Photocleavable fluorescent nucleotides for DNA sequencing on a chip constructed by site-specific coupling chemistry

DNA sequencing by synthesis on a solid surface offers new paradigms to overcome limitations of electrophoresis-based sequencing methods. Here we report DNA sequencing by synthesis using photocleavable (PC) fluorescent nucleotides [dUTP-PC-4,4-difluoro-4-bora-3 alpha,4 alpha-diaza-s-indacene (Bodipy)-FL-510, dCTP-PC-Bodipy-650, and dUTP-PC-6-carboxy-X-rhodamine (ROX)] on a glass chip constructed by 1,3-dipolar azide-alkyne cycloaddition coupling chemistry. Each nucleotide analogue consists of a different fluorophore attached to the base through a PC 2-nitrobenzyl linker. We constructed a DNA microarray by using the 1,3-dipolar cycloaddition chemistry to site-specifically attach azido-modified DNA onto an alkyne-functionalized glass chip at room temperature under aqueous conditions. After verifying that the polymerase reaction could be carried out successfully on the above-described DNA array, we then performed a sequencing reaction on the chip by using a self-primed DNA template. In the first step, we extended the primer using DNA polymerase and dUTP-PC-Bodipy-FL-510, detected the fluorescent signal from the fluorophore Bodipy-FL-510, and then cleaved the fluorophore using 340 nm UV irradiation. This process was followed by extension of the primer with dCTP-PC-Bodipy-650 and the subsequent detection of the fluorescent signal from Bodipy-650 and its photocleavage. The same procedure was also performed by using dUTP-PC-ROX. The entire process was repeated five times by using the three fluorescent nucleotides to identify 7 bases in the DNA template. These results demonstrate that the PC nucleotide analogues can be incorporated accurately into a growing DNA strand during polymerase reaction on a chip, and the fluorophore can be detected and then efficiently cleaved using near-UV irradiation, thereby allowing the continuous identification of the template sequence.     

Four-color DNA sequencing by synthesis on a chip using photocleavable fluorescent nucleotides

We report four-color DNA sequencing by synthesis (SBS) on a chip, using four photocleavable fluorescent nucleotide analogues (dGTP-PC-Bodipy-FL-510, dUTP-PC-R6G, dATP-PC-ROX, and dCTP-PC-Bodipy-650) (PC, photocleavable; Bodipy, 4,4-difluoro-4-bora-3alpha,4alpha-diaza-s-indacene; ROX, 6-carboxy-X-rhodamine; R6G, 6-carboxyrhodamine-6G). Each nucleotide analogue consists of a different fluorophore attached to the 5 position of the pyrimidines and the 7 position of the purines through a photocleavable 2-nitrobenzyl linker. After verifying that these nucleotides could be successfully incorporated into a growing DNA strand in a solution-phase polymerase reaction and the fluorophore could be cleaved using laser irradiation ( approximately 355 nm) in 10 sec, we then performed an SBS reaction on a chip that contains a self-priming DNA template covalently immobilized by using 1,3-dipolar azide-alkyne cycloaddition. The DNA template was produced by PCR, using an azido-labeled primer, and the self-priming moiety was attached to the immobilized DNA template by enzymatic ligation. Each cycle of SBS consists of the incorporation of the photocleavable fluorescent nucleotide into the DNA, detection of the fluorescent signal, and photocleavage of the fluorophore. The entire process was repeated to identify 12 continuous bases in the DNA template. These results demonstrate that photocleavable fluorescent nucleotide analogues can be incorporated accurately into a growing DNA strand during a polymerase reaction in solution and on a chip. Moreover, all four fluorophores can be detected and then efficiently cleaved using near-UV irradiation, thereby allowing continuous identification of the DNA template sequence. Optimization of the steps involved in this SBS approach will further increase the read-length.     

Direct Effect of Endodontic Sealers on Trigeminal Neuronal Activity

Introduction

Endodontic sealers are selected on the basis of their antimicrobial properties and ability to provide a tight seal. Sealer extrusions, whether intentional or unintentional, are common during obturation procedures. Such events have been correlated with increased postoperative discomfort and persistent pain states. However, the mechanisms underlying this phenomenon are largely unknown. Thus, we sought to evaluate the effect of commonly used endodontic sealers on peripheral nociceptors. We hypothesized that endodontic sealers can directly activate trigeminal nociceptors in a concentration-dependent manner, resulting in release of calcitonin gene-related peptide (CGRP), a potent modulator of neurogenic inflammation.

Methods

Rat trigeminal sensory neurons were exposed in vitro to vehicle, zinc oxide-eugenol (ZOE)–based sealer, AH Plus, EndoSequence BC sealer, or RealSeal SE. Neuronal activation was measured by quantification of neuropeptide (CGRP) release. In addition, cultured neurons were also subjected to the set form of all 4 sealers. The concentration of CGRP released was quantified by using a radioimmunoassay. Data were analyzed by using one-way analysis of variance with Newman-Keuls multiple comparison post hoc test.

Results

Both ZOE-based sealer and AH Plus in their fresh form evoked greater CGRP release than the control groups. Conversely, EndoSequence BC and RealSeal sealers both reduced basal GCRP release at all concentrations tested. Evaluation of the set sealers revealed that only ZOE-based sealer evoked significant CGRP release compared with its control group.

Conclusions

Overall, our results suggest that sealers can directly activate trigeminal nociceptors, leading to a robust release of CGRP, and may therefore lead to pain and neurogenic inflammation. This direct activation along with the immunologic response may underlie the symptoms and flare-up occurrences often seen with sealer extrusions.     

Systemic lupus erythematosus presenting as acute lupus pneumonitis in a young female

Acute lupus pneumonitis is a rare initial presentation of systemic lupus erythematosus (SLE). We report a 19-year-old female presenting with fever and recurrent hemoptysis with radiological evidence of parenchymal lung involvement with mild pleural effusion. Subsequent development of malar and discoid rash with anti-nuclear antibodies (ANA) and anti-dsDNA positivity clinched the diagnosis. Her clinical signs and symptoms resolved with a course of intravenous pulse methyl-prednisolone along with radiological resolution.KEY WORDS: Acute lupus pneumonitis, hemoptysis, systemic lupus erythematosus     

STABILIZATION OF THORACOLUMBAR SPINAL INJURIES

Spinal Cord injury cases are being managed in Base Hospital Delhi Cantt since Oct. 97. 27 cases of thoracolumbar injuries were admitted in this hospital during the period Oct 97 to Aug 99. 20 patients underwent surgical treatment (9 thoracic and 11 lumbar) and 7 were treated conservatively. All these operations were done within 3 weeks following trauma, and methylprednisolone therapy was instituted in those who reached the hospital early. Contraindications to surgery included stable fracture, bed sores, any focus of sepsis and generalized bone disorders. Transpedicular fixation with Dyna-lok system was done in 10 cases, universal spinal system was applied in 6 cases and Harrington instrumentation was carried out in 4 cases. Decompression laminectomy was done in all cases. Patients with incomplete cord injury showed neurological improvement and early rehabilitation was possible after surgery.KEY WORDS: Harrington instrumentation, Pedicle screw and plate, Spine trauma, Thoracolumbar fractures     

Cognitive impairment after transurethral resection of the prostate (TURP)

Purpose: It has been suggested that cognitive impairment may occur following transurethral resection of the prostate (TURP) operations due to the effects of anaesthesia or hyponatraemia or both. The aim of the study was to investigate whether TURP was associated with long-term memory complaints or cognitive impairment. Method: Patients who had received a TURP or transurethral resection of a bladder tumour (TURT) in the previous 10 months were sent a questionnaire on memory problems and mood. Seventy-six TURP patients and 38 TURT patients returned the completed questionnaires. A sample of each patient group (30 TURP, 29 TURT) was assessed on standardized tests of cognitive abilities. Results: There was no significant difference between the TURP and TURT patients on the Everyday Memory Questionnaire completed by themselves or by a family member (p?>?0.05). On formal cognitive testing there were no significant differences between the groups, except on the overall grading of the Kendrick Assessment Scales of Cognitive Ageing, in which TURP patients performed at a significantly lower level than TURT patients. Conclusions: The results suggest that patients are no more likely to complain of memory problems following a TURP than with any other operative procedure. There was a slight difference in cognitive abilities but this did not indicate significant impairment of cognitive function.