The strain SH-10 with maximum Zn solubilization zone of 24 mm on Zn3 (PO4)2ore and strain SH-17 with maximum Zn solubilization zone of 14–15 mm on ZnO and ZnCO3ores were selected for further studies. These two strains solubilized phosphorous (P) and potassium (K) in vitro with a solubilization zone of 38–46 mm and 47–55 mm respectively. The strains also suppressed economically important rice pathogens Pyricularia oryzae and Fusarium moniliforme by 22–29% and produced various biocontrol determinants in vitro. The strains enhanced Zn translocation toward grains and increased yield of basmati-385 and super basmati rice varieties by 22–49% and 18–47% respectively. The Zn solubilizing strains were identified as Bacillus sp. and Bacillus cereus by 16S rRNA gene analysis.Plant associated rhizobacteria prevailing in different agro-ecosystems exhibit multiple traits which could be utilized in various aspect of sustainable agriculture. Two hundred thirty four isolates were obtained from the roots of basmati-385 and basmati super rice varieties growing in clay loam and saline soil at different locations of Punjab (Pakistan). Out of 234 isolates, 27 were able to solubilize zinc (Zn) from different Zn ores like zinc phosphate [Zn Rice is an important cereal crop growing across the world and is a major staple food of Asian population. In current scenario of intensive farming, soils are continuously depleting in macro and micro nutrients especially in wheat-rice cropping system 2, ZnCO3, and ZnO  by fixing it from the atmosphere, phosphorous (P), and potassium (K) by solubilizing them from the soil 2, and ZnCO3 was tested on Bunt and Rovira agar medium , ZnO, ZnTwo strains exhibiting maximum potential to solubilize Zn from various ores were selected for morphological and biochemical characterization. For each test, the strains were freshly grown in LB broth overnight and normalized to the OD-600 of 0.5 before inoculation on respective plate.The Zn solubilizing strains were characterized for Gram reaction and intrinsic resistance to antibiotics following the method of Vincent . BrieflyAbility of strains to solubilize the major plant nutrients  were tested on Pikovskaya agar containing tricalcium phosphate as insoluble phosphate source and Aleksandrov agar having potassium aluminum silicate as source of insoluble inorganic potassium respectively  was added in the cavity and observed for change in color. Appearance of pink halo zone around the cavity indicated production of IAA by the bacterial culture. Qualitative production of siderophores by the bacterial strains was detected on the Chrome-azurol S (CAS) medium  production by the Zn solubilizers was qualitatively determined by growing the strains on LB agar plates supplemented with 100 μg mLPyricularia oryzae causing rice blast and Fusarium moniliforme causing bakanae disease of rice were used as test strains to study the antagonistic potential of potent Zn solubilizers. The antagonism was tested by dual culture assay as described by Spence et al.  of the fungus growing in the control plate and T = mycelium diameter (cm) of the fungus growing in the bacterial treated plates. The experiment was repeated twice with three biological replicates each time.−1. A piece of filter paper having diameter 6 cm was dipped in solution consisting of 0.5% picric acid, 1% Na2CO3 and placed in the upper lid of each petri plates. The plates were wrapped with parafilm and incubated at 28 ± 2°C for 48–72 h. The change in color of filter paper from yellow to brown was used as indicator for HCN production.Production of HCN was determined by following the method of Miller and Higgins  with cerHydrolytic enzymes production such as protease, cellulase, and glucanase were detected on the agar plates containing skim milk, carboxy methylcellulase and laminarin respectively , 10 mM of each primer, and 1.0–1.5 U of Taq polymerase (Fermentas) was amplified in thermocycler (Peq lab Germany) with the amplifying conditions; initial denaturation at 95°C for 5 min, 25 cycles  followed by final extension at 72°C for 5 min  and K were applied in single dose before sowing the plants while N was applied in three split doses. The experiment was laid out in a completely randomized design (CRD) with three replications per treatment. Zinc sulfate (ZnSO4) at the rate of 7.5 mg kg −1 of soil was used as Zn in respective treatments. There were eight treatments viz. T1 = un-inoculated plants (Negative control), T2 = Zn (positive control), T3 = Zn solubilizing strains SH-10, T4 = Zn solubilizing strains SH-17, T5 = Consortium of Zn solubilizing strains SH-10 and SH-17, T6 = Zn solubilizing strains SH-10 + Zn, T7 = Zn solubilizing strains SH-17 + Zn, T8 = Consortium of Zn solubilizing strains + Zn.Effect of Zn solubilizing strains inoculation on plant growth, yield and grain Zn concentration on two rice varieties basmati 385 and basmati super was examined in cleaned earthen pots (20 cm × 30 cm) under net house conditions. The pots were filled with 5 Kg sterilized clay loam soil. NPK fertilizer was applied at the rate of 40, 30, and 20 mg kg−1, 28 ± 2°C for overnight. The cells were pelleted by centrifugation and dissolved in 0.85% saline with a cell OD = 0.45 (~109 CFU mL−1). One mL of this cell suspension was applied near the root of each seedling. Sterile saline without bacteria was applied in negative control. The pots were kept in net house during the months of July to October . The plants were irrigated when needed until maturity. A second dose of bio inoculants was applied after 45 days of 1st inoculation. The plants were harvested during the month of October and observed for all agronomic traits like number of tillers, plant height, panicle length, thousand grain weight and yield except the leaf chlorophyll content which was measured at anthesis stage. Three leaves per plant were randomly selected and chlorophyll content of each leaf was measured by SPAD meter  from different places , Zn (T2), consortium of strains (T5) and consortium of strains along with Zn (T8) were selected for determining the Zn content in shoot and grains. The Zn analysis was carried-out commercially by the Nuclear Institute for food and Agriculture (NIFA), Peshawar, KPK, Pakistan. Zn translocation index (ZTI) toward rice grains was calculated by using the formula  , tetracycline (30 μg), vanlomycine (30 μg), and minocycline (30 μg) while strain SH-17 showed resistance against all the antibiotics. The strains solubilized P and K with a solubilization zone of 38–46 mm and 47–55 mm respectively but did not produce IAA and HCN. They also inhibited the growth of Zn solubilizing strains significantly increased the yield and yield components of rice variety basmati-385 Table . MaximumEffect of Zn solubilizing bacteria on the yield and yield components of rice variety super basmati was significant Table . HighestEffect of Zn solubilizers on the ZTI of both varieties was similar Table . HighestBacillus sp. and Bacillus cereus on the basis of 16S rRNA gene analysis. The sequences of strains were submitted to NCBI Gene Bank database under accession numbers KT380823 and KT380824.The potent Zn solubilizers were identified as in vitro and in vivo to enhance the growth, yield and Zn content of rice varieties.PGPR inhabit wide range of crops growing under varying agricultural practices and commonly used as bio inoculants. In addition to their synergistic effect on plant's growth and yield, they have strong potential to enhance the Zn content of cereals  and K are the macro nutrients required by rice and the other field crops for growth and optimum yield −3 is dependent on secretion of acids such as gluconic acids, lactic acid, malic acid and oxalic acid etc (Estrada et al., −3] are released from their insoluble compounds by following the same basic mechanism of acidification and thus correlate the metabolism of each other.Ability of rhizobacteria to solubilize Zn, K, and (POP. oryzae and F. moniliforme and produce several secondary metabolites involved in antagonistic activity of the PGPR. These metabolites include siderophores which chelate iron and deprive the pathogen from an important source of nutrition, thereby inhibiting the pathogen by creating a competitive environment (Aznar and Dellagi, in vitro which are similar to the earlier findings (El-Sayed et al., Gluconacetobacter diazotrophicus. The siderophores produced by the antagonistic bacteria chelate iron and play major role in their antagonistic activity. On the other hand, Fe +3 oxidation under aerobic soil conditions limits the Zn availability (Gao, A PGPR could be an ideal candidate for the development of bioformulation provided it possesses multiple characteristics for plant growth promotion. The Zn solubilizers suppress growth of economically important pathogens of rice ity Gao, . This faIn vivo evaluation of the strains exhibiting multiple plant growth promoting properties in laboratory conditions is necessary to develop an effective bio inoculum. Hence, the potent strains were inoculated on two rice varieties, basmati 385 and super basmati grown in net house under the natural growth conditions. The strains inoculated in consortium along with chemical Zn significantly enhanced the yield and related parameters in present research. Effect of PGPR on rice yield has already been reported (Estrada et al., in vivo they may serve as useful bio inoculum.In addition to increase yield, the Zn solubilizing rhizobacteria also enhanced Zn translocation to the rice grains in similar way as that of chemical Zn. These findings were similar to earlier studies where PGPRs translocated Zn toward rice grains (Tariq et al., Bacillus sp. and Bacillus cereus. These findings are similar to that of earlier reports in which zinc solubilizers have been found to be recurrent among various bacterial taxa (He et al., B. cereus are opportunistic human pathogens but numerous strains isolated from plant rhizoplane exhibit PGPR traits and their potential as bio fertilizer is well documented (Niu et al., Bacillus sp. capable to solubilize nutrients, produce siderophores and antagonize the pathogens by different mechanisms could be used as effective bio inoculants.The potent Zn solubilizers capable to enhance growth, yield and grain Zn content of rice were identified as The Zn solubilizing bacteria associated with indigenous host enhance the growth, yield and Zn content by providing nutrition as well as disease protection. Such strains could be ideal candidate to develop bioformulation to improve not only rice yield but also produce Zn fortified rice grains. Moreover, these findings could help researchers to explore the mechanisms involved in PGPR mediated Zn translocation in cereals. The Zn solubilizing strains reported in this study would be made available upon request as per institutional material transfer agreement policy.The 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.


Sod1−/−), a mouse model of elevated oxidative stress that exhibits accelerated loss of muscle mass, which recapitulates many phenotypes of sarcopenia as early as 5 months of age. We found that young adult Sod1−/− mice display a considerable reduction in hind limb skeletal muscle mass and strength when compared to age-matched wild-type mice. These changes are accompanied by gross alterations in neuromuscular junction (NMJ) morphology, including reduced occupancy of the motor endplates by axons, terminal sprouting and axon thinning and irregular swelling. Surprisingly however, the average density of acetylcholine receptors in endplates is preserved. Using in vivo electromyography and ex vivo electrophysiological studies of hind limb muscles in Sod1−/− mice, we found that motor axons innervating the extensor digitorum longus (EDL) and gastrocnemius muscles release fewer synaptic vesicles upon nerve stimulation. Recordings from individually identified EDL NMJs show that reductions in neurotransmitter release are apparent in the Sod1−/− mice even when endplates are close to fully innervated. However, electrophysiological properties, such as input resistance, resting membrane potential and spontaneous neurotransmitter release kinetics (but not frequency) are similar between EDL muscles of Sod1−/− and wild-type mice. Administration of the potassium channel blocker 3,4-diaminopyridine, which broadens the presynaptic action potential, improves both neurotransmitter release and muscle strength. Together, these results suggest that ROS-associated motor nerve terminal dysfunction is a contributor to the observed muscle changes in Sod1−/− mice.Elevated reactive oxygen species (ROS) production and ROS-dependent protein damage is a common observation in the pathogenesis of many muscle wasting disorders, including sarcopenia. However, the contribution of elevated ROS levels to –a breakdown in neuromuscular communication and muscle atrophy remains unknown. In this study, we examined a copper zinc superoxide dismutase [CuZnSOD (Sod1)] knockout mouse ( There is a delicate balance between reactive oxygen species (ROS) production and detoxification resulting in a limited amount of free ROS. At low levels, ROS have been shown to exert a modulatory action on a number of intracellular signaling pathways and ion channels Sod1−/−) results in very high levels of oxidative stress and oxidative damage in all tissues and an acceleration of age-related loss of skeletal muscle mass Experimentally reducing cellular antioxidant capacity by disruption of the Cu/Zn superoxide dismutase gene in mice  display hind limb atrophy and weakness, and reduced compound muscle action potential (CMAP) parameters when compared to age-matched wild-type controls. Consistent with reduced CMAP measurements, electrophysiological recordings from individual muscle fibers reveal a substantial decrease in neurotransmitter release. Morphological analyses of NMJs performed together with the electrophysiological recordings suggest that changes in synaptic release likely precede NMJ denervation. In agreement with a presynaptic deficit hypothesis, administration of 3,4-diaminopyridine, a potassium channel blocker which increases action potential duration, improves synaptic transmission and contributes to an increase in muscle strength.In this study, we utilized Guide for the Care and Use of Laboratory Animals. Experimental protocols were approved by the Institutional Animal Care and Use Committee at the University of Texas Health Science Center at San Antonio (IACUC # 08080z) and the Audie L. Murphy VA Hospital.All experiments were performed in accordance with the National Institutes of Health Sod1−/−) were originally derived in Dr. Charles Epstein's laboratory at the University of California, San Francisco Sod1−/− mice to generate Thy1-YFPtg/+::Sod1+/+and Thy1-YFPtg/+::Sod1−/− mice for NMJ imaging experiments. Mice were fed a standard rodent chow diet ad libitum and kept in a temperature and humidity controlled environment with artificial lighting (12h dark/12h light). All experiments were performed using young adult animals aged between 4 and 10 months old. The average age of mice used for each experiment is noted in the figure legends. Tail clips were used for DNA isolation and genotyping PCR.CuZnSOD deficient mice ), where the membrane capacitance factor (f) was fixed at 0.8, appropriate for mouse muscle fibers. QC was calculated by dividing mean EPPcorrected amplitude by mean mEPP amplitude. Rundown analysis was obtained by recording EPPs at 10 Hz and 40 Hz for 5 seconds and EPP(n) amplitude was quantified using Mini analysis  and expressed as either raw amplitude (mV) or normalized to the initial EPP(1).Mice were euthanized and either the left or right hind limb was shaved and skinned. After removal of the biceps femoris muscle and sciatic nerve dissection, a complex of the EDL and tibialis anterior (TA) muscles was dissected out and pinned by the tendons into a 35 mm Sylgard coated dishes containing oxygenated perfused Ringer's solution. For electrical stimulation of the NMJ, the common peroneal nerve was drawn into a glass suction electrode and stimulated at the desired frequency. µ-conotoxin GIIIB  was used to block muscle contractions. Electrophysiological recordings of individual muscle fibers were made using a 10X water-immersion objective of a BX51WI Olympus microscope. Micropipettes were filled with a mixture of KCl and K-acetate solution. A 1∶1 mixture of 3 M KCl and K-acetate solutions was used because chloride ions leak from the pipette into the muscle, making the resting membrane potential recordings unstable. Inclusion of an additional ion other than chloride (acetate/citrate) improves the stability of recordings. To make the recordings, each electrode was connected to an Axoclamp 900A amplifier through 0.1 gain headstages . Data were digitized , acquired and analyzed with Chart 5.5.6 software (AD Instruments software and hardware). On average 5 NMJs were sampled per muscle per experimental condition and at least 5 end-plate potentials (EPPs) and 30 miniature EPPs (mEPPs) were sampled per NMJ. The calculation of quantal content (QC) was done according to McLachlan and Martin Thy1-YFPtg/+::Sod1+/+and Thy1-YFPtg/+::Sod1−/− mice were obtained by first identifying each nerve terminal by YFP fluorescence. Images were captured to be used to identify the morphology of the NMJ after fixation and staining with fluorescent conjugated α-bungarotoxin (method described below).Individual recordings from the Thy1-YFPtg/+::Sod1+/+ and Thy1-YFPtg/+::Sod1−/− mice, pinned on a Sylgard dish and fixed in 4% paraformaldehyde solution for 30 minutes. After several washes with PBS buffer, muscles were incubated with α-bungarotoxin, Alexa Fluor 594 conjugate  for 30 minutes to stain post-synaptic acetylcholine receptors. The whole muscles were then washed a few times with PBS, thin filets of muscles were dissected off and mounted with Prolong-Gold mounting media. The innervation quantification was scored manually under an epi-fluorescent microscope following criteria as we described previously Gastrocnemius and EDL muscles were dissected from 2 after calibration. Overlap pixels were obtained by superimposing the axon on AChR binary images. Occupancy values were derived by dividing the number of overlap pixels by AChR pixels for each NMJ. AChR average density estimates (int/AreaF) were produced by calculating the total integrated pixel intensity (intF) in 16-bit background-corrected average intensity z-projections for α-bungarotoxin-Alexa Fluor 594 staining, and normalizing them to AChR area (Area). In some instances, physical positioning of the invading axon and the presynaptic bouton might lead to some degree of overestimation of overlap. Obstructed NMJs were excluded from the analysis.Z-slice images were captured using Olympus FV-1000 Confocal MultiPhoton Spectral Laser Scanning Microscope. Motor axon/nerve terminals and nAChRs were visualized by YFP fluorescence and Alexa Fluor 594 respectively. Image analysis to quantify AChR area of an NMJ and overlap between the axon and AChRs was performed in ImageJ t-test (two tailed) unless otherwise mentioned. The group mean value was calculated from mean values for individual animals shown as N. The total number of records for all animals is designated as n.Electrophysiology data and NMJ morphological parameters are expressed as group mean ± standard error of the mean, and analyzed using paired student Sod1−/−), a model of oxidative stress and sarcopenia, have significantly increased levels of oxidative damage biomarkers, such as F2-isoprostanes, protein carbonyls and 8-oxo-dG in various tissues including skeletal muscle Sod1−/− mice also fail to sustain their muscle mass in various hind limb muscles from early adulthood. Sod1−/− and wild-type (WT) mice. Both Sod1−/− muscles were smaller than those in WT with the gastrocnemius muscle affected more than the EDL. As anticipated, muscle strength in Sod1−/− mice was also diminished, which is demonstrated by poor performance in grip strength and wire hanging tests  elicited by stimulation of the sciatic nerve revealed that both CMAP area and amplitude were diminished in the gastrocnemius muscle in Sod1−/− mice . This decrease in CMAP parameters in Sod1−/− mice could be due to a decrease in the size of muscle fibers, functional muscle fiber denervation and/or inhibition of synaptic release. We also found that there was a significant increase in tail distal motor latency (TDML) and a reduction in sciatic nerve motor nerve conduction velocity . However, sensory nerve conduction velocity (NCV) remained unchanged . The repetitive nerve stimulation (RNS) test was also performed to determine whether the reduction in muscle contractile force is caused by a deficit in neurotransmission at the NMJ. As expected, in WT mice, there was no reduction in CMAP amplitude when the sciatic nerve was stimulated with a train of ten stimuli at either 0.2 Hz or 10 Hz stimulation frequencies. In contrast, Sod1−/− mice displayed a significant decrement (12%) in CMAP amplitude when stimulated at 10 Hz but no obvious deficit at 0.2 Hz . In addition, we performed electromyography tests to assess the spontaneous activities of the muscle without nerve stimulation. Consistent with our predictions of NMJ denervation, we observed increased insertional activity, fibrillation potential and positive sharp waves, which are all characteristics of irregular spontaneous muscle discharge (data not shown). These results indicate that loss of CuZnSOD has a selective effect on motor axons and NMJs relative to the sensory nerve.To examine the effects of oxidative stress on the peripheral nerve and muscle Sod1−/− mice showed a reduction in both amplitude and area of CMAP recorded in both gastrocnemius and TA muscles in Sod1−/− mice, which could be the result of changes in synaptic release at individual NMJs. To test this, we studied synaptic transmission in 5-6 month old Sod1−/− and WT mice using the EDL muscle-peroneal nerve preparation due to the similarities in fiber composition with gastrocnemius. On average, EDL muscles from both genotypes had similar resting membrane potential and input resistance values, see Sod1−/− mice than in WT mice . To further investigate the functional impairment of neurotransmitter release at the NMJ in Sod1−/− mice, we examined sustained release properties by applying 5 s trains of high frequency stimulation. At both 10 Hz and 40 Hz, the rundown of EPP amplitude was faster in Sod1−/− mice than in WT control mice  Sod1−/− mice. AChR was labeled using Alexa Fluor 594 conjugated α-bungarotoxin, and presynaptic axon and nerve terminal was visualized with YFP expression (Thy1-YFP). Consistent with previous studies Sod1−/− muscles, including axon thinning and irregular swelling, complete or partial withdrawal of nerve terminals from the endplate, terminal sprouting, and discontinuous AChRs  in fixed and α-bungarotoxin labeled EDL muscles from WT and Sod1−/− mice that express YFP in neurons (Thy1-YFPtg/+::Sod1+/+ and Thy1-YFPtg/+::Sod1−/− mice respectively). Suprisingly, we found that there was no difference in the average NMJ area between Sod1−/− and WT EDL muscles  calculated as integrated fluorescence intensity of Alexa Fluor 594 normalized to the total endplate area was not significantly different between WT and Sod1−/− endplate . Moreover, we recorded both EPPs and mEPPs from individually identified NMJs in Sod1−/− and WT EDL muscles, and also, measured post-fixation their endplate occupancy and area values. In general, smaller occupancies were associated with smaller EPP amplitudes in Sod1−/− mice , defined as the degree of overlap between the innervating axon terminal and acetylcholine receptor (AChR) field of a muscle fiber might be responsible for the observed decreases in both EPP amplitude and mEPP frequency in in vivo, and ex-vivo electrophysiological recordings from single muscle fibers indicate that the cause of muscle fiber denervation in Sod1−/− mice is likely to be a defect in neurotransmitter release. Muscle fiber denervation is manifest as reduced axonal endplate occupancy and muscle weakness. Such changes might be reversed, and muscle performance might be improved, if neurotransmitter release can be enhanced. We therefore tested the acute effects of 3,4-diaminopyridine (DAP), a non-selective potassium channel blocker that broadens the presynaptic action potential and allows more calcium to enter the terminal. DAP (administered i.p.) significantly diminished the CMAP decrement at 10 Hz in Sod1−/− mice , whereas it did not alter CMAP assessment of muscle function in WT mice. Moreover, acute administration of DAP augmented grip strength compared to saline treatment in Sod1−/− mice , while it did not change grip strength in WT mice  mouse, a model of accelerated oxidative stress and sarcopenia that shows muscle atrophy and weakness as early as 5 months of age Sod1−/− hind limb muscles is markedly impaired  or morphological denervation Sod1−/− mice, NMJ morphology and neurotransmission are fully rescued Sod1−/− mice was sufficient to reverse the NMJ degeneration and loss of muscle mass Similar values of mEPP amplitudes, mEPP time constants and muscle input resistances in Sod1−/− mice is independent of NMJ axonal occupancy  a reduced synaptic vesicle pool size, 2) a decrease in active zone number, or, 3) a change in the probability of release. The unchanged average area and density of AChRs in Sod1−/− mice  Sod1−/− mice was mitigated by an acute administration of DAP  and Lambert-Eaton syndrome (LES). Both of these syndromes involve autoimmune reactions against NMJ components which lead to a marked impairment in sustained neurotransmission and inhibition of muscle action potential initiation. DAP, a potassium channel blocker has been successfully used in several clinical trials in LES patients resulting in some improvements in muscle strength. Experimentally, it has also been shown to improve neurotransmission in a MuSK antibody-induced mouse model of MG both acutely and chronically Sod1−/− mice which may contribute to reduced muscle strength and loss of muscle mass. Oxidative stress due to the lack of CuZnSOD specifically in the presynaptic neurons and nerve terminals likely gives rise to the abnormal neurotransmitter release. A better understanding of the interplay between the NMJ functional parameters and NMJ morphological maintenance in genetic mouse models will lead to further insights into synaptic plasticity in diseases and aging.In conclusion, we demonstrated that at the synaptic level there is profound functional impairment in neurotransmission in young adult


Interstitial extravillous cytotrophoblasts invade the decidua, while endovascular extravillous cytotrophoblasts are involved in re-modelling uterine spiral arteries. We tested the idea that sodium butyrate (an epigenetic modulator) induces trophoblast differentiation in early gestation rhesus monkey trophoblasts through activation of the Wnt/β-catenin pathway. The results show that syncytiotrophoblast formation was increased by butyrate, accompanied by nuclear accumulation of β-catenin, and increased expression of EnvV2 and galectin-1 (two factors thought to be involved in trophoblast fusion). Surprisingly, the expression of GCM1 and syncytin-2 was not affected by sodium butyrate. When trophoblasts were incubated with lithium chloride, a GSK3 inhibitor that mimics Wnt activation, nuclear accumulation of β-catenin also occurred but differentiation into syncytiotrophoblast was not observed. Instead the cells differentiated to mononucleated spindle-shaped cells and showed molecular and behavioral characteristics of endovascular trophoblasts. Another highly specific inhibitor of GSK3, CHIR99021, failed to induce endovascular trophoblast characteristics. These observations suggest that activation of the Wnt/β-catenin pathway correlates with both trophoblast differentiation pathways, but that additional factors determine specific cell fate decisions. Other experiments suggested that the differential effects of sodium butyrate and lithium chloride might be explained by their effects on TNFα production. The results provide valuable tools to manipulate trophoblast differentiation in vitro and to better understand the differentiation pathways that occur during early gestation.Trophoblast differentiation during early placental development is critical for successful pregnancy and aberrant differentiation causes preeclampsia and early pregnancy loss. During the first trimester, cytotrophoblasts are exposed to low oxygen tension (equivalent to~2%-3% O Trophoblast differentiation during early placental development proceeds along an extravillous pathway (giving rise to invasive extravillous cytotrophoblasts) and a villous pathway (giving rise to multinucleated syncytiotrophoblast). Interstitial extravillous cytotrophoblasts invade the decidua, interacting with decidual cells, while endovascular extravillous cytotrophoblasts enter the uterine spiral arteries and remodel vessel walls. The origin of these different trophoblast lineages remains uncertain and it is not clear whether a single progenitor gives rise to all phenotypes or whether more than one kind of progenitor cell exists. James et al provided evidence in favor of two populations of progenitor cells for villous and extravillous trophoblasts, respectively ,while otThe Wnt/β-catenin pathway is known to play a role in trophoblast differentiation along both the villous and extravillous pathways , 5. ActiSodium butyrate has diverse effects on cells including induction of differentiation, inhibition of proliferation, modulation of immune response, and inhibition of inflammation –13. MostIn the present paper we tested the idea that sodium butyrate induces trophoblast differentiation in early gestation rhesus monkey trophoblasts through activation of the Wnt/β-catenin pathway. Although sodium butyrate induced syncytiotrophoblast formation and caused nuclear accumulation of β-catenin, another Wnt activator, lithium chloride (LiCl), failed to induce syncytiotrophoblast formation and instead induced extravillous trophoblast differentiation. Thus, the Wnt pathway is involved in both trophoblast differentiation pathways but additional factors determine specific cell fate decisions. Other experiments suggest that the differential effects of sodium butyrate and lithium chloride might be explained by their differential effects on TNFα production.Macaca mulatta) were performed in accordance with the NIH Guide for the Care and Use of Laboratory Animals and under the approval of the University of California Davis, Animal Care and Use Committee. This species was chosen because of certain known similarities in development to that of the human and our knowledge of the implantation process in these species. Studies of this kind are not possible with first trimester human placental tissue.All procedures involving Rhesus monkeys  on the University of California Davis campus. Animals were caged with a 0600–1800 hours light cycle and at a temperature maintained at 25–27°C. Animals were allowed to socialize by being housed in pairs during the day from approximately 0800–1400 hours. Animals were fed a diet of Purina Monkey Chow and water ad libitum. Seasonal produce, seeds and cereal were offered as supplements for environmental enrichment.2, and breathing pattern were continuously monitored. After uterotomy, the placenta, fetus and associated membranes were separated from the endometrial wall and removed intact. After closure of the uterotomy, oxytocin was administered. Isoflurane or constant infusion anesthetics were turned off and the endotracheal tube was removed. The animal was returned to the recovery cage and monitored every 15 minutes until able to sit up. Post-surgery analgesics were administered for at least 48 h following the procedure. Depending on the condition of the animal as assessed by the veterinary staff, either narcotic  or non-narcotic  analgesics were used. Animals were then evaluated daily by veterinary staff to decide whether treatment should be continued or discontinued. Animals were monitored daily for food intake, fecal consistency and output, hydration, and activity. Discomfort was scored daily for 3 days and sutures are assessed daily for 7 days post-surgery. Experimental treatments were only performed in vitro on isolated cells and tissues obtained from this surgical procedure. Since no experimental treatments of animals were involved, only post-operative discomfort was expected. No animals were sacrificed and no complications resulting from surgery were reported.Timed pregnancies were obtained by mating the animals (4–8 years old) twice, 2 days apart, at the anticipated time of ovulation based on records of previous menstrual cycles, with the second mating designated as day of pregnancy. On day 16 of pregnancy, the presence of a conceptus was confirmed by ultrasound diagnosis. Placentas and endometrial tissue was collected at 52–55 days of pregnancy by surgical embryectomy which involved removal of the entire conceptus and surrounding endometrial tissue. Prior to surgery animals were fasted overnight. Pre-anesthetic analgesics such as oxymorphone or buprenorphine were administered as deemed necessary by veterinary staff. Other pre-anesthetic medications such as atropine, ketamine or glycopyrrolate were also used as needed. After endotracheal intubation anesthesia was induced using inhalant isoflurane or constant rate infusion of propofol, ketamine, fentanyl, lidocaine or dexmedetomidine. Heart rate, respiratory rate, oxygen saturation, blood pressure, C0Four rhesus placentas were obtained at 52–55 days of gestation (full gestation in this species is 170 days) and used for cell isolations. Trophoblasts were isolated from rhesus placental villous tissue using a modification of serial trypsin digestion methods originally described for human trophoblasts . Our proCharacterization of the isolated trophoblasts was carried out by immunocytochemical staining for the trophoblast marker cytokeratin 7 (CK7) and the mesenchyme marker vimentin (Vim). Nuclei were stained using DAPI. Freshly isolated cells were plated into LabTek culture chamber slides and double-stained with the antibodies 24 h after plating. Images of double-stained cells were captured and superimposed using Adobe Photoshop to allow quantitation of total number of nuclei, total number of CK7+ cells, and the total number of Vim+ cells. Using these three sets of values, the numbers of CK7+Vim- cells, CK7+Vim+ cells, CK7-Vim+ cells and CK7-Vim- cells were derived and expressed as a percentage of the total number of cells. Cells from four different placentas were analyzed. For Fraction B 100–200 cells were counted per field of view. For some Fraction A samples less than 100 cells were counted per field of view. This was due to the low cell recovery and the poor plating efficiency in this fraction.Cells were maintained in trophoblast medium in a hypoxic glove box (Coy Laboratories) at 2% oxygen and 5% CO2. The concentration of oxygen was selected to mimic the approximate oxygen tension encountered by trophoblasts during the early first trimester , 20. ForHuman uterine microvascular endothelial cells  were purchased from Lonza  and maintained in endothelial basal medium-2  supplemented with EGM-2MV Single-Quots  exactly as described by the manufacturer. Cells between passages 5–7 were used for experiments.The antibodies used along with their dilutions and sources are shown in For immunofluorescence staining, adherent cells on 2-chamber glass LabTek culture slides were fixed with ice-cold 3.7% paraformaldehyde for 5 min and permeabilized using 0.2% Triton X-100. The slides were then blocked in PBS containing 0.2% gelatin and incubated overnight at 4°C with primary antibodies. The slides were then washed three times using PBS/gelatin and incubated with the appropriate AlexaFluor-conjugated secondary antibodies for 30 min. Nuclei were stained using 4',6-diamidino-2-phenylindole (DAPI). The slides were coverslipped using glycerol-based medium containing anti-fade reagent and viewed using a widefield microscope. Controls consisted of using matched mouse or rabbit immunoglobulin in place of the primary antibodies. Identical image capture settings were used for test and control samples. Captured images were pseudocolorized using Adobe Photoshop.The formation of multinucleated syncytiotrophoblast was assessed as we have described previously –24. TropTotal RNA was extracted from trophoblast cells using RNeasyplus kit (Qiagen) and cDNAs were obtained by reverse transcription (RT) using SuperscriptII reverse transcriptase . Real-time RT-PCR experiments were performed in triplicate using SYBR Green chemistry using StepOneplus Real-time PCR system . StepOneplus software was used to calculate ΔCt values normalized against a GAPDH control. Fold change was calculated using the Livak ΔΔCt method . The priTotal lysates were obtained by incubating cells in RIPA lysis buffer (ThermoScientific) supplemented with 1% Protease Inhibitor Cocktail  for 1 h at 4 . The supernatant was mixed with NuPAGE LDS sample buffer (Invitrogen) containing DTT and heated at 70°C for 10 min. The samples were centrifuged at 13,000 x g for 2 min and loaded on 4–12% Bis-Tris SDS-NuPAGE gels (Invitrogen) in MES buffer  and MOPS buffer for analyzing other proteins at 10 μg protein per lane. After electrophoresis the proteins were transferred to PVDF . The membrane was blocked for 1 h in 5% Blotto in TBST . The blocked membrane was incubated overnight with primary antibody in 5% Blotto in TBST, then washed and incubated with secondary antibody labeled with horseradish peroxidase (ThermoScientific). After further washing, the membrane was incubated with chemiluminescent substrate  and then exposed to a Kodak imager . Densitometry was performed using Kodak Carestream Molecular Imaging Softwareand normalized to GAPDH control.Gelatin zymography was used to measure the activity of MMP2 and MMP9. 5μl of culture supernatant was added to 5μl of 2X Tis-glycine SDS sample buffer  and incubated for 10 min at room temperature. The samples were loaded onto a 10% zymogram gel supplemented with 1% gelatin . Electrophoresis, renaturing and development of the gels were performed according to the manufacturer's instructions . The developed gels were stained with Simply Safe blue stain . The gels were photographed and the proteolytic activity was determined by densitometric analysis using Image J software.In order to correctly interpret the results of the invasion assay (see next paragraph) it was first necessary to test the effect of LiCl on cell proliferation. To test the effect of LiCl on cell proliferation equal numbers of trophoblasts were incubated in the presence either NaCl (control) or LiCl for 7 days after which DNA was quantified using a CyQuant Proliferation assay kit  following the manufacturer’s instructions.An invasion assay was performed using the CytoSelect 24 well, 8μm invasion assay kit . Briefly, trophoblast cells were cultured in the inserts and treated for 7 days in the presence of sodium chloride or lithium chloride. The inserts were removed from the medium and any cells on the lower surface were removed by using a sterile cotton swab. The inserts were placed in complete medium in the lower chamber and medium with no FBS was added to the upper chamber. 72h after incubation, invasion was assessed as described by the manufacturer.For trophoblast/endothelial cell co-culture experiments, UtMVECS were cultured to confluence in 2-well LabTek permanox slides under 2% oxygen. Trophoblasts that had been treated for 7 days in the presence of lithium chloride or sodium chloride were trypsinized and resuspended in trophoblast medium. Cells were counted and 10,000 cells were added per well. Slides were cultured in trophoblast medium and photographed, fixed and stained at different time points.t test for all other data using the Prism software program . Data are expressed as means ± SEMs and differences were considered significant if p < 0.05.All experiments were repeated at least 3 times. Statistical analysis was performed by two-way ANOVA with repeated measures for the time-course experiments and by Student’s -Vim+.+ cells and the majority of cells expressed CK7 only consistent with trophoblasts  we found significantly increased expression of α5-integrin and PECAM-1 consistent with extravillous trophoblast formation . Howeverin vitro conditions. We therefore compared the behavior of LiCl-treated trophoblasts and control trophoblasts when added and cocultured on top of adherent endothelial cells. When trophoblasts which had been incubated in the presence of LiCl for 7 days were added to confluent cultures of endothelial cells, large rounded colonies of trophoblasts could be distinguished from surrounding endothelial cells by their distinctive refractive properties  show reduced expression of PECAM-1 . This suoperties . In contoperties .When the cocultures that contained LiCl-treated trophoblasts were stained using antibodies against CK7 (trophoblast marker) and VE-cadherin , the trophoblast colonies appeared to be in “gaps” devoid of underlying VE-cadherin staining but closely abutted adjacent endothelial cells . EndotheNext we incubated trophoblasts with CHIR99021, a more highly specific inhibitor of GSK3. Unlike LiCl, this compound did not induce the appearance of spindle-shaped cells  nor did TNFα is known to inhibit villous trophoblast differentiation  as well Trophoblast differentiation is a complex and incompletely understood process that is essential for normal placental development and successful pregnancy outcome. In primates, two main pathways have been identified. The villous pathway results in the formation of multinucleated syncytiotrophoblast whereas the extravillous pathway results in the appearance of invasive trophoblasts (extravillous trophoblasts) . Cells tPlacentation in the rhesus monkey shows many similarities to that in the human  and we hAlthough the rhesus monkey trophoblasts expressed syncytin-2 and GCM1, two other genes with major roles in syncytiotrophoblast formation in the human, we found no evidence that sodium butyrate altered the expression of these genes at the transcriptional level in rhesus trophoblasts. However, this does not rule out changes at the translational level nor does it rule out their involvement in syncytiotrophoblast formation. We were unable to assess translational changes due to lack of antibodies reactive with rhesus monkey GCM1, syncytin-2, and EnvV2. Although GCM1 plays a major role in regulating the expression of syncytins  it is noper se does not determine specific cell fate decisions.While the effect of sodium butyrate reported here provides a useful tool for manipulating syncytiotrophoblast formation in vitro, its mechanism of action remains elusive. Sodium butyrate has a wide range of effects on cells –13. Manyin vitro studies support the idea that α5-integrin is directly involved in regulating trophoblast invasion [When cultured in the absence of LiCl (or butyrate) rhesus monkey trophoblasts showed significantly increased expression of α5-integrin and PECAM-1 over 7 days in culture consistent with acquisition of an extravillous phenotype. Surprisingly, although lithium chloride failed to induce syncytiotrophoblast formation, as described above, it significantly reduced the expression of α5-integrin, PECAM1, E-cadherin, and NCAM1 compared to control cells. The appearance of spindle-shaped cells was also observed in the presence of LiCl. Although trophoblasts also expressed αV-integrin , expression was not significantly altered by exposure to lithium chloride. A large number of immunohistochemical studies of first trimester human placental tissues have demonstrated increased expression of α5-integrin as extravillous trophoblasts change from the proliferative phase to the more invasive stage –57. Alsoinvasion . Invasivinvasion , 60. In invasion . The expinvasion –63. Incrinvasion , 64. Takin vitro coculture studies have also shown that trophoblasts attach to and erode/displace underlying endothelial cells [The idea that lithium chloride induced the formation an endovascular trophoblast phenotype is supported by the observation that lithium chloride-treated trophoblasts behaved differently from control trophoblasts when added to confluent endothelial cell monolayers. The lithium-treated trophoblasts formed large rounded colonies that attached to the substrate in intercellular gaps that formed within the endothelial monolayers in regions of trophoblast-endothelial cell contact. These gaps were devoid of endothelial cells since staining for VE-cadherin was negative in these areas. The gaps in the endothelial cell monolayers only appeared in the presence of trophoblasts and were not observed when endothelial cells were exposed to trophoblast conditioned-medium. Although the latter observation suggests that the endothelial gaps were not caused by factors secreted by the trophoblasts, it is possible that close contact of trophoblasts and endothelial cells creates a localized microenvironment in which high concentrations of secreted factors accumulate. Previous al cells –69. It hThe mechanism by which lithium chloride induced these changes in trophoblast cells does not seem to be directly related to its ability to inhibit GSK3 because of the results obtained using another GSK3 inhibitor, CHIR99021. This compound is highly specific for GSK3 and, unlike LiCl, failed to alter the acquisition of extravillous trophoblast adhesion molecules and also failed to induce the formation of spindle-shaped cells. Together, these results suggest that the effects of LiCl are not mediated through direct inhibition of GSK3 and not directly through activation of canonical Wnt signaling. Further experiments will be required to better understand these observations.While recognizing that it is difficult to uncover the mechanisms of action of sodium butyrate and lithium chloride on trophoblast differentiation, a possible explanation of their differential effects was suggested by the experiments using TNFα. We found that incubation of trophoblasts with TNFα, like LiCl, reduced the expression of PECAM1 and induced the formation of spindle-shaped cells. We therefore speculate that the effects of LiCl on trophoblasts might be related to increased TNFα production. LiCl has been reported to increase TNFα expression in neutrophils . The inhin vitro and may also assist in uncovering how trophoblast cell fate decisions are regulated during early pregnancy. Finally, it is suggested that future studies address the pathway-selective effects of butyrate and LiCl and that these may be related to their differential effects on TNFα production.In summary, the results presented here show that sodium butyrate and lithium chloride have differential effects on the differentiation of rhesus monkey trophoblasts although both induce nuclear accumulation of β-catenin. The ability of sodium butyrate and lithium chloride to induce syncytiotrophoblast formation and endovascular trophoblast formation, respectively, provides useful tools for manipulating trophoblast differentiation S1 TableCells were incubated with NaCl (control) or sodium butyrate (NaBu) and then fixed and stained for E-cadherin and DAPI as described in Methods. Plac1, Plac2, and Plac3 represent results from three different placentas. Three replicates were analyzed within each experiment.(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.S6 TableAssessment of trophoblast proliferation, MMP secretion and invasion was carried out as described in Methods. Results from 4 different experiments are shown.(XLSX)Click here for additional data file.S7 Table(XLSX)Click here for additional data file.S8 Table(XLSX)Click here for additional data file.S1 FigCells were stained for cytokeratin 7 (CK7), vimentin and DAPI as described in Methods and (TIF)Click here for additional data file.S2 FigCells were stained for cytokeratin 7 (CK7), vimentin and DAPI as described in Methods and (TIF)Click here for additional data file.S3 FigCells were fixed and stained for immunofluorescence detection of Factor VIII as described in Methods. The antibody against Factor VIII was obtained from Santa Cruz Biotechnologies (sc27647). Nuclei were stained using DAPI. Each row shows factor VIII staining and the respective DAPI stain of the same field of view. Each row represents samples from different placentas. As a control, cells were incubated with non-immune goat Ig.(TIF)Click here for additional data file.S4 FigCells were fixed and stained for immunofluorescence detection of CD34 and glycodelin as described in Methods. Antibody against CD34 was from Abcam (ab30375). Antibody against glycodelin was from Santa Cruz Biotechnologies (sc57511). Nuclei were detected using DAPI. Images show staining from cells from two different placentas in each case. Each row shows DAPI staining and the respective antibody stain. Note that the level of staining was similar to the background staining seen using control mouse Ig in place of the primary antibodies.(TIF)Click here for additional data file.S5 FigControl cells were incubated in the absence of NaBu as described in Methods and (TIF)Click here for additional data file.S6 FigControl cells were incubated with NaBu as described in Methods and levels. QPCR data and densitometry data were obtained as described in Methods.(TIF)Click here for additional data file.S7 FigTrophoblasts were incubated with NaCl (control) or NaBu and then stained with anti-beta-catenin antibody and DAPI as described in Methods and (TIF)Click here for additional data file.S8 FigTotal RNA was obtained from trophoblast cells using the RNeasy Plus Mini kit . cDNA synthesized from 1μg of RNA using Superscript II Reverse transcriptase  was used in PCR reactions using primers shown below. PCR reactions were performed using AccuPower PCR premix  at an annealing temperature of 60°C.(TIF)Click here for additional data file.S9 FigCells were incubated in the presence of NaCl (control) or LiCl for 7 days as described in Methods and (TIF)Click here for additional data file.S10 FigCocultures were established as described in Methods and (TIF)Click here for additional data file.S11 FigCells were incubated in the presence of TNFalpha, CHIR, CHIR plus TNF, or DMSO (vehicle control) as described in Methods and (TIF)Click here for additional data file.


Brucella abortus (BA) model and treated with or without high molecular weight intravenous iron. 4h after BA injection followed by 2h after intravenous iron treatment, inflammatory cytokines were upregulated by BA, but not enhanced by iron treatment. In long term experiments, mice were fed a regular or an iron deficient diet and then treated with intravenous iron or saline 14 days after BA injection. Iron treatment in mice with BA-induced AI was effective 24h after iron administration. In contrast, mice with IDA (on iron deficiency diet) prior to BA-IA required 7d to recover from AI. In these experiments, inflammatory markers were not further induced in iron-treated compared to vehicle-treated BA-injected mice. These results demonstrate that intravenous iron supplementation effectively treated the murine BA-induced AI without further enhancement of the inflammatory response. Studies in humans have to reveal treatment options for AI in patients.Intravenous iron supplementation is an effective therapy in iron deficiency anemia (IDA), but controversial in anemia of inflammation (AI). Unbound iron can be used by bacteria and viruses for their replication and enhance the inflammatory response. Nowadays available high molecular weight iron complexes for intravenous iron substitution, such as ferric carboxymaltose, might be useful in AI, as these pharmaceuticals deliver low doses of free iron over a prolonged period of time. We tested the effects of intravenous iron carboxymaltose in murine AI: Wild-type mice were exposed to the heat-killed  Anemia is with more than 2 billion people affected worldwide one of the major public health burdens. The most common form of anemia is iron deficiency anemia (IDA). The second most common form is called anemia of inflammation (AI) or anemia of chronic disease. AI can develop in a previously healthy individual or in addition to an already existing IDA in response to increased cytokine levels and inflammation , 2. The In intact iron homeostasis, high serum iron levels induce hepcidin , 11, 12.A decrease in serum iron concentrations and impairment of erythropoiesis are known as protective mechanisms of the body during inflammation. Iron can be used by virus, bacteria or parasites for their replication and/or amplification and thereby might enhance the infectious disease , 17.In patients with AI, treatment of the underlying disease is priority. The use of oral iron in inflammatory states is problematic, as oral iron cannot be absorbed from the gut or released from iron stores. Second, oral iron substitution might promote infections by delivering unbound iron or production of oxidative stress. A study in an endemic region for malaria in Zanzibar was interrupted, as routine oral iron and folate acid substitution caused an increase in overall mortality . If indiThe goal of the presented study was to investigate the efficiency of intravenous ferric carboxymaltose, a so called “type I” iron complex, to treat AI and the change of the inflammatory reaction in a murine model of AI. According to Geisser et al. type I complexes are described as robust with a long elimination half-time of 7–12h, strong in their kinetic and thermodynamic variability and should release labile iron in low amounts only . HypotheBrucella abortus (BA) AI mouse model which presents features similar to human AI, provoking a transient inflammation and AI .Erythroid maturation in the bone marrow was assessed by flow cytometry in C57BL/6 mice fed a regular iron diet or an iron deficient diet. Mice were then treated once with BA until anemic at 14d, followed by intravenous iron treatment for 24h or 7d. (TIF)Click here for additional data file.S4 Fig(A) fed a regular diet (198 ppm iron) or (B) iron deficient diet (5ppm) 4 weeks prior to the administration of 5x108 particles per mouse BA. After 14d mice were treated with a single dose (0.015 mg/g i.v.) iron carboxymaltose.Bodyweight of WT mice were either (TIFF)Click here for additional data file.S1 Table(TIF)Click here for additional data file.


Current findings suggest that mother's marital status indicating father's absence or conflicting relationship to father may be specifically related to dissociation and other stress-related symptoms.We have assessed relationships of mother's marital status, dissociative symptoms, and other psychopathological manifestations in a sample of 19 years’ old young adults (N = 364) participating in European longitudinal study .The results show clinically significant manifestations of dissociative symptoms in young adult men whose mothers were fatherless and in women whose mothers were re-married. Other psychopathological symptoms did not reach clinically significant manifestations.The results suggest that significant factor related to high level of dissociative symptoms in men growing in fatherless families might be linked with disturbed and conflicting attachment to a father's figure and pathological dependent attachment to mother. In women dissociative symptoms likely are linked to conflicting relationship between mother and daughter associated with stepfather’ presence in the family. Other factors influencing child's mental health related to parental separation represent parental relationships after divorce and quality of contacts that children may have with a parent living out of the family. In this context, there is evidence that divorce and destructive couple conflict represent major risk factors for many forms of dysfunction and psychopathological manifestations in children.5 In addition, there is evidence that children from single parent or blended families have increased vulnerability to traumatic and other stressful life events.8Recent empirical data indicate that relationships between parental divorce and children's emotional and behavioral problems are predominantly associated with psychological factors and there is a relationship between fatherlessness and children's emotional and behavioral problems.Although numerous findings show that conflicting attachment and disrupted family structure play a significant role in child psychopathology, there is no evidence indicating a relationship between disrupted family structure and dissociative symptoms that frequently occur as a response to traumatic or stressful experiences. Together these findings on stress influences related to conflicting attachment suggest a hypothesis that mother's marital status indicating father's absence in a family is related to dissociative psychopathology and other stress-related manifestations. With the aim to test the hypothesis, we have assessed whether mother's marital status will be related to dissociative symptoms, symptoms of traumatic stress, anxiety and depression, in a sample of young adults participating in the European longitudinal study  studying parental and environmental influences on children development.Within the framework of European Longitudinal Study of Parenthood and Childhood (ELSPAC) data of 364 Czech young adults who within period of 2 months responded to invitation for participation in the study were collected. All participants included in the study were 19 years’ old, born in 1992 in the interval of 6 months with a high school education . All participants lived in the city of Brno, with similar social and economical status. The ELSPAC study started in 1992 in few European countries and its organization centers were in the United Kingdom and Czech Republic. The participants included in the study were selected randomly from the population based on voluntary agreement provided by parents awaiting newborn children.To find potential mental health risks related to stress influences associated with mother's marital status, we have assessed dissociative symptoms, symptoms of traumatic stress, depression, and anxiety. All the participants gave written informed consent and the research based on collaboration of ELSPAC and Center for Neuropsychiatric Research of Traumatic Stress was approved by Charles University (First Faculty of Medicine) ethical committee.10 The DES is a self-reported scale with 28 items, asking respondents to indicate their response on 100-mm scale to what extent they experience typical dissociative phenomena in daily life . Normative score of the scale defines increased probability for manifestations of dissociative disorders for total scores >30. Dissociative phenomena for example include feelings of depersonalization, derealization, psychogenic amnesia, and others. DES as well as other psychometric measures used in this study were translated into Czech language from the English original and then back-translated into English. The resulting documents were compared with the originals by a native English speaker and all the tests have good psychometric properties and equivalent quality to test occurrence of the symptoms as their English originals.Symptoms of dissociation were assessed using the Dissociative Experiences Scale (DES).12 Normative score of the scale defines significant occurrence of somatoform dissociative symptoms for scores >30. Somatoform dissociative symptoms represent alterations in sensations of pain , alterations of perception, loss of motor control, gastrointestinal symptoms, etc. Subjects indicate the degree of their experience on 5-point Likert scale .Somatoform dissociative symptoms were measured using 20-items self-reported Somatoform Dissociation Questionnaire (SDQ-20).13 was used. The TSC-40 is a self-reported 40-item questionnaire rated on a 4-point Likert scale. TSC-40 evaluates symptomatology in adults associated with childhood or adult traumatic experiences and measures aspects of posttraumatic stress and other symptom clusters found in some traumatized individuals . Normative score of the scale assumes likely occurrence of traumatic events for scores >70.13For investigation of childhood traumas, the Trauma Symptom Checklist (TSC-40)14 was used. The BDI-II represents 21-items questionnaire for assessing depression . Subjects indicate degree of their experience of depressive symptoms on a 4-point Likert scale. Normative score of the scale defines significant level of depression for scores >30.14For the assessment of depressive symptoms, the Beck Depression Inventory (BDI-II)15 The SAS is a 20-item self-reporting questionnaire focused on the most common general anxiety symptoms. Each question is scored on 4-point Likert scale from 1 to 4. Normative score of the scale defines significant level of anxiety for scores >30.15Levels of anxiety symptoms were assessed using the Zung's Self-Rating Anxiety Scale (SAS) .Statistical evaluations of psychometric measures included means, standard deviations, Kruskal–Wallis analysis of variance (ANOVA), Mann–Whitney test for independent samples, and power analysis. All the methods of statistical evaluation were performed using the software package Statistica, version 6.The group of participants (N = 364) was divided into 3 subgroups according to the mother's marital status. The first Fatherless subgroup contained 60 participants and included 20 men whose mother was divorced (N = 16) or unmarried (N = 4), and 40 women whose mother was divorced (N = 36) or unmarried (N = 4). The second subgroup contained 273 participants whose mother was married and included 114 men and 159 women. The third group contained 31 participants whose mother was re-married and included 17 men and 14 women.P < 0.0002, z > 4.23 and SDQ-20 at P < 0.002, z > 3.69), symptoms of traumatic stress , depression , and anxiety .In the statistical analysis, all differences between these 3 subgroups using Kruskal–Wallis ANOVA for the whole group including women and men Table  and sepaP < 0.002, z > 0.76) and women .The statistical analysis shows significant clinical values of psychological dissociative symptoms measured by DES Table . All difP < 0.01; between Fatherless and Re-married subgroup is 0.74, P < 0.01; whereas the effect size between Married and Re-married subgroup was not statistically significant (population SD = 8.71). The effect size for DES Means in women between Remarried and Married subgroup is 0.45, P < 0.01; between Remarried and Fatherless subgroup is 0.43, P < 0.01; whereas the effect size between Married and Fatherless subgroup is not significant.Because Kruskal–Wallis ANOVA has not well established and standardized methods of power analysis, we have estimated standardized effect sizes for the mean values characterizing differences of dissociative symptoms measured by DES between the subgroups of men and women Table  that werP < 0.01), traumatic stress (TSC-40) , depression (BDI-II) , and anxiety (SAS) .The results also indicate that psychological dissociative symptoms measured by DES are significantly correlated to symptoms of somatoform dissociation (SDQ-20) , anxiety (SAS), and traumatic symptoms (TSC-40) (18 Key results of this study represent significantly increased dissociation in young adult men whose mothers were fatherless (unmarried or divorced) and increased dissociation in women whose mothers were re-married.The results indicate that fatherless conditions in single parent families in boys and stepfather influence in families with girls are significantly associated with development of psychological dissociative symptoms that have clinically significant values. Other psychopathological manifestations related to mother's marital status did not reach clinical values, but may represent latent vulnerability that may manifest later in life because of specific influences of stressful experiences on brain development.21 and some data also suggest that stepfather–daughter erotic attachment and sexual abuse is more prevalent than the abuse by biological fathers.25 In addition, mother–daughter attachment due to stepfather–daughter erotic transference (or partnership) can result to competition and conflict between mother and daughter, which may seriously affect mental disintegration and dissociative symptoms.In this context of differences in dissociative symptoms between women and men, results of this study show that girls and boys respond to the same mother's marital status and to limited father's presence or absence differently. Daughters of remarried mothers have pathologically increased dissociation in comparison with sons. This result is in agreement with findings suggesting that girls had more difficulties interacting with stepfathers than sons34 In addition, in the case of boys without fathers or step-fathers, there is an increased risk of compensatory erotic transference that significantly increases pathologically dependent attachment between mother and son.36 For example, recent data indicate that more than two-thirds of African American children are born to unmarried mothers,37 which may represent a significant factor of criminal behavior.39 These data seem to support several psychoanalytical data and interpretations regarding mental disintegration associated with pathologically dependent mother-son relationships and violence.39 Further research is warranted to explain to which extent psychodynamic factors play significant roles in these family processes associated with dissociation.On the contrary, boys living with unmarried or divorced (not re-married) mothers, have clinically significant dissociation. The reason for this difference might be that, in comparison with girls, boys need a specific kind of separateness from mothers to find male identity for which they need father or father's figure.


VHL) gene. In contrast, inactivation of VHL has been previously observed in only a minority of sporadic hemangioblastomas, suggesting an alternative genetic etiology. We performed deep-coverage DNA sequencing on 32 sporadic hemangioblastomas , followed by analysis of clonality, copy number alteration, and somatic mutation. We identified somatic mutation, loss of heterozygosity and/or deletion of VHL in 8 of 10 discovery cohort tumors. VHL inactivating events were ultimately detected in 78% (25/32) of cases. No other gene was significantly mutated. Overall, deep-coverage sequence analysis techniques uncovered VHL alterations within the neoplastic fraction of these tumors at higher frequencies than previously reported. Our findings support the central role of VHL inactivation in the molecular pathogenesis of both familial and sporadic hemangioblastomas.Hemangioblastomas consist of 10-20% neoplastic “stromal” cells within a vascular tumor cell mass of reactive pericytes, endothelium and lymphocytes. Familial cases of central nervous system hemangioblastoma uniformly result from mutations in the Von Hippel-Lindau (The online version of this article (doi:10.1186/s40478-014-0167-x) contains supplementary material, which is available to authorized users. VHL on chromosome 3, with subsequent inactivation of the second allele by somatic mutation or gene loss in the neoplastic “stromal” cell. The clinical spectrum of VHL disease includes other neoplastic lesions, including retinal angiomatosis, pheochromocytoma and clear cell renal carcinoma.Hemangioblastomas comprise 1–2.5% of primary intracranial and 7-10% of spinal cord tumors, with a benign pathology notable for neoplastic “stromal” cells embedded in a dense network of vascular channels , and havVHL somatic mutations have been clearly identified in a fraction of sporadic tumors [VHL , (2) lack of family history of hemangioblastomas or VHL disease, and/or (3) lack of other stigmata of VHL disease, such as renal cancer, pheochromocytoma, or retinal angiomas. Pathology was verified by the Departments of Pathology at MGH and BWH  and tumor rich areas were microdissected.This study was approved by the Institutional Review Board at the hospitals providing specimens. Written informed consent was obtained for all samples undergoing whole exome sequencing. The discovery and validation cohorts of sporadic hemangioblastomas involving the cerebellum and spinal cord were obtained from patients treated at Massachusetts General Hospital , Brigham and Women’s Hospital , and Seoul National University (South Korea). Patients were identified as likely sporadic hemangioblastomas based on (1) prior testing that was negative for germline DNA was extracted from FFPE tumor specimens and matching peripheral blood samples using standard techniques  and quantified by PicoGreen dye (Invitrogen). Whole exome sequencing was performed by hybrid capture and next-generation sequencing as described previously . In brieVHL and ARID1B. The captures were sequenced on the Illumina HiSeq 2500 in Rapid Run Mode.The validation cohort was sequenced for 560 cancer-associated genes and 39 translocations previously implicated in cancer  pathology specimens, we applied a filtering algorithm to reduce the number of artifactual mutations introduced by FFPE [Somatic variant, germline variant, and small deletions and insertion calling was performed within the Firehose environment at the Broad Institute with the previously published MuTect, MapReduce, and Indelocator algorithms -30. Pind by FFPE . The FFP by FFPE .Segmented copy data was obtained using copy number ratios. These were calculated as the ratio of tumor read depth to the average read depth observed in a panel of normal samples. Subsequently we converted the copy ratio data to allelic ratio, which allows for detection of copy neutral LOH and gives another constraint on copy number alterations. ABSOLUTE  was usedVHL in our validation cohort was deleted or in copy neutral loss of heterozygosity, we examined the distribution of allele fractions of heterozygous mutations in the mix of reported somatic and germline variants. In a diploid state (ploidy = 2) germline heterozygous sites should be represented by 50% allele fraction. When samples gain or lose a copy, the allele fraction of those sites will shift in relationship to the purity of the tumor. In order to determine the copy state of each sample we identified peaks in the probability density function (pdf) of allele fractions:To assess whether af is the true allele fraction between 0 and 1, and alt is the alternate allele counts, ref is the reference allele count, and m is each single nucleotide variant detected along chromosome 3p. Samples demonstrating allelic fractions with bimodal distributions were called positive for a deletion or copy neutral loss of heterozygosity.Where T) was further estimated from the allele fraction of the upper allele (χA) in the two following scenarios.T) is given by the following equation:if the LOH event represents a copy neutral event, then the tumor purity , VEGF , or PDGFR-β  by protocols described in the product data sheets. IHC was reviewed and scored by MPH.VHL mutations, indicating that these patients do not have VHL disease and that the tumors we evaluated are bona fide sporadic hemangioblastomas. The exomes of tumor specimens in the discovery cohort were covered at a mean depth of 116×  of 10 sporadic hemangioblastomas for our discovery cohort and targeted sequencing of 22 sporadic hemangioblastomas for our validation cohort. Estimation of neoplastic stromal cell content was 10-30% at pathology review  specimens, which are characterized by artificially induced C > T . These aVHL was mutated in 5 samples with an allele fraction ranging from 18-21% and cancer cell fraction from 76-100%, suggesting a clonal somatic mutation carried by the subpopulation of neoplastic “stromal” cells. VHL was the only gene found as statistically significant with respect to the false discovery rate by MutSigCV [The sequence data was then post-processed with MuTect  to identMutSigCV   than exons 2 (213×) and 3 (51×)  and 39 translocations previously implicated in cancer were sequenced at an average depth of 182x. The validation cohort revealed LOH of chromosome 3p in 15 of the 22 samples  or platelet derived growth factor B (PDGF B) [Biallelic (PDGF B) . Accordi(PDGF B) ,40. GenoVHL in 47% and an inactivating alteration in at least one allele in 78% of sporadic hemangioblastomas. We detected these alterations in specimens with tumor cell fractions as low as 9%. It is possible that we could have captured an even higher prevalence of VHL inactivation via somatic mutation or LOH with deeper sequencing across VHL in its entirety.As predicted by the benign clinico-pathologic features, hemangioblastomas were notable for a relatively low frequency of mutations. Within this context of infrequent mutations and copy number alterations, we found a markedly high prevalence of recurrent somatic mutations (50%) and LOH of the gene locus on chromosome 3p (72%). Together, these alterations account for biallelic inactivation of VHL in 10 to 44% of sporadic hemangioblastoma [VHL in only 1 of 13 sporadic hemangioblastomas [VHL by comparative genomic hybridization in only 2 of 16 sporadic hemangioblastomas [VHL mutant allele fractions confined to the neoplastic “stromal” cells in sporadic hemangioblastomas, as compared to familial tumors that are characterized by heterozygous germline VHL alterations.Prior studies have reported the presence of mutations in blastoma ,3. One sARID1B, which behaves as a tumor suppressor in other cancers [Mutations in  cancers -44, were cancers ,46.VHL inactivation in the pathogenesis of sporadic hemangioblastoma mirrors the VHL alterations seen in benign renal cysts, which may serve as precursors of clear cell renal cancer [VHL inactivation in renal tubular epithelial cells; whereas, clear cell renal cancer is additionally characterized by mutations in genes involved in the PTEN-mTOR pathway [BAP1, SETD2, and PBRM1 implicated in clear cell renal carcinoma.l cancer . These c pathway ,14. SimiVHL inactivation in these tumors parallels “stromal” cell content estimated by IHC, rather than representing a homogenous genome that would characterize a tumor cell mass entirely derived from an ancestral hemangioblast clone. This important distinction lends further support to the model that stromal cells are genetically unique in VHL deficiency and stimulate reactive angiogenesis by recruiting normal endothelial cells via pseudo-hypoxia mediated paracrine signaling. This model contrasts with the alternate proposed pathway of an embryologically arrested “hemangioblast” ancestral clone that trans-differentiates into the various cell types contained in the tumor [The analysis of tumor purity by ABSOLUTE also demonstrates that the extent of he tumor ,25,36.VHL is highly recurrent and characteristic of the majority of sporadic hemangioblastomas. In addition, we do not detect other common recurrent somatic mutations in these tumors. These data therefore confirm the central role of VHL inactivation in all hemangioblastomas, suggesting that treatment strategies for both familial and sporadic cases could be uniformly considered, as VHL loss in the neoplastic subpopulation likely orchestrates the architecture of these tumors. For example, VEGF inhibitors have been used in cases of multifocal familial hemangioblastomas with treatment responses [In summary, we find that inactivation of esponses ,49; theiVHL inactivation is more highly prevalent in this disease process than previously noted and that no other gene alteration was identified at a statistically significant level in these tumors. High depth sequencing was required to unmask the low allelic fraction of VHL mutations and copy number alterations found in the neoplastic “stromal” cells, which are present only in a fraction of cells comprising the overall tumor mass. The central role of VHL in the formation of sporadic hemangioblastomas suggests that the approach to these tumors can be universally considered as a single entity together with familial cases, which arise as a result of VHL disease.In this study, we performed whole exome sequencing of matched tumor-normal specimens of sporadic hemangioblastoma and found that


Phenylketonuria (PKU) is one of the most common inherited metabolic disorders. The molecular pathway of neurological damage is not yet sufficiently understood. To date, there is a lack information about cardiac involvement related to the disease. This study aims to characterize cardiac morphology and function in adult patients with PKU using cardiovascular magnetic resonance (CMR).28 patients with PKU (age 30 ± 9 years/mean ± SD) underwent a comprehensive CMR protocol at a 1.5T CMR scanner  including assessment of left ventricular (LV) volume and mass. In addition, T1 measurements pre- and post-administration of gadolinium for evaluation of extra corpuscular volume (ECV) and tagging for quantitative analysis of left ventricular circumferential strain (Ecc) were performed. 8 healthy age-matched volunteers underwent a similar protocol and served as controls for the ECV values. LV parameters and Ecc were compared to reference values from previous studies with similar data setup [CMR exams were successfully performed in all patients. As shown in Figure The results of this study indicate that in PKU patients compared to healthy controls LV mass indexed to BSA is reduced to the reference values; we found increased average Ecc parameters and normal ECV values. Further investigations in larger patient groups and older PKU patients are necessary for evaluation of cardiac involvement of the disease over time and its consequences.None.


In vitro assays showed that SRF promotes survival and filopodia formation and is required for normal asymmetric neurite outgrowth, indicating that its activation favors dendrite enlargement versus branching. In turn, in vivo experiments demonstrated that SRF-dependent regulation of neuronal morphology has important consequences in the developing cortex and retina, affecting neuronal migration, dendritic and axonal arborization and cell positioning in these laminated tissues. Overall, our results show that the controlled and timely activation of SRF is essential for the coordinated growth of neuronal processes, suggesting that this event regulates the switch between neuronal growth and branching during developmental processes.The stimulus-regulated transcription factor Serum Response Factor (SRF) plays an important role in diverse neurodevelopmental processes related to structural plasticity and motile functions, although its precise mechanism of action has not yet been established. To further define the role of SRF in neural development and distinguish between cell-autonomous and non cell-autonomous effects, we bidirectionally manipulated SRF activity through gene transduction assays that allow the visualization of individual neurons and their comparison with neighboring control cells.  The assembly of neuronal networks is accomplished through a precise sequence of developmental processes that are highly dependent on cellular motile functions, including cell migration, axon versus dendrite specification, neurite outgrowth, axon guidance and synaptic targeting. It is thought that the stimulus-regulated transcription factor (TF) Serum Response Factor (SRF) plays a role in these processes by regulating actin dynamics134Srf impairs tangential cell migration along the rostral migratory stream and causes the ectopic accumulation of progenitor cells in the subventricular zone (SVZ)Srf loss in forebrain neurons abolishes mossy fiber segregation and causes ectopic fiber growth in the hippocampus131415in vivo, are still unknown.From the early gastrulation defects observed in conventional knockout (KO) mice to the most specific phenotypes found in tissue-restricted conditional mutants, the deficits associated with SRF’s loss-of-function (LOF) have been consistently related to cytoskeletal perturbations127in vitro and in vivo experiments in which SRF activity was bidirectionally manipulated. Our experiments in neuronal cultures reveal that SRF controls the asymmetric outgrowth of neurites favoring primary dendrite enlargement versus branching. In turn, this cellular function influences different neurodevelopmental processes, including neuronal migration, tissue lamination and circuit assembly, which highlights the critical importance of fine-tuned SRF activity in the development of the nervous system.Here, we investigated the cell-autonomous role of SRF in neuronal growth and neurodevelopment by combining To precisely investigate the impact of chronic enhancement or interference of SRF function in neuronal growth, we produced lentivirus-based bicistronic constructs that co-express GFP with different SRF variants under the control of the neuronal-specific synapsin promoter . For gaiper se toxic. Moreover, as previously reported2021We next combined lentiviral infection of primary hippocampal cultures from E17.5 mouse embryos with the transfection of a plasmid encoding dsRed . The lowin vivo, we transduced the lentiviral constructs described above in the somatosensory cortex of E14.5 embryos by in utero electroporation and examined the brain of electroporated mice at P20  , panel cSlit2, Dcc, Nrp1, Rnd2, Alcam, Epha7, Epha3 and Epha5.In order to identify candidate effector genes downstream of SRF responsible for these phenotypes, we turned to the transcriptomics screen presented inArc and Egr1 promoters  assays in infected hippocampal cultures that confirmed the direct binding to the romoters . For othromoters . Consistromoters , these eromoters . Finally neurons  and RGCs neurons .in vitro and in the formation of neuronal circuits in vivo. Our results are congruent with previous LOF studies in SRF deficient mice indicating that interfering with SRF function alters neuronal growth and neurodevelopment1011in vitro GOF experiments indicating that enhancing SRF function promotes neuronal growth1011212021in vitro and in vivo. Axonal arborization was also severely reduced both in cortex and retina. This altered neuronal polarity, in turn, affected neuronal migration and the correct positioning of the soma in laminated structures like the cortical plate and the retina. Consistent with the cell-autonomous effect of caSRF expression reported here, recent studies have shown that ectopic expression of a different constitutive active form of SRF caused the mislamination of the photoreceptor layer in the retina of young transgenic animalsWe demonstrate here that the manipulation of SRF activity in neurons has important consequences in cell morphology, survival and growth Arc, transgelin (Tagln) and several members of the Egr family , we identified a number of novel downstream genes related to the various phenotypes associated with enhanced SRF-driven transcription  encoded by Slc8a2, a low affinity, high capacitance calcium anti-porter membrane protein that can switch to ‘reverse mode’ under excitotoxicity and other forms of cellular stress, is a likely candidate to mediate the neuroprotective effect of caSRFSlc8a2 and Abca8a  are two of the genes specifically expressed in the cortical subplate at E15.5Other genes whose function in neurons is less understood can also contribute to the altered morphology. For instance, the downregulation in caSRF-expressing neurons of the small Rho GTPase encoded by as Net1A , which lIn conclusion, although the list of putative transcriptional targets and their biological role are highly consistent with the phenotypes associated with SRF manipulations, the direct binding to most of these targets remains to be determined and unspecific caSRF-DNA interactions are not excluded. It is expected that several effector genes downstream of SRF redundantly or synergically contribute to the alterations associated with enhanced SRF activity. Functional assays for each of these candidate genes overexpressed either individually or in combination would be required to determine the precise molecular underpinning of the different phenotypes described here.in vivo. During development, dendrites and axons of central nervous system neurons grow and refine by addition and retraction of thin branches. This process, reproduced in neuronal cultures, is highly dynamic and only a small fraction of newly added branches remain as part of the mature dendritic and axonal trees. It is thought that the growth rates of axons and dendrites result from the interplay between pre-programmed TFs levels and intrinsic length-sensing mechanismsLittle is still known about how the precise regulation of neurite outgrowth by intrinsic and extrinsic signals leads to the intricate branching pattern unique to each neuronal class observed To generate the caSRF variant, the DNA binding domain (DBD) of SRF was fused with the acidic transactivation domain of viral protein 16 (VP16) of herpes simplex virusin utero electroporation experiments in the cerebral cortex, whereas B6D2F1/J mice were used for in utero electroporation experiments in the retina because pigmented eyes are easier targets for DNA injection. All mice were maintained according to animal care standards and experimental protocols were approved by the Institutional Animal Care and Use Committee.TetO-caSRF transgenic mice were generated by microinjection of the linear construct as previously describedin vitro 1 (DIV1). Neurons were incubated for at least 3 additional days (DIV4) before they were used in any experiment. Primary hippocampal neurons were infected at the times indicated by adding the necessary volume of the concentrated viral preparation to achieve an effective multiplicity of infection of 1–10, which provided a percentage of neuronal infection close to 100%. In co-infection experiments, equal volumes of each virus were mixed. In transfection experiments, the plasmid pDsRed-Express2-C1 (Clontech 632538) driving dsRed expression was transfected using Lipofectamine 2000 (Invitrogen) as described by the manufacturer.Primary hippocampal neurons were obtained from E17.5–E18.5 embryos. Hippocampi were dissected and processed as describedFor luciferase assays, HEK293 cells were grown in 90% DMEM 10% fetal calf serum supplemented with 2 mm glutamine and penicillin/streptomycin (100 U/ml to 100 μg/ml) (Invitrogen). HEK293 cells were transfected using Lipofectamine 2000 (Invitrogen) with efficiency larger than 90%. Plasmids were transfected in a ratio of 500:50:1 (construct of interest: Firefly reporter: Renilla reporter) with a total DNA amount of 1 μg per 100.000 cells. The Firefly and Renilla luciferase reporter plasmids used in this study were pSRE-Luc (Clontech 631911) and pRL-SV40 (Promega E2231), respectively.Luciferase activity was measured at 24 h after transfection, using the Dual Luciferase Assay kit (Promega) as reported beforein utero electroporations were performed in timed-pregnant ICR mice anesthetized with isofluorane; their abdominal cavity was cut open, and the uterine horns exposed. Approximately 1–2 μl of DNA solution (1 μg/μl) was injected into the lateral ventricle of E14.5 or E16.5 embryos using a pulled glass micropipette. In co-electroporation experiments we used the same amount of total DNA . Each embryo was placed between tweezers-type electrodes . The angle of inclination of the electrode paddles with respect to the horizontal plane of the brain was zero for targeting the cortical ventricular zone (VZ). Square electric pulses  were passed five times at 950 ms intervals using an electroporator . Retina in utero electroporation experiments were performed in E13.5 B6D2F1/J embryos as described inCortex Immunostainings in primary hippocampal cultures were conducted as described inTotal RNA from hippocampal cultures was extracted with TRI reagent (Sigma-Aldrich) and reverse transcribed to cDNA using the RevertAid First-Strand cDNA Synthesis kit (Fermentas). qPCR was performed in an Applied Biosystems 7300 real-time PCR unit using Eva Green qPCR reagent mix. The primer sequences used in the RT-qPCR assays are shown in 6) were scraped and resuspended in 400 μl 1% SDS, 10 mM EDTA, 50 mM Tris-HCl (pH 8.1), and protease inhibitors and incubated for 10 min on ice. The cell suspension was sonicated using a Branson sonicator at 10% of capacity, 6 cycles of 15 sec. Chromatin was used for ChIP by incubation for 16 h at 4 °C with 5 μg specific anti-VP16 (from Sigma V4388) or isotype-matched controls Abs, followed by 2.5 h incubation at 4 °C with 30 μl protein G Dynabeads. The DNA was resuspended in 30 μl of water for subsequent PCR analysis as a template to evaluate the presence of regulatory regions of caSRF targets. The primer sequences used in the ChIP-qPCR assays are listed in t-tests were used to determine the statistical significance between values.Chromatin immunoprecipitation (ChIP) experiments were conducted as previously describedHow to cite this article: Scandaglia, M. et al. Fine-tuned SRF activity controls asymmetrical neuronal outgrowth: implications for cortical migration, neural tissue lamination and circuit assembly. Sci. Rep.5, 17470; doi: 10.1038/srep17470 (2015).