Synthesis and molecular modeling of novel non-sulfonylureas as hypoglycemic agents and selective ALR2 inhibitors

Novel non-sulfonylureas derivatives bearing an acetamide linker between a spirohydantoin scaffold and a phenyl ring were prepared and their hypoglycemic activity was estimated in vivo. Their abilities to discriminate in vitro between aldehyde reductase (ALR1) and aldose reductase (ALR2) were determined. The molecular docking and the in silico prediction studies were performed to rationalize the obtained biological results and to predict the physicochemical properties and drug-likeness scores of the new compounds. N-(2,4-Dichlorophenyl)-2-(2′,4′-dioxospiro[fluorene-9,5′-imidazolidine]-3′-yl)acetamide (3e) displayed an 84% reduction in blood glucose level superior to that of repaglinide 66% and showed an IC₅₀ value of 0.37 μM against ALR2 that is superior to that of sorbinil 3.14 µM. Compound (3e) was selective 96 fold towards ALR2 which is closely related to serious diabetic complications. Based on the identification of this hit candidate, a new generation of safe and effective antidiabetic agents could be designed.     

Covalent reaction intermediate revealed in crystal structure of the Geobacillus stearothermophilus carboxylesterase Est30

Est30 is a thermophilic carboxylesterase cloned from Geobacillus stearothermophilus that showed optimal hydrolysis of esters with short acyl chains at 70 degrees C. Est30 is a member of a new family of carboxylesterases with representatives in other Gram-positive bacteria. The crystal structure has been determined at 1.63A resolution using multiple anomalous dispersion data. The two-domain crystal structure showed a large domain with a modified alpha/beta hydrolase core including a seven, rather than an eight-stranded beta sheet, and a smaller cap domain comprising three alpha helices. The catalytic triad consists of residues Ser94, Asp193, and His223. A 100Da tetrahedral ligand was observed to be covalently bound to the side-chain of Ser94. The propyl acetate ligand represents the first tetrahedral intermediate in the reaction mechanism. Therefore, this Est30 crystal structure will help understand the mode of action of all enzymes in the serine hydrolase superfamily.     

Protective Efficacy of Recombinant Modified Vaccinia Virus Ankara Delivering Middle East Respiratory Syndrome Coronavirus Spike Glycoprotein

Middle East respiratory syndrome coronavirus (MERS-CoV) causes severe respiratory disease in humans. We tested a recombinant modified vaccinia virus Ankara (MVA) vaccine expressing full-length MERS-CoV spike (S) glycoprotein by immunizing BALB/c mice with either intramuscular or subcutaneous regimens. In all cases, MVA-MERS-S induced MERS-CoV-specific CD8⁺ T cells and virus-neutralizing antibodies. Vaccinated mice were protected against MERS-CoV challenge infection after transduction with the human dipeptidyl peptidase 4 receptor. This MERS-CoV infection model demonstrates the safety and efficacy of the candidate vaccine.     

Trans-spinal direct current stimulation modulates motor cortex-induced muscle contraction in mice

The present study investigated the effect of trans-spinal direct current (tsDC) on the firing rate, pattern, and amplitude of spontaneous activity of the tibial nerve and on the magnitude of cortically elicited triceps surae (TS) muscle contractions. The effect of combined tsDC and repetitive cortical electrical stimulation (rCES) on the amplitude of cortically elicited TS twitches was also investigated. Stimulation was applied by two disk electrodes (0.79 cm(2)): one was located subcutaneously over the vertebral column (T(10)-L(1)) and was used to deliver anodal DC (a-tsDC) or cathodal DC (c-tsDC) (density range: ± 0.64 to ± 38.2 A/m(2)), whereas the other was located subcutaneously on the lateral aspect of the abdomen and served as a reference. While the application of a-tsDC significantly increased the spike frequency and amplitude of spontaneous discharges compared with c-tsDC, c-tsDC made the spontaneous discharges more rhythmic. Cortically elicited TS twitches were depressed during a-tsDC and potentiated after termination. Conversely, cortically elicited TS twitches were enhanced during c-tsDC and depressed after termination. While combined a-tsDC and rCES produced similar effects as a-tsDC alone, combined c-tsDC and rCES showed the greatest increase in cortically elicited TS twitches. tsDC appears to be a powerful neurostimulation tool that can differentially modulate spinal cord excitability and corticospinal transmission.     

PepFect 14, a novel cell-penetrating peptide for oligonucleotide delivery in solution and as solid formulation

Numerous human genetic diseases are caused by mutations that give rise to aberrant alternative splicing. Recently, several of these debilitating disorders have been shown to be amenable for splice-correcting oligonucleotides (SCOs) that modify splicing patterns and restore the phenotype in experimental models. However, translational approaches are required to transform SCOs into usable drug products. In this study, we present a new cell-penetrating peptide, PepFect14 (PF14), which efficiently delivers SCOs to different cell models including HeLa pLuc705 and mdx mouse myotubes; a cell culture model of Duchenne's muscular dystrophy (DMD). Non-covalent PF14-SCO nanocomplexes induce splice-correction at rates higher than the commercially available lipid-based vector Lipofectamine 2000 (LF2000) and remain active in the presence of serum. Furthermore, we demonstrate the feasibility of incorporating this delivery system into solid formulations that could be suitable for several therapeutic applications. Solid dispersion technique is utilized and the formed solid formulations are as active as the freshly prepared nanocomplexes in solution even when stored at an elevated temperatures for several weeks. In contrast, LF2000 drastically loses activity after being subjected to same procedure. This shows that using PF14 is a very promising translational approach for the delivery of SCOs in different pharmaceutical forms.     

Biodegradation of chicken feathers waste directed by Bacillus subtilis recombinant cells: scaling up in a laboratory scale fermentor

Biodegradation of chicken feathers waste directed by Bacillus subtilis DB 100 (p5.2) cells was successfully carried out in 14 L Bio Flo 110 laboratory scale fermentor. Seven liters of feathers-based modified basal medium II, feathers-based tap water and feathers-based distilled water separately in the fermentor were inoculated with activated bacterial cells. The fermentation processes were conducted at 37 °C, 700 rpm agitation speed and 0.7 vvm air flow rate in the absence of kanamycin. Highest net levels of released feathers hydrolysis end products [soluble proteins and NH₂-free amino groups] and keratinolytic alkaline protease activity in the fermentor were greatly comparable to those of shake flasks. Interestingly, the plasmid (p5.2) inside the recombinant B. subtilis cells growing in the fermentor displayed 100% stability till the fifth day of incubation and this presents a great challenge. Data certainly would encourage the transfer to larger scale fermentors to carry out feathers biodegradation process.     

Nde1-mediated inhibition of ciliogenesis affects cell cycle re-entry

The primary cilium is an antenna-like organelle that is dynamically regulated during the cell cycle. Ciliogenesis is initiated as cells enter quiescence, whereas resorption of the cilium precedes mitosis. The mechanisms coordinating ciliogenesis with the cell cycle are unknown. Here we identify the centrosomal protein Nde1 (nuclear distribution gene E homologue 1) as a negative regulator of ciliary length. Nde1 is expressed at high levels in mitosis, low levels in quiescence and localizes at the mother centriole, which nucleates the primary cilium. Cells depleted of Nde1 have longer cilia and a delay in cell cycle re-entry that correlates with ciliary length. Knockdown of Nde1 in zebrafish embryos results in increased ciliary length, suppression of cell division, reduction of the number of cells forming the Kupffer's vesicle and left-right patterning defects. These data suggest that Nde1 is an integral component of a network coordinating ciliary length with cell cycle progression and have implications for understanding the transition from a quiescent to a proliferative state.     

BBS4 Is Necessary for Ciliary Localization of TrkB Receptor and Activation by BDNF

Primary cilia regulate an expanding list of signaling pathways in many different cell types. It is likely that identification of the full catalog of pathways associated with cilia will be necessary to fully understand their role in regulation of signaling and the implications for diseases associated with their dysfunction, ciliopathies. Bardet-Biedl Syndrome (BBS) is one such ciliopathy which is characterized by a spectrum of phenotypes. These include neural defects such as impaired cognitive development, centrally mediated hyperphagia and peripheral sensory defects. Here we investigate potential defects in a signaling pathway associated with neuronal function, brain derived neurotrophic factor (BDNF) signaling. Upon loss of BBS4 expression in cultured cells, we observed decreased phosphorylation and activation by BDNF of its target receptor, TrkB. Assessment of ciliary localization revealed that, TrkB localized to the axonemes or basal bodies of cilia only in the presence of BDNF. Axonemal localization, specifically, was abrogated with loss of BBS4. Finally, we present evidence that loss of the ciliary axoneme through depletion of KIF3A impedes activation of TrkB. Taken together, these data suggest the possibility of a previously uninvestigated pathway associated with perturbation of ciliary proteins.     

Assessing the potential use of chitosan scaffolds for the sustained localized delivery of vitamin D

Vitamin D is a commonly used bone modulator in regenerative medicine. Several modalities have been explored for the delivery of vitamin D including nanoparticles and scaffold. The present study aimed to assess the potential use of a bio-degradable chitosan scaffold for the delivery of vitamin D. The objectives included fabrication of a bio-degradable chitosan scaffold, integration of vitamin D into the scaffold, characterization of the vitamin D integrated scaffold. Characterization was carried out using, X-ray diffraction, Fourier transform infrared spectroscopy, and differential scanning calorimetry. The structure of the scaffold was assessed by scanning electron microscopy. The scaffold was placed in phosphate buffer saline and the release duration of vitamin D was observed using UV spectrophotometry. Dental pulp mesenchymal stem cells were added to the scaffold to study the scaffold associated toxicity and the functionality of the scaffold released vitamin D. The vitamin D release period from the scaffold was estimated to be for 80 hrs. MTT assay of the stem cells was comparable to that of the control group (stem cells cultured in media) inferring that the scaffold is not toxic towards the stem cells. The positive alizarin red S staining, a higher expression of alkaline phosphatase, osteocalcin, and RunX2 confirmed the functional capability (osteogenic differentiation of the stem cells) of the released vitamin D. Based on the data from the present study, it can be inferred that chitosan scaffold can be used for the sustained delivery of functional vitamin D for 3–5 days.     

Spectrofluorimetric study on fluorescence quenching of tyrosine and l‐tryptophan by the aniracetam cognition enhancer drug: quenching mechanism using Stern–Volmer and double‐log plots

A novel approach on fluorescence quenching of tyrosine and l ‐tryptophan is presented for spectrofluorimetric determination of aniracetam in drug substances and products. The quenching mechanism was investigated using Stern–Volmer plots and ultraviolet spectra figures of quencher–fluorophore mixtures. Binding constant and stoichiometry were calculated using double‐log plots. The spectrofluorimetric method was optimized for the experimental conditions affecting fluorescence quenching including fluorophore concentration, diluent, and reaction time. Moreover, the pH‐rate profile of aniracetam was studied using simple kinetics and found to be stable within the pH range 5–8. Fluorescence quenching of tyrosine and l ‐tryptophan were observed on addition of aniracetam in aqueous medium at pH 5.5–6.5. Aniracetam quenched the fluorescence of tyrosine and l ‐tryptophan in the concentration range 1–20 μg/ml and 0.3–20 μg/ml, respectively, with binomial relationships between quenching values (ΔF) and aniracetam concentration. Limits of detection were found to be 0.10 μg/ml for tyrosine–aniracetam and 0.14 μg/ml for l ‐tryptophan–aniracetam. Method validation was performed as per ICH guidelines and demonstrated that the developed spectrofluorimetric method was accurate, precise, specific, and suitable for analysis of aniracetam in routine quality control laboratories. All experimental materials and solvents used are eco‐friendly, indicating that the cited spectrofluorimetric procedure is an excellent green method.