A key cytosolic iron-sulfur cluster synthesis protein localizes to the mitochondrion of Toxoplasma gondii.

Iron-sulfur (Fe-S) clusters are prosthetic groups on proteins that function in a range of enzymatic and electron transfer reactions. Fe-S cluster synthesis is essential for the survival of all eukaryotes. Independent Fe-S cluster biosynthesis pathways occur in the mitochondrion, plastid, and cytosolic compartments of eukaryotic cells. Little is known about the cytosolic Fe-S cluster biosynthesis in apicomplexan parasites, the causative agents of diseases such as malaria and toxoplasmosis. NBP35 serves as a key scaffold protein on which cytosolic Fe-S clusters assemble, and has a cytosolic localization in most eukaryotes studied thus far. Unexpectedly, we found that the NBP35 homolog of the apicomplexan Toxoplasma gondii (TgNBP35) localizes to the outer mitochondrial membrane, with mitochondrial targeting mediated by an N-terminal transmembrane domain. We demonstrate that TgNBP35 is critical for parasite proliferation, but that, despite its mitochondrial localization, it is not required for Fe-S cluster synthesis in the mitochondrion. Instead, we establish that TgNBP35 is important for the biogenesis of cytosolic Fe-S proteins. Our data are consistent with TgNBP35 playing a central and specific role in cytosolic Fe-S cluster biosynthesis, and imply that the assembly of cytosolic Fe-S clusters occurs on the cytosolic face of the outer mitochondrial membrane in these parasites.

Targeting NLRP3 and Staphylococcal pore-forming toxin receptors in human-induced pluripotent stem cell-derived macrophages.

Staphylococcus aureus causes necrotizing pneumonia by secreting toxins such as leukocidins that target front-line immune cells. The mechanism by which leukocidins kill innate immune cells and trigger inflammation during S. aureus lung infection, however, remains unresolved. Here, we explored human-induced pluripotent stem cell-derived macrophages (hiPSC-dMs) to study the interaction of the leukocidins Panton-Valentine leukocidin (PVL) and LukAB with lung macrophages, which are the initial leukocidin targets during S. aureus lung invasion. hiPSC-dMs were susceptible to the leukocidins PVL and LukAB and both leukocidins triggered NLPR3 inflammasome activation resulting in IL-1ß secretion. hiPSC-dM cell death after LukAB exposure, however, was only temporarily dependent of NLRP3, although NLRP3 triggered marked cell death after PVL treatment. CRISPR/Cas9-mediated deletion of the PVL receptor, C5aR1, protected hiPSC-dMs from PVL cytotoxicity, despite the expression of other leukocidin receptors, such as CD45. PVL-deficient S. aureus had reduced ability to induce lung IL-1ß levels in human C5aR1 knock-in mice. Unexpectedly, inhibiting NLRP3 activity resulted in increased wild-type S. aureus lung burdens. Our findings suggest that NLRP3 induces macrophage death and IL-1ß secretion after PVL exposure and controls S. aureus lung burdens.

Benchmarking single cell RNA-sequencing analysis pipelines using mixture control experiments.

Single cell RNA-sequencing (scRNA-seq) technology has undergone rapid development in recent years, leading to an explosion in the number of tailored data analysis methods. However, the current lack of gold-standard benchmark datasets makes it difficult for researchers to systematically compare the performance of the many methods available. Here, we generated a realistic benchmark experiment that included single cells and admixtures of cells or RNA to create 'pseudo cells' from up to five distinct cancer cell lines. In total, 14 datasets were generated using both droplet and plate-based scRNA-seq protocols. We compared 3,913 combinations of data analysis methods for tasks ranging from normalization and imputation to clustering, trajectory analysis and data integration. Evaluation revealed pipelines suited to different types of data for different tasks. Our data and analysis provide a comprehensive framework for benchmarking most common scRNA-seq analysis steps.

Elucidating the mitochondrial proteome of Toxoplasma gondii reveals the presence of a divergent cytochrome c oxidase.

The mitochondrion of apicomplexan parasites is critical for parasite survival, although the full complement of proteins that localize to this organelle has not been defined. Here we undertake two independent approaches to elucidate the mitochondrial proteome of the apicomplexan Toxoplasma gondii. We identify approximately 400 mitochondrial proteins, many of which lack homologs in the animals that these parasites infect, and most of which are important for parasite growth. We demonstrate that one such protein, termed TgApiCox25, is an important component of the parasite cytochrome c oxidase (COX) complex. We identify numerous other apicomplexan-specific components of COX, and conclude that apicomplexan COX, and apicomplexan mitochondria more generally, differ substantially in their protein composition from the hosts they infect. Our study highlights the diversity that exists in mitochondrial proteomes across the eukaryotic domain of life, and provides a foundation for defining unique aspects of mitochondrial biology in an important phylum of parasites.

scPipe: A flexible R/Bioconductor preprocessing pipeline for single-cell RNA-sequencing data.

Single-cell RNA sequencing (scRNA-seq) technology allows researchers to profile the transcriptomes of thousands of cells simultaneously. Protocols that incorporate both designed and random barcodes have greatly increased the throughput of scRNA-seq, but give rise to a more complex data structure. There is a need for new tools that can handle the various barcoding strategies used by different protocols and exploit this information for quality assessment at the sample-level and provide effective visualization of these results in preparation for higher-level analyses. To this end, we developed scPipe, an R/Bioconductor package that integrates barcode demultiplexing, read alignment, UMI-aware gene-level quantification and quality control of raw sequencing data generated by multiple protocols that include CEL-seq, MARS-seq, Chromium 10X, Drop-seq and Smart-seq. scPipe produces a count matrix that is essential for downstream analysis along with an HTML report that summarises data quality. These results can be used as input for downstream analyses including normalization, visualization and statistical testing. scPipe performs this processing in a few simple R commands, promoting reproducible analysis of single-cell data that is compatible with the emerging suite of open-source scRNA-seq analysis tools available in R/Bioconductor and beyond. The scPipe R package is available for download from https://www.bioconductor.org/packages/scPipe.

Legionella pneumophila Strain 130b Evades Macrophage Cell Death Independent of the Effector SidF in the Absence of Flagellin.

The human pathogen Legionella pneumophila must evade host cell death signaling to enable replication in lung macrophages and to cause disease. After bacterial growth, however, L. pneumophila is thought to induce apoptosis during egress from macrophages. The bacterial effector protein, SidF, has been shown to control host cell survival and death by inhibiting pro-apoptotic BNIP3 and BCL-RAMBO signaling. Using live-cell imaging to follow the L. pneumophila-macrophage interaction, we now demonstrate that L. pneumophila evades host cell apoptosis independent of SidF. In the absence of SidF, L. pneumophila was able to replicate, cause loss of mitochondria membrane potential, kill macrophages, and establish infections in lungs of mice. Consistent with this, deletion of BNIP3 and BCL-RAMBO did not affect intracellular L. pneumophila replication, macrophage death rates, and in vivo bacterial virulence. Abrogating mitochondrial cell death by genetic deletion of the effectors of intrinsic apoptosis, BAX, and BAK, or the regulator of mitochondrial permeability transition pore formation, cyclophilin-D, did not affect bacterial growth or the initial killing of macrophages. Loss of BAX and BAK only marginally limited the ability of L. pneumophila to efficiently kill all macrophages over extended periods. L. pneumophila induced killing of macrophages was delayed in the absence of capsase-11 mediated pyroptosis. Together, our data demonstrate that L. pneumophila evades host cell death responses independently of SidF during replication and can induce pyroptosis to kill macrophages in a timely manner.

Novel Characteristics of Photodamage to PSII in a High-Light-Sensitive Symbiodinium Phylotype.

Dinoflagellates from the genus Symbiodinium form symbiotic relationships with many marine invertebrates, including reef-building corals. Symbiodinium is genetically diverse, and acquiring suitable Symbiodinium phylotypes is crucial for the host to survive in habitat environments, such as high-light conditions. The sensitivity of Symbiodinium to high light differs among Symbiodinium phylotypes, but the mechanism that controls light sensitivity has not yet been fully resolved. In the present study using high-light-tolerant and -sensitive Symbiodinium phylotypes, we examined what determines sensitivity to high light. In growth experiments under different light intensities, Symbiodinium CS-164 (clade B1) and CCMP2459 (clade B2) were identified as high-light-tolerant and -sensitive phylotypes, respectively. Measurements of the maximum quantum yield of photosystem II (PSII) and the maximum photosynthetic oxygen production rate after high-light exposure demonstrated that CCMP2459 is more sensitive to photoinhibition of PSII than CS-164, and tends to lose maximum photosynthetic activity faster. Measurement of photodamage to PSII under light of different wavelength ranges demonstrated that PSII in both Symbiodinium phylotypes was significantly more sensitive to photodamage under shorter wavelength regions of light spectra (<470 nm). Importantly, PSII in CCMP2459, but not CS-164, was also sensitive to photodamage under the regions of light spectra around 470-550 and 630-710 nm, where photosynthetic antenna proteins of Symbiodinium have light absorption peaks. This finding indicates that the high-light-sensitive CCMP2459 has an extra component of photodamage to PSII, resulting in higher sensitivity to high light. Our results demonstrate that sensitivity of PSII to photodamage differs among Symbiodinium phylotypes and this determines their sensitivity to high light.

Gradient nanofiber scaffolds for tissue engineering.

Scaffolds are one of the key factors for the success of tissue engineering, in particular when dealing with anchorage-dependent cells. The concept of using scaffolds in tissue engineering lies in mimicking the physical, chemical and biological features of native extracellular matrix (ECM) in order to support cell function, which in turn regulates cellular microenvironment that directs cell growth and subsequent tissue formation. Nanofibers fabricated from both synthetic and natural polymers are being used as scaffolds in many tissue engineering applications. At the molecular level, native ECM is made up of a gradient of fibrous proteins and polysaccharides that are nanoscale structures. The gradient cues of ECM, directs critical cell behaviors such as alignment, motility and differentiation, particularly in the region between soft and hard tissues called interfacial tissue. Therefore, it is essential to develop gradient nanofiber scaffolds particularly for interfacial tissue engineering applications. Keeping these points in view, in this article, we review the recent developments of gradient nanofiber scaffolds, their design strategies, and their applications in tissue engineering.

Draft assembly of the Symbiodinium minutum nuclear genome reveals dinoflagellate gene structure.

BACKGROUND: Dinoflagellates are known for their capacity to form harmful blooms (e.g., "red tides") and as symbiotic, photosynthetic partners for corals. These unicellular eukaryotes have permanently condensed, liquid-crystalline chromosomes and immense nuclear genome sizes, often several times the size of the human genome. Here we describe the first draft assembly of a dinoflagellate nuclear genome, providing insights into its genome organization and gene inventory. RESULTS: Sequencing reads from Symbiodinium minutum were assembled into 616 Mbp gene-rich DNA regions that represented roughly half of the estimated 1,500 Mbp genome of this species. The assembly encoded ∼42,000 protein-coding genes, consistent with previous dinoflagellate gene number estimates using transcriptomic data. The Symbiodinium genome contains duplicated genes for regulator of chromosome condensation proteins, nearly one-third of which have eukaryotic orthologs, whereas the remainder have most likely been acquired through bacterial horizontal gene transfers. Symbiodinium genes are enriched in spliceosomal introns (mean = 18.6 introns/gene). Donor and acceptor splice sites are unique, with 5' sites utilizing not only GT but also GC and GA, whereas at 3' sites, a conserved G is present after AG. All spliceosomal snRNA genes (U1-U6) are clustered in the genome. Surprisingly, the Symbiodinium genome displays unidirectionally aligned genes throughout the genome, forming a cluster-like gene arrangement. CONCLUSIONS: We show here that a dinoflagellate genome exhibits unique and divergent characteristics when compared to those of other eukaryotes. Our data elucidate the organization and gene inventory of dinoflagellates and lay the foundation for future studies of this remarkable group of eukaryotes.

Controlled release of drugs from gradient hydrogels for high-throughput analysis of cell-drug interactions.

In this paper, we report a method to fabricate microengineered hydrogels that contain a concentration gradient of a drug for high-throughput analysis of cell-drug interactions. A microfluidic gradient generator was used to create a concentration gradient of okadaic acid (OA) as a model drug within poly(ethylene glycol) diacrylate hydrogels. These hydrogels were then incubated with MC3T3-E1 cell seeded glass slides to investigate the cell viability through the spatially controlled release of OA. The drug was released from the hydrogel in a gradient manner and induced a gradient of the cell viability. The drug concentration gradient containing hydrogels developed in this study have the potential to be used for drug discovery and diagnostics applications due to their ability to simultaneously test the effects of different concentrations of various chemicals.

SHP2 tyrosine phosphatase converts parafibromin/Cdc73 from a tumor suppressor to an oncogenic driver.

Deregulation of SHP2 is associated with malignant diseases as well as developmental disorders. Although SHP2 is required for full activation of RAS signaling, other potential roles in cell physiology have not been elucidated. Here we show that SHP2 dephosphorylates parafibromin/Cdc73, a core component of the RNA polymerase II-associated factor (PAF) complex. Parafibromin is known to act as a tumor suppressor that inhibits cyclin D1 and c-myc by recruiting SUV39H1 histone methyltransferase. However, parafibromin can also act in the opposing direction by binding ß-catenin, thereby activating promitogenic/oncogenic Wnt signaling. We found that, on tyrosine dephosphorylation by SHP2, parafibromin acquires the ability to stably bind ß-catenin. The parafibromin/ß-catenin interaction overrides parafibromin/SUV39H1-mediated transrepression and induces expression of Wnt target genes, including cyclin D1 and c-myc. Hence, SHP2 governs the opposing functions of parafibromin, deregulation of which may cause the development of tumors or developmental malformations.

A microfluidic-based neurotoxin concentration gradient for the generation of an in vitro model of Parkinson's disease.

In this study, we developed a miniaturized microfluidic-based high-throughput cell toxicity assay to create an in vitro model of Parkinson's disease (PD). In particular, we generated concentration gradients of 6-hydroxydopamine (6-OHDA) to trigger a process of neuronal apoptosis in pheochromocytoma PC12 neuronal cell line. PC12 cells were cultured in a microfluidic channel, and a concentration gradient of 6-OHDA was generated in the channel by using a back and forth movement of the fluid flow. Cellular apoptosis was then analyzed along the channel. The results indicate that at low concentrations of 6-OHDA along the gradient (i.e., approximately less than 260 µM), the neuronal death in the channel was mainly induced by apoptosis, while at higher concentrations, 6-OHDA induced neuronal death mainly through necrosis. Thus, this concentration appears to be useful for creating an in vitro model of PD by inducing the highest level of apoptosis in PC12 cells. As microfluidic systems are advantageous in a range of properties such as throughput and lower use of reagents, they may provide a useful approach for generating in vitro models of disease for drug discovery applications.

Gradient biomaterials for soft-to-hard interface tissue engineering.

Interface tissue engineering (ITE) is a rapidly developing field that aims to fabricate biological tissue alternates with the goal of repairing or regenerating the functions of diseased or damaged zones at the interface of different tissue types (also called "interface tissues"). Notable examples of the interface tissues in the human body include ligament-to-bone, tendon-to-bone and cartilage-to-bone. Engineering interface tissues is a complex process, which requires a combination of specialized biomaterials with spatially organized material composition, cell types and signaling molecules. Therefore, the use of conventional biomaterials (monophasic or composites) for ITE has certain limitations to help stimulate the tissue integration or recreating the structural organization at the junction of different tissue types. The advancement of micro- and nanotechnologies enable us to develop systems with gradients in biomaterials properties that encourage the differentiation of multiple cell phenotypes and subsequent tissue development. In this review we discuss recent developments in the fabrication of gradient biomaterials for controlling cellular behavior such as migration, differentiation and heterotypic interactions. Moreover, we give an overview of potential uses of gradient biomaterials in engineering interface tissues such as soft tissues (e.g. cartilage) to hard tissues (e.g. bone), with illustrated experimental examples. We also address fundamentals of interface tissue organization, various gradient biomaterials used in ITE, micro- and nanotechnologies employed for the fabrication of those gradients, and certain challenges that must be met in order for ITE to reach its full potential.

Recombination system based on cre alpha complementation and leucine zipper fusions.

In a previous study, a recombination system based on the alpha complementation of cre recombinase and protein transduction was established. This system relied on the transient expression of the inactive, self-excisable C-terminal (beta) and the transduction of the N-terminal (alpha) cre fragments to cells as a purified protein. This recombination system potentially results in a less invasive and more controllable cre recombination in mammalian cells. In this study, we have employed a more efficient complementation triggering sequence using more than only the overlapping amino acids to help the alpha and beta fragments reassociate. In order to increase the association efficiency of the complementing fragments of cre recombinase, we chose to use a fusion of cre fragments to a self-heterodimerizing pair of proteins to trigger their binding and thus increase the efficiency of the restored enzymatic activity. For this purpose, the leucine zipper motifs (bJun and bFos) of the AP-1 transcription were fused to cre fragments (alpha and beta, respectively). This resulted in an increased reassociation efficiency of the fragments and a two times more efficient recombination system compared with the previous study.

Novel recombination system using Cre recombinase alpha complementation.

A major limitation for the use of Cre recombinase is its toxicity and a lack of temporal control over its activity. We have developed a new recombination system using Cre recombinase alpha-complementation. Cre recombinase was divided and one fragment (beta) was introduced into cells between two loxP sites with a CMV promoter in the upstream. The gene of interest (EGFP) was positioned just downstream of this construct. Cre recombinase activity was recovered by adding the other part of the molecule (alpha) to cells as a protein fragment, as evidenced by the expression of EGFP under the control of the CMV promoter. The activity of fragmented cre reached 68% of that of the wild type enzyme at 1 microM alpha-protein.