Selective Formation of S4- and T-Symmetric Supramolecular Tetrahedral Cages and Helicates in Polar Media Assembled via Cooperative Action of Coordination and Hydrogen Bonds.

We report on the synthesis and self-assembly study of novel supramolecular monomers encompassing quadruple hydrogen-bonding motifs and metal-coordinating 2,2'-bipyridine units. When mixed with metal ions such as Fe2+ or Zn2+, the tetrahedron cage complexes are formed in quantitative yields and full diastereoselectivity, even in highly polar acetonitrile or methanol solvents. The symmetry of the complexes obtained has been shown to depend critically on the flexibility of the ligand. Restriction of the rotation of the hydrogen-bonding unit with respect to the metal-coordinating site results in a T-symmetric cage, whereas introducing flexibility either through a methylene linker or rotating benzene ring allows the formation of S4-symmetric cages with self-filled interior. In addition, the possibility to select between tetrahedral cages or helicates and to control the dimensions of the aggregate has been demonstrated with a three-component assembly using external hydrogen-bonding molecular inserts or by varying the radius of the metal ion (Hg2+ vs Fe2+). Self-sorting studies of individual Fe2+ complexes with ligands of different sizes revealed their inertness toward ligand scrambling.

Seasonal and dietary effects on Vitamin D deficiencies detected in wild boar from mid-western Spain.

Vitamin D (VitD) is involved in important mammalian physiological mechanisms, such as Ca-P metabolism, bone development and immunological response. VitD deficiencies are frequently detected in domestic animals and related to various health problems (e.g., rickets, bone deformation). However, knowledge about the status of VitD in wildlife species, such as the wild boar, is scarce. The aims of this work were to explore VitD status in wild boar populations from mid-western Spain and to elucidate the influence of daylight exposure and food supplementation in levels of VitD. Serum concentration of VitD (measured as 25-hydroxivitaminD) was assessed in 276 wild boar from 27 game estates located in mid-western Spain using a commercial ELISA kit. In 19 out of 27 estates, the staff supplied a specific VitD-enriched food (2,000 UI/Kg) ad libitum throughout the year, while in the remaining estates (8), no food was supplied. Blood samples were extracted from hunted animals (198) between October and February of hunting seasons 2016/2017 and 2017/2018, and from live wild boar (78) that were captured, sampled and released (March-September of 2017). The percentage of animals with VitD deficiency (<20 ng/ml), VitD insufficiency (20-30 ng/ml) and VitD sufficiency (>30 ng/ml) was estimated, and the relationship of these levels to factors like sex, age and season was assessed using chi-square tests. Furthermore, associations between daylight exposure and supplemental food with VitD levels were explored using linear models. Of the studied wild boar population, 82.2% showed a VitD deficiency or insufficiency. VitD deficiencies were more frequent in animals sampled in winter and spring. Furthermore, levels of VitD positively correlated with daylight exposure and supplemental food intake. Ad libitum supplementation with VitD-enriched food was insufficient to prevent VitD deficiencies in wild boar from November to April, probably because food consumption is lower during this period.

Molecular Basis for the Recognition of Higher Fullerenes into Ureidopyrimidinone-Cyclotriveratrylene Self-Assembled Capsules.

Fullerenes C60 , C70 , and C84 may be readily encaged within a hydrogen-bonded dimeric capsule, based on two concave cyclotriveratrylene (CTV) scaffolds, each containing three self-complementary 2-ureido-4-[1H]-pyrimidinone (UPy) subunits. NMR spectroscopy and circular dichroism studies, complemented by dispersion-corrected DFT calculations, are reported with the aim of characterizing such capsule-fullerene complexes both structurally and energetically. Six fullerenes are considered: in agreement with experiments, calculations find that encapsulation is most favorable for C84 (on a par with C90 ), and follows the trend C60

Spoligotype diversity and 5-year trends of bovine tuberculosis in Extremadura, southern Spain.

Bovine tuberculosis (bTB) causes significant losses to farming economies worldwide. A better understanding on the epidemiology of this disease and the role that the different hosts develop in the maintenance and spread of bTB is vital to control this zoonotic disease. This study reports the spoligotype diversity and temporal evolution of Mycobacterium tuberculosis Complex (MTBC) isolates obtained from Extremadura (southern Spain). Genotyping data of Mycobacterium bovis (n = 2102) and Mycobacterium caprae (n = 96) isolates from cattle and wildlife species, collected between 2008 and 2012, were used in this study. The isolates resulted clustered into 88 spoligotypes which varied largely in frequency and occurrence in the three hosts. The 20 most frequent patterns represented 91.99 % of the isolates, the spoligotype SB0121 being the clearly predominant and most widely dispersed geographically. The major variety of the spoligotype patterns (78 out of 88) was isolated from the cattle, in fact 50 (56.83 %) of the patterns were found only in this species. Within the spoligotypes shared between the cattle and wildlife species, 17 patterns (1747 isolates) were shared with wild boar and Iberian red deer, 10 patterns (308 isolates) were exclusively shared with wild boar, and only one pattern (two isolates) was shared exclusively with Iberian red deer. The significant number of spoligotypes shared between the three hosts (79.49 %) highlights the components of the multi-host system that allows the bTB maintenance in our study area. The greater percentage of isolates shared by the wild boar and cattle (93.50 %) supports the role of wild boar as main maintenance host for bTB in cattle. These results could be extrapolated to areas with a similar epidemiological scenario and could be helpful for other countries where wild reservoirs represent a handicap for the successful eradication of bTB from livestock.

Cellular Antisense Activity of PNA-Oligo(bicycloguanidinium) Conjugates Forming Self-Assembled Nanoaggregates.

A series of peptide nucleic acid-oligo(bicycloguanidinium) (PNA-BGn ) conjugates were synthesized and characterized in terms of cellular antisense activity by using the pLuc750HeLa cell splice correction assay. PNA-BG4 conjugates exhibited low micromolar antisense activity, and their cellular activity required the presence of a hydrophobic silyl terminal protecting group on the oligo(BG) ligand and a minimum of four guanidinium units. Surprisingly, a nonlinear dose-response with an activity threshold around 3-4 µM, indicative of large cooperativity, was observed. Supported by light scattering and electron microscopy analyses, we propose that the activity, and thus cellular delivery, of these lipo-PNA-BG4 conjugates is dependent on self-assembled nanoaggregates. Finally, cellular activity was enhanced by the presence of serum. Therefore we conclude that the lipo-BG-PNA conjugates exhibit an unexpected mechanism for cell delivery and are of interest for further in vivo studies.

FATAL CASE OF STREPTOCOCCUS SUIS INFECTION IN A YOUNG WILD BOAR (SUS SCROFA) FROM SOUTHWESTERN SPAIN.

Streptococcus suis is a recognized pathogen that may cause important diseases in pigs and humans. This microorganism has been repeatedly isolated from wild boar (Sus scrofa). However, its health implications for this wild species are still unknown. This article reports a detailed description of a fatal case of septicemia by S. suis affecting a young wild boar. The affected animal, about 15 days old, was found near death and exhibiting neurologic signs at a wild boar estate in southwestern Spain. Postmortem examination showed generalized congestion, brain hemorrhages and lobular pneumonia. Histopathological evaluation demonstrated the presence of meningitis and encephalitis with marked congestion and suppurative bronchopneumonia. Streptococcus suis serotype 2 isolates exhibiting important virulence factors (extracellular factor, muramidase-released protein, and suylisin) were isolated from the affected animal. This study confirms the presence of potentially virulent and zoonotic strains of S. suis in wild boar from Spain.

Enhancing molecular recognition in electron donor-acceptor hybrids via cooperativity.

Herein, we report the synthesis of guanidinium bis-porphyrin tweezers 1 and fullerene carboxylate 3, their assembly into a novel supramolecular 1@3 electron donor-acceptor hybrid, and its characterization. In solution, the binding constant affording 1@3 is exceptionally high. 1@3, which features a highly confined topography, builds up from a combination of guanidinium-carboxylate hydrogen bonding and π-π stacking/charge-transfer motifs. The latter is governed by interactions between the electron-donating porphyrin and the electron-accepting fullerene. Importantly, positive cooperativity between the applied binding motifs is corroborated by a number of experimental techniques, such as NMR, absorption, fluorescence, etc. In addition, transient absorption experiments shed light onto electron-transfer processes taking place in the ground state and upon photoexcitation. In fact, porphyrin excitation powers an electron transfer to the fullerene yielding charge separated state lifetimes in the nanosecond regime.

Genotypic characterization by spoligotyping and VNTR typing of Mycobacterium bovis and Mycobacterium caprae isolates from cattle of Tunisia.

This work is an approach to the molecular epidemiology of Mycobacterium tuberculosis complex (MTBC) bovine infections in Tunisia. A total of 35 MTBC isolates from both lateral retropharyngeal lymph node samples of cattle slaughtered in different Tunisian regions were genotyped by spoligotyping and variable number tandem repeat typing (VNTR)-typing. Spoligotyping allowed to identify two profiles not previously registered, namely SB2024, a Mycobacterium caprae isolate from Nabeul Region (North East Tunisia), the first description of this species in the country, and SB2025 (Mycobacterium bovis) from Sfax Region (Southern Tunisia). A second M. caprae isolate with a spoligotyping profile previously described in Europe mainland, SB0418, was also isolated from a bovine of Sfax region. Both isolates suggest the possibility of a widespread distribution of this species in the country. The predominant spoligotype was SB0120, present in all Tunisian regions selected for the study but Nabeul. Molecular typing also allowed to describe a mixed infection caused by two different M. bovis isolates (SB0120 and SB0848) in the same animal. VNTR typing was highly discriminant by testing a panel of six loci. Loci QUB3232 and QUB11b were the most discriminant, whereas ETR-D and QUB11a had the lower diversity index. The value of allelic diversity can significantly vary among countries; thus, it is important to standardize a panel of loci for future inter-laboratory comparisons. Although VNTR typing proved to be useful for an efficient discrimination among MTBC isolates, especially in combination with spoligotyping, further studies are needed in order to assess the genetic diversity of the MTBC in Tunisia.

Population structure of Mycobacterium tuberculosis in El Oro: A first insight into Ecuador-Peru tuberculosis transmission.

BACKGROUND: Tuberculosis (TB) is a major public health concern in Ecuador and Peru, both settings of high burden of drug resistance TB. Molecular epidemiology tools are important to understand the transmission dynamics of Mycobacterium tuberculosis Complex (MTBC) and to track active transmission clusters of regional importance. This study is the first to address the transmission of TB between Peru and Ecuador through the population structure of MTBC lineages circulating in the Ecuadorian border province of "El Oro". METHODS: A total number of 56 MTBC strains from this province for years 2012-2015 were included in the study and analyzed by 24-loci MIRU-VNTR and spoligotyping. RESULTS: Genotyping revealed a high degree of diversity for MTBC in "El Oro", without active transmission clusters. MTBC L4 was predominant, with less than 2% of strains belonging to MTBC L2-Beijing. CONCLUSIONS: These results may suggest that TB dynamics in this rural and semi-urban area would not be linked to highly transmitted strains like MTBC L2-Beijing from Peru, but related to TB relapse; although further studies with larger MTBC cultures collection from recent years are needed. Nevertheless, we recommend to reinforce TB surveillance programs in remote rural settings and border regions in Ecuador.

A first insight into tuberculosis transmission at the border of Ecuador and Colombia: a retrospective study of the population structure of Mycobacterium tuberculosis in Esmeraldas province.

Objective: Tuberculosis (TB) is a major public health concern in Ecuador and Colombia, considering that both countries are high-burden TB settings. Molecular epidemiology is crucial to understand the transmission dynamics of Mycobacterium tuberculosis complex (MTBC) and to identify active transmission clusters of regional importance. Methods: We studied the potential transmission of TB between Colombia and Ecuador through the analysis of the population structure of MTBC lineages circulating in the Ecuadorian province of Esmeraldas at the border with Colombia. A total of 105 MTBC strains were characterized by 24-loci MIRU-VNTR and spoligotyping. Results: MTBC lineage 4 is only present in Esmeraldas; no MTBC strains belonging to Lineage 2-sublineage Beijing were found despite its presence in other provinces of Ecuador and, in Colombia. Genotyping results revealed a high degree of diversity for MTBC in Esmeraldas: Neither active transmission clusters within this province nor including MTBC strains from Colombia or other provinces of Ecuador were found. Conclusion: Our data suggest that tuberculosis dynamics in this rural and isolated area may be not related to highly transmitted strains but could be influenced by other health determinants that favor TB relapse such as poverty and poor health system access. Further studies including a larger number of MTBC strains from Esmeraldas are necessary to test this hypothesis.

Rapid and accurate identification and differentiation of Mycobacterium tuberculosis and non-tuberculous mycobacteria using PCR kits available in a high-burden setting.

Infections caused by mycobacteria, including Mycobacterium tuberculosis complex (MTBC) and non-tuberculous mycobacteria (NTM), are a major public health issue worldwide. An accurate diagnosis of mycobacterial species is a challenge for surveillance and treatment, particularly in high-burden settings usually associated with low- and middle-income countries. In this study, we analyzed the clinical performance of two commercial PCR kits designed for the identification and differentiation of MTBC and NTM, available in a high-burden setting such as Ecuador. A total of 109 mycobacteria isolates were included in the study, 59 of which were previously characterized as M. tuberculosis and the other 59 as NTM. Both kits displayed great clinical performance for the identification of M. tuberculosis, with 100% sensitivity. On the other hand, for NTM, one of the kits displayed a good clinical performance with a sensitivity of 94.9% (CI 95%: 89-100%), while the second kit had a reduced sensitivity of 77.1% (CI 95%: 65-89%). In conclusion, one of the kits is a fast and reliable tool for the identification and discrimination of MTBC and NTM from clinical isolates.

Prevalence, drug resistance, and genotypic diversity of the RDRio subfamily of Mycobacterium tuberculosis in Ecuador: a retrospective analysis for years 2012-2016.

Introduction: A major sublineage within the Mycobacterium tuberculosis (MTB) LAM family characterized by a new in-frame fusion gene Rv3346c/55c was discovered in Rio de Janeiro (Brazil) in 2007, called RDRio, associated to drug resistance. The few studies about prevalence of MTB RDRio strains in Latin America reported values ranging from 3% in Chile to 69.8% in Venezuela, although no information is available for countries like Ecuador. Methods: A total of 814 MTB isolates from years 2012 to 2016 were screened by multiplex PCR for RDRio identification, followed by 24-loci MIRU-VNTR and spoligotyping. Results: A total number of 17 MTB RDRio strains were identified, representing an overall prevalence of 2.09% among MTB strains in Ecuador. While 10.9% of the MTB isolates included in the study were multidrug resistance (MDR), 29.4% (5/17) of the RDRio strains were MDR. Discussion: This is the first report of the prevalence of MTB RDRio in Ecuador, where a strong association with MDR was found, but also a very low prevalence compared to other countries in Latin America. It is important to improve molecular epidemiology tools as a part of MTB surveillance programs in Latin America to track the transmission of potentially dangerous MTB stains associated to MDR TB like MTB RDRio.

Mapping the risk of exposure to Crimean-Congo haemorrhagic fever virus in the Iberian Peninsula using Eurasian wild boar (Sus scrofa) as a model.

Crimean-Congo haemorrhagic fever (CCHF) virus (CCHFV) is a tick-borne zoonotic pathogen that can cause a lethal haemorrhagic disease in humans. Although the virus appears to be endemically established in the Iberian Peninsula, CCHF is an emerging disease in Spain. Clinical signs of CCHFV infection are mainly manifested in humans, but the virus replicates in several animal species. Understanding the determinants of CCHFV exposure risk from animal models is essential to predicting high-risk exposure hotspots for public health action. With this objective in mind, we designed a cross-sectional study of Eurasian wild boar (Sus scrofa) in Spain and Portugal. The study analysed 5,291 sera collected between 2006 and 2022 from 90 wild boar populations with a specific double-antigen ELISA to estimate CCHFV serum prevalence and identify the main determinants of exposure probability. To do so, we statistically modelled exposure risk with host- and environment-related predictors and spatially projected it at a 10 × 10 km square resolution at the scale of the Iberian Peninsula to map foci of infection risk. Fifty-seven (63.3 %) of the 90 populations had at least one seropositive animal, with seroprevalence ranging from 0.0 to 88.2 %. Anti-CCHFV antibodies were found in 1,026 of 5,291 wild boar (19.4 %; 95 % confidence interval: 18.3-20.5 %), with highest exposure rates in southwestern Iberia. The most relevant predictors of virus exposure risk were wild boar abundance, local rainfall regime, shrub cover, winter air temperature and soil temperature variation. The spatial projection of the best-fit model identified high-risk foci as occurring in most of western and southwestern Iberia and identified recently confirmed risk foci in eastern Spain. The results of the study demonstrate that serological surveys of CCHFV vector hosts are a powerful, robust and highly informative tool for public health authorities to take action to prevent human cases of CCHF in enzootic and emergency settings.

Dynamic multivalency for carbohydrate-protein recognition through dynamic combinatorial libraries based on Fe(II)-bipyridine complexes.

Molecular recognition: Dynamic combinatorial libraries (DCLs) exploiting multivalency effects and metal coordination have been employed for carbohydrate-protein recognition. The interaction of a three-component DCL based on 2,2'-bipyridine (bipy)-Fe(II) complexes with concanavalin A (ConA; see scheme) results in enhanced binding by multivalent presentation with a bias towards mannose-containing library components.

Ocular lesions associated with Chlamydia suis in a wild boar piglet (Sus scrofa) from a semi-free range population in Spain.

The role of the wild boar (Sus scrofa) as a reservoir for a large number of pathogens that can affect both domestic animals and humans has been widely studied in the last few years. However, the impact of some of these pathogens on the health of wild boar populations is still being determined. This article presents a clinical case of severe bilateral keratoconjunctivitis affecting a 2-mo-old piglet from a semi-free range population in Spain. Histopathologic and microbiologic analysis revealed lesions in the cornea, choroid, and optical nerve, and Chlamydia suis was detected in the eyes bilaterally. The visual handicap resulting from this type of lesion greatly affects the survival of this affected piglet.

Isolation of Aeromonas hydrophila in the respiratory tract of wild boar: pathologic implications.

Aeromonas hydrophila has been repeatedly reported as an animal pathogen. This study describes a case of a wild boar piglet in Spain with severe purulent pneumonia caused by A. hydrophila. To confirm the presence of A. hydrophila in the respiratory tract of wild boars from the same region, lung samples from 34 adult hunted animals and nasal samples from 12 live animals were collected and cultured in selective medium. Lung lesions were studied in hunted wild boars to determine the presence of A. hydrophila and to assess its role as a possible respiratory pathogen in wild boars. A. hydrophila was isolated in 10.87% of the animals studied (8.82% of the dead animals and 16% of the live animals). However, its presence in the lungs of adults could not be correlated with the lesions found. Thus, the role of A. hydrophila as a respiratory pathogen is likely to be influenced by other factors, such as age, immunologic status, or coinfection with other pathogens. As a zoonotic pathogen, the presence of A. hydrophila in wild boars may pose a potential risk to people who consume their flesh.

A tetrahedron from homooxacalix[3]arene, the fifth Platonic polyhedron from calixarenes and uranyl.

A self-assembled tetrahedral cage results from two C 3-symmetry building blocks, namely, homooxacalix[3]arene tricarboxylate and uranyl cation, as demonstrated by X-ray crystallography. In the cage, four metals coordinate at the lower rim with the phenolic and ether oxygen atoms to shape the macrocycle with appropriate dihedral angles for tetrahedron formation, whereas four additional uranyl cations further coordinate at the upper-rim carboxylates to finalize the assembly. Counterions dictate the filling and porosity of the aggregates, whereas potassium induces highly porous structures, and tetrabutylammonium yields compact, densely packed frameworks. The tetrahedron metallo-cage complements our previous report (Pasquale et al., Nat. Commun., 2012, 3, 785) on uranyl-organic frameworks (UOFs) from calix[4]arene and calix[5]arene carboxylates (octahedral/cubic and icosahedral/dodecahedral giant cages, respectively) and completes the assembly of all five Platonic solids from just two chemical components.

Combination with Annual Deworming Treatments Does Not Enhance the Effects of PCV2 Vaccination on the Development of TB in Wild Boar Populations.

Vaccination against PCV2 has been proven to be an effective measure to reduce the severity of TB in wild boar. The combination of this measure with strategies focused on treating other key concomitant pathogens, such as nematodes, could be a useful strategy. This study assesses whether a combination of deworming treatments and PCV2 vaccination may reduce the prevalence and severity of TB in wild boar. The study was conducted on five game estates in mid-western Spain where four groups of wild boar were produced: control, vaccinated, dewormed and vaccinated-dewormed. Wild boars from all groups were hunted between 2017 and 2020, and all of them received a TB diagnosis based on pathological and microbiological tests. Generalised linear models were used to explore the effect of deworming and PCV2 vaccination on TB prevalence and severity. PCV2-vaccinated animals showed lower probabilities of suffering severe TB lesions. However, no differences regarding TB severity were found between dewormed and non-dewormed wild boar. PCV2 vaccination reduces TB severity in wild boar. However, annual deworming does not produce a long-term parasitological reduction that can influence the development of TB in wild boar, nor does it improve the effect of PCV2 vaccination on TB.

Anion-responsive diguanidinium-based chiral organogelators.

The chiral bicyclic diguanidinium chloride 1 forms gels in aromatic apolar solvents. The gels were characterized at different levels of organization, from the macroscopic to the molecular level by using microscopy, spectroscopy, and powder X-ray diffraction. The dependency on chirality has been highlighted by circular dichroism and electron microscopy. Furthermore, the gel has been shown to be effectively responsive to anionic stimuli, thus allowing the reversible control of the organic-phase gelation in contact with different salted aqueous solutions.

Non-peptidic cell-penetrating agents: synthesis of oligomeric chiral bicyclic guanidinium vectors.

Polycationic oligo(chiral bicyclic guanidines) constitute useful non-peptidic penetrating agents for cell uptake and protein surface recognition. We report herein improved and selective procedures for the preparation of oligoguanidinium scaffolds linked through thioether bonds, with similar or different groups and functions at both ends of the chain. Two synthetic strategies were developed to obtain these compounds in relatively good yields from a common thioacetate precursor: generation of a disulfide intermediate or thiolate formation. Thus, tetraguanidinium intermediates 8 and 22 are best synthesized by the disulfide route, whereas hexamer 29, octamer 31, and trimer 37 arise from a combination of both the disulfide and the thioacetate routes. Finally, tetramer 28 can be readily obtained from either strategy.