We used the SH-SY5Y, U373, and BV2 cell lines related to neurons, astrocytes, and microglia, respectively. Scopolamine or lipopolysaccharide (LPS) was used to antagonize the muscarinic receptors or induce inflammatory responses, respectively. The concentration of intracellular Ca2+ was measured using Fura-2 AM. Treatment with scopolamine and LPS significantly increased SOCE in the neuron-like cells and microglia but not in the scopolamine-pretreated astrocytes. LA significantly reduced SOCE in the scopolamine-pretreated neuron-like cells and microglia exposed to LPS, which was partially inhibited by the Na+-K+ ATPase inhibitor ouabain and the Na+/Ca2+ exchanger (NCX) inhibitor Ni2+. Notably, SOCE was significantly reduced in the LPS plus scopolamine-pretreated cells mixed with astrocytes and microglia, with a two-fold increase in the applied number of astrocytes. LA may be useful in protecting neurons and microglia by reducing elevated SOCE that is induced by inflammatory responses and inhibiting the muscarinic receptors via Na+-K+ ATPase and the forward mode of NCX. Astrocytes may protect microglia by reducing increased SOCE under the conditions of inflammation and a muscarinic receptor blockade.Inflammation and loss of cholinergic transmission are involved in neurodegenerative diseases, but possible interactions between them within neurons, astrocytes, and microglia have not yet been investigated. We aimed to compare store-operated Ca LA was  L.) oil , ulcerat L.) oil , and rhe L.) oil . LA has ar cells . It alsogenation . HoweverHere, we compared SOCE in the SH-SY5Y, U373, and BV2 cell lines related to neurons, astrocytes, and microglia, respectively. The cells were pretreated with scopolamine to block the muscarinic receptors, with or without an LPS for inflammatory stimulus, and were investigated for effects of LA on these processes.p = 0.001) (p = 0.008) cells compared with the corresponding control (CON) group (p = 0.001)  A,B and BN) group E,F. In t= 0.001) G,H. Thes2O2-induced oxidative stress has been shown to stimulate the Ca2+ release-activated Ca2+ current i was significantly higher in the deletion of the NCX gene in the hippocampal neurons than the presence of the NCX gene in hippocampal neurons [+/K+ ATPase can interact with NCX to regulate [Ca2+]i in the central nervous system, preventing Ca2+ overload i was significantly increased in those neurons pretreated with hydrogen superoxide i among the LPS plus scopolamine-pretreated astrocytes. Therefore, we aimed to investigate the possible protective effect of astrocytes against an increase in SOCE induced by LPS and scopolamine. A two-fold increase in the applied number of astrocytes significantly inhibited SOCE in the LPS plus scopolamine-treated cells mixed with neuron-like cells, astrocytes, and microglia via the forward mode of the NCX, which was comparable to the effects of LA. The NCX plays an important role in maintaining Na+ and Ca2+ homeostasis by mediating Ca2+ entry into cells under inflammatory conditions i levels under an inflammatory stimulus, indicating that the astrocytes had a neuroprotective effect i levels and interleukin-1β secretion in cultured mice microglia, leading to microglial activation [2+]i in microglia under the conditions of inflammation and a muscarinic receptor blockade.More importantly, we showed for the first time that astrocytes have protective effects against an increase in SOCE induced by LPS and scopolamine in microglia via a two-fold increase in the applied number of astrocytes in the mixed cells (of astrocytes and microglia). Microglial activation can trigger chronic neurodegeneration through its ability to cause the release of neurotoxic factors, such as ROS . The amyansients , and Aβ tivation . Therefo2 atmosphere. [Ca2+]i was measured in the SH-SY5Y, U373, and BV2 cells, and in a mixture of these three cell types. In the latter experiments, the SH-SY5Y, U373, and BV2 cells were cultured at a 7:2:1 ratio, reflecting the proportion of neurons, astrocytes, and microglia, respectively, in the human central nervous system [2+]i, we pretreated the cells with 0.1 mM scopolamine for 1 h to sufficiently inhibit the muscarinic receptors and then exposed the cells to 1 μg/mL LPS for 200 s to induce inflammatory responses i was determined as previously described i was measured at an emission wavelength of 510 nm with fluorescence detection at alternating excitation wavelengths of 340 nm and 380 nm using a fluorescence spectrometer . The maximum 340/380 nm fluorescence (Rmax) was measured in the presence of 5 µM ionomycin, a Ca2+ ionophore, and minimum 340/380 nm fluorescence (Rmin) was determined after adding 10 mM ethylene glycol-bis(β-aminoethylether)-N,N,N’,N’-tetraacetic acid (EGTA), a Ca2+ chelator. The composition of the extracellular fluid was 150 mM NaCl, 10 mM glucose, 10 mM HEPES, 6 mM KCl, 1.5 mM CaCl2, and 1 mM MgCl2, with the final pH adjusted to pH 7.4 with NaOH. Ca2+-free extracellular fluid was prepared by adding 2 mM EGTA.[Caescribed . Briefly2+ by adding 30 µM 2′,5′-di (tert-butyl)-1,4-benzohydroquinone (BHQ) to the Ca2+-free solution, and then adding 1.5 mM Ca2+ to activate store-operated Ca2+ channels [SOCE was induced by depleting the endoplasmic reticulum (ER) of Cachannels .2+, nifedipine, and dimethylsulfoxide (DMSO) were obtained from Sigma-Aldrich . Fura-2 AM, BHQ, and LA were dissolved in DMSO, and scopolamine was dissolved in 0.9% saline. All other regents were dissolved in distilled water.The DMEM used for cell culture was purchased from Welgene . FBS and penicillin/streptomycin were purchased from Biowest , and Fura-2 AM (purity > 95%) was obtained from Invitrogen . LA, VitC, scopolamine, EGTA, BHQ, LPS, ionomycin, ouabain, Nit-test, and comparisons among multiple groups were analyzed using and one-way analysis of variance followed by least significant deviation posthoc tests, if necessary. Differences were considered statistically significant at p-values < 0.05.All results are displayed as means ± standard error of the mean (SEM). All statistical analyses were performed using SPSS Statistics version 22 software . Differences between the groups were analyzed by Student’s +/K+ ATPase and the forward mode of NCX, which is attributable to inflammatory responses and the muscarinic receptor blockade. A two-fold increase in the applied number of astrocytes attenuated the increase in SOCE caused by an inflammatory stimulus and inhibited the muscarinic receptors in the mixed cells of the astrocytes and microglia. Collectively, these findings suggest that LA may have therapeutic efficacy in neurons and microglia under the conditions of inflammation and muscarinic receptor blockade. Also, the enhancement of astrocytes can be a promising strategy for preventing neurodegenerative diseases caused by inflammation and muscarinic receptor blockade.This study is the first to compare SOCE with and without an inflammatory stimulus under muscarinic receptor blockade conditions in neuron-like cells, astrocytes, and microglia to investigate the effects of LA on this process and its underlying mechanisms. Our findings suggest that microglia respond to inflammation, and neuron-like cells respond to muscarinic receptor blockade, with each case causing elevations in SOCE but resulting in differences between the cells. LA decreased SOCE in the neuron-like cells and microglia via the activation of Na


How environmental cues influence peroxisome proliferation, particularly through organelles, remains largely unknown. Yeast peroxisomes metabolize fatty acids (FA), and methylotrophic yeasts also metabolize methanol. NADH and acetyl-CoA, produced by these pathways enter mitochondria for ATP production and for anabolic reactions. During the metabolism of FA and/or methanol, the mitochondrial oxidative phosphorylation (OXPHOS) pathway accepts NADH for ATP production and maintains cellular redox balance. Remarkably, peroxisome proliferation in Pichia pastoris was abolished in NADH-shuttling- and OXPHOS mutants affecting complex I or III, or by the mitochondrial uncoupler, 2,4-dinitrophenol (DNP), indicating ATP depletion causes the phenotype. We show that mitochondrial OXPHOS deficiency inhibits expression of several peroxisomal proteins implicated in FA and methanol metabolism, as well as in peroxisome division and proliferation. These genes are regulated by the Snf1 complex (SNF1), a pathway generally activated by a high AMP/ATP ratio. In OXPHOS mutants, Snf1 is activated by phosphorylation, but Gal83, its interacting subunit, fails to translocate to the nucleus. Phenotypic defects in peroxisome proliferation observed in the OXPHOS mutants, and phenocopied by the Δgal83 mutant, were rescued by deletion of three transcriptional repressor genes  controlled by SNF1 signaling. Our results are interpreted in terms of a mechanism by which peroxisomal and mitochondrial proteins and/or metabolites influence redox and energy metabolism, while also influencing peroxisome biogenesis and proliferation, thereby exemplifying interorganellar communication and interplay involving peroxisomes, mitochondria, cytosol, and the nucleus. We discuss the physiological relevance of this work in the context of human OXPHOS deficiencies. Like other subcellular organelles, peroxisomes divide and segregate to endow daughter cells with peroxisomes. Cells stringently regulate organelle number, volume, size, and content in response to environmental signals. Regulation of these properties, together with organelle dynamics and homeostasis, allows cells to endure metabolic or environmental stress, cope with the needs of cell division or differentiation, remove excess or damaged organelles by turnover, correct imbalances in organelle segregation during cell division, or repopulate organelles with different enzymes upon switching to a new environment. The division of some organelles, such as the nucleus or the Golgi apparatus, is coupled to the cell cycle . HoweverIn yeast and other eukaryotes, three major pathways control peroxisome number. First, in constitutively dividing cells, peroxisomes divide by fission of pre-existing peroxisomes, a process we refer to simply as peroxisome division . This prIn yeasts, peroxisome division occurs both during constitutive growth and cell division. Peroxisome size and number are sensitive to peroxisomal metabolic pathways and the metabolites available . ChangesSaccharomyces cerevisiae, while Pex11 promotes the division of peroxisomes already present in the cell, Pex25 initiates remodeling at the peroxisomal membrane to allow proliferation and Pex27 counters this activity   .In contrast, the addition of glucose to cells (glucose repression) results in a reduction in ADP levels that causes the Glc7-Reg1 protein phosphatase to dephosphorylate T210 and thereby inactivate Snf1 .Notably, this entire description of peroxisome induction and proliferation involves transactions of proteins and metabolites residing in three compartments – the cytosol, nucleus, and peroxisomes, with no direct involvement of mitochondria. Yet, it is clear that peroxisomes contact other subcellular compartments, including mitochondria . FurtherP. pastoris. During growth in glucose, most P. pastoris cells possess a few, small peroxisomes, with a limited lumenal content, but when cells are grown in oleate, numerous, small peroxisomes proliferate and are distributed throughout the cells, whereas in methanol medium the peroxisomes are large, less numerous, and clustered , Pex3-GFP or GFP-Pex36, in several mutant strains grown in different carbon sources  shuttles, during growth in glucose  and one G3P dehydrogenase (GpdA). We recently reported that MdhA and GpdA are predominantly mitochondrial enzymes in all media analyzed, whereas MdhB is cytosolic during growth in glucose or methanol, but is cytosolic/peroxisomal during growth in oleate   . We obseBFP-SKL) . Interesroxisome . The resP. pastoris uses methanol as a sole carbon source for carbon assimilation and energy production .The similar phenotypes of the NADH-shuttling and P. pastoris, the NADH shuttled to the mitochondria is oxidized by the respiratory chain. The NADH dehydrogenase complex I (CI) is the main entry point for electrons into the respiratory chain. It directly oxidizes NADH to NAD+ and concomitantly reduces coenzyme Q , which causes H+ to be pumped out across the inner mitochondrial membrane, to contribute to the proton gradient that drives ATP synthesis  encoding a downstream component of the OXPHOS complex, but not directly implicated in NADH oxidation. Like the CI mutants, the Δcyt1 mutant also blocked peroxisome proliferation in both media  after 4 hr of induction in oleate medium  during growth in glucose, glycerol, and methanol media    and the ae Adr1)  , but not for peroxisome proliferation, by a mechanism resembling Pip2-Oaf1 activation  is identical with that in human AMPKα and S. cerevisiae Snf1  analysis and confirmed that expected . However+/NADH sensors, and also measured oxygen consumption rates (OCR), which reflect mitochondrial ATP production ; however, the peroxisome sensors are transported to the peroxisomes by the PTS1 pathway, and mislocalization of the sensors will affect the assay. Oleate medium was chosen because the microscopy data from gpdA ΔmdhB.To confirm our conclusions about the redox and ATP status in mitochondria, peroxisome, NADH shuttles, and SNF1 mutants during growth in peroxisome proliferation conditions, we developed cytosolic and peroxisome NADoduction . Due to + biosensors were recently developed, providing insight into NAD+ dynamics. We choose one of these sensors, SoNar, to measure NAD +/NADH ratios  which should not increase because NADH cannot shuttle out of the peroxisomes, and this was also corroborated by this assay (We predicted that during growth in peroxisome proliferation conditions (oleate or methanol), dysfunctional peroxisomes, due to OXPHOS, SNF1, NADH- or acetyl-CoA-shuttling mutants or mutants lacking peroxisomal β-oxidation enzymes, such as Pot1, should not produce peroxisomal NADH, thereby decreasing the cytosolic pool of NADH, which should increase the cytosolic ratio of NADng SoNar . Likewisis assay .In yeast, during growth on nonfermentable carbon sources, most if not all the oxygen, is consumed by OXPHOS to produce ATP. To assess the consequence of our mutants on ATP production during growth in peroxisome proliferation conditions we measured OCR using the Seahorse XF analyzer . In addiAs Snf1 is phosphorylated in cells with dysfunctional mitochondria, we hypothesized that OXPHOS mutations might affect peroxisomal gene expression downstream of Snf1 activation. We started by analyzing the role of Mit1 , which is the putative kinase for the inhibitory phosphorylation of the S. cerevisiae homolog of Mxr1 (Adr1), and overexpressed Mit1 (OE-Mit1) from the strong, constitutive GAPDH promoter  did not affect the fitness of cells, but like OE-Mit1 or PKA inhibition, did not rescue the OXPHOS or SNF1 deficiencies  strongly derepressed Aox1 under glycerol-repression conditions, but not during glucose derepression, even when transcription activators (Mit1 and Prm1) were artificially activated (MIG1 and MIG2 significantly upregulated the binding of Mit1 at the AOX1 promoter (In ctivated . This mapromoter .mig1 Δmig2Δnrg1 strain (NUGM or GAL83 (control) genes. Most of the peroxisome proliferation defects we observed in the OXPHOS and gal83 mutants, such as peroxisome proliferation, as well as Pot1 and Aox1 expression, were rescued when cells were induced in either oleate or methanol  behaved similarly in both media. Additionally, peroxisome proliferation was identical to that in WT and the triple mutant (Δmig1 Δmig2Δnrg1) strains , with Gal83 being the subunit responsible for the nuclear localization of the complex .Transcriptional repressors and activators shuttle between the cytoplasm and the nucleus in a glucose-dependent manner. There is no doubt that the SNF1 complex needs to be localized in the nucleus to inactivate the repressors that occlude the promoters of glucose-repressed genes, but there is little to no evidence for Mxr1/Adr1 activation in the nucleus. Instead, if Mxr1/Adr1 indeed can be activated within the cytosol, a deficient nuclear enrichment of SNF1 in the OXPHOS mutant during glucose derepression could explain the rescue of peroxisome biogenesis defects in the ΔnugM mutant on Gal83 nuclear localization by using a Gal83-GFP fusion expressed from the native GAL83 promoter. WT and mutant cells were grown on abundant glucose, and the nuclear enrichment of Gal83-GFP was stimulated by shifting the cells to oleate for 30 min or methanol for 2 hr in the presence of leptomycin B  medium (nonfermentable carbon source), that WT cells showed higher levels of Pex11, Aox1, and Pot1 proteins compared to glucose depletion or to cultivation in methanol or oleate , and upon using DNP, peroxisome proliferation, division, and biogenesis of several peroxisome-associated proteins, but not peroxisomal matrix protein import  also affects peroxisome proliferation, as seen for the OXPHOS mutants   . Consequ A (CoA) . Acetyl- and H2O .P. pastoris, the nuclear translocation of the SNF1–Gal83 complex requires OXPHOS and ATP production, as shown here. Peroxisome-associated proteins, such as the division protein, Pex11, and the matrix proteins, Pot1 and Aox1, are regulated negatively by transcriptional repressors, that compete with transcriptional activators, such as Mit1 and Mxr1 . Snf1 activation in the cytosol and SNF1–Gal83 entry into the nucleus removes, by phosphorylation of the appropriate proteins, the repression of expression of the peroxisome-associated proteins, while also activating the transcriptional activators. This simultaneous action of SNF1–Gal83 turns on the biogenesis of peroxisome-associated proteins, peroxisome proliferation, as well as division. In the ΔnugM, Δndufa9, Δgal83, Δpot1, Δaox1 aox2, and Δpex5 mutants of P. pastoris, or in the presence of the mitochondrial uncoupler, DNP, peroxisome proliferation, division, and the biogenesis of certain peroxisome-associated proteins in compromised.The SNF1/AMPK complex, which is sensitive to the cellular AMP:ATP ratio, maintains the balance between ATP production and consumption in all eukaryotic cells . GlucoseIn conclusion, since ATP production by mitochondrial OXPHOS is impaired in many human diseases involving over 150 genes , includiMethods availability statement – Strains and plasmids are described in https://blink.ucsd.edu/research/conducting-research/mta/index.html).P. pastoris strains were prototrophic without requiring amino acid supplements or yeast extract and were cultivated in minimal media containing different carbon sources. Supplementation of media with amino acids did not change the results of this study. YPD , glucose medium , oleate medium , and methanol medium . Amino acids were supplemented when indicated using 0.79 g/l complete synthetic medium of amino acids and supplements . DNP  was dissolved in methanol, used at a final concentration of 0.25 mM and added after switching cells from glucose to methanol (1%). LMB solution  was added to the methanol medium at final concentration of 200 ng/ml.https://nebuilder.neb.com/#!/). DNA was amplified from WT genomic DNA by PCR using Advantage 2 Polymerase . Plasmid backbones were double digested with the necessary restriction enzymes and purified using the Qiagen DNA purification kit . DNA inserts were cloned into the digested vectors using the NEBuilder Hifi DNA Assembly Mastermix .Plasmids were constructed by Gibson Assembly for which primers were designed using NEBuilder (Plasmids were linearized with the appropriate restriction enzyme before transformation of competent yeast cells by electroporation . The tra600/ml), washed twice with sterile water, and then transferred to glucose or peroxisome proliferation (methanol or oleate) media. With the goal of keeping cells at exponential phase, different starting OD600 were used for different strains, depending on the strain’s cell doubling time in the respective media. Cells were grown in indicated media in 250 ml flasks at 30°C and shaken at 250 rpm. Cells were pelleted, washed twice with sterile water and 1.5 μl of cells were mixed with 1% low melting point agarose and placed on a glass slide with a cover slip and imaged using ×63 or ×100 magnification on a Carl Zeiss Axioskop fluorescence microscope. Images were taken on an AxioCam HR digital camera, no digital gain was used, exposition for peroxisome markers was kept constant. Images processed on AxioVision software are representative results from experiments conducted at least in triplicate.Cells were grown in YPD at 30°C until exponential phase , Anti-Snf1 , Anti-Rabbit HRP , Anti-Mouse HRP , and Anti-Rat HRP . Additionally, the following antibodies were generated in-house: Anti-Pex2, Anti-Pex3, Anti-Pot1, and Anti-Aox1.The same conditions described for fluorescence microscopy assays were used to grow cells. Five ODqRT-PCR – RNA extraction was performed using Trizol Reagent  following the manufacturer’s instructions with some modifications. Twenty OD600 of frozen cells, grown in the conditions described, were resuspended with 1 ml of Trizol and 250 μl of acid-washed glass beads (425–600 μm). The suspension was vortexed for 30 s, followed by chilling on ice for 30 s. This step was repeated three times. The extraction was completed according to the protocol described by the kit manufacturer and was followed by DNAse I treatment . The RNA quantity and quality were determined using the 260/280 nm ratio of approximately 1.8–2.0 for good RNA quality. The RNA samples were stored at (add symbol 80° C). cDNA was synthesized from 1 μg of total RNA using an Invitrogen two-step kit with SuperScript III  as the reverse transcriptase (RT) enzyme with random hexamer. This was followed by RNAse treatment. The cDNAs were stored at add symbol 80°C. One μl of cDNA , 300 nM of primers listed in -ΔΔCT method  following the manufacturer’s instructions . Briefly, cells were grown in lactate medium, washed and resuspended in high lactose osmotic stabilizing solution (YMAS), containing digitonin . After 1 hr, the permeabilized cells were seeded into 96-well PM1 MicroPlates containing different potential energy substrates. The colorimetric assay was initiated by adding Redox Dye Mix MC (Biolog #74353). The PM1 MicroPlate was then loaded into the OmniLog PM-M system  for kinetic reading at 30°C. The MitoPlates were read for 24 hr at 15 min intervals at OD590. OmniLog rate values were calculated using Data Analysis 1.7 software before being transferred to a Microsoft Excel sheet for reformatting. The software calculates the maximal rate change from the gradient of the reading output during the 24 hr time period. These data are presented as raw OD590 (A.U.).4 cells, coming from 15 to 16 hr methanol cultures, were added to a poly-lysine coated Seahorse XF96 PDL Cell Culture Microplate  in 50 μl of assay medium  supplemented with 6 mM glutamine solution  and 1% methanol). The plate was centrifuged at 3000 × g for 20 min with gentle acceleration and deceleration. Then 130 μl of assay medium were added to each well and incubated at 30°C in a non-CO2 incubator for 1 hr. Immediately following completion of the incubation, OCR and ECAR was measured three times every 10 min and an additional three times every 30 min after the injection of glucose . All samples were analyzed in triplicate, normalized by cell number and the third stable reading of every condition was plotted in respective graphs.OCR and extracellular acidification rate were determined in a Seahorse XF96 analyzer. In brief, for the OCR and ECAR analysis, between 5–10 × 10+/NADH ratio was measured using the genetically encoded sensor, SoNar and its control cpYFP. Cells were pregrown in oleate medium for 8 hr, washed twice and resuspended in 2× YNB supplemented with 0.04 mg/l biotin. Between 2–3 × 107 cells in 200 μl were seeded in a 96 well black/clear plate  and end point fluorescence intensities were measured in a NOVOstar Microplate Reader . The SoNar sensor/cpYFP was excited at 420 and 485 nm and the emission at 520 nm was detected to obtain the ratiometric measurement. NAD+/NADH ratios were obtained from the emission value of the 420 nm excitation (F420) and 485 nm excitation (F485). After background subtraction and normalization for cell number, obtained from absorbance at 600 nm in the same plate reader, the ratios of NAD+/NADH were calculated as F420/F480. All samples were analyzed in triplicate.NAD This paper elegantly describes how the assembly, division and proliferation of peroxisomes are controlled by proteins/metabolites residing in 4 different subcellular compartments. Mitochondrial energy production is assisted by peroxisomal proteins and metabolites, which shuttle via the cytosol to mitochondria, where ATP is produced. ATP availability is transmitted by the nuclear shuttling of cytosolic proteins to regulate genes controlling peroxisome assembly, division and proliferation. In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Review Commons.] Zhao, Y., Hu, Q., Cheng, F., Su, N., Wang, A., Zou, Y., Hu, H., Chen, X., Zhou, H. M., Huang, X., Yang, K., Zhu, Q., Wang, X., Yi, J., Zhu, L., Qian, X., Chen, L., Tang, Y., Loscalzo, J., and Yang, Y. (2015) SoNar, a highly responsive NADEMBO Rep 22, e51606.[2] Liu, S., Liu, S., He, B., Li, L., Li, L., Wang, J., Cai, T., Chen, S., and Jiang, H. (2021) OXPHOS deficiency activates global adaptation pathways to maintain mitochondrial membrane potential, mig1 Δmig2 mutant reveals their roles in methanol catabolism, peroxisome biogenesis and autophagy in methylotrophic yeast Pichia pastoris, Genes Genomics 40, 399-412.[3] Shi, L., Wang, X., Wang, J., Zhang, P., Qi, F., Cai, M., Zhang, Y., and Zhou, X. (2018) Transcriptome analysis of ΔAOX1 promoter in methylotrophic yeast Pichia pastoris, Biotechnol Lett 38, 291-298.[4] Wang, X., Cai, M., Shi, L., Wang, Q., Zhu, J., Wang, J., Zhou, M., Zhou, X., and Zhang, Y. (2016) PpNrg1 is a transcriptional repressor for glucose and glycerol repression of Virulence 2, 538-541.[5] Nguyen, L. N., Gacser, A., and Nosanchuk, J. D. (2011) Secreted lipases supply fatty acids for yeast growth in the absence of de novo fatty acid synthesis, AOX1 promoter-regulated expression cassettes leads to transcription attenuation of the methanol metabolism in Pichia pastoris, Sci Rep 7, 44302.[6] Camara, E., Landes, N., Albiol, J., Gasser, B., Mattanovich, D., and Ferrer, P. (2017) Increased dosage of Pichia pastoris, J Biol Chem 271, 18973-18980.[7] Wiemer, E. A., Luers, G. H., Faber, K. N., Wenzel, T., Veenhuis, M., and Subramani, S. (1996) Isolation and characterization of Pas2p, a peroxisomal membrane protein essential for peroxisome biogenesis in the methylotrophic yeast Biochim Biophys Acta 1864, 1518-1528.[8] McCartney, R. R., Garnar-Wortzel, L., Chandrashekarappa, D. G., and Schmidt, M. C. (2016) Activation and inhibition of Snf1 kinase activity by phosphorylation within the activation loop, J Biol Chem 276, 36460-36466.[9] McCartney, R. R., and Schmidt, M. C. (2001) Regulation of Snf1 kinase. 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HLA-mismatched hematopoietic stem cell micro-transplantation (MST) is an effective treatment for older patients (≥60 years) with acute myeloid leukemia. Donor selection for MST is broad, ranging from HLA fully mismatched unrelated donors to HLA partially matched related donors. However, the influence of HLA haplotype homozygous donors such donors on MST has not been studied. Such donors has been reported to be associated with a higher risk of graft-versus-host disease (GVHD) in transfusion and cord blood transplantation (CBT). Additionally, sustained complete donor chimerism is rare in MST and usually accompanied by severe acute GVHD and death. Herein, we report the first case of MST using an HLA haplotype homozygous donor. The patient developed persistent complete donor chimerism (donor cells>95%) for 7 months and prolonged isolated thrombocytopenia (PT) for 3 months, after receiving MST from his HLA homozygous son. Grade I acute GVHD presented on day 12 post-MST and it was controlled by timely immunosuppressive treatment. Then he maintained complete molecular remission, complete donor chimerism and mild GVHD for 5 months. However, moderate overlapping GVHD with skin, oral, eyes, and intestinal involvement developed after he self-discontinued Tacrolimus treatment. Fortunately, the GVHD was controlled after intensive anti-rejection therapy and Tacrolimus is now being continued for prophylaxis. This case underscores that HLA haplotype homozygous donors might not be a good choice for MST and GVHD prophylactic should be administrated if such donors have to be selected. Although allogeneic hematopoietic stem cell transplantation  is a potentially curative therapy for acute myeloid leukemia (AML), older patients ≥60 years) are susceptible to transplant-related toxicity and severe graft-versus-host disease (GVHD) . Infusio0 years aHLA - mismatched donors are generally selected for MST and the efficacy of unrelated donor-derived MST is similar to or higher than that of related donor-derived MST , 3. HLA 9/L, and hemoglobin (Hb) of 82 g/dL. Bone marrow (BM) aspirates showed 65% blasts by histology and 64.8% blasts by flow cytometry that were positive for CD123, CD117, CD13, CD33, HLA-DR, and CD45, and negative for CD34. Cytogenetics demonstrated normal male chromosomes. Next-Generation Sequencing (NGS) revealed mutations of KMT2A-PTD, DNMT3A, IDH2, and STAG2. A diagnosis of AML with KMT2A-PTD (high risk) was established. The patient received two cycles of induction chemotherapy with idarubicin and cytarabine (IA) and achieved partial remission (PR). He then was admitted to our hospital for further treatment. Complete peripheral blood (PB) cell count showed a leukocyte count of1.18 × 109/L, Hb of 75 g/L, and platelets of 64 × 109/L. Blast count of BM was 7% by histology and 6.41% by flow cytometry, blasts were positive for CD117, CD13, CD33, CD38, HLA-DR, and CD45, and negative for CD34, CD19, and CD10. Chromosome analysis showed 46,XY [20]. The mutations of KMT2A-PTD, DNMT3A, IDH2, and STAG2 were reconfirmed by NGS. He then underwent reinduction of decitabine plus venetoclax and achieved complete remission (CR). Minimal residual disease (MRD) based on flow cytometry was negative (<1.1×10-4). He then received one cycle of consolidation with the same regimen  count of 1.3 ×10 regimen Figure 18 cells/kg of body weight from his son whose HLA typing was homozygous at HLA-A, B, C, DR, and DQ loci , cytarabine (Ara-C), aclarubicin (Acla), and G-CSF) . Periphe DQ loci Table 1 +stem cells, NK cells, T lymphocytes, Neutrophils, and B lymphocytes, respectively (-5). The mutations were also not detected by quantitative polymerase chain reaction (qPCR) and PCR-pair end sequencing. PCR-STR of sorted BM cells revealed donor chimerism 98.16% and 86.34% for CD34+stem cells and T lymphocytes, respectively  and mild GVHD for 5 months. His platelets recovered about 120 days after the infusion of G-PBSCs. However, it’s noteworthy that he developed moderate overlapping GVHD with skin, oral, eyes, and intestinal involvement after he self-discontinued Tacrolimus 5 months post-MST. Then Methylprednisolone, Tacrolimus, Ruxolitinib, and CD25 monoclonal antibody were given and the GVHD was controlled. Now the patient is still in complete molecular remission with no infection, rash, diarrhea, or abnormal liver function. Tacrolimus is now being continued for prophylaxis due to complete donor chimerism.MST is an important treatment for patients who cannot tolerate standard allo-HSCT or lack suitable donors –6. Donor+ cells in the graft is a critical factor in neutrophil and platelet recovery, and the latter appears to be more important  for the publication of any potentially identifiable images or data included in this article.LL was the main writer of the case report, contributing to all of the sections of the report. XT, QC, ML, ZL, and SC provided direct patient care while the patient was hospitalized. XT, QC, and YM helped write part of the case report and edited the case report. JH was in charge of HLA typing. All authors read and approved the final manuscript.The authors would like to thank all members of the study team, the patient and their families.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Alzheimer’s disease (AD) and ischemic stroke (IS) are common neurological disorders, and the comorbidity of these two brain diseases is often seen. Although AD and IS were regarded as two distinct disease entities, in terms of different etiologies and clinical presentation, recent genome-wide association studies (GWASs) revealed that there were common risk genes between AD and IS, indicating common molecular pathways and their common pathophysiology. In this review, we summarize AD and IS risk single nucleotide polymorphisms (SNPs) and their representative genes from the GWAS Catalog database, and find thirteen common risk genes, but no common risk SNPs. Furthermore, the common molecular pathways associated with these risk gene products are summarized from the GeneCards database and clustered into inflammation and immunity, G protein-coupled receptor, and signal transduction. At least seven of these thirteen genes can be regulated by 23 microRNAs identified from the TargetScan database. Taken together, the imbalance of these molecular pathways may give rise to these two common brain disorders. This review sheds light on the pathogenesis of comorbidity of AD and IS, and provides molecular targets for disease prevention, manipulation, and brain health maintenance. In the elderly, brain health is mainly jeopardized by dementia or stroke . About 6Clinically, AD is characterized by the inability to recall recent events, changes in personality, and difficulty in solving problems, at the disease’s early stage. AD patients may develop behavioral changes, confusion, disorientation, communication impairment, and diminished social skills at a later stage, and eventually, the patients may have difficulties in speaking, swallowing, and walking. Pathologically, AD is mainly characterized by hippocampal neuronal loss, amyloid β (Aβ) extracellular deposition forming senile plaques, and the formation of intracellular neurofibrillary tangles. Clinically, IS can present with a variety of physical and cognitive manifestations depending on the brain areas affected and associated with neuron lysis and death . MultiplGenetic risk factors play an important susceptible role in both AD and IS . Genome-https://www.ebi.ac.uk/gwas/, accessed on 14 September 2022 from the database’s inception to 14 September 2022. The GWAS Catalog data was extracted from the published GWASs, identified through a weekly PubMed search . In the database, the search terms used were: “Alzheimer’s disease” and “ischemic stroke”. The information on risk SNPs and their mapped genes was extracted and aligned. If the SNP was located within a gene, that gene was listed, with multiple overlapping genes separated into several genes. If the gene was intergenic, the upstream and downstream genes were listed.The literature was reviewed by searching the GWAS Catalog database . The section “pathways” provides links to pathways according to information extracted from Cell Signaling Technology, R&D Systems, GeneGo (Thomson Reuters), Reactome, Sino Biological, Tocris Bioscience, PharmGKB, Qiagen, GeneTex, BosterBio, MedChemExpress, WikiPathways and PubChem, SuperPathways from PathCards, the protein-protein interaction network was derived based on the STRING database . We identified the miRNAs that regulate the target genes through the TargetScan database . The molecular selection processes are shown in a flow chart , presenilin1 (PSEN1), and presenilin 2 (PSEN2), respectively. The Apolipoprotein E (ApoE) gene is a major player in sporadic AD. The major hereditary IS diseases are cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), cerebral autosomal recessive arteriopathy/arteriosclorosis with subcortical infarcts and leukoencephalopathy (CARASIL), and Fabry disease, which are caused by mutations in the notch receptor 3 (Notch3), htrA serine peptidase 1 (HTRA1), and galactosidase alpha (GLA) genes, respectively.APP, PSEN1, or PSEN2 mutations on the risk of IS. However, they may influence the risk of IS through their effects on lipid metabolism [No study has found a direct effect of tabolism . When evtabolism . Lipid ptabolism . Statinstabolism .ApoE has been consistently considered as the major genetic risk for sporadic AD and cognitive decline post-IS [ApoE ε4 is associated with an increased number of Aβ plaques, but we did not find ApoE related to IS in the GWAS database, although ApoE has been shown to be associated with IS risk conditions, such as diabetes [ post-IS . The Apodiabetes  and low diabetes , which mIt has been suggested that Notch3 and HTRA1 are associated with AD ,31. AmonALDH1A2, ANKRD22, ANTXR1, DIO2-AS1, HDAC9, JH3, KCNN3, LNC-LBCS, MMP3, MMP12, PCSK6, RBMS3, and RNU6-909P. Among them, ten genes are protein coding genes, including ALDH1A2, ANKRD22, ANTXR1, HDAC9, JPH3, KCNN3, MMP3, MMP12, PCSK6, and RBMS3. The shared genetic studies of AD and IS are summarized in HDAC9, MMP3, and MMP12 have been relatively well studied in AD and IS.We found there were thirteen common mapped genes in AD and IS, although no common risk SNPs were found. The common genes are HDAC9 promoter and increased the transcription capacity, which was related to higher HDAC9 mRNA levels [Histone deacetylase 9 (HDAC9) is responsible for the lysine deacetylation residues on the N-terminal part of the core histones. Histone deacetylation is involved in epigenetic repression, and plays a significant role in transcriptional regulation, cell cycle progression, and some developmental events. In AD, the expression of HDAC9 was significantly reduced in the sampled prefrontal and visual cortices . HoweverA levels . PathwayA levels . Thus, tMatrix metalloproteinase 3 (MMP3) and matrix metalloproteinase 12 (MMP12) are two members of the matrix metalloproteinases (MMPs) family, which are both involved in the breakdown of the extracellular matrix (ECM). Microglia surround the Aβ plaques, provoke an inflammatory response, and contribute to neuronal cell loss. Aβ1-42 induces the upregulation of MMP3 and MMP12 in microglia, which further enhances the inflammatory processes and accelerates the progression of AD . The risALDH1A2), DIO2 antisense RNA 1 (DIO2-AS1), JH3, potassium calcium-activated channel subfamily N member 3 (KCNN3), long noncoding RNAs bladder and prostate cancer suppressor (LNC-LBCS), RNA binding motif single stranded interacting protein 3 (RBMS3) and RNA, U6 small nuclear 909, and pseudogene (RNU6-909P) genes, and AD, IS were only observed in populational studies and have not been confirmed in molecular studies yet.ANTXR Cell Adhesion Molecule 1 (ANTXR1) is a type I transmembrane protein and a tumor-specific endothelial marker. The higher peptide from ANTXR1 showed a significant ability to discriminate AD patients from healthy controls, which suggested that the risk allele of rs7561207 may increase the risk of AD through enhancing the expression of ANTXR1 . AnkyrinTo address the common AD and IS risk genes products in AD important molecules, a STRING analysis was performed. The thirteen common AD and IS risk genes in the AD important molecules network were identified. MMP3 and MMP12 links with ApoE, indicating that ApoE may associate with IS through connecting with MMP3 and MMP12 .HDAC9, MMP3, and MMP12 gene products are involved in the G protein-coupled receptors (GPCR) pathway. Both HDAC9 and MMP3 gene products participate in the macrophage-migration inhibitory factor (MIF) mediated glucocorticoid regulation pathway, both MMP3 and MMP12 gene products are involved in the transendothelial migration of leukocytes pathway, and the two pathways are both immune related pathways. MMP3, ALDH1A2, HDAC9, and PCSK6 gene products participate in the signal transduction pathway; ALDH1A2 and MMP3 gene products are involved in the estrogen receptor (ESR)-mediated signaling and signaling by nuclear receptors pathways; ALDH1A2 and HDAC9 gene products are involved in the ethanol effects on histone modifications pathway; both MMP3 and MMP12 gene products participate in the urokinase-type plasminogen activator (uPA) and urokinase plasminogen activator receptor (uPAR)-mediated signaling and UPA-UPAR pathway, and these six pathways are signal transduction pathways , and overcomes glucocorticoid’s inhibition of T-cell proliferation . MIF mayThe inflammatory responses are initiated locally under aberrant local conditions in both AD and IS, and elicited by the immune system . TherefoGPCRs are the largest membrane protein family of seven-transmembrane receptors in humans. They are involved in neuronal signal transduction in response to various extracellular signals such as hormones and neurotransmitters . GPCRs mNR1H3 gene was shown to reduce the risk of AD, and the soluble Aβ42 levels were significantly reduced in the temporal cortex of patients with the CC genotype [The occurrence of both AD and IS are tightly regulated by a multitude of signal transduction pathways. Nuclear receptors generally act as ligand-activated transcription factors . They regenotype . In an Agenotype . The TT genotype . Ex vivogenotype . In addigenotype . Peroxisgenotype . The PPAgenotype . The PPAgenotype . The nucgenotype  in AD migenotype . The absgenotype .ApoE -/- mice samples, assay results indicated that estrogen prevented atherosclerosis through upregulating ESRα expression to induce ESRα-mediated activation of autophagy and reduce inflammation and cell pyroptosis [ApoE -/- mice model, it was demonstrated that ESRα inhibited the synthesis and secretion of proprotein convertase subtilisin/kexin type 9 (PCSK9) and subsequently lowered the accumulation of cholesterol and triglyceride to prevent post-menopausal atherosclerosis [In AD hypothalamic medial mamillary (MMN), a higher nuclear ESRα intensity was significantly associated with larger nuclear and perikaryal sizes, indicating nuclear ESRα may mediate extra activation in MMN that acts as a unique brain area to prevent neurodegeneration . In AD hroptosis . In addiclerosis . Hyperinclerosis .Urokinase-type plasminogen gene rs2227564 C-positive genotype (CC+CT) has been reported to associate with a higher risk of developing AD . NeuronaMIR204 gene, regulating ALDH1A2, ANKRD22, ANTXR1, HDAC9, JH3, KCNN3, MMP3, and RBMS3. Hsa-miR-664b-3p is the product of MIR664B, regulating ALDH1A2, ANKRD22, HDAC9, KCNN3, MMP3, MMP12, PCSK6, and RBMS3. Heavy metals like Pb acetate increased the expression of has-miR-204-5p, deteriorated the cognitive functions, and were associated with the overexpression of tau via pathways of neurodegeneration-multiple diseases [APP, PSEN1, MAPT, and HTRA1 (At least nine of these thirteen genes can be regulated by 23 miRNAs identified from the TargetScan database. Among the 23 miRNAs, four miRNAs regulate eight genes, including hsa-miR-204-5p, hsa-miR-211-5p, hsa-miR-548c-3p, and hsa-miR-660-3p; and the other miRNAs regulate seven genes. Among them, we found that thirteen miRNAs were reported through a Pubmed search using the key words of each miRNA . Howeverdiseases . Has-miRdiseases . Hsa-miRdiseases . It was diseases . It may nd HTRA1 .Compared with other studies , our stuIn this study, we used genetic data from the GWAS Catalog database and PubMed to determine whether the same genetic loci contribute to AD and IS. We identified thirteen common risk genes, which are involved in three common molecular pathways: immunity, GPCR, and signaling transduction pathways. Furthermore, we identified 23 miRNAs that regulated at least seven of the common risk genes. In summary, there are some common genetic factors and pathways shared by AD and IS, which might be the molecular targets for maintaining good brain health jeopardized by AD or IS.


Anopheles gambiae, and discovered that an infection upregulates multiple components of the immune deficiency (IMD) and c-Jun N-terminal kinase (JNK) pathways in the heart with periostial haemocytes. This upregulation is greater in the heart with periostial haemocytes than in the circulating haemocytes or the entire abdomen. RNA interference-based knockdown then showed that the IMD and JNK pathways drive periostial haemocyte aggregation and alter phagocytosis and melanization on the heart, thereby demonstrating that these pathways regulate the functional integration between the immune and circulatory systems. Understanding how insects fight infection lays the foundation for novel strategies that could protect beneficial insects and harm detrimental ones.The immune and circulatory systems of animals are functionally integrated. In mammals, the spleen and lymph nodes filter and destroy microbes circulating in the blood and lymph, respectively. In insects, immune cells that surround the heart valves (ostia), called periostial haemocytes, destroy pathogens in the areas of the body that experience the swiftest haemolymph (blood) flow. An infection recruits additional periostial haemocytes, amplifying heart-associated immune responses. Although the structural mechanics of periostial haemocyte aggregation have been defined, the genetic factors that regulate this process remain less understood. Here, we conducted RNA sequencing in the African malaria mosquito,  These gambiae –9, heart gambiae –12. Furt gambiae .Escherichia coli but not Staphylococcus aureus [In mosquitoes, pattern recognition receptors in the thioester-containing protein family, together with adhesion and phagocytosis factors in the Nimrod protein family, positively regulate immune responses on the heart ,15. Howes aureus . The Jaks aureus ,23, and s aureus ,25.A. gambiae. Using an unbiased RNA sequencing (RNAseq) approach that was followed by empirical testing with RNA interference (RNAi), we uncovered that the IMD and JNK pathways drive periostial haemocyte aggregation and immune responses on the heart, thereby regulating the functional integration between the immune and circulatory systems of mosquitoes.The goal of this study was to identify genes and regulatory pathways that drive periostial haemocyte aggregation in . 2. 2.1E. coli or Gram(+) S. aureus . Three tissues were isolated at 4 h after treatment: (i) the heart containing periostial haemocytes, (ii) the haemolymph containing circulating haemocytes and (iii) the entire abdomen. Mosquitoes were assayed at 4 h after treatment because the number of periostial haemocytes approximately doubles within the first hour of infection, and plateaus by 4 h after infection [E. coli and S. aureus infection, respectively .To identify genes that may drive periostial haemocyte aggregation, we undertook an unbiased RNAseq approach where we sequenced the transcriptome of tissues from mosquitoes that were naive, injured or infected with Gram(–) GFP-nfection . In thesp < 0.05) in the heart and periostial haemocytes of mosquitoes infected with GFP-E. coli and S. aureus, respectively (d). Because periostial haemocyte aggregation is a dynamic immune response that occurs during any bacterial infection, we hypothesized that its core molecular drivers must be preferentially upregulated in periostial haemocytes following both E. coli and S. aureus infection. We found that 55 genes met this overlap criterion . Comparing the heart and periostial haemocyte RNAseq data to RNAseq data from (i) circulating haemocytes and (ii) the entire abdomen then revealed that infection upregulates more genes in the heart with periostial haemocytes than in the circulating haemocytes or the abdomen .RNAseq revealed that, relative to the heart and periostial haemocytes of naive and injured mosquitoes, 73 and 132 genes are upregulated more than fourfold (ectively d. Becausf), and strikingly, six are part of IMD and JNK pathway: the peptidoglycan recognition protein PGRP-LA [IAP2 [rel2 [cec3 [def1 [JNK3 [PGRP-LA and IAP2, and downstream elements from both branches of the pathway, with the classical IMD cascade represented by rel2, cec3 and def1, and the bifurcation into the JNK pathway represented by JNK3 [eater [TEP1, which is involved in phagocytosis and periostial haemocyte aggregation [Drosophila inhibit the IMD pathway [TGase3—negatively regulates periostial haemocyte aggregation and has infection-dependent effects on the heart rate [draper [TEP1 [CLIPB17, DDC, SRPN16, CLIPB11, PGRP-LB and upd3a—do not participate in a common pathway.We then scrutinized the reported or predicted function of the 55 candidate genes. Twelve have immune function f, and st PGRP-LA , the inhLA [IAP2 , the IMDP2 [rel2 ,29, the c3 [def1 ,31 and tf1 [JNK3 ,33. Mult by JNK3 ,34,35 . Five of these genes were shared between the heart with periostial haemocytes and the circulating haemocytes, but none of the 43 genes have a classical role in immunity or carry a function suggestive of involvement in haemocyte aggregation. Therefore, based on the RNAseq analyses, we hypothesized that the main driver of periostial haemocyte aggregation is the IMD and JNK pathway.Because the downregulation of genes could also impact haemocyte aggregation, we assessed the genes that were downregulated after both . 2.2rel2, and its negative regulator, caspar, and achieved RNAi-based silencing of 43% and 37%, respectively, compared to the dsR)bla. Moreover, because Tep1 is (i) activated by the Rel2 arm of the IMD pathway [rel2 and caspar RNAi mosquitoes. Silencing of rel2 did not alter TEP1 mRNA abundance but silencing of caspar increased it. Because cec3 and def1 are also activated by Rel2 [rel2 decreased cec3 and def1 mRNA abundance whereas silencing of caspar increased them . Overall, these data are consistent with the role that the Rel2 arm of the IMD pathway plays in regulating TEP1, cec3 and def1 expression.To determine whether the IMD pathway regulates periostial haemocyte aggregation, we synthesized double-stranded RNA (dsRNA) to target the IMD cascade transcription factor,  pathway ,45 and bla(Ap control mosquitoes (figures R)blabla. At 24 h post-infection, the effect of rel2 RNAi on periostial haemocyte aggregation was diminished. When we instead knocked down caspar, the number of periostial haemocytes increased by 39% in uninfected mosquitoes, and infection for 4 and 24 h increased the number of periostial haemocytes by 30% and 19%, respectively, relative to similarly treated dsR)bla. Knockdown of rel2 or caspar did not alter the number of non-periostial sessile haemocytes in uninfected mosquitoes or mosquitoes infected for 4 h. However, at 24 h following infection, caspar RNAi decreased non-periostial sessile haemocytes by 22%. These results demonstrate that the IMD pathway regulates periostial haemocyte aggregation while having a minimal effect on the rest of the sessile haemocytes.To determine whether the IMD pathway specifically affects periostial haemocytes and not sessile haemocytes in general, we counted the number of sessile haemocytes outside of the periostial regions in the tergum of abdominal segments 4 and 5 in the same mosquitoes examined for periostial haemocytes c. Knockd-E. coli fluorescence pixel area in the periostial regions of infected mosquitoes . At 4 h following infection, the GFP-E. coli pixel area was similar regardless of dsRNA treatment. At 24 h after infection, this area decreased in dsR)bla. Melanin was absent in uninfected mosquitoes and mosquitoes that were infected for 4 h. However, at 24 h following infection, rel2 RNAi increased melanin deposits at the periostial regions whereas caspar RNAi had no effect.Periostial haemocytes phagocytose bacteria, leading to their accumulation at the periostial regions ,6. To debacteria ,6, and oria [DDC ,47—was uE. coli and melanin in rel2 RNAi mosquitoes at 24 h after infection could be due to either (i) enhanced phagocytosis by periostial haemocytes or (ii) higher bacterial proliferation in the haemocoel, which places increased pressure on the phagocytosis response. To differentiate between these two scenarios, we measured the systemic GFP-E. coli infection intensity and observed that, at 4 h after infection, the bacterial intensity was similar for all dsRNA treatments, but at 24 h, knockdown of rel2 resulted in a higher infection intensity than treatment with dscaspar or dsR)bla(Ap (c). This suggests that rel2 is essential for proper bacterial killing in the haemocoel, and therefore, knocking it down increases infection intensity in a manner that leads to increased phagocytosis in the periostial regions. However, because silencing rel2 and caspar had opposite effects on periostial haemocyte aggregation at 4 h, a time when dsRNA treatment does not impact infection intensity, we conclude that the IMD pathway is a positive regulator of periostial haemocyte aggregation.The increased accumulation of GFP-bla(ApR) c. This sDrosophila haemocytes [f). Therefore, we tested the involvement of PGRP-LA in periostial haemocyte aggregation. PGRP-LA has three splice forms, so to knock it down, we synthesized dsPGRP-LA-RARB to target the RA and RB splice forms, and dsPGRP-LA-RC to target the RC splice form. Using these dsRNAs, we achieved RNAi-based silencing that ranged from 56% to 77%, compared to the dsR)bla. PGRP-LA knockdown with any dsRNA did not alter the number of periostial haemocytes, the number of non-periostial sessile haemocytes, melanin accumulation at the periostial regions or systemic infection intensity . However, knockdown of PGRP-LA-RC increased phagocytosis of GFP-E. coli in the periostial regions, which suggests that the RC splice form is involved in the phagocytosis response .PGRP-LC is the canonical activator of the IMD pathway , but PGRemocytes  and . Because of high sequence identity between JNK1 and JNK3, dsJNK1/3 simultaneously targeted both JNK genes. RNAi-based knockdown resulted in 33%, 44% and 31% reduction in mRNA abundance of JNK1, JNK3 and puc, respectively . Moreover, because the JNK pathway has also been implicated in the expression of TEP1 [TEP1, cec3 and def1 in JNK1/JNK3 and puc RNAi mosquitoes. Manipulating the JNK pathway did not have a clear impact on effector gene expression . The lack of clarity on how the JNK pathway controls the expression of TEP1, cec3 or def1 could be due to their co-regulation by other pathways, or a consequence of incomplete gene silencing of JNK1/3 and puc.We next tested whether the JNK pathway regulates periostial haemocyte aggregation. and JNK3 . Therefo of TEP1 , we measJNK1/3 and puc RNAi mosquitoes, relative to dsR)bla(Ap control mosquitoes (figures JNK1/3 and dsR)bla. Knockdown of puc, however, increased the number of periostial haemocytes 1.6-fold in uninfected mosquitoes, and 1.4- and 1.5-fold at 4 and 24 h following GFP-E. coli infection, respectively, relative to similarly treated dsR)bla. JNK1/3 RNAi mosquitoes showed a trend of fewer non-periostial sessile haemocytes than control mosquitoes regardless of infection status. Treatment with dspuc, however, increased the number of non-periostial sessile haemocytes for all treatments. This demonstrates that the JNK pathway positively regulates both periostial and non-periostial sessile haemocyte abundance.To assess whether the JNK pathway drives heart-associated immune responses, we compared the number and activity of periostial haemocytes in  figures  and 5. Reatments a,b. Knocsquitoes c. JNK1/3a; electronic supplementary material, figure S8). At 4 h following infection, the GFP-E. coli pixel area was similar regardless of dsRNA treatment. However, at 24 h following infection, periostial haemocytes in dspuc mosquitoes phagocytosed more GFP-E. coli than the dsR)bla. Melanin deposits were undetectable in uninfected mosquitoes and mosquitoes at 4 h post infection; however, at 24 h following GFP-E. coli infection, more melanin was present in dspuc mosquitoes than in dsR)bla enhanced phagocytosis by periostial haemocytes or (ii) higher bacterial proliferation in the haemocoel. To differentiate between these two scenarios, we quantified the systemic E. coli infection intensity and found that the bacterial intensity was similar regardless of dsRNA treatment (c). Therefore, this suggests that puc negatively regulates haemocyte adhesion and phagocytic activity, and demonstrates that the JNK pathway is a positive regulator of periostial immune responses.The difference in GFP-reatment c. Theref. 3Previous microarray and RNAseq analyses revealed genes and signalling pathways that are active in mosquito haemocytes –58, inclrel2, decreases infection-induced periostial haemocyte aggregation, whereas knockdown of the negative regulator of the IMD pathway, caspar, increases the number of periostial haemocytes. We hypothesize that the IMD pathway drives periostial haemocyte aggregation via two cascades that are also driving phagocytosis: the TEP1-TEP3-LRP1-CED6L and TEP4-BINT2-CED2L-CED5L pathways [TEP1 and TEP4 [rel2, bacteria proliferated uncontrollably during the later stages of infection. We hypothesize that knocking down rel2 initially suppresses periostial haemocyte aggregation, but as the infection progresses and the intensity increases, the necessity of periostial immune responses also increases, and this pressure recruits haemocytes to the heart and enhances their collective phagocytic activity. Overall, our data show that the IMD pathway drives periostial haemocyte aggregation during the early stages of infection and limits systemic infection intensity during the later stages of infection.The IMD pathway controls the production of antimicrobial peptides ,27, but pathways . The IMDand TEP4 ,31,68, aand TEP4 . Moreoveand TEP4 –71, and and TEP4 ,68,72–78puc, increases the number of periostial and adjacent sessile haemocytes. This suggests that the JNK pathway positively regulates haemocyte adhesion. Indeed, the Drosophila orthologue of mosquito JNK1 and JNK3, called basket, is involved in the formation of actin-rich and focal adhesion kinase-rich placodes in haemocytes [Drosophila, producing large lamellocytes that adhere and encapsulate parasitoid eggs [E. coli and melanin on the heart, suggesting that the JNK pathway positively regulates the phagocytosis and melanization responses. Indeed, JNK1 positively regulates melanization in mosquitoes that are refractory to malaria [JNK1 and JNK3 play in periostial haemocyte aggregation. However, simultaneous knockdown of both resulted in phenotypes that were opposite of puc knockdown, strongly suggesting that JNK1 or JNK3—or both—positively regulates periostial haemocyte aggregation.The JNK pathway modulates mosquito longevity, regulates oviposition, and limits infection with malaria parasites and viruses ,51,79–82emocytes . The JNKoid eggs . We foun malaria . We hypo malaria ,15. The  malaria ,80,86, a malaria ,87–89. ITEP1 [Drosophila, draper and the BINT2 orthologue (Integrin βν) function independently [Because both the IMD and JNK pathways induce the production of TEP1 ,33,68,80TEP1 ,35. Secoendently , and in endently ,67,70,86upd3a by periostial haemocytes, which is a ligand for the receptor of the JAK/STAT pathway [TEP1 [Bacteria, malaria parasites and fungal components all induce periostial haemocyte aggregation, and this process is structurally conserved across the entire insect lineage ,6,13. Th pathway . The Tol pathway ,93 and tay [TEP1 , which pay [TEP1 . Beyond ay [TEP1 . TherefoDrosophila, called Branchless, is expressed in the heart and pericardial cells, and regulates haemocyte differentiation in the lymph glands of the larval heart [Drosophila cardiac extracellular matrix (ECM) proteins, Pericardin and Laminin A, disrupts the formation of the cardiac ECM and lowers the number of haemocytes on the heart [To date, we have tested the involvement of several haemocyte-produced factors on periostial haemocyte aggregation. However, the heart itself may produce complementary factors that drive this immune response. For example, a fibroblast growth factor is highly regulated in the heart and periostial haemocytes. Its orthologue in al heart ,96. Morehe heart ,97. ThesDrosophila, transglutaminase negatively regulates the IMD pathway by (i) crosslinking the transcription factor, Relish, into a polymer and (ii) incorporating natural primary amines into the DNA binding site of Relish [A. gambiae, and found that TGase3—but not TGase1 or TGase2—negatively regulates periostial haemocyte aggregation during the early stages of infection and the sequestration of melanin by periostial haemocytes during the later stages of infection [TGase2 and TGase3 has infection-dependent effects on the heart rate [The mosquito genome encodes three transglutaminase genes, and our RNAseq experiment revealed that two of them are upregulated in the heart and periostial haemocytes following infection. In f Relish ,39. We rnfection . Moreoveart rate . CombineA. gambiae sheds light on how this medically important insect and other insects of societal importance fight the pathogens that invade them. Indeed, the TEP gene family, the Nimrod gene family  and the JNK pathway are conserved amongst insects [Heart-associated immune responses occur in insects from at least 16 different taxonomic orders, including insects that are of agricultural, urban and medical importance . Underst insects ,98–100,  insects –104. The. 4. 4.1Anopheles gambiae, Giles sensustricto , were maintained at 27°C, 75% relative humidity, and a 12 h : 12 h light : dark photoperiod [E. coli  and S. aureus (RN6390) were grown in Luria-Bertani (LB) and tryptic soy broth, respectively, at 37°C in a shaking incubator. Dilutions of the bacterial cultures were injected at the thoracic anepisternal cleft using a Nanoject III . The infection dose was determined by plating the cultures and counting the colony forming units (CFUs).toperiod . ExperimIn this study, a biological replicate is an independent experiment that uses mosquitoes from an independent egg batch. A technical replicate is an experimental resampling of a biological replicate. All data collected were included in the analysis; no data were excluded.. 4.2E. coli (approx. 75 779 CFUs) and (iv) infected by injecting 69 nl of S. aureus (approx. 39 451 CFUs). At 4 h after treatment, three tissues were isolated: (i) the heart with periostial haemocytes, (ii) haemolymph with circulating haemocytes and (iii) the entire abdomen containing all its internal organs and tissues . To isolate the heart, mosquitoes were bisected along the coronal plane in RNase-free PBS, and the heart with the periostial haemocytes was resected by severing the alary muscles and detaching it from the cuticle [Six-day-old mosquitoes were randomly divided into four groups: (i) naive (unmanipulated), (ii) injured by injecting 69 nl of sterile LB, (iii) infected by injecting 69 nl of GFP- cuticle . The abdTo isolate RNA from hearts or abdomens, samples were homogenized in TRIzol Reagent , extracted following the TRIzol protocol, and resuspended in Buffer RLT  with 2% 2-mercaptoethanol. The RNA was further purified using the RNeasy Micro Kit (Qiagen), DNase treated, and eluted in RNase-free water. To isolate RNA from circulating haemocytes, perfused haemolymph was collected in Buffer RLT and 2% 2-mercaptoethanol, and RNA was isolated using the RNeasy Micro Kit as above.a priori statistical method was used to pre-determine sample sizes. The number of RNAseq samples and biological replicates was determined prior to the initiation of the study, and was based on our determination that three biological replicates is a conservative approach to detect statistical differences (if any) in gene expression. Three biological replicates were conducted; each heart sample contained 72 hearts, each haemolymph sample contained perfusate from 108 mosquitoes, and each abdomen sample contained 36 abdomens.No The integrity and quantity of RNA was assessed on a 2100 Bioanalyzer  using an RNA 6000 Series II Nano kit for abdomen samples and a Pico kit for the heart and haemolymph samples. The library for sequencing was prepared using 1 µg of RNA and the NEBNext Ultra kit , according to the protocol for low-input samples. Library quality and concentration were assessed using a DNA 1000 Series II kit on the 2100 Bioanalyzer.. 4.3A. gambiae genome (AgamP4.7) by STAR [2 fold change ≥ 2 or log2 fold change ≤ –2 and the Benjamini–Hochberg adjusted p < 0.05. RNAseq results are presented in electronic supplementary material, dataset S1, which includes read counts and log2 fold changes. Using fourfold expression difference as the criterion, together with experimental replication and statistical validation, is a conservative approach that is proven to yield accurate and reliable results [https://www.ncbi.nlm.nih.gov) as SRA data PRJNA730047.All 36 samples—four treatments, three tissue types and three biological replicates—were sequenced across three lanes on an Illumina HiSeq 3000  at Vanderbilt University's Vantage facility. Reads were mapped to the  by STAR ,108. The by STAR . Genes w results . RNAseq . 4.4rel2, caspar, PGRP-LA, JNK1 and JNK3, and puc. Anopheles gambiae cDNA was amplified by PCR using gene-specific primers with T7 promoter tags . dsRNA was synthesis using the MEGAscript T7 Kit (Applied Biosystems) as described [R)bla containing the pET-46 plasmid as template  [Double-stranded RNA was synthesized for escribed ,15. As aown, NJ) ,15.E. coli (approx. 16 528 CFUs). Injured mosquitoes were not included in the experiments because injury does not induce periostial haemocyte aggregation [TEP1, cec3 and def1 was determined by qPCR [CT−ΔΔ method, with RpS7 as the reference and RpS17 as a control [Two- or three-day-old mosquitoes were intrathoracically injected 300 ng of dsRNA to initiate systemic gene silencing. Four days later, mosquitoes were divided into two groups for phenotypic analyses: (i) uninfected and (ii) infected with GFP-regation –6. RNAi  by qPCR . Briefly by qPCR ,15. qPCR control . Two to . 4.5Haemocytes were labelled with the Vybrant CM-DiI Cell-Labeling Solution (Invitrogen) as we described . Briefly. 4.6Z-stacks for bright field, red fluorescence (haemocytes), green fluorescence (GFP-E. coli) and blue fluorescence (nuclei) were acquired using a linear encoded Z-motor. Specific channels were selected and all images within a stack were combined into a two-dimensional image using the extended depth of focus (EDF) function.Specimens were imaged on a Nikon Eclipse Ni-E compound microscope connected to a Nikon Digital Sight DS-Qi1 camera and Advanced Research NIS Elements software . . 4.7Z-stack [R)bla. Data for RNAi phenotypic experiments are from individual mosquitoes, which were sampled across three to four biological replicates. The exception is experiments for PGRP-LA, where mosquitoes were sampled across two biological replicates.Haemocytes, labelled with both CM-DiI and Hoechst 33342, were counted manually by examining all images within a Z-stack . A cell Z-stack . PeriostZ-stack ,8. Data . 4.8E. coli at the periostial regions was calculated by measuring the area of pixels with intensities above a threshold that distinguished GFP-E. coli from background fluorescence [R)blabla(Ap mosquitoes as the reference. For rel2 and caspar RNAi mosquitoes, data were first log2 transformed to achieve normality, and then analysed by two-way ANOVA.Mosquitoes that were infected with tetracycline resistant GFP-


Alterations in neural pathways that regulate appetitive motivation may contribute to increased obesity risk in offspring born to mothers fed a high fat (HF) diet. However, current findings on the impact of maternal obesity on motivation in offspring are inconclusive, and there is no information about the long-lasting effects in aged animals. This study examined the longitudinal effect of perinatal and chronic postnatal HF intake on appetitive motivation in young and aged offspring. Female C57Bl/6 were fed either a control (C) or HF diet before mating through to lactation. At weaning, offspring were maintained on the C or HF diet, generating the following four diet groups: C/C, C/HF, HF/C, and HF/HF based on the pre/post weaning diet. At 6 months, motivation was higher in HF/C females, but lower in male and female C/HF and HF/HF mice. By 12 months, this difference was lost, as C-fed animals became less motivated, while motivation increased in HF-fed mice. The mRNA levels of dopamine receptor 1 and 2 increased with age, while cannabinoid receptor 1 and μ-opioid receptor expression remained stable or decreased in mesolimbic and mesocortical dopaminergic pathways. Results from this study suggest that perinatal and chronic postnatal HF feeding produced opposite effects on appetitive motivation in young adult offspring mice, which was also reflected in the shift in motivation over time. These results have significant implications for patterns of hedonic eating across the life course and the relative risk of obesity at different time points. The latest estimates of global obesity suggest that more than 37% of men and 30% of women are overweight or obese . Rates oTotal fats account for 30–45% of total dietary intake among adults across the USA and Europe ,16. CurrDopamine (DA) is a key regulator of motivation for hedonistic rewards and signals. This is mediated primarily by dopaminergic signaling within the mesolimbic and neocortical pathways. The mesolimbic pathway sends projections from the DA cell bodies in the ventral tegmental area (VTA) and innervates the nucleus accumbens (NAc), caudate–putamen (CPu), and limbic lobe, while the mesocortical pathway innervates the prefrontal cortex (PFC). Modulation of DA concentrations and activation of dopamine receptors 1 and 2 (D1R and D2R) within these pathways mediate different aspects of the “liking” and “wanting” of palatable foods. For example, anticipation and consumption of a food reward is associated with increased DA release in the PFC, but not in the NAc ,31. By cIn addition to DA, motivation for appetitive reward is also influenced by the endocannabinoid (eCB) and opioid systems. The eCBs contribute to the regulation of DA signaling within the mesocortical pathway via a negative feedback loop. In this loop, dopaminergic activation of GABAergic interneurons in the PFC suppresses the activity of glutamatergic neurons that innervate the VTA. The GABA receptor activation stimulates the release of eCBs, which retroactively diffuse across the synapse and bind to cannabinoid receptor 1 (CB1) on presynaptic GABA neurons, thereby blocking GABA release . The endSeparate to the modulation of DA, opioids also directly regulate food intake. Administration of opioid agonists into the NAc stimulates the ingestion of palatable foods, and μ-opioid receptor activation contributes to both the hedonistic and incentive value of rewarding stimuli . InteresPrevious studies have reported inconsistent effects of both pre- and postnatal HF feeding on DA, opioid, and eCB markers. For example, levels of D1R and D2R, CB1, and μ-opioid receptor have been found to be unchanged, upregulated, and decreased in the VTA, NAc, and/or PFC of HF-fed rodents and offspring born to mothers fed a HF diet ,39,40,41The purpose of this study was to characterize the effect of perinatal and postnatal HF diet exposure on appetitive motivation in male and female mice longitudinally across their lifespan and to characterize any related effects on the expression of DA, opioid, and eCB markers in associated brain areas.n = 34) or high fat ; Special Diet Services, UK, n = 39) diet for 4 weeks before mating (when dams were 18–27 weeks old). Dams were kept on the diet during gestation and lactation. Macro minerals, vitamins, and amino acid composition was matched between diets, as previously described  containing a protease inhibitor cocktail . Homogenates were centrifuged , and supernatants were frozen at −80 °C until further use. Proteins (15 μg) were separated by gel electrophoresis using 4–20% Tris-glycine gels , and then transferred onto a nitrocellulose membrane. Membranes were incubated overnight at 4 °C with anti-dopamine transporter antibody . Protein loading was confirmed by stripping and re-probing membranes with anti-glyceraldehyde-3-phosphate dehydrogenase . Data was generated from replicates of two blots. Densitometry of immunoblots was quantified using Image J  and calculated as an optical density ratio of protein levels normalized to GAPDH levels.Western blotting—Frozen brain tissues . Data represent mean ± SEM unless otherwise specified, and p < 0.05 was considered to be statistically significant.Statistical analysis—Data with After 4 weeks of feeding, HF-fed dams weighed significantly more than C-fed dams, and this was maintained during gestation and after weaning A. No difp = 0.05; To determine the long-term effects of peri- and postnatal obesity on motivation for an appetitive reward, male and female offspring were tested in the PR2 task at 6 months of age and again when they were 12 months old. In young animals, breakpoint was significantly lower in both male and female C/HF and HF/HF mice compared to C/C and HF/C animals, respectively A,B. LateAt 12 months of age, breakpoints were similar between all diet groups in both male and female offspring D–F. RewaPerformance over time showed a decrease in breakpoint between 6 and 12 months of age in male C/C and HF/C mice, although this was only significant in the HF/C group G. By conIn females, breakpoint remained stable between 6- and 12-month-old C/C animals, while changes in the other diet groups had a similar but non-significant pattern to that of male offspring H. LatencTo determine if differences in appetitive motivation were related to differential taste preferences, fully fed 9- and 15-month-old mice were given ad lib access to water, milkshake, and sucrose for 24 h after completion of the behavioural tasks. All animals drank significantly less water than sucrose or milkshake, regardless of age or diet group A–F. BothAt 15 months of age, male mice drank the same amount of milkshake and sucrose regardless of pre- and postnatal diet D. In femA summary of offspring body weight, motivation, and sucrose preference in young and aged offspring is shown in Cnr1, Drd1a, Drd2, and Oprm1 were evaluated in the NAc, CPu and PFC of 10- and 16-month-old offspring. Because DAT mRNA expression is largely limited to the ventral tegmental area (45), DAT protein expression was determined in the NAc, CPu and PFC by western blotting.To evaluate the impact of pre- and postnatal HF diet on expression of genes in neural circuits associated with reward and motivation, mRNA levels of Cnr1 expression in HF/HF males compared to C/HF animals (Cnr1 were observed between female diet groups (Drd1a, Drd2, and Oprm1 were also unchanged between diet groups (In 10-month-old mice, mRNA levels of the genes of interest were generally unaltered between diet groups. Within the NAc, there was a significant increase in  animals A. No dift groups A. Levelst groups . The DATt groups A, while t groups A.Drd2 expression was significantly increased in HF/HF males vs. HF/C males but did not differ between other male diet groups or between female groups (Drd1a, Cnr1, and Oprm1 in the CPu (Drd1a, Drd2, Cnr1, or Oprm1 expression between diet groups in the PFC (In the CPu, e groups B. Diet d the CPu . Similar the CPu B. There  the PFC . The DAT the PFC C.Drd1a were increased in the NAc of HF/C mice vs. C/C males (Drd2, Crn1, and Oprn1 in the NAc was not altered by diet in either male or female offspring (Drd1a, Drd2, Oprn1, or Crn1 levels between diet groups in the CPu of males or female mice (Drd1a levels were significantly elevated by 4–5-fold in C/HF mice compared to C/C females (Oprm1 and Cnr1 levels were unaltered between diet groups in the PFC (In 16-month-old animals, levels of /C males C. A simiffspring . No diffale mice . In the ale mice . In fema females E. The Op the PFC . The DAT the PFC A–C.Crn1, Drd1a, Drd2, Oprm1 (Drd2 levels were significantly higher in female C/C and HF/C mice compared to males in the same groups (Drd1a, Cnr1, and Oprm1 (Drd1a, Cnr1, or Oprm1 expression between male and female offspring in the PFC (Drd2 expression was increased in HF/HF females compared to HF/HF males; however, this bordered significance (p = 0.05, In 10-month-old animals, no differences were observed between male and female offspring in the expression of 2, Oprm1 , or DAT 2, Oprm1 A. In thee groups B. Howevend Oprm1 . Similar the PFC . The DrdDrd1a, Drd2, and Crn1 in the NAc were similar between male and female offspring (Oprm1 were significantly higher in HF/C females compared to males in this diet group (Drd1a than male counterparts (Drd2, Oprn1, or Crn1 in the aged CPu (Drd1a levels were significantly elevated by 4–5-fold in C/HF females compared to C/HF males (Drd2 levels was also noted in the PFC of C/HF females vs. C/HF males (Oprm1 and Cnr1 levels were unaltered between males and females (In 16-month-old animals, levels of ffspring , but levet group D. In theterparts . There waged CPu . In the HF males E. A signHF males F. Furthe females . There w females A–C.Drd1a levels in aged C/C males, but no significant differences in other male diet groups or between female groups (Drd2 levels were significantly upregulated in all aged male groups except C/C mice, including a 19-fold increase in the HF/HF mice (Drd2 expression also significantly increased by 5- to 11-fold between 10 and 16 months of age in females in all diet groups (Crn1 expression in the NAc was stable between ages, while Oprm1 levels were decreased in 16-month-old HF/C and HF/HF males, although this was only significant in HF/HF animals (Evaluation of changes in gene expression in the NAc between 10 and 16 months found a significant decrease in e groups A. By con/HF mice B. The Drt groups B. The Cr animals .Drd1a were significantly increased in all aged female animals and in all aged male diet groups except C/HF mice (Drd2 expression was also upregulated in 16-month-old C/C and HF/C males and in C/HF female mice (Oprm1 and Crn1. The Oprm1 levels were significantly decreased in 16-month-old C/C, C/HF, and HF/C males, with a similar non-significant trend observed in HF/HF males (Crn1 expression was also decreased in aged C/C and C/HF male mice (Oprm1 in all diet groups and a significant decrease in Crn1 in all aged diet groups, except in C/HF animals (In the CPu, levels of /HF mice C. The Drale mice D. An oppHF males E. The Crale mice F. In fem animals E,F.Drd2 levels were decreased by 3-fold in aged C/HF males and by 4-fold in C/C females (Drd1a mRNA; however, the differences were not statistically significant (Oprm1 levels in the PFC were stable across time (Crn1 expression was decreased in 16-month-old HF/HF males and in aged C/C, HF/C, and HF/HF females (p = 0.05, In the PFC,  females G. A siminificant . While Ooss time , Crn1 ex females H. The DA females A,B. In tDrd1a and Drd2 mRNA expression, and stable or decreased expression of Crn1 and Oprm1 in the mesolimbic and mesocortical DA pathways.Although maternal HF feeding is known to have lasting effects on the offspring brain, few studies have examined the impact of prenatal HF feeding on offspring behaviour beyond early adulthood. In addition, most postnatal diet-induced obesity studies use relatively short feeding periods that do not model human patterns of food consumption. This study examined the impact of perinatal exposure to HF diet alone and in combination with chronic postnatal HF intake on motivation in young and aged offspring and changes in the associated neurobiology. Our results suggest that young adult female, but not male, HF/C mice had higher levels of motivation than offspring born to C-fed dams. Male and female C/HF offspring had significantly decreased motivation, irrespective of maternal diet. However, these differences were no longer observed at 12-months of age, due to a general decrease in motivation in C-fed animals and an increase in motivation in HF-fed mice. In parallel, there was an age-related increase in Initial studies using rat dams fed a 20–30% fat diet before and/or during gestation and lactation found that young adult male offspring were more motivated for a food reward than offspring of dams fed a 5% fat diet ,24. HoweDrd2 mRNA levels were significantly higher in the CPu of 10-month-old HF/C females vs. HF/C males. Prolonged DA release can induce desensitization of D2R autoreceptors in the CPu and NAc, leading to burst firing of dopaminergic neurons [By contrast, appetitive motivation was not altered in male HF/C offspring. This provides further evidence of sexual dimorphism in the sensitivity of the fetal brain to maternal diet ,46,47,48 neurons ,52. MoreParental dietary habits are known to influence their children’s food preferences and eating patterns, which contribute to risk of obesity ,54. Our Drd1a, Drd2, Cnr1, and Oprm1 were similar in the PFC, NAc, and CPu of 10-month-old animals. As DAT regulates the reuptake of DA, the observed decrease in DAT in the NAc of C/HF and HF/HF mice may reflect a compensatory response to blunted DA transmission in response to a food reward. Previous findings have shown that cafeteria or HF feeding over a 15–16-week period attenuated DA release in the NAc and CPu in response to intake of low-calorie foods [Drd2 were unaltered in C/HF or HF/HF animals. However, HF feeding has been inconsistently associated with both up- and downregulation of D1R and D2R expression across the VTA, CPu, NAc, and PFC [Based on the role of DA, opioids, and eCBs in the regulation of motivation and consumption of rewarding stimuli ,31,37, wie foods ,58. Moreie foods . This is and PFC ,40, incl and PFC . Taken tOprm1 and Slc6a3, which encodes the DAT protein, in the NAc of offspring born to dams fed a control or cafeteria diet switched between 6 weeks and 3 months of age. Taken together, these data suggest that alterations in DA signaling in adolescent or young adult offspring born to HF-fed mothers may not persist into old age. Additional longitudinal studies using aged offspring are needed to confirm this hypothesis.Evidence from human studies suggest that dietary patterns established in early childhood are retained into adolescence and young adulthood ,59,60,61Contrary to the C-fed mice, motivation increased between 6- and 12-months of age in C/HF and HF/HF mice. Interestingly, the increase in breakpoint in aged HF/HF mice was approximately 50% of that in the C/HF animals. This suggests that adaptations to perinatal HF exposure more strongly counterbalance the effects of postnatal HF diet in older animals. Alterations in satiety hormones, resulting from either increased adiposity or diet-induced depletion, have been proposed to contribute to decreased appetitive motivation following prolonged HF feeding ,57. As tDrd1a and Drd2 gene expression in the CPu in C/HF and HF/HF animals may have contributed to the increased motivation in these animals. However, as this upregulation was observed across all diet groups, other factors must also contribute to the age-related difference in motivation between C and HF groups. Food restriction itself is known to increase the sensitivity of D2R activation and induce burst firing of DA neurons in response to cocaine [Coactivation of striatal D1R and D2R and hypersensitivity of D1R in the CPu contribute to sensitization to drugs of addiction ,70. Ther cocaine ,72. It a cocaine ,74. Alth cocaine , the extIn conclusion, results from this study suggest that perinatal and chronic postnatal HF feeding produced opposite effects on appetitive motivation in young adult offspring mice, and this was also reflected in the shift in motivation over time. The impact of maternal HF on offspring motivation varied across the lifespan and was influenced by postnatal diet, age, sex, and possibly frequency or order of reward presentation. These results also indicate that caution is needed when extrapolating findings of the impact of perinatal and/or short-term HF feeding on the persistence of offspring behaviour beyond early adulthood, and highlights the need for more studies into the long-term effects of perinatal diet across the lifespan of the offspring.


Primary hypertrophic osteoarthropathy (PHO) is a rare autosomal recessive inherited multi-system disorder characterized by a triad of pachydermia, periostosis, and clubbing. PHO was revealed to be caused by the HPGD gene producing 15-prostaglandin dehydrogenase and the SLCO2A1 gene expressing one kind of prostaglandin transporter. It is primarily a benign disorder, but coexisting myelofibrosis can lead to clinically significant cytopenias. In this case report, we present the case of a 21-year-old boy with a history of transfusion-dependent anemia and a progressive increase in transfusion requirements over the course of seven years. On basis of the patient’s medical history, family history, and clinical examination genetic testing was done. The patient was found to have homozygous c.664G>A (p. Gly222Arg) mutation in the SLCO2A1 gene; confirming the diagnosis of PHO. Primary hypertrophic osteoarthropathy (PHO), also known as Touraine Solente Gole (TSG) syndrome or Pachy-dermoperiostosis (PDP), is an inherited, autosomal recessive, rare, multi-system disorder characterized by a triad of thickening of the skin (pachydermia), abnormal growth of bone and connective tissue (periostosis) and digital clubbing (acropachia) . In 2008A 21-year-old male, a resident of Punjab, Pakistan, a tailor by profession, presented to a local hospital with a seven-year history of intermittent low-grade fever (not associated with rigors and chills), lethargy, generalized weakness, excessive sweating, body aches and joint pains affecting large joints i.e., bilateral wrist and knee joints. The patient had five siblings and the parents were in a consanguineous marriage . His blood complete picture (CP) showed, Hemoglobin (Hb): 4.7 g/dl, mean corpuscular volume (MCV): 96 fl, white blood cell (WBC): 3.1 x103/L, absolute neutrophil count (ANC): 2.6 x103/L, platelets: 47 x109/L on Sysmex Hematology Analyzer . The peripheral blood smear revealed anisocytosis, microcytosis, hypochromia, tear drop cells, two nucleated red blood cells (NRBC) per 100 WBCs, and a reticulocyte count of 1.5%. A diluted bone marrow sample was received with no evaluable fragment, whereas a trephine biopsy showed a hypercellular marrow with overall cellularity of ~80% and megakaryocytes forming clusters. A grade 2 marrow fibrosis (as per WHO marrow fibrosis grading), suggestive of overt myelofibrosis was also observed. Myelofibrosis was further examined for its underlying etiology. Polymerase chain reaction (PCR) for JAK2 (V617F), CALR (exon 9), cMPL (exon 10), and BCR-ABL1 were carried out, and no mutation was detected. The autoimmune profile  was negative. Bone scan showed diffusely increased tracer uptake in the appendicular skeleton. All these findings were consistent with myelofibrosis. On the basis of the patient’s history, family history, and clinical examination suspicion of hypertrophic osteoarthropathy (HOA) was raised and genetic testing was advised. The patient was found to be homozygous for the c.664G>A (p. Gly222Arg) mutation in SLCO2A1 gene. Additional variants of uncertain significance were found in genes COL10A1, IFT172, MESDC2, PCNT, RUNX2, SQSTM1, TONSL, TRIM37, WDR35, and WDR60. The family was also screened for the detected mutation and the results are shown in Table Based on all the above features, a diagnosis of primary idiopathic PHO with an unusually severe complication of myelofibrosis was determined. He was started on erythropoietin and cyclooxygenase-2 (COX 2) inhibitors, causing a reduction of transfusion dependency.PHO is a rare genetic disorder majorly characterized by abnormal bone formation and dermatosis and is poorly understood . The patAccording to the best of our knowledge, only 22 PHO cases with myelofibrosis have been reported to date, nine 40.9%) were reported from Asian countries % were re.Although PHO is primarily a benign disorder, but the coexisting myelofibrosis can lead to clinically significant cytopenia. To avoid missing the diagnosis, we recommend taking detailed history notes from such patients along with regular clinical and laboratory examinations on a regular basis.