Biomarkers for Duchenne muscular dystrophy progression: impact of age in the mdx tongue spared muscle.

BACKGROUND: Duchenne muscular dystrophy (DMD) is a severe form of muscular dystrophy without an effective treatment, caused by mutations in the DMD gene, leading to the absence of dystrophin. DMD results in muscle weakness, loss of ambulation, and death at an early age. Metabolomics studies in mdx mice, the most used model for DMD, reveal changes in metabolites associated with muscle degeneration and aging. In DMD, the tongue muscles exhibit unique behavior, initially showing partial protection against inflammation but later experiencing fibrosis and loss of muscle fibers. Certain metabolites and proteins, like TNF-α and TGF-ß, are potential biomarkers for dystrophic muscle characterization. METHODS: To investigate disease progression and aging, we utilized young (1 month old) and old (21-25 months old) mdx and wild-type tongue muscles. Metabolite changes were analyzed using 1H nuclear magnetic resonance, while TNF-α and TGF-ß were assessed using Western blotting to examine inflammation and fibrosis. Morphometric analysis was conducted to assess the extent of myofiber damage between groups. RESULTS: The histological analysis of the mid-belly tongue showed no differences between groups. No differences were found between the concentrations of metabolites from wild-type or mdx whole tongues of the same age. The metabolites alanine, methionine, and 3-methylhistidine were higher, and taurine and glycerol were lower in young tongues in both wild type and mdx (p < 0.001). The metabolites glycine (p < 0.001) and glutamic acid (p = 0.0018) were different only in the mdx groups, being higher in young mdx mice. Acetic acid, phosphocreatine, isoleucine, succinic acid, creatine, and the proteins TNF-α and TGF-ß had no difference in the analysis between groups (p > 0.05). CONCLUSIONS: Surprisingly, histological, metabolite, and protein analysis reveal that the tongue of old mdx remains partially spared from the severe myonecrosis observed in other muscles. The metabolites alanine, methionine, 3-methylhistidine, taurine, and glycerol may be effective for specific assessments, although their use for disease progression monitoring should be cautious due to age-related changes in the tongue muscle. Acetic acid, phosphocreatine, isoleucine, succinate, creatine, TNF-α, and TGF-ß do not vary with aging and remain constant in spared muscles, suggesting their potential as specific biomarkers for DMD progression independent of aging.

Biomarkers for Duchenne muscular dystrophy progression: impact of age in the mdx tongue spared muscle.

Background: Duchenne muscular dystrophy (DMD) is a severe form of muscular dystrophy without an effective treatment, caused by mutations in the DMD gene, leading to the absence of dystrophin. DMD results in muscle weakness, loss of ambulation and death at an early age. Metabolomics studies in mdx mice, the most used model for DMD, reveal changes in metabolites associated with muscle degeneration and aging. In DMD, the tongue muscles exhibit unique behavior, initially showing partial protection against inflammation but later experiencing fibrosis and loss of muscle fibers. Certain metabolites and proteins, like TNF-α and TGF-ß, are potential biomarkers for dystrophic muscle characterization. Methods: To investigate disease progression and aging, we utilized young (1-month old) and old (21-25 months old) mdx and wild-type mice. Metabolite changes were analyzed using 1-H Nuclear Magnetic Resonance, while TNF-α and TGF-ß were assessed using Western blotting to examine inflammation, and fibrosis. Morphometric analysis was conducted to assess the extent of myofiber damage between groups. Results: The histological analysis of the tongue showed no differences between groups. No differences were found between the concentrations of metabolites from wild type or mdx animals of the same age. The metabolites alanine, methionine, 3-methylhistidine were higher, and taurine and glycerol were lower in young animals in both wild type and mdx (p < 0.001). The metabolites glycine (p < 0.001) and glutamic acid (p = 0.0018) were different only in the mdx groups, being higher in young mdx mice. Acetic acid, phosphocreatine, isoleucine, succinic acid, creatine and the proteins TNF-α and TGF-ß had no difference in the analysis between groups (p > 0.05). Conclusions: Surprisingly, histological and protein analysis reveals that the tongue of young and old mdx animals is protected from severe myonecrosis observed in other muscles. The metabolites alanine, methionine, 3-methylhistidine, taurine, and glycerol may be effective for specific assessments, although their use for disease progression monitoring should be cautious due to age-related changes. Acetic acid, phosphocreatine, isoleucine, succinate, creatine, TNF-α, and TGF-ß do not vary with aging and remain constant in spared muscles, suggesting their potential as specific biomarkers for DMD progression independent of aging.

5-Chloro-8-nitro-1-naphthoyl (NNap): A Selective Protective Group for Amines and Amino Acids.

The synthesis of 5-chloro-8-nitro-1-naphthoyl chloride and its use as a protective group for amines is described. Protection is carried out with an auxiliary amine or under mild Schotten-Baumann conditions in high yield (>86%), while deprotection can be achieved easily under gentle reducing conditions due to the large steric tension between C-1 and C-8 naphthalene substituents. The reaction has been successfully tested in dipeptide synthesis and amino alcohols protection, and it has proved selective for the ε-amine group of lysine.

A Reciprocal Transplant Experiment Confirmed Mite-Resistance in a Honey Bee Population from Uruguay.

In the past few years there has been an increasing interest for the study of honey bee populations that are naturally resistant to the ectoparasitic mite Varroa destructor, aiming to identify the mechanisms that allow the bees to limit the reproduction of the mite. In eastern Uruguay there are still bees resistant to mites that survive without acaricides. In order to determine if the differential resistance to V. destructor was maintained in other environments, a reciprocal transplant experiment was performed between the mite-resistant bee colonies and the mite-susceptible bee colonies from the east and the west of the country, respectively, infesting bees with local mites. In both regions, the mite-resistant colonies expressed a higher hygienic behavior and presented a higher phoretic mites/reproductive mites and mites in drone cells/mites in worker cells ratio than the mite-susceptible colonies. All the mite-susceptible colonies died during fall-winter, while a considerable number of mite-resistant colonies survived until spring, especially in the east of the country. This study shows that the bees in the east of the country maintain in good measure the resistance to V. destructor in other regions and leaves open the possibility that the mites of the two populations have biases in the reproductive behavior.

Tryptophan association in water driven by charge-transfer interactions with electron-deficient aromatic haptens.

The ability of a series of electron-deficient aromatic compounds to form charge-transfer complexes with tryptophan in water has been evaluated by X-ray diffraction studies, UV-vis spectra and NMR. As dinitrophenyl (DNP) ligands are well-known to generate antibody-mediated responses and the π-π stacking interactions with tryptophan residues of the antibody Fab fragment have been reported, most of the aromatic receptors studied here are nitro derivatives. Charge-transfer interactions between the rich indole ring of tryptophan and the electron-deficient aromatic receptors have been observed in the solid state, as four crystal structures of the complexes were obtained. The aromatic donor-acceptor interactions in solution were also verified by UV-vis and NMR spectroscopy. The association of the tripeptide Trp-Gly-Trp, a motif found in antigen Ag43, with the electron-deficient aromatic diimide was also studied by UV-vis and NMR spectroscopy. Our results show that these simple electron-deficient molecules could potentially behave as novel haptens and be incorporated in more elaborated drugs targeting protein-protein interactions, due to the synergistic effect of multiple non-covalent interactions.

Transesterification of Non-Activated Esters Promoted by Small Molecules Mimicking the Active Site of Hydrolases.

The synthesis of small molecules able to mimic the active site of hydrolytic enzymes has been largely pursued in recent decades. The high reaction rates and specificity shown by natural hydrolases present an attractive target, and yet the preparation of suitable small-molecule mimics remains challenging, requiring activated substrates to achieve productive outcomes. Here we present small synthetic artificial enzymes which mimic the catalytic site and the oxyanion hole of chymotrypsin and N-terminal hydrolases and are able to perform, for the first time, the transesterification of a non-activated ester such as ethyl acetate with methanol under mild and neutral reaction conditions.

Geographic Area of Collection Determines the Chemical Composition and Antimicrobial Potential of Three Extracts of Chilean Propolis.

The biological properties of chilean propolis have been described and include antibacterial, antifungal and antibiofilm activities. Propolis has a strong antimicrobial potential. Clinical experiences with synthetic antibiotics indicated the need to discover new sources of bioactive compounds associated with ethnopharmacological knowledge or natural sources such as propolis. The microscopic analysis of pollen grains from plants allows us to determine the botanical origin of the propolis samples. In Angol, sample pollen grains were obtained from fodder plants (Sorghum bicolor; Lotus sp.) and trees, such as Acacia sp., Pinus radiata, Eucalyptus sp. and Salix babylonica. Propolis from the Maule region contains pollen grains from endemic plants such as Quillaja saponaria. Finally, the sample obtained from Melipilla presented a wider variety of pollen extracted from vegetable species.Colorimetric assays performed to quantify the total polyphenols present in Chilean propolis samples established that PCP2 (Angol sample) showed high amounts of phenolics compounds, with significant statistical differences in comparison with the other samples. The main compounds identified were pinocembrin, quercetin and caffeic acid phenethyl ester (CAPE). The Angol sample showed a high content of polyphenols.Studies that determine the influence of geographical and floral variables on the chemical composition of propolis are a valuable source of information for the study of its biological properties.

When a Tritrophic Interaction Goes Wrong to the Third Level: Xanthoxylin From Trees Causes the Honeybee Larval Mortality in Colonies Affected by the River Disease.

The "River Disease" (RD), a disorder impacting honeybee colonies located close to waterways with abundant riparian vegetation (including Sebastiania schottiana, Euphorbiaceae), kills newly hatched larvae. Forager bees from RD-affected colonies collect honeydew excretions from Epormenis cestri (Hemiptera: Flatidae), a planthopper feeding on trees of S. schottiana. First-instar honeybee larvae fed with this honeydew died. Thus, we postulated that the nectars of RD-affected colonies had a natural toxin coming from either E. cestri or S. schottiana. An untargeted metabolomics characterization of fresh nectars extracts from colonies with and without RD allowed to pinpoint xanthoxylin as one of the chemicals present in higher amounts in nectar from RD-affected colonies than in nectars from healthy colonies. Besides, xanthoxylin was also found in the aerial parts of S. schottiana and the honeydew excreted by E. cestri feeding on this tree. A larva feeding assay where xanthoxylin-enriched diets were offered to 1st instar larvae showed that larvae died in the same proportion as larvae did when offered enriched diets with nectars from RD-colonies. These findings demonstrate that a xenobiotic can mimic the RD syndrome in honeybee larvae and provide evidence of an interspecific flow of xanthoxylin among three trophic levels. Further, our results give information that can be considered when implementing measures to control this honeybee disease.

Unraveling Honey Bee-Varroa destructor Interaction: Multiple Factors Involved in Differential Resistance between Two Uruguayan Populations.

The ectoparasite Varroa destructor is the greatest biotic threat of honey bees Apis mellifera in vast regions of the world. Recently, the study of natural mite-resistant populations has gained much interest to understand the action of natural selection on the mechanisms that limit the mite population. In this study, the components of the A. mellifera-V. destructor relationship were thoroughly examined and compared in resistant and susceptible honey bee populations from two regions of Uruguay. Mite-resistant honey bees have greater behavioral resistance (hygienic and grooming behaviors) than susceptible honey bees. At the end of the summer, resistant honey bees had fewer mites and a lower deformed wing virus (DWV) viral load than susceptible honey bees. DWV variant A was the only detected variant in honey bees and mites. Molecular analysis by Short Tandem Repeat showed that resistant honey bees were Africanized (A. m. scutellata hybrids), whereas susceptible honey bees were closer to European subspecies. Furthermore, significant genetic differentiation was also found between the mite populations. The obtained results show that the natural resistance of honey bees to V. destructor in Uruguay depends on several factors and that the genetic variants of both organisms can play a relevant role.

Diversity of Bee Assemblage (Family Apidae) in Natural and Agriculturally Intensified Ecosystems in Uruguay.

Bees (Family Apidae) hold a key role as pollinators in a wide range of angiosperm communities. South America suffered strong modifications during the last decade due to increasing anthropic activities and the expansion of agricultural areas, particularly the boom of soybean. The goal of this research was to know the current diversity of bees in South and Low Littoral regions in Uruguay. Specimens were collected in the seasons 2015-2016 and 2016-2017 on natural meadows, cultivated grasslands, soybean fields, among others flowering communities. Collected specimens were classified following taxonomic keys. Forty-five bee species or taxa were distinguished belonging to the subfamilies Megachilinae (11), Apinae (22), Halictinae (7), Colletinae (2), and Andreninae (3). Most taxa were classified up to genus and 14 to species level. Bee diversity was higher for the South region, with traditional agriculture and rangelands, than that of the Low Littoral region where an increasing agricultural land use took place during the last decade, particularly soybean. In addition, this research aimed to study the presence of native bees and the introduced Apis mellifera (Apinae) in a soybean field at four transects located at 0, 50, 100, and 200 m from the crop border in both seasons. Native bee species were only present at 0 and 50 m, pointing to the relevance of natural edge plant communities for their preservation. This is the first survey on native bee diversity in Uruguay to be regarded as a baseline and the setup of conservation strategies.

Epormenis cestri secretions in Sebastiania schottiana trees cause mass death of honey bee Apis mellifera larvae in Uruguay.

For more than 60 years, sporadic cases of massive summer honey bee larvae mortality in colonies located near freshwater systems with abundant riparian vegetation have been reported in Uruguay. This odd phenomenon, known as "River disease" by beekeepers, can lead to colony death by depopulation. The aim of this study was to detect the causes of larvae death. Different experiments and analyses were performed using affected apiaries located between two important water courses. 1 day old larvae were the most susceptible and substances that killed the larvae were present in the nectar but not in the pollen. A palynological analysis of nectar samples showed that bees collect this resource from commonly pollinated floral species in the country. However, abundant fungi spores and conidia were found, which indicates that the bees also collected honeydews. In the riparian vegetation, bees were observed collecting the secretions of the planthopper Epormenis cestri on Sebastiania schottiana trees. It was found that the mortality period of larvae overlaps with the presence of E. cestri. Larvae maintained in the laboratory were fed (i) nectar from healthy colonies, (ii) nectar from affected colonies, and (iii) secretions of E. cestri. The mortality of the larvae that received nectar from colonies affected with River disease and secretions of E. cestri was higher than the mortality of those receiving nectar from healthy colonies. This represents the first report of planthopper honeydew causing mass larval mortality in honey bees.

Control programme for cystic echinococcosis in Uruguay

Cystic echinococcosis is a highly endemic parasitic zoonosis that is present in the Southern Cone countries of America. For several decades, various prevention and control programmes have been implemented in different countries and regions, with varying results. In Uruguay, a new control programme was implemented in 2006 that employed new strategies for canine diagnosis and treatment, dog population control, diagnosis in humans, epidemiological surveillance, and health education, including community participation. The control programme in Uruguay addresses the control and surveillance of the disease from a holistic perspective based on Primary Health Care, which has strengthened the community’s participation in developing and coordinating activities in an interdisciplinary manner. Similarly, the control programme that is currently implemented is based on a risk-focused approach. The surveillance and control measures were focused on small villages and extremely poor urban areas. In this study, the strategies used and the results obtained from 2008-2013 are analysed and discussed.

Control programme for cystic echinococcosis in Uruguay.

Cystic echinococcosis is a highly endemic parasitic zoonosis that is present in the Southern Cone countries of America. For several decades, various prevention and control programmes have been implemented in different countries and regions, with varying results. In Uruguay, a new control programme was implemented in 2006 that employed new strategies for canine diagnosis and treatment, dog population control, diagnosis in humans, epidemiological surveillance, and health education, including community participation. The control programme in Uruguay addresses the control and surveillance of the disease from a holistic perspective based on Primary Health Care, which has strengthened the community's participation in developing and coordinating activities in an interdisciplinary manner. Similarly, the control programme that is currently implemented is based on a risk-focused approach. The surveillance and control measures were focused on small villages and extremely poor urban areas. In this study, the strategies used and the results obtained from 2008-2013 are analysed and discussed.

Seasonal Variation of Honeybee Pathogens and its Association with Pollen Diversity in Uruguay.

Honeybees are susceptible to a wide range of pathogens, which have been related to the occurrence of colony loss episodes reported mainly in north hemisphere countries. Their ability to resist those infections is compromised if they are malnourished or exposed to pesticides. The aim of the present study was to carry out an epidemiological study in Uruguay, South America, in order to evaluate the dynamics and interaction of honeybee pathogens and evaluate their association with the presence of external stress factors such as restricted pollen diversity and presence of agrochemicals. We monitored 40 colonies in two apiaries over 24 months, regularly quantifying colony strength, parasite and pathogen status, and pollen diversity. Chlorinated pesticides, phosphorus, pyrethroid, fipronil, or sulfas were not found in stored pollen in any colony or season. Varroa destructor was widespread in March (end of summer-beginning of autumn), decreasing after acaricide treatments. Viruses ABPV, DWV, and SBV presented a similar trend, while IAPV and KBV were not detected. Nosema ceranae was detected along the year while Nosema apis was detected only in one sample. Fifteen percent of the colonies died, being associated to high V. destructor mite load in March and high N. ceranae spore loads in September. Although similar results have been reported in north hemisphere countries, this is the first study of these characteristics in Uruguay, highlighting the regional importance. On the other side, colonies with pollen of diverse botanical origins showed reduced viral infection levels, suggesting that an adequate nutrition is important for the development of healthy colonies.

Essential oil from Eupatorium buniifolium leaves as potential varroacide.

Beekeeping has experienced a great expansion worldwide. Nowadays, several conventional pesticides, some organic acids, and essential oil components are the main means of chemical control used against Varroa destructor, an ectoparasite that may contribute to the colony collapse disorders. Varroa resistance against conventional pesticides has already been reported; therefore it is imperative to look for alternative control agents to be included in integrated pest management programs. A good alternative seems to be the use of plant essential oils (EOs) which, as natural products, are less toxic and leave fewer residues. Within this context, a bioprospecting program of the local flora searching for botanical pesticides to be used as varroacides was launched. A primary screening (driven by laboratory assays testing for anti-Varroa activity, and safety to bees) led us to select the EOs from Eupatorium buniifolium (Asteraceae) for follow up studies. We have chemical characterized EOs from twigs and leaves collected at different times. The three E. buniifolium EOs tested were active against Varroa in laboratory assays; however, there are differences that might be attributable to chemical differences also found. The foliage EO was selected for a preliminary field trial (on an experimental apiary with 40 hives) that demonstrated acaricidal activity when applied to the hives. Although activity was less than that for oxalic acid (the positive control), this EO was less toxic to bees than the control, encouraging further studies.

Screening of Uruguayan plants for deterrent activity against insects.

We evaluated the anti-insectan activity of extracts from different vegetative parts of ten plant species native to Uruguay. The selected plants belong to five families: Bignoniaceae: Clytostoma callistegioides, Dolichandra cynanchoides, Macfadyena unguis-cati; Sapindaceae: Dodonaea viscosa, Allophylus edulis, Serjania meridionalis; Lamiaceae: Salvia procurrens, Salvia guaranitica; Solanaceae: Lycium cestroides; and Phytolaccaceae: Phytolacca dioica. The extracts were evaluated in independent bioassays against four insect pests and one beneficial insect. Aphid settling inhibition was evaluated with a grass specialist, Rhopalosiphum padi, and a feeding generalist, Myzus persicae (both Hemiptera: Aphididae). Antifeedant activity was tested with adults of the specialist Epilachna paenulata (Coleoptera: Coccinellidae) and larvae of the generalist Spodoptera littoralis (Lepidoptera: Noctuidae). Finally, contact toxicity was assessed with honey bees, Apis mellifera (Hymenoptera: Apidae). Strong settling inhibition (SI) activity (expressed as %SI, where 100% means complete inhibition by the extract) was found only for the twig extracts of A. edulis (Sapindaceae) against M. persicae (% SI = 77 +/- 4). Antifeedant activity (expressed as % of feeding reduction (FR), where 100% means no consumption on extract-treated diet) against E. paenulata was significant for the leaf extracts of L. cestroides (Solanaceae) (% FR = 100 +/- 0) as well as of all Bignoniaceae and Sapindaceae species. No extracts were active against S. littoralis larvae, and most of them were innocuous to honey bees, with the exception of L. cestroides and S. meridionalis leaf extracts.