Combined In Silico,In Vivo,and In Vitro Studies Shed Insights into the Acute Inflammatory Response in Middle-Aged Mice

We combined in silico, in vivo, and in vitro studies to gain insights into age-dependent changes in acute inflammation in response to bacterial endotoxin (LPS). Time-course cytokine, chemokine, and NO₂ ^−/NO₃ ⁻ data from “middle-aged” (6–8 months old) C57BL/6 mice were used to re-parameterize a mechanistic mathematical model of acute inflammation originally calibrated for “young” (2–3 months old) mice. These studies suggested that macrophages from middle-aged mice are more susceptible to cell death, as well as producing higher levels of pro-inflammatory cytokines, vs. macrophages from young mice. In support of the in silico-derived hypotheses, resident peritoneal cells from endotoxemic middle-aged mice exhibited reduced viability and produced elevated levels of TNF-α, IL-6, IL-10, and KC/CXCL1 as compared to cells from young mice. Our studies demonstrate the utility of a combined in silico, in vivo, and in vitro approach to the study of acute inflammation in shock states, and suggest hypotheses with regard to the changes in the cytokine milieu that accompany aging.     

In Vitro,In Silico and In Vivo Studies of Ursolic Acid as an Anti-Filarial Agent

As part of our drug discovery program for anti-filarial agents from Indian medicinal plants, leaves of Eucalyptus tereticornis were chemically investigated, which resulted in the isolation and characterization of an anti-filarial agent, ursolic acid (UA) as a major constituent. Antifilarial activity of UA against the human lymphatic filarial parasite Brugia malayi using in vitro and in vivo assays, and in silico docking search on glutathione-s-transferase (GST) parasitic enzyme were carried out. The UA was lethal to microfilariae (mf; LC₁₀₀: 50; IC₅₀: 8.84 µM) and female adult worms (LC₁₀₀: 100; IC50: 35.36 µM) as observed by motility assay; it exerted 86% inhibition in MTT reduction potential of the adult parasites. The selectivity index (SI) of UA for the parasites was found safe. This was supported by the molecular docking studies, which showed adequate docking (LibDock) scores for UA (−8.6) with respect to the standard antifilarial drugs, ivermectin (IVM −8.4) and diethylcarbamazine (DEC-C −4.6) on glutathione-s-transferase enzyme. Further, in silico pharmacokinetic and drug-likeness studies showed that UA possesses drug-like properties. Furthermore, UA was evaluated in vivo in B. malayi-M. coucha model (natural infection), which showed 54% macrofilaricidal activity, 56% female worm sterility and almost unchanged microfilaraemia maintained throughout observation period with no adverse effect on the host. Thus, in conclusion in vitro, in silico and in vivo results indicate that UA is a promising, inexpensive, widely available natural lead, which can be designed and developed into a macrofilaricidal drug. To the best of our knowledge this is the first ever report on the anti-filarial potential of UA from E. tereticornis, which is in full agreement with the Thomson Reuter''s ‘Metadrug’ tool screening predictions.     

Acetazolamide Inhibits the Level of Tyrosinase and Melanin: An Enzyme Kinetic,In Vitro,In Vivo,and In Silico Studies

Melanin is the major factor that determines skin color and protects from ultraviolet radiation. In present study we evaluated the anti‐melanogenesis effect of acetazolamide (ACZ) using four different approaches: enzyme kinetic, in vitro, in vivo and in silico. ACZ demonstrated significant inhibitory activity (IC₅₀ 7.895 ± 0.24 μm ) against tyrosinase as compared to the standard drug kojic acid (IC₅₀ 16.84 ± 0.64 μm ) and kinetic analyses showed that ACZ is a non‐competitive inhibitor without cytotoxic effect. In in vitro experiments, A375 human melanoma cells were treated with 20 or 40 μm of ACZ with or without 50 μm of l ‐DOPA. Western blot results showed that ACZ significantly (P < 0.05) decreased the expression level of tyrosinase at 40 μm . Zebrafish embryos were treated with 10, 20 or 40 μm of ACZ and of positive control kojic acid. At 72 h of treatment with ACZ and kojic acid, ACZ significantly (P < 0.001) decreased the embryos pigmentation to 40.8% of untreated embryos at the dose of 40 μm of ACZ while kojic acid decreased only 25.0% of pigmentation at the same dose of kojic acid. In silico docking were performed against tyrosinase using PyRx tool. Docking studies suggested that His244, Asn260 and His85 are the major interacting residues in the binding site of the protein. In conclusion, our results suggest that ACZ is a good candidate for the inhibition of melanin and it could be used as a lead for developing the drugs for hyperpigmentary disorders and skin whitening.     

Synthetic Oxoisoaporphine Alkaloids: In Vitro,In Vivo and In Silico Assessment of Antileishmanial Activities

Leishmaniasis is a growing health problem worldwide. As there are certain drawbacks with the drugs currently used to treat human leishmaniasis and resistance to these drugs is emerging, there is a need to develop novel antileishmanial compounds, among which isoquinoline alkaloids are promising candidates. In this study, 18 novel oxoisoaporphine derivatives were synthesized and their possible antileishmanial activity was evaluated. The in vitro activity of these derivatives against Leishmania amazonensis axenic amastigotes was first evaluated, and the selected compounds were then tested in an inhibition assay with promastigotes of L. infantum, L. braziliensis, L. amazonensis and L. guyanensis, and with intracellular amastigotes of L. infantum and L. amazonensis. Finally, the most active compounds, OXO 1 (2,3-dihydro-7H-dibenzo[de,h]quinolin-7-one) and OXO 13 (2,3,8,9,10,11-hexahydro-7H-dibenzo[de,h]quinolin-7-one), were tested in BALB/c mice infected with L. infantum. Treatment of mice at a dose of 10 mg/kg with OXO 1 yielded significant reductions (p<0.05) in parasite burden in liver and spleen (99% and 78%, respectively) whereas with OXO 13 were not significant. Although previous reports suggest that this family of molecules displays inhibitory activity against monoamine oxidase A and acetylcholinesterase, these enzymes were not confirmed as targets for antileishmanial activity on the basis of the present results. However, after development of a new bioinformatics model to analyze the Leishmania proteome, we were able to identify other putative targets for these molecules. The most promising candidates were four proteins: two putative pteridine reductase 2 (1MXF and 1MXH), one N-myristoyltransferase (2WUU) and one type I topoisomerase (2B9S).     

GrowMatch: An Automated Method for Reconciling In Silico/In Vivo Growth Predictions

Genome-scale metabolic reconstructions are typically validated by comparing in silico growth predictions across different mutants utilizing different carbon sources with in vivo growth data. This comparison results in two types of model-prediction inconsistencies; either the model predicts growth when no growth is observed in the experiment (GNG inconsistencies) or the model predicts no growth when the experiment reveals growth (NGG inconsistencies). Here we propose an optimization-based framework, GrowMatch, to automatically reconcile GNG predictions (by suppressing functionalities in the model) and NGG predictions (by adding functionalities to the model). We use GrowMatch to resolve inconsistencies between the predictions of the latest in silico Escherichia coli (iAF1260) model and the in vivo data available in the Keio collection and improved the consistency of in silico with in vivo predictions from 90.6% to 96.7%. Specifically, we were able to suggest consistency-restoring hypotheses for 56/72 GNG mutants and 13/38 NGG mutants. GrowMatch resolved 18 GNG inconsistencies by suggesting suppressions in the mutant metabolic networks. Fifteen inconsistencies were resolved by suppressing isozymes in the metabolic network, and the remaining 23 GNG mutants corresponding to blocked genes were resolved by suitably modifying the biomass equation of iAF1260. GrowMatch suggested consistency-restoring hypotheses for five NGG mutants by adding functionalities to the model whereas the remaining eight inconsistencies were resolved by pinpointing possible alternate genes that carry out the function of the deleted gene. For many cases, GrowMatch identified fairly nonintuitive model modification hypotheses that would have been difficult to pinpoint through inspection alone. In addition, GrowMatch can be used during the construction phase of new, as opposed to existing, genome-scale metabolic models, leading to more expedient and accurate reconstructions.     

Brain Amino Acids During Hyponatremia In Vivo: Clinical Observations and Experimental Studies

Hyponatremia is a highly morbid condition, present in a wide range of human pathologies, that exposes patients to encephalopathic complication and the risk of permanent brain damage and death. Treating hyponatremia has proved to be difficult and still awaits safe management, avoiding the morbid sequelae of demyelinizing and necrotic lesions associated with the use of hypertonic solutions. During acute and chronic hyponatremia in vivo, the brain extrudes the excessive water by decreasing its content of electrolytes and organic osmolytes. At the cellular level, a similar response occurs upon cell swelling. Among the organic osmolytes involved in both responses, free amino acids play a prominent role because of the large intracellular pools often found in nerve cells. An overview of the changes in brain amino acid content during hyponatremia in vivo is presented and the contribution of these changes to the adaptive cell responses involved in volume regulation discussed. Additionally, new data are provided concerning changes in amino acid levels in different regions of the central nervous system after chronic hyponatremia. Results favor the role of taurine, glutamine, glutamate, and aspartate as the main amino acid osmolytes involved in the brain adaptive response to hyponatremia in vivo. Deeper knowledge of the adaptive overall and cellular brain mechanisms activated during hyponatremia would lead to the design of safer therapies for the hyponatremic patient.     

In Vivo,In Vitro,and In Silico Characterization of Peptoids as Antimicrobial Agents

Bacterial resistance to conventional antibiotics is a global threat that has spurred the development of antimicrobial peptides (AMPs) and their mimetics as novel anti-infective agents. While the bioavailability of AMPs is often reduced due to protease activity, the non-natural structure of AMP mimetics renders them robust to proteolytic degradation, thus offering a distinct advantage for their clinical application. We explore the therapeutic potential of N-substituted glycines, or peptoids, as AMP mimics using a multi-faceted approach that includes in silico, in vitro, and in vivo techniques. We report a new QSAR model that we developed based on 27 diverse peptoid sequences, which accurately correlates antimicrobial peptoid structure with antimicrobial activity. We have identified a number of peptoids that have potent, broad-spectrum in vitro activity against multi-drug resistant bacterial strains. Lastly, using a murine model of invasive S. aureus infection, we demonstrate that one of the best candidate peptoids at 4 mg/kg significantly reduces with a two-log order the bacterial counts compared with saline-treated controls. Taken together, our results demonstrate the promising therapeutic potential of peptoids as antimicrobial agents.     

AP液抗衰老作用的实验研究

Ｄ－半乳糖皮下注射致Ｗｉｓｔａｒ大鼠亚急性衰老模型,观察ＡＰ液对大鼠衰老指标的影响。实验结果显示,ＡＰ液能明显增加胸腺重／体重的比值,提高血清ＳＯＤ活性和降低血清ＬＰＯ活性。ＡＰ液抗衰老组血清ＳＯＤ活性比衰老模型组增加２５．２９％,血清ＬＰＯ活性下降６．２４％,提示ＡＰ液可能具有抗衰老作用。     

Central Role for MCP-1/CCL2 in Injury-Induced Inflammation Revealed by In Vitro,In Silico,and Clinical Studies

The translation of in vitro findings to clinical outcomes is often elusive. Trauma/hemorrhagic shock (T/HS) results in hepatic hypoxia that drives inflammation. We hypothesize that in silico methods would help bridge in vitro hepatocyte data and clinical T/HS, in which the liver is a primary site of inflammation. Primary mouse hepatocytes were cultured under hypoxia (1% O₂) or normoxia (21% O₂) for 1–72 h, and both the cell supernatants and protein lysates were assayed for 18 inflammatory mediators by Luminex™ technology. Statistical analysis and data-driven modeling were employed to characterize the main components of the cellular response. Statistical analyses, hierarchical and k-means clustering, Principal Component Analysis, and Dynamic Network Analysis suggested MCP-1/CCL2 and IL-1α as central coordinators of hepatocyte-mediated inflammation in C57BL/6 mouse hepatocytes. Hepatocytes from MCP-1-null mice had altered dynamic inflammatory networks. Circulating MCP-1 levels segregated human T/HS survivors from non-survivors. Furthermore, T/HS survivors with elevated early levels of plasma MCP-1 post-injury had longer total lengths of stay, longer intensive care unit lengths of stay, and prolonged requirement for mechanical ventilation vs. those with low plasma MCP-1. This study identifies MCP-1 as a main driver of the response of hepatocytes in vitro and as a biomarker for clinical outcomes in T/HS, and suggests an experimental and computational framework for discovery of novel clinical biomarkers in inflammatory diseases.     

Controlled Release of Dutasteride from Biodegradable Microspheres: In Vitro and In Vivo Studies

The aim of the present work was to study the in vitro/in vivo characteristics of dutasteride loaded biodegradable microspheres designed for sustained release of dutasteride over four weeks. An O/W emulsion-solvent evaporation method was used to incorporate dutasteride, which is of interest in the treatment of benign prostatic hyperplasia (BPH), into poly(lactide-co-glycolide) (PLGA). A response surface method (RSM) with central composite design (CCD) was employed to optimize the formulation variables. A prolonged in vitro drug release profile was observed, with a complete release of the entrapped drug within 28 days. The pharmacokinetics study showed sustained plasma drug concentration-time profile of dutasteride loaded microspheres after subcutaneous injection into rats. The in vitro drug release in rats correlated well with the in vivo pharmacokinetics profile. The pharmacodynamics evaluated by determination of the BPH inhibition in the rat models also showed a prolonged pharmacological response. These results suggest the potential use of dutasteride loaded biodegradable microspheres for the management of BPH over long periods.     

Systemic Inflammation Decreases Pain Threshold in Humans In Vivo

Background

Hyperalgesia is a well recognized hallmark of disease. Pro-inflammatory cytokines have been suggested to be mainly responsible, but human data are scarce. Changes in pain threshold during systemic inflammation evoked by human endotoxemia, were evaluated with three quantitative sensory testing methods.

Methods and Results

Pressure pain thresholds, electrical pain thresholds and tolerance to the cold pressor test were measured before and 2 hours after the intravenous administration of 2 ng/kg purified E. coli endotoxin in 27 healthy volunteers. Another 20 subjects not exposed to endotoxemia served as controls. Endotoxemia led to a rise in body temperature and inflammatory symptom scores and a rise in plasma TNF-α, IL-6, IL-10 and IL-1RA. During endotoxemia, pressure pain thresholds and electrical pain thresholds were reduced with 20±4 % and 13±3 %, respectively. In controls only a minor decrease in pressure pain thresholds (7±3 %) and no change in electrical pain thresholds occurred. Endotoxin-treated subjects experienced more pain during the cold pressor test, and fewer subjects were able to complete the cold pressor test measurement, while in controls the cold pressor test results were not altered. Peak levels and area under curves of each individual cytokine did not correlate to a change in pain threshold measured by one of the applied quantitative sensory testing techniques.

Conclusions and Significance

In conclusion, this study shows that systemic inflammation elicited by the administration of endotoxin to humans, results in lowering of the pain threshold measured by 3 quantitative sensory testing techniques. The current work provides additional evidence that systemic inflammation is accompanied by changes in pain perception.     

Antioxidant Activity of a New Xanthone Derivative from Aspidistra Letreae: In Vitro and In Silico Studies

A new xanthone derivative, aspidxanthone A ( 1 ), and three known compounds ((2S)-1-(β-D-galactopyranosyloxy)-3-(hexadecanoyloxy)propan-2-yl (9Z,12Z)-octadeca-9,12-dienoate ( 2 ), (25S)-spirostane-1β,3α,5β-triol ( 3 ), and asparenyldiol ( 4 )) were isolated from the whole of the endemic species Aspidistra letreae in Vietnam. Their structures were elucidated by means of extensive spectroscopic analyses and comparison with published data. In this study, we report the isolation and structure elucidation of a new compound aspidxanthone A, antioxidant activities of the extract and isolates 1 – 4 , and in silico molecular docking of aspidxanthone A. The ethyl acetate extract had good antioxidant activity with an IC₅₀ value of 26.3 μg mL⁻¹. Among the isolates, aspidxanthone A exhibited DPPH reduction activity with an IC₅₀ value of 11.2 μM, which is in the same range as that of the positive control, ascorbic acid. The mechanism of action of aspidxanthone A on the tyrosinase and xanthine oxidase proteins have been clarified by in silico studies.     

In Silico Prediction and In Vivo Validation of Daphnia pulex Micrornas

Daphnia pulex, the crustacean with the first sequenced genome, is an important organism that has been widely used in ecological and toxicological research. MicroRNAs (miRNAs) are 21–25 nucleotide small non-coding RNAs that are involved in a myriad of physiological processes. In this research, we predicted 75 D. pulex miRNAs by sequence homology and secondary structure identification from the full genome sequence. Fourteen predicted miRNAs were selected for quantitative real time polymerase chain reaction (RT-PCR) validation. Out of these, eight (mir-8, mir-9, mir-12, mir-92, mir-100, mir-133, mir-153 and mir-283) were successfully amplified and validated. Next, expression levels were quantified at three different life stages (days 4, 8 and 12 of age) using U6 spliceosomal RNA as a reference gene. The expression of mir-8, mir-9, mir-12, mir-92 and mir-100 significantly differed across time suggesting these microRNAs might play a critical role during D. pulex development. This is the first study to identify and validate miRNAs in D. pulex, which is an important first step in further studies that evaluate their roles in development and response to environmental and ecological stimuli.     

Radiation-Induced DNA Methylation Disorders: In Vitro and In Vivo Studies

Biology Bulletin - Phenomenological aspects and mechanisms of DNA methylation disorders (changes in the total level of DNA methylation and in genome repetitive elements, locus-specific methylation...     

Compound Ex Vivo and In Silico Method for Hemodynamic Analysis of Stented Arteries

Hemodynamic factors such as low wall shear stress have been shown to influence endothelial healing and atherogenesis in stent-free vessels. However, in stented vessels, a reliable quantitative analysis of such relations has not been possible due to the lack of a suitable method for the accurate acquisition of blood flow. The objective of this work was to develop a method for the precise reconstruction of hemodynamics and quantification of wall shear stress in stented vessels. We have developed such a method that can be applied to vessels stented in or ex vivo and processed ex vivo. Here we stented the coronary arteries of ex vivo porcine hearts, performed vascular corrosion casting, acquired the vessel geometry using micro-computed tomography and reconstructed blood flow and shear stress using computational fluid dynamics. The method yields accurate local flow information through anatomic fidelity, capturing in detail the stent geometry, arterial tissue prolapse, radial and axial arterial deformation as well as strut malapposition. This novel compound method may serve as a unique tool for spatially resolved analysis of the relationship between hemodynamic factors and vascular biology. It can further be employed to optimize stent design and stenting strategies.