CRISPR-Cas9 screens reveal common essential miRNAs in human cancer cell lines.

BACKGROUND: Genome-wide functional screening using the CRISPR-Cas9 system is a powerful tool to uncover tumor-specific and common genetic dependencies across cancer cell lines. Current CRISPR-Cas9 knockout libraries, however, primarily target protein-coding genes. This limits functional genomics-based investigations of miRNA function. METHODS: We designed a novel CRISPR-Cas9 knockout library (lentiG-miR) of 8107 distinct sgRNAs targeting a total of 1769 human miRNAs and benchmarked its single guide RNA (sgRNA) composition, predicted on- and off-target activity, and screening performance against previous libraries. Using a total of 45 human cancer cell lines, representing 16 different tumor entities, we performed negative selection screens to identify miRNA fitness genes. Fitness miRNAs in each cell line were scored using a combination of supervised and unsupervised essentiality classifiers. Common essential miRNAs across distinct cancer cell lines were determined using the 90th percentile method. For subsequent validation, we performed knockout experiments for selected common essential miRNAs in distinct cancer cell lines and gene expression profiling. RESULTS: We found significantly lower off-target activity for protein-coding genes and a higher miRNA gene coverage for lentiG-miR as compared to previously described miRNA-targeting libraries, while preserving high on-target activity. A minor fraction of miRNAs displayed robust depletion of targeting sgRNAs, and we observed a high level of consistency between redundant sgRNAs targeting the same miRNA gene. Across 45 human cancer cell lines, only 217 (12%) of all targeted human miRNAs scored as a fitness gene in at least one model, and fitness effects for most miRNAs were confined to small subsets of cell lines. In contrast, we identified 49 common essential miRNAs with a homogenous fitness profile across the vast majority of all cell lines. Transcriptional profiling verified highly consistent gene expression changes in response to knockout of individual common essential miRNAs across a diverse set of cancer cell lines. CONCLUSIONS: Our study presents a miRNA-targeting CRISPR-Cas9 knockout library with high gene coverage and optimized on- and off-target activities. Taking advantage of the lentiG-miR library, we define a catalogue of miRNA fitness genes in human cancer cell lines, providing the foundation for further investigation of miRNAs in human cancer.

Biological Characterization and Genomic Analysis of Three Novel Serratia- and Enterobacter-Specific Virulent Phages.

Due to the high microbiological contamination of raw food materials and the increase in the incidence of multidrug-resistant bacteria, new methods of ensuring microbiological food safety are being sought. One solution may be to use bacteriophages (so-called phages) as natural bacterial enemies. Therefore, the aim of this study was the biological and genomic characterization of three newly isolated Serratia- and Enterobacter-specific virulent bacteriophages as potential candidates for food biocontrol. Serratia phage KKP_3708 (vB_Sli-IAFB_3708), Serratia phage KKP_3709 (vB_Sma-IAFB_3709), and Enterobacter phage KKP_3711 (vB_Ecl-IAFB_3711) were isolated from municipal sewage against Serratia liquefaciens strain KKP 3654, Serratia marcescens strain KKP 3687, and Enterobacter cloacae strain KKP 3684, respectively. The effect of phage addition at different multiplicity of infection (MOI) rates on the growth kinetics of the bacterial hosts was determined using a Bioscreen C Pro growth analyzer. The phages retained high activity in a wide temperature range (from -20 °C to 60 °C) and active acidity values (pH from 3 to 12). Based on transmission electron microscopy (TEM) imaging and whole-genome sequencing (WGS), the isolated bacteriophages belong to the tailed bacteriophages from the Caudoviricetes class. Genomic analysis revealed that the phages have linear double-stranded DNA of size 40,461 bp (Serratia phage KKP_3708), 67,890 bp (Serratia phage KKP_3709), and 113,711 bp (Enterobacter phage KKP_3711). No virulence, toxins, or antibiotic resistance genes were detected in the phage genomes. The lack of lysogenic markers indicates that all three bacteriophages may be potential candidates for food biocontrol.

Unveiling the mysteries: Functional insights into hypothetical proteins from Bacteroides fragilis 638R.

Humans benefit from a vast community of microorganisms in their gastrointestinal tract, known as the gut microbiota, numbering in the tens of trillions. An imbalance in the gut microbiota known as dysbiosis, can lead to changes in the metabolite profile, elevating the levels of toxins like Bacteroides fragilis toxin (BFT), colibactin, and cytolethal distending toxin. These toxins are implicated in the process of oncogenesis. However, a significant portion of the Bacteroides fragilis genome consists of functionally uncharacterized and hypothetical proteins. This study delves into the functional characterization of hypothetical proteins (HPs) encoded by the Bacteroides fragilis genome, employing a systematic in silico approach. A total of 379 HPs were subjected to a BlastP homology search against the NCBI non-redundant protein sequence database, resulting in 162 HPs devoid of identity to known proteins. CDD-Blast identified 106 HPs with functional domains, which were then annotated using Pfam, InterPro, SUPERFAMILY, SCANPROSITE, SMART, and CATH. Physicochemical properties, such as molecular weight, isoelectric point, and stability indices, were assessed for 60 HPs whose functional domains were identified by at least three of the aforementioned bioinformatic tools. Subsequently, subcellular localization analysis was examined and the gene ontology analysis revealed diverse biological processes, cellular components, and molecular functions. Remarkably, E1WPR3 was identified as a virulent and essential gene among the HPs. This study presents a comprehensive exploration of B. fragilis HPs, shedding light on their potential roles and contributing to a deeper understanding of this organism's functional landscape.

The nuclear and mitochondrial genome assemblies of Tetragonisca angustula (Apidae: Meliponini), a tiny yet remarkable pollinator in the Neotropics.

BACKGROUND: The field of bee genomics has considerably advanced in recent years, however, the most diverse group of honey producers on the planet, the stingless bees, are still largely neglected. In fact, only eleven of the ~ 600 described stingless bee species have been sequenced, and only three using a long-read (LR) sequencing technology. Here, we sequenced the nuclear and mitochondrial genomes of the most common, widespread and broadly reared stingless bee in Brazil and other neotropical countries-Tetragonisca angustula (popularly known in Brazil as jataí). RESULTS: A total of 48.01 Gb of DNA data were generated, including 2.31 Gb of Pacific Bioscience HiFi reads and 45.70 Gb of Illumina short reads (SRs). Our preferred assembly comprised 683 contigs encompassing 284.49 Mb, 62.84 Mb of which (22.09%) corresponded to 445,793 repetitive elements. N50, L50 and complete BUSCOs reached 1.02 Mb, 91 contigs and 97.1%, respectively. We predicted that the genome of T. angustula comprises 17,459 protein-coding genes and 4,108 non-coding RNAs. The mitogenome consisted of 17,410 bp, and all 37 genes were found to be on the positive strand, an unusual feature among bees. A phylogenomic analysis of 26 hymenopteran species revealed that six odorant receptor orthogroups of T. angustula were found to be experiencing rapid evolution, four of them undergoing significant contractions. CONCLUSIONS: Here, we provided the first nuclear and mitochondrial genome assemblies for the ecologically and economically important T. angustula, the fourth stingless bee species to be sequenced with LR technology thus far. We demonstrated that even relatively small amounts of LR data in combination with sufficient SR data can yield high-quality genome assemblies for bees.

Drought-induced molecular changes in crown of various barley phytohormone mutants.

One of the main signal transduction pathways that modulate plant growth and stress responses, including drought, is the action of phytohormones. Recent advances in omics approaches have facilitated the exploration of plant genomes. However, the molecular mechanisms underlying the response in the crown of barley, which plays an essential role in plant performance under stress conditions and regeneration after stress treatment, remain largely unclear. The objective of the present study was the elucidation of drought-induced molecular reactions in the crowns of different barley phytohormone mutants. We verified the hypothesis that defects of gibberellins, brassinosteroids, and strigolactones action affect the transcriptomic, proteomic, and hormonal response of barley crown to the transitory drought influencing plant development under stress. Moreover, we assumed that due to the strong connection between strigolactones and branching the hvdwarf14.d mutant, with dysfunctional receptor of strigolactones, manifests the most abundant alternations in crowns and phenotype under drought. Finally, we expected to identify components underlying the core response to drought which are independent of the genetic background. Large-scale analyses were conducted using gibberellins-biosynthesis, brassinosteroids-signaling, and strigolactones-signaling mutants, as well as reference genotypes. Detailed phenotypic evaluation was also conducted. The obtained results clearly demonstrated that hormonal disorders caused by mutations in the HvGA20ox2, HvBRI1, and HvD14 genes affected the multifaceted reaction of crowns to drought, although the expression of these genes was not induced by stress. The study further detected not only genes and proteins that were involved in the drought response and reacted specifically in mutants compared to the reaction of reference genotypes and vice versa, but also the candidates that may underlie the genotype-universal stress response. Furthermore, candidate genes involved in phytohormonal interactions during the drought response were identified. We also found that the interplay between hormones, especially gibberellins and auxins, as well as strigolactones and cytokinins may be associated with the regulation of branching in crowns exposed to drought. Overall, the present study provides novel insights into the molecular drought-induced responses that occur in barley crowns.

AnnoView enables large-scale analysis, comparison, and visualization of microbial gene neighborhoods.

The analysis and comparison of gene neighborhoods is a powerful approach for exploring microbial genome structure, function, and evolution. Although numerous tools exist for genome visualization and comparison, genome exploration across large genomic databases or user-generated datasets remains a challenge. Here, we introduce AnnoView, a web server designed for interactive exploration of gene neighborhoods across the bacterial and archaeal tree of life. Our server offers users the ability to identify, compare, and visualize gene neighborhoods of interest from 30 238 bacterial genomes and 1672 archaeal genomes, through integration with the comprehensive Genome Taxonomy Database and AnnoTree databases. Identified gene neighborhoods can be visualized using pre-computed functional annotations from different sources such as KEGG, Pfam and TIGRFAM, or clustered based on similarity. Alternatively, users can upload and explore their own custom genomic datasets in GBK, GFF or CSV format, or use AnnoView as a genome browser for relatively small genomes (e.g. viruses and plasmids). Ultimately, we anticipate that AnnoView will catalyze biological discovery by enabling user-friendly search, comparison, and visualization of genomic data. AnnoView is available at http://annoview.uwaterloo.ca.

The chromosome-level genome and functional database accelerate research about biosynthesis of secondary metabolites in Rosa roxburghii.

Rosa roxburghii Tratt, a valuable plant in China with long history, is famous for its fruit. It possesses various secondary metabolites, such as L-ascorbic acid (vitamin C), alkaloids and poly saccharides, which make it a high nutritional and medicinal value. Here we characterized the chromosome-level genome sequence of R. roxburghii, comprising seven pseudo-chromosomes with a total size of 531 Mb and a heterozygosity of 0.25%. We also annotated 45,226 coding gene loci after masking repeat elements. Orthologs for 90.1% of the Complete Single-Copy BUSCOs were found in the R. roxburghii annotation. By aligning with protein sequences from public platform, we annotated 85.89% genes from R. roxburghii. Comparative genomic analysis revealed that R. roxburghii diverged from Rosa chinensis approximately 5.58 to 13.17 million years ago, and no whole-genome duplication event occurred after the divergence from eudicots. To fully utilize this genomic resource, we constructed a genomic database RroFGD with various analysis tools. Otherwise, 69 enzyme genes involved in L-ascorbate biosynthesis were identified and a key enzyme in the biosynthesis of vitamin C, GDH (L-Gal-1-dehydrogenase), is used as an example to introduce the functions of the database. This genome and database will facilitate the future investigations into gene function and molecular breeding in R. roxburghii.

TFTG: A comprehensive database for human transcription factors and their targets.

Transcription factors (TFs) are major contributors to gene transcription, especially in controlling cell-specific gene expression and disease occurrence and development. Uncovering the relationship between TFs and their target genes is critical to understanding the mechanism of action of TFs. With the development of high-throughput sequencing techniques, a large amount of TF-related data has accumulated, which can be used to identify their target genes. In this study, we developed TFTG (Transcription Factor and Target Genes) database (http://tf.liclab.net/TFTG), which aimed to provide a large number of available human TF-target gene resources by multiple strategies, besides performing a comprehensive functional and epigenetic annotations and regulatory analyses of TFs. We identified extensive available TF-target genes by collecting and processing TF-associated ChIP-seq datasets, perturbation RNA-seq datasets and motifs. We also obtained experimentally confirmed relationships between TF and target genes from available resources. Overall, the target genes of TFs were obtained through integrating the relevant data of various TFs as well as fourteen identification strategies. Meanwhile, TFTG was embedded with user-friendly search, analysis, browsing, downloading and visualization functions. TFTG is designed to be a convenient resource for exploring human TF-target gene regulations, which will be useful for most users in the TF and gene expression regulation research.

A Principal Components Analysis and Functional Annotation of Differentially Expressed Genes in Brain Regions of Gray Rats Selected for Tame or Aggressive Behavior.

The process of domestication, despite its short duration as it compared with the time scale of the natural evolutionary process, has caused rapid and substantial changes in the phenotype of domestic animal species. Nonetheless, the genetic mechanisms underlying these changes remain poorly understood. The present study deals with an analysis of the transcriptomes from four brain regions of gray rats (Rattus norvegicus), serving as an experimental model object of domestication. We compared gene expression profiles in the hypothalamus, hippocampus, periaqueductal gray matter, and the midbrain tegmental region between tame domesticated and aggressive gray rats and revealed subdivisions of differentially expressed genes by principal components analysis that explain the main part of differentially gene expression variance. Functional analysis (in the DAVID (Database for Annotation, Visualization and Integrated Discovery) Bioinformatics Resources database) of the differentially expressed genes allowed us to identify and describe the key biological processes that can participate in the formation of the different behavioral patterns seen in the two groups of gray rats. Using the STRING- DB (search tool for recurring instances of neighboring genes) web service, we built a gene association network. The genes engaged in broad network interactions have been identified. Our study offers data on the genes whose expression levels change in response to artificial selection for behavior during animal domestication.

Protein-Coding Gene Families in Prokaryote Genome Comparisons.

The identification of orthologous genes is relevant for comparative genomics, phylogenetic analysis, and functional annotation. There are many computational tools for the prediction of orthologous groups as well as web-based resources that offer orthology datasets for download and online analysis. This chapter presents a simple and practical guide to the process of orthologous group prediction, using a dataset of 10 prokaryotic proteomes as example. The orthology methods covered are OrthoMCL, COGtriangles, OrthoFinder2, and OMA. The authors compare the number of orthologous groups predicted by these various methods, and present a brief workflow for the functional annotation and reconstruction of phylogenies from inferred single-copy orthologous genes. The chapter also demonstrates how to explore two orthology databases: eggNOG6 and OrthoDB.

Comparative Analyses of Bacteriophage Genomes.

Bacterial viruses (bacteriophages or phages) are the most abundant and diverse biological entities on Earth. There is a renewed worldwide interest in phage-centered research motivated by their enormous potential as antimicrobials to cope with multidrug-resistant pathogens. An ever-growing number of complete phage genomes are becoming available, derived either from newly isolated phages (cultivated phages) or recovered from metagenomic sequencing data (uncultivated phages). Robust comparative analysis is crucial for a comprehensive understanding of genotypic variations of phages and their related evolutionary processes, and to investigate the interaction mechanisms between phages and their hosts. In this chapter, we present a protocol for phage comparative genomics employing tools selected out of the many currently available, focusing on complete genomes of phages classified in the class Caudoviricetes. This protocol provides accurate identification of similarities, differences, and patterns among new and previously known complete phage genomes as well as phage clustering and taxonomic classification.

The Landscape and Perspectives of the Human Gut Metaproteomics.

The human gut microbiome is closely associated with human health and diseases. Metaproteomics has emerged as a valuable tool for studying the functionality of the gut microbiome by analyzing the entire proteins present in microbial communities. Recent advancements in liquid chromatography and tandem mass spectrometry (LC-MS/MS) techniques have expanded the detection range of metaproteomics. However, the overall coverage of the proteome in metaproteomics is still limited. While metagenomics studies have revealed substantial microbial diversity and functional potential of the human gut microbiome, few studies have summarized and studied the human gut microbiome landscape revealed with metaproteomics. In this article, we present the current landscape of human gut metaproteomics studies by re-analyzing the identification results from 15 published studies. We quantified the limited proteome coverage in metaproteomics and revealed a high proportion of annotation coverage of metaproteomics-identified proteins. We conducted a preliminary comparison between the metaproteomics view and the metagenomics view of the human gut microbiome, identifying key areas of consistency and divergence. Based on the current landscape of human gut metaproteomics, we discuss the feasibility of using metaproteomics to study functionally unknown proteins and propose a whole workflow peptide-centric analysis. Additionally, we suggest enhancing metaproteomics analysis by refining taxonomic classification and calculating confidence scores, as well as developing tools for analyzing the interaction between taxonomy and function.

Complete chloroplast genomes of three copal trees (Bursera: Bullockia): comparative analysis and phylogenetic relationships.

BACKGROUND: Bursera trees are conspicuous elements of the tropical dry forests in the Neotropics that have significant cultural value due to their fragrant resins (incense), wood sources (handcrafts), and ecological benefits. Despite their relevance, genetic resources developed for the genus are scarce. METHODS AND RESULTS: We obtained the complete chloroplast (Cp) genome sequence, analyzed the genome structure, and performed functional annotation of three Bursera species of the Bullockia section: Bursera cuneata, B. palmeri, and B. bipinnata. The Cp genome sizes ranged from 159,824 to 159,872 bp in length, including a large single-copy (LSC) region from 87,668 to 87,656 bp, a small single-copy (SSC) from 18,581 to 18,571 bp, and two inverted repeats regions (IRa and IRb) of 26,814 bp each. The three Cp genomes consisted of 135 genes, of which 90 were protein-coding, 37 tRNAs, and 8 rRNAs. The Cp genomes were relatively conserved, with the LSC region exhibiting the greatest nucleotide divergence (psbJ, trnQ-UCC, trnG-UCC, and petL genes), whereas few changes were observed in the IR border regions. Between 589 and 591 simple sequence repeats were identified. Analysis of phylogenetic relationships using our data for each Cp region (LSC, SSC, IRa, and IRb) and of seven species within Burseraceae confirmed that Commiphora is the sister genus of Bursera. Only the phylogenetic trees based on the SSC and LSC regions resolved the close relationship between B. bipinnata and B. palmeri. CONCLUSION: Our work contributes to the development of Bursera's genomic resources for taxonomic, evolutionary, and ecological-genetic studies.

The updated genome of the Hungarian population of Aedes koreicus.

Vector-borne diseases pose a potential risk to human and animal welfare, and understanding their spread requires genomic resources. The mosquito Aedes koreicus is an emerging vector that has been introduced into Europe more than 15 years ago but only a low quality, fragmented genome was available. In this study, we carried out additional sequencing and assembled and characterized the genome of the species to provide a background for understanding its evolution and biology. The updated genome was 1.1 Gbp long and consisted of 6099 contigs with an N50 value of 329,610 bp and a BUSCO score of 84%. We identified 22,580 genes that could be functionally annotated and paid particular attention to the identification of potential insecticide resistance genes. The assessment of the orthology of the genes indicates a high turnover at the terminal branches of the species tree of mosquitoes with complete genomes, which could contribute to the adaptation and evolutionary success of the species. These results could form the basis for numerous downstream analyzes to develop targets for the control of mosquito populations.

Analyzing the Diversity of MYB Family Response Strategies to Drought Stress in Different Flax Varieties Based on Transcriptome Data.

The MYB transcription factor family has numerous members, and is involved in biological activities, such as ABA signaling, which plays an important role in a plant's resistance to abiotic stresses such as drought. However, the diversity of MYB members that respond to drought stress and their regulatory mechanisms in different flax varieties were unclear. In this study, we obtained 855.69 Gb of clean data from 120 flax root samples from 20 flax (Linum usitatissimum L.) varieties, assembled 92,861 transcripts, and identified 434 MYB family members in each variety. The expression profiles of the MYB transcription factor family from 20 flax varieties under drought stress were analyzed. The results indicated that there are four strategies by which the MYB family responds to drought stress in these 20 flax varieties, each of which has its own specific processes, such as development, reproduction, and localization processes. The four strategies also include common biological processes, such as stimulus responses, metabolic processes, and biological regulation. The WGCNA method was subsequently employed to identify key members of the MYB family involved in response strategies to drought stress. The results demonstrated that a 1R-MYB subfamily gene co-expression network is significantly related to the gibberellin response and cytokinin-activated signaling pathway processes in the 'Strategy 4' for MYB family response to drought, identifying core genes such as Lus.scaffold70.240. Our results showed a diversity of MYB family responses to drought stress within flax varieties, and these results contribute to deciphering the mechanisms of the MYB family regulation of drought resistance. This will promote the more accurate breeding development of flax to adapt to agricultural production under drought conditions.

Full-length transcriptome characterization of Platycladus orientalis based on the PacBio platform.

As a unique and native conifer in China, Platycladus orientalis is widely used in soil erosion control, garden landscapes, timber, and traditional Chinese medicine. However, due to the lack of reference genome and transcriptome, it is limited to the further molecular mechanism research and gene function mining. To develop a full-length reference transcriptome, tissues from five different parts of P. orientalis and four cone developmental stages were sequenced and analyzed by single-molecule real-time (SMRT) sequencing through the PacBio platform in this study. Overall, 37,111 isoforms were detected by PacBio with an N50 length of 2,317 nt, an average length of 1,999 bp, and the GC content of 41.81%. Meanwhile, 36,120 coding sequences, 5,645 simple sequence repeats (SSRs), 1,201 non-coding RNAs (lncRNAs), and 182 alternative splicing (AS) events with five types were identified using the results obtained from the PacBio transcript isoforms. Furthermore, 1,659 transcription factors (TFs) were detected and belonged to 51 TF families. A total of 35,689 transcripts (96.17%) were annotated through the NCBI nr, KOG, Swiss-Prot and KEGG databases, and 385 transcript isoforms related to 8 types of hormones were identified incorporated into plant hormone signal transduction pathways. The assembly and revelation of the full-length transcriptome of P. orientalis offer a pioneering insight for future investigations into gene function and genetic breeding within Platycladus species.

Incorporating functional annotation with bilevel continuous shrinkage for polygenic risk prediction.

BACKGROUND: Genetic variants can contribute differently to trait heritability by their functional categories, and recent studies have shown that incorporating functional annotation can improve the predictive performance of polygenic risk scores (PRSs). In addition, when only a small proportion of variants are causal variants, PRS methods that employ a Bayesian framework with shrinkage can account for such sparsity. It is possible that the annotation group level effect is also sparse. However, the number of PRS methods that incorporate both annotation information and shrinkage on effect sizes is limited. We propose a PRS method, PRSbils, which utilizes the functional annotation information with a bilevel continuous shrinkage prior to accommodate the varying genetic architectures both on the variant-specific level and on the functional annotation level. RESULTS: We conducted simulation studies and investigated the predictive performance in settings with different genetic architectures. Results indicated that when there was a relatively large variability of group-wise heritability contribution, the gain in prediction performance from the proposed method was on average 8.0% higher AUC compared to the benchmark method PRS-CS. The proposed method also yielded higher predictive performance compared to PRS-CS in settings with different overlapping patterns of annotation groups and obtained on average 6.4% higher AUC. We applied PRSbils to binary and quantitative traits in three real world data sources (the UK Biobank, the Michigan Genomics Initiative (MGI), and the Korean Genome and Epidemiology Study (KoGES)), and two sources of annotations: ANNOVAR, and pathway information from the Kyoto Encyclopedia of Genes and Genomes (KEGG), and demonstrated that the proposed method holds the potential for improving predictive performance by incorporating functional annotations. CONCLUSIONS: By utilizing a bilevel shrinkage framework, PRSbils enables the incorporation of both overlapping and non-overlapping annotations into PRS construction to improve the performance of genetic risk prediction. The software is available at https://github.com/styvon/PRSbils .

Charge cluster occurrence in land plants' mitochondrial proteomes with functional and structural insights.

The Charge Clusters (CCs) are involved in key functions and are distributed according to the organism, the protein's type, and the charge of amino acids. In the present study, we have explored the occurrence, position, and annotation as a first large-scale study of the CCs in land plants mitochondrial proteomes. A new python script was used for data curation. The Finding Clusters Charge in Protein Sequences Program was performed after adjusting the reading window size. A 44316 protein sequences belonging to 52 species of land plants were analysed. The occurrence of Negative Charge Clusters (NCCs) (1.2%) is two times more frequent than the Positive Charge Clusters (PCCs) (0.64%). Moreover, 39 and 30 NCCs were conserved in 88 and 41 proteins in intra and in inter proteomes respectively, while 14 and 21 PCCs were conserved in 53 and 85 protein sequences in intra and inter proteomes consecutively. Sequences carrying mixed CCs are rare (0.12%). Despite this low abundance, CCs play a crucial role in protein function. The CCs tend to be located mainly in the terminal regions of proteins which guarantees specific protein targeting and import into the mitochondria. In addition, the functional annotation of CCs according to Gene Ontology shows that CCs are involved in binding functions of either proteins or macromolecules which are deployed in different metabolic and cellular processes such as RNA editing and transcription. This study may provide valuable information while considering the CCs in understanding the environmental adaptation of plants.Communicated by Ramaswamy H. Sarma.

Annotating microbial functions with ProkFunFind.

Analyzing microbial genomes has become an essential part of microbiology research, giving valuable insights into the functions and evolution of microbial species. Identifying genes of interest and assigning putative annotations to those genes is a central task in genome analysis, and a plethora of tools and approaches have been developed for this task. The ProkFunFind tool was developed to bridge the gap between these various annotation approaches, providing a flexible and customizable search approach to annotate microbial functions. ProkFunFind is designed around hierarchical definitions of biological functions, where individual genes can be identified using heterogeneous search terms consisting of sequences, profile hidden Markov models, protein domains, and orthology groups. This flexible and customizable search approach allows for searches to be tailored to specific biological functions, and the search results are output in multiple formats to facilitate downstream analyses. The utility of the ProkFunFind search tool was demonstrated through its application in searching for bacterial flagella, which are complex organelles composed of multiple genes. Overall, ProkFunFind provides an accessible and flexible way to integrate multiple types of annotation and sequence data while annotating biological functions in microbial genomes.IMPORTANCEGenome sequencing and analysis are increasingly important parts of microbiology, providing a way to predict metabolic functions, identify virulence factors, and understand the evolution of microbes. The expanded use of genome sequencing has also brought an abundance of search and annotation methods, but integrating the information from these different methods can be challenging and is often done through ad hoc approaches. To bridge the gap between different types of annotations, we developed ProkFunFind, a flexible and customizable search tool incorporating multiple search approaches and annotation types to annotate microbial functions. We demonstrated the utility of ProkFunFind by searching for gene clusters encoding flagellar genes using a combination of different annotation types and searches. Overall, ProkFunFind provides a reproducible and flexible way to identify gene clusters of interest, facilitating the meaningful analysis of new and existing microbial genomes.

In silico exploration of hypothetical proteins in Neisseria gonorrhoeae for identification of therapeutic targets.

Neisseria gonorrhoeae, a World Health Organization (WHO) declared superbug and the second-most frequent cause of bacterial sexually transmitted infections worldwide is responsible for gonorrhea. Hypothetical proteins are gene products that are predicted to be encoded by a particular gene based on the DNA sequence, but their specific functions and characteristics have not been experimentally determined or verified. In the context of this research, annotating hypothetical proteins is crucial for identifying their potential as therapeutic targets. Without proper annotation, these proteins would remain vague, hindering efforts to understand their roles in disease. The methodology used aims to bridge this gap by employing algorithm-based tools and software to annotate hypothetical proteins and assess their suitability as therapeutic targets based on factors such as essentiality, virulence, subcellular localization, and druggability. Out of 716 N. gonorrhoeae hypothetical proteins reported in UniProt, assessment of crucial pathogenic factors, including essentiality, virulence, subcellular localization, and druggability, effectively filtered and prioritized the hypothetical proteins for further therapeutic exploration and lead to 5 proteins being chosen as targets. The molecular docking studies conducted identified 10 hits targeting the five targets. Conclusively, this study aided in identification of targets and hit compounds for therapeutic targeting of gonorrhea disease. Supplementary Information: The online version contains supplementary material available at 10.1007/s40203-023-00186-w.