Microbiome characterization by high-throughput transfer RNA sequencing and modification analysis.

Advances in high-throughput sequencing have facilitated remarkable insights into the diversity and functioning of naturally occurring microbes; however, current sequencing strategies are insufficient to reveal physiological states of microbial communities associated with protein translation dynamics. Transfer RNAs (tRNAs) are core components of protein synthesis machinery, present in all living cells, and are phylogenetically tractable, which make them ideal targets to gain physiological insights into environmental microbes. Here we report a direct sequencing approach, tRNA-seq, and a software suite, tRNA-seq-tools, to recover sequences, abundance profiles, and post-transcriptional modifications of microbial tRNA transcripts. Our analysis of cecal samples using tRNA-seq distinguishes high-fat- and low-fat-fed mice in a comparable fashion to 16S ribosomal RNA gene amplicons, and reveals taxon- and diet-dependent variations in tRNA modifications. Our results provide taxon-specific in situ insights into the dynamics of tRNA gene expression and post-transcriptional modifications within complex environmental microbiomes.

RNA modification landscape of the human mitochondrial tRNALys regulates protein synthesis.

Post-transcriptional RNA modifications play a critical role in the pathogenesis of human mitochondrial disorders, but the mechanisms by which specific modifications affect mitochondrial protein synthesis remain poorly understood. Here we used a quantitative RNA sequencing approach to investigate, at nucleotide resolution, the stoichiometry and methyl modifications of the entire mitochondrial tRNA pool, and establish the relevance to human disease. We discovered that a N1-methyladenosine (m1A) modification is missing at position 58 in the mitochondrial tRNALys of patients with the mitochondrial DNA mutation m.8344 A > G associated with MERRF (myoclonus epilepsy, ragged-red fibers). By restoring the modification on the mitochondrial tRNALys, we demonstrated the importance of the m1A58 to translation elongation and the stability of selected nascent chains. Our data indicates regulation of post-transcriptional modifications on mitochondrial tRNAs is finely tuned for the control of mitochondrial gene expression. Collectively, our findings provide novel insight into the regulation of mitochondrial tRNAs and reveal greater complexity to the molecular pathogenesis of MERRF.

Queuosine modification protects cognate tRNAs against ribonuclease cleavage.

Eukaryotic transfer RNAs (tRNA) contain on average 13 modifications that perform a wide range of roles in translation and in the generation of tRNA fragments that regulate gene expression. Queuosine (Q) modification occurs in the wobble anticodon position of tRNAs for amino acids His, Asn, Tyr, and Asp. In eukaryotes, Q modification is fully dependent on diet or on gut microbiome in multicellular organisms. Despite decades of study, cellular roles of Q modification remain to be fully elucidated. Here we show that in human cells, Q modification specifically protects its cognate tRNAHis and tRNAAsn against cleavage by ribonucleases. We generated cell lines that contain completely depleted or fully Q-modified tRNAs. Using these resources, we found that Q modification significantly reduces angiogenin cleavage of its cognate tRNAs in vitro. Q modification does not change the cellular abundance of the cognate full-length tRNAs, but alters the cellular content of their fragments in vivo in the absence and presence of stress. Our results provide a new biological aspect of Q modification and a mechanism of how Q modification alters small RNA pools in human cells.

Pseudouridines have context-dependent mutation and stop rates in high-throughput sequencing.

The abundant RNA modification pseudouridine (Ψ) has been mapped transcriptome-wide by chemically modifying pseudouridines with carbodiimide and detecting the resulting reverse transcription stops in high-throughput sequencing. However, these methods have limited sensitivity and specificity, in part due to the use of reverse transcription stops. We sought to use mutations rather than just stops in sequencing data to identify pseudouridine sites. Here, we identify reverse transcription conditions that allow read-through of carbodiimide-modified pseudouridine (CMC-Ψ), and we show that pseudouridines in carbodiimide-treated human ribosomal RNA have context-dependent mutation and stop rates in high-throughput sequencing libraries prepared under these conditions. Furthermore, accounting for the context-dependence of mutation and stop rates can enhance the detection of pseudouridine sites. Similar approaches could contribute to the sequencing-based detection of many RNA modifications.

Determination of tRNA aminoacylation levels by high-throughput sequencing.

Transfer RNA (tRNA) decodes mRNA codons when aminoacylated (charged) with an amino acid at its 3' end. Charged tRNAs turn over rapidly in cells, and variations in charged tRNA fractions are known to be a useful parameter in cellular responses to stress. tRNA charging fractions can be measured for individual tRNA species using acid denaturing gels, or comparatively at the genome level using microarrays. These hybridization-based approaches cannot be used for high resolution analysis of mammalian tRNAs due to their large sequence diversity. Here we develop a high-throughput sequencing method that enables accurate determination of charged tRNA fractions at single-base resolution (Charged DM-tRNA-seq). Our method takes advantage of the recently developed DM-tRNA-seq method, but includes additional chemical steps that specifically remove the 3'A residue in uncharged tRNA. Charging fraction is obtained by counting the fraction of A-ending reads versus A+C-ending reads for each tRNA species in the same sequencing reaction. In HEK293T cells, most cytosolic tRNAs are charged at >80% levels, whereas tRNASer and tRNAThr are charged at lower levels. These low charging levels were validated using acid denaturing gels. Our method should be widely applicable for investigations of tRNA charging as a parameter in biological regulation.

Selective Enzymatic Demethylation of N2 ,N2 -Dimethylguanosine in RNA and Its Application in High-Throughput tRNA Sequencing.

The abundant Watson-Crick face methylations in biological RNAs such as N1 -methyladenosine (m1 A), N1 -methylguanosine (m1 G), N3 -methylcytosine (m3 C), and N2 ,N2 -dimethylguanosine (m22 G) cause significant obstacles for high-throughput RNA sequencing by impairing cDNA synthesis. One strategy to overcome this obstacle is to remove the methyl group on these modified bases prior to cDNA synthesis using enzymes. The wild-type E. coli AlkB and its D135S mutant can remove most of m1 A, m1 G, m3 C modifications in transfer RNA (tRNA), but they work poorly on m22 G. Here we report the design and evaluation of a series of AlkB mutants against m22 G-containing model RNA substrates that we synthesize using an improved synthetic method. We show that the AlkB D135S/L118V mutant efficiently and selectively converts m22 G modification to N2 -methylguanosine (m2 G). We also show that this new enzyme improves the efficiency of tRNA sequencing.

ALKBH1-Mediated tRNA Demethylation Regulates Translation.

tRNA is a central component of protein synthesis and the cell signaling network. One salient feature of tRNA is its heavily modified status, which can critically impact its function. Here, we show that mammalian ALKBH1 is a tRNA demethylase. It mediates the demethylation of N1-methyladenosine (m1A) in tRNAs. The ALKBH1-catalyzed demethylation of the target tRNAs results in attenuated translation initiation and decreased usage of tRNAs in protein synthesis. This process is dynamic and responds to glucose availability to affect translation. Our results uncover reversible methylation of tRNA as a new mechanism of post-transcriptional gene expression regulation.

tRNA base methylation identification and quantification via high-throughput sequencing.

Eukaryotic transfer RNAs contain on average 14 modifications. Investigations of their biological functions require the determination of the modification sites and the dynamic variations of the modification fraction. Base methylation represents a major class of tRNA modification. Although many approaches have been used to identify tRNA base methylations, including sequencing, they are generally qualitative and do not report the information on the modification fraction. Dynamic mRNA modifications have been shown to play important biological roles; yet, the extent of tRNA modification fractions has not been reported systemically. Here we take advantage of a recently developed high-throughput sequencing method (DM-tRNA-seq) to identify and quantify tRNA base methylations located at the Watson-Crick face in HEK293T cells at single base resolution. We apply information derived from both base mutations and positional stops from sequencing using a combination of demethylase treatment and cDNA synthesis by a thermophilic reverse transcriptase to compile a quantitative "Modification Index" (MI) for six base methylations in human tRNA and rRNA. MI combines the metrics for mutational and stop components from alignment of sequencing data without demethylase treatment, and the modifications are validated in the sequencing data upon demethylase treatment. We identify many new methylation sites in both human nuclear and mitochondrial-encoded tRNAs not present in the RNA modification databases. The potentially quantitative nature of the MI values obtained from sequencing is validated by primer extension of several tRNAs. Our approach should be widely applicable to identify tRNA methylation sites, analyze comparative fractional modifications, and evaluate the modification dynamics between different samples.

Crystal structure of tris-(di-methyl-amido-κN)-bis-(di-methyl-amine-κN)-zirconium(IV) iodide.

Zirconium amides have become increasingly popular and useful due to their widespread use as precursors to other zirconium complexes and their use in the production of solid oxide fuel cells (SOFCs). Herein we report the mol-ecular structure of tris-(di-methyl-amido)-bis-(di-methyl-amine)-zirconium(IV) iodide, [Zr(C2H6N)3(C2H7N)2]I. The bond lengths and bond angles are consistent with a slightly distorted trigonal-bipyramidal coordination geometry around the metal atom. N⋯I contacts of 3.6153 (15) and 3.5922 (14) Šare consistent with the presence of N-H⋯I inter-actions. These N-H⋯I inter-actions link the complex cations and iodide anions into extended chains that propagate parallel to the a axis.

The dynamic N(1)-methyladenosine methylome in eukaryotic messenger RNA.

Gene expression can be regulated post-transcriptionally through dynamic and reversible RNA modifications. A recent noteworthy example is N(6)-methyladenosine (m(6)A), which affects messenger RNA (mRNA) localization, stability, translation and splicing. Here we report on a new mRNA modification, N(1)-methyladenosine (m(1)A), that occurs on thousands of different gene transcripts in eukaryotic cells, from yeast to mammals, at an estimated average transcript stoichiometry of 20% in humans. Employing newly developed sequencing approaches, we show that m(1)A is enriched around the start codon upstream of the first splice site: it preferentially decorates more structured regions around canonical and alternative translation initiation sites, is dynamic in response to physiological conditions, and correlates positively with protein production. These unique features are highly conserved in mouse and human cells, strongly indicating a functional role for m(1)A in promoting translation of methylated mRNA.

A dual fluorescent reporter for the investigation of methionine mistranslation in live cells.

In mammalian cells under oxidative stress, the methionyl-tRNA synthetase (MetRS) misacylates noncognate tRNAs at frequencies as high as 10% distributed among up to 28 tRNA species. Instead of being detrimental for the cell, misincorporation of methionine residues in the proteome reduces the risk of oxidative damage to proteins, which aids the oxidative stress response. tRNA microarrays have been essential for the detection of the full pattern of misacylated tRNAs, but have limited capacity to investigate the misacylation and mistranslation mechanisms in live cells. Here we develop a dual-fluorescence reporter to specifically measure methionine misincorporation at glutamic acid codons GAA and GAG via tRNA(Glu) mismethionylation in human cells. Our method relies on mutating a specific Met codon in the active site of the fluorescent protein mCherry to a Glu codon that renders mCherry nonfluorescent when translation follows the genetic code. Mistranslation utilizing mismethionylated tRNA(Glu) restores fluorescence in proportion to the amount of misacylated tRNA(Glu). This cellular approach works well for both transient transfection and established stable HEK293 lines. It is rapid, straightforward, and well suited for high-throughput activity analysis under a wide range of physiological conditions. As a proof of concept, we apply this method to characterize the effect of human tRNA(Glu) isodecoders on mistranslation and discuss the implications of our findings.

Efficient and quantitative high-throughput tRNA sequencing.

Despite its biological importance, tRNA has not been adequately sequenced by standard methods because of its abundant post-transcriptional modifications and stable structure, which interfere with cDNA synthesis. We achieved efficient and quantitative tRNA sequencing in HEK293T cells by using engineered demethylases to remove base methylations and a highly processive thermostable group II intron reverse transcriptase to overcome these obstacles. Our method, DM-tRNA-seq, should be applicable to investigations of tRNA in all organisms.

Utility of noninvasive transcutaneous measurement of postoperative hemoglobin in total joint arthroplasty patients.

This study prospectively evaluated the clinical utility of a noninvasive transcutaneous device for postoperative hemoglobin measurement in 100 total hip and knee arthroplasty patients. A protocol to measure hemoglobin noninvasively, prior to venipuncture, successfully avoided venipuncture in 73% of patients. In the remaining 27 patients, there were a total of 48 venipunctures performed during the postoperative hospitalization period due to reasons including transcutaneous hemoglobin measurement less than or equal to 9 g/dL (19), inability to obtain a transcutaneous hemoglobin measurement (8), clinical signs of anemia (3), and noncompliance with the study protocol (18). Such screening protocols may provide a convenient and cost-effective alternative to routine venipuncture for identifying patients at risk for blood transfusion after elective joint arthroplasty.

Toward molecular rotors: tetra-N-heterocyclic carbene Ag(I)-halide cubane-type clusters.

1,3-Bis(3'-butylimidazol-1'-yl)benzene diiodide (2a), 1,3-bis(3'-but-3''-enyl-imidazolium-3'-yl)benzene diiodide (2b), 1,3-bis(3'-pent-4''-enyl-imidazolium-3'-yl)benzene diiodide (2c), 1,3-bis(4'-butyl-1',2',4'-triazolium-1'-yl)benzene diiodide (2d), or 1,3-bis(4'-butyl-1',2',4'-triazolium-1'-yl)benzene dibromide (2e) was reacted with Ag2O yielding unprecedented tetra-N-heterocyclic carbene-Ag(I)-X cubane-type clusters. These were characterized by (1)H and (13)C NMR spectroscopy, ESI-TOF MS, elemental analysis, and X-ray crystallography. Results from VT (13)C NMR spectroscopy and cross-over experiments are consistent with intramolecular exchange suggesting that the Ag(I) complexes are molecular rotors.

Alternatives to a routine follow-up visit for early medical abortion.

OBJECTIVE: To evaluate the ability of women and their providers to assess abortion outcome without the routine use of ultrasonography. METHODS: This multicenter trial enrolled 4,484 women seeking medical abortion at 10 clinics in the United States. Women received the standard medical abortion care with mifepristone-misoprostol in those clinics and blinded clinical assessments before follow-up ultrasonography. Data were collected prospectively on abortion outcomes, receipt of additional treatment, and clinical, laboratory, and ultrasound assessments associated with the procedure. We constructed five model algorithms for evaluating women's postabortion status, each using a different assortment of data. Four of the algorithms (algorithms 1-4) rely on data collected by the woman and on the results of the low-sensitivity pregnancy test. Algorithm 5 relies on the woman's assessment, the results of the pregnancy test, and follow-up physician assessment (sometimes including bimanual or speculum examination). RESULTS: A total of 3,054 women received medical abortion and had adequate data for evaluation. Twenty women (0.7%) had an ongoing pregnancy; 26 (0.9%) received curettage for retained tissue, empiric treatment for possible infection, or both; and 55 (1.8%) received additional uterotonics or other medical abortion-related care. Screening algorithms including patient-observed outcomes, a low-sensitivity pregnancy test, and nonsonographic clinical evaluation were as effective as sonography in identifying women who received interventions at or after the follow-up visit. CONCLUSION: Relying on women's observations, a low-sensitivity pregnancy test, and clinical examination, women and their providers can accurately assess whether follow-up care is required after medical abortion without routine ultrasonography. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov, www.clinicaltrials.gov, NCT00120224. LEVEL OF EVIDENCE: II.

Can mifepristone medical abortion be simplified? A review of the evidence and questions for future research.

Mifepristone medical abortion has been a valuable addition to the reproductive health options of women. Aspects of its provision have however sometimes limited its accessibility and use. This article summarizes existing evidence for simplifying the provision of medical abortion and thus increasing its availability. We identify three ways through which medical abortion provision might be simplified based on existing evidence and suggest five additional simplifications that require further research to confirm their safety and efficacy.

Medication abortion employing routine sequential measurements of serum hCG and sonography only when indicated.

OBJECTIVES: This study was designed to demonstrate the safety and efficacy of providing medication abortion in a primary care site without routine use of pre- and postprocedure transvaginal sonography. METHODS: We performed a retrospective record review of 172 consecutive patients choosing medication abortion at our clinic. Our protocol used sonography only as needed for specific indications. All patients were intended to be followed up with serum human chorionic gonadotropin (hCG) testing pre- and posttreatment. RESULTS: Of the 151 patients not lost to follow-up, 96 (63%) had pretreatment sonography according to protocol or physician preference and 55 did not. Ninety-nine percent (95/96) of those receiving initial sonography had a successful, and uneventful, medication abortion treatment, while 98.2% (54/55) of those not receiving an initial sonography did so. This difference was not statistically significant (.597 by one-sided Fisher's Exact Test). All 119 of the women who did not receive postabortion sonography aborted completely. Only 4 of the 91 women who had both pre- and postprocedure hCG measurements, all of whom aborted successfully, had follow-up-to-initial hCG ratios of greater than 0.2 (20%). CONCLUSION: Using a clinical protocol that involves obtaining pre- and posttreatment serum hCG measurements, with sonograms only when indicated, has similar outcomes to a protocol that uses mandatory pre- and posttreatment sonograms.

Adolescents in Vietnam: looking beyond reproductive health.

The research that has been conducted to date on Vietnamese adolescents has focused on unprotected and unsanctioned sexual activity and its health consequences, specifically abortion and sexually transmitted diseases, especially HIV. The question we pose in this article is whether this concern is warranted. Is the population community justified in limiting research on this population to early sexual activity and HIV risk? Even if the sexual behavior of young people can be considered problematic, are there perhaps other aspects of young peoples' lives to which more attention should be devoted? The literature on adolescent sexual behavior in Vietnam is reviewed and data on premarital sex and reproductive behavior are analyzed from a 1999 survey conducted in six provinces among nearly 1,500 adolescent boys and girls aged 15-22. Descriptive data on schooling and work are included in order to put the information on sexual activity in perspective. The data analysis reveals that, at least currently, the sexual behavior of unmarried adolescents in Vietnam is not what jeopardizes their health and well-being.

The decline of female circumcision in Egypt: evidence and interpretation.

Female circumcision is widespread in Egypt. Research suggests that the practice persists because of a belief that circumcision will moderate female sexuality, that it will assure a girl's marriagability, and that it is sanctioned by Islam. Using data from a nationally representative survey of adolescents, this paper investigates the prevalence and social correlates of circumcision among girls aged 10-19, the circumstances surrounding the procedure, and the attitudes of adolescents towards it. While the vast majority of adolescents are circumcised, a life table analysis indicates that girls today are at least 10 percentage points less likely to undergo female circumcision than were their mothers. Circumcision may have begun to decline prior to the time when the current cohort of girls were at risk; however, the data hint at a temporal association between the decline and the 1994 International Conference on Population and Development (ICPD) in Cairo, a time when the campaign against circumcision gained momentum. Over half of circumcised girls reported that the procedure was performed by a physician or nurse rather than a traditional practitioner. This represents a substantial increase over rates of "medicalized" circumcision found among earlier cohorts of Egyptian women. Even among circumcised girls, support for the practice is by no means universal, with 14 percent saying they think the procedure is unnecessary and a further 28 percent expressing ambivalence. A multivariate analysis indicates that girls who have been or are currently in school, who live in urban governorates, and who are older are more likely to believe that circumcision is not obligatory. When the analysis includes boys as well as uncircumcised girls, a large gender gap emerges, with boys considerably more supportive of the practice than are their female counterparts.