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.

A new reduced-order observer for the synchronization of nonlinear chaotic systems: An application to secure communications.

This paper deals with the master-slave synchronization scheme for partially known nonlinear chaotic systems, where the unknown dynamics is considered as the master system and we propose the slave system structure which estimates the unknown states. It introduced a new reduced order observer, using the concept of Algebraic Observability; we applied the results to a Sundarapandian chaotic system, and by means of some numerical simulations we show the effectiveness of the suggested approach. Finally, the proposed observer is utilized for encryption, where encryption key is the master system and decryption key is the slave system.

Multisynchronization of chaotic oscillators via nonlinear observer approach.

The goal of this work is to synchronize a class of chaotic oscillators in a master-slave scheme, under different initial conditions, considering several slaves systems. The Chen oscillator is employed as a benchmark model and a nonlinear observer is proposed to reach synchronicity between the master and the slaves' oscillators. The proposed observer contains a proportional and integral form of a bounded function of the synchronization error in order to provide asymptotic synchronization with a satisfactory performance. Numerical experiments were carried out to show the operation of the considered methodology.

Fatal leptospirosis in endangered Barbary macaques (Macaca sylvanus) kept in captivity: Assessing the role of sympatric rodents.

Between December 2020 and January 2021, an outbreak of acute mortality in endangered Barbary macaques (Macaca sylvanus) kept in captivity was detected in a zoo in Spain. The main findings observed in the two fatally affected animals at post-mortem evaluation were jaundice, renal tubular necrosis and interstitial nephritis. Leptospira spp. infection was confirmed by real time PCR (qPCR) in different tissues in both individuals. Analyses of secY gene from a positive individual showed 100% homology with a previously published sequence corresponding to Leptospira interrogans serovar Copenhageni. Free-living sympatric brown rats (Rattus norvegicus) from the affected zoo were also analyzed, and showed a prevalence and seroprevalence of Leptospira spp. of 18.2% (4/22; 95% CI: 2.1-34.3) and 41.9% (26/62; 95% CI: 29.7-54.2), respectively. We detected seropositive sera to five different serovars of Leptospira spp. (Copenhageni, Grippotyphosa, Pomona, Canicola and Hardjo) in the rodent population, with L. Copenhageni being the predominant one. This study describes for first time an outbreak of fatal leptospirosis in captive non-human primates in Europe. Our results show that Barbary macaques, an endangered species, are highly susceptible to Leptospira spp. infection, with sympatric wild rodents being the most likely reservoir animals involved in transmission in this outbreak. Our results suggest that rodent control could be an effective measure for minimizing exposure to Leptospira spp. in zoological collections. Given the potential implications for conservation, animal and public health, non-human primates and rodents should be included in surveillance programs for Leptospira spp. in zoos.

Supramolecular DNA-based catalysis in organic solvents.

The distinct folding accompanied by its polymorphic character renders DNA G-quadruplexes promising biomolecular building blocks to construct novel DNA-based and supramolecular assemblies. However, the highly polar nature of DNA limits the use of G-quadruplexes to water as a solvent. In addition, the archetypical G-quadruplex fold needs to be stabilized by metal-cations, which is usually a potassium ion. Here, we show that a noncovalent PEGylation process enabled by electrostatic interactions allows the first metal-free G-quadruplexes in organic solvents. Strikingly, incorporation of an iron-containing porphyrin renders the self-assembled metal-free G-quadruplex catalytically active in organic solvents. Hence, these "supraG4zymes" enable DNA-based catalysis in organic media. The results will allow the broad utilization of DNA G-quadruplexes in nonaqueous environments.

[Neurosurgical importance of the superior petrosal venous complex]. / Importancia neuroquirúrgica del complejo venoso petroso superior.

OBJECTIVE: To study the microsurgical anatomy of the superior petrosal venous complex (SPVC). MATERIAL AND METHODS: We conducted a descriptive and prospective study. Six injected specimens were used (12 sides). The microsurgical anatomy of the SPVC was studied by means of an anterior, retrosigmoid and transpetrosal approach. Neurosurgical equipment, 0-degree rigid endoscopy and OPMI-1 surgical microscope with 6× to 20× magnification were all used in this study. The venous drainage pattern toward the superior petrosal sinus was analysed, as were the formation of tributary veins, the relationship with the trigeminal nerve and the anatomical variants of SPVC. RESULTS: The SPVC was present in all cases. A tributary, cerebellopontine fissure vein was identified in 100% of cases. The venous drainage pattern of the SPVC was divided into medial, intermediate and lateral with respect to the suprameatal crest. The SPVC was simple in 8 sides and duplicate in 4 sides. A triangle formed by the tentorium, the SPVC and part of the tentorial and petrosal surface of the cerebellum was also observed in the study. This triangle was called the petrosal-tentorial triangle. CONCLUSIONS: It is important to understand the microsurgical anatomy of the SPVC. Therefore, we propose the petrosal-tentorial triangle as a neurosurgical route for the management of pathologies from the cerebellopontine angle to the superior petroclival region.

A fractional PI observer for incommensurate fractional order systems under parametric uncertainties.

The problem of state observation in incommensurate fractional order systems has been poorly studied. Currently some observers that have been proposed are based on a copy of the system, which causes them to be highly dependent on the system parameters, additionally they are redundant (estimate variables that are available). So this paper proposes a novel fractional observer against parametric uncertainties for a certain type of incommensurate fractional order systems. The fractional observer design is based on a property concerning observability in incommensurate fractional order systems which allows us to construct the observer only considering the available output and its fractional derivatives. On the other hand, the convergence analysis of the observation error is carried out using a particular approach of fractional order systems related to the Global Mittag-Leffler boundedness. We prove that there is a compact set GMLA (Globally Mittag-Leffler Attractive, according to Definition 4) where the system that represents the observation error dynamics is attractive and we also prove that the observation error is uniformly bounded. Additionally, the fractional observer is model-free i.e., a system copy is not required, this gives robustness in spite of parametric uncertainties and it is also reduced order therefore one observer must be designed for each variable that we want to estimate consequently the observer is non-redundant (no estimation of variables that are already available). Moreover, our proposed fractional observer can be designed for commensurate fractional order systems and we also show that if we consider integer derivative order, the proposed fractional observer presents certain properties. Finally, in order to show the effectiveness of the proposed fractional observer, an incommensurate fractional order Rössler hyperchaotic system is considered as a numerical example and an incommensurate fractional model of the COVID-19 pandemic as a real-world application.

Microsurgical and endoscopic-assisted supraorbital keyhole approach for intra-suprasellar cysticercosis.

Background: Treatment for intra/suprasellar cysticercosis can be challenging and may result in visual disturbances if not managed properly. Despite its limited knowledge, an effective surgical option exists to treat this condition. This article presents three cases of sellar cysticercosis, comprising one female and two male patients, managed with microsurgical supraorbital keyhole approach (mSKA) and endoscopic-assisted supraorbital keyhole approach (eaSKA). Case Description: The first patient is a 35-year-old man with no prior medical history who suffered from memory deficits and visual disturbances due to a sellar cyst pushing the orbitofrontal gyrus treated with mSKA. The second case involved a 52-year-old man who experienced visual deficits caused by a rostral sellar cyst with posterior displacement of the pituitary gland treated with eaSKA. The third case was a 46-year-old woman who experienced decreased visual acuity and memory loss due to multifocal neurocysticercosis (NCC) with sellarsuprasellar cyst extension treated with mSKA. All case diagnoses were confirmed by neuropathology department. Conclusion: The authors confidently suggest that the SKA is an effective surgical option and could be considered for removing sellar cystic lesions with suprasellar extension. With endoscopic assistance, it improves adequate neurovascular structure visualization.

One enzyme, many faces: urease is also canatoxin.

Ureases catalyze the hydrolysis of urea into carbamate and ammonia. Well-conserved proteins, most plant ureases are hexamers of a single chain subunit, like the most abundant isoform of the jack bean (Canavalia ensiformis) urease (JBU). Canatoxin (CNTX) was originally isolated from these seeds as a neurotoxic protein, and later characterized as an isoform of JBU with lower molecular mass and enzyme activity. Inactive CNTX oligomers form upon storage and stabilization of CNTX was achieved by treatment with low concentration of formaldehyde, avoiding its oligomerization. Here, nano-LC-MS/MS-based peptide analysis of CNTX revealed 804 amino acids identical to those of JBU's sequence (840 amino acids). De novo sequencing of CNTX revealed 15 different peptides containing substitution of amino acid residues, denoting CNTX as a product of a paralog gene of JBU. The MS/MS analysis of formaldehyde-treated CNTX showed that amino acid residues located at the trimer-trimer interface of JBU's hexamer were modified. The data confirmed that CNTX is an isoform of JBU and elucidated that stabilization by formaldehyde treatment occurs by modification of amino acids at the protein's surface that prevents the formation of the hexamer and of higher molecular mass inactive aggregates. HIGHLIGHTSCanatoxin (CNTX) is an isoform of jack bean urease (JBU, hexamer of 90 kDa chains)MS/MS sequencing of CNTX showed 804 amino acids identical in JBU (840 residues)Formaldehyde treatment of CNTX stabilizes its toxicity and avoids oligomerizationModified amino acid residues in CNTX are at the trimer-trimer interface of JBUCommunicated by Ramaswamy H. Sarma.

Biochemical and structural studies on native and recombinant Glycine max UreG: a detailed characterization of a plant urease accessory protein.

Urea is the nitrogen fertilizer most utilized in crop production worldwide. Understanding all factors involved in urea metabolism in plants is an essential step towards assessing and possibly improving the use of urea by plants. Urease, the enzyme responsible for urea hydrolysis, and its accessory proteins, necessary for nickel incorporation into the enzyme active site and concomitant activation, have been extensively characterized in bacteria. In contrast, little is known about their plant counterparts. This work reports a detailed characterization of Glycine max UreG (GmUreG), a urease accessory protein. Two forms of native GmUreG, purified from seeds, were separated by metal affinity chromatography, and their properties (GTPase activity in absence and presence of Ni(2+) or Zn(2+), secondary structure and metal content) were compared with the recombinant protein produced in Escherichia coli. The binding affinity of recombinant GmUreG (rGmUreG) for Ni(2+) and Zn(2+) was determined by isothermal titration calorimetry. rGmUreG binds Zn(2+) or Ni(2+) differently, presenting a very tight binding site for Zn(2+) (K (d) = 0.02 ± 0.01 µM) but not for Ni(2+), thus suggesting that Zn(2+) may play a role on the plant urease assembly process, as suggested for bacteria. Size exclusion chromatography showed that Zn(2+) stabilizes a dimeric form of the rGmUreG, while NMR measurements indicate that rGmUreG belongs to the class of intrinsically disordered proteins. A homology model for the fully folded GmUreG was built and compared to bacterial UreG models, and the possible sites of interaction with other accessory proteins were investigated.

The "ePTFE Sleeve" for Microvascular Decompression of the Facial Nerve.

Hemifacial spasm is a cranial nerve compression syndrome caused by the anteroinferior cerebellar artery (AICA) and posteroinferior cerebellar artery (PICA), characterized by involuntary tonic/clonic contractions of the muscles. The refractory hemifacial spasm can be treated with microvascular decompression, and multivessel compression could require more than conventional microvascular decompression. Multivessel compression may be challenging, and placement of conventional materials may not be sufficient and risks migration. Some transposition techniques for the vertebral artery may even increase the risk of injuring some perforators. Our circumferential expanded polytetrafluoroethylene (ePTFE) (IMPRA, Tempe, Arizona, USA) sleeve technique is unique and accessible, and it could solve this issue. The 2-dimensional Video 1 demonstrates the case of a 38-year-old woman who presented with debilitating left hemifacial spasm for 11 years. On physical examination, she exhibited involuntary, recurrent twitches of left facial muscles and loss of sensory taste. Magnetic resonance imaging revealed touching of the left dominance dolicoectasic vertebral artery, with compression of the PICA and AICA over cranial nerve VII. Conservative measures were recommended. Nevertheless, progressive worsening symptoms presented despite botulinum toxin injections. Left retrosigmoid craniotomy was performed, first placing a 3/4 ePTFE sleeve for decompression of the cisternal portion and the second ePTFE sleeve at the root entry zone of cranial nerve VII to optimize decompression of the offending vessel. In this case, we decided to use ePTFE prosthetic material. The semirigidity and semielastic property force brings an adequate decompression of the nerve and isolates it from the offending artery (AICA-PICA and vertebral artery). Without an uneventful postoperative course, the patient remained neurologically intact with immediate recovery after surgery without facial spasms or facial paresis. At 14 months' follow-up, the patient was without any alteration. The circumferential ePTFE Sleeve is an effective option for microvascular decompression. This technique offers semielastic continuous isolation of the nerve keeping it away from the offending artery. In addition, the circumferential ePTFE fit between neurovascular structures could avoid migration out of position. No cases using this technique have been reported; beyond that, we would like to illustrate this procedure, which is not widely available as a video article.