Alpha-mannosidosis in Tunisian consanguineous families: Potential involvement of variants in GHR and SLC19A3 genes in the variable expressivity of cognitive impairment

Alpha-Mannosidosis (AM) is an ultra-rare storage disorder caused by a deficiency of lysosomal alpha-mannosidase encoded by the MAN2B1 gene. Clinical presentation of AM includes mental retardation, recurrent infections, hearing loss, dysmorphic features, and motor dysfunctions. AM has never been reported in Tunisia. We report here the clinical and genetic study of six patients from two Tunisian families with AM. The AM diagnosis was confirmed by an enzymatic activity assay. Genetic investigation was conducted by Sanger sequencing of the mutational hotspots for the first family and by ES analysis for the second one. In the first family, a frameshift duplication p.(Ser802GlnfsTer129) was identified in the MAN2B1 gene. For the second family, ES analysis led to the identification of a missense mutation p.(Arg229Trp) in the MAN2B1 gene in four affected family members. The p.(Ser802GlnfsTer129) mutation induces a premature termination codon which may trigger RNA degradation by the NMD system. The decrease in the levels of MAN2B1 synthesis could explain the severe phenotype observed in the index case. According to the literature, the p.(Arg229Trp) missense variant does not have an impact on MAN2B1 maturation and transportation, which correlates with a moderate clinical sub-type. To explain the intra-familial variability of cognitive impairment, exome analysis allowed the identification of two likely pathogenic variants in GHR and SLC19A3 genes potentially associated to cognitive decline. The present study raises awareness about underdiagnosis of AM in the region that deprives patients from accessing adequate care. Indeed, early diagnosis is critical in order to prevent disease progression and to propose enzyme replacement therapy.

Antibacterial Activity of Caffeic Acid Combined with Ultraviolet-A Light against Escherichia coli O157:H7, Salmonella Typhimurium and Listeria monocytogenes

The aim of this study was to evaluate the antibacterial activity of caffeic acid (CA) which is a natural polyphenol, combined with Ultraviolet-A (UV-A) light against the representative foodborne bacteria, Escherichia coli O157:H7, Salmonella Typhimurium and Listeria monocytogenes. The inactivation results were obtained depending on the CA concentration, light wavelength and light dose.E. coli O157:H7 and S. Typhimurium were reduced to the detection limit when treated with 3 mM CA and UV-A for 3 J/cm2 and 4 J/cm2 respectively, and 5 J/cm2 treatment induced 3.10 log reduction in L. monocytogenes. To investigate the inactivation mechanism, measurement of polyphenol uptake, membrane damage assessment, enzymatic activity assay, and transmission electron microscopy (TEM) were conducted against S. Typhimurium and L. monocytogenes. It was revealed that CA was significantly (P < 0.05) absorbed by bacterial cells, and UV-A light allowed a higher uptake of CA for both pathogens. Additionally, CA + UV-A treatment induced significant (P < 0.05) cell membrane damage. In the enzymatic activity assay, both pathogens showed a reduction in their activity by CA and a higher reduction occurred by CA + UV-A. Moreover, TEM images indicated that CA + UV-A treatment notably destroyed the intercellular structure. In addition, antibacterial activity was also observed in commercial apple juice, which showed similar results to those obtained from PBS, resulting in a significant (P < 0.05) reduction for all three pathogens without any changes in color parameters (L*, a*, and b*), total phenolic compounds and DPPH (2,2-diphenyl-1-picrylhydrazyl) free radical scavenging activity. IMPORTANCE Photodynamic inactivation (PDI), which involves photoactivation of a photosensitizer (PS), is an emerging field of study as it effectively reduces various kinds of microorganisms. Although there are several PSs that have been used for PDI, there is a need to find naturally occurring PSs for safer application in food industry. Caffeic acid, a natural polyphenol found in most of fruits and vegetables, has recently been studied for its potential to act as a novel photosensitizer. However, no studies have been conducted regarding its antibacterial activity depending on treatment conditions and its antibacterial mechanism. In this study we closely examined the effectiveness of caffeic acid in combination with UV-A light for inactivating representative foodborne bacteria in liquid medium. Therefore, the results of this research are expected to be utilized as basic data for future application of caffeic acid in PDI, especially when controlling pathogens in liquid food processing.

Identification of Small Molecule Inhibitors of the Deubiquitinating Activity of the SARS-CoV-2 Papain-Like Protease: in silico Molecular Docking Studies and in vitro Enzymatic Activity Assay

COVID-19 is an ongoing pandemic caused by the SARS-CoV-2 virus with important political, socio-economic, and public health consequences. Inhibiting replication represents an important antiviral approach, and in this context two viral proteases, the SARS-CoV-2 main and papain-like proteases (PLpro), which cleave pp1a and pp1ab polypeptides, are critical. Along with protease activity, the PLpro possesses deubiquitinating activity, which is important in immune regulation. Naphthalene-based inhibitors, such as the well-investigated GRL-0617 compound, have been shown to possess dual effects, inhibiting both protease and deubiquitinating activity of the PLpro. Rather than binding to the canonical catalytic triad, these type of non-covalent inhibitors target an adjacent pocket, the naphthalene-inhibitor binding site. Using a high-throughput screen, we have previously identified the dietary hypericin, rutin, and cyanidin-3-O-glucoside compounds as potential protease inhibitors targeting the naphthalene-inhibitor binding site. Here, our aim was to investigate the binding characteristics of these compounds to the PLpro, and to evaluate deubiquitinating activity, by analyzing seven different PLpro crystal structures. Molecular docking highlighted the relatively high affinity of GRL-0617 and dietary compounds. In contrast binding of the small molecules was abolished in the presence of ubiquitin in the palm subdomain of the PLpro. Further, docking the small molecules in the naphthalene-inhibitor binding site, followed by protein-protein docking revealed displacement of ubiquitin in a conformation inconsistent with functional activity. Finally, the deubiquitinating activity was validated in vitro using an enzymatic activity assay. The findings indicated that the dietary compounds inhibited deubiquitinase activity in the micromolar range with an order of activity of GRL-0167, hypericin &gt;&gt; rutin, cyanidin-3-O-glucoside &gt; epigallocatechin gallate, epicatechin gallate, and cefotaxime. Our findings are in accordance with mechanisms and potential antiviral effects of the naphthalene-based, GRL-0617 inhibitor, which is currently progressing in preclinical trials. Further, our findings indicate that in particular hypericin, rutin, and cyanidin-3-O-glucoside, represent suitable candidates for subsequent evaluation as PLpro inhibitors.

Discovery of indolyl-containing peptides as novel antibacterial agents targeting tryptophanyl-tRNA synthetase

Background: There is an urgent need for antibiotics with novel structures and unexploited targets to counteract bacterial resistance. Methodology & results: Novel tryptophanyl-tRNA synthetase inhibitors were discovered based on virtual screening, surface plasmon resonance binding, enzymatic activity assay and antibacterial activity evaluation. Of the 29 peptide derivatives tested for antibacterial activity, some inhibited the growth of both Staphylococcus aureus and Staphylococcus epidermidis. A13 and A15 exhibited antibacterial activity against methicillin-resistant S. aureus NRS384 at an 8 μg/ml minimum inhibitory concentration. A13 snugly docked into the active site, explaining its improved inhibitory activity. Conclusion: Our results provide us with new structural clues to develop more potent tryptophanyl-tRNA synthetase inhibitors and lay a solid foundation for future drug design efforts.

Biodegradation of recalcitrant compounds and phthalates by a consortium of Liometopum apiculatum gut microbiota

Liometopum apiculatum ants species are widely distributed in arid and semi-arid ecosystems where there is a relative food shortage. They have established an ecological balance involving symbiotic interactions, which have allowed them to survive through mechanisms that are still unknown. The aim of this study was to explore the metabolic potential of isolated bacteria from L. apiculatum using substrate assimilation and enzymatic activity assay. Results revealed a complex bacteria consortium belonging to Proteobacteria, Firmicutes, and Actinobacteria phylum. These symbionts showed activities associated with biopolymers degradation, suggesting their important role in nutrients release stored in plant-derived material. By other hand, some microorganisms had a high capacity for degradation of phthalate esters. These results not only revealed the important contribution of the symbionts in L apiculatum ants feeding habits, but also they showed a promising source of enzymes with high potential for biotechnological applications such as lignocellulosic biomass hydrolysis and bioremediation.

Droplet Combinations: A Scalable Microfluidic Platform for Biochemical Assays

Droplet-based microfluidics holds enormous potential for transforming high-throughput drug screening. Miniaturization through droplets in combination with automation contributes to reduce reagent use and analysis time as well as minimizing or eliminating labor-intensive steps leading to associated reductions in cost. In this paper, we demonstrate the potential of automated and cost-effective microfluidic droplet-generating technology in the context of an enzymatic activity assay for screening collagenase inhibitors. Experimental results show reproducible and accurate creation and mixing of droplet combinations resulting in biochemical data comparable to data produced by an industry standard instrument. This microfluidic platform that can generate and combine multiple droplets represents a promising tool for high-throughput drug screening.

Preparation of “Open/closed” pores of PLGA-microsphere for controlled release of protein drug

Poly(D,L-lactic-co-glycolic acid) has been extensively used as a controlled release carrier for drug delivery due to its good biocompatibility, biodegradability, and mechanical strength. In this study, porous PLGA microspheres were fabricated by an emulsion-solvent evaporation technique using poly ethylene glycol (PEG) as an extractable porogen and loaded with protein (lysozyme) by suspending them in protein solution. For controlled release of protein, porous microspheres containing lysozyme were treated with water-miscible solvents in aqueous phase for production of pore-closed microspheres. The surface morphology of microspheres were investigated using scanning electron microscopy (SEM) for confirmation of its porous microstructure structure. Protein property after release was observed by enzymatic activity assay. The pore-closing process resulted in nonporous microspheres which exhibited sustained release patterns over an extended period.