Tandem Placement Research Articles

Stability and Function of Hybrid Protein Domains of Dystrophin Created by Exon Shuffling

The dystrophin gene is the largest human gene, at ~2.5 Mbp, and one of the most complex. Its 79 exons are processed to a ~14 kbp mRNA resulting in the 427 kDa full length dystrophin. This protein is largely composed of 24 tandem copies of the spectrin-type-repeat, STR a ~110 amino acid motif that forms a triple α-helical rodshaped bundle. Tandem placement of many STR in this protein produces a long rod. The exon structure of this gene is such that every second exon junction very nearly coincides with STR junctions, while intervening exon junctions fall ~0.43 through each. Each STR is thus very nearly coded for by two exons. Recently it has been shown that dystrophin mRNA is alternatively processed in a complex, tissue dependent fashion. Many of these result in the excision of multiple exons, and the most common type of deletion observed start and end at junctions in the middle of STRs, rather than at junctions coincident with STR boundaries. If translated into protein (something that has not been demonstrated) this would result in novel, hybrid domains made up of two heterologous halves fused together. It is unclear whether such shuffling would result in viable protein structures. We have tested this in one case, reproducing an observed brain-specific deletion in a recombinant dystrophin fragment, and determining the resulting hybrid's stability by thermal and urea denaturation. We find that this hybrid structure, while slightly less stable than the native structure thermodynamically, is well folded and forms a viable and stable protein. Other measures of biological stability, including resistance to proteolysis, also show that is it stable, and in fact more so than the full-length structure.

Tandem application with transvaginal ultrasound guidance

Purpose Intrauterine tandem placement can be difficult in brachytherapy for cervical cancer. A prospective study was planned to investigate transvaginal ultrasound (TVU) guidance for intrauterine tandem insertions in the treatment of cervix cancer. Methods and materials Between January 1997 and January 2002, TVU was used after pelvic external beam radiotherapy in 48 cervical cancer patients with a high complication risk because the cervical canal orifice could not be identified and/or the patients could not undergo hysterometry. The TVU findings were graded as 1–4, with a higher number indicating greater difficulty in implantation. The tandem was inserted using the guidance of the TVU findings. Tandem application difficulties were graded in accordance with the number of implantation trials. The relations between tandem applicability and possible factors were analyzed. Results By TVU, the difficulties in tandem insertion were graded as Grade 1 in 5 cases, Grade 2 in 23 cases, Grade 3 in 17 cases, and Grade 4 in 3 cases. The tandem was inserted in 19 patients on the first try, 12 patients on the second try, 12 patients on the third try, and 2 patients on the fourth try. However, the cervical canal could not be opened in 3 patients. Factors such as median age, stage, chemotherapy, diameter of the tandem applicators, time between external beam radiotherapy and brachytherapy, and the rate of tumor regression were not statistically significant. The ultrasound grade ( p = 0.02) and diameter of the tandem applicator ( p = 0.007) were statistically significant. Perforation and sepsis were not observed. Conclusion TVU guidance before brachytherapy has a positive contributory effect in reducing the morbidity rate of patients considered high risk.

Uterine perforation at the time of brachytherapy for carcinoma of the cervix

Objective The reported incidence of uterine perforation by tandems at the time of vaginal brachytherapy for management of cervical cancer ranges from 1.75% to 10% per application. The potential ramifications of afterloading a tandem that is outside the uterine cavity within the pelvis are sufficiently severe, in terms of bowel injury, that a reliable means of determining tandem positioning at the time of placement is essential. Methods We examined a representative patient that underwent tandem placement for Federation Internationale of Gynecologic Oncologist (FIGO) stage Ib1 cervical Squamous Cell Carcinoma (SCCA). The information provided by conventional radiographic assessment and clinical impression was not helpful in ultimately determining tandem placement. Results Direct endoscopic visualization provided accurate and irrefutable evidence of tandem location and positioning prior to leaving the operating room. Conclusion A variety of techniques have been described to assist in accurate placement of the uterine tandem prior to afterloading, when utilizing definitive chemoradiation in the management of cervical carcinoma. These include postoperative plain films or computerized tomography, versus intraoperative transabdominal or transrectal ultrasound guidance at the time of placement. Postoperative techniques that confirm perforation necessitate a second visit to the operating room, a second general anesthetic, as well as a treatment delay that may impact negatively upon central control rates in the long term. Intraoperative techniques have their limitations as well, particularly in the obese patient. With difficult or questionable insertions, direct endoscopic evaluation at the time of tandem insertion is the only means of knowing definitively the location of the tandem prior to leaving the operating room.

Tandem Placement of a Coronavirus Promoter Results in Enhanced mRNA Synthesis from the Downstream-Most Initiation Site

Insertion of the 17-nucleotide promoter region for the bovine coronavirus N gene as part of a 27-nucleotide cassette into the open reading frame of a cloned synthetic defective-interfering (DI) RNA resulted in synthesis of subDI RNA transcripts from the replicating DI RNA genome. Duplicating and triplicating the promoter sequence in tandem caused a progressive increase in the efficiency of subgenomic mRNA synthesis despite a concurrent decrease in the rate of DI RNA accumulation that was not specific to the promoter sequences being added. Although initiation of transcription (leader fusion) occurred at each of the three promoter sites in the tandem construct, almost all of the transcripts were found as a product of the most downstream (3′-most on the genome) promoter. These results show that enhancement of subgenomic mRNA synthesis is a property that can reside within sequence situated near the promoter. A possible role for the plus strand in the downstream promoter choice is suggested.

Major geometric variations between multiple high-dose-rate applications of brachytherapy in cancer of the cervix: frequency and types of variation.

To evaluate major geometric variations in multiple intracavitary applications for carcinoma of the cervix. Orthogonal radiographs were reviewed of 17 consecutive patients with carcinoma of the cervix treated with 70 applications of high-dose-rate brachytherapy. In seven patients, conscious sedation was used for all applications. In 10 patients, general anesthesia was used for the first application and conscious sedation for subsequent applications. Major geometric variation between applications in axis, length, and slippage in tandem placement and separation, packing, and slippage in colpostats placement were reviewed. A major variation was defined as more than 1.0-cm deviation. Major variations between applications occurred more commonly in colpostats placement than in tandem placement. For tandems, the rates of variation were 5.7% in axis, 4.3% in length, and 1.4% in slippage. For colpostats, rates of variation were 7.1% in separation, 25.7% in vaginal packing, and 7.1% in slippage. No consistent pattern of variation was found between applications except in vaginal packing. Awareness of geometric variations should improve proper placement of intracavitary applicators for brachytherapy.

Intraoperative guidance for intrauterine procedures with transrectal sonography.

Transrectal sonography was used for intraoperative guidance in intrauterine tandem placement for intracavitary radiation therapy and in dilation and curettage procedures. The authors describe the method and three representative cases in which it was applied. It is concluded that the technique may prevent complications such as uterine perforation or bladder injury in tandem placement, and that it can facilitate dilation and curettage in complicated cases.

Real-time ultrasound as an aid in tandem placement in intracavitary therapy of gynecologic malignancies

Real-time ultrasound as an aid in tandem placement in intracavitary therapy of gynecologic malignancies

Ultrasound for diagnosing and preventing malplacement of intrauterine tandems

Postoperative B-mode ultrasound was used to evaluate final tandem position in 50 consecutive placements (28 patients). In 34% (17 of 50), the tandem was found to be suboptimally positioned; in 24% (12 of 50), it penetrated the myometrium; and in 10% (five of 50), it frankly perforated the uterus. The uterine fundus was the region most commonly perforated, and the anterior myometrium was the site most frequently penetrated. In all cases in which postoperative ultrasound showed malpositioning, the clinical and radiographic assessment indicated proper intracavitary placement. Ultrasound affected clinical management in 42% (21 of 50) of the placements involving 61% (17 of 28) of the patients. To improve tandem placement, we used intraoperative real-time ultrasound to guide 73 consecutive surgical insertions. Ultrasound clearly visualized the procedure, allowing tandems to be positioned with confidence even in the most difficult cases. The immediate feedback from intraoperative ultrasound eliminated malplacements and thus the need for a second anesthesia to reposition the tandem.

Ultrasound used for assessing the in situ position of intrauterine tandems

Intracavitary radiation is an integral part of the treatment in many gynecologic cancers. The intrauterine tandem has a well-established role in treating both cervical and endometrial malignancies. The effectiveness of this therapy and its complications are directly related to the proper intracavitary position of the tandem. To date, there has been no objective technique appropriate for routine use in assessing the precise in situ location of these devices, apart from plain abdominal films. As such, little is known about the incidence or types of procedural complications and the sequelae associated with improper tandem placement. Here it is reported that ultrasound is an effective tool for evaluating the in situ position of the intrauterine tandem. Two cases are presented demonstrating important applications of this method.