Inducible nuclear import by TetR aptamer-controlled 3' splice site selection.

Correct cellular localization is essential for the function of many eukaryotic proteins and hence cell physiology. Here, we present a synthetic genetic device that allows the control of nuclear and cytosolic localization based on controlled alternative splicing in human cells. The device is based on the fact that an alternative 3' splice site is located within a TetR aptamer that in turn is positioned between the branch point and the canonical splice site. The novel splice site is only recognized when the TetR repressor is bound. Addition of doxycycline prevents TetR aptamer binding and leads to recognition of the canonical 3' splice site. It is thus possible to produce two independent splice isoforms. Since the terminal loop of the aptamer may be replaced with any sequence of choice, one of the two isoforms may be extended by the respective sequence of choice depending on the presence of doxycycline. In a proof-of-concept study, we fused a nuclear localization sequence to a cytosolic target protein, thus directing the protein into the nucleus. However, the system is not limited to the control of nuclear localization. In principle, any target sequence can be integrated into the aptamer, allowing not only the production of a variety of different isoforms on demand, but also to study the function of mislocalized proteins. Moreover, it also provides a valuable tool for investigating the mechanism of alternative splicing in human cells.

Over a decade of single-center experience with thoracoscopic sympathicolysis for primary palmar hyperhidrosis: a case series.

BACKGROUND: Primary palmar hyperhidrosis is a severely debilitating condition that can affect patients of any age. We report our experience with thoracoscopic sympathicolysis in a large cohort of children less than 14 years of age. METHODS: All children who underwent thoracoscopic sympathicolysis from April 2005 through January 2017 were evaluated retrospectively. The procedure entailed bilateral bipolar fulguration of the second and third thoracic ganglia with transverse disruption of collateral nerve fibers along the third and fourth rib. Demographic information, as well as postoperative outcome, complications, and satisfaction were analyzed. RESULTS: Over the 12 year study interval, a total of 102 children underwent thoracoscopic sympathicolysis for palmar hyperhidrosis. Complete follow-up was available for 98 patients (median age 12 [range 5-14] years; 38 boys [39%]). Median follow-up was 4 [range 2-12] years. Complete palmar dryness was achieved in 93 (95%) cases. One patient suffered postoperative unilateral ptosis, 6 reported gustatory sweating, and 65 experienced compensatory sweating. Average postoperative rating on a 1 (lowest) to 10 (highest) rating scale was 9, with 97 (99%) patients saying that they would undergo the procedure again. CONCLUSION: Our technique of thoracoscopic sympathicolysis in children was associated with very high postoperative satisfaction, despite a high rate of compensatory sweating and occasional autonomic gustatory sweating. Other more severe complications in this age group were rare.

Robust gene expression control in human cells with a novel universal TetR aptamer splicing module.

Fine-tuning of gene expression is desirable for a wide range of applications in synthetic biology. In this context, RNA regulatory devices provide a powerful and highly functional tool. We developed a versatile, robust and reversible device to control gene expression by splicing regulation in human cells using an aptamer that is recognized by the Tet repressor TetR. Upon insertion in proximity to the 5' splice site, intron retention can be controlled via the binding of TetR to the aptamer. Although we were able to demonstrate regulation for different introns, the genomic context had a major impact on regulation. In consequence, we advanced the aptamer to develop a splice device. Our novel device contains the aptamer integrated into a context of exonic and intronic sequences that create and maintain an environment allowing a reliable and robust splicing event. The exon-born, additional amino acids will then be cleaved off by a self-cleaving peptide. This design allows portability of the splicing device, which we confirmed by demonstrating its functionality in different gene contexts. Intriguingly, our splicing device shows a high dynamic range and low basal activity, i.e. desirable features that often prove a major challenge when implementing synthetic biology in mammalian cell lines.

Laparoscopic histological mapping for the determination of the length of aganglionic segment in children with Hirschsprung disease.

BACKGROUND: Modern approach to the surgical treatment of Hirschsprung's disease (HD) consists in the earliest possible repair and reduction of the number of surgical interventions. Primary one-stage transanal endorectal pull-through (TEPT) technique requires preoperative determination of the length of aganglionic segment. The efficacy of the standard method - contrast enema - is questionable in patients with a poorly defined transitional zone. OBJECTIVES: To present the proposed laparoscopic method for the management pathway in patients with HD, in whom the determination of the length of aganglionic segment with contrast enema was not possible. MATERIAL AND METHODS: A retrospective analysis of the diagnostic and therapeutic management employed in 14 patients, from 2 weeks to 55 months of age, with diagnosed HD, treated between January 2013 and May 2020. Laparoscopic histological mapping was performed with the use of 3 laparoscopic ports of 3-5 mm diameter. RESULTS: In all patients, laparoscopic mapping allowed for the determination of the length of aganglionic segment and the mode of surgical treatment. Four children with determined short-segment disease underwent TEPT, while 2 underwent temporary colostomy formation using the Duhamel-Martin-Ikeda method. Five patients with long-segment HD underwent laparoscopic-assisted TEPT. One patient with long-segment disease was treated with a temporary double-barrel colostomy and definitive surgery was performed 3 months later using the Duhamel-Martin-Ikeda method. In 2 patients with an initial diagnosis of HD established using current diagnostic pathway, HD pathology was later excluded based on the results of laparoscopic mapping and repeat rectal suction biopsy. No complications related to the laparoscopic procedure were identified. CONCLUSIONS: The method of laparoscopic mapping is effective in the determination of the length of aganglionic segment in children with diagnosed HD. In doubtful cases, it can be the preferred option in establishing the final mode of surgical treatment.

The application of the percutaneous suturing technique in thoracoscopic repair of congenital diaphragmatic hernia.

BACKGROUND: Thoracoscopic surgery of congenital diaphragmatic hernia (CDH) is connected with a higher incidence of recurrence than open repair is. This is usually caused by the dehiscence of sutures in the lateral part of the defect. This area is characterized by increased tension on proximate tissues and difficult thoracoscopic suturing. For more effective repair, the authors adopted a variant of percutaneous internal ring suturing (PIRS) technique. OBJECTIVES: To present and evaluate the efficacy of the PIRS technique for the repair of CDH. MATERIAL AND METHODS: The study is based on retrospective analysis of the medical data of patients with CDH treated in the Department of Pediatric Surgery of the Jagiellonian University Medical College (Kraków, Poland) from January 2013 to July 2019. The PIRS technique was applied when thoracoscopic repair under acceptable tension appeared impossible. RESULTS: Fifty-one patients were identified. Of these, 11 children died before surgery and 1 after, leaving 39 (76%) who were operated on and survived. Thoracoscopy was used in 27 cases (69%), with 3 conversions. The recurrence rate in patients who underwent thoracoscopic closure of the defect with intermittent sutures was 27% (3 out of 11 children), while in the group that underwent thoracoscopic repair with the additional use of percutaneous suturing, the recurrence rate was 6.25% (1 out of 16 patients). The follow-up periods ranged from 4 months to 6 years. CONCLUSIONS: The applied PIRS technique permits safe and effective closure of intermediate-size diaphragmatic defects under acceptable tension. The method is feasible and can be listed among countermeasures against recurrence.