Mapping and characterizing N6-methyladenine in eukaryotic genomes using single-molecule real-time sequencing.

N6-Methyladenine (m6dA) has been discovered as a novel form of DNA methylation prevalent in eukaryotes; however, methods for high-resolution mapping of m6dA events are still lacking. Single-molecule real-time (SMRT) sequencing has enabled the detection of m6dA events at single-nucleotide resolution in prokaryotic genomes, but its application to detecting m6dA in eukaryotic genomes has not been rigorously examined. Herein, we identified unique characteristics of eukaryotic m6dA methylomes that fundamentally differ from those of prokaryotes. Based on these differences, we describe the first approach for mapping m6dA events using SMRT sequencing specifically designed for the study of eukaryotic genomes and provide appropriate strategies for designing experiments and carrying out sequencing in future studies. We apply the novel approach to study two eukaryotic genomes. For green algae, we construct the first complete genome-wide map of m6dA at single-nucleotide and single-molecule resolution. For human lymphoblastoid cells (hLCLs), it was necessary to integrate SMRT sequencing data with independent sequencing data. The joint analyses suggest putative m6dA events are enriched in the promoters of young full-length LINE-1 elements (L1s), but call for validation by additional methods. These analyses demonstrate a general method for rigorous mapping and characterization of m6dA events in eukaryotic genomes.

Immunotherapy using algal-produced Ara h 1 core domain suppresses peanut allergy in mice.

Peanut allergy is an IgE-mediated adverse reaction to a subset of proteins found in peanuts. Immunotherapy aims to desensitize allergic patients through repeated and escalating exposures for several months to years using extracts or flours. The complex mix of proteins and variability between preparations complicates immunotherapy studies. Moreover, peanut immunotherapy is associated with frequent negative side effects and patients are often at risk of allergic reactions once immunotherapy is discontinued. Allergen-specific approaches using recombinant proteins are an attractive alternative because they allow more precise dosing and the opportunity to engineer proteins with improved safety profiles. We tested whether Ara h 1 and Ara h 2, two major peanut allergens, could be produced using chloroplast of the unicellular eukaryotic alga, Chlamydomonas reinhardtii. C. reinhardtii is novel host for producing allergens that is genetically tractable, inexpensive and easy to grow, and is able to produce more complex proteins than bacterial hosts. Compared to the native proteins, algal-produced Ara h 1 core domain and Ara h 2 have a reduced affinity for IgE from peanut-allergic patients. We further found that immunotherapy using algal-produced Ara h 1 core domain confers protection from peanut-induced anaphylaxis in a murine model of peanut allergy.

Alga-produced malaria transmission-blocking vaccine candidate Pfs25 formulated with a human use-compatible potent adjuvant induces high-affinity antibodies that block Plasmodium falciparum infection of mosquitoes.

A vaccine to prevent the transmission of malaria parasites from infected humans to mosquitoes is an important component for the elimination of malaria in the 21st century, yet it remains neglected as a priority of malaria vaccine development. The lead candidate for Plasmodium falciparum transmission-blocking vaccine development, Pfs25, is a sexual stage surface protein that has been produced for vaccine testing in a variety of heterologous expression systems. Any realistic malaria vaccine will need to optimize proper folding balanced against cost of production, yield, and potentially reactogenic contaminants. Here Chlamydomonas reinhardtii microalga-produced recombinant Pfs25 protein was formulated with four different human-compatible adjuvants (alum, Toll-like receptor 4 [TLR-4] agonist glucopyranosal lipid A [GLA] plus alum, squalene-oil-in-water emulsion, and GLA plus squalene-oil-in-water emulsion) and compared for their ability to induce malaria transmission-blocking antibodies. Alga-produced recombinant Pfs25 plus GLA plus squalene-oil-in-water adjuvant induced the highest titer and avidity in IgG antibodies, measured using alga-produced recombinant Pfs25 as the enzyme-linked immunosorbent assay (ELISA) antigen. These antibodies specifically reacted with the surface of P. falciparum macrogametes and zygotes and effectively prevented parasites from developing within the mosquito vector in standard membrane feeding assays. Alga-produced Pfs25 in combination with a human-compatible adjuvant composed of a TLR-4 agonist in a squalene-oil-in-water emulsion is an attractive new vaccine candidate that merits head-to-head comparison with other modalities of vaccine production and administration.

Developing inexpensive malaria vaccines from plants and algae.

Malaria is a parasitic, mosquito-borne, infectious disease that threatens nearly half of the global population. The last decade has seen a dramatic drop in the number of malaria-related deaths because of vector control methods and anti-malarial drugs. Unfortunately, this strategy is not sustainable because of the emergence of insecticide-resistant mosquitoes and drug-resistant Plasmodium parasites. Eradication of malaria will ultimately require low-cost easily administered vaccines that work in concert with current control methods. Low cost and ease of administration will be essential components of any vaccine, because malaria endemic regions are poor and often lack an adequate healthcare infrastructure. Recently, several groups have begun addressing these issues using inexpensive photosynthetic organisms for producing vaccine antigens and exploring oral delivery strategies. Immune responses from plant-based injectable malaria vaccines are promising, but attempts to adapt these for oral delivery suggest we are far from a feasible strategy. Here, we review examples of these technologies and discuss the progress and potential of this research, as well as the obstacles ahead.

New therapies on the horizon: Targeted protein degradation in neuroscience.

This minireview explores the burgeoning field of targeted protein degradation (TPD) and its promising applications in neuroscience and clinical development. TPD offers innovative strategies for modulating protein levels, presenting a paradigm shift in small-molecule drug discovery and therapeutic interventions. Importantly, small-molecule protein degraders specifically target and remove pathogenic proteins from central nervous system cells without the drug delivery challenges of genomic and antibody-based modalities. Here, we review recent advancements in TPD technologies, highlight proteolysis targeting chimera (PROTAC) protein degrader molecules with proximity-induced degradation event-driven and iterative pharmacology, provide applications in neuroscience research, and discuss the high potential for translation of TPD into clinical settings.

Nutrient Intake, Performance, and Body Composition of Preseason Wrestlers.

College athletes, especially in weight class sports, often experience energy deficits. Athletes competing in weight class sports such as wrestling are at greatest risk for deficiencies and little is known about the relationships between body composition, nutrient intake, and performance in these athletes. The purposes of this study were to (1) quantify macronutrient and micronutrient intake of pre-season male collegiate wrestlers and compare to estimated needs, and (2) examine relationships among nutritional intakes, body composition, and performance measurements of strength and anaerobic capacity. Male Division I wrestlers (n = 11, age: 21.3 ± 1.7 years, wrestling experience: 14.9 ± 2.5 years) were recruited during pre-season. Nutrient intake was collected from a 3-day food diary. A 7-site skinfold assessment determined fat-free mass (FFM) to estimate total daily energy expenditure (eTEE). Isokinetic and isometric strength were evaluated by a Biodex dynamometer. Anaerobic capacity was tested on a stationary cycle ergometer. Eight of eleven wrestlers were energy deficient based on estimated needs. Mean intake of four micronutrients fell below the Recommended Dietary Allowances (RDA). Significant correlations were found between dietary intake, strength and anaerobic performance variables (r = 0.603 - 0.902, p = 0.0001 - 0.05). However, after accounting for FFM, these relationships were no longer significant. Nutrient intake in tandem with body composition affects performance for weight class athletes. Achieving high FFM during the preseason may be advantageous for wrestling performance. Nutrient intake and body composition should be monitored so coaches and health professionals can create individualized recommendations to help athletes optimize performance.

Alga-produced cholera toxin-Pfs25 fusion proteins as oral vaccines.

Infectious diseases disproportionately affect indigent regions and are the greatest cause of childhood mortality in developing countries. Practical, low-cost vaccines for use in these countries are paramount to reducing disease burdens and concomitant poverty. Algae are a promising low-cost system for producing vaccines that can be orally delivered, thereby avoiding expensive purification and injectable delivery. We engineered the chloroplast of the eukaryotic alga Chlamydomonas reinhardtii to produce a chimeric protein consisting of the 25-kDa Plasmodium falciparum surface protein (Pfs25) fused to the ß subunit of the cholera toxin (CtxB) to investigate an alga-based whole-cell oral vaccine. Pfs25 is a promising malaria transmission-blocking vaccine candidate that has been difficult to produce in traditional recombinant systems due to its structurally complex tandem repeats of epidermal growth factor-like domains. The noncatalytic CtxB domain of the cholera holotoxin assembles into a pentameric structure and acts as a mucosal adjuvant by binding GM1 ganglioside receptors on gut epithelial cells. We demonstrate that CtxB-Pfs25 accumulates as a soluble, properly folded and functional protein within algal chloroplasts, and it is stable in freeze-dried alga cells at ambient temperatures. In mice, oral vaccination using freeze-dried algae that produce CtxB-Pfs25 elicited CtxB-specific serum IgG antibodies and both CtxB- and Pfs25-specific secretory IgA antibodies. These data suggest that algae are a promising system for production and oral delivery of vaccine antigens, but as an orally delivered adjuvant, CtxB is best suited for eliciting secretory IgA antibodies for vaccine antigens against pathogens that invade mucosal surfaces using this strategy.

Heterologous expression of the C-terminal antigenic domain of the malaria vaccine candidate Pfs48/45 in the green algae Chlamydomonas reinhardtii.

Malaria is a widespread and infectious disease that is a leading cause of death in many parts of the world. Eradication of malaria has been a major world health goal for decades, but one that still remains elusive. Other diseases have been eradicated using vaccination, but traditional vaccination methods have thus far been unsuccessful for malaria. Infection by Plasmodium species, the causative agent of malaria, is currently treated with drug-based therapies, but an increase in drug resistance has led to the need for new methods of treatment. A promising strategy for malaria treatment is to combine transmission blocking vaccines (TBVs) that prevent spread of disease with drug-based therapies to treat infected individuals. TBVs can be developed against surface protein antigens that are expressed during parasite reproduction in the mosquito. When the mosquito ingests blood from a vaccinated individual harboring the Plasmodium parasite, the antibodies generated by vaccination prevent completion of the parasites life-cycle. Animal studies have shown that immunization with Pfs48/45 results in the production of malaria transmission blocking antibodies; however, the development of this vaccine candidate has been hindered by poor expression in both prokaryotic and eukaryotic hosts. Recently, the chloroplast of Chlamydomonas reinhardtii has been used to express complex recombinant proteins. In this study, we show that the C-terminal antigenic region of the Pfs48/45 antigen can be expressed in the chloroplast of the green algae C. reinhardtii and that this recombinant protein has a conformation recognized by known transmission blocking antibodies. Production of this protein in algae has the potential to scale to the very large volumes required to meet the needs of millions at risk for contracting malaria.

Cell Type-Specific In Vitro Gene Expression Profiling of Stem Cell-Derived Neural Models.

Genetic and genomic studies of brain disease increasingly demonstrate disease-associated interactions between the cell types of the brain. Increasingly complex and more physiologically relevant human-induced pluripotent stem cell (hiPSC)-based models better explore the molecular mechanisms underlying disease but also challenge our ability to resolve cell type-specific perturbations. Here, we report an extension of the RiboTag system, first developed to achieve cell type-restricted expression of epitope-tagged ribosomal protein (RPL22) in mouse tissue, to a variety of in vitro applications, including immortalized cell lines, primary mouse astrocytes, and hiPSC-derived neurons. RiboTag expression enables depletion of up to 87 percent of off-target RNA in mixed species co-cultures. Nonetheless, depletion efficiency varies across independent experimental replicates, particularly for hiPSC-derived motor neurons. The challenges and potential of implementing RiboTags in complex in vitro cultures are discussed.

The induction of macrophage gene expression by LPS predominantly utilizes Myd88-independent signaling cascades.

Myeloid differentiation protein-88 (MyD88) is a signal adaptor protein required for cytokine production following engagement of Toll-like receptors (TLRs) by their cognate ligands. Activation of both TLR-3 and TLR-4, however, can engage signaling events independent of MyD88 expression. The relative importance of these MyD88-dependent and -independent signaling pathways in the macrophage response to lipopolysaccharide (LPS) is unknown. Here we define these events using microarray expression profiling of LPS-stimulated macrophages taken from MyD88-null and wild-type mice. Of the 1,055 genes found to be LPS responsive, only 21.5% were dependent on MyD88 expression, with MyD88-independent genes constituting 74.7% of the genetic response. This MyD88-independent gene expression was predominantly transcriptionally regulated, as it was unaffected by cycloheximide blockade of new protein synthesis. A previously undescribed group of LPS-regulated genes (3.8%), whose induction or repression was significantly greater in the absence of MyD88, was also identified by these studies. The regulation of these genes suggested that MyD88 could serve as a molecular brake, constraining gene activity in a subset of LPS-responsive genes. The findings generated with LPS stimulation were recapitulated by exposure of macrophages to live Escherichia coli. These expression-profiling studies redefine the current dogma of TLR-4 signaling and establish that MyD88, although essential for some of the best-characterized macrophage responses to LPS, is not required for the regulation of the majority of genes engaged by macrophage exposure to endotoxin or live bacteria.

Algae-produced Pfs25 elicits antibodies that inhibit malaria transmission.

Subunit vaccines are significantly more expensive to produce than traditional vaccines because they are based primarily on recombinant proteins that must be purified from the expression system. Despite the increased cost, subunit vaccines are being developed because they are safe, effective, and can elicit antibodies that confer protection against diseases that are not currently vaccine-preventable. Algae are an attractive platform for producing subunit vaccines because they are relatively inexpensive to grow, genetically tractable, easily scaled to large volumes, have a short generation time, and are devoid of inflammatory, viral, or prion contaminants often present in other systems. We tested whether algal chloroplasts can produce malaria transmission blocking vaccine candidates, Plasmodium falciparum surface protein 25 (Pfs25) and 28 (Pfs28). Antibodies that recognize Pfs25 and Pfs28 disrupt the sexual development of parasites within the mosquito midgut, thus preventing transmission of malaria from one human host to the next. These proteins have been difficult to produce in traditional recombinant systems because they contain tandem repeats of structurally complex epidermal growth factor-like domains, which cannot be produced in bacterial systems, and because they are not glycosylated, so they must be modified for production in eukaryotic systems. Production in algal chloroplasts avoids these issues because chloroplasts can fold complex eukaryotic proteins and do not glycosylate proteins. Here we demonstrate that algae are the first recombinant system to successfully produce an unmodified and aglycosylated version of Pfs25 or Pfs28. These antigens are structurally similar to the native proteins and antibodies raised to these recombinant proteins recognize Pfs25 and Pfs28 from P. falciparum. Furthermore, antibodies to algae-produced Pfs25 bind the surface of in-vitro cultured P. falciparum sexual stage parasites and exhibit transmission blocking activity. Thus, algae are promising organisms for producing cysteine-disulfide-containing malaria transmission blocking vaccine candidate proteins.

Cellular architecture mediates DivIVA ultrastructure and regulates min activity in Bacillus subtilis.

UNLABELLED: The assembly of the cell division machinery at midcell is a critical step of cytokinesis. Many rod-shaped bacteria position septa using nucleoid occlusion, which prevents division over the chromosome, and the Min system, which prevents division near the poles. Here we examined the in vivo assembly of the Bacillus subtilis MinCD targeting proteins DivIVA, a peripheral membrane protein that preferentially localizes to negatively curved membranes and resembles eukaryotic tropomyosins, and MinJ, which recruits MinCD to DivIVA. We used structured illumination microscopy to demonstrate that both DivIVA and MinJ localize as double rings that flank the septum and first appear early in septal biosynthesis. The subsequent recruitment of MinCD to these double rings would separate the Min proteins from their target, FtsZ, spatially regulating Min activity and allowing continued cell division. Curvature-based localization would also provide temporal regulation, since DivIVA and the Min proteins would localize to midcell after the onset of division. We use time-lapse microscopy and fluorescence recovery after photobleaching to demonstrate that DivIVA rings are highly stable and are constructed from newly synthesized DivIVA molecules. After cell division, DivIVA rings appear to collapse into patches at the rounded cell poles of separated cells, with little or no incorporation of newly synthesized subunits. Thus, changes in cell architecture mediate both the initial recruitment of DivIVA to sites of cell division and the subsequent collapse of these rings into patches (or rings of smaller diameter), while curvature-based localization of DivIVA spatially and temporally regulates Min activity. IMPORTANCE: The Min systems of Escherichia coli and Bacillus subtilis both inhibit FtsZ assembly, but one key difference between these two species is that whereas the E. coli Min proteins localize to the poles, the B. subtilis proteins localize to nascent division sites by interaction with DivIVA and MinJ. It is unclear how MinC activity at midcell is regulated to prevent it from interfering with FtsZ engaged in medial cell division. We used superresolution microscopy to demonstrate that DivIVA and MinJ, which localize MinCD, assemble double rings that flank active division sites and septa. This curvature-based localization mechanism holds MinCD away from the FtsZ ring at midcell, and we propose that this spatial organization is the primary mechanism by which MinC activity is regulated to allow division at midcell. Curvature-based localization also conveys temporal regulation, since it ensures that MinC localizes after the onset of division.

Transposon assisted gene insertion technology (TAGIT): a tool for generating fluorescent fusion proteins.

We constructed a transposon (transposon assisted gene insertion technology, or TAGIT) that allows the random insertion of gfp (or other genes) into chromosomal loci without disrupting operon structure or regulation. TAGIT is a modified Tn5 transposon that uses Kan(R) to select for insertions on the chromosome or plasmid, beta-galactosidase to identify in-frame gene fusions, and Cre recombinase to excise the kan and lacZ genes in vivo. The resulting gfp insertions maintain target gene reading frame (to the 5' and 3' of gfp) and are integrated at the native chromosomal locus, thereby maintaining native expression signals. Libraries can be screened to identify GFP insertions that maintain target protein function at native expression levels, allowing more trustworthy localization studies. We here use TAGIT to generate a library of GFP insertions in the Escherichia coli lactose repressor (LacI). We identified fully functional GFP insertions and partially functional insertions that bind DNA but fail to repress the lacZ operon. Several of these latter GFP insertions localize to lacO arrays integrated in the E. coli chromosome without producing the elongated cells frequently observed when functional LacI-GFP fusions are used in chromosome tagging experiments. TAGIT thereby faciliates the isolation of fully functional insertions of fluorescent proteins into target proteins expressed from the native chromosomal locus as well as potentially useful partially functional proteins.

Bacillus subtilis MinC destabilizes FtsZ-rings at new cell poles and contributes to the timing of cell division.

Division site selection in rod-shaped bacteria depends on nucleoid occlusion, which prevents division over the chromosome and MinCD, which prevent division at the poles. MinD is thought to localize MinC to the cell poles where it prevents FtsZ assembly. Time-lapse microscopy demonstrates that in Bacillus subtilis transient polar FtsZ rings assemble adjacent to recently completed septa and that in minCD strains these persist and are used for division, producing a minicell. This suggests that MinC acts when division proteins are released from newly completed septa to prevent their immediate reassembly at new cell poles. The minCD mutant appears to uncouple FtsZ ring assembly from cell division and thus shows a variable interdivisional time and a rapid loss of cell cycle synchrony. Functional MinC-GFP expressed from the chromosome minCD locus is dynamic. It is recruited to active division sites before septal biogenesis, rotates around the septum, and moves away from completed septa. Thus high concentrations of MinC are found primarily at the septum and, more transiently, at the new cell pole. DivIVA and MinD recruit MinC to division sites, rather than mediating the stable polar localization previously thought to restrict MinC activity to the pole. Together, our results suggest that B. subtilis MinC does not inhibit FtsZ assembly at the cell poles, but rather prevents polar FtsZ rings adjacent to new cell poles from supporting cell division.