Nuclear RNA-related processes modulate the assembly of cytoplasmic RNA granules.

Stress granules (SGs) are cytoplasmic assemblies formed under various stress conditions as a consequence of translation arrest. SGs contain RNA-binding proteins, ribosomal subunits and messenger RNAs (mRNAs). It is well known that mRNAs contribute to SG formation; however, the connection between SG assembly and nuclear processes that involve mRNAs is not well established. Here, we examine the effects of inhibiting mRNA transcription, splicing and export on the assembly of SGs and the related cytoplasmic P body (PB). We demonstrate that inhibition of mRNA transcription, splicing and export reduces the formation of canonical SGs in a eukaryotic initiation factor 2α phosphorylation-independent manner, and alters PB size and quantity. We find that the splicing inhibitor madrasin promotes the assembly of stress-like granules. We show that the addition of synthetic mRNAs directly to the cytoplasm is sufficient for SG assembly, and that the assembly of these SGs requires the activation of stress-associated protein synthesis pathways. Moreover, we show that adding an excess of mRNA to cells that do not have active splicing, and therefore have low levels of cytoplasmic mRNAs, promotes SG formation under stress conditions. These findings emphasize the importance of the cytoplasmic abundance of newly transcribed mRNAs in the assembly of SGs.

Glucocorticoids enhance chemotherapy-driven stress granule assembly and impair granule dynamics, leading to cell death.

Stress granules (SGs) can assemble in cancer cells upon chemotoxic stress. Glucocorticoids function during stress responses and are administered with chemotherapies. The roles of glucocorticoids in SG assembly and disassembly pathways are unknown. We examined whether combining glucocorticoids such as cortisone with chemotherapies from the vinca alkaloid family, which dismantle the microtubule network, affects SG assembly and disassembly pathways and influences cell viability in cancer cells and human-derived organoids. Cortisone augmented SG formation when combined with vinorelbine (VRB). Live-cell imaging showed that cortisone increased SG assembly rates but reduced SG clearance rates after stress, by increasing protein residence times within the SGs. Mechanistically, VRB and cortisone signaled through the integrated stress response mediated by eIF2α (also known as EIF2S1), yet induced different kinases, with cortisone activating the GCN2 kinase (also known as EIF2AK4). Cortisone increased VRB-induced cell death and reduced the population of cells trapped in mitotic catastrophe. These effects were mediated by the core SG proteins G3BP1 and G3BP2. In conclusion, glucocorticoids induce SG assembly and cell death when administered with chemotherapies, suggesting that combining glucocorticoids with chemotherapies can enhance cancer cell chemosensitivity.

Mutagenesis of the melon Prv gene by CRISPR/Cas9 breaks papaya ringspot virus resistance and generates an autoimmune allele with constitutive defense responses.

The majority of plant disease resistance (R) genes encode nucleotide binding-leucine-rich repeat (NLR) proteins. In melon, two closely linked NLR genes, Fom-1 and Prv, were mapped and identified as candidate genes that control resistance to Fusarium oxysporum f.sp. melonis races 0 and 2, and to papaya ringspot virus (PRSV), respectively. In this study, we validated the function of Prv and showed that it is essential for providing resistance against PRSV infection. We generated CRISPR/Cas9 [clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein 9] mutants using Agrobacterium-mediated transformation of a PRSV-resistant melon genotype, and the T1 progeny proved susceptible to PRSV, showing strong disease symptoms and viral spread upon infection. Three alleles having 144, 154, and ~3 kb deletions, respectively, were obtained, all of which caused loss of resistance. Interestingly, one of the Prv mutant alleles, prvΔ154, encoding a truncated product, caused an extreme dwarf phenotype, accompanied by leaf lesions, high salicylic acid levels, and defense gene expression. The autoimmune phenotype observed at 25 °C proved to be temperature dependent, being suppressed at 32 °C. This is a first report on the successful application of CRISPR/Cas9 to confirm R gene function in melon. Such validation opens up new opportunities for molecular breeding of disease resistance in this important vegetable crop.

Contemporary patterns of labor in nulliparous and multiparous women.

BACKGROUND: Controversy surrounds the definition of "normal" and "abnormal" labor. OBJECTIVE: In this study, we used contemporary labor charts to explore labor patterns in large obstetric population (2011-2016). STUDY DESIGN: Detailed information from electronic medical records of live singleton deliveries at term (≥37 weeks of gestation) was extracted. Cases of elective cesarean deliveries, nonvertex presentation, and cesarean deliveries during the first stage of labor were excluded. RESULTS: Overall, 35,146 deliveries were included, of whom 15,948 deliveries (45.3%) were of nulliparous women. Median cervical dilation at admission was not significantly different between nulliparous (median, 4 cm; interquartile range, 3-5 cm) and multiparous women (median, 4 cm; interquartile range, 3-6 cm). In all, 99.3% of the women delivered vaginally. For nulliparous women, the median duration of the first stage of labor was 274 minutes (interquartile range, 145-441 minutes; 95th percentile, 747.5 minutes). Likewise, for multiparous women, the corresponding duration was 133 minutes (interquartile range, 56-244 minutes; 95th percentile, 494 minutes). During the latent phase (cervical dilation at admission, ≤4 cm), the time elapsed to the second stage of labor was 120-140 minutes longer in nulliparous women, whereas the gap between the groups decreased dramatically with advanced cervical dilation on admission. Nulliparous and multiparous women appeared to progress at a similar pace during the latent phase; however, after 5 cm, labor accelerated faster in multiparous women. Epidural anesthesia lengthens duration first and second stages of labor in all parities. Partograms according to cervical dilation at presentation are proposed. CONCLUSION: Cervical dilation rate is relatively constant between nulliparous and multiparous pregnant women during the latent phase. Time interval of the first stage was far slower than previously described, which allowed labor to continue for a longer period during this stage. These findings may reduce the rate of intrapartum iatrogenic interventions.

Red Sea corals under Artificial Light Pollution at Night (ALAN) undergo oxidative stress and photosynthetic impairment.

Coral reefs represent the most diverse marine ecosystem on the planet, yet they are undergoing an unprecedented decline due to a combination of increasing global and local stressors. Despite the wealth of research investigating these stressors, Artificial Light Pollution at Night (ALAN) or "ecological light pollution" represents an emerging threat that has received little attention in the context of coral reefs, despite the potential of disrupting the chronobiology, physiology, behavior, and other biological processes of coral reef organisms. Scleractinian corals, the framework builders of coral reefs, depend on lunar illumination cues to synchronize their biological rhythms such as behavior, reproduction and physiology. While, light pollution (POL) may mask and lead de-synchronization of these biological rhythms process. To reveal if ALAN impacts coral physiology, we have studied two coral species, Acropora eurystoma and Pocillopora damicornis, from the Gulf of Eilat/Aqaba, Red Sea, which is undergoing urban development that has led to severe POL at night. Our two experimental design data revealed that corals exposed to ALAN face an oxidative stress condition, show lower photosynthesis performances measured by electron transport rate (ETR), as well as changes in chlorophyll and algae density parameters. Testing different lights such as Blue LED and White LED spectrum showed more extreme impact in comparison to Yellow LEDs on coral physiology. The finding of this work sheds light on the emerging threat of POL and the impacts on the biology and ecology of Scleractinian corals, and will help to formulate specific management implementations to mitigate its potentially harmful impacts.

Correction to: Landraces of snake melon, an ancient Middle Eastern crop, reveal extensive morphological and DNA diversity for potential genetic improvement.

Following publication of the original article [1], the authors reported the need for a more detailed acknowledgement of the source of the samples that were analyzed and their coordinates, which are discussed in the 'Methods' section of the article. This Correction provides an addition to the 'Methods' section, and a subsequently revised 'Acknowledgements' and 'Availability of data and materials' section.

Landraces of snake melon, an ancient Middle Eastern crop, reveal extensive morphological and DNA diversity for potential genetic improvement.

BACKGROUND: Snake melon (Cucumis melo var. flexuosus, "Faqqous") is a traditional and ancient vegetable in the Mediterranean area. A collection of landraces from 42 grower fields in Israel and Palestinian territories was grown and characterized in a "Common Garden" rain-fed experiment, at the morphological-horticultural and molecular level using seq-DArT markers. RESULTS: The different landraces ("populations") showed extensive variation in morphology and quantitative traits such as yield and femaleness, and clustered into four horticultural varieties. Yield was assessed by five harvests along the season, with middle harvests producing the highest yields. Yield correlated with early vigor, and with femaleness, but not with late vigor. At the molecular level, 2784 SNP were produced and > 90% were mapped to the melon genome. Populations were very polymorphic (46-72% of the markers biallelic in a 4 individuals sample), and observed heterozygosity was higher than the expected, suggesting gene flow among populations and extensive cross pollination among individuals in the field. Genetic distances between landraces were significantly correlated with the geographical distance between collecting sites, and with long term March precipitation average; variation in yield correlated with April temperature maxima. CONCLUSIONS: The extensive variation suggests that selection of local snake melon could result in yield improvement. Correlations between traits and climatic variables could suggest local adaptation of landraces to the diverse environment in which they evolved. This study stresses the importance of preserving this germplasm, and its potential for breeding better snake melons as an heirloom crop in our region.

Estimate of within population incremental selection through branch imbalance in lineage trees.

Incremental selection within a population, defined as limited fitness changes following mutation, is an important aspect of many evolutionary processes. Strongly advantageous or deleterious mutations are detected using the synonymous to non-synonymous mutations ratio. However, there are currently no precise methods to estimate incremental selection. We here provide for the first time such a detailed method and show its precision in multiple cases of micro-evolution. The proposed method is a novel mixed lineage tree/sequence based method to detect within population selection as defined by the effect of mutations on the average number of offspring. Specifically, we propose to measure the log of the ratio between the number of leaves in lineage trees branches following synonymous and non-synonymous mutations. The method requires a high enough number of sequences, and a large enough number of independent mutations. It assumes that all mutations are independent events. It does not require of a baseline model and is practically not affected by sampling biases. We show the method's wide applicability by testing it on multiple cases of micro-evolution. We show that it can detect genes and inter-genic regions using the selection rate and detect selection pressures in viral proteins and in the immune response to pathogens.

Converging evolution leads to near maximal junction diversity through parallel mechanisms in B and T cell receptors.

T and B cell receptor (TCR and BCR) complementarity determining region 3 (CDR3) genetic diversity is produced through multiple diversification and selection stages. Potential holes in the CDR3 repertoire were argued to be linked to immunodeficiencies and diseases. In contrast with BCRs, TCRs have practically no Dß germline genetic diversity, and the question emerges as to whether they can produce a diverse CDR3 repertoire. In order to address the genetic diversity of the adaptive immune system, appropriate quantitative measures for diversity and large-scale sequencing are required. Such a diversity method should incorporate the complex diversification mechanisms of the adaptive immune response and the BCR and TCR loci structure. We combined large-scale sequencing and diversity measures to show that TCRs have a near maximal CDR3 genetic diversity. Specifically, TCR have a larger junctional and V germline diversity, which starts more 5' in Vß than BCRs. Selection decreases the TCR repertoire diversity, but does not affect BCR repertoire. As a result, TCR is as diverse as BCR repertoire, with a biased CDR3 length toward short TCRs and long BCRs. These differences suggest parallel converging evolutionary tracks to reach the required diversity to avoid holes in the CDR3 repertoire.

The restricted DH gene reading frame usage in the expressed human antibody repertoire is selected based upon its amino acid content.

The Ab repertoire is not uniform. Some variable, diversity, and joining genes are used more frequently than others. Nonuniform usage can result from the rearrangement process, or from selection. To study how the Ab repertoire is selected, we analyzed one part of diversity generation that cannot be driven by the rearrangement mechanism: the reading frame usage of DH genes. We have used two high-throughput sequencing methodologies, multiple subjects and advanced algorithms to measure the DH reading frame usage in the human Ab repertoire. In most DH genes, a single reading frame is used predominantly, and inverted reading frames are practically never observed. The choice of a single DH reading frame is not limited to a single position of the DH gene. Rather, each DH gene participates in rearrangements of differing CDR3 lengths, restricted to multiples of three. In nonproductive rearrangements, there is practically no reading frame bias, but there is still a striking absence of inversions. Biases in DH reading frame usage are more pronounced, but also exhibit greater interindividual variation, in IgG(+) and IgA(+) than in IgM(+) B cells. These results suggest that there are two developmental checkpoints of DH reading frame selection. The first occurs during VDJ recombination, when inverted DH genes are usually avoided. The second checkpoint occurs after rearrangement, once the BCR is expressed. The second checkpoint implies that DH reading frames are subjected to differential selection. Following these checkpoints, clonal selection induces a host-specific DH reading frame usage bias.

Cell specialization in cyanobacterial biofilm development revealed by expression of a cell-surface and extracellular matrix protein.

Cyanobacterial biofilms are ubiquitous and play important roles in diverse environments, yet, understanding of the processes underlying the development of these aggregates is just emerging. Here we report cell specialization in formation of Synechococcus elongatus PCC 7942 biofilms-a hitherto unknown characteristic of cyanobacterial social behavior. We show that only a quarter of the cell population expresses at high levels the four-gene ebfG-operon that is required for biofilm formation. Almost all cells, however, are assembled in the biofilm. Detailed characterization of EbfG4 encoded by this operon revealed cell-surface localization as well as its presence in the biofilm matrix. Moreover, EbfG1-3 were shown to form amyloid structures such as fibrils and are thus likely to contribute to the matrix structure. These data suggest a beneficial 'division of labor' during biofilm formation where only some of the cells allocate resources to produce matrix proteins-'public goods' that support robust biofilm development by the majority of the cells. In addition, previous studies revealed the operation of a self-suppression mechanism that depends on an extracellular inhibitor, which supresses transcription of the ebfG-operon. Here we revealed inhibitor activity at an early growth stage and its gradual accumulation along the exponential growth phase in correlation with cell density. Data, however, do not support a threshold-like phenomenon known for quorum-sensing in heterotrophs. Together, data presented here demonstrate cell specialization and imply density-dependent regulation thereby providing deep insights into cyanobacterial communal behavior.

Rep-Seq: uncovering the immunological repertoire through next-generation sequencing.

Recent scientific discoveries fuelled by the application of next-generation DNA and RNA sequencing technologies highlight the striking impact of these platforms in characterizing multiple aspects in genomics research. This technology has been used in the study of the B-cell and T-cell receptor repertoire. The novelty of immunosequencing comes from the recent rapid development of techniques and the exponential reduction in cost of sequencing. Here, we describe some of the technologies, which we collectively refer to as Rep-Seq (repertoire sequencing), to portray achievements in the field and to present the essential and inseparable role of next-generation sequencing to the understanding of entities in immune response. The large Rep-Seq data sets that should be available in the near future call for new computational algorithms to segue the transition from 'classic' molecular-based analysis to system-wide analysis. The combination of new algorithms with high-throughput data will form the basis for possible new clinical implications in personalized medicine and deeper understanding of immune behaviour and immune response.

Immune-induced evolutionary selection focused on a single reading frame in overlapping hepatitis B virus proteins.

Viruses employ various means to evade immune detection. Reduction of CD8(+) T cell epitopes is one of the common strategies used for this purpose. Hepatitis B virus (HBV), a member of the Hepadnaviridae family, has four open reading frames, with about 50% overlap between the genes they encode. We computed the CD8(+) T cell epitope density within HBV proteins and the mutations within the epitopes. Our results suggest that HBV accumulates escape mutations that reduce the number of epitopes. These mutations are not equally distributed among genes and reading frames. While the highly expressed core and X proteins are selected to have low epitope density, polymerase, which is expressed at low levels, does not undergo the same selection. In overlapping regions, mutations in one protein-coding sequence also affect the other protein-coding sequence. We show that mutations lead to the removal of epitopes in X and surface proteins even at the expense of the addition of epitopes in polymerase. The total escape mutation rate for overlapping regions is lower than that for nonoverlapping regions. The lower epitope replacement rate for overlapping regions slows the evolutionary escape rate of these regions but leads to the accumulation of mutations more robust in the transfer between hosts, such as mutations preventing proteasomal cleavage into epitopes.

Modulation of intrinsic inhibitory checkpoints using nano-carriers to unleash NK cell activity.

Natural killer (NK) cells provide a powerful weapon mediating immune defense against viral infections, tumor growth, and metastatic spread. NK cells demonstrate great potential for cancer immunotherapy; they can rapidly and directly kill cancer cells in the absence of MHC-dependent antigen presentation and can initiate a robust immune response in the tumor microenvironment (TME). Nevertheless, current NK cell-based immunotherapies have several drawbacks, such as the requirement for ex vivo expansion of modified NK cells, and low transduction efficiency. Furthermore, to date, no clinical trial has demonstrated a significant benefit for NK-based therapies in patients with advanced solid tumors, mainly due to the suppressive TME. To overcome current obstacles in NK cell-based immunotherapies, we describe here a non-viral lipid nanoparticle-based delivery system that encapsulates small interfering RNAs (siRNAs) to gene silence the key intrinsic inhibitory NK cell molecules, SHP-1, Cbl-b, and c-Cbl. The nanoparticles (NPs) target NK cells in vivo, silence inhibitory checkpoint signaling molecules, and unleash NK cell activity to eliminate tumors. Thus, the novel NP-based system developed here may serve as a powerful tool for future NK cell-based therapeutic approaches.

The Endosymbiotic Coral Algae Symbiodiniaceae Are Sensitive to a Sensory Pollutant: Artificial Light at Night, ALAN.

Artificial Light at Night, ALAN, is a major emerging issue in biodiversity conservation, which can negatively impact both terrestrial and marine environments. Therefore, it should be taken into serious consideration in strategic planning for urban development. While the lion's share of research has dealt with terrestrial organisms, only a handful of studies have focused on the marine milieu. To determine if ALAN impacts the coral reef symbiotic algae, that are fundamental for sustainable coral reefs, we conducted a short experiment over a period of one-month by illuminating isolated Symbiodiniaceae cell cultures from the genera Cladocopium (formerly Clade C) and Durusdinium (formerly Clade D) with LED light. Cell cultures were exposed nightly to ALAN levels of 0.15 µmol quanta m-2 s-1 (∼4-5 lux) with three light spectra: blue, yellow and white. Our findings showed that even in very low levels of light at night, the photo-physiology of the algae's Electron Transport Rate (ETR), Non-Photochemical Quenching, (NPQ), total chlorophyll, and meiotic index presented significantly lower values under ALAN, primarily, but not exclusively, in Cladocopium cell cultures. The findings also showed that diverse Symbiodiniaceae types have different photo-physiology and photosynthesis performances under ALAN. We believe that our results sound an alarm for the probable detrimental effects of an increasing sensory pollutant, ALAN, on the eco-physiology of symbiotic corals. The results of this study point to the potential effects of ALAN on other organisms in marine ecosystem such as fish, zooplankton, and phytoplankton in which their biorhythms is entrained by natural light and dark cycles.

Early Life Experience Shapes Male Behavior and Social Networks in Drosophila.

Living in a group creates a complex and dynamic environment in which behavior of individuals is influenced by and affects the behavior of others. Although social interaction and group living are fundamental adaptations exhibited by many organisms, little is known about how prior social experience, internal states, and group composition shape behavior in groups. Here, we present an analytical framework for studying the interplay between social experience and group interaction in Drosophila melanogaster. We simplified the complexity of interactions in a group using a series of experiments in which we controlled the social experience and motivational states of individuals to compare behavioral patterns and social networks of groups under different conditions. We show that social enrichment promotes the formation of distinct group structure that is characterized by high network modularity, high inter-individual and inter-group variance, high inter-individual coordination, and stable social clusters. Using environmental and genetic manipulations, we show that visual cues and cVA-sensing neurons are necessary for the expression of social interaction and network structure in groups. Finally, we explored the formation of group behavior and structure in heterogenous groups composed of flies with distinct internal states and documented emergent structures that are beyond the sum of the individuals that constitute it. Our results demonstrate that fruit flies exhibit complex and dynamic social structures that are modulated by the experience and composition of different individuals within the group. This paves the path for using simple model organisms to dissect the neurobiology of behavior in complex social environments.

Neuronal regulated ire-1-dependent mRNA decay controls germline differentiation in Caenorhabditis elegans.

Understanding the molecular events that regulate cell pluripotency versus acquisition of differentiated somatic cell fate is fundamentally important. Studies in Caenorhabditis elegans demonstrate that knockout of the germline-specific translation repressor gld-1 causes germ cells within tumorous gonads to form germline-derived teratoma. Previously we demonstrated that endoplasmic reticulum (ER) stress enhances this phenotype to suppress germline tumor progression(Levi-Ferber et al., 2015). Here, we identify a neuronal circuit that non-autonomously suppresses germline differentiation and show that it communicates with the gonad via the neurotransmitter serotonin to limit somatic differentiation of the tumorous germline. ER stress controls this circuit through regulated inositol requiring enzyme-1 (IRE-1)-dependent mRNA decay of transcripts encoding the neuropeptide FLP-6. Depletion of FLP-6 disrupts the circuit's integrity and hence its ability to prevent somatic-fate acquisition by germline tumor cells. Our findings reveal mechanistically how ER stress enhances ectopic germline differentiation and demonstrate that regulated Ire1-dependent decay can affect animal physiology by controlling a specific neuronal circuit.

Transcriptome Analysis of NPFR Neurons Reveals a Connection Between Proteome Diversity and Social Behavior.

Social behaviors are mediated by the activity of highly complex neuronal networks, the function of which is shaped by their transcriptomic and proteomic content. Contemporary advances in neurogenetics, genomics, and tools for automated behavior analysis make it possible to functionally connect the transcriptome profile of candidate neurons to their role in regulating behavior. In this study we used Drosophila melanogaster to explore the molecular signature of neurons expressing receptor for neuropeptide F (NPF), the fly homolog of neuropeptide Y (NPY). By comparing the transcription profile of NPFR neurons to those of nine other populations of neurons, we discovered that NPFR neurons exhibit a unique transcriptome, enriched with receptors for various neuropeptides and neuromodulators, as well as with genes known to regulate behavioral processes, such as learning and memory. By manipulating RNA editing and protein ubiquitination programs specifically in NPFR neurons, we demonstrate that the proper expression of their unique transcriptome and proteome is required to suppress male courtship and certain features of social group interaction. Our results highlight the importance of transcriptome and proteome diversity in the regulation of complex behaviors and pave the path for future dissection of the spatiotemporal regulation of genes within highly complex tissues, such as the brain.

Coral Gametogenesis Collapse under Artificial Light Pollution.

Artificial light at night (ALAN) can have negative impacts on the health of humans and ecosystems.1-4 Marine organisms, including coral reefs in particular, rely on the natural light cycles of sunlight and moonlight to regulate various physiological, biological, and behavioral processes.5-8 Here, we demonstrate that light pollution caused delayed gametogenesis and unsynchronized gamete release in two coral species, Acropora millepora and Acropora digitifera, from the Indo-Pacific Ocean. Given the urbanization along major coasts, light pollution could thus further threaten coral communities' populations, which are already under severe degradation. A worldwide-modeled light pollution impact assessment is provided, which can help incorporate an important variable in coral reef conservation planning.

Deep neural network recognition of shallow water corals in the Gulf of Eilat (Aqaba).

We describe the application of the computerized deep learning methodology to the recognition of corals in a shallow reef in the Gulf of Eilat, Red Sea. This project is aimed at applying deep neural network analysis, based on thousands of underwater images, to the automatic recognition of some common species among the 100 species reported to be found in the Eilat coral reefs. This is a challenging task, since even in the same colony, corals exhibit significant within-species morphological variability, in terms of age, depth, current, light, geographic location, and inter-specific competition. Since deep learning procedures are based on photographic images, the task is further challenged by image quality, distance from the object, angle of view, and light conditions. We produced a large dataset of over 5,000 coral images that were classified into 11 species in the present automated deep learning classification scheme. We demonstrate the efficiency and reliability of the method, as compared to painstaking manual classification. Specifically, we demonstrated that this method is readily adaptable to include additional species, thereby providing an excellent tool for future studies in the region, that would allow for real time monitoring the detrimental effects of global climate change and anthropogenic impacts on the coral reefs of the Gulf of Eilat and elsewhere, and that would help assess the success of various bioremediation efforts.