Striga parasitizes transgenic hairy roots of Zea mays and provides a tool for studying plant-plant interactions

Background Striga species are noxious root hemi-parasitic weeds that debilitate cereal production in sub-Saharan Africa (SSA). Control options for Striga are limited and developing Striga resistant crop germplasm is regarded as the best and most sustainable control measure. Efforts to improve germplasm for Striga resistance by a non-Genetic Modification (GM) approach, for example by exploiting natural resistance, or by a GM approach are constrained by limited information on the biological processes underpinning host-parasite associations. Additionaly, a GM approach is stymied by lack of availability of candidate resistance genes for introduction into hosts and robust transformation methods to validate gene functions. Indeed, a majority of Striga hosts, the world’s most cultivated cereals, are recalcitrant to genetic transformation. In maize, the existing protocols for transformation and regeneration are tedious, lengthy, and highly genotype-specific with low efficiency of transformation. Results We used Agrobacterium rhizogenes strain K599 carrying a reporter gene construct, Green Fluorescent Protein (GFP), to generate transgenic composite maize plants that were challenged with the parasitic plant Striga hermonthica. Eighty five percent of maize plants produced transgenic hairy roots expressing GFP. Consistent with most hairy roots produced in other species, transformed maize roots exhibited a hairy root phenotype, the hallmark of A. rhizogenes mediated transformation. Transgenic hairy roots resulting from A. rhizogenes transformation were readily infected by S. hermonthica. There were no significant differences in the number and size of S. hermonthica individuals recovered from either transgenic or wild type roots. Conclusions This rapid, high throughput, transformation technique will advance our understanding of gene function in parasitic plant-host interactions

Genetically engineered East African highland bananas – proximate analysis and effect of cooking on the enhanced provitamin A levels

Micronutrient deficiency is a major challenge in the developing world. This is mainly attributed to over-reliance on starchy staples foods such as cassava, rice and banana among others, which are deficient in micronutrients such as vitamin A and iron. Strategies put in place to provide a solution to micronutrient deficiencies such as dietary supplementation of vitamin A and food fortification have not been successful in the developing world due to high costs and unreliable supply chains (food and medical). Biofortification of the easily accessible staple foods could help reduce this problem associated with micronutrient deficiency. On this account, the Biofortification project in Uganda under the National Banana Program developed transgenic East African Highland Bananas (EAHBs) (M9 and Nakitembe) with enhanced levels of provitamin A (PVA) using the Fe’i banana-derived phytoene synthase 2a (MtPsy2a) gene. To determine the nutritional quality of the transgenic bananas, an analysis of the proximate composition of the biofortified East African Highland Bananas was carried out. The effect of cooking on retention of provitamin A carotenoids (pVAC), was assessed using two cooking methods; boiling and steaming (most common methods of preparing cooking banana meals in Uganda). It was observed that there were no significant (P≤0.05) differences in moisture content (P=0.4287), carbohydrate (P=0.3966), crude fat (P=0.4051), crude fiber (P=0.3214), protein (P=0.0858) and ash content P=0.1336) between transgenic and non-transformed bananas. It was found that steaming, as a cooking method allowed for retention of more provitamin A carotenoids compared to boiling. Comparison of the cultivars on their retention of provitamin A carotenoids, results indicated that Nakitembe was superior to M9. Genetic engineering of bananas by biofortification has no effect on major food components in EAHBs (M9 and Nakitembe) and, therefore, genetically modified M9 and Nakitembe are substantially equivalent to the non-transgenic controls and the biofortified bananas can provide the necessary nutrients even after cooking. This  data will inform subsequent steps for the commercialization of biofortified EAHBs

RECOVERY OF amiRNA3-PARP1 TRANSGENIC MAIZE PLANTS USING A BINARY VECTOR HAVING THE BIOSAFE PMI GENE

Positive plant selectable marker genes are commonly used in plant transformation because they not only enhance the frequency of generation transgenic tissues but are considered biosafe, unlike antibiotic or herbicide resistance genes. In this study, the binary vector pNOV2819-ubiamiRNA3PARP1, harbouring the phosphomannose isomerase (pmi) gene was developed and used in recovery of transgenic maize ( Zea mays L.) plants containing the drought tolerance gene, amiRNA3-PARP1. The pre-amiRNA3-PARP1 and Tnos transgenes were sequentially PCR-cloned upstream the ubiquitine promoter in the Ubi/NC1300 plasmid. The pre-amiRNA3-PARP1 expression cassette was transferred into the pmi gene-containing pNOV2819 plasmid to produce the pNOV2819-ubiamiRNA3PARP1 vector. Transgenic IL3 and A188 plants containing pre-amiRNA3-PARP1 were generated through transformation with LBA4404 harbouring the pNOV2819-ubiamiRNA3PARP1 vector. The plants were confirmed transgenic by PCR. It is clear that the developed vectors are effective in recovery of amiRNA3-PARP1 transgenic tissues and plants containing the pmi gene, which has been shown to have no negative environmental or health effects.Les marqueurs g\ue9n\ue9tiques de s\ue9lection positive de plantes sont commun\ue9ment utilis\ue9s dans la transformation des plantes parce que, non seulement ils augmentent la fr\ue9quence de la g\ue9n\ue9ration des tissus transg\ue9niques, mais aussi sont consid\ue9r\ue9s comme biosains, \ue0 l\u2019inverse des g\ue8nes de r\ue9sistance aux antibiotiques et herbicides. Dans cette \ue9tude, le vecteur binaire pNOV2819-ubiamiRNA3PARP1 portant le g\ue8ne isom\ue9rase phosphomannose (pmi) a \ue9t\ue9 d\ue9velopp\ue9 et utilis\ue9 dans le recouvrement transg\ue9nique des plantes du ma\uefs ( Zea mays L.) contenant le g\ue8ne to tol\ue9rance \ue0 la s\ue9cheresse amiRNA3-PARP1. Les transg\ue8nes pre-amiRNA3-PARP1 et Tnos \ue9taient s\ue9quentiellement clon\ue9s par PCR dans la partie sup\ue9rieure du promoteur ubiquitine dans le plasmide Ubi/NC1300. L\u2019expression de la cassette de la pr\ue9-amiRNA3-PARP1 \ue9tait transf\ue9r\ue9e dans le g\ue8ne pmi contenant le plasmide pNOV2819 pour produire le vecteur pNOV2819-ubiamiRNA3PARP1. Les plants transg\ue9niques IL3 et A188 contenant le pre-amiRNA3-PARP1 \ue9taient g\ue9n\ue9r\ue9s \ue0 travers la transformation avec LBA4404 portant le vecteur pNOV2819-ubiamiRNA3PARP1. Les plants \ue9taient confirm\ue9es transg\ue9niques par PCR. Il est clair que les vecteurs d\ue9velopp\ue9s sont efficaces dans le recouvrement des tissus transg\ue9niques amiRNA3-PARP1 et les plants contenant le g\ue8ne pmi qui ne pr\ue9sentent aucun effet n\ue9gatif sur l\u2019environnement et la sant\ue9

Disseminated tuberculosis presenting with polymorphonuclear effusion and septic shock in an HIV-seropositive patient: a case report

<p>Abstract</p> <p>Introduction</p> <p>Because a substantial number of patients present with few or atypical symptoms, the recognition of tuberculosis remains challenging. Disseminated tuberculosis presenting with septic shock has already been described in some case reports, but, to the best of our knowledge, it has never been associated with polymorphonuclear effusion.</p> <p>Case presentation</p> <p>We describe the case of a 27-year-old man from western Africa who was seropositive for human immunodeficiency virus. He presented with pleural and abdominal polymorphonuclear effusions and quickly developed septic shock due to disseminated <it>Mycobacterium tuberculosis </it>infection leading to multiple organ failure and death.</p> <p>Conclusion</p> <p>In high-risk patients, <it>Mycobacterium tuberculosis </it>infection should be considered even in exceptional clinical presentations, such as septic shock and polymorphonuclear effusions.</p

Inhibition of SOC/Ca2+/NFAT pathway is involved in the anti-proliferative effect of sildenafil on pulmonary artery smooth muscle cells

<p>Abstract</p> <p>Background</p> <p>Sildenafil, a potent phosphodiesterase type 5 (PDE5) inhibitor, has been proposed as a treatment for pulmonary arterial hypertension (PAH). The mechanism of its anti-proliferative effect on pulmonary artery smooth muscle cells (PASMC) is unclear. Nuclear translocation of nuclear factor of activated T-cells (NFAT) is thought to be involved in PASMC proliferation and PAH. Increase in cytosolic free [Ca²⁺] ([Ca²⁺]_i) is a prerequisite for NFAT nuclear translocation. Elevated [Ca²⁺]_iin PASMC of PAH patients has been demonstrated through up-regulation of store-operated Ca²⁺channels (SOC) which is encoded by the transient receptor potential (TRP) channel protein. Thus we investigated if: 1) up-regulation of TRPC1 channel expression which induces enhancement of SOC-mediated Ca²⁺influx and increase in [Ca²⁺]_iis involved in hypoxia-induced PASMC proliferation; 2) hypoxia-induced promotion of [Ca²⁺]_ileads to nuclear translocation of NFAT and regulates PASMC proliferation and TRPC1 expression; 3) the anti-proliferative effect of sildenafil is mediated by inhibition of this SOC/Ca²⁺/NFAT pathway.</p> <p>Methods</p> <p>Human PASMC were cultured under hypoxia (3% O₂) with or without sildenafil treatment for 72 h. Cell number and cell viability were determined with a hemocytometer and MTT assay respectively. [Ca²⁺]_iwas measured with a dynamic digital Ca²⁺imaging system by loading PASMC with fura 2-AM. TRPC1 mRNA and protein level were detected by RT-PCR and Western blotting respectively. Nuclear translocation of NFAT was determined by immunofluoresence microscopy.</p> <p>Results</p> <p>Hypoxia induced PASMC proliferation with increases in basal [Ca²⁺]_iand Ca²⁺entry via SOC (SOCE). These were accompanied by up-regulation of TRPC1 gene and protein expression in PASMC. NFAT nuclear translocation was significantly enhanced by hypoxia, which was dependent on SOCE and sensitive to SOC inhibitor SKF96365 (SKF), as well as cGMP analogue, 8-brom-cGMP. Hypoxia-induced PASMC proliferation and TRPC1 up-regulation were inhibited by SKF and NFAT blocker (VIVIT and Cyclosporin A). Sildenafil treatment ameliorated hypoxia-induced PASMC proliferation and attenuated hypoxia-induced enhancement of basal [Ca²⁺]_i, SOCE, up-regulation of TRPC1 expression, and NFAT nuclear translocation.</p> <p>Conclusion</p> <p>The SOC/Ca²⁺/NFAT pathway is, at least in part, a downstream mediator for the anti-proliferative effect of sildenafil, and may have therapeutic potential for PAH treatment.</p

Phosphodiesterase type 4 expression and anti-proliferative effects in human pulmonary artery smooth muscle cells

BACKGROUND: Pulmonary arterial hypertension is a proliferative vascular disease, characterized by aberrant regulation of smooth muscle cell proliferation and apoptosis in distal pulmonary arteries. Prostacyclin (PGI(2)) analogues have anti-proliferative effects on distal human pulmonary artery smooth muscle cells (PASMCs), which are dependent on intracellular cAMP stimulation. We therefore sought to investigate the involvement of the main cAMP-specific enzymes, phosphodiesterase type 4 (PDE4), responsible for cAMP hydrolysis. METHODS: Distal human PASMCs were derived from pulmonary arteries by explant culture (n = 14, passage 3–12). Responses to platelet-derived growth factor-BB (5–10 ng/ml), serum, PGI(2 )analogues (cicaprost, iloprost) and PDE4 inhibitors (roflumilast, rolipram, cilomilast) were determined by measuring cAMP phosphodiesterase activity, intracellular cAMP levels, DNA synthesis, apoptosis (as measured by DNA fragmentation and nuclear condensation) and matrix metalloproteinase-2 and -9 (MMP-2, MMP-9) production. RESULTS: Expression of all four PDE4A-D genes was detected in PASMC isolates. PDE4 contributed to the main proportion (35.9 ± 2.3%, n = 5) of cAMP-specific hydrolytic activity demonstrated in PASMCs, compared to PDE3 (21.5 ± 2.5%), PDE2 (15.8 ± 3.4%) or PDE1 activity (14.5 ± 4.2%). Intracellular cAMP levels were increased by PGI(2 )analogues and further elevated in cells co-treated with roflumilast, rolipram and cilomilast. DNA synthesis was attenuated by 1 μM roflumilast (49 ± 6% inhibition), rolipram (37 ± 6%) and cilomilast (30 ± 4%) and, in the presence of 5 nM cicaprost, these compounds exhibited EC(50 )values of 4.4 (2.6–6.1) nM (Mean and 95% confidence interval), 59 (36–83) nM and 97 (66–130) nM respectively. Roflumilast attenuated cell proliferation and gelatinase (MMP-2 and MMP-9) production and promoted the anti-proliferative effects of PGI(2 )analogues. The cAMP activators iloprost and forskolin also induced apoptosis, whereas roflumilast had no significant effect. CONCLUSION: PDE4 enzymes are expressed in distal human PASMCs and the effects of cAMP-stimulating agents on DNA synthesis, proliferation and MMP production is dependent, at least in part, on PDE4 activity. PDE4 inhibition may provide greater control of cAMP-mediated anti-proliferative effects in human PASMCs and therefore could prove useful as an additional therapy for pulmonary arterial hypertension

Impact of external factors on the size of investment in private forest companies in Poland

The article presents an analysis of selected external factors influencing the level of investment in the private forestry companies providing services to the State Forests. The scope of research included: the characteristics of forest companies and competition in the forestry services market, the investments being undertaken by these companies, as well as the external conditions for the activities of forest companies and their impact on the level of investment. The data on the financial performance of the administration of forest districts and the size of their tasks in the field of timber harvest was obtained from the General Directorate of the State Forests in Warsaw, while information in the field of competitiveness on the forestry labour market was gained from the materials provided by the Forest Research Institute in Sękocin Stary. The data necessary to analyse the influence of the selected factors on the level of investment in private forestry companies were obtained from the questionnaire survey conducted at the turn of 2006 and 2007. The survey concerned the financial and investment situation of the companies in the period of 2004−2006 and the strategy for further action. Out of all the companies working for the State Forests, 1,009 companies responded to the questionnaire, of which, in 2008, 269 companies were selected that showed the level of investments in excess of 5%. The questionnaire addressed to forest entrepreneurs concerned the assessment of the services market, competition, investment efficiency, as well as the motives which guided the entrepreneurs in making investment decisions. Nine external factors related to the recipients of the services (forest districts), barriers to the introduction of modern technical means, the situation on the labour market and competition in the forestry services market were subjected to statistical verification. The completed investigations have shown no effect on the level of investment of the factors directly related to the main recipient of their services – forest districts, i.e. the financial result of the forest districts, the length of the period for which forest districts intended to hire forest companies, and the size of the demand (the volume of harvested timber). The investigations have demonstrated a link between the companies' problems with the recruitment of qualified staff and the level of investments neither. There was no confirmation either of the effect of the competition on the forestry services market on the level of investment. The low rates for the services and the high prices of the specialist equipment were the only external factors that had a major influence on the amount of resources allocated for investments in private forest companies

Organizational information creation through a design game : A sensemaking perspective

Information creation taking place in the use of an organizational design card game, Topaasia® is discussed. Using 18 video-recorded play sessions, the researchers analyzed the ways in which players make propositions based on cards and raise points or further development in the organization, as well as discuss contextual factors. The sessions were analyzed on topical turn-taking and by using the Systems Intelligence Inventory. Through these processes, the study shows the importance of breaking organizational communication genre conventions for the goal of revealing tacit information and for the creation of new information. The research thereby contributes to studies of organizational information creation and sensemaking.publishedVersionPeer reviewe

KSTP 94, an open-pollinated maize variety has postattachment resistance to purple witchweed (Striga hermonthica)

Striga spp. are obligate root hemiparasites that constrain cereal production in sub-Saharan Africa. Although purple witchweed [ Striga hermonthica (Delile) Benth.] and Asiatic witchweed [ Striga asiatica (L.) Kuntze] infect all cereal crops, maize ( Zea mays L.) is particularly vulnerable to their infestations. A sustainable control strategy for Striga would be to breed crops with host-based resistance as part of an integrated management plan. In maize, the open-pollinated variety Kakamega Striga -tolerant population of the year 1994 (‘KSTP 94’) has been popularized as a Striga -tolerant/resistant variety. This resistance was earlier reported to result from production of low amounts of sorgomol, a less potent strigolactone. To determine whether KSTP 94 harbors postattachment resistance, we used a soil-free assay based on observation chambers called rhizotrons. We found that the size of Striga seedlings attached to ‘CML 144’ (a susceptible maize inbred line) were 2.5-fold longer than those on KSTP 94. In addition, KSTP 94 had significantly fewer Striga attachments, which corresponded to significantly lower biomass (2.6-fold) compared with CML 144. Histological analysis revealed that the low Striga growth and development while infecting KSTP 94 was due the parasite’s inability to penetrate the host’s endodermis and make effective xylem–xylem connections. We therefore conclude that in addition to preattachment resistance, KSTP 94 exhibits postattachment resistance to S. hermonthica and could therefore be a good genetic source for postattachment resistance breeding