Heterologous promoter recognition leading to high-level expression of cloned foreign genes in Bombyx mori cell lines and larvae

The baculovirus expression system using the Autographa californica nuclear polyhedrosis virus (AcNPV) has been extensively utilized for high-level expression of cloned foreign genes, driven by the strong viral promoters of polyhedrin (polh) and p10 encoding genes. A parallel system using Bombyx mori nuclear polyhedrosis virus (BmNPV) is much less exploited because the choice and variety of BmNPV-based transfer vectors are limited. Using a transient expression assay, we have demonstrated here that the heterologous promoters of the very late genes polh and p10 from AcNPV function as efficiently in BmN cells as the BmNPV promoters. The location of the cloned foreign gene with respect to the promoter sequences was critical for achieving the highest levels of expression, following the order +35 > +1 > -3 > -8 nucleotides (nt) with respect to the polh or p10 start codons. We have successfully generated recombinant BmNPV harboring AcNPV promoters by homeologous recombination between AcNPV-based transfer vectors and BmNPV genomic DNA. Infection of BmN cell lines with recombinant BmNPV showed a temporal expression pattern, reaching very high levels in 60-72 h post infection. The recombinant BmNPV harboring the firefly luciferase-encoding gene under the control of AcNPV polh or p10 promoters, on infection of the silkworm larvae led to the synthesis of large quantities of luciferase. Such larvae emanated significant luminiscence instantaneously on administration of the substrate luciferin resulting in 'glowing silkworms'. The virus-infected larvae continued to glow for several hours and revealed the most abundant distribution of virus in the fat bodies. In larval expression also, the highest levels were achieved when the reporter gene was located at +35 nt of the polh.

A Pulsewidth Modulated Control of Induction Motor Drive Using Multilevel 12-Sided Polygonal Voltage Space Vectors

In this paper, a novel 12-sided polygonal space vector structure is proposed for an induction motor drive. The space vector pattern presented in this paper consists of two 12-sided concentric polygons with the outer polygon having a radius double the inner one. As compared to previously reported 12-sided polygonal space vector structures, this paper subdivides the space vector plane into smaller sized triangles. This helps in reducing the switching frequency of the inverters without deteriorating the output voltage quality. It also reduces the device ratings and dv/dt stress on the devices to half. At the same time, other benefits obtained from the existing 12-sided space vector structure, such as increased linear modulation range and complete elimination of 5th and 7th order harmonics in the phase voltage, are also retained in this paper. The space vector structure is realized by feeding an open-end induction motor with two conventional three-level neutral point clamped (NPC) inverters with asymmetric isolated dc link voltage sources. The neutral point voltage fluctuations in the three-level NPC inverters are eliminated by utilizing the switching state multiplicities for a space vector point. The pulsewidth modulation timings are calculated using sampled reference waveform amplitudes and are explained in detail in this paper. Experimental verification on a laboratory prototype shows that this configuration may be considered suitable for high power drives.

Non-segmented negative sense RNA viruses as vectors for vaccine development

This article intends to cover two aspects of non-segmented negative sense RNA viruses. In the initial section, the strategy employed by these viruses to replicate their genomes is discussed. This would help in understanding the later section in which the use of these viruses as vaccine vectors has been discussed. For the description of the replication strategy which encompasses virus genome transcription and genome replication carried out by the same RNA dependent RNA polymerase complex, a member of the prototype rhabdovirus family - Chandipura virus has been chosen as an example to illustrate the complex nature of the two processes and their regulation. In the discussion on these viruses serving as vectors for carrying vaccine antigen genes, emphasis has been laid on describing the progress made in using the attenuated viruses as vectors and a description of the systems in which the efficiency of immune responses has been tested.

Translational Up-Regulation and High-Level Protein Expression from Plasmid Vectors by mTOR Activation via Different Pathways in PC3 and 293T Cells

Background: Though 293T cells are widely used for expression of proteins from transfected plasmid vectors, the molecular basis for the high-level expression is yet to be understood. We recently identified the prostate carcinoma cell line PC3 to be as efficient as 293T in protein expression. This study was undertaken to decipher the molecular basis of high-level expression in these two cell lines. Methodology/Principal Findings: In a survey of different cell lines for efficient expression of platelet-derived growth factor-B (PDGF-B), beta-galactosidase (beta-gal) and green fluorescent protein (GFP) from plasmid vectors, PC3 was found to express at 5-50-fold higher levels compared to the bone metastatic prostate carcinoma cell line PC3BM and many other cell lines. Further, the efficiency of transfection and level of expression of the reporters in PC3 were comparable to that in 293T. Comparative analyses revealed that the high level expression of the reporters in the two cell lines was due to increased translational efficiency. While phosphatidic acid (PA)-mediated activation of mTOR, as revealed by drastic reduction in reporter expression by n-butanol, primarily contributed to the high level expression in PC3, multiple pathways involving PA, PI3K/Akt and ERK1/2 appear to contribute to the abundant reporter expression in 293T. Thus the extent of translational upregulation attained through the concerted activation of mTOR by multiple pathways in 293T could be achieved through its activation primarily by the PA pathway in PC3. Conclusions/Significance: Our studies reveal that the high-level expression of proteins from plasmid vectors is effected by translational up-regulation through mTOR activation via different signaling pathways in the two cell lines and that PC3 is as efficient as 293T for recombinant protein expression. Further, PC3 offers an advantage in that the level of expression of the protein can be regulated by simple addition of n-butanol to the culture medium.

Central Limit Theorem for truncated heavy tailed Banach valued random vectors

In this paper the question of the extent to which truncated heavy tailed random vectors, taking values in a Banach space, retain the characteristic features of heavy tailed random vectors, is answered from the point of view of the central limit theorem.

Baculovirus mediated high-level expression of luciferase in silkworm cells and larvae

Bombyx mori nuclear polyhedrosis virus (BmNPV)-based baculovirus expression system exploits silkworm larvae as an economical alternative to large-scale cell cultures for production of biomolecules. To generate recombinant BmNPV at high efficiency, we have achieved high efficiency transfection of B. mori cells, BmN, through lipofection. Optimal conditions for lipofection were standardized by quantification of the transient expression level of firefly luciferase (luc) reporter gene under control of an immediate early gene promoter of BmNPV Lipofection was 50-fold and 100-fold more efficient than the calcium phosphate method for transfecting BmN and Sf9 cells, respectively. Lipofection enabled us to generate a recombinant BmNPV (vBmluc), harboring luc under control of the strong polyhedrin promoter On infection with vBmluc, luciferase was expressed at very high levels, 170 mu g/10(6) BmN cells or 13 mg/larva. Expression of luciferase in vBmluc-infected larvae was visualized by luminescence emission instantaneously following luciferin injection generating ''glowing silkworms''.

Advantage of the ether linkage between the positive charge and the cholesteryl skeleton in cholesterol-based amphiphiles as vectors for gene delivery

Twelve novel cationic cholesterol derivatives with different linkage types between the cationic headgroup and the cholesteryl backbone have been developed. These have been tested for their efficacies as gene transfer agents as mixtures with dioleoyl phosphatidylethanolamine (DOPE). A pronounced improvement in transfection efficiency was observed when the cationic center was linked to the steroid backbone using an ether type bond. Among these, cholest-5-en-3b-oxyethane-N, N,N-trimethylammonium bromide (2a) and cholest-5-en-3b-oxyethane-N, N-dimethyl-N-2-hydroxyethylammonium bromide (3d) showed transfection efficiencies considerably greater than commercially available reagents such as Lipofectin or Lipofectamine. To achieve transfection, 3d did not require DOPE. Increasing hydration at the headgroup level for both ester- and ether-linked amphiphiles resulted in progressive loss of transfection efficiency. Transfection efficiency was also greatly reduced when a 'disorder'-inducing chain like an oleyl (cis-9-octadecenyl) segment was added to these cholesteryl amphiphiles. Importantly, the transfection ability of 2a with DOPE in the presence of serum was significantly greater than for a commercially available reagent, Lipofectamine. This suggests that these novel cholesterol-based amphiphiles might prove promising in applications involving liposome-mediated gene transfection. This investigation demonstrates the importance of structural features at the molecular level for the design of cholesterol-based gene delivery reagents that would aid the development of newer, more efficient formulations based on this class of molecules.

Generation of Minimum Leakage Input Vectors with Constrained NBTI Degradation

Technology scaling has caused Negative Bias Temperature Instability (NBTI) to emerge as a major circuit reliability concern. Simultaneously leakage power is becoming a greater fraction of the total power dissipated by logic circuits. As both NBTI and leakage power are highly dependent on vectors applied at the circuit’s inputs, they can be minimized by applying carefully chosen input vectors during periods when the circuit is in standby or idle mode. Unfortunately input vectors that minimize leakage power are not the ones that minimize NBTI degradation, so there is a need for a methodology to generate input vectors that minimize both of these variables.This paper proposes such a systematic methodology for the generation of input vectors which minimize leakage power under the constraint that NBTI degradation does not exceed a specified limit. These input vectors can be applied at the primary inputs of a circuit when it is in standby/idle mode and are such that the gates dissipate only a small amount of leakage power and also allow a large majority of the transistors on critical paths to be in the “recovery” phase of NBTI degradation. The advantage of this methodology is that allowing circuit designers to constrain NBTI degradation to below a specified limit enables tighter guardbanding, increasing performance. Our methodology guarantees that the generated input vector dissipates the least leakage power among all the input vectors that satisfy the degradation constraint. We formulate the problem as a zero-one integer linear program and show that this formulation produces input vectors whose leakage power is within 1% of a minimum leakage vector selected by a search algorithm and simultaneously reduces NBTI by about 5.75% of maximum circuit delay as compared to the worst case NBTI degradation. Our paper also proposes two new algorithms for the identification of circuit paths that are affected the most by NBTI degradation. The number of such paths identified by our algorithms are an order of magnitude fewer than previously proposed heuristics.

Fast Direct Torque Control of an Open-End Induction Motor Drive Using 12-Sided Polygonal Voltage Space Vectors

A torque control scheme, based on a direct torque control (DTC) algorithm using a 12-sided polygonal voltage space vector, is proposed for a variable speed control of an open-end induction motor drive. The conventional DTC scheme uses a stator flux vector for the sector identification and then the switching vector to control stator flux and torque. However, the proposed DTC scheme selects switching vectors based on the sector information of the estimated fundamental stator voltage vector and its relative position with respect to the stator flux vector. The fundamental stator voltage estimation is based on the steady-state model of IM and the synchronous frequency of operation is derived from the computed stator flux using a low-pass filter technique. The proposed DTC scheme utilizes the exact positions of the fundamental stator voltage vector and stator flux vector to select the optimal switching vector for fast control of torque with small variation of stator flux within the hysteresis band. The present DTC scheme allows full load torque control with fast transient response to very low speeds of operation, with reduced switching frequency variation. Extensive experimental results are presented to show the fast torque control for speed of operation from zero to rated.

On the structure of analytic vectors for the Schrodinger representation

This article deals with the structure of analytic and entire vectors for the Schrodinger representations of the Heisenberg group. Using refined versions of Hardy's theorem and their connection with Hermite expansions we obtain very precise representation theorems for analytic and entire vectors.

Badly approximable numbers and vectors in Cantor-like sets

We show that a large class of Cantor-like sets of R-d, d >= 1, contains uncountably many badly approximable numbers, respectively badly approximable vectors, when d >= 2. An analogous result is also proved for subsets of R-d arising in the study of geodesic flows corresponding to (d+1)-dimensional manifolds of constant negative curvature and finite volume, generalizing the set of badly approximable numbers in R. Furthermore, we describe a condition on sets, which is fulfilled by a large class, ensuring a large intersection with these Cantor-like sets.

A Hybrid Multilevel Inverter System Based on Dodecagonal Space Vectors for Medium Voltage IM Drives

Dodecagonal (12-sided) space vector pulsewidth modulation (PWM) schemes are characterized by the complete absence of (6n +/- 1)th-order harmonics (for odd n) in the phase voltages, within the linear modulation range and beyond, including over-modulation. This paper presents a new topology suitable for the realization of such multilevel inverter schemes for induction motor (IM) drives, by cascading two-level inverters with flying-capacitor-inverter fed floating H-bridge cells. Now, any standard IM may be used to get the dodecagonal operation which hitherto was possible only with open-end winding IM. To minimize the current total harmonic distortion (THD), a strategy for synchronous PWM is also proposed. It is shown that the proposed method is capable of obtaining better THD figures, compared to conventional dodecagonal schemes. The topology and the PWM strategy are validated through analysis and subsequently verified experimentally.

Medium Voltage Drive for Induction Motors Using Multilevel Octadecagonal Voltage Space Vectors

Multilevel inverters with hexagonal and dodecagonal voltage space vector structures have improved harmonic profile compared to two-level inverters. Further improvement in the quality of the waveform is possible using multilevel octadecagonal (18-sided polygon) voltage space vectors. This paper proposes an inverter circuit topology capable of generating multilevel octadecagonal voltage space vectors, by cascading two asymmetric three-level inverters. By the proper selection of dc-link voltages and the resultant switching states for the inverters, voltage space vectors, whose tips lie on three concentric octadecagons, are obtained. The advantages of octadecagonal voltage space vector-based pulsewidth modulation (PWM) techniques are the complete elimination of fifth, seventh, eleventh, and thirteenth harmonics in phase voltages and the extension of linear modulation range. In this paper, a simple PWM timing calculation method is also proposed. Experimental results have been presented in this paper to validate the proposed concept.

A Multilevel Inverter Scheme With Dodecagonal Voltage Space Vectors Based on Flying Capacitor Topology for Induction Motor Drives

This paper presents a multilevel inverter topology suitable for the generation of dodecagonal space vectors instead of hexagonal space vectors as in the case of conventional schemes. This feature eliminates all the 6n +/- 1 (n = odd) harmonics from the phase voltages and currents in the entire modulation range with an increase in the linear modulation range. The topology is realized by flying capacitor-based three-level inverters feeding from two ends of an open-end winding induction motor with asymmetric dc links. The flying capacitor voltages are tightly controlled throughout the modulation range using redundant switching states for any load power factor. A simple and fast carrier-based space-vector pulsewidth modulation (PWM) scheme is also proposed for the topology which utilizes only the sampled amplitudes of the reference wave for the PWM timing computation.

Development of a New Generation of Vectors for Gene Expression, Gene Replacement, and Protein-Protein Interaction Studies in Mycobacteria

Escherichia coli-mycobacterium shuttle vectors are important tools for gene expression and gene replacement in mycobacteria. However, most of the currently available vectors are limited in their use because of the lack of extended multiple cloning sites (MCSs) and convenience of appending an epitope tag(s) to the cloned open reading frames (ORFs). Here we report a new series of vectors that allow for the constitutive and regulatable expression of proteins, appended with peptide tag sequences at their N and C termini, respectively. The applicability of these vectors is demonstrated by the constitutive and induced expression of the Mycobacterium tuberculosis pknK gene, coding for protein kinase K, a serine-threonine protein kinase. Furthermore, a suicide plasmid with expanded MCS for creating gene replacements, a plasmid for chromosomal integrations at the commonly used L5 attB site, and a hypoxia-responsive vector, for expression of a gene(s) under hypoxic conditions that mimic latency, have also been created. Additionally, we have created a vector for the coexpression of two proteins controlled by two independent promoters, with each protein being in fusion with a different tag. The shuttle vectors developed in the present study are excellent tools for the analysis of gene function in mycobacteria and are a valuable addition to the existing repertoire of vectors for mycobacterial research.