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SETD5 haploinsufficiency affects mitochondrial compartment in neural cells
Molecular Autism volume 14, Article number: 20 (2023)
Neurodevelopmental disorders (NDDs) are heterogeneous conditions due to alterations of a variety of molecular mechanisms and cell dysfunctions. SETD5 haploinsufficiency leads to NDDs due to chromatin defects. Epigenetic basis of NDDs has been reported in an increasing number of cases while mitochondrial dysfunctions are more common within NDD patients than in the general population.
We investigated in vitro neural stem cells as well as the brain of the Setd5 haploinsufficiency mouse model interrogating its transcriptome, analyzing mitochondrial structure, biochemical composition, and dynamics, as well as mitochondrial functionality.
Mitochondrial impairment is facilitated by transcriptional aberrations originated by the decrease of the SETD5 enzyme. Low levels of SETD5 resulted in fragmented mitochondria, reduced mitochondrial membrane potential, and ATP production both in neural precursors and neurons. Mitochondria were also mislocalized in mutant neurons, with reduced organelles within neurites and synapses.
We found several defects in the mitochondrial compartment; however, we can only speculate about their position in the hierarchy of the pathological mechanisms at the basis of the disease.
Our study explores the interplay between chromatin regulation and mitochondria functions as a possible important aspect of SETD5-associated NDD pathophysiology. Our data, if confirmed in patient context, suggest that the mitochondrial activity and dynamics may represent new therapeutic targets for disorders associated with the loss of SETD5.
A functional mitochondrial compartment is mandatory to support proper cell physiology and metabolism. This is particularly true for highly metabolic-demanding cells such as neurons. Accordingly, impairments in mitochondrial biogenesis, turnover, and specific functions of the organelle, such as oxidative phosphorylation, calcium handling, and ATP production, are associated with several neurological disorders including Parkinson’s disease, ataxia, epilepsy, and others [1, 2]. Interestingly, mitochondrial dysfunctions have been observed in a slice of cases of neurodevelopmental disorders (NDD) [3,4,5,6], for example, the prevalence of mitochondrial disease in children affected by autism spectrum disorders (ASD) was at least 5%, much higher than in general population (0.01%) [7, 8]. However, the etiology and the actual impact of mitochondrial dysfunction in NDD are unclear and poorly investigated. NDDs represent a galaxy of heterogeneous diseases in which genetic causes, when either present or identified, rely mainly on genes encoding for synaptic components, epigenetic modifiers, and transcriptional regulators [9,10,11]. De novo and familiar mutations in the SETD5 gene, eventually resulting in genetic haploinsufficiency, have been associated with NDD, specifically ASD and intellectual disability (ID) [10, 12,13,14,15,16,17,18,19,20,21]
SETD5 codifies for a protein containing a SET (Su(var)3–9, Enhancer-of-zeste, Trithorax) domain, a member of the histone-modifying protein family . The direct function of SETD5 as histone methyltransferase activity is debated [23,24,25]. SETD5 may interact with HDAC-containing complexes regulating access to chromatin [23, 26,27,28]. Experimental Setd5 haploinsufficiency in neural stem cells (NSC) and mouse brain affects RNA polymerase elongation rate with consequent alterations on gene transcription at multiple levels including expression level and alternative splicing . Phenotypically, low levels of the protein impact brain development dynamics, synaptic transmission, and potentiation as well as animal behavior possibly resembling what was observed in humans [23, 24, 26]
In this study, we investigated the affinity of SETD5 for mitochondrial-associated genes and identified impairment of mitochondrial compartment in different experimental models of Setd5 gene loss including NSC and cerebral cortex. We report that a defect in the control of gene transcription leads to alterations in the maturation, dynamics, and functionality of mitochondria that possibly contribute to the etiology of SETD5-associated NDDs.
Neural stem cells culture
Neural stem cells (NSCs) were derived from Telencephalic cortex of embryos at 14.5 days of gestation. Embryonic cortices were dissociated, fragmented in Hank’s Balanced Salt Solution (HBSS, Life Technologies) with 1% Penicillin/Streptomycin (Sigma-Aldrich) and digested with papain (10 U/ml, Worthington Biochemical) and cysteine (1 mM, Sigma-Aldrich) in HBSS with 0.5 mM EDTA at 37 °C. The obtained NSCs were routinely cultured in suspension as neurospheres.
Cells were cultured in Neural inducing medium (NIM) composed of: DMEM/F12 (Sigma-Aldrich) supplemented with Hormon Mix (DMEM/F12, 0.6% Glucose (Sigma-Aldrich) (30%), Insulin (Sigma-Aldrich) 250 µg/ml, putrescin powder (Sigma-Aldrich) 97 µg/ml, apotransferrin powder (Sigma Aldrich), sodium selenite 0.3 µM, progesterone 0.2 µM), 1 mg/ml penicillin/streptomycin (Sigma-Aldrich), 2 mM glutamine (Sigma-Aldrich), 0.66% Glucose (30% in phosphate buffer salt (PBS) (Euroclone)), Heparin 4 µg/ml, 10 ng/ml bFGF (basic fibroblast growth factor) (ThermoFisher Scientific) and 10 ng/ml EGF (epithelial growth factor) (10 ng/ml) (ThermoFisher Scientific).
To culture NSCs in adhesion, cells were seeded in coverslips coated with 1:100 Matrigel.
For low oxygen culture condition, cells were maintained in an incubator with 5% oxygen concentration. For high oxygen culture, cells were cultured in an incubator with 21% oxygen concentration.
Mutant Setd5+/− NSCs were generated as previously explained using Crispr-Cas9 technology .
Glucose-free NSCs culture
NSCs were cultured in normal NIM using DMEM without glucose (GIBCO) instead of normal DMEM. The media was supplemented with 25 mM galactose, Hormon Mix which contains glucose was replaced by N2 supplement, the rest of the ingredients remained the same of NIM.
RNA extraction with RNAeasy mini kit and RNA sequencing of High oxygen NSCs
To perform RNA extraction, cells were seeded in adherent condition (see above) in 15 mm Petrie dish at approximately 60–70% of confluence and were grown until they reached almost 90% of confluence. Cells were then scraped and pelleted in cold (4 °C) and sterile PBS. RNA extraction was performed using RNAeasy mini kit (Qiagen).
Each sample was lysed using 600 µl of RLT buffer of RLT buffer and 1 volume of 70% ethanol was added and to the cell lysate. Then up to 700 µl of the sample, were transferred to an RNAeasy spin column placed in 2-ml collection tube and centrifuged for 15 s at 10,000 rpm and the flow trough was discarded. If the volume of the sample exceeded 700 µl the centrifuge is repeated a second time with the rest of the sample.
After this step, DNAse digestion is performed to eliminate DNA contaminants. First wash with 350 µl of RW1 is performed centrifuging at 10,000 rpm for 15 s, after the wash a mix compose of 10 µl of DNAse and 70 µl of buffer RDD are added to each column and incubation at room temperature for 15 min is performed, then after incubation another wash with 350 µl of RW1 is performed centrifuging at the same speed and time of the first wash. Buffer RPE (500 µl) is then added to the column and centrifuge for 15 s at 10,000 rpm, the flow through is then discarded. Another wash step with RPE is then performed, centrifuge step this time is for 2 min at the same speed. Then spin column is then placed on a new 2-ml collection tube and 1 min centrifuge at full speed is performed to eliminate RPE buffer carryover.
Finally, to elute the RNA a new 1.5-ml collection tube is positioned under the spin column, and 30 µl are gently posed on the filter and 1 min centrifuge is performed to elute the RNA.
An RNA sequencing (RNA-seq) with a coverage of 30 million reads (paired end) was performed on control samples and on mutant heterozygous Setd5 clones. RNA was extracted as described below.
The sequencing was performed by the Centre for Translational Genomics and Bioinformatics at San Raffaele Hospital. Libraries have been prepared using Tru Seq RNA v2 kit (Illumina) starting from total RNA. Sequencing was performed using HiSeq 2500 (Illumina).
RNA sequencing analysis
Fastq files, after adaptors trimming using Trimmomatic , were aligned to the 10-mm mouse reference genomes using Bowtie2 . Differential gene expression and Functional enrichment analyses were performed with DESeq2  and GSEA , respectively. Statistical and downstream bioinformatics analysis were performed within the R environment. RNA-seq of NSCs cultured in low oxygen condition was performed as described in our previous work .
The data were deposited in the NCBI Gene Expression Omnibus and are accessible through GSE103912 (NSCs low oxygen) and GSE222188 (NSCs high oxygen).
RNA extraction with trizol
To perform RNA extraction, NSCs were firstly seeded in adherent condition using matrigel 1:100 in 6 wells at a density of at least 200,000 per well in NIM.
After 2–3 days, medium was removed, cells were washed once with PBS and were dissolved using 1 ml of Trizol (ThermoFisher Scientific) 5 min at room temperature, directly in the well. After dissolving, samples were collected in 1.5-ml tubes (Eppendorf) and 0.2 ml of chlorophorm were added, tubes were then shaken vigorously and then incubated 2–3 min at room temperature. Tubes were subsequently centrifuged at 13,000 rpm for 15 min at 4°. An aqueous phase appears at the top of the liquid volume, that is collected and transfer to a new tube, the rest is discarded.
To precipitate RNA Isopropyl alcohol is added, 0.5 ml per 1 ml of Tryzol, shake and incubated for 10 min at room temperature before centrifuge 10 min at 13,000 rpm at 4°. After this passage, a pellet is often visible, supernatant is removed and 1 ml of 75% ethanol per ml of Tryzol is added, then samples are vortexed and centrifuged at no more than 7500 rpm for 5 min at 4°. Finally, ethanol is removed, and RNA pellet is dried at room temperature, then a suitable volume of nuclease free water is added, and samples are posed at 55° for a maximum time of 10 min to allow complete resuspension of RNA.
Retrotranscription and real-time quantitative PCR
RNA retrotranscription was performed using ImProm II reverse transcription kit (Promega).
Briefly, the reaction is divided in two parts. The first reaction is a denaturation step of a mix composed of 1 µg of RNA, random primers (0.5 μg/reaction) and water up to 5 μl, for 5′ at 70 °C. Then after chilling the first mix on ice for 5′, a second retronscription mix (4 μl of ImProm II 5 × buffer, 3.8 μl MgCl2 (final concentration 2.5 mM), 1 μl dNTP Mix (final concentration 0.5 mM each dNTP), 1 μl ImProm II reverse transcriptase, 5.2 of nuclease free water) is added to first mix, and then reverse transcription is performed with the following reaction profile:
4° × 30″
25° × 5′
42 × 1 h
70° × 15′
16 × ∞
After c-DNA synthesis real-time quantitative PCR was performed (RT-qPCR) using CFX 96 Real-Time System (Biorad) thermocycler.
The real-time TITAN (Bioatlas) mix was composed as follows for each reaction samples:
2 μl of c-DNA
9.8 μl of H2O
3.2 μl of TITAN HotTaq EvaGreen qPCR Mix (Titan hotTaq DNA polymerase, 5 × qPCR buffer E, 12.5 mM MgCl2)
The amplification profile used was the following:
95° × 15′
(95° × 15″, 60° × 20″, 72° × 20″) × 40
RT-qPCR data were analyzed using CFX Manager software (Biorad) and the differential gene expression was calculated using the ∆∆ct methods. Used primers are listed in the Additional file 3: Table S3.
Mitochondrial fragmentation analysis in NSCs
NSCs were plated on Matrigel-coated glass coverslips (13 mm) and were fixed for 20 min on ice in 4% paraformaldehyde (PFA, Sigma), solution in phosphate-buffered saline (PBS, Euroclone). Two washes were performed afterwards with PBS, cells were then permeabilized for 30 min in blocking solution, containing 0.1% Triton X-100 (Sigma-Aldrich) and 5% donkey serum (Sigma-Aldrich), and incubated overnight at 4 °C with antibody against transporter of outer mitochondrial membrane 20 (TOMM-20) diluted in blocking solution. The next day, cells were washed 3 times with PBS for 5 min and incubated for 1 h at room temperature with Hoechst 33,342 (ThermoFischer Scientific) and specific secondary antibodies (ThermoFisher Scientific) in blocking solution.
Images were acquired using confocal microscope (Leica Sp5). Fragmentation level was assessed based on circularity calculated using ImageJ plugin mitochondrial morphology (http://imagejdocu.tudor.lu/doku.php?id=plugin:morphology:mitochondrial_morphology_macro_plug-in:start).
Transmission electron microscopy (TEM) was performed at Advanced Light and Electron Microscopy BioImaging Center (ALEMBIC) at San Raffaele Hospital. One NSC WT sample and one Setd5 heterozygous NSCs sample were cultured in adherent condition in 10 mm Petrie dish till reaching 90% of confluence. Cells were then pelleted at 1200 rpm for 5′ and then included in TEM fixative solution for further analysis.
For adult mice, the brain of wild type and Setd5+/− were extracted from the skull after brief perfusion with Sodium Chloride 0.9% (S.A.L.F) and then included in TEM fixative solution for further analysis.
Major axis of mitochondria was measured using ImageJ software. Qualitative evaluation of mitochondria was performed manually on each image.
To perform Western Blot analysis cells were seeded in adherent condition in 15 mm Petrie dish till 90% of confluence. Cells were lysed in RIPA buffer (50 mM Tris–Hcl ph 7.4, 150 mM NaCl, 0.1% SDS, 1% Triton X-100, 2% complete protease inhibitor cocktail (Roche), 10%complete phosphatase inhibitor cocktail) for 30 min at 4°. After the lysis samples were centrifuged at 13,000 rpm for 30 min and the supernatant was rescued. Protein samples were then quantified using BCA protein assay kit (ThermoFisher).
After quantification protein samples containing 30 µg of protein, sample buffer (Tris–Hcl ph 6.8 150 mM, glycerol 45%, SDS 7,50%, Bromophenol blue 0.08%) and H2O up to 24 µl each were prepared and boiled for 5 min at 95°.
Samples were loaded on poly-acrylamide gel and after proper run were transferred on nitrocellulose membranes for 90–120 min at constant intensity of 350 mA.
Blocking reaction was performed using 5% milk in PBS-T (PBS with 0.1% tween) for 1 h at room temperature. Incubation with primary antibody (Additional file 3: Table S3) was performed over/night at 4° in blocking solution at the proper dilution. The next day membranes were washed 3 times, for 15–20 min in PBS-T and incubation with secondary antibody (Additional file 3: Table S3) conjugated with horseradish peroxidase (HRP) was performed at the proper dilution in blocking solution for 1 h at room temperature.
Finally, to reveal the signal ECL prime kit (GE healthcare) has been used and images were acquired using a chemiluminescent reader.
Densitometry was calculated using ImageLab software (Biorad).
MitoTracker Orange and MitoTracker Green FM cellular staining
NSCs were analyzed by fluorescence activating cell sorter (FACS) using LSRFortessa analyzer (BD Bioscience), using two different fluorescent dyes, MitoTracker® Green FM (ThermoFisher) and MitoTracker® Orange CMTMRos (ThermoFisher). The first one stains mitochondria regardless of the membrane potential, the second one stains mitochondria depending on the membrane potential (Fig. 2). Using both at the same time allows to determine if there is a difference in the membrane potential and in the total mitochondria mass in the whole cell population.
Cells were seeded at a density of approximately 500,000 cells per well in 6 wells in adherent condition the day before the analysis. The next day cells were stained with MitoTracker Green FM (stock 1 mM in DMSO) and MitoTracker Orange CMTMRos (stock 1 mM in DMSO) at a final concentration of 50 nM diluted in NIM in both cases. Each sample was single and double stained with both dyes.
Measurement of membrane potential using TMRM
To perform live imaging on NSCs with TMRM, cells were seeded in adherent condition on glass coverslips at a density of 30,000 cells per well in 24 wells plate. The die was diluted at a final concentration of 250 nM and then cells were incubated at 37° for 10′ or 20′ before image acquisition.
To measure the maximum level of membrane potential cells were treated with complex V inhibitor oligomycin at concentration of 1 µM. To measure the background level of TMRM fluorescence, NSCs were treated with decoupling agent FCCP at concentration of 0.7 µM.
After incubation with the fluorescent dyes, coverslips were mounted on cover glasses using mounting medium, without performing fixation and images were immediately acquired using epifluorescence microscopy. Images were quantified and analyzed using ImageJ software, using gray scale to quantify the fluorescence intensity of the cells for TMRM.
Cells ATP concentration analysis
Cellular ATP concentration was determined using ATP-Lite assay kit (Perkin-Elmer), an assay that use luminescence to determine cellular ATP concentration.
Cells were seeded in 96 wells plate coated with Matrigel 24 h before the experiment at a density of 30,000 cells per well. The next day cells were lysed using the Mammalian cells lysis solution provided by the kit, then the plate is shaken at 700 rpm for 5 min. The lysates are then transferred to a dark adapt plate and the substrate solution is added and the plate is shaken for 5 min avoiding light exposure, then after ten minutes the plate can be read using a luminometer.
Lactate medium concentration determination
Lactate concentration in the medium were measured EnzyChrom™ L-Lactate Assay Kit. Cells were seeded in a Matrigel-coated 96 wells plate at a density of 30,000 cells per well. After 72 h 20 µl of medium are taken for each well and 80 µl (60 µl of assay buffer, 1 µl of Enzyme A, 1 µl of Enzyme B, 10 µl of NAD and 14 µl of MTT). Optical density (OD) is read at 565 nm at time 0 and at time 20, 20 min after adding the working solution. OD values obtained at time 0 are subtracted to time OD obtained at time 20 and it’s possible to calculate the lactate concentration building a standard curve using, the range of detection varies depending on the medium or samples that is measure. For cell cultured in phenol red containing media, the range of concentrations detectable spans from 0.1 to 1 mM.
NAD/NADH cell concentration measurement
To quantify the total intracellular concentration of NAD/NADH, NSCs were seeded in a Matrigel-coated 96 wells plate at a density of 30,000 cells per well 24 h before the experiment.
The NAD/NADH-Glo™ Assay (Promega) kit was used according to the manufacturer’s instructions.
Measurement of oxidative stress with DCF and DHE
To perform live imaging on NSCs with H2DCFDA and DHE, cells were seeded in adherent condition on glass coverslips at a density of 30,000 cells per well in 24 wells plate. Both dyes were diluted in PBS at a concentration of 10 µM for H2DCFDA and 3.2 µM for DHE, together with Hoechst to stain nuclei. NSCs were then incubated 15′ for H2DCFDA and for 10′ for DHE at 37°. After incubation with the fluorescent dyes, coverslips were mounted on cover glasses using mounting medium, without performing fixation and images were immediately acquired using epifluorescence microscopy. Images were quantified and analyzed using ImageJ software, using gray scale to quantify the fluorescence intensity of the cells for H2DCFDA, or the nuclei for DHE.
Mitochondria analysis in cortical neurons using mito-dsRed
Cortical Neurons were obtained by dissecting telencephalic cortex of embryos, wild type and Setd5+/−, at day 17.5 post-conception from wild type and Setd5+/−. Briefly, after dissection, the cortex was digested 30′ with trypsin. After 3 washes with HBSS medium, the tissue was gently dissociated mechanically by using pipette, DNAse was added to avoid cell clumping and cells were centrifuged 5′ at 1200 rpm. Subsequently, cells were plated at density of 400,000 in an Ibidi, µ-Dish 35 mm, high chamber for live imaging coated the day before with poly-L lysine. The next day cells were infected with CMV-mito-dsRed lentivirus. After 7 days in culture, live imaging was performed using the in vivo imaging setup of Leica sp8 confocal microscope.
For Mdvi-1 rescue experiment, the compound was resuspended in DMSO as vehicle and added directly to the culture media at final concentration of 25 um the day after plating. The treatment was maintained throughout the whole culture period before performing image acquisition.
Mitochondrial fragmentation level was assessed based on circularity calculated using ImageJ plugin mitochondrial morphology (http://imagejdocu.tudor.lu/doku.php?id=plugin:morphology:mitochondrial_morphology_macro_plug-in:start). Other measures were performed using image J software.
In vivo ATP quantification
Mitochondria were isolated as previously published [34, 35], from the cortex of 3 month old wild-type and Setd5+/− mice. In brief, mice cortex was homogenized in an appropriate isotonic buffer [0.25 m sucrose, 20 mm 3-(N-morpholino) propane sulfonic acid (MOPS), pH 7.2, 1 mm EDTA, 0.1% BSA fatty acid free and digitonin 0.1 mg/ml] using a glass-Teflon homogenizer. Cell debris and nuclei were pelleted twice by centrifugation at 2500 g for 10 min at 4 °C. Supernatants were centrifuged at 12,500 g for 30 min at 4 °C and the mitochondrial pellet was resuspended in an isotonic buffer (0.5 m sucrose, 20 mm MOPS, pH 7.2, 1 mm EDTA). Isolated mitochondria were incubated at 37 °C for 30 min in a respiratory buffer (0.25 m sucrose, 20 mm MOPS, 1 mm EDTA, 5 mm inorganic phosphate, 0.1% BSA fatty acid free, and 1 mm ADP, pH 7.4) containing specific substrates of the respiratory chain complexes. By providing pyruvate/malate (5 and 1 mm, respectively) and glutamate/malate (5 and 1 mm, respectively), we stimulated ATP synthesis. ATP production was measured by luminometric assay.
Synaptosomes were isolated from cortical brain tissue of Setd5+/+ and Setd5+/− by using the Syn-PER reagent (Thermo Fisher). Briefly, the cortical tissue was isolated and disaggregated into a glass Dounce homogenizer filled up with 4 mL of Syn-PER reagent. After 15 gently strokes, the homogenate was poured in a 15 mL falcon and centrifuged at 1200× g for 10 m at 4 °C. The supernatant was then centrifuged at 15,000× g for 20 m at 4 °C. The pellet containing the purified synaptosome fraction was weighted for subsequent treatments.
All statistical analyses were performed using GraphPad PRISM 8.0.2 software, calculating for each sample the arithmetic mean and error, calculated as standard error mean (SEM). In experiments with only two test items (TI), statistical significance was calculated using unpaired T-Test or Mann–Whitney test were indicated.
In experiments with more than two TI, one-way analysis of variance (ANOVA) and Dunn multiple comparison test was performed to determine the statistical significance of the experiments.
In experiments with more than one factor analyze and more than two TI, two-way analysis of variance (ANOVA) and Sidak, Bonferroni or Tuckey multiple comparison test was performed to determine the statistical significance of the experiments.
SETD5 controls the transcription of nuclear-encoded mitochondrial genes
Murine Setd5 haploinsufficient neural stem cells (NSCs) normally cultured at 5% O2, to respect the condition found in the embryo and stem cells niches, showed wide downregulation of mitochondrial-associated genes as well as the increase of response of hypoxia including glycolysis by RNA-seq (Additional file 1: Table S1, Additional file 4: Fig. S1A, B) . We reasoned that culturing the NSCs upon ambient air condition (21% O2) possibly uncover mitochondrial impairment in Setd5 haploinsufficiency (Additional file 4: Fig. S1C). We found that high oxygen further exacerbated the deregulation of the mitochondrial gene subset (Fig. 1A, Additional file 1: Table S1, Additional file 4: Fig. S1D, E) being the mutant cells unable to properly react to the oxygen increase, at least at the transcriptional level (Fig. 1B). Interestingly, ~ 30% of the mitochondrial-associated genes were among the SETD5 target genes identified by ChIP-seq in the same cells (Fig. 1C, Additional file 2: Table S2) . Of note, the SETD5 binding on gene bodies was particularly relevant on mitochondrial genes compared to both all genes and a random subset of the same size as indicated by the density plot (Fig. 1D, Additional file 2: Table S2). In accordance with the showed SETD5 binding preference, the mitochondrial subset encloses genes that are both rich in exons, highly expressed and long, features that emerged as correlated with a high probability of SETD5 genomic association (Fig. 1E) .
Together these observations highlight the importance of correct levels of SETD5 to promote the transcription of mitochondrial-associated genes especially when those genes are specifically required, i.e., in response to high O2 tension.
Setd5 haploinsufficient NSCs show impaired mitochondria
Since the observed transcriptional deregulation, we investigated the mitochondrial compartment in both wild-type (WT) and Setd5 mutant NSCs upon high O2 levels. Initially, we evaluated the fusion/fission dynamic through both immunocytochemistry and electron microscopy. TOMM20 immunostaining indicated that mitochondria in Setd5 mutant NSCs were more fragmented compared to the WT (Fig. 2A) while transmission electron microscopy confirmed the smaller size of mitochondria and showed the altered ultrastructural morphology with a high number of organelles with aberrant cristae structure (Fig. 2B). Interestingly, among genes downregulated in our RNA-seq dataset, we found genes encoding for important regulators of mitochondrial dynamics (Mfn1 and Opa1) and biogenesis (Pgc1α, Polg) that indeed were decreased in mutant cells compared to the WT (Additional files 4, 5: Figs. S1C, E, S2A).
Then, we evaluated the amount of several mitochondrial proteins as a proxy of the complexes of oxidative phosphorylation (OXPHOS), also known as the electron transport chain, which is the biochemical core of the mitochondrial functionality. In line with our transcriptional (Fig. 1) and structural data (Fig. 2A, B), Setd5 mutant mitochondria were deficient in many protein subunits of the different complexes (Fig. 2C). Since the OXPHOS complexes I, II, and IV are proton pumps, we sought defects in the generation of mitochondrial membrane potential (ΔΨm) in mutant cells. Using cell-permeant fluorescent dyes that are either insensitive (Mitotracker green) or sensitive (Mitotracker orange and TMRM) to mitochondrial membrane potential we showed, as expected, lower ΔΨm in Setd5 mutant cells compared to control despite the total mitochondrial masses were equal (Fig. 2D, E, Additional file 5: Fig. S2B). Mitochondrial membrane potential (measured as Tetramethylrhodamine, Methyl Ester, Perchlorate (TMRM) signal) was then challenged by adding to the medium chemical compounds to induce either hyper- (using oligomycin) or de-polarization (carbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP)) . Interestingly, the mutant cells were not able to properly increase their potential, having only a mild response to oligomycin while the uncoupling between the respiratory chain and phosphorylation system due to FCCP was effective (Fig. 3A, Additional file 6: Fig. S3A). Low ΔΨm and blunt reaction to the hyperpolarizing agent would suggest an impairment to generate ATP by OXPHOS. We measured a decrease in total ATP content in mutant cells compared to the control, supporting an energetic defect due to Setd5 haploinsufficiency (Fig. 3B). Accordingly, the levels of nicotinamide adenine dinucleotide (NAD) were decreased (Fig. 3C). However, the mitochondria of mutated cells seemed not to experience electron leakiness since the evaluation of reactive oxygen species (ROS) resulted in diminished in the mutant cells (Additional file 6: Fig. S3B, C).
We hypothesized that low SETD5 levels may hamper the mitochondrial maturation, and thus the related energy supply, forcing the cells to use glycolysis to survive, as the transcriptional profile already suggested (Fig. 1, Additional file 4: Fig. S1). To investigate this issue, we measured the lactate, a byproduct of glycolysis, that was indeed increased in mutant NSCs (Fig. 3D). To further analyze this aspect, we have grown both WT and mutant NSCs in a culture medium in which the glucose was replaced by galactose as the carbon source for metabolic reaction; the galactose can enter in the glycolytic pathway but without a net gain of energy; moreover, it cannot enter in the pyruvate-lactate anaerobic pathway. Thus, forcing the cells to rely only on mitochondrial respiration, to produce ATP . WT NSCs behaved similarly in glucose and galactose media while the mutant cells, that with glucose proliferated more than the WT, tended to die (Fig. 3E).
These data indicate that low SETD5 levels lead to metabolic reprogramming due to an inefficient mitochondrial dynamic, maturation, and function.
Setd5 haploinsufficiency affects mitochondrial machinery and localization in neurons
To better circumstantiate our findings in regard to neurological pathologies associated with SETD5 mutations, we moved to analyze Setd5+/− animal model. We first evaluated the abundance of OXPHOS complexes. In line with NSCs data, we showed that Setd5+/− brains presented a decrease in OXPHOS proteins particularly evident in complex I (Additional file 7: Fig. S4A). Evaluation of ATP content in freshly isolated mitochondria from control and Setd5+/− brains sustained the in vivo hypo-functionality of oxidative phosphorylation since isolated mitochondria from mutants produced less ATP than their normal counterpart, specifically using glutamate as the substrate for mitochondrial respiration (Fig. 4A).
Using electron microscopy, we retrieved small mitochondria in mutant brains compared to control littermates possibly confirming the defects in fission/fusion dynamics seen in NSCs (Fig. 4B). To better analyze this aspect, we moved to cultured primary neurons. Live staining using the mito-dsRed mitochondrial marker, showed that neurons from E17.5 Setd5+/− cortices displayed more fragmented mitochondria compared to controls (Fig. 4C). Interestingly, in mutant neurons, the mitochondria were located primarily in cell soma while were reduced in neurites (Fig. 4C). We reasoned that mitochondria may be stacked in the cell body, with few possibilities to travel along neurites to reach synaptic sites within neurons. To investigate this issue, we isolated synaptosomes containing fraction from Setd5+/+ and Setd5+/− cortices and estimated mitochondria content showing that mutant synapses contained fewer mitochondria (Fig. 4D).
The quinazolinone derivative mitochondrial division inhibitor 1 (Mdiv1), reversibly inhibits DRP1, a master regulator of fission machinery . Its action has been linked with increased mitochondrial fusion, attenuation of apoptosis, and cytoprotection [38,39,40,41,42]. We decided to use Mdiv1 in treating Setd5 mutant neurons. Mdiv1 was effective to increase mitochondrial length in mutant neurons as well as mitochondrial distribution inside the mutant cell soma and neurites (Fig. 5A).
These data indicate that Setd5 haploinsufficiency in neuronal cells impairs mitochondrial dynamics, functionality, and localization that can be at least in part rescued by the DRP1 inhibitor Mdiv1.
In this study, we revealed mitochondrial dysfunctions as possible contributors to the pathogenesis of neurodevelopmental disorders caused by mutations in the SETD5 gene. Setd5 haploinsufficiency in mice is leading to a decrease in mitochondrial membrane potential and subsequent ATP production as well as impairment in organelle dynamics in mutant neuronal cells. Our data suggest that the mutant mitochondria are hypo-functional and mislocalized due to transcriptional defects downstream to low SETD5 activity. Both the consequent metabolic reprogramming of mutant cells and the mitochondrial impoverished neurites and synapses may contribute, at least in part to the pathogenesis of SETD5-associated diseases.
Brain development and related disorders have been associated with mitochondrial functions/dysfunctions [6, 43]. Recent studies have suggested that a large part of ASD patients showed high levels of lactate, pyruvate, and alanine in body fluids and decrease the activity of a variety of mitochondrial respiratory complexes in brain regions as well as in peripheral organs, particularly evident at young age, that eventually were restored in adulthood [44,45,46,47,48]. Interestingly, mutations in the mtDNA have been associated with ASD cases and other NDDs [49, 50]. These observations support the idea that metabolic changes due to mitochondrial impairment may either contribute to or induce those neurodevelopmental defects at the basis of NDDs. For instance, OXPHOS aberrations lead to abnormal neuronal migration , while the PolG mutator mouse, a model of mtDNA mutations and respiratory dysfunction due to lack of POLG activity, revealed neural stem cell dysfunctions with decreased capabilities to self-renew and differentiate .
Deregulation of the fission/fusion processes is intimately implicated in neurological disorders. The strong association between increased mitochondrial fragmentation and neurodegenerative diseases (e.g., Parkinson’s, Alzheimer’s, and Huntington’s diseases) underlines the importance of mitochondrial dynamics in neuronal function and survival . Moreover, mutations in genes encoding for key regulators of fission/fusion, such as DRP1, MFN1, MFN2, and OPA1 genes among others, also induce neurodevelopmental defects such as brain abnormalities, decrease the number of neurites and synapses up to neuronal death both in humans and animal models [53,54,55,56,57]. One of the proposed mechanisms at the basis of the pathological onset is the fact that fragmented mitochondria become refracting to movements within polarized cells as neurons, possibly because small organelles may not arrange enough molecular adaptors for transport, as Miro/Milton, for their correct mobility [57, 58]. As consequence, the organelles are not available in highly demanding cellular compartments, such as synapses, causing low ATP supply and Ca2+ buffering with functional impairment for the neuron. Accordingly, we report that Setd5 animal models presented fragmented mitochondria stalled in the cell body that eventually contribute to the loss of synapses and neuronal hypo-functionality that characterize these mice [24, 26].
A recent report proposed that SETD5 regulates glycolysis through the regulation of EP300/HIF-1a co-activators upon the hypoxic condition in breast cancer stem-like cells . In our models, we did not find evidence for such function in regulating glycolytic genes. In fact, glycolysis seemed to sustain the ATP production in Setd5 haploinsufficient cells. Further investigations are needed to understand whether a direct effect on glycolysis is cell-, environmental-specific, or coupled and eventually masked by mitochondrial impairment.
The interplay between epigenome and mitochondria is bidirectional: mitochondrial are essential for the post-translational histone modifications for instance supplying acetyl-CoA and S-adenosyl methionine (SAM) for acetylation and methylation, and, conversely, epigenetic changes influence mitochondrial biogenesis and function . Compelling examples of the latter are given by: (1) the inhibition of SET containing protein SETD7, a histone methyltransferase to H3K4me1, promotes mitogenesis through activation of PGC1A ; (2) the activity of LSD1, the first identified H3K4me1/2 demethylase, also regulates mitochondrial activity at different extent depending the system [34, 35, 62,63,64]; (3) the depletion of H3K4 methylase SMYD1 leads to a reduction in mitochondrial energetics in the heart due to PGC1A repression .
One limitation of the study is that we used only a murine experimental setup, while species-specific differences may occur and the phenotype in human cells and organisms be different. Further experiments will clarify this point. Another important issue is that we can only speculate about the possible primary causative role of mitochondrial impairment in the establishment of the SETD5-related NDDs. Indeed, as for other disorders related to brain development that have been associated with mitochondrial damage, it remains challenging to claim that mitochondria-associated phenotypes are the major driver of the diseases. Specifically, for SETD5 its broad impact on gene transcription not related with mitochondria is contributing to the disease, as previously suggested.
In summary, our data indicate that massive deregulation of transcription, including mitochondrial master regulators such as Pgc1a and Polg, is at the basis of a variety of mitochondrial aberrations both at the levels of neural precursors and adult neuronal cells which carry Setd5 haploinsufficiency. These impairments ranging from hypo-functionality of the OXPHOS machinery up to mitochondrial miss-localization and fission alterations. Despite no report or evidences link SETD5 patients with mitochondrial defects, our work suggests that those may contribute to SETD5-related pathologies. In fact, this kind of defects may be overlooked especially if they occur in inaccessible developmental stages. Further work is needed to better determine the extent of the pathology that is properly caused by mitochondrial impairment and, consequently, if the mitochondria dysfunctions should be considered novel therapeutic targets for SETD5-related NDDs.
Availability of data materials
RNA-seq raw data are available at GSE103912 for NSCs cultured in low oxygen condition and GSE222188 for NSCs cultured in high oxygen condition.
Autism spectrum disorder
Mitochondrial division inhibitor 1
Nicotinamide adenine dinucleotide
Neural stem cell
Reactive oxygen species
Tetramethylrhodamine, methyl ester, perchlorate
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We thank the members of the Broccoli laboratory for helpful discussion.
This work was supported by Telethon Grant (GGP15096) to A.S. and A.Z., the Italian Ministry of Health to A.S (Young investigator Grant # GR-2013-02355540) and to A.Z. (Young investigator Grant # GR-2011-02351172) and the Italian Ministry of Education, Universities and Research (MUR-PRIN-2017M95WBA) to V.B and A.Z.
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
: Table S1. RNA-seq normalized counts, functional enrichment data for High & Low Oxygen condition.
: Table S2. Gene list of SETD5 targets, mitochondrial genes and random dataset used to perform comparison in Fig. 1.
: Table S3. List of primers and antibodies used in this work.
: Figure S1. Level of oxygen and mitochondrial genes in Setd5+/− NSCs.Enrichment plots from gene set enrichment analysis for Glycolysis, electron transport chain, Mitochondria and Hypoxia, between Setd5+/−and controlNSCs cultured at lowand high oxygenlevel.Western blot analysis for SETD5 protein level in NSCs cultured at 21% oxygen. Actin was used as loading control.RT-qPCR validation of transcription level of the indicated mitochondrial-related genes between Setd5+/−and controlNSCs cultured at lowand high oxygenlevel. All data are presented as mean values ± SEM.
: Figure S2. Mitochondrial membrane potential is altered in Setd5+/− NSCs.Western blot quantification of Opa1and Mfn1protein level in NSCs normalized on Tomm-20. Statistics, Opa1, one-way ANOVA, multiple comparison Dunnet test; Mfn1, one-way ANOVA, multiple comparison Dunnet test.Quantification of mitochondrial membrane potential using TMRM in live imaging. The signal level is calculated by performing a ratio between the basal fluorescence intensity and the fluorescence after FCCP treatment. Statistics, one-way ANOVA, multiple comparison Dunnet test. All data are presented as mean values +/− SEM.
: Figure S3. Mitochondrial function alteration in Setd5+/− NSCs.TMRM live staining on control and Setd5+/− NSCs. From the left, basal condition, oligomycin treatment and last two images FCCP treatment.Reactive oxygen species quantification in control and Setd5+/− NSCs performing live staining of DHEand DCF. Statistics, DHE, one-way ANOVA, multiple comparison Dunnet test; DCF, one-way ANOVA, multiple comparison Dunnet test. All data are presented as mean values +/− SEM.
: Figure S4. Electron transport chain alteration in mouse cortex.Western blot analysis on whole protein lysate from Setd5+/+ and Setd5+/− mouse cortex for the indicated mitochondrial protein, normalized on total Tomm-20 level. Statistics, NDUFB8, T-test,; SDHB,; UQCRIII,; MTCO1,; ATP5A,. All data are presented as mean values +/− SEM.
: Figure S5. Uncropped Western blot images.The images refer to Fig. 2C.The images refer to Fig. 4D.The images refer to the figure S1C.The images refer to the figure S2A, B.The images refer to the figure S4A.
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Zaghi, M., Longo, F., Massimino, L. et al. SETD5 haploinsufficiency affects mitochondrial compartment in neural cells. Molecular Autism 14, 20 (2023). https://doi.org/10.1186/s13229-023-00550-9


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Maybe you have a distant memory of going to a “fancy” restaurant when relatives were in town or a cousin was off to college. There were white tablecloths, a jacketed waiter and “continental’ specials. The kitchen somehow came up with kid’s plates that weren’t on the menu, and the staff had been there forever. Oh, yes, and the adults could order a handmade cocktail for the occasion. It was family-friendly in a kind of stuffy way. You dressed up a little and Shirley Temples were a house drink.
Very few such places exist any more, but Carpineteria, yes, that little town 10 minutes south of Montecito and lacking an upscale hotel, pier or restaurant scene of much description, has one. It’s not in its 60th year of operation, however, only its third, helmed by James Sly, a long time chef who made Montecito’s Lucky’s steakhouse what it is today. You kind of only find this place if you live near there or someone tells you about it. And don’t worry, it won’t be closing due to lack of interest. Sly’s (www.slysonline.com) has its followers who make sure the place is not empty. Here is where you’ll get Blue Plate specials of American classics like pot pie and meat loaf every night of the week, very good steak (hand cut and aged in house), locally-sourced wines, produce and breads. Their grilled artichoke is delicious and straight from Castroville, of course.
The low key town means low key rent, one suspects, and thus a lot more value goes on your plate than you might expect. No, it’s not cheap, but it’s not crazy pricewise either. We took our 6 year old there a couple of Sundays ago and enjoyed the aforementioned artichokes and herbed scallops to start. My husband had his favorite Bloody Mary, ever, and perfectly pan-roasted chicken. I had the prix fixe menu of the day, which had a Gruyere souffle, a more than decent steak with a carrot puree, and a petite tarte tatin to finish. My souffle was fallen, I must say, and the waiter blinked a bit when I asked about it. (Update: From James Sly: “The soufflé suissesse is unmolded, and served in cream, baked with gruyere over.” So it turns out that the souffle wasn’t fallen after all. However, that waiter should have known that.)
Sounds much better than fallen! It’s baked in a half inch deep mold.
He said it came that way. Hmmm. Well, it tasted good. And it was $34 for all three courses.
As a more than decent home cook, Top Chef addict, farmer’s market shopper and all-around food enthusiast, I am well aware most restaurants use shortcuts to streamline operations and pare costs. It’s an admirable business model – if you don’t care that the food be special and personal. Sly’s does indeed care about special and personal. On a brunch visit, we asked how they get the artichoke just so. We were advised how to steam them using vegatable broth for 45 minutes, chill them in the fridge for at least an hour, trim them properly and then grill them. I’m not so sure Morton’s, McCormick and Schmicks’ et.al. are doing it that way or would take the time to tell us about it.
The Los Angeles, and, casting the net wider, southern California, food scene is really world class these days. You can get well-made food from eager culinary grads who’ve cut their teeth in New York, Paris and Miami. You want deconstructed Italian, cutting edge Vietnamese, fusion Korean? Seriously amazing.
But no frills delicious is much harder than that. Deceptively simple is all about showcasing ingredients and adding salt, pepper, butter, herbs and a huge dash of service. Sly’s has it.
By the way, one of the Bloody Mary’s key ingredients is fresh squeezed juice of a whole lemon. As for the rest, sorry, you’ll have to go to the corner of Linden and Seventh in Carpinteria for the recipe.

domain: food


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Month 3: bye LOMBOK (November 2023)
It’s hard to believe, but after 2 months, we finally bid farewell to LOMBOK! But not for long, silly! In Indonesia, you can only extend your 30-day visa on arrival once. So, after 2 months, it was time for what they call a visa run! Although we still loved the lifestyle here, we also welcomed a break from the work and surf routine. Something different. So, after 2 months, it was time to say goodbye to Lombok for a few weeks.
Quite decadent, doing a visa run
That sounds rather fancy, doesn’t it? Just doing a visa run. As if it’s the most ordinary thing in the world. We flew off to Bangkok for a few days because we needed to exit the country for our visa but then return. MAN! Incredibly decadent. And we’re well aware of it. Despite working hard for this lifestyle, we’re two incredibly lucky individuals. The fortune of doing this together and being born in a country where this is even an option, we truly recognize.
Indulging in Bangkok’s street food
What a hardship, stuffing ourselves for a few days with the tastiest street food in Bangkok. We found a craft beer café around the corner and thought it was the perfect time to satisfy our craft beer cravings, considering they’re almost impossible to fulfill in Lombok. Other than that, we didn’t do much except spending an hour going wild in the arcade. From Guitar Hero to my favorite, the dance machine. It becomes painfully clear that we might need to work on our dance moves a bit more.
Diving at Nusa
After taking the ferry from Lombok to Bali three times (still a solid tip), we decide this time to travel back via Nusa Lembongan and the Gili Islands to Lombok. Nusa Lembongan because we’re eager to dive again. It’s been 5 years, so we start with a refresher mini-course in the pool. Even though we’ve done quite a few dives, I feel like a complete dive loser. It’s great that we managed this before getting into the water for real.
The next day, we’re ready for the real deal. Coral Bay and Manta Bay are on the itinerary. It’s pouring rain, and the sea is ROUGH! We’re champions at choosing the most impossible moments for these trips. But we’re here, and there’s no way back.
At Coral Bay, we mostly stay near the entrance because diving into the current doesn’t seem like the best idea after 5 years without diving. Soon enough, a turtle casually swims by. But mostly, it’s just wonderful being back in the water.
Then onto Manta Bay, which we secretly anticipate. Diving with Manta Rays. The sea gets even rougher, and despite quickly taking motion sickness pills – Desi shoves one in her mouth – within minutes, everything comes up. I NEED off that boat. I dive into the sea like a maniac, hoping it’s less turbulent. It’s slightly better, but even after getting off the boat, the waves are intense. We start the dives, and underwater, it’s a left-right struggle. Even Leroy, who NEVER gets seasick, is barely holding it together. To make matters worse, the visibility is maybe 3-4 meters.
But within 5 minutes, the first manta ray glides past, then another and another. They keep coming, and it’s truly MAGICAL! They’re unbelievably large and incredibly graceful. While we float around like two fools in the water, these giants elegantly swim by as if nothing’s wrong.
It might not be the dive we hoped for, but it’s still extraordinary.
Chilling on the Gili Islands
After a few dives and touring Nusa Lembongan, we take the fast boat to Gili Air. We had plans to join some parties, but the island is VERY quiet. The beach is a stone’s throw away from our accommodation, and mostly, we just open our laptops occasionally.
Back to Kuta Lombok
From Gili Air, we take the local ferry to Bangsal and drive straight to Kuta Lombok! We arrive at Loka, where we rent a small room per month, and are greeted by the dachshund and the stray dog who have made this place their home. It feels so good! We resume our now “normal” life, mostly consisting of the question “where are we eating tonight?” and “what time are we surfing?”

domain: travel


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- Rand Paul: ‘Disingenuous’ Obama Can Stop NSA Spying Any Time He Wants 2015-05-26 22:11
- Wealthy Installing “Safe Rooms” to Prepare for Civil Unrest? 2015-05-26 21:34
- Obama Usurps Local Police With Fake “Ban” on Militarization 2015-05-26 21:28
- RIP: Over 100 newspapers dumped in year, ads down 50%, circulation hits bottom 2015-05-26 01:36
Is the Georgian government going to take the knock on forests?2009-06-18 16:48
Ministry of Economic Development of Georgia announced the timber processing license auction sale. The environmental specialists were the first to speak against it; however, they may also be followed by the oppositionists. At the end of last year, the intended transfer of the forest administration into private hands aroused such a vehement protest that the auction sale was postponed. GeorgiaTimes is talking to economical expert Ditrikh Muller.
Non-parliament organization Green Alternative is protesting against the decision taken by Ministry of Economic Development of Georgia, GHN reports. The environmental specialists are indignant at the fact of "encroachment upon the right to take part in the decision-making process related to the environment, and the country legislation being violated". They fear lest the secret decision-making should encourage corruption and creation of more trouble spots in society.
The environmental specialists are right: this is not a time for touching upon the sore point of forest sale at the heat of confrontation between the authorities and the opposition, even if the Georgian state budget is lacking funds to cover its obligations. Last December, an attempt to announce such kind of auction sale failed due to the opposition's activity. At that time, the point was the leasing of 162 thousand hectares of forest for 20 years with the right of deforestation.
It was the Labourite party that came out in protest against the forest sale. It was planning to introduce a bill into the parliament in order to prohibit the forest sale in Georgia once and for all. As Kakha Dzagania promised, the forests "would have been used only by the local residents". According to him, the Constitution of the country does not allow the sale of natural resources.
Nino Burjanadze's Democratic Movement - United Georgia also criticized the government's intention to sale out the forests. "Such kind of auctions is nothing but an attempt of some officials in the outgoing government to "catch it"; this is not an urgent need. Such absolute exploitation and realization of the Georgian forests is going on under the aegis of replenishing the budget", - leader of the party's economical block Temur Murgulia stated.
The oppositionists managed to save the forests. "As the new minister, despite my implicit trust in my predecessor Eka Sharashidze, I have to see into this matter and make out whether the economical policy has been worked out correctly", - Minister of Economical Development Lasha Zhvania quoted by Nasha Abkhazia (Our Abkhazia) explained the decision to postpone the auction sale.

domain: news


Tata 45X Premium Hatchback's Production Version Confirmed For Geneva Debut, Video Teased
Tata Motors earlier confirmed to launch a new premium hatchback based on the 45X concept in the second quarter of next fiscal.
Tata 45X Premium Hatchback. (Image: Tata Motors)
Tata Motors has confirmed to unveil the production version of the upcoming premium hatchback 45X at the Geneva Motorshow 2019. Tata has posted a video on YouTube, teasing the 45X ahead of its global debut in the production guise. Tata Motors previewed 45X Concept at the 2018 Auto Expo in India. The description on video reads – “We are here to reset the rules! Bringing the marvel of engineering for the young and tech-savvy! Stay tuned for the grand unveil of the production avatar of the 45X concept premium urban car at the Geneva International Motor Show, 2019. Redefining Driving.”
Tata Motors earlier confirmed to launch a new premium hatchback based on the 45X concept in the second quarter of next fiscal. The upcoming hatchback, based on 45X, will rival Maruti Suzuki's Baleno. "We are going to launch it after six-seven months, in quarter second," Mayank Pareek, president of passenger vehicle business unit of Tata Motors told reporters when asked about the launch of 45X.
Pareek, who was here for the launch of Harrier, said the company has registered significant growth in the last three years after it introduced new vehicles with attractive designs and features and robust safety aspects.
"Against the car industry growth of 4.4 per cent from April to December in the current fiscal, Tata Motor's growth was 21.4 per cent. In Rajasthan also, the company grew at 30 per cent against the industry growth of 6-7 per cent," he said.
Tata Motors has expanded its sales points across the country, with Rajasthan being one of the focus markets having huge potential for the passenger car market, he said. The Tata Group company has a plan of having nearly 2,000 sales points across the country by 2021-22, he said. It has increased the number of showrooms to 800 from 400 three years ago.
With Inputs from PTI
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Updated: Jun 18, 2021
The Secret Battle For Absorption Occurring in You Multivitamin or Nutritional Supplement.
Each micronutrient, whether essential or nonessential, vitamin or mineral, differs in form, function and in the amount needed by the body to produce or maintain our individual health. They are absorbed by the body by attaching to receptor sites, or absorption pathways, which act as docking locations for specific micronutrients. These receptor sites are found throughout the entire gastrointestinal tract. For the most part, the majority of micronutrients are absorbed in the small intestine. However, due to something called micronutrient competition the process of absorption can be more like an epic battle scene than a harmonious event. Unfortunately, most supplements companies simply overlook this battle (competition) for absorption during the formulation process of their multivitamins and nutritional supplements.
Science has shown that there are four types of competition that can occur between micronutrients, they are chemical, biochemical, physiological, and clinical competition. Research published in the American Journal of Clinical Nutrition (AJCN), the British Journal of Nutrition (BJN), the Harvard Health Letter as well as many other nutritional and medical based peer-reviewed journals shows that competitions for absorption occur between many of the vitamins and minerals typically found in popular multivitamins. This makes it so that unless specially formulated to prevent micronutrient competition, many of the vitamins and minerals in the typical multivitamin and/or nutritional supplement are simply not absorbed.
INTRODUCING THE WEB OF COMPETITIONS
“When spider webs unite, they can tie up a lion.” ~ Ethiopian Proverb
As you can see, micronutrient competition can greatly reduce your body’s ability to absorb the vitamin and minerals in the typical multivitamin or nutritional supplement. In fact, nearly 90% of the common micronutrients evaluated above are affected by micronutrient competition in some way. To make matters worse, when key micronutrients don’t get absorbed, the ability of other micronutrients to perform specific functions in the body can be greatly diminished or even eliminated. We call this the domino effect. Regardless of which kind of the four types of micronutrient competition is present, taking a supplement that is not formulated to eliminate these competitions can be like throwing money down the toilet and can leave you vulnerable to micronutrient deficiency.
“Teamwork is the ability to work together toward a common vision…It is the fuel that allows common people to attain uncommon results.” ~ Andrew Carnegie
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domain: health


text:
Critics have accused the Obama administration of being unwilling to exploit federal lands for energy. But the Interior Department is now taking aggressive action to promote green-power development in areas it controls.
You are here
SEIA is the solar energy industry’s go-to source for the latest coverage on solar power, including U.S. and international policy, research and polls, business and financing trends, and more. Our staff strives to support the media covering solar energy issues and guide our members on effective media outreach with clear statements, background materials, news and multimedia resources.
SEIA is committed to informing policymakers, the media, and the American public about the benefits of solar energy for today’s communities, our economy, and our country.
Learn more from our statements and industry news below.
With the announcement today that California’s San Onofre Nuclear Generating Station will be shut down permanently, Rhone Resch, SEIA president & CEO, issued the following statement:
The Solar Energy Industries Association has joined the U.S. Chamber of Commerce, the National Association of Manufacturers and other leading business groups to protest discriminatory trade policies by India.
Across Japan, technology firms and private investors are racing to install devices that until recently they had little interest in: solar panels. Massive solar parks are popping up by the dozen, and companies are mounting panels atop warehouse and factory rooftops as part of a rapid buildup that one developer likened to an “explosion.”
Massachusetts’ second-in-the-nation ranking in clean technology is a tribute to the green energy policies the state has implemented and encouraged over the last several years. As environmental concerns grow along with the perils of global warming, those policies will pay greater dividends -- as long as the state doesn’t stray from that path.
Georgia Power has agreed to buy more power from solar-equipped homes and businesses as part of its plan to boost the amount of solar power it sells, the utility told regulators Thursday.
On Wednesday Balfour Beatty Communities, LLC and SolarCity® announced plans to provide up to 13.2 megawatts of solar energy on 4,700 military homes at Balfour Beatty-managed residential communities at Fort Bliss, Texas, and the adjacent White Sands Missile Range (WSMR) in New Mexico.
The temperature in Massachusetts may be getting increasingly colder as winter approaches, but the state's solar energy industry continues to heat up thanks in part to available financial incentives. After all, it's not by coincidence that the Bay State has had the 10th fastest growing market for photovoltaic power since late 2009, according to the Solar Energy Industries Association (SEIA).
Last Friday, DOE also said it would distribute a $62 million grant to a handful of companies researching and developing concentrated solar power projects for electric utilities. CSP uses mirrors to collect and focus sunlight on a surface to produce steam for electric power generation.
Just because Veteran's Day has already passed does not mean it's time to forget the work of current and former military personnel. In fact, the solar energy industry has been instrumental in powering operations for all branches of the military and in providing reliable and cost-effective electricity for when veterans return home from deployment.

domain: politics


Container Ship, IMO 9379387
The current position of CLAIRE A is at Black Sea reported 1 min ago by AIS. The vessel arrived at the port of Poti Anch., Georgia on Nov 29, 07:33 UTC. The vessel CLAIRE A (IMO 9379387, MMSI 271001092) is a Container Ship built in 2008 (15 years old) and currently sailing under the flag of Turkey.
Position & Voyage Data
|Distance / Time||-|
|Course / Speed|
|Current draught||7.8 m|
|Navigation Status||At anchor|
1 min ago
|IMO / MMSI||9379387 / 271001092|
|Length / Beam||185 / 24 m|
Recent Port Calls
|Vessel Name||Registered Owner||Year|
|CLAIRE A.||Arkas Denizcilik ve Nakliyat AS||2008|
CLAIRE A current position and history of port calls are received by AIS. Technical specifications, tonnages and management details are derived from VesselFinder database. The data is for informational purposes only and VesselFinder is not responsible for the accuracy and reliability of CLAIRE A data.


Most CAD systems can create thread geometry and that is done for this detail model of the strain in the threads of a cast part that is created by the tension in a power screw (not shown) that is threaded into the hole in the cast part. This model was created to estimate the tension that would break off a thread in this casting of a brittle material.
The thread faces have frictional contact defined and the coefficient of friction and the pitch of the screw ensures that the screw will not slip with no applied torque when the tension in the screw is applied. The picture below shows a section view through a cast part where the screw is hidden. This shows that as the load is increased, the first thread will fracture, since the strain at fracture is 0.01 for this material. Above that value, the strain is colored red.
The second image shows higher force in the screw and how the second thread will fail next.
The material used in this simulation is described in this post.


text:
The successful implementation of the integrity management toolbox in Bangladesh attracted media attention:
The cewas Open Working days are the best opportunity to get to know the team of cewas and learn about the current projects. Every two weeks, everyone is kindly invited to meet the cewas Team at the Willisau Office. The team of cewas is looking forward to fruitful discussions.
Lawrence Afere from Nigeria is the winner of the first cewas Start-up Competition. His business idea “Green Pads” convinced both the jury and the audience. As a banana farmer Lawrence was looking for opportunities to use the banana leaves and stems. He came up with the idea of producing pulp and thereof sanitary pads for the Nigerian market. It’s a win win win solution. It financially supports banana farmers, increases resource efficiency, and improves the hygiene situation of women in Nigeria. The lack of affordable sanitary pads in Nigeria currently leads to stigmatization of menstruating women. Usually, girls would not go to school when menstruating. Some girls even drop out of the school system due to poor attendance. So, the jury saw a great potential for “Green Pads” to improve the situation of low income women and girls in Nigeria. “Green Pads” received a scholarship to participate in the upcoming Start-up program plus CHF 5000.- to launch the business.
The Swiss Water Partnership rewards organizations or projects worldwide that have made considerable efforts and can demonstrate results in achieving Water Integrity. For further information check out the flyer and application form.
To apply please fill in the apllication form and submit it to firstname.lastname@example.org by 31st October 2017.
Das Schweizer Nachhaltigkeitsforum eco.ch hat am 31. März 2017 im Theater Basel vor rund 500 Besuchenden die Schweizer Nachhaltigkeitspreise verliehen. Der Schweizer Nachhaltigkeitspreis prix eco.ch 2017 geht in der Kategorie Hauptpreis an das Projekt Gravit’eau der Eawag und der Hochschule für Life Sciences FHNW für ihr herausragendes Engagement für nachhaltige Wasseraufbereitung. Ebenfalls überzeugen konnte das Projekt AquAero mit ihrer Technologie, die Wasser energieeffizient aus der Luft gewinnt, und den Publikumspreis erhielt. Ein Sonderpreis ging an Viva con Agua Schweiz. Wasserbotschafter und Expeditionsschwimmer Ernst Bromeis ist diesjähriger Gewinner in der Kategorie “Hoffnungsträger”.
Die Preisverleihung wurde in Partnerschaft mit dem Wasser-Start-Up-Programm cewas, der Swiss Bluetec Bridge und der Direktion für Entwicklung und Zusammenarbeit (DEZA) durchgeführt.
In the annual conference of the Swiss Humanitarian Aid Unit on March 18th 2016 in Winterthur, Switzerland, the Swiss Federal Councillor Didier Burkhalter, Head of the Federal Department of Foreign Affairs, especially mentioned the cewas Middle East programme as a innovative and exemplary initiative to mitigate the water and humanitarian crisis in the region. As Mr. Burkhalter explained, the cewas training programme fosters entrepreneurial solutions for local water problems, offering also possibilities for employment for the popublation affected by the crisis.
The Middle East, especially the neighbouring countries of war-struck Syria, has recently come into focus of cewas, the world’s first start-up incubator focused on water and sanitation issues. Besides the devastating civil war, the region is also the most water scarce region of the world in dire need of new, sustainable solutions to manage dwindling resources under difficult conditions. In the coming years, cewas will run a large training and start-up support programme for the water and sanitations sector in Lebanon, Jordan, Kurdistan Iraq and Palestine to offer innovative solutions and employment opportunities in the region.
cewas is currently preparing for its 6th start-up programme commencing in August 2016 in Willisau, Switzerland to support young, innovative graduates with fresh start-up ideas for the water sector. Besides its programme in Switzerland, cewas has also been active in supporting water start-ups across the world, namely in India, Southern Africa and South America.
Congratulations to the cewas start-up SedaConsult which won the Swiss Environmental Prize 2015 in the category “youth promotion”. The start-up is awarded with CHF 10’000 for its aim to improve the transparency, integrity and sustainability of the textile industry.
The cewas subsidiary “cewas Middle East” updated the design of its online presence and added further contents. The offers to entrepreneurs from Arab countries are awesome: Start-up programmes in Lebanon, Palestine and Jordan; plus access to trainings and tools in the fields of water and sanitation.
“The jury awarded MoSan with the Award for Social Entrepreneurship 2016. The Zurich company developed a toilet that separates urin from faeces and thereby can prevent the spread of diseases. The verdict of the Jury: a simple but effective solution that can help billions of people who do not have access to toilets, yet. The mobile toilet could also be used in refugee camps.”
The Integrity Management Toolbox, co-developed by cewas and Water Integrity Network, supports water sector organizations like utilities, small and medium-sized enterprises and public institutions in making integrity a part of their strategic plans, business models, and daily practices to reduce corruption risks and improve performance.
The online IM Toolbox provides:
The Toolbox is NOW ONLINE.


The second generation 2018 Maruti Suzuki Ertiga will be launched in India on November 21; gets heavily revised exterior and interior along with new petrol engine
Maruti Suzuki India Limited (MSIL) has the new generation Ertiga MPV as its next big launch in the domestic market and it has been spotted running trials numerous times over the course of last several months. The Indo-Japanese automaker has already begun production of the second-gen Ertiga and it will go on sale on November 21.
The upcoming Ertiga made its global debut at the 2018 IIMS (Indonesia International Motor Show) in April, and it is gracing India to hold its stead as the best-selling MPV in its segment. The existing Ertiga constantly posts good volumes for Maruti Suzuki and none of its rivals could even come close to its domination as they struggle in three digit figures.
Over the last one year, it has averaged around 4,500 units every month but Maruti Suzuki wanted to up the ante in the segment especially considering the arrival of Mahindra Marazzo that garnered 10,000 bookings in just 45 days, and is diligent priced between Ertiga and Toyota Innova Crysta. Based on the lightweight Heartect platform that underpins Baleno and Swift, it does have dimensional changes over the outgoing model.
Despite having the same wheelbase of 2,740 mm, the new-gen Ertiga is 99 mm longer, 40 mm wider and 5 mm taller amidst sacrificing 32 litres of bootspace and 5 mm ground clearance in comparison. The heavily revised exterior boasts a new set of sleeker headlights, and chrome treated grille ensuring bolder front end.
It also gets 15-inch wheels, XC90-inspired wraparound LED tail lights, sportier front bumper with wider airdam, restyled bonnet, etc. The nearly undisguised spy pictures we have included here show the presence of chrome boot applique. As we reported earlier, the new in-house 1.5-litre diesel powertrain will only be introduced with BSVI compliance and the DDiS badge seen on the fender stands evident to the existing 1.3-litre unit staying put.
It produces 89 bhp and 200 Nm. However, the 1.4-litre petrol engine will make for the newly developed 1.5-litre K15B four-cylinder SHVS unit that makes 105 PS at 6,000 rpm and 138 Nm of peak torque delivered at 4,400 rpm.
It is mated to either a standard five-speed manual or four-speed automatic transmission. Sharing several components with latest Maruti parts bin, the new Ertiga will gain a new Smartplay touchscreen infotainment system mounted in the middle, light coloured dash with faux wooden trim, etc.


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Elite Gold Drop
Add extra glitz to any property with our elegant ornament drops. Our eye-catching 8' Elite Gold Drops are adorned with shiny and durian gold ornamentation, frosted snowflakes, and exquisite gold finials for a beautiful sparkle that will bring brilliance both day and night!
Color or size subject to slight change depending upon availability of ornamentation.

domain: design


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32 Teams/32 Days: Day 31
Steven Jackson to void contract after season, become free agent. (cbssports.com)
submitted 2 years ago by Billsdoitforthepeople
view the rest of the comments →
[–][deleted] 16 points17 points18 points 2 years ago (8 children)
I hope he doesn't test the market.
However, even if he does he will always be in the heart of Ram fans. He's been on a lot of shitty teams and didn't complain once. He just took the ball and ran with it like he's suppose to. No matter where he goes, I will always wear my #39 shirt with pride.
[–]RavensBALTIM0R0N 11 points12 points13 points 2 years ago (6 children)
Even if he goes to ...
...remind me who the Rams' biggest rival is?
[–][deleted] 9 points10 points11 points 2 years ago (0 children)
The 49ers used to be. They've had trouble both being good at the same time.
[–][deleted] 4 points5 points6 points 2 years ago (2 children)
We really don't have a bitter rival. Rooted for the Seahawks in the playoffs when they beat the Saints, rooted for the Cards during their run to the Superbowl (mainly because of Kurt), and rooted for the 49ers in the playoffs last year.
Hell, I still rooted for Isaac Bruce when he went to San Francisco.
[–]RavensBALTIM0R0N 2 points3 points4 points 2 years ago (1 child)
No bitter rival? That's the saddest thing I ever heard! I'll be your rival, little orphan boy!
[–][deleted] 2 points3 points4 points 2 years ago (0 children)
No! I like the Ravens. Ray Lewis and Ed Reed are two of my favorite non-Rams of all time.
Sorry we don't have rivals! We love everybody (except the Saints. I hate those fuckers).
[–]Chargersanteros86 2 points3 points4 points 2 years ago (0 children)
[–]Ramsthetasigma1355 1 point2 points3 points 2 years ago (0 children)
That team that beats us regularly... oh wait...
[–]PanthersAssumptionBulltron 0 points1 point2 points 2 years ago (0 children)
This is a great attitude. I have a lot of respect for Steven Jackson as a player because even on those really bad Rams teams of the last couple years, he has always seemed, at least to me, to play as hard as he possibly could anytime he's in the game. That's got to be tough to do when you're the #1 target of the other team's defense on every snap you play, but he does it pretty much without complaint.
REDDIT and the ALIEN Logo are registered trademarks of reddit inc.
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domain: sports


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domain: geography


text:
The Judge of Nudge
It is rare for a book about regulation to get much press. But this week saw the publication of one such book: Simpler: The Future of Government, by Cass Sunstein, former regulatory “czar” to President Obama. Sunstein served until the end of the first term, and was responsible for coordinating and reviewing regulations on behalf of the President. A Harvard Law School professor and prolific author, Sunstein has been promoting this book for months. As I awaited my copy, the only question in my mind would be the content: Would the book be a memoir, a defense of the President and his regulations, or a plug for policymakers to use behavioral economics? The answer, it turns out, is a bit of each.
As a memoir, the book is rather light. We learn of Sunstein’s introduction to Barack Obama, his relatively rough Senate confirmation battle (thank you, Glenn Beck), some info about his colleagues in the White House (fellow OMB official Jeff Zients can throw a tight football spiral), and a couple of instances where his views clashed with others (e.g., the phaseout of Primatene Mist by FDA ). He also provides his perspective on important topics well-known to readers of his other books (e.g., the precautionary principle, cost-benefit analysis). No Bob Woodward-esque moments of revelation here.
As a defense of the President and his policies, Sunstein comes across as the ultimate team player. Much of the book is spent complimenting the foresight of the President to adopt policies that help people (e.g., requiring for-profit colleges to disclose job placement rates). Sunstein admits that our regulatory system needs improvement, but the book is bereft of any admission of regulatory mistakes. For example, Sunstein takes pride in defending the President’s decision to reject EPA’s draft final rule establishing a more stringent (and expensive) ozone standard, but he fails to mention his acquiescence in having the rule get that far to begin with, and the political fallout that occurred as a result of the President’s last-minute rejection of the rule.
Although I cannot recommend the book as a memoir or as a complete and objective assessment of President Obama’s regulatory efforts (and I doubt Sunstein himself would characterize his book in this way), I can recommend the book for its penetrating insight into the practice of behavioral economics by the federal government. In a 2008 book, Nudge, Sunstein and co-author Richard Thayer introduce the field of behavioral economics, the concept of choice architecture, and the myriad ways that people can be manipulated (nudged) toward making better choices for themselves. Whereas Nudge could be described as a hypothesis in need of greater application, Simpler is its logical sequel: nudges in practice.
And without question, the Obama Administration has taken the concept of nudging to the next level. The book includes many examples, which is impressive enough, but Sunstein takes the time to delve deeply into the examples to show the research and theory that support the policies. Not that these policies have proven to be a success (indeed, Sunstein argues for randomized trials to better determine if proposed policies might work, and for retrospective review to determine if established policies did work), but rather that their design was based on careful consideration of the latest research suggesting effectiveness. (Why didn’t Sunstein employ transparency—a nudge—by requiring regulatory agencies to make publicly available the underlying data for its cost-benefit analyses. Such a move, it seems to me, would improve the objectivity of analysis used to justify rulemaking.)
To his credit, Sunstein goes to great lengths to present arguments against nudges. He then responds to each in a mostly effective manner. And yet, I found myself not-so-convinced on some of the Obama nudges. For example, the mandate for the placement of graphic images on cigarette packages is defended by Sunstein, and yet it still raises important policy questions for me. If this kind of “nudge” is correct, then why doesn’t Sunstein argue for replacing current regulations that prevent a product from entering commerce with a mandate for more salient information disclosure? On the other hand, if a product is so bad that the government has to go to extremes to convince people not to buy it, then why shouldn’t the government just ban the product altogether?
Sunstein recognizes the “slippery slope” argument against nudges, and he has much to say on this topic. He presents a list of potential nudges, ranging from those considered relatively “soft” to those considered rather “hard”. He lands firmly on the “soft” side. He also talks about “means” paternalism versus “ends” paternalism, and prefers “means” paternalism. Sunstein is right to define the boundaries of government nudges.
As I read the book, it occurred to me that private sector nudges represent a new arena for government regulation. Sunstein acknowledges that marketers of products use nudges in ways that are misleading and unfair. (Just try to unsubscribe from classmates.com; I dare you.) He points out that the FTC has started investigating such “negative option marketing”. But I do not think he carries this idea to its logical conclusion. It seems to me that federal regulatory agencies will be regulating private sector nudges with greater frequency as “bad” nudges become more widely used in the marketplace.
Which raises in my mind a question: How will conservatives react to Sunstein’s book? On one hand, they may hate it because it suggests not only a new type of tool for governmental intervention (i.e., a good nudge), but also an excuse for governmental regulation of the free market (i.e., to prevent a bad nudge). On the other hand, it seems only a matter of time before conservatives recommend the replacement of existing governmental mandates with less-intrusive governmental nudges.
Cass Sunstein is to be commended for writing a book that honestly and deeply examines the practice of nudging. He had a birds-eye view, and his perspective is both informative and thought-provoking.
Perhaps he will one day write (1) a memoir of his time in the Obama White House and (2) a book about the many regulatory mandates imposed during his watch. Consider this suggestion a nudge, Cass.

domain: literature