32 research outputs found
Analyzing the Efficiency of Inserting GP10 into a Planar Bilayer Membrane Dopped with a Carboxylated Lipid
No full textThe phi29 connector, GP10, is a channel protein from the bacteriophage phi29, a virus that can infect bacteria. The protein provides a channel that allows the phi29 genomic DNA to go through it during the infection process in-vivo. It has been successfully demonstrated that the connector can be inserted into a planar lipid bilayer membrane in-vitro, and thus the channel protein could potentially be used as a single-molecule platform to detect individual molecules (both small molecules and macromolecules), to monitor chemical and biochemical reactions in real time, and to perform DNA/RNA sequencing. Understanding the process of inserting the protein translocation the planar bilayer membranes will allow scientists to design a platform for the novel biosensor. However, the current problem encountered is the difficulty in achieving stable and efficient insertions of the protein into a synthetic lipid, 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC). For this purpose, in the research, we used molecular biology methods to prepare the protein, GP10 and employed a new strategy to optimize used lipids for efficient orthogonal integration of the phi29 connector into bilayer lipid membranes. Utilizing the precise knowledge of the 3-D structure of the phi29 connector, the bilayer membrane is optimized by tuning reactivity in the bilayer using the DPhPC doped with a carboxylated lipid, N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (PAcPC). The addition of the lipid into the bilayer would expectedly result in electrostatic attractive forces between the exposed basic amino acids on the surface of stalk in the phi29 connector and tails of the carboxylated lipid molecules in the core of the bilayer membrane. We will compare differences between the three particular factors that the data would give: the insertion efficiency, which is the total amount of insertions per sample time; the time of each insertion; and the total amount of insertions per sample batch. Furthermore, we will employ an independent two-tailed t-test to determine whether the differences are significant, thereby implying if the modified carboxylated lipid molecule does indeed stabilize the channel.http://opus.ipfw.edu/stu_symp2014/1055/thumbnail.jp
Dimethyl Fumarate Attenuates Microglia Activation and Long-Term Memory Deficits Following Systemic Immune Challenge
No full textPeripheral systemic inflammation is associated with increased cognitive decline and higher risk of developing Alzheimer’s disease. Microglia activated during systemic inflammation can cause exaggerated neuroinflammatory responses and trigger chronic neurodegeneration. Dimethyl fumarate is a fumaric acid ester approved by the Food and Drug Administration as a treatment for multiple sclerosis. Here, in vitro or in vivo dimethyl fumarate was shown to suppress classical activation of microglia induced by lipopolysaccharide, via reducing co-stimulatory surface molecules and inflammatory mediators. Although the modulation of the inflammatory microglia phenotype through dimethyl fumarate is correlated to activation of the nuclear factor erythroid-2-related factor 2, it was further shown that dimethyl fumarate suppressed the nuclear translocation of nuclear factor kappa beta pathway induced by lipopolysaccharide through a nuclear factor erythroid-2-related factor 2 independent manner. Further investigation showed that dimethyl fumarate alleviated the hyperactive inflammatory microglial response through the downregulation of the inflammatory mediators tumor necrosis factor alpha, interleukin-1 beta, and chemokine ligand 2. Through the modulation of inflammatory microglia, dimethyl fumarate prevented neuronal cell death after exposure to conditioned media generated from lipopolysaccharide-primed microglia. Importantly, dimethyl fumarate treatment rescued long-term memory deficits induced by peripheral lipopolysaccharide challenge. In summary, the results demonstrate a direct effect of DMF on modulation of the microglial phenotype, and dimethyl fumarate treatment protects neurons against toxic microenvironments produced by reactive microglia in response to systemic inflammation
Messianic Jews in Israel: Identity and Attitudes to Israeli Statehood - Case Study of the Community Beit Hallel in Ashdod.
No full textHlavním důvodem pro napsání této práce byl nedostatek prostoru, který současné vědecké práce věnují během výzkumu fenoménu mesiánského judaismu mesiánským komunitám v Izraeli. Pouze okrajově věnují pozornost oblasti, která je podle autora klíčem ke správnému pochopení dané problematiky. Tato práce proto zkoumá náboženskou identitu mesiánského judaismu v Izraeli a jeho vztah ke Státu Izrael. Práce je rozdělena do dvou částí odlišné povahy. První část je teoretická se zaměřením na fenomén mesiánského judaismu v Izraeli obecně. Zabývá se vznikem, historií a narativem hnutí, dále zkoumá mesiánský judaismus v Izraeli v současné podobě a následně rolí náboženství v Izraeli, náboženskou identitou izraelských Židů a pozici mesiánských Židův izraelské společnosti. Druhá část práce obsahuje případovou studii konkrétní mesiánské komunity Beit Hallel v Ašdodu. Výzkum je veden pomocí online polostrukturovaného dotazníku složeného z 10 otázek, které rozvíjejí a doplńují teze teoretické části.ObhájenoThe main reason for writing this work was the lack of space that contemporary scientific work devotes during the research of the phenomenon of Messianic Judaism to Messianic communities in Israel. They only marginally pay attention to the area which, according to the author, is key to a correct understanding of the issue. Therefore this thesis examines religious identity of Messianic Judaism in Israel and its relationship with the State of Israel. The work is divided into two parts of different nature. The first part is theoretical with a focus on the phenomenon of Messianic Judaism in Israel in general. It deals with the origin, history and narrative of the movement, further examines Messianic Judaism in Israel in its current form and subsequently the role of religion in Israel, the religious identity of Israeli Jews and the position of Messianic Jews in Israeli society. The second part of the work contains a case study of a specific messianic community Beit Hallel in Ashdod. The research is conducted using an online semi-structured survey consisting of 10 questions that develop and supplement the thesis of the theoretical part
Highly Efficient Integration of the Viral Portal Proteins from Different Types of Phages into Planar Bilayers for the Black Lipid Membrane Analysis
No full textThe planar lipid bilayer technology is a technique that yields incredibly useful structural function information about a single channel protein. It is also currently actively utilized as a powerful platform using biological protein nanopores for the development of single-molecule nanopore sensing technology, as well as ultrafast DNA sequencing technology. The portal protein, GP10, from the bacteriophage f29 was the first phage portal protein shown to be successfully inserted into planar bilayer membranes, thereby it may inspire more researchers to apply the techniques to portal proteins from the other bacteriophages.
However, the technology is far from perfect since the insertion of the channel proteins into planar bilayer membranes is not only technically difficult but also time-consuming. For the fusion of phage portal proteins, vesicles are typically needed to be reconstituted with the portal proteins to form proteoliposomes. However, most of the phage portal proteins have low solubility, and may self-aggregate during the preparation of the proteoliposomes. Furthermore, the fusion of the formed proteoliposomes is sporadic, unpredictable and varied from person to person. Due to the lack of experimental consistency between labs, the results from different methodologies reported for generating fusible proteoliposomes are highly variable.
In this research, we propose a new method for the preparation of the fusible proteoliposomes containing portal proteins from bacteriophages, to circumvent the problems aforementioned. Compared to the conventional methods, this method was able to avoid the protein aggregation issues during the vesicle preparation by eliminating the need for detergents and the subsequent time-consuming step for detergent removal. The proteoliposomes prepared by the method were shown to be more efficiently and rapidly inserted into planar bilayer membranes bathed in different conducting buffer solutions including those with nonelectrolytes such as glycerol and PEG. In addition, the method of forming proteoliposoomes has significantly extended the shelf life of the proteoliposomes. To further explore its potentials, we have successfully applied the method to the insertion of a mutant portal protein, GP20, from T4 bacteriophage, a hydrophobic portal protein that has not been explored using the planar lipid bilayer membrane technique. The results suggest that this method could be used to prepare proteoliposomes formed by hydrophobic portal proteins from other bacteriophages
Analysis of Cerebral Small Vessel Changes in an APOE4 Knock-In AD Mouse Model
No full textIntroduction:
Alzheimer’s disease (AD) is a complex neurodegenerative disorder and the leading cause of dementia worldwide. Recently approved monoclonal antibody therapy has shown increased instances of amyloid-related imaging abnormalities (ARIA) in patients with the APOE4 allele compared to those with the APOE3 allele. Although it is well established that AD adversely affects cerebral vasculature, the differential pathology between alleles is not fully understood. This study aims to explore and quantify the changes of cerebral small vessels in a human APOE4 vs. APOE3 knock-in AD mouse model.
Methods:
Brains were collected from APOE3: APP-SAA and APOE4: APP-SAA mouse cohorts at 8 months. Sectioning and staining were completed with immunofluorescence imaging of beta-amyloid (6E10), blood vessel (CD31), and microglia (Iba1) biomarkers. Vessel density, diameter, signal intensity, and vessel-plaque colocalization were analyzed using NIH Fiji software, and t-tests were performed to compare averages between cohorts.
Results:
Two-tail t tests revealed a vessel density difference of -0.4956±0.4590% (t(8)=1.080;p=0.3118) and a CD31 intensity difference of -269881±169413au (t(8)=1.593;p=0.1498) showing no statistical significance. Further testing showed a vessel diameter difference of -0.4043±0.02431 um (t(700)=16.63;p<0.0001). Additionally, testing showed a plaque-vessel colocalization difference of 16.34±6.307% (t(51)=2.591;p=0.0124). The qualitative assessment showed higher levels of microglial activation, a marker of neuroinflammation, in APOE4: APP-SAA brain samples.
Discussion:
The APOE4 allele is associated with adverse changes in cerebral small vessels in a controlled APOE3 vs. APOE4 APP-SAA model. The observed pathology of increased neuroinflammation, decreased vessel diameter, and heightened amyloid-beta localization to cerebral small vessels may elucidate the mechanisms by which monoclonal antibody therapy targeting plaque removal results in increased pathological side effects related to vessel damage. These findings warrant further studies on vascular changes responding to AD progression across ages and investigations into how this novel model may respond to monoclonal antibody therapy experiments. 
Investigating the Functional Impacts of Metabolic Disease Associated Immune-Vascular Interactions in Alzheimer’s Disease
No full textBackground and Hypothesis:Alzheimer’s disease (AD) is an irreversible neurodegenerative disorder with undefined etiology and is the fifth leading cause of death worldwide. AD pathology is characterized by amyloid-beta (Aβ) plaques. Previous work demonstrated that alterations in the microvasculature are some of the earliest recognizable changes in AD, and that most patients with dementia have mixed vascular pathologies. We investigated the functional impacts of metabolic disease associated immune-vascular perturbation on the underlying mechanisms of AD.
Methods:Adult male Leprdb/J (db/db) were obtained from the Jackson Laboratory. Activated microglia and brain vessel density levels were assessed using immunofluorescence. Cerebral microvessels were isolated for RNA examination using qPCR, and FACS-based analysis of brain endothelial cells. Immunofluorescence of hAβ42 transport in microvessels were observed via confocal microscope. Quantification of images were performed using Fiji (NIH) software.
Results:Db/db mice brains displayed higher levels of activated microglia with increased soma area and decreased circularity (p<0.05). This confirms early vascular stress leads to increased immune cell activation. Brain vessel density analysis revealed a non-statistically significant trend with decreased density in db/db mice. Given that functional changes occur before structural changes, we shifted our examination to the microvasculature. Brain microvessels were isolated and validated and both qPCR and FACS results demonstrated increased levels of inflammatory mediators and cell adhesion molecules in db/db mice (p<0.05), confirming microvessel dysfunction and neuroinflammation. Finally, quantification of luminal area fluorescence demonstrated decreased hAβ42 transport in db/db mice (p<0.01), validating functional disturbance in the cerebral microvasculature.
Conclusion and Impact:The vascular risk factors of metabolic disease can lead to dysfunction and inflammation in cerebral microvasculature, causing accelerated progression of AD. Our results emphasize the contributory role of cerebral small vessel health in the origin and evolution of AD and present an opportunity for novel development of surrogate biomarkers and therapeutic treatments
Ischemia-induced endogenous Nrf2/HO-1 axis activation modulates microglial polarization and restrains ischemic brain injury
No full textCerebral ischemic stroke accounts for more than 80% of all stroke cases. During cerebral ischemia, reactive oxygen species produced in the ischemic brain induce oxidative stress and inflammatory responses. Nrf2 is a transcription factor responsible for regulating cellular redox balance through the induction of protective antioxidant and phase II detoxification responses. Although the induction of endogenous Nrf2/HO-1 axis activation has been observed in the ischemic brain, whether ischemia-induced endogenous Nrf2/HO-1 axis activation plays a role in modulating microglia (MG) phenotypes and restraining ischemic brain injury is not characterized and requires further exploration. To investigate that, we generated mice with Nrf2 knockdown specifically in MG to rigorously assess the role of endogenous Nrf2 activation in ischemic brain injury after stroke. Our results showed that MG-specific Nrf2 knockdown exacerbated ischemic brain injury after stroke. We found that Nrf2 knockdown altered MG phenotypes after stroke, in which increased frequency of inflammatory MG and decreased frequency of anti-inflammatory MG were detected in the ischemic brain. Moreover, we identified attenuated Nrf2/HO-1 axis activation led to increased CD68/IL-1β and suppressed CD206 expression in MG, resulting in aggravated inflammatory MG in MG-specific Nrf2 knockdown mice after stroke. Intriguingly, using type II diabetic preclinical models, we revealed that diabetic mice exhibited attenuated Nrf2/HO-1 axis activation in MG and exacerbated ischemic brain injury after stroke that phenocopy mice with MG-specific Nrf2 knockdown. Finally, the induction of exogenous Nrf2/HO-1 axis activation in MG through pharmacological approaches ameliorated ischemic brain injury in diabetic mice. In conclusion, our findings provide cellular and molecular insights demonstrating ischemia-induced endogenous Nrf2/HO-1 axis activation modulates MG phenotypes and restrains ischemic brain injury. These results further strengthen the therapeutic potential of targeting Nrf2/HO-1 axis in MG for the treatment of ischemic stroke and diabetic stroke
Immunoresponsive gene 1 modulates the severity of brain injury in cerebral ischaemia
No full textInflammatory stimuli induce immunoresponsive gene 1 expression that in turn catalyses the production of itaconate through diverting cis-aconitate away from the tricarboxylic acid cycle. The immunoregulatory effect of the immunoresponsive gene 1/itaconate axis has been recently documented in lipopolysaccharide-activated mouse and human macrophages. In addition, dimethyl itaconate, an itaconate derivative, was reported to ameliorate disease severity in the animal models of psoriasis and multiple sclerosis. Currently, whether immunoresponsive gene 1/itaconate axis exerts a modulatory effect in ischaemic stroke remains unexplored. In this study, we investigated whether immunoresponsive gene 1 plays a role in modulating ischaemic brain injury. In addition, the molecular mechanism underlying the protective effects of immunoresponsive gene 1 in ischaemic stroke was elucidated. Our results showed that immunoresponsive gene 1 was highly induced in the ischaemic brain following ischaemic injury. Interestingly, we found that IRG1-/- stroke animals exhibited exacerbated brain injury, displayed with enlarged cerebral infarct, compared to wild-type stroke controls. Furthermore, IRG1-/- stroke animals presented aggravated blood-brain barrier disruption, associated with augmented Evans blue leakage and increased immune cell infiltrates in the ischaemic brain. Moreover, IRG1-/- stroke animals displayed elevated microglia activation, demonstrated with increased CD68, CD86 and Iba1 expression. Further analysis revealed that immunoresponsive gene 1 was induced in microglia after ischaemic stroke, and deficiency in immunoresponsive gene 1 resulted in repressed microglial heme oxygenase-1 expression and exacerbated ischaemic brain injury. Notably, the administration of dimethyl itaconate to compensate for the deficiency of immunoresponsive gene 1/itaconate axis led to enhanced microglial heme oxygenase-1 expression, alleviated ischaemic brain injury, improved motor function and decreased mortality in IRG1-/- stroke animals. In summary, we demonstrate for the first time that the induction of immunoresponsive gene 1 in microglia following ischaemic stroke serves as an endogenous protective mechanism to restrain brain injury through heme oxygenase-1 up-regulation. Thus, our findings suggest that targeting immunoresponsive gene 1 may represent a novel therapeutic approach for the treatment of ischaemic stroke
The connections of the Torah-Psalm 119 to the fifth Psalter of David (Ps 138–145)
No full textThe following exposition goes back to my lecture, ‘The Ways of YHWH and the Ways of the
supplicant in the fifth and last Psalter of David’, at the convention, ‘The Torah in the psalms
and the prophecy’, held at Munich on 13–14 July 2007. The first part of the lecture, which dealt
with the composition of the fifth Psalter of David, has appeared in an excursus (on the concept
of the fifth Psalter of David) in the psalm commentary by Erich Zenger and me (Author). It was
for this reason that the second part, which dealt with the Way-motif in Psalm 119 in the fifth
Psalter of David, was extended to the current subject of the article.
INTRADISCIPLINARY AND/OR INTERDISCIPLINARY IMPLICATIONS : The psalms and the Torah belong to
two different parts of the Hebrew canon. This means that the intertextual relations between
them rest on the interdisciplinary relationship between the two corpuses. The connections
between Psalm 119 and David’s fifth Psalter relate with the autonomous theologies of the fifth
Psalter of David (Ps 138–145) and the final Hallel (Ps 146–150). Psalm 119 had both groups of
psalms in front of it, and it respected the graduated psalm endings or rather the final redactions
in both instances. The implication for the redaction study of the psalms ishttp://www.ve.org.zaam2020Old Testament Studie
Additional file 1: of Dimethyl fumarate attenuates reactive microglia and long-term memory deficits following systemic immune challenge
No full textFigure S1. Characterization of microglial cultures, cell toxicity of DMF, and enriched astrocyte cultures. (PDF 134Â kb
