Mitochondrial function is key for maintaining cellular health, while mitochondrial failure is associated with various pathologies, including inherited metabolic disorders and age‐related diseases. In order to maintain mitochondrial quality, several pathways of mitochondrial quality control have evolved. These systems monitor mitochondrial integrity through antioxidants, DNA repair systems, and chaperones and proteases involved in the mitochondrial unfolded protein response. Additional regulation of mitochondrial function involves dynamic exchange of components through mitochondrial fusion and fission. Sustained stress induces a selective autophagy – termed mitophagy – and ultimately leads to apoptosis. Together, these systems form a network that acts on the molecular, organellar, and cellular level. In this review, we highlight how these systems are regulated in an integrated context‐ and time‐dependent network of mitochondrial quality control that is implicated in healthy aging. ERendoplasmic reticulumETCelectron transport chainIMMinner mitochondrial membranemtDNAmitochondrial DNAnDNAnuclear DNAOMMouter mitochondrial membraneOXPHOSoxidative phosphorylationROSreactive oxygen speciesmtUPRmitochondrial unfolded protein responseUPSubiquitin‐proteasome systemAlphaproteobacteriac.Mitochondria are double membrane‐enclosed organelles that execute many metabolic functions, including ATP generation through oxidative phosphorylation (OXPHOS). Next to their role as energy suppliers, mitochondria are also involved in synthesis of biomolecules, maintenance of calcium homeostasis, production of reactive oxygen species (ROS), and apoptosis The significance of maintaining mitochondrial integrity is underscored by various diseases associated with mitochondrial dysfunction. These include inherited mitochondrial diseases caused by mutations in mtDNA or nDNA, resulting in mitochondrial defects that severely affect cells/tissues with high energy demands such as brain, muscle, liver, and kidney 2−· is the primary ROS, and is produced when molecular oxygen is reduced by a single electron, which occurs at seven or more sites by IMM‐associated proteins, in particular complexes I and III of the electron transport chain (ETC) 2−· is converted to H2O2 by the superoxide dismutases MnSOD in the matrix or CuZnSOD in the intermembrane space and cytosol. Mitochondrial H2O2 is then enzymatically scavenged by peroxiredoxins (Prxs) and glutathione peroxidases (GPxs) The superoxide radical O2O2 is involved in signaling pathways that maintain cellular function 2O2 is membrane‐permeable and has a relatively long half‐life: therefore, it can diffuse to the cytosol to alter protein activity through reversible oxidation of sulfur‐containing methionine and active site cysteine residues Caenorhabditis elegans glucose restriction increased mitochondrial respiration and ROS generation, but at the same time improved ROS scavenging capacity, and ultimately extended worm lifespan. Of note, the outcome of ROS exposure depended on the ROS levels, because the administration of antioxidants prohibited the glucose restriction‐mediated extension of lifespan ROS levels are not only maintained at low level to prevent damage, they are also tightly controlled because Hm), but also depends on the presequence translocase‐associated motor (PAM), and the ATPase activity of mitochondrial heat shock protein 70 (mtHsp70) Given that only ∼1% of all mitochondrial proteins are mtDNA‐encoded, the majority of the mitochondrial proteins have to be imported in a tightly regulated manner mt) The mitochondrial proteolytic system consists of subcompartment‐specific proteases and the ubiquitin‐proteasome system (UPS) that together regulate mitochondrial protein turnover In the inter‐membrane space (IMS), protein quality is controlled by the membrane‐bound protease i‐AAA and soluble protease HtrA2/Omi The IMM houses two other proteases that have essential functions in mitochondrial quality control. First, metallopeptidase OMA1 has similar functions as the membrane‐bound AAA proteases More recently, the major cytosolic proteolytic system – the UPS – was described to act in mitochondrial quality control as well mt, a mitochondria‐to‐nucleus adaptive signaling involving attenuation of protein translation and induced expression of protein folding and proteolytic machineries mt exists in mammalian cells following disruption of the stoichiometric balance between nDNA‐ and mtDNA‐encoded proteins, or overexpression of a mutated form of the matrix protein ornithine transcarbamylase C. elegansmt upon mitochondrial stress elicits expression of mitochondrial chaperones HSP‐6 and HSP‐60 in C. elegans  mt signal transduction pathway using hsp‐6 and hsp‐60 reporter worms mt is initiated by the accumulation of unfolded proteins that activates the matrix protease CLPP‐1 Mitochondria may suffer from proteotoxic stress when the protein folding capacity is exceeded, for instance due to excessive ROS, mutations, or heat. Sustained proteotoxic stress induces the UPRmt, showing that mitochondrial stress triggered the upregulation of mitochondrial chaperones Hsp60, Hsp10, mtDnaJ, and the mitochondrial protease ClpP, while the levels of ER‐specific chaperones remained the same mt is not yet defined, although it requires ClpP activity and involves PKR‐mediated eIF2α phosphorylation mt responsive genes mt induction as well through the activation of c‐Jun and/or JNK2  flanking the CHOP‐C/EBPß binding site mt genes mt mechanisms, some major questions remain. For example, how do mitochondria prevent import of a signaling protein such as ATFS‐1, yet allow import of chaperones, proteases, and other UPRmt‐induced proteins? Further research should elucidate the temporal regulation of these signaling pathways in which chaperones are upregulated, while protein import and translation are attenuated.The initial work in mammalian cell culture demonstrated that disturbances of mitochondrial protein balance elicited UPRmt has been found to increase lifespan in worms and flies C. elegans impaired complex IV activity due to knockdown of cco‐1 during larval development induced the UPRmt, which was maintained in adulthood and required for longevity mrps‐5 disturbed mitonuclear protein balance enough to induce the UPRmt and extend worm lifespan, a mechanism that was conserved in mammalian models C. elegans and D. melanogaster local perturbation of ETC function in brain, intestine, or muscle cells during larval development induces a systemic hormetic response leading to lifespan extension mt requires further investigation. In addition to spatial regulation, the timing of UPRmt induction is key, as illustrated by the fact that ETC disturbance during adulthood does not promote longevity mt activates adaptive responses that may be remembered through epigenetic alterations and contribute to an increased lifespan. The causal relation between the two mitohormetic pathways – UPRmt and antioxidants – is not fully elucidated. In several studies, UPRmt and lifespan extension are not attenuated by supplementation of antioxidants mt‐dependent lifespan extension mt and antioxidants Mitochondrial dysfunction is associated with aging and common age‐related diseases Although mitochondria are often depicted as individual rod‐shaped organelles, they actually exist in interconnected networks that are highly dynamic. As a consequence, the number, shape, and localization of mitochondria are constantly changing. This dynamic character is a result of the continuous alternation between fusion and fission events. Fusion results in a more interconnected mitochondrial network c, thereby protecting cells from apoptotic cell death c‐mediated apoptosis c release and concomitant induction of apoptosis Mitochondrial fusion in mammals requires three membrane bound GTPases, the mitofusins Mfn1 and Mfn2 (Fzo1 in yeast) for OMM fusion, and optic atrophy 1  for IMM fusion The regulatory mechanism of the multiple proteins involved in OMM fusion is still poorly understood S‐nitrosylation  An equally significant aspect of mitochondrial dynamics regulation is how the alternating fusion and fission events are coordinated. This is substantiated by several diseases related to imbalances in mitochondrial fusion and fission , cytoplasmic components are sequestered in a double membrane vesicle (autophagosome) in a non‐selective manner. The autophagosome then fuses with a lysosome, causing its contents to be degraded. Autophagy is an important cellular quality control process that permits the cell to remove and recycle its cell content upon starvation m, and involves the kinase PINK1 and the E3 ligase Parkin m hampers PINK1 import, causing it to accumulate at the OMM Mitophagy is induced upon loss of ΔΨPINK and PARK2 (encoding Parkin) genes have been found mutated in early‐onset hereditary Parkinson's disease mt and mitophagy. These stress responses are both activated upon dissipation of ΔΨm and extramitochondrial accumulation of signaling proteins, i.e. ATFS‐1 and PINK1, respectively. In unstressed conditions, these proteins are constitutively imported and degraded in the mitochondrion, but stress‐induced loss of ΔΨm impairs mitochondrial protein import, alleviating the protein folding load and facilitating ATFS‐1 and PINK1‐dependent induction of the UPRmt and mitophagy mt and mitophagy, if both signaling proteins are accumulating extramitochondrially following ΔΨm dissipation. While both may indeed be induced upon mitochondrial depolarization, the kinetics of the two responses could be different, for instance requiring a prolonged activation state or changes in mitochondrial morphology mt and mitophagy may require secondary signals or processes to fully engage their protective effects. Along these lines, it was recently shown that PINK1/Parkin are also involved in an emerging quality control system involving the release of mitochondria‐derived vesicles (MDVs), that bud off from mitochondria and deliver damaged content to lysosomes for degradation In addition to questions pertaining to the molecular regulation of mitophagy, it will be interesting to assess how PINK1 and Parkin regulate mitophagy in more physiological conditions, as experimental systems often rely on potent uncoupling agents such as CCCP. Given that both mt and mitochondrial dynamics in mammals, as these processes have mostly been studied in worms and yeast. Also, how the network of quality control pathways is cross‐regulated should receive more attention, with particular focus on post‐translational modifications such as ubiquitination that seems to regulate all the systems of mitochondrial quality control. Moreover, getting a better grip on how mitohormesis is regulated may establish the mitochondrion and its quality control system as target in future therapeutic interventions. These may range from dietary, exercise‐related, and pharmaceutical approaches that relieve mitochondrial dysfunction in disease, or create mitochondrial stress to induce a (mito‐) hormetic response and eventually promote longevity. A better understanding of how these systems are coordinated holds the promise of potential future applications.Mitochondrial quality control pathways play a central role in mitochondrial health, which has major potential to improve health and lifespan. In the past decade, tremendous progress has been made in this field with the identification of various quality control pathways. The different systems of mitochondrial quality control are often described as a highly regulated and hierarchical network. In this classical view, each system has a maximum capacity, and crosstalk between them permits induction of the next system when the previous one is overwhelmed. In recent years, however, it has become evident that mitochondrial quality control pathways are not hierarchical, and that the sequence of events is highly dependent on various factors such as stress stimuli, stress duration, activation of auxiliary proteins, and degree of mitochondrial damage Fig. . Future


MRI) abnormalities and clinical weakness, and to evaluate the value of brachial plexus MRI in predicting disease course and response to treatment in multifocal motor neuropathy (MMN), Lewis‐Sumner syndrome (LSS) and chronic inflammatory demyelinating polyradiculoneuropathy (CIDP).The main objective of this study was to evaluate the correlation between the distribution of brachial plexus magnetic resonance imaging (STIR) MRI of the brachial plexus. We obtained clinical follow‐up data and scored all MRIs for abnormalities and the symmetry of their distribution.Sixty‐seven patients with an inflammatory neuropathy diagnosed at our tertiary referral center for neuromuscular diseases had undergone bilateral T2‐weighted short tau inversion recovery .Brachial plexus STIR brachial plexus MRI abnormalities correspond with the distribution of neurological deficits in inflammatory neuropathies, but do not correlate with specific clinical characteristics, response to treatment or disease course.T2  Asymmetry was defined as a minimum difference of two Medical research Council (MRC) points of tested muscle groups per limb. Sensory deficits were scored as being present or absent for each individual limb. Clinical response to treatment was defined as clinical improvement reported by the patient, or improving in muscle strength by gaining at least 1 MRC point in a muscle group . Time to diagnosis was significantly longer in MMN and LSS than for CIDP (p < .001).Multifocal motor neuropathy patients were significantly younger than CIDP patients at the time of diagnosis  patients Table . AbnormaEvaluation of the correlation between clinical and radiological asymmetry revealed that asymmetric MRI abnormalities were associated with asymmetric weakness in 12 of 14 patients with MMN (86%) and in all five patients with LSS (100%) Table . Similar3.3No significant differences in clinical presentation, response to treatment and follow‐up data were detected between patients with and without abnormalities on MRI per diagnosis Table .Follow‐up studies of all 67 patients who underwent an MRI revealed that the diagnosis was altered only twice, namely from MMN into CIDP.3.4p < .001, phi 0.791).Pooled data analysis Table  showed t4This study shows that (a)symmetry of abnormalities on brachial plexus MR imaging is associated with the distribution of clinical findings in patients with an inflammatory neuropathy. Hence, symmetrical distribution of MRI abnormalities is strongly associated with CIDP, whereas asymmetry suggests a diagnosis of MMN or LSS. Asymmetry of brachial plexus MRI abnormalities is associated with persisting asymmetry of weakness at follow‐up indicating that radiological asymmetry is not a snapshot in time in the evolution to a symmetric disease, that is CIDP. Moreover, presence of (patterns of) MRI abnormalities do not predict a beneficial response to treatment with immunoglobulins.Brachial plexus MRI is an established technique in the diagnostic work‐up of patients suspected of an inflammatory neuropathy, but its predictive value for disease course or the relevance of specific patterns of weakness have not been studied. Moreover, comparative studies combining MR data of MMN, LSS and CIDP patients have not been performed. Symmetric brachial plexus abnormalities have been described using high‐resolution ultrasound (HRUS) in asymmetric inflammatory neuropathies , which were considered incompatible with MMN. The other patient had asymmetric weakness and MRI abnormalities at onset, but this developed rapidly into symmetrical weakness with prominent proximal leg weakness, and he showed remission of symptoms after plasmapheresis and corticosteroids; these findings are all compatible with a diagnosis of CIDP.As a result of the rarity of inflammatory neuropathies, a drawback of our study are the relatively low number of inclusions and its retrospective nature, possibly giving rise to a selected study population. Indeed, the number of patients diagnosed with probable and possible MMN compared with definite MMN were higher in this study than in the Dutch population‐based MMN cohort, , The Netherlands Organization for Health Research and Development ). W.L. van der Pol receives research support from the Prinses Beatrix Spierfonds; The Netherlands ALS Foundation; Stichting Spieren voor Spieren.


The polymerization‐induced self‐assembly (PISA) process is a useful synthetic tool for the efficient synthesis of polymeric nanoparticles of different morphologies. Recently, studies on visible light initiated PISA processes have offered a number of key research opportunities that are not readily accessible using traditional thermally initiated systems. For example, visible light mediated PISA (Photo‐PISA) enables a high degree of control over the dispersion polymerization process by manipulation of the wavelength and intensity of incident light. In some cases, the final nanoparticle morphology of a single formulation can be modulated by simple manipulation of these externally controlled parameters. In addition, temporal  control over the Photo‐PISA process can be achieved in most cases. Exploitation of the mild room temperature polymerizations conditions can enable the encapsulation of thermally sensitive therapeutics to occur without compromising the polymerization rate and their activities. Finally, the Photo‐PISA process can enable further mechanistic insights into the morphological evolution of nanoparticle formation such as the effects of temperature on the self‐assembly process. The purpose of this mini‐review is therefore to examine some of these recent advances that have been made in Photo‐PISA processes, particularly in light of the specific advantages that may exist in comparison with conventional thermally initiated systems. This process involves the in situ production of self‐assembled polymeric nanoparticles by the chain extension of a living solvophilic polymer precursor with a monomer which forms a solvophobic polymer. Such a process can take place under dispersion or emulsion polymerization conditions and has been successfully demonstrated in a range of different solvent systems such as water,Whilst conventional self‐assembly techniques are typically performed under relatively dilute conditions (<1 wt%), the PISA technique (under dispersion or emulsion conditions) allows for the production of nanomaterials under relatively concentrated conditions (10–50 wt%). This approach is therefore less sensitive to issues of scale. Furthermore, the PISA technique allows for more facile access to a range of higher order morphologies in a more reproducible manner compared to conventional self‐assembly. It should be noted that currently, the vast majority of PISA polymerizations are conducted under reversible addition‐fragmentation chain transfer (RAFT) dispersion or emulsion polymerization conditions using thermally initiated radical sources such as azo‐initiators. However, there are some reports on the use of other techniques for initiating a PISA process including atom transfer radical polymerization (ATRP),] nitroxide mediated polymerization (NMP),Some of the earliest works by Hawkett et al. using RAFT emulsion polymerization demonstrated that polymer latexes could be synthesized from in situ self‐assembled micelles although a controlled monomer feed was required to prevent monomer droplet formation.A number of parameters are known to strongly influence the morphology of nanoparticles synthesized using a PISA approach. For example, higher order morphologies are generally favoured by increasing the length of the solvophobic block, increasing monomer concentration, using relatively low molecular weight macroRAFT agents and improving the mobility of the solvophobic polymer chains. Generally, however, morphological transitions can be difficult to predict owing to a broad number of kinetic and thermodynamic factors which may influence the necessary rearrangement of polymer chains. For example, most RAFT emulsion (and some dispersion) polymerizations form exclusively spherical particles that only increase in diameter during the polymerization and do not rearrange into higher order morphologies as expected from an increased packing parameter. This phenomenon is generally attributed to the limited mobility of the core forming polymer chains which results in so‐called kinetically trapped spheres.In order to improve the general reproducibility of the PISA technique, it is possible to generate phase diagrams as a means to assist in the targeting of different nanoparticle morphologies.] Typically, these PISA based phase diagrams are generated by varying the target degree of polymerization (DP) (y‐axis) against the total solids content (x‐axis) and observing the predominant nanoparticle morphology (or morphologies for mixed phases) by TEM. For example, a comprehensive study by Blanazs et al. demonstrated that a phase diagram generated using a relatively long solvophilic polymer was exclusively populated by spherical morphologies.[[qv: 1s]] In contrast, the use of a shorter stabilizing polymer under a similar range of reaction conditions allowed the formation of higher order morphologies. These phase diagrams therefore provide a useful insight into the qualitative trends of the RAFT dispersion process by acting as a “roadmap” for nanoparticle synthesis. Several excellent reviews on the state of the current PISA literature are available elsewhere.]2Historically, the use of thermally activated radical sources has been the most common method to initiate CLRP techniques such as RAFT. However, in the last few years, there has been intense interest in using alternate forms of initiation to drive CLRP polymerizations. For example, several alternative techniques have recently been reported for initiating a RAFT‐type polymerization such as (photo)redox catalysts,As an alternative to other initiation methods , the use of visible or NIR light for controlling a CLRP is particularly attractive owing to their relatively low energy requirements (particularly the use of light‐emitting diodes (LEDs)), less side reactions and potential to exert temporal and spatial control over the polymerization.Although light mediated CLRP techniques based on ATRP,3N‐vinylpyrrolidone) (PVP) as a stabilizer.visible light mediated PISA approaches, particularly when used in the synthesis of nanoparticles with well‐defined morphologies.Some early works demonstrated the use of UV light to initiate conventional free radical dispersion polymerizations. For example, Chen et al. demonstrated that microspheres could be obtained at relatively rapid rates when using UV activated photoinitiators (Darocur 1173) in the presence of poly absorb only in the UV region of the spectrum, there are a number of photoinitiators capable of generating radicals under visible light irradiation.] In the presence of a suitable RAFT agent, photoinitiators have been used to synthesize well defined polymers under a range of both homogenous and heterogenous conditions.]Photoinitiators are light absorbing compounds which under UV or visible light generate species  capable of initiating polymerization. For the purposes of this review, we will consider only photoinitiators that generate radical species for initiating radical polymerization of vinyl monomers. They are typically classified according to whether they generate radicals in a unimolecular homolytic cleavage reaction (Type I) or bimolecular reaction with a hydrogen donor such as an amine or thiol (Type II).N‐(2‐aminoethyl) acrylamide (AEAM) as a comonomer (under acidic conditions to prevent RAFT aminolysis) allowed for the installation of metal binding motifs via post‐polymerization modification. More recently, the same photopolymerization technique was used to synthesize an unusual collection of morphologies such as silk‐like films, ribbons, interlinked vesicles and nanotubes.The first example of a visible light initiated Photo‐PISA process yielding self‐assembled nanoparticles was reported by Cai and co‐workers in 2015 using SPTP as a water soluble radical photoinitiator and a 400 W mercury lamp source (with a 400 nm filter).113‐b‐PHPMA differs qualitatively from the same block copolymer synthesis performed at 50 °C by Armes and coworkers. It is likely that this effect is due to either the varying polymerization rates and/or the differing polymerization temperatures affecting the degree of solvent/monomer partitioning. However, it should be noted that the difference in RAFT agent structure and fraction of residual unesterified PEG  would not be completely negligible. The addition of silica nanoparticles (at 30% solids) to the initial polymerization mixture allowed for the in situ encapsulation of the inorganic particles into the lumen of the polymer vesicles. During this loading process, the added silica had no effect on the polymerization kinetics and unencapsulated silica could be removed by gentle centrifugation. This facile in situ loading process was further explored to load bovine serum albumin (BSA) as a model protein  in conjunction with the photoinitiator, SPTP to polymerize 2‐hydroxypropyl methacrylate (HPMA) from a poly(ethylene glycol) (PEG) based macroRAFT agent. Under aqueous RAFT dispersion conditions, this Photo‐PISA approach led to a diverse set of morphologies with S, WLM and V (unilamellar and multilamellar) morphologies obtained by varying the target DP of HPMA and the total solids content.[[qv: 1r]] Using the same photoinitiator reported in Cai's work (SPTP), ultrafast kinetics were observed with quantitative monomer conversions achievable within 30 min at room temperature. In comparison, a typical aqueous RAFT dispersion initiated using the azoinitiator, 4,4′‐azobis at 70 °C typically requires greater than 3 hours to achieve quantitative conversions.] Importantly, the full range of nanoparticle morphologies was readily accessible and comprehensive experiments yielded the first report of a PISA phase diagram synthesized at room temperature. Interestingly, the phase diagram generated here for PEGThe same group has also explored the use of other water soluble macroRAFT stabilizers such as those based on thermoresponsive poly(ethylene glycol) methacrylates.] Copolymerization of ethylene glycol based methacrylates can yield thermoresponsive polymers with well‐defined and tuneable LCST behaviour in water. The use of a stabilizer block with thermoresponsive behaviour is difficult to achieve under typical thermally initiated PISA conditions (70 °C), owing to a loss of particle stability close to the LCST. Interestingly, in this study the authors determined that the observed LCST of the thermoresponsive macroRAFT was further lowered in the presence of the HPMA monomer by as much as 29 °C and thereby prevented its controlled polymerization at 20 °C. However, by lowering the polymerization temperature to 15 °C (or lower) the dispersion polymerization proceeded with quantitative monomer conversion within 15 min of irradiation with a reasonable degree of control being maintained over the polymerization. This process is only possible due to the relative temperature insensitivity of radical production by SPTP under visible light. Importantly, the full range of nanoparticle morphologies  could be obtained even at the lowered polymerization temperature. Spherical nanoparticles were shown to retain the thermoresponsive behaviour of the stabilizing macroRAFT agent, and underwent a decrease in size from 85 nm to 61 nm as the temperature was increased above the LCST. DLS was used to demonstrate the stability of these particles at temperatures above the LCST however the morphology at different temperatures or the reversibility of this process was not reported. Nonetheless, this study demonstrates a key advantage of photopolymerization in performing dispersion polymerizations below room temperature.2 resulted in a decrease in particle size or in some cases complete solvation of the polymer chains . Finally, the authors demonstrated the loading of BSA into vesicles under mild photopolymerization conditions and its subsequent release in the presence of dissolved CO2  within 30 min due to the fast decomposition lifetime of TPO (compared to AIBN at 70 °C). Despite the ultrafast kinetics, the alcoholic dispersion polymerization was well‐controlled (Đ < 1.3) and the full range of nanoparticle morphologies could be accessed by varying the monomer concentration and target DP.Recently, the same group studied the effect of incorporating a minor fraction of the tertiary amine containing monomer, 2‐(dimethylamino)ethyl methacrylate (DMAEMA) into the HPMA core‐forming polymer block.ns Figure3A. FiFigureApart from the use of redox initiators,] RAFT photopolymerization is an attractive option to  decouple the polymerization temperature from the radical initiation process. RAFT photopolymerization may therefore be used to study the effect of temperature on the polymerization process. For example, Tan et al. extended their work on low temperature Photo‐PISA[[qv: 6b]] to study the effect of variable temperature on nanoparticle evolution during the Photo‐PISA process.4.2photo‐initiator‐transfer agent‐terminator) mechanism as originally proposed by Otsu.n‐butyltrithiocarbonate) propionic acid (BTPA) appears to require a much longer reaction time for the polymerization of acrylates, , as well as a relatively long inhibition period.]It is well known that UV light can be used to directly activate a RAFT type polymerization by causing photolytic cleavage of C–S bond yielding carbon centred radicals for initiating polymerization. This approach is attractive since it removes the need for exogenous catalysts/initiators to be added to initiate polymerization and has been studied intensively by a number of groups.] However, the use of UV light generally results in limited control over the polymerization particularly at high conversion owing to gradual degradation of the RAFT agent. Recently, research by our group,[[qv: 36c]] Qiao's group] and others] has suggested that visible light can instead be used to directly activate the RAFT agent  leading to a higher degree of livingness compared to UV initiated approaches. This process is possible due to the weak n to π* absorption of some thiocarbonylthio species in the visible spectrum and has been proposed to proceed according to the photoiniferter  or green light  using a PISA approach.–1), the isolation of WLM could still be obtained by this method of viscosity monitoring suggesting its use as a metric to identify the WLM phase prior to conventional TEM analysis  under either visible blue 3]Cl2 (at ppm concentrations) can be used to directly activate RAFT polymerization under visible light. This process was named photoinduced electron/energy transfer – reversible addition‐fragmentation chain transfer (PET‐RAFT) and proposed to proceed according to the mechanism provided in Figure Figuremax = 465 nm, 0.7 mW cm−2) was studied using [Ru(bpy)3]Cl2 as a photoredox catalyst owing to its excellent solubility in polar solvents.] Although the room temperature polymerization was relatively slow (α ≈ 70% in 24 h), the formation of different morphologies  could be observed by varying a combination of solvent quality (MeCN:EtOH mixtures) and total solids content. The addition of MeCN was hypothesised to aid in morphological transformation by plasticising the core forming polymer and enabling micelle fusion into WLM.[[qv: 1s]] As in the visible light mediated photoiniferter system described above, the formation of WLM could be readily monitored at intermediate conversions due to the in situ viscous transition caused by worm entanglements. Furthermore, the photopolymerization could be temporally controlled by turning the LED source ON and OFF providing an additional means of synthetic control over the nanoparticle morphology. Such a fine degree of ON/OFF control over nanoparticle morphology is not possible under the typical conditions of a thermally initiated PISA process.In 2014, our group demonstrated that photoredox catalysts such as Ir(ppy)3]Cl2 photoredox catalyst, the phase separation behaviour of this system could be readily studied using fluorescence microscopy.Szymański and Pérez‐Mercader utilized a similar approach for the aqueous polymerization of HPMA under blue LED light. Owing to the intrinsic fluorescent properties of the ] The use of longer wavelengths of visible light is advantageous due to the lower degree of nanoparticle induced scattering during the polymerization and potential for decreased side reactions particularly in the presence of sensitive moieties such as drugs or proteins. Furthermore, initiation using different wavelengths of light opens the possibility of utilising monomers with distinct absorptions in the visible spectrum. As in our previous studies on blue and green light mediated dispersion polymerization,FigureMore recently, we demonstrated that Photo‐PISA could be extended to longer visible light wavelengths (red and yellow light) by the addition of the metalloporphyrin, 5,10,15,20‐tetraphenyl‐21H,23H‐porphine zinc (ZnTPP) which can activate the dispersion polymerization of benzyl methacrylate under low energy red light (λism.[qv: 2b] The u−2).FigureThe ability to perform polymerization in a non‐deoxygenated vessel opens up the possibility of performing synthesis in ultralow volumes in non‐traditional reactor systems.5The application of the PISA technique is generally favoured for its ease of setup along with the ability to produce different nanoparticle morphologies at high solids content. Such a process is much more amenable to scale up especially compared to conventional self‐assembly techniques. However, the use of visible light or UV light to initiate polymerization (in either homogenous or heterogenous conditions), poses a number of challenges in terms of scale compared to thermally or redox initiated systems. In particular, the attenuation of UV or visible light through the reaction mixture , can induce a strong dependence of the polymerization rate/kinetics on the geometry, size and materials of the vessel itself.6/λ4, it is likely that the effects of light scattering will become more apparent as the nanoparticles increase in size (and change morphology) during the polymerization.In a dispersion polymerization, the role of light scattering on the dynamic polymerization rate is likely to be more complex than that observed under homogenous conditions. Since Rayleigh scattering increases proportionally to dFigureAs an alternative to traditional batch reactors, the high surface area to volume ratio offered by continuous flow reactors is attractive for overcoming issues of scale in Photo‐PISA. In addition, the distribution of light intensity throughout a flow reactor is essentially uniform minimising the presence of local inhomogeneities Figure9. WorkThis flow polymerization setup was recently studied in a thermally initiated PISA system and is likely to have significant benefits for Photo‐PISA systems particularly in terms of production rates and uniform light penetration.Some of the earliest work on photopolymerization in CLRP systems demonstrated the ability to temporally control the polymerization by modulation of the light source.The facile conditions of visible light photopolymerization have proven to be highly compatible with biologically active species such as proteins or drugs. It should be noted that low temperature thermal initiators also enable mild polymerization conditions to be achieved, however the rate of radical generation is generally slow and highly sensitive to temperature unlike most photoinitiation systems. Using a Photo‐PISA approach, the encapsulation of BSA as a model protein has been demonstrated along with its subsequent triggered release under a carbon dioxide stimulus.Finally, the oxygen tolerant nature of some visible light mediated polymerizations can be applied to perform PISA synthesis without specialized equipment . This process can allow for nanoparticle synthesis to be performed in ultralow volumes and high throughput which lends itself to the more efficient production of PISA phase diagrams which can require extensive experimentation time and resources to produce. In addition, the simplifying of a typical polymerization setup should enable more facile access of the PISA technique  to non‐experts in the field.The authors declare no conflict of interest.


The majority of floral displays simultaneously broadcast signals from multiple sensory modalities, but these multimodal displays come at both a metabolic cost and an increased conspicuousness to floral antagonists. Why then do plants invest in these costly multimodal displays? The efficacy backup hypothesis suggests that individual signal components act as a backup for others in the presence of environmental variability. Here, we test the efficacy backup hypothesis by investigating the ability of bumblebees to differentiate between sets of artificial flowers in the presence of either chemical interference or high wind speeds, both of which have the potential to impede the transmission of olfactory signals. We found that both chemical interference and high wind speeds negatively affected forager learning times, but these effects were mitigated in the presence of a visual signal component. Our results suggest that visual signals can act as a backup for olfactory signals in the presence of chemical interference and high wind speeds, and support the efficacy backup hypothesis as an explanation for the evolution of multimodal floral displays. These displays are complex in that they broadcast visual, olfactory, tactile, gustatory, electrostatic and even acoustic information –3. TheseSchizocosa ocreata) where males, which use both visual and vibratory signals during courtship displays, were observed using more visual signals while on substrates which inhibit the transmission of seismic signals [Bombus impatiens [Many non-exclusive hypotheses have been developed to explain the ubiquity of multimodal or multicomponent signals in nature –13, incl signals . This ‘bmpatiens . Floral Plants produce a range of volatile organic compounds (VOCs), which mediate interactions between plants and their pollinators, seed dispersers and potential antagonists ,18. ThesVOCs can be affected by environmental noise during their production by the plant, or during their transfer through the air, or during their reception and processing by the receiver . Here, wChemical interference occurs if different plant species share VOCs making the desired flowers more difficult to localize , or when−1, but positively correlated at wind speeds above 2.7 m s−1 [Air turbulence, which is defined as random fluctuations in the velocity of a fluid  and is h.7 m s−1 . These i.7 m s−1  highlighet al. [We argue here that bumblebees have the capacity to use visual information as a backup for olfactory information in the presence of environmental noise. Previous tests of the efficacy backup hypothesis have shown bumblebees to use scents to back up colour signals in low-light conditions , giving et al. , who sug2.2.1.Bombus terrestris dalmatinus colonies  via a transparent gated tube which could be manually manipulated to regulate which bees, and how many, could enter or leave the flight arena. Forty-six Sylvania Activa 172 Professional 36 W fluorescent tubes (Germany) on a 12 L : 12 D regime were used to simulate natural illumination. Bees were fed 30% sucrose solution daily after experiments had taken place and pollen was added directly to the colony three days a week. A total of ten colonies were used over the three experiment types —in analysis, we assumed that there was sufficient behavioural difference between individuals to ignore any colony effects, as is discussed in Thomson & Chittka [All three experiment types were carried out in wooden framed 72 × 104 × 30 cm flight arenas topped with UV-transparent Perspex with the floor covered in Advance Green gaffer tape . Flight arenas were connected to the plastic nesting box of flower naive  Chittka . Foragin2.2.Ten white Perspex discs  were used as foraging stimuli  during each experiment. Each disc had 43 holes (2 mm diameter) in a hexagonal pattern , with a Within treatments which incorporated scented flowers, five of the ten artificial flowers had a lavender oil solution added using a pipette  in a hexagonal arrangement  with a 30% sucrose solution (20 µl) reward placed in the central wells of each disc of one group, and into the central well of the other group 20 µl of distilled water was added as an unrewarding stimulus. Each disc was placed on top of an upturned plastic container  and distributed randomly throughout the flight arena.2.4.Pelargonium graveolens, bog myrtle Myrica gale, juniperberry Juniperus communis and camomile Roman Anthemis nobilis , which were applied using a pipette. Group A had no additional scents in the flight arena. This wide range of essential oils, rather than comparatively simpler single odorants, was used as both floral recognition cues and chemical interference to better simulate the complexity of odours which pollinators are presented with in the wild.Foragers were randomly allocated to one of four groups : (A) with no chemical interference and unimodal-scented flowers (control treatment), used to gain a baseline level of foraging efficiency when using olfactory cues in an interference-free environment; (B) with chemical interference and unimodal-scented flowers, used to see the effects of the introduced interference; (C) with chemical interference and bimodal scented and visual flowers, used to see if effects caused by interference (as demonstrated by group B) could be mitigated with the addition of a visual cue; and (D) with chemical interference and unimodal visual flowers, used to demonstrate if there are effects to foraging efficiency when purely visual artificial flowers are presented alongside chemical interference. The three groups with chemical interference , which was used to reduce the reliability of recognition cues, had four sets of two upturned Eppendorf lids, each set with 200 µl of one of four essential oils distributed throughout the flight arena. The scents used were essential oils from geranium Individual marked bees which were naive to both visual and olfactory stimuli, but had experienced drinking from Eppendorf lid wells, were then allowed entry into the flight arena. The sequence of lands on rewarding or non-rewarding artificial flowers was recorded as well as whether the forager drank after landing or abandoned the flower before drinking. Visits to the same flower without visiting another flower in between were not recorded. Flowers which had been visited had their sucrose or water refilled and after each foraging bout the artificial flowers were removed and wiped with ethanol to remove visual cues and foraging pheromones . After t2.4.1.Foragers were assumed to have satisfactorily learned to discriminate between the two flower groups when eight of the last ten drinks were from rewarding flowers, not counting visits which did not lead to drinks. Only visits which led to drinks were included in this count as it was clearer in these instances that bees made correct choices (positive drinks) or incorrect choices (unrewarding drinks); this was less clear in visits which did not lead to drinks. However, as fine-tuned discrimination of flower odours is known to occur post-landing, and as these visits have implications in terms of benefits to the plant ,45, we i2.5.Foragers were randomly allocated to one of four groups  with either: (A) no air movement and unimodal-scented flowers (control treatment); (B) air movement and unimodal-scented flowers; (C) air movement and bimodal scented and visual flowers; or (D) air movement and unimodal visual flowers. These treatment groups were used for the same comparative purposes as those used in experiment 1. All groups had two opposite slats of the flight arena removed and replaced with a metallic mesh held in place with gaffer tape. Groups B, C and D had a fan  placed on the outside of one of the mesh openings facing into the flight arena creating a corridor where air can pass into the flight arena, pass over the artificial flowers and out of the flight arena from the far opening . This ne−1 (using a Kestrel 4500 pocket weather tracker), which passes the threshold at which odour source finding is compromised in tsetse flies Glossina pallidipes [At the beginning of the experiment, the fan was turned onto its highest setting with wind speed measured at mean ± s.d. = 1.07 ± 0.86 m sllidipes . Individ2.6.Scent preference tests were also undertaken to investigate if naive bees had an innate preference to peppermint or lavender. Within these preference tests naive forager bees were presented with 10 artificial flowers . Post hoc comparisons revealed that foragers presented with chemical interference and unimodal scented flowers took more flower visits to reach the learning criterion compared with foragers presented with no chemical interference and unimodal scented flowers (p = 0.0117) and those with chemical interference and bimodal scented and coloured flowers (p = 0.0015). There was no difference in the number of flower visits taken to reach the learning criterion between the rewarding scents (lavender: learning time = 18.07 ± 7.96 flower visits (mean ± s.d.), peppermint: learning time = 21.44 ± 8.75 flower visits, 1N = 27,2N = 27,U = 267.5, p = 0.094) or rewarding colours .The number of flower visits taken to reach the learning criterion was different between the treatments and control . Post hoc comparisons revealed that foragers presented with chemical interference and unimodal scented flowers drank from fewer rewarding flowers compared with foragers presented with no chemical interference and unimodal scented flowers (p = 0.0016), those presented with chemical interference and unimodal coloured flowers (p = 0.0136) and those with chemical interference and bimodal scented and coloured flowers (p < 0.0001). Foragers presented with chemical interference and unimodal coloured flowers also drank from fewer rewarding flowers compared with those presented with chemical interference and bimodal scented and coloured flowers (p = 0.014).The total number of drinks from rewarding flowers was different between the treatments and control . Post hoc comparisons revealed that foragers presented with both chemical interference and unimodal scented flowers and those presented with chemical interference and unimodal coloured flowers made fewer correct choices after landing compared with foragers presented with no chemical interference and unimodal scented flowers  and those with chemical interference and bimodal scented and coloured flowers .The number of correct choices made after landing was different between the treatments and control . Post hoc comparisons revealed that foragers presented with wind and unimodal scented flowers took significantly more flower visits to reach the learning criterion compared with foragers presented with no wind simulation and unimodal scented flowers (p = 0.004), those presented with wind simulation and bimodal scented and coloured flowers (p = 0.007) and those with wind simulation and unimodal coloured flowers (p = 0.005). There was no difference in the number of flower visits taken to reach the learning criterion between the rewarding scents  or rewarding colours .The number of flower visits taken to reach the learning criterion was different between the treatments and control . Post hoc comparisons demonstrated that forager bees exposed to wind simulation and unimodal scented flowers made fewer consecutive rewarding drinks compared with foragers presented with no wind simulation and unimodal scented flowers (p = 0.001), those presented with wind simulation bimodal scented and coloured flowers (p < 0.001) and those with wind simulation and unimodal coloured flowers (p < 0.001).The total number of drinks from rewarding flowers was different between the treatments and control . Post hoc comparisons revealed that foragers presented with wind and unimodal scented flowers made fewer correct choices after landing compared with foragers presented with no wind simulation and unimodal scented flowers (p = 0.002), those presented with wind and unimodal coloured flowers (p = 0.003) and those with wind and bimodal scented and coloured flowers (p < 0.0001).The number of correct choices made after landing was different between the treatments and control .Foragers had no innate preference to either peppermint or lavender scents (peppermint: 8.73 ± 2.74 (mean ± s.d.), lavender: 11.27 ± 2.74, 4.B. terrestris in scented unimodal flowers, but the inclusion of an additional visual signal component to floral displays negated these effects. This suggests that in scenarios where olfactory communication is compromised, visual signal components can act as a backup, supporting the efficacy backup hypothesis as an explanation for the evolution and maintenance of multimodal floral signals.We examined bumblebee foraging behaviour in the presence of one of two types of environmental noise which affect the transfer of volatiles: chemical interference and high wind speeds. Our results clearly show that both chemical interference and high wind speeds have detrimental effects on the foraging behaviour of a and 4b). These results correspond with the claim by Wilson et al. [Manduca sexta to correctly navigate an odour plume to its source. The detrimental effects on learning time, the total number of rewarding drinks and the number of correct choices made after landing were mitigated in foragers presented with bimodal floral displays and either interference type. The increase in post-landing accuracy also complements a previous study [Both chemical interference and high wind speeds caused foragers to take more flower visits to reach the learning criterion in unimodal scented flowers compared with foragers presented with unimodal scented flowers without either interference type figures a and 4b.n et al.  that olfn et al.  in whichus study  where thThese detrimental effects to foraging efficiency are likely to have negative consequences to both plant and pollinator. Reductions in total number of rewarding drinks would limit the net energy gained by individual bees during foraging and subsequently the colony, negatively affecting survival, growth and reproductive output . A decreB. terrestris. This effect on learning may have detrimental effects to the level of flower constancy a forager reaches, lessening benefits to the plant, and potentially increase the costs associated with switching plant species for the pollinator [Deilephila elpenor, which can use colour vision to discriminate between coloured stimuli in light condition equivalent to dim starlight [Adverse effects to forager learning times also suggest that both chemical and wind interference can affect associative learning in llinator ,53. Thisllinator –57. Althtarlight .Curiously, chemical interference also impeded foraging on purely visual artificial flowers . This coConsidering the results, chemical interference has the potential to affect plant–animal interactions in multiple ways. Flowering plants and pollinators inhabiting environments with high plant species richness, such as tropical forests  or MeditPollinators may mitigate these effects of olfactory noise through perceptual filtering, whereby only particular odours present in complex odour blends are detected by the antenna and only a select few of these detected volatiles elicit behavioural responses –69. Thiset al. [et al. [These effects of chemical interference also relate to atmospheric pollution, which affects all terrestrial ecosystems and potentially affects VOC transfer at a global level . McFredeet al.  propose  [et al.  suggest  [et al. ,73.It is worth noting that in some instances, the inclusion of additional blends of floral volatiles may not contribute towards noise or decreases in the efficacy of a learned VOC blend. In cases where odour blends have no chemical overlap or have shared compounds that are not perceived by the forager, it is unlikely that there would be any detrimental effects to signal transmission. It has also been speculated that in some instances, the presence of additional and contrasting VOC blends may enhance a response to a learned VOC blend by highlighting the perceptual contrast . HoweverIn terms of interference through high wind speeds, we saw detrimental effects to learning speeds, nectar collection rate and post-landing accuracy, mirroring the negative effects found in previous studies ,37. ThisB. terrestris in scented unimodal flowers, but the inclusion of an additional visual signal component to the floral displays negated these negative effects. This suggests that visual signal components can act as a backup in environments where olfactory communication is compromised, benefitting both plant and pollinator, and supporting the efficacy backup hypothesis as an explanation for the evolution and maintenance of multimodal floral signals. It is our hope that this study provides a proof of concept for these effects on the transmission of olfactory signals and inspires future research into which areas these interference types are most (or least) likely to occur, and at what spatial scales.Communication between plants and their visitors is of extreme importance to all terrestrial ecosystems and by understanding the factors which influence the evolution and effectiveness of this information transfer we gain a great insight into how this relationship can be affected in a changing world. Our results suggest that both chemical interference and high wind speeds have negative effects on the foraging behaviour of bumblebees


Embryonal carcinomas (ECs), the stem cell population of the non‐seminomas, is pluri‐ to totipotent and able to differentiate into cells of all three germ layers, giving rise to teratomas or tumours mimicking extraembryonic tissues . With regard to gene expression, (epi)genetics and histology, seminomas are highly similar to GCNIS and primordial germ cells, but limited in development. It remains elusive, whether this block in differentiation is controlled by cell intrinsic mechanisms or by signals from the surrounding microenvironment. Here, we reviewed the recent literature emphasizing the plasticity of GCCs, especially of seminomas. We propose that this plasticity is controlled by the microenvironment, allowing seminomas to transit into an EC or mixed non‐seminoma and vice versa. We discuss several mechanisms and routes of reprogramming that might be responsible for this change in the cell fate. We finally integrate this plasticity into a new model of GCC pathogenesis, allowing for an alternative view on the dynamics of GCC development and progression.So far, the understanding of germ cell cancer (GCC) pathogenesis is based on a model, where seminomas and non‐seminomas represent distinct entities although originating from a common precursor termed germ cell neoplasia  NANOG, STELLA, BCAT1 and GDF3A defective primordial germ cell (PGC) development is thought to be the origin of a lesion termed GCNIS, which itself is the precursor of testicular type II GCCs In clinics, GCCs present as pure or mixed tumours. The finding that seminoma and GCNIS cells are highly similar to each other and ECs are more closely related to ESCs led to the hypothesis that formation of a seminoma is the default developmental pathway for GCNIS cells and that ECs develop through reprogramming of GCNIS cells The testis is an immune‐privileged organ, where a blood–testis barrier shields the germ cells from harmful outer effects, allowing for the generation of healthy sperm and faithful transmission of the genetic material to the next generation. GCCs normally grow within this microenvironment until they either penetrate the testis confines during invasive growth or spread to other sites in the body. Generally, GCCs metastasize along the body mid‐line via the lymphatic vessels, giving rise to GCCs in the brain, where they are termed germinomas (a seminoma‐like tumour) In general, a microenvironment is a specialized and isolated biophysical entity that influences its surroundings by specific signalling molecules, mitogens, enzymes, hormones, etc., provided by the cells growing within. The tumour microenvironment is defined as the specific location in which tumour cells exist and interact with their surrounding environment. It includes blood vessels, immune cells, fibroblast, lymphocytes and an extracellular matrix Seminoma cells quickly initiate cell death upon disruption of their microenvironment in a process termed ‘anoikis’, suggesting that survival of seminomas critically depends on the microenvironment GCNIS and seminomas seem to be limited in their developmental capacity. It remains elusive, whether this block in differentiation is controlled by cell intrinsic mechanisms or by signals from the surrounding microenvironment.in vitro and in vivo experiments utilizing the seminoma‐derived cell line TCam‐2. TCam‐2 is the only available cell line, which reliably resembles a seminoma / GCNIS / PGC in vitroCellular plasticity describes the ability of cells to undergo a transition into another cell type, thereby fully adapting the newly acquired cell fate OCT3/4, NANOG and LIN28, but lack expression of the core pluripotency factor SOX2SOX17In vitro, TCam‐2 is resistant to many differentiation‐inducing stimuli, like all‐trans‐retinoic acid (ATRA), the demethylating agent 5‐aza‐2‐deoxycytidine (5aza), the monoaminooxidase inhibitor tranylcypromine or a combination of all three NANOG and the seminoma / PGC marker TFAP2C had no differentiation‐inducing effect NANOG expression Like seminomas and similar to ECs, TCam‐2 cells express pluripotency markers, like Orthotopic injection of TCam‐2 cells into the seminiferous tubules of the murine testis leads to a GCNIS‐ / seminoma‐like growth. However, TCam‐2 cells reprogramme into an EC‐like fate after transplantation into the murine flank or brain SOX2 and down‐regulation of SOX17. SOX2 triggers the induction of typical EC, pluripotency and epigenetic reprogramming factors, like GDF3, DPPA3, NODAL, ZIC3 and ZFP42 (REX1), whereas PGC / GCNIS / seminoma markers are down‐regulated  In observing this remarkable and fast reprogramming of TCam‐2 cells, the molecular mechanisms had to be determined. It was obvious to look at the activity of receptors and their signalling molecules first. Interestingly, these studies revealed that BMP (Bone Morphogenetic Protein) signalling is inhibited after transplantation into the flank. In consequence, this leads to up‐regulation of Of note, DNA methylation levels strongly increase during the reprogramming, but the changes in DNA methylation follow the deregulations in gene expression in vivo, the NODAL signalling cascade remains inactive LEFTY1/2 and CRIPTO, but not NODAL itself In vitro, treatment of TCam‐2 cells with the BMP signalling inhibitor NOGGIN led to up‐regulation of SOX2SOX2 must be induced upon repression of BMP signalling. Treatment of TCam‐2 (no SOX2 expression) with recombinant NODAL did not lead to establishment of the NODAL signalling loop SOX2 and establishment of the NODAL signalling cascade. Thus, SOX2 is the driving force behind the reprogramming of seminomas to an EC‐like state.The role of SOX2 in the reprogramming of TCam‐2 was tested by a loss‐of‐function approach. SOX2‐deficient TCam‐2 cells, when being xenografted into the flank of nude mice, do not undergo this reprogramming et al. demonstrated that in TCam‐2 cells, SOX2 is repressed by the polycomb repressive complex and the H3K27me3 chromatin mark enriched at the promotor in vivo reprogramming, whether BMP signalling is involved in establishment of these marks in vitro and whether these regulatory mechanisms can also be found in seminoma tissues.Recently, Kushwaha PRAME, whereas non‐seminomas lack PRAME expression (SOX17 ‐) PRAME is down‐regulated during in vivo reprogramming of TCam‐2 cells into an EC PRAME expression correlates to SOX17 expression and can be associated with a PGCs / seminoma cell fate. It has been proposed that PRAME regulates the pluripotency programme in seminomas / TCam‐2 cells and represses somatic and germ cell‐like differentiation processes by acting downstream of SOX17 It has been shown that PGCs / seminomas / TCam‐2 cells (SOX17 +) express the cancer/testis‐antigen In vivo a subpopulation of SOX2‐deficient cells initiated differentiation into a cell type resembling a mixed non‐seminoma indicated by up‐regulation of germ layer differentiation markers HAND1, PAX6, CDX1 and FOXA2, the trophoblast stem cell / choriocarcinoma marker EOMES and the yolk sac tumour marker AFPin vitro differentiation of TCam‐2 into a mixed non‐seminoma in vitroin vitro differentiation FOXA2, which is up‐regulated during the differentiation of TCam‐2 into a mixed non‐seminoma and was predicted to interact with many differentiation markers, such as AFP, HAND1 and EOMESFOXA2 during the differentiation of TCam‐2 into a mixed non‐seminoma are unknown.Further studies have to identify the factors that drive the development of seminomas into a mixed non‐seminoma. An interesting candidate gene is et al. derived human PGC‐like cells from ESCs cultured in ‘4i’ medium , rendering the ESCs germ cell competent et al. identified SOX17, which acts upstream of BLIMP1 and TFAP2C, as a critical specifier of the PGC fate The studies described so far indicate that the microenvironment seems to be able to trigger reprogramming of seminomas into a pluripotent EC or directly into a mixed non‐seminoma. In consequence, but not proven yet, a transition of ECs into a seminoma seems likely. Future studies have to identify factors able to drive reprogramming of ECs into a seminoma‐like state. Recently, Irie Reprogramming of teratomas back to an EC‐like state or even seminoma‐like state seems unlikely due to the terminally differentiated nature of mature teratomas. Choriocarcinomas consist of cells resembling syncytio‐ and cytotrophoblast cells, which derive from cells of the extraembryonic trophectoderm. These extraembryonic trophectoderm / trophoblast cells can be reprogrammed to pluripotent stem cells by introducing the four Yamanaka factors  e.g. culture conditions, reprogramming factors, mitogens) or routes  are known, each island can be reached. Sometimes more than one route is available, some routes or islands remain uncharted and returning to an island might be (im)possible. Compared to the existing model of GCC pathogenesis, our model reflects the plasticity and dynamics of GCC development more accurately.Based on the studies described in this article and on Sieweke's analogy of reprogramming processes as a map of James Cook's journeys, we propose an alternative view on the development of GCCs Fig.  51. TherIn summary, the development of the type II GCC entities seems to be a highly plastic process strongly influenced by the cellular microenvironment, allowing reprogramming of seminomas into a pluripotent EC or direct differentiation into a mixed non‐seminoma. Importantly, no additional genetic aberration, like a mutation seems to be necessary for switching the cell fate of GCCs.The initial progression of GCCs from GCNIS relies on acquiring the ‘12p gain’ (by mutation), rendering the cells more aggressive. The ‘12p gain’ might also be the reason why seminomas and ECs cannot revert back to the GCNIS fate (Fig. These findings might also have implications for the therapy of GCCs. Patients initially diagnosed with a seminoma might develop an EC or mixed non‐seminoma during invasive growth or metastasis, when the tumour cells are confronted with a different microenvironment. As ECs grow more aggressive and require a harsher treatment than seminomas, an adaptation of the therapeutic strategy might be necessary to avoid an ineffective therapy.in vivo.Future studies should address whether ECs are able to adopt a seminoma cell fate, which factors drive the differentiation of seminomas into a mixed non‐seminoma, or whether reprogramming of a choriocarcinoma / yolk sac tumour to an EC might be possible. Also, albeit demonstrated in a murine xenotransplantation model, it remains elusive whether such reprogramming processes occur The authors confirm that there are no conflicts of interest.


Lacerta agilis), hypothesizing that physiological and behavioral costs resulting from elevated baseline glucocorticoids function in maintenance of honest signal expression. We found that large‐badged males had higher corticosterone titer, with this relationship apparent at the end of the season and absent early in the season. Large‐badged males also suffered higher ectoparasite load (number of tick nymphs), despite being in better condition than small‐badged males. Ectoparasite load was positively related to corticosterone titer early in the season at the time of badge formation. High‐condition individuals had lower corticosterone and lower numbers of ectoparasites than low‐condition individuals, suggestive of conditional variation in ability to withstand costs of corticosterone. We found an opposing negative relationship between corticosterone titer and endoparasite load. Corticosterone titer was also negatively associated with male mobility, a fitness‐determining behavior in this species. Because badge size is involved in mediating agonistic social interactions in this species, our results suggest that badge‐dependent variation in corticosterone is likely reflective of variation in social conditions experienced over the course of the season. Our results implicate corticosterone in maintenance of signal honesty, both early in the season through enforcement of physiological costs (ectoparasite load) and during the season through behavioral costs . We propose that socially modulated variation in corticosterone critically functions in mediation of signal honesty without requiring a direct role for corticosterone in trait expression.The mechanisms underlying honest signal expression remain elusive and may involve the integration of social and physiological costs. Corticosterone is a socially modulated metabolic hormone that mediates energy investment and behavior and may therefore function to deter dishonest signal expression. We examined the relationship between corticosterone and green badge coloration in male sand lizards ( Sexually selected ornaments accurately reflect individual “quality” in most systems (Searcy and Nowicki Zonotrichia leucophrys gambelii (white‐crowned sparrows) induced both an increase in aggression from challengers and an increase in baseline corticosterone . The bright green “badge,” present on the sides of male sand lizards during the breeding season  function in maintenance of honest signal expression in male sand lizards. We examine covariation between badge size, CORT, and costly parasite load both early in the season, at the time of badge development, and late in the season, when we assume social dominance hierarchies to be well established. By doing so, we aim to differentiate between potential direct effects of CORT on trait production vs. indirect associations with trait intensity arising from a correlated third variable, social aggression. We ask (1) whether badge size is related to baseline CORT and predictive of costly parasite infection; (2) whether variation in body condition and both ecto‐ or endoparasite load are related to CORT, indicative of physiological costs of elevated CORT, and whether these potential costs are experienced equally by high‐ versus low‐condition individuals; and (3) whether elevated CORT is behaviorally costly, related to expression of fitness‐enhancing reproductive behavior . More mobile males are expected to encounter and acquire a greater number of mates. As a consequence, mobility positively predicts reproductive success in this species  and weighed to the nearest 0.001 g. Males were scanned on a flatbed scanner, and badge size was estimated as the proportion of green coloration versus the remaining side of the body  collected on our field population, while measurements of endoparasite counts and male behavior (mobility) are restricted to samples collected in 1998. Plasma CORT concentrations were measured in 1998 and in 2004 and, as with morphological variables, were collected from individuals at the time of first observation in the population each season. We therefore classified these as “early‐season” measurements of CORT. To determine ectoparasite load, we counted the number of nymphs of the tick s Olsson  which grs Olsson .N = 21) or a silastic implant containing crystallized testosterone , were involved in a hormone manipulation experiment. On the day following capture, males were anaesthetized and given either an empty silastic control implant (μL) were spiked with tritiated CORT for recovery determination and diluted with distilled water to a total volume of 400 μL. Samples were then extracted overnight with 4 mL redistilled dichloromethane, dried under nitrogen, and resuspended in 500 μL phosphate‐buffered saline. Samples were assayed in duplicate, and CORT recovery was 88–95% after extraction.Plasma CORT levels for samples collected in 1998 were measured with a single radioimmunoassay (RIA) at Gothenburg University following protocols established by B. Silverin Silverin ; for detμL) were spiked with tritiated CORT for recovery determination and extracted with dichloromethane. Dried and phosphate‐buffered saline reconstituted samples were assayed in duplicate, and CORT measurements were within the range of detectability. All samples were corrected for an average CORT recovery of 87.5%.Samples collected in 2004 were measured at University of Wollongong in a single assay following a similar direct RIA protocol , and late‐season measurements of morphology, ectoparasite and endoparasite load, male mobility, and CORT . We additionally assessed the relationship between body condition, ectoparasite load, and badge size across our 10‐year observational dataset (N = 336). Early‐season measurements of ectoparasite load, body condition , badge size, and CORT taken during 1998 were assessed at the beginning of experimentation and are thus unaffected by treatment (T vs. control). The potential influence of T treatment on all late‐season measurements has been statistically accounted for by inclusion (and subsequent exclusion if nonsignificant) in statistical models. Specifically, T treatment had no effect on late‐season badge size, body condition, CORT, or ectoparasite load , and thus, these data have been combined across treatments  and have therefore either been standardized for year or the effect of year has been controlled for by inclusion of year as a random factor in a mixed‐effect model. Time of capture and blood sampling was recorded for early‐season samples collected in 1998 only and was not correlated with measurements of CORT . Date of capture is included as a potential predictive factor in analyses of early‐season badge size, but has no detectable effect on late‐season measurements of badge size  and has therefore been excluded. Date of capture also had no influence on early‐ and late‐season CORT nor on early‐season ectoparasites. In order to meet assumptions of normality, we log‐transformed both early‐ and late‐season CORT, ectoparasite load, and badge size .Both early‐season CORT and ectoparasite load differed between years  with stepwise backward elimination to select the best fitted minimal models, eliminating factors and interactions at We adjusted for multiple comparisons within each group of analyses  using the false discovery rate procedure  . Larger‐badged males also had higher CORT late in the breeding season, but there was no relationship between CORT and badge size early in the season .Badge size was positively associated with body condition .Neither early‐ nor late‐season CORT varied with body condition  nor to badge size .Male mobility was negatively related to late‐season CORT Table ; Fig. 4 While carrying a large badge appears to be costly for male sand lizards in terms of associations with elevated CORT and ectoparasite load, only males in good condition produce large badges Olsson . The phyLarger‐badged males have more ectoparasites     social aggression, it appears to direct energy usage away from a behavior that functions primarily in mate acquisition . The negative correlation between CORT and male mobility presented here is similar to findings in some (DeNardo and Sinervo We add to a growing body of literature implicating a role for glucocorticoids in mediation of signal honesty (Rubenstein and Hauber 10.5061/dryad.67136Data will be deposited in Dryad Digital Repository doi: None declared.


Exactly one year ago we celebrated the first Journal Impact Factor of Advanced Science with the publication of an exclusive Review Special Issue. This year, there is again reason to celebrate: we are happy to announce that the impact factor of Advanced Science has increased by more than 50% to a value of 9.034 . That is an amazing result for this young journal and clearly an achievement of the strong international editorial team in combination with an excellent advisory board. Executive advisory board member Ali Khademhosseini  recently stated: “Advanced Science has become a premier journal that publishes a broad spectrum of scientific breakthroughs. Thus the journal fits an important need given that such advances are increasingly interdisciplinary.”In this issue we have once again compiled a variety of overview articles that demonstrate the broad range of topics covered in the journal: From polymer science to robotics, from computer simulations of supercapacitors to photodynamic therapy, from batteries to aggregation‐induced emission – Advanced Science has it all.Marco Rolandi and co‐workers  summarize their latest efforts on developing bio‐protonic devices that control H+ currents at the palladium hydride contact solution interface to monitor and control biological reactions. Cyrille Boyer  highlights recent advances in the field of polymerization‐induced self‐assembly (PISA) with a focus on the visible‐light‐initiated process (photo‐PISA). Alessandro Chiolerio and Marco B. Quadrelli  provide an initial assessment of existing capabilities of liquid robotics with many future applications, such as space exploration in extreme environments, post‐disaster search, compliant wearable devices, and in vivo medical applications. Yat Li et al.  summarize recent advances in the area of paper‐based electrodes, with a particular focus on the methodology of fabricating these novel functional electrodes, and discuss some key challenges and opportunities in this rapidly growing field. Mingbo Wu, Jianzhang Zhao, and co‐workers  have written a review article that summarizes a variety of strategies for modulating the singlet oxygen quantum yield and thus enhancing photodynamic therapy efficiency. The fundamental scientific principle, structure, and possible classification of battery–supercapacitor hybrid devices are discussed in the review article by Yuanyuan Li and Jinping Liu . Qianqian Li and Zhen Li  elucidate the important role of molecular packing modes on aggregation‐induced‐emission (AIE) materials. In a more theoretical contribution, De‐en Jiang and co‐workers  present a detailed overview of progress made in the past decade using computer simulations to understand supercapacitors. Finally, the issue contains a review article by Zhou Li  on biomechanical energy harvesters, nanosensors, and stimulators in the biomedical field.I would like to thank all authors who have contributed their fantastic articles to this issue and I hope that you will enjoy this collection. If you are interested in contributing your own open access article that anyone can freely access, do not hesitate to contact the editorial team. We are happy to discuss any potential topic with you.On behalf of the entire editorial team,


AHRR gene , cg03636183 in the F2RL3 gene , cg21566642 and cg05951221 in 2q37.1 , cg06126421 in 6p21.33  and cg23387569 in 12q14.1 . For cg05951221 and cg23387569 the strength of association was virtually identical in never and current smokers. For all these CpGs except for cg23387569, the methylation levels were different across smoking categories in controls , were lowest for current smokers and increased with time since quitting for former smokers. We observed a gain in discrimination between cases and controls measured by the area under the ROC curve of at least 8%  in former smokers by adding methylation at the 6 CpGs into risk prediction models including smoking status and number of pack‐years. Our findings provide convincing evidence that smoking and possibly other factors lead to DNA methylation changes measurable in peripheral blood that may improve prediction of lung cancer risk.DNA methylation changes are associated with cigarette smoking. We used the Illumina Infinium HumanMethylation450 array to determine whether methylation in DNA from pre‐diagnostic, peripheral blood samples is associated with lung cancer risk. We used a case‐control study nested within the EPIC‐Italy cohort and a study within the MCCS cohort as discovery sets . We validated the top signals in 429 case‐control pairs from another 3 studies. We identified six CpGs for which hypomethylation was associated with lung cancer risk: cg05575921 in the  What's new?It is well known that smoking can cause lung cancer but the concept that it might do so by changing DNA methylation is only emerging. Here the authors identify six sites of methylation (CpGs), where methylation levels were associated with lung cancer risk after adjusting for smoking, current or former. Methylation of five of the CpGs was lowest in current smokers and increased in former smokers with time since quitting, supporting the growing evidence that smoking may lead to DNA methylation changes measurable in peripheral blood and useful as predictive markers for lung cancer risk, especially in former smokers. Smoking is the main cause of lung cancer with attributable risks of at least 85% for men and 60% for women,AHRR and F2RL3 genes are associated with lung cancer riskF2RL3 was also reported in a recent German studyAlterations of the methylation profile of DNA from peripheral blood associated with cigarette smoking have been recently describedIn order to identify novel DNA methylation changes associated with lung cancer risk and better understand the mechanisms underlying these associations, we conducted a further analysis of the four epigenome‐wide association studies (EWAS) in NOWAC, MCCS, NSHDS, EPIC‐Heidelberg and in a new, independent EWAS in the EPIC‐Italy cohort that became available recently. In all these five case‐control studies nested within prospective cohorts, we investigated associations between methylation of DNA from pre‐diagnostic, peripheral blood samples and lung cancer risk accounting for reported smoking habits.AHRR and F2RL3 genes. For all studies one control was individually matched to each case. In MCCS and NSHDS controls were matched to cases by reported smoking status at blood draw using five categories  in EPIC‐Heidelberg they were matched by reported smoking status in two categories (current and former) and number of pack‐years. In EPIC‐Italy and NOWAC controls were not matched by smoking. Further details for each study are provided in the Supplementary Materials.To test our hypotheses regarding the relationship between DNA methylation and lung cancer risk, we used data from a new EWAS within the Italian component of the European Prospective Investigation into CancerLaboratory methods for DNA extraction, quality control, bisulphite conversion and Illumina Infinium HumanMethylation450 BeadChip assays as well as details about data pre‐processing and quality control are described in detail in the Supplementary Materials and were broadly similar across studies. Exceptions are noted explicitly. For the MCCS some DNA samples were extracted from dried blood spots on Guthrie cards using a method developed in‐houseNormalisation procedures of the methylation measures were applied to perform colour channel and probe type correction as described in the Supplementary Materials.M‐values of methylation calculated as log2(beta/(1‐beta))M‐value of methylation at each CpG that we obtained by dividing the M‐values into quartiles according to the distribution in the control group and assigning to each category the within‐quartile median value. We estimated odds ratios per 1 standard deviation (SD) of the pseudo‐continuous variable and the corresponding 95% confidence intervals (95% CI).For all analyses we used p values lower than 10−4 for at least one study. For these CpGs we calculated pooled MCCS and EPIC‐Italy estimates and selected for further analyses and replication 6 CpGs whose combined estimate had a p values lower than 10−5. For the selected 6 CpGs and for the previously identified cg03636183 in the F2RL3 gene, we estimated the ORs for lung cancer separately for MCCS, EPIC‐Italy and the three replication studies. For this set of seven CpGs, we estimated pooled ORs by combining the study‐specific estimates fitting fixed effect models overall and for different categories of smoking . We adjusted the estimates for current and former smokers for number of cigarettes and duration of smoking and the estimates for former smokers also for time since quitting smoking. We estimated ORs for different categories of time to diagnosis and used a likelihood ratio test to test for heterogeneity. We assessed the possible effect of cell composition on the results by adding into the models the proportions of different cell types  calculated using the method suggested by HousemanWe ranked the CpGs according to the p‐values of the corresponding ORs, separately for MCCS and EPIC‐Italy, and identified 34 CpGs with a M‐values of methylation and reported smoking for the control group by fitting a linear mixed effect model with slide (i.e. chip) nested within plate fitted as random effects and gender, age at blood collection and smoking fitted as fixed effects. We used likelihood ratio tests to test the association between methylation and smoking.We also estimated the association between We evaluated separately for the MCCS and EPIC‐Italy the additional contribution of DNA methylation at the CpGs associated with lung cancer risk to the ability of the model to discriminate between cases and controls using area under the curve (AUC) statistics obtained from unconditional logistic regression models adjusted for the matching variables. We accounted for the contribution of smoking by including among the covariates smoking status and the number of pack‐years of cigarettes smoked.p values of <10−7, we investigated graphically the association between DNA methylation and lung cancer risk in the 100 kilobase region around each CpG site, by plotting the pooled MCCS and EPIC ORs versus the CpG location.Finally, for the CpGs whose pooled estimates had an OR with a Relative to the total number of CpGs investigated across the genome, the proportion of CpGs with methylation levels inversely associated with lung cancer risk was 55% in the new EPIC‐Italy study and 53% in MCCS. Overall, for 48% of the CpGs we observed concordant associations between methylation level and lung cancer risk in MCCS and EPIC‐Italy (either both negative or both positive) and of these 58% were concordant negative.p values lower than 10−4 in at least one of the two studies , and these are presented in Table AHRR gene on chromosome 5 and cg06126421 on chromosome 6. Table F2RL3 on chromosome 19, that, together with cg05575921 in the AHRR gene we previously reported to be associated with lung cancer riskWe identified 34 CpGs for which the smoking‐adjusted association with lung cancer risk corresponds to a p = 0.01) were concordant in the two studies and, of these, 79%  were concordant negative .Of the associations corresponding to the 34 CpGs listed in Table p values lower than 10−5 ; cg21566642 ; cg05575921 ; cg06126421 ; cg23387569 ; cg03636183   the pooled ORs for lung cancer across EPIC‐Italy and MCCS had a −7). DNA methylation levels were lowest for current smokers while average levels for former smokers were intermediate between those for current and never smokers; DNA methylation levels for former smokers increased with increasing time since quitting  were strongly associated with reported smoking history in the control groups  overall; 11.1% (p = 0.011) in former smokers and 1.2% in current smokers (p = 0.52). We obtained similar results from the MCCS in which the overall gain from including all the CpGs combined was 5.5% (p = 0.002) relative to the model including smoking history  and number of pack‐years; the gain was 7.6% (p = 0.004) in former smokers and 3.3% (p = 0.28) in current smokers.For EPIC‐Italy, the value of the AUC for the model including reported smoking status  and the number of pack years was 79%. Adding methylation levels for each CpG individually resulted in gains of 1.8% for cg05575921, 1.3% for cg06126421 and <1% for each of the other selected CpGs  and the pseudogene ECEL1P2 whose methylation has been found to increase through developmentBoth cg05951221 and cg21566642 are located in a CpG island on chromosome 2q37.1 in which a region with differential methylation between cases and controls is clearly visible  that codes for a component of the exocyst complex and its antisense RNA (EXOC3‐AS1).We observed no significant correlation between cg05575921, located within the MDC1), cellular growth and division (DHX16), protection of cells from Fas‐ or tumor necrosis factor type alpha‐induced apoptosis (IER3), cytoskeleton regulation and membrane traffic . In particular, FLOT1 mRNA expression has been shown to be upregulated in non‐small cell lung cancer tissuelncRNAs) map in the region: MDC1‐AS1 and LINC00243.The probe cg06126421 on chromosome 6p21.33 is flanked by a region extending approximately 200 bases containing another 5 CpGs whose methylation levels correlate with methylation levels of cg06126421 (correlations ranging from 0.44 to 0.67) . One of them codes for the cyclin‐dependent kinase 4, a member of the Ser/Thr protein kinase family that is important for cell cycle G1–S transition by the RB1–CCND1–CDKN2A pathway that is known to be damaged in lung cancer.A region of ∼300 bases extends around cg23387569 on chromosome 12q14.1 as methylation levels for the six CpG sites in this region quite strongly correlate with methylation level at cg23387569 (correlations ranging from 0.52 to 0.87)  and were targeted because of the established strong association between cigarette smoking and methylation at this site.Our previous report that DNA methylation changes at cg05575921 in the AHRR and F2RL3 genes; consequently, the results reported here for all other CpGs can be considered original and independent from those previously published. To investigate the possible role of smoking in explaining the observed associations between DNA methylation and lung cancer risk, we deliberately oversampled cases of former and never smokers from some of the cohorts.Relative to our previous reportThe associations we observed between DNA methylation and lung cancer risk are relatively strong (ORs for 1 SD increase in DNA methylation are between 0.74 and 0.50), they are not limited to current smokers and they remained strong after adjusting for smoking duration and intensity: this suggests that the associations between DNA methylation and lung cancer risk are unlikely to be explained by residual confounding by smoking. The observation that for all the identified CpGs except one the methylation levels are lower in current smokers and rise to the levels of never smokers with increasing time since quitting suggests that smoking contributes to the methylation status of these CpGs, although it might not be the only determinant and disentangling the relation between smoking, methylation and lung cancer risk might be challengingAHRR geneInterestingly, for all the six CpGs identified the ORs for lung cancer for former smokers are consistently lower than ORs for current smokers. This observation, consistent across all five studies, is intriguing but difficult to explain. The analyses by smoking have been adjusted for smoking intensity, duration and time since quitting in former smokers, but we cannot exclude that the result is due to misreported smoking habits or residual confounding. It has been recently reported that inflammation processes such as those caused by smoking induce changes in the methylation profile of natural killer cells including hypomethylation in the Lung diseases, including lung cancer, may trigger an immune response and alter the prevalence of specific cell types in the bloodThe observations that at cg23387569 the association between methylation levels and smoking history was not evident or at least not as strong as for the other CpGs and that for all CpGs the associations between DNA methylation levels and lung cancer were not limited to current smokers suggest that DNA methylation changes at these CpGs may play a role in pathways to lung cancer that are independent of smoking. Further studies specifically designed to increase the number of lung cancer cases in never smokers are necessary to provide convincing evidence to support this hypothesis.The use of the Illumina Infinium HumanMethylation 450 BeadChip allowed us to obtain epigenome‐wide data at single CpG resolution for a relatively large number of cases and controls but these microarray data do not permit the systematic investigation of the regions surrounding the CpGs identified to evaluate whether the observed associations may be regional and thus have greater predictive power should a more comprehensive measure of methylation be possible. In the region on chromosome 2q.37.1, for example, two measured CpGs (cg21566642 and cg05951221) were both strongly associated with lung cancer risk and others in the same region show suggestive evidence of association. It is possible that methylation at other unmeasured sites in this region might be even more strongly associated with lung cancer risk. It is, therefore, important that further studies, for example based on targeted bisulphite sequencing, are conducted to finely map methylation in these regions.AHRR gene both in lung tumour tissue from current smokers and in mouse models of exposure to cigarette smokingTo further investigate the functional relevance of the observed associations it would be important to test whether methylation in the CpGs identified alter the expression of proximal genes. We could not do this directly in our cohorts as we do not have gene expression data but we have investigated it in other datasets and available public data. In a previous study we showed that methylation at cg05575921 was associated with decreased expression of the AHRR; cg23387569 in AGAP2; cg03636183 in F2RL3) we investigated the correlation between methylation and expression using TCGA (http://cancergenome.nih.gov/) and HapMap (http://hapmap.ncbi.nlm.nih.gov/) dataF2RL3‐probe were available. In brief, AHRR‐probe methylation seems to be inversely correlated with AHRR expression in lung tumour tissue from TCGA. F2RL3‐probe methylation does not show methylation‐expression correlation in TCGA data but HapMap data suggest a weak inverse correlation . AGAP2‐probe methylation seems to be positively correlated with AGAP2 expression in both lung tumour tissue from adenocarcinomas and squamous cell carcinomas .For the three probes that map within a gene sequence In the study within the ESTHER cohort, the authors estimated that the gain in ability to discriminate between cases and controls by adding methylation levels in cg03636183 in the Supporting InformationClick here for additional data file.Supporting Figure LegendClick here for additional data file.Supporting Figure 1Click here for additional data file.Supporting Figure 2Click here for additional data file.Supporting Figure 3Click here for additional data file.Supporting Figure 4Click here for additional data file.Supporting Table 1Click here for additional data file.Supporting Table 2Click here for additional data file.Supporting Table 3Click here for additional data file.