Cell-type-specific alternative splicing in the Arabidopsis germline.

During sexual reproduction in flowering plants, the two haploid sperm cells (SCs) embedded within the cytoplasm of a growing pollen tube are carried to the embryo sac for double fertilization. Pollen development in flowering plants is a dynamic process that encompasses changes at transcriptome and epigenome levels. While the transcriptome of pollen and SCs in Arabidopsis (Arabidopsis thaliana) is well documented, previous analyses have mostly been based on gene-level expression. In-depth transcriptome analysis, particularly the extent of alternative splicing (AS) at the resolution of SC and vegetative nucleus (VN), is still lacking. Therefore, we performed RNA-seq analysis to generate a spliceome map of Arabidopsis SCs and VN isolated from mature pollen grains. Based on our de novo transcriptome assembly, we identified 58,039 transcripts, including 9,681 novel transcripts, of which 2,091 were expressed in SCs and 3,600 in VN. Four hundred and sixty-eight genes were regulated both at gene and splicing levels, with many having functions in mRNA splicing, chromatin modification, and protein localization. Moreover, a comparison with egg cell RNA-seq data uncovered sex-specific regulation of transcription and splicing factors. Our study provides insights into a gamete-specific AS landscape at unprecedented resolution.

Impact of the SnRK1 protein kinase on sucrose homeostasis and the transcriptome during the diel cycle.

SNF1-related Kinase 1 (SnRK1) is an evolutionarily conserved protein kinase with key functions in energy management during stress responses in plants. To address a potential role of SnRK1 under favorable conditions, we performed a metabolomic and transcriptomic characterization of rosettes of 20-d-old Arabidopsis (Arabidopsis thaliana) plants of SnRK1 gain- and loss-of-function mutants during the regular diel cycle. Our results show that SnRK1 manipulation alters the sucrose and trehalose 6-phosphate (Tre6P) relationship, influencing how the sucrose content is translated into Tre6P accumulation and modulating the flux of carbon to the tricarboxylic acid cycle downstream of Tre6P signaling. On the other hand, daily cycles of Tre6P accumulation were accompanied by changes in SnRK1 signaling, leading to a maximum in the expression of SnRK1-induced genes at the end of the night, when Tre6P levels are lowest, and to a minimum at the end of the day, when Tre6P levels peak. The expression of SnRK1-induced genes was strongly reduced by transient Tre6P accumulation in an inducible Tre6P synthase (otsA) line, further suggesting the involvement of Tre6P in the diel oscillations in SnRK1 signaling. Transcriptional profiling of wild-type plants and SnRK1 mutants also uncovered defects that are suggestive of an iron sufficiency response and of a matching induction of sulfur acquisition and assimilation when SnRK1 is depleted. In conclusion, under favorable growth conditions, SnRK1 plays a role in sucrose homeostasis and transcriptome remodeling in autotrophic tissues and its activity is influenced by diel fluctuations in Tre6P levels.

Inferring Tree-Shaped Single-Cell Trajectories with Totem.

Single-cell transcriptomics allows unbiased characterization of cell heterogeneity in a sample by profiling gene expression at single-cell level. These profiles capture snapshots of transient or steady states in dynamic processes, such as cell cycle, activation, or differentiation, which can be computationally ordered into a "flip-book" of cell development using trajectory inference methods. However, prediction of more complex topology structures, such as multifurcations or trees, remains challenging. In this chapter, we present two user-friendly protocols for inferring tree-shaped single-cell trajectories and pseudotime from single-cell transcriptomics data with Totem. Totem is a trajectory inference method that offers flexibility in inferring both nonlinear and linear trajectories and usability by avoiding the cumbersome fine-tuning of parameters. The QuickStart protocol provides a simple and practical example, whereas the GuidedStart protocol details the analysis step-by-step. Both protocols are demonstrated using a case study of human bone marrow CD34+ cells, allowing the study of the branching of three lineages: erythroid, lymphoid, and myeloid. All the analyses can be fully reproduced in Linux, macOS, and Windows operating systems (amd64 architecture) with >8 Gb of RAM using the provided docker image distributed with notebooks, scripts, and data in Docker Hub (elolab/repro-totem-ti). These materials are shared online under open-source license at https://elolab.github.io/Totem-protocol .

Single-cell RNA sequencing and analysis of rodent blood stage Plasmodium.

Bulk RNA sequencing of Plasmodium spp., the causative parasite of malaria, fails to discriminate developmental-stage-specific gene regulation. Here, we provide a protocol that uses single-cell RNA sequencing of FACS-sorted Plasmodium-chabaudi-chabaudi-AS-infected red blood cells (iRBCs) to characterize developmental-stage-specific modulation of gene expression during malaria blood stage. We describe steps for infecting mice, monitoring disease progression, preparing iRBCs, and single-cell sequencing iRBCs. We then detail procedures for analyzing scRNA-seq data. For complete details on the use and execution of this protocol, please refer to Ramos et al.1.

PIM kinases regulate early human Th17 cell differentiation.

The serine/threonine-specific Moloney murine leukemia virus (PIM) kinase family (i.e., PIM1, PIM2, and PIM3) has been extensively studied in tumorigenesis. PIM kinases are downstream of several cytokine signaling pathways that drive immune-mediated diseases. Uncontrolled T helper 17 (Th17) cell activation has been associated with the pathogenesis of autoimmunity. However, the detailed molecular function of PIMs in human Th17 cell regulation has yet to be studied. In the present study, we comprehensively investigated how the three PIMs simultaneously alter transcriptional gene regulation during early human Th17 cell differentiation. By combining PIM triple knockdown with bulk and scRNA-seq approaches, we found that PIM deficiency promotes the early expression of key Th17-related genes while suppressing Th1-lineage genes. Further, PIMs modulate Th cell signaling, potentially via STAT1 and STAT3. Overall, our study highlights the inhibitory role of PIMs in human Th17 cell differentiation, thereby suggesting their association with autoimmune phenotypes.

A hypometabolic defense strategy against malaria.

Hypoglycemia is a clinical hallmark of severe malaria, the often-lethal outcome of Plasmodium falciparum infection. Here, we report that malaria-associated hypoglycemia emerges from a non-canonical resistance mechanism, whereby the infected host reduces glycemia to starve Plasmodium. This hypometabolic response is elicited by labile heme, a byproduct of hemolysis that induces illness-induced anorexia and represses hepatic glucose production. While transient repression of hepatic glucose production prevents unfettered immune-mediated inflammation, organ damage, and anemia, when sustained over time it leads to hypoglycemia, compromising host energy expenditure and adaptive thermoregulation. The latter arrests the development of asexual stages of Plasmodium via a mechanism associated with parasite mitochondrial dysfunction. In response, Plasmodium activates a transcriptional program associated with the reduction of virulence and sexual differentiation toward the generation of transmissible gametocytes. In conclusion, malaria-associated hypoglycemia represents a trade-off of a hypometabolic-based defense strategy that balances parasite virulence versus transmission.

Self-renewal of double-negative 3 early thymocytes enables thymus autonomy but compromises the ß-selection checkpoint.

T lymphocyte differentiation in the steady state is characterized by high cellular turnover whereby thymocytes do not self-renew. However, if deprived of competent progenitors, the thymus can temporarily maintain thymopoiesis autonomously. This bears a heavy cost, because prolongation of thymus autonomy causes leukemia. Here, we show that, at an early stage, thymus autonomy relies on double-negative 3 early (DN3e) thymocytes that acquire stem-cell-like properties. Following competent progenitor deprivation, DN3e thymocytes become long lived, are required for thymus autonomy, differentiate in vivo, and include DNA-label-retaining cells. At the single-cell level, the transcriptional programs of thymopoiesis in autonomy and the steady state are similar. However, a new cell population emerges in autonomy that expresses an aberrant Notch target gene signature and bypasses the ß-selection checkpoint. In summary, DN3e thymocytes have the potential to self-renew and differentiate in vivo if cell competition is impaired, but this generates atypical cells, probably the precursors of leukemia.

The Response of Estuarine Ammonia-Oxidizing Communities to Constant and Fluctuating Salinity Regimes.

Aerobic nitrification is a fundamental nitrogen biogeochemical process that links the oxidation of ammonia to the removal of fixed nitrogen in eutrophicated water bodies. However, in estuarine environments there is an enormous variability of water physicochemical parameters that can affect the ammonia oxidation biological process. For instance, it is known that salinity can affect nitrification performance, yet there is still a lack of information on the ammonia-oxidizing communities behavior facing daily salinity fluctuations. In this work, laboratory experiments using upstream and downstream estuarine sediments were performed to address this missing gap by comparing the effect of daily salinity fluctuations with constant salinity on the activity and diversity of ammonia-oxidizing microorganisms (AOM). Activity and composition of AOM were assessed, respectively by using nitrogen stable isotope technique and 16S rRNA gene metabarcoding analysis. Nitrification activity was negatively affected by daily salinity fluctuations in upstream sediments while no effect was observed in downstream sediments. Constant salinity regime showed clearly higher rates of nitrification in upstream sediments while a similar nitrification performance between the two salinity regimes was registered in the downstream sediments. Results also indicated that daily salinity fluctuation regime had a negative effect on both ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA) community's diversity. Phylogenetically, the estuarine downstream AOM were dominated by AOA (0.92-2.09%) followed by NOB (0.99-2%), and then AOB (0.2-0.32%); whereas NOB dominated estuarine upstream sediment samples (1.4-9.5%), followed by AOA (0.27-0.51%) and AOB (0.01-0.23%). Analysis of variance identified the spatial difference between samples (downstream and upstream) as the main drivers of AOA and AOB diversity. Our study indicates that benthic AOM inhabiting different estuarine sites presented distinct plasticity toward the salinity regimes tested. These findings help to improve our understanding in the dynamics of the nitrogen cycle of estuarine systems by showing the resilience and consequently the impact of different salinity regimes on the diversity and activity of ammonia oxidizer communities.

Diversity of Bacterial Biosynthetic Genes in Maritime Antarctica.

Bacterial natural products (NPs) are still a major source of new drug leads. Polyketides (PKs) and non-ribosomal peptides (NRP) are two pharmaceutically important families of NPs and recent studies have revealed Antarctica to harbor endemic polyketide synthase (PKS) and non-ribosomal peptide synthetase (NRPS) genes, likely to be involved in the production of novel metabolites. Despite this, the diversity of secondary metabolites genes in Antarctica is still poorly explored. In this study, a computational bioprospection approach was employed to study the diversity and identity of PKS and NRPS genes to one of the most biodiverse areas in maritime Antarctica-Maxwell Bay. Amplicon sequencing of soil samples targeting ketosynthase (KS) and adenylation (AD) domains of PKS and NRPS genes, respectively, revealed abundant and unexplored chemical diversity in this peninsula. About 20% of AD domain sequences were only distantly related to characterized biosynthetic genes. Several PKS and NRPS genes were found to be closely associated to recently described metabolites including those from uncultured and candidate phyla. The combination of new approaches in computational biology and new culture-dependent and -independent strategies is thus critical for the recovery of the potential novel chemistry encoded in Antarctica microorganisms.

Distinct Temporal Succession of Bacterial Communities in Early Marine Biofilms in a Portuguese Atlantic Port.

Marine biofilms are known to influence the corrosion of metal surfaces in the marine environment. Despite some recent research, the succession of bacterial communities colonizing artificial surfaces remains uncharacterized in some temporal settings. More specifically, it is not fully known if bacterial colonizers of artificial surfaces are similar or distinct in the different seasons of the year. In particular the study of early biofilms, in which the bacterial cells communities first adhere to artificial surfaces, are crucial for the development of the subsequent biofilm communities. In this work, we used amplicon-based NGS (next-generation sequencing) and universal 16S rRNA bacterial primers to characterize the early biofilm bacterial communities growing on 316 L stainless steel surfaces in a Northern Portugal port. Sampling spanned 30-day periods in two distinct seasons (spring and winter). Biofilm communities growing in steel surfaces covered with an anti-corrosion paint and planktonic communities from the same location were also characterized. Our results demonstrated that distinct temporal patterns were observed in the sampled seasons. Specifically, a significantly higher abundance of Gammaproteobacteria and Mollicutes was found on the first days of biofilm growth in spring (day 1 to day 4) and a higher abundance of Alphaproteobacteria during the same days of biofilm growth in winter. In the last sampled day (day 30), the spring biofilms significantly shifted toward a dominance of photoautotrophic groups (mostly diatoms) and were also colonized by some macrofouling communities, something not observed during the winter sampling. Our results revealed that bacterial composition in the biofilms was particularly affected by the sampled day of the specific season, more so than the overall effect of the season or overall sampling day of both seasons. Additionally, the application of a non-fouling-release anti-corrosion paint in the steel plates resulted in a significantly lower diversity compared with plates without paint, but this was only observed during spring. We suggest that temporal succession of marine biofilm communities should be taken in consideration for future antifouling/anti-biofilm applications.

Actinobacteria and Cyanobacteria Diversity in Terrestrial Antarctic Microenvironments Evaluated by Culture-Dependent and Independent Methods.

Bacterial diversity from McMurdo Dry Valleys in Antarctica, the coldest desert on earth, has become more easily assessed with the development of High Throughput Sequencing (HTS) techniques. However, some of the diversity remains inaccessible by the power of sequencing. In this study, we combine cultivation and HTS techniques to survey actinobacteria and cyanobacteria diversity along different soil and endolithic micro-environments of Victoria Valley in McMurdo Dry Valleys. Our results demonstrate that the Dry Valleys actinobacteria and cyanobacteria distribution is driven by environmental forces, in particular the effect of water availability and endolithic environments clearly conditioned the distribution of those communities. Data derived from HTS show that the percentage of cyanobacteria decreases from about 20% in the sample closest to the water source to negligible values on the last three samples of the transect with less water availability. Inversely, actinobacteria relative abundance increases from about 20% in wet soils to over 50% in the driest samples. Over 30% of the total HTS data set was composed of actinobacterial strains, mainly distributed by 5 families: Sporichthyaceae, Euzebyaceae, Patulibacteraceae, Nocardioidaceae, and Rubrobacteraceae. However, the 11 actinobacterial strains isolated in this study, belonged to Micrococcaceae and Dermacoccaceae families that were underrepresented in the HTS data set. A total of 10 cyanobacterial strains from the order Synechococcales were also isolated, distributed by 4 different genera (Nodosilinea, Leptolyngbya, Pectolyngbya, and Acaryochloris-like). In agreement with the cultivation results, Leptolyngbya was identified as dominant genus in the HTS data set. Acaryochloris-like cyanobacteria were found exclusively in the endolithic sample and represented 44% of the total 16S rRNA sequences, although despite our efforts we were not able to properly isolate any strain from this Acaryochloris-related group. The importance of combining cultivation and sequencing techniques is highlighted, as we have shown that culture-dependent methods employed in this study were able to retrieve actinobacteria and cyanobacteria taxa that were not detected in HTS data set, suggesting that the combination of both strategies can be usefull to recover both abundant and rare members of the communities.

Potential of dissimilatory nitrate reduction pathways in polycyclic aromatic hydrocarbon degradation.

This study investigates the potential of an indigenous estuarine microbial consortium to degrade two polycyclic aromatic hydrocarbons (PAHs), naphthalene and fluoranthene, under nitrate-reducing conditions. Two physicochemically diverse sediment samples from the Lima Estuary (Portugal) were spiked individually with 25 mg L-1 of each PAH in laboratory designed microcosms. Sediments without PAHs and autoclaved sediments spiked with PAHs were run in parallel. Destructive sampling at the beginning and after 3, 6, 12, 30 and 63 weeks incubation was performed. Naphthalene and fluoranthene levels decreased over time with distinct degradation dynamics varying with sediment type. Next-generation sequencing (NGS) of 16 S rRNA gene amplicons revealed that the sediment type and incubation time were the main drivers influencing the microbial community structure rather than the impact of PAH amendments. Predicted microbial functional analyses revealed clear shifts and interrelationships between genes involved in anaerobic and aerobic degradation of PAHs and in the dissimilatory nitrate-reducing pathways (denitrification and dissimilatory nitrate reduction to ammonium - DNRA). These findings reinforced by clear biogeochemical denitrification signals (NO3- consumption, and NH4+ increased during the incubation period), suggest that naphthalene and fluoranthene degradation may be coupled with denitrification and DNRA metabolism. The results of this study contribute to the understanding of the dissimilatory nitrate-reducing pathways and help uncover their involvement in degradation of PAHs, which will be crucial for directing remediation strategies of PAH-contaminated anoxic sediments.