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The Mechanosensitive Ion Channel Piezo1 Significantly Mediates In Vitro Ultrasonic Stimulation of Neurons

Summary Ultrasound brain stimulation is a promising modality for probing brain function and treating brain disease non-invasively and with high spatiotemporal resolution. However, the mechanism underlying its effects remains unclear. Here, we examine the role that the mouse piezo-type mechanosensitive ion channel component 1 (Piezo1) plays in mediating the in vitro effects of ultrasound in mouse primary cortical neurons and a neuronal cell line. We show that ultrasound alone could activate heterologous and endogenous Piezo1, initiating calcium influx and increased nuclear c-Fos expression in primary neurons but not when pre-treated with a Piezo1 inhibitor. We also found that ultrasound significantly increased the expression of the important proteins phospho-CaMKII, phospho-CREB, and c-Fos in a neuronal cell line, but Piezo1 knockdown significantly reduced this effect. Our findings demonstrate that the activity of mechanosensitive ion channels such as Piezo1 stimulated by ultrasound is an important contributor to its ability to stimulate cells in vitro.

INTRODUCTION
Neuromodulation technologies developed recently, such as magnetic, electric, and optogenetic stimulation methods, have provided unprecedented ways to study brain function and treat its diseases and conditions. Ultrasound is another modality being studied for its neuro-stimulation abilities. Ultrasound beams can be focused on millimeter-sized areas in sub-cortical regions through the intact skull (King et al., 2013;Legon et al., 2014;Tufail et al., 2010). It has been demonstrated to be capable of generating neural responses from the human visual cortex (Lee et al., 2016), somatosensory cortex (Lee et al., 2015;Legon et al., 2014), motor cortex (Legon et al., 2018b), and even the thalamus (Legon et al., 2018a) without obvious side effects. These features have spurred research into ultrasonic neuromodulation, with attempts being made to use it therapeutically for conditions such as Parkinson disease, epilepsy, and depression (Leinenga et al., 2016).
The observed effects of ultrasound, a physical stimulus, in the brain points to central nervous system cells possessing mechanosensitivity by various mechanisms (reviewed by Tyler (2012)). At the low intensities required to apply ultrasound in the brain through the intact skull safely and successfully (Duck, 2007;Fry and Goss, 1980;Tyler et al., 2008), the thermal and cavitation effects of ultrasound are likely to be minor (Dinno et al., 1989;Kim et al., 2014;Tyler et al., 2018). Another possible mechanism of ultrasound is through its action on mechanoresponsive components of cellular machinery, which sense physical forces and initiate cellular signaling. Given the observed timescale of ultrasound's stimulation effects, a plausible explanation is offered by the activity of mechanosensitive ion channels (Bystritsky et al., 2011;Fomenko et al., 2018;Tyler et al., 2018). Such channels are implicated in sensing a wide variety of physical stimuli, including sound, membrane stretch, and shear forces (Martinac, 2012). Some mechanosensitive ion channels have been shown to be activated by ultrasound, but these studies lack the context of brain stimulation, studying either non-mammalian channels (Ibsen et al., 2015;Kubanek et al., 2018;Ye et al., 2018) or mammalian channels expressed in non-neuronal cells (Kubanek et al., 2016). (C) Acoustic mapping profile of the controllable ultrasound field generated using the customized setup.
(D) A schematic illustration of the ultrasound stimulation parameters used in our experiments, with a peak-to-peak acoustic pressure of 0.3 MPa used as an example.
(E) The expression of functional mouse Piezo1 in Piezo1-transfected cells (''Piezo1''), compared with a vector control (''Ctrl''), was verified in three ways. Left: qRT-PCR was performed for mouse Piezo1, normalized to b-actin and expressed as a fold change. Bar charts represent the mean G SEM of three independent experiments. n = 3, ***p < 0.0001, unpaired two-tailed t test. A representative Western blot of the two groups is also shown. Piezo1 functionality was verified by stimulating cells with 1 mM Piezo1 agonist Yoda1 and performing calcium imaging with Cal-590. Middle: a representative time course of Yoda1 stimulation on the two groups is shown. Right: bar chart shows the mean G SEM of three independent Ca 2+ imaging experiments. n = 10, ***p < 0.0001, unpaired two-tailed t test.
10 pN (Wu et al., 2016). Although it allows cations to permeate cells in general, it is reported to exhibit a preference for calcium ions (Ca 2+ (Coste et al., 2010). Heterologously expressed Piezo1 could be activated by ultrasound in non-neuronal cells (Gao et al., 2017;Pan et al., 2018;Prieto et al., 2018) but only with the use of very high frequencies or with microbubbles. It remains to be shown whether endogenous Piezo1 in neurons can be activated by ultrasound alone and what role this interaction could play in neuro-stimulation by ultrasound.
In the present study we confirm endogenous Piezo1 expression in mouse primary neurons, and in a neuronal cell line, and show that these channels play a role in neuronal activation in vitro. We demonstrate that low-intensity low-frequency ultrasound alone can activate heterologously expressed HEK293T cells, as well as endogenous Piezo1 channels, initiating Ca 2+ influx and increasing levels of c-Fos. We also show ultrasound is capable of significantly affecting the levels of downstream Ca 2+ signaling proteins crucially involved in neuronal function and that knocking down Piezo1 significantly decreased this effect. Thus, we show that Piezo1 activation plays a major role in the mechanotransduction of ultrasound and that ultrasound alone is capable of significantly affecting the function and activation of neurons in vitro.

Customized In Vitro Ultrasound Stimulation Setup
We developed a customized in vitro ultrasound stimulation system incorporated with a calcium imaging system ( Figure 1A). Briefly, the calcium imaging system consisted of a modified upright epifluorescence microscope. The excitation light was generated by a dual-color LED, filtered and delivered to the sample to illuminate the calcium sensor. Signals from the cells were collected by a water immersion objective, passed through a dual-filter wheel, and captured by a camera. To minimize phototoxic effects, the LEDs were triggered at 1 Hz and synchronized with sCMOS time-lapse imaging. Ultrasound was delivered through a triangle waveguide attached to the transducer placed below the culture dish at a 45-degree angle. The waveguide was also attached to an acoustic absorber to minimize acoustic reverberation. This design helped us to minimize the generation of standing waves and created a strong water-air interface on the bottom of the dish. This results in a controllable ultrasound field, as measured by an acoustic pressure mapping system consisting of a needle hydrophone and high-precision 3D motor ( Figure 1B). The obtained acoustic pressure map is displayed in Figure 1C, using an acoustic pressure of 0.3 MPa for illustration. Each stimulus was composed of 200 tone burst pulses at a center frequency of 500 kHz with a duty cycle of 40% at a pulse repetition frequency (PRF) of 1 kHz, at low acoustic intensities ( Figure 1D). These parameters corresponded to very short bursts of ultrasound stimulation, helping to minimize any resultant thermal effects (Kubanek et al., 2018).

Heterologously Expressed Piezo1 Can Be Activated by Ultrasound and Induce Ca 2+ Influx in HEK293T Cells
To confirm that ultrasound could activate Piezo1 using our customized setup, we overexpressed mouse Piezo1 heterologously in HEK293T cells, known to show minimal response to mechanical stimulation (Coste et al., 2010;Syeda et al., 2015). We transfected cells with a pcDNA3.1-mPiezo1-IRES-GFP plasmid (described in Coste et al. (2010)) or a vector control (described in Schaefer et al. (2008)) and treated them with a Piezo1-specific agonist, Yoda1 (Syeda et al., 2015). We verified the overexpression using qRT-PCT and Western blotting for Piezo1, and calcium imaging revealed that 1 mM Yoda1 induced significantly more Ca 2+ influx in the Piezo1-transfected cells, compared with the control ( Figure 1E). We thus confirmed the expression and functionality of transfected mouse Piezo1 channels in HEK293T cells.
We proceeded to perform Ca 2+ using ultrasound at acoustic pressures corresponding to a range previously reported to have elicited responses (Tufail et al., 2010). We found that increasing ultrasound pressure on Piezo1-transfected cells resulted in increasing Ca 2+ influx into cells, with 0.3 MPa ultrasound showing a significant increase, whereas the control remained largely unchanged ( Figure 1E). To exclude the possibility Right: the bar chart shows the mean G SEM of three independent Ca 2+ imaging experiments. n = 9, ***p < 0.0001, two-tailed unpaired t test with Holm-Sidak correction. All statistically significant differences are shown. See also Figure S1.
that the fluorescence changes were due to cells being out-of-focus due to ultrasound perturbation, we also performed ratiometric Ca 2+ imaging. We observed that the fluorescence ratio (340/380) of control cells showed no response, whereas the Piezo1-expressing cells showed a robust and large response to 0.1 MPa ultrasound ( Figure S1). Pre-treating the Piezo1-expressing cells with a Piezo1-specific inhibitor, GsMTx-4 (Bae et al., 2011), at 40 mM reduced the cells' response to the same level as the control cells (Figure S1). This finding confirmed that the elevated cytoplasmic Ca 2+ levels seen upon ultrasound stimulation were not experimental artifacts. Thus, our ultrasound stimulation setup could successfully activate Piezo1 to allow Ca 2+ influx at low, physiologically relevant acoustic pressures.

Mouse Primary Neurons Are Activated by Ultrasound in a Piezo1 Activity-Dependent Manner
We next tested the effects of ultrasound on primary cortical neurons harvested from embryonic mice to test the feasibility of stimulating live neurons with ultrasound. We first probed whether primary neurons cultured using our protocol could express any functional Piezo1. Treating primary neurons at DIV 10 with 10 mM Yoda1 stimulated Ca 2+ influx, whereas pre-treatment with 40 mM GsMTx-4 significantly reduced it ( Figure 2A). The inhibitory effect of GsMTx-4 upon Yoda1-induced Ca 2+ influx was consistent with the known mechanism of its action as a Piezo1 gating modifier (Bae et al., 2011;Gnanasambandam et al., 2017). Immunofluorescent staining revealed that primary neurons at day in vitro (DIV) 10 expressed Piezo1 endogenously ( Figure 2B). Thus, primary neurons at DIV 10 expressed some functional Piezo1, and we proceeded to evaluate the effects of ultrasound on these cells. We also acutely isolated mouse cortical neurons from mouse pups at P3, to evaluate in vivo expression of Piezo1 in neurons of approximately equal maturity. These neurons influxed Ca 2+ when stimulated with Yoda1, but this effect was almost completely abrogated in presence of the mechanosensitive ion channel antagonist Ruthenium red ( Figure S2). We thus found that mature neurons, both in vitro and in vivo, expressed functional Piezo1 with detectable effects.
Ultrasound stimulation resulted in dose-dependent Ca 2+ influx into the neurons, and this influx was abrogated when the cells were pre-treated with 40 mM GsMTx-4 ( Figure 2C). Ultrasound at 0.3 MPa and above was able to induce significant Ca 2+ influx, so we evaluated the effects of ultrasound on neuron activation by immunofluorescent staining of c-Fos, a well-established molecular marker of neuronal activation that is responsive to Ca 2+ influx (Chaudhuri et al., 2000;Ghosh et al., 1994;Sheng and Greenberg, 1990). Untreated cells, cells treated with 0.3 MPa ultrasound for 20 min and cells pre-treated with 20 mM GsMTx-4 before ultrasound inside a standard cell culture incubator (setup illustrated in Figure 2D), were compared. We found that c-Fos expression in the nuclei of neurons (identified by MAP2 staining) significantly increased upon US treatment compared with the untreated control and reduced significantly with GsMTx-4 pre-treatment ( Figure 2E). Hence, low-intensity ultrasound could activate primary neurons in vitro by opening the Piezo1 channel to allow Ca 2+ influx.

Ultrasound Requires Piezo1 to Induce Calcium-Dependent Downstream Signaling in a Neuronal Cell Line
We were interested in exploring the signaling implications of Piezo1-mediated ultrasound effects on neurons in greater depth, to help elucidate the possible downstream effects of Ca 2+ influx through ultrasoundactivated Piezo1 channels. We chose the mouse hippocampal cell line mHippoE-18 (CLU199) as an in vitro representation of normal (non-cancerous) neuronal cells to enable better quantification of these effects. Expression of Piezo1 in CLU199 cells was confirmed through semi-quantitative RT-PCR, with HeLa cells as a positive control (McHugh et al., 2010) and Western blot ( Figure 3A). To evaluate the treatment's effects on cell signaling downstream of the observed Ca 2+ influx, we investigated levels of the phosphorylated (activated) forms of the Ca 2+ /calmodulin-dependent protein kinase type II (p-CaMKII) and the transcription factor CREB (p-CREB). Both these proteins are crucial players in neuronal Ca 2+ signaling. CaMKII is directly regulated by calmodulin, a good indicator of the level of Ca 2+ inside the cell, and both CaMKII and CREB are involved in critical neuronal functions such as neurotransmitter secretion, plasticity, transcription regulation, learning, and memory (Hardingham et al., 2001;Yamauchi, 2005). We observed the levels of these activated proteins, along with c-Fos, by Western blot to gauge whether ultrasound could affect aspects of neuronal function and activation downstream of Ca 2+ influx. Ultrasound treatment inside an incubator for 20 min increased the levels of p-CaMKII, p-CREB, and c-Fos in a dose-dependent manner, with 0.3 and 0.5 MPa inducing significant increases ( Figure 3B). We then evaluated Piezo1's contribution to these effects by siRNA knockdown. We were able to achieve over 50% knockdown of Piezo1 and found that this significantly reduced the 1 mM Yoda1-induced Ca 2+ influx compared with cells treated with non-targeting siRNA (''Ctrl'') ( Figure 3C). CLU199 cells with Piezo1 knockdown also displayed no significant upregulation of p-CaMKII,  p-CREB, and c-Fos than the control when treated with 0.3 MPa ultrasound compared with the control (Figure 3D). Thus, we determined that ultrasound stimulation significantly affects the levels of important proteins in CLU199 neuronal cells, and these effects were significantly mediated by the Piezo1 channel.

DISCUSSION
In the present study, we show that Piezo1 expressed in neurons is an important mediator of ultrasound's in vitro effects. We observed that heterologous Piezo1 expression in cells could enable them to respond to ultrasound (HEK293T). We showed that mouse neuronal cells express Piezo1 endogenously and that ultrasound alone can open these channels, allowing Ca 2+ influx into them. We also demonstrated that activation of primary neurons by ultrasound in vitro can be significantly reduced by inhibiting Piezo1's activity. Our experiments used low-intensity low-frequency ultrasound, without microbubbles, allowing us to minimize the thermal and cavitation effects of ultrasound. We manipulated endogenous Piezo1 on a functional level, using an inhibitor drug, and at the expression level, using siRNA, and found that the effects of ultrasound on proteins crucially involved in neuronal activation and calcium signaling were significantly reduced in both cases. Blocking the activity of heterologous Piezo1 could also suppress ultrasound-induced Ca 2+ influx. Thus, we see an important role played by Piezo1 activation in enabling neurons to respond to ultrasound stimulation.
In addition to increased c-Fos expression in primary neurons, our results in a neuronal cell line also showed that the expression of the activated forms of CaMKII and CREB can be increased by ultrasound stimulation, along with increasing c-Fos expression. These proteins are involved in complex neuronal functions such as neuronal plasticity, learning, and memory. Although results from a cell line must not be overinterpreted, previous studies of ultrasound have also shown increases of calmodulin-dependent kinases in primary neurons (Liu et al., 2017) and increases in the crucial neuroprotective protein BDNF in mouse and rat brains treated with ultrasound (Chen et al., 2018;Su et al., 2017;Tufail et al., 2010). The observed Piezo1-dependence of these effects in our experiments also helps to pinpoint their source to the ultrasound stimulation. Such results at the cellular level also provide evidence of the basis for non-auditory effects of ultrasound.
Our results help to bolster the idea that ultrasound treatment can have therapeutic effects in the brain and could contribute to identifying which conditions could benefit most from such a treatment.
The obvious next step would be to demonstrate Piezo1-dependence of ultrasound effects in vivo, by upregulating or downregulating it in rodent brains. It will likely be challenging to upregulate Piezo1 in vivo, due to its size and complexity (Coste et al., 2010(Coste et al., , 2015, but also to downregulate it, as knocking it out totally in mice embryos can be lethal (Li et al., 2015;Ranade et al., 2014). However, delivering a Piezo1 inhibitor or Piezo1 knockdown reagents in rodents is feasible and is a promising approach for further understanding the effects of ultrasound. Transgenic mice with conditional knockout of Piezo1 in the brain are also a viable option. Concurrently, research about the Piezo1 molecule itself could contribute to both evaluating and enhancing the role of brain-based Piezo1 in mediating ultrasound's effects. Studies comparing the different segments show some domains to be more mechanosensitive than other (Wu et al., 2016). This raises the possibility that the effects of ultrasound on such domains could be studied instead of the entire molecule, making it easier to tailor ultrasound parameters to activate Piezo1 more efficiently. Knowledge of this kind could also have significance in other fields, as Piezo1 plays crucial roles in other systems, such as the development of blood vessels (Li et al., 2015;Ranade et al., 2014) or T-cell activation . Thus, understanding ultrasound's mechanism of action on Piezo1 could have implications for usefully modulating neuronal activity, as well as that of a host of other cell types.

Limitations of the Study
Although we aimed to demonstrate that ultrasound opens the Piezo1 channel, we could not use the standard method of patch clamping to determine this. This is because the recording pipette in our system was found to be incompatible with low-frequency ultrasound, a phenomenon also reported elsewhere (Prieto et al., 2018). We thus used intracellular ratiometric Ca 2+ imaging to measure the ion influx resulting from ultrasound opening Piezo1, which helped us work around the vibration issue. It is also important to note that the cells used in this study were cultured on lysine-coated glass, a hard substrate, as this can complicate data interpretation. Ultrasound could cause vibrations in the base of the dish, amplifying the observed effects (Kubanek et al., 2016), leading to an underestimation of the acoustic pressure required to activate Piezo1. Secondly, neurons develop divergently depending upon the stiffness of their surroundings (Chang et al., 2017;Koser et al., 2016), and substrate stiffness is also reported to affect Piezo1's activity (Pathak et al., 2014). A possible solution would be to coat plates with a soft gel that mimics the mechanical properties of brain tissue. However, there is no standard method to achieve this, which is why we opted to maintain a simple culture protocol to avoid unnecessary complications. An aim of future studies ought to be to solve such issues in vitro, to gain more physiologically relevant data.
Finally, we did not examine the role of other mechanosensitive ion channels that may be involved in mediating the cellular response to ultrasound, because we were primarily interested in whether low-intensity ultrasound stimulation could activate our chosen Piezo1 channel. For all three issues, in the context of the present study, we based our conclusions upon comparison to controls, with lower Piezo1 expression or with a Piezo1 blocker, which were also exposed to ultrasound but showed significantly lower (but not abrogated) responses. We thus argue that Piezo1 mediated ultrasound's effects in our cells to a considerable degree, a finding that could be adapted and applied to in vivo conditions as appropriate. However, future studies with in vivo Piezo1 knockout/ knockdown in brains and using sensitive genetically encoded Ca 2+ or voltage sensors for real-time tracking of ultrasound's effects will be crucial to truly establish the role of channels as Piezo1 in this context.

METHODS
All methods can be found in the accompanying Transparent Methods supplemental file.

DATA AND CODE AVAILABILITY
The authors confirm that the data supporting the findings of this study are available within the article and its Supplemental Information. reduces apoptosis in a mouse model of traumatic brain injury. Brain Stimul. 10, 1032-1041.

SUPPLEMENTAL INFORMATION
Syeda, R., Xu, J., Dubin, A.E., Coste, B., Mathur, J., Huynh, T., Matzen, J., Lao, J., Tully, D.C., Engels, I.H., et al. (2015).  Neurons were isolated acutely from the cortices of mouse pups at P3, and treated with Piezo1 agonist Yoda1, with or without the mechanosensitive ion channel blocker Ruthenium red (RR) and imaged using the ratiometric dye Fura-2. (A) Representative pictures of phase contrast and Ca 2+ imaging using the ratiometric dye Fura-2, showing the same cells prior to and after the addition of 10 µM Yoda1. (B) Representative Ca 2+ imaging time-course of single neurons upon the addition of 10 µM Yoda1, with and without RR. (C) Quantified Ca 2+ imaging results using Yoda1 at various concentrations, with and without the addition of RR. Bars represent mean ± SEM of 2 independent experiments. n for each condition is indicated above the bar; ** P < 0.01, **** P < 0.0001, unpaired one-way ANOVA with post-hoc Tukey test. All statistically-significant relationships are shown.


extfieldsofstudy: Biology Medicine

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HMGB proteins are required for sexual development in Aspergillus nidulans

Aspergillus nidulans has three high mobility group box (HMGB) proteins, HmbA, HmbB and HmbC that are chromatin-associated architectural proteins involved in DNA-related functions. By creating and studying deletion strains in both veA+ and veA1 background, we have characterized the role of HmbA, HmbB and HmbC in sexual development. Expression of the mating-type MAT1-1 and MAT1-2 coding genes were found to be extremely down-regulated in all three mutants on day 4 of sexual development, which results in deficient ascospore production and/or ascospore viability in the mutants. In addition, we found that HmbA and HmbB play also a role in sensing of and response to environmental signals, while HmbC functionally interacts with VeA, a key regulator of the coordination of asexual and sexual development, as well as of secondary metabolism.

Introduction
Beside linker histones (H1 and H5), B-type high mobility group box domain proteins (HMGB) (for nomenclature of HMG proteins see [1]) are also important architectural components of chromatin. These proteins can bind to linker DNA, and induce or repress gene expression. They are able to interact with both DNA and protein components of chromatin through their high mobility group box (HMG-box) domains (reviewed in [2][3][4][5][6][7]). The HMGbox domain (comprising three α-helices) folds up into an L-shape three dimensional form, and binds to the minor grove of DNA-helix, distorting the DNA's backbone (reviewed in [4]). In HMGB proteins the amino acid that precedes the second α-helix of the HMG-box has nonpolar characteristics, which endows the HMG-box with the ability to bind to the DNA with no or little sequence-specificity [3,8]. HMGB proteins (e.g. human HMGB proteins, Drosophila DSP1, yeast HMO1 and HMO2) are generally composed of two or more copies of HMGbox domains, although single-copy HMG-box containing HMGB proteins also exist [4,9,10]. Three architectural HMGB proteins, namely HmbA (AN2885), HmbB (AN1267) and HmbC (AN10103), were identified in Aspergillus nidulans according to their diagnostic non-polar amino acids that precede the second α-helix in their canonical HMG-box [11]. Their closest characterized fungal homologues are Nhp6A/Bp, Abf2p and Hmo1p in Saccharomyces cerevisiae (homologues of HmbA, HmbB and HmbC, respectively) and mtHMG1 in Podospora anserina (homologue of HmbB) [9][10][11][12][13][14][15]. Of these three A. nidulans proteins, the physiological role of the dually localized mitochondrial/nuclear HmbB was previously characterized in details [11,16]. Deletion of hmbB has pleiotropic effects (S1 Table). The hmbBΔ mutant is viable, but displays severe fitness loss due to the drastic reduction in the viability of conidiospores and ascospores (0.4% viable) [11]. Besides its role in spore viability, HmbB is also involved in diverse biological processes such as sugar metabolism during conidia germination, sterigmatocystin production, redox homeostasis or maintaining mitochondrial DNA copy number [11,16]. The latter correlates with the mitochondrial localization of HmbB, whereas the other functions might be associated with the nuclear localization of the protein. This indicates that HmbB may play a role in the structure and function of nuclear chromatin [11]. This hypothesis is further supported by the altered transcription level of numerous nuclear genes in hmbBΔ [11,16]. Regarding HmbA and HmbC, we have limited knowledge about their function in terms of their physiological roles and transcriptional regulation, however, based on the functions of their yeast homologues, it can be assumed that they modulate the expression of a wide range of genes [10,14,17,18].
In S. cerevisiae, five HMGB proteins were identified, namely Nhp6Ap, Nhp6Bp, Hmo1p, Hmo2p, and Abf2p (physiological functions are summarized in S1 Table). The functionally redundant paralogue proteins, Nhp6Ap and Nhp6Bp (Nhp6A/Bp, homologues of HmbA), are composed of a single HMG-box domain, and act in the form of homodimers. Double deletion of nhp6A and nhp6B exerts a pleiotropic physiological effect. Specifically, the mutant shows morphological and cytoskeletal defects, such as sensitivity to starvation and growth defect at high temperature settings, which can be suppressed by adding an osmotic stabilizer in the medium [19]. At the molecular level, the functions of Nhp6A/Bp proteins are similar to that of mammalian HMGB1. Specifically, they regulate the transcription of Pol II-dependent genes at the genome-scale level through different mechanisms. They modulate the interaction of TATA-binding protein (TBP) to the promoter sites and the subsequent formation of the TBP/ TFIIA/DNA complexes [20][21][22]. They also interact with the yeast FACT complex, which ensures transcription elongation by RNA Pol II through nucleosomal templates by removing histone H2A-H2B dimers [23]. In addition, they also interact with transcription activators or repressors [24]. The Nhp6A/Bp proteins are also important mediators of the expression of RNA Pol III-transcribed genes [25].
Prominent examples of HMGB proteins having two copies of HMG-box domains are Hmo1p, Hmo2p and Abf2p. Deletion of the Hmo1p (homologue of HmbC) coding gene results in a severe growth defect (with extremely reduced colony size), reduced plasmid stability, hypersensitivity to micrococcal nuclease, and a decrease in transcription executed by RNA Pol I and II [9]. At the molecular level, Hmo1p promotes the transcription of rRNA genes [10], as well as interacts with Fhl1p (regulator of ribosomal protein genes) [26], TBP and TFIID [17]. Deletion of the Hmo2p coding gene results in the weakening of the DNA doublestrand break repair mechanisms mediated by INO80 complex and γ-H2A.X [27]. At the molecular level, Hmo2p protects broken DNA from exonucleolytic cleavage by recognizing and binding to DNA ends [28], and recruits the INO80 complex to the damage-induced phosphorylated γ-H2A.X [29]. Deletion of the Abf2p (homologue of HmbB) coding gene has pleiotropic effects including the loss of compaction of the mitochondrial nucleoid, the decrease of mitochondrial recombination frequency and the decrease of mitochondrial genome stability on fermentable carbon sources. These effects are the consequences of DNA bending and wrapping, as well as the promotion and stabilization of Holliday-junction recombination intermediates [30][31][32].
P. anserina has five HMGB proteins (PaHMG2, PaHMG3, PaHMG4, PaHMG6 and mtHMG1) (physiological functions are summarized in S1 Table) and two putative HMGB proteins (PaHMG7 and PaHMG9) [33]. Of these seven proteins, mtHMG1 localizes to mitochondria (similarly to yeast Abf2p and HmbB of A. nidulans) and is essential for the maintenance of mitochondrial genome. In the presence of an extra copy of this gene, the 'premature death' phenotype originating from mitochondrial DNA rearrangements in an AS1-4 context is partially suppressed [12]. The mthmg1 deletion strain displays altered germination, growth and fertility, as well as reduced life span in AS1 + context [12]. All but one (PaHMG2) of the P. anserina HMGB type proteins were shown to be implicated in certain aspects of sexual development [33]. Two of them, PaHMG6 and mtHMG1 (orthologues of A. nidulans HmbA and HmbB, respectively), govern the expression of transcription factors required for sexual development, such as the α-box mating-type FMR1, the HMG-box mating-type transcription factor FPR1 and mating-type HMG-box domain containing transcription factors PaHMG5, PaHMG8 and PaHMG9 [33].
The sexual development of A. nidulans (homothallic, self-fertile) is regulated by various environmental factors (light, oxygen level, nitrogen source, pH), and requires the contribution of regulatory proteins, such as the velvet family protein VeA and mating-type factors MAT1-1 and MAT1-2 [34,35]. Deletion of the veA gene results in a complete loss of the ability to undergo sexual development, while the veA1 mutation (start codon ATG mutated to ATT) results in an N-terminal truncated protein devoid of nuclear localization signal and evokes a light-independent functioning for the protein [36]. The veA1 mutant strains produce conidia abundantly even in dark conditions, produce few aerial hyphae and form fruiting bodies regardless of light conditions, although at a reduced level compared to that of veA + strains [37].
During the sexual development of A. nidulans, thick-walled Hülle cells are formed, which surround an aggregated hyphal mass (primordium) composed of differentiated cells of the pericarp (outside) and ascogenous hyphae (inside). Subsequently, a tiny immature cleistothecium (μ-cleistothecium) develops from the primordium, which is composed of non-or lightlypigmented pericarp cells and ascogenous hyphae with ascus mother cells. At later stages of the development, immature asci are formed with 8 non-or lightly-pigmented ascospores [38]. During maturation, the immature cleistothecium transforms to mature cleistothecium by the accumulation of a dark pigment in the wall cells and binucleate ascospores having an oval shape [38]. In our current research we have investigated HmbA, HmbB and HmbC of A. nidulans, orthologues of PaHMG6, mtHMG1 and PaHMG4 of P. anserina [33]. By studying the sexual structures of hmbA, hmbB and hmbC deletion mutants and analyzing gene expression of mating-type genes, we have revealed that the HMGB proteins play an essential role in ascospore production and viability, possibly via their modulation of the expression of mating-type genes that influence the late-stage sexual development of A. nidulans.

Strains, media and growth conditions
The A. nidulans strains used in this study are listed in S2 Table. Standard genetic markers, complete medium (CM) and minimal medium (MM) are described at the following URL: http://www.fgsc.net/Aspergillus/gene_list/. Media were supplemented with vitamins (www. fgsc.net) according to the requirements of each auxotrophic strain. Agar for minimal media was obtained from BD/Difco. For total DNA extraction, 10 8 conidiospores were inoculated to 100 ml liquid MM and incubated at 37˚C for 14-16 h with 180 rpm shaking. Sexually developing mycelia for RNA extraction were prepared according to Zheng et al. [39]. 10 8 conidiospores were inoculated to 300 ml MM and incubated at 37˚C for 24 h with 180 rpm shaking. For the induction of sexual development, the mycelia from the 24 h cultures were transferred to solid MM plates and sealed tightly with scotch tape. The plates were incubated in complete darkness at 37˚C for 48 h and 96 h prior to RNA extraction.

Construction of deletion and complementation strains
Deletion of hmbB had been reported previously [11]. Deletions of hmbA and hmbC were obtained by the transformation of tripartite gene-substitution cassettes constructed by the double-joint PCR method [40] as described previously [11]. The cassettes were composed of "A", "B" and "C" components. The flanking "A" and "C" components contained a 2-3 kb fragments of genomic region upstream and downstream to the target locus, respectively, while the middle "B" components contained the selection marker gene riboB + (in case of hmbA deletion) or a pabaA + (in case of hmbC deletion). Used primers are listed in S3 Table. The "A", "B" and "C" components of the hmbA substitution cassette were amplified with the "hmbA up frw"-"hmbA up rev", "hmbA ribo chim frw"-hmbA ribo chim rev" and "hmbA down frw"-"hmbA down rev" primer pairs, respectively. The 3,428 bp, 2,208 bp and 3,058 bp long "A", "B" and "C" components were assembled to a 7,827 bp long substitution cassette using "hmbA up nest frw" and "hmbA down nest rev" primers. The substitution cassette was transformed into HZS.120 and 30 riboflavin prototrophic strains were selected and pre-screened for the deletion by PCR using "HmbA frw" and "HmbA rev" primers. Single copy integration mutants were selected on the basis of Southern blot analysis using the DIG-labeled "C" component as DNA probe on XbaI digested total DNA (S1 Fig). One single copy integration mutant (HZS.205) was used in genetic crosses to obtain veA + and veA1 hmbAΔ strains (HZS.521 and HZS.239, respectively) for further studies. Reconstitution of hmbA deletion was obtained by cloning the hmbA product (amplified by "hmbA prom NotI frw" and "hmbA term NheI rev" primers) into the NheI/NotI sites of pAN-HZS-1 vector [11] (see more in S1 Materials and methods) and transforming the developed pAN-HZS-9 vector into the recipient strains HZS.320 (hmbAΔ, veA1) and HZS.655 (hmbAΔ, veA + ). Out of 50 transformants of HZS.320 and 19 transformants of HZS.655, 4 carried single copy integration (cross-checked by qPCR with "hmbA ReTi frw" and "hmbA ReTi rev" primers, respectively). The single copy integration strains HZS.621 (reconstituted hmbAΔ, veA1) and HZS.678 (reconstituted hmbAΔ, veA + ) were used in further studies.
The "A", "B" and "C" components of the hmbC substitution cassette were amplified with the "hmbC up frw"-"hmbC up rev", "hmbC paba chim frw"-"hmbC paba chim rev" and "hmbC down frw"-"hmbC down rev" primer pairs, respectively. The 2,634 bp, 3,846 bp and 2,320 bp long "A", "B" and "C" components were assembled to a 8,069 bp long substitution cassette using "hmbC up nest frw" and "hmbC down nest rev" primers. The substitution cassette was transformed into HZS.314 and 30 p-amino-benzoic acid prototroph strains were selected and pre-screened for the deletion by PCR using "HmbC frw" and "HmbC rev" primers. Single copy integration mutants were selected on the basis of Southern blot analysis using the DIG-labeled "C" component as DNA probe on EcoRV digested total DNA (S2 Fig). One single copy integration mutant (HZS.338) was used in genetic crosses to obtain prototrophic veA + and veA1 hmbCΔ strains for the further studies. Reconstitution of hmbC deletion was obtained by cloning the hmbC PCR product (obtained by using "hmbC NcoI frw" and "hmbC BamHI rev" primers) into the NcoI/BamHI sites of pAN-HZS-1 vector [11] (see more in S1 Materials and methods) and transforming the obtained pAN-HZS-10 into the recipient strains HZS.338 (hmbCΔ, veA1) and HZS.658 (hmbCΔ, veA + ). Out of 16 transformants of HZS.338 and 13 transformants of HZS.658, 2 and 3 carried single copy integration (checked by qPCR with "hmbC ReTi frw" and "hmbC ReTi rev" primers), respectively. The single copy integration strains HZS.676 (reconstituted hmbCΔ, veA1) and HZS.679 (reconstituted hmbCΔ, veA + ) were used in further studies.

DNA manipulations
DNA was prepared from A. nidulans as described by Specht et al. [41], and hybond-N membranes (Amersham/GE Healthcare) were used for Southern blots [42]. Southern hybridizations were done by DIG DNA Labeling and Detection Kit (Roche) according to the manufacturer's instructions. Transformations of A. nidulans protoplasts were performed as described by Antal et al. [43]. The protoplasts were prepared from mycelia grown over cellophane [44,45] using a 1% solution of Glucanex (Novozymes, Switzerland) in 0.7 M KCl. Transformation of 5x10 7 protoplasts was carried out with 100-500 ng of fusion PCR products or plasmid vectors.

Genetic crosses
Heterozygotic crosses were done according to Pontecorvo [38]. The veA allele of each veA + progeny selected for further work was checked by using the "veA DM frw"-"veA DM rev" and "veA frw"-"veA rev" primer pairs [46] and also sequenced with the "veA seq frw" primer. Homozygotic crosses were always self-crosses (selfing or inbreeding) made by sealing (in the case of hmbBΔ selfing the plates were not sealed for obtaining normal-sized fruiting bodies) of 2-day-old CM agar cultures with scotch tape. Inbreeding plates were inoculated in replicates and kept in complete darkness at 37˚C for different time periods. Each day following the fourth day of incubation one of the replicate plates was opened and the sexual structures were studied under stereo-and light microscopes.

Microscopy
Cleistothecia were taken from colonies by a needle and the samples were mounted on slides directly or after their cleaning (by rolling them on the surface of an agar plate to eliminate mycelia, conidiospores and Hülle cells) using 1x PBS buffer. After crushing the samples between the slide and cover slip, the sexual structures were documented. DIC images were taken by using Olympus BX51 microscope.

Quantitative PCRs and data analysis
To assess the copy number of integration events in the transformant strains, total DNAs were extracted [41] and used in quantitative real-time PCR (qPCR) using hmbA, hmbB, hmbC and reference gene actA (AN6542) specific primer pairs (S3 Table). For reverse transcription-qPCR (RT-qPCR), total RNA was isolated from sexually developing mycelia [39] by using RNeasy Plus Mini Kit (Qiagen) or TRIsure reagent (Bioline) with RNase-Free DNase Set (Qiagen) according to the manufacturer's instructions (for further details, see S1 Materials and methods). cDNA synthesis was carried out on 1 μg RNAs with a mixture of oligo-dT and random primers using the RevertAid First Strand cDNA Synthesis Kit (Fermentas). Quantitative PCRs were performed in a CFX96 Real Time PCR System (BioRad) with SYBR Green/ Fluorescein qPCR Master Mix (Fermentas) reaction mixture and specific primers listed in S3 Table. Five reference genes (actA, histone H3 hhtA/AN0733, beta-tubulin tubC/AN6838, Glyceraldehyde-3-phosphate dehydrogenase gpdA/AN8041 and Elongation factor 3 eEF-3/ AN6700) were tested by using geNorm (https://genorm.cmgg.be). The gpdA and tubC genes were selected to obtain a 'gene expression normalization factor' (for further details, see S1 Materials and methods). Transcript levels were estimated according to the relative standard curve method [47], using three technical replicates of three biological samples.

Determination of the size of cleistothecia and germination ability of ascospores
In order to determine the size of the cleistothecia, a mass of cleistothecia was taken out from one spot of the colonies. Ten individual cleistothecia that represented the frequency of the observed sizes were purified and documented in the presence of a size ruler by a camera. The average sizes of the cleistothecia were determined by conducting image analysis on individual cleistothecia using Adobe Photoshop. The germination ability of the ascospores was determined by counting colony-forming units after inoculating 100 ascospores on solid medium. Dilutions of 10 5 −10 7 ascospores were plated onto MM agar plates and incubated at 37˚C for 2-4 days. The numbers of colonies were counted and normalized to 100 ascospores. All experiments were performed on at least ten biological replicates in three technical replicates.

Statistical analysis
All statistical analysis was performed using GraphPad Prism version 5.02 for OSX (GraphPad Software, San Diego, CA). The significant differences between sets of data were determined by one-way ANOVA test, two-way ANOVA or Mann-Whitney U-test according to the data.

Generation of deletion strains and analysis of the vegetative development
In order to study the role of the three HMGB proteins of A. nidulans in sexual development, we generated hmbAΔ, hmbBΔ and hmbCΔ deletion strains both with a veA + and a veA1 genetic background. In case of hmbB, we used a previously generated mutant strain, the hmbBΔ veA1 (HZS.212) [11]. By performing genetic crossing, we obtained prototrophic hmbBΔ veA1 strain (HZS.280) and hmbBΔ veA + strain (HZS.495). The hmbA and hmbC deletion mutants were developed in a veA1 genetic background (HZS.205 and HZS.338, respectively) via the transformation of gene-substitution cassettes constructed by the Double-Joint PCR method [40] into the appropriate recipient strains (HZS.120 and HZS.314, respectively) (for details see Materials and methods and S1 and S2 Figs). We developed the prototrophic or auxotrophic hmbAΔ and hmbCΔ strains with veA1 (HZS.239 and HZS.338) and veA + genetic backgrounds (HZS.521 and HZS.531) by performing genetic crosses. In trans expression of P hmbA -hmbA, P gpdA -hmbB and P gpdA -hmbC in the corresponding hmbA, hmbB and hmbC deletion strains (for details see the Materials and methods section and S1 Materials and methods) mitigated but did not completely abolish the deletion phenotype with respect to colony macromorphology and ascospore production/viability (see below). The reason for this partial complementation may be the in trans expression of the constructs and/or usage of the constitutive gpdA promoter (P gpdA ) in the case of complementation of hmbB and hmbC deletion.
Macromorphological analysis of the mutant strains revealed that the hmbCΔ strain grown on MM or CM (Materials and methods) was not different from the hmbC + control (Fig 1 and  S3 Fig). The hmbAΔ strains formed smaller colonies compared to those of the hmbA + controls, due to slower growth rate (detailed description of the hmbAΔ phenotype will be described elsewhere) (Fig 1 and S3 Fig). The hmbBΔ strains also formed smaller colonies compared to the controls, explained by delayed conidiospore germination rather than by a slower growth rate as it was described earlier [11] (Fig 1 and S3 Fig).

Time course of sexual development and intracolonial distribution of cleistothecia
The sexual development of veA + and veA1 control strains, along with that of the hmbAΔ, hmbBΔ and hmbCΔ strains with both veA + and veA1 background were documented on a daily basis from day 5 to day 12 of incubation on selfing cultures (Fig 1) (for details see Materials and methods). All the deletion mutants were able to produce Hülle cells and cleistothecia with various severities of defects in ascospore formation and ascospore viability. This phenomenon was accompanied by the appearance of a reddish granular amorphous (RGA) material, which resembles to that observed in the barren cleistothecia of MAT1-1 and MAT1-2 protein coding gene deletion strains [35]. The comparative study of the time course of sexual development, intracolonial distribution, size and ascospore content of the cleistothecia revealed the role of  Table. Panel B: Schematic representation of oxygen regulated distribution pattern of cleistothecia in control and hmbAΔ, hmbBΔ and hmbCΔ strains. "Sealed" and "non-sealed" refer to conditions where plates are sealed with scotch tape or kept without sealing, respectively. "Limited oxygen availability" refers to the nonsealed condition with the applied medium almost entirely filling up the Petri dish. Small-sized grey dots mark micro-sized cleistothecia, black dots indicate normal-sized cleistothecia. Panel C: Time course of sexual structure formation in control and hmbA, hmbB and hmbC deletion strains. Selfing was done on CM at 37˚C in complete darkness. In the case of hmbBΔ strains (both veA + and veA1) the plates remained non-sealed and the medium almost entirely filled up the Petri dish. The rest of the selfing plates were sealed by scotch tape. Samples from the areas showing sexual development were taken each day from day 5 until day 11, and were studied without purification by using microscope. Cleistothecia collected from day 12 were purified on a sterile agar plate prior to mounting. HmbA, HmbB and HmbC proteins in sensing of and response to environmental factors, the production and viability of ascospores and their possible functional interaction with VeA functions. The observed phenotypes of the mutants are summarized in Table 1.
The control veA + strain formed primordia on day 5 (image A on Fig 1) and formed mature ascospores on day 10 of incubation (image B on Fig 1). The veA1 control developed more rapidly than the veA + control and μ-cleistothecia formation was observed on day 5 of incubation (image C on Fig 1) and mature free ascospores were present from day 6 of incubation (image D on Fig 1).
The time course of the sexual development of the hmbCΔ veA + strain was not different from that of the veA + control, however the primordium formation of the hmbAΔ veA + and hmbBΔ veA + strains was delayed by a day or two compared to that of the veA + control (images E, K and L on Fig 1).
Development of the sexual structures in the hmbAΔ veA1 and hmbBΔ veA1 strains were found to occur earlier (1-2 days) than in their veA + counterparts (images F and M on Fig 1), however the veA1 background did not accelerate sexual development in the hmbCΔ veA1 mutant. The primordium and μ-cleistothecia formation of hmbCΔ veA1 were observed one day later compared to the hmbCΔ veA + strain (image R on Fig 1).
The mature hmbAΔ cleistothecia, collected on days 11 and 12 of incubation, were practically empty in both the veA + (images G and H on Fig 1) and veA1 background (images I and J on Fig 1). These cleistothecia contained RGA material, nearly zero amounts of ascogenous hyphae and sporadically detected ascospores (magnified sections of images H and J on Fig 1). On day 12, the mature veA + and veA1 hmbBΔ cleistothecia contained ascogenous hyphae, RGA material, a few asci in different maturation stages and a few matured, free ascospores (images N and O on Fig 1). Cleistothecia with somewhat improved quality and quantity of internal content could only be observed under highly specific conditions (detailed below). On day 12, the veA + hmbCΔ cleistothecia still contained immature asci with transparent ascospores among the released, mature ascospores (image P on Fig 1). The veA1 hmbCΔ mutant frequently produced barren cleistothecia without any ascospores. However, when the veA1 hmbCΔ cleistothecia contained ascospores, these ascospores were always mature (image Q on Fig 1). RGA material was detected in both veA + and veA1 hmbCΔ cleistothecia, but this was more pronounced in the veA1 background.
In the case of the colonies of veA + and veA1 controls, cleistothecia were accumulated in the middle of the colony and were equally distributed in the rest of the area regardless whether the plates were sealed or not (Fig 1). The size of the cleistothecia in veA + and veA1 controls was in the ranges of 183-304 μm and 216-304 μm, respectively (Fig 2, S4 Fig). The distribution pattern of the hmbAΔ cleistothecia was detected to depend on oxygen availability. Oxygen-deprivation (via sealing the plates) resulted in a wild type-like distribution pattern both in the veA + and the veA1 hmbAΔ strains (Fig 1). However, when the plates were not sealed and thereby air exchange was not restricted, fruiting body formation was mainly exclusive to the central part of the colony in both the veA + and the veA1 hmbAΔ strains (Fig 1). The size of cleistothecia in the veA + and veA1 hmbAΔ strains was in the ranges of 166-270 μm and 95-200 μm, respectively (Fig 2, S4 Fig).
Selfing of the hmbBΔ strains (both veA + and veA1) provided colonies with barren microsized cleistothecia and/or few small-sized cleistothecia (79-95 μm, Fig 2, S4 Fig) with a low ascospore content (~100 ascospores/cleistothecia) in nearly all cases. However, selfing plates with few normal-sized cleistothecia (up to 200-225 μm, Fig 2, S4 Fig) with an improved ascospore content were also detected sporadically. A systematic combination of environmental factors (light and oxygen availability) revealed that the hmbBΔ strain favors a medium-level of oxygen restriction. Neither the sealed, nor the non-sealed plates provided normal-sized cleistothecia. We found that the height of the medium (40-45 ml) in the Petri dish (9 cm diameter) combined with keeping the plates unsealed was a determinant factor of normal-sized cleistothecia. Based on this phenomenon, we propose that the hmbBΔ strain needs a certain level of oxygen for the optimal sexual development in both the veA + and the veA1 backgrounds. The distribution of sterile micro-sized cleistothecia resembled the wild type, however when the environmental parameters favored the production of normal-sized cleistothecia, they were always formed at the perimeter of the colonies (Fig 1).
The distribution and the size of the veA + hmbCΔ cleistothecia were similar to that of the veA + control (195-354 μm), whilst veA1 hmbCΔ produced equally-distributed, medium-sized (112-200 μm) cleistothecia in lower abundance compared to its veA + counterpart (Fig 1, Fig 2  and S4 Fig). The veA1 hmbCΔ colonies tended to form conidia-depleted sectors, where the density of cleistothecia was significantly increased (shown in S5 Fig and indicated in Fig 1).
The size of cleistothecia was restored only in the veA1 hmbB complemented strain (Fig 2). None of the other reconstitutions provided any improvement (veA + hmbB, veA1 hmbA and veA1 hmbC) or even decreased (veA + hmbA and veA + hmbC) the size of cleistothecia (Fig 2), most probably due to the in trans expression of the cognate genes and/or the usage of a constitutive promoter in the case of hmbB and hmbC reconstitution.

Ascospore content of cleistothecia and viability of ascospores in veA + background
The veA + control contained 10 5 -10 6 ascospores per cleistothecium, and 32% of them were able to form colonies (Fig 3, S1 Data). We estimated the number of ascospores in the hmbAΔ veA + cleistothecia (�10 ascospores/cleistothecium) by counting the visible ascospores in crushed cleistothecia samples (Fig 3). The frequency of viable hmbAΔ veA + ascospores could not be calculated based on counting the colony forming units (CFU), because the extremely low (�10) number of CFUs might also originate from mycelium-, conidiospore-or Hülle cell contamination of the cleaned cleistothecium samples. We therefore decided to monitor the germination rate of the hmbAΔ veA + ascospores using a microscope that does not necessarily reflect colony forming ability (viability). We collected and pooled 25 hmbAΔ veA + cleistothecia, and the total content was incubated in liquid MM on the surface of cover slips. Remarkably, we found that at least 30% of these ascospores were viable (Fig 3), although we have no proof that the germinating ascospores can also form colonies. Complementation of the hmbAΔ deletion phenotype (�10 ascospores per cleistothecium) by in trans expression of hmbA under the control of its native promoter resulted in~10 5 ascospores per cleistothecium (with 38% viability rate), which was significantly lower than that of the wild-type control, however, it approached the productivity level of the control. The ascospore content of hmbBΔ veA + cleistothecia increased with the age of the colony (10 3 ascospores per cleistothecium at week 2, 10 4 ascospores per cleistothecium at week 4 and 10 5 ascospores per cleistothecium at week 6). However, the viability of these ascospores decreased with aging (1% at week 2, 0.06% at week 4 and 0% at week 6). Complementation of  The strains are color-coded as follows: blue denotes control; red denotes deletion, green denotes complementation (comp.) strains originating from the corresponding deletion strains. The bubbles mark the number of ascospores; the size of the bubbles is proportional to the viability of the corresponding ascospores. An estimated rate of germination was used as a proxy of viability in the case of hmbAΔ (explained in the main text). The number of ascospores were estimated based on 10 independent cleistothecia per strain. An estimated ascospore number was used in the case of hmbAΔ (explained in the main text). Mann-Whitney U-test was used to assess the differences between the numbers of ascospores of the mutant and the control strains. � / �� / ��� indicates p < 0.01/0.001/0.0001. Panel B: Graphical representation of the viability of ascospores. The boxplot shows the viability of the ascospores across the hmbA, hmbB and hmbC mutant and control strains in a veA + genetic background. Viability was calculated by counting the number of colony-forming ascospores on solid medium. An estimated rate of germination was used as a proxy of viability in the case of hmbAΔ (explained in the main text). The strains are color-coded as follows: blue denotes control; red denotes deletion, green denotes complementation (comp.) strains originating from the corresponding deletion strains. Centre lines indicate the median viability of ascospores collected from 10 independent cleistothecia per strain. Mann-Whitney U-test was used to assess size differences between the mutant strains and the control in the corresponding genetic background. the hmbBΔ deletion phenotype (~4×10 4 ascospores per cleistothecium) by in trans expression of hmbB under the control of the constitutive gpdA promoter resulted in an ascospore productivity approaching that of the wild type (~1.6×10 5 ascospores per cleistothecium) with a viability rate of 31% (Fig 3). Even though ascospore productivity was significantly lower than that of the wild-type control, the difference was marginal. Ascospore productivity of the hmbCΔ veA + strain was also similar to that of the wild type (~2×10 5 ascospores/cleistothecium); however, only 7% of them was found to be viable (Fig 3). Complementation of the hmbCΔ deletion phenotype (~2×10 5 ascospores per cleistothecium) by in trans expression of hmbC under the control of the constitutive gpdA promoter resulted in significantly decreased ascospore productivity (~6.3×10 4 ascospores/cleistothecium) compared to that of the deleted strain, however the viability of the ascospores increased more than two-fold (18.6%) compared to that of the deletion strain (Fig 3).

Mating-type MAT1-1 and MAT1-2 coding genes are extremely downregualted in the veA + hmbAΔ, hmbBΔ and hmbCΔ mutants
The expression of the mating-type MAT1-1 and MAT1-2 coding matB and matA genes starts at the late stage of sexual development (four days after the induction of sexual development), when Hülle cells and immature cleistothecia are already formed [35]. We determined the mRNA levels of matB and matA on day 4 of sexual development (96 h after the induction of sexual development) and found that both mating-type genes were extremely down-regulated in all of the deletion mutants (showing a 4.9-23.6-fold negative change) (Fig 4). As discussed in details below, this extremely high degree of down-regulation might indicate the aberrant ascospore development in cleistothecia observed in the mutants.

Discussion
According to the results presented above, HmbA, HmbB and HmbC architectural chromatin components of A. nidulans are required for normal sexual development, especially for the formation and viability of ascospores and the spatial distribution of cleistothecia ( Table 2, S1  Table). By studying hmbA, hmbB and hmbC deletions in both veA + and veA1 background, we have investigated the possible functional interaction of these HMGB proteins with VeA, the master regulator of sexual development ( Table 2, S1 Table).
The hmbAΔ, hmbBΔ and hmbCΔ strains were found to show defect in ascospore production and viability to various extent ( Table 2). The hmbA deletion resulted in nearly sterile cleistothecia with less than 10 ascospores inside, which were remarkably able to germinate with an estimated 30% rate, however the colony forming ability of these germinating ascospores was not confirmed. The deletion of hmbB and hmbC resulted in a decreased ascospore productivity, accompanied by a severe (in hmbBΔ) and a less-severe (in hmbCΔ) decrease of ascospore viability. RGA material was detected in all three deleted mutants; amongst them the hmbCΔ strain accumulated RGA material to a greater extent, which was more pronounced in the veA1 background. The veA + hmbCΔ cleistothecia invariably contained asci and ascospores, however, these asci were frequently immature. Remarkably, the veA1 hmbCΔ cleistothecia were either barren (devoid of ascospores) or fertile (containing ascospores). The fertile veA1 hmbCΔ cleistothecia mostly contained mature, free ascospores and an increased amount of RGA material. Furthermore, the abundance of the hmbCΔ cleistothecia approached that of the wild type in the veA + background, whereas it was reduced in the veA1 background. Both hmbA and hmbB deletions caused a delay in the time course of sexual development, independently of VeA, while hmbC deletion had no effect on sexual development in the veA + , but caused a pronounced delay in the veA1 background. Considering all the above described hmbCΔ phenotypes, we propose that HmbC functionally interacts with VeA ( Table 2, S1 Table).
HmbA and HmbB might be involved in sensing of and responding to the changes of environmental oxygen levels. The wild type-like distribution of the cleistothecia was found to depend on the strict deprivation of oxygen in the hmbAΔ mutants, while normal-sized cleistothecia formation in the hmbBΔ strain required a medium level of oxygen deprivation ( Table 2, S1 Table).
Mating-type protein functions in heterothallic fungi are essential for pheromone-signalling and recognition of the mating partner [48,49]. However, mating-type proteins of the homothallic A. nidulans (MAT1-1 encoded by matB and MAT1-2 encoded by matA) are essential for the fertility of the formed cleistothecia by contributing to ascospore production [35]. Deletion of matA or matB does not affect Hülle cell formation and cleistothecium production, however the cleistothecia are devoid of ascospores and contain only granular amorphous materials [35]. The MAT gene deletion phenotype is somewhat similar to the ascospore production-and viability-related phenotypes of the hmbA, hmbB and hmbC deleted strains. As  [47], were normalized to an 'expression normalization factor' calculated from two selected reference genes (tubC and gpdA) (detailed in S1 Materials and methods). Standard deviations of three technical replicates of three biological samples are shown. The stars above the columns indicate the significance of the differences compared to the veA + control. Significant differences between the mutants and the control were determined by using a two-way ANOVA test. ���� indicates p < 0.0001. The following strains were used in the experiment: veA + (HZS.450), hmbAΔ veA + (HZS.521), hmbBΔ veA + (HZS.495) and hmbCΔ veA + (HZS.531). The cultivation settings were as follows: approximately 10 6 conidiospores per strain were inoculated into liquid MM, and were grown for 24 hours at 37˚C with 180 rpm shaking. Then the vegetatively grown mycelia were transferred onto solid MM covered with cellophane, sealed carefully with scotch tape and incubated for 96 hours at 37˚C in complete darkness. After the incubation period, total RNA was extracted and processed.
https://doi.org/10.1371/journal.pone.0216094.g004 expected, HmbA, HmbB and HmbC were found to be critical for the normal expression of the MAT genes on day 4 after the initiation of sexual development (Fig 4, Table 2). We excluded the possibility that all three HMGB linker proteins are required for the direct activation of MAT gene expression. Instead, we propose an alternative explanation for the observed changes: HmbA, HmbB and HmbC might operate on upstream MAT gene regulator(s) that is (are) most probably involved in the sensing of environmental and/or intracellular factors and/ or the transduction of related signals that affect the activation of the MAT genes. Future research should elucidate the potential role of HMGB proteins in the upstream regulation of MAT genes. HmbA, HmbB and HmbC orthologue proteins of the heterothallic fungus P. anserina (PaHMG6, mtHMG1 and PaHMG4, respectively) do not contribute equally to the regulation of mating-type transcription factors (coded by fmr1 and fpr1, and being essential for the regulation of mating-type specific genes and the recognition of the mating partner at the pheromone-signalling stage) [48]. Only the HmbA orthologue protein, PaHMG6 regulates positively fmr1 in a mat − strain (in a mat + strain the regulation bypasses fpr1), while the MAT genes are negatively regulated by the HmbB orthologue protein mtHMG1 and unaffected by the HmbC orthologue protein PaHMG4 [33] (S1 Table).
Although all three HMGB proteins play a role in the expression of the mating-type genes, neither the hmbB, nor the hmbC deletion phenocopied the matBΔ or matAΔ phenotypes completely. The deletion phenotypes of hmbBΔ (both veA + and veA1) and veA + hmbCΔ resembled to that of the matBΔ or matAΔ regarding RGA material accumulation inside the cleistothecia, however, ascospores were produced, although with deficient viability. Only the hmbAΔ (in both veA + and veA1) phenotype shared all the characteristics of the matBΔ and matAΔ phenotypes. The MAT gene deletion phenotype was frequently, but not always observed in the veA1 hmbCΔ strain, which indicates that HmbC functionally interacts with VeA. On the other hand, the random occurrence of barren cleistothecia amongst fertile cleistothecia might reflect a sporadic compensation for the loss of hmbC in the veA1 background. We cannot exclude the possibility that HmbA, HmbB and HmbC directly influence the gene expression of MAT-regulated genes, and that they can provide the functional backups for each other's loss. Such a compensatory effect had already been reported in the case of other types of linker proteins. Mice contain eight subtypes of the linker H1 histone that are differentially expressed during development [50][51][52]. These H1 histone variants can compensate for each other's loss in homozygous knockout mouse models (e.g. H1c, H1d and H1e variants can compensate for the homozygous deletion of H1(0)) [53]. Considering all of the phenotypes of the hmbAΔ, hmbBΔ and hmbCΔ strains, it is reasonable to suppose that besides the pronounced down-regulation of MAT genes, a qualitatively different perturbation in the expression of other genes might further contribute to the hmbAΔ-, hmbBΔ-and hmbCΔ-specific phenotypes, however, investigation of these changes are beyond the scope of this work.
Based on the orthologous relation of HmbA, HmbB and HmbC proteins with PaHMG6, mtHMG1 and PaHMG4 proteins of P. anserina, respectively, we compared the roles of the orthologous proteins to assess functional relations (S1 Table). The orthologue of HmbA in P. anserina, PaHMG6, is required for achieving a normal-sized colony [33]. This function is qualitatively similar to that seen in the case of HmbA of A. nidulans (S3 Fig). In a homozygous cross, the Pahmg6Δ mutant produced fruiting bodies with 50 times less abundance, with smaller body and larger neck compared to the wild type, and began to eject ascospores several days later in comparison to the wild type. Although both A. nidulans hmbAΔ and P. anserina Pahmg6Δ mutants showed a delay in the time course of sexual development in homozygotic crosses, an analogy between their functions in the aspect of sexual competency cannot be concluded.
The absence of the orthologue of HmbB in P. anserina AS1 + strain (mthmg1Δ, AS1 + ) does not result in the loss of the ascospores' ability for germination (ascospores germinate slowly with a spindly phenotype [12]), thus we cannot establish an analogy between the functions of mtHMG1 and HmbB. Although the dual localization of the mtHMG1 protein of P. anserina has not been studied yet, we previously revealed a dual localization of HmbB, and found that the orthologous HmbB and mtHMG1 share a third HMG-box domain, called Shadow-HMGbox, which is characteristic to the HmbB orthologues across Pezizomycotina [11]. The structural similarity between HmbB and mtHMG1, as well as the fact that both proteins modulate the expression of nuclear genes [11,33] makes it reasonable to suppose that mtHMG1 fulfils nuclear-localization-linked functions as we suggested previously for HmbB [11].
The HmbC counterpart in P. anserina, PaHMG4, was found to be required for the normal distribution of the fruiting bodies [33]. The PaHMG4 deletion mutant produced five times more spermatia (with wild type-like viability), whereas the deletion had no effect on female fertility [33]. Thus, PaHMG4 function differs from that of HmbC, thereby the two proteins are functionally diverged.
Some of the physiological functions of HMGB proteins we revealed are specific for A. nidulans compared to yeast and P. anserina (summarized in S1 Table). This includes that HmbA and HmbB play a role in sensing of and/or response to environmental signals. By revealing the functional connections of HmbA and HmbB with signal transduction pathways, one would gain a deeper understanding of the regulatory machinery that governs physiological responses to environmental changes. On the other hand, we found that HmbC functionally interacts with VeA, a key regulator of the coordination of asexual and sexual development, as well as of secondary metabolism. By revealing the functional interactions of HmbC, one would gain a deeper insight into the regulation of these biological processes. Finally, HmbA, HmbB and HmbC are equally important in the positive regulation of mating-type genes, and thereby have a great impact on ascospore production in A. nidulans. The knowledge on the regulation of fungal mating-type genes is scarce, thereby clarifying, whether these HMGB proteins influence matA/matB expression directly or indirectly (e.g. via the modulation of upstream regulatory factors) would be of great interest. Additionally, future works should elucidate the geneexpression modulatory role of the HMGB proteins on a genome-scale that might lead to a more detailed characterization of the physiological roles of HmbA, HmbB and HmbC.
Supporting information S1


extfieldsofstudy: Chemistry Medicine

s2fieldsofstudy:
Biology
Materials Science
Chemistry

text:
Reduction-Triggered Paclitaxel Release Nano-Hybrid System Based on Core-Crosslinked Polymer Dots with a pH-Responsive Shell-Cleavable Colorimetric Biosensor

Herein, we describe the fabrication and characterization of carbonized disulfide core-crosslinked polymer dots with pH-cleavable colorimetric nanosensors, based on diol dye-conjugated fluorescent polymer dots (L-PD), for reduction-triggered paclitaxel (PTX) release during fluorescence imaging-guided chemotherapy of tumors. L-PD were loaded with PTX (PTX loaded L-PD), via π–π stackings or hydrophobic interactions, for selective theragnosis by enhanced release of PTX after the cleavage of disulfide bonds by high concentration of glutathione (GSH) in a tumor. The nano-hybrid system showed fluorescence quenching behavior with less than 2% of PTX released under physiological conditions. However, in a tumor microenvironment, the fluorescence recovered at an acidic-pH, and PTX (approximately 100% of the drug release) was released efficiently out of the matrix by reduction caused by the GSH level in the tumor cells, which improved the effectiveness of the cancer treatment. Therefore, the colorimetric nanosensor showed promising potential in distinguishing between normal and cancerous tissues depending on the surrounding pH and GSH concentrations so that PTX can be selectively delivered into cancer cells for improved cancer diagnosis and chemotherapy.

Introduction
Drug delivery innovation has been rapidly advancing, but it requires continuous development to improve therapeutic performance. However, current drug delivery nano-based systems are limited by the need for high dosages, the poor availability of drugs at disease sites, and the unwanted side effects [1]. It is therefore necessary to develop controlled drug delivery with the ability to transport drugs effectively and efficiently to tumor sites [2]. Taking into account the fact that the extracellular microenvironment of tumor tissue is acidic at approximately pH 6.5-6.9 as compared to normal tissue (pH 7.2-7.4) [3], a pH-sensitive nanosystem could be beneficial in distinguishing between tumor and healthy tissues to solve the drawbacks in drug delivery. Additionally, tumor cells contain a reducing substance in the form of glutathione (GSH), which has been proved to be at least four times higher than that in the normal cells [4,5]. Considering both facts, a dual pH and redox-sensitive drug delivery system (DDS) may have great potential for the controlled release of drugs for efficient cancer therapy especially hydrophobic drugs [6].
Polymer-based nano-hybrid delivery systems, especially in cancer therapy, have been extensively investigated as a way to achieve temporal and spatial control for targeting drug delivery at disease sites such as lipid based vesicular, micelle-like structure or nanogel-based DDS systems [2,[7][8][9][10][11]. However, most of organic compound-based nano-hybrid systems still need to be improved due to a lack of real-time monitoring properties; they also show low drug-loading percentages and poor stability during blood circulation, thus hindering their performance, which remains a noticeable challenge [12]. Recently, sp 2 -riched carbon hybrid nanocomposites such as graphene oxide (GO) and carbon nanotubes (CNTs) were employed as drug carriers for better stability. However, the nano-based system still showed problems, especially during the drug-release phase [13]. The π-π stackings and hydrophobic interactions hindered the release of the drug during delivery, making the controlled release of a hydrophobic drug to the desired tumor site difficult [14]. Therefore, a drug nano-hybrid matrix containing both hydrophobic and hydrophilic sides and that has a structure similar to that of graphene would improve the stability and drug release during delivery [15]. In recent times, fluorescent polymer dots (PD) have been studied owing to their nanoparticle size, easy preparation, desirable biocompatibility, and sensitivity to environmental conditions such as pH, redox potential, light, and temperature after modifications [16][17][18]. PD have been reported to possess a lattice structure similar to that of GO and the ability to form core-shell structures under aqueous conditions due to rich sp 2 bonds in the core and hydrophilic segments in the shell, which can be easily loaded with hydrophobic drugs. As compared with conventional systems, PD has performed lower systemic toxicity with prolong drug circulation time that also improved drug solubility and targeting ability. These characteristics suggest that PD could act as stable drug carriers with real-time monitoring properties that would meet the criteria for an intelligent, stimuli-responsive drug carrier.
In this study, we designed a dual-sensitive drug delivery system based on a carbonized form of disulfide-crosslinked pluronic-grafted-2-(dimethylamino) ethyl methacrylate-2-hydroxyethyl methacrylate (B/S-Pluronic) to achieve pH-and redox-responsive fluorescence PD (B-PD). These were then conjugated with fluorescent dyes to form L-PD by utilizing the boron-diol interaction to turn the fluorescence "off", which hypothetically could recover under acidic pH. Next, the L-PD was loaded with paclitaxel (PTX; PTX loaded L-PD) through π-π stacking or hydrophobic interactions for simultaneous diagnosis and cancer therapy by controlled release of PTX after cleaving disulfide bonds by GSH. It is hypothesized that the designed DDS could show fluorescence quenching behavior at physiological pH and low drug release amounts without GSH. Oppositely, the fluorescence could be recovered by tumor acidic environment and PTX could be sustainably released from the matrices in a time manner to start therapeutic procedure responded quickly to reductive GSH concentration in the cancer cell to enhance the therapeutic efficacy of anticancer drugs. Therefore, the distinguishing characteristic of the proposed system may be the selectivity of a PTX loaded L-PD system in response to surrounding pH and GSH concentrations, thus showing its promising potential for an improved cancer diagnosis and therapy.

Synthesis and Characterization of PTX Loaded L-PD
The synthesis method and principle of simultaneous diagnosis and therapy of PTX loaded L-PD are shown in Scheme 1. The matrices were formed by grafting 2-(dimethylamino) ethyl methacrylate (DMA) and 2-hydroxyethyl methacrylate (HEMA) to Pluronic F-127 to obtain B/S-Pluronic (Scheme S1). To achieve structural stability and redox-responsive functional groups, disulfide bonds (-S-S-) were introduced by allowing the polymer matrices to react with cysteamine; the disulfide-crosslinked polymers spontaneously formed within 48 h. Once B-PD was achieved by carbonization using an oxidative reaction with concentrated sulfuric acid (H 2 SO 4 ), B-PD was conjugated with fluorescent dye (DYE) utilizing boron-diol chemistry to achieve fluorescence quenching and pH-sensitive nanoparticles (L-PD). Paclitaxel (PTX) was then loaded into the nano-hybrid matrices by optimizing the π-π stackings or hydrophobic interactions between the aromatic group of PTX and the sp 2 group of the L-PD to form PTX loaded L-PD [19]. In a tumor environment (acidic pH), it is expected that the disintegration of boronate ester bonds could recover the fluorescence intensity that would subsequently discharge DYE and facilitate further cleavage of disulfide bonds by high concentrations of GSH, releasing PTX to kill cancer cells [20]. The chemical structure of the PTX loaded L-PD nanoparticles was analyzed using FT-IR spectra. As shown in Figure S1, the presence of peak at 1020 cm −1 of B-PD, which is specific to B-O-H functional groups was observed initially, which then disappear after DYE was introduced, indicating successful conjugation of DYE to B-PD (L-PD) [21]. Moreover, UV-vis spectrophotometry was used to confirm the synthesis results of each nanoparticles. As displayed in Figure S2a, the B-PD displayed broad absorbance peaks between 200-300 nm, which represents a typical absorbance range for sp 2 carbon that could act as a fluorophore of the nano-hybrid system [22]. However, the absorbance peak shifted to longer wavelengths after the conjugation with DYE, demonstrating successful attachment of DYE to the B-PD (L-PD). Further confirmation of the conjugation of DYE-PD analysis was obtained using 1 H-NMR. As shown in Figure 1a, the conjugation of DYE to the B-PD was observed at 7-9 ppm, corresponding to the aromatic peaks of DYE, confirming attachment of DYE onto the PD shells [23]. Additionally, the surface charge properties during synthesis were investigated ( Figure S3). The B/S-Pluronic surface charge was 14.8 ± 1.3 mV, which decreased to 7.5 ± 0.9 mV after carbonization owing to the presence of carboxylic groups (B-PD). After DYE was introduced, the zeta potentials slightly shifted to 5.1 ± 0.9 mV due to the −SO 3 functional groups of DYE molecules (L-PD) and insignificantly dropped to 4.5 ± 0.8 mV after loaded with PTX. As we hypothesized that the disulfide linkages could be cleaved in response to high concentrations of GSH, XPS analysis before and after GSH treatment under different pH conditions was performed (Figure 1b and Figure S6). Without GSH at physiological pH, PTX loaded L-PD showed a strong disulfide (-S-S-) bond peak at 164 eV as compared to the thiol group (-S-H; 162 eV), as displayed in Figure 1b. In contrast, the disulfide bond peak significantly decreased whereas the thiol peak remarkably increased after GSH treatment, confirming the cleavage of -S-S-bonds by GSH [17,24]. Real-time monitoring based on the fluorescence OFF/ON nano-hybrid system has promising potential in simultaneous diagnosis and therapy [25,26]; hence, photoluminescence analysis was conducted. As presented in Figure 2a, the fluorescence intensity of the B-PD was high, described as fluorescence "ON", which then greatly decreased when conjugated with DYE (L-PD), described as fluorescence "OFF" (Figure S2b,c). This decrease was due to the formation of boronate ester bonds, causing the Förster resonance energy transfer (FRET) phenomenon [23,27,28]. Additionally, the fluorescence remained quenched when the PTX was introduced, indicating no fluorescence recovery when the hydrophobic drug was loaded ( Figure S2d). In the UV-vis spectra of the PTX loaded L-PD, we observed broad absorbance peaks in the 300-400 nm region for π-π bonding of PD with a PTX absorption peak at 230 nm, which represents loading PTX into the core of the PD ( Figure S2a) [29]. Moreover, the average size distribution of nanoparticles in the aqueous state at each step was observed using dynamic light scattering (DLS; Figure S4). The B/S-Pluronic nanoparticles size was 252.06 nm with a polydispersity index (PDI) value at 0.361 suggesting the monodispersity of the samples. After carbonization process, the size of B-PD in aqueous solution become 169.24 nm (PDI = 0.261), which elevated to 238.52 nm (PDI = 0.310) due to the conjugation between the DYE nanoparticles and the polymer dots (boron-ester). Furthermore, the successful loading of PTX was observed after the particle size increased to 245.66 nm (PDI = 0.365) [30]. To investigate the stability of the hydrophobic drug-loaded matrix in biological fluid before reaching the cancer cells, DLS and UV-vis spectra analyses were performed at different times. Figure 2b displays the hydrodynamic sizes and absorption intensities of the PTX loaded L-PD in different solutions over 4 days. No change in size is observed in media and BSA solutions, indicating the high stability of the nano-hybrid system in physiological conditions. TEM images of PTX loaded L-PD before treatment, shown in Figure 2c, exhibited a dot-like morphology with a diameter of around 220 nm and a lattice spacing similar to that of graphene (0.28-0.34 nm) [29]. However, the size decreased to around 45 nm when treated with 10 mM GSH at pH 6.8, which is consistent with the DLS results. The size of the PTX loaded L-PD decreased even more to 37.618 nm in the presence of 10 mM GSH at acidic pH, contributing to the further cleavage of disulfide bonds in the core and DYE disembarkment from the shell [23]. Moreover, the fluorescence decay of the PTX loaded L-PD was analyzed to understand the quenching phenomena caused by DYE nanoparticles. The fluorescence lifetime of the B-PD decreased when DYE was introduced to the nanoparticles (L-PD). Additionally, fluorescence recovery was observed after treatment with pH 6.8 and 10 mM GSH, further confirming the cleavage of boron-diol conjugation, as shown in Figure 2d [31].
As shown in Figure 3a, for PTX loaded L-PD at physiological pH without GSH, the fluorescent intensity of the PTX loaded L-PD did not show any significant increase due to the non-responsive behavior of the boron-diol conjugation at pH 7.4, even after GSH treatment, confirming the stability of the fluorescent quenching state of the nano-hybrid system. Next, the pH of the solution was varied from 6.0 to 7.4 to study the dot sizes and fluorescent intensity changes. The photoluminescence (PL) signal of the PTX loaded L-PD at λ = 360 nm displays specific enhancement in acidic pH depending on the reaction time [32]. In addition, changes in the PL profile under different pH conditions evaluated after 10 mM treatment shows that all the fluorescence intensities were higher, not just those subjected to acid treatment ( Figure 3b). Furthermore, the combination of both acidic pH and 10 mm GSH showed higher fluorescence recovery rather than single treatments. High GSH concentration in cancer cells confirm the possibility of using reduction-oxidation to change the disulfide bonds into thiol bonds, whereas acidic pH values in tumor cells induce hydrolysis of the L-PD boron-diol covalent bond [33]. The simultaneous GSH and acid treatments supported effective splitting of the PTX loaded L-PD nanoparticles to release PTX, resulting in much smaller particles and distinguish fluorescence response. In similar experimental groups and preparation techniques, the hydrodynamic diameter change was observed using DLS measurements over time, as shown in Figure 3c,d. These figures also confirm that high GSH concentrations and low pH values resulted in greater cleavage of the disulfide cross-linked sites to generate smaller particles, whereas no significant change was observed at low levels of GSH [17]. The same phenomenon was observed in the L-PD when treated with different pH values and GSH concentrations ( Figure S5).

Stimuli-Responsive Drug Release and Anticancer Efficacy Evaluation of PTX Loaded L-PD
The release of PTX in response to acidic and GSH levels in cancer cells were evaluated by UV-vis spectroscopy over time. The loading content (%LC) and encapsulation efficiency (%EE) of PTX were 7.708% and 83.52%, respectively. The successful loading of PTX in the nano-hybrid matrices was dependent on π-π stacking and hydrophobic interactions between PTX and the L-PD. However, the attachment of DYE to the B-PD was determined by the boron-diol conjugation of both types of nanoparticles. The PTX and DYE release profile were conducted at absorbance values 230 nm and 335 nm, respectively, as depicted in Figure 4a,b, respectively. As shown in Figure 4a, the amount of PTX released was less than 5% in the absence of GSH regardless whether pH was at the physiological or the acidic level, suggesting that there was no breakage of L-PD crosslinking and stable hydrophobic interactions. However, burst release was observed after treatment with GSH, which was related to the cleavage of disulfide bonds in the L-PD core. Specifically, 31.8% and 32.1% of PTX amount was released from the PTX loaded L-PD cargo during the first 60 min and 100% after 24 h in the presence of GSH. Disintegration of boron-diol covalent bond of B-PD and DYE was observed in the form of the release profile of DYE (Figure 4b). At pH 7.4 and GSH concentrations of either 0 mM or 10 mM, less than 5% of DYE was released, even after 24 h, suggesting no boron-diol cleavage on the shells of the L-PD. Additionally, we investigated DYE release under acidic pH conditions and GSH concentrations of 0 mM and 10 mM. The results showed a high percentage of DYE, corresponding to the pH effect on the nano-hybrid matrix that induced hydrolysis of the boron-diol covalent bond between DYE and the drug carrier. It should be noted that 23.7% (0 mM GSH) and 25.3% (10 mM GSH) of DYE released from the PTX loaded L-PD matrices in the first 60 min after pH treatment. To understand the efficient delivery, therapeutic performance, and biocompatibility, MTT assay was performed using Madin-Darby canine kidney (MDCK) and breast cancer cell lines (MDA-MB-231) cells. The L-PD nanoparticles demonstrated excellent biocompatibility across a wide range of concentrations from 0.01 to 1.5 mg/mL, as displayed in Figure 4c. Moreover, the viability of the cells remained high for the MDCK cells, even with the presence of PTX, indicating no release or leakage during incubation. However, the number of dead cells remarkably increased for the MDA-MB-231 cells, which had a cell survival of only 37%, which further supported the anticancer effect of the released PTX after cleaving of boron-diol covalent bonds by pH and disulfide bonds by GSH. These phenomena suggest the selectivity of PTX loaded L-PD to the pH environment and GSH concentrations that effectively promoted the therapeutic performance of the nano-hybrid system.

Fluorescence OFF/ON Behavior Based on Confocal Imaging
Further investigation to confirm the fluorescence OFF/ON behavior to distinguish normal and cancer conditions was performed using confocal images. Figure 5a

Apoptosis/Necrosis Analysis and Live/Dead Assay
Further study of PTX loaded L-PD cytotoxicity was proven to be necessary, and therefore flow cytometry was conducted to investigate cellular apoptosis/necrosis using annexin V and propidium iodide (PI). The MDCK cells demonstrated excellent survivability when treated with PTX loaded L-PD as no cells reached late apoptosis and necrosis (Figure 6a). However, a higher percentage of apoptosis cells were counted when the MDA-MB-231 cells were treated with PTX loaded L-PD, corresponding to the release of the drug from the nano-hybrid matrices and the therapeutic effect of PTX. To monitor the cytotoxicity of the PTX loaded L-PD, an in vitro live and dead assay was performed using calcein AM and PI staining. After 3 h incubation with the nanoparticles, confocal images of the MDCK cells (Figure 6b) revealed that most of the cells survived (green), which was in contrast to the MDA-MB-231 cells, which were killed (red), indicating the high cytotoxicity of the PTX loaded L-PD.

Synthesis of Carbonized
To synthesize carbonized disulfide crosslinked B/S-Pluronic, 2 g of the obtained powder (B/S-Pluronic) was dissolved in 1% DMSO in 10 mL of deionized water (DDW) and stirred at 25 • C in air. After 48 h, 10 mL of concentrated sulfuric acid was added and reacted for 1 min, followed by the addition of 185 mL of DDW. The product was then dialyzed (MWCO: 3500 Da) for 1 day, freeze-dried, and was labeled as B-PD. (82% Yield).

Synthesis of the Diol-Conjugated Fluorescent Probe-B-PD (L-PD)
The carbonized disulfide-crosslinked B/S-Pluronic (100 mg) and DYE (0.001 mg/mL) were dissolved in 10 mL of Tris-buffered saline (TBS) at pH 12.0. The mixture was stirred for 24 h at room temperature. The solution was dialyzed (MWCO 1 kDa) against water overnight, freeze-dried, and labeled as L-PD (67% Yield).

Drug Loading into L-PD (PTX Loaded L-PD) and Release Profiles
First, 100 mg of the L-PD in 20 mL of PBS (pH 7.4) and 5 mg of PTX in ethanol were stirred together overnight at 24-25 • C. The sample was then purified by dialysis (MWCO 1 kDa) for 24 h and freeze-dried to achieve PTX loaded L-PD (76% yield). The PTX loading content (%LC) and PTX encapsulation efficiency (%EE) were measured with a UV-vis spectrophotometer at 225 nm and calculated using following equations [24]: %LC = weight of drug into the carrier weight of nanoparticles × 100.
(1) %EE = weight of PTX loaded in nanoparticles weight of PTX in Feed × 100.
The drug release profile was performed at different concentrations of GSH (0, 3, 6, and 10 mM) inside the chamber while soaked in approximately 30 mL of release solutions at pH values of 6.8 and 7.4. The sample was positioned inside a shaking incubator at 37 • C and evaluated with UV-vis spectrophotometry to quantify the release of PTX.

Cytotoxicity Assay
To evaluate the cytotoxicity of the PTX loaded L-PD, MDCK and MDA-MB-231 cells at densities of 1 × 10 5 cells/mL were cultured in a 37 • C incubator (humidified 5% CO 2 atmosphere) for 24 h. A stock solution containing the PTX loaded L-PD in RPMI-1640 medium (1.5 mg/mL) was prepared and diluted to 0.01 mg/mL serially. The cell media was replaced with stock solution and incubated overnight. The MTT solution was added after the removal of the stock solution. The cell viability was evaluated by using a microplate reader with an absorbance wavelength of 570 nm [35].

Flow Cytometry
The flow cytometry was conducted by culturing MDCK and MDA-MB-231 cells in six-well plates at densities of 1 × 10 6 cells/mL for 24 h at 37 • C (5% CO 2 ). The medium was then changed with a stock solution (dissolved PTX loaded L-PD in media) followed by incubation at the designated time. The samples were then collected and dissolved with 1% FBS in PBS (pH 7.4) and evaluated using filter at excitation wavelength of 405 nm and an emission wavelength of 440/50 nm.
For apoptosis assays, the cells were directly evaluated after stained with annexin V and PI using an excitation wavelength of 488 nm and emission wavelengths of 530/30 nm and 574/26 nm, respectively.

Confocal Imaging
The time-dependent internalization of the PTX loaded L-PD was investigated using confocal laser-scanning microscope (magnification 20×). First, the cells were cultured then treated with sample (1 mg/mL) in medium (different incubation time). The medium was then removed, washed with PBS (pH 7.4), stained with LysoTracker Green (acidic compartment) for 30 min and SYTO 61 (nucleic acid) for 15 min, and observed using a confocal microscope.
For live/dead cell analysis, cultured MDCK and MDA-MB-231 cells were incubated with PTX loaded L-PD (0.2 mg/mL) for 3 h. Then, the prepared cells were stained with calcein AM and PI and placed under confocal laser-scanning microscope (magnification, 10×) to evaluate the stained cells (live or dead).

Conclusions
In conclusion, a dual-sensitive fluorescent polymer dots (PD) as a paclitaxel (PTX) drug carrier was successfully developed based on a disulfide bond as core-crosslinked and boron-ester as shell-cleavable parts. The PTX loaded L-PD nano-hybrid system core part utilized disulfide bond as a redox-responsive site and for stabilizing the inner structure, which avoid the leaks of PTX drugs during treatment. Meanwhile, the attachment of DYE on the shell part of PD was beneficial for a pH-sensitive bioimaging probe due to the fluorescence quenching at the alkali condition, which recovered at acidic pH owing to boron-ester hydrolysis. The PTX loaded L-PD demonstrated low fluorescence intensity at physiological pH, which recovered after the pH become acidic. Additionally, the release of PTX, which was expected to show distinguish result during different GSH concentrations, was proven to be the same as hypothesized with burst released detected at high concentration of GSH. Moreover, time dependent MDCK treated PTX loaded L-PD confocal images displayed no fluorescent signal while MDA-MB-231 started to showed a fluorescence signal at 30 min treatment. Hence, this nanosystem represents an improved system for differentiating between normal and tumor sites depending on the surrounding pH and GSH concentrations, which could enable controlled release of drugs with bioimaging abilities, showing its high potential for further applications in cancer diagnosis and treatment.