The enormous repetitive Antarctic krill genome reveals environmental adaptations and population insights.

Antarctic krill (Euphausia superba) is Earth's most abundant wild animal, and its enormous biomass is vital to the Southern Ocean ecosystem. Here, we report a 48.01-Gb chromosome-level Antarctic krill genome, whose large genome size appears to have resulted from inter-genic transposable element expansions. Our assembly reveals the molecular architecture of the Antarctic krill circadian clock and uncovers expanded gene families associated with molting and energy metabolism, providing insights into adaptations to the cold and highly seasonal Antarctic environment. Population-level genome re-sequencing from four geographical sites around the Antarctic continent reveals no clear population structure but highlights natural selection associated with environmental variables. An apparent drastic reduction in krill population size 10 mya and a subsequent rebound 100 thousand years ago coincides with climate change events. Our findings uncover the genomic basis of Antarctic krill adaptations to the Southern Ocean and provide valuable resources for future Antarctic research.

The Alarm Clock Against the Sun: Trends in Google Trends Search Activity Across the Transitions to and from Daylight Saving Time.

The human circadian timing system depends on the light/dark cycle as its main cue to synchronize with the environment, and thus with solar time. However, human activities depend also on social time, i.e. the set of time conventions and restrictions dictated by society, including Daylight Saving Time (DST), which adds an hour to any degree of desynchrony between social and solar time. Here, we used Google Trends as a data source to analyze diurnal variation, if any, and the daily peak in the relative search volume of 26 Google search queries in relation to the transitions to/from DST in Italy from 2015 to 2020. Our search queries of interest fell into three categories: sleep/health-related, medication and random non sleep/health-related. After initial rhythm and phase analysis, 11 words were selected to compare the average phase of the 15 days before and after the transition to/from DST. We observed an average phase advance after the transition to DST, and a phase delay after the transition to civil time, ranging from 25 to 60 minutes. Advances or delays shorter than 60 minutes, which were primarily observed in the sleep/health-related category, may suggest that search timing for these queries is at least partially driven by the endogenous circadian rhythm. Finally, a significant trend in phase anticipation over the years was observed for virtually all words. This is most likely related to an increase in age, and thus in earlier chronotypes, amongst Google users.

Modulation of miR-210 alters phasing of circadian locomotor activity and impairs projections of PDF clock neurons in Drosophila melanogaster.

Single microRNAs are usually associated with hundreds of putative target genes that can influence multiple phenotypic traits in Drosophila, ranging from development to behaviour. We investigated the function of Drosophila miR-210 in circadian behaviour by misexpressing it within circadian clock cells. Manipulation of miR-210 expression levels in the PDF (pigment dispersing factor) positive neurons affected the phase of locomotor activity, under both light-dark conditions and constant darkness. PER cyclical expression was not affected in clock neurons, however, when miR-210 was up-regulated, a dramatic alteration in the morphology of PDF ventral lateral neuron (LNv) arborisations was observed. The effect of miR-210 in shaping neuronal projections was confirmed in vitro, using a Drosophila neuronal cell line. A transcriptomic analysis revealed that miR-210 overexpression affects the expression of several genes belonging to pathways related to circadian processes, neuronal development, GTPases signal transduction and photoreception. Collectively, these data reveal the role of miR-210 in modulating circadian outputs in flies and guiding/remodelling PDF positive LNv arborisations and indicate that miR-210 may have pleiotropic effects on the clock, light perception and neuronal development.

Sleep, Circadian Rhythmicity and Response to Chronotherapy in University Students: Tips from Chronobiology Practicals.

Chronobiology is not routinely taught to biology or medical students in most European countries. Here we present the results of the chronobiology practicals of a group of students of the University of Padova, with a view to highlight some interesting features of this group, and to share a potentially interesting cross-faculty teaching experience. Thirty-eight students (17 males; 22.9 ± 1.6 yrs) completed a set of self-administered electronic sleep quality [Pittsburgh Sleep Quality Index (PSQI)], chronotype and sleepiness [Epworth Sleepiness Scale (ESS)] questionnaires. They then went on to complete sleep diaries for two weeks. Sixteen also wore an actigraph, 8 wore wireless sensors for skin temperature, and 8 underwent a course of chronotherapy aimed at anticipating their sleep-wake timing. Analyses were performed as practicals, together with the students. Average PSQI score was 5.4 ± 1.9, with 15 (39%) students being poor sleepers. Average ESS score was 6.5 ± 3.3, with 3 (8%) students exhibiting excessive daytime sleepiness. Seven classified themselves as definitely/moderately morning, 25 as intermediates, 6 as moderately/definitely evening. Students went to bed/fell asleep significantly later on weekends, it took them less to fall asleep and they woke up/got up significantly later. Diary-reported sleep onset time coincided with the expected decrease in proximal skin temperature. Finally, during chronotherapy they took significantly less time to fall asleep. In conclusion, significant abnormalities in the sleep-wake patterns of a small group of university students were observed, and the students seemed to benefit from chronotherapy. We had a positive impression of our teaching experience, and the chronobiology courses obtained excellent student feedback.

Leigh syndrome in Drosophila melanogaster: morphological and biochemical characterization of Surf1 post-transcriptional silencing.

Leigh Syndrome (LS) is the most common early-onset, progressive mitochondrial encephalopathy usually leading to early death. The single most prevalent cause of LS is occurrence of mutations in the SURF1 gene, and LS(Surf1) patients show a ubiquitous and specific decrease in the activity of mitochondrial respiratory chain complex IV (cytochrome c oxidase, COX). SURF1 encodes an inner membrane mitochondrial protein involved in COX assembly. We established a Drosophila melanogaster model of LS based on the post-transcriptional silencing of CG9943, the Drosophila homolog of SURF1. Knockdown of Surf1 was induced ubiquitously in larvae and adults, which led to lethality; in the mesodermal derivatives, which led to pupal lethality; or in the central nervous system, which allowed survival. A biochemical characterization was carried out in knockdown individuals, which revealed that larvae unexpectedly displayed defects in all complexes of the mitochondrial respiratory chain and in the F-ATP synthase, while adults had a COX-selective impairment. Silencing of Surf1 expression in Drosophila S2R(+) cells led to selective loss of COX activity associated with decreased oxygen consumption and respiratory reserve. We conclude that Surf1 is essential for COX activity and mitochondrial function in D. melanogaster, thus providing a new tool that may help clarify the pathogenic mechanisms of LS.

Functional characterization of drim2, the Drosophila melanogaster homolog of the yeast mitochondrial deoxynucleotide transporter.

The CG18317 gene (drim2) is the Drosophila melanogaster homolog of the Saccharomyces cerevisiae Rim2 gene, which encodes a pyrimidine (deoxy)nucleotide carrier. Here, we tested if the drim2 gene also encodes for a deoxynucleotide transporter in the fruit fly. The protein was localized to mitochondria. Drosophila S2R(+) cells, silenced for drim2 expression, contained markedly reduced pools of both purine and pyrimidine dNTPs in mitochondria, whereas cytosolic pools were unaffected. In vivo drim2 homozygous knock-out was lethal at the larval stage, preceded by the following: (i) impaired locomotor behavior; (ii) decreased rates of oxygen consumption, and (iii) depletion of mtDNA. We conclude that the Drosophila mitochondrial carrier dRIM2 transports all DNA precursors and is essential to maintain mitochondrial function.

The circadian protein BMAL1 supports endothelial cell cycle during angiogenesis.

AIMS: The circadian clock is an internal biological timer that co-ordinates physiology and gene expression with the 24-h solar day. Circadian clock perturbations have been associated to vascular dysfunctions in mammals, and a function of the circadian clock in angiogenesis has been suggested. However, the functional role of the circadian clock in endothelial cells (ECs) and in the regulation of angiogenesis is widely unexplored. METHODS AND RESULTS: Here, we used both in vivo and in vitro approaches to demonstrate that ECs possess an endogenous molecular clock and show robust circadian oscillations of core clock genes. By impairing the EC-specific function of the circadian clock transcriptional activator basic helix-loop-helix ARNT like 1 (BMAL1) in vivo, we detect angiogenesis defects in mouse neonatal vascular tissues, as well as in adult tumour angiogenic settings. We then investigate the function of circadian clock machinery in cultured EC and show evidence that BMAL and circadian locomotor output cycles protein kaput knock-down impair EC cell cycle progression. By using an RNA- and chromatin immunoprecipitation sequencing genome-wide approaches, we identified that BMAL1 binds the promoters of CCNA1 and CDK1 genes and controls their expression in ECs. CONCLUSION(S): Our findings show that EC display a robust circadian clock and that BMAL1 regulates EC physiology in both developmental and pathological contexts. Genetic alteration of BMAL1 can affect angiogenesis in vivo and in vitro settings.

Persistent and transient olfactory deficits in COVID-19 are associated to inflammation and zinc homeostasis.

Introduction: The Coronavirus Disease 2019 (COVID-19) is mainly a respiratory syndrome that can affect multiple organ systems, causing a variety of symptoms. Among the most common and characteristic symptoms are deficits in smell and taste perception, which may last for weeks/months after COVID-19 diagnosis owing to mechanisms that are not fully elucidated. Methods: In order to identify the determinants of olfactory symptom persistence, we obtained olfactory mucosa (OM) from 21 subjects, grouped according to clinical criteria: i) with persistent olfactory symptoms; ii) with transient olfactory symptoms; iii) without olfactory symptoms; and iv) non-COVID-19 controls. Cells from the olfactory mucosa were harvested for transcriptome analyses. Results and discussion: RNA-Seq assays showed that gene expression levels are altered for a long time after infection. The expression profile of micro RNAs appeared significantly altered after infection, but no relationship with olfactory symptoms was found. On the other hand, patients with persistent olfactory deficits displayed increased levels of expression of genes involved in the inflammatory response and zinc homeostasis, suggesting an association with persistent or transient olfactory deficits in individuals who experienced SARS-CoV-2 infection.

Toward a Molecular Approach to Chronotype Assessment.

The aim of the present study was to develop a Polygenic Score-based model for molecular chronotype assessment. Questionnaire-based phenotypical chronotype assessment was used as a reference. In total, 54 extremely morning/morning (MM/M; 35 females, 39.7 ± 3.8 years) and 44 extremely evening/evening (EE/E; 20 females, 27.3 ± 7.7 years) individuals donated a buccal DNA sample for genotyping by sequencing of the entire genetic variability of 19 target genes known to be involved in circadian rhythmicity and/or sleep duration. Targeted genotyping was performed using the single primer enrichment technology and a specifically designed panel of 5526 primers. Among 2868 high-quality polymorphisms, a cross-validation approach lead to the identification of 83 chronotype predictive variants, including previously known and also novel chronotype-associated polymorphisms. A large (35 single-nucleotide polymorphisms [SNPs]) and also a small (13 SNPs) panel were obtained, both with an estimated predictive validity of approximately 80%. Potential mechanistic hypotheses for the role of some of the newly identified variants in modulating chronotype are formulated. Once validated in independent populations encompassing the whole range of chronotypes, the identified panels might become useful within the setting of both circadian public health initiatives and precision medicine.

A thorough annotation of the krill transcriptome offers new insights for the study of physiological processes.

The krill species Euphausia superba plays a critical role in the food chain of the Antarctic ecosystem. Significant changes in climate conditions observed in the Antarctic Peninsula region in the last decades have already altered the distribution of krill and its reproductive dynamics. A deeper understanding of the adaptation capabilities of this species is urgently needed. The availability of a large body of RNA-seq assays allowed us to extend the current knowledge of the krill transcriptome. Our study covered the entire developmental process providing information of central relevance for ecological studies. Here we identified a series of genes involved in different steps of the krill moulting cycle, in the reproductive process and in sexual maturation in accordance with what was already described in previous works. Furthermore, the new transcriptome highlighted the presence of differentially expressed genes previously unknown, playing important roles in cuticle development as well as in energy storage during the krill life cycle. The discovery of new opsin sequences, specifically rhabdomeric opsins, one onychopsin, and one non-visual arthropsin, expands our knowledge of the krill opsin repertoire. We have collected all these results into the KrillDB2 database, a resource combining the latest annotation of the krill transcriptome with a series of analyses targeting genes relevant to krill physiology. KrillDB2 provides in a single resource a comprehensive catalog of krill genes; an atlas of their expression profiles over all RNA-seq datasets publicly available; a study of differential expression across multiple conditions. Finally, it provides initial indications about the expression of microRNA precursors, whose contribution to krill physiology has never been reported before.

Seasonal gene expression profiling of Antarctic krill in three different latitudinal regions.

The Antarctic krill, Euphausia superba, has evolved seasonal rhythms of physiology and behaviour to survive under the extreme photoperiodic conditions in the Southern Ocean. However, the molecular mechanisms generating these rhythms remain far from understood. The aim of this study was to investigate seasonal differences in gene expression in three different latitudinal regions (South Georgia, South Orkneys/Bransfield Strait, Lazarev Sea) and to identify genes with potential regulatory roles in the seasonal life cycle of Antarctic krill. The RNA-seq data were analysed (a) for seasonal differences between summer and winter krill sampled from each region, and (b) for regional differences within each season. A large majority of genes showed an up-regulation in summer krill in all regions with respect to winter krill. However, seasonal differences in gene expression were less pronounced in Antarctic krill from South Georgia, most likely due to the milder seasonal conditions of the lower latitudes of this region, with a less extreme light regime and food availability between summer and winter. Our results suggest that in the South Orkneys/Bransfield Strait and Lazarev Sea region, Antarctic krill entered a state of metabolic depression and regressed development (winter quiescence) in winter. Moreover, seasonal gene expression signatures seem to be driven by a photoperiodic timing system that may adapt the flexible behaviour and physiology of Antarctic krill to the highly seasonal environment according to the latitudinal region. However, at the lower latitude South Georgia region, food availability might represent the main environmental cue influencing seasonal physiology.

New miRNA labeling method for bead-based quantification.

BACKGROUND: microRNAs (miRNAs) are small single-stranded non-coding RNAs that act as crucial regulators of gene expression. Different methods have been developed for miRNA expression profiling in order to better understand gene regulation in normal and pathological conditions. miRNAs expression values obtained from large scale methodologies such as microarrays still need a validation step with alternative technologies. RESULTS: Here we have applied with an innovative approach, the Luminex xMAP technology validate expression data of differentially expressed miRNAs obtained from high throughput arrays. We have developed a novel labeling system of small RNA molecules (below 200 nt), optimizing the sensitive cloning method for miRNAs, termed miRNA amplification profiling (mRAP). The Luminex expression patterns of three miRNAs (miR-23a, miR-27a and miR-199a) in seven different cell lines have been validated by TaqMan miRNA assay. In all cases, bead-based meas were confirmed by the data obtained by TaqMan and microarray technologies. CONCLUSIONS: We demonstrate that the measure of individual miRNA by the bead-based method is feasible, high speed, sensitive and low cost. The Luminex xMAP technology also provides flexibility, since the central reaction can be scaled up with additional miRNA capturing beads, allowing validation of many differentially expressed miRNAs obtained from microarrays in a single experiment. We propose this technology as an alternative method to qRT-PCR for validating miRNAs expression data obtained with high-throughput technologies.

Circadian regulation of diel vertical migration (DVM) and metabolism in Antarctic krill Euphausia superba.

Antarctic krill (Euphausia superba) are high latitude pelagic organisms which play a key ecological role in the ecosystem of the Southern Ocean. To synchronize their daily and seasonal life-traits with their highly rhythmic environment, krill rely on the implementation of rhythmic strategies which might be regulated by a circadian clock. A recent analysis of krill circadian transcriptome revealed that their clock might be characterized by an endogenous free-running period of about 12-15 h. Using krill exposed to simulated light/dark cycles (LD) and constant darkness (DD), we investigated the circadian regulation of krill diel vertical migration (DVM) and oxygen consumption, together with daily patterns of clock gene expression in brain and eyestalk tissue. In LD, we found clear 24 h rhythms of DVM and oxygen consumption, suggesting a synchronization with photoperiod. In DD, the DVM rhythm shifted to a 12 h period, while the peak of oxygen consumption displayed a temporal advance during the subjective light phase. This suggested that in free-running conditions the periodicity of these clock-regulated output functions might reflect the shortening of the endogenous period observed at the transcriptional level. Moreover, differences in the expression patterns of clock gene in brain and eyestalk, in LD and DD, suggested the presence in krill of a multiple oscillator system. Evidence of short periodicities in krill behavior and physiology further supports the hypothesis that a short endogenous period might represent a circadian adaption to cope with extreme seasonal photoperiodic variability at high latitude.

Analysis of the circadian transcriptome of the Antarctic krill Euphausia superba.

Antarctic krill (Euphausia superba) is a high latitude pelagic organism which plays a central role in the Southern Ocean ecosystem. E. superba shows daily and seasonal rhythms in physiology and behaviour, which are synchronized with the environmental cycles of its habitat. Recently, the main components of the krill circadian machinery have been identified and characterized. However, the exact mechanisms through which the endogenous timing system operates the control and regulation of the overt rhythms remains only partially understood. Here we investigate the involvement of the circadian clock in the temporal orchestration of gene expression by using a newly developed version of a krill microarray platform. The analysis of transcriptome data from krill exposed to both light-dark cycles (LD 18:6) and constant darkness (DD), has led to the identification of 1,564 putative clock-controlled genes. A remarkably large proportion of such genes, including several clock components (clock, period, cry2, vrille, and slimb), show oscillatory expression patterns in DD, with a periodicity shorter than 24 hours. Energy-storage pathways appear to be regulated by the endogenous clock in accordance with their ecological relevance in daily energy managing and overwintering. Our results provide the first representation of the krill circadian transcriptome under laboratory, free-running conditions.

Functional validation of miRNAs targeting genes of DNA double-strand break repair to radiosensitize non-small lung cancer cells.

DNA-Double strand breaks (DSBs) generated by radiation therapy represent the most efficient lesions to kill tumor cells, however, the inherent DSB repair efficiency of tumor cells can cause cellular radioresistance and impact on therapeutic outcome. Genes of DSB repair represent a target for cancer therapy since their down-regulation can impair the repair process making the cells more sensitive to radiation. In this study, we analyzed the combination of ionizing radiation (IR) along with microRNA-mediated targeting of genes involved in DSB repair to sensitize human non-small cell lung cancer (NSCLC) cells. MicroRNAs are natural occurring modulators of gene expression and therefore represent an attractive strategy to affect the expression of DSB repair genes. As possible IR-sensitizing targets genes we selected genes of homologous recombination (HR) and non-homologous end joining (NHEJ) pathway (i.e. RAD51, BRCA2, PRKDC, XRCC5, LIG1). We examined these genes to determine whether they may be real targets of selected miRNAs by functional and biological validation. The in vivo effectiveness of miRNA treatments has been examined in cells over-expressing miRNAs and treated with IR. Taken together, our results show that hsa-miR-96-5p and hsa-miR-874-3p can directly regulate the expression of target genes. When these miRNAs are combined with IR can decrease the survival of NSCLC cells to a higher extent than that exerted by radiation alone, and similarly to radiation combined with specific chemical inhibitors of HR and NHEJ repair pathway.

KrillDB: A de novo transcriptome database for the Antarctic krill (Euphausia superba).

Antarctic krill (Euphausia superba) is a key species in the Southern Ocean with an estimated biomass between 100 and 500 million tonnes. Changes in krill population viability would have catastrophic effect on the Antarctic ecosystem. One looming threat due to elevated levels of anthropogenic atmospheric carbon dioxide (CO2) is ocean acidification (lowering of sea water pH by CO2 dissolving into the oceans). The genetics of Antarctic krill has long been of scientific interest for both for the analysis of population structure and analysis of functional genetics. However, the genetic resources available for the species are relatively modest. We have developed the most advanced genetic database on Euphausia superba, KrillDB, which includes comprehensive data sets of former and present transcriptome projects. In particular, we have built a de novo transcriptome assembly using more than 360 million Illumina sequence reads generated from larval krill including individuals subjected to different CO2 levels. The database gives access to: 1) the full list of assembled genes and transcripts; 2) their level of similarity to transcripts and proteins from other species; 3) the predicted protein domains contained within each transcript; 4) their predicted GO terms; 5) the level of expression of each transcript in the different larval stages and CO2 treatments. All references to external entities (sequences, domains, GO terms) are equipped with a link to the appropriate source database. Moreover, the software implements a full-text search engine that makes it possible to submit free-form queries. KrillDB represents the first large-scale attempt at classifying and annotating the full krill transcriptome. For this reason, we believe it will constitute a cornerstone of future approaches devoted to physiological and molecular study of this key species in the Southern Ocean food web.

Functional characterization of the circadian clock in the Antarctic krill, Euphausia superba.

Antarctic krill (Euphausia superba) is a key species in Southern Ocean ecosystem where it plays a central role in the Antarctic food web. Available information supports the existence of an endogenous timing system in krill enabling it to synchronize metabolism and behavior with an environment characterized by extreme seasonal changes in terms of day length, food availability, and surface ice extent. A screening of our transcriptome database "KrillDB" allowed us to identify the putative orthologues of 20 circadian clock components. Mapping of conserved domains and phylogenetic analyses strongly supported annotations of the identified sequences. Luciferase assays and co-immunoprecipitation experiments allowed us to define the role of the main clock components. Our findings provide an overall picture of the molecular mechanisms underlying the functioning of the endogenous circadian clock in the Antarctic krill and shed light on their evolution throughout crustaceans speciation. Interestingly, the core clock machinery shows both mammalian and insect features that presumably contribute to an evolutionary strategy to cope with polar environment's challenges. Moreover, despite the extreme variability characterizing the Antarctic seasonal day length, the conserved light mediated degradation of the photoreceptor EsCRY1 suggests a persisting pivotal role of light as a Zeitgeber.

The opsin repertoire of the Antarctic krill Euphausia superba.

The Antarctic krill Euphausia superba experiences almost all marine photic environments throughout its life cycle. Antarctic krill eggs hatch in the aphotic zone up to 1000m depth and larvae develop on their way to the ocean surface (development ascent) and are exposed to different quality (wavelength) and quantity (irradiance) of light. Adults show a daily vertical migration pattern, moving downward during the day and upward during the night within the top 200m of the water column. Seawater acts as a potent chromatic filter and animals have evolved different opsin photopigments to perceive photons of specific wavelengths. We have investigated the transcriptome of E. superba and, using a candidate gene approach, we identified six novel opsins. Five are r-type visual opsins: four middle-wavelength-sensitive (EsRh2, EsRh3, EsRh4 and EsRh5) and one long-wavelength-sensitive (EsRh6). Moreover, we have identified a non-visual opsin, the EsPeropsin. All these newly identified opsin genes were significantly expressed in compound eyes and brain, while only EsPeropsin and EsRh2 were clearly detected also in the abdomen. A temporal modulation in the transcription of these novel opsins was found, but statistically significant oscillations were only observed in EsRrh3 and EsPeropsin. Our results contribute to the dissection of the complex photoreception system of E. superba, which enables this species to respond to the daily and seasonal changes in irradiance and spectral composition in the Southern Ocean.

The Antarctic krill Euphausia superba shows diurnal cycles of transcription under natural conditions.

BACKGROUND: Polar environments are characterized by extreme seasonal changes in day length, light intensity and spectrum, the extent of sea ice during the winter, and food availability. A key species of the Southern Ocean ecosystem, the Antarctic krill (Euphausia superba) has evolved rhythmic physiological and behavioral mechanisms to adapt to daily and seasonal changes. The molecular organization of the clockwork underlying these biological rhythms is, nevertheless, still only partially understood. METHODOLOGY/PRINCIPAL FINDINGS: The genome sequence of the Antarctic krill is not yet available. A normalized cDNA library was produced and pyrosequenced in the attempt to identify large numbers of transcripts. All available E. superba sequences were then assembled to create the most complete existing oligonucleotide microarray platform with a total of 32,217 probes. Gene expression signatures of specimens collected in the Ross Sea at five different time points over a 24-hour cycle were defined, and 1,308 genes differentially expressed were identified. Of the corresponding transcripts, 609 showed a significant sinusoidal expression pattern; about 40% of these exibithed a 24-hour periodicity while the other 60% was characterized by a shorter (about 12-hour) rhythm. We assigned the differentially expressed genes to functional categories and noticed that those concerning translation, proteolysis, energy and metabolic process, redox regulation, visual transduction and stress response, which are most likely related to daily environmental changes, were significantly enriched. Two transcripts of peroxiredoxin, thought to represent the ancestral timekeeping system that evolved about 2.5 billion years ago, were also identified as were two isoforms of the EsRh1 opsin and two novel arrestin1 sequences involved in the visual transduction cascade. CONCLUSIONS: Our work represents the first characterization of the krill diurnal transcriptome under natural conditions and provides a first insight into the genetic regulation of physiological changes, which occur around the clock during an Antarctic summer day.