Lichens, encompassing 20,000 known species, are symbioses between specialized fungi (mycobionts), mostly ascomycetes, and unicellular green algae or cyanobacteria (photobionts). Here we describe the ...first parallel genomic analysis of the mycobiont Cladonia grayi and of its green algal photobiont Asterochloris glomerata. We focus on genes/predicted proteins of potential symbiotic significance, sought by surveying proteins differentially activated during early stages of mycobiont and photobiont interaction in coculture, expanded or contracted protein families, and proteins with differential rates of evolution. A) In coculture, the fungus upregulated small secreted proteins, membrane transport proteins, signal transduction components, extracellular hydrolases and, notably, a ribitol transporter and an ammonium transporter, and the alga activated DNA metabolism, signal transduction, and expression of flagellar components. B) Expanded fungal protein families include heterokaryon incompatibility proteins, polyketide synthases, and a unique set of G-protein α subunit paralogs. Expanded algal protein families include carbohydrate active enzymes and a specific subclass of cytoplasmic carbonic anhydrases. The alga also appears to have acquired by horizontal gene transfer from prokaryotes novel archaeal ATPases and Desiccation-Related Proteins. Expanded in both symbionts are signal transduction components, ankyrin domain proteins and transcription factors involved in chromatin remodeling and stress responses. The fungal transportome is contracted, as are algal nitrate assimilation genes. C) In the mycobiont, slow-evolving proteins were enriched for components involved in protein translation, translocation and sorting. The surveyed genes affect stress resistance, signaling, genome reprogramming, nutritional and structural interactions. The alga carries many genes likely transferred horizontally through viruses, yet we found no evidence of inter-symbiont gene transfer. The presence in the photobiont of meiosis-specific genes supports the notion that sexual reproduction occurs in Asterochloris while they are free-living, a phenomenon with implications for the adaptability of lichens and the persistent autonomy of the symbionts. The diversity of the genes affecting the symbiosis suggests that lichens evolved by accretion of many scattered regulatory and structural changes rather than through introduction of a few key innovations. This predicts that paths to lichenization were variable in different phyla, which is consistent with the emerging consensus that ascolichens could have had a few independent origins.
Introduction: Site-specific gene correction of the point mutation causing sickle cell disease (SCD) in hematopoietic stem cells (HSCs) constitutes a precise strategy to generate a life-long source of ...gene-corrected erythrocytes that do not sickle. However, low efficiency of homology-directed repair (HDR) in primitive reconstituting HSCs is currently a limit to the use of therapeutic genome editing for treatment of severe genetic blood disorders. To identify the mechanism(s) that underlie decreased HDR efficacy in primitive HSCs relative to that in more mature progenitor populations, we assessed: efficiency of gene delivery and expression after electroporation of in vitro transcribed mRNA; functional ZFN-mediated endonuclease activity; cell cycle status; gene expression of key HDR genes; and cytotoxic responses; in the following immunophenotypically-defined human cell populations: HSCs (CD34+/CD38-/CD90+CD45RA-); multipotent progenitors (MPPs) (CD34+/CD38-/CD45RA-/CD90-); and progenitor cells (CD34+/CD38+).
Methods: CD34+ cells were enriched from human G-CSF-mobilized peripheral blood and cultured for 1-3 days prior to electroporation of in vitro transcribed mRNA encoding GFP or a pair of zinc finger nucleases (ZFN). The ZFNs, designed to target the sickle mutation in exon 1 of the human beta-globin gene, were co-delivered with one of the homologous donor templates containing the corrective base (A/T): an integrase-deficient lentiviral vector (IDLV) or a 101bp single-stranded oligodeoxynucleotide (oligo). Percentages of alleles containing insertions/deletions (indels) and/or HDR-mediated gene correction were analyzed by high throughput sequencing (HTS). Acute cytotoxicity was determined by flow cytometry, identifying viable cells as 7AAD/AnnexinV neg. cells. To assess HDR-mediated gene correction in vivo after three months, gene-edited cells were transplanted (>1E6 viable CD34+ cells/mouse, I.V.) one day after electroporation into irradiated (250cGy) NOD/SCID/IL2R gamma-/- (NSG) mice.
Results: In HSCs, MPPs and progenitor populations, no differences were observed in delivery and expression from electroporated GFP mRNA %GFP(+) and MFI. To assess the activity of ZFN mRNA in the stem and progenitor populations, ZFNs were delivered to CD34+ cells through electroporation of in vitrotranscribed mRNA. The CD34+ cells were then FACS-sorted into the respective populations and HTS was used to determine the percentage of alleles containing indels; the frequencies of indels were equivalent among the populations indicating equivalent ZFN mRNA activity. To evaluate the efficacy of site-specific HDR in HSCs and progenitor cells, ZFN mRNA was co-delivered with either an IDLV or an oligodeoxynucleotide donor template to modify the single base-pair involved in SCD. We observed lower percentage of HDR-mediated gene modification in the HSC population compared to progenitors with all donor templates.
Due to the cell cycle phase restriction of HDR, we pre-stimulated CD34+ cells for 1-3 days prior to electroporation of ZFN mRNA and the oligo donor, and analyzed the cell cycle phases at the time of electroporation, and the frequencies of HDR and NHEJ produced by HTS. Only a small percentage of the immunophenotypic HSCs were in S/G2 phase after 24 hours of pre-stimulation; no HDR modification was observed in these cells. After 2-3 days of pre-stimulation, the HDR levels increased as the percentage of HSCs in S/G2 phase reached 20%. Importantly, assessment of relative cytotoxicity of the genome editing procedure (electroporation of ZFN mRNA and oligo donor) revealed a heightened sensitivity of HSCs/MPPs compared to progenitors, resulting in ~80% cell death in HSC vs. ~30% in progenitors under the conditions we are using. Transient expression of BCL-2 mRNA, co-electroporated with the genome editing reagents, improved HSC survival and significantly increased the numbers of HDR gene-corrected HSCs both in vitro and in vivo.
Conclusions: These data indicate an elevated sensitivity to cytotoxicity from the gene editing process for HSCs compared to the mature progenitor cells under our conditions, which may explain the lower levels of gene modification seen using in vivo compared to in vitro assays. Transient overexpression of BCL-2 mRNA preserves HSC survival after HDR-based gene editing, increasing the frequency of gene-corrected HSCs.
Bjurström:UCLA: Patents & Royalties: 2016-290. Holmes:Sangamo BioSciences Inc: Employment.
Purpose Digital technologies over time are becoming increasingly pervasive and relatively affordable, finding a large diffusion in Small and Medium Enterprises (SMEs) also for internationalization ...purposes. However, less is known about the specific mechanisms by which this can be achieved. Specifically, we focus on how SMEs can face the international environment, leveraging digital technologies and thanks to their intellectual capital (IC). Design/methodology/approach We analyze the relationship between digital technologies and the internationalization of SMEs, exploring the mediating role of IC in its three dimensions: human, relational and innovation capital, and assessing the possible moderating effects posed by international institutional conditions, specifically the Sino-US trade frictions. The relationships are tested using a sample of companies listed on China’s A-share Growth Enterprise Market (GEM) from 2010 to 2021. Findings Digital technologies help to internationalize SMEs. However, this positive relationship is affected (mediated) by the presence of an already consolidated IC. In addition, the institutional conditions of the international market, such as the Sino-US trade friction, moderate the components of IC differently. Specifically, the overall mediating effect of human and relational capital is boosted, while this does not happen for innovation capital. Originality/value First, this study contributes to the literature on organizational resilience, especially digital resilience, confirming its validity in the context of internationalization and, in particular, those processes adopted by SMEs. Second, we clarify the mechanisms through which digital technologies exert their impact on the process of internationalization and in particular the prominent necessity of having IC. Third, our conclusions enrich the understanding of how IC components react to turbulence in international markets.
Secretory and alga-specific genes are induced during gamete and zygote development in Chlamydomonas reinhardtii, concurrent with a dramatic increase in chloroplast cytosine methylation.
The green ...alga
Chlamydomonas reinhardtii
undergoes gametogenesis and mating upon nitrogen starvation. While the steps involved in its sexual reproductive cycle have been extensively characterized, the genome-wide transcriptional and epigenetic changes underlying different life cycle stages have yet to be fully described. Here, we performed transcriptome and methylome sequencing to quantify expression and DNA methylation from vegetative and gametic cells of each mating type and from zygotes. We identified 361 gametic genes with mating type-specific expression patterns and 627 genes that are specifically induced in zygotes; furthermore, these sex-related gene sets were enriched for secretory pathway and alga-specific genes. We also examined the
C. reinhardtii
nuclear methylation map with base-level resolution at different life cycle stages. Despite having low global levels of nuclear methylation, we detected 23 hypermethylated loci in gene-poor, repeat-rich regions. We observed mating type-specific differences in chloroplast DNA methylation levels in plus versus minus mating type gametes followed by chloroplast DNA hypermethylation in zygotes. Lastly, we examined the expression of candidate DNA methyltransferases and found three,
DMT1a
,
DMT1b
, and
DMT4
, that are differentially expressed during the life cycle and are candidate DNA methylases. The expression and methylation data we present provide insight into cell type-specific transcriptional and epigenetic programs during key stages of the
C. reinhardtii
life cycle.
Sluggish market demand can deteriorate the financial situation of a company and affect a shareholder’s decision to adopt environmental, social, and governance criteria (ESG). According to the ...socioemotional wealth theory, family firms place significant emphasis on sustainable development and long-term orientation, but this emphasis can be either internally or externally driven according to the type of involvement chosen by the owning family. Therefore, this study uses listed family firms to explore the relationship between different types of family involvement (i.e., family ownership and control, the influence of market competition, and the institutionalisation level of the environment in which a firm decides to pursue ESG criteria). We performed a multivariate regression analysis on a sample of 1,151 Chinese companies to test these relationships and found that both family ownership and control are positively related to ESG scores. Market competition negatively moderates the influence of both family ownership and control on the adoption of ESG criteria. Moreover, the influence of family control is negatively moderated by the institutional environment. Thus, types of family involvement seem to be relevant for the firm’s engagement with ESG criteria.
When growing budding yeast under continuous, nutrient-limited conditions, over half of yeast genes exhibit periodic expression patterns. Periodicity can also be observed in respiration, in the timing ...of cell division, as well as in various metabolite levels. Knowing the transcription factors involved in the yeast metabolic cycle is helpful for determining the cascade of regulatory events that cause these patterns.
Transcription factor activities were estimated by linear regression using time series and genome-wide transcription factor binding data. Time-translation matrices were estimated using least squares and were used to model the interactions between the most significant transcription factors. The top transcription factors have functions involving respiration, cell cycle events, amino acid metabolism and glycolysis. Key regulators of transitions between phases of the yeast metabolic cycle appear to be Hap1, Hap4, Gcn4, Msn4, Swi6 and Adr1.
Analysis of the phases at which transcription factor activities peak supports previous findings suggesting that the various cellular functions occur during specific phases of the yeast metabolic cycle.
MicroRNAs (miRs) are known to play an important role in mRNA regulation, often by binding to complementary sequences in "target" mRNAs. Recently, several methods have been developed by which existing ...sequence-based target predictions can be combined with miR and mRNA expression data to infer true miR-mRNA targeting relationships. It has been shown that the combination of these two approaches gives more reliable results than either by itself. While a few such algorithms give excellent results, none fully addresses expression data sets with a natural ordering of the samples. If the samples in an experiment can be ordered or partially ordered by their expected similarity to one another, such as for time-series or studies of development processes, stages, or types, (e.g. cell type, disease, growth, aging), there are unique opportunities to infer miR-mRNA interactions that may be specific to the underlying processes, and existing methods do not exploit this. We propose an algorithm which specifically addresses partially ordered expression data and takes advantage of sample similarities based on the ordering structure. This is done within a Bayesian framework which specifies posterior distributions and therefore statistical significance for each model parameter and latent variable. We apply our model to a previously published expression data set of paired miR and mRNA arrays in five partially ordered conditions, with biological replicates, related to multiple myeloma, and we show how considering potential orderings can improve the inference of miR-mRNA interactions, as measured by existing knowledge about the involved transcripts.
The crystal structure of the complex between the cross-reacting antigen Guinea fowl lysozyme and the Fab from monoclonal antibody F9.13.7, raised against hen egg lysozyme, has been determined by ...x-ray diffraction to 3-A resolution. The antibody interacts with exposed residues of an alpha-helix and surrounding loops adjacent to the lysozyme active site cleft. The epitope of lysozyme bound by antibody F9.13.7 overlaps almost completely with that bound by antibody HyHEL10; the same 12 residues of the antigen interact with the two antibodies. The antibodies, however, have different combining sites with no sequence homology at any of their complementarity-determining regions and show a dissimilar pattern of cross-reactivity with heterologous antigens. Side chain mobility of epitope residues contributes to confer steric and electrostatic complementarity to differently shaped combining sites, allowing functional mimicry to occur. The capacity of two antibodies that have different fine specificities to bind the same area of the antigen emphasizes the operational character of the definition of an antigenic determinant. This example demonstrates that degenerate binding of the same structural motif does not require the existence of sequence homology or other chemical similarities between the different binding sites.
A remarkable exception to the large genetic diversity often observed for bacteriophages infecting a specific bacterial host was found for the Cutibacterium acnes (formerly Propionibacterium acnes) ...phages, which are highly homogeneous. Phages infecting the related species, which is also a member of the Propionibacteriaceae family, Propionibacterium freudenreichii, a bacterium used in production of Swiss-type cheeses, have also been described and are common contaminants of the cheese manufacturing process. However, little is known about their genetic composition and diversity. We obtained seven independently isolated bacteriophages that infect P. freudenreichii from Swiss-type cheese samples, and determined their complete genome sequences. These data revealed that all seven phage isolates are of similar genomic length and GC% content, but their genomes are highly diverse, including genes encoding the capsid, tape measure, and tail proteins. In contrast to C. acnes phages, all P. freudenreichii phage genomes encode a putative integrase protein, suggesting they are capable of lysogenic growth. This is supported by the finding of related prophages in some P. freudenreichii strains. The seven phages could further be distinguished as belonging to two distinct genomic types, or 'clusters', based on nucleotide sequences, and host range analyses conducted on a collection of P. freudenreichii strains show a higher degree of host specificity than is observed for the C. acnes phages. Overall, our data demonstrate P. freudenreichii bacteriophages are distinct from C. acnes phages, as evidenced by their higher genetic diversity, potential for lysogenic growth, and more restricted host ranges. This suggests substantial differences in the evolution of these related species from the Propionibacteriaceae family and their phages, which is potentially related to their distinct environmental niches.
The access of transcription factors and the replication machinery to DNA is regulated by the epigenetic state of chromatin. In eukaryotes, this complex layer of regulatory processes includes the ...direct methylation of DNA, as well as covalent modifications to histones. Using next-generation sequencers, it is now possible to obtain profiles of epigenetic modifications across a genome using chromatin immunoprecipitation followed by sequencing (ChIP-seq). This technique permits the detection of the binding of proteins to specific regions of the genome with high resolution. It can be used to determine the target sequences of transcription factors, as well as the positions of histones with specific modification of their N-terminal tails. Antibodies that selectively bind methylated DNA may also be used to determine the position of methylated cytosines. Here, we present a data analysis pipeline for processing ChIP-seq data, and discuss the limitations and idiosyncrasies of these approaches.
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