Sequence Similarity Research Articles

Identification of multiple inter- and intra-genotype reassortment mammalian orthoreoviruses from Japanese black cattle in a beef cattle farm

Mammalian orthoreoviruses (MRVs), belonging to the genus Orthoreovirus in the family Spinareoviridae, possess a double-stranded RNA segmented genome. Due to the segmented nature of their genome, MRVs are prone to gene reassortment, which allows for evolutionary diversification. Recently, a genotyping system for each MRV gene segment was proposed based on nucleotide differences. In the present study, MRVs were isolated from the fecal samples of Japanese Black cattle kept on a farm in Japan. Complete genome sequencing and analysis of 41 MRV isolates revealed that these MRVs shared almost identical sequences in the L1, L2, L3, S3, and S4 gene segments, while two different sequences were found in the S1, M1, M2, M3, and S2 gene segments. By plaque cloning, at least six genetic constellation patterns were identified, indicating the occurrence of multiple inter- (S1 and M2) and intra- (M1, M3, and S2) reassortment events. This paper represents the first report describing multiple reassortant MRVs on a single cattle farm. These MRV gene segments exhibited sequence similarity to those of MRVs isolated from cattle in the U.S. and China, rather than to MRVs previously isolated in Japan. Genotypes consisting solely of bovine MRVs were observed in the L1, M1, and M2 segments, suggesting that they might have evolved within the cattle population.

Molecular characterization of a novel gammapartitivirus infecting the fungus Nigrospora oryzae.

Here, we identified a new mycovirus infecting the phytopathogenic fungus Nigrospora oryzae, which we have designated "Nigrospora oryzae partitivirus 2" (NoPV2). The genome of NoPV2 consists of two dsRNA segments (dsRNA 1 and dsRNA 2), measuring 1771 and 1440 bp in length, respectively. dsRNA 1 and dsRNA 2 each contain a single open reading frame (ORF) that encodes the RNA-dependent RNA polymerase (RdRp) and capsid protein (CP), respectively. A BLASTp search showed that the RdRp of NoPV2 had significant sequence similarity to the RdRps of other partitiviruses, including Nigrospora sphaerica partitivirus 1 (75.61% identity) and Magnaporthe oryzae partitivirus 1 (67.53% identity). Phylogenetic analysis revealed that NoPV2 is a new member of the genus Gammapartitivirus in the family Partitiviridae. This study provides important information for understanding the diversity of mycoviruses in N. oryzae.

Development of a novel crAss-like phage detection method with a broad spectrum for microbial source tracking

CrAssphage has been recognized as the most abundant and human-specific bacteriophage in the human gut. Consequently, crAssphage has been used as a microbial source tracking (MST) marker to monitor human fecal contamination. Many crAss-like phages (CLPs) have been recently discovered, expanding the classification into the new order Crassvirales. This study aims to assess CLP prevalence in South Korea and develop a detection system for MST applications. Thirteen CLPs were identified in six human fecal samples and categorized into seven genera via metagenomic analysis. The major head protein (MHP) displayed increased sequence similarity within each genus. Eight PCR primer candidates, designed from MHP sequences, were evaluated in animal and human feces. CLPs were absent in animal feces except for those from raccoons, which hosted genera VI, VIIa, and VIIb. CLPs were detected in 91.52% (54/59) of humans, with genus VI (38 out of 59) showing the highest prevalence, nearly double that of p-crAssphage in genus I (22 out of 59). This study highlights genus VI as a potent MST marker, broadening the detection range for CLPs. Human-specific and selectively targeted MST markers can significantly impact hygiene regulations, lowering public health costs through their application in screening liver, sewage, wastewater, and various environmental samples.

Comparative chloroplast genome analyses of cultivated and wild Capsicum species shed light on evolution and phylogeny

The chloroplast (cp.) genome, also known as plastome, plays crucial roles in plant survival, adaptation, and evolution. The stable genetic structure of cp. genomes provides an ideal system for investigating species evolution. We sequenced three complete cp. genome sequences of Capsicum species and analyzed them using sequences of various Capsicum species retrieved from the NCBI database. The cp. genome of Capsicum species maintains a well-preserved quadripartite structure consisting of two inverted repeats (IRs) flanked by a large single copy (LSC) region and a small single copy (SSC) region. The sizes of cp. genome sequences ranged from 156,583 bp (C. lycianthoides) to 157,390 bp (C.pubescens). A total of 127–132 unique genes, including 83–87 protein-coding, 36–37 tRNA, and eight rRNA genes, were predicted. Comparison of cp. genomes of 10 Capsicum species revealed high sequence similarity in genome-wide organization and gene arrangements. Fragments of trnT-UGU/trnL-UAA, ccsA, ndhD, rps12, and ycf1 were identified as variable regions, and nucleotide variability of LSC and SSC was higher than that of IR. Phylogenetic speciation analysis showed that the major domesticated C. annuum species were the most extensively divergent species and closely related to C. tovarii and C. frutescens. Analysis of divergent times suggested that a substantial range of speciation events started occurring ~ 25.79 million years ago (Mya). Overall, comparative analysis of cp. genomes of Capsicum species not only offers new insights into their genetic variation and phylogenetic relationships, but also lays a foundation for evolutionary history, genetic diversity, conservation, and biological breeding of Capsicum species.

Genome-wide identification of phytosulfokine (PSK) peptide family reveals TaPSK1 gene involved in grain development of wheat (Triticum aestivum L.)

BackgroundPhytosulfokine (PSK) functions as a plant peptide growth factor that plays important and diverse roles in plant development and stress responses. Nevertheless, PSKs have not been systematically analyzed in wheat.ResultsA genome-wide comparative analysis of PSK genes in wheat was conducted and 15 TaPSKs were identified and divided into four subgroups in the wheat genome based on sequence similarity. The examination of motif compositions of TaPSK genes revealed the presence of the YIYTQ signature motif in the C-terminus of all TaPSK polypeptide precursors, with a highly conserved feature across different species. Exogenous application of TaPSK peptide promoted root growth in wheat. Quantitative RT-PCR analysis revealed that the TaPSKs exhibited preferential or tissue-specific expression patterns in wheat. In particular, three homologs of the TaPSK1 genes were specifically expressed in grains, with the strongest expression observed in the developing grains at 15 days after anthesis. Compared with wild type, transgenic rice lines overexpressing TaPSK1-A exhibited larger grain size and higher thousand-grain weight. The promoter region and genomic sequence of the wheat TaPSK1-A gene were cloned. Sequence polymorphism showed that five single-nucleotide polymorphisms (SNPs) were identified in the promoter region of TaPSK1-A. A Kompetitive Allele-Specific PCR (KASP) marker was developed for TaPSK1-A based on –806 bp SNP (C/T transition), and two haplotypes, TaPSK1-A-HapI and TaPSK1-A-HapII were detected in 260 wheat accessions collected from different regions. The expression of TaPSK1-A, promoter activity, and thousand-grain weight (TGW) in the TaPSK1-A-HapII haplotype were higher than those in the TaPSK1-A-HapI haplotype. Furthermore, yeast one-hybrid assays revealed the binding of TaNF-YB1 and TaERF39 to the promoter regions of the TaPSK1-A gene, and TaMADS29 could bind to the promoters of TaPSK1-B and TaPSK1-D genes.ConclusionsComparative genome-wide analysis of TaPSK peptide family revealed that the TaPSK1 gene is involved in wheat grain development, and the developed TaPSK1-A-KASP marker could be utilized for marker-assisted selection breeding of wheat.Graphical

Biosynthesis of the benzylpyrrolidine precursor in anisomycin by a unique ThDP-dependent enzyme

Anisomycin (compound 1), a multifunctional pyrrolidine antibiotic, primarily inhibits protein biosynthesis by binding to the ribosome. Upon binding to the ribosome, the para-phenol moiety of anisomycin inserts completely into the hydrophobic crevice of the A-site and blocks the access of the incoming aminoacyl-tRNAs, disrupting peptide bond formation. Hence, the para-methoxyphenyl group serves as a starting point for developing novel anisomycin analogs with potent antifungal and insecticidal properties. However, the activation and condensation mechanism of phenylpyruvic acid has not yet been elucidated. In this study, genetic manipulations of aniP and its homologue siAniP confirmed their indispensable role in 1 biosynthesis. Bioinformatics analysis suggested that AniP and siAniP function as transketolase. siAniP was found to catalyzed condensation between 4-hydroxyphenylpyruvic acid (3) and glyceraldehyde (GA), initiating pyrrolidine synthesis. siAniP was specific for aromatic keto acids and tolerant of aliphatic and aromatic aldehydes, and was able to catalyze the asymmetric intermolecular condensation of two keto acids, leading to the formation of 24 α-hydroxy ketone. To the best of our knowledge, siAniP is the first TK that catalyzes the transfer of a C2 ketol and symmetrical intermolecular coupling using aromatic keto acids as donor substrates. Structural analysis, docking model construction, and site-directed mutagenesis identified that I220, H275, R322 and W391 were crucial for substrate binding. Moreover, sequence similarity network (SSN)-based genome neighborhood network (GNN) analyses of AniP suggested the widespread occurrence of the AniP-like-mediated reaction in the biosynthesis of 1 and its analogs, particularly in the assembly of benzylpyrrolidine. These findings not only expand the repertoire of TKs but also provide a potent biocatalyst that could be used for the structural innovation of 1 and its derivatives.

Molecular and phylogenetic analysis of transmissible gastroenteritis virus strain VET-16, isolated from piglets in Vietnam.

Porcine transmissible gastroenteritis virus (TGEV) is a major pathogen that causes viral enteritis and severe diarrhea in newborn piglets. TGEV strains have been isolated in the USA, Europe, and China, and their molecular characteristics are well known. However, there have been few reports of molecular analysis of TGEV strains isolated in Southeast Asia. In 2016, we isolated TGEV strain VET-16 from fecal samples collected from piglets in Vietnam and determined its complete genome sequence by Sanger sequencing. We found that, while the full genome of the VET-16 strain was 92.4-99.9% identical to those of other TGEV strains, the ORF3 gene showed very little sequence similarity. Phylogenetic analysis suggested that the VET-16 strain belongs to the Purdue subgroup. Comparison of the predicted amino acid (aa) sequence of the spike protein of strain VET-16 with those of other TGEV strains revealed three aa substitutions (V378L, S379T, and D380N) and a 3-aa insertion (F383_F387insWEK) in antigenic site D of the VET-16 strain. Also, a single aa deletion (∆F1413) was found in the transmembrane domain of the spike gene of VET-16. Like the ORF3 gene from the TGEV Miller M60 vaccine strain, the VET-16 strain has a large deletion (∆725 nt) in the ORF3 gene. Previous studies have suggested that these mutations in the spike and ORF3 genes might be associated with a reduction in pathogenicity. The data from this study will facilitate further genetic analysis and research into the evolution of TGEV in pigs in Vietnam.

A new isolate of mungbean yellow mosaic India virus in Vigna mungo L. reported from a Dayalbagh field, Agra.

Black gram (Vigna mungo L.) plants showing yellow mosaic symptoms during 2019-2022 crop seasons were collected randomly from a Dayalbagh field, Agra Region of Uttar Pradesh, India. Total genomic DNA was isolated from the infected leaf samples by the Cetyltrimethylammonium bromide (CTAB) method and subjected to PCR. After viral confirmation, the viral genome was amplified by rolling circle amplification following the standard protocol. The DNA A and DNA B subgenomes were cloned individually as a PstI and BamHI fragment in the pUC18 vector. Positive clones were subjected to DNA sequencing. The results revealed that DNA A and DNA B show the closest nucleotide identity with "mungbean yellow mosaic India virus-[Mungbean], DNA-A, the complete sequence" (GeneBank Accession No AF416742.1) with 98.14% identity, and "mungbean yellow mosaic India virus isolate Mu1-Dholi segment DNA-B, the complete sequence" (GeneBank Accession No MW814723.1) with 97.94% identity, respectively. The new isolate of mungbean yellow mosaic India virus (MYMIV) shows sequence similarity with the coat protein gene of various strains of MYMIV. In the new isolate of MYMIV, a point mutation wasobserved at the 2036th nucleotide of DNA B, which disrupts the reading frame to introduce a stop codon and thus leading to a decrease in the size of the movement protein gene. In the present study we are reporting the whole genome sequence of the MYMIV Dayalbagh isolate for the first time.

Computational detection of antigen-specific B cell receptors following immunization

B cell receptors (BCRs) play a crucial role in recognizing and fighting foreign antigens. High-throughput sequencing enables in-depth sampling of the BCRs repertoire after immunization. However, only a minor fraction of BCRs actively participate in any given infection. To what extent can we accurately identify antigen-specific sequences directly from BCRs repertoires? We present a computational method grounded on sequence similarity, aimed at identifying statistically significant responsive BCRs. This method leverages well-known characteristics of affinity maturation and expected diversity. We validate its effectiveness using longitudinally sampled human immune repertoire data following influenza vaccination and SARS-CoV-2 infections. We show that different lineages converge to the same responding Complementarity Determining Region 3, demonstrating convergent selection within an individual. The outcomes of this method hold promise for application in vaccine development, personalized medicine, and antibody-derived therapeutics.

Comparative and phylogenetic analyses of plastid genomes of the medicinally important genus Alisma (Alismataceae).

Alisma L. is a medicinally important genus of aquatic and wetland plants consisting of c. 10 recognized species. However, largely due to polyploidy and limited taxon and gene sampling, the phylogenomic relationships of Alisma remain challenging. In this study, we sequenced 34 accessions of Alismataceae, including eight of the ten species of Alisma, one species of Echinodorus and one species of Luronium, to perform comparative analyses of plastid genomes and phylogenetic analyses. Comparative analysis of plastid genomes revealed high sequence similarity among species within the genus. Our study analyzed structural changes and variations in the plastomes of Alisma, including IR expansion or contraction, and gene duplication or loss. Phylogenetic results suggest that Alisma is monophyletic, and constitutes four groups: (1) A. lanceolatum and A. canaliculatum; (2) the North American clade of A. subcordatum and A. triviale; (3) A. wahlenbergii and A. gramineum; and (4) A. plantago-aquatica from Eurasia and northern Africa with the eastern Asian A. orientale nested within it. Hence the results challenge the recognition of A. orientale as a distinct species and raise the possibility of treating it as a synonym of the widespread A. plantago-aquatica. The well-known Alismatis Rhizoma (Zexie) in Chinese medicine was likely derived from the morphologically variable Alisma plantago-aquatica throughout its long history of cultivation in Asia. The plastome phylogenetic results also support the tetraploid A. lanceolatum as the likely maternal parent of the hexaploid eastern Asian A. canaliculatum.

Evolutionary Insights from the Mitochondrial Genome of Oikopleura dioica: Sequencing Challenges, RNA Editing, Gene Transfers to the Nucleus, and tRNA Loss.

Sequencing the mitochondrial genome of the tunicate Oikopleura dioica is a challenging task due to the presence of long poly-A/T homopolymer stretches, which impair sequencing and assembly. Here, we report on the sequencing and annotation of the majority of the mitochondrial genome of O. dioica by means of combining several DNA and amplicon reads obtained by Illumina and MinIon Oxford Nanopore Technologies with public RNA sequences. We document extensive RNA editing, since all homopolymer stretches present in the mitochondrial DNA correspond to 6U-regions in the mitochondrial RNA. Out of the 13 canonical protein-coding genes, we were able to detect eight, plus an unassigned open reading frame that lacked sequence similarity to canonical mitochondrial protein-coding genes. We show that the nad3 gene has been transferred to the nucleus and acquired a mitochondria-targeting signal. In addition to two very short rRNAs, we could only identify a single tRNA (tRNA-Met), suggesting multiple losses of tRNA genes, supported by a corresponding loss of mitochondrial aminoacyl-tRNA synthetases in the nuclear genome. Based on the eight canonical protein-coding genes identified, we reconstructed maximum likelihood and Bayesian phylogenetic trees and inferred an extreme evolutionary rate of this mitochondrial genome. The phylogenetic position of appendicularians among tunicates, however, could not be accurately determined.

Walrycin B, as a novel separase inhibitor, exerts potent anticancer efficacy in a mouse xenograft model

Proper chromosome segregation during cell division relies on the timely dissolution of chromosome cohesion. Separase (EC3.4.22.49), a cysteine protease, plays a critical role in mitosis by cleaving the kleisin subunit of cohesin, thereby presenting a promising target for cancer therapy. However, challenges in isolating active human separase suitable for high-throughput screening have limited the identification of effective inhibitors. Here, we conducted a high-throughput screening of small-molecule inhibitors using the protease domain of Chaetomium thermophilum separase (ctSPD), which not only shares significant sequence similarity with human separase but is also readily available. After conducting a primary screening of a library containing 9,172 compounds and subsequent validation using human separase, we identified walrycin B and its analogs, toxoflavin, 3-methyltoxoflavin, and 3-phenyltoxoflavin, as potent inhibitors of human separase. Subsequent microscale thermophoresis assays and molecular dynamics simulations revealed that walrycin B binds to the active site of separase and competes with substrates for binding. Additionally, cell-based studies showed that walrycin B and its analogs effectively induce cell cycle arrest at the M phase, activate apoptosis, and ultimately lead to cell death in mitosis. Finally, in a mouse xenograft model, walrycin B exhibited significant antitumor efficacy with minimal side effects. Together, these findings highlight the therapeutic potential of walrycin B for cancer treatment and its utility as a chemical tool in future studies involving separase.

Sequence analysis of isolated strains of herpes zoster virus among patients with shingles.

Herpes zoster, or shingles, is caused by the varicella-zoster virus (VZV), which initially presents as chickenpox in children. VZV is a global health concern, especially in winter and spring, affecting 10-20% of adults over 50 and posing a 30% risk for the general population. This study used PCR to detect VZV, confirming results with duplicated DNA samples and identifying 234 bp fragments by targeting the gpB gene. This study examined 50 herpes zoster cases from October 2020 to April 2021, involving 30 males and 20 females aged 10 to 90, diagnosed by dermatologists. Data were collected via a questionnaire. PCR detected VZV by amplifying the gpB and MCP genes from skin lesion samples. Six positive 234-bp PCR products were sequenced at Macrogen Inc. in Seoul, South Korea. Six DNA samples with 234 bp amplicons were sequenced, showing 99-100% similarity to human alpha herpesvirus sequences in the gpB gene. NCBI BLAST matched these sequences to a reference (GenBank acc. MT370830.1), assigning accession numbers LC642111, LC642112, and LC642113. Eight nucleic acid substitutions caused amino acid changes in the gpB protein: isoleucine to threonine, serine to isoleucine, and threonine to Proline. These variants were deposited in NCBI GenBank as gpB3 samples. The study found high sequence similarity to known VZV sequences, identifying six nucleic acid variations and eight SNPs. Notable amino acid changes in the gpB protein were deposited in NCBI GenBank as the gpB3 sample.

The Feline calicivirus Leader of the Capsid (LC) Protein Contains a Putative Transmembrane Domain, Binds to the Cytoplasmic Membrane, and Exogenously Permeates Cells.

Feline calicivirus (FCV), an important model for studying the biology of the Caliciviridae family, encodes the leader of the capsid (LC) protein, a viral factor known to induce apoptosis when expressed in a virus-free system. Our research has shown that the FCV LC protein forms disulfide bond-dependent homo-oligomers and exhibits intrinsic toxicity; however, it lacked a polybasic region and a transmembrane domain (TMD); thus, it was initially classified as a non-classical viroporin. The unique nature of the FCV LC protein, with no similarity to other proteins beyond the Vesivirus genus, has posed challenges for bioinformatic analysis reliant on sequence similarity. In this study, we continued characterizing the LC protein using the AlphaFold 2 and the recently released AlphaFold 3 artificial intelligence tools to predict the LC protein tertiary structure. We compared it to other molecular modeling algorithms, such as I-Tasser's QUARK, offering new insights into its putative TMD. Through exogenous interaction, we found that the recombinant LC protein associates with the CrFK plasmatic membrane and can permeate cell membranes in a disulfide bond-independent manner, suggesting that this interaction might occur through a TMD. Additionally, we examined its potential to activate the intrinsic apoptosis pathway in murine and human ovarian cancer cell lines, overexpressing survivin, an anti-apoptotic protein. All these results enhance our understanding of the LC protein's mechanism of action and suggest its role as a class-I viroporin.

FAM210A: An emerging regulator of mitochondrial homeostasis.

Mitochondrial homeostasis serves as a cornerstone of cellular function, orchestrating a delicate balance between energy production, redox status, and cellular signaling transduction. This equilibrium involves a myriad of interconnected processes, including mitochondrial dynamics, quality control mechanisms, and biogenesis and degradation. Perturbations in mitochondrial homeostasis have been implicated in a wide range of diseases, including neurodegenerative diseases, metabolic syndromes, and aging-related disorders. In the past decades, the discovery of numerous mitochondrial proteins and signaling has led to a more complete understanding of the intricate mechanisms underlying mitochondrial homeostasis. Recent studies have revealed that Family with sequence similarity 210 member A (FAM210A) is a novel nuclear-encoded mitochondrial protein involved in multiple aspects of mitochondrial homeostasis, including mitochondrial quality control, dynamics, cristae remodeling, metabolism, and proteostasis. Here, we review the function and physiological role of FAM210A in cellular and organismal health. This review discusses how FAM210A acts as a regulator on mitochondrial inner membrane to coordinate mitochondrial dynamics and metabolism.

Rufibacter psychrotolerans sp. nov., a Cold-Tolerating Novel Species Isolated from Desert Soil.

Strain SYSU D00308T, a short-rod-shaped bacterium, was isolated from a sandy soil collected from the Gurbantunggut Desert, Xinjiang, PR China. Strain SYSU D00308T was Gram-stain-negative, aerobic, pink-pigmented, non-motile, catalase- and oxidase-positive. The strain grew at 4-37 ℃, pH 5.0-8.0 and 0-1.5% (w/v) NaCl. 16S rRNA gene sequencing analyses demonstrated that strain SYSU D00308T belonged to the genus Rufibacter and exhibited the highest sequence similarity (97.4%) to Rufibacter glacialis MDT1-10-3T. Summed features 3, 4, and iso-C15:0 were the major fatty acids, and menaquinone 7 (MK-7) was the sole respiratory menaquinone. The polar lipid profiles comprised phosphatidylethanolamine, an unidentified glycolipid, an unidentified phospholipid, two unidentified aminophospholipids, and two unidentified lipids. The genome size and DNA G + C content of strain SYSU D00308T were 5,176,683 bp and 54.8%, respectively. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between SYSU D00308T and members of the genus Rufibacter were 77.7-81.8% and 20.4-23.4% respectively, which were less than the corresponding thresholds (ANI: 95-96%; dDDH: 70%) for prokaryotic species definition. According to the genotypic, phenotypic and phylogenetic characteristics, strain SYSU D00308T represents a novel species of the genus Rufibacter. We propose the name, Rufibacter psychrotolerans sp. nov., with SYSU D00308T (= CGMCC 1.18621T = KCTC 82275T = MCCC 1K04970T) as the type strain.

Fam49b dampens TCR signal strength to regulate survival of positively selected thymocytes and peripheral T cells.

The fate of developing T cells is determined by the strength of T cell receptor (TCR) signal they receive in the thymus. This process is finely regulated through the tuning of positive and negative regulators in thymocytes. The Family with sequence similarity 49 member B (Fam49b) protein is a newly discovered negative regulator of TCR signaling that has been shown to suppress Rac-1 activity in vitro in cultured T cell lines. However, the contribution of Fam49b to the thymic development of T cells is unknown. To investigate this important issue, we generated a novel mouse line deficient in Fam49b (Fam49b-KO). We observed that Fam49b-KO double positive (DP) thymocytes underwent excessive negative selection, whereas the positive selection stage was unaffected. Fam49b deficiency impaired the survival of single positive thymocytes and peripheral T cells. This altered development process resulted in significant reductions in CD4 and CD8 single-positive thymocytes as well as peripheral T cells. Interestingly, a large proportion of the TCRγδ+ and CD8αα+TCRαβ+ gut intraepithelial T lymphocytes were absent in Fam49b-KO mice. Our results demonstrate that Fam49b dampens thymocytes TCR signaling in order to escape negative selection during development, uncovering the function of Fam49b as a critical regulator of the selection process to ensure normal thymocyte development and peripheral T cells survival.

Candidatus Midichloria mitochondrii can be vertically transmitted in Hyalomma anatolicum

In this study, a tick intracellular symbiont, Candidatus Midichloria mitochondrii, was detected in Hyalomma anatolicum from Xinjiang, China. Morphological identification and cytochrome oxidase subunit I sequence alignment were used for molecular identification of the tick species. PCR detection further revealed the presence of endosymbiont C. M. mitochondrii in the tick. Specific primers were designed for Groel and 16S rRNA genes of C. M. mitochondrii for PCR amplification and phylogenetic analysis. To further investigate the vertical transmission characteristics of C. M. mitochondrii, specific primers were designed based on the FabⅠ gene fragment to detect C. M. mitochondrii in different developmental stages and organs of the tick using qPCR. Of the 336 tick specimens collected from the field, 266 samples were identified as H. anatolicum on the basis of morphological characteristics. The gene fragment alignment results of COI confirmed that these ticks were H. anatolicum. The phylogenetic analysis showed that Groel gene of C. M. mitochondrii clustered with Midichloria strains detected in Ixodes ricinus ticks from Italy and Ixodes holocyclus ticks from Australia, with 100% sequence similarity. Furthermore, the 16S rRNA gene of C. M. mitochondrii clusters with the strains isolated from Hyalomma rufipes ticks in Italy, exhibiting the highest degree of homology. qPCR results showed that C. M. mitochondrii was present at all developmental stages of H. anatolicum, with the highest relative abundance in eggs, and lower relative abundance in nymphs and unfed males. With female tick blood feeding, the relative abundance of C. M. mitochondrii increased, and a particularly high relative abundance was detected in the ovaries of engorged female ticks. This study provides information for studying the survival adaptability of H. anatolicum, and provides data for further investigation of the mechanisms regulating tick endosymbionts in ticks, enriching the reference materials for comprehensive prevention and control of tick-borne diseases.

Homology-based characterization of the cis-regulatory elements modulate flavone induction of CYP321A1 in Helicoverpa armigera

Xenobiotic response element (XRE) to flavone was the cis- regulatory elements that mediates the induction of the allelochemical-metabolizing CYP321A1 gene from Helicoverpa zea. However, it was unknown whether the XRE-Fla element existed in other species. Recently we have identified and cloned the CYP321A1 gene with promoter region in a related species, Helicoverpa armigera. Sequence similarity of two orthologous CYP321A1 genes was 97.27%, but the promoter sequence similarity was only 56.32%. Sequence alignment showed the XRE-Fla like element owns three mutations in H. armigera compared with H. zea. Progressive 5′ deletions and internal mutation indicated that H. armigera XRE-Fla was the essential element of CYP321A1 gene in response to flavone. XRE-Fla mutations and EMSA analysis confirmed that the H. armigera XRE-Fla element binding factor was stronger than H. zea. The findings indicate the XRE element mutations mainly contribute to the differences between the flavone-induced expressions of two CYP321A1 genes, which improve the flexibility and adaptability for allelochemical response of H. armigera.

Water-medicated specifically targeting the S1 pockets among serine proteases using an arginine analogue

Because of the high similarity in structure and sequence, it is challenging to distinguish the S1 pocket among serine proteases, primarily due to the only variability at residue 190 (A190 and S190). Peptide or protein-based inhibitors typically target the negatively charged S1 pocket using lysine or arginine as the P1 residue, yet neither discriminates between the two S1 pocket variants. This study introduces two arginine analogues, L-4-guanidinophenylalanine (12) and L-3-(N-amidino-4-piperidyl)alanine (16), as novel P1 residues in peptide inhibitors. 16 notably enhances affinities across all tested proteases, whereas 12 specifically improved affinities towards proteases possessing S190 in the S1 pocket. By crystallography and molecular dynamics simulations, we discovered a novel mechanism involving a water exchange channel at the bottom of the S1 pocket, modulated by the variation of residue 190. Additionally, the specificity of 12 towards the S190-presenting S1 pocket is dependent on this water channel. This study not only introduces novel P1 residues to engineer inhibitory potency and specificity of peptide inhibitors targeting serine proteases, but also unveils a water-mediated molecular mechanism of targeting serine proteases.