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Clustal W and Clustal X version 2.0
... The graphical output displays the base pair identity within a sliding window of 75-bp, ranging from 50 to 100%. The upper shows the FT1-like gene structure and the bottom with red box shows longan-specific conserved segments in the multiple sequence alignment of different Sapindaceae species by ClustalW2 (Larkin et al. 2007). The approximately 2.5-kb upstream sequence of the ATG in lychee, longan, and rambutan showed a high degree of conservation, marked by blue dotted box (b) Venn diagram assessing the count of CREs disparately among DlFT1 pro Del−321 (2.5-kb upstream from the ATG of DlFT1 out of longan-specific 321-bp insertion), DlFT1 pro ...
... (Brudno et al. 2003). The conserved regions were aligned using ClustalW2 (V.2.1) (Larkin et al. 2007). ...
Many species of Sapindaceae, such as lychee, longan, and rambutan, provide nutritious and delicious fruit. Understanding the molecular genetic mechanisms that underlie the regulation of flowering is essential for securing flower and fruit productivity. Most endogenous and exogenous flowering cues are integrated into the florigen encoded by FLOWERING LOCUS T. However, the regulatory mechanisms of flowering remain poorly understood in Sapindaceae. Here, we identified 60 phosphatidylethanolamine-binding protein-coding genes from six Sapindaceae plants. Gene duplication events led to the emergence of two or more paralogs of the FT gene that have evolved antagonistic functions in Sapindaceae. Among them, the FT1-like genes are functionally conserved and promote flowering, while the FT2-like genes likely serve as repressors that delay flowering. Importantly, we show here that the natural variation at nucleotide position − 1437 of the lychee FT1 promoter determined the binding affinity of the SVP protein (LcSVP9), which was a negative regulator of flowering, resulting in the differential expression of LcFT1, which in turn affected flowering time in lychee. This finding provides a potential molecular marker for breeding lychee. Taken together, our results reveal some crucial aspects of FT gene family genetics that underlie the regulation of flowering in Sapindaceae.
Supplementary Information
The online version contains supplementary material available at 10.1186/s43897-024-00104-4.
... The amino acid sequences of these genes were aligned using CLUSTALW2.0 [34] with default settings and manual adjustments. Gaps were introduced for proper alignment. ...
Background
Viola philippica and V. prionantha develop chasmogamous (CH) flowers under ≤ 12-h daylight conditions and cleistogamous (CL) flowers under long daylight (> 12-h daylight) conditions (LD), whereas V. cornuta develops CH flowers regardless of the daylight conditions. APETALA3 (AP3) is a major floral B-function gene that regulates the organ identity and development of stamens and petals. Evolutionary changes in AP3 orthologous genes might involve in the dimorphic flower formation. In the present study, we compared AP3 orthologous genes among three Viola species.
Results
The AP3 sequences were highly conserved, and obligate AP3-PISTILLATA heterodimers were universally formed. However, the floral expression of VphAP3 in V. philippica and VprAP3 in V. prionantha changed in response to the photoperiod. Their expression was significantly higher under 12-h daylight conditions than under 16-h daylight conditions. In contrast, VcoAP3 expression in the floral buds of V. cornuta was comparable among photoperiods. In accordance with these variations in expression, correlated sequence divergences were observed in the putative regulatory regions of Viola AP3 orthologous genes.
Conclusions
Developmental inhibition of petals and stamens may result from AP3 downregulation by LD, which thereby induces CL flowers. Our study provides insight into the molecular basis underlying the developmental evolution of environmentally dependent mating systems in dimorphic CL plants.
... To analyze sequencing data obtained from Sanger sequencing, CDSs were identified via multiple alignment of the seven landraces against the Heinz1706 reference gene (Tomato version SL4.0) [14] using ClustalX 2.1 software [22]. To assess the identification of novel allele variants in the C gene, identified CDS of each landrace were realigned against those carrying known SNP mutations [8,9] To evaluate whether SNP mutations detected by sequencing could influence protein function, Ensembl Variant Effect Predictor online tool [23] was queried. ...
The genetic diversity of tomato in Italy and the growing interest in high-quality food products highlight the importance of establishing varietal distinctiveness through molecular strategies to ensure agrifood product quality and traceability. In this study, four Italian potato-like leaf (PL) landraces were analyzed: “Spagnoletta di Formia e di Gaeta” (SPA) from southern Lazio, “Giagiù” (GIA) and “Patanara” (PTN) from Campania, and “Pomodoro di Mola” (MOL) from Apulia. These landraces were genotyped for the potato leaf gene (C), with two PL American genotypes and a non-allelic PL mutant line included as outgroups. Nagcarlang served as control. An allelism test confirmed C as determinant gene. The SCAR marker for C revealed that the Italian landraces presented determinants other than the most representative one responsible for PL. Whole-genome sequencing of SPA identified a private novel nonsense SNP variant allele, confirmed through dCAPS marker analysis. Additionally, two novel PL alleles responsible for missense variations were identified in GIA/PTN and MOL. In silico protein analysis suggested that novel C alleles could be functional determinants for the protein activity. Overall, PL mutations identified for the first time could serve as molecular tools for agrifood chain traceability, enabling early differentiation and recognition of genotypically similar varieties.
... The maximum likelihood phylogenetic analysis was inferred using IQ-TREE2 [62,63] with parameters '-wbtl -bb 1000'. Sequence alignment between SEV1-VP4 and SBV-GP06 was performed by ClustalW [64] and the results was displayed by ESPript 3.0 [65]. The alpha-helices of the two proteins predicted by AlphaFold described below were also showed by ESPript. ...
Archaeal viruses are well known for their diverse morphologies and extreme stability. In this study, we used cryo-electron tomography to analyze the structure of SEV1 and its infection strategies in its native state. The results show that SEV1 nucleocapsid adopts a ‘coil-stacking’ architecture which displays a degree of flexibility. VP4, whose homologues are widespread in the thermo-acidic environment globally, is identified as the major capsid protein and binds genomic DNA forming a “beads-on-a-string” arrangement. Simulations in various extreme environments indicate that the envelope of SEV1 is crucial to the thermostability. SEV1 infects the host by membrane fusion revealed by the membrane fusion assay. The infected cell undergoes cytoplasm condensation to form a “viral factory”, leading to the successive production of nascent virions. A series of assembly intermediates of SEV1 are identified revealing an integrated picture of the virus assembly process. The nascent virions are found to be released through virus-associated pyramids (VAPs), composed of unique proteins encoded by SEV1 distinct from other known VAP proteins. Our study provides novel insights to the survival strategies of SEV1, a flexible and enveloped archaeal virus, by using the unique “coil-stacking” architecture and the characteristic infection strategies.
... 48 or UniProt (https://www.uniprot.org/). The sequences of the four mtAP2s and their homologous proteins were aligned using ClustalW 49 . The phylogenetic tree was generated by neighbor-joining, and the resulting tree in Newick format was visualized using MEGA 11. ...
The phylum Apicomplexa comprises eukaryotic parasites that cause fatal diseases affecting millions of people and animals worldwide. Their mitochondrial genomes have been significantly reduced, leaving only three protein-coding genes and highly fragmented mitoribosomal rRNAs, raising challenging questions about mitoribosome composition, assembly and structure. Our study reveals how Toxoplasma gondii assembles over 40 mt-rRNA fragments using exclusively nuclear-encoded mitoribosomal proteins and three lineage-specific families of RNA-binding proteins. Among these are four proteins from the Apetala2/Ethylene Response Factor (AP2/ERF) family, originally known as transcription factors in plants and Apicomplexa, now repurposed as essential mitoribosome components. Cryo-EM analysis of the mitoribosome structure demonstrates how these AP2 proteins function as RNA binders to maintain mitoribosome integrity. The mitoribosome is also decorated with members of lineage-specific RNA-binding proteins belonging to RAP (RNA-binding domain abundant in Apicomplexa) proteins and HPR (heptatricopeptide repeat) families, highlighting the unique adaptations of these parasites. Solving the molecular puzzle of apicomplexan mitoribosome could inform the development of therapeutic strategies targeting organellar translation.
... Sequence alignments were built using ClustalW2 with default settings 40 . Maximum likelihood tree shown in Supplementary Fig. 1b and Supplementary Fig. 1 were determined using MEGAX 41 . ...
Salicylic acid (SA) production in Brassicaceae plants is uniquely accelerated from isochorismate by EPS1, a newly identified enzyme in the BAHD acyltransferase family. We present crystal structures of EPS1 from Arabidopsis thaliana in both its apo and substrate-analog-bound forms. Integrating microsecond-scale molecular dynamics simulations with quantum mechanical cluster modeling, we propose a pericyclic rearrangement lyase mechanism for EPS1. We further reconstitute the isochorismate-derived SA biosynthesis pathway in Saccharomyces cerevisiae, establishing an in vivo platform to examine the impact of active-site residues on EPS1 functionality. Moreover, stable transgenic expression of EPS1 in soybean increases basal SA levels, highlighting the enzyme’s potential to enhance defense mechanisms in non-Brassicaceae plants lacking an EPS1 ortholog. Our findings illustrate the evolutionary adaptation of an ancestral enzyme’s active site to enable a novel catalytic mechanism that boosts SA production in Brassicaceae plants.
... The resulting nucleotide sequences were reviewed and edited using the Clustal W software [51]. Next, for confirmation to taxa and genes, the obtained sequences were blasted in the NCBI database [52]. ...
The Russian Far East is a region of unique biodiversity, with numerous plant species, including Reynoutria japonica and Reynoutria sachalinensis. These plants are considered a serious threat to biodiversity and are classified as threatened species. However, Reynoutria plants synthesize and accumulate a variety of metabolites that are valued for their positive effects on human health. The main objective of this study is to quantitatively and qualitatively evaluate the content of secondary metabolites in different parts of R. japonica and R. sachalinensis plants. In this study, the results of phylogenetic analysis of the ITS2, matK, and rps16 genes showed that samples collected in the Sakhalin region were closest to R. sachalinensis, while samples collected in Primorsky krai were closer to R. japonica. The high-performance chromatography and mass spectrometry (HPLC-MS/MS) method was used to identify the compounds. As a result of the identification of metabolites in the leaves, stem, and roots of R. japonica and R. sachalinensis, we showed the presence of a total of 31 compounds, including stilbenes, phenolic acids, flavan-3-ols, flavones and flavonols, naphthalene derivatives, anthraquinones and derivatives, and phenylpropanoid disaccharide esters. The root of R. japonica was shown to be a rich source of stilbenes (up to 229.17 mg/g DW), which was 8.5 times higher than that of R. sachalinensis root (up to 27.04 mg/g DW). The root also contained high amounts of emodin derivatives and vanicoside B. Quercetin and its derivatives were the major metabolites in the leaves and stems of both Reynoutria species. In R. japonica leaves, quercetin-3-O-pentoside was the major compound, reaching a total of 7 mg/g DW, accounting for 34% of all compounds analyzed. In contrast, in R. sachalinensis leaves, quercitrin was the major compound (up to 13.96 mg/g DW), accounting for 62% of all compounds and 12.7 times higher than in R. japonica leaves. In turn, R. japonica leaves also contained high amounts of phenolic acids (up to 10 mg/g DW). Thus, the obtained results showed significant differences in the qualitative and quantitative composition of metabolites between R. japonica and R. sachalinensis plants. Additionally, in this work, a cell culture of R. japonica was obtained and tested for its ability to synthesize and accumulate stilbenes.
... Comparative analyses were performed using sequences from both the Mediterranean/Eastern Atlantic and the Indo-Pacific region for the genera Loimia, Palola, Subadyte and Trypanosyllis. Gene sequences were aligned with ClustalX v. 2.1 (Larkin et al., 2007), and alignments were edited using the program BIOEDIT v. 7.2.5 (Hall, 1999). The program jModelTest 2.1.6 ...
Sampling activities conducted in the Tyre Coast Nature Reserve, southern Lebanon, to study the marine annelid fauna of the area, allowed for the revision of the diversity of this group in Lebanon. We particularly focused on non-indigenous species (NIS), which were characterised from morphological and molecular points of view. A total of 116 taxa were collected; 10 species are reported here for the first time in the Mediterranean Sea, while 6 species with presumable Indo-Pacific affinity are likely new to science. Seventy-three taxa were native, while 43 taxa were NIS; among the latter, 24 species are reported for the first time in Lebanon. Molecular data were obtained for 28 NIS, representing the first data from the Mediterranean Sea for 23 of them. Non-indigenous annelids occurring along the coast of Lebanon mainly have Indo-Pacific affinity. However, molecular data highlighted inconsistencies between the sampled material and the sequences available in public repositories, suggesting the widespread occurrence of species complexes in these taxa. These results suggest that further research on Indo-Pacific annelids is needed to understand their diversity patterns and invasion pathways. Additionally, the large number of new records of annelids in Lebanese waters indicate that further studies are needed to explore their diversity in comparison with neighbouring Mediterranean regions.
... (Kanagawa, Japan). The phylogenetic tree was constructed by the neighbor-joining method [32] with the ClustalX package [33]. ...
A new bioactive substance was identified from a leaf-litter actinomycete strain by screening for antibacterial activity against Neisseria gonorrhoeae. The thiazolyl peptide antibiotic, named thiazoplanomicin, was isolated from the secondary metabolites of the leaf-litter actinomycetes Actinoplanes sp. MM794L-181F6 by extraction with n-butanol, silica gel column chromatography, Sephadex LH-20 column chromatography, and preparative HPLC. Thiazoplanomicin was characterized by LC-HR-ESI-MS, NMR, and X-ray analyses, along with analysis of the degradation products and chemical derivatives, and determined to be a nocathiacin-like multiple macrocyclic thiazolyl peptide. Thiazoplanomicin showed potent antimicrobial activity against gonococcal strains, including those resistant to known anti-gonococcal compounds such as telithromycin, azithromycin, and ceftriaxone, with MIC values ranging from 0.0312 to 0.125 µg ml-1. Such anti-gonococcal activity has not been reported on nocathiacin-like thiazolyl peptide antibiotic so far. Similar to other thiazolyl peptide antibiotics, thiazoplanomicin also showed potent antibacterial activity against Gram-positive bacteria with MIC values ranging from 0.0005 to 0.0156 µg ml-1 but showed no antibacterial activity against Escherichia coli.
... The original tree was visualized via an interactive tree of life (iTOL) (https://itol.embl.de/, accessed on 19 March 2024) [30,62]. Dicots, monocots, and model plants were selected as outgroup species; gene conserved domains were downloaded from the NCBI Conserved Domains Database (CDD) (https://www.ncbi.nlm.nih.gov/cdd/, ...
The trithorax group (TrxG) complex is an important protein in the regulation of plant histone methylation. The ABSENT, SMALL, OR HOMEOTIC DISCS 1 (ASH1) gene family, as important family members of the TrxG complex, has been shown to regulate tolerance to abiotic stress and growth and development in many plants. In this study, we identified nine GhASH1s in upland cotton. Bioinformatics analysis revealed that GhASH1s contain a variety of cis-acting elements related to stress resistance and growth and development. The transcriptome expression profiles revealed that GhASHH1.A and GhASHH2.A genes expression were upregulated in flower organs and in response to external temperature stress. The results of virus-induced gene silencing (VIGS) indicated that GhASHH1.A and GhASHH2.A genes silencing reduced the ability of cotton to adapt to temperature stress and delayed the development of the flowering phenotype. We also showed that the silencing of these two target genes did not induce early flowering at high temperature (32 °C), suggesting that GhASHH1.A and GhASHH2.A might regulate cotton flowering in response to temperature. These findings provide genetic resources for future breeding of early-maturing and temperature-stress-tolerant cotton varieties.
... IncX plasmids multiple sequence alignment was done using BLASTn 52 and visualized using EasyFig version 2.2.2 53 . VirBR protein homologs alignments were done using CLUSTALW 54 and visualized using ESPript 3.0 55 . ...
IncX3 plasmids carrying the New Delhi metallo-β-lactamase-encoding gene, blaNDM-5, are rapidly spreading globally in both humans and animals. Given that carbapenems are listed on the WHO AWaRe watch group and are prohibited for use in animals, the drivers for the successful dissemination of Carbapenem-Resistant Enterobacterales (CRE) carrying blaNDM-5-IncX3 plasmids still remain unknown. We observe that E. coli carrying blaNDM-5-IncX3 can persist in chicken intestines either under the administration of amoxicillin, one of the largest veterinary β-lactams used in livestock, or without any antibiotic pressure. We therefore characterise the blaNDM-5-IncX3 plasmid and identify a transcription regulator, VirBR, that binds to the promoter of the regulator gene actX enhancing the transcription of Type IV secretion systems (T4SS); thereby, promoting conjugation of IncX3 plasmids, increasing pili adhesion capacity and enhancing the colonisation of blaNDM-5-IncX3 transconjugants in animal digestive tracts. Our mechanistic and in-vivo studies identify VirBR as a major factor in the successful spread of blaNDM-5-IncX3 across one-health AMR sectors. Furthermore, VirBR enhances the plasmid conjugation and T4SS expression by the presence of copper and zinc ions, thereby having profound ramifications on the use of universal animal feeds.
... et al. (2013). Clustal X 2.1 (Larkin et al., 2007) was used to align the sequences. The best evolution model of nucleotide substitution was selected by jModelTest v2.1.10 ...
Sillago muktijoddhai, S. mengjialensis, S. sihama and Sillaginops macrolepis (Perciformes, Sillaginidae) are common coastal sand borers. The first three species are found in the Northeastern Indian Ocean, and the fourth one is located in the Western Central Pacific Ocean only. Molecular data applying in the prior investigations on systematics and phylogenetic relationships were not only incomplete but also uncertain for these fishes. This study sequenced their complete mitochondrial genomes using Sanger Dideoxy DNA sequencing for the first time. Their complete mitochondrial genome was a circular molecule of 17,022, 16,624, 16,825 and 16,502bp in length for S. muktijoddhai, S. mengjialensis, S. sihama and Sillaginops macrolepis, respectively. Most protein-coding genes (PCGs) were initiated with the typical ATG codon and terminated with the TAA or TAG codon and the incomplete termination codon T/TA could be detected in the four species. The majority of AT-skew and GC-skew values of the entire mitogenomes among the four species were negative. The Ka/Ks ratio analyses indicated 13 PCGs were suffering strong purifying selection. In the phylogenetic analysis, S. muktijoddhai, S. mengjialensis, and S. sihama were placed with relative species of the genus Sillago supporting morphological phylogeny. However, S. macrolepis was situated in the clade of genus Sillago that contrasted to morphological phylogeny. Divergence time analysis showed that Sillaginidae species diverged around 61 million years ago. Bangladesh J. Sci. Ind. Res. 59(2), 87-104, 2024
... In addition, the proteins with a tandem kinase -vWA structure were included in the phylogenetic analysis of kinase domains. Multiple sequence alignments were carried out with ClustalW software with default settings 70 . The conserved motifs in the Kin I and Kin II domains were previously annotated by Klymiuk et al. 43 . ...
Powdery mildew is a devastating disease that affects wheat yield and quality. Wheat wild relatives represent valuable sources of disease resistance genes. Cloning and characterization of these genes will facilitate their incorporation into wheat breeding programs. Here, we report the cloning of Pm57, a wheat powdery mildew resistance gene from Aegilops searsii. It encodes a tandem kinase protein with putative kinase-pseudokinase domains followed by a von Willebrand factor A domain (WTK-vWA), being ortholog of Lr9 that mediates wheat leaf rust resistance. The resistance function of Pm57 is validated via independent mutants, gene silencing, and transgenic assays. Stable Pm57 transgenic wheat lines and introgression lines exhibit high levels of all-stage resistance to diverse isolates of the Bgt fungus, and no negative impacts on agronomic parameters are observed in our experimental set-up. Our findings highlight the emerging role of kinase fusion proteins in plant disease resistance and provide a valuable gene for wheat breeding.
... Sequence alignments were carried out by ClustalW 2.1 web server (https://www.genome.jp/tools-bin/clustalw) (117,118), and the result was then processed by the ESPript 3.0 web server (https://espript.ibcp.fr/ESPript/cgi-bin/ESPript.cgi) (119). ...
A riboswitch generally regulates the expression of its downstream genes through conformational change in its expression platform (EP) upon ligand binding. The cyclic diguanosine monophosphate (c-di-GMP) class I riboswitch Bc1 is widespread and conserved among Bacillus cereus group species. In this study, we revealed that Bc1 has a long EP with two typical ρ-independent terminator sequences 28 bp apart. The upstream terminator T1 is dominant in vitro, while downstream terminator T2 is more efficient in vivo. Through mutation analysis, we elucidated that Bc1 exerts a rare and incoherent “transcription-translation” dual regulation with T2 playing a crucial role. However, we found that Bc1 did not respond to c-di-GMP under in vitro transcription conditions, and the expressions of downstream genes did not change with fluctuation in intracellular c-di-GMP concentration. To explore this puzzle, we conducted SHAPE-MaP and confirmed the interaction of Bc1 with c-di-GMP. This shows that as c-di-GMP concentration increases, T1 unfolds but T2 remains almost intact and functional. The presence of T2 masks the effect of T1 unwinding, resulting in no response of Bc1 to c-di-GMP. The high Shannon entropy values of EP region imply the potential alternative structures of Bc1. We also found that zinc uptake regulator can specifically bind to the dual terminator coding sequence and slightly trigger the response of Bc1 to c-di-GMP. This work will shed light on the dual-regulation riboswitch and enrich our understanding of the RNA world.
IMPORTANCE
In nature, riboswitches are involved in a variety of metabolic regulation, most of which preferentially regulate transcription termination or translation initiation of downstream genes in specific ways. Alternatively, the same or different riboswitches can exist in tandem to enhance regulatory effects or respond to multiple ligands. However, many putative conserved riboswitches have not yet been experimentally validated. Here, we found that the c-di-GMP riboswitch Bc1 with a long EP could form a dual terminator and exhibit non-canonical and incoherent “transcription-translation” dual regulation. Besides, zinc uptake regulator specifically bound to the coding sequence of the Bc1 EP and slightly mediated the action of Bc1. The application of SHAPE-MaP to the dual regulation mechanism of Bc1 may establish the foundation for future studies of such complex untranslated regions in other bacterial genomes.
... All the representing model and cryo-EM density maps were generated using UCSF Chimera 39 and PyMOL 44 . Programs Clustal X 45 and ESPript 46 were used to align multiple sequences. ...
Type II topoisomerases are ubiquitous enzymes that play a pivotal role in modulating the topological configuration of double-stranded DNA. These topoisomerases are required for DNA metabolism and have been extensively studied in both prokaryotic and eukaryotic organisms. However, our understanding of virus-encoded type II topoisomerases remains limited. One intriguing example is the African swine fever virus, which stands as the sole mammalian-infecting virus encoding a type II topoisomerase. In this work, we use several approaches including cryo-EM, X-ray crystallography, and biochemical assays to investigate the structure and function of the African swine fever virus type II topoisomerase, pP1192R. We determine the structures of pP1192R in different conformational states and confirm its enzymatic activity in vitro. Collectively, our results illustrate the basic mechanisms of viral type II topoisomerases, increasing our understanding of these enzymes and presenting a potential avenue for intervention strategies to mitigate the impact of the African swine fever virus.
... (https://blast.ncbi.nlm.nih.gov/Blast.cgi). Clustalx 2.1 was used to align those obtained protein sequences (23). Phylogenetic analysis was performed using the MEGA 5.2 program to elucidate the relationship of MoxB to other flavin reductases (24). ...
Microbial synthesis is a desirable approach to produce indirubin but suffers from low synthetic efficiency. Insufficient supply of reduced flavins is one major factor limiting synthetic efficiency. To address this, a novel flavin reductase, MoxB, was discovered through screening of the metagenomic library. MoxB showed a strong preference for NADH over NADPH as the electron source for FMN/FAD reduction and exhibited the highest activity at pH 8.0 and 30°C. It displayed remarkable thermostability by maintaining 80% of full activity after incubation at 60°C for 1 h. Furthermore, MoxB showed great organic solvent tolerance and its activity could be significantly increased by bivalent metal ions. In addition, heterologous expression of the moxB gene in the indirubin-producing E. coli significantly improved indirubin production up to 15.12-fold. This discovery expands the understanding of flavin reductases and provides a promising catalytic tool for microbial indirubin production.
IMPORTANCE
Much effort has been exerted to produce indirubin using engineered Escherichia coli, but high-level production has not been achieved so far. Insufficient supply of reduced flavins is one key factor limiting the catalytic efficiency. However, the flavin reductases involved in indirubin biosynthesis have not been hitherto reported. Discovery of the novel flavin reductase MoxB provides a useful tool for enhancing indirubin production by E. coli. Overexpression of MoxB in indirubin-producing E. coli increased indirubin production by 15.12-fold in comparison to the control strain. Our results document the function of flavin reductase that reduces flavins during indirubin biosynthesis and provide an important foundation for using the flavin reductases to improve indirubin production by engineered microorganisms.
... In the 3D models, the non-conserved regions, including the leader peptides with chloroplast-targeting motifs and transit peptides, were excluded from the models. For the phylogenetic analyses, a protein alignment of 158 fulllength ZEP proteins was produced using ClustalW [49] and manually refined using GeneDoc software version 2.7.000 (h p://nrbsc.org/gfx/genedoc, accessed on 19 February 2024). ...
Carotenoids are pigments that have a range of functions in human health. The carotenoid diatoxanthin is suggested to have antioxidant, anti-inflammatory and chemo-preventive properties. Diatoxanthin is only produced by a few groups of microalgae, where it functions in photoprotection. Its large-scale production in microalgae is currently not feasible. In fact, rapid conversion into the inactive pigment diadinoxanthin is triggered when cells are removed from a high-intensity light source, which is the case during large-scale harvesting of microalgae biomass. Zeaxanthin epoxidase (ZEP) 2 and/or ZEP3 have been suggested to be responsible for the back-conversion of high-light-accumulated diatoxanthin to diadinoxanthin in low-light diatoms. Using CRISPR/Cas9 gene editing technology, we knocked out the ZEP2 and ZEP3 genes in the marine diatom Phaeodactylum tricornutum to investigate their role in the diadinoxanthin–diatoxanthin cycle and determine if one of the mutant strains could function as a diatoxanthin production line. Light-shift experiments proved that ZEP3 encodes the enzyme converting diatoxanthin to diadinoxanthin in low light. Loss of ZEP3 caused the high-light-accumulated diatoxanthin to be stable for several hours after the cultures had been returned to low light, suggesting that zep3 mutant strains could be suitable as commercial production lines of diatoxanthin.
... gov/ refseq/, accessed January 17, 2023). The pairwise sequence alignment was performed using the ClustalW algorithm [33,34], and multiple sequence alignment was performed using Molecular Evolutionary Genetics Analysis (MEGA) version 11 [35]. GBLOCKS program was used to filter the alignments to eliminate potential false positive results [36]. ...
Background
Genome stability is maintained by the DNA damage repair (DDR) system composed of multiple DNA repair pathways of hundreds of genes. Germline pathogenic variation (PV) in DDR genes damages function of the affected DDR genes, leading to genome instability and high risk of diseases, in particular, cancer. Knowing evolutionary origin of the PVs in human DDR genes is essential to understand the etiology of human diseases. However, answer to the issue remains largely elusive. In this study, we analyzed evolutionary origin for the PVs in human DDR genes.
Methods
We identified 169 DDR genes by referring to various databases and identified PVs in the DDR genes of modern humans from ClinVar database. We performed a phylogenetic analysis to analyze the conservation of human DDR PVs in 100 vertebrates through cross-species genomic data comparison using the phyloFit program of the PHAST package and visualized the results using the GraphPad Prism software and the ggplot module. We identified DDR PVs from over 5000 ancient humans developed a database to host the DDR PVs (https://genemutation.fhs.um.edu.mo/dbDDR-AncientHumans). Using the PV data, we performed a molecular archeological analysis to compare the DDR PVs between modern humans and ancient humans. We analyzed evolution selection of DDR genes across 20 vertebrates using the CodeML in PAML for phylogenetic analysis.
Results
Our phylogenic analysis ruled out cross-species conservation as the origin of human DDR PVs. Our archeological approach identified rich DDR PVs shared between modern and ancient humans, which were mostly dated within the last 5000 years. We also observed similar pattern of quantitative PV distribution between modern and ancient humans. We further detected a set of ATM, BRCA2 and CHEK2 PVs shared between human and Neanderthals.
Conclusions
Our study reveals that human DDR PVs mostly arose in recent human history. We propose that human high cancer risk caused by DDR PVs can be a by-product of human evolution.
Inflammation-associated perturbations of the gut microbiome are well characterized, but poorly understood. Here, we demonstrate that disparate taxa recapitulate the metabolism of the oxidized sugars glucarate and galactarate, utilizing enzymatically divergent, yet functionally equivalent, gud/gar pathways. The divergent pathway in commensals includes a putative 5-KDG aldolase (GudL) and an uncharacterized ABC transporter (GarABC) that recapitulate the function of their non-homologous counterparts in pathogens. A systematic bioinformatic search for the gud/gar pathway in gut microbes identified 887 species putatively capable of metabolizing oxidized sugars. Previous studies showed that inflammation-derived nitrate, formed by nitric oxide reacting with superoxide, promotes pathogen growth. Our findings reveal a parallel phenomenon: oxidized sugars, also produced from reactions with nitric oxide, serve as alternative carbon sources for commensal microbes. Previously considered a pathogen virulence factor, oxidized sugar metabolism is also present in specific commensals and may contribute to their increased relative abundance in gastrointestinal inflammation.
Hybridization between wild Musa species and subspecies from Southeast Asia is at the origin of cultivated bananas. The genomes of these cultivars are complex mosaics involving nine genetic groups, including two previously unknown contributors. This study provides continuous genome assemblies for six wild genetic groups, one of which represents one of the unknown ancestor, identified as M.acuminata ssp. halabanensis. The second unknown ancestor partially present in a seventh assembly appears related to M. a. ssp. zebrina. These assemblies provide key resources for banana genetics and for improving cultivar assemblies, including that of the emblematic triploid Cavendish. Comparative and phylogenetic analyses reveal an ongoing speciation process within Musa, characterised by large chromosome rearrangements and centromere differentiation through the integration of different types of repeated sequences, including rDNA tandem repeats. This speciation process may have been favoured by reproductive isolation related to the particular context of climate and land connectivity fluctuations in the Southeast Asian region.
Transmembrane AMPA receptor regulatory proteins (TARPs) are claudin-like proteins that tightly regulate AMPA receptors (AMPARs) and are fundamental for excitatory neurotransmission. With cryo-electron microscopy (cryo-EM) we reconstruct the 36 kDa TARP subunit γ2 to 2.3 Å, which points to structural diversity among TARPs. Our data reveals critical motifs that distinguish TARPs from claudins and define how sequence variations within TARPs differentiate subfamilies and their regulation of AMPARs.
Probiotic strains, the main component of which are beneficial lactic acid bacteria, are of considerable interest in biotechnological production. Among various probiotic microorganisms, special attention is paid to Lactobacillus, which have many positive properties. The object of the study is lactic acid bacteria of the genus Lactobacillus isolated from biological samples, as well as collection strains of the Biobank of the Republican Collection of Microorganisms. The aim of the study is to isolate and characterize the probiotic properties of lactic acid bacteria for the development of probiotics. In the course of work, 15 probiotically active strains of lactic acid bacteria were screened and four most active cultures (Lactobacillus casei 2LB, Lactobacillus brevis 3LB, Lactobacillus fermentum BV-4, Lactobacillus plantarum 5LB) were selected among the strains of the Biobank of the Republican Collection of Microorganisms. A consortium of starter cultures of these strains was created and the optimal variant was selected for inclusion in the probiotic under development. The probiotic potential of bacteria was studied, including resistance to low pH values, osmotic pressure, such properties as autoaggregation and coaggregation to opportunistic microorganisms. Studies were conducted to investigate the survival of Lactobacillus in the aggressive environment of the gastrointestinal tract, their adhesive properties, ability to produce antimicrobial substances and modulate the immune response. The results showed that Lactobacillus strains can effectively suppress pathogenic microflora, colonize the intestine and stimulate immune processes. This holds promise for the development of novel Lactobacillus-based probiotic preparations to improve human health. These results emphasize the importance of studying the probiotic potential of Lactobacillus and their strain-specific properties, which are essential for the development of functional products.
The genomes of many plants, animals, and fungi frequently comprise dispensable B chromosomes that rely upon various chromosomal drive mechanisms to counteract the tendency of non-essential genetic elements to be purged over time. The B chromosome of rye – a model system for nearly a century – undergoes targeted nondisjunction during first pollen mitosis, favouring segregation into the generative nucleus, thus increasing their numbers over generations. However, the genetic mechanisms underlying this process are poorly understood. Here, using a newly-assembled, ~430 Mb-long rye B chromosome pseudomolecule, we identify five candidate genes whose role as trans-acting moderators of the chromosomal drive is supported by karyotyping, chromosome drive analysis and comparative RNA-seq. Among them, we identify DCR28, coding a microtubule-associated protein related to cell division, and detect this gene also in the B chromosome of Aegilops speltoides. The DCR28 gene family is neo-functionalised and serially-duplicated with 15 B chromosome-located copies that are uniquely highly expressed in the first pollen mitosis of rye.
Genetic incompatibility occurs when a mismatched pair of plant immune components mounts autoimmune responses in hybrids. Highly diversified NLR receptors are main culprits of the genetic conflict, recognizing host proteins from different origin as immune trigger. Here, we report the molecular mechanism underlying a DANGEROUS MIX (DM) autoimmunity, comprising DM2h/RPP1 NLR and its incompatible partner DM3, an alpha/beta hydrolase. Cryo-electron microscopy reveals the oligomeric nature of two natural DM3 variants in a trimer of dimer configuration. The polymorphism triggering autoimmunity is located at the dimer interface, resulting in drastic structural differences such that dimerizing helix and loop reinforcing the interface is lost and disordered. Structure-function analysis shows that integrity of the dimer interface, but neither maintenance of hexamer nor its enzymatic activity, is the key factor contributing to autoimmunity. Our finding pinpoints checkpoints embedded in the oligomeric configuration of a host enzyme that controls the switching mechanism of NLR activity.
Oxidosqualene cyclases (OSCs) are important regulatory enzymes involved in cyclization reactions of 2, 3-oxidosqualene to form triterpenes and sterols. This study presents the identification and characterization of three OSC genes, a β – amyrin synthase (VuβAS), a lupeol synthase (VuLUS) and a cycloartenol synthase (VuCAS) in Vigna unguiculata, an edible leguminous plant with high nutritional and nutraceutical value. Phylogenetic analysis showed that the VuβAS, VuLUS and VuCAS were clustered within the clades of previously characterized β amyrin synthases, lupeol synthases and cycloartenol synthases. Heterologous expression in Saccharomyces cerevisiae and Gas Chromatography – Mass Spectrometry (GC – MS) analysis in different plant stages confirmed their specific functions. VuβAS showed higher expression in roots from early germinating seedlings to older plants (4day to 28-day), while VuLUS expression levels were higher in the roots of older plants only (14-day to 28-day). VuCAS expression was increased in all the tissues of 4-day seedlings, with a peak in stem and leaves and a lower accumulation in radicles. These findings revealed the presence and function of OSC genes in V. unguiculata, and future research could lead to the discovery of promising biologically active compounds.
T-even type bacteriophages are virulent phages commonly used as model organisms, playing a crucial role in understanding various biological processes. One such process involves the regulation of DNA topology during phage replication upon host infection, governed by type IIA DNA topoisomerases. In spite of various studies on prokaryotic and eukaryotic counterparts, viral topoisomerase II remains insufficiently understood, especially the unique domain composition of T4 phage. In this study, we determine the cryo-EM structures of topoisomerase II from T4 and T6 phages, including full-length structures of both apo and DNA-binding states which have never been determined before. Together with other conformational states, these structures provide an explicit blueprint of mechanisms of phage topoisomerase II. Particularly, the asymmetric dimeric interactions observed in cryo-EM structures of T6 phage topoisomerase II ATPase domain and central domain bound with DNA shed light on the asynchronous ATP usage and asynchronous cleavage of the G-segment DNA, respectively. The elucidation of phage topoisomerase II’s structures and functions not only enhances our understanding of mechanisms and evolutionary parallels with prokaryotic and eukaryotic homologs but also highlights its potential as a model for developing type IIA topoisomerase inhibitors.
TRIM25 is an RNA-binding ubiquitin E3 ligase with central but poorly understood roles in the innate immune response to RNA viruses. The link between TRIM25’s RNA binding and its role in innate immunity has not been established. Thus, we utilized a multitude of biophysical techniques to identify key RNA-binding residues of TRIM25 and developed an RNA-binding deficient mutant (TRIM25-m9). Using iCLIP2 in virus-infected and uninfected cells, we identified TRIM25’s RNA sequence and structure specificity, that it binds specifically to viral RNA, and that the interaction with RNA is critical for its antiviral activity.
Regeneration in many animals involves the formation of a blastema, which differentiates and organizes into the appropriate missing body parts. Although the mechanisms underlying blastema formation are often fundamental to regeneration biology, information on the cellular and molecular basis of blastema formation remains limited. Here, we focus on a fragmenting potworm (Enchytraeus japonensis), which can regenerate its whole body from small fragments. We find soxC and mmpReg as upregulated genes in the blastema. RNAi of soxC and mmpReg reduce the number of blastema cells, indicating that soxC and mmpReg promote blastema formation. Expression analyses show that soxC-expressing cells appear to gradually accumulate in blastema and constitute a large part of the blastema. Additionally, similar expression dynamics of SoxC orthologue genes in frog (Xenopus laevis) are found in the regeneration blastema of tadpole tail. Our findings provide insights into the cellular and molecular mechanisms underlying blastema formation across species.
Photosynthesis converting solar energy to chemical energy is one of the most important chemical reactions on earth. In cyanobacteria, light energy is captured by antenna system phycobilisomes (PBSs) and transferred to photosynthetic reaction centers of photosystem II (PSII) and photosystem I (PSI). While most of the protein complexes involved in photosynthesis have been characterized by in vitro structural analyses, how these protein complexes function together in vivo is not well understood. Here we implemented STAgSPA, an in situ structural analysis strategy, to solve the native structure of PBS–PSII supercomplex from the cyanobacteria Arthrospira sp. FACHB439 at resolution of ~3.5 Å. The structure reveals coupling details among adjacent PBSs and PSII dimers, and the collaborative energy transfer mechanism mediated by multiple super-PBS in cyanobacteria. Our results provide insights into the diversity of photosynthesis-related systems between prokaryotic cyanobacteria and eukaryotic red algae but are also a methodological demonstration for high-resolution structural analysis in cellular or tissue samples.
Methanogenic hydrocarbon degradation can be carried out by archaea that couple alkane oxidation directly to methanogenesis, or by syntrophic associations of bacteria with methanogenic archaea. However, metagenomic analyses of methanogenic environments have revealed other archaea with potential for alkane degradation but apparent inability to form methane, suggesting the existence of other modes of syntrophic hydrocarbon degradation. Here, we provide experimental evidence supporting the existence of a third mode of methanogenic degradation of hydrocarbons, mediated by syntrophic cooperation between archaeal partners. We collected sediment samples from a hot spring sediment in Tengchong, China, and enriched Hadarchaeota under methanogenic conditions at 60 °C, using hexadecane as substrate. We named the enriched archaeon Candidatus Melinoarchaeum fermentans DL9YTT1. We used ¹³C-substrate incubations, metagenomic, metatranscriptomic and metabolomic analyses to show that Ca. Melinoarchaeum uses alkyl-coenzyme M reductases (ACRs) to activate hexadecane via alkyl-CoM formation. Ca. Melinoarchaeum likely degrades alkanes to carbon dioxide, hydrogen and acetate, which can be used as substrates by hydrogenotrophic and acetoclastic methanogens such as Methanothermobacter and Methanothrix.
Environmental remediation through biological methods is one of the main concerns of nations today. Benzopyrene (BP) is the main component of polycyclic aromatic hydrocarbons, and has been frequently found in the air, surface water, soil, and sediments. BP is resistant to remediation because of their low water solubility. Laccases are one of the best materials that could be employed for degradation of BP. Bacterial laccases are a high thermostable enzyme that can endure a vast range of pH; however, due to their low redox potential and activity, they have not been studied thoroughly. In this work, site specific mutation at a site near the Cu1, which is the primary electron acceptor in laccase-catalyzed oxidation, was applied to a laccase from Bacillus subtilis to increase laccase activity. The mutated gene cotA laccase was cloned in pET22b(+) plasmid and transformed in expression host Escherichia coli BL21 (DE3). The recombinant protein was produced under optimum induction (37 °C), purified and then analyzed through SDS-PAGE method. Enzyme activity was analyzed by specific laccase substrate (ABTS). further analysis was done for enzyme properties in different temperature, pH, and salt concentration. The results showed that the recombinant enzyme had a higher activity than native enzyme in the same condition. Determining biological degradation characteristics of BP using the recombinant laccase indicated that the recombinant laccase was more stable in different conditions and had a high enzyme potential. The functionality assays of these enzymes imply that the mutant-type is more potent given its affinity with the benzo[a]pyrene as the corresponding ligand and ABTS oxidation as a substrate under the in-silico and in-vitro conditions respectively. Furthermore, the Km and Vmax of the purified enzymes were determined in line with their activities, so that the mutant-type revealed 54% decrease in Km and 5.5-fold increase in Kcat and an increase in Kcat/Km parameter. In addition, through HPLC technique, the comparison of BP degradation by the natural and mutant enzymes confirmed the superior capability of the mutant enzyme in BP removal.
The increase in the use of molecular methodologies in systematics has driven the necessity for a comprehensive understanding of the limitations of different genetic markers. Not every marker is optimal for all species, which has led to multiple approaches in the study of the taxonomy and systematics of polyclad flatworms. The present study evaluates base-substitution rates of nuclear ribosomal (18S rDNA and 28S rDNA), mitochondrial ribosomal (16S rDNA), and protein-codifying (cytb, cox1) markers for this taxonomic group, with the main objective of assessing the robustness of these different markers for phylogenetic studies. Mutation rates and Ti/Tv ratios of the other markers were assessed for the first time. We estimated substitution rates and found cytb to be the most variable, while 18S rDNA was the least variable among them. On the other hand, the transition to transversion (Ti/Tv) ratio of the different genes revealed differences between the markers, with a higher number of transitions in the nuclear gene 28S and a higher number of transversions in the mitochondrial genes. Lastly, we identified that the third codon position of the studied protein codifying genes was highly variable and that this position was saturated in the cox1 marker but not in cytb. We conclude that it is important to assess the markers employed for different phylogenetic levels for future studies, particularly in the order Polycladida. We encourage the use of mitochondrial genes cytb and 16S for phylogenetic studies at suborder, superfamily, and family levels and species delimitation in polyclads, in addition to the well-known 28S and cox1.
The emergence of new structures can often be linked to the evolution of novel cell types that follows the rewiring of developmental gene regulatory subnetworks. Vertebrates are characterized by a complex body plan compared to the other chordate clades and the question remains of whether and how the emergence of vertebrate morphological innovations can be related to the appearance of new embryonic cell populations. We previously proposed, by studying mesoderm development in the cephalochordate amphioxus, a scenario for the evolution of the vertebrate head mesoderm. To further test this scenario at the cell population level, we used scRNA-seq to construct a cell atlas of the amphioxus neurula, stage at which the main mesodermal compartments are specified. Our data allowed us to validate the presence of a prechordal-plate like territory in amphioxus. Additionally, the transcriptomic profile of somite cell populations supports the homology between specific territories of amphioxus somites and vertebrate cranial/pharyngeal and lateral plate mesoderm. Finally, our work provides evidence that the appearance of the specific mesodermal structures of the vertebrate head was associated to both segregation of pre-existing cell populations, and co-option of new genes for the control of myogenesis.
Endosperm is the main storage organ in cereal grain and determines grain yield and quality. The molecular mechanisms of heat shock proteins in regulating starch biosynthesis and endosperm development remain obscure.
Here, we report a rice floury endosperm mutant flo24 that develops abnormal starch grains in the central starchy endosperm cells. Map‐based cloning and complementation test showed that FLO24 encodes a heat shock protein HSP101, which is localized in plastids. The mutated protein FLO24T296I dramatically lost its ability to hydrolyze ATP and to rescue the thermotolerance defects of the yeast hsp104 mutant.
The flo24 mutant develops more severe floury endosperm when grown under high‐temperature conditions than normal conditions. And the FLO24 protein was dramatically induced at high temperature. FLO24 physically interacts with several key enzymes required for starch biosynthesis, including AGPL1, AGPL3 and PHO1. Combined biochemical and genetic evidence suggests that FLO24 acts cooperatively with HSP70cp‐2 to regulate starch biosynthesis and endosperm development in rice.
Our results reveal that FLO24 acts as an important regulator of endosperm development, which might function in maintaining the activities of enzymes involved in starch biosynthesis in rice.
Wild bumble bees (Hymenoptera: Apidae) play a vital role in agro-ecosystems as important pollinators. However, they are threatened by virus pathogens that are widespread in honey bees. Previous studies have reported that viruses were able to be transmitted across bee genera and caused potential danger to wild bumble bees. China is a global biodiversity hotspot for bumble bees. However, the impact of viruses on the wild bumble bee communities remains elusive. Black queen cell virus (BQCV) is one of the most common honey bee viruses. Here, a total of 72 wild bumble bee samples from 17 geographic regions of China were tested for BQCV. Thirteen positive samples were identified and sequence comparison of partial capsid genes demonstrated a genetic identity of 99.69% to 100%. A phylogenetic tree analysis also showed a close relationship between 13 BQCV isolates and others from a variety of recorded hosts in China. Meanwhile, a distinct evolutionary branch of China isolates was formed when clustering isolates from worldwide bumble bee species. A correlation between BQCV and their geographic locations were observed (P < 0.05). This study not only provides the first evidence of widespread BQCV in wild bumble bee communities in China but also detects a distinct set of genetically identical or closely related BQCV variants that circulate and evolutionarily differ from other countries.
MicroRNAs (miRNAs) play a key role in regulating gene expression and their biogenesis is precisely controlled through modulating the activity of microprocessor. Here, we report that CWC15, a spliceosome-associated protein, acts as a positive regulator of miRNA biogenesis. CWC15 binds the promoters of genes encoding miRNAs (MIRs), promotes their activity, and increases the occupancy of DNA-dependent RNA polymerases at MIR promoters, suggesting that CWC15 positively regulates the transcription of primary miRNA transcripts (pri-miRNAs). In addition, CWC15 interacts with Serrate (SE) and HYL1, two key components of microprocessor, and is required for efficient pri-miRNA processing and the HYL1-pri-miRNA interaction. Moreover, CWC15 interacts with the 20 S proteasome and PRP4KA, facilitating SE phosphorylation by PRP4KA, and subsequent non-functional SE degradation by the 20 S proteasome. These data reveal that CWC15 ensures optimal miRNA biogenesis by maintaining proper SE levels and by modulating pri-miRNA levels. Taken together, this study uncovers the role of a conserved splicing-related protein in miRNA biogenesis.
Malaria control in South-East Asia remains a challenge, underscoring the importance of accurately identifying malaria mosquitoes to understand transmission dynamics and improve vector control. Traditional methods such as morphological identification require extensive training and cannot distinguish between sibling species, while molecular approaches are costly for extensive screening. Matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS) has emerged as a rapid and cost-effective tool for Anopheles species identification, yet its current use is limited to few specialized laboratories. This study aimed to develop and validate an online reference database for MALDI-TOF MS identification of Southeast Asian Anopheles species. The database, constructed using the in-house data analysis pipeline MSI2 (Sorbonne University), comprised 2046 head mass spectra from 209 specimens collected at the Thailand-Myanmar border. Molecular identification via COI and ITS2 DNA barcodes enabled the identification of 20 sensu stricto species and 5 sibling species complexes. The high quality of the mass spectra was demonstrated by a MSI2 median score (min-max) of 61.62 (15.94-77.55) for correct answers, using the best result of four technical replicates of a test panel. Applying an identification threshold of 45, 93.9% (201/214) of the specimens were identified, with 98.5% (198/201) consistency with the molecular taxonomic assignment. In conclusion, MALDI-TOF MS holds promise for malaria mosquito identification and can be scaled up for entomological surveillance in Southeast Asia. The free online sharing of our database on the MSI2 platform represents an important step towards the broader use of MALDI-TOF MS in malaria vector surveillance.
Appropriate diagnoses of parasites of apex marine predators are crucial to understand their biodiversity, host specificity, biogeography, and life cycles. Such diagnoses are also informative of ecological and biological characteristics of both host and environment in which the hosts and their parasites live. We here (i) investigate the parasite fauna of a bluntnose sixgill shark Hexanchus griseus (Bonnaterre, 1788) obtained from the Gulf of Naples (Tyrrhenian Sea), (ii) characterize molecularly all its metazoan parasites, and (iii) resurrect and report the main morphological features and phylogenetic position of Grillotia acanthoscolex, a cestode species previously synonymized with Grillotia adenoplusia. A rich parasite fauna represented by eight different taxa was found, including two monogeneans (Protocotyle grisea and Protocotyle taschenbergi), one digenean (Otodistomum veliporum), four cestodes (Crossobothrium dohrnii, Clistobothrium sp., G. acanthoscolex, and G. adenoplusia), and one copepod (Protodactylina pamelae). Sequencing of these samples accounts for an important molecular baseline to widen the knowledge on the parasitic fauna of bluntnose sixgill sharks worldwide and to reconstruct their correct food chains. The bluntnose sixgill shark was found to be a definitive host for all endoparasites found here, confirming that it occupies an apex trophic level in the Mediterranean Sea. The taxa composition of the trophic parasite fauna confirms that the bluntnose sixgill shark mostly feeds on teleost fish species. However, the occurrence of two phillobothrid cestodes (C. dohrnii and Clistobothrium sp.) suggests that it also feeds on squids. Finally, we emphasize the importance of using integrative taxonomic approaches in the study of parasites from definitive and intermediate hosts to elucidate biology and ecology of taxa generally understudied in the Mediterranean Sea.
Present-day geographic and phylogenetic patterns often reflect the geological and climatic history of the planet. Neontological distribution data is often sufficient to unravel a lineage’s biogeographic history, yet ancestral range inferences can be at odds with fossil evidence. Here, I use the Fossilized Birth Death process and the Dispersal Extinction Cladogenesis model to jointly infer the dated phylogeny and range evolution of the tree fern order Cyatheales. I use data for 101 fossil and 442 extant tree ferns to reconstruct the biogeographic history of the group over the last 220 million years. Fossil-aware reconstructions evince a prolonged occupancy of Laurasia over the Triassic–Cretaceous by Cyathealean tree ferns, which is evident in the fossil record but hidden from analyses relying on neontological data alone. Nonetheless, fossil-aware reconstructions are affected by uncertainty in fossils’ phylogenetic placement, taphonomic biases, and specimen sampling, and are sensitive to interpretation of palaeodistributions and how these are scored. The present results highlight the need and challenges of incorporating fossils into joint inferences of phylogeny and biogeography to improve the reliability of ancestral geographic range estimation.
Blue denim, a billion-dollar industry, is currently dyed with indigo in an unsustainable process requiring harsh reducing and alkaline chemicals. Forming indigo directly in the yarn through indican (indoxyl-β-glucoside) is a promising alternative route with mild conditions. Indican eliminates the requirement for reducing agent while still ending as indigo, the only known molecule yielding the unique hue of blue denim. However, a bulk source of indican is missing. Here, we employ enzyme and process engineering guided by techno-economic analyses to develop an economically viable drop-in indican synthesis technology. Rational engineering of PtUGT1, a glycosyltransferase from the indigo plant, alleviated the severe substrate inactivation observed with the wildtype enzyme at the titers needed for bulk production. We further describe a mild, light-driven dyeing process. Finally, we conduct techno-economic, social sustainability, and comparative life-cycle assessments. These indicate that the presented technologies have the potential to significantly reduce environmental impacts from blue denim dyeing with only a modest cost increase.
The effect of pathogens on host diversity has attracted much attention in recent years, yet how the influence of pathogens on individual plants scales up to affect community‐level host diversity remains unclear.
Here, we assessed the effects of foliar fungal pathogens on plant growth and species richness using allometric growth theory in population‐level and community‐level foliar fungal pathogen exclusion experiments. We calculated growth scaling exponents of 24 species to reveal the intraspecific size‐dependent effects of foliar fungal pathogens on plant growth. We also calculated the intercepts to infer the growth rates of relatively larger conspecific individuals.
We found that foliar fungal pathogens inhibited the growth of small conspecific individuals more than large individuals, resulting in a positive allometric growth. After foliar fungal pathogen exclusion, species‐specific growth scaling exponents and intercepts decreased, but became positively related to species' relative abundance, providing a growth advantage for individuals of abundant species with a higher growth scaling exponent and intercept compared with rare species, and thus reduced species diversity.
By adopting allometric growth theory, we elucidate the size‐dependent mechanisms through which pathogens regulate species diversity and provide a powerful framework to incorporate antagonistic size‐dependent processes in understanding species coexistence.
C-type lectin receptors (CLRs) are a family of pattern recognition receptors, which detect a broad spectrum of ligands via small carbohydrate recognition domains (CRDs). CLEC12A is an inhibitory CLR that recognizes crystalline structures such as monosodium urate crystals. CLEC12A also recognizes mycolic acid, a major component of mycobacterial cell walls, and suppresses host immune responses. Although CLEC12A could be a therapeutic target for mycobacterial infection, structural information on CLEC12A was not available. We report here the crystal structures of human CLEC12A in ligand-free form and in complex with 50C1, its inhibitory antibody. 50C1 recognizes human-specific residues on the top face of hCLEC12A CRD. A comprehensive alanine scan demonstrated that the ligand-binding sites of mycolic acid and monosodium urate crystals may overlap with each other, suggesting that CLEC12A utilizes a common interface to recognize different types of ligands. Our results provide atomic insights into the blocking and ligand-recognition mechanisms of CLEC12A and leads to the design of CLR-specific inhibitors.
Heveins are one of the most important groups of plant antimicrobial peptides. So far, various roles in plant growth and development and in response to biotic and abiotic stresses have reported for heveins. The present study aimed to identify and characterize the hevein genes in two-row and six-row cultivars of barley. In total, thirteen hevein genes were identified in the genome of two-row and six-row cultivars of barley. The identified heveins were identical in two-row and six-row cultivars of barley and showed a high similarity with heveins from other plant species. The hevein coding sequences produced open reading frames (ORFs) ranged from 342 to 1002 bp. Most of the identified hevein genes were intronless, and the others had only one intron. The hevein ORFs produced proteins ranged from 113 to 333 amino acids. Search for conserved functional domains showed CBD and LYZ domains in barley heveins. All barley heveins comprised extracellular signal peptides ranged from 19 to 35 amino acids. The phylogenetic analysis divided barley heveins into two groups. The promoter analysis showed regulatory elements with different frequencies between two-row and six-row cultivars. These cis-acting elements included elements related to growth and development, hormone response, and environmental stresses. The expression analysis showed high expression level of heveins in root and reproductive organs of both two-row and six-row cultivars. The expression analysis also showed that barley heveins is induced by both biotic and abiotic stresses. The results of antimicrobial activity prediction showed the highest antimicrobial activity in CBD domain of barley heveins. The findings of the current study can improve our knowledge about the role of hevein genes in plant and can be used for future studies.
We conducted a study on interpopulation variation of colour patterns in two congeneric chameleon species, which have an analogous life history. Both species are able to rapidly change colour pattern, and their context-dependent colour patterns often vary across a wide geographical range. Specifically, we tested four hypotheses that can explain the observed interpopulation variation of colour patterns by a series of behavioural field trials where the colour patterns of individuals were recorded and later analysed by a deep neural network algorithm. We used redundancy analysis to relate genetic, spectral and behavioural predictors to interpopulation colour pattern distance. Our results showed that both isolation by distance (IBD) and alternative mating tactics were significant predictors for interpopulation colour pattern variation in Chamaeleo chamaeleon males. By contrast, in Chamaeleo dilepis, the interpopulation colour pattern variation was largely explained by IBD, and evidence for alternative mating tactics was absent. In both chameleon species, the environmental colours showed no evidence of influencing chameleon interpopulation colour pattern variation, regardless of sex or behavioural context. This contrasting finding suggests that interpopulation context-dependent colour pattern variations in each species are maintained under a different set of selective pressures or circumstances.
A strategy is described for the rapid alignment of many long nucleic acid or protein sequences on a microcomputer. The program described can handle up to 100 sequences of 1200 residues each. The approach is based on progressively aligning sequences according to the branching order in an initial phylogenetic tree. The results obtained using the package appear to be as sensitive as those from any other available method.
CLUSTAL X is a new windows interface for the widely-used progressive multiple sequence alignment program CLUSTAL W. The new
system is easy to use, providing an integrated system for performing multiple sequence and profile alignments and analysing
the results. CLUSTAL X displays the sequence alignment in a window on the screen. A versatile sequence colouring scheme allows
the user to highlight conserved features in the alignment. Pull-down menus provide all the options required for traditional
multiple sequence and profile alignment. New features include: the ability to cut-and-paste sequences to change the order
of the alignment, selection of a subset of the sequences to be realigned, and selection of a sub-range of the alignment to
be realigned and inserted back into the original alignment. Alignment quality analysis can be performed and low-scoring segments
or exceptional residues can be highlighted. Quality analysis and realignment of selected residue ranges provide the user with
a powerful tool to improve and refine difficult alignments and to trap errors in input sequences. CLUSTAL X has been compiled
on SUN Solaris, IRIX5.3 on Silicon Graphics, Digital UNIX on DECstations, Microsoft Windows (32 bit) for PCs, Linux ELF for
x86 PCs, and Macintosh PowerMac.
A multiple sequence alignment program, MAFFT, has been developed. The CPU time is drastically reduced as compared with existing methods. MAFFT includes two novel techniques. (i) Homo logous regions are rapidly identified by the fast Fourier transform (FFT), in which an amino acid sequence is converted to a sequence composed of volume and polarity values of each amino acid residue. (ii) We propose a simplified scoring system that performs well for reducing CPU time and increasing the accuracy of alignments even for sequences having large insertions or extensions as well as distantly related sequences of similar length. Two different heuristics, the progressive method (FFT-NS-2) and the iterative refinement method (FFT-NS-i), are implemented in MAFFT. The performances of FFT-NS-2 and FFT-NS-i were compared with other methods by computer simulations and benchmark tests; the CPU time of FFT-NS-2 is drastically reduced as compared with CLUSTALW with comparable accuracy. FFT-NS-i is over 100 times faster than T-COFFEE, when the number of input sequences exceeds 60, without sacrificing the accuracy.
We describe a new method (T-Coffee) for multiple sequence alignment that provides a dramatic improvement in accuracy with a modest sacrifice in speed as compared to the most commonly used alternatives. The method is broadly based on the popular progressive approach to multiple alignment but avoids the most serious pitfalls caused by the greedy nature of this algorithm. With T-Coffee we pre-process a data set of all pair-wise alignments between the sequences. This provides us with a library of alignment information that can be used to guide the progressive alignment. Intermediate alignments are then based not only on the sequences to be aligned next but also on how all of the sequences align with each other. This alignment information can be derived from heterogeneous sources such as a mixture of alignment programs and/or structure superposition. Here, we illustrate the power of the approach by using a combination of local and global pair-wise alignments to generate the library. The resulting alignments are significantly more reliable, as determined by comparison with a set of 141 test cases, than any of the popular alternatives that we tried. The improvement, especially clear with the more difficult test cases, is always visible, regardless of the phylogenetic spread of the sequences in the tests.
The CLUSTAL package of multiple sequence alignment programs has been completely rewritten and many new features added. The new software is a single program called CLUSTAL V, which is written in C and can be used on any machine with a standard C compiler. The main new features are the ability to store and reuse old alignments and the ability to calculate phylogenetic trees after alignment. The program is simple to use, completely menu driven and on-line help is provided.
An approach for performing multiple alignments of large numbers of amino acid or nucleotide sequences is described. The method is based on first deriving a phylogenetic tree from a matrix of all pairwise sequence similarity scores, obtained using a fast pairwise alignment algorithm. Then the multiple alignment is achieved from a series of pairwise alignments of clusters of sequences, following the order of branching in the tree. The method is sufficiently fast and economical with memory to be easily implemented on a microcomputer, and yet the results obtained are comparable to those from packages requiring mainframe computer facilities.
The sensitivity of the commonly used progressive multiple sequence alignment method has been greatly improved for the alignment
of divergent protein sequences. Firstly, individual weights are assigned to each sequence in a partial alignment in order
to downweight near-duplicate sequences and up-weight the most divergent ones. Secondly, amino acid substitution matrices are
varied at different alignment stages according to the divergence of the sequences to be aligned. Thirdly, residue-specific
gap penalties and locally reduced gap penalties in hydrophilic regions encourage new gaps in potential loop regions rather
than regular secondary structure. Fourthly, positions in early alignments where gaps have been opened receive locally reduced
gap penalties to encourage the opening up of new gaps at these positions. These modifications are incorporated into a new
program, CLUSTAL W which is freely available.
We describe MUSCLE, a new computer program for creating multiple alignments of protein sequences. Elements of the algorithm
include fast distance estimation using kmer counting, progressive alignment using a new profile function we call the log‐expectation score, and refinement using tree‐dependent
restricted partitioning. The speed and accuracy of MUSCLE are compared with T‐Coffee, MAFFT and CLUSTALW on four test sets
of reference alignments: BAliBASE, SABmark, SMART and a new benchmark, PREFAB. MUSCLE achieves the highest, or joint highest,
rank in accuracy on each of these sets. Without refinement, MUSCLE achieves average accuracy statistically indistinguishable
from T‐Coffee and MAFFT, and is the fastest of the tested methods for large numbers of sequences, aligning 5000 sequences
of average length 350 in 7 min on a current desktop computer. The MUSCLE program, source code and PREFAB test data are freely
available at http://www.drive5. com/muscle.