Multiple Sequence Research Articles

Application of Magnetic Resonance Imaging Report Combined With VI-RADS Bi-Parametric and Multi-Parametric Scoring Systems in Bladder Cancer Diagnosis

To verify by retrospective analysis the performance of applying dual-parametric (T2 weighted image [T2WI] and diffusion weighted image [DWI]) and multi-parametric (T2WI, DWI, and dynamic contrast enhance [DCE]) evaluation systems of Vesical Imaging-Reporting and Data System (VI-RADS) in the magnetic resonance imaging (MRI) diagnosis of bladder cancer. The imaging and clinical data of bladder cancer patients who underwent bladder MRI examination in the Department of Urology of our hospital between January 2020 and December 2021 were collected. A total of 215 bladder cancer patients, among whom there were 183 males and 32 females with an average age of (67.60±11.42) years, were included. The bladder cancer diagnosis of all the cases was verified by pathology analysis of tissue samples. Two radiologists, who were double-blinded, scored multiple sequences separately. Then, a comparative analysis was made on the diagnostic performance of dual-parametric and multi-parametric VI-RADS diagnostic scores. The diagnostic test with receiver operator characteristic (ROC) curves and Cohen's Kappa were used to evaluate the diagnostic efficacy and consistency. The area under the curve (AUC) and 95% confidence interval (CI) of the multi-parametric VI-RADS evaluation system by the two radiologists in the overall population were 0.878 (0.830-0.925) and 0.856 (0.805-0.907), while those for the bi-parametric VI-RADS evaluation system were 0.889 (0.844-0.934) and 0.856 (0.805-0.907), showing no statistically significant difference. No significant difference was observed in the subgroup analysis of patients who underwent transurethral resection of bladder tumor (TURBT). Furthermore, the Kappa values for inter-rater agreement between of the two radiologists were 0.694 and 0.546 (with the VI-RADS score≥3 defined as being positive) and 0.693 and 0.712 (with the VI-RADS score≥4 defined as being positive) in multi-parametric and bi-parametric evaluation, respectively (all P<0.001). The bi-parametric VI-RADS scoring system can achieve a diagnostic efficacy comparable to that of the multi-parametric scoring system, offering an alternative to patients who are unable to undergo contrast-enhanced MRI due to allergic reaction to contrasts.

Automated evaluation of multiple sequence alignment methods to handle third generation sequencing errors

Automated evaluation of multiple sequence alignment methods to handle third generation sequencing errors

Integrated subtractive genomics and structure-based approach to unravel the therapeutic drug target of Leishmania species.

Leishmaniasis is a complex vector-borne disease caused by intracellular protozoan parasites of the Leishmania genus. It presents a significant public health challenge in tropical and subtropical regions globally. As resistance to treatment increases, managing and controlling Leishmaniasis becomes more challenging, necessitating innovative approaches. To address this challenge, our study utilized subtractive genomics and structure-based approaches to identify common drug targets and combat antimicrobial resistance (AMR) across five Leishmania species strains. The subtractive genomics approach unraveled Glutamate Dehydrogenase (GDH) as a promising drug target for treating Leishmania infections. The investigation considered established methodologies observed in analogous studies, orthologous group, and druggability tests. Multiple sequence alignment revealed conserved sequences in GDH, while phylogenetic tree analysis provided insights into the evolutionary origin and close relationships of GDH across Leishmania species. Conserved sequences in GDH along with its function in pathogenicity provided insights into the close relationships of GDH across Leishmania species. Using a structure-based approach, our study showed the molecular interactions between GDH and three ligands-Bithionol, GW5074, and Hexachlorophene-through molecular docking and 100ns molecular dynamics (MD) simulations. GW5074 exhibited a significant affinity for GDH, as indicated by stable RMSD values, a more compact conformation, and a higher number of hydrogen bonds than Bithionol. MMPBSA analysis confirmed the superior binding energy of the GW5074-GDH complex, emphasizing its potential as a potent ligand for drug development. This comprehensive analysis identified GW5074 as a promising candidate for inhibiting GDH activities in Leishmania species, contributing to the development of effective therapeutics against Leishmania infections.

Predicting Mutation-Induced Allosteric Changes in Structures and Conformational Ensembles of the ABL Kinase Using AlphaFold2 Adaptations with Alanine Sequence Scanning.

Despite the success of AlphaFold2 approaches in predicting single protein structures, these methods showed intrinsic limitations in predicting multiple functional conformations of allosteric proteins and have been challenged to accurately capture the effects of single point mutations that induced significant structural changes. We examined several implementations of AlphaFold2 methods to predict conformational ensembles for state-switching mutants of the ABL kinase. The results revealed that a combination of randomized alanine sequence masking with shallow multiple sequence alignment subsampling can significantly expand the conformational diversity of the predicted structural ensembles and capture shifts in populations of the active and inactive ABL states. Consistent with the NMR experiments, the predicted conformational ensembles for M309L/L320I and M309L/H415P ABL mutants that perturb the regulatory spine networks featured the increased population of the fully closed inactive state. The proposed adaptation of AlphaFold can reproduce the experimentally observed mutation-induced redistributions in the relative populations of the active and inactive ABL states and capture the effects of regulatory mutations on allosteric structural rearrangements of the kinase domain. The ensemble-based network analysis complemented AlphaFold predictions by revealing allosteric hotspots that correspond to state-switching mutational sites which may explain the global effect of regulatory mutations on structural changes between the ABL states. This study suggested that attention-based learning of long-range dependencies between sequence positions in homologous folds and deciphering patterns of allosteric interactions may further augment the predictive abilities of AlphaFold methods for modeling of alternative protein sates, conformational ensembles and mutation-induced structural transformations.

MOLECULAR CHARACTERIZATION AND PATHOGENICITY TEST ON CITRUS FRUITS BY GREEN MOLD AND BLUE MOLD

Citrus fruits are essential for preventing various health conditions, including diabetes, neurological diseases, and cancer. Among the postharvest diseases affecting citrus fruits, Penicillium digitatum and Penicillium italicum are particularly significant. This study aimed to characterize Penicillium spp. isolated from three citrus varieties: orange (Citrus sinensis), small orange, and Malta. Pathogenicity tests confirmed that these Penicillium isolates were capable of infecting the tested citrus fruits. For molecular characterization, PCR amplification of the D1/D2 domain of 26S rDNA was performed using universal primers, which target the conserved regions of the nucleotide sequence. The PCR products were inserted into the pGEM-T Easy vector and transformed into E. coli Dh5α. The presence of the D1/D2 domain was verified by endonuclease digestion with EcoR1. Sequencing was conducted using the T7 promoter primer, and the resulting DNA sequences were analyzed with the DNAMAN analysis system. Sequence analysis revealed that the D1/D2 region of 26S rDNA from the orange isolate showed 99% similarity with Penicillium sp., while the D1/D2 region from the small orange isolate had 99.84% similarity with P. digitatum strain CBS 112082. The D1/D2 region from the Malta isolate showed 100% similarity with P. digitatum. Multiple sequence alignments among the three Penicillium isolates revealed a 98.81% identity. This study highlights the use of molecular techniques for understanding the pathogenicity of Penicillium spp. in citrus fruits.

Vehicle trajectory prediction method integrating spatiotemporal relationships with hybrid time-step scene interaction

In vehicle trajectory prediction, constructing the interactive relationships among vehicles within the traffic environment poses a significant challenge. Existing models predominantly focus on temporal dependencies within vehicle histories and spatial correlations among neighboring vehicles, overlooking the continuous influence of historical vehicle states on the current time step and the interplay of multiple sequences over time. To address these limitations, we propose a method for multimodal vehicle trajectory prediction that integrates Hybrid Time-step Scene Interaction (HTSI) into the spatiotemporal relationships. Firstly, we introduce the HTSI module, comprising Multi-step Temporal Information Aggregation (MTIA) and Single-step Temporal Information Aggregation (STIA) methods. MTIA utilizes multi-head attention mechanisms to capture temporal dependencies between consecutive frames, thereby generating new time series amalgamating the ongoing influence of historical time states on the current timestamp. Simultaneously, STIA employs multi-head attention mechanisms to capture the spatial dimension weights of multiple time series and, by aggregating spatial interaction features at each timestamp, generates new time series fused with spatial interaction influences. Subsequently, feature extraction is performed through LSTM layers. Moreover, we propose an improved DIPM pooling module, improving the model’s long-term prediction capability by selectively reusing historical hidden states. Ultimately, based on training results from the HighD and NGSIM datasets, our model demonstrates significant advantages in long-term prediction compared to other state-of-the-art trajectory prediction models. Specifically, within the 5 s prediction window, the model achieved a root mean square error (RMSE) of 2.79 m on the NGSIM dataset, representing a 33.62% improvement over the baseline model’s average accuracy. Additionally, on the HighD dataset, the model attained an RMSE of 2.16 m, reflecting a 33.43% enhancement. The crucial code can be obtained from the provided link: https://github.com/gyhhq/Prediction-trajectory .

Fluorescent In Situ Hybridization Chain Reaction for RNA in the Drosophila Embryonic and Larval Central Nervous System.

In the Drosophila nerve cord, much is known about the generation of neurons from neuronal stem cells. Over the lifetime of a neuron, the cumulative expression of genes within that neuron determines its fate. Furthermore, gene expression in mature neurons determines their functional characteristics. It is therefore useful to visualize neural gene expression, which is often done via staining with antibodies to a protein of interest. In cases where there is no antibody to a desired gene product, or when it is useful to detect RNA rather than protein products, fluorescent in situ hybridization chain reaction for RNA (HCR RNA-FISH, or HCR for this protocol) can be used to detect and quantify RNA expression. RNA molecules reside predominantly in the cell soma, so HCR can facilitate determining neuron identity because somata position within the nerve cord is stereotyped across animals. HCR provides high-amplitude, high-fidelity signals. In principle, HCR can be broken down into a detection/hybridization stage and an amplification stage. During detection/hybridization, a probe set hybridizes to multiple sequences within a target gene. In the amplification step, concatemerized fluorescent hairpins bind to the hybridized probes. This two-step process increases the specificity of the fluorescent signal and helps reduce the likelihood of background fluorescence compared to traditional in situ hybridization techniques where the hybridizing probe itself contains the fluorescent signal. Here, we describe a protocol for using HCR to study gene expression in the Drosophila embryonic and larval nerve cord. We also describe how to combine HCR with immunofluorescence staining.

Guidelines in Designing a Universal Primer Mixture to Probe and Quantify Antibiotic-Resistant Genes Using the Polymerase Chain Reaction (PCR).

Multidrug resistance efflux pumps (MDREPs) in biofilm communities have become an increasingly expensive problem in clinical settings. Polymerase chain reaction (PCR)-based detection can be used to diagnose and characterize these genes, but this requires effective primer design to minimize false positives and negatives in test conclusions. A universal primer approach has previously been used to detect conserved core genes but not for accessory genes such as MDREPs. This study describes a guideline for the design of primers used in the detection of MDREP genes and an optimization approach for creating primers by using multiple sequence alignments to target conserved regions in silico, progressing from in silico to in vitro to generate working primers. Using thisapproach, this paper was able to generate primers to target sugE, a small multidrug resistance (SMR) protein found in microbial species. Primers were tested positively against synthetic DNA sequences but were inconsistent with DNA extracted from the organism of interest. Primer design informs the shortfalls of this detection technique and the difficulty in characterizing such genomic elements.

Chloroplast Genome Variation and Phylogenetic Relationships of Autochthonous Varieties of Vitis vinifera from the Don Valley.

The autochthonous grape varieties of the Don Valley, situated in southern Russia, constitute a distinctive element of regional cultural heritage. These varieties have been adapted over centuries to the region's specific local climatic and soil conditions. For the most part, these varieties are not imported from other countries. They are closely related to varieties found in Crimea and the North Caucasus. In this study, we obtained the first complete, unfragmented sequences of the chloroplast genomes of eight autochthonous varieties from the Don Valley and one from Crimea. We also performed a comparative analysis of their genomic features. The size of Vitis vinifera chloroplast genome sequences varied from 160,925 to 160,991 bp, depending on the cultivar, with a uniform GC ratio of 37.38%. Each genome consists of four subregions: a single copy region (LSC) ranging from 89,158 to 89,336 bp, a small single copy region (SSC) ranging from 19,070 to 19,073 bp, and a pair of inverted repeat regions (IRa and IRb) in the range of 26,292 to 26,353 bp. The chloroplast genomes of the studied V. vinifera varieties contained 130 genes, including 85 protein-coding genes, 8 rRNA genes, and 37 tRNA genes. The sequence divergence analysis has enabled the identification of four highly variable regions, which may be utilized as potential markers for phylogenetic analysis. The analysis revealed the presence of 58 to 61 SSRs and multiple long repeated sequences in the chloroplast genomes of these varieties. The phylogenetic analyses of the sequences obtained and complete chloroplast genomes available from public databases indicated that the majority of autochthonous V. vinifera varieties do not have a direct origin from any European variety.

Phylogenetic analysis and morphological characteristics of laccate Ganoderma specimens in Finland

ABSTRACT The Ganoderma lucidum complex includes fungi with similar morphologies but which are thought to represent different species. The lack of available type material and associated absence of multiple locus sequence data has complicated identification of these fungi. The aim of this study was to clarify the identity of the laccate Ganoderma species occurring in Finland by inferring a phylogeny using DNA sequences from available boreal-temperate material. DNA from Finnish isolates together with an older G. lucidum isolate originating from the United Kingdom was sequenced, and the morphological features of the Finnish specimens were examined. The phylogenetic analysis of the internal transcribed spacer region (ITS), the elongation factor 1-α (tef1), RNA polymerase II subunit (rpb2), and partial β-tubulin (β-tub) genes revealed that the G. lucidum isolate from the United Kingdom did not fall within a well-supported clade with other G. lucidum sequences or related species. The Finnish isolates were closely related to the G. tsugae lineage in tef1, rpb2, and β-tub phylogenies. However, G. tsugae appears morphologically distinct from the Finnish material. The results suggest that G. tsugae, or a species phylogenetically closely related to it, may occur in Finland. But further investigation into the relationship between G. tsugae and G. lucidum from Europe will be needed to clarify the identity of the laccate Ganoderma species in Finland.

Unraveling barley's PAL gene family: a genome-wide study on defense mechanisms against Puccinia graminis f. sp. tritici

Phenylalanine ammonia lyase (PAL) is a pivotal enzyme bridging primary and secondary phenylpropanoid metabolism, influencing plant growth, development, and stress responses. Despite extensive studies on PAL genes across various plant species, their investigation in barley, a critical staple food globally, has been relatively scarce. In this study, we have successfully identified 10 HvPAL genes, designated as HvPAL genes, in Hordeum vulgare (barley). These HvPAL genes were categorized based on their conserved sequences, which revealed patterns through MEME analysis and multiple sequence alignment. Interestingly, we found cis elements related to stress in the promoter regions of HvPAL genes, indicating their involvement in the response to pathogens. Furthermore, these gene promoters contained components associated with light, development, and hormone responsiveness. This suggests that they may play a role in hormonal developmental processes. MicroRNAs were also identified as regulators of the HvPAL genes we identified highlighting their significance in barley. To further investigate these gene expression patterns, we analyzed the RNA-seq data revealed the upregulating of HvPAL 2, HvPAL3, and HvPAL8, and downregulating HvPAL 5, HvPAL 6, and HvPAL9 genes in this study. This study focused on the regulation of PAL genes in response to 23 different races of Puccinia graminis f. sp. tritici in barley. These results suggest ways to improve traits and develop barley varieties that are resistant to pathogens by selectively increasing the expression of certain HvPAL genes that were not previously regulated. This thorough investigation aims to expand our knowledge of the versatility of the PAL gene family, providing insights for advancements in host -pathogen genetics.

Research on multi-sequence MR image synthesis CT algorithm based on unsupervised method

BackgroundUsing multi-sequence MRI to synthesize CT, combining different MRI sequences, and processing synthetic CT (sCT) based on unsupervised algorithms, to conduct more in-depth and comprehensive research on MR-based radiotherapy planning. MethodsFirst, we compare and analyze the effects of single-sequence MRI synthesis to CT to determine the quality of the synthesis and find a set of optimal sequence combinations. In addition, the study introduces a Staged Multi-Sequence Fusion Network (SMSF-Net) that employs an unsupervised approach to effectively fuse information from multiple MRI sequences. The staged sequence fusion module can efficiently extract information from different sequences and combine complementary information from different sequences. Finally, image encoding is processed using a multi-layer convolutional network to generate synthetic CT images. ResultsIn this study, the mean absolute error (MAE), root mean square error (RMSE), peak signal-to-noise ratio (PSNR), and structural similarity (SSIM) of the SMSF-Net are 18.85, 84.62, 34.31, and 0.962, which are superior to other single-sequence models and multi-sequence models. Experiments show that the improvement of SMSF-Net is statistically significant. In the qualitative comparison experiment, sCT synthesized by SMSF-Net is superior to other models in detail in the synthesis of complex areas of bones and nasal cavities. ConclusionThis study proposes a Staged SMSF-Net. As a multi-sequence MRI synthesis sCT algorithm based on unsupervised learning, SMSF-Net can use the complementary information between multiple sequences to improve the quality of synthetic sCT. This study has reference value for MRI-only radiotherapy planning.

Blocking uncertain mispriming errors of PCR

The polymerase chain reaction (PCR) plays a central role in genetic engineering and is routinely used in various applications, from biological and medical research to the diagnosis of viral infections. PCR is an extremely sensitive method for detecting target DNA sequences, but it is substantially error prone. In particular, the mishybridization of primers to contaminating sequences can result in false positives for virus tests. The blocker method, also called the clamping method, has been developed to suppress mishybridization errors. However, its application is limited by the requirement that the contaminating template sequence be known in advance. Here, we demonstrate that a mixture of multiple blocker sequences effectively suppresses the amplification of contaminating sequences even in the presence of uncertainty. The blocking effect was characterized by a simple model validated by experiments. Furthermore, the modeling allowed us to minimize the errors by optimizing the blocker concentrations. The results highlighted an inherent robustness of the blocker method in that fine-tuning the blocker concentrations is not necessary. Our method extends the applicability of PCR and other hybridization-based techniques, including genome editing, RNA interference, and DNA nanotechnology, by improving their fidelity.

Genome-wide identification of oxidosqualene cyclase genes regulating natural rubber in Taraxacum kok-saghyz.

Nine TkOSC genes have been identified by genome-wide screening. Among them, TkOSC4-6 might be more crucial for natural rubber biosynthesis in Taraxacum kok-saghyz roots. Taraxacum kok-saghyz Rodin (TKS) roots contain large amounts of natural rubber, inulin, and valuable metabolites. Oxidosqualene cyclase (OSC) is a key member for regulating natural rubber biosynthesis (NRB) via the triterpenoid biosynthesis pathway. To explore the functions of OSC on natural rubber producing in TKS, its gene family members were identified in TKS genome via genome-wide screening. Nine TkOSCs were identified, which were mainly distributed in the cytoplasm. Their family genes experienced a neutral selection during the evolution process. Overall sequence homology analysis OSC proteins revealed 80.23% similarity, indicating a highly degree of conservation. Pairwise comparisons showed a multiple sequence similarity ranging from 57% to 100%. Protein interaction prediction revealed that TkOSCs may interact with baruol synthase, sterol 1,4-demethylase, lupeol synthase and squalene epoxidase. Phylogenetic analysis showed that OSC family proteins belong to two branches. TkOSC promoter regions contain cis-acting elements related to plant growth, stress response, hormones response and light response. Protein accumulation analysis demonstrated that TkOSC4, TkOSC5 and TkOSC6 proteins had strong expression levels in the root, latex and plumular axis. Comparison of gene expression patterns showed TkOSC1, TkOSC4, TkOSC5, TkOSC6, TkOSC7, TkOSC8 and TkOSC9 might be important in regulating NRB. Combination of gene and protein results revealed TkOSC4-6 might be more crucial, and the data might contribute to a more profound understanding of the roles of OSCs for NRB in TKS roots.

AARS Online: A collaborative database on the structure, function, and evolution of the aminoacyl-tRNA synthetases.

The aminoacyl-tRNA synthetases (aaRS) are a large group of enzymes that implement the genetic code in all known biological systems. They attach amino acids to their cognate tRNAs, moonlight in various translational and non-translational activities beyond aminoacylation, and are linked to many genetic disorders. The aaRS have a subtle ontology characterized by structural and functional idiosyncrasies that vary from organism to organism, and protein to protein. Across the tree of life, the 22 coded amino acids are handled by 16 evolutionary families of Class I aaRS and 21 families of Class II aaRS. We introduce AARS Online, an interactive Wikipedia-like tool curated by an international consortium of field experts. This platform systematizes existing knowledge about the aaRS by showcasing a taxonomically diverse selection of aaRS sequences and structures. Through its graphical user interface, AARS Online facilitates a seamless exploration between protein sequence and structure, providing a friendly introduction to the material for non-experts and a useful resource for experts. Curated multiple sequence alignments can be extracted for downstream analyses. Accessible at www.aars.online, AARS Online is a free resource to delve into the world of the aaRS.

Magnetic resonance imaging of the dromedary camel carpus

BackgroundThe dromedary camel (Camelus dromedarius) carpal joint presents multiple joints and constitutes several bones and soft tissues. Radiography and/or ultrasonography of the carpus are challenging due to structural superimposition. High-field magnetic resonance imaging (MRI) technique precludes superimposed tissues and offers high soft tissue contrast in multiple sequences and planes. Hence, understanding the normal MRI anatomy is crucial during clinical investigations. Magnetic resonance imaging is highly sensitive for investigation of soft tissues and articular cartilage; therefore, it is extensively used for outlining joint anatomy and evaluation of a wide range of musculoskeletal conditions. MRI images of a specific anatomical region acquired by using multiple sequences in various planes are necessary for a complete MRI examination. Given the dearth of information on the MRI features of the dromedary camel carpus, the current study demonstrates the MRI appearance of the clinically significant structures in the camel carpus in various sequences and planes using a high-field 1.5 Tesla superconducting magnet. For this purpose, twelve cadaveric forelimbs, obtained from 6 clinically sound lameness free adult dromedary camels, were examined.ResultsThe cortex and medulla of the radius, carpal bones and metacarpus were evaluated. Articular cartilage of the carpal joints was depicted and showed intermediate intensity. Carpal tendons expressed lower signal intensity in all pulse sequences. The collateral and inter-carpal ligaments showed mixed signal intensity.ConclusionsThe obtained data outlines the validation of MRI for investigation of the camel carpus and could set as a reference for interpretation in clinical patients.

Engineering of Substrate-Binding Domain to Improve Catalytic Activity of Chondroitin B Lyase with Semi-Rational Design.

Dermatan sulfate and chondroitin sulfate are dietary supplements that can be utilized as prophylactics against thrombus formation. Low-molecular-weight dermatan sulfate (LMWDS) is particularly advantageous due to its high absorbability. The enzymatic synthesis of low-molecular-weight dermatan sulfates (LMWDSs) using chondroitin B lyase is a sustainable and environmentally friendly approach to manufacturing. However, the industrial application of chondroitin B lyases is severely hampered by their low catalytic activity. To improve the activity, a semi-rational design strategy of engineering the substrate-binding domain of chondroitin B lyase was performed based on the structure. The binding domain was subjected to screening of critical residues for modification using multiple sequence alignments and molecular docking. A total of thirteen single-point mutants were constructed and analyzed to assess their catalytic characteristics. Out of these, S90T, N103C, H134Y, and R159K exhibited noteworthy enhancements in activity. This study also examined combinatorial mutagenesis and found that the mutant H134Y/R159K exhibited a substantially enhanced catalytic activity of 1266.74 U/mg, which was 3.21-fold that of the wild-type one. Molecular docking revealed that the enhanced activity of the mutant could be attributed to the formation of new hydrogen bonds and hydrophobic interactions with the substrate as well as neighbor residues. The highly active mutant would benefit the utilization of chondroitin B lyase in pharmaceuticals and functional foods.

K3PO4–Mediated Synthesis of Chromeno–[3,4‐c]isoxazole from 3–Nitro–2H–chromene and α–Chloro Aldehyde via Michael Addition/C−Cl Bond Cleavage/Deformylation as Key Fragmentation Sequences

AbstractHere we report a transition metal– and oxidant–free annulation cascade method for the synthesis of chromeno–isoxazoles. The annulation reaction involves easily accessible starting materials, 3‐nitro‐2H‐chromene and α‐chloro aldehyde, yielding the structurally intriguing framework, chromeno[3,4‐c]isoxazoles employed by tripotassium phosphate as a base. The isoxazole ring construction over the 2H–chromene unit is believed to occur via a series of multiple reaction sequences. Here, the enolate generation/Michael addition/C−Cl bond cleavage/oxy–anionic addition to reactive tethered‐aldehyde/deformylation via retro–C−C and N−O bond cleavages are the most possible sequences which furnish the desired end–product in good chemical yields. The multiple reaction sequences occur in one‐pot at mild conditions, supported by experimental results and a few control experiments including DFT studies. This process exhibits a broad substrates scope illustrating good functional group compatibility towards both the nitrochromenes and aldehydes, and thus prove to be a general and efficient protocol for the synthesis of chromeno[3,4–c]isoxazoles. Overall, the use of nitrochromene as the limiting reagent, noteworthy functional group tolerance, scalability including great applications like chiral pool strategy towards the synthesis of enantioenriched chromeno‐isoxazole are the additional features.

Unveiling the genetic landscape of infectious laryngotracheitis virus in Switzerland: Evidence for vaccine-like and wild-type strains

Infectious laryngotracheitis (ILT) is a respiratory disease affecting chickens worldwide. Unlike many countries, Switzerland does not vaccinate against ILT. This study analysed ILT samples from 21 natural outbreaks in Switzerland using restriction fragment length polymorphism (RFLP) and multiple gene sequencing. Chicken embryo origin (CEO) and tissue culture origin (TCO) vaccine strains were included as references. Both vaccine strains were distinguishable, and 14 out of 21 samples resembled the CEO vaccine. Additionally, four distinct non-vaccine-like groups were identified. Sequencing of three genes from selected Swiss samples and those from neighbouring countries revealed four phylogenetic clades. Notably, four Swiss field strains formed two unique clades, not closely related to vaccine strains or ILTV from neighbouring countries. Overall, RFLP results were supported by sequencing data. This study demonstrates the presence of both vaccine-like and wild-type ILT viruses in Switzerland, where vaccination is de facto prohibited.

A putative scenario of how de novo protein-coding genes originate in the Saccharomyces cerevisiae lineage

BackgroundNovel protein-coding genes were considered to be born by re-organization of pre-existing genes, such as gene duplication and gene fusion. However, recent progress of genome research revealed that more protein-coding genes than expected were born de novo, that is, gene origination by accumulating mutations in non-genic DNA sequences. Nonetheless, the in-depth process (scenario) for de novo origination is not well understood.ResultsWe have conceived bioinformatic analysis for sketching a scenario for de novo origination of protein-coding genes. For each de novo protein-coding gene, we firstly identified an edge of a given phylogenetic tree where the gene was born based on parsimony. Then, from a multiple sequence alignment of the de novo gene and its orthologous regions, we constructed ancestral DNA sequences of the gene corresponding to both end nodes of the edge. We finally revealed statistical features observed in evolution between the two ancestral sequences. In the analysis of the Saccharomyces cerevisiae lineage, we have successfully sketched a putative scenario for de novo origination of protein-coding genes. (1) In the beginning was GC-rich genome regions. (2) Neutral mutations were accumulated in the regions. (3) ORFs were extended/combined, and then (4) translation signature (Kozak consensus sequence) was recruited. Interestingly, as the scenario progresses from (2) to (4), the specificity of mutations increases.ConclusionTo the best of our knowledge, this is the first report outlining a scenario of de novo origination of protein-coding genes. Our bioinformatic analysis can capture events that occur during a short evolutionary time by directly observing the evolution of the ancestral sequences from non-genic to genic. This property is suitable for the analysis of fast evolving de novo genes.