Amino Acid Sequence Identity Research Articles

Identification of the Csr global regulatory system mediated by small RNA decay in Aeromonas salmonicida.

We investigated the presence and functionality of the carbon storage regulator (Csr) system in Aeromonas salmonicida SWSY-1.411. CsrA, an RNA-binding protein, shared 89% amino acid sequence identity with Escherichia coli CsrA. CsrB/C sRNAs exhibited a typical stem-loop structure, with more GGA motifs, which bind CsrA, than E. coli. CsrD had limited sequence identity with E. coli CsrD; however, it contained the conserved GGDEF and EAL domains. Functional analysis in E. coli demonstrated that the Csr system of A. salmonicida influences glycogen biosynthesis, biofilm formation, motility, and stability of both CsrB and CsrC sRNAs. These findings suggest that in A. salmonicida, the Csr system affects phenotypes like its E. coli counterpart. In A. salmonicida, defects in csr homologs affected biofilm formation, motility, and chitinase production. However, glycogen accumulation and protease production were unaffected. The expression of flagellar-related genes and chitinase genes was suppressed in the csrA-deficient A. salmonicida. Northern blot analysis indicated the stabilization of CsrB and CsrC in the csrD-deficient A. salmonicida. Similar to that in E. coli, the Csr system in A. salmonicida comprises the RNA-binding protein CsrA, the sRNAs CsrB and CsrC, and the sRNA decay factor CsrD. This study underscores the conservation and functionality of the Csr system and raises questions about its regulatory targets and mechanisms in A. salmonicida.

Genomic variation in pepper vein yellows viruses in Australia, including a new putative variant, PeVYV-10.

Since the first identification and full sequence of the polerovirus pepper vein yellows virus in Australia in 2016, virus surveys of crops and weeds have sporadically identified PeVYV in different hosts and locations. Genomic comparisons of 14 PeVYV-like isolates using RT-PCR products spanning the 3' end of the RdRp region (ORF 2), the intergenic region, ORF 3a, ORF 4, and ORF 3 (1388 nt) showed that four of the PeVYV isolates might be a new variant or PeVYV-like virus. From six PeVYV-positive plants, eight PeVYV-like sequences were obtained by high-throughput sequencing, as two hosts, 5352 and 5634, contained two slightly different PeVYV-like isolates. Three of the PeVYV-like isolates were most closely related to PeVYV-6 and PeVYV-5, and two isolates were closely related to PeVYV-9 and PeVYV-2. The other three isolates shared only 69-74% nucleotide sequence identity across the whole genome with any of the other PeVYVs, despite sharing 73-98%, 87-91%, and 84-87% amino acid sequence identity in ORF 3a, ORF 3, and the RdRp (ORF 2), respectively, suggesting that this virus is a new PeVYV-like virus, which we have tentatively called PeVYV-10. This is also the first report of a PeVYV-like virus infecting garlic.

The totality of evidence approach in the development of AVT02 (adalimumab), a biosimilar to Humira.

The development of a biosimilar is based on comparative structural, physicochemical, functional and clinical assessments. The sum of these analyses encompasses the 'totality of evidence', which demonstrates no clinically meaningful differences between the biosimilar and the reference product (RP). Once biosimilarity has been established, provided there is suitable scientific justification, clinical data may be extrapolated to other indications of the RP. AVT02 has been developed as a biosimilar to high-concentration, low-volume Humira (adalimumab), an anti-tumour necrosis factor-alpha monoclonal antibody approved for various chronic inflammatory indications. The totality of evidence for AVT02 is described, supporting its approval as an adalimumab biosimilar for all approved indications globally. Analytical similarity assessments using mass spectrometry methods demonstrated identical amino acid sequences for AVT02 and the RP, with high similarity in terms of primary structure, post-translational modifications and higher-order structural attributes. The mechanism of action was assessed by various cell-based potency assays and binding assays, and the results demonstrated that AVT02 is highly similar to the RP. No clinically meaningful differences in terms of purity, potency and safety were observed, and minor differences in a few physiochemical attributes did not impact the in vitro biologic activity and were not considered clinically relevant. Clinical similarity was demonstrated by comparing the pharmacokinetic, efficacy, safety and immunogenicity profiles of AVT02 with those of the RP. Clinical studies supported similar pharmacokinetic and comparable immunogenicity profiles between AVT02 and the RP in healthy participants and participants with moderate-to-severe chronic plaque psoriasis, with no new safety signals detected. The totality of evidence described demonstrates the biosimilarity of AVT02 to the RP, thereby fulfilling the scientific and regulatory requirements for AVT02 as a high-concentration biosimilar for the treatment of chronic plaque psoriasis and all approved indications of the RP.

Novel Selective Quantification of Zinpentraxin Alfa Biotherapeutic in the Presence of Endogenous Isomer in Plasma Samples of Idiopathic Pulmonary Fibrosis Patients Using Immunoaffinity LC–MS

Idiopathic pulmonary fibrosis (IPF) is a progressive fatal interstitial lung disease that affects three million patients worldwide and currently without an effective cure. Zinpentraxin alfa, a recombinant human pentraxin-2 (rhPTX-2) protein, has been evaluated as a potential drug candidate for the treatment of IPF. Clinical pharmacokinetic analysis of zinpentraxin alfa has been challenging historically due to interference from serum amyloid P component (SAP), an endogenous human pentraxin-2 protein. These molecules share an identical primary amino acid sequence and glycan composition; however, zinpentraxin alfa possesses α2,3-linked terminal sialic acid residues while SAP is an α2,6-linked isomer. By taking advantage of this only structural difference, we developed a novel assay strategy where α2,3-sialidase was used to selectively hydrolyze α2,3-linked sialic acid residues, resulting in desialylated zinpentraxin alfa versus unchanged sialylated SAP, following an immunoaffinity capture step. Subsequent tryptic digestion produced a unique surrogate asialo-glycopeptide from zinpentraxin alfa and allowed specific quantification of the biotherapeutic in human plasma. In addition, a common peptide shared by both molecules was selected as a surrogate to determine total hPTX-2 concentrations, i.e., sum of zinpentraxin alfa and SAP. The quantification methods for both zinpentraxin alfa and total hPTX-2 were validated and used in pharmacokinetic assessment in IPF patients. The preliminary results suggest that endogenous SAP levels remained largely constant in IPF patients throughout the treatment with zinpentraxin alfa. Our novel approach provides a general bioanalytical strategy to selectively quantify α2,3-sialylated glycoproteins in the presence of their corresponding α2,6-linked isomers.Graphical

Molecular characterization and functional study of a galectin-9 from a teleost fish, Boleophthalmus pectinirostris

Galectin-9, a tandem-repeat galectin, plays an important role in the regulation of innate immune response against various microbial infections. Here, galectin-9 from mudskipper (Boleophthalmus pectinirostris) was identified and named as BpGal-9. Putative BpGal-9 contains two conserved carbohydrate recognition domains (CRDs), one CRD within N-terminal (N-CRD) and the other one within C-terminal (C-CRD). Multi-alignment analysis indicated that BpGal-9 shared the highest amino acid sequence identity of 64.3 % with that of Southern platyfish (Xiphophorus maculatus). Phylogenetic analysis showed that BpGal-9 grouped tightly with other teleosts galectin-9 and was most closely related to that of Southern platyfish. BpGal-9 transcripts were more abundant in the intestine, and its expression upregulated significantly in the intestine, kidney, spleen, gills, and skin after Edwardsiella tarda infection. Meanwhile, BpGal-9 expression significantly increased in hemocytes and serum of mudskipper infected by E. tarda. The recombinant BpGal-9 (rBpGal-9) and rBpGal-9C-CRD could agglutinate all tested bacteria, whereas rBpGal-9N-CRD could only agglutinate three kinds of bacteria. When targeting the same bacteria, rBpGal-9 showed stronger agglutinating activities than rBpGal-9C-CRD or rBpGal-9N-CRD. In addition, the induction effect of three recombinant proteins on the mRNA expression of anti-inflammatory cytokines (BpIL-10 and BpTGF-β) was better than that on the pro-inflammatory cytokines (BpIL-1β and BpTNF-α). Our result suggested that the N-CRD and C-CRD of galectin-9 contribute differently to its multiple functions in innate immunity in teleosts.

CYP108N14: A Monoterpene Monooxygenase from Rhodococcus globerulus

Rhodococcus globerulus (R. globerulus) was isolated from the soil beneath a Eucalypt tree. Metabolic growth studies revealed that R. globerulus was capable of living on certain monoterpenes, including 1,8-cineole and p-cymene, as sole sources of carbon and energy. Multiple P450 genes were identified in the R. globerulus genome that shared homology to known bacterial, monoterpene hydroxylating P450s. To date, two of these P450s have been expressed and characterised as 1,8-cineole (CYP176A1) and p-cymene (CYP108N12) monooxygenases that are believed to initiate the biodegradation of these terpenes. In this work, another putative P450 gene (CYP108N14) was identified in R. globerulus genome. Given its amino acid sequence identity to other monoterpene hydroxylating P450s it was hypothesised to catalyse monoterpene hydroxylation. These include CYP108A1 from Pseudomonas sp. (47 % identity, 68 % similarity) which hydroxylates α-terpineol, and CYP108N12 also from R. globerulus (62 % identity, 77 % similarity). Also present in the operon containing CYP108N14 were putative ferredoxin and ferredoxin reductase genes, suggesting a typical Class I P450 system. CYP108N14 was successfully over-expressed heterologously and purified, resulting in a good yield of CYP108N14 holoprotein. However, neither the ferredoxin nor ferredoxin reductase could be produced heterologously. Binding studies with CYP108N14 revealed a preference for the monoterpenes p-cymene, (R)-limonene, (S)-limonene, (S)-α-terpineol and (S)-4-terpineol. An active catalytic system was reconstituted with the non-native redox partners cymredoxin (from the CYP108N12 system) and putidaredoxin reductase (from the CYP101A1 system). CYP108N14 when supported by these redox partners was able to catalyse the hydroxylation of the five aforementioned substrates selectively at the methyl benzylic/allylic positions.

Effects of Ionic Strength on the Morphology, Scattering, and Mechanical Response of Neurofilament-Derived Protein Brushes.

Protein brushes not only play a key role in the functionality of neurofilaments but also have wide applications in biomedical materials. Here, we investigate the effect of ionic strength on the morphology of protein brushes using continuous-space self-consistent field theory. A coarse-grained multiblock charged macromolecular model is developed to capture the chemical identity of amino acid sequences. For neurofilament heavy (NFH) brushes at pH 2.4, we predict three morphological regimes: swollen brushes, condensed brushes, and coexisting brushes, which consist of both a dense inner layer and a diffuse outer layer. The brush height predicted by our theory is in good agreement with the experimental data for a wide range of ionic strengths. The dramatic height decrease is a result of the electrostatic screening-induced transition from the overlapping state to the isolated state of the coexisting brushes. We also studied the evolution of the scattering and mechanical responses accompanying the morphological change. The oscillation in the reflectivity spectra characterizes the existence and microstructure of the inner condensed layer, whereas the shoulder in the force spectra signifies a swollen morphology.

Complete genome sequencing and construction of full-length infectious cDNA clone of papaya ringspot virus-HYD isolate and its efficient in planta expression.

Papaya ringspot virus (PRSV) is a devastating Potyvirus that causes papaya ringspot disease in Carica papaya plantations globally. In this study, the complete genome sequence of a PRSV isolate from Shankarpalli, Telangana, India, was reported and designated as PRSV-HYD (KP743981.1). The genome is a single-stranded positive-sense RNA comprising 10,341 nucleotides. Phylogenetic analysis revealed that PRSV-HYD is closely related to PRSV Pune (Aundh) isolate with 92 and 95% nucleotide and amino acid sequence identity, respectively. To develop infectious cDNA (icDNA), the complete nucleotide sequence of PRSV-HYD was cloned between the right and left borders in the binary vector pCB301 using BglII and XmaI restriction sites. Cauliflower mosaic virus (CaMV) double promoter (35S) was fused at the 5'-end and Avocado sunblotch viroid (ASBVd) ribozyme (RZ) sequence was fused to the 3' end to generate an authentic 3' viral end in the transcribed mRNAs. The icDNA generated was mobilized into the Agrobacterium tumefaciens EHA 105, and the agrobacterial cultures were infiltrated into the natural host C. papaya and a non-host Nicotiana benthamiana plants; both did not show any symptoms. In RT-PCR analysis of RNAs isolated from N. benthamiana, we could detect viral genes as early as 3 days and continued up to 28 days post infiltration. Alternatively, virion particles were purified from agroinfiltrated N. benthamiana plants and introduced into C. papaya by mechanical inoculation as well as by pinprick method. In both cases, we could see visible systemic symptoms similar to that of wild type by 40 days. Additionally, we studied the expression patterns of the genes related to plant defense, transcription factors (TFs), and developmental aspects from both C. papaya and N. benthamiana.

THE COMPLETE NUCLEOTIDE SEQUENCE OF THE GENOMIC RNA OF BEAN COMMON MOSAIC VIRUS STRAIN NL4

The complete nucleotide sequence of the genomic RNA of the NL4 strain of Bean common mosaic virus (BCMV-NL4) was determined. The viral genome is 10037 nucleotides in length, excluding the 3’ terminal poly (A) tail, and contains a single open reading frame (ORF) of 9666 nucleotides encoding a polyprotein of 3222 amino acids. The ORF is flanked by 5’ and 3’ untranslated regions (UTRs) of 133 and 235 nucleotides, respectively. Comparative analyses of the predicted BCMV-NL4 polyprotein with other species of the genus Potyvirus revealed nine cleavage sites resulting in ten functional proteins. Nucleotide and amino acid sequence identities indicated a close relationship between BCMV-NL4 and BCMV-NL1. Blast comparisons using coat protein (CP) and 3’UTR sequences showed 100% identity between BCMV-NL4 and the Mexican variant (US6) of this strain.

Identification and complete genome sequence of a novel sadwavirus discovered in chrysanthemum (Chrysanthemum morifolium Ramat.).

The complete genome sequence of a putative novel member of the genus Sadwavirus was determined by high-throughput sequencing of a chrysanthemum from an orchard of the Tongxiang Agricultural Science Institute in Tongxiang, Zhejiang province. The complete genome sequence was confirmed using RT-PCR and the rapid amplification of cDNA ends (RACE) method. The predicted genome of the putative virus is composed of two RNA molecules, 7016 and 6772 nucleotides in length, excluding their poly-A tails. The new virus was tentatively named "chrysanthemum sadwavirus" (ChSV). The Pro-Pol region of RNA1 and the CP region of RNA2 of ChSV shared the highest amino acid sequence identity (53.01% and 36.40%, respectively) with the corresponding sequences of lettuce secovirus 1 (LSV-1). Phylogenetic analysis showed that ChSV clustered with members of the subgenus Stramovirus (genus Sadwavirus). Taken together, these results suggest that ChSV is a new member of the genus Sadwavirus.

Complete genome sequence of Paris polyphylla chlorotic mottle virus infecting Paris polyphylla var. yunnanensis in southwest China.

A novel virus infecting a Paris polyphylla var. yunnanensis plant, tentatively named "Paris polyphylla chlorotic mottle virus" (PpCMV), was discovered in the city of Lijiang, Yunnan Province, China. Its genome consists of 6384 nucleotides (nt), excluding the 3'-terminal poly(A) tail, and contains two open reading frames: ORF1 and ORF2. ORF1 is 6150 nt in length, encoding a large 2050-aa polyprotein with at least two conserved regions encoding a replication-associated protein and a coat protein, the latter of which is located at the 3' end of ORF1. ORF2, consisting of 1185 nt, is located within ORF1 but has a different reading frame. It encodes a 394-aa-long putative movement protein. Phylogenetic analysis based on amino acid sequences revealed that the newly discovered virus exhibited the closest relationship to Hobart betaflexivirus 1 and rhodiola betaflexivirus 1, both of which belong to the genus Capillovirus, sharing 48.8% and 36.5% amino acid sequence identity, respectively, in the structural protein. This is the first report of the complete genome sequence of PpCMV in China.

Combined analytical assays for the characterization of drugs binding to human IgE: Applicability to omalizumab-bearing biosimilar candidates assessment

Analytical and functional comparison is key for substantiating the level of convergence (essential sameness) or divergence between versions or variants of a given biological medicine. Accordingly, an overlapping biological activity between products meant to be equal probably reflects a highly similar structure and anticipates a comparable pharmacodynamic behavior. We developed an orthogonal approach to compare the human IgE binding features of different lots and versions of Xolair® (omalizumab), an anti-human IgE monoclonal antibody. The IgE binding affinity and kinetics were measured by surface plasmon resonance. Ability to prevent mast cell activity was assessed in vitro and in vivo in mast cell-based models. The variability of monoclonal antibodies with identical amino acid sequences produced either in Chinese hamster ovarian cells or in human HEK293 cells, was compared. Monoclonal antibodies from the two sources exhibited slightly different human IgE binding and neutralizing features. A known variant exhibiting a three amino acid replacement in the Fab region had lower IgE binding affinity than the original omalizumab. The lower binding affinity translated into reduced IgE neutralizing capacity and, in turn, a difference in the ability to prevent mast cell activation in vitro and in vivo. The proposed set of analytical and functional assays was sensitive enough to detect Fab-linked differences between anti-IgE antibody versions exhibiting an identical aminoacid sequence. In addition to add value to the comparative assessment of biosimilar candidates bearing omalizumab, these methods can aid pre-assessments of new anti-IgE agents that aim to improve therapeutic performance.

Genotype analysis of rotaviruses isolated from children during a phase III clinical trial with the hexavalent rotavirus vaccine in China

The oral hexavalent live human-bovine reassortant rotavirus vaccine (RV6) developed by Wuhan Institute of Biological Products Co., Ltd (WIBP) has finished a randomized, placebo-controlled phase III clinical trial in four provinces of China in 2021. The trail demonstrated that RV6 has a high vaccine efficacy against the prevalent strains and is safe for use in infants. During the phase III clinical trial (2019–2021), 200 rotavirus-positive fecal samples from children with RV gastroenteritis (RVGE) were further studied. Using reverse transcription-polymerase chain reaction and high-throughput sequencing, VP7 and VP4 sequences were obtained and their genetic characteristics, as well as the differences in antigenic epitopes of VP7, were analyzed in detail. Seven rotavirus genotypes were identified. The predominant rotavirus genotype was G9P [8] (77.0%), followed by prevalent strains G8P [8] (8.0%), G3P [8] (3.5%), G3P [9] (1.5%), G1P [8] (1.0%), G2P [4] (1.0%), and G4P [6] (1.0%). The amino acid sequence identities of G1, G2, G3, G4, G8, and G9 genotypes of isolates compared to the vaccine strains were 98.8%, 98.2%–99.7%, 88.4%–99.4%, 98.2%, 94.2%–100%, and 93.9%–100%, respectively. Notably, the vaccine strains exhibited high similarity in amino acid sequence, with only minor differences in antigenic epitopes compared to the Chinese endemic strains. This supports the potential application of the vaccine in preventing diseases caused by rotaviruses.

Molecular characterization of a novel cytorhabdovirus infecting Plumbago indica L.

In 2021, Plumbago indica plants with necrotic spots on their leaves were observed in Beijing, China. Through high-throughput sequencing, we discovered a putative novel member of the genus Cytorhabdovirus, which was provisionally named "plumbago necrotic spot-associated virus" (PNSaV). The full-length negative-sense single-stranded RNA genome of this virus is 13,180 nucleotides in length and contains eight putative open reading frames (ORFs), in the order 3' leader-N-(P')-P-P3-M-G-P6-L-5' trailer. Phylogenetic analysis and pairwise comparisons suggested that PNSaV is most closely related to pastinaca cytorhabdovirus 1, with 59.2% nucleotide sequence identity in the complete genome and 56.4% amino acid sequence identity in the L protein. These findings suggest that PNSaV should be considered a new member of the genus Cytorhabdovirus.

Development of a Novel L-Asparaginase with Enhanced Properties Using Ancestral Sequence Reconstruction

The overall survival of children with acute lymphoblastic leukemia (ALL) now exceeds 90% due to the precise disease risk stratification and intensification of multi-agent chemotherapy regimens. L-asparaginase (L-ASNase) is an indispensable biotherapeutic enzyme used in treatment of pediatric ALL. Discontinuation of L-ASNase treatment due to immunological or adverse reactions leads to significantly inferior disease-free survival (73% ±7% vs 92% ± 2%; P <0.01, Gupta et al. JCO 2020). Clinical L-ASNases are bacterial in origin, derived from either Escherichia coli or Erwinia chrysanthemi and are therefore highly immunogenic. Additionally, emerging data suggests that the concurrent glutaminase activity seen in bacterial asparaginases exacerbates its liver and pancreatic toxicity, especially in adults thereby limiting its extensive use beyond children. Therefore, finding alternative sources with preferable biochemical features remains paramount. The inferior catalytic properties of human L-ASNase restricts its functionality in ALL treatment. However, characterization has shown that guinea pig (GP) L-ASNase demonstrates significant anti-tumor properties and improved enzyme kinetics despite a 69.8% amino acid sequence identity with human L-ASNase, as compared to 30% for bacterial L-ASNases. Additionally, guinea pig L-ASNase has no glutaminase activity. Thus, to overcome the major deficiencies of bacterial L-ASNase and develop a more humanized form of L-ASNase we performed ancestral sequence reconstruction (ASR). ASR is a protein drug discovery / optimization platform that predicts ancient DNA and protein sequences from extant sequences. This approach permits higher-resolution mapping potentially highlighting functional resides. The objective therefore is to exploit functional diversity refined by natural selection using ASR to identify therapeutic variants with superior properties. Recent work on guinea pig (GP) L-ASNase (Schalk et al. J Biol Chem 2014) identified a low micromolar (μM) Michaelis constant (Km) at 57.7 ± 6.4 μM essential for hydrolyzing the extra-cellular pool of asparagine ~50 μM in human blood; thus, equipping us with the ideal mammalian L-ASNase ortholog to serve as a template to resurrect ancient ASNase sequences. Ancestral L-ASNase sequences were generated utilizing 54 extant L- asparaginase sequences, aligned using MUSCLE and an evolutionary tree inferred using MrBayes. A total of 53 ancestral nodes were identified, and the ten ancestral L-ASNase variants spanning the ancient primate and guinea pig (GP) lineage were resurrected. The sequence identity of these ancestral L-ASNase variants ranged from 81 to 98% when compared to human L-ASNase. E. coli codon optimized complementary DNA (cDNA) sequences were synthesized and subcloned intoan expression vector. Correctly cloned plasmids were transformed into E. coli BL21 (DE3) cells for protein expression. An-ASNase candidates were isolated by Ni 2+ affinity chromatography and then further purified by size exclusion chromatography. Asparaginase activity of the An-ASNase candidates were determined via a modified Nessler's reagent assay by using a continuous spectroscopic enzyme-coupled assay. Three of the ancestral L-ASNase candidates, An-88, An-104 and An-107 demonstrated excellent asparaginase activity when tested at an enzyme concentration of 0.1 mg/mL and an excess substrate concentration, with activity within a comparable range to the clinically relevant E. coli and Erwinia asparaginases. An-88 had 81% similarity whereas both An-104 and An-107 ASNases had 88% identity to human L-ASNase, which is in stark contrast to the only 30% similarity seen with bacterial L-ASNases. Detailed enzymatic characterization of these three lead drug candidates is currently being performed. Preliminary cytotoxicity data testing the ancestral L-ASNase candidates against a T-cell ALL cell line CCRF-CEM showed an expected dose response, with An-107 showing the highest cytotoxicity. Our findings demonstrate that ASR is an efficient protein drug design/optimization platform that we have now extended beyond our previous work with recombinant coagulation factors (Zakas et al. Nat Biotech 2017, Knight et al. Blood Adv 2021) to therapeutic enzymes; thereby, enabling the creation of novel recombinant L-ASNases with translational implications due to improved enzymatic specificity and reduced toxicity.

Tree shrew cytochrome P450 2E1 is a functional enzyme that metabolises chlorzoxazone and p-nitrophenol

Cytochromes P450 (CYPs or P450s) are important enzymes for drug metabolism. Tree shrews are non-primate animal species used in various fields of biomedical research, including infection (especially hepatitis viruses), depression, and myopia. A recent tree shrew genome analysis indicated that the sequences and the numbers of P450 genes are similar to those of humans; however, P450s have not been adequately identified and analysed in this species. In this study, a novel CYP2E1 was isolated from tree shrew liver and was characterised in comparison with human, dog, and pig CYP2E1. Tree shrew CYP2E1 and human CYP2E1 showed high amino acid sequence identity (83%) and were closely related in a phylogenetic tree. Gene and genome structures of CYP2E1 were generally similar in humans, dogs, pigs, and tree shrews. Tissue expression patterns showed that tree shrew CYP2E1 mRNA was predominantly expressed in liver, just as for dog and pig CYP2E1 mRNAs. In tree shrews, recombinant CYP2E1 protein and liver microsomes metabolised chlorzoxazone and p-nitrophenol, probe substrates of human CYP2E1, just as they do in dogs and pigs. These results suggest that tree shrew CYP2E1 encodes a functional drug-metabolising enzyme that plays a role in the liver, similar to human CYP2E1.

Whole Genomic Constellation of Avian Reovirus Strains Isolated from Broilers with Arthritis in North Carolina, USA.

Avian reovirus (ARV) is an emerging pathogen which causes significant economic challenges to the chicken and turkey industry in the USA and globally, yet the molecular characterization of most ARV strains is restricted to a single particular gene, the sigma C gene. The genome of arthrogenic reovirus field isolates (R18-37308 and R18-38167), isolated from broiler chickens in North Carolina (NC), USA in 2018, was sequenced using long-read next-generation sequencing (NGS). The isolates were genotyped based on the amino acid sequence of sigma C (σC) followed by phylogenetic and amino acid analyses of the other 11 genomically encoded proteins for whole genomic constellation and genetic variation detection. The genomic length of the NC field strains was 23,494 bp, with 10 dsRNA segments ranging from 3959 bp (L1) to 1192 bp (S4), and the 5' and 3' untranslated regions (UTRs) of all the segments were found to be conserved. R18-37308 and R18-38167 were found to belong to genotype (G) VI based on the σC analysis and showed nucleotide and amino acid sequence identity ranging from 84.91-98.47% and 83.43-98.46%, respectively, with G VI strains. Phylogenetic analyses of individual genes of the NC strains did not define a single common ancestor among the available completely sequenced ARV strains. Nevertheless, most sequences supported the Chinese strain LY383 as a probable ancestor of these isolates. Moreover, amino acid analysis revealed multiple amino acid substitution events along the entirety of the genes, some of which were unique to each strain, which suggests significant divergence owing to the accumulation of point mutations. All genes from R18-37308 and R18-38167 were found to be clustered within genotypic clusters that included only ARVs of chicken origin, which negates the possibility of genetic pooling or host variation. Collectively, this study revealed sequence divergence between the NC field strains and reference ARV strains, including the currently used vaccine strains could help updating the vaccination regime through the inclusion of these highly divergent circulating indigenous field isolates.

Four closely related endornaviruses each with a low incidence in the phytopathogenic fungi Exserohilum turcicum or Bipolaris maydis

Endornaviruses are known to occur widely in plants, fungi, and oomycetes, but our understanding of their diversity and distribution is limited. In this study, we report the discovery of four endornaviruses tentatively named Setosphaeria turcica endornavirus 1 (StEV1), Setosphaeria turcica endornavirus 2 (StEV2), Bipolaris maydis endornavirus 1 (BmEV1), and Bipolaris maydis endornavirus 2 (BmEV2). StEV1 and StEV2 infect Exserohilum turcicum, while BmEV1 and BmEV2 infect Bipolaris maydis. The four viruses encode a polyprotein with less than 40 % amino acid sequence identity to other known endornaviruses, indicating that they are novel, previously undescribed endornaviruses. However, StEV1 and BmEV1 share a sequence identity of 78 % at the full-genome level and 87 % at the polyprotein level, suggesting that they may belong to the same species. Our study also found that each of the four endornaviruses has an incidence of approximately 3.5 % to 5.5 % in E. turcicum or B. maydis. Interestingly, BmEV1 and BmEV2 were found to be unable to transmit between hosts of different vegetative incompatibility groups, which may explain their low incidence.

Anthriscus sylvestris Deoxypodophyllotoxin Synthase Involved inthe Podophyllotoxin Biosynthesis.

Tetrahydrofuran ring formation from dibenzylbutyrolactone lignans is a key step in the biosynthesis of aryltetralin lignans including deoxypodophyllotoxin and podophyllotoxin. Previously, Fe(II)- and 2-oxoglutarate-dependent dioxygenase (2-ODD) from Podophyllum hexandrum (Himalayan mayapple, Berberidaceae) was found to catalyze the cyclization of a dibenzylbutyrolactone lignan, yatein, to give deoxypodophyllotoxin and designated as deoxypodophyllotoxin synthase (DPS). Recently, we reported that the biosynthesis of deoxypodophyllotoxin and podophyllotoxin evolved in a lineage-specific manner in phylogenetically unrelated plant species such as P. hexandrum and Anthriscus sylvestris (cow parsley, Apiaceae). Therefore, a comprehensive understanding of the characteristics of DPSs that catalyze the cyclization of yatein to deoxypodophyllotoxin in various plant species is important. However, for plant species other than P. hexandrum, the isolation of the DPS enzyme gene and the type of the enzyme, e.g.whether it is 2-ODD or another type of enzyme such as cytochrome P-450, have not been reported. In this study, we report the identification and characterization of A. sylvestris DPS (AsDPS). Phylogenetic analysis showed that AsDPS belonged to the 2-ODD superfamily and shared moderate amino acid sequence identity (40.8%) with P. hexandrum deoxypodophyllotoxin synthase (PhDPS). Recombinant protein assay indicated that AsDPS and PhDPS differ in terms of the selectivity of substrate enantiomers. Protein modeling using AlphaFold2 and site-directed mutagenesis indicated that the Tyr305 residue of AsDPS probably contributes to substrate recognition. This study advances our understanding of the podophyllotoxin biosynthetic pathway in A. sylvestris and provides new insight into 2-ODD involved in plant secondary (specialized) metabolism.

Structural identification of catalytic His158 of PtMAC2p from Pseudozyma tsukubaensis, an acyltransferase involved in mannosylerythritol lipids formation.

Mannosylerythritol lipids (MELs) are extracellular glycolipids produced by the basidiomycetous yeast strains. MELs consist of the disaccharide mannosylerythritol, which is acylated with fatty acids and acetylated at the mannose moiety. In the MEL biosynthesis pathway, an acyltransferase from Pseudozyma tsukubaensis, PtMAC2p, a known excellent MEL producer, has been identified to catalyze the acyl-transfer of fatty acid to the C3'-hydroxyl group of mono-acylated MEL; however, its structure remains unclear. Here, we performed X-ray crystallography of recombinant PtMAC2p produced in Escherichia coli and homogeneously purified it with catalytic activity in vitro. The crystal structure of PtMAC2p was determined by single-wavelength anomalous dispersion using iodide ions. The crystal structure shows that PtMAC2p possesses a large putative catalytic tunnel at the center of the molecule. The structural comparison demonstrated that PtMAC2p is homologous to BAHD acyltransferases, although its amino acid-sequence identity was low (<15%). Interestingly, the HXXXD motif, which is a conserved catalytic motif in the BAHD acyltransferase superfamily, is partially conserved as His158-Thr159-Leu160-Asn161-Gly162 in PtMAC2p, i.e., D in the HXXXD motif is replaced by G in PtMAC2p. Site-directed mutagenesis of His158 to Ala resulted in more than 1,000-fold decrease in the catalytic activity of PtMAC2p. These findings suggested that His158 in PtMAC2p is the catalytic residue. Moreover, in the putative catalytic tunnel, hydrophobic amino acid residues are concentrated near His158, suggesting that this region is a binding site for the fatty acid side chain of MEL (acyl acceptor) and/or acyl-coenzyme A (acyl donor). To our knowledge, this is the first study to provide structural insight into the catalytic activity of an enzyme involved in MEL biosynthesis.