An enhanced protocol for expression and purification of heat-stable enterotoxin of enterotoxigenic Escherichia coli

We present an improved protocol for expression and purification of heat-stable enterotoxin (STa) of enterotoxigenic Escherichia coli (ETEC). In this protocol, controlled growth conditions at different pHs (7.4, 8.0, and 8.6) were adopted using a bioreactor. In addition, specific adsorbent resins, methacrylate, were used for STa purification. The bioreactor provided optimal ETEC growth at pH 7.4 with high STa production. Furthermore, methacrylate bounded specifically to STa and dramatically enhanced the purification process of STa. The STa-specific activity was high (8.9 × 10(6) units/mg protein), and the minimal effective dose of STa required for production of gut weight to remaining body weight ratio ≥ 0.083 was recorded as less than 0.2 ng in 2-3 days old suckling mice. The protocol presented, produces highly purified STa as documented by matrix-assisted laser desorption ionization-time of flight mass spectroscopy/. Also, as compared with the traditional methods, this procedure is trouble-free and practical for scale-up production and purification of STa peptides.     

Electrochemical-based biosensors for detection of Mycobacterium tuberculosis and tuberculosis biomarkers

Abstract

Early detection of tuberculosis (TB) reduces the interval between infection and the beginning of treatment. However, commercially available tests cannot discriminate between BCG-vaccinated healthy persons and patients. Also, they are not suitable to be used for immunocompromised persons. In recent years, biosensors have attracted great attention due to their simple utility, accessibility, and real-time outputs. These sensors are increasingly being considered as pioneering tools for point-of-care diagnostics in communities with a high burden of TB and limited accessibility to reference laboratories. Among other types of biosensors, the electrochemical sensors have the advantages of low-cost operation, fast processing, simultaneous multi-analyte analyzing, operating with turbid samples, comparable sensitivity and readily available miniaturization. Electrochemical biosensors are sub-divided into several categories including: amperometric, impedimetric, potentiometric, and conductometric biosensors. The biorecognition element in electrochemical biosensors is usually based on antibodies (immunosensors), DNAs or PNAs (genosensors), and aptamers (aptasensors). In either case, whether an interaction of the antigen–antibody/aptamer or the hybridization of probe with target mycobacterial DNA is detected, a change in the electrical current occurs that is recorded and displayed as a plot. Therefore, impedimetric-based methods evaluate resistance to electron transfer toward an electrode by a Nyquist plot and amperometric/voltammetric-based methods weigh the electrical current by means of cyclic voltammetry, square wave voltammetry, and differential pulse voltammetry. Electrochemical biosensors provide a promising scope for the new era of diagnostics. As a consequence, they can improve detection of Mycobacterium tuberculosis traces even in attomolar scales.     

The comparative efficacy of some botanicals for the control of the seed weevil Caryedon serratus Olivier (Col., Bruchidae) in seeds of Acacia seyal Del.

A comparative study on the efficacy of powder and oil extracts from leaves and seed kernels of Azadirachta indica Juss. and Eucalyptus camaldulensis Dehn. against the seed weevil Caryedon serratus Olivier infesting seeds of Acacia seyal Del. was carried out. EO 1, EO 5 and NSKO5% were comparable to BHC 3% in significantly reducing egg laying by C. serratus. With the exception of ELP and NLP, all botanicals significantly reduced egg hatching. Botanicals significantly reduced larval development with the exception of ELP and NLP. EO and NSKO at 3 and 5% were significantly more effective than BHC in reducing larval development by 95–97%. All botanicals had no significant effect on adult emergence which was only affected by BHC. Botanicals caused 4.3–43% mortality of C. serratus adults and the maximum mortality occurred in EO 3% as compared to BHC. It is apparent that oils extracted from E. camaldulensis had a fumigant and knock down effect on adult C. serratus. Extracted oils were more effective than leaf or seed powder. Oils extracted from leaves of E. camaldulensis were volatile and had a knock down effect on adult C. serratus indicating the possibility of using them as alternative fumigants in seed stores.     

Efficacy of crude neem seed kernel oil (NSKO) in controlling the tree locust (Anacridium melanorhodon melanorhodon Walker), a serious pest of the gum arabic producing tree (Acacia senegal L. Willd.) in the Sudan

The present study was carried out at Al Semih (western Sudan) (13° 14′ N: 30° 27′ E: Alt. 550 m). The area is part of the gum arabic belt which is heavily populated with farmers who practice shifting cultivation and/or agroforestry where Acacia senegal L. Willd (the main gum arabic producing tree) is dominant. The tree locust (Anacridium melanorhodon melanorhodon Walker (Orthoptera, Acrididae) is a serious pest of A. senegal. Farmers practice agroforestry within the gum belt where they cultivate their crops e.g. millet, groundnut, sesame and sorghum between A. senegal trees. Gum Arabic is an important source of revenue for farmers. Chemical pesticides have been used to control the tree locust. Although the gum belt (where A. senegal grows) is heavily populated, pesticides are used to control the tree locust in the area. The present study was undertaken with the objective of using alternative environmentally safe pesticides. Several doses of neem seed kernel crude oil (NSKO) were tested against the 4^th, 5^th, 6^th instars and the mature tree locust (A. melanorhodon melanorhodon) in a completely randomized block design. Experiments were carried out in cages placed on part of the gum arabic belt. NSKO at high doses (5 and 10% v/v) significantly reduced feeding, moulting and ovipositing of the tree locust and significantly increased the mortality of all developmental stages tested. However, low doses of NSKO had no significant effect on the tree locust.     

Phage typing,PCR amplification for mecA gene,and antibiotic resistance patterns as epidemiologic markers in nosocomial outbreaks of methicillin resistant Staphylococcus aureus

Staphylococcus aureus is one of the major causes of community and hospital-acquired infections. Bacteriophage considered as a major risk factor acquires S. aureus new virulence genetic elements. A total number of 119 S. aureus isolated from different specimens obtained from (RKH) were distinguished by susceptibility to 19 antimicrobial agents, phage typing, and PCR amplification for mecA gene. All of MRSA isolates harbored mecA gene, except three unique isolates. The predominant phage group is belonging to the (mixed group). Phage group (II) considered as an epidemiological marker correlated to β-lactamase hyper producer isolates. MRSA isolates indicated high prevalence of phage group (II) with highly increase for phage types (Ø3A), which were correlated to the skin. Phage types (Ø80/Ø81) played an important roll in Community Acquired Methicillin Resistant S. aureus (CAMRSA). Three outpatients MRSA isolates had low multiresistance against Bacitracin (Ba) and Fusidic acid (FD), considered as CAMRSA isolates. It was detected that group I typed all FD-resistant MSSA isolates. Phage groups (M) and (II) were found almost to be integrated for Gentamycin (GN) resistance especially phage type (Ø95) which relatively increased up to 20% in MRSA. Tetracycline (TE) resistant isolates typed by groups (II) and (III) in MSSA. Only one isolate resistant to Sulphamethoxazole/Trimethoprim (SXT) was typed by (III/V) alone in MSSA. MRSA isolates resistant to Chloramphenicol (C) and Ba were typed by all groups except (V). It could be concluded that (PERSA) S. aureus isolates from the wound that originated and colonized, and started to build up multi-resistance against the topical treatment antibiotics. In this study, some unique sporadic isolates for both MRSA and MSSA could be used as biological, molecular and epidemiological markers such as prospective tools.     

Comparison of different techniques for detection of Gal-GalNAc, an early marker of colonic neoplasia

The tumor marker, D-galactose-beta [1-3]-N-acetyl-D-galactosamine (Gal-GalNAc, also known as T-antigen) can be identified by a very simple galactose oxidase-Schiff's (GOS) reaction either on tissues or on rectal mucus samples from patients with colorectal neoplasms. Gal-GalNAc is expressed in the neoplastic mucosa as well as the remote non-neoplastic mucosa. It is, however, not expressed in colonic mucosa of normal subjects. We studied the expression of Gal-GalNAc by GOS reaction, lectin reactivity and immunocytochemistry in 10 normal, .45 precancerous [5 Crohn's disease, 15 ulcerative colitis (5 without dysplasia and 10 with dysplasia), 25 tubular adenomas], and 25 adenocarcinoma cases. Normal mucosa remote from tubular adenoma and adenocarcinoma was also studied. The GOS method was compared with reactivity of the lectin jacalin and immunostaining with antibody to T antigen (Anti-Tag Ab). GOS reaction was negative in all of the 10 normal specimens. Of the 5 Crohn's disease specimens, 2 were positive and 3 negative. In the 5 ulcerative colitis cases without dysplasia, positive reaction was seen in 2 cases and negative in 3. Of the 10 cases of ulcerative colitis with dysplasia, 5 showed positivity in dysplastic areas, and 3 of these were also positive in remote non dysplastic mucosa. Twenty of 25 tubular adenomas yielded a positive reaction in the adenoma, 14 of them showing positivity also in remote mucosa; 3 cases showed a positive reaction only in remote mucosa. Of the 25 adenocarcinomas, 21 showed a positive reaction in the adenocarcinoma as well as the remote mucosa. GOS reaction was intense in well differentiated adenocarcinoma and weak in poorly differentiated adenocarcinoma. Intense reaction was also seen in the intracellular mucus of some aberrant crypts and morphologically normal crypts remote from adenocarcinoma and tubular adenoma. GOS reaction showed an overall sensitivity of 75.7% and specificity of 100% for cancer and precancerous lesions. Jacalin reactivity was slightly more sensitive (84.3%) but less specific (80%) and Tag Ab reactivity even less sensitive (50%) but as specific (100%) for neoplastic and dysplastic mucosa. We conclude that the detection of the carbohydrate moiety Gal-GalNAc varies with the technique used. Compared to other techniques, GOS reaction is extremely simple and has a high degree of sensitivity and specificity. It can be used for detection of this tumor marker in remote non-neoplastic mucosa of patients with neoplasia or at risk of developing neoplasia. It, therefore, could be used as a cost effective screening test in rectal biopsy specimens of such patients.     

Expression of a foreign gene in electroporated pollen grains of tobacco

Summary The incorporation of genetically engineered DNA into pollen and subsequent fertilization of eggs by the transformed pollen would be a convenient method for producing genetically engineered seed. This method of pollen transformation would circumvent the need for other types of gene transfer methods such as the use of Agrobacterium tumefaciens, which has a limited host range and thus a limited capability for genetically engineering plants. It would also avoid the problems associated with the regeneration of some plants from tissue, cell, or protoplast culture after receiving foreign DNA. To this end, the genetically engineered plasmid DNA vector pBI221 containing the gene encoding -glucuronidase (GUS) was introduced by electroporation into germinating pollen grains of tobacco (Nicotiana gossei L.). Transient expression of the GUS gene was demonstrated by the presence of GUS activity in fluorometric assays of pollen extracts 24 h after the introduction of pBI221 via electroporation. Intact pBI221 was detected by Southern blotting procedures as a distinct DNA band in pollen extracts 1 h after electroporation. In addition, pBI221 was detected as a diffuse band of higher molecular weight DNA 24 h after electroporation, suggesting that some of the pBI221 was incorporated into the genome of the pollen.     

A comparative study in young male animals of 10 species of the distribution of alkaline phosphatase activity in small arteries

Summary The distribution of alkaline phosphatase activity in the walls of small arteries measuring 100–200 in internal diameter in the collapsed state was investigated in nine organs in male animals of each of ten species employing the Gomori and azo-coupling techniques. Alkaline phosphatase activity was high in relation to the arterial endothelium of the chick, hen, turkey, rabbit, frog and man but was related to the arterial adventitia and not to the endothelium in the rat. In the guinea pig and hamster arterial alkaline phosphatase activity was uniformly low or absent. Within nine of these ten species the pattern of organ distribution of arterial alkaline phosphatase activity in vessels of comparable size was uniform. In the cat however, enzyme activity was confined to the adventitia of small cerebral arteries and to the media of the smallest arteries (arterioles) in all organs except the testis from which arterial activity was absent.The distribution of alkaline phosphatase activity in 100–200 , arteries in the species investigated was not directly related to the presence of capillary endothelial activity which was detected in all the organs examined. The activity in the walls of the smallest arteries (arterioles) was located in different sites in different species.United Arab Republic Research Fellow.