Correction to: BMC Infect Dishttps://doi.org/10.1186/s12879-017-2622-3After publication of the original article , the autThus, the authors have conducted trial sequential analysis with new version of the software for the two continuous end-points. All corrections are following:1. “v0.9.5.5” in the Trial sequential analysis (methods section) is corrected to “v0.9.5.10”.2. The estimated required information size of Length of hospitalization  is corrected to “7242”.3. Fig. The conclusions are as same as published in the original article. Referenc


Alterations in the corticostriatal system have been implicated in numerous substance use disorders, including alcohol use disorder (AUD). Adaptations in this neural system are associated with enhanced drug-seeking behaviors following exposure to cues predicting drug availability. Therefore, understanding how potential treatments alter neural activity in this system could lead to more refined and effective approaches for AUD. Local field potentials (LFPs) were acquired simultaneously in the prefrontal cortex (PFC) and nucleus accumbens (NA) of both alcohol preferring (P) and Wistar rats engaged in a Pavlovian conditioning paradigm wherein a light cue signaled the availability of ethanol (EtOH). On test days, the catechol-o-methyl-transferase (COMT) inhibitor tolcapone was administered prior to conditioning. Stimulus-evoked voltage changes were observed following the presentation of the EtOH cue in both strains and were most pronounced in the PFC of P rats. Phase analyses of LFPs in the θ band (5–11 Hz) revealed that PFC-NA synchrony was reduced in P rats relative to Wistars but was robustly increased during drinking. Presentation of the cue resulted in a larger phase reset in the PFC of P rats but not Wistars, an effect that was attenuated by tolcapone. Additionally, tolcapone reduced cued EtOH intake in P rat but not Wistars. These results suggest a link between corticostriatal synchrony and genetic risk for excessive drinking. Moreover, inhibition of COMT within these systems may result in reduced attribution of salience to reward paired stimuli via modulation of stimulus-evoked changes to cortical oscillations in genetically susceptible populations. Alcoholism is highly heritable and genetic vulnerability is associated with increased likelihood of alcohol use-related problems. Presentation of environmental stimuli paired with alcohol are capable of inducing craving and relapse in alcohol-dependent individuals. The work described here using a rodent model of cued ethanol (EtOH) availability suggests that altered corticostriatal activity may be associated with genetic vulnerability to abuse alcohol. Additionally, our data suggest that inhibition of the catechol-o-methyl-transferase (COMT) enzyme activity may reduce the influence that alcohol-conditioned stimuli (CSs) elicit on behavior, resulting in a reduction of cued alcohol seeking in genetically susceptible individuals.Environmental stimuli associated with drugs of abuse acquire motivational properties  capable Theta oscillations  have been shown to facilitate plasticity , as wellAlcohol preferring (P) rats are a validated preclinical model of AUD . P rats The catechol-o-methyl-transferase (COMT) enzyme metabolizes catecholamine’s including DA in cortical regions . Using tN = 10, 37 datasets; Indiana University) and Wistar  rats. All animals arrived from both respective outside vendors within the same week to ensure habituation and rearing conditions were identical for both strains. Animals were single housed, weighed ∼250–300 g at the start of experiment, maintained on a reverse light dark cycle and supplied with food and water ad libitum. All animals initially received four weeks of intermittent two bottle access for 20% EtOH to pre-expose animals to EtOH  or vehicle  as described previously , 2018.x-, y-coordinates in separate voltage traces. These voltage traces were then used to compute the velocity of the animal’s movement  fed through silica tubing and then secured to custom-made head caps via epoxy. Wires were externally referenced to a stainless-steel skull screw over the cerebellum. Probes were implanted over the mPFC  and NA x¯t=HxThen the analytic signal is projected on the unit circle (3)zt=ζtζThe following widely used measure of the strength of phase locking between these two signals was calculated:  (4)γ=1N∑strength . To explTo investigate the impact of CS presentation on the phase of the signals, we analyzed the phase delay. Phase delay was calculated by first computing the averaged time difference between peaks of LFP phases for 1 s before CS on for each brain region . Then, the time difference between the first peak right after the CS and the last peak right before the CS was computed. Phase delay was derived by computing the difference between the first value and second value. Positive values indicate that the CS causes a delay of peak while negative values suggest that the CS causes the advance of the peak.To measure the CS-evoked precision of phases across trials in the θ band, ITPC was calculated. Drinking trials were analyzed for 200 ms before, and 1 s after the CS. ITPC was calculated per After calculating ITPC for each trial, averages across trials were taken in the θ band (5–11 Hz) for each dataset. Statistics were performed on the dataset-averaged time series.https://www.r-project.org/). Unless specified otherwise, all comparisons were first subjected to ANOVA testing or mixed-design ANOVA (a mixture of between subject factor and within subject factors) followed by post hoc tests for all multiple comparison procedures.All analyses were performed in MATLAB (MathWorks) and R  = 4.56, p = 5.70 × 10−5; t test, t(36) = 9.71 × 10−1, p > 0.05; F = 15.5, p = 8.21 × 10−5) and non-drinking trials  = 1972, p = 9.21 × 10−6), trial × strain interaction  = 2.90 × 101, p = 7.46 × 10−8; Consistent with our previous findings , behavioF = 1.39, p = 1.05 × 10−5; F = 2.78 × 102, p < 1.00 × 10−16; main effect of time F = 3.15, p < 1.00 × 10−16; On trials that animals drank, a robust change in voltage of the mPFC and NA was observed following the presentation of the CS in both strains of animals . The chaF = 6.75, p = 4.6 × 10−6; P rat, trial × time interaction, F = 6.82, p = 4.1 × 10−6). The increase in mPFC-NA synchrony was more robust in P rats  = 1.39 × 101, p = 1.26 × 10−3), indicating that, overall, synchrony differed for trials in which animals chose to drink versus trials where animals abstained.Phase synchrony during the US but not before the CS was observed A–C. To dF = 2.42, p = 2.97 × 10−3; F = 4.00, p > 0.05) or epoch  = 2.63 × 10−1, p > 0.05) were observed. Additionally, no two-way or three-way interactions were detected (ps > 0.05). Collectively, these data indicate that, on non-drinking trials, the observed synchrony is influenced by trial number but not strain or epoch.Synchrony was next analyzed for drinking and non-drinking trials separately. A mixed-design ANOVA on non-drinking trials revealed a significant main effect of trial number  = 4.64, p = 3.97 × 10−2), epoch  = 1.50 × 102, p = 5.45 × 10−6), and trial number  = 4.61, p = 8.72 × 10−8) were observed. Post hoc tests indicated that phase synchrony for Wistars was significantly higher overall compared to P rats  and immediately after the sipper was removed from the chamber . Additionally there were significant strain × epoch  = 1.10 × 101, p = 8.69 × 10−5), and strain × trial  = 2.89, p = 3.52 × 10−4) interactions, indicating the pattern of phase synchrony across trials and epochs was different across rat strain. Therefore, drinking trials were analyzed separately for Wistar and P rats to determine differences in synchrony across each epoch and trial.On drinking trials, significant main effects of strain  = 5.18, p = 3.48 × 10−8), but neither a main effect of epoch  = 1.76 × 10−1, p > 0.05) nor an epoch × trial interaction  = 1.33, p > 0.05) were observed  = 1.40, p > 0.05). However, synchrony differed by epoch  = 2.62 × 101, p = 1.82 × 10−7; Post hoc tests indicated that synchrony during drinking (US) was significantly higher than synchrony before CS presentation  and after drinking . There was no difference in synchrony before CS on and after drinking . In summary, consistent with wavelet coherence analyses, increases in θ band synchrony were observed when alcohol was available during drinking trials  = 4.37, p = 4.54 × 10−2) but not brain region  = 3.48, p > 0.05) or trial number  = 5.97 × 10−1, p > 0.05). Both two-way and three-way interactions were not significant (ps > 0.05). Next, the difference of the phase delays was assessed for both strains in each brain region separately. In the mPFC, the phase delay of P rats was significantly longer compared to Wistars . During non-drinking trials, a mixed-design ANOVA was employed to investigate the change in the phase of the signals due to CS presentation. There was no main effect of strain  = 1.36 × 10−1, p > 0.05), brain region  = 1.51 × 10−1, p > 0.05) or trial number  = 1.39, p > 0.05; ps > 0.05). Collectively these analyses indicate that the phase resets are most robust in P rats in the mPFC on drinking trials.To determine how the presentation of the CS influenced oscillatory dynamics, changes in θ phase over time in the mPFC and NA were assessed . To deteF = 3.02, p = 5.50 × 10−6) and P rats  = 3.73, p = 0.1 × 10−6). CS-evoked changes in ITPC were assessed by comparing ITPC values 200 ms before stimulus onset to values 1 s after CS presentation  = 1.47 × 101, p = 9.01 × 10−4) but no effect of strain  = 7.23 × 10−1, p > 0.05). There was also a significant strain × treatment interaction  = 4.39, p = 4.79 × 10−2). Post hoc test indicated that tolcapone decreased intake of EtOH . In particular, tolcapone decreased intake in P rats  but not Wistars  = 6.19, p = 2.28 × 10−2) was observed, but no main effect of strain  = 1.14, p > 0.05) or strain × treatment interaction  = 8.39 × 10−1, p > 0.05) were observed. Post hoc testing indicated that treatment with tolcapone resulted in fewer drinking trials compared to treatment with saline . To investigate the effects of tolcapone on mPFC-NA synchrony (γ), we performed a four-way ANOVA with treatment , strain, epoch, and drinking status  as factors. There were significant main effects of treatment  = 3.16 × 101, p = 2.27 × 10−8), strain  = 1.72 × 102, p < 1.0 × 10−16), and drinking status  = 5.24, p = 2.21 × 10−2), but not epoch  = 1.47, p > 0.05). There was also a significant treatment × strain interaction  = 2.83 × 101, p = 1.17 × 10−7), while all other two-way, three-way, or four-way interactions were not significant (ps > 0.05). Post hoc tests indicated that administration of tolcapone resulted in reduced mPFC-NA synchrony  but not P rats . Consistent with non-treatment sessions, overall mPFC-NA synchrony in Wistars was higher compared to P rats . Moreover, mPFC-NA synchrony during drinking trials was significantly higher than during non-drinking trials .In the following analyses, data are collapsed across trial number due to the few drinking trials observed following tolcapone treatment in P rats. There was no effect of tolcapone on CS-evoked changes in voltage  in eitheF = 7.31, p = 6.96 × 10−3). Post hoc testing indicated that the phase delays of P rats were significantly shorter compared to Wistars  = 3.07 × 10−1, p > 0.05), drinking  = 1.37, p > 0.05) or brain region  = 2.30 × 10−1, p > 0.05). However, there was a significant treatment × strain interaction  = 4.24, p = 3.97 × 10−2). Post hoc testing indicated that tolcapone reduced the phase delay in the mPFC of Prats during drinking trials . Similar to non-treatment sessions, a main effect of strain was observed  = 1.08 × 101, p = 1.08 × 10−3) with post hoc test indicating a shorter phase delay in P rats, relative to Wistars .Phase-response curves were computed to assess CS-induced phase resets following treatment and were collapsed across trials. A four-way ANOVA  was performed. There was a significant main effect of strain  = 1.09, p = 0.37), whereas a main effect of time was observed following tolcapone administration  = 3.00, p = 1.04 × 10−4; F = 2.95, p = 7.41 × 10−5), but not tolcapone  = 1.30, p = 0.178).To further determine how changes CS-evoked synchrony were influenced by treatment, ITPC was assessed following saline and tolcapone injections on drinking trials. In Wistars, no main effect of time was observed following saline administration (repeated-measures ANOVA, These experiments investigated the neural mechanisms by which drug paired stimuli elicit reward seeking in a rodent model of addiction vulnerability. Relative to Wistars, P rats exhibited an increase in mPFC-NA synchrony when drinking and a greater CS-induced phase delay in mPFC. Following tolcapone administration, a reduction in EtOH intake in P but not Wistar rats was observed. While no effect of tolcapone on synchrony in P rats was observed, a tolcapone-mediated suppression of the mPFC phase delay was observed in P rats but not Wistars during drinking trials only. These data suggest a novel neural mechanism whereby tolcapone suppresses drinking in genetically susceptible populations. These data also further elucidate the modulatory role of catecholamines in the corticostriatal system on alcohol seeking behaviors.Theta oscillations in the mPFC are hypothesized to play a critical role in encoding reward value. MPFC θ phase locking was shown to be present during both reinforced and non-reinforced licks in a high-value context while phase locking was notably weaker during both reinforced and non-reinforced licks in a low-value context, suggesting that the strength of mPFC θ activity corresponds to reward value . StrongeIn the present experiment, P rats exhibited lower mPFC-NA θ synchrony than Wistars overall D,E. Low Alcohol is hypothesized to disrupt the structure of oscillations by making them asynchronous . AlcoholA wealth of literature supports the hypothesis that PFC-NA connections are important for reward seeking, especially in the presence of CSs . In the A robust θ phase reset was observed in mPFC following cue presentation B2. PhaseAlcohol associated cues elicit increases in the frontal BOLD response  and the Strain differences in tolcapone’s ability to reduce the phase reset may be associated with differences in basal DA tone and subsequent differences in cue-evoked DA transmission. Alcohol-paired cues elicit increases in DA concentrations in both clinical  and precThe results obtained here highlight the heterogeneity of factors which may contribute to excessive drinking phenotypes. Whereas alterations in corticostriatal synchrony may be associated with excessive drinking, catecholamine activity in the PFC appears to more strongly influence attribution of motivational salience to drug paired stimuli in addiction vulnerable organisms. These results highlight the role that catecholamines play on shaping how motivating stimuli are processed by PFC. Furthermore, these results suggest that targeting the catecholamine system in PFC may provide an effective way to blunt the incentive motivational properties of drug-associated stimuli. In this way, novel treatments that target this system might be effective in preventing craving and relapse.


In SCA1, excessive excretion of S100β by reactive astrocytes and its uptake by Purkinje cells has been demonstrated previously. Under pathological conditions, excessive extracellular concentration of S100β stimulates the production of proinflammatory cytokines and induces apoptosis. We modeled astrogliosis by S100β injections into cerebellar cortex in mice. Injections of S100β led to significant changes in Bergmann glia (BG) cortical organization and affected their processes. S100β also changed morphology of the Purkinje cells (PCs), causing a significant reduction in the dendritic length. Moreover, the short-term synaptic plasticity and depolarization-induced suppression of synaptic transmission were disrupted after S100β injections. We speculate that these effects are the result of Ca2+-chelating properties of S100β protein. In summary, exogenous S100β induced astrogliosis in cerebellum could lead to neuronal dysfunction, which resembles a natural neurodegenerative process. We suggest that astrocytes play an essential role in SCA1 pathology, and that astrocytic S100β is an important contributor to this process.Astrogliosis is a pathological process that affects the density, morphology, and function of astrocytes. It is a common feature of brain trauma, autoimmune diseases, and neurodegeneration including spinocerebellar ataxia type 1 (SCA1), a poorly understood neurodegenerative disease. S100β is a Ca No. 2532) was obtained from Tocris. Concentrated stock solutions of FC and TBOA were initially prepared and diluted in artificial spinocerebellar fluid (ACSF) to their final concentrations before use.Twelve-week-old (P90-P100) WT CD1 mice were anaesthetized by intraperitoneal injection of chloral hydrate solution (400 mg/kg of body weight). Then, 2.5 μL of 50 μM S100β in phosphate buffered saline (PBS) or PBS was stereotaxically injected into the cortex of cerebellar vermis (lobule VI) using a 10 μL Hammilton syringe. To reach the injection point in the vermis, we used the coordinates relative to bregma: AP: −2.5 mm, ML: 0 mm, DV: 2 mm. Mice were used 24 h after the injection.3, 2.5 mM KCl, 1.25 mM NaH2PO4, 11 mM glucose, 10 mM MgSO4, and 0.5 mM CaCl2 0.5; pH 7.4, continuously oxygenated with 95% O2 and 5% CO2. Parasagittal slices of cerebellar vermis were made using a microslicer . The slices were maintained in an extracellular solution containing the following: 125 mM NaCl, 2.5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 1.25 mM NaH2PO4, 26 mM NaHCO3, 10 mM D-glucose, and 0.05–0.1 mM picrotoxin bubbled by 95% O2/5% CO2 gas mix at room temperature for 1h before starting the electrophysiological experiments. For current clamp whole-cell recordings from Purkinje cells (PCs), we used K-gluconate-based intracellular solution containing the following: 130 mM K-gluconate, 4 mM KCl, 20 mM HEPES, 1 mM MgCl2, 4 mM MgATP, 1 mM NaGTP, and 0.4 mM EGTA (pH 7.3 adjusted with KOH). For voltage clamp whole-cell recordings from Purkinje cells (PCs), we used intracellular solution containing the following: 140 mM Cs-gluconate, 8 mM KCl, 10 mM HEPES, 1 mM MgCl2, 2 mM MgATP, 0.4 mM NaGTP, and 0.4 mM EGTA (pH 7.3 adjusted with CsOH). Passive electrical properties of the PCs were estimated using averaged traces of ~10 current responses to hyperpolarising voltage pulses . Fast capacitance component was automatically compensated; the signal was sampled at 50 kHz and low-pass filtered at 10 kHz. No correction was made for liquid junction potentials. Analysis of electrophysiological data was performed using pClamp10 (Molecular Devices), Pachmaster software (HEKA), and Clampfit 10.5 (Axon instruments).Cerebellar slices (250 μm thick) were prepared, and whole-cell recordings were conducted as described previously . BrieflyPCs were voltage-clamped at −70 mV to record excitatory postsynaptic currents after irritation of parallel fibers (PF EPSCs). Selective stimulation of PFs was confirmed by paired-pulse facilitation of EPSC amplitudes .2+ channels (VGCC) and releases endocannabinoids, which presynaptically decrease glutamate release and suppress amplitude of PF EPSC [To examine depolarization induced suppression of excitation (DSE), PF EPSCs were recorded every 3 s. After monitoring basal PF EPSCs for 1 min, a single depolarizing pulse (5s from −70 to 0 mV) was applied to the recorded PC. This opens the voltage gated Ca PF EPSC . Amplituv/v) normal donkey serum, 0.1% (v/v) Triton X-100, and 0.05% NaN3.For immunohistochemistry (IHC), anesthetized mice were perfused transcardially with 4% paraformaldehyde in 0.1 M phosphate buffer. The brain was postfixed in the same fixative overnight. The cerebellar vermis was cut into 50 µm sagittal sections. The sections were treated with rabbit monoclonal anti-calbindin D-28 k , chicken polyclonal anti-GFAP antibodies , and rabbit polyclonal anti-S100β . Secondary antibodies were Alexa Fluor 514-conjugated donkey anti-rabbit IgG , Alexa Fluor 647-conjugated donkey anti-chicken IgG , and Alexa Fluor 488-conjugated donkey anti-rabbit IgG . Antibodies were dissolved in PBS solution containing 2% . Images were recorded as Z-stacks of 0.25 μm thickness with a ×10 lense, numerical aperture of 1.0, zoom ×6, and 1024 × 1024 resolution. In all groups, the cerebellar lobes 6 and 7 of the vermis cerebellum were used for comparison . For douThe thickness and number of BG processes were measured on confocal images of sagittal cerebellar slices. The number of radial glial processes for 100 µm of the molecular layer  was counar layer , visualiQuantitative morphological analysis was performed in the three-dimensional (3D) mode. Using a confocal laser scanning microscope , anti-GFAP-labeled BG was scanned in Z-stacks . For Scholl analysis, Z-stacks mages of soma and processes of BG were traced on focal planes using ImageJ software. We used the Sholl method of concentric circles using an ImageJ regime  [p < 0.05.Pooled data are expressed as the mean ± standard error of mean (SEM). Statistical analyses of differences between the groups were performed using the unpaired t-test and Mann–Whitney U test. The influence of FC and TBOA on the EPSC recovery was estimated with a one-way analysis of varaiance (ANOVA) test. Differences were considered significant at We estimated the dendritic and somatic capacitance by optimization of the two-term exponential series to the current response curve to the voltage step to find the time constants ep=10 mV :DSE was analyzed using dual exponential waveform Equation (1).τ1” and “τ2”—the half-times for the EPSC to reach the minimum and to recover to the initial 100%, respectively. The parametric bootstrap technique was used to obtain the 95% confidence intervals for the parameters A, τ1, and τ2. This analysis was performed using the Python 3 package.This curve is convenient for the prediction of the conduction changes in synapses . It contp = 1.25 × 10−13, unpaired t-test  in comparison with PBS-injected areas . On images of digitally traced BG processes A, we anaThese data show that excessive extracellular S100β protein in the cerebellar cortex leads to significant changes in BG morphology.n = 12 areas from 3 mice) compared with 150.7 ± 6.3 µm, n = 14 areas from 3 mice in PBS-injected mice  and 513.5 ± 27.1 pF (n = 52 cells from 10 mice) in the PBS-injected group. . A slow component predominantly reflects the size of neuronal dendrites. We found a significant difference between the two groups. The capacitances of PCs’ dendrites in S100β injected mice were 359.4 ± 37.5 pF  ratio in PF–PC synapses of S100β and PBS-injected mice. The PPF ratio in S100β injected mice was 1.8 ± 0.4, n = 35 cells from 8 mice versus 1.9 ± 0.1, n = 39 cells from 9 mice in PBS-injected mice , compared with 2.3 ± 0.1 ms (n = 36 cells from 9 mice) in PBS-injected mice , while it increased to 21.6 ± 1.5 ms (n = 37 cells from 9 mice) in PBS-injected mice , compared with WT mice  and an increase in the intracellular Ca2+. Ca2+ triggers endocannabinoid release from the postsynaptic cell with consecutive activation of CB1 receptors on the presynaptic terminal, leading to a reduction in release of glutamate. We examined dynamic of PF-EPSC amplitude after 5 s of depolarization from −70 to 0 mV. The stimulus intensity was adjusted to reach an EPSC amplitude of approximately 150 pA before DSE induction. In control mice, DSE protocol reduced EPSC by 67.3% ± 3.5% (n = 11 cells from 4 mice), which was similar to that in mice pre-injected with S100β  is significantly smaller in S100β-injected mice with 29.7 s  in comparison with 64.5 s  in the PBS group, p < 0.05.The double waveform model fit (1) confirmed the slowing of the recovery kinetics by S100β. The amplitude reduction in the DSE protocol was not significantly different in S100β-injected mice compared with the PBS group: 45.5%  versus 38.1% , respectively. Using the fitting protocol, we calculate that the half-time for the recovery of the parameter  and in PBS-injected mice, to 64.4% ± 9.9% of the control  . The rise time of PF-EPSC in PBS- and S100β-injected mice increased after FC treatment. In the PBS group, it increased from 2.3 ± 0.2 ms to 2.7 ± 0.2 ms  A,B. Ten q/11-proteins and trigger PLC activation [n = 9 cells from 4 mice), which was not statistically different from that registered without FC in S100β-injected mice . However, this comparison is compromised by the direct impact of FC on PF-induced EPSCs and should be interpreted with care.It was shown previously that astrocytes also contain CB1 receptors and could modulate the synaptic plasticity . CB1 rectivation  and reletivation . This phtivation ,40. Howeted mice E  and 46.23%  for PBS and S100β, respectively.p = 0.025, unpaired t-test; p = 0.181, unpaired t-test , while after TBOA, they reached approximately the same values . Application of nonselective EAAT blocker DL-TBOA 500 μM did not significantly change PF-EPSC amplitudes and rise time in the cerebellum of both PBS- and S100β-injected mice (data not shown). TBOA did not affect the PPF ratio of PF-EPSC in PBS-injected mice , while in S100β-injected mice, it resulted in a slight, but significant PPF ratio reduction from 1.9 ± 0.2 to 1.76 ± 0.1 , which was not statistically different from that before application . Furthermore, the recovery of the PCs amplitude was not affected by TBOA . Also, the recovery of the PCs amplitude was unaffected by TBOA. The double waveform model yielded in the PF-EPSC amplitudes reduction during DSE as 45.71%  and 32.11%  for PBS and S100β cells, respectively. The restoration half-time We examined endocannabinoid short-term plasticity in PF–PC synapses after treatment of cerebellar slices with 500 μM TBOA. In PBS-injected mice, DSE protocol was not affected by TBOA. EPSC was reduced by 59.2% ± 5.4%;  by TBOA C,D, nor  the DSE C,D.These results demonstrate that the slowdown of glutamate uptake predictably affects PF-EPSC decay time and does not change DSE .It was shown previously that activated astrocytes secrete large amounts of S100β ,18. PCs The thickness of the molecular layer correlates with the length of the PC dendritic tree . To exam2+ chelation, which affects neuronal sodium channels [2+ chelation by S100β in intercellular space leads to Na+ current enhancement and makes them fire action potentials in bursts, rather than single AP. It is very likely that this opens NMDA receptors and brings more Ca2+ inside the neurons [2+ overload of neurons, potentially contributing to neurotoxicity in our model.It is interesting that S100β may modulate sodium channels in neurons via Cachannels , which m neurons . Such a The dendritic tree of each PC has thousands of synaptic connections with parallel fibers and 1–2 synapses with climbing fibers . Hence, At the same time, astrogliosis and neurodegeneration is characterized by suppression of EAAT1 and EAAT2 function and accumulation of extracellular glutamate, leading to excitotoxicity . It high2+-dependent release of endocannabinoids from PCs, which retrogradely activate the CB1 receptors on the terminals of PFs [The effect of S100β on short-term synaptic plasticity was assessed using DSE. In DSE, strong depolarization leads to Cas of PFs . Activats of PFs , but obvOur modelling also confirmed that while DSE protocol depolarization was sufficient to decrease PF-EPSC amplitude up to 67%–69% of control in S100β- and PBS-injected mice A, there In summary, elevated extracellular S100β leads to reorganization of glia/neuron morphology and disturbs synaptic transmission. Our findings are reminiscent of the early stage of a neurodegenerative process in cerebellar cortex, such as that seen from three-old-week SCA1 mice . The cha


Vibrio tritonius strain AM2 can perform efficient hydrogen production with a molar yield of 1.7 mol H2/mol mannitol, which corresponds to 85% theoretical molar yield of H2 production, under saline conditions. With a view to maximizing the hydrogen production using marine biomass, it is important to accumulate knowledge on the effects of salts on the hydrogen production kinetics. Here, we show the kinetics in batch hydrogen production of V. tritonius strain AM2 to investigate the response to various NaCl concentrations. The modified Han–Levenspiel model reveals that salt inhibition in hydrogen production using V. tritonius starts precisely at the point where 10.2 g/L of NaCl is added, and is critically inhibited at 46 g/L. NaCl concentration greatly affects the substrate consumption which in turn affects both growth and hydrogen production. The NaCl-dependent behavior of fermentative hydrogen production of V. tritonius compared to that of Escherichia coli JCM 1649 reveals the marine-adapted fermentative hydrogen production system in V. tritonius. V. tritonius AM2 is capable of producing hydrogen from seaweed carbohydrate under a wide range of NaCl concentrations (5 to 46 g/L). The optimal salt concentration producing the highest levels of hydrogen, optimal substrate consumption and highest molar hydrogen yield is at 10 g/L NaCl (1.0% (w/v)).Biohydrogen is one of the most suitable clean energy sources for sustaining a fossil fuel independent society. The use of both land and ocean bioresources as feedstocks show great potential in maximizing biohydrogen production, but sodium ion is one of the main obstacles in efficient bacterial biohydrogen production.  Surveys of the complete genome of V. tritonius strain AM2 shows a single 24-kb gene cluster containing 21 genes that is responsible for the formation of a formate hydrogen lyase (FHL) complex on the large chromosome. This vibrio FHL complex is structurally similar to the hyf gene cluster (hyfABCDEFGHIJ genes) found in E. coli K-12 with the amino acid identity of each homolog ranging from 45 to 68% , fhlA-type transcriptional activator, and hydrogenase maturation proteins (hyp) which together form a “super-gene-set” of the FHL-Hyp gene cluster. The V. tritonius hydrogenase is also classified as a [NiFe]-hydrogenase because of typical motifs coordinating the [NiFe] center at the active site of its hyfG, the large subunit of FHL complex  NaCl. Optimum growth of the bacterium was observed in 3.0% NaCl (w/v)  . Thus, tchnology . The ideeedstock . Previounditions . These rove salt .V. tritonius concentrated on the effects of different pH levels, temperatures and substrates on higher hydrogen yields  was maintained on a marine agar slant at 15 °C and sub-cultured 3 times a month. The cells were then pre-cultured aerobically in marine broth to reach a cell density of 0.8 to 1.0 ± 0.1 OD620 measured by a microplate reader , which corresponds to 1.4 to 1.7 ± 0.1 OD620 using a UV/Visible spectrophotometer , with shaking at 130 rpm  at 30 °C. The pre-culture medium was a marine broth composed of 0.5% (w/v) polypeptone, 0.1% (w/v) yeast extract, and 75% (v/v) artificial seawater (ASW) composed of 0.7 g/L KCl, 5.3 g/L MgSO4 ⋅ 7H2O, 10.8 g/L MgCl2 ⋅ 6H2O, 1.3 g/L CaSO4 ⋅ 2H2O, and 30 g/L NaCl at pH 7.5.Pure culture of Escherichia coli strain JCM 1649 was used as a reference. Similar to the experiment using V. tritonius AM2, the pure culture E. coli was stored on LB agar slant (1% (w/v) tryptone, 0.5% (w/v) yeast extract, 0.5% (w/v) NaCl, 1.5% (w/v) agar) at 15 °C and sub-cultured three times a month. Pre-culture was prepared using LB broth following a similar method to that used for V. tritonius.In a separate experiment, V. tritonius strain AM2 inoculated in a basal marine media made of 0.5% (w/v) polypeptone, 0.1% (w/v) yeast extract, 100 mM MES , and 75% (v/v) basal salt solution composed of 0.7 g/L KCl, 5.3 g/L MgSO4 ⋅ 7H2O, 10.8 g/L MgCl2 ⋅ 6H2O, and 1.3 g/L CaSO4 ⋅ 2H2O. To study the effects of Na+ concentration, the amount of NaCl was adjusted accordingly to constitute 0.5%, 1.0%, 2.25%, 3.0% and 5.0% (w/v) of the culture media. One mL of the precultured inoculum was then added to 95 mL marine broth containing 5% (w/v) mannitol in a glass bottle reactor. Batch culture was performed at 37 °C and pH 6.0 with gentle agitation using a magnetic stirrer . The pH was maintained using a pH controller  attached to an autoclavable electrode  with the addition of 5 N NaOH. The experiment was repeated in triplicate and samplings of culture and gas were done at 12 h-intervals.Batch culture was performed using E. coli strain JCM 1649, the bacteria was cultured on three types of media namely LB broth (1% (w/v) tryptone, 0.5% (w/v) yeast extract, 0.5% (w/v) NaCl), LB broth prepared using 60% (w/v) artificial seawater  and denoted as LB broth with 60% ASW) and marine broth (containing 2.25% (w/v) NaCl). 100 mM MES  was supplemented in all media. The strain JCM 1649 was precultured under the same conditions as the strain AM2 culture, which gave a cell density of 1.3 OD620 after 24 h incubation. In these batch culture experiments, broth containing 5% (w/v) glucose was used for strain JCM 1649, due to its unsuitability in utilizing mannitol. Other culture conditions were set the same as those for the strain AM2. For both experiments using V. tritonius strain AM2 and E. coli strain JCM 1649, neither external reducing agents nor sparging with inert gas is required because both bacteria are capable of self-installing and maintaining anaerobiosis for hydrogen production using sugars as carbohydrate  and measured using the water displacement method. Hydrogen content in the biogas was determined using gas chromatography  equipped with thermal conductivity detector (TCD) and ShinCarbon ST packed column  with Argon (Ar) as the carrier gas. The operational temperatures of the injection port, oven and detector were 60 °C, 40 °C and 60 °C respectively. Extracellular ethanol was measured using the same gas chromatography equipment together with a flame ionization detector (FID) and HP-Blood Alcohol Capillary column . Helium (He) was used as the carrier gas. The temperatures of the column, oven and detector were 60 °C, 40 °C and 60 °C, respectively.p-toluenesulfonic acid was used as the mobile phase with the flow rate at 0.8 mL/min. Mannitol concentration was measured by HPLC with a reflective index detector (RID) and TSKgel Amide-80 column . 75% acetonitrile was used as the mobile phase with the flow rate at 1.0 mL/min. The optical density (OD) of cell growth was measured using a microplate reader  at a wavelength of 620 nm.Organic acids produced, such as formate, acetate, lactate and succinate were measured using high performance liquid chromatography (HPLC) with a conductivity detector and tandem connected to two Shim-pack SCR-102H columns . 5 mmol/L P < 0.05) were considered statistically significant.Molar yields of the fermentative products including hydrogen were analyzed using one-way analysis of variance (ANOVA) with Duncan’s multiple range tests. All statistical analyses were performed using the Statistical Analysis System (SAS) University Edition. Probabilities of less than 5% (H (mL), in batch tests can also be described using the following modified Gompertz model: The progress of cumulative hydrogen production, α.Xmax with P as hydrogen production potential (mL), and α.μmax by R m, as maximum hydrogen production rate (mL h−1), S0 – S as the amount of substrate consumed in g/L; Smax as the maximum substrate consumed and Rms as the maximum rate of substrate consumed (in g/L/h).Replacing tz model : (3)\docλ is the lag time (h) and e is equal to 2.718. All parameters in r (mL h−1) of AM2 grown at various salt levels was calculated following Hydrogen production rate, The hydrogen production rate was also denoted as HPR.r is the hydrogen production rate (mL/h); r max is the maximum hydrogen production rate; C is the added Na+ concentration (g/L); C crit is the critical Na+ concentration at which H2 production ceases (g/L) and n is the degree of inhibition.Response of the hydrogen production rate to different NaCl concentrations was calculated using a modified Han–Levenspiel model as described in A is the logarithmic increase of bacterial population; λ is the lag time; μ is the maximal growth rate; and t is the time.In order to describe the microbial growth in a batch culture, the Gompertz equation was again modified following 2 production using AM2. Substrate fermentation took place for 72 h and at the end of the process, the culture in 0.5 and 1.0% (w/v) NaCl produced the highest amounts of H2, i.e., 7,831 ± 295 and 8,561 ± 602 mL/L reactor volume on average, respectively. On the other hand, cells grown in media supplemented with 2.25% (w/v) NaCl produced slightly less hydrogen at 6,693 ± 443 mL/L reactor volume followed by 3.0% and 5.0% (w/v) NaCl at 1,808 ± 79 and 3 ± 2 mL/L reactor volume, respectively. P as hydrogen production potential (mL), R m, as maximum hydrogen production rate (mL h−1) and λ (h) of the batch system in response to various salt levels. The results imply the strain AM2 is capable of hydrogen production under a wide range of NaCl concentrations, i.e., 0.5 to 3.0% (w/v). However, the amount of hydrogen produced dropped as NaCl concentrations increased. Maximum H2 production potential, P, was observed when 0.5 and 1.0% (w/v) NaCl was supplemented in the media followed by the addition of 2.25% (w/v) NaCl and showed further decreases at higher NaCl concentrations in the media.The cumulative hydrogen production at different NaCl concentrations over time was fitted with P < 0.05) to one another. We also calculate the maximum number of organisms, X max, the maximum growth rate, µmax [ln(N/N0) h−1] and the lag time, λ (h) of the batch system in response to various salt levels. The maximum specific growth rate, µmax, was insignificant at 0.5 to 2.25% (w/v) with average µmax = 0.8 ln(N/N0)/h but inhibited at 3.0 and 5.0% (w/v) with an average of µmax = 0.5 ln(N/N0)/h. V. tritonius showed a classical growth trend under different salt concentrations and the average lag time, λ was at 2.3 h. The cells enter a stationary growth phase after between approximately 14 to 23 h of fermentation and then plateaued until the end of the fermentation period. 2 produced reveals that hydrogen production started during the exponential phase in which cell growth and H2 formation may occur simultaneously. Later, hydrogen evolution continues during the stationary phases of bacterial growth and most of the hydrogen was produced during this period.The efficiency of fermentative biohydrogen is strongly connected to the presence of active cells . Differer for each NaCl concentration tested was estimated using r values of 0.5 and 1.0% NaCl were indistinguishable (r2 = 0.99) at n = 2 to demonstrate the relationship of the hydrogen production rate to different NaCl concentrations using a modified Han–Levenspiel model. The model demonstrates that the maximum hydrogen production rate was 374 mL H2/h. 2 production by AM2 is promoted with the addition of 5 to 10.2 g/L (= 0.5 to 1.02% (w/v)) NaCl to the fermentation media. H2 production is inhibited under higher NaCl concentrations and is critically reduced, Ccrit at 46 g/L (=4.6% (w/v)) NaCl. Media containing 22.5 and 30 g/L NaCl resulted in 15 and 78% reduction of the original amount of H2 produced in 5 g/L NaCl containing media, respectively. The molar yields of hydrogen produced at the end of fermentation (72 h) were compared and presented as mol H2 per mol mannitol. Statistical analysis by Duncan’s multiple range test revealed that the H2 yield by cells grown in 5, 10 and 22.5 g/L NaCl supplemented media were not significantly different to each other. The yields were greatly reduced as the NaCl concentration increases.The hydrogen production rate (HPR), uishable , 2.25 anuishable ,  were estimated by dividing the amount of mannitol consumed at a particular time by the amount of initial mannitol (expressed as a percentage). P > 0.05) among 0.5 to 3.0% NaCl tested with the highest, 2.25% NaCl had achieved 42.3 mM formate. At the end, the average of 10.7 mM formate was left unconsumed/imported in 0.5 to 2.25% NaCl containing media. For both 3.0 and 5.0% NaCl containing media, 20.8 mM formate remained.E. coli strain JCM 1649. Maximum formate production was observed after 6 h-cultivation in normal LB broth which contains 0.5% NaCl and LB broth supplemented with 60% artificial water corresponding to 2.3% NaCl. The formate produced was recorded at 12.2 mM/OD620 in 0.5% NaCl LB broth and 11.0 mM/OD620 in LB broth with 60% ASW  helped us to see that hydrogen production by V. tritonius was much more stable than that produced from glucose by the E. coli strain JCM 1649 in the seawater-based media. V. tritonius offers high hydrogen production in saline environments and prolongs stable fermentation in a wide range of NaCl concentrations up to 46 g/L NaCl.Vibrios are phylogenetically related to eriaceae , and pre E. coli . It is i+ is one of the important factors in bacterial growth  and increased acidity  and LB broth prepared with 60% ASW (23 g/L NaCl) were not significantly different but the hydrogen produced showed significant decreases in the latter medium. Furthermore, the formate production of E. coli in marine broth occurs at much slower rates than those in LB broths but after 24 h fermentation, more formate was detected and the amount was much higher than in those produced in the two LB broths  V. tritonius AM2 is a better candidate for fermentative bioH2 production in saline conditions than E. coli. Hypothetically, the more substrate consumed (converted to formate) the more hydrogen will be produced. The actual mechanism by which the hydrogen production machineries of vibrios adapt to salt is not yet understood. Careful examination, however, on the V. tritonius system revealed that the pattern of hydrogen production (V. tritonius AM2. The results support the hypothesis that the highest substrate conversion efficiency (SCE) of 94% and 97% were observed in media containing 5 to 10 g/L NaCl, respectively, which both resulted in the highest hydrogen production rate (HPR) of 240 mL/h hydrogen. The results were in agreement with Clostridium amygdalinum strain C9 in their study.Up to this point in the study, the observations suggest; (1) the strain AM2 is capable of producing hydrogen under a wide range of NaCl levels, i.e., 5 to 46 g/L NaCl in the seawater-based broth; (2) the amount of formate exported out of the AM2 cells within the first 24 h of incubation is not affected by increasing salt levels up to 30 g/L NaCl but the formate re-import drops significantly at ≥22.5 g/L NaCl; (3) the reduced ability of oduction  closely oduction . The obsV. tritonius strain AM2 via FHL- complex. In conclusion, our data suggests that higher NaCl levels have an overall inhibitory effect on the fermentation system yet V. tritonius is still capable of producing hydrogen under a wide range of NaCl concentrations up to 46 g/L NaCl. The fermentative system of V. tritonius is generally not affected in seawater-based media containing 5 to 22.5 g/L NaCl, an amount that is relatively higher than common systems of other bacteria. The tolerance to a wider range of NaCl concentrations is a promising attribute towards a better fermentative system when seawater-based feedstocks or waste biomasses with high salt concentrations are used. This behaviour could also be beneficial in developing continuous batch culture for commercial-scale production. In addition to that, data presented herein  demonstrates the need for global transcriptome studies that would provide insights in the interaction of cellular reducing power or energy level on hydrogen production. As a result, our data in general suggests the use of bacteria of marine origin, V. tritonius, for marine-based fermentative hydrogen production due to its attributes being superior to those of other bacteria.Current research is underway to understand the NaCl-dependent behavior of fermentative hydrogen production of


Artemisia rupestris L. aerial parts against the booklice Liposcelis bostrychophila Badonnel and isolation of insecticidal and repellent constituents from the essential oil. The essential oil of A. rupestris was obtained by hydrodistillation and analyzed by GC-MS. A total of 30 components of the essential oil of A. rupestris was identified and the principal compounds in the essential oil were α-terpinyl acetate (37.18%), spathulenol (10.65%), α-terpineol (10.09%), and linalool (7.56%), followed by 4-terpineol (3.92%) and patchoulol (3.05%). Based on bioactivity-guided fractionation, the four active constituents were isolated from the essential oil and identified as α-terpineol, α-terpinyl acetate, 4-terpineol and linalool. The essential oil of A. rupestris exhibited contact toxicity against L. bostrychophila with LD50 value of 414.48 µg/cm2. α-Terpinyl acetate (LD50 = 92.59 µg/cm2) exhibited stronger contact toxicity against booklice than α-terpineol (LD50 = 140.30 µg/cm2), 4-terpineol (LD50 = 211.35 µg/cm2), and linalool (LD50 = 393.16 µg/cm2). The essential oil of A. rupestris (LC50 = 6.67 mg/L air) also possessed fumigant toxicity against L. bostrychophila while the four constituents, 4-terpineol, α-terpineol, α-terpinyl acetate and linalool had LC50 values of 0.34, 1.12, 1.26 and 1.96 mg/L air, respectively. α-Terpinol and α-terpinyl acetate showed strong repellency against L. bostrychophila, while linalool and 4-terpinol exhibited weak repellency. The results indicate that the essential oil of A. rupestris aerial parts and its constituent compounds have potential for development into natural insecticides or fumigants as well as repellents for control of insects in stored grains.The aim of this research was to determine the chemical composition and insecticidal and repellent activity of the essential oil of  Liposcelis bostrychophila Badonnel; Psocoptera: Liposcelididae) have a worldwide distribution infesting domestic premises, raw material stores, manufacturing factories, and historical documents in museums  in cereal grains. However, new evidence indicates that psocids are perhaps the most important emerging pests in stored grains and related commodities due to their small size, and resistance to chemicals  and AMX500 [500 MHz (1H)] instruments using CDCl3 as the solvent with TMS as internal standard. EIMS were determined on a ThermoQuest Trace 2000 mass spectrometer at 70 eV (probe). Components of the essential oil of A. rupestris aerial parts were separated and identified by gas chromatography-flame ionization detection (GC-FID) and gas chromatography–mass spectrometry (GC–MS) on an Agilent 6890N gas chromatograph equipped with a flame ionization detector connected to an Agilent 5973N mass selective detector. The same capillary column (HP-5MS 30 m × 0.25 mm × 0.25 μm) and analysis conditions were used for both GC-FID and GC-MS.A. rupestris (5 kg) were purchased from Anguo Chinese Medicinal Herbs Market . The plant was identified by Dr. Liu, QR  and a voucher specimen (CMH-Xinjiangyizhihao-Xinjiang-2013-04) was deposited in the museum of Department of Entomology, China Agricultural University. The sample was ground to a powder using a grinding mill . The powder was subjected to hydrodistillation using a modified Clevenger-type apparatus for 6 h and extracted with n-hexane. Anhydrous sodium sulphate was used to remove water after extraction. The essential oil was stored in airtight containers in a refrigerator at 4 °C for subsequent experiments.The dried aerial parts of L. bostrychophila were obtained from laboratory cultures in the dark in incubators at 28–30 °C and 70%–80% relative humidity and was reared on a 1:1:1 mixture, by mass, of milk powder, active yeast, and flour. All the containers housing insects and the Petri dishes used in experiments were made escape proof with a coating of polytetrafluoroethylene . Laboratory bioassays were done within one week after adult collections.Booklice, −1 to 180 °C where it was held for 1 min, and then ramped at 20 °C min−1 to 280 °C and held there for 15 min. The injector temperature was maintained at 270°C. The samples  were injected, with a split ratio of 1:10. The carrier gas was helium at flow rate of 1.0 mL·min−1. Spectra were scanned from 20 to 550 m/z at 2 scans s−1. Most constituents were identified by gas chromatography by comparison of their retention indices with those of the literature or with those of authentic compounds available in our laboratories. The retention indices were determined in relation to a homologous series of n-alkanes (C8–C24) under the same operating conditions. Further identification was made by comparison of their mass spectra with those stored in NIST 05  and Wiley 275 libraries  or with mass spectra from the literature , 136 (58), 121 (100), 105 (22), 93 (86), 91 (46), 77 (26), 67 (37), 51 (21). The data matched with previous reports . A . A A. ruA. rupestris aerial parts and its four constituent compounds have potential for development into natural insecticides/fumigants and repellents for control of insects in stored grains.The study indicates that the essential oil of


NRPS) clusters are spread across the chromosomes, where several modifying enzyme-encoding genes typically flank one NRPS. However, a recent study showed that the octapeptide fusaoctaxin A is tandemly synthesized by two NRPSs in Fusarium graminearum. Here, we illuminate parts of the biosynthetic route of fusaoctaxin A, which is cleaved into the tripeptide fusatrixin A and the pentapeptide fusapentaxin A during transport by a cluster-specific ABC transporter with peptidase activity. Further, we deleted the histone H3K27 methyltransferase kmt6, which induced the production of fusaoctaxin A.Fungal non-ribosomal peptide synthetase ( The final module typically contains a thioesterase domain (TE) or a reductase domain  obtained from Stratagene . Uracil-specific excision reagent (USER) friendly cloning was carried out using chemically competent E. coli JM109 genotype: e14– (McrA–) recA1 endA1 gyrA96 thi-1 hsdR17 (rK– mK+) supE44 relA1 ∆(lac-proAB) [F´ traD36 proAB lacIqZ∆M15] obtained from Stratagene. Agrobacterium tumefaciens mediated transformation (ATMT) was carried out using ElectroMAXTMA. tumefaciens LBA4404 cells that were obtained from Invitrogen, Thermo Fisher Scientific Inc. catalog number: 18313-015, Hvidovre, Denmark. F. graminearum PH-1 (NRRL31084) was obtained from the Agricultural Research Service Culture Collection, National Center for Agricultural Utilisation Research. All of the primers were obtained from MWG Biotech,   . All theF. graminearum chromosome III (accession number: HG970334.1) into Antismash 3.0 Fungal Version [Fusarium species containing the gene cluster were imported into CLC Main Workbench 7  and used to generate a Synteny plot.The  Version , where t Version . The genHierarchical clustering of the fusaoctaxin A gene cluster and neighbouring genes was performed in CLC Genomics Workbench 6 using 175 microarray samples from 55 experiments, as previously described .F. graminearum PH-1 genomic DNA with the primer pairs FGSG_15795_5F//FGSG_15795_5R and FGSG_15795_3F//FGSG_15795_3R, respectively. The hygromycin B resistance cassette was amplified while using the primer pair hph-F and hph-R from pCSN44. The Saccharomyces cerevisiae strain FY834 [NRPS5::NRPS9 mutant was constructed using a U-GOTL template vector [NRPS5 mutant. The NRPS9 gene was amplified using NRPS9 (tubulin) forward and reverse primers . 15N chemical shifts were indirectly referenced using Ξ = 0.10132912. The assignment and chemical shift of 13C were reviewed by CSEARCH Robot [All of the NMR experiments were recorded on a BRUKER AVIII-600 MHz NMR spectrometer that was equipped with a CPP-TCI cryogenically cooled probe. Spectra were recorded and processed with TopSpin 3.5pl6. The compound was dissolved in DMSO-d6, and the following NMR spectra were recorded at 308.1 K: CH Robot .NRPS5, OE::NRPS9, ΔNRPS9, and OE::NRPS5::NRPS9. Genomic DNA from 5 mg freeze-dried mycelium was extracted with the FastDNA SPIN Kit for Soil  and subsequently cleaned with Agencourt AMPure XP beads  using a 1.7 bead:sample ratio. A Nextera paired-end DNA library (Illumina) was prepared according to the Nextera DNA Library Prep Reference Guide (Document # 15027987 v01), with the modification of using Agencourt AMPure XP beads (0.7 bead:sample ratio) for the post-tagmentation clean-up. DNA quality (A260/A280 and A260/A230), size-distributions, and concentration were assessed on the NanoDrop ND-1000 (Thermo Scientific), D1000 ScreenTape system , and using the Qubit dsDNA HS assay kit (Thermo Scientific), respectively. Each paired-end library was sequenced to approximately 200× coverage on the Illumina HiSeq 2500 system while using the Rapid SBS Kit v2 (2 × 250 cycles). Low quality reads, ambiguous nucleotides, and adaptors were trimmed (custom trim settings) in CLC Genomics Workbench v. 9.4.2 . Reads were subsequently mapped to the corresponding mutant reference genome in CLC Genomics Workbench while using the default settings. High-molecular weight (HMW) genomic DNA were extracted from 250 mg freeze-dried mycelia by incubation in 10 mL Qiagen lysis buffer C2 (Qiagen) for 2 h at 50 °C and in the presence of 10 mg RNase A (Qiagen) and 6 mg Proteinase K (Qiagen). Following two rounds of 1:1:1 (v/v) chloroform:phenol:isoamyl extraction , HMW DNA was precipitated with 1:1 (v/v) room-temperature isopropanol. DNA was spooled into new tubes and washed twice with 70% ethanol. Following the removal of residual ethanol, DNA was allowed to re-dissolve in 10 mM Tris pH 8.5 for a minimum of 48 h. DNA concentration, quality, and size-distribution were assessed, as previously described. Approximately 400 ng of HMW DNA was used as the input for the Rapid Barcoding kit protocol (SQK-RBK004) . Sequencing was carried out on a R9.4 FLO-MIN106 flow-cell in the MinION sequencer (Oxford Nanopore Technologies), with approximately 10–30× coverage. Base-calling and barcode sorting was performed in Albacore v. 2.2.7. The reads were subsequently mapped to the corresponding mutant reference genome in CLC Genomics Workbench.Illumina Sequencing of OE::F. graminearum infections, the highly susceptible (FHB) USU-Apogee full-dwarf hard red spring wheat  was used. Type II infections were carried out by inoculating two spikelets per wheat ear with 10 μL of a 105 spores/mL solution. As a control, the MOCK ears were inoculated with water instead of the spore suspension. Five ears per F. graminearum strain were inoculated, as well as five MOCK ears. After each treatment, the ears were covered in moistened plastic bags for the first 24 h to provide high humidity. The incubation conditions were set to 60% humidity, 20 °C for 16 h (day) and 18 °C for 6 h (night). The F. graminearum infection progress was monitored by counting the infected spikelets over a time period of 10 days.For F. graminearum, OE::NRPS5, OE::NRPS9, and OE::NRPS5OE::NRPS9 that were grown in triplicates for two weeks at 25 °C in the dark on solid yeast extract sucrose (YES) media in 90 mm petri dishes [v:v:v 3:2:1 ethyl acetate:dichloromethane:methanol  with 1% formic acid  and sonicated for 45 min. The extracts were decanted into new tubes and dried under a flow of N2 gas. The dried samples were re-dissolved in 400 µL methanol and centrifuged for 5 min. at 14.1k rcf. The supernatants were transferred to 2 mL HPLC vials. The samples were analyzed for secondary metabolites by HPLC-DAD-HRMS while using a Hitachi Elite LaChrom HPLC system, which was equipped with a C6-phenyl  column kept at 40 °C and coupled by a 5:95 flow splitter to a high-resolution mass spectrometer  with an electrospray source  that was operated in positive mode. The injection volume was set to 20 µL and separated using a 1 mL/min gradient initiating at 10% solvent A  with 0.1% v/v formic acid ) in 90% solvent B  with 0.1% v/v formic acid ), increasing linearly to 100% solvent A over 20 min. and then held for 10 min. After 0.2 min. from sample injection, 2 µL calibrant  in 1:1 water :isopropanol) was directly injected to the mass analyzer and was used for internal spectra calibration. Mass spectrometric instrument control, data recording, and data processing were performed using Compass DataAnalysis 4.2 SR2 (Bruker). The metabolite extract from OE::NRPS5::NRPS9 was additionally analysed by bbCID fragmentation that was acquired at 20 eV.The secondary metabolites were extracted from Wt i dishes . FifteenF. graminearum, OE::NRPS5::NRPS9, Δkmt6, Δfgm5+Δkmt6, and ΔFGSG_10982+Δkmt6 were grown in triplicates on solid YES agar medium for 12 days at 25 °C in the dark. The metabolites were extracted from six plugs that were covered with 3 mL extraction solvent A and sonicated for 40 min., otherwise following same procedure, as previously described. HPLC-HRMS analysis was acquired, as previously described, except the gradient was held at 100% solvent A for 5 min.Wild type F. graminearum, OE::NRPS5, OE::NRPS9, OE::NRPS5:NRPS9, ΔNRPS9, and a MOCK were analyzed for secondary metabolites. Extraction was performed on approx. 0.2 g grinded barley powder using 1 mL of acetonitrile:water (82:18 v/v). The samples were gently mixed and sonicated for 25 min. The solvent was centrifuged at 14.1k rfc and transferred to a 2 mL HPLC vial. HPLC-HRMS analysis was performed, as described above.Barley that was infected with wild type NRPS5::NRPS9 that was grown on 25 YES agar plates at 25 °C in the dark for 14 days. The fungi were diced to roughly 5X5 mm and covered with 400 mL of extraction solvent A and sonicated for 45 min. The solvent was filtered through Miracloth (Merck) and subsequently evaporated using a rotary evaporator at 40 °C. The dried metabolites were re-dissolved in 1 mL methanol , centrifuged at 14.1k rcf, and then transferred to a 2 mL HPLC vial. Fusatrixin A was purified from 120 YES agar plates following the same procedure as for fusaoctaxin A, except 1.2 L of ethyl acetate  with 1% formic acid  was used as the extraction solvent and sonication was performed for 1 h. The dried metabolites were re-dissolved in 8 mL methanol  and subsequently reduced to 2.5 mL by a flow of nitrogen gas. The semi-preparative HPLC was conducted on an Agilent 1260 infinity LC system (Agilent Technologies) with a diode array detector (190–900 nm). 100 µL sample was injected and separated on a 150 × 10.0 mm Gemini 5 µm C6-Phenyl 110 Å column using a flow of 5 mL/min and with a linear gradient of water (MilliQ)-acetonitrile , with both being supplemented with 50 ppm trifluoroacetic acid (Sigma-Aldrich). The gradient initiated at 10% acetonitrile increasing to 100% over 12 min. and held for 2 min. before returning to initial conditions over the final 2 min. The peaks of interest were detected by UV at 195 nm and then collected.Fusaoctaxin A was purified from OE::6 to 250 µg. Full hydrolysis was performed in 2.4 mL of 1:3 DMSO-d6:8M HCl at 110 °C for 24 h under a nitrogen atmosphere. The hydrolyzed sample was diluted 1:10 with Milli-Q water, flash-frozen in liquid nitrogen, and lyophilised. The dried sample was re-dissolved in 200 µL Milli-Q water and derivatized with N-α--l-alaninamid (Sigma-Aldrich), together with commercially bought amino acid standards  [Fusaoctaxin A was quantified by a PULCON NMR experiment in DMSO-deucinol) . The derFusarium graminearum and Fusarium Solani exposed to chemical synthesized fusaoctaxin A, fusapentaxin A, and fusatrixin A in the concentration range 0–100 µM in real-time. Briefly, growth was monitored at 25 °C in Nunc Edge 2.0 96-Well plates (Thermo Fischer Scientific) containing 200 µL sterile-filtered YPG media  d-(+)-glucose), 5000 spores and 0 µM (0.5% ethanol control), 12.5 µM, 25 µM, 50 µM, or 100 µM fusaoctaxin A’s. Sterile medium (8 mL) was added to the edge-reservoirs to minimize the sample evaporation. Focus and illumination level was manually adjusted following a 0.5 h spore settling-time. Two image-strips (11.43 µm image distance) per. well were automatically acquired in UniExplorer version 7.3.0.1057  .We used the OcelloScope optical detection system  ,39 to mo


NUBPL (nucleotide binding protein-like), encoding an assembly factor of complex I, which are potentially pathogenic. To help assign pathogenicity and learn more about the function of NUBPL, amino acid substitutions were recreated in the homologous Ind1 protein of the yeast model Yarrowia lipolytica. Leu102Pro destabilized the Ind1 protein, leading to a null-mutant phenotype. Asp103Tyr, Leu191Phe and Gly285Cys affected complex I assembly to varying degrees, whereas Gly136Asp substitution in Ind1 did not impact on complex I levels nor dNADH:ubiquinone activity. Blue-native polyacrylamide gel electrophoresis and immunolabelling of the structural subunits NUBM and NUCM revealed that all Ind1 variants accumulated a Q module intermediate of complex I. In the Ind1 Asp103Tyr variant, the matrix arm intermediate was virtually absent, indicating a dominant effect. Dysfunction of Ind1, but not absence of complex I, rendered Y. lipolytica sensitive to cold. The Ind1 Gly285Cys variant was able to support complex I assembly at 28°C, but not at 10°C. Our results indicate that Ind1 is required for progression of assembly from the Q module to the full matrix arm. Cold sensitivity could be developed as a phenotype assay to demonstrate pathogenicity of NUBPL mutations and other complex I defects.Complex I deficiency is a common cause of mitochondrial disease, resulting from mutations in genes encoding structural subunits, assembly factors or defects in mitochondrial gene expression. Advances in genetic diagnostics and sequencing have led to identification of several variants in  Defects in respiratory chain function underlie a large share of mitochondrial disorders, of which a third are due to complex I deficiency OMIM 252010) . Clinica2010 . ClYarrowia lipolytica where it was named Ind1 (iron–sulfur protein required for NADH dehydrogenase)  clusters in complex I based on its homology to the cytosolic FeS cluster assembly factors Nbp35 and Cfd1, corresponding to NUBP1 and NUBP2, respectively, in human  was first identified in the aerobic yeast ogenase) . Genomicin human . HoweverIND1 gene is present in almost all eukaryotes, and closely matches the distribution of complex I , is unstable, and NUBPL protein is almost undetectable in patient fibroblasts  with likely clinically relevant mutations in iagnosed . Patient familiesiagnosed . In addithe gene . The c.8Y. lipolytica has been used extensively in biotechnology, but it has also been developed as a model organism to study complex I biology , of which Asn198Tyr and Val182Ala occur at a relatively high allele frequency  variant with an allele frequency of 6.73 × 10-4 does affect a conserved amino acid, corresponding to Gly136 in Y. lipolytica Ind1, and was included in this study. G138D is predicted to be damaging to NUBPL   . The L10pe, cWT) .L102P protein were due to decreased transcript levels, expression of IND1 was assessed by reverse transcription–polymerase chain reaction (RT-PCR)  . While tIND1 have approximately 28% of fully active complex I compared with wild type  and N271Qfs*31 (nubmΔ) displayed no complex I activity, in agreement with the FMN cofactor of NUBM being the site of NADH oxidation. Another complex I mutant, where a key catalytic residue of the NUCM subunit has been substituted (Y144F) (IND1 (It has previously been shown that cells lacking ild type . In cell71Qfs*31 . The L19 (Y144F) , displayTo measure the oxidoreductase activity of the matrix arm of complex I, two different spectrophotometric assays were used. Electron transfer from NADH to the artificial electron acceptor hexaammineruthenium(III) chloride (HAR) involves only the primary electron acceptor FMN bound to the NUBM subunit of complex I and, like NADH:NBT staining, serves as a proxy for complex I content. The L102P, D103Y, L191F and N271Qfs*31 variants showed a significant decrease in NADH:HAR activity, whereas G136D and G285C were similar to cWT .nucmΔ + NUCMY144F mutant had low dNADH:DBQ activity  to n-decylubiquinone (DBQ) was measured, encompassing all cofactors in the matrix arm of complex I. A significant decrease in electron transfer was seen with the L102P, D103Y, L191F and N271Qfs*31 variants, but not in G136D and G285C . The nucactivity , despiteactivity . CompariIND1 deletion. The L191F substitution caused a mild decrease, whereas G136D and G285C appeared to have no significant effect on complex I levels under standard growth conditions.Taken together, the data from three different methods are in agreement and indicate that the L102P and D103Y substitutions in Ind1 cause a defect in complex I assembly of similar severity as NUBPL, a subcomplex representing part of the membrane arm was observed  in the N module. NUCM  and NUKM  are in the Q module . AssembD103Y accumulated relatively more Q module intermediate and very little of the 500 kDa intermediate. For intermediates containing the NUBM subunit, variants with low levels of complex I  closely resembled the empty vector control, whereas those with near normal complex I levels  more closely resemble cWT  or cold (10°C) conditions. Ind1 variants, except for G136D, also display this temperature-dependent growth defect.ind1Δ mutant . Since the allele frequency for G138D and c.815-27T>C are moderately high , there was no significant effect on complex I levels and activities, although accumulation of the Q module assembly intermediate was observed  were found to be deleterious to complex I and one (G138D) was found to exhibit a mild defect. Along with other studies  was used to analyze mutant versions of IND1 and has been described previously (IND1 gene (YALI0B18590g) including its native promoter (1 kb upstream of the ATG) was reintroduced using the pUB4 plasmid . The nubmΔ, nucmΔ, nucmΔ + NUCMY144F and nukmΔ were as previously described (Y. lipolytica cells were grown in rich medium containing 1% (w/v) yeast extract and 1% (w/v) glucose (Y-1) was added for selection of cells transformed with the pUB4 plasmid.The eviously . The INDne YALI0B590g inclescribed ,38 but tIND1 sequence from pUB4-IND1-strep  and plasmidpLYS. Colonies were grown overnight at 37°C in Lysogeny broth (LB) media, with required antibiotics, and then diluted 50× in 100 ml Terrific broth  and grown until OD600 = 0.6. Protein expression was induced by addition of 1 ml l-1 benzylalcohol, 50 μm Fe–ammonium citrate, 100 μm L-cysteine and 1.2 ml l-1 isopropyl β-D-1-thiogalactopyranoside (IPTG). Cells were grown at 20°C overnight, then harvested by centrifugation at 5000 × g and flash frozen in liquid nitrogen. Cells were then resuspended in buffer W  with 0.2% (w/v) dodecyl maltoside. The cell suspension was sonicated and separated into soluble and insoluble fractions by centrifugation at 16100 × g.h et al. . BrieflyY. lipolytica cells from 0.5 l overnight culture were harvested by centrifugation at 3500 × g. Pellet weights were typically between 2–8 g. Cells were washed with dH20. Per gram cells, 2 g fine glass beads, 2 mm PMSF and 2 ml ice cold mito-membrane buffer  were added. Cells were disrupted by 15 rounds of 1 min vortexing and 1 min incubation in ice. Differential centrifugation was performed at 3500 × g for 10 min and 40000 × g for 120 min at 4°C. Pellets were resuspended in mito-membrane buffer and stored at -80°C. For BN-PAGE, unsealed mitochondrial membranes were mixed with solubilization buffer  and incubated on ice for 5 min followed by centrifugation at 16100 × g, at 4°C, for 10 min. The supernatant containing solubilized membrane proteins were diluted to 0.25 μg μl-1 protein (Bradford assay) with solubilization buffer and 0.25% (w/v) Coomassie G250. Typically, 6.25 μg protein was loaded per lane. 5 μl NativeMark™ was used as a molecular weight marker. All BN-PAGE performed used NativePage™ 4–16% Bis-Tris gels , with 1 mm spacers. The anode buffer was 50 mm Bis-Tris–HCl, pH7.0; cathode buffer was 50 mm Tricine, 15 mm Bis-Tris–HCl, pH7.0. Gels were run in a XCell SureLock™ system  at 4°C, for the first 45 min at 100 V (max 10 mA) with cathode buffer containing 0.02% (w/v) Coomassie G250, then for ∼2.5 h at 250 V (max 15 mA) with cathode buffer containing 0.002% (w/v) Coomassie G250, until the dye front exited the bottom.Unsealed mitochondrial membranes were isolated as published , with miBN gels or PVDF membrane were stained in Coomassie solution  for 1–12 h. Gels were destained using a solution of 45% (v/v) methanol and 5% (v/v) acetic acid.m Tris–HCl pH 6.8, 10% (w/v) glycerol, 0.04% (v/v) β-mercaptoethanol, and 0.002% (w/v) bromophenol blue] and separated on a 15% SDS-PAGE gel. Proteins were transferred under semi-dry conditions to nitrocellulose membrane (Protran™). Equal loading and transfer was confirmed using Ponceau S stain. Proteins were labelled with antibodies and detected using secondary horseradish peroxidase-conjugated antibodies and chemiluminescence. Mouse monoclonal antibodies against the NUBM and NUCM subunit complex I were a gift from Volker Zickermann. Rabbit polyclonal antibodies against Sdh2 and Aco1 were raised against recombinant Saccharomyces cerevisiae proteins and previously reported  SDS, 125 mreported ; antibodProtein was transferred from the BN-PAGE gel to a PVDF membrane  using wet transfer and BN cathode buffer, for 600 min at 40 mA (maximum voltage 100 V). After transfer, immunolabelling was carried out as described above.m NADH and 0.1% (w/v) NBT. After the desired staining intensity was reached, the reaction was stopped using a solution of 45% (v/v) MeOH and 5% (v/v) acetic acid. Destaining using a solution of 45% (v/v) MeOH and 5% (v/v) acetic acid was performed for optimal enzyme stain visualization.BN gels were stained for complex I, as described in Sabar et al. . Brieflym HEPES–NaOH pH 8.0, 2 mm NaN3, 0.2 mm NADH, 2 mm HAR and 40 μg ml-1 alamethicin. Measurements were made in a 1-ml quartz cuvette using a UV/Vis spectrophotometer  and started after addition of HAR.NADH:HAR oxidoreductase activity was assayed as described . Activitm MOPS–NaOH pH 7.4, 50 mm NaCl, 2 mm KCN, 0.1 mm dNADH and 0.1 mm DBQ. Measurement started after addition of DBQ. 0.01 mm of the complex I inhibitor Piericidin A was added at the end of the reaction to ensure that dNADH:DBQ oxidoreductase activity is due to complex I.dNADH:DBQ oxidoreductase activity was measured as dNADH oxidation activity in the presence of the ubiquinone analogue n-decylubiquinone (DBQ) as an electron acceptor. Activity for 50 μg mitochondria was measured in a 1 ml volume of 20 mSupplementary DataClick here for additional data file.


Scaptotrigona depilis requires the brood cells-associated fungus Zygosaccharomyces sp. as steroid source for metamorphosis. Besides the presence of Zygosaccharomyces sp., other fungi inhabit S. depilis brood cells, but their biological functions are unknown. Here we show that Candida sp. and Monascus ruber, isolated from cerumen of S. depilis brood provisions, interact with Zygosaccharomyces sp. and modulate its growth. Candida sp. produces volatile organic compounds (VOCs) that stimulate Zygosacchromyces sp. development. Monascus ruber inhibits Zygosacchromyces sp. growth by producing lovastatin, which blocks steroid biosynthesis. We also observed that in co-cultures M. ruber inhibits Candida sp. through the production of monascin. The modulation of Zygosaccharomyces sp. growth by brood cell-associated fungi suggests their involvement in S. depilis larval development. This tripartite fungal community opens new perspectives in the research of microbial interactions with bees.The Brazilian stingless bee  Scaptotrigona depilis   requires the fungus Zygosaccharomyces sp. to develop. This fungus, which start to grow inside brood cells after eggs hatch, is ingested by the larvae and is an essential source of ergosterol for ecdysteroid production [de novo and all steroids they need are obtained from the food source, including those requested to produce ecdysteroids for pupation [The Brazilian stingless bee s Moure, 942  were responsible for growth stimulation  did not change (1.7x104 CFU/mL)  Figs . HoweverCP2 Figs . AnalyseCP2 Figs . Analyse as well . Isoamyl as well . These rAureobasidium pullulans stimulated the growth of Armillaria mellea, and ethanol was the unique VOC detected by GC-MS analysis [Boletus virigatus produced ethanol that stimulated the growth of the root pathogenic fungi Phytophthora cinnamomi and Fomes annosus [Saccharomyces cerevisiae secrets aromatic alcohols involved in quorum-sensing signaling that promote its filamentous growth [Ethanol has been reported to stimulate microbial growth in other studies ,20. Cellanalysis . In anots growth .Candida sp. SDCP2 decreases the pH of the medium while growing. When cultured in Potato Dextrose Broth (PDB), this fungus decreased the pH from 5.0 to pH 3.0, after 3 days of culturing. Zygosaccharomyces sp. SDBC30G1 grows better in acidic pH . This result indicates that Candida sp. SDCP2 could also facilitate Zygosaccharomyces sp. growth by decreasing the pH of the media.Additionally, we observed that cidic pH , which iCandida sp. SDCP2 and M. ruber SDCP1, we also performed co-cultures. We observed that Candida sp. stimulated M. ruber SDCP1 pigment production  of 50 μg/mL and minimal fungicidal concentration (MFC) of 100 μg/mL against Candida sp. SDCP2.Monascinol, also known as monascuspiloin, and monascin are found in  of Asia ,23. Thes of Asia . We testMonascus ruber SDCP1 starts to produce monascin and monascinol on the second day in co-culture, and on the third day of co-culturing, the cells of Candida sp. SDCP2 are no longer viable, as confirmed by microscopy , the coexistence of Candida sp. and M. ruber is possible inside S. depilis colonies.croscopy  and cultMonascus spp. are also known to produce lovastatin (monacolin K), which reduces endogenous cholesterol biosynthesis. This natural product inhibits 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase), which catalyzes the production of mevalonate, a key intermediate for sterol biosynthesis [Zygosaccharomyces sp. is an essential source of sterols for S. depilis larvae [M. ruber SDCP1 produces lovastatin when cultured in monoculture using a 30% glucose medium . The collections were made from 2013 to 2016. Samples from brood cells cerumen were carefully collected and aseptically plated on Potato Dextrose Agar (PDA) containing streptomycin and penicillin (12.5 g/L). For Zygosaccharomyces sp. SDBC30G1 isolation, samples from the white microorganism growing inside S. depilis brood cells were plated aseptically on 30G agar medium (pH 6.0) [18SF (5’-ATTGGAGGGCAAGTCTGGTG-3’) and 18SR (5’-CCGATCCCTAGTCGGCATAG-3’) were used for Candida sp. SDCP2. For Monascus ruber SDCP1 and Zygosaccharomyces sp. SDBC30G1, we used the primers NS1 (5’-GTAGTCATATGCTTGTCTC-3’) and NS4 (5’-CTTCCGTCAATTCCTTTAAG-3’). Data are available under accession numbers KX999554, KX999555 and KX999557 in the National Center for Biotechnology Information (NCBI).DNA extractions from isolated strains were performed using FastDNA SPIN Kit for soil (MP), according the manufacturer instructions and quantified using NanoDrop. The isolated fungi were identified by 18S rRNA gene sequencing. The primers Candida sp. SDCP2 was reactivated in PDB medium for 48 h at 120 rpm and 30°C. After reactivation, an inoculum of OD600 0.06 was prepared. Monascus ruber SDCP1 was reactivated in PDA medium for seven days at 30°C. The spores of this fungus were collected and counted with the aid of a Neubauer chamber to prepare an inoculum containing 1x106 spores/mL. Zygosaccharomyces sp. SDBC30G1 was reactivated in 30G agar medium (pH 6.0) for 10–15 days at 30°C. After this time, an inoculum of OD600 0.06 was prepared. For all microorganisms, 2 μL of the inoculum were transferred to the culture media. The plates were incubated at 30°C for seven days. The experiments were made in duplicate.Co-cultures were performed in 15GF agar medium (pH 6.0 and pH 4.5) [Candida sp. SDCP2 was cultured in duplicate in 100 mL of PDB  at 30°C for 3 days. The pH of the supernatant was measured using a direct reading pH stick. The pH of S. depilis larval food was measured using a pH meter (GEHAKA). The result was expressed as the average of the pH from four samples of larval food collected in different S. depilis colonies.Candida sp. SDCP2 was grown in 100 mL of 30G broth (pH 6.0) [600 0.002) for 10 days. Then, the culture was centrifuged for 10 min (6000 rpm at 4°C) and filtered sterilized to obtain the cell-free supernatant.A monoculture of Candida sp. SDCP2 on Zygosaccharomyces sp. SDBC30G1 growth, we spotted 10 and 20 μL of this supernatant on top of a fresh inoculated culture of Zygosaccharomyces sp. SDBC30G1 in a Petri dish containing 25 mL of 30G agar medium, pH 6.0. Zygosaccharomyces sp. SDBC30G1 inoculum was prepared from a 10–15 days-old culture in a Petri dish containing 30G agar medium, pH 6.0 (100 μL inoculum of OD600 0.1), and plated using an inoculating loop. The control was made by replacing the cell-free supernatant from Candida sp. SDCP2 for 30G broth (pH 6.0). The Petri dishes were incubated at 30°C for 3–5 days.To test the stimulating capacity of the cell-free supernatant from Candida sp. SDCP2 was poured inside small Petri dishes (10 x 35 mm). In other Petri dishes containing 3 mL of 30G agar medium (pH 6.0) each, 25 μL of Zygosaccharomyces sp. SDBC30G1 inoculum (OD600 0.02) were plated using an inoculating loop. This inoculum was prepared from a 24 h culture of Zygosaccharomyces sp. SDBC30G1 in 2 mL of 30G broth (pH 6.0) at 200 rpm shaking and 30°C . The plates with Zygosaccharomyces sp. SDBC30G1 were placed on top of plates containing Candida sp. SDCP2 supernatant, the plates were sealed together with parafilm and incubated individually inside plastic bags at 30°C [Candida sp. SDCP2 supernatant, was used as a control. After 3–5 days, the growth of Zygosaccharomyces in the presence of Candida sp. SDCP2 supernatant was compared to the control. This experiment was made in triplicate.For VOCs experiments, the supernatant (3 mL) from  at 30°C . SterileCandida sp. SDCP2 supernatant was performed at Small Molecules Mass Spectrometry Facility (Faculty of Arts and Sciences Harvard University) using an Agilent 6890 gas chromatograph and CTC CombiPAL autosampler. A DB-624UI column (30m x 0.32mm x 1.80um) was used, at flow rate of 2.3 mL/min, injection temperature of 220°C, interface at 220°C, ion source of 200°C, and m/z 10–500. Initial temperature was set at 30°C for 6 min, then increased at 10°C/min to 150°C, and then at 50°C/min to 250°C, and kept at 250°C for 15 min. The split ratio was 200 and headspace method adopted was 80°C for 10 min. The reads were compared to the NIST MS search 2.0 library.Headspace analysis of Scaptotrigona depilis brood cells headspace analysis was performed at Research Support Center in Natural and Synthetic Products (NPPNS), University of São Paulo, using a Shimadzu GC-2010 Plus gas chromatograph and the auto sampler AOC 5000 with 2.5 mL syringe. A ZB-624 column (30m x 0.32mm x 1.80um) was used, at flow rate of 2.68 mL/min, injection temperature of 260°C, interface at 220°C, ion source of 200°C, and m/z 10–200. Initial temperature was set at 30°C for 6 min, then increased at 10°C/min to 150°C, and then at 50°C/min to 250°C, and kept at 250°C for 15 min. The split ratio was 5 and headspace method adopted was 80°C for 45 min. The reads were compared to the NIST 11 library.Monascus ruber SDCP1 and Candida sp. SDCP2, isolated from cerumen of S. depilis brood cells, were grown in PDA at 30°C for 7 days. After this period, two fungal-agar fragments (diameter of approximately 6 mm) were transferred to 50 mL Falcon tubes containing 20 mL of Potato Dextrose Broth (PDB). The cultures were incubated at 30°C with 120 rpm shaking and, after 48 h, were combined in Erlenmeyer flasks (500 mL) containing 150 mL of PDB. Monocultures of each microorganism were performed as controls. To analyze the production of metabolites, 10 mL of each culture were collected daily, centrifuged and filtered, and a sample of 1 mL was extracted with solid phase extraction (SPE) cartridges  using 1 mL of methanol. The organic extracts were filtered and 4 μL were analyzed by UPLC 1290 Infinity (Agilent Technologies) coupled to 6530 ESI-Q-TOF (Agilent Technologies) operating in the positive mode. A 10 x 4.6 mm column, Luna 5 mm C18 (Phenomenex) was used, with acetonitrile:water gradient as mobile phase, flow rate of 0.7 mL/min .Candida sp. SDCP2 cultures were incubated in Petri dishes containing PDA for 48 h at 30°C. Cultures of C. albicans ATCC MYA—2876 were reactivated in Petri dishes containing LB, Miller agar [Candida sp. SDCP2 and LB-Miller for C. albicans ATCC MYA—2876. When OD600 of the turbid solution was 0.08, 67 μL were transferred to 933 μL of culture medium. This final volume (1.0 mL) was transferred to another tube containing 9.0 mL of culture medium and vortexed. Then, 2.0 mL were transferred to a tube containing 10 mL of culture medium, and these inoculums were used in the treatments.The experiments were carried out according to the conditions recommended by CLSI (Formerly NCCLS) . Candidaler agar  for 24 hCandida sp. SDCP2, and clotrimazole (Sigma-Aldrich) was used for C. albicans ATCC MYA—2876. To prepare the positive controls solutions, 3.2 mg of each antifungal were dissolved in 0.5 mL of dimethyl sulfoxide (DMSO). Then, 16 μL were transferred to 144 μL of PDB or LB-Miller media and vortexed. Finally, 80 μL were transferred to the first well and serially diluted.Cycloheximide (Sigma-Aldrich) was used as a positive control for inhibiting M. ruber SDCP1 culture  and characterized by NMR and ESI-HRMS , a sample of each well was removed with sterile toothpicks and transferred to Petri dishes containing PDA or LB agar culture media. The plates were incubated for 48 h at 30°C or 24 h at 37°C for M. ruber SDCP1 was cultured in 30G agar plates for seven days at 30°C. After this period, the culture was extracted with ethyl acetate, the solvent was dried using rotary evaporator, yielding a crude extract. The HRESIMS analysis of the extract was carried out using a Bruker Maxis Impact HD LC-q-TOF Mass Spectrometer equipped with an uHPLC system showing lovastatin as the major compound . The production of lovastatin by M. ruber SDCP1 was further confirmed by retention time comparison with lovastatin standard (Sigma-Aldrich) using a Quadrupole 6130 (Agilent Technologies) equipped with HPLC-DAD-MS 1200 series (Agilent Technologies).Zygosaccharomyces sp. SDBC30G1 growth. Stock solutions were prepared in ethanol  and 5 μL of each solution were transferred to the corresponding wells in a 6-well plate containing 4995 μL of freshly inoculated Zygosaccharomyces sp. SDBC30G1 cultures  in 15GF pH 4.5 medium [Lovastatin standard (Sigma-Aldrich) was used to determine its effects on S1 Fig(PDF)Click here for additional data file.S2 Fig(PDF)Click here for additional data file.S3 Fig(PDF)Click here for additional data file.S4 Fig(PDF)Click here for additional data file.S5 Fig(PDF)Click here for additional data file.S6 Fig(PDF)Click here for additional data file.S7 Fig(PDF)Click here for additional data file.S8 Fig(PDF)Click here for additional data file.S1 Video(MP4)Click here for additional data file.S1 Data(ZIP)Click here for additional data file.