Small-molecule control of antibody N-glycosylation in engineered mammalian cells.

N-linked glycosylation in monoclonal antibodies (mAbs) is crucial for structural and functional properties of mAb therapeutics, including stability, pharmacokinetics, safety and clinical efficacy. The biopharmaceutical industry currently lacks tools to precisely control N-glycosylation levels during mAb production. In this study, we engineered Chinese hamster ovary cells with synthetic genetic circuits to tune N-glycosylation of a stably expressed IgG. We knocked out two key glycosyltransferase genes, α-1,6-fucosyltransferase (FUT8) and ß-1,4-galactosyltransferase (ß4GALT1), genomically integrated circuits expressing synthetic glycosyltransferase genes under constitutive or inducible promoters and generated antibodies with concurrently desired fucosylation (0-97%) and galactosylation (0-87%) levels. Simultaneous and independent control of FUT8 and ß4GALT1 expression was achieved using orthogonal small molecule inducers. Effector function studies confirmed that glycosylation profile changes affected antibody binding to a cell surface receptor. Precise and rational modification of N-glycosylation will allow new recombinant protein therapeutics with tailored in vitro and in vivo effects for various biotechnological and biomedical applications.

Risk factors of infected sternal wounds versus sterile wound dehiscence.

BACKGROUND: Sterile sternal dehiscence (SSD) and sternal wound infections (SWIs) are two complications of median sternotomy with high rates of morbidity. Sternal wound complications also carry significant economic burden, almost tripling patients' hospital costs and are considered a nonreimbursable "never event" for Medicare. Historically, SDD and SWI have been recognized as discrete entities, but nonetheless continue to be categorized as a singular complication in literature. The purpose of this study was to determine specific patient demographic and perioperative predictors of SSD and SWI. MATERIALS AND METHODS: An institutional review board-approved, retrospective study of 8098 consecutive patients who underwent cardiac surgery at Columbia University Medical Center between January 2008 and December 2013 was conducted. Patients were categorized into three groups: no sternal wound complication, SSD, or SWI. Statistical analysis was performed using univariate and multivariate logistic regression analysis. RESULTS: Of 8098 patients, there were 73 patients (0.9%) with SSD and 40 (0.5%) with SWI who required plastic surgical consultation, debridement, and flap closure. In univariate analysis of SSD, positive predictors (i.e., "risk" factors) were age >42 years, prior surgery this admission, ≥2 arterial conduits, internal mammary artery (IMA) grafting with or without previous IMA grafting, body mass index (BMI) >30 (obese), CHF, diabetes requiring medication, respiratory failure, and unplanned cardiac reoperation; negative predictors (i.e., "protective" factors) were no arterial conduits and extubation within 24 h. In univariate analysis of SWI, positive predictors were IMA grafting with or without previous IMA grafting, postoperative hematocrit urgent/emergent surgical priority, BMI >30 (obese), cardiac ejection fraction <40%, and respiratory failure; negative predictors were no arterial conduits and elective surgical priority. In multivariate regression, BMI >30, diabetes requiring medication, and respiratory failure were determined to be significant positive predictors of SSD, and IMA grafting with or without prior IMA grafting and respiratory failure were significant positive predictors for SWI; no significant negative predictors were identified. CONCLUSIONS: This study found that SSD and SWI have many common significant predictors consistent with findings that increased BMI, use of IMA grafts, poor cardiac reserve, and postoperative respiratory failure confer increased risk of sternal wound complications. Additionally, this study also found that there were predictors unique to each entity supporting that SSD and SWI may be related but are not a singular entity. Recognition and prevention of significant positive and negative predictors of SSD and SWI may be valuable in preoperative counseling, operative planning, and postoperative management. Although sternal wound complications can be successfully managed by plastic surgical intervention, preventing the development of median sternotomy complications may curb costs incurred by both patients and health care systems.

The "Superior Ledge": a Modification of the Standard Superomedial Pedicle Reduction Mammoplasty to Accentuate Nipple-Areola Complex Projection.

BACKGROUND: The superomedial pedicle parenchymal excision pattern for reduction mammaplasty has the benefits of a reproducible breast shape and improved superomedial fullness, but is limited by a susceptibility to nipple retraction. The senior author of this paper has formalized the "superior ledge" modification of the superomedial pedicle technique (SL-SMP) to address these limitations. OBJECTIVE: To describe the technical details of the SL-SMP breast reduction technique and to analyze patient outcomes. METHODS: The technique involves only partial-thickness parenchymal excision superolateral to the pedicle, thereby leaving a "superior ledge" of parenchyma on top of which the nipple-areola complex (NAC) rests in a tension-free manner. Postoperative photographs were recorded; and patient demographics, intraoperative details, complications, and outcomes were recorded and analyzed. RESULTS: One hundred seven patients underwent SL-SMP reduction mammaplasty between 2007 and 2013. Complications included wound-healing complications (9.3 %), infection (2.8 %), seroma (1.9 %), and hematoma (1.9 %). Mean follow-up was 44.6 months (Range: 17-72), and during that period no incidence of clinically relevant NAC retraction was noted by either the patient or surgical team. CONCLUSIONS: Maintenance of a distinct superior ledge underlying the final position of the NAC is an important modification to stress, to prevent nipple retraction. Importantly, the height of the ledge can be personalized for each patient. We feel it is a valuable addition to the plastic surgeon's armamentarium to optimize outcomes for patients seeking relief from excessive breast tissue. LEVEL OF EVIDENCE V: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Alloplastic Augmentation of the Asian Face: A Review of 215 Patients.

BACKGROUND: Asian aesthetic surgery has become increasingly popular over the last decade, especially augmentation of characteristically flattened facial features. Alloplastic implants are an option for facial augmentation, however many avoid their use due to concerns for morbidity associated with their use. OBJECTIVES: To validate our hypothesis that when used properly, alloplastic implants have a low complication profile and provides excellent aesthetic results. METHODS: A retrospective review was performed of all Asian patients undergoing alloplastic facial augmentation between 2009 and 2013 by a single surgeon. Procedures included augmentation of the forehead, nasal dorsum, midface, and chin. Charts were reviewed for outcomes including infection, extrusion, malposition, and operative revision. RESULTS: Two hundred and fifteen patients had 243 implants placed. Of 141 nasal augmentations, there were 2 infections (1.4%), 1 extrusion (0.7%), 7 malpositions (4.9%), and 16 revisions (11.3%), 5 for malposition, 2 for contour irregularity, and 9 for aesthetic change. Augmentation genioplasty was performed in 40 patients with 1 malposition (2.5%) and 6 revisions (15%), 4 for under-correction and 2 for aesthetic change. Thirty-one midface and 31 forehead augmentations were performed without complications. One patient (3.2%) had forehead implant removal for aesthetic change. Overall infection and extrusion rates were 0.8% and 0.4%, respectively. CONCLUSIONS: By utilizing surgical techniques such as creation of a precise sub-periosteal pocket, placing the implant away from the incision site, and leaving well-vascularized soft tissue coverage under minimal tension, alloplastic implants can safely be used as a first-line option for Asian facial augmentation. LEVEL OF EVIDENCE: 4 Therapeutic.

Biochemical evidence for an alternate pathway in N-linked glycoprotein biosynthesis.

Asparagine-linked glycosylation is a complex protein modification conserved among all three domains of life. Herein we report the in vitro analysis of N-linked glycosylation from the methanogenic archaeon Methanococcus voltae. Using a suite of synthetic and semisynthetic substrates, we show that AglK initiates N-linked glycosylation in M. voltae through the formation of α-linked dolichyl monophosphate N-acetylglucosamine, which contrasts with the polyprenyl diphosphate intermediates that feature in both eukaryotes and bacteria. Notably, AglK has high sequence homology to dolichyl phosphate ß-glucosyltransferases, including Alg5 in eukaryotes, suggesting a common evolutionary origin. The combined action of the first two enzymes, AglK and AglC, afforded an α-linked dolichyl monophosphate glycan that serves as a competent substrate for the archaeal oligosaccharyl transferase AglB. These studies provide what is to our knowledge the first biochemical evidence revealing that, despite the apparent similarity of the overall pathways, there are actually two general strategies to achieve N-linked glycoproteins across the domains of life.

Oral rehabilitation outcomes after free fibula reconstruction of the mandible without condylar restoration.

PURPOSE: Resection of the posterior mandible for tumor or osteonecrosis may include the mandibular condyle, an integral part of the temporomandibular joint (TMJ). Condylar reconstruction, including use of prostheses, the native condylar head, or part of the fibula, all have associated drawbacks including skull base erosion and the potential for ankylosis and TMJ dysfunction as well as the increased difficulty associated with trying to recapitulate the TMJ with high fidelity. We report our experience leaving a single side of the reconstructed mandible unsecured to the glenoid fossa, allowing the mandible to "hang." We hypothesized that a good functional recovery may be achieved with this simple approach while avoiding the potential for ankylosis and TMJ dysfunction. METHODS: A retrospective chart review of all patients undergoing free fibula reconstruction of the mandible with condylar removal was performed. Outcomes were determined by maximum interincisal opening, occlusion, and diet after full recovery. RESULTS: Six patients were studied. Two had condylar reconstruction with a contoured fibular head secured to the glenoid fossa. One of them had progressive postoperative trismus and ankylosis. One patient was reconstructed with the native condyle rigidly fixed to the fibula flap, complicated by avascular necrosis requiring condylar resection, with good function afterward. Three patients were left to "hang." All 3 had either normal or improved function after surgery. Two had slight ipsilateral deviation on mouth opening. CONCLUSIONS: Function can reliably be reestablished after segmental mandibulectomy and condylectomy with a vascularized fibula flap whose distal end is not precisely contoured or actively seated in the glenoid fossa, as a valid alternative to condylar reconstruction.

The second step of the nitric oxide synthase reaction: evidence for ferric-peroxo as the active oxidant.

Nitric oxide synthase (NOS) is a P450 mono-oxygenase that catalyzes the oxidation of l-arginine to citrulline and NO through the stable intermediate N(G)-hydroxy-l-arginine (NHA). The oxidation of NHA by NOS is unique. There is little direct evidence in support of the nature of the heme bound oxidant [i.e., ferric-peroxo vs Fe(IV)O(por(*+))] responsible for this transformation. Previous work characterizing the H(2)O(2)-driven oxidation of NHA by NOS showed the formation of citrulline and the side product N(delta)-cyanoornithine (CN-orn). This led to the proposed involvement of a ferric-peroxo intermediate in the oxidation of NHA to citrulline. To test this hypothesis we used this model reaction to study the effects of pH, heme substitution, active site mutagenesis, and a fluorinated substrate analogue on the product distribution. Further, the oxidation of 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) by H(2)O(2) and iNOS(heme) was used to probe the protein-catalyzed breakdown of peroxide to the Fe(IV)O(por(*+)) intermediate. At pH 6.5, 7.5, and 8.5 the peroxide shunt reaction forms 26 +/- 2, 36 +/- 1, and 51 +/- 1% citrulline, respectively. The rate of peroxidase activity, however, was negatively correlated to pH, with a peroxide breakdown rate of 13.1 +/- 0.3, 8.3 +/- 0.2, and 4.2 +/- 0.1 M(-1) s(-1) at pH 6.5, 7.5, and 8.5, respectively. Mutation of active site valine 346 to an alanine shifted the product distribution to 5.2 +/- 0.5% citrulline while enhancing the peroxide cleavage rate to 14.3 +/- 0.7 M(-1) s(-1). Substitution of the heme cofactor with iron mesoporphyrin IX (Fe-MPIX) alters the product distribution from 36 +/- 1% citrulline to 22 +/- 3% citrulline. Metal substitution with Mn results in the formation of 64.7 +/- 0.8% citrulline. Conversely, the electrophilic 4,4-difluoro-N(G)-hydroxy-l-arginine substrate analogue shifted the product distribution to 68.6 +/- 0.6% 4,4-difluorocitrulline. The peroxidase data provide insight into the chemical features of NOS that control the processing of the ferric-peroxo species to the Fe(IV)O(por(*+)) intermediate and help interpret the product distributions observed for the peroxide shunt under various conditions. In all cases, the ability of the protein to break down peroxide is negatively correlated with the formation of citrulline by the peroxide shunt. These results support the high valent Fe(IV)O(por(*+)) intermediate as the species responsible for CN-orn formation and are consistent with the involvement of the ferric-peroxo intermediate in the oxidation of NHA to citrulline.

Chemoenzymatic Synthesis and Applications of Prokaryote-Specific UDP-Sugars.

This method describes the chemoenzymatic synthesis of several nucleotide sugars, which are essential substrates in the biosynthesis of prokaryotic N- and O-linked glycoproteins. Protein glycosylation is now known to be widespread in prokaryotes and proceeds via sequential action of several enzymes, utilizing both common and modified prokaryote-specific sugar nucleotides. The latter, which include UDP-hexoses such as UDP-diNAc-bacillosamine (UDP-diNAcBac), UDP-diNAcAlt, and UDP-2,3-diNAcManA, are also important components of other bacterial and archaeal glycoconjugates. The ready availability of these "high-value" intermediates will enable courses of study into inhibitor screening, glycoconjugate biosynthesis pathway discovery, and unnatural carbohydrate incorporation toward metabolic engineering.

N-Linked Glycans Are Assembled on Highly Reduced Dolichol Phosphate Carriers in the Hyperthermophilic Archaea Pyrococcus furiosus.

In all three domains of life, N-glycosylation begins with the assembly of glycans on phosphorylated polyisoprenoid carriers. Like eukaryotes, archaea also utilize phosphorylated dolichol for this role, yet whereas the assembled oligosaccharide is transferred to target proteins from dolichol pyrophosphate in eukaryotes, archaeal N-linked glycans characterized to date are derived from a dolichol monophosphate carrier, apart from a single example. In this study, glycan-charged dolichol phosphate from the hyperthermophile Pyrococcus furiosus was identified and structurally characterized. Normal and reverse phase liquid chromatography-electrospray ionization mass spectrometry revealed the existence of dolichol phosphate charged with the heptasaccharide recently described in in vitro studies of N-glycosylation on this species. As with other described archaeal dolichol phosphates, the α- and ω-terminal isoprene subunits of the P. furiosus lipid are saturated, in contrast to eukaryal phosphodolichols that present only a saturated α-position isoprene subunit. Interestingly, an additional 1-4 of the 12-14 isoprene subunits comprising P. furiosus dolichol phosphate are saturated, making this lipid not only the longest archaeal dolichol phosphate described to date but also the most highly saturated.

The prevention and genetics of pancreatic cancer: a programmatic approach.

Pancreatic cancer (PC) is typically a fatal disease due to its rapid growth and the lack of early diagnostic -techniques. Because approximately 10% of PCs are attributable to a hereditary susceptibility, identifying and studying patients with a family history of PC or known genetic predisposition to PC can improve the prevention, diagnosis, and treatment of PC. A skilled team of study investigators, physicians, genetic counselors, and data managers must work with patients and families to confidentially store and organize data from PC patients and high-risk patients. This data, collected in conjunction with patients' tissue and blood specimens, will contribute to the understanding of the biology, etiology, and epidemiology of PC, and can ultimately improve screening and management for patients with an underlying hereditary predisposition to PC.

Outcomes of surgical bleb revision for late-onset bleb leaks after trabeculectomy.

PURPOSE: To describe the outcome of surgical bleb revision for late-onset bleb leaks after trabeculectomy. PATIENTS AND METHODS: Appropriate cases were identified. Qualified and complete success required intraocular pressure of 21 mm Hg or less with and without glaucoma medication use, respectively. Bleb survival was determined using Kaplan-Meier survival analysis, and overall success rate was defined as qualified success at last follow-up. Preoperative and postoperative ocular parameters were compared using the signed-rank test. Age, sex, ethnicity, time between leak and revision, and surgeon type (attending vs. surgeons in training) were entered into a logistic regression analysis to assess the impact on surgical outcome. RESULTS: Seventy-eight eyes of 75 patients were included. The overall rate of successful bleb revision was 77%, and qualified and complete success at 24 months was 71% and 34%, respectively. Postoperative complications included early and late bleb leaks in 6% and 9% of the eyes, respectively; bleb-related infections in 4% of the eyes; and the need for additional glaucoma surgery in 10% of the eyes. There was no difference in preoperative and postoperative visual acuity (P=0.34) but there was an increase in intraocular pressure (P<0.0001) and the number of medications used (P<0.0001). The number of eyes that did not require glaucoma medication decreased (P=0.002). None of the variables examined had a significant impact on successful surgical outcome. CONCLUSION: Bleb revision showed a high success rate. About two-thirds of eyes required medication, 10% of eyes required additional glaucoma surgery, and there was a low risk for bleb-related infection following bleb revision.