Improved protocol for Bst polymerase and reverse transcriptase production and application to a point-of-care diagnostics system.

The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has raised awareness in the scientific community about the importance of being prepared for sanitary emergencies. Many measures implemented during the COVID pandemic are now being expanded to other applications. In the field of molecular and immunological diagnostics, the need to massively test the population worldwide resulted in the application of a variety of methods to detect viral infection. Besides gold standard reverse transcription quantitative polymerase chain reaction (RT-qPCR), the use of reverse transcription loop-mediated isothermal amplification (RT-LAMP) arose as an alternative and sensitive method to amplify and detect viral genetic material. We have used openly available protocols and have improved the protein production of RT-LAMP enzymes Bst polymerase and HIV-reverse transcriptase. To optimize enzyme production, we tested different protein tags, and we shortened the protein purification protocol, resulting in reduced processing time and handling of the enzymes and, thus, preserved the protein activity with high purity. The enzymes showed significant stability at 4 °C and 25 °C, over 60 days, and were highly reliable when used as a one-step RT-LAMP reaction in a portable point-of-care device with clinical samples. The enzymes and the reaction setup can be further expanded to detect other infectious diseases agents.

Genome-wide translation control analysis of developing human neurons.

During neuronal differentiation, neuroprogenitor cells become polarized, change shape, extend axons, and form complex dendritic trees. While growing, axons are guided by molecular cues to their final destination, where they establish synaptic connections with other neuronal cells. Several layers of regulation are integrated to control neuronal development properly. Although control of mRNA translation plays an essential role in mammalian gene expression, how it contributes temporarily to the modulation of later stages of neuronal differentiation remains poorly understood. Here, we investigated how translation control affects pathways and processes essential for neuronal maturation, using H9-derived human neuro progenitor cells differentiated into neurons as a model. Through Ribosome Profiling (Riboseq) combined with RNA sequencing (RNAseq) analysis, we found that translation control regulates the expression of critical hub genes. Fundamental synaptic vesicle secretion genes belonging to SNARE complex, Rab family members, and vesicle acidification ATPases are strongly translationally regulated in developing neurons. Translational control also participates in neuronal metabolism modulation, particularly affecting genes involved in the TCA cycle and glutamate synthesis/catabolism. Importantly, we found translation regulation of several critical genes with fundamental roles regulating actin and microtubule cytoskeleton pathways, critical to neurite generation, spine formation, axon guidance, and circuit formation. Our results show that translational control dynamically integrates important signals in neurons, regulating several aspects of its development and biology.

Translational Control during Mammalian Neocortex Development and Postembryonic Neuronal Function.

The control of mRNA translation has key roles in the regulation of gene expression and biological processes such as mammalian cellular differentiation and identity. Methodological advances in the last decade have resulted in considerable progress towards understanding how translational control contributes to the regulation of diverse biological phenomena. In this review, we discuss recent findings in the involvement of translational control in the mammalian neocortex development and neuronal biology. We focus on regulatory mechanisms that modulate translational efficiency during neural stem cells self-renewal and differentiation, as well as in neuronal-related processes such as synapse, plasticity, and memory.

Structural features and development of an assay platform of the parasite target deoxyhypusine synthase of Brugia malayi and Leishmania major.

Deoxyhypusine synthase (DHS) catalyzes the first step of the post-translational modification of eukaryotic translation factor 5A (eIF5A), which is the only known protein containing the amino acid hypusine. Both proteins are essential for eukaryotic cell viability, and DHS has been suggested as a good candidate target for small molecule-based therapies against eukaryotic pathogens. In this work, we focused on the DHS enzymes from Brugia malayi and Leishmania major, the causative agents of lymphatic filariasis and cutaneous leishmaniasis, respectively. To enable B. malayi (Bm)DHS for future target-based drug discovery programs, we determined its crystal structure bound to cofactor NAD+. We also reported an in vitro biochemical assay for this enzyme that is amenable to a high-throughput screening format. The L. major genome encodes two DHS paralogs, and attempts to produce them recombinantly in bacterial cells were not successful. Nevertheless, we showed that ectopic expression of both LmDHS paralogs can rescue yeast cells lacking the endogenous DHS-encoding gene (dys1). Thus, functionally complemented dys1Δ yeast mutants can be used to screen for new inhibitors of the L. major enzyme. We used the known human DHS inhibitor GC7 to validate both in vitro and yeast-based DHS assays. Our results show that BmDHS is a homotetrameric enzyme that shares many features with its human homologue, whereas LmDHS paralogs are likely to form a heterotetrameric complex and have a distinct regulatory mechanism. We expect our work to facilitate the identification and development of new DHS inhibitors that can be used to validate these enzymes as vulnerable targets for therapeutic interventions against B. malayi and L. major infections.

Identification of genes regulated by oleic acid in Jurkat cells by suppressive subtractive hybridization analysis.

In this study, the effect of oleic acid (50 microM) on gene expression of Jurkat cells (human T lymphocytes cell line) was examined using the suppressive subtractive hybridization approach. This technique allowed us to identify genes with higher or lower expression after cell treatment with oleic acid as compared to untreated cells. Oleic acid upregulated the expression of the translation elongation factor alpha 1 and ATP synthase 8 and downregulated gp96 (human tumor rejection antigen gp96), heat-shock protein 60 and subtilisin-like protein 4. These results suggest that oleic acid, at plasma physiological concentration, can regulate the expression of important genes to maintain the machinery that ensures cell functioning.

The generation and utilization of a cancer-oriented representation of the human transcriptome by using expressed sequence tags.

Whereas genome sequencing defines the genetic potential of an organism, transcript sequencing defines the utilization of this potential and links the genome with most areas of biology. To exploit the information within the human genome in the fight against cancer, we have deposited some two million expressed sequence tags (ESTs) from human tumors and their corresponding normal tissues in the public databases. The data currently define approximately 23,500 genes, of which only approximately 1,250 are still represented only by ESTs. Examination of the EST coverage of known cancer-related (CR) genes reveals that <1% do not have corresponding ESTs, indicating that the representation of genes associated with commonly studied tumors is high. The careful recording of the origin of all ESTs we have produced has enabled detailed definition of where the genes they represent are expressed in the human body. More than 100,000 ESTs are available for seven tissues, indicating a surprising variability of gene usage that has led to the discovery of a significant number of genes with restricted expression, and that may thus be therapeutically useful. The ESTs also reveal novel nonsynonymous germline variants (although the one-pass nature of the data necessitates careful validation) and many alternatively spliced transcripts. Although widely exploited by the scientific community, vindicating our totally open source policy, the EST data generated still provide extensive information that remains to be systematically explored, and that may further facilitate progress toward both the understanding and treatment of human cancers.

Caracterizaçäo celular e molecular dos efeitos do ácido retinóico sobre as células ST1 de glioma de rato / Cellular and molecular characterization of retinoic acid effects on rat glioma ST1 cells

Os gliomas säo os tumores mais fatais do sistema nervoso central, para os quais ainda näo há tratamento eficaz. Para analisar as bases celulares e moleculares da açäo do agente diferenciador e antitumoral ácido retinóico (ATRA) sobre gliomas, foi utilizado, como modelo, a linhagem ST1 de glioma de rato. Propôs-se: a) analisar os efeitos de ATRA sobre a morfologia, proliferaçäo e morte celular; b) isolar, identificar e caracterizar genes induzidos por ATRA em células ST1. Verificou-se que o tratamento com ATRA promove achatamento celular e inibiçäo da síntese de DNA e do crescimento em suspensäo de agarose, caracteizando um completa reversäo fenotípica tumoral-normal, a qual näo é acompanhada de induçäo de apoptose...