Characterization of Precursor PfHsp60 in Plasmodium falciparum Cytosol during Its Asexual Development in Human Erythrocytes

Mitochondrial heat shock protein 60 (Hsp60) is a nuclear encoded gene product that gets post-translationally translocated into the mitochondria. Using multiple approaches such as immunofluorescence experiments, isoelectric point analysis with two-dimensional gel electrophoresis, and mass spectrometric identification of the signal peptide, we show that Hsp60 from Plasmodium falciparum (PfHsp60) accumulates in the parasite cytoplasm during the ring, trophozoite, and schizont stages of parasite development before being imported into the parasite mitochondria. Using co-immunoprecipitation experiments with antibodies specific to cytoplasmic PfHsp90, PfHsp70-1, and PfHsp60, we show association of precursor PfHsp60 with cytoplasmic chaperone machinery. Metabolic labeling involving pulse and chase indicates translocation of the precursor pool into the parasite mitochondrion during chase. Analysis of results obtained with Geldanamycin treatment confirmed precursor PfHsp60 to be one of the clients for PfHsp90. Cytosolic chaperones bind precursor PfHsp60 prior to its import into the mitochondrion of the parasite. Our data suggests an inefficient co-ordination in the synthesis and translocation of mitochondrial PfHsp60 during asexual growth of malaria parasite in human erythrocytes.

Synthesizing Racy Tests

Subtle concurrency errors in multithreaded libraries that arise because of incorrect or inadequate synchronization are often difficult to pinpoint precisely using only static techniques. On the other hand, the effectiveness of dynamic race detectors is critically dependent on multithreaded test suites whose execution can be used to identify and trigger races. Usually, such multithreaded tests need to invoke a specific combination of methods with objects involved in the invocations being shared appropriately to expose a race. Without a priori knowledge of the race, construction of such tests can be challenging. In this paper, we present a lightweight and scalable technique for synthesizing precisely these kinds of tests. Given a multithreaded library and a sequential test suite, we describe a fully automated analysis that examines sequential execution traces, and produces as its output a concurrent client program that drives shared objects via library method calls to states conducive for triggering a race. Experimental results on a variety of well-tested Java libraries yield 101 synthesized multithreaded tests in less than four minutes. Analyzing the execution of these tests using an off-the-shelf race detector reveals 187 harmful races, including several previously unreported ones.

Mapping Key Residues of ISD11 Critical for NFS1-ISD11 Subcomplex Stability IMPLICATIONS IN THE DEVELOPMENT OF MITOCHONDRIAL DISORDER, COXPD19

Biogenesis of the iron-sulfur (Fe-S) cluster is an indispensable process in living cells. In mammalian mitochondria, the initial step of the Fe-S cluster assembly process is assisted by the NFS1-ISD11 complex, which delivers sulfur to scaffold protein ISCU during Fe-S cluster synthesis. Although ISD11 is an essential protein, its cellular role in Fe-S cluster biogenesis is still not defined. Our study maps the important ISD11 amino acid residues belonging to putative helix 1 (Phe-40), helix 3 (Leu-63, Arg-68, Gln-69, Ile-72, Tyr-76), and C-terminal segment (Leu-81, Glu-84) are critical for in vivo Fe-S cluster biogenesis. Importantly, mutation of these conserved ISD11 residues into alanine leads to its compromised interaction with NFS1, resulting in reduced stability and enhanced aggregation of NFS1 in the mitochondria. Due to altered interaction with ISD11 mutants, the levels of NFS1 and Isu1 were significantly depleted, which affects Fe-S cluster biosynthesis, leading to reduced electron transport chain complex (ETC) activity and mitochondrial respiration. In humans, a clinically relevant ISD11 mutation (R68L) has been associated in the development of a mitochondrial genetic disorder, COXPD19. Our findings highlight that the ISD11 R68A/R68L mutation display reduced affinity to form a stable subcomplex with NFS1, and thereby fails to prevent NFS1 aggregation resulting in impairment of the Fe-S cluster biogenesis. The prime affected machinery is the ETC complex, which showed compromised redox properties, causing diminished mitochondrial respiration. Furthermore, the R68L ISD11 mutant displayed accumulation of mitochondrial iron and reactive oxygen species, leading to mitochondrial dysfunction, which correlates with the phenotype observed in COXPD19 patients.

Evidence for N-7 guanine methyl transferase activity encoded within the modular domain of RNA-dependent RNA polymerase L of a Morbillivirus

Post-transcriptional modification of viral mRNA is essential for the translation of viral proteins by cellular translation machinery. Due to the cytoplasmic replication of Paramyxoviruses, the viral-encoded RNA-dependent RNA polymerase (RdRP) is thought to possess all activities required for mRNA capping and methylation. In the present work, using partially purified recombinant RNA polymerase complex of rinderpest virus expressed in insect cells, we demonstrate the in vitro methylation of capped mRNA. Further, we show that a recombinant C-terminal fragment (1717-2183 aa) of L protein is capable of methylating capped mRNA, suggesting that the various post-transcriptional activities of the L protein are located in independently folding domains.

Robust Glyoxalase activity of Hsp31, a ThiJ/DJ-1/PfpI Family Member Protein, Is Critical for Oxidative Stress Resistance in Saccharomyces cerevisiae

Methylglyoxal (MG) is a reactive metabolic intermediate generated during various cellular biochemical reactions, including glycolysis. The accumulation of MG indiscriminately modifies proteins, including important cellular antioxidant machinery, leading to severe oxidative stress, which is implicated in multiple neurodegenerative disorders, aging, and cardiac disorders. Although cells possess efficient glyoxalase systems for detoxification, their functions are largely dependent on the glutathione cofactor, the availability of which is self-limiting under oxidative stress. Thus, higher organisms require alternate modes of reducing the MG-mediated toxicity and maintaining redox balance. In this report, we demonstrate that Hsp31 protein, a member of the ThiJ/DJ-1/PfpI family in Saccharomyces cerevisiae, plays an indispensable role in regulating redox homeostasis. Our results show that Hsp31 possesses robust glutathione-independent methylglyoxalase activity and suppresses MG-mediated toxicity and ROS levels as compared with another paralog, Hsp34. On the other hand, glyoxalase-defective mutants of Hsp31 were found highly compromised in regulating the ROS levels. Additionally, Hsp31 maintains cellular glutathione and NADPH levels, thus conferring protection against oxidative stress, and Hsp31 relocalizes to mitochondria to provide cytoprotection to the organelle under oxidative stress conditions. Importantly, human DJ-1, which is implicated in the familial form of Parkinson disease, complements the function of Hsp31 by suppressing methylglyoxal and oxidative stress, thus signifying the importance of these proteins in the maintenance of ROS homeostasis across phylogeny.

Epigenetics and miRNA during bacteria-induced host immune responses

Various cellular processes including the pathogen-specific immune responses, host-pathogen interactions and the related evasion mechanisms rely on the ability of the immune cells to be reprogrammed accurately and in many cases instantaneously. In this context, the exact functions of epigenetic and miRNA-mediated regulation of genes, coupled with recent advent in techniques that aid such studies, make it an attractive field for research. Here, we review examples that involve the epigenetic and miRNA control of the host immune system during infection with bacteria. Interestingly, many pathogens utilize the epigenetic and miRNA machinery to modify and evade the host immune responses. Thus, we believe that global epigenetic and miRNA mapping of such host-pathogen interactions would provide key insights into their cellular functions and help to identify various determinants for therapeutic value.

PLUTO plus : Near-Complete Modeling of Affine Transformations for Parallelism and Locality

Affine transformations have proven to be very powerful for loop restructuring due to their ability to model a very wide range of transformations. A single multi-dimensional affine function can represent a long and complex sequence of simpler transformations. Existing affine transformation frameworks like the Pluto algorithm, that include a cost function for modern multicore architectures where coarse-grained parallelism and locality are crucial, consider only a sub-space of transformations to avoid a combinatorial explosion in finding the transformations. The ensuing practical tradeoffs lead to the exclusion of certain useful transformations, in particular, transformation compositions involving loop reversals and loop skewing by negative factors. In this paper, we propose an approach to address this limitation by modeling a much larger space of affine transformations in conjunction with the Pluto algorithm's cost function. We perform an experimental evaluation of both, the effect on compilation time, and performance of generated codes. The evaluation shows that our new framework, Pluto+, provides no degradation in performance in any of the Polybench benchmarks. For Lattice Boltzmann Method (LBM) codes with periodic boundary conditions, it provides a mean speedup of 1.33x over Pluto. We also show that Pluto+ does not increase compile times significantly. Experimental results on Polybench show that Pluto+ increases overall polyhedral source-to-source optimization time only by 15%. In cases where it improves execution time significantly, it increased polyhedral optimization time only by 2.04x.

Falcon: A Graph Manipulation Language for Heterogeneous Systems

Graph algorithms have been shown to possess enough parallelism to keep several computing resources busy-even hundreds of cores on a GPU. Unfortunately, tuning their implementation for efficient execution on a particular hardware configuration of heterogeneous systems consisting of multicore CPUs and GPUs is challenging, time consuming, and error prone. To address these issues, we propose a domain-specific language (DSL), Falcon, for implementing graph algorithms that (i) abstracts the hardware, (ii) provides constructs to write explicitly parallel programs at a higher level, and (iii) can work with general algorithms that may change the graph structure (morph algorithms). We illustrate the usage of our DSL to implement local computation algorithms (that do not change the graph structure) and morph algorithms such as Delaunay mesh refinement, survey propagation, and dynamic SSSP on GPU and multicore CPUs. Using a set of benchmark graphs, we illustrate that the generated code performs close to the state-of-the-art hand-tuned implementations.

PolyMage: Automatic Optimization for Image Processing Pipelines

This paper presents the design and implementation of PolyMage, a domain-specific language and compiler for image processing pipelines. An image processing pipeline can be viewed as a graph of interconnected stages which process images successively. Each stage typically performs one of point-wise, stencil, reduction or data-dependent operations on image pixels. Individual stages in a pipeline typically exhibit abundant data parallelism that can be exploited with relative ease. However, the stages also require high memory bandwidth preventing effective utilization of parallelism available on modern architectures. For applications that demand high performance, the traditional options are to use optimized libraries like OpenCV or to optimize manually. While using libraries precludes optimization across library routines, manual optimization accounting for both parallelism and locality is very tedious. The focus of our system, PolyMage, is on automatically generating high-performance implementations of image processing pipelines expressed in a high-level declarative language. Our optimization approach primarily relies on the transformation and code generation capabilities of the polyhedral compiler framework. To the best of our knowledge, this is the first model-driven compiler for image processing pipelines that performs complex fusion, tiling, and storage optimization automatically. Experimental results on a modern multicore system show that the performance achieved by our automatic approach is up to 1.81x better than that achieved through manual tuning in Halide, a state-of-the-art language and compiler for image processing pipelines. For a camera raw image processing pipeline, our performance is comparable to that of a hand-tuned implementation.

SMO: An Integrated Approach to Intra-array and Inter-array Storage Optimization

The polyhedral model provides an expressive intermediate representation that is convenient for the analysis and subsequent transformation of affine loop nests. Several heuristics exist for achieving complex program transformations in this model. However, there is also considerable scope to utilize this model to tackle the problem of automatic memory footprint optimization. In this paper, we present a new automatic storage optimization technique which can be used to achieve both intra-array as well as inter-array storage reuse with a pre-determined schedule for the computation. Our approach works by finding statement-wise storage partitioning hyper planes that partition a unified global array space so that values with overlapping live ranges are not mapped to the same partition. Our heuristic is driven by a fourfold objective function which not only minimizes the dimensionality and storage requirements of arrays required for each high-level statement, but also maximizes inter statement storage reuse. The storage mappings obtained using our heuristic can be asymptotically better than those obtained by any existing technique. We implement our technique and demonstrate its practical impact by evaluating its effectiveness on several benchmarks chosen from the domains of image processing, stencil computations, and high-performance computing.