Lysosome Dynamic Properties during Neuronal Stem Cell Differentiation Studied by Spatiotemporal Fluctuation Spectroscopy and Organelle Tracking

We investigated lysosome dynamics during neuronal stem cell (NSC) differentiation by two quantitative and complementary biophysical methods based on fluorescence: imaging-derived mean square displacement (iMSD) and single-particle tracking (SPT). The former extracts the average dynamics and size of the whole population of moving lysosomes directly from imaging, with no need to calculate single trajectories; the latter resolves the finest heterogeneities and dynamic features at the single-lysosome level, which are lost in the iMSD analysis. In brief, iMSD analysis reveals that, from a structural point of view, lysosomes decrement in size during NSC differentiation, from 1 \u3bcm average diameter in the embryonic cells to approximately 500 nm diameter in the fully differentiated cells. Concomitantly, iMSD analysis highlights modification of key dynamic parameters, such as the average local organelle diffusivity and anomalous coefficient, which may parallel cytoskeleton remodeling during the differentiation process. From average to local, SPT allows mapping heterogeneous dynamic responses of single lysosomes in different districts of the cells. For instance, a dramatic decrease of lysosomal transport in the soma is followed by a rapid increase of transport in the projections at specific time points during neuronal differentiation, an observation compatible with the hypothesis that lysosomal active mobilization shifts from the soma to the newborn projections. Our combined results provide new insight into the lysosome size and dynamics regulation throughout NSC differentiation, supporting new functions proposed for this organelle

Association of \u3ci\u3eEscherichia coli\u3c/i\u3e O157:H7 \u3ci\u3etir\u3c/i\u3e polymorphisms with human infection

Background: Emerging molecular, animal model and epidemiologic evidence suggests that Shigatoxigenic Escherichia coli O157:H7 (STEC O157) isolates vary in their capacity to cause human infection and disease. The translocated intimin receptor (tir) and intimin (eae) are virulence factors and bacterial receptor-ligand proteins responsible for tight STEC O157 adherence to intestinal epithelial cells. They represent logical genomic targets to investigate the role of sequence variation in STEC O157 pathogenesis and molecular epidemiology. The purposes of this study were (1) to identify tir and eae polymorphisms in diverse STEC O157 isolates derived from clinically ill humans and healthy cattle (the dominant zoonotic reservoir) and (2) to test any observed tir and eae polymorphisms for association with human (vs bovine) isolate source. Results: Five polymorphisms were identified in a 1,627-bp segment of tir. Alleles of two tir polymorphisms, tir 255 T\u3eA and repeat region 1-repeat unit 3 (RR1-RU3, presence or absence) had dissimilar distributions among human and bovine isolates. More than 99% of 108 human isolates possessed the tir 255 T\u3eA T allele and lacked RR1-RU3. In contrast, the tir 255 T\u3eA T allele and RR1-RU3 absence were found in 55% and 57%, respectively, of 77 bovine isolates. Both polymorphisms associated strongly with isolate source (p \u3c 0.0001), but not by pulsed field gel electrophoresis type or by stx1 and stx2 status (as determined by PCR). Two eae polymorphisms were identified in a 2,755-bp segment of 44 human and bovine isolates; 42 isolates had identical eae sequences. The eae polymorphisms did not associate with isolate source. Conclusion: Polymorphisms in tir but not eae predict the propensity of STEC O157 isolates to cause human clinical disease. The over-representation of the tir 255 T\u3eA T allele in human-derived isolates vs the tir 255 T\u3eA A allele suggests that these isolates have a higher propensity to cause disease. The high frequency of bovine isolates with the A allele suggests a possible bovine ecological niche for this STEC O157 subset

Association of Escherichia coli O157:H7 tir polymorphisms with human infection

<p>Abstract</p> <p>Background</p> <p>Emerging molecular, animal model and epidemiologic evidence suggests that Shiga-toxigenic <it>Escherichia coli </it>O157:H7 (STEC O157) isolates vary in their capacity to cause human infection and disease. The translocated intimin receptor (<it>tir</it>) and intimin (<it>eae</it>) are virulence factors and bacterial receptor-ligand proteins responsible for tight STEC O157 adherence to intestinal epithelial cells. They represent logical genomic targets to investigate the role of sequence variation in STEC O157 pathogenesis and molecular epidemiology. The purposes of this study were (1) to identify <it>tir </it>and <it>eae </it>polymorphisms in diverse STEC O157 isolates derived from clinically ill humans and healthy cattle (the dominant zoonotic reservoir) and (2) to test any observed <it>tir </it>and <it>eae </it>polymorphisms for association with human (vs bovine) isolate source.</p> <p>Results</p> <p>Five polymorphisms were identified in a 1,627-bp segment of <it>tir</it>. Alleles of two <it>tir </it>polymorphisms, <it>tir </it>255 T>A and repeat region 1-repeat unit 3 (RR1-RU3, presence or absence) had dissimilar distributions among human and bovine isolates. More than 99% of 108 human isolates possessed the <it>tir </it>255 T>A T allele and lacked RR1-RU3. In contrast, the <it>tir </it>255 T>A T allele and RR1-RU3 absence were found in 55% and 57%, respectively, of 77 bovine isolates. Both polymorphisms associated strongly with isolate source (p < 0.0001), but not by pulsed field gel electrophoresis type or by <it>stx</it>1 and <it>stx</it>2 status (as determined by PCR). Two <it>eae </it>polymorphisms were identified in a 2,755-bp segment of 44 human and bovine isolates; 42 isolates had identical <it>eae </it>sequences. The <it>eae </it>polymorphisms did not associate with isolate source.</p> <p>Conclusion</p> <p>Polymorphisms in <it>tir </it>but not <it>eae </it>predict the propensity of STEC O157 isolates to cause human clinical disease. The over-representation of the <it>tir </it>255 T>A T allele in human-derived isolates vs the <it>tir </it>255 T>A A allele suggests that these isolates have a higher propensity to cause disease. The high frequency of bovine isolates with the A allele suggests a possible bovine ecological niche for this STEC O157 subset.</p

Probing labeling-induced lysosome alterations in living cells by imaging-derived mean squared displacement analysis

Lysosomes are not merely degradative organelles but play a central role in nutrient sensing, metabolism and cell-growth regulation. Our ability to study their function in living cells strictly relies on the use of lysosome-specific fluorescent probes tailored to optical microscopy applications. Still, no report thus far quantitatively analyzed the effect of labeling strategies/procedures on lysosome properties in live cells. We tackle this issue by a recently developed spatiotemporal fluctuation spectroscopy strategy that extracts structural (size) and dynamic (diffusion) properties directly from imaging, with no a-priori knowledge of the system. We highlight hitherto neglected alterations of lysosome properties upon labeling. In particular, we demonstrate that Lipofectamine reagents, used to transiently express lysosome markers fused to fluorescent proteins (FPs) (e.g. LAMP1-FP or CD63-FP), irreversibly alter the organelle structural identity, inducing a ∼2-fold increase of lysosome average size. The organelle structural identity is preserved, instead, if electroporation or Effectene are used as transfection strategies, provided that the expression levels of the recombinant protein marker are kept low. This latter condition can be achieved also by generating cell lines stably expressing the desired FP-tagged marker. Reported results call into question the interpretation of a massive amount of data collected so far using fluorescent protein markers and suggest useful guidelines for future studies

Time-lapse confocal imaging datasets to assess structural and dynamic properties of subcellular nanostructures

Time-lapse optical microscopy datasets from living cells can potentially afford an enormous amount of quantitative information on the relevant structural and dynamic properties of sub-cellular organelles/structures, provided that both the spatial and temporal dimensions are properly sampled during the experiment. Here we provide exemplary live-cell, time-lapse confocal imaging datasets corresponding to three sub-cellular structures of the endo-lysosomal pathway, i.e. early endosomes, late endosomes and lysosomes, along with detailed guidelines to produce analogous experiments. Validation of the datasets is conducted by means of established analytical tools to extract the structural and dynamic properties at the sub-cellular scale, such as Single Particle Tracking (SPT) and imaging derived Mean Square Displacement (iMSD) analyses. In our aim, the present work would help other researchers in the field to reuse the provided datasets for their own scopes, and to combine their creative approaches/analyses to similar acquisitions

Data descriptor: time-lapse confocal imaging datasets to assess structural and dynamic properties of subcellular nanostructures

Time-lapse optical microscopy datasets from living cells can potentially afford an enormous amount of quantitative information on the relevant structural and dynamic properties of sub-cellular organelles/ structures, provided that both the spatial and temporal dimensions are properly sampled during the experiment. Here we provide exemplary live-cell, time-lapse confocal imaging datasets corresponding to three sub-cellular structures of the endo-lysosomal pathway, i.e. early endosomes, late endosomes and lysosomes, along with detailed guidelines to produce analogous experiments. Validation of the datasets is conducted by means of established analytical tools to extract the structural and dynamic properties at the sub-cellular scale, such as Single Particle Tracking (SPT) and imaging derived Mean Square Displacement (iMSD) analyses. In our aim, the present work would help other researchers in the field to reuse the provided datasets for their own scopes, and to combine their creative approaches/analyses to similar acquisitions

Dynamic fingerprinting of sub-cellular nanostructures by image mean square displacement analysis

Here we provide demonstration that image mean square displacement (iMSD) analysis is a fast and robust platform to address living matter dynamic organization at the level of sub-cellular nanostructures (e.g. endocytic vesicles, early/late endosomes, lysosomes), with no a-priori knowledge of the system, and no need to extract single trajectories. From each iMSD, a unique triplet of average parameters (namely: diffusivity, anomalous coefficient, size) are extracted and represented in a 3D parametric space, where clustering of single-cell points readily defines the structure "dynamic fingerprint", at the whole-cell-population level. We demonstrate that different sub-cellular structures segregate into separate regions of the parametric space. The potency of this approach is further proved through application to two exemplary, still controversial, cases: i) the intracellular trafficking of lysosomes, comprising both free diffusion and directed motion along cytoskeletal components, and ii) the evolving dynamic properties of macropinosomes, passing from early to late stages of intracellular trafficking. We strongly believe this strategy may represent a flexible, multiplexed platform to address the dynamic properties of living matter at the sub-cellular level, both in the physiological and pathological state

Distribution of Shiga-Toxigenic Escherichia coli O157 in the Gastrointestinal Tract of Naturally O157-Shedding Cattle at Necropsy▿

Shiga-toxigenic Escherichia coli (STEC) O157 occurrence was determined along the entire gastrointestinal tract (GIT) of each of four naturally shedding cattle and at three sites in 61 slaughter cattle. STEC O157 was distributed along the entire GIT, though interanimal distribution was variable. Neither feces nor rectoanal-junction samples accurately predicted the STEC O157-negative status of any particular animal