New helicase-primase inhibitors as drug candidates for the treatment of herpes simplex disease

The vast majority of the world population is infected with at least one member of the human herpesvirus family. Herpes simplex virus (HSV) infections are the cause of cold sores and genital herpes as well as life-threatening or sight-impairing disease mainly in immunocompromized patients, pregnant women and newborns. Since the milestone development in the late 1970s of acyclovir (Zovirax), a nucleosidic inhibitor of the herpes DNA polymerase, no new non-nucleosidic anti-herpes drugs have been introduced. Here we report new inhibitors of the HSV helicase-primase with potent in vitro anti-herpes activity, a novel mechanism of action, a low resistance rate and superior efficacy against HSV in animal models. BAY 57-1293 (N-[5-(aminosulfonyl)-4-methyl-1,3-thiazol-2-yl]-N-methyl-2-[4-(2-pyridinyl)phenyl]acetamide), a well-tolerated member of this class of compounds, significantly reduces time to healing, prevents rebound of disease after cessation of treatment and, most importantly, reduces frequency and severity of recurrent disease. Thus, this class of drugs has significant potential for the treatment of HSV disease in humans, including those resistant to current medications.     

Towards Long Term Cultivation of Drosophila Wing Imaginal Discs In Vitro

The wing imaginal disc of Drosophila melanogaster is a prominent experimental system for research on control of cell growth, proliferation and death, as well as on pattern formation and morphogenesis during organogenesis. The precise genetic methodology applicable in this system has facilitated conceptual advances of fundamental importance for developmental biology. Experimental accessibility and versatility would gain further if long term development of wing imaginal discs could be studied also in vitro. For example, culture systems would allow live imaging with maximal temporal and spatial resolution. However, as clearly demonstrated here, standard culture methods result in a rapid cell proliferation arrest within hours of cultivation of dissected wing imaginal discs. Analysis with established markers for cells in S- and M phase, as well as with RGB cell cycle tracker, a novel reporter transgene, revealed that in vitro cultivation interferes with cell cycle progression throughout interphase and not just exclusively during G1. Moreover, quantification of EGFP expression from an inducible transgene revealed rapid adverse effects of disc culture on basic cellular functions beyond cell cycle progression. Disc transplantation experiments confirmed that these detrimental consequences do not reflect fatal damage of imaginal discs during isolation, arguing clearly for a medium insufficiency. Alternative culture media were evaluated, including hemolymph, which surrounds imaginal discs during growth in situ. But isolated larval hemolymph was found to be even less adequate than current culture media, presumably as a result of conversion processes during hemolymph isolation or disc culture. The significance of prominent growth-regulating pathways during disc culture was analyzed, as well as effects of insulin and disc co-culture with larval tissues as potential sources of endocrine factors. Based on our analyses, we developed a culture protocol that prolongs cell proliferation in cultured discs.     

Heterochrony in Silurian phacopid trilobites as suggested by the ontogeny of Acernaspis

New material of early growth stages of the Silurian (Llandovery) trilobite Acernaspis is described. Pre-adult ontogenetic stages of this genus closely resemble adults of the post-Llandovery genus ***Ananaspis. A heterochronic descent of Ananaspis from Acernaspis is proposed. ***Ananaspis is interpreted as pae-domorphic, having arisen largely through neoteny. Neotenic changes already appear in the lineage in the last Acernaspis species, and Ananaspis then underwent continuous ncotcnic change throughout its known Silurian history. ▭ Heterochrony, neoteny, Silurian, Trilobita, Phacopidae, Acernaspis. Ananaspis.     

Validity and reliability of agility tests in junior Australian football players

Young, W, Farrow, D, Pyne, D, McGregor, W, and Handke, T. Validity and reliability of agility tests in junior Australian football players. J Strength Cond Res 25(12): 3399-3403, 2011-The importance of sport-specific stimuli in reactive agility tests (RATs) compared to other agility tests is not known. The purpose of this research was to determine the validity and reliability of agility tests. Fifty junior Australian football players aged 15-17 years, members of either an elite junior squad (n = 35) or a secondary school team (n = 15), were assessed on a new RAT that involved a change of direction sprint in response to the movements of an attacking player projected in life size on a screen. These players also underwent the planned Australian Football League agility test, and a subgroup (n = 13) underwent a test requiring a change of direction in response to a left or right arrow stimulus. The elite players were significantly better than the school group players on the RAT (2.81 ± 0.08 seconds, 3.07 ± 0.12 seconds; difference 8.5%) but not in the arrow stimulus test or planned agility test. The data were log transformed and the reliability of the new RAT estimated using typical error (TE) expressed as a coefficient of variation. The TE for the RAT was 2.7% (2.0-4.3, 90% confidence interval) or 0.07 seconds (0.5-1.0), with an intraclass correlation coefficient (ICC) of 0.33. For the test using the arrow stimulus, the TE was 3.4% (2.4-6.2), 0.09 (0.06-0.15) seconds, and ICC was 0.10. The sport-specific stimulus provided by the new RAT is a crucial component of an agility test; however, adoption of the new RAT for routine testing is likely to require more accessible equipment and several familiarization trials to improve its reliability.     

西双版纳尚勇自然保护区野生印支虎及其三种主要有蹄类猎物种群现状调查

中国野生印支虎及其猎物种群状况的野外实地研究一直处于空白。本研究使用足迹鉴别法、粪堆计数法,首次对西双版纳尚勇自然保护区野生印支虎种群数量现状及该区域内的虎猎物种群状况进行了调查研究。结果显示:2004 ～ 2009 年间,确认西双版纳保护区存在3 只成年印支虎个体(2 雌1 雄),西双版纳尚勇保护区拥有比较丰富的有蹄类种群,其中虎的主要猎物:水鹿平均密度为7.63 (7.40 ～ 9.23)只/ km² ;赤麂平均密度为17. 39 (11.33 ～24.94)只/ km² ,野猪平均密度为10.26 (7.69 ～ 14.51) 只/ km² ,该区域虎猎物生物量为1 715. 74 kg/ km² 。本研究还探讨了该区域印支虎种群的保护前景以及中国境内开展虎种群调查的适用办法等。     

Amino acid balancing for the prediction and evaluation of protein concentrations in cell-free protein synthesis systems

Cell-free protein synthesis (CFPS) systems are an attractive method to complement the usual cell-based synthesis of proteins, especially for screening approaches. The literature describes a wide variety of CFPS systems, but their performance is difficult to compare since the reaction components are often used at different concentrations. Therefore, we have developed a calculation tool based on amino acid balancing to evaluate the performance of CFPS by determining the fractional yield as the ratio between theoretically achievable and experimentally achieved protein molar concentration. This tool was applied to a series of experiments from our lab and to various systems described in the literature to identify systems that synthesize proteins very efficiently and those that still have potential for higher yields. The well-established Escherichia coli system showed a high efficiency in the utilization of amino acids, but interestingly, less considered systems, such as those based on Vibrio natriegens or Leishmania tarentolae, also showed exceptional fractional yields of over 70% and 90%, respectively, implying very efficient conversions of amino acids. The methods and tools described here can quickly identify when a system has reached its maximum or has limitations. We believe that this approach will facilitate the evaluation and optimization of existing CFPS systems and provides the basis for the systematic development of new CFPS systems.     

Genetic characterization of the complete genome of a highly divergent simian T-lymphotropic virus (STLV) type 3 from a wild Cercopithecus mona monkey

Background

RNA interference is a gene regulatory mechanism that employs small RNA molecules such as microRNA. Previous work has shown that HIV-1 produces TAR viral microRNA. Here we describe the effects of the HIV-1 TAR derived microRNA on cellular gene expression.

Results

Using a variation of standard techniques we have cloned and sequenced both the 5' and 3' arms of the TAR miRNA. We show that expression of the TAR microRNA protects infected cells from apoptosis and acts by down-regulating cellular genes involved in apoptosis. Specifically, the microRNA down-regulates ERCC1 and IER3, protecting the cell from apoptosis. Comparison to our cloned sequence reveals possible target sites for the TAR miRNA as well.

Conclusion

The TAR microRNA is expressed in all stages of the viral life cycle, can be detected in latently infected cells, and represents a mechanism wherein the virus extends the life of the infected cell for the purpose of increasing viral replication.     

A DNA vaccine against tuberculosis based on the 65 kDa heat-shock protein differentially activates human macrophages and dendritic cells

Background

Application of plasmid DNA for immunization of food-producing animals established new standards of food safety. The addition of foreign products e.g. pDNA into the food chain should be carefully examined to ensure that neither livestock animals nor consumers develop unpredicted or undesirable side-effects.

Methods

A quantitative real-time PCR (QRTPCR) methodology was developed to study the biodistribution and persistence of plasmid DNA vaccine pDNAX (pVAX-Hsp60 TM814) in mice and beef cattle. The linear quantification range and the sensitivity of the method was found to be 10 – 10⁹ copies per reaction (500 ng/gDNA) and 3 copies per reaction, respectively.

Results

Persistence of pDNAX in mice muscle tissue was restricted to injection site and the amount of pDNAX showed delivery formulation dependent (naked pDNA, electroporation, cationic liposome complexes) and mouse age-dependent clearance form injection site but pDNAX was still detectable even after 365 days. The QRTPCR analysis of various muscle tissue samples of vaccinated beef bulls performed 242–292 days after the last revaccination proved that residual pDNAX was found only in the injection site. The highest plasmid levels (up to 290 copies per reaction) were detected in the pDNAX:CDAN/DOPE group similarly to mice model. No pDNA was detected in the samples from distant muscles and draining lymph nodes.

Conclusion

Quantitative real-time PCR (QRTPCR) assay was developed to assess the residual pDNA vaccine pVAX-Hsp60 TM814 in mice and beef cattle. In beef cattle, ultra low residual level of pDNA vaccine was only found at the injection site. According to rough estimation, consumption of muscles from the injection site represents almost an undetectable intake of pDNA (400 fg/g muscle tissue) for consumers. Residual plasmid in native state will hardly be found at measurable level following further meat processing. This study brings supportive data for animal and food safety and hence for further approval of pDNA vaccine field trials.     

Serological detection of Plasmodium vivax malaria using recombinant proteins corresponding to the 19-kDa C-terminal region of the merozoite surface protein-1

BACKGROUND: Serological tests to detect antibodies specific to Plasmodium vivax could be a valuable tool for epidemiological studies, for screening blood donors in areas where the malaria is not endemic and for diagnosis of infected individuals. Because P. vivax cannot be easily obtained in vitro, ELISA assays using total or semi-purified antigens are rarely used. Based on this limitation, we tested whether recombinant proteins representing the 19 kDa C-terminal region of the merozoite surface protein-1 of P. vivax (MSP119) could be useful for serological detection of malaria infection. METHODS: Three purified recombinant proteins produced in Escherichia coli (GST-MSP119, His6-MSP119 and His6-MSP119-PADRE) and one in Pichia pastoris (yMSP119-PADRE) were compared for their ability to bind to IgG antibodies of individuals with patent P. vivax infection. The method was tested with 200 serum samples collected from individuals living in the north of Brazil in areas endemic for malaria, 53 serum samples from individuals exposed to Plasmodium falciparum infection and 177 serum samples from individuals never exposed to malaria. RESULTS: Overall, the sensitivity of the ELISA assessed with sera from naturally infected individuals was 95%. The proportion of serum samples that reacted with recombinant proteins GST-MSP119, His6-MSP119, His6-MSP119-PADRE and yMSP119-PADRE was 90%, 93.5%, 93.5% and 93.5%, respectively. The specificity values of the ELISA determined with sera from healthy individuals and from individuals with other infectious diseases were 98.3% (GST-MSP119), 97.7% (His6-MSP119 and His6-MSP119-PADRE) or 100% (yMSP119-PADRE). CONCLUSIONS: Our study demonstrated that for the Brazilian population, an ELISA using a recombinant protein of the MSP119 can be used as the basis for the development of a valuable serological assay for the detection of P. vivax malaria.