Isolation and Functional Characterisation of a fads2 in Rainbow Trout (Oncorhynchus mykiss) with Δ5 Desaturase Activity

Rainbow trout, Oncorhynchus mykiss, are intensively cultured globally. Understanding their requirement for long-chain polyunsaturated fatty acids (LC-PUFA) and the biochemistry of the enzymes and biosynthetic pathways required for fatty acid synthesis is important and highly relevant in current aquaculture. Most gnathostome vertebrates have two fatty acid desaturase (fads) genes with known functions in LC-PUFA biosynthesis and termed fads1 and fads2. However, teleost fish have exclusively fads2 genes. In rainbow trout, a fads2 cDNA had been previously cloned and found to encode an enzyme with Δ6 desaturase activity. In the present study, a second fads2 cDNA was cloned from the liver of rainbow trout and termed fads2b. The full-length mRNA contained 1578 nucleotides with an open reading frame of 1365 nucleotides that encoded a 454 amino acid protein with a predicted molecular weight of 52.48 kDa. The predicted Fads2b protein had the characteristic traits of the microsomal Fads family, including an N-terminal cytochrome b5 domain containing the heme-binding motif (HPPG), histidine boxes (HDXGH, HFQHH and QIEHH) and three transmembrane regions. The fads2b was expressed predominantly in the brain, liver, intestine and pyloric caeca. Expression of the fasd2b in yeast generated a protein that was found to specifically convert eicosatetraenoic acid (20:4n-3) to eicosapentaenoic acid (20:5n-3), and therefore functioned as a Δ5 desaturase. Therefore, rainbow trout have two fads2 genes that encode proteins with Δ5 and Δ6 desaturase activities, respectively, which enable this species to perform all the desaturation steps required for the biosynthesis of LC-PUFA from C₁₈ precursors.     

Combined and Singular Effects of Dietary PrimaLac® and Potassium Diformate (KDF) on Growth Performance and Some Physiological Parameters of Rainbow Trout (Oncorhynchus mykiss)

The present study was conducted to assess the effects of combined and singular dietary administration of PrimaLac® and potassium diformate (KDF) on growth performance, feed utilization, digestive enzymes activity, and some physiological parameters of rainbow trout (Oncorhynchus mykiss) juvenile. Three hundred sixty rainbow trout juveniles (25 ± 1.8 g) were randomly stocked in 300-L tanks (30 fish/tank), and fed three times daily on a basal diet (control), diets incorporated with 12 g kg⁻¹ KDF (FT₁), 1.5 g kg⁻¹ PrimaLac® (FT₂), and combination of 1.5 g kg⁻¹ probiotic and 12 g kg⁻¹ KDF (FT₃) in triplicates, for 8 weeks. At the end of feeding trial, growth performance, body composition, digestive enzymes, liver enzymes, and biochemical parameters were measured. Our results revealed that combined administration of PrimaLac® and KDF (FT₃) exhibited significantly higher weight gain and specific growth rate (SGR) compared to other groups (P < 0.05). Glucose and cortisol levels showed no significant differences between fish fed different test diets (P > 0.05). The highest lipase, protease and amylase activity were observed in group of fish fed FT₃ followed by FT₂ and FT₁. Besides, the diets FT₂ and FT₃ led to significantly lower of ALP, ALT, and AST compared to control group. The present results indicated that combined administration of PrimaLac® and KDF can be considered as a beneficial feed additive and growth promotor for O. mykiss juvenile.

     

Protein Retention Assessment of Four Levels of Poultry By-Product Substitution of Fishmeal in Rainbow Trout (Oncorhynchus mykiss) Diets Using Stable Isotopes of Nitrogen (δ15N) as Natural Tracers

This is second part from an experiment where the nitrogen retention of poultry by-product meal (PBM) compared to fishmeal (FM) was evaluated using traditional indices. Here a quantitative method using stable isotope ratios of nitrogen (δ¹⁵N values) as natural tracers of nitrogen incorporation into fish biomass is assessed. Juvenile rainbow trout (Oncorhynchus mykiss) were fed for 80 days on isotopically distinct diets in which 0, 33, 66 and 100% of FM as main protein source was replaced by PBM. The diets were isonitrogenous, isolipidic and similar in gross energy content. Fish in all treatments reached isotopic equilibrium by the end of the experiment. Two-source isotope mixing models that incorporated the isotopic composition of FM and PBM as well as that of formulated feeds, empirically derived trophic discrimination factors and the isotopic composition of fish that had reached isotopic equilibrium to the diets were used to obtain a quantitative estimate of the retention of each source of nitrogen. Fish fed the diets with 33 and 66% replacement of FM by PBM retained poultry by-product meal roughly in proportion to its level of inclusion in the diets, whereas no differences were detected in the protein efficiency ratio. Coupled with the similar biomass gain of fishes fed the different diets, our results support the inclusion of PBM as replacement for fishmeal in aquaculture feeds. A re-feeding experiment in which all fish were fed a diet of 100% FM for 28 days indicated isotopic turnover occurred very fast, providing further support for the potential of isotopic ratios as tracers of the retention of specific protein sources into fish tissues. Stable isotope analysis is a useful tool for studies that seek to obtain quantitative estimates of the retention of different protein sources.     

Transforming growth factor-β1b: A second TGF-β1 paralogue in the rainbow trout (Oncorhynchus mykiss) that has a lower constitutive expression but is more responsive to immune stimulation

The rainbow trout (Oncorhynchus mykiss) TGF-β1 sequence was one of the first fish cytokines described. Studies of its expression suggest it is constitutively expressed but displays refractory inducibility. Here we describe a second TGF-β1 (TGF-β1b) gene that is novel in several respects. TGF-β1b possesses typical TGF-β features, including a CXC motif and an integrin binding site, a tetrabasic cut site and a mature peptide of 112 amino acids (aa) containing nine conserved cysteine residues. The mature peptide is 83% identical to the first TGF-β1 sequence described in rainbow trout, that we designate TGF-β1a, and relative to TGF-β1a shows higher homology to Atlantic salmon TGF-β1b, zebrafish TGF-β1a, and sea bass and seabream TGF-β1. The gene organisation of salmonid TGF-β1b genes, as inferred from Atlantic salmon whole genome shotgun contigs, is a 6 exon/5 intron structure with exons 3 and 4 of salmonid TGF-β1a genes apparently fused together. The two trout TGF-β1 genes have a wide distribution in vivo, with highest expression found in immune tissues for both isoforms indicating that TGF-β1 has a predominant role in immunity of fish. Expression of both genes was also seen during the ontogeny of trout, with TGF-β1a relatively constant in expression level but TGF-β1b increasing over time. Immune responses in head kidney (HK) macrophages induced by pathogen associated molecular patterns (PAMPs), pro-inflammatory cytokines, mitogens and pathway activators highly elevated the expression level of TGF-β1b but not that of TGF-β1a. TGF-β1b expression was also increased by polyinosinic:polycytidylic acid (poly(I:C)) and/or lipopolysaccharide (LPS) stimulation in three different trout cell lines studied. Finally we show that TGF-β1b is potentially involved in defense against infection with viral haemorrhagic septicemia virus (VHSV), which had no effect on TGF-β1a expression. Thus, it is likely the TGF-β1b gene represents a copy which fulfils the major immune orchestrating functions of TGF-β1 as seen in other vertebrates.     

Rapid Fishery Assessment by Market Survey (RFAMS) – An Improved Rapid-Assessment Approach to Characterising Fish Landings in Developing Countries

The complex multi-gear, multi-species tropical fisheries in developing countries are poorly understood and characterising the landings from these fisheries is often impossible using conventional approaches. A rapid assessment method for characterising landings at fish markets, using an index of abundance and estimated weight within taxonomic groups, is described. This approach was developed for contexts where there are no detailed data collection protocols, and where consistent data collection across a wide range of fisheries types and geographic areas is required, regardless of the size of the site and scale of the landings. This methodology, which was demonstrated at seven fish landing sites/fish markets in southern Indonesia between July 2008 and January 2011, provides a rapid assessment of the abundance and diversity in the wild catch over a wide variety of taxonomic groups. The approach has wider application for species-rich fisheries in developing countries where there is an urgent need for better data collection protocols, monitoring future changes in market demographics, and evaluating health of fisheries.     

Differential Allocation by Female Zebrafish (Danio rerio) to Different-Sized Males – An Example in a Fish Species Lacking Parental Care

Organisms allocate resources to reproduction in response to the costs and benefits of current and future reproductive opportunities. According to the differential allocation hypothesis, females allocate more resources to high-quality males. We tested whether a fish species lacking parental care (zebrafish, Danio rerio) expresses male size-dependent differential allocation in monogamous spawning trials. In addition, we tested whether reproductive allocation by females is affected by previous experience of different-quality males, potentially indicating plasticity in mate choice. To that end, females were conditioned to large, small or random-sized males (controls) for 14 days to manipulate females'' expectations of the future mate quality. Females showed a clear preference for large males in terms of spawning probability and clutch size independent of the conditioning treatment. However, when females experienced variation in male size (random-sized conditioning treatment) they discriminated less against small males compared to females conditioned to large and small males. This might suggest that differential allocation and size-dependent sexual selection is of less relevance in nature than revealed in the present laboratory study.     

Lipid Metabolism as Related to the Functional Activity of the Photosynthetic Apparatus in the Studies by G.L. Klyachko-Gurvich (1929–2003)

Russian Journal of Plant Physiology -     

Pre-eclampsia – a disease of oxidative stress resulting from the catabolism of DNA (primarily fetal) to uric acid by xanthine oxidase in the maternal liver: A hypothesis

Pre-eclamptic toxaemia or toxaemia has become outdated terminology for the disease of pregnancy called pre-eclampsia (PE) but, according to this hypothesis, these may be more relevant. This hypothesis is that PE is a toxaemia or poisoning of the blood that results in multi-organ dysfunction and injury, putting at risk the lives of both the infant and the mother. Yet these dysfunctions and injuries are reversible with the cessation of the pregnancy and the disease can be reduced with vitamins (antioxidants) and aspirin.This hypothesis is that the PE cascade starts with excessive shedding/embolisation of trophoblast from the placenta into the maternal venous circulation. This trophoblast embolisation (‘deportation’) is secondary either to an excessively large amount of trophoblast tissue (‘hyperplacentosis’) or to vascular trophoblast injury from a faulty uteroplacental circulation. The deported nuclear rich trophoblast is largely filtered out of the circulation in the lungs, and breaks down releasing fetal DNA. Accordingly, the level of fetal DNA in the maternal circulation rises. This DNA is then broken down in the maternal liver with the hepatocytes being presented with excessive amounts of purines for catabolism. In the hepatocytes of patients who subsequently develop PE, there is activation of xanthine oxidase (XO), the more toxic isoenzyme of xanthine oxidoreductase (XOR), with the generation of superoxide anion (O₂⁻) as a by-product. Excessive superoxide production overwhelms the normal antioxidant ability of the tissues to produce oxidative stress.In the hepatocytes, the excessive superoxide causes the peroxidation of polyunsaturated lipids to form microvesicular fat deposition. Excessive superoxide also causes hepatocellular damage with leakage of enzymes, lipids, DNA and superoxide into the circulation. In the circulation, oxidative injury of the blood corpuscles occurs releasing more DNA and accelerating purine catabolism and oxidative stress.The toxins, superoxide and the other reactive oxygen species (ROS), then travel in the arterial blood to the peripheral circulation where the microvasculature, the arterioles, capillaries, endothelial cells and venules, is injured. The damaged microvasculature leaks intravascular fluid into the extravascular compartment causing an intravascular dehydration and tissue oedema. In the kidneys, protein leaks through the damaged glomerular capillaries causing proteinuria. ROS causes arteriolar vasospasm and impairs vasorelaxation, mechanisms of hypertension. Micro-haemorrhages can occur and in the brain these, in combination with hypertension and oedema, can result in seizures or eclampsia.     

NMK-BH2, a novel microtubule-depolymerising bis (indolyl)-hydrazide-hydrazone,induces apoptotic and autophagic cell death in cervical cancer cells by binding to tubulin at colchicine – site

BackgroundMicrotubules, the key components of the eukaryotic cytoskeleton and mitotic spindle, are one of the most sought-after targets for cancer chemotherapy, especially due to their indispensible role in mitosis. Cervical cancer is a prevalent malignancy among women of developing countries including India. In spite of the remarkable therapeutic advancement, the non-specificity of chemotherapeutic drugs adversely affect the patients' survival and well-being, thus, necessitating the quest for novel indole-based anti-microtubule agent against cervical cancer, with high degree of potency and selectivity.MethodsFor in vitro studies, we used MTT assay, confocal microscopy, fluorescence microscopy, flow cytometry and Western blot analysis. Study in cell free system was accomplished by spectrophotometry, fluorescence spectroscopy and TEM and computational analysis was done by AutodockTools 1.5.6.ResultsNMK-BH2 exhibited significant and selective anti-proliferative activity against cervical cancer HeLa cells (IC₅₀ = 1.5 μM) over normal cells. It perturbed the cytoskeletal and spindle microtubules of HeLa cells leading to mitotic block and cell death by apoptosis and autophagy. Furthermore, NMK-BH2 targeted the tubulin-microtubule system through fast and strong binding to the αβ-tubulin heterodimers at colchicine-site.ConclusionThis study identifies and characterises NMK-BH2 as a novel anti-microtubule agent and provides insights into its key anti-cancer mechanism through two different cell death pathways: apoptosis and autophagy, which are mutually independent.General significanceIt navigates the potential of the novel bis (indolyl)-hydrazide-hydrazone, NMK-BH2, to serve as lead for development of new generation microtubule-disrupting chemotherapeutic with improved efficacy and remarkable selectivity towards better cure of cervical cancer.     

From high symmetry to high resolution in biological electron microscopy: a commentary on Crowther (1971) ‘Procedures for three-dimensional reconstruction of spherical viruses by Fourier synthesis from electron micrographs’

Elucidation of the structure of biological macromolecules and larger assemblies has been essential to understanding the roles they play in living processes. Methods for three-dimensional structure determination of biological assemblies from images recorded in the electron microscope were therefore a key development. In his paper published in Philosophical Transactions B in 1971, Crowther described new computational procedures applied to the first three-dimensional reconstruction of an icosahedral virus from images of virus particles preserved in negative stain. The method for determining the relative orientation of randomly oriented particles and combining their images for reconstruction exploited the high symmetry of the virus particle. Computational methods for image analysis have since been extended to include biological assemblies without symmetry. Further experimental advances, combined with image analysis, have led to the method of cryomicroscopy, which is now used by structural biologists to study the structure and dynamics of biological machines and assemblies in atomic detail. This commentary was written to celebrate the 350th anniversary of the journal Philosophical Transactions of the Royal Society.     

The Type III Secreted Effector DspE Is Required Early in Solanum tuberosum Leaf Infection by Pectobacterium carotovorum to Cause Cell Death,and Requires Wx(3–6)D/E Motifs

Pectobacterium species are enterobacterial plant-pathogens that cause soft rot disease in diverse plant species. Unlike hemi-biotrophic plant pathogenic bacteria, the type III secretion system (T3SS) of Pectobacterium carotovorum subsp. carotovorum (P. carotovorum) appears to secrete only one effector protein, DspE. Previously, we found that the T3SS regulator HrpL and the effector DspE are required for P. carotovorum pathogenesis on leaves. Here, we identified genes up-regulated by HrpL, visualized expression of dspE in leaves, and established that DspE causes host cell death. DspE required its full length and WxxxE-like motifs, which are characteristic of the AvrE-family effectors, for host cell death. We also examined expression in plant leaves and showed that hrpL is required for the expression of dspE and hrpN, and that the loss of a functional T3SS had unexpected effects on expression of other genes during leaf infection. These data support a model where P. carotovorum uses the T3SS early in leaf infection to initiate pathogenesis through elicitation of DspE-mediated host cell death.     

Compliance to Oseltamivir among Two Populations in Oxfordshire,United Kingdom Affected by Influenza A(H1N1)pdm09, November 2009 – A Waste Water Epidemiology Study

Antiviral provision remains the focus of many pandemic preparedness plans, however, there is considerable uncertainty regarding antiviral compliance rates. Here we employ a waste water epidemiology approach to estimate oseltamivir (Tamiflu®) compliance. Oseltamivir carboxylate (oseltamivir''s active metabolite) was recovered from two waste water treatment plant (WWTP) catchments within the United Kingdom at the peak of the autumnal wave of the 2009 Influenza A (H1N1)pdm09 pandemic. Predictions of oseltamivir consumption from detected levels were compared with two sources of national government statistics to derive compliance rates. Scenario and sensitivity analysis indicated between 3–4 and 120–154 people were using oseltamivir during the study period in the two WWTP catchments and a compliance rate between 45–60%. With approximately half the collected antivirals going unused, there is a clear need to alter public health messages to improve compliance. We argue that a near real-time understanding of drug compliance at the scale of the waste water treatment plant (hundreds to millions of people) can potentially help public health messages become more timely, targeted, and demographically sensitive, while potentially leading to less mis- and un-used antiviral, less wastage and ultimately a more robust and efficacious pandemic preparedness plan.