In vitro selection of DNA aptamers that bind L-tyrosinamide

We have applied SELEX (Systematic Evolution of Ligands by EXponential enrichment), a combinatorial method that employs biopolymers for drug discovery, to identify single stranded DNA sequences able to bind L-Tyrosinamide, a simple mimic of Tyrosine, an amino acid essential to the catalytic activity of several enzymes of pharmaceutical interest. After 15 SELEX cycles using L-Tyrosinamide immobilized on an affinity chromatography column, the percentage of aptamers specifically eluted from the affinity column with free L-Tyrosinamide was 55% of the total. Aptamers were subcloned and sequenced, allowing the identification of a highly conserved consensus sequence, and showed a K(d) value for L-Tyrosinamide of 45 microM. The identified aptamer sequence will constitute the basis for further in vitro evolution protocols and structure-based drug design.     

Adipoparacrinology--vascular periadventitial adipose tissue (tunica adiposa) as an example

Human adipose tissue is partitioned into two large depots (subcutaneous and visceral), and many small depots associated with internal organs, e.g. heart, blood vessels, major lymph nodes, pancreas, prostate gland and ovaries. Since the adipose 'Big Bang' led to the discovery of leptin (Zhang, Proenca, Maffei, Barone, Leopold and Friedman, Nature 1994;372:425-32), adipose tissue has been seen not merely as a lipid store, but as a secretory - endocrine and paracrine - organ, particularly in the pathogenesis of a number of diseases. Accordingly, two major sub-fields of adipobiology have emerged, viz. adipoendocrinology and adipoparacrinology, the latter herein being illustrated by PAAT (periadventitial adipose tissue) in vascular walls. A long-standing paradigm holds that the vascular wall consists of three coats, tunica intima, tunica media and tunica adventitia. It is now imperative that 'to further elucidate vascular function, we should no longer, as hitherto, separate adventitia and PAAT from the vascular wall, but keep them attached and in place, and subject to thorough examination' (Chaldakov, Fiore, Ghenev, Stankulov and Aloe, Int Med J 2000;7:43-9; Chaldakov, Stankulov and Aloe, Atherosclerosis 2001;154:237-8; Chaldakov GN, Stankulov IS, Fiore M, Ghenev PI and Aloe L, Atherosclerosis 2001;159:57-66). From the available data, we propose that it is time to rethink about vascular wall composition, and suggest that the PAAT may be considered the fourth and outermost vascular coat, hence, tunica adiposa (regarding the proximal segment of coronary artery, it is the innermost part of the EAT (epicardial adipose tissue) situated around the coronary adventitia). Its significance in the pathogenesis and therapy of CMDs (cardiometabolic diseases), particularly atherosclerosis and hypertension, requires further basic, translational and clinical studies.     

Specific cellular localization of tyrosinase mRNA during Ciona intestinalis larval development

A Ciona intestinalis cDNA clone that encodes a protein highly homologous to other tyrosinases was isolated. Northern blot analysis showed that expression of Ciona tyrosinase starts at the early neurula stage and continues throughout the tail-bud and tadpole larval stages. The earliest tyrosinase expression was detected, by in situ hybridization, at the neural plate stage, in pigment precursor cells located along the two neural folds, in the animal region of the embryo. In the course of embryonic development the strong hybridization signal was always localized, within the rostral part of the developing brain, in the pigment precursor cells and was later detected in the otolith and ocellus. These results are discussed in relation to tyrosinase as an early marker of neural induction.     

Preferred proline puckerings in cis andtrans peptide groups: Implications for collagen stability

The interplay between side-chain and main-chain conformations is a distinctive characteristic of proline residues. Here we report the results of a statistical analysis of proline conformations using a large protein database. In particular, we found that proline residues with the preceding peptide bond in the cis state preferentially adopt a down puckering. Indeed, out of 178 cis proline residues, as many as 145 (81%) are down. By analyzing the 1-4 and 1-5 nonbonding distances between backbone atoms, we provide a structural explanation for the observed trend. The observed correlation between proline puckering and peptide bond conformation suggests a new mechanism to explain the reported shift of the cis-trans equilibrium in proline derivatives. The implications of these results for the current models of collagen stability are also discussed.     

Moving towards the detection of frailty with biomarkers: A population health study

Aging adults experience increased health vulnerability and compromised abilities to cope with stressors, which are the clinical manifestations of frailty. Frailty is complex, and efforts to identify biomarkers to detect frailty and pre-frailty in the clinical setting are rarely reproduced across cohorts. We developed a predictive model incorporating biological and clinical frailty measures to identify robust biomarkers across data sets. Data were from two large cohorts of older adults: “Invecchiare in Chianti (Aging in Chianti, InCHIANTI Study”) (n = 1453) from two small towns in Tuscany, Italy, and replicated in the Atherosclerosis Risk in Communities Study (ARIC) (n = 6508) from four U.S. communities. A complex systems approach to biomarker selection with a tree-boosting machine learning (ML) technique for supervised learning analysis was used to examine biomarker population differences across both datasets. Our approach compared predictors with robust, pre-frail, and frail participants and examined the ability to detect frailty status by race. Unique biomarker features identified in the InCHIANTI study allowed us to predict frailty with a model accuracy of 0.72 (95% confidence interval (CI) 0.66–0.80). Replication models in ARIC maintained a model accuracy of 0.64 (95% CI 0.66–0.72). Frail and pre-frail Black participant models maintained a lower model accuracy. The predictive panel of biomarkers identified in this study may improve the ability to detect frailty as a complex aging syndrome in the clinical setting. We propose several concrete next steps to keep research moving toward detecting frailty with biomarker-based detection methods.     

The first genetic engineered system for ovothiol biosynthesis in diatoms reveals a mitochondrial localization for the sulfoxide synthase OvoA

Diatoms represent one of the most abundant groups of microalgae in the ocean and are responsible for approximately 20% of photosynthetically fixed CO₂ on Earth. Due to their complex evolutionary history and ability to adapt to different environments, diatoms are endowed with striking molecular biodiversity and unique metabolic activities. Their high growth rate and the possibility to optimize their biomass make them very promising ‘biofactories’ for biotechnological applications. Among bioactive compounds, diatoms can produce ovothiols, histidine-derivatives, endowed with unique antioxidant and anti-inflammatory properties, and occurring in many marine invertebrates, bacteria and pathogenic protozoa. However, the functional role of ovothiols biosynthesis in organisms remains almost unexplored. In this work, we have characterized the thiol fraction of Phaeodactylum tricornutum, providing the first evidence of the presence of ovothiol B in pennate diatoms. We have used P. tricornutum to overexpress the 5-histidylcysteine sulfoxide synthase ovoA, the gene encoding the key enzyme involved in ovothiol biosynthesis and we have discovered that OvoA localizes in the mitochondria, a finding that uncovers new concepts in cellular redox biochemistry. We have also obtained engineered biolistic clones that can produce higher amount of ovothiol B compared to wild-type cells, suggesting a new strategy for the eco-sustainable production of these molecules.     

Anti‐inflammatory properties of lemon‐derived extracellular vesicles are achieved through the inhibition of ERK/NF‐κB signalling pathways

Chronic inflammation is associated with the occurrence of several diseases. However, the side effects of anti‐inflammatory drugs prompt the identification of new therapeutic strategies. Plant‐derived extracellular vesicles (PDEVs) are gaining increasing interest in the scientific community for their biological properties. We isolated PDEVs from the juice of Citrus limon L. (LEVs) and characterized their flavonoid, limonoid and lipid contents through reversed‐phase high‐performance liquid chromatography coupled to electrospray ionization quadrupole time‐of‐flight mass spectrometry (RP‐HPLC–ESI‐Q‐TOF‐MS). To investigate whether LEVs have a protective role on the inflammatory process, murine and primary human macrophages were pre‐treated with LEVs for 24 h and then were stimulated with lipopolysaccharide (LPS). We found that pre‐treatment with LEVs decreased gene and protein expression of pro‐inflammatory cytokines, such as IL‐6, IL1‐β and TNF‐α, and reduced the nuclear translocation and phosphorylation of NF‐κB in LPS‐stimulated murine macrophages. The inhibition of NF‐κB activation was associated with the reduction in ERK1‐2 phosphorylation. Furthermore, the ability of LEVs to decrease pro‐inflammatory cytokines and increase anti‐inflammatory molecules was confirmed ex vivo in human primary T lymphocytes. In conclusion, we demonstrated that LEVs exert anti‐inflammatory effects both in vitro and ex vivo by inhibiting the ERK1‐2/NF‐κB signalling pathway.