'Seeing' the electromagnetic spectrum: spotlight on the cryptochrome photocycle.

Cryptochromes are widely dispersed flavoprotein photoreceptors that regulate numerous developmental responses to light in plants, as well as to stress and entrainment of the circadian clock in animals and humans. All cryptochromes are closely related to an ancient family of light-absorbing flavoenzymes known as photolyases, which use light as an energy source for DNA repair but themselves have no light sensing role. Here we review the means by which plant cryptochromes acquired a light sensing function. This transition involved subtle changes within the flavin binding pocket which gave rise to a visual photocycle consisting of light-inducible and dark-reversible flavin redox state transitions. In this photocycle, light first triggers flavin reduction from an initial dark-adapted resting state (FADox). The reduced state is the biologically active or 'lit' state, correlating with biological activity. Subsequently, the photoreduced flavin reoxidises back to the dark adapted or 'resting' state. Because the rate of reoxidation determines the lifetime of the signaling state, it significantly modulates biological activity. As a consequence of this redox photocycle Crys respond to both the wavelength and the intensity of light, but are in addition regulated by factors such as temperature, oxygen concentration, and cellular metabolites that alter rates of flavin reoxidation even independently of light. Mechanistically, flavin reduction is correlated with conformational change in the protein, which is thought to mediate biological activity through interaction with biological signaling partners. In addition, a second, entirely independent signaling mechanism arises from the cryptochrome photocycle in the form of reactive oxygen species (ROS). These are synthesized during flavin reoxidation, are known mediators of biotic and abiotic stress responses, and have been linked to Cry biological activity in plants and animals. Additional special properties arising from the cryptochrome photocycle include responsivity to electromagnetic fields and their applications in optogenetics. Finally, innovations in methodology such as the use of Nitrogen Vacancy (NV) diamond centers to follow cryptochrome magnetic field sensitivity in vivo are discussed, as well as the potential for a whole new technology of 'magneto-genetics' for future applications in synthetic biology and medicine.

Advanced prescription of injectable anticancer drugs: Safety assessment in a European Comprehensive Cancer Centre using the risk matrix approach.

AIMS: The purpose of this work was to assess failures in the advanced prescription of parenteral anticancer agents in an adult day oncology care unit with more than 100 patients per day. METHODS: An a priori descriptive analysis was carried out by using the risk matrix approach. After defining the scope in a multidisciplinary meeting, we determined at each step the failure modes (FMs), their effects (E) and their associated causes (C). A severity score (S) was assigned to all effects and a probability of occurrence (O) to all causes. These S and O indicators, were used to obtain a criticality index (CI) matrix. We assessed the risk control (RC) of each failure in order to define a residual criticality index (rCI) matrix. RESULTS: During risk analysis, 14 FMs were detected, and 61 scenarios were identified considering all possible effects and causes. Nine situations (15%) were highlighted with the maximum CI, 18 (30%) with a medium CI, and 34 (55%) with a negligible CI. Nevertheless, among all these critical situations, only three (5%) had an rCI to process (i.e., missed dose adjustment, multiple prescriptions and abnormal biology data); the others required monitoring only. Clinicians' and pharmacists' knowledge of these critical situations enables them to manage the associated risks. CONCLUSIONS: Advanced prescription of injectable anticancer drugs appears to be a safe practice for patients when combined with risk management. The major risks identified concerned missed dose adjustment, prescription duplication and lack of consideration for abnormal biology data.

Residual chemotherapy drugs after flushing infusion lines.

INTRODUCTION: During administration of chemotherapies, disconnection presents risks for nurses. Thus, it is recommended to flush the infusion line with solvent to reduce this risk and ensure that the entire dose is administered. Objectives of this study were to evaluate flushing practices and to investigate the efficiency of flushing, according to the type of hospitalization, in hospitalization (HU) or day-care unit (DCU), for three drugs. METHODS: Twenty secondary infusion lines were collected in five HU and 20 in two DCU. Flushing volumes were estimated by weighing solvent bags. The amount of residual drug was measured for secondary lines by mass spectrometry coupled with high-performance liquid chromatography. RESULTS: Chemotherapies were administered by 26 nurses. All of infusion lines contained chemotherapy after flushing. Flushing volumes, residual concentrations and flushing efficiencies were significantly different between these two types of units. In contrast, flushing volumes administrated did not differ between chemotherapy drugs. CONCLUSIONS: Local recommendations are fully implemented in HU and partially in DCU. The use of small volumes in DCU is related to the patient length of stay, it may, also, be due to omitting the average tubing volume. All infusion lines still contained chemotherapy, including those with a flush volume much higher than recommended, showing that the risk of exposure persists. To achieve a rinse volume greater than 50 mL, it is necessary to use at least 100 mL. It is also important to insist on personal protective equipment and to consider closed safety system for administration.

Cryptochrome and quantum biology: unraveling the mysteries of plant magnetoreception.

Magnetoreception, the remarkable ability of organisms to perceive and respond to Earth's magnetic field, has captivated scientists for decades, particularly within the field of quantum biology. In the plant science, the exploration of the complicated interplay between quantum phenomena and classical biology in the context of plant magnetoreception has emerged as an attractive area of research. This comprehensive review investigates into three prominent theoretical models: the Radical Pair Mechanism (RPM), the Level Crossing Mechanism (LCM), and the Magnetite-based MagR theory in plants. While examining the advantages, limitations, and challenges associated with each model, this review places a particular weight on the RPM, highlighting its well-established role of cryptochromes and in-vivo experiments on light-independent plant magnetoreception. However, alternative mechanisms such as the LCM and the MagR theory are objectively presented as convincing perspectives that permit further investigation. To shed light on these theoretical frameworks, this review proposes experimental approaches including cutting-edge experimental techniques. By integrating these approaches, a comprehensive understanding of the complex mechanisms driving plant magnetoreception can be achieved, lending support to the fundamental principle in the RPM. In conclusion, this review provides a panoramic overview of plant magnetoreception, highlighting the exciting potential of quantum biology in unraveling the mysteries of magnetoreception. As researchers embark on this captivating scientific journey, the doors to deciphering the diverse mechanisms of magnetoreception in plants stand wide open, offering a profound exploration of nature's adaptations to environmental cues.

Near-Infrared Light Exposure Triggers ROS to Downregulate Inflammatory Cytokines Induced by SARS-CoV-2 Spike Protein in Human Cell Culture.

The leading cause of mortality from SARS-CoV-2 is an exaggerated host immune response, triggering cytokine storms, multiple organ failure and death. Current drug- and vaccine-based therapies are of limited efficacy against novel viral variants. Infrared therapy is a non-invasive and safe method that has proven effective against inflammatory conditions for over 100 years. However, its mechanism of action is poorly understood and has not received widespread acceptance. We herein investigate whether near-infrared (NIR) light exposure in human primary alveolar and macrophage cells could downregulate inflammatory cytokines triggered by the SARS-CoV-2 spike (S) protein or lipopolysaccharide (LPS), and via what underlying mechanism. Our results showed a dramatic reduction in pro-inflammatory cytokines within days of NIR light treatment, while anti-inflammatory cytokines were upregulated. Mechanistically, NIR light stimulated mitochondrial metabolism, induced transient bursts in reactive oxygen species (ROS) and activated antioxidant gene transcription. These, in turn, downregulated ROS and inflammatory cytokines. A causal relationship was shown between the induction of cellular ROS by NIR light exposure and the downregulation of inflammatory cytokines triggered by SARS-CoV-2 S. If confirmed by clinical trials, this method would provide an immediate defense against novel SARS-CoV-2 variants and other inflammatory infectious diseases.

SNAC3 Transcription Factor Enhances Arsenic Stress Tolerance and Grain Yield in Rice (Oryza sativa L.) through Regulating Physio-Biochemical Mechanisms, Stress-Responsive Genes, and Cryptochrome 1b.

Arsenic (As) is one of the toxic heavy metal pollutants found in the environment. An excess of As poses serious threats to plants and diminishes their growth and productivity. NAC transcription factors revealed a pivotal role in enhancing crops tolerance to different environmental stresses. The present study investigated, for the first time, the functional role of SNAC3 in boosting As stress tolerance and grain productivity in rice (Oryza sativa L.). Two SNAC3-overexpressing (SNAC3-OX) and two SNAC3-RNAi transgenic lines were created and validated. The wild-type and transgenic rice plants were exposed to different As stress levels (0, 25, and 50 µM). The results revealed that SNAC3 overexpression significantly improved rice tolerance to As stress and boosted grain yield traits. Under both levels of As stress (25 and 50 µM), SNAC3-OX rice lines exhibited significantly lower levels of oxidative stress biomarkers and OsCRY1b (cryptochrome 1b) expression, but they revealed increased levels of gas exchange characters, chlorophyll, osmolytes (soluble sugars, proteins, proline, phenols, and flavonoids), antioxidant enzymes (SOD, CAT, APX, and POD), and stress-tolerant genes expression (OsSOD-Cu/Zn, OsCATA, OsCATB, OsAPX2, OsLEA3, OsDREB2B, OsDREB2A, OsSNAC2, and OsSNAC1) in comparison to wild-type plants. By contrast, SNAC3 suppression (RNAi) reduced grain yield components and reversed the aforementioned measured physio-biochemical and molecular traits. Taken together, this study is the first to demonstrate that SNAC3 plays a vital role in boosting As stress resistance and grain productivity in rice through modulating antioxidants, photosynthesis, osmolyte accumulation, and stress-related genes expression, and may be a useful candidate for further genetic enhancement of stress resistance in many crops.

Exposure to 1.8 GHz radiofrequency field modulates ROS in human HEK293 cells as a function of signal amplitude.

The modern telecommunications industry is ubiquitous throughout the world, with a significant percentage of the population using cellular phones on a daily basis. The possible physiological consequences of wireless emissions in the GHz range are therefore of major interest, but remain poorly understood. Here, we show that exposure to a 1.8 GHz carrier frequency in the amplitude range of household telecommunications induces the formation of ROS (Reactive Oxygen Species) in human HEK293 cultured cells. The ROS concentrations detected by fluorescent imaging techniques increased significantly after 15 minutes of RF field exposure, and were localized to both nuclear and cytosolic cellular compartments. qPCR analysis showed altered gene expression of both anti-oxidative (SOD, GPX, GPX, and CAT) and oxidative (Nox-2) enzymes. In addition, multiple genes previously identified as responsive to static magnetic fields were found to also be regulated by RF, suggesting common features in response mechanisms. By contrast, many RF effects showed evidence of hormesis, whereby biological responsivity does not occur linearly as a function of signal amplitude. Instead, biphasic dose response curves occur with 'blind' spots at certain signal amplitudes where no measureable response occurs. We conclude that modulation of intracellular ROS can be a direct consequence of RF exposure dependent on signal frequency and amplitude. Since changes in intracellular ROS may have both harmful and beneficial effects, these could provide the basis for many reported physiological effects of RF exposure.

Infrared light therapy relieves TLR-4 dependent hyper-inflammation of the type induced by COVID-19.

The leading cause of mortality from COVID-19 infection is respiratory distress due to an exaggerated host immune response, resulting in hyper-inflammation and ensuing cytokine storms in the lungs. Current drug-based therapies are of limited efficacy, costly, and have potential negative side effects. By contrast, photobiomodulation therapy, which involves periodic brief exposure to red or infrared light, is a noninvasive, safe, and affordable method that is currently being used to treat a wide range of diseases with underlying inflammatory conditions. Here, we show that exposure to two 10-min, high-intensity periods per day of infrared light causes a marked reduction in the TLR-4 dependent inflammatory response pathway, which has been implicated in the onset of cytokine storms in COVID-19 patients. Infrared light exposure resulted in a significant decline in NFkB and AP1 activity as measured by the reporter gene assay; decreased expression of inflammatory marker genes IL-6, IL-8, TNF-alpha, INF-alpha, and INF-beta as determined by qPCR gene expression assay; and an 80% decline in secreted cytokine IL6 as measured by ELISA assay in cultured human cells. All of these changes occurred after only 48 hours of treatment. We suggest that an underlying cellular mechanism involving modulation of ROS may downregulate the host immune response after Infrared Light exposure, leading to decrease in inflammation. We further discuss technical considerations involving light sources and exposure conditions to put these observations into potential clinical use to treat COVID-19 induced mortality.

Therapeutic application of light and electromagnetic fields to reduce hyper-inflammation triggered by COVID-19.

COVID-19 - related morbidity is associated with exaggerated inflammation and cytokine production in the lungs, leading to acute respiratory failure. The cellular mechanisms underlying these so-called 'cytokine storms' are regulated through the Toll-like receptor 4 (TLR4) signaling pathway and by ROS (Reactive Oxygen Species). Both light (Photobiomodulation) and magnetic fields (e.g., Pulsed Electro Magnetic Field) stimulation are noninvasive therapies known to confer anti-inflammatory effects and regulate ROS signaling pathways. Here we show that daily exposure to two 10-minute intervals of moderate intensity infra-red light significantly lowered the inflammatory response induced via the TLR4 receptor signaling pathway in human cell cultures. Anti-inflammatory effects were likewise achieved by electromagnetic field exposure of cells to daily 10-minute intervals of either Pulsed Electromagnetic Fields (PEMF), or to Low-Level static magnetic fields. Because current illumination and electromagnetic field therapies have no known side effects, and are already approved for some medical uses, we have here developed protocols for verification in clinical trials of COVID-19 infection. These treatments are affordable, simple to implement, and may help to resolve the acute respiratory distress of COVID-19 patients both in the home and in the hospital.

Effect of temperature on the Arabidopsis cryptochrome photocycle.

Cryptochromes are blue light-absorbing photoreceptors found in plants and animals with many important signalling functions. These include control of plant growth, development, and the entrainment of the circadian clock. Plant cryptochromes have recently been implicated in adaptations to temperature variation, including temperature compensation of the circadian clock. However, the effect of temperature directly on the photochemical properties of the cryptochrome photoreceptor remains unknown. Here we show that the response to light of purified Arabidopsis Cry1 and Cry2 proteins was significantly altered by temperature. Spectral analysis at 15°C showed a pronounced decrease in flavin reoxidation rates from the biologically active, light-induced (FADH°) signalling state of cryptochrome to the inactive (FADox) resting redox state as compared to ambient (25°C) temperature. This result indicates that at low temperatures, the concentration of the biologically active FADH° redox form of Cry is increased, leading to the counterintuitive prediction that there should be an increased biological activity of Cry at lower temperatures. This was confirmed using Cry1 cryptochrome C-terminal phosphorylation as a direct biological assay for Cry activation in vivo. We conclude that enhanced cryptochrome function in vivo at low temperature is consistent with modulation by temperature of the cryptochrome photocycle.

HEK293 cell response to static magnetic fields via the radical pair mechanism may explain therapeutic effects of pulsed electromagnetic fields.

PEMF (Pulsed Electromagnetic Field) stimulation has been used for therapeutic purposes for over 50 years including in the treatment of memory loss, depression, alleviation of pain, bone and wound healing, and treatment of certain cancers. However, the underlying cellular mechanisms mediating these effects have remained poorly understood. In particular, because magnetic field pulses will induce electric currents in the stimulated tissue, it is unclear whether the observed effects are due to the magnetic or electric component of the stimulation. Recently, it has been shown that PEMFs stimulate the formation of ROS (reactive oxygen species) in human cell cultures by a mechanism that requires cryptochrome, a putative magnetosensor. Here we show by qPCR analysis of ROS-regulated gene expression that simply removing cell cultures from the Earth's geomagnetic field by placing them in a Low-Level Field condition induces similar effects on ROS signaling as does exposure of cells to PEMF. This effect can be explained by the so-called Radical Pair mechanism, which provides a quantum physical means by which the rates and product yields (e.g. ROS) of biochemical redox reactions may be modulated by magnetic fields. Since transient cancelling of the Earth's magnetic field can in principle be achieved by PEMF exposure, we propose that the therapeutic effects of PEMFs may be explained by the ensuing modulation of ROS synthesis. Our results could lead to significant improvements in the design and therapeutic applications of PEMF devices.

Twenty-year trends in patient referrals throughout the creation and development of a regional memory clinic network.

INTRODUCTION: Memory clinics (MCs) are the main model for dementia diagnosis and care. Following the development of a MC network in Northern France, our objectives were to assess its impact on patient characteristics over 20 years. METHODS: The characteristics of new consultants were studied from 1997 to 2016. RESULTS: New consultants increased from 774 per year in 1997 to 26258 per year in 2016, as the number of MCs increased from 12 to 29. Over time, patients were progressively older and less educated, and more were living alone. A greater proportion of patients were referred by specialists. Referral delay and home-to-MC distance kept decreasing. The oldest patients were referred at a progressively less-severe stage. The proportion of young patients kept increasing in the tertiary referral center. DISCUSSIONS: The development of a region-wide MC network led to increased referral of vulnerable patients and differentiation of the tertiary referral center over time.

Sirtuin 7 promotes 45S pre-rRNA cleavage at site 2 and determines the processing pathway.

In humans, ribosome biogenesis mainly occurs in nucleoli following two alternative pre-rRNA processing pathways differing in the order in which cleavages take place but not by the sites of cleavage. To uncover the role of the nucleolar NAD+-dependent deacetylase sirtuin 7 in the synthesis of ribosomal subunits, pre-rRNA processing was analyzed after sirtinol-mediated inhibition of sirtuin 7 activity or depletion of sirtuin 7 protein. We thus reveal that sirtuin 7 activity is a critical regulator of processing of 45S, 32S and 30S pre-rRNAs. Sirtuin 7 protein is primarily essential to 45S pre-rRNA cleavage at site 2, which is the first step of processing pathway 2. Furthermore, we demonstrate that sirtuin 7 physically interacts with Nop56 and the GAR domain of fibrillarin, and propose that this could interfere with fibrillarin-dependent cleavage. Sirtuin 7 depletion results in the accumulation of 5' extended forms of 32S pre-rRNA, and also influences the localization of fibrillarin. Thus, we establish a close relationship between sirtuin 7 and fibrillarin, which might determine the processing pathway used for ribosome biogenesis.

Low-intensity electromagnetic fields induce human cryptochrome to modulate intracellular reactive oxygen species.

Exposure to man-made electromagnetic fields (EMFs), which increasingly pollute our environment, have consequences for human health about which there is continuing ignorance and debate. Whereas there is considerable ongoing concern about their harmful effects, magnetic fields are at the same time being applied as therapeutic tools in regenerative medicine, oncology, orthopedics, and neurology. This paradox cannot be resolved until the cellular mechanisms underlying such effects are identified. Here, we show by biochemical and imaging experiments that exposure of mammalian cells to weak pulsed electromagnetic fields (PEMFs) stimulates rapid accumulation of reactive oxygen species (ROS), a potentially toxic metabolite with multiple roles in stress response and cellular ageing. Following exposure to PEMF, cell growth is slowed, and ROS-responsive genes are induced. These effects require the presence of cryptochrome, a putative magnetosensor that synthesizes ROS. We conclude that modulation of intracellular ROS via cryptochromes represents a general response to weak EMFs, which can account for either therapeutic or pathological effects depending on exposure. Clinically, our findings provide a rationale to optimize low field magnetic stimulation for novel therapeutic applications while warning against the possibility of harmful synergistic effects with environmental agents that further increase intracellular ROS.

Exploring the Microbiota of Diabetic Foot Infections With Culturomics.

The purpose of this prospective observational study was to evaluate the richness and diversity of bacteria in samples from diabetic foot infections using a culturomics approach. Bacterial culture findings from wound samples were analyzed together with clinical characteristics and treatment outcomes. We included 43 patients admitted to a French referral center with a moderate to severe diabetic foot infection. The 30,000 colonies identified yielded 53 different bacterial species. The global α-Shannon diversity was 3.34 and the bacterial richness per patient was 4 ± 2. Of all the identified bacterial species, 19 (35.8%) had never been previously cultured or identified by molecular methods from diabetic foot ulcers. Most of the samples were polymicrobial (N = 38; 88.3%). Of all the isolated species, the most prevalent were Staphylococcus aureus (N = 28; 52.8%), Enterococcus faecalis (N = 24; 45.2%), Enterobacter cloacae (N = 12; 22.6%), Staphylococcus lugdunensis (N = 10; 18.7%), Staphylococcus epidermidis (N = 6; 11.3%), Proteus mirabilis (N = 6; 11.3%), and Finegoldia magna (N = 5; 9.4%). The only factor associated with wound improvement after a 1-month follow-up was the presence of E. faecalis (p = 0.012) when compared with patients without wound improvement. This study confirms the complementary role of culturomics in the exploration of complex microbiota. Further studies on a larger scale are needed to fully understand the clinical importance of the microbiota of diabetic foot infections.

Blue-light induced biosynthesis of ROS contributes to the signaling mechanism of Arabidopsis cryptochrome.

Cryptochromes are evolutionarily conserved blue light receptors with many roles throughout plant growth and development. They undergo conformational changes in response to light enabling interaction with multiple downstream signaling partners. Recently, it has been shown that cryptochromes also synthesize reactive oxygen species (ROS) in response to light, suggesting the possibility of an alternate signaling mechanism. Here we show by fluorescence imaging and microscopy that H202 and ROS accumulate in the plant nucleus after cryptochrome activation. They induce ROS-regulated transcripts including for genes implicated in pathogen defense, biotic and abiotic stress. Mutant cryptochrome alleles that are non-functional in photomorphogenesis retain the capacity to induce ROS-responsive phenotypes. We conclude that nuclear biosynthesis of ROS by cryptochromes represents a new signaling paradigm that complements currently known mechanisms. This may lead to novel applications using blue light induced oxidative bursts to prime crop plants against the deleterious effects of environmental stresses and toxins.

Blue-light induced accumulation of reactive oxygen species is a consequence of the Drosophila cryptochrome photocycle.

Cryptochromes are evolutionarily conserved blue-light absorbing flavoproteins which participate in many important cellular processes including in entrainment of the circadian clock in plants, Drosophila and humans. Drosophila melanogaster cryptochrome (DmCry) absorbs light through a flavin (FAD) cofactor that undergoes photoreduction to the anionic radical (FAD•-) redox state both in vitro and in vivo. However, recent efforts to link this photoconversion to the initiation of a biological response have remained controversial. Here, we show by kinetic modeling of the DmCry photocycle that the fluence dependence, quantum yield, and half-life of flavin redox state interconversion are consistent with the anionic radical (FAD•-) as the signaling state in vivo. We show by fluorescence detection techniques that illumination of purified DmCry results in enzymatic conversion of molecular oxygen (O2) to reactive oxygen species (ROS). We extend these observations in living cells to demonstrate transient formation of superoxide (O2•-), and accumulation of hydrogen peroxide (H2O2) in the nucleus of insect cell cultures upon DmCry illumination. These results define the kinetic parameters of the Drosophila cryptochrome photocycle and support light-driven electron transfer to the flavin in DmCry signaling. They furthermore raise the intriguing possibility that light-dependent formation of ROS as a byproduct of the cryptochrome photocycle may contribute to its signaling role.

Sharing of mitotic pre-ribosomal particles between daughter cells.

Ribosome biogenesis is a fundamental multistep process initiated by the synthesis of 90S pre-ribosomal particles in the nucleoli of higher eukaryotes. Even though synthesis of ribosomes stops during mitosis while nucleoli disappear, mitotic pre-ribosomal particles persist as observed in pre-nucleolar bodies (PNBs) during telophase. To further understand the relationship between the nucleolus and the PNBs, the presence and the fate of the mitotic pre-ribosomal particles during cell division were investigated. We demonstrate that the recently synthesized 45S precursor ribosomal RNAs (pre-rRNAs) as well as the 32S and 30S pre-rRNAs are maintained during mitosis and associated with the chromosome periphery together with pre-rRNA processing factors. Maturation of the mitotic pre-ribosomal particles, as assessed by the stability of the mitotic pre-rRNAs, is transiently arrested during mitosis by a cyclin-dependent kinase (CDK)1-cyclin-B-dependent mechanism and can be restored by CDK inhibitor treatments. At the M-G1 transition, the resumption of mitotic pre-rRNA processing in PNBs does not induce the disappearance of PNBs; this only occurs when functional nucleoli reform. Strikingly, during their maturation process, mitotic pre-rRNAs localize in reforming nucleoli.

Blue-light dependent ROS formation by Arabidopsis cryptochrome-2 may contribute toward its signaling role.

Cryptochromes are blue-light absorbing flavoproteins with many important signaling roles in plants, including in de-etiolation, development, and stress response. They interact with downstream signaling partners such as transcription factors and components of the proteasome, and thereby alter regulation of nuclear gene expression in a light dependent manner. In a prior study, it has also been shown that Arabidopsis cry1 activation by blue light results in direct enzymatic conversion of molecular oxygen (O2) to ROS (reactive oxygen species) in vivo leading to cell death in overexpressing lines. Here we extend these observations to show that Atcry2 is translocated from the cytosol to the nucleus in response to blue light illumination, resulting in nuclear accumulation of ROS in expressing insect cell cultures. These observations suggest that ROS formation may represent a novel means of signaling by Atcry2 distinct from, and perhaps complementary to, the currently known mechanism of light-mediated conformational change.

Blue-light dependent reactive oxygen species formation by Arabidopsis cryptochrome may define a novel evolutionarily conserved signaling mechanism.

Cryptochromes are widespread blue-light absorbing flavoproteins with important signaling roles. In plants they mediate de-etiolation, developmental and stress responses resulting from interaction with downstream signaling partners such as transcription factors and components of the proteasome. Recently, it has been shown that Arabidopsis cry1 activation by blue light also results in direct enzymatic conversion of molecular oxygen (O2 ) to reactive oxygen species (ROS) and hydrogen peroxide (H2 O2 ) in vitro. Here we explored whether direct enzymatic synthesis of ROS by Arabidopsis cry1 can play a physiological role in vivo. ROS formation resulting from cry1 expression was measured by fluorescence assay in insect cell cultures and in Arabidopsis protoplasts from cryptochrome mutant seedlings. Cell death was determined by colorimetric assay. We found that ROS formation results from cry1 activation and induces cell death in insect cell cultures. In plant protoplasts, cryptochrome activation results in rapid increase in ROS formation and cell death. We conclude that ROS formation by cryptochromes may indeed be of physiological relevance and could represent a novel paradigm for cryptochrome signaling.