Robust and stable transcriptional repression in Giardia using CRISPRi.

Giardia lamblia is a binucleate protistan parasite causing significant diarrheal disease worldwide. An inability to target Cas9 to both nuclei, combined with the lack of nonhomologous end joining and markers for positive selection, has stalled the adaptation of CRISPR/Cas9-mediated genetic tools for this widespread parasite. CRISPR interference (CRISPRi) is a modification of the CRISPR/Cas9 system that directs catalytically inactive Cas9 (dCas9) to target loci for stable transcriptional repression. Using a Giardia nuclear localization signal to target dCas9 to both nuclei, we developed efficient and stable CRISPRi-mediated transcriptional repression of exogenous and endogenous genes in Giardia. Specifically, CRISPRi knockdown of kinesin-2a and kinesin-13 causes severe flagellar length defects that mirror defects with morpholino knockdown. Knockdown of the ventral disk MBP protein also causes severe structural defects that are highly prevalent and persist in the population more than 5 d longer than defects associated with transient morpholino-based knockdown. By expressing two guide RNAs in tandem to simultaneously knock down kinesin-13 and MBP, we created a stable dual knockdown strain with both flagellar length and disk defects. The efficiency and simplicity of CRISPRi in polyploid Giardia allows rapid evaluation of knockdown phenotypes and highlights the utility of CRISPRi for emerging model systems

Evaluation of different heat transfer conditions on an automotive turbocharger

This paper presents a combination of theoretical and experimental investigations for determining the main heat fluxes within a turbocharger. These investigations consider several engine speeds and loads as well as different methods of conduction, convection, and radiation heat transfer on the turbocharger. A one-dimensional heat transfer model of the turbocharger has been developed in combination with simulation of a turbocharged engine that includes the heat transfer of the turbocharger. Both the heat transfer model and the simulation were validated against experimental measurements. Various methods were compared for calculating heat transfer from the external surfaces of the turbocharger, and one new method was suggested. The effects of different heat transfer conditions were studied on the heat fluxes of the turbocharger using experimental techniques. The different heat transfer conditions on the turbocharger created dissimilar temperature gradients across the turbocharger. The results show that changing the convection heat transfer condition around the turbocharger affects the heat fluxes more noticeably than changing the radiation and conduction heat transfer conditions. Moreover, the internal heat transfers from the turbine to the bearing housing and from the bearing housing to the compressor are significant, but there is an order of magnitude difference between these heat transfer rates.The Swedish Energy Agency and KTH Royal Institute of Technology sponsored this work within the Competence Centre for Gas Exchange (CCGEx).Aghaali, H.; Angström, H.; Serrano Cruz, JR. (2015). Evaluation of different heat transfer conditions on an automotive turbocharger. International Journal of Engine Research. 16(2):137-151. doi:10.1177/1468087414524755S137151162Romagnoli, A., & Martinez-Botas, R. (2012). Heat transfer analysis in a turbocharger turbine: An experimental and computational evaluation. Applied Thermal Engineering, 38, 58-77. doi:10.1016/j.applthermaleng.2011.12.022Romagnoli, A., & Martinez-Botas, R. (2009). Heat Transfer on a Turbocharger Under Constant Load Points. Volume 5: Microturbines and Small Turbomachinery; Oil and Gas Applications. doi:10.1115/gt2009-59618Baines, N., Wygant, K. D., & Dris, A. (2010). The Analysis of Heat Transfer in Automotive Turbochargers. Journal of Engineering for Gas Turbines and Power, 132(4). doi:10.1115/1.3204586Serrano, J. R., Olmeda, P., Páez, A., & Vidal, F. (2010). An experimental procedure to determine heat transfer properties of turbochargers. Measurement Science and Technology, 21(3), 035109. doi:10.1088/0957-0233/21/3/035109Bohn, D., Heuer, T., & Kusterer, K. (2005). Conjugate Flow and Heat Transfer Investigation of a Turbo Charger. Journal of Engineering for Gas Turbines and Power, 127(3), 663-669. doi:10.1115/1.1839919Galindo, J., Luján, J. M., Serrano, J. R., Dolz, V., & Guilain, S. (2006). Description of a heat transfer model suitable to calculate transient processes of turbocharged diesel engines with one-dimensional gas-dynamic codes. Applied Thermal Engineering, 26(1), 66-76. doi:10.1016/j.applthermaleng.2005.04.010Sirakov, B., & Casey, M. (2012). Evaluation of Heat Transfer Effects on Turbocharger Performance. Journal of Turbomachinery, 135(2). doi:10.1115/1.4006608Serrano, J., Olmeda, P., Arnau, F., Reyes-Belmonte, M., & Lefebvre, A. (2013). Importance of Heat Transfer Phenomena in Small Turbochargers for Passenger Car Applications. SAE International Journal of Engines, 6(2), 716-728. doi:10.4271/2013-01-0576Larsson, P.-I., Westin, F., Andersen, J., Vetter, J., & Zumeta, A. (2009). Efficient turbo charger testing. MTZ worldwide, 70(7-8), 16-21. doi:10.1007/bf03226965Aghaali, H., & Ångström, H.-E. (2012). Turbocharged SI-Engine Simulation With Cold and Hot-Measured Turbocharger Performance Maps. Volume 5: Manufacturing Materials and Metallurgy; Marine; Microturbines and Small Turbomachinery; Supercritical CO2 Power Cycles. doi:10.1115/gt2012-68758Leufven, O., & Eriksson, L. (2012). Investigation of compressor correction quantities for automotive applications. International Journal of Engine Research, 13(6), 588-606. doi:10.1177/146808741243901

Compound C prevents Hypoxia-Inducible Factor-1α protein stabilization by regulating the cellular oxygen availability via interaction with Mitochondrial Complex I

The transcription factor Hypoxia-Inducible Factor-1α is a master regulator of the cellular response to low oxygen concentration. Compound C, an inhibitor of AMP-activated kinase, has been reported to inhibit hypoxia dependent Hypoxia-Inducible Factor-1α activation via a mechanism that is independent of AMP-activated kinase but dependent on its interaction with the mitochondrial electron transport chain. The objective of this study is to characterize the interaction of Compound C with the mitochondrial electron transport chain and to determine the mechanism through which the drug influences the stability of the Hypoxia-Inducible Factor-1α protein

Mosquito Feeding Affects Larval Behaviour and Development in a Moth

Organisms are attacked by different natural enemies present in their habitat. While enemies such as parasitoids and predators will kill their hosts/preys when they successfully attack them, enemies such as micropredators will not entirely consume their prey. However, they can still have important consequences on the performance and ecology of the prey, such as reduced growth, increased emigration, disease transmission

Effectiveness of manual therapy compared to usual care by the general practitioner for chronic tension-type headache: design of a randomised clinical trial

<p>Abstract</p> <p>Background</p> <p>Patients with Chronic Tension Type Headache (CTTH) report functional and emotional impairments (loss of workdays, sleep disturbances, emotional well-being) and are at risk for overuse of medication. Manual therapy may improve symptoms through mobilisation of the spine, correction of posture, and training of cervical muscles.</p> <p>We present the design of a randomised clinical trial (RCT) evaluating the effectiveness of manual therapy (MT) compared to usual care by the general practitioner (GP) in patients with CTTH.</p> <p>Methods and design</p> <p>Patients are eligible for participation if they present in general practice with CTTH according to the classification of the International Headache Society (IHS).</p> <p>Participants are randomised to either usual GP care according to the national Dutch general practice guidelines for headache, or manual therapy, consisting of mobilisations (high- and low velocity techniques), exercise therapy for the cervical and thoracic spine and postural correction. The primary outcome measures are the number of headache days and use of medication. Secondary outcome measures are severity of headache, functional status, sickness absence, use of other healthcare resources, active cervical range of motion, algometry, endurance of the neckflexor muscles and head posture. Follow-up assessments are conducted after 8 and 26 weeks.</p> <p>Discussion</p> <p>This is a pragmatic trial in which interventions are offered as they are carried out in everyday practice. This increases generalisability of results, but blinding of patients, GPs and therapists is not possible.</p> <p>The results of this trial will contribute to clinical decision making of the GP regarding referral to manual therapy in patients with chronic tension headache.</p

Low back pain in 17 year olds has substantial impact and represents an important public health disorder: a cross-sectional study

<p>Abstract</p> <p>Background</p> <p>Prevalence of low back pain (LBP) rises rapidly during adolescence, reaching adult levels by the age of 18. It has been suggested that adolescent LBP is benign with minimal impact, despite limited evidence.</p> <p>Methods</p> <p>The aim of this study was to investigate the impact of LBP and the influence of chronicity, gender and presence of other spinal pain comorbidities at age 17. Subjects (n = 1283) were categorised according to experiencing current and chronic LBP, gender and presence of other areas of spinal pain. LBP impact was ascertained via questions regarding seeking professional assistance, using medication, missing school/work, limited normal or recreational physical activity and health related quality of life (HRQOL).</p> <p>Results</p> <p>12.3% of participants reported current but not chronic LBP, while 19.9% reported current chronic LBP. LBP was more commonly reported by females than males. Other spinal pain comorbidities were common in the LBP groups. Impact was greater in subjects with chronic LBP, in females and in those with other spinal pain comorbidities.</p> <p>Conclusion</p> <p>LBP, and particularly chronic LBP, has a significant negative impact at 17 years. It is commonly associated with care seeking, medication use, school absenteeism, and reduced HRQOL. These findings support that adolescent LBP is an important public health issue that requires attention.</p

Coordinate and redox interactions of epinephrine with ferric and ferrous iron at physiological pH

Coordinate and redox interactions of epinephrine (Epi) with iron at physiological pH are essential for understanding two very different phenomena - the detrimental effects of chronic stress on the cardiovascular system and the cross-linking of catecholamine-rich biopolymers and frameworks. Here we show that Epi and Fe3+ form stable high-spin complexes in the 1:1 or 3:1 stoichiometry, depending on the Epi/Fe3+ concentration ratio (low or high). Oxygen atoms on the catechol ring represent the sites of coordinate bond formation within physiologically relevant bidentate 1:1 complex. Redox properties of Epi are slightly impacted by Fe3+. On the other hand, Epi and Fe2+ form a complex that acts as a strong reducing agent, which leads to the production of hydrogen peroxide via O-2 reduction, and to a facilitated formation of the Epi-Fe3+ complexes. Epi is not oxidized in this process, i.e. Fe2+ is not an electron shuttle, but the electron donor. Epi-catalyzed oxidation of Fe2+ represents a plausible chemical basis of stress-related damage to heart cells. In addition, our results support the previous findings on the interactions of catecholamine moieties in polymers with iron and provide a novel strategy for improving the efficiency of cross-linking.Supplementary material: [http://cherry.chem.bg.ac.rs/handle/123456789/3040

Long-Term IGF-I Exposure Decreases Autophagy and Cell Viability

A reduction in IGF-I signaling has been found to increase lifespan in multiple organisms despite the fact that IGF-I is a trophic factor for many cell types and has been found to have protective effects against multiple forms of damage in acute settings. The increase in longevity seen in response to reduced IGF-I signaling suggests that there may be differences between the acute and chronic impact of IGF-I signaling. We have examined the possibility that long-term stimulation with IGF-I may have a negative impact at the cellular level using quiescent human fibroblasts. We find that fibroblast cells exposed to IGF-I for 14 days have reduced long-term viability as judged by colony forming assays, which is accompanied by an accumulation of senescent cells. In addition we observe an accumulation of cells with depolarized mitochondria and a reduction in autophagy in the long-term IGF-I treated cultures. An examination of mice with reduced IGF-I levels reveals evidence of enhanced autophagy and fibroblast cells derived from these mice have a larger mitochondrial mass relative to controls indicating that changes in mitochondrial turnover occurs in animals with reduced IGF-I. The results indicate that chronic IGF-I stimulation leads to mitochondrial dysfunction and reduced cell viability

Identification and characterization of 27 conserved microRNAs in citrus

MicroRNAs (miRNAs) are a class of non-protein-coding small RNAs. Considering the conservation of many miRNA genes in different plant genomes, the identification of miRNAs from non-model organisms is both practicable and instrumental in addressing miRNA-guided gene regulation. Citrus is an important staple fruit tree, and publicly available expressed sequence tag (EST) database for citrus are increasing. However, until now, little has been known about miRNA in citrus. In this study, 27 known miRNAs from Arabidopsis were searched against citrus EST databases for miRNA precursors, of which 13 searched precursor sequences could form fold-back structures similar with those of Arabidopsis. The ubiquitous expression of those 13 citrus microRNAs and other 13 potential citrus miRNAs could be detected in citrus leaf, young shoot, flower, fruit and root by northern blotting, and some of them showed differential expression in different tissues. Based on the fact that miRNAs exhibit perfect or nearly perfect complementarity with their target sequences, a total of 41 potential targets were identified for 15 citrus miRNAs. The majority of the targets are transcription factors that play important roles in citrus development, including leaf, shoot, and root development. Additionally, some other target genes appear to play roles in diverse physiological processes. Four target genes have been experimentally verified by detection of the miRNA-mediated mRNA cleavage in Poncirus trifoliate. Overall, this study in the identification and characterization of miRNAs in citrus can initiate further study on citrus miRNA regulation mechanisms, and it can help us to know more about the important roles of miRNAs in citrus

Detailed dimethylacetal and fatty acid composition of rumen content from lambs fed lucerne or concentrate supplemented with soybean oil

Articles in International JournalsLipid metabolism in the rumen is responsible for the complex fatty acid profile of rumen outflow compared with the dietary fatty acid composition, contributing to the lipid profile of ruminant products. A method for the detailed dimethylacetal and fatty acid analysis of rumen contents was developed and applied to rumen content collected from lambs fed lucerne or concentrate based diets supplemented with soybean oil. The methodological approach developed consisted on a basic/ acid direct transesterification followed by thin-layer chromatography to isolate fatty acid methyl esters from dimethylacetal, oxo- fatty acid and fatty acid dimethylesters. The dimethylacetal composition was quite similar to the fatty acid composition, presenting even-, odd- and branched-chain structures. Total and individual odd- and branched-chain dimethylacetals were mostly affected by basal diet. The presence of 18:1 dimethylacetals indicates that biohydrogenation intermediates might be incorporated in structural microbial lipids. Moreover, medium-chain fatty acid dimethylesters were identified for the first time in the rumen content despite their concentration being relatively low. The fatty acids containing 18 carbon-chain lengths comprise the majority of the fatty acids present in the rumen content, most of them being biohydrogenation intermediates of 18:2n26 and 18:3n23. Additionally, three oxo- fatty acids were identified in rumen samples, and 16-O-18:0 might be produced during biohydrogenation of the 18:3n23