Biodegradation of aliphatic and aromatic hydrocarbons by bacteria isolated from Bahregan area.

Environmental pollution with aromatic and aliphatic hydrocarbons caused by oil and petrochemical industries has very toxic and carcinogenic effects on living organisms and should be removed from the environment. In this research, after analyzing the oil sludge of the Bahregan area, it was found that most aliphatic paraffin compounds are related to octadecane, most liquid aliphatic compounds are related to hexadecane, and most aromatic compounds are related to naphthalene, phenanthrene, fluoranthene, and anthracene. Then, we investigated the ability of native bacteria from this area, such as Thalassospira, Chromohalobacter, and a bacterial consortium, to biodegrade the dominant aromatic and aliphatic hydrocarbons found in oil sludge. The results of Gas Chromatography-Mass Spectrometry analysis showed that among the tested hydrocarbon sources, Thalassospira can completely remove octadecane and hexadecane, and Chromohalobacter can reduce hexadecane from 15.9 to 9.9%. The bacterial consortium can completely remove octadecane and reduce hexadecane from 15.9 to 5.1%, toluene from 25.6 to 0.6%, and phenanthrene from 12.93 to 6%. According to the obtained results, the bacterial consortium effectively plays a role in the biodegradation of aromatic and aliphatic hydrocarbons, making it a viable solution for treating hydrocarbon pollutants in various environments.

Improved heat shock method for extracting total RNA from nasopharyngeal swab samples even with low viral load.

This study aimed to improve the heat shock method as a cost-effective and time-efficient for total RNA extraction. We compared the effectiveness of two total RNA extraction methods by using Real-Time PCR for nasopharynx swabs. Include: I; use of a commercial total RNA extraction kit as a standard. II; utilized a modified heat shock method (MHS). Time, centrifuge speed and duration, proteinase K, and RNA carrier were optimized. The optimized parameters included treating the sample with 5 µg/µL at 56°C for 5 minutes, heating at 95°C for 5 minutes followed by thermal shock in ice for 3 minutes, adding 4 µg/µL RNA carrier at room temperature for 3 minutes, and centrifuging at 7000 rpm for 10 minutes. This optimization demonstrated a sensitivity and specificity of 100% (CI: 95%) even in samples with low viral load. Our in-house method presents a rapid, and cost-effective alternative for total RNA extraction.

Cytotoxic effect of pyocyanin on human pancreatic cancer cell line (Panc-1).

OBJECTIVES: Pyocyanin, a blue-green pigment produced by Pseudomonas aeruginosa, interferes with host redox cycles, which can lead to generation of reactive oxygen species and progressive cellular oxidative damage. The aim of this study was to assess the influence of pyocyanin on human pancreatic cancer cell line. MATERIALS AND METHODS: Polymerase Chain Reaction (PCR) was applied to confirm the existence of a specific pyocyanin producing gene (phzM). The pigment was then characterized by UV-visible, FT-IR, and HPLC analysis. Panc-1 cells were treated by different concentrations of pyocyanin and their cytotoxic effect as well as the induction of apoptosis/necrosis were evaluated by XTT assay and flow cytometry. RESULTS: An overnight pyocyanin treatment resulted in significant cell reduction in a concentration-dependent manner. Inhibition rate of 6 mg.ml-1 pyocyanin (the highest concentration) extracted from clinical and soil isolates of P. aeruginosa were 98.69±0.23 and 89.88±1.86%, respectively, which decreased as the pyocyanin concentration lessened. Pyocyanin could also induce dose-dependent apoptosis/necrosis in Panc-1 cells after 24 hr. CONCLUSION: We reported, for the first time, cytotoxic effects of pyocyanin against human pancreatic cancer cell line. Considering this effect of the pigment, study on pyocyanin as a potential anti-tumor biodrug requires further studies.

Pasteurella multocida inactivated with ferric chloride and adjuvanted with bacterial DNA is a potent and efficacious vaccine in Balb/c mice.

PURPOSE: Pasteurella multocida (P. multocida) is a principal pathogen of domestic animals and an opportunistic pathogen of humans. It is the causative agent of pneumonia and haemorrhagic septicaemia in cattle, sheep and goats, fowl cholera in chickens and progressive atrophic rhinitis in swine. In this study, we investigated the humoral and cellular immune responses and protective immunity conferred by an iron-inactivated vaccine with bacterial DNA (IIV+bDNA) as an adjuvant in mice. METHODOLOGY: P. multocida was grown in BHI broth, inactivated with formalin and FeCl3 and adjuvanted with alum and bDNA. Mice were immunized with two whole-cell inactivated vaccine doses 2 weeks apart. The animals were challenged 4 weeks after booster immunization. Immunogens (vaccines and bDNA) posed no safety problems when mice were injected subcutaneously (s/c) with these preparations. The serum antibody titres were tested by ELISA. At 28 days post immunization, cell-mediated immunity responses were determined. The responses were measured by assay of IL-6 and IL-12 in lymphocyte spleen culture supernatants. RESULTS: ELISA results showed that the levels of antibodies in iron inactivated with bDNA adjuvant groups were higher than in the formalin inactivated with alum adjuvant vaccine group. The protection rate of IIV+bDNA adjuvant vaccine was superior to that of the other vaccines and it protected 100 % of the challenge group mice. Following immunization, bDNA promoted increased production of interleukins compared to the control groups. CONCLUSION: These studies indicate that bDNA is effective as an immune adjuvant, and along with stimulatory bDNA represent promising new humoral and cellular immune enhancers for vaccination applications. In addition, this vaccine is able to provide long-term protection against infection.

Evaluation of The Number of CD4(+) CD25(+) FoxP3(+) Treg Cells in Normal Mice Exposed to AFB1 and Treated with Aged Garlic Extract.

OBJECTIVE: Aflatoxin B1 (AFB1) suppresses the immune system. To decrease such suppressive effects on the immune system, a wide range of herbal medicines like garlic are utilized. Biological activities of garlic in vitro and in vivo have also been verified. Our previous studies demonstrated that aged garlic (dry garlic bulbs preserved in the freezer for six months at -20˚C) have increased immunostimulator fractions and reduced immunosuppressor fractions. This study focuses on the immunosuppressor activity of AFB1 and immunostimulator activity of aged garlic extract (AGE) through the evaluation of CD4(+) CD25(+) FoxP(+) regulator cell (Treg) counts and the pattern of cytokine production in Balb/c normal mice. MATERIALS AND METHODS: In this experimental research, AFB1 was separated from Aspergillus flavus (PTCC 5004) by HPLC and AGE prepared using the Mantis method. The Delayed-Type Hypersensitivity (DTH) test was carried out to determinate the effectiveness of different doses of AGE and AFB1, which can both have an effect on the immune system. Subsequent experiments were carried out on 20 Balb/c mice to estimate the effects of AGE and AFB1 on the number of Treg cell in 4 groups: 10 µl/kg/day of AFB1 and AGE diluents were administered for 4 consecutive days to group 1. AFB1, 2. control, 3. AGE + AFB1 and 4. AGE via intraperitoneal (IP) route, respectively. Mice were sacrificed and splenocytes harvested and the percentage of splenic Treg cells was measured by flow cytometry analysis. The ELISA method was utilized to measure Cytokine production. RESULTS: The findings reveal that AGE increased the level of IFN-λ and IL-4 cytokines produced by splenocytes stimulated by specific tumor antigen and decreased the number of Treg cells in the spleen (p<0.05). AFB1 increased the number Treg cells in the spleen and decreased cytokine production (p<0.05). In groups 2 (control) and 4 (AGE) the number of Treg cells decreased (p value<0.05) whereas in groups 1 and 3 the number of Treg cells increased (p<0.05). CONCLUSION: This study indicated that AGE is able to alter the cytokine production in normal mice into a Th1 protective pattern which is beneficial to the immune system in general and anti-tumor immunity in particular. AFB1 is able to alter the cytokine production into a Th2 protective pattern. Therefore, AGE might be used as herbal medicine with few side effects as compared to chemotherapy in treating cancers caused by substances like AFB1.

Effect of Culture Conditions on the Production of an Extracellular Protease by Bacillus sp. Isolated from Soil Sample of Lavizan Jungle Park.

Soil samples of Tehran jungle parks were screened for proteolytic Bacilli. Among eighteen protease producers one of the isolates obtained from Lavizan park, in north east of Tehran, was selected for further experimental studies. This isolate was identified as Bacillus sp. strain CR-179 based on partial sequencing of 16S rRNA. Various nutritional and environmental parameters affected protease production by Bacillus sp. strain CR-179. Protease production by this Bacillus cultivated in liquid cultures reached a maximum at 24 h, with levels of 340.908 U/mL. Starch and maltose were the best substrates for enzyme production while some pure sugars such as fructose, glucose, and sucrose could not influence production of protease. Among various organic nitrogen sources corn steep liquor, which is commercial, was found as the best substrate followed by yeast extract, whey protein, and beef extract. The optimal pH and optimal temperature of enzyme production were 8.0 and 45°C, respectively. Studies on enzymatic characterization revealed that crude protease showed maximum activity at pH 9.0 and 60°C, which is indicating the enzyme to be thermoalkaline protease.

Cost-Effective Production and Optimization of Alkaline Xylanase by Indigenous Bacillus mojavensis AG137 Fermented on Agricultural Waste.

A xylanase producer Bacillus mojavensis strain, called AG137, isolated from cotton farm (Kashan-Iran). The optimal xylanase activity reached at 55°C & pH 9.0. Enzyme yield was studied using a medium with different agricultural wastes as inducers. Xylanase production of about 249.308 IU/mL was achieved at pH 8 and 37°C, within 48 h submerged fermentation in enzyme production medium supplemented with 2% (w/v) oat bran as an optimum carbon source. A mixture of 1% (w/v) yeast extract and 1% (w/v) tryptone as optimum nitrogen sources, agitation speed 200 rpm, and inoculum size 2% (v/v) were the optimums for maximum production. Accordingly, xylanase yield from 194.68 IU/mL under non-optimized fermentation condition enhanced to 302.466 IU/mL in optimized condition. Screened xylanase is thermostable, presenting 70% stability at 60°C during 30 min. Further enzyme incubation in higher temperature caused a decrease in the residual enzyme activity, yet it retained 68%-50% of its activity after 1 hour from 45°C to 55°C. Besides, it is stable in pH 9 and 10, maintaining over 70% of its activity for 2 h. The enzyme also could preserve 71% and 63% of its initial activity after 3 hours of pre-incubation in the same alkaline condition. Produced xylanase therefore was introduced as an alkaline-active and stable one, displaying suitable thermostability feature, confirmed by HPLC analysis. Hence, all xylanase properties highlight its promising uses in industrial scale.