Multiple modes of active center communication in thiamin diphosphate-dependent enzymes

Detection of interaction between cofactors at the active centers of homodimeric and homotetrameric enzymes is usually elusive by steady-state kinetic approaches and requires protein variants where such interactions are diminished or exaggerated. In this Account, evidence for active-center interactions will be presented for the following thiamin diphosphate-dependent enzymes: yeast pyruvate decarboxylase, benzoylformate decarboxylase, and examples from the 2-oxoacid dehydrogenase multienzyme complex class. The dissymmetry of active sites is especially evident in the X-ray structures of these enzymes with substrate/substrate analogues bound. Perturbations that reveal active center communication include use of chromophoric substrates and substitutions of active center residues on putative pathways.     

Antioxidant and cardioprotective activities of phenolic extracts from fruits of Chilean blackberry Aristotelia chilensis (Elaeocarpaceae), Maqui

The methanol extract from mature fruits of Aristotelia chilensis (Mol) Stuntz (Elaeocarpaceae) showed antioxidant activities and cardioprotective effects on acute ischemia/reperfusion performed in rat heart in vivo. This extract protected animals from heart damage by the incidence of reperfusion dysrythmias, and the no-recovery of sinus rhythm. On the other hand, the MeOH extract of the fruit was able to prevent these harmful events in the animal’s heart by diminishing lipid oxidation and reducing the concentration of thiobarbituric acid reactive substances (TBARS), a lipid peroxidation index. In addition, MeOH extract of A. chilensis was evaluated for DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging, crocin radical scavenging, oxygen radical absorption capacity (ORAC), ferric reducing antioxidant power (FRAP), an estimation of lipid peroxidation in liposomes through the inhibition of formation of TBARS. MeOH extract was found to have IC₅₀ of 1.62 ppm against DPPH and 2.51 ppm against TBARS, compared with the juice, whose IC₅₀ was 12.1 ppm and 9.58 ppm against DPPH and TBARS formation, respectively. Antioxidant activities of MeOH extract were strongly correlated with total polyphenol content. Consistent with this finding, MeOH had the greatest ORAC and FRAP values as percentage of activity. These results show that these fruits could be useful as antioxidant, cardioprotective and nutraceutical sources.     

Food protozoa safety assessment and risk in school restaurants in Armenia,Colombia

This work assessed the risk of protozoa in 10 school restaurants in Armenia (Quindío, Colombia) by analyzing the presence of Cryptosporidium spp, Giardia duodenalis, Blastocystis, and Cyclospora cayetanensis DNA in the food, water, and living and inert surfaces of school restaurants and in stools of children who ate at these restaurants. Of the 213 food, water, and surface samples, 6.6% were positive using PCR to test DNA for Blastocystis; 3.8% for Cryptosporidium spp; 0.9% for G. duodenalis; and 0% for C. cayetanensis. In 187 stool samples analyzed via microscopy from children who attended the restaurants, 40 (21.4%) were positive for Blastocystis and 21 (11.2%) were positive for Giardia spp. Via PCR, 20 (10.7%) were positive for Cryptosporidium and 0 (0%) for C. cayetanensis. A higher positivity in children's stools for Blastocystis spp was correlated with lower compliance in property conditions and for higher positivity of Giardia spp in children's stool was related to lower knowledge by food manipulators. Inspection scores can identify restaurants with higher risk for protozoa infection.     

Critical water activity for the preservation of Lactobacillus bulgaricus by vacuum drying

Lactobacillus delbrueckii subsp. bulgaricus was dried under vacuum at different temperatures and its preservation evaluated analyzing the evolution of three parameters throughout the process: lag time, percentage of membrane damage and zeta potential. Microorganisms were dehydrated at 30, 45 and 70 degrees C in a vacuum centrifuge for different times. The aw achieved for each time of drying was correlated with the cell recovery at all the temperatures assayed. The recovery of microorganisms was evaluated by means of: a) kinetics of growth in milk after drying, as a measure of the global damage; b) quantification of the membrane damage using the fluorescent dyes SYTO 9 and PI; c) determination of changes in the superficial charges (zeta potential) as measured of the increase in the hydrophobic residues exposed in the bacterial surface after dehydration. These changes correlate well with the bacterial damage occurred during the dehydration process. The Page's equation allowed fitting of aw and time of drying, thus making possible the determination of the appropriate dehydration conditions (time-temperature ratios) for which no cell damage occurs. The evaluation of three parameters (lag time, percentage of membrane damage and zeta potential) allowed us to conclude that at the lowest temperature of dehydration, the first target of damage is the cell membrane. However, this damage is not decisive for the bacterial recovery after rehydration, as are the increase in the lag time and the changes in the zeta potential, as was observed for L. bulgaricus dehydrated at 45 and 70 degrees C for larger times.     

Mesenchymal Stem Cell-Based Therapy as an Alternative to the Treatment of Acute Respiratory Distress Syndrome: Current Evidence and Future Perspectives

Acute respiratory distress syndrome (ARDS) represents a current challenge for medicine due to its incidence, morbidity and mortality and, also, the absence of an optimal treatment. The COVID-19 outbreak only increased the urgent demand for an affordable, safe and effective treatment for this process. Early clinical trials suggest the therapeutic usefulness of mesenchymal stem cells (MSCs) in acute lung injury (ALI) and ARDS. MSC-based therapies show antimicrobial, anti-inflammatory, regenerative, angiogenic, antifibrotic, anti-oxidative stress and anti-apoptotic actions, which can thwart the physiopathological mechanisms engaged in ARDS. In addition, MSC secretome and their derived products, especially exosomes, may reproduce the therapeutic effects of MSC in lung injury. This last strategy of treatment could avoid several safety issues potentially associated with the transplantation of living and proliferative cell populations and may be formulated in different forms. However, the following diverse limitations must be addressed: (i) selection of the optimal MSC, bearing in mind both the heterogeneity among donors and across different histological origins, (ii) massive obtention of these biological products through genetic manipulations of the most appropriate MSC, (iii) bioreactors that allow their growth in 3D, (iv) ideal culture conditions and (v) adequate functional testing of these obtaining biological products before their clinical application.     

Glucocorticoid-Dependent Mechanisms of Brain Tolerance to Hypoxia

Adaptation of organisms to stressors is coordinated by the hypothalamic-pituitary-adrenal axis (HPA), which involves glucocorticoids (GCs) and glucocorticoid receptors (GRs). Although the effects of GCs are well characterized, their impact on brain adaptation to hypoxia/ischemia is still understudied. The brain is not only the most susceptible to hypoxic injury, but also vulnerable to GC-induced damage, which makes studying the mechanisms of brain hypoxic tolerance and resistance to stress-related elevation of GCs of great importance. Cross-talk between the molecular mechanisms activated in neuronal cells by hypoxia and GCs provides a platform for developing the most effective and safe means for prevention and treatment of hypoxia-induced brain damage, including hypoxic pre- and post-conditioning. Taking into account that hypoxia- and GC-induced reprogramming significantly affects the development of organisms during embryogenesis, studies of the effects of prenatal and neonatal hypoxia on health in later life are of particular interest. This mini review discusses the accumulated data on the dynamics of the HPA activation in injurious and non-injurious hypoxia, the role of the brain GRs in these processes, interaction of GCs and hypoxia-inducible factor HIF-1, as well as cross-talk between GC and hypoxic signaling. It also identifies underdeveloped areas and suggests directions for further prospective studies.     

Metabolic Glycoengineering in hMSC-TERT as a Model for Skeletal Precursors by Using Modified Azide/Alkyne Monosaccharides

Metabolic glycoengineering enables a directed modification of cell surfaces by introducing target molecules to surface proteins displaying new features. Biochemical pathways involving glycans differ in dependence on the cell type; therefore, this technique should be tailored for the best results. We characterized metabolic glycoengineering in telomerase-immortalized human mesenchymal stromal cells (hMSC-TERT) as a model for primary hMSC, to investigate its applicability in TERT-modified cell lines. The metabolic incorporation of N-azidoacetylmannosamine (Ac₄ManNAz) and N-alkyneacetylmannosamine (Ac₄ManNAl) into the glycocalyx as a first step in the glycoengineering process revealed no adverse effects on cell viability or gene expression, and the in vitro multipotency (osteogenic and adipogenic differentiation potential) was maintained under these adapted culture conditions. In the second step, glycoengineered cells were modified with fluorescent dyes using Cu-mediated click chemistry. In these analyses, the two mannose derivatives showed superior incorporation efficiencies compared to glucose and galactose isomers. In time-dependent experiments, the incorporation of Ac₄ManNAz was detectable for up to six days while Ac₄ManNAl-derived metabolites were absent after two days. Taken together, these findings demonstrate the successful metabolic glycoengineering of immortalized hMSC resulting in transient cell surface modifications, and thus present a useful model to address different scientific questions regarding glycosylation processes in skeletal precursors.     

Effects of Agaricus bisporus on gel properties of chicken myofibrillar protein

The gel-forming ability of myofibrillar protein (MP) is highly correlated with the characteristics of emulsified meat products. Incorporation of Agaricus bisporus (Ab) powder into MP gels may enhance its gel properties to facilitate the development of a novel and safe meat product. Therefore, this study investigated the effects of Ab powder on gel strength, water holding capacity (WHC), texture, rheological behaviour, LF-NMR spin–spin relaxation (T₂), microstructure and protein secondary structure of the MP gel system. The results indicated that the gel strength, WHC, G' value and G" value were significantly improved when the addition of Ab powder increased from 0% to 6% (P < 0.05). Meanwhile, the T₂ relaxation time was shortened, and free water was transformed into immobilised water. The texture of the gel was improved when 1%–4% Ab powder was added compared to the control. Furthermore, Ab filled in the gel network and promoted the unfolding of MP α-helix and the formation of MP β-sheet during the thermal denaturation of MP, leading to a dense aggregated network structure. The study suggested that Ab could be a promising ingredient in improving chicken MP's gel properties and developing fat-reduced meat products.