Evaluation of greenness and analytical performances of separative methods for chiral separation of novel lactam-based P2RX7-antagonists.

In this work, a preparative supercritical fluid chromatography (SFC) method was first developed to separate a series of chiral compounds evaluated as lactam-based P2RX7 antagonists. Subsequently, high-performance liquid chromatography, SFC, and capillary electrophoresis (CE) were comparatively investigated as QC tools to determine the enantiomeric purity of the separated isomers, including analytical performance and greenness. The screening of the best conditions was carried out in liquid and SFC on the nine derivatives and the amylose tris(3,5-dimethylphenylcarbamate)-based chiral stationary phase was found to be highly efficient. The same screening was carried out in CE and very different conditions, either in acidic or basic background electrolyte and different cyclodextrins used as chiral selectors, allowed the separation of six of the nine derivatives. 1-((3,4-Dichlorophenyl)carbamoyl)-5-oxopyrrolidine-2-carboxylic acid (compound 1) was chosen as a probe, and its semi-preparative separation by SFC and enantiomeric verification using the three techniques are presented. Its limit of detection and limit of quantification are calculated for each method. Finally, the greenness of each quality control method was evaluated.

BrainPepPass: A Framework Based on Supervised Dimensionality Reduction for Predicting Blood-Brain Barrier-Penetrating Peptides.

Peptides that pass through the blood-brain barrier (BBB) not only are implicated in brain-related pathologies but also are promising therapeutic tools for treating brain diseases, e.g., as shuttles carrying active medicines across the BBB. Computational prediction of BBB-penetrating peptides (B3PPs) has emerged as an interesting approach because of its ability to screen large peptide libraries in a cost-effective manner. In this study, we present BrainPepPass, a machine learning (ML) framework that utilizes supervised manifold dimensionality reduction and extreme gradient boosting (XGB) algorithms to predict natural and chemically modified B3PPs. The results indicate that the proposed tool outperforms other classifiers, with average accuracies exceeding 94% and 98% in 10-fold cross-validation and leave-one-out cross-validation (LOOCV), respectively. In addition, accuracy values ranging from 45% to 97.05% were achieved in the independent tests. The BrainPepPass tool is available in a public repository for academic use (https://github.com/ewerton-cristhian/BrainPepPass).

Characterizing Non-covalent Protein Complexes Using Asymmetrical Flow Field-Flow Fractionation On-Line Coupled to Native Mass Spectrometry.

We report an online analytical platform based on the coupling of asymmetrical flow field-flow fractionation (AF4) and native mass spectrometry (nMS) in parallel with UV-absorbance, multi-angle light scattering (MALS), and differential-refractive-index (UV-MALS-dRI) detectors to elucidate labile higher-order structures (HOS) of protein biotherapeutics. The technical aspects of coupling AF4 with nMS and the UV-MALS-dRI multi-detection system are discussed. The "slot-outlet" technique was used to reduce sample dilution and split the AF4 effluent between the MS and UV-MALS-dRI detectors. The stability, HOS, and dissociation pathways of the tetrameric biotherapeutic enzyme (anticancer agent) l-asparaginase (ASNase) were studied. ASNase is a 140 kDa homo-tetramer, but the presence of intact octamers and degradation products with lower molecular weights was indicated by AF4-MALS/nMS. Exposing ASNase to 10 mM NaOH disturbed the equilibrium between the different non-covalent species and led to HOS dissociation. Correlation of the information obtained by AF4-MALS (liquid phase) and AF4-nMS (gas phase) revealed the formation of monomeric, tetrameric, and pentameric species. High-resolution MS revealed deamidation of the main intact tetramer upon exposure of ASNase to high pH (NaOH and ammonium bicarbonate). The particular information retrieved from ASNase with the developed platform in a single run demonstrates that the newly developed platform can be highly useful for aggregation and stability studies of protein biopharmaceuticals.

The quorum sensing peptide EntF* promotes colorectal cancer metastasis in mice: a new factor in the host-microbiome interaction.

BACKGROUND: Colorectal cancer, one of the most common malignancies worldwide, is associated with a high mortality rate, mainly caused by metastasis. Comparative metagenome-wide association analyses of healthy individuals and cancer patients suggest a role for the human intestinal microbiota in tumor progression. However, the microbial molecules involved in host-microbe communication are largely unknown, with current studies mainly focusing on short-chain fatty acids and amino acid metabolites as potential mediators. Quorum sensing peptides are not yet considered in this context since their presence in vivo and their ability to affect host cells have not been reported so far. RESULTS: Here, we show that EntF*, a metabolite of the quorum sensing peptide EntF produced by Enterococcus faecium, is naturally present in mice bloodstream. Moreover, by using an orthotopic mouse model, we show that EntF* promotes colorectal cancer metastasis in vivo, with metastatic lesions in liver and lung tissues. In vitro tests suggest that EntF* regulates E-cadherin expression and consequently the epithelial-mesenchymal transition, via the CXCR4 receptor. In addition, alanine-scanning analysis indicates that the first, second, sixth, and tenth amino acid of EntF* are critical for epithelial-mesenchymal transition and tumor metastasis. CONCLUSION: Our work identifies a new class of molecules, quorum sensing peptides, as potential regulators of host-microbe interactions. We prove, for the first time, the presence of a selected quorum sensing peptide metabolite in a mouse model, and we demonstrate its effects on colorectal cancer metastasis. We believe that our work represents a starting point for future investigations on the role of microbiome in colorectal cancer metastasis and for the development of novel bio-therapeutics in other disease areas.

Degradation kinetics of artesunate for the development of an ex-tempore intravenous injection.

BACKGROUND: Artesunate is recommended by the World Health Organization (WHO) for parenteral treatment of severe Plasmodium falciparum malaria. However, artesunate is inherently unstable in an aqueous solution and hydrolyses rapidly after its preparation for injection. Therefore, the aim of the study was to evaluate the stabilizing effects of phosphate buffer and mannitol against short-term (ex-tempore) artesunate hydrolysis. METHODS: A HPLC-UV isocratic method was developed using a reversed-phase fused core column (HALO RP-C18) and a mobile phase consisting of a mixture of 45% ammonium formate 10 mM in water (pH 4.5) and 55% methanol. Artesunate was formulated as aqueous solutions using a design of experiment (DOE) to investigate the artesunate stabilizing effects of pH (8-10), phosphate buffer strength (0.3-0.5 M), and mannitol (0-0.22 mmol/mL). The solutions were incubated at predefined temperatures (5, 25, and 40 °C) with subsequent analysis. Arrhenius equation was applied to model and evaluate the stability results. RESULTS: The developed HPLC-based method using fused-core stationary phase allowed to selectively quantify artesunate in the presence of its main hydrolysis degradants; namely ß-dihydroartemisinin (ß-DHA) and α-dihydroartemisinin (α-DHA) within 10 min. By applying the Arrhenius equation, the rate of hydrolysis of the drug increased approximately by 3.4 as the temperature raised by 10 °C. Buffer strength was found to be the main factor affecting the hydrolysis rate constants at 5 and 25 °C (p < 0.05), the activation energy (p = 0.009), and the frequency factor (p = 0.045). However, the effect of the buffer was predominant on the activation energy and hydrolysis rate constants, revealing its stabilizing effect on the drug at lower buffer strength (0.3 M). Within the investigated range (pH = 8-10), pH was found to influence the activation energy, with a positive stabilizing effect in the pH range of 8-9. The addition of mannitol as stabilizing agent into artesunate aqueous formulation did not show an improved response. CONCLUSION: Phosphate buffer was the main stability determining factor of artesunate in the aqueous intravenous (i.v.) formulation and was found to be more effective in stabilizing artesunate at a buffer strength of 0.3 M in pH 8-9, while mannitol lacked stabilizing effect.

Statin Intake and All-Cause Mortality among Older Nursing Home Residents.

BACKGROUND: Statins are progressively accepted as being associated with reduced mortality. However, few real-world statin studies have been conducted on statin use in older people and especially the most frail, that is, the nursing home residents. OBJECTIVE: The aim of this study was to evaluate the impact of statin intake in nursing home residents on all-cause mortality. METHOD: This is a cross-sectional study of 1,094 older people residing in 6 nursing homes in Flanders (Belgium) between March 1, 2020 and May 30, 2020. We considered all residents who were taking statins for at least 5 days as statin users. All-cause mortality during the 3 months of data collection was the primary outcome. Propensity score overlap-weighted logistic regression models were applied with age, sex, functional status, diabetes, and cardiac failure/ischemia as potential confounders. RESULTS: 185 out of 1,094 residents were on statin therapy (17%). The statin intake was associated with decreased all-cause mortality: 4% absolute risk reduction; adjusted odds ratio 0.50; CI 0.31-0.81, p = 0.005. CONCLUSIONS: The statin intake was associated with decreased all-cause mortality in older people residing in nursing homes. More in-depth studies investigating the potential geroprotector effect of statins in this population are needed.

Dietary muramidase degrades bacterial peptidoglycan to NOD-activating muramyl dipeptides and reduces duodenal inflammation in broiler chickens.

Muramidases constitute a superfamily of enzymes that hydrolyse peptidoglycan (PGN) from bacterial cell walls. Recently, a fungal muramidase derived from Acremonium alcalophilum has been shown to increase broiler performance when added as a feed additive. However, the underlying mechanisms of action are not yet identified. Here, we investigated the hypothesis that this muramidase can cleave PGN to muramyl dipeptide (MDP), activating nucleotide-binding oligomerisation domain-containing protein 2 (NOD2) receptors in eukaryotic cells, potentially inducing anti-inflammatory host responses. Using Micrococcus luteus as a test bacterium, it was shown that muramidase from A. alcalophilum did not display antimicrobial activity, while it could cleave fluorescently labelled PGN. It was shown that the muramidase could degrade PGN down to its minimal bioactive structure MDP by using UPLC-MS/MS. Using HEK-Blue™-hNOD2 reporter cells, it was shown that the muramidase-treated PGN degradation mixture could activate NOD2. Muramidase supplementation to broiler feed increased the duodenal goblet cell and intraepithelial lymphocyte abundance while reducing duodenal wall CD3+ T lymphocyte levels. Muramidase supplementation to broiler feed only had moderate effects on the duodenal, ileal and caecal microbiome. It was shown that the newly discovered muramidase hydrolysed PGN, resulting in MDP that activates NOD2, potentially steering the host response for improved intestinal health.

BBPpred: Sequence-Based Prediction of Blood-Brain Barrier Peptides with Feature Representation Learning and Logistic Regression.

Blood-brain barrier peptides (BBPs) have a large range of biomedical applications since they can cross the blood-brain barrier based on different mechanisms. As experimental methods for the identification of BBPs are laborious and expensive, computational approaches are necessary to be developed for predicting BBPs. In this work, we describe a computational method, BBPpred (blood-brain barrier peptides prediction), that can efficiently identify BBPs using logistic regression. We investigate a wide variety of features from amino acid sequence information, and then a feature learning method is adopted to represent the informative features. To improve the prediction performance, seven informative features are selected for classification by eliminating redundant and irrelevant features. In addition, we specifically create two benchmark data sets (training and independent test), which contain a total of 119 BBPs from public databases and the literature. On the training data set, BBPpred shows promising performances with an AUC score of 0.8764 and an AUPR score of 0.8757 using the 10-fold cross-validation. We also test our new method on the independent test data set and obtain a favorable performance. We envision that BBPpred will be a useful tool for identifying, annotating, and characterizing BBPs. BBPpred is freely available at http://BBPpred.xialab.info.

Apoptosis Imaging in Oncology by Means of Positron Emission Tomography: A Review.

To date, a wide variety of potential PET-apoptosis imaging radiopharmaceuticals targeting apoptosis-induced cell membrane asymmetry and acidification, as well as caspase 3 activation (substrates and inhibitors) have been developed with the purpose of rapidly assessing the response to treatment in cancer patients. Many of these probes were shown to specifically bind to their apoptotic target in vitro and their uptake to be enhanced in the in vivo-xenografted tumours in mice treated by means of chemotherapy, however, to a significantly variable degree. This may, in part, relate to the tumour model used given the fact that different tumour cell lines bear a different sensitivity to a similar chemotherapeutic agent, to differences in the chemotherapeutic concentration and exposure time, as well as to the different timing of imaging performed post-treatment. The best validated cell membrane acidification and caspase 3 targeting radioligands, respectively 18F-ML-10 from the Aposense family and the radiolabelled caspase 3 substrate 18F-CP18, have also been injected in healthy individuals and shown to bear favourable dosimetric and safety characteristics. However, in contrast to, for instance, the 99mTc-HYNIC-Annexin V, neither of both tracers was taken up to a significant degree by the bone marrow in the healthy individuals under study. Removal of white and red blood cells from the bone marrow through apoptosis plays a major role in the maintenance of hematopoietic cell homeostasis. The major apoptotic population in normal bone marrow are immature erythroblasts. While an accurate estimate of the number of immature erythroblasts undergoing apoptosis is not feasible due to their unknown clearance rate, their number is likely substantial given the ineffective quote of the erythropoietic process described in healthy subjects. Thus, the clinical value of both 18F-ML-10 and 18F-CP18 for apoptosis imaging in cancer patients, as suggested by a small number of subsequent clinical phase I/II trials in patients suffering from primary or secondary brain malignancies using 18F-ML-10 and in an ongoing trial in patients suffering from cancer of the ovaries using 18F-CP18, remains to be proven and warrants further investigation.

LC-MS Compatible Antiadsorption Diluent for Peptide Analysis.

Analytical method development for peptides often proves challenging since these molecules can adsorb to the plastic or glass consumables used in the analysis. This adsorption causes considerable loss and unreliable results, especially in the lower concentration range. Therefore, a variety of antiadsorption strategies have previously been developed to cope with this adsorption, often however incompatible with direct liquid chromatography-mass spectrometry (LC-MS) analysis. Here, a novel antiadsorption diluent is introduced, based on controlled hydrolysis and precipitation of bovine serum albumin. This diluent considerably decreases the adsorption of certain peptides to glass. Moreover, it is LC-MS compatible and can also be used in combination with formic acid and/or acetonitrile addition.

Structural study of L-ascorbic acid 2-phosphate magnesium, a raw material in cell and tissue therapy.

L-ascorbic acid 2-phosphate magnesium (APMg) salt is a vitamin C derivative frequently used as a raw material in cell and tissue therapy. APMg is not only used as a replacement of the unstable ascorbate, but also shows additional cell-biological functionalities. However, its unknown structural characteristics hamper the mechanistic elucidation of its biological role. Therefore, different techniques were applied for APMg structure characterization. Firstly, the stoichiometric composition was characterized by its solvent, ligand and magnesium content. No crystals of APMg could be obtained; however, a single crystal of APNa, the sodium salt of l-ascorbic acid 2-phosphate, was successfully obtained and its crystal structure was elucidated. FT-IR was applied to further clarify the structure of solid APMg. Finally, the structure of APMg in aqueous solution was explored by potentiometric titration as well as FT-IR.

Development of a dissolution method for lumefantrine and artemether in immediate release fixed dose artemether/lumefantrine tablets.

BACKGROUND: Dissolution of artemether (ART) and lumefantrine (LUM) active pharmaceutical ingredients (APIs) in fixed dose combination (FDC) ART/LUM tablets is one of the critical quality attributes. Thus, the verification of the release profile of ART and LUM from FDC ART/LUM tablets using a robust and discriminatory dissolution method is crucial. Therefore, the aim of this study was to develop and validate an appropriate dissolution method for quality control of FDC ART/LUM tablets. METHODS: The dissolution medium was selected based on saturation solubility data and sink conditions. The effect of agitation speed, pH and surfactant concentration on the release of ART and LUM was evaluated by employing a two-level factorial experiment. The resulting final method was validated for linearity, precision, robustness and API stability. In addition, the discriminatory power of the method was evaluated using expired and unexpired FDC ART/LUM products. RESULTS: A suitable dissolution profile of FDC ART/LUM tablets was obtained in 900 ml HCl (0.025 N, pH 1.6) with 1%Myrj 52 using paddle method at 100 rpm and 37 °C. ART and LUM were analysed using a HPLC method with UV detection at wavelengths of 210 and 335 nm, respectively. The results from the stability study showed that ART and LUM were sufficiently stable in HCl (0.025 N, pH 1.6) with 1%Myrj 52 at 37 °C. The method was linear (r2 = 0.999) over the concentration range of 6.25-100 µg/ml. The results for precision were within the acceptance limit (%RSD < 2). The percent relative standard deviation (< 2%) and statistically non-significant (p > 0.05) difference in release of ART and LUM observed between deliberately changed dissolution method settings (pH = 1.6 ± 0.2 or agitation speed = 100 ± 2) and optimized dissolution conditions revealed the robustness of the dissolution method. The method was capable to discriminate among different FDC ART/LUM products with different quality. CONCLUSIONS: The developed dissolution method is robust and discriminatory. It can be used in the quality evaluation of FDC ART/LUM tablets.

Blood-brain barrier transport kinetics of NOTA-modified proteins: the somatropin case.

BACKGROUND: Chemical modifications such as PEG, polyamine and radiolabeling on proteins can alter their pharmacokinetic behavior and their blood-brain barrier (BBB) transport characteristics. NOTA, i.e. 1,4,7-triazacyclononane-1,4,7-triacetic acid, is a bifunctional chelating agent that has attracted the interest of the scientific community for its high complexation constant with metals like gallium. Until now, the comparative BBB transport characteristics of NOTA-modified proteins versus unmodified proteins are not yet described. METHODS: Somatropin (i.e. recombinant human growth hormone), NOTA-conjugated somatropin and gallium-labelled NOTA-conjugated somatropin were investigated for their brain penetration characteristics (multiple time regression and capillary depletion [CD]) in an in vivo mice model to determine the blood-brain transfer properties. RESULTS: The three compounds showed comparable initial brain influx, with Kin=0.38±0.14 µL/(g×min), 0.36±0.16 µL/(g×min) and 0.28±0.18 µL/(g×min), respectively. CD indicated that more than 80% of the influxed compounds reached the brain parenchyma. All three compounds were in vivo stable in serum and brain during the time frame of the experiments. CONCLUSIONS: Our results show that modification of NOTA as well as gallium chelation onto proteins, in casu somatropin, does not lead to a significantly changed pharmacokinetic profile at the blood-brain barrier.

Therapeutic treatment of Zika virus infection using a brain-penetrating antiviral peptide.

Zika virus is a mosquito-borne virus that is associated with neurodegenerative diseases, including Guillain-Barré syndrome1 and congenital Zika syndrome2. As Zika virus targets the nervous system, there is an urgent need to develop therapeutic strategies that inhibit Zika virus infection in the brain. Here, we have engineered a brain-penetrating peptide that works against Zika virus and other mosquito-borne viruses. We evaluated the therapeutic efficacy of the peptide in a lethal Zika virus mouse model exhibiting systemic and brain infection. Therapeutic treatment protected against mortality and markedly reduced clinical symptoms, viral loads and neuroinflammation, as well as mitigated microgliosis, neurodegeneration and brain damage. In addition to controlling systemic infection, the peptide crossed the blood-brain barrier to reduce viral loads in the brain and protected against Zika-virus-induced blood-brain barrier injury. Our findings demonstrate how engineering strategies can be applied to develop peptide therapeutics and support the potential of a brain-penetrating peptide to treat neurotropic viral infections.

Quality of fixed dose artemether/lumefantrine products in Jimma Zone, Ethiopia.

BACKGROUND: Malaria caused by Plasmodium vivax and Plasmodium falciparum is among the major public health problems in most endemic areas of the world. Artemisinin-based combination therapy (ACT) has been recommended as a first-line treatment for uncomplicated Plasmodium falciparum malaria almost in all endemic regions. Since ineffectively regulated medicines in resource limited settings could favour infiltration of poor quality anti-malarial medicines into pharmaceutical supply chain and jeopardize a positive treatment outcome, regular monitoring of the quality of anti-malarial medicines is critical. Thus, the aim of this study was to assess the quality of fixed dose combination (FDC) artemether (ART)/lumefantrine (LUM) tablets available in Jimma zone, Ethiopia. METHODS: This study was conducted in Jimma zone, Ethiopia. A total of 74 samples of FDC ART/LUM (20 mg ART/120 mg LUM) tablets were collected from 27 public facilities. All samples were subjected to visual inspection and the relevant information was recorded. The samples were transported to Jimma University Laboratory of Drug Quality (JuLaDQ) and stored at ambient temperature (20 °C to 25 °C) until analysis. The Pharmacopoeial conform/non-conform methods and the risk-based Derringer's desirability function approach were employed to assess the pharmaceutical quality of the investigated products. RESULTS: The visual inspection results revealed that there were no signs of falsified in the investigated products. Identification test results of samples indicated that all samples contained the stated active pharmaceutical ingredients (APIs). The results of uniformity of mass indicated that all samples complied with International Pharmacopoeial specification limits. The assay results, expressed as percent label claim (%lc) of ART (89.8 to 108.8%, mean ± SD = 99.1 ± 3.9%) and LUM (90.0 to 111.9%, mean ± SD = 98.2 ± 3.8%) revealed that, all samples complied with International Pharmacopoeia acceptance specification limits (i.e. 90-110%lc), except one generic product (IPCA Laboratories Ltd., India) which contains excessive LUM (111.9 ± 1.7%lc). The risk priority number (RPN) results revealed that assay (RPN = 392) is relatively the most critical quality attribute followed by identity (RPN = 280) and mass uniformity (40). Quality evaluation based on psycho-physical Harrington's scale revealed that more than 96% of samples were within the acceptable ranges (D ≥ 0.7-1.0). CONCLUSIONS: Both Pharmacopoeial and risk-based desirability function approaches to quality evaluation applied to the investigated products revealed that above 96% FDC ART/LUM tablets circulating in public settings of Jimma zone are of good quality.

Methylation of selected CpG islands involved in the transcription of myeloperoxidase and superoxide dismutase 2 in neutrophils of periparturient and mid-lactation cows.

It is generally accepted that intracellular killing of microorganisms by production of reactive oxygen species (ROS) in the phagosome of the neutrophil is an important arm of innate defense. High-producing dairy cows are prone to periparturient metabolic and infectious diseases. Both myeloperoxidase (MPO) activity and ROS production decrease the day of parturition. Several studies have demonstrated changes in the expression of genes involved in, for example, metabolism and defense in the circulating neutrophil during peripartum. In this study, we wanted to further characterize the periparturient neutrophil in terms of its oxidative killing capacity by analyzing the oxidative burst at 3 levels. First, the ROS phenotype was evaluated using chemiluminescence. The cows (sampled within 24 h after parturition and at 135 d in milk) showed a significantly slower production of ROS at parturition. Both primiparous (n = 13) and multiparous (n = 12) cows were included in this study, but parity did not affect the kinetics of ROS production. Second, the expression of 11 genes involved in ROS production was measured in the same cows: cytochrome b-245 α and ß chain (CYBA, CYBB; coding for membrane-bound constituents of NADPH oxidase); neutrophil cytosolic factors 1, 2, and 4 (NCF1, NCF2, and NCF4); Rac family small GTPase 1 and 2 (RAC1 and RAC2; coding for regulatory proteins of NADPH oxidase); superoxide dismutase 2 (SOD2); catalase (CAT); myeloperoxidase (MPO; coding for enzymes involved in metabolizing downstream ROS); and spleen-associated tyrosine kinase (SYK; involved in signaling). During peripartum, a shift in expression in the oxidative killing pathway was observed, characterized by a downregulation of MPO and a simultaneous upregulation of the genes coding for NADPH oxidase. Third, as total DNA methylation is known to change during pregnancy, we investigated whether the observed differences were due to different methylation patterns. Promotor regions initiate transcription of particular genes; therefore, we analyzed the methylation status in annotated CpG islands of MPO and SOD2, 2 genes with a significant difference in expression between both lactation stages. The differences in methylation of these CpG islands were nonsignificant. High-throughput techniques may be necessary to obtain more detailed information on the total DNA methylation dynamics in bovine neutrophils and increase our understanding of how gene expression is controlled in neutrophils.

PSMA-Targeting Positron Emission Agents for Imaging Solid Tumors Other Than Non-Prostate Carcinoma: A Systematic Review.

Despite its name, prostate-specific membrane antigen (PSMA) has been shown using immunohistochemistry (IHC) to also be over-expressed in the tumor neovasculature of a wide variety of solid tumors other than prostate carcinoma. Accordingly, positron-emitting radiolabeled small molecules targeting PSMA, initially developed for positron emission tomography in prostate carcinomas, are currently being explored for their staging and restaging potential as an alternative imaging modality in other solid tumor types where 18-F-fluorodeoxyglucose (FDG)-PET imaging has low diagnostic accuracy. In this paper, the currently available literature in this field is reviewed. Preliminary, mainly retrospective studies are encouraging, with evidence of improved diagnostic sensitivity and specificity in clear cell renal carcinoma, glioma, and hepatocellular carcinoma, leading to a change in patient management in several patients. However, the results published thus far warrant confirmation by larger prospective studies additionally assessing the longitudinal impact on patient outcomes.

Disbiome database: linking the microbiome to disease.

BACKGROUND: Recent research has provided fascinating indications and evidence that the host health is linked to its microbial inhabitants. Due to the development of high-throughput sequencing technologies, more and more data covering microbial composition changes in different disease types are emerging. However, this information is dispersed over a wide variety of medical and biomedical disciplines. DESCRIPTION: Disbiome is a database which collects and presents published microbiota-disease information in a standardized way. The diseases are classified using the MedDRA classification system and the micro-organisms are linked to their NCBI and SILVA taxonomy. Finally, each study included in the Disbiome database is assessed for its reporting quality using a standardized questionnaire. CONCLUSIONS: Disbiome is the first database giving a clear, concise and up-to-date overview of microbial composition differences in diseases, together with the relevant information of the studies published. The strength of this database lies within the combination of the presence of references to other databases, which enables both specific and diverse search strategies within the Disbiome database, and the human annotation which ensures a simple and structured presentation of the available data.

Reversible myocardial perfusion defects in patients not suffering from obstructive epicardial coronary artery disease as assessed by coronary angiography.

In approximately 10-30% of patients presenting with angina complaints, normal or non-obstructive coronary arteries are found on angiography. In this review paper, available literature on the underlying pathophysiological substrate explaining these discrepancies is reviewed. Both histological studies as well as studies using intravascular ultrasound e.g. the PROSPECT trial, show that epicardial coronary vessel significant lumen stenosis may be delayed until a plaque occupies 40% of the internal elastic lamina area. Limited available data suggest that these angiographically undetectable plaques are associated with an abnormal vasodilation capacity of the coronary circulation and may results in reversible perfusion defects on myocardial perfusion imaging (MPI). Organic non-atherosclerotic causes of epicardial coronary artery disease such as anomalous coronary arteries that course between the aorta and pulmonary artery, myocardial bridging and coronary vasospasm may also contribute to MPI results suggesting the presence of ischemia in the presence of normal coronary arteries on coronary angiography. Additional causes of reversible perfusion defects on MPI in the presence of a normal coronary angiogram are intraventricular conduction disturbances. The existence of reversible perfusion defects in the anteroseptal region in most of the patients suffering from left bundle branch block (LBBB) on MPI following physical exercise as stressor is well documented. As the observed reduced septal uptake of both 201Tl and 99mTc-sestamibi/tetrofosmin in LBBB reflects coronary autoregulation in response to lower oxygen demands, not surprisingly, dipyridamole which uniformly exploits flow reserve, has proven more accurate for the diagnosis of coronary artery disease (CAD) in patients suffering from LBBB. Although patients with a permanent ventricular pacemaker have a similar conduction abnormality as patients presenting with a LBBB, most of the defects found on MPI imaging in this patient population (in up to 78% of patients with a normal coronary angiogram that area continuously paced) are localized in the inferoposterior (71%), apical (50%) and inferoseptal (28%) wall; coronary flow velocities in the left anterior descending (LAD) and dominant coronary artery and coronary flow reserve are also significantly lower when compared to a control group. Contrary to what is seen in LBBB patients, dipyridamole stress does not significantly reduce the incidence of abnormalities found but limits the defects to the inferior wall. Furthermore, the frequency of abnormalities found on MPI increases over time with right ventricular outflow tract pacing. Previous histologic studies have shown that microvessel disease is often accompanied by a slow-flow phenomenon reflecting decreased resting flow velocity. Thus, not surprisingly, MPI reversible abnormalities in the presence of a normal coronary angiogram have been reported in a wide variety of diseases characterized by microvessel disease such as diabetes, systemic lupus erythematosus, Behçet's disease and metabolic syndrome. In these patients, low adiponectin and high lipoprotein(a) levels are found which are known to be associated with endothelial dysfunction, atherosclerosis and coronary artery disease. Furthermore, in these patients, limited available data suggest that reversible perfusion defects on MPI confer a significantly poorer prognosis both in terms of hard event rate (MI and cardiac death) and total event rate (MI, cardiac death or late revascularization). It is thus suggested that MPI could discriminate patients with a more severe prognosis. Finally, physical training in patients with primary microvascular angina appears to be associated with reduction of myocardial perfusion abnormalities.

Regulatory status of N-alkylamide containing health products.

N-alkylamides (NAAs) are secondary metabolites occurring in more than 25 plant families. Plants containing NAAs are traditionally used in food for flavouring, tingling, pungent and saliva-enhancing properties but also to treat various diseases. NAA containing products are abundantly available on the market as food, cosmetics, medical devices and medicinal products. However, no unambiguous legal product classification is applied for these products. In this study, the different health product classes from a European viewpoint are discussed in relation to the pharmacokinetic and pharmacodynamic properties of the NAAs, their applied dosage and claimed usage.