Debottlenecking 4-hydroxybenzoate hydroxylation in Pseudomonas putida KT2440 improves muconate productivity from p-coumarate.

The transformation of 4-hydroxybenzoate (4-HBA) to protocatechuate (PCA) is catalyzed by flavoprotein oxygenases known as para-hydroxybenzoate-3-hydroxylases (PHBHs). In Pseudomonas putida KT2440 (P. putida) strains engineered to convert lignin-related aromatic compounds to muconic acid (MA), PHBH activity is rate-limiting, as indicated by the accumulation of 4-HBA, which ultimately limits MA productivity. Here, we hypothesized that replacement of PobA, the native P. putida PHBH, with PraI, a PHBH from Paenibacillus sp. JJ-1b with a broader nicotinamide cofactor preference, could alleviate this bottleneck. Biochemical assays confirmed the strict preference of NADPH for PobA, while PraI can utilize either NADH or NADPH. Kinetic assays demonstrated that both PobA and PraI can utilize NADPH with comparable catalytic efficiency and that PraI also efficiently utilizes NADH at roughly half the catalytic efficiency. The X-ray crystal structure of PraI was solved and revealed absolute conservation of the active site architecture to other PHBH structures despite their differing cofactor preferences. To understand the effect in vivo, we compared three P. putida strains engineered to produce MA from p-coumarate (pCA), showing that expression of praI leads to lower 4-HBA accumulation and decreased NADP+/NADPH ratios relative to strains harboring pobA, indicative of a relieved 4-HBA bottleneck due to increased NADPH availability. In bioreactor cultivations, a strain exclusively expressing praI achieved a titer of 40 g/L MA at 100% molar yield and a productivity of 0.5 g/L/h. Overall, this study demonstrates the benefit of sampling readily available natural enzyme diversity for debottlenecking metabolic flux in an engineered strain for microbial conversion of lignin-derived compounds to value-added products.

Functional insights into Brucella transcriptional regulator ArsR.

ArsR-family transcriptional factors regulates diverse physiological functions necessary for Brucella adaptation to environmental changes. However, whether the ArsR-family transcriptional regulator are related to virulence, and the precise determination of ArsR direct targets in Brucella are still unknown. Therefore, we created a 2308ΔArsR6 mutant of B. abortus 2308 (S2308). Virulence assay was performed using a murine macrophage cell line (RAW 264.7). We performed chromatin immunoprecipitation of ArsR6 followed by next-generation sequencing (ChIP-seq). We also selected the target gene pobA (BAB2_0600), and created the mutant (2308ΔpobA). The survival capability of 2308ΔpobA strain in RAW 264.7 was detected and the levels of tumor necrosis factor-α (TNF-α), interferon-gamma (IFN-γ), interleukin-12 (IL-12) and interleukin-18 (IL-18) were also measured. The results showed that 2308ΔArsR6 reduced survival capability in RAW 264.7. We detected 40 intergenic ChIP-seq peaks of ArsR6 binding distributed across the Brucella genome. 2308ΔpobA was significantly reduced survival capability in RAW 264.7. After the macrophages were infected with 2308ΔpobA, the levels of TNF-α, IFN-γ, IL-12 and IL-18 were decreased and were significantly lower than that for the S2308-infected group, indicating that the 2308ΔpobA could reduce the secretion of inflammatory cytokines. Taken together, the research provided new insights into the functionality of ArsR6 and great significance to clarify the function of ArsR6.

The phytopathogen Xanthomonas campestris utilizes the divergently transcribed pobA/pobR locus for 4-hydroxybenzoic acid recognition and degradation to promote virulence.

Xanthomonas campestris pv. campestris (Xcc) is the causal agent of black rot in crucifers. Our previous findings revealed that Xcc can degrade 4-hydroxybenzoic acid (4-HBA) via the ß-ketoadipate pathway. This present study expands on this knowledge in several ways. First, we show that infective Xcc cells induce in situ biosynthesis of 4-HBA in host plants, and Xcc can efficiently degrade 4-HBA via the pobA/pobR locus, which encodes a 4-hydroxybenzoate hydroxylase and an AraC-family transcription factor respectively. Next, the transcription of pobA is specifically induced by 4-HBA and is positively regulated by PobR, which is constitutively expressed in Xcc. 4-HBA directly binds to PobR dimers, resulting in activation of pobA expression. Point mutation and subsequent isothermal titration calorimetry and size exclusion chromatography analysis identified nine key conserved residues required for 4-HBA binding and/or dimerization of PobR. Furthermore, overlapping promoters harboring fully overlapping -35 elements were identified between the divergently transcribed pobA and pobR. The 4-HBA/PobR dimer complex specifically binds to a 25-bp site, which encompasses the -35 elements shared by the overlapping promoters. Finally, GUS histochemical staining and subsequent quantitative assay showed that both pobA and pobR genes are transcribed during Xcc infection of Chinese radish, and the strain ΔpobR exhibited compromised virulence in Chinese radish. These findings suggest that the ability of Xcc to survive the 4-HBA stress might be important for its successful colonization of host plants.

Hybrid Approach: Combining Surgical Thrombectomy and AngioJet™ Aspirational Thrombectomy in Limb Graft Occlusion Post-FEVAR with Fenestrated Anaconda™ and in ePTFE Bypass Graft Occlusion.

Acute limb ischemia due to limb-graft occlusion (LGO) after fenestrated endovascular aneurysm repair (FEVAR) and acute bypass graft occlusion with an ePTFE graft pose critical challenges, necessitating prompt intervention to prevent limb loss. This paper discusses two cases of acute limb ischemia treated with a hybrid approach using the AngioJet™ Ultra Thrombectomy System as an adjunct to Fogarty thrombectomy. Case I involved a 69-year-old male post-FEVAR with contralateral iliac limb graft occlusion of the fenestrated Anaconda™, while Case II featured a 70-year-old male (ASA IV) post-bypass surgery (iliopopliteal arterial bypass with ePTFE Graft) with acute bypass graft occlusion. Both cases underwent successful recanalization using the AngioJet™ Ultra Thrombectomy System (ZelanteDVT™ 8F catheter, Solent™ Proxi 6F catheter) (Boston Scientific, Marlborough, MA, USA), combined with adjunctive techniques including Fogarty thrombectomy, balloon angioplasty, stenting, and local lysis. Immediate postoperative and follow-up assessments after 6 months revealed restored limb perfusion and improved clinical outcomes, with palpable pulses and improved ulcer healing. The aim of this treatment strategy is not only to alleviate limb ischemia but also to preserve future options in the event of graft failure. The use of the AngioJet™ Thrombectomy System in cases of LGO aims not only to clear the thrombus load but also to avoid the need for graft relining. In the case of acute arterial bypass graft occlusion in a patient with ASA IV, the goal of using the thrombectomy device is to preserve the native vessels for future procedures, such as long infragenual bypass, in addition to limb salvage. These cases demonstrate the efficacy of a hybrid surgical approach in managing acute limb ischemia following graft occlusion following FEVAR and bypass surgery. Long-term follow-up will further elucidate the durability of these interventions and their impact on limb salvage and overall patient outcomes. By combining mechanical thrombectomy with adjunctive techniques, such as balloon angioplasty and stenting, this hybrid approach offers a comprehensive solution to acute limb ischemia, addressing both the underlying occlusive pathology and ensuring optimal limb perfusion. Furthermore, the utilization of the AngioJet™ Ultra Thrombectomy System provides a minimally invasive yet effective method for thrombus removal, reducing procedural time and potential complications associated with open surgical techniques. As such, this approach represents a valuable addition to the armamentarium of treatments for acute limb ischemia, particularly in cases of graft occlusion following complex endovascular and bypass procedures.

Use of Computed Tomography Coronary Calcium Score for Coronary Artery Disease Risk Stratification During Liver Transplant Evaluation.

Background: End-stage liver disease (ESLD) is not considered a risk factor for atherosclerotic cardiovascular disease (ASCVD). However, lifestyle characteristics commonly associated with increased ASCVD risk are highly prevalent in ESLD. Emerging literature shows a high burden of asymptomatic coronary artery disease (CAD) in patients with ESLD and a high ASCVD risk in liver transplantation (LT) recipients. Coronary artery calcium score (CAC) is a noninvasive test providing reliable CAD risk stratification. We implemented an LT evaluation protocol with CAC playing a central role in triaging and determining the need for further CAD assessment. Here, we inform our results from this early experience. Methods: Patients with ESLD referred for LT evaluation were prospectively studied. We compared accuracy of CAC against that of CAD risk factors/scores, troponin I, dobutamine stress echocardiogram (DSE), and single-photon emission computed tomography (SPECT) to detect coronary stenosis ≥70 (CAD ≥ 70) per left heart catheterization (LHC). Thirty-day post-LT cardiac outcomes were also analyzed. Results: One hundred twenty-four of 148 (84%) patients underwent CAC, 106 (72%) DSE/SPECT, and 50 (34%) LHC. CAC ≥ 400 was found in 35 (28%), 100 to 399 in 17 (14%), and <100 in 72 (58%). LHC identified CAD ≥ 70% in 8 of 29 (28%), 2 of 9 (22%), and 0 of 4, respectively. Two acute coronary syndromes occurred after LT in a patient with CAC 811 (CAD < 70%), and one with CAC 347 (CAD ≥ 70%). No patients with CAC < 100 presented with acute coronary syndrome after LT. When using CAD ≥ 70% as primary endpoint of LT evaluation, CAC ≥ 346 was the only test showing predictive usefulness (negative predictive value 100%). Conclusions: CAC is a promising tool to guide CAD risk stratification and need for LHC during LT evaluation. Patients with a CAC < 100 can safely undergo LT without the need for LHC or cardiac stress testing, whereas a CAC < 346 accurately rules out significant CAD stenosis (≥70%) on LHC, outperforming other CAD risk-stratification strategies.

Comparative pathological findings between coronary bare-metal stent implantation and balloon angioplasty over 16 years.

A 68-year old man underwent bare metal stent (BMS; Palmaz-Shatz stent) implantation in the distal right coronary artery (RCA; #4PL) and balloon angioplasity (BA) in the proximal left circumflex coronary artery (LCX; #11) for stable angina. After 5 years after initial stent implantation, de novo lesion located in mid RCA was treated by BMS (NIR stent) implantation additionally. After 16 years from first BMS implantation, the patient died from heart failure due to cardiac amyloidosis, and pathologic examination was performed for the specimen of stented RCA segment and LCX BA segment. An autopsy demonstrated that the lesions of stented site at RCA and BA site LCX were histopathologically different. In the stented segment, severe luminal stenosis is observed due to marked proliferation of the neointima in all stented sites. In addition, neovascularization and spotty calcification with mild lymphocyte infiltration were observed especially around the struts. In contrast, at the BA site of the proximal LCX lesion, in the neointima, the smooth muscle cells were rather atrophic and abundant collagen fibers were evident in the intercellular spaces, which showed very stable findings. Compared with these BMS implanted portions, the site of BA was associated with a trend for smaller late lumen loss. These findings suggested that in the stented site, chronic inflammatory reaction to the stent struts could evoke continuous proliferation of neointima resulting in severe late lumen loss. .

Comparison of Clinical Outcomes With Drug Coated Balloons Versus Plain Balloon Angioplasty In Chronic Limb-Threatening Ischemia.

OBJECTIVE: The use of drug coated balloon (DCB) for angioplasty has shown superior efficacy against plain balloons for treating complex infrainguinal arterial disease. We report and compare the clinical outcomes following application of DCB(Paclitaxel) and plain angioplasty (POBA) in our tertiary care centre. METHODS: A retrospective, single centre analysis of 301 patients with chronic limb-threatening ischemia involving the infrainguinal segment was conducted between September 2014 and September 2018, after approval from the Institutional review board. We analyzed clinical outcomes by measuring postoperative ABI improvement, restenosis requiring reintervention procedure, minor and major amputations at the end of 18 months. . To find the association between the group variables (POBA and DCB) and other risk variables, Chi-square test/Fisher's exact test was used. Multivariable logistic regression analysis was used. RESULTS: Patients who underwent treatment with plain balloon (POBA) and DCB(Paclitaxel) angioplasty were 246(81.7%) and 55(18.3%) respectively. Our study group was predominantly male (Male: Female = 6.7:1), most patients were more than 50 years of age (n = 251, 83.4%). Smoking (n = 199, 66.1%) and diabetes (n = 210, 69.8%) were the most common atherosclerotic risk factors. Postoperative Ankle Brachial Pressure Index (ABI) improvement were similar in both groups (POBA = 57.7%; DCB = 69.8%; p = 0.103). Minor and major amputations following POBA were 26% and 22%; and DCB were 12.7% and 16.4% respectively. Re-stenosis requiring a re-interventional procedure within 18 months was 15%, (n = 37) following POBA; and 12.7% (n = 7) following DCB (p = 0.661). CONCLUSIONS: This retrospective study shows similar clinical limb related outcomes following POBA and DCB at 18 months. However, our comparative analysis between the POBA and DCB groups was totally unadjusted and not adjusted for common confounders such as age and sex. Hence, for one to draw definitive conclusions leading to changes in clinical practice; a randomized, prospective study with a larger patient cohort is needed.

Long-term Outcome following Percutaneous Intervention of Intra-stent Coronary Occlusion and Evaluating the Different Treatment Modalities.

BACKGROUND: Angioplasty for ISR remains a challenge with relatively high rates of recurrence. Although there is a plethora of data on ISR, there is relatively less data on intra-stent-CTO. In this study, we explore the long-term clinical outcomes following angioplasty to intra-stent CTO and study the differences in clinical outcomes between three treatment-arms: POBA vs. DES vs. DCB. METHODS AND RESULTS: We evaluated all patients who underwent PCI to intra-stent CTO between 2011 and 2017. The endpoints used were: cardiac-death, TVMI, TLR, TVR, and MACE.During the study period, 403-patients with a mean age of 69.2 years had successful PCI to intra-stent CTO. 50% were diabetic, 38% had CKD and 32% had left ventricular dysfunction. 93% of cases were stable angina. 22% (n = 88) received only POBA, 28% (n = 113) received DCB and 50% (n = 202) received DES. During the median follow-up of 48-months, cardiac-death occurred in 5.8% (n = 23), TVMI in 4% (n = 16), TLR in 45.6% (n = 182), TVR in 48.7% (n = 194) and MACE of 46%. There were no differences in the hard endpoints between the 3treatment arms. However, the TLR and overall MACE were better in DCB and DES-groups as compared to POBA (TLR: 33%vs.42%vs.49%; p = 0.06); MACE (34% vs. 45% vs. 52%; p = 0.05). CONCLUSION: This is the first study that has focussed on the outcomes following angioplasty to intra-stent CTOs with a very long-term follow-up. The hard endpoints were low, although the TLR rates were high. In regards to treatment strategy, the DCB and DES provide relatively better outcomes than POBA.

Usefulness of Venous Pressure Measurement in Endovascular Treatment of Budd-Chiari Syndrome: A Retrospective Cohort Study.

Objective Therapeutic predictors derived from the venous pressure before therapy have not been identified for Budd-Chiari syndrome (BCS). The aim of this study was to determine whether or not measuring the distal pressure or pressure gradient was useful for predicting treatment efficacy in BCS. Methods We retrospectively analyzed seven consecutive patients diagnosed with symptomatic BCS at our hospital between 2008 and 2017. Distal and proximal venous pressures at occlusion sites of BCS were measured before treatment in all cases. The pressure gradient was defined as the difference between distal and proximal venous pressures. A receiver operating characteristics (ROC) analysis was performed for venous pressures. Results Percutaneous old balloon angioplasty (POBA) was performed in seven cases, with technical success achieved in all cases (100%). No complications were encountered. The median primary patency was 574 (interquartile range, 439.5-1,056.5) days. The 1-year primary patency rate was 71.73%. Six cases (85.7%) showed resolution of symptoms, representing clinical success. The ROC analysis revealed a high distal pressure (area under the ROC curve = 0.83, cut-off=12 mmHg) as a predictor of treatment efficacy of POBA for symptomatic BCS. In addition, the pressure gradient was considered significant from a clinical perspective, because the 6 successful cases with resolution of symptoms showed a large pressure gradient (range, 8-21 mmHg) before treatment, whereas the failed case showed a relatively small pressure gradient (7 mmHg). Conclusion High distal pressure and a large pressure gradient might predict the treatment efficacy of balloon angioplasty for symptomatic BCS.

Structural comparison of p-hydroxybenzoate hydroxylase (PobA) from Pseudomonas putida with PobA from other Pseudomonas spp. and other monooxygenases.

The crystal structure is reported of p-hydroxybenzoate hydroxylase (PobA) from Pseudomonas putida, a possible drug target to combat tetracycline resistance, in complex with flavin adenine dinucleotide (FAD). The structure was refined at 2.2 Šresolution with four polypeptide chains in the asymmetric unit. Based on the results of pairwise structure alignments, PobA from P. putida is structurally very similar to PobA from P. fluorescens and from P. aeruginosa. Key residues in the FAD-binding and substrate-binding sites of PobA are highly conserved spatially across the proteins from all three species. Additionally, the structure was compared with two enzymes from the broader class of oxygenases: 2-hydroxybiphenyl 3-monooxygenase (HbpA) from P. nitroreducens and 2-methyl-3-hydroxypyridine-5-carboxylic acid oxygenase (MHPCO) from Mesorhizobium japonicum. Despite having only 14% similarity in their primary sequences, pairwise structure alignments of PobA from P. putida with HbpA from P. nitroreducens and MHPCO from M. japonicum revealed local similarities between these structures. Key secondary-structure elements important for catalysis, such as the ßαß fold, ß-sheet wall and α12 helix, are conserved across this expanded class of oxygenases.

Drug-eluting balloons with provisional bail-out or adjunctive stenting in de novo coronary artery lesions-a systematic review and meta-analysis.

BACKGROUND: Efficacy of drug-eluting balloons (DEB) for treatment of de novo coronary lesions remains controversial. The present systematic review and meta-analysis of randomised controlled trials assessed DEB with bare-metal stents (BMS) and also DEB with provisional bail-out stents ('DEB-only' strategy), to other conventional options: plain-old balloon angioplasty (POBA), BMS and drug-eluting stents (DES). METHODS: A systematic literature search from January 2000 until May 2017 was conducted. Primary outcome measure, late lumen loss (LLL); and secondary outcomes; binary restenosis, major adverse cardiac events (MACE), target lesion revascularization (TLR), myocardial infarction (MI), cardiovascular death and stent thrombosis were analysed. RESULTS: Seventeen RCTs were included with 2,616 patients. Several comparative groups showed significant differences. DEB with BMS were inferior to DES for LLL [mean difference (MD) =0.12 mm; 95% confidence interval (CI), 0.03 to 0.22; P=0.01]; and binary restenosis [risk ratio (RR) =1.89; (CI, 1.13 to 3.18); P=0.02]. DEB with BMS was superior to BMS for LLL [MD =-0.27 mm; (-0.45 to -0.10); P=0.002]; and MACE [RR =0.64; (0.46 to 0.90); P=0.010]. Finally, DEB alone was superior to POBA for LLL [MD =-0.39 mm; (-0.67 to -0.11); P=0.006] and binary restenosis [RR =0.20; (0.05 to 0.85); P=0.03] in bifurcation lesions. CONCLUSIONS: The results of this meta-analysis showed that whilst DEB with BMS is superior to BMS alone, the combination is inferior to DES for treatment of de novo coronary lesions. Thus, DEB + BMS should not be applied in de novo lesions unless in patients who have absolute contraindications to DES. DEB alone, however, should be considered for relative contraindications to DES such as small vessel disease and bifurcation lesions.