Measuring the burden of hundreds of BioBricks defines an evolutionary limit on constructability in synthetic biology.

Engineered DNA will slow the growth of a host cell if it redirects limiting resources or otherwise interferes with homeostasis. Populations of engineered cells can rapidly become dominated by "escape mutants" that evolve to alleviate this burden by inactivating the intended function. Synthetic biologists working with bacteria rely on genetic parts and devices encoded on plasmids, but the burden of different engineered DNA sequences is rarely characterized. We measured how 301 BioBricks on high-copy plasmids affected the growth rate of Escherichia coli. Of these, 59 (19.6%) negatively impacted growth. The burden imposed by engineered DNA is commonly associated with diverting ribosomes or other gene expression factors away from producing endogenous genes that are essential for cellular replication. In line with this expectation, BioBricks exhibiting burden were more likely to contain highly active constitutive promoters and strong ribosome binding sites. By monitoring how much each BioBrick reduced expression of a chromosomal GFP reporter, we found that the burden of most, but not all, BioBricks could be wholly explained by diversion of gene expression resources. Overall, no BioBricks reduced the growth rate of E. coli by >45%, which agreed with a population genetic model that predicts such plasmids should be "unclonable" because escape mutants will take over during growth of a bacterial colony or small laboratory culture from a transformed cell. We made this model available as an interactive web tool for synthetic biology education and added our burden measurements to the iGEM Registry descriptions of each BioBrick.

Vasopressin for Post-kidney Transplant Hypotension.

Introduction: Hypotension after deceased donor kidney transplant (DDKT) is a risk factor for delayed graft function (DGF) and poor graft survival (GS). We hypothesize that vasopressin use in hypotensive DDKT recipients (DDKTRs) to increase blood pressure (BP) reduces DGF rates and is safe without increasing mortality. Methods: Group with vasopressin "study group" (n = 45) was defined as DDKTRs between 2012 and 2017 who required vasopressin for hypotension systolic BP (SBP) <120 mm Hg or diastolic BP (DBP) <60 mm Hg. DDKTRs with no-vasopressin "comparison group" (n = 90) were propensity score-matched DDKTRs between 2012 and 2017 without vasopressin use. Primary outcomes were GS, creatinine and allograft biopsy rate at 1 year, DGF rate, and death during transplant hospitalization. Results: Vasopressin group had lower mean maximum and minimum SBP and DBP in the operating room (OR). Median vasopressin start time post-DDKT was 2 hours (interquartile range [IQR] 1-6), and duration of use was 42 hours (IQR 24-63). DGF, creatinine at 1 year, and allograft biopsy rates were comparable. No deaths occurred during transplant hospitalization. Multivariable analysis did not find an effect of vasopressin use on GS. Conclusion: Treatment of hypotensive DDKTRs with vasopressin is safe and facilitated similar graft function and survival with that of nonhypotensive patients. In the absence of a randomized control trial, our study supports the safety of vasopressin therapy to prevent the adverse effects of hypotension.

Nondirected Living Kidney Donation and Recipient Outcomes in the United States: A 20-Year Review.

Introduction: Nondirected donation (NDD) of the kidneys is a growing practice where donors who do not have any genetic or emotional relationship are selected to donate to a wide variety of recipients with a range of selection criteria and decisions which are left up to individual transplant centers. Methods: We review all adult living kidney donor-recipient (DR) pairs and outcomes from NDDs who were recorded in United Network for Organ Sharing (UNOS) database as code 10 (anonymous) from October 1997 to September 2017 for demographics and outcomes. Results: A total of 2174 DR pairs were identified. The number of NDDs increased from 18 in 2000 to 256 in 2016. Survival analysis showed higher death-censored-graft survival (DC-GS) when recipient was 20 years or more older than donor followed by recipient-donor within 20 years of age and lowest when donor was 20 years or more older than recipient (P = 0.0114). Conclusion: Overall, the number of NDDs has increased significantly in the 20-year review period. Transplants from NDDs have excellent long-term outcomes. Better matching of controllable DR factors, such as age and body mass index (BMI), could further improve GS. Further research is needed to incorporate these DR factors into paired kidney donation programs potentially enhancing the utility and beneficence of this invaluable donation.

A National Survey of Practice Patterns for Accepting Living Kidney Donors With Prior COVID-19.

INTRODUCTION: A critical question facing transplant programs is whether, when, and how to safely accept living kidney donors (LKDs) who have recovered from COVID-19 infection. The purpose of the study is to understand current practices related to accepting these LKDs. METHODS: We surveyed US transplant programs from 3 September through 3 November 2020. Center level and participant level responses were analyzed. RESULTS: A total of 174 respondents from 115 unique centers responded, representing 59% of US LKD programs and 72.4% of 2019 and 72.5% of 2020 LKD volume (Organ Procurement and Transplantation Network-OPTN 2021). In all, 48.6% of responding centers had received inquiries from such LKDs, whereas 44.3% were currently evaluating. A total of 98 donors were in the evaluation phase, whereas 27.8% centers had approved 42 such donors to proceed with donation. A total of 50.8% of participants preferred to wait >3 months, and 91% would wait at least 1 month from onset of infection to LD surgery. The most common reason to exclude LDs was evidence of COVID-19-related AKI (59.8%) even if resolved, followed by COVID-19-related pneumonia (28.7%) and hospitalization (21.3%). The most common concern in accepting such donors was kidney health postdonation (59.2%), followed by risk of transmission to the recipient (55.7%), donor perioperative pulmonary risk (41.4%), and donor pulmonary risk in the future (29.9%). CONCLUSION: Practice patterns for acceptance of COVID-19-recovered LKDs showed considerable variability. Ongoing research and consensus building are needed to guide optimal practices to ensure safety of accepting such donors. Long-term close follow-up of such donors is warranted.

Bioassay for Determining the Concentrations of Caffeine and Individual Methylxanthines in Complex Samples.

Caffeine and other methylxanthines are stimulant molecules found in formulated beverages, including sodas and energy drinks, and in brewed beverages, such as coffee and teas. Previously, we developed a bioassay for caffeine that involves monitoring the growth of a ΔguaB mutant of Escherichia coli defective in de novo guanine biosynthesis. When supplemented with a plasmid expressing the genes for an N-demethylation pathway from Pseudomonas putida CBB5, these bacteria demethylate caffeine (1,3,7-trimethylxanthine) and other methylxanthines into xanthine, which is then converted into guanine to support cell growth. A major limitation of this bioassay was that it could only measure the total concentration of all methylxanthines in a mixture. Therefore, it could not be used to measure the caffeine content of beverages like teas, which contain substantial quantities of multiple methylxanthines. To overcome this limitation, we created seven new plasmids containing all subsets of the three demethylase genes (ndmA, ndmB, and ndmC). We show that strains of ΔguaBE. coli containing each plasmid are able to demethylate specific subsets of methylxanthines and that they can be used to determine the concentrations of individual methylxanthines in complex mixtures containing multiple methylxanthines, including coffee doped with an additional methylxanthine. While validating this assay, we also discovered an unexpected demethylation event at the 1-methyl position when NdmB and NdmC were expressed in the absence of NdmA. The improved cell-based bioassay is inexpensive, is easy to use, and gives results comparable to standard high-performance liquid chromatography methods for measuring methylxanthine concentrations.IMPORTANCE Caffeine (1,3,7-trimethylxanthine) is the dominant neurostimulant found in coffee, teas, sodas, and energy drinks. Measuring the amount of caffeine and other methylxanthines in these beverages is important for quality assurance and safety in food science. Methylxanthines are also used in medicine and as performance-enhancing drugs, two contexts in which accurately determining their concentrations in bodily fluids is important. Liquid chromatography is the standard method for measuring methylxanthine concentrations in a sample, but it requires specialized equipment and expertise. We improved a previous bioassay that links E. coli growth to methylxanthine demethylation so that it can now be used to determine the amounts of individual methylxanthines in complex mixtures or beverages, such as coffee.

Synthetic Genome Defenses against Selfish DNA Elements Stabilize Engineered Bacteria against Evolutionary Failure.

Mobile genetic elements drive evolution by disrupting genes and rearranging genomes. Eukaryotes have evolved epigenetic mechanisms, including DNA methylation and RNA interference, that silence mobile elements and thereby preserve the integrity of their genomes. We created an artificial reprogrammable epigenetic system based on CRISPR interference to give engineered bacteria a similar line of defense against transposons and other selfish elements in their genomes. We demonstrate that this CRISPR interference against mobile elements (CRISPRi-ME) approach can be used to simultaneously repress two different transposon families in Escherichia coli, thereby increasing the evolutionary stability of costly protein expression. We further show that silencing a transposon in Acinetobacter baylyi ADP1 reduces mutation rates by a factor of 5, nearly as much as deleting all copies of this element from its genome. By deploying CRISPRi-ME on a broad-host-range vector, we have created a generalizable platform for stabilizing the genomes of engineered bacterial cells for applications in metabolic engineering and synthetic biology.

Early Fatal Cutaneous Lower Extremity Angiosarcoma Associated with Deep Venous Thrombosis in a Renal Transplant Recipient.

Angiosarcomas are extremely rare malignant tumors of vascular origin. We describe a 63-year-old recipient after a kidney transplant who had an angiosarcoma in the lower extremity that presented after new-onset deep venous thrombosis and was not associated with any fistula. There was rapid progression to metastasis and death. We reviewed the literature of this rare malignant tumor in kidney transplant patients.

Rapid and Inexpensive Evaluation of Nonstandard Amino Acid Incorporation in Escherichia coli.

By introducing engineered tRNA and aminoacyl-tRNA synthetase pairs into an organism, its genetic code can be expanded to incorporate nonstandard amino acids (nsAAs). The performance of these orthogonal translation systems (OTSs) varies greatly, however, with respect to the efficiency and accuracy of decoding a reassigned codon as the nsAA. To enable rapid and systematic comparisons of these critical parameters, we developed a toolkit for characterizing any Escherichia coli OTS that reassigns the amber stop codon (TAG). It assesses OTS performance by comparing how the fluorescence of strains carrying plasmids encoding a fused RFP-GFP reading frame, either with or without an intervening TAG codon, depends on the presence of the nsAA. We used this kit to (1) examine nsAA incorporation by seven different OTSs, (2) optimize nsAA concentration in growth media, (3) define the polyspecificity of an OTS, and (4) characterize evolved variants of amberless E. coli with improved growth rates.

Re-transplants compared to primary kidney transplants recipients: a mate kidney paired analysis of the OPTN/UNOS database.

Outcomes of kidney re-transplant recipients (RTR) were compared to primary recipients (FTR) from paired donor kidneys. Organ Procurement and Transplantation Network (OPTN) database was used to identify deceased donors (n = 6266) who donated one kidney to an RTR and the mate kidney to an FTR between January 2000 to December 2010. As compared to FTR, RTR were younger (45 vs. 52 yr, p < 0.001) and had higher proportion of plasma reactive antibody >80 (25% vs 7%, p < 0.001). There were higher 0 mismatches in RTR (19% vs. 16%, p < 0.001). There were more pre-emptive transplants in RTR (24% vs. 21%, p = 0.002). Delayed graft function (28% vs. 25%, p = 0.007) was higher in RTR. Patient survival was similar in FTR and RTR groups at one, three, and five yr (95.7%, 90.2%, and 82.5% vs. 95.2%, 89.8% and 82.7%). Allograft survival rates were higher in FTR group compared to RTR group at one, three, and five yr (91.1%, 82.4%, and 70.9% vs. 87.8%, 77.4%, and 66.1% p < 0.001). Death-censored allograft survival rates were higher in FTR group at one, three, and five yr (91.3%, 82.7% and 71.4% vs. 88%, 77.7% and 66.5% p < 0.001). In today's era of modern immunosuppression, graft survival in RTR has improved but remains inferior to FTR when controlling for donor factors.

Automated physics-based design of synthetic riboswitches from diverse RNA aptamers.

Riboswitches are shape-changing regulatory RNAs that bind chemicals and regulate gene expression, directly coupling sensing to cellular actuation. However, it remains unclear how their sequence controls the physics of riboswitch switching and activation, particularly when changing the ligand-binding aptamer domain. We report the development of a statistical thermodynamic model that predicts the sequence-structure-function relationship for translation-regulating riboswitches that activate gene expression, characterized inside cells and within cell-free transcription-translation assays. Using the model, we carried out automated computational design of 62 synthetic riboswitches that used six different RNA aptamers to sense diverse chemicals (theophylline, tetramethylrosamine, fluoride, dopamine, thyroxine, 2,4-dinitrotoluene) and activated gene expression by up to 383-fold. The model explains how aptamer structure, ligand affinity, switching free energy and macromolecular crowding collectively control riboswitch activation. Our model-based approach for engineering riboswitches quantitatively confirms several physical mechanisms governing ligand-induced RNA shape-change and enables the development of cell-free and bacterial sensors for diverse applications.

Long-term outcomes of transplant recipients referred for angiography for suspected transplant renal artery stenosis.

Our aim was to study the long-term outcomes of all transplant recipients who underwent angiography for suspected TRAS at our institution. The patients were divided into TRAS+ve and TRAS-ve groups based upon angiographically confirmed results. TRAS was confirmed in 58.1% of 74 patients with median time of 8.9 months. Primary angioplasty alone was performed in 56% of patients with TRAS, while the remaining had PTA with stent (PTAS). There was reduction in systolic and diastolic BP (165 ± 19-136 ± 15 mmHg and 82 ± 14 mmHg to 68 ± 12 mmHg; p < 0.05) and number of antihypertensive drugs (3.5 ± 0.9-2.7 ± 1.0; p < 0.05). Overall, graft survival and patient survival from time of transplant were similar in both groups. Graft function was similar for the patients with treated TRAS+ve as compared to TRAS-ve over time. Graft survival and patient survival when compared to an age- and year of transplant-matched cohort control group were also similar. In conclusion, angiography for suspected TRAS is more likely to yield a confirmatory result early in the transplant course as compared to late. Treatment of TRAS in these patients had sustained long-term graft function. Alternative etiologies of HTN and graft dysfunction should be sought for recipients further out from transplant.

Predicting the Genetic Stability of Engineered DNA Sequences with the EFM Calculator.

Unwanted evolution can rapidly degrade the performance of genetically engineered circuits and metabolic pathways installed in living organisms. We created the Evolutionary Failure Mode (EFM) Calculator to computationally detect common sources of genetic instability in an input DNA sequence. It predicts two types of mutational hotspots: deletions mediated by homologous recombination and indels caused by replication slippage on simple sequence repeats. We tested the performance of our algorithm on genetic circuits that were previously redesigned for greater evolutionary reliability and analyzed the stability of sequences in the iGEM Registry of Standard Biological Parts. More than half of the parts in the Registry are predicted to experience >100-fold elevated mutation rates due to the inclusion of unstable sequence configurations. We anticipate that the EFM Calculator will be a useful negative design tool for avoiding volatile DNA encodings, thereby increasing the evolutionary lifetimes of synthetic biology devices.

Pre-transplant angiotensin receptor II type 1 antibodies and risk of post-transplant focal segmental glomerulosclerosis recurrence.

Post-kidney transplant recurrence of focal segmental glomerulosclerosis (FSGS) is a major problem. AT1R is expressed on podocyte; its expression is elevated in the proteinuric state. Using an ELISA, we tested pre-transplant sera of 28 patients with history of idiopathic FSGS for anti-AT1R levels and serum soluble urokinase-type plasminogen activator receptor (suPAR) as a biomarker for risk of recurrence of FSGS. Sera from 11 patients with polycystic kidney disease (PKD) were used as controls. Twelve patients had biopsy proven post-transplant FSGS recurrence at 1.5 months. No difference was found in the pre-transplant suPAR levels of FSGS patients (5993 ± 2292 pg/mL) vs. PKD (7334 ± 4538 pg/mL) (p = 0.23). Serum suPAR levels in patients with FSGS recurrence (5786 ± 1899 pg/mL) vs. no FSGS recurrence (6149 ± 2598 pg/mL) (p = 0.69) were not different. Anti-AT1R levels in patients with FSGS were 12.66 ± 11.85 U/mL vs. 8.69 ± 6.52 U/mL in PKD (p = 0.32); however, a difference was found in patients with and without FSGS recurrence 20.41 ± 14.36 U/mL 6.84 ± 4.181 U/mL, respectively (p < 0.01). Area under curve for suPAR and anti-AT1R to predict post-transplant FSGS recurrence was 0.51 and 0.84, respectively. Pre-transplant anti-AT1R levels appear to be a helpful biomarker in identifying patients at high risk of post-transplant FSGS recurrence.

A family of synthetic riboswitches adopts a kinetic trapping mechanism.

Riboswitches are sequences of RNA that control gene expression via RNA-ligand interactions, without the need for accessory proteins. Riboswitches consist of an aptamer that recognizes the ligand and an expression platform that couples ligand binding to a change in gene expression. Using in vitro selection, it is possible to screen large (∼ 10(13) members) libraries of RNA sequences to discover new aptamers. However, limitations in bacterial transformation efficiency make screening such large libraries for riboswitch function in intact cells impractical. Here we show that synthetic riboswitches function in an E. coli S30 extract in a manner similar to how they function in intact E. coli cells. We discovered that, although this family of riboswitches regulates the initiation of protein translation, the fate of whether an RNA message is translated is determined during transcription. Thus, ligand binding does not bias a population of rapidly equilibrating RNA structures, but rather, co-transcriptional ligand binding kinetically traps the RNA in a conformation that supports efficient translation. In addition to providing new insights into the mechanisms of action of a family of synthetic riboswitches, our experiments suggest that it may be possible to perform selections for novel synthetic riboswitches in an in vitro system.

Correlation between CT-based measured renal volumes and nuclear-renography-based split renal function in living kidney donors. Clinical diagnostic utility and practice patterns.

INTRODUCTION: Living donor evaluation involves imaging to determine the choice of kidney for nephrectomy. Our aim was to study the diagnostic accuracy and correlation between CT-based volume measurements and split renal function (SRF) as measured by nuclear renography in potential living donors and its impact on kidney selection decision. METHODS: We analyzed 190 CT-based volume measurements in healthy donors, of which 65 donors had a radionuclide study performed to determine SRF. RESULTS: There were no differences in demographics, anthropometric measurements, total volumes, eGFR, creatinine clearances between those who required a nuclear scan and those who did not. There was a significant correlation between CT-volume-measurement-based SRF and nuclear-scan-based SRF (Pearson coefficient r 0.59; p < 0.001). Furthermore, selective nuclear-based SRF allowed careful selection of donor nephrectomy, leaving the donor with the higher functioning kidney in most cases. There was also a significantly higher number of right-sided nephrectomies selected after nuclear-based SRF studies. CONCLUSION: CT-based volume measurements in living donor imaging have sufficient correlation with nuclear-based SRF. Selective use of nuclear-scan-based SRF allows careful selection for donor nephrectomy.

The effectiveness of the combination of rituximab and high-dose immunoglobulin in the immunomodulation of sensitized kidney transplant candidates.

Kidney transplantation faces many challenges not the least of which is the presence of pre-formed HLA antibodies. At our institution, we have used a combination of methods to immunomodulate sensitized patients. Most recently, this has been attempted with a combination of immunoglobulin (IVIG) and rituximab (Rituxan; Genetech, CA, USA). A total of 31 patients were followed for up to one yr following treatment with IVIG (2 gm/kg on day 1 and day 30) and rituximab (1 g - day 15). Antibody levels were followed serially at designated time points via solid-phase single-antigen beads (SAB) method (One Lambda, Inc., Canoga Park, CA, USA). Concentration of antibodies was based on median fluorescence intensity (MFI). The majority of patients had both class I and class II antibodies (79%). Our results showed that this protocol appeared to be patient and antibody specific. The most pronounced MFI reduction in antibodies occurred within the 30- to 100-d period post-treatment. Calculated panel-reactive antibodies decreased but rebound tended to occur by 104 d after antibody MFI nadir. Because of this rebound, it can be inferred that the patients did not show a durable increase in their potential for transplantation. The search for a more effective method to immunomodulate patients continues.

Early findings of prospective anti-HLA donor specific antibodies monitoring study in pancreas transplantation: Indiana University Health Experience.

The significance of donor-specific antibodies (DSA) is not well known in the setting of pancreas transplantation. Since December 2009, we prospectively followed pancreas transplant patients with single-antigen-luminex-bead testing at one, two, three, six, and then every six months for the first two yr. Thirty-five of the 92 patients that underwent pancreas transplantation (13 pancreas-alone [PTA], 20 with a kidney [SPK], and two after a kidney [PAK]) agreed to participate in study. Median age at transplant was 45 yr and follow-up was 23 months. Majority were Caucasian (n = 33) and male (n = 18). Rabbit anti-thymocyte globulin induction was used. Median HLA-mismatch was 4.2 ± 1.1. Eight patients (7SPK, 1PAK) developed post-transplant DSA at median follow-up of 76 d (26-119), 1 SPK had pre-formed DSA. Seven patients had both class I and class II DSA, one with class I and one with class II only. Mean peak class I DSA-MFI was 3529 (±1456); class II DSA-MFI was 5734 (±3204) whereas cumulative DSA MFI (CI + CII) was 9264 (±4233). No difference was observed in the patient and donor demographics among patients with and without DSA. One patient in non-DSA group developed acute cellular rejection of pancreas. From our data it appears that post-transplant DSA in pancreas allograft recipients may not impact the early-pancreatic allograft outcomes. The utility of prospective DSA monitoring in pancreatic transplant patients needs further evaluation and long-term follow-up.