Late-Stage Modification of Aminoglycoside Antibiotics Overcomes Bacterial Resistance Mediated by APH(3') Kinases.

The continuous emergence of antimicrobial resistance is causing a threat to patients infected by multidrug-resistant pathogens. In particular, the clinical use of aminoglycoside antibiotics, broad-spectrum antibacterials of last resort, is limited due to rising bacterial resistance. One of the major resistance mechanisms in Gram-positive and Gram-negative bacteria is phosphorylation of these amino sugars at the 3'-position by O-phosphotransferases [APH(3')s]. Structural alteration of these antibiotics at the 3'-position would be an obvious strategy to tackle this resistance mechanism. However, the access to such derivatives requires cumbersome multi-step synthesis, which is not appealing for pharma industry in this low-return-on-investment market. To overcome this obstacle and combat bacterial resistance mediated by APH(3')s, we introduce a novel regioselective modification of aminoglycosides in the 3'-position via palladium-catalyzed oxidation. To underline the effectiveness of our method for structural modification of aminoglycosides, we have developed two novel antibiotic candidates overcoming APH(3')s-mediated resistance employing only four synthetic steps.

Aptamer protective groups tolerate different reagents and reactions for regioselective modification of neomycin B.

The aptameric protective group strategy for the one-step regioselective transformation of aminoglycoside antibiotics was found to be compatible with diverse reagents and reaction conditions. New derivatives of neomycin B were synthesized with regioselectivities of >99%. This result extends the scope of applicability of APGs facilitating access to novel aminoglycoside derivatives.

COMET: a multicomponent home-based disease-management programme versus routine care in severe COPD.

The COPD Patient Management European Trial (COMET) investigated the efficacy and safety of a home-based COPD disease management intervention for severe COPD patients.The study was an international open-design clinical trial in COPD patients (forced expiratory volume in 1 s <50% of predicted value) randomised 1:1 to the disease management intervention or to the usual management practices at the study centre. The disease management intervention included a self-management programme, home telemonitoring, care coordination and medical management. The primary end-point was the number of unplanned all-cause hospitalisation days in the intention-to-treat (ITT) population. Secondary end-points included acute care hospitalisation days, BODE (body mass index, airflow obstruction, dyspnoea and exercise) index and exacerbations. Safety end-points included adverse events and deaths.For the 157 (disease management) and 162 (usual management) patients eligible for ITT analyses, all-cause hospitalisation days per year (mean±sd) were 17.4±35.4 and 22.6±41.8, respectively (mean difference -5.3, 95% CI -13.7 to -3.1; p=0.16). The disease management group had fewer per-protocol acute care hospitalisation days per year (p=0.047), a lower BODE index (p=0.01) and a lower mortality rate (1.9% versus 14.2%; p<0.001), with no difference in exacerbation frequency. Patient profiles and hospitalisation practices varied substantially across countries.The COMET disease management intervention did not significantly reduce unplanned all-cause hospitalisation days, but reduced acute care hospitalisation days and mortality in severe COPD patients.

Antiadhesive polymer brush coating functionalized with antimicrobial and RGD peptides to reduce biofilm formation and enhance tissue integration.

This paper describes the synthesis and characterization of polymer-peptide conjugates to be used as infection-resistant coating for biomaterial implants and devices. Antiadhesive polymer brushes composed of block copolymer Pluronic F-127 (PF127) were functionalized with antimicrobial peptides (AMP), able to kill bacteria on contact, and arginine-glycine-aspartate (RGD) peptides to promote the adhesion and spreading of host tissue cells. The antiadhesive and antibacterial properties of the coating were investigated with three bacterial strains: Staphylococcus aureus, Staphylococcus epidermidis, and Pseudomonas aeruginosa. The ability of the coating to support mammalian cell growth was determined using human fibroblast cells. Coatings composed of the appropriate ratio of the functional components: PF127, PF127 modified with AMP, and PF127 modified with RGD showed good antiadhesive and bactericidal properties without hampering tissue compatibility.

Comparison of Ultrasound Measurements for Diaphragmatic Mobility, Diaphragmatic Thickness, and Diaphragm Thickening Fraction with Each Other and with Lung Function in Patients with Chronic Obstructive Pulmonary Disease.

Purpose: To compare ultrasound measurements of diaphragmatic mobility, diaphragm thickness, and diaphragm thickening fraction with one another and also with lung function parameters in patients with chronic obstructive pulmonary disease (COPD). Patients and Methods: We conducted a prospective, observational study from 2015 to 2018. A total of 140 patients were randomly selected for this study. Diaphragmatic thickness was measured at deep expiration and deep inspiration with a linear 10-MHz ultrasound probe. Diaphragm thickening fraction was calculated as the ratio between diaphragm thickness at deep inspiration and end expiratory diaphragm thickness. Diaphragmatic mobility was measured with a 3.5-MHz curved probe. Forced expiratory volume in one second (FEV1), residual lung volume, Pimax, and P0.1max were also measured. Sonographic results were compared to FEV1 and other lung function parameters. Results: There was a significant positive correlation between diaphragmatic mobility and the following measurements: FEV1 (P < 0.01), diaphragm thickening fraction (P = 0.013), and lung function parameters reflecting ventilatory muscle strength such as Pimax (P < 0.017) and P0.1/Pimax (P < 0.01). There was a significant negative correlation between diaphragmatic mobility and both residual volume (P < 0.01) and diaphragmatic thickness (P = 0.022). In contrast, there was no correlation between diaphragmatic thickness and FEV1, Pimax, and P0.1/Pimax. Diaphragm thickening fraction had a significant correlation with FEV1 (P = 0.041). Conclusion: In patients with COPD, diaphragm mobility measured sonographically correlates with different lung function parameters and also with sonographically measured diaphragm thickness (negative correlation) and diaphragm thickening fraction (positive correlation).

Endoscopic Lung Volume Reduction Results in Improvement of Diaphragm Mobility as Measured by Sonography.

Background: Hyperinflation in patients with pulmonary emphysema is an important cause of reduced diaphragm mobility. We investigated whether endoscopic lung volume reduction (ELVR) could improve diaphragm mobility. Methods: Diaphragm mobility data obtained by sonography from 44 patients were compared before and 3-6 months after ELVR therapy with a Spiration™ valve system. These patients were asked whether they wanted this procedure again after they had learned of their treatment outcome; this was a subjective indicator of outcome. Lung function parameters and blood gases were also measured. Results: After ELVR, 30 patients (82%) developed atelectasis of ≥50% of the targeted lung lobe. These patients had a diaphragm mobility increase of 28.97 ± 15.93 mm, while the remaining patients experienced an improvement in diaphragm mobility of 16.07 ± 21.17 mm; this difference was significant (p = 0.030). All 30 patients with atelectasis and additional 6 patients without radiologically demonstrated atelectasis perceived an improved outcome after ELVR. Their diaphragm mobility increased by 28.89 ± 17.26 mm. Conversely, the patients with no perceived improvement in outcome had a diaphragm mobility increase of 6.75 ± 12.76 mm; this difference was significant (p = 0.001). Conclusion: ELVR can improve diaphragm mobility, and this improvement is correlated with a perceived positive outcome in patients.

Hypercapnia at Hospital Admission as a Predictor of Mortality.

INTRODUCTION: Hypercapnia is an indicator of ventilatory exhaustion. There is some disagreement regarding whether hypercapnia is also a predictor of mortality. In this prospective study, we aimed to investigate whether hypercapnia can predict in-hospital and 1-year mortality rates in patients with dyspnea or pulmonary diseases. PATIENTS AND METHODS: All patients with dyspnea or pulmonary diseases underwent routine blood gas analysis at hospital admission. During the 12-month enrollment period, 2710 patients were enrolled, and 588 patients with hypercapnia at admission were identified. Of the 1626 normocapnic patients, 62 were randomly selected as controls. In-hospital and 1-year mortality rates were determined. RESULTS: There were significant increases in mortality rate between acute hypercapnic patients and both chronic hypercapnic patients and normocapnic controls. Their in-hospital mortality rates were 17%, 6.7% and 3.2%, respectively. Their 1-year mortality rates were 32%, 20.2% and 14.5%, respectively. The 1-year mortality rates of hypercapnic patients with different underlying diseases were 24.6% (chronic obstructive pulmonary disease), 28.4% (congestive heart disease), 1.6% (obstructive sleep apnea syndrome/obesity hypoventilation syndrome), 50.9% (pneumonia), 0% (suppressed central respiratory drive, primarily due to opiate abuse) and 22.8% (other conditions). DISCUSSION: The 1-year mortality rate of patients with acute hypercapnia at hospital admission was 32%, with significant differences compared to chronic hypercapnic patients (20.2%) and normocapnic patients (14.5%). There was a wide range of 1-year mortality rates between the hypercapnic patients with different underlying diseases.

Nasal high-flow versus noninvasive ventilation in patients with chronic hypercapnic COPD.

Background: Despite the encouraging results of noninvasive ventilation (NIV) in chronic hypercapnic COPD patients, it is also evident that some patients do not tolerate NIV or do not benefit from it. We conducted a study in which COPD patients with stable, chronic hypercapnia were treated with NIV and nasal high-flow (NHF) to compare effectiveness. Methods: In a multi-centered, randomized, controlled, cross-over design, patients received 6 weeks of NHF ventilation followed by 6 weeks of NIV ventilation or vice-versa (TIBICO) between 2011 and 2016. COPD patients with stable daytime hypercapnia (pCO2≥50 mmHg) were recruited from 13 German centers. The primary endpoint was pCO2 changes from baseline blood gas, lung function, quality of life (QoL), the 6 min walking test, and duration of device use were secondary endpoints. Results: A total of 102 patients (mean±SD) age 65.3±9.3 years, 61% females, body mass index 23.1±4.8 kg/m2, 90% GOLD D, pCO2 56.5±5.4 mmHg were randomized. PCO2 levels decreased by 4.7% (n=94; full analysis set; 95% CI 1.8-7.5, P=0.002) using NHF and 7.1% (95% CI 4.1-10.1, P<0.001) from baseline using NIV (indistinguishable to intention-to-treat analysis). The difference of pCO2 changes between the two devices was -1.4 mmHg (95% CI -3.1-0.4, P=0.12). Both devices had positive impact on blood gases and respiratory scores (St. George's Respiratory Questionnaire, Severe Respiratory Insufficiency Questionnaire). Conclusions: NHF may constitute an alternative to NIV in COPD patients with stable chronic hypercapnia, eg, those not tolerating or rejecting NIV with respect to pCO2 reduction and improvement in QoL.

Open-Source Selective Laser Sintering (OpenSLS) of Nylon and Biocompatible Polycaprolactone.

Selective Laser Sintering (SLS) is an additive manufacturing process that uses a laser to fuse powdered starting materials into solid 3D structures. Despite the potential for fabrication of complex, high-resolution structures with SLS using diverse starting materials (including biomaterials), prohibitive costs of commercial SLS systems have hindered the wide adoption of this technology in the scientific community. Here, we developed a low-cost, open-source SLS system (OpenSLS) and demonstrated its capacity to fabricate structures in nylon with sub-millimeter features and overhanging regions. Subsequently, we demonstrated fabrication of polycaprolactone (PCL) into macroporous structures such as a diamond lattice. Widespread interest in using PCL for bone tissue engineering suggests that PCL lattices are relevant model scaffold geometries for engineering bone. SLS of materials with large powder grain size (~500 µm) leads to part surfaces with high roughness, so we further introduced a simple vapor-smoothing technique to reduce the surface roughness of sintered PCL structures which further improves their elastic modulus and yield stress. Vapor-smoothed PCL can also be used for sacrificial templating of perfusable fluidic networks within orthogonal materials such as poly(dimethylsiloxane) silicone. Finally, we demonstrated that human mesenchymal stem cells were able to adhere, survive, and differentiate down an osteogenic lineage on sintered and smoothed PCL surfaces, suggesting that OpenSLS has the potential to produce PCL scaffolds useful for cell studies. OpenSLS provides the scientific community with an accessible platform for the study of laser sintering and the fabrication of complex geometries in diverse materials.

Expression of beta-defensin 1 and 2 in nasal epithelial cells and alveolar macrophages from HIV-infected patients.

BACKGROUND: The incidence of respiratory infection is high in HIV-infected patients. beta-defensins are anti-microbial peptides derived from epithelia on the mucosal surfaces of the respiratory, gastrointestinal and urinary tract. Nothing is known about the rate of expression of beta-defensin 1 and 2 mRNAs in nasal epithelial cells and alveolar macrophages in HIV-infected patients. METHODS: Semiquantitative rt-PCR measurement of beta-defensins 1 and 2 and beta-actin were carried out on nasal epithelial cells of 109 patients (76 HIV-infected) and alveolar macrophages from 56 patients (18 HIV-infected). RESULTS: The levels of beta-defensin 1 and 2 mRNAs in nasal epithelial cells did not differ significantly between HIV-infected and non-infected patients. In the nasal epithelial cells of HIV-negative patients who suffered from respiratory infections beta-defensin levels were decreased. beta-defensin 1 mRNA expression was significantly reduced in alveolar macrophages from HIV infected patients. beta-defensin 2 mRNA expression in alveolar macrophages was very low. beta-defensins 1 and 2 mRNA expression did not correlate with CD 4 cell numbers in the blood of HIV-infected patients. CONCLUSION: HIV infection and CD 4 cell numbers in the blood do not influence beta-defensin 1 and 2 expressions in nasal epithelial cells. In alveolar macrophages, beta-defensin 1 expression is decreased in HIV-infected patients.

Sonographic evaluation of diaphragmatic dysfunction in COPD patients.

BACKGROUND: Diaphragmatic dysfunction is an important reason for dyspnea in COPD patients. But diaphragmatic dysfunction is difficult to evaluate. Ultrasound is an option. We measure sonographically the up- and downward movement of the lung silhouette on both hemidiaphragms. The aim of this prospective investigation was to compare this method with another sonographic method that visualizes the right hemidiaphragm directly and to compare the sonographic results with lung function parameters. METHODS AND PATIENTS: Eighty participants - 20 healthy persons and 60 COPD patients - three groups each with 20 patients with COPD GOLD II, III, and IV - were investigated. The sonographic measurements of the diaphragms were performed. Lung function parameters, blood gases, and 6-minute walk test were also collected and compared to the sonographic results. RESULTS: The sonographic measurement of the lung silhouette was easy to perform in all study participants. The correlation between the sonographic methods measuring the right hemidiaphragmatic movement was strong (r=0.85). There was also a strong correlation between the demonstrated sonographic measurement of the up- and downward movement of the lung silhouette and the forced expiratory volume in the first second (r=0.83). CONCLUSION: We demonstrated that the sonographic measurement of the movement of the lung silhouette is an easy way to establish diaphragmatic dysfunction in COPD patients; it can be done in all patients with reliable results for the right and the left hemidiaphragm.

Increased abundance of cyclooxygenase-2 correlates with vascular endothelial growth factor-A abundance and tumor angiogenesis in gastric cancer.

To understand the role of cyclooxygenase-2 (COX-2) in gastric cancer, we examined the abundance of COX-2, vascular endothelial growth factor-A (VEGF-A), and CD34 in 45 surgically resected human gastric cancers and paired normal gastric mucosa by immunohistochemical analysis. In addition, the message RNA (mRNA) expression of COX-2 and VEGF-A was evaluated in ten fresh surgically resected human gastric cancers and paired normal gastric mucosas using semi-quantitative reverse transcriptional polymerase chain reaction analysis. Our results confirmed an increased abundance of COX-2 and VEGF-A, and the microvessel density, which was assessed by CD34 abundance, in gastric cancer tissues compared with normal paired mucosa. Abundance of COX-2 and VEGF-A was significantly associated with tumor-node-metastasis (TNM) stage (P<0.05) and lymph node metastasis (P<0.001). In addition, abundance of VEGF-A associates with distance metastasis. A significant correlation was found between COX-2 and VEGF-A abundances (P<0.001). Both abundance of COX-2 and VEGF-A were significantly correlated with microvessel density (P<0.001, respectively). In six of ten cancerous tissues and in one of ten paired normal mucosas, the mRNA expression of COX-2 and VEGF-A was detected in the same specimen. In one other cancerous tissue, only COX-2 mRNA was detected. This study indicates that COX-2 is related to tumor angiogenesis in gastric cancer. VEGF-A might play a main role in the COX-2 angiogenic pathway.

Inhibition of cytosolic phospholipase A2 mRNA expression: a novel mechanism for acetylsalicylic acid-mediated growth inhibition and apoptosis in colon cancer cells.

Acetylsalicylic acid (ASA) has been confirmed to inhibit proliferation and to induce apoptosis in human colorectal cancer cells in vitro. However, the mechanism by which ASA exhibits antiproliferative and proapoptotic effects in cyclooxygenase 2 (COX-2)-negative cells remains to be further elucidated. In the present study, SW480, a COX-2-negative colon cancer cell line, was treated with various concentrations of ASA (0, 2.5, 5, and 10 mM). The antiproliferative and proapoptotic effects of ASA were confirmed by MTT assay, flow cytometry of propidium iodide (PI)-stained cells, and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) assay. After treatment with ASA, intracellular cyclic AMP (cAMP) levels were increased and the production of prostaglandin E2 (PGE2) was decreased. RT-PCR analysis revealed that treatment of ASA induced a concentration-dependent downregulation of cytosolic phospholipase A2 (cPLA2) mRNA expression in SW480 cells and also in two other colorectal cancer cell lines, Colo320 and HT-29 cells. Intracellular calcium levels were unaffected by ASA treatment. Our results indicate that the ASA-induced downregulation of cytosolic phospholipase A2 mRNA expression might be a novel mechanism for ASA-mediated growth inhibition and apoptosis in colon cancer cells.

UVA1-induced decrease in dermal neuron-specific enolase (NSE) in acrosclerosis.

Besides its role in small-cell carcinoma of the lung, elevated serum levels of neuron-specific enolase (NSE) have recently been reported to be associated with autoimmune rheumatic disorders such as systemic sclerosis. Serum NSE seems to correlate with disease activity as well as Rodnan skin score. The aim of the study was to assess the neuromodulatory effects of conventional UVA1 phototherapy on acrosclerosis as an additional mechanism besides an assumed T cell apoptosis, collagenase induction and angiogenesis. Punch skin biopsies of acrosclerotic skin lesions taken before and after treatment from four patients were evaluated immunohistochemically for the presence of NSE, S100 and neurofilament. Immunolabeling revealed a UVA-induced decrease in dermal NSE expression. In contrast, no alteration in neurofilament+ cells could be detected. In line with the findings of a previous investigation, a high number of S100+ cells were detected in most specimens. We demonstrated a UVA1-induced reduction in dermal NSE levels correlating with a softening of former sclerotic lesions. Even though the origin and the functional mechanisms remain obscure, NSE might be relevant directly within sclerotic skin lesions and may possibly be used as a diagnostic marker at least in SSc-associated acrosclerotic skin.

Inhibition of adenovirus infection by the bronchoalveolar lavage supernatant in vitro.

BACKGROUND: Gene transfer using the adenovirus has been discouraging, since the level of recombinant gene expression was always low. Non-specific and specific immune responses prevent infection or kill infected cells. Most of the specific immune responses against the adenovirus are well known and can be eluded at least in animal models. The non-specific immune response is not so well investigated. We have previously demonstrated a strong anti-adenoviral effect of bronchoalveolar lavage (BAL) supernatants of different patients independent of their content of specific anti-adenoviral immunoglobulins of the subclasses IgA, IgG, and IgM. In this paper we examine the influence of defensins and immunoglobulins within the epithelial lining fluid of the lung on the infection of adenovirus type 5. METHODS: Pooled BAL supernatants were separated by gelchromatography; IgA and IgG, respectively were removed from the BAL supernatants by anti-IgA- and anti-IgG affinity chromatography. RESULTS: The anti-adenoviral capacity could be assigned to the high molecular weight portion including the immunoglobulin- and at a much lower degree the albumin-fractions. All fractions from affinity chromatography; the IgA fraction, the resulting BAL depleted from IgA, the IgG fraction, and the resulting BAL depleted from IgG were highly inhibitory on adenoviral infectivity. CONCLUSION: The main anti-adenoviral component in BAL is part of a high molecular weight complex, either being a large protein itself, or being a small peptide bound unspecifically to different bigger proteins. Defensins are not important factors of anti-adenoviral infectivity in the epithelial linig fluid of our patients.

Fibromatosis of the hand associated with EMO syndrome: a case report.

BACKGROUND: EMO syndrome, defined as a triad including exophthalmus, pretibial myxedema and osteoarthropathia, is a rare condition in patients suffering from hyperthyreosis. CASE PRESENTATION: We here describe an interesting case of EMO syndrome associated with unilateral fibromatosis of the hand and an initial stage of generalized myxedema of the skin. To our knowledge a similar case has not yet been described in literature though reports about associated fibromatosis, e.g. located retroperitoneally, already exist. Familiar explanations include its initiation by autoimmune processes or aberrant T-cell cytokine stimulation leading to an overwhelming production of glycosaminoglycans. CONCLUSION: Interpreting our case in context with previous reports we conclude that associated fibromatosis induced by autoimmune processes may affect a variety of different localizations and therefore requires careful monitoring. A therapeutical attempt by using UVA1 irridation for pretibial myxedema remained without a satisfying regression.

Probing the shielding properties of aptameric protective groups.

Site-specific derivatization of chemically equivalent functional groups has recently been facilitated by the introduction of high-affinity aptamers as non-covalent protective groups. More specifically, a series of RNA aptamers have proven to be highly efficient in enhancing the regioselectivity of reactions with the aminoglycoside antibiotic neomycin B, which carries several chemically indistinguishable amino and hydroxy groups. Since small-molecule targets tend to exhibit multiple modes of binding with a single aptamer, the impact of secondary binding sites on the regioselectivity should be considered. To address this issue, we investigated a series of well-characterized RNA aptamers that bind neomycin B and propose a mechanism that accounts for the regioselective outcome of these transformations. We further demonstrate that the regioselectivity induced by non-covalent aptamer protective groups is determined by the number of binding sites, their affinity, and the mode of interaction with the guest molecule.

A surface-bound molecule that undergoes optically biased Brownian rotation.

Developing molecular systems with functions analogous to those of macroscopic machine components, such as rotors, gyroscopes and valves, is a long-standing goal of nanotechnology. However, macroscopic analogies go only so far in predicting function in nanoscale environments, where friction dominates over inertia. In some instances, ratchet mechanisms have been used to bias the ever-present random, thermally driven (Brownian) motion and drive molecular diffusion in desired directions. Here, we visualize the motions of surface-bound molecular rotors using defocused fluorescence imaging, and observe the transition from hindered to free Brownian rotation by tuning medium viscosity. We show that the otherwise random rotations can be biased by the polarization of the excitation light field, even though the associated optical torque is insufficient to overcome thermal fluctuations. The biased rotation is attributed instead to a fluctuating-friction mechanism in which photoexcitation of the rotor strongly inhibits its diffusion rate.

Selective transformations of complex molecules are enabled by aptameric protective groups.

Emerging trends in drug discovery are prompting a renewed interest in natural products as a source of chemical diversity and lead structures. However, owing to the structural complexity of many natural compounds, the synthesis of derivatives is not easily realized. Here, we demonstrate a conceptually new approach using oligonucleotides as aptameric protective groups. These block several functionalities by non-covalent interactions in a complex molecule and enable the highly chemo- and regioselective derivatization (>99%) of natural antibiotics in a single synthetic step with excellent conversions of up to 83%. This technique reveals an important structure-activity relationship in neamine-based antibiotics and should help both to accelerate the discovery of new biologically active structures and to avoid potentially costly and cumbersome synthetic routes.