Emerging Relations among Cognitive Constructs and Cochlear Implant Outcomes: A Systematic Review and Meta-Analysis.

OBJECTIVE: Hearing loss has a detrimental impact on cognitive function. However, there is a lack of consensus on the impact of cochlear implants on cognition. This review systematically evaluates whether cochlear implants in adult patients lead to cognitive improvements and investigates the relations of cognition with speech recognition outcomes. DATA SOURCES: A literature review was performed following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines. Studies evaluating cognition and cochlear implant outcomes in postlingual, adult patients from January 1996 to December 2021 were included. Of 2510 total references, 52 studies were included in qualitative analysis and 11 in meta-analyses. REVIEW METHODS: Proportions were extracted from studies of (1) the significant impacts of cochlear implantation on 6 cognitive domains and (2) associations between cognition and speech recognition outcomes. Meta-analyses were performed using random effects models on mean differences between pre- and postoperative performance on 4 cognitive assessments. RESULTS: Only half of the outcomes reported suggested cochlear implantation had a significant impact on cognition (50.8%), with the highest proportion in assessments of memory & learning and inhibition-concentration. Meta-analyses revealed significant improvements in global cognition and inhibition-concentration. Finally, 40.4% of associations between cognition and speech recognition outcomes were significant. CONCLUSION: Findings relating to cochlear implantation and cognition vary depending on the cognitive domain assessed and the study goal. Nonetheless, assessments of memory & learning, global cognition, and inhibition-concentration may represent tools to assess cognitive benefit after implantation and help explain variability in speech recognition outcomes. Enhanced selectivity in assessments of cognition is needed for clinical applicability.

Feasibility and Cost of Telehealth Head and Neck Cancer Survivorship Care: A Systematic Review.

OBJECTIVE: Evaluate the feasibility and cost-effectiveness of telehealth head and neck cancer (HNC) survivorship care. DATA SOURCES: Ovid MEDLINE, Embase, Scopus, CINAHL. REVIEW METHODS: A systematic search for peer-reviewed feasibility studies on telehealth models for HNC survivorship care published between 2005 and 2021 was conducted using the terms "head and neck cancer" and "telehealth" and their synonyms. Inclusion criteria were studies on telehealth survivorship program interventions for HNC patients with quantitative feasibility outcome measures (eg, enrollment, retention, attrition/dropout rate, adherence/task completion rate, patient satisfaction, cost). RESULTS: Thirty-eight studies out of 1557 identified met inclusion criteria and were included for analysis. Feasibility outcomes evaluated were enrollment and attrition rates, adherence/task completion rates, patient satisfaction, and user feedback surveys in different survivorship domains. Patient enrollment ranged from 20.8% to 85.7%, while attrition ranged from 7% to 47.7%. Overall, adherence was 30.2% higher in the intervention group than in the control group (46.8% vs 16.6%). Studies with cost analysis found telehealth models of care to be statistically significantly less expensive and more cost-efficient than the standard model of care, with a $642.30 saving per patient (n = 3). Telehealth models also substantially reduced work time saving per visit (on average, 7 days per visit). CONCLUSION: While telehealth survivorship programs are feasible and cost-effective and are associated with improved patient outcomes, they might not be ideal for every patient. Further investigations are needed to understand the role of telehealth in survivorship care, given the variability in study design, reporting, measures, and methodological quality.

The design and synthesis of green emissive iridium(III) complexes guided by calculations of the vibrationally-resolved emission spectra.

A key challenge in developing emissive materials for organic light-emitting diodes is to optimize their colour saturation, which means targeting narrowband emitters. In this combined theoretical and experimental study, we investigate the use of heavy atoms in the form of trimethylsilyl groups as a tool to reduce the intensity of the vibrations in the 2-phenylpyridinato ligands of emissive iridium(III) complexes that contribute to the vibrationally coupled modes that broaden the emission profile. An underutilised computational technique, Frank-Condon vibrationally coupled electronic spectral modelling, was used to identify the key vibrational modes that contribute to the broadening of the emission spectra in known benchmark green-emitting iridium(III) complexes. Based on these results, a family of eight new green-emitting iridium complexes containing trimethylsilyl groups substituted at different positions of the cyclometalating ligands has been prepared to explore the impact that these substituents have on reducing the intensity of the vibrations and the resulting reduction in the contribution of vibrationally coupled emission modes to the shape of the emission spectra. We have demonstrated that locating a trimethylsilyl group at the N4 or N5 position of the 2-phenylpyridine ligand damps the vibrational modes of the iridium complex and provides a modest narrowing of the emission spectrum of 8-9 nm (or 350 cm-1). The strong correlation between experimental and calculated emission spectra highlights the utility of this computational method to understand how the vibrational modes contribute to the profile of the emission spectra in phosphorescent iridium(III) emitters.

Breast Implant-Associated Anaplastic Large-Cell Lymphoma: Updated Systematic Review and Analysis of Treatment Strategies.

BACKGROUND: Although guidelines have been published on treatment of breast implant-associated anaplastic large-cell lymphoma (BIA-ALCL), there has been no comprehensive analysis of BIA-ALCL treatment variation based on the available literature. The authors sought to assess current treatment strategies of BIA-ALCL relative to current guidelines. METHODS: Database searches were conducted in June of 2020. Included articles were case reports and case series with patient-level data. Collected variables included clinicopathologic features, implant characteristics, diagnostic tests, ALCL characteristics, treatment, and details of follow-up and outcome. Treatment data from before and after 2017 were compared with National Cancer Center Network guidelines. RESULTS: A total of 89 publications were included and 178 cases of BIA-ALCL were identified. Most patients presented with seroma ( n = 114, 70.4 percent), followed by a mass ( n = 14, 8.6 percent), or both ( n = 23, 14.2 percent). Treatment included en bloc capsulectomy of the affected implant in 122 out of 126 cases with treatment details provided (96.8 percent). Radiation therapy was given in 38 cases (30.2 percent) and chemotherapy was given in 71 cases (56.3 percent). Practitioners used less chemotherapy for local disease after treatment guideline publication in 2017 ( p < 0.001), whereas treatment for advanced disease remained unchanged ( p = 0.3). There were 10 recurrences and eight fatalities attributable to BIA-ALCL, which were associated with advanced presentation (29 versus 2.1 percent; OR, 19.4; 95 percent CI, 3.9 to 96.3; p < 0.001). CONCLUSIONS: BIA-ALCL remains a morbid but treatable condition. Current guidelines focus treatment for local disease and reduce nonsurgical interventions with radiation or chemotherapy. Patients presenting with advanced BIA-ALCL experience higher rates of recurrence and mortality.

Evaluation of a suicide loss support training program for organ procurement personnel.

OBJECTIVE: To evaluate the impact of a 6-h traumatic loss training program on organ procurement personnel attitudes, confidence, and knowledge about suicide loss and best practices in supporting survivors. Organ procurement personnel may be uniquely positioned to deliver supportive programming as they have early access to suicide loss survivors (i.e., within hours or days of a loss) and are trained in a variety of backgrounds that include skills in supportive service delivery (i.e., nursing and social work). METHOD: Sixty-four personnel participated in the 6-h Traumatic Loss Support Program (TLSP) training and completed measures of attitudes, confidence, and knowledge about supporting suicide loss survivors prior to and following the training. RESULTS: Ratings of the usefulness of the training were high and mean scores on all measures of attitudes, confidence, and knowledge increased with moderate to large effect sizes. CONCLUSIONS: Results of the current study demonstrate that the TLSP training may be an effective way of increasing suicide loss supportive services provided by organ procurement personnel; however, further research is needed to determine the extent to which these interactions influence the bereavement of those who have lost loved ones to suicide and the fidelity in which the TLSP is implemented.

Pediatric Extracorporeal Cardiopulmonary Resuscitation: A Systematic Review.

OBJECTIVES: This systematic review aims to summarize the body of available literature on pediatric extracorporeal cardiopulmonary resuscitation in order to delineate current utilization, practices, and outcomes, while highlighting gaps in current knowledge. DATA SOURCES: PubMed, Embase, Scopus, Cochrane Library, and ClinicalTrials.gov databases. STUDY SELECTION: We searched for peer-reviewed original research publications on pediatric extracorporeal cardiopulmonary resuscitation (patients < 18 yr old) and were inclusive of all publication years. DATA EXTRACTION: Our systematic review used the structured Preferred Reporting Items for Systematic Reviews and Meta-Analyses methodology. Our initial literature search was performed on February 11, 2019, with an updated search performed on August 28, 2019. Three physician reviewers independently assessed the retrieved studies to determine inclusion in the systematic review synthesis. Using selected search terms, a total of 4,095 publications were retrieved, of which 96 were included in the final synthesis. Risk of bias in included studies was assessed using the Risk of Bias in Non-Randomized Studies of Interventions-I tool. DATA SYNTHESIS: There were no randomized controlled trials of extracorporeal cardiopulmonary resuscitation use in pediatrics. A vast majority of pediatric extracorporeal cardiopulmonary resuscitation publications were single-center retrospective studies reporting outcomes after in-hospital cardiac arrest. Most pediatric extracorporeal cardiopulmonary resuscitation use in published literature is in cardiac patients. Survival to hospital discharge after extracorporeal cardiopulmonary resuscitation for pediatric in-hospital cardiac arrest ranged from 8% to 80% in included studies, and there was an association with improved outcomes in cardiac patients. Thirty-one studies reported neurologic outcomes after extracorporeal cardiopulmonary resuscitation, of which only six were prospective follow-up studies. We summarize the available literature on: determination of candidacy, timing of activation of extracorporeal cardiopulmonary resuscitation, staffing/logistics, cannulation strategies, outcomes, and the use of simulation for training. CONCLUSIONS: This review highlights gaps in our understanding of best practices for pediatric extracorporeal cardiopulmonary resuscitation. We summarize current studies available and provide a framework for the development of future studies.

Evidence of a Monoclonal Origin for Bilateral Serous Tubal Intraepithelial Neoplasia.

Serous tubal intraepithelial carcinoma (STIC) is found in 10% to 60% of cases of tuboovarian high-grade serous carcinoma (HGSC) and is presumed to be the site of origin, linking many HGSCs to the fallopian tube. Bilateral STIC is present in ∼20% of cases. Because clonal Tp53 mutations are a defining feature of HGSC, including their associated STICs, we analyzed 4 cases of bilateral serous tubal intraepithelial neoplasia (STIN), including STIC and Tp53-mutated serous tubal intraepithelial lesions (STILs), associated with HGSC to determine whether they contained the same or different p53 mutations. Extracted DNA from STINs, concurrent HGSCs and control tissues was analyzed for mutations in all exons of Tp53. Sequencing was successful in 3 of the 4 cases, and an identical Tp53 mutation was detected in the HGSC and bilateral STINs in 2 of these 3 cases. One STIN was morphologically a STIL. These findings confirm that a subset of bilateral STINs share the same Tp53 mutation, implying that at least one of the STINs is an intraepithelial metastasis from either the contralateral STIN or HGSC. This study complements others addressing the multiple origins of STIN in the setting of existing HGSC. It further underscores the fact that potential overlap in biologic behavior between STILs and STICs as well as timing and direction of metastatic spread has yet to be resolved.

Evidence for lineage continuity between early serous proliferations (ESPs) in the Fallopian tube and disseminated high-grade serous carcinomas.

The distal Fallopian tube is a site of origin for many 'ovarian' high-grade serous carcinomas (HGSCs) with intraepithelial carcinomas (STICs) that share identical TP53 mutations with metastatic tumors. TP53 mutation-positive early serous proliferations (ESPs) comprise a spectrum including p53 signatures and serous tubal intraepithelial lesions (STILs) and are not considered malignant; however, ESPs are often the only abnormality found in Fallopian tubes of women with metastatic HGSC. The purpose of this study was to determine if a relationship exists between isolated ESPs and concurrent metastatic HGSCs in the absence of STIC. Fallopian tubes from 32 HGSCs without a co-existing STIC/HGSC in the endosalpinx were exhaustively sectioned. The presence of either STIC/HGSC or ESP in the endosalpinx was documented and DNA from tissues containing ESPs, concurrent HGSC, and control epithelia were interrogated for TP53 mutations by targeted amplicon-based sequencing with average coverage reads >4000 across DNA replicate samples. Serial sectioning revealed a previously unrecognized STIC/HGSC in 3 of 32 (9.3%) and ESPs in 13 (40.6%). Twelve contained TP53 mutations. Nine (75%) shared identical TP53 mutations with concurrent HGSCs, four at high (≥ 5%) and five at low (< 5%) allele frequency. All control epithelia were TP53 mutation-negative. This study, for the first time, indicates lineage identity between ESPs in the distal tube and some metastatic HGSCs via a shared site-specific TP53 mutation. It supports a novel serous carcinogenic sequence in which an ESP could eventually culminate in a metastatic serous cancer via 'precursor escape' and would explain the apparent sudden onset of cancers without co-existing STICs. This paradigm for serous cancer development underscores the likelihood that multiple precursor types in the Fallopian tube contribute to serous cancer development with implications for the evolution, pathologic classification, and prevention of this lethal malignancy. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Candida albicans and Early Childhood Caries: A Systematic Review and Meta-Analysis.

Oral Candida albicans has been detected in children with early childhood caries (ECC) and has demonstrated cariogenic traits in animal models of the disease. Conversely, other studies found no positive correlation between C. albicans and caries experience in children, while suggesting it may have protective effects as a commensal organism. Thus, this study aimed to examine whether oral C. albicans is associated with ECC. Seven electronic databases were searched. The data from eligible studies were extracted, and the risk of bias was evaluated. A fixed effects model (Mantel-Haenszel estimate) was used for meta-analysis, and the summary effect measure was calculated by odds ratio (OR) and 95% confidence interval (CI). Fifteen cross-sectional studies were included for the qualitative assessment and 9 studies for meta-analysis. Twelve studies revealed higher oral C. albicans prevalence in ECC children than in caries-free children, while 2 studies indicated an equivalent prevalence. A pooled estimate, with OR = 6.51 and 95% CI = 4.94-8.57, indicated a significantly higher ECC experience in children with oral C. albicans than those without C. albicans (p < 0.01). The odds of experiencing ECC in children with C. albicans versus children without C. albicans were 5.26 for salivary, 6.69 for plaque, and 6.3 for oral swab samples. This systematic review indicates that children with oral C. albicans have >5 times higher odds of having ECC compared to those without C. albicans. Further prospective cohort studies are needed to determine whether C. albicans could be a risk factor for ECC, and whether it is dependent on different sample sources (saliva/plaque).

KLF15 Establishes the Landscape of Diurnal Expression in the Heart.

Circadian rhythms offer temporal control of anticipatory physiologic adaptations in animals. In the mammalian cardiovascular system, the importance of these rhythms is underscored by increased cardiovascular disease in shift workers, findings recapitulated in experimental animal models. However, a nodal regulator that allows integration of central and peripheral information and coordinates cardiac rhythmic output has been elusive. Here, we show that kruppel-like factor 15 (KLF15) governs a biphasic transcriptomic oscillation in the heart with a maximum ATP production phase and a remodeling and repair phase corresponding to the active and resting phase of a rodent. Depletion of KLF15 in cardiomyocytes leads to a disorganized oscillatory behavior without phasic partition despite an intact core clock. Thus, KLF15 is a nodal connection between the clock and meaningful rhythmicity in the heart.

Hidden specificity in an apparently nonspecific RNA-binding protein.

Nucleic-acid-binding proteins are generally viewed as either specific or nonspecific, depending on characteristics of their binding sites in DNA or RNA. Most studies have focused on specific proteins, which identify cognate sites by binding with highest affinities to regions with defined signatures in sequence, structure or both. Proteins that bind to sites devoid of defined sequence or structure signatures are considered nonspecific. Substrate binding by these proteins is poorly understood, and it is not known to what extent seemingly nonspecific proteins discriminate between different binding sites, aside from those sequestered by nucleic acid structures. Here we systematically examine substrate binding by the apparently nonspecific RNA-binding protein C5, and find clear discrimination between different binding site variants. C5 is the protein subunit of the transfer RNA processing ribonucleoprotein enzyme RNase P from Escherichia coli. The protein binds 5' leaders of precursor tRNAs at a site without sequence or structure signatures. We measure functional binding of C5 to all possible sequence variants in its substrate binding site, using a high-throughput sequencing kinetics approach (HITS-KIN) that simultaneously follows processing of thousands of RNA species. C5 binds different substrate variants with affinities varying by orders of magnitude. The distribution of functional affinities of C5 for all substrate variants resembles affinity distributions of highly specific nucleic acid binding proteins. Unlike these specific proteins, C5 does not bind its physiological RNA targets with the highest affinity, but with affinities near the median of the distribution, a region that is not associated with a sequence signature. We delineate defined rules governing substrate recognition by C5, which reveal specificity that is hidden in cellular substrates for RNase P. Our findings suggest that apparently nonspecific and specific RNA-binding modes may not differ fundamentally, but represent distinct parts of common affinity distributions.

Protein-precursor tRNA contact leads to sequence-specific recognition of 5' leaders by bacterial ribonuclease P.

Bacterial ribonuclease P (RNase P) catalyzes the cleavage of 5' leader sequences from precursor tRNAs (pre-tRNAs). Previously, all known substrate nucleotide specificities in this system are derived from RNA-RNA interactions with the RNase P RNA subunit. Here, we demonstrate that pre-tRNA binding affinities for Bacillus subtilis and Escherichia coli RNase P are enhanced by sequence-specific contacts between the fourth pre-tRNA nucleotide on the 5' side of the cleavage site (N(-4)) and the RNase P protein (P protein) subunit. B. subtilis RNase P has a higher affinity for pre-tRNA with adenosine at N(-4), and this binding preference is amplified at physiological divalent ion concentrations. Measurements of pre-tRNA-containing adenosine analogs at N(-4) indicate that specificity arises from a combination of hydrogen bonding to the N6 exocyclic amine of adenosine and steric exclusion of the N2 amine of guanosine. Mutagenesis of B. subtilis P protein indicates that F20 and Y34 contribute to selectivity at N(-4). The hydroxyl group of Y34 enhances selectivity, likely by forming a hydrogen bond with the N(-4) nucleotide. The sequence preference of E. coli RNase P is diminished, showing a weak preference for adenosine and cytosine at N(-4), consistent with the substitution of Leu for Y34 in the E. coli P protein. This is the first identification of a sequence-specific contact between P protein and pre-tRNA that contributes to molecular recognition of RNase P. Additionally, sequence analyses reveal that a greater-than-expected fraction of pre-tRNAs from both E. coli and B. subtilis contains a nucleotide at N(-4) that enhances RNase P affinity. This observation suggests that specificity at N(-4) contributes to substrate recognition in vivo. Furthermore, bioinformatic analyses suggest that sequence-specific contacts between the protein subunit and the leader sequences of pre-tRNAs may be common in bacterial RNase P and may lead to species-specific substrate recognition.

Binding of C5 protein to P RNA enhances the rate constant for catalysis for P RNA processing of pre-tRNAs lacking a consensus (+ 1)/C(+ 72) pair.

The RNA subunit of the ribonucleoprotein enzyme ribonuclease P (RNase P (P RNA) contains the active site, but binding of Escherichia coli RNase P protein (C5) to P RNA increases the rate constant for catalysis for certain pre-tRNA substrates up to 1000-fold. Structure-swapping experiments between a substrate that is cleaved slowly by P RNA alone (pre-tRNA(f-met605)) and one that is cleaved quickly (pre-tRNA(met608)) pinpoint the characteristic C(+1)/A(+72) base pair of initiator tRNA(f-met) as the sole determinant of slow RNA-alone catalysis. Unlike other substrate modifications that slow RNA-alone catalysis, the presence of a C(+1)/A(+72) base pair reduces the rate constant for processing at both correct and miscleavage sites, indicating an indirect but nonetheless important role in catalysis. Analysis of the Mg(2)(+) dependence of apparent catalytic rate constants for pre-tRNA(met608) and a pre-tRNA(met608) (+1)C/(+72)A mutant provides evidence that C5 promotes rate enhancement primarily by compensating for the decrease in the affinity of metal ions important for catalysis engendered by the presence of the CA pair. Together, these results support and extend current models for RNase P substrate recognition in which contacts involving the conserved (+1)G/C(+72) pair of tRNA stabilize functional metal ion binding. Additionally, these observations suggest that C5 protein has evolved to compensate for tRNA variation at positions important for binding to P RNA, allowing for tRNA specialization.

Suicide survivors seeking mental health services: a preliminary examination of the role of an active postvention model.

Archival data from suicide survivors presenting for treatment from 1999-2005 at the Baton Rouge Crisis Intervention Center (BRCIC) were used to examine differences in those who received an active model of postvention (APM; n=150) compared to those who received a traditional passive postvention (PP, n=206). APM presented sooner for treatment (48 days) than PP (97 days). APM survivors were more likely to have been the survivor of a violent suicide. APM were more likely than PP to attend survivor support group meetings and APM attended more groups. To better understand the efficacy of APM in helping survivors, a prospective evaluation is needed.

Evidence that substrate-specific effects of C5 protein lead to uniformity in binding and catalysis by RNase P.

The ribonucleoprotein enzyme RNase P processes all pre-tRNAs, yet some substrates apparently lack consensus elements for recognition. Here, we compare binding affinities and cleavage rates of Escherichia coli pre-tRNAs that exhibit the largest variation from consensus recognition sequences. These results reveal that the affinities of both consensus and nonconsensus substrates for the RNase P holoenzyme are essentially uniform. Comparative analyses of pre-tRNA and tRNA binding to the RNase P holoenzyme and P RNA alone reveal differential contributions of the protein subunit to 5' leader and tRNA affinity. Additionally, these studies reveal that uniform binding results from variations in the energetic contribution of the 5' leader, which serve to compensate for weaker tRNA interactions. Furthermore, kinetic analyses reveal uniformity in the rates of substrate cleavage that result from dramatic (> 900-fold) contributions of the protein subunit to catalysis for some nonconsensus pre-tRNAs. Together, these data suggest that an important biological function of RNase P protein is to offset differences in pre-tRNA structure such that binding and catalysis are uniform.

RNA-dependent folding and stabilization of C5 protein during assembly of the E. coli RNase P holoenzyme.

The pre-tRNA processing enzyme ribonuclease P is a ribonucleoprotein. In Escherichia coli assembly of the holoenzyme involves binding of the small (119 amino acid residue) C5 protein to the much larger (377 nucleotide) P RNA subunit. The RNA subunit makes the majority of contacts to the pre-tRNA substrate and contains the active site; however, binding of C5 stabilizes P RNA folding and contributes to high affinity substrate binding. Here, we show that RNase P ribonucleoprotein assembly also influences the folding of C5 protein. Thermal melting studies demonstrate that the free protein population is a mixture of folded and unfolded conformations under conditions where it assembles quantitatively with the RNA subunit. Changes in the intrinsic fluorescence of a unique tryptophan residue located in the folded core of C5 provide further evidence for an RNA-dependent conformational change during RNase P assembly. Comparisons of the CD spectra of the free RNA and protein subunits with that of the holoenzyme provide evidence for changes in P RNA structure in the presence of C5 as indicated by previous studies. Importantly, monitoring the temperature dependence of the CD signal in regions of the holoenzyme spectra that are dominated by protein or RNA structure permitted analysis of the thermal melting of the individual subunits within the ribonucleoprotein. These analyses reveal a significantly higher Tm for C5 when bound to P RNA and show that unfolding of the protein and RNA are coupled. These data provide evidence for a general mechanism in which the favorable free energy for formation of the RNA-protein complex offsets the unfavorable free energy of structural rearrangements in the RNA and protein subunits.

An active postvention program.

The Active Postvention Model (APM) described in this article demonstrates how survivors of suicide can be an installation of hope for the newly bereaved and an effective referral resource for support in the grief process. The benefit of active postvention compared to passive postvention results in a dramatic reduction in the elapsed time between death and seeking help for the survivors who receive the active postvention. Other benefits of the APM are discussed as well.

Pre-steady-state and stopped-flow fluorescence analysis of Escherichia coli ribonuclease III: insights into mechanism and conformational changes associated with binding and catalysis.

To better understand substrate recognition and catalysis by RNase III, we examined steady-state and pre-steady-state reaction kinetics, and changes in intrinsic enzyme fluorescence. The multiple turnover cleavage of a model RNA substrate shows a pre-steady-state burst of product formation followed by a slower phase, indicating that the steady-state reaction rate is not limited by substrate cleavage. RNase III catalyzed hydrolysis is slower at low pH, permitting the use of pre-steady-state kinetics to measure the dissociation constant for formation of the enzyme-substrate complex (K(d)=5.4(+/-0.6) nM), and the rate constant for phosphodiester bond cleavage (k(c)=1.160(+/-0.001) min(-1), pH 5.4). Isotope incorporation analysis shows that a single solvent oxygen atom is incorporated into the 5' phosphate of the RNA product, which demonstrates that the cleavage step is irreversible. Analysis of the pH dependence of the single turnover rate constant, k(c), fits best to a model for two or more titratable groups with pK(a) of ca 5.6, suggesting a role for conserved acidic residues in catalysis. Additionally, we find that k(c) is dependent on the pK(a) value of the hydrated divalent metal ion included in the reaction, providing evidence for participation of a metal ion hydroxide in catalysis, potentially in developing the nucleophile for the hydrolysis reaction. In order to assess whether conformational changes also contribute to the enzyme mechanism, we monitored intrinsic tryptophan fluorescence. During a single round of binding and cleavage by the enzyme we detect a biphasic change in fluorescence. The rate of the initial increase in fluorescence was dependent on substrate concentration yielding a second-order rate constant of 1.0(+/-0.1)x10(8) M(-1) s(-1), while the rate constant of the second phase was concentration independent (6.4(+/-0.8) s(-1); pH 7.3). These data, together with the unique dependence of each phase on divalent metal ion identity and pH, support the hypothesis that the two fluorescence transitions, which we attribute to conformational changes, correlate with substrate binding and catalysis.