A transgenic RNAi approach for developing tomato plants immune to Pepino mosaic virus

RNA interference (RNAi) or gene silencing is a natural defence response of plants to invading viruses. Here, we applied this approach against pepino mosaic virus (PepMV) isolates in their natural host, tomato. PepMV isolates differ in their genetic sequences, the severity of the disease they induce, and their worldwide distribution. PepMV causes heavy crop losses, mainly due to impaired tomato fruit quality. Resistant varieties are not yet available, despite many years of resistance breeding efforts within the tomato seed industry. To generate broad resistance to PepMV strains, conserved sequences from three different strains of PepMV (US1, LP, and CH2) were synthesized as a single insert and cloned in a hairpin configuration into a binary vector, which was used to transform tomato plants. Transgenic tomato lines that expressed a high level of transgene-siRNA exhibited immunity to PepMV strains, including a new Israeli isolate. This immunity was maintained even after graft inoculation, in which a transgenic scion was grafted onto nontransgenic infected rootstocks. However, an immune transgenic rootstock was unable to induce resistance in a nontransformed scion. These results provide the first example of engineered immunity to diverse PepMV strains in transgenic tomato based on gene silencing.     

Der Baobab (Adansonia digitata L.): Wildobst aus Afrika für Deutschland und Europa?!

In Germany and Europe exotic fruits from distant places are becoming more and more important. In the tropics and subtropics an enormous variety of edible fruits exists. In 2008 the dry fruit pulp of the baobab (Adansonia digitata L.) was approved as novel food for the EU and thus entered the formal food market. Hence, an increasing number of products became available in Europe. The baobab is an impressive tree in the savannas of Africa. Due to its dense and wide spreading root system and its enormous water storing capacity in the stem, the tree can grow under very dry environmental conditions. In many regions of Africa it is a multipurpose indigenous fruit tree species which offers shelter and provides food, fodder and medicine as well as raw material for many purposes. However, even in Europe a wide range of products is now available as shown in the present study, which identified more than 300 baobab products or products with baobab as an ingredient. In Germany, not only the natural baobab pulp but also ready-made foodstuffs such as soft drinks, sandwich spreads, cereal bars, sweets and chocolates are available. Furthermore, a diverse range of pharmaceutical and cosmetic products with extracts from the baobab is available in the market. The products range from after shave and perfume to shampoo and shower gel to leg lotion and foot spray. In Germany baobab saplings and seeds are offered for planting and cultivation as an indoor pot plant. Commercially marketed baobab bonsai seedlings for the European market are produced in Senegal. In Africa the baobab fruits are mainly harvested from wild stands. However, the commercial production of baobab fruits in orchards could be an important future opportunity for income generation to many local households. More research on baobab, including the domestication of this wild fruit tree species, and the conservation of its natural stands as important genetic resource are prerequisites for sustainably increased fruit production.     

Evaluation of the Local Analgesic Effects of a Commercial Aqueous Extract of Sarracenia purpurea and Ammonium Sulfate in the Equine Abaxial Sesamoid Block Model

Sarracenia purpurea is a carnivorous plant whose aqueous extracts have been proposed to exert analgesic effects by neurolytical action on peripheral nerves. The aim of this study was to determine the local analgesic effects of a commercially available aqueous extract solution of S. purpurea (P-Bloc, St. Joseph, Missouri) and a 1% ammonium sulfate solution, using the horse abaxial sesamoid block model. Twenty horses were randomly assigned to two groups. The first group (n = 10) was used to evaluate the effect of P-Bloc. The horses received a bilateral (medial and lateral) abaxial sesamoid block with 5 mL of P-Bloc in one random forelimb, while in the contralateral forelimb, they received either 5 mL of 2% lidocaine as a positive control (n = 5) or 5 mL of 0.9% NaCl as a negative control (n = 5). The second group (n = 10) was treated as the first but received 5 mL of 1% ammonium sulfate in NaCl (0.9% NaCl) solution instead of P-Bloc. The period of hoof withdrawal reflex latency (HWRL, in seconds) was measured using a custom-made heat projector lamp as a source of a noxious skin heating stimulus applied to the pastern. Lidocaine (2%) prolonged (P ≤ .05) the HWRL period, returning to the negative control basal values after 240 minutes. Neither the treatment with P-Bloc nor 1% ammonium sulfate modified the HWRL period. The lack of effect of these compounds in this model reinforces the results reported elsewhere and suggests a nonlocal anesthetic mechanism of action for the aqueous extract of S. purpurea in the horse.     

Synthesis and Antiproliferative Activity of 2,5-bis(3′-Indolyl)pyrroles,Analogues of the Marine Alkaloid Nortopsentin

2,5-bis(3′-Indolyl)pyrroles, analogues of the marine alkaloid nortopsentin, were conveniently prepared through a three step procedure in good overall yields. Derivatives 1a and 1b exhibited concentration-dependent antitumor activity towards a panel of 42 human tumor cell lines with mean IC₅₀ values of 1.54 μM and 0.67 μM, respectively. Investigating human tumor xenografts in an ex-vivo clonogenic assay revealed selective antitumor activity, whereas sensitive tumor models were scattered among various tumor histotypes.     

CLINICAL,IMAGING, AND PATHOLOGIC CHARACTERISTICS OF GURLTIA PARALYSANS MYELOPATHY IN DOMESTIC CATS FROM CHILE

Gurltia paralysans is a rare metastrongylid nematode of domestic cats that is found mainly in the veins of the spinal cord subarachnoid space and parenchyma. Endemic regions for G. paralysans mainly include Chile and Argentina. The ante mortem diagnosis of gurltiosis is difficult and based primarily on neurological signs, epidemiological factors, and the exclusion of other causes of feline myelopathies. The purpose of this retrospective case series was to describe clinical, imaging, and pathologic characteristics in nine domestic cats naturally infected with G. paralysans. Imaging tests included radiography, myelography, computed tomographic myelography (myelo‐CT), and magnetic resonance imaging (MRI). Neurological signs included paraparesis, paraplegia, pelvic limb ataxia and proprioceptive deficits, pelvic limb tremors, lumbosacral hyperesthesia, and tail trembling or atony. Complete blood count findings included a decrease in the mean corpuscular hemoglobin concentration value in eight cats. Eosinophilia in peripheral blood was observed in three cats, and thrombocytopenia was observed in three cats. Cerebrospinal fluid analysis revealed mononuclear pleocytosis in five cases. Myelo‐CT showed diffuse enlargement of the spinal cord at the midthoracic, lumbar, and sacral regions in all cats. Magnetic resonance image findings in the thoracic and lumbar region demonstrated multiple small nodular areas of T2 hyperintensity in the periphery of the spinal cord parenchyma. Localized intraparenchymal areas of increased T2 intensity were also observed in the thoracolumbar spinal cord and lumbosacral conus medullaris. In conclusion, G. paralysans should be considered as a differential diagnosis for domestic cats in endemic regions that have this combination of clinical and imaging characteristics.     

Adaptation of ostriches to transport‐induced stress: Physiometabolic response

This study assessed the effects of five different transport periods on physiometabolic responses and gas exchange in ostriches. It included 138 ostriches that were assigned to five experimental groups. G₁ included 78 birds that were set aside as a reference group (RG). Each one of the four remaining experimental groups included 20 ostriches, which were transported in the following manner: G₁ on one occasion for a period of 1 h; G₂ on one occasion for 2 h; and G₃ on one occasion for 3 h; G_4a was made up of the ostriches from G₁ but they were shipped on a second occasion, for 2 h (making a total of 3 h); finally, G_4b was that same group, but after a third transport period, on this occasion during 3 h (for a total of 6 h). Groups G₂ and G₃ presented the most marked blood alterations (P < 0.05), including an increase in pH, hypocapnia (27.8 ± 0.80 mmHg), hypernatremia (171.75 ± 1.84 mmol/L), hypocalcemia (0.95 ± 0.03 mmol/L), and hyperglycemia (224.05 ± 3.94 mg/dL). Also, group G₃ presented the lowest hematocrit values (26.5 ± 0.47 HTC%). Therefore, transporting ostriches with no prior experience for 3 h caused the most pronounced physiometabolic changes.     

Structural and sensory characterization of key pungent and tingling compounds from black pepper (Piper nigrum L.)

To gain a more comprehensive knowledge on whether, besides the well-known piperine, other compounds are responsible for the pungent and tingling oral impression imparted by black pepper, an ethanol extract prepared from black pepper (Piper nigrum L.) was screened for its key sensory-active nonvolatiles by application of taste dilution analysis (TDA). Purification of the compounds perceived with the highest sensory impact, followed by LC-MS and 1D/2D NMR experiments as well as synthesis, led to the structure determination of 25 key pungent and tingling phytochemicals, among which the eight amides 1-(octadeca-2E,4E,13Z-trienyl)piperidine, 1-(octadeca-2E,4E,13Z-trienyl)pyrrolidine, (2E,4E,13Z)-N-isobutyl-octadeca-2,4,13-trienamide, 1-(octadeca-2E,4E,12Z-trienoyl)-pyrrolidine, 1-(eicosa-2E,4E,15Z-trienyl)piperidine, 1-(eicosa-2E,4E,15Z-trienyl)pyrrolidine, (2E,4E,15Z)-N-isobutyl-eicosa-2,4,15-trienamide, and 1-(eicosa-2E,4E,14Z-trienoyl)-pyrrolidine were not yet reported in literature. Sensory studies by means of a modified half-tongue test revealed recognition thresholds ranging from 3.0 to 1150.2 nmol/cm2 for pungency and from 520.6 to 2162.1 nmol/cm2 for the tingling orosensation depending on their chemical structure.     

Apigenin-induced suicidal erythrocyte death

Apigenin, a flavone in fruits and vegetables, stimulates apoptosis and thus counteracts cancerogenesis. Erythrocytes may similarly undergo suicidal cell death or eryptosis, characterized by cell shrinkage and phosphatidylserine exposure at the cell surface. Triggers of eryptosis include increase of cytosolic Ca(2+) activity ([Ca(2+)](i)), ceramide formation and ATP depletion. The present study explored the effect of apigenin on eryptosis. [Ca(2+)](i) was estimated from Fluo3-fluorescence, cell volume from forward scatter, phosphatidylserine exposure from annexin V binding, hemolysis from hemoglobin release, ceramide utilizing antibodies, and cytosolic ATP with luciferin-luciferase. A 48 h exposure to apigenin significantly increased [Ca(2+)](i) (≥ 1 μM), increased ceramide formation (15 μM), decreased ATP concentration (15 μM), decreased forward scatter (≥ 1 μM), and increased annexin V binding (≥ 5 μM) but did not significantly modify hemolysis. The effect of 15 μM apigenin on annexin V binding was blunted by Ca(2+) removal. The present observations reveal novel effects of apigenin, i.e. stimulation of Ca(2+) entry, ceramide formation and ATP depletion in erythrocytes with subsequent triggering of suicidal erythrocyte death, paralleled by cell shrinkage and phosphatidylserine exposure.     

Computational Assessment of Chito-Oligosaccharides Interactions with Plasma Proteins

It is widely rec ognized that chitin and chitosan are potential sources of bioactive materials and that their oligosaccharides reveal various biological activities (including antimicrobial) that are correlated with their structures and physicochemical properties. This study uses the molecular docking approach to assess the interactions of small chito-oligosaccharides (MW< 1500 Da) with plasma proteins in order to obtain information regarding their fate of distribution in the human organism. There are favorable interactions of small chito-oligomers with plasma proteins, the interactions with human serum albumin being stronger than those with α-1-acid glycoprotein. The interaction energies increase with increasing the molecular weight, decrease with increasing deacetylation degrees and are reliant on the deacetylation pattern. This study could inform the application of chito-oligosaccharides with varying molecular weights, degrees, and patterns of deacetylation in human health.     

Evidence of exotoxin secretion of Piscirickettsia salmonis,the causative agent of piscirickettsiosis

Piscirickettsia salmonis is the aetiological agent of piscirickettsiosis, a disease which affects a variety of teleost species and that is particularly severe in salmonid fish. Bacterial‐free supernatants, obtained from cultures of three isolates of Piscirickettsia salmonis, were inoculated in Atlantic salmon, Salmo salar L., and in three continuous cell lines in an effort to determine the presence of secretion of extracellular products (ECPs) by this microorganism. Although steatosis was found in some liver samples, no mortalities or clinical signs occurred in the inoculated fish. Clear cytotoxicity was observed after inoculation in the cell lines CHSE‐214 and ASK, derived from salmonid tissues, but not in MDBK, which is of mammalian origin. The degree of cytotoxicity of the ECPs was different among the P. salmonis isolates tested. The isolate that evidenced the highest cytotoxicity in its ECPs exhibited only an intermediate virulence level after challenging fish with bacterial suspensions of the three P. salmonis isolates. Almost complete inhibition of the cytotoxic activity of ECPs was seen after proteinase K treatment, indicating their peptidic nature, and a total preclusion of the cytotoxicity was shown after their incubation at 50 °C for 30 min. Results show that P. salmonis can produce ECPs and at least some of them are thermolabile exotoxins that probably play a role in the pathogenesis of piscirickettsiosis.