Toxic cyanobacterial blooms are a major contaminant in inland aquatic ecosystems. Furthermore, toxic blooms are carried downstream by rivers and waterways to estuarine and coastal ecosystems. Concerning marine and estuarine animal species, very little is known about how these species are affected by the exposure to freshwater cyanobacteria and cyanotoxins. So far, most of the knowledge has been gathered from freshwater bivalve molluscs. This work aimed to infer the sensitivity of the marine mussel Mytilus galloprovincialis to single as well as mixed toxic cyanobacterial cultures and the underlying molecular responses mediated by toxic cyanobacteria. For this purpose, a mussel exposure experiment was outlined with two toxic cyanobacteria species, Microcystis aeruginosa and Chrysosporum ovalisporum at 1 × 105 cells/mL, resembling a natural cyanobacteria bloom. The estimated amount of toxins produced by M. aeruginosa and C. ovalisporum were respectively 0.023 pg/cell of microcystin-LR (MC-LR) and 7.854 pg/cell of cylindrospermopsin (CYN). After 15 days of exposure to single and mixed cyanobacteria, a depuration phase followed, during which mussels were fed only non-toxic microalga Parachlorella kessleri. The results showed that the marine mussel is able to filter toxic cyanobacteria at a rate equal or higher than the non-toxic microalga P. kessleri. Filtration rates observed after 15 days of feeding toxic microalgae were 1773.04 mL/ind.h (for M. aeruginosa), 2151.83 mL/ind.h (for C. ovalisporum), 1673.29 mL/ind.h (for the mixture of the 2 cyanobacteria) and 2539.25 mL/ind.h (for the non-toxic P. kessleri). Filtering toxic microalgae in combination resulted in the accumulation of 14.17 ng/g dw MC-LR and 92.08 ng/g dw CYN. Other physiological and biochemical endpoints (dry weight, byssus production, total protein and glycogen) measured in this work did not change significantly in the groups exposed to toxic cyanobacteria with regard to control group, suggesting that mussels were not affected with the toxic microalgae. Nevertheless, proteomics revealed changes in metabolism of mussels related to diet, specially evident in those fed on combined cyanobacteria. Changes in metabolic pathways related with protein folding and stabilization, cytoskeleton structure, and gene transcription/translation were observed after exposure and feeding toxic cyanobacteria. These changes occur in vital metabolic processes and may contribute to protect mussels from toxic effects of the toxins MC-LR and CYN.

Heteroptera, or true bugs populate many climate zones, coping with different environmental conditions. The aim of this study was the evaluation of their thermal limits and derived traits, as well as climatological parameters which might influence their distribution. We assessed the thermal limits (critical thermal maxima, CTmax, and minima, CTmin) of eight seed bug species (Lygaeidae, Pyrrhocoridae) distributed over four Köppen-Geiger climate classification types (KCC), approximately 6° of latitude, and four European countries (Austria, Italy, Croatia, Bulgaria). In test tubes, a temperature ramp was driven down to -5 °C for CTmin and up to 50 °C for CTmax (0.25 °C/min) until the bugs' voluntary, coordinated movement stopped. In contrast to CTmin, CTmax depended significantly on KCC, species, and body mass. CTmax showed high correlation with bioclimatic parameters such as annual mean temperature and mean maximum temperature of warmest month (BIO5), as well as three parameters representing temperature variability. CTmin correlated with mean annual temperature, mean minimum temperature of coldest month (BIO6), and two parameters representing variability. Although the derived trait cold tolerance (TC = BIO6 - CTmin) depended on several bioclimatic variables, heat tolerance (TH = CTmax - BIO5) showed no correlation. Seed bugs seem to have potential for further range shifts in the face of global warming.

The Western diet can lead to alterations in cardiac function and increase cardiovascular risk, which can be reproduced in animal models by implementing a high-fat diet (HFD). However, the mechanism of these alterations is not fully understood and may be dependent on alterations in heart lipid composition. The aim of this study was to evaluate the effect of an HFD on the fatty acid (FA) composition of total lipids, as well as of various lipid fractions in the heart, and on heart function. C57BL/6 mice were fed an HFD or standard laboratory diet. The FA composition of chow, serum, heart and skeletal muscle tissues was measured by gas chromatography-mass spectrometry. Cardiac function was evaluated by ultrasonography. Our results showed an unexpected increase in polyunsaturated FAs (PUFAs) and a significant decrease in monounsaturated FAs (MUFAs) in the heart tissue of mice fed the HFD. For comparison, no such effects were observed in skeletal muscle or serum samples. Furthermore, we found that the largest increase in PUFAs was in the sphingolipid fraction, whereas the largest decrease in MUFAs was in the phospholipid and sphingomyelin fractions. The hearts of mice fed an HFD had an increased content of triacylglycerols. Moreover, the HFD treatment altered aortic flow pattern. We did not find significant changes in heart mass or oxidative stress markers between mice fed the HFD and standard diet. The above results suggest that alterations in FA composition in the heart may contribute to deterioration of heart function. A possible mechanism of this phenomenon is the alteration of sphingolipids and phospholipids in the fatty acid profile, which may change the physical properties of these lipids. Since phospho- and sphingolipids are the major components of cell membranes, alterations in their structures in heart cells can result in changes in cell membrane properties.

Aging-induced cognitive dysfunction can be regulated by probiotics through bidirectional communication with the brain. This study aimed to investigate whether Capsosiphon fulvescens glycoproteins (Cf-hGP) enhanced probiotic-induced improvement of memory in aged rats and the underlying mechanism in the dorsal hippocampus. Cf-hGP were isolated using lectin resin. Cf-hGP (15 mg/kg/day) and/or Lactobacillus plantarum (L. plantarum) (109 CFU/rat/day) were orally administered once a day for 4 weeks. Co-treatment with Cf-hGP and L. plantarum synergistically improved spatial memory in aged rats, which was overturned by functional blocks of brain-derived neurotrophic factor (BDNF) signaling. Increases in BDNF expression and nuclear factor erythroid 2-related factor 2 (Nrf2) phosphorylation were accompanied by mono- and/or co-administration in the dorsal hippocampus, while c-Jun N-terminal kinase (JNK) phosphorylation and glucose-regulated protein 78 expression were decreased. These synergistic effects were downregulated by blocks of BDNF/Nrf2-mediated signaling. In particular, co-treatment, not mono-treatment, reduced phosphorylation of eukaryotic elongation factor 2 (eEF2) regulated by eEF2 kinase and protein phosphatase 2A. Additionally, co-treatment downregulated the interaction between eEF2 kinase and JNK. These data demonstrated that cognitive impairment in aged rats was synergistically diminished by co-treatment with Cf-hGP and L. plantarum through BDNF-mediated regulation of Nrf2 and eEF2 signaling pathways in the dorsal hippocampus.

Azorhizobium caulinodans is a symbiotic nitrogen-fixing bacterium that forms both root and stem nodules on Sesbania rostrata. During nodule formation, bacteria have to withstand organic peroxides that are produced by plant. Previous studies have elaborated on resistance to these oxygen radicals in several bacteria; however, to the best of our knowledge, none have investigated this process in A. caulinodans. In this study, we identified and characterised the organic hydroperoxide resistance gene ohr (AZC_2977) and its regulator ohrR (AZC_3555) in A. caulinodans ORS571. Hypersensitivity to organic hydroperoxide was observed in an ohr mutant. While using a lacZ-based reporter system, we revealed that OhrR repressed the expression of ohr. Moreover, electrophoretic mobility shift assays demonstrated that OhrR regulated ohr by direct binding to its promoter region. We showed that this binding was prevented by OhrR oxidation under aerobic conditions, which promoted OhrR dimerization and the activation of ohr. Furthermore, we showed that one of the two conserved cysteine residues in OhrR, Cys11, was critical for the sensitivity to organic hydroperoxides. Plant assays revealed that the inactivation of Ohr decreased the number of stem nodules and nitrogenase activity. Our data demonstrated that Ohr and OhrR are required for protecting A. caulinodans from organic hydroperoxide stress and play an important role in the interaction of the bacterium with plants. The results that were obtained in our study suggested that a thiol-based switch in A. caulinodans might sense host organic peroxide signals and enhance symbiosis.

Structural alterations of pericytes in microvessels are important features of diabetic retinopathy. Although capillary pericytes had been known not to have α-smooth muscle actin (αSMA), a recent study revealed that a specific fixation method enabled the visualization of αSMA along retinal capillaries. In this study, we applied snap-fixation in wild type and streptozotocin-induced diabetic mice to evaluate the differences in vascular smooth muscle cells of the retina and the choroid. Mice eyeballs were fixed in ice-cold methanol to prevent the depolymerization of filamentous actin. Snap-fixated retina showed αSMA expression in higher-order branches along the capillaries as well as the arterioles and venules, which were not detected by paraformaldehyde fixation. In contrast, most choriocapillaris, except those close to the arterioles, were not covered with αSMA-positive perivascular mural cells. Large choroidal vessels were covered with more αSMA-positive cells in the snap-fixated eyes. Diabetes induced less coverage of αSMA-positive perivascular mural cells overall, but they reached higher-order branches of the retinal capillaries, which was prominent in the aged mice. More αSMA-positive pericytes were observed in the choroid of diabetic mice, but the αSMA-positive expression reduced with aging. This study suggests the potential role of smooth muscle cells in the pathogenesis of age-related diabetic retinopathy and choroidopathy.

7-Methylguanine (7-MG), a natural compound that inhibits DNA repair enzyme poly(ADP-ribose) polymerase 1 (PARP-1), can be considered as a potential anticancer drug candidate. Here we describe a study of 7-MG inhibition mechanism using molecular dynamics, fluorescence anisotropy and single-particle Förster resonance energy transfer (spFRET) microscopy approaches to elucidate intermolecular interactions between 7-MG, PARP-1 and nucleosomal DNA. It is shown that 7-MG competes with substrate NAD+ and its binding in the PARP-1 active site is mediated by hydrogen bonds and nonpolar interactions with the Gly863, Ala898, Ser904, and Tyr907 residues. 7-MG promotes formation of the PARP-1-nucleosome complexes and suppresses DNA-dependent PARP-1 automodification. This results in nonproductive trapping of PARP-1 on nucleosomes and likely prevents the removal of genotoxic DNA lesions.

Cuscuta reflexa Roxb. is traditionally used by the indigenous communities of Bangladesh to treat different diseases, such as pain, edema, tumor, jaundice, and skin infections. This study tested neuro-pharmacological, anti-nociceptive, and antidiarrheal activities by in vivo and in silico experiments for the metabolites extracted (methanol) from the leaves of Cuscuta reflexa (MECR). During the anxiolytic evaluation analyzed by elevated plus maze and hole board tests, MECR (200 and 400 mg/kg) exhibited a significant dose-dependent reduction of anxiety-like behavior in mice. Similarly, mice treated with MECR demonstrated a dose-dependent decrease in the time of immobility in both forced swimming and tail suspension tests. In addition, anti-nociceptive activity was assessed by the chemical-induced (acetic acid and formalin) pain models. In both cases, 400 mg/kg was found to be most effective and significantly (p < 0.001) inhibited acetic acid stimulated writhing and formalin-induced licking (pain response) in mice. Furthermore, antidiarrheal efficacy determined by the castor-oil induced diarrheal model manifested an evident inhibition of diarrheal stool frequency. In parallel, previously isolated bioactive compounds were documented based on the biological activities and subjected to in silico studies to correlate with the current pharmacological outcomes. The selected isolated compounds (15) displayed favorable binding affinities to potassium channels, human serotonin receptor, COX-1, COX-2, M3 muscarinic acetylcholine receptor, and 5-HT3 receptor proteins. Additionally, the ADME/T and toxicological properties were justified to unveil their drug-like properties and toxicity level. Overall, Cuscuta reflexa is bioactive and could be a potential source for the development of alternative medicine.

Members of the Reoviridae family assemble virus factories within the cytoplasm of infected cells to replicate and assemble virus particles. Bluetongue virus (BTV) forms virus inclusion bodies (VIBs) that are aggregates of viral RNA, certain viral proteins, and host factors, and have been shown to be sites of the initial assembly of transcriptionally active virus-like particles. This study sought to characterize the formation, composition, and ultrastructure of VIBs, particularly in relation to virus replication. In this study we have utilized various microscopic techniques, including structured illumination microscopy, and virological assays to show for the first time that the outer capsid protein VP5, which is essential for virus maturation, is also associated with VIBs. The addition of VP5 to assembled virus cores exiting VIBs is required to arrest transcriptionally active core particles, facilitating virus maturation. Furthermore, we observed a time-dependent association of the glycosylated non-structural protein 3 (NS3) with VIBs, and report on the importance of the two polybasic motifs within NS3 that facilitate virus trafficking and egress from infected cells at the plasma membrane. Thus, the presence of VP5 and the dynamic nature of NS3 association with VIBs that we report here provide novel insight into these previously less well-characterized processes.

The MET oncogene encodes a tyrosine kinase receptor involved in the control of a complex network of biological responses that include protection from apoptosis and stimulation of cell growth during embryogenesis, tissue regeneration, and cancer progression. We previously developed an antagonist antibody (DN30) inducing the physical removal of the receptor from the cell surface and resulting in suppression of the biological responses to MET. In its bivalent form, the antibody displayed a residual agonist activity, due to dimerization of the lingering receptors, and partial activation of the downstream signaling cascade. The balance between the two opposing activities is variable in different biological systems and is hardly predictable. In this study, we generated and characterized two single-chain antibody fragments derived from DN30, sharing the same variable regions but including linkers different in length and composition. The two engineered molecules bind MET with high affinity but induce different biological responses. One behaves as a MET-antagonist, promoting programmed cell death in MET "addicted" cancer cells. The other acts as a hepatocyte growth factor (HGF)-mimetic, protecting normal cells from doxorubicin-induced apoptosis. Thus, by engineering the same receptor antibody, it is possible to generate molecules enhancing or inhibiting apoptosis either to kill cancer cells or to protect healthy tissues from the injuries of chemotherapy.

The active targeting to alveolar macrophages (AM) is an attractive strategy to improve the therapeutic efficacy of 'old' drugs currently used in clinical practice for the treatment of pulmonary tuberculosis. Previous studies highlighted the ability of respirable solid lipid nanoparticle assemblies (SLNas), loaded with rifampicin (RIF) and functionalized with a novel synthesized mannose-based surfactant (MS), both alone and in a blend with sodium taurocholate, to efficiently target the AM via mannose receptor-mediated mechanism. Here, we present the in vivo biodistribution of these mannosylated SLNas, in comparison with the behavior of both non-functionalized SLNas and bare RIF. SLNas biodistribution was assessed, after intratracheal instillation in mice, by whole-body real-time fluorescence imaging in living animals and RIF quantification in excised organs and plasma. Additionally, SLNas cell uptake was determined by using fluorescence microscopy on AM from bronchoalveolar lavage fluid and alveolar epithelium from lung dissections. Finally, histopathological evaluation was performed on lungs 24 h after administration. SLNas functionalized with MS alone generated the highest retention in lungs associated with a poor spreading in extra-pulmonary regions. This effect could be probably due to a greater AM phagocytosis with respect to SLNas devoid of mannose on their surface. The results obtained pointed out the unique ability of the nanoparticle surface decoration to provide a potential more efficient treatment restricted to the lungs where the primary tuberculosis infection is located.

The delivery of therapeutic genes in sites of articular cartilage lesions using non-invasive, scaffold-guided gene therapy procedures is a promising approach to stimulate cartilage repair while protecting the cargos from detrimental immune responses, particularly when targeting chondroreparative bone marrow-derived mesenchymal stromal cells in a natural microenvironment like marrow aspirates.

Clinical intervention studies support the efficacy and safety of exercise programs as a treatment modality for non-small-cell lung cancer (NSCLC) during adjuvant/palliative treatment, but the effectiveness of real-world oncogeriatric services is yet to be established. We aimed to examine the effects of a 10-week structured and individualized multicomponent exercise program on physical/cognitive functioning and mental wellness in elderly patients with NSCLC under adjuvant therapy or palliative treatment. A non-randomized, opportunistic control, longitudinal-design trial was conducted on 26 patients with NSCLC stage I-IV. Of 34 eligible participants, 21 were allocated into two groups: (i) control group (n = 7) received usual medical care; and (ii) intervention group (n =19) received multicomponent program sessions, including endurance, strength, balance, coordination and stretching exercises. Tests included the Short Physical Performance Battery (SPPB), 5-m habitual Gait Velocity Test (GVT), Timed Up & Go Test (TUG), 6-Min Walk Test (6MWT), independence of activities in daily living (IADL), muscular performance, cognitive function, and quality of life, which were measured at baseline and after 10 weeks of the program. Results revealed a significant group×time interaction for SPPB (p = 0.004), 5-m GVT (p = 0.036), TUG (p = 0.007), and muscular performance (chest and leg power; p < 0.001). Similarly, significant changes were observed between groups for cognitive functioning (p = 0.021) and quality of life for EUROQoL 5D (p = 0.006). Our findings confirm that a multicomponent exercise program improves measures of physical/cognitive functioning and quality of life in the elderly with NSCLC under adjuvant therapy or palliative treatment. This is an interesting and important study that adds to our current body of knowledge on the safety of exercise interventions, especially in the elderly with solid tumors.

low vitamin D status in pregnancy can influence the offspring's lung function and contribute to childhood asthma development. The objective of this study was to examine the influence of neonatal vitamin D status on the development of asthma among children age 3-9 years in a large population sample.

Transient receptor potential (TRP) ion channels are polymodal receptors that have been implicated in a variety of pathophysiologies, including pain, obesity, and cancer. The capsaicin and heat sensor TRPV1, and the menthol and cold sensor TRPM8, have been shown to be modulated by the membrane protein PIRT (Phosphoinositide-interacting regulator of TRP). The emerging mechanism of PIRT-dependent TRPM8 regulation involves a competitive interaction between PIRT and TRPM8 for the activating phosphatidylinositol 4,5-bisphosphate (PIP2) lipid. As many PIP2 modulated ion channels also interact with calmodulin, we investigated the possible interaction between PIRT and calmodulin. Using microscale thermophoresis (MST), we show that calmodulin binds to the PIRT C-terminal α-helix, which we corroborate with a pull-down experiment, nuclear magnetic resonance-detected binding study, and Rosetta-based computational studies. Furthermore, we identify a cholesterol-recognition amino acid consensus (CRAC) domain in the outer leaflet of the first transmembrane helix of PIRT, and with MST, show that PIRT specifically binds to a number of cholesterol-derivatives. Additional studies identified that PIRT binds to cholecalciferol and oxytocin, which has mechanistic implications for the role of PIRT regulation of additional ion channels. This is the first study to show that PIRT specifically binds to a variety of ligands beyond TRP channels and PIP2.

Multiple sclerosis (MS) is a chronic immunemediated disease of the central nervous system with a highly variable clinical presentation and disease progression. In this study, we investigate the metabolomics profile of patients affected by relapsing-remitting MS (RRMS)and primary progressive MS (PPMS), in order to find potential biomarkers to distinguish between the two forms.

Berberine (BBR) is an isoquinoline alkaloid which exhibits a variety of biological and therapeutic properties, and has been reported by some to block replication of the influenza virus. However, contradictory results have also been presented, and the mechanistic explanation is lacking.

Gene expression analysis by quantitative real-time polymerase chain reaction (RT-qPCR) is routinely used in biomedical studies. The reproducibility and reliability of the data fundamentally depends on experimental design and data interpretation. Despite the wide application of this assay, there is significant variation in the validation process of gene expression data from research laboratories. Since the validity of results depends on appropriate normalisation, it is crucial to select appropriate reference gene(s), where transcription of the selected gene is unaffected by experimental setting. In this study we have applied geNorm technology to investigate the transcription of 12 'housekeeping' genes for use in the normalisation of RT-qPCR data acquired using a widely accepted HepaRG hepatic cell line in studies examining models of pre-clinical drug testing. geNorm data identified a number of genes unaffected by specific drug treatments and showed that different genes remained invariant in response to different drug treatments, whereas the transcription of 'classical' reference genes such as GAPDH (glyceralde- hyde-3-phosphate dehydrogenase) was altered by drug treatment. Comparing data normalised using the reference genes identified by geNorm with normalisation using classical housekeeping genes demonstrated substantial differences in the final results. In light of cell therapy application, RT-qPCR analyses has to be carefully evaluated to accurately interpret data obtained from dynamic cellular models undergoing sequential stages of phenotypic change.

We report the discovery and functional characterization of αM-Conotoxin MIIIJ, a peptide from the venom of the fish-hunting cone snail Conus magus. Injections of αM-MIIIJ induced paralysis in goldfish (Carassius auratus) but not mice. Intracellular recording from skeletal muscles of fish (C. auratus) and frog (Xenopus laevis) revealed that αM-MIIIJ inhibited postsynaptic nicotinic acetylcholine receptors (nAChRs) with an IC50 of ~0.1 μM. With comparable potency, αM-MIIIJ reversibly blocked ACh-gated currents (IACh) of voltage-clamped X. laevis oocytes exogenously expressing nAChRs cloned from zebrafish (Danio rerio) muscle. αM-MIIIJ also protected against slowly-reversible block of IACh by α-bungarotoxin (α-BgTX, a snake neurotoxin) and α-conotoxin EI (α-EI, from Conus ermineus another fish hunter) that competitively block nAChRs at the ACh binding site. Furthermore, assessment by fluorescence microscopy showed that αM-MIIIJ inhibited the binding of fluorescently-tagged α-BgTX at neuromuscular junctions of X. laevis,C. auratus, and D. rerio. (Note, we observed that αM-MIIIJ can block adult mouse and human muscle nAChRs exogenously expressed in X. laevis oocytes, but with IC50s ~100-times higher than those of zebrafish nAChRs.) Taken together, these results indicate that αM-MIIIJ inhibits muscle nAChRs and furthermore apparently does so by interfering with the binding of ACh to its receptor. Comparative alignments with homologous sequences identified in other fish hunters revealed that αM-MIIIJ defines a new class of muscle nAChR inhibitors from cone snails.

Hepatocellular carcinoma (HCC) is the strongest independent predictor of mortality in non-alcoholic steatohepatitis (NASH)-related cirrhosis. The effects and mechanisms of combination of sodium-dependent glucose cotransporter inhibitor and canagliflozin (CA) and dipeptidyl peptidase-4 inhibitor and teneligliptin (TE) on non-diabetic NASH progression were examined. CA and TE suppressed choline-deficient, L-amino acid-defined diet-induced hepatic fibrogenesis and carcinogenesis. CA alone or with TE significantly decreased proinflammatory cytokine expression. CA and TE significantly attenuated hepatic lipid peroxidation. In vitro studies showed that TE alone or with CA inhibited cell proliferation and TGF-β1 and α1 (I)-procollagen mRNA expression in Ac-HSCs. CA+TE inhibited liver fibrogenesis by attenuating hepatic lipid peroxidation and inflammation and by inhibiting Ac-HSC proliferation with concomitant attenuation of hepatic lipid peroxidation. Moreover, CA+TE suppressed in vivo angiogenesis and oxidative DNA damage. CA or CA+TE inhibited HCC cells and human umbilical vein endothelial cell (HUVEC) proliferation. CA+TE suppressed vascular endothelial growth factor expression and promoted increased E-cadherin expression in HUVECs. CA+TE potentially exerts synergistic effects on hepatocarcinogenesis prevention by suppressing HCC cell proliferation and angiogenesis and concomitantly reducing oxidative stress and by inhibiting angiogenesis with attenuation of oxidative stress. CA+TE showed chemopreventive effects on NASH progression compared with single agent in non-diabetic rat model of NASH, concurrent with Ac-HSC and HCC cell proliferation, angiogenesis oxidative stress, and inflammation. Both agents are widely, safely used in clinical practice; combined treatment may represent a potential strategy against NASH.