Comparative analysis of the accuracy of periapical radiography and cone-beam computed tomography for diagnosing complex endodontic pathoses using a gold standard reference - A prospective clinical study.

AIM: To compare the diagnostic accuracy of periapical radiography (PR) and cone-beam computed tomography (CBCT) for detecting pathoses of endodontic origin using the gold standard of direct surgical visualization. METHODOLOGY: Seventy-four patients (112 teeth) underwent radiographic examination by periapical radiography and CBCT. The presence of periapical lesions, root perforations and their locations, root fractures, different types of external root resorptive defects, apicomarginal bone defects, through and through bone defects, buccal bone status and proximity of root apices and lesions to vital anatomical structures were assessed. These same parameters were assessed intraoperatively by direct surgical visualization which served as the gold standard reference for the radiographic assessments. Sensitivity, specificity, positive predictive values, negative predictive values were determined for comparison of diagnostic accuracy between two modalities. RESULTS: There were no significant differences between the two imaging modalities for root resorptive defects and root fractures. However, CBCT accurately detected periapical lesions, root perforations, apicomarginal bone defects and through and through bone defects. The overall accuracy of CBCT varied from 91% to 96% in detecting dehiscence and fenestration of buccal cortical plates. CONCLUSION: Overall, CBCT had a higher diagnostic accuracy in complex endodontic pathoses compared to PR. Nevertheless, CBCT failed to diagnose apicomarginal bone defects in 33% teeth. In evaluating the status of buccal cortical plate from CBCT images, observers could detect absence of bone better than its presence. Thus, limited FOV CBCT should be considered for selective cases where periapical radiography has diagnostic ambiguity.

Over-expression of the peroxisomal ascorbate peroxidase (SbpAPX) gene cloned from halophyte Salicornia brachiata confers salt and drought stress tolerance in transgenic tobacco.

Salicornia brachiata Roxb., an extreme halophyte, is a naturally adapted higher plant model for additional gene resources to engineer salt tolerance in plants. Ascorbate peroxidase (APX) plays a key role in protecting plants against oxidative stress and thus confers abiotic stress tolerance. A full-length SbpAPX cDNA, encoding peroxisomal ascorbate peroxidase, was cloned from S. brachiata. The open reading frame encodes for a polypeptide of 287 amino acid residues (31.3-kDa protein). The deduced amino acid sequence of the SbpAPX gene showed characteristic peroxisomal targeting sequences (RKRAI) and a C-terminal hydrophobic region of 39 amino acid residues containing a transmembrane domain (TMD) of 23 amino acid residues. Northern blot analysis showed elevated SbpAPX transcript in response to salt, cold, abscisic acid and salicylic acid stress treatments. The SbpAPX gene was transformed to tobacco for their functional validation under stresses. Transgenic plants over-expressing SbpAPX gene showed enhanced salt and drought stress tolerance compared to wild-type plants. Transgenic plants showed enhanced vegetative growth and germination rate both under normal and stressed conditions. Present study revealed that the SbpAPX gene is a potential candidate, which not only confers abiotic stress tolerance to plants but also seems to be involved in plant growth.

Ectopic over-expression of peroxisomal ascorbate peroxidase (SbpAPX) gene confers salt stress tolerance in transgenic peanut (Arachis hypogaea).

Peroxisomal ascorbate peroxidase gene (SbpAPX) of an extreme halophyte Salicornia brachiata imparts abiotic stress endurance and plays a key role in the protection against oxidative stress. The cloned SbpAPX gene was transformed to local variety of peanut and about 100 transgenic plants were developed using optimized in vitro regeneration and Agrobacterium mediated genetic transformation method. The T0 transgenic plants were confirmed for the gene integration; grown under controlled condition in containment green house facility; seeds were harvested and T1 plants were raised. Transgenic plants (T1) were further confirmed by PCR using gene specific primers and histochemical GUS assay. About 40 transgenic plants (T1) were selected randomly and subjected for salt stress tolerance study. Transgenic plants remained green however non-transgenic plants showed bleaching and yellowish leaves under salt stress conditions. Under stress condition, transgenic plants continued normal growth and completed their life cycle. Transgenic peanut plants exhibited adequate tolerance under salt stress condition and thus could be explored for the cultivation in salt affected areas for the sustainable agriculture.

Ovary-specific novel peroxisome proliferator activated receptors-gamma transcripts in buffalo.

In the present study, we describe the isolation and characterization of the transcripts encoding peroxisome proliferator-activated receptor gamma (PPARγ1 and PPARγ2) in buffalo ovary. 5' RACE experiments and sequence analysis showed that these transcripts (PPARγ1a, PPARγ1b and PPARγ2) were transcribed by the different promoter usage and alternative splicing of terminal 5'-exon. The distribution of these isoforms of PPARγ transcripts in different tissues (ovary, mammary gland, spleen, liver, lung, adipose tissue) was investigated using quantitative real time analysis. Tissue- and transcript-specific expression analyses showed that a transcript, transcribed from distal promoter, not only expressed preferentially in ovary but contributes predominantly to PPAR gamma expression in ovary. Western blot analysis of both, in vivo and in vitro, experiments also supported that PPARγ1 predominantly expressed in ovary. In buffalo granulosa cells culture, the isolated transcripts were found to be up-regulated by both natural (CLA) and synthetic (Rosiglitazone) ligands and effect was reversed by PPARγ antagonist GW9662. In conclusion, the present study identified an ovary-specific novel transcript, transcribed by distal promoter, predominantly expressed in ovary which could have functional relevance in buffalo ovary.

Cloning and characterization of buffalo NANOG gene: alternative transcription start sites, splicing, and polyadenylation in embryonic stem cell-like cells.

NANOG is a critical homeodomain transcription factor responsible for maintaining embryonic stem cell (ESC) self-renewal and pluripotency. In the present study, we isolated, sequenced, and characterized the NANOG gene in buffalo ESC-like cells. Here, we demonstrated that NANOG mRNA is expressed as multiple isoforms and uses four alternative transcriptional start sites (TSSs) and five different polyadenylation sites. The TSSs identified by 5'-RNA ligase-mediated rapid amplification of cDNA ends (RLM-5'-RACE) were positioned at 182, 95, 35, and 17 nucleotides upstream relative to the translation initiation codon. 3'-RACE experiment revealed the presence of tandem polyadenylation signals, which leads to the expression of at least five different 3'-untranslated regions (269, 314, 560, 566, and 829 nucleotides). Expression analysis showed that these alternatively polyadenylated transcripts expressed differentially. Sequence analysis showed that the open reading frame of buffalo NANOG codes for a 300-amino-acid-long protein. Further, results showed that alternative splicing leads to the expression of two types of transcript variants encoded by four and five exons. In silico analysis of cloned 5'-flanking region (3366 nucleotides upstream of translation start codon) identified several putative transcription factors binding sites in addition to a TATA box and CAAT box at -30 and -139 bp (upstream to the distal most TSS), respectively, in the buffalo NANOG promoter.

Characterization of POU5F1 (OCT4) gene and its promoter in buffalo ESC-like cells identifies multiple transcription start sites and expression of four pseudogenes.

In the present study, we cloned and characterized the buffalo (Bubalus bubalis) OCT4 ortholog expressed in embryonic stem cell (ESC) like cells and its promoter region. The 5'- and 3'-RACE experiments were conducted to analyze the transcription initiation site and regulatory regions. The comparative analysis of buffalo OCT4 promoter with other mammalian orthologs revealed high conservation. Among the regulatory regions highest similarity was observed between buffalo, bovine and sheep. Interestingly, buffalo OCT4 promoter exhibited a 78 bp deletion between two proximal enhancers (PE-1A and PE-1B) when compared to other mammalian orthologs. 5'-RACE revealed four different transcription start sites for OCT4 gene. As far as we know there is no previous report regarding multiple transcription initiation sites for OCT4 gene in any species. In addition, we identified expression of four pseudogenes in buffalo ESC-like cells. Among the multiple transcripts characterized, we found four cDNA clones (1083 bp) derived from ESC-like cells sharing 96.9-99.3% sequence homology with the parent gene and having the capacity of encoding 139, 206, 206 and 324 amino acid long truncated proteins. Multiple pseudogenes have been proposed for OCT4 which might contribute to the false detection of this gene during expression studies. However, only few of them were reported to be transcribed and none were reported to be translated in stem cells. Western blot analysis of OCT4 protein using ESC-like cells revealed multiple bands, indicating that some of the hypothetical pseudogenes are being translated. These novel pseudogenes or their protein products may have some important regulatory functions.