Four-color flow cytometric detection of retrovirally expressed red, yellow, green, and cyan fluorescent proteins

Flow cytometric procedures are described to detect a "humanized" version of a new red fluorescent protein (DsRed) from the coral Discosoma sp. in conjunction with various combinations of three Aequorea victoria green fluorescent protein (GFP) variants--EYFP, EGFP, and ECFP. In spite of overlapping emission spectra, the combination of DsRed with EYFP, EGFP, and ECFP generated fluorescence signals that could be electronically compensated in real time using dual-laser excitation at 458 and 568 nm. Resolution of fluorescence signals from DsRed, EYFP, and EGFP was also readily achieved by single-laser excitation at 488 nm. Since many flow cytometers are equipped with an argon-ion laser that can be tuned to 488 nm, the DsRed/EYFP/EGFP combination is expected to have broad utility for facile monitoring of gene transfer and expression in mammalian cells. The dual-laser technique is applicable for use on flow cytometers equipped with tunable multiline argon-ion and krypton-ion lasers, providing the framework for studies requiring simultaneous analysis of four fluorescent gene products within living cells.     

Study of physio-biochemical responses elicited by potassium phosphite in downy mildew-infected cucumber plants

Potassium phosphite (KPhi) is widely used as a resistance inducer to protect plants against fungal pathogens. In the present study, the effect of KPhi on the activation of defence-related enzymes and biochemicals in Pseudoperonospora cubensis-challenged cucumber plants was investigated. Cucumber plants were treated with KPhi before or after inoculation with P. cubensis and leaf samples were collected at different time courses for physiological and biochemical assessments. Results revealed that the activity of reactive oxygen species (ROS)-scavenging enzymes like catalase, guaiacol peroxidase, superoxide dismutase and ascorbate peroxidase as well as proline and total carbohydrates contents were significantly increased by KPhi application, while hydrogen peroxide (H₂O₂) concentration, as a disease damage indicator was reduced. The maximum activity of ROS-scavenging system was achieved 3–4 days after KPhi application. These findings suggest that KPhi application prior to pathogen infection efficiently triggers plant defence responses which may reduce the disease severity.     

Measurement of ion beam angular distribution at different helium gas pressures in a plasma focus device by large-area polycarbonate detectors

The paper presents an experimental study and analysis of full helium ion density angular distributions in a 4-kJ plasma focus device (PFD) at pressures of 10, 15, 25, and 30 mbar using large-area polycarbonate track detectors (PCTDs) (15-cm etchable diameter) processed by 50-Hz-HV electrochemical etching (ECE). Helium ion track distributions at different pressures, in particular, at the main axis of the PFD are presented. Maximum ion track density of ~4.4 × 10⁴ tracks/cm² was obtained in the PCTD placed 6 cm from the anode. The ion distributions for all pressures applied are ring-shaped, which is possibly due to the hollow cylindrical copper anode used. The large-area PCTD processed by ECE proves, at the present state-of-theart, a superior method for direct observation and analysis of ion distributions at a glance with minimum efforts and time. Some observations of the ion density distributions at different pressures are reported and discussed.     

The roles of FOX proteins in virus-associated cancers

Forkhead box (FOX) proteins play a crucial role in regulating the expression of genes involved in multiple biological processes, such as metabolism, development, differentiation, proliferation, apoptosis, migration, invasion, and longevity. Deregulation of FOX proteins is commonly associated with cancer initiation, progression, and chemotherapeutic drug resistance in many human tumors. FOX proteins deregulate through genetic events and the perturbation of posttranslational modification. The purpose of the present review is to describe the deregulation of FOX proteins by oncoviruses. Oncoviruses utilize various mechanisms to deregulate FOX proteins, including alterations in posttranslational modifications, cellular localization independently of posttranslational modifications, virus-encoded miRNAs, activation or suppression of a series of cell signaling pathways. This deregulation can affect proliferation, metastasis, chemotherapy resistance, and immunosuppression in virus-induced cancers and help to chronic viral infection, development of gluconeogenic responses, and inflammation. Since the PI3K/Akt/mTOR signaling pathway is the upstream FOXO, suppressing it can cause FOXO function to return, and this can be one of the reasons for patients to recover from the infection of the viruses used to treat these inhibitors. Hence, FOX proteins could serve as prognosis markers and target therapy specifically in cancers caused by oncoviruses.     

Of PCOS Symptoms,Hirsutism Has the Most Significant Impact on the Quality of Life of Iranian Women

Introduction

Polycystic ovary syndrome is a common endocrine disorder affecting women both physically and psychologically and can lead to a poor quality of life compared to their normal counterparts. The aim of the present study was to assess the impact of various clinical features of polycystic ovary syndrome on the health-related quality of life of Iranian women diagnosed with this syndrome.

Materials and Methods

A total of 796 women diagnosed with polycystic ovary syndrome, aged 15–49 years, completed the questionnaires, interviews, and medical assessments required for this study. A reliable and validated Persian version of the health-related quality of life questionnaire for polycystic ovary syndrome patients was used. Linear regression models were used to assess the association between the symptoms of polycystic ovary syndrome and health-related quality of life.

Results

The mean age of participants was 28.02 years. 35.4% of the subjects were classified as overweight or obese. Hirsutism, was reported to have the strongest impact on the patients’ health-related quality of life, followed in descending order by body mass index, irregular menses and infertility. The relative level of hirsutism was directly proportional to decrease in health-related quality of life score (p<0.001).

Conclusions

The results of the study found that hirsutism had the strongest impact on the health-related quality of life measures in Iranian women diagnosed with polycystic ovary syndrome. Health care officials need to evaluate in depth the effect of each clinical feature of polycystic ovary syndrome separately and design management strategies, keeping in mind the psychological and physical manifestations.     

Preparation of Effective and Safe Gene Carriers by Grafting Alkyl Chains to Generation 5 Polypropyleneimine

Gene therapy is a novel method to treat a variety of diseases including genetic disorders and cancer. Nonviral gene carriers have now gained considerable attention as gene carrier systems. Polyamidoamine (PAMAM) and polypropyleneimine (PPI) are the two most widely used denderimers in gene delivery studies. The aim of the current study was to investigate the effects of modification of generation 5 polypropyleneimine (G5 PPI) dendrimers with alkanoate groups as hydrophobic moieties on DNA transfection and cytotoxicity. Six, 10, and 16 carbon derivatives of bromoalkanoic acids were conjugated onto PPI with 10%, 30%, and 50% of surface amine grafting. Ethidium bromide exclusion assay results proved the ability of modified carriers to condense DNA. Transfection assay showed higher DNA delivery potential for 30% and 50% grafting with decanoate moieties compared to native G5 PPI and Superfect^TM. 3-(4,5-Dimethylthiazol-2-yl)-2,5-di phenyltetrazolium bromide (MTT) and apoptosis experiments showed lower toxicity for modified carriers compared to unmodified PPI. The hemolytic effect of grafted carriers was not significantly different from G5 PPI. Size and zeta potential measurements revealed that polyplex size was less than 200 nm and electrical charges were in the range 14–25 mV. The hydrophobic modifications improved transfection activity and toxicity of G5 PPI without negatively affecting hemocompatibility. These modified carriers are therefore promising candidates for further in vivo investigations.KEY WORDS: gene delivery, hydrophobic modification, nonviral vector, polypropyleneimine, transfection     

Effect of temperature on development time and life table parameters of Nephus hiekei Fürsch,the important predator of Phenacoccus solenopsis Tinsley

The cotton mealybug, Phenacoccus solenopsis Tinsley (Hemiptera: Pseudococcidae) has become a serious threat to agricultural and ornamental plants in Southwest Iran. Pest-control studies have found Nephus hiekei Fürsch (Coleoptera: Coccinellidae), in large numbers on Phenacoccus solenopsis Tinsley, as a potential predator. However, there has been no investigation on this predator. In this research, the biology and age-stage, two-sex life table parameters of N. hiekei feeding on Ph. solenopsis were investigated at three constant temperatures (27, 32 and 37 ± 1 °C, 65 ± 5% RH and a photoperiod of 16: 8 (L: D) h. The duration of total pre-adult stage was found to decrease raising temperature from 26.98 ± 0.36 days at 27 °C to 14.24 ± 0.27 days at 37 °C. The oviposition period lasted 63.95 ± 3.14, 66.42 ± 3.34 and 25.20 ± 1.59 days at 27, 32, and 37 °C, respectively. Females laid an average of 365.68 ± 22.36, 543.79 ± 27.27 and 106.25 ± 6.38 eggs, at these three temperatures, respectively. The intrinsic rate of increase (r_m = 0.1333 ± 0.0050 d^–1), finite rate of increase (λ = 1.1423 ± 0.0057 d^–1) and net reproductive rate (R₀ = 188.89 ± 28.34 offspring) as well as gross reproductive rate (GRR = 308.31 ± 39.54 offspring) were greatest at 32 °C. The shortest mean generation time (T = 26.74 ± 0.65 days) was recorded at 37 °C and the longest at 27 °C (T = 56.48 ± 1.0 days). These results indicate that N. hiekei can successfully survive and reproduce at temperatures of around 32 °C, and has good potential to be an effective biological control agent of Ph. solenopsis.     

Molecular docking based virtual screening of compounds for inhibiting sortase A in L.monocytogenes

Listeriosis is considered as an important public health issue. Sortase A (srtA) is an enzyme with catalytic role in L. monocytogenes that breaks the junction between threonine and glycine in the LPXTG motif (a key motif in internalin A (InlA) that plays an important role in listeriosis). Inactivation of srtA was shown to inhibit anchoring of the invasion protein InIA. This is in addition to inhibiting peptidoglycan-associated LPXTG proteins. Therefore, it is of interest to inhibit strA using potential molecules. Here, we describe the design of an inhibitor with high binding affinity to srtA with the ability to prevent the attachment of srtA to the LPXTG proteins such as InIJ. A homology model of Listeria monocytogenes Sortase A was developed using MODELLER (version 9.12). We screened StrA to 100,000 drug-like ligands from the Zinc database using Molecular docking and virtual screening tool PyRX). Pharmacokinetic analysis using the FAFDrugs³ web server along with ADME and toxicity analysis based on Lipinski rule of five were adopted for the screening exercise followed by oral toxicity check using PROTOX (a server) for every 10 successive hits. The results from PROTOX server indicated that Lig #1 (with LD50 of 2000mg/kg) and Lig #7 (with LD50 of 2000mg/kg) have toxicity class 4 and Lig #3 (with LD50 of 14430mg/kg) has toxicity class 6. Subsequent modifications of these structures followed by FAFDrugs³ analysis for high bioavailability value selected Lig #7 according to Lipinski rules of five. Thus, Lig #7 with IUPAC name ((R)-4-{(S)-1-[(S)-2-Amino-4-methylvaleryl]-2-pyrrolidinyl}-1-[(S)-1-(ethylamino) carbonyl-propylamino] -2-propyl-1, 4- butanedione) is suggested as a potential candidate for srtA inhibition for further consideration.