Sequence Alignment Research Articles

Advanced Bioluminescence Reporter with Engineered Gaussia Luciferase via Sequence-Guided Mutagenesis

Gaussia luciferase (GLuc) is the preeminent secreted luciferase widely used in cell-based reporter assays. By employing sequence-guided mutagenesis informed by alignments of diverse copepod luciferase sequences, we identified key amino acids that significantly enhance bioluminescence (BL) intensity. Among the mutated proteins expressed in bacteria, five individual mutations (M60L, K88Q, F89Y, I90L, or S103T) independently increased BL intensity by 1.8 to 7.5-fold compared to wild-type GLuc in the presence of coelenterazine substrates. Remarkably, the combination of all five mutations in GLuc (designated as GLuc5) resulted in an unexpected 29-fold enhancement in BL intensity. Subsequent evaluation of the GLuc5-secreted reporter in transfected mammalian cells confirmed its superior BL performance across multiple cell lines. These findings suggest that the mutated residues are likely crucial for enhancing BL intensity in GLuc, supporting its potential to serve as a highly sensitive biosensor or reporter for a wide range of biological applications.

Molecular characterization, spatio-temporal expression patterns of crtc2 gene and its immune roles in yellow catfish (Pelteobagrus fulvidraco)

cAMP response element binding (CREB) protein 2 (CRTC2) is a transcriptional coactivator of CREB and plays an important role in the immune system. Thus far, the physiological roles of Crtc2 in teleost are still poorly understood. In this study, the crtc2 gene was identified and characterized from yellow catfish (Pelteobagrus fulvidraco; therefore, the gene is termed as pfcrtc2), and its evolutionary and molecular characteristics as well as potential immunity-related roles were investigated. Our results showed that the open reading frame of pfcrtc2 was 2,346 bp in length, encoding a protein with 781 amino acids. Gene structure analysis revealed its existence of 14 exons and 13 introns. A phylogenetic analysis proved that the tree of crtc2 was clustered into five groups, exhibiting a similar evolutionary topology with species evolution. Multiple protein sequence alignment demonstrated high conservation of the crtc2 in various vertebrates with similar structure. Syntenic and gene structural comparisons further established that crtc2 was highly conserved, implying its similar roles in diverse vertebrates. Tissue distribution pattern detected by quantitative real-time PCR showed that the pfcrtc2 gene was almost expressed in all detected tissues except for eyes, with the highest expression levels in the gonad, indicating that Crtc2 may play important roles in various tissues. In addition, pfcrtc2 was transcribed at all developmental stages in yellow catfish, showing the highest expression levels at 12 h after fertilization. Finally, the transcriptional profiles of crtc2 were significantly increased in yellow catfishes injected with Aeromonas hydrophila or Poly I:C, which shared a consistent change pattern with four immune-related genes including IL-17A, IL-10, MAPKp38, and NF-κBp65, suggesting pfCrtc2 may play critical roles in preventing both exogenous bacteria and virus invasion. Anyway, our findings lay a solid foundation for further studies on the functions of pfcrtc2, and provide novel genetic loci for developing new strategies to control disease outbreak in teleost.

Establishment of the auxin inducible degron system for Babesia duncani: a conditional knockdown tool to study precise protein regulation in Babesia spp.

BackgroundBabesia duncani is a pathogen within the phylum Apicomplexa that causes human babesiosis. It poses a significant threat to public health, as it can be transmitted not only through tick bites but also via blood transfusion. Consequently, an understanding of the gene functions of this pathogen is necessary for the development of drugs and vaccines. However, the absence of conditional gene knockdown tools has hindered the research on this pathogen. The auxin-inducible degron (AID) system is a rapid, reversible conditional knockdown system widely used in gene function studies. Thus, there is an urgent need to establish the AID system in B. duncani to study essential gene functions.MethodsThe endogenous genes of the Skp1-Cullin-F-box (SCF) complex in B. duncani were identified and confirmed through multiple sequence alignment and conserved domain analysis. The expression of the F-box protein TIR1 from Oryza sativa (OsTIR1) was achieved by constructing a transgenic parasite strain using a homologous recombination strategy. Polymerase chain reaction (PCR), western blot, and indirect immunofluorescence assay (IFA) were used to confirm the correct monoclonal parasite strain. The degradation of enhanced green fluorescent protein (eGFP) tagged with an AID degron was detected through western blot and live-cell fluorescence microscopy after treatment of indole-3-acetic acid (IAA).ResultsIn this study, Skp1, Cul1, and Rbx1 of the SCF complex in B. duncani were identified through sequence alignment and domain analysis. A pure BdTIR1 strain with expression of the OsTIR1 gene was constructed through homologous recombination and confirmed. This strain showed no significant differences from the wild type (WT) in terms of growth rate and proportions of different parasite forms. The eGFP tagged with an AID degron was successfully induced for degradation using 500 μM IAA. Grayscale analysis of western blot indicated a 61.3% reduction in eGFP expression levels, while fluorescence intensity analysis showed a 77.5% decrease in fluorescence intensity. Increasing the IAA concentration to 2 mM accelerated eGFP degradation and enhanced the extent of degradation.ConclusionsThis study demonstrated the functionality of the AID system in regulating protein levels by inducing rapid degradation of eGFP using IAA, providing an important research tool for studying essential gene functions related to invasion, egress, and virulence of B. duncani. Moreover, it also offers a construction strategy for apicomplexan parasites that have not developed an AID system.Graphical

Floral Developmental Morphology and Biochemical Characteristics of Male Sterile Mutants of Lagerstroemia indica

Male sterility is a common phenomenon in higher plants and often plays an important role in the selection of superior offspring. ‘Xiang Yun’ is a mutant of Lagerstroemia indica that does not bear fruit after flowering, and its flowering period is significantly longer than that of normal L. indica. To explore the timing and molecular mechanisms of sterility in ‘Xiang Yun’, this study determined the period of sterility through anatomical observation and compared the content of nutrients and the activity of antioxidative enzymes at different stages of flower development. Finally, sequence alignment and qPCR were used to analyze the differences in pollen development genes between ‘Xiang Yun’ and ‘Hong Ye’. The results showed that the anthers of ‘Xiang Yun’ dispersed pollen normally, but the pollen grains could not germinate normally. Observations with scanning electron microscopy revealed that the pollen grains were uneven in size and shriveled in shape. Further observation of anther sections found that abnormal development of the microspores began at the S2 stage, with the callose wall between microspores of ‘Xiang Yun’ being thicker than that of ‘Hong Ye’. In addition, during the flower development of ‘Xiang Yun’, the contents of soluble sugar, soluble protein, free proline, and triglycerides were deficient to varying degrees, and the activities of POD, SOD, and MDA were lower. Sequence alignment and qPCR showed that there were several mutations in EFD1, TPD1, and DEX1 of ‘Xiang Yun’ compared with ‘Hong Ye’, and the expression levels of these genes were abnormally elevated in the later stages of development. Our results clarified the timing and phenotype of male sterility in ‘Xiang Yun’. This provides solid and valuable information for further research on the molecular mechanism of sterility in ‘Xiang Yun’ and the genetic breeding of crape myrtle.

Molecular Cloning, Characterization, and Application of a Novel Multifunctional Isoamylase (MIsA) from Myxococcus sp. Strain V11

A novel multifunctional isoamylase, MIsA from Myxococcus sp. strain V11, was expressed in Escherichia coli BL21(DE3). Sequence alignment revealed that MIsA is a typical isoamylase that belongs to glycoside hydrolase family 13 (GH 13). MIsA can hydrolyze the α-1,6-branches of amylopectin and pullulan, as well as the α-1,4-glucosidic bond in amylose. Additionally, MIsA demonstrates 4-α-D-glucan transferase activity, enabling the transfer of α-1,4-glucan oligosaccharides between molecules, particularly with linear maltooligosaccharides. The Km, Kcat, and Vmax values of the MIsA for amylopectin were 1.22 mM, 40.42 µmol·min–1·mg–1, and 4046.31 mM·min–1. The yields of amylopectin and amylose hydrolyzed into oligosaccharides were 10.16% and 11.70%, respectively. The hydrolysis efficiencies were 55%, 35%, and 30% for amylopectin, soluble starch, and amylose, respectively. In the composite enzyme hydrolysis of amylose, the yield of maltotetraose increased by 1.81-fold and 2.73-fold compared with that of MIsA and MTHase (MCK8499120) alone, respectively.

PhyKIT: A Multitool for Phylogenomics.

Multiple sequence alignments and phylogenetic trees are rich in biological information and are fundamental to research in biology. PhyKIT is a tool for processing and analyzing the information content of multiple sequence alignments and phylogenetic trees. Here, we describe how to use PhyKIT for diverse analyses, including (i) constructing a phylogenomic supermatrix, (ii) detecting errors in orthology inference, (iii) quantifying biases in phylogenomic data sets, (iv) identifying radiation events or lack of resolution using gene support frequencies, and (v) conducting evolution-based screens to facilitate gene function prediction. Several PhyKIT functions that streamline multiple sequence alignment and phylogenetic processing-such as renaming FASTA entries or tree tips-are also discussed. These protocols demonstrate how simple command-line operations in the unified framework of PhyKIT facilitate diverse phylogenomic data analysis and processing, from supermatrix construction and diagnosis to gaining clues about gene function. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Installing PhyKIT and syntax for usage Basic Protocol 2: Constructing a phylogenomic supermatrix Basic Protocol 3: Detecting anomalies in orthology relationships Basic Protocol 4: Quantifying biases in phylogenomic data matrices and related measures Basic Protocol 5: Identifying polytomies Basic Protocol 6: Assessing gene-gene coevolution as a genetic screen.

Unraveling antibiotic resistance in Achromobacter mucicolens IA strain: genomic insights, structural analysis, and prospects for targeted therapeutics

ABSTRACT The mortality rate of infectious diseases caused by Achromobacter mucicolens is increasing. The enhanced antibiotic resistance among bacterial species through genetic transfer and mutations in the efflux mediating genes has made the treatment quite challenging. A. mucicolens is an aerobic, gram-negative, and non-fermenting opportunistic pathogen found in immunocompromised patients. A. mucicolens shows resistance against beta-lactams and other antibiotics through intrinsic resistance mechanisms, including multi-drug efflux pumps and beta-lactamases. In this study, the clinical isolate whole genome sequencing of A. mucicolens data was analyzed to identify the genes and mutations responsible for antimicrobial resistance. The identified genes and their mutants were then subjected to structural analysis to better understand the impact of mutations on the protein structure, and domain analysis was performed to investigate the role of domains in antibiotic resistance. A total of 4 genes, acrR, macB, msbA, and tolC , were identified with significant mutations, whereas macB was shortlisted for further analysis based on the conserved regions, sequence alignment, and the maximum number of mutations. All the mutants of the macB gene contain the two common domains, the ABC transporter-like ATP-binding domain and the AAA + ATPase domain. These domains are crucial in efflux mediating drug transport and can be targeted to design novel drugs for treating infections caused by A. mucicolens . IMPORTANCE Achromobacter species represent a significant threat as opportunistic pathogens, particularly in healthcare settings. Their resilience to antibiotics, demonstrated by strains like A. mucicolens , poses a serious challenge in treating infections, especially in immunocompromised patients. This study emphasizes the critical need for heightened vigilance among healthcare professionals regarding Achromobacter infections. By analyzing the whole genome sequencing data of A. mucicolens , the study sheds light on the genetic basis of antimicrobial resistance, aiding in more targeted treatment strategies. Furthermore, structural and domain analyses offer insights into how mutations impact protein structure and function, crucial for developing effective interventions. Ultimately, implementing rigorous sanitation measures and antibiotic stewardship protocols is needed to mitigate the spread of Achromobacter and safeguard vulnerable patient populations.

Cry1Ac toxin binding in the velvetbean caterpillar Anticarsia gemmatalis: study of midgut aminopeptidases N

The velvetbean caterpillar Anticarsia gemmatalis is one of the main soybean defoliators in Brazil. Currently, the main biopesticide used to control insect pests worldwide is the bacteria Bacillus thuringiensis (Bt), which produces entomopathogenic Crystal toxins (Cry) that act in the midgut of susceptible insects, leading them to death. The mode of action of Cry toxins in the midgut involves binding to specific receptors present on the brush border of epithelial cells such as aminopeptidase N (APN), alkaline phosphatase (ALP), cadherin, and others. Mutations in these receptors, among other factors, may be involved in the development of resistance; identification of functional Cry receptors in the midgut of A. gemmatalis is crucial to develop effective strategies to overcome this possible scenario. This study’s goal is to characterize APNs of A. gemmatalis and identify a receptor for Cry1Ac in the midgut. The interaction of Bt spores with the midgut epithelium was observed in situ by immunohistochemistry and total aminopeptidase activity was estimated in brush border membrane vesicle (BBMV) samples, presenting higher activity in challenged individuals than in control ones. Ten APN sequences were found in a A. gemmatalis’ transcriptome and subjected to different in silico analysis, such as phylogenetic tree, multiple sequence alignment and identification of signal peptide, activity domains and GPI-anchor signal. BBMV proteins from 5th instar larvae were submitted to a ligand blotting using activated Cry1Ac toxin and a commercial anti-Cry polyclonal antibody; corresponding bands of proteins that showed binding to Cry toxin were excised from the SDS-PAGE gel and subjected to mass spectrometry analysis, which resulted in the identification of seven of those APNs. Quantitative PCR was realized to compare expression levels between individuals subjected to sublethal infection with Bt spores and control ones, presenting up- and downregulations upon Bt infection. From these results, we can infer that aminopeptidases N in A. gemmatalis could be involved in the mode of action of Cry toxins in its larval stage.

Multiple Function Merging for Code Size Reduction

Resource-constrained environments, such as embedded devices, have limited amounts of memory and storage. Practical programming languages such as C++ and Rust tend to output multiple similar functions by monomorphizing polymorphic functions. An optimization technique called Function Merging, which merges similar functions into a single function, has been studied. However, in the state-of-the-art approach, the number of functions that can be merged at once is limited to two; thus, efficiently merging three or more functions, which are often generated from polymorphic functions, has been impossible. In this study, we propose Multiple Function Merging optimization, which targets merging three or more similar functions into a single function using a multiple sequence alignment algorithm. With multiple aligned information, Multiple Function Merging can increase merge opportunities and reduce extra branching overheads at the code generation stage. We evaluated it using the SPEC CPU benchmark suite and some large-scale C/C++ programs, and the results show that it reduces code size by as much as 7.61% compared with the state-of-the-art approach.

Evaluating Sequence Alignment Tools for Antimicrobial Resistance Gene Detection in Assembly Graphs

Antimicrobial resistance (AMR) is an escalating global health threat, often driven by the horizontal gene transfer (HGT) of resistance genes. Detecting AMR genes and understanding their genomic context within bacterial populations is crucial for mitigating the spread of resistance. In this study, we evaluate the performance of three sequence alignment tools—Bandage, SPAligner, and GraphAligner—in identifying AMR gene sequences from assembly and de Bruijn graphs, which are commonly used in microbial genome assembly. Efficiently identifying these genes allows for the detection of neighboring genetic elements and possible HGT events, contributing to a deeper understanding of AMR dissemination. We compare the performance of the tools both qualitatively and quantitatively, analyzing the precision, computational efficiency, and accuracy in detecting AMR-related sequences. Our analysis reveals that Bandage offers the most precise and efficient identification of AMR gene sequences, followed by GraphAligner and SPAligner. The comparison includes evaluating the similarity of paths returned by each tool and measuring output accuracy using a modified edit distance metric. These results highlight Bandage’s potential for contributing to the accurate identification and study of AMR genes in bacterial populations, offering important insights into resistance mechanisms and potential targets for mitigating AMR spread.

Biochemical Characterization of an Arabinoside Monophosphate Specific 5′-Nucleotidase-like Enzyme from Streptomyces antibioticus

To investigate the function of the gene penF in the pentostatin and vidarabine (Ara-A) biosynthetic gene cluster in Streptomyces antibioticus NRRL 3238, PenF was recombinantly expressed and characterized. Enzymatic characterization of the enzyme demonstrated that PenF exhibited metal-dependent nucleoside 5ʹ-monophosphatase activity, showing a substrate preference for arabinose nucleoside 5ʹ-monophosphate over 2ʹ-deoxyribonucleoside 5ʹ-monophosphate and ribonucleoside 5ʹ-monophosphate. Metal ions such as Mg2+ and Mn2+ significantly enhanced enzyme activity, whereas Zn2+, Cu2+, and Ca2+ inhibited it. For vidarabine 5′-monophosphate, the Km and kcat values were determined to be 71.5 μM and 33.9 min−1, respectively. The kcat/Km value was 474.1 mM−1·min−1 for vidarabine 5-monophosphate and was 68-fold higher than that for 2′-deoxyadenosine 5′-monophosphate. Comparative sequence alignment and structural studies suggested that residues outside the primary substrate-binding site are responsible for this substrate specificity. In conclusion, PenF’s activity toward vidarabine 5ʹ-monophosphate likely plays a role in the dephosphorylation of precursors during Ara-A biosynthesis.

Deciphering the 4',7-Dihydroxy-3'-Methylflavone from Boerhavia, Agonist/Antagonist Interactions Against 5-HT2 Receptors using Homology Modeling, Molecular Docking, and Dynamic Simulation Studies.

Seizures, depression, and anxiety are neurological disorders that affected innumerable people worldwide. Recent research has revealed that targeting 5-hydroxytryptamine 2 (5-HT2) receptors can help suppress these conditions. An in-depth literature study has identified that phytocompounds from the Boerhavia genus could reduce seizures. Therefore, homology models of 5-HT2 receptors were generated and validated using techniques such as the alignment of amino acid sequences and the Ramachandran plot. Later, a comparison of modeled structures was made with a non-redundant set of PDB structures. The pharmacokinetics, drug-likeness, blood-brain barrier (BBB) permeability, and Lipinski's rule of five shed light on 22 phytocompounds, which are the potential candidates for molecular docking among 127 Boerhavia's bioactive. Notably, molecular docking analysis revealed 4',7-dihydroxy-3'-methylflavone as the most potent lead compound, which has a strong binding affinity to all modeled receptors. Additionally, with a remarkably high docking score of -9.1 kcal/mol, 4',7-dihydroxy-3'-methylflavone showed promising interactions, particularly with 5-HT2A receptor, as seen from the RMSD, SASA, Rg, and number of hydrogen bonds during 100 ns molecular dynamic (MD) simulation. Principal component analysis (PCA) and Molecular Mechanics-Poisson-Boltzmann Surface Area (MM-PBSA) further confirmed that 4',7-dihydroxy-3'-methylflavone is the best novel phytocompound in Boerhavia genus for 5-HT2 receptor as agonist/antagonist activity against seizures.

Prokaryotic expression, purification, and activity of the inositol polyphosphate 5-phosphatase Gs5PTase8 from wild soybean

Inositol polyphosphate-5-phosphatase (5PTase) is a key enzyme in the inositol signaling pathway. It hydrolyzes the 5-phosphate on the inositol ring of inositol phosphate (IP) or phosphatidylinositol phosphate (PIP). However, there is limited reports on the homologous genes in soybean. This study cloned the salt tolerant gene Gs5PTase8 from wild soybean (Glycine soja S. & Z.) and explored its substrate. Gs5PTase8 encodes 493 amino acid residues. The sequence alignment and phylogenetic tree showed that this gene was conserved in plants. RT-qPCR was employed to determine the expression of Gs5PTase8 in different tissues of soybean and the results showed that Gs5PTase8 was mainly expressed in soybean roots. To investigate the hydrolytic substrates, we constructed pET28a-Gs5PTase8 and pGEX4T1-Gs5PTase8 for the Escherichia coli expression system and only obtained the recombinant protein GST-Gs5PTase8. The induction conditions for the protein expression including the isopropyl beta-d-thiogalactopyranoside (IPTG) concentration and temperature (16 ℃, 30 ℃, and 37 ℃) were optimized. The expression level was highest when the expression was induced overnight with 0.2 mmol/L IPTG at 16 ℃. The SDS-PAGE results showed that the recombinant protein had a relative molecular weight of 75 kDa and presented a single band after purification, with the purity reaching over 95%. The yield of the recombinant protein determined by the BCA method was 4.9 mg/L LB. The hydrolytic substrates of this enzyme in vitro included IP3 [inositol(1, 4, 5)trisphosphate], IP4 [inositol(1, 3, 4, 5)tetrakisphosphate], PI(4, 5)P2 [phosphatidylinositol(4, 5) bisphosphate] and PI(3, 4, 5)P3 [phosphatidylinositol(3, 4, 5)trisphosphate]. This study provides a scientific basis for further research on the molecular mechanism of Gs5PTase8 involved in salt tolerance.

Identification and Antibacterial Activity Study of a Terrestrial Strain of Brevibacterium aureus 431

This study isolated and purified strain 431 from an animal probiotic product. Through staining and microscopic examination, colony morphology analysis, biochemical reaction tests, and 16S rDNA sequence alignment, the strain was identified and named Brevibacterium aureus 431. The study focused on the production of biosurfactants by strain 431, and antibacterial activity tests were conducted on the strain and its secondary metabolites. The results showed that strain 431 exhibited no resistance to 10 commonly used drugs, and its concentrated secondary metabolites were highly sensitive to the indicator bacterium Escherichia coli. Oral administration of strain 431 to BALB/c mice resulted in normal mental state, diet, and bowel movements, with no signs of illness or death, indicating that strain 431 is highly safe and non-pathogenic to mice. The study suggests that Brevibacterium aureus 431 has significant research value as a new source of actinomycetes and that its secondary metabolites have potential application value in the development of antibacterial drugs.

First Description of Loreto Virus in Three Culicidae Species from the Atlantic Forest, Bahia, Brazil

Loreto virus (LORV) is an insect-specific virus classified into the proposed taxon Negevirus. It was originally described in Iquitos, Peru, in 1977. Here, we describe three novel LORV genomes obtained from the isolates of three pooled samples of Trichoprosopon digitatum, Aedes (Ochlerotatus) fulvus, and Limatus durhamii collected in Ilhéus—Bahia, 2014. Samples were submitted to RNA sequencing on the Illumina platform to recover the LORV genome. The genomes presented, on average, 81.5% nucleotide identity and 92.6% global amino acid identity with the LORV reference genome (NC_034158). Subsequently, phylogenetic analysis was performed based on a multiple sequence alignment of the concatenated amino acid sequences predicted for the three ORFs of the Negevirus genomes, and the target sequences were clustered within the LORV clade. The taxon Negevirus is in constant expansion of its species content and host range. New data about insect specific negeviruses are important for virus evolution studies, along with those approaching interactions with the hosts and their influence in the transmission of arboviruses. Also, the assessment of these data may allow the development of biologic control strategies for arboviral vectors. This is the original report of the identification of LORV in Brazil, infecting three Culicidae species hosts native to the Atlantic Forest biome.

Diversification of sphingolipid synthase activities in kinetoplastid protozoa

Phosphosphingolipids (PSL) are essential components of eukaryotic membranes. The major PSL in fungi and protists is inositol phosphorylceramide (IPC), while sphingomyelin (SM), and to a lesser extent ethanolamine phosphorylceramide (EPC) predominate in mammals. Most kinetoplastid protozoa have a syntenic locus that encodes a single sphingolipid synthase (SLS) gene. Uniquely, among the kinetoplastids, the salivarian (African) trypanosomes have expanded this locus from a single gene in Trypanosoma vivax (TvSLS) to four genes in T. brucei (TbSLS1-4). We have previously shown that one of these is an IPC synthase, while the others are SM/EPC synthases, and that specificity is controlled by a single signature residue (IPC, serine; SM/EPC, phenylalanine). This residue is serine in T. cruzi and Leishmania major SLSs, both of which are demonstrated IPC synthases. However, T. vivax has a tyrosine at this residue raising the issue of specificity. Using a liposome-supplemented in vitro translation system we now show that T. vivax SLS is an SM/EPC synthase, and that the basal kinetoplastid Bodo saltans SLS is an IPC synthase (serine). We use these data, and a multiple alignment of available sequences, to discuss the evolution of kinetoplastid SLSs and their unique expansion in T. brucei and related salivarian trypanosomes.

Characterization of the Coriolopsis gallica DyP for Its Potential to Biotransform Various Fluoroquinolones

Coriolopsis gallica (Cga) is a white-rot fungus renowned for its ability to secrete ligninolytic enzymes that are capable of oxidizing phenolic compounds. This study aimed to investigate the biochemical characteristics of a dye-decolorizing peroxidase named CgaDyP1 and test its ability to biotransform antibiotics. CgaDyP1 was cloned and heterologously expressed in Escherichia coli. We fully characterized the biochemical properties of CgaDyP1 and evaluated its dye-decolorizing potential to confirm that it belongs to the DyP class of enzymes. We also tested its fluoroquinolone antibiotic biotransformation potential for possible biotechnological applications. Alignment of the primary amino acid sequence with DyP homolog sequences showed that CgaDyP1 has high similarity with other fungal DyPs. The recombinant CgaDyP1 exhibited activity on substrates such as ABTS and 2,6-dimethoxyphenol (DMP) with optimal performance at a pH of 3, although activity at pH 2.5, pH 4, and pH5 diminished over time. Thermostability tests indicated that the enzyme remains stable at temperatures between 30 °C and 50 °C and retains 70% of its initial activity after 180 min at 50 °C. Tests on the effect of hydrogen peroxide on CgaDyP1 activity found peak activity at 0.25 mM H2O2. CgaDyP1 decolorized five industrial dyes, and kinetics data confirmed that it belongs to the DyP class of enzymes. CgaDyP1 was shown to biotransform some of the 7 recalcitrant fluoroquinolone antibiotics tested here, including levofloxacin, moxifloxacin, and norfloxacin, and thus holds potential for biotechnological applications.

Comparative Analysis of Heat Shock Protein Sequences in Crop Species: Unravelling Phylogenetic Relationships Through Multiple Sequence Alignment

Comparative Analysis of Heat Shock Protein Sequences in Crop Species: Unravelling Phylogenetic Relationships Through Multiple Sequence Alignment

BIMSA: Accelerating Long Sequence Alignment Using Processing-In-Memory.

Recent advances in sequencing technologies have stressed the critical role of sequence analysis algorithms and tools in genomics and healthcare research. In particular, sequence alignment is a fundamental building block in many sequence analysis pipelines and is frequently a performance bottleneck both in terms of execution time and memory usage. Classical sequence alignment algorithms are based on dynamic programming and often require quadratic time and memory with respect to the sequence length. As a result, classical sequence alignment algorithms fail to scale with increasing sequence lengths and quickly become memory-bound due to data-movement penalties. Processing-In-Memory (PIM) is an emerging architectural paradigm that seeks to accelerate memory-bound algorithms by bringing computation closer to the data to mitigate data-movement penalties. This work presents BIMSA (Bidirectional In-Memory Sequence Alignment), a PIM design and implementation for the state-of-the-art sequence alignment algorithm BiWFA (Bidirectional Wavefront Alignment), incorporating new hardware-aware optimizations for a production-ready PIM architecture (UPMEM). BIMSA supports aligning sequences up to 100K bases, exceeding the limitations of state-of-the-art PIM implementations. First, BIMSA achieves speedups up to 22.24 × (11.95× on average) compared to state-of-the-art PIM-enabled implementations of sequence alignment algorithms. Second, achieves speedups up to 5.84 × (2.83× on average) compared to the highest-performance multicore CPU implementation of BiWFA. Third, BIMSA exhibits linear scalability with the number of compute units in memory, enabling further performance improvements with upcoming PIM architectures equipped with more compute units and achieving speedups up to 9.56 × (4.7× on average). Code and documentation are publicly available at https://github.com/AlejandroAMarin/BIMSA.

Integrative taxonomic revision of the grasshopper genera Parapetasia Bolívar, 1884, and Loveridgacris Rehn, 1954 (Orthoptera, Pyrgomorphidae), with description of a new species of Loveridgacris

The taxonomic status of the Pyrgomorphid genera Parapetasia Bolívar, 1884, and Loveridgacris Rehn, 1954 is complex and challenging. Here, we use a combination of morphological, distributional, and genetic data to revise the two genera and provide new information on their diversity. We describe a new species, Loveridgacris tectiferussp. nov., from Tanzania and formally resurrect the status of Parapetasia rammei as a valid species within Parapetasia, resulting in two species in Parapetasia (P. femorata and P. rammei) and two in Loveridgacris (L. impotens and L. tectiferussp. nov.). We also sequenced the COI and 16S genes of 10 Pyrgomorphidae species and provided the first phylogeny of the group. Our data show that all species are clearly distinct and represent molecular operational taxonomic units (mOTUs), with the exceptions of L. impotens and L. tectiferussp. nov., which are morphologically clearly distinct but for which the concatenated sequence alignments of the two individual gene datasets (COI and 16S) do not provide sufficient information. In addition, high interspecific distances were found between Parapetasia and Loveridgacris. Moreover, the complete mitogenomes of L. impotens and L. tectiferussp. nov. were sequenced using next-generation sequencing technology. The total lengths of the assembled mitogenomes were 15,592 bp and 15,737 bp, representing 13 protein-coding genes, 22 transfer RNA genes, two ribosomal RNA genes and one D-loop region, respectively. To aid in identification, we present a key for the two genera, including a key to species. This study provides insights into the morphology, distribution, and phylogeny of Pyrgomorphidae in Africa.