Speeding Up Ecological and Evolutionary Computations in R; Essentials of High Performance Computing for Biologists

Computation has become a critical component of research in biology. A risk has emerged that computational and programming challenges may limit research scope, depth, and quality. We review various solutions to common computational efficiency problems in ecological and evolutionary research. Our review pulls together material that is currently scattered across many sources and emphasizes those techniques that are especially effective for typical ecological and environmental problems. We demonstrate how straightforward it can be to write efficient code and implement techniques such as profiling or parallel computing. We supply a newly developed R package (aprof) that helps to identify computational bottlenecks in R code and determine whether optimization can be effective. Our review is complemented by a practical set of examples and detailed Supporting Information material (S1–S3 Texts) that demonstrate large improvements in computational speed (ranging from 10.5 times to 14,000 times faster). By improving computational efficiency, biologists can feasibly solve more complex tasks, ask more ambitious questions, and include more sophisticated analyses in their research.

This is part of the PLOS Computational Biology Education collection.

     

Keys to Lipid Selection in Fatty Acid Amide Hydrolase Catalysis: Structural Flexibility,Gating Residues and Multiple Binding Pockets

The fatty acid amide hydrolase (FAAH) regulates the endocannabinoid system cleaving primarily the lipid messenger anandamide. FAAH has been well characterized over the years and, importantly, it represents a promising drug target to treat several diseases, including inflammatory-related diseases and cancer. But its enzymatic mechanism for lipid selection to specifically hydrolyze anandamide, rather than similar bioactive lipids, remains elusive. Here, we clarify this mechanism in FAAH, examining the role of the dynamic paddle, which is formed by the gating residues Phe432 and Trp531 at the boundary between two cavities that form the FAAH catalytic site (the “membrane-access” and the “acyl chain-binding” pockets). We integrate microsecond-long MD simulations of wild type and double mutant model systems (Phe432Ala and Trp531Ala) of FAAH, embedded in a realistic membrane/water environment, with mutagenesis and kinetic experiments. We comparatively analyze three fatty acid substrates with different hydrolysis rates (anandamide > oleamide > palmitoylethanolamide). Our findings identify FAAH’s mechanism to selectively accommodate anandamide into a multi-pocket binding site, and to properly orient the substrate in pre-reactive conformations for efficient hydrolysis that is interceded by the dynamic paddle. Our findings therefore endorse a structural framework for a lipid selection mechanism mediated by structural flexibility and gating residues between multiple binding cavities, as found in FAAH. Based on the available structural data, this exquisite catalytic strategy for substrate specificity seems to be shared by other lipid-degrading enzymes with similar enzymatic architecture. The mechanistic insights for lipid selection might assist de-novo enzyme design or drug discovery efforts.     

Activation of Type I and III Interferon Response by Mitochondrial and Peroxisomal MAVS and Inhibition by Hepatitis C Virus

Sensing viruses by pattern recognition receptors (PRR) triggers the innate immune system of the host cell and activates immune signaling cascades such as the RIG-I/IRF3 pathway. Mitochondrial antiviral-signaling protein (MAVS, also known as IPS-1, Cardif, and VISA) is the crucial adaptor protein of this pathway localized on mitochondria, peroxisomes and mitochondria-associated membranes of the endoplasmic reticulum. Activation of MAVS leads to the production of type I and type III interferons (IFN) as well as IFN stimulated genes (ISGs). To refine the role of MAVS subcellular localization for the induction of type I and III IFN responses in hepatocytes and its counteraction by the hepatitis C virus (HCV), we generated various functional and genetic knock-out cell systems that were reconstituted to express mitochondrial (mito) or peroxisomal (pex) MAVS, exclusively. Upon infection with diverse RNA viruses we found that cells exclusively expressing pexMAVS mounted sustained expression of type I and III IFNs to levels comparable to cells exclusively expressing mitoMAVS. To determine whether viral counteraction of MAVS is affected by its subcellular localization we employed infection of cells with HCV, a major causative agent of chronic liver disease with a high propensity to establish persistence. This virus efficiently cleaves MAVS via a viral protease residing in its nonstructural protein 3 (NS3) and this strategy is thought to contribute to the high persistence of this virus. We found that both mito- and pexMAVS were efficiently cleaved by NS3 and this cleavage was required to suppress activation of the IFN response. Taken together, our findings indicate comparable activation of the IFN response by pex- and mitoMAVS in hepatocytes and efficient counteraction of both MAVS species by the HCV NS3 protease.     

Inhibition of mTORC1 Enhances the Translation of Chikungunya Proteins via the Activation of the MnK/eIF4E Pathway

Chikungunya virus (CHIKV), the causative agent of a major epidemic spanning five continents, is a positive stranded mRNA virus that replicates using the cell’s cap-dependent translation machinery. Despite viral infection inhibiting mTOR, a metabolic sensor controls cap-dependent translation, viral proteins are efficiently translated. Rapalog treatment, silencing of mtor or raptor genes, but not rictor, further enhanced CHIKV infection in culture cells. Using biochemical assays and real time imaging, we demonstrate that this effect is independent of autophagy or type I interferon production. Providing in vivo evidence for the relevance of our findings, mice treated with mTORC1 inhibitors exhibited increased lethality and showed a higher sensitivity to CHIKV. A systematic evaluation of the viral life cycle indicated that inhibition of mTORC1 has a specific positive effect on viral proteins, enhancing viral replication by increasing the translation of both structural and nonstructural proteins. Molecular analysis defined a role for phosphatidylinositol-3 kinase (PI3K) and MAP kinase-activated protein kinase (MnKs) activation, leading to the hyper-phosphorylation of eIF4E. Finally, we demonstrated that in the context of CHIKV inhibition of mTORC1, viral replication is prioritized over host translation via a similar mechanism. Our study reveals an unexpected bypass pathway by which CHIKV protein translation overcomes viral induced mTORC1 inhibition.     

A Plasmodium Phospholipase Is Involved in Disruption of the Liver Stage Parasitophorous Vacuole Membrane

The coordinated exit of intracellular pathogens from host cells is a process critical to the success and spread of an infection. While phospholipases have been shown to play important roles in bacteria host cell egress and virulence, their role in the release of intracellular eukaryotic parasites is largely unknown. We examined a malaria parasite protein with phospholipase activity and found it to be involved in hepatocyte egress. In hepatocytes, Plasmodium parasites are surrounded by a parasitophorous vacuole membrane (PVM), which must be disrupted before parasites are released into the blood. However, on a molecular basis, little is known about how the PVM is ruptured. We show that Plasmodium berghei phospholipase, PbPL, localizes to the PVM in infected hepatocytes. We provide evidence that parasites lacking PbPL undergo completely normal liver stage development until merozoites are produced but have a defect in egress from host hepatocytes. To investigate this further, we established a live-cell imaging-based assay, which enabled us to study the temporal dynamics of PVM rupture on a quantitative basis. Using this assay we could show that PbPL-deficient parasites exhibit impaired PVM rupture, resulting in delayed parasite egress. A wild-type phenotype could be re-established by gene complementation, demonstrating the specificity of the PbPL deletion phenotype. In conclusion, we have identified for the first time a Plasmodium phospholipase that is important for PVM rupture and in turn for parasite exit from the infected hepatocyte and therefore established a key role of a parasite phospholipase in egress.     

ROS-Induced JNK and p38 Signaling Is Required for Unpaired Cytokine Activation during Drosophila Regeneration

Upon apoptotic stimuli, epithelial cells compensate the gaps left by dead cells by activating proliferation. This has led to the proposal that dying cells signal to surrounding living cells to maintain homeostasis. Although the nature of these signals is not clear, reactive oxygen species (ROS) could act as a signaling mechanism as they can trigger pro-inflammatory responses to protect epithelia from environmental insults. Whether ROS emerge from dead cells and what is the genetic response triggered by ROS is pivotal to understand regeneration of Drosophila imaginal discs. We genetically induced cell death in wing imaginal discs, monitored the production of ROS and analyzed the signals required for repair. We found that cell death generates a burst of ROS that propagate to the nearby surviving cells. Propagated ROS activate p38 and induce tolerable levels of JNK. The activation of JNK and p38 results in the expression of the cytokines Unpaired (Upd), which triggers the JAK/STAT signaling pathway required for regeneration. Our findings demonstrate that this ROS/JNK/p38/Upd stress responsive module restores tissue homeostasis. This module is not only activated after cell death induction but also after physical damage and reveals one of the earliest responses for imaginal disc regeneration.     

In Vivo Evidence for Lysosome Depletion and Impaired Autophagic Clearance in Hereditary Spastic Paraplegia Type SPG11

Hereditary spastic paraplegia (HSP) is characterized by a dying back degeneration of corticospinal axons which leads to progressive weakness and spasticity of the legs. SPG11 is the most common autosomal-recessive form of HSPs and is caused by mutations in SPG11. A recent in vitro study suggested that Spatacsin, the respective gene product, is needed for the recycling of lysosomes from autolysosomes, a process known as autophagic lysosome reformation. The relevance of this observation for hereditary spastic paraplegia, however, has remained unclear. Here, we report that disruption of Spatacsin in mice indeed causes hereditary spastic paraplegia-like phenotypes with loss of cortical neurons and Purkinje cells. Degenerating neurons accumulate autofluorescent material, which stains for the lysosomal protein Lamp1 and for p62, a marker of substrate destined to be degraded by autophagy, and hence appears to be related to autolysosomes. Supporting a more generalized defect of autophagy, levels of lipidated LC3 are increased in Spatacsin knockout mouse embryonic fibrobasts (MEFs). Though distinct parameters of lysosomal function like processing of cathepsin D and lysosomal pH are preserved, lysosome numbers are reduced in knockout MEFs and the recovery of lysosomes during sustained starvation impaired consistent with a defect of autophagic lysosome reformation. Because lysosomes are reduced in cortical neurons and Purkinje cells in vivo, we propose that the decreased number of lysosomes available for fusion with autophagosomes impairs autolysosomal clearance, results in the accumulation of undegraded material and finally causes death of particularly sensitive neurons like cortical motoneurons and Purkinje cells in knockout mice.     

SIRT5 regulation of ammonia-induced autophagy and mitophagy

In liver the mitochondrial sirtuin, SIRT5, controls ammonia detoxification by regulating CPS1, the first enzyme of the urea cycle. However, while SIRT5 is ubiquitously expressed, urea cycle and CPS1 are only present in the liver and, to a minor extent, in the kidney. To address the possibility that SIRT5 is involved in ammonia production also in nonliver cells, clones of human breast cancer cell lines MDA-MB-231 and mouse myoblast C2C12, overexpressing or silenced for SIRT5 were produced. Our results show that ammonia production increased in SIRT5-silenced and decreased in SIRT5-overexpressing cells. We also obtained the same ammonia increase when using a new specific inhibitor of SIRT5 called MC3482. SIRT5 regulates ammonia production by controlling glutamine metabolism. In fact, in the mitochondria, glutamine is transformed in glutamate by the enzyme glutaminase, a reaction producing ammonia. We found that SIRT5 and glutaminase coimmunoprecipitated and that SIRT5 inhibition resulted in an increased succinylation of glutaminase. We next determined that autophagy and mitophagy were increased by ammonia by measuring autophagic proteolysis of long-lived proteins, increase of autophagy markers MAP1LC3B, GABARAP, and GABARAPL2, mitophagy markers BNIP3 and the PINK1-PARK2 system as well as mitochondrial morphology and dynamics. We observed that autophagy and mitophagy increased in SIRT5-silenced cells and in WT cells treated with MC3482 and decreased in SIRT5-overexpressing cells. Moreover, glutaminase inhibition or glutamine withdrawal completely prevented autophagy. In conclusion we propose that the role of SIRT5 in nonliver cells is to regulate ammonia production and ammonia-induced autophagy by regulating glutamine metabolism.     

Effects of particulate matter exposure on blood 5-hydroxymethylation: results from the Beijing truck driver air pollution study

Previous studies have reported epigenetic changes induced by environmental exposures. However, previous investigations did not distinguish 5-methylcytosine (5mC) from a similar oxidative form with opposite functions, 5-hydroxymethylcytosine (5hmC). Here, we measured blood DNA global 5mC and 5hmC by ELISA and used adjusted mixed-effects regression models to evaluate the effects of ambient PM₁₀ and personal PM_2.5 and its elemental components—black carbon (BC), aluminum (Al), calcium (Ca), potassium (K), iron (Fe), sulfur (S), silicon (Si), titanium (Ti), and zinc (Zn)—on blood global 5mC and 5hmC levels. The study was conducted in 60 truck drivers and 60 office workers in Beijing, China from The Beijing Truck Driver Air Pollution Study at 2 exams separated by one to 2 weeks. Blood 5hmC level (0.08%) was ∼83-fold lower than 5mC (6.61%). An inter-quartile range (IQR) increase in same-day PM₁₀ was associated with increases in 5hmC of 26.1% in office workers (P = 0.004), 20.2% in truck drivers (P = 0.014), and 21.9% in all participants combined (P < 0.001). PM₁₀ effects on 5hmC were increasingly stronger when averaged over 4, 7, and 14 d preceding assessment (up to 132.6% for the 14-d average in all participants, P < 0.001). PM₁₀ effects were also significant after controlling for multiple testing (family-wise error rate; FWER < 0.05). 5hmC was not correlated with personal measures of PM_2.5 and elemental components (FWER > 0.05). 5mC showed no correlations with PM₁₀, PM_2.5, and elemental components measures (FWER > 0.05). Our study suggests that exposure to ambient PM₁₀ affects 5hmC over time, but not 5mC. This finding demonstrates the need to differentiate 5hmC and 5mC in environmental studies of DNA methylation.     

Probiotic characteristics and in vitro compatibility of a combination of Bifidobacterium breve M-16 V,Bifidobacterium longum subsp. infantis M-63 and Bifidobacterium longum subsp. longum BB536

The consumption of probiotic-based products has risen greatly in recent decades. Due to their probiotic characteristics, microorganisms such as lactobacilli and bifidobacteria are in daily use in the production of food supplements. In the present study, three bifidobacterial strains (Bifidobacterium breve M-16 V, Bifidobacterium longum subsp. infantis M-63 and Bifidobacterium longum subsp. longum BB536) were tested for growth compatibility, resistance to antimicrobial agents, antibacterial activity against pathogens, resistance to gastric acidity, bile salt hydrolysis and adhesion to the human intestinal epithelial cell line HT29. All of these strains were resistant to gentamycin, but none showed in vitro growth incompatibility or the presence of known resistance determinants. B. breve M-16 V had the best probiotic characteristics and, indeed, was the only strain possessing antibacterial activity against Escherichia coli and Klebsiella pneumoniae. All strains were resistant to simulated gastric juice, while only B. longum subsp. longum BB536 and B. breve M-16 V showed a bile salt hydrolytic activity. Interestingly, a strong adhesion to HT29 cells was observed in all Bifidobacterium strains. In conclusion, B. breve M-16 V, B. longum subsp. longum BB536 and B. longum subsp. infantis M-63 showed several promising characteristics as probiotic strains.