A rapid, efficient, and economical inverse polymerase chain reaction-based method for generating a site saturation mutant library

With the development of deep sequencing methodologies, it has become important to construct site saturation mutant (SSM) libraries in which every nucleotide/codon in a gene is individually randomized. We describe methodologies for the rapid, efficient, and economical construction of such libraries using inverse polymerase chain reaction (PCR). We show that if the degenerate codon is in the middle of the mutagenic primer, there is an inherent PCR bias due to the thermodynamic mismatch penalty, which decreases the proportion of unique mutants. Introducing a nucleotide bias in the primer can alleviate the problem. Alternatively, if the degenerate codon is placed at the 5' end, there is no PCR bias, which results in a higher proportion of unique mutants. This also facilitates detection of deletion mutants resulting from errors during primer synthesis. This method can be used to rapidly generate SSM libraries for any gene or nucleotide sequence, which can subsequently be screened and analyzed by deep sequencing. (C) 2013 Elsevier Inc. All rights reserved.

PMV model is insufficient to capture subjective thermal response from Indians

A controlled laboratory experiment was carried out on forty Indian male college students for evaluating the effect of indoor thermal environment on occupants' response and thermal comfort. During experiment, indoor temperature varied from 21 degrees C to 33 degrees C, and the variables like relative humidity, airflow, air temperature and radiant temperature were recorded along with subject's physiological parameters (skin (T-sk) and oral temperature (T-c)) and subjective thermal sensation responses (TSV). From T-sk and T-c, body temperature (T-b) was evaluated. Subjective Thermal Sensation Vote (TSV) was recorded using ASHRAE 7-point scale. In PMV model, Fanger's T-sk equation was used to accommodate adaptive response. Step-wise regression analysis result showed T-b was better predictor of TSV than T-sk and T-c. Regional skin temperature response, suppressed sweating without dipping, lower sweating threshold temperature and higher cutaneous threshold for sweating were observed as thermal adaptive responses. These adaptive responses cannot be considered in PMV model. To incorporate subjective adaptive response, mean skin temperature (T-sk) is considered in dry heat loss calculation. Along with these, PMV-model and other two methodologies are adopted to calculate PMV values and results are compared. However, recent literature is limited to measure the sweat rate in Indians and consideration of constant Ersw in PMV model needs to be corrected. Using measured T-sk in PMV model (Method(1)), thermal comfort zone corresponding to 0.5 <= PMV <= 0.5 was evaluated as (22.46-25.41) degrees C with neutral temperature of 23.91 degrees C, similarly while using TSV response, wider comfort zone was estimated as (23.25-26.32) degrees C with neutral temperature at 24.83 degrees C, which was further increased to with TSV-PPDnew, relation. It was observed that PMV-model overestimated the actual thermal response. Interestingly, these subjects were found to be less sensitive to hot but more sensitive to cold. A new TSV-PPD relation (PPDnew) was obtained from the population distribution of TSV response with an asymmetric distribution of hot-cold thermal sensation response from Indians. The calculations of human thermal stress according to steady state energy balance models used on PMV model seem to be inadequate to evaluate human thermal sensation of Indians. Relevance to industry: The purpose of this paper is to estimate thermal comfort zone and optimum temperature for Indians. It also highlights that PMV model seems to be inadequate to evaluate subjective thermal perception in Indians. These results can be used in feedback control of HVAC systems in residential and industrial buildings. (C) 2014 Elsevier B.V. All rights reserved.

Parallel Flow-Sensitive Pointer Analysis by Graph-Rewriting

Precise pointer analysis is a problem of interest to both the compiler and the program verification community. Flow-sensitivity is an important dimension of pointer analysis that affects the precision of the final result computed. Scaling flow-sensitive pointer analysis to millions of lines of code is a major challenge. Recently, staged flow-sensitive pointer analysis has been proposed, which exploits a sparse representation of program code created by staged analysis. In this paper we formulate the staged flow-sensitive pointer analysis as a graph-rewriting problem. Graph-rewriting has already been used for flow-insensitive analysis. However, formulating flow-sensitive pointer analysis as a graph-rewriting problem adds additional challenges due to the nature of flow-sensitivity. We implement our parallel algorithm using Intel Threading Building Blocks and demonstrate considerable scaling (upto 2.6x) for 8 threads on a set of 10 benchmarks. Compared to the sequential implementation of staged flow-sensitive analysis, a single threaded execution of our implementation performs better in 8 of the benchmarks.

Risk-sensitive control of continuous time Markov chains

We study risk-sensitive control of continuous time Markov chains taking values in discrete state space. We study both finite and infinite horizon problems. In the finite horizon problem we characterize the value function via Hamilton Jacobi Bellman equation and obtain an optimal Markov control. We do the same for infinite horizon discounted cost case. In the infinite horizon average cost case we establish the existence of an optimal stationary control under certain Lyapunov condition. We also develop a policy iteration algorithm for finding an optimal control.

Male Takeover and Infanticide in Nilgiri Langurs Semnopithecus johnii in the Western Ghats, India

Male takeover and infanticide are a widespread phenomenon among non-human primates, observed mostly in species with a relatively longer lactation in relation to gestation. In this study, we report for the first time an episode of male takeover and infanticide, and the rarely reported occurrence of an all-male band and female dispersal, in Nilgiri langurs, Semnopithecus johnii, in the Western Ghats, India. The new male was a member of an all-male band. After the takeover, the resident male and 3 juvenile males left the group and joined the all-male band. A female whose infant was killed was found missing after some days. There were significant changes in the patterns of social interactions among the resident group females soon after the male takeover, wherein the females spent less time on social interactions as compared to before and after the episode of takeover. The new male rarely interacted with the females soon after the takeover. We also observed that the resident group shifted its home range to a poorer quality habitat. (C) 2014 S. Karger AG, Basel

Selective and Sensitive Turn-on Chemosensor for Arsenite Ion at the ppb Level in Aqueous Media Applicable in Cell Staining

A newly designed and structurally characterized cell permeable diformyl-p-cresol based receptor (HL) selectively senses the AsO33- ion up to ca. 4.1 ppb in aqueous media over the other competitive ions at biological pH through an intermolecular H-bonding induced CHEF (chelationenhanced fluorescence) process, established by detailed experimental and theoretical studies. This biofriendly probe is highly competent in recognizing the existence of AsO33- ions in a living organism by developing an image under a fluorescence microscope and useful to estimate the amount of arsenite ions in various water samples.

Highly Sensitive Carbon Nanotubes Coated Etched Fiber Bragg Grating Sensor for Humidity Sensing

The sensing of relative humidity (RH) at room temperature has potential applications in several areas ranging from biomedical to horticulture, paper, and textile industries. In this paper, a highly sensitive humidity sensor based on carbon nanotubes (CNTs) coated on the surface of an etched fiber Bragg grating (EFBG) sensor has been demonstrated, for detecting RH over a wide range of 20%-90% at room temperature. When water molecules interact with the CNT coated EFBG, the effective refractive index of the fiber core changes, resulting in a shift in the Bragg wavelength. It has been possible to achieve a high sensitivity of similar to 31 pm/% RH, which is the highest compared with many of the existing FBG-based humidity sensors. The limit of detection in the CNT coated EFBG has been found to be similar to 0.03 RH. The experimental data shows a linear response of Bragg wavelength shift with increase in humidity. This novel method of incorporating CNTs on to the FBG sensor for humidity sensing has not been reported before.

Nanocomposite Based Organic-Inorganic Cu3BiS3 High Sensitive Hybrid Photonic Devices

We report the synthesis and application Cu3BiS3 nanorods in infrared photodectection. Cu3BiS3 nano rods were characterized structurally, optically and electrically. The detailed IR photodectection properties in terms of photo response were demonstrated with IA lamp and 1064 nm laser illuminations. The rapid photocurrent time constants followed by the slower components, resulting due to the defect states. The photo detecting properties for different concentrations of nanorods blended with the conjugate polymer devices were demonstrated. Further the photocurrent was enhanced to threefold increase from 3.47 x 10(-7) A to 2.37 x 10(-3) A at 1 V for 10 mg nanorods embedded in the polymer device. Responsivity of hybrid device was enhanced from 0.0158 NW to 102 NW. The detailed trap assisted space charge transport properties were studied considering the different regimes. Hence Cu3BiS3 can be a promising candidate in the nano switchable near IA photodetectors.

Chaperone protein HYPK interacts with the first 17 amino acid region of Huntingtin and modulates mutant HTT-mediated aggregation and cytotoxicity

Huntington's disease is a polyglutamine expansion disorder, characterized by mutant HTT-mediated aggregate formation and cytotoxicity. Many reports suggests roles of N-terminal 17 amino acid domain of HTT (HTT-N17) towards subcellular localization, aggregate formation and subsequent pathogenicity induced by N-terminal HTT harboring polyQ stretch in pathogenic range. HYPK is a HTT-interacting chaperone which can reduce N-terminal mutant HTT-mediated aggregate formation and cytotoxicity in neuronal cell lines. However, how HYPK interacts with N-terminal fragment of HTT remained unknown. Here we report that specific interaction of HYPK with HTT-N17 is crucial for the chaperone activity of HYPK. Deletion of HTT-N17 leads to formation of tinier, SDS-soluble nuclear aggregates formed by N-terminal mutant HTT. The increased cytotoxicity imparted by these tiny aggregates might be contributed due to loss of interaction with HYPK. (C) 2014 Elsevier Inc. All rights reserved.

Sensitive detection of C-reactive protein using optical fiber Bragg gratings

An accurate and highly sensitive sensor platform has been demonstrated for the detection of C-reactive protein (CRP) using optical fiber Bragg gratings (FBGs). The CRP detection has been carried out by monitoring the shift in Bragg wavelength (Delta lambda(B)) of an etched FBG (eFBG) coated with an anti-CRP antibody (aCRP)-graphene oxide (GO) complex. The complex is characterized by Fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy and atomic force microscopy. A limit of detection of 0.01 mg/L has been achieved with a linear range of detection from 0.01 mg/L to 100 mg/L which includes clinical range of CRP. The eFBG sensor coated with only aCRP (without GO) show much less sensitivity than that of aCRP-GO complex coated eFBG. The eFBG sensors show high specificity to CRP even in the presence of other interfering factors such as urea, creatinine and glucose. The affinity constant of similar to 1.1 x 10(10) M-1 has been extracted from the data of normalized shift (Delta lambda(B)/lambda(B)) as a function of CRP concentration. (C) 2014 Elsevier B.V. All rights reserved.

Raman Spectroscopic Studies on Screening of Myopathies

Myopathies are among the major causes of mortality in the world. There is no complete cure for this heterogeneous group of diseases, but a sensitive, specific, and fast diagnostic tool may improve therapy effectiveness. In this study, Raman spectroscopy is applied to discriminate between muscle mutants in Drosophila on the basis of associated changes at the molecular level. Raman spectra were collected from indirect flight muscles of mutants, upheld1 (up1), heldup(2) (hdp(2)), myosin heavy chain7 (Mhc7), actin88F(KM88) (Act88F(KM88)), upheld101 (up101), and Canton-S (CS) control group, for both 2 and 12 days old flies. Difference spectra (mutant minus control) of all the mutants showed an increase in nucleic acid and beta-sheet and/or random coil protein content along with a decrease in a-helix protein. Interestingly, the 12th day samples of up1 and Act88F(KM88) showed significantly higher levels of glycogen and carotenoids than CS. A principal components based linear discriminant analysis classification model was developed based on multidimensional Raman spectra, which classified the mutants according to their pathophysiology and yielded an overall accuracy of 97% and 93% for 2 and 12 days old flies, respectively. The up1 and Act88F(KM88) (nemaline-myopathy) mutants form a group that is clearly separated in a linear discriminant plane from up101 and hdp2 (cardiomyopathy) mutants. Notably, Raman spectra from a human sample with nemaline-myopathy formed a cluster with the corresponding Drosophila mutant (up1). In conclusion, this is the first demonstration in which myopathies, despite their heterogeneity, were screened on the basis of biochemical differences using Raman spectroscopy.

Highly sensitive and rapid detection of acetylcholine using an ITO plate modified with platinum-graphene nanoparticles

Determining the concentrations of acetylcholine (ACh) and choline (Ch) is clinically important. ACh is a neurotransmitter that acts as a key link in the communication between neurons in the spinal cord and in nerve skeletal junctions in vertebrates, and plays an important role in transmitting signals in the brain. A bienzymatic sensor for the detection of ACh was prepared by co-immobilizing choline oxidase (ChO) and acetylcholinesterase (AChE) on graphene matrix/platinum nanoparticles, and then electrodepositing them on an ITO-coated glass plate. Graphene nanoparticles were decorated with platinum nanoparticles and were electrodeposited on a modified ITO-coated glass plate to form a modified electrode. The modified electrode was characterized by scanning electron microscopy (SEM), cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) studies. The optimum response of the enzyme electrode was obtained at pH 7.0 and 35 degrees C. The response time of this ACh-sensing system was shown to be 4 s. The linear range of responses to ACh was 0.005-700 mu M. This biosensor exhibits excellent anti-interferential abilities and good stability, retaining 50% of its original current even after 4 months. It has been applied for the detection of ACh levels in human serum samples.

Path Sensitive Cache Analysis Using Cache Miss Paths

Cache analysis plays a very important role in obtaining precise Worst Case Execution Time (WCET) estimates of programs for real-time systems. While Abstract Interpretation based approaches are almost universally used for cache analysis, they fail to take advantage of its unique requirement: it is not necessary to find the guaranteed cache behavior that holds across all executions of a program. We only need the cache behavior along one particular program path, which is the path with the maximum execution time. In this work, we introduce the concept of cache miss paths, which allows us to use the worst-case path information to improve the precision of AI-based cache analysis. We use Abstract Interpretation to determine the cache miss paths, and then integrate them in the IPET formulation. An added advantage is that this further allows us to use infeasible path information for cache analysis. Experimentally, our approach gives more precise WCETs as compared to AI-based cache analysis, and we also provide techniques to trade-off analysis time with precision to provide scalability.

Curcumin Pyrazole and its derivative (N-(3-Nitrophenylpyrazole) Curcumin inhibit aggregation, disrupt fibrils and modulate toxicity of Wild type and Mutant alpha-Synuclein

Accumulating evidence suggests that deposition of neurotoxic a-synuclein aggregates in the brain during the development of neurodegenerative diseases like Parkinson's disease can be curbed by anti-aggregation strategies that either disrupt or eliminate toxic aggregates. Curcumin, a dietary polyphenol exhibits anti-amyloid activity but the use of this polyphenol is limited owing to its instability. As chemical modifications in curcumin confiscate this limitation, such efforts are intensively performed to discover molecules with similar but enhanced stability and superior properties. This study focuses on the inhibitory effect of two stable analogs of curcumin viz. curcumin pyrazole and curcumin isoxazole and their derivatives against a-synuclein aggregation, fibrillization and toxicity. Employing biochemical, biophysical and cell based assays we discovered that curcumin pyrazole (3) and its derivative N-(3-Nitrophenylpyrazole) curcumin (15) exhibit remarkable potency in not only arresting fibrillization and disrupting preformed fibrils but also preventing formation of A11 conformation in the protein that imparts toxic effects. Compounds 3 and 15 also decreased neurotoxicity associated with fast aggregating A53T mutant form of a-synuclein. These two analogues of curcumin described here may therefore be useful therapeutic inhibitors for the treatment of a-synuclein amyloidosis and toxicity in Parkinson's disease and other synucleinopathies.