We review the key steps leading to an improved analysis of thermal protein unfolding. Thermal unfolding is a dynamic cooperative process with many short-lived intermediates. Protein unfolding has been measured by various spectroscopic techniques that reveal structural changes, and by differential scanning calorimetry (DSC) that provides the heat capacity change C_p(T). The corresponding temperature profiles of enthalpy ΔH(T), entropy ΔS(T), and free energy ΔG(T) have thus far been evaluated using a chemical equilibrium two-state model. Taking a different approach, we demonstrated that the temperature profiles of enthalpy ΔH(T), entropy ΔS(T), and free energy ΔG(T) can be obtained directly by a numerical integration of the heat capacity profile C_p(T). DSC thus offers the unique possibility to assess these parameters without resorting to a model. These experimental parameters now allow us to examine the predictions of different unfolding models. The standard two-state model fits the experimental heat capacity peak quite well. However, neither the enthalpy nor entropy profiles (predicted to be almost linear) are congruent with the measured sigmoidal temperature profiles, nor is the parabolic free energy profile congruent with the experimentally observed trapezoidal temperature profile. We introduce three new models, an empirical two-state model, a statistical–mechanical two-state model and a cooperative statistical-mechanical multistate model. The empirical model partially corrects for the deficits of the standard model. However, only the two statistical–mechanical models are thermodynamically consistent. The two-state models yield good fits for the enthalpy, entropy and free energy of unfolding of small proteins. The cooperative statistical–mechanical multistate model yields perfect fits, even for the unfolding of large proteins such as antibodies. Full article

Milk oligosaccharides are a complex class of carbohydrates that act as bioactive factors in numerous defensive and physiological functions, including brain development. Early nutrition can modulate nervous system development and can lead to epigenetic imprinting. We attempted to increase the sialylated oligosaccharide content of zebrafish yolk reserves, with the aim of evaluating any short-term effects of the treatment on mortality, locomotor behavior, and gene expression. Wild-type embryos were microinjected with saline solution or solutions containing sialylated milk oligosaccharides extracted from human and bovine milk. The results suggest that burst activity and larval survival rates were unaffected by the treatments. Locomotion parameters were found to be similar during the light phase between control and treated larvae; in the dark, however, milk oligosaccharide-treated larvae showed increased test plate exploration. Thigmotaxis results did not reveal significant differences in either the light or the dark conditions. The RNA-seq analysis indicated that both treatments exert an antioxidant effect in developing fish. Moreover, sialylated human milk oligosaccharides seemed to increase the expression of genes related to cell cycle control and chromosomal replication, while bovine-derived oligosaccharides caused an increase in the expression of genes involved in synaptogenesis and neuronal signaling. These data shed some light on this poorly explored research field, showing that both human and bovine oligosaccharides support brain proliferation and maturation. Full article

Microcirculatory and mitochondrial dysfunction are considered the main mechanisms of septic shock. Studies suggest that statins modulate inflammatory response, microcirculation, and mitochondrial function, possibly through their action on peroxisome proliferator-activated receptor alpha (PPAR-α). The aim of this study was to examine the effects of pravastatin on microcirculation and mitochondrial function in the liver and colon and the role of PPAR-α under septic conditions. This study was performed with the approval of the local animal care and use committee. Forty Wistar rats were randomly divided into 4 groups: sepsis (colon ascendens stent peritonitis, CASP) without treatment as control, sepsis + pravastatin, sepsis + PPAR-α-blocker GW6471, and sepsis + pravastatin + GW6471. Pravastatin (200 µg/kg s.c.) and GW6471 (1 mg/kg) were applied 18 h before CASP-operation. 24 h after initial surgery, a relaparotomy was performed, followed by a 90 min observation period for assessment of microcirculatory oxygenation (μHbO₂) of the liver and colon. At the end of the experiments, animals were euthanized, and the colon and liver were harvested. Mitochondrial function was measured in tissue homogenates using oximetry. The ADP/O ratio and respiratory control index (RCI) for complexes I and II were calculated. Reactive oxygen species (ROS) production was assessed using the malondialdehyde (MDA)-Assay. Statistics: two-way analysis of variance (ANOVA) + Tukey’s/Dunnett’s post hoc test for microcirculatory data, Kruskal–Wallis test + Dunn’s post hoc test for all other data. In control septic animals µHbO₂ in liver and colon deteriorated over time (µHbO₂: −9.8 ± 7.5%* and −7.6 ± 3.3%* vs. baseline, respectively), whereas after pravastatin and pravastatin + GW6471 treatment μHbO₂ remained constant (liver: µHbO₂ pravastatin: −4.21 ± 11.7%, pravastatin + GW6471: −0.08 ± 10.3%; colon: µHbO₂ pravastatin: −0.13 ± 7.6%, pravastatin + GW6471: −3.00 ± 11.24%). In both organs, RCI and ADP/O were similar across all groups. The MDA concentration remained unchanged in all groups. Therefore, we conclude that under septic conditions pravastatin improves microcirculation in the colon and liver, and this seems independent of PPAR-α and without affecting mitochondrial function. Full article

The reproductive stage of plant development has the most critical impact on yield. Flowering is highly sensitive to abiotic stress, and increasing temperatures and drought harm crop yields. Salicylic acid is a phytohormone that regulates flowering and promotes stress resilience in plants. However, the exact molecular mechanisms and the level of protection are far from understood and seem to be species-specific. Here, the effect of salicylic acid was tested in a field experiment with Pisum sativum exposed to heat stress. Salicylic acid was administered at two different stages of flowering, and its effect on the yield and composition of the harvested seeds was followed. Plants treated with salicylic acid produced larger seed pods, and a significant increase in dry weight was found for the plants with a delayed application of salicylic acid. The analyses of the seed proteome, lipidome, and metabolome did not show any negative impact of salicylic treatment on seed composition. Identified processes that could be responsible for the observed improvement in seed yields included an increase in polyamine biosynthesis, accumulation of storage lipids and lysophosphatidylcholines, a higher abundance of components of chromatin regulation, calmodulin-like protein, and threonine synthase, and indicated a decrease in sensitivity to abscisic acid signaling. Full article

Wheat allergies are potentially life-threatening and, therefore, have become a major health concern at the global level. It is largely unknown at present whether genetic variation in allergenicity potential exists among hexaploid, tetraploid and diploid wheat species. Such information is critical in establishing a baseline allergenicity map to inform breeding efforts to identify hyper-, hypo- and non-allergenic varieties. We recently reported a novel mouse model of intrinsic allergenicity using the salt-soluble protein extract (SSPE) from durum, a tetraploid wheat (Triticum durum). Here, we validated the model for three other wheat species [hexaploid common wheat (Triticum aestivum), diploid einkorn wheat (Triticum monococcum), and the ancient diploid wheat progenitor, Aegilops tauschii], and then tested the hypothesis that the SSPEs from wheat species will exhibit differences in relative allergenicities. Balb/c mice were repeatedly exposed to SSPEs via the skin. Allergic sensitization potential was assessed by specific (s) IgE antibody responses. Oral anaphylaxis was quantified by the hypothermic shock response (HSR). The mucosal mast cell response (MMCR) was determined by measuring mast cell protease in the blood. While T. monococcum elicited the least, but significant, sensitization, others were comparable. Whereas Ae. taushcii elicited the least HSR, the other three elicited much higher HSRs. Similarly, while Ae. tauschii elicited the least MMCR, the other wheats elicited much higher MMCR as well. In conclusion, this pre-clinical comparative mapping strategy may be used to identify potentially hyper-, hypo- and non-allergenic wheat varieties via crossbreeding and genetic engineering methods. Full article

Heparan sulfate proteoglycans (HSPGs) possess various functions driving malignancy of tumors. However, their impact on tumor cell sensitivity to cytotoxic treatment is far less understood. Aiming to investigate this, we depleted HSPGs by downregulating Exostosin 1 (EXT1), a key enzyme in HS formation, or upregulating heparanase in human MV3 human melanoma cells, and investigated their response to cytotoxic drugs. Cytotoxicity of trametinib, doxorubicin, and mitoxantrone was detected by MTT assay. Insights into intracellular signaling was provided by kinome protein profiler array, and selected kinases were inhibited to investigate their impact on cell sensitization and migratory dynamics. EXT1 knockdown (EXT1kd) in MV3 cells affected the activity of doxorubicin and mitoxantrone, significantly increasing EC₅₀ values two- or fourfold, respectively. Resistance formation was scarcely related to HSPG deficiency, suggested by enzymatic cleavage of HSPG in control cells. Notably, EXT1kd induced an upregulation of EGFR signaling via JNK and MEK/ERK, and hence blocking these kinases returned resistance to a sensitive level. JNK appeared as a key signal component, also inducing higher migratory activity of EXT1kd cells. Furthermore, EXT1kd upregulated thrombotic properties of MV3 cells, indicated by tissue factor and PAR-1 expression, functionally reflected by a stronger activation of platelet aggregation. EXT1 was confirmed to act as a tumor suppressor, shown here for the first time to affect chemosensitivity of melanoma cells. Full article

The lifetime effects of space irradiation (IR) on left ventricular (LV) function are unknown. The cardiac effects induced by space-type IR, specifically 5-ion simplified galactic cosmic ray simulation (simGCRsim), are yet to be discovered. Three-month-old, age-matched, male C57BL/6J mice were irradiated with ¹³⁷Cs gamma (γ; 100, 200 cGy) and simGCRsim (50 and 100 cGy). LV function was assessed via transthoracic echocardiography at 14 and 28 days (early), and at 365, 440, and 660 (late) days post IR. We measured the endothelial function marker brain natriuretic peptide in plasma at three late timepoints. We assessed the mRNA expression of the genes involved in cardiac remodeling, fibrosis, inflammation, and calcium handling in LVs harvested at 660 days post IR. All IR groups had impaired global LV systolic function at 14, 28, and 365 days. At 660 days, 50 cGy simGCRsim-IR mice exhibited preserved LV systolic function with altered LV size and mass. At this timepoint, the simGCRsim-IR mice had elevated levels of cardiac fibrosis, inflammation, and hypertrophy markers Tgfβ1, Mcp1, Mmp9, and βmhc, suggesting that space-type IR may induce the cardiac remodeling processes that are commonly associated with diastolic dysfunction. IR groups showing statistical significance were modeled to calculate the Relative Biological Effectiveness (RBE) and Radiation Effects Ratio (RER). The observed dose-response shape did not indicate a lower threshold at these IR doses. A single full-body IR at doses of 100–200 cGy for γ-IR, and 50–100 cGy for simGCRsim-IR decreases the global LV systolic function in WT mice as early as 14 and 28 days after exposure, and at 660 days post IR. Interestingly, there is an intermediate time point (365 days) where the impairment in LV function is observed. These findings do not exclude the possibility of increased acute or degenerative cardiovascular disease risks at lower doses of space-type IR, and/or when combined with other space travel-associated stressors such as microgravity. Full article

Genome damage has been related to the induction of autoimmune processes, chronic inflammation, and apoptosis. Recent studies suggest that some rheumatological diseases are associated with overall genomic instability in the T cell compartment. However, no data regarding leucocyte abnormalities in synovial fluid (SF) and their relationship with inflammation are available. The aim of this study was to investigate cellular phenotypes in SF collected from patients with different inflammatory arthropathies, including rhematoid arthritis (RA), psoriatic arthritis (PsA), crystal-induced arthritis (CIA), and non-inflammatory arthropathies, such as osteoarthritis (OA). We found high percentage of micronuclei in SF from CIA compared to the other groups and a high frequency of pyknotic cell in RA and CIA patients. A correlation between pyknosis and immature polymorphonuclear cells with local inflammatory indices was observed. The study of the apoptosis process revealed an increased BAX expression in CIA and RA compared to OA and PsA, while Bcl-2 was higher in CIA. Caspase-3 activity was increased in SF from RA patients and correlates with inflammatory and anti-inflammatory cytokines. In conclusion, our results showed that inflammatory SF is associated with genomic instability and abnormal cell subsets. Full article

The paper aims to investigate the antitumor activity of a series of phenothiazine derivatives in order to establish a structure–antitumor activity relationship. To this end, PEGylated and TEGylated phenothiazine have been functionalized with formyl units and further with sulfonamide units via dynamic imine bonds. Their antitumor activity was monitored in vitro against seven human tumors cell lines and a mouse one compared to a human normal cell line by MTS assay. In order to find the potential influence of different building blocks on antitumor activity, the antioxidant activity, the ability to inhibit farnesyltransferase and the capacity to bind amino acids relevant for tumor cell growth were investigated as well. It was established that different building blocks conferred different functionalities, inducing specific antitumor activity against the tumor cells. Full article

Drug-induced gingival overgrowth (DIGO) is one of the side effects produced by therapeutic agents, most commonly phenytoin, nifedipine and cyclosporin A. However, the precise mechanism of DIGO is not entirely understood. A literature search of the MEDLINE/PubMed databases was conducted to identify the mechanisms involved in DIGO. The available information suggests that the pathogenesis of DIGO is multifactorial, but common pathogenic sequelae of events emerge, i.e., sodium and calcium channel antagonism or disturbed intracellular handling of calcium, which finally lead to reductions in intracellular folic acid levels. Disturbed cellular functions, mainly in keratinocytes and fibroblasts, result in increased collagen and glycosaminoglycans accumulation in the extracellular matrix. Dysregulation of collagenase activity, as well as integrins and membrane receptors, are key mechanisms of reduced degradation or excessive synthesis of connective tissue components. This manuscript describes the cellular and molecular factors involved in the epithelial–mesenchymal transition and extracellular matrix remodeling triggered by agents producing DIGO. Full article

Reproductive dysfunction and urogenital malignancies represent a serious health concern in men. This is in part as a result of the absence of reliable non-invasive tests of diagnosis/prognosis. Optimizing diagnosis and predicting the patient’s prognosis will affect the choice of the most appropriate treatment and therefore increase the chances of success and the result of therapy, that is, it will lead to a more personalized treatment of the patient. This review aims firstly to critically summarize the current knowledge of the reproductive roles played by extracellular vesicle small RNA components, which are typically altered in diseases affecting the male reproductive tract. Secondly, it aims to describe the use of semen extracellular vesicles as a non-invasive source of sncRNA-based biomarkers for urogenital diseases. Full article

Therapeutic peptides are oligomers or short polymers of amino acids used for various medical purposes. Peptide-based treatments have evolved considerably due to new technologies, stimulating new research interests. They have been shown to be beneficial in a variety of therapeutic applications, notably in the treatment of cardiovascular disorders such as acute coronary syndrome (ACS). ACS is characterized by coronary artery wall damage and consequent formation of an intraluminal thrombus obstructing one or more coronary arteries, leading to unstable angina, non-ST elevated myocardial infarction, and ST-elevated myocardial infarction. One of the promising peptide drugs in the treatment of these pathologies is eptifibatide, a synthetic heptapeptide derived from rattlesnake venom. Eptifibatide is a glycoprotein IIb/IIIa inhibitor that blocks different pathways in platelet activation and aggregation. In this narrative review, we summarized the current evidence on the mechanism of action, clinical pharmacology, and applications of eptifibatide in cardiology. Additionally, we illustrated its possible broader usage with new indications, including ischemic stroke, carotid stenting, intracranial aneurysm stenting, and septic shock. Further research is, however, required to fully evaluate the role of eptifibatide in these pathologies, independently and in comparison to other medications. Full article

Candida albicans is the main causal pathogen of fungal infections in human beings. Although diverse anti-C. albicans drugs have been explored, the drug resistance and side effects of these drugs are intensifying. Thus, it is urgent to explore new anti-C. albicans compounds from natural products. In this study, we identified trichoderma acid (TA), a compound from Trichoderma spirale with a strong inhibitory effect on C. albicans. Transcriptomic and iTRAQ-based proteomic analyses of TA-treated C. albicans in combination with scanning electronic microscopy and reactive oxygen species (ROS) detection were performed to investigate the potential targets of TA. The most significant differentially expressed genes and proteins after TA treatment were verified through Western blot analysis. Our results revealed that mitochondrial membrane potential, endoplasmic reticulum, ribosomes in the mitochondria, and cell walls were disrupted in TA-treated C. albicans, leading to the accumulation of ROS. The impaired enzymatic activities of superoxide dismutase further contributed to the increase in ROS concentration. The high concentration of ROS led to DNA damage and cell skeleton destruction. The expression levels of Rho-related GTP-binding protein RhoE (RND3), asparagine synthetase (ASNS), glutathione S-transferase, and heat shock protein 70 were significantly up-regulated in response to apoptosis and toxin stimulation. These findings suggest that RND3, ASNS, and supereoxide dismutase 5 are the potential targets of TA, as further demonstrated through Western blot analysis. The combination of transcriptomic, proteomic, and cellular analyses would provide clues for the anti-C. albicans mechanism of TA and the defensive response mechanism of C. albicans. TA is thus recognized as a promising new anti-C. albicans leading compound that alleviates the hazard of C. albicans infection in human beings. Full article

Rice is one of the most economically important staple food crops in the world. Soil salinization and drought seriously restrict sustainable rice production. Drought aggravates the degree of soil salinization, and, at the same time, increased soil salinity also inhibits water absorption, resulting in physiological drought stress. Salt tolerance in rice is a complex quantitative trait controlled by multiple genes. This review presents and discusses the recent research developments on salt stress impact on rice growth, rice salt tolerance mechanisms, the identification and selection of salt-tolerant rice resources, and strategies to improve rice salt tolerance. In recent years, the increased cultivation of water-saving and drought-resistance rice (WDR) has shown great application potential in alleviating the water resource crisis and ensuring food and ecological security. Here, we present an innovative germplasm selection strategy of salt-tolerant WDR, using a population that is developed by recurrent selection based on dominant genic male sterility. We aim to provide a reference for efficient genetic improvement and germplasm innovation of complex traits (drought and salt tolerance) that can be translated into breeding all economically important cereal crops. Full article

Selenium (Se) is an essential micronutrient of fundamental importance to human health and the main Se source is from plant-derived foods. Plants mainly take up Se as selenate (SeO₄²⁻), through the root sulfate transport system, because of their chemical similarity. The aims of this study were (1) to characterize the interaction between Se and S during the root uptake process, by measuring the expression of genes coding for high-affinity sulfate transporters and (2) to explore the possibility of increasing plant capability to take up Se by modulating S availability in the growth medium. We selected different tetraploid wheat genotypes as model plants, including a modern genotype, Svevo (Triticum turgidum ssp. durum), and three ancient Khorasan wheats, Kamut, Turanicum 21, and Etrusco (Triticum turgidum ssp. turanicum). The plants were cultivated hydroponically for 20 days in the presence of two sulfate levels, adequate (S = 1.2 mM) and limiting (L = 0.06 mM), and three selenate levels (0, 10, 50 μM). Our findings clearly showed the differential expression of genes encoding the two high-affinity transporters (TdSultr1.1 and TdSultr1.3), which are involved in the primary uptake of sulfate from the rhizosphere. Interestingly, Se accumulation in shoots was higher when S was limited in the nutrient solution. Full article