Iodoquinazoline-derived VEGFR-2 and EGFRT790M dual inhibitors: Design, synthesis, molecular docking and anticancer evaluations.

Herein, we report the synthesis of a series of new fourteen iodoquinazoline derivatives 7a-c to 13a-e and their evaluation as potential anticancer agents via dual targeting of EGFRT790M and VEGFR-2. The new derivatives were designed according to the target receptors structural requirements. The compounds were evaluated for their cytotoxicity against HepG2, MCF-7, HCT116 and A549 cancer cell lines using MTT assay. Compound 13e showed the highest anticancer activities with IC50 = 5.70, 7.15, 5.76 and 6.50 µM against HepG2, MCF-7, HCT116 and A549 cell lines correspondingly. Compounds 7c, 9b and 13a-d exhibited very good anticancer effects against the tested cancer cell lines. The highly effective six derivatives 7c, 10, 13b, 13c, 13d and 13e were examined against VERO normal cell lines to estimate their cytotoxic capabilities. Our conclusion revealed that compounds 7c, 10, 13b, 13c, 13d and 13e possessed low toxicity against VERO normal cells with IC50 prolonging from 41.66 to 53.99 µM. Also compounds 7a-c to 13a-e were further evaluated for their inhibitory activity against EGFRT790M and VEGFR-2. Also, their ability to bind with both EGFR and VEGFR-2 receptors was examined by molecular modeling. Compounds 13e, 13d, 7c and 13c excellently inhibited VEGFR-2 activity with IC50 = 0.90, 1.00, 1.25 and 1.50 µM respectively. Moreover, Compounds 13e, 7c, 10 and 13d excellently inhibited EGFRT790M activity with IC50 = 0.30, 0.35, 0.45 and 0.47 µM respectively. Finally, our derivatives 7b, 13d and 13e showed good in silico calculated ADMET profile.

Design, synthesis, docking, ADMET and anticancer evaluations of N-alkyl substituted iodoquinazoline derivatives as dual VEGFR-2 and EGFR inhibitors.

Fifteen new 1-alkyl-6-iodoquinazoline derivatives 5a-d to 9a-e were designed and synthesized and their anticancer activities were evaluated against HepG2, MCF-7, HCT116 and A549 cancer cell lines via dual targeting of EGFR and VEGFR-2. The newly synthesized compounds were designed based on the structure requirements of the target receptors and were confirmed using spectral data. Compound 9c showed the highest anticancer activities with EC50 = 5.00, 6.00, 5.17 and 5.25 µM against HepG2, MCF-7, HCT116 and A549 cell lines correspondingly. Moreover, compounds 5d, 8b, 9a, 9b, 9d, and 9e exhibited very good anticancer effects against the tested cancer cell lines. The highly effective seven derivatives 5d, 8b, 9a-e were examined against VERO normal cell lines to estimate their cytotoxic capabilities. Compounds 9c, 9b, 9d, 9a, 9e and 5d excellently inhibited VEGFR-2 activity with IC50 = 0.85, 0.90, 0.90, 1.00, 1.20 and 1.25 µM respectively. Moreover, compounds 9c, 9d, 9e, 5d, 8b and 9b excellently inhibited EGFRT790M activity with IC50 = 0.22, 0.26, 0.30, 0.40, 0.45 and 0.50 µM respectively. Also, compounds 9c, 9d and 9e excellently inhibited EGFRWT activity with IC50 = 0.15, 0.20 and 0.25 µM respectively. As planned, compound 9c showed excellent dual EGFR/VEGFR-2 inhibitory activities. Consonantly, ADMET study was calculated in silico for the supreme three worthwhile compounds 9b, 9c and 9e in contrast to sorafenib and erlotinib as reference drugs. The obtained results concluded that, our compounds might be useful as prototype for design, optimization, adaptation and investigation to have more powerful and selective dual VEGFR-2/EGFRT790M inhibitors with higher antitumor activity.

Productivity and Phytochemicals of Asclepias curassavica in Response to Compost and Silver Nanoparticles Application: HPLC Analysis and Antibacterial Activity of Extracts.

The application of compost and metallic nanoparticles has a significant impact on the productivity and chemical composition of horticulture plants. In two subsequent growing seasons, 2020 and 2021, the productivity of Asclepias curassavica L. plants treated with various concentrations of silver nanoparticles (AgNPs) and compost was assessed. In the pot experiments, the soil was amended with 25% or 50% compost, and the plants were sprayed with 10, 20, and 30 mg/L of AgNPs. Scanning electron microscopy (SEM), transmission electron microscopy (TEM), X-ray diffraction analysis (XRD), and dynamic light scattering (DLS) were used to characterize AgNPs. The TEM measurements of AgNPs showed that the particles had spherical forms and ranged in size from roughly 5 to 16 nm. Leaf methanol extracts (LMEs) were prepared from the treated plants and assayed against the growth of two soft rot bacteria, Dickeya solani and Pectobacterium atrosepticum. The maximum plant height, diameter, number of branches/plant, total fresh weight (g), total dry weight (g), and leaf area (cm2) was recorded when levels of 25% compost + AgNPs 20 mg/L, 25% compost, or 50% + AgNPs 20 mg/L, 25% compost + AgNPs 30 mg/L or 50% compost + AgNPs 20 mg/L, 50% compost + AgNPs 20 mg/L, 50% compost + AgNPs 30 or 20 mg/L, and 25% compost + AgNPs 30 mg/L, respectively, were applied. The plants treated with 25% or 50% compost + 30 mg/L AgNPs showed a high chlorophyll content, while the plants treated with 50% compost + AgNPs 30 mg/L or 20 mg/L showed the highest extract percentages. The highest inhibition zones (IZs), 2.43 and 2.2 cm, against the growth of D. solani were observed in the LMEs (4000 mg/L) extracted from the plants treated with compost (v/v) + AgNPs (mg/L) at the levels of 50% + 30 and 25% + 30, respectively. The highest IZs, 2.76 and 2.73 cm, against the growth of P. atrosepticum were observed in the LMEs (4000 mg/L) extracted from the plants treated at the levels of 50% + 30 and 25% + 30, respectively. Several phenolic compounds such as syringic acid, p-coumaric acid, chlorogenic acid, cinnamic acid, ellagic acid, caffeic acid, benzoic acid, gallic acid, ferulic acid, salicylic acid, pyrogallol, and catechol, as well as flavonoid compounds such as 7-hydroxyflavone, naringin, rutin, apigenin, quercetin, kaempferol, luteolin, hesperidin, catechin, and chrysoeriol, were identified in the LMEs as analyzed by HPLC with different concentrations according to the treatment of compost + AgNPs used for the plants. In conclusion, the specific criteria that were utilized to measure the growth of A. curassavica revealed the novelty of compost and AgNPs combination treatments, particularly at a concentration of 50% compost + AgNPs 30 mg/L or 20 mg/L, which is better for the growth and phytochemical production of A. curassavica in the field.

Memantine mitigates ROS/TXNIP/NLRP3 signaling and protects against mouse diabetic retinopathy: Histopathologic, ultrastructural and bioinformatic studies.

Diabetic retinopathy (DRET) triggers vision loss in adults, however, little therapeutic options are existing. Memantine is an anti-Alzheimer drug that antagonizes the activity of glutamate at N-methyl-D-aspartate (NMDA) receptors. Glutamate and thioredoxin-interacting protein (TXNIP) are known to be overexpressed in diabetic retinas and can produce activation of NOD-like receptor protein 3 (NLRP3) with subsequent secretion of interlukin-1ß. This study repurposed memantine for its neuroprotective effect in experimental DRET and tested its impact on ROS/TXNIP/NLRP3. In addition, KEGG pathway database and STRING database identified the protein-protein interaction between glutamate receptors and TXNIP/NLRP3. Male Swiss albino mice received alloxan (180 mg/kg) to induce DRET. After 9 weeks, we divided the mice into groups: (a) saline, (ii) DRET, (iii and iv) DRET + oral memantine (5 or 10 mg per kg) for 28 days. Then, mice were euthanized, and eyeballs were removed. Retinal samples were utilized for biochemical, histopathological, and electron microscopy studies. Retinal levels of glutamate, TXNIP, NLRP3 and interlukin-1ß were estimated using ELISA technique as well as retinal malondialdehyde. Histopathological and ultrastructural examination demonstrated that oral memantine attenuated vacuolization and restored normal retinal cell layers. Moreover, memantine reduced TXNIP, NLRP3, interleukin-1ß and MDA concentrations. These results provide evidence demonstrating memantine' efficacy in alleviating DRET via suppressing reactive oxygen species/TXNIP/NLRP3 signaling cascade. Therefore, memantine might serve as a potential therapy for retinopathy after adequate clinical research.

New Approaches for the Synthesis N-alkylated Benzo[b]thiophene Derivatives together with their Antiproliferative and Molecular Docking Studies.

BACKGROUND: 2-Amino thiophene derivatives are important compounds not only for their uses in many heterocyclic reactions but also due to their wide range of pharmaceutical and biological activities. OBJECTIVE: The aim of this work was to explore a number of new heterocyclic derivatives, studying their inhibitions toward cancer cell lines and studying their structure activity relation ship. METHODS: Alkylation of 2-amino-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carbonitrile was achieved through its reaction with chloroacetone and 2-bromo-1-(4-aryl)ethanone derivatives to give compounds 3 and 11a-c. The produced compoumds were subjected to further heterocylization reactions and cytotoxic evaluation against the three cancer cell lines MCF-7, NCI-H460 and SF-268, together with the normal cell line WI 38. Further evaluations were obtained through studying their inhibitions against cancer cell lines classified according to the disease. Anticancer screening against hepatocellular carcinoma HepG2 and cervical carcinoma HeLa cell lines for all compounds together with the molecular docking of 12c, 12d, 12e and 12f were studied. RESULTS: Anti-proliferative evaluations and inhibitions for all of the synthesized compounds showed that many compounds exhibited high inhibitions. CONCLUSION: Toward the three cancer cell lines, compounds 3, 5a, 7a, 9a, 9b, 11b, 12b, 12d, 12e, 12f, 14c, 14e, 14f, 15e, 15f, 16e, 16f, 17c, 18b, 22a and 22c were the most cytotoxic compounds. The high activities of some compounds were attributed to the presence of the electronegative CN and or Cl groups within the molecule. Most of the tested compounds exhibited inhibitions higher than the reference doxorubicin toward hepatocellular carcinoma HepG2 and cervical carcinoma HeLa cell lines. The score of binding energy of compounds 12c, 12d, 12e and 12f was close to the reference Foretinib which appeared through the molecular docking results of such compounds.

Expression of CD3 and CD20 in Antistreptolysin-O Titer Seropositive and Seronegative Children with Chronic Tonsillitis.

Background: Chronic tonsillitis (CT) is a common inflammatory illness in children, and serum antistreptolysin O titer (ASOT) is a common investigation performed for these cases and considered a perfect sign for tonsillectomy. Objective: To evaluate the expression of tonsillar T-and B-lymphocytes markers in relation to seropositive or seronegative ASOT in cases of CT. Materials and Methods: Thirty children (15 males and 15 females) aged 6-10 years were divided equally into two groups: Group A seropositive ASOT (≥400 IU) and Group B seronegative ASOT (<400 IU). Both performed bilateral tonsillectomy. Specimens from the removed tonsils were taken and prepared for light microscopic examination and immunohistochemical evaluation of CD20 and CD3 expression. Results: Seropositive ASOT group showed significant histopathologic changes in the form of hyperplasia of the stratified squamous nonkeratinized epithelium, Urgas's abscess, and severe lymphocytic infiltration. Immunohistochemical results of seropositive ASOT group showed marked expression of CD3 and CD20, while seronegative ASOT group showed mild expression of CD3 and CD20. Conclusion: Seropositive ASOT CT, in addition to histopathological changes, is associated with significant increase in both B-lymphocytes (CD20 expression) and T-lymphocytes (CD3 expression) markers.

Photosynthetic Pigments and Biochemical Response of Zucchini (Cucurbita pepo L.) to Plant-Derived Extracts, Microbial, and Potassium Silicate as Biostimulants Under Greenhouse Conditions.

There are many technological innovations in the field of agriculture to improve the sustainability of farmed products by reducing the chemicals used. Uses of biostimulants such as plant extracts or microorganisms are a promising process that increases plant growth and the efficient use of available soil resources. To determine the effects of some biostimulants' treatments on the photosynthetic pigments and biochemicals composition of zucchini plants, two experiments were conducted in 2019 and 2020 under greenhouse conditions. In this work, the effects of beneficial microbes (Trichoderma viride and Pseudomonas fluorescens), as well as three extracts from Eucalyptus camaldulensis leaf extract (LE), Citrus sinensis LE, and Ficus benghalensis fruit extract (FE) with potassium silicate (K2SiO3) on productivity and biochemical composition of zucchini fruits, were assessed as biostimulants. The results showed that E. camaldulensis LE (4,000 mg/L) + K2SiO3 (500 mg/L) and T. viride (106 spore/ml) + K2SiO3 (500 mg/L) gave the highest significance yield of zucchini fruits. Furthermore, the total reading response of chlorophylls and carotenoids was significantly affected by biostimulants' treatments. The combination of K2SiO3 with E. camaldulensis LE increased the DPPH scavenging activity and the total phenolic content of zucchini fruits, in both experiments. However, the spraying with K2SiO3 did not observe any effects on the total flavonoid content of zucchini fruits. Several phenolic compounds were identified via high-performance liquid chromatography (HPLC) from the methanol extracts of zucchini fruits such as syringic acid, eugenol, caffeic acid, pyrogallol, gallic acid, ascorbic acid, ferulic acid, α-tocopherol, and ellagic acid. The main elemental content (C and O) analyzed via energy-dispersive X-ray spectroscopy (EDX) of leaves was affected by the application of biostimulants. The success of this work could lead to the development of cheap and easily available safe biostimulants for enhancing the productivity and biochemical of zucchini plants.

Simultaneous quantitative determination of insulin aspart and insulin degludec in human plasma using simple shoot LC method.

Combining short-acting and long-acting insulin analogs was a real challenge that was overcome by NovoNordisk through the co-formulation of insulin aspart and insulin degludec in single-dosage form. The proposed study provides a simple, short, and reliable HPLC method with diode array detection that is developed and validated for the simultaneous determination of insulin aspart and insulin degludec in human plasma. The proposed method achieved good separation between the two analytes utilizing a C8 column at 35°C in a very short run time (6 min), with a simple, low-cost, and reliable extraction method through precipitation of plasma protein. Gradient elution was applied using a mobile phase consisting of 0.1 M sodium sulfate (pH 3.4) and acetonitrile. The method was validated according to EMA Guideline on Bioanalytical Validation. The proposed method had a linear range from 3.0 to 300 µg/mL for insulin aspart and from 3.5 to 300 µg/mL for insulin degludec. The intra- and inter-day precision of insulin aspart were 0.36-3.33% and 1.59-8.84%, respectively, and accuracy was between 10.06 and 3.09% The intra- and inter-day precision of insulin degludec were 0.29-1.93% and 0.89-5.14%, respectively, and accuracy was between -5.29 and 3.91%.

Characterization of Phytoconstituents from Alcoholic Extracts of Four Woody Species and Their Potential Uses for Management of Six Fusarium oxysporum Isolates Identified from Some Plant Hosts.

BACKGROUND: Trees are good sources of bioactive compounds as antifungal and antioxidant activities. METHODS: Management of six molecularly identified Fusarium oxysporum isolates (F. oxy 1, F. oxy 2, F. oxy 3, F. oxy 4, F. oxy 5 and F. oxy 6, under the accession numbers MW854648, MW854649, MW854650, MW854651, and MW854652, respectively) was assayed using four extracts from Conium maculatum leaves, Acacia saligna bark, Schinus terebinthifolius wood and Ficus eriobotryoides leaves. All the extracts were analyzed using HPLC-VWD for phenolic and flavonoid compounds and the antioxidant activity was evaluated using 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging and ß-carotene-linoleic acid (BCB) bleaching assays. RESULTS: In mg/kg extract, the highest amounts of polyphenolic compounds p-hydroxy benzoic, benzoic, gallic, and rosmarinic acids, with 444.37, 342.16, 311.32 and 117.87, respectively, were observed in C. maculatum leaf extract; gallic and benzoic acids with 2551.02, 1580.32, respectively, in A. saligna bark extract; quinol, naringenin, rutin, catechol, and benzoic acid with 2530.22, 1224.904, 798.29, 732.28, and 697.73, respectively, in S. terebinthifolius wood extract; and rutin, o-coumaric acid, p-hydroxy benzoic acid, resveratrol, and rosmarinic acid with 9168.03, 2016.93, 1009.20, 1156.99, and 574.907, respectively, in F. eriobotryoides leaf extract. At the extract concentration of 1250 mg/L, the antifungal activity against the growth of F. oxysporum strains showed that A. saligna bark followed by C. maculatum leaf extracts had the highest inhibition percentage of fungal growth (IPFG%) against F. oxy 1 with 80% and 79.5%, F. oxy 2 with 86.44% and 78.9%, F. oxy 3 with 86.4% and 84.2%, F. oxy 4 with 84.2, and 82.1%, F. oxy 5 with 88.4% and 86.9%, and F. oxy 6 with 88.9, and 87.1%, respectively. For the antioxidant activity, ethanolic extract from C. maculatum leaves showed the lowest concentration that inhibited 50% of DPPH free radical (3.4 µg/mL). Additionally, the same extract observed the lowest concentration (4.5 µg/mL) that inhibited BCB bleaching. CONCLUSIONS: Extracts from A. saligna bark and C. maculatum leaves are considered potential candidates against the growth of F. oxysporum isolates-a wilt pathogen-and C. maculatum leaf as a potent antioxidant agent.

A comprehensive stability assessment of insulin degludec using New customized validated RP-HPLC and SEC-HPLC methods in an orthogonal testing protocol.

Stress testing of biopharmaceuticals plays an important role in preparation of their stability profiles through investigation of possible degradation pathways and identification of degradation products, so in this study Insulin Degludec which is a new generation ultra-long-acting basal insulin is subjected to stress conditions as different temperatures, different pH, oxidation, mechanical agitation, and repeated freeze and thaw cycles to generate possible degradation products and aggregation that are investigated by two new validated RP-HPLC and SEC-HPLC methods in addition to dynamic light scattering (DLS) and native polyacrylamide gel electrophoresis (Nu-PAGE). SEC-HPLC was used to investigate formation of aggregates whose results were correlated with those obtained from DLS and Nu-PAGE, while RP-HPLC was used to investigate any possible chemical modifications. The Proposed RP-method had limit of detection (LOD) and limit of quantitation (LOQ) of 0.012 mg/mL and 0.045 mg/mL respectively and accuracy of 99.22 ± 1.07 %, while the SEC methods had limit of detection (LOD) and limit of quantitation (LOQ) of 0.012 mg/mL and 0.031 mg/mL, respectively. The degradation pattern due to high temperature effect and oxidation is investigated by LC- tandem mass spectrometry. Results showed that Insulin degludec is highly stable under low temperature, mechanical agitation and repeated freeze and thaw stress conditions but elevated temperature and high acidic condition lead to formation of aggregates and also chemical modifications including deamidation, isomerization and oxidation. Such different chemical degradation pathways are due to presence of variable reactive moieties in Insulin degludec structure. Insulin degludec is highly vulnerable to oxidation at the sulfur containing cysteine residue in B chain in position B7 forming trioxidation derivative when exposed to hydrogen peroxide. Formation of A21-Asp and A18-Asp deamidated variants as well as B3-Asp and B3-isoAsp deamidated variants are prominent degradation pathways at neutral pH but at elevated temperature.

The Uses of Dimedone for the Synthesis of Thiophene, Thiazole and Annulated Derivatives with Antitumor, Pim-1 Kinase Inhibitions, PAINS Evaluations and Molecular Docking.

BACKGROUND: Dimedone is considered as one of the most important classes of compounds belonging to cyclohexan-1,3-dione. Such groups of compounds were considered as precursors for many pharmaceutically active heterocyclic compounds. OBJECTIVE: The target molecules in this work were synthesized from arylhydrazones of dimedone with different substituents enhancing the study of their structure-activity relationship. METHODS: Arylhydrazones of dimedones were subjected to a series of heterocyclization reactions affording annulated compounds. The anti-proliferative activities of the synthesized molecules were evaluated against six cancer cell lines. In addition, inhibitions toward tyrosine kinases, Pim-1 kinases and PAINS of the most active compounds were also studied. c-Met enzymatic inhibitions and molecular docking studies were carried out for three compounds. RESULTS: Anti-cancer evaluations together with tyrosine and Pim-1 kinases of most of the synthesized compounds were carried out through this work. The study revealed that changing of substituents had a strong impact on the activity of the molecule. CONCLUSION: Many of the synthesized compounds exhibited high inhibitions towards the six cancer cell lines. This will encourage further work through the synthesis of target molecules with the same ring systems. The three compounds 7b, 8c and 12b that revealed excellent inhibitions were tested against c-Met kinase and their molecular modelling was expressed.

Multi-Component Reactions of Cyclohexan-1,3-dione: Synthesis of Fused Pyran, Pyridine, Thiophene and Pyrazole Derivatives with c-Met, Anti-Proliferative Activities.

BACKGROUND: Recently, products of Multi-Component Reactions (MCR's) acquired special attention due to their wide range of pharmacological activities especially therapeutic activities. In the market it was found that many pharmacological drugs containing the pyran and pyridine nucleus that were produced through MCR's were found. OBJECTIVE: This work aims to synthesize target molecules not only possess anti-tumour activities but also c-Met and prostate cancer inhibitors. The target molecules were obtained starting from cyclohexan-1,3-dione through its multi-component reactions to produce anticancer target molecules. METHODS: Cyclohexane-1,3-dione underwent different multi-component reactions to produce fused pyran, pyridine and thiophene derivatives. The anti-proliferative activity of the newly synthesized compounds among the synthesized compounds toward the six cancer cell lines, namely A549, H460, HT-29, MKN-45, U87MG, and SMMC-7721 was studied. In addition, inhibitions toward c-Met kinase and prostate cancer cell line were studied. Antitumor evaluations toward seventeen cancer cell lines subpanel, for certain compounds, were also demonstrated according to the diseases. Pim-1 kinase inhibitions of the most active compounds were also measured. RESULTS: Anti-proliferative evaluations, c-Met and Pim-1 kinase inhibitions were performed for most of the synthesized compounds where the varieties of substituent through the aryl ring and the heterocyclic ring afforded compounds with high activities. CONCLUSION: Compounds 4b, 6b, 8b, 9a, 11b, 12b, 17b, 18b, 19, 22c, 23b, 25b and 26b were the most cytotoxic compounds toward the six cancer cell lines. Inhibitions toward c-Met kinase and prostate cancer cells showed that the presence of the electronegative Cl group within the molecule were responsible for its high activity. In addition, inhibitions toward Pim-1 kinase exhibited that most of tested compounds showed high inhibitions.

Evaluating Flinders Technology Associates card for transporting bacterial isolates and retrieval of bacterial DNA after various storage conditions.

BACKGROUND AND AIM: Flinders Technology Associates (FTA) cards simplify sample storage, transport, and extraction by reducing cost and time for diagnosis. This study evaluated the FTA suitability for safe transport and storage of Gram-positive and Gram-negative bacterial cells of animal origin on its liquid culture form and from organ impression smears (tissues) under the same routine condition of microbiological laboratory along with detecting their nucleic acid over different storage conditions. MATERIALS AND METHODS: Increase in bacterial count from 104 to 107 (colony-forming units/mL) of 78 isolates representing seven bacterial species was applied onto cards. FTA cards were grouped and inoculated by these bacteria and then stored at different conditions of 24-27°C, 4°C, and -20°C for 24 h, for 2 weeks, for 1 and 3 month storage, respectively. Bacteriological examination was done, after which bacterial DNA was identified using specific primers for each bacterial type and detected by polymerase chain reaction (PCR). RESULTS: The total percentage of recovered bacteria from FTA cards was 66.7% at 24-27°C for 24 h, the detection limit was 100% in Gram-positive species, while it was 57.4% in Gram-negative ones. Regarding viable cell detection from organ impression smears, it was successful under the previous conditions. No live bacterial cells were observed by bacteriological isolation rather than only at 24-27°C for 24 h storage. All bacterial DNA were sufficiently confirmed by the PCR technique at different conditions. CONCLUSION: Overall, the FTA card method was observed to be a valid tool for nucleic acid purification for bacteria of animal origin in the form of culture or organ smears regardless of its Gram type and is used for a short time only 24 h for storage and transport of live bacteria specifically Gram-positive type. Moreover, the bacterial nucleic acid was intact after storage in -20°C for 3 months and was PCR amplifiable.

Influences of Dual-Task Training on Walking and Cognitive Performance of People With Relapsing Remitting Multiple Sclerosis: Randomized Controlled Trial.

OBJECTIVE: We sought to investigate whether there is any additional effect of coupled cognitive and physical rehabilitation compared to exercise training alone on walking and cognitive performance in individuals with relapsing remitting multiple sclerosis (RRMS). METHODS: A randomized controlled trial was conducted from March to November 2015 with 30 individuals with RRMS (aged 20 to 50 years; 21 women, 9 men), who underwent detailed medical and neurologic examination. They were randomly allocated using sealed envelopes to either the study group, who received physical and cognitive rehabilitation (dual-task training), or the control group, who received physical rehabilitation alone. Participants (in both groups) were assessed twice (8 weeks apart), before and after rehabilitation. Assessment tools were the Mini-Mental State Examination (MMSE), the Expanded Disability Status Scale (EDSS), neuropsychological evaluation (using RehaCom), and walking tests. RESULTS: After training, the control group significantly improved regarding MMSE, attention/concentration test, and 10-meter walking test, whereas the scores of the study group significantly improved in all studied parameters (Expanded Disability Status Scale, MMSE, logical reasoning, and attention/concentration and walking tests). The differential (delta) scores from before to after rehabilitation were significantly higher in the study group for logical reasoning, attention/concentration, and 2-minute walking distance scores. CONCLUSIONS: Coupled physical and cognitive (dual-task) training showed concurrent improvement in cognitive and walking abilities in individuals with RRMS which exceeded that achieved by physical training alone.

Foliar Application of Bio-Stimulants Enhancing the Production and the Toxicity of Origanum majorana Essential Oils Against Four Rice Seed-Borne Fungi.

In the present study, the enhancement of the production of Origanum majorana essential oils (EOs) was studied by treating plants with ascorbic acid (AA) and tryptophan (Trp) at concentrations of 100, 200 and 300 mg/L and Moringa oleifera leaf extract (MLE) at 2.5%, 5% and 10% as foliar applications during the seasons 2018-2019. The toxicities of the EOs were assayed against four seed-borne fungi (Bipolaris orzyae, Curvularia lunata, Fusarium verticilliodies and F. graminearum) isolated from rice grains (Oryzae sativa). Vegetative growth parameters and EO production were enhanced by the application of AA, Trp and MLE in both seasons. Analysis of the EOs by Gas chromatography-mass spectrometry (GC-MS) showed that the main chemical constituents were terpineol (cis-ß-(1-terpinenol)), terpinen-4-ol, 4-thujanol (sabinene hydrate), α-terpineol, cymene and sabinene. The highest fungal mycelial growth inhibition (FMGI) percentages against F. verticilliodies were 94.57% and 92.63% as MLE at 5% and 10%, respectively, was applied to plants and 85.60% and 82.19% against F. graminearum as Trp was applied to plants at 300 and 200 mg/L, respectively. EOs from the treated plant with MLE (10%) observed the highest FMGI (84.46%) against B. oryzae, and EOs from plants treated with AA as foliar application at 300 and 200 mg/L showed the highest FMGI values of 81.11% and 81.85%, respectively, against the growth of C. lunata. Application of EOs extracted from plants treated with Trp, AA and MLE at 300 mg/L, 300 mg/L and 10%, respectively, or untreated plants to rice seeds inhibited or decreased the fungal infection percentage from 82.5% (naturally infected grains) to 1.75%, 10.5%, 17.5% and 18.5%, respectively. In conclusion, the extracted EOs affected by the foliar application of O. majorana plants with Trp, AA, and MLE could be useful as a biofungicide against rice seed-borne fungi.

Antibacterial activity of three essential oils and some monoterpenes against Ralstonia solanacearum phylotype II isolated from potato.

The present study describes the possibility of using some essential oils and monoterpens as bioagents against the growth of Ralstonia solanacearum, a causal bacterium of potato brown rot disease. Eight isolates of the bacterium were recovered from infected potato tubers, showing typical symptoms of the disease, Isolates were identified as R.solanacearum phylotype II, based on biochemical and physiological characteristics, as well as, at the molecular level through PCR analysis. Three essential oils extracted from Corymbia citriodora (leaves), Cupressus sempervirens (aerial parts), and Lantana camara (aerial parts) were evaluated for their antibacterial activity against eight isolates of R. solanacearum phylotype II. Results demonstrated that L. camara essential oil (concentration 5000 µg/mL) had the highest effects against the RsMo2, RsSc1 and Rs48, with inhibition zone (IZ) values of 17.33, 16.33, and 17.50 mm, respectively, also against Rs2 (IZ 14.33 mm), and RsIs2 (IZ 16 mm). C. citriodora oil showed the highest activity against RsBe2 (IZ 14 mm), RsFr4 (IZ 13.66 mm) and RsNe1 (IZ 13.66 mm). Gas Chromatography-Mass Spectrometry (GC-MS-FID) analyzed the chemical composition of these essential oils. It was proved that L. camara leaves contains mainly trans-caryophyllene (16.24%) and α-humulene (9.55%), in C. citriodora oil were α-citronellal (56.55%), α-citronellol (14.89%), and citronellol acetate (13.04%), and in Cup. sempervirens aerial parts were cedrol (22.17%), and Δ3-carene (18.59%). Five monoterpenes were evaluated against the most resistance Ralstonia isolate RsFr5 to the three studied essential oils and found that limonene had the highest effect against it compared with the lowest thymol. The results proved the strong bio effects of the essential oil from L. camara leaves as a natural product contained monoterpenes that can inhibit the growth of tested R. solanacearum phylotype II isolates.

Antibacterial activity of the bioactive compounds identified in three woody plants against some pathogenic bacteria.

Three bacterial isolates were identified from infected potato tubers showing soft and blackleg like symptoms as well as one isolate from infected pear tree showing crown gall symptom. Conventional and molecular identification proved that bacterial isolates belonging to Pectobacterium carotovorum subsp. carotovorum, Pectobacterium atrosepticum, Dickeya solani and Agrobacterium tumefaciens. The above plant bacterial isolates and human pathogenic bacteria Escherichia coli, Sarcina lutea, and Staphylococcus aureus were used for the bioassay. The chloroform leaf extracts from Duranta plumieri variegata, Lantana camara, and Citharexylum spinosum were assayed for their antibacterial activity by measuring the inhibition zones and minimum inhibitory concentrations (MICs). The suggested chemical compositions of extracts were analyzed using GC/MS apparatus. The main compounds in leaf extract of L. camara were 5,8-diethyl-dodecane, pyrimidin-2-one, 4-[N-methylureido]-1-[4-methylaminocarbonyloxymethyl, oleic acid,3-(octadecyloxy)propyl ester; in D. plumieri were 4,7-dimethoxy-2-methylindan-1-one and 5-(hexadecyloxy)-2-pentadecyl-,trans-1,3-dioxane; and in C. spinosum were N-[5-(3-hydroxy-2-methylpropenyl)-1,3,4,5-tetrahydrobenzo[cd]indol-3-yl]-N-methylacetamide. Promising activity was found against A. tumefaciens, E. coli, P. carotovorum, Sar. lutea, and Staph. aureus with MIC values of 8, 128, 64, 500 and 500 µg/mL, respectively, as L. camara leaf extract was applied. D. plumieri leaf extract showed good activity against D. solani and P. atrosepticum with MIC values of 16 µg/mL and 128 µg/mL, respectively. On the other hand, weak bioactivity was found with leaf extract from C. spinosum. It could be concluded that leaf extracts from D. plumieri and L. camara have a promising antibacterial agents.

Antigenic and pathogenicity activities of Ralstonia solanacearum race 3 biovar 2 molecularly identified and detected by indirect ELISA using polyclonal antibodies generated in rabbits.

Eight molecular-characterized isolates of Ralstonia solanacearum from potato belonging to race 3 biovar 2, their virulence were evaluated on potato cv. Lady Rosette, tomato cv. Strain B, eggplant cv. Balady and pepper cv. Balady and showed high virulence on potato and tomato, and lower virulence on eggplant and pepper. A laboratory study conducted to produce polyclonal antibodies against the potato brown rot bacterium; R. solanacearum cells were generated in female New Zealand white rabbits. A modification were made on the technique of indirect enzyme-linked immunosorbent assay (ELISA) to improve the sensitivity of detection, including antigenic and sensitivity to R. solanacearum race 3 biovar 2 isolates. Determination of the optimum period to collect the antiserum (including, polyclonal antibodies) showed that the best collection dates were at 14, 3 and 7 days, in that order. The efficiency of the antiserum was compared among 42 isolates that cause potato brown rot disease; our polyclonal antiserum (14 days) reacted positively with all tested isolates at a dilution of 1:6.4 × 103. Data indicated the different reactions of eight R. solanacearum isolates at various dilutions (1:1.6 × 103 to 1:5.12 × 106) at 14 days against polyclonal antiserumat a concentration of approximately 1 × 108 CFU/mL and we found the lowest detection level by the indirect ELISA technique was 106 CFU/mL. Finally we recommended the reasonable sensitivity results of the ELISA technique to detect the bacterial pathogen given than the cost of this technique if much lower than that of other expensive molecular techniques.

Stability assessment of antibody-drug conjugate Trastuzumab emtansine in comparison to parent monoclonal antibody using orthogonal testing protocol.

Antibody-drug conjugates (ADC) represent an emerging, novel class of biopharmaceuticals. The heterogeneity originating from the sophisticated structure requires orthogonal analytical techniques for quality and stability assessment of ADC to ensure safety and efficacy. In this study, the stability of Trastuzumab (recombinant humanized IgG1 mAb, targeting HER2 receptor) and its ADC with DM1 (anti-tubulin anticancer drug), Trastuzumab emtansine (T-DM1) were studied. SE-HPLC was used to monitor formation of aggregates and/or fragments of the monoclonal antibodies (mAb). Correlation with the results of reducing and non-reducing sodium dodecyl sulphate - polyacrylamide gel electrophoresis (SDS-PAGE) and dynamic light scattering (DLS) were performed to interpret the obtained results. RP-HPLC was used for assessment of the stability of DM1 in ADC while spectrophotometry was employed to determine drug antibody ratio (DAR) . The studied drugs were subjected to several stress conditions including pH, temperature, mechanical agitation and repeated freeze and thaw to generate possible degradation products and ensure suitability of the assay protocol. The degradation pattern and extent were demonstrated under the indicated stress conditions. The correlation between the results of SE-HPLC and those of SDS-PAGE and DLS ensured the validity of the orthogonal assay protocol and indicated aggregates that were not detected using SE-HPLC. Results showed clearly that T-DM1 is relatively less stable than its parent mAb. This was attributed to the presence of the drug-linker part that is attached to the mAb. RP-HPLC showed that the cytotoxic drug moiety is liable for degradation under the studied conditions resulting in alteration of DAR as well as formation of degradation products. This confirmed the need for more robust coupling chemistries for production of safe and effective ADC and highlighted the importance of orthogonal testing protocols for quality assessment. The assay protocol should be applicable for quality and stability assessment of various ADC.

Comparative Assessment of the Effect of Hyper-glycosylation on the Pattern and Kinetics of Degradation of Darbepoetin Alfa using a Stability-Indicating Orthogonal Testing Protocol.

Darbepoetin alfa (DA); hyper-glycosylated Erythropoietin alfa (EPO) is an essential treatment of anemia in patients with chronic kidney failure and cancer. In this study, DA and EPO were subjected to physicochemical stress factors that might be encountered during production, transport and storage (pH, temperature, agitation, repeated freeze-thaw and oxidation). An orthogonal stability-indicating assay protocol comprised of SE-HPLC, RP-HPLC, ELISA and SDS-PAGE was developed and validated to investigate the effect of further glycosylation of DA on the pattern and kinetics of degradation. Results showed a relatively higher stability and lower tendency to form high molecular weight aggregates in the case of DA when compared to EPO, under equivalent stress conditions. Dimers and aggregates were formed for both drugs across the whole pH range and following incubation at temperatures higher than 2-8°C or repeated freeze/thaw. The same observation was noted upon agitation of standard samples prepared in the formulation buffers at high speed and upon oxidation with hydrogen peroxide. The agreement between SE-HPLC, supported with spectral purity data and ELISA confirmed the specificity of both techniques for the intact drugs. Results of RP-HPLC and SDS-PAGE indicated that dimerization occurred through disulfide and bi-tyrosine covalent bonds in the case of pH and oxidation, respectively. It was evident that aggregation was significantly suppressed upon increasing the glycan size and under any of the studied stress factors loss of the glycan has not been observed. These observations supported with the slow kinetics of degradation confirmed the superiority of glyco-engineering over chemical pegylation to enhance the stability of EPO. Formation of such potentially immunogenic product-related impurities at all tested stress factors confirmed the need for orthogonal testing protocols to investigate the complex pattern of degradation of such sensitive products.