Quality Control of Widely Used Therapeutic Recombinant Proteins by a Novel Real-Time PCR Approach

Background:

Existence of bacterial host-cell DNA contamination in biopharmaceutical products is a potential risk factor for patients receiving these drugs. Hence, the quantity of contamination must be controlled under the regulatory standards. Although different methods such as hybridization assays have been employed to determine DNA impurities, these methods are labor intensive and rather expensive. In this study, a rapid real-time PCR test was served as a method of choice to quantify the E. coli host- cell DNA contamination in widely used recombinant streptokinase (rSK), and alpha interferon (IFN-α) preparations.

Methods:

A specific primer pair was designed to amplify a sequence inside the E. coli 16S rRNA gene. Serial dilutions of DNA extracted from E. coli host cells, along with DNA extracted from Active Pharmaceutical Ingredients of rSK, and IFN-α samples were subjected to an optimized real-time PCR assay based on SYBR Green chemistry.

Results:

The test enabled us to detect a small quantity of genomic DNA contamination as low as 0.0002 pg in recombinant protein-based drugs. For the first time, this study showed that DNA contamination in rSK and IFN-α preparation manufactured in Pasteur Institute of Iran is much lower than the safety limit suggested by the US FDA.

Conclusion:

Real-time PCR is a reliable test for rapid detection of host-cell DNA contamination, which is a major impurity of therapeutic recombinant proteins to keep manufacturers’ minds on refining drugs, and provides consumers with safer biopharmaceuticals. Key Words: DNA contamination, Real-time PCR, Streptokinase, Interferon-alpha (IFN-α)     

Organo-silane compounds in medium density fiberboard:physical and mechanical properties

We studied the effects of nanoparticles of organo-silane(NOS) compounds in the size range of20–80 nm on physical and mechanical properties in medium density fiberboard,and used NOS at four consumption levels of 0,50,100,and 150 g kg-1dry wood fibers.Density of all treatments was kept constant at 0.67 g cm-3.The water-repellent property of organo-silane significantly reduced water absorption(WA) and thickness swelling but mechanical properties declined due to the reduced proportion of wood-fiber as organo-silane was added to the matrix:the compression ratio of MDF panels and the integrity among wood-fibers both declined,resulting in reduced mechanical properties.We recommend use of 50 g of NOS/kg wood-fiber to improve WA and thickness swelling while retaining acceptable mechanical properties.     

EFFECTS OF WATER SALINITY ON GROWTH INDICES AND PHYSIOLOGICAL PARAMETERS IN SOME PISTACHIO ROOTSTOCKS

Pistachio is one of the most important horticultural crops in Iran. The majority of the pistachio producing regions is located in arid and semi-arid areas with saline conditions. Therefore, selection of suitable rootstocks is important for increasing yield efficiency of this important nut crop. In this study, the effect of four water salinity levels (0.75, 5, 10 and 15 ds m^?1) on growth indices and physiological parameters of four Pistacia vera L. rootstocks (Badami-e-Zarand A, Badami-e-Zarand B, Qazvini, and Sarakhs) were investigated under greenhouse conditions. After treatment for three months, leaf dry weight was reduced by about 30-50% at an irrigation water electrical conductivity (EC_w) of 10 ds m^?1. Badami-e-Zarand B was the most vigorous rootstock at the highest EC. Decreases in root and stem dry weight (average of all rootstocks combined) occurred at water salinity of 10 ds m^?1. Chemical analysis of shoot and root indicated that the salinity affected the concentration and distribution of sodium (Na⁺), potassium (K⁺), and calcium (Ca²⁺) in pistachio rootstocks. The concentrations of Na⁺ and K⁺ increased with a rise in water salinity levels. Comparison between Na⁺ concentration of shoot and root showed that all examined rootstocks limited the Na⁺ transportation to shoot tissue up-to 15 ds m^-1, and retained it in the roots. However, this ability was less in the Sarakhs rootstock. Based on measured parameters, Badami-e-Zarand B and Sarakhs could be considered as tolerant and sensitive pistachio rootstocks to water salinity, respectively.     

Effects of Artificial Pollination Using Pollen Suspension Spray on Nut and Kernel Quality of Pistachio Cultivar Owhadi

Yield and nut quality in pistachio are highly dependent on cultural practices especially pollination. This study was conducted to investigate the effects of pollen suspension spray containing 0.01% and 0.02% boric acid combined with 1% (w/v) pollen on nut and kernel quality of pistachio (Pistacia vera L. cv. Owhadi). The experiment was carried out as RCBD with three replications over 2011–12 period. The results showed that application of medium containing agar (A) and 0.01% boric acid (B1) combined with 1% (w/v) pollen decreased fruit set in both years compared to control whereas nut splitting was increased during 2012. Pollen suspension sprays unaffected nut and kernel dimensions in both years but increased kernel proteins and decreased oil during 2011. According to this study, it could be postulated that artificial pollination in pistachio using pollen suspension spray is possible.     

Effect of different concentrations of ginger root powder and its essential oil on growth performance,serum metabolites and antioxidant status in broiler chicks under heat stress

1. This study was carried out to evaluate the impact of ginger (Zingiber officinale) feed supplementation on growth performance, antioxidant status, carcass characteristics and blood parameters in broiler chicks under conditions of heat stress (32 ± 2ºC for 8 h per d).

2. A total of 336 d-old male broiler chicks (Cobb-500) were randomly assigned to one of 6 dietary groups representing: basal diet with no supplement as control, basal diet containing 100 mg/kg vitamin E as positive control, basal diets containing either 7.5 or 15 g/kg of ginger root powder, and diets containing 75 or 150 mg/kg of ginger essential oil.

3. The results indicated that at 22 d of age, the group receiving 7.5 g/kg of ginger root powder experienced significantly increased body weight (BW) and body weight gain (BWG) compared to the control group. There were no significant difference among the diet groups regarding BW, BWG, feed intake (FI) or feed conversion ratio (FCR) at 42 and 49 d of age.

4. The inclusion of powder and essential oil of ginger in broiler diets did not affect carcass characteristics and blood parameters of the chickens. However, in the group receiving 150 mg/kg ginger essential oil, the total superoxide dismutase (TSOD) activity in liver increased compared to the control group. Malondialdehyde (MDA) concentrations in liver also decreased in the groups receiving ginger powder and essential oil compared to that in the control group. There were no significant difference between experimental groups regarding glutathione peroxidise (Gpx), TSOD and catalase (CAT) enzymes in red blood cells. All dietary groups increased total antioxidant capacity (TAC) and decreased MDA concentration in serum compared to the control group.

5. The results of this study suggest that ginger powder and essential oils may be a suitable replacement for synthetic antioxidants in broiler diets. Results also suggest that ginger powder might be better than extracted essential oil for improving antioxidant status in broilers.     

Effect of Piriformospora indica and Azospirillum strains from saline or non-saline soil on mitigation of the effects of NaCl

Salinity toxicity is a worldwide agricultural and eco-environmental problem. The intent of this study was to determine the salt tolerance of Piriformospora indica and strains of Azospirillum, isolated from non-saline and saline soil, as well as to determine their affect on the tolerance of wheat to soil salinity. In this study, an experiment was conducted to investigate the salt stress tolerance abilities of the endophytic fungi, P. indica, and Azospirillum strains, isolated from non-saline and saline soil, at five NaCl levels (0, 0.1, 0.2, 0.3, 0.4, 0.5 mol L^?1). Additionally, a greenhouse experiment was conducted to test the effects of these selected microorganisms under increasing salinity levels on seedling growth, solute accumulation (proline and sugars), and photosynthetic pigments (Chl a, b, ab) of seedling wheat. Azospirillum strains were isolated in Iran from the root of field-grown maize from non-saline soil with an EC = 0.7 dS m^?1 and from saline soil with an EC = 4.7 dS m^?1. Plants were irrigated with non-saline water–tap water with an electrical conductivity water (ECw) value of 0.2 dS m^?1, as well as low, moderate and severe saline water-irrigation with saline water with an ECw of 4 dS m^?1, 8 dS m^?1 and 12 dS m^?1, respectively. The upper threshold of P. indica salinity tolerance was 0.4 mol L^?1 NaCl in both liquid and solid broth medium. The upper thresholds of the salt adapted and non-adapted Azospirillum strains were 0.2 and 0.4 mol L^?1 NaCl, respectively. The results indicated a positive influence of the organisms on salinity tolerance, more with the saline-adapted Azospirillum strains than the non-adapted strains. P. indica was more effective than the Azospirillum strains. These results could be related to a better water status, higher photosynthetic pigment contents and proline accumulation in wheat seedlings inoculated with P. indica. The benefits of both isolates and P. indica depended on two factors: water salinity and growth stage of the host plant. Inoculation with the two isolates increased salinity tolerance of wheat plants; the saline-adapted Azospirillum strains showed better performance with respect to improved fresh and dry weights at 80 and 100 days after sowing under both non-saline and saline conditions. When compared to plants inoculated with non-saline-adapted Azospirillum strains, those inoculated with adapted Azospirillum strains had much better performance with respect to the presence of photosynthetic pigment (Chl a, b and ab) and proline accumulation. Overall, these results indicate that the symbiotic association between P. indica fungus and wheat plants improved wheat growth, regardless of the salinity. It is concluded that the mechanisms for protecting plants from the detrimental effects of salinity by P. indica fungus and Azospirillum strains may differ in their salinity tolerance and influence the uptake of water, photosynthetic pigment contents and proline accumulation in wheat seedlings.     

Including 793/B type avian infectious bronchitis vaccine in 1-day-old chicken increased the protection against QX genotype

Tropical Animal Health and Production - Infectious bronchitis virus (IBV) is a highly infectious pathogen, which affects the respiratory tract, reproductive system, and kidney of chickens. Many...     

Vulnerability and Adaptation of Livestock Producers to Climate Variability and Change

Rangeland-based animal husbandry has been frequently exposed to the vagaries of weather. While the current rates of climate variability are unprecedented in arid and semiarid regions, climate change is expected to put further pressure on rangelands with medium- and poor-quality forages and increase the vulnerability of households who mainly depend on livestock production. Therefore, it is imperative to ensure that livestock producers increase their resilience to climate variability or change. However, few field-based studies have focused on simultaneous investigation of both vulnerability and adaptation of livestock keepers to climate-related risks. To fill this gap, a field-based research study was conducted in southwest Iran. A survey of 274 herder families, selected using a cluster sampling technique, revealed low, medium and high levels of vulnerability, which were principally distinguished by various degrees of exposure, sensitivity, and adaptive capacity. Further, this study identified the main adaptation strategies employed by the herders of this region (i.e., purchasing fodder; rotational grazing; raising a mixed-herd, on-farm occupation; and postharvest grazing). Results indicated that most herder families applied traditional adaptation strategies in response to climate variability. Findings revealed that herders had to reduce their livestock due to low adaptability. Loan term, purchasing insurance, level of exposure, income, experience, response efficacy, and knowledge were the major determinants of the herders’ adaptation decisions. To increase the resilience of livestock producers against climate change, restructuring traditional livestock production systems, producing participatory knowledge and information for sustainable management of rangelands, and designing or redesigning effective adaptation strategies are required.     

Effects of supplemental conjugated linoleic acids (CLA) on fresh and post‐thaw sperm quality of Holstein bulls

This study was designed to investigate the effects of feeding‐protected conjugated linoleic acid (CLA) on the semen production and sperm freezability in Holstein bulls. Twelve bulls were randomly assigned to two groups (n = 6 per group). Bulls received the normal diet (control group) or the normal diet top‐dressed with 50 g of CLA (treated group) for 10 weeks. The control group received 40 g/day calcium soap of fatty acid. Fresh and post‐thaw semen quality was assessed on ejaculates collected at the 0, 4, 6, 8 and 10 week of supplementation. Semen evaluations including sperm concentration, motion characteristics (subjective and computer‐assisted), viability (Eosin–Nigrosin), membrane integrity (hypo‐osmotic swelling test) and abnormality were conducted. Semen volume, sperm concentration and total sperm output were not affected by dietary treatment (p > .05). The proportion of spermatozoa with abnormal morphology in fresh semen significantly increased (p < .05) in the CLA‐fed group compared to control group. Also, in CLA‐fed group, the proportion of post‐thaw spermatozoa with abnormal morphology at week 10 of trial was significantly higher in CLA than control group (p < .05). Progressive motility tended to be increased in the CLA‐fed group, although dietary supplementation did not affect other CASA parameters or viability in fresh and frozen‐thawed sperm. In this study, CLA supplementation had little positive effect on fresh or post‐thaw sperm quality of Holstein bulls.     

Seasonal,tissue and age influences on frequency and biodiversity of endophytic fungi of Citrus sinensis in Iran

A total of 4,009 endophytic fungal isolates were recovered from healthy leaves, bark and xylem of Citrus sinensis in different seasons and age‐classes. Corresponding to those factors, the majorities of fungal isolates were recovered from leaves, 2‐yr‐old trees and winter, respectively. Fungal isolates were initially categorized based on cultural and morphological characters, and representatives of each morphotype were subjected to molecular identifications based on ITS‐rDNA and β‐tubulin sequences. All isolates obtained in this study belonged to the phyla Ascomycota and Basidiomycota assigned to 30 fungal taxa. The most abundant fungal species were Alternaria spp. Species diversity indices of Margalef richness and Shannon–Wiener revealed a high diversity of fungal taxa recovered from leaf and during winter. Frequency (%) of fungal endophytes was highest in winter followed by summer, autumn, and spring significantly, whereas the corresponding sequence was winter, autumn, summer and spring for the diversity of endophytic fungi. Overall, the results of this study based on the community and diversity of endophytic fungal species in C. sinensis showed that the type of plant tissue, season of sampling and age of tree play a driving role on their abundance while their diversity was mainly dependent on the type of plant tissue, season of sampling than on the age of tree.