Escherichia coli. Temperature, pH, and osmotic pressure showed little effect. At commonly used concentrations, most tested oxidants, including hydrogen peroxide, tert-butyl hydroperoxide, hypochlorous acid, and diamide, did not affect iSS, but carbonyl cyanide m-chlorophenyl hydrazone increased iSS. For reductants, 10 mM dithiothreitol significantly decreased iSS, but tris(2-carboxyethyl)phosphine did not. Among different sulfur-bearing compounds, NaHS, cysteine, S2O32− and diallyl disulfide increased iSS, of which only S2O32− did not inhibit E. coli growth at 10 mM or less. Thus, with the improved method, we have identified reagents that may be used to change iSS in E. coli and other organisms, providing tools to further study the physiological functions of iSS.Sulfane sulfur is a class of compounds containing zero-valent sulfur. Most sulfane sulfur compounds are reactive and play important signaling roles. Key enzymes involved in the production and metabolism of sulfane sulfur have been characterized; however, little is known about how to change intracellular sulfane sulfur (iSS) levels. To accurately measure iSS, we optimized a previously reported method, in which reactive iSS reacts with sulfite to produce thiosulfate, a stable sulfane sulfur compound, before detection. With the improved method, several factors were tested to influence iSS in  Therpiration . Reagentpiration . CCCP ispiration , which ipiration . The reapiration . CommonlEscherichia coli [Saccharomyces cerevisiae can convert thiosulfate to iSS which is then reduced to sulfide for Cys biosynthesis [Schizosaccharomyces pombe directly uses sulfane sulfur to produce Cys [The production of iSS also affects its homeostasis. The metabolism of Cys is the main source of sulfane sulfur in hia coli . Cys is hia coli . Sulfateynthesis . Furtherduce Cys . Garlic duce Cys . The sup32− is used to react with unstable iSS to produce stable S2O32− under hot and alkaline conditions . S2O32− is then derived with mono-bromobimane (mBBr) to form bimane-S2O32−, which is detected by using high-performance liquid chromatography (HPLC) with a fluorescence detector. The thiosulfate content of the samples incubated in the control buffer without sulfite is the blank sample that is subtracted from the test samples [sulfite-dependent sulfane sulfur detection method (SdSS), and it is sensitive to detect active sulfane sulfur in bacteria, plants, and animals [Recently, we have developed a simple and sensitive method to detect total iSS from biological samples. SO samples . Thiosul samples , but it  samples ,35. This animals ,31,33,36 animals .2O32− and glutathione (GSH) partially overlapped, affecting the detection sensitivity of bimane-S2O32−; the washing process of preparing the bacterium samples might cause iSS loss; a fraction of iSS was converted to thiosulfate without sulfite during heating. Here, we addressed these issues and revised the method. With this improved method, we investigated the factors that could affect iSS in E. coli. The key findings included that thiosulfate is a good reagent to increase iSS without affecting bacterial growth.However, after extensive use, we noticed several shortcomings that affect the accuracy and sensitivity of this method. The peaks of mBBr derivatized SEscherichia coli BL21(DE3) was grown in the lysogeny broth (LB) at 37 °C. Sodium sulfite, sodium thiosulfate, sodium sulfate, GSH, Cys, NaHS, DTT, mBBr, tBH, DADS, TCEP, diamide (CAS: 10465-78-8), and CCCP were purchased from Sigma-Aldrich . Diethylenetriaminepentaacetic acid (DTPA), FeSO4·7H2O, acetic acid, sodium hypochlorite, and H2O2 were purchased from Bio Basic Inc . TritonX-100 was purchased from Sangon Biotech . Rosup is a compound mixture of oxidative reagents, and it is used as a positive control in the Reactive Oxygen Species Assay Kit .E. coli BL21(DE3) were picked up and cultured in LB medium overnight. The cultures were transferred into a fresh medium at an initial OD600 of 0.05 for aerobic cultures or at an initial OD600 of 0.02 for anaerobic cultures. Cells were at defined time intervals, and the OD600 value was measured with the spectrophotometer UV1800 . Three different procedures were used to harvest cells. Option 1 (No-wash): E. coli cells were transferred into a microfuge tube and harvested by centrifuging at 3300× g for 5 min at 4 °C. The supernatants were carefully removed with a pipettor, and the collected cells were used for iSS measurement. Option 2 (Double-centrifugation): after the cells were harvested and the supernatant was removed, the tubes with pellets were centrifuged again at 3300× g for 1 min. The residual supernatant was carefully removed with a pipettor. Option 3 (Wash-once): after the cells were harvested and the supernatant was removed, the collected pellets were washed once with 1 mL 100 mM Tris-HCl buffer (pH = 7.4) to remove the residual supernatant. Wash-once is the reported procedure [Colonies of 2S, avoiding the spontaneous oxidation of sulfane sulfur to thiosulfate during subsequent steps. One mL of cells at OD600 of 1.0 was harvested with double-centrifugation and resuspended in 100 μL of the reaction buffer or the control buffer. The resuspended cells were incubated at 95 °C for 10 min. The samples were then centrifuged at 15,700× g for 3 min; 50 μL of the supernatant was mixed with 5 μL of 25 mM mBBr and incubated in the dark at room temperature for 25 min to convert S2O32− into bimane-S2O32− adduct. 110 μL of an acetic acid and acetonitrile mixture  was added to stop the reaction and denature proteins. The mixtures were centrifuged to precipitate cell debris and denatured proteins at 15,700× g for 3 min.The previously reported SdSS method is designed to measure iSS . The SdS2O32− adduct in the supernatant was determined by using HPLC  with a fluorescence detector . The gain value and sensitivity of this fluorescence detector were set as “medium” and “×16”, respectively. Briefly, 5 μL supernatant was injected onto a reverse-phase C18 column  with a guard column  through an autosampler . The column was maintained at 38 °C in a column thermostat , and eluted with a gradient solution A  and solution B (0.25% acetic acid and 90% methanol in distilled water) from 8% B to 40% B in 7 min, 40% B for 5 min, 40% B to 100% in 0.1 min, 100% B for 6 min at a flow rate of 0.8 mL/min. The adduct was detected by a fluorescence detector with an optimized excitation wavelength (Ex) and emission wavelength (Em) at 380 nm and 466 nm, respectively. The bimane-S2O32− adduct was normally detected at a retention time of 13.0 min.The bimane-SE. coli BL21(DE3) were transferred into a fresh LB medium and incubated at 37 °C until OD600 reached about 1.0. The cultures were aliquoted and incubated under various conditions, including different temperatures, pH, and osmolarities, or spiked with sulfur-containing compounds , oxidants  and reductants (DTT and TCEP). DADS and CCCP were dissolved in absolute ethanol and DMSO, respectively. The other reagents were dissolved in distilled water. Fenton reagent was prepared by mixing 10 mM H2O2 with 10 mM FeSO4·7H2O. The final concentration of each reagent was given in the results. After incubation for 30 min or as specified in the text [The cells of the text , the celE. coli cells was also assayed with cells cultured under aerobic and anaerobic conditions. For aerobic growth, the cells were cultured in 50 mL of LB medium. For anaerobic growth, the cells were cultured in 100 mL serum bottles sealed with butyl rubber stoppers. 70 mL nitrogen deoxygenated LB medium was filled into the bottles before sterilization. The cells were inoculated in the bottles by using a syringe. Cells were taken at defined time intervals given in the results. The iSS content was detected by using our optimized method.The iSS content in The detection sensitivity for sulfane sulfur was improved by optimizing HPLC conditions.2O32−, which is derivatized with mBBr to bimane-S2O32− for detection [2O32− and GSH partially overlapped within the chromatogram  with or without E. coli BL21(DE3) at OD600 of 1.0 was determined. With the cells, the baseline was increased, but the detection of added S2O32− was not affected  and the control buffer (without SO32−), and heating was used to convert iSS and SO32− to S2O32− [2O32− during the heating process was tested by using DTT to reduce iSS to H2S, as DTT readily reduces reactive sulfane sulfur, such as organic persulfide, to the corresponding thiol and H2S [E. coli cells were treated in the control buffer. When 0.2 mM DTT or more was added into the control buffer, S2O32− in the control group decreased from 8.5 × 10−2 nmol/mL/OD to 1.5 × 10−2 nmol/mL/OD  in the LB medium (−3 nmol/mL/OD) (−3·nmol/mL/OD) (−3·nmol/mL/OD)  A. When tl/mL/OD) A. The unl/mL/OD) A. This dE. coli BL21(DE3) culture at OD600 were used to detect iSS contents. With the revised method, iSS concentrations could be accurately detected with 1 mL of E. coli BL21(DE3) cells at OD600 of 1.0, but the accuracy was reduced with sample volume smaller than 1 mL (Various volumes of an han 1 mL B. This oE. coli BL21(DE3) in LB medium were incubated at different temperatures for up to 30 min, the iSS contents were not significantly changed  cultures to test if they affected iSS. Cys, NaHS, DADS, and S2O32− increased the iSS content of E. coli, but GSH, SO32− and SO42− did not  culture to observe whether they were toxic to cells at different concentrations. In a closed environment, all of these sulfur compounds except S degrees . S2O32− relieved , likely l growth .2O32− at 2 mM was added into the cell cultures of E. coli in LB medium to observe the change of iSS during growth. The iSS content greatly increased in comparison with the control  are signaling molecules, and their signaling pathways may overlap ,60,61. Oe stress , and it ess [199 . Other ress [199 . Two mem and iSS ,12. Furtli cells A. The re2O32− is the only tested sulfur donor that could increase the iSS content without affecting bacterial growth (E. coli or other organisms to evaluate the effect of elevated iSS on cells. Although S2O32− may be used with other organisms to increase iSS, its concentration should be tested. For example, Saccharomyces cerevisiae is partially inhibited by 10 mM S2O32−, as high concentrations of S2O32− directly inhibit cytochrome c oxidase of the electron transport chain in the yeast mitochondria [E. coli is not inhibited by 10 mM S2O32− (2O32− in E. coli: one is catalyzed by CysM that uses S2O32− to produce Cys [2O32− to sulfane sulfur [E. coli contains eight proteins carrying RHOD domains [2O32− to sulfane sulfur.Sl growth , and it chondria . E. coliM S2O32− , likely duce Cys ,66, and duce Cys ; the othe sulfur . E. coli domains . FurtherE. coli as a model to extensively investigate the effects of different stress factors and reagents on iSS homeostasis. This work not only provides a better method for analyzing iSS in E. coli and possibly other biological samples but also investigated several factors possibly affecting iSS homeostasis, which facilitates further studies of the physiological functions of iSS.In summary, we optimized the SdSS method and used


C4A) gene expression was significantly increased following acute IFNγ exposure. This study builds on our previous work by showing that IFNγ-mediated disruption of relevant synaptic proteins can occur at early stages of neuronal development, potentially contributing to neurodevelopmental disorder phenotypes.Human epidemiological data links maternal immune activation (MIA) during gestation with increased risk for psychiatric disorders with a putative neurodevelopmental origin, including schizophrenia and autism. Animal models of MIA provide evidence for this association and suggest that inflammatory cytokines represent one critical link between maternal infection and any potential impact on offspring brain and behavior development. However, to what extent specific cytokines are necessary and sufficient for these effects remains unclear. It is also unclear how specific cytokines may impact the development of specific cell types. Using a human cellular model, we recently demonstrated that acute exposure to interferon-γ (IFNγ) recapitulates molecular and cellular phenotypes associated with neurodevelopmental disorders. Here, we extend this work to test whether IFNγ can impact the development of immature glutamatergic neurons using an induced neuronal cellular system. We find that acute exposure to IFNγ activates a signal transducer and activator of transcription 1 (STAT1)-pathway in immature neurons, and results in significantly increased major histocompatibility complex I (MHCI) expression at the mRNA and protein level. Furthermore, acute IFNγ exposure decreased synapsin I/II protein in neurons but did not affect the expression of synaptic genes. Interestingly, complement component 4A ( Althougophrenia , 13, redophrenia , and a mophrenia .One key feature of the maternal immune response that shapes these phenotypes is the elevation of numerous cytokines in the maternal serum, placenta and fetal brain , 17. Conin vitro manipulations of MHCI expression inversely affecting the density of both GABAergic and glutamatergic synapses in rat and mouse cultures  and neurons derived from human induced pluripotent stem cells (iPSCs) to IFNγ results in gene expression changes in genes associated with schizophrenia and autism, and altered neuronal morphology in exposed neurons . In partcultures . In a mocultures . Genome-cultures , 34. Forcultures .NGN2-iNs)  term “synapses” in exposed iPSC-neurons . Given tGN2-iNs) , we findThe ioGlutamatergic male neurotypical stem cell line  was obtaTM tissue culture plates  at a density of 500,000 cells/well, with dedicated wells for treatment and vehicle treatments. The day after plating, the cells were exposed to 25 ng/ml IFNγ or vehicle and incubated for 24-h before sample collection.In parallel, the 127_CTM_01 human iPSC male neurotypical line  was diffNGN2-iNs were prepared from day 3 cells following treatment. Cells were lysed in RIPA buffer , 5 mM EDTA, 0.1% SDS (weight/volume), 1% Triton X-100 (volume/volume), 1% deoxycholate (weight/volume), and inhibitors), before being sonicated with 10 short bursts. Sample buffer was added to all samples, which were then denatured for 5 min at 95°C and stored at −80°C until used further. All samples (5 μg) were subsequently separated by SDS-PAGE and analyzed by Western Blotting with antibodies against phospho-STAT1, phosphor-ERK1/2, ERK1/2, and GAPDH , and then fixed with ice cold Methanol at 4°C for 10 min, then washed 2× with DPBS. Cells were permeabilized and blocked using 2% normalized goat serum (NGS) in DPBS with 0.1% triton x-100 for 2 h. Antibody solutions  were preCoverslips were imaged using a Leica SP5 confocal microscope. The gain and other imaging parameters were set using the vehicle control and were not changed during subsequent imaging of the control and IFNγ exposed coverslips with 246.5x246.5 μm regions imaged. The Z-stack thickness was kept at 0.5 μm and Z-stacks were then maximally projected to form a single image in FIJI. Prior to measuring fluorescent intensity, the background of each image and channel was measured in FIJI by selection of 10 25×25-pixel areas of background and measuring the mean and standard deviation (SD) of staining intensity of each area. The mean of these measurements + 2SD was then subtracted from the image. Cell Profiler  was used2O. A nanodrop spectrophotometer was used to measure RNA concentration and quality.Cells for RNA extraction were lysed in TRI Reagent  for 5 min at room temperature and RNA was extracted from TRI Reagent according to the manufacturer's protocol. Isolated RNA was cleaned by precipitation with 3% sodium acetate in ethanol at −80°C overnight, washed as in the isolation protocol, and resuspended in HFor cDNA synthesis, a mixture of 1 μl of oligo(dT)20 (50 μM) , 2 μg total RNA, 1 μl 10 mM dNTP Mix (10 mM each) , and water to make up a total of 13 μl per sample was heated to 65°C for 5 min and incubated on ice for 1 min. Next, superscript mastermix  was added to each sample , 1 μl of SuperScript III RT (200 units/μl)) and the mixture was incubated at 50°C for 50 min and then 70°C for 15 min. qPCR was done in a 348 well-plate, with two technical replicates per sample, and also a blank well-containing no cDNA for each primer pair . Three h−ΔΔCt method  were analyzed, where each replicate is the same cell line but with a different passage number and differentiated on a different day. The number of replicates was decided prior to the conducting of the experiments. Statistical analysis was done in Prism 9.0.2. The exposed and control mean intensity values (ICC) or log (2−ΔΔCt) values (qPCR) were compared using multiple 2-tailed unpaired t-tests, and corrected for multiple comparisons using the Holm-Šídák method.For both the ICC and qPCR experiments, three biological replicates (n = 12) were annotated in the presynapse cluster with four genes enriched for the synaptic vesicle membrane term. These results suggest that acute IFNγ exposure leads to the downregulation of 18 genes that exert presynaptic functions and regulate synaptic vesicle mechanisms in iPSC-neurons.In the RNA sequencing data from our previous study, we found downregulation of genes related to the GO term “synapses” in human iPSC-NPCs exposed to IFNγ for 24 h . To explNGN2 optimized inducible overexpression to allow for rapid and reliable generation of NGN2-induced neurons (NGN2-iNs) upon treatment with doxycycline  and excitatory presynaptic marker vesicular glutamate transporter 1 (VGLUT1) , 42NGN2 (VGLUT1) . After 2(VGLUT1) . This is(VGLUT1) . Analysith cones , suitablNGN2-iNs using immunocytochemistry (ICC) and quantitative PCR (qPCR). We stained for the post-mitotic neuron marker neuronal nuclei antigen (NeuN) and mature neuron marker microtubule-associated protein 2 (MAP2) and the neuroprogenitor markers nestin (NES) and PAX6. In addition, staining was conducted for the early neuron/late progenitor marker Class III β-Tubulin (TUBB3). Qualitatively, all imaged Day 4 NGN2-iNs expressed both the neuroprogenitor markers nestin and PAX6 and the neuronal markers NeuN and MAP2 = 27.97, P = 0.00001) was significantly increased in the exposed neurons -dependent pathway, which in turn regulates the transcription of target genes . We thusNGN2-iNs . First, exposure . No increxposure , we furtith IFNγ . IFNγ trobserved . We furt neurons . TogetheNGN2-iNs following treatment with IFNγ for 24 h. Under baseline conditions, MHCI localized to the cell body, processes, and growth cones of all Day 4 NGN2-iNs (NGN2-iNs compared to the control ((43)= 2.920, P < 0.0001); increased in the cytoplasm by 56.3% (t(40)= 4.723, P < 0.0001); cell body by 34.6% (t(40)= 3.819, P = 0.0005); and in neurite processes by 52.5% (t(40)= 4.331, P < 0.0001) ((43)= 1.937, P = 0.06). No change in neurite morphology was observed 24 h after IFNγ exposure  . MHC I iexposure .NGN2-iNs. Synapsin was selected as an early synaptic marker, since this protein is expressed in NPCs and colocalizes with constitutively recycling vesicles along the whole surface of developing axons that then localize to forming synapses = 2.303, P = 0.0261), cell body by 23.7% (t(43)= 2.300, P = 0.0263), and cytoplasm by 31.1% (t(43)= 3.339, P = 0.0017) ((43)= 1.840, P = 0.0726, unpaired t-test) , which did not change  in the IFNγ-exposed condition .In contrast to the effects on MHCI, synapsin I/II appeared to be decreased in IFNγ-exposed neurons. Specifically, the asymmetrically localized clusters of synapsin I/II vesicles in the shaft and cell body appeared reduced in some exposed neurons, while others had intensity that is similar to control neurons . Quantif 0.0017) . The mea t-test) . These rSYN1, DLG4, SV2A, and GRIN1 were not significantly different from vehicle conditions = 11.84, P = 0.000291). We validated these findings using dual SMAD inhibition differentiated 127_CTM iPSC line NPCs exposed to IFNγ at day 26 to ensure that the observed effects were not cell line specific. As seen in treated NGN2-iNs, IFNγ caused an increase in HLA-B (t(4)= 22.53, P = 0.000023) and C4A expression (t(4)= 6.466, P = 0.002947) in treated NPCs   . Future C4A after acute IFNγ exposure. C4A mRNA levels are increased in post-mortem cortical brain tissue from individuals with schizophrenia and C4A variants are associated with elevated risk for schizophrenia  Centre grant (MR/N026063/1). AP and RM are in receipt of the MRC-Sackler Ph.D. Programme studentship as part of the MRC Centre for Neurodevelopmental Disorders . LS was supported by the UK Medical Research Council (MR/N013700/1) and King's College London member of the MRC Doctoral Training Partnership in Biomedical Sciences. LD was supported by a research grant from the University of Pennsylvania Autism Spectrum Program of Excellence awarded to DS. DS was also supported by an Independent Researcher Award from the Brain and Behavior Foundation  (Grant No. 25957). AV acknowledges financial support for this study from the National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC/S001506/1).bit.bio. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.Authors AV and DS receive research funding from All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Sophoraflavescens, has been shown to regulate immunity and protect neurons in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis and ON. However, the protective mechanism of MAT on RGCs is largely unknown. In this study, we show that MAT treatment significantly reduced the degree of inflammatory infiltration and demyelination of the optic nerve and increased the survival rate of RGCs. The expression of Sirtuin 1 (SIRT1), a member of an evolutionarily conserved gene family (sirtuins), was upregulated, as well as its downstream molecules Nrf2 and PGC-1α. The percentage of TOMM20-positive cells was also increased remarkably in RGCs after MAT treatment. Thus, our results indicate that MAT protects RGCs from apoptosis, at least in part, by activating SIRT1 to regulate PGC-1α and Nrf2, which, together, promote mitochondrial biosynthesis and reduce the oxidative stress of RGCs.Optic neuritis (ON), characterized by inflammation of the optic nerve and apoptosis of retinal ganglion cells (RGCs), is one of the leading causes of blindness in patients. Given that RGC, as an energy-intensive cell, is vulnerable to mitochondrial dysfunction, improving mitochondrial function and reducing oxidative stress could protect these cells. Matrine (MAT), an alkaloid derived from  Optic neuritis (ON), a common cause of visual defects in young people, is an inflammatory disease of the optic nerve, which is characterized by immune cell infiltration and subsequent demyelination . Patientvia regulating the expression of peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), a metabolic coactivator that induces mitochondrial biogenesis and respiration by interacting with transcription factors  . Because factors . SIRT1 a factors , a basic factors .Matrine (MAT), a natural alkaloid component extracted from the herb Radix Sophorae Flavescentis, with a molecular weight (MW) of 258.43 (C15H24N2O), has been widely used in the clinical treatment of human hepatitis B and leukocytopenia with very few adverse reactions . PreviouFemale Wistar rats  were purchased from the Jinan Pengyue Experimental Animal Breeding Co., Ltd., China, and raised under the condition of specific pathogens at the Laboratory Animal Center of the Henan Academy of Chinese Medicine, China. EAE was induced as described previously . BrieflyThis study was approved by the Ethics Committee of Scientific Research of Henan Academy of Chinese Medicine; the ethical review number is HNTCMDW-20170601. All of the protocols were approved, and every effort was made to ensure minimal animal suffering.n = 10, each group)  : EAE rath group)  (Jiangsuh group) ; and nonh group) . From thThe rats were sacrificed on day 17 p.i. After extensive perfusion with physiological saline solution, the optic nerves and retinas were removed and post-fixed with FAS eyeball fixative . The tissue was then embedded in paraffin and cut into 2–5 μm thick sections, dewaxed in xylene, and rehydrated. Hematoxylin–eosin (H&E) staining was used to detect inflammatory infiltration, and Luxol fast blue (LFB) staining was used to detect demyelination. The degree of infiltration of inflammatory cells and demyelination in the optic nerve was assessed by a double-blind investigator, similar to the open standard. For inflammation, 0 = no infiltration, 1 = a little cell infiltration of the optic nerve or optic nerve sheath, 2 = moderate infiltration, 3 = serious infiltration, and 4 = substantial infiltration. For demyelination, 0 = no demyelination, 1 = scattered demyelinating lesions, 2 = partial demyelinating lesion, and 3 = large number of demyelinating lesions. Scores of inflammation, infiltration, and demyelination were evaluated by Image-Pro Plus 6.0 software. Each experiment was repeated three times, and the results were averaged.Briefly, non-specific binding was blocked with 3% bovine serum albumin (BSA)  and permeabilized with 0.3% Triton X-100 in 1% BSA-PBS for 30 min. The sections were then incubated in blocking solution at 4°C overnight with primary antibodies specific for rabbit anti-Nrf2 (1:100), rabbit anti-SIRT1 (1:100), rabbit anti-PGC-1α (1:300), and rabbit anti-TOMM20 (1:250)  and then incubated with secondary antibody donkey anti-rabbit FITC  at room temperature (RT) for 2 h. After being permeabilized with 1% BSA-PBS for 3 × 5 min, the sections were incubated with rabbit anti-Brn3a  specific primary antibody in blocking solution overnight at 4°C, then incubated with secondary antibody donkey anti-rabbit Cy3  at RT for 2 h, mounted with 4’,6-diamidino-2-phenylindole , washed with PBS, cover-slipped, and examined under a fluorescence microscope . As a negative control, additional sections were treated similarly, but the primary antibodies were omitted. All pictures were captured by a confocal microscope (Olympus Fluoview FV1000). For each group, ten sections were examined in a blinded fashion. Image-Pro Plus 6.0 software was used to calculate the quantification of target protein expression.2 at 37°C. RGC-5 cells were seeded into 6-well plates. Cells were treated in four groups: for the control group, 2 ml complete medium was added. For the TNF-α group, 2 ml of TNF-α  at a concentration of 50 ng/ml was added to the medium. For the MAT group, 2 ml (50 ng/ml) of TNF-α and 100 μM of MAT were added to the medium. For the MAT + Ex-527 group, 2 ml (50 ng/ml) of TNF-α, 100 μM of MAT, and 38 nM of EX527  were added. All groups were cultured for 48 h before harvest.Rat retinal ganglion cells (RGC-5) were purchased from iCell Bioscience Inc., Shanghai, China. Cells were maintained in Dulbecco’s modified eagle medium  supplemented with 10% heat-inactivated fetal bovine serum  and in a humidified atmosphere containing 5% COAfter treatment, total protein from both cell lysate and the supernatant was isolated from N9 cells with RIPA lysis buffer  including the protease and phosphatase inhibitor . For Western blot analysis, 8%–10% SDS-PAGE was used to resolve equal amounts of protein samples from both cell lysate and supernatant. The gel was transferred onto polyvinylidene fluoride (PVDF) membranes , and the membranes were blocked with 5% BSA in Tris-buffered saline containing 0.05% Tween-20 (TBST). Membranes were probed with primary antibodies at 4°C overnight. The following day, the incubated membranes were washed four times with TBST and then incubated with the horseradish peroxidase (HRP)-conjugated secondary antibodies. The antigen–antibody complex was screened by chemiluminescence using the Supersignal West Dura ECL reagent . Protein bands were detected with a densitometer . Band density analysis was performed with ImageJ software .® easyCyteTM). Rhodamine 123 is a cationic fluorescent dye. Treated cells were harvested and washed once with PBS and incubated with 500 μl of Rho123 dye (2 μM) for 30 min at a 37°C shaker incubator. After incubation, cells were washed with PBS and analyzed immediately by an FACS analyzer.For the measurement of mitochondrial membrane potential (ΔΨM), cells were harvested and stained with JC-1  and Rhodamine 123  and were quantified by flow cytometry analysis as described previously . JC-1 dyp < 0.05 was considered significant. Given that ON can occur bilaterally or unilaterally in either eye  was used for statistical analyses; ther eye , each eyAs we reported before, MAT effectively suppresses CNS inflammation, demyelination, and axonal loss in optic nerves, as well as RGC apoptosis in EON . BrieflyTo investigate the effect of MAT treatment on SIRT1 expression, we tested the number of cells that colocalized SIRT1 with Brn3a (RGCs marker) by immunofluorescence double staining. As shown in Nrf2 is a key factor in the oxidative defense system, and SIRT1 is involved in the expression and activation of Nrf2. We, therefore, studied Nrf2 expression on the retina by immunofluorescence double staining to illustrate the effect of MAT on oxidative stress in ON . The expThe PGC-1α expression has been considered to play a non-negligible role in mitochondrial biosynthesis. Therefore, we used immunofluorescence double staining to study the PGC-1α in the retina on the ON model . Our resTo confirm that MAT could prevent RGC death through the SIRT1-PCG-1α/Nrf2 pathway, we used the RGC-5 cell line to perform the experiment. The PCG-1α and Nrf2 protein expression in RGC treated by TNF-α were attenuated compared to the control group, as well as the SIRT1 expression. MAT co-treatment could improve the expression of SIRT1, PCG-1α, and Nrf2 significantly. This effect could be revised by EX527, a SIRT1 inhibitor .Previous studies had shown that MAT protected mitochondrial membrane integrity by inhibiting cytochrome c (Cyt c) release . TherefoMitochondrial membrane potential (ΔѰM) reflects the functional status of the mitochondrion. We used JC-1 and Rho123 for the qualitative measurement of ΔѰM in RGC-5 cells. Cells with healthy mitochondria show high ΔѰM, whereas cells with damaged mitochondria show low ΔѰM. FACS analysis showed that, with the exposure of TNF-α to RGC-5 cells for 48 h, a significant decrease in ΔѰM was observed, and this was revised by 100 μM MAT, whose effect could be attenuated by Ex527 . To suppAxons of RGCs, which form the optic nerve, are demyelinated in various optic neuropathies, including ON , and havin vivo and in vitro, suggesting that MAT may regulate mitochondrial function by upregulating the SIRT1 expression of RGCs, thereby preventing their loss in ON.Mitochondria are cytoplasmic organelles responsible for producing adenosine triphosphate (ATP), and they play an important role in regulating cellular calcium metabolism, reactive oxygen species (ROS) production, and apoptosis . It has Mitochondrial oxidative stress is generally considered to be the major agent of many neurodegenerative diseases such as MS and ON . Nrf2 isIt has been shown that the activity of Nrf1 and Nrf2 can be induced by the PGC-1 transcriptional coactivators family, composed of PGC-1α, PGC-1β, and PGC-1-related coactivators. This activity leads to the transactivation of many genes encoding mitochondrial biogenic specific proteins . PGC-1α In conclusion, our study shows that MAT can effectively inhibit the disease progression of ON and protect RGCs. MAT activates the SIRT1, PGC-1α, and Nrf2 expression in the retina to promote mitochondrial biosynthesis and reduce oxidative stress, which could be revised by the SIRT1 inhibitor. Thus, we believe that MAT treatment could protect RGCs from apoptosis in ON by the SIRT1-PGC-1α/Nrf2 pathway.


Trichoderma harzianum (F1), Trichoderma asperellum (F2) and Trichoderma viride (F3), to improve the growth and physiology of Zea mays L. plants grown on soil contaminated with Cu and Ni. Initially, a biosorption trial was conducted to test the HM removal efficiency of species F1, F2 and F3. Among them, F2 sp. showed the maximum Cu and Ni removal efficiency. Then, a pot study was conducted with two cultivars (spring corn and footer corn) having eleven treatments with three replicates. The results demonstrated a significant genotypic variation among both cultivars under applied HM stress. The maximum decreases in leaf Chl a. (53%), Chl b. (84%) and protein (63%) were reported in footer corn with applied Cu stress. The combined application of biochar and F2 increased leaf CAT (96%) in spring corn relative to Cu stress. Altogether, it was found that BC + F2 treatment showed the maximum efficiency in combatting Cu and Ni stress in spring corn.Crop cultivation in heavy metal (HM)-polluted soils is a routine practice in developing countries that causes multiple human health consequences. Hence, two independent studies have been performed to investigate the efficiency of rice husk biochar (BC) and three fungal species,  According to the Pakistan Metrological Department, the average maximum and minimum temperature is 42 °C and 25 °C, respectively, and the annual rainfall is about 626 mm at the region of study site. Bhal soil was used for the experiment and characterized as [Soil texture (clay loam), Sand (37%), Silt (34%), Clay (35%), organic matter (0.88%) ,55, pH (−1) , Cation boratory .T. asp, T5 = BC (2%), T6 = T. asperellum + Cu (300 ppm), T7 = T. asperellum + Ni (100 ppm), T8 = BC (2%) + Cu (300 ppm), T9 = BC (2%) + Ni (100 ppm), T10 = T. asperellum + BC (2%) + Cu (300 ppm) and T11 = T. asperellum + BC (2%) + Ni(100 ppm) applied in the pots, each with three kg of soil. Each treatment was replicated three times and an adequate amount of water was applied to all pots to maintain soil field capacity.Two maize cultivars “spring corn” (V1) and “footer corn” (V2) were selected for the experiment. There were in total 11 treatments: T1 = Control, T2 = Cu (300 ppm), T3 = Ni (100 ppm), T4 = 7 conidia mL−1 using a Neubauer haemocytometer. This dilution was added in soil @ 10% per 1 Kg of soil to carry out the pot experiment. Different proportions of treatments were added into the pots for both cultivars. A conidial suspension was prepared using a scraping method in sterile distilled water, as fungal cultures could not be applied directly into soil. For this purpose, the fungal mycelium was collected by adding 5 mL autoclaved water to each Petri dish and the surface was scraped with a spatula. The conidia number per mL was adjusted to 1.0 × 10After the 2 days of applying treatments, 6–7 seeds of maize were sown in each pot. After 8 days of sowing, Urea and DAP were added in the pots, respectively. After 3 months, the plants were harvested; roots and shoots of plants were carefully extracted and washed with distilled water to remove unwanted materials. Plants and soil samples were stored in appropriately labelled zipper bags. Then, soil and plants were carried into the laboratory for further analysis.g for 5 min. Absorbance was recorded using UV/visible spectrophotometer  at 663 nm and 645 nm.The length and fresh weight of roots and shoots were recorded using standard measuring tape and a weighing balance. To analyze dry weight, fresh plants were first dried in open air for 2 days followed by in a hot oven at 65 °C for 24 h. The proposed method of Du  was used2O (1800 µL) and 2 mL of Bradford reagent was prepared and incubated at room temperature for 10–20 min. After incubation, absorbance was recorded using a UV/visible spectrophotometer  at 595 nm.The concentrations of protein in root, stem and leaves of maize plants were determined using the method of Metwally . A total2O (1580 µL), Na2CO3 (300 µL) and 100 µL of Folin-Coicâlteu’s reagent was prepared and incubated in the dark at room temperature for 2 h. A spectrophotometer was used to measure the absorbance at 760 nm after incubation.The concentrations of total phenolics in root, stem and leaves of maize plants were determined using the method of Ahmed . A reactThe Di-Acid digestion method was used to analyse the concentrations of HMs such as Cu and Ni in maize and soil. Concentrations of HMs were analysed using a multi-sequential AAS (iCE 3000 SERIES) against prepared standards of known concentration .g for 15 min. In a dry oven, a reaction mixture containing sample extract, TCA and TBA (thiobarbituric acid) was heated for 30 min at 95 °C and then cooled at freezing temperature. The absorbance was recorded at 532 nm and 600 nm using a UV/visible spectrophotometer . The concentration of MDA was determined using Beer and Lambert’s equation [MDA concentrations were analysed using the protocol of Kanwal . For thiequation .2O2, fresh maize samples (0.5 g) of each plant part were ground with 5 mL of Trichloroacetic acid (TCA) (0.1%) solution [g for 15 min. After centrifugation, a reaction mixture was prepared using 1 mL of supernatant, 1 mL 10 mM K-P buffer (pH 7.0) and 2 mL 1M potassium iodide (KI) solution. The concentration of H2O2 was calculated by recording the absorbance of the mixture at 390 nm.To determine Hsolution . The mixg at 4 °C for 20 min. Supernatant (sample extract) was used for further analysis [To calculate the concentration of Superoxide Dismutase (SOD), an Ascorbate peroxidase (APX), Catalase (CAT) and Guaiacol peroxidase (POD) enzyme assay was prepared using 1 g of fresh root, stem and leaf in potassium phosphate buffer followed by centrifugation at 15,000× analysis .For SOD, a reaction mixture was prepared using sample extract (70 μL), 1630 μL of sodium carbonate buffer (pH = 10.2), 500 μL of nitroblue tetrazolium (24 μM), 100 μL of ethylenediamine tetraacetic acid (EDTA) (0.1 mM), 100 μL of hydroxylamine hydrochloride (1 mM) and 100 μL of Triton-X-100 (0.03%). To express the activity of SOD, absorbance was recorded at 560 nm using a spectrophotometer  .2O2. To express the activity of APX, absorbance was recorded at 290 nm using a spectrophotometer  [2O2 (12 mM). To express the activity of POD, absorbance was recorded at 436 nm using a spectrophotometer  [For APX, a reaction mixture was prepared containing sample extract, 50 mM K-P buffer (pH 7.0), 0.5 mM Ascorbate and 0.5 mM H, Japan) . To calc, Japan) . For POD, Japan) .2O following the protocol of Hailegnaw [Soil pH was measured by forming a soil suspension of 50 g soil sample in 50 mL d.Hailegnaw . After 1ailegnaw . Soil mo® and Statistix 8.1®  tools were used to analyze the data. A two-factor factorial design  was used to statistically analyze the datasets of the above-mentioned treatments. To compare the mean groups, the Least Significant Difference (LSD) test was applied through Statistix 8.1® [n = 3) with standard error ± (SE).Microsoft Excel 2019tix 8.1® . To findtix 8.1® . The resT. asperellum species were screened out initially from the biosorption trial on the basis of their performance against Cu and Ni stresses; latterly, T. asperellum species with BC provided significant results by improving physiological and biochemical parameters of maize cultivars against Cu stress. Hence, applications of BC with interaction of fungi proved to be a cost-effective and eco-friendly soil conditioner in developing countries including Pakistan to limit the deleterious effects of HM toxicity.There was significant genotypic variation shown by both maize cultivars, and spring corn proved to be a tolerant cultivar, compared to footer corn, against applied HM stress.


Depsides and tridepsides are secondary metabolites found in lichens. In the last 10 years, there has been a growing interest in the pharmacological activity of these compounds. This review aims to discuss the research findings related to the biological effects and mechanisms of action of lichen depsides and tridepsides. The most studied compound is atranorin, followed by gyrophoric acid, diffractaic acid, and lecanoric acid. Antioxidant, cytotoxic, and antimicrobial activities are among the most investigated activities, mainly in in vitro studies, with occasional in silico and in vivo studies. Clinical trials have not been conducted using depsides and tridepsides. Therefore, future research should focus on conducting more in vivo work and clinical trials, as well as on evaluating the other activities. Moreover, despite the significant increase in research work on the pharmacology of depsides and tridepsides, there are many of these compounds which have yet to be investigated . Usnea species has been used in many cultures around the world for its antiseptic, wound healing, antibacterial, and anti-inflammatory properties. Traditional knowledge and confirmed activity studies led to the improvement of its pharmacological potential by new pharmaceutical formulations. Popovici et al. have developed bioadhesive oral films with Usnea barbata extract in Canola oil as an effective oral formulation   .50 values > 50 µg/mL), Hela cells , and A549 cells and LS174 cells  [Studies on the cytotoxic activity of lecanoric acid showed that this depside has moderate activity against colon HCT116 cancer cells and reduced cell colony formation by decreasing Axin2 expression and M phase arrest  ,96. Leca0 μg/mL) . Moreove0 μg/mL) .50 value of 350 µM [50 31 μM) [Finally, in vitro studies revealed that lecanoric acid exhibited effective α-glucosidase inhibitory activity (85.9% of inhibition) with an ICf 350 µM  and mode0 31 μM) .Bacillus cereus  and Candida albicans . Furthermore, methyl evernate had low DPPH radical scavenging activity  and higher reducing power than acetone extracts of Ramalina spp. [50 value of 46.45 μg/mL), the human lung carcinoma A549 cell line , and the human colon carcinoma LS174 cell line .Methyl evernate has displayed antimicrobial activity against bacteria and fungi and is especially active against ina spp. . MoreoveKlebsiella pneumoniae, Pseudomonas aeruginosa, and Pseudomonas syringae) and against 7 of 11 species of fungi  [Olivetoric acid has antimicrobial activity against a wide range of bacteria, yeast, and fungi. This compound was active against 12 of 15 species of bacteria and yeast (it was inactive in raminis) .50 value of 17.55 mg/L) and primary rat cerebral cortex (PRCC) cells  via oxidative stress-induction, as evidenced by the lactate dehydrogenase (LDH) activity and oxidative DNA damage [Olivetoric acid showed slight to moderate antioxidant properties in cultured human amnion fibroblasts  and in cultured human lymphocytes (HLs)  ,101. FinA damage . MoreoveA damage . FurtherA damage .Staphylococcus aureus strains, with an MIC90 value of 32 µg/mL, and it showed a synergic action with gentamicin and an antagonism action with levofloxacin [Perlatolic acid has good antimicrobial properties against methicillin-resistant floxacin .50 6.8 μM) [50 0.4 µM), 5-lipoxygenase (IC50 1.8 µM for cell-based assay and IC50 0.4 µM for purified enzyme), tumor necrosis factor alpha-induced nuclear factor kappa B (IC50 7 µM), and leukocyte recruitment [This compounds also exerted neurobiological processes such as neuroprotection, neurotrophicity, and neurogenesis. Hence, this depside acts as a neurotrophic and proneurogenic agent (125.34 μm in neurite outgrowth at 0.5 μM) by inducing the upregulation of neurotrophic genes (BDNF and NGF). This neurotrophic activity is also related to the increased histone acetylation of H3 and H4 protein in a mouse neuroblastoma (Neuro2A) cell line. Moreover, this secondary metabolite was a potent acetylcholinesterase (AChE) inhibitor (IC 6.8 μM) . Moreoveruitment ,107. Furruitment .50 value of 324.61 μg/mL and reducing power of 0.0142 at 125 µg/mL) [50 values of 43.24 μg/mL, 93.98 μg/mL, and 74.28 μg/mL [Ramalic acid/Obtusatic acid was active as an antimicrobial agent against five bacteria and ten fungal species with MIC values from 0.125 to 1 mg/mL . Moreove28 μg/mL ,76.Escherichia coli (78% inhibition), moderate activity against Streptococcus mutans, Staphylococcus aureus, and Streptomyces viridochromogenes , and low activity against Bacillus subtilis (15% inhibition) [50 5.69 µg/mL) and a respiratory syncytial virus A2 strain (IC50 7.7 µg/mL) by interfering with viral replication at a viral post-entry step. Its antiviral action is even higher than the reference compound ribavirin [50 value of 41.5 µg/mL), ferric ion assay , DPPH assay , and SOR assay ; this is attributed to the three hydroxyl groups of its structure [Sekikaic acid displayed maximum antimicrobial activity against ibition) . In addiibavirin . Moreovetructure ,109,111.tructure .Staphylococcus aureus (MIC = 1250.0 mg/mL) [50 value of >200 µM) [Squamatic acid showed weak antimicrobial activity against 0 mg/mL) . Moreove>200 µM) .Alternaria alternata, Aspergillus fumigatus, and Sclerotium rolfsii, with MIC values of 400, 400, and 200 µg/mL, respectively), yeast , and bacteria  [50 value of >200 µM) [Thamnolic acid showed antimicrobial activity against fungi (0 µg/mL) . On the >200 µM) .The biological activities and tridepside chemical structures have been gathered in B. subtilis [Bacillus cereus and Bacillus subtilis and the fungi Candida albicans and Candida glabrata [Culiseta longiareolata, with LC (50) and LC (90) values of 0.41 ppm and 1.93 ppm, respectively [Gyrophoric acid is a potent antimicrobial agent against a wide range of bacteria and fungi, with MIC values from 0.019 mg/mL for subtilis . Moreoveglabrata . Furtherectively .50 values for DPPH and superoxide anion scavenging were 105.7 µg/mL and 196.6 µg/mL, respectively, and its reducing power value was 1.32 at 1000 µg/mL [Gyrophoric acid has also demonstrated potent antioxidant properties. Hence, the IC00 µg/mL .Gyrophoric acid has been investigated for its role as a cytotoxic agent against different cancer cells. This compound reduced the cell viability of human ovarian carcinoma (A2780 cells), human promyelocytic leukemia (HL-60 cells), human T cell lymphocyte leukemia (Jurkat cells), malignant melanoma (Fem-x cells), and chronic myelogenous leukemia (K562 cells) ,117. In 50 value of 1.7 µM by a cytostatic mechanism [Other studies, based on the antiproliferative capacity of gyrophoric acid, have investigated its effect on skin cells for therapeutic purposes for psoriasis. This tridepside significantly inhibited the growth of the human keratinocyte HaCaT cell line, with the ICechanism . Moreoveechanism ,121. Antechanism .50 value of 3.6 μM, making it a drug candidate for type 2 diabetes and obesity [Gyrophoric acid has been shown to have antihypertensive properties by acting as an angiotensin II type-1 receptor (AT1) antagonist by interacting with residues ARG167, TRP84, and VAL108 . Moreove obesity . Finally obesity .50 152.6 μM), the human cancer colon WIDR cell line (ED50 95.9 μM), and the human cancer pancreas PANC-1 cell line (ED50 87.9 μM), which seems to be related to its ability to inhibit 5-lipoxygenase activity [50 100 µM) [Tenuiorin acid showed weak to moderate antiproliferative action against the human cancer breast T-47D cell line (EDactivity . Using a 100 µM) .50 value of 173 nM. Moreover, this tridepside improved the insulin-stimulated glucose uptake through the insulin receptor (IR)/IRS/Akt/GLUT2 pathway in the human liver HepG2 cancer cell line. Furthermore, in vivo studies demonstrated the beneficial effects of trivaric acid as an antidiabetic agent at doses of 5 mg/kg and 50 mg/kg through significantly improving lipid and glycemic profiles [The tridepside trivaric acid has resulted in being a promising antidiabetic agent. In silico and in vitro studies revealed that this compound inhibited protein tyrosine phosphatase 1B (PTP1B) by blocking its active site with an ICprofiles ,126.50 value of 1.8 µM) [In another study, trivaric acid exerted a potent anti-inflammatory action by significantly inhibiting human leukocyte elastase (IC 1.8 µM) .Most of the works on the pharmacological activity of depsides and tridepsides have been published in the last 10 years, which shows the growing therapeutic interest in the secondary metabolites of lichens. Most of these works are in vitro studies, with the occasional in silico and in vivo studies.Bacillus and Staphylococcus, followed by Mycobacterium, Streptococcus, and Enterococcus. Among the Gram-negative bacteria, the genera Escherichia and Proteus were the most investigated. In general terms, depsides and tridepsides showed weak to moderate antimicrobial activity, being more potent against Gram-positive bacteria. In addition, the antifungal activity of several of these compounds  has mainly been studied against Candida spp. Moreover, some depsides displayed good antiparasitic activity against Plasmodium falciparum and Schistosoma mansoni.Lichens have been investigated for their ability to inhibit bacterial growth. The most common Gram-positive bacteria genera studied on depsides and tridepsides are Moreover, antioxidant activity has also been widely investigated using different in vitro techniques, such as DPPH assay, hydroxyl radical scavenging activity, superoxide radical scavenging activity, and reducing power assay. Depsides and tridepsides have a phenolic structure that provides antioxidant properties. The compounds with the greatest capacity to scavenge free radicals are sekikaic acid and atranorin.The cytotoxic activity of depsides and tridepsides has been extensively investigated in in vitro studies. The mechanisms involved in the cytotoxicity of these compounds imply oxidative stress induction (ROS overproduction), apoptosis induction , cell-cycle arrest, and 5-lipoxygenase antagonist therapy.Despite the interesting activities that have been compiled in this review, the information seems to stop after the in vitro assays with diverse cell lines. Only a few studies have continued with the in vivo model. The great variety of activities indicate low specificity, which must be deepened with regard to structure–activity studies and toxicological studies. Preclinical and clinical studies should focus on identifying the molecular targets for the action and the nontoxic doses in humans. Furthermore, there are also several compounds for which there is no study of pharmacological activity, such as hiascic acid, lassalic acid, ovoic acid, crustinic acid, and hypothamnolic acid, all being potential metabolites to be investigated. On the other hand, new technology advances will allow the improvement of growing yields and compound extraction, solving the current problem that limits the study with these interesting compounds


Solanum tuberosum L.) is vulnerable to high temperatures, which are expected to increase in frequency and duration due to climate change. Nondestructive phenotyping techniques represent a promising technology for helping the adaptation of agriculture to climate change. In this study, three potato cultivars  were grown under four temperature treatments: 20/15 °C (T1), 25/20 °C (T2), 30/25 °C (T3), and 35/30 °C (T4). Multispectral and chlorophyll fluorescence imaging, 3D multispectral scanning, and gas exchange analysis were used to study the effect of moderate heat stress on potato morphology and physiology and select phenotypic traits most responsive to increased temperatures. The most responsive morphological traits to increased temperatures are related to decreased leaf area, which were detected already at T2. Increased temperatures  also changed leaf spectral characteristics, indicated by increased red, green, and blue reflectance and decreased far-red reflectance and anthocyanin index (ARI). Regarding chlorophyll fluorescence, increasing temperatures (T2) caused an increase in minimal fluorescence of both dark-adapted (F0) and light-adapted (F0’) plants. Stomatal conductance, transpiration rate, photosynthetic rate, instantaneous water use efficiency (WUE), and intrinsic water use efficiency increased from T1 to T3 and decreased again in T4. Using recursive partitioning analysis, the most responsive potato phenotypic traits to increased temperature were leaf area projected (LAP), ARI, F0, and WUE. These traits could be considered marker traits for further studying potato responses to increased temperatures.Potato ( Solanum tuberosum L.) represents one of the most important food crops in the world, along with wheat (Triticum aestivum L.), maize (Zea mays L.), and rice (Oryza sativa L.) [Potato (tiva L.) . Grown wtiva L.)  and has tiva L.) , which mtiva L.) . Howevertiva L.) . In additiva L.) . Potato tiva L.) , and it tiva L.) .Tuber initiation, induction, set, and tuber bulking are delayed or even inhibited as the temperature increases from 15 to 23 °C . In addiOne of the critical steps for adapting agriculture to climate change and new growing conditions will be adopting and implementing new technologies ,18. ProxThis study aims to evaluate the effect of increased temperatures on potato morphology and physiology using nondestructive plant phenotyping techniques. Specific aims were: (i) to quantify traits obtained by nondestructive phenotyping techniques  in potato under increasing temperatures, (ii) to compare these groups of traits in their efficiency to discriminate among potato plants grown under different temperatures, and (iii) to select phenotypic traits of potato that are most responsive to increased temperatures.A list of all measured traits with the description is given in After scanning plants with a 3D multispectral scanner, 3D plant models were built from 3D point clouds. Plant 3D models were then used for the quantification of different morphological parameters. Results for all measured morphological traits are shown in Average plant height increased with increasing temperatures (from T1 to T4), which was most pronounced for cv. Desiree and least pronounced for cv. Bellarosa b. The opThe recursive partitioning model based on morphological traits (MORPH) correctly assigned 84.6% of the plants to their respective temperature treatments. Variables selected by this model were LAP, DV, LANG, and LPD/PH a. This mPotato multispectral analysis was performed by capturing whole-plant top view images in red (R), green (G), specific green (SpcGrn), blue (B), far-red (FR), and near-infrared (NIR) wavelengths and specific wavelengths for the chlorophyll. These reflectance analyses were used to calculate HUE, SAT, and VAL, and different vegetation indices  .Temperature treatments significantly affected all measured multispectral traits except SAT . SignifiIncreased temperatures (from T1 to T4) generally increased the reflectance in R, G, B, SpcGrn, and FR. For the reflectance in B, the highest increase was found for cv. Agria. The reflectance in NIR was not affected by temperature in Bellarosa, while it increased from T1 to T2 and decreased in T3 and T4 in cv. Agria, and increased from T1 to T3 and then decreased at T4 for cv. Desiree . A decrease in most vegetation indices accompanied these changes (increases) in reflection found at higher temperatures. However, a significant decrease in CHI was found only for Desiree in T4 b, whereaUsing multispectral traits, recursive partitioning correctly assigned 89.3% of potato plants into their respective temperature treatments. Variables selected by this model were ARI, SpcGrn, and GLI b. This m0 and Fs’ compared to Fm and Fm’, which caused a decrease in Fv/Fm and Fq’/Fm’, although only at higher-temperature treatments. Namely, a significant decrease in Fv/Fm was detected only for cv. Agria in T3 and T4 and for Bellarosa in T4, whereas Fq’/Fm’ significantly decreased in T3 and T4 for cv. Desiree, and in T4 for cv. Agria and cv. Bellarosa , recursive partitioning correctly assigned 90.5% of the plants to their respective temperature treatments. Variables selected by this model were F and ETR a. This m from T1 a.A significant effect of temperature treatments and cultivars was found for all gas exchange traits. In addition, the cultivar × treatment interaction was significant for all gas exchange traits except the Ci . Despite a significant cultivar × treatment interaction, generally, stomatal conductance, transpiration rate, and photosynthesis rate increased with increasing temperatures from T1 to T3 and decreased at T4 . In addiThe highest classification success, 100% correctly assigned potato plants to their respective temperature treatments using recursive partitioning, was achieved using gas exchange traits b. VariabIn this study, three commercial potato cultivars  were grown over four different day/night temperature treatments , aiming to evaluate the effect of increased temperatures on potato morphology and physiology using nondestructive plant phenotyping techniques. Although the results of this study show a significant effect of the cultivar and treatment x cultivar interaction for most studied traits, a general effect of increased temperatures that alter potato physiological processes and growth could be noticed. Similarly, Tang et al.  studied In addition to leaf area reduction, increased temperatures affected leaf spectral properties. With the increasing temperatures from T1 to T4, reflectance in R, G, B, SpcGrn, NIR, and FR significantly increased. Higher reflectance, especially in R and B, indicates lower light absorption, which is often related to the lower chlorophyll content . However0) and light-adapted (F0’) plants, which increased already in T2. This rise in minimal fluorescence indicates the inactivation of PSII reaction centers [m, there was a less pronounced effect on the reduction in Fv/Fm. Moreover, although Fv/Fm significantly decreased in T3 and T4, values higher than >0.8 indicate a good fitness of all plants in terms of PSII [q’/Fm’ and ETR), and an increase in thermal dissipation . It was previously shown that the transfer of potato leaves from 25 °C to temperatures between 30 and 35 °C does not negatively affect PSII activity, whereas temperatures above 38 °C caused an inhibition in the PSII function [0, this model enabled the discrimination of plants grown in T4 from T1 and T2.Regarding CFT, increasing temperatures had the most profound effect on the minimal fluorescence of both dark-adapted  decreased the gs to levels such as those found in T1. This drop in gs is probably related to an increased water vapor pressure deficit (WVPD), which increased up to 1.95 kPa under T4. The photosynthetic rate decreased at T4 due to stomatal limitation. However, increased temperatures had a stronger effect on E and gs than on A, and thus, WUE and WUEi decreased with increasing temperatures from T1 to T3, and then WUEi increased again at T4. The fact that increased temperatures had a stronger effect on gs and E compared to A was further confirmed by the recursive partitioning model, which correctly assigned plants to the respective temperature treatment groups based on WUE, gs, and E. Using only WUE, this model enabled discrimination of plants from T1 and T2 against those from T3 and T4. Recursive partitioning based on gas exchange traits achieved the best assignment success. This result is probably caused by the gas exchange measurement technique, which was always performed on a single leaf of the same size and development stage, compared to the whole-plant imaging and scanning, which captured the leaf/plant spatial variability. Similarly, increased gs and A under well-watered conditions in potatoes acclimated to moderately high temperatures (up to 30 °C) were obtained by Hancock et al. . These aOne of the aims was to select phenotypic traits most responsive to increased temperatures using recursive partitioning models. The recursive partitioning for all groups of traits did not include the effect of the cultivar. Thus, it can be assumed that the traits selected by these models are the ones that had the most uniform response to increased temperature over the tested cultivars. Because the studied cultivars showed similar reactions to the temperature treatments, and although these results were similar to those previously described ,9,16,27,Thus, these results should be confirmed using a higher number of cultivars and include cultivars with different responses to heat stress (heat-tolerant vs. heat-sensitive). Moreover, this study was performed on young potato plants, and measurements were performed after ten days of acclimation, which could also affect the traits selected by recursive partitioning models. In addition to the genotypic variability, the magnitude of the stress response depends on the severity, duration, and the plants’ growth stage ,. Young −2 s−1 provided by light-emitting-diode (LED) lights PhenoLight3 . Light intensity was determined at the upper side of the tube (at substrate level) using an LI-180 spectrometer . Plants were regularly irrigated with distilled water, keeping the substrate volumetric water content (VWC) at 35% throughout the experiment. The substrate VWC was regularly monitored using a substrate-calibrated WET Sensor and HH2 Moisture Meter . After emergence, all plants were thinned to the single main stem. Two weeks after the emergence, seven plants per cultivar  were subjected to four different temperature treatments (thermoperiods): 20/15 °C (T1), 25/20 °C (T2), 30/25 °C (T3), and 35/30 °C (T4). Temperature treatments lasted for ten days, during which all other environmental factors were kept the same as during the emergency and initial growth phases.The experiment was conducted in growth chambers 2 × 3 × 2.5 m (width × length × height)  located at the Faculty of Agriculture, Zagreb, Croatia. Three potato cultivars, cv. Agria, cv. Bellarosa, and cv. Desiree, were planted in plastic tubes  filled with 2.5 kg of Potting Substrate . One tuber (average fresh weight 50 g) per tube was planted at 7 cm depth. Emergency and early growths were conducted at 16/8 h photoperiod, 20/15 °C thermoperiod, 65% relative air humidity, and 250 µmol PAR mAll measurements were performed ten (10) days after the onset of temperature treatments. A list of all measured traits, along with equations, is shown in 2), total leaf area , digital volume , leaf area index , leaf angle , and light penetration depth . Due to changes in plant height under different temperature treatments, which also affected light penetration depth, the LPD parameter was replaced by a parameter calculated as LPD/PH.Morphological traits were measured nondestructively using the PlantEye F500 multispectral 3D scanner . A detailed description of the PlantEye F500, along with the resolution and the analyzed wavelengths, is given in Lazarević et al. . In brieTM . The CropReporterTM is located in the room next to the growth chambers. Plants were manually transferred to the CropReporterTM room and were left for dark adaptation for 3 h. During the period of dark adaptation and subsequent measurements, the imaging room temperature was kept constant at 23 °C. Thus, plants were not subjected to temperature treatments during this period. A detailed description of the CropReporterTM and the protocol used for chlorophyll fluorescence imaging is given in Lazarević et al. [−2 s−1, for 800 ms) was used. Minimum chlorophyll fluorescence (F0) was measured at 10 μs and maximum chlorophyll fluorescence (Fm) was measured after saturation (800 ms). After these measurements, plants were relaxed for 15 s in the dark and were then light-adapted for five minutes under the 250 μmol m−2 s−1 (PPFD). After light adaptation, a saturation pulse (4500 μmol m−2 s−1 for 800 ms) was applied again. Steady-state fluorescence (Fs’) was measured at the onset of the saturating pulse and maximum chlorophyll fluorescence (Fm’) was measured at saturation. After the measurement, the actinic light was turned off, and in the presence of far-red light, the minimal fluorescence yield of the illuminated plant (F0’) was estimated. Collected images were processed by DATM software . From the measured F0, Fm, Fs’, Fm’, and F0’, different chlorophyll fluorescence parameters were calculated: v/Fm) = (Fm − F0)/Fm [Maximum quantum yield of PSII (F− F0)/Fm .q’/Fm’) = (Fm’ − Fs’)/Fm’ [Effective quantum yield of PSII (FFs’)/Fm’ .q’/Fm’ × PPFD × 0.5 × 0.83 [Electron transport rate (ETR) = F5 × 0.83 .m − Fm’)/Fm’ [Non-photochemical quenching (NPQ) = (FFm’)/Fm’ .m’ − Fs)/Fv [Coefficient of Photochemical Quenching (qP) = (F− Fs)/Fv .m’ − F0’)/(Fm − F0) [Coefficient of Non-photochemical Quenching (qN) = 1 − (FFm − F0) .m’ − Fs’) × F0’))/((Fm’ − F0’) × Fs’)) [Estimation of ‘open’ Reaction Centers based on a Lake Model (qL) = ((F × Fs’)) .m/F0 − 1)) [Quantum Yield of Non-regulated Non-Photochemical Energy Loss in PSII (ɸnq) = 1/(NPQ + 1 + qL(FF0 − 1)) .m/F0 − 1)) [Quantum yield of Regulated Non-Photochemical Energy Loss in PSII (ɸnpq) = 1 − ɸpsII − 1/(NPQ + 1 + qL(FF0 − 1)) .Chlorophyll fluorescence imaging was performed on the whole plant using CropReporterć et al. . Brieflyć et al. . For theExamples of generated chlorophyll fluorescence images are shown in −2 s−1) using CropReporterTM. Reflectance images in different spectral wavelengths, red (R) (640 nm), green (G) (550 nm), specific green (SpcGrn) (510–590 nm), blue (B) (475 nm), far-red (FR) (710 nm), and near-infrared (NIR) (769 nm), and specific wavelengths for the chlorophyll (730 nm), were collected. Information about the absolute spectral reflectance and different vegetation indices was calculated by DATM software. Color analysis was presented as an absolute reflection in R, G, and B and as hue (HUE), saturation (SAT), and value (VAL). HUE was calculated from R, G, and B values and is represented as one channel arranged in a rainbow color chart with values 0–360°. The saturation (SAT) of each color represents its intensity , and the value (VAL) shows if the color is dark or bright.Multispectral images were obtained following the chlorophyll fluorescence imaging under the actinic light  = (NIR − Red)/(NIR + Red) .Chlorophyll index (CHI) = (R730)-1 − (R769)-1 .Anthocyanin index (ARI) = (R550)-1 − (R700)-1 .Green Leaf Index (GLI) = (2 × Green − Red − Blue)/(2 × Green + Red + Blue) .In addition to color parameters, different vegetation indices were calculated:Examples of generated multispectral images are shown in −2 s−1 and 90:10% red-to-blue wavelength ratio, provided by the small light source 6800-02 . The cuvette temperature was set to meet the applied daytime treatment temperature regimes (T1–T4): 20, 25, 30, and 35 °C, respectively. The cuvette relative humidity was set at 70%, giving a leaf-to-air vapor pressure difference between 0.85 and 1.95 kPa, T1 to T4, respectively. The concentration of CO2 was set at 400 µmol mol−1 and the flow rate was set at 600 µmol s−1. Recordings were performed after CO2 and H2O stability criteria were met, and automatic matching was performed after each record.Gas exchange measurements were conducted using the portable photosynthesis system LI-6800  at the main leaflet of the youngest fully developed leaf (fourth leaf from the top of the canopy). Measurements were performed using a 3 × 3 cm, 6800-02P clear-top chamber . Measurements were recorded under a saturating light of 1200 µmol PAR m® Pro 16 . The model included the fixed effects of treatments (T1–T4), cultivars , and treatment × cultivar interactions. Individual plants were used as pseudo-replications and were treated as a random factor. In the case of the significant F test, pairwise comparisons were performed using Tukey’s Honest Significant Difference (HSD) post hoc test.The analysis of variance (ANOVA) using the general linear model (GLM) was performed in JMPRecursive partitioning was used to identify the traits within each group  that are most responsive to increased temperatures . This me0, and WUE) to increased temperatures. These traits could be considered as marker traits for the identification of heat stress in potatoes and in screening for heat tolerance among potato germplasms. This set of traits could also be used to increase the throughput in phenotyping studies.Potato is very sensitive to heat, and its acclimation to high temperatures involves complex changes in the physiological and morphological levels. Nondestructive plant phenotyping techniques enable the study of these complex reactions and represent technological solutions that will have a significant role in the adaptation of agriculture to climate change and the new growing conditions they bring. Although these results should be confirmed using a higher number of potato cultivars and including the cultivars with different responses to heat stress (heat-tolerant vs. heat-sensitive), as well as under the field conditions after a prolonged period of adaptation to increased temperatures, we selected the most responsive phenotypic traits (LAP, ARI, F


Furthermore, high-throughput DNA sequencing and coimmunoprecipitation experiments indicated that Cbx1, PurB, and Sp3 also bound together as a complex and were necessary to localize nucleosomes to cardiomyocyte genes on the chromosome. Finally, we found that the expression of these genes led to nucleosome modification via H3K27me3 (trimethylated histone-H3 lysine-27) deposition through an interaction with the polycomb repressive PRC2 complex. In summary, we conclude that Cbx1, PurB, and Sp3 control cell fate by actively repressing lineage-specific genes.miRNA-based cellular fate reprogramming offers an opportunity to investigate the mechanisms of long-term gene silencing. To further understand how genes are silenced in a tissue-specific manner, we leveraged our miRNA-based method of reprogramming fibroblasts into cardiomyocytes. Through screening approaches, we identified three proteins that were downregulated during reprogramming of fibroblasts into cardiomyocytes: heterochromatin protein Cbx1, transcriptional activator protein PurB, and transcription factor Sp3. We show that knockdown of Cbx1, PurB, and Sp3 was sufficient to induce cardiomyocyte gene expression in fibroblasts. Similarly, gene editing to ablate Cbx1, PurB, and Sp3 expression induced fibroblasts to convert into cardiomyocytes  Of these ten candidates, Cbx1, Csde1, Ddx5, Egr1, PurB, Sp3, and Zfp36 were found to be targets of miR combo .We have previously demonstrated that a combination of four miRNAs , which we call miR combo, reprograms fibroblasts into cardiomyocytes . ConsideFig. 1C). Following knockdown of the candidate repressor in fibroblasts, expression levels of cardiomyocyte-specific genes were measured. With the exception of Csde1, knockdown of each candidate repressor induced the expression of at least one cardiomyocyte-specific gene (D). Of the ten potential repressors, knockdown of Cbx1, PurB, or Sp3 increased the expression of >90% of the measured cardiomyocyte-specific genes  as well as sarcomere formation (F).Knockdown of Cbx1, PurB, or Sp3 was sufficient to reprogram fibroblasts into cardiomyocyte-like cells as evidenced by expression of the cardiomyocyte protein Actn2 E as wellormation F.In contrast to cardiac fibroblasts, knockdown of Cbx1, PurB, or Sp3 did not induce cardiomyocyte gene expression in lung or tail-tip fibroblasts .A). This result suggested that the fibroblasts were indeed exiting the fibroblast phenotype. Similarly, loss of Cbx1, PurB, and Sp3 reduced endothelial gene expression (A). Generally, neuronal markers were not expressed in fibroblasts or expressed at low levels. Despite the apparent induction of a few neuronal markers, loss of Cbx1, PurB, and Sp3 generally reduced neuronal gene expression (A).To further investigate the role of Cbx1, PurB, and Sp3 as repressors, we investigated the effect of their knockdown on fibroblast, endothelial, and neuronal specific gene expression. As expected, knockdown of Cbx1, PurB, and Sp3 strongly induced cardiomyocyte-specific gene expression. In contrast, loss of Cbx1, PurB, and Sp3 reduced the expression of fibroblast-specific genes A. This rpression A. Generapression A.Figure B). To demonstrate that the loss of Cbx1, PurB, and Sp3 was not because of a general loss of expression, we also measured the expression of Sox6; a transcription factor expressed during cardiomyocyte differentiation (B).If Cbx1, PurB, and Sp3 play an important role in repressing cardiomyocyte-specific genes, their expression should decrease during cardiomyocyte development. In support of this hypothesis, we found that inducible pluripotent stem (iPS) cell differentiation to cardiomyocytes was associated with a significant decrease in the expression of Cbx1, PurB, and Sp3 B. To demntiation . As expentiation B.et al. . Again, Sox6 expression increased (C). PurB levels were not measured in the Pavlovic study; however, in all 12 human iPS cell lines, cardiomyocyte differentiation was associated with significant loss of Cbx1 and Sp3 expression (D). Cbx1 and Sp3 expression were similar in iPS-derived cardiomyocytes and the heart tissue from which the iPS cells were generated (D). In contrast, Sox6 expression increased during iPS cell differentiation to cardiomyocytes in all 12 lines (D).To verify these results, we utilized publically available RNA-Seq data. We chose two separate RNA-Seq studies that measured mRNA changes in human iPS cells undergoing differentiation into cardiomyocytes. Churko et al.  provided . CRISPR–CRISPR-associated protein 9 (Cas9) gene editing was employed to ablate Cbx1, PurB, and Sp3 expression in vivo. To identify functional guide RNAs (gRNAs), 2 to 3 gRNAs for the first exon of Cbx1, PurB, or Sp3 were introduced individually into cultured cardiac fibroblasts along with Cas9. Immunoblotting indicated that the gRNAs were effective in ablating Cbx1, PurB, and Sp3 expression (C). To test efficacy of knockout in vivo, the gRNAs for the three proteins as well as Cas9 were subsequently packaged into lentivirus particles and injected into the mouse heart. Seven days later, cardiac tissue was analyzed for the expression of Cbx1, PurB, and Sp3. In control cardiac tissue, Cbx1, PurB, and Sp3 expression was robust and localized to the nucleus (D). However, expression of the three proteins was absent in cardiac tissue isolated from mice receiving the repressor gRNAs (D). Having demonstrated the efficacy of the approach, control and repressor targeting gRNAs were delivered into the hearts of Fsp1-Cre:tdTomato fibroblast lineage-tracing mice. In these Fsp1-Cre:tdTomato fibroblast lineage-tracing mice, fibroblasts are permanently marked with tdTomato (E). In contrast, following the ablation of Cbx1, PurB, and Sp3 expression, ∼10% of cardiomyocytes in the vicinity of the injection site were tdTomato+; indicating fibroblast conversion into cardiomyocyte-like cells (E).We wanted to determine if Cbx1, PurB, and Sp3 also repressed cardiomyocyte genes in myocytes , A and Bpression C. To tes nucleus D. Howeveor gRNAs D. HavingtdTomato . In conttdTomato E. In conke cells E.Figure via high-throughput sequencing. Analysis of the dataset indicated that Cbx1, PurB, and Sp3 shared many of the same targets . In contrast, PurB-binding sites were only present within the coding sequence (B). Sp3-binding sites were found in both promoter and within the coding sequence (B).To understand the mechanism by which these repressors actively repress the cardiomyocyte phenotype in fibroblasts, we first employed ChIP-Seq. Chromatin derived from mouse cardiac fibroblasts was incubated with Cbx1, PurB, or Sp3 antibodies, and the resulting immunoprecipitated DNA was sequenced lusively B. In consequence B. Sp3-bisequence B.Figure C; full GO analysis is provided in Fig. 4C; full GO analysis is provided in Gene Ontology (GO) analysis of repressor-bound genes gave further support to notion that Cbx1, PurB, and Sp3 play an important role in regulating the cardiomyocyte phenotype. Significant GO terms included those for cation transport, formation of the action potential, and muscle contraction . The MNase-digested samples were then submitted for high-throughput sequencing. The nucleosome architecture of active eukaryotic genes comprises of a nucleosome-free region just upstream of the transcription start site and an array of regularly spaced nucleosomes over the gene (B). This suggests that in fibroblasts, the majority of genes are silent. Gene silencing appears to require Cbx1 and PurB as the loss of either protein induced nucleosome-free regions to appear (B). Loss of Sp3 differs in that the nucleosome architecture of control cells is retained (B). However, seeing as the read density was higher in the Sp3 siRNA-transfected cells, the data suggest that Sp3 plays a role in histone binding (B). At the level of individual genes, in control fibroblasts, cardiomyocyte-specific genes such as Ryr2 and Actn2 contain a large number of nucleosomes (C). Following knockdown of Cbx1, PurB, or Sp3, these nucleosomes disappear (C). In contrast, knockdown of Cbx1, PurB, or Sp3 had no effect on nucleosome patterning in noncardiomyocyte genes (D).We hypothesized that Cbx1, PurB, and Sp3 inhibited cardiomyocyte-specific genes in fibroblasts by modifying the nucleosome architecture. To test this hypothesis, we performed MNase-Seq. MNase-Seq is used to map nucleosomes. Nucleosomes are the basic unit of DNA compaction and a fundamental component of chromatin. Cardiac fibroblasts were transfected with either a control nontargeting siRNA or an siRNA that targets Cbx1, PurB, or Sp3. Chromatin was isolated 7 days later and incubated MNase. As shown in Figure 5the gene . In contthe gene B. This so appear B. Loss oretained B. Howeve binding B. At theleosomes C. Followisappear C. In conte genes D.Figure A). Similarly, Sp3 was also highly enriched in PurB immunoprecipitates (A). The data suggest shared protein complexes, with Cbx1–Sp3 and PurB–Sp3 dimers being readily apparent. Cbx1 binding to PurB is somewhat unclear as binding was apparent when the Cbx1 antibody was used but not when the PurB antibody was used instead (A). This may be due to steric inhibition between the PurB antibody and the Cbx1 protein.The ChIP-Seq data suggested that Cbx1, PurB, and Sp3 may act as a complex. To investigate complex formation, coimmunoprecipitation experiments were performed. Cbx1 immunoprecipitates were found to be highly enriched in Sp3 A. Similaipitates A. The da instead A. This mB). The formation of H3K27me3 is dependent upon the PRC2 complex, which comprises catalytic  and regulatory  subunits (C).Having identified binding between the repressors, we wanted to determine how Cbx1, PurB, and Sp3 regulated gene activity. The MNase-Seq data suggested that Cbx1, PurB, and Sp3 were necessary for nucleosome patterning on cardiomyocyte-specific genes especially on gene promoters. Considering their role as repressors, we hypothesized that Cbx1, PurB, and Sp3 were important for the formation of inhibitory nucleosomes. Based on our prior miR combo studies, we further hypothesized that these inhibitory nucleosomes contained H3K27me3. Indeed, the combined knockdown of Cbx1, PurB, and Sp3 was found to reduce H3K27me3 levels B. The fosubunits . We founsubunits C.D).The coimmunoprecipitation experiments suggested that Cbx1–Sp3 and PurB–Sp3 are two nonoverlapping complexes. In further support of this notion, knockdown of PurB was found to have no effect on Cbx1–Sp3 complex formation D.Tissue-specific genes are mostly silent. They are typically silent during early development and remain so in most cell types, only undergoing reactivation in their tissues of expression. A number of mechanisms have been proposed for long-term gene silencing including sequence-dependent repression factors, DNA methylation, timing of replication, and histone modifications , 14, 15.in vivo, as well as knockdown in vitro, was sufficient to induce the expression of cardiomyocyte-specific genes in fibroblasts. Based on the data obtained, it appears that Cbx1, PurB, and Sp3 mediate tissue-specific gene silencing by modifying the nucleosome architecture as well as regulating the deposition of silencing histone modifications. Cardiomyocyte genes in fibroblasts were found to contain a significant number of nucleosomes. A large number of nucleosomes may act to compact the gene and prevent expression. However, following the knockdown of Cbx1, PurB, or Sp3, these nucleosomes were no longer present. Nucleosome-free genes are typically transcriptionally active. The effects on nucleosome patterning were restricted as Cbx1, PurB, or Sp3 knockdown had no effect on nucleosomes in fibroblast genes such as S100a4. How Cbx1, PurB, and Sp3 binding induces nucleosome formation on cardiomyocyte genes is an open question. Cbx1-, PurB-, and Sp3-binding sites within cardiomyocyte genes were distinct and often separated by more than 100 nucleotides. However, coimmunoprecipitation studies suggested shared protein complexes between the three proteins. Complex formation would suggest that Cbx1, PurB, and Sp3 are causing DNA to loop. DNA looping has been invoked as the explanation for the ability of enhancers to increase transcription. It is possible that DNA looping induced by Sp3–Cbx1 and Sp3–PurB dimers acts as a scaffold for nucleosome binding.This study suggests that the long-term silencing of tissue-specific genes is regulated by Cbx1, PurB, and Sp3. ChIP-Seq indicated that Cbx1, PurB, and Sp3 were specifically localized to cardiomyocyte genes. Their role appears to gene silencing as genetic ablation of these three proteins The influence of nucleosomes on gene transcription is both simple and complex , 25, 26.et al.  were derived from 1-day-old neonate C57BL6 mice and cultured according to the established protocols .Human cardiac fibroblasts were acquired from Cell Applications, Inc (306-05f) and were cultured according to the manufacturer’s instructions.et al.  were used for each repressor. The siRNA that gave rise to the highest level of knockdown was used for future experiments: Cbx1 siRNA 4 , Ddx5 siRNA 2 , Egr1 siRNA 1 , Fhl2 siRNA 4 , Fli1 siRNA 1 , PurB siRNA 2 , Sp3 siRNA 2 , Tcf4 siRNA 6 , and Zfp36 siRNA 5 . A nontargeting siRNA was used as a control . For transfection, cardiac fibroblasts were seeded into 12-well plates at 22,500 cells per well 1 day prior to transfection. On the day of transfection, siRNAs (0.75 μl) were diluted in serum-free Dulbecco's modified Eagle's medium . In a separate tube, 0.75 μl of Dharmafect-I (Dharmacon) was diluted with 99.25 μl Opti-MEM serum-free media. After 5 min of incubation, the two solutions were combined. After 20 min, complexes were added to cells along with complete media (550 μl), and the transfection complexes were added to the cells. Knockdown was verified 4 days post-transfection. When used in conjunction with miRNA transfection, siRNA and miRNA transfection complexes were set up independently as described and then added to the cells together. When siRNA and miRNA were used in conjunction, the amount of complete media was reduced (250 μl).et al. , with transfection reagent plus nontargeting miRNAs (negmiR), or with transfection reagent plus our previously reported combination of cardiac reprogramming miRNAs .Mouse (C57BL/6) neonatal cardiac fibroblasts were isolated from 2-day-old mouse neonates according to the method outlined in the study by Jayawardena et al. . FollowiTotal RNA was extracted using a Quick-RNA MiniPrep Kit according to the manufacturer’s instructions (Zymo Research). Total RNA (50–100 ng) was converted to complementary DNA (cDNA) using a High-Capacity cDNA Reverse Transcription kit (Applied Biosystems). cDNA was used in a standard qPCR involving FAM-conjugated gene-specific primers (Thermo Fisher Scientific) and TaqMan Gene Expression Master Mix (Thermo Fisher Scientific). Primers were acquired from Thermo Fisher Scientific, and the following are the assay ID numbers: Actn2 Mm00473657_m1; Cacna1c Mm00437917_m1; Gapdh Mm99999915_m1; Kcna4 Mm01336166_m1; Kcnh2 Mm00465377_mH; Kcnj2 Mm00434616_m1; Kcnk6 Mm01176312_g1; Myh6 Mm00440359_m1; Myoz2 Mm00469639_m1; Nebl Mm00503886_m1; Ryr2 Mm00465877_m1; Scn5a Mm01342518_m1; Tnnc1 Mm00437111_m1; Tnni1 Mm00502426_m1; Tnni3 Mm00437164_m1; Tnnt2 Mm01290256_m1; and Ttn Mm00621005_m1.6 individual reads per sample. Individual bioinformatics programs within the Galaxy suite were used for sequence alignment, peak calling, and peak comparisons. Adaptors were removed, and sequences were then aligned to mouse reference genome mm10 using Bowtie2 (p < 0.01. Peaks present in both duplicate samples were identified.Isolated mouse (C57BL/6) neonatal cardiac fibroblasts  were seeded into T150 flasks in growth media. Where necessary, the next day, cells were transfected with a nontargeting control siRNA or an siRNA targeting Cbx1, PurB, or Sp3 as described previously. Seven days after seeding, chromatin was isolated with a SimpleChIP Plus Enzymatic Chromatin IP Kit  according to the manufacturer’s instructions. Once isolated, chromatin was digested with the supplied MNase  according to the manufacturer’s instructions. The amount of MNase was empirically determined to digest chromatin to one nucleosome in length. MNase-digested chromatin was then used for MNase-Seq. MNase-digested chromatin was also used for ChIP-Seq. Here, MNase-digested chromatin  was incubated overnight with 8 μg of control immunoglobulin G, Cbx1 , PurB , or Sp3  antibodies. High-throughput sequencing was performed by the Duke Genomic Core. In total, five independent experiments were performed, and libraries were generated with a NovaSeq 6000 kit (Illumina). Libraries were pooled and run in duplicate (50 bp paired end) with an Illumina NovaSeq 6000. Sequencing depth was >25 × 10 Bowtie2 . For MNaSense and antisense strands for each gRNA (see later) were resuspended in water (100 μM).The gRNA sequences are as follows:5′CACCGCAAAACAAGAAGAAAGTGG3′Cbx1 Sense: 5′AAACCCACTTTCTTCTTGTTTTGC3′Cbx1 Antisense: 5′CACCATCCGCCAGACGGTGAACCG3′PurB Sense: 5′AAACCGGTTCACCGTCTGGCGGAT3′PurB Antisense: 5′CACCGCTGCCTTGGACGTGGACGG3′Sp3 Sense: 5′AAACCCGTCCACGTCCAAGGCAGC3′Sp3 Antisense: −1. Phosphorylated and annealed gRNAs were then cloned into an pSpCas9(BB)-2A-GFP plasmid . The following ligation reaction was set up for each gRNA: 100 ng pSpCas9(BB)-2A-GFP, 20 nmol gRNA, 2 μl Tango buffer (Thermo Fisher Scientific), 1 μl 10 mM DTT , 1 μl of 10 mM ATP (NEB), 1 μl Bbsl (Thermo Fisher Scientific), and 1 μl T4 DNA ligase (NEB) in a final volume of 20 μl. The ligation reaction was incubated for 1 h at 4 °C. Following ligation, the mixture was used (5 μl) to transform DH5α Competent Cells (NEB) as per the manufacturer’s protocol. Standard overnight DNA minipreps and DNA sequencing were used to ensure correct insertion of the gRNAs.Following resuspension, the sense and antisense strands were annealed and phosphorylated. Phosphorylation of the strands (100 nmol per strand) was catalyzed by T4 polynucleotide kinase (New England BioLabs [NEB]). After a 30 min of incubation at 37 °C, the enzyme was inactivated by heating the reaction at 95 °C for 5 min. Annealing of the two strands was then carried out by ramping the temperature down to 25 °C at 5 °C min2 in growth media (15% FBS and 1% penicillin/streptomycin). The next day, cells were transfected with 1 μg plasmid DNA using the transfection reagent Lipofectamine 2000 (Thermo Fisher Scientific) as per the manufacturer’s protocol. After 24 h, transfection complexes were removed and replaced with growth media. Three days after transfection, puromycin  was added daily for a total of 7 days to select for transfected cells. Cells were then harvested, and protein was isolated for immunoblotting.3T3 cells were seeded at 5625/cmvia Lenti-Pac HIV Expression Packaging kit (GeneCopoeia) according to the manufacturer’s instructions. Viral particles (2 × 106 plaque-forming units in 40 μl) were injected into the left ventricle at two sites.The lentiviral CRISPR–Cas9 plasmid (1 μg), LentiCRISPRv2  was digested for 2 h at 37 °C with 3 μl BsmBI (NEB) in a 60 μl reaction containing FastAP , NEBuffer r3.1 , and 100 mM DTT . Digested plasmid was subsequently gel purified using a QIAquick Gel Extraction kit (Qiagen). Following purification, digested plasmid DNA (50 ng) was ligated with the phosphorylated and annealed gRNAs (1 nmol) in a 10 μl reaction containing T4 DNA ligase . Ligations were performed overnight at 4 °C. The next day, ligated products (5 μl) were transformed into DH5α Competent Cells as per the manufacturer’s protocol. Plasmid constructs were verified by restriction digest and DNA sequencing. Lentivirus was generated Images were processed with CorelDraw and Zeiss software (Axiovision Rel 4.8 and Zen Blue).t tests were used for studies with two groups. For more than two groups, one-way ANOVAs were used. For ANOVA, Bonferroni post hoc tests were used to determine significance between groups. Individual data points and the mean are shown in all graphs. A p value of less than 0.05 was considered significant.All statistical analyses were performed using GraphPad . Two-tailed Experiments using animals were approved by the Duke University Division of Laboratory Animals and the Duke Institutional Animal Care and Use Committee.Raw sequencing data can be found at the Single Read Archive (accession number: SAMN12628632). All other data are contained within the article.This article contains V. J. D. and C. P. H. are cofounders of Recardia Therapeutics. This company is focused on developing miRNAs that reprogram fibroblasts into cardiomyocytes. All other authors declare that they have no conflicts of interest with the contents of this article.