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Microscopy and mass spectrometry (MS) are complementary techniques: The former provides spatiotemporal information in living cells, but only for a handful of recombinant proteins at a time, whereas the latter can detect thousands of endogenous proteins simultaneously, but only in lysed samples. Here, we introduce technology that combines these strengths by offering spatially and temporally resolved proteomic maps of endogenous proteins within living cells. Our method relies on a genetically targetable peroxidase enzyme that biotinylates nearby proteins, which are subsequently purified and identified by MS. We used this approach to identify 495 proteins within the human mitochondrial matrix, including 31 not previously linked to mitochondria. The labeling was exceptionally specific and distinguished between inner membrane proteins facing the matrix versus the intermembrane space (IMS). Several proteins previously thought to reside in the IMS or outer membrane, including protoporphyrinogen oxidase, were reassigned to the matrix by our proteomic data and confirmed by electron microscopy. The specificity of peroxidase-mediated proteomic mapping in live cells, combined with its ease of use, offers biologists a powerful tool for understanding the molecular composition of living cells.

Calcium (Ca²⁺) uptake into the mitochondrial matrix is critically important to cellular function. As a regulator of matrix Ca²⁺ levels, this flux influences energy production and can initiate cell death. If large, this flux could potentially alter intracellular Ca²⁺ ([Ca²⁺]i) signals. Despite years of study, fundamental disagreements on the extent and speed of mitochondrial Ca²⁺ uptake still exist. Here, we review and quantitatively analyze mitochondrial Ca²⁺ uptake fluxes from different tissues and interpret the results with respect to the recently proposed mitochondrial Ca²⁺ uniporter (MCU) candidate. This quantitative analysis yields four clear results: (i) under physiological conditions, Ca²⁺ influx into the mitochondria via the MCU is small relative to other cytosolic Ca²⁺ extrusion pathways; (ii) single MCU conductance is ~6-7 pS (105 mM [Ca²⁺]), and MCU flux appears to be modulated by [Ca²⁺]i, suggesting Ca²⁺ regulation of MCU open probability (Po); (iii) in the heart, two features are clear: the number of MCU channels per mitochondrion can be calculated, and MCU probability is low under normal conditions; and (iV) in skeletal muscle and liver cells, uptake per mitochondrion varies in magnitude but total uptake per cell still appears to be modest. Based on our analysis of available quantitative data, we conclude that although Ca²⁺ critically regulates mitochondrial function, the mitochondria do not act as a significant dynamic buffer of cytosolic Ca²⁺ under physiological conditions. Nevertheless, with prolonged (superphysiological) elevations of [Ca²⁺]i, mitochondrial Ca²⁺ uptake can increase 10- to 1,000-fold and begin to shape [Ca²⁺]i dynamics.

The transport of pyruvate, the end product of glycolysis, into mitochondria is an essential process that provides the organelle with a major oxidative fuel. Although the existence of a specific mitochondrial pyruvate carrier (MPC) has been anticipated, its molecular identity remained unknown. We report that MPC is a heterocomplex formed by two members of a family of previously uncharacterized membrane proteins that are conserved from yeast to mammals. Members of the MPC family were found in the inner mitochondrial membrane, and yeast mutants lacking MPC proteins showed severe defects in mitochondrial pyruvate uptake. Coexpression of mouse MPC1 and MPC2 in Lactococcus lactis promoted transport of pyruvate across the membrane. These observations firmly establish these proteins as essential components of the MPC.

The influence of the opposite phases of ENSO on the frequency of extreme rainfall events over South America is analyzed for each month of the ENSO cycle on the basis of a large set of daily station rainfall data and compared with the influence of ENSO on the monthly total rainfall. The analysis is carried out with station data and their gridded version and the results are consistent. Extreme events are defined as 3-day mean precipitation above the 90th percentile. The mean frequencies of extreme events are determined for each month and for each category of year (El Niño, La Niña, and neutral), and the differences between El Niño and neutral years and La Niña and neutral years are computed. Changes in the mean intensity of extreme events are also investigated. Significant ENSO signals in the frequency of extreme events are found over extensive regions of South America during different periods of the ENSO cycle. Although ENSO-related changes in intensity show less significance and spatial coherence, there are some robust changes in several regions, especially in southeastern South America. The ENSO-related changes in the frequency of extreme rainfall events are generally coherent with changes in total monthly rainfall quantities. However, significant changes in extremes are much more extensive than the corresponding changes in monthly rainfall because the highest sensitivity to ENSO seems to be in the extreme range of daily precipitation. This is important, since the most dramatic consequences of climate variability result from changes in extreme events. The pattern of frequency changes produced by El Niño and La Niña episodes with respect to neutral years is roughly symmetric, but there are several examples of nonlinearity in the ENSO regional teleconnections.

Significance Optimal performance of super resolution fluorescence localization microscopy relies on a clear understanding of the photo-physical properties of photoactivatable (photo-switchable) fluorescent proteins [PA(PS)-FPs] at the single-molecule level. Our comparative study of Dronpa and a novel variant, rsKame, demonstrates the crucial role of photo-switching kinetics in super resolution imaging. rsKame, with its superior properties, significantly broadens the green PA(PS)-FP palette. We demonstrate the efficacy of rsKame and our two-color super resolution imaging method (paired with PAmCherry1) by visualizing the inner and outer mitochondrial membranes and in situ structural parameters of dynamin related protein 1 helical rings during mitochondrial fission. Our two-color super resolution imaging method presented here is a reliable and user-friendly technique without complicated sample preparation.

We study the problem of locating disaster response and relief facilities in the city of Istanbul, where a massively destructive earthquake is expected to occur in the near future. The Metropolitan Municipality of Istanbul decided to establish facilities to preposition relief aid and execute post-disaster response operations. We propose a two-tier distribution system that utilizes existing public facilities locally in addition to the new facilities that will act as regional supply points. We develop mathematical models to decide on the locations of the new facilities with the objectives of minimizing the average-weighted distance between casualty locations and closest facilities, and opening a small number of facilities, subject to distance limits and backup requirements under regional vulnerability considerations. We analyze the trade-offs between these two objectives under various disaster scenarios and investigate the solutions for several modelling extensions. The results demonstrate that a small number of facilities will be sufficient and their locations are robust to various parameter and modelling changes.

Modular variation, whereby the relative degree of connectivity varies within a system, is thought to evolve through a process of selection that favors the integration of certain traits and the decoupling of others. In this way, modularity may facilitate the pace of evolution and determine evolvability. Alternatively, conserved patterns of modularity may act to constrain the rate and direction of evolution by preventing certain functions from evolving. A comprehensive understanding of the potential interplay between these phenomena will require knowledge of the inheritance and the genetic basis of modularity. Here we explore these ideas in the cichlid mandible by investigating patterns of modularity at the clade and species levels and through the introduction of a new approach, the individual level. Specifically, we assessed patterns of covariation in Lake Malawi cichlid species that employ alternate “biting” and “suction-feeding” modes of feeding and in a hybrid cross between these two ecotypes. Across the suction-feeding clade, patterns of modularity were largely conserved and reflected a functionally based pattern. In contrast, the biting species displayed a pattern of modularity that more closely matched developmental modules. The pattern of modularity present in our F2 population was very similar to the pattern exhibited by the biter, suggesting a role for dominant inheritance. We demonstrate that our individual-level metric of modularity (IMM) is a valid quantitative trait that has a nonlinear relationship with shape. IMMs for each model were used as quantitative characters to map quantitative trait loci (QTL) that underlie modularity. Our QTL analysis offers new insights into the genetic basis of modularity in these fishes that may eventually lead to the discovery of the genetic processes that delineate particular modules. In all, our findings suggest that modularity is both a constraining and an evolvable force in cichlid evolution, as distinct patterns occur between species and variation exists among individuals.

1. Zoonotic disease control presents significant costs and challenges in human and wildlife populations. Although spatial variability and temporal variability in host populations play a significant role influencing the spread and persistence of pathogens, their impact on the effectiveness of disease control are not well understood. 2. Field studies are impractical for many zoonotic diseases; thus, simulation modelling is an alternative. Some research has experimented with metapopulation models of host-pathogen systems, with discrete host populations distributed on a network of connections or on a one-dimensional transect of contiguous cells. Little attention has been paid to treating geographic space as a fine-grained two-dimensional continuum, a more appropriate spatial model for many generalist host and vector species. 3. Using raccoon rabies as an example, we apply an individual-based spatially explicit stochastic simulation model to evaluate effectiveness of vaccination barrier strategies to control rabies. Barrier width and immunization levels are varied over landscapes with habitats of varying quality and spatial heterogeneity, resulting in varying degrees of host connectivity. 4. Our results demonstrate that spatial heterogeneity in the landscape does affect vaccination efficacy. The probability that rabies will breach a vaccination barrier is greater and rabies incidence is higher in landscapes with (i) overall good-quality homogeneous habitat and (ii) overall poor-quality habitat with high spatial heterogeneity, than in landscapes with overall good-quality habitat and high spatial heterogeneity. The influence of landscape conditions on disease dynamics decreases with increasing population immunity. 5. Synthesis and applications. Using a spatially explicit stochastic simulation model, we demonstrated that landscape spatial heterogeneity and vaccination control will interact to influence the success of controlling infectious disease outbreaks. Further, under some landscape conditions, insufficient vaccination is counter-productive because immunized individuals (i) reduce the number of disease transmitting contacts, preventing the disease from growing rapidly thus depleting the susceptible population; and (ii) survive to replenish the stock of susceptible animals through reproduction, facilitating disease persistence.

Achieving sustained student engagement with practice in computer-based writing strategy training can be a challenge. One potential solution is to foster engagement by embedding practice in educational games; yet there is currently little research comparing the effectiveness of game-based practice versus more traditional forms of practice. In this study, the ARCS model (Keller, Perform Instr 26(8):1–7, 1987b) was used to investigate the motivational characteristics of different practice conditions. To this end, 175 students were randomly assigned to one of four experimental conditions: game-based, question-based, model-based, and writing-based practice. All students first learned strategies to write an essay introduction. Subsequently, students practiced using the strategies in the four different conditions. Game-based practice was expected to positively affect ARCS-related motivation toward practice. Results showed that students perceived game-based practice as significantly more interesting and engaging than question-based practice. However, although game-based practice was perceived more positively, only model-based and question-based practice demonstrated a beneficial impact on students' ability to implement the writing strategies. These results underline the necessity of interconnecting motivational and instructional design when developing practice methods for computer-based writing strategy training.

Mitochondria maintain tight regulation of inner mitochondrial membrane (IMM) permeability to sustain ATP production. Stressful events cause cellular calcium (Ca 2+ ) dysregulation followed by rapid loss of IMM potential known as permeability transition (PT), which produces osmotic shifts, metabolic dysfunction, and cell death. The molecular identity of the mitochondrial PT pore (mPTP) was previously unknown. We show that the purified reconstituted c-subunit ring of the F O of the F 1 F O ATP synthase forms a voltage-sensitive channel, the persistent opening of which leads to rapid and uncontrolled depolarization of the IMM in cells. Prolonged high matrix Ca 2+ enlarges the c-subunit ring and unhooks it from cyclophilin D/cyclosporine A binding sites in the ATP synthase F 1 , providing a mechanism for mPTP opening. In contrast, recombinant F 1 beta-subunit applied exogenously to the purified c-subunit enhances the probability of pore closure. Depletion of the c-subunit attenuates Ca 2+ -induced IMM depolarization and inhibits Ca 2+ and reactive oxygen species-induced cell death whereas increasing the expression or single-channel conductance of the c-subunit sensitizes to death. We conclude that a highly regulated c-subunit leak channel is a candidate for the mPTP. Beyond cell death, these findings also imply that increasing the probability of c-subunit channel closure in a healthy cell will enhance IMM coupling and increase cellular metabolic efficiency.