Designing Direct and Indirect Factor Xa Inhibitors

Anticoagulants are the basis for treatment and prevention of thrombotic diseases. The currently available medicines are associated with a wide range of adverse reactions that mandates developing new anticoagulants. Several lines of evidence support the superiority of factor Xa (FXa) as a promising target to develop novel anticoagulants. This work focuses on the design of direct and indirect FXa inhibitors using an interdisciplinary approach. As indirect FXa inhibitors, a focused library of tetrasulfated N–arylacyl tetrahydroisoquinoline (THIQ) nonsaccharide allosteric antithrombin activators was designed, synthesized, and biochemically evaluated to establish their structure–activity relationship (SAR). An N–arylacyl THIQ analog having carboxylate at position–3, two sulfate groups at positions–5 and –8 of THIQ moiety, butanoyl linker, and two sulfate groups at positions–2 and –5 of the phenolic monocyclic moiety was identified as the most promising nonsaccharide antithrombin activator with KD of 1322 ± 237 μM and acceleration potential of 80–fold. Its biochemical profile indicates a strong possibility that it activates antithrombin by the pre–equilibrium pathway rather than the induced–fit mechanism utilized by heparin analogs. A similar interdisciplinary approach was exploited to design direct FXa inhibitors that possess high selectivity and are potentially orally bioavailable. Structurally, the designed direct FXa inhibitors are neutral THIQ dicarboxamides. THIQ dicarboxamide is a privileged structure with a semi–rigid character, a structural feature that potentially offers high selectivity for targeting FXa over other coagulation and digestive proteases. It can also be thought of as an amino acid–like structure, which affords accessibility to a large number of compounds using well established peptide chemistry. Mechanistically, the designed inhibitors were expected to bind to FXa in the active site and function as orthosteric inhibitors. These direct FXa active site inhibitors are also likely to inhibit clot–bound enzyme. Nearly 60 THIQ dicarboxamides were synthesized and biochemically evaluated. Through detailed SAR analysis, the most potent analog was designed and found to exhibit an IC50 of 270 nM (Ki = 135 nM), an improvement of more than 207–fold over the first inhibitor synthesized in the study. The most potent inhibitor displayed at least 1887–fold selectivity for FXa over other coagulation enzymes and a selectivity index of at least 279–fold over the digestive serine proteases. This analog doubled plasma clotting times at 17–20 μM, which are comparable to those of agents being currently studied in clinical trials. Overall, allosteric and orthosteric approaches led to the design of indirect and direct small molecule inhibitors of FXa based on the THIQ scaffold. This work introduces two promising molecules, a tetrasulfated N–arylacyl THIQ analog as a heparin mimetic and a neutral THIQ dicarboxamide as a potent, selective, and potentially bioavailable peptidomimetic, for further advanced medicinal chemistry studies

Allosteric Inhibition of Factor XIIIa. Non-Saccharide Glycosaminoglycan Mimetics, but Not Glycosaminoglycans, Exhibit Promising Inhibition Profile

Factor XIIIa (FXIIIa) is a transglutaminase that catalyzes the last step in the coagulation process. Orthostery is the only approach that has been exploited to design FXIIIa inhibitors. Yet, allosteric inhibition of FXIIIa is a paradigm that may offer a key advantage of controlled inhibition over orthosteric inhibition. Such an approach is likely to lead to novel FXIIIa inhibitors that do not carry bleeding risks. We reasoned that targeting a collection of basic amino acid residues distant from FXIIIa’s active site by using sulfated glycosaminoglycans (GAGs) or non-saccharide GAG mimetics (NSGMs) would lead to the discovery of the first allosteric FXIIIa inhibitors. We tested a library of 22 variably sulfated GAGs and NSGMs against human FXIIIa to discover promising hits. Interestingly, although some GAGs bound to FXIIIa better than NSGMs, no GAG displayed any inhibition. An undecasulfated quercetin analog was found to inhibit FXIIIa with reasonable potency (efficacy of 98%). Michaelis-Menten kinetic studies revealed an allosteric mechanism of inhibition. Fluorescence studies confirmed close correspondence between binding affinity and inhibition potency, as expected for an allosteric process. The inhibitor was reversible and at least 9-fold- and 26-fold selective over two GAG-binding proteins factor Xa (efficacy of 71%) and thrombin, respectively, and at least 27-fold selective over a cysteine protease papain. The inhibitor also inhibited the FXIIIa-mediated polymerization of fibrin in vitro. Overall, our work presents the proof-of-principle that FXIIIa can be allosterically modulated by sulfated non-saccharide agents much smaller than GAGs, which should enable the design of selective and safe anticoagulants

Toxicity of Bacillus thuringiensis β-exotoxins and δ-endotoxins to Drosophila melanogaster, Ephestia kuhniella and human erythrocytes

A total of 73 Bacillus thuringiensis (Bt) strains were screened for the presence of non-hemolytic insecticidal β-exotoxin-free δ-endotoxins. Out of them, 45 Bt strains produced δ-endotoxins with specific insecticidal activity against Drosophila melanogaster and/or Ephestia kuhniella larvae. The thermostable β-exotoxin was observed only in 15 Bt strains and appeared to exhibit dual non-specific insecticidal activity against both D. melanogaster and E. kuhniella larvae and showed in vitro hemolysis for human erythrocytes. It was found that β-exotoxin was produced by Bt strains belonging to five serovars (israelensis, kenyae, kurstaki, pakistani, and tohokuensis) and two non-serotypable strains. This result suggests that β-exotoxin production is a strain-specific property rather than a serovar-specific property. To our knowledge, this is the first study that demonstrates β-exotoxins production association with Bt strains belonging to serovars israelensis, pakistani, and tohokuensis. The plasmid DNA profiles of some β-exotoxin producing Bt strains shared large plasmid patterns which may have the common β-exotoxin regulatory gene(s). It was found that 16 local Bt strains, 15 of which belonged to five serovars (aizawai, israelensis, kurstaki, morrisoni, and pakistani) and one was autoagglutinated strain, produced non-hemolytic insecticidal β-exotoxin-free δ-endotoxins. Based on random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR), the genotypic relatedness among these 16 Bt strains was investigated. The strains were grouped into two clusters. Bt strains within serovars israelensis were grouped in two subclusters, Bt strains within serovars aizawai were genomically homogeneous and clustered together, while the other serovars were grouped together in one subcluster. The autoagglutinated strain was clustered within serovar israelensis. Thus, these δ-endotoxins can be developed for the use in Bt-based insecticidal preparations.Keywords: Endotoxin, exotoxin, hemolytic, serovar, thuringiensi

Dynamics of confrontation between popular mobilization and state In Jordan: An analytical social conflict approach

This study aimed at analyzing the dynamics of confrontation between popular mobilization and state in Jordan from the sociological perspective of an analytical social conflict. It also concentrated on the factors that led to the emergence of popular mobilization and its Legitimacy and the variables that guided the peaceful confrontation between mobilization and state. The study results showed that economic deprivation was the main factor behid the emergence of the popular mobilization, and the political demands were emerged when the state didn’t achieve the economical Reforms , and the mobilization derives its legitimacy From its objective economical demands, the Arab revolutions and the concessions of the state which mean recognition of the Mobilization and its demands. Then the results showed that the mobilization was Fall back because of its lack of organization leadership and Ideology and the cleavages in the structure of national identity , but the conscious of the mobilization and its legitimacy may intense it again. Besides, The results indicated that the confrontation was included dialectical Relationship between the peaceful oriention of the state toward the mobilizations and the peaceful orientation of the mobilization from the other hand. This dialectical relationship constituted moral treaty between the two partisans stand out as safty valve against violence and polarization. After that, the study showed some Factors which, sometimes , provoked violence such as : the partisans, usage of violent power, and arrestation of mobilization’s activists . Then, the study showed that the confrontation was realistic , but didn’t occur substantial change in the social structure, However, it escalated the level of freedom, Raised the power of the street, and obtained the state and the society more flexibility and democracy. With regard to the theoretical approach, This study showed that the perspectives of analytical conflict are of complementarily nature, and it could be syenthesized to produce a new theoretical perspective

Influence of cupping treatment on high-intensity anaerobic performance

The use of cupping therapy prior to sports events has increased in popularity, with limited evidence to support its efficacy. The purpose of this study was to evaluate the efficacy of dry and wet cupping therapy on subsequent Wingate anaerobic test (WAnT) performance. Twelve trained men participated in this repeated measures randomized crossover study (age 24.9 ± 4.8 years; body mass index 27.6 ± 14.3 kg.m-2). Participants were familiarized with the ergometer and the Wingate anaerobic test on three separate occasions. They then randomly performed three experimental Wingate tests separated by 48-72 h after either dry cupping (DRY), wet cupping (WET), or no treatment (CON). Repeated measures ANOVA and Pearson’s correlation coefficient were used to analyze data and determine the relationships between WAnT and peak lactate and heart rate (HR). Peak power (PP), mean power (MP), and fatigue index (FI) were similar in all treatments (p=.47-.72). Heart rate (HR) and lactate increased similarly at all time points in all treatments (p<.001 for all comparisons). Post-WAnT peak HR was moderately negatively correlated with PP in all treatments and MP in CON only (p<.05 for all correlations). No other significant correlations were detected. The present findings demonstrate no beneficial effects of wet and dry cupping therapy, and hence do not support its use prior to high-intensity anaerobic sports events

Progress in diagnosing primary ciliary dyskinesia: The North American perspective

Primary Ciliary Dyskinesia (PCD) is a rare, under-recognized disease that affects respiratory ciliary function, resulting in chronic oto-sino-pulmonary disease. The PCD clinical phenotype overlaps with other common respiratory conditions and no single diagnostic test detects all forms of PCD. In 2018, PCD experts collaborated with the American Thoracic Society (ATS) to create a clinical diagnostic guideline for patients across North America, specifically considering the local resources and limitations for PCD diagnosis in the United States and Canada. Nasal nitric oxide (nNO) testing is recommended for first-line testing in patients ≥5 years old with a compatible clinical phenotype; however, all low nNO values require confirmation with genetic testing or ciliary electron micrograph (EM) analysis. Furthermore, these guidelines recognize that not all North American patients have access to nNO testing and isolated genetic testing is appropriate in cases with strong clinical PCD phenotypes. For unresolved diagnostic cases, referral to a PCD Foundation accredited center is recommended. The purpose of this narrative review is to provide insight on the North American PCD diagnostic process, to enhance the understanding of and adherence to current guidelines, and to promote collaboration with diagnostic pathways used outside of North America

Isolation, characterization, and hydrolytic activities of Geobacillus species from Jordanian hot springs

The present study was conducted to isolate, identify, characterize and to determine the enzymatic activities of the thermophilic Geobacillus species from five Jordanian hot springs. Based on phenotypic characters, eight thermophilic isolates were identified and belonged to the genus Geobacillus. The Geobacillus isolates were abundant in all investigated hot springs. The optimal temperature for growth of the isolates was 60 to 65°C and the optimal pH was 6 to 8. Colonies were light yellow circular to rhizoid. The bacterial cells were Gram positive rods and endospore forming. All isolates produced amylase, caseinase, alkaline and acid phosphatases, esterase (C4), esterase lipase (C8), α-Galactosidase, β-Glucuronidase, β-Glucosidase, and N-Acetyl-β-glucosaminidase. Seven isolates produced leucine and valine arylamidases and five isolates produced naphthol-AS-B1- phsphohydrolase. Lipase (C14) activity from two isolates and α-chymotrypsin activity from three isolates were also detected. The phenotypic characterization of those isolates was confirmed by genotypic method using 16S rDNA sequence analysis. Maximal homology of all eight  isolates to genus Geobacillus was observed. Five of these isolates showed greater than 98% homology with Geobacillus stearothermophilus and one isolate showed 100% homology with Geobacillus thermoglucosidasius. Therefore, 16S rRNA gene sequence analysis can be considered as a valuable genotypic tool for the identification and characterization of thermophilic bacteria at genus level. Moreover, enzymatic products of those isolates could receive considerable attention due to their potential applications in biotechnology.Keywords: Thermophiles, Geobacillus, hydrolytic enzymes, hot spring, 16S rRNA

Photosynthesis-controlled calcification in a hypersaline microbial mat

We investigated the hypothesis that sulfate reduction rather than oxygenic photosynthesis promotes calcification in a hypersaline microbial mat by increasing the ion concentration product: ICP 5 [Ca2+] X [CO32-]. Pore‐water 3 calcium concentration profiles directly measured with microsensors show that calcium concentration in the photic zone decreased in illuminated mats and increased slightly in dark mats. High pH values in the photic zone of illuminated mats resulted in higher carbonate concentrations (2.25 mmol L-1) than in dark mats (0.75 mmol L-1), although the dissolved inorganic carbon (DIC) pore‐water concentration in the former was much lower (5.9 mmol L-1) than in the latter (9.9 mmol L-1). The pH‐induced rise in carbonate concentration in the light was the main factor influencing the ICP, while changes in Ca-1 concentration played a subsidiary role. Sulfate reduction did not result in a net pH increase in these mats, as rates in the photic zone were comparable between illuminated and dark mats (4 and 5 nmol cm-2 h-1, respectively), and pH increased in illuminated mats but not in dark mats. Calcium carbonate precipitation in the photic zone of these hypersaline mats is primarily controlled by photosynthesisinduced pH and carbonate concentration increases. However, heterotrophic bacteria, including sulfate reducers, play an important complementary role in calcification because they maintain high concentrations of DIC in the mat pore water