Highly Specific Voltammetric Detection of Cephalexin in Tablet Formulations and Human Urine Samples Using a Poly(2,4,6-2',4',6'-hexanitrodiphenylamine)-Modified Glassy Carbon Electrode.

ß-Lactam antibiotics are employed to treat bacterial illnesses. Despite a high level of clinical success, they have encountered serious resistance that demands a high-dose regimen and a new pharmacokinetic combination. This requires continuous monitoring of their levels in pharmaceutical and biological samples. In this study, an electrochemical sensor was developed for the determination of cephalexin (CLN) in pharmaceutical formulations and biological fluid samples. The sensors were developed by modifying a glassy carbon electrode (GCE) using a conducting polymer (dipicrylamine) by potentiodynamic electropolymerization. Characterization (using cyclic voltammetry and electron impedance spectroscopy) results revealed modification of the electrode surface, leading to an enhanced effective electrode surface area and their conductivity. The appearance of an irreversible oxidative peak at much-reduced potential with 5-fold current enhancement at a poly(dipicrylamine)-modified glassy carbon electrode (poly(DPA)/GCE) verified the electrocatalytic role toward CLN. Under optimized conditions, a wider linear concentration range (5 × 10-8 to 3.0 × 10-4 M), lowest limit of detection (LoD) (2.5 nM), detected amount of each tablet brand above 97.00% of the labeled value (showing excellent agreement between the detected amount and company label), and excellent % recovery results in pharmaceutical and biological samples were obtained with an excellent interference recovery error of less than 4.05%. Its excellent accuracy, selectivity, reproducibility, and stabilities and only requiring a simple electrode modification step combined with its readily available and nontoxic modifier, which sets it apart from most previously reported methods, have validated the present method's potential applicability for determining CLN in biological and pharmaceutical samples.

Dietary practice and associated factors among elderly people in Northwest Ethiopia, 2022: Community based mixed design.

BACKGROUND: The planet's population is aging at an incredible speed. Poor dietary practices are a major problem among the elderly. However, literature is scarce on dietary practices among elders in the study area. Therefore, the results of this study may give information to decision-makers. OBJECTIVE: This study aimed to assess dietary practices and associated factors among elderly people in Northwest Ethiopia, in 2022. METHODS: A community-based mixed study design was employed among elderly people from May 20 to July 2, 2022. Systematic random sampling and purposive sampling techniques were used for selecting study participants in quantitative and qualitative studies, respectively. Data were collected using an interviewer-administered structured questionnaire for the quantitative part and an interview guide for the qualitative part. Binary logistic regression analysis was used. A P-value less than or equal to 0.05 was used to declare statistically significant variables. A crude odds ratio and an adjusted odds ratio with a 95% confidence interval were used to measure the strength of the association. Thematic analysis was used for qualitative data analysis. RESULTS: A total of 422 participants were recruited for the study. Twenty-six in-depth interviews were done. The prevalence of adequate dietary practice was only 54.5% [95% CI: (49.8, 59.2)]. It was significantly associated with being aged between 65 to 74 years (AOR: 8.32; 95 CI: 3.9, 18.1), being aged between 75 to 84 years (AOR: 2.90; 95% CI: 1.1, 7.9), eating sometimes alone (AOR: 1.86; 95% CI: 1.03, 3.4), eating always with family members (AOR: 4.96; 95% CI: 2.6, 9.4), and food security (AOR: 3.13; 95% CI: 1.8, 5.4). Thematic analysis revealed three themes that interfere with the dietary practices of elders. A majority of in-depth interviewees mentioned that there were taboos and cultural beliefs which favor inadequate dietary practices of the elderly; the study participants reported that individual, economic, societal, and physiological factors are barriers affecting the dietary practices of the elderly, and all respondents have no experiences regarding elderly dietary practices. CONCLUSION: The prevalence of adequate dietary practice was low. It was significantly associated with age, with whom feeding, and household food security status. Taboos and cultural beliefs, barriers, and experiences hampered the dietary practices of elders. Therefore, improving the dietary practices of elders focusing on advanced age, loneliness, food security, taboos, cultural beliefs, barriers, and experiences regarding dietary practices should be done.

Novel insecticidal proteins from ferns resemble insecticidal proteins from Bacillus thuringiensis.

Lepidopterans affect crop production worldwide. The use of transgenes encoding insecticidal proteins from Bacillus thuringiensis (Bt) in crop plants is a well-established technology that enhances protection against lepidopteran larvae. Concern about widespread field-evolved resistance to Bt proteins has highlighted an urgent need for new insecticidal proteins with different modes or sites of action. We discovered a new family of insecticidal proteins from ferns. The prototype protein from Pteris species (Order Polypodiales) and variants from two other orders of ferns, Schizaeales and Ophioglossales, were effective against important lepidopteran pests of maize and soybean in diet-based assays. Transgenic maize and soybean plants producing these proteins were more resistant to insect damage than controls. We report here the crystal structure of a variant of the prototype protein to 1.98 Å resolution. Remarkably, despite being derived from plants, the structure resembles the 3-domain Cry proteins from Bt but has only two out of three of their characteristic domains, lacking the C-terminal domain which is typically required for their activities. Two of the fern proteins were effective against strains of fall armyworm that were resistant to Bt 3-domain Cry proteins Cry1Fa or Cry2A.127. This therefore represents a novel family of insecticidal proteins that have the potential to provide future tools for pest control.

Selective Determination of Norfloxacin in Pharmaceutical Formulations and Human Urine Samples Using Poly(8-aminonaphthaline-2-sulfonic Acid)-Modified Glassy Carbon Electrodes.

In this study, a glassy carbon electrode was modified potentiodynamically with poly(8-aminonaphthaline-2-sulfonic acid) [poly(ANSA)/GCE] for the detection of norfloxacin (NFN) in tablet formulations and human urine samples. Improvement of the effective surface area of the modified electrode and decreased charge-transfer resistance confirmed surface modification of the GCE by a conductive poly(ANSA) film. The appearance of an oxidative peak without a reductive peak in the reverse scan direction showed the irreversibility of the electrochemical oxidation of NFN in both the bare GCE and poly(ANSA)/GCE. A better coefficient of determination for the peak current on the square root of the scan rate (R2 = 0.99514) than the scan rate (R2 = 0.97109), indicating the oxidation of NFN at the poly(ANSA)/GCE, was predominantly diffusion mass transport-controlled. Under optimized pH and square wave parameters, the voltammetric current response of NFN at the poly(ANSA)/GCE showed linear dependence on the concentration, ranging from 1.0 × 10-8 to 4.0 × 10-4 M with a limit of detection of 4.1 × 10-10. The NFN level in the studied tablet brands was in the range of 90.30-103.3% of their labeled values. Recovery results in tablet and urine samples ranged from 98.35 to 101.20% and 97.75 to 99.60%, respectively, and interference recovery results were less than 2.13% error in the presence of ampicillin, chloroquine phosphate, and cloxacillin. The present method had a better performance for the determination of NFN in tablet formulations and urine samples as compared with recently reported voltammetric methods due to its requirement of a simple electrode modification step, which provides the least limit of detection and a reasonably wider linear dynamic range.

Structural journey of an insecticidal protein against western corn rootworm.

The broad adoption of transgenic crops has revolutionized agriculture. However, resistance to insecticidal proteins by agricultural pests poses a continuous challenge to maintaining crop productivity and new proteins are urgently needed to replace those utilized for existing transgenic traits. We identified an insecticidal membrane attack complex/perforin (MACPF) protein, Mpf2Ba1, with strong activity against the devastating coleopteran pest western corn rootworm (WCR) and a novel site of action. Using an integrative structural biology approach, we determined monomeric, pre-pore and pore structures, revealing changes between structural states at high resolution. We discovered an assembly inhibition mechanism, a molecular switch that activates pre-pore oligomerization upon gut fluid incubation and solved the highest resolution MACPF pore structure to-date. Our findings demonstrate not only the utility of Mpf2Ba1 in the development of biotechnology solutions for protecting maize from WCR to promote food security, but also uncover previously unknown mechanistic principles of bacterial MACPF assembly.

Characterization and Application of a Synthesized Novel Poly(chlorobis(1,10-phenanthroline)resorcinolcobalt(II) chloride)-Modified Glassy Carbon Electrode for Selective Voltammetric Determination of Cefadroxil in Pharmaceutical Formulations, Human Urine, and Blood Serum Samples.

Cefadroxil belongs to the ß-lactam antibiotics, mainly used for the treatment of various bacterial infections, caused by Gram-positive and Gram-negative bacteria. However, it is also encountering serious bacterial resistance, necessitating continuous monitoring of its level in pharmaceutical and biological samples. This study presents a selective, accurate, and precise square-wave voltammetric method based on a novel poly(chlorobis(1,10-phenanthroline)resorcinolcobalt(II)chloride)-modified glassy carbon electrode (poly(CP2RCoC)/GCE) for determination of cefadroxil (CDL). UV-vis spectroscopy, FT-IR spectroscopy, metal and halide estimation, CHN elemental analysis, and electrolytic conductivity measurement results confirmed the synthesis of the title complex modifier. Electrode characterization results revealed modification of the surface of the electrode by an electroactive and a conductive polymer film (poly(CP2RCoC)/GCE), leading to an improved effective electrode surface area. In contrast to the bare electrode, the appearance of an irreversible oxidative peak at a much reduced potential with a 7-fold current enhancement at poly(CP2RCoC)/GCE showed the catalytic effect of the modifier toward oxidation of CDL. The square-wave voltammetric current response of poly(CP2RCoC)/GCE showed a linear dependence on the concentration of CDL in the range of 1 × 10-7-3.0 × 10-4 M with a detection limit of 4.3 × 10-9. The CDL level in the selected two tablet brands was in the range of 97.25-100.00% of their labeled values. The spike recovery results in tablet, human blood serum, and urine samples were 98.85-101.30, 99.20-100.39, and 98.10-99.99%, respectively. Interference recovery results with a less than 4.74% error, lower LoD, and wider dynamic range than the previously reported methods validated the potential applicability of the present method with excellent accuracy and sensitivity based on the novel mixed-ligand complex-modified GCE (poly(CP2RCoC)/GCE) for determination of CDL in various real samples with a complex matrix.

Electrochemical Determination of Chloroquine Phosphate in Real Samples Using a Diresorcinate-1,10-phenanthrolinecobalt(II)-Modified Glassy Carbon Electrode.

Chloroquine phosphate (CQP) is used for malaria treatment. As it is facing increasing resistance, it needs continuous monitoring using sensitive and specific detection methods. In this work, a voltammetric sensor was prepared by electropolymerization of a diresorcinate-1,10-phenanthrolinecobalt(II) complex on a glassy carbon electrode (poly(DHRPCo)/GCE) which was followingly characterized. Compared with a bare GCE, CQP showed single well shaped irreversible oxidative peak at the poly(DHRPCo)/GCE. The peak current showed excellent linearity with CQP concentration in the range of 0.005-300.0 µm with a detection limit of 0.39 nm. The response of CQP at poly(DHRPCo)/GCE was not influenced by the presence of amoxicillin, ciprofloxacillin and paracetamol in addition to its high stability and reproducibility. It was applied for detection of CQP in various real samples, including three brands of tablets, human blood serum, and urine samples. The detected amount in tablets were in the range 98.4-103.2 % of their labeled value. Spike recovery results in human blood serum, urine, and tablet samples were 99.35-100.28 %, 99.03-100.32 %, and 98.40-100.41 %, respectively. Interference recovery results with less than 4.60 % error, the lower limit of detection and the wider dynamic range than most of the previously reported methods validate the potential applicability of the proposed method for CQP determination in various real samples with complex matrices.

Synthesis of a novel diaquabis(1,10-phenanthroline)copper(II)chloride complex and its voltammetric application for detection of amoxicillin in pharmaceutical and biological samples.

A one step facile synthesis of the novel diaquabis(1,10-phenanthroline)copper(II)chloride (A2P2CuC) complex is demonstrated. Cyclic voltammetric and electrochemical impedance spectroscopic results revealed potentiodynamic deposition of a conductive electroactive poly(A2P2CuC) film on the glassy carbon electrode surface increasing its effective surface area. In contrast to the unmodified glassy carbon electrode, appearance of an oxidative peak at a reduced potential with over two fold current for amoxicillin at poly(A2P2CuC)/GCE demonstrated its electrocatalytic property attributed to reduce charge transfer resistance and the improved surface area of the electrode surface. Better correlation of the oxidative peak current with square root of scan rate (R2 = 0.99779) than with scan rate (R2 = 0.96953) supplemented by slope of 0.58 for log(current) versus log(scan rate) confirmed diffusion controlled irreversible oxidation of amoxicillin. At optimized solution and SWV parameters, current response of poly(A2P2CuC)/GCE showed linear dependence on concentration of amoxicillin (2.0-100.0 µM) with LoD 0.0115 µM. While no amoxicillin was detected in the human blood serum sample, an amount 89.40-100.55% of the nominal level was detected in the analyzed eight tablet brands. Spike recovery in tablet samples (98.90-101.95%) and blood serum sample (102.20-101.37%); interference with an error (%RSD) of 0.00-4.51% in tablet and 0.00-2.10% in serum samples; excellent stability and reproducible results, added with the wide dynamic range and low LoD validated the method for amoxicillin determination in pharmaceutical formulations and human urine samples.

Potentiodynamic Poly(resorcinol)-Modified Glassy Carbon Electrode as a Voltammetric Sensor for Determining Cephalexin and Cefadroxil Simultaneously in Pharmaceutical Formulation and Biological Fluid Samples.

This study covers the development of a fast, selective, sensitive, and stable method for the simultaneous determination of cephalosporins (cephalexin (CLN) and cefadroxil (CFL)) in biological fluids and tablet samples using potentiodynamic fabrication of a poly(resorcinol)-modified glassy carbon electrode (poly(reso)/GCE). The results of cyclic voltammetry and electrochemical impedance spectroscopy supported the modification of the GCE by a polymer layer that raised the electrode surface area and conductivity. At the poly(reso)/GCE, an irreversible oxidative peak with four- and fivefold current enhancement for CLN and CFL, respectively, at a substantially lower potential demonstrated the catalytic action of the modifier. Under optimized solution and parameters, the peak current response at the poly(reso)/GCE revealed a linear dependence on the concentration of CLN and CFL within the range 0.1-300 and 0.5-300 µM, respectively, with a limit of detection (LoD) of 3.12 and 8.7 nM, respectively. The levels of CLN in four selected tablet brands and CFL in two tablet brands were in the vicinity of 91.00-103.65% and 97.7-98.83%, respectively, of their nominal values. The recovery results for CLN in pharmaceutical samples were in the range of 99.00-100.67% and for CFL 97.9-99.75% and for blood serum and urine samples 99.55-100.55% and 99.33-100.34% for CLN and 97.13-100.60% and 96.73-102.50% for CFL, respectively. Interference recovery results with errors less than 4.81%, lower LoD, wider dynamic range, excellent recovery results, and good stability of the modifier compared to those for the previously reported methods validated the use of the poly(reso)/GCE for determining CLN and CFL simultaneously in various real samples.

A Random-Screening Approach to Identify RNAi Targets for the Control of Western Corn Rootworm (Diabrotica. virgifera virgifera Le Conte).

Identification of active target genes in bioassay screening is the first important step for application of RNA interference (RNAi) for pest control. Here, we describe the methodology for performing high-throughput RNAi target screening against important agriculture pest, Western corn rootworm in 96-well microplate. Two approaches are presented to identify active targets from random-cDNA library or testing a certain group of specific targets via in silico sequence analysis. Methods of PCR primer design, DNA template preparation, and dsRNA production described here can be applied for other pests.

Poly(4-amino-3-hydroxynaphthalene-1-sulfonic acid) modified glassy carbon electrode for square wave voltammetric determination of amoxicillin in four tablet brands.

BACKGROUND: Amoxicillin (AMX), which is one of the ß-lactam antibiotics used in the treatment of bacterial infections, is known to have a serious mechanism of resistance necessitating continuous monitoring of its level in pharmaceutical and serum samples. RESULTS: In this study, we presented selective, accurate, and precise square wave voltammetric method based on poly(4-amino-3-hydroxynaphthalene-1-sulfonic acid) modified glassy carbon electrode (poly(AHNSA/GCE)) for determination of amoxicillin in four selected tablet brands. Appearance of a peak in the oxidative scan direction without a peak in the reductive direction of cyclic voltammograms of both bare GCE and poly(AHNSA/GCE) with four folds current and much reduced potential on the modified electrode showed catalytic property of the modifier towards oxidation of AMX. While cyclic voltammetric studies of effect of scan rate showed predominantly diffusion controlled oxidation of AMX with one electron participation, effect of pH revealed participation of protons and electrons in a 1:1 ratio. The square wave voltammetric peak current response of the modified electrode for AMX showed linear dependence on the concentration of the spiked standard AMX in the range 10-150 µmol L-1 with 9.9 nmol L-1 LOD. The AMX content of the studied tablet brands were found in the range 97.84-100.78% of the labeled value. Spike recovery results of 99.6-100.5%, and interference recovery results of 95.4-100.8% AMX in the presence of 50-200% of ampicillin and cloxicillin validated the applicability of the method for determination of amoxicillin in tablet formulation. CONCLUSION: In contrast to the previously reported works on determination of amoxicillin, the present method showed an excellent performance making it a potential method for determination of amoxicillin in real samples including serum samples.

Functional validation of DvABCB1 as a receptor of Cry3 toxins in western corn rootworm, Diabrotica virgifera virgifera.

Western corn rootworm (WCR), Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae), is a serious insect pest in the major corn growing areas of North America and in parts of Europe. WCR populations with resistance to Bacillus thuringiensis (Bt) toxins utilized in commercial transgenic traits have been reported, raising concerns over their continued efficacy in WCR management. Understanding the modes of action of Bt toxins is important for WCR control and resistance management. Although different classes of proteins have been identified as Bt receptors for lepidopteran insects, identification of receptors in WCR has been limited with no reports of functional validation. Our results demonstrate that heterologous expression of DvABCB1 in Sf9 and HEK293 cells conferred sensitivity to the cytotoxic effects of Cry3A toxins. The result was further validated using knockdown of DvABCB1 by RNAi which rendered WCR larvae insensitive to a Cry3A toxin. However, silencing of DvABCB2 which is highly homologous to DvABCB1 at the amino acid level, did not reduce the sensitivity of WCR larvae to a Cry3A toxin. Furthermore, our functional studies corroborate different mode-of-actions for other insecticidal proteins including Cry34Ab1/35Ab1, Cry6Aa1, and IPD072Aa against WCR. Finally, reduced expression and alternatively spliced transcripts of DvABCB1 were identified in a mCry3A-resistant strain of WCR. Our results provide the first clear demonstration of a functional receptor in the molecular mechanism of Cry3A toxicity in WCR and confirmed its role in the mechanism of resistance in a mCry3A resistant strain of WCR.

Green synthesized silver nanoparticle modified carbon paste electrode for SWAS voltammetric simultaneous determination of Cd(II) and Pb(II) in Bahir Dar Textile discharged effluent.

The principal objective of this research was to demonstrate the sensitivity and selectivity of carbon paste electrode modified with Ocimum Sanctum leaf extract synthesized silver nanoparticles for simultaneous determination of Cd(II) and Pb(II) in discharged textile effluent. While UV-Vis, XRD and FT-IR were used to fully characterize the green synthesized silver nanoparticles, cyclic voltammetry was used to evaluate the electrochemical behavior of the two metals at the modified electrode relative to the unmodified electrode. Square wave anodic stripping (SWAS) voltammetric current showed linear dependence on the concentration in the range 5-160 ppm with determination coefficients (R2) of 0.9976 and 0.9996 for Cd(II) and Pb(II), respectively. The method also showed extremely low detection limit (0.0891ppm for Cd(II) and 0.048 ppm for Pb(II)) making the method superior over the previously reported methods. Recovery results of 94.3 for Cd(II) and 101.0% for Pb(II) validated the applicability of the method for simultaneous determination of the two metals in a complex matrix textile effluent sample. While levels of Pb(II) and Cd(II) in the untreated sample were 117.0 and 128.3 ppm, their levels in the treated sample were 17.7 and 101.4 ppm, respectively confirming the low efficiency of the treatment plant the factory claims to have. The level of the studied metals in the discharged effluent is much higher than the permissible limit indicating extent of pollution of the water system to which the effluent is discharged.

Identification and Evaluations of Novel Insecticidal Proteins from Plants of the Class Polypodiopsida for Crop Protection against Key Lepidopteran Pests.

Various lepidopteran insects are responsible for major crop losses worldwide. Although crop plant varieties developed to express Bacillus thuringiensis (Bt) proteins are effective at controlling damage from key lepidopteran pests, some insect populations have evolved to be insensitive to certain Bt proteins. Here, we report the discovery of a family of homologous proteins, two of which we have designated IPD083Aa and IPD083Cb, which are from Adiantum spp. Both proteins share no known peptide domains, sequence motifs, or signatures with other proteins. Transgenic soybean or corn plants expressing either IPD083Aa or IPD083Cb, respectively, show protection from feeding damage by several key pests under field conditions. The results from comparative studies with major Bt proteins currently deployed in transgenic crops indicate that the IPD083 proteins function by binding to different target sites. These results indicate that IPD083Aa and IPD083Cb can serve as alternatives to traditional Bt-based insect control traits with potential to counter insect resistance to Bt proteins.

Knockdown of RNA interference pathway genes impacts the fitness of western corn rootworm.

Western corn rootworm (Diabrotica virgifera virgifera) is a serious agricultural pest known for its high adaptability to various management strategies, giving rise to a continual need for new control options. Transgenic maize expressing insecticidal RNAs represents a novel mode of action for rootworm management that is dependent on the RNA interference (RNAi) pathways of the insect for efficacy. Preliminary evidence suggests that western corn rootworm could develop broad resistance to all insecticidal RNAs through changes in RNAi pathway genes; however, the likelihood of field-evolved resistance occurring through this mechanism remains unclear. In the current study, eight key genes involved in facilitating interference in the microRNA and small interfering RNA pathways were targeted for knockdown in order to evaluate impact on fitness of western corn rootworm. These genes include drosha, dicer-1, dicer-2, pasha, loquacious, r2d2, argonaute 1, and argonaute 2. Depletion of targeted transcripts in rootworm larvae led to changes in microRNA expression, decreased ability to pupate, reduced adult beetle emergence, and diminished reproductive capacity. The observed effects do not support evolution of resistance through changes in expression of these eight genes due to reduced insect fitness.

A selective insecticidal protein from Pseudomonas mosselii for corn rootworm control.

The coleopteran insect western corn rootworm (WCR, Diabrotica virgifera virgifera) is an economically important pest in North America and Europe. Transgenic corn plants producing Bacillus thuringiensis (Bt) insecticidal proteins have been useful against this devastating pest, but evolution of resistance has reduced their efficacy. Here, we report the discovery of a novel insecticidal protein, PIP-47Aa, from an isolate of Pseudomonas mosselii. PIP-47Aa sequence shows no shared motifs, domains or signatures with other known proteins. Recombinant PIP-47Aa kills WCR, two other corn rootworm pests (Diabrotica barberi and Diabrotica undecimpunctata howardi) and two other beetle species (Diabrotica speciosa and Phyllotreta cruciferae), but it was not toxic to the spotted lady beetle (Coleomegilla maculata) or seven species of Lepidoptera and Hemiptera. Transgenic corn plants expressing PIP-47Aa show significant protection from root damage by WCR. PIP-47Aa kills a WCR strain resistant to mCry3A and does not share rootworm midgut binding sites with mCry3A or AfIP-1A/1B from Alcaligenes that acts like Cry34Ab1/Cry35Ab1. Our results indicate that PIP-47Aa is a novel insecticidal protein for controlling the corn rootworm pests.

Control of Western Corn Rootworm (Diabrotica virgifera virgifera) Reproduction through Plant-Mediated RNA Interference.

RNA interference (RNAi) in transgenic maize has recently emerged as an alternative mode of action for western corn rootworm (Diabrotica virgifera virgifera) control which can be combined with protein-based rootworm control options for improved root protection and resistance management. Currently, transgenic RNAi-based control has focused on suppression of genes that when silenced lead to larval mortality. We investigated control of western corn rootworm reproduction through RNAi by targeting two reproductive genes, dvvgr and dvbol, with the goal of reducing insect fecundity as a new tool for pest management. The results demonstrated that exposure of adult beetles, as well as larvae to dvvgr or dvbol dsRNA in artificial diet, caused reduction of fecundity. Furthermore, western corn rootworm beetles that emerged from larval feeding on transgenic maize roots expressing dvbol dsRNA also showed significant fecundity reduction. This is the first report of reduction of insect reproductive fitness through plant-mediated RNAi, demonstrating the feasibility of reproductive RNAi as a management tool for western corn rootworm.

An Alcaligenes strain emulates Bacillus thuringiensis producing a binary protein that kills corn rootworm through a mechanism similar to Cry34Ab1/Cry35Ab1.

Crops expressing Bacillus thuringiensis (Bt)-derived insecticidal protein genes have been commercially available for over 15 years and are providing significant value to growers. However, there remains the need for alternative insecticidal actives due to emerging insect resistance to certain Bt proteins. A screen of bacterial strains led to the discovery of a two-component insecticidal protein named AfIP-1A/1B from an Alcaligenes faecalis strain. This protein shows selectivity against coleopteran insects including western corn rootworm (WCR). Transgenic maize plants expressing AfIP-1A/1B demonstrate strong protection from rootworm injury. Surprisingly, although little sequence similarity exists to known insecticidal proteins, efficacy tests using WCR populations resistant to two different Cry proteins show that AfIP-1A/1B and mCry3A differ in their mode of action while AfIP-1A/1B and the binary Cry34Ab1/Cry35Ab1 protein share a similar mode. These findings are supported by results of competitive binding assays and the similarity of the x-ray structure of AfIP-1A to Cry34Ab1. Our work indicates that insecticidal proteins obtained from a non-Bt bacterial source can be useful for developing genetically modified crops and can function similarly to familiar proteins from Bt.

Production of thermotolerant entomopathogenic fungal conidia on millet grain.

Thermotolerance of entomopathogenic (insect-killing) fungi should be seriously considered before industrialization. This work describes the feasibility of millet grain as a substrate for production of thermotolerant Beauveria bassiana (Bb) GHA and ERL1170 and Metarhizium anisopliae (Ma) ERL1171 and ERL1540 conidia. First, conidial suspensions of the Bb isolates, produced on millet grain in polyethylene bags, were exposed to five temperatures (43-47 °C) at 15-min intervals for up to 120 min (experiment I). Agar-based quarter-strength (») Sabouraud dextrose agar supplemented with yeast extract (SDAY) and whey permeate media served as controls. Millet-grain-based culture was superior in producing the most thermotolerant Bb conidia, followed by whey permeate agar and »SDAY-based cultures. Secondly, to compare the thermotolerance of conidia produced at the same conditions, the Bb isolates were then produced on agar-based millet powder medium, with »SDAY and whey permeate agar media as controls, and the two Ma isolates were added (experiment II). They were then exposed to the same temperatures as above. More thermotolerant Bb and Ma conidia were produced on millet powder agar than on whey permeate agar and »SDAY overall. These results suggest that millet grain can be used as a substrate to produce thermotolerant conidia in a mass production system.

Effect of Bt-176 maize pollen on first instar larvae of the Peacock butterfly (Inachis io) (Lepidoptera; Nymphalidae).

More than 10 years after registration of the first Bt maize cultivar in Europe, there still exists a remarkable lack of data on effects on Lepidoptera which would be necessary for a complete and comprehensive environmental risk assessment. So far only very few European butterfly species have been tested in this aspect. In our study the effect of transgenic Bacillus thuringiensis (Bt) maize pollen (event Bt-176) on the development and survival of neonate larvae of the Peacock butterfly, Inachis io (L.) was for the first time shown. The results of our study suggest that the Peacock butterfly may serve as a model organism for assessing potential side effects of new developed transgenic Bt crops on non-target butterflies in a GMO environmental risk assessment. The study was done under laboratory conditions by exposing larvae of the Peacock butterfly to various pollen doses of transgenic maize event Bt-176 (cv. PACTOL CB) or the conventional isogenic maize (cv. PACTOL) using a no-choice test. Larvae feeding for 48 h on nettle plants (Urtica dioica) that were contaminated with higher pollen concentrations from Bt-176 maize (205 and 388 applied pollen.cm⁻²) suffered a significantly higher mortality rate (68 and 85% respectively) compared to larvae feeding on leaves with no pollen (11%), or feeding on leaves with pollen from conventional maize (6 to 25%). At lower Bt maize pollen doses (23-104 applied pollen.cm⁻²),mortality ranged from 11-25% and there were no apparent differences among treatments. The corresponding LC50-and LC90-values for neonate larvae of the Peacock butterfly were 187 and 448 applied pollen grains.cm⁻² of Bt-176, respectively.Weight of larvae surviving consumption of Bt-176 maize pollen declined between 10 and 81% with increased pollen doses (r = -0.95). The highest weight reduction (81%) corresponded to the highest pollen concentration (388 pollen grains applied.cm⁻²). Ingestion of pollen from the conventional maize hybrid did not have negative effects on larval weight gain or survival rate.