Additional file 2 of Dual-screw versus single-screw cephalomedullary nails for intertrochanteric femoral fractures: a systematic review and meta-analysis

Additional file 2. PRISMA 2020 for Abstract Checklist

Translation Enhancing ACA Motifs and Their Silencing by a Bacterial Small Regulatory RNA

<div><p>GcvB is an archetypal multi-target small RNA regulator of genes involved in amino acid uptake or metabolism in enteric bacteria. Included in the GcvB regulon is the <i>yifK</i> locus, encoding a conserved putative amino acid transporter. GcvB inhibits <i>yifK</i> mRNA translation by pairing with a sequence immediately upstream from the Shine-Dalgarno motif. Surprisingly, we found that some target sequence mutations that disrupt pairing, and thus were expected to relieve repression, actually lower <i>yifK</i> expression and cause it not to respond to GcvB variants carrying the corresponding compensatory changes. Work prompted by these observations revealed that the GcvB target sequence in <i>yifK</i> mRNA includes elements that stimulate translation initiation. Replacing each base of an ACA trinucleotide near the center of the target sequence, by any other base, caused <i>yifK</i> expression to decrease. Effects were additive, with some triple replacements causing up to a 90% reduction. The enhancer activity did not require the ACA motif to be strictly positioned relative to the Shine-Dalgarno sequence, nor did it depend on a particular spacing between the latter and the initiating AUG. The <i>dppA</i> mRNA, another GcvB target, contains four ACA motifs at the target site. Quite strikingly, replacement of all four ACAs by random trinucleotide sequences yielded variants showing over 100-fold reduction in expression, virtually inactivating the gene. Altogether, these data identify the ACA motif as a translation-enhancing module and show that GcvB's ability to antagonize the enhancer function in target mRNAs is quintessential to the regulatory effectiveness of this sRNA.</p></div

Additional file 4 of Dual-screw versus single-screw cephalomedullary nails for intertrochanteric femoral fractures: a systematic review and meta-analysis

Additional file 4. Figure S1 Forest plot of meta-analysis of operative time. Figure S2 Forest plot of meta-analysis of fluoroscopy time. Figure S3 Forest plot of meta-analysis of intraoperative blood loss. Figure S4 Forest plot of meta-analysis of length of hospital stay. Figure S5 Forest plot of meta-analysis of femoral neck shortening. Figure S6 Forest plot of meta-analysis of time to full bearing. Figure S7 Forest plot of meta-analysis of 6-month Harris Hip Score. Figure S8 Forest plot of meta-analysis of Harris Hip Score at last follow-up. Figure S9 Forest plot of meta-analysis of femoral shaft fracture. Figure S10 Forest plot of meta-analysis of cut-out. Figure S11 Forest plot of meta-analysis of screw migration. Figure S12 Forest plot of meta-analysis of varus collapse. Figure S13 Forest plot of meta-analysis of non-union. Figure S14 Forest plot of meta-analysis of infection. Figure S15 Forest plot of meta-analysis of deep venous thrombosis. Figure S16 Forest plot of meta-analysis of mortality. Figure S17 Sensitivity analysis of operative time using the “Leave-one-out” method

Additional file 1 of Dual-screw versus single-screw cephalomedullary nails for intertrochanteric femoral fractures: a systematic review and meta-analysis

Additional file 1. PRISMA 2020 Checklist

Additional file 3 of Dual-screw versus single-screw cephalomedullary nails for intertrochanteric femoral fractures: a systematic review and meta-analysis

Additional file 3. Table S1 List of excluded articles and their reasons. Table S2 Outcomes reported in studies of the meta-analysis. Table S3 Risk of bias assessment of randomized controlled trials. Table S4 Quality assessment of observational studies using GRACE checklist. Table S5 Results of subgroup analyses

Toeprinting analysis of <i>yifK</i> mRNA.

<p>30S ribosomal toeprinting was carried out in the absence or in the presence of GcvB RNA or of RyhB RNA as described in the <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004026#s4" target="_blank">Materials and Methods</a>. “+” and “−” signs denote the presence of absence of indicated components. The decline and disappearance of the toeprint (shown by arrows) at increasing GcvB concentrations (50 nM, 100 nM and 500 nM), is indicative of interference with the 30S subunit binding to <i>yifK</i> mRNA. Failure of RyhB to do so at the concentration of 6.0 µM (lane “R”) confirms the specificity of the effect.</p

Randomizing ACA motifs in the ribosome binding site of <i>dppA</i> gene.

<p>A <i>dppA-lacZ</i> translational fusion was constructed by converting a KanR insertion derived from plasmid pKD13 <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004026#pgen.1004026-Datsenko1" target="_blank">[47]</a> to a <i>lac</i> fusion using plasmid pCE40 <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004026#pgen.1004026-Camacho1" target="_blank">[56]</a>. A <i>tetAR</i> insertion deleting the 15 bp ACA-encoding segment was constructed using a fragment amplified with primer pair ppN82/ppN83 (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004026#pgen.1004026.s007" target="_blank">Tables S2</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004026#pgen.1004026.s008" target="_blank">S3</a>). Next, the <i>tetAR</i> insert was replaced with a PCR-amplified fragment (reciprocal priming of ppN85/ppN86) containing a randomized sequence in the ACA-encoding portion. Tetracycline-sensitive recombinants were selected as described (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004026#pgen.1004026-Bochner1" target="_blank">[52]</a>, see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004026#s4" target="_blank">Materials and Methods</a>) and subsequently screened on MacConkey-lactose indicator plates. A number of isolates were characterized by DNA sequence analysis and ß-galactosidase assays. The activity of the wild-type strain was set to 100.</p

Northern blot analysis of <i>yifK</i> transcription.

<p>All strains used as source of RNA carried -33 the promoter “up” mutation. The RNAse E mutant carried the temperature sensitive (ts) <i>rne-3071</i> allele <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004026#pgen.1004026-FigueroaBossi2" target="_blank">[28]</a>. In experiments involving this strain, bacteria were grown at 30°C and shifted to 43°C 15 min prior to RNA extraction. RNA was separated on a 1% agarose-formaldehyde gel and probed with ³²P-labeled DNA oligonucleotides complementary to a sequence near the 5′ end of <i>yifK</i> mRNA (ppF16; probe 1 above) or to a sequence in the <i>argX-hisR</i> intercistronic region (ppH27, probe 2). The blot in A and B was initially probed with ppF16 (A), then stripped and re-probed with ppH27 (B). Probing for the SsrA RNA (pp813) served as loading control. The blot in C was probed simultaneously with ppF16 and pp813. “SD⁻” denotes a G to C change in the Shine-Dalgarno sequence (+59), which causes an about 10-fold reduction in <i>yifK</i> expression (construct n. 2 in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004026#pgen-1004026-g009" target="_blank">Figure 9</a>).</p

Effect of <i>hfq</i> and <i>gcvB</i> deletions on the expression of a <i>yifK</i>-<i>lacZ</i> fusion.

<p>Deleting <i>gcvB</i> causes <i>yifK</i> expression to increase approximately five-fold in exponential cultures (OD600≈0.4) and less than three-fold in stationary overnight cultures (OD600≈2.0). A greater increase is observed in the <i>hfq</i> deletion mutant, suggesting the involvement of a separate Hfq-dependent step in <i>yifK</i> regulation. Strains used were MA8020 (wt), MA8021 (Δ<i>hfq</i>), MA10377 (Δ<i>gcvB</i>) and MA10403 (Δ<i>hfq</i> Δ<i>gcvB</i>). All strains carry the <i>yifK</i>::MudK lac fusion. Their full genotypes are listed in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004026#pgen.1004026.s006" target="_blank">Table S1</a>.</p

Increasing the distance between the enhancer and the translation initiation region.

<p>Constructs were made as described in the legend to <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004026#pgen-1004026-g005" target="_blank">Figure 5</a> using strain MA11594 (<i>yifK</i>-<i>lacZY</i> Δ<i>gcvB</i>) as recipient and fragments amplified by reciprocal priming of oligonucleotides described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004026#pgen.1004026.s007" target="_blank">Table S2</a>. A 7-nt segment (boxed in grey), duplicating the SD sequence (boxed in green), was inserted between the ACA triplets (underlined in red) and the SD sequence (construct n. 3). A G to C change was then introduced in either copy of the SD (constructs n. 4 and 5; construct n. 2 shows the effect of this change in a strain with a single SD). The upstream ACA was converted to GGG in the constructs carrying either SD sequence mutated (constructs n. 6 and 7). The same procedure was used to replace the SD-AUG interval of <i>yifK</i> with the corresponding segment from the <i>chiP</i> gene (constructs n. 8 and 9). ß-galactosidase activity was measured as described in the legend to <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004026#pgen-1004026-g008" target="_blank">Figure 8</a>. The activity of the wild-type strain (construct n. 1) was set to 100. Standard deviations were less than 5% of the mean in all cases. The data (see also strains' phenotypes on MacConkey-lactose plates) show that the upstream ACA maintains its enhancer effect when placed further upstream from the initiation region, independent of the spacing between the SD sequence and the starting AUG.</p