Expression and purification of sea raven type II antifreeze protein from Drosophila melanogaster S2 cells

We present a system for the expression and purification of recombinant sea raven type II antifreeze protein, a cysteine-rich, C-type lectin-like globular protein that has proved to be a difficult target for recombinant expression and purification. The cDNAs encoding the pro- and mature forms of the sea raven protein were cloned into a modified pMT Drosophila expression vector. These constructs produced N-terminally His(6)-tagged pro- and mature forms of the type II antifreeze protein under the control of a metallothionein promoter when transfected into Drosophila melanogaster S2 cells. Upon induction of stable cell lines the two proteins were expressed at high levels and secreted into the medium. The proteins were then purified from the cell medium in a simple and rapid protocol using immobilized metal affinity chromatography and specific protease cleavage by tobacco etch virus protease. The proteins demonstrated antifreeze activity indistinguishable from that of wild-type sea raven antifreeze protein purified from serum as illustrated by ice affinity purification, ice crystal morphology, and their ability to inhibit ice crystal growth. This expression and purification system gave yields of 95 mg/L of fully active mature sea raven type II AFP and 9.6 mg/L of the proprotein. This surpasses all previous attempts to express this protein in Escherichia coli, baculovirus-infected fall armyworm cells and Pichia pastoris and will provide sufficient protein for structural analysis.     

A cyanobacterial bloom prevents fish trophic cascades

1. We experimentally compared the impacts of visually feeding zooplanktivorous fish and filter‐feeding omnivorous fish in shallow tropical Dakar Bango reservoir, Senegal. We provoked a cyanobacterial Anabaena bloom under mesotrophic to eutrophic N‐limited conditions in 18 enclosures assigned to six Nile tilapia life‐stage treatments, at typical biomasses: fishless control (C), zooplanktivorous fry (Z), omnivorous juveniles (O), herbivorous fingerlings (H) and two combinations (OZ, OH). 2. All fish grew well, but as prevalent inedible phytoplankton dampened fish effects, community‐level trophic cascades did not occur. Planktivore types acted independently and affected differentially the biomasses of total zooplankton, cyclopoids, nauplii, cladocerans, invertebrate carnivores, large herbivores, colonial cyanobacteria and Chlorophyta. They neither influenced the total biomass of phytoplankton, nor most water chemistry characteristics. Responses were apparently not fish‐biomass related. The bloom collapsed synchronously in all enclosures, coinciding with enrichment ending, with a return to clear water within 12 days. 3. Our results support the hypothesis that excess nutrients and prevalent inedible cyanobacteria inhibit the cascading effects of natural biomass levels of both visually feeding zooplanktivores and filter‐feeding omnivores. In N‐limited meso‐eutrophic shallow tropical lakes with predominantly small herbivorous zooplankton, neither the type nor the biomass of planktivorous fish present seems likely to prevent the transient outburst of cyanobacterial blooms. Such fragile ecosystems may thus not sustain a trophic state suitable for drinking water production, unless human impacts are restricted. The generality of restoration approaches based on ecological engineering should be further explored.     

Changes in antioxidant activities and phenol content in tomato plants subjected to partial root drying and regulated deficit irrigation

Abstract

Partial rootzone drying (PRD) and regulated deficit irrigation (RDI) are water saving irrigation systems that have been developed to increase water use efficiency (WUE) without significant yield reduction. To examine whether tomato responded differently to RDI and PRD, we compared the changes in antioxidative defenses in tomato plants using a split-root system. Tomato plants were grown for 21 days under controlled conditions with their roots separated equally between two soil compartments. Three irrigation treatments were imposed: Control, receiving an amount of water equivalent to 100% of plant transpiration; PRD in which one compartment was watered with 50% of the amount of water supplied to the controls, allowing one-half of the root system to be exposed to dry soil, and switching irrigation between sides weekly; RDI in which 50% of the amount of water given to the controls was supplied, half to each side of the root system. Relative water content (RWC), midday leaf Ψ and chlorophyll content decreased largely in RDI-treated plants, whereas the PRD plants exhibited relatively higher Ψ and RWC values. An enhanced level of lipid peroxidation in both roots and leaves indicated that PRD and RDI caused oxidative stress in tomato plants. In leaves, superoxide dismutase (SOD), soluble peroxidase (POX) and polyphenol oxidase (PPO) activities showed an increase in the early phase of water deficit, and then decreased in the remaining phase of the drying cycle. However, the increase was more pronounced under RDI. Catalase (CAT) activity declined continuously from the onset of PRD and RDI treatments to below the control level, and the reduction was less under PRD than RDI. POX cell-wall associated activities exceeded the control level by 450% and 230%, respectively, under RDI and PRD. At the root level, while CAT activity also decreased under both PRD and RDI, the activities of SOD, POX and PPO significantly increased and their activities showed an alternating increase/decrease paralleling the alternating irrigation in PRD-treated roots. As a result of the difference in POX and PPO activities between the two water treatments applied, PRD-treated plants accumulated more soluble and cell-wall bound phenolic compounds.     

Loss of Tankyrase-mediated destruction of 3BP2 is the underlying pathogenic mechanism of cherubism

Cherubism is an autosomal-dominant syndrome characterized by inflammatory destructive bony lesions resulting in symmetrical deformities of the facial bones. Cherubism is caused by mutations in Sh3bp2, the gene that encodes the adaptor protein 3BP2. Most identified mutations in 3BP2 lie within the peptide sequence RSPPDG. A mouse model of cherubism develops hyperactive bone-remodeling osteoclasts and systemic inflammation characterized by expansion of the myelomonocytic lineage. The mechanism by which cherubism mutations alter 3BP2 function has remained obscure. Here we show that Tankyrase, a member of the poly(ADP-ribose)polymerase (PARP) family, regulates 3BP2 stability through ADP-ribosylation and subsequent ubiquitylation by the E3-ubiquitin ligase RNF146 in osteoclasts. Cherubism mutations uncouple 3BP2 from Tankyrase-mediated protein destruction, which results in its stabilization and subsequent hyperactivation of the SRC, SYK, and VAV signaling pathways.     

DockRank: Ranking docked conformations using partner‐specific sequence homology‐based protein interface prediction

Selecting near‐native conformations from the immense number of conformations generated by docking programs remains a major challenge in molecular docking. We introduce DockRank, a novel approach to scoring docked conformations based on the degree to which the interface residues of the docked conformation match a set of predicted interface residues. DockRank uses interface residues predicted by partner‐specific sequence homology‐based protein–protein interface predictor (PS‐HomPPI), which predicts the interface residues of a query protein with a specific interaction partner. We compared the performance of DockRank with several state‐of‐the‐art docking scoring functions using Success Rate (the percentage of cases that have at least one near‐native conformation among the top m conformations) and Hit Rate (the percentage of near‐native conformations that are included among the top m conformations). In cases where it is possible to obtain partner‐specific (PS) interface predictions from PS‐HomPPI, DockRank consistently outperforms both (i) ZRank and IRAD, two state‐of‐the‐art energy‐based scoring functions (improving Success Rate by up to 4‐fold); and (ii) Variants of DockRank that use predicted interface residues obtained from several protein interface predictors that do not take into account the binding partner in making interface predictions (improving success rate by up to 39‐fold). The latter result underscores the importance of using partner‐specific interface residues in scoring docked conformations. We show that DockRank, when used to re‐rank the conformations returned by ClusPro, improves upon the original ClusPro rankings in terms of both Success Rate and Hit Rate. DockRank is available as a server at http://einstein.cs.iastate.edu/DockRank/ . Proteins 2014; 82:250–267. © 2013 Wiley Periodicals, Inc.     

Potentiality of Different Seed Priming Agents to Mitigate Cold Stress of Winter Rice Seedling

Seed priming has proved to be an effective pre-germination seed invigoration technique for different crops to improve seed and seedling performance under different abiotic stresses. In Bangladesh, winter rice is very often exposed to cold waves just after sowing in the nursery bed resulting in poor seed germination and seedling emergence, yellowish and thin seedlings production, and a very low survival rate. Seed priming may mitigate the cold stress during seed germination and seedling emergence and helps in the quality seedling production of winter rice. To evaluate the efficacy of different seed priming techniques in increasing seedling emergence, growth, vigor and survivability of winter rice cultivars under cold stress, a pot experiment was conducted at the Department of Agronomy, Bangladesh Agricultural University during December 2018 to January 2019. The experiment comprised two factors, (A) Winter rice variety namely, i) BRRI dhan29 and ii) BRRI dhan36; (B) Seed priming agent namely i) Control (no priming), ii) 20000 ppm NaCl, iii) 30000 ppm NaCl, iv) 20000 ppm KCl, v) 30000 ppm KCl, vi) 20000 ppm CaCl₂, vii) 30000 ppm CaCl₂, viii) 50 ppm CuSO₄, ix) 75 ppm CuSO₄, x) 10000 ppm ZnSO₄, xi) 15000 ppm ZnSO₄, xii) 2 ppm Na₂MoO₄, xiii) 3 ppm Na₂MoO₄, xiv) 100 ppm PEG (Polyethylene glycol 4000) and xv) 150 ppm PEG. Seeds were sown on two different dates viz., 1st December and 1st January so that seedlings are exposed to cold stress at different stages. The experiment was laid out in a completely randomized design (CRD) with three replications. Results indicated that (in most of the cases) seed priming has a positive impact on seedling emergence rate (%), root length, shoot length, root shoot ratio, root dry weight, shoot dry weight, seedling dry weight and survival rate (%). Among the priming agents, KCl and CaCl₂ performed best; while priming with NaCl and PEG showed no advantages over no priming for both the sowing dates. In general, BRRI dhan36 performed better than BRRI dhan29 in terms of seedling growth because of its higher tolerance to cold stress. But, both the varieties performed similarly in terms of emergence rate and survival rate. Thus, priming is an effective tool to increase seed germination, better seedling growth, and higher seedling survivability of winter rice under cold stress, and KCl (20000 ppm) or CaCl₂ (20000 ppm) can be considered as a viable priming agent.     

The Primacy of Moringa (<i>Moringa oleifera</i> Lam.) in Boosting Nutrition Status and Immunity Defence Amidst the COVID-19 Catastrophe: A Perspective

A severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) led novel coronavirus disease (COVID-19) outbreak spread through China has become the biggest global public health challenge today. The virus upon several mutations has led to the resurgence of more infectious and lethal variants infecting over 298 million people with more than 5.46 million deaths worldwide by the end of December, 2021. Though vaccines are available, various preventive measures particularly a high body immunity is still extremely important which determines the likelihood of disease severity and subsequent recovery in the current and future pandemics. This review acknowledges the potentiality of miraculous Moringa oleifera Lam. against recently evolved novel coronavirus and accompanying health complications. Moringa a well-proven super-food, densely packed with an abundant quantity of 92 minerals, several vitamins, 46 antioxidants, and numerous bioactive compounds, thus own a massive therapeutic potential for healing all levels of nutritional deficiencies and poor immunities and cure above 300 diseases. Moringa acts as anti-asthmatic, anti-cancerous, anti-diabetic, anti-inflammatory, hypotensive, hepatic, renal and cardio-protective, and anti-viral in nature. Thus it may reduce the severity of COVID-19 infections and associated serious medical emergencies. In addition, self-isolation at home or the workplace has put people at increased risk of physical and mental sicknesses, which could be simply addressed by integrating this wonderful plant into everyday diet. Furthermore, the immune-modulatory properties and viral inhibiting nature of moringa contribute to reduced risk of COVID-19 infection and quicker recovery from its symptoms. As per the existing pieces of literature, it is a great time to harness the esteemed moringa for safeguarding people from the terrible ongoing COVID-19 situation and other future pandemics.     

Recent Developments to Mitigate Selenium Deficiency in Agricultural Eco-Systems

Under changing climate, trace elements like selenium (Se) have emerged as vital constituent of agro-ecosystems enabling crop plants to off-set the adverse effects of suboptimal growth conditions. The available form of selenium is important for boosting its bioavailability to crop plants having varied agro-botanical traits and root architectural systems. As compared to selenite, the selenate has a weaker soil bonding, higher absorption in the soil solution which results in a comparatively absorption by plant roots. Various factors including dry climate, high pH, optimal ambient air temperature, less accumulation of water, and low concentration of organic matter in the soil tend to boost the selenate ratio in the soil. The use of selenium pelleted seeds has emerged as an interesting and viable alternative to alleviate selenium deficiency in agricultural eco-systems. Similarly, the co-inoculation of a mixture of Selenobacteria and Arbuscular mycorrhizal fungi represents an evolving promising strategy for the bio-fortification of wheat plants to produce selenium-rich flour to supplement human dietary needs. Furthermore, in-depth research is required to assure the effectiveness of biological fertilization procedures in field conditions as well as to explore and increase our understanding pertaining to the underlying main mechanisms and channels of selenium absorption in plants. The focus of this review is to synthesize the recent developments on Se dynamics in soil-plant systems and emerging promising strategies to optimize its levels for crop plants. Recent developments regarding the use of micro-organisms as a biotechnological mean to enhance plant nutrition and crop quality have been objectively elaborated. The study becomes even more pertinent for arid and semi-arid agro-ecosystems owing to the potential role of selenium in providing stress tolerance to crop plants. Moreover, this review synthesizes and summarizes the recent developments on climate change and bioavailability, and the protective role of selenium in crop plants.

     

Thymol and Carvacrol Prevent Cisplatin‐Induced Nephrotoxicity by Abrogation of Oxidative Stress,Inflammation, and Apoptosis in Rats

The aim of the present study is to assess the possible protective effects of thymol and carvacrol against cisplatin (CP)‐induced nephrotoxicity. A single dose of CP {6 mg/kg, intraperitoneally (i.p.)} injected to male rats revealed significant increases in serum urea, creatinine, and tumor necrosis factor alpha levels. It also increased kidney contents of malondialdehyde and caspase‐3 activity with significant reduction in serum albumin, kidney content of reduced glutathione as well as catalase, and superoxide dismutase activity as compared to that of the control group. In contrast, administration of thymol {20 mg/kg, orally (p.o.)} and/or carvacrol (15 mg/kg, p.o.) for 14 days before CP injection and for 7 days after CP administration restored the kidney function and examined oxidative stress parameters. In conclusion, thymol was more effective nephroprotective than carvacrol. Moreover, a combination of thymol and carvacrol had a synergistic nephroprotective effect that might be attributed to antioxidant, anti‐inflammatory, and antiapoptotic activities.