Drug efflux and lipid A modification by 4-L-aminoarabinose are key mechanisms of polymyxin B resistance in the sepsis pathogen Enterobacter bugandensis.

OBJECTIVES: A concern with the ESKAPE pathogen, Enterobacter bugandensis, and other species of the Enterobacter cloacae complex, is the frequent appearance of multidrug resistance against last-resort antibiotics, such as polymyxins. METHODS: Here, we investigated the responses to polymyxin B (PMB) in two PMB-resistant E. bugandensis clinical isolates by global transcriptomics and deletion mutagenesis. RESULTS: In both isolates, the genes of the CrrAB-regulated operon, including crrC and kexD, displayed the highest levels of upregulation in response to PMB. ∆crrC and ∆kexD mutants became highly susceptible to PMB and lost the heteroresistant phenotype. Conversely, heterologous expression of CrrC and KexD proteins increased PMB resistance in a sensitive Enterobacter ludwigii clinical isolate and in the Escherichia coli K12 strain, W3110. The efflux pump, AcrABTolC, and the two component regulators, PhoPQ and CrrAB, also contributed to PMB resistance and heteroresistance. Additionally, the lipid A modification with 4-L-aminoarabinose (L-Ara4N), mediated by the arnBCADTEF operon, was critical to determine PMB resistance. Biochemical experiments, supported by mass spectrometry and structural modelling, indicated that CrrC is an inner membrane protein that interacts with the membrane domain of the KexD pump. Similar interactions were modeled for AcrB and AcrD efflux pumps. CONCLUSION: Our results support a model where drug efflux potentiated by CrrC interaction with membrane domains of major efflux pumps combined with resistance to PMB entry by the L-Ara4N lipid A modification, under the control of PhoPQ and CrrAB, confers the bacterium high-level resistance and heteroresistance to PMB.

Phage treatment of Pseudomonas aeruginosa yields a phage-resistant population with different susceptibility to innate immune responses and mild effects on metabolic profiles.

In this study, we have investigated innate immune activation capacity and metabolic features of a population of P. aeruginosa PAO1 phage-resistant mutants with diverse genetic modification (large genomic deletions and point mutations) arising after exposure to phages targetting lipopolysaccharide (LPS) or Type-4 pili (T4P). Deletions led to the loss of genes involved in LPS synthesis, cell envelope permeability, efflux systems, biofilm production, oxidative stress tolerance, and DNA repair. Loss of LPS O antigen resulted in bacterial sensitivity to serum complement and stimulation of inflammatory cascades but did not cause increased phagocytosis, while T4P phage-resistant mutants were more effectively phagocytized than LPS-defective mutants. Changes in the utilization of different carbon, nitrogen, sulphur, and phosphorus sources were identified, especially in mutants where the two phage DNA persisted in the bacterial population (pseudolysogeny). However, the metabolic changes did not directly correlate with single-gene mutations or the large gene deletions, suggesting they reflect adaptive changes to the gene modifications that arise during the selection of resistant mutants. In contrast, phage-resistant mutants were susceptible to humoral innate immune responses, suggesting that phage resistance may be a beneficial outcome of phage therapy.

Late-onset methylmalonic acidemia and homocysteinemia (cblC disease): systematic review.

INTRODUCTION: Combined methylmalonic acidemia and homocystinuria, cblC type is an inborn error of intracellular cobalamin metabolism and the most common one. The age of onset ranges from prenatal to adult. The disease is characterised by an elevation of methylmalonic acid (MMA) and homocysteine and a decreased production of methionine. The aim is to review existing scientific literature of all late onset cblC patients in terms of clinical symptoms, diagnosis, and outcome. METHODS: A bibliographic database search was undertaken in PubMed (MEDLINE) complemented by a reference list search. We combined search terms regarding cblC disease and late onset. Two review authors performed the study selection, data extraction and quality assessment. RESULTS: Of the sixty-five articles included in this systematic review, we collected a total of 199 patients. The most frequent clinical symptoms were neuropathy/myelopathy, encephalopathy, psychiatric symptoms, thrombotic microangiopathy, seizures, kidney disease, mild to severe pulmonary hypertension with heart failure and thrombotic phenomena. There were different forms of supplementation used in the different studies collected and, within these studies, some patients received several treatments sequentially and/or concomitantly. The general outcome was: 64 patients recovered, 78 patients improved, 4 patients did not improve, or the disease progressed, and 12 patients died. CONCLUSIONS: Most scientific literature regarding the late onset cblC disease comes from case reports and case series. In most cases treatment initiation led to an improvement and even recovery of some patients. The lack of complete recovery underlines the necessity for increased vigilance in unclear clinical symptoms for cblC disease.

Systemic amyloidosis as a cause of chronic diarrhea after gastric surgery. Literature review.

Amyloidosis with gastrointestinal involvement is a rare cause of chronic diarrhoea, and should be considered especially in adult patients with intestinal malabsorption and extra GI manifestations. We present the case of a male patient who, after an oncological gastrectomy, presented with chronic diarrhea that did not respond to treatment and, after study, the cause of the diarrhea was finally found. Primary systemic light chain amyloidosis (AL) initially presents as chronic diarrhea and weight loss after gastrectomy. Immunohistochemistry was completely negative for amyloid A, which virtually rules out AA amyloidosis. With regard to the immunoglobulin chains, an amyloid signal was observed for both light chains, with a predominance of lambda light chain but not entirely conclusive. This situation is not uncommon since amyloid, whatever its chemical nature, can annex serum proteins, including immunoglobulin chains. In the case of chronic diarrhea, the possibility of amyloidosis should be kept in mind, especially in the case of weight loss.

Grapevine Shoot Extract Rich in Trans-Resveratrol and Trans-ε-Viniferin: Evaluation of Their Potential Use for Cardiac Health.

A grapevine shoot extract (GSE) was obtained using ultrasound-assisted extraction and characterized. The main phenolic constituents were identified as stilbenoids. Among them, trans-resveratrol and trans-ε-viniferin stood out. The GSE was administered to an isoproterenol-induced myocardial injury animal model. The extract alleviated the associated symptoms of the administration of the drug, i.e., the plasma lipid profile was improved, while the disturbed plasma ion concentration, the cardiac dysfunction markers, the DNA laddering, and the necrosis of myocardial tissue were diminished. This effect could be related to the anti-oxidative potential of GSE associated with its antioxidant properties, the increased levels of endogenous antioxidants (glutathione and enzymatic antioxidants), and the diminished lipid peroxidative markers in the heart. The results also revealed angiotensin-converting enzyme (ACE)-inhibitory activity, which indicated the potential of GSE to deal with cardiovascular disease events. This work suggests that not only trans-resveratrol has a protective role in heart function but also GSE containing this biomolecule and derivatives. Therefore, GSE has the potential to be utilized in the creation of innovative functional ingredients.

Hepatopreventive properties of hydroxytyrosol and mannitol-rich extracts obtained from exhausted olive pomace using green extraction methods.

Exhausted olive pomace (EOP) is produced in olive-pomace oil extractors as a by-product. However, the obtention of bioactive compounds from EOP can reinsert it into the economy as a new bioresource before applying other exploitation ways. The objective of the present study was to investigate the phytochemical differences between aqueous and aqueous acetonic extracts from EOP (AE-EOP and AAE-EOP, respectively) obtained by hydrothermal and ultrasound-assisted extraction, respectively. The in vitro antioxidant activities and the in vivo hepatopreventive potential were evaluated. Using RP-HPLC-ESI-QTOF-MS, the chemical profile revealed that AE-EOP and AAE-EOP showed similar qualitative profiles, with some quantitative differences. Hydroxytyrosol and mannitol were the major compounds of the extracts. The investigation of antioxidant properties in vitro highlighted that AE-EOP was slightly more efficient in scavenging DPPH, ABTS, superoxide, and hydrogen peroxide radicals, when compared to AAE-EOP. Additionally, AE-EOP and AAE-EOP showed dose-dependent suppressive effects on pancreatic lipase activity. In vivo studies showed that AE-EOP and AAE-EOP presented interesting hepatopreventive capacities against CCl4 induced liver injury, as evidenced by (i) the preventive effects against DNA damage, (ii) the normalised hepatic biomarker parameters (ALT, AST, GGT, and LDH) and (iii) the normalised lipid profile (LDL-C, TC, TG, and HDL-C) through diminishing their levels, (iv) which was supported by Oil Red O analysis. Furthermore, AE-EOP and AAE-EOP reduced the oxidative stress in liver tissue by inhibiting lipid peroxidation together with the enhancement of the hepatic antioxidant activities (CAT, SOD and GPx) and GSH content. Additionally, AE-EOP and AAE-EOP exhibited an antifibrotic effect, which was clearly demonstrated by the histopathological examination using Picrosirius red staining. The obtained results support the use of EOP extracts in industries without further purification as antioxidants and against free radical induced damage.

Optimization of Microwave-Assisted Water Extraction to Obtain High Value-Added Compounds from Exhausted Olive Pomace in a Biorefinery Context.

Microwave-assisted water extraction (MAWE) was evaluated to obtain the valuable bioactive compounds hydroxytyrosol and mannitol from exhausted olive pomace (EOP). The influence of the operational parameters solid loading (3-15%, w/v), temperature (40-100 °C), and extraction time (4-40 min) was studied using an experimental design. The optimized conditions maximizing their joint extraction were 12% w/v solid loading, 100 °C temperature, and 16 min. It was possible to solubilize 5.87 mg of hydroxytyrosol/g EOP and 46.70 mg mannitol/g EOP. The extracts were also further characterized by liquid chromatography-mass spectrometry, which detected other hydroxytyrosol derivatives such as oleacein, verbascoside, and oleuropein. Moreover, the applied MAWE conditions promoted the co-extraction of proteinaceus material, which was also evaluated. In order to carry out an integral valorization of this waste, the extracted EOP solid was further evaluated chemically and microscopically before recovering the bioactive triterpenes. In particular, maslinic acid and oleanolic acid were obtained, 9.54 mg/g extracted solid and 3.60 mg/g extracted solid, respectively. Overall, MAWE can be applied as a first stage in the fractionation of EOP to support its valorization in a biorefinery framework.

Enrichment of Refined Olive Oils with Phenolic Extracts of Olive Leaf and Exhausted Olive Pomace.

Refined olive oils (ROOs) are commonly enriched with synthetic antioxidants. Antioxidant extracts obtained from natural products can be used to improve the stability of these oils. In this study, ROOs were enriched through the addition of phenolic extracts from olive leaves (OLs) and exhausted olive pomace (EOP). In addition to replacing synthetic antioxidants with natural ones, this results in the valorization of these olive-derived biomasses. The most suitable method for mixing and enriching refined oils was probe-type ultrasonication using lecithin as the emulsifier. Thereafter, the change in the content of antioxidant compounds and the antioxidant capacity of the oils at 25, 35, and 45 °C were studied over 28 and 50 days of storage. The experimental results were fitted using a pseudo-first-order kinetic model. The oxidative stability index of the ROO enriched with a 2 g/L OL extract (70 h) was higher than that of a commercial ROO (46.8 h). Moreover, the oxidative stability index of the refined olive pomace oil (ROPO) enriched with a 2 g/L EOP extract (44.1 h) was higher than that of a commercial ROPO (38.9 h). In addition, the oxidative stabilities and antioxidant capacities of the oils were significantly correlated.

Importance of immunohistochemistry in the differential diagnosis of gastric stromal tumors: GIST vs. schwannoma.

The intention of this work is to present a recent case treated in our center. A 77-year-old woman who underwent a laparoscopic subtotal gastrectomy due to the suspicion of a GIST tumor (gastrointestinal stromal tumor), in which the anatomy study the definitive pathology showed the finding of gastric Shawnnoma.

Diets for inflammatory bowel disease: What do we know so far?

In recent years, the role of diet in the pathogenesis of inflammatory bowel disease (IBD) has gained great interest within the scientific community. Eating habits from industrialised countries (the so-called western diet or WD) have been associated with a higher incidence of IBD in observational studies, although the dietary factors responsible for the development of the disease are still to be elucidated. Some components of the diet with proinflammatory potential may cause changes in immunity and intestinal microbiota, leading to the inflammatory reaction that causes IBD-related lesions. The quality of available evidence is low, due to methodological issues, such as the lack of intervention studies, small sample size and heterogeneity of studies. For this reason, scientific societies have offered their recommendations using clinical practice guidelines and consensus documents, in order to establish a common criterion in the nutritional treatment of patients with IBD. The objective of this review was to summarise the data published regarding diet in IBD and review the recommendations given by scientific societies.

Evaluation of patients with advanced epithelial ovarian cancer before primary treatment: correlation between tumour burden assessed by [18F]FDG PET/CT volumetric parameters and tumour markers HE4 and CA125.

OBJECTIVES: Accurate assessment of disease extent is required to select the best primary treatment for advanced epithelial ovarian cancer patients. Estimation of tumour burden is challenging and it is usually performed by means of a surgical procedure. Imaging techniques and tumour markers can help to estimate tumour burden non-invasively. 2-[18F]FDG PET/CT allows the evaluation of the whole-body disease. This study aimed to correlate HE4 and CA125 serum concentrations with tumour burden evaluated by volumetric 2-[18F]FDG PET/CT parameters in advanced high-grade epithelial ovarian cancer. METHODS: We included 66 patients who underwent 2-[18F]FDG PET/CT and serum tumour markers determination before primary treatment. Volumes of interest were delimited in every pathological uptake. Whole-body metabolic tumour volume (wb_MTV) and total lesion glycolysis (wb_TLG) were calculated summing up every VOI's MTV value. SUVmax thresholds were set at 40% (MTV40 and TLG40) and 50% (MTV50 and TLG50). In addition, four VOI subgroups were defined: peritoneal carcinomatosis, retroperitoneal nodes, supradiaphragmatic nodes, and distant metastases. MTV and TLG were calculated for each group by adding up the corresponding MTV values. TLG was calculated likewise. RESULTS: wb_MTV and wb_TLG were found to be significantly correlated with serum CA125 and HE4 concentrations. The strongest correlation was observed between HE4 and wb_MTV40 (r = 0.62, p < 0.001). Pearson's correlation coefficients between peritoneal carcinomatosis MTV40 and tumour markers were 0.61 (p < 0.0001) and 0.29 (p = 0.02) for HE4 and CA125 respectively. None of these tumour markers showed a positive correlation with tumour load outside the abdominal cavity assessed by volumetric parameters. CONCLUSION: HE4 performs better than CA125 to predict metabolic tumour burden in high-grade epithelial ovarian cancer before primary treatment. 2-[18F]FDG PET/CT volumetric parameters arise as feasible tools for the objective assessment of tumour load and its anatomical distribution. These results support the usefulness of HE4 and PET/CT to improve the stratification of these patients in clinical practice. KEY POINTS: • In patients with high-grade advanced ovarian epithelial carcinoma, both CA125 and HE4 correlate to whole-body tumour burden assessed by PET/CT before primary treatment. • HE4 estimates peritoneal disease much better than CA125. • PET/CT volumetric parameters arise as feasible tools for the objective assessment of tumour load and its anatomical distribution.

Sequential Extraction of Hydroxytyrosol, Mannitol and Triterpenic Acids Using a Green Optimized Procedure Based on Ultrasound.

Olive-derived biomasses contain bioactive compounds with health promoting effects as well as antioxidant and sweet-tasting properties. However, their sequential extraction has not been attained. In the present study, firstly antioxidants and mannitol were extracted from exhausted olive pomace (EOP) by an eco-friendly method, ultrasound-assisted water extraction (UAWE). The amplitude (20-80%), extraction time (2-18 min) and solid loading (2-15%, w/v) were evaluated according to a Box-Behnken experimental design. Using the response surface methodology, the optimal conditions for extraction were obtained: 80% amplitude, 11.5% solid loading and 16 min. It enabled the multi-response optimization of the total phenolic content (TPC) (40.04 mg/g EOP), hydroxytyrosol content (6.42 mg/g EOP), mannitol content (50.92 mg/g EOP) and antioxidant activity (ferric reducing power or FRAP, 50.95 mg/g EOP; ABTS, 100.64 mg/g EOP). Moreover, the phenolic profile of the extracts was determined by liquid chromatography-UV and mass spectrometry, identifying hydroxytyrosol as the main phenolic compound and other minor derivatives could be characterized. Scanning electron microscopy was used to analyze the morphological changes produced in the cellular structure of EOP after UAWE. In addition, the chemical composition of the extracted EOP solid was characterized for further valorization. Then, a second extraction step was performed in order to extract bioactive triterpenes from the latter solid. The triterpenes content in the extract was determined and the effect of the previous UAWE step on the triterpenes extraction was evaluated. In this case, the use of ultrasound enhanced the extraction of maslinic acid and oleanolic acid from pelletized EOP with no milling requirement. Overall, UAWE can be applied to obtain antioxidant compounds and mannitol as first extraction step from pelletized EOP while supporting the subsequent recovery of triterpenic acids.

Recovery of Bioactive Compounds from Industrial Exhausted Olive Pomace through Ultrasound-Assisted Extraction.

Exhausted olive pomace (EOP) is the main agro-industrial waste of the olive pomace extracting industries. It contains phenolic compounds and mannitol, so the extraction of these bioactive compounds should be considered as a first valorization step, especially if EOP is used as biofuel. Therefore, EOP was subjected to bath-type ultrasound-assisted extraction (UAE), and the effects of the acetone concentration (20-80%, v/v), solid load (2-15%, w/v), and extraction time (10-60 min) on the extraction of antioxidant compounds were evaluated according to a Box-Behnken experimental design. By means of the response surface methodology, the optimum conditions were obtained: 40% acetone, 8.6% solids, and 43 min. For all the extracts, the total phenolic content (TPC), flavonoid content (TFC), and antioxidant activity (DPPH, ABTS, and FRAP) were determined. With the aim of shortening the extraction time, a two-level factorial experiment design was also carried out using a probe-type UAE, keeping the solid load at 8.6% (w/v) and the acetone concentration at 40% (v/v), while the amplitude (30-70%) and the extraction time (2-12 min) were varied to maximize the aforementioned parameters. Finally, a maximum of phenolic compounds was reached (45.41 mg GAE/g EOP) at 12 min and 70% amplitude. It was comparable to that value obtained in the ultrasonic bath (42.05 mg GAE/g EOP), but, remarkably, the extraction time was shortened, which translates into lower costs at industrial scale. Moreover, the bioactive compound hydroxytyrosol was found to be the major phenolic compound in the extract, i.e., 5.16 mg/g EOP (bath-type UAE) and 4.96 mg/g EOP (probe-type UAE). Other minor phenolic compounds could be detected by capillary zone electrophoresis and liquid-chromatography-mass spectrometry. The sugar alcohol mannitol, another bioactive compound, was also found in the extract, and its content was determined. Thus, the use of this technology can support the valorization of this waste to obtain bioactive compounds, including mannitol, hydroxytyrosol, and other derivatives, before being applied for other uses.

Correction: Gómez-Cruz et al. Valorisation of Exhausted Olive Pomace by an Eco-Friendly Solvent Extraction Process of Natural Antioxidants. Antioxidants 2020, 9, 1010.

The author wishes to make the following correction to this paper [...].

Defining chaperone-usher fimbriae repertoire in Serratia marcescens.

Chaperone-usher (CU) fimbriae are surface organelles particularly prevalent among the Enterobacteriaceae. Mainly associated to their adhesive properties, CU fimbriae play key roles in biofilm formation and host cell interactions. Little is known about the fimbriome composition of the opportunistic human pathogen Serratia marcescens. Here, by using a search based on consensus fimbrial usher protein (FUP) sequences, we identified 421 FUPs across 39 S. marcescens genomes. Further analysis of the FUP-containing loci allowed us to classify them into 20 conserved CU operons, 6 of which form the S. marcescens core CU fimbriome. A new systematic nomenclature is proposed according to FUP sequence phylogeny. We also established an in vivo transcriptional assay comparing CU promoter expression between an environmental and a clinical isolate of S. marcescens, which revealed that promoters from 3 core CU operons (referred as fgov, fpo, and fps) are predominantly expressed in the two strains and might represent key core adhesion appendages contributing to S. marcescens pathogenesis.

Olive Pomace-Derived Biomasses Fractionation through a Two-Step Extraction Based on the Use of Ultrasounds: Chemical Characteristics.

Olive-derived biomass is not only a renewable bioenergy resource but also it can be a source of bioproducts, including antioxidants. In this study, the antioxidant composition of extracted olive pomace (EOP) and a new byproduct, the residual fraction from olive pit cleaning (RFOPC or residual pulp) was characterized and compared to olive leafy biomass, which have been extensively studied as a source of antioxidants and other bioactive compounds with pharmacological properties. The chemical characterization showed that these byproducts contain a high amount of extractives; in the case of EOP, it was even higher (52.9%) than in olive leaves (OL) and olive mill leaves (OML) (35.8-45.1%). Then, ultrasound-assisted extraction (UAE) was applied to recover antioxidants from the extractive fraction of these biomasses. The solubilization of antioxidants was much higher for EOP, correlating well with the extractives content and the total extraction yield. Accordingly, this also affected the phenolic richness of the extracts and the differences between all biomasses were diminished. In any case, the phenolic profile and the hydroxytyrosol cluster were different. While OL, OML, and EOP contained mainly hydroxytyrosol derivatives and flavones, RFOPC presented novel trilignols. Other compounds were also characterized, including secoiridoids, hydroxylated fatty acids, triterpenoids, among others, depending on the bioresource. Moreover, after the UAE extraction step, alkaline extraction was applied recovering a liquid and a solid fraction. While the solid fraction could of interest for further valorization as a biofuel, the liquid fraction contained proteins, sugars, and soluble lignin, which conferred antioxidant properties to these extracts, and whose content depended on the biomass and conditions applied.

Fused deposition modelling for the development of drug loaded cardiovascular prosthesis.

Cardiovascular diseases constitute a number of conditions which are the leading cause of death globally. To combat these diseases and improve the quality and duration of life, several cardiac implants have been developed, including stents, vascular grafts and valvular prostheses. The implantation of these vascular prosthesis has associated risks such as infection or blood clot formation. In order to overcome these limitations medicated vascular prosthesis have been previously used. The present paper describes a 3D printing method to develop medicated vascular prosthesis using fused deposition modelling (FDM) technology. For this purpose, rifampicin (RIF) was selected as a model molecule as it can be used to prevent vascular graft prosthesis infection. Thermoplastic polyurethane (TPU) and RIF were combined using hot melt extrusion (HME) to obtain filaments containing RIF concentrations ranging between 0 and 1% (w/w). These materials are capable of providing RIF release for periods ranging between 30 and 80 days. Moreover, TPU-based materials containing RIF were capable of inhibiting the growth of Staphylococcus aureus. This behaviour was observed even for TPU-based materials containing RIF concentrations of 0.1% (w/w). TPU containing 1% (w/w) of RIF showed antimicrobial properties even after 30 days of RIF release. Alternatively, these methods were used to prepare dipyridamole containing TPU filaments. Finally, using a dual extrusion 3D printer vascular grafts containing both drugs were prepared.