Escherichia coli expression and characterization of alfa-amylase from Geobacillus thermodenitrificans DSM-465 / Expressão e caracterização de Escherichia coli de alfa-amilase de Geobacillus thermodenitrificans DSM-465

Alpha amylase, catalyzing the hydrolysis of starch is a ubiquitous enzyme with tremendous industrial applications. A 1698 bp gene coding for 565 amino acid amylase was PCR amplified from Geobacillus thermodenitrificans DSM-465, cloned in pET21a (+) plasmid, expressed in BL21 (DE3) strain of E. coli and characterized. The recombinant enzyme exhibited molecular weight of 63 kDa, optimum pH 8, optimum temperature 70°C, and KM value of 157.7µM. On pilot scale, the purified enzyme efficiently removed up to 95% starch from the cotton fabric indicating its desizing ability at high temperature. 3D model of enzyme built by Raptor-X and validated by Ramachandran plot appeared as a monomer having 31% α-helices, 15% β-sheets, and 52% loops. Docking studies have shown the best binding affinity of enzyme with amylopectin (∆G -10.59). According to our results, Asp 232, Glu274, Arg448, Glu385, Asp34, Asn276, and Arg175 constitute the potential active site of enzyme.

A alfa-amilase, que catalisa a hidrólise do amido, é uma enzima ubíqua com imensas aplicações industriais. Um gene de 1698 pb que codifica a amilase de 565 aminoácidos foi amplificado por PCR, a partir de Geobacillus thermodenitrificans DSM-465, clonado no plasmídeo pET21a (+), expresso na cepa BL21 (DE3) de E. coli e caracterizado. A enzima recombinante exibiu peso molecular de 63 kDa, pH ótimo igual a 8, temperatura ótima de 70° C e valor KM de 157,7 µM. Em escala piloto, a enzima purificada removeu com eficiência até 95% de amido do tecido de algodão, indicando sua capacidade de desengomagem em alta temperatura. O modelo 3D da enzima construída por Raptor-X e validada por Ramachandran plot apareceu como um monômero com 31% de hélices alfa, 15% de folhas beta e 52% de loops. Os estudos de docking mostraram melhor afinidade de ligação da enzima com amilopectina (∆G: - 10,59). De acordo com nossos resultados, Asp 232, Glu274, Arg448, Glu385, Asp34, Asn276 e Arg175 constituem o sítio ativo potencial da enzima.

Lentivirus vectors fail to deliver transgenes into bovine zygotes after co-incubation with sperm during in vitro fertilization / [Lentivírus falham na entrega de transgenes em zigotos bovinos após coincubação com espermatozoide, durante fertilização in vitro]

As vantagens dos animais transgênicos têm sido demonstradas em diferentes aplicações, entretanto muitas metodologias usadas para gerar animais geneticamente modificados (GM) apresentam baixas taxas de eficiência. O objetivo deste estudo foi avaliar a entrega dos vetores lentivirais (VLs) em zigotos durante a fertilização in vitro (FIV), para gerar embriões GM, com o gene da proteína verde fluorescente (GFP) ou do fator IX de coagulação humana (FIX). Vetores lentivirais com os genes GFP (pLGW-GFP-LV) ou FIX (pLWE2-FIX-LV) foram utilizados na FIV ou na cultura de embriões in vitro (CIV). A coincubação de pLWE2-FIX-LV com espermatozoides e complexos oócitos-células do cumulus (COCs) durante a FIV diminuiu (P<0,05) as taxas de clivagem e de blastocistos, enquanto com pLGW-GFP-LV diminuiu (P<0,05) a taxa de blastocisto quando se comparou ao controle sem VL. A coincubação de pLWE2-FIX-LV e pLGW-GFP-LV com presumíveis zigotos durante a CIV não afetou (P>0,05) o desenvolvimento embrionário. A expressão da proteína GFP não foi detectada em embriões após a coincubação de FIV ou CIV, embora as células do cumulus expressassem a proteína até o dia oito de cultivo in vitro. Reações em cadeia da polimerase (PCR) não detectaram os genes GFP ou FIX em embriões, mas ambos foram detectados em células do cumulus. Assim, a coincubação de VL com espermatozoide bovino e COCs não é eficaz para produzir embriões geneticamente modificados por meio de FIV.(AU)

In-Situ Gel-Free Plasmid Reassembling for Rapid Gene Subcloning and Truncation

Abstract Gene subcloning, a process in which the nucleotide sequence of interest is excised from on plasmid and inserted into another, seems to be an easy task to done. However, not all subcloning attempts are successful, even when the insert sequence and the double digested target plasmid are successfully purified form agarose gel and thought to be ready for subsequent ligation. In the current study we introduce a reliable, easy, and time consuming method for gene subcloning and also truncation. The stages are all carried out in a single microtube without any running on a gel, making it possible to accomplish a successful gene subcloning or truncation even with low concentrations of DNA molecules. Summarily, subcloning is achieved by mixing the plasmids of interest in a microtube and subjecting to restriction enzymes whose restriction sites flank the segment that is going to be subcloned. Digestion mixture is precipitated in the same microtube using isopropanol and the resultant DNA molecules are allowed to take part in a ligation reaction. The recombinant plasmids of interest are screened by colony PCR. Truncation is achieved by double- digestion of the plasmid of interest using a restriction enzyme whose restriction site flanks the segment that is going to be cut out.

Characterisation of alternative expression vectors for recombinant Bacillus Calmette-Guérin as live bacterial delivery systems

BACKGROUND Bacillus Calmette-Guérin (BCG) is considered a promising live bacterial delivery system. However, several proposals for rBCG vaccines have not progressed, mainly due to the limitations of the available expression systems. OBJECTIVES To obtain a set of mycobacterial vectors using a range of promoters with different strengths based on a standard backbone, previously shown to be stable. METHODS Mycobacterial expression vectors based on the pLA71 vector as backbone, were obtained inserting different promoters (PAN, PαAg, PHsp60, PBlaF* and PL5) and the green fluorescence protein (GFP) as reporter gene, to evaluate features such as their relative strengths, and the in vitro (inside macrophages) and in vivo stability. FINDINGS The relative fluorescence observed with the different vectors showed increasing strength of the promoters: PAN was the weakest in both Mycobacterium smegmatis and BCG and PBlaF* was higher than PHsp60 in BCG. The relative fluorescence observed in a macrophage cell line showed that PBlaF* and PHsp60 were comparable. It was not possible to obtain strains transformed with the extrachromosomal expression vector containing the PL5 in either species. MAIN CONCLUSION We have obtained a set of potentially stable mycobacterial vectors with a arrange of expression levels, to be used in the development of rBCG vaccines.

Relationship between PI3K/mTOR/RhoA pathway-regulated cytoskeletal rearrangements and phagocytic capacity of macrophages

The objective of this study was to investigate the relationship between PI3K/mTOR/RhoA signaling regulated cytoskeletal rearrangements and phagocytic capacity of macrophages. RAW264.7 macrophages were divided into four groups; blank control, negative control, PI3K-RNAi, and mTOR-RNAi. The cytoskeletal changes in the macrophages were observed. Furthermore, the phagocytic capacity of macrophages against Escherichia coli is reported as mean fluorescence intensity (MFI) and percent phagocytosis. Transfection yielded 82.1 and 81.5% gene-silencing efficiencies against PI3K and mTOR, respectively. The PI3K-RNAi group had lower mRNA and protein expression levels of PI3K, mTOR, and RhoA than the blank and negative control groups (Р<0.01). The mTOR-RNAi group had lower mRNA and protein levels of mTOR and RhoA than the blank and the negative control groups (Р<0.01). Macrophages in the PI3K-RNAi group exhibited stiff and inflexible morphology with short, disorganized filopodia and reduced number of stress fibers. Macrophages in the mTOR-RNAi group displayed pronounced cellular deformations with long, dense filopodia and an increased number of stress fibers. The PI3K-RNAi group exhibited lower MFI and percent phagocytosis than blank and negative control groups, whereas the mTOR-RNAi group displayed higher MFI and percent phagocytosis than the blank and negative controls (Р<0.01). Before and after transfection, the mRNA and protein levels of PI3K were both positively correlated with mTOR and RhoA (Р<0.05), but the mRNA and protein levels of mTOR were negatively correlated with those of RhoA (Р<0.05). Changes in the phagocytic capacity of macrophages were associated with cytoskeletal rearrangements and were regulated by the PI3K/mTOR/RhoA signaling pathway.

Respuesta inmune y terapia gó©nica con vectores virales adenoasociados / Immune response and gene therapy with adenoassociated viral vectors

En los ó;ºltimos aó;±os la terapia gó;©nica se ha posicionado como una opció;n real y segura en el desarrollo de alternativas terapó;©uticas para la cura y la prevenció;n de diferentes enfermedades. Consiste en la inserció;n de material genó;©tico en un tejido o có;©lula defectuosa, mediante el uso de un vector. Existen varias consideraciones para seleccionar el vector más apropiado, incluyendo el potencial de unió;n y entrada a la có;©lula diana, la capacidad de transferencia del material genó;©tico al nó;ºcleo, la habilidad de expresió;n del inserto y la ausencia de toxicidad. En el panorama actual, los vectores virales más utilizados son los derivados de los virus adenoasociados (AAV). Características como su bioseguridad, baja toxicidad y tropismo selectivo, han posibilitado su evaluació;n como opció;n terapó;©utica en un amplio nó;ºmero de enfermedades monogó;©nicas o complejas. A pesar de sus ventajas, los vectores AAV presentan inconvenientes, siendo el más importante la respuesta inmune del paciente al vector, especialmente la respuesta mediada por anticuerpos neutralizantes (NAb). Los NAb disminuyen la transducció;n del vector e impiden la expresió;n del gen que transporta, limitando su aplicació;n clínica. Por lo tanto, identificar y cuantificar la presencia y actividad de los NAbs, es el primer paso en cualquier protocolo de terapia gó;©nica con vectores AAV. La presencia de NAb depende principalmente de la exposició;n al virus en la naturaleza y varía drásticamente segó;ºn edad, localizació;n geográfica y estado de salud de la persona evaluada.

In recent years, gene therapy has been positioned as a real and safe option in the development of therapeutic alternatives for the cure and prevention of different diseases. It consists in the insertion of genetic material in a defective tissue or cell, through the use of a vector. There are several considerations for selecting the most appropriate vector, including the potential for binding and entry to the target cell, the ability of the genetic material to transfer to the nucleus, the ability to express the insert, and the absence of toxicity. In the current scenario, the most commonly used viral vectors are those derived from adeno-associated viruses (AAV). Characteristics such as biosafety, low toxicity and selective tropism have enabled its evaluation as a therapeutic option in many monogenic or complex diseases. Despite their advantages, AAV vectors have drawbacks, the most important being the patient’s immune response to the vector, especially the response mediated by neutralizing antibodies (NAb). NAbs decrease the transduction of the vector and prevent the expression of the gene it transports, limiting its clinical application. Therefore, identifying and quantifying the presence and activity of NAbs is the first step in any gene therapy protocol with AAV vectors. The presence of NAbs depends mainly on exposure to the virus in nature and varies drastically according to age, geographic location and health status of the person evaluated.

Neurogenesis and gliogenesis modulation in cerebral ischemia by CDK5 RNAi-based therapy / Modulación de la neurogénesis y la gliogénesis en la isquemia cerebral mediante una terapia basada en la interferencia de CDK5

Abstract Introduction: Cerebral ischemia is the third cause of death risk in Colombia and the first cause of physical disability worldwide. Different studies on the silencing of the cyclin-dependent kinase 5 (CDK5) have shown that reducing its activity is beneficial in ischemic contexts. However, its effect on neural cell production after cerebral ischemia has not been well studied yet. Objective: To evaluate CDK5 silencing on the production of neurons and astrocytes after a focal cerebral ischemia in rats. Materials and methods: We used 40 eight-week-old male Wistar rats. Both sham and ischemia groups were transduced at CA1 hippocampal region with an adeno-associated viral vector using a noninterfering (shSCRmiR) and an interfering sequence for CDK5 (shCDK5miR). We injected 50 mg/kg of bromodeoxyuridine intraperitoneally from hour 24 to day 7 post-ischemia. We assessed the neurological abilities during the next 15 days and we measured the immunoreactivity of bromodeoxyuridine (BrdU), doublecortin (DCX), NeuN, and glial fibrillary acid protein (GFAP) from day 15 to day 30 post-ischemia. Results: Our findings showed that CDK5miR-treated ischemic animals improved their neurological score and presented increased BrdU+ cells 15 days after ischemia, which correlated with higher DCX and lower GFAP fluorescence intensities, and, although mature neurons populations did not change, GFAP immunoreactivity was still significantly reduced at 30 days post-ischemia in comparison with untreated ischemic groups. Conclusion: CDK5miR therapy generated the neurological recovery of ischemic rats associated with the induction of immature neurons proliferation and the reduction of GFAP reactivity at short and longterm post-ischemia.

Resumen Introducción. La isquemia cerebral es la tercera causa de riesgo de muerte en Colombia y la primera causa de discapacidad física en el mundo. En diversos estudios en los que se silenció la cinasa 5 dependiente de la ciclina (CDK5) se ha demostrado que la reducción de su actividad es beneficiosa frente a la isquemia. Sin embargo, su efecto sobre la neurogénesis después de la isquemia no se ha dilucidado suficientemente. Objetivo. Evaluar el silenciamiento de la CDK5 en la neurogénesis y la gliogénesis después de la isquemia cerebral focal en ratas. Materiales y métodos. Se usaron 40 machos de rata Wistar de ocho semanas de edad. Los grupos de control y los isquémicos sometidos a transducción en la región del hipocampo CA1, se inyectaron intraperitonealmente por estereotaxia con 50 mg/kg de bromodesoxiuridina (BrdU) a partir de las 24 horas y hasta el día 7 después de la isquemia, con un vector viral asociado a adenovirus usando una secuencia no interferente (SCRmiR) y una interferente (CDK5miR). Se evaluó la capacidad neurológica durante los quince días siguientes y se detectó la capacidad de inmunorreacción para la BrdU, la proteína doblecortina (DCX), los núcleos neuronales (NeuN), y la proteína fibrilar acídica de la glía (Glial Fibrillary Acidic Protein, GFAP) a los 15 y 30 días de la isquemia. Resultados. Los animales isquémicos tratados con CDK5miR mejoraron su puntuación neurológica y presentaron un incremento de la BrdU+ a los 15 días de la isquemia, lo cual se correlacionó con una mayor intensidad de la DCX+ y una menor de la GFAP+. No hubo modificación de los NeuN+, pero sí una reducción significativa de la GFAP+ a los 30 días de la isquemia en los animales tratados comparados con los animales isquémicos no tratados. Conclusión. La terapia con CDK5miR generó la recuperación neurológica de ratas isquémicas asociada con la inducción de la neurogénesis y el control de la capacidad de reacción de la proteína GFAP a corto y largo plazo después de la isquemia.

Methods for the genetic manipulation of marine bacteria

Genetic manipulation of bacteria is a procedure necessary to obtain new strains that express peculiar and defined genetic determinants or to introduce genetic variants responsible for phenotypic modifications. This procedure can be applied to explore the biotechnological potential associated with environmental bacteria and to utilize the functional properties of specific genes when inserted into an appropriate host. In the past years, marine bacteria have received increasing attention because they represent a fascinating reservoir of genetic and functional diversity that can be utilized to fuel the bioeconomy sector. However, there is an urgent need for an in-depth investigation and improvement of the genetic manipulation tools applicable to marine strains because of the paucity of knowledge regarding this. This review aims to describe the genetic manipulation methods hitherto used in marine bacteria, thus highlighting the limiting factors of the different techniques available today to increase manipulation efficiency. In particular, we focus on methods of natural and artificial transformations (especially electroporation) and conjugation because they have been successfully applied to several marine strains. Finally, we emphasize that, to avoid failure, future work should be carried out to establish tailored methodologies for marine bacteria.

Improving adenoviral vectors and strategies for prostate cancer gene therapy

Gene therapy has been evaluated for the treatment of prostate cancer and includes the application of adenoviral vectors encoding a suicide gene or oncolytic adenoviruses that may be armed with a functional transgene. In parallel, versions of adenoviral vector expressing the p53 gene (Ad-p53) have been tested as treatments for head and neck squamous cell carcinoma and non-small cell lung cancer. Although Ad-p53 gene therapy has yielded some interesting results when applied to prostate cancer, it has not been widely explored, perhaps due to current limitations of the approach. To achieve better functionality, improvements in the gene transfer system and the therapeutic regimen may be required. We have developed adenoviral vectors whose transgene expression is controlled by a p53-responsive promoter, which creates a positive feedback mechanism when used to drive the expression of p53. Together with improvements that permit efficient transduction, this new approach was more effective than the use of traditional versions of Ad-p53 in killing prostate cancer cell lines and inhibiting tumor progression. Even so, gene therapy is not expected to replace traditional chemotherapy but should complement the standard of care. In fact, chemotherapy has been shown to assist in viral transduction and transgene expression. The cooperation between gene therapy and chemotherapy is expected to effectively kill tumor cells while permitting the use of reduced chemotherapy drug concentrations and, thus, lowering side effects. Therefore, the combination of gene therapy and chemotherapy may prove essential for the success of both approaches.

The effect of downregulation of Stathmin gene on biological behaviors of U373 and U87-MG glioblastoma cells

BACKGROUND: Stathmin as a critical protein involved in microtubule polymerization, is necessary for survival of cancer cells. However, extremely little is known about Stathmin in glioblastoma. So, this study was designed to elucidate the function of Stathmin gene in the tumorigenesis and progression of glioblastoma cells. METHOD: The lentiviral interference vector pLV3-si-Stathmin targeting Stathmin gene and the control vector pLV3-NC were established for the co-transfection of 293T cells together with the helper plasmids. Viral titer was determined via limiting dilution assay. Then pLV3-si-Stathmin and pLV3-NC were stably co-transfected into U373 and U87-MG glioblastoma cells. Expression levels of Stathmin protein in each group were determined by using Western Blot, and the proliferation and migration ability of the cells with downregulated Stathmin were evaluated through CCK8 assay and transwell invasion assay, respectively. Cell cycles and cell apoptosis were detected with flow cytometry. Finally, the effect of Stathmin in tumor formation was determined in nude mice. RESULT: DNA sequencing and viral titer assay indicated that the lentiviral interference vector was successfully established with a viral titer of 4 × 108 TU/ml. According to the results from Western Blotting, Stathmin protein expression level decreased significantly in the U373 and U87-MG cells after transfected with pLV3-si-Stathmin, respectively, compared with those transfected with pLV3-NC. In glioblastoma cells, the cell proliferation and migration were greatly inhibited after the downregulation of Stathmin protein. Flow cytometry showed that much more cells were arrested in G2/M phasein Stathmin downregulated group, compared with the non-transfection group and NC group. But Stathmin downregulation did not induce significant cell apoptosis. Tumor formation assay in nude mice showed that tumor formation was delayed after Stathmin downregulation, with a reduction in both tumor formation rate and tumor growth velocity. CONCLUSION: Stathmin downregulation affected the biological behaviors of U373 and U87-MG glioblastoma cells, inhibiting the proliferation and migration of tumor cells. Stathmin gene may serve as a potential target in gene therapy for glioblastoma.

A novel expression vector for the secretion of abaecin in Bacillus subtilis

ABSTRACT This study aimed to describe a Bacillus subtilis expression system based on genetically modified B. subtilis. Abaecin, an antimicrobial peptide obtained from Apis mellifera, can enhance the effect of pore-forming peptides from other species on the inhibition of bacterial growth. For the exogenous expression, the abaecin gene was fused with a tobacco etch virus protease cleavage site, a promoter Pglv, and a mature beta-glucanase signal peptide. Also, a B. subtilis expression system was constructed. The recombinant abaecin gene was expressed and purified as a recombinant protein in the culture supernatant. The purified abaecin did not inhibit the growth of Escherichia coli strain K88. Cecropin A and hymenoptaecin exhibited potent bactericidal activities at concentrations of 1 and 1.5 µM. Combinatorial assays revealed that cecropin A and hymenoptaecin had sublethal concentrations of 0.3 and 0.5 µM. This potentiating functional interaction represents a promising therapeutic strategy. It provides an opportunity to address the rising threat of multidrug-resistant pathogens that are recalcitrant to conventional antibiotics.

Development of a novel vector for cloning and expressing extremely toxic genes in Escherichia coli

Background: Escherichia coli has been widely used as a host to clone and express heterologous genes. However, there are few vectors available for cloning and expressing extremely toxic genes, which limits further basic and applied research on extremely toxic proteins. Results: In this study, a novel vector pAU10 was constructed in E. coli. pAU10 utilizes the combination of the efficient but highly repressible T7-lacO promoter/operator and the strong rrnBT2 transcriptional terminator upstream of the T7 promoter to strictly control unwanted transcription of the extremely toxic gene; in addition, the trp promoter/operator is oriented opposite to the T7 promoter to control the production of the antisense RNA that may block the translation of leaky mRNA. Without the supplementation of IPTG and L-tryptophan in the culture medium, transcription of the extremely toxic gene by the T7 promoter is highly repressed, and the trp promoter produces the antisense RNA, which strictly prevents unwanted expression of the extremely toxic protein in E. coli. With the supplementation of IPTG and L-tryptophan, the T7 promoter efficiently transcribes the extremely toxic gene, and the trp promoter does not produce the antisense RNA, ensuring efficient expression of the extremely toxic protein in E. coli. Tight regulation and efficiency of expression of an extremely toxic gene cloned in the vector pAU10 were confirmed by cloning and expressing the restriction endonuclease-encoding gene bamHI without its corresponding methylase gene in E. coli JM109(DE3). Conclusion: pAU10 is a good vector used for cloning and expressing extremely toxic genes in E. coli.

Improving the expression of recombinant pullulanase by increasing mRNA stability in Escherichia coli

Background: Pullulanase production in both wild-type strains and recombinantly engineered strains remains low. The Shine-Dalgarno (SD) sequence and stem-loop structure in the 5' or 3' untranslated region (UTR) are well-known determinants of mRNA stability. This study investigated the effect of mRNA stability on pullulanase heterologous expression. Results: We constructed four DNA fragments, pulA, SD-pulA, pulA-3t, and SD-pulA-3t, which were cloned into the expression vector pHT43 to generate four pullulanase expression plasmids. The DNA fragment pulA was the coding sequence (CDS) of pulA in Klebsiella variicola Z-13. SD-pulA was constructed by the addition of the 5' SD sequence at the 5' UTR of pulA. pulA-3t was constructed by the addition of a 3' stem-loop structure at the 3' UTR of pulA. SD-pulA-3t was constructed by the addition of the 5' SD sequence at the 5' UTR and a 3' stem-loop structure at the 3' UTR of pulA. The four vectors were transformed into Escherichia coli BL21(DE3). The pulA mRNA transcription of the transformant harboring pHT43-SD-pulA-3t was 338.6%, 34.9%, and 79.9% higher than that of the other three transformants, whereas the fermentation enzyme activities in culture broth and intracellularly were 107.0 and 584.1 times, 1.2 and 2.0 times, and 62.0 and 531.5 times the amount of the other three transformants (pulA, SD-pulA, and pulA-3 t), respectively. Conclusion: The addition of the 5' SD sequence at the 5' UTR and a 3' stem-loop structure at the 3' UTR of the pulA gene is an effective approach to increase pulA gene expression and fermentation enzyme activity.

Gene therapy: advances, challenges and perspectives / Terapia gênica: avanços, desafios e perspectivas

ABSTRACT The ability to make site-specific modifications to the human genome has been an objective in medicine since the recognition of the gene as the basic unit of heredity. Thus, gene therapy is understood as the ability of genetic improvement through the correction of altered (mutated) genes or site-specific modifications that target therapeutic treatment. This therapy became possible through the advances of genetics and bioengineering that enabled manipulating vectors for delivery of extrachromosomal material to target cells. One of the main focuses of this technique is the optimization of delivery vehicles (vectors) that are mostly plasmids, nanostructured or viruses. The viruses are more often investigated due to their excellence of invading cells and inserting their genetic material. However, there is great concern regarding exacerbated immune responses and genome manipulation, especially in germ line cells. In vivo studies in in somatic cell showed satisfactory results with approved protocols in clinical trials. These trials have been conducted in the United States, Europe, Australia and China. Recent biotechnological advances, such as induced pluripotent stem cells in patients with liver diseases, chimeric antigen receptor T-cell immunotherapy, and genomic editing by CRISPR/Cas9, are addressed in this review.

RESUMO A habilidade de fazer modificações pontuais no genoma humano tem sido o objetivo da medicina desde o conhecimento do DNA como unidade básica da hereditariedade. Entende-se terapia gênica como a capacidade do melhoramento genético por meio da correção de genes alterados (mutados) ou modificações sítio-específicas, que tenham como alvo o tratamento terapêutico. Este tipo de procedimento tornou-se possível por conta dos avanços da genética e da bioengenharia, que permitiram a manipulação de vetores para a entrega do material extracromossomal em células-alvo. Um dos principais focos desta técnica é a otimização dos veículos de entrega (vetores) que, em sua maioria, são plasmídeos, nanoestruturados ou vírus − sendo estes últimos os mais estudados, devido à sua excelência em invadir as células e inserir seu material genético. No entanto, existe grande preocupação referente às respostas imunes exacerbadas e à manipulação do genoma, principalmente em linhagens germinativas. Estudos em células somáticas in vivo apresentaram resultados satisfatórios, e já existem protocolos aprovados para uso clínico. Os principais trials têm sido conduzidos nos Estados Unidos, Europa, Austrália e China. Recentes avanços biotecnológicos empregados para o aprimoramento da terapia gênica, como células-tronco pluripotentes induzidas em pacientes portadores de doenças hepáticas, imunoterapia com células T do receptor do antígeno quimera e edição genômica pelos sistema CRISPR/Cas9, são abordados nesta revisão.

Construction of an RNAi vector for knockdown of GM-ACS genes in the cotyledonary nodes of soybean

Background: Ethylene plays an important role in the regulation of floral organ development in soybean, and 1-aminocyclopropane-1-carboxylate synthase (ACS) is a rate-limiting enzyme for ethylene biosynthesis. However, whether ACS also regulates floral organ differentiation in soybean remains unknown. To address this, we constructed an RNAi vector to inhibit ACS expression in cotyledonary nodes. Linear DNA cassettes of RNAi-ACS obtained by PCR were used to transform soybean cotyledonary nodes. Results: In total, 131 of 139 transiently transformed plants acquired herbicide resistance and displayed GUS activities in the new buds. In comparison to untransformed seedling controls, a greater number of flower buds were differentiated at the cotyledonary node; GM-ACS1 mRNA expression levels and ethylene emission in the transformed buds were reduced. Conclusion: These results indicate that the cotyledonary node transient transformation system may be suitable for stable transformation and that the inhibition of ACS expression may be an effective strategy for promoting floral organ differentiation in soybean.

Stable expression of Mycobacterium bovis antigen 85B in auxotrophic M. bovis bacillus Calmette-Guérin

BACKGROUND Bovine tuberculosis (TB) is a zoonotic disease caused by Mycobacterium bovis, responsible for causing major losses in livestock. A cost effective alternative to control the disease could be herd vaccination. The bacillus Calmette-Guérin (BCG) vaccine has a limited efficacy against bovine TB, but can improved by over-expression of protective antigens. The M. bovis antigen 85B demonstrates ability to induce protective immune response against bovine TB in animal models. However, current systems for the construction of recombinant BCG expressing multiple copies of the gene result in strains of low genetic stability that rapidly lose the plasmid in vivo. Employing antibiotic resistance as selective markers, these systems also compromise vaccine safety. We previously reported the construction of a stable BCG expression system using auxotrophic complementation as a selectable marker. OBJECTIVES The fundamental aim of this study was to construct strains of M. bovis BCG Pasteur and the auxotrophic M. bovis BCG ΔleuD expressing Ag85B and determine their stability in vivo. METHODS Employing the auxotrophic system, we constructed rBCG strains that expressed M. bovis Ag85B and compared their stability with a conventional BCG strain in mice. Stability was measured in terms of bacterial growth on the selective medium and retention of antigen expression. FINDINGS The auxotrophic complementation system was highly stable after 18 weeks, even during in vivo growth, as the selective pressure and expression of antigen were maintained comparing to the conventional vector. MAIN CONCLUSION The Ag85B continuous expression within the host may generate a stronger and long-lasting immune response compared to conventional systems.

Knockdown of ubiquitin-specific peptidase 39 inhibited the growth of osteosarcoma cells and induced apoptosis in vitro

BACKGROUND: Ubiquitin specific peptidase 39 (USP39), an essential factor in the assembly of the mature spliceosome complex, has an aberrant expression in several cancer. However, its function and the corresponding mechanism on human osteosarcoma has not been fully explored yet. METHODS: The mRNA and DNA copies of USP39 were increased in osteosarcoma cancer tissues compared with the one in human normal tissues according to datasets from the publicly available Oncomine database. A further western blot analysis also demonstrated an aberrant endogenous expression of USP39 in three different osteosarcoma cells. Then lentivirus-mediated short hairpin RNA (shRNA) was designed to silence USP39 in human osteosarcoma cell line U2OS, which is used to test the impact of USP39-silencing on cellular proliferation, colony formation, cell cycle distribution and apoptosis. RESULTS: Knockdown of USP39 expression in U2OS cell significantly decreased cell proliferation, impaired colony formation ability. A further analysis indicated suppression of USP39 arrested cell cycle progression at G2/M phase via p21 dependent way. In addition, the results of Annexin V/7-AAD staining suggested the knockdown of USP39 could promote U2OS cell apoptosis through PARP cleavage. CONCLUSIONS: These results uncover the critical role of USP39 in regulating cancer cell mitosis and indicate USP39 is critical for osteosarcoma tumorigenesis.

Long-term restoration of alpha-L-iduronidase activity in fibroblasts from patients with mucopolysaccharidosis type I after non-viral gene transfer

Mucopolysaccharidosis type I (MPS I) is a lysosomal storage disorder caused by deficiency of alpha-L-iduronidase (IDUA). Limitations such as the need for weekly injections, high morbidity and mortality, and high cost of current treatments show that new approaches to treat this disease are required. In this study, we aimed to correct fibroblasts from a patient with MPS I using non-viral gene therapy. Using a plasmid encoding the human IDUA cDNA, we achieved stable high IDUA levels in transfected fibroblasts up to 6 months of treatment. These results serve as proof of concept that a non-viral approach can correct the enzyme deficiency in cells of patients with lysosomal storage disorders, which can be used as a research tool for a series of disease aspects. Future studies should focus on showing if this approach can be useful in small animals and clinical trials (AU)

A broad pH range and processive chitinase from a metagenome library

Chitinases are hydrolases that degrade chitin, a polymer of N-acetylglucosamine linked β(1-4) present in the exoskeleton of crustaceans, insects, nematodes and fungal cell walls. A metagenome fosmid library from a wastewater-contaminated soil was functionally screened for chitinase activity leading to the isolation and identification of a chitinase gene named metachi18A. The metachi18A gene was subcloned and overexpressed in Escherichia coli BL21 and the MetaChi18A chitinase was purified by affinity chromatography as a 6xHis-tagged fusion protein. The MetaChi18A enzyme is a 92-kDa protein with a conserved active site domain of glycosyl hydrolases family 18. It hydrolyses colloidal chitin with an optimum pH of 5 and temperature of 50°C. Moreover, the enzyme retained at least 80% of its activity in the pH range from 4 to 9 and 98% at 600 mM NaCl. Thin layer chromatography analyses identified chitobiose as the main product of MetaChi18A on chitin polymers as substrate. Kinetic analysis showed inhibition of MetaChi18A activity at high concentrations of colloidal chitin and 4-methylumbelliferyl N,N′-diacetylchitobiose and sigmoid kinetics at low concentrations of colloidal chitin, indicating a possible conformational change to lead the chitin chain from the chitin-binding to the catalytic domain. The observed stability and activity of MetaChi18A over a wide range of conditions suggest that this chitinase, now characterized, may be suitable for application in the industrial processing of chitin.