Correction: Characterization of a Polyethylene Glycol-Amphotericin B Conjugate Loaded with Free AMB for Improved Antifungal Efficacy.

[This corrects the article DOI: 10.1371/journal.pone.0152112.].

Wide Resection for Pediatric Extremity Vascular Malformation-A Tertiary Hospital's Experience.

BACKGROUND: Vascular malformations of the extremities (VM) are common benign lesions that tend to grow throughout the patient's lifetime. They can cause various issues like pain, swelling, and even limb length discrepancies. Sclerotherapy was the preferred treatment choice in previous studies. However, sclerotherapy and many other treatments have the potential to result in higher recurrence rates. Surgical treatment has been shown to be effective and safe in many cases. Hence, this study aims to evaluate the suitability of wide resection surgery for VM to reduce recurrence. METHODS: Fort-seven VM cases that underwent wide resection were identified retrospectively in the institution of study. Demographics, depth of malformation, whether malformations were local or diffuse, location and size of malformations, and histology records were taken note of. Records of recurrence and postoperative function were also gathered. We utilized self-reported questionnaires, QuickDASH and Lower Extremity Functional Scale, to determine patients' postsurgical physical function. RESULTS: Out of 47 cases that underwent wide resection, we found a recurrence rate of 2.1%. No patients sustained any loss of function postsurgery, with few patients experiencing minor complications like tenderness, hypertrophic scars/keloids, as well as numbness. Good functionality posttreatment was also seen through self-reported questionnaires, with an average score of 2.12 for QuickDASH and 99.96% for LEFS. CONCLUSION: Where margins can be obtained without functional impairment, surgical-wide resection for VM is a viable treatment option to minimize recurrence. LEVEL OF EVIDENCE: Level-IV.

Combined multivariate statistical and flux balance analyses uncover media bottlenecks to the growth and productivity of Chinese hamster ovary cell cultures.

Chinese hamster ovary (CHO) cells are widely used for producing recombinant proteins. To enhance their productivity and product quality, media reformulation has been a key strategy, albeit with several technical challenges, due to the myriad of complex molecular mechanisms underlying media effects on culture performance. Thus, it is imperative to characterize metabolic bottlenecks under various media conditions systematically. To do so, we combined partial least square regression (PLS-R) with the flux balance analysis of a genome-scale metabolic model to elucidate the physiological states and metabolic behaviors of human alpha-1 antitrypsin producing CHO-DG44 cells grown in one commercial and another two in-house media under development. At the onset, PLS-R was used to identify metabolite exchanges that were correlated to specific growth and productivity. Then, by comparing metabolic states described by resultant flux distributions under two of the media conditions, we found suboptimal level of four nutrients and two metabolic wastes, which plausibly hindered cellular growth and productivity; mechanistically, lactate and ammonia recycling were modulated by glutamine and asparagine metabolisms in the media conditions, and also by hitherto unsuspected folate and choline supplements. Our work demonstrated how multivariate statistical analysis can be synergistically combined with metabolic modeling to uncover the mechanistic elements underlying differing media performance. It thus paved the way for the systematic identification of nutrient targets for medium reformulation to enhance recombinant protein production in CHO cells.

Using the radiocoronoid line for diagnosis of elbow dislocation.

The radiocapitellar line (RCL) has been widely used to diagnose elbow dislocation. However, there are limitations to the RCL, with the cartilaginous portion of bone making interpretation of radiographs difficult. The study aims to show that the radiocoronoid line, which connects two points on the medial aspect of the radius, proximal to the radial tuberosity, is more suited to diagnose elbow dislocations in the anterior-posterior projection. This study also observes factors affecting accuracy of the radiocapitellar line. The radiographs of 50 normal and 17 laterally dislocated elbows were obtained. An unbiased independent reviewer drew the radiocoronoid and radiocapitellar line (RCL). Four other blinded independent reviewers drew the RCL and the radiocoronoid line for 20 radiographs and repeated the process a week later. The accuracy of the RCL was assessed using distance away from bisection point of capitellum, and ratio (distance from the point where line crosses capitellum to lateral aspect of capitellum over the total width of capitellum). The relationship of the radio-coronoid line and the lateral aspect of coronoid fossa was assessed, with dislocation being the line lateral to it and normal being medial to or on it. The radiocoronoid line had a higher accuracy (95.5%) compared to RCL (32.8%), higher specificity (94%) compared to RCL (10%) as well as higher positive predictive value (85%) compared to RCL (27.4%). There was no intra- or inter-observer variability for the radio-coronoid line. Skeletal age statistically predicted the ratio for the male population ( P < 0.05), however, the independent variables did not statistically predict the dependent variables for the female and total population. The radiocoronoid line serves as an additional method to assess radiocapitellar joint lateral dislocation. It is more accurate and reliable than the radiocapitellar line in the anterior-posterior projection. Sex and skeletal age also influence the accuracy of the radiocapitellar line with the radiocapitellar line nearing the bisection point as skeletal age in males increases.

Health-Related Quality of Life After Diagnosis and Treatment of Differentiated Thyroid Cancer and Association With Type of Surgical Treatment.

Importance: Concerns around possible overdiagnosis and overtreatment of differentiated thyroid cancer (DTC) have been raised. Issues concerning health-related quality of life (HRQOL) after diagnosis and treatment of DTC are understudied in this patient group. Objective: To better understand the range of HRQOL outcomes, including possible adverse effects of treatment, associated with diagnosis and treatment of DTC and whether these outcomes vary by type of surgery received. Design, Setting, and Participants: This content analysis assessed responses to an open-ended question about outcomes and concerns after DTC diagnosis and treatment among patients ascertained from the major postsurgical thyroid cancer treatment center and the population-based Cancer Registry in Queensland, Australia. Participants were aged 18 to 79 years and recently diagnosed with throid cancer. Responses underwent analysis to identify and code emergent themes to describe HRQOL issues and adverse effects of treatment experienced. Quantitative analysis was used to explore whether surgery type was associated with HRQOL issues and/or adverse effects of treatment. Of 1416 eligible patients, 1005 (71.0%) participated. Data were collected from July 1, 2013, through August 31, 2016, and analyzed from January 11 through April 9, 2018. Main Outcomes and Measures: Issues concerning HRQOL. Results: The analysis included 1005 patients (72.2% female [n = 726]; mean [SD] age, 52 [14.0 years) with DTC. Most patients were diagnosed with papillary thyroid cancer (889 of 1003 [88.6%]), had tumors smaller than 2 cm in size (564 of 1000 [56.4%]), and received a total thyroidectomy (791 of 1005 [78.7%]). Overall, 775 patients (77.1%) reported HRQOL issues after diagnosis and treatment of DTC. The following 4 main themes emerged from content analysis of patient responses: physical (663 [66.0%]), psychological (187 [18.6%]), lifestyle (82 [8.2%]), and no issue or adverse effect (246 [24.5%]). Patients who had a total thyroidectomy (without neck dissection) were 1.5 times (odds ratio, 1.49; 95% CI, 1.04-2.12) more likely to report an HRQOL issue or an adverse effect of treatment compared with patients who underwent a hemithyroidectomy. Conclusions and Relevance: According to results of this study, patients diagnosed with DTC report wide-ranging HRQOL issues; these are more prevalent among patients who have total thyroidectomies rather than hemithyroidectomies. For patients with small, localized DTCs, hemithyroidectomy may offer fewer adverse effects of treatment and better HRQOL outcomes than total thyroidectomy. It appears that issues with HRQOL should be considered by patients and physicians when deciding on the best treatment approach after a diagnosis of DTC.

Mammalian Systems Biotechnology Reveals Global Cellular Adaptations in a Recombinant CHO Cell Line.

Effective development of host cells for therapeutic protein production is hampered by the poor characterization of cellular transfection. Here, we employed a multi-omics-based systems biotechnology approach to elucidate the genotypic and phenotypic differences between a wild-type and recombinant antibody-producing Chinese hamster ovary (CHO) cell line. At the genomic level, we observed extensive rearrangements in specific targeted loci linked to transgene integration sites. Transcriptional re-wiring of DNA damage repair and cellular metabolism in the antibody producer, via changes in gene copy numbers, was also detected. Subsequent integration of transcriptomic data with a genome-scale metabolic model showed a substantial increase in energy metabolism in the antibody producer. Metabolomics, lipidomics, and glycomics analyses revealed an elevation in long-chain lipid species, potentially associated with protein transport and secretion requirements, and a surprising stability of N-glycosylation profiles between both cell lines. Overall, the proposed knowledge-based systems biotechnology framework can further accelerate mammalian cell-line engineering in a targeted manner.

Characterization of a Polyethylene Glycol-Amphotericin B Conjugate Loaded with Free AMB for Improved Antifungal Efficacy.

Amphotericin B (AMB) is a highly hydrophobic antifungal, whose use is limited by its toxicity and poor solubility. To improve its solubility, AMB was reacted with a functionalized polyethylene glycol (PEG), yielding soluble complex AmB-PEG formulations that theoretically comprise of chemically conjugated AMB-PEG and free AMB that is physically associated with the conjugate. Reverse-phase chromatography and size exclusion chromatography methods using HPLC were developed to separate conjugated AMB-PEG and free AmB, enabling the further characterization of these formulations. Using HPLC and dynamic light scattering analyses, it was observed that the AMB-PEG 2 formulation, having a higher molar ratio of 2 AMB: 1 PEG, possesses more free AMB and has relatively larger particle diameters compared to the AMB-PEG 1 formulation, that consists of 1 AMB: 1 PEG. The identity of the conjugate was also verified using mass spectrometry. AMB-PEG 2 demonstrates improved antifungal efficacy relative to AMB-PEG 1, without a concurrent increase in in vitro toxicity to mammalian cells, implying that the additional loading of free AMB in the AMB-PEG formulation can potentially increase its therapeutic index. Compared to unconjugated AMB, AMB-PEG formulations are less toxic to mammalian cells in vitro, even though their MIC50 values are comparatively higher in a variety of fungal strains tested. Our in vitro results suggest that AMB-PEG 2 formulations are two times less toxic than unconjugated AMB with antifungal efficacy on Candida albicans and Cryptococcus neoformans.

Production of functional soluble Dectin-1 glycoprotein using an IRES-linked destabilized-dihydrofolate reductase expression vector.

Dectin-1 (CLEC7A) is a C-type lectin receptor that binds to ß-glucans found in fungal cell walls to act as a major pattern recognition receptor (PRR). Since ß-glucans epitope is not present in human cells, we are of the opinion that Dectin-1 can have therapeutic functions against fungal infections. We thus set out to produce a soluble extracellular domain of murine Dectin-1 (called sDectin-1) in sufficient titers to facilitate such studies in mouse models. Since sDectin-1 has previously been shown to be glycosylated, we chose to produce this protein using Chinese Hamster Ovary (CHO) cells, a mammalian host cell line suitable for the high-titer production of recombinant glycoproteins. To ensure a high titer production of sDectin-1 and minimize the effects of gene fragmentation, we constructed a mammalian expression vector with a PEST-destabilized dhfr amplifiable marker downstream of an attenuated IRES element, which was in turn downstream of the sDectin-1 gene and a CMV IE promoter. Stably transfected and MTX-amplified cell pools were generated using this vector, and maximum sDectin-1 titers of 246 mg/l and 598 mg/l were obtained in shake flask batch culture and bioreactor fed-batch culture respectively. The purified recombinant sDectin-1 was shown to be glycosylated. Protein functionality was also demonstrated by its ability to bind to zymosan particles and to the cell wall of Saccharomyces cerevisiae. We describe for the first time the use of an attenuated IRES-linked PEST-destabilized dhfr amplifiable marker for the production of recombinant proteins with stably amplified cell pools. With our process, we reached the highest reported titer for producing recombinant proteins smaller than 50 kDa in cell pools. sDectin-1 protein produced is glycosylated and functional. This vector design can thus be used efficiently for the high-titer production of functional recombinant proteins.