Identification and function analysis of BCL2 in immune response of Pteria penguin.

B-cell lymphoma/leukemia-2 (BCL2), an anti-apoptotic factor in the mitochondrial regulatory pathway of apoptosis, is critically important in immune defenses. In this study, a novel BCL2 gene was characterized from Pteria penguin (P. penguin). The PpBCL2 was 1482 bp long, containing an open reading frame (ORF) of 588 bp encoding 195 amino acids. Four highly conserved BCL-2 homology (BH) domains were found in PpBCL2. Amino acid alignment and phylogenetic tree showed that PpBCL2 had the highest similarity with BCL2 of Crassostrea gigas at 65.24 %. Tissue expression analysis showed that PpBCL2 had high constitutive expression in gill, digestive diverticulum and mantle, and was significantly increased 72 h of Vibrio parahaemolyticus (V. parahaemolyticus) challenge in these immune tissues. Furthermore, PpBCL2 silencing significantly inhibited antimicrobial activity of hemolymph supernatant by 1.4-fold, and significantly reduced the survival rate by 51.7 % at 72 h post infection in P. penguin. These data indicated that PpBCL2 played an important role in immune response of P. penguin against V. parahaemolyticus infection.

Novel C-type lectin mediated non-specific cytotoxic cells killing activity through NCCRP-1 in nile tilapia (Oreochromis niloticus).

Non-specific cytotoxic cells (NCCs) are vital immune cells involved in teleost's non-specific immunity. As a receptor molecule on the NCCs' surface, the non-specific cytotoxic cell receptor protein 1 (NCCRP-1) is known to play a crucial role in mediating their activity. Nevertheless, there have been limited studies on the signal molecule that transmits signals via NCCRP-1. In this study, a yeast two-hybrid (Y2H) library of tilapia liver and head kidney was constructed and subsequently screened with the bait vector NCCRP-1 of Oreochromis niloticus (On-NCCRP-1) to obtain a C-type lectin (On-CTL) with an interacting protein sequence. Consequently, the full-length sequence of On-CTL was cloned and analyzed. The expression analysis revealed that On-CTL is highly expressed in the liver and is widely distributed in other tissues. Furthermore, On-CTL expression was significantly up-regulated in the brain, intestine, and head kidney following a challenge with Streptococcus agalactiae. A point-to-point Y2H method was also used to confirm the binding between On-NCCRP-1 and On-CTL. The recombinant On-CTL (rOn-CTL) protein was purified. In vitro experiments demonstrated that rOn-CTL can up-regulate the expression of killer effector molecules in NCCs via its interaction with On-NCCRP-1. Moreover, activation of NCCs by rOn-CTL resulted in a remarkable enhancement in their ability to eliminate fathead minnow cells, indicating that rOn-CTL effectively modulates the killing activity of NCCs through the NCC receptor molecule On-NCCRP-1. These findings significantly contribute to our comprehension of the regulatory mechanisms governing NCC activity, paving the way for future research in this field.

Identification and functional characterization of a superoxide dismutase (CuZnSOD) from Pinctada fucata martensii.

Copper/zinc superoxide dismutase (Cu/Zn-SOD) can effectively eliminate reactive oxygen species (ROS)，avoid damage from O2 to the body, and maintain O2 balance. In this study, multi-step high-performance liquid chromatography (HPLC), combined with Mass Spectrometry (MS), was used to isolate and identify Cu/Zn-SOD from the serum of Pinctada fucata martensii (P. f. martensii) and was designated as PmECSOD. With a length of 1864 bp and an open reading frame (ORF) of 1422 bp, the cDNA encodes a 473 amino acid protein. The PmECSOD transcript was detected in multiple tissues by quantitative real-time PCR (qRT-PCR), with its highest expression level being in the gills. Additionally, the temporal expression of PmECSOD mRNA in the hemolymph was highest at 48 h after in vivo stimulation with Escherichia coli and Micrococcus luteus. The results from this study provide a valuable base for further exploration of molluscan innate immunity and immune response.

The dehydration-responsive protein PpFAS1.3 in moss Physcomitrium patens plays a regulatory role in lipid metabolism.

Moss plants appear in the early stages of land colonization and possess varying degrees of dehydration tolerance. In this study, a protein called PpFAS1.3 was identified, which contains a fasciclin 1-like domain and is essential for the moss Physcomitrium patens' response to short-term rapid dehydration. When the FAS1.3 protein was knocked out, leafyshoots showed a significant decrease in tolerance to rapid dehydration, resulting in accelerated water loss and increased membrane leakage. Phylogenetic analysis suggests that PpFAS1.3 and its homologous proteins may have originated from bacteria and are specifically found in non-vascular plants like mosses and liverworts. As a dehydration-related protein, FAS1.3 plays a significant role in regulating lipid metabolism, particularly in the synthesis of free fatty acids (FFA) and the metabolism of two phospholipids, PC and PA. This discovery highlights the close connection between PpFAS1.3 and lipid metabolism, providing new insights into the molecular mechanisms underlying plant adaptation to stresses.

CgSHIP2 negatively regulates the mRNA expressions of CgIL-17s in response to Vibrio splendidus stimulation in Crassostrea gigas.

SH2 domain containing inositol polyphosphate5-phosphatase-2 (SHIP2) is a member of the 5-phosphatase family, acting as a vital negative regulator of immune response in vertebrates. In the present study, a SHIP2 homologue (designed as CgSHIP2) was identified from Pacific oyster, Crassostrea gigas. There was a SH2 domain, an IPPc domain and a SAM domain in CgSHIP2. The mRNA transcripts of CgSHIP2 were widely expressed in all the tested tissues with the highest expression in haemolymph. The mRNA expressions of CgSHIP2 in haemocytes increased significantly at 6, 12, 48 and 72 h after Vibrio splendidus stimulation. The positive green signals of CgSHIP2 protein were mainly located in cytoplasm of haemocytes. After the expression of CgSHIP2 was inhibited by RNA interference, the mRNA transcripts of interleukin 17s (CgIL-17-1, CgIL-17-2, CgIL-17-3 and CgIL-17-6) in the haemocytes increased significantly at 24 h after V. splendidus stimulation, which were 8.15-fold (p < 0.001), 3.44-fold (p < 0.05), 2.15-fold (p < 0.01) and 4.63-fold (p < 0.05) compared with that in NC-RNAi group, respectively. Obvious branchial swelling and cilium shedding in gills were observed in CgSHIP2-RNAi group at 24 h after V. splendidus stimulation. The results suggested that CgSHIP2 played an important role in controlling inflammatory response induced by bacteria in oysters.

Identification of toll-like receptor family and the immune function of new Sptlr-6 gene of Scylla paramamosain.

As a crucial member of pattern-recognition receptors (PRRs), the Tolls/Toll-like receptors (TLRs) gene family has been proven to be involved in innate immunity in crustaceans. In this study, nine members of TLR gene family were identified from the mud crab (Scylla paramamosain) transcriptome, and the structure and phylogeny of different SpTLRs were analyzed. It was found that different SpTLRs possessed three conserved structures in the TIR domain. Meanwhile, the expression patterns of different Sptlr genes in examined tissues detected by qRT-PCR had wide differences. Compared with other Sptlr genes, Sptlr-6 gene was significantly highly expressed in the hepatopancreas and less expressed in other tissues. Therefore, the function of Sptlr-6 was further investigated. The expression of the Sptlr-6 gene was up-regulated by Poly I: C, PGN stimulation and Vibrio parahaemolyticus infection. In addition, the silencing of Sptlr-6 in hepatopancreas mediated by RNAi technology resulted in the significant decrease of several conserved genes involved in innate immunity in mud crab after V. parahaemolyticus infection, including relish, myd88, dorsal, anti-lipopolysaccharide factor (ALF), anti-lipopolysaccharide factor 2 (ALF-2) and glycine-rich antimicrobial peptide (glyamp). This study provided new knowledge for the role of the Sptlr-6 gene in defense against V. parahaemolyticus infection in S. paramamosain.

Molecular cloning and functional analyses of C-C motif chemokine ligand 3 (CCL3) in mandarin fish Siniperca chuatsi.

Chemokines are critical molecules involved in immune reaction and immune system homeostasis, and some chemokines play a role in antiviral immunity. It is not known if the C-C motif chemokine ligand 3 (CCL3), a member of the CC chemokine family, possesses antiviral properties in fish. In this study, a ccl3 was cloned from the mandarin fish (Siniperca chuatsi), and it has an open reading frame (ORF) of 276 base pairs, which are predicted to encode a 91-amino acid peptide. Mandarin fish CCL3 revealed conserved sequence features with four cysteine residues and closely relationships with the CCL3s from other vertebrates based on the sequence alignment and phylogenetic analysis. The transcripts of ccl3 were notably enriched in immune-related organs, such as spleen and gills in healthy mandarin fish, and the ccl3 was induced in the isolated mandarin fish brain (MFB) cells following infection with infectious spleen and kidney necrosis virus (ISKNV). Moreover, in MFB cells, overexpression of CCL3 induced immune factors, such as IL1ß, TNFα, MX, IRF1 and IFNh, and exhibited antiviral activity against ISKNV. This study sheds light on the immune role of CCL3 in immune response of mandarin fish, and its antiviral defense mechanism is of interest for further investigation.

The tick Ixodes scapularis has five different GPCRs specifically activated by ACP (adipokinetic hormone/corazonin-related peptide).

Insects have about 50 neuropeptide genes and about 70 genes, coding for neuropeptide G protein-coupled receptors (GPCRs). An important, but small family of evolutionarily related insect neuropeptides consists of adipokinetic hormone (AKH), corazonin, and AKH/corazonin-related peptide (ACP). Normally, insects have one specific GPCR for each of these neuropeptides. The tick Ixodes scapularis is not an insect, but belongs to the subphylum Chelicerata, which comprises ticks, scorpions, mites, spiders, and horseshoe crabs. Many of the neuropeptides and neuropeptide GPCRs occurring in insects, also occur in chelicerates, illustrating that insects and chelicerates are evolutionarily closely related. The tick I. scapularis is an ectoparasite and health risk for humans, because it infects its human host with dangerous pathogens during a blood meal. Understanding the biology of ticks will help researchers to prevent tick-borne diseases. By annotating the I. scapularis genome sequence, we previously found that ticks contain as many as five genes, coding for presumed ACP receptors. In the current paper, we cloned these receptors and expressed each of them in Chinese Hamster Ovary (CHO) cells. Each expressed receptor was activated by nanomolar concentrations of ACP, demonstrating that all five receptors were functional ACP receptors. Phylogenetic tree analyses showed that the cloned tick ACP receptors were mostly related to insect ACP receptors and, next, to insect AKH receptors, suggesting that ACP receptor genes and AKH receptor genes originated by gene duplications from a common ancestor. Similar duplications have probably occurred for the ligand genes, during a process of ligand/receptor co-evolution. Interestingly, chelicerates, in contrast to all other arthropods, do not have AKH or AKH receptor genes. Therefore, the ancestor of chelicerates might have lost AKH and AKH receptor genes and functionally replaced them by ACP and ACP receptor genes. For the small family of AKH, ACP, and corazonin receptors and their ligands, gene losses and gene gains occur frequently between the various ecdysozoan clades. Tardigrades, for example, which are well known for their survival in extreme environments, have as many as ten corazonin receptor genes and six corazonin peptide genes, while insects only have one of each, or none.

Novel structure of secreted small molecular weight antigen Mtb12 from Mycobacterium tuberculosis.

Mtb12, a small protein secreted by Mycobacterium tuberculosis, is known to elicit immune responses in individuals infected with the pathogen. It serves as an antigen recognized by the host's immune system. Due to its immunogenic properties and pivotal role in tuberculosis (TB) pathogenesis, Mtb12 is considered a promising candidate for TB diagnosis and vaccine development. However, the structural and functional properties of Mtb12 are largely unexplored, representing a significant gap in our understanding of M. tuberculosis biology. In this study, we present the first structure of Mtb12, which features a unique tertiary configuration consisting of four beta strands and four alpha helices. Structural analysis reveals that Mtb12 has a surface adorned with a negatively charged pocket adjacent to a central cavity. The features of these structural elements and their potential effects on the function of Mtb12 warrant further exploration. These findings offer valuable insights for vaccine design and the development of diagnostic tools.

Substrate displacement of CK1 C-termini regulates kinase specificity.

CK1 kinases participate in many signaling pathways, and their regulation is of meaningful biological consequence. CK1s autophosphorylate their C-terminal noncatalytic tails, and eliminating these tails increases substrate phosphorylation in vitro, suggesting that the autophosphorylated C-termini act as inhibitory pseudosubstrates. To test this prediction, we comprehensively identified the autophosphorylation sites on Schizosaccharomyces pombe Hhp1 and human CK1ε. Phosphoablating mutations increased Hhp1 and CK1ε activity toward substrates. Peptides corresponding to the C-termini interacted with the kinase domains only when phosphorylated, and substrates competitively inhibited binding of the autophosphorylated tails to the substrate binding grooves. Tail autophosphorylation influenced the catalytic efficiency with which CK1s targeted different substrates, and truncating the tail of CK1δ broadened its linear peptide substrate motif, indicating that tails contribute to substrate specificity as well. Considering autophosphorylation of both T220 in the catalytic domain and C-terminal sites, we propose a displacement specificity model to describe how autophosphorylation modulates substrate specificity for the CK1 family.

Staphylococcal peroxidase inhibitor (SPIN): Residue-level investigation of the helical bundle domain.

Myeloperoxidase is a critical component of the antibacterial arsenal of neutrophils, whereby it consumes H2O2 as an oxidant to convert halogen and pseudohalogen anions into cytotoxic hypohalous acids. Following phagocytosis by neutrophils, the human pathogen Staphylococcus aureus secretes a potent myeloperoxidase inhibitory protein, called SPIN, as part of its immune evasion repertoire. The matured S. aureus SPIN polypeptide consists of only 73 residues yet contains two functional domains: whereas the 60 residue C-terminal helical bundle domain is responsible for MPO binding, the 13 residue N-terminal domain is required to inhibit MPO. Previous studies have informed understanding of the SPIN N-terminal domain, but comparatively little is known about the helical domain insofar as the contribution of individual residues is concerned. To address this limitation, we carried out a residue-level structure/function investigation on the helical bundle domain of S. aureus SPIN. Using sequence conservation and existing structures of SPIN bound to human MPO as a guide, we selected residues L49, E50, H51, E52, Y55, and Y75 for interrogation by site-directed mutagenesis. We found that loss of L49 or E52 reduced SPIN activity by roughly an order of magnitude, but that loss of Y55 or H51 caused progressively greater loss of inhibitory potency. Direct binding studies by SPR showed that loss of inhibitory potency in these SPIN mutants resulted from a diminished initial interaction between the inhibitor and MPO. Together, our studies provide new insights into the structure/function relationships of SPIN and identify positions Y55 and H51 as critical determinants of SPIN function.

The importance of input sequence set to consensus-derived proteins and their relationship to reconstructed ancestral proteins.

A protein sequence encodes its energy landscape-all the accessible conformations, energetics, and dynamics. The evolutionary relationship between sequence and landscape can be probed phylogenetically by compiling a multiple sequence alignment of homologous sequences and generating common ancestors via Ancestral Sequence Reconstruction or a consensus protein containing the most common amino acid at each position. Both ancestral and consensus proteins are often more stable than their extant homologs-questioning the differences between them and suggesting that both approaches serve as general methods to engineer thermostability. We used the Ribonuclease H family to compare these approaches and evaluate how the evolutionary relationship of the input sequences affects the properties of the resulting consensus protein. While the consensus protein derived from our full Ribonuclease H sequence alignment is structured and active, it neither shows properties of a well-folded protein nor has enhanced stability. In contrast, the consensus protein derived from a phylogenetically-restricted set of sequences is significantly more stable and cooperatively folded, suggesting that cooperativity may be encoded by different mechanisms in separate clades and lost when too many diverse clades are combined to generate a consensus protein. To explore this, we compared pairwise covariance scores using a Potts formalism as well as higher-order sequence correlations using singular value decomposition (SVD). We find the SVD coordinates of a stable consensus sequence are close to coordinates of the analogous ancestor sequence and its descendants, whereas the unstable consensus sequences are outliers in SVD space.

Discovery of Borosin Catalytic Strategies and Function through Bioinformatic Profiling.

Borosins are ribosomally synthesized and post-translationally modified peptides (RiPPs) containing backbone α-N-methylations. These modifications confer favorable pharmacokinetic properties including increased membrane permeability and resistance to proteolytic degradation. Previous studies have biochemically and bioinformatically explored several borosins, revealing (1) numerous domain architectures and (2) diverse core regions lacking conserved sequence elements. Due to these characteristics, large-scale computational identification of borosin biosynthetic genes remains challenging and often requires additional, time-intensive manual inspection. This work builds upon previous findings and updates the genome-mining tool RODEO to automatically evaluate borosin biosynthetic gene clusters (BGCs) and identify putative precursor peptides. Using the new RODEO module, we provide an updated analysis of borosin BGCs identified in the NCBI database. From our data set, we bioinformatically predict and experimentally characterize a new fused borosin domain architecture, in which the modified natural product core is encoded N-terminal to the methyltransferase domain. Additionally, we demonstrate that a borosin precursor peptide is a native substrate of shewasin A, a reported aspartyl peptidase with no previously identified substrates. Shewasin A requires post-translational modification of the leader peptide for proteolytic maturation, a feature not previously observed in RiPPs. Overall, this work provides a user-friendly and open-access tool for the analysis of borosin BGCs and we demonstrate its utility to uncover additional biosynthetic strategies within the borosin class of RiPPs.

A conserved motif in the immune-subdominant RAP-1 related antigen of Babesia bovis contains a B-cell epitope recognized by antibodies from protected cattle.

Introduction: Babesia bovis, a tick-borne apicomplexan parasite causing bovine babesiosis, remains a significant threat worldwide, and improved and practical vaccines are needed. Previous studies defined the members of the rhoptry associated protein-1 (RAP-1), and the neutralization-sensitive rhoptry associated protein-1 related antigen (RRA) superfamily in B. bovis, as strong candidates for the development of subunit vaccines. Both RAP-1 and RRA share conservation of a group of 4 cysteines and amino acids motifs at the amino terminal end (NT) of these proteins. Methods and results: Sequence comparisons among the RRA sequences of several B. bovis strains and other Babesia spp parasites indicate a high level of conservation of a 15-amino acid (15-mer) motif located at the NT of the protein. BlastP searches indicate that the 15-mer motif is also present in adenylate cyclase, dynein, and other ATP binding proteins. AlphaFold2 structure predictions suggest partial exposure of the 15-mer on the surface of RRA of three distinct Babesia species. Antibodies in protected cattle recognize a synthetic peptide representing the 15-mer motif sequence in iELISA, and rabbit antibodies against the 15-mer react with the surface of free merozoites in immunofluorescence. Discussion and conclusion: The presence of the 15-mer-like regions in dynein and ATP-binding proteins provides a rationale for investigating possible functional roles for RRA. The demonstrated presence of a surface exposed B-cell epitope in the 15-mer motif of the B. bovis RRA, which is recognized by sera from protected bovines, supports its inclusion in future subunit epitope-based vaccines against B. bovis.

Mass spectrometric analysis of Odonthobuthus Doriae scorpion venom and its non-neutralized fractions after interaction with commercial antivenom.

It is believed that antivenoms play a crucial role in neutralizing venoms. However, uncontrolled clinical effects appear in patients stung by scorpions after the injection of antivenom. In this research, non-neutralized components of the venom of the Iranian scorpion Odonthobuthus doriae were analyzed after interacting with the commercial antivenom available in the market. The venom and antivenom interaction was performed, then centrifuged, and the supernatant was analyzed by high-performance liquid chromatography (HPLC). Two peaks of Odonthobuthus doriae venom were observed in the chromatogram of the supernatant. Two components were isolated by HPLC and analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) instruments. Peptide sequencing was done by Liquid Chromatography Quadrupole Time-of-Flight Tandem Mass Spectrometry (LC-Q-TOF MS/MS). Results indicate that the components of scorpion venom mainly have a molecular weight below 10 kDa, consisting of toxic peptides that disrupt the function of sodium and potassium channels. The MALDI-TOF MS results show that two toxic peptides with molecular masses of 6941 Da and 6396 Da were not neutralized by the antivenom. According to the MS/MS sequencing data, the components have been related to peptides A0A5P8U2Q6_MESEU and A0A0U4FP89_ODODO, which belong to the sodium and potassium channels toxins family, respectively.

Predicting TCR sequences for unseen antigen epitopes using structural and sequence features.

T-cell receptor (TCR) recognition of antigens is fundamental to the adaptive immune response. With the expansion of experimental techniques, a substantial database of matched TCR-antigen pairs has emerged, presenting opportunities for computational prediction models. However, accurately forecasting the binding affinities of unseen antigen-TCR pairs remains a major challenge. Here, we present convolutional-self-attention TCR (CATCR), a novel framework tailored to enhance the prediction of epitope and TCR interactions. Our approach utilizes convolutional neural networks to extract peptide features from residue contact matrices, as generated by OpenFold, and a transformer to encode segment-based coded sequences. We introduce CATCR-D, a discriminator that can assess binding by analyzing the structural and sequence features of epitopes and CDR3-ß regions. Additionally, the framework comprises CATCR-G, a generative module designed for CDR3-ß sequences, which applies the pretrained encoder to deduce epitope characteristics and a transformer decoder for predicting matching CDR3-ß sequences. CATCR-D achieved an AUROC of 0.89 on previously unseen epitope-TCR pairs and outperformed four benchmark models by a margin of 17.4%. CATCR-G has demonstrated high precision, recall and F1 scores, surpassing 95% in bidirectional encoder representations from transformers score assessments. Our results indicate that CATCR is an effective tool for predicting unseen epitope-TCR interactions. Incorporating structural insights enhances our understanding of the general rules governing TCR-epitope recognition significantly. The ability to predict TCRs for novel epitopes using structural and sequence information is promising, and broadening the repository of experimental TCR-epitope data could further improve the precision of epitope-TCR binding predictions.

1H, 13C, and 15N resonance assignments of the La Motif of the human La-related protein 1.

Human La-related protein 1 (HsLARP1) is involved in post-transcriptional regulation of certain 5' terminal oligopyrimidine (5'TOP) mRNAs as well as other mRNAs and binds to both the 5'TOP motif and the 3'-poly(A) tail of certain mRNAs. HsLARP1 is heavily involved in cell proliferation, cell cycle defects, and cancer, where HsLARP1 is significantly upregulated in malignant cells and tissues. Like all LARPs, HsLARP1 contains a folded RNA binding domain, the La motif (LaM). Our current understanding of post-transcriptional regulation that emanates from the intricate molecular framework of HsLARP1 is currently limited to small snapshots, obfuscating our understanding of the full picture on HsLARP1 functionality in post-transcriptional events. Here, we present the nearly complete resonance assignment of the LaM of HsLARP1, providing a significant platform for future NMR spectroscopic studies.

A new point mutation in the HC-Pro of potato virus Y is involved in tobacco vein necrosis.

Tobacco vein necrosis (TVN) is a complex phenomenon regulated by different genetic determinants mapped in the HC-Pro protein (amino acids N330, K391 and E410) and in two regions of potato virus Y (PVY) genome, corresponding to the cytoplasmic inclusion (CI) protein and the nuclear inclusion protein a-protease (NIa-Pro), respectively. A new determinant of TVN was discovered in the MK isolate of PVY which, although carried the HC-Pro determinants associated to TVN, did not induce TVN. The HC-Pro open reading frame (ORF) of the necrotic infectious clone PVY N605 was replaced with that of the non-necrotic MK isolate, which differed only by one amino acid at position 392 (T392 instead of I392). The cDNA clone N605_MKHCPro inoculated in tobacco induced only weak mosaics at the systemic level, demostrating that the amino acid at position 392 is a new determinant for TVN. No significant difference in accumulation in tobacco was observed between N605 and N605_MKHCPro. Since phylogenetic analyses showed that the loss of necrosis in tobacco has occurred several times independently during PVY evolution, these repeated evolutions strongly suggest that tobacco necrosis is a costly trait in PVY.

Silencing Ditylenchus destructor cathepsin L-like cysteine protease has negative pleiotropic effect on nematode ontogenesis.

Ditylenchus destructor is a migratory plant-parasitic nematode that severely harms many agriculturally important crops. The control of this pest is difficult, thus efficient strategies for its management in agricultural production are urgently required. Cathepsin L-like cysteine protease (CPL) is one important protease that has been shown to participate in various physiological and pathological processes. Here we decided to characterize the CPL gene (Dd-cpl-1) from D. destructor. Analysis of Dd-cpl-1 gene showed that Dd-cpl-1 gene contains a signal peptide, an I29 inhibitor domain with ERFNIN and GNFD motifs, and a peptidase C1 domain with four conserved active residues, showing evolutionary conservation with other nematode CPLs. RT-qPCR revealed that Dd-cpl-1 gene displayed high expression in third-stage juveniles (J3s) and female adults. In situ hybridization analysis demonstrated that Dd-cpl-1 was expressed in the digestive system and reproductive organs. Silencing Dd-cpl-1 in 1-cell stage eggs of D. destructor by RNAi resulted in a severely delay in development or even in abortive morphogenesis during embryogenesis. The RNAi-mediated silencing of Dd-cpl-1 in J2s and J3s resulted in a developmental arrest phenotype in J3 stage. In addition, silencing Dd-cpl-1 gene expression in female adults led to a 57.43% decrease in egg production. Finally, Dd-cpl-1 RNAi-treated nematodes showed a significant reduction in host colonization and infection. Overall, our results indicate that Dd-CPL-1 plays multiple roles in D. destructor ontogenesis and could serve as a new potential target for controlling D. destructor.

Scoring alignments by embedding vector similarity.

Sequence similarity is of paramount importance in biology, as similar sequences tend to have similar function and share common ancestry. Scoring matrices, such as PAM or BLOSUM, play a crucial role in all bioinformatics algorithms for identifying similarities, but have the drawback that they are fixed, independent of context. We propose a new scoring method for amino acid similarity that remedies this weakness, being contextually dependent. It relies on recent advances in deep learning architectures that employ self-supervised learning in order to leverage the power of enormous amounts of unlabelled data to generate contextual embeddings, which are vector representations for words. These ideas have been applied to protein sequences, producing embedding vectors for protein residues. We propose the E-score between two residues as the cosine similarity between their embedding vector representations. Thorough testing on a wide variety of reference multiple sequence alignments indicate that the alignments produced using the new $E$-score method, especially ProtT5-score, are significantly better than those obtained using BLOSUM matrices. The new method proposes to change the way alignments are computed, with far-reaching implications in all areas of textual data that use sequence similarity. The program to compute alignments based on various $E$-scores is available as a web server at e-score.csd.uwo.ca. The source code is freely available for download from github.com/lucian-ilie/E-score.