Hydroxylation of polypropylene using the monooxygenase mutant 139-3 from Bacillus megaterium BM3

Enzymatic hydroxylation of polypropylene (PP) was investigated in order to increase hydrophilicity. A mutant (139-3) of the P450monooxygenase from Bacillus megaterium expressed in E. coli DH5α was purified using anion exchange chromatography. Hydroxylation of PP fabrics led to a dramatic increase of hydrophilicity as indicated by a water drop dissipation time of below 1 s compared to the hydrophobic reference material. Likewise, a 4.9 cm increase of rising height was measured which remained consistent after 144 h of storage. Similarly, enzymatic hydroxylation of PP films lead to a decrease of the WCA from 104.6° to 77.3° with no major change after exposure to air for 6 days. Using X-ray photoelectron spectroscopy, an increase in normalized atomic concentrations of oxygen from 1.40 to 4.98% for the CO-inhibited and enzyme treated sample, respectively, was measured confirming enzymatic hydroxylation.This study was performed within Austrian Centre of Industrial Biotechnology ACIB, the MacroFun project and COST Action 868. This work has been supported by the Federal Ministry of Economy, Family and Youth (BMWFJ), the Federal Ministry of Traffic, Innovation and Technology (bmvit), the Styrian Business Promotion Agency SFG, the Standortagentur Tirol and ZIT - Technology Agency of the City of Vienna through the COMET- Funding Program managed by the Austrian Research Promotion Agency FFG

Targeting cancer metabolism: a therapeutic window opens

Genetic events in cancer activate signalling pathways that alter cell metabolism. Clinical evidence has linked cell metabolism with cancer outcomes. Together, these observations have raised interest in targeting metabolic enzymes for cancer therapy, but they have also raised concerns that these therapies would have unacceptable effects on normal cells. However, some of the first cancer therapies that were developed target the specific metabolic needs of cancer cells and remain effective agents in the clinic today. Research into how changes in cell metabolism promote tumour growth has accelerated in recent years. This has refocused efforts to target metabolic dependencies of cancer cells as a selective anticancer strategy.Burroughs Wellcome FundSmith Family FoundationStarr Cancer ConsortiumDamon Runyon Cancer Research FoundationNational Institutes of Health (U.S.

Target enzyme mutations are the molecular basis for resistance towards pharmacological inhibition of nicotinamide phosphoribosyltransferase

<p>Abstract</p> <p>Background</p> <p>Inhibitors of nicotinamide phosphoribosyltransferase (NAMPT) are promising cancer drugs currently in clinical trials in oncology, including APO866, CHS-828 and the CHS-828 prodrug EB1627/GMX1777, but cancer cell resistance to these drugs has not been studied in detail.</p> <p>Methods</p> <p>Here, we introduce an analogue of CHS-828 called TP201565 with increased potency in cellular assays. Further, we describe and characterize a panel of cell lines with acquired stable resistance towards several NAMPT inhibitors of 18 to 20,000 fold compared to their parental cell lines.</p> <p>Results</p> <p>We find that 4 out of 5 of the resistant sublines display mutations of NAMPT located in the vicinity of the active site or in the dimer interface of NAMPT. Furthermore, we show that these mutations are responsible for the resistance observed. All the resistant cell lines formed xenograft tumours <it>in vivo</it>. Also, we confirm CHS-828 and TP201565 as competitive inhibitors of NAMPT through docking studies and by NAMPT precipitation from cellular lysate by an analogue of TP201565 linked to sepharose. The NAMPT precipitation could be inhibited by addition of APO866.</p> <p>Conclusion</p> <p>We found that CHS-828 and TP201565 are competitive inhibitors of NAMPT and that acquired resistance towards NAMPT inhibitors can be expected primarily to be caused by mutations in NAMPT.</p

Detection of human neutrophil elastase (HNE) on wound dressings as marker of inflammation

Chronic wound fluids have elevated concentration of human neutrophil elastase (HNE) which can be used as inflammation/infection marker. Our goal is to develop functional materials for fast diagnosis of wound inflammation/infection by using HNE as a specific marker. For that, fluorogenic peptides with a HNE-specific cleavage sequence were incorporated into traditional textile dressings, to allow real-time detection of the wound status. Two different fluorogenic approaches were studied in terms of intensity of the signal generated upon HNE addition: a fluorophore 7-amino-4-trifluormethylcoumarin (AFC) conjugated to a HNE-specific peptide and two fluorophore/quencher pairs (FAM/Dabcyl and EDANS/Dabcyl) coupled to a similar peptide as a Förster resonance energy transfer (FRET) strategy. Also, two immobilization methods were tested: sonochemistry immobilization onto a cotton bandage and glutaraldehyde (GTA)-assisted chemical crosslinking onto a polyamide dressing. The immobilized fluorogenic AFC peptide showed an intense fluorescence emission in the presence of HNE. HNE also induced an enhanced fluorescent signal with the EDANS/Dabcyl FRET peptide which showed to be a more sensitive and effective strategy than the AFC peptide. However, its chemical immobilization onto the polyamide dressing greatly decreased its detection, mainly due to the more difficult access of the enzyme to the cleavage sequence of the immobilized peptide. After optimization of the in situ immobilization, it will be possible to use these fluorescence-functionalized dressings for an effective and specific monitoring of chronic wounds by simply using a portable ultraviolet (UV) light source. We envision that the development of this point-of-care medical device for wound control will have a great impact on patients life quality and reduction of costs on health care system.This study was funded by the European project InFact-Functional materials for fast diagnosis of wound infection (FP7-NMP-2013-SME-7-grant agreement no. 604278). The work done at Centre of Biological Engineering (CEB) was also supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2013 unit, COMPETE 2020 (POCI-01-0145-FEDER-006684) and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by European Regional Development Fund under the scope of Norte 2020-Programa Operacional Regional do Norte

Separation and purification of curcumin using novel aqueous two-phase micellar systems composed of amphiphilic copolymer and cholinium ionic liquids

Novel aqueous two-phase micellar systems (ATPMS) composed of Pluronic F68, a triblock amphiphilic copolymer, and cholinium-based ionic liquids (ILs) were formulated and applied for separation/purification of curcumin (CCM). CCM stability in the presence of ATPMS components was also evaluated. CCM is stable up to 24 h in copolymer (1.0 10.0 wt%) and ILs (0.1 3.0 M) aqueous solutions. Very mild phase separation conditions (close to room temperature) were achieved by adding cholinium ILs to the Pluronic F68 + McIlvaine buffer at pH 6.0 solution. The decrease of cloud-point temperature is dependent on the relative hydrophobicity of IL anion, [Hex] > [But] > [Prop] > [Ac] > Cl. ATPMS composed of more hydrophobic ILs ([Ch][Hex] > [Ch][But] > [Ch][Prop]) are most efficient in the partition of commercial CCM into polymeric micelles-rich phase. The best ATPMS (0.70 M [Ch][But] and 0.60 M [Ch][Hex]-based ATPMS) were then used to purify CCM from a crude extract of Curcuma longa L. Both systems were very selective to separate CCM from protein-based contaminants (selectivity values 25; purification yields 12-fold). Pluronic F68-based ATPMS are promising for selective separation of hydrophobic biomolecules by using cholinium-based ILs as adjuvants to adjust phase separation temperatures and biomolecules partition.This study was funded by the Coordination for Higher Level Graduate Improvements (CAPES/Brazil, finance code 001), National Council for Scientific and Technological Development (CNPq/Brazil) and the State of São Paulo Research Foundation (FAPESP/Brazil, processes #2014/16424-7, #2017/10789-1, #2018/10799-0, #2018/05111-9; #2019/05624-9, and #2019/08549-8). A.M. Lopes and J.F.B. Pereira are grateful for the language revision of native speaker H.S. Pacheco Neto.info:eu-repo/semantics/publishedVersio

Pharmacological Inhibition of Nicotinamide Phosphoribosyltransferase/Visfatin Enzymatic Activity Identifies a New Inflammatory Pathway Linked to NAD

Nicotinamide phosphoribosyltransferase (NAMPT), also known as visfatin, is the rate-limiting enzyme in the salvage pathway of NAD biosynthesis from nicotinamide. Since its expression is upregulated during inflammation, NAMPT represents a novel clinical biomarker in acute lung injury, rheumatoid arthritis, and Crohn's disease. However, its role in disease progression remains unknown. We report here that NAMPT is a key player in inflammatory arthritis. Increased expression of NAMPT was confirmed in mice with collagen-induced arthritis, both in serum and in the arthritic paw. Importantly, a specific competitive inhibitor of NAMPT effectively reduced arthritis severity with comparable activity to etanercept, and decreased pro-inflammatory cytokine secretion in affected joints. Moreover, NAMPT inhibition reduced intracellular NAD concentration in inflammatory cells and circulating TNFα levels during endotoxemia in mice. In vitro pharmacological inhibition of NAMPT reduced the intracellular concentration of NAD and pro-inflammatory cytokine secretion by inflammatory cells. Thus, NAMPT links NAD metabolism to inflammatory cytokine secretion by leukocytes, and its inhibition might therefore have therapeutic efficacy in immune-mediated inflammatory disorders

The role of specific biomarkers, as predictors of post-operative complications following flexible ureterorenoscopy (FURS), for the treatment of kidney stones: a single-centre observational clinical pilot-study in 37 patients

The number of patients diagnosed and subsequently treated for kidney stones is increasing, and as such the number of post-operative complications is likely to increase. At present, little is known about the role of specific biomarkers, following flexible ureterorenoscopy (FURS) for the surgical treatment of kidney stones. The main aim of the study was to evaluate the role of kidney and infection biomarkers, in patients undergoing FURS. Methods: Included were 37 patients (24 males, 13 females), who underwent elective FURS, for the treatment of kidney stones. Venous blood samples were collected from each patient: pre-operatively, and at 30 min, 2 and 4 h post-operatively. Changes to kidney (NGAL, Cystatin-C) and infection (MPO, PCT) biomarkers was quantified by means of ELISA, Biomerieux mini-vidas and Konelab 20 analysers. Results: Four patients developed post-operative complications (3 - UTIs with urinary retention, 1 - urosepsis. NGAL concentration increased significantly following FURS (p = 0.034). Although no significant changes were seen in Cystatin C, MPO and PCT (p ≥ 0.05) some key clinical observation were noted. Limiting factors for this study were the small number of patients recruited and restriction in blood sampling beyond 4 h. Conclusions: Although not confirmative, changes seen to biomarkers such as Cystatin C, NGAL and MPO in our observational clinical pilot-study may warrant further investigation, involving larger cohorts, to fully understand the role of these biomarkers and their potential association with post-operative complications which can develop following FURS