Background: Of the 200 million patients worldwide affected by peripheral arterial disease (PAD), 4% will inevitably require major limb amputation. Previous systematic reviews presented a conflicting ...body of evidence in terms of vascular endothelial growth factor (VEGF) family member effects upon PAD natural progression. Despite that, modulation of intrinsic angiogenesis mechanisms targeting the VEGF family members still confers an attractive therapeutic target. The aim of the present study was to evaluate current evidence of VEGF modulation in the context of PAD. Methods: This is a systematic literature review conducted according to the PRISMA guidelines and registered under PROSPERO database CRD42021285988. Independent literature search was performed up to April 1, 2022, on six databases. A total of 22 eligible studies were identified N: 3, interventional patient studies; N: 19, animal studies. Animal studies were appraised by the SYRCLE risk of bias tool, while human participant studies were assessed by the Newcastle Ottawa scale. Overall, quality of evidence was deemed fair for both animal and human studies. Main study outcomes were percentage change of injured vessel lumen stenosis and neointimal area formation upon VEGF modulation (inhibition or activation) in comparison with control group. Findings: Nineteen animal models and three human participant studies were included in the systematic review and assessed separately. Positive modulation of VEGF-A in animal models resulted in a median decrease of 65.58% 95% CI 45.2; 71.87 in lumen stenosis 14 studies. Furthermore, positive modulation of VEGF-A was found to reduce neointimal area proliferation by a median decrease of 63.41% 95% CI 41.6; 79.59 14 studies. Median end of study duration was 28 days range: 14–84 days. Data were insufficient to assess these outcomes with respect to VEGF-B or VEGF-C modulation. The limited number of available human studies presented inadequate outcome assessment despite their overall fair NOS grading. Interpretation: VEGF-A-positive modulation decreases lumen stenosis and neointimal hyperplasia in PAD simulation animal models. Previously identified variability among outcomes was found to strongly stem from the variability of experimental designs. Clinical applicability and safety profile of VEGF-A in the context of PAD remain to be defined by a robust and uniformly designed body of further animal model-based experiments.
The COVID-19 pandemic has affected the healthcare systems worldwide since the dawn of 2020. In March 2020, the United Kingdom government announced the first national lockdown which severely disturbed ...all National Health Service (NHS) healthcare elective services. Our aim is to assess the long-term impact of COVID-19 related disruption of NHS elective services on emergency major lower limb amputations (MLLAs).
Patients' data for emergency MLLA for critical limb-threatening ischemia and diabetic foot infections performed at Aberdeen Royal Infirmary was collected through Trakcare and divided into the control prepandemic group (April 2018-March 2020) and the pandemic group (April 2020-March 2022). The statistical analysis was conducted using the IBM SPSS software (v28.0.1.1 14).
A total of 358 patients underwent MLLA and 206 (57.5%) of these had diabetes mellitus. There was a 17% increase in the number of urgent referrals and every 1 in 5 of these finally underwent an amputation. There was an increase in the absolute number of Above- and Below-Knee amputations. There was a statistically significant increase by 33% in emergency MLLAs during the pandemic period (P < 0.05). A total of 165 postoperative deaths up to December 2022 were recorded with 30-day mortality rate of 7.26% (n = 26).
NHS vascular management groups should update themselves with evolving technologies to optimize the care provided during future unprecedented times. Furthermore, more effective measures should also be implemented to avoid delayed presentations, which can potentially lead to higher rates of major limb amputations.
Inferior mesenteric artery (IMA) aneurysms represent the minority of visceral aneurysm presentations. A 57-year-old female was admitted with a symptomatic IMA aneurysm secondary to atherosclerotic ...disease. She was treated with open excision which revealed a contained ruptured of a true aneurysm. This case highlights the challenges of an accurate preoperative diagnosis of IMA aneurysm and the correct position of the recent guidelines on visceral aneurysms issued by the Society of Vascular Surgery (SVS).
Purpose:
To determine the feasibility of synchronous superficial venous surgery and on-table subintimal angioplasty in the treatment of venous ulcers with arterial compromise.
Methods:
Between ...January 1992 and December 2004, 9 patients (8 women; median age 83 years, range 72–92) underwent the synchronous procedure. No patient had deep venous reflux; 4 patients presented with rest pain and 2 were diabetic. Their medical records were retrospectively reviewed to gather data on clinical assessment, ankle-brachial index (ABI), lower limb arterial and venous duplex scans, and treatment variables.
Results:
Angioplasty was technically successful (median ABI improvement 0.31, range 0.23–0.34) in all procedures, which lasted a median 85 minutes (range 60–160). The procedure was performed under general anesthesia in 7 patients, local in 1, and spinal in 1. Superficial venous surgery was performed first in 8 patients: 6 had great saphenous vein (GSV) ligation without stripping, followed by subintimal angioplasty, and 1 also had small saphenous vein (SSV) ligation. One patient underwent SSV ligation first, followed by angioplasty; another had GSV disconnection and stripping, followed by angioplasty. In 1 patient, angioplasty preceded GSV surgery.
One patient required repeat angioplasty 1 week later for re-occlusion. All ulcers healed (median 4 months, range 1–36). Five patients died during a median 32-month follow-up (range 4–82) from unrelated causes; there was 1 ulcer recurrence at 3 years in a patient who declined further investigation and treatment.
Conclusions:
Simultaneous superficial venous surgery and on-table subintimal angioplasty is a safe and novel strategy in the management of a select subgroup of patients with venous ulcers and coexistent arterial compromise.
Synthetic biology efforts for the production of valuable chemicals are frequently hindered by the structure and regulation of the native metabolic pathways of the chassis. This is particularly ...evident in the case of monoterpenoid production in Saccharomyces cerevisiae, where the canonical terpene precursor geranyl diphosphate is tightly coupled to the biosynthesis of isoprenoid compounds essential for yeast viability. Here, we establish a synthetic orthogonal monoterpenoid pathway based on an alternative precursor, neryl diphosphate. We identify structural determinants of isomeric substrate selectivity in monoterpene synthases and engineer five different enzymes to accept the alternative substrate with improved efficiency and specificity. We combine the engineered enzymes with dynamic regulation of metabolic flux to harness the potential of the orthogonal substrate and improve the production of industrially-relevant monoterpenes by several-fold compared to the canonical pathway. This approach highlights the introduction of synthetic metabolism as an effective strategy for high-value compound production.
One application of synthetic biology is the redesign of existing biological systems to acquire new functions. In this context, expanding the chemical code underlying key biosynthetic pathways will ...lead to the synthesis of compounds with new structures and potentially new biological activities. Terpenoids are a large group of specialized metabolites with numerous applications. Yet, being synthesized from five-carbon units, they are restricted to distinct classes that differ by five carbon atoms (C10, C15, C20, etc.). To expand the diversity of terpenoid structures, we engineered yeast cells to synthesize a noncanonical building block with 11 carbons, and produced 40 C11 terpene scaffolds that can form the basis for an entire terpenoid class. By identifying a single-residue switch that converts C10 plant monoterpene synthases to C11-specific enzymes, we engineered dedicated synthases for C11 terpene production. This approach will enable the systematic expansion of the chemical space accessed by terpenoids.
Synthetic biology approaches achieving the reconstruction of specific plant natural product biosynthetic pathways in dedicated microbial “chassis” have provided access to important industrial ...compounds (e.g., artemisinin, resveratrol, vanillin). However, the potential of such production systems to facilitate elucidation of plant biosynthetic pathways has been underexplored. Here we report on the application of a modular terpene production platform in the characterization of the biosynthetic pathway leading to the potent antioxidant carnosic acid and related diterpenes in Salvia pomifera and Rosmarinus officinalis. Four cytochrome P450 enzymes are identified (CYP76AH24, CYP71BE52, CYP76AK6, and CYP76AK8), the combined activities of which account for all of the oxidation events leading to the biosynthesis of the major diterpenes produced in these plants. This approach develops yeast as an efficient tool to harness the biotechnological potential of the numerous sequencing datasets that are increasingly becoming available through transcriptomic or genomic studies.
Terpenoids constitute a large family of natural products, attracting commercial interest for a variety of uses as flavours, fragrances, drugs and alternative fuels. Saccharomyces cerevisiae offers a ...versatile cell factory, as the precursors of terpenoid biosynthesis are naturally synthesized by the sterol biosynthetic pathway.
S. cerevisiae wild type yeast cells, selected for their capacity to produce high sterol levels were targeted for improvement aiming to increase production. Recyclable integration cassettes were developed which enable the unlimited sequential integration of desirable genetic elements (promoters, genes, termination sequence) at any desired locus in the yeast genome. The approach was applied on the yeast sterol biosynthetic pathway genes HMG2, ERG20 and IDI1 resulting in several-fold increase in plant monoterpene and sesquiterpene production. The improved strains were robust and could sustain high terpenoid production levels for an extended period. Simultaneous plasmid-driven co-expression of IDI1 and the HMG2 (K6R) variant, in the improved strain background, maximized monoterpene production levels. Expression of two terpene synthase enzymes from the sage species Salvia fruticosa and S. pomifera (SfCinS1, SpP330) in the modified yeast cells identified a range of terpenoids which are also present in the plant essential oils. Co-expression of the putative interacting protein HSP90 with cineole synthase 1 (SfCinS1) also improved production levels, pointing to an additional means to improve production.
Using the developed molecular tools, new yeast strains were generated with increased capacity to produce plant terpenoids. The approach taken and the durability of the strains allow successive rounds of improvement to maximize yields.
Carnosic acid (CA) is a phenolic diterpene with anti-tumour, anti-diabetic, antibacterial and neuroprotective properties that is produced by a number of species from several genera of the Lamiaceae ...family, including Salvia fruticosa (Cretan sage) and Rosmarinus officinalis (Rosemary). To elucidate CA biosynthesis, glandular trichome transcriptome data of S. fruticosa were mined for terpene synthase genes. Two putative diterpene synthase genes, namely SfCPS and SfKSL, showing similarities to copalyl diphosphate synthase and kaurene synthase-like genes, respectively, were isolated and functionally characterized. Recombinant expression in Escherichia coli followed by in vitro enzyme activity assays confirmed that SfCPS is a copalyl diphosphate synthase. Coupling of SfCPS with SfKSL, both in vitro and in yeast, resulted in the synthesis miltiradiene, as confirmed by 1D and 2D NMR analyses (1H, 13C, DEPT, COSY H-H, HMQC and HMBC). Coupled transient in vivo assays of SfCPS and SfKSL in Nicotiana benthamiana further confirmed production of miltiradiene in planta. To elucidate the subsequent biosynthetic step, RNA-Seq data of S. fruticosa and R. officinalis were searched for cytochrome P450 (CYP) encoding genes potentially involved in the synthesis of the first phenolic compound in the CA pathway, ferruginol. Three candidate genes were selected, SfFS, RoFS1 and RoFS2. Using yeast and N. benthamiana expression systems, all three where confirmed to be coding for ferruginol synthases, thus revealing the enzymatic activities responsible for the first three steps leading to CA in two Lamiaceae genera.
Monoterpenes have an established use in the food and cosmetic industries and have recently also found application as advanced biofuels. Although metabolic engineering efforts have so far achieved ...significant yields of larger terpenes, monoterpene productivity is lagging behind. Here, we set out to establish a monoterpene-specific production platform in Saccharomyces cerevisiae and identified the sequential reaction mechanism of the yeast farnesyl diphosphate synthase Erg20p to be an important factor limiting monoterpene yield. To overcome this hurdle, we engineered Erg20p into a geranyl diphosphate synthase and achieved a significant increase in monoterpene titers. To further improve production, we converted the engineered geranyl diphosphate synthase into a dominant negative form, so as to decrease the ability of the endogenous Erg20p to function as a farnesyl diphosphate synthase, without entirely abolishing sterol biosynthesis. Fusion of the synthetic dominant negative Erg20p variant with the terpene synthase, combined with yeast strain engineering, further improved monoterpene yields and achieved an overall 340-fold increase in sabinene yield over the starting strain. The design described here can be readily incorporated to any dedicated yeast strain, while the developed plasmid vectors and heterozygous ERG20 deletion yeast strain can also be used as a plug-and-play system for enzyme characterization and monoterpene pathway elucidation.
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