Manipulation of adenovirus early region 1 rescue plasmids by homologous recombination in Saccharomyces cerevisiae

The manipulation of large (>10 kb) plasmid systems amplifies problems common to traditional cloning strategies. Unique or rare restriction enzyme recognition sequences are uncommon and very rarely located in opportunistic locations. Making site-specific deletions and insertions in larger plasmids consequently leads to multiple step cloning strategies that are often limited by time-consuming, low efficiency linker insertions or blunt-end cloning strategies. Manipulation ofthe adenovirus genome and the genomes ofother viruses as bacterial plasmids are systems that typify such situations. Recombinational cloning techniques based on homologous recombination in Saccharomyces cerevisiae that circumvent many ofthese common problems have been developed. However, these techniques are rarely realistic options for such large plasmid systems due to the above mentioned difficulties associated with the addition ofrequired yeast DNA replication, partitioning and selectable marker sequences. To determine ifrecombinational cloning techniques could be modified to simplify the manipulation of such a large plasmid system, a recombinational cloning system for the creation of human adenovirus EI-deletion rescue plasmids was developed. Here we report for the first time that the 1,456 bp TRP1/ARS fragment ofYRp7 is alone sufficient to foster successful recombinational cloning without additional partitioning sequences, using only slight modifications of existing protocols. In addition, we describe conditions for efficient recombinational cloning involving simultaneous deletion of large segments ofDNA (>4.2 kb) and insertion of donor fragment DNA using only a single non-unique restriction site. The discovery that recombinational cloning can foster large deletions has been used to develop a novel recombiliational cloillng technique, selectable inarker 'kilockouf" recombinational cloning, that uses deletion of a yeast selectable marker coupled with simultaneous negative and positive selection to reduce background transformants to undetectable levels. The modification of existing protocols as described in this report facilitates the use of recombinational cloning strategies that are otherwise difficult or impractical for use with large plasmid systems. Improvement of general recombinational cloning strategies and strategies specific to the manipulation ofthe adenovirus genome are considered in light of data presented herein.

The impact of a training program in phonological awareness on children's early writing

This study examined the effects that a training program in phonological awareness had on the early writing skills of children in a Grade One class in the Lincoln County Separate school system. The intent of the training program was to provide consistent and systematic practice in the manipulation of the phonological structure of language. The games and activities of the training program were related to a framework of developmental phonological skills and practised in a group setting during an unstructured period of the regular classroom schedule. The training program operated three days in a six-day cycle for approximately twenty minutes a day, from November until mid-March. All children were tested at the outset and conclusion of the study to determine level of functioning in letter identification, word recognition, verbal intelligence, phonological awareness and spelling. Results of the pre-tests and post-tests were compared to determine differences between the experimental and control groups over time. In addition, a systematic analysis of the children's writing looked at the development of the spelling of regular and irregular words. The results of this study provided strong support for the hypothesis that the treatment group would progress through the stages of early writing development more quickly than children without such training. On the basis of differences between the groups over time, it was evident that training in phonological awareness had a direct positive effect on the spelling of regular words for children during the early stages of writing. The training program did not have a significant effect on the spelling of irregular words. Test results evaluating phonological awareness indicated a significant difference within each group over time but no significance between the groups during the experimental period. It would appear that the results of these tests reflect maturational changes in the child rather than causal effects of the training program. Nor did the effects of the training program transfer significantly to other aspects of language. Although some of the hypotheses considered were not supported by the study, the results do indicate that children during the early stages of writing development can benefit from a training program in phonological awareness. The theoretical direction for effective programming as a result of this study is discussed. The educational implications of training phonological awareness concurrent to beginning efforts in writing are considered.

Warning Patients’ Relatives of Genetic Risks: Policy Approaches

As an increasing number of genetic tests for specific early- and late-onset disorders move from research to the clinical setting, health care professionals are faced with new challenges or, alternatively, with novel twists on age-old ethical dilemmas. A finding that an individual carries a deleterious mutation can indicate that his or her relatives are at an increased risk of being affected by the same genetic disorder.

Manipulation of the ubiquitin-proteasome system by HIV-1 : role of the accessory protein Vpr

Le virus de l’immunodéficience humaine de type 1 (VIH-1), l’agent étiologique du SIDA, est un rétrovirus complexe arborant plusieurs protéines accessoires : Nef, Vif, Vpr, et Vpu. Celles-ci sont impliquées dans la modulation de la réplication virale, dans l’évasion immunitaire et dans la progression de la pathogenèse du SIDA. Dans ce contexte, il a été démontré que la protéine virale R (Vpr) induit un arrêt de cycle cellulaire en phase G2. Le mécanisme par lequel Vpr exerce cette fonction est l’activation, ATR (Ataxia telangiectasia and Rad3 related)-dépendante, du point de contrôle de dommage à l’ADN, mais les facteurs et mécanismes moléculaires directement impliqués dans cette activité demeurent inconnus. Afin d’identifier de nouveaux facteurs cellulaires interagissant avec Vpr, nous avons utilisé une purification d’affinité en tandem (TAP) pour isoler des complexes protéiques natifs contenant Vpr. Nous avons découvert que Vpr s’associait avec CRL4A(VprBP), un complexe cellulaire d’E3 ubiquitine ligase, comprenant les protéines Cullin 4A, DDB1 (DNA damage-binding protein 1) et VprBP (Vpr-binding protein). Nos études ont mis en évidence que le recrutement de la E3 ligase par Vpr était nécessaire mais non suffisant pour l’induction de l’arrêt de cycle cellulaire en G2, suggérant ainsi que des événements additionnels seraient impliqués dans ce processus. À cet égard, nous apportons des preuves directes que Vpr détourne les fonctions de CRL4A(VprBP) pour induire la polyubiquitination de type K48 et la dégradation protéosomale de protéines cellulaires encore inconnues. Ces événements d’ubiquitination induits par Vpr ont été démontrés comme étant nécessaire à l’activation d’ATR. Finalement, nous montrons que Vpr forme des foyers ancrés à la chromatine co-localisant avec VprBP ainsi qu’avec des facteurs impliqués dans la réparation de l’ADN. La formation de ces foyers représente un événement essentiel et précoce dans l’induction de l’arrêt de cycle cellulaire en G2. Enfin, nous démontrons que Vpr est capable de recruter CRL4A(VprBP) au niveau de la chromatine et nous apportons des preuves indiquant que le substrat inconnu ciblé par Vpr est une protéine associée à la chromatine. Globalement, nos résultats révèlent certains des ménanismes par lesquels Vpr induit des perturbations du cycle cellulaire. En outre, cette étude contribue à notre compréhension de la modulation du système ubiquitine-protéasome par le VIH-1 et son implication fonctionnelle dans la manipulation de l’environnement cellulaire de l’hôte.

STUDIES ON TAXONOMY, DISTRIBUTION, ECOLOGY AND REPRODUCTIVE POTENTIAL OF ROTIFERS FROM SELECTED CENTRES IN COCHIN BACKWATER SYSTEM, KERALA

The studies were conducted in nine stations with varying ecological characteristics along Cochin backwaters and adjoining canals. Many workers opined that the distribution of rotifers is cosmopolitan. The significance of rotifers as first food for early larvae was indicated by Fujita. Aquaculture is a fast growing field in fisheries sector and it is gaining more importance as the fish landings and supply are getting irregular. A consistent supply of fish/shellfish can only be achieved through aquaculture. The success of any culture activity depends on the timely production of seeds of finfishes/shellfishes. The availability of wild seed is seasonal and erratic. So, a dependable source of seed of fishes and shellfishes is possible only through large scale production in hatchery. A successful seed production activity depends on the availability of a variety of suitable live feed organisms in sufficient quantities at the proper time for use in the larval stages. As the live feeds promote high growth rates, easy digestion, assimilation and the quality of not contaminating the culture water when compared to other artificial feeds, make the culture of live feed organisms the principal means of providing food for the larvae of finfishes and shellfishes. Rotifers are considered to be an excellent and indispensable food for larvae of many finfishes and crustaceans. It (1960) was the first to culture Brachionus plicatilis for feeding marine fish larvae, and now it is being extensively used as live feed in hatcheries all over the world. They are a group of microscopic organisms coming under the Phylum Rotifera which comprises of about 2000 species. Their slow swimming habits, ability to tolerate a wide range of salinities, parthenogenetic mode of reproduction and ability to get enriched easily, make rotifers an ideal live feed organism. The major factors such as temperature, salinity and food that influence the reproductive potential and thereby the population size of rotifer, Salinity is one of the most important aspect influencing the reproductive rate of rotifers. The feed type and feed concentration play a vital role in influencing the reproductive rate of rotifers. For culture of rotifers, the commonly used micro algae belong to Chlorella, Nannochloropsis, Isochrysis and Tetraselmis. While some studies have suggested that, algal diet has little effect on reproductive rates in 1979 while using the rotifer, Brachionus plicatilis as feed for the larvae of red sea bream, Pagrus major. It is generally accepted that rotifers play a pivotal role in the successful rearing of marine fish larvae.

Criminal justice system in pondicherry - a comparative critical study of the past and the presen

The Union Territory of Pondicherry prior to its merger with the Indian Union was a French Colony. The erstwhile territory of Pondicherry along with its hamlets, namely, KARAIKKAL, MAHE and YANAM was administered by the French Regime. Before it was established by French in 1 6 74 A.D. it was part of Vijayanagara Empire. Prior to this, Pondicherry was a part of the Kingdom of Chola and Pallava Kings. During French Regime, the laws which were in force in France in relation to administration of civil and criminal justice were extended to the erstwhile Territory of Pondicherry. Thus while Pondicherry stood influenced by the Inquisitorial system since the beginning of the 18th century, the neighboring states forming part of the Indian Union since Independence came under the Influence of the British system, viz. accusatorial system. The territory of Pondicherry, for administrative reasons, came to be merged with the Indian Union in the early 60's. Following the merger, the Indian administration sought to extent its own laws from time to time replacing erstwhile French Laws, however, subject to certain savings. Thus the transitional period witnessed consequential changes in the administration of the territory, including the sphere of judicial system. Since I 963, the Union Territory of Pondicherry was brought under the spell of the Indian Legal System The people in Pondicherry ' thus have had the benefit of experiencing both the svstems. Their experiences will be of much help to those who undertake comparative studies in law. The plus and minus points of the respective systems help one to develop a detachment that helps independent evaluation of the svstents. The result of these studies could be relevant in revitalising our criminal systems.The present system is evaluated in the light of the past system. New dimensions are added by way' of an empirical study also.

Biomarker pigment signatures in Cochin back water system e A tropical estuary south west coast of India

Sedimentary biomarker pigments around Cochin estuary situated in the southwest coast of India were determined by HPLC. Fucoxanthin, an indicator of diatom was observed to be the most abundant carotenoid pigment in the estuary. Dinoflagellate derived carotenoid pigment peridinin was confined in the southern part of estuary and zeaxanthin pigment indicative of cyanobacteria were more found in sites influenced by anthropogenic activities. One compound having close similarity to fucoxanthin was also detected. Alloxanthin (cryptophyceae), chl b (green algae), canthaxanthin, neoxanthin, lutein and peridinin isomer were also detected by spectra and corresponding algal class were identified. The highest concentration of chl a (11.01 mg g 1) found near to the anthropogenic affected area while the lowest chl a (0.65 mg g 1) was recorded in industrial area. Degradation products of chl a, such as pheophorbide and pheophytin were observed and principal mode of mechanism of degradation were derived. Higher pheopigments content than chl a, reflects a density trapping of dead cells and early degradation of phytopigments from grazing activities

Legume rotation effects on early growth and rhizosphere microbiology of sorghum in West African soils

Cereal yield increases in legume rotations on west African soils were the subject of much recent research aiming at the development of more productive cropping systems for the mainly subsistence-oriented agriculture in this region. However, little has been done to elucidate the possible contribution of soil microbiological factors to these rotation effects. Therefore a pot trial was conducted using legume rotation and continuous cereal soils each from one site in Burkina Faso and two sites in Togo where cropping system experiments had been conducted over 4 yrs. All soils were planted with seedlings of sorghum (Sorghum bicolor L. Moench). From 21 days after sowing onwards relative growth rates in rotation soils were higher than in the continuous cereal soils, resulting in between 69 and 500% higher shoot dry matter of rotation sorghum compared to sorghum growing in continuous cereal soils. Across sites rotation soils were characterized by higher pH, higher microbial N and a lower microbial biomass C/N ratio and, with the exception of one site, a higher fungal biomass in the rhizosphere. The bacterial and eukaryal community structure in the soil, assessed by denaturing gradient gel electrophoresis (DGGE), differed between sites. However, only at one site differed the bacterial and the eukaryal community structure in the rotation soil significantly from that in the continuous cereal soil. Although the results of this study confirmed the marked plantgrowth differences between sub-Saharan legume-rotation soils and their continuous cereal counterparts they also showed the difficulties to differentiate possible microbiological causes from their effects.

Assessing the users’ need for a spatial decision support system of smallholder farming in Kenya

Accurate data of the natural conditions and agricultural systems with a good spatial resolution are a key factor to tackle food insecurity in developing countries. A broad variety of approaches exists to achieve precise data and information about agriculture. One system, especially developed for smallholder agriculture in East Africa, is the Farm Management Handbook of Kenya. It was first published in 1982/83 and fully revised in 2012, now containing 7 volumes. The handbooks contain detailed information on climate, soils, suitable crops and soil care based on scientific research results of the last 30 years. The density of facts leads to time consuming extraction of all necessary information. In this study we analyse the user needs and necessary components of a system for decision support for smallholder farming in Kenya based on a geographical information system (GIS). Required data sources were identified, as well as essential functions of the system. We analysed the results of our survey conducted in 2012 and early 2013 among agricultural officers. The monitoring of user needs and the problem of non-adaptability of an agricultural information system on the level of extension officers in Kenya are the central objectives. The outcomes of the survey suggest the establishment of a decision support tool based on already available open source GIS components. The system should include functionalities to show general information for a specific location and should provide precise recommendations about suitable crops and management options to support agricultural guidance on farm level.

Lower Bounds for Achieving Synchronous Early Stopping Consensus with Orderly Crash Failures

In this paper, we discuss the consensus problem for synchronous distributed systems with orderly crash failures. For a synchronous distributed system of n processes with up to t crash failures and f failures actually occur, first, we present a bivalency argument proof to solve the open problem of proving the lower bound, min (t + 1, f + 2) rounds, for early-stopping synchronous consensus with orderly crash failures, where t < n - 1. Then, we extend the system model with orderly crash failures to a new model in which a process is allowed to send multiple messages to the same destination process in a round and the failing processes still respect the order specified by the protocol in sending messages. For this new model, we present a uniform consensus protocol, in which all non-faulty processes always decide and stop immediately by the end of f + 1 rounds. We prove that the lower bound of early stopping protocols for both consensus and uniform consensus are f + 1 rounds under the new model, and our proposed protocol is optimal.

Polimorfismos del gen Butirilcolinesterasa responsables de reacciones adversas en pacientes consumidores de “cocaína”

La butirilcolinesterasa humana (BChE; EC 3.1.1.8) es una enzima polimórfica sintetizada en el hígado y en el tejido adiposo, ampliamente distribuida en el organismo y encargada de hidrolizar algunos ésteres de colina como la procaína, ésteres alifáticos como el ácido acetilsalicílico, fármacos como la metilprednisolona, el mivacurium y la succinilcolina y drogas de uso y/o abuso como la heroína y la cocaína. Es codificada por el gen BCHE (OMIM 177400), habiéndose identificado más de 100 variantes, algunas no estudiadas plenamente, además de la forma más frecuente, llamada usual o silvestre. Diferentes polimorfismos del gen BCHE se han relacionado con la síntesis de enzimas con niveles variados de actividad catalítica. Las bases moleculares de algunas de esas variantes genéticas han sido reportadas, entre las que se encuentra las variantes Atípica (A), fluoruro-resistente del tipo 1 y 2 (F-1 y F-2), silente (S), Kalow (K), James (J) y Hammersmith (H). En este estudio, en un grupo de pacientes se aplicó el instrumento validado Lifetime Severity Index for Cocaine Use Disorder (LSI-C) para evaluar la gravedad del consumo de “cocaína” a lo largo de la vida. Además, se determinaron Polimorfismos de Nucleótido Simple (SNPs) en el gen BCHE conocidos como responsables de reacciones adversas en pacientes consumidores de “cocaína” mediante secuenciación del gen y se predijo el efecto delos SNPs sobre la función y la estructura de la proteína, mediante el uso de herramientas bio-informáticas. El instrumento LSI-C ofreció resultados en cuatro dimensiones: consumo a lo largo de la vida, consumo reciente, dependencia psicológica e intento de abandono del consumo. Los estudios de análisis molecular permitieron observar dos SNPs codificantes (cSNPs) no sinónimos en el 27.3% de la muestra, c.293A>G (p.Asp98Gly) y c.1699G>A (p.Ala567Thr), localizados en los exones 2 y 4, que corresponden, desde el punto de vista funcional, a la variante Atípica (A) [dbSNP: rs1799807] y a la variante Kalow (K) [dbSNP: rs1803274] de la enzima BChE, respectivamente. Los estudios de predicción In silico establecieron para el SNP p.Asp98Gly un carácter patogénico, mientras que para el SNP p.Ala567Thr, mostraron un comportamiento neutro. El análisis de los resultados permite proponer la existencia de una relación entre polimorfismos o variantes genéticas responsables de una baja actividad catalítica y/o baja concentración plasmática de la enzima BChE y algunas de las reacciones adversas ocurridas en pacientes consumidores de cocaína.

Dental training system using haptic technology

In the U.K., dental students require to perform training and practice on real human tissues at the very early stage of their courses. Currently, the human tissues, such as decayed teeth, are mounted in a human head like physical model. The problems with these models in teaching are; (1) every student operates on tooth, which are always unique; (2) the process cannot be recorded for examination purposes and (3) same training are not repeatable. The aim of the PHATOM Project is to develop a dental training system using Haptic technology. This paper documents the project background, specification, research and development of the first prototype system. It also discusses the research in the visual display, haptic devices and haptic rendering. This includes stereo vision, motion parallax, volumetric modelling, surface remapping algorithms as well as analysis design of the system. A new volumetric to surface model transformation algorithm is also introduced. This paper includes the future work on the system development and research.

Interpreting the response of a dryland river system to Late Quaternary climate change

A U-series calcrete chronology has been constructed for three Late Quaternary terrace units, termed the D1, D2 and D3 terraces in age descending order, from the Rio Aguas river system of the Sorbas basin, southeast Spain. The D1 terrace formed between 30,300 +/- 4400 year BP and 12,140 +/- 360 year BP, correlating well with the Last Glacial Maximum when rates of sediment supply would have increased greatly, because of higher rates of weathering, reduced vegetation cover and weak soil development. The D2 terrace formed between 12,800 +/- 1100 year BP and 9,600 +/- 530 year BP, correlating well with the Younger Dryas event. The D3 terrace could only be poorly constrained to the early Holocene and no unequivocal cause could be assigned to this period of aggradation. The sedimentology and geomorphology of the D2 terrace suggests, however, that the aggradation of this unit was a response to diapirism/karstic processes occurring within the underlying Messinian gypsum strata and the subsequent damming of the Aguas system. Therefore, despite its coincident occurrence with the Younger Dryas, aggradation of the D2 terrace is unrelated to climate change. The style of this response, controlled predominantly by the characteristics of the underlying bedrock, makes correlating the terrace record of the Aguas with other systems in the Mediterranean unreliable. This study, therefore, highlights the problems of correlating fluvial sequences in regions of variable tectonics, climatic history and bedrock geology and emphasises the need to properly understand the main controls on individual fluvial systems before any attempt is made to correlate their depositional histories. (C) 2004 Elsevier Ltd. All rights reserved.

The Upper Valanginian (Early Cretaceous) positive carbon-isotope event recorded in terrestrial plants

Our understanding of the ancient ocean-atmosphere system has focused on oceanic proxies. However, the study of terrestrial proxies is equally necessary to constrain our understanding of ancient climates and linkages between the terrestrial and oceanic carbon reservoirs. We have analyzed carbon-isotope ratios from fossil plant material through the Valanginian and Lower Hauterivian from a shallow-marine, ammonite-constrained succession in the Crimean Peninsula of the southern Ukraine in order to determine if the Upper Valanginian positive carbon-isotope excursion is expressed in the atmosphere. delta(13)C(plant) values fluctuate around -23% to -22% for the Valanginian-Hauterivian, except during the Upper Valanginian where delta(13)C(plant) values record a positive excursion to similar to-18%. Based upon ammonite biostratigraphy from Crimea, and in conjunction with a composite Tethyan marine delta(13)C(carb) curve, several conclusions can be drawn: (1) the delta(13)C(plant) record indicates that the atmospheric carbon reservoir was affected; (2) the defined ammonite correlations between Europe and Crimea are synchronous; and (3) a change in photosynthetic carbon-isotope fractionation, caused by a decrease in atmospheric PCO2, occurred during the Upper Valanginian Positive delta(13)C excursion. Our new data, combined with other paleoenvironmental and paleoclimatic information, indicate that the Upper Valanginian was a cool period (icehouse) and highlights that the Cretaceous period was interrupted by periods of cooling and was not an equable climate as previously thought. (C) 2005 Elsevier B.V. All rights reserved.

An X-ray micro-tomography system optimised for the low-dose study of living organisms

An X-ray micro-tomography system has been designed that is dedicated to the low-dose imaging of radiation sensitive living organisms and has been used to image the early development of the first few days of plant development immediately after germination. The system is based on third-generation X-ray micro-tomography system and consists of an X-ray tube, two-dimensional X-ray detector and a mechanical sample manipulation stage. The X-ray source is a 50 kVp X-ray tube with a silver target with a filter to centre the X-ray spectrum on 22 keV.A 100 mm diameter X-ray image intensifier (XRII) is used to collect the two-dimensional projection images. The rotation tomography table incorporates a linear translation mechanism to eliminate ring artefact that is commonly associated with third-generation tomography systems' Developing maize seeds (Triticum aestivum) have been imaged using the system with a cubic voxel linear dimension of 100 mum, over a diameter of 25 mm and the root lengths and volumes measured. The X-ray dose to the plants was also assessed and found to have no effect on the plant root development. (C) 2003 Elsevier Science Ltd. All rights reserved.