The CXCR4 chemokine receptor has an important role in cancer cell metastasis. The CXCR4 antagonist, AMD3100, has limited efficacy in controlling metastasis. HuR, an RNA-binding protein, regulates CXCR4 in cancer cells. We therefore investigated whether targeting HuR using a siRNA-based nanoparticle plus AMD3100 would suppress CXCR4 and inhibit lung cancer metastasis. We treated human H1299 lung cancer cells with HuR-specific siRNA contained in a folate-targeted lipid nanoparticle (HuR-FNP) plus AMD3100, and compared this with AMD3100 alone, HuR-FNP alone and no treatment. HuR-FNP plus AMD3100 treatment produced a G1 phase cell cycle arrest and reduced cell viability above and beyond the effects of AMD3100 alone. HuR and CXCR4 mRNA and protein expression levels were markedly reduced in all treatment groups. Phosphorylated (p) AKT(S473) protein was also reduced. P27 protein expression increased with HuR-FNP and combination treatment. Promoter-based reporter studies showed that the combination inhibited CXCR4 promoter activity more than did either treatment alone. Cell migration and invasion was significantly reduced with all treatments; the combination provided the most inhibition. Reduced matrix metalloprotease (MMP)-2 and -9 expression was associated with reduced invasion in all treatment groups. Thus, we found that combined HuR and CXCR4 targeting effectively controlled lung cancer metastasis.

Manganese superoxide dismutase (MnSOD) promotes invasive and migratory activities by upregulating Forkhead box protein M1 (FoxM1) expression. The present study investigated whether modulation of MnSOD and FoxM1 expression was responsible for the antitumor effects of genistein on cancer stem-like cells (CSLCs) derived from non-small cell lung cancer cells (NSCLCs). Spheroids prepared from H460 or A549 cells were defined as lung cancer stem-like cells (LCSLCs) and were treated with genistein. The Cell Counting Kit-8 assay was performed to assess human lung fibroblast IMR-90 cell proliferation, as well as NSCLC H460 and A549 cell proliferation following treatment with genistein. MnSOD, FoxM1, cluster of differentiation (CD)133, CD44, BMI1 proto-oncogene, polycomb ring finger (Bmi1) and Nanog homeobox (Nanog) protein expression levels were examined via western blotting. The sphere formation assay was conducted to evaluate LCSLC self-renewal potential, and LSCLC migratory and invasive activities were analyzed using the wound healing and Transwell invasion assays, respectively. Knockdown and overexpression of MnSOD and FOXM1 via short hairpin-RNA or cDNA transfection were performed. The results indicated that genistein (80 and 100 µM) suppressed H460 and A549 cell viability compared with IMR-90 cells. Sub-cytotoxic concentrations of genistein (20 and 40 µM) inhibited sphere formation activity and decreased the protein expression levels of CD133, CD44, Bmi1 and Nanog in LCSLCs compared with the control group. Genistein also suppressed the migratory and invasive activities of LCSLCs compared with the control group. MnSOD and FoxM1 overexpression antagonized the effects of genistein (40 µM), whereas MnSOD and FoxM1 knockdown enhanced the inhibitory effects of genistein (20 µM) on CSLC characteristics of LCSLCs. Overall, the results suggested that genistein suppressed lung cancer cell CSLC characteristics by modulating MnSOD and FoxM1 expression levels.

Most of the fascinating phenomena studied in cell biology emerge from interactions among highly organized multimolecular structures embedded into complex and frequently dynamic cellular morphologies. For the exploration of such systems, computer simulation has proved to be an invaluable tool, and many researchers in this field have developed sophisticated computational models for application to specific cell biological questions. However, it is often difficult to reconcile conflicting computational results that use different approaches to describe the same phenomenon. To address this issue systematically, we have defined a series of computational test cases ranging from very simple to moderately complex, varying key features of dimensionality, reaction type, reaction speed, crowding, and cell size. We then quantified how explicit spatial and/or stochastic implementations alter outcomes, even when all methods use the same reaction network, rates, and concentrations. For simple cases, we generally find minor differences in solutions of the same problem. However, we observe increasing discordance as the effects of localization, dimensionality reduction, and irreversible enzymatic reactions are combined. We discuss the strengths and limitations of commonly used computational approaches for exploring cell biological questions and provide a framework for decision making by researchers developing new models. As computational power and speed continue to increase at a remarkable rate, the dream of a fully comprehensive computational model of a living cell may be drawing closer to reality, but our analysis demonstrates that it will be crucial to evaluate the accuracy of such models critically and systematically.

Whether DNA methyltransferase 1 (DNMT1)/miR-34a/FoxM1 signaling promotes the stemness of liver cancer stem cells (LCSCs) remains unclear. This study aimed to assess whether methylation-based silencing of miR-34a by DNMT1 contributes to stemness features via FoxM1 upregulation in LCSCs.

Corticotropin-releasing factor (CRF) and CRF-related neuropeptides are involved in the regulation of stress-related physiology and behavior. Members of the CRF family of neuropeptides bind to two known receptors, the CRF type 1 (CRF₁) receptor, and the CRF type 2 (CRF₂) receptor. Although the distribution of CRF₂ receptor mRNA expression has been extensively studied, the distribution of CRF₂ receptor protein has not been characterized. An area of the brain known to contain high levels of CRF₂ receptor mRNA expression and CRF₂ receptor binding is the dorsal raphe nucleus (DR). In the present study we investigated in detail the distribution of CRF₂ receptor immunoreactivity throughout the rostrocaudal extent of the DR. CRF₂ receptor-immunoreactive perikarya were observed throughout the DR, with the highest number and density in the mid-rostrocaudal DR. Dual immunofluorescence revealed that CRF₂ receptor immunoreactivity was frequently co-localized with tryptophan hydroxylase, a marker of serotonergic neurons. This study provides evidence that CRF₂ receptor protein is expressed in the DR, and that CRF₂ receptors are expressed in topographically organized subpopulations of cells in the DR, including serotonergic neurons. Furthermore, these data are consistent with the hypothesis that CRF₂ receptors play an important role in the regulation of stress-related physiology and behavior through actions on serotonergic and non-serotonergic neurons within the DR.

Gene expression of connective tissue growth factor (CTGF) is induced in activated hepatic stellate cells (HSC), the major effectors in hepatic fibrosis, and production of extracellular matrix (ECM) is consequently increased. We previously reported that curcumin, the yellow pigment in curry, suppressed ctgf expression, leading to decreased production of ECM by HSC. The purpose of this study is to evaluate signal transduction pathways involved in the curcumin suppression of ctgf expression in HSC.

Manganese superoxide dismutase (MnSOD) upregulating FoxM1 have previously been demonstrated promoting lung cancer stemness. Isovitexin exhibits antitumor activities in various cancers. This study aimed to assess whether isovitexin inhibits hepatic carcinoma stem-like cells (HCSLCs) features via regulating MnSOD and FoxM1 expression.

Midbrain neurons of the centrally projecting Edinger-Westphal nucleus (EWcp) are activated by alcohol, and enriched with stress-responsive neuropeptide modulators (including the paralog of corticotropin-releasing factor, urocortin-1). Evidence suggests that EWcp neurons promote behavioral processes for alcohol-seeking and consumption, but a definitive role for these cells remains elusive. Here we combined targeted viral manipulations and gene array profiling of EWcp neurons with mass behavioral phenotyping in C57BL/6 J mice to directly define the links between EWcp-specific urocortin-1 expression and voluntary binge alcohol intake, demonstrating a specific importance for EWcp urocortin-1 activity in escalation of alcohol intake.

Subpopulations of neurons display increased activity during memory encoding and manipulating the activity of these neurons can induce artificial formation or erasure of memories. Thus, these neurons are thought to be cellular engrams. Moreover, correlated activity between pre- and postsynaptic engram neurons is thought to lead to strengthening of their synaptic connections, thus increasing the probability of neural activity patterns occurring during encoding to reoccur at recall. Therefore, synapses between engram neurons can also be considered as a substrate of memory, or a synaptic engram. One can label synaptic engrams by targeting two complementary, non-fluorescent, synapse-targeted GFP fragments separately to the pre- and postsynaptic compartment of engram neurons; the two GFP fragments reconstitute a fluorescent GFP at the synaptic cleft between the engram neurons, thereby highlighting synaptic engrams. In this work we explored a transsynaptic GFP reconstitution system (mGRASP) to label synaptic engrams between hippocampal CA1 and CA3 engram neurons identified by different Immediate-Early Genes: cFos and Arc. We characterized the expression of the cellular and synaptic labels of the mGRASP system upon exposure to a novel environment or learning of a hippocampal-dependent memory task. We found that mGRASP under the control of transgenic ArcCreERT2 labeled synaptic engrams more efficiently than when controlled by viral cFostTA, possibly due to differences in the genetic systems rather than the specific IEG promoters.

Models of artificial intelligence (AI) that have billions of parameters can achieve high accuracy across a range of tasks1,2, but they exacerbate the poor energy efficiency of conventional general-purpose processors, such as graphics processing units or central processing units. Analog in-memory computing (analog-AI)3-7 can provide better energy efficiency by performing matrix-vector multiplications in parallel on 'memory tiles'. However, analog-AI has yet to demonstrate software-equivalent (SWeq) accuracy on models that require many such tiles and efficient communication of neural-network activations between the tiles. Here we present an analog-AI chip that combines 35 million phase-change memory devices across 34 tiles, massively parallel inter-tile communication and analog, low-power peripheral circuitry that can achieve up to 12.4 tera-operations per second per watt (TOPS/W) chip-sustained performance. We demonstrate fully end-to-end SWeq accuracy for a small keyword-spotting network and near-SWeq accuracy on the much larger MLPerf8 recurrent neural-network transducer (RNNT), with more than 45 million weights mapped onto more than 140 million phase-change memory devices across five chips.

We identified ventrolateral medullary nuclei in which thyrotropin-releasing hormone (TRH) regulates glucose metabolism by modulating autonomic activity. Immunolabeling revealed dense prepro-TRH-containing fibers innervating the rostroventrolateral medulla (RVLM) and nucleus ambiguus (Amb), which contain, respectively, pre-sympathetic motor neurons and vagal motor neurons. In anesthetized Wistar rats, microinjection of the stable TRH analog RX77368 (38-150 pmol) into the RVLM dose-dependently and site-specifically induced hyperglycemia and hyperinsulinemia. At 150 pmol, blood glucose reached a peak of 180+/-18 mg% and insulin increased 4-fold. The strongest hyperglycemic effect was induced when RX77368 was microinjected into C1 area containing adrenalin cells. Spinal cord transection at cervical-7 abolished the hyperglycemia induced by RVLM RX77368, but not the hyperinsulinemic effect. Bilateral vagotomy prevented the rise in insulin, resulting in a prolonged hyperglycemic response. The hyperglycemic and hyperinsulinemic effects of the TRH analog in the RVLM was peptide specific, since angiotensin II or a substance P analog at the same dose had weak or no effects. Microinjection of RX77368 into the Amb stimulated insulin secretion without influencing glucose levels. In conscious type 2 diabetic Goto-Kakizaki (GK) rats, intracisternal injection of RX77368 induced a remarkably amplified hyperglycemic effect with suppressed insulin response compared to Wistar rats. RX77368 microinjected into the RVLM of anesthetized GK rats induced a significantly potentiated hyperglycemic response and an impaired insulin response, compared to Wistar rats. These results indicate that the RVLM is a site at which TRH induces sympathetically-mediated hyperglycemia and vagally-mediated hyperinsulinemia, whereas the Amb is mainly a vagal activating site for TRH. Hyperinsulinemia induced by TRH in the RVLM is not secondary to the hyperglycemic response. The potentiated hyperglycemic and suppressed hyperinsulinemic responses in diabetic GK rats indicate that an unbalanced "sympathetic-over-vagal" activation by TRH in brainstem RVLM contributes to the pathophysiology of impaired glucose homeostasis in type 2 diabetes.

Urocortins are the endogenous ligands for the corticotropin-releasing factor receptor type 2 (CRFR2), which is implicated in regulating energy balance and/or glucose metabolism. We determined the effects of chronic CRFR2 activation on metabolism in vivo, by generating and phenotyping transgenic mice overproducing the specific CRFR2 ligand urocortin 3.

Glucagon-like peptide 1 (GLP-1), a kind of gut hormone, is used in the treatment of type 2 diabetes (T2D). Emerging evidence indicates that GLP-1 has anti-inflammatory activity. Chronic inflammation in the adipose tissue of obese individuals is a cause of insulin resistance and T2D. We hypothesized that GLP-1 analogue therapy in patients with T2D could suppress the inflammatory response of macrophages, and therefore inhibit insulin resistance. Our results showed that GLP-1 agonist (exendin-4) not only attenuated macrophage infiltration, but also inhibited the macrophage secretion of inflammatory cytokines including TNF-β, IL-6, and IL-1β. Furthermore, we observed that lipopolysaccharide (LPS)-induced macrophage conditioned media could impair insulin-stimulated glucose uptake. This effect was compensated by treatment with the conditioned media from macrophages treated with the combination of LPS and exendin-4. It was also observed that exendin-4 directly inhibited the activation of NF-κB in macrophages. In conclusion, our results indicated that GLP-1 improved inflammatory macrophage-derived insulin resistance by inhibiting NF-κB pathway and secretion of inflammatory cytokines in macrophages. Furthermore, our observations suggested that the anti-inflammatory effect of GLP-1 on macrophages can contribute to GLP-1 analogue therapy of T2D.

Recent studies showed that macrophages co-cultured with ovarian cancer stem-like cells (OCSLCs) induced SKOV3 cell stemness via IL-8/STAT3 signaling. Genistein (GEN) demonstrates chemopreventive activity in inflammation-associated cancers. The present study aimed to examine whether and if GEN inhibits the stemness of SKOV3 and OVCA-3R cells induced by co-culture of THP-1 macrophages and SKOV3-derived OCSLCs.

We have previously reported that 8-bromo-7-methoxychrysin (BrMC), a novel synthetic derivative of chrysin, was demonstrated anti-tumor activities against several human cancers, including lung cancer. Interaction between inflammation and cancer stem cell are recently increasingly recognized in tumorigenesis and progression. The purpose of this study was to investigate whether BrMC inhibits lung cancer stemness of H460 cells induced by inflammatory factors (TGF-β combined with TNF-α) and its potential mechanism. Our results showed that BrMC inhibited lung cancer stemness, as validated by enhanced self-renewal ability, higher in vitro tumorigenicity, and increased expression of CD133, CD44, Bmi1 and Oct4 in H460 cells administered TNF-α after prolonged induction by TGF-β, in a concentration-dependent manner. Both NF-κB inhibition by SN50 and FoxM1 suppression by thiostrepton (THI) prompted the inhibition of BrMC on lung CSCs. Conversely, overexpression of NF-κBp65 significantly antagonized the above effects of BrMC. Meanwhile, overexpression of FoxM1 also significantly compromised BrMC function on suppression of FoxM1 and NF-κBp65 as well as stemness of lung CSCs. Our results suggest that activation of NF-κB and FoxM1 by cytokines facilitate the acquisition CSCs phenotype, and compromise the chemical inhibition, which may represent an effective therapeutic target for treatment of human lung cancer. Moreover, BrMC may be a potential promising candidate for targeting NF-κB/ FoxM1 to prevent the tumorigenesis under inflammatory microenvironment.

Viral vector mediated gene therapy has become commonplace in clinical trials for a wide range of inherited disorders. Successful gene transfer depends on a number of factors, of which tissue tropism is among the most important. To date, definitive mapping of the spatial and temporal distribution of viral vectors in vivo has generally required postmortem examination of tissue. Here we present two methods for radiolabeling adeno-associated virus (AAV), one of the most commonly used viral vectors for gene therapy trials, and demonstrate their potential usefulness in the development of surrogate markers for vector delivery during the first week after administration. Specifically, we labeled adeno-associated virus serotype 10 expressing the coding sequences for the CLN2 gene implicated in late infantile neuronal ceroid lipofuscinosis with iodine-124. Using direct (Iodogen) and indirect (modified Bolton-Hunter) methods, we observed the vector in the murine brain for up to one week using positron emission tomography. Capsid radioiodination of viral vectors enables non-invasive, whole body, in vivo evaluation of spatial and temporal vector distribution that should inform methods for efficacious gene therapy over a broad range of applications.

Transplantation of cellular components of the permissive peripheral nerve environment in some types of spinal cord injury holds great promise to support regrowth of axons through the site of injury. In the present study, Schwann cell grafts were positioned between transected stumps of adult rat thoracic spinal cord to test their efficacy to serve as bridges for axonal regeneration. Schwann cells were purified in culture from adult rat sciatic nerve, suspended in Matrigel: DMEM (30:70), and drawn into polymeric guidance channels 8 mm long at a density of 120 x 10(6) cells ml-1. Adult Fischer rat spinal cords were transected at the T8 cord level and the next caudal segment was removed. Each cut stump was inserted 1 mm into the channel. One month later, a bridge between the severed stumps had been formed, as determined by the gross and histological appearance and the ingrowth of propriospinal axons from both stumps. Propriospinal neurons (mean, 1064 +/- 145 SEM) situated as far away as levels C3 and S4 were labelled by retrograde tracing with Fast Blue injected into the bridge. Near the bridge midpoint there was a mean of 1990 +/- 594 myelinated axons and eight times as many nonmyelinated, ensheathed axons. Essentially no myelinated or unmyelinated axons were observed in control Matrigel-only grafts. Brainstem neurons were not retrogradely labelled from the graft, consistent with growth of immunoreactive serotonergic and noradrenergic axons only a short distance into the rostral end of the graft, not far enough to reach the tracer placed at the graft midpoint. Anterograde tracing with PHA-L introduced rostral to the graft demonstrated that axons extended the length of the graft but essentially did not leave the graft. This study demonstrates that Schwann cell grafts serve as bridges that support (1) regrowth of both ascending and descending axons across a gap in the adult rat spinal cord and (2) limited regrowth of serotonergic and noradrenergic fibers from the rostral stump. Regrowth of monoaminergic fibres into grafts was not seen in an earlier study of similar grafts placed inside distally capped rather than open-ended channels. Additional intervention will be required to foster growth of the regenerated axons from the graft into the distal cord tissue.

Central blockade of mineralocorticoid receptors (MRs) or angiotensin II type 1 receptors (AT1Rs) attenuates aldosterone (aldo)-salt induced hypertension. We examined the role of the subfornical organ (SFO), aldo synthesized locally in the brain, and MR and AT1R specifically in the paraventricular nucleus (PVN) in aldo-salt hypertension. Wistar rats were treated with subcutaneous aldo (1 μg/h) plus saline as drinking fluid, and gene expression was assessed by real-time qPCR. Other sets of rats received chronic intra-cerebroventricular (icv) infusion of aldo synthase (AS) inhibitor FAD286, MR blocker eplerenone or vehicle, electrolytic or sham lesions of the SFO, or intra-PVN infusion of AAV-MR-siRNA or AAV-AT1aR-siRNA. Infusion of aldo had no effect on 11βHSD2, MR and AT1R mRNA in different nuclei but increased CYP11B2 mRNA in the SFO, and serum and glucocorticoid-kinase 1 (Sgk1) and epithelial sodium channel (ENaC) γ subunit mRNA in the SFO and supraoptic nucleus (SON). MR-siRNA decreased both MR and AT1R mRNA in the PVN by ∼ 60%, but AT1aR-siRNA only decreased AT1R mRNA. SFO lesion, blockade of brain AS or MR, or knockdown of MR or AT1R in the PVN similarly attenuated aldosterone-induced saline intake by ∼ 50% and hypertension by ∼ 70%. These results suggest that an increase in circulating aldosterone may via MR and AT1R in the SFO increase local aldosterone production in hypothalamic nuclei such as the SON and PVN, and via MR enhance AT1R signaling in the PVN. This central aldosterone-MR-AT1R neuro-modulatory pathway appears to play a major role in the progressive hypertension.

The integral selectivity characteristic of the blood brain barrier (BBB) limits therapeutic options for many neurologic diseases and disorders. Currently, very little is known about the mechanisms that govern the dynamic nature of the BBB. Recent reports have focused on the development and application of human brain organoids developed from neuro-progenitor cells. While these models provide an excellent platform to study the effects of disease and genetic aberrances on brain development, they may not model the microvasculature and BBB of the adult human cortex. To date, most in vitro BBB models utilize endothelial cells, pericytes and astrocytes. We report a 3D spheroid model of the BBB comprising all major cell types, including neurons, microglia and oligodendrocytes, to recapitulate more closely normal human brain tissue. Spheroids show expression of tight junctions, adherens junctions, adherens junction-associated proteins and cell specific markers. Functional assessment using MPTP, MPP+ and mercury chloride indicate charge selectivity through the barrier. Junctional protein distribution was altered under hypoxic conditions. Our spheroid model may have potential applications in drug discovery, disease modeling, neurotoxicity and cytotoxicity testing.

Interferon regulatory factor-4 binding protein (IBP) is a novel upstream activator of Rho GTPases. Our previous studies have shown that ectopic expression of IBP was correlated with malignant behaviors of human breast cancer cells, and invasive human breast cancer had high expression of IBP that promoted the proliferation of these cells. However, it remains unknown whether autophagy inhibition contributes to IBP-mediated tumorigenesis. In this study, we for the first time, reported that upregulation of IBP expression significantly suppressed the autophagy of breast cancer cells, and downregulation of IBP expression markedly induced autophagy of these cells. Further investigation revealed that IBP effectively counteracted autophagy by directly activating mammalian target of rapamycin complex 2 (mTORC2) and upregulating phosphorylation of Akt on ser473 and FOXO3a on Thr32. Moreover, IBP-mediated suppression of autophagy was dependent on mTORC2/Akt/FOXO3a signaling pathway. Finally, our results demonstrated that IBP-mediated breast cancer cell growth in vitro and in vivo was strongly correlated with suppression of mTORC2-dependent autophagy. These findings suggest that the anti-autophagic property of IBP has an important role in IBP-mediated tumorigenesis, and IBP may serve as an attractive target for treatment of breast cancer.