Noura S. Dosoky

Engineering Lipid Bilayer Membranes for Protein Studies

Lipid membranes regulate the flow of nutrients and communication signaling between cells and protect the sub-cellular structures. Recent attempts to fabricate artificial systems using nanostructures that mimic the physiological properties of natural lipid bilayer membranes (LBM) fused with transmembrane proteins have helped demonstrate the importance of temperature, pH, ionic strength, adsorption behavior, conformational reorientation and surface density in cellular membranes which all affect the incorporation of proteins on solid surfaces. Much of this work is performed on artificial templates made of polymer sponges or porous materials based on alumina, mica, and porous silicon (PSi) surfaces. For example, porous silicon materials have high biocompatibility, biodegradability, and photoluminescence, which allow them to be used both as a support structure for lipid bilayers or a template to measure the electrochemical functionality of living cells grown over the surface as in vivo. The variety of these media, coupled with the complex physiological conditions present in living systems, warrant a summary and prospectus detailing which artificial systems provide the most promise for different biological conditions. This study summarizes the use of electrochemical impedance spectroscopy (EIS) data on artificial biological membranes that are closely matched with previously published biological systems using both black lipid membrane and patch clamp techniques.

Volatile constituents of Pinus roxburghii from Nepal.

Background: Pinus roxburghii Sarg. Is one of 3 species of pine found in Nepal, the oil of which is traditionally used to treat cuts, wounds, boils, and blisters. Objective: To obtain, analyze, and examine the anti-microbial and cytotoxic activities of the essential oils of P. roxburghii. Materials and Methods: Three plant parts (cone, needle, and bark) of Pinus roxburghii were collected in Biratnagar, Nepal. The essential oils were obtained by hydrodistillation, and the chemical compositions were determined by GC-MS. The needle and cone essential oils were screened for anti-microbial activity against Bacillus cereus, Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, and Aspergillus niger; brine shrimp (Artemia salina) lethality; and in-vitro cytotoxicity against MCF-7 cells. Results: GC-MS analysis for the cone oil revealed 81 compounds with 78 components being identified (95.5% of the oil) while 98.3% of needle oil was identified to contain 68 components and 98.6% of the bark oil (38 components) was identified. The 3 essential oils were dominated by sesquiterpenes, particularly (E)-caryophyllene (26.8%-34.5%) and α-humulene (5.0%-7.3%) as well as monoterpene alcohols terpinen-4-ol (4.1%-30.1%) and α-terpineol(2.8%-5.0%). The monoterpene δ-3-carene was present only in needle and cone essential oils (2.3% and 6.8%, respectively). Bio-activity assays of the cone essential oil of P. roxburghii showed remarkable cytotoxic activity (100% killing of MCF-7 cells at 100 μg/mL) along with notable brine shrimp lethality (LC50 =11.8 μg/mL). The cone essential oil did not show anti-bacterial activity, but it did exhibit anti-fungal activity against Aspergillus niger (MIC=39 μg/mL). Conclusion: The bioactivity of P. roxburghii essential oil is consistent with its traditional medicinal use.

Biological Activities and Volatile Constituents of Aegle marmelos (L.) Corrêa from Nepal

Aegle marmelos, a deciduous fruit tree that grows in Nepal, has numerous uses in traditional medicine, being used as an anti-inflammatory, antidiarrheal, heart disease treatment, and in birth control. In this study, leaves were collected to obtain, analyze, and examine the bioactivity of the essential oil of A. marmelos, which is often referred to as bael tree in Nepal. The essential oil from leaves was obtained by hydrodistillation. The chemical composition, determined by GC-MS, revealed 82 compounds, with 81 components being identified. The major component was limonene (64.1%), with the other two abundant components being (E)-β-ocimene (9.7%) and germacrene B (4.7%). Bioassay screening of the essential oil indicated marginal toxicity against MCF-7 human breast adenocarcinoma cells (LC50 = 98.2 μg mL), but good larvicidal activity against mosquitoes (Culex pipiens) (LC50 = 2.15 μg mL), nematocidal activity against Caenorhabditis elegans, (LC50 = 113 μg mL) and insecticidal activity against termites (Reticulitermes virginicus), fruit flies (Drosophila melanogaster), and fire ants (Solenopsis invica × richteri). The essential oil of A. marmelos showed remarkable brine shrimp lethality. Antimicrobial and antifungal activity of the leaf oil was negligible. Most of the observed biological activity was apparently due to the relatively high level of limonene (64.1%) in the essential of the leaf essential oil from A. marmelos. INTRODUCTION Aegle marmelos (L.) Corr. Serr. (Rutaceae) is one of only three species in genus Aegle (Sharma et al., 2011), is a subtropical fruit-bearing, deciduous tree that grows throughout hills and plains of the sub-Himalayan countries of Nepal, India, Sri Lanka, and Bangladesh (Sekar et al., 2011). The species, which is normally characterized by a thick trunk with spiny branches and a soft, flaky bark (Sekar et al., 2011), grows to a height of 7 to 8 m and has pale-green leaves with 3-5 leaflets that release an aroma when bruised. The plant has greenish-white flowers that bloom in late spring and produce an oval-shaped, sweet-tasting, soft fruit covered in a woody shell that turns yelow when the fruit ripens (Dhankhar et al., 2011; Sekar et al., 2011). A. marmelos has several common names, and in Nepal, is referred to as the bael tree. The plant is often seen growing in temple gardens of Nepal where the leaves are used in prayers to Lord Shiva and an important fertility ritual, Bel biha (Sekar et al., 2011). Considered one of the most important medicinal plants in southern and southeastern Asia, the entire plant is traditionally used to treat an array of ailments. Various tissues of A. marmelos have demonstrated positive effects on heart diseases (root bark) (Kakiuchi et al., 1991), dysentery and inflammation (leaves) (Arul et al., 2005), diabetes (leaf extract) (Kumar et al., 2009), and ulcers (Udupa et al., 1994). Satyal et al.: Biological Activities and Volatile Constituents of Aegle marmelos

Electrophysiology of Epithelial Sodium Channel (ENaC) Embedded in Supported Lipid Bilayer Using a Single Nanopore Chip

Nanopore-based technologies are highly adaptable supports for developing label-free sensor chips to characterize lipid bilayers, membrane proteins, and nucleotides. We utilized a single nanopore chip to study the electrophysiology of the epithelial Na+ channel (ENaC) incorporated in supported lipid membrane (SLM). An isolated nanopore was developed inside the silicon cavity followed by fusing large unilamellar vesicles (LUVs) of DPPS (1,2-dipalmitoyl-sn-glycero-3-phosphoserine) and DPPE (1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine) to produce a solvent-free SLM with giga-ohm (GΩ) sealed impedance. The presence and thickness of SLM on the nanopore chip were confirmed using atomic force spectroscopy. The functionality of SLM with and without ENaC was verified in terms of electrical impedance and capacitance by sweeping the frequency from 0.01 Hz to 100 kHz using electrochemical impedance spectroscopy. The nanopore chip exhibits long-term stability for the lipid bilayer before (144 h) and after (16 h) incorporation of ENaC. Amiloride, an inhibitor of ENaC, was utilized at different concentrations to test the integrity of fused ENaC in the lipid bilayer supported on a single nanopore chip. The developed model presents excellent electrical properties and improved mechanical stability of SLM, making this technology a reliable platform to study ion channel electrophysiology.

Chemical Diversity, Biological Activity, and Genetic Aspects of Three Ocotea Species from the Amazon

Ocotea species present economic importance and biological activities attributed to their essential oils (EOs) and extracts. For this reason, various strategies have been developed for their conservation. The chemical compositions of the essential oils and matK DNA sequences of O. caudata, O. cujumary, and O. caniculata were subjected to comparison with data from O. floribunda, O. veraguensis, and O. whitei, previously reported. The multivariate analysis of chemical composition classified the EOs into two main clusters. Group I was characterized by the presence of α-pinene (9.8–22.5%) and β-pinene (9.7–21.3%) and it includes O. caudata, O. whitei, and O. floribunda. In group II, the oils of O. cujumary and O. caniculata showed high similarity due amounts of β-caryophyllene (22.2% and 18.9%, respectively). The EO of O. veraguensis, rich in p-cymene (19.8%), showed minor similarity among all samples. The oils displayed promising antimicrobial and cytotoxic activities against Escherichia coli (minimum inhibitory concentration (MIC) < 19.5 µg·mL−1) and MCF-7 cells (median inhibitory concentration (IC50) ≅ 65.0 µg·mL−1), respectively. The analysis of matK gene displayed a good correlation with the main class of chemical compounds present in the EOs. However, the matK gene data did not show correlation with specific compounds.

Lipid Bilayer Membrane in a Silicon Based Micron Sized Cavity Accessed by Atomic Force Microscopy and Electrochemical Impedance Spectroscopy

Supported lipid bilayers (SLBs) are widely used in biophysical research to probe the functionality of biological membranes and to provide diagnoses in high throughput drug screening. Formation of SLBs at below phase transition temperature (Tm) has applications in nano-medicine research where low temperature profiles are required. Herein, we report the successful production of SLBs at above—as well as below—the Tm of the lipids in an anisotropically etched, silicon-based micro-cavity. The Si-based cavity walls exhibit controlled temperature which assist in the quick and stable formation of lipid bilayer membranes. Fusion of large unilamellar vesicles was monitored in real time in an aqueous environment inside the Si cavity using atomic force microscopy (AFM), and the lateral organization of the lipid molecules was characterized until the formation of the SLBs. The stability of SLBs produced was also characterized by recording the electrical resistance and the capacitance using electrochemical impedance spectroscopy (EIS). Analysis was done in the frequency regime of 10−2–105 Hz at a signal voltage of 100 mV and giga-ohm sealed impedance was obtained continuously over four days. Finally, the cantilever tip in AFM was utilized to estimate the bilayer thickness and to calculate the rupture force at the interface of the tip and the SLB. We anticipate that a silicon-based, micron-sized cavity has the potential to produce highly-stable SLBs below their Tm. The membranes inside the Si cavity could last for several days and allow robust characterization using AFM or EIS. This could be an excellent platform for nanomedicine experiments that require low operating temperatures.

The Chemical Compositions of the Volatile Oils of Garlic (Allium sativum) and Wild Garlic (Allium vineale)

Garlic, Allium sativum, is broadly used around the world for its numerous culinary and medicinal uses. Wild garlic, Allium vineale, has been used as a substitute for garlic, both in food as well as in herbal medicine. The present study investigated the chemical compositions of A. sativum and A. vineale essential oils. The essential oils from the bulbs of A. sativum, cultivated in Spain, were obtained by three different methods: laboratory hydrodistillation, industrial hydrodistillation, and industrial steam distillation. The essential oils of wild-growing A. vineale from north Alabama were obtained by hydrodistillation. The resulting essential oils were analyzed by gas chromatography-flame ionization detection (GC-FID) and gas chromatography-mass spectrometry (GC-MS). Both A. sativum and A. vineale oils were dominated by allyl polysulfides. There were minor quantitative differences between the A. sativum oils owing to the distillation methods employed, as well as differences from previously reported garlic oils from other geographical locations. Allium vineale oil showed a qualitative similarity to Allium ursinum essential oil. The compositions of garlic and wild garlic are consistent with their use as flavoring agents in foods as well as their uses as herbal medicines. However, quantitative differences are likely to affect the flavor and bioactivity profiles of these Allium species.

Antimicrobial, Antioxidant, and Cytotoxic Activities of Ocimum forskolei and Teucrium yemense (Lamiaceae) Essential Oils

Background: Ocimum forskolei and Teucrium yemense (Lamiaceae) are used in traditional medicine in Yemen. Methods: The chemical composition, antimicrobial, antioxidant and cytotoxic activities of the essential oils isolated from the leaves of Ocimum forskolei Benth. (EOOF) and two different populations of Teucrium yemense Deflers., one collected from Dhamar province (EOTY-d), and another collected from Taiz (EOTY-t) were investigated. The antimicrobial activities of the oils were evaluated against several microorganisms with the disc diffusion test or the broth microdilution test. The essential oils were screened for in-vitro cytotoxic activity against human tumor cells. EOOF and EOTY-d were screened for free-radical-inhibitory activity using the DPPH radical scavenging assay. Results: Sixty-four compounds were identified in (EOOF) representing 100% of the oil content with endo-fenchol (31.1%), fenchone (12.2%), τ-cadinol (12.2%), and methyl (E)-cinnamate (5.1%) as the major compounds. In EOTY-d, 67 compounds were identified, which made up 91% of the total oil. The most abundant constituents were (E)-caryophyllene (11.2%), α-humulene (4.0.%), γ-selinene (5.5%), 7-epi-α-selinene (20.1%), and caryophyllene oxide (20.1%), while the major compounds in EOTY-t were α-pinene (6.6%), (E)-caryophyllene (19.1%) α-humulene (6.4%), δ-cadinene (6.5%), caryophyllene oxide (4.3%), α-cadinol (9.5%), and shyobunol (4.6%). The most sensitive microorganisms for EOOF were B. subtilis, S. aureus, and C. albicans with inhibition zones of 34, 16, and 24 mm and MIC values of, 4.3 mg/mL, 4.3 mg/mL, and 8.6 mg/mL, respectively. EOTY-t showed antimicrobial activity against S. aureus, B. cereus, A. niger, and B. cinerea with MIC values of 0.156, 0.156, 0.313 and 0.313 mg/mL, respectively. Neither essential oil showed remarkable radical inhibition (IC50 = 31.55 and 31.41 μL/mL). EOTY-d was active against HT-29 human colorectal adenocarcinoma cell lines with IC50 = 43.7 μg/mL. Consistent with this, EOTY-t was active against both MCF-7 and MDA-MB-231 human breast adenocarcinoma cells. Conclusions: The antimicrobial activity of Ocimum forskolei essential oil against B. subtilis and C. albicans is consistent with its traditional use in Yemeni traditional medicine to treat skin infections. Both O. forskolei and T. yemense show wide variations in their respective essential oil compositions; there remains a need to investigate both species botanically, genetically, and phytochemically more comprehensively.

Composition and Biological Activities of Murraya paniculata (L.) Jack Essential Oil from Nepal

Murraya paniculata (L.) Jack, a small tropical evergreen shrub growing in Nepal, has numerous uses in traditional medicine for treatment of abdominal pain, diarrhea, stomach ache, headache, edema, thrombosis, and blood stasis. The present study investigated the chemical composition and bioactivities of the leaf essential oil from M. paniculata from Nepal. The essential oil from leaves was obtained by hydrodistillation and a detailed chemical analysis was conducted by gas chromatography-mass spectrometry (GC-MS). The essential oil was screened for antimicrobial activity using the microbroth dilution test, for nematicidal activity against Caenorhabditis elegans, and for lethality against brine shrimp (Artemia salina). A total of 76 volatile components were identified from the essential oil. The major components were methyl palmitate (11.1%), isospathulenol (9.4%), (E,E)-geranyl linalool (5.3%), benzyl benzoate (4.2%), selin-6-en-4-ol (4.0%), β-caryophyllene (4.0%), germacrene B (3.6%), germacrene D (3.4%), and γ-elemene (3.2%). The essential oil showed no antibacterial activity, marginal antifungal activity against Aspergillus niger (MIC = 313 μg/mL), a moderate activity against A. salina (LC50 = 41 μg/mL), and a good nematicidal activity against C. elegans (LC50 = 37 μg/mL).

The Genus Conradina (Lamiaceae): A Review

Conradina (Lamiaceae) is a small genus of native United States (US) species limited to Florida, Alabama, Mississippi, Tennessee and Kentucky. Three species of Conradina are federally listed as endangered and one is threatened while two are candidates for listing as endangered. The purpose of the present review is to highlight the recent advances in current knowledge on Conradina species and to compile reports of chemical constituents that characterize and differentiate between Conradina species.