Deepak Saxena

Insecticidal toxin in root exudates from Bt corn

Bt corn is corn (Zea mays) that has been genetically modified to express insecticidal toxins derived from the bacterium Bacillus thuringiensis to kill lepidopteran pests feeding on these plants. Here we show that Bt toxin is released into the rhizosphere soil in root exudates from Bt corn.

Bacillus thuringiensis (Bt) toxin released from root exudates and biomass of Bt corn has no apparent effect on earthworms, nematodes, protozoa, bacteria, and fungi in soil

There were no significant differences in the percent mortality and weight of earthworms (Lumbricus terrestris) after 40 days in soil planted with Bt (NK4640Bt) or non-Bt corn or after 45 days in soil amended with biomass of Bt or non-Bt corn. The toxin was present in the guts and casts of earthworms in soil planted with Bt corn or amended with biomass of Bt corn, but it was cleared within 2‐3 days from the guts after placing in fresh soil. There were no significant differences in the colony-forming units of culturable bacteria (including actinomycetes) and fungi and in the numbers of protozoa and nematodes between rhizosphere soil of Bt and non-Bt corn or between soil amended with biomass of Bt and non-Bt corn. The Cry1Ab protein in root exudates and biomass of Bt corn appears not to be toxic to earthworms, nematodes, protozoa, bacteria, and fungi. The presence of the toxin in the guts and casts of earthworms confirmed that the toxin released in root exudates and from transgenic biomass was bound on surface-active particles in soil, which protected the toxin from biodegradation, as has been observed in this laboratory with purified toxin. q 2001 Elsevier Science Ltd. All rights reserved.

Bt corn has a higher lignin content than non-Bt corn

Bt corn has been genetically modified to express the Cry1Ab protein of Bacillus thuringiensis to kill lepidopteran pests. Fluorescence microscopy and staining with toluidine blue indicated a higher content of lignin in the vascular bundle sheaths and in the sclerenchyma cells surrounding the vascular bundle in all ten Bt corn hybrids, representing three different transformation events, studied than of their respective non-Bt isolines. Chemical analysis confirmed that the lignin content of all hybrids of Bt corn, whether grown in a plant growth room or in the field, was significantly higher (33-97% higher) than that of their respective non-Bt isolines. As lignin is a major structural component of plant cells, modifications in lignin content may have ecological implications.

Genetic Profiling of the Oral Microbiota Associated with Severe Early-Childhood Caries

ABSTRACT The determination of the composition of the microbial community in the oral cavity is usually based on cultivation methods; however, nearly half of the bacteria in the saliva and the dental plaque are not cultivable. In this study, we evaluated the difference in oral microbial diversity between children with severe early-childhood caries (S-ECC) and caries-free (CF) controls by means of a cultivation-independent approach called denaturing gradient gel electrophoresis (DGGE). Pooled dental plaque samples were collected from 20 children aged 2 to 8 years. Total microbial genomic DNA was isolated from those subjects, and a portion of the 16S rRNA gene locus was PCR amplified by using universal primers. We observed that the mean species richness of the bacterial population was greater in the CF children (n = 12) (42 ± 3.7) than in the S-ECC children (n = 8) (35 ± 4.3); the difference was statistically significant (P = 0.005). The overall diversity of plaque samples as measured by the Shannon index was 3.5 for the S-ECC group and 3.7 for the CF group (P = 0.004). Differences in DGGE profiles were distinguished on the basis of a cluster analysis. Sequence analysis of excised DGGE bands consisted of 2.7 phylotypes, on average. After adjusting for the number of observed bands, we estimated that the S-ECC group exhibited 94.5 total phylotypes and that the CF group exhibited 113.4. These results suggest that the microbial diversity and complexity of the microbial biota in dental plaque are significantly less in S-ECC children than in CF children.

Survey of Oral Microbial Diversity using PCR-based Denaturing Gradient Gel Electrophoresis

Polymicrobial biofilms in the human oral cavity exhibit marked diversity. PCR-based denaturing gradient gel electrophoresis (PCR-DGGE) surveys microbial diversity by displaying PCR-generated 16S rDNA fragments that migrate at different distances, reflecting the differences in the base-pair (i.e., % G+C) composition of the fragment. This study examined DGGE-generated diversity profiles of cultivable bacteria from individuals with different caries status. Initially, we developed a set of PCR-DGGE running conditions appropriate to oral bacteria. Next, we assessed migration standards from known oral bacterial reference strains. To test the methods, we profiled 20 bacterial saliva samples cultivated from young adults. The study produced a battery of species-specific 16S rDNA amplicons that could be used as a migration distance standard necessary for computer-assisted profile analysis. From the clinical samples, we found a significantly greater diversity of oral microbes in caries-free individuals compared with caries-active individuals (P = 0.01). These findings suggest thtat a portion of oral microbiota of caries-active individuals may be absent, suppressed, or replaced.

Comparison of oral microbiota in tumor and non-tumor tissues of patients with oral squamous cell carcinoma

BackgroundBacterial infections have been linked to malignancies due to their ability to induce chronic inflammation. We investigated the association of oral bacteria in oral squamous cell carcinoma (OSCC/tumor) tissues and compared with adjacent non-tumor mucosa sampled 5 cm distant from the same patient (n = 10). By using culture-independent 16S rRNA approaches, denaturing gradient gel electrophoresis (DGGE) and cloning and sequencing, we assessed the total bacterial diversity in these clinical samples.ResultsDGGE fingerprints showed variations in the band intensity profiles within non-tumor and tumor tissues of the same patient and among the two groups. The clonal analysis indicated that from a total of 1200 sequences characterized, 80 bacterial species/phylotypes were detected representing six phyla, Firmicutes, Bacteroidetes, Proteobacteria, Fusobacteria, Actinobacteria and uncultivated TM7 in non-tumor and tumor libraries. In combined library, 12 classes, 16 order, 26 families and 40 genera were observed. Bacterial species, Streptococcus sp. oral taxon 058, Peptostreptococcus stomatis, Streptococcus salivarius, Streptococcus gordonii, Gemella haemolysans, Gemella morbillorum, Johnsonella ignava and Streptococcus parasanguinis I were highly associated with tumor site where as Granulicatella adiacens was prevalent at non-tumor site. Streptococcus intermedius was present in 70% of both non-tumor and tumor sites.ConclusionsThe underlying changes in the bacterial diversity in the oral mucosal tissues from non-tumor and tumor sites of OSCC subjects indicated a shift in bacterial colonization. These most prevalent or unique bacterial species/phylotypes present in tumor tissues may be associated with OSCC and needs to be further investigated with a larger sample size.

Microbial diversity in saliva of oral squamous cell carcinoma

In the oral cavity, chronic inflammation has been observed at various stages of oral squamous cell carcinomas (OSCC). Such inflammation could result from persistent mucosal or epithelial cell colonization by microorganisms. There is increasing evidence of the involvement of oral bacteria in inflammation, warranting further studies on the association of bacteria with the progression of OSCC. The objective of this study was to evaluate the diversity and relative abundance of bacteria in the saliva of subjects with OSCC. Using 454 parallel DNA sequencing, ∼58,000 PCR amplicons that span the V4-V5 hypervariable region of rRNAs from five subjects were sequenced. Members of eight phyla (divisions) of bacteria were detected. The majority of classified sequences belonged to the phyla Firmicutes (45%) and Bacteroidetes (25%). Further, 52 different genera containing approximately 860 (16.51%) known species were identified and 1077 (67%) sequences belonging to various uncultured bacteria or unclassified groups. The species diversity estimates obtained with abundance-based coverage estimators and Chao1 were greater than published analyses of other microbial profiles from the oral cavity. Fifteen unique phylotypes were present in all three OSCC subjects.

Predicting risk for bisphosphonate-related osteonecrosis of the jaws: CTX versus radiographic markers

BACKGROUND AND OBJECTIVE The most common risk factor for bisphosphonate-related osteonecrosis of the jaws (BRONJ) is dentoalveolar surgery. It has been suggested that reduced serum C-terminal telopeptide (CTX) can determine the degree of osteoclast suppression and may predict the development of BRONJ after dentoalveolar surgery. Although there are many radiographic appearances associated with BRONJ, there are little data that describes changes preceding dentoalveolar surgery. The objective of this retrospective study was: 1) to investigate if reduced serum CTX values (i.e., <150 pg/mL) were associated with BRONJ after dentoalveolar surgery; and 2) to determine if specific radiographic changes are associated with teeth that develop BRONJ after extraction. STUDY DESIGN A retrospective review of radiographic and/or serum CTX data was performed for 68 patients with a history of bisphosphonate therapy who either underwent dental extraction or were diagnosed with BRONJ in the Department of Oral and Maxillofacial Surgery during the period 2007-2009. Postoperative healing was assessed for 26 patients with reduced serum CTX levels (<150 pg/mL) who either underwent dental extraction or treatment for BRONJ. Preoperative radiographs were evaluated for 55 patients who either healed normally or developed BRONJ after dental extraction. RESULTS All 26 patients (100%) who had serum CTX levels <150 pg/mL healed successfully after dentoalveolar surgery (20 patients) or after treatment for BRONJ (6 patients). Among the 55 patients who underwent radiographic evaluation, 24 patients (83%) with BRONJ exhibited periodontal ligament (PDL) widening associated with extracted teeth, whereas only 3 patients (11%) who healed normally demonstrated PDL widening. CONCLUSION These data suggest that radiographic PDL widening may be a more sensitive indicator than CTX testing in predicting risk of BRONJ. Current guidelines that recommend minimal surgical intervention may need to be revised to include alternative strategies for the elimination or management of this pathology.

Pancreatic cancer, inflammation, and microbiome.

AbstractPancreatic cancer is one of the most lethal cancers worldwide. No effective screening methods exist, and available treatment modalities do not effectively treat the disease. Inflammatory conditions such as pancreatitis represent a well-known risk factor for pancreatic cancer development. Yet only in the past 2 decades has pancreatic cancer been recognized as an inflammation-driven cancer, and the precise mechanisms underlying the pathogenic role of inflammation are beginning to be explored in detail. A substantial amount of preclinical and clinical evidence suggests that bacteria are likely to influence this process by activating immune receptors and perpetuating cancer-associated inflammation. The recent explosion of investigations of the human microbiome have highlighted how perturbations of commensal bacterial populations can promote inflammation and promote disease processes, including carcinogenesis. The elucidation of the interplay between inflammation and microbiome in the context of pancreatic carcinogenesis will provide novel targets for intervention to prevent and treat pancreatic cancer more efficiently. Further studies toward this direction are urgently needed.

Bt toxin is not taken up from soil or hydroponic culture by corn, carrot, radish, or turnip

The culture of transgenic Bt corn (Zea mays L.) has resulted in concern about the uptake of the Cry1Ab protein toxin by crops subsequently grown in soils in which Bt corn has been grown. The toxin released to soil in root exudates of Bt corn, from the degradation of the biomass of Bt corn, or as purified toxin, was not taken up from soil, where the toxin is bound on surface-active particles (e.g. clays and humic substances), or from hydroponic culture, where the toxin is not bound on particles, by non-Bt corn, carrot (Daucus carota L.), radish (Raphanus sativus L.), and turnip (Brassica rapa L.). The persistence of the toxin in soil for 90 days after its addition in purified form or for 120–180 days after its release in exudates or from biomass, the longest times evaluated, confirmed that the toxin was bound on surface-active particles in soil, which protected the toxin from biodegradation. The greater toxicity of the toxin in soil amended with 9% montmorillonite or kaolinite than in soil amended with 3% of these clay minerals indicated that the binding and persistence of the toxin increased as the clay concentration was increased.