A Major Role for the Plasmodium falciparum ApiAP2 Protein PfSIP2 in Chromosome End Biology

The heterochromatic environment and physical clustering of chromosome ends at the nuclear periphery provide a functional and structural framework for antigenic variation and evolution of subtelomeric virulence gene families in the malaria parasite Plasmodium falciparum. While recent studies assigned important roles for reversible histone modifications, silent information regulator 2 and heterochromatin protein 1 (PfHP1) in epigenetic control of variegated expression, factors involved in the recruitment and organization of subtelomeric heterochromatin remain unknown. Here, we describe the purification and characterization of PfSIP2, a member of the ApiAP2 family of putative transcription factors, as the unknown nuclear factor interacting specifically with cis-acting SPE2 motif arrays in subtelomeric domains. Interestingly, SPE2 is not bound by the full-length protein but rather by a 60kDa N-terminal domain, PfSIP2-N, which is released during schizogony. Our experimental re-definition of the SPE2/PfSIP2-N interaction highlights the strict requirement of both adjacent AP2 domains and a conserved bipartite SPE2 consensus motif for high-affinity binding. Genome-wide in silico mapping identified 777 putative binding sites, 94% of which cluster in heterochromatic domains upstream of subtelomeric var genes and in telomere-associated repeat elements. Immunofluorescence and chromatin immunoprecipitation (ChIP) assays revealed co-localization of PfSIP2-N with PfHP1 at chromosome ends. Genome-wide ChIP demonstrated the exclusive binding of PfSIP2-N to subtelomeric SPE2 landmarks in vivo but not to single chromosome-internal sites. Consistent with this specialized distribution pattern, PfSIP2-N over-expression has no effect on global gene transcription. Hence, contrary to the previously proposed role for this factor in gene activation, our results provide strong evidence for the first time for the involvement of an ApiAP2 factor in heterochromatin formation and genome integrity. These findings are highly relevant for our understanding of chromosome end biology and variegated expression in P. falciparum and other eukaryotes, and for the future analysis of the role of ApiAP2-DNA interactions in parasite biology

Symbiodinium Transcriptomes: Genome Insights into the Dinoflagellate Symbionts of Reef-Building Corals

Dinoflagellates are unicellular algae that are ubiquitously abundant in aquatic environments. Species of the genus Symbiodinium form symbiotic relationships with reef-building corals and other marine invertebrates. Despite their ecologic importance, little is known about the genetics of dinoflagellates in general and Symbiodinium in particular. Here, we used 454 sequencing to generate transcriptome data from two Symbiodinium species from different clades (clade A and clade B). With more than 56,000 assembled sequences per species, these data represent the largest transcriptomic resource for dinoflagellates to date. Our results corroborate previous observations that dinoflagellates possess the complete nucleosome machinery. We found a complete set of core histones as well as several H3 variants and H2A.Z in one species. Furthermore, transcriptome analysis points toward a low number of transcription factors in Symbiodinium spp. that also differ in the distribution of DNA-binding domains relative to other eukaryotes. In particular the cold shock domain was predominant among transcription factors. Additionally, we found a high number of antioxidative genes in comparison to non-symbiotic but evolutionary related organisms. These findings might be of relevance in the context of the role that Symbiodinium spp. play as coral symbionts

Identification and Genome-Wide Prediction of DNA Binding Specificities for the ApiAP2 Family of Regulators from the Malaria Parasite

The molecular mechanisms underlying transcriptional regulation in apicomplexan parasites remain poorly understood. Recently, the Apicomplexan AP2 (ApiAP2) family of DNA binding proteins was identified as a major class of transcriptional regulators that are found across all Apicomplexa. To gain insight into the regulatory role of these proteins in the malaria parasite, we have comprehensively surveyed the DNA-binding specificities of all 27 members of the ApiAP2 protein family from Plasmodium falciparum revealing unique binding preferences for the majority of these DNA binding proteins. In addition to high affinity primary motif interactions, we also observe interactions with secondary motifs. The ability of a number of ApiAP2 proteins to bind multiple, distinct motifs significantly increases the potential complexity of the transcriptional regulatory networks governed by the ApiAP2 family. Using these newly identified sequence motifs, we infer the trans-factors associated with previously reported plasmodial cis-elements and provide evidence that ApiAP2 proteins modulate key regulatory decisions at all stages of parasite development. Our results offer a detailed view of ApiAP2 DNA binding specificity and take the first step toward inferring comprehensive gene regulatory networks for P. falciparum

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure fl ux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defi ned as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (inmost higher eukaryotes and some protists such as Dictyostelium ) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the fi eld understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation it is imperative to delete or knock down more than one autophagy-related gene. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways so not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field

Effect of functional anisotropy on the local dynamics of polymer grafted nanoparticles

End-functionalised polymer grafted nanoparticles (PGNs) form bonds when their coronas overlap. The bonded interactions between the overlapping PGNs depend on the energy of the bonds (L1). In the present study, oscillatory deformation imposed on a simple system with interacting PGNs placed on the vertices of a triangle is employed to examine the local dynamics as a function of energy of the bonds and the frequency of oscillation relative to the characteristic rupture frequency, omega(0) = 2(pi)nu exp(-U/k(B)T), of the bonds. In particular, the effect of functional anisotropy is studied by introducing bonds of two different energies between adjacent PGNs. A multicomponent model developed by Kadre and lyer, Macromol. Theory Simul., 2021, 30, 2100005, that combines the features of effective interactions between PGNs, self-consistent field theory and master equation approach to study bond kinetics is employed to obtain the local dynamics. The resulting force-strain curves are found to exhibit a simple broken symmetry where F-x (gamma,(gamma)over dot) not equal -F-x (-gamma,(gamma)over dot) and F-gamma (gamma,(gamma)over dot) not equal F-gamma (-gamma, -(gamma)over dot) in systems with functional anisotropy. Fourier analysis of the dynamic response reveals that functional anisotropy leads to finite even harmonic terms and systematic variation of both the elastic and dissipative response from that of the isotropic systems. Furthermore, the intra-cycle variations in the strain stiffening and shear thickening ratios obtained from the analysis indicate that functional anisotropy leads to anisotropic nonlinear response

Computational study of pair interactions between functionalized polymer grafted nanoparticles

End functionalized polymer grafted nanoparticles (PGNs) interact to form bonds when their coronas overlap. The number of bonds formed at equilibrium depends on structure of PGN, polymer-solvent interactions and nature of bonds. Here, we examine the effect of grafting density, bond energy, solvent quality and length of grafted arm on pair interaction between PGNs. We employ a multicomponent model to capture the interaction between PGNs under varying conditions. Via computer simulations, based on the multicomponent model, we examine evolution of number of bonds formed between interacting functionalized PGNs. Based on the simulations we show that grafting density and bond energy have an opposing effect on the number of bonds formed at equilibrium. Furthermore, we show that the number of bonds formed at equilibrium changes non-monotonically with decrease in solvent quality. Specifically, for a transition from good to athermal solvent the number of bonds formed at equilibrium decreases, while a further decrease in solvent quality causes interacting PGNs to form excessive bonds. Finally, we also show that a change in length of grafted arms has a negligible effect on equilibrium bond formatio

Modeling Local Oscillatory Shear Dynamics of Functionalized Polymer Grafted Nanoparticles

This study examines local oscillatory shear dynamics of spherical polymer grafted nanoparticles (PGNs) composed of a rigid nanoparticle core of fixed radius, (Formula presented.) nm, and coronas of varying number of grafted polymer arms, (Formula presented.). The grafted polymer arms are end-functionalized such that the interacting PGNs form labile bonds when their coronas overlap. The effect of PGN structure on local dynamics is examined by imposing controlled oscillatory displacement on simple systems constructed using three identical interacting PGNs with bonded interactions under mechanical equilibrium. The displacement-induced bond evolution between PGNs in this system is captured employing a master equation for determining probability of finding n bonds between PGNs as a function of oscillation frequency ((Formula presented.)). A multi-component model combined with master equation approach is employed for modeling the effect of varying number of grafted arms ((Formula presented.) and 1200) and strain amplitude ((Formula presented.)) on the local shear response of the PGN system. The resulting shear response is analyzed using both linear and non-linear rheological measures obtained using the Chebyshev polynomial framework. The rheological measures thus obtained are found to vary strongly with the number of grafted arms and strain amplitude

Computational Study of Polymer Grafted Nanoparticles Cross-linked by Labile Bonds

Functionalized polymer grafted nanoparticles (PGNs) cross-linked via labile bonds are known to form polymer-particle hybrid networks [1]. The mechanical properties of such PGN networks are expected to depend both on repulsive-cohesive and bond-scale interactions between pair PGNs. The interactions in turn depend on physical parameters like number of grafted polymer arms (f), bond energy (U), solvent quality (χ) and length of grafted polymer arms (N). A systematic study of the effect of these parameters on pair interactions between PGNs is essential for better understanding of network properties. Here, we investigate, via computer simulations, one such aspect of PGN pair interactions viz. the number of bonds (Nb) formed between two interacting spherical PGNs at equilibrium. The simulations are based on a multi-component model that allows for variation of parameters, f, U, χ and N. Using the results from simulation we construct state diagrams for bond formation between PGNs as a function of number of grafted arms (f) for varying bond energy (U), solvent quality (χ) and chain length (N). We infer from the state diagrams that the number of bonds formed between PGNs at equilibrium (Nbeq): (1) decreases with increase in number of grafted polymer arms, (2) increases with bond energy, (3) increases for good solvent condition compared to athermal and leads to excessive bond formation and jamming at theta condition and (4) remains unaltered with change in length of grafted polymer arms

Complex Fluids Meeting (CompFlu@Hyd 2016) HYDERABAD, INDIA DECEMBER 12-14, 2016

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