Biology Direct

Ten years ago, it was predicted that the multi-omics revolution would also revolutionize space pharmacogenomics. Current barriers related to the findable, accessible, interoperable, and reproducible use of space-flown pharmaceutical data have contributed to a lack of progress beyond application of earth-based principles. To directly tackle these challenges, we have produced a novel database of all the drugs flown into space, compiled from publicly available ontological and spaceflight-related datasets, to exemplify analyses for describing significant spaceflight-related targets. By focusing on mechanisms perturbed by spaceflight, we have provided a novel avenue for identifying the most relevant changes within the drug absorption, distribution, metabolism, and excretion pathways. We suggest a set of space genes, by necessity limited to available tissue types, that can be expanded and modified based on future tissue-specific and mechanistic-specific high-throughput assays. In sum, we provide the justification and a definitive starting point for pharmacogenomics guided spaceflight as a foundation of precision medicine, which will enable long-term human habitation of the Moon, Mars, and beyond. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=147 SRC="FIGDIR/small/575951v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@48f739org.highwire.dtl.DTLVardef@5ecdb0org.highwire.dtl.DTLVardef@121c93org.highwire.dtl.DTLVardef@1122b3f_HPS_FORMAT_FIGEXP M_FIG C_FIG

In cyanobacteria DNA supercoiling varies over the diurnal light/dark cycle and is integrated with the circadian transcription program and (Woelfle et al. [2007], Vijayan et al. [2009], PNAS). Specifically, Woelfle et al. have reported that DNA supercoiling of an endogenous plasmid became progressively higher during prolonged dark phases in Synechococcus elongatus PCC 7942. This is counterintuitive, since higher levels of negative DNA supercoiling are commonly associated with exponential growth and high metabolic flux. Vijayan et al. then have reverted the interpretation of plasmid mobility on agarose gels supplemented with chloroquine diphosphate (CQ), but not further discussed the differences. Here, we set out to clarify this open issue in cyanobacterial DNA supercoiling dynamics. We first re-capitulate Kellers band counting method (1975, PNAS) using CQ instead of ethidium bromide as the intercalating agent. A 500x-1000x higher CQ concentration is required in the DNA relaxation reaction (topoisomerase I) than in the agarose gel buffer to quench all negative supercoiling of pUC19 extracted from Escherichia coli. This is likely due to the dependence of both, the DNA binding affinity of CQ and the induced DNA unwinding angle, on the ionic strength of the buffer. Lower levels of CQ were required to fully relax in vivo pUC19 supercoiling than were used by Woelfle et al. Next, we analyzed the in vivo supercoiling of endogenous plasmids of Synechocystis sp. PCC 6803, at two different CQ concentrations. These experiments indicate that negative supercoiling of plasmids does not increase but decreases in the dark phase, and progressively decreases further in prolonged darkness.

Pyridoxal-phosphate binding proteins (PLPBP) are involved in the homeostasis of B6 vitamers and amino/keto acids, share a high degree of sequence conservation and are represented in all three domains of life. Despite the obligate presence of the catalyst cofactor PLP, attempts to show enzymatic activity have been unsuccessful. Instead, evidence of RNA binding activity has been provided for several members of the family. Here we use PipY, one of the few PLBPB members studied so far, as a model system to address the phenotypic impact in the cyanobacterium Synechococcus elongatus of mutations K26A, P63L and R210Q, which respectively prevent PLP binding or are equivalent to those conferring B6-dependent epilepsy in humans with a recessive inheritance pattern. We found that while mutation K26A at the PLP-binding residue abrogated all phenotypes associated to PipY overexpression and toxicity, P63L and R210Q behaved as dominant gain-of-function mutations that inhibited bacterial growth. We provide in vivo evidence of PipY performing PLP-independent functions, in which mutant variant PipYK26A but not PipYP63L or PipYR210Q would be defective. A model integrating our observations with previous data from other organims and PLPBP variants is discussed.

Synthetic prostaglandin analogues, such as latanoprost, are first-line treatments to reduce intraocular pressure (IOP) in the management of glaucoma, treating millions of patients daily. Glaucoma is a leading cause of blindness, characterized by progressive optic neuropathy, with elevated IOP being the sole modifiable risk factor. Despite this importance, the underlying latanoprost mechanism is still not well defined, being associated with both acute and long term activities, and ocular side effects. Prostaglandins are eicosanoid lipid mediators. Yet, there has not been a comprehensive assessment of small lipid mediators in glaucomatous eyes. Here we performed a lipidomic screen of aqueous humour sampled from glaucoma patients or healthy control eyes. The resulting signature was surprisingly focused on significantly elevated levels of arachidonic acid (AA) and the potent proresolving mediator, lipoxin A4 (LXA4) in glaucoma eyes. Subsequent experiments revealed that this response is due to latanoprost actions, rather than a consequence of elevated IOP. We demonstrated that increased LXA4 inhibits pro-inflammatory cues and promotes TGF-{beta}3 mediated tissue remodeling in the anterior chamber. In concert, an autocrine prostaglandin circuit mediates rapid IOP-lowering. This work reveals parallel mechanisms underlying acute and long-term latanoprost activities during the treatment of glaucoma.

Photosynthetic organisms coordinate their metabolism and growth with diurnal light, which can range in intensity from limiting to inhibitory. To gain a comprehensive understanding of how diurnal regulatory circuits interface with sensing and response to various light intensities, we performed a systems analysis of synchronized Chlamydomonas populations acclimated to low, moderate, and high diurnal light. Transcriptomic and proteomic data revealed that the Chlamydomonas rhythmic gene expression program is resilient to limiting and excess light. Although gene expression and photodamage are dynamic over the diurnal cycle, Chlamydomonas populations acclimated to low and high diurnal light exhibit constitutive phenotypes with respect to photosystem abundance, thylakoid architecture, and non-photochemical quenching that persist through the night. This suggests that cells "remember" or anticipate the daylight environment. The integrated data constitute an excellent resource for understanding gene regulatory mechanisms and photoprotection in eukaryotes under environmentally relevant conditions.

Nitrogen is a key macro-nutrient required for the metabolism and growth of biological systems. Although multiple nitrogen sources can serve this purpose, they are all converted into ammonium/ammonia as a first step of assimilation. It is thus reasonable to expect that molecular parts involved in the transport of ammonium/ammonia across biological membranes (i.e. catalysed by AMT transporters) connect with the regulation of both nitrogen and central carbon metabolism. In order to test this hypothesis, we applied both (1) genetic (i.e. {Delta}amt mutation) and (2) environmental treatments to a target biological system, the cyanobacterium Anabaena sp. PCC 7120. Cyanobacteria have a key role in the global nitrogen cycle and thus represent a useful model system. The aim was to both (1) perturb sensing and low-affinity uptake of ammonium/ammonia and (2) induce multiple inner N states, followed by targeted quantification of key proteins, metabolites and enzyme activities, with experiments intentionally designed over a longer time-scale than the available studies in literature. We observed that the absence of AMT transporters triggered a substantial response at a whole-system level, affecting enzyme activities and the quantity of both proteins and metabolites, spanning both N and C metabolism. Moreover, the absence of AMT transporters left a molecular fingerprint indicating N-deficiency even under N replete conditions (i.e. greater GS activity, lower 2-OG content and faster nitrogenase activation upon N deprivation). Contrasting with all of the above dynamic adaptations was the striking near-complete lack of any externally measurable phenotype (i.e. growth, photosynthesis, pigments, metabolites). We thus conclude that this species evolved a highly robust and adaptable molecular network to maintain homeostasis, resulting in substantial internal but minimal external perturbations. The analytical data highlights several internal adaptations, including increased N assimilation (i.e. greater GS activity) and nitrogenase activity (i.e. faster activation upon N deprivation) together with altered amino acids metabolism, as indicated by changes in Gln, Glu and 2-OG, indicating an altered C/N balance. The analyses provides evidence for an active role of AMT transporters in the regulatory/signalling network of N metabolism in this biological system, and the existence of a novel fourth IF7A-independent regulatory mechanism controlling GS activity.

Circadian clocks regulate biological activities, providing organisms a fitness advantage under diurnal changing conditions by allowing them to anticipate and adapt to recurring external changes. In recent years attention was drawn to the entrainment by intracellular cycles. Photosynthetic Cyanobacteria coordinate their gene expression, metabolism, and other activities in a circadian fashion. Solely, three proteins, KaiA, KaiB, and KaiC, constitute the well-studied circadian clock of the cyanobacterial model, Synechococcus elongatus PCC 7942. It remained inconclusive for a long time whether Synechocystis sp. PCC 6803, an important organism for biotechnological applications, can also maintain circadian rhythms under continuous illumination. Using an approach, which does not require genetic modification, we investigated the growth behavior of Synechocystis via non-invasive online backscattering measurement and verified all three criteria for true circadian oscillators: temperature compensation, entrainment by external stimuli, and a self-sustained freerunning period of about 24 hours. Since manipulation of the circadian clock (Synechocystis {Delta}kaiA1B1C1) led to a significant reduction in glycogen content, disruption of glycogen synthesis (Synechocystis {Delta}glgC) entirely inhibited glycogen formation and both mutants lost oscillations, we hypothesize that the oscillations reflect glycogen metabolism. Significance StatementMonitoring circadian rhythms in cyanobacteria usually requires genetically modified reporter strains or intensive sampling for downstream analysis. Even for the main cyanobacterial model Synechocystis sp. PCC 6803 it was debated for years to which extent undamped circadian oscillations are really present until a suitable reporter strain was developed. We applied online backscatter measurements as an alternative readout to monitor circadian oscillations in cyanobacteria. In Synechocystis the temperature-compensated kaiA1B1C1-driven 24 h metabolic oscillations did not require light-dark entrainment, highlighting the relevance of the clock for the carbon metabolism even under continuous light, an aspect which should be considered for industrial set-ups. Our method opens the possibility to extend circadian analysis to non-GMO and monitor metabolic rhythmicity during high-density cultivation.

Levacetylleucine (AqneursaTM), a chemically modified amino acid, is the only US Food and Drug Administration-approved monotherapy for the treatment of Niemann-Pick disease type C (NPC) (Beninger, 2024; Mullard, 2024; van Gool et al., 2025). This acetylated derivative of L-leucine functions as a pro-drug, with the acetyl group rendering it a substrate for the monocarboxylate transporter (MCT) family of transporters to allow appreciable penetration of the blood-brain barrier and its efficient uptake into cells (Churchill et al., 2021). Inside cells, levacetylleucine undergoes metabolism catalysed by acylases, and the resultant high quantities of L-leucine enter metabolic pathways which enhance mitochondrial bioenergetics and, as previously demonstrated, indirectly ameliorate lysosomal function (Kaya et al., 2020). Here, we show a novel aspect of levacetylleucines mechanism of action, demonstrating a direct effect on lysosomal function through its rapid modulation of the translocation of the transcription factor TFEB, a master regulator of lysosomal biogenic and autophagic genes (Napolitano and Ballabio, 2016), from cytoplasm to nucleus. Uniquely, we have demonstrated a biphasic action whereby levacetylleucine normalizes TFEB activity, consistent with levacetylleucines previously shown ability to regulate cellular homeostasis: in wild-type HeLa cells, levacetylleucine enhances and activates the translocation of TFEB to the nucleus. In contrast, in cellular models of NPC type 1 disease, where TFEB is already over-expressed in the nucleus (as the cell attempts to compensate for the primary defect by activating TFEB as a natural cellular response to the lysosomal substrate accumulation and associated cellular stress), treatment with levacetylleucine down-regulates and restores the distribution of TFEB to a more normalized cytoplasmic: nuclear ratio. Importantly, both effects of levacetylleucine occur at concentrations consistent with plasma concentrations in therapeutic dosing (Churchill et al., 2020). The effects were also confirmed to be stereospecific to the L-enantiomer, as neither the D-enantiomer (N-acetyl-D-leucine) or racemate (N-acetyl-DL-leucine) had any effect, The presence of the D-enantiomer in the racemic mixture inhibited the ability of levacetylleucine to promote TFEB bidirectional translocation, consistent with previous studies, which have established antagonism of N-acetyl-L-leucine by N-acetyl-D-leucine in the racemic mixture (rendering the racemic mixture without effect). This bidirectional mechanism of action of levacetylleucine to impact lysosomal function directly and normalize, either by activating basal TFEB signalling or reducing aberrant TFEB function in NPC1 knockout cells, thereby modulating lysosomal and autophagic functions, lends itself to the treatment of a broad range of neurological and neurodevelopment disorders.

The Antarctic green alga Chlamydomonas priscuii is an obligate psychrophile and an emerging model for photosynthetic adaptation to extreme conditions. Endemic to the ice-covered Lake Bonney, this alga thrives at highly unusual light conditions characterized by very low light irradiance (<15 mol m-2 s-1), a narrow wavelength spectrum enriched in blue light, and an extreme photoperiod. Genome sequencing of C. priscuii exposed an unusually large genome, with hundreds of highly similar gene duplicates and expanded gene families, some of which could be aiding its survival in extreme conditions. In contrast to the described expansion in the genetic repertoire in C. priscuii, here we suggest that the gene family encoding for photoreceptors is reduced when compared to related green algae. This alga also possesses a very small eyespot and exhibits an aberrant phototactic response, compared to the model Chlamydomonas reinhardtii. We also investigated the genome and behaviour of the closely related psychrophilic alga Chlamydomonas sp. ICE-MDV, that is found throughout the photic zone of Lake Bonney and is naturally exposed to higher light levels. Our analyses revealed a photoreceptor gene family and a robust phototactic response similar to those in the model Chlamydomonas reinhardtii. These results suggest that the aberrant phototactic response in C. priscuii is a result of life under extreme shading rather than a common feature of all psychrophilic algae. We discuss the implications of these results on the evolution and survival of shade adapted polar algae.

In cyanobacteria DNA supercoiling varies over the diurnal light/dark cycle and is integrated with temporal programs of transcription and replication. We manipulated DNA supercoiling in Synechocystis sp. PCC 6803 by CRISPRi-based knock-down of gyrase subunits and overexpression of topoisomerase I (TopoI), and characterized the phenotypes. Cell division was blocked, most likely due to inhibition of genomic but not plasmid DNA replication. Cell growth continued to 4-5x of the wildtype cell volume, and metabolic flux was redirected towards glycogen in the TopoI overexpression strain. TopoI induction initially lead to down-regulation of GC-rich and up-regulation of AT-rich genes. The response quickly bifurcated and four diurnal co-expression cohorts (dawn, noon, dusk and night) all responded differently, in part with a circadian ({approx} 24 h) pattern. A GC-rich region - 50 bp of transcription start sites is differentially enriched in these four cohorts. We suggest a model where energy- and gyrase-gated transcription of growth genes at the dark/light transition (dawn) generates DNA supercoiling which then facilitates DNA replication and initiates the diurnal transcriptome program.

Spinal cord injury (SCI) affects between 250,000 to 500,000 individuals annually. After the initial injury, a delayed secondary cascade of cellular responses occurs causing progressive degeneration and permanent disability. One part of this secondary process is disturbance of ionic homeostasis. The K+ channel blocker, 4-aminopyridine (4-AP), is used clinically to alleviate symptoms of multiple sclerosis (MS). Several ongoing studies are being conducted to explore additional areas where 4-AP may have an effect, including stroke, traumatic brain injury, and nervous system recovery after SCI. The goal of our study was to determine whether 4-AP affects recovery from SCI in zebrafish (Danio rerio). Using the transgenic line Tg(gfap:EGFP), we created a spinal transection and tracked swim recovery. We found that constant treatment with 10 {micro}M 4-AP increases swimming distance 40%. Live imaging demonstrated that treatment with 4-AP increases radial glial cells bridging at the site of injury in the presence of 4-AP. We conclude that 10 {micro}M 4-AP is pro-regenerative after SCI. Significance StatementIn this study, we found that 4-AP can enhance locomotor recovery in zebrafish after spinal cord injury. Our findings indicate that inhibition of K+ channels with 4-AP may promote glial remodeling that is pro-regenerative.

Mitochondrial pyruvate carrier (MPC) inhibition was found protective in models of neurodegenerative diseases, such as Alzheimers and Parkinsons. However, little is known about MPC as a potential therapeutic target in Huntingtons disease (HD), a neurodegenerative disorder with dysregulation of the pro-survival pathway integrated stress response (ISR). Here, we investigate if MPC inhibition modulates the ISR and mitigates mutant huntingtin (mut-Htt) proteotoxicity in a cellular HD model. We treated cells expressing N-terminal fragments of wild-type- (wt-) or mut-Htt with two MPC inhibitors (mitoglitazone and UK5099) or solvent control. Metabolism was assessed analysing resazurin reduction, oxygen consumption, extracellular acidification, and ATP levels. ISR activation and huntingtin proteostasis were assessed using western-blot and filter-trap assays. Mut-Htt-expressing cells showed decreased resazurin reduction and ATP levels, and increased eIF2 phosphorylation, indicating metabolic stress and ISR activation. MPC inhibitors (100 {micro}M) increased resazurin reduction and decreased respiration. The latter was rescued by the membrane-permeant methyl pyruvate, which bypasses MPC inhibition. In wt-Htt-expressing cells, MPC inhibitors increased levels of ATP and ISR markers, suggesting metabolic adaptation and ISR activation. In mut-Htt-expressing cells, MPC inhibitors preserved ATP levels and attenuated mut-Htt-induced eIF2 phosphorylation but without changing soluble or aggregated mut-Htt levels. This work showed that MPC inhibition differentially modulates the ISR: it activates ISR in control cells and attenuates overactive ISR in mut-Htt-expressing cells. However, MPC inhibition did not impact the proteostasis of N-terminal fragment mut-Htt. Further studies are essential to explore MPC inhibition in less severe full-length mut-Htt-expressing models to better understand its therapeutic potential in HD.

As primary contributors to oxygenic photosynthesis, cyanobacteria intricately regulate their metabolic pathways during the diurnal cycle to ensure survival and growth. Under dark conditions, breakdown of stored energy reserves of glycogen replenishes the intermediates, especially the downstream glycolytic metabolites necessary for photosynthetic initiation upon light irradiation. The intracellular level of the intermediates is maintained throughout the dark period. However, it remains unclear how their accumulation is maintained in the dark despite the limited availability of glycogen. Here, we showed that the metabolite accumulation stability is ensured by the low activities of phosphoenolpyruvate (PEP) converting enzymes, namely PEP carboxylase and pyruvate kinase, during the dark period. Overexpression of these enzymes significantly decreased the accumulation of glycolytic intermediates after dark incubation. The oxygen evolution ability simultaneously decreased in the overexpressing strains, indicating that the dark limitation of the PEP-consuming pathway facilitates photosynthetic initiation through the maintenance of glycolytic intermediates. This finding shed light on the importance of controlling cataplerotic flux during the dark for maintaining stable operation of the Calvin cycle.

Hyperornithinaemia with gyrate atrophy of choroid and retina (HOGA) is a recessive metabolic disease caused by dysfunction of the ornithine aminotransferase (OAT) gene, leading to ornithine accumulation and a complex metabolic imbalance. This causes retinal degeneration that ultimately evolve to blindness. However, the mechanisms of this degeneration remain unknown. Here, we have conducted untargeted metabolomic analysis in patient-derived induced pluripotent stem cells and their isogenic counterparts. Mutant cells show altered levels of ornithine-related metabolites, including low creatine, proline and glutamate, and elevated arginine and citrulline. The untargeted metabolomics approach revealed changes in the urea cycle and polyamine synthesis pathways with a significant intracellular accumulation of gamma-aminobutyric acid (GABA). Hence, we propose GABA as a key player in the disease pathogenicity, potentially affecting neuronal function in the eye.

The microbiome-gut-brain axis has been implicated in multiple bodily systems and pathologies, and intentional manipulation of the gut-microbiome has yielded clinically significant results. Here, we examined the effects of bi-directional fecal microbial transplants (FMT) between methylone-induced hyperthermic tolerant (MHT) and methylone-naive (MN) rats. Rats treated with methylone once per week developed tolerance to methylone-induced hyperthermia by the fourth week. Once tolerant, daily bi-directional FMT between the two groups were performed for seven days prior to the next methylone treatment. The FMT abated the developed tolerance in the MHT group. When treated with methylone for the first time following FMT, recipient MN rats displayed significant tolerance to hyperthermia despite it being their initial drug treatment. Post-FMT, MHT rats displayed elevations in norepinephrine and expression of UCP1, UCP3 and TGR5 in brown adipose tissue, with reductions in expression of TGR5 and UCP3 in skeletal muscle. The pre- and post-FMT methylone tolerance phenotypes of transplant recipients are concurrent with changes in the relative abundance of several Classes of Proteobacteria, most evident for Gammaproteobacter and Alphaproteobacter. MHT recipients demonstrated a marked increase in the relative proportion of the Firmicutes Class Erysipelotrichia. These findings suggest that transplantation of gut-microbiomes can confer phenotypic responses to a drug.

Nuclear factor erythroid 2-related factor 2 (NRF2) is a master regulator of anti-oxidative and detoxifying cell responses. In addition, it plays important roles in host cell defenses against pathogenic viruses, and small molecules that activate NRF2 signaling can exert potent antiviral effects. We recently found that the NRF2 activators 4-octyl itaconate (4OI), bardoxolone (BARD), and sulforaphane (SFN) interfere with influenza A virus replication by blocking the nuclear export factor exportin 1 (XPO1), which did not require NRF2 signaling. Here, we have assessed their potential to inhibit highly pathogenic (SARS-CoV-2) and seasonal (hCoV-229E) coronaviruses and begun to elucidate the involved mechanisms of action. Using human cell lines and iPSC-derived vascular endothelial cells, we find that NRF2 knock-out or knock-down enhances infection by both viruses, indicating that physiologic NRF2 signaling restricts human coronavirus infection. 4OI, BARD, SFN, as well as the XPO1 blocker Selinexor (SEL), greatly limit infection by both viruses, but in an NRF2-independent manner. Strikingly, the compounds (particularly 4OI) downregulate ACE2 and TMPRSS2 mRNA and protein in Calu3 cells, leading to a >10-fold reduction in viral cell entry by 4OI and SEL, as assessed using SARS-CoV-1 and -2 spike protein VSV pseudotypes. A cycloheximide chase experiment revealed that 4OI dramatically reduces ACE2 half-life, which requires the E3 ligases NEDD4L and MCM1, suggesting that 4OI targets ACE2 for destruction by the proteasome. Moreover, 4OI and SEL reduce XPO1 protein levels, and all compounds reduce XPO1 mRNA levels. Co-incubation experiments of 4OI and the transcription blocker actinomycin D in A549 cells suggest that 4OI acts primarily by interfering with transcription of the XPO1 gene. XPO1 knock-down markedly reduces 229E replication. All four compounds interfere with 229E infection, but do not alter expression of ANPEP, the cellular receptor for this virus. Their anti-229E efficacy depends on expression of XPO1 in host cells in the order of SEL (most dependent) >4OI >SFN >BARD (least dependent), suggesting that especially BARD interferes with 229E infectivity via yet another, unknown, target. Taken together, these results suggest that "NRF2 activators" act as potent antivirals against human coronaviruses by targeting diverse host factors which are critical for viral infectivity. Author summaryHost-directed antiviral compounds act by a variety of mechanisms. For instance, they stimulate cellular antiviral immune responses and target host cell factors which are required for the viral life cycle. Pharmacologic activation of the NRF2 signaling pathway is a particularly attractive antiviral strategy, as this pathway restricts replication of a variety of viruses and also protects cells from excessive inflammation and oxidative stress resulting from accumulation of reactive oxygen species. In our previous study of the NRF2 activators bardoxolone, sulforaphane, and 4-octyl itaconate as host-directed treatments for influenza A virus infection, we found that these compounds interfered with replication of the virus. Unexpectedly, this antiviral activity was completely independent of NRF2 signaling, but resulted from blocking the nuclear export factor XPO1. In the present study, we find that these compounds limit infection by SARS-CoV-2 and hCoV-229E and that, again, this antiviral effect is NRF-independent. Instead, it depends to a large extent on downregulating ACE2 and TMPRSS2 (the major host cell receptors for SARS-CoV-1 and 2) and blocking/downregulating XPO1. Our results underscore the potential of "NRF2 activators" as adjunct treatments for viral infections, as they protect the host by anti-oxidative, anti-inflammatory, and cytoprotective mechanisms and also interfere with diverse host factors required for the viral life cycle.

Cyanobacteria utilize type IV pili for many behavioural responses, such as phototaxis, aggregation, floating, and DNA uptake. Type IV pilus-dependent functions are regulated by the nucleotide second messengers, c-di-GMP and cAMP. In this study, we investigated the role of a recently identified c-di-GMP receptor (CdgR) in cyanobacteria that harbours a ComFB domain. ComFB-domain proteins are widespread in cyanobacteria and are also present in heterotrophic bacteria. We demonstrated that the CdgR homolog from the cyanobacterium Synechocystis sp. PCC 6803, a model organism for studying type IV pilus-dependent functions, specifically binds to c-di-GMP. Genetic and phenotypic analyses revealed that Synechocystis CdgR is involved in phototactic motility and natural competence. Inactivation of cdgR resulted in altered expression of specific sets of minor pilins, which are essential for motility or natural competence. We identified interactions between CdgR and the CRP-family transcription factors, SyCRP1 and SyCRP2. Disruption of these CdgR-SyCRP1 and CdgR/SyCRP2 complexes is initiated by elevated c-di-GMP levels. Moreover, the assembly and stability of these complexes are influenced by other cyclic nucleotides, such as cAMP and c-di-AMP. These observed interactions imply a complex regulatory mechanism by which CdgR influences gene expression in response to cyclic nucleotide messenger signalling, particularly c-di-GMP. The present findings highlight the importance of CdgR in c-di-GMP signalling and its role in regulating type IV pilus-dependent functions in Synechocystis. The modulation of the expression of specific minor pilin genes by CdgR, through interactions with the transcription factors SyCRP1 and SyCRP2, contributes to the establishment of multiple type IV pilus functions and adaptive behaviours of cyanobacteria.

Heteromeric TASK1/3 channels play a fundamental role in oxygen-sensing by carotid body type 1 cells, where hypoxia-induced inhibition of TASK3 and/or TASK1/3 potassium currents leads to depolarisation, voltage-gated calcium entry, exocytotic transmitter release and increases in carotid body afferent input responses that initiate corrective changes in breathing patterns. However, the mechanism by which hypoxia leads to TASK-1/3 channel inhibition is still debated. It had been proposed that the AMP-activated protein kinase (AMPK) might directly phosphorylate and inhibit TASK channels, in particular TASK-3, although subsequent studies on rat type I cells argued against this view. Here we report on the effects of novel, highly selective AMPK activators on recombinant human TASK-3 potassium channels. Sequence alignment identified an AMPK recognition motif in TASK-3, but not TASK-1, with Ser55 representing a potential site for AMPK-dependent phosphorylation in TASK-3. However, neither of the AMPK activators, AICAR or MK-8722, caused a significant reduction of human TASK-3 current amplitude. By contrast, high concentrations of the AMPK activator A-769662 (100-500 {micro}M) inhibited human TASK-3 currents in a concentration-dependent manner. Importantly, A-769662 (300 {micro}M) also inhibited human TASK-3 channels in HEK293 cells that stably over-expressed an AMPK-{beta}1 subunit mutant (S108A) that renders AMPK insensitive to activators binding the Allosteric Drug and Metabolite (ADaM) site, such as A-769662. We therefore identify A-769662 as a novel human TASK-3 channel inhibitor and provide conclusive evidence that AMPK does not regulate TASK-3 channel currents.

Abstract Regulator-of-gene-silencing calmodulins (rgsCaM) represent a phylogenetic subfamily of calmodulin-like calcium sensors that are targets of viral induced suppression of posttranscriptional gene silencing by secondary siRNAs. The present work shows that a stress (hypoxia) that induces mRNP granule formation also induces the relocalization of rgsCaM to cytosolic granule-like foci that interact with the surface of stress granule and processing body structures. Co-expression of rgsCaM and its binding protein Suppressor of Gene Silencing 3 causes re-localization and integration of rgsCaM into stress granule structures. RgsCaMs contain a conserved topology that consists for four EF hand like domains (three functional and one divergent) that are separated into two calcium binding lobes with an extended amino terminal region. RgsCaM also contains an "ATG8 family interacting motif" (AIM) within its amino-terminal domain that is characteristic of selective autophagy cargo receptors. Co-localization experiments and ratiometric BiFC analyses in Nicotiana benthamiana support the hypothesis that rgsCaM binds directly to ATG8e through this conserved AIM domain, and the two proteins co-localize with mRNP granule markers. Previous reports show that rgsCaM mediates the suppression of gene silencing, at least in part, via turnover of SGS3 via autophagy. A model is proposed for rgsCaM-like proteins as potential mediators of selective autophagy of RNA granules in response to biotic and abiotic stresses.

The skin microenvironment created by keratinocytes (KC) influences stress responses of melanocytes (MC) to UVB insult. Here, we investigated paracrine factors involved in the regulatory role of microenvironment created by KC in UVB-mediated MC responses using RNA sequencing analysis as well as in vitro and in vivo models. RNA-Seq showed that G-CSF and CCL20 genes were highly upregulated in UVB-irradiated KC and their levels best correlated with paracrine protective effects of KC on stress responses of MC to UVB. Recombinant G-CSF and CCL20 treatment revealed the strongest modulatory effects on UVB-induced MC responses by mitigating apoptosis and ROS formation and upregulating tyrosinase and tyrosinase-related protein-1 (TRP-1) involved in the melanogenic pathway. A similar correlation between G-CSF and CCL20 expression in KC and the tyrosinase level in MC was also observed in the UVB-irradiated mouse skin. Our study reports for the first time that G-CSF and CCL20 might play a regulatory role in the KCs paracrine effects on UVB-mediated MC damage and also provides translational insights for the development of biomarkers for predicting susceptibility to photodamage. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=185 SRC="FIGDIR/small/523939v1_ufig1.gif" ALT="Figure 1"> View larger version (58K): org.highwire.dtl.DTLVardef@113588org.highwire.dtl.DTLVardef@1d1af40org.highwire.dtl.DTLVardef@1489df0org.highwire.dtl.DTLVardef@7935c5_HPS_FORMAT_FIGEXP M_FIG C_FIG