Characterizing circular peptides in mixtures: sequence fragment assembly of cyclotides from a violet plant by MALDI-TOF/TOF mass spectrometry

Cyclotides are a very abundant class of plant peptides that display significant sequence variability around a conserved cystine-knot motif and a head-to-tail cyclized backbone conferring them with remarkable stability. Their intrinsic bioactivities combined with tools of peptide engineering make cyclotides an interesting template for the design of novel agrochemicals and pharmaceuticals. However, laborious isolation and purification prior to de novo sequencing limits their discovery and hence their use as scaffolds for peptide-based drug development. Here we extend the knowledge about their sequence diversity by analysing the cyclotide content of a violet species native to Western Asia and the Caucasus region. Using an experimental approach, which was named sequence fragment assembly by MALDI-TOF/TOF, it was possible to characterize 13 cyclotides from Viola ignobilis, whereof ten (vigno 1-10) display previously unknown sequences. Amino acid sequencing of various enzymatic digests of cyclotides allowed the accurate assembly and alignment of smaller fragments to elucidate their primary structure, even when analysing mixtures containing multiple peptides. As a model to further dissect the combinatorial nature of the cyclotide scaffold, we employed in vitro oxidative refolding of representative vigno cyclotides and confirmed the high dependency of folding yield on the inter-cysteine loop sequences. Overall this work highlights the immense structural diversity and plasticity of the unique cyclotide framework. The presented approach for the sequence analysis of peptide mixtures facilitates and accelerates the discovery of novel plant cyclotides

Investigation of Polar and Nonpolar Cyclotides Separation from Violet Extract Through Microfluidic Chip

Cyclotides (CTs) as a cyclic peptide obtained from different groups of plants have been very attractable field of research for scientists because of their specific properties like their natural function as host defense agents. CTs are bioactive peptides from plants that characterized by their head-to-tail cyclic backbone and knotted arrangement of their three conserved disulfide bonds. Their natural function is thought to be as host defense agents and a single plant can express dozens to hundreds of CTs. CTs stand out as a family of antimicrobial peptides (AMPs) because of their exceptional stability, structural plasticity, unique biochemical target, and Gram-negative selective antimicrobial action. These features together with recent advancements in the methods of production of CTs make them an intriguing prospect from a drug development perspective. To accomplish this aim, as part of a separation, detection and research of anti-cancer properties CTs study, we investigate the separation of cyclotides in violets into polar and non-polar groups by microfluidic chips

Global investments in pandemic preparedness and COVID-19: development assistance and domestic spending on health between 1990 and 2026

Background The COVID-19 pandemic highlighted gaps in health surveillance systems, disease prevention, and treatment globally. Among the many factors that might have led to these gaps is the issue of the financing of national health systems, especially in low-income and middle-income countries (LMICs), as well as a robust global system for pandemic preparedness. We aimed to provide a comparative assessment of global health spending at the onset of the pandemic; characterise the amount of development assistance for pandemic preparedness and response disbursed in the first 2 years of the COVID-19 pandemic; and examine expectations for future health spending and put into context the expected need for investment in pandemic preparedness. Methods In this analysis of global health spending between 1990 and 2021, and prediction from 2021 to 2026, we estimated four sources of health spending: development assistance for health (DAH), government spending, out-of-pocket spending, and prepaid private spending across 204 countries and territories. We used the Organisation for Economic Co-operation and Development (OECD)'s Creditor Reporting System (CRS) and the WHO Global Health Expenditure Database (GHED) to estimate spending. We estimated development assistance for general health, COVID-19 response, and pandemic preparedness and response using a keyword search. Health spending estimates were combined with estimates of resources needed for pandemic prevention and preparedness to analyse future health spending patterns, relative to need. Findings In 2019, at the onset of the COVID-19 pandemic, US$9·2 trillion (95$7·3 trillion (95% UI 7·2–7·4) in 2019; 293·7 times the $24·8 billion (95$43·1 billion in development assistance was provided to maintain or improve health. The pandemic led to an unprecedented increase in development assistance targeted towards health; in 2020 and 2021, $1·8 billion in DAH contributions was provided towards pandemic preparedness in LMICs, and$37·8 billion was provided for the health-related COVID-19 response. Although the support for pandemic preparedness is 12·2% of the recommended target by the High-Level Independent Panel (HLIP), the support provided for the health-related COVID-19 response is 252·2% of the recommended target. Additionally, projected spending estimates suggest that between 2022 and 2026, governments in 17 (95% UI 11–21) of the 137 LMICs will observe an increase in national government health spending equivalent to an addition of 1% of GDP, as recommended by the HLIP. Interpretation There was an unprecedented scale-up in DAH in 2020 and 2021. We have a unique opportunity at this time to sustain funding for crucial global health functions, including pandemic preparedness. However, historical patterns of underfunding of pandemic preparedness suggest that deliberate effort must be made to ensure funding is maintained

Manganese Oxide Particles as Cytoprotective, Oxygen Generating Agents

Cell culture and cellular transplant therapies are adversely affected by oxidative species and radicals. Herein, we present the production of bioactive manganese oxide nanoparticles for the purpose of radical scavenging and cytoprotection. Manganese comprises the core active structure of somatic enzymes that perform the same function, in vivo. Formulated nanoparticles were characterized structurally and surveyed for maximal activity (superoxide scavenging, hydrogen peroxide scavenging with resultant oxygen generation) and minimal cytotoxicity (48-h direct exposure to titrated manganese oxide concentrations). Cytoprotective capacity was tested using cell exposure to hydrogen peroxide in the presence or absence of the nanoparticles. Several ideal compounds were manufactured and utilized that showed complete disproportionation of superoxide produced by the xanthine/xanthine oxidase reaction. Further, the nanoparticles showed catalase–like activity by completely converting hydrogen peroxide into the corresponding concentration of oxygen. Finally, the particles protected cells (murine β-cell insulinoma) against insult from hydrogen peroxide exposure. Based on these observed properties, these particles could be utilized to combat oxidative stress and inflammatory response in a variety of cell therapy applications

Analysis of cyclotides in viola ignobilis by nano liquid chromatography fourier transform mass spectrometry

Cyclotides are macrocyclic knotted peptides originating from plants. They are extremely stable and have a range of bioactivities including anti-HIV and insecticidal activity. Given the stability of the cyclotide framework, there is interest in using these peptides as scaffolds in drug design. In the current study, we have shown that nano-LC Fourier transform mass spectrometry (FTMS) is an effective method of analyzing cyclotides in plants. In addition, we have used this technique to find cyclotides in a novel species, Viola ignobilis (Violaceae plant family), which was collected from the East Azerbaijan province of Iran. Varv peptide A, cycloviolacin B2, and cycloviolacin O8 were found in this species. This study provides a novel method for directly analyzing cyclotide sequences without enzymatic digestion and further information regarding the distribution of cyclotides in plant species

Effect of exogenous nitric oxide on germination and some of biochemical characteristics of purple coneflower (Echinacea purpurea L.) in saline condition

To find out the role and effects of exogenous nitric oxide (NO) application in reducing oxidative damage induced by salinity stress in purple coneflower (Echinacea purpurea L.) seed germination, a compeletly randomized design in factorial arrangment with three replications was conducted in Plant Physiology Laboratory of the Genetics and Agricultural Biotechnology Institute of Tabarestan, Sari Agricultural Sciences and Natural Resources University in 2012. The crop seeds were pre-soaked in 0.1 mM of sodium nitroprusside (SNP 0.1 as nitric oxide donor) solution as well as in distilled water (SNP 0 as control) for 20 hs just before the onset of the experiment. Then, pre-treated seeds were subjected to different levels of salinity (0, 50, 75, 100, 125, 150 mM NaCl solution) to germinate. Results showed that the SNP0.1 treatment in saline conditions had significant effect on germination percentage. Different levels of salinity significantly reduced germination rate, germination vigor and index. Pretreated with exogenous NO increased the activity of SOD, POD and APX as compared with SNP0. Accordingly, the highest activity of SOD (116.3 μM g-1 FW), POD (1.7 μM g-1 FW. min.) and APX (50.1 μM g-1 FW. min.) was related to the 125, 50 and 125 Mm NaCl, respectively. Significant and adverse effects of NO were seen on CAT activity. Exogenous NO, as an antioxidant, also reduced peroxidation of membrane lipids (MDA) and delayed the oxidation of proteins. Overall, it could be concluded that sodium nitroprusside as NO donor could improve coneflower seed germination characteristics in saline condition and increase salinity tolerance by means of scavenging of free ROS radicals

Viola plant cyclotide vigno 5 induces mitochondria-mediated apoptosis via cytochrome C release and caspases activation in cervical cancer cells

Cyclotides describe a unique cyclic peptide family that displays a broad range of biological activities including uterotonic, anti -bacteria, anti -cancer and anti -HIV. The vigno cyclotides consist of vigno 1-10 were reported recently from Viola ignobilis. In the present study, we examined the effects of vigno 5, a natural cyclopeptide from V. ignobilis, on cervical cancer cells and the underlying mechanisms. We found that vigno 5 -treated Hela cells were killed off by apoptosis in a dose -dependent manner within 24 h, and were characterized by the appearance of nuclear shrinkage, cleavage of poly (ADP -ribose) polymerase (PARP) and DNA fragmentation. The mitochondrial pathway of apoptosis revealed that cytochrome C is released from mitochondria to cytosol, associated with the activation of caspase-9 and -3, and the cleavage of poly (ADP -ribose) polymerase (PARP). Overall, the results indicate that vigno 5 induces apoptosis in part via the mitochondrial pathway, which is associated with a release of cytochrome C and elevated activity of caspase-9 and -3 in Hela cells. (C) 2016 Elsevier B.V. All rights reserved