Synthetic intrinsically disordered protein fusion tags that enhance protein solubility.

We report the de novo design of small (<20 kDa) and highly soluble synthetic intrinsically disordered proteins (SynIDPs) that confer solubility to a fusion partner with minimal effect on the activity of the fused protein. To identify highly soluble SynIDPs, we create a pooled gene-library utilizing a one-pot gene synthesis technology to create a large library of repetitive genes that encode SynIDPs. We identify three small (<20 kDa) and highly soluble SynIDPs from this gene library that lack secondary structure and have high solvation. Recombinant fusion of these SynIDPs to three known inclusion body forming proteins rescue their soluble expression and do not impede the activity of the fusion partner, thereby eliminating the need for removal of the SynIDP tag. These findings highlight the utility of SynIDPs as solubility tags, as they promote the soluble expression of proteins in E. coli and are small, unstructured proteins that minimally interfere with the biological activity of the fused protein.

Non-operative adjuncts for the prevention of mastectomy skin flap necrosis: a systematic review and meta-analysis.

BACKGROUND: Native skin flap necrosis is a potentially devastating complication following skin-sparing or nipple-sparing mastectomy with a reported incidence of as high as 30%. Treatment depends on the depth and extent of tissue necrosis and can range from dressings to surgical debridement and further reconstruction. This can have implications on patient physical and psychological wellbeing as well as cost of treatment. This study aims to identify and appraise cost-effective non-surgical adjuncts for the prevention of native skin flap necrosis. METHODS: A systematic review was performed using the preferred reporting items for systematic reviews and meta-analyses (PRISMA) statement and structured around existing recommended guidelines. A search of MEDLINE, EMBASE, Cumulative Index to Nursing and Allied Health Literature and ClinicalTrials.gov was performed with the medical subject headings 'mastectomy' and 'flap necrosis'. After exclusion, 12 articles were selected for review and analysed. RESULTS: A total of 8439 mastectomies were performed on 7895 patients. Preventative non-surgical adjuncts that demonstrated statistically significant reduction in mastectomy flap necrosis included topical nitroglycerin ointment (P = 0.000), closed-Incision negative pressure wound therapy (P = 0.000), topical dimethylsulfoxide ointment (P = 0.03), oral cilostazol (P = 0.032), and local heat pre-conditioning (P = 0.047). CONCLUSIONS: This study identifies multiple adjuncts that may aid in preventing mastectomy skin flap necrosis, especially in high-risk patients. Further studies could aim to define standardized protocols and compare the various adjuncts in different circumstances.

Antibiotic Prophylaxis Prescribing Practice in Head and Neck Tumor Resection and Free Flap Reconstruction.

BACKGROUND: Adherence to guidelines for antibiotic prophylaxis is often poor and is an important target for antimicrobial stewardship programs. Prescribing audits that suggested poor adherence to guidelines in a plastic surgery department led to a targeted education program to bring antibiotic prescriptions in line with hospital guidelines. We reviewed whether this intervention was associated with changed perioperative prescribing and altered surgical outcomes, including the rate of surgical site infections, specifically looking at clean-contaminated head and neck tumor resections with free flap reconstruction. METHODS: A retrospective cohort study was performed on 325 patients who underwent clean-contaminated head and neck tumor resection and free flap reconstruction from January 1, 2013, to February 19, 2019. Patients were divided into 2 groups, those before (pre-intervention) and after (postintervention) the education campaign. We analyzed patient demographic and disease characteristics, intraoperative and postoperative factors, and surgical outcomes. RESULTS: Patients pre-intervention were prescribed longer courses of prophylactic antibiotics (median [interquartile range], 9 [8] vs 1 [1]; P < .001), more topical chloramphenicol ointment (21.82% vs 0%; P < .001), and more oral nystatin (36.9% vs 12.2%; P < .001). Patients postintervention had higher rates of recipient infections (36.11% vs 17.06%; P < .001) and donor site infections (6.94% vs 1.19%; P = .006). CONCLUSIONS: Following the education campaign, patients were prescribed shorter courses of prophylactic antibiotics, more of the recommended cefazolin-metronidazole regimen, and fewer topical antibiotics. However, patients also had a higher rate of surgical site infections.

Risk factors for surgical site infection in free-flap reconstructive surgery for head and neck cancer: Retrospective Australian cohort study.

BACKGROUND: Surgical site infections (SSI) are common complications of free-flap reconstruction for head and neck cancer defects. This study aimed to identify risk factors for SSI following a significant change in local antibiotic prophylaxis practice. METHODS: A retrospective cohort study was conducted of 325 patients receiving free-flap reconstruction for head and neck cancer defects at a tertiary hospital in Melbourne, Australia between 2013 and 2019. Charts were queried for recipient SSI (primary outcome), donor SSI, other infections, antibiotic use, hospital length of stay, and mortality. RESULTS: Risk factors for SSI included female sex, T-classification, hardware insertion, clindamycin prophylaxis, and operative duration. There was a trend toward increased SSI with shorter ≤24 h prophylaxis (OR: 0.43). CONCLUSION: Antibiotic duration and type were associated with SSI. Complexity of surgery, T-classification, hardware use, and operative duration were also independently associated with SSI. A prospective trial is indicated to elicit optimal prophylactic antibiotic duration.

A novel technique for proximal interphalangeal joint fracture-dislocations: the second toe middle phalanx osteochondral graft.

Many different surgical techniques have been used to treat unstable dorsal proximal interphalangeal joint fracture-dislocations. The authors have used the base of the middle phalanx of the second toe base as an alternative autograft to treat this type of injury. This retrospective study assessed the clinical outcomes of this procedure in 11 patients. Range of motion, grip strength, Disability of the Arm, Shoulder and Hand score and donor site morbidity were assessed at regular intervals postoperatively. Nine patients had acute injuries and two had chronic injuries. The mean range of motion in the proximal interphalangeal joint at final review was 65° for patients with acute injuries and 41° for patients with chronic injuries. Other outcomes were satisfactory and there were no complications.Level of evidence: IV.

Quality-of-life outcomes after operative management of primary and secondary lymphoedema: a systematic review.

BACKGROUND: Lymphoedema is an incurable and progressive disease that affects not only physical function but overall quality of life. Surgical treatment options for the management of lymphoedema are being increasingly performed. This study aims to review post-operative health-related quality of life (HRQOL) following surgical treatment of lymphoedema. METHODS: A systematic search of the PubMed and Medline databases was performed from the date of their inception until September 2018 to evaluate HRQOL following different surgical options for the treatment of lymphoedema. RESULTS: One hundred and thirteen articles were identified. Twenty-one articles were included in the final review, comprising a total of 736 patients. HRQOL improvements appear to be sustained for at least 6-12 months post-operatively. In particular, major benefits were noted in the domains based around physical functioning. Patient satisfaction similarly mirrors HRQOL improvements, following an initial dip in the immediate post-operative period. CONCLUSION: All surgical treatment modalities for the management of lymphoedema confer significant HRQOL improvements across a diverse range of health domains, with this critical outcome of surgery an important pre-operative consideration. Recommendations for ongoing research are suggested.

Virtual surgical planning in fibula free flap head and neck reconstruction: A systematic review and meta-analysis.

BACKGROUND: The traditional approach to head and neck reconstruction is considered challenging, requiring a subjective assessment of an often-complex defect followed by careful modelling of a bony flap to match this. The introduction of Virtual Surgical Planning (VSP) has provided the surgeon with a means to increase efficiency, precision and overall patient outcomes. This study aims to compare VSP and traditional head and neck reconstructions utilising fibula free flaps with regards surgical efficiency and patient outcomes. METHODS: A systematic search of the PubMed and Medline databases was performed from the date of their inception through to August 2018 to evaluate and compare VSP and non-VSP cohorts in the context of fibula free flap head and neck reconstruction. Primary comparative outcomes included operative and ischaemic time, with secondary outcomes including complications rates, measures of accuracy and financial benefits. RESULTS: One hundred and fifty-three articles were identified. Twenty-three articles were included in the review, comprising a total of 713 patients. VSP was associated with significantly decreased intraoperative time (Standardised Mean Difference -1.01; 95% CI -1.23 to 0.80; p = 0.000) and ischaemic time (Standardised Mean Difference -1.55; 95% CI -1.87 to -1.23, p = 0.002). VSP was also associated with reduced orthognathic deviation from an ideal outcome when compared to conventional techniques. No statistically significant differences in complication rates between conventional and VSP techniques were identified. CONCLUSION: The results of this meta-analysis suggests that VSP confers significant benefits with respect to improved orthognathic accuracy, ischaemic times and intraoperative times without any significant increase in complications. Recommendations for ongoing research are suggested.

Juxtanodin in retinal pigment epithelial cells: Expression and biological activities in regulating cell morphology and actin cytoskeleton organization.

Juxtanodin (JN, also known as ermin) was initially identified as an actin cytoskeleton-related oligodendroglial protein in the rat central nervous system. It was subsequently also found in the rat olfactory neuroepithelium, especially at the apical junctional belt of the sustentacular cells. We further examined JN expression and functional roles in the retina using fluorescence histochemistry, confocal microscopy, immuno-electron microscopy, molecular biology, and cell culture. Prominent JN expression was found in the photoreceptor-supporting retinal pigment epithelium (RPE), especially in a zone corresponding to the apices of RPE cells, at the roots of the RPE microvilli, and at the base of RPE cells next to the Bruch's membrane. Partial co-localization of JN immunoreactivity with F-actin (labeled with phalloidin) was observed at the apices and bases of RPE cells. No JN was detected in other cell types of the retina. In cultured human RPE cell line ARPE-19, expression of extrinsic JN up-regulated formation of actin cytoskeleton stress fibers, caused redistribution of more F-actin fibers to the cell periphery, and promoted spreading/enlargement of transfected cells. These findings suggest possible roles of JN in RPE molecular transport, phagocytosis and formation of outer blood-retinal barrier, or possible involvement of JN expression perturbations in pathogenesis of such retinal disorders as proliferative vitreoretinopathy and age-related macular degeneration.

Combinatorial codon scrambling enables scalable gene synthesis and amplification of repetitive proteins.

Most genes are synthesized using seamless assembly methods that rely on the polymerase chain reaction (PCR). However, PCR of genes encoding repetitive proteins either fails or generates nonspecific products. Motivated by the need to efficiently generate new protein polymers through high-throughput gene synthesis, here we report a codon-scrambling algorithm that enables the PCR-based gene synthesis of repetitive proteins by exploiting the codon redundancy of amino acids and finding the least-repetitive synonymous gene sequence. We also show that the codon-scrambling problem is analogous to the well-known travelling salesman problem, and obtain an exact solution to it by using De Bruijn graphs and a modern mixed integer linear programme solver. As experimental proof of the utility of this approach, we use it to optimize the synthetic genes for 19 repetitive proteins, and show that the gene fragments are amenable to PCR-based gene assembly and recombinant expression.

DNA synthesis, assembly and applications in synthetic biology.

The past couple of years saw exciting new developments in microchip-based gene synthesis technologies. Such technologies hold the potential for significantly increasing the throughput and decreasing the cost of gene synthesis. Together with more efficient enzymatic error correction and genome assembly methods, these new technologies are pushing the field of synthetic biology to a higher level.

Parallel on-chip gene synthesis and application to optimization of protein expression.

Low-cost, high-throughput gene synthesis and precise control of protein expression are of critical importance to synthetic biology and biotechnology. Here we describe the development of an on-chip gene synthesis technology, which integrates on a single microchip the synthesis of DNA oligonucleotides using inkjet printing, isothermal oligonucleotide amplification and parallel gene assembly. Use of a mismatch-specific endonuclease for error correction results in an error rate of ~0.19 errors per kb. We applied this approach to synthesize pools of thousands of codon-usage variants of lacZα and 74 challenging Drosophila protein antigens, which were then screened for expression in Escherichia coli. In one round of synthesis and screening, we obtained DNA sequences that were expressed at a wide range of levels, from zero to almost 60% of the total cell protein mass. This technology may facilitate systematic investigation of the molecular mechanisms of protein translation and the design, construction and evolution of macromolecular machines, metabolic networks and synthetic cells.