainly composed of two polyproteins, pp62 and pp220. Both play an important role in the correct assembly of ASFV core shell and viral replication (Andrés et al. 1997; Suárez et al. 2010). The pp62, encoded by the CP530R gene, is located in the viral factory and can be proteolytically cleaved into mature proteins p35 and p15 by the ASFV cysteine protease pS273R in the late stage of infection (Simón-Mateo et al. 1997; Andrés et al. 2002; Jia et al. 2017). The pp62 derivatives p35 and p15 are important structural proteins of ASFV, which participate in the formation of icosahedral capsid structure. Moreover, pp62 was demonstrated to be a late protein that is incredibly immunogenic and is highly conserved among different ASFV strains (Simón-Mateo et al. 1997). Considering the important properties of pp62 and its critical role in the ASFV replication cycle, development of monoclonal antibodies (mAbs) against pp62 can provide valuable tools for the diagnosis and basic research of ASFV.

In this study, multiple bioinformatics analyses of pp62 showed that its 1−180 amino acid (aa) region has good antigenicity and solubility, and contains abundant antigenic epitopes (Appendix A). Therefore, this region was prokarytically expressed as a His-tagged fusion protein (designated pp62_(1−180aa)-His) by constructing a recombinant plasmid pET-28a(+)-pp62_(1−180aa)-His, transforming it into E. coli Rosette (DE3) cells and then inducing with isopropyl β-D-1-thiogalactopyranoside. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed that a protein band of ~21 kDa was detected in the supernatant of transformed cell lysates (Appendix B), and its size was consistent with the theoretical calculation. The soluble pp62_(1−180aa)-His was affinity-purified from the supernatants of bacterial lysates using Ni-NTA agarose under native conditions. SDS-PAGE analysis indicated that the purity of purified pp62_(1−180aa)-His was about 95%, and western blot (WB) analysis further revealed that the protein could be recognized by mouse anti-His-tag mAb and swine ASFV antiserum (Fig. 1-A). The purified pp62_(1−180aa)-His was used to immunize 6-week-old BALB/c mice subcutaneously using 60 μg protein emulsified in Freund's complete adjuvant, followed by two booster immunizations with 30 μg protein emulsified in Freund's incomplete adjuvant at two-week interval. Splenocytes from the immunized mice were fused with SP2/0 cells. By means of indirect ELISA screening combined with three rounds of subcloning, two monoclonal hybridoma cell clones (4B5-19-7 and 4C5-11-6) stably secreting anti-pp62 mAbs were obtained, and then intraperitoneally injected into BALB/c mice to produce ascites. The titers of 4B5-19-7 and 4C5-11-6 in ascites were 1:204800 and 1:409600, respectively, as determined by indirect ELISA (Appendix C). The isotypes of mAbs 4B5-19-7 and 4C5-11-6 were determined to be IgG2b/κ and IgG1/κ, respectively (Appendix D). Furthermore, RT-PCR was performed to amplify the heavy- and light-chain variable regions of each mAb using the RNA extracted from hybridoma cells as the template and with the specific primers (Appendix E, F). The amplicons were sequenced and the results were submitted to the GenBank database under accession numbers PQ037594−PQ037597 (Table 1). The corresponding aa sequences were annotated using the Kabat Antibody Numbering Scheme online tool to show the complementarity-determining regions (CDR), including CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3. The results showed that aa sequences of the heavy-chain CDR regions of pp62 mAbs are different from each other, while their light-chain CDR region aa sequences are completely identical (Table 1). Next, the reaction of mAbs with pp62 protein was analyzed by an indirect immunofluorescence assay (IFA), which showed that both mAbs react well with the natural pp62 protein in ASFV-infected WSL cells (Fig. 1-B). However, the two mAbs showed no cross reactivity with other important porcine pathogens, such as porcine reproductive and respiratory syndrome virus and Getah virus (Appendix G), indicating that they have good specificity. WB analysis further demonstrated that pp62 mAbs could recognize a ~62 kDa protein band in ASFV-infected WSL cells (Fig. 1-C), which matches the theoretical molecular weight of the natural pp62. Considering that the protein denaturation condition was used when analyzing the reactivity of mAbs with pp62 by WB, it was concluded that the epitopes recognized by two mAbs are linear. Notably, the expression of pp62 was detectable from 12 h post-infection onward, which is consistent with the previous reports that pp62 is a late protein (Andrés et al. 2002; Simón-Mateo et al. 1997).

To our knowledge, there are very few reports on preparation of mAbs against the ASFV pp62 protein. Bai and colleagues prepared eighteen mAbs against pp62, of which fifteen recognize p15 and the remaining three recognize p35 (Bai et al. 2020). In addition, Chu et al. generated four mAbs against pp62, of which two recognize pp62 and the remaining recognize p15 and p35, respectively (Chu et al. 2022). Interestingly, when using WB to analyze the reaction of pp62 mAbs with the total proteins of ASFV-infected WSL cells, we found that in addition to producing a clear protein band at ~62 kDa, the 4B5-19-7 also produced a clear band at ~15 kDa, while 4C5-11-6 at ~35 kDa (Fig. 1-D). This suggests that the epitopes recognized by pp62 mAbs should be located at different positions in pp62. Previous studies have shown that pp62 needs to be proteolytically cleaved into two mature structural proteins, p15 and p35, when assembled into the core shell in the late stage of ASFV infection (Simón-Mateo et al. 1997; Andrés et al. 2002; Jia et al. 2017). The cleavage of pp62 has been demonstrated to be mediated by the ASFV pS273R, with cleavage sites located at G158-G159 and G463-G464 (Appendix H). The resultant p15 and p35 are composed of aa at positions 1−158 and 159−462 of pp62, respectively (Simón-Mateo et al. 1997; Suárez et al. 2010). To verify whether 4B5-19-7 and 4C5-11-6 recognize p15 and p35, respectively, the eukaryotic recombinant plasmids pCMV-HA-p15 and pCMV-HA-p35 constructed with the primers shown in Appendix F were individually transfected into WSL cells, which were then analyzed by IFA. The results showed that 4B5-19-7 only reacted with p15, while 4C5-11-6 only reacted with p35 (Appendix I). This indicates that pp62 mAbs recognize different antigenic epitopes.

To identify the epitopes recognized by pp62 mAbs, a series of recombinant prokaryotic plasmids respectively expressing GST-tagged pp62_(1−180aa) and 19 additional truncated pp62_(1−180aa) were constructed (Appendix F, J). Fig. 1-E shows that only the truncated proteins containing 95–105 aa (SYTGVKLEVEK) were able to react with both 4B5-19-7 and GST polyclonal antibody, while those lacking this domain could not. This indicate that the epitope recognized by 4B5-19-7 is located at 95–105 aa of pp62. Similarly, the epitope recognized by 4C5-11-6 was shown to be located at 171–180 aa (YTPRTRIAIE). As illustrated in Appendix H, the epitopes recognized by mAbs 4B5-19-7 and 4C5-11-6 are located exactly within the aa range of p15 and p35.

To analyze whether the epitopes recognized by pp62 mAbs are conserved, a total of 36 pp62 aa sequences of representative ASFV strains of different genotypes currently available in the GenBank database were downloaded and analyzed. These ASFV strains used for analysis originate from 21 countries in Africa, Europe, and Asia, involving 12 genotypes. The results showed that the pp62 is highly conserved among ASFV strains of different genotypes, and the epitopes recognized by pp62 mAbs are more conserved, without any mutations in the aa regions where the epitopes are located (Fig. 1-F).

In summary, we successfully prepared two mAbs that can specifically recognize the ASFV pp62. Among them, 4B5-19-7 and 4C5-11-6 can further recognize the p15 and p35 of pp62 cleavage products, respectively. The epitopes recognized by 4B5-19-7 and 4C5-11-6 are highly conserved and located at amino acids 95−105 (SYTGVKLEVEK) and 171−180 (YTPRTRIAIE) of pp62, respectively. The mAbs provide valuable materials for pp62 functional study and development of improved serological diagnostic agents and assays.