Order, Please! Uncertainty in the Ordinal-Level Classification of Chlorophyceae

Background. Chlorophyceae is one of three most species-rich green algal classes and also the only class in core Chlorophyta whose monophyly remains uncontested as gene and taxon sampling improves. However, some key relationships within Chlorophyceae are less clear-cut and warrant further investigation. The present study combined genome-scale chloroplast data and rich sampling in an attempt to resolve the ordinal classification in Chlorophyceae. The traditional division into Sphaeropleales and Volvocales (SV), and a clade containing Oedogoniales, Chaetopeltidales, and Chaetophorales (OCC) was of particular interest with the addition of deeply branching members of these groups, as well as the placement of several incertae sedis taxa. Methods. We sequenced 18 chloroplast genomes across Chlorophyceae to compile a data set of 58 protein-coding genes of a total of 68 chlorophycean taxa. We analyzed the concatenated nucleotide and amino acid datasets in the Bayesian and Maximum Likelihood frameworks, supplemented by analyses to examine potential discordant signal among genes. We also examined gene presence and absence data across Chlorophyceae. Results. Concatenated analyses yielded at least two well-supported phylogenies: nucleotide data supported the traditional classification with the inclusion of the enigmatic Treubarinia into Sphaeropleales sensu lato. However, amino acid data yielded equally strong support for Sphaeropleaceae as sister to Volvocales, with the rest of the taxa traditionally classified in Sphaeropleales in a separate clade, and Treubarinia as sister to all of the above. Single-gene and other supplementary analyses indicated that the data have low phylogenetic signal at these critical nodes. Major clades were supported by genomic structural features such as gene losses and trans-spliced intron insertions in the plastome. Discussion. While the sequence and gene order data support the deep split between the SV and OCC lineages, multiple phylogenetic hypotheses are possible for Sphaeropleales s.l. Given this uncertainty as well as the higher-taxonomic disorder seen in other algal groups, dwelling on well-defined, strongly supported Linnaean orders is not currently practical in Chlorophyceae and a less formal clade system may be more useful in the foreseeable future. For example, we identify two strongly and unequivocally supported clades: Treubarinia and Scenedesminia, as well as other smaller groups that could serve a practical purpose as named clades. This system does not preclude future establishment of new orders, or emendment of the current ordinal classification if new data support such conclusions

Order, Please! Uncertainty in the Ordinal-Level Classification of Chlorophyceae

Background. Chlorophyceae is one of three most species-rich green algal classes and also the only class in core Chlorophyta whose monophyly remains uncontested as gene and taxon sampling improves. However, some key relationships within Chlorophyceae are less clear-cut and warrant further investigation. The present study combined genome-scale chloroplast data and rich sampling in an attempt to resolve the ordinal classification in Chlorophyceae. The traditional division into Sphaeropleales and Volvocales (SV), and a clade containing Oedogoniales, Chaetopeltidales, and Chaetophorales (OCC) was of particular interest with the addition of deeply branching members of these groups, as well as the placement of several incertae sedis taxa. Methods. We sequenced 18 chloroplast genomes across Chlorophyceae to compile a data set of 58 protein-coding genes of a total of 68 chlorophycean taxa. We analyzed the concatenated nucleotide and amino acid datasets in the Bayesian and Maximum Likelihood frameworks, supplemented by analyses to examine potential discordant signal among genes. We also examined gene presence and absence data across Chlorophyceae. Results. Concatenated analyses yielded at least two well-supported phylogenies: nucleotide data supported the traditional classification with the inclusion of the enigmatic Treubarinia into Sphaeropleales sensu lato. However, amino acid data yielded equally strong support for Sphaeropleaceae as sister to Volvocales, with the rest of the taxa traditionally classified in Sphaeropleales in a separate clade, and Treubarinia as sister to all of the above. Single-gene and other supplementary analyses indicated that the data have low phylogenetic signal at these critical nodes. Major clades were supported by genomic structural features such as gene losses and trans-spliced intron insertions in the plastome. Discussion. While the sequence and gene order data support the deep split between the SV and OCC lineages, multiple phylogenetic hypotheses are possible for Sphaeropleales s.l. Given this uncertainty as well as the higher-taxonomic disorder seen in other algal groups, dwelling on well-defined, strongly supported Linnaean orders is not currently practical in Chlorophyceae and a less formal clade system may be more useful in the foreseeable future. For example, we identify two strongly and unequivocally supported clades: Treubarinia and Scenedesminia, as well as other smaller groups that could serve a practical purpose as named clades. This system does not preclude future establishment of new orders, or emendment of the current ordinal classification if new data support such conclusions

Assessing Combinability of Phylogenomic Data Using Bayes Factors

With the rapid reduction in sequencing costs of high-throughput genomic data, it has become commonplace to use hundreds of genes to infer phylogeny of any study system. While sampling a large number of genes has given us a tremendous opportunity to uncover previously unknown relationships and improve phylogenetic resolution, it also presents us with new challenges when the phylogenetic signal is confused by differences in the evolutionary histories of sampled genes. Given the incorporation of accurate marginal likelihood estimation methods into popular Bayesian software programs, it is natural to consider using the Bayes Factor (BF) to compare different partition models in which genes within any given partition subset share both tree topology and edge lengths. We explore using marginal likelihood to assess data subset combinability when data subsets have varying levels of phylogenetic discordance due to deep coalescence events among genes (simulated within a species tree), and compare the results with our recently described phylogenetic informational dissonance index (D) estimated for each data set. BF effectively detects phylogenetic incongruence and provides a way to assess the statistical significance of D values. We use BFs to assess data combinability using an empirical data set comprising 56 plastid genes from the green algal order Volvocales. We also discuss the potential need for calibrating BFs and demonstrate that BFs used in this study are correctly calibrated

Consensus guidelines for the definition, detection and interpretation of immunogenic cell death.

Cells succumbing to stress via regulated cell death (RCD) can initiate an adaptive immune response associated with immunological memory, provided they display sufficient antigenicity and adjuvanticity. Moreover, multiple intracellular and microenvironmental features determine the propensity of RCD to drive adaptive immunity. Here, we provide an updated operational definition of immunogenic cell death (ICD), discuss the key factors that dictate the ability of dying cells to drive an adaptive immune response, summarize experimental assays that are currently available for the assessment of ICD in vitro and in vivo, and formulate guidelines for their interpretation

Combined Tumor Cell-Based Vaccination and Interleukin-12 Gene Therapy Polarizes the Tumor Microenvironment in Mice

Tumor progression depends on tumor milieu, which influences neovasculature formation and immunosuppression. Combining immunotherapy with antiangiogenic/antivascular therapy might be an effective therapeutic approach. The aim of our study was to elaborate an anticancer therapeutic strategy based on the induction of immune response which leads to polarization of tumor milieu. To achieve this, we developed a tumor cell-based vaccine. CAMEL peptide was used as a B16-F10 cell death-inducing agent. The lysates were used as a vaccine to immunize mice bearing B16-F10 melanoma tumors. To further improve the therapeutic effect of the vaccine, we combined it with interleukin (IL)-12 gene therapy. IL-12, a cytokine with antiangiogenic properties, activates nonspecific and specific immune responses. We observed that combined therapy is significantly more effective (as compared with monotherapies) in inhibiting tumor growth. Furthermore, the tested combination polarizes the tumor microenvironment, which results in a switch from a proangiogenic/immunosuppressive to an antiangiogenic/immunostimulatory one. The switch manifests itself as a decreased number of tumor blood vessels, increased levels of tumor-infiltrating CD4+, CD8+ and NK cells, as well as lower level of suppressor lymphocytes (Treg). Our results suggest that polarizing tumor milieu by such combined therapy does inhibit tumor growth and seems to be a promising therapeutic strategy

Enhanced Growth and Osteogenic Differentiation of Human Osteoblast-Like Cells on Boron-Doped Nanocrystalline Diamond Thin Films

Intrinsic nanocrystalline diamond (NCD) films have been proven to be promising substrates for the adhesion, growth and osteogenic differentiation of bone-derived cells. To understand the role of various degrees of doping (semiconducting to metallic-like), the NCD films were deposited on silicon substrates by a microwave plasma-enhanced CVD process and their boron doping was achieved by adding trimethylboron to the CH4:H2 gas mixture, the B∶C ratio was 133, 1000 and 6700 ppm. The room temperature electrical resistivity of the films decreased from >10 MΩ (undoped films) to 55 kΩ, 0.6 kΩ, and 0.3 kΩ (doped films with 133, 1000 and 6700 ppm of B, respectively). The increase in the number of human osteoblast-like MG 63 cells in 7-day-old cultures on NCD films was most apparent on the NCD films doped with 133 and 1000 ppm of B (153,000±14,000 and 152,000±10,000 cells/cm2, respectively, compared to 113,000±10,000 cells/cm2 on undoped NCD films). As measured by ELISA per mg of total protein, the cells on NCD with 133 and 1000 ppm of B also contained the highest concentrations of collagen I and alkaline phosphatase, respectively. On the NCD films with 6700 ppm of B, the cells contained the highest concentration of focal adhesion protein vinculin, and the highest amount of collagen I was adsorbed. The concentration of osteocalcin also increased with increasing level of B doping. The cell viability on all tested NCD films was almost 100%. Measurements of the concentration of ICAM-1, i.e. an immunoglobuline adhesion molecule binding inflammatory cells, suggested that the cells on the NCD films did not undergo significant immune activation. Thus, the potential of NCD films for bone tissue regeneration can be further enhanced and tailored by B doping and that B doping up to metallic-like levels is not detrimental for cells

Molecular mechanisms of cell death: recommendations of the Nomenclature Committee on Cell Death 2018.

Over the past decade, the Nomenclature Committee on Cell Death (NCCD) has formulated guidelines for the definition and interpretation of cell death from morphological, biochemical, and functional perspectives. Since the field continues to expand and novel mechanisms that orchestrate multiple cell death pathways are unveiled, we propose an updated classification of cell death subroutines focusing on mechanistic and essential (as opposed to correlative and dispensable) aspects of the process. As we provide molecularly oriented definitions of terms including intrinsic apoptosis, extrinsic apoptosis, mitochondrial permeability transition (MPT)-driven necrosis, necroptosis, ferroptosis, pyroptosis, parthanatos, entotic cell death, NETotic cell death, lysosome-dependent cell death, autophagy-dependent cell death, immunogenic cell death, cellular senescence, and mitotic catastrophe, we discuss the utility of neologisms that refer to highly specialized instances of these processes. The mission of the NCCD is to provide a widely accepted nomenclature on cell death in support of the continued development of the field