High-throughput screening to identify potential inhibitors of the Zα domain of the adenosine deaminase 1 (ADAR1)

Adenosine deaminases acting on RNA 1 (ADAR1) are enzymes involved in editing adenosine to inosine in the dsRNAs of cells associated with cancer development. The p150 isoform of ADAR1 is the only isoform containing the Zα domain that binds to both Z-DNA and Z-RNA. The Zα domain is suggested to modulate the immune response and could be a suitable target for antiviral treatment and cancer immunotherapy. In this study, we aimed to identify potential inhibitors for ADAR1 protein that bind the Zα domain using molecular docking and simulation tools. Virtual docking and molecular dynamics simulation approaches were used to screen the potential activity of 2115 FDA-approved compounds on the Zα domain of ADAR1 and filtered for to obtain the top-scoring hits. The top three compounds with the best XP Gscore—namely alendronate (−7.045), etidronate (−6.923), and zoledronate (−6.77)—were subjected to 50 ns simulations to characterize complex stability and identify the fundamental interactions that contribute to inhibition of the ADAR1 Zα domain. The three compounds were shown to interact with Lys169, Lys170, Asn173, and Tyr177 of the Zα domain-like helical backbone of Z-RNA. The study provides a comprehensive and novel insights of repurposes drugs for the inhibition of ADAR1 function.     

Response surface methodology-based optimization of glucose acylation bio-catalyzed by immobilized lipase

This paper describes in detail the selection and optimization of immobilized lipases for enhanced regioselective acylation of glucose into glucose monolaurate (GlcML). Initially, nature of biocatalyst, immobilization approach, reaction media, glucose, and lauric acid concentration were screened out. Finally, lipases from Rhizopus arrhizus immobilized on dead mycelia were investigated under various reaction conditions (Temperature, shaking speed, enzyme dose, and water content) following a fully rotatable central composite design (FRCCD) to optimize the activity of lipases. The immobilized lipases-based biocatalysts in the presence of polar solvents (tertiary alcohols) and higher concentrations of substrates i.e. glucose and lauric acid (100 and 300?mmol?L^?1, respectively) offered conversion rate of 1.5 mmolmin^?1?L^?1. Moreover, optimization of reaction conditions revealed that 162.5 lipase units/100mL at 31.25?°C, 3% water content, and 105?RPM shaking speed enhanced the conversion rate by 0.5 mmolmin^?1?L^?1 rendering the reaction more economical. Hence, lipases-based immobilized biocatalysts may provide an intelligent and green choice for commercial scale synthesis of GlcML for food and pharmaceutical industries.     

Increased efficacy of HIV-1 neutralization by antibodies at low CCR5 surface concentration

It has been observed that some antibodies, including the CD4-induced (CD4i) antibody IgG X5 and the gp41-specific antibody IgG 2F5, exhibit higher neutralizing activity in PBMC-based assays than in cell line based assays [J.M. Binley, T. Wrin, B. Korber, M.B. Zwick, M. Wang, C. Chappey, G. Stiegler, R. Kunert, S. Zolla-Pazner, H. Katinger, C.J. Petropoulos, D.R. Burton, Comprehensive cross-clade neutralization analysis of a panel of anti-human immunodeficiency virus type 1 monoclonal antibodies, J. Virol. 78 (2004) 13232-13252]. It has been hypothesized that the lower CCR5 concentration on the surface of the CD4 T lymphocytes compared to that on cell lines used for the neutralization assays could be a contributing factor to the observed differences in neutralizing activity. To test this hypothesis and to further elucidate the contribution of CCR5 concentration differences on antibody neutralizing activity, we used a panel of HeLa cell lines with well-defined and differential surface concentrations of CCR5 and CD4 in a pseudovirus-based assay. We observed that the CCR5 cell surface concentration but not the CD4 concentration had a significant effect on the inhibitory activity of X5 and several other CD4i antibodies including 17b and m9, as well as that of the gp41-specifc antibodies 2F5 and 4E10 but not on that of the CD4 binding site antibody (CD4bs), b12. The 50% inhibitory concentration (IC50) decreased up to two orders of magnitude in cell lines with low CCR5 concentration corresponding to that in CD4 T cells used in PBMC-based assays (about 10(3) per cell) compared to cell lines with high CCR5 concentration (about 10(4) or more). Our results suggest that the CCR5 cell surface concentration could be a contributing factor to the high neutralizing activities of some antibodies in PBMC-based-assays but other factors could also play an important role. These findings could have implications for development of vaccine immunogens based on the epitopes of X5 and other CD4i antibodies, for elucidation of the mechanisms of HIV-1 neutralization by antibodies, and for design of novel therapeutic approaches.     

Efficient tumor killing and minimal cytokine release with novel T-cell agonist bispecific antibodies

T-cell-recruiting bispecific antibodies (T-BsAbs) have shown potent tumor killing activity in humans, but cytokine release-related toxicities have affected their clinical utility. The use of novel anti-CD3 binding domains with more favorable properties could aid in the creation of T-BsAbs with improved therapeutic windows. Using a sequence-based discovery platform, we identified new anti-CD3 antibodies from humanized rats that bind to multiple epitopes and elicit varying levels of T-cell activation. In T-BsAb format, 12 different anti-CD3 arms induce equivalent levels of tumor cell lysis by primary T-cells, but potency varies by a thousand-fold. Our lead CD3-targeting arm stimulates very low levels of cytokine release, but drives robust tumor antigen-specific killing in vitro and in a mouse xenograft model. This new CD3-targeting antibody underpins a next-generation T-BsAb platform in which potent cytotoxicity is uncoupled from high levels of cytokine release, which may lead to a wider therapeutic window in the clinic.     

2-Deoxy-D-glucose reverses the Indian red scorpion venom-induced cardiopulmonary abnormalities in anesthetized rats

Role of 2-Deoxy-D-glucose (2-DG) in reversing the Indian red scorpion (Mesobuthus tamulus concanesis Pocock, MBT) venom-induced toxicity was examined. Femoral arterial pressure, ECG and respiratory movements were recorded in urethane anesthetized rats. Plasma glucose and serum insulin levels were also estimated. Intravenous injection of 5 mg/kg MBT venom produced immediate decrease in mean arterial pressure, heart rate and respiratory frequency followed by an increase and subsequent progressive decrease. ECG pattern exhibited ischaemic changes. There was hyperinsulinemia after venom without corresponding decrease in plasma glucose. The animals died within 37 +/- 9 min and demonstrated significant increase in pulmonary water content. 2-DG pretreatment (0.5 g/kg, iv) improved the cardiopulmonary abnormalities induced by venom and the animals survived for nearly 120 min. There was no hyperinsulinemia and increased pulmonary water content in these animals. In insulin (2 IU/kg) treated rats, the MBT venom-induced cardiopulmonary abnormalities were attenuated and ECG abnormalities were reversed. The pulmonary water content in these animals exhibited a decreasing trend and the animals survived for 120 min. Repaglinide (10 microg/kg, iv) pretreatment failed to reverse the venom-induced cardiopulmonary changes including the increased pulmonary water content. The survival time was similar to venom only group. The present results reveal that 2-DG reverses the venom-induced cardiopulmonary toxicity probably by restoring insulin sensitivity.     

History shaped the geographic distribution of genomic admixture on the island of Puerto Rico

Contemporary genetic variation among Latin Americans human groups reflects population migrations shaped by complex historical, social and economic factors. Consequently, admixture patterns may vary by geographic regions ranging from countries to neighborhoods. We examined the geographic variation of admixture across the island of Puerto Rico and the degree to which it could be explained by historic and social events. We analyzed a census-based sample of 642 Puerto Rican individuals that were genotyped for 93 ancestry informative markers (AIMs) to estimate African, European and Native American ancestry. Socioeconomic status (SES) data and geographic location were obtained for each individual. There was significant geographic variation of ancestry across the island. In particular, African ancestry demonstrated a decreasing East to West gradient that was partially explained by historical factors linked to the colonial sugar plantation system. SES also demonstrated a parallel decreasing cline from East to West. However, at a local level, SES and African ancestry were negatively correlated. European ancestry was strongly negatively correlated with African ancestry and therefore showed patterns complementary to African ancestry. By contrast, Native American ancestry showed little variation across the island and across individuals and appears to have played little social role historically. The observed geographic distributions of SES and genetic variation relate to historical social events and mating patterns, and have substantial implications for the design of studies in the recently admixed Puerto Rican population. More generally, our results demonstrate the importance of incorporating social and geographic data with genetics when studying contemporary admixed populations.     

Rapid intra‐operative diagnosis of kidney cancer by attenuated total reflection infrared spectroscopy of tissue smears

Herein, a technique to analyze air‐dried kidney tissue impression smears by means of attenuated total reflection infrared (ATR‐IR) spectroscopy is presented. Spectral tumor markers—absorption bands of glycogen—are identified in the ATR‐IR spectra of the kidney tissue smear samples. Thin kidney tissue cryo‐sections currently used for IR spectroscopic analysis lack such spectral markers as the sample preparation causes irreversible molecular changes in the tissue. In particular, freeze‐thaw cycle results in degradation of the glycogen and reduction or complete dissolution of its content. Supervised spectral classification was applied to the recorded spectra of the smears and the test spectra were classified with a high accuracy of 92% for normal tissue and 94% for tumor tissue, respectively. For further development, we propose that combination of the method with optical fiber ATR probes could potentially be used for rapid real‐time intra‐operative tissue analysis without interfering with either the established protocols of pathological examination or the ordinary workflow of operating surgeon. Such approach could ensure easier transition of the method to clinical applications where it may complement the results of gold standard histopathology examination and aid in more precise resection of kidney tumors.      

Mitochondria play an important role in 17beta-estradiol attenuation of H(2)O(2)-induced rat endothelial cell apoptosis

Studies have shown salutary effects of 17beta-estradiol following trauma-hemorrhage on different cell types. 17beta-Estradiol also induces improved circulation via relaxation of the aorta and has an anti-apoptotic effect on endothelial cells. Because mitochondria play a pivotal role in apoptosis, we hypothesized that 17beta-estradiol will maintain mitochondrial function and will have protective effects against H(2)O(2)-induced apoptosis in endothelial cells. Endothelial cells were isolated from rats' aorta and cultured in the presence or absence of H(2)O(2), a potent inducer of apoptosis. In additional studies, endothelial cells were pretreated with 17beta-estradiol. Flow cytometry analysis revealed H(2)O(2)-induced apoptosis in 80.9% of endothelial cells; however, prior treatment of endothelial cells with 17beta-estradiol resulted in an approximately 40% reduction in apoptosis. This protective effect of 17beta-estradiol was abrogated when endothelial cells were cultured in the presence ICI-182780, indicating the involvement of estrogen receptor (ER). Fluorescence microscopy revealed a 17beta-estradiol-mediated attenuation of H(2)O(2)-induced mitochondrial condensation. Western blot analysis demonstrated that H(2)O(2)-induced cytochrome c release from mitochondrion to cytosol and the activation of caspase-9 and -3 were decreased by 17beta-estradiol. These findings suggest that 17beta-estradiol attenuated H(2)O(2)-induced apoptosis via ER-dependent activation of caspase-9 and -3 in rat endothelial cells through mitochondria.     

Liver cytokine production and ICAM-1 expression following bone fracture, tissue trauma, and hemorrhage in middle-aged mice

Although studies have indicated that hemorrhagic shock and resuscitation produces hepatic damage by mechanisms involving adhesion molecules in endothelial cells and hepatocytes, it is not known if there is any difference in the extent of hepatic damage following bone fracture, soft tissue trauma, and hemorrhage (Fx-TH) between young and middle-aged animals. To study this, young (6-8 wk) and middle-aged (approximately 12 mo) C3H/HeN male mice were subjected to a right lower leg fracture, soft tissue trauma, (i.e., midline laparotomy), and hemorrhage (blood withdrawal to decrease the blood pressure to 35 +/- 5 mmHg for 90 min) followed by resuscitation with four times the shed blood volume in the form of lactated Ringer solution. Mice were euthanized 24 h later, and liver tissues were harvested. Total bilirubin levels in the hepatocyte extract increased markedly following Fx-TH in both groups of mice; however, the increase in middle-aged mice was significantly higher compared with young mice. TNF-alpha and IL-6 levels in the hepatocyte extract following Fx-TH increased significantly in middle-aged mice but remained unchanged in young mice. IL-10 levels significantly decreased in middle-aged mice following Fx-TH but remained unchanged in young mice. Kupffer cells from middle-aged mice produced significantly higher IL-6 and IL-10 levels compared with young mice. Protein levels and mRNA expression of ICAM-1 in hepatocytes were also significantly higher in middle-aged mice compared with young mice following Fx-TH. These results collectively suggest that the extent of hepatic damage following Fx-TH is dependent on the age of the subject.