Combination of error-prone PCR (epPCR) and Circular Polymerase Extension Cloning (CPEC) for improving the coverage of random mutagenesis libraries.

Random mutagenesis, such as error-prone PCR (epPCR), is a technique capable of generating a wide variety of a single gene. However, epPCR can produce a large number of mutated gene variants, posing a challenge in ligating these mutated PCR products into plasmid vectors. Typically, the primers for mutagenic PCRs incorporate artificial restriction enzyme sites compatible with chosen plasmids. Products are cleaved and ligated to linearized plasmids, then recircularized by DNA ligase. However, this cut-and-paste method known as ligation-dependent process cloning (LDCP), has limited efficiency, as the loss of potential mutants is inevitable leading to a significant reduction in the library's breadth. An alternative to LDCP is the circular polymerase extension cloning (CPEC) method. This technique involves a reaction where a high-fidelity DNA polymerase extends the overlapping regions between the insert and vector, forming a circular molecule. In this study, our objective was to compare the traditional cut-and-paste enzymatic method with CPEC in producing a variant library from the gene encoding the red fluorescent protein (DsRed2) obtained by epPCR. Our findings suggest that CPEC can accelerate the cloning process in gene library generation, enabling the acquisition of a greater number of gene variants compared to methods reliant on restriction enzymes.

Course on Breeding, Handling, and Experimental Procedures of Zebrafish (Danio rerio): Training, Refinement, Paradigm Shift, and Dissemination of Knowledge from Brazil to the World.

Currently, in Brazil, all researchers involved in animal experimentation must undergo training in laboratory animal science to stay updated on biology, methodology, ethics, and legal considerations related to the use of animals. The training program presented in this study not only aims to fulfill a legal obligation but also intends to train students and professionals to effectively care for their biomodels. It seeks to help them understand the importance of this care, both for the welfare of the animals and for the results of their projects. In total, 58 participants were present at the event (pre-event and full-time course). These participants consisted students and professionals from 11 institutions and 5 different countries. These numbers demonstrate the successful attainment of the desired capillarity in the scientific community and the posterior dissemination of knowledge. Through this course, it was possible to train the participants and raise their awareness about the importance of applying scientific knowledge in their daily practices to maintain the animals, ensuring the welfare of the models and refining the research. Finally, the program presented in this study, as well as the strategies adopted, can serve as a model for other institutions aiming to achieve similar results.

Red Fluorescent Protein Variant with a Dual-Peak Emission of Fluorescence.

The marine environment is a rich reservoir of diverse biological entities, many of which possess unique properties that are of immense value to biotechnological applications. One such example is the red fluorescent protein derived from the coral Discosoma sp. This protein, encoded by the DsRed gene, has been the subject of extensive research due to its potential applications in various fields. In the study, a variant of the red fluorescent protein was generated through random mutagenesis using the DsRed2 gene as a template. The process employed error-prone PCR (epPCR) to introduce random mutations, leading to the isolation of twelve gene variants. Among these, one variant stood out due to its unique spectral properties, exhibiting dual fluorescence emission at both 480 nm (green) and 550 nm (red). This novel variant was expressed in both Escherichia coli and zebrafish (Danio rerio) muscle, confirming the dual fluorescence emission in both model systems. One of the immediate applications of this novel protein variant is in ornamental aquaculture. The dual fluorescence can serve as a unique marker or trait, enhancing the aesthetic appeal of aquatic species in ornamental settings.

Effects of chemotherapeutic drugs on the antioxidant capacity of human erythroleukemia cells with MDR phenotype.

In this work, we identified that different chemotherapeutic drugs may select cells with different antioxidant capacities. For this, we evaluated the sensitivity of two multidrug-resistant (MDR) erythroleukemia cell lines: Lucena (resistant to vincristine, VCR) and FEPS (resistant to daunorubicin, DNR) derived from the same sensitive cell K562 (non-MDR) to hydrogen peroxide. In addition, we evaluated how the cell lines respond to the oxidizing agent in the absence of VCR/DNR. In absence of VCR, Lucena drastically decreases cell viability when exposed to hydrogen peroxide, while FEPS is not affected even without DNR. To analyze whether selection by different chemotherapeutic agents may generate altered energetic demands, we analyzed the production of reactive oxygen species (ROS) and the relative expression of the glucose transporter 1 gene (glut1). We observed that the selection through DNR apparently generates a higher energy demand than VCR. High levels of transcription factors genes expression (nrf2, hif-1α, and oct4) were kept even when the DNR is withdrawn from the FEPS culture for one month. Together, these results indicate that DNR selects cells with greater ability to express the major transcription factors related to the antioxidant defense system and the main extrusion pump (ABCB1) related to the MDR phenotype. Taking into account that the antioxidant capacity of tumor cells is closely related to resistance to multiple drugs, it is evident that endogenous antioxidant molecules may be targets for the development of new anticancer drugs.

Growth Hormone Overexpression Induces Hyperphagia and Intestinal Morphophysiological Adaptations to Improve Nutrient Uptake in Zebrafish.

The excess of circulating growth hormone (GH) in most transgenic animals implies mandatory growth resulting in higher metabolic demand. Considering that the intestine is the main organ responsible for the digestion, absorption, and direction of dietary nutrients to other tissues, this study aimed to investigate the mechanisms by which gh overexpression modulates the intestine to support higher growth. For this purpose, we designed an 8-weeks feeding trial to evaluate growth parameters, feed intake, and intestinal morphometric indices in the adult gh-transgenic zebrafish (Danio rerio) model. To access the sensitivity of the intestine to the excess of circulating GH, the messenger RNA (mRNA) expression of intestine GH receptors (GHRs) (ghra and ghrb) was analyzed. In addition, the expression of insulin-like growth factor 1a (igf1a) and genes encoding for di and tripeptide transporters (pept1a and pept1b) were assessed. Gh-transgenic zebrafish had better growth performance and higher feed intake compared to non-transgenic sibling controls. Chronic excess of GH upregulates the expression of its cognate receptor (ghrb) and the main growth factor related to trophic effects in the intestine (igf1a). Moreover, transgenic zebrafish showed an increased intestinal absorptive area and higher expression of crucial genes related to the absorption of products from meal protein degradation. These results reinforce the ability of GH to modulate intestinal morphology and the mechanisms of assimilation of nutrients to sustain the energy demand for the continuous growth induced by the excess of circulating GH.

First Record of Clinostomum sp. (Digenea: Clinostomidae) in Danio rerio (Actinopterygii: Cyprinidae) and the Implication of Using Zebrafish from Pet Stores on Research.

Many scientific studies still use zebrafish from pet stores as animal models, even cutting-edge researches. However, these animals differ genotypically and phenotypically between them. The importance of the use of standardized models is widely recognized. Besides that, another consequence of using zebrafish from unknown origins is the acquisition of parasitized animals. This study aimed to relate the infection by Clinostomum sp. in zebrafish. Animals sold as "high standard" were acquired from a commercial company. Swimming alterations and superficial yellow dots were observed in five zebrafish with clinical signs, which were isolated, euthanized, and necropsied. Muscular yellow cysts with metacercaria associated with lesions were observed. The muscular cysts were responsible for the superficial yellow dots as well as the swimming alterations. The prevalence was 2.5%, and the mean infection intensity was 7 digeneans/host. The cysts measured a mean of 1251.43 µm long × 784.28 µm wide. Metacercariae measured a mean of 4847 µm long × 1353 µm wide. This first report about infection by Clinostomum sp. in zebrafish is globally relevant since the host and the parasite genus currently overlap worldwide. Furthermore, this study sheds light on the importance of the specific pathogen-free commercial creations or laboratory-reared zebrafish for research.

Zebrafish (Danio rerio) ability to activate ABCC transporters after exposure to glyphosate and its formulation Roundup Transorb®.

The emergent demand for food production has increased the widespread use of pesticides, especially glyphosate-based herbicides as they can protect different types of crops, especially transgenic ones. Molecules of glyphosate have been found in water bodies around the world, and its presence can cause negative effects on non-target organisms, such as fish. Glyphosate toxicity appears to be systemic in fish but does not affect their organs equally. Also, its formulations can be more toxic than pure glyphosate. In this sense, we investigated if these variations in toxicity could be related to ATP binding cassette subfamily C (ABCC) transporters and the cellular detoxification capacity, following exposure to herbicides. Thus, adults of Danio rerio were exposed (24 and 96 h) to glyphosate and Roundup Transorb® (RT) at an environmental concentration of 0.1 mg/L, and the activity of ABCC proteins and gene expression of five isoforms of ABCC were analyzed. Glyphosate and RT exposure increased ABCC protein activity and gene expression up to 3-fold when compared to controls, indicating the activation of detoxification mechanisms. Only in the brain of D. rerio, the exposure to RT did not stimulate the activity of ABCC proteins, neither the expression of genes abcc1 and abcc4 that responded to the exposure to pure glyphosate. These results may suggest that the brain is more sensitive to RT than the other target-tissues since the mechanism of detoxification via ABCC transporters were not activated in this tissue as it was in the other.

Probiotic expressing heterologous phytase improves the immune system and attenuates inflammatory response in zebrafish fed with a diet rich in soybean meal.

Although aquaculture is among the fastest growing food production sectors in the world, one of the bottlenecks for the continuity of its expansion is the dependence of animal protein on commercial feed formulations. Vegetable proteins are an alternative due to the low cost and high availability. However, this protein source is accompanied by a series of antinutritional and pro-inflammatory compounds, including phytate. Phytases can be added in feed for phytate degradation and increase nutrient availability. However, the use of purified phytases significantly increases the production costs. An interesting alternative is to use probiotics genetically modified as bioreactors for phytase production. In the present study, a strain of Bacillus subtilis secreting a fungal phytase was used to evaluate the effect of a feed with high content of soybean meal on zebrafish (Danio rerio). We analysed the condition factor (K) of fish, and the expression of genes related to the immune system, inflammatory response and oxidative. stress. The results obtained demonstrate that the transgenic probiotic was efficient in improving the fish condition factor, stimulating the immune system, reducing the inflammatory response and oxidative stress. Thus, probiotics acting as phytase bioreactors can be considered an interesting tool for the adaptation of commercial species to feed of lower cost.

A comparison of classifiers for predicting the class color of fluorescent proteins.

Fluorescent proteins have been applied in a wide variety of fields ranging from basic science to industrial applications. Apart from the naturally occurring fluorescent proteins, there is a growing interest in genetically modified variants that emit light in a specific wavelength. Genetically modifying a protein is not an easy task, especially because the exchange of one residue by other has to achieve the desired property while maintaining protein stability. To help in the choice of residue exchange, computational methods are applied to predict function and stability of proteins. In this work we have prepared a dataset composed by 109 fluorescent proteins and tested four classical supervised classification algorithms: artificial neural networks (ANNs), decision trees (DTs), support vector machines (SVMs) and random forests (RFs). This is the first time that algorithms are compared in this task. Results of comparing the algorithm's performance shows that DT, SVM and RF were significantly better than ANNs, and RF was the best method in all the scenarios. However, the interpretability of DTs is highly relevant and can provide important clues about the mechanisms involved in protein color emission. The results are promising and indicate that the use of in silico methods can greatly reduce the time and cost of the in vitro experiments.

Synechococcus elongatus as a model of photosynthetic bioreactor for expression of recombinant ß-glucosidases.

BACKGROUND: The production of glucose from cellulose requires cellulases, which are obtained from decomposing microorganisms such as fungi and bacteria. Among the cellulases, ß-glucosidases convert cellobiose to glucose and have low concentration in commercial cocktails used for the production of second-generation (2G) ethanol. Genetic engineering can be used to produce recombinant ß-glucosidases, and cyanobacteria may be interesting bioreactors. These photosynthetic microorganisms can be cultured using CO2 emitted from the first-generation ethanol (1G) industry as a carbon source. In addition, vinasse, an effluent of 1G ethanol production, can be used as a source of nitrogen for cyanobacteria growth. Thus, photosynthetic bioreactors cannot only produce cellulases at a lower cost, but also reduce the environmental impact caused by residues of 1G ethanol production. RESULTS: In the present work, we produced a strain of Synechococcus elongatus capable of expressing high levels of a heterologous ß-glucosidase from a microorganism from the Amazonian soil. For this, the pET system was cloned into cyanobacteria genome. This system uses a dedicated T7 RNA polymerase for the expression of the gene of interest under the control of a nickel-inducible promoter. The results showed that the pET system functions efficiently in S. elongatus, once nickel induced T7 RNA polymerase expression which, in turn, induced expression of the gene of the microbial ß-glucosidase at high levels when compared with non-induced double transgenic strain. ß-glucosidase activity was more than sevenfold higher in the transformed cyanobacteria than in the wild-type strain. CONCLUSIONS: The T7 system promotes high expression levels of the cloned gene in S. elongatus, demonstrating that the arrangement in which an exclusive RNA polymerase is used for transcription of heterologous genes may contribute to high-level gene expression in cyanobacteria. This work was the first to demonstrate the use of cyanobacteria for the production of recombinant ß-glucosidases. This strategy could be an alternative to reduce the release of 1G ethanol by-products such as CO2 and vinasse, not only contributing to decrease the cost of ß-glucosidase production, but also mitigating the environmental impacts of ethanol industrial plants.

Comparing methods of genetic manipulation in Bacillus subtilis for expression of recombinant enzyme: Replicative or integrative (CRISPR-Cas9) plasmid?

The present study evaluated the stability of Bacillus subtilis strains transformed with a replicative or integrative plasmid (via CRISPR-Cas9) to express a recombinant phytase. Both transformation methods did not affect the growth of B. subtilis, but the stability of the construct and the enzymatic activity was reduced in the strain transformed with the replicative plasmid.

New Mechanistic Insight on the PIM-1 Kinase Inhibitor AZD1208 Using Multidrug Resistant Human Erythroleukemia Cell Lines and Molecular Docking Simulations.

BACKGROUND: PIM-1 is a kinase which has been related to the oncogenic processes like cell survival, proliferation, and multidrug resistance (MDR). This kinase is known for its ability to phosphorylate the main extrusion pump (ABCB1) related to the MDR phenotype. OBJECTIVE: In the present work, we tested a new mechanistic insight on the AZD1208 (PIM-1 specific inhibitor) under interaction with chemotherapy agents such as Daunorubicin (DNR) and Vincristine (VCR). MATERIALS AND METHODS: In order to verify a potential cytotoxic effect based on pharmacological synergism, two MDR cell lines were used: Lucena (resistant to VCR) and FEPS (resistant to DNR), both derived from the K562 non-MDR cell line, by MTT analyses. The activity of Pgp was ascertained by measuring accumulation and the directional flux of Rh123. Furthermore, we performed a molecular docking simulation to delve into the molecular mechanism of PIM-1 alone, and combined with chemotherapeutic agents (VCR and DNR). RESULTS: Our in vitro results have shown that AZD1208 alone decreases cell viability of MDR cells. However, co-exposure of AZD1208 and DNR or VCR reverses this effect. When we analyzed the ABCB1 activity AZD1208 alone was not able to affect the pump extrusion. Differently, co-exposure of AZD1208 and DNR or VCR impaired ABCB1 activity, which could be explained by compensatory expression of abcb1 or other extrusion pumps not analyzed here. Docking analysis showed that AZD1208 is capable of performing hydrophobic interactions with PIM-1 ATP- binding-site residues with stronger interaction-based negative free energy (FEB, kcal/mol) than the ATP itself, mimicking an ATP-competitive inhibitory pattern of interaction. On the same way, VCR and DNR may theoretically interact at the same biophysical environment of AZD1208 and also compete with ATP by the PIM-1 active site. These evidences suggest that AZD1208 may induce pharmacodynamic interaction with VCR and DNR, weakening its cytotoxic potential in the ATP-binding site from PIM-1 observed in the in vitro experiments. CONCLUSION: Finally, the current results could have a pre-clinical relevance potential in the rational polypharmacology strategies to prevent multiple-drugs resistance in human leukemia cancer therapy.

Modeling drug-drug interactions of AZD1208 with Vincristine and Daunorubicin on ligand-extrusion binding TMD-domains of multidrug resistance P-glycoprotein (ABCB1).

In the present study, the molecular docking mechanism based on pharmacodynamic interactions between the ligands AZD1208 and recognized chemotherapy agents (Vincristine and Daunorubicin) with human ATP-binding cassette (ABC) transporters (ABCB1) was investigated. For the first time, were combined an in silico approaches like molecular docking and ab initio computational simulation based on Density Functional Theory (DFT) to explain the drug-drug interaction mechanism of aforementioned chemotherapy ligands with the transmembrane ligand extrusion binding domains (TMDs) of ABCB1. In this regard, the theoretical pharmacodynamic interactions were characterized by using the Gibbs free energy (FEB, kcal/mol) from the best ABCB1-ligand docking complexes. The molecular docking results pointing that for the three chemotherapy ABCB1-ligand complexes are mainly based in non-covalent hydrophobic and hydrogen-bond interactions showing a similar toxicodynamic behavior in terms of strength of interaction (FEB, kcal/mol) and very close free binding energies when compared with the FEB-values of the ABCB1 specific-inhibitor (Rhodamine B) = -6.0 kcal/mol used as theoretical docking control to compare with FEB (AZD1208-ABCB1) ∼ FEB (Vincristine-ABCB1) ∼ FEB (Daunorubicin-ABCB1) -6.2 kcal/mol as average. Ramachandran plot suggests that the 3D-crystallographic structure from ABCB1 transporter can be efficiently-modeled with conformationally-favored Psi versus Phi dihedral angles for all key TMDs-residues. Though, the results of DFT-simulation corroborate the existence of drug-drug interaction between (AZD1208/Vincristine) > (AZD1208/Daunorubicin). These theoretical pieces of evidence have preclinical relevance potential in the design of the new drugs to understand the polypharmacology influence in the molecular mechanism of multiple-drugs resistance, contributing with a higher success in chemotherapy and prognosis of cancer patients.

Expression profile of glucose transport-related genes under chronic and acute exposure to growth hormone in zebrafish.

The brain is a highly demanding organ in terms of energy requirements, and precise regulatory mechanisms must operate to ensure adequate energy delivery to maintain normal neuronal activity. Of the energy-promoting substrates present in the circulation, glucose is preferred by the brain, and as with all other substrates, its utilization depends on the presence of humoral factors such as hormones including growth hormone (GH). Glucose enters the cells though specific transport proteins. Among all transporter families and subtypes described to date, the most studied ones are the glucose transporters (GLUTs). The aim of this study is to determine a possible relationship between GH and GLUTs. Therefore, we evaluated the effect of GH-transgenesis and recombinant GH injections upon GLUT expression in the brain of male zebrafish. Overall, the results demonstrated that increasing the GH concentrations above the normal level, via transgenesis or injection, in the fish may impair energy uptake by the brain. This appeared to occur through downregulation of most of the analyzed GLUTs.

Complete genome sequence of native Bacillus cereus strains isolated from intestinal tract of the crab Ucides sp.

Bacillus cereus is a gram positive bacterium with sporulation capacity. Here, we report the complete genome sequence of two native B. cereus strains (#25 and #29) isolated from intestinal tract of the crab Ucides sp. from Pacoti River in the State of Ceará, Brazil. The findings of this study might increase the molecular information for Bacillus strains. The data can be used in comparative analyses, origin and distribution, as well support for genetic engineering.

Fullerene (C60) particle size implications in neurotoxicity following infusion into the hippocampi of Wistar rats.

The buckminsterfullerene (C60) is considered as a relevant candidate for drug and gene delivery to the brain, once it has the ability to cross the blood-brain barrier. However, the biological implications of this nanomaterial are not fully understood, and its safety for intracerebral delivery is still debatable. In this study, we investigated if C60 particle size could alter its biological effects. For this, two aqueous C60 suspensions were used with maximum particle size up to 200nm and 450nm. The suspensions were injected in the hippocampus, the main brain structure involved in memory processing and spatial localization. In order to assess spatial learning, male Wistar rats were tested in Morris water maze, and the hippocampal BDNF protein levels and gene expression were analyzed. Animals treated with C60 up to 450nm demonstrated impaired spatial memory with a significant decrease in BDNF protein levels and gene expression. However, an enhanced antioxidant capacity was observed in both C60 treatments. A decrease in reactive oxygen species levels was observed in the treatments with suspensions containing particles measuring with up to 450nm. Thiobarbituric acid reactive substances, glutamate cysteine ligase, and glutathione levels showed no alterations among the different treatments. In conclusion, different particle sizes of the same nanomaterial can lead to different behavioral outcomes and biochemical parameters in brain tissue.

Reproductive parameters of double transgenic zebrafish (Danio rerio) males overexpressing both the growth hormone (GH) and its receptor (GHR).

Growth hormone (GH) transgenesis presents a high potential application in aquaculture. However, excess GH may have serious consequences due to pleiotropic actions. In order to study these effects in zebrafish (Danio rerio), two transgenic lines were developed. The first expresses GH ubiquitously and constitutively (F0104 line), while the second expresses the GH receptor in a muscle-specific manner (Myo-GHR line). Results from the F0104 line showed accelerated growth but increased reproductive difficulties, while Myo-GHR did not show the expected increase in muscle mass. Since the two lines appeared to display complementary characteristics, a double transgenic (GH/GHR) was created via crossing between them. This double transgenic displayed accelerated growth, however reproductive parameters remained uncertain. The objective of the present study was to determine the reproductive capacity of males of this new line, by evaluating sperm parameters, expression of spermatogenesis-related genes, and reproductive tests. Double transgenics showed a strong recovery in almost all sperm parameters analyzed when compared to the F0104 line. Gene expression analyses revealed that Anti-Müllerian Hormone gene (amh) appeared to be primarily responsible for this recovery. Reproductive tests showed that double transgenic males did not differ from non-transgenics. It is possible that GHR excess in the muscle tissues of double transgenics may have contributed to lower circulating GH levels and thus reduced the negative effects of this hormone with respect to reproduction. Therefore, it is clear that GH-transgenesis technology should take into account the need to obtain adequate levels of circulating hormone in order to achieve maximum growth with minimal negative side effects.

Effects of learning on mTOR pathway gene expression in the brain of zebrafish (Danio rerio) of different ages.

Target of rapamycin (TOR) is a protein kinase involved in the modulation of mRNA translation and, therefore, in the regulation of protein synthesis. In neurons, the role of TOR is particularly important in the consolidation of long-term memory (LTM). One of the modulators of TOR is brain-derived neurotrophic factor (BDNF), which activates the TOR signaling pathway to promote protein synthesis, synapse strengthening, and the creation of new neural networks. We investigated the gene expression pattern of this pathway during memory consolidation in zebrafish of different ages. Our findings demonstrate that TOR activation in old animals occurs in the early phase of consolidation, and follows a pattern identical to that of BDNF expression. In younger animals, this increase in activation did not occur, and changes in BDNF expression were also not so remarkable. Furthermore, the expression of the main proteins regulated by the synthesis of TOR (i.e., 4EBP and p70S6K) remained identical to that of TOR in all age groups.

GH indirectly enhances the regeneration of transgenic zebrafish fins through IGF2a and IGF2b.

The somatotropic axis, composed essentially of the growth hormone (GH) and insulin-like growth factors (IGFs), is the main regulator of somatic growth in vertebrates. However, these protein hormones are also involved in various other major physiological processes. Although the importance of IGFs in mechanisms involving tissue regeneration has already been established, little is known regarding the direct effects of GH in these processes. In this study, we used a transgenic zebrafish (Danio rerio) model, which overexpresses GH from the beta-actin constitutive promoter. The regenerative ability of the caudal fin was assessed after repeated amputations, as well as the expression of genes related to the GH/IGF axis. The results revealed that GH overexpression increased the regenerated area of the caudal fin in transgenic fish after the second amputation. Transgenic fish also presented a decrease in gene expression of the GH receptor (ghrb), in opposition to the increased expression of the IGF1 receptors (igf1ra and igf1rb). These results suggest that transgenic fish have a higher sensitivity to IGFs than to GH during fin regeneration. With respect to the different IGFs produced locally, a decrease in igf1a expression and a significant increase in both igf2a and igf2b expression was observed, suggesting that igf1a is not directly involved in fin regeneration. Overall, the results revealed that excess GH enhances fin regeneration in zebrafish through igf2a and igf2b expression, acting indirectly on this major physiological process.