Laboratory Models of Infection and Transmission of Mycobacterium ulcerans; causative agent of Buruli ulcer disease

Identification of the environmental reservoir of Mycobacterium ulcerans, the etiological agent of Buruli ulcer, within the aquatic ecosystem has been a salient research area within the last five years. Based on extensive environmental sampling and elegant laboratory models, associations have been made between the bacterial DNA and aquatic invertebrates, biofilms, plants, fish and detritus material captured on 0.2μm pore filters. These studies have suggested that M. ulcerans is widely distributed within many functional feeding groups and may be concentrated through different trophic links; however, the specific route of transmission to humans remains a mystery. In this study we have used laboratory models of infection to ascertain the role of aquatic invertebrates and fish in M. ulcerans transmission. A biologically relevant infection model in which M. ulcerans-infected mosquito larvae were fed to a species of predaceous hemiptera (African Belostomatidae) was used to demonstrate the persistent colonization of M. ulcerans and subsequent transmission of bacteria to naïve prey. The association of M. ulcerans with specific anatomical compartments showed that M. ulcerans accumulates preferentially on the exoskeleton. No difference was found between the ability of wild-type M. ulcerans and an M. ulcerans isogenic mycolactone-negative mutant to colonize belostomatids. These data show that African belostomatids can successfully be colonized by M. ulcerans and support the trophic transfer of M. ulcerans within the environment. We have shown that M. ulcerans with or without the toxin is not lethal to fish (Medaka) even at high doses following direct inoculation. Over time (23wks), infected Medaka do not exhibit any visible signs of infection or toxicity and histopathological sections do not reveal significant gross pathogenesis. M. ulcerans also appears not to replicate in infected Medaka. We also show that fish monocytes are susceptible to nanogram amounts of purified mycolactone. This is the first study to demonstrate the possibility of fish as a reservoir for M. ulcerans within the aquatic environment

The Role of Employment Protection During An Exogenous Shock To An Economy

This paper explores the role of employment protection when powerful external crises reduce demand for products. We first present a theoretical framework that shows that employment protection has a U-shaped effect on abnormal unemployment during a negative exogenous shock to an economy. Using data from the 33 OECD countries, we analyze how the level of employment protection affected the stability of unemployment rates during the recent global economic crisis. The results suggest that countries with an intermediate level of employment protection will have more stable unemployment rates during a world crisis. The policy implication of our paper is that countries should seek a medium level of employment protection that may act as an automatic stabilizer of the economy on the macro level.

The voluntary intake and digestibility of combinations of cereal crop residues and legume hay for sheep

Examines the effects of supplementing diets based on common Ethiopian crop residues (teff, wheat and oat straws and maize stover) with different proportions of hay made from a common local clover, Triofolium tembense

A re-examination of value-creation through strategic alliances

This paper uses a sample of 335 firms participating in strategic alliances in order to re-examine the value creation through strategic alliances. We show that the immediate positive response of stock markets to new strategic alliances is followed by negative abnormal returns. Twenty days after announcements, cumulative positive abnormal return is only evident for the firms with the highest stock market’s response to the announcement. We relate the positive abnormal returns reported in previous research to the presence of short-run over-reaction in stock markets and conclude in the market’s ability to identify the more valuable alliances.Strategic alliance; over-reaction; momentum

Optimal policy for FDI incentives : an auction theory approach

A multinational corporation’s (MNC) entry into a host country brings benefits to the economy of that country, some direct (such as increasing production and employment) and some indirect (such as productivity spin-off). Governments that view MNCs as engines for growth and regional development have begun to encourage the flow of foreign investment into their country in hopes of increased local employment, market production and export capacity. MNCs consider first the maximization of profit when selecting a site to establish their subsidiaries. An MNC examines possible investment sites and indicates those that are best fitted for the investment. The countries that remain at this stage are similar in terms of their economic characteristics, and they compete with each other for receiving the foreign investment. In this paper we use tools from auction theory to analyze the competition between host countries and MNCs and investigate the existence of Nash equilibrium strategies. The characteristics of this equilibrium are considered and assessed. We developed a general model for examining the incentive competition between two countries and then apply it for several subgroups according to the number of MNCs and the availability of information. It turns out that the characteristics of the equilibrium depend on the number of MNCs as well as on the structure of their contribution to the host country economy.peer-reviewe

Trapping and Characterization of the Reaction Intermediate in Cyclodextrin Glycosyltransferase by Use of Activated Substrates and a Mutant Enzyme

Cyclodextrin glycosyltransferases (CGTases) catalyze the degradation of starch into linear or cyclic oligosaccharides via a glycosyl transfer reaction occurring with retention of anomeric configuration. They are also shown to catalyze the coupling of maltooligosaccharyl fluorides. Reaction is thought to proceed via a double-displacement mechanism involving a covalent glycosyl-enzyme intermediate. This intermediate can be trapped by use of 4-deoxymaltotriosyl α-fluoride (4DG3αF). This substrate contains a good leaving group, fluoride, thus facilitating formation of the intermediate, but cannot undergo the transglycosylation step since the nucleophilic hydroxyl group at the 4-position is missing. When 4DG3αF was reacted with wild-type CGTase (Bacillus circulans 251), it was found to be a slow substrate (kcat = 2 s-1) compared with the parent glycosyl fluoride, maltotriosyl R-fluoride (kcat = 275 s-1). Unfortunately, a competing hydrolysis reaction reduces the lifetime of the intermediate precluding its trapping and identification. However, when 4DG3αF was used in the presence of the presumed acid/base catalyst mutant Glu257Gln, the intermediate could be trapped and analyzed because the first step remained fast while the second step was further slowed (kcat = 0.6 s-1). Two glycosylated peptides were identified in a proteolytic digest of the inhibited enzyme by means of neutral loss tandem mass spectrometry. Edman sequencing of these labeled peptides allowed identification of Asp229 as the catalytic nucleophile and provided evidence for a covalent intermediate in CGTase. Asp229 is found to be conserved in all members of the family 13 glycosyl transferases.

Zavajajoče dojemanje in gospodarska učinkovitost regionalnega sistema

Novost, ki jo prinaša ta članek, je uporaba učinka napačnega dojemanja na modelu središče-obrobje. Na podlagi teoretičnega modela bomo pokazali, da na prostem trgu ob uvedbi učinka napačnega dojemanja v model pride do napačne porazdelitve podjetij med središčem in obrobjem. Anketa z vprašalnikom je potrdila, da obstaja napačno dojemanje kakovosti proizvodnih dejavnikov, in sicer v korist središča. To napačno dojemanje lahko najdemo tako v pogledih središča na obrobje kot v pogledih obrobja nase. Za spremembo teh napačnih dojemanj bi se moralo obrobje bolj tržiti, obenem pa tudi krepiti svojo vlogo in svoj položaj v državi

Misleading perceptions and economic efficiency in a regional system

This paper’s innovation is the introduction of a misperception effect to the core-periphery model. Using a theoretical model, we show that the free market will lead to distorted spatial distribution of firms between the core and the periphery when a misperception effect is introduced into the model. Based on a questionnaire, we found that a misperception regarding the quality of the production factor exists in favour of the core compared to the periphery. This misperception is found not only in the eyes of the core with regard to the periphery, but also in the periphery regarding itself. We suggest marketing and empowerment of the periphery as a policy tool to alter these misperceptions

Géographie de la santé à la croisée de la géographie des maladies et de la géographie de soins: Geography of health at the crossroads of disease and care geography

No Abstrac

Probing mechanisms by which cerebral vascular disease may influence cognitive impairment and dementia

Introduction Vascular cognitive impairment (VCI) describes a full spectrum of cognitive deficits caused by underlying cerebral vascular alterations, regardless of the specific mechanisms involved. Several factors such as ageing, stroke, hypertension and cerebral hypoperfusion are associated with an increased risk of developing VCI. Vascular dementia (VaD) is the second most common cause of dementia after Alzheimer’s disease (AD). It is now recognised that considerable overlaps exist between the features of VaD and AD. Key pathological and neuroimaging features including cerebral amyloid angiopathy (CAA), white matter lesions (WML), microinfarcts and microbleeds are evident in both VaD and AD. Furthermore, brain infarction has been reported to influence the presence and severity of clinical expressions such as cognitive performance of AD, suggesting a common pathophysiological mechanism that contributes to the development of cognitive deficits. However, gaps remain in understanding the exact mechanisms by which vascular risk factors contribute to cognitive decline and neurodegenerative processes leading to dementia. Given the importance of blood supply to the brain for maintaining its structural and functional integrity, it has been proposed that vascular risk factors may affect the cerebral haemodynamic and alter the vascular function resulting in damages to the brain. These changes may involve altered neurovascular coupling that is a critical mechanism for regulating the dynamic changes of local cerebral circulation. Further, impaired vascular function, amyloid-β (Aβ) accumulation in the cerebral vasculature and disrupted neurovascular unit are found in VCI. The glymphatic pathway, a clearance route for removing soluble waste from the brain to periphery, has been proposed to play a role in the pathogenesis of VCI. Mounting evidence has suggested that cerebral hypoperfusion, by large vessel occlusion and stenosis, is emerging as a major contributor to cognitive impairment. This has led to the development of mouse models of bilateral common carotid stenosis (BCAS), a model narrowing both common carotid arteries by placing microcoils. The BCAS model has been reported to produce many features of VCI, including white matter damage, microglial activation, gliovascular disruption, increased oxidative stress (by increased NADPH oxidase 2 (NOX2) levels) as well as memory impairment. To investigate whether there is an interaction between cerebral hypoperfusion and microvascular Aβ accumulation, a mixed model that demonstrates both microvascular amyloid (Tg-SwDI mouse model) and BCAS has been developed. In this thesis, it is hypothesized that the complex interaction of Aβ and BCAS leads to cognitive impairment via an impaired glymphatic function in addition to perfusion deficits that promote vascular related lesions and neurodegenerative changes. Second to this, given clear links between NOX2, hypoperfusion and amyloid accumulation, it was further hypothesised that NOX2 is a central mechanism leading to VCI. Methods Mice were given BCAS surgery to mimic cerebral hypoperfusion for a period of 3 months. In vivo laser speckle imaging was performed to evaluate the changes in cortical blood flow. This was followed by additional CBF measurements using arterial spin labelling (ASL)-based magnetic resonance imaging (MRI), which gave a non-invasive access to CBF information in the cerebral cortex, hippocampus and thalamus. Neurovascular coupling was assessed by performing whisker stimulation. Barnes maze was used to assess the spatial learning and memory function at 3 months following BCAS or sham surgery. For the examination of glymphatic function, in vivo intracisternal injection and ex vivo imaging of CSF fluorescent tracers were performed. Histological assessment and immunohistochemistry were used to examine vascular related pathology, Aβ burden, astrogliosis and basement membrane changes following BCAS. Results Part 1: To examine the effect of BCAS on cerebral perfusion deficits, glymphatic function and cognition in Tg-SwDI mice compared to wild-type mice. The first studies in the thesis sought to examine the effect of BCAS and microvascular amyloid on the extent of cerebral perfusion deficits and cognitive impairment. The first step was to validate whether the BCAS model has an effect on cerebral perfusion. Cortical cerebral blood flow (CBF) was examined by laser speckle imaging. This revealed sustained reductions of CBF at 24 hours, 1 and 3 months following the establishment of BCAS (p<0.001) but no effect of the microvascular Aβ was found to affect cortical perfusion (p>0.05). To further explore the CBF changes in other brain regions following BCAS, Arterial spin labelling (ASL), a technique widely used in clinical imaging, was performed. A significant effect of BCAS was confirmed in the dorsolateral cortex and hippocampus (p<0.001, respectively) but no genotype effect of the microvascular Aβ or any interaction was found (p>0.05, respectively). In order to investigate whether long-term carotid stenosis has a further effect on cognitive function in the experimental animals, assessment of Barnes maze demonstrated that BCAS mice spent longer escape latency than the sham mice in both wild-type and Tg-SwDI animals (p<0.05, respectively) indicating visuo-spatial learning was significantly impaired at 3 months following BCAS. To determine the effect of BCAS and Aβ on long-term memory, a probe test was taken to examine whether mice remember the previous training target after a period of time. This test revealed that all groups spent a significantly higher percentage of time than chance (25%). Exclusively in wild-type BCAS mice, the percentage of time spent in the target quadrant was significantly lower than by chance (p<0.05). In addition, there was no significant effect of BCAS or Aβ on the percentage of time spent in the correct quadrant (p>0.05, respectively). These results suggested long-term memory was not impaired in BCAS and the presence of amyloid. Further to enhance the detection of spatial learning and memory impairment, reversal trials were taken to evaluate the ability of experimental animals to learn a new location. Compared to wild-type mice that still learned the new tests showing significantly improved performance over time (p<0.05), both the Tg-SwDI sham and BCAS mice no longer learned the task (p>0.05). The long-term memory tested in reversal tests showed impairment in both wildtype and Tg-SwDI BCAS as well as in the presence of amyloid after increasing the difficulties in reversal probe tests. The results indicated the only mice from wild-type sham (37.40 ± 12.63) (p<0.05) spent a significantly higher percentage of time by 12.40 (95%CI, 1.84 to 22.96) than by chance, t(7)=2.8, p=0.027 and a significantly higher percentage of time than Tg-SwDI BCAS mice (p<0.05) with all the other groups spending a lower percentage of time than chance (wild-type BCAS: 27.57 ± 11.12%, Tg-SwDI sham: 20.36 ± 15.50%, Tg-SwDI BCAS: 26.79 ± 16.79%). To further explore the potential mechanisms by which BCAS causes cognitive impairment, the glymphatic entry was further assessed. This revealed that the global influx of CSF tracers was different across the anatomical levels (p<0.001) but unaltered post-BCAS in wild-type and Tg-SwDI mice (p>0.05, respectively). To explore whether BCAS influences CSF glymphatic influx, ex vivo images of the CSF tracer influx in the dorsolateral cortex (DL CTX) and hippocampus (CA1-DG molecular layer) on the D-3 tracer were measured. The results showed in both regions, altered CSF influx was found in the BCAS and Tg-SwDI mice due to the main effect of BCAS (p=0.037 and p=0.011, DL CTX and CA-DG regions respectively) but not Aβ (p>0.05, respectively). Taken together, these first studies support the original hypothesis that BCAS causes cognitive impairment via reduced cerebral perfusion and impaired glymphatic function. However, there was no exacerbation of these effects in Tg-SwDI mice. Part 2: To examine the effect of BCAS on neurovascular function, degenerative changes and amyloid accumulation in Tg-SwDI mice compared to wild-type mice. To begin with, responses of cortical blood vessels to whisker stimulation were recorded and quantified as the mean CBF percentage increase from the baseline. There was a significant effect of BCAS (p<0.001), whereby impaired neurovascular coupling was observed in the BCAS mice from both wild-type and Tg-SwDI mice. However, there was no significant effect of Aβ in these mice (p>0.05). Vascular related lesions including microinfarcts and microbleeds were compared by measuring the frequency in experimental animals. No vascular lesions were detectable in wild-type and Tg-SwDI sham mice. 4/10 mice were found to have vascular lesions in the wild-type BCAS mice following 3 months of surgery. 6/10 mice were identified with vascular lesions in the Tg-SwDI mice. No significant difference in proportions (p>0.05) was found between Tg-SwDI BCAS and wild-type BCAS mice. To discern the mechanisms by which BCAS and microvascular amyloid may impact on the glymphatic function, the extent of astrogliosis was further studied. GFAP immunostaining was undertaken to investigate the extent of reactive gliosis post-BCAS. Increased astrogliosis following BCAS was found (p<0.05), but no effect of Aβ or interaction was found in the dorsolateral cortex. The hippocampal CA1-DG molecular layer was further analysed, and this showed a significant effect of Aβ (p=0.002) but no effect of BCAS (p>0.05) and interactions (p>0.05) on astrogliosis. Further, Aβ load was evaluated in the cortex and colabelled with collagen 4 (COL4) (a marker of the basement membrane of blood vessels) to enable the assessment of microvascular amyloid in the Tg-SwDI mouse model. A significant increase in the total amount of amyloid as well as the percentage of vascular amyloid was detected post-stenosis (p<0.05, respectively). No changes of COL4 levels were found in the mice post-BCAS (p>0.05). In summary, these results demonstrated that BCAS impaired neurovascular coupling and promoted amyloid accumulation in the cerebral microvasculature. Part 3: To determine whether targeting NOX2 has an effect on cerebral perfusion, degenerative changes and cognitive impairment in Tg-SwDI mice compared to wildtype mice. The third aim of the thesis was to determine the effect of NOX inhibitor (apocynin) on the previously reported cerebral hypoperfusion, impaired neurovascular coupling, development of neurodegenerative pathologies and cognitive deficits caused by BCAS in the Tg-SwDI mice. Following BCAS surgery, mice were immediately fed with either apocynin or vehicle in their drinking water for 3 months. Cortical CBF changes after the treatment of apocynin were assessed using laser speckle imaging, in apocynin treated mice, a recovery of CBF from the BCAS apocynin group after 3 months of treatment was found. The further investigation of neurovascular coupling revealed that apocynin restored vascular function following carotid stenosis. A significant interaction between BCAS surgery and apocynin treatment (p<0.05) was found after 3 months of treatment. The mice that received 3 months of apocynin treatment showed a robust response during the stimulation. The frequency of vascular lesions was counted to compare whether inhibiting NOX activity could provide any beneficial effect on the development of vascular pathology. However, there was no significant difference in proportions between the mice treated with vehicle and apocynin (p>0.05). The cortical amyloid load was assessed by double labelling of COL4 and 6E10. The results revealed no effect of treatment on the Aβ burden and vessel densities compared to vehicle treated group (p>0.05). Finally, the cognitive function was assessed using Barnes maze. It demonstrated that apocynin did not improve spatial learning and memory in the behavioural tests (p>0.05, respectively). Conclusions The findings in this thesis demonstrate novel evidence of how carotid stenosis damages the cerebral microcirculation and structure, contributing to the pathogenesis of cognitive impairment. Specifically, long-term BCAS caused chronic cerebral hypoperfusion and impaired glymphatic function, which is likely to contribute to the accumulation of Aβ in the microvasculature. Additionally, carotid stenosis caused sustained cerebral hypoperfusion and led to impaired neurovascular coupling, neurodegenerative changes and cognitive deficits. However, despite evidence supporting a basis for targeting NADPH oxidase, there was only a modest beneficial effect of the NOX inhibitor on neurovascular function. Collectively, this thesis provides evidence that following the carotid stenosis, while reducing cerebral perfusion, the glymphatic drainage pathway may be affected, leading to cognitive impairment. This new data adds credence to a growing body of human studies that alternate mechanisms may exist in addition to cerebral hypoperfusion leading to VCI. The treatment with non-selective NOX inhibitor successfully restored blood perfusion and vascular function with no ultimate improvement in cognitive function, suggesting a limited role by targeting NOX to restore the full pathological processes in VCI. Thus, further studies using more specific method targeting post-carotid stenosis events will help to understand the proposed mechanisms and provide a therapeutic strategy