Pharmacologic fibroblast reprogramming into photoreceptors restores vision.

Photoreceptor loss is the final common endpoint in most retinopathies that lead to irreversible blindness, and there are no effective treatments to restore vision1,2. Chemical reprogramming of fibroblasts offers an opportunity to reverse vision loss; however, the generation of sensory neuronal subtypes such as photoreceptors remains a challenge. Here we report that the administration of a set of five small molecules can chemically induce the transformation of fibroblasts into rod photoreceptor-like cells. The transplantation of these chemically induced photoreceptor-like cells (CiPCs) into the subretinal space of rod degeneration mice (homozygous for rd1, also known as Pde6b) leads to partial restoration of the pupil reflex and visual function. We show that mitonuclear communication is a key determining factor for the reprogramming of fibroblasts into CiPCs. Specifically, treatment with these five compounds leads to the translocation of AXIN2 to the mitochondria, which results in the production of reactive oxygen species, the activation of NF-κB and the upregulation of Ascl1. We anticipate that CiPCs could have therapeutic potential for restoring vision.

Multiomics in the central Arctic Ocean for benchmarking biodiversity change.

Multiomics approaches need to be applied in the central Arctic Ocean to benchmark biodiversity change and to identify novel species and their genes. As part of MOSAiC, EcoOmics will therefore be essential for conservation and sustainable bioprospecting in one of the least explored ecosystems on Earth.

Resting cell formation in the marine diatom Thalassiosira pseudonana.

Resting cells represent a survival strategy employed by diatoms to endure prolonged periods of unfavourable conditions. In the oceans, many diatoms sink at the end of their blooming season and therefore need to endure cold and dark conditions in the deeper layers of the water column. How they survive these conditions is largely unknown. We conducted an integrative analysis encompassing methods from histology, physiology, biochemistry, and genetics to reveal the biological mechanism of resting-cell formation in the model diatom Thalassiosira pseudonana. Resting-cell formation was triggered by a decrease in light and temperature with subsequent catabolism of storage compounds. Resting cells were characterised by an acidic and viscous cytoplasm and altered morphology of the chloroplast ultrastructure. The formation of resting cells in T. pseudonana is an energy demanding process required for a biophysical alteration of the cytosol and chloroplasts to endure the unfavourable conditions of the deeper ocean as photosynthetic organisms. However, most resting cells (> 90%) germinate upon return to favorable growth conditions.

Growth performance and feed utilisation of Australian hybrid abalone (Haliotis rubra × Haliotis laevigata) fed increasing dietary protein levels at three water temperatures.

Determining the macronutrient requirements for commercially valuable aquaculture species remains crucial for maximising production efficiency. Yet, such information is lacking for Australian hybrid abalone (Haliotis rubra × Haliotis laevigata), particularly with respect to life stage and water temperatures. The present study aimed to evaluate the effect of dietary protein inclusion level on the growth performance, nutrient utilisation and nutritional quality of juvenile (3·3 g) Australian hybrid abalone reared at three different temperatures representative of winter (12°C), average annual (17°C) and summer (22°C) grow-out periods and fed five diets containing graded dietary protein levels of 35, 38, 41, 44 and 47 %. Abalone growth increased with increasing water temperature with weight gains of approximately 100, 280 and 380 % of their initial weight at 12, 17 and 22°C, respectively. Furthermore, the present study clearly demonstrated that higher dietary protein inclusion levels (41 %) than those currently used commercially (35 %) would significantly improve the growth performance when water temperatures are ≥17°C without any adverse impacts on nutrient utilisation, nutrient deposition or nutritional quality of the abalone soft tissue. For example, at 22°C abalone fed a diet containing 41 % protein obtained a significantly higher weight gain percentage (421 %) compared with those fed a diet containing 35 % protein (356 %). Lastly, it is suggested that maintaining a dietary protein inclusion level of 35 % or implementing a 'least cost' feeding approach during cooler seasons, or where water temperatures are ∼12°C, may be beneficial, considering only marginal growth improvements were observed during these periods of slow growth.

Efficient gene replacement by CRISPR/Cas-mediated homologous recombination in the model diatom Thalassiosira pseudonana.

CRISPR/Cas enables targeted genome editing in many different plant and algal species including the model diatom Thalassiosira pseudonana. However, efficient gene targeting by homologous recombination (HR) to date is only reported for photosynthetic organisms in their haploid life-cycle phase. Here, a CRISPR/Cas construct, assembled using Golden Gate cloning, enabled highly efficient HR in a diploid photosynthetic organism. Homologous recombination was induced in T. pseudonana using sequence-specific CRISPR/Cas, paired with a dsDNA donor matrix, generating substitution of the silacidin, nitrate reductase and urease genes by a resistance cassette (FCP:NAT). Up to c. 85% of NAT-resistant T. pseudonana colonies screened positive for HR by nested PCR. Precise integration of FCP:NAT at each locus was confirmed using an inverse PCR approach. The knockout of the nitrate reductase and urease genes impacted growth on nitrate and urea, respectively, while the knockout of the silacidin gene in T. pseudonana caused a significant increase in cell size, confirming the role of this gene for cell-size regulation in centric diatoms. Highly efficient gene targeting by HR makes T. pseudonana as genetically tractable as Nannochloropsis and Physcomitrella, hence rapidly advancing functional diatom biology, bionanotechnology and biotechnological applications targeted on harnessing the metabolic potential of diatoms.

Cold-induced [Ca2+]cyt elevations function to support osmoregulation in marine diatoms.

Diatoms are a group of microalgae that are important primary producers in a range of open ocean, freshwater, and intertidal environments. The latter can experience substantial long- and short-term variability in temperature, from seasonal variations to rapid temperature shifts caused by tidal immersion and emersion. As temperature is a major determinant in the distribution of diatom species, their temperature sensory and response mechanisms likely have important roles in their ecological success. We examined the mechanisms diatoms use to sense rapid changes in temperature, such as those experienced in the intertidal zone. We found that the diatoms Phaeodactylum tricornutum and Thalassiosira pseudonana exhibit a transient cytosolic Ca2+ ([Ca2+]cyt) elevation in response to rapid cooling, similar to those observed in plant and animal cells. However, [Ca2+]cyt elevations were not observed in response to rapid warming. The kinetics and magnitude of cold-induced [Ca2+]cyt elevations corresponded with the rate of temperature decrease. We did not find a role for the [Ca2+]cyt elevations in enhancing cold tolerance but showed that cold shock induces a Ca2+-dependent K+ efflux and reduces mortality of P. tricornutum during a simultaneous hypo-osmotic shock. As intertidal diatom species may routinely encounter simultaneous cold and hypo-osmotic shocks during tidal cycles, we propose that cold-induced Ca2+ signaling interacts with osmotic signaling pathways to aid in the regulation of cell volume. Our findings provide insight into the nature of temperature perception in diatoms and highlight that cross-talk between signaling pathways may play an important role in their cellular responses to multiple simultaneous stressors.

Diatom Molecular Research Comes of Age: Model Species for Studying Phytoplankton Biology and Diversity.

Diatoms are the world's most diverse group of algae, comprising at least 100,000 species. Contributing ∼20% of annual global carbon fixation, they underpin major aquatic food webs and drive global biogeochemical cycles. Over the past two decades, Thalassiosira pseudonana and Phaeodactylum tricornutum have become the most important model systems for diatom molecular research, ranging from cell biology to ecophysiology, due to their rapid growth rates, small genomes, and the cumulative wealth of associated genetic resources. To explore the evolutionary divergence of diatoms, additional model species are emerging, such as Fragilariopsis cylindrus and Pseudo-nitzschia multistriata Here, we describe how functional genomics and reverse genetics have contributed to our understanding of this important class of microalgae in the context of evolution, cell biology, and metabolic adaptations. Our review will also highlight promising areas of investigation into the diversity of these photosynthetic organisms, including the discovery of new molecular pathways governing the life of secondary plastid-bearing organisms in aquatic environments.

Evolutionary genomics of the cold-adapted diatom Fragilariopsis cylindrus.

The Southern Ocean houses a diverse and productive community of organisms. Unicellular eukaryotic diatoms are the main primary producers in this environment, where photosynthesis is limited by low concentrations of dissolved iron and large seasonal fluctuations in light, temperature and the extent of sea ice. How diatoms have adapted to this extreme environment is largely unknown. Here we present insights into the genome evolution of a cold-adapted diatom from the Southern Ocean, Fragilariopsis cylindrus, based on a comparison with temperate diatoms. We find that approximately 24.7 per cent of the diploid F. cylindrus genome consists of genetic loci with alleles that are highly divergent (15.1 megabases of the total genome size of 61.1 megabases). These divergent alleles were differentially expressed across environmental conditions, including darkness, low iron, freezing, elevated temperature and increased CO2. Alleles with the largest ratio of non-synonymous to synonymous nucleotide substitutions also show the most pronounced condition-dependent expression, suggesting a correlation between diversifying selection and allelic differentiation. Divergent alleles may be involved in adaptation to environmental fluctuations in the Southern Ocean.

The diatom Fragilariopsis cylindrus: A model alga to understand cold-adapted life.

Diatoms are significant primary producers especially in cold, turbulent, and nutrient-rich surface oceans. Hence, they are abundant in polar oceans, but also underpin most of the polar food webs and related biogeochemical cycles. The cold-adapted pennate diatom Fragilariopsis cylindrus is considered a keystone species in polar oceans and sea ice because it can thrive under different environmental conditions if temperatures are low. In this perspective paper, we provide insights into the latest molecular work that has been done on F. cylindrus and discuss its role as a model alga to understand cold-adapted life.

Integrative analysis of chloroplast DNA methylation in a marine alga-Saccharina japonica.

KEY MESSAGE: We applied an integrative approach using multiple methods to verify cytosine methylation in the chloroplast DNA of the multicellular brown alga Saccharina japonica. Cytosine DNA methylation is a heritable process which plays important roles in regulating development throughout the life cycle of an organism. Although methylation of nuclear DNA has been studied extensively, little is known about the state and role of DNA methylation in chloroplast genomes, especially in marine algae. Here, we have applied an integrated approach encompassing whole-genome bisulfite sequencing, methylated DNA immunoprecipitation, gene co-expression networks and photophysiological analyses to provide evidence for the role of chloroplast DNA methylation in a marine alga, the multicellular brown alga Saccharina japonica. Although the overall methylation level was relatively low in the chloroplast genome of S. japonica, gametophytes exhibited higher methylation levels than sporophytes. Gene-specific bisulfite-cloning sequencing provided additional evidence for the methylation of key photosynthetic genes. Many of them were highly expressed in sporophytes whereas genes involved in transcription, translation and biosynthesis were strongly expressed in gametophytes. Nucleus-encoded photosynthesis genes were co-expressed with their chloroplast-encoded counterparts potentially contributing to the higher photosynthetic performance in sporophytes compared to gametophytes where these co-expression networks were less pronounced. A nucleus-encoded DNA methyltransferase of the DNMT2 family is assumed to be responsible for the methylation of the chloroplast genome because it is predicted to possess a plastid transit peptide.

A PP4 holoenzyme balances physiological and oncogenic nuclear factor-kappa B signaling in T lymphocytes.

Signal transduction to nuclear factor-kappa B (NF-κB) involves multiple kinases and phosphorylated target proteins, but little is known about signal termination by dephosphorylation. By RNAi screening, we have identified protein phosphatase 4 regulatory subunit 1 (PP4R1) as a negative regulator of NF-κB activity in T lymphocytes. PP4R1 formed part of a distinct PP4 holoenzyme and bridged the inhibitor of NF-κB kinase (IKK) complex and the phosphatase PP4c, thereby directing PP4c activity to dephosphorylate and inactivate the IKK complex. PP4R1 expression was triggered upon activation and proliferation of primary human T lymphocytes and deficiency for PP4R1 caused sustained and increased IKK activity, T cell hyperactivation, and aberrant NF-κB signaling in NF-κB-addicted T cell lymphomas. Collectively, our results unravel PP4R1 as a previously unknown activation-associated negative regulator of IKK activity in lymphocytes whose downregulation promotes oncogenic NF-κB signaling in a subgroup of T cell lymphomas.

Single-base methylome profiling of the giant kelp Saccharina japonica reveals significant differences in DNA methylation to microalgae and plants.

Brown algae have convergently evolved plant-like body plans and reproductive cycles, which in plants are controlled by differential DNA methylation. This contribution provides the first single-base methylome profiles of haploid gametophytes and diploid sporophytes of a multicellular alga. Although only c. 1.4% of cytosines in Saccharina japonica were methylated mainly at CHH sites and characterized by 5-methylcytosine (5mC), there were significant differences between life-cycle stages. DNA methyltransferase 2 (DNMT2), known to efficiently catalyze tRNA methylation, is assumed to methylate the genome of S. japonica in the structural context of tRNAs as the genome does not encode any other DNA methyltransferases. Circular and long noncoding RNA genes were the most strongly methylated regulatory elements in S. japonica. Differential expression of genes was negatively correlated with DNA methylation with the highest methylation levels measured in both haploid gametophytes. Hypomethylated and highly expressed genes in diploid sporophytes included genes involved in morphogenesis and halogen metabolism. The data herein provide evidence that cytosine methylation, although occurring at a low level, is significantly contributing to the formation of different life-cycle stages, tissue differentiation and metabolism in brown algae.

Adaptive divergence across Southern Ocean gradients in the pelagic diatom Fragilariopsis kerguelensis.

The Southern Ocean is characterized by longitudinal water circulations crossed by strong latitudinal gradients. How this oceanographic background shapes planktonic populations is largely unknown, despite the significance of this region for global biogeochemical cycles. Here, we show, based on genomic, morphometric, ecophysiological and mating compatibility data, an example of ecotypic differentiation and speciation within an endemic pelagic inhabitant, the diatom Fragilariopsis kerguelensis. We discovered three genotypic variants, one present throughout the latitudinal transect sampled, the others restricted to the north and south, respectively. The latter two showed reciprocal monophyly across all three genomes and significant ecophysiological differences consistent with local adaptation, but produced viable offspring in laboratory crosses. The third group was also reproductively isolated from the latter two. We hypothesize that this pattern originated by an adaptive expansion accompanied by ecotypic divergence, followed by sympatric speciation.

Ocean acidification increases iodine accumulation in kelp-based coastal food webs.

Kelp are main iodine accumulators in the ocean, and their growth and photosynthesis are likely to benefit from elevated seawater CO2 levels due to ocean acidification. However, there are currently no data on the effects of ocean acidification on iodine metabolism in kelp. As key primary producers in coastal ecosystems worldwide, any change in their iodine metabolism caused by climate change will potentially have important consequences for global geochemical cycles of iodine, including iodine levels of coastal food webs that underpin the nutrition of billions of humans around the world. Here, we found that elevated pCO2 enhanced growth and increased iodine accumulation not only in the model kelp Saccharina japonica using both short-term laboratory experiment and long-term in situ mesocosms, but also in several other edible and ecologically significant seaweeds using long-term in situ mesocosms. Transcriptomic and proteomic analysis of S. japonica revealed that most vanadium-dependent haloperoxidase genes involved in iodine efflux during oxidative stress are down-regulated under increasing pCO2 , suggesting that ocean acidification alleviates oxidative stress in kelp, which might contribute to their enhanced growth. When consumed by abalone (Haliotis discus), elevated iodine concentrations in S. japonica caused increased iodine accumulation in abalone, accompanied by reduced synthesis of thyroid hormones. Thus, our results suggest that kelp will benefit from ocean acidification by a reduction in environmental stress however; iodine levels, in kelp-based coastal food webs will increase, with potential impacts on biogeochemical cycles of iodine in coastal ecosystems.

Endogenous biosynthesis of n-3 long-chain PUFA in Atlantic salmon.

A more efficient utilisation of marine-derived sources of dietary n-3 long-chain PUFA (n-3 LC PUFA) in cultured Atlantic salmon (Salmo salar L.) could be achieved by nutritional strategies that maximise endogenous n-3 LC PUFA synthesis. The objective of the present study was to quantify the extent of n-3 LC PUFA biosynthesis and the resultant effect on fillet nutritional quality in large fish. Four diets were manufactured, providing altered levels of dietary n-3 substrate, namely, 18 : 3n-3, and end products, namely, 20 : 5n-3 and 22 : 6n-3. After 283 d of feeding, fish grew in excess of 3000 g and no differences in growth performance or biometrical parameters were recorded. An analysis of fatty acid composition and in vivo metabolism revealed that endogenous production of n-3 LC PUFA in fish fed a diet containing no added fish oil resulted in fillet levels of n-3 LC PUFA comparable with fish fed a diet with added fish oil. However, this result was not consistent among all treatments. Another major finding of this study was the presence of abundant dietary n-3 substrate, with the addition of dietary n-3 end product (i.e. fish oil) served to increase final fillet levels of n-3 LC PUFA. Specifically, preferential ß-oxidation of dietary C18 n-3 PUFA resulted in conservation of n-3 LC PUFA from catabolism. Ultimately, this study highlights the potential for endogenous synthesis of n-3 LC PUFA to, partially, support a substantial reduction in the amount of dietary fish oil in diets for Atlantic salmon reared in seawater.

APOB-associated cholesterol deficiency in Holstein cattle is not a simple recessive disease.

In 2015, cholesterol deficiency (CD) was reported for the first time as a new recessive defect in Holstein cattle. After GWAS mapping and identification of a disease-associated haplotype, a causative loss-of-function variant in APOB was identified. CD-clinically affected APOB homozygotes showed poor development, intermittent diarrhea and hypocholesterolemia and, consequently, a limited life expectation. Herein, we present a collection of 18 cases clinically diagnosed as CD-affected APOB heterozygotes. CD-clinically affected heterozygotes show reduced cholesterol and triglyceride blood concentrations. The differences in total blood cholesterol and triglycerides between nine CD-clinically affected and 36 non-affected heterozygotes were significant. As only some APOB heterozygotes show the clinical CD phenotype, we assume that the penetrance is reduced in heterozygotes compared to the fully penetrant effect observed in homozygotes. We conclude that APOB-associated CD represents most likely an incomplete dominant inherited metabolic disease with incomplete penetrance in heterozygotes.

Algal genomes reveal evolutionary mosaicism and the fate of nucleomorphs.

Cryptophyte and chlorarachniophyte algae are transitional forms in the widespread secondary endosymbiotic acquisition of photosynthesis by engulfment of eukaryotic algae. Unlike most secondary plastid-bearing algae, miniaturized versions of the endosymbiont nuclei (nucleomorphs) persist in cryptophytes and chlorarachniophytes. To determine why, and to address other fundamental questions about eukaryote-eukaryote endosymbiosis, we sequenced the nuclear genomes of the cryptophyte Guillardia theta and the chlorarachniophyte Bigelowiella natans. Both genomes have >21,000 protein genes and are intron rich, and B. natans exhibits unprecedented alternative splicing for a single-celled organism. Phylogenomic analyses and subcellular targeting predictions reveal extensive genetic and biochemical mosaicism, with both host- and endosymbiont-derived genes servicing the mitochondrion, the host cell cytosol, the plastid and the remnant endosymbiont cytosol of both algae. Mitochondrion-to-nucleus gene transfer still occurs in both organisms but plastid-to-nucleus and nucleomorph-to-nucleus transfers do not, which explains why a small residue of essential genes remains locked in each nucleomorph.

Finding a partner in the ocean: molecular and evolutionary bases of the response to sexual cues in a planktonic diatom.

Microalgae play a major role as primary producers in aquatic ecosystems. Cell signalling regulates their interactions with the environment and other organisms, yet this process in phytoplankton is poorly defined. Using the marine planktonic diatom Pseudo-nitzschia multistriata, we investigated the cell response to cues released during sexual reproduction, an event that demands strong regulatory mechanisms and impacts on population dynamics. We sequenced the genome of P. multistriata and performed phylogenomic and transcriptomic analyses, which allowed the definition of gene gains and losses, horizontal gene transfers, conservation and evolutionary rate of sex-related genes. We also identified a small number of conserved noncoding elements. Sexual reproduction impacted on cell cycle progression and induced an asymmetric response of the opposite mating types. G protein-coupled receptors and cyclic guanosine monophosphate (cGMP) are implicated in the response to sexual cues, which overall entails a modulation of cell cycle, meiosis-related and nutrient transporter genes, suggesting a fine control of nutrient uptake even under nutrient-replete conditions. The controllable life cycle and the genome sequence of P. multistriata allow the reconstruction of changes occurring in diatoms in a key phase of their life cycle, providing hints on the evolution and putative function of their genes and empowering studies on sexual reproduction.

Biotic interactions as drivers of algal origin and evolution.

Contents 670 I. 671 II. 671 III. 676 IV. 678 678 References 678 SUMMARY: Biotic interactions underlie life's diversity and are the lynchpin to understanding its complexity and resilience within an ecological niche. Algal biologists have embraced this paradigm, and studies building on the explosive growth in omics and cell biology methods have facilitated the in-depth analysis of nonmodel organisms and communities from a variety of ecosystems. In turn, these advances have enabled a major revision of our understanding of the origin and evolution of photosynthesis in eukaryotes, bacterial-algal interactions, control of massive algal blooms in the ocean, and the maintenance and degradation of coral reefs. Here, we review some of the most exciting developments in the field of algal biotic interactions and identify challenges for scientists in the coming years. We foresee the development of an algal knowledgebase that integrates ecosystem-wide omics data and the development of molecular tools/resources to perform functional analyses of individuals in isolation and in populations. These assets will allow us to move beyond mechanistic studies of a single species towards understanding the interactions amongst algae and other organisms in both the laboratory and the field.