In Vitro Imaging and Molecular Characterization of Ca2+ Flux Modulation by Nanosecond Pulsed Electric Fields.

In recent years, the application of pulsed electric fields with very short durations (nanoseconds) and extremely high amplitudes (MV/m) has been investigated for novel medical purposes. Various electric protocols have been explored for different objectives, including the utilization of fractionated pulse doses to enhance cell electrosensitization to the uptake of different markers or an increase in apoptosis. This study focused on the use of fluorescence imaging to examine molecular calcium fluxes induced by different fractionated protocols of short electric pulses in neuroblastoma (SH-SY5Y) and mesenchymal stem cells (HaMSCs) that were electroporated using nanosecond pulsed electric fields. In our experimental setup, we did not observe cell electrosensitization in terms of an increase in calcium flux following the administration of fractionated doses of nanosecond pulsed electric fields with respect to the non-fractionated dose. However, we observed the targeted activation of calcium-dependent genes (c-FOS, c-JUN, EGR1, NURR-1, ß3-TUBULIN) based on the duration of calcium flux, independent of the instantaneous levels achieved but solely dependent on the final plateau reached. This level of control may have potential applications in various medical and biological treatments that rely on calcium and the delivery of nanosecond pulsed electric fields.

Autism-related KLHL17 and SYNPO act in concert to control activity-dependent dendritic spine enlargement and the spine apparatus.

Dendritic spines, the tiny and actin-rich protrusions emerging from dendrites, are the subcellular locations of excitatory synapses in the mammalian brain that control synaptic activity and plasticity. Dendritic spines contain a specialized form of endoplasmic reticulum (ER), i.e., the spine apparatus, required for local calcium signaling and that is involved in regulating dendritic spine enlargement and synaptic plasticity. Many autism-linked genes have been shown to play critical roles in synaptic formation and plasticity. Among them, KLHL17 is known to control dendritic spine enlargement during development. As a brain-specific disease-associated gene, KLHL17 is expected to play a critical role in the brain, but it has not yet been well characterized. In this study, we report that KLHL17 expression in mice is strongly regulated by neuronal activity and KLHL17 modulates the synaptic distribution of synaptopodin (SYNPO), a marker of the spine apparatus. Both KLHL17 and SYNPO are F-actin-binding proteins linked to autism. SYNPO is known to maintain the structure of the spine apparatus in mature spines and contributes to synaptic plasticity. Our super-resolution imaging using expansion microscopy demonstrates that SYNPO is indeed embedded into the ER network of dendritic spines and that KLHL17 is closely adjacent to the ER/SYNPO complex. Using mouse genetic models, we further show that Klhl17 haploinsufficiency and knockout result in fewer dendritic spines containing ER clusters and an alteration of calcium events at dendritic spines. Accordingly, activity-dependent dendritic spine enlargement and neuronal activation (reflected by extracellular signal-regulated kinase (ERK) phosphorylation and C-FOS expression) are impaired. In addition, we show that the effect of disrupting the KLHL17 and SYNPO association is similar to the results of Klhl17 haploinsufficiency and knockout, further strengthening the evidence that KLHL17 and SYNPO act together to regulate synaptic plasticity. In conclusion, our findings unravel a role for KLHL17 in controlling synaptic plasticity via its regulation of SYNPO and synaptic ER clustering and imply that impaired synaptic plasticity contributes to the etiology of KLHL17-related disorders.

Occurrence of plasmid-mediated fosfomycin resistance (fos genes) among Escherichia coli isolates, Portugal.

OBJECTIVES: To evaluate the occurrence of plasmid-mediated fos genes among fosfomycin-resistant Escherichia coli isolates collected from patients in Lisbon, Portugal, and characterize the fos-positive strains. METHODS: A total of 19 186 E. coli isolates were prospectively collected between April 2022 and January 2023 from inpatients and outpatients at a private laboratory in Lisbon. Fosfomycin resistance was initially assessed by semi-automated systems and further confirmed by the disc diffusion method. Resistant isolates were investigated for plasmid-mediated fos genes (fosA1-fosA10, fosC and fosL1-fosL2) and extended-spectrum beta-lactamases (ESBLs) by PCR and sequencing. Multilocus sequence typing was performed to evaluate the clonal relationship among fos-carrying isolates. RESULTS: Out of the 19 186 E. coli isolates, 100 were fosfomycin-resistant (0.5%), out of which 15 carried a fosA-like gene (15%). The most prevalent fosfomycin-resistant determinant was fosA3 (n = 11), followed by fosA4 (n = 4). Among the 15 FosA-producing isolates, 10 co-produced an ESBL (67%), being either of CTX-M-15 (n = 8) or CTX-M-14 (n = 2) types. The fosA3 gene was carried on IncFIIA-, IncFIB-, and IncY-type plasmids, whereas fosA4 was always located on IncFIB-type plasmids. Most FosA4-producing isolates belonged to a single sequence type ST2161, whereas isolates carrying the fosA3 gene were distributed into nine distinct genetic backgrounds. CONCLUSION: The prevalence of fosfomycin-resistant E. coli isolates is still low in Portugal. Notably, 15% of fosfomycin-resistant isolates harbour a transferable fosA gene, among which there is a high rate of ESBL producers, turning traditional empirical therapeutical options used in Portugal (fosfomycin and amoxicillin-clavulanic acid) ineffective.

The Role of C1473G Polymorphism in Mouse Triptophan Hydroxylase 2 Gene in the Acute Effects of Ethanol on the c-fos Gene Expression and Metabolism of Biogenic Amines in the Brain.

Tryptophan hydroxylase 2 is a key enzyme in the synthesis of the neurotransmitter serotonin, which plays an important role in the regulation of behavior and various physiological functions. We studied the effect of acute ethanol administration on the expression of the early response c-fos gene and metabolism of serotonin and catecholamines in the brain structures of B6-1473C and B6-1473G congenic mouse strains differing in the single-nucleotide substitution C1473G in the Tph2 gene and activity of the encoded enzyme. Acute alcoholization led to a significant upregulation of the c-fos gene expression in the frontal cortex and striatum of B6-1473G mice and in the hippocampus of B6-1473C mice and caused a decrease in the index of serotonin metabolism in the nucleus accumbens in B6-1473C mice and in the hippocampus and striatum of B6-1473G mice, as well as to the decrease in the norepinephrine level in the hypothalamus of B6-1473C mice. Therefore, the C1473G polymorphism in the Tph2 gene has a significant effect of acute ethanol administration on the c-fos expression pattern and metabolism of biogenic amines in the mouse brain.

Quanty-cFOS, a Novel ImageJ/Fiji Algorithm for Automated Counting of Immunoreactive Cells in Tissue Sections.

Analysis of neural encoding and plasticity processes frequently relies on studying spatial patterns of activity-induced immediate early genes' expression, such as c-fos. Quantitatively analyzing the numbers of cells expressing the Fos protein or c-fos mRNA is a major challenge owing to large human bias, subjectivity and variability in baseline and activity-induced expression. Here, we describe a novel open-source ImageJ/Fiji tool, called 'Quanty-cFOS', with an easy-to-use, streamlined pipeline for the automated or semi-automated counting of cells positive for the Fos protein and/or c-fos mRNA on images derived from tissue sections. The algorithms compute the intensity cutoff for positive cells on a user-specified number of images and apply this on all the images to process. This allows for the overcoming of variations in the data and the deriving of cell counts registered to specific brain areas in a highly time-efficient and reliable manner. We validated the tool using data from brain sections in response to somatosensory stimuli in a user-interactive manner. Here, we demonstrate the application of the tool in a step-by-step manner, with video tutorials, making it easy for novice users to implement. Quanty-cFOS facilitates a rapid, accurate and unbiased spatial mapping of neural activity and can also be easily extended to count other types of labelled cells.

Toll-Like Receptor Signaling in the Pathogenesis of Chronic Dacryocystitis: Implication of c-FOS Transcription Factor and its Downstream Effector Chemokine Genes CCL2, CCL4, CXCL3, CXCR4 with a Shift of the M1/M2 Macrophage Phenotype.

INTRODUCTION: TLRs are fundamental elements in the orchestration of the innate immune system. These receptors seem to be responsible for the inflammation and fibrosis in chronic dacryocystitis. The aim of the present study was to investigate the role of the toll-Like receptors (TLR2 and TLR4) signaling pathway and its downstream effector chemokine genes in the pathogenesis of chronic dacryocystitis. METHODS: This study was conducted on 20 patients diagnosed with chronic dacryocystitis and underwent external dacryocystorhinostomy. Estimation of gene expression of TLR2, TLR4, CCL2, CCL4, CXCL3, CXCR4, and c-FOS genes in the lacrimal sac tissues was performed together with the assessment of the inflammatory markers TNFα, IL-1ß, IFN-γ, and IL-22. Histopathological examination of the lacrimal sac walls using hematoxylin and eosin (H&E) stain, in addition to immunohistochemical staining of the CD68 and CD163 macrophage markers, was also performed. RESULTS: Our results showed that TLR2, TLR4, and c-FOS gene expressions were significantly increased in the chronic dacryocystitis group with a subsequent increase in their downstream effector chemokine genes CCL2, CCL4, and CXCL3. This up-regulation of genes was accompanied by macrophage shift of polarization toward the M1 pro-inflammatory phenotype (increased CD68 and decreased CD163 expression), leading to increased levels of the pro-inflammatory cytokines (TNF- α, IL-1ß and IFN-γ) and decreased anti-inflammatory marker IL-22 with chronic dacryocystitis. CONCLUSION: It is essential to fine-tune TLR activation through emerging therapeutic approaches. Targeting TLR signaling at the level of receptors or downstream adaptor molecules represents a new challenge for treating chronic dacryocystitis.

HSF1 Can Prevent Inflammation following Heat Shock by Inhibiting the Excessive Activation of the ATF3 and JUN&FOS Genes.

Heat Shock Factor 1 (HSF1), a transcription factor frequently overexpressed in cancer, is activated by proteotoxic agents and participates in the regulation of cellular stress response. To investigate how HSF1 level affects the response to proteotoxic stress, we integrated data from functional genomics analyses performed in MCF7 breast adenocarcinoma cells. Although the general transcriptional response to heat shock was impaired due to HSF1 deficiency (mainly chaperone expression was inhibited), a set of genes was identified, including ATF3 and certain FOS and JUN family members, whose stress-induced activation was stronger and persisted longer than in cells with normal HSF1 levels. These genes were direct HSF1 targets, suggesting a dual (activatory/suppressory) role for HSF1. Moreover, we found that heat shock-induced inflammatory response could be stronger in HSF1-deficient cells. Analyses of The Cancer Genome Atlas data indicated that higher ATF3, FOS, and FOSB expression levels correlated with low HSF1 levels in estrogen receptor-positive breast cancer, reflecting higher heat shock-induced expression of these genes in HSF1-deficient MCF7 cells observed in vitro. However, differences between the analyzed cancer types were noted in the regulation of HSF1-dependent genes, indicating the presence of cell-type-specific mechanisms. Nevertheless, our data indicate the existence of the heat shock-induced network of transcription factors (associated with the activation of TNFα signaling) which includes HSF1. Independent of its chaperone-mediated cytoprotective function, HSF1 may be involved in the regulation of this network but prevents its overactivation in some cells during stress.

FOS gene associated immune infiltration signature in perivascular adipose tissues of abdominal aortic aneurysm.

Abdominal aortic aneurysms (AAA) are pathological dilations in local aortic wall. The inflammatory infiltrates of the perivascular adipose tissue (PAT) surrounding AAAs were associated with AAAs and have been shown to contribute vascular pathology. However, the mechanism by which PAT inflammation contributes to vascular pathology in AAA remains to be clarified. This study aimed to explore the association between immune cell infiltration and key gene expression profile in PAT of AAA. For that, a gene expression dataset of human dilated perivascular adipose tissue (dPAT), non-dilated perivascular adipose tissue (ndPAT), subcutaneous abdominal fat (SAF) and omental-visceral fat (OVF) samples, as well as another microarray dataset of the abdominal perivascular adipose tissue in peripheral artery disease patients were downloaded from GEO database for analysis in this study. The CIBERSORT algorithm, weighted gene co-expression network analysis (WGCNA) and LASSO algorithm were used for the identification of immune infiltration, immune-related genes and the development of diagnostic signature. Our data discovered a significant higher proportion of activated mast cells and follicular helper T (Tfh) cells in dPAT than ndPAT, OVT and SAF samples. Moreover, AP-1 family members (FOS, FOSB, ATF3, JUN and JUNB) were found to compose the hub genes of purple module in WGCNA. Among them, FOS gene acts as a higher efficient marker to discriminate dPAT from ndPAT, OVT and SAF in AAA. Meanwhile, the expression profiles of the AP-1 family members are all significantly positive correlated with activated mast cell, plasma cell and Tfh cell infiltration in dPAT of AAA. Therefore, in the PAT surrounding AAA, the signature of inflammatory infiltration might be represented by a FOS-dominated cell network consist of activated mast cell, plasma cell and Tfh cell. Given the complicated etiology of AAA, our results are likely to shed new light on the pathophysiologic mechanism of AAA influenced by the local dPAT.

Dense functional and molecular readout of a circuit hub in sensory cortex.

Although single-cell transcriptomics of the neocortex has uncovered more than 300 putative cell types, whether this molecular classification predicts distinct functional roles is unclear. We combined two-photon calcium imaging with spatial transcriptomics to functionally and molecularly investigate cortical circuits. We characterized behavior-related responses across major neuronal subclasses in layers 2 or 3 of the primary somatosensory cortex as mice performed a tactile working memory task. We identified an excitatory intratelencephalic cell type, Baz1a, that exhibits high tactile feature selectivity. Baz1a neurons homeostatically maintain stimulus responsiveness during altered experience and show persistent enrichment of subsets of immediately early genes. Functional and anatomical connectivity reveals that Baz1a neurons residing in upper portions of layers 2 or 3 preferentially innervate somatostatin-expressing inhibitory neurons. This motif defines a circuit hub that orchestrates local sensory processing in superficial layers of the neocortex.

Fos regulates macrophage infiltration against surrounding tissue resistance by a cortical actin-based mechanism in Drosophila.

The infiltration of immune cells into tissues underlies the establishment of tissue-resident macrophages and responses to infections and tumors. Yet the mechanisms immune cells utilize to negotiate tissue barriers in living organisms are not well understood, and a role for cortical actin has not been examined. Here, we find that the tissue invasion of Drosophila macrophages, also known as plasmatocytes or hemocytes, utilizes enhanced cortical F-actin levels stimulated by the Drosophila member of the fos proto oncogene transcription factor family (Dfos, Kayak). RNA sequencing analysis and live imaging show that Dfos enhances F-actin levels around the entire macrophage surface by increasing mRNA levels of the membrane spanning molecular scaffold tetraspanin TM4SF, and the actin cross-linking filamin Cheerio, which are themselves required for invasion. Both the filamin and the tetraspanin enhance the cortical activity of Rho1 and the formin Diaphanous and thus the assembly of cortical actin, which is a critical function since expressing a dominant active form of Diaphanous can rescue the Dfos macrophage invasion defect. In vivo imaging shows that Dfos enhances the efficiency of the initial phases of macrophage tissue entry. Genetic evidence argues that this Dfos-induced program in macrophages counteracts the constraint produced by the tension of surrounding tissues and buffers the properties of the macrophage nucleus from affecting tissue entry. We thus identify strengthening the cortical actin cytoskeleton through Dfos as a key process allowing efficient forward movement of an immune cell into surrounding tissues.

Chronic exposure to cyclohexane induces stereotypic circling, hyperlocomotion, and anxiety-like behavior associated with atypical c-Fos expression in motor- and anxiety-related brain regions.

Recreational abuse of solvents continues, despite cyclohexane (CHX) is used as a safe replacement in gasoline or adhesive formulations. Increasing evidence indicates that CHX inhalation affects brain functioning; however, scanty information is available about its effects on behavior and brain activity upon drug removal. In this study, we used CD1 adult mice to mimic an intoxication period of recreational drugs for 30 days. During the CHX exposure (~30,000 ppm), we analyzed exploratory and biphasic behaviors, stereotypic circling, and locomotion. After CHX removal (24 h or a month later), we assessed anxiety-like behaviors and quantified c-Fos cells in motor- and anxiety-related brain regions. Our findings indicate that the repeated inhalation of CHX produced steady hyperactivity and reduced ataxia, sedation, and seizures as the exposure to CHX progressed. Also, CHX decreased grooming and rearing behaviors. In the first week of CHX inhalation, a stereotypic circling behavior emerged, and locomotion increased gradually. One month after CHX withdrawal, mice showed low activity in the center zone of the open field and more buried marbles. Twenty-four hours after CHX removal, c-Fos expression was low in the dorsal striatum, ventral striatum, motor cortex, dorsomedial prefrontal cortex, basolateral amygdala, lateral hypothalamus, and ventral hippocampus. One month later, c-Fos expression remained low in the ventral striatum and lateral hypothalamus but increased in the dorsomedial prefrontal cortex and primary motor cortex. This study provides a comprehensive behavioral characterization and novel histological evidence of the CHX effects on the brain when is administered in a recreational-like mode.

2E-Decene-4,6-diyn-1-ol-acetate inhibits osteoclastogenesis through mitogen-activated protein kinase-c-Fos-NFATc1 signalling pathways.

An imbalance of osteoclasts and osteoblasts can result in a variety of bone-related diseases, including osteoporosis. Thus, decreasing the activity of osteoclastic bone resorption is the main therapeutic method for treating osteoporosis. 2E-Decene-4, 6-diyn-1-ol-acetate (DDA) is a natural bioactive compound with anti-inflammatory and anti-cancer properties. However, its effects on osteoclastogenesis are unknown. Murine bone marrow-derived macrophages (BMMs) or RAW264.7 cells were treated with DDA, followed by evaluation of cell viability, RANKL-induced osteoclast differentiation, and pit formation assay. Effects of DDA on RANKL-induced phosphorylation of MAPKs were assayed by western blot analysis. Expression of osteoclast-specific genes was examined with reverse transcription-PCR (RT-PCR) and western blot analysis. In this study, DDA significantly inhibited RANKL-induced osteoclast differentiation in RAW264.7 cells as well as in BMMs without cytotoxicity. DDA also strongly blocked the resorbing capacity of BMM on calcium phosphate-coated plates. DDA inhibited RANKL-induced phosphorylation of ERK, JNK and p38 MAPKs, as well as expression of c-Fos and NFATc1, which are essential transcription factors for osteoclastogenesis. In addition, DDA decreased expression levels of osteoclastogenesis-specific genes, including matrix metalloproteinase-9 (MMP-9), tartrate-resistant acid phosphatase (TRAP), and receptor activator of NF-κB (RANK) in RANKL-induced RAW264.7 cells. Collectively, these findings indicated that DDA attenuates RANKL-induced osteoclast formation by suppressing the MAPKs-c-Fos-NFATc1 signalling pathway and osteoclast-specific genes. These results indicate that DDA may be a potential candidate for bone diseases associated with abnormal osteoclast formation and function.

MiR-744 Functions as an Oncogene Through Direct Binding to c-Fos Promoter and Facilitates Non-small Cell Lung Cancer Progression.

Metastasis is the leading cause of death in non-small cell lung cancer (NSCLC) patients. Previously, we reported that miR-744 exerted proto-oncogenic function in nasopharyngeal carcinoma, but the role of miR-744 during NSCLC development has not been established. We focused on the function and molecular mechanism of miR-744 in NSCLC. The clinical cohort data from TCGA were analyzed for the correlation of miR-744 and outcomes in NSCLC patients. Gain- and loss-of-function experiment was performed by transfection with miR-744 agomir or antagomir in NSCLC cell lines. The expression of mRNA and protein were analyzed by qPCR assays and Western blotting respectively. Cellular proliferation, migration, and invasion were analyzed by CCK8 assays, wound healing, and transwell assays, respectively. Promoter activities and gene transcription were analyzed by luciferase reporter assays. Xenograft model was applied for in vivo study. High miR-744 expression correlated with lymph node metastasis and poor prognosis in NSCLC patient. MiR-744 aggravated the growth, invasion, and metastasis of NSCLC cells eventually induced the malignant phenotype and promotes radio/chemoresistance in vitro. The -1195 to -1227 and -298 to -323 bp upstream of c-FOS gene was observed to bind with miR-744. Lastly, miR-744 acted as a tumor promoter in lung cancer growth and metastasis in vivo. Taken together, our results indicated that miR-744 up-regulated c-Fos by binding with its promoter contributed to development of NSCLC cells malignant phenotype. Our findings highlight the potential value of miR-744, which may serve as a possible therapeutic target for NSCLC.

Arginine vasopressin in the medial amygdala causes greater post-stress recruitment of hypothalamic vasopressin neurons.

Arginine vasopressin (AVP) is expressed in both hypothalamic and extra-hypothalamic neurons. The expression and role of AVP exhibit remarkable divergence between these two neuronal populations. Polysynaptic pathways enable these neuronal groups to regulate each other. AVP neurons in the paraventricular nucleus of the hypothalamus increase the production of adrenal stress hormones by stimulating the hypothalamic-pituitary-adrenal axis. Outside the hypothalamus, the medial amygdala also contains robust amounts of AVP. Contrary to the hypothalamic counterpart, the expression of extra-hypothalamic medial amygdala AVP is sexually dimorphic, in that it is preferentially transcribed in males in response to the continual presence of testosterone. Male gonadal hormones typically generate a negative feedback on the neuroendocrine stress axis. Here, we investigated whether testosterone-responsive medial amygdala AVP neurons provide negative feedback to hypothalamic AVP, thereby providing a feedback loop to suppress stress endocrine response during periods of high testosterone secretion. Contrary to our expectation, we found that AVP overexpression within the posterodorsal medial amygdala increased the recruitment of hypothalamic AVP neurons during stress, without affecting the total number of AVP neurons or the number of recently activated neurons following stress. These observations suggest that the effects of testosterone on extra-hypothalamic AVP facilitate stress responsiveness through permissive influence on the recruitment of hypothalamic AVP neurons.

Histone modification of pain-related gene expression in spinal cord neurons under a persistent postsurgical pain-like state by electrocautery.

Chronic postsurgical pain (CPSP) is a serious problem. We developed a mouse model of CPSP induced by electrocautery and examined the mechanism of CPSP. In this mouse model, while both incision and electrocautery each produced acute allodynia, persistent allodynia was only observed after electrocautery. Under these conditions, we found that the mRNA levels of Small proline rich protein 1A (Sprr1a) and Annexin A10 (Anxa10), which are the key modulators of neuropathic pain, in the spinal cord were more potently and persistently increased by electrocautery than by incision. Furthermore, these genes were overexpressed almost exclusively in chronic postsurgical pain-activated neurons. This event was associated with decreased levels of tri-methylated histone H3 at Lys27 and increased levels of acetylated histone H3 at Lys27 at their promoter regions. On the other hand, persistent allodynia and overexpression of Sprr1a and Anxa10 after electrocautery were dramatically suppressed by systemic administration of GSK-J4, which is a selective H3K27 demethylase inhibitor. These results suggest that the effects of electrocautery contribute to CPSP along with synaptic plasticity and epigenetic modification.

Olfactory learning and memory in the greater short-nosed fruit bat Cynopterus sphinx: the influence of conspecifics distress calls.

This study was designed to test whether Cynopterus sphinx distress calls influence olfactory learning and memory in conspecifics. Bats were exposed to distress calls/playbacks (PBs) of distress calls/modified calls and were then trained to novel odors. Bats exposed to distress calls/PBs made significantly fewer feeding attempts and bouts of PBs exposed to modified calls, which significantly induced the expression of c-Fos in the caudomedial neostriatum (NCM) and the amygdala compared to bats exposed to modified calls and trained controls. However, the expression of c-Fos in the hippocampus was not significantly different between the experimental groups. Further, protein phosphatase-1 (PP-1) expression was significantly lower, and the expression levels of E1A homologue of CREB-binding protein (CBP) (P300), brain-derived neurotrophic factor (BDNF) and its tyrosine kinase B1 (TrkB1) receptor were significantly higher in the hippocampus of control/bats exposed to modified calls compared to distress calls/PBs of distress call-exposed bats. Exposure to the call possibly alters the reciprocal interaction between the amygdala and the hippocampus, accordingly regulating the expression levels of PP1, P300 and BDNF and its receptor TrkB1 following training to the novel odor. Thus, the learning and memory consolidation processes were disrupted and showed fewer feeding attempts and bouts. This model may be helpful for understanding the contributions of stressful social communications to human disorders.

CCL2 associated with CD38 expression during ex vivo expansion in human cord blood-derived hematopoietic stem cells.

To date, different experimental strategies have been developed for the ex vivo expansion of human hematopoietic stem cells (HSCs) for clinical applications. However, differences in the genomic function of expanded HSCs under different culture systems remain unclear. In this study, we compared the gene expression profiles of HSCs in ex vivo expanded serum (10% FBS, fetal bovine serum) and serum-free culture systems and analyzed the molecular functions of differentially expressed genes using microarray chips. We identified 839 differentially expressed genes between the two culture systems. These genes were enriched in the TNF -regulated inflammatory pathway in an FBS culture system. In addition, the mRNA expression of CCL2 (C-C motif chemokine ligand 2), TNF (tumor necrosis factor) and FOS (FBJ murine osteosarcoma viral oncogene homolog) was validated by RT-qPCR. Our data revealed that ex vivo expansion of HSCs using the FBS culture system induces an inflammatory response and high CD38 expression, indicating that this system might activate an inflammatory pathway and induce expression of the cancer marker CD38 during ex vivo expansion of HSCs. This study provides a transcriptional profile and new insights into the genomic functions of HSCs under different expanded cultures.

Dynamic Change of Endocannabinoid Signaling in the Medial Prefrontal Cortex Controls the Development of Depression After Neuropathic Pain.

Many patients with chronic pain conditions suffer from depression. The mechanisms underlying pain-induced depression are still unclear. There are critical links of medial prefrontal cortex (mPFC) synaptic function to depression, with signaling through the endocannabinoid (eCB) system as an important contributor. We hypothesized that afferent noxious inputs after injury compromise activity-dependent eCB signaling in the mPFC, resulting in depression. Depression-like behaviors were tested in male and female rats with traumatic neuropathy [spared nerve injury (SNI)], and neuronal activity in the mPFC was monitored using the immediate early gene c-fos and in vivo electrophysiological recordings. mPFC eCB Concentrations were determined using mass spectrometry, and behavioral and electrophysiological experiments were used to evaluate the role of alterations in eCB signaling in depression after pain. SNI-induced pain induced the development of depression phenotypes in both male and female rats. Pyramidal neurons in mPFC showed increased excitability followed by reduced excitability in the onset and prolonged phases of pain, respectively. Concentrations of the eCBs, 2-arachidonoylglycerol (2-AG) in the mPFC, were elevated initially after SNI, and our results indicate that this resulted in a loss of CB1R function on GABAergic interneurons in the mPFC. These data suggest that excessive release of 2-AG as a result of noxious stimuli triggers use-dependent loss of function of eCB signaling leading to excessive GABA release in the mPFC, with the final result being behavioral depression.SIGNIFICANCE STATEMENT Pain has both somatosensory and affective components, so the complexity of mechanisms underlying chronic pain is best represented by a biopsychosocial model that includes widespread CNS dysfunction. Many patients with chronic pain conditions develop depression. The mechanism by which pain causes depression is unclear. Although manipulation of the eCB signaling system as an avenue for providing analgesia per se has not shown much promise in previous studies. An important limitation of past research has been inadequate consideration of the dynamic nature of the connection between pain and depression as they develop. Here, we show that activity-dependent synthesis of eCBs during the initial onset of persistent pain is the critical link leading to depression when pain is persistent.

Mu-opioid receptor agonism differentially alters social behaviour and immediate early gene expression in male adolescent rats prenatally exposed to valproic acid versus controls.

Mu-opioid receptors (MOPs) mediate and modulate social reward and social interaction. However, few studies have examined the functionality of this system in rodent models of social impairment. Deficits in social motivation and cognition are observed in rodents following pre-natal exposure to the anti-epileptic valproic acid (VPA), however it is not known whether MOP functionality is altered in these animals. The present study examined the effects of acute administration of the prototypical MOP agonist morphine (1 mg/kg) on social behavioural responding in the 3-chamber test and immediate early gene expression in adolescent rats (postnatal day 28-43) prenatally exposed to VPA vs saline-exposed controls. Pharmacokinetic analysis of morphine concentration, MOP binding and expression were also examined. The data revealed that sociability and social novelty preference in the 3-chamber test were reduced in rats prenatally exposed to VPA compared to saline-exposed control counterparts. Morphine reduced both sociability and social novelty preference behaviour in saline-, but not VPA-, exposed rats. Analysis of immediate early gene expression revealed that morphine reduced the expression of cfos in the prefrontal cortex of both saline- and VPA-exposed rats and reduced expression of cfos and junb in the hippocampus of VPA-exposed rats only. Pharmacokinetic analysis revealed similar concentrations of morphine in the plasma and brain of both saline- and VPA-exposed rats and similar thalamic MOP occupancy levels. Gene and protein expression of MOP in prefrontal cortex and hippocampus did not differ between saline and VPA-exposed rats. These data indicate differential effects of morphine on social responding and immediate early gene expression in the hippocampus of VPA-exposed rats compared with saline-exposed controls. This study provides support for altered MOP functionality in rats prenatally exposed to VPA, which may underlie the social deficits observed in the model.