Development of New Biological Nanopores and Their Application for Biosensing and Disease Detection

Nanopore technology has recently emerged as a new real-time single molecule sensing method. The current dominant technologies, such as mass spectrometry and immunoassay, for protein analysis is still slow and complex, which can’t meet the urgent need and fields of use. Development of a highly simple, portable and sensitive detection system for pathogen detection, disease diagnosis, and environmental monitoring is in great need. Membrane embedded Phi29 connector nanopore, the first protein nanopore coming from bacteriophage, was mainly focusing on DNA and RNA translocation in previous studies. Here, Phi29 connector nanopore was first time established for antibody detection by engineering Epithelial Cell Adhesion Molecule peptide as a probe. The results demonstrate that the specific antibody can be detected in presence of diluted serum or non-specific antibody. To enable detecting more different types of analytes with high sensitivity, developing new nanopore with various properties, such as size, charge, hydrophilic/hydrophobic and physical dimension, is needed. In this work, besides Phi29 nanopore, several new protein nanopores that derived from T3, T4, and SPP1 bacteriophages were developed. A shared property of three step conformational change among these portal channel has been discovered. Elucidating the sequence and oligomeric states of proteins and peptides is critical for understanding their biological functions. Here, SPP1 nanopore was used to characterize the translocation of TAT peptide with dimer and monomer forms. Translocation of the peptide was confirmed by optical single molecule imaging for the first time, and analyzed quantitatively. The dynamics of peptide oligomeric states were clearly differentiated based on their characteristic electronic signatures. Main challenge for probing protein structure, folding, detection and sequencing using nanopore is the ultra-fast translocation speed which normally beyond electronic detection limit. In this work, the peptides translocation was slowed in SPP1 nanopore by changing the charge shielding of the channel. A 500-fold reduction was observed for TAT peptide translocation. By using this method, arginine chain peptide as short as two arginine can be detected first time. Further improving the bandwidth may lead to single amino acid detection and has the potential for protein sequencing. Compared with protein nanopore, de novo designed nanopore can provide numerous advantages, such as tunable size and functionality, ease of construction, scale up and modification. In the final study, an RNA-based biomimetic nanopore was first time constructed. The insertion of RNA nanopore into lipid bilayer and cell membrane were characterized and translocation of short amino acids through RNA nanopore was detected. This new artificial nanopore has the potential to be used for sensing, disease diagnosis, and even protein sequencing

The Essence of Foreign Language Learning in Today’s Globalizing World: Benefits and Hindrances

This paper highlights the essence of foreign language learning in today’s globalizing world as well as underscores its benefits and hindrances. It emphasizes that foreign language does collectively bind us together as human beings predicated upon our irresistible interdependence in various ways. Rationally speaking, being knowledgeable of a second or foreign language is extremely pertinent because it provides numerous life time opportunities. Yielding the dividends of studying abroad, employment, personal development, security, political and economic benefits among others make foreign language learning absolutely beneficial. Consequently, this paper sees foreign language conversance as a key that could unlock the door of your life and it must not be ignored. However, despite its overarching benefits, foreign language learning is continually constrained with the challenges of complexity, ignorance of its importance, learner-native speaker gaps, weak policies and inadequate support. Some of these challenges are related to the constructs of motivational beliefs and strategies which are vital to foreign language learning because learners must believe in themselves, value tasks and use appropriate strategies if they are to succeed. Finally, it recommends a collective concerted effort against monolingualism, but supports the formulation of salient policies and programs including the inclusion of foreign languages in the curricula, increased financial and moral supports, and early teaching of foreign languages to kids. Key words: foreign language, language benefits and barriers, motivational beliefs, language learning strategies, globalizing world

Lipid Bilayer-Integrated SPP1 Connector Protein Nanopore and SPP1 Connector Protein Variants for Use as Lipid Bilayer-Integrated Nanopore

A conductive channel-containing membrane includes a membrane layer, and a SPPl connector polypeptide variant that is incorporated into the membrane layer to form an aperture through which conductance can occur when an electrical potential is applied across the membrane. A method of sensing a molecule, such as a polypeptide or nucleic acid molecule, makes use of the conductive channel- containing membrane. A method of DNA sequence makes use of the conductive channel-containing membrane

TNF-α inhibits glucose-induced insulin secretion in a pancreatic β-cell line (INS-1)

AbstractRecent studies suggest that TNF-α affects various biochemical and physiological processes which may be linked to the etiology of non-insulin-dependent diabetes mellitus (NIDDM). For example, TNF-α interferes with the signaling of the insulin receptor and the metabolism of glucose transporters. The possibility that TNF-α might directly reduce glucose-stimulated insulin secretion in pancreatic β-cells was examined by using an established pancreatic β-cell line (INS-1). TNF-α did not affect glucose-induced acute insulin secretion (30 min). However, over a longer time period (24 h), TNF-α decreased glucose-induced insulin secretion without affecting the total amount of insulin in the cell. In the presence of TNF-α levels of 0, 10, 100 and 1000 U/ml, the respective 20 mM glucose-induced insulin secretion was 1.736 ± 0.166, 1.750 ± 0.302, 1.550 ± 0.200, and 1.400 ± 0.112 mU/ml per 3 × 105 cells in 24 h

Differential RhoA Dynamics in Migratory and Stationary Cells Measured by FRET and Automated Image Analysis

Genetically-encoded biosensors based on fluorescence resonance energy transfer (FRET) have been widely applied to study the spatiotemporal regulation of molecular activity in live cells with high resolution. The efficient and accurate quantification of the large amount of imaging data from these single-cell FRET measurements demands robust and automated data analysis. However, the nonlinear movement of live cells presents tremendous challenge for this task. Based on image registration of the single-cell movement, we have developed automated image analysis methods to track and quantify the FRET signals within user-defined subcellular regions. In addition, the subcellular pixels were classified according to their associated FRET signals and the dynamics of the clusters analyzed. The results revealed that the EGF-induced reduction of RhoA activity in migratory HeLa cells is significantly less than that in stationary cells. Furthermore, the RhoA activity is polarized in the migratory cells, with the gradient of polarity oriented toward the opposite direction of cell migration. In contrast, there is a lack of consistent preference in RhoA polarity among stationary cells. Therefore, our image analysis methods can provide powerful tools for high-throughput and systematic investigation of the spatiotemporal molecular activities in regulating functions of live cells with their shapes and positions continuously changing in time

Developing Further Versatility in Benzoxazine Synthesis via Hydrolytic Ring-Opening

In this study, 2-(aminomethyl)phenol and its derivatives, the reactants for 2-substituted 1,3-benzoxazines, are synthesized by HCl hydrolysis from the typical benzoxazines. The phenol/ aniline-based mono-oxazine benzoxazine, PH-a, and the bisphenol A/aniline-based bis-oxazine benzoxazine, BA-a, are used as examples to demonstrate the feasibility of this new approach. Their chemical structures are characterized by nuclear magnetic resonance (NMR), Fourier transform infrared (FTIR) and Raman spectroscopies, and are further verified by elementary analysis. Their thermal properties are studied by differential scanning calorimetry (DSC). These two 2-(aminomethyl) phenolic derivatives are reacted with paraformaldehyde to close the oxazine rings. A benzoxazine with a phenyl substituent at the 2-position of the oxazine ring is obtained from the 2-(phenylamino)methyl)phenol (hPH-a) and benzaldehyde. All these results highlight the success of the HCl hydrolysis and the formation of stable intermediates, namely 2-(aminomethyl) phenolic derivatives, from readily available benzoxazine monomers. This further demonstrates the feasibility of using these intermediates as reactants for a novel benzoxazine synthesis.Fil: Cui, Shaoying. Sichuan University; ChinaFil: Arza, Carlos R.. Case Western Reserve University; Estados UnidosFil: Froimowicz, Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Tecnología en Polímeros y Nanotecnología. Universidad de Buenos Aires. Facultad de Ingeniería. Instituto de Tecnología en Polímeros y Nanotecnología; Argentina. Case Western Reserve University; Estados UnidosFil: Ishida, Hatsuo. Case Western Reserve University; Estados Unido

Formation stage and controlling factors of the paleo-uplifts in the Tarim Basin: A further discussion

AbstractVarious types of paleo-uplifts with different characteristics are developed in the Tarim Basin. Previously, there were multiple opinions on the pale-uplifts origins and structural evolution, so the oil and gas exploration ideas and deployment in the Tarim Basin were not developed smoothly. In this paper, regional seismic interpretation and structural analysis were carried out on the deep marine carbonate rocks in this basin based on the new seismic and drilling data. Then combined with the structural denudation results, the paleo-structural frameworks were reconstructed. And finally, the formation stage and main controlling factors of paleo-uplifts were discussed. It is shown that the Middle Ordovician is the key period when regional extension was converted to compression in this basin, so stratigraphic, sedimentary and structural differences occurred. Before the deposition of Yijianfang Fm in late Middle Ordovician, three carbonate paleo-uplifts (i.e., the Northern, Central and SW Tarim paleo-uplifts) begun to appear, and they were all broad-folded paleo-uplifts of nearly E–W striking and were formed at the same stage. The distribution and development of the Phanerozoic uplifts in this basin are restricted by the Northern and Southern Tarim basement pale-uplifts of nearly E–W striking which were developed during the Precambrian. It is indicated that all the three paleo-uplifts are compressional paleo-uplifts originated from the convergence of the southern plate margin based on the basement paleo-uplifts and they are all characterized by similar structural characteristics and inherited formation and evolution. The current differences of paleo-uplifts are controlled by multi-stage intense structural reformation since the Silurian. It is concluded that the oil and gas exploration potential is immense in the carbonate reservoirs of well-preserved deep paleo-structural zones in a larger area