The Impact of Second-Shell Nucleotides on Ligand Specificity in Cyclic Dinucleotide Riboswitches.

Ligand specificity is an essential requirement for all riboswitches. Some variant riboswitches utilize a common structural motif, yet through subtle sequence differences, they are able to selectively respond to different small molecule ligands and regulate downstream gene expression. These variants discriminate between structurally and chemically similar ligands. Crystal structures provide insight into how specificity is achieved. However, ligand specificity cannot always be explained solely by nucleotides in direct contact with the ligand. The cyclic dinucleotide variant family contains two classes, cyclic-di-GMP and cyclic-AMP-GMP riboswitches, that were distinguished based on the identity of a single nucleotide in contact with the ligand. Here we report a variant riboswitch with a mutation at a second ligand-contacting position that is promiscuous for both cyclic-di-GMP and cyclic-AMP-GMP despite a predicted preference for cyclic-AMP-GMP. A high-throughput mutational analysis, SMARTT, was used to quantitatively assess thousands of sites in the first- and second-shells of ligand contact for impacts on ligand specificity and promiscuity. In addition to nucleotides in direct ligand contact, nucleotides more distal from the binding site, within the J1/2 linker and the terminator helix, were identified that impact ligand specificity. These findings provide an example of how nucleotides outside the ligand binding pocket influence the riboswitch specificity. Moreover, these distal nucleotides could be used to predict promiscuous sequences.

Translation regulation by a guanidine-II riboswitch is highly tunable in sensitivity, dynamic range, and apparent cooperativity.

Riboswitches function as important translational regulators in bacteria. Comprehensive mutational analysis of transcriptional riboswitches has been used to probe the energetic intricacies of interplay between the aptamer and expression platform, but translational riboswitches have been inaccessible to massively parallel techniques. The guanidine-II (gdm-II) riboswitch is an exclusively translational class. We have integrated RelE cleavage with next-generation sequencing to quantify ligand-dependent changes in translation initiation for all single and double mutations of the Pseudomonas aeruginosa gdm-II riboswitch, a total of more than 23,000 variants. This extensive mutational analysis is consistent with the prominent features of the bioinformatic consensus. These data indicate, unexpectedly, that direct sequestration of the Shine-Dalgarno sequence is dispensable for riboswitch function. Additionally, this comprehensive data set reveals important positions not identified in previous computational and crystallographic studies. Mutations in the variable linker region stabilize alternate conformations. The double mutant data reveal the functional importance of the previously modeled P0b helix formed by the 5' and 3' tails that serves as the basis for translational control. Additional mutations to GU wobble base pairs in both P1 and P2 reveal how the apparent cooperativity of the system involves an intricate network of communication between the two binding sites. This comprehensive examination of a translational riboswitch's expression platform illuminates how the riboswitch is precisely tuned and tunable with regard to ligand sensitivity, the amplitude of expression between ON and OFF states, and the cooperativity of ligand binding.

How Do Bacteria "See" Molecules Inside Themselves?

RNA, like its close cousin DNA, is used to store information in the cell. Unlike DNA, it is really good at folding up into interesting shapes, which makes it good at lots of other important jobs. Some kinds of RNA, called riboswitches, can sense what is going on inside a cell. Each riboswitch fits a specific small molecule. When the riboswitch and small molecule interact it changes what the cell does. For example, if the small molecule is harmful the cell might start making a protein that will get rid of it. Recently, scientists discovered some riboswitches that look very similar to each other but recognize very different small molecules. We used X-ray crystallography to get pictures of these riboswitches. We saw how changing just one piece of the riboswitch changed which small molecule it recognized. This shows us how RNA can gain new functions as an organism evolves.

Dynamic Response of Donor-Derived Cell-Free DNA Following Treatment of Acute Rejection in Kidney Allografts.

Background: The quantification of rejection treatment efficacy has been insufficient using traditional markers due, in part, to the lagging response of serum creatinine and histologic alterations on biopsy specimens. Donor-derived cell-free DNA (dd-cfDNA) is a molecular marker of injury that may assess allograft injury after rejection. Methods: Retrospective review of the DART study identified 70 patients who had a clinically indicated biopsy, simultaneous dd-cfDNA measurement, and at least one follow-up dd-cfDNA within 3 months post-treatment. Thirty-five patients had no biopsy-proven rejection and no rejection treatment (NR), 16 patients had no biopsy-proven rejection but did receive rejection treatment (CR), 9 patients had diagnosis of ABMR/mixed rejection on biopsy and received rejection treatment (ABMR), and 10 patients had diagnosis of TCMR and received rejection treatment (TCMR). The CR, ABMR, and TCMR groups combined to form a rejection (R) group. Results: In the R group, median dd-cfDNA values at baseline and 1 month were 0.62% and 0.35% (n=21 pairs, p=0.34), and at baseline and 2-3 months were 0.77% and 0.21% (n=23 pairs, p=0.002). In TCMR, median dd-cfDNA values at baseline and 1 month were 1.13% and 0.37% (n=5 pairs, p=0.63), and at baseline and 2-3 months were 0.25% and 0.12% (n=9 pairs, p=0.004). In ABMR, median dd-cfDNA values at baseline and 1 month were 1.61% and 1.2 % (n=6 pairs, p>0.99), and at baseline and 2-3 months were 3.85% and 1.32% (n=6 pairs, p=0.09). In CR, median dd-cfDNA values at baseline and 1 month were 0.31% and 0.29% (n=10 pairs, p=0.38), and at baseline and 2-3 months were 0.38% and 0.17% (n=8 pairs, p=0.31). Lastly, in NR, median dd-cfDNA values at baseline and 1 month were 0.23% and 0.18% (n=21 pairs, p=0.10), and at baseline and 2-3 months were 0.33% and 0.17% (n=26 pairs, p=0.003). Changes in serum creatinine across 1 month and 2-3 months following rejection were similar. Conclusions: dd-cfDNA may be a useful dynamic biomarker to assess the health of the kidney allograft following rejection treatment.

The Positively Charged Active Site of the Bacterial Toxin RelE Causes a Large Shift in the General Base pKa.

The bacterial toxin RelE cleaves mRNA in the ribosomal A site. Although it shares a global fold with other microbial RNases, the active site contains several positively charged residues instead of histidines and glutamates that are typical of ribonucleases. The pH dependences of wild-type and mutant RelE indicate it uses general acid-base catalysis, but either the general acid (proposed to be R81) or the general base must have a substantially downshifted pKa. However, which group is shifted cannot be determined using available structural and biochemical data. Here, we use a phosphorothiolate at the scissile phosphate to remove the need for a general acid. We show this modification rescues nearly all of the defect of the R81A mutation, supporting R81 as the general acid. We also find that the observed pKa of the general base is dependent on the charge of the side chain at position 81. This indicates that positive charge in the active site contributes to a general base pKa downshifted by more than 5 units. Although this modestly reduces the effectiveness of general acid-base catalysis, it is strongly supplemented by the role of the positive charge in stabilizing the transition state for cleavage. Furthermore, we show that the ribosome is required for cleavage but not binding of mRNA by RelE. Ribosome functional groups do not directly contact the scissile phosphate, indicating that positioning and charge interactions dominate RelE catalysis. The unusual RelE active site catalyzes phosphoryl transfer at a rate comparable to those of similar enzymes, but in a ribosome-dependent fashion.

The asymmetry and cooperativity of tandem glycine riboswitch aptamers.

Glycine riboswitches utilize both single- and tandem-aptamer architectures. In the tandem system, the relative contribution of each aptamer toward gene regulation is not well understood. To dissect these contributions, the effects of 684 single mutants of a tandem ON switch from Bacillus subtilis were characterized for the wild-type construct and binding site mutations that selectively restrict ligand binding to either the first or second aptamer. Despite the structural symmetry of tandem aptamers, the response to these mutations was frequently asymmetrical. Mutations in the first aptamer often significantly weakened the K1/2, while several mutations in the second aptamer improved the amplitude. These results demonstrate that this ON switch favors ligand binding to the first aptamer. This is in contrast to the tandem OFF switch variant from Vibrio cholerae, which was previously shown to have preferential binding to its second aptamer. A bioinformatic analysis of tandem glycine riboswitches revealed that the two binding pockets are differentially conserved between ON and OFF switches. Altogether, this indicates that tandem ON switch variants preferentially utilize binding to the first aptamer to promote helical switching, while OFF switch variants favor binding to the second aptamer. The data set also revealed a cooperative glycine response when both binding pockets were maximally stabilized with three GC base pairs. This indicates a cooperative response may sometimes be obfuscated by a difference in the affinities of the two aptamers. This conditional cooperativity provides an additional layer of tunability to tandem glycine riboswitches that adds to their versatility as genetic switches.

Gene expression profiling and racial disparities in outcomes after heart transplantation.

BACKGROUND: African Americans (AAs) have lower survival rates after heart transplantation (HTx) than Caucasians. The aim of this analysis was to evaluate racial differences in gene expression and their associations with survival and the composite outcome of death, retransplant, rejection with hemodynamic compromise, and graft dysfunction in the Outcomes AlloMap Registry. METHODS: Registry participants included low-risk Caucasian and AA heart transplant recipients with a baseline and at least 1 follow-up gene expression test (AlloMap(C)) within the first year after HTx. The Kaplan-Meier method with delayed entry was used to describe differences in outcomes. Multivariable Cox hazard regression was used to evaluate the associations of overall gene expression profiling score, MARCH8 and FLT3 expression, and tacrolimus levels with each outcome, and stratified Cox models were developed to quantify race-specific associations. RESULTS: Among 933 eligible recipients, 737 (79%) were Caucasian and 196 (21%) were AA. Compared with Caucasians, AAs were significantly younger (55 vs 59 years, p < 0.001), with higher rates of non-ischemic cardiomyopathy (68% vs 50%, p < 0.001), sensitization (>10% panel reactive antibody, 16% vs 9.1%, p = 0.009), and human leukocyte antigen mismatches (7 vs 7, p = 0.01), but less frequent primary cytomegalovirus serostatus mismatch (14.31% vs 27.3%, p < 0.001). Overall, AAs had an increased adjusted mortality risk (hazard ratio [HR] 4.13, p = 0.007). Higher tacrolimus levels were associated with decreased mortality in AAs (HR 0.62, p = 0.009). Overall gene expression profiling score was associated with increased mortality among Caucasians (HR 1.21, p = 0.048). In Caucasians, but not AAs, overexpression of MARCH8 was associated with increased mortality (HR 2.90, p = 0.001). FLT3 upregulation was associated with increased mortality (HR 2.42, p = 0.033) in AAs. There was an inverse relationship between FLT3 expression and tacrolimus levels (-0.029 and -0.176, respectively) in Caucasians and AAs. CONCLUSIONS: AAs have a significantly higher mortality risk after HTx than Caucasians, even in the low-risk Outcomes AlloMap Registry population. AAs and Caucasians had differential outcomes based upon the varying expression of MARCH8 and FLT3 genes following HTx.

Noninvasive detection of graft injury after heart transplant using donor-derived cell-free DNA: A prospective multicenter study.

Standardized donor-derived cell-free DNA (dd-cfDNA) testing has been introduced into clinical use to monitor kidney transplant recipients for rejection. This report describes the performance of this dd-cfDNA assay to detect allograft rejection in samples from heart transplant (HT) recipients undergoing surveillance monitoring across the United States. Venous blood was longitudinally sampled from 740 HT recipients from 26 centers and in a single-center cohort of 33 patients at high risk for antibody-mediated rejection (AMR). Plasma dd-cfDNA was quantified by using targeted amplification and sequencing of a single nucleotide polymorphism panel. The dd-cfDNA levels were correlated to paired events of biopsy-based diagnosis of rejection. The median dd-cfDNA was 0.07% in reference HT recipients (2164 samples) and 0.17% in samples classified as acute rejection (35 samples; P = .005). At a 0.2% threshold, dd-cfDNA had a 44% sensitivity to detect rejection and a 97% negative predictive value. In the cohort at risk for AMR (11 samples), dd-cfDNA levels were elevated 3-fold in AMR compared with patients without AMR (99 samples, P = .004). The standardized dd-cfDNA test identified acute rejection in samples from a broad population of HT recipients. The reported test performance characteristics will guide the next stage of clinical utility studies of the dd-cfDNA assay.

Consequences of Increasing Complexity in Anorectal Surgery Performed at an Academic Center.

BACKGROUND: Anorectal surgery encompasses a wide range of procedures with varying complexity. The Accreditation Council for Graduate Medical Education Review Committee for Colon and Rectal Surgery recommends minimum case numbers (60) for 1-year specialty trainees in 6 categories of anorectal surgery, with definitions for procedural complexity. OBJECTIVE: The purpose of this study was to assess the scope of anorectal procedures and propose a stratification of procedures based on a consensus of levels of difficulty, as well as to identify a predictive charge cutoff suggestive of procedural complexity. DESIGN: This was a retrospective review. SETTINGS: The study was conducted at a tertiary academic center. PATIENTS: Patients undergoing anorectal procedures between January 2011 and December 2014 identified by Current Procedural Terminology coding were entered into 6 categories. Codes were stratified as routine or complex based on an assessment of perioperative care and technical expertise required. Patients with an abdominal portion to any procedure were excluded. MAIN OUTCOMES MEASURES: The study measured distribution of complexity in anorectal surgical procedures and procedural charge associated with differentiating routine from complex procedures. RESULTS: Seven colorectal surgeons performed 2483 anorectal procedures (mean = 620 per year). Mean age was 48 ± 16 years. Forty six (64%) of 71 procedures were classified as routine and 25 (36%) of 71 as complex. Most disease processes had subsets with routine or complex procedures, whereas all of the procedures performed for fecal incontinence or advanced anorectal techniques were considered complex. Fistula procedures and transanal excisions were most heterogeneous, with a high procedural complexity rate (37% and 50%). After a procedural complexity rating, intraclass correlation by 6 surgeons was 0.70, demonstrating good correlation. Receiver operating curve assessments of consensus categorization according to billing codes revealed $553 as the optimal cutoff between routine and complex procedures. LIMITATIONS: This was a single-institution retrospective review. CONCLUSIONS: Colorectal residents may benefit from anorectal case stratification, because it serves as a dialogue for those interested in complex anorectal surgery during training. Surgeon categorization of procedures correlates well with a charge-based model of complexity. See Video Abstract at http://links.lww.com/DCR/A806.

Donor-derived Cell-free DNA Identifies Antibody-mediated Rejection in Donor Specific Antibody Positive Kidney Transplant Recipients.

BACKGROUND: Elevated levels of donor-derived cell-free DNA (dd-cfDNA) in the plasma of renal allograft recipients indicates organ injury and an increased probability of active rejection. Donor-specific antibodies (DSA) to HLA antigens are associated with risk of antibody-mediated rejection (ABMR). This study assessed the combined use of dd-cfDNA and DSA testing to diagnose active ABMR. METHODS: Donor-derived cell-free DNA was assayed in 90 blood samples with paired DSA and clinically indicated biopsies from 87 kidney transplant patients. Sixteen cases met criteria for active ABMR. Performance characteristics of dd-cfDNA for diagnosis of active ABMR were determined for samples with prior or current positive DSA (DSA+, n = 33). RESULTS: The median level of dd-cfDNA (2.9%) in DSA+ patients with active ABMR was significantly higher than the median level (0.34%) in DSA+ patients without ABMR (P < 0.001). The median level of dd-cfDNA in DSA- patients was 0.29%. The positive predictive value of dd-cfDNA (at 1%) to detect active ABMR in DSA+ patients was 81%, whereas the negative predictive value was 83%. The positive predictive value for DSA+ alone was 48%. CONCLUSIONS: The combined use of dd-cfDNA and DSA testing may improve the noninvasive diagnosis of active ABMR in kidney transplant patients. Patients with dd-cfDNA+/ DSA+ results have a high probability of active ABMR.

Gene regulation by a glycine riboswitch singlet uses a finely tuned energetic landscape for helical switching.

Riboswitches contain structured aptamer domains that, upon ligand binding, facilitate helical switching in their downstream expression platforms to alter gene expression. To fully dissect how riboswitches function requires a better understanding of the energetic landscape for helical switching. Here, we report a sequencing-based high-throughput assay for monitoring in vitro transcription termination and use it to simultaneously characterize the functional effects of all 522 single point mutants of a glycine riboswitch type-1 singlet. Mutations throughout the riboswitch cause ligand-dependent defects, but only mutations within the terminator hairpin alter readthrough efficiencies in the absence of ligand. A comprehensive analysis of the expression platform reveals that ligand binding stabilizes the antiterminator by just 2-3 kcal/mol, indicating that the competing expression platform helices must be extremely close in energy to elicit a significant ligand-dependent response. These results demonstrate that gene regulation by this riboswitch is highly constrained by the energetics of ligand binding and conformational switching. These findings exemplify the energetic parameters of RNA conformational rearrangements driven by binding events.

Cell-Free DNA and Active Rejection in Kidney Allografts.

Histologic analysis of the allograft biopsy specimen is the standard method used to differentiate rejection from other injury in kidney transplants. Donor-derived cell-free DNA (dd-cfDNA) is a noninvasive test of allograft injury that may enable more frequent, quantitative, and safer assessment of allograft rejection and injury status. To investigate this possibility, we prospectively collected blood specimens at scheduled intervals and at the time of clinically indicated biopsies. In 102 kidney recipients, we measured plasma levels of dd-cfDNA and correlated the levels with allograft rejection status ascertained by histology in 107 biopsy specimens. The dd-cfDNA level discriminated between biopsy specimens showing any rejection (T cell-mediated rejection or antibody-mediated rejection [ABMR]) and controls (no rejection histologically), P<0.001 (receiver operating characteristic area under the curve [AUC], 0.74; 95% confidence interval [95% CI], 0.61 to 0.86). Positive and negative predictive values for active rejection at a cutoff of 1.0% dd-cfDNA were 61% and 84%, respectively. The AUC for discriminating ABMR from samples without ABMR was 0.87 (95% CI, 0.75 to 0.97). Positive and negative predictive values for ABMR at a cutoff of 1.0% dd-cfDNA were 44% and 96%, respectively. Median dd-cfDNA was 2.9% (ABMR), 1.2% (T cell-mediated types ≥IB), 0.2% (T cell-mediated type IA), and 0.3% in controls (P=0.05 for T cell-mediated rejection types ≥IB versus controls). Thus, dd-cfDNA may be used to assess allograft rejection and injury; dd-cfDNA levels <1% reflect the absence of active rejection (T cell-mediated type ≥IB or ABMR) and levels >1% indicate a probability of active rejection.

Mycofumigation through production of the volatile DNA-methylating agent N-methyl-N-nitrosoisobutyramide by fungi in the genus Muscodor.

Antagonistic microorganisms produce antimicrobials to inhibit the growth of competitors. Although water-soluble antimicrobials are limited to proximal interactions via aqueous diffusion, volatile antimicrobials are able to act at a distance and diffuse through heterogeneous environments. Here, we identify the mechanism of action of Muscodor albus, an endophytic fungus known for its volatile antimicrobial activity toward a wide range of human and plant pathogens and its potential use in mycofumigation. Proposed uses of the Muscodor species include protecting crops, produce, and building materials from undesired fungal or bacterial growth. By analyzing a collection of Muscodor isolates with varying toxicity, we demonstrate that the volatile mycotoxin, N-methyl-N-nitrosoisobutyramide, is the dominant factor in Muscodor toxicity and acts primarily through DNA methylation. Additionally, Muscodor isolates exhibit higher resistance to DNA methylation compared with other fungi. This work contributes to the evaluation of Muscodor isolates as potential mycofumigants, provides insight into chemical strategies that organisms use to manipulate their environment, and provokes questions regarding the mechanisms of resistance used to tolerate constitutive, long-term exposure to DNA methylation.

Biological Variation of Donor-Derived Cell-Free DNA in Renal Transplant Recipients: Clinical Implications.

BACKGROUND: Previous studies have demonstrated that donor-derived cell-free DNA (dd-cfDNA) found in circulating blood of transplant recipients may serve as a noninvasive biomarker of allograft rejection. To better interpret the clinical meaning of dd-cfDNA, it is essential to understand the biological variation of this biomarker in stable healthy recipients. This report establishes the biological variation and clinical reference intervals of dd-cfDNA in renal transplant recipients by using an analytically validated assay that has a CV of 6.8%. METHODS: We sampled venous blood at patient surveillance visits (typically at posttransplant months 1-4, 6, 9, and 12) in a 14-center observational study. Patients with stable renal allograft function spanning ≥3 serial visits were selected. We used AlloSure®, a targeted next-generation sequencing-based approach, to measure dd-cfDNA in the plasma and computed the intraindividual CV (CVI) and interindividual CV (CVG), the index of individuality (II), and reference change value (RCV). RESULTS: Of 93 patients, 61% were men, 56% were Caucasian, mean age was 49 years, and 63% were deceased donor kidney recipients. Of 380 blood samples, the dd-cfDNA median value was 0.21% (interquartile range 0.12%-0.39%) and the 97.5th percentile was 1.20%. In 18 patients with an average of 4.1 tests, the CVI was 21%, CVG was 37%, II was 0.57, and RCV was 61%. CONCLUSIONS: In a renal transplant recipient, a dd-cfDNA level above 1.2% is out of range and potentially abnormal. A serial increase of up to 61% in level of dd-cfDNA in a patient may be attributable to biological variation.Clinicaltrials.gov Identifier: NCT02424227.

Robotic Rectopexy for Rectal Prolapse in Pediatric Patients.

Rectal prolapse is the protrusion of the rectum out of the anus. Surgical correction can be accomplished via open and minimally invasive abdominal approaches, as well as from the perineum. Robotic rectopexy is an option for minimally invasive treatment of rectal prolapse. There are no studies that have established the efficacy of robotic rectopexy for rectal prolapse in the pediatric population. The aim of this study was to review the experience of robotic rectopexy at a single institution. This is a retrospective review of our pediatric robotic rectopexy experience from 2012 to 2015. Information was obtained from chart review of both operative notes and clinic visits. Four pediatric patients underwent a robotic rectopexy for rectal prolapse from 2012 to 2015. Three patients were male and one was female. The mean age was 15.5 years (range 13-17). Two patients had rectal prolapse with chronic constipation. One patient had rectal prolapse from Ehlers Danlos syndrome, and the last had rectal prolapse after imperforate anus repair as an infant. Three patients received a bowel preparation. Three patients were completed robotically, and one patient required conversion to an open procedure. The average postoperative length of stay was 3.25 days (range 2-4). There were no episodes of recurrent prolapse. Two patients had improvement in constipation, one had no improvement, and one had no documented change. Average postoperative follow-up was 11.5 months (range 3-29). This study was a review of one institution's experience with pediatric robotic rectopexy. With short-term follow-up, there was no recurrence of prolapse. Robotic rectopexy provided a safe, reliable, and short-term resolution of rectal prolapse in pediatric patients.

Validation of a Clinical-Grade Assay to Measure Donor-Derived Cell-Free DNA in Solid Organ Transplant Recipients.

The use of circulating cell-free DNA (cfDNA) as a biomarker in transplant recipients offers advantages over invasive tissue biopsy as a quantitative measure for detection of transplant rejection and immunosuppression optimization. However, the fraction of donor-derived cfDNA (dd-cfDNA) in transplant recipient plasma is low and challenging to quantify. Previously reported methods to measure dd-cfDNA require donor and recipient genotyping, which is impractical in clinical settings and adds cost. We developed a targeted next-generation sequencing assay that uses 266 single-nucleotide polymorphisms to accurately quantify dd-cfDNA in transplant recipients without separate genotyping. Analytical performance of the assay was characterized and validated using 1117 samples comprising the National Institute for Standards and Technology Genome in a Bottle human reference genome, independently validated reference materials, and clinical samples. The assay quantifies the fraction of dd-cfDNA in both unrelated and related donor-recipient pairs. The dd-cfDNA assay can reliably measure dd-cfDNA (limit of blank, 0.10%; limit of detection, 0.16%; limit of quantification, 0.20%) across the linear quantifiable range (0.2% to 16%) with across-run CVs of 6.8%. Precision was also evaluated for independently processed clinical sample replicates and is similar to across-run precision. Application of the assay to clinical samples from heart transplant recipients demonstrated increased levels of dd-cfDNA in patients with biopsy-confirmed rejection and decreased levels of dd-cfDNA after successful rejection treatment. This noninvasive clinical-grade sequencing assay can be completed within 3 days, providing the practical turnaround time preferred for transplanted organ surveillance.