Divalent cations stimulate DNA repair activities of bacterial (6-4) photolyases

Die (6-4) Photolyasen der bakteriellen Cryptochrom/Photolyase (BCP) -Gruppe gehören zu einer Familie von Flavoproteinen, die als Reparaturenzyme für UV-B-induzierte DNA-Läsionen (Photolyasen) oder als Blaulicht-Photorezeptoren (Cryptochrome) fungieren. Die (6-4) BCP-Proteine kommen ausschließlich in Prokaryoten vor. In dieser Arbeit wurden 3 Mitglieder der (6-4) BCP-Gruppe, PhrB von Agrobacterium fabrum, CryB von Rhodobacter sphaeroides und Proma-PL von Prochlorococcus marinus mit einer eukaryotischen (6-4) Photolyase von Ostreococcus tauri OtCPF1 und einem Mitglied der Klasse III CPD Photolyasen PhrA von Agrobacterium fabrum verglichen. Es zeigte sich, dass die DNA-Reparatur-Effizienz von (6-4) BCP-Proteinen durch Mg2+ oder andere zweiwertige Kationen stimuliert wird, während bei OtCPF1 und PhrA keine Wirkung von zweiwertigen Kationen beobachtet wurde. Der Einfluss zweiwertiger Kationen auf die Photoreduktion bei verschiedenen Photolyasen wurde ebenfalls untersucht. Die Photoreduktion von PhrB wurde durch Mg2+ negativ beeinflusst, während bei PhrA Mg2+ einen stimulierenden Effekt hatte. Es stellte sich klar heraus, dass die Abhängigkeit von Mg2+ bei der DNA-Reparatur der (6-4) BCP und nicht bei der Photoreduktion zu suchen ist. Die veranlasste uns zu der Annahme, dass Mg2+ die DNA-Bindung und -Reparatur in (6-4) BCP-Proteinen beeinflusst. Zusammen mit den Strukturdaten und der Sequenzanalyse fanden wir eine vorgeschlagene Mg2+ Bindungsposition neben der DNA-Läsion und modifizierten die beiden betreffenden Aminosäuren durch ortsgerichtete Mutagenese. Der Effekt von Mg2+ ging für beide Mutanten verloren, während die Basisreparaturaktivität ohne Mg2+ von der Mutation nicht beeinflusst wurde. Vermutlich fördert Mg2+ die Bindung der (6-4) Läsion und erhöht die Elektronen-Affinität des Substrats. Außerdem wird die Barriere für Elektronentransfer verringert, wodurch und die Reaktion reibungsloser verläuft. Ich untersuchte auch die Reparatureffizienz für verschiedene Längen einzelsträngiger und doppelsträngiger DNA. Je länger die DNA war, desto schneller war die Reparatur. Bei einer Länge der DNA von 12 nt oder mehr änderte sich allerdings mit Zunahme der Länge die Geschwindigkeit der Reparatur nicht mehr. In dieser Arbeit werden auch die Mutanten PhrB-Y424F und PhrB-I51W vorgestellt. Tyr424 von PhrB ist Teil der DNA-Bindungsstelle und könnte eine Elektronenverbindung zum Fe-S-Cluster bilden. Die PhrB-Y424F-Mutante zeigte eine starke Verringerung der Bindung von Läsions-DNA und DNA-Reparatur. Die Mutante PhrB-I51W ist durch den Verlust des DMRL-Chromophors, reduzierte Photoreduktion und reduzierte DNA-Reparaturkapazität gekennzeichnet. Die Kristallstrukturen zeigen eine hohe Übereinstimmung mit der Wildtyp-Struktur, somit beeinflussen Mutationen nur lokale Proteinumgebungen. Die Photoreduktion von PhrB sich unterscheidet von dem typischen Muster, da die dem FAD benachbarte Aminosäure der Elektronenkaskade ein Tyrosin (Tyr391) ist, während Photolyasen und Cryptochrome anderer Gruppen ein Tryptophan als direkten Elektronendonor von FAD besitzen. In einer Mutante, in der Tyr391 durch Tryptophan ersetzt wurde, ging der Cofaktor-FAD verloren und die PhrB-Struktur war instabil. Trp342 und Trp390 sind für den Ladungstransfer essentiell sind. Trp342 befindet sich an der Peripherie von PhrB. Die Rolle von Tyr391, die zwischen Trp390 und FAD liegt, war jedoch unklar, da der Ersatz durch Phenylalanin die Photoreduktion nicht blockierte. Bei der Substitution von Tyr391 durch Ala wurde die Photoreduktion blockiert, was darauf hindeutet, dass Tyr391 ein Teil der Elektronentransferkette ist und zeigt, dass der Ladungstransfer über die Triade 342-391-390 erfolgt. Diese Ergebnisse deuten auf ein tunneling (Elektronentransfer ohne Ladungsänderung) zwischen Trp390 und FAD hin

Characterization of transcriptome dynamics during watermelon fruit development: sequencing, assembly, annotation and gene expression profiles

<p>Abstract</p> <p>Background</p> <p>Cultivated watermelon [<it>Citrullus lanatus </it>(Thunb.) Matsum. & Nakai var. <it>lanatus</it>] is an important agriculture crop world-wide. The fruit of watermelon undergoes distinct stages of development with dramatic changes in its size, color, sweetness, texture and aroma. In order to better understand the genetic and molecular basis of these changes and significantly expand the watermelon transcript catalog, we have selected four critical stages of watermelon fruit development and used Roche/454 next-generation sequencing technology to generate a large expressed sequence tag (EST) dataset and a comprehensive transcriptome profile for watermelon fruit flesh tissues.</p> <p>Results</p> <p>We performed half Roche/454 GS-FLX run for each of the four watermelon fruit developmental stages (immature white, white-pink flesh, red flesh and over-ripe) and obtained 577,023 high quality ESTs with an average length of 302.8 bp. <it>De novo </it>assembly of these ESTs together with 11,786 watermelon ESTs collected from GenBank produced 75,068 unigenes with a total length of approximately 31.8 Mb. Overall 54.9% of the unigenes showed significant similarities to known sequences in GenBank non-redundant (nr) protein database and around two-thirds of them matched proteins of cucumber, the most closely-related species with a sequenced genome. The unigenes were further assigned with gene ontology (GO) terms and mapped to biochemical pathways. More than 5,000 SSRs were identified from the EST collection. Furthermore we carried out digital gene expression analysis of these ESTs and identified 3,023 genes that were differentially expressed during watermelon fruit development and ripening, which provided novel insights into watermelon fruit biology and a comprehensive resource of candidate genes for future functional analysis. We then generated profiles of several interesting metabolites that are important to fruit quality including pigmentation and sweetness. Integrative analysis of metabolite and digital gene expression profiles helped elucidating molecular mechanisms governing these important quality-related traits during watermelon fruit development.</p> <p>Conclusion</p> <p>We have generated a large collection of watermelon ESTs, which represents a significant expansion of the current transcript catalog of watermelon and a valuable resource for future studies on the genomics of watermelon and other closely-related species. Digital expression analysis of this EST collection allowed us to identify a large set of genes that were differentially expressed during watermelon fruit development and ripening, which provide a rich source of candidates for future functional analysis and represent a valuable increase in our knowledge base of watermelon fruit biology.</p

HIV Antigen Incorporation within Adenovirus Hexon Hypervariable 2 for a Novel HIV Vaccine Approach

Adenoviral (Ad) vectors have been used for a variety of vaccine applications including cancer and infectious diseases. Traditionally, Ad-based vaccines are designed to express antigens through transgene expression of a given antigen. However, in some cases these conventional Ad-based vaccines have had sub-optimal clinical results. These sub-optimal results are attributed in part to pre-existing Ad serotype 5 (Ad5) immunity. In order to circumvent the need for antigen expression via transgene incorporation, the “antigen capsid-incorporation” strategy has been developed and used for Ad-based vaccine development in the context of a few diseases. This strategy embodies the incorporation of antigenic peptides within the capsid structure of viral vectors. The major capsid protein hexon has been utilized for these capsid incorporation strategies due to hexon's natural role in the generation of anti-Ad immune response and its numerical representation within the Ad virion. Using this strategy, we have developed the means to incorporate heterologous peptide epitopes specifically within the major surface-exposed domains of the Ad capsid protein hexon. Our study herein focuses on generation of multivalent vaccine vectors presenting HIV antigens within the Ad capsid protein hexon, as well as expressing an HIV antigen as a transgene. These novel vectors utilize HVR2 as an incorporation site for a twenty-four amino acid region of the HIV membrane proximal ectodomain region (MPER), derived from HIV glycoprotein gp41 (gp41). Our study herein illustrates that our multivalent anti-HIV vectors elicit a cellular anti-HIV response. Furthermore, vaccinations with these vectors, which present HIV antigens at HVR2, elicit a HIV epitope-specific humoral immune response

Adenovirus Gene Transfer to Amelogenesis Imperfecta Ameloblast-Like Cells

To explore gene therapy strategies for amelogenesis imperfecta (AI), a human ameloblast-like cell population was established from third molars of an AI-affected patient. These cells were characterized by expression of cytokeratin 14, major enamel proteins and alkaline phosphatase staining. Suboptimal transduction of the ameloblast-like cells by an adenovirus type 5 (Ad5) vector was consistent with lower levels of the coxsackie-and-adenovirus receptor (CAR) on those cells relative to CAR-positive A549 cells. To overcome CAR -deficiency, we evaluated capsid-modified Ad5 vectors with various genetic capsid modifications including “pK7” and/or “RGD” motif-containing short peptides incorporated in the capsid protein fiber as well as fiber chimera with the Ad serotype 3 (Ad3) fiber “knob” domain. All fiber modifications provided an augmented transduction of AI-ameloblasts, revealed following vector dose normalization in A549 cells with a superior effect (up to 404-fold) of pK7/RGD double modification. This robust infectivity enhancement occurred through vector binding to both αvβ3/αvβ5 integrins and heparan sulfate proteoglycans (HSPGs) highly expressed by AI-ameloblasts as revealed by gene transfer blocking experiments. This work thus not only pioneers establishment of human AI ameloblast-like cell population as a model for in vitro studies but also reveals an optimal infectivity-enhancement strategy for a potential Ad5 vector-mediated gene therapy for AI

Stability Research of an SEIR Model with Distinct General Contact Rates and Infectious Force in Latent and Recovered Period

In this work, an SEIR infectious model with distinct general contact rates and infectious force in latent and recovered period is established, and the stability of the model is studied using theoretical and numerical methods. First, we derive the basic reproduction number R0, which determines whether the disease is extinct or not. Second, using the LaSalle’s invariance principle, we show that the disease-free equilibrium is globally asymptotically stable and the disease always dies out when R01. Third, through the method of autonomous convergence theorem, we obtain that the unique endemic equilibrium is globally asymptotically stable and the disease persists at this endemic equilibrium when R0>1. Finally, numerical simulations are carried out to confirm the theoretical analysis

The Design of AC Regulated Power Supply Based on PWM Chopping Series Compensation Technology

Abstract: Area for the fast load variation, the power grid voltage fluctuations is very serious. In order to supply stable voltage for the small power user, a unipolar chopping compensation AC regulated power supply is designed based on bidirectional H PWM chopping bridge which can realize the bidirectional flow of energy when the voltage is boosted and step-down. It adopts voltage feedback control. The initial chopping duty ratio is determined by proportion regulation according to difference of the input voltage and the standard voltage; it can improve the voltage response speed. The initial duty ratio is corrected according to digital PID regulation based on the output voltage feedback, which ensures the stability of the output voltage. The STC12C5A62AD single chip is adopted for the regulated power supply that have 8 AD inputs and PWM output, it make the system structure simple. The 12.8 kHz sampling frequency and high precision voltage sampling technology ensures the precision of sampling. The 10 kHz frequency of the chopper reduces the harmonic component and it is easy to filter. Experimental results show that dynamic response speed of the regulated power supply is fast and precision of it is high. It has great practical value

Boundary Value Problems of Nonlinear Mixed-Type Fractional Differential Equations

In this paper, by means of a fixed point theorem for monotone decreasing operators on a cone, we discuss the existence of positive solutions for boundary value problems of nonlinear fractional singular differential equation. The proof of the main result is based on Gatica–Oliker–Waltman fixed-point theorem. At last, an example is given to illustrate our main conclusion

Effect of interface structure regulation caused by variation of imidization rate on conduction current characteristics of PI/nano-Al2O3 three-layer composite films

A series of sandwich structure PI films were prepared by different imidization process, with pure PI film as the interlayer and PI/Al2O3 composite films as outer layers. The imidization rate of the film with different cured processes was calculated by characterizing by infrared spectrum (FT-IR), and the morphology of interlayer interface with different imidization rates by scanning electron microscope (SEM). When the imidization conditions of the first and second films were 260 °C/120 min, the composite films displayed better interface structure and higher imidization rate (ID) than others. Moreover, results also showed that the conduction current of three-layer composite film steadily improved with increased ID and temperature, and was higher than that of the pure film. At the temperature of 30 °C, the electrical aging threshold at different ID was obtained. When the ID reached the maximum value of 78.9%, the electrical aging threshold reached the maximum 41.69 kV/mm. Keywords: Polyimides, Composite films, Interfaces, Conduction current, Electrical degradation threshol

Analysis on Composition and Content of Glucosinolates in Broccoli Flowers and Leaves

Glucosinolate composition and content were evaluated in flowers and leaves of 12 different broccoli varieties. The results indicated that there were 9 glucosinolates in broccoli, namely Glucoiberin (IBE); Progoitrin (PRO); Sinigrin (SIN); Glucoraphanin (RAA); Gluconapin (NAP); 4-Hydroxyglucobrassicin (4OH); Glucobrassicin (GBC); 4-Methoxyglucobrassicin (4ME); Neoglucobrassicin (NEO). Total glucosinolate content in flowers was 1-5 times higher than in leaves. The predominant glucosinolate in broccoli was glucoraphanin