The Utility of Sirolimus Eluting Balloons in the Setting of Chronic Limb Threatening Ischaemia in Asian Patients from Singapore - 12 Months Results of the PRISTINE Registry.

PURPOSE: The aim of PRISTINE was to evaluate the 6 and 12 months safety and efficacy of the Selution Sustained Limus Release (SLR)™ sirolimus-coated balloon for treatment of complex lower limb occlusive lesions (TASC II C & D) in patients with chronic limb threatening ischemia (CLTI) from Singapore. METHODS: PRISTINE was a prospective, non-randomized, single arm, observational, multi-investigator, single-center clinical study. Complication-free survival at 30 days was the safety clinical endpoint. Immediate technical success (ability to cross and dilate the lesion and achieve residual angiographic stenosis < 30%), 6-month primary vessel patency, limb salvage, clinically driven target lesion revascularization (TLR) and amputation free survival (AFS) were the efficacy endpoints of interest. RESULTS: Seventy five patients were included. There were 50 (68.0%) males; mean age, 69.0 ± 10.7 years. CLTI severity was based on the Rutherford Scale (R5 = 51; R6 = 17). Significant co-morbidities included diabetes mellitus (n = 68; 91.0%) and end-stage renal failure (n = 28; 37.0%). 112 atherosclerotic lesions were treated (TASC II D = 58 (52%); 76 (67%) de novo). There was 100% technical success. Mean lesion length treated was 22.4 ± 13.9 cm. Primary vessel patencies at 6 and 12 months were 64/86 (74%) and 43/74 (58%) and freedom from clinically driven TLR were 72/86 (84%) and 55/74 (74%) respectively. AFS was 61/73 (84.0%; five deaths and seven major lower extremity amputation) at 6-months. Mean Rutherford score improved from 5.1 ± 0.55 at baseline to 1.1 ± 2.05 (p < 0.05) at one year and there was a wound healing rate of 38/48 (79%) at the same timepoint. CONCLUSIONS: The Selution SLR™ drug eluting balloon is safe and efficacious in treating highly complex infra-inguinal atherosclerotic lesions in an otherwise challenging frail population of CLTI patients with a high incidence of diabetes and end-stage renal failure. It is associated with highly satisfactory acute technical and clinical success, 12-month target lesion patency and AFS. LEVEL OF EVIDENCE: Level 2b, Individual Cohort Study.

Cell Free Expression in Proteinosomes Prepared from Native Protein-PNIPAAm Conjugates.

Towards the goal of building synthetic cells from the bottom-up, the establishment of micrometer-sized compartments that contain and support cell free transcription and translation that couple cellular structure to function is of critical importance. Proteinosomes, formed from crosslinked cationized protein-polymer conjugates offer a promising solution to membrane-bound compartmentalization with an open, semi-permeable membrane. Critically, to date, there has been no demonstration of cell free transcription and translation within water-in-water proteinosomes. Herein, a novel approach to generate proteinosomes that can support cell free transcription and translation is presented. This approach generates proteinosomes directly from native protein-polymer (BSA-PNIPAAm) conjugates. These native proteinosomes offer an excellent alternative as a synthetic cell chassis to other membrane bound compartments. Significantly, the native proteinosomes are stable under high salt conditions that enables the ability to support cell free transcription and translation and offer enhanced protein expression compared to proteinosomes prepared from traditional methodologies. Furthermore, the integration of native proteinosomes into higher order synthetic cellular architectures with membrane free compartments such as liposomes is demonstrated. The integration of bioinspired architectural elements with the central dogma is an essential building block for realizing minimal synthetic cells and is key for exploiting artificial cells in real-world applications.

Compartmentalized Cell-Free Expression Systems for Building Synthetic Cells.

One of the grand challenges in bottom-up synthetic biology is the design and construction of synthetic cellular systems. One strategy toward this goal is the systematic reconstitution of biological processes using purified or non-living molecular components to recreate specific cellular functions such as metabolism, intercellular communication, signal transduction, and growth and division. Cell-free expression systems (CFES) are in vitro reconstitutions of the transcription and translation machinery found in cells and are a key technology for bottom-up synthetic biology. The open and simplified reaction environment of CFES has helped researchers discover fundamental concepts in the molecular biology of the cell. In recent decades, there has been a drive to encapsulate CFES reactions into cell-like compartments with the aim of building synthetic cells and multicellular systems. In this chapter, we discuss recent progress in compartmentalizing CFES to build simple and minimal models of biological processes that can help provide a better understanding of the process of self-assembly in molecularly complex systems.

Ribozyme activity modulates the physical properties of RNA-peptide coacervates.

Condensed coacervate phases are now understood to be important features of modern cell biology, as well as valuable protocellular models in origin-of-life studies and synthetic biology. In each of these fields, the development of model systems with varied and tuneable material properties is of great importance for replicating properties of life. Here, we develop a ligase ribozyme system capable of concatenating short RNA fragments into long chains. Our results show that the formation of coacervate microdroplets with the ligase ribozyme and poly(L-lysine) enhances ribozyme rate and yield, which in turn increases the length of the anionic polymer component of the system and imparts specific physical properties to the droplets. Droplets containing active ribozyme sequences resist growth, do not wet or spread on unpassivated surfaces, and exhibit reduced transfer of RNA between droplets when compared to controls containing inactive sequences. These altered behaviours, which stem from RNA sequence and catalytic activity, constitute a specific phenotype and potential fitness advantage, opening the door to selection and evolution experiments based on a genotype-phenotype linkage.

Statistical mechanics of biomolecular condensates via cavity methods.

Physical mechanisms of phase separation in living systems play key physiological roles and have recently been the focus of intensive studies. The strongly heterogeneous nature of such phenomena poses difficult modeling challenges that require going beyond mean-field approaches based on postulating a free energy landscape. The pathway we take here is to calculate the partition function starting from microscopic interactions by means of cavity methods, based on a tree approximation for the interaction graph. We illustrate them on the binary case and then apply them successfully to ternary systems, in which simpler one-factor approximations are proved inadequate. We demonstrate the agreement with lattice simulations and contrast our theory with coacervation experiments of associative de-mixing of nucleotides and poly-lysine. Different types of evidence are provided to support cavity methods as ideal tools for modeling biomolecular condensation, giving an optimal balance between the consideration of spatial aspects and fast computational results.

Programmable synthetic cell networks regulated by tuneable reaction rates.

Coupled compartmentalised information processing and communication via molecular diffusion underpin network based population dynamics as observed in biological systems. Understanding how both compartmentalisation and communication can regulate information processes is key to rational design and control of compartmentalised reaction networks. Here, we integrate PEN DNA reactions into semi-permeable proteinosomes and characterise the effect of compartmentalisation on autocatalytic PEN DNA reactions. We observe unique behaviours in the compartmentalised systems which are not accessible under bulk conditions; for example, rates of reaction increase by an order of magnitude and reaction kinetics are more readily tuneable by enzyme concentrations in proteinosomes compared to buffer solution. We exploit these properties to regulate the reaction kinetics in two node compartmentalised reaction networks comprised of linear and autocatalytic reactions which we establish by bottom-up synthetic biology approaches.

Accessory Vein Obliteration and Balloon-Assisted Maturation for Immature Arteriovenous Fistulas for Haemodialysis: A Systematic Review and Meta-Analysis.

PURPOSE: This study aims to examine outcomes of immature arteriovenous fistula salvage using balloon angioplasty (PTA) without and with accessory vein obliteration (PTA + VO). MATERIALS AND METHODS: PubMed and Embase were accessed on 21 September 2020 to retrieve cohort studies on adult patients with end-stage renal failure (ESRF) requiring dialysis. Risk of bias was assessed using Newcastle-Ottawa Scale. Studies were pooled into PTA or PTA + VO arms, with outcomes (technical/clinical success, primary/secondary post-intervention patency until 12 months) reported as event rates with 95% confidence intervals. Random-effects model and maximum likelihood meta-regression were used for meta-analysis. RESULTS: Fourteen studies (1030 participants) were included. The between-subgroup difference in outcomes was largely non-significant (p > 0.050). CONCLUSION: The evidence does not support balloon angioplasty with concomitant accessory vein obliteration for immature fistula salvage.

Utility of sirolimus coated balloons in the peripheral vasculature - a review of the current literature.

Sirolimus-coated balloons (SCB) have demonstrated much promise as an alternative drug eluting device to the existing paclitaxel coated balloon platforms for the treatment of peripheral arterial disease (PAD). They have been well tested pre-clinically and have demonstrated anti-restenotic effects as well as clinical safety in its use for treatment of coronary artery disease. The existing approved SCBs have thus far demonstrated good short-term patency (12-months) and did not exhibit any major adverse events or device related shortcomings in its use for treatment of PAD. There are several studies ongoing which aim to further investigate the efficacy of existing SCBs and establish a direct comparison of its outcomes compared with plain balloon angioplasty. Also, SCB utility to salvage failing arteriovenous fistulas for haemodialysis patients has also been explored. We review the current progress made in the establishment of SCB in the treatment of PAD as well as highlight ongoing studies investigating the role of SCB in various settings.

Characterization of RNA content in individual phase-separated coacervate microdroplets.

Condensates formed by complex coacervation are hypothesized to have played a crucial part during the origin-of-life. In living cells, condensation organizes biomolecules into a wide range of membraneless compartments. Although RNA is a key component of biological condensates and the central component of the RNA world hypothesis, little is known about what determines RNA accumulation in condensates and to which extend single condensates differ in their RNA composition. To address this, we developed an approach to read the RNA content from single synthetic and protein-based condensates using high-throughput sequencing. We find that certain RNAs efficiently accumulate in condensates. These RNAs are strongly enriched in sequence motifs which show high sequence similarity to short interspersed elements (SINEs). We observe similar results for protein-derived condensates, demonstrating applicability across different in vitro reconstituted membraneless organelles. Thus, our results provide a new inroad to explore the RNA content of phase-separated droplets at single condensate resolution.

Working Memory Content Is Distorted by Its Use in Perceptual Comparisons.

Visual information around us is rarely static. To perform a task in such a dynamic environment, we often have to compare current visual input with our working memory (WM) representation of the immediate past. However, little is known about what happens to a WM representation when it is compared with perceptual input. To test this, we asked young adults (N = 170 total in three experiments) to compare a new visual input with a WM representation prior to reporting the WM representation. We found that the perceptual comparison biased the WM report, especially when the input was subjectively similar to the WM representation. Furthermore, using computational modeling and individual-differences analyses, we found that this similarity-induced memory bias was driven by representational integration, rather than incidental confusion, between the WM representation and subjectively similar input. Together, our findings highlight a novel source of WM distortion and suggest a general mechanism that determines how WM interacts with new visual input.

Charge-density reduction promotes ribozyme activity in RNA-peptide coacervates via RNA fluidization and magnesium partitioning.

It has long been proposed that phase-separated compartments can provide a basis for the formation of cellular precursors in prebiotic environments. However, we know very little about the properties of coacervates formed from simple peptides, their compatibility with ribozymes or their functional significance. Here we assess the conditions under which functional ribozymes form coacervates with simple peptides. We find coacervation to be most robust when transitioning from long homopeptides to shorter, more pre-biologically plausible heteropeptides. We mechanistically show that these RNA-peptide coacervates display peptide-dependent material properties and cofactor concentrations. We find that the interspacing of cationic and neutral amino acids increases RNA mobility, and we use isothermal calorimetry to reveal sequence-dependent Mg2+ partitioning, two critical factors that together enable ribozyme activity. Our results establish how peptides of limited length, homogeneity and charge density facilitate the compartmentalization of active ribozymes into non-gelating, magnesium-rich coacervates, a scenario that could be applicable to cellular precursors with peptide-dependent functional phenotypes.

Cell-Free Gene Expression Dynamics in Synthetic Cell Populations.

The ability to build synthetic cellular populations from the bottom-up provides the groundwork to realize minimal living tissues comprising single cells which can communicate and bridge scales into multicellular systems. Engineered systems made of synthetic micron-sized compartments and integrated reaction networks coupled with mathematical modeling can facilitate the design and construction of complex and multiscale chemical systems from the bottom-up. Toward this goal, we generated populations of monodisperse liposomes encapsulating cell-free expression systems (CFESs) using double-emulsion microfluidics and quantified transcription and translation dynamics within individual synthetic cells of the population using a fluorescent Broccoli RNA aptamer and mCherry protein reporter. CFE dynamics in bulk reactions were used to test different coarse-grained resource-limited gene expression models using model selection to obtain transcription and translation rate parameters by likelihood-based parameter estimation. The selected model was then applied to quantify cell-free gene expression dynamics in populations of synthetic cells. In combination, our experimental and theoretical approaches provide a statistically robust analysis of CFE dynamics in bulk and monodisperse synthetic cell populations. We demonstrate that compartmentalization of CFESs leads to different transcription and translation rates compared to bulk CFE and show that this is due to the semipermeable lipid membrane that allows the exchange of materials between the synthetic cells and the external environment.

Ultrasound-guided foam sclerotherapy as a therapeutic modality in venous ulceration.

OBJECTIVES: The objective of this systematic review and meta-analysis was to evaluate rates of ulcer healing following ultrasound-guided foam sclerotherapy (UGFS). METHODS: The MEDLINE, CENTRAL and Embase databases were used to search for relevant studies using the terms ' (sclerotherapy AND ulcer) OR (vein AND ulcer) OR (sclerotherapy AND vein)'. Heterogeneity between studies was quantified using the I2 statistic. A random effects model was used to calculate risk ratios where substantial heterogeneity was found. RESULTS: The initial search yielded 8266 articles. 8 studies were included in the qualitative synthesis and 3 in the meta-analysis. Superior complete ulcer healing rates were noted in patients treated with foam sclerotherapy versus compression therapy alone (pooled OR 6.41, 95% CI = 0.3-148.2, p = 0.246, random effects method). A marked degree of heterogeneity was observed between studies (I2 = 81%). CONCLUSION: A prospective, trial is warranted in order to determine the true merits of UGFS in the setting of venous ulceration.

Non-equilibrium conditions inside rock pores drive fission, maintenance and selection of coacervate protocells.

Key requirements for the first cells on Earth include the ability to compartmentalize and evolve. Compartmentalization spatially localizes biomolecules from a dilute pool and an evolving cell, which, as it grows and divides, permits mixing and propagation of information to daughter cells. Complex coacervate microdroplets are excellent candidates as primordial cells with the ability to partition and concentrate molecules into their core and support primitive and complex biochemical reactions. However, the evolution of coacervate protocells by fusion, growth and fission has not yet been demonstrated. In this work, a primordial environment initiated the evolution of coacervate-based protocells. Gas bubbles inside heated rock pores perturb the coacervate protocell distribution and drive the growth, fusion, division and selection of coacervate microdroplets. Our findings provide a compelling scenario for the evolution of membrane-free coacervate microdroplets on the early Earth, induced by common gas bubbles within heated rock pores.

Enhanced Ribozyme-Catalyzed Recombination and Oligonucleotide Assembly in Peptide-RNA Condensates.

The ability of RNA to catalyze RNA ligation is critical to its central role in many prebiotic model scenarios, in particular the copying of information during self-replication. Prebiotically plausible ribozymes formed from short oligonucleotides can catalyze reversible RNA cleavage and ligation reactions, but harsh conditions or unusual scenarios are often required to promote folding and drive the reaction equilibrium towards ligation. Here, we demonstrate that ribozyme activity is greatly enhanced by charge-mediated phase separation with poly-L-lysine, which shifts the reaction equilibrium from cleavage in solution to ligation in peptide-RNA coaggregates and coacervates. This compartmentalization enables robust isothermal RNA assembly over a broad range of conditions, which can be leveraged to assemble long and complex RNAs from short fragments under mild conditions in the absence of exogenous activation chemistry, bridging the gap between pools of short oligomers and functional RNAs.

Registry to investigate the efficacy and safety of the VenaBlock© VeIn SEaling system for VaRicose veins in SingApore - Six months results of the RIVIERA trial.

BACKGROUND: The Venablock© Venous Closure System (Invamed, Ankara, Turkey) is a novel cyanoacrylate-based non-thermal non-tumescent embolization device to block refluxing truncal veins for chronic venous insufficiency and varicose veins. The aim was to prospectively evaluate the safety and 6 months efficacy of Venablock© for the treatment of primary great saphenous vein (GSV) and small saphenous vein (SSV) incompetency in a multi-ethnic cohort from Singapore. METHODS: This was a single arm, single investigator prospective study of 29 patients (39 limbs, 39 truncal veins) recruited over a 5-month period (August 2019 to February 2020), who were treated with the Venablock© device at a tertiary vascular unit in Singapore. Patients with symptomatic varicose veins (C2-6) and had truncal reflux > 0.5 second on venous Duplex ultrasound were included. Follow-up occurred at 2 weeks, 3 and 6 months with dedicated quality of life questionnaires and a targeted Duplex ultrasound performed to check for continued venous occlusion. RESULT: Mean age was 61.4 (±11.0) years and mean BMI was 26.2 (±5.7) kg/m2. 11/29 (37.9%) were males. Most common CEAP class treated was 2 (12/29, 41.3%). Mean diameter of treated GSV was 5.7 (±2.0) mm, 4.8 (±1.7) mm and 4.2 (±1.3) mm for the proximal, mid and distal above knee segments respectively. Mean time from access puncture to sheath removal was 23.4 (±10.0) mins. Vein occlusion at 2 weeks, 3 and 6 months was 39/39 (100%), 39/39 (100%) and 36/37 (97.2%) respectively. 5/29 (17.2%) developed puncture site infections, of which 3/29 (7.7%) required formal surgical drainage. 3/29 (7.7%) developed phlebitis. At 6 months, revised Venous Clinical Severity Score improved from 5.2 (±3.5) to 2.1 (±2.9; p < .001); EuroQol-5 Dimension score, from 7.4 (±2.1) to 5.7 (±1.4; p < .001); Aberdeen Varicose Vein Questionnaire score, from 18.1 (±15.5) to 7.9 (±8.9; p = .007); and Chronic Venous Insufficiency Questionnaire, from 18.6 (±16.2) to 4.5 (±6.3; p < .001). CONCLUSION: Venablock© is a safe and efficacious option of treating truncal venous insufficiency in a multi-ethnic Asian cohort from Singapore in the short term. There is a significant improvement in QoL. Longer follow-up is required to assess the durability of this technique, in particular the higher puncture site infection rates observed compared to other glue-based therapies.