Functionalizing DNA Origami by Triplex-Directed Site-Specific Photo-Cross-Linking.

Here, we present a cross-linking approach to covalently functionalize and stabilize DNA origami structures in a one-pot reaction. Our strategy involves adding nucleotide sequences to adjacent staple strands, so that, upon assembly of the origami structure, the extensions form short hairpin duplexes targetable by psoralen-labeled triplex-forming oligonucleotides bearing other functional groups (pso-TFOs). Subsequent irradiation with UVA light generates psoralen adducts with one or both hairpin staples leading to site-specific attachment of the pso-TFO (and attached group) to the origami with ca. 80% efficiency. Bis-adduct formation between strands in proximal hairpins further tethers the TFO to the structure and generates "superstaples" that improve the structural integrity of the functionalized complex. We show that directing cross-linking to regions outside of the origami core dramatically reduces sensitivity of the structures to thermal denaturation and disassembly by T7 RNA polymerase. We also show that the underlying duplex regions of the origami core are digested by DNase I and thus remain accessible to read-out by DNA-binding proteins. Our strategy is scalable and cost-effective, as it works with existing DNA origami structures, does not require scaffold redesign, and can be achieved with just one psoralen-modified oligonucleotide.

Equilibration time improves the sperm variables of wild ruminant ejaculated and epididymal sperm cryopreserved by ultra-rapid freezing.

This work examines the effect of equilibration time with extender on ultra-rapidly frozen-thawed wild ruminant epididymal (origin: Iberian ibex) and ejaculated (origin: mouflon) sperm variables. Sperm samples were prepared either without prior equilibration, or equilibrated for 30 min before freezing. Higher quality (p < 0.05) frozen-thawed spermatozoa were obtained when equilibration was allowed, for ejaculated sperm in terms of sperm motility, acrosome apical ridge integrity, sperm viability, and percentage of normal cells, and for epididymal sperm in terms of linearity and straightness of sperm movement. The sperm head area, head perimeter, head length and head width were smaller (p < 0.01) in the equilibrated than non-equilibrated frozen-thawed epididymal sperm; no such dimensional changes were recorded for ejaculated sperm. In conclusion, equilibration prior to ultra-rapid freezing improves the cryoresistance of sperm cells, although viable sperm cells can be obtained without equilibration. The epididymal sperm showed greater cryoresistance, supporting the idea that it is more resistant to freeze-thawing than ejaculated sperm.

An algorithm for the robust estimation of the COVID-19 pandemic's population by considering undetected individuals.

Due to the current COVID-19 pandemic, much effort has been put on studying the spread of infectious diseases to propose more adequate health politics. The most effective surveillance system consists of doing massive tests. Nonetheless, many countries cannot afford this class of health campaigns due to limited resources. Thus, a transmission model is a viable alternative to study the dynamics of the pandemic. The most used are the Susceptible, Infected and Removed type models (SIR). In this study, we tackle the population estimation problem of the A-SIR model, which takes into account asymptomatic or undetected individuals. By means of an algebraic differential approach, we design a model-free (no copy system) reduced-order estimation algorithm (observer) to determine the different non-measured population groups. We study two types of estimation algorithms: Proportional and Proportional-Integral. Both shown fast convergence speed, as well as a minimal estimation error. Additionally, we introduce random fluctuations in our analysis to represent changes in the external conditions and which result in poor measurements. The numerical results reveal that both model-free estimators are robust despite the presence of these fluctuations. As a point of reference, we apply the classical Luenberger type observer to our estimation problem and compare the results. Finally, we consider real data of infected individuals in Mexico City, reported from February 2020 to March 2021, and estimate the non-measured populations. Our work's main goal is to proportionate a simple and therefore, an accessible methodology to estimate the behavior of the COVID-19 pandemic from the available data, such that the competent authorities can propose more adequate health politics.

A rapid fluorescent indicator displacement assay and principal component/cluster data analysis for determination of ligand-nucleic acid structural selectivity.

We describe a rapid fluorescence indicator displacement assay (R-FID) to evaluate the affinity and the selectivity of compounds binding to different DNA structures. We validated the assay using a library of 30 well-known nucleic acid binders containing a variety chemical scaffolds. We used a combination of principal component analysis and hierarchical clustering analysis to interpret the results obtained. This analysis classified compounds based on selectivity for AT-rich, GC-rich and G4 structures. We used the FID assay as a secondary screen to test the binding selectivity of an additional 20 compounds selected from the NCI Diversity Set III library that were identified as G4 binders using a thermal shift assay. The results showed G4 binding selectivity for only a few of the 20 compounds. Overall, we show that this R-FID assay, coupled with PCA and HCA, provides a useful tool for the discovery of ligands selective for particular nucleic acid structures.

In Vitro and In Vivo Studies of Spironolactone as an Antischistosomal Drug Capable of Clinical Repurposing.

Schistosomiasis is a parasitic flatworm disease that infects over 200 million people worldwide, especially in poor communities. Treatment and control of the disease rely on just one drug, praziquantel. Since funding for drug development for poverty-associated diseases is very limited, drug repurposing is a promising strategy. In this study, from a screening of 13 marketed diuretics, we identified that spironolactone, a potassium-sparing diuretic, had potent antischistosomal effects on Schistosoma mansoniin vitro and in vivo in a murine model of schistosomiasis. In vitro, spironolactone at low concentrations (<10 µM) is able to alter worm motor activity and the morphology of adult schistosomes, leading to parasitic death. In vivo, oral treatment with spironolactone at a single dose (400 mg/kg) or daily for five consecutive days (100 mg/kg/day) in mice harboring either patent or prepatent infections significantly reduced worm burden, egg production, and hepato- and splenomegaly (P < 0.05 to P < 0.001). Taken together, with the safety profile of spironolactone, supported by its potential to affect schistosomes, these results indicate that spironolactone could be a potential treatment for schistosomiasis and make it promising for repurposing.

Stay Gathered Rather than Scattered: Strategies of Common Marmosets during Agonistic Interactions with Birds in the Atlantic Rain Forest.

To reduce the vulnerability of their small body size, common marmosets live in large and cohesive social groups. Thus, we hypothesized that in order to compensate for small body size and predation risk, individuals of common marmosets will stay gathered rather than scattered when foraging for eggs and/or nestling birds. Furthermore, in order to avoid costly injuries and eventual predation risks, for both sides, the majority of interactions among common marmosets and small birds will not involve direct physical contact. The study was developed in a small fragment of Atlantic Forest in the northeast of Brazil. We recorded a total of 115 interactions between common marmosets (Callithrix jacchus) with 7 different bird species. As expected, agonistic interactions were significantly more frequent when the marmosets were gathered. Also, most agonistic interactions by the birds toward common marmosets involved overflights without physical contact. Apparently, the set of avoidance behavior leads to a reduced predation risk for both sides. It appears that dispersed marmosets do not represent an imminent threat that justifies an agonistic reaction by the birds as the latter appear to avoid exposing themselves to unnecessary danger during agonistic interactions, especially when the marmosets are gathered.

G-Quadruplex Secondary Structure Obtained from Circular Dichroism Spectroscopy.

A curated library of circular dichroism spectra of 23 G-quadruplexes of known structure was built and analyzed. The goal of this study was to use this reference library to develop an algorithm to derive quantitative estimates of the secondary structure content of quadruplexes from their experimental CD spectra. Principal component analysis and singular value decomposition were used to characterize the reference spectral library. CD spectra were successfully fit to obtain estimates of the amounts of base steps in anti-anti, syn-anti or anti-syn conformations, in diagonal or lateral loops, or in other conformations. The results show that CD spectra of nucleic acids can be analyzed to obtain quantitative structural information about secondary structure content in an analogous way to methods used to analyze protein CD spectra.

Effects of topical insulin on second-intention wound healing in the red-eared slider turtle (Trachemys scripta elegans) - a controlled study.

BACKGROUND: Compared with mammals, wound healing in reptiles is characterized by reduced wound contraction and longer healing times. The aim of this study is to describe the clinical and histopathological effects of topical insulin on second-intention healing of experimentally induced wounds in skin without dermal bony plates of Trachemys scripta elegans exposed to daily variations in ambient temperature and in an aquatic environment. Forty-four healthy adult females were assigned to two groups: Group 1 (n = 24) was used to assess clinical features such as wound contraction; Group 2 (n = 20) was used for histological evaluation and morphometric analysis. Topical porcine insulin (5 IU/ml diluted in glycerol) was applied daily 1 week. For each control time (2, 7, 14, 21 and 28 days post-wounding), re-epithelisation and wound remodelling were evaluated histologically and the number of main inflammatory cells (heterophils, macrophages, lymphocytes and fibroblasts) was scored. RESULTS: Mean wound contraction was higher in the insulin-treated group at each time point and differences were significant at day 28 (P < 0.0001). Histologically, these clinical findings were associated with better re-epithelisation, inflammatory response, collagen synthesis and remodelling of the wounds. Morphometrically, insulin-treated wounds had significantly higher mean counts of heterophils (day 7), macrophages (days 2, 7 and 14) and fibroblasts (days 14 and 21), whereas lymphocyte counts were significantly lower at day 21. These results demonstrate that topical insulin modifies the inflammatory response of turtle skin up-regulating inflammatory cells at early stages and promoting wound healing. CONCLUSIONS: Topical insulin is a potentially useful therapy in skin wounds of Trachemys scripta and should be evaluated in non-experimental wounds of turtles and other reptiles.

FATAL ACINETOBACTER BAUMANNII INFECTION IN THE CRITICALLY ENDANGERED EUROPEAN MINK (MUSTELA LUTREOLA).

The present study reports the first case of fatal Acinetobacter baumannii infection in the critically endangered European mink ( Mustela lutreola ). Gross examination revealed a severe, diffuse hemorrhagic pneumonia and generalized congestion as main features. Microscopically, the main lesions were an acute, severe fibrinous-hemorrhagic pneumonia associated with proliferation of coccobacilli and generalized acute-subacute congestion. Cultures yielded A. baumannii ; the species was confirmed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and the strain presented a multidrug-resistant pattern. The results are not only of conservation concern but also of public health concern given A. baumannii is one of the most important pathogens implicated in nosocomial infections in humans.

Clinical and histological findings of cutaneous wound healing in the red-eared slider turtle (Trachemys scripta elegans) housed in unheated outdoor enclosures.

BACKGROUND: Cutaneous wounds are common in chelonians. The clinical and histological features of wound healing in these species are not well described and this prevents evaluation of new therapies. OBJECTIVES: To describe clinical and histopathological features of cutaneous wound healing in the red-eared slider turtle (Trachemys scripta elegans). ANIMALS: Twenty four healthy adult females housed in outdoor facilities with free access to water and exposed to daily variations in temperature. METHODS: Full thickness 6 mm skin biopsy punch wounds were created in the rear limbs. The turtles were assigned to Group 1 (n = 12 for clinical evaluation) and Group 2 (n = 12 for microscopic study). Group 1 was photographed on Day 1 and weekly, until 28 days post wounding. Wound retraction was expressed as the percentage of perimeter reduction. For Group 2, three skin wounds were sampled at 2, 7, 14, 21, 28, 42, 60 and 135 days post wounding for histological study. The avidin-biotin-peroxidase (ABC) staining method was used to evaluate five commercial antibodies. RESULTS: Wound contraction was limited; crust persisted at least 28 days. Re-epithelialization was complete by Day 14 in many animals; active inflammation persisted until 28 days; connective tissue re-constitution and remodelling was achieved from 42 to 135 days. Antibodies AE1/AE3, Factor VIII, MAC 387, CD3 and NCL-MSA showed cross reactivity with the cell counterpart in turtle tissues. CONCLUSIONS AND CLINICAL IMPORTANCE: Second intention wound healing progressed slowly and with an indolent behaviour. Microscopically there was marked overlapping of the inflammatory and proliferative phases over a long time period.