Mismatched Postsurgical Opioid Prescription to Liver Transplant Patients: A Retrospective Cohort Study From a Single High-volume Transplant Center.

BACKGROUND: Improper opioid prescription after surgery is a well-documented iatrogenic contributor to the current opioid epidemic in North America. In fact, opioids are known to be overprescribed to liver transplant patients, and liver transplant patients with high doses or prolonged postsurgical opioid use have higher risks of graft failure and death. METHODS: This is a retrospective cohort study of 552 opioid-naive patients undergoing liver transplant at an academic center between 2012 and 2019. The primary outcome was the discrepancy between the prescribed discharge opioid daily dose and each patient's own inpatient opioid consumption 24 h before discharge. Variables were analyzed with Wilcoxon and chi-square tests and logistic regression. RESULTS: Opioids were overprescribed in 65.9% of patients, and 54.3% of patients who required no opioids the day before discharge were discharged with opioid prescriptions. In contrast, opioids were underprescribed in 13.4% of patients, among whom 27.0% consumed inpatient opioids but received no discharge opioid prescription. The median prescribed opioid daily dose was 333.3% and 56.3% of the median inpatient opioid daily dose in opioid overprescribed and underprescribed patients, respectively. Importantly, opioid underprescribed patients had higher rates of opioid refill 1 to 30 and 31 to 90 d after discharge, and the rate of opioid underprescription more than doubled from 2016 to 2019. CONCLUSIONS: Opioids are both over- and underprescribed to liver transplant patients, and opioid underprescribed patients had higher rates of opioid refill. Therefore, we proposed to prescribe discharge opioid prescriptions based on liver transplant patients' inpatient opioid consumption to provide patient-centered opioid prescriptions.

A systematic review of opioid use and multimodal strategies in solid organ transplant recipients and living donors.

The opioid epidemic has impacted analgesia in the postoperative period for solid organ transplant (SOT) donors and recipients. However, optimal pain management and opioid stewardship strategies have not been identified across this unique population. The purpose of this systematic review was to evaluate the impact of perioperative opioid use and to describe multimodal analgesic strategies to reduce opiate use in SOT recipients and living donors. A systematic review was conducted. Electronic searches were performed in Medline, Embase, Google Scholar, and Web of Science through December 31, 2021. Title and abstracts were screened. Relevant articles underwent full-text review. Literature was separated into effects of opioid exposure on post-transplant outcomes, recipient pain management strategies, and living donor pain management strategies. Search yielded 25,190 records, and 63 were ultimately included. The impact of opioid use on post-transplant outcomes was assessed in 19 publications. The risk of graft loss in pretransplant opioid users was assessed in six reports and was found to be higher in the majority (66%) of publications. Opioid minimization strategies were reported in 20 studies in transplant recipients. Twenty-four studies evaluated pain management strategies in living donors. Both populations used a combination of multimodal strategies to minimize opioid use throughout the hospitalization and on discharge. Opioids are associated with select negative outcomes in post-transplant recipients. To minimize their use while also maintaining appropriate analgesia, multimodal pain regimens should be considered in SOT recipients and donors.

Nirmatrelvir/ritonavir Use With Tacrolimus in Lung Transplant Recipients: A Single-center Case Series.

BACKGROUND: Limited data and guidelines exist for using nirmatrelvir/ritonavir in solid organ transplant recipients stabilized on tacrolimus for the treatment of mild-to-moderate coronavirus disease. Concern exists regarding the impact of utilizing a 5-d course of nirmatrelvir/ritonavir with calcineurin inhibitors because of significant drug-drug interactions between ritonavir, a potent cytochrome P450 3A inhibitor, and other cytochrome P450 3A substrates, such as tacrolimus. METHODS: We report the successful use of nirmatrelvir/ritonavir in 12 outpatient lung transplant recipients with confirmed severe acute respiratory syndrome coronavirus 2 infection stabilized on tacrolimus immunosuppression. All patients stopped tacrolimus and started nirmatrelvir/ritonavir 10 to 14 h after the last dose of tacrolimus. Tacrolimus was withheld and then reinitiated at a modified dose 48 h following the completion of nirmatrelvir/ritonavir therapy. Tacrolimus trough levels were checked during nirmatrelvir/ritonavir therapy and tacrolimus reinitiation. RESULTS: Ten (10/12) patients were able to resume their original tacrolimus dose within 4 d of completing nirmatrelvir/ritonavir therapy and maintain therapeutic levels of tacrolimus. No patients experienced tacrolimus toxicity or acute rejection during the 30-d postcompletion of nirmatrelvir/ritonavir therapy. CONCLUSIONS: In this cohort of lung transplant recipients on tacrolimus, we demonstrated that nirmatrelvir/ritonavir can be safely used with close monitoring of tacrolimus levels and appropriate dose adjustments of tacrolimus. Further confirmatory studies are needed to determine the appropriate use of therapeutic drug monitoring and tacrolimus dose following completion of nirmatrelvir/ritonavir in the solid organ transplant population.

Bacterial chemotaxis in static gradients quantified in a biopolymer membrane-integrated microfluidic platform.

Chemotaxis is a fundamental bacterial response mechanism to changes in chemical gradients of specific molecules known as chemoattractant or chemorepellent. The advancement of biological platforms for bacterial chemotaxis research is of significant interest for a wide range of biological and environmental studies. Many microfluidic devices have been developed for its study, but challenges still remain that can obscure analysis. For example, cell migration can be compromised by flow-induced shear stress, and bacterial motility can be impaired by nonspecific cell adhesion to microchannels. Also, devices can be complicated, expensive, and hard to assemble. We address these issues with a three-channel microfluidic platform integrated with natural biopolymer membranes that are assembled in situ. This provides several unique attributes. First, a static, steady and robust chemoattractant gradient was generated and maintained. Second, because the assembly incorporates assembly pillars, the assembled membrane arrays connecting nearby pillars can be created longer than the viewing window, enabling a wide 2D area for study. Third, the in situ assembled biopolymer membranes minimize pressure and/or chemiosmotic gradients that could induce flow and obscure chemotaxis study. Finally, nonspecific cell adhesion is avoided by priming the polydimethylsiloxane (PDMS) microchannel surfaces with Pluronic F-127. We demonstrated chemotactic migration of Escherichia coli as well as Pseudomonas aeruginosa under well-controlled easy-to-assemble glucose gradients. We characterized motility using the chemotaxis partition coefficient (CPC) and chemotaxis migration coefficient (CMC) and found our results consistent with other reports. Further, random walk trajectories of individual cells in simple bright field images were conveniently tracked and presented in rose plots. Velocities were calculated, again in agreement with previous literature. We believe the biopolymer membrane-integrated platform represents a facile and convenient system for robust quantitative assessment of cellular motility in response to various chemical cues.

Iron availability enhances the cellular energetics of aerobic Escherichia coli cultures while upregulating anaerobic respiratory chains.

Aerobic Escherichia coli growth at restricted iron concentrations (≤ 1.75 ± 0.04 µM) is characterized by lower biomass yield, higher acetate accumulation and higher activation of the siderophore iron-acquisition systems. Although iron homeostasis in E. coli has been studied intensively, previous studies focused only on understanding the regulation of the iron import systems and the iron-requiring enzymes. Here, the effect of iron availability on the energy metabolism of E. coli has been investigated. It was established that aerobic cultures growing under limiting iron conditions showed lower ATP yield per glucose, lower growth rate and lower TCA cycle activity and respiration, at the same time as increased glucose consumption, acetate and pyruvate accumulation, practically mimicking microaerobic growth. However, at excess iron, independent of oxygen availability, the cultures showed high cellular energetics (5.8 ATP/mol of glucose) by using pathways requiring iron-rich complex proteins found in the TCA cycle and respiratory chain. In conditions of iron excess, some iron-requiring terminal reductases of the respiratory chain, that were thought to function only under anaerobiosis, were used by the E. coli, when in aerobic conditions, to maintain high respiratory activity. This allowed it to produce more biomass and more reactive oxygen species that were controlled by the higher activity of the antioxidant defenses (SOD, peroxidase and catalase) and the iron-sulfur cluster repair systems.

rAAV Production and Titration at the Microscale for High-Throughput Screening.

In the literature, there are few high-throughput screens or even methods for high-throughput screens of recombinant adeno-associated virus (rAAV) production despite potential benefits to research and production. In this study, a generalizable high-throughput relative rAAV titration method is examined within the context of an siRNA screen as siRNA knockdown is a common means of pathway engineering in bioproduction. Crude samples generated from transfected HEK293T/17 cultures were subjected to quantitative PCR (qPCR) and used to transduce COS7 cells to assess relative differences in genomic and infectious rAAV titer, respectively, at the 384-well scale, evaluating both supernatant and lysed samples. To evaluate relevant differences in titer for conditions that could be used in an actual screen, cultures subjected to an siRNA reverse transfection and subsequent rAAV forward transfection were also tested. The delayed forward rAAV triple-plasmid transfection was not seen to affect the siRNA activity of tested controls, while siRNA transfection was shown to measurably impact rAAV titer. Effective differentiation between infectious titer levels was dependent upon the choice of sample dilution, but trends between qPCR and infectious titer assays were consistent across sample sets.

Affecting HEK293 Cell Growth and Production Performance by Modifying the Expression of Specific Genes.

The HEK293 cell line has earned its place as a producer of biotherapeutics. In addition to its ease of growth in serum-free suspension culture and its amenability to transfection, this cell line's most important attribute is its human origin, which makes it suitable to produce biologics intended for human use. At the present time, the growth and production properties of the HEK293 cell line are inferior to those of non-human cell lines, such as the Chinese hamster ovary (CHO) and the murine myeloma NSO cell lines. However, the modification of genes involved in cellular processes, such as cell proliferation, apoptosis, metabolism, glycosylation, secretion, and protein folding, in addition to bioprocess, media, and vector optimization, have greatly improved the performance of this cell line. This review provides a comprehensive summary of important achievements in HEK293 cell line engineering and on the global engineering approaches and functional genomic tools that have been employed to identify relevant genes for targeted engineering.

Mechanisms and management of drug-induced hyperkalemia in kidney transplant patients.

Hyperkalemia is a common and potentially life-threatening complication following kidney transplantation that can be caused by a composite of factors such as medications, delayed graft function, and possibly potassium intake. Managing hyperkalemia after kidney transplantation is associated with increased morbidity and healthcare costs, and can be a cause of multiple hospital admissions and barriers to patient discharge. Medications used routinely after kidney transplantation are considered the most frequent culprit for post-transplant hyperkalemia in recipients with a well-functioning graft. These include calcineurin inhibitors (CNIs), pneumocystis pneumonia (PCP) prophylactic agents, and antihypertensives (angiotensin-converting enzyme inhibitors, angiotensin receptor blockers, beta blockers). CNIs can cause hyperkalemic renal tubular acidosis. When hyperkalemia develops following transplantation, the potential offending medication may be discontinued, switched to another agent, or dose-reduced. Belatacept and mTOR inhibitors offer an alternative to calcineurin inhibitors in the event of hyperkalemia, however should be prescribed in the appropriate patient. While trimethoprim/sulfamethoxazole (TMP/SMX) remains the gold standard for prevention of PCP, alternative agents (e.g. dapsone, atovaquone) have been studied and can be recommend in place of TMP/SMX. Antihypertensives that act on the Renin-Angiotensin-Aldosterone System are generally avoided early after transplant but may be indicated later in the transplant course for patients with comorbidities. In cases of mild to moderate hyperkalemia, medical management can be used to normalize serum potassium levels and allow the transplant team additional time to evaluate the function of the graft. In the immediate post-operative setting following kidney transplantation, a rapidly rising potassium refractory to medical therapy can be an indication for dialysis. Patiromer and sodium zirconium cyclosilicate (ZS-9) may play an important role in the management of chronic hyperkalemia in kidney transplant patients, although additional long-term studies are necessary to confirm these effects.

Standardizing Discharge Opioid Prescriptions in Kidney Transplant Patients Decreases Opioid Usage.

BACKGROUND: Kidney transplant recipients are frequently prescribed excess opioids at discharge relative to their inpatient requirements. Recipients who fill prescriptions after transplant have an increased risk of death and graft loss. This study examined the impact of standardized prescriptions on discharge amount and number of outpatient refills. MATERIALS AND METHODS: A historical cohort (Group 1) was compared to a cohort without patient-controlled analgesia (Group 2) and a cohort in which providers prescribed no opioids to patients who required none on the day prior to discharge, and 10 pills to those who required opioids on the day prior (Group 3). Demographics, oral morphine equivalents (OMEs) prescribed on the day prior to and at discharge, and outpatient refills were collected. RESULTS: 270 recipients were included. There was a nonsignificant trend towards lower OMEs on the day prior to discharge in Groups 2 and 3. Nonopioid adjunct use increased (P < 0.001). Discharge OMEs significantly decreased (mean 87.2 in Group 1, 62.8 in Group 2, 26.6 in Group 3, P< 0.001). The number of patients discharged without opioids increased (23.8% of Group 1, 37.5% of Group 2, 60.6% of Group 3, P < 0.001). Group 3, Asian descent, and lower OMEs on the day prior were factors significantly associated with decreased discharge OMEs on multivariable linear regression. Twelve percent of Group 2 and 2% of Group 3 patients received an outpatient refill (P = 0.02). CONCLUSIONS: A protocol targeting discharge opioids significantly reduced the amount of opioids prescribed in kidney transplant recipients; most patients subsequently received no opioids at discharge.

Novel approaches to management of hyperkalaemia in kidney transplantation.

PURPOSE OF REVIEW: Medications used frequently after kidney transplantation, including calcineurin inhibitors, angiotensin-converting enzyme inhibitors, angiotensin receptor blockers, beta blockers and antimicrobials, are considered the leading culprit for posttransplant hyperkalaemia in recipients with a well functioning allograft. Other risk factors include comorbidities such as diabetes, hypertension and heart failure; and consumption of a potassium-enriched diet. We review the mechanisms for hyperkalaemia following kidney transplantation that are addressed using nonpharmacological and pharmacological interventions. We also discuss emerging therapeutic approaches for the management of recurrent hyperkalaemia in solid organ transplantation, including newer potassium binding therapies. RECENT FINDINGS: Patiromer and sodium zirconium cyclosilicate are emerging potassium binders approved for the treatment of hyperkalaemia. Patiromer is a polymer that exchanges potassium for calcium ions. In contrast, sodium zirconium cyclosilicate is a nonpolymer compound that exchanges potassium for sodium and hydrogen ions. Both agents are efficacious in the treatment of chronic or recurrent hyperkalaemia and may result in fewer gastrointestinal side effects than older potassium binders such as sodium polystyrene sulfonate and calcium polystyrene sulfonate. Large-scale clinical studies have not been performed in kidney transplant patients. Patiromer may increase serum concentrations of tacrolimus, but not cyclosporine. Sodium zirconium cyclosilicate does not appear to compromise tacrolimus pharmacokinetics, although it may have a higher sodium burden. SUMMARY: Patiromer and sodium zirconium cyclosilicate may be well tolerated options to treat asymptomatic hyperkalaemia and have the potential to ease potassium dietary restrictions in kidney transplant patients by maintaining a plant-dominant, heart-healthy diet. Their efficacy, better tolerability and comparable cost with respect to previously available potassium binders make them an attractive therapeutic option in chronic hyperkalaemia following kidney transplantation.

Electrochemical measurement of serotonin by Au-CNT electrodes fabricated on microporous cell culture membranes.

Gut-brain axis (GBA) communication relies on serotonin (5-HT) signaling between the gut epithelium and the peripheral nervous system, where 5-HT release patterns from the basolateral (i.e., bottom) side of the epithelium activate nerve afferents. There have been few quantitative studies of this gut-neuron signaling due to a lack of real-time measurement tools that can access the basolateral gut epithelium. In vitro platforms allow quantitative studies of cultured gut tissue, but they mainly employ offline and endpoint assays that cannot resolve dynamic molecular-release patterns. Here, we present the modification of a microporous cell culture membrane with carbon nanotube-coated gold (Au-CNT) electrodes capable of continuous, label-free, and direct detection of 5-HT at physiological concentrations. Electrochemical characterization of single-walled carbon nanotube (SWCNT)-coated Au electrodes shows increased electroactive surface area, 5-HT specificity, sensitivity, and saturation time, which are correlated with the CNT film drop-cast volume. Two microliters of CNT films, with a 10-min saturation time, 0.6 µA/µM 5-HT sensitivity, and reliable detection within a linear range of 500 nM-10 µM 5-HT, can be targeted for high-concentration, high-time-resolution 5-HT monitoring. CNT films (12.5 µL) with a 2-h saturation time, 4.5 µA/µM 5-HT sensitivity, and quantitative detection in the linear range of 100 nM-1 µM can target low concentrations with low time resolution. These electrodes achieved continuous detection of dynamic diffusion across the porous membrane, mimicking basolateral 5-HT release from cells, and detection of cell-released 5-HT from separately cultured RIN14B cell supernatant. Electrode-integrated cell culture systems such as this can improve in vitro molecular detection mechanisms and aid in quantitative GBA signaling studies.

3D-Printed electrochemical sensor-integrated transwell systems.

This work presents a 3D-printed, modular, electrochemical sensor-integrated transwell system for monitoring cellular and molecular events in situ without sample extraction or microfluidics-assisted downstream omics. Simple additive manufacturing techniques such as 3D printing, shadow masking, and molding are used to fabricate this modular system, which is autoclavable, biocompatible, and designed to operate following standard operating protocols (SOPs) of cellular biology. Integral to the platform is a flexible porous membrane, which is used as a cell culture substrate similarly to a commercial transwell insert. Multimodal electrochemical sensors fabricated on the membrane allow direct access to cells and their products. A pair of gold electrodes on the top side of the membrane measures impedance over the course of cell attachment and growth, characterized by an exponential decrease (~160% at 10 Hz) due to an increase in the double layer capacitance from secreted extracellular matrix (ECM) proteins. Cyclic voltammetry (CV) sensor electrodes, fabricated on the bottom side of the membrane, enable sensing of molecular release at the site of cell culture without the need for downstream fluidics. Real-time detection of ferrocene dimethanol injection across the membrane showed a three order-of-magnitude higher signal at the membrane than in the bulk media after reaching equilibrium. This modular sensor-integrated transwell system allows unprecedented direct, real-time, and noninvasive access to physical and biochemical information, which cannot be obtained in a conventional transwell system.

Engineering Escherichia coli for enhanced sensitivity to the autoinducer-2 quorum sensing signal.

The autoinducer-2 (AI-2) quorum sensing system is involved in a range of population-based bacterial behaviors and has been engineered for cell-cell communication in synthetic biology systems. Investigation into the cellular mechanisms of AI-2 processing has determined that overexpression of uptake genes increases AI-2 uptake rate, and genomic deletions of degradation genes lowers the AI-2 level required for activation of reporter genes. Here, we combine these two strategies to engineer an Escherichia coli strain with enhanced ability to detect and respond to AI-2. In an E. coli strain that does not produce AI-2, we monitored AI-2 uptake and reporter protein expression in a strain that overproduced the AI-2 uptake or phosphorylation units LsrACDB or LsrK, a strain with the deletion of AI-2 degradation units LsrF and LsrG, and an "enhanced" strain with both overproduction of AI-2 uptake and deletion of AI-2 degradation elements. By adding up to 40 µM AI-2 to growing cell cultures, we determine that this "enhanced" AI-2 sensitive strain both uptakes AI-2 more rapidly and responds with increased reporter protein expression than the others. This work expands the toolbox for manipulating AI-2 quorum sensing processes both in native environments and for synthetic biology applications.

A platform of genetically engineered bacteria as vehicles for localized delivery of therapeutics: Toward applications for Crohn's disease.

For therapies targeting diseases of the gastrointestinal tract, we and others envision probiotic bacteria that synthesize and excrete biotherapeutics at disease sites. Toward this goal, we have engineered commensal E. coli that selectively synthesize and secrete a model biotherapeutic in the presence of nitric oxide (NO), an intestinal biomarker for Crohn's disease (CD). This is accomplished by co-expressing the pore forming protein TolAIII with the biologic, granulocyte macrophage-colony stimulating factor (GM-CSF). We have additionally engineered these bacteria to accumulate at sites of elevated NO by engineering their motility circuits and controlling pseudotaxis. Importantly, because we have focused on in vitro test beds, motility and biotherapeutics production are spatiotemporally characterized. Together, the targeted recognition, synthesis, and biomolecule delivery comprises a "smart" probiotics platform that may have utility in the treatment of CD. Further, this platform could be modified to accommodate other pursuits by swapping the promoter and therapeutic gene to reflect other disease biomarkers and treatments, respectively.

An immune magnetic nano-assembly for specifically amplifying intercellular quorum sensing signals.

Quorum sensing (QS) enables intercellular communication after bacterial cells sense the autoinducers have reached or exceeded a critical concentration. Selectively amplifying specific bacterial "quorum" activity at a lower cell density is still a challenge. Here, we propose a novel platform of immune magnetic nano-assembly to amplify specific bacterial QS signaling via improving the bioavailability of autoinducers-2 (AI-2, furanosyl borate) from sender (wide-type, WT cells) to receiver (reporter cells). Antibody coated magnetic nanoparticle (MNPAB) was fabricated with an average diameter of 12 nm and a specific surface area of 96.5 m2/g. The distribution efficiency of the antibody on the surface was 25.8 µg/m2 of magnetic nanoparticles. It was found that more than 3 × 108 of K12 serotype Escherichia coli (E. coli) reporter or WT cells were collected using 1 mg fabricated MNPAB at a saturated condition. The MNPAB not only captured E. coli WT cells but also brought them into proximity of E. coli (CT104, pCT6+pET-DsRed) reporter cells via magnetic attraction. The amplified QS signaling of the reporter cells by this immune magnetic nano-assembly was approximately 3 times higher than the nature QS signaling in cell suspension at optical density (OD) 0.08. This study foresees potential applications in amplifying specific biological QS signals based on a preprogrammed design.

Development of Cell-Based Sentinels for Nitric Oxide: Ensuring Marker Expression and Unimodality.

We generated "sentinel" bacteria that respond to the biomarker nitric oxide (NO) and produce a homogeneous and strong fluorescent response. Our dual-plasmid system consists of a signal "relay" vector that employs an NO-responsive promoter that amplifies the native signal (via expression of T7 Polymerase (T7Pol)) to a second vector responsible for GFP expression. Importantly, to achieve an optimal "sentinel" response, we developed strategies that balance the transcriptional load within cells by altering (i) translation and (ii) activity of the T7Pol. Our optimized genetic circuitry was then used to transform commensal E. coli Nissle, as a proof-of-concept toward an ingestible cell-based sensor for Crohn's disease (CD) that, in turn, is marked by elevated levels of intestinal NO. Thus, the "biosensors" demonstrated here may serve as a simple diagnostic tool, contrasting the standard of care including colonoscopies or biopsies.

Focusing quorum sensing signalling by nano-magnetic assembly.

Quorum sensing (QS) exists widely among bacteria, enabling a transition to multicellular behaviour after bacterial populations reach a particular density. The coordination of multicellularity enables biotechnological application, dissolution of biofilms, coordination of virulence, and so forth. Here, a method to elicit and subsequently disperse multicellular behaviour among QS-negative cells is developed using magnetic nanoparticle assembly. We fabricated magnetic nanoparticles (MNPs, ∼5 nm) that electrostatically collect wild-type (WT) Escherichia coli BL21 cells and brings them into proximity of bioengineered E. coli [CT104 (W3110 lsrFG- luxS- pCT6 + pET-DsRed)] reporter cells that exhibit a QS response after receiving autoinducer-2 (AI-2). By shortening the distance between WT and reporter cells (e.g., increasing local available AI-2 concentrations), the QS response signalling was amplified four-fold compared to that in native conditions without assembly. This study suggests potential applications in facilitating intercellular communication and modulating multicellular behaviours based on user-specified designs.

Biofabricating Functional Soft Matter Using Protein Engineering to Enable Enzymatic Assembly.

Biology often provides the inspiration for functional soft matter, but biology can do more: it can provide the raw materials and mechanisms for hierarchical assembly. Biology uses polymers to perform various functions, and biologically derived polymers can serve as sustainable, self-assembling, and high-performance materials platforms for life-science applications. Biology employs enzymes for site-specific reactions that are used to both disassemble and assemble biopolymers both to and from component parts. By exploiting protein engineering methodologies, proteins can be modified to make them more susceptible to biology's native enzymatic activities. They can be engineered with fusion tags that provide (short sequences of amino acids at the C- and/or N- termini) that provide the accessible residues for the assembling enzymes to recognize and react with. This "biobased" fabrication not only allows biology's nanoscale components (i.e., proteins) to be engineered, but also provides the means to organize these components into the hierarchical structures that are prevalent in life.

Site-specific immobilization of endoglycosidases for streamlined chemoenzymatic glycan remodeling of antibodies.

Chemoenzymatic glycan remodeling of antibodies using an endoglycosidase and its mutant is emerging as an attractive approach for producing homogeneous antibody glycoforms. We report in this paper a site-specific covalent immobilization of the endoglycosidases (Endo-S2 and its glycosynthase mutant D184M) using a recombinant microbial transglutaminase (MTG) and evaluation of the immobilized enzymes in deglycosylation and glycosylation of a therapeutic antibody. The site-specific covalent immobilization was achieved by introduction of a Q-tag at the C-terminus of the recombinant enzymes followed by conjugation of the enzymes to a primary amine-containing solid support through MTG-catalyzed transglutamination. Using rituximab as a model system, we found that the Endo-S2 wild-type and D184M glycosynthase mutant immobilized by this approach were efficient in the two step antibody glycan remodeling to generate homogeneous antibody glycoforms. Notably using the covalently immobilized enzymes can efficiently avoid the need of intermediate purification and eliminate the residual contamination of wild type enzyme for product hydrolysis, thus streamlining the chemoenzymatic Fc glycan remodeling of antibodies.

A simple and reusable bilayer membrane-based microfluidic device for the study of gradient-mediated bacterial behaviors.

We have developed a user-friendly microfluidic device for the study of gradient-mediated bacterial behaviors, including chemotaxis. This device rapidly establishes linear concentration gradients by exploiting solute diffusion through porous membranes in the absence of convective flows. As such, the gradients are created rapidly and can be sustained for long time periods (e.g., hours), sufficient to evaluate cell phenotype. The device exploits a unique simple bilayer configuration that enables rapid setup and quick reproducible introduction of cells. Its reusability represents an additional advantage in that it need not be limited to settings with microfluidics expertise. We have successfully demonstrated the applicability of this tool in studying the chemotactic response of Escherichia coli to glucose. When coupled with our recent Python program, quantified metrics such as speed, ratio of tumble to run, and effective diffusivity can be obtained from slow frame rate videos. Moreover, we introduce a chemotaxis partition coefficient that conveniently scores swimming behavior on the single-cell level.