Protecting human subjects participating in research.

OBJECTIVES: Institutions conducting research involving human subjects establish institutional review boards (IRBs) and/or human research protection programs to protect human research subjects. Our objectives were to develop performance metrics to measure human research subject protections and to assess how well IRBs and human research protection programs are protecting human research subjects. METHODS: A set of five performance metrics for measuring human research subject protections was developed and data were collected through annual audits of informed consent documents and human research protocols at 107 Department of Veterans Affairs research facilities from 2010 through 2021. RESULTS: The proposed performance metrics were: local adverse events that were serious, unanticipated, and related or probably related to research, including those that resulted in hospitalization or death; where required informed consent was not obtained; required Heath Insurance Portability and Accountability Act authorization was not obtained; non-exempt research was conducted without IRB approval; and research activities were continued during a lapse in IRB continuing reviews. Analysis of these performance metric data from 2010 through 2021 revealed that incident rates of all five performance metrics were very low; three showed a statistically significant trend of improvement ranging from 70% to 100%; and none of these five performance metrics deteriorated. CONCLUSIONS: Department of Veterans Affairs human research protection programs appeared to be effective in protecting human research subjects and showed improvement from 2010 through 2021. These proposed performance metrics will be useful in monitoring the effectiveness of human research protection programs in protecting human research subjects.

Impact of Federal Regulatory Changes on Clinical Pharmacology and Drug Development: the Common Rule and the 21st Century Cures Act.

The Federal Policy for the Protection of Human Subjects, generally referred to as the "Common Rule," is the basis for the human research protection policies of 16 signatory federal agencies and governs virtually all federally funded research involving humans. The Common Rule was originally published in 1991. It has been recognized that changes to the Common Rule are needed to accommodate changes in the research environment and advances in information technology. The Department of Health & Human Services (HHS) issued an Advance Notice of Proposed Rulemaking in the Federal Register in 2011 and a Notice of Proposed Rulemaking in 2015. The final rule was published on January 19, 2017, just prior to the change in presidential administrations. The long gestation of the new Common Rule reflects the difficulty of obtaining consensus on a number of controversial issues. HHS received more than 2100 public comments on the proposed rule. The revised rule introduces important changes that may be particularly relevant to clinical pharmacology research and drug development. These include: (1) revised informed consent requirements, (2) procedures for "broad consent" to facilitate secondary research use of identifiable private information and/or biological specimens, (3) a mandate to promote review by a single institutional review board (IRB) for oversight of federally funded domestic cooperative research involving multiple institutions, (4) expansion of the categories of exempt research, and (5) removal of the requirement for annual continuing IRB review of research in which the remaining activities are limited to data analysis or accessing clinical follow-up data. Also noteworthy are proposed revisions not included in the final rule, including one to extend the Common Rule to multicenter studies that are not federally funded and one to require informed consent for research use of de-identified biological specimens. Major changes could also be coming for approval of new drugs by the Food and Drug Administration (FDA), although it is not a signatory to the Common Rule. The 21st Century Cures Act, which became law in December 2016, enables faster drug approvals by expanding the kinds of evidence, beyond traditional clinical trials, that the FDA can consider when reviewing new drug applications. For example, the law allows greater use of surrogate markers and data from "real-world experience" to evaluate a drug's efficacy. The Cures Act requires HHS and the FDA to harmonize differences between the Common Rule and FDA regulations for protection of human subjects in research.

LabHEART: an interactive computer model of rabbit ventricular myocyte ion channels and Ca transport.

An interactive computer program, LabHEART, was developed to simulate the action potential (AP), ionic currents, and Ca handling mechanisms in a rabbit ventricular myocyte. User-oriented, its design allows switching between voltage and current clamp and easy on-line manipulation of key parameters to change the original formulation. The model reproduces normal rabbit ventricular myocyte currents, Ca transients, and APs. We also changed parameters to simulate data from heart failure (HF) myocytes, including reduced transient outward (I(to)) and inward rectifying K currents (I(K1)), enhanced Na/Ca exchange expression, and reduced sarcoplasmic reticulum Ca-ATPase function, but unaltered Ca current density. These changes caused reduced Ca transient amplitude and increased AP duration (especially at lower frequency) as observed experimentally. The model shows that the increased Na/Ca exchange current (I(NaCa)) in HF lowers the intracellular [Ca] threshold for a triggered AP from 800 to 540 nM. Similarly, the decrease in I(K1) reduces the threshold to 600 nM. Changes in I(to) have no effect. Combining enhanced Na/Ca exchange with reduced I(K1) (as in HF) lowers the threshold to trigger an AP to 380 nM. These changes reproduce experimental results in HF, where the contributions of different factors are not readily distinguishable. We conclude that the triggered APs that contribute to nonreentrant ventricular tachycardia in HF are due approximately equally (and nearly additively) to alterations in I(NaCa) and I(K1). A free copy of this software can be obtained at http://www.meddean.luc.edu/lumen/DeptWebs/physio/bers.html.

Aminoglycoside resistance with homogeneous and heterogeneous populations of antibiotic-resistant ribosomes.

Aminoglycosides bind to rRNA in the small subunit of the bacterial ribosome. Mutations in the decoding region of 16S rRNA confer resistance to specific subsets of aminoglycoside antibiotics. The two major classes of 2-deoxystreptamine aminoglycosides are the 4,5- and the 4,6-disubstituted antibiotics. Antibiotics of the 4,5-disubstituted class include neomycin, paromomycin, and ribostamycin. Gentamicins and kanamycins belong to the 4,6-disubstituted class of aminoglycosides. Structural studies indicated the potential importance of position 1406 (Escherichia coli numbering) in the binding of ring III of the 4,6-disubstituted class of aminoglycosides to 16S rRNA. We have introduced a U1406-to-A mutation in a plasmid-encoded copy of E. coli 16S rRNA which has been expressed either in a mixture with wild-type ribosomes or in a strain in which all rRNA is transcribed from the plasmid-encoded rrn operon. High-level resistance to many of the 4,6-disubstituted aminoglycosides is observed only when all the rRNA contains the U1406-to-A mutation. In contrast to the partial dominance of resistance observed with other mutations in the decoding region, there is a dominance of sensitivity with the 1406A mutation. Chemical footprinting experiments indicate that resistance arises from a reduced affinity of the antibiotic for the rRNA target. These results demonstrate that although position 1406 is an important determinant in the binding and action of the 4,6-disubstituted aminoglycosides, other rRNA mutations that perturb the binding of ring I of both classes of 2-deoxystreptamine aminoglycosides confer higher levels of resistance as well as a partial dominance of resistance.

The Mount Sinai Hospital clinicalpathological conference: a 45-year-old man with Pompe's disease and dilated cardiomyopathy.

This is an unusual case of a 45-year-old man, born in Ecuador, with evidence of profound left ventricular dysfunction, dilated cardiomyopathy and marked myocardial hypertrophy. Preceding events were advanced atrioventricular block (necessitating pacemaker implantation) and atrial flutter. The diagnosis of Pompe's disease was established by endomyocardial biopsy and appropriate staining, which indicated abnormal glycogen storage.

Solution structure of the A loop of 23S ribosomal RNA.

The A loop is an essential RNA component of the ribosome peptidyltransferase center that directly interacts with aminoacyl (A)-site tRNA. The A loop is highly conserved and contains a ubiquitous 2'-O-methyl ribose modification at position U2552. Here, we present the solution structure of a modified and unmodified A-loop RNA to define both the A-loop fold and the structural impact of the U2552 modification. Solution data reveal that the A-loop RNA has a compact structure that includes a noncanonical base pair between C2556 and U2552. NMR evidence is presented that the N3 position of C2556 has a shifted pKa and that protonation at C2556-N3 changes the C-U pair geometry. Our data indicate that U2552 methylation modifies the A-loop fold, in particular the dynamics and position of residues C2556 and U2555. We compare our structural data with the structure of the A loop observed in a recent 50S crystal structure [Ban, N., Nissen, P., Hansen, J., Moore, P. B. & Steitz, T. A. (2000) Science 289, 905--920; Nissen, P., Hansen, J., Ban, N., Moore, P. B. & Steitz, T. A. (2000) Science 289, 920--930]. The solution and crystal structures of the A loop are dramatically different, suggesting that a structural rearrangement of the A loop must occur on docking into the peptidyltransferase center. Possible roles of this docking event, the shifted pKa of C2556 and the U2552 2'-O-methylation in the mechanism of translation, are discussed.

Structure of a eukaryotic decoding region A-site RNA.

The aminoglycoside antibiotics target a region of highly conserved nucleotides in the aminoacyl-tRNA site (A site) of 16 S RNA on the 30 S subunit. The structures of a prokaryotic decoding region A-site oligonucleotide free in solution and bound to the aminoglycosides paromomycin and gentamicin C1A have been determined. Here, the structure of a eukaryotic decoding region A-site oligonucleotide has been determined using homonuclear and heteronuclear NMR spectroscopy, and compared to the unbound prokaryotic rRNA structure. The two structures are similar, with a U1406-U1495 base-pair, a C1407-G1494 Watson-Crick base-pair, and a G1408-A1493 base-pair instead of the A1408-A1493 base-pair of the prokaryotic structure. The two structures differ in the orientation of the 1408 position with respect to A1493; G1408 is rotated toward the major groove, which is the binding pocket for aminoglycosides. The structures also differ in the stacking geometry of G1494 on A1493, which could have slight long-range conformational effects.

Structural origins of aminoglycoside specificity for prokaryotic ribosomes.

Aminoglycoside antibiotics, including paromomycin, neomycin and gentamicin, target a region of highly conserved nucleotides in the decoding region aminoacyl-tRNA site (A site) of 16 S rRNA on the 30 S subunit. Change of a single nucleotide, A1408 to G, reduces the affinity of many aminoglycosides for the ribosome; G1408 distinguishes between prokaryotic and eukaryotic ribosomes. The structures of a prokaryotic decoding region A-site oligonucleotide free in solution and bound to the aminoglycosides paromomycin and gentamicin C1a were determined previously. Here, the structure of a eukaryotic decoding region A-site oligonucleotide bound to paromomycin has been determined using NMR spectroscopy and compared to the prokaryotic A-site-paromomycin structure. A conformational change in three adenosine residues of an internal loop, critical for high-affinity antibiotic binding, was observed in the prokaryotic RNA-paromomycin complex in comparison to its free form. This conformational change is not observed in the eukaryotic RNA-paromomycin complex, disrupting the binding pocket for ring I of the antibiotic. The lack of the conformational change supports footprinting and titration calorimetry data that demonstrate approximately 25-50-fold weaker binding of paromomycin to the eukaryotic decoding-site oligonucleotide. Neomycin, which is much less active against Escherichia coli ribosomes with an A1408G mutation, binds non-specifically to the oligonucleotide. These results suggest that eukaryotic ribosomal RNA has a shallow binding pocket for aminoglycosides, which accommodates only certain antibiotics.

RNAPack: an integrated NMR approach to RNA structure determination.

Over the last decade, a vast number of useful nuclear magnetic resonance (NMR) experiments have been developed and successfully employed to determine the structure and dynamics of RNA oligonucleotides. Despite this progress, high-resolution RNA structure determination by NMR spectroscopy still remains a lengthy process and requires programming and extensive calibrations to perform NMR experiments successfully. To accelerate RNA structure determination by NMR spectroscopy, we have designed and programmed a package of RNA NMR experiments, called RNAPack. The user-friendly package contains a set of semiautomated single, double, and triple resonance NMR experiments, which are fully optimized for high-resolution RNA solution structure determination on Varian NMR spectrometers. RNAPack provides an autocalibration feature that allows rapid calibration of all NMR experiments in a single step and thereby speeds up the NMR data collection and eliminates user errors. In our laboratory, we have successfully employed this technology to solve RNA solution structures of domains of the internal ribosome entry site of the genomic hepatitis C viral RNA in less than 3 months. RNAPack therefore makes NMR spectroscopy an attractive and rapid structural tool and allows integration of atomic resolution structural information into biochemical studies of large RNA systems.

Structures of two RNA domains essential for hepatitis C virus internal ribosome entry site function.

Translation of the hepatitis C virus (HCV) polyprotein is initiated at an internal ribosome entry site (IRES) element in the 5' untranslated region of HCV RNA. The HCV IRES element interacts directly with the 40S subunit, and biochemical experiments have implicated RNA elements near the AUG start codon as required for IRES-40S subunit complex formation. The data we present here show that two RNA stem loops, domains IIId and IIIe, are involved in IRES-40S subunit interaction. The structures of the two RNA domains were solved by NMR spectroscopy and reveal structural features that may explain their role in IRES function.

Approaching translation at atomic resolution.

Atomic resolution structures of 50S and 30S ribosomal particles have recently been solved by X-ray diffraction. These ribosomal structures show often unusual folds of ribosomal RNAs and proteins, and provide molecular explanations for fundamental aspects of translation. In the 50S structure, the active site for peptide bond formation was localized and found to consist of RNA. The ribosome is thus a ribozyme. In the 30S structures, tRNA binding sites were located, and molecular mechanisms for ribosomal fidelity were proposed. The 30S subunit particle has three globular domains, and relative movements of these domains may be required for translocation of the ribosome during protein synthesis. The structures are consistent with and rationalize decades of biochemical analysis of translation and usher in a molecular age in understanding the ribosome.

Interaction of translation initiation factor IF1 with the E. coli ribosomal A site.

Initiation Factor 1 (IF1) is required for the initiation of translation in Escherichia coli. However, the precise function of IF1 remains unknown. Current evidence suggests that IF1 is an RNA-binding protein that sits in the A site of the decoding region of 16 S rRNA. IF1 binding to 30 S subunits changes the reactivity of nucleotides in the A site to chemical probes. The N1 position of A1408 is enhanced, while the N1 positions of A1492 and A1493 are protected from reactivity with dimethyl sulfate (DMS). The N1-N2 positions of G530 are also protected from reactivity with kethoxal. Quantitative footprinting experiments show that the dissociation constant for IF1 binding to the 30 S subunit is 0.9 microM and that IF1 also alters the reactivity of a subset of Class III sites that are protected by tRNA, 50 S subunits, or aminoglycoside antibiotics. IF1 enhances the reactivity of the N1 position of A1413, A908, and A909 to DMS and the N1-N2 positions of G1487 to kethoxal. To characterize this RNA-protein interaction, several ribosomal mutants in the decoding region RNA were created, and IF1 binding to wild-type and mutant 30 S subunits was monitored by chemical modification and primer extension with allele-specific primers. The mutations C1407U, A1408G, A1492G, or A1493G disrupt IF1 binding to 30 S subunits, whereas the mutations G530A, U1406A, U1406G, G1491U, U1495A, U1495C, or U1495G had little effect on IF1 binding. Disruption of IF1 binding correlates with the deleterious phenotypic effects of certain mutations. IF1 binding to the A site of the 30 S subunit may modulate subunit association and the fidelity of tRNA selection in the P site through conformational changes in the 16 S rRNA.

The ribosome revealed.

Several recently reported structures reveal the details of ribosome architecture and provide new insights into the mechanism of protein synthesis.

Ca(2+) influx through Ca(2+) channels in rabbit ventricular myocytes during action potential clamp: influence of temperature.

Ca(2+) influx via Ca(2+) current (I(Ca)) during the action potential (AP) was determined at 25 degrees C and 35 degrees C in isolated rabbit ventricular myocytes using AP clamp. Contaminating currents through Na(+) and K(+) channels were eliminated by using Na(+)- and K(+)-free solutions, respectively. DIDS (0.2 mmol/L) was used to block Ca(2+)-activated chloride current (I(Cl(Ca))). When the sarcoplasmic reticulum (SR) was depleted of Ca(2+) by preexposure to 10 mmol/L caffeine, total Ca(2+) entry via I(Ca) during the AP was approximately 12 micromol/L cytosol (at both 25 degrees C and 35 degrees C). Similar Ca(2+) influx at 35 degrees C and 25 degrees C resulted from a combination of higher and faster peak I(Ca), offset by more rapid I(Ca) inactivation at 35 degrees C. During repeated AP clamps, the SR gradually fills with Ca(2+), and consequent SR Ca(2+) release accelerates I(Ca) inactivation during the AP. During APs and contractions in steady state, total Ca(2+) influx via I(Ca) was reduced by approximately 50% but was again unaltered by temperature (5.6+/-0.2 micromol/L cytosol at 25 degrees C, 6.0+/-0.2 micromol/L cytosol at 35 degrees C). Thus, SR Ca(2+) release is responsible for sufficient I(Ca) inactivation to cut total Ca(2+) influx in half. However, because of the kinetic differences in I(Ca), the amount of Ca(2+) influx during the first 10 ms, which presumably triggers SR Ca(2+) release, is much greater at 35 degrees C. I(Ca) during a first pulse, given just after the SR was emptied with caffeine, was subtracted from I(Ca) during each of 9 subsequent pulses, which loaded the SR. These difference currents reflect I(Ca) inactivation due to SR Ca(2+) release and thus indicate the time course of local [Ca(2+)] in the subsarcolemmal space near Ca(2+) channels produced by SR Ca(2+) release (eg, maximal at 20 ms after the AP activation at 35 degrees C). Furthermore, the rate of change of this difference current may reflect the rate of SR Ca(2+) release as sensed by L-type Ca(2+) channels. These results suggest that peak SR Ca(2+) release occurs within 2.5 or 5 ms of AP upstroke at 35 degrees C and 25 degrees C, respectively. I(Cl(Ca)) might also indicate local [Ca(2+)], and at 35 degrees C in the absence of DIDS (when I(Cl(Ca)) is prominent), peak I(Cl(Ca)) also occurred at a time comparable to the peak I(Ca) difference current. We conclude that SR Ca(2+) release decreases the Ca(2+) influx during the AP by approximately 50% (at both 25 degrees C and 35 degrees C) and that changes in I(Ca) (and I(Cl(Ca))), which depend on SR Ca(2+) release, provide information about local subsarcolemmal [Ca(2+)].

Recognition of the codon-anticodon helix by ribosomal RNA.

Translational fidelity is established by ribosomal recognition of the codon-anticodon interaction within the aminoacyl-transfer RNA (tRNA) site (A site) of the ribosome. Experiments are presented that reveal possible contacts between 16S ribosomal RNA and the codon-anticodon complex. N1 methylation of adenine at position 1492 (A1492) and A1493 interfered with A-site tRNA binding. Mutation of A1492 and A1493 to guanine or cytosine also impaired A-site tRNA binding. The deleterious effects of A1492G or A1493G (or both) mutations were compensated by 2'fluorine substitutions in the mRNA codon. The results suggest that the ribosome recognizes the codon-anticodon complex by adenine contacts to the messenger RNA backbone and provide a mechanism for molecular discrimination of correct versus incorrect codon-anticodon pairs.

Basis for prokaryotic specificity of action of aminoglycoside antibiotics.

The aminoglycosides, a group of structurally related antibiotics, bind to rRNA in the small subunit of the prokaryotic ribosome. Most aminoglycosides are inactive or weakly active against eukaryotic ribosomes. A major difference in the binding site for these antibiotics between prokaryotic and eukaryotic ribosomes is the identity of the nucleotide at position 1408 (Escherichia coli numbering), which is an adenosine in prokaryotic ribosomes and a guanosine in eukaryotic ribosomes. Expression in E.coli of plasmid-encoded 16S rRNA containing an A1408 to G substitution confers resistance to a subclass of the aminoglycoside antibiotics that contain a 6' amino group on ring I. Chemical footprinting experiments indicate that resistance arises from the lower affinity of the drug for the eukaryotic rRNA sequence. The 1408G ribosomes are resistant to the same subclass of aminoglycosides as previously observed both for eukaryotic ribosomes and bacterial ribosomes containing a methylation at the N1 position of A1408. The results indicate that the identity of the nucleotide at position 1408 is a major determinant of specificity of aminoglycoside action, and agree with prior structural studies of aminoglycoside-rRNA complexes.