Summary
The rod‐shaped bacterium Escherichia coli grows by insertion of peptidoglycan into the lateral wall during cell elongation and synthesis of new poles during cell division. The monofunctional ...transpeptidases PBP2 and PBP3 are part of specialized protein complexes called elongasome and divisome, respectively, which catalyse peptidoglycan extension and maturation. Endogenous immunolabelled PBP2 localized in the cylindrical part of the cell as well as transiently at midcell. Using the novel image analysis tool Coli‐Inspector to analyse protein localization as function of the bacterial cell age, we compared PBP2 localization with that of other E. coli cell elongation and division proteins including PBP3. Interestingly, the midcell localization of the two transpeptidases overlaps in time during the early period of divisome maturation. Försters Resonance Energy Transfer (FRET) experiments revealed an interaction between PBP2 and PBP3 when both are present at midcell. A decrease in the midcell diameter is visible after 40% of the division cycle indicating that the onset of new cell pole synthesis starts much earlier than previously identified by visual inspection. The data support a new model of the division cycle in which the elongasome and divisome interact to prepare for cell division.
We use Richardson-Lucy (RL) deconvolution to combine multiple images of a simulated object into a single image in the context of modern fluorescence microscopy techniques. RL deconvolution can merge ...images with very different point-spread functions, such as in multiview light-sheet microscopes,1, 2 while preserving the best resolution information present in each image. We show that RL deconvolution is also easily applied to merge high-resolution, high-noise images with low-resolution, low-noise images, relevant when complementing conventional microscopy with localization microscopy. We also use RL deconvolution to merge images produced by different simulated illumination patterns, relevant to structured illumination microscopy (SIM)3, 4 and image scanning microscopy (ISM). The quality of our ISM reconstructions is at least as good as reconstructions using standard inversion algorithms for ISM data, but our method follows a simpler recipe that requires no mathematical insight. Finally, we apply RL deconvolution to merge a series of ten images with varying signal and resolution levels. This combination is relevant to gated stimulated-emission depletion (STED) microscopy, and shows that merges of high-quality images are possible even in cases for which a non-iterative inversion algorithm is unknown.
The quality of super resolution images obtained by stochastic single-molecule microscopy critically depends on image analysis algorithms. We find that the choice of background estimator is often the ...most important determinant of reconstruction quality. A variety of techniques have found use, but many have a very narrow range of applicability depending upon the characteristics of the raw data. Importantly, we observe that when using otherwise accurate algorithms, unaccounted background components can give rise to biases on scales defeating the purpose of super-resolution microscopy. We find that a temporal median filter in particular provides a simple yet effective solution to the problem of background estimation, which we demonstrate over a range of imaging modalities and different reconstruction methods.
Genetically encoded biosensors based on Förster resonance energy transfer (FRET) can visualize responses of individual cells in real time. Here, we evaluated whether FRET-based biosensors provide ...sufficient contrast and specificity to measure activity of G-protein-coupled receptors. The four histamine receptor subtypes (H1R, H2R, H3R, and H4R) respond to the ligand histamine by activating three canonical heterotrimeric G-protein-mediated signaling pathways with a reported high degree of specificity. Using FRET-based biosensors, we demonstrate that H1R activates Gαq. We also observed that H1R activates Gαi, albeit at a 10-fold lower potency. In addition to increasing cAMP levels, most likely via Gαs, we found that the H2R induces Gαq-mediated calcium release. The H3R and H4R activated Gαi with high specificity and a high potency. We demonstrate that a number of FRET sensors provide sufficient contrast to: 1) analyze the specificity of the histamine receptor subtypes for different heterotrimeric G-protein families with single-cell resolution, 2) probe for antagonist specificity, and 3) allow the measurement of single-cell concentration-response curves.
Summary
The rod‐shaped bacterium
E
scherichia coli
grows by insertion of peptidoglycan into the lateral wall during cell elongation and synthesis of new poles during cell division. The monofunctional ...transpeptidases
PBP
2 and
PBP
3 are part of specialized protein complexes called elongasome and divisome, respectively, which catalyse peptidoglycan extension and maturation. Endogenous immunolabelled
PBP
2 localized in the cylindrical part of the cell as well as transiently at midcell. Using the novel image analysis tool
C
oli‐
I
nspector to analyse protein localization as function of the bacterial cell age, we compared
PBP
2 localization with that of other
E
. coli
cell elongation and division proteins including
PBP
3. Interestingly, the midcell localization of the two transpeptidases overlaps in time during the early period of divisome maturation.
F
örsters
R
esonance
E
nergy
T
ransfer (
FRET
) experiments revealed an interaction between
PBP
2 and
PBP
3 when both are present at midcell. A decrease in the midcell diameter is visible after 40% of the division cycle indicating that the onset of new cell pole synthesis starts much earlier than previously identified by visual inspection. The data support a new model of the division cycle in which the elongasome and divisome interact to prepare for cell division.
Abstract Introduction The use of improvised explosive devices is a frequent method of insurgents to inflict harm on deployed military personnel. Consequently, lower extremity injuries make up the ...majority of combat related trauma. The wounding pattern of an explosion is not often encountered in a civilian population and can lead to substantial disability. It is therefore important to study the impact of these lower extremity injuries and their treatment (limb salvage versus amputation) on functional outcome and quality of life. Patients and methods All Dutch repatriated service members receiving treatment for wounds on the lower extremity sustained in the Afghan theater between august 2005 and August 2014, were invited to participate in this observational cohort study. We conducted a survey regarding their physical and mental health using the Short Form health survey 36, EuroQoL 6 dimensions and Lower Extremity Functional Scale questionnaires. Results were collated in a specifically designed electronic database combined with epidemiology and hospital statistics gathered from the archive of the Central Military Hospital. Statistical analyses were performed to identify differences between combat and non-combat related injuries and between limb salvage treatment and amputation. Results In comparison with non-battle injury patients, battle casualties were significantly younger of age, sustained more severe injuries, needed more frequent operations and clinical rehabilitation. Their long-term outcome scores in areas concerning well-being, social and cognitive functioning, were significantly lower. Regarding treatment, amputees experienced higher physical well-being and less pain compared to those treated with limb salvage surgery. Conclusion Sustaining a combat injury to the lower extremity can lead to partial or permanent dysfunction. However, wounded service members, amputees included, are able to achieve high levels of activity and participation in society, proving a remarkable resilience. These long-term results demonstrate that amputation is not a failure for casualty and surgeon, and strengthen a life before limb (damage control surgery) mindset in the initial phase. For future research, we recommend the use of adequate coding and injury scoring systems to predict outcome and give insight in the attributes that are supportive for the resilience that is needed to cope with a serious battle injury.
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