Sequence Comparison Research Articles

Molecular Characterization of Native Isolates of Metarhizium rileyi (Farlow) Samson (Ascomycetes: Hypocreales) from Major Cropping Ecosystems of Northeastern Karnataka, India

Aims: In India, commercial biopesticides have been recommended against different insect pests on different crops, but their application has shown only a reduced efficiency attributed to differences in susceptibility of target insect pests or non-adaptability to Indian agro-climatic conditions. Therefore, there is a need to isolate region-specific biopesticide strains to enhance the efficacy of such biopesticides for pest management. The current study aimed to explore the new isolates of the entomopathogenic fungus, Metarhizium rileyi (Farlow) Samson species across the major cropping ecosystems of kalyana Karnataka. M. rileyi is an important entomopathogenic fungus of more than 30 species of Lepidoptera larvae. The aim of this research was to characterize the isolate of M. rileyi from Northeastern Karnataka by using morphological and molecular approaches. Methodology: The fungus was isolated from the naturally infected cadavers collected from different cropping ecosystems and it was cultured on Sabouraud’s Maltose Yeast Extract Agar (SMAY) media for morphological characterization, SMY media used for molecular characterization. The vegetative hyphae of fungi were 2-3 µm in diameter and hyaline, septate. The conidia ellipsoidal, smooth, and pale green were observed under the optical microscope. The DNA was isolated using the CTAB method and an internal transcribed spacer (ITS1, ITS4) was used as the primers for the amplification. The amplified products of 650 bp were purified and sequenced at ClustaL W in both directions using the above primers. A consensus sequence was obtained by alignment of the forward and reverse sequences for this region and deposited in GenBank (UASRBC Mr-24, UASRBC Mr-3, UASRBC Mr-20, UASRBC Mr-29, UASRBC Mr-7, UASRBC Mr-18). Result: A BLAST search of the sequence in GenBank revealed a 50-100 percent identity with several strains of M. rileyi (Eg. JQ686668, MN907775 and KJ725726 etc.,). Conclusion: The phylogenetic relationship was derived from comparisons of the ITS gene sequences and a Dendrogram was constructed. The homology and variations based on amino acid sequences among the isolates showed 71 per cent (OK184897) and 51 per cent similarity with four isolates (OK184899, OK177835, OK178862 and OK184898).

Comprehensive analysis of microbiome biodiversity in popular date palm (Phoenix dactylifera L.) fruit varieties

Due to its nutritional value and health benefits, the date palm (Phoenix dactylifera L.) is an essential dietary food crop throughout Middle Eastern and African countries. Consumers are concerned about the possible microbial contamination of dates, especially since most dates arriving in local markets are unprocessed. The absence of processing increases the possibility of microbial contamination, which raises the probability of microbial contamination. This study aims to analyze and evaluate the variability of fungal and bacterial microbiota identified in the most popular date palm fruits in Saudi Arabia. The study assessed ten date variety fruits from the most popular date palm varieties for consumption in Saudi Arabia and analyzed the microbial count. Morphological and molecular characterization and comparison of nuclear ribosomal DNA internal transcribed spacer (ITS) sequences identified 78 fungi, including 36 distinct species across 15 fungal genera. Alternaria, Fusarium, Curvilaria, Aspergillus, and Penicillium were the most frequent genera among the ten fruit cultivars studied, according to ITS-rDNA sequence analysis. Furthermore, 36 bacterial isolates were obtained from ten date varieties studied, each with a unique colony morphology. These isolates were identified based on sequence alignment and comparison of their 16S rDNA internal spacer regions to those available in public databases. The results showed that the bacterial isolates included 15 species from five bacterial genera. The results suggested that Bacillus, Stenotrophomonas, and Brucella were the prevailing genera among the ten tested fruit varieties. Some bacterial genera, such as Brucella, Achromobacter, and Stenotrophomonas, are well-known potential human pathogens. Chaetomium globosum was also recognized as air pollution causing adverse health effects such as allergies and as the causal agent of human fungal infections among the tested date varieties; the Rashodiah type exhibited the highest fungal contamination, whereas the Sagai variety displayed the lowest fungal contamination. Conversely, the Sukkari, Barhi, and Mejdool varieties were the most contaminated with bacteria among the ten tested varieties, while the Khalas variety showed the least bacterial contamination. To the best of the authors' knowledge, this study provides the initial comprehensive account of the molecular and morphological identification of all fungal and bacterial genera associated with date palm (P. dactylifera) fruits.

Engineering of Phycourobilin Synthase: PubS to a Two-Electron Reductase.

Phycourobilin:ferredoxin oxidoreductase (PubS) belongs to the ferredoxin-dependent bilin reductase (FDBR) family and catalyzes the reduction of the C15=C16 double bond, followed by the C4=C5 double bond of biliverdin IXα to produce phycourobilin. Among the diverse FDBR enzymes that catalyze site-specific reduction reactions of bilins, PubS lineage is the only one that reduces the C4=C5 double bond. This family can be broadly divided into four-electron reduction enzymes, which catalyze two successive two-electron reductions, such as PubS, and two-electron reduction enzymes, which catalyze a single two-electron reduction. The crystal structures of diverse FDBRs, excluding PubS, have unraveled that there are two distinct binding modes in the substrate-binding pocket. In this study, we focused on the arginine (Arg) residues that is considered crucial for substrate-binding mode in two-electron reduction enzymes. Through sequence alignment and comparison with the predicted structure of PubS, we identified a residue in PubS that corresponds to the Arg residue in the two-electron reduction enzymes. We further introduced mutations to avoid the steric hindrance associated with changes in the binding mode. Biochemical characterization of these variants showed that we successfully modified PubS from a four-electron reduction enzyme to a two-electron reduction enzyme with the accumulation of radicals. Our results provide insight into the molecular mechanisms of the chromophore binding mode and proton donation from acidic residues.

A Microcosm Analysis of Species-Specific Responses of Chironomidae on Heavy Metal Pollution in The Nyanza Gulf of Lake Victoria

The Chironomidae family, known as "non-biting midges" in their adult stage and "bloodworms" in their larval stage, consists of diverse dipteran insects inhabiting various global aquatic environments. Despite extensive global research, data on Chironomidae in the polluted Nyanza Gulf of Lake Victoria, Kenya, is scarce, and molecular identification methods have not been explored. This study aimed to quantify heavy metal concentrations in water, sediment, and insect samples and assess their impact on Chironomid species identified using mitochondrial DNA barcoding of the cytochrome oxidase subunit 1 (COI) gene. Analysis of Variance was used to determine if there were any statistically significant differences in heavy metal concentrations across different sample types or locations along the pollution gradient. Chironomids were collected from Nyanza Gulf, focusing on a pollution gradient. Results showed that concentrations of arsenic (As), lead (Pb), and cadmium (Cd) in insect, water, and sediment samples exceeded standard limits, while mercury (Hg) concentrations were within limits. Significant variations (p ≤ 0.05) in Pb levels were observed in water samples, and heavy metal concentrations in sediment samples varied significantly (p ≤ 0.05), with Pb showing the highest variation (p ≤ 0.0001). Insect samples exhibited significant differences (p ≤ 0.0001) in As and Hg contents. Genetic analysis identified two known species: Chironomus transvaalensis at the heavily polluted Kisumu station and Chironomus pseudothummi at the moderately polluted Kendu Bay and Homa Bay stations. Additionally, a unique Chironomus species was found on Ndere Island, a relatively clean site with restricted human activities. Sequence comparisons indicated proximity to global data but also highlighted the evolutionary significance and uniqueness of the identified species. This study demonstrated the potential use of genetic methods in determining Chironomid species diversity, community structure, and abundance in relation to heavy metal concentration. It suggests that heavy metal pollution may act as a selective pressure, driving the evolution of Chironomid species. The study recommends combining genetic approaches with other pollution sources for a comprehensive understanding of using this species in monitoring pollution

Molecular characterization of avian metapneumovirus subtype C associated with hydrosalpinx fluid and egg drop syndrome in Jinding ducks.

A disease called "hydrosalpinx fluid and egg drop syndrome" (HFEDS) was observed in four flocks of Jinding ducks (Anas platyrhynchos domesticus) in Northeast China during the years 2022 to 2023. Here, we investigated the possible involvement of avian metapneumovirus (AMPV) infection. Full-length genome sequencing and sequence analysis of two AMPV strains showed that they belong to Eurasian lineage of AMPV subtype C. Based on surface glycoprotein (G) sequence comparisons, the Eurasian lineage can be divided into two sublineages (E1 and E2), and sublineage E2 is circulating in Jinding duck populations in Northeast China.

Identification and characterization of NHX gene family for their role under salt stress in Vigna mungo.

In the current study, we have performed a comprehensive analysis of the Sodium Hydrogen Exchanger (NHX) gene family in Vigna mungo, and a total of 44 NHX genes were identified. A bimodal distribution based on domains, gene structure and phylogenetic analysis was evident. All intronpoor and intron-rich genes were clustered in clades I and II, respectively. Interestingly, all genes of subclade IIb were localized to vacuoles and possess only the NHX domain. The isoelectric point and trans-membrane domain analysis reflect the wide distribution of the NHX genes. Interestingly, Vm_NHX2 and Vm_NHX3 lacked trans-membrane domain but were found to interact with other NHX genes as well as vital salinity pathway genes, including calcium-mediated salt-responsive genes. The comparison of the mRNA sequences with that of V. marina, a halophytic species, reflects their independent evolution, majorly supporting the convergent evolution. The Ka/Ks ratio reflects the abundance of purifying selection supporting their conserved function during evolution. In our analysis, several abiotic stress and hormone-responsive elements and transcription factor binding sites were present in the promoter of the NHX genes. Further, the ion partitioning of a tolerant (K90) and a susceptible (K49) variety of V. mungo suggested that K90 managed the Na+/K+ ratio more affluently, which was also supported by profiling of superoxide radicals, hydrogen peroxide, phenol, peroxidase activity and superoxide dismutase activity. From the expression, we identified five candidate Vm_NHX genes, four of which, i.e. Vm_NHX16, Vm_NHX17, Vm_NHX29 and Vm_NHX33, were localized to the vacuolar and lysosomal membrane.

Cryptosporidium spp. in German wildlife: Detection, regional occurrence and diversity in wild boar, roe, red and fallow deer

Cryptosporidium is a cause of diarrheal infections responsible for a loss of human and animal welfare worldwide. The impact of the parasite is underestimated and the reported sources of infection are diverse, as it occurs in a wide variety of hosts. Wildlife has been reported as a notifiable source, but few studies are available on its occurrence in European wild boar and cervid species. To determine the occurrence of Cryptosporidium in game in Brandenburg, Germany, a molecular survey was conducted during the 2017 to 2020 hunting seasons. A total of 562 fecal samples from wild boar (Sus scrofa, n = 262), roe deer (Capreolus capreolus, n = 174), red deer (Cervus elaphus, n = 62), fallow deer (Dama dama, n = 51) and 13 samples of unspecified species were analyzed for both 18S ribosomal RNA (18S rRNA) and Cryptosporidium oocyst wall protein (COWP) gene sequence regions. PCR results showed that 21.2 % of the samples (n = 119/562) were positive for at least one target gene (18S rRNA: n = 114; COWP: n = 14), but differences in Cryptosporidium occurrence were observed within species and hunting seasons, with variations ranging from 1.8 % to 41.7 % (roe deer), respectively. Analysis of Sanger sequences of the 18S rRNA and COWP PCR products indicated that the C. sp. deer genotype was predominant in deer (roe deer: 86.7 %, red deer: 66.7 %, fallow deer: 58.8 %), while C. suis and C. scrofarum were mainly detected in wild boar (88.5 %). The human pathogenic species C. parvum was detected in only 1.2 % (n = 7) of the samples analyzed, but without a clear indication of a specific wild animal host. The highest Cryptosporidium diversity was found in wild boar and roe deer with five and four different species, respectively. Comparison of the 18S rRNA sequences with the designated reference revealed minor variations at several nucleotide positions in some isolates, possibly indicating evolutionary adaptations and the development of new subtypes. In conclusion, wildlife represents a reservoir for a diverse spectrum of Cryptosporidium species and may thus contribute to their environmental spread and the transmission to humans.

PQEB1: a hospital outbreak plasmid lineage carrying bla KPC-2.

While conducting genomic surveillance of carbapenemase-producing Enterobacteriaceae (CPE) from patient colonisation and clinical infections at Birmingham's Queen Elizabeth Hospital (QE), we identified an N-type plasmid lineage, pQEB1, carrying several antibiotic resistance genes, including the carbapenemase gene bla KPC-2. The pQEB1 lineage is concerning due to its conferral of multidrug resistance, its host range and apparent transmissibility, and its potential for acquiring further resistance genes. Representatives of pQEB1 were found in three sequence types (STs) of Citrobacter freundii, two STs of Enterobacter cloacae, and three species of Klebsiella. Hosts of pQEB1 were isolated from 11 different patients who stayed in various wards throughout the hospital complex over a 13 month period from January 2023 to February 2024. At present, the only representatives of the pQEB1 lineage in GenBank were carried by an Enterobacter hormaechei isolated from a blood sample at the QE in 2016 and a Klebsiella pneumoniae isolated from a urine sample at University Hospitals Coventry and Warwickshire (UHCW) in May 2023. The UHCW patient had been treated at the QE. Long-read whole-genome sequencing was performed on Oxford Nanopore R10.4.1 flow cells, facilitating comparison of complete plasmid sequences. We identified structural variants of pQEB1 and defined the molecular events responsible for them. These have included IS26-mediated inversions and acquisitions of multiple insertion sequences and transposons, including carriers of mercury or arsenic resistance genes. We found that a particular inversion variant of pQEB1 was strongly associated with the QE Liver speciality after appearing in November 2023, but was found in different specialities and wards in January/February 2024. That variant has so far been seen in five different bacterial hosts from six patients, consistent with recent and ongoing inter-host and inter-patient transmission of pQEB1 in this hospital setting.

Comparative Whole-Genome Sequencing Analysis of In-situ and Invasive Acral Lentiginous Melanoma: Markedly Increased Copy Number Gains of GAB2 , PAK1 , UCP2 , and CCND1 are Associated with Melanoma Invasion.

Acral lentiginous melanoma (ALM) is the most common subtype of acral melanoma. Even though recent genetic studies are reported in acral melanomas, the genetic differences between in-situ and invasive ALM remain unclear. We aimed to analyze specific genetic changes in ALM and compare genetic differences between in-situ and invasive lesions to identify genetic changes associated with the pathogenesis and progression of ALM. We performed whole genome sequencing of 71 tissue samples from 29 patients with ALM. Comparative analyses were performed, pairing in-situ ALMs with normal tissues and, furthermore, invasive ALMs with normal and in-situ tissues. Among 21 patients with in-situ ALMs, 3 patients (14.3%) had SMIM14 , SLC9B1 , FRG1 , FAM205A , ESRRA , and ESPN mutations, and copy number (CN) gains were identified in only 2 patients (9.5%). Comparing 13 invasive ALMs with in-situ tissues, CN gains were identified in GAB2 in 8 patients (61.5%), PAK1 in 6 patients (46.2%), and UCP2 and CCND1 in 5 patients (38.5%). Structural variants were frequent in in-situ and invasive ALM lesions. Both in-situ and invasive ALMs had very low frequencies of common driver mutations. Structural variants were common in both in-situ and invasive ALMs. Invasive ALMs had markedly increased CN gains, such as GAB2 , PAK1 , UCP2 , and CCND1 , compared with in-situ lesions. These results suggest that they are associated with melanoma invasion.

Polaribacter ponticola sp. nov., isolated from seawater, reclassification of Polaribacter undariae as a later heterotypic synonym of Polaribacter sejongensis, and emended description of Polaribacter sejongensis Kim et al. 2013.

A Gram-stain-negative, yellow-pigmented, and strictly aerobic bacterium, designated as strain MSW5T, was isolated from seawater of the Yellow Sea in South Korea. The cells were non-motile rods exhibiting oxidase- and catalase-positive activities. Growth was observed at 15-25 °C (optimum, 25 °C) and pH 5.0-9.0 (optimum, pH 7.0-8.0) and in the presence of 1.0-5.0% (w/v) NaCl (optimum, 2.0%). Menaquinone-6 was the sole respiratory quinone, and iso-C15 : 0, summed feature 3 (C16 : 1 ω7c and/or C16 : 1 ω6c), iso-C15 : 0 3-OH, and C15 : 1 ω6c were the major cellular fatty acids. Major polar lipids included phosphatidylethanolamine, two unidentified aminolipids, and three unidentified lipids. Phylogenetic analyses based on 16S rRNA gene sequences and 92 concatenated core protein sequences revealed that strain MSW5T formed a distinct lineage within the genus Polaribacter. The genome of strain MSW5T was 3582 kb in size with a 29.1 mol% G+C content. Strain MSW5T exhibited the highest similarity to Polaribacter atrinae WP25T, with a 97.9% 16S rRNA gene sequence similarity. However, the average nucleotide identity and digital DNA-DNA hybridization values were 79.4 and 23.3%, respectively, indicating that strain MSW5T represents a novel species. Based on its phenotypic, chemotaxonomic, and phylogenetic characteristics, strain MSW5T is proposed to represent a novel species, with the name Polaribacter ponticola sp. nov. The type strain is MSW5T (=KACC 22340T=NBRC 116025T). In addition, whole genome sequence comparisons and phenotypic features suggested that Polaribacter sejongensis and Polaribacter undariae belong to the same species, with P. undariae proposed as a later heterotypic synonym of P. sejongensis. An emended description of Polaribacter sejongensis is also proposed.

Identification of a Novel Parvovirus in the Arctic Wolf (Canis lupus arctos).

A novel virus, temporarily named "Arctic wolf parvovirus" (AWPV), was discovered in a pharyngeal metagenomic library derived from an Arctic wolf (Canis lupus arctos) in China. The genome sequence was assigned GenBase accession number C_AA071902.1. AWPV has a genome comprised of 4,920 base pairs with a nucleotide composition of 36.4% A, 23.4% T, 18.2% G, and 22.0% C, with a GC content of 40.2%. Its structure resembles parvoviruses, containing two open reading frames: the nonstructural (NS) region encoding replication enzymes and the structural (VP) region encoding capsid protein. Pairwise sequence comparison and phylogenetic analysis suggest AWPV may represent a novel species within the genus Protoparvovirus. This discovery enhances our understanding of mammalian virus ecology and potential future infectious diseases.

Identification of putative coral pathogens in endangered Caribbean staghorn coral using machine learning.

Coral diseases contribute to the rapid decline in coral reefs worldwide, and yet coral bacterial pathogens have proved difficult to identify because 16S rRNA gene surveys typically identify tens to hundreds of disease-associate bacteria as putative pathogens. An example is white band disease (WBD), which has killed up to 95% of the now-endangered Caribbean Acropora corals since 1979, yet the pathogen is still unknown. The 16S rRNA gene surveys have identified hundreds of WBD-associated bacterial amplicon sequencing variants (ASVs) from at least nine bacterial families with little consensus across studies. We conducted a multi-year, multi-site 16S rRNA gene sequencing comparison of 269 healthy and 143 WBD-infected Acropora cervicornis and used machine learning modelling to accurately predict disease outcomes and identify the top ASVs contributing to disease. Our ensemble ML models accurately predicted disease with greater than 97% accuracy and identified 19 disease-associated ASVs and five healthy-associated ASVs that were consistently differentially abundant across sampling periods. Using a tank-based transmission experiment, we tested whether the 19 disease-associated ASVs met the assumption of a pathogen and identified two pathogenic candidate ASVs-ASV25 Cysteiniphilum litorale and ASV8 Vibrio sp. to target for future isolation, cultivation, and confirmation of Henle-Koch's postulate via transmission assays.

Automated Photogrammetric Tool for Landslide Recognition and Volume Calculation Using Time-Lapse Imagery

Digital photogrammetry has attracted widespread attention in the field of geotechnical and geological surveys due to its low-cost, ease of use, and contactless mode. In this work, with the purpose of studying the progressive block surficial detachments of a landslide, we developed a monitoring system based on fixed multi-view time-lapse cameras. Thanks to a newly developed photogrammetric algorithm based on the comparison of photo sequences through a structural similarity metric and the computation of the disparity map of two convergent views, we can quickly detect the occurrence of collapse events, determine their location, and calculate the collapse volume. With the field data obtained at the Perarolo landslide site (Belluno Province, Italy), we conducted preliminary tests of the effectiveness of the algorithm and its accuracy in the volume calculation. The method of quickly and automatically obtaining the collapse information proposed in this paper can extend the potential of landslide monitoring systems based on videos or photo sequence and it will be of great significance for further research on the link between the frequency of collapse events and the driving factors.

Genomic insights into cpb2-positive Clostridium perfringens and the potential biological function of cpb2 gene

Clostridium perfringens, capable of causing intestinal infections in both animals and humans, represents a significant public health concern. This study aimed to assess the occurrence of the beta2 toxin-coding gene cpb2 in C. perfringens from various host species and to explore the genetic contexts of this gene. The results showed an enrichment of cpb2 in pig-derived C. perfringens. A comparative analysis of the detection rates of cpb2 and pCP13-like plasmids revealed that the cpb2 gene itself, rather than the pCP13-like plasmids, caused the enrichment. Sequence comparison of cpb2-positive pCP13-like plasmids showed that cpb2 was located on the cpb2-hp-transcriptional regulator (PadR family) segment. Despite the diverse plasmid structures of pCP13-like plasmids, the cpb2-hp-transcriptional regulator (PadR family) segment was consistently observed in all cpb2-positive C. perfringens strains, suggesting the potential transmission of the cpb2 gene on this specific genetic segment. Additionally, phylogenetic analysis of the C. perfringens strains harboring pCP13-like plasmids, as well as 31 pCP13-like plasmids, indicated that cpb2 did not affect the evolutionary relationship of either pCP13-like plasmids or C. perfringens. Genetic markers, particularly those located on mobile genetic elements (MGEs), that can help bacteria survive in external environments are more readily enriched in the population. The high prevalence of cpb2 in pig-derived strains indicated that it might confer a selective advantage, enhancing the survival and persistence of C. perfringens in the pig intestine. In conclusion, our study elucidated the genetic context, host tropism and potential biological functions of cpb2, which can provide references for further research.

The archaeal family 3 polyphosphate kinase reveals a function of polyphosphate as energy buffer under low energy charge.

Inorganic polyphosphate, a linear polymer of orthophosphate residues linked by phosphoanhydride bonds, occurs in all three domains of life and plays a diverse and prominent role in metabolism and cellular regulation. While the polyphosphate metabolism and its physiological significance have been well studied in bacteria and eukaryotes including human, there are only few studies in archaea available so far. In Crenarchaeota including members of Sulfolobaceae , the presence of polyphosphate and degradation via exopolyphosphatase has been reported and there is some evidence for a functional role in metal ion chelation, biofilm formation, adhesion and motility, however, the nature of the crenarchaeal polyphosphate kinase is still unknown. Here we used the crenarchaeal model organism Sulfolobus acidocaldarius to study the enzymes involved in polyphosphate synthesis. The two genes annotated as thymidylate kinase ( saci_2019 and saci_2020 ), localized downstream of the exopolyphosphatase, were identified as the missing polyphosphate kinase in S. acidocaldarius ( Sa PPK3). Thymidylate kinase activity was confirmed for Saci_0893. Notably Saci_2020 showed no polyphosphate kinase activity on its own but served as regulatory subunit (rPPK3) and was able to enhance polyphosphate kinase activity of the catalytically active subunit Saci_2019 (cPPK3). Heteromeric polyphosphate kinase activity is reversible and shows a clear preference for polyP-dependent nucleotide kinase activity, i.e. polyP-dependent formation of ATP from ADP (12.4 U/mg) and to a lower extent of GDP to GTP whereas AMP does not serve as substrate. PPK activity in the direction of ATP-dependent polyP synthesis is rather low (0.25 U/mg); GTP was not used as phosphoryl donor. A combined experimental modelling approach using quantitative 31 P NMR allowed to follow the reversible enzyme reaction for both ATP and polyP synthesis. PolyP synthesis was only observed when the ATP/ADP ratio was kept high, using an ATP recycling system. In absence of such a recycling system, all incubations with polyP and PPK would reach an equilibrium state with an ATP/ADP ratio between 3 and 4, independent of the initial conditions. Structural and sequence comparisons as well as phylogenetic analysis reveal that the S. acidocaldarius PPK is a member of a new PPK family, named PPK3, within the thymidylate kinase family of the P-loop kinase superfamily, clearly separated from PPK2. Our studies show that polyP, in addition to its function as phosphate storage, has a special importance for the energy homeostasis of S. acidocaldarius and due to its reversibility serves as energy buffer under low energy charge enabling a quick response to changes in cellular demand.

Rational Design of High Affinity Interaction Between CC Chemokine Binding Protein vCCI and CCL17/TARC.

The poxvirus-derived protein vCCI (viral CC chemokine inhibitor) binds almost all members of the CC chemokine family with nanomolar affinity, inhibiting their pro-inflammatory actions. Understanding the affinity and specificity of vCCI could lead to new anti-inflammatory therapeutics. CCL17, also known as TARC, is unusual among CC chemokines by having only micromolar binding to vCCI. We have used sequence analysis and molecular simulations to determine the cause of this weak binding, which identified several locations in CCL17 where mutations seemed likely to improve binding to vCCI. Based on the aforementioned analysis, we expressed and tested multiple mutants of CCL17. We found two single point mutants V44K and Q45R that increased binding affinity to vCCI by 2-3-fold and, in combination, further improved affinity by 7-fold. The CCL17 triple mutant G17R/V44K/Q45R yielded a Kd of 0.25 ± 0.13 μM, a 68-fold improvement in affinity compared to the complex with wild-type CCL17. A quadruple mutant G17R/V44K/Q45R/R57W showed high affinity (0.59 ± 0.09 μM) compared to the wild type but lower affinity than the triple mutant. This work demonstrates that sequence comparisons and molecular simulations can predict chemokine mutations that increase the level of binding to vCCI, an important first step in developing engineered chemokine inhibitors useful for anti-inflammatory therapy.

Conserved amino acid residues and gene expression patterns associated with the substrate preferences of the competing enzymes FLS and DFR.

Flavonoids, an important class of specialized metabolites, are synthesized from phenylalanine and present in almost all plant species. Different branches of flavonoid biosynthesis lead to products like flavones, flavonols, anthocyanins, and proanthocyanidins. Dihydroflavonols form the branching point towards the production of non-colored flavonols via flavonol synthase (FLS) and colored anthocyanins via dihydroflavonol 4-reductase (DFR). Despite the wealth of publicly accessible data, there remains a gap in understanding the mechanisms that mitigate competition between FLS and DFR for the shared substrate, dihydroflavonols. An angiosperm-wide comparison of FLS and DFR sequences revealed the amino acids at positions associated with the substrate specificity in both enzymes. A global analysis of the phylogenetic distribution of these amino acid residues revealed that monocots generally possess FLS with Y132 (FLSY) and DFR with N133 (DFRN). In contrast, dicots generally possess FLSH and DFRN, DFRD, and DFRA. DFRA, which restricts substrate preference to dihydrokaempferol, previously believed to be unique to strawberry species, is found to be more widespread in angiosperms and has evolved independently multiple times. Generally, angiosperm FLS appears to prefer dihydrokaempferol, whereas DFR appears to favor dihydroquercetin or dihydromyricetin. Moreover, in the FLS-DFR competition, the dominance of one over the other is observed, with typically only one gene being expressed at any given time. This study illustrates how almost mutually exclusive gene expression and substrate-preference determining residues could mitigate competition between FLS and DFR, delineates the evolution of these enzymes, and provides insights into mechanisms directing the metabolic flux of the flavonoid biosynthesis, with potential implications for ornamental plants and molecular breeding strategies.

Optimizing protein sequence classification: integrating deep learning models with Bayesian optimization for enhanced biological analysis

Efforts to enhance the accuracy of protein sequence classification are of utmost importance in driving forward biological analyses and facilitating significant medical advancements. This study presents a cutting-edge model called ProtICNN-BiLSTM, which combines attention-based Improved Convolutional Neural Networks (ICNN) and Bidirectional Long Short-Term Memory (BiLSTM) units seamlessly. Our main goal is to improve the accuracy of protein sequence classification by carefully optimizing performance through Bayesian Optimisation. ProtICNN-BiLSTM combines the power of CNN and BiLSTM architectures to effectively capture local and global protein sequence dependencies. In the proposed model, the ICNN component uses convolutional operations to identify local patterns. Captures long-range associations by analyzing sequence data forward and backwards. In advanced biological studies, Bayesian Optimisation optimizes model hyperparameters for efficiency and robustness. The model was extensively confirmed with PDB-14,189 and other protein data. We found that ProtICNN-BiLSTM outperforms traditional categorization models. Bayesian Optimization’s fine-tuning and seamless integration of local and global sequence information make it effective. The precision of ProtICNN-BiLSTM improves comparative protein sequence categorization. The study improves computational bioinformatics for complex biological analysis. Good results from the ProtICNN-BiLSTM model improve protein sequence categorization. This powerful tool could improve medical and biological research. The breakthrough protein sequence classification model is ProtICNN-BiLSTM. Bayesian optimization, ICNN, and BiLSTM analyze biological data accurately.

Improved functions for nonlinear sequence comparison using SEEKR.

SEquence Evaluation through k-mer Representation (SEEKR) is a method of sequence comparison that uses sequence substrings called k-mers to quantify the nonlinear similarity between nucleic acid species. We describe the development of new functions within SEEKR that enable end-users to estimate P-values that ascribe statistical significance to SEEKR-derived similarities, as well as visualize different aspects of k-mer similarity. We apply the new functions to identify chromatin-enriched lncRNAs that contain XIST-like sequence features, and we demonstrate the utility of applying SEEKR on lncRNA fragments to identify potential RNA-protein interaction domains. We also highlight ways in which SEEKR can be applied to augment studies of lncRNA conservation, and we outline the best practice of visualizing RNA-seq read density to evaluate support for lncRNA annotations before their in-depth study in cell types of interest.

Identification of a Major QTL for Seed Protein Content in Cultivated Peanut (Arachis hypogaea L.) Using QTL-Seq.

Peanut (Arachis hypogaea L.) is a great plant protein source for human diet since it has high protein content in the kernel. Therefore, seed protein content (SPC) is considered a major agronomic and quality trait in peanut breeding. However, few genetic loci underlying SPC have been identified in peanuts, and the underlying regulatory mechanisms remain unknown, limiting the effectiveness of breeding for high-SPC peanut varieties. In this study, a major QTL (qSPCB10.1) controlling peanut SPC was identified within a 2.3 Mb interval in chromosome B10 by QTL-seq using a recombinant inbred line population derived from parental lines with high and low SPCs, respectively. Sequence comparison, transcriptomic analysis, and annotation analysis of the qSPCB10.1 locus were performed. Six differentially expressed genes with sequence variations between two parents were identified as candidate genes underlying qSPCB10.1. Further locus interaction analysis revealed that qSPCB10.1 could not affect the seed oil accumulation unless qOCA08.1XH13 was present, a high seed oil content (SOC) allele for a major QTL underlying SOC. In summary, our study provides a basis for future investigation of the genetic basis of seed protein accumulation and facilitates marker-assisted selection for developing high-SPC peanut genotypes.