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

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

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

Plasmin Regulation through Allosteric, Sulfated, Small Molecules

Plasmin, a key serine protease, plays a major role in clot lysis and extracellular matrix remodeling. Heparin, a natural polydisperse sulfated glycosaminoglycan, is known to allosterically modulate plasmin activity. No small allosteric inhibitor of plasmin has been discovered to date. We screened an in-house library of 55 sulfated, small glycosaminoglycan mimetics based on nine distinct scaffolds and varying number and positions of sulfate groups to discover several promising hits. Of these, a pentasulfated flavonoid-quinazolinone dimer 32 was found to be the most potent sulfated small inhibitor of plasmin (IC50 = 45 μM, efficacy = 100%). Michaelis-Menten kinetic studies revealed an allosteric inhibition of plasmin by these inhibitors. Studies also indicated that the most potent inhibitors are selective for plasmin over thrombin and factor Xa, two serine proteases in coagulation cascade. Interestingly, different inhibitors exhibited different levels of efficacy (40%–100%), an observation alluding to the unique advantage offered by an allosteric process. Overall, our work presents the first small, synthetic allosteric plasmin inhibitors for further rational design

Anti-cancer Prodrugs-Three Decades of Design

The conventional old treatment method for cancer therapy is associated with severe side effects along with several limitations. Therefore, searching and developing new methods for cancer became crucial. This mini review was devoted on the design and synthesis of prodrugs for cancer treatment. The methods discussed include targeted prodrugs which are depending on the presence of unique cellular conditions at the desired target, especially the availability of certain enzymes and transporters at these target sites, antibody directed enzyme prodrug therapy (ADEPT), gene-directed enzyme prodrug therapy (GDEPT) which is considered one of the important strategies for the treatment of cancer and prodrugs based on enzyme models that have been advocated to understand enzyme catalysis. In this approach, a design of prodrugs is accomplished using computational calculations based on molecular orbital and molecular mechanics methods. Correlations between experimental and calculated rate values for some intramolecular processes provided a tool to predict thermodynamic and kinetic parameters for intramolecular processes that can be utilized as prodrugs linkers. This approach does not require any enzyme to catalyze the prodrug interconversion. The interconversion rate is solely dependent on the factors govern the limiting step of the intramolecular process

Reef sponges facilitate the transfer of coral-derived organic matter to their associated fauna via the sponge loop

The high biodiversity of coral reefs results in complex trophic webs where energy and nutrients are transferred between species through a multitude of pathways. Here, we hypothesize that reef sponges convert the dissolved organic matter released by benthic primary producers (e.g. corals) into particulate detritus that is transferred to sponge-associated detritivores via the sponge loop pathway. To test this hypothesis, we conducted stable isotope (13C and15N) tracer experiments to investigate the uptake and transfer of coral-derived organic matter from the sponges Mycale fistulifera and Negombata magnifica to 2 types of detritivores commonly associated with sponges: ophiuroids (Ophiothrix savignyi and Ophiocoma scolopendrina) and polychaetes (Polydorella smurovi). Findings revealed that the organic matter naturally released by the corals was indeed readily assimilated by both sponges and rapidly released again as sponge detritus. This detritus was subsequently consumed by the detritivores, demonstrating transfer of coral-derived organic matter from sponges to their associated fauna and confirming all steps of the sponge loop. Thus, sponges provide a trophic link between corals and higher trophic levels, thereby acting as key players within reef food webs

Effect of Various Local Anthropogenic Impacts on the Diversity of Coral Mucus-Associated Bacterial Communities

The global continued decline in coral reefs is intensifying the need to understand the response of corals to local environmental stressors. Coral-associated bacterial communities have been suggested to have a swift response to environmental pollutants. This study aims to determine the variation in the bacterial communities associated with the mucus of two coral species, Pocillopora damicornis (Linnaeus, 1758) and Stylophora pistillata (Esper, 1792), and the coral-surrounding seawater from three areas exposed to contamination at the Jordanian coast of the Gulf of Aqaba (Red Sea), and also explores the antibacterial activity of these bacteria. Corals were collected from three contaminated zones along the coast, and the bacteria were quantified and identified by conventional morphological and biochemical tests, as well as 16S rRNA gene sequencing. The average number of bacteria significantly varied among the coral mucus from the sampling zones and between the coral mucus and the surrounding seawater. The P. damicornis mucus-associated bacterial community was dominated by members of the classes Gammaproteobacteria, Cytophagia, and Actinomycetia, while the mucus of S. pistillata represented higher bacterial diversity, with the dominance of the bacterial classes Gammaproteobacteria, Actinomycetia, Alphaproteobacteria, and Bacilli. The effects of local anthropogenic impacts on coral mucus bacterial communities were represented in the increased abundance of bacterial species related to coral diseases. Furthermore, the results demonstrated the existence of bacterial isolates with antibacterial activity that possibly acted as a first line of defense to protect and maintain the coral host against pathogens. Indeed, the dynamics of coral-associated microbial communities highlight the importance of holistic studies that focus on microbial interactions across the coral reef ecosystem

Engineering rotating apical-out airway organoid for assessing respiratory cilia motility

Motile cilia project from the airway apical surface and directly interface with inhaled external environment. Owing to cilia\u27s nanoscale dimension and high beating frequency, quantitative assessment of their motility remains a sophisticated task. Here we described a robust approach for reproducible engineering of apical-out airway organoid (AOAO) from a defined number of cells. Propelled by exterior-facing cilia beating, the mature AOAO exhibited stable rotational motion when surrounded by Matrigel. We developed a computational framework leveraging computer vision algorithms to quantify AOAO rotation and correlated it with the direct measurement of cilia motility. We further established the feasibility of using AOAO rotation to recapitulate and measure defective cilia motility caused by chemotherapy-induced toxicity and by CCDC39 mutations in cells from patients with primary ciliary dyskinesia. We expect our rotating AOAO model and the associated computational pipeline to offer a generalizable framework to expedite the modeling of and therapeutic development for genetic and environmental ciliopathies

Mutation of CFAP57, a protein required for the asymmetric targeting of a subset of inner dynein arms in Chlamydomonas, causes primary ciliary dyskinesia

Primary ciliary dyskinesia (PCD) is characterized by chronic airway disease, reduced fertility, and randomization of the left/right body axis. It is caused by defects of motile cilia and sperm flagella. We screened a cohort of affected individuals that lack an obvious axonemal defect for pathogenic variants using whole exome capture, next generation sequencing, and bioinformatic analysis assuming an autosomal recessive trait. We identified one subject with an apparently homozygous nonsense variant [(c.1762C\u3eT), p.(Arg588*)] in the uncharacterized CFAP57 gene. Interestingly, the variant results in the skipping of exon 11 (58 amino acids), which may be due to disruption of an exonic splicing enhancer. In normal human nasal epithelial cells, CFAP57 localizes throughout the ciliary axoneme. Nasal cells from the PCD patient express a shorter, mutant version of CFAP57 and the protein is not incorporated into the axoneme. The missing 58 amino acids include portions of WD repeats that may be important for loading onto the intraflagellar transport (IFT) complexes for transport or docking onto the axoneme. A reduced beat frequency and an alteration in ciliary waveform was observed. Knockdown of CFAP57 in human tracheobronchial epithelial cells (hTECs) recapitulates these findings. Phylogenetic analysis showed that CFAP57 is highly conserved in organisms that assemble motile cilia. CFAP57 is allelic with the BOP2/IDA8/FAP57 gene identified previously in Chlamydomonas reinhardtii. Two independent, insertional fap57 Chlamydomonas mutant strains show reduced swimming velocity and altered waveforms. Tandem mass tag (TMT) mass spectroscopy shows that FAP57 is missing, and the g inner dyneins (DHC7 and DHC3) and the d inner dynein (DHC2) are reduced, but the FAP57 paralog FBB7 is increased. Together, our data identify a homozygous variant in CFAP57 that causes PCD that is likely due to a defect in the inner dynein arm assembly process