The branched Entner-Doudoroff pathway in hyperthermophilic archaea

Vergleichende Studien der Glykolyse in hyperthermophilen Archaeen haben eine Vielzahl an Variationen der klassischen bakteriellen und eukaryontischen Wege offen gelegt, namentlich des Entner-Doudoroff (ED) Weg und des Embden-Meyerhof-Parnas (EMP) Weg. Während im aeroben Sulfolobus solfataricus der ED Weg die einzige Option für den Glukose Katabolismus darstellt, nutzt der anaerobe Thermoproteus tenax neben diesem zusätzlich einen reversiblen EMP Weg. Es wurden bislang zwei Modifikationen des ED Weg identifiziert, (i) der semi-phosphorylative (sp) ED Weg für halophile Archaea und (ii) der nicht-phosphorylative (np) ED Weg in thermophilen und hyperthermophilen Archaea. Durch einen Ansatz, basierend auf vergleichender Genomanalyse wurde ein ED Gencluster in den Genomen von T. tenax und S. solfataricus ausgemacht, was die Präsenz des sp ED Weg in diesen Organismen nahe legt. Das ED Gencluster umfasst Gene (i) einer putativen Glukonat Dehydratase (gad), (ii) einer 2-Keto-3-deoxy-glukonat Aldolase (kdgA), welche zuvor in S. solfataricus charakterisiert wurde [BUCHANAN et al., 1999], (iii) einer Zucker (KDG) Kinase, (iv) in T. tenax einer Glukan-1,4-α-Glukosidase (gaa gene) und (v) in S. solfataricus einer nicht phosphorylierenden Glycerinaldehyd-3-phosphat Dehydrogenase (GAPN, gapN). Northern Blot und Primer Extension zeigten eine koordinierte Transkription der für die Synthese und Degradation von 2-Keto-3-deoxy-glukonat (KDG) und 2-Keto-3-deoxy-6-phosphoglukonat (KDPG) kodierenden ED Gene. In T. tenax verfügt das ED Gencluster über ein gad Gen und das kdgA-kdgK-gaa Operon und in S. solfataricus findet sich ein gad-kdgA-kdgK Operon und das gapN Gen. Um die vorhergesagten Enzymaktivitäten zu bestätigen, wurden die entsprechenden Gene kloniert (für S. solfataricus geschah die Klonierung in Zusammenarbeit mit Prof. Dr. John van der Oost, Universität Wageningen, Niederlande), rekombinant exprimiert und für die Bestimmung der Enzymaktivitäten gereinigt bzw. angereichert. Zusammenfassend offenbart diese Arbeit (i) einen neuen Typ der Glukonat Dehydratase, (ii) eine bifunktionale 2-Keto-3-deoxy-(6-phospho)-glukonat Aldolase, (iii) eine 2-Keto-3-deoxy-glukonat Kinase und (iv) in S. solfataricus eine nicht phosphorylierende Glycerinaldehyd-3-phosphat Dehydrogenase (GAPN). Die Glukonat Dehydratase (GAD) katalysiert die Dehydratation von Glukonat zu KDG. Obwohl die GAD aus verschiedenen bakteriellen Quellen gereinigt und charakterisiert wurde, war das kodierende Gen nie zuvor identifiziert worden und stellt damit ein fehlendes Schlüsselelement im zentralen Kohlenhydratmetabolismus dar. Für T. tenax wurde die entsprechende Enzymspezifität mit Glukonat nachgewiesen, wohingegen für das Enzym aus S. solfataricus zusätzliche Aktivität mit Galaktonat nachgewiesen werden konnte [LAMBLE et al., 2004] Entgegen vorangehenden Studien der KDG Aldolase aus S. solfataricus [LAMBLE et al., 2003; HENDRY, et al., 2000], wurde sowohl für dieses Enzym, als auch für das Enzym aus T. tenax, Aktivität nicht nur mit den nicht-phosphorylierten Substraten, sondern auch mit den phosphorylierten Substraten, nachgewisen. Demnach ist das Enzym eine echte bifunktionale KD(P)G Aldolase und katalysiert die reversible Spaltung von KDG und KDPG. Für das Enzym aus S. solfataricus konnte gezeigt werden, dass es unspezifisch für KDG/KDPG und 2-Keto-3-Desoxygalaktonat/2-Keto-3-Desoxy-6-Phosphogalaktonat (KDGal/KDPGal) ist [LAMBLE et al., 2005; THEODOSSIS et al., 2004; LAMBLE et al., 2003]. Bislang gibt es noch keine Information über die Stereoselektivität für das Enzym aus T. tenax. KDG Kinase vermittelt die ATP abhängige Phosphorylierung von KDG zu KDPG und stellt somit ein Schlüsselenzym für den sp ED Weg dar. Für das Substrat KDG folgt das Enzym der Michaelis-Menten Kinetik. Das Enzym aus T. tenax ist damit die erste identifizierte und charakterisierte archaeale KDG kinase. Die Aktivität der nicht phosphorylierenden Glycerinaldehyd-3-phosphat Dehydrogenase (GAPN) aus S. solfataricus wurde mittels eines kontinuierlichen Tests bestimmt, indem die Bildung von NADPH oder NADH gemessen wurde. Das Enzym folgt der Michaelis Menten Kinetik für NADP+ und DL-GAP. In Gegenwart von NAD+ zeigte sich lediglich eine geringe Aktivität. Außerdem weist das Enzym allosterische Eigenschaften auf, wobei für Glukose 1-phosphat der größte Effekt beobachtet wurde. Zusammenfassend kann festgehalten werden, dass sowohl die enzymatischen Studien, als auch die in vivo Identifizierung von Enzymaktivitäten in Rohextrakten, sowie Experimente zur in vitro Rekonstruktion, die Präsenz des semi-phosphorylativen ED Weg und des nicht-phosphorylativen ED Weg und damit eines verzweigten ED Weg in T. tenax und S. solfataricus belegen. Verfügbare Genomdaten, ebenso wie biochemische Daten zeigen darüber hinaus die Anwesenheit des verzweigten ED Wege in thermophilen Mitgliedern der Thermoproteales (T. acidophilum, T. volcanium, P. torridus und F. acidiphilum). Folglich stellt der verzweigte ED Weg die ED Modifikation in thermophilen und hyperthermophilen Archaea dar. Darüber hinaus legen BLAST Analysen eine noch weit größere Verbreitung dieses archaealen Typs der ED-ähnlichen Pfade auch bei den Bacteria und Eukarya nahe und helfen daher Komplexität und Flexibilität des zentralen Kohlenhydratmetabolismus in allen drei Domänen des Lebens zu entschlüsseln

Glycerate kinase of the hyperthermophilic archaeon Thermoproteus tenax: new insights into the phylogenetic distribution and physiological role of members of the three different glycerate kinase classes

<p>Abstract</p> <p>Background</p> <p>The presence of the branched Entner-Doudoroff (ED) pathway in two hyperthermophilic Crenarchaea, the anaerobe <it>Thermoproteus tenax </it>and the aerobe <it>Sulfolobus solfataricus</it>, was suggested. However, so far no enzymatic information of the non-phosphorylative ED branch and especially its key enzyme – glycerate kinase – was available. In the <it>T. tenax </it>genome, a gene homolog with similarity to putative hydroxypyruvate reductase/glycerate dehydrogenase and glycerate kinase was identified.</p> <p>Results</p> <p>The encoding gene was expressed in <it>E. coli </it>in a recombinant form, the gene product purified and the glycerate kinase activity was confirmed by enzymatic studies. The enzyme was active as a monomer and catalyzed the ATP-dependent phosphorylation of D-glycerate forming exclusively 2-phosphoglycerate. The enzyme was specific for glycerate and highest activity was observed with ATP as phosphoryl donor and Mg²⁺as divalent cation. ATP could be partially replaced by GTP, CTP, TTP and UTP. The enzyme showed high affinity for D-glycerate (K_m0.02 ± 0.01 mM, V_maxof 5.05 ± 0.52 U/mg protein) as well as ATP (K_mof 0.03 ± 0.01 mM, V_maxof 4.41 ± 0.04 U/mg protein), although at higher glycerate concentrations, substrate inhibition was observed. Furthermore, the enzyme was inhibited by its product ADP via competitive inhibition. Data bank searches revealed that archaeal glycerate kinases are members of the MOFRL (multi-organism fragment with rich leucine) family, and homologs are found in all three domains of life.</p> <p>Conclusion</p> <p>A re-evaluation of available genome sequence information as well as biochemical and phylogenetic studies revealed the presence of the branched ED pathway as common route for sugar degradation in Archaea that utilize the ED pathway. Detailed analyses including phylogenetic studies demonstrate the presence of three distinct glycerate kinase classes in extant organisms that share no common origin. The affiliation of characterized glycerate kinases with the different enzyme classes as well as their physiological/cellular function reveals no association with particular pathways but a separate phylogenetic distribution. This work highlights the diversity and complexity of the central carbohydrate metabolism. The data also support a key function of the conversion of glycerate to 2- or 3-phosphoglycerate via glycerate kinase in funneling various substrates into the common EMP pathway for catabolic and anabolic purposes.</p

EFFECT OF INNOVATIVE INTERVENTION IN ELIMINATING STIGMA AND DISCRIMINATION AMONG PEOPLE LIVING WITH AIDS IN KHARTOUM STATE, SUDAN 2013

Since the outbreak of the epidemic, almost 78 million people have been infected with the HIV virus and about 39 million people have died of HIV. Globally, 35.0 million people were recorded to be living with HIV at the end of 2013. Sub-Saharan Africa remains the most severely affected region, and accounts for almost 71% of the people living with HIV worldwide12. HIV/AIDS cases in Sudan increases gradually from one case in 1986 to 10444 cases in 2009, to about 0.67% 13. People living with the disease are the main source through which HIV is spreading. They suffer social stigma and community discrimination. Information, Education, and Communication (IEC) are public health approach aim in changing or reinforcing health-related behaviors to a target audience through communication methods and principles 1. A quasi-experimental study was conducted in Khartoum State, which aim to study the effect of information, education, and communication in reducing stigmatization and discrimination among people living with AIDS/HIV (PLWAIDS/HIV). 25 participants of the total registered members of PLWAIDS/HIV in the society have been selected by systematic random sampling. An intensive intervention using IEC was conducted for six months. Data which were collected before and after the intervention were compared and analyzed using Chi-square test through a computer using SPSS Program. Furthermore, this was presented in tables and graphs. The study revealed that there was a significant elimination of social stigma and a slight decrease in the feeling of community discrimination. The IEC strategy has been proven to be effective in a significant elimination of social stigma and has slightly decreased the feeling of community discrimination

EFFECT OF INNOVATIVE INTERVENTION IN ELIMINATING STIGMA AND DISCRIMINATION AMONG PEOPLE LIVING WITH AIDS IN KHARTOUM STATE, SUDAN 2013

Since the outbreak of the epidemic, almost 78 million people have been infected with the HIV virus and about 39 million people have died of HIV. Globally, 35.0 million people were recorded to be living with HIV at the end of 2013. Sub-Saharan Africa remains the most severely affected region, and accounts for almost 71% of the people living with HIV worldwide12. HIV/AIDS cases in Sudan increases gradually from one case in 1986 to 10444 cases in 2009, to about 0.67% 13. People living with the disease are the main source through which HIV is spreading. They suffer social stigma and community discrimination. Information, Education, and Communication (IEC) are public health approach aim in changing or reinforcing health-related behaviors to a target audience through communication methods and principles 1. A quasi-experimental study was conducted in Khartoum State, which aim to study the effect of information, education, and communication in reducing stigmatization and discrimination among people living with AIDS/HIV (PLWAIDS/HIV). 25 participants of the total registered members of PLWAIDS/HIV in the society have been selected by systematic random sampling. An intensive intervention using IEC was conducted for six months. Data which were collected before and after the intervention were compared and analyzed using Chi-square test through a computer using SPSS Program. Furthermore, this was presented in tables and graphs. The study revealed that there was a significant elimination of social stigma and a slight decrease in the feeling of community discrimination. The IEC strategy has been proven to be effective in a significant elimination of social stigma and has slightly decreased the feeling of community discrimination

Improvement estimating of project cost and design for a hospital project by using (3D&5D) simulation

Building Information Modeling (BIM) is an approach of managing and generating building data during its life cycle, dimensional (2D) drawings and later developed to (3D). The scope of BIM 3D, 4D, 5D, in this research 3D model to understand the process project special for beginner's engineers and give idea about all steps the project. 5D the cost component helps create estimates. Estimator is consuming 70% of a cost estimator’s time which required to a project. BIM can provide the capability to create takeoffs the materials, measurements and counts immediately from a model. Building Information models are also more and more used by Owners, Designers, Contractors and Engineer during the project lifecycle. Planning and cost estimation used in design phases for huge project to detect errors before start in the work , through account the time required to set up a hospital and reduce the time needed to build the project through overlapping relationships and getting shorter period to build the project utilizing MS Project software and to detect estimated 5D costing of total construction project, BIM can support cost estimating, the period spent by the estimator on quantification differ by project, using BIM for takeoff or cost estimating, the removal of manual takeoffs saves cost ,time and minimize potential for human error.

Safety management in private construction project in Iraq

The construction industry consider one of the most dynamic industries due to the unique features such as the change in the project site and condition. These features make the application of specifications and codes more difficult. Occupational Safety requirements in project is also difficult to be implemented and monitored due to the unique features of this industry. The aim of the study is to propose a new method that will determine the performance and management of the occupational safety in the construction site, in order to increase the safety at work in the private construction sector in Iraq. Another purpose of this evaluation is to establish a fundamental point that can be graded according to the safety index for the Iraqi construction industry. The information has been collected through a checklist and a questionnaire; the study focused on data collection paradigm and framework based on a fuzzy logic approach, which is especially able to evaluate the efficiency and monitoring of management defects of private contractors in the fields of occupational safety management. The results show a high security management performance in the private construction sector in Iraq, and the survey results show that there is a great deficiency in training on job security, also study found out that the top three elements of occupational safety management in Iraq are: First Aid-medical needs, Safety committees, Hazard prevention and control

Transmission System On –Line Fault Location Using ArtificialNeural Network

In this work, protection systems for overhead transmission lines areinvestigated and an efficient technique for on –line fault location based onArtificial Neural Network(ANN ) is suggested. First, Studying and investigatingthe power transmission lines short circuit modeling and analysis, and thendeveloping a MATLAB programs to calculate fault currents and voltages fordifferent fault types depending on the location of the fault in the transmission lineand finding the location of this fault. The proposed technique for the faultlocation is the two -end data technique. The pre-fault data plus the fault dataconstruct a training set for the neural network programs which contain two types,one for fault detection and classification, and the other for the fault location. Then,these programs are applied on the Iraqi super grid (400 kV)

Role of Health Education in Raising Tetanus Toxoid Vaccination Coverage among Pregnant Women and Mothers with Newborns in Khartoum North-Sudan, 2019

Tetanus is an acute disease caused by the toxin of a bacterium called Clostridium tetani which is often found in soil. It is a life-threatening bacterial disease, which causes severe mortality among maternal, neonatal, and young infant. It is so serious even it can waste pregnancy, harm her baby during pregnancy or after delivery. A quasi experimental community based study was conducted among pregnant women and mothers with newborns in Khartoum North Locality from 2015-2019 with the aim to assess the effectiveness of health education intervening in raising tetanus toxoid vaccination coverage among pregnant women and mothers with new born. 266 participants were selected by using a systematic random sampling technique. The intervention that continued for six months includes; health talks, home visits, video show, presentations with power points, focus groups discussion, exhibitions, distribution of printed materials, pamphlets, brochures and posters. The data was statistically analyzed by using the (SPSS) and significant t and c2 - tests. The result showed a significant increase in the participants’ knowledge after the intervention towards tetanus toxoid at P value 0.00, the mode of infection with tetanus at P value 0.00, vulnerable group to tetanus at p value is 0.00, the seriousness of the tetanus at P value 0.00, neonatal tetanus at P value 0.00. There was a significant positive modification of the participants’ attitudes towards the easiness to communicate with the health workers at P value 0.00. The result showed a significant change in the participants’ practices towards vaccination against tetanus toxic at P value 0.00 and significant change towards taking vaccine dose regularly at P value 0.00. The study recommended that Federal Ministry of Health has to conduct many health education interventions to raise the tetanus toxoid vaccination coverage

New cost control techniques in mega construction projects

The construction industry in the U.K. is a vital sector of the country's economy. However, this industry is faced by various types of uncertainties and risks due to their dynamic environment, particularly for megaprojects. The study aimed to determine the modern and effective cost control techniques that are utilized in for mega construction projects in the U.K. A mixed-method approach that involves both qualitative and quantitative approaches were adopted in this research for data collection and analysis. Five semi-structured interviews with construction professionals were conducted first to find out modern and effective techniques used for projects cost control. This is followed by carrying out Delphi survey twice with 42 and 39 construction professionals. Results revealed that the top five techniques that can be utilized for controlling megaproject costs were: Activity-based costing method, Contract Variance – Unit Costing, To-Complete Performance Index (TCPI), Cost-Value Reconciliation and BIM

A comparative study between X-chromosome mapping of Sudanese and Egyptian Anopheles pharoensis theobald (Diptera: Culicidae) strains

Background: Current literature lacks information regarding Anopheles pharoensis malaria transmission patterns. Anopheles pharoensis succeeded in transmitting Malaria in Egypt. However, it was unsuccessful in doing the same in Sudan. From here arises these important questions: Why does it transmit malaria in Egypt but not in the Sudan or other Countries? Is it a Sibling species or a Sub-species? This investigation aimed to answer these questions by studying the genetics of Anopheles pharoensis collected from Egypt and Sudan.Methods: A comparative study was conducted in Egypt and Sudan to determine the population genetic structure of Anopheles pharoensis species based on chromosomal inversion of karyotypes. Fourth-stage larvae of Anopheles pharoensis were collected from Egypt (Faiyoum government) and from Sudan (Khartoum, Gezira and Sennar states).Result: Significant levels of differentiation were observed among the species studied.Conclusion: Investigations suggest that Anopheles pharoensis species collected from Egypt can transmit malaria due to the presence of an inversion in the X-chromosome. The lack of the aforementioned inversion probably prevented the transmission of malaria by Anopheles pharoensis in Sudan. The results above provide insight into malaria transmission patterns by Anopheles pharoensis. However, more needs to be done and hence it is recommended to undergo further research on genetics and morphological studies using molecular biological tool son Anopheles pharoensis in Sudan