a. javadmanesh

Excessive use of antibiotics to treat diseases such as mastitis in dairy cows caused by Escherichia coli has led to the emergence of antibiotic-resistant bacteria, which is considered a threat to public health. Nowadays, antimicrobial peptides such as bacteriocins have attracted more attention as potential antimicrobial agents. Therefore, the study of their stability and antibacterial activity in physiological conditions is very important. In order to investigate the thermal stability of Enterocin P peptide (EntP), the third structure of EntP was first predicted using the I-TASSER server. Then, the stability of this structure was evaluated by dynamic conditions for this purpose, the simulation was performed using GROMACS software in 400 ns at cow body temperature (312 °K). Finally, the root-mean-square deviation (RMSD) graph of this peptide was plotted against time along the simulation trajectory. ClusPro online server was used to study the interaction of peptide with LptD surface protein from Escherichia coli bacterium causing mastitis. In the in vitro phase of this study, the minimum inhibitory concentration (MIC) and minimum dissociation concentration (MBC) of EntP peptide were estimated based on the microbroth method on Escherichia coli. The results showed that the best predicted structure of EntP peptide by I-TASSER server had a C-score of 1.4 which was selected. The values reported by the ERRAT and Verify3D plots indicated that the tertiary structure predicted for the EntP peptide was satisfactory. The RMSD graph related to the thermal stability of EntP at bovine body temperature showed that the structure of EntP peptide is stable after 400 ns of the simulation trajectory. The results of molecular docking also showed that this peptide was correctly attached to the LptD antigen. After measuring the MIC, the bacteria were cultured and the MBC was obtained, which were estimated at 48.25 and 96.5 μg/ml, respectively. This peptide could be used as an alternative to antibiotics or in combination with them for the treatment of infections with the origin of Escherichia coli.

Silver nanoparticles (AgNPs) have a broad spectrum of uses, therefore, AgNPs will be released from those products into many different ecosystems. In the last decades, AgNPs have received substantial attention due to their distinctive physical and chemical properties such as high thermal and electrical conductivity, chemical stability, catalytic activity and antimicrobial properties against microbes such as bacteria, fungi, and viruses.  There are many parameters for assessment effect of toxicity due to AgNPs but soil microbial community is one of which considered being an important target for assessing the impact of manufactured nano-materials on the terrestrial environment. Toxicity of AgNPs is due to the physical interaction of AgNPs with microorganisms and the production of reactive oxygen species (ROS). Although as we have been known harmful effects of AgNPs on the soil bacterial community, but the most information about antimicrobial properties of AgNPs come from the routine lab instructions such as soil respiration, substrate induced respiration and microbial biomass and colony forming unite. So, the objective of this paper was to study the effects of silver nanoparticles on microbial activity using the routine lab instructions and compare with the obtained data from the molecular genetic techniques. In this paper, the quantitate population of soil bacterial was estimated using Real time qPCR with the MIQE guidelines.  In order to study the effect of silver nanoparticles on microbial activity and bacterial population in a calcareous soil, an experiment was conducted as a completely randomized design based on factorial arrangement with three replications. Experimental factors included silver slat forms (AgNPs and AgNO3), Ag concentrations (0, 0.5, 5, 10, 50, and 100 mg Ag kg-1 dry soil) and incubation time (7 and 42 days). Soil samples (Typic Haplicambids) with clay loam texture and seven percent of calcium carbonate was collected from Research Field of Ferdowsi University of Mashhad, Mashhad, Khorasan Razavi, Iran. The soil samples were amended with different concentrations of AgNPs and incubated at 25oC for 42 days. The water content of soil samples was adjusted at 70% WHC during the incubation time. After 7 and 42 days of incubation, the soil substrate-induced respiration (SIR), heterotrophic plate count (HPC), and soil urease and dehydrogenase activities were measured. Finally, based on the obtained data, the soil biological quality index was estimated using the soil biological parameters. In order to quantify the total bacterial population, DNA was extracted from soil samples and was estimated using the relative concentration of 16S rDNA gene by a quantitative Polymerase Chain Reaction (qPCR), with a minimum information for publication of quantitative real-time PCR experiments (MIQE) guidelines.  The results showed that with increasing the concentration of both AgNPs and AgNO3, the activity of dehydrogenase and urease in soil samples decreased during the incubation times. Microbial substrate induced respiration (SIR) and the total bacterial population in soil samples considerably declined at the end of experiment. Bacterial population in AgNPs treatments decreased compared to AgNO3 treatments but the reduction was not statistically significant. Over time, soil dehydrogenase activity and soil SIR decreased in both AgNPs and AgNO3 treatments, while soil urease activity and heterotrophic bacterial populations improved but again in heterotrophic bacterial populations was not statistically significant. The soil biological quality index was estimated from the soil biological data. AgNO3 treatments reduced the soil biological quality index compared to AgNPs treatments. In other words, the results showed that AgNO3 was more toxic to soil bacteria activity compared to AgNPs. The lowest soil urease and dehydrogenase enzyme activity and soil biological quality index were observed in the treatment of 100 mg kg-1 dry soil AgNO3 after 7 days of incubation. The application of 0.5, 5, 10, 50, and 100 mg Ag kg-1 dry soil decreased relative soil bacterial population by 22%, 40%, 59%, 73%, and 82% in AgNO3 treatment and 10%, 30%, 68%, 76%, and 86% in AgNO3 treatment compared to control after 42 days of incubation, respectively.  The results of this study showed that silver nanoparticles can negatively affect the enzymes involved in the nitrogen and carbon cycle. The AgNPs had less toxicity effect on the soil microbial activity compared to AgNO3. However, AgNPs was more toxic to soil bacteria populations compared to AgNO3. Different behavior AgNPs and AgNO3 in calcareous soil needs more investigations but there is no doubt that AgNPs is as an emerging contaminant and it has high toxicity potential for soil microbial community.

Mastitis is one of the most important diseases in dairy cattle. Administration of synthetic antibiotics for treatment of mastitis leads to drug-resistance bacteria or fungi. Hence, the use of natural alternatives such as antimicrobial peptides and plant extracts has been considered as alternatives to common antibiotics. The aim of this study was to evaluate the effect of Thanatin peptide and Chinaberry extract against fungal factors of bovine mastitis such as Candida parapsilosis, Candida albicans and Mucor spp in vitro. The minimum inhibitory concentration (MIC) and the minimum fungicidal concentration (MFC) were determined based on broth micro-dilution method by 96-well plate with four replications using Chinaberry (Melia azedarach) extract and thanatin peptide on three mastitis-causing fungi. The results showed that the thanatin was effective on growth inhibition of all three fungi. Thanatin was more effective against Candida albicans and parapsilosis. Chinaberry extract with MIC of 6250 µg / ml and MFC of 12500 µg / ml had the greatest effect on Candida parapsilosis. Applied Chinaberry extract had no effect on Mucor spp fungi while thanatin showed an antifungal activity against this species. This study proved that Chinaberry extract and Thanatin peptide could show suitable antifungal activity against factors for mastitis includes Candida parasilosis, Candida albicans and Mucor spp in vitro although the antifungal activity of this peptide should be investigated in clinical trials in the future.

Calf diarrhea is a commonly reported disease and a major problem to the livestock industry. Although, antibiotics are used to treat diarrhea in calves, they are not effective against viruses as well as they may cause some other problems such as antibiotic resistance. As a result, natural antibodies such as immunoglobulin Y (IgY) from egg yolk could be a possible replacement for traditional antibiotics with less side effects. The objective of this research was production of specific IgY againt major calf diarrhea pathogens such as coronavirus, rotavirus, E. coli, and Clostridium perfringens in the egg yolk. For this purpose, this experiment was performed with two treatments (control and vaccinated group with the trivalent vaccine of coronavirus, rotavirus, and E. coli as well as Clostridium perfringens vaccine) and seven replicates in each group on W-36 Hy-Line laying hens. SDS-PAGE and Bradford methods were used for quality and quantity evaluations of IgY, respectively. Finally, the effect of freeze-drying and spray drying on IgY degradation was assessed by SDS-PAGE. The results showed that heavy and light chains of IgY were visible in the treatment group. The control group did not show any visible IgY fragments. Furthermore, the quantity of IgY secreted in egg yolk was estimated at 4.729 mg/ml. Finally, the comparison between two drying methods showed that freeze-drying did not damage IgY although spray-drying caused a substantial damage. Furthur experiments are required to validate the biological activity of the specific IgY produced in this study.

To investigate the effects of dietary supplementation with industrial vinegar (IV) and waste date vinegar (WDV) on growth performance, intestinal morphology, and immune response of broilers, five hundred Ross 308 chickens were randomly divided into 50 cages in a 42-day breeding period. The experiment consisted of ten treatments with five replications in each treatment including; control 1 (standard feed without  WDV or IV), control 2 (control 1 + 2% water into the feed), 1, 2, and 3% of WDV and 2% industrial vinegar (IV) into the feed and 0.5, 1, and 1.5% of WDV and 1% industrial vinegar (IV) into the water. One chicken from each replicate was slaughtered on days 24 and 42 to investigate gastrointestinal tissue growth as well as intestinal morphology. The results showed that growth performance was not affected by treatments in any period. The height and width of the villus in the treatments containing 1% of WDV into the water and 2% of WDV into the feed increased with time compared to the control treatments. On day 42 of the experiment, the crypt depth was also higher in the treatment containing 1% of WDV into the feed compared to the other treatments. The ileal coliforms were also affected by WDV addition into the feed at 42 days of age compared to other groups (P < 0.05). The highest amount of IgM and IgG were found to levels of 2% and 1% of WDV into the diet, respectively (P < 0.05). However, SRBC, ND titter was not affected by treatments. Also, no difference was found between industrial vinegar in water or feed in most of the studied parameters. The results of this study showed that supplementation of the diet with WDV had a positive effect on intestinal morphology and immune system of broilers compared to industrial vinegar.

This study was performed to investigate the effects of dietary threonine (Thr). level on performance, metabolizable energy, intestinal morphology, and immune system in coccidian–infected broiler chickens. The diets contained: 88%, 100% (Non challenged (NC) and challenged control (PC)), 112%, 124%, and 136% of Thr requirement according to Cobb 500 recommendation and fed during grower (pre challenged) and finisher (post challenged) periods. On d 23 (end of grower period), each bird received 0.5 mL of distilled water or received around 24000 sporulated oocysts. On d 23 and 31, one bird per replicate was slaughtered to measure the performance criteria. Mean dietary apparent metabolizable energy corrected for nitrogen (AMEn) and digestible energy were greater in NC birds than the challenged birds fed on 88% or 100% Thr diets. Feed intake and blood parameters were not significantly influenced by increasing levels of Thr in the diet. Compared with unchallenged birds with coccidia (NC), the growth performance, morphological parameters (not crypt depth), and immune responses decreased (P < 0.05) in the birds (PC) that were challenged with coccidia, and oocyte numbers were enhanced. Growth performance, jejunal morphology, and immune responses improved and oocyte count decreased in coccidian- challenged birds fed on the diets with greater levels of Thr (P < 0.05). Feeding the challenged birds with the diet containing greater levels of Thr improved (P < 0.05) their growth performance, morphology, and immune responses and decreased oocyte number. The birds fed on the diet with 124% Thr demonstrated a similar response as the NC birds. Increased diet Thr level linearly increased average daily gain and decreased feed conversion ratio in the grower and the whole experimentation periods. The AMEn and digestibility of crude protein were enhanced linearly Thr level increased in coccidian-challenged birds. It is concluded that diets containing 124% of Thr recommendation led to the greatest efficacy on the intestinal immuneresponse and normal growth maintenance of the birds contaminated with coccidia.

Lysine is one of the essential amino acids not synthesized biologically in the human body and mammals so should be supplied through diets. Among the industrially important amino acids, L-lysine is in 1st position, which is used in pharmaceuticals, animals, human feeds and precursors for the production of peptides or agrochemicals. As L-lysine has large applications, the demand for it is increasing constantly year by year. To minimize the gap between increasing demand and production of L-lysine, it has to be produced in large scale. Corynebacterium species especially Corynebacterium glutamicum is widely used for the industrial production of amino acids especially L-glutamate and L- lysine. The C. glutamicum from long period has been used for the industrial production of various amino acids, primary metabolites and nucleotides. This organism is an aerobic gram positive, rod shaped and non-sporulating bacteria which used for the industrial production of amino acids of L-lysine and L-glutamate. This bacterium uses a variety of carbohydrates, alcohols and organic acids as single sources of carbon and energy for growth and also for the amino acid production. The quantity of lysine production by wild (natural) type of Corynebacterium glutamycum is very low, and its cultivation and propagation cannot provide the amino acid required by markets, therefor the wild type of this bacterium is not suitable and cost-effective for industrial purposes. To minimize the gap between increasing demand and production of L-lysine, it has to be produced in large scale. Aspartate kinase (AK) is one of the key enzymes in the biosynthesis of aspartate-derived amino acids such as lysine. This enzyme catalyzes the transfer of the C-phosphate group of ATP to aspartic acid. In most bacteria, the reaction is the first step of branched biosynthetic pathway for lysine, threonine, isoleucine and methionine and is known to be regulated by the end metabolites through feedback inhibition. For example, aspartate kinase from Corynebacterium glutamicum is concertedly inhibited by lysine and threonine, while aspartate kinase I and III from Escherichia coli is inhibited by threonine and lysine, respectively. Due to industrial production of lysine amino acid by using Corynebacterium species, a lot of researches have been done to improve the genetic modification of these microorganisms. Today, bioinformatics tools are available as online access through web-based databases and software, which can be used to study best structures of aspartate kinase enzyme with the least cost and time.  Also due to high laboratory costs, the use of bioinformatics methods will be important in obtaining the final result. The aim of this study was to investigate the bioinformatics structure of aspartate kinase enzyme in different species of Corynebacterium by authenticated bioinformatics databases to suggest best bioinformatics structure of the aspartate kinase enzyme for applying in laboratory cloning and production of lysine amino acid for industrial purposes.

The amino acid sequences of the aspartic kinase enzyme from 33 species of the Corynebacterium was obtained from the NCBI (https://www.ncbi.nlm.nih.gov/protein) and stored as FASTA. In order to study the genetic distances and similarities in 33 species of Corynebacterium a phylogenetic tree was constructed using the Neighbor Joining method using the Mega software (MEGA 6). (A bootstrap check with 1000 replications was also conducted to obtain a confidence level for the branches) ClC main work bench5 software was used to investigate genetic similarities using protein sequences. The Evolutionary properties, physiological and physicochemical properties of aspartate kinase enzyme were studied and investigated in 33 different species of Corynebacterium through valid databases and software of NCBI, MEGA, ProtScale and ProtParam. In order to predict the second structure, two proteins selected from the psipred server were used (http://bioinf.cs.ucl.ac.uk/psipred). For this purpose, the protein sequences of aspartate kinase enzyme in Corynebacterium glutamicum with access numbers of CAO00530.1 and SJM57548.1 introduced into the psipred and their second structure was mapped. Afterward, three-dimensional structure of mentioned protein was modeled using Swiss-model server (https://swissmodel.expasy.org) Then the quality of the two predicted models evaluated by the Rampage server (http://mordred.bioc.cam.ac.uk/ ~ rapper / rampage2.php), and in the next step its ramachandran plot mapped.

The results of evolutionary tree analysis in Corynebacterium species showed that derivation time of aspartate kinase protein in these 33 species is very close. The results of the ProtScale and ProtParam databases showed that the aspartatekinase enzyme of Corynebacterium glutamicum with the access number of CAO00530.1 and SJM57548.1 have the best physicochemical and maximum stability among 33 different species of study. Afterward, with further in silico investigation by the Swiss-Model server and Rampage tool, it was found that the two access numbers of CAO00530.1 and SJM57548.1 had the best three-dimensional structure. From the results of in silico analyses, it can be inferred that the aspartate kinase enzyme with the two access numbers of CAO00530.1 and SJM57548.1 have the best physicochemical properties, the most stable and also the best three-dimensional structure and therefore could be offered for laboratory cloning and production of lysine amino acid for industrial purposes.

Due to wide applications and importance of lysine production in our country and also the necessity of selecting appropriate strain of Corynebacterium for genetic engineering and industrial production, this bioinformatics study was done to predict best structure of aspartate kinase enzyme and best strain of Corynebacterium. Based on the results of our in silico analysis, it is suggested that corynebacterium glutamicum has the best protein structure of aspartate kinase enzyme and may be beneficial to increase Industrial lysine amino acid production.