BioProtIS: Streamlining protein-ligand interaction pipeline for analysis in genomic and transcriptomic exploration.

The identification of protein-ligand interactions plays a pivotal role in elucidating biological processes and discovering potential bioproducts. Harnessing the capabilities of computational methods in drug discovery, we introduce an innovative Inverted Virtual Screening (IVS) pipeline. This pipeline Integrated molecular dynamics and docking analyses to ensure that protein structures are not only energetically favorable but also representative of stable conformations. The primary objective of this pipeline is to automate and streamline the analysis of protein-ligand interactions at both genomic and transcriptomic scales. In the contemporary post-genomic era, high-throughput computational screening for bioproducts, biological systems, and therapeutic drugs has become a cornerstone practice. This approach offers the promise of cost-effectiveness, time efficiency, and optimization of laboratory work. Nevertheless, a notable deficiency persists in the availability of efficient pipelines capable of automating the virtual screening process, seamlessly integrating input and output, and leveraging the full potential of open-source tools. To bridge this critical gap, we have developed a versatile pipeline known as BioProtIS. This tool seamlessly integrates a suite of state-of-the-art tools, including Modeller, AlphaFold, Gromacs, FPOCKET, and AutoDock Vina, thus facilitating the streamlined docking of ligands with an expansive repertoire of proteins sourced from genomes and transcriptomes, and substrates. To assess the pipeline's performance, we employed the transcriptomes of Cereus jamacaru (a cactus species) and Aspisoma lineatum (firefly), along with the genome of Homo sapiens. This integration not only improves the accuracy of ligand-protein interactions by minimizing replicability deviations but also optimizes the discovery process by enabling the simultaneous evaluation of multiple substrates. Furthermore, our pipeline accommodates distinct testing scenarios, such as blind docking or site-specific targeting, which are invaluable in applications ranging from drug repositioning to the exploration of new allosteric binding sites and toxicity assessments. BioProtIS has been designed with modularity at its core. This inherent flexibility empowers users to make custom modifications directly within the source code, tailoring the pipeline to their specific research needs. Moreover, it lays the foundation for seamless integration of diverse docking algorithms in future iterations, promising ongoing advancements in the field of computational biology. This pipeline is available for free distribution and can be download at: https://github.com/BBMDO/BioProtIS.

Opsin diversity and evolution in the Elateroidea superfamily: Insights from transcriptome data.

Vision plays a vital biological role in organisms, which depends on the visual pigment molecules (opsin plus chromophore). The expansion or reduction of spectral channels in the organisms is determined by distinct opsin classes and copy numbers resulting from duplication or loss. Within Coleoptera, the superfamily Elateroidea exhibits a great diversity of morphological and physiological characteristics, such as bioluminescence, making this group an important model for opsin studies. While molecular and physiological studies have been conducted in Lampyridae and Elateridae, other families remain unexplored. Here, we reused transcriptome datasets from Elateroidea species, including members of Elateridae, Lampyridae, Phengodidae, Rhagophthalmidae, Cantharidae, and Lycidae, to detect the diversity of putative opsin genes in this superfamily. In addition, we tested the signature of sites under positive selection in both ultraviolet (UV)- and long-wavelength (LW)-opsin classes. Although the visual system in Elateroidea is considered simple, we observed events of duplication in LW- and UV-opsin, as well as the absence of UV-opsin in distinct families, such as larval Phengodidae individuals. We detected different copies of LW-opsins that were highly expressed in the eyes of distinct tribes of fireflies, indicating the possible selection of each copy during the evolution of the sexual mating to avoid spectrum overlapping. In Elateridae, we found that the bioluminescent species had a distinct LW-opsin copy compared with the non-bioluminescent species, suggesting events of duplication and loss. The signature of positive selection showed only one residue associated with the chromophore binding site in the Elateroidea, which may produce a bathochromic shift in the wavelength absorption spectra in this family. Overall, this study brings important content and fills gaps regarding opsin evolution in Elateroidea.

Signal Amplification for Cell-Free Biosensors, an Analog-to-Digital Converter.

Toehold switches are biosensors useful for the detection of endogenous and environmental RNAs. They have been successfully engineered to detect virus RNAs in cell-free gene expression reactions. Their inherent sequence programmability makes engineering a fast and predictable process. Despite improvements in the design, toehold switches suffer from leaky translation in the OFF state, which compromises the fold change and sensitivity of the biosensor. To address this, we constructed and tested signal amplification circuits for three toehold switches triggered by Dengue and SARS-CoV-2 RNAs and an artificial RNA. The serine integrase circuit efficiently contained leakage, boosted the expression fold change from OFF to ON, and decreased the detection limit of the switches by 3-4 orders of magnitude. Ultimately, the integrase circuit converted the analog switches' signals into digital-like output. The circuit is broadly useful for biosensors and eliminates the hard work of designing and testing multiple switches to find the best possible performer.

Exploring Phylogenetic Relationships and Divergence Times of Bioluminescent Species Using Genomic and Transcriptomic Data.

Next-generation sequencing (NGS) has dominated the scene of genomics and evolutionary biology as a great amount of genomic data have been accumulated for a diverse set of species. At the same time, phylogenetic approaches and programs are in development to allow better use of such large-size datasets. Phylogenomics appears as a promising field to accommodate and explore all the information of NGS data in phylogenetic methods, being an important approach to investigate the evolution of bioluminescence in different organisms. To guarantee accurate results in phylogenomic studies, it is mandatory to correctly identify orthologous genes in phylogenetic reconstruction. Here, we show a simplified step-by-step framework to perform phylogenetic analysis along with divergence time estimation, beginning with an orthologous search. As empirical data, we exemplify transcriptome sequences of six species of the Elateroidea superfamily (Coleoptera). We introduce several bioinformatics tools for handling genomic data, especially those available in the software OrthoFinder, IQTREE, BEAST2, and TreePL.

RNA-Seq analysis of the blue light-emitting Orfelia fultoni (Diptera: Keroplatidae) suggest photoecological adaptations at the molecular level.

Bioluminescence in Diptera is found in the Keroplatidae family, within Arachnocampininae and Keroplatinae subfamilies, with reported occurrences in Oceania, Eurasia, and Americas. Larvae of Orfelia fultoni, which inhabit stream banks in the Appalachian Mountains, emit the bluest bioluminescence among insects, using it for prey attraction, similarly to Arachnocampa spp. Although bioluminescence has a similar prey attraction function, the systems of Arachonocampininae and Keroplatinae subfamilies are morphologically/biochemically distinct, indicating different evolutionary origins. To identify the possible coding genes associated with physiological control, ecological adaptations, and origin/evolution of bioluminescence in the Keroplatinae subfamily, we performed the RNA-Seq analysis of O. fultoni larvae during day and night and compared it with the transcriptomes of Arachnocampa luminosa, and reanalyzed the previously published proteomic data of O. fultoni against the RNA-Seq dataset. The abundance of chaperones/heat-shock and hexamerin gene products at night and in luciferase enriched fractions supports their possible association and participation in bioluminescence. The low diversity of copies/families of opsins indicate a simpler visual system in O. fultoni. Noteworthy, gene products associated with silk protein biosynthesis in Orfelia were more similar to Lepidoptera than to the Arachnocampa, indicating that, similarly to the bioluminescent systems, at some point, the biochemical apparatus for web construction may have evolved independently in Orfelia and Arachnocampa.

A new brilliantly blue-emitting luciferin-luciferase system from Orfelia fultoni and Keroplatinae (Diptera).

Larvae of O. fultoni (Keroplatidae: Keroplatinae), which occur along river banks in the Appalachian Mountains in Eastern United States, produce the bluest bioluminescence among insects from translucent areas associated to black bodies, which are  located mainly in the anterior and posterior parts of the body. Although closely related to Arachnocampa spp (Keroplatidae: Arachnocampininae), O.fultoni has a morphologically and biochemically distinct bioluminescent system which evolved independently, requiring a luciferase enzyme, a luciferin, a substrate binding fraction (SBF) that releases luciferin in the presence of mild reducing agents, molecular oxygen, and no additional cofactors. Similarly, the closely related Neoceroplatus spp, shares the same kind of luciferin-luciferase system of Orfelia fultoni. However, the molecular properties, identities and functions of luciferases, SBF and luciferin of Orfelia fultoni and other  luminescent members of the Keroplatinae subfamily still remain to be fully elucidated. Using O. fultoni as a source of luciferase, and the recently discovered non-luminescent cave worm Neoditomiya sp as the main source of luciferin and SBF, we isolated and initially characterized these compounds. The luciferase of O. fultoni is a stable enzyme active as an apparent trimer (220 kDa) composed of ~70 kDa monomers, with an optimum pH of 7.8. The SBF, which is found in the black bodies in Orfelia fultoni and in smaller dark granules in Neoditomiya sp, consists of a high molecular weight complex of luciferin and proteins, apparently associated to mitochondria. The luciferin, partially purified from hot extracts by a combination of anion exchange chromatography and TLC, is a very polar and weakly fluorescent compound, whereas its oxidized product displays blue fluorescence with an emission spectrum matching the bioluminescence spectrum (~460 nm), indicating that it is oxyluciferin. The widespread occurrence of luciferin and SBF in both luminescent and non-luminescent Keroplatinae larvae indicate an additional important biological function for the substrate, and therefore the name keroplatin.

Neoceroplatus betaryiensis nov. sp. (Diptera: Keroplatidae) is the first record of a bioluminescent fungus-gnat in South America.

Blue shining fungus gnats (Diptera) had been long reported in the Waitomo caves of New Zealand (Arachnocampa luminosa Skuse), in stream banks of the American Appalachian Mountains (Orfelia fultoni Fisher) in 1939 and in true spore eating Eurasiatic Keroplatus Bosc species. This current report observes that similar blue light emitting gnat larvae also occur nearby the Betary river in the buffer zone of High Ribeira River State Park (PETAR) in the Atlantic Forest of Brazil, where the larvae were found when on fallen branches or trunks enveloped in their own secreted silk. The new species is named Neoceroplatus betaryiensis nov. sp. (Diptera: Keroplatidae: Keroplatinae: Keroplatini) based on a morphological analysis. Neoceroplatus betaryiensis nov. sp. larvae emit blue bioluminescence that can be seen from their last abdominal segment and from two photophores located laterally on the first thoracic segment. When touched, the larvae can actively stop its luminescence, which returns when it is no longer being agitated. The in vitro bioluminescence spectrum of N. betaryiensis nov. sp. peaks at 472 nm, and cross-reactivity of hot and cold extracts with the luciferin-luciferase from Orfelia fultoni indicate significant similarity in both enzyme and substrate of the two species, and that the bioluminescence system in the subfamily Keroplatinae is conserved.

Phylogenomic analyses and divergence time estimation of Elateroidea (Coleoptera) based on RNA-Seq data.

Bioluminescence, the emission of visible light in a living organism, is an intriguing phenomenon observed in different species and environments. In terrestrial organisms, the bioluminescence is observed mainly in beetles of the Elateroidea superfamily (Coleoptera). Several phylogenetic studies have been used different strategies to propose a scenario for the origin and evolution of the bioluminescence within this group, however some of them showed incongruences, mainly about the relationship of the bioluminescent families. In order to increase the number of molecular markers available for Elateroidea species and to propose a more accurate phylogeny, with high supported topology, we employed the Next-Generation Sequencing (NGS) methodology to perform the RNA-Seq analysis of luminescent (Elateridae, Phengodidae, Rhagophthalmidae, and Lampyridae) and non-luminescent (Cantharidae) species of Neotropical beetles. We used the RNA-Seq data to construct a calibrated phylogeny of Elateroidea superfamily using a large number of nuclear molecular markers. The results indicate Lampyridae and Phengodidae/Rhagophthalmidae as sister-groups, suggesting that the bioluminescence evolved later in Elateridae than other families (Lampyridae, Phengodidae, and Rhagophthalmidae), and indicating the Upper Cretaceous as the period for the main diversification of Elateroidea bioluminescent species.

Comparison of the Malpighian tubules and fat body transcriptional profiles of Zophobas morio larvae (Coleoptera: Tenebrionidae).

The Malpighian tubules in insects play an essential role in osmoregulation, through the transport of ions during excretion, whereas the fat body is usually associated with the intermediary metabolism. The tubules also are involved in excretion of organic solutes and xenobiotics. However, with the exception of a preliminary transcriptional survey of the Zophobas morio (Tenebrionidae) larval tubules, there are no detailed transcriptional analysis of this organ in Coleoptera. A luciferase-like enzyme that displays weak luminescence activity in the presence of firefly D-luciferin and ATP was cloned from the tubules of Z. morio larvae. In order to better understand the molecular physiology of Malpighian tubules and fat body in Coleoptera larvae, and to investigate the occurrence and functions of AMP-CoA ligases in these tissues, we performed a comparative transcriptional analysis of these tissues using Z. morio giant-mealworms. As expected, the tubules displayed organic and inorganic transporters, xenobiotic metabolism enzymes, V-ATPases, channels, and pumps. The fat body showed proteins that are synthesized in this tissue and secreted to the hemolymph, as well as enzymes involved in lipid and carbohydrate metabolism. These tissues are also involved in common pathways, such as nitrogen metabolism to degradation/excretion, eye pigments biosynthesis, immunity, and detoxification. The presence of coumarate-CoA ligase-like enzymes in these tissues suggest their involvement in the degradation of coumaric acid derivatives obtained from the diet, or alternatively, in the biosynthesis of compounds structurally related to coumaric acids such as eye pigments. Our results confirm to the physiological versatility of tubules and fat body in larval Coleoptera.

Orfelia-type luciferin and its associated storage protein in the non-luminescent cave worm Neoditomyia sp. (Diptera: Keroplatidae) from the Atlantic rainforest: biological and evolutionary implications.

Bioluminescence in Diptera is found in the family Keroplatidae, in the glowworms of the genera Arachnocampa, Orfelia and Keroplatus. Despite belonging to the same family, Arachnocampa spp. and Orfelia fultoni display morphologically and biochemically distinct bioluminescence systems: Arachnocampa spp. produce light by the terminal ends of Malpighian tubules using ATP, a luciferin and a luciferase, whereas Orfelia fultoni produces light by translucent areas associated with rows of black bodies in the anterior and posterior parts of the body, using a 140 kDa luciferase and a luciferin which do not cross-react with the Arachnocampa luciferin-luciferase system, and a substrate binding fraction (SBF) which apparently releases luciferin in the presence of reductants. While several other keroplatids are not luminescent, we recently discovered a non-luminescent web-constructing keroplatid larva living in the roofs of caves in the Atlantic rainforest in Brazil, which noteworthily has a compound with Orfelia luciferin-like activity and its associated binding protein (SBF). Both the Neoditomyia luciferin-like compound and SBF cross-react with purified Orfelia luciferase to produce light in the same blue region of the Orfelia luciferin-luciferase system (479 nm). We also checked for the presence of Orfelia-type luciferin in Arachnocampa luminosa and Aedes aegytpi larval bodies, but no traces were found. Molecular studies indicate that Neoditomyia sp. is phylogenetically closer to Keroplatus and Orfelia than to Arachnocampa species. The presence of luciferin and its associated binding protein in this non-bioluminescent keroplatid larva indicates that luciferin may display another important biochemical function in keroplatid larvae and suggests that bioluminescence could be a recently evolved trait in Keroplatidae.

Revisiting Coleoptera a + T-rich region: structural conservation, phylogenetic and phylogeographic approaches in mitochondrial control region of bioluminescent Elateridae species (Coleoptera).

The control region (CR) or A + T-rich region in Coleoptera mt genome is poorly characterized, including the Elateroidea bioluminescent species. Here, we provided the first attempt to characterize and compare the structure and organization of the CR of different species within Elateridae. We also revisited some sequenced Coleoptera CR and observed consensus T-stretches, non-conserved sequences near the stem-loop and unusual inner tRNAs-like sequences. All these features are probably involved in the replication start of the mt genome. The phylogenetic relationships in Elateridae bioluminescent groups using partial sequence of CR showed the monophyly of Pyrearinus pumilus group and Pyrearinus as a polyphyletic genus, corroborating our previous results. The wider genetic variation obtained by CR analysis could separate two different lineages that occur within P. termitilluminans populations. In Elateridae, the CR exhibited high polymorphism within and between populations, which was also observed in other Coleoptera species, suggesting that the CR could be described as a suitable molecular marker to be applied in phylogenetic and phylogeographic studies.

A new orange emitting luciferase from the Southern-Amazon Pyrophorus angustus (Coleoptera: Elateridae) click-beetle: structure and bioluminescence color relationship, evolutional and ecological considerations.

Bioluminescent click-beetles display a wide variation of bioluminescence colors ranging from green to orange, including an unusual intra-specific color variation in the Jamaican Pyrophorus plagiophthalamus. Recently, we collected individuals of the Pyrophorus angustus species from the Southern Amazon forest, in Brazil, which displays an orange light emitting abdominal lantern. This species was also previously described from Central America, but displaying a bioluminescence spectrum from 536 nm (dorsal) to 578 nm (ventral). The biogeographic variation of the bioluminescence color in this species could be an adaptation to environmental reflectance and inter/intraspecific sexual competition. Here, we cloned, sequenced, characterized and performed site-direct mutagenesis of this new orange emitting luciferase. The in vitro luciferase spectrum displayed a peak at 594 nm, KM values for ATP and d-luciferin of 160 µM and 17 µM, respectively, and an optimum pH of approximately 8.5. Comparative multialignment and site-directed mutagenesis using different color emitting click-beetle luciferases from P. angustus, Fulgeochlizus bruchi and Pyrearinus termitilluminans luciferases cloned by our group showed an integral role of residue 247 in bioluminescence color modulation.

Organization and comparative analysis of the mitochondrial genomes of bioluminescent Elateroidea (Coleoptera: Polyphaga).

Mitochondrial genome organization in the Elateroidea superfamily (Coleoptera), which include the main families of bioluminescent beetles, has been poorly studied and lacking information about Phengodidae family. We sequenced the mitochondrial genomes of Neotropical Lampyridae (Bicellonycha lividipennis), Phengodidae (Brasilocerus sp.2 and Phrixothrix hirtus) and Elateridae (Pyrearinus termitilluminans, Hapsodrilus ignifer and Teslasena femoralis). All species had a typical insect mitochondrial genome except for the following: in the elaterid T. femoralis genome there is a non-coding region between NADH2 and tRNA-Trp; in the phengodids Brasilocerus sp.2 and P. hirtus genomes we did not find the tRNA-Ile and tRNA-Gln. The P. hirtus genome showed a ~1.6kb non-coding region, the rearrangement of tRNA-Tyr, a new tRNA-Leu copy, and several regions with higher AT contents. Phylogenetics analysis using Bayesian and ML models indicated that the Phengodidae+Rhagophthalmidae are closely related to Lampyridae family, and included Drilus flavescens (Drilidae) as an internal clade within Elateridae. This is the first report that compares the mitochondrial genomes organization of the three main families of bioluminescent Elateroidea, including the first Neotropical Lampyridae and Phengodidae. The losses of tRNAs, and translocation and duplication events found in Phengodidae mt genomes, mainly in P. hirtus, may indicate different evolutionary rates in these mitochondrial genomes. The mitophylogenomics analysis indicates the monophyly of the three bioluminescent families and a closer relationship between Lampyridae and Phengodidae/Rhagophthalmidae, in contrast with previous molecular analysis.

A transcriptional survey of the cDNA library of Macrolampis sp2 firefly lanterns (Coleoptera: Lampyridae).

The biochemistry of firefly bioluminescence is well understood; however, the molecular physiology of the lanterns is still poorly studied, especially the biosynthesis and origin of beetle luciferin which are almost unknown. Using a cDNA library previously constructed from Macrolampis sp2 lanterns, we randomly selected and sequenced 572 cDNAs in order to have a first transcriptional profile of the most represented messages found in the lanterns and therefore to better understand their molecular physiology. As expected, high percentage of the gene products (~22%) displayed high similarity with Coleoptera genome products. About 7% represented mitochondrial genes, including several copies of cytochrome oxidase, which are also expected for this tissue. Luciferase genes were especially abundant, representing ca 2% of the products. Gene products involved with cysteine and sulfur metabolism such as the cystathionine ß-lyase and the S-adenosylmethionine synthetase were abundant. Noteworthy, an abundance of proteins involved with hormone metabolism was found, suggesting a possible link between bioluminescence and hormone metabolism.

Luciferase from Fulgeochlizus bruchi (Coleoptera:Elateridae), a Brazilian click-beetle with a single abdominal lantern: molecular evolution, biological function and comparison with other click-beetle luciferases.

Bioluminescent click-beetles emit a wide range of bioluminescence colors (λ(Max) = 534-594 nm) from thoracic and abdominal lanterns, which are used for courtship. Only the luciferases from Pyrophorus and Pyrearinus species were cloned and sequenced. The Brazilian Fulgeochlizus bruchi click-beetle, which inhabits the Central-west Cerrado (Savannas), is noteworthy because, differently from other click-beetles, the adult stage displays only a functional abdominal lantern, which produces a bright green bioluminescence for sexual attraction purposes, and lacks functional thoracic lanterns. We cloned the cDNA for the abdominal lantern luciferase of this species. Notably, the primary sequence of this luciferase showed slightly higher identity with the green emitting dorsal lantern luciferases of the Pyrophorus genus instead of the abdominal lanterns luciferases. This luciferase displays a blue-shifted spectrum (λ(Max) = 540 nm), which is pH-insensitive from pH 7.5 to 9.5 and undergoes a slight red shift and broadening above this pH; the lowest K(M) for luciferin among studied click-beetle luciferases, and the highest optimum pH (9.0) ever reported for a beetle luciferase. At pH 9.0, the K(M) for luciferin increases, showing a decrease of affinity for this substrate, despite the higher activity. The slow luminescence decay rate of F. bruchi luciferase in vitro reaction could be an adaptation of this luciferase for the long and sustained in vivo luminescence display of the click-beetle during the courtship, and could be useful for in vivo intracellular imaging.