En bloc resection of urothelium carcinoma of the bladder (EBRUC): a European multicenter study to compare safety, efficacy, and outcome of laser and electrical en bloc transurethral resection of bladder tumor.

PURPOSE: En bloc resection of bladder tumors (ERBT) may improve staging quality and perioperative morbidity and influence tumor recurrence. This study was designed to evaluate the safety, efficacy, and recurrence rates of electrical versus laser en bloc resection of bladder tumors. METHODS: This European multicenter study included 221 patients at six academic hospitals. Transurethral ERBT was performed with monopolar/bipolar current or holmium/thulium laser energy. Staging quality measured by detrusor muscle involvement, various perioperative parameters, and 12-month follow-up data was analyzed. RESULTS: Electrical and laser ERBT were used to treat 156 and 65 patients, respectively. Median tumor size was 2.1 cm; largest tumor was 5 cm. Detrusor muscle was present in 97.3 %. A switch to conventional TURBT was significantly more frequent in the electrical ERBT group (26.3 vs. 1.5 %, p < 0.001). Median operation duration (25 min), postoperative irrigation (1 day), catheterization time (2 days), and hospitalization (3 days) were similar. Overall complication rate was low (Clavien ≥ 3, n = 6 [2.7 %]). Hemoglobin was significantly lower after electrical ERBT (p = 0.0013); however, overall hemoglobin loss was not clinically relevant (0.38 g/dl). Patients (n = 148) were followed for 12 months; 33 (22.3 %) had recurrences. In total, 63.6 % recurrences occurred outside the ERBT resection field. No difference was noted between ERBT groups. CONCLUSIONS: ERBT is safe and reliable regardless of the energy source and provides high-quality resections of tumors >1 cm. Recurrence rates did not differ between groups, and the majority of recurrences occurred outside the ERBT resection field.

Biomechanical comparison of prophylactic medial malleolar fixation in total ankle arthroplasty.

The purpose of this study was to compare the biomechanical properties of two common forms of prophylactic fixation of the medial malleolus during total ankle arthroplasty (TAA). Ten matched pairs of cadaveric lower extremities were fixed with a single cannulated cortical screw or a one-third tubular plate after preparation for TAA. Ten unmatched single cadaver lower extremities were used as the control. A transverse load was applied to the medial malleolus. Bone mineral density, peak loads at fracture, and fracture pattern were noted. There was no statistically significant difference in peak load to failure between either fixation cohorts or between the fixation cohort and the control cohort. There was a moderate positive correlation between bone mineral density and peak load for all three experimental groups. The peak load was significantly (p = .035) increased in male cadavers. Fractures occurred almost exclusively at the apex of the tibial and malleolar cuts with a vertical fracture pattern.

Glutamine synthetase (GS) knockout (KO) using CRISPR/Cpf1 diversely enhances selection efficiency of CHO cells expressing therapeutic antibodies.

The glutamine synthetase (GS)-based Chinese hamster ovary (CHO) selection system is an attractive approach to efficiently identify suitable clones in the cell line generation process for biologics manufacture, for which GS-knockout (GS-KO) CHO cell lines are commonly used. Since genome analysis indicated that there are two GS genes in CHO cells, deleting only 1 GS gene could potentially result in the activation of other GS genes, consequently reducing the selection efficiency. Therefore, in this study, both GS genes identified on chromosome 5 (GS5) and 1 (GS1) of CHO-S and CHO-K1, were deleted using CRISPR/Cpf1. Both single and double GS-KO CHO-S and K1 showed robust glutamine-dependent growth. Next, the engineered CHO cells were tested for their efficiency of selection of stable producers of two therapeutic antibodies. Analysis of pool cultures and subclones after a single round of 25 µM methionine sulfoxinime (MSX) selection indicated that for CHO-K1 the double GS5,1-KO was more efficient as in the case of a single GS5-KO the GS1 gene was upregulated. In CHO-S, on the other hand, with an autologously lower level of expression of both variants of GS, a single GS5-KO was more robust and already enabled selection of high producers. In conclusion, CRISPR/Cpf1 can be efficiently used to knock out GS genes from CHO cells. The study also indicates that for the generation of host cell lines for efficient selection, the initial characterisation of expression levels of the target gene as well as the identification of potential escape mechanisms is important.

Age, microbiology and prognostic scores help to differentiate between secondary and tertiary peritonitis.

BACKGROUND AND AIMS: Tertiary peritonitis is a severe persisting intra-abdominal infection and associated with high mortality. The aim was to find significant risk factors for mortality and tertiary peritonitis including the Mannheim Peritonitis Index (MPI), the Acute Physiology and Chronic Health Evaluation (APACHE) II score, and a sumscore of both. MATERIALS AND METHODS: In this retrospective single-center cohort study, 122 patients were treated at the Surgical Department of a University Hospital. RESULTS: Sixty-nine patients (56.6%) developed tertiary peritonitis. Nineteen patients (27.5%), who suffered from tertiary peritonitis, died in contrast to eight patients (15.1%) with secondary peritonitis (P = 0.101). Patients with tertiary peritonitis had significantly higher APACHE II (P < 0.001), MPI (P = 0.035), and combined APACHE II and MPI scores (P < 0.001) than patients with secondary peritonitis. Age (P = 0.035), fungal infections (P = 0.025), and infections with more than one microbial organism (P = 0.047) were predictive for tertiary peritonitis. Combined APACHE II and MPI scores detected tertiary peritonitis better than the MPI (P = 0.014). Detection of mortality was comparable in all evaluated prognostic scores. CONCLUSION: Prognostic scores besides age and fungal infections are risk factors for mortality and help to differentiate between secondary and tertiary peritonitis. The combination of prognostic scores is comparable to the APACHE II and superior compared to the MPI in regard to detection of tertiary peritonitis.

Increased growth rate and productivity following stable depletion of miR-7 in a mAb producing CHO cell line causes an increase in proteins associated with the Akt pathway and ribosome biogenesis.

Cell line engineering using microRNAs represents a desirable route for improving the efficiency of recombinant protein production by CHO cells. In this study we generated stable CHO DP12 cells expressing a miR-7 sponge transcript which sequesters miR-7 from its endogenous targets. Depletion of miR-7 results in a 65% increase in cell growth and >3-fold increase in yield of secreted IgG protein. Quantitative labelfree LC-MS/MS proteomic profiling was carried out to identify the targets of miR-7 and understand the functional drivers of the improved CHO cell phenotypes. Subcellular enrichment and total proteome analysis identified more than 3000 proteins per fraction resulting in over 5000 unique proteins identified per timepoint analysed. Early stage culture analysis identified 117 proteins overexpressed in miR-7 depleted cells. A subset of these proteins are involved in the Akt pathway which could be the underlying route for cell density improvement and may be exploited more specifically in the future. Late stage culture identified 160 proteins overexpressed in miR-7 depleted cells with some of these involved in ribosome biogenesis which may be causing the increased productivity through improved translational efficiency. This is the first in-depth proteomic profiling of the IgG producing CHO DP12 cell line stably depleted of miR-7. SIGNIFICANCE: Chinese hamster ovary (CHO) cells are the mammalian cell expression system of choice for production of recombinant therapeutic proteins. There is much research ongoing to characterise CHO cell factories through the application of systems biology approaches that will enable a fundamental understanding of CHO cell physiology, and as a result, a better knowledge and understanding of recombinant protein production. This study profiles the proteomic effects of microRNA-7 depletion on the IgG producing CHO DP12 cell line. This is one of the very few studies that attempts to identify the functioning proteins driving improved CHO cell phenotypes resulting from microRNA manipulation. Using subcellular enrichment and total proteome analysis we identified over 5000 unique proteins in miR-7 depleted CHO cells. This work has identified a cohort of proteins involved in the Akt pathway and ribosome biogenesis. These proteins may drive improved CHO cell phenotypes and are of great interest for future work.

Torsional stiffness after subtalar arthrodesis using second generation headless compression screws: Biomechanical comparison of 2-screw and 3-screw fixation.

BACKGROUND: Subtalar joint arthrodesis is a common operative treatment for symptomatic subtalar arthrosis. Because excessive relative motion between the talus and calcaneus can delay or prohibit fusion, fixation should be optimized, particularly in patients at risk for subtalar arthrodesis nonunion. Tapered, fully-threaded, variable pitch screws are gaining popularity for this application, but the mechanical properties of joints fixed with these screws have not been characterized completely. We quantified the torsion resistance of 2-screw and 3-screw subtalar joint fixation using this type of screw. METHODS: Ten pairs of cadaveric subtalar joints were prepared for arthrodesis and fixed using Acutrak 2-7.5 screws. One specimen from each pair was fixed with two diverging posterior screws, and the contralateral joint was fixed using two posterior screws and a third screw directed through the anterior calcaneus into the talar neck. Internal and external torsional loads were applied and joint rotation and torsional stiffness were measured at two torque levels. FINDINGS: Internal rotation was significantly less in specimens fixed with three screws. No difference was detectable between 2-screw and 3-screw fixation in external rotation or torsional stiffness in either rotation direction. Both 2-screw and 3-screw fixation exhibited torsion resistance surpassing that reported previously for subtalar joints fixed with two diverging conventional lag screws. INTERPRETATION: Performance of the tapered, fully threaded, variable pitch screws exceeded that of conventional lag screws regardless of whether two or three screws were used. Additional resistance to internal rotation afforded by a third screw placed anteriorly may offer some advantage in patients at risk for nonunion.

The influence of patient age and bone mineral density on osteotomy fixation stability after hallux valgus surgery: A biomechanical study.

BACKGROUND: Oblique osteotomies of the first metatarsal are common surgical treatments for moderate to severe hallux valgus deformity. Osteotomy fixation integrity is important to minimize interfragment motion and maintain correction during healing, and our clinical observations suggest that patient age and bone quality affect fixation stability and ultimately the clinical outcome. Accordingly, this study correlated these patient factors with key mechanical measures of osteotomy angulation resistance in a cadaver hallux valgus correction model. METHODS: Standard Ludloff osteotomies were created in 31 fresh-frozen first metatarsals and fixed with two cannulated, dual-pitch headless screws. Each specimen underwent 1000 plantar-to-dorsal bending loads while monitoring bending stiffness and distal fragment dorsal angulation. Donor age and bone mineral density were then correlated with each mechanical measure at selected cycling increments. FINDINGS: We found significant positive correlation between bone mineral density and osteotomy fixation stiffness for all evaluated load cycles. Moderate negative correlation between bone density and angulation was identified, significant for load cycle 500. There was a weak, nonsignificant negative correlation between donor age and osteotomy bending stiffness, with r ranging from -0.134 to -0.243 between the first and 1000th loads. Little correlation was demonstrable between age and angulation. INTERPRETATION: Because low bone density correlates with decreased osteotomy site stiffness and increased angulation under load, patient compliance and protected weight bearing in the early postoperative phase are particularly important if bone mineral density is exceptionally low. Correspondingly, patients with especially high bone mineral density may be considered candidates for earlier weight bearing and active physical therapy.