Allelic frequency and genotypes of prion protein at codon 136 and 171 in Iranian Ghezel sheep breeds

PrP genotypes at codons 136 and 171 in 120 Iranian Ghezel sheep breeds were studied using allele-specific PCR amplification and compared with the well-known sheep breeds in North America, the United States and Europe. The frequency of V allele and VV genotype at codon 136 of Ghezel sheep breed was significantly lower than AA and AV. At codon 171, the frequency of allele H was significantly lower than Q and R. Despite the similarities of PrP genotypes at codons 136 and 171 between Iranian Ghezel sheep breeds and some of the studied breeds, significant differences were found with others. Planning of effective breeding control and successful eradication of susceptible genotypes in Iranian Ghezel sheep breeds will not be possible unless the susceptibility of various genotypes in Ghezel sheep breeds to natural or experimental scrapie has been elucidated.Key words: scrapie, Ghezel sheep breed, PrP genotyping, allele specific amplification, codon 136, codon 171Scrapie was first described in England in 1732,¹ and it is an infectious neurodegenerative fatal disease of sheep and goats belonging to the group of transmissible subacute spongiform encephalopathies (TSEs), along with bovine spongiform encephalopathy (BSE), chronic wasting disease and Creutzfeldt-Jakob disease.^2,3 The term prion, proteinaceous infectious particles, coined by Stanley B. Prusiner, was introduced, and he presents the idea that the causal agent is a protein.⁴ Prion proteins are discovered in two forms, the wild-type form (PrP^c) and the mutant form (PrP^Sc).⁵ Although scrapie is an infectious disease, the susceptibility of sheep is influenced by genotypes of the prion protein (PrP) gene.^2,6 Researchers have found that the PrP allelic variant alanine/arginine/arginine (ARR) at codons 136, 154 and 171 is associated with resistance to scrapie in several breeds.^7–14 Most of the sheep populations in the Near East and North African Region (84% of the total population of 255 million) are raised in Iran, Turkey, Pakistan, Sudan, Algeria, Morocco, Afghanistan, Syria and Somalia.¹⁵ In 2003, the Iranian sheep population was estimated at 54,000,000 head. The Ghezel sheep breed, which also is known as Kizil-Karaman, Mor-Karaman, Dugli, Erzurum, Chacra, Chagra, Chakra, Gesel, Gezel, Kazil, Khezel, Khizel, Kizil, Qezel, Qizil and Turkish Brown, originated in northwestern Iran and northeastern Turkey. By considering sheep breeds as one of the main sources of meat, dairy products and related products, a global screening attempt is started in different areas. In compliance with European Union Decision 2003/100/EC, each member state has introduced a breeding program to select for resistance to TSEs in sheep populations to increase the frequency of the ARR allele. A similar breeding program is established in United States and Canada. The Near East and North African Region still needs additional programs to help the global plan of eradication of scrapie-susceptible genotypes. The current study was the first to assess the geographical and molecular variation of codons 136 and 171 polymorphism between Iranian Ghezel sheep breed and well-known sheep breeds.Polymorphism at codon 136 is associated with susceptibility to scrapie in both experimental and natural models.^10,11,13,16 17 and Austrian Carynthian sheep.¹⁸ Swiss White Alpine showed higher frequency of allele V at position 136 than Swiss Oxford Down, Swiss Black-Brown Mountain and Valais Blacknose.¹⁹ Comparison of polymorphism at codon 136 in the current study with some of other breeds (20 some flock of Hampshire sheep²¹ with current study, but the frequency of it is higher than that of some other breeds.

Table 1

Comparison of PrP allelic and genotype frequencies at codon 136 in different breeds

PEG-PLA nanoparticles decorated with small-molecule PSMA ligand for targeted delivery of galbanic acid and docetaxel to prostate cancer cells

Prostate cancer (PCa) is one of the most prevalent non-drug delivery system cutaneous malignancies. Undoubtedly, introducing novel treatment options to achieve higher therapeutic index will be worthwhile. In this study, we report for the first time, a novel targeted self-assembled based on PEG-PLA nanoparticles (PEG-PLA NPs) containing galbanic acid (GBA) and docetaxel, which was targeted using ((S)-2-(3-((S)-5-amino-1-carboxypentyl) ureido) pentanedioic acid (ACUPA), a small molecule inhibitor targeting prostate-specific membrane antigen (PSMA), in prostate cancer cell line. The prepared NPs were characterized by different analytical methods. The MTT assay was used to compare the anti-proliferation of drugs-loaded PEG-PLA NPs and ACUPA-PEG-PLA against LNCaP (PSMA⁺) and PC3 (PSMA⁻) cells. PEG-PLA NPs with an average size of 130–140 nm had an enhanced release of GBA and docetaxel at pH 5.5 compared with pH 7.5. Spectrofluorometric analysis suggested that ACUPA-modified PEG-PLA could effectively enhance the drug uptake in PSMA⁺ prostate cancer cells. Cytotoxicity studies showed that the targeted NPs loaded with different concentrations of GBA and fixed concentration of docetaxel (4 nM) have shown higher toxicity (IC₅₀ 30 ± 3 µM) than both free GBA (80 ± 4.5 µM) and nontargeted NPs (IC₅₀ 40 ± 4.6 µM) in LNCaP cells. Collectively, these findings suggest that ACUPA-conjugated PEG-PLA nanosystem containing GBA and docetaxel is a viable delivery carrier for various cancer-targeting PSMA that suffer from short circulation half-life and limited therapeutic efficacy.     

Structure-activity relationships of 3-aminoquinazolinediones, a new class of bacterial type-2 topoisomerase (DNA gyrase and topo IV) inhibitors

A series of 3-aminoquinazolinediones was synthesized and evaluated for its antibacterial and DNA gyrase activity. The SAR around the quinazolinedione core was explored and the optimal substitutions were combined to give two compounds, 2r and 2s, with exceptional enzyme potency (IC50 = 0.2 microM) and activity against gram-positive organisms (MIC's = 0.015-0.06 microg/mL).     

A novel human bone morphogenetic protein-7 variant with an enriched heparin-binding site

BMPs are osteoinductive proteins which are used in treatment of acute fractures. Large quantities of recombinant proteins are usually needed to achieve efficacy in the clinic. This translates to severe complications and high costs. Different strategies have been developed to improve the efficacy and safety of BMPs. Modification of the heparin-binding site in order to increase the local retention time of the morphogen is one of these approaches. Aiming at further improvement in properties of BMP-7, a novel form of this protein was designed and expressed successfully in Chinese Hamster Ovarian (CHO) cells. Substitution of the Bone morphogenetic protein-7 N-terminus by the heparin-binding site of Bone morphogenetic protein-2 was carried out to increase the heparin binding capacity of the novel protein. It was found that the novel variant, retained its in vitro biological activity and the heparin binding capacity of this protein was approximately 20% higher than that of the wild-type at a protein concentration of 100 ng/mL. The novel protein as the first variant of hBMP-7 with the enriched heparin-binding site may offer more advantages in clinical use as compared to the existing commercial form.     

Determining the most effective traits to improve saffron (<Emphasis Type="Italic">Crocus sativus</Emphasis> L.) yield

To determine the effective traits to improve saffron yield, a split plot design based on RBCD was done in Mashhad region in Iran for three years (2012–2014). The results showed that all traits except number of daughter corm, fresh weight of daughter corm and dry leaf weight had low general heritability. Results of genotypic and phenotypic coefficients of variation and genetic advance demonstrated that the majority of traits had a low diversity and the selection did not have any effect in improving the traits. As a result, the best way to increase saffron yield is improvement of farm management. It was also found that saffron yield had the highest phenotypic and genotypic correlations with fresh and dry weight of daughter corm and dry and fresh flower weight. Therefore, the efforts to improve these traits will increase saffron yield. According to the present study 5-Jun to 5-Jul was found to be the best sowing date for planting saffron. Also, the Mashhad and Torbat ecotypes were the best ecotypes in this study. Phenotypic and genotypic path analysis showed that in the first step three traits number of daughter corm, fresh flower weight and flower number and in the second step traits fresh weight of daughter corm, dry flower weight and dry leaf weight interred to the regression model and had the highest positive direct and indirect effects on saffron yield. Mainly, it can be derived that the implementation of correct farm management including appropriate sowing date, saffron ecotypes, proper density, bigger and higher quality saffron corm can play an important role in improving yield components and subsequently increasing saffron yield.     

Formulation and evaluation of anticancer and antiangiogenesis efficiency of PLA–PEG nanoparticles loaded with galbanic acid in C26 colon carcinoma,in vitro and in vivo

Galbanic acid (GBA) is an active sesquiterpene coumarin derivative, with various medicinal benefits, including anticancer properties. However, the low solubility of GBA is the main limitation of its clinical applications. In this study, we used a nanosystem based on poly (D, l -lactide)–polyethylene glycol (PLA–PEG), for the delivery of GBA to C26 colon carcinoma cells. The physicochemical characteristics of nanoparticles (NPs) prepared by the emulsification–evaporation method were evaluated. MTT assay was used to compare the anticell proliferation of GBA and PLA–PEG–GBA against C26 cell lines. PLA–PEG-NPs with an average size of about 140 nm had an enhanced release of GBA at a pH of 5.5 compared with a pH of 7.4. Cytotoxicity studies showed that the IC ₅₀ of the PLA–PEG–GBA NPs (8 µM) was significantly lower than free GBA (15 µM). In the in vivo study, PLA–PEG–GBA NPs exhibited remarkable efficacy and reduced in vivo toxicity in C26 colon carcinoma tumor-bearing female BALB/c mice. To study the antiangiogenesis effect of the NPs, tumor sections were stained with an anti CD34 antibody. The results show the CD34 (+) vessels were decreased in the GBA and PLA–PEG–GBA treated mice by more than 75% and 90%, respectively. These results suggest that the encapsulation of GBA into the PLA–PEG could potentially be used for the treatment of colorectal cancer.     

Proteomics screening of molecular targets of curcumin in mouse brain

Aims

Curcumin is one of the most important constituent of Curcuma longa L. with antioxidant, anti-inflammatory and anticancer effects. In this study, we investigated potential intracellular targets of curcumin by affinity chromatography based on target deconvolution. Identification of curcumin interacting proteins may help in evaluating biological and side effects of this natural compound.

Main methods

Curcumin was immobilized through a linker to sepharose beads as solid matrix. Pull down assay was performed by passing tissue lysate of mouse brain through the column to enrich and purify curcumin interacting proteins. Then proteins were separated using two-dimensional gel electrophoresis and identified using MALDI/TOF/TOF mass spectrometry.

Key findings

Our results show that curcumin physically binds to a wide range of cellular proteins including structural proteins, metabolic enzymes and proteins involved in apoptosis pathway.

Significance

Finding curcumin interacting proteins may help in understanding a part of curcumin pharmacological effects.     

Prolonged viral shedding and antibody persistence in patients with COVID-19

SARS-CoV-2 as a new global threat has affected global population for one year. Despite the great effort to eradicate this infection, there are still some challenges including different viral presentation, temporal immunity in infected individuals and variable data of viral shedding. We studied 255 COVID-19 suspected individuals to assess the viral shedding duration and also the antibody development against SARS-CoV-2 among the cases. Real Time RT-PCR assay was applied to determine the virus presence and SARS-CoV-2 antibodies were evaluated using SARS-CoV-2 IgM and IgG kits. 113 patients were confirmed for COVID-19 infection. The patients were followed until negative PCR achieved. The median viral shedding among studied population was obtained 34.16 (±17.65) days which was not significantly associated with age, sex and underlying diseases. Shiver and body pain were found in prolonged form of the infection and also patients who had gastrointestinal problems experienced longer viral shedding. Moreover, IgG was present in 84% of patients after 150 days. According to this data, the median viral shedding prolongation was 34.16 days which indicates that 14 days isolation might not be enough for population. In addition, IgG profiling indicated that it is persistent in a majority of patients for nearly 6 months which has brought some hopes in vaccine efficacy and application.     

Homology Modeling of an Alternative Splice Variant of Human Granulocyte Colony-Stimulating Factor,G-CSF Isoform D,and Study of Its Binding Properties by Molecular Docking

International Journal of Peptide Research and Therapeutics - Granulocyte colony-stimulating factor (G-CSF) is known as the major mediator of granulopoiesis. However, overexpression of G-CSF has...