bluuebunny/crossref_metadata_2025 huggingface dataset:
A pilot study evaluating the use of cervical ultrasound to confirm endotracheal intubation in dogs
Authors: Herreria‐Bustillo Vicente J. , Kuo Kendon W. , Burke Pierre J. , Cole Robert , Bacek Lenore M.
Published: July 2016

Objective

To assess the accuracy and usability of cervical ultrasound as a means to confirm endotracheal (ET) intubation in dogs.

Design

Randomized pilot study.

Setting

University teaching hospital.

Animals

Six recently euthanized cadaver dogs.

Interventions

Endotracheal and esophageal intubations were randomly performed. The investigators performing the ultrasound examinations were blinded to the type of intubation. Ultrasound examinations were performed in right and left lateral recumbency. The time taken to obtain the images was recorded.

Measurements and Main Results

The sensitivity, specificity, positive predictive value, and negative predictive value with their correspondent 95% confidence intervals (CI) were 91.7% (CI, 82.7–96.7), 72.7% (CI, 60.4–84.0%), 78.6% (CI, 68.3–86.8%), and 88.9% (CI, 77.4–95.8%), respectively. The overall test accuracy with its correspondent 95% CI was 82.8% (CI, 75.4–88.1%). The mean time for confirmation with ultrasound was 20.2 seconds (standard deviation, 14.3 s). Cervical ultrasound was significantly more accurate at recognizing ET intubations than esophageal intubations (odds ratio, 4.52; 95% CI, 1.43–14.27; P = 0.010). There was a significant relationship between increase in body weight and accuracy, indicating that the test is more accurate in larger dogs (odds ratio, 1.04; 95% CI, 1.00–1.08; P = 0.042).

Conclusions

Cervical ultrasound has high sensitivity and moderate specificity for ET intubation, and may be a useful tool to confirm ET intubation in dogs.
PRACTICAL PRODUCTION LAYOUT DESIGN FOR MULTI-PRODUCT AND SMALL-LOT-SIZE PRODUCTION: A CASE STUDY
Authors: Nyeoh Cheng Ying, , Ab-Samat Hasnida , Kamaruddin Shahrul
Published: July 2016

Facility layout design has an important function in manufacturing systems because it affects manufacturing costs, work in progress, lead times, and production output. This study presents facility layout designs for multi-product and small-lot-sized production lines. This research focuses on the development and analysis of layout alternatives on the basis of performance measures and aims to improve production efficiency. Thus, the tools and techniques available for the layout designed were investigated. Related data were collected, and alternative layouts were developed using the WITNESS simulation software. Finally, the alternative layouts were ana1yzed and evaluated using the analytic hierarchy process to identify the best possible layout. Two important parameters observed in the alternative layouts: the ability to produce a desired output and the flexibility of each layout coherent with the fluctuation of product demands in the industry. Results from analysis shows that suggested Model 3 with the combination of flow line and job shop configurations is the most suitable layout. This model has the highest machine utilization rate and the highest labour utilization rate yet requires only 21 operators, the lowest number of workers. For future work, this type of layout should be tested with different variation in lot sizes.
COMPARATIVE STUDIES OF CELL GROWTH OF FRESHWATER MICROALGA CHLORELLA SP. IN PHOTOAUTOTROPHIC, HETEROTROPHIC AND MIXOTROPHIC CULTURES
Authors: Joannes Costantine , Mansa Rachel Fran , Md. Yasir Suhaimi , Dayou Jedol
Published: July 2016

Lately, research on biodiesel production as a renewable and sustainable energy has become increasingly apparent due to the fact that fossil fuel is decreasing and the concern of global warming issues. The third generation of biofuel, which is microalgae-based biodiesel had gained interest over the last decade. The ability of microalgae to grow in various conditions is one of its advantages as the potential and promising feedstock for biodiesel. Microalgae can be cultivated in three modes such as photoautotrophic, heterotrophic and mixotrophic culture mode. Unlike photoautotrophic mode where light is required, the heterotrophic mode mainly utilized carbon compounds to grow. On the other hand, the mixotrophic mode is the condition where light and carbon compounds are supplied for microalgae culturing. This paper investigates the cell growth of Chlorella sp. cultivated in photoautotrophic, heterotrophic and mixotrophic culture mode. It was found that Chlorella sp. was capable of producing the highest cell concentration of 6.67 ± 0.56 x 106 cell mL-1 in the photoautotrophic mode for 23 days of cultivation period. This was 1.3 times and 3.2 times greater than the cell concentration in mixotrophic (5.02 ± 0.49 x 106 cell mL-1) and heterotrophic (2.03 ± 0.29 x 106 cell mL-1) culture, respectively. On the contrary, the highest specific growth rate obtained in the study was from heterotrophic mode (0.32 ± 0.04 day-1) followed by photoautotrophic and mixotrophic mode with 0.26 ± 0.05 day-1 and 0.20 ± 0.04 day-1, respectively. Chlorella sp. cell grew well under the photoautotrophic and mixotrophic mode. However, the insufficient of glucose level had contributed to lower cells productivity in the heterotrophic culture. Therefore, the mixotrophic mode could also be an alternative pathway in microalgae cultivation for biodiesel production if the glucose supplied was adequate and at the suitable level.
ISOLATION AND CHARACTERIZATION OF POLYHYDROXYALKANOATES (PHAS) PRODUCING BACTERIA FROM BRACKISH STREAM
Authors: Mohd Zain Nor Azimah , Ali Zargoun Laila Muftah , Elias Nur Fatihah , Abdul-Wahab Mohd Firdaus , Suhaimi Mohd Suardi
Published: July 2016

Polyhydroxyalkanoates (PHAs) are biopolymers which have similar characteristics with petrochemical plastics but a step better due to its biodegradable property. A total of 23 strains were isolated from two different brackish sources. In order to detect the PHAs granules, the PHAs producing bacteria were first screened with Sudan Black B staining. Twenty strains were observed with lipid granules and were subjected to further confirmation with Nile blue staining.  From the Nile blue staining, only 10 strains have the ability in producing PHAs and 2 were identified as strong PHAs producers. This study focuses on the 2 strains named S1 and L1. Further identification procedure was carried out and found that strain S1 and L1 belongs to Pseudomonas sp. L1 strain was found to be promising for PHAs production since it accumulated PHAs for about 88.3%. The PHAs produced by this strain was analyzed using Fourier Transform Infrared Spectroscopy (FT-IR) and Nuclear Magnetic Resonance (NMR) analysis and was identified as poly-3-hydroxybutyrate (P-3HB).
ONLINE PEER-TO-PEER TRAFFIC IDENTIFICATION BASED ON COMPLEX EVENTS PROCESSING OF TRAFFIC EVENT SIGNATURES
Authors: Stephen Bassi Joseph , Hui Ru Loo , Ismail Ismahani , Mohammed Khammas Ban , Marsono Muhammad Nadzir
Published: July 2016

Peer-to-Peer (P2P) applications are bandwidth-heavy and lead to network congestion. The masquerading nature of P2P traffic makes conventional methods of its identification futile. In order to manage and control P2P traffic efficiently preferably in the network, it is necessary to identify such traffic online and accurately.  This paper proposes a technique for online P2P identification based on traffic events signatures. The experimental results show that it is able to identify P2P traffic on the fly with an accuracy of 97.7%, precision of 98% and recall of 99.2%.
ARID HYDROLOGICAL MODELING AT WADI ALAQIQ, MADINAH, SAUDI ARABIA
Authors: Abd Rahman Norhan , Taher Saud , Alahamdi Fahad , Mohd Nasir Kamarul Azlan
Published: July 2016

Madinah is one of the urban centers that have experienced several devastating floods during the past 50 years.  The objectives of this study are to estimate the flood hydrograph and peak discharge in the Wadi Alaqiq in Madinah and its major sub-catchments based on daily rainfall distribution. Daily annual maximum records are chosen for rainfall distribution in the study area. Wadi Alaqiq is located in the western part of the Madinah city and consists of five major sub-catchments, namely, Aqiq, Ruwawah, Reem, Al-Yutmah and Annaqi. The HEC-HMS and modified Talbot models are applied to estimate design flood for various average recurrence intervals (ARI). The analysis involves two stages, where stage one is concerned with modeling of the Wadi Alaqiq sub-catchment and comparison of the peak flow values obtained by previous studies and empirical formulae based on rainfall distribution analysis. In the second stage, the HEC-HMS is run separately for the five sub-catchments of Wadi Alaqiq. The model parameter values are then used to simulate for 25-year, 50-year and 100-year flood hydrographs. The HEC-HMS model is used to analyze the hydrologic behaviour of the wadi catchments. Initial and constant rate loss method is used to determine the hydrologic loss and the excess rainfall was transformed using the Clark’s Unit Hydrograph. The peak flow simulations are very close with reported values and those derived by modified Talbot. Also, the peak discharges are applied to model discharges from the HEC-HMS and modified Talbot equations of the five sub-catchments for a set of selected return periods. The direct runoff ratio for Madinah region in wadis Al-Yutmah, Annaqi, Aqiq, Reem and Ruwawah ranged from 25% to 28% of the total rainfall. This is due to topography condition, wadi catchment area and the use of predicted maximum daily rainfall results. The model results are reasonable for rainfall-runoff application and can be used for wadi corridor management in arid region.
INDUCED PHOTODEGRADATION EFFECT ON THE FUNCTIONALIZED FE(III) COMPLEX ADDITIVE-POLY(VINYL CHLORIDE) THIN FILM
Authors: Yousif Emad , Haddad Raghad , Yahya Badrul Hisyam , Aizad Syazwan , Zainal Abidin Noraziani , Omar Hazreen , Ab Kadir Ili Afiqa , Zaman Huri Hasniza , Abdullah Bashar Mudhaffar , Zubairi Saiful Irwan
Published: July 2016

A complex of Fe(III) with 4-amino-5-(pyridyl)-4H-1,2,4-triazole-3-thiol was prepared and evaluated as a photodegradation for polyvinyl chloride (PVC).  Polyvinyl chloride  was dissolved in THF solvent (5%, w/w) and functionalized with Fe (III) complex to form PVC films by means of solvent-casting method with a thickness of 40 μm. To study the effectiveness of Fe (III) complex additive, different concentrations (0.01, 0.02, 0.03, 0.04 and 0.05 g) were introduced into the PVC solution. The films’ photodegradation was investigated; the photodegradation activity of the compounds was determined by monitoring the carbonyl group, weight loss method and morphological study with irradiation time. The obtained experimental results showed that  the additive behaved successfully as a photoinducer for the degradation process for the PVC films and the photooxidation rate increased with the increase of additive concentration.
DEVELOPMENT OF ROBUST VELOCIMETER FOR NATURAL WATER FLOW MONITORING
Authors: Shirakashi Masataka , Yeo Kye Wei , Koide Mizuyasu , Takahashi Tsutomu , Shaikh Salim Sheikh Ahmad Zaki
Published: July 2016

The ring-velocimeter coupled with a hot wire/film probe was developed and has been applied to wind and water tunnel experiments in Fluids Engineering Laboratory of Nagaoka University of Technology.  In this study, the hot-wire/film probe is replaced by a cantilever attached by a strain gauge to detect the drag acting on the ring.  The vortex shedding frequency from the ring is determined from the drag fluctuation by applying the spectrum analysis, and the flow velocity in turn since it is proportional with the vortex shedding frequency.  This technique for flow velocity measurement is robust in the sense that it is strong against the noise or decay of the detected signal since the dominant frequency is insensitive to such disturbances, and that the detecting probe is strong against the contaminants or particles/objects carried by the fluid.  These advantages, together with its simple and cheap characteristics, make it possible to apply to natural water flow with severe conditions.
READINESS FOR INDUSTRIALIZED BUILDING SYSTEM IMPLEMENTATION AMONG MALAYSIAN ARCHITECTURAL FIRMS’ MEMBERS
Authors: Hanafi Mohd Hanizun , Abas Arniza , Ibrahim Fazdliel Aswad , Abdullah Shardy
Published: July 2016

The Malaysian construction industry has lags behind other industries in terms of performance that had drawn the attention to implement innovative construction method like Industrial Building System (IBS). However, the implementation of IBS in Malaysia remains low although the Government had commenced various efforts. The architects have been perceived as the prime mover for construction innovations by integrating their roles as designer, manager, and professional advisor with their strength in design. Therefore, this paper is designed to assess readiness for IBS implementation among organizational members of architectural firms by examining the influence of demographic backgrounds. The study distributes questionnaires to randomly selected respondents who represent firms that are located in highly developed states in Malaysia. Various analysis techniques have been used namely Cronbach’s Alpha coefficients, frequency, mean, standard deviation, one-way ANOVA, and independent samples t-test towards achieving the research objective. The findings disclose that working experience in tender and construction stage (more than 18 years) indicate high likelihood of readiness to implement IBS. This research also reveals that demographic variables (job designation, working experience in design stage, and working experience in tender and construction stage) has significant impacts on the decision maker category, whereas decision maker is the most influential aspect for organizational readiness to implement IBS.
THE DEVELOPMENT OF MATERIAL FLOW ARCHITECTURE USING RFID TECHNOLOGY IN REAL INDUSTRIAL ENVIRONMENT
Authors: Zulkifli Che Zalina , Abd Malek Shafawati , Mohd Nor Nursyahida , Jaffer Siti Suraya
Published: July 2016

The development of Radio Frequency Identification (RFID) technology is widely used to monitor pallets, cases or individual items in industrial manufacturing fields. However, this method was implemented only at the warehouse where it captured raw materials that need to undergo with manufacturing process until it becomes products but it was not monitor during the process in production line. Our proposed is to build the RFID system that can capture real-time data and monitoring during the entire production work flow where the transmission, tracing, storage and retrieval of overall process can be done.  This technology is associate with unique electronic identity that are embedded to raw materials throughout the manufacturing cycle where it was registered to the RFID reader at every workstation so that the information for the start and end at each job can be transmitted to the main server at real-time for storage, categorizing, tracing, processing and analyzing. By these technology systems, we can promptly update of production-line information with real-time visibility of the whole work in progress production management among offices and sites in a construction supply chain environment.
EXPERIMENTAL EVALUATION OF CO2 LASER CUTTING QUALITY OF UHSS USING OXYGEN AS AN ASSISTED GAS
Authors: Mohamad Tahir Abdul Fattah , Rashid Ahmad Razelan
Published: July 2016

Development of new material known as Ultra High Strength Steel (UHSS) able to improve the vehicle mass thus reflecting better fuel consumption. Transformation into high strength steel has been a significant drawback in trimming the UHSS into its final shape thus laser cutting process appeared to be the solution. This study emphasizes the relationship between Carbon Dioxide (CO2) laser cutting input parameters on 22MnB5 boron steel focusing on the kerf width formation and Heat Affected Zone (HAZ). Experimental research with variation of laser power, cutting speed and assisted gas pressure were executed to evaluate the responses. Metrological and metallographic evaluation of the responses were made on the outputs that are the kerf width formation and HAZ.  Positive correlation for power and negative interaction for cutting speed were found as the major factors on formation of the kerf. For the HAZ formation, thicker HAZ were formed as bigger laser power were applied to the material. Cutting speed and gas pressure does not greatly influence the HAZ formation for 22MnB5 boron steel.
A CLUSTERED WIRELESS SENSOR NETWORK BASED AIR POLLUTION MONITORING SYSTEM WITH SWARM INTELLIGENCE BASED DATA AGGREGATION
Authors: S. Murali, , N. Jaisankar,
Published: July 2016

Air pollution has significant influence on the concentration of constituents in the atmosphere leading to effects like global warming and acid rains. The disproportion of the constituents in the air or atmosphere is monitored using the air pollution monitoring system. The classical air pollution monitoring system uses the costly instruments for monitoring the environment at fixed locations. Most of the traditional monitoring system is coarse- grained and costlier during the real time implementation. Some system have problems such as communication overhead, time and power consuming. The efficient clustering based data aggregation is proposed in this paper for reducing the communication overhead and efficiently monitoring the environment. The sensor nodes in the networks are grouped into clusters and the cluster head is selected using the optimization algorithm such as firefly algorithm. The data aggregation using the hybrid genetic algorithm is also proposed in this paper for efficient data transmission by reducing the communication overhead. The simulation results shows that the performance of the proposed methodology is better than the existing one and the proposed system collects the reliable source of real time fine-grain pollution data.
HUBUNGAN AMALAN PENGURUSAN KUALITI DAN PRESTASI ORGANISASI DALAM INDUSTRI PEMBUATAN DI MALAYSIA
Authors: Jamaluddin Zaharuzaman , Razali Ahmad Mahir , Mustafa Zainol
Published: July 2016

Measurement of quality management practices (QMP) is one of requirements which should be emphasized by the organizations to be competitive in challenging market. Therefore, this study examines the relationship between quality management practices and performance in the manufacturing industry using constructs namely management commitment, quality tools, continuous improvement, process management, training and organisational performance. A framework was developed based on the literature review and assessed using Structural Equation Modeling (SEM) with AMOS software used for modeling analysis. A total of 480 questionnaires were distributed, and 210 questionnaires were valid for analysis. The findings show significant relationship by the management commitment to process management and training. Also, training affects the quality tools, continuous improvement and process management. Similarly, quality tools influence on continuous improvement and process management. Furthermore process management affects the continuous improvement Next, continuous improvement gives impact to organisational performance. The results of the study can be used by managers in manufacturing companies to consider and adapt their QMPs and performance assessments toward increasing competitiveness.
REVIEW ON DECOMPOSED FUZZY PID STRUCTURE FOR POWER INVERTERS REGULATION
Authors: Arif M. S. , Ayob S. M. , Salam Z.
Published: July 2016

The aim of this paper is to critically review prominent decomposed Fuzzy PID control structures. Structural construction and output control laws of these controllers will be discussed.  Their merits and drawbacks are highlighted.  Based on the critical discussions, a new structure of Fuzzy PID controller is proposed.  It is based on cascaded structure, which yields simpler design flow and parameters tuning.  Other advantages of the proposed Fuzzy PID structure are the reduction of tuning parameters and rules of the Fuzzy controller. In addition, the proposed structure allows the usage of signed distance method. The application of the method reduces the computation burden significantly as the power inverter regulation needs very fast and precise computations.
THE ANALYSIS OF TWO-PROFILE ENVIRONMENT GROUND CLUTTER STATISTICS MEASURED USING FORWARD SCATTER RADAR WITH VHF AND UHF BANDS
Authors: Ismail N. N. , Rashid N. E. A. , Ismail Khan Z. , Ripin N. , Abdul Rashid M. F.
Published: July 2016

In this paper, a FSR two-profile environment ground clutter-measured signal with very high frequency (VHF) and ultra high frequency (UFH) at a border of dense forest and free space area are presented. Statistical distribution method is used to model the clutter signal, namely Weibull, Gamma, Log-Logistic and Log-Normal distribution. Two goodness-of-fit (GOF) tests are used to calculate the error between the amplitude of the clutter data and the statistical model, which are the root mean square error (RMSE) and chi-square (CS). At the end of this analysis, Weibull model was found to be the best fit for 64 MHz clutter signal while Gamma model is best fitted at 151 MHz carrier frequency. Another model known as Log-Logistic model fits well to a clutter signal measured with 434 MHz carrier frequency.
POINT-TO-POINT (PTP) CONTROL PERFORMANCES OF AN UPPER LIMB ROBOTIC ARM
Authors: Md Ghazaly Mariam , Teo Ting Huan , A/L Regeev Vivek , A/L Vijayan Kartikesu , Shin Hong Chong , Che Amran Aliza , Abdullah Zulkeflee , Md Ali Mohd Amran
Published: July 2016

The objective of this paper is to design a controller which is able to control the output angle for an upper limb of a robotic arm, for precision motion and high speed response.  The aim is to optimize the best controller for an upper limb robotic arm system for precision motion, in which improper motion will results in injuries/ fatality and loss of production in manufacturing system. In this research, a robotic arm prototype with a 1 degree-of-freedom (DOF) was designed and fabricated, in which the DC geared motor was implemented.  Studies are carried out based on previous research to investigate the suitable type of controller. PID controller and fuzzy logic controller are chosen and compared in terms of their performances such as the steady-state error, settling time, rise time and overshoot. The equipment’s used are Micro-Box 2000/2000C, Cytron DC geared motor, motor driver circuit. Micro-Box module acts as the interface between hardware component and MATLAB R2009a. Open-loop simulations are carried out to obtain the transfer function of the motor and substituted into the system for further simulation analysis. Simulation for the uncompensated system is carried out to observe the close-loop system characteristic without the controller. After that, the close-loop point-to-point (PTP) trajectory control for simulations & experiments are carried out for the compensated systems using PID controller based on the Ziegler-Nichols frequency response method. Analyses are made based on the results obtained and the best type of controller is chosen for achieving precise motion control for the upper limb robotic arm. In this paper, the PID controller shows better performances compared to the Fuzzy Logic controller (FLC) with the steady state error of less than 0.010 and settling time of 0.5s; for the input reference of 150  respectively.
PALEOENVIRONMENT OF BROWN COAL FROM SANGATTA COAL MINES, EAST BORNEO, INDONESIA
Authors: Zetra Yulfi , B. Sosrowidjojo Imam , Burhan R. Y. Perry
Published: July 2016

Organic geochemical studies on brown coal (lignite) located in Inul area were carried out, an area that is located in Sangatta coal mines, East Borneo, which is a part of the Balikpapan formation, aged as old as Middle Miocene to Late Miocene. Aliphatic hydrocarbon biomarker compounds which were identified by using the GC-MS analysis showed a presence of n-alkane (n-C12-n-C36) with bimodal distribution, which described the sources of organic compounds that originated from two different sources. These sources of organic compounds are phytoplankton, zooplankton and higher plants terrestrial. The Pr/Ph ratio of 1.3, the dominance of C31-homohopane and a high ratio of Tm/Ts indicated that the process of peat depositions took place in an oxic environment.  The existence of hopanoid contents showed that there were dominant activities of microbes and biolipids organic compounds such as bb-hopane and hop-17(21)-ene inside the samples of olean-12-ene and a-cadinene, which inidicated that they are organic compounds that came from higher plants such as Angiosperm and immature coals. The existence of 24-ethylcholestene and 5b-cholest-24-ene found in the coal samples, which are categorised as steranes, also indicated a low level of carbonization from the coal itself.
Role of astrocyte–synapse interactions in CNS disorders
Authors: Blanco‐Suárez Elena , Caldwell Alison L. M. , Allen Nicola J.
Published: July 2016

Astrocytes comprise half of the cells in the brain. Although astrocytes have traditionally been described as playing a supportive role for neurons, they have recently been recognized as active participants in the development and plasticity of dendritic spines and synapses. Astrocytes can eliminate dendritic spines, induce synapse formation, and regulate neurotransmission and plasticity. Dendritic spine and synapse impairments are features of many neurological disorders, including autism spectrum disorder, schizophrenia, and Alzheimer's disease. In this review we will present evidence from multiple neurological disorders demonstrating that changes in astrocyte–synapse interaction contribute to the pathologies. Genomic analysis has connected altered astrocytic gene expression with synaptic deficits in a number of neurological disorders. Alterations in astrocyte‐secreted factors have been implicated in the neuronal morphology and synaptic changes seen in neurodevelopmental disorders, while alteration in astrocytic glutamate uptake is a core feature of multiple neurodegenerative disorders. This evidence clearly demonstrates that maintaining astrocyte–synapse interaction is crucial for normal central nervous system functioning. Obtaining a better understanding of the role of astrocytes at synapses in health and disease will provide a new avenue for future therapeutic targeting.

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Speech intelligibility in rooms: Effect of prior listening exposure interacts with room acoustics
Authors: Zahorik Pavel , Brandewie Eugene J.
Published: July 2016

There is now converging evidence that a brief period of prior listening exposure to a reverberant room can influence speech understanding in that environment. Although the effect appears to depend critically on the amplitude modulation characteristic of the speech signal reaching the ear, the extent to which the effect may be influenced by room acoustics has not been thoroughly evaluated. This study seeks to fill this gap in knowledge by testing the effect of prior listening exposure or listening context on speech understanding in five different simulated sound fields, ranging from anechoic space to a room with broadband reverberation time (T60) of approximately 3 s. Although substantial individual variability in the effect was observed and quantified, the context effect was, on average, strongly room dependent. At threshold, the effect was minimal in anechoic space, increased to a maximum of 3 dB on average in moderate reverberation (T60 = 1 s), and returned to minimal levels again in high reverberation. This interaction suggests that the functional effects of prior listening exposure may be limited to sound fields with moderate reverberation (0.4 ≤ T60 ≤ 1 s).
Robust classification of bacterial and viral infections via integrated host gene expression diagnostics
Authors: Sweeney Timothy E. , Wong Hector R. , Khatri Purvesh
Published: July 2016

An 18-gene panel distinguishes patients with bacterial and viral infections as well as those with inflammation not caused by infection.
In Vitro
Exposure to PC-1005 and Cervicovaginal Lavage Fluid from Women Vaginally Administered PC-1005 Inhibits HIV-1 and HSV-2 Infection in Human Cervical Mucosa
Authors: Villegas Guillermo , Calenda Giulia , Zhang Shimin , Mizenina Olga , Kleinbeck Kyle , Cooney Michael L. , Hoesley Craig J. , Creasy George W. , Friedland Barbara , Fernández-Romero José A. , Zydowsky Thomas M. , Teleshova Natalia
Published: July 2016

Our recent phase 1 trial demonstrated that PC-1005 gel containing 50 μM MIV-150, 14 mM zinc acetate dihydrate, and carrageenan (CG) applied daily vaginally for 14 days is safe and well tolerated. Importantly, cervicovaginal lavage fluid samples (CVLs) collected 4 or 24 h after the last gel application inhibited HIV-1 and human papillomavirus (HPV) in cell-based assays in a dose-dependent manner (MIV-150 for HIV-1 and CG for HPV). Herein we aimed to determine the anti-HIV and anti-herpes simplex virus 2 (anti-HSV-2) activity of PC-1005 in human cervical explants after
in vitro
exposure to the gel and to CVLs from participants in the phase 1 trial. Single HIV-1
BaL
infection and HIV-1
BaL
–HSV-2 coinfection explant models were utilized. Coinfection with HSV-2 enhanced tissue HIV-1
BaL
infection.
In vitro
exposure to PC-1005 protected cervical mucosa against HIV-1
BaL
(up to a 1:300 dilution) in single-challenge and cochallenge models. CG gel (PC-525) provided some barrier effect against HIV-1
BaL
at the 1:100 dilution in a single-challenge model but not in the cochallenge model. Both PC-1005 and PC-525 at the 1:100 dilution inhibited HSV-2 infection, pointing to a CG-mediated protection. MIV-150 and CG in CVLs inhibited HIV (single-challenge or cochallenge models) and HSV-2 infections in explants in a dose-dependent manner (
P
< 0.05). Stronger inhibition of HIV-1 infection by CVLs collected 4 h after the last gel administration was observed compared to infection detected in the presence of baseline CVLs. The anti-HIV and anti-HSV-2 activity of PC-1005 gel
in vitro
and CVLs in human ectocervical explants supports the further development of PC-1005 gel as a broad-spectrum on-demand microbicide.
Aspergillus fumigatus Intrinsic Fluconazole Resistance Is Due to the Naturally Occurring T301I Substitution in Cyp51Ap
Authors: Leonardelli Florencia , Macedo Daiana , Dudiuk Catiana , Cabeza Matias S. , Gamarra Soledad , Garcia-Effron Guillermo
Published: July 2016

Aspergillus fumigatus
intrinsic fluconazole resistance has been demonstrated to be linked to the
CYP51A
gene, although the precise molecular mechanism has not been elucidated yet. Comparisons between
A. fumigatus
Cyp51Ap and
Candida albicans
Erg11p sequences showed differences in amino acid residues already associated with fluconazole resistance in
C. albicans
. The aim of this study was to analyze the role of the natural polymorphism I301 in
Aspergillus fumigatus
Cyp51Ap in the intrinsic fluconazole resistance phenotype of this pathogen. The I301 residue in
A. fumigatus
Cyp51Ap was replaced with a threonine (analogue to T315 at
Candida albicans
fluconazole-susceptible Erg11p) by changing one single nucleotide in the
CYP51A
gene. Also, a
CYP51A
knockout strain was obtained using the same parental strain. Both mutants' antifungal susceptibilities were tested. The I301T mutant exhibited a lower level of resistance to fluconazole (MIC, 20 μg/ml) than the parental strain (MIC, 640 μg/ml), while no changes in MIC were observed for other azole- and non-azole-based drugs. These data strongly implicate the
A. fumigatus
Cyp51Ap I301 residue in the intrinsic resistance to fluconazole.
Broad-Spectrum Antimicrobial Effects of Photocatalysis Using Titanium Dioxide Nanoparticles Are Strongly Potentiated by Addition of Potassium Iodide
Authors: Huang Ying-Ying , Choi Hwanjun , Kushida Yu , Bhayana Brijesh , Wang Yuguang , Hamblin Michael R.
Published: July 2016

Photocatalysis describes the excitation of titanium dioxide nanoparticles (a wide-band gap semiconductor) by UVA light to produce reactive oxygen species (ROS) that can destroy many organic molecules. This photocatalysis process is used for environmental remediation, while antimicrobial photocatalysis can kill many classes of microorganisms and can be used to sterilize water and surfaces and possibly to treat infections. Here we show that addition of the nontoxic inorganic salt potassium iodide to TiO
2
(P25) excited by UVA potentiated the killing of Gram-positive bacteria, Gram-negative bacteria, and fungi by up to 6 logs. The microbial killing depended on the concentration of TiO
2
, the fluence of UVA light, and the concentration of KI (the best effect was at 100 mM). There was formation of long-lived antimicrobial species (probably hypoiodite and iodine) in the reaction mixture (detected by adding bacteria after light), but short-lived antibacterial reactive species (bacteria present during light) produced more killing. Fluorescent probes for ROS (hydroxyl radical and singlet oxygen) were quenched by iodide. Tri-iodide (which has a peak at 350 nm and a blue product with starch) was produced by TiO
2
-UVA-KI but was much reduced when methicillin-resistant
Staphylococcus aureus
(MRSA) cells were also present. The model tyrosine substrate
N
-acetyl tyrosine ethyl ester was iodinated in a light dose-dependent manner. We conclude that UVA-excited TiO
2
in the presence of iodide produces reactive iodine intermediates during illumination that kill microbial cells and long-lived oxidized iodine products that kill after light has ended.
Rifamycin Resistance in Clostridium difficile Is Generally Associated with a Low Fitness Burden
Authors: Dang Uyen T. , Zamora Idalia , Hevener Kirk E. , Adhikari Sudip , Wu Xiaoqian , Hurdle Julian G.
Published: July 2016

We characterized clinically occurring and novel mutations in the β subunit of RNA polymerase in
Clostridium difficile
(
Cd
RpoB), conferring rifamycin (including rifaximin) resistance. The Arg
505
Lys substitution did not impose an
in vitro
fitness cost, which may be one reason for its dominance among rifamycin-resistant clinical isolates. These observations were supported through the structural modeling of
Cd
RpoB. In general, most mutations lacked
in vitro
fitness costs, suggesting that rifamycin resistance may in some cases persist in the clinic.
Infectivity of Plasmodium falciparum in Malaria-Naive Individuals Is Related to Knob Expression and Cytoadherence of the Parasite
Authors: Stanisic Danielle I. , Gerrard John , Fink James , Griffin Paul M. , Liu Xue Q. , Sundac Lana , Sekuloski Silvana , Rodriguez Ingrid B. , Pingnet Jolien , Yang Yuedong , Zhou Yaoqi , Trenholme Katharine R. , Wang Claire Y. T. , Hackett Hazel , Chan Jo-Anne A. , Langer Christine , Hanssen Eric , Hoffman Stephen L. , Beeson James G. , McCarthy James S. , Good Michael F.
Published: July 2016

Plasmodium falciparumis the most virulent human malaria parasite because of its ability to cytoadhere in the microvasculature. Nonhuman primate studies demonstrated relationships among knob expression, cytoadherence, and infectivity. This has not been examined in humans. Cultured clinical-gradeP. falciparumparasites (NF54, 7G8, and 3D7B) andex vivo-derived cell banks were characterized. Knob and knob-associated histidine-rich protein expression, CD36 adhesion, and antibody recognition of parasitized erythrocytes (PEs) were evaluated. Parasites from the cell banks were administered to malaria-naive human volunteers to explore infectivity. For the NF54 and 3D7B cell banks, blood was collected from the study participants forin vitrocharacterization. All parasites were infectivein vivo. However, infectivity of NF54 was dramatically reduced.In vitrocharacterization revealed that unlike other cell bank parasites, NF54 PEs lacked knobs and did not cytoadhere. Recognition of NF54 PEs by immune sera was observed, suggestingP. falciparumerythrocyte membrane protein 1 expression. Subsequent recovery of knob expression and CD36-mediated adhesion were observed in PEs derived from participants infected with NF54. Knobless cell bank parasites have a dramatic reduction in infectivity and the ability to adhere to CD36. Subsequent infection of malaria-naive volunteers restored knob expression and CD36-mediated cytoadherence, thereby showing that the human environment can modulate virulence.
A Combined Computational and Genetic Approach Uncovers Network Interactions of the Cyanobacterial Circadian Clock
Authors: Boyd Joseph S. , Cheng Ryan R. , Paddock Mark L. , Sancar Cigdem , Morcos Faruck , Golden Susan S.
Published: July 2016

Two-component systems (TCS) that employ histidine kinases (HK) and response regulators (RR) are critical mediators of cellular signaling in bacteria. In the model cyanobacterium
Synechococcus elongatus
PCC 7942, TCSs control global rhythms of transcription that reflect an integration of time information from the circadian clock with a variety of cellular and environmental inputs. The HK CikA and the SasA/RpaA TCS transduce time information from the circadian oscillator to modulate downstream cellular processes. Despite immense progress in understanding of the circadian clock itself, many of the connections between the clock and other cellular signaling systems have remained enigmatic. To narrow the search for additional TCS components that connect to the clock, we utilized direct-coupling analysis (DCA), a statistical analysis of covariant residues among related amino acid sequences, to infer coevolution of new and known clock TCS components. DCA revealed a high degree of interaction specificity between SasA and CikA with RpaA, as expected, but also with the phosphate-responsive response regulator SphR. Coevolutionary analysis also predicted strong specificity between RpaA and a previously undescribed kinase, HK0480 (herein CikB). A knockout of the gene for CikB (
cikB
) in a
sasA cikA
null background eliminated the RpaA phosphorylation and RpaA-controlled transcription that is otherwise present in that background and suppressed cell elongation, supporting the notion that CikB is an interactor with RpaA and the clock network. This study demonstrates the power of DCA to identify subnetworks and key interactions in signaling pathways and of combinatorial mutagenesis to explore the phenotypic consequences. Such a combined strategy is broadly applicable to other prokaryotic systems.

IMPORTANCE
Signaling networks are complex and extensive, comprising multiple integrated pathways that respond to cellular and environmental cues. A TCS interaction model, based on DCA, independently confirmed known interactions and revealed a core set of subnetworks within the larger HK-RR set. We validated high-scoring candidate proteins via combinatorial genetics, demonstrating that DCA can be utilized to reduce the search space of complex protein networks and to infer undiscovered specific interactions for signaling proteins
in vivo
. Significantly, new interactions that link circadian response to cell division and fitness in a light/dark cycle were uncovered. The combined analysis also uncovered a more basic core clock, illustrating the synergy and applicability of a combined computational and genetic approach for investigating prokaryotic signaling networks.
Analyzing Neisseria gonorrhoeae Pilin Antigenic Variation Using 454 Sequencing Technology
Authors: Rotman Ella , Webber David M. , Seifert H. Steven
Published: July 2016

Many pathogens use homologous recombination to vary surface antigens in order to avoid immune surveillance.
Neisseria gonorrhoeae
, the bacterium responsible for the sexually transmitted infection gonorrhea, achieves this in part by changing the sequence of the major subunit of the type IV pilus in a process termed pilin antigenic variation (Av). The
N. gonorrhoeae
chromosome contains one expression locus (
pilE
) and many promoterless, partial-coding silent copies (
pilS
) that act as reservoirs for variant pilin information. Pilin Av occurs by high-frequency gene conversion reactions, which transfer
pilS
sequences into the
pilE
locus. We have developed a 454 sequencing-based assay to analyze the frequency and characteristics of pilin Av that allows a more robust analysis of pilin Av than previous assays. We used this assay to analyze mutations and conditions previously shown to affect pilin Av, confirming many but not all of the previously reported phenotypes. We show that mutations or conditions that cause growth defects can result in Av phenotypes when analyzed by phase variation-based assays. Adapting the 454 sequencing to analyze pilin Av demonstrates the utility of this technology to analyze any diversity generation system that uses recombination to develop biological diversity.

IMPORTANCE
Measuring and analyzing complex recombination-based systems constitute a major barrier to understanding the mechanisms used to generate diversity. We have analyzed the contributions of many gonococcal mutations or conditions to the process of pilin antigenic variation.
Primary and Secondary Sequence Structure Requirements for Recognition and Discrimination of Target RNAs by Pseudomonas aeruginosa RsmA and RsmF
Authors: Schulmeyer Kayley H. , Diaz Manisha R. , Bair Thomas B. , Sanders Wes , Gode Cindy J. , Laederach Alain , Wolfgang Matthew C. , Yahr Timothy L.
Published: July 2016

CsrA family RNA-binding proteins are widely distributed in bacteria and regulate gene expression at the posttranscriptional level.
Pseudomonas aeruginosa
has a canonical member of the CsrA family (RsmA) and a novel, structurally distinct variant (RsmF). To better understand RsmF binding properties, we performed parallel systematic evolution of ligands by exponential enrichment (SELEX) experiments for RsmA and RsmF. The initial target library consisted of 62-nucleotide (nt) RNA transcripts with central cores randomized at 15 sequential positions. Most targets selected by RsmA and RsmF were the expected size and shared a common consensus sequence (CANGGAYG) that was positioned in a hexaloop region of the stem-loop structure. RsmA and RsmF also selected for longer targets (≥96 nt) that were likely generated by rare PCR errors. Most of the long targets contained two consensus-binding sites. Representative short (single consensus site) and long (two consensus sites) targets were tested for RsmA and RsmF binding. Whereas RsmA bound the short targets with high affinity, RsmF was unable to bind the same targets. RsmA and RsmF both bound the long targets. Mutation of either consensus GGA site in the long targets reduced or eliminated RsmF binding, suggesting a requirement for two tandem binding sites. Conversely, RsmA bound long targets containing only a single GGA site with unaltered affinity. The RsmF requirement for two binding sites was confirmed with
tssA1
, an
in vivo
regulatory target of RsmA and RsmF. Our findings suggest that RsmF binding requires two GGA-containing sites, while RsmA binding requirements are less stringent.

IMPORTANCE
The CsrA family of RNA-binding proteins is widely conserved in bacteria and plays important roles in the posttranscriptional regulation of protein synthesis.
P. aeruginosa
has two CsrA proteins, RsmA and RsmF. Although RsmA and RsmF share a few RNA targets, RsmF is unable to bind to other targets recognized by RsmA. The goal of the present study was to better understand the basis for differential binding by RsmF. Our data indicate that RsmF binding requires target RNAs with two consensus-binding sites, while RsmA recognizes targets with just a single binding site. This information should prove useful to future efforts to define the RsmF regulon and its contribution to
P. aeruginosa
physiology and virulence.
Shutdown of HIV-1 Transcription in T Cells by Nullbasic, a Mutant Tat Protein
Authors: Jin Hongping , Li Dongsheng , Sivakumaran Haran , Lor Mary , Rustanti Lina , Cloonan Nicole , Wani Shivangi , Harrich David
Published: July 2016

Nullbasic is a derivative of the HIV-1 transactivator of transcription (Tat) protein that strongly inhibits HIV-1 replication in lymphocytes. Here we show that lentiviral vectors that constitutively express a Nullbasic-ZsGreen1 (NB-ZSG1) fusion protein by the eEF1α promoter led to robust long-term inhibition of HIV-1 replication in Jurkat cells. Although Jurkat-NB-ZSG1 cells were infected by HIV-1, no virus production could be detected and addition of phorbol ester 12-myristate 13-acetate (PMA) and JQ1 had no effect, while suberanilohydroxamic acid (SAHA) modestly stimulated virus production but at levels 300-fold lower than those seen in HIV-1-infected Jurkat-ZSG1 cells. Virus replication was not recovered by coculture of HIV-1-infected Jurkat-NB-ZSG1 cells with uninfected Jurkat cells. Latently infected Jurkat latent 6.3 and ACH2 cells treated with latency-reversing agents produced measurable viral capsid (CA), but little or none was made when they expressed NB-ZSG1. When Jurkat cells chronically infected with HIV-1 were transduced with lentiviral virus-like particles conveying NB-ZSG1, a >3-log reduction in CA production was observed. Addition of PMA increased virus CA production but at levels 500-fold lower than those seen in nontransduced Jurkat cells. Transcriptome sequencing analysis confirmed that HIV-1 mRNA was strongly inhibited by NB-ZSG1 but indicated that full-length viral mRNA was made. Analysis of HIV-1-infected Jurkat cells expressing NB-ZSG1 by chromatin immunoprecipitation assays indicated that recruitment of RNA polymerase II (RNAPII) and histone 3 lysine 9 acetylation were inhibited. The reduction of HIV-1 promoter-associated RNAPII and epigenetic changes in viral nucleosomes indicate that Nullbasic can inhibit HIV-1 replication by enforcing viral silencing in cells.

IMPORTANCE
HIV-1 infection is effectively controlled by antiviral therapy that inhibits virus replication and reduces measurable viral loads in patients below detectable levels. However, therapy interruption leads to viral rebound due to latently infected cells that serve as a source of continued viral infection. Interest in strategies leading to a functional cure of HIV infection by permanent viral suppression, which may be achievable, is growing. Here we show that a mutant form of the HIV-1 Tat protein, referred to as Nullbasic, can inhibit HIV-1 transcription in infected Jurkat T cell to undetectable levels. Analysis shows that Nullbasic alters the epigenetic state of the HIV-1 long terminal repeat promoter, inhibiting its association with RNA polymerase II. This study indicates that key cellular proteins and pathways targeted here can silence HIV-1 transcription. Further elucidation could lead to functional-cure strategies by suppression of HIV transcription, which may be achievable by a pharmacological method.