Probing the Role of the Conserved Arg174 in Formate Dehydrogenase by Chemical Modification and Site-Directed Mutagenesis

The reactive adenosine derivative, adenosine 5′-O-[S-(4-hydroxy-2,3-dioxobutyl)]-thiophosphate (AMPS-HDB), contains a dicarbonyl group linked to the purine nucleotide at a position equivalent to the pyrophosphate region of NAD⁺. AMPS-HDB was used as a chemical label towards Candida boidinii formate dehydrogenase (CbFDH). AMPS-HDB reacts covalently with CbFDH, leading to complete inactivation of the enzyme activity. The inactivation kinetics of CbFDH fit the Kitz and Wilson model for time-dependent, irreversible inhibition (K_D = 0.66 ± 0.15 mM, first order maximum rate constant k₃ = 0.198 ± 0.06 min⁻¹). NAD⁺ and NADH protects CbFDH from inactivation by AMPS-HDB, showing the specificity of the reaction. Molecular modelling studies revealed Arg174 as a candidate residue able to be modified by the dicarbonyl group of AMPS-HDB. Arg174 is a strictly conserved residue among FDHs and is located at the Rossmann fold, the common mononucleotide-binding motif of dehydrogenases. Arg174 was replaced by Asn, using site-directed mutagenesis. The mutant enzyme CbFDHArg174Asn was showed to be resistant to inactivation by AMPS-HDB, confirming that the guanidinium group of Arg174 is the target for AMPS-HDB. The CbFDHArg174Asn mutant enzyme exhibited substantial reduced affinity for NAD⁺ and lower thermostability. The results of the study underline the pivotal and multifunctional role of Arg174 in catalysis, coenzyme binding and structural stability of CbFDH.     

Physiological and Immunological Changes Associated with Oral Microbiota When Using a Thermoplastic Retainer

Background: The study examined the oral microbiota, physiological and immunological changes in patients using thermoplastic retainers during three months of use. Methods: The study included several steps. Firstly, 10 swabs were collected from the buccal and palatal surfaces of the teeth of the patients, approximately 2 mL of saliva was collected from the same patients and 2 mL of saliva was collected from 10 healthy people to measure the pH and secretory IgA level. This was followed by the isolation and identfication of the bacterial isolates in the patient samples. Then, isolate susceptibility toward chlorhexidine (CHX) and their adhesion ability to thermoplastic retainer surfaces was measured. In addition to that the study estimated the numbers of Lactobacillus and Streptooccus mutans colonies during three months and finally, a comparsion of pH acidity and IgA level between the patients and healthy people was performed. The results showed the predominant bacteria during the three months were Lactobacillus spp. and Streptococcus spp. followed by different rates of other bacteria. Raoultella ornithinolytica showed more resistance to CHX while Lactobacillus spp. showed more sensitivity. Streptococcus mutans colony levels were higher than Lactobacillus spp. colonies during the three months, also S. mutans had the highest value in adherence to retainer thermoplastic. Finally, pH acidity showed a highly significant difference (p ≤ 0.05) in the third month, like IgA levels (p ≤ 0.05). Conclusions: According to the results obtained from the current study, the researchers noted that the thermoplastic retainers helped change the oral cavity environment.     

Biosynthesis of Zinc Oxide Nanoparticles from Acacia nilotica (L.) Extract to Overcome Carbapenem-Resistant Klebsiella Pneumoniae

Recently, concerns have been raised globally about antimicrobial resistance, the prevalence of which has increased significantly. Carbapenem-resistant Klebsiella pneumoniae (KPC) is considered one of the most common resistant bacteria, which has spread to ICUs in Saudi Arabia. This study was established to investigate the antibacterial activity of biosynthesized zinc oxide nanoparticles (ZnO-NPs) against KPC in vitro and in vivo. In this study, we used the aqueous extract of Acacia nilotica (L.) fruits to mediate the synthesis of ZnO-NPs. The nanoparticles produced were characterized by UV-vis spectroscopy, zetasizer and zeta potential analyses, X-ray diffraction (XRD) spectroscopy, Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDX), and transmission electron microscopy (TEM). The antimicrobial activity of ZnO-NPs against KPC was determined via the well diffusion method, and determining minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC), the results showed low MIC and MBC when compared with the MIC and MBC of Imipenem and Meropenem antibiotics. The results of in vitro analysis were supported by the results upon applying ZnO-NP ointment to promote wound closure of rats, which showed better wound healing than the results with imipenem ointment. The biosynthesized ZnO-NPs showed good potential for use against bacteria due to their small size, applicability, and low toxicity to human cells.     

Bimetallic Iron–Palladium Catalyst System as a Lewis-Acid for the Synthesis of Novel Pharmacophores Based Indole Scaffold as Anticancer Agents

The Friedel–Crafts reaction between substituted indoles as nucleophiles with chalcones-based benzofuran and benzothiophene scaffolds was carried out by employing a highly efficient bimetallic iron–palladium catalyst system. This catalytic approach produced the desired bis-heteroaryl products with low catalyst loading, a simple procedure, and with acceptable yield. All synthesized indole scaffolds 3a–3s were initially evaluated for their cytotoxic effect against human fibroblast BJ cell lines and appeared to be non-cytotoxic. All non-cytotoxic compounds 3a–3s were then evaluated for their anticancer activities against cervical cancer HeLa, prostate cancer PC3, and breast cancer MCF-7 cell lines, in comparison to standard drug doxorubicin, with IC₅₀ values 1.9 ± 0.4 µM, 0.9 ± 0.14 µM and 0.79 ± 0.05 µM, respectively, and appeared to be moderate to weak anticancer agents. Fluoro-substituted chalcone moiety-containing compounds, 3b appeared to be the most active member of the series against cervical HeLa (IC₅₀ = 8.2 ± 0.2 µM) and breast MCF-7 cancer cell line (IC₅₀ = 12.3 ± 0.04 µM), whereas 6-fluroindol-4-bromophenyl chalcone-containing compound 3e (IC₅₀ = 7.8 ± 0.4 µM) appeared to be more active against PC3 prostate cancer cell line.     

Design,Synthesis, and Validation of a Novel [11C]Promethazine PET Probe for Imaging Abeta Using Autoradiography

Promethazine, an antihistamine drug used in the clinical treatment of nausea, has been demonstrated the ability to bind Abeta in a transgenic mouse model of Alzheimer’s disease. However, so far, all of the studies were performed in vitro using extracted tissues. In this work, we report the design and synthesis of a novel [¹¹C]promethazine PET radioligand for future in vivo studies. The [¹¹C]promethazine was isolated by RP-HPLC with radiochemical purity >95% and molar activity of 48 TBq/mmol. The specificity of the probe was demonstrated using human hippocampal tissues via autoradiography.     

Exploiting the Chiral Ligands of Bis(imidazolinyl)- and Bis(oxazolinyl)thiophenes—Synthesis and Application in Cu-Catalyzed Friedel–Crafts Asymmetric Alkylation

Five new C₂-symmetric chiral ligands of 2,5-bis(imidazolinyl)thiophene (L1–L3) and 2,5-bis(oxazolinyl)thiophene (L4 and L5) were synthesized from thiophene-2,5-dicarboxylic acid (1) with enantiopure amino alcohols (4a–c) in excellent optical purity and chemical yield. The utility of these new chiral ligands for Friedel–Crafts asymmetric alkylation was explored. Subsequently, the optimized tridentate ligand L5 and Cu(OTf)₂ catalyst (15 mol%) in toluene for 48 h promoted Friedel–Crafts asymmetric alkylation in moderate to good yields (up to 76%) and with good enantioselectivity (up to 81% ee). The bis(oxazolinyl)thiophene ligands were more potent than bis(imidazolinyl)thiophene analogues for the asymmetric induction of the Friedel–Crafts asymmetric alkylation.     

The Nephroprotective Effect of Zizyphus lotus L. (Desf.) Fruits in a Gentamicin-Induced Acute Kidney Injury Model in Rats: A Biochemical and Histopathological Investigation

Zizyphus lotus L. (Desf.) (Z. lotus) is a medicinal plant largely distributed all over the Mediterranean basin and is traditionally used by Moroccan people to treat many illnesses, including kidney failure. The nephrotoxicity of gentamicin (GM) has been well documented in humans and animals, although the preventive strategies against it remain to be studied. In this investigation, we explore whether the extract of Zizyphus lotus L. (Desf.) Fruit (ZLF) exhibits a protective effect against renal damage produced by GM. Indeed, twenty-four Wistar rats were separated into four equal groups of six each (♂/♀ = 1). The control group was treated orally with distilled water (10 mL/kg); the GM treated group received distilled water (10 mL/kg) and an intraperitoneal injection of GM (80 mg/kg) 3 h after; and the treated groups received ZLF extract orally at the doses 200 or 400 mg/kg and injected intraperitoneally with the GM. All treatments were given daily for 14 days. At the end of the experiment, the biochemical parameters and the histological observation related the kidney function was explored. ZLF treatment has significantly attenuated the nephrotoxicity induced by the GM. This effect was indicated by its capacity to decrease significantly the serum creatinine, uric acid, urea, alkaline phosphatase, gamma-glutamyl-transpeptidase, albumin, calcium, sodium amounts, water intake, urinary volume, and relative kidney weight. In addition, this effect was also shown by the increase in the creatinine clearance, urinary creatinine, uric acid, and urea levels, weight gain, compared to the rats treated only with the GM. The hemostasis of oxidants/antioxidants has been significantly improved with the treatment of ZLF extract, which was shown by a significant reduction in malondialdehydes levels. Histopathological analysis of renal tissue was correlated with biochemical observation. Chemical analysis by HPLC-DAD showed that the aqueous extract of ZLF is rich in phenolic compounds such as 3-hydroxycinnamic acid, catechin, ferulic acid, gallic acid, hydroxytyrosol, naringenin, p- coumaric Acid, quercetin, rutin, and vanillic acid. In conclusion, ZLF extract improved the nephrotoxicity induced by GM, through the improvement of the biochemical and histological parameters and thus validates its ethnomedicinal use.     

Discovery of New Coumarin-Based Lead with Potential Anticancer,CDK4 Inhibition and Selective Radiotheranostic Effect: Synthesis, 2D & 3D QSAR,Molecular Dynamics,In Vitro Cytotoxicity,Radioiodination, and Biodistribution Studies

Novel 6-bromo-coumarin-ethylidene-hydrazonyl-thiazolyl and 6-bromo-coumarin-thiazolyl-based derivatives were synthesized. A quantitative structure activity relationship (QSAR) model with high predictive power r² = 0.92, and RMSE = 0.44 predicted five compounds; 2b, 3b, 5a, 9a and 9i to have potential anticancer activities. Compound 2b achieved the best ΔG of –15.34 kcal/mol with an affinity of 40.05 pki. In a molecular dynamic study 2b showed an equilibrium at 0.8 Å after 3.5 ns, while flavopiridol did so at 0.5 Å after the same time (3.5 ns). 2b showed an IC₅₀ of 0.0136 µM, 0.015 µM, and 0.054 µM against MCF-7, A-549, and CHO-K1 cell lines, respectively. The CDK4 enzyme assay revealed the significant CDK4 inhibitory activity of compound 2b with IC₅₀ of 0.036 µM. The selectivity of the newly discovered lead compound 2b toward localization in tumor cells was confirmed by a radioiodination biological assay that was done via electrophilic substitution reaction utilizing the oxidative effect of chloramine-t. ¹³¹I-2b showed good in vitro stability up to 4 h. In solid tumor bearing mice, the values of tumor uptake reached a height of 5.97 ± 0.82%ID/g at 60 min p.i. ¹³¹I-2b can be considered as a selective radiotheranostic agent for solid tumors with promising anticancer activity.     

Surface Polymers on Multiwalled Carbon Nanotubes for Selective Extraction and Electrochemical Determination of Rhodamine B in Food Samples

In this study, we combine magnetic solid phase extraction (MSPE), with the screen-printed carbon electrode (SPCE) modified by a molecular imprinted polymer (MIP) for sensitive and selective extraction and electrochemical determination of Rhodamine B in food samples. A magnetic solid phase extraction (MSPE) was carried out using magnetic poly(styrene-co-divinylbenzene) (PS-DVB) and magnetic nanoparticles (MNPs) synthetized on the surface of multiwalled carbon nanotubes (MWCNTs). An MIP was prepared on the surface of MWCNTs in the presence of titanium oxide nanoparticles (TiO₂NPs) modifying the SPCE for the rapid electrochemical detection of Rhodamine B. The MIPs synthesis was optimized by varying the activated titanium oxide (TiO₂) and multiwalled carbon nanotubes (MWCNTs) amounts. The MSPE and electrochemical detection conditions were optimized as well. The present method exhibited good selectivity, high sensitivity, and good reproducibility towards the determination of Rhodamine B, making it a suitable method for the determination of Rhodamine B in food samples.