Glycosylation plays important roles in cellular function and endows protein therapeutics with beneficial properties. However, constructing biosynthetic pathways to study and engineer precise glycan structures on proteins remains a bottleneck. Here, we report a modular, versatile cell-free platform for glycosylation pathway assembly by rapid in vitro mixing and expression (GlycoPRIME). In GlycoPRIME, glycosylation pathways are assembled by mixing-and-matching cell-free synthesized glycosyltransferases that can elaborate a glucose primer installed onto protein targets by an N-glycosyltransferase. We demonstrate GlycoPRIME by constructing 37 putative protein glycosylation pathways, creating 23 unique glycan motifs, 18 of which have not yet been synthesized on proteins. We use selected pathways to synthesize a protein vaccine candidate with an α-galactose adjuvant motif in a one-pot cell-free system and human antibody constant regions with minimal sialic acid motifs in glycoengineered Escherichia coli. We anticipate that these methods and pathways will facilitate glycoscience and make possible new glycoengineering applications.

High-throughput quantification of the posttranslational modification of many individual protein samples is challenging with current label-based methods. This paper demonstrates an efficient method that addresses this gap by combining Escherichia coli-based cell-free protein synthesis (CFPS) and self-assembled monolayers for matrix-assisted laser desorption/ionization mass spectrometry (SAMDI-MS) to analyze intact proteins. This high-throughput approach begins with polyhistidine-tagged protein substrates expressed from linear DNA templates by CFPS. Here, we synthesized an 87-member library of the E. coli Immunity Protein 7 (Im7) containing an acceptor sequence optimized for glycosylation by the Actinobacillus pleuropneumoniae N-glycosyltransferase (NGT) at every possible position along the protein backbone. These protein substrates were individually treated with NGT and then selectively immobilized to self-assembled monolayers presenting nickel-nitrilotriacetic acid (Ni-NTA) complexes before final analysis by SAMDI-MS to quantify the conversion of substrate to glycoprotein. This method offers new opportunities for rapid synthesis and quantitative evaluation of intact glycoproteins.

OBJECTIVES This study was conducted to determine if gender bias explains the worse outcomes in women than in men who undergo mitral valve surgery for degenerative mitral regurgitation. METHODS Patients who underwent mitral valve surgery for degenerative mitral regurgitation with or without concomitant ablation surgery for atrial fibrillation were identified from the Cardiovascular Research Database of the Clinical Trial Unit of the Bluhm Cardiovascular Institute at Northwestern Memorial Hospital and were defined according to the Society of Thoracic Surgery National Adult Cardiac Surgery Database. Of the 1004 patients (33% female, mean age 62.1 ± 12.4 years; 67% male, mean age 60.1 ± 12.4 years) who met this criteria, propensity score matching was utilized to compare sex-related differences. RESULTS Propensity score matching of 540 patients (270 females, mean age 61.0 ± 12.2; 270 males, mean age 60.9 ± 12.3) demonstrated that 98% of mitral valve surgery performed in both groups was mitral valve repair and 2% was mitral valve replacement. Preoperative CHA2DS2-VASc scores were higher in women and fewer women were discharged directly to their homes. Before surgery, women had smaller left heart chambers, lower cardiac outputs, higher diastolic filling pressures and higher volume responsiveness than men. However, preoperative left ventricular and right ventricular strain values, which are normally higher in women, were similar in the 2 groups, indicating worse global strain in women prior to surgery. CONCLUSIONS The worse outcomes reported in women compared to men undergoing surgery for degenerative mitral regurgitation are misleading and not based on gender bias except in terms of referral patterns. Men and women who present with the same type and degree of mitral valve disease and similar comorbidities receive the same types of surgical procedures and experience similar postoperative outcomes. Speckle-tracking echocardiography to assess global longitudinal strain of the left and right ventricles should be utilized to monitor for myocardial dysfunction related to chronic mitral regurgitation.

Glycosylation plays important roles in cellular function and endows protein therapeutics with beneficial properties. However, constructing biosynthetic pathways to study and engineer protein glycosylation remains a bottleneck. To address this limitation, we describe a modular, versatile cell-free platform for glycosylation pathway assembly by rapid in vitro mixing and expression (GlycoPRIME). In GlycoPRIME, crude cell lysates are enriched with glycosyltransferases by cellfree protein synthesis and then glycosylation pathways are assembled in a mix-and-match fashion to elaborate a single glucose priming handle installed by an N-linked glycosyltransferase. We demonstrate GlycoPRIME by constructing 37 putative protein glycosylation pathways, creating 23 unique glycan motifs. We then use selected pathways to design a one-pot cell-free system to synthesize a vaccine protein with an α-galactose motif and engineered Escherichia coli strains to produce human antibody constant regions with minimal sialic acid motifs. We anticipate that our work will facilitate glycoscience and make possible new glycoengineering applications.

Objective:To analyze publicly available content on a major social media outlet related to anterior cervical discectomy and fusion (ACDF) surgery based on perspective, location, timing, content, tone, and patient satisfaction.Summary of Background Data: Social media offers a powerful platform for sharing the patient experience with the public through an unfiltered perspective. Social media content may influence future perceptions around surgical care and postings around ACDF have not been previously reported.Methods: A query of content from a major social media outlet was performed for the study period January 1, 2018, to January 1, 2020, and returned 6500 publicly available posts. Content was identified by the hashtags "#acdf" or "#acdfsurgery." Content was ranked by number of "likes." Of the 1500 most popular posts, 1136 related to ACDF surgery and were included. Post content was characterized and classified.Results: Patients created 85% of ACDF-related content and spine surgeons created 11.8%. Most posts portrayed the patient experience in the postoperative period and depicted patients performing activities of daily living, participating in sports, or completing work activities (54.4%). The connotation of posts was deemed to be positive in 79.2% of cases. In regard to their care or state of health as it related to ACDF surgery, 59.8% of patients expressed satisfaction, whereas 14.1% expressed dissatisfaction. Female patients were > 4 times as likely to express dissatisfaction (odds ratio = 4.16, P = < 0.0001), with their clinical course compared with their male counterparts. Conclusions: Patients were the source of most posts on a major social media outlet that pertained to ACDF surgery with a majority reporting positive tone and satisfaction. These mechanisms of communication offer surgeons unique insights into patient experience and may provide an opportunity for surgeons to assess patient feedback, influence patient perceptions, and enhance delivery of cervical spine care.