Nurul Karim

A Rapid and Precise Diagnostic Method for Detecting the Pinewood Nematode Bursaphelenchus xylophilus by Loop-Mediated Isothermal Amplification

ABSTRACT Bursaphelenchus xylophilus is the causal agent of pine wilt disease, which is a major forest disease in Japan, Korea, China, Taiwan, and Portugal. A diagnostic method which is rapid, precise, and simple could greatly help the proper management of this disease. Here, we present a novel detection method using loop-mediated isothermal amplification (LAMP) targeting the internal transcribed spacer region of ribosomal DNA of the nematode. Specificity of the primers and LAMP was confirmed using DNA from various nematode species related to B. xylophilus. Our experimental results suggest that LAMP can detect B. xylophilus faster and with higher sensitivity than the traditional diagnostic method. Moreover, because it does not require expensive equipment or specialized techniques, this LAMP-based diagnostic method has the potential to be used under field conditions.

Organophosphorus and Carbamate Pesticide Residues Detected in Water Samples Collected from Paddy and Vegetable Fields of the Savar and Dhamrai Upazilas in Bangladesh

Several types of organophosphorous and carbamate pesticides have been used extensively by the farmers in Bangladesh during the last few decades. Twenty seven water samples collected from both paddy and vegetable fields in the Savar and Dhamrai Upazilas in Bangladesh were analyzed to determine the occurrence and distribution of organo-phosphorus (chlorpyrifos, malathion and diazinon) and carbamate (carbaryl and carbofuran) pesticide residues. A high performance liquid chromatograph instrument equipped with a photodiode array detector was used to determine the concentrations of these pesticide residues. Diazinon and carbofuran were detected in water samples collected from Savar Upazila at 0.9 μg/L and 198.7 μg/L, respectively. Malathion was also detected in a single water sample at 105.2 μg/L from Dhamrai Upazila. Carbaryl was the most common pesticide detected in Dhamrai Upazila at 14.1 and 18.1 μg/L, while another water sample from Dhamrai Upazila was contaminated with carbofuran at 105.2 μg/L. Chlorpyrifos was not detected in any sample. Overall, the pesticide residues detected were well above the maximum acceptable levels of total and individual pesticide contamination, at 0.5 and 0.1 μg/L, respectively, in water samples recommended by the European Economic Community (Directive 98/83/EC). The presence of these pesticide residues may be attributed by their intense use by the farmers living in these areas. Proper handling of these pesticides should be ensured to avoid direct or indirect exposure to these pesticides.

Identification of putative expansin-like genes from the pine wood nematode, Bursaphelenchus xylophilus, and evolution of the expansin gene family within the Nematoda

We report the cloning and characterisation of genes encoding expansin-like proteins from the pine wood nematodes, Bursaphelenchus xylophilus and B. mucronatus. A small family of genes is present in both species and the Bursaphelenchus genes are most similar to expansins and expansin-like proteins from the potato cyst nematode Globodera rostochiensis and root-knot nematodes. Molecular modelling suggests that the genes could encode a protein with a structure similar to that of functionally characterised expansins. Expression analysis showed that the Bursaphelenchus expansin-like genes are expressed solely in the pharyngeal gland cells, implying a role in the host-parasite interaction, most likely in assisting migration through the plant. Some G. rostochiensis and root-knot nematode expansins are composed of a carbohydrate-binding domain coupled to an expansin domain but no carbohydrate binding domain is present on any of the Bursaphelenchus sequences. We suggest a model for evolution of the expansin gene family within the plant-parasitic nematodes of the Tylenchida and Aphelenchida.

Analysis of expressed sequence tags and identification of genes encoding cell-wall-degrading enzymes from the fungivorous nematode Aphelenchus avenae

BackgroundThe fungivorus nematode, Aphelenchus avenae is widespread in soil and is found in association with decaying plant material. This nematode is also found in association with plants but its ability to cause plant disease remains largely undetermined. The taxonomic position and intermediate lifestyle of A. avenae make it an important model for studying the evolution of plant parasitism within the Nematoda. In addition, the exceptional capacity of this nematode to survive desiccation makes it an important system for study of anhydrobiosis. Expressed sequence tag (EST) analysis may therefore be useful in providing an initial insight into the poorly understood genetic background of A. avenae.ResultsWe present the generation, analysis and annotation of over 5,000 ESTs from a mixed-stage A. avenae cDNA library. Clustering of 5,076 high-quality ESTs resulted in a set of 2,700 non-redundant sequences comprising 695 contigs and 2,005 singletons. Comparative analyses indicated that 1,567 (58.0%) of the cluster sequences had homologues in Caenorhabditis elegans, 1,750 (64.8%) in other nematodes, 1,321(48.9%) in organisms other than nematodes, and 862 (31.9%) had no significant match to any sequence in current protein or nucleotide databases. In addition, 1,100 (40.7%) of the sequences were functionally classified using Gene Ontology (GO) hierarchy. Similarity searches of the cluster sequences identified a set of genes with significant homology to genes encoding enzymes that degrade plant or fungal cell walls. The full length sequences of two genes encoding glycosyl hydrolase family 5 (GHF5) cellulases and two pectate lyase genes encoding polysaccharide lyase family 3 (PL3) proteins were identified and characterized.ConclusionWe have described at least 2,214 putative genes from A. avenae and identified a set of genes encoding a range of cell-wall-degrading enzymes. This EST dataset represents a starting point for studies in a number of different fundamental and applied areas. The presence of genes encoding a battery of cell-wall-degrading enzymes in A. avenae and their similarities with genes from other plant parasitic nematodes suggest that this nematode can act not only as a fungal feeder but also a plant parasite. Further studies on genes encoding cell-wall-degrading enzymes in A. avenae will accelerate our understanding of the complex evolutionary histories of plant parasitism and the use of genes obtained by horizontal gene transfer from prokaryotes.

A model of chlorpyrifos distribution and its biochemical effects on the liver and kidneys of rats.

This study investigated the main target sites of chlorpyrifos (CPF), its effect on biochemical indices, and the pathological changes observed in rat liver and kidney function using gas chromatography/mass spectrometry. Adult female Wistar rats (n = 12) were randomly assigned into two groups (one control and one test group; n = 6 each). The test group received CPF via oral gavage for 21 days at 5 mg/kg daily. The distribution of CPF was determined in various organs (liver, brain, heart, lung, kidney, ovary, adipose tissue, and skeletal muscle), urine and stool samples using GCMS. Approximately 6.18% of CPF was distributed in the body tissues, and the highest CPF concentration (3.80%) was found in adipose tissue. CPF also accumulated in the liver (0.29%), brain (0.22%), kidney (0.10%), and ovary (0.03%). Approximately 83.60% of CPF was detected in the urine. CPF exposure resulted in a significant increase in plasma transaminases, alkaline phosphatase, and total bilirubin levels, a significant reduction in total protein levels and an altered lipid profile. Oxidative stress due to CPF administration was also evidenced by a significant increase in liver malondialdehyde levels. The detrimental effects of CPF on kidney function consisted of a significant increase in plasma urea and creatinine levels. Liver and kidney histology confirmed the observed biochemical changes. In conclusion, CPF bioaccumulates over time and exerts toxic effects on animals.

Determination of Carbamate and Organophosphorus Pesticides in Vegetable Samples and the Efficiency of Gamma-Radiation in Their Removal

In the present study, the residual pesticide levels were determined in eggplants (Solanum melongena) (n = 16), purchased from four different markets in Dhaka, Bangladesh. The carbamate and organophosphorus pesticide residual levels were determined by high performance liquid chromatography (HPLC), and the efficiency of gamma radiation on pesticide removal in three different types of vegetables was also studied. Many (50%) of the samples contained pesticides, and three samples had residual levels above the maximum residue levels determined by the World Health Organisation. Three carbamates (carbaryl, carbofuran, and pirimicarb) and six organophosphates (phenthoate, diazinon, parathion, dimethoate, phosphamidon, and pirimiphos-methyl) were detected in eggplant samples; the highest carbofuran level detected was 1.86 mg/kg, while phenthoate was detected at 0.311 mg/kg. Gamma radiation decreased pesticide levels proportionately with increasing radiation doses. Diazinon, chlorpyrifos, and phosphamidon were reduced by 40–48%, 35–43%, and 30–45%, respectively, when a radiation strength of 0.5 kGy was utilized. However, when the radiation dose was increased to 1.0 kGy, the levels of the pesticides were reduced to 85–90%, 80–91%, and 90–95%, respectively. In summary, our study revealed that pesticide residues are present at high amounts in vegetable samples and that gamma radiation at 1.0 kGy can remove 80–95% of some pesticides.

Identification of potential drug targets by subtractive genome analysis of Escherichia coli O157:H7: an in silico approach.

Bacterial enteric infections resulting in diarrhea, dysentery, or enteric fever constitute a huge public health problem, with more than a billion episodes of disease annually in developing and developed countries. In this study, the deadly agent of hemorrhagic diarrhea and hemolytic uremic syndrome, Escherichia coli O157:H7 was investigated with extensive computational approaches aimed at identifying novel and broad-spectrum antibiotic targets. A systematic in silico workflow consisting of comparative genomics, metabolic pathways analysis, and additional drug prioritizing parameters was used to identify novel drug targets that were essential for the pathogen’s survival but absent in its human host. Comparative genomic analysis of Kyoto Encyclopedia of Genes and Genomes annotated metabolic pathways identified 350 putative target proteins in E. coli O157:H7 which showed no similarity to human proteins. Further bio-informatic approaches including prediction of subcellular localization, calculation of molecular weight, and web-based investigation of 3D structural characteristics greatly aided in filtering the potential drug targets from 350 to 120. Ultimately, 44 non-homologous essential proteins of E. coli O157:H7 were prioritized and proved to have the eligibility to become novel broad-spectrum antibiotic targets and DNA polymerase III alpha (dnaE) was the top-ranked among these targets. Moreover, druggability of each of the identified drug targets was evaluated by the DrugBank database. In addition, 3D structure of the dnaE was modeled and explored further for in silico docking with ligands having potential druggability. Finally, we confirmed that the compounds N-coeleneterazine and N-(1,4-dihydro-5H-tetrazol-5-ylidene)-9-oxo-9H-xanthene-2-sulfon-amide were the most suitable ligands of dnaE and hence proposed as the potential inhibitors of this target protein. The results of this study could facilitate the discovery and release of new and effective drugs against E. coli O157:H7 and other deadly human bacterial pathogens.

Antioxidant Properties of Popular Turmeric (Curcuma longa) Varieties from Bangladesh

We investigated the aqueous and ethanolic extracts of different forms (local names: mura and chora) of turmeric (Curcuma longa) from the Khulna and Chittagong divisions of Bangladesh for their antioxidant properties and polyphenol, flavonoid, tannin, and ascorbic acid contents. The antioxidant activity was determined using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical-scavenging activity and ferric reducing antioxidant power (FRAP) values. The ethanolic extract of Chittagong’s mura contained the highest concentrations of polyphenols (16.07%), flavonoids (9.66%), and ascorbic acid (0.09 mg/100 g) and chora resulted in high yields (17.39%). The ethanolic extract of Khulna’s mura showed a higher DPPH radical-scavenging activity with the lowest 50% inhibitory concentration (IC50) (1.08 μg/mL), while Khulna’s chora had the highest FRAP value (  μM Fe per 100 g). Overall, the ethanolic extract had higher antioxidant properties than those in the aqueous extract. However, the tannin concentration was lower in the ethanolic extract. We conclude that the turmeric varieties investigated in this study are useful sources of natural antioxidants, which confer significant protection against free radical damage.

Sundarban Honey Confers Protection against Isoproterenol-Induced Myocardial Infarction in Wistar Rats

The present study was designed to investigate the cardioprotective effects of Sundarban honey (SH) in rats with isoproterenol- (ISO-) induced myocardial infarction. Adult male Wistar Albino rats were pretreated with Sundarban honey (5 g/kg) daily for a period of 6 weeks. After the treatment period, ISO (85 mg/kg) was subcutaneously injected into the rats at 24 h intervals for 2 days. ISO-induced myocardial damage was indicated by increased serum cardiac specific troponin I levels and cardiac marker enzyme activities including creatine kinase-MB, lactate dehydrogenase, aspartate transaminase, and alanine transaminase. Significant increases in serum total cholesterol, triglycerides, and low-density lipoprotein-cholesterol levels were also observed, along with a reduction in the serum high-density lipoprotein-cholesterol level. In addition to these diagnostic markers, the levels of lipid peroxide products were significantly increased. The activities of antioxidant enzymes such as superoxide dismutase, glutathione peroxidase, and glutathione reductase were significantly decreased in the hearts after ISO-induced myocardial infarction. However, pretreatment of ischemic rats with Sundarban honey brought the biochemical parameters to near normalcy, indicating the protective effect of Sundarban honey against ISO-induced ischemia in rats. Histopathological findings of the heart tissues further confirmed the biochemical findings, indicating that Sundarban honey confers protection against ISO-induced oxidative stress in the myocardium.

Minerals, Toxic Heavy Metals, and Antioxidant Properties of Honeys from Bangladesh

The study reports on major and trace elements as well as antioxidant properties of honey samples from Bangladesh. Four major cationic elements, seven trace elements, and three heavy metals were determined in the 12 honey samples using atomic absorption spectrophotometer. Nutritional values in these honey samples were further investigated according to their antioxidant properties. The content of major elements was in the range of 62.75–616.58, 579.48–2219.43, 69.42–632.25, and 0.13–1.20 mg/kg for sodium, potassium, magnesium, and calcium, respectively. The trace elements varied in the range of 0.41–28, 0.12–3.54, 1.54–2.85, 0.29–0.59, 0.02–0.35, and 0.01–0.06 mg/kg for iron, zinc, copper, nickel, cobalt, and cadmium, respectively. Among the heavy metals, only lead (0.17–2.19 mg/kg) was detected. The results of antioxidant analysis based on phenolics, flavonoids, ascorbic acid, reducing sugar, and proteins (as nonphenolic antioxidants) revealed that multifloral raw honey samples contain significantly higher levels of reducing agents than monofloral and commercial brand honeys. The study provides a useful insight on the minerals, heavy metals, and antioxidant properties of honey samples commonly consumed in Bangladesh and found to be rich source of antioxidants and minerals. Some samples might pose some risk to the health due to lead contamination.