Aptamer-based diagnostic and therapeutic approaches in animals: Current potential and challenges

Fast and precise diagnosis of infectious and non-infectious animal diseases and their targeted treatments are of utmost importance for their clinical management. The existing biochemical, serological and molecular methods of disease diagnosis need improvement in their specificity, sensitivity and cost and, are generally not amenable for being used as points-of-care (POC) device. Further, with dramatic changes in environment and farm management practices, one should also arm ourselves and prepare for emerging and re-emerging animal diseases such as cancer, prion diseases, COVID-19, influenza etc. Aptamer – oligonucleotide or short peptides that can specifically bind to target molecules – have increasingly become popular in developing biosensors for sensitive detection of analytes, pathogens (bacteria, virus, fungus, prions), drug residues, toxins and, cancerous cells. They have also been proven successful in the cellular delivery of drugs and targeted therapy of infectious diseases and physiological disorders. However, the in vivo application of aptamer-mediated biosensing and therapy in animals has been limited. This paper reviews the existing reports on the application of aptamer-based biosensors and targeted therapy in animals. It also dissects the various modifications to aptamers that were found to be successful in in vivo application of the aptamers in diagnostics and therapeutics. Finally, it also highlights major challenges and future directions in the application of aptamers in the field of veterinary medicine.     

Circannual rhythm of plasmatic vitamin D levels and the association with markers of psychophysical stress in a cohort of Italian professional soccer players

Adequate plasmatic Vitamin D levels are crucial to maintain calcium homeostasis and bone metabolism both in the general population and in athletes. Correct dietary supply and a regular sun exposure are fundamental for allowing the desired and effective fitness level. Past studies highlighted a scenario of Vitamin D insufficiency among professional soccer players in several countries, especially in North Europe, whilst a real deficiency in athletes is rare. The typical seasonal fluctuations of Vitamin D are wrongly described transversally in athletes belonging to teams that play at different latitudes and a chronobiologic approach studying the Vitamin D circannual rhythm in soccer players has not been described yet. Therefore, we studied plasma vitamin D, cortisol, testosterone, and creatin kinase (CK) concentrations in three different Italian professional teams training at the same latitude during a period of two consecutive competitive seasons (2013 and 2014). In this retrospective observational study, 167 professional soccer players were recruited (mean age at sampling 25.1 ± 4.7 years) and a total of 667 blood drawings were carried out to determine plasma 25(OH)D, serum cortisol, serum testosterone and CK levels. Testosterone to cortisol ratio (TC) was calculated based as a surrogate marker of overtraining and psychophysical stress and each athlete was drawn until a maximum of 5 times per season. Data extracted by a subgroup of players that underwent at least 4 sample drawings along a year (N = 45) were processed with the single and population mean cosinor tests to evaluate the presence of circannual rhythms: the amplitude (A), acrophase (Φ) and the MESOR (M) are described. In total, 55 players (32.9%) had an insufficient level of 25(OH)D during the seasons and other 15 athletes (9.0%) showed, at least once, a deficiency status of Vitamin D. The rhythmometric analyses applied to the data of Vitamin D revealed the presence of a significant circannual rhythm (p < 0.001) with the acrophase that occurred in August; the rhythms of Vitamin D levels were not different neither among the three soccer teams nor between competitive seasons. Cortisol, testosterone and TC showed significant circannual rhythms (p < 0.001): cortisol registered an acrophase during winter (February) while testosterone and TC registered their peaks in the summer months (July). On the contrary, CK did not display any seasonal fluctuations. In addition, we observed weak but significant correlations between 25(OH)D versus testosterone (r = 0.29 and p < 0.001), cortisol (r = ?0.27 and p < 0.001) and TC (r = 0.37 and p < 0.001). No correlation was detected between Vitamin D and CK. In conclusion, the correct chronobiologic approach in the study of annual variations of Vitamin D, cortisol and testosterone could be decisive in the development of more specific supplementation and injury prevention strategies by athletic trainers and physicians.     

Development of the fetal neural retina in vitro and in ectopic transplants in vivo

Investigation of the developmental potential of immature tissues is important for novel approaches to human regenerative medicine. Development of the fetal neural retina has therefore been investigated in two experimental systems. Retinas were microsurgically isolated from 20-days-old rat fetuses and cultivated in vitro for 12 days or transplanted in vivo under the kidney capsule of adult males for as long as 6 months. Shedding of the photoreceptor outer segment which is a process occurring at the terminal stage of photoreceptor differentiation was observed in culture by transmission electron microscopy (TEM). In transplants, no photoreceptors were found although markers of terminal neural and glial differentiation (e,g. synaptophysin, chromogranin and glial fibrilary acidic protein--GFAP) along with the molecules involved in the process of differentiation (guidance molecule semaphorin IIIA and chondroitin sulfate proteoglycan) were expressed. Semaphorin was differentially expressed being absent from older transplants. Proliferating cell nuclear antigen and nestin (marker of undifferentiated neural cells) were still weakly expressed even in six-months-old transplants. We could conclude that in both our experimental systems fetal neural retina proceeded to differentiate further on. However, even in long-term ectopic transplants a small population of cells still retained the potential for proliferation and has not yet reached the stage of terminal differentiation.     

Is Body Mass Index a Measure of Adiposity in Elderly Women?

Objective: To evaluate the accuracy of body mass index (BMI) as a predictor of body fat in elderly women. Research Methods and Procedures: A total of 1423 women aged 67 ± 5 (mean ± SD, range: 60 to 88) years were consecutively enrolled into the study. Fat mass (FM) was measured using DXA. Results: BMI explained 72.9% of FM variance (p < 0.0001), with a root mean square error of estimate (RMSE) of 3.5 kg. After standardization of RMSE on the dependent variable as RMSE%, the prediction error equaled 15%. BMI explained 54.8% of FM% variance (p < 0.0001), with an RMSE of 4.1%, corresponding to an RMSE% of 11%. Discussion: The relatively high RMSE% of the FM and FM%‐BMI associations caution against the use of BMI as an adiposity index in individual elderly women. However, an error corresponding to 11% of FM% may be accepted for population studies of body fat in elderly women.     

Thiamin biosynthesis in prokaryotes

Twelve genes involved in thiamin biosynthesis in prokaryotes have been identified and overexpressed. Of these, six are required for the thiazole biosynthesis (thiFSGH, thiI, and dxs), one is involved in the pyrimidine biosynthesis (thiC), one is required for the linking of the thiazole and the pyrimidine (thiE), and four are kinase genes (thiD, thiM, thiL, and pdxK). The specific reactions catalyzed by ThiEF, Dxs, ThiDM, ThiL, and PdxK have been reconstituted in vitro and ThiS thiocarboxylate has been identified as the sulfur source. The X-ray structures of thiamin phosphate synthase and 5-hydroxyethyl-4-methylthiazole kinase have been completed. The genes coding for the thiamin transport system (thiBPQ) have also been identified. Remaining problems include the cloning and characterization of thiK (thiamin kinase) and the gene(s) involved in the regulation of thiamin biosynthesis. The specific reactions catalyzed by ThiC (pyrimidine formation), and ThiGH and ThiI (thiazole formation) have not yet been identified. Received: 23 August 1998 / Accepted: 16 January 1999     

Characterization of endothelin-converting enzyme-2. Implication for a role in the nonclassical processing of regulatory peptides

Most neuroendocrine peptides are generated by proteolysis of the precursors at basic residue cleavage sites. Prohormone convertases belonging to the subtilisin family of serine proteases are primarily responsible for processing at these "classical sites." In addition to the classical cleavages, a subset of bioactive peptides is generated by processing at "nonclassical" sites. The proteases responsible for these cleavages have not been well explored. Members of several metalloprotease families have been proposed to be involved in nonclassical processing. Among them, endothelin-converting enzyme-2 (ECE-2) is a good candidate because it exhibits a neuroendocrine distribution and an acidic pH optimum. To examine the involvement of this protease in neuropeptide processing, we purified the recombinant enzyme and characterized its catalytic activity. Purified ECE-2 efficiently processes big endothelin-1 to endothelin-1 by cleavage between Trp(21) and Val(22) at acidic pH. To characterize the substrate specificity of ECE-2, we used mass spectrometry with a panel of 42 peptides as substrates to identify the products. Only 10 of these 42 peptides were processed by ECE-2. A comparison of residues around the cleavage site revealed that ECE-2 exhibits a unique cleavage site selectivity that is related to but distinct from that of ECE-1. ECE-2 tolerates a wide range of amino acids in the P1-position and prefers aliphatic/aromatic residues in the P1'-position. However, only a small fraction of the aliphatic/aromatic amino acid-containing sites were cleaved, indicating that there are additional constraints beyond the P1- and P1'-positions. The enzyme is able to generate a number of biologically active peptides from peptide intermediates, suggesting an important role for this enzyme in the biosynthesis of regulatory peptides. Also, ECE-2 processes proenkephalin-derived bovine adrenal medulla peptides, and this processing leads to peptide products known to have differential receptor selectivity. Finally, ECE-2 processes PEN-LEN, an endogenous inhibitor of prohormone convertase 1, into products that do not inhibit the enzyme. Taken together, these results are consistent with an important role for ECE-2 in the processing of regulatory peptides at nonclassical sites.     

A facile Er(OTf)3-catalyzed synthesis of 2,3-unsaturated O- and S-glycosides

Er(OTf)(3) is a useful catalyst for the Ferrier rearrangement furnishing high yields of O- and S-glycosides. The transformation has wide applicability, cleaner reaction profiles, mild reaction conditions, and high stereoselectivity and the catalyst, which is also commercially available, can be recovered and reused.     

Knocking Out Sigma-1 Receptors Reveals Diverse Health Problems

Cellular and Molecular Neurobiology - Sigma-1 receptor (Sig-1R) is a protein present in several organs such as brain, lung, and heart. In a cell, Sig-1R is mainly located across the membranes of...     

Carotid Stiffness and Physical Activity in Elderly—A Short Report of the SAPALDIA 3 Cohort Study

Introduction

Regular physical activity has been shown to reduce cardiovascular disease risk in the general population. While smaller studies in specified groups (highly trained versus untrained individuals) indicate a certain dose-dependent effect of physical activity on the reduction of carotid stiffness (an indicator of subclinical vascular disease), it is unclear whether this association is present in a representative sample. Thus, we investigated this question cross-sectionally in participants from the population-based Swiss Cohort Study on Air Pollution And Lung and Heart Diseases In Adults (SAPALDIA).

Methods

Self-reported total, moderate and vigorous physical activity and distensibility as a measure of local arterial stiffness among 1636 participants aged 50 to 81 years without clinically manifest diseases were evaluated. Mixed regression models were used to examine associations of physical activity intensity with distensibility.

Results

Vigorous physical activity, but not total nor moderate physical activity, was significantly associated with increased distensibility (= reduced carotid stiffness) in univariate analyses (percent change in the geometric mean and 95% confidence interval per 1 standard deviation increment in vigorous physical activity = 2.54 (0.69; 4.43), p<0.01; in total physical activity = 1.62 (-0.22; 3.50), p = 0.08; in moderate physical activity = 0.70 (-1.12; 2.56), p = 0.45). These associations disappeared when we additionally adjusted for age.

Conclusion

After adjustment for the most important confounders and risk factors, we found no evidence for an association of physical activity with carotid stiffness in the general middle aged to elderly population.     

Long-Term Exposure to Ambient Air Pollution and Metabolic Syndrome in Adults

Air pollutants (AP) play a role in subclinical inflammation, and are associated with cardiovascular morbidity and mortality. Metabolic syndrome (MetS) is inflammatory and precedes cardiovascular morbidity and type 2 diabetes. Thus, a positive association between AP and MetS may be hypothesized. We explored this association, (taking into account, pathway-specific MetS definitions), and its potential modifiers in Swiss adults. We studied 3769 participants of the Swiss Cohort Study on Air Pollution and Lung and Heart Diseases in Adults, reporting at least four-hour fasting time before venepuncture. AP exposures were 10-year mean residential PM₁₀ (particulate matter <10μm in diameter) and NO₂ (nitrogen dioxide). Outcomes included MetS defined by World Health Organization (MetS-W), International Diabetes Federation (MetS-I) and Adult Treatment Panel-III (MetS-A) using four- and eight-hour fasting time limits. We also explored associations with individual components of MetS. We applied mixed logistic regression models to explore these associations. The prevalence of MetS-W, MetS-I and MetS-A were 10%, 22% and 18% respectively. Odds of MetS-W, MetS-I and MetS-A increased by 72% (51-102%), 31% (11-54%) and 18% (4-34%) per 10μg/m³ increase in 10-year mean PM₁₀. We observed weaker associations with NO₂. Associations were stronger among physically-active, ever-smokers and non-diabetic participants especially with PM₁₀ (p<0.05). Associations remained robust across various sensitivity analyses including ten imputations of missing observations and exclusion of diabetes cases. The observed associations between AP exposure and MetS were sensitive to MetS definitions. Regarding the MetS components, we observed strongest associations with impaired fasting glycemia, and positive but weaker associations with hypertension and waist-circumference-based obesity. Cardio-metabolic effects of AP may be majorly driven by impairment of glucose homeostasis, and to a less-strong extent, visceral adiposity. Well-designed prospective studies are needed to confirm these findings.