VPg-based bidirectional synthetic mRNA circuits enable orthogonal protein regulation for high-resolution cell separation.

Synthetic mRNA circuits commonly sense input to produce binary output signals for cell separation. Based on virus-origin cap-independent translation initiation machinery and RBP-aptamer interaction, we designed smart synthetic mRNA-based circuits that sense single input molecules to bidirectionally tune output signals in an orthogonal manner, enabling high-resolution separation of cell populations.

Multiplex miRNA reporting platform for real-time profiling of living cells.

Accurately characterizing cell types within complex cell structures provides invaluable information for comprehending the cellular status during biological processes. In this study, we have developed an miRNA-switch cocktail platform capable of reporting and tracking the activities of multiple miRNAs (microRNAs) at the single-cell level, while minimizing disruption to the cell culture. Drawing on the principles of traditional miRNA-sensing mRNA switches, our platform incorporates subcellular tags and employs intelligent engineering to segment three subcellular regions using two fluorescent proteins. These designs enable the quantification of multiple miRNAs within the same cell. Through our experiments, we have demonstrated the platform's ability to track marker miRNA levels during cell differentiation and provide spatial information of heterogeneity on outlier cells exhibiting extreme miRNA levels. Importantly, this platform offers real-time and in situ miRNA reporting, allowing for multidimensional evaluation of cell profile and paving the way for a comprehensive understanding of cellular events during biological processes.

Dual Input-Controlled Synthetic mRNA Circuit for Bidirectional Protein Expression Regulation.

Synthetic mRNA circuits manipulate cell fate by controlling output protein expression via cell-specific input molecule detection. Most current circuits either repress or enhance output production upon input binding. Such binary input-output mechanisms restrict the fine-tuning of protein expression to control complex cellular events. Here we designed mRNA circuits using enhancer/repressor modules that were independently controlled by different input molecules, resulting in bidirectional output regulation; the maximal enhancement over maximal repression was 57 fold. The circuit either enhances or represses protein production in different cells based on the difference in the expression of two microRNAs. This study examined novel bidirectional circuit designs capable of fine-tuning protein production by sensing multiple input molecules. It also broadened the scope of cell manipulation by synthetic mRNA circuits, facilitating the development of mRNA circuits for precise cell manipulation and providing cell-based solutions to biomedical problems.

Intracellular Molecules Induced Extracellular Peptide Self-Assembly for Efficient and Effective In Situ Cell Purification.

Synthetic messenger RNA (mRNA) switches are powerful tools for in situ cell purification, especially for cells derived from stem cells. However, the retention effectiveness of the target cells is limited by the leaky expression of toxic protein. The elimination efficiency of non-target cells is also constrained due to the lack of signal amplification. In this study, we designed a novel approach that uses synthetic mRNA switch to convey intracellular marker molecule information into spatially controlled extracellular toxic assembly formation. The approach bypasses the use of toxic protein to ensure high target cell recovery effectiveness. Meanwhile, the marker molecule information is amplified at multiple levels to ensure high non-target cell elimination effectiveness. Our approach can be tailored to meet various in situ cell purification needs, promising high-quality in situ cell purification for a wide range of biomedical applications.

Cytidine-containing tails robustly enhance and prolong protein production of synthetic mRNA in cell and in vivo.

Synthetic mRNAs are rising rapidly as alternative therapeutic agents for delivery of proteins. However, the practical use of synthetic mRNAs has been restricted by their low cellular stability as well as poor protein production efficiency. The key roles of poly(A) tail on mRNA biology inspire us to explore the optimization of tail sequence to overcome the aforementioned limitations. Here, the systematic substitution of non-A nucleotides in the tails revealed that cytidine-containing tails can substantially enhance the protein production rate and duration of synthetic mRNAs both in vitro and in vivo. Such C-containing tails shield synthetic mRNAs from deadenylase CCR4-NOT transcription complex, as the catalytic CNOT proteins, especially CNOT6L and CNOT7, have lower efficiency in trimming of cytidine. Consistently, these enhancement effects of C-containing tails were observed on all synthetic mRNAs tested and were independent of transfection reagents and cell types. As the C-containing tails can be used along with other mRNA enhancement technologies to synergically boost protein production, we believe that these tails can be broadly used on synthetic mRNAs to directly promote their clinical applications.

Literature review of the causes of pain following total knee replacement surgery: prosthesis, inflammation and arthrofibrosis.

Adverse knee pain occurs in 10-34% of all total knee replacements (TKR), and 20% of TKR patients experience more pain post-operatively than pre-operatively. Knee pain is amongst the top five reasons for knee replacement revision in the United Kingdom. The number of TKRs is predicted to continue increasing due to the ageing population.A narrative literature review was performed on the different causes of pain following TKR. A database search on Scopus, PubMed, and Google Scholar was conducted to look for articles related to TKR, pain, and cause. Articles were selected based on relevance, publication date, quality of research and validation. Relevant sections were added to the review.One hundred and fourteen articles were identified and potential causes of TKR pain included: arthrofibrosis, aseptic loosening, avascular necrosis, central sensitization, component malpositioning, infection, instability, nerve damage, overstuffing, patellar maltracking, polyethylene wear, psychological factors and unresurfaced patella.It is important to tailor our approach to address the individual causes of pain. Certain controllable risk factors can be managed pre-operatively to minimize post-operative pain. Risk factors help to predict adverse pain outcomes and identify specific causes.There are multiple causes of pain following TKR. Some factors will require further extensive studies, and as pain is a commonly attributed reason for TKR revision, its underlying aetiologies should be explored. Understanding these factors helps to develop effective methods for diagnosis, prevention and management of TKR pain, which help to improve patient outcomes. Cite this article: EFORT Open Rev 2020;5:534-543. DOI: 10.1302/2058-5241.5.200031.

Quantification of intra-articular fibrosis in patients with stiff knee arthroplasties using metal-reduction MRI.

AIMS: Stiffness is a common complication after total knee arthroplasty (TKA). Pathogenesis is not understood, treatment options are limited, and diagnosis is challenging. The aim of this study was to investigate if MRI can be used to visualize intra-articular scarring in patients with stiff, painful knee arthroplasties. METHODS: Well-functioning primary TKAs (n = 11), failed non-fibrotic TKAs (n = 5), and patients with a clinical diagnosis of fibrosis1 (n = 8) underwent an MRI scan with advanced metal suppression (Slice Encoding for Metal Artefact Correction, SEMAC) with gadolinium contrast. Fibrotic tissue (low intensity on T1 and T2, low-moderate post-contrast enhancement) was quantified (presence and tissue thickness) in six compartments: supra/infrapatella, medial/lateral gutters, and posterior medial/lateral. RESULTS: Fibrotic tissue was identified in all patients studied. However, tissue was significantly thicker in fibrotic patients (4.4 mm ± 0.2 mm) versus non-fibrotic (2.5 mm ± 0.4 mm) and normal TKAs (1.9 mm ± 0.2 mm, p = < 0.05). Significant (> 4 mm thick) tissue was seen in 26/48 (54%) of compartments examined in the fibrotic group, compared with 17/30 (57%) non-fibrotic, and 10/66 (15%) normal TKAs. Although revision surgery did improve range of movement (ROM) in all fibrotic patients, clinically significant restriction remained post-surgery. CONCLUSION: Stiff TKAs contain intra-articular fibrotic tissue that is identifiable by MRI. Studies should evaluate whether MRI is useful for surgical planning of debridement, and as a non-invasive measurement tool following interventions for stiffness caused by fibrosis. Revision for stiffness can improve ROM, but outcomes are sub-optimal and new treatments are required. Cite this article: Bone Joint J 2020;102-B(10):1331-1340.

The use of 5% lidocaine medicated plaster for acute postoperative pain after gynecological surgery: A pilot randomized controlled feasibility trial.

OBJECTIVES: To examine the feasibility and potential efficacy of 5% lidocaine medicated plaster for acute postoperative pain in a parallel, blinded, randomized controlled pilot trial. METHODS: Twenty-eight women undergoing elective gynecological surgery with midline incisions were randomly allocated 5% lidocaine medicated patch (Lignopad) or placebo plasters. Postoperative pain at rest and on movement at 24 hours were the primary study endpoints, with secondary endpoints of postoperative pain within the first 48 hours, cumulative morphine consumption (mg), predicted peak flow rate (PFR) (%) and adverse effects. We assessed pain scores at rest and on movement using the visual analogue scale (0-100). RESULTS: The lidocaine patch group had lower postoperative pain scores at rest at 24 hours (mean difference [MD] -15.1, 95% confidence interval [95% CI] -28.3 to -2.0; P = .024) but not on movement at 24 hours (MD -6.4, 95% CI -22.7 to 9.9; P = .445). Compared to placebo, lidocaine may slightly lower cumulative morphine consumption (mg) over time (MD -3.4, 95% CI -6.9 to 0.2; group*time interaction P = .065). The difference in improvement in the PFR over time after surgery between groups appeared small (group*time P = .0980). No adverse effects occurred. CONCLUSIONS: Lidocaine patch may provide a clinically important reduction in postoperative pain intensity. A larger trial to confirm the efficacy and safety of lidocaine patch is feasible after modifying the inclusion criteria and collecting patient-centered outcomes, such as quality of recovery and patient satisfaction.

A hospital clinic-based survey on traditional Chinese medicine usage among chronic hepatitis B patients.

OBJECTIVES: To survey the pattern of traditional Chinese medicine usage among chronic hepatitis B patients. DESIGN: Self-administered questionnaire survey. SETTING: Hepatitis clinic at a university hospital in Hong Kong. MAIN OUTCOME MEASURES: Proportion of chronic hepatitis B patients who have ever used traditional Chinese medicine for the treatment of chronic hepatitis B and factors associated with the use. RESULTS: Three hundred and sixty-two patients completed the survey (response rate 93%). One hundred and sixteen (32%) patients reported history of traditional Chinese medicine usage. One hundred and five (91%) patients felt that Chinese medicine had few or no side effects. Most (81%) patients did not inform their physicians on Chinese medicine usage. On multivariate analysis, recent travel to Mainland China, perceived active hepatitis and family members with chronic hepatitis B were independent factors associated with the use of Chinese medicine. CONCLUSIONS: Chronic hepatitis B patients commonly use traditional Chinese medicine. As patients seldom inform the physicians about the use of Chinese medicine, doctors should explicitly enquire about this because of potential therapeutic implications.