Experimental infection of Flavobacterium psychrophilum in fins of Atlantic salmon Salmo salar revealed by scanning electron microscopy

Infections caused by Flavobacterium psychrophilum include 'bacterial coldwater disease' (BCWD) and 'rainbow trout fry syndrome' (RTFS), which are severe diseases that can cause high mortality and significant losses in hatchery-reared salmonids worldwide. Usually, these conditions start with necrosis along the edge of the fins. As the infection progresses, both the fish surface and the internal organs can be involved. The aetiological agent produces a Ca-dependent protease that can be responsible for some of the pathogenic responses, although the precise nature of the response remains to be elucidated. Atlantic salmon Salmo salar were experimentally infected by F. psychrophilum in order to investigate the bacterial invasion in the fin tissues by scanning electron microscopy. The images showed numerous bacteria embedded in the mucous layer when this remained on the tegument. In other zones without mucus, it was observed that bacteria were present on the axis of fin rays, but not on the epidermal surface. The material on these axes was largely eroded by tubular boreholes, and bacterial rods could be seen in these perforations. EDX (Energy Dispersive X-ray) microanalysis of the axis of the fin rays showed significant amounts of P and Ca, revealing the ossification of the ray axis. The protease activity could explain the formation of the tubular boreholes, allowing the bacteria the necessary Ca for the activation of the enzyme. The erosion pattern suggests that the gliding motility of F. psychrophilum could be involved in this burrowing ability.     

Left main stem disease in a patient with Takayasu’s arteritis

Takayasu’s arteritis is a chronic vasculitis of unknown aetiology involving the aorta and its main branches, the pulmonary and coronary tree. Women are affected more often than men (80 to 90% of the cases) with an age onset between 10 and 40 years. This case report demonstrates the limitations of exercise testing and stress echocardiography in diagnosing the extent of coronary artery disease in patients with inflammatory disease in the left main stem coronary artery. (Neth Heart J 2007;15:260-2.)     

Centrocestus formosanus (Opisthorchiida: Heterophyidae) as a cause of death in gray tilapia fry Oreochromis niloticus (Perciforme: Cichlidae) in the dry Pacific of Costa Rica

Centrocestusformosanus is a zoonotic trematode from Asia and has been mainly associated as cause of death of cultured fish. To identify pathogen trematode species in tilapia fry (Oreochromis niloticus) and to determine mollusks hosting these parasites, freshwater mollusks were collected from tilapia cultured ponds and experimental infections were carried out with tilapia fries and different mollusk species. A total of 907 freshwater mollusks were obtained from tilapia ponds and were identified to species level, four gastropods and one bivalve were determined: Melania tuberculata, Melanoides turricula, Pomacea flagellata, Haitia cubensis and Anodontiles luteola. For the first time, the presence of M. turricula and H. cubensis are reported in Costa Rica. Seven morphotypes of cercariae (Xifiodiocercaria, Equinostoma, Oftalmocercaria, Parapleurolofocercus, Cistocerca, Furcocercaria and Leptocercaria) parasitizing all five species of mollusks were found, all of distome type. Experimental exposure of tilapia fry to M. tuberculata demonstrated that the parapleurolofocercus morphotype found in the mollusk is in accordance with the finding of C. formosanus in tilapia fry. An abundance and mean intensity of 1018-1027 digeneans per gill in each exposed fish was determined. Centrocestus formosanus is reported for the first time in Costa Rica, for which the primary and secondary intermediate hosts were also determined.     

In Silico Design of an Oseltamivir Derivative with Increased Affinity against Wild-Type and Mutant Variants of Neuraminidase and Hemagglutinin of Influenza A H1N1 Virus

Antiviral resistance has turned into a world concern nowadays. Influenza A H1N1 emerged as a problem at the world level due to the neuraminidase (NA) mutations. The NA mutants conferred resistance to oseltamivir and zanamivir. Several efforts were conducted to develop better anti-influenza A H1N1 drugs. Our research group combined in silico methods to create a compound derived from oseltamivir to be tested in vitro against influenza A H1N1. Here we show the results of a new compound derived from oseltamivir but with specific chemical modifications, with significant affinity either on NA (in silico and in vitro assays) or HA (in silico) from influenza A H1N1 strain. We include docking and molecular dynamics (MD) simulations of the oseltamivir derivative at the binding site onto NA and HA of influenza A H1N1. Additionally, the biological experimental results show that oseltamivir derivative decreases the lytic-plaque formation on viral susceptibility assays, and it does not show cytotoxicity. Finally, oseltamivir derivative assayed on viral NA showed a concentration-dependent inhibition behavior at nM, depicting a high affinity of the compound for the enzyme, corroborated with the MD simulations results, placing our designed oseltamivir derivative as a potential antiviral against influenza A H1N1.     

Expression of a Rhizopus oryzae lipase in Pichia pastoris under control of the nitrogen source-regulated formaldehyde dehydrogenase promoter

A Rhizopus oryzae lipase gene has been expressed in Pichia pastoris as a reporter using the formaldehyde dehydrogenase 1 promoter (PFLD1) of this organism, which has been reported to be strongly and independently induced by either methanol as sole carbon source or methylamine as sole nitrogen source. Levels of lipase expressed and secreted under the control of the PFLD1 at different induction conditions have been compared to those obtained with the commonly used alcohol oxidase 1 promoter (PAOX1) in small (shake flask) and 1l bioreactor batch cultures. PFLD1-controlled heterologous gene expression was strongly repressed by excess of either glycerol or glucose-but not sorbitol-during growth using methylamine both as sole nitrogen source and inducing substrate. Co-induction of PFLD1 with methanol and methylamine resulted in a synergistic effect on extracellular lipase expression levels. In all tested conditions, the substitution of ammonium for methylamine as carbon source provoked a clear decrease in the specific growth rate and yield of biomass per gram of carbon source. Overall, this study demonstrates that the PFLD1 promoter is at least as efficient as the PAOX1 for extracellular expression of heterologous proteins in P. pastoris bioreactor cultures and provides a first basis for the further design of methanol-free high cell density fed-batch cultivation strategies for controlled overproduction of foreign proteins in P. pastoris.     

Effect of ultraviolet radiation on chlorophyll,carotenoid, protein and proline contents of some annual desert plants

Investigation was carried out to find whether enhanced ultraviolet radiation influences the Malva parviflora L., Plantago major L., Rumex vesicarius L. and Sismbrium erysimoids Desf. of some annual desert plants. The seeds were grown in plastic pots equally filled with a pre-sieved normal sandy soil for 1 month. The planted pots from each species were randomly divided into equal groups (three groups). Plants of the first group exposed to white-light tubes (400–700 nm) 60 w and UV (365 nm) 8 w tubes. The second group was exposed to white-light tubes (400–700 nm) 60 w and UV (302 nm) 8 w tubes. The third group was exposed to white-light tubes (400–700 nm) 60 w and UV (254 nm) 8 w tubes, respectively, for six days. The results indicated that the chlorophyll contents were affected by enhanced UV radiation. The chlorophyll a, b, and total contents were decreased compared with the control values and reduced with the enhanced UV radiation, but the carotenoid was increased compared with the control and also reduced with the enhanced UV radiation. So, the contents of chlorophylls varied considerably. M. parviflora showed the highest constitutive levels of accumulated chlorophyll a, b, and total chlorophyll (0.463, 0.307 and 0.774 mg g⁻¹ f w) among the investigated plant species. P. major showed the lowest constitutive levels of the chloroplast pigments, 0.0036, 0.0038 and 0.0075 mg g⁻¹ f w for chlorophyll a, b, and total chlorophyll at UV-365 nm, respectively. The protein content was decreased significantly in both root and shoot systems compared with the control values but, it was increased with increasing wave lengths of UV-radiation of all tested plants. R. vesicarius showed the highest protein contents among the investigated plants; its content was 3.8 mg g⁻¹ f w at UV-365 nm in shoot system. On the other hand, decreasing ultraviolet wave length induced a highly significant increase in the level of proline in both root and shoot of all tested plants. From the results obtained, it is suggested that proline can protect cells against damage induced by ultraviolet radiation. Statistically, the variations of the studied metabolic activities were significant due to UV radiation treatment in shoot and root system of all investigated plant species.     

Phenotypic switching of vascular smooth muscle cells in atherosclerosis,hypertension, and aortic dissection

Vascular smooth muscle cells (VSMCs) play a critical role in regulating vasotone, and their phenotypic plasticity is a key contributor to the pathogenesis of various vascular diseases. Two main VSMC phenotypes have been well described: contractile and synthetic. Contractile VSMCs are typically found in the tunica media of the vessel wall, and are responsible for regulating vascular tone and diameter. Synthetic VSMCs, on the other hand, are typically found in the tunica intima and adventitia, and are involved in vascular repair and remodeling. Switching between contractile and synthetic phenotypes occurs in response to various insults and stimuli, such as injury or inflammation, and this allows VSMCs to adapt to changing environmental cues and regulate vascular tone, growth, and repair. Furthermore, VSMCs can also switch to osteoblast-like and chondrocyte-like cell phenotypes, which may contribute to vascular calcification and other pathological processes like the formation of atherosclerotic plaques. This provides discusses the mechanisms that regulate VSMC phenotypic switching and its role in the development of vascular diseases. A better understanding of these processes is essential for the development of effective diagnostic and therapeutic strategies.     

Successful synthesis of a glial-specific blood–brain barrier shuttle peptide following a fragment condensation approach on a solid-phase resin

Successful manual synthesis of the TD2.2 peptide acting as a blood–brain barrier shuttle was achieved. TD2.2 was successfully synthesised by sequential condensation of four protected peptide fragments on solid-phase settings, after several unsuccessful attempts using the stepwise approach. These fragments were chosen to minimise the number of demanding amino acids (in terms of coupling, Fmoc removal) in each fragment that are expected to hamper the overall synthetic process. Thus, the hydrophobic amino acids as well as Arg(Pbf) were strategically spread over multiple fragments rather than having them congested in one fragment. This study shows how a peptide that shows big challenges in the synthesis using the common stepwise elongation methodology can be synthesised with an acceptable purity. It also emphasises that choosing the right fragment with certain amino acid constituents is key for a successful synthesis. It is worth highlighting that lower amounts of reagents were required to synthesise the final peptide with an identical purity to that obtained by the automatic synthesiser.     

The conserved Xanthomonas campestris pv. vesicatoria effector protein XopX is a virulence factor and suppresses host defense in Nicotiana benthamiana

Nicotiana benthamiana leaves display a visible plant cell death response when infiltrated with a high titer inoculum of the non-host pathogen, Xanthomonas campestris pv. vesicatoria (Xcv). This visual phenotype was used to identify overlapping cosmid clones from a genomic cosmid library constructed from the Xcv strain, GM98-38. Individual cosmid clones from the Xcv library were conjugated into X. campestris pv. campestris (Xcc) and exconjugants were scored for an altered visual high titer inoculation response in N. benthamiana. The molecular characterization of the cosmid clones revealed that they contained a novel gene, xopX, that encodes a 74-kDa type III secretion system (TTSS) effector protein. Agrobacterium-mediated transient expression of XopX in N. benthamiana did not elicit the plant cell death response although detectable XopX protein was produced. Interestingly, the plant cell death response occurred when the xopX Agrobacterium-mediated transient expression construct was co-inoculated with strains of either XcvDeltaxopX or Xcc, both lacking xopX. The co-inoculation complementation of the plant cell death response also depends on whether the Xanthomonas strains contain an active TTSS. Transgenic 35S-xopX-expressing N. benthamiana plants also have the visible plant cell death response when inoculated with the non-xopX-expressing strains XcvDeltaxopX and Xcc. Unexpectedly, transgenic 35S-xopX N. benthamiana plants displayed enhanced susceptibility to bacterial growth of Xcc as well as other non-xopX-expressing Xanthomonas and Pseudomonas strains. This result is also consistent with the increase in bacterial growth on wild type N. benthamiana plants observed for Xcc when XopX is expressed in trans. Furthermore, XopX contributes to the virulence of Xcv on host pepper (Capsicum annuum) and tomato (Lycopersicum esculentum) plants. We propose that the XopX bacterial effector protein targets basic innate immunity in plants, resulting in enhanced plant disease susceptibility.