Identification and validation of fusidic acid and flufenamic acid as inhibitors of SARS-CoV-2 replication using DrugSolver CavitomiX.

In this work, we present DrugSolver CavitomiX, a novel computational pipeline for drug repurposing and identifying ligands and inhibitors of target enzymes. The pipeline is based on cavity point clouds representing physico-chemical properties of the cavity induced solely by the protein. To test the pipeline's ability to identify inhibitors, we chose enzymes essential for SARS-CoV-2 replication as a test system. The active-site cavities of the viral enzymes main protease (Mpro) and papain-like protease (Plpro), as well as of the human transmembrane serine protease 2 (TMPRSS2), were selected as target cavities. Using active-site point-cloud comparisons, it was possible to identify two compounds-flufenamic acid and fusidic acid-which show strong inhibition of viral replication. The complexes from which fusidic acid and flufenamic acid were derived would not have been identified using classical sequence- and structure-based methods as they show very little structural (TM-score: 0.1 and 0.09, respectively) and very low sequence (~ 5%) identity to Mpro and TMPRSS2, respectively. Furthermore, a cavity-based off-target screening was performed using acetylcholinesterase (AChE) as an example. Using cavity comparisons, the human carboxylesterase was successfully identified, which is a described off-target for AChE inhibitors.

Invasiveness of decompression surgery affects modeled lumbar spine kinetics in patients with degenerative spondylolisthesis.

Introduction: The surgical treatment of degenerative spondylolisthesis with accompanying spinal stenosis focuses mainly on decompression of the spinal canal with or without additional fusion by means of a dorsal spondylodesis. Currently, one main decision criterion for additional fusion is the presence of instability in flexion and extension X-rays. In cases of mild and stable spondylolisthesis, the optimal treatment remains a subject of ongoing debate. There exist different opinions on whether performing a fusion directly together with decompression has a potential benefit for patients or constitutes overtreatment. As X-ray images do not provide any information about internal biomechanical forces, computer simulation of individual patients might be a tool to gain a set of new decision criteria for those cases. Methods: To evaluate the biomechanical effects resulting from different decompression techniques, we developed a lumbar spine model using forward dynamic-based multibody simulation (FD_MBS). Preoperative CT data of 15 patients with degenerative spondylolisthesis at the level L4/L5 who underwent spinal decompression were identified retrospectively. Based on the segmented vertebrae, 15 individualized models were built. To establish a reference for comparison, we simulated a standardized flexion movement (intact) for each model. Subsequently, we performed virtual unilateral and bilateral interlaminar fenestration (uILF, bILF) and laminectomy (LAM) by removing the respective ligaments in each model. Afterward, the standardized flexion movement was simulated again for each case and decompression method, allowing us to compare the outcomes with the reference. This comprehensive approach enables us to assess the biomechanical implications of different surgical approaches and gain valuable insights into their effects on lumbar spine functionality. Results: Our findings reveal significant changes in the biomechanics of vertebrae and intervertebral discs (IVDs) as a result of different decompression techniques. As the invasiveness of decompression increases, the moment transmitted on the vertebrae significantly rises, following the sequence intact ➝ uILF ➝ bILF ➝ LAM. Conversely, we observed a reduction in anterior-posterior shear forces within the IVDs at the levels L3/L4 and L4/L5 following LAM. Conclusion: Our findings showed that it was feasible to forecast lumbar spine kinematics after three distinct decompression methods, which might be helpful in future clinical applications.

A randomized controlled trial of lorcaserin and lifestyle counselling for weight loss maintenance: changes in emotion- and stress-related eating, food cravings and appetite.

Anti-obesity medication may help people maintain diet-induced reductions in appetite. The present exploratory analysis assessed the effects of lorcaserin on changes at 24 weeks post-randomization in emotion- and stress-related eating, food cravings and other measures of appetite (i.e. binge eating, cognitive restraint, disinhibition, hunger, preoccupation with eating and fullness). The parent study investigated the efficacy of combined lorcaserin and behavioural treatment in facilitating weight loss maintenance (WLM) in 137 adults (mean age = 46.1 years, 86.1% female, 68.6% black) who had lost ≥5% of initial weight during a 14-week, low-calorie diet (LCD) run-in. Participants were randomly assigned to lorcaserin or placebo and were provided with group WLM counselling sessions. Emotion- and stress-related eating, food cravings and appetite were measured at the start of the LCD (week -14), randomization (0) and week 24. From randomization, lorcaserin-treated participants had significantly greater improvements in emotion- and stress-related eating compared to placebo-treated participants (P = 0.04). However, groups did not differ significantly after randomization in changes in the frequency of food cravings, binge eating or other measures of appetite (Ps > 0.05). Compared to placebo, lorcaserin may improve emotion- and stress-related eating.

Computer simulation and image guidance for individualised dynamic spinal stabilization.

PURPOSE: Dynamic implants for the human spine are used to re-establish regular segmental motion. However, the results have often been unsatisfactory and complications such as screw loosening are common. Individualisation of appliances and precision implantation are needed to improve the outcome of this procedure. Computer simulation, virtual implant optimisation and image guidance were used to improve the technique. METHODS: A human lumbar spine computer model was developed using multi-body simulation software. The model simulates spinal motion under load and degenerative changes. After virtual degeneration of a L4/5 segment, virtual pedicle screw-based implants were introduced. The implants' positions and properties were iteratively optimised. The resulting implant positions were used as operative plan for image guidance and finally implemented in a physical spine model. RESULTS: In the simulation, the introduction and optimisation of virtually designed dynamic implants could partly compensate for the effects of virtual lumbar segment degeneration. The optimised operative plan was exported to two different image-guidance systems for transfer to a physical spine model. CONCLUSION: Three-dimensional computer graphic simulation is a feasible means to develop operative plans for dynamic spinal stabilization. These operative plans can be transferred to commercially available image-guidance systems for use in implantation of physical implants in a spine model. This concept has important potential in the design of operative plans and implants for individualised dynamic spine stabilization surgery.

Atherogenic versus non-atherogenic lipoprotein profiles in healthy individuals. is there a need to change our approach to diagnosing dyslipidemia?

The electrophoretic separation of lipoproteins on polyacrylamide gels enables the quantification of nonatherogenic and atherogenic plasma lipoproteins including small dense low density lipoprotein (sdLDL) particles, which represent the atherogenic lipoprotein subpopulations in plasma. This methodology could help distinguish between nonatherogenic hyperlipidemia, normolipidemia with an atherogenic lipoprotein profile, non-atherogenic normolipidemia, and atherogenic hyperlipidemia. According to our pilot research of a normolipidemic population, the atherogenic lipoprotein profile might be present in about 6% of normolipidemic young healthy individuals. Therefore, if confirmed by other studies, it will be necessary to consider a different diagnostic approach and risk stratification for patients with atherogenic normolipidemia (as well as non-atherogenic hypercholesterolemia).

Zinc deficiency adversely influences interleukin-4 and interleukin-6 signaling.

Zinc deficiency is accompanied by a severe impairment of the immune system, causing a high risk for infections and autoimmune diseases due to altered functionality of B- and T- cells. The influence of zinc deficiency on T- and B- cells via alteration of cytokine expression is well established. The aim of this study was to examine potential direct effects of zinc deficiency on the reactivity of B- and T- cells. Zinc deficient B- and T- cells revealed divergent reaction patterns compared to zinc sufficienT-cells. This was manifested by a stronger proliferative response following IL-6 and IL-2 stimulation on the one hand, but less proliferation following IL-4 stimulation on the other hand. Moreover, these results were supported by the finding that the B- and T-cell signaling cascades activated by IL-4 or IL-6, respectively, were affected directly by zinc deficiency, resulting in reduced Stat6 phosphorylation and increased Stat3 phosphorylation. Whereas the transcription factor Stat6 is involved in IL-4 signaling, Stat3 is activated by IL-6 signaling. Consequently, these results show opposing effects of zinc deficiency on IL-4 and IL-6/IL-2 signaling pathways, thus underlying the importance of zinc for proper immune function.

Acute anterior uveitis is associated with depression and reduction of general health.

AIMS: This study explored the association of active acute anterior uveitis (AAU) with depressive feelings and patients' general health. METHODS: This case-control study comprised 80 participants: 35 patients with an active case of AAU and 45 healthy peers were analysed to compare their depressive moods (Beck depression inventory (BDI), Zerssen Mood Scale (ZMS)) and general health (Short form-8 health survey (SF8)). Furthermore, patients were questioned regarding the presence of distress, occupational satisfaction and impairment in performing daily life tasks. RESULTS: During an acute attack, AAU patients were shown to suffer from depressive moods more often than their healthy peers (BDI 54% vs 9%, respectively; ZMS 2.7-fold shift in mood levels towards depression) and experienced a reduction in mental and physical scores (SF8). Patients felt impaired reading (94%), driving (69%) and working (69%). In all, 74% of the patients believed that distress was a probable trigger. Lack of occupational satisfaction was associated with a persistence of depressive feelings and a reduction in physical health. CONCLUSIONS: AAU patients exhibited a significant psychopathology towards depression, a reduction of general health and impairment of performing daily life tasks. Patients who were unsatisfied at work seemed to suffer even more. The results underline the importance of offering psychological support to the patients and providing information to patients and close social contacts.

Simulating self-organized molecular patterns using interaction-site models.

Molecular building blocks interacting at the nanoscale organize spontaneously into stable monolayers that display intriguing long-range ordering motifs on the surface of atomic substrates. The patterning process, if appropriately controlled, represents a viable route to manufacture practical nanodevices. With this goal in mind, we seek to capture the salient features of the self-assembly process by means of an interaction-site model. The geometry of the building blocks, the symmetry of the underlying substrate, and the strength and range of interactions encode the self-assembly process. By means of Monte Carlo simulations, we have predicted an ample variety of ordering motifs which nicely reproduce the experimental results. Here, we explore in detail the phase behavior of the system in terms of the temperature and the lattice constant of the underlying substrate.

Extracellular serine proteases from Stenotrophomonas maltophilia: Screening, isolation and heterologous expression in E. coli.

A large strain collection comprising antagonistic bacteria was screened for novel detergent proteases. Several strains displayed protease activity on agar plates containing skim milk but were inactive in liquid media. Encapsulation of cells in alginate beads induced protease production. Stenotrophomonas maltophilia emerged as best performer under washing conditions. For identification of wash-active proteases, four extracellular serine proteases called StmPr1, StmPr2, StmPr3 and StmPr4 were cloned. StmPr2 and StmPr4 were sufficiently overexpressed in E. coli. Expression of StmPr1 and StmPr3 resulted in unprocessed, insoluble protein. Truncation of most of the C-terminal domain which has been identified by enzyme modeling succeeded in expression of soluble, active StmPr1 but failed in case of StmPr3. From laundry application tests StmPr2 turned out to be a highly wash-active protease at 45°C. Specific activity of StmPr2 determined with suc-L-Ala-L-Ala-L-Pro-l-Phe-p-nitroanilide as the substrate was 17±2U/mg. In addition we determined the kinetic parameters and cleavage preferences of protease StmPr2.

Isometric non-machine-based prevention training program: effects on the cross-sectional area of the paravertebral muscles on magnetic resonance imaging.

PURPOSE: The purpose of this study was to determine potential effects of isometric non-machine-based training on the cross-sectional area (CSA) of the paravertebral muscles in volunteers who were participants in a prevention program. An increase in the CSA of back muscles after various machine-based exercises have been reported but non-machine-based training programs have not been adressed before. MATERIALS AND METHODS: In the study 14 volunteers, who were participants of a company internal prevention program, underwent a magnetic resonance (MR) examination before and after a 3 months training program to improve back muscle strength. The MRI protocol consisted of T1-weighted and T2-weighted images aligned to the intervertebral disc spaces. The CSAs of the erector spinae and quadratus lumborum muscles were assessed twice by 2 operators at the levels L3/4, L4/5 and L5/S1. RESULTS: Out of 14 subjects 11 completed the training. The recorded CSA values exhibited an intrarater and intrarater correlation coefficient ranging from 0.949 to 0.989. There was an increase in CSA in all subjects after the training period (mean increase 8%). CONCLUSIONS: A 3-month isometric training program is sufficient to effect measurable increases in back muscles volume. The study demonstrated the usefulness of MRI to quantify such changes as a measure of training efficacy and compliance into the training program. These results represent a rationale for further studies to determine the effect of different training methods on the CSA of back muscles and to correlate structural changes with clinical symptoms in chronic low back pain syndrome.

Distress, depression and coping in HLA-B27-associated anterior uveitis with focus on gender differences.

BACKGROUND/AIMS: To evaluate depression, coping with disease and stress, and the subjective impression of distress and/or life events as triggers for recurrences in HLA-B27-associated anterior uveitis (B27-AU), with attention to gender-specific characteristics. METHODS: 171 patients with a history of B27-AU responded to a postal survey performed between January 2006 and April 2008 using standardised psychological questionnaires: Beck Depression Inventory, Freiburg Questionnaire on Coping with Illness, and Stress Coping Inventory. RESULTS: Patients with B27-AU differed from healthy controls showing more depressive symptoms (Beck Depression Inventory, 31.6%), applying characteristic disease coping as well as negative stress coping strategies. Female B27-AU patients tended to react with depression and male patients to use negative stress coping strategies. 57.9% of patients believed that psychological distress was a trigger for relapses, and 34.5% stated specific life events. Together, this group of patients achieved higher depression scores and used more negative disease and stress coping styles than patients without perception of distress. CONCLUSION: Patients with B27-AU patients exhibited significant psychopathology concerning depression and disease coping. Distress and life events were subjectively suspected to be a trigger. By imparting knowledge to the patients on probable development of depressive moods and the role of stress/life events as trigger for relapses, as well as offering behaviour therapy to optimise coping, may help patients to cope better with B27-AU.

C-terminal truncation of a metagenome-derived detergent protease for effective expression in E. coli.

Recently, a new alkaline protease named HP70 showing highest homology to extracellular serine proteases of Stenotrophomonas maltophilia and Xanthomonas campestris was found in the course of a metagenome screening for detergent proteases (Niehaus et al., submitted for publication). Attempts to efficiently express the enzyme in common expression hosts had failed. This study reports on the realization of overexpression in Escherichia coli after structural modification of HP70. Modelling of HP70 resulted in a two-domain structure, comprising the catalytic domain and a C-terminal domain which includes about 100 amino acids. On the basis of the modelled structure the enzyme was truncated by deletion of most of the C-terminal domain yielding HP70-C477. This structural modification allowed effective expression of active enzyme using E. coli BL21-Gold as the host. Specific activity of HP70-C477 determined with suc-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide as the substrate was 30 ± 5 U/mg compared to 8 ± 1 U/mg of the native enzyme. HP70-C477 was most active at 40°C and pH 7-11; these conditions are prerequisite for a potential application as detergent enzyme. Determination of kinetic parameters at 40°C and pH=9.5 resulted in K(M)=0.23 ± 0.01 mM and k(cat)=167.5 ± 3.6s(-1). MS-analysis of peptide fragments obtained from incubation of HP70 and HP70-C477 with insulin B indicated that the C-terminal domain influences the cleavage preferences of the enzyme. Washing experiments confirmed the high potential of HP70-C477 as detergent protease.

Molecular jigsaw: pattern diversity encoded by elementary geometrical features.

Scanning tunneling microscopy (STM) images of self-organized monolayers of Frechet dendrons display a variety of two-dimensional ordering motifs, which are influenced by engineering the molecular interactions. An interaction-site model condenses the essential molecular properties determined by molecular mechanics modeling, which in a Monte Carlo approach successfully predicts the various ordering motifs. This confirms that geometry as well as a few salient weak interaction sites encode these structural motifs.

T helper 2 biased de novo immune response to Keyhole Limpet Hemocyanin in humans.

BACKGROUND: Allergen-specific T helper 2 (Th2) cells play an important role in the pathogenesis of atopic disorders. To date, no model system exists in humans that would allow the monitoring of a developing de novo Th2 immune response in vivo. OBJECTIVE: The aim of the experiment was to establish an immunization protocol inducing a de novo Th2 response in humans using Keyhole Limpet Hemocyanin (KLH) as neo-antigen. METHODS: The double-blind placebo-controlled, parallel-group study was conducted in two groups of subjects (16 healthy volunteers and 16 patients with allergic rhinitis). Subjects received three i.m. injections of 100 microg KLH adsorbed to aluminium hydroxide or matching placebo (alum alone) in intervals of 2 weeks. On day 43, KLH alone (10 microg) was given intra-dermally (i.d.) to all subjects to assess immediate and late-phase skin responses. RESULTS: The immunization protocol was well tolerated, highly specific and efficient. Antigen-specific production of Th2-cytokines (mainly IL-5 and IL-13) by PBMCs suggested a Th2 pattern, as did the presence of KLH-specific IgG4 in sera. Intra-dermal KLH challenge induced an immediate-type of response predominantly in atopic subjects followed by a late-phase skin reaction. The latter was accompanied by the presence of IgE(+) cells, eosinophils and a strong up-regulation of IL-4 and IL-13 along with the absence of Th1 transcripts in biopsies taken from the site of antigen challenge. IL-17 and IL-22 transcripts were detected only in healthy subjects skin following KLH challenge, while IL-1beta and IL-33 expression did not differ between the healthy and the atopics. CONCLUSIONS: The immunization protocol resulted in the elicitation of a local and peripheral Th2 immune response in both healthy and atopic individuals. This may permit the investigation and monitoring of novel immunomodulatory strategies aiming to interfere with Th2 responses in man. The relevance of lack of Th17 cells in atopic skin in this model remains to be determined.

Heterologous expression and characterization of choline oxidase from the soil bacterium Arthrobacter nicotianae.

In the course of a microbial screening of soil samples for new oxidases, different enrichment strategies were carried out. With choline as the only carbon source, a microorganism was isolated and identified as Arthrobacter nicotianae. From this strain, a gene coding for a choline oxidase was isolated from chromosomal DNA. This gene named codA was cloned in Escherichia coli BL21-Gold and the protein (An_CodA) heterologously overexpressed as a soluble intracellular protein of 59.1 kDa. Basic biochemical characterization of purified protein revealed a pH optimum of 7.4 and activity over a broad temperature range (15-70 degrees C). Specific activities were determined toward choline chloride (4.70 +/- 0.12 U/mg) and the synthetic analogs bis(2-hydroxyethyl)-dimethylammonium chloride (0.05 +/- 0.45 x 10(-2) U/mg) and tris-(2-hydroxyethyl)-methylammonium methylsulfate (0.01 +/- 0.12 x 10(-2) U/mg). With increasing number of oxidizable groups, a significant decrease in activity was noted. Determination of kinetic parameters in atmorspheric oxygen resulted in K (M) = 1.51 +/- 0.09 mM and V (max) = 42.73 +/- 0.42 mU/min for choline chloride and K (M) = 4.77 +/- 0.76 mM and V (max) = 48.40 +/- 2.88 mU/min for the reaction intermediate betaine aldehyde respectively. Nuclear magnetic resonance spectroscopic analysis of the products formed during the enzyme reaction with choline chloride showed that in vitro the intermediate betaine aldehyde exists also free in solution.

Early human milk feeding is associated with a lower risk of necrotizing enterocolitis in very low birth weight infants.

BACKGROUND: Necrotizing enterocolitis (NEC) is a frequent cause of mortality and morbidity in very low birth weight (VLBW) infants. Human milk (HM) feeding has been associated with lower risk of NEC. However, mothers of VLBW infants often experience insufficient milk production, resulting in mixed feedings of HM and formula. Moreover, medical complications often limit the volume of feeding they can be given. OBJECTIVE: To determine if high proportions of (50% or greater) HM enteral feeding within the first 14 days of life are protective against NEC. METHOD: This was a prospective cohort study of VLBW infants who were grouped according to the HM proportion of enteral feeding in the first 14 days: <50% (low human milk, LHM, n=46) and > or =50% (high human milk, HHM, n=156). The outcome of interest was development of NEC (Bell stage 2 or 3). Logistic regression was used to estimate odds ratios (OR) and 95% confidence intervals (CI) and to assess potential confounding due to perinatal risk factors. RESULT: Two hundred and two infants were studied. Confirmed NEC occurred in 5/46 (10.6%) of the LHM group, as compared with 5/156 (3.2%) of the HHM. Gestational age was the only perinatal factor associated with risk of NEC. After adjustment for gestational age, HHM was associated with a lower risk of NEC ((OR=0.17, 95% CI: 0.04 to 0.68), P=0.01). CONCLUSION: Enteral feeding containing at least 50% HM in the first 14 days of life was associated with a sixfold decrease in the odds of NEC.

The hydroxynitrile lyase from almond: a lyase that looks like an oxidoreductase.

BACKGROUND: Cyanogenesis is a defense process of several thousand plant species. Hydroxynitrile lyase, a key enzyme of this process, cleaves a cyanohydrin into hydrocyanic acid and the corresponding aldehyde or ketone. The reverse reaction constitutes an important tool in biocatalysis. Different classes of hydroxynitrile lyases have convergently evolved from FAD-dependent oxidoreductases, alpha/beta hydrolases, and alcohol dehydrogenases. The FAD-dependent hydroxynitrile lyases (FAD-HNLs) carry a flavin cofactor whose redox properties appear to be unimportant for catalysis. RESULTS: We have determined the crystal structure of a 61 kDa hydroxynitrile lyase isoenzyme from Prunus amygdalus (PaHNL1) to 1.5 A resolution. Clear electron density originating from four glycosylation sites could be observed. As concerns the overall protein fold including the FAD cofactor, PaHNL1 belongs to the family of GMC oxidoreductases. The active site for the HNL reaction is probably at a very similar position as the active sites in homologous oxidases. CONCLUSIONS: There is strong evidence from the structure and the reaction product that FAD-dependent hydroxynitrile lyases have evolved from an aryl alcohol oxidizing precursor. Since key residues implicated in oxidoreductase activity are also present in PaHNL1, it is not obvious why this enzyme shows no oxidase activity. Similarly, features proposed to be relevant for hydroxy-nitrile lyase activity in other hydroxynitrile lyases, i.e., a general base and a positive charge to stabilize the cyanide, are not obviously present in the putative active site of PaHNL1. Therefore, the reason for its HNL activity is far from being well understood at this point.