Protocol for the establishment of a serine integrase-based platform for functional validation of genetic switch controllers in eukaryotic cells.

Serine integrases (Ints) are a family of site-specific recombinases (SSRs) encoded by some bacteriophages to integrate their genetic material into the genome of a host. Their ability to rearrange DNA sequences in different ways including inversion, excision, or insertion with no help from endogenous molecular machinery, confers important biotechnological value as genetic editing tools with high host plasticity. Despite advances in their use in prokaryotic cells, only a few Ints are currently used as gene editors in eukaryotes, partly due to the functional loss and cytotoxicity presented by some candidates in more complex organisms. To help expand the number of Ints available for the assembly of more complex multifunctional circuits in eukaryotic cells, this protocol describes a platform for the assembly and functional screening of serine-integrase-based genetic switches designed to control gene expression by directional inversions of DNA sequence orientation. The system consists of two sets of plasmids, an effector module and a reporter module, both sets assembled with regulatory components (as promoter and terminator regions) appropriate for expression in mammals, including humans, and plants. The complete method involves plasmid design, DNA delivery, testing and both molecular and phenotypical assessment of results. This platform presents a suitable workflow for the identification and functional validation of new tools for the genetic regulation and reprogramming of organisms with importance in different fields, from medical applications to crop enhancement, as shown by the initial results obtained. This protocol can be completed in 4 weeks for mammalian cells or up to 8 weeks for plant cells, considering cell culture or plant growth time.

DeepReg: a deep learning hybrid model for predicting transcription factors in eukaryotic and prokaryotic genomes.

Deep learning models (DLMs) have gained importance in predicting, detecting, translating, and classifying a diversity of inputs. In bioinformatics, DLMs have been used to predict protein structures, transcription factor-binding sites, and promoters. In this work, we propose a hybrid model to identify transcription factors (TFs) among prokaryotic and eukaryotic protein sequences, named Deep Regulation (DeepReg) model. Two architectures were used in the DL model: a convolutional neural network (CNN), and a bidirectional long-short-term memory (BiLSTM). DeepReg reached a precision of 0.99, a recall of 0.97, and an F1-score of 0.98. The quality of our predictions, the bias-variance trade-off approach, and the characterization of new TF predictions were evaluated and compared against those produced by DeepTFactor, as well as against experimental data from three model organisms. Predictions based on our DLM tended to exhibit less variance and bias than those from DeepTFactor, thus increasing reliability and decreasing overfitting.

Reactivity of DENV-positive sera against recombinant envelope proteins produced in bacteria and eukaryotic cells.

Dengue is a mosquito-borne disease endemic in many tropical and subtropical countries. It is caused by the dengue virus (DENV) that can be classified into 4 different serotypes (DENV-1-4). Early diagnosis and management can reduce morbidity and mortality rates of severe forms of the disease, as well as decrease the risk of larger outbreaks. Hiperendemicity in some regions of the world and the possibility that some people develop a more severe form of disease after a secondary infection caused by antibody-dependent enhancement justify the need to understand more thoroughly the antibody response induced against the virus. Here, we successfully produced a recombinant DENV-2 envelope (E) protein and its domains (EDI/II and EDIII) in two distinct expression systems: the Drosophila S2 insect cell system and the BL21 (DE3) pLySs bacterial system. We then evaluated the reactivity of sera from patients previously infected with DENV to each recombinant protein and to each domain separately. Our results show that the E protein produced in Drosophila S2 cells is recognized more frequently than the protein produced in bacteria. However, the recognition of E protein produced in bacteria correlates better with the DENV-2 sera neutralization capacity. The results described here emphasize the differences observed when antigens produced in bacteria or eukaryotic cells are used and may be useful to gain more insight into the humoral immune responses induced by dengue infection.

TRAP-SEQ of Eukaryotic Translatomes Applied to the Detection of Polysome-Associated Long Noncoding RNAs.

Translating ribosome affinity purification (TRAP) technology allows the isolation of polysomal complexes and the RNAs associated with at least one 80S ribosome. TRAP consists of the stabilization and affinity purification of polysomes containing a tagged version of a ribosomal protein. Quantitative assessment of the TRAP RNA is achieved by direct sequencing (TRAP-SEQ), which provides accurate quantitation of ribosome-associated RNAs, including long noncoding RNAs (lncRNAs). Here we present an updated procedure for TRAP-SEQ, as well as a primary analysis guide for identification of ribosome-associated lncRNAs. This methodology enables the study of dynamic association of lncRNAs by assessing rapid changes in their transcript levels in polysomes at organ or cell-type level, during development, or in response to endogenous or exogenous stimuli.

Protein Dynamics in Phosphoryl-Transfer Signaling Mediated by Two-Component Systems.

The ability to perceive the environment, an essential attribute in living organisms, is linked to the evolution of signaling proteins that recognize specific signals and execute predetermined responses. Such proteins constitute concerted systems that can be as simple as a unique protein, able to recognize a ligand and exert a phenotypic change, or extremely complex pathways engaging dozens of different proteins which act in coordination with feedback loops and signal modulation. To understand how cells sense their surroundings and mount specific adaptive responses, we need to decipher the molecular workings of signal recognition, internalization, transfer, and conversion into chemical changes inside the cell. Protein allostery and dynamics play a central role. Here, we review recent progress on the study of two-component systems, important signaling machineries of prokaryotes and lower eukaryotes. Such systems implicate a sensory histidine kinase and a separate response regulator protein. Both components exploit protein flexibility to effect specific conformational rearrangements, modulating protein-protein interactions, and ultimately transmitting information accurately. Recent work has revealed how histidine kinases switch between discrete functional states according to the presence or absence of the signal, shifting key amino acid positions that define their catalytic activity. In concert with the cognate response regulator's allosteric changes, the phosphoryl-transfer flow during the signaling process is exquisitely fine-tuned for proper specificity, efficiency and directionality.

Virus Control of Cell Metabolism for Replication and Evasion of Host Immune Responses.

Over the last decade, there has been significant advances in the understanding of the cross-talk between metabolism and immune responses. It is now evident that immune cell effector function strongly depends on the metabolic pathway in which cells are engaged in at a particular point in time, the activation conditions, and the cell microenvironment. It is also clear that some metabolic intermediates have signaling as well as effector properties and, hence, topics such as immunometabolism, metabolic reprograming, and metabolic symbiosis (among others) have emerged. Viruses completely rely on their host's cell energy and molecular machinery to enter, multiply, and exit for a new round of infection. This review explores how viruses mimic, exploit or interfere with host cell metabolic pathways and how, in doing so, they may evade immune responses. It offers a brief outline of key metabolic pathways, mitochondrial function and metabolism-related signaling pathways, followed by examples of the mechanisms by which several viral proteins regulate host cell metabolic activity.

Exploring the Remote Ties between Helitron Transposases and Other Rolling-Circle Replication Proteins.

Rolling-circle replication (RCR) elements constitute a diverse group that includes viruses, plasmids, and transposons, present in hosts from all domains of life. Eukaryotic RCR transposons, also known as Helitrons, are found in species from all eukaryotic kingdoms, sometimes representing a large portion of their genomes. Despite the impact of Helitrons on their hosts, knowledge about their relationship with other RCR elements is still elusive. Here, we compared the endonuclease domain sequence of Helitron transposases with the corresponding region from RCR proteins found in a wide variety of mobile genetic elements. To do that, we used a stepwise alignment approach followed by phylogenetic and multidimensional scaling analyses. Although it has been suggested that Helitrons might have originated from prokaryotic transposons or eukaryotic viruses, our results indicate that Helitron transposases share more similarities with proteins from prokaryotic viruses and plasmids instead. We also provide evidence for the division of RCR endonucleases into three groups (Y1, Y2, and Yx), covering the whole diversity of this protein family. Together, these results point to prokaryotic elements as the likely closest ancestors of eukaryotic RCR transposons, and further demonstrate the fluidity that characterizes the boundaries separating viruses, plasmids, and transposons.

N-glycosylation Triggers a Dual Selection Pressure in Eukaryotic Secretory Proteins.

Nearly one third of the eukaryotic proteome traverses the secretory pathway and most of these proteins are N-glycosylated in the lumen of the endoplasmic reticulum. N-glycans fulfill multiple structural and biological functions, and are crucial for productive folding of many glycoproteins. N-glycosylation involves the attachment of an oligosaccharide to selected asparagine residues in the sequence N-X-S/T (X ≠ P), a motif known as an N-glycosylation'sequon'. Mutations that create novel sequons can cause disease due to the destabilizing effect of a bulky N-glycan. Thus, an analogous process must have occurred during evolution, whenever ancestrally cytosolic proteins were recruited to the secretory pathway. Here, we show that during evolution N-glycosylation triggered a dual selection pressure on secretory pathway proteins: while sequons were positively selected in solvent exposed regions, they were almost completely eliminated from buried sites. This process is one of the sharpest evolutionary signatures of secretory pathway proteins, and was therefore critical for the evolution of an efficient secretory pathway.

Shock Wave-Induced Damage and Poration in Eukaryotic Cell Membranes.

Shock waves are known to permeabilize eukaryotic cell membranes, which may be a powerful tool for a variety of drug delivery applications. However, the mechanisms involved in shock wave-mediated membrane permeabilization are still poorly understood. In this study, the effects on both the permeability and the ultrastructural features of two human cell lineages were investigated after the application of underwater shock waves in vitro. Scanning Electron Microscopy of cells derived from a human embryo kidney (HEK)-293 and Michigan Cancer Foundation (MCF)-7 cells, an immortalized culture derived from human breast adenocarcinoma, showed a small amount of microvilli (as compared to control cells), the presence of hole-like structures, and a decrease in cell size after shock wave exposure. Interestingly, these effects were accompanied by the permeabilization of acid and macromolecular dyes and gene transfection. Trypan blue exclusion assays indicated that cell membranes were porated during shock wave treatment but resealed after a few seconds. Deformations of the cell membrane lasted for at least 5 min, allowing their observation in fixed cells. For each cell line, different shock wave parameters were needed to achieve cell membrane poration. This difference was correlated to successful gene transfection by shock waves. Our results demonstrate, for the first time, that shock waves induce transient micro- and submicrosized deformations at the cell membrane, leading to cell transfection and cell survival. They also indicate that ultrastructural analyses of cell surfaces may constitute a useful way to match the use of shock waves to different cells and settings.

Mediator: A key regulator of plant development.

Mediator is a multiprotein complex that regulates transcription at the level of RNA pol II assembly, as well as through regulation of chromatin architecture, RNA processing and recruitment of epigenetic marks. Though its modular structure is conserved in eukaryotes, its subunit composition has diverged during evolution and varies in response to environmental and tissue-specific inputs, suggesting different functions for each subunit and/or Mediator conformation. In animals, Mediator has been implicated in the control of differentiation and morphogenesis through modulation of numerous signaling pathways. In plants, studies have revealed roles for Mediator in regulation of cell division, cell fate and organogenesis, as well as developmental timing and hormone responses. We begin this review with an overview of biochemical mechanisms of yeast and animal Mediator that are likely to be conserved in all eukaryotes, as well as a brief discussion of the role of Mediator in animal development. We then present a comprehensive review of studies of the role of Mediator in plant development. Finally, we point to important questions for future research on the role of Mediator as a master coordinator of development.

Gene regulation and noise reduction by coupling of stochastic processes.

Here we characterize the low-noise regime of a stochastic model for a negative self-regulating binary gene. The model has two stochastic variables, the protein number and the state of the gene. Each state of the gene behaves as a protein source governed by a Poisson process. The coupling between the two gene states depends on protein number. This fact has a very important implication: There exist protein production regimes characterized by sub-Poissonian noise because of negative covariance between the two stochastic variables of the model. Hence the protein numbers obey a probability distribution that has a peak that is sharper than those of the two coupled Poisson processes that are combined to produce it. Biochemically, the noise reduction in protein number occurs when the switching of the genetic state is more rapid than protein synthesis or degradation. We consider the chemical reaction rates necessary for Poisson and sub-Poisson processes in prokaryotes and eucaryotes. Our results suggest that the coupling of multiple stochastic processes in a negative covariance regime might be a widespread mechanism for noise reduction.

Boron nitride nanotubes chemically functionalized with glycol chitosan for gene transfection in eukaryotic cell lines.

Nanostructured materials have been widely studied concerning their potential biomedical applications, primarily to selectively carry specific drugs or molecules within a tissue or organ. In this context, boron nitride nanotubes (BNNTs) have generated considerable interest in the scientific community because of their unique properties, presenting good chemical inertness and high thermal stability. Among the many applications proposed for BNNTs in the biomedical field in recent years, the most important include their use as biosensors, nanovectors for the delivery of proteins, drugs, and genes. In the present study, BNNTs were synthesized, purified, and functionalized with glycol chitosan through a chemical process, yielding the BNNT-GC. The size of BNNT-GC was reduced using an ultrasound probe. Two samples with different sizes were selected for in vitro assays. The nanostructures were characterized by transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FTIR), thermal analysis (TGA), and dynamic light scattering (DLS). The in vitro assays MTT and neutral red (NR) were performed with NIH-3T3 and A549 cell lines and demonstrated that this material is not cytotoxic. Furthermore, the BNNT-GC was applied in gene transfection of plasmid pIRES containing a gene region that express a green fluorescent protein (GFP) in NIH-3T3 and A549 cell lines. The gene transfection was characterized by fluorescent protein produced in the cells and pictured by fluorescent microscopy. Our results suggest that BNNT-GC has moderate stability and presents great potential as a gene carrier agent in nonviral-based therapy, with low cytotoxicity and good transfection efficiency.

Construction of a eukaryotic expression vector for pEGFP-FST and its biological activity in duck myoblasts

Background Follistatin (FST), a secreted glycoprotein, is intrinsically linked to muscle hypertrophy. To explore the function of duck FST in myoblast proliferation and differentiation, the pEGFP-FST eukaryotic expression vector was constructed and identified. The biological activities of this vector were analyzed by transfecting pEGFP-FST into cultured duck myoblasts using Lipofectamine™ 2000 and subsequently determining the mRNA expression profiles of FST and myostatin (MSTN). Results The duck pEGFP-FST vector was successfully constructed and was confirmed to have high liposome-mediated transfection efficiency in duck myoblasts. Additionally, myoblasts transfected with pEGFP-FST had a higher biological activity. Significantly, the overexpression of FST in these cells significantly inhibited the mRNA expression of MSTN (a target gene that is negatively regulated by FST). Conclusions The duck pEGFP-FST vector has been constructed successfully and exhibits biological activity by promoting myoblast proliferation and differentiation in vitro.

Differential effects of CSF-1R D802V and KIT D816V homologous mutations on receptor tertiary structure and allosteric communication.

The colony stimulating factor-1 receptor (CSF-1R) and the stem cell factor receptor KIT, type III receptor tyrosine kinases (RTKs), are important mediators of signal transduction. The normal functions of these receptors can be compromised by gain-of-function mutations associated with different physiopatological impacts. Whereas KIT D816V/H mutation is a well-characterized oncogenic event and principal cause of systemic mastocytosis, the homologous CSF-1R D802V has not been identified in human cancers. The KIT D816V oncogenic mutation triggers resistance to the RTK inhibitor Imatinib used as first line treatment against chronic myeloid leukemia and gastrointestinal tumors. CSF-1R is also sensitive to Imatinib and this sensitivity is altered by mutation D802V. Previous in silico characterization of the D816V mutation in KIT evidenced that the mutation caused a structure reorganization of the juxtamembrane region (JMR) and facilitated its departure from the kinase domain (KD). In this study, we showed that the equivalent CSF-1R D802V mutation does not promote such structural effects on the JMR despite of a reduction on some key H-bonds interactions controlling the JMR binding to the KD. In addition, this mutation disrupts the allosteric communication between two essential regulatory fragments of the receptors, the JMR and the A-loop. Nevertheless, the mutation-induced shift towards an active conformation observed in KIT D816V is not observed in CSF-1R D802V. The distinct impact of equivalent mutation in two homologous RTKs could be associated with the sequence difference between both receptors in the native form, particularly in the JMR region. A local mutation-induced perturbation on the A-loop structure observed in both receptors indicates the stabilization of an inactive non-inhibited form, which Imatinib cannot bind.

Use of the harmonic mean to the determination of dissociation constants of stereoisomeric mixtures of biologically active compounds.

Herein we introduce the derivation of a mathematical expression to evaluate the dissociation constant of a mixture of stereoisomers in equal amounts (KdMIX), when the corresponding dissociation constants (Kd) or medium response (MR50) of the pure stereoisomers are known; the final equation takes the form of the harmonic mean. In order to validate the equation, we carried out a bibliographic search of experimental data of enantiomeric molecules with biological activity, considering the Kd's or MR50's of the isolated enantiomers as well as that of the racemate. The comparisons between the experimental dissociation constants of the mixtures (KdEXP or MR50EXP) and the calculated values (KdMIX or MR50MIX) were consistent; the similarity between these values is supported through statistical analyses of group comparison and simple linear correlation. The equation we obtained, which corresponds to the harmonic mean, was used to predict the values of KdMIX (or MR50MIX) or Kd (or MR50) in systems when only two of the experimental values are known: either the dissociation constants of both enantiomers or the Kd (or MR50) of one of the enantiomers and dissociation constant of the racemate.

Preferential duplication of intermodular hub genes: an evolutionary signature in eukaryotes genome networks.

Whole genome protein-protein association networks are not random and their topological properties stem from genome evolution mechanisms. In fact, more connected, but less clustered proteins are related to genes that, in general, present more paralogs as compared to other genes, indicating frequent previous gene duplication episodes. On the other hand, genes related to conserved biological functions present few or no paralogs and yield proteins that are highly connected and clustered. These general network characteristics must have an evolutionary explanation. Considering data from STRING database, we present here experimental evidence that, more than not being scale free, protein degree distributions of organisms present an increased probability for high degree nodes. Furthermore, based on this experimental evidence, we propose a simulation model for genome evolution, where genes in a network are either acquired de novo using a preferential attachment rule, or duplicated with a probability that linearly grows with gene degree and decreases with its clustering coefficient. For the first time a model yields results that simultaneously describe different topological distributions. Also, this model correctly predicts that, to produce protein-protein association networks with number of links and number of nodes in the observed range for Eukaryotes, it is necessary 90% of gene duplication and 10% of de novo gene acquisition. This scenario implies a universal mechanism for genome evolution.

Toxicity of bovicin HC5 against mammalian cell lines and the role of cholesterol in bacteriocin activity.

Bacteriocins are ribosomally synthesized antimicrobial peptides produced by Bacteria and some Archaea. The assessment of the toxic potential of antimicrobial peptides is important in order to apply these peptides on an industrial scale. The aim of the present study was to investigate the in vitro cytotoxic and haemolytic potential of bovicin HC5, as well as to determine whether cholesterol influences bacteriocin activity on model membranes. Nisin, for which the mechanism of action is well described, was used as a reference peptide in our assays. The viability of three distinct eukaryotic cell lines treated with bovicin HC5 or nisin was analysed by using the MTT assay and cellular morphological changes were determined by light microscopy. The haemolytic potential was evaluated by using the haemoglobin liberation assay and the role of cholesterol on bacteriocin activity was examined by using model membranes composed of DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine) and DPoPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine). The IC(50) of bovicin HC5 and nisin against Vero cells was 65.42 and 13.48 µM, respectively. When the MTT assay was performed with MCF-7 and HepG2 cells, the IC(50) obtained for bovicin HC5 was 279.39 and 289.30 µM, respectively, while for nisin these values were 105.46 and 112.25 µM. The haemolytic activity of bovicin HC5 against eukaryotic cells was always lower than that determined for nisin. The presence of cholesterol did not influence the activity of either bacteriocin on DOPC model membranes, but nisin showed reduced carboxyfluorescein leakage in DPoPC membranes containing cholesterol. In conclusion, bovicin HC5 only exerted cytotoxic effects at concentrations that were greater than the concentration needed for its biological activity, and the presence of cholesterol did not affect its interaction with model membranes.

Adaptive horizontal transfer of a bacterial gene to an invasive insect pest of coffee.

Horizontal gene transfer (HGT) involves the nonsexual transmission of genetic material across species boundaries. Although often detected in prokaryotes, examples of HGT involving animals are relatively rare, and any evolutionary advantage conferred to the recipient is typically obscure. We identified a gene (HhMAN1) from the coffee berry borer beetle, Hypothenemus hampei, a devastating pest of coffee, which shows clear evidence of HGT from bacteria. HhMAN1 encodes a mannanase, representing a class of glycosyl hydrolases that has not previously been reported in insects. Recombinant HhMAN1 protein hydrolyzes coffee berry galactomannan, the major storage polysaccharide in this species and the presumed food of H. hampei. HhMAN1 was found to be widespread in a broad biogeographic survey of H. hampei accessions, indicating that the HGT event occurred before radiation of the insect from West Africa to Asia and South America. However, the gene was not detected in the closely related species H. obscurus (the tropical nut borer or "false berry borer"), which does not colonize coffee beans. Thus, HGT of HhMAN1 from bacteria represents a likely adaptation to a specific ecological niche and may have been promoted by intensive agricultural practices.

BC047440 antisense eukaryotic expression vectors inhibited HepG2 cell proliferation and suppressed xenograft tumorigenicity

The biological functions of the BC047440 gene highly expressed by hepatocellular carcinoma (HCC) are unknown. The objective of this study was to reconstruct antisense eukaryotic expression vectors of the gene for inhibiting HepG2 cell proliferation and suppressing their xenograft tumorigenicity. The full-length BC047440 cDNA was cloned from human primary HCC by RT-PCR. BC047440 gene fragments were ligated with pMD18-T simple vectors and subsequent pcDNA3.1(+) plasmids to construct the recombinant antisense eukaryotic vector pcDNA3.1(+)BC047440AS. The endogenous BC047440 mRNA abundance in target gene-transfected, vector-transfected and naive HepG2 cells was semiquantitatively analyzed by RT-PCR and cell proliferation was measured by the MTT assay. Cell cycle distribution and apoptosis were profiled by flow cytometry. The in vivo xenograft experiment was performed on nude mice to examine the effects of antisense vector on tumorigenicity. BC047440 cDNA fragments were reversely inserted into pcDNA3.1(+) plasmids. The antisense vector significantly reduced the endogenous BC047440 mRNA abundance by 41 percent in HepG2 cells and inhibited their proliferation in vitro (P < 0.01). More cells were arrested by the antisense vector at the G1 phase in an apoptosis-independent manner (P = 0.014). Additionally, transfection with pcDNA3.1(+)BC047440AS significantly reduced the xenograft tumorigenicity in nude mice. As a novel cell cycle regulator associated with HCC, the BC047440 gene was involved in cell proliferation in vitro and xenograft tumorigenicity in vivo through apoptosis-independent mechanisms.