Cost-effectiveness of immediate versus delayed sequential bilateral cataract surgery in the Netherlands (the BICAT-NL study): study design of a prospective multicenter randomised controlled trial.

BACKGROUND: Cataract surgery is one of the most frequently performed types of surgery. Most patients suffer from bilateral cataract and while cataract surgery of only one eye is effective in restoring functional vision, second-eye surgery leads to further improvements in health-related quality of life, and is cost-effective. At present, most patients undergo cataract surgery in both eyes on separate days as recommended in national guidelines, referred to as delayed sequential bilateral cataract surgery (DSBCS). An alternative procedure involves operating both eyes on the same day, but as separate procedures, known as immediately sequential bilateral cataract surgery (ISBCS). The aim of this study is to evaluate the effectiveness and costs of ISBCS compared to DSBCS, in order to test the hypothesis that ISBCS is non-inferior to DSBCS in terms of effectiveness and superior to ISBCS in terms of cost-effectiveness. METHODS/DESIGN: Multicenter non-inferiority randomised controlled clinical trial. Patients (18 years or older) with bilateral cataract and an indication for bilateral cataract surgery with an expected uncomplicated intraoperative and postoperative course are included in the study. Patients are randomly assigned to either ISBCS or DSBCS. The primary endpoint is the proportion of patients with a refractive outcome in the second eye within 1.0 dioptre from the target refraction, at 4 weeks after surgery. Secondary outcomes include corrected and uncorrected distance visual acuity, complications, patient reported outcomes (PROMs), cost-effectiveness, and budget impact. Follow-up visits are planned at 1 week after first-eye surgery and 4 weeks after second-eye surgery. At 3 months after first-eye surgery, the occurrence of complications is checked and patients fill in a final questionnaire. DISCUSSION: This study protocol describes the design of a multicenter non-inferiority randomised controlled trial. Current studies on ISBCS often lack information on safety regarding refractive outcomes. In addition, there is a lack of well-designed cost-effectiveness studies using established methods. The BICAT-NL study will provide more insight in refractive and cost-effectiveness outcomes for ISBCS compared to DSBCS. TRIAL REGISTRATION: This study was prospectively registered at Clinicaltrials.gov on January 17th 2018. (Identifier: NCT03400124 .

Cold-temperature induction of Escherichia coli polynucleotide phosphorylase occurs by reversal of its autoregulation.

When Escherichia coli cells are shifted to low temperatures (e.g. 15 degrees C), growth halts while the 'cold shock response' (CSR) genes are induced, after which growth resumes. One CSR gene, pnp, encodes polynucleotide phosphorylase (PNPase), a 3'-exoribonuclease and component of the RNA degradosome. At 37 degrees C, ribonuclease III (RNase III, encoded by rnc) cleaves the pnp untranslated leader, whereupon PNPase represses its own translation by an unknown mechanism. Here, we show that PNPase cold-temperature induction involves several post-transcriptional events, all of which require the intact pnp mRNA leader. The bulk of induction results from reversal of autoregulation at a step subsequent to RNase III cleavage of the pnp leader. We also found that pnp translation occurs throughout cold-temperature adaptation, whereas lacZ(+) translation was delayed. This difference is striking, as both mRNAs are greatly stabilized upon the shift to 15 degrees C. However, unlike the lacZ(+) mRNA, which remains stable during adaptation, pnp mRNA decay accelerates. Together with other evidence, these results suggest that mRNA is generally stabilized upon a shift to cold temperatures, but that a CSR mRNA-specific decay process is initiated during adaptation.

The widely conserved Era G-protein contains an RNA-binding domain required for Era function in vivo.

Era is a small G-protein widely conserved in eubacteria and eukaryotes. Although essential for bacterial growth and implicated in diverse cellular processes, its actual function remains unclear. Several lines of evidence suggest that Era may be involved in some aspect of RNA biology. The GTPase domain contains features in common with all G-proteins and is required for Era function in vivo. The C-terminal domain (EraCTD) bears scant similarity to proteins outside the Era subfamily. On the basis of sequence comparisons, we argue that the EraCTD is similar to, but distinct from, the KH RNA-binding domain. Although both contain the consensus VIGxxGxxI RNA-binding motif, the protein folds are probably different. We show that bacterial Era binds RNA in vitro and can form higher-order RNA-protein complexes. Mutations in the VIGxxGxxI motif and other conserved residues of the Escherichia coli EraCTD decrease RNA binding in vitro and have corresponding effects on Era function in vivo, including previously described effects on cell division and chromosome partitioning. Importantly, mutations in L-66, located in the predicted switch II region of the E. coli Era GTPase domain, also perturb binding, leading us to propose that the GTPase domain regulates RNA binding in response to unknown cellular cues. The possible biological significance of Era RNA binding is discussed.

Molecular characterisation of the pifC gene encoding translation initiation factor 3, which is required for normal photosynthetic complex formation in Rhodobacter sphaeroides NCIB 8253.

In order to determine whether translation initiation events play a selective role in regulating the expression of photosynthetic complexes in the photosynthetic bacterium Rhodobacter sphaeroides, we have undertaken an initial study to investigate the potential role of translation initiation factor IF3, which also behaves as a pleiotropic regulatory factor in some bacteria. Following the isolation and purification of a 24-kDa IF3-like protein (PifC) from R. sphaeroides, we used nested PCR to clone and characterise the encoding gene, pifC (photosynthesis-affecting initiation factor). The 545-bp pifC encodes a protein exhibiting 60% identity (78.6% similarity) with the Escherichia coli IF3 (InfC) protein and, in common with all other IF3 genes identified to date, pifC possesses a rare initiation codon (AUA). Furthermore, in common with IF3, PifC was shown here to perform a discriminatory function towards CUG start codons, confirming its role and function as an IF3 in R. sphaeroides. Insertion of a kanamycin resistance cassette into the 5' end of pifC resulted in a viable phenotype which exhibits growth rates similar to wild type but which possesses reduced bacteriochlorophyll and photosynthetic complexes in semi-aerobic cultures. It is shown here that the mutant is still able to produce a PifC protein but that it possesses reduced IF3 activity. This may account for the viable nature of the mutant strain, and may indicate that the effect of the mutation on photosynthesis can be more severe than shown in the present study. The mechanisms by which PifC may exert its selective regulatory effect on photosynthesis expression are discussed.

Regulation of the "tetCD" genes of transposon Tn10.

In addition to the genes involved in tetracycline resistance, the loop region of the composite transposon Tn10 contains two other known genes, tetC and tetD, whose functions are unclear. Using primarily a genetic approach, we examined tetCD gene expression and regulation. The tetC gene product, TetC, is a diffusible repressor of both tetC and tetD transcription. Despite an earlier claim by others, we do not detect induction of either tetC or tetD by tetracycline (Tc) or several of its analogs. Although the 5' ends of the tetC and tetD messages overlap due to transcription from convergent promoters, we find no evidence for anti-sense RNA control. The operator for the TetC repressor has been localized. We also demonstrate that transcription from the tetD promoter probably terminates within IS10-Right and does not apparently interfere with Tn10 or IS10-Right transposition or its regulation.

Escherichia coli RNase III (rnc) autoregulation occurs independently of rnc gene translation.

Control of mRNA stability is an established means of regulating gene expression. However, the detailed mechanisms by which such control is achieved are only now emerging. In particular, there remains a question about the involvement of translation. Escherichia coli ribonuclease III (RNase III) negatively autoregulates expression of its own gene (rnc) approximately 10-fold, by cleaving the untranslated leader and initiating approximately 10-fold more rapid decay of the rnc mRNA, after which RNase III plays no further role. Here, we define the mechanism of this control further. Mutations that increase rnc gene translation abolish autoregulation by increasing the stability of the RNase III-cleaved transcript RNA approximately 10-fold, with no effect on the uncleaved species. Mutations that decrease translation destabilize the rnc mRNA in the presence or absence of RNase III. In so doing, they reveal a pathway of rnc transcript decay distinct from the RNase III-dependent pathway. Stability of a 'mini-rnc' transcript containing the rnc leader and only the first two codons of the rnc gene is unaffected by decreased translation, presumably because sequences required for this pathway were removed. Importantly, this mini-rnc transcript is regulated normally by RNase III. Moreover, rnc transcripts synthesized in vitro do not decay in cell-free extracts lacking ribosomes, unless they are first cleaved by RNase III. These two results show that RNase III cleavage can initiate rnc transcript decay independently of rnc gene translation, unambiguously establishing that control of mRNA decay need not involve changes in translation. How rnc gene translation is optimized for efficient autoregulation will also be discussed.

RNase III autoregulation: structure and function of rncO, the posttranscriptional "operator".

Expression of the Escherichia coli rnc-era-recO operon is regulated posttranscriptionally by ribonuclease III (RNase III), encoded in the rnc gene. RNase III initiates rapid decay of the rnc operon mRNA by cleaving a double-stranded region of the rnc leader. This region, termed rncO, is portable, conferring stability and RNase III regulation to heterologous RNAs. Here, we report the detailed analysis of rncO structure and function. The first 215 nt of the rnc leader are sufficient for its function. Dimethylsulfate (DMS) modification in vivo revealed distinct structural elements in this region: a 13-nt single-stranded 5' leader, followed by a 6-bp stem-loop structure (I), a larger stem-loop structure (II) containing the RNase III site, a single-stranded region containing the rnc translation initiation site, and a small stem-loop structure (III) at the 3' terminus of rncO, wholly within the rnc coding region. Genetic analysis revealed the function of these structural elements. The single-stranded leader is not required for stability or RNase III control, stem-loop II is required only for RNase III control, and both stem-loops I and III are required for stability. Stem-loop II effectively serves only as the site at which RNase III cleaves to remove stem-loop I and thereby initiates decay, after which RNase III plays no role. Mutations at the cleavage site underscore the importance of base pairing for efficient RNase III attack. When stem-loops I and II were replaced with an artificial hairpin structure, stability was restored only partially, but was restored almost fully when a single-stranded leader was also added.

Expression and regulation of the rnc and pdxJ operons of Escherichia coli.

Escherichia coli rnc-era-recO operon (rnc operon) expression is negatively autoregulated at the level of message stability by ribonuclease III (RNase III), which is encoded by the rnc gene. RNase III, a double-stranded RNA-specific endoribonuclease involved in rRNA and mRNA processing and degradation, cleaves a stemloop structure in the 5' untranslated leader, initiating rapid decay of the rnc operon mRNA. Here, we examine rnc operon expression and regulation in greater detail. Northern, primer extension, and lacZ fusion analyses show that a single promoter (rncP) specifies two principal mRNAs: the 1.9 kb rnc-era transcript and the less-abundant 3.7 kb RNA encoding rnc-era-recO and the downstream pdxJ and acpS genes. A 1.3 kb pdxJ-acpS RNA is transcribed from a promoter (pdxP) located within recO. About 70% of pdxJ transcription depends on transcription from rncP. Both promoters were characterized genetically. RNase III reduces 1.9 kb and 3.7 kb transcript levels and stability, and corresponding effects are seen with genetic fusions. These detailed studies enabled us to show that the first 378 nucleotides of the rnc transcript comprise a portable RNA stability element (rncO) that contains all of the cis-acting elements required for RNase III-initiated decay of the rnc mRNA as well as the heterologous lacZ transcript. Moreover, mutations in rncO that block RNase III cleavage also block control, showing that RNase III initiates mRNA decay by cleaving at a single site.

Escherichia coli translation initiation factor 3 discriminates the initiation codon in vivo.

In a genetic selection designed to isolate Escherichia coli mutations that increase expression of the IS 10 transposase gene (tnp), we unexpectedly obtained viable mutants defective in translation initiation factor 3 (IF3). Several lines of evidence led us to conclude that transposase expression, per se, was not increased. Rather, these mutations appear to increase expression of the tnp'-'lacZ gene fusions used in this screen, by increasing translation initiation at downstream, atypical initiation codons. To test this hypothesis we undertook a systematic analysis of start codon requirements and measured the effects of IF3 mutations on initiation from various start codons. Beginning with an efficient translation initiation site, we varied the AUG start codon to all possible codons that differed from AUG by one nucleotide. These potential start codons fall into distinct classes with regard to translation efficiency in vivo: Class I codons (AUG, GUG, and UUG) support efficient translation; Class IIA codons (CUG, AUU, AUC, AUA, and ACG) support translation at levels only 1-3% that of AUG; and Class IIB codons (AGG and AAG) permit levels of translation too low for reliable quantification, importantly, the IF3 mutations had no effect on translation from Class I codons, but they increased translation from Class II codons 3-5-fold, and this same effect was seen in other gene contexts. Therefore, IF3 is generally able to discriminate between efficient and inefficient codons in vivo, consistent with earlier in vitro observations. We discuss these observations as they relate to IF3 autoregulation and the mechanism of IF3 function.

Structure and regulation of the Salmonella typhimurium rnc-era-recO operon.

The Escherichia coli rnc-era-recO operon encodes ribonuclease III (RNase III; a dsRNA endonuclease involved in rRNA and mRNA processing and decay), Era (an essential G-protein of unknown functions and RecO (involved in the RecF homologous recombination pathway). Expression of the rnc and era genes is negatively autoregulated: RNase III cleaves the rncO 'operator' in the untranslated leader, destabilizing the operon mRNA. As part of a larger effort to understand RNase III and Era structure and function, we characterized rnc operon structure, function and regulation in the closely related bacterium Salmonella typhimurium. Construction of a S typhimurium strain conditionally defective for RNase III and Era expression showed that Era is essential for cell growth. This mutant strain also enabled selection of recombinant clones containing the intact S typhimurium rnc-era-recO operon, whose nucleotide sequence, predicted protein sequence, and predicted rncO RNA secondary structure were all highly conserved with those of E coli. Furthermore, genetic and biochemical analysis revealed that S typhimurium rnc gene expression is negatively autoregulated by a mechanism very similar or identical to that in E coli, and that the cleavage specificities of RNase IIIs.t. and RNase IIIE.c. are indistinguishable with regard to rncO cleavage and S typhimurium 23S rRNA fragmentation in vivo.

Control of translation by mRNA secondary structure: the importance of the kinetics of structure formation.

RNA secondary structure is important in a wide variety of biological processes, but relatively little is known about the pathways and kinetics of RNA folding. When the IS10 transposase (tnp) gene is transcribed from a promoter outside the element, little increase in tnp expression is observed. This protection from outside transcription (pot) occurs at the translational level, presumably resulting from mRNA secondary structure proposed to sequester the tnp ribosome-binding site. Here, we confirm the pot RNA structure and show that it blocks 30S ribosomal subunit binding in vitro. Point mutations that abolish protection in vivo map to the pot structure. Surprisingly, these pot mutations do not severely alter the pot secondary structure or increase 30S subunit binding in vitro, except in one case. Using an oligonucleotide hybridization assay, we show that most of the pot mutations slow the kinetics of pot structure formation, with little or no effect on the inhibitory function of the final structure. Moreover, a suppressor mutation reverses this effect. We propose a pathway for pot mRNA folding that is consistent with the mutations and implicates the formation of important kinetic intermediates. The significance of these observations for the RNA folding problem in general is discussed.

Decay of the IS10 antisense RNA by 3' exoribonucleases: evidence that RNase II stabilizes RNA-OUT against PNPase attack.

RNA-OUT, the 69-nucleotide antisense RNA that regulates Tn10/IS10 transposition folds into a simple stem-loop structure. The unusually high metabolic stability of RNA-OUT is dependent, in part, on the integrity of its stem-domain: mutations that disrupt stem-domain structure (Class II mutations) render RNA-OUT unstable, and restoration of structure restores stability. Indeed, there is a strong correlation between the thermodynamic and metabolic stabilities of RNA-OUT. We show here that stem-domain integrity determines RNA-OUT's resistance to 3' exoribonucleolytic attack: Class II mutations are almost completely suppressed in Escherichia coli cells lacking its principal 3' exoribonucleases, ribonuclease II (RNase II) and polynucleotide phosphorylase (PNPase). RNase II and PNPase are individually able to degrade various RNA-OUT species, albeit with different efficiencies: RNA-OUT secondary structure provides greater resistance to RNase II than to PNPase. Surprisingly, RNA-OUT is threefold more stable in wild-type cells than in cells deficient for RNase II activity, suggesting that RNase II somehow lessens PNPase attack on RNA-OUT. We discuss how this might occur. We also show that wild-type RNA-OUT stability changes only two-fold across the normal range of physiological growth temperatures (30-44 degrees C) in wild-type cells, which has important implications for IS10 biology.

Antisense RNA control in bacteria, phages, and plasmids.

Antisense RNA control is now recognized as an efficient and specific means of regulating gene expression at the posttranscriptional level. Almost all naturally occurring cases have been found in prokaryotes, often in their accessory genetic elements. Several antisense RNA systems are now well-understood, and these display a spectrum of mechanisms of action, binding pathways, and kinetics. This review summarizes antisense RNA control in prokaryotes, emphasizing the biology of the systems involved.

Chromosomal supercoiling in Escherichia coli.

The Escherichia coli chromosome is compacted into 40-50 negatively supercoiled domains. It has been proposed that these domains differ in superhelical density. Here, we present evidence that this is probably not the case. A modified Tn10 transposable element was inserted at a number of locations around the E. coli chromosome. This element, mTn10-plac-lacZ+, contains the lac operon promoter, plac, whose activity increases with increasing superhelical density, fused to a lacZ+ reporter gene. Although mTn10-plac-lacZ+ fusion expression varies as much as approximately threefold at different insertion sites, the relative levels of expression from these elements are unaffected by replacing plac with the gyrA promoter, pgyrA, which has a reciprocal response to changes in superhelical density. Importantly, topoisomerase mutations and coumermycin, which inhibits DNA gyrase activity, alter mTn10-plac-lacZ+ and mTn10-pgyrA-lacZ+ fusion expression in expected ways, showing that the elements remain responsive to supercoiling and that topoisomerase activity is required for maintaining superhelical density. Fusion expression is not affected by anaerobic growth or osmotic shock, two physiological conditions thought to alter supercoiling. The approximately threefold difference in mTn10-plac-lacZ+ and mTn10-pgyrA-lacZ+ fusion expression observed at different sites may be explained by regional differences in chromosomal copy number that arise from bidirectional replication. Together, these results strongly suggest that the E. coli chromosomal domains do not differ in functional superhelical density.

DNA from diverse sources manifests cryptic low-level transcription in Escherichia coli.

We present evidence that DNA from diverse prokaryotic and eukaryotic sources gives rise to low-level fusion expression in Escherichia coli promoter-probe vectors. This expression may be as high as approximately 10% of the E. coli lacUV5 promoter. Although expression does not correlate with the presence of obvious E. coli promoter-like sequences, it is blocked by transcriptional terminators. Furthermore, transcription across the fusion junction is detected at levels that correlate with fusion expression. We suggest that this 'low-level transcription' (LLT) results from infrequent initiation by RNA polymerase at random sites and/or weak promoters. We propose that LLT has biological significance. In some instances, it may provide an advantageous basal level of gene expression, and we suggest that this may be true for the E. coli lacY gene. In other instances, LLT may be detrimental, in which case it may be blocked by mechanisms such as RNA secondary structure or transcriptional polarity. We present evidence to show that activation of the IS10 transposase gene by LLT is blocked at the translational level.

Vectors for constructing kan gene fusions: direct selection of mutations affecting IS10 gene expression.

We describe several vectors for constructing translational fusions to the kan gene of Tn5. Fusions are constructed in vitro using multi-copy vectors containing unique cloning sites situated between upstream transcriptional terminators and a downstream kan gene lacking transcriptional and translational start signals. Multi-copy fusions can be converted to single-copy chromosomal fusions by in vivo recombination with specific phage lambda vectors and vice versa. We find that kan fusions are often more suitable than lacZ fusions for the direct selection of mutations that increase fusion expression. These vectors were developed for isolating mutations that increase IS10 transposase expression; we describe strategies used to isolate such mutations, which map to IS10 or the Escherichia coli himA, himD(hip), dam or infC genes.

The IS10 transposase mRNA is destabilized during antisense RNA control.

RNA stability is an important component of gene expression, and antisense RNAs have been proposed to alter target RNA stability. We show here that the IS10 transposase mRNA, RNA-IN, is rendered unstable during control by the IS10 antisense RNA, RNA-OUT. Destabilization requires RNA-OUT/RNA-IN pairing and ribonuclease III cleavage. Independent of such cleavage, RNA-OUT is rendered unstable through disruption of its secondary structure. Pairing has no other obvious effects on RNA-IN transcription or stability. Nevertheless, RNA-IN destabilization is not required for antisense control in vivo. In the accompanying paper [Ma,C. and Simons, R.W. (1990) EMBO J., 9, 1267-1274 we show that pairing blocks ribosome binding to RNA-IN. Were it not for control at this level, destabilization would play a more prominent role.

The IS10 antisense RNA blocks ribosome binding at the transposase translation initiation site.

Transposase (tnp) expression from insertion sequence IS10 is controlled, in part, by an antisense RNA, RNA-OUT, which pairs to the translation initiation region of the tnp mRNA, RNA-IN. Genetic experiments suggest that control occurs post-transcriptionally. Here, we present evidence that bears on the control mechanism. Specific ribosome binding at the tnp translation initiation site is demonstrated in vitro. Two mutations that alter tnp translation in vivo are shown to have corresponding effects in vitro. Most importantly, RNA-OUT/RNA-IN pairing is shown to block ribosome binding. In conjunction with the work described in the accompanying paper, we propose that inhibition of ribosome binding also occurs in vivo, and that it is sufficient to account for control. Implications for translational control in analogous systems are discussed.

Insertion sequence IS10 anti-sense pairing initiates by an interaction between the 5' end of the target RNA and a loop in the anti-sense RNA.

Transposition of insertion sequence IS10 is regulated by an anti-sense RNA which inhibits transposase expression when IS10 is present in multiple copies per cell. The anti-sense RNA (RNA-OUT) consists of a stem domain topped by a flexibly paired loop; the 5' end of the target molecule, RNA-IN, is complementary to the top of the loop, and complementarity extends for 35 base-pairs down one side of RNA-OUT. We present here genetic evidence that anti-sense pairing, both in vitro and in vivo, initiates by interaction of the 5' end of RNA-IN and the loop domain of RNA-OUT; other features of the reaction are discussed. In the context of this model, we discuss features of this anti-sense system which are important for its biological effectiveness, and suggest that IS10 provides a convenient model for design of efficient artificial anti-sense RNA molecules.

The unusual stability of the IS10 anti-sense RNA is critical for its function and is determined by the structure of its stem-domain.

IS10 transposition is regulated by an approximately 70 nt anti-sense RNA, RNA-OUT. RNA-OUT folds into a duplex 'stem-domain' topped by a loosely paired 'loop-domain'. The loop-domain is critical for RNA-RNA pairing per se; pairing initiates by interaction of the RNA-OUT loop with the 5' end of the target mRNA. We show here that RNA-OUT is unusually stable in vivo (half-life 60 min) and that this stability is conferred by specific features of the RNA-OUT stem-domain. One critical feature is stable base-pairing: mutations that disrupt stem pairing destabilize RNA-OUT in vivo and abolish anti-sense control; combinations of mutations that restore pairing also restore both stability and control. We propose that the stem renders RNA-OUT resistant to 3' exoribonucleases. Other features of the stem-domain prevent this essential duplex from being an effective substrate for double-strand nucleases: two single base mutations disrupt antisense control by making RNA-OUT susceptible to RNase III. Mutations in the loop region have little effect on RNA-OUT stability. Implications for IS10 biology and the design of efficient anti-sense RNAs are discussed.