Metabolomics-Based Study of Logarithmic and Stationary Phases of Promastigotes in Leishmania major by 1H NMR Spectroscopy

Background:

Cutaneous leishmaniasis is one of the most important parasitic diseases in humans. In this disease, one of the responsible organisms is Leishmania major, which is transmitted by sandfly vector. There are specific differences in biochemical profiles and metabolite pathways in logarithmic and stationary phases of Leishmania parasites. In the present study, ¹H NMR spectroscopy was used to examine the metabolites outliers in the logarithmic and stationary phases of promastigotes in L. major to enlighten more about the transmission mechanism in metacyclogenesis of L. major.

Methods:

Promastigote was cultured, logarithmic and stationary phases were separated by the peanut agglutinin, and cell metabolites were extracted. ¹H NMR spectroscopy was applied, and outliers were analyzed using principal component analysis.

Results:

The most altered metabolites in stationary and logarithmic phases were limited to citraconic acid, isopropylmalic acid, L-leucine, ornithine, caprylic acid, capric acid, and acetic acid.

Conclusion:

¹H NMR spectroscopy could play an important role in the characterization of metabolites in biochemical pathways during a metacyclogenesis process. These metabolites and their pathways can help in exploiting a transmission mechanism in metacyclogenesis, and outcoming data might be used in the metabolic network reconstruction of L. major modeling. Key Words: Leishmania major, Metabolomics, Principal component analysis     

Effect of salinity and arbuscular mycorrhizal fungi on growth and some physiological parameters of Citrus jambheri

Arbuscular mycorrhizal (AM) fungi alleviate the unfavorable effects of salinity stress on plant growth. A pot study was conducted to determine the effects of AM fungi and salt on growth and some physiological parameters of Citrus jambheri rootstock. Four levels of salinity (2, 4, 6, and 8 dS m^?1 as NaCl) and three mycorrhizal treatments (Glomus etunicatum, Glomus intraradices and non-mycorrhizal (NM) control) were used. As salinity increased, all measured characteristics of plants after 4.5-month growth except Na uptake, proline content, and electrolyte leakage decreased. Shoot dry weight and K uptake were significantly higher in G. intraradices-colonized seedlings than NM controls at all salinity levels. Root dry weight and shoot P uptake were significantly higher in G. etunicatum-colonized seedlings than NM controls at all salinity levels. G. intraradices-colonized seedlings had significantly higher proline content than NM controls and G. etunicatum-colonized seedlings at salinity levels of 4, 6 and 8 dS m^?1. The electrolyte leakage percentage was significantly lower in G. intraradices-colonized seedlings than NM controls at all salinity levels. The data demonstrated that mycorrhizal citrus seedlings exhibited greater tolerance to salt stress than NM seedlings and the enhanced proline content seems to be one of the mechanisms involved.     

Approaches for developing molecular markers associated with virus resistances in potato (Solanum tuberosum)

Journal of Plant Diseases and Protection - Potato, (Solanum tuberosum), is one of the most common and important food sources for humans. It is not only essential as a food source but also has many...     

Targeted Deletion of Los1 Homologue Affects the Production of a Recombinant Model Protein in Pichia pastoris

Background:The methylotrophic yeast Pichia pastoris is an appealing production host for a variety of recombinant proteins, including biologics. In this sense, various genetic- and non-genetic-based techniques have been implemented to improve the production efficiency of this expression platform. Los1 (loss of supression) encodes a non-essential nuclear tRNA exporter in Saccharomyces cerevisiae, which its deletion extends RLS. Herein, a los1-deficient strain of P. pastoris was generated and characterized. Methods:A gene disruption cassette was prepared and transformed into an anti-CD22-expressing strain of P. pastoris. A δ los1 mutant was isolated and confirmed. The drug sensitivity of the mutant was also assessed. The growth pattern and the level of anti-CD22 ScFv expression were compared between the parent and mutant strains.Results:The los1 homologue was found to be a non-essential gene in P. pastoris. Furthermore, the susceptibility of los1 deletion strain to protein synthesis inhibitors was altered. This strain showed an approximately 1.85-fold increase in the extracellular level of anti-CD22 scFv (p < 0.05). The maximum concentrations of total proteins secreted by δ los1 and parent strains were 125 mg/L and 68 mg/L, respectively.Conclusion:The presented data suggest that the targeted disruption of los1 homologue in P. pastoris can result in a higher expression level of our target protein. Findings of this study may improve the current strategies used in optimizing the productivity of recombinant P. pastoris strains. Key Words: Aging, Longevity, Pichia pastoris, Recombinant proteins     

Transplanting P75-Suppressed Bone Marrow Stromal Cells Promotes Functional Behavior in a Rat Model of Spinal Cord Injury

Background: Bone marrow stromal cells (BMSC) have been successfully employed for movement deficit recovery in spinal cord injury (SCI) rat models. One of the unsettled problems in cell transplantation is the relative high proportion of cell death, specifically after neural differentiation. According to our previous studies, p75 receptor, known as the death receptor, is only expressed in BMSC in a time window of 6-12 hours following neural induction. Moreover, we have recently reported a decreased level of apoptosis in p75-suppressed BMSC in vitro. Therefore, our objective in this research was to explore the functional effects of transplanting p75:siRNA expressing BMSC in SCI rats. Methods: Laminectomy was performed at L1 vertebra level to expose spinal cord for contusion using weight-drop method. PBS-treated SCI rats (group one) were used as negative controls, in which cavitations were observed 10 weeks after SCI. pRNA-U6.1/Hygro- (group two, as a mock) and pRNA-U6.1/Hygro-p75 shRNA- (group three) transfected BMSC were labeled with a fluorescent dye, CM-DiI, and grafted into the lesion site 7 days after surgery. The Basso-Beattie-Bresnehan locomotor rating scale was performed weekly for 10 weeks. Results: There was a significant difference (P≤0.05) between all groups of treated rats regarding functional recovery. Specifically, the discrepancy among p75 siRNA and mock-transfected BMSC was statistically significant. P75 siRNA BMSC also revealed a higher level of in vivo survival compared to the mock BMSC. Conclusion: Our data suggest that genetically modified BMSC that express p75:siRNA could be a more suitable source of cells for treatment of SCI. Key Words: Spinal cord injury (SCI), Apoptosis, Bone marrow stromal cells (BMSC)     

Genetic identity and relationships of Iranian apple (Malus?×?domestica Borkh.) cultivars and landraces, wild Malus species and representative old apple cultivars based on simple sequence repeat (SSR) marker analysis

In order to shed light on the role of Iran in apple evolution and domestication, we chose to investigate the relationships of a collection of 159 accessions of wild and domesticated apples including Iranian indigenous apple cultivars and landraces, selected wild species, and old apple scion and rootstock cultivars from different parts of the world. The majority of the wild species belonged to M. sieversii, which is widely believed to be the main maternal wild ancestor of domestic apples, from Kazakhstan and M. orientalis, which is one of the probable minor ancestors of domestic apples, from Turkey and Russia located on the east and west of Iran, respectively. The accessions were assigned into six arbitrary populations for the purpose of generating information on genetic parameters. Nine simple sequence repeat (SSR) loci selected from previous studies in apple were screened over DNA extracted from all the accessions. Results showed that all SSR loci displayed a very high degree of polymorphism with 11–25 alleles per locus. In total, there were 153 alleles across all loci with an average of 17 alleles per locus. The SSR allelic data were then used for estimation of population genetic parameters, including genetic variation statistics, F-statistics, gene flow, genetic identity, genetic distance and then cluster analysis using POPGENE 1.32 software. The F-statistics and gene flow in particular, showed that there was more intra-population than between population variation. The genetic identity and genetic distance estimates, and the dendrogram generated from the un-weighted pair group arithmetic average (UPGMA) method of cluster analysis showed that the Iranian cultivars and landraces were more closely related to M. sieversii from Central Asia (east of Iran) and M. orientalis native to Turkey and Russia than to other accessions of Malus species. Also, the old apple cultivars from different parts of the world have a closer genetic relationship to M. sieversii, M. orientalis and the Iranian apples, than to other wild species. Based on these results, we suggest that the Iranian apples may occupy an intermediate position between the domesticated varieties and wild species. We propose that Iran could be one of the major players in apples’ domestication and transfer from Central Asia to the western countries.     

Influence of superabsorbent polymer rates on growth of saffron replacement corms

Mother corm size is the most important factor for production of replacement corms, flower, and stigma yield in saffron. In semi-arid regions, water shortage is a deterrent agent for appropriate corm growth of saffron. Therefore, in this experiment the effects of six levels of superabsorbent polymer (SAP) including 0, 10, 20, 30, 40, and 50 kg ha^-1 was investigated on growth indices of replacement corms of saffron. For this purpose, an experiment was evaluated at the research farm of the Saffron Research Group, Sarayan Faculty of Agriculture, University of Birjand, Iran, during 2013-2015. Results showed that SAP application did not considerable effect on corm growth indices in the first life cycle of saffron. Nevertheless, consumption of 30-40 kg ha^-1 SAP somewhat improved the number and total weight of replacement corms per clone. In the second life cycle of saffron, the positive impact of SAP was clearly observed on the growth of replacement corms. The application of 40 kg ha^-1 SAP increased the amount of number of replacement corms per clone by 13%, total weight of replacement corms per clone by 36%, and scale weight of corms per clone by 50% compared with control. In addition, the amount of mean weight of replacement corms and mean number of buds per corm in treatment of application of 40 kg ha^-1 SAP were 29 and 27% higher than the control treatment, respectively. SAP application decreased the amount of non-standard (< 8 g) corm production while significantly increased the percentage of corms with standard weight. Overall, the increasing effect of SAP application on corm weight, bud number, and standard corm yield was higher than the number of produced corms per clone. In addition, water-use efficiency in SAP application treatments (6.1 kg standard corm m^-3) was more than the control (4.34 kg m^-3) treatment. In total, SAP application is an appropriate strategy for production of standard saffron corms in semi-arid regions.     

Changes of vitamins A, E, and C and lipid peroxidation status of breeding and pregnant sheep during dry seasons on medium-to-low quality forages

This study assessed the changes of plasma vitamin A, E, and C and the lipid peroxidation status of sheep during breeding and pregnancy under drought conditions. The study was conducted on 105 cross-bred fat tailed ewes, 3–5 years old with body condition scores (BCS) of 2.5 to 3.5. The ewes were grazing on medium-to-low quality forages during summer and low quality forages within the succeeding months and had ad libitum access to a mixture of alfalfa hay (40%) and wheat straw (60%) in the afternoons. From 3 weeks before breeding till 1 month after the introduction of rams, 300 g of barley grain/head/day was offered to the ewes and then the supplemental grain was reduced to 100 g/head/day. For better synchronization of estrus cycles in ewes, they were isolated from the rams for at least 2 months and then kept in close proximity of the rams for 1 week before the introduction of the rams to the ewe flock. Then, whole blood samples were collected on days 1, 7, 21, and 120 after ram introduction. Vitamins A, E, and C were measured in plasma. Malondialdehyde (MDA) was measured in the hemolysate as a biomarker of lipid peroxidation. Plasma progesterone (P4) was measured in the samples of day 120 for assessing pregnancy status of the ewes. Vitamins A and C showed continuous and significant declines (P < 0.05) through days 1 to 120. Vitamin E declined only during the first 21 days of the study and remained almost constant till day 120. MDA concentration increased significantly at day 21. An elevated concentration of MDA was also detected at day 120. The difference between days 21 and 120 was not significant (P > 0.05). A positive correlation between vitamins E and C was detected at day 120 (r = 0.349, P < 0.01). Age and BCS did not affect the patterns of changes. Assuming that the ewes with P4 concentrations ≥2.5 ng/ml were pregnant, 95 out of 105 ewes (90.5%) were pregnant at day 120 of the study. Under the conditions of the present study with medium-to-low quality pastures as the main sources of feed, ewes of various ages and body conditions may suffer from oxidative stress during breeding and pregnancy.     

Fumigant toxicity of Carum copticum and Vitex pseudo-negundo essential oils against eggs, larvae and adults of Callosobruchus maculatus

Fumigant activity of essential oil vapors distilled from Carum copticum C. B. Clarke and Vitex pseudo-negundo Hand I. MZT. was tested against eggs, larvae and adults of Callosobruchus maculatus (F.). Fumigant toxicity was assessed at 27 ± 1°C and 60 ± 5% RH, in dark condition. The influence of different concentrations of the essential oil vapors on egg hatchability, larval and adult mortality was significant. Data probit analysis showed that lethal concentration of the essential oil to kill 50% of the population (LC₅₀) for egg, larvae and adult were found to be 1.01, 2.50 and 0.90 μl/l air of C. copticum oil, followed by 2.20, 8.42 and 9.39 μl/l air essential oil of V. pseudo-negundo, respectively. Between these essential oils, C. copticum was almost more toxic than V. pseudo-negundo on all growth stages of C. maculatus. The present study suggests that essential oils from these medicinal plants may be potential grain protectants as botanical alternative fumigants and could be used in the management of various life stages of C. maculatus.     

The selection of superior walnut (<Emphasis Type="Italic">Juglans regia</Emphasis> L.) genotypes as revealed by morphological characterization

Walnut (Juglans regia L.), is one of the main economic fruit species in the world and is rich in nutritious substances. In order to obtain superior walnut genotypes, a set of seedling walnut genotypes were evaluated and their results were recorded. The range of nut length was between 25.57 and 47.77 mm. Also, the ranges of nut weight, kernel weight and kernel percentage varied from 5.35 to 21.31 g, 2.49 to 11.15 g and 37.27 to 66.29%, respectively. Simple correlation analysis showed significant positive and negative correlations between some of the measured traits. Principal component analysis reduced the evaluated traits into the seven main components, which explained 71.71% of the total variance. Bi-plot analysis performed using the first two components, distributed the genotypes into four sides of the plot. Based on the present results, a high variation was observed among the trees, indicating a high potential of the studied germplasm to select the superior genotypes. According to the present research, 57 genotypes with having the highest nut weight and kernel weight, as well as bright color of the kernel, were recognized as superior genotypes and can be used to establish the new orchards or improve cultivars.