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.

Propylthiouracil-Induced Skin Vasculitis.

The use of propylthiouracil (PTU) is associated with the development of autoantibodies, namely, antineutrophil cytoplasmic antibodies (ANCAs), which are associated with the pathogenesis of ANCA-associated systemic vasculitis, most often related to the myeloperoxidase subtype (ANCA-MPO). The authors report the case of a 61-year-old woman on PTU for one year who was referred to Internal Medicine for a three-month evolution of painless non-blanching purple patches, non-pruriginous, involving the chest and legs. The autoimmunity revealed ANCA antibody positivity, with a cutaneous biopsy compatible with leukocytoclastic vasculitis/necrotizing vasculitis with involvement of small and medium-sized vessels. Clinical improvement was noted after the drug was discontinued, with the resolution of the analytical changes.

Isoniazid-Induced Acute Liver Failure Requiring Liver Transplantation.

Tuberculosis (TB) is a contagious disease caused by the bacilli Mycobacterium tuberculosis complex. Currently, about a quarter of the world's population is infected with Mycobacterium tuberculosis. According to the World Health Organization, in 2018, Portugal had a TB incidence rate of about 24 cases per 100,000 inhabitants. It is estimated that 5-15% of individuals with latent TB infection progress to active TB. Isoniazid is one of the most widely used drugs for the treatment of active and latent TB. However, care must be taken with the possible development of toxicity, particularly hepatic. The authors report a case of a 60-year-old woman diagnosed with latent TB who started therapy with isoniazid. Five months later, she developed acute liver failure secondary to the drug, requiring liver transplantation, with a favorable clinical outcome. Thus, we intend to alert to the potential toxicities of isoniazid, establishing follow-up strategies in patients on this therapy.

Genetic switches designed for eukaryotic cells and controlled by serine integrases.

Recently, new serine integrases have been identified, increasing the possibility of scaling up genomic modulation tools. Here, we describe the use of unidirectional genetic switches to evaluate the functionality of six serine integrases in different eukaryotic systems: the HEK 293T cell lineage, bovine fibroblasts and plant protoplasts. Moreover, integrase activity was also tested in human cell types of therapeutic interest: peripheral blood mononuclear cells (PBMCs), neural stem cells (NSCs) and undifferentiated embryonic stem (ES) cells. The switches were composed of plasmids designed to flip two different genetic parts driven by serine integrases. Cell-based assays were evaluated by measurement of EGFP fluorescence and by molecular analysis of attL/attR sites formation after integrase functionality. Our results demonstrate that all the integrases were capable of inverting the targeted DNA sequences, exhibiting distinct performances based on the cell type or the switchable genetic sequence. These results should support the development of tunable genetic circuits to regulate eukaryotic gene expression.

Nasal Vaccines Against Hepatitis B: An Update.

After several decades of immunization against hepatitis B virus, the question still remains whether a new vaccine could avoid the limitations of the current vaccine similar to those associated with its injectable form or ineffectiveness on chronic hepatitis B disease. A hypothesis to overcome first limitation is the development of an intranasal vaccine, self-administered, able to achieve not only systemic immunity, but also sIgA on the vaginal mucosa which would be a great advantage to prevent the sexually transmitted disease cases. Injectable hepatitis B vaccines that are already available in the market led to achieving protection mainly through a strong antibody-mediated response. For chronic hepatitis, a strong cellular immune response would also be required. The aim of this review is to give an overview of the work done in recent years, with the objective of developing a vaccine that can be administered by intranasal route. A discussion of the leading studies is presented, focusing not only on potential antigens, but also on promising adjuvants for the hepatitis B antigen. The results of the immune response generated with different formulations are summarized in tables. It is important to note that almost all studies claimed the induction of specific mucosal immune response (sIgA) and a balanced cellular and humoral Th1/Th2 or a Th1-type immune response. The further evaluation of these formulations, using a laboratory animal model of viral hepatitis B, would allow scientific community to conclude about the utility of these new adjuvants, particularly on a combined immunotherapy strategy for chronic hepatitis B.

Parameter estimation for blind and non-blind deblurring using residual whiteness measures.

Image deblurring (ID) is an ill-posed problem typically addressed by using regularization, or prior knowledge, on the unknown image (and also on the blur operator, in the blind case). ID is often formulated as an optimization problem, where the objective function includes a data term encouraging the estimated image (and blur, in blind ID) to explain the observed data well (typically, the squared norm of a residual) plus a regularizer that penalizes solutions deemed undesirable. The performance of this approach depends critically (among other things) on the relative weight of the regularizer (the regularization parameter) and on the number of iterations of the algorithm used to address the optimization problem. In this paper, we propose new criteria for adjusting the regularization parameter and/or the number of iterations of ID algorithms. The rationale is that if the recovered image (and blur, in blind ID) is well estimated, the residual image is spectrally white; contrarily, a poorly deblurred image typically exhibits structured artifacts (e.g., ringing, oversmoothness), yielding residuals that are not spectrally white. The proposed criterion is particularly well suited to a recent blind ID algorithm that uses continuation, i.e., slowly decreases the regularization parameter along the iterations; in this case, choosing this parameter and deciding when to stop are one and the same thing. Our experiments show that the proposed whiteness-based criteria yield improvements in SNR, on average, only 0.15 dB below those obtained by (clairvoyantly) stopping the algorithm at the best SNR. We also illustrate the proposed criteria on non-blind ID, reporting results that are competitive with state-of-the-art criteria (such as Monte Carlo-based GSURE and projected SURE), which, however, are not applicable for blind ID.

Deconvolving images with unknown boundaries using the alternating direction method of multipliers.

The alternating direction method of multipliers (ADMM) has recently sparked interest as a flexible and efficient optimization tool for inverse problems, namely, image deconvolution and reconstruction under non-smooth convex regularization. ADMM achieves state-of-the-art speed by adopting a divide and conquer strategy, wherein a hard problem is split into simpler, efficiently solvable sub-problems (e.g., using fast Fourier or wavelet transforms, or simple proximity operators). In deconvolution, one of these sub-problems involves a matrix inversion (i.e., solving a linear system), which can be done efficiently (in the discrete Fourier domain) if the observation operator is circulant, i.e., under periodic boundary conditions. This paper extends ADMM-based image deconvolution to the more realistic scenario of unknown boundary, where the observation operator is modeled as the composition of a convolution (with arbitrary boundary conditions) with a spatial mask that keeps only pixels that do not depend on the unknown boundary. The proposed approach also handles, at no extra cost, problems that combine the recovery of missing pixels (i.e., inpainting) with deconvolution. We show that the resulting algorithms inherit the convergence guarantees of ADMM and illustrate its performance on non-periodic deblurring (with and without inpainting of interior pixels) under total-variation and frame-based regularization.

Blind and semi-blind deblurring of natural images.

A method for blind image deblurring is presented. The method only makes weak assumptions about the blurring filter and is able to undo a wide variety of blurring degradations. To overcome the ill-posedness of the blind image deblurring problem, the method includes a learning technique which initially focuses on the main edges of the image and gradually takes details into account. A new image prior, which includes a new edge detector, is used. The method is able to handle unconstrained blurs, but also allows the use of constraints or of prior information on the blurring filter, as well as the use of filters defined in a parametric manner. Furthermore, it works in both single-frame and multiframe scenarios. The use of constrained blur models appropriate to the problem at hand, and/or of multiframe scenarios, generally improves the deblurring results. Tests performed on monochrome and color images, with various synthetic and real-life degradations, without and with noise, in single-frame and multiframe scenarios, showed good results, both in subjective terms and in terms of the increase of signal to noise ratio (ISNR) measure. In comparisons with other state of the art methods, our method yields better results, and shows to be applicable to a much wider range of blurs.