Chloroplast localization of Cry1Ac and Cry2A protein- an alternative way of insect control in cotton

BACKGROUND: Insects have developed resistance against Bt-transgenic plants. A multi-barrier defense system to weaken their resistance development is now necessary. One such approach is to use fusion protein genes to increase resistance in plants by introducing more Bt genes in combination. The locating the target protein at the point of insect attack will be more effective. It will not mean that the non-green parts of the plants are free of toxic proteins, but it will inflict more damage on the insects because they are at maximum activity in the green parts of plants. RESULTS: Successful cloning was achieved by the amplification of Cry2A, Cry1Ac, and a transit peptide. The appropriate polymerase chain reaction amplification and digested products confirmed that Cry1Ac and Cry2A were successfully cloned in the correct orientation. The appearance of a blue color in sections of infiltrated leaves after 72 hours confirmed the successful expression of the construct in the plant expression system. The overall transformation efficiency was calculated to be 0.7%. The amplification of Cry1Ac-Cry2A and Tp2 showed the successful integration of target genes into the genome of cotton plants. A maximum of 0.673 µg/g tissue of Cry1Ac and 0.568 µg/g tissue of Cry2A was observed in transgenic plants. We obtained 100% mortality in the target insect after 72 hours of feeding the 2nd instar larvae with transgenic plants. The appearance of a yellow color in transgenic cross sections, while absent in the control, through phase contrast microscopy indicated chloroplast localization of the target protein. CONCLUSION: Locating the target protein at the point of insect attack increases insect mortality when compared with that of other transgenic plants. The results of this study will also be of great value from a biosafety point of view.

Cloning and chloroplast-targeted expression studies of insect-resistant gene with ricin fusion-gene under chloroplast transit peptide in cotton

Background: Transgenic plants inhabiting single Bt gene are prone to develop insect resistance and this resistance has been reported in case of some important yield-devastating insect larvae of commercial crops, such as cotton and rice. Therefore, it has become essential to adapt new strategies to overcome the problem of insect resistance and these new strategies should be sophisticated enough to target such resistant larvae in broad spectrum. Among these, plants may be transformed with Bt gene tagged with some fusion-protein gene that possesses lectin-binding capability to boost the binding sites for crystal protein gene within insect mid-gut in order to overcome any chances of insect tolerance against Bt toxin. Enhanced chloroplast-targeted Bt gene expression can also help in the reduction of insect resistance. Results: In the present investigation, a combined effect of both these strategies was successfully used in cotton (G. hirsutum). For this purpose, plant expression vector pKian-1 was created, after a series of cloning steps, carrying Cry1Ac gene ligated with chloroplast transit peptide towards N-terminal and Ricin B-Chain towards C-terminal, generating TP-Cry1Ac-RB construct. Conclusions: Efficacy of pKian-1 plasmid vector was confirmed by in-planta Agrobacterium-mediated leaf GUS assay in tobacco. Cotton (G. hirsutum) local variety MNH-786 was transformed with pKian-1 and the stable integration of TP-Cry1Ac-RB construct in putative transgenic plants was confirmed by PCR; while fusion-protein expression in cytosol as well as chloroplast was substantiated by Western blot analysis. Whereas, confocal microscopy of leaf-sections of transgenic plants exposed that hybrid-Bt protein was expressing inside chloroplasts.

In vitro susceptibility of chloramphenicol against methicillin-resistant staphylococcus aureus

To determine the in vitro susceptibility of chloramphenicol against methicillin-resistant Staphylococcus aureus. Cross-sectional study. Department of Microbiology, Armed Forces Institute of Pathology, Rawalpindi, from January to June 2012. One hundred and seventy four isolates of methicillin-resistant Staphylococcus aureus were included in this study using cefoxitin [30 microg] disc for detection. Minimum inhibitory concentration [MIC] of chloramphenicol against MRSA was determined by using E-strip [AB BIO DISK]. The susceptibility was determined by swabbing the Mueller-Hinton agar [MHA] plates with the resultant saline suspension of MRSA and applying E-strip of chloramphenicol from AB Biodisk Sweden and determining the MIC of chloramphenicol [in microg/ml]. Clinical and Laboratory Standards Institute [CLSI] recommendations of /= 32 microg/ml as resistant were followed in interpreting the results. Out of the 174 MRSA isolates, 132 [75.86%] isolates were susceptible to chloramphenicol with MICs of A[2] 8 microg/ml, 38 [21.84%] were resistant >/= 32 microg/ml while 4 [2.30%] were in intermediate range with MIC of 16 microg/ml. Chloramphenicol has shown good in vitro activity against MRSA and is likely to have a key role in the treatment of MRSA infections providing us a good alternative to newer expensive antimicrobials in resource limited countries

Susceptibility pattern of extended specturm beta-lactamase producing isolates in various clinical specimens

To determine the susceptibility pattern of extended spectrum betal-lactamase [ESBL] producing Gram negative isolates from various clinical specimens. Descriptive study. Microbiology Department, Armed Forces Institute of Pathology, Rawalpindi, from January 2008 to January 2009. A total of 308 ESBL producing isolates from various clinical specimens sent to AFIP for culture and sensitivity were identified using standard microbiological techniques and tested for antimicrobial susceptibility. At the same time screening for ESBL production was also done. ESBL production was confirmed by combination disc synergy method. The susceptibility pattern of isolates was then recorded in frequency percentages. Out of the 308 ESBL producing isolates more than 99% were susceptible to carbapenems, 84% to tazobactam/ piperacillin, 81% to sulbactam/cefoperazone, 12% to fluoroquinolones, 13% to cotrimoxazole, 59% to amikacin and 18% to gentamicin. Among the urinary isolates 49% were susceptible to Nitrofurontoin and only 5% to Pipemidic acid. Antibiotic choices in case of ESBL producing isolates are limited and at present only carbapenems can be regarded as treatment of choice. As empirical agents, beta-lactam/beta lactamase inhibitor combinations should be used cautiously for serious infections. Fluoroquinolones showed very poor efficacy. Amikacin can be used alternatively in such cases. Nitrofurantoin is still a good oral agent for treating UTI

In vitro efficacy of piperacillin/sulbactam, piperacillin/ tazobactam and cefoperazone/sulbactam against clinical isolates of pseudomonas aeruginosa - a comparative study

To determine the in vitro efficacy of Piperacillin/Sulbactam, Piperacillin/Tazobactam and Cefoperazone/Sulbactam against clinical isolates of Pseudomonas aeruginosa. Cross-sectional study Department of Microbiology, Armed Forces Institute of Pathology from January 2010 to September 2010. A total of 287 isolates of Pseudomonas aeruginosa recovered from various clinical specimens were taken under consideration. Routine microbiological methods were used to identify the organism. Susceptibility of the isolates was carried out by modified Kirby-Bauer disc diffusion method against piperacillin 100/sulbactam 30 [SPR130micro g], cefoperazone 75/sulbactam 30 [SCF105micro g] and piperacillin 100/tazobactam 10 [TZP110micro g], according to the guidelines provided by Clinical and Laboratory Standards Institute [CLSI]. The highest numbers of Pseudomonas aeruginosa isolates were found in pus swabs, followed by urine and endobronchial washings. Seventy five percent of clinical isolates of P. aeruginosa were susceptible to tazobactam/piperacillin, 71% to piperacillin/sulbactam and 70% to cefoperazone/sulbactam. The difference between the susceptibility of isolates to these three antimicrobials was statistically not significant [p>0.05]. We conclude that there was very little difference in the antimicrobial susceptibility of P. aeruginosa to the three beta-lactam/beta-lactamase inhibitor combination drugs studied. Periodic susceptibility testing should be carried out over a period of two to three years, to detect the current resistance trends. Moreover, a rational strategy on the limited and prudent use of anti-Pseudomonal agents is urgently required

Non-dermatophyte moulds as pathogens of onychomycosis

To determine the role and pattern of non-dermatophyte moulds as causative agents of onychomycosis. Case series. Department of Microbiology, Armed Forces Institute of Pathology [AFIP], Rawalpindi, from November 2009 to July 2010. Nail clippings and nail scrapings were obtained from abnormal looking nails with treatment and detection failure for onychomycosis. Microscopic [40% potassium hydroxide mounts] examination and culture on Sabouraud's dextrose agar [SDA], SDA containing chloramphenicol, and SDA containing actidione and chloramphenicol were used for species identification. Non-dermatophyte moulds were isolated from 32 out of the total 47 culture positive cases [68%]. Alternaria alternata was the commonest species [46%]. Dermatophytes were isolated from only 7 patients [15%] belonging to genus Trichophyton. Yeasts were isolated in 8 [17%]. There was no fungal growth in 53% of cases. The non-dermatophytes should be considered important in evaluating the culture negative cases for dermatophytes as well as those cases ending up in treatment failure after empirical treatment for dermatophyte infections

Spectrum and antimicrobial susceptibility of bacterial organisms isolated from patients in a bone marrow transplant centre-A review of 6 years

To determine the spectrum and antimicrobial susceptibility pattern of pathogens isolated from bone marrow transplant centre and to examine the evolution of bacterial resistance to antibiotics in these patients. It was a retrospective study conducted in the department of Microbiology, Armed Forces institute of Pathology [AFIP] from January 2004 to December 2009. Isolation, identification and antimicrobial susceptibilities were performed by standard methods. Gram negative bacterie accounted for 50.4% of the total isolates and Gram positives 49.6%. Escherichia coli was the most common isolate among the gram negative rods followed by Pseudomonas spp and Acinetobacter spp. Amongst the Gram positive organisms staphylococcus aureus and coagulase negative staphylococci followed by enterococci were the predominant organisms. The susceptibility of methicillin resistant staphylococcus aureus remained 100% for Vancomycin and Linezolid, while those of minocycline, fusidic acid and doxycycline were at an acceptable level of above 60%. Imipenem remained the most effective antibiotic for Gram negative rods. Acinetobacter spp. was the most problematic organism as only 62% isolates were susceptible to imipenem. Linezolid, vancomycin and tigecycline were very effective for enterococci. Ciprofloxacin sensitivity has been reduced among most of the bacteria isolated. The spectrum of isolates from neutropenic patients in our population appears to be changing with a shift towards Gram-positive microorganisms. At the same time resistance to most of the commonly used antimicrobials is increasing. Continuous surveillance of their susceptibility patterns is essential for formulation of empiric therapeutic regimens for these patients

Antimicrobial activity of tigecycline against methicillin resistant staphylococcus aureus in a tertiary care setting

To determine the in vitro efficacy of tigecycline against methicillin resistant Staphylococcus aureus [MRSA]. Department of Microbiology Army Medical College and Armed Forces Institute of Pathology Rawalpindi, from Feb 2008 to Jan 2009. One hundred clinical isolates of MRSA were taken, Detection of MRSA was done using 30 microg disc of cefoxitin as recommended by Clinical laboratory Standard Institute [CLSI]. Susceptibility of the isolates to tigecycline was done by employing modified Kirby Bauer disk diffusion technique, according to the guidelines provided by the Food and Durg Administration [FDA]. Minimum inhibitory concentrations [MICs] of the isolates were determined by using E-strips [bioMerieux] of tigecycline. Results were interpreted according to FDA recommendations. All MRSA isolates were susceptible to tigecycline by disc diffusion method. The MICs of tigecycline revealed that all MRSA isolates were in sensitive range. In an era of rapidly growing antibiotic resistance, tigecycline has been found to have very good in vitro efficacy against MRSA isolates

Comparison of two different sensitivity testing agars for detecting methicillin resistance in Staphylococcus aureus

To compare the accuracy of Mueller-Hinton agar and Isosensitest agar using cefoxitin disc for detecting methicillin resistant Staphylococcus aureus using mecA gene PCR assay as gold standard. Comparative study. Department of Microbiology, Armed Forces Institute of Pathology, Rawalpindi, from May 2006 to January 2007. One hundred clinical isolates of Staphylococcus aureus were evaluated; 64 MRSA [methicillin resistant Staphylococcus aureus] and 36 MSSA [methicillin sensitive Staphylococcus aureus] by mecA PCR assay All the isolates were tested with cefoxitin 30 micro g disc using semi-confluent growth on Mueller-Hinton agar as well as on Iso-sensitest agar in ambient air at 35-37°C after an overnight incubation as per recommendations of Clinical and Laboratory Standard Institute. Following diameters provided the best sensitivity and specificity without substantial overlapping between the zones of resistant and sensitive isolates; Mueller-Hinton agar: R /= 22 mm [sensitivity 97.2% and specificity 100%], and Iso-sensitest agar: R /= 26 mm [sensitivity 100% and specificity 100%]. High accuracy was obtained with cefoxitin disc on both media. Performance of both media was equally convincing for reliable prediction of methicillin resistance in Stapfylococcus aureus by placing cefoxitin 30 micro g disc on either of these in routine susceptibility testing

Insect resistance and risk assessment studies of advanced generations of basmati rice expressing two genes of Bacillus thuringiensis

Advanced generations of different transgenic lines of indica basmati rice (Basmati-370) expressing two unrelated Bt genes, cry1Ac and cry2A were evaluated for resistance to Yellow Stem Borer (YSB) and Rice Leaf Folder (RLF) under field conditions compared to control lines over three years (2003-2005). Homozygous lines were selected and analyzed for insect resistance, morphological, physiochemical properties and risk assessment studies. After artificial infestation of target insects, the transgenic plants showed significant resistance. Data were recorded in terms of dead hearts and white heads at vegetative and flowering stage respectively. Transgenic lines showed up to 100 and 96 percent resistance against yellow stem borer at vegetative and flowering stages, respectively. Natural damage of rice leaf folder was also observed during the year 2005. The transgenic plants were 98 percent more resistant as compared to untransformed control plants. Variations in some morphological characteristics, e.g., the average number of tillers, plant height and maturity were also observed. Transgenic lines produced 40 percent more grains than control plants. All these characteristics were stably inherited in advanced generations. The transgenic lines had no significant effect on non-target insects (insects belonging to orders other than Lepidoptera and Diptera) in field or under storage conditions. Chances of pollen-mediated gene flow were recorded at a rate of 0.14 percent.

effect of essential hypertension on serum uric acid level

Patient with arterial hypertension and no definable cause are said to have essential, primary or idiopathic hypertension. A total of 100 patients suffering from essential hypertension and 100 control subjects with almost same mean age and sex distribution were enrolled in this study. Levels of serum uric acid, total cholesterol, TG, and HDL-c were determined by enzymatic colorimetric method. Serum LDL-c and VLDL-c was calculated by using Friedewald and Wilson's formulae. The study was aimed to find out the levels of serum uric acid and lipid profile in patients with essential hypertension and to compare them with levels of normal healthy individuals. Results show increased level of serum uric acid and lipid parameters except for HDL-c, which was significantly, decreased in patients with essential hypertension as compared to the control subjects. Variations in aforementioned parameters were also observed on comparing the obese and the non-obese patients. It can be concluded from the present study that the essential hypertension is associated with abnormalities in the levels of serum uric acid and lipid profile

Genus and species-specific IgG and IgM antibodies for pulmonary tuberculosis

To evaluate three different enzyme immunoassays for serological diagnosis of pulmonary tuberculosis and to compare their diagnostic accuracy in different combinations. Design: A non-interventional comparative study. Place and Duration of Study: The study was carried out at the Department of Microbiology, Armed Forces Institute of Pathology, Rawalpindi between April and September 2001. Subjects and Sera from patients suffering from pulmonary tuberculosis [n=94] with sputum positive for acid fast bacilli [AFB] and sera from control group of healthy individuals [n=90] with sputum negative for AFB were tested by Pathozyme-Myco G EIA, Pathozyme-TB Complex Plus EIA and Pathozyme Myco M EIA kits for the genus-specific IgG and IgM, and the species-specific IgG antibodies against antigens of Mycobacterium tuberculosis. The detection of IgG against genus-specific antigens by Pathozyme-Myco G had a sensitivity of 46% and a specificity of 93%, of IgG against species-specific antigens by Pathozyme-TB Complex Plus had a sensitivity of 64% and specificity of 97% and of IgM against genus-specific antigens by Pathozyme Myco M had a sensitivity of 67% and specificity of 98%. When the results of these immunoassays were evaluated in combination, their sensitivity improved. Combination of genus-specific IgM and species-specific IgG yielded best results with a sensitivity of 87% and specificity of 93%. The sensitivity of serological diagnosis of tuberculosis is low, but it can be increased by utilizing a combination of several antigens

Prevalence of anti HCV in hemodialysis patients

Eighty two patients. 55 males and 27 females, aged 18 to 80 years [mean 42.32 +/- 14.95 years] on chronic hemodialysis were screened for sero- markers of hepatitis B and C and their Liver function was evaluated. Fifty one patients [62.2%] showed anti Hepatitis C antibodies [anti HCV] in their serum. Seven patients [8.5%] were Hepatitis B surface antigen positive; live of them were positive for both anti HCV and HBsAg. Liver functions tests were performed in 70 patients; 22 of them had abnormal liver function. Out of these, 18 patients [81.8%] were anti HCV positive and 4 cases [18.2%] were negative for anti HCV antibodies [P<0.05]. Eighteen of anti HCV positive [39.1%] and 4 of anti HCV negative [16.7%] had abnormal liver functions. Out of 51 Anti HCV Positive patients 43 had received blood transfusions [84.3%]while 8 patients [15.7%] did not have any transfusion [P<0.05]. The patients, who were Anti HCV positive, had more blood transfusions and were on hemodialysis for longer periods as compared to Anti HCV negative patient but the difference in our study was not statistically significant

Effectiveness of recombinant hepatitis B vaccine

The study was carried out to see the efficacy of recombinant hepatitis B vaccine in two high risk groups viz; chronic renal failure patients on maintenance haemodialysis [n = 20] and medical staff [n = 25]. The pre-vaccination status of these individuals was assessed by HBsAg, Anti-HBs and LFTs. Only those subjects were included in this study who were negative for HBsAg and Anti-HBs. The patients were given 2cc while the staff 1cc dose of the vaccine intramuscularly at the schedule of 0,1 and 6 months. Quantitative estimation of antibody titre [seroconversion] was noted by ELISA method 15 days after the last dose. This became positive in 84% of the medical staff members and 65% of the patients in a concentration of >/= 10 IU/L. No significant side effects were noted in any of the subject

Hospital associated hyponatremia

Out of 7843 hospitalized patients 342 [4.36%] have serum electrolytes abnormalities. Hyponatremia was the most common which was seen in 3.45% of all patients and 79.2% of 342 patients with serum electrolyte abnormalities. 73.4% had mild hyponatremia [serum sodium concentration 121-130 mE[q]/L] 53 patients [9.6%] had moderate hyponatremia, [serum sodium concentration 111-120 mE[q]/L] and 19 patients [7%] had severe hyponatremia [serum sodium 110 mE[q]/L or less]. Advanced renal failure was the most common cause while gastrointestinal problems with vomiting diarrhea, congestive heart failure, liver disease, post-operative states, and i.v. fluid administration were other frequent causes. Mildly hyponatremic patients were usually asymptomatic but progressive symptomatology appeared with further decline of serum sodium concentration. Cerebral symptoms were seen in 8% of patients with mild hyponatremia 53% of cases with moderate and 84% of cases with severe hyponatremia. 9 patients with hyponatremia died, mortality being 3.3%

Effectiveness of recombinant hepatitis B vaccine

The study was carried out to see the efficacy of recombinant hepatitis B vaccine [Engerix 'B' by SK and F] in two high risk groups viz; chronic renal failure patients on maintenance haemodialysis [n=20] and medical staff [n-25]. The prevaccination status of these individuals was assessed by HB[s]A[g], Anti-HB[s] and LFT[s]. Only those subjects were included in this study who were negative for HB[s]A[g] and Anti-HB[s]. The patients were given 2cc while the staff 1cc dose of the vaccine intramuscularly at the schedule of 0, 1 and 6 months. Quantitative estimation of antibody titre [seroconversion] was noted by ELISA method 15 days after the last dose. This become positive in 84% of the medical staff members and 65% of the patients in a concentration of >/= 10IU/L

Hospital associated hyponatremia

Out of 7843 hospitalized patients admitted in Shaikh Zayed Hospital between April to August, 1988, 342 [4.36%] [had serum electrolytes abnormalities. Hyponatremia was the most common and was seen in 3.45% of all patients and 79.2% of 34.2 patients, with serum electrolyte abnormalities. 73.4% had mild hyponatremia [serum concentration 121-130 mEq/I] 19.6% patients had moderate hyponatremia, [serum sodium concentration 111-120mEq/L] and 7% had severe hyponatremia [serum sodium 110 mEg/L or less]. Advanced renal failure was the most common cause while gastrointestinal problems with vomiting, diarrhoea, congestive heart failure, liver diseases, postoperative states, and i. v. fluid administration were other frequent causes. Mildly hyponatremic patients were usually asyrnptomatic but progressive symptomatology appeared with further decline of serum sodium concentration. Cererbral symptoms were seen in 8% of patients with mild hypomatremia, 53% of cases with moderate and 84% of cases with severe hyponatremia. Nine patients with hyponatremia died, mortality being 3.3%