Mitochondrial Rejuvenation After Induced Pluripotency

Background: As stem cells of the early embryo mature and differentiate into all tissues, the mitochondrial complement undergoes dramatic functional improvement. Mitochondrial activity is low to minimize generation of DNA-damaging reactive oxygen species during pre-implantation development and increases following implantation and differentiation to meet higher metabolic demands. It has recently been reported that when the stem cell type known as induced pluripotent stem cells (IPSCs) are re-differentiated for several weeks in vitro, the mitochondrial complement progressively re-acquires properties approximating input fibroblasts, suggesting that despite the observation that IPSC conversion ‘‘resets’ ’ some parameters of cellular aging such as telomere length, it may have little impact on other age-affected cellular systems such as mitochondria in IPSC-derived cells. Methodology/Principal Findings: We have examined the properties of mitochondria in two fibroblast lines, corresponding IPSCs, and fibroblasts re-derived from IPSCs using biochemical methods and electron microscopy, and found a dramatic improvement in the quality and function of the mitochondrial complement of the re-derived fibroblasts compared to input fibroblasts. This observation likely stems from two aspects of our experimental design: 1) that the input cell lines used were of advanced cellular age and contained an inefficient mitochondrial complement, and 2) the re-derived fibroblasts were produced using an extensive differentiation regimen that may more closely mimic the degree of growth and maturatio

SMC complexes differentially compact mitotic chromosomes according to genomic context

Structural maintenance of chromosomes (SMC) protein complexes are key determinants of chromosome conformation. Using Hi-C and polymer modelling, we study how cohesin and condensin, two deeply conserved SMC complexes, organize chromosomes in the budding yeast Saccharomyces cerevisiae. The canonical role of cohesin is to co-align sister chromatids, while condensin generally compacts mitotic chromosomes. We find strikingly different roles for the two complexes in budding yeast mitosis. First, cohesin is responsible for compacting mitotic chromosome arms, independently of sister chromatid cohesion. Polymer simulations demonstrate that this role can be fully accounted for through cis-looping of chromatin. Second, condensin is generally dispensable for compaction along chromosome arms. Instead, it plays a targeted role compacting the rDNA proximal regions and promoting resolution of peri-centromeric regions. Our results argue that the conserved mechanism of SMC complexes is to form chromatin loops and that distinct SMC-dependent looping activities are selectively deployed to appropriately compact chromosomes

Effectiveness of propensity score methods with density estimation in identifying overlap for causal inference

For causal inference, sufficient overlap is needed. It is possible to use propensity scores with the positivity assumption to ensure overlap is present. However, positivity is not enough to properly identify the region of overlap. For this, propensity scores need to be used in combination with density estimation. This project aims to evaluate this method, discovering in which scenarios it performs well or fails in identifying the region of overlap. More specifically, how it scales with more features or outliers, and how using different classifiers affects the performance. The method was tested with samples from a simulated dataset. The predicted overlap was compared with the true overlap of the known distributions.Following the experiments, the method seems to perform best when the treatment and control groups share one region of overlap. In this case, logistic regression works best out of the classifiers that were tested. The overall performance drops when the two groups have multiple regions of overlap. For this, the random forest classifier performs best instead. Throughout all scenarios, the performance of the model drops with increasing dimensionality. Furthermore, having a small percentage of outliers only slightly affects the model. With more outliers, logistic regression is the only classifier further affected.CSE3000 Research ProjectComputer Science and Engineerin

Muscle structure and mitochondrial volume density in HA thoracic muscle.

<p>(A) and (B): Typical electron micrographs of thoracic muscle from control (NC) and hypoxia-selected flies (HA) (scale  = 1 µm). No significant modifications on myofiber architecture were determined in the HA muscle as compared to that of NC control. However, a reduced myofiber size (C) (n = 69, *p<0.01) and an increased mitochondrial volume density (volume fraction) (D) (n = 11, *p<0.01) were found in the thoracic muscle of HA flies.</p

Ultrastructural modifications in the mitochondria of HA flies.

<p>(A) Slice through a tomographic volume of a normoxic mitochondrion found in thoracic muscle sliced longitudinally. Normal morphology is observed, including stacks of ordered cristae with light intracristal space and darker matrix. Scale  = 200 nm. (B) Segmented volume of the mitochondrial membrane structure. The cristae (various colors) were all found to be lamellar in form, as expected. This mitochondrial volume contained 42 cristae. Scale  = 200 nm. (C) Oblique view of the mitochondrion viewed partially through the outer membrane (blue) made translucent to observe the cristae arrangement. Same scale as B. (D) Slice through a tomographic volume of hypoxic thoracic muscle. This large mitochondrion displays a few regions devoid of cristae (arrowheads) and more notably, small regions where the cristae have been fractured (boxed example and shown expanded in G-I). Scale  = 1000 nm. (E) Top view of the membrane segmentation of the volume. This mitochondrial volume contained 641 cristae. Scale  = 1000 nm. (F) Oblique view of the mitochondrion viewed partially through the outer membrane to observe the cristae motif. Same scale as E. (G) Expanded view of the boxed region in D. This area and others in (H) and (I) from different slices of the volume show views of rod-like dense cores between fractured cristae. The edge of the rod is the fracture curve of the cristae (black arrowheads) where the cristae in a localized region fractured almost uniformly (black lines in H) and then the ends annealed, i.e., the cristae membrane closed around the break. The dense core (white arrowhead) appears to be the degenerated cristae (black outline in I) that are separated by a white (translucent) band from the annealed cristae membrane curve. Scale  = 100 nm. (J) Top view of the segmented region of I showing the dense core (blue) surrounded by the cristae fragments (red). Scale  = 100 nm. (K) Side view perpendicular to the view in J, down the axis of the dense core appearing almost as an avenue with the cristae as spaced sentinels. Scale  = 100 nm. (L) Examples of two lamellar cristae in the hypoxic mitochondrion of panel D. These cristae were not as extensive as those in the control and often exhibited fenestrations (arrowhead). A greater percentage of cristae were similar to the highly branched and severely fenestrated crista shown in (M) (* p<0.01). Scale  = 500 nm. (N) The cristae surface area, normalized to the mitochondrial outer membrane surface area, was significantly greater in the hypoxic thoracic muscle as was the cristae number, normalized to the mitochondrial cross-sectional area (O) (* p<0.01).</p

Ultrastructural Modifications in the Mitochondria of Hypoxia-Adapted <i>Drosophila melanogaster</i>

<div><p>Chronic hypoxia (CH) occurs under certain physiological or pathological conditions, including in people who reside at high altitude or suffer chronic cardiovascular or pulmonary diseases. As mitochondria are the predominant oxygen-consuming organelles to generate ATP through oxidative phosphorylation in cells, their responses, through structural or molecular modifications, to limited oxygen supply play an important role in the overall functional adaptation to hypoxia. Here, we report the adaptive mitochondrial ultrastructural modifications and the functional impacts in a recently generated hypoxia-adapted <i>Drosophila melanogaster</i> strain that survives severe, otherwise lethal, hypoxic conditions. Using electron tomography, we discovered increased mitochondrial volume density and cristae abundance, yet also cristae fragmentation and a unique honeycomb-like structure in the mitochondria of hypoxia-adapted flies. The homeostatic levels of adenylate and energy charge were similar between hypoxia-adapted and naïve control flies and the hypoxia-adapted flies remained active under severe hypoxia as quantified by negative geotaxis behavior. The equilibrium ATP level was lower in hypoxia-adapted flies than those of the naïve controls tested under severe hypoxia that inhibited the motion of control flies. Our results suggest that the structural rearrangement in the mitochondria of hypoxia-adapted flies may be an important adaptive mechanism that plays a critical role in preserving adenylate homeostasis and metabolism as well as muscle function under chronic hypoxic conditions.</p></div

Honeycomb-like structures in the mitochondria of HA flies.

<p>(A) Slice through another tomographic reconstruction of a hypoxic mitochondrion in thoracic muscle oriented in cross-section (transverse) to the myofibrils. A honeycomb-like pattern of light, roughly circular regions with a dense center (4 circled) was found. Scale  = 200 nm. (B–E) The segmented honeycomb-like pattern (various colors) shows the spatial distribution and 3D extent of these structures. The full complement of rod-like structures having a dense core that is also rod-shaped, yet narrower is shown in B (top view) and C (oblique view). This mitochondrial volume contained 84 rods in the honeycomb-like structures. A subset of rods (23 total) is shown in D (top) and E (oblique) to note that these rods do not extend completely through the volume (height) of the mitochondrion. Scale  = 500 nm.</p

Increased sensitivity to oxidative stress in HA flies.

<p>A significantly shortened lifespan was determined in the HA flies (red) as compared to NC controls (blue) with 5 mM paraquat treatment under room air condition. The 50% survival of HA was 4.9±1.1 days, and the 50% survival of NC controls was 9.7±3.0 days (summarized from 6 separate experiments, p<0.01).</p

Medium journey time for hypoxia-selected and control flies to climb 2.5 cm distance in room air and 2 kPa O₂ hypoxic environment.

<p>Medium journey time for hypoxia-selected and control flies to climb 2.5 cm distance in room air and 2 kPa O₂ hypoxic environment.</p