Amino Acid Sequence Homology Research Articles

Preclinical screen for protection efficacy of chlamydial antigens that are immunogenic in humans.

To search for subunit vaccine candidates, immunogenic chlamydial antigens identified in humans were evaluated for protection against both infection and pathology in a mouse genital tract infection model under three different immunization regimens. The intramuscular immunization regimen was first used to evaluate 106 chlamydial antigens, which revealed that two antigens significantly reduced while 11 increased genital chlamydial burden. The two infection-reducing antigens failed to prevent pathology and 23 additional antigens even exacerbated pathology. Thus, intranasal mucosal immunization was tested next since intranasal inoculation with live Chlamydia muridarum prevented both genital infection and pathology. Two of the 29 chlamydial antigens evaluated were found to prevent genital infection but not pathology and three exacerbate pathology. To further improve protection efficacy, a combinational regimen (intranasal priming + intramuscular boosting + a third intraperitoneal/subcutaneous boost) was tested. This regimen identified four infection-reducing antigens, but only one of them prevented pathology. Unfortunately, this protective antigen was not advanced further due to its amino acid sequence homology with several human molecules. Two pathology-exacerbating antigens were also found. Nevertheless, intranasal mucosal priming with viable C. muridarum in control groups consistently prevented both genital infection and pathology regardless of the subsequent boosters. Thus, screening 140 different chlamydial antigens with 21 repeated multiple times in 17 experiments failed to identify a subunit vaccine candidate but demonstrated the superiority of viable chlamydial organisms in inducing immunity against both genital infection and pathology, laying the foundation for developing a live-attenuated Chlamydia vaccine.

Amino acid sequence homology of monoclonal serum free light chain dimers and tissue deposited light chains in AL amyloidosis: a pilot study.

Diagnosis of light chain amyloidosis (AL) requires demonstration of amyloid deposits in a tissue biopsy followed by appropriate typing. Previous studies demonstrated increased dimerization of monoclonal serum free light chains (FLCs) as a pathological feature of AL. To further examine the pathogenicity of FLC, we aimed at testing amino acid sequence homology between circulating and deposited light chains (LCs). Matched tissue biopsy and serum of 10 AL patients were subjected to tissue proteomic amyloid typing and nephelometric FLC assay, respectively. Serum FLC monomers (M)and dimers (D)were analyzed by Western blotting (WB) and mass spectrometry (MS). WB of serum FLCs showed predominance of either κ or λ type, in agreement with the nephelometric assay data. Abnormal FLC M-D patterns typical of AL amyloidosis were demonstrated in 8 AL-λ patients and in one of two AL-κ patients: increased levels of monoclonal FLC dimers, high D/M ratio values of involved FLCs, and high ratios of involved to uninvolved dimeric FLCs. MS of serum FLC dimers showed predominant constant domain sequences, in concordance with the tissue proteomic amyloid typing. Most importantly, variable domain sequence homology between circulating and deposited LC species was demonstrated, mainly in AL-λ cases. This is the first study to demonstrate homology between circulating FLCs and tissue-deposited LCs in AL-λ amyloidosis. The applied methodology can facilitate studying the pathogenicity of circulating FLC dimers in AL amyloidosis. The study also highlights the potential of FLC monomer and dimer analysis as a non-invasive screening tool for this disease.

More than just pattern recognition: Prediction of uncommon protein structure features by AI methods

The CASP14 experiment demonstrated the extraordinary structure modeling capabilities of artificial intelligence (AI) methods. That result has ignited a fierce debate about what these methods are actually doing. One of the criticisms has been that the AI does not have any sense of the underlying physics but is merely performing pattern recognition. Here, we address that issue by analyzing the extent to which the methods identify rare structural motifs. The rationale underlying the approach is that a pattern recognition machine tends to choose the more frequently occurring motifs, whereas some sense of subtle energetic factors is required to choose infrequently occurring ones. To reduce the possibility of bias from related experimental structures and to minimize the effect of experimental errors, we examined only CASP14 target protein crystal structures determined to a resolution limit better than 2 Å, which lacked significant amino acid sequence homology to proteins of known structure. In those experimental structures and in the corresponding models, we track cis peptides, π-helices, 310-helices, and other small 3D motifs that occur in the PDB database at a frequency of lower than 1% of total amino acid residues. The best-performing AI method, AlphaFold2, captured these uncommon structural elements exquisitely well. All discrepancies appeared to be a consequence of crystal environment effects. We propose that the neural network learned a protein structure potential of mean force, enabling it to correctly identify situations where unusual structural features represent the lowest local free energy because of subtle influences from the atomic environment.

BnGLO4 gene cloning and bioinformatics analysis in Brassica napus L

High oleic acid rapeseed oil offers superior nutritional and health benefits, but its mass production is limited due to poor resistance and slightly lower yield. Photosynthesis serves as the foundation of biological survival and closely correlates with crop resistance to stresses, yield, and quality. To identify photosynthesis-related genes, transcriptome sequencing was conducted on high oleic acid rapeseed Gaoyousuan No. 1 and low oleic acid rapeseed Xiangyou 15 resulting in the identification of a total of 9396 differentially expressed genes (4669 up-regulated and 4727 down-regulated). From these genes nine candidate genes were screened using GO and KEGG analysis with BnGLO4 being selected for cloning purposes. The BnGLO4 gene fragment has a length of 1161 bp with an ORF sequence of 1092 bp encoding a theoretical isoelectric point of pI 7.60; it encodes an unstable lipid-soluble protein localized in peroxisomes without transmembrane structural domains or signal peptides. Its amino acid sequence homology was highest with that of BnaA01G0355200ZS, BnaA01G0355600ZS, BnaA05G0410400ZS, BnaC01G0441800ZS, and Brassica rapa (XP_0091171 45.1), Brassica napus (CDY39100.1), Brassica cretica (KAF3533604.1) and Brassica oleracea var. oleracea (XP_0136106 44.1) belonging to the same cruciferous family.The BnGLO4 gene may be associated with responses to abiotic stresses such as salt, drought, and temperature extremes along with photosynthesis and growth in rapeseed plants. The expression levels of the GLO4 gene (BnaA01G0355200 ZS) were highest in filaments while being higher at each stage in seeds and siliques with the highest expression level at day 14 in seeds. Expression was significantly upregulated after 3 ​h of salt stress treatment, reaching a maximum at 12 ​h before slightly decreasing at 24 ​h. The findings of this study lay a foundation for further investigation on photosynthesis and stress response in high oleic rapeseed.

Deconstructing the Potency and Cell-Line Selectivity of Membranolytic Anticancer Peptides.

Current cancer treatments damage healthy cells and tissues, causing short-term and long-term side effects. New treatments are desired that show greater selectivity toward cancer cells and evade the common mechanisms of multidrug resistance. Membranolytic anticancer peptides (mACPs) hold promise against cancer and multidrug resistance. Amphipathicity, hydrophobicity, and net charge of mACPs participate in their respective interactions with cell membranes and their overall inhibition of cancer cells. To support the design of cell-line selective mACPs, we investigated the relationships that amino acid composition, physicochemical properties, sequence motifs, and sequence homology could have with their potency and selectivity towards several healthy and cancer cell lines. Sequence length and net charge are known to affect the selectivity of mACPs between cancer and healthy cell lines. Our study reveals that increasing the net charge or flexibility (i. e., small and aliphatic residues) influences their selectivity between cancer cell lines with comparable lipid compositions.

Are scattered microsatellites weak chromosomal markers? Guided mapping reveals new insights into Trachelyopterus (Siluriformes: Auchenipteridae) diversity.

The scattered distribution pattern of microsatellites is a challenging problem in fish cytogenetics. This type of array hinders the identification of useful patterns and the comparison between species, often resulting in over-limited interpretations that only label it as "scattered" or "widely distributed". However, several studies have shown that the distribution pattern of microsatellites is non-random. Thus, here we tested whether a scattered microsatellite could have distinct distribution patterns on homeologous chromosomes of closely related species. The clustered sites of 18S and 5S rDNA, U2 snRNA and H3/H4 histone genes were used as a guide to compare the (GATA)n microsatellite distribution pattern on the homeologous chromosomes of six Trachelyopterus species: T. coriaceus and Trachelyopterus aff. galeatus from the Araguaia River basin; T. striatulus, T. galeatus and T. porosus from the Amazonas River basin; and Trachelyopterus aff. coriaceus from the Paraguay River basin. Most species had similar patterns of the (GATA)n microsatellite in the histone genes and 5S rDNA carriers. However, we have found a chromosomal polymorphism of the (GATA)n sequence in the 18S rDNA carriers of Trachelyopterus galeatus, which is in Hard-Weinberg equilibrium and possibly originated through amplification events; and a chromosome polymorphism in Trachelyopterus aff. galeatus, which combined with an inversion polymorphism of the U2 snRNA in the same chromosome pair resulted in six possible cytotypes, which are in Hardy-Weinberg disequilibrium. Therefore, comparing the distribution pattern on homeologous chromosomes across the species, using gene clusters as a guide to identify it, seems to be an effective way to further the analysis of scattered microsatellites in fish cytogenetics.

Insights from a chum salmon (Oncorhynchus keta) genome assembly regarding whole-genome duplication and nucleotide variation influencing gene function.

Chum salmon are ecologically important to Pacific Ocean ecosystems and commercially important to fisheries. To improve the genetic resources available for this species, we sequenced and assembled the genome of a male chum salmon using Oxford Nanopore read technology and the Flye genome assembly software (contig N50: ∼2 Mbp, complete BUSCOs: ∼98.1%). We also resequenced the genomes of 59 chum salmon from hatchery sources to better characterize the genome assembly and the diversity of nucleotide variants impacting phenotype variation. With genomic sequences from a doubled haploid individual, we were able to identify regions of the genome assembly that have been collapsed due to high sequence similarity between homeologous (duplicated) chromosomes. The homeologous chromosomes are relics of an ancient salmonid-specific genome duplication. These regions were enriched with genes whose functions are related to the immune system and responses to toxins. From analyzing nucleotide variant annotations of the resequenced genomes, we were also able to identify genes that have increased levels of variants thought to moderately impact gene function. Genes related to the immune system and the detection of chemical stimuli (olfaction) had increased levels of these variants based on a gene ontology enrichment analysis. The tandem organization of many of the enriched genes raises the question of why they have this organization.

TEMPROT: protein function annotation using transformers embeddings and homology search

BackgroundAlthough the development of sequencing technologies has provided a large number of protein sequences, the analysis of functions that each one plays is still difficult due to the efforts of laboratorial methods, making necessary the usage of computational methods to decrease this gap. As the main source of information available about proteins is their sequences, approaches that can use this information, such as classification based on the patterns of the amino acids and the inference based on sequence similarity using alignment tools, are able to predict a large collection of proteins. The methods available in the literature that use this type of feature can achieve good results, however, they present restrictions of protein length as input to their models. In this work, we present a new method, called TEMPROT, based on the fine-tuning and extraction of embeddings from an available architecture pre-trained on protein sequences. We also describe TEMPROT+, an ensemble between TEMPROT and BLASTp, a local alignment tool that analyzes sequence similarity, which improves the results of our former approach.ResultsThe evaluation of our proposed classifiers with the literature approaches has been conducted on our dataset, which was derived from CAFA3 challenge database. Both TEMPROT and TEMPROT+ achieved competitive results on F_{max }, S_{min }, AuPRC and IAuPRC metrics on Biological Process (BP), Cellular Component (CC) and Molecular Function (MF) ontologies compared to state-of-the-art models, with the main results equal to 0.581, 0.692 and 0.662 of F_{max } on BP, CC and MF, respectively.ConclusionsThe comparison with the literature showed that our model presented competitive results compared the state-of-the-art approaches considering the amino acid sequence pattern recognition and homology analysis. Our model also presented improvements related to the input size that the model can use to train compared to the literature methods.

ZIP4 is required for normal progression of synapsis and for over 95% of crossovers in wheat meiosis.

Tetraploid (AABB) and hexaploid (AABBDD) wheat have multiple sets of similar chromosomes, with successful meiosis and preservation of fertility relying on synapsis and crossover (CO) formation only taking place between homologous chromosomes. In hexaploid wheat, the major meiotic gene TaZIP4-B2 (Ph1) on chromosome 5B, promotes CO formation between homologous chromosomes, whilst suppressing COs between homeologous (related) chromosomes. In other species, ZIP4 mutations eliminate approximately 85% of COs, consistent with loss of the class I CO pathway. Tetraploid wheat has three ZIP4 copies: TtZIP4-A1 on chromosome 3A, TtZIP4-B1 on 3B and TtZIP4-B2 on 5B. Here, we have developed single, double and triple zip4 TILLING mutants and a CRISPR Ttzip4-B2 mutant, to determine the effect of ZIP4 genes on synapsis and CO formation in the tetraploid wheat cultivar 'Kronos'. We show that disruption of two ZIP4 gene copies in Ttzip4-A1B1 double mutants, results in a 76-78% reduction in COs when compared to wild-type plants. Moreover, when all three copies are disrupted in Ttzip4-A1B1B2 triple mutants, COs are reduced by over 95%, suggesting that the TtZIP4-B2 copy may also affect class II COs. If this is the case, the class I and class II CO pathways may be interlinked in wheat. When ZIP4 duplicated and diverged from chromosome 3B on wheat polyploidization, the new 5B copy, TaZIP4-B2, could have acquired an additional function to stabilize both CO pathways. In tetraploid plants deficient in all three ZIP4 copies, synapsis is delayed and does not complete, consistent with our previous studies in hexaploid wheat, when a similar delay in synapsis was observed in a 59.3 Mb deletion mutant, ph1b, encompassing the TaZIP4-B2 gene on chromosome 5B. These findings confirm the requirement of ZIP4-B2 for efficient synapsis, and suggest that TtZIP4 genes have a stronger effect on synapsis than previously described in Arabidopsis and rice. Thus, ZIP4-B2 in wheat accounts for the two major phenotypes reported for Ph1, promotion of homologous synapsis and suppression of homeologous COs.

Expression and purification of 15N-labeled Fra a 1, a strawberry allergen, to prepare samples for NMR measurements

Raw strawberries contain allergens that cause oral allergic syndrome. Fra a 1 is one of the major allergens in strawberries and might decrease their allergenicity by heating, likely due to structural changes in the allergen leading to decreased recognition of the allergens in the oral cavity. In the present study, to understand the relationship between allergen structure and allergenicity, the expression and purification of 15N-labeled Fra a 1 were examined and the sample was used for NMR analysis. Two isoforms, Fra a 1.01 and Fra a 1.02, were used and expressed in E. coli BL21(DE3) in M9 minimal medium. Fra a 1.02 was purified as a single protein by using the GST tag approach, whereas histidine × 6-tag (his6-tag) Fra a 1.02 was obtained both as the full-length (∼20 kDa) and a truncated (∼18 kDa) form. On the other hand, his6-tag Fra a 1.01 was purified as a homogeneous protein. 1⁵N-labeled HSQC NMR spectra suggested that Fra a 1.02 was thermally denatured at lower temperatures than Fra a 1.01, despite the high amino acid sequence homology (79.4%) of these isoforms. Furthermore, the samples in the present study allowed us to analyze ligand binding that probably affects structural stability. In conclusion, GST tag was effective for obtaining a homogeneous protein when his6-tag failed to give a single form, and the present study provided a sample that could be used for NMR studies of the details of the allergenicity and structure of Fra a 1.

T-DNA insertion mutagenesis in Penicillium brocae results in identification of an enolase gene mutant impaired in secretion of organic acids and phosphate solubilization.

Penicillium brocae strain P6 is a phosphate-solubilizing fungus isolated from farmland in Guangdong Province, China. To gain better insights into the phosphate solubilization mechanisms of strain P6, a T-DNA insertion population containing approximately 4500 transformants was generated by Agrobacterium tumefaciens-mediated transformation. The transformation procedure was optimized by using a Hybond N membrane for co-cultivation of A. tumefaciens and P. brocae. A mutant impaired in phosphate solubilization (named MT27) was obtained from the T-DNA insertion population. Thermal asymmetric interlaced PCR was then used to identify the nucleotide sequences flanking the T-DNA insertion site. The T-DNA in MT27 was inserted into the fourth exon of an enolase gene, which shows 90.8 % nucleotide identity with enolase mRNA from Aspergillus neoniger. Amino acid sequence homology analysis indicated that the enolase is well conserved among filamentous fungi and Saccharomyces cerevisiae. Complementation tests with the MT27 mutant confirmed that the enolase gene is involved in phosphate solubilization. Analysis of organic acids in culture supernatants indicated reduced levels of oxalic acid and lactic acid for the MT27 mutant compared to the parent strain P6 or the complementation strain. In conclusion, we suggest that the identified enolase gene of P. brocae is involved in production of specific organic acids, which, when secreted, act as phosphate solubilizing agents.

High-efficient production of mushroom polyketide compounds in a platform host Aspergillus oryzae

BackgroundOrsellinic acid (2,4-dihydroxy-6-methylbenzoic acid, OA) and its structural analog o-Orsellinaldehyde, have become widely used intermediates in clinical drugs synthesis. Although the research on the biosynthesis of such compounds has made significant progress, due to the lack of suitable hosts, there is still far from the industrial production of such compounds based on synthetic biology.ResultsWith the help of genome mining, we found a polyketide synthase (PKS, HerA) in the genome of the Hericium erinaceus, which shares 60% amino acid sequence homology with ArmB from Armillaria mellea, an identified PKS capable of synthesizing OA. To characterize the function of HerA, we cloned herA and heterologously expressed it in Aspergillus oryzae, and successfully detected the production of OA. Subsequently, the introduction of an incomplete PKS (Pks5) from Ustilago maydis containing only three domains (AMP-ACP-R), which was into herA-containing A. oryzae, the resulted in the production of o-Orsellinaldehyde. Considering the economic value of OA and o-Orsellinaldehyde, we then optimized the yield of these compounds in A. oryzae. The screening showed that when maltose was used as carbon source, the yields of OA and o-Orsellinaldehyde were 57.68 mg/L and 15.71 mg/L respectively, while the yields were 340.41 mg/Kg and 84.79 mg/Kg respectively in rice medium for 10 days.ConclusionsHerein, we successfully expressed the genes of basidiomycetes using A. oryzae heterologous host. As a fungus of ascomycetes, which not only correctly splices genes of basidiomycetes containing multiple introns, but also efficiently produces their metabolites. This study highlights that A. oryzae is an excellent host for the heterologous production of fungal natural products, and has the potential to become an efficient chassis for the production of basidiomycete secondary metabolites in synthetic biology.Graphical

Atomic force microscopic analysis of the light-harvesting complex 2 from purple photosynthetic bacterium Thermochromatium tepidum.

Structural information on the circular arrangements of repeating pigment-polypeptide subunits in antenna proteins of purple photosynthetic bacteria is a clue to a better understanding of molecular mechanisms for the ring-structure formation and efficient light harvesting of such antennas. Here, we have analyzed the ring structure of light-harvesting complex 2 (LH2) from the thermophilic purple bacterium Thermochromatium tepidum (tepidum-LH2) by atomic force microscopy. The circular arrangement of the tepidum-LH2 subunits was successfully visualized in a lipid bilayer. The average top-to-top distance of the ring structure, which is correlated with the ring size, was 4.8 ± 0.3nm. This value was close to the top-to-top distance of the octameric LH2 from Phaeospirillum molischianum (molischianum-LH2) by the previous analysis. Gaussian distribution of the angles of the segments consisting of neighboring subunits in the ring structures of tepidum-LH2 yielded a median of 44°, which corresponds to the angle for the octameric circular arrangement (45°). These results indicate that tepidum-LH2 has a ring structure consisting of eight repeating subunits. The coincidence of an octameric ring structure of tepidum-LH2 with that of molischianum-LH2 is consistent with the homology of amino acid sequences of the polypeptides between tepidum-LH2 and molischianum-LH2.

Cytosolic domain regulates the calcium sensitivity and surface expression of BEST1 channels in the HEK293 cells

BEST family is a class of Ca2+-activated Cl- channels evolutionary well conserved from bacteria to human. The human BEST paralogs (BEST1-BEST4) share significant amino acid sequence homology in the N-terminal region, which forms the transmembrane helicases and contains the direct calcium-binding site, Ca2+-clasp. But the cytosolic C-terminal region is less conserved in the paralogs. Interestingly, this domain-specific sequence conservation is also found in the BEST1 orthologs. However, the functional role of the C-terminal region in the BEST channels is still poorly understood. Thus, we aimed to understand the functional role of the C-terminal region in the human and mouse BEST1 channels by using electrophysiological recordings. We found that the calcium-dependent activation of BEST1 channels can be modulated by the C-terminal region. The C-terminal deletion hBEST1 reduced the Ca2+-dependent current activation and the hBEST1-mBEST1 chimera showed a significantly reduced calcium sensitivity to hBEST1 in the HEK293 cells. And the C-terminal domain could regulate cellular expression and plasma membrane targeting of BEST1 channels. Our results can provide a basis for understanding the C-terminal roles in the structure-function of BEST family proteins. [BMB Reports 2023; 56(3): 172-177].

Abstract A021: Divergent roles for BNIP3 and NIX in tumor progression and metastasis

Abstract BNIP3 and NIX (BNIP3L) are mitochondrial cargo receptors (MCRs) induced by physiological stresses, including hypoxia and nutrient deprivation, to promote turnover of mitochondria at the autolysosome in a process known as mitophagy. We and others have shown that BNIP3 and NIX function similarly in mitophagy by integrating into the outer mitochondrial membrane (OMM) via their conserved C-terminal transmembrane domain while interacting via conserved N-terminal LIR motifs with processed LC3 at nascent phagophores. To address functional roles of BNIP3 and NIX in tumorigenesis and metastasis in vivo, we examined the effect of loss of BNIP3 and/or NIX in two different mouse models: (1) the 4T1 orthotopic model of mammary tumorigenesis, and (2) the KRasG12D driven autochthonous model of pancreatic ductal adenocarcinoma (PDAC). Using CRISPR/Cas9 gene editing to delete BNIP3 (DBNIP3), NIX (DNIX) or both BNIP3 and NIX (DKO) in 4T1 cells, we showed that while loss of BNIP3 increased tumor growth, promoted invasion/migration and progression to metastasis in vivo, loss of NIX decreased primary tumor growth, inhibited invasion/migration and markedly attenuated metastasis to the lungs. Intriguingly, the effect of NIX loss was dominant over the effect of BNIP3 loss, such that DKO 4T1 cells phenocopied DNIX 4T1 cells in exhibiting decreased growth, invasion/migration properties and inability to metastasize from the mammary gland. Similar findings were observed in Pdx1-Cre;LSL-KRasG12D (KC) mice lacking BNIP3 and/or NIX with KC-DBNIP3 mice showing accelerated progression from PanIN to PDAC, increased organoid growth in vitro and increased metastasis to the liver compared to control KC mice, while KC-DNIX and KC-DKO mice showed increased tumor latency and absence of metastases. Thus, despite 55% homology in primary amino acid sequence and a shared role in mitophagy, our work indicates that BNIP3 and NIX do not function analogously in vivo in tumorigenesis and metastasis. In summary, our work shows that BNIP3 suppresses tumorigenesis in mouse models of breast, pancreas (and liver) cancer and that loss of BNIP3 correlates with worse prognosis in several human cancers. Conversely, NIX is required for progression to malignancy in models of both breast and pancreatic ductal adenocarcinoma (PDAC), and elevated NIX levels correlated with worse overall patient survival. These opposing roles for BNIP3 and NIX in progression to malignancy were unexpected and work presented will discuss mechanistic insight to their differential roles in tumorigenesis and metastasis, including a novel role for BNIP3 in suppressing RAS pathway signaling and a new role for NIX in promoting EMT. These novel functions are in addition to their canonical roles in mitophagy. Citation Format: Damian Berardi, Kay Macleod. Divergent roles for BNIP3 and NIX in tumor progression and metastasis [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr A021.

Identification of a novel linear B-cell epitope in porcine deltacoronavirus nucleocapsid protein.

Porcine deltacoronavirus (PDCoV) is an emerging swine enteropathogenic coronavirus that caused diarrhea and/or vomiting in neonatal piglets worldwide. Coronaviruses nucleocapsid (N) protein is the most conserved structural protein for viral replication and possesses good antigenicity. In this study, three monoclonal antibodies (mAbs), 3B4, 4D3, and 4E3 identified as subclass IgG2aκ were prepared using the lymphocytic hybridoma technology against PDCoV N protein. Furthermore, the B-cell epitope recognized by mAb 4D3 was mapped by dozens of overlapping truncated recombinant proteins based on the western blotting. The polypeptide 28QFRGNGVPLNSAIKPVE44 (EP-4D3) in the N-terminal of PDCoV N protein was identified as the minimal linear epitope for binding mAb 4D3. And the EP-4D3 epitope's amino acid sequence homology study revealed that PDCoV strains are substantially conserved, with the exception of the Alanine43 substitution Valine43 in the China lineage, the Early China lineage, and the Thailand, Vietnam, and Laos lineage. The epitope sequences shared high similarity (94.1%) with porcine coronavirus HKU15-155 (PorCoV HKU15), Asian leopard cats coronavirus (ALCCoV), sparrow coronavirus HKU17 (SpCoV HKU17), and sparrow deltacoronavirus. In contrast, the epitope sequences shared a very low homology (11.8 to 29.4%) with other porcine CoVs (PEDV, TGEV, PRCV, SADS-CoV, PHEV). Overall, the study will enrich the biological function of PDCoV N protein and provide foundational data for further development of diagnostic applications. KEY POINTS: • Three monoclonal antibodies against PDCoV N protein were prepared. • Discovery of a novel B-cell liner epitope (28QFRGNGVPLNSAIKPVE44) of PDCoV N protein. • The epitope EP-4D3 was conserved among PDCoV strains.

Inference of Polyploid Origin and Inheritance Mode from Population Genomic Data.

Whole-genome duplications yield varied chromosomal pairing patterns, ranging from strictly bivalent to multivalent, resulting in disomic and polysomic inheritance modes. In the bivalent case, homeologous chromosomes form pairs, where in a multivalent pattern all copies are homologous and are therefore free to pair and recombine. As sufficient sequencing data is more readily available than high-quality cytological assessments of meiotic behavior or population genetic assessment of allelic segregation, especially for non-model organisms, bioinformatics approaches to infer origins and inheritance modes of polyploids using short-read sequencing data are attractive. Here we describe two such approaches, where the first is based on distributions of allelic read depth at heterozygous sites within an individual, as the expectations of such distributions are different for disomic and polysomic inheritance modes. The second approach is more laborious and based on a phylogenetic assessment of partially phased haplotypes of a polyploid in comparison to the closest diploid relatives. We discuss the sources of deviations from expected inheritance patterns, advantages and pitfalls of both methods, effects of mating types on the performance of the methods, and possible future developments.

Features and Possible Applications of Plant Lipid-Binding and Transfer Proteins.

In plants, lipid trafficking within and inside the cell is carried out by lipid-binding and transfer proteins. Ligands for these proteins are building and signaling lipid molecules, secondary metabolites with different biological activities due to which they perform diverse functions in plants. Many different classes of such lipid-binding and transfer proteins have been found, but the most common and represented in plants are lipid transfer proteins (LTPs), pathogenesis-related class 10 (PR-10) proteins, acyl-CoA-binding proteins (ACBPs), and puroindolines (PINs). A low degree of amino acid sequence homology but similar spatial structures containing an internal hydrophobic cavity are common features of these classes of proteins. In this review, we summarize the latest known data on the features of these protein classes with particular focus on their ability to bind and transfer lipid ligands. We analyzed the structural features of these proteins, the diversity of their possible ligands, the key amino acids participating in ligand binding, the currently known mechanisms of ligand binding and transferring, as well as prospects for possible application.

Structure elucidation of Staphylococcus capitis lipase. Molecular dynamics simulations to investigate the effects of calcium and zinc ions on the structural stability

Cold-adapted and organic solvent tolerant lipases have significant potential in a wide range of synthetic reactions in industry. But there are no sufficient studies on how these enzymes interacts with their substrates. Herein, the predicted structure and function of the Staphylococcus capitis lipase (SCL) are studied. Given the high amino acid sequence homology with the Staphylococcus simulans lipase (SSL), 3D structure models of closed and open forms of the S. capitis lipase were built using the structure of SSL as template. The models suggested the presence of a main lid and a second lid that may act with the former as a double door to control the access to the active site. The SCL models also allowed us to identify key residues involved in binding substrates, calcium or zinc ions. By following this model and utilizing molecular dynamics (MD) simulations, the stability of the S. capitis lipase at low temperatures could be explained in the presence and in the absence of calcium and zinc. Due to its thermolability, the SCL is extremely valuable for different biotechnological applications in a wide variety of industries from molecular biology to detergency to food and beverage preparation. Communicated by Ramaswamy H. Sarma

Meiosis and male fertility in F1 interspecific hybrids (Passiflora vitifolia vs. Passiflora hatschbachii)

Interspecific hybrids can be studied using methodologies in which the male gamete with high reproduction potential, viability, and fertility is prioritized. Passiflora species, with lush, showy, and exotic colors, have great potential for the ornamental plant market. In addition, artificial Passiflora hybrids were developed without many difficulties because of weak reproductive barriers. Thus, meiotic and post-meiotic behaviors were analyzed with 2% acetic carmine staining. Confirmation of interspecific hybridization was performed using SSR markers and GISH technique was used to detect genomic differentiation. The pollen viability of the parental and hybrids genotypes was tested using Alexander`s solution, fluorescein diacetate and in vitro germination tests were performed using culture medium. The meiotic behavior was regular and displayed haploid number n = 9 with nine bivalent (II) chromosomal, and pairing in 90% of the cells in diakinesis. There was a significant difference (p < 0.05) in terminal and interstitial chiasma frequencies. Meiotic irregularities observed were as follows: early and/or delayed chromosomes, disorientation of spindle fibers, transverse spindles, tripolar spindles, and asynchrony; and consequently irregular post-meiotic products were observed: monads, dyads, triads, and polyads. GISH was used in the interspecific hybrids and pairing between homeologous chromosomes, and bivalent and tetravalent formation were observed. From this study, we could conclude that hybrid genotypes are fertile and pollen grains are viable and can be used in breeding programs.