Insertional mutagenesis and marker rescue in a protozoan parasite: cloning of the uracil phosphoribosyltransferase locus from Toxoplasma gondii.

Nonhomologous integration vectors have been used to demonstrate the feasibility of insertional mutagenesis in haploid tachyzoites of the protozoan parasite Toxoplasma gondii. Mutant clones resistant to 5-fluorouracil were identified at a frequency of approximately 10(-6) (approximately 2 x 10(-5) of the stable transformants). Four independent mutants were isolated, all of which were shown to lack uracil phosphoribosyl-transferase (UPRT) activity and harbor transgenes integrated at closely linked loci, suggesting inactivation of the UPRT-encoding gene. Genomic DNA flanking the insertion point (along with the integrated vector) was readily recovered by bacterial transformation with restriction-digested, self-ligated total genomic DNA. Screening of genomic libraries with the recovered fragment identified sequences exhibiting high homology to known UPRT-encoding genes from other species, and cDNA clones were isolated that contain a single open reading frame predicted to encode the 244-amino acid enzyme. Homologous recombination vectors were exploited to create genetic knock-outs at the UPRT locus, which are deficient in enzyme activity but can be complemented by transient transformation with wild-type sequences--formally confirming identification of the functional UPRT gene. Mapping of transgene insertion points indicates that multiple independent mutants arose from integration at distinct sites within the UPRT gene, suggesting that nonhomologous integration is sufficiently random to permit tagging of the entire parasite genome in a single transformation.

Bridging grafts and transient nerve growth factor infusions promote long-term central nervous system neuronal rescue and partial functional recovery.

Grafts of favorable axonal growth substrates were combined with transient nerve growth factor (NGF) infusions to promote morphological and functional recovery in the adult rat brain after lesions of the septohippocampal projection. Long-term septal cholinergic neuronal rescue and partial hippocampal reinnervation were achieved, resulting in partial functional recovery on a simple task assessing habituation but not on a more complex task assessing spatial reference memory. Control animals that received transient NGF infusions without axonal-growth-promoting grafts lacked behavioral recovery but also showed long-term septal neuronal rescue. These findings indicate that (i) partial recovery from central nervous system injury can be induced by both preventing host neuronal loss and promoting host axonal regrowth and (ii) long-term neuronal loss can be prevented with transient NGF infusions.

Rescue, propagation, and partial purification of a helper virus-dependent adenovirus vector.

Adenoviral vectors are widely used as highly efficient gene transfer vehicles in a variety of biological research strategies including human gene therapy. One of the limitations of the currently available adenoviral vector system is the presence of the majority of the viral genome in the vector, resulting in leaky expression of viral genes particularly at high multiplicity of infection and limited cloning capacity of exogenous sequences. As a first step to overcome this problem, we attempted to rescue a defective human adenovirus serotype 5 DNA, which had an essential region of the viral genome (L1, L2, VAI + II, pTP) deleted and replaced with an indicator gene. In the presence of wild-type adenovirus as a helper, this DNA was packaged and propagated as transducing viral particles. After several rounds of amplification, the titer of the recombinant virus reached at least 4 x 10(6) transducing particles per ml. The recombinant virus could be partially purified from the helper virus by CsCl equilibrium density-gradient centrifugation. The structure of the recombinant virus around the marker gene remained intact after serial propagation, while the pBR sequence inserted in the E1 region was deleted from the recombinant virus. Our results suggest that it should be possible to develop a helper-dependent adenoviral vector, which does not encode any viral proteins, as an alternative to the currently available adenoviral vector systems.

Partial rescue of retinal function in chronically hypoglycemic mice

Purpose. Mice rendered hypoglycemic by a null mutation in the glucagon receptor gene Gcgr display late-onset retinal degeneration and loss of retinal sensitivity. Acute hyperglycemia induced by dextrose ingestion does not restore their retinal function, which is consistent with irreversible loss of vision. The goal of this study was to establish whether long-term administration of high dietary glucose rescues retinal function and circuit connectivity in aged Gcgr−/− mice. Methods. Gcgr−/− mice were administered a carbohydrate-rich diet starting at 12 months of age. After 1 month of treatment, retinal function and structure were evaluated using electroretinographic (ERG) recordings and immunohistochemistry. Results. Treatment with a carbohydrate-rich diet raised blood glucose levels and improved retinal function in Gcgr−/− mice. Blood glucose increased from moderate hypoglycemia to euglycemic levels, whereas ERG b-wave sensitivity improved approximately 10-fold. Because the b-wave reflects the electrical activity of second-order cells, we examined for changes in rod-to-bipolar cell synapses. Gcgr−/− retinas have 20% fewer synaptic pairings than Gcgr+/− retinas. Remarkably, most of the lost synapses were located farthest from the bipolar cell body, near the distal boundary of the outer plexiform layer (OPL), suggesting that apical synapses are most vulnerable to chronic hypoglycemia. Although treatment with the carbohydrate-rich diet restored retinal function, it did not restore these synaptic contacts. Conclusions. Prolonged exposure to diet-induced euglycemia improves retinal function but does not reestablish synaptic contacts lost by chronic hypoglycemia. These results suggest that retinal neurons have a homeostatic mechanism that integrates energetic status over prolonged periods of time and allows them to recover functionality despite synaptic loss.

Natural Compounds from Saffron and Bear Bile Prevent Vision Loss and Retinal Degeneration

All retinal disorders, regardless of their aetiology, involve the activation of oxidative stress and apoptosis pathways. The administration of neuroprotective factors is crucial in all phases of the pathology, even when vision has been completely lost. The retina is one of the most susceptible tissues to reactive oxygen species damage. On the other hand, proper development and functioning of the retina requires a precise balance between the processes of proliferation, differentiation and programmed cell death. The life-or-death decision seems to be the result of a complex balance between pro- and anti-apoptotic signals. It has been recently shown the efficacy of natural products to slow retinal degenerative process through different pathways. In this review, we assess the neuroprotective effect of two compounds used in the ancient pharmacopoeia. On one hand, it has been demonstrated that administration of the saffron constituent safranal to P23H rats, an animal model of retinitis pigmentosa, preserves photoreceptor morphology and number, the capillary network and the visual response. On the other hand, it has been shown that systemic administration of tauroursodeoxycholic acid (TUDCA), the major component of bear bile, to P23H rats preserves cone and rod structure and function, together with their contact with postsynaptic neurons. The neuroprotective effects of safranal and TUDCA make these compounds potentially useful for therapeutic applications in retinal degenerative diseases.

Quarter bills for Samuel Stearns signed by Steward Caleb Gannett, 1793-1794

Six printed quarter bills for Samuel Stearns (Harvard AB 1794) with Steward and Commons, Sizings, Study and Cellar Rent, Instruction, Library, French Instruction, Medical Instruction, Books, Sweepers, Catalogues and Commencement Dinner, Repairs, Wood, and Punishment totals. Bills signed by Steward Caleb Gannett.

[Letter from Samuel Stearns to President Kirkland], 1813 December 7

Letter to President Kirkland regarding a student named Hartshorn.

Letter from Charles Stearns to Edward Wigglesworth, 1781 November 8, and receipt signed by Stephen Sewall, 1781 November 16

In a letter to Wigglesworth, acting president of Harvard and Hollis Professor of Divinity, Stearns resigns his tutorship and writes that he will return an unnamed silver tankard and various books to the College. The folder also includes as a receipt for those objects upon their return, signed by Harvard Professor Stephen Sewall.

Norway and the Bear. Norwegian defence policy- lessons for the Baltic Sea region. OSW Point of View Number 38, January 2014

Norway is currently the only Western European state and ‘old’ NATO member that strongly relies on the traditional dimension of NATO's collective defence. It is also the only ally in Western Europe which perceives Russia as a threat to its military security, in the so-called High North. In order to successfully deal with the potential challenges and threats in the region, Norway has been pursuing a defence policy based on cooperation and deterrence. Cooperation means improving collaboration with Russia in cross-border relations, in the petroleum sector and in the military sphere. The deterrent measures include maintaining NATO’s credibility as a collective defence alliance; increasing military cooperation with the United States; building up Norway’s own military capabilities; and developing military cooperation across Northern Europe. The primary objective of Oslo’s defence policy is to minimise the likelihood of crises and conflicts emerging in the High North which could prove too ‘big’ for Norway but too ‘small’ for NATO.

The bear watches the dragon. The Russian debate on China. OSW Point of View Number 31, February 2013

Most participants in the Russian public debate seem to agree in their evaluation of the present condition of Russian-Chinese relations. There is awareness of increasing inequality between these two powers and Russia’s weakening position as compared to China.Those who share the optimistic view see co-operation with China as an opportunity for the Russian economy and a key element of Russia’s multi-directional foreign policy, an opportunity for Russia to avoid unilateral dependence on the West. The pessimists view the deepening co-operation with China through the prism of threats resulting from the increasing imbalance in bilateral relations. The greatest source of concern is the model of economic relations, which is often referred to as neo-colonial, where Russia’s role is reduced to that of a supplier of raw materials to China. The possible consequences are evaluated in different ways, ranging from the political subordination of Russian interests the Chinese ones to real loss of control over the Russian Far East. Those who share such views believe that Moscow should slow down its rapprochement with China and search for other partners in Eastern Asia, relations with whom could counterbalance the Chinese influence.

The Economics of State Aid for the Rescue and Restructuring of Firms in Difficulty: Theoretical Considerations, Empirical Analysis and Proposals for Reform. Bruges European Economic Research (BEER) Papers 27/September 2013

State aid for rescue and restructuring (R&R) of companies in difficulty causes a significant distortion of competition. It prevents the market from eliminating inefficient companies. Because of this, the European Commission has to be specially strict when it assesses rescue or restructuring aid. This paper examines recent cases of corporate restructuring partly funded with public money. It explains the main aspects of the current guidelines which are applicable to R&R State Aid and establishes a theoretical framework for the economic assessment of R&R aid. It then analyses decisions adopted by the European Commission concerning R&R state aid during the period 2000-2013. It finds that there is little economic rationale in the granting of R&R aid. The paper concludes by applying the lessons drawn from the empirical analysis to the anticipated revision of the R&R guidelines in the context of the State Aid Modernisation process.

Is a Concordat over Ukraine now conceivable with Mishka the bear? CEPS Commentary, 21 December 2014

With the tumultuous year of ever-changing episodes in Ukraine coming to a close – from Yanukovich reneging at Vilnius last November, to the new Maidan, to Yanukovich fleeing for his life, to Russia’s annexation of Crimea and incursions into the eastern Donbass, the election of pro-European President Poroshenko, the war with over 4,000 dead, the election of a new pro-European parliament and now the crash of the rouble – Michael Emerson sees at last a possible the end-game in sight. In this commentary, he sketches the essential elements of a Concordat to be struck between Russia, Ukraine and the West that would allow the eastern Donbass to be drawn into more normal processes of political and economic negotiation and the badly wounded Russia to gradually return to more normal international relations.