Mutational analysis of active-site residues in the Mycobacterium leprae RecA intein, a LAGLIDADG homing endonuclease: Asp(122) and Asp(193) are crucial to the double-stranded DNA cleavage activity whereas Asp(218) is not

Mycobacterium leprae recA harbors an in-frame insertion sequence that encodes an intein homing endonuclease (PI-MleI). Most inteins (intein endonucleases) possess two conserved LAGLIDADG (DOD) motifs at their ctive center. A common feature of LAGLIDADG-type homing endonucleases is that they recognize and cleave the same or very similar DNA sequences. However, PI-MleI is distinctive from other members of the family of LAGLIDADG-type HEases for its modular structure with functionally separable domains for DNA-binding and cleavage, each with distinct sequence preferences. Sequence alignment analyses of PI-MleI revealed three putative LAGLIDADG motifs; however, there is conflicting bioinformatics data in regard to their identity and specific location within the intein polypeptide. To resolve this conflict and to determine the active-site residues essential for DNA target site recognition and double-stranded DNA cleavage, we performed site-directed mutagenesis of presumptive catalytic residues in the LAGLIDADG motifs. Analysis of target DNA recognition and kinetic parameters of the wild-type PI-MleI and its variants disclosed that the two amino acid residues, Asp(122) (in Block C) and Asp(193) (in functional Block E), are crucial to the double-stranded DNA endonuclease activity, whereas Asp(218) (in pseudo-Block E) is not. However, despite the reduced catalytic activity, the PI-MleI variants, like the wild-type PI-MleI, generated a footprint of the same length around the insertion site. The D122T variant showed significantly reduced catalytic activity, and D122A and D193A mutations although failed to affect their DNA-binding affinities, but abolished the double-stranded DNA cleavage activity. On the other hand, D122C variant showed approximately twofold higher double-stranded DNA cleavage activity, compared with the wild-type PI-MleI. These results provide compelling evidence that Asp(122) and Asp(193) in DOD motif I and II, respectively, are bona fide active-site residues essential for DNA cleavage activity. The implications of these results are discussed in this report.

Novel matrix metalloproteinases in intestinal inflammation and in cancer of the gastrointestinal tract

Matrix metalloproteinases (MMPs) comprise a family of 23 zinc-dependent human endopeptidases that can degrade virtually all components of the extracellular matrix (ECM). They are classified into eight subgroups according to their structure and into six subgroups based on their substrate-specificity. MMPs have been implicated in inflammation, tissue destruction, cell migration, arthritis, vascular remodeling, angiogenesis, and tumor growth and invasion. MMPs are inhibited by their natural inhibitors, tissue inhibitors of metalloproteinases (TIMPs). Different MMPs function in the same tasks depending on the tissue or cancer subtype. I investigated the role of recently discovered MMPs, especially MMPs-19 and -26, in intestinal inflammation, in intestinal and cutaneous wound healing, and in intestinal cancer. Several MMPs and TIMPs were studied to determine their exact location at tissue level and to obtain information on possible functions of MMPs in such tissues and diseases as the healthy intestine, inflammatory bowel disease (IBD), neonatal necrotizing enterocolitis (NEC), pyoderma gangrenosum (PG), and colorectal as well as pancreatic cancers. In latent celiac disease (CD), I attempted to identify markers to predict later onset of CD in children and adolescents. The main methods used were immunohistochemistry, in situ hybridization, and Taqman RT-PCR. My results show that MMP-26 is important for re-epithelialization in intestinal and cutaneous wound healing. In colon and pancreatic cancers, MMP-26 seems to be a marker of invasive potential, although it is not itself expressed at the invasive front. MMP-21 is upregulated in pancreatic cancer and may be associated with tumor differentiation. MMPs-19 and -28 are associated with normal tissue turnover in the intestine, but they disappear in tumor progression as if they were protective markers . MMP-12 is an essential protease in intestinal inflammation and tissue destruction, as seen here in NEC and in previous CD studies. In patients with type 1 diabetes (T1D), MMPs-1, -3, and -12 were upregulated in the intestinal mucosa. Furthermore, MMP-7 was strongly elevated in NEC. In a model of aberrant wound repair, PG, MMPs-8, -9, and 10 and TNFα may promote ECM destruction, while absence of MMP-1 and MMP-26 from keratinocytes retards re-epithelialization. Based on my results, I suggest MMP-26 to be considered a putative marker for poor prognosis in pancreatic and colon cancer. However, since it functions differently in various tissues and tumor subtypes, this use cannot be generalized. Furthermore, MMP-26 is a beneficial marker for wound healing if expressed by migrating epithelial cells. MMP-12 expression in latent CD patients warrants research in a larger patient population to confirm its role as a specific marker for CD in pathologically indistinct cases. MMP-7 should be considered one of the most crucial proteases in NEC-associated tissue destruction; hence, specific inhibitors of this MMP are worth investigating. In PG, TNFα inhibitors are potential therapeutic agents, as shown already in clinical trials. In conclusion, studies of several MMPs in specific diseases and in healthy tissues are needed to elucidate their roles at the tissue level. MMPs and TIMPs are not exclusively destructive or reparative in tissues. They seem to function differently in different tissues. To identify selective MMP inhibitors, we must thoroughly understand the MMP profile (degradome) and their functions in various organs not to interfere with normal reparative functions during wound repair or beneficial host-response effects during cancer initiation and growth.

Robust superhydrophobicity of hierarchical ZnO hollow microspheres fabricated by two-step self-assembly

Superhydrophobic and superhydrophilic surfaces have been extensively investigated due to their importance for industrial applications. It has been reported, however, that superhydrophobic surfaces are very sensitive to heat, ultraviolet (UV) light, and electric potential, which interfere with their long-term durability. In this study, we introduce a novel approach to achieve robust superhydrophobic thin films by designing architecture-defined complex nanostructures. A family of ZnO hollow microspheres with controlled constituent architectures in the morphologies of 1D nanowire networks, 2D nanosheet stacks, and 3D mesoporous nanoball blocks, respectively, was synthesized via a two-step self-assembly approach, where the oligomers or the constituent nanostructures with specially designed structures are first formed from surfactant templates, and then further assembled into complex morphologies by the addition of a second co-surfactant. The thin films composed of two-step synthesized ZnO hollow microspheres with different architectures presented superhydrophobicities with contact angles of 150°-155°, superior to the contact angle of 103° for one-step synthesized ZnO hollow microspheres with smooth and solid surfaces. Moreover, the robust superhydrophobicity was further improved by perfluorinated silane surface modification. The perfluorinated silane treated ZnO hollow microsphere thin films maintained excellent hydrophobicity even after 75 h of UV irradiation. The realization of environmentally durable superhydrophobic surfaces provides a promising solution for their long-term service under UV or strong solar light irradiations.

An algebraic formulation of static isotropy and design of statically isotropic 6-6 Stewart platform manipulators

In this paper, we present an algebraic method to study and design spatial parallel manipulators that demonstrate isotropy in the force and moment distributions. We use the force and moment transformation matrices separately, and derive conditions for their isotropy individually as well as in combination. The isotropy conditions are derived in closed-form in terms of the invariants of the quadratic forms associated with these matrices. The formulation is applied to a class of Stewart platform manipulator, and a multi-parameter family of isotropic manipulators is identified analytically. We show that it is impossible to obtain a spatially isotropic configuration within this family. We also compute the isotropic configurations of an existing manipulator and demonstrate a procedure for designing the manipulator for isotropy at a given configuration. (C) 2008 Elsevier Ltd. All rights reserved.

A numerical study of crack tip constraint in ductile single crystals

In this work, the effect of crack tip constraint on near-tip stress and deformation fields in a ductile FCC single crystal is studied under mode I, plane strain conditions. To this end, modified boundary layer simulations within crystal plasticity framework are performed, neglecting elastic anisotropy. The first and second terms of the isotropic elastic crack tip field, which are governed by the stress intensity factor K and T-stress, are prescribed as remote boundary conditions and solutions pertaining to different levels of T-stress are generated. It is found that the near-tip deformation field, especially, the development of kink or slip shear bands, is sensitive to the constraint level. The stress distribution and the size and shape of the plastic zone near the crack tip are also strongly influenced by the level of T-stress, with progressive loss of crack tip constraint occurring as T-stress becomes more negative. A family of near-tip fields is obtained which are characterized by two terms (such as K and T or J and a constraint parameter Q) as in isotropic plastic solids.

Evolutionary Significance of Self-acylation Property in Acyl Carrier Proteins

Acyl carrier protein is an integral component of many cellular metabolic processes. A number of studies have reported self-acylation behavior in acyl carrier proteins. Although AM exhibit high levels of similarity in their primary and tertiary structures, self-acylation behavior is restricted to only some ACPs that can be classified into two major families based on their function. The first family of ACPs is involved in polyketide biosynthesis, whereas the second family participates in fatty acid synthesis. Facilitated by the growing number of genome sequences available for analyses, large-scale phylogenetic studies were used in these studies to uncover as to how self-acylation behavior of acyl carrier proteins is linked with the evolution of metabolic pathways in organisms. These studies show that self-acylation behavior in acyl carrier proteins was lost during the course of evolution, with certain organisms and organelles viz. plastids, retaining it for specified functions. (C) 2009 IUBMB IUBMB Life, 61(8): 853-859, 2009

Two elementary proofs of the Wigner theorem on symmetry in quantum mechanics

In quantum theory, symmetry has to be defined necessarily in terms of the family of unit rays, the state space. The theorem of Wigner asserts that a symmetry so defined at the level of rays can always be lifted into a linear unitary or an antilinear antiunitary operator acting on the underlying Hilbert space. We present two proofs of this theorem which are both elementary and economical. Central to our proofs is the recognition that a given Wigner symmetry can, by post-multiplication by a unitary symmetry, be taken into either the identity or complex conjugation. Our analysis often focuses on the behaviour of certain two-dimensional subspaces of the Hilbert space under the action of a given Wigner symmetry, but the relevance of this behaviour to the larger picture of the whole Hilbert space is made transparent at every stage.

The partition of unity finite element method for elastic wave propagation in Reissner-Mindlin plates

This paper reports a numerical method for modelling the elastic wave propagation in plates. The method is based on the partition of unity approach, in which the approximate spectral properties of the infinite dimensional system are embedded within the space of a conventional finite element method through a consistent technique of waveform enrichment. The technique is general, such that it can be applied to the Lagrangian family of finite elements with specific waveform enrichment schemes, depending on the dominant modes of wave propagation in the physical system. A four-noded element for the Reissner-indlin plate is derived in this paper, which is free of shear locking. Such a locking-free property is achieved by removing the transverse displacement degrees of freedom from the element nodal variables and by recovering the same through a line integral and a weak constraint in the frequency domain. As a result, the frequency-dependent stiffness matrix and the mass matrix are obtained, which capture the higher frequency response with even coarse meshes, accurately. The steps involved in the numerical implementation of such element are discussed in details. Numerical studies on the performance of the proposed element are reported by considering a number of cases, which show very good accuracy and low computational cost. Copyright (C)006 John Wiley & Sons, Ltd.

PE_PGRS Antigens of Mycobacterium tuberculosis Induce Maturation and Activation of Human Dendritic Cells

Mycobacterium tuberculosis, the causative agent of pulmonary tuberculosis, infects one-third of the world's population. Activation of host immune responses for containment of mycobacterial infections involves participation of innate immune cells, such as dendritic cells (DCs). DCs are sentinels of the immune system and are important for eliciting both primary and secondary immune responses to pathogens. In this context, to understand the molecular pathogenesis of tuberculosis and host response to mycobacteria and to conceive prospective vaccine candidates, it is important to understand how cell wall Ags of M.tuberculosis and, in particular, the proline-glutamic acid-polymorphicguanine-cytosine-rich sequence (PE_PGRS) family of proteins modulate DC maturation and function. In this study, we demonstrate that two cell wall-associated/secretory PE_PGRS proteins, PE_PGRS 17 (Rv0978c) and PE_PGRS 11 (Rv0754), recognize TLR2, induce maturation and activation of human DCs, and enhance the ability of DCs to stimulate CD4(+) T cells. We further found that PE_PGRS protein-mediated activation of DCs involves participation of ERK1/2, p38 MAPK, and NF-kappa B signaling pathways. Priming of human DCs with IFN-gamma further augmented PE_PGRS 17 or PE_PGRS 11 Ag-induced DC maturation and secretion of key proinflammatory cytokines. Our results suggest that by activating DCs, PE_PGRS proteins, important mycobacterial cell wall Ags, could potentially contribute in the initiation of innate immune responses during tuberculosis infection and hence regulate the clinical course of tuberculosis. The Journal of Immunology, 2010, 184: 3495-3504.

The emerging role of microRNAs in resistance to lung cancer treatments

One of the major challenges in the treatment of lung cancer is the development of drug resistance. This represents a major obstacle in the treatment of patients, limiting the efficacy of both conventional chemotherapy and biological therapies. Deciphering the mechanisms of resistance is critical to further understanding the multifactorial pathways involved, and in developing more specific targeted treatments. To date, numerous studies have reported the potential role of microRNAs (miRNAs) in resistance to various cancer treatments. MicroRNAs are a family of small non-coding RNAs that regulate gene expression by sequence-specific targeting of mRNAs causing translational repression or mRNA degradation. More than 1200 validated human miRNAs have been identified to date. While as little as one miRNA can regulate hundreds of targets, a single target can also be affected by multiple miRNAs. Evidence suggests that dysregulation of specific miRNAs may be involved in the acquisition of resistance to a number of cancer treatments, thereby modulating the sensitivity of cancer cells to such therapies. Therefore, targeting miRNAs may be an attractive strategy for developing novel and more effective individualized therapies, improving drug efficiency, and for predicting patient response to different treatments. In this review, we provide an overview on the role of miRNAs in resistance to current lung cancer therapies and novel biological agents.

PE_PGRS Antigens of Mycobacterium tuberculosis Induce Maturation and Activation of Human Dendritic Cells

Mycobacterium tuberculosis, the causative agent of pulmonary tuberculosis, infects one-third of the world's population. Activation of host immune responses for containment of mycobacterial infections involves participation of innate immune cells, such as dendritic cells (DCs). DCs are sentinels of the immune system and are important for eliciting both primary and secondary immune responses to pathogens. In this context, to understand the molecular pathogenesis of tuberculosismand host response to mycobacteria and to conceive prospective vaccine candidates, it is important to understand how cell wall Ags of M. tuberculosis and, in particular, the proline-glutamic acid-polymorphic guanine-cytosine-rich sequence (PE_PGRS) family of proteins modulate DC maturation and function. In this study, we demonstrate that two cell wall-associated/secretory PE_PGRS proteins, PE_PGRS 17 (Rv0978c) and PE_PGRS 11 (Rv0754), recognize TLR2, induce maturation and activation of human DCs, and enhance the ability of DCs to stimulate CD4(+) T cells. We further found that PE_PGRS protein-mediated activation of DCs involves participation of ERK1/2, p38 MAPK, and NF-kappa B signaling pathways. Priming of human DCs with IFN-gamma further augmented PE_PGRS 17 or PE_PGRS 11 Ag-induced DC maturation and secretion of key proinflammatory cytokines. Our results suggest that by activating DCs, PE_PGRS proteins, important mycobacterial cell wall Ags, could potentially contribute in the initiation of innate immune responses during tuberculosis infection and hence regulate the clinical course of tuberculosis. The Journal of Immunology, 2010, 184: 3495-3504.

What does “futility” mean? An empirical study of doctors’ perceptions

Objective(s) To describe how doctors define and use the terms “futility” and “futile treatment” in end-of-life care. Design, Setting, Participants A qualitative study using semi-structured interviews with 96 doctors across a range of specialties who treat adults at the end of life. Doctors were recruited from three large Australian teaching hospitals and were interviewed from May to July 2013. Results Doctors’ conceptions of futility focused on the quality and chance of patient benefit. Aspects of benefit included physiological effect, weighing benefits and burdens, and quantity and quality of life. Quality and length of life were linked, but many doctors discussed instances when benefit was determined by quality of life alone. Most doctors described the assessment of chance of success in achieving patient benefit as a subjective exercise. Despite a broad conceptual consensus about what futility means, doctors noted variability in how the concept was applied in clinical decision-making. Over half the doctors also identified treatment that is futile but nevertheless justified, such as short-term treatment as part of supporting the family of a dying person. Conclusions There is an overwhelming preference for a qualitative approach to assessing futility, which brings with it variation in clinical decision-making. “Patient benefit” is at the heart of doctors’ definitions of futility. Determining patient benefit requires discussions with patients and families about their values and goals as well as the burdens and benefits of further treatment.

Simulation of wavelength/time multiple-pulses-per-row fiber-optic CDMA network

Passive wavelength/time fiber-optic code division multiple access (WIT FO-CDMA) network is a viable option for highspeed access networks. Constructions of 2-D codes, suitable for incoherent WIT FO-CDMA, have been proposed to reduce the time spread of the 1-D sequences. The 2-D constructions can be broadly classified as 1) hybrid codes and 2) matrix codes. In our earlier work [141, we had proposed a new family of wavelength/time multiple-pulses-per-row (W/T MPR) matrix codes which have good cardinality, spectral efficiency and at the same time have the lowest off-peak autocorrelation and cross-correlation values equal to unity. In this paper we propose an architecture for a WIT MPR FO-CDAM network designed using the presently available devices and technology. A complete FO-CDMA network of ten users is simulated, for various number of simultaneous users and shown that 0 --> 1 errors can occur only when the number of interfering users is at least equal to the threshold value.

Novel peptides of therapeutic promise from Indian Conidae

Highly structured small peptides are the major toxic constituents of the venom of cone snails, a family of widely distributed predatory marine molluscs. These animals use the venom for rapid prey immobilization. The peptide components in the venom target a wide variety of membrane-bound ion channels and receptors. Many have been found to be highly selective for a diverse range of mammalian ion channels and receptors associated with pain-signaling pathways. Their small size, structural stability, and target specificity make them attractive pharmacologic agents. A select number of laboratories mainly from the United States, Europe, Australia, Israel, and China have been engaged in intense drug discovery programs based on peptides from a few snail species. Coastal India has an estimated 20-30% of the known cone species; however, few serious studies have been reported so far. We have begun a comprehensive program for the identification and characterization of peptides from cone snails found in Indian Coastal waters. This presentation reviews our progress over the last 2 years. As expected from the evolutionary history of these venom components, our search has yielded novel peptides of therapeutic promise from the new species that we have studied.

On the effect of matrix cracks in composite helicopter rotor blade

This paper investigates the feasibility of an on-line damage detection capability for helicopter main rotor blades made of composite material. Damage modeled in the composite is matrix cracking. A box-beam with stiffness properties similar to a hingeless rotor blade is designed using genetic algorithm for the typical [+/-theta(m)/90(n)](s) family of composites. The effect of matrix cracks is included in an analytical model of composite box-beam. An aeroelastic analysis of the helicopter rotor based on finite elements in space and time is used to study the effects of matrix cracking in the rotor blade in forward flight. For global fault detection, rotating frequencies, tip bending and torsion response, and blade root loads are studied. It is observed that the effect of matrix cracking on lag bending and elastic twist deflection at the blade tip and blade root yawing moment is significant and these parameters can be monitored for online health monitoring. For implementation of local fault detection technique, the effect on axial and shear strain, for matrix cracks in the whole blade as well as matrix cracks occurring locally is studied. It is observed that using strain measurement along the blade it is possible to locate the matrix cracks as well as to predict density of matrix cracks. (C) 2004 Elsevier Ltd. All rights reserved.