Structural basis for reduced ribosomal A-site fidelity in response to P-site codon-anticodon mismatches.

Rapid and accurate translation is essential in all organisms to produce properly folded and functional proteins. mRNA codons that define the protein-coding sequences are decoded by tRNAs on the ribosome in the aminoacyl (A) binding site. The mRNA codon and the tRNA anticodon interaction is extensively monitored by the ribosome to ensure accuracy in tRNA selection. While other polymerases that synthesize DNA and RNA can correct for misincorporations, the ribosome is unable to correct mistakes. Instead, when a misincorporation occurs, the mismatched tRNA-mRNA pair moves to the peptidyl (P) site and, from this location, causes a reduction in the fidelity at the A site, triggering post-peptidyl transfer quality control. This reduced fidelity allows for additional incorrect tRNAs to be accepted and for release factor 2 (RF2) to recognize sense codons, leading to hydrolysis of the aberrant peptide. Here, we present crystal structures of the ribosome containing a tRNALys in the P site with a U•U mismatch with the mRNA codon. We find that when the mismatch occurs in the second position of the P-site codon-anticodon interaction, the first nucleotide of the A-site codon flips from the mRNA path to engage highly conserved 16S rRNA nucleotide A1493 in the decoding center. We propose that this mRNA nucleotide mispositioning leads to reduced fidelity at the A site. Further, this state may provide an opportunity for RF2 to initiate premature termination before erroneous nascent chains disrupt the cellular proteome.

Disruption of evolutionarily correlated tRNA elements impairs accurate decoding.

Bacterial transfer RNAs (tRNAs) contain evolutionarily conserved sequences and modifications that ensure uniform binding to the ribosome and optimal translational accuracy despite differences in their aminoacyl attachments and anticodon nucleotide sequences. In the tRNA anticodon stem-loop, the anticodon sequence is correlated with a base pair in the anticodon loop (nucleotides 32 and 38) to tune the binding of each tRNA to the decoding center in the ribosome. Disruption of this correlation renders the ribosome unable to distinguish correct from incorrect tRNAs. The molecular basis for how these two tRNA features combine to ensure accurate decoding is unclear. Here, we solved structures of the bacterial ribosome containing either wild-type [Formula: see text] or [Formula: see text] containing a reversed 32-38 pair on cognate and near-cognate codons. Structures of wild-type [Formula: see text] bound to the ribosome reveal 23S ribosomal RNA (rRNA) nucleotide A1913 positional changes that are dependent on whether the codon-anticodon interaction is cognate or near cognate. Further, the 32-38 pair is destabilized in the context of a near-cognate codon-anticodon pair. Reversal of the pairing in [Formula: see text] ablates A1913 movement regardless of whether the interaction is cognate or near cognate. These results demonstrate that disrupting 32-38 and anticodon sequences alters interactions with the ribosome that directly contribute to misreading.

The abundant DNA adduct N7-methyl deoxyguanosine contributes to miscoding during replication by human DNA polymerase η.

Aside from abasic sites and ribonucleotides, the DNA adduct N7-methyl deoxyguanosine (N7 -CH3 dG) is one of the most abundant lesions in mammalian DNA. Because N7 -CH3 dG is unstable, leading to deglycosylation and ring-opening, its miscoding potential is not well-understood. Here, we employed a 2'-fluoro isostere approach to synthesize an oligonucleotide containing an analog of this lesion (N7 -CH3 2'-F dG) and examined its miscoding potential with four Y-family translesion synthesis DNA polymerases (pols): human pol (hpol) η, hpol κ, and hpol ι and Dpo4 from the archaeal thermophile Sulfolobus solfataricus We found that hpol η and Dpo4 can bypass the N7 -CH3 2'-F dG adduct, albeit with some stalling, but hpol κ is strongly blocked at this lesion site, whereas hpol ι showed no distinction with the lesion and the control templates. hpol η yielded the highest level of misincorporation opposite the adduct by inserting dATP or dTTP. Moreover, hpol η did not extend well past an N7-CH3 2'-F dG:dT mispair. MS-based sequence analysis confirmed that hpol η catalyzes mainly error-free incorporation of dC, with misincorporation of dA and dG in 5-10% of products. We conclude that N7-CH3 2'-F dG and, by inference, N7-CH3 dG have miscoding and mutagenic potential. The level of misincorporation arising from this abundant adduct can be considered as potentially mutagenic as a highly miscoding but rare lesion.

Function and origin of mistranslation in distinct cellular contexts.

Mistranslation describes errors during protein synthesis that prevent the amino acid sequences specified in the genetic code from being reflected within proteins. For a long time, mistranslation has largely been considered an aberrant cellular process that cells actively avoid at all times. However, recent evidence has demonstrated that cells from all three domains of life not only tolerate certain levels and forms of mistranslation, but actively induce mistranslation under certain circumstances. To this end, dedicated biological mechanisms have recently been found to reduce translational fidelity, which indicates that mistranslation is not exclusively an erroneous process and can even benefit cells in particular cellular contexts. There currently exists a spectrum of mistranslational processes that differ not only in their origins, but also in their molecular and cellular effects. These findings suggest that the optimal degree of translational fidelity largely depends on a specific cellular context. This review aims to conceptualize the basis and functional consequence of the diverse types of mistranslation that have been described so far.

The multiple flavors of GoU pairs in RNA.

Wobble GU pairs (or GoU) occur frequently within double-stranded RNA helices interspersed within the standard G═C and A─U Watson-Crick pairs. However, other types of GoU pairs interacting on their Watson-Crick edges have been observed. The structural and functional roles of such alternative GoU pairs are surprisingly diverse and reflect the various pairings G and U can form by exploiting all the subtleties of their electronic configurations. Here, the structural characteristics of the GoU pairs are updated following the recent crystallographic structures of functional ribosomal complexes and the development in our understanding of ribosomal translation.

Limitations of pulmonary embolism ICD-10 codes in emergency department administrative data: let the buyer beware.

BACKGROUND: Administrative data is a useful tool for research and quality improvement; however, validity of research findings based on these data depends on their reliability. Diagnoses assigned by physicians are subsequently converted by nosologists to ICD-10 codes (International Statistical Classification of Diseases and Related Health Problems, 10th Revision). Several groups have reported ICD-9 coding errors in inpatient data that have implications for research, quality improvement, and policymaking, but few have assessed ICD-10 code validity in ambulatory care databases. Our objective was to evaluate pulmonary embolism (PE) ICD-10 code accuracy in our large, integrated hospital system, and the validity of using these codes for operational and health services research using ED ambulatory care databases. METHODS: Ambulatory care data for patients (age ≥ 18 years) with a PE ICD-10 code (I26.0 and I26.9) were obtained from the records of four urban EDs between July 2013 to January 2015. PE diagnoses were confirmed by reviewing medical records and imaging reports. In cases where chart diagnosis and ICD-10 code were discrepant, chart review was considered correct. Physicians' written discharge diagnoses were also searched using 'pulmonary embolism' and 'PE', and patients who were diagnosed with PE but not coded as PE were identified. Coding discrepancies were quantified and described. RESULTS: One thousand, four hundred and fifty-three ED patients had a PE ICD-10 code. Of these, 257 (17.7%) were false positive, with an incorrectly assigned PE code. Among the 257 false positives, 193 cases had ambiguous ED diagnoses such as 'rule out PE' or 'query PE', while 64 cases should have had non-PE codes. An additional 117 patients (8.90%) with a PE discharge diagnosis were incorrectly assigned a non-PE ICD-10 code (false negative group). The sensitivity of PE ICD-10 codes in this dataset was 91.1% (95%CI, 89.4-92.6) with a specificity of 99.9% (95%CI, 99.9-99.9). The positive and negative predictive values were 82.3% (95%CI, 80.3-84.2) and 99.9% (95%CI, 99.9-99.9), respectively. CONCLUSIONS: Ambulatory care data, like inpatient data, are subject to coding errors. This confirms the importance of ICD-10 code validation prior to use. The largest proportion of coding errors arises from ambiguous physician documentation; therefore, physicians and data custodians must ensure that quality improvement processes are in place to promote ICD-10 coding accuracy.

Investigating the consequences of mRNA modifications on protein synthesis using in vitro translation assays.

The ribosome translates the information stored in the genetic code into functional proteins. In this process messenger RNAs (mRNAs) serve as templates for the ribosome, ensuring that amino acids are linked together in the correct order. Chemical modifications to mRNA nucleosides have the potential to influence the rate and accuracy of protein synthesis. Here, we present an in vitro Escherichia coli translation system utilizing highly purified components to directly investigate the impact of mRNA modifications on the speed and accuracy of the ribosome. This system can be used to gain insights into how individual chemical modifications influence translation on the molecular level. While the fully reconstituted system described in this chapter requires a lengthy time investment to prepare experimental materials, it is highly verstaile and enables the systematic assessment of how single variables influence protein synthesis by the ribosome.

Animal-Encounter Fatalities, United States, 1999-2016: Cause of Death and Misreporting.

OBJECTIVES: Errors and misreporting on death certificates are common, along with potential inaccuracies in cause-of-death coding. We characterized and compared fatalities by animal-encounter mentions reported as underlying cause of death (UCD) with animal-encounter mentions reported as multiple cause of death (MCD) to determine factors associated with misreporting UCD. METHODS: We analyzed fatality data from 1999-2016 from the Centers for Disease Control and Prevention Wide-ranging ONline Data for Epidemiologic Research by UCD and MCD animal-encounter mentions (International Classification of Diseases, 10th Revision codes W53-59, X20-27 and X29, T63.0-63.6, T63.8-63.9, and T78.2-78.4). We examined differences in reporting by age, sex, race, autopsy (yes, no, unknown), allergic reactions, and toxicities. RESULTS: The number of animal-encounter mentions by UCD was 3638 (202 average per year) and by MCD was 4280 (238 average per year), a difference of 18% (n = 642; 36 average per year) by MCD analysis. The number of nonvenomous animal-encounter mentions increased 20% (from 2138 UCD to 2567 MCD), and the number of venomous animal-encounter mentions increased 14% (from 1500 UCD to 1713 MCD). Decedents aged ≥65 had the highest additional number of animal-encounter mentions among all age groups, primarily encounters with other reptiles (n = 113), other mammals (n = 71), and dogs (n = 42). Of 642 MCD additional animal-encounter mentions, heart disease (n = 211, 33%) and infections (n = 146, 23%) represented more than half of the UCD. Of 553 dog-encounter fatalities, 165 (30%) were among children aged ≤4. CONCLUSIONS: Animal-encounter fatalities, analyzed by UCD alone, may be underreported. An initiating animal injury, complicated by comorbidities and fatality, may obscure the causal chain, resulting in misreporting UCD. Ongoing training for medical certifiers is recommended, highlighting accurate identification of UCD and contributing causes in the causal chain of death.

A Population-Based Study on Myelodysplastic Syndromes in the Lazio Region (Italy), Medical Miscoding and 11-Year Mortality Follow-Up: the Gruppo Romano-Laziale Mielodisplasie Experience of Retrospective Multicentric Registry.

Data on Myelodysplastic Syndromes (MDS) are difficult to collect by cancer registries because of the lack of reporting and the use of different classifications of the disease. In the Lazio Region, data from patients with a confirmed diagnosis of MDS, treated by a hematology center, have been collected since 2002 by the Gruppo Romano-Laziale Mielodisplasie (GROM-L) registry, the second MDS registry existing in Italy. This study aimed at evaluating MDS medical miscoding during hospitalizations, and patients' survival. For these purposes, we selected 644 MDS patients enrolled in the GROM-L registry. This cohort was linked with two regional health information systems: the Hospital Information System (HIS) and the Mortality Information System (MIS) in the 2002-2012 period. Of the 442 patients who were hospitalized at least once during the study period, 92% had up to 12 hospitalizations. 28.5% of patients had no hospitalization episodes scored like MDS, code 238.7 of the International Classification of Disease, Ninth Revision, Clinical Modification (ICD-9-CM). The rate of death during a median follow-up of 46 months (range 0.9-130) was 45.5%. Acute myeloid leukemia (AML) was the first cause of mortality, interestingly a relevant portion of deaths is due to cerebro-cardiovascular events and second tumors. This study highlights that MDS diagnosis and treatment, which require considerable healthcare resources, tend to be under-documented in the HIS archive. Thus we need to improve the HIS to better identify information on MDS hospitalizations and outcome. Moreover, we underline the importance of comorbidity in MDS patients' survival.

Alterations in ribosomal protein L19 that decrease the fidelity of translation.

Ribosomal protein L19 is an essential ribosomal protein and is a component of bridge B8, one of the protein-RNA bridges linking the large and small ribosomal subunits. Bridge B8 also contributes to the accuracy of translation by affecting GTPase activation by ribosome-bound aminoacyl tRNA-EF-Tu•GTP ternary complexes. Previous work has identified a limited number of accuracy-altering alterations in protein L19 of Salmonella enterica and Thermus thermophilus. Here, we have targeted the Escherichia coli rplS gene encoding L19 for mutagenesis and have screened for mutants with altered levels of miscoding. We have recovered 14 distinct L19 mutants, all of which promote increased stop codon readthrough, but do not have major effects on subunit association or cell growth. Examination of the E. coli 70S ribosome structure indicates that the amino acid substitutions cluster in three distinct regions of L19 and thereby potentially affect its interactions with L14 and 16S rRNA.

Testing quality of a self-monitoring blood glucose sensor with an auto-coding mechanism when used by patients versus technicians.

BACKGROUND: In response to the problem of erroneous readings due to miscoding when performing self-monitoring blood glucose (SMBG), this study introduces a user-friendly SMBG biosensor with an innovative auto-coding module on the meter and strip. Actual users characterized the performance of the SMBG systems. METHODS: A total of 105 patients were incorporated in the study and Clarke error grid analysis (EGA) was administered to evaluate the clinical accuracy of the results obtained by the patients versus the technicians. All patients used the questionnaires to comment on the use of the auto-coding sensor. RESULTS: In the imprecision test, the total CV of the 5 BG levels was 2.1%. In the EGA plot, the results of the auto-coding sensor were 96.2%, both lots A and B, in zone A for the patients and 99.0% and 97.1% for the technician. The paired t-test demonstrated no statistically significant difference between the patient and technician measurements. Regression analysis also demonstrated that the measurements taken by the patients agreed with those obtained using the laboratory method. CONCLUSIONS: The patients achieved satisfactory performance using the auto-coding SMBG sensor and derived similar results with both laboratory reference and operation by a technician.