High pain reported at 3 months post-total knee arthroplasty often persists for the next 3 years and is associated with reduced function and quality of life.

BACKGROUND AND AIMS: Five to ten percent of people having a knee arthroplasty for osteoarthritis continue to experience high pain levels more than 3 months after surgery. The primary aim of this study was to determine the relative risk (RR) of having high pain at 12 and 36 months based on the presence of high pain at 3 and 12 months, respectively. METHODS: A retrospective analysis of data from a prospective study of participants who had a total knee arthroplasty for osteoarthritis. A score of ≤14 on the Oxford Knee Pain Subscale was defined as 'High Pain', and RRs were calculated comparing those with high or low pain. RESULTS: There were 718 participants and 13.8% reported high pain at any time point, 2.5% reported high pain at all time points and 10.3% and 4.7% and 6.6% reported high pain at 3-, 12- and 36-months, respectively, post-surgery. For participants with high pain at 3 months, 33.8% had high pain at 12 months with a RR of 24.2 (95% CI 11.7-49.8, p < 0.001) and 35.1% had high pain at 36 months with a RR of 10.8 (95% CI 6.4-18.2, p < 0.001). For participants with high pain at 12 months, 67.6% had high pain at 36 months, with a RR of 19.3 (95% CI 12.2-30.4, p < 0.01). CONCLUSIONS: Although high pain rates are low overall following knee arthroplasty, once high pain is established there is an elevated RR of it persisting at 12- and 36 months post-surgery. TRIAL REGISTRATION: The data were collected in the Evidence-based Processes and Outcomes of Care (EPOC) study, ClinicalTrials.gov Identifier: NCT01899443.

Staying Active with Multimorbidity In Acute hospital settings (StAMInA) trial: protocol for a feasibility randomised controlled trial of allied health assistant mobility rehabilitation for patients with multimorbidity.

INTRODUCTION: Key to improving outcomes for patients with multimorbidity is increasing mobility through prescription of a physical activity programme, but this can be difficult to achieve in acute hospital settings. One approach that would assist physiotherapists to increase levels of physical activity is delegation of rehabilitation to allied health assistants. We aim to conduct a randomised controlled trial to determine the feasibility of an allied health assistant providing daily inpatient mobility rehabilitation for patients with multimorbidity. METHODS AND ANALYSIS: Using a parallel group randomised controlled design, participants will be allocated to allied health assistant mobility rehabilitation or physiotherapist mobility rehabilitation. Adult inpatients (n=60) in an acute hospital with a diagnosis of multimorbidity who walked independently preadmission will be included. The experimental group will receive routine mobility rehabilitation, including daily mobilisation, from an allied health assistant under the supervision of a physiotherapist. The comparison group will receive routine rehabilitation from a physiotherapist. Feasibility will be determined using the following areas of focus in Bowen's feasibility framework: Acceptability (patient satisfaction); demand (proportion of patients who participate); implementation (time allied health assistant/physiotherapist spends with participant, occasions of service); and practicality (cost, adverse events). Staff involved in the implementation of allied health assistant rehabilitation will be interviewed to explore their perspectives on feasibility. Secondary outcomes include: Physical activity (daily time spent walking); daily mobilisation (Y/N); discharge destination; hospital readmission; falls; functional activity (Modified Iowa Level of Assistance Scale); and length of stay. Descriptive statistics will be used to describe feasibility. Secondary outcomes will be compared between groups using Poisson or negative binomial regression, Cox proportional hazards regression, survival analysis, linear regression or logistic regression. ETHICS AND DISSEMINATION: Ethics approval was obtained from Peninsula Health (HREC/97 431/PH-2023). Findings will be disseminated in peer-reviewed journals and conference presentations. TRIAL REGISTRATION NUMBER: Australian and New Zealand Clinical Trial Registry ACTRN12623000584639p.

Is inpatient rehabilitation a predictor of a lower incidence of persistent knee pain 3-months following total knee replacement? A retrospective, observational study.

BACKGROUND: Moderate to severe levels of persistent knee pain have been estimated to affect up to 25% of people 3-months or more after a total knee replacement. It is unknown whether the type of rehabilitation pathway is associated with persistent high pain after surgery. Using a prospectively followed Australian cohort who underwent total knee replacement for knee osteoarthritis, this study aimed to i) report the incidence of high-intensity knee pain (defined as a score ≤ 15 on the Oxford Knee Score pain subscale) across time and ii) identify whether referral to inpatient rehabilitation was one of the predictors of persistent pain at 3-months post-surgery. METHODS: A retrospective analysis of a large prospective study was conducted using the Oxford Knee Score pain subscale to determine if participants had high pain at 3-months, 12-months and 36-months post-surgery. Relative risks for high pain at 3-, 12- and 36-months between the type of rehabilitation pathway were determined using Poisson multivariable regression with robust standard errors. The same technique was also employed to determine potential predictors, including rehabilitation pathway, of high pain at 3 months. RESULTS: The incidence of high pain in all participants was 73% pre-surgery and 10, 5 and 6% at 3-, 12- and 36-months respectively following knee replacement. There was a significant interaction between time and rehabilitation pathway, suggesting that the effect of the rehabilitation pathway varied across time. The incidence of high pain at 3-months did not significantly differ between those who attended inpatient rehabilitation (11.6%) and those discharged directly home (9.5%). Multivariable Poisson regression analysis identified the pre-surgical presence of high pain, co-morbid low back pain or other lower limb problem, younger age and having a major complication within 3-months following surgery as significant predictors of persistent pain whilst discharge to inpatient rehabilitation was not. CONCLUSION: A small but clinically significant minority of people continued to have high pain levels at 3-, 12- and 36-months following a primary total knee replacement for osteoarthritis. Participation in an inpatient rehabilitation program does not appear to be an important predictor of ongoing knee pain. TRIAL REGISTRATION: The data were collected in the Evidence-based Processes and Outcomes of Care (EPOC) study, ClinicalTrials.gov Identifier: NCT01899443.

Extensive regulation of enzyme activity by phosphorylation in Escherichia coli.

Protein serine/threonine/tyrosine (S/T/Y) phosphorylation is an essential and frequent post-translational modification in eukaryotes, but historically has been considered less prevalent in bacteria because fewer proteins were found to be phosphorylated and most proteins were modified to a lower degree. Recent proteomics studies greatly expanded the phosphoproteome of Escherichia coli to more than 2000 phosphorylation sites (phosphosites), yet mechanisms of action were proposed for only six phosphosites and fitness effects were described for 38 phosphosites upon perturbation. By systematically characterizing functional relevance of S/T/Y phosphorylation in E. coli metabolism, we found 44 of the 52 mutated phosphosites to be functional based on growth phenotypes and intracellular metabolome profiles. By effectively doubling the number of known functional phosphosites, we provide evidence that protein phosphorylation is a major regulation process in bacterial metabolism. Combining in vitro and in vivo experiments, we demonstrate how single phosphosites modulate enzymatic activity and regulate metabolic fluxes in glycolysis, methylglyoxal bypass, acetate metabolism and the split between pentose phosphate and Entner-Doudoroff pathways through mechanisms that include shielding the substrate binding site, limiting structural dynamics, and disrupting interactions relevant for activity in vivo.

Prescription Opioids Higher Among Knee Arthroplasty Recipients Randomized to Inpatient Rehabilitation.

OBJECTIVE: To determine whether the purchase of prescription opioids was lower among people randomized to inpatient rehabilitation (IR) compared with those discharged directly home following total knee arthroplasty (TKA). METHOD: A secondary analysis of a previous clinical trial in which participants were randomized 3 to 5 days after -surgery to 10 days of IR and a home program or to a home program alone. The primary outcome for this secondary analysis was the purchase of opioid-based pain relief up to 10-weeks after surgery, which was captured via patient diaries. Between-group differences were analyzed using a χ2 test and relative risk (RR) (95% confidence interval [CI]). We report this outcome alongside the main outcomes observed at 10 weeks for the original study (6-minute walk test, index joint pain, and function) for context. RESULTS: At 10 weeks, 158 participants were available for follow-up; 120 (76%) provided diaries, with 113 providing generic or brand names for the pain relief purchased. In the IR group, 60% (33/55) reported the purchase of opioid-based medications after discharge compared with 34% (20/58) in the home group (χ2 = 7.4; P = 0.007); thus, the risk of purchasing opioids for those in the IR group was almost double (RR, 1.7 [95% CI, 1.1-2.6]). No significant or meaningful between-group differences in index joint pain, function, or mobility were observed. CONCLUSION: Contrary to what was hypothesized, IR is a strong driver of opioid purchase after discharge from the hospital following TKA.

Protecting Linear DNA Templates in Cell-Free Expression Systems from Diverse Bacteria.

Recent advances in cell-free systems have opened up new capabilities in synthetic biology from rapid prototyping of genetic circuits and metabolic pathways to portable diagnostics and biomanufacturing. A current bottleneck in cell-free systems, especially those employing non-E. coli bacterial species, is the required use of plasmid DNA, which can be laborious to construct, clone, and verify. Linear DNA templates offer a faster and more direct route for many cell-free applications, but they are often rapidly degraded in cell-free reactions. In this study, we evaluated GamS from λ-phage, DNA fragments containing Chi-sites, and Ku from Mycobacterium tuberculosis for their ability to protect linear DNA templates in diverse bacterial cell-free systems. We show that these nuclease inhibitors exhibit differential protective activities against endogenous exonucleases in five different cell-free lysates, highlighting their utility for diverse bacterial species. We expect these linear DNA protection strategies will accelerate high-throughput approaches in cell-free synthetic biology.

Genome and sequence determinants governing the expression of horizontally acquired DNA in bacteria.

While horizontal gene transfer is prevalent across the biosphere, the regulatory features that enable expression and functionalization of foreign DNA remain poorly understood. Here, we combine high-throughput promoter activity measurements and large-scale genomic analysis of regulatory regions to investigate the cross-compatibility of regulatory elements (REs) in bacteria. Functional characterization of thousands of natural REs in three distinct bacterial species revealed distinct expression patterns according to RE and recipient phylogeny. Host capacity to activate foreign promoters was proportional to their genomic GC content, while many low GC regulatory elements were both broadly active and had more transcription start sites across hosts. The difference in expression capabilities could be explained by the influence of the host GC content on the stringency of the AT-rich canonical σ70 motif necessary for transcription initiation. We further confirm the generalizability of this model and find widespread GC content adaptation of the σ70 motif in a set of 1,545 genomes from all major bacterial phyla. Our analysis identifies a key mechanism by which the strength of the AT-rich σ70 motif relative to a host's genomic GC content governs the capacity for expression of acquired DNA. These findings shed light on regulatory adaptation in the context of evolving genomic composition.

Multiplex transcriptional characterizations across diverse bacterial species using cell-free systems.

Cell-free expression systems enable rapid prototyping of genetic programs in vitro. However, current throughput of cell-free measurements is limited by the use of channel-limited fluorescent readouts. Here, we describe DNA Regulatory element Analysis by cell-Free Transcription and Sequencing (DRAFTS), a rapid and robust in vitro approach for multiplexed measurement of transcriptional activities from thousands of regulatory sequences in a single reaction. We employ this method in active cell lysates developed from ten diverse bacterial species. Interspecies analysis of transcriptional profiles from > 1,000 diverse regulatory sequences reveals functional differences in promoter activity that can be quantitatively modeled, providing a rich resource for tuning gene expression in diverse bacterial species. Finally, we examine the transcriptional capacities of dual-species hybrid lysates that can simultaneously harness gene expression properties of multiple organisms. We expect that this cell-free multiplex transcriptional measurement approach will improve genetic part prototyping in new bacterial chassis for synthetic biology.

Metagenomic mining of regulatory elements enables programmable species-selective gene expression.

Robust and predictably performing synthetic circuits rely on the use of well-characterized regulatory parts across different genetic backgrounds and environmental contexts. Here we report the large-scale metagenomic mining of thousands of natural 5' regulatory sequences from diverse bacteria, and their multiplexed gene expression characterization in industrially relevant microbes. We identified sequences with broad and host-specific expression properties that are robust in various growth conditions. We also observed substantial differences between species in terms of their capacity to utilize exogenous regulatory sequences. Finally, we demonstrate programmable species-selective gene expression that produces distinct and diverse output patterns in different microbes. Together, these findings provide a rich resource of characterized natural regulatory sequences and a framework that can be used to engineer synthetic gene circuits with unique and tunable cross-species functionality and properties, and also suggest the prospect of ultimately engineering complex behaviors at the community level.

Principles for designing synthetic microbial communities.

Advances in synthetic biology to build microbes with defined and controllable properties are enabling new approaches to design and program multispecies communities. This emerging field of synthetic ecology will be important for many areas of biotechnology, bioenergy and bioremediation. This endeavor draws upon knowledge from synthetic biology, systems biology, microbial ecology and evolution. Fully realizing the potential of this discipline requires the development of new strategies to control the intercellular interactions, spatiotemporal coordination, robustness, stability and biocontainment of synthetic microbial communities. Here, we review recent experimental, analytical and computational advances to study and build multi-species microbial communities with defined functions and behavior for various applications. We also highlight outstanding challenges and future directions to advance this field.

Transfer of noncoding DNA drives regulatory rewiring in bacteria.

Understanding the mechanisms that generate variation is a common pursuit unifying the life sciences. Bacteria represent an especially striking puzzle, because closely related strains possess radically different metabolic and ecological capabilities. Differences in protein repertoire arising from gene transfer are currently considered the primary mechanism underlying phenotypic plasticity in bacteria. Although bacterial coding plasticity has been extensively studied in previous decades, little is known about the role that regulatory plasticity plays in bacterial evolution. Here, we show that bacterial genes can rapidly shift between multiple regulatory modes by acquiring functionally divergent nonhomologous promoter regions. Through analysis of 270,000 regulatory regions across 247 genomes, we demonstrate that regulatory "switching" to nonhomologous alternatives is ubiquitous, occurring across the bacterial domain. Using comparative transcriptomics, we show that at least 16% of the expression divergence between Escherichia coli strains can be explained by this regulatory switching. Further, using an oligonucleotide regulatory library, we establish that switching affects bacterial promoter architecture. We provide evidence that regulatory switching can occur through horizontal regulatory transfer, which allows regulatory regions to move across strains, and even genera, independently from the genes they regulate. Finally, by experimentally characterizing the fitness effect of a regulatory transfer on a pathogenic E. coli strain, we demonstrate that regulatory switching elicits important phenotypic consequences. Taken together, our findings expose previously unappreciated regulatory plasticity in bacteria and provide a gateway for understanding bacterial phenotypic variation and adaptation.