Isolation and molecular identification of halophilic protease producing actinomycete and evaluation of effective factors for maximal enzyme production

Introduction: Proteases are one of the most applicable hydrolyzing enzymes especially in food and beverage industries. Isolation of thermophilic or halophilic proteases was aimed by many investigations. The present study was designed to screen soil samples for halophilic actinomycetes capable to produce protease and evaluation of factors affect on the enzyme production.  Methods: Twenty soil samples were collected from salty land around Kerman, Iran and aseptically transferred to biotechnology lab and allowed to dry. Ten grams of each soil was extracted using NaCl (0.9%) sterile solution and added to Casein Glycerol Agar (CGA) or nutrient agar medium containing skim milk (1%) and NaCl (10%) and incubated at 30◦C untill the halo zone of protease activity was formed. The ratio of halo zone/colony size of isolated actinomyctes was used as an index to select the most suitable strains. Morphological and biochemical tests as well as molecular identification using 16S rDNA technique were then applied for identification of the strain. Evaluation of chemical factors such as carbon sources, nitrogen sources and inorganic salts as well as physical factors such as temperature and pH on protease production of the selected strain was performed using one factor at a time approach. Results: Seven protease-producing actinomycetes were isolated in preliminary studies among which one isolate (identified as Nocardiopsis sp.) was the most efficient one able to produce 650 U/mL protease after 5 days incubation in CG medium containing 10% NaCl. Evaluation of factors is now conducting to obtain the maximum protease production. Conclusion: one halophilic actinomycete able to produce protease was isolated in the present study and evaluation of factors affect on the enzyme production is now performing

Efficacy of Myrtus communis L. to inactivate the hydatid cyst protoscoleces

Purpose: The present study aims to investigate the scolicidal effects of Myrtus communis L. essential oil against protoscoleces of hydatid cysts and also its toxicity in mice model. Materials and Methods: Protoscoleces were aseptically aspirated from sheep livers having hydatid cysts. Various concentrations of the essential oil (12.5–100 μl/ml) were used for 5–30 min. Viability of protoscoleces was confirmed using eosin exclusion test (0.1% eosin staining). Moreover, 48 male NMRI mice were used to determine the acute and sub-acute toxicity of M. communis essential oil. One-way ANOVA with Tukey’s post-hoc test was used to assess differences between experimental groups. Results: Findings of the present study demonstrated that the M. communis essential oil at the concentration of 100 μl/ml after 5 min of exposure killed 100% protoscoleces. Similarly, the mean mortality rate of protoscoleces after 10 min of exposure to concentration of 50 μl/ml was 100%. However, lower concentrations (12.5 and 25 μl/ml) of M. communis essential oil provoked a delayed protoscolicidal effects. The LD50 values of intraperitoneal injection of the M. communis essential oil was 2.23 mL/kg body wt. No significant difference (p > .05) was observed in the clinical chemistry and hematological parameters following oral administrations of M. communis essential oil at the doses 0.05, 0.1, 0.2, and 0.4 mL/kg for 14 days. Conclusion: The results showed potent scolicidal activity of M. communis with no significant toxicity, which might be used as a natural scolicidal agent in hydatid cyst surgery

Antifungal Activity of Selenium Nanoparticles Synthesized by Bacillus species Msh-1 Against Aspergillus fumigatus and Candida albicans

Background: Fungal infections affect various parts of the body and can be difcult to treat. Aspergillus infection causes a spectrum of diverse diseases particularly in lung according to host immunity. The two major entities are invasive pulmonary aspergillosis and chronic pulmonary aspergillosis. Candida infections can be superfcial or invasive. Superfcial infections often affect the skin or mucous membranes. However, invasive fungal infections are often life-threatening. Advances in nanotechnology have opened new horizons in nanomedicine, allowing the synthesis of nanoparticles that can be assembled into complex architectures. Novel studies and technologies are devoted to understanding the mechanisms of disease for the design of new drugs. Objectives: In the present study, the antifungal activity of biogenic selenium nanoparticles (Se NPs) against Aspergillus fumigatus and Candida albicans was investigated. Materials and Methods: Se-reducing bacteria previously identifed as Bacillus sp. MSh-1 were used for the intracellular biosynthesis of elemental Se NPs. The shape, size, and purity of the extracted NPs were determined with various instrumental techniques. The nanoparticles antifungal characterization mainly derives from the following pathways: (i) to generate sustained flux of nano-ions from the compounds that deposited on special substrates or imbedded in colloidal or semisolid matrices. (ii) To transport active those ions to sensitive targets on plasma membrane of fungi. Results: The results of energy-dispersive X-ray demonstrated that the purifed NPs consisted of only Se. In addition, transmission electron micrographs showed that 120- to 140-nm spherical Se NPs were the most common. An antifungal assay was performed with a standard Clinical and Laboratory Standards Institute broth microdilution method. Minimum inhibitory concentration (MIC) measurements of the antifungal activity of the Se NPs against C. albicans (70 μg/mL) and A. fumigatus (100 μg/mL) showed that yeast cells were more sensitive than mold cells. Conclusions: The MICs against A. fumigatus (100 μg/mL) and C. albicans (70 μg/mL) showed that biogenic Se NPs are useful antifungal agents

In Vitro Study of Leishmanicidal Activity of Biogenic Selenium Nanoparticles against Iranian Isolate of Sensitive and Glucantime- Resistant Leishmania tropica

Background: Sensitive and glucantime (MA) resistance Leishmania tropica are referred to those isolates, which are responsive, or non-responsive to one or two full courses of treatment by MA systematically and/or intra-lesionally, respectively. In this study, we evaluated the antileishmanial activity of biogenic selenium nanoparticles (Se NPs) alone and in combination with MA against sensitive and glucantimeresistant L. tropica on in vitro model. Methods: The Se NPs were synthesized by employing the Bacillus sp. MSh-1. The antileishmanial effects of Se NPs alone and in combination with MA on promastigote and amastigote stages of sensitive and glucantime-resistant L. tropica strains have been investigated using a colorimetric MTT assay and in a macrophage model. In addition hemolytic activity in type O+ human red blood cells and infectivity rate of the promastigotes before and after treatment with the Se NPs was evaluated. Results: In the promastigote stage, various concentrations of Se NPs significantly inhibited (P<0.05) the growth of promastigotes of both strains in a dose-dependent manner. Similarly, Se NPs especially in combination with MA significantly reduced the mean number of amastigotes of both strains in each macrophage. Se NPs showed no hemolytic effect on human RBCs at low concentrations. Moreover, infection rate of macrophages by promastigotes significantly (P<0.05) was reduced when promastigotes pre-treated with Se NPs. Conclusion: The findings of this study suggest a first step in the search of Se NPs as a new antileishmanial agent. Further experiments are needed to investigate antileishmanial effects of biogenic Se NPs on L. tropica using a clinical setting

Anti-biofilm activityofbiogenicseleniumnanoparticlesandselenium dioxide againstclinicalisolatesof Staphylococcus aureus, Pseudomonas aeruginosa, and Proteus mirabilis

The aimofthepresentstudywastoinvestigatetheanti-biofilmactivityofbiologicallysynthesizedsele- nium nanoparticles(SeNPs)againstthebiofilmproducedbyclinicallyisolatedbacterialstrainscompared to thatofseleniumdioxide.Thirtystrainsof Staphylococcus aureus, Pseudomonas aeruginosa, and Proteus mirabilis were isolatedfromvariousspecimensofthepatientshospitalizedindifferenthospitals(Ker- man, Iran).Quantificationofthebiofilmusingmicrotiterplateassaymethodintroduced30%of S. aureus, 13% of P. aeruginosa and 17%of P. mirabilis isolates asseverelyadherentstrains.Transmissionelectron micrograph (TEM)ofthepurifiedSeNPs(producedby Bacillus sp. MSh-1)showedindividualandspher- ical nano-structureinthesizerangeof80–220nm.Obtainedresultsofthebiofilmformationrevealed that seleniumnanoparticlesinhibitedthebiofilmof S. aureus, P. aeruginosa, and P. mirabilis by 42%,34.3%, and 53.4%,respectively,comparedtothatofthenon-treatedsamples.EffectoftemperatureandpHon the biofilmformationinthepresenceofSeNPsandSeO2 was alsoevaluated

Anti-leishmanial activities of selenium nanoparticles and selenium dioxide on Leishmania infantum

Leishmania infantum is one of the important causes of visceral leishmaniasis in many countries. There are different complications for treatment of leishmaniasis such as toxicity and drug resistant. So far, there isn’t any information about the effects of selenium nanoparticles and selenium dioxide (chemical form of selenium) on Leishmania parasites; hence, the aim of the present study is to investigate in vitro effects of six dilutions of these drugs on L. infantum. Anti-leishmanial activities were studied by adding different dilutions of 2.5, 5, 10, 25, 50, and 100 μg/ml of the drugs into promastigote cultures. Promastigote cytotoxicity was tested using the colorimetric MTT assay. Anti-amastigote activity was assessed in peritoneal macrophages of BALB/c mice. Also, cytotoxic effect of these drugs was evaluated on uninfected macrophages. The results showed that both of drugs have dose-dependent anti-leishmanial activities. Selenium NPs have more growth-inhibitory effect on promastigotes than SeO2; while the IC50 (50 % inhibitory concentration) was determined to be 25 and 50 μg/ml, respectively. The mean numbers of amastigotes per macrophage in selenium NPs-treated groups were less than SeO2-treated and control groups. The IC50 of selenium NPs was 10 μg/ml and SeO2 was 25 μg/ml for amastigotes. Also, the IC50 of selenium NPs and SeO2 for uninfected macrophages were calculated to be 100 and 50 μg/ml, respectively. In addition, selenium NPs has less cytotoxic effect than SeO2 on uninfected macrophages. These findings suggest that selenium NPs have more anti-leishmanial properties and less cytotoxic effects than SeO2 against L. infantum

In Vitro Antiparasitic and Apoptotic Effects of Antimony Sulfide Nanoparticles on Leishmania infantum

Visceral leishmaniasis is one of the most important sever diseases in tropical and subtropical countries. In the present study the effects of antimony sulfide nanoparticles on Leishmania infantum in vitro were evaluated. Antimony sulfide NPs (Sb2S5) were synthesized by biologicalmethod fromSerratia marcescens bacteria. Then the cytotoxicity effects of different concentrations (5, 10, 25, 50, and 100 μg/mL) of this nanoparticle were assessed on promastigote and amastigote stages of L. infantum. MTTmethodwas used for verification results of promastigote assay. Finally, the percentages of apoptotic, necrotic, and viable cells were determined by flow cytometry. The results indicated the positive effectiveness of antimony sulfide NPs on proliferation of promastigote form. The IC50 (50% inhibitory concentration) of antimony sulfide NPs on promastigotes was calculated 50 μg/mL. The cytotoxicity effect was dose-dependent means by increasing the concentration of antimony sulfide NPs, the cytotoxicity curve was raised and the viability curve of the parasite dropped simultaneously. Moreover, the IC50 of antimony sulfide NPs on amastigote stage was calculated 25 μg/mL. On the other hand, however, antimony sulfide NPs have a low cytotoxicity effect on uninfected macrophages but it can induce apoptosis in promastigote stage at 3 of 4 concentrations

Effect of selenium supplementation with sodium selenite and selenium nanoparticles on iron homeostasis and transferrin gene expression in sheep: A preliminary study

The present research aimed at evaluating the effects of sodium selenite and selenium nanoparticles (Se NPs) on iron homeostasis and the expression of transferrin and its receptor-binding protein genes. Twenty one Lori–Bakhtiary sheep were randomly allocated into 3 groups. Groups 1 and 2 orally received Se NPs and sodium selenite (1 mg kg�1) for 10 consecutive days, respectively. Group 3 served as the control. Blood and sternal bone marrow samples were collected at different supplementation intervals. Various factors such as serum iron concentration, total iron binding capacity (TIBC), and transferrin saturation percent were determined. The expression of transferrin and transferrin binding receptor genes was also studied. Results showed a decreasing trend in serum iron concentration particularly during the early and middle stages of supplementation (0–20 days) with Se NPs or selenium ions. Conversely, the TIBC level increased in sera especially during these periods (0–20 days) in animals that received selenium NPs or selenium ions. Our results also showed that expression of transferrin and its receptor genes was considerably increased during supplementation of the animals by both selenium compounds for 10 or 20 days. After this period, the expression of the mentioned genes significantly decreased, especially in animals that received selenium ions

Biosynthesis and recovery of selenium nanoparticles and the effects on matrix metalloproteinase-2 expression

Today, green synthesis of nanoparticles is attracting increasing attention. In the present study, the Bacillus sp. MSh-1 was isolated from the Caspian Sea (located in the northern part of Iran) and identified by various identification tests and 16S ribosomal DNA analysis. The reduction time course study of selenium ion (Se4+) reduction by using this test strain was performed in a liquid culture broth. Then, the intracellular NPs (nanoparticles) were released by the liquid nitrogen disruption method and thoroughly purified using an n-octyl alcohol water extraction system. Characterization of the separated NPs on features such as particle shape, size and purity was carried out with different devices. The energy dispersive X-ray and X-ray diffraction patterns showed that the purified NPs consisted of only selenium and are amorphous respectively. In addition, the transmission electron micrograph showed that the separated NPs were spherical and 80–220 nm in size. Furthermore, the cytotoxicity effect of these extracted biogenic selenium (Se) NPs on the fibrosarcoma cell line (HT-1080) proliferation and the inhibitory effect of the Se NPs on MMP-2 (matrix metalloproteinase-2) expression were studied using the MTT [3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyl-2H-tetrazolium bromide] assay and gelatin zymography. Biogenic Se NPs showed a moderately inhibitory effect on MMP-2 expression