Evidence from human and animal studies suggests that chronic behavioral stress and resulting activation of the sympathetic nervous system may influence initiation and progression of tumors. However, the underlying mechanisms for these observations are poorly understood. The purpose of this study is to explore the effects of adrenergic signaling on cell line models derived from normal cells presumed to originate epithelial ovarian cancers. Here we explored the effects of the stress-related hormone, norepinephrine, on the transcriptional program of normal immortalized ovarian (iOSE) and fallopian tube (iFTSEC) surface epithelial cells. Analysis of RNA-Seq data of treated and untreated cells revealed a significant overlap between the responses in iOSE and iFTSEC cells. Most genes modulated by norepinephrine in ovarian and fallopian tube epithelial cells are already expressed in normal ovarian and fallopian tissue and cells. For several genes, expression changes were reflected at the protein level. Genes in immune-related and developmental pathways were enriched in the set of genes modulated by norepinephrine. We identified HOXA5, SPIB, REL, SRF, SP1, NFKB1, MEF2A, E2F1, and EGR1 transcription factor binding sites to be highly enriched in our dataset. These data represent the early transcriptional response to norepinephrine in cells postulated to originate epithelial ovarian cancer.

Neuregulin 1 (NRG1) is required for development of the central and peripheral nervous system and regulates neurotransmission in the adult. NRG1 and the gene encoding its receptor, ERBB4, are risk genes for schizophrenia, although how alterations in these genes disrupt their function has not been fully established. Studies of knockout and transgenic mice have yielded conflicting results, with both gain and loss of function resulting in similar behavioral and electrophysiological phenotypes. Here, we used high affinity antibodies to NRG1 and ErbB4 to perturb the function of the endogenous proteins in adult mice. Treatment with NRG1 antibodies that block receptor binding caused behavioral alterations associated with schizophrenia, including, hyper-locomotion and impaired pre-pulse inhibition of startle (PPI). Electrophysiological analysis of brain slices from anti-NRG1 treated mice revealed reduced synaptic transmission and enhanced paired-pulse facilitation. In contrast, mice treated with more potent ErbB4 function blocking antibodies did not display behavioral alterations, suggesting a receptor independent mechanism of the anti-NRG1-induced phenotypes. We demonstrate that anti-NRG1 causes accumulation of the full-length transmembrane protein and increases phospho-cofilin levels, which has previously been linked to impaired synaptic transmission, indicating enhancement of non-canonical NRG1 signaling could mediate the CNS effects.

The effects of MC-3129, a synthetic cyclohexene derivative, on cell viability and apoptosis have been investigated in human leukemia cells. Exposure of leukemia cells to MC-3129 led to the inhibition of cell viability and induction of apoptosis through the dephosphorylation and mitochondrial translocation of cofilin. A mechanistic study revealed that interruption of the RhoA/ROCK1/PTEN/PI3K/Akt signaling pathway plays a crucial role in the MC-3129-mediated dephosphorylation and mitochondrial translocation of cofilin and induction of apoptosis. Our in vivo study also showed that the MC-3129-mediated inhibition of the tumor growth in a mouse leukemia xenograft model is associated with the interruption of ROCK1/PTEN/PI3K/Akt signaling and apoptosis. Molecular docking suggested that MC-3129 might activate the RhoA/ROCK1 pathway by targeting LPAR2. Collectively, these findings suggest a hierarchical model, in which the induction of apoptosis by MC-3129 primarily results from the activation of RhoA/ROCK1/PTEN and inactivation of PI3K/Akt, leading to the dephosphorylation and mitochondrial translocation of cofilin, and culminating in cytochrome c release, caspase activation, and apoptosis. Our study reveals a novel role for RhoA/ROCK1/PTEN/PI3K/Akt signaling in the regulation of mitochondrial translocation of cofilin and apoptosis and suggests MC-3129 as a potential drug for the treatment of human leukemia.

To extend the knowledge on the microbial diversity of manganese rich environments, we performed a clone library based study using metagenomic approach. Pyrosequencing based analysis of 16S rRNA genes were carried out on an Illumina platform to gain insights into the bacterial community inhabiting in a manganese mining site and the taxonomic profiles were correlated with the inherent capacities of these strains to solubilise manganese. The application of shot gun sequencing in this study yielded results which revealed the highest prevalence of Proteobacteria (42.47%), followed by Actinobacteria (23.99%) in the area of study. Cluster of orthologous group (COG) functional category has 85,066 predicted functions. Out of which 11% are involved in metabolism of amino acid, 9% are involved in production and conversion of energy while Keto Encyclopedia of Gene and Genomes (KEGG) functional category has 107,388 predicted functions, out of which 55% are involved in cellular metabolism, 15% are environmental and information processing and 12% are genetic information processing in nature. The isolated microbial consortia demonstrated visible growth in presence of high concentrations of Mn. Solubilisation studies resulted in 86% of manganese recovery after 20 days. The result presented in this study has important implications in understanding the microbial diversity in manganese contaminated mine tailings and their role in natural geochemical cycling of Mn.

The architecture of the genome influences the functions of DNA from bacteria to eukaryotes. Intrinsically disordered regions (IDR) of eukaryotic histones have pivotal roles in various processes of gene expression. IDR is rare in bacteria, but interestingly, mycobacteria produce a unique histone-like protein, MDP1 that contains a long C-terminal IDR. Here we analyzed the role of IDR in MDP1 function. By employing Mycobacterium smegmatis that inducibly expresses MDP1 or its IDR-deficient mutant, we observed that MDP1 induces IDR-dependent DNA compaction. MDP1-IDR is also responsible for the induction of growth arrest and tolerance to isoniazid, a front line tuberculosis drug that kills growing but not growth-retardated mycobacteria. We demonstrated that MDP1-deficiency and conditional knock out of MDP1 cause spreading of the M. smegmatis genome in the stationary phase. This study thus demonstrates for the first time a C-terminal region-dependent organization of the genome architecture by MDP1, implying the significance of IDR in the function of bacterial histone-like protein.

Alzheimer's disease (AD) is a dreadful neurodegenerative disease that leads to severe impairment of cognitive function, leading to a drastic decline in the quality of life. The primary pathological features of AD include senile plaques (SPs) and intracellular neurofibrillary tangles (NFTs), comprising aggregated amyloid β (Aβ) and hyperphosphorylated tau protein, respectively, in the hippocampus of AD patients. Histone deacetylase 6 (HDAC6) is a key enzyme in this neurodegenerative disease, in particular, as it relates to tau hyperphosphorylation. This study aimed to investigate the protective effects and mechanism of the novel HDAC6 inhibitor, MPT0G211, using an AD model. Our results indicated that MPT0G211 significantly reduced tau phosphorylation and aggregation, the processes highly correlated with the formation of NFTs. This HDAC6 inhibitory activity resulted in an increase in acetylated Hsp90, which decreased Hsp90 and HDAC6 binding, causing ubiquitination of phosphorylated tau proteins. In addition, a significant increase of phospho-glycogen synthase kinase-3β (phospho-GSK3β) on Ser9 (the inactive form) through Akt phosphorylation was associated with the inhibition of phospho-tau Ser396 in response to MPT0G211 treatment. In AD in vivo models, MPT0G211 appeared to ameliorate learning and memory impairment in animals. Furthermore, MPT0G211 treatment reduced the amount of phosphorylated tau in the hippocampal CA1 region. In summary, MPT0G211 treatment appears to be a promising strategy for improving the AD phenotypes, including tau hyperphosphorylation and aggregation, neurodegeneration, and learning and memory impairment, making it a valuable agent for further investigation.

Danggui-Sayuk-Ga-Osuyu-Saenggang-Tang (DSGOST in Korean, Danggui-Sini-Jia-Wuzhuyu-Shengian-Tang in Chinese, and Tokishigyakukagoshuyushokyoto (TJ-38) in Japanese), a well-known traditional Korean/Chinese/Japanese medicine, has long been used to treat vascular diseases such as Raynaud's phenomenon (RP). However, anticancer effect of DSGOST remains elusive. In this study, we checked if DSGOST has an anticancer effect against gastric cancer cells, and investigated the mechanisms underlying DSGOST resistance. Moreover, DSGOST regulates chemoresistance in cisplatin-treated gastric cancer cells. Interestingly, DSGOST treatment induced the accumulation of GFP-LC3 puncta and increased the level of autophagy markers, such as LC3-II, ATG5, and Beclin-1, indicating activated autophagy. Furthermore, DSGOST could activate epithelial-to-mesenchymal transition (EMT) and exosomes via induction of autophagy. DSGOST in combination with TGFβ also induced autophagy and EMT. However, autophagy inhibition induces DSGOST-mediated cell death in gastric cancer cells. In addition, autophagy inhibition blocks the activation of DSGOST-mediated EMT markers including N-cadherin, Snail, Slug, vimentin, β-catenin, p-Smad2, and p-Smad3. Taken together, these findings indicated that prosurvival autophagy was one of the mechanisms involved in the resistance of gastric cancer to DSGOST. Targeting the inhibition of autophagy could be an effective therapeutic approach to overcome resistance to DSGOST in gastric cancer.

The "non-specific" ventromedial thalamic nucleus (VM) has long been considered a candidate for mediating cortical arousal due to its diffuse, superficial projections, but direct evidence was lacking. Here, we show in mice that the activity of VM calbindin1-positive matrix cells is high in wake and REM sleep and low in NREM sleep, and increases before cortical activity at the sleep-to-wake transition. Optogenetic stimulation of VM cells rapidly awoke all mice from NREM sleep and consistently caused EEG activation during slow wave anesthesia, while arousal did not occur from REM sleep. Conversely, chemogenetic inhibition of VM decreased wake duration. Optogenetic activation of the "specific" ventral posteromedial nucleus (VPM) did not cause arousal from either NREM or REM sleep. Thus, matrix cells in VM produce arousal and broad cortical activation during NREM sleep and slow wave anesthesia in a way that accounts for the effects classically attributed to "non-specific" thalamic nuclei.

Macrophages release iron into the bloodstream via a membrane-bound iron export protein, ferroportin (FPN). The hepatic iron-regulatory hormone hepcidin controls FPN internalization and degradation in response to bacterial infection. Salmonella typhimurium can invade macrophages and proliferate in the Salmonella-containing vacuole (SCV). Hepcidin is reported to increase the mortality of Salmonella-infected animals by increasing the bacterial load in macrophages. Here we assess the iron levels and find that hepcidin increases iron content in the cytosol but decreases it in the SCV through FPN on the SCV membrane. Loss-of-FPN from the SCV via the action of hepcidin impairs the generation of bactericidal reactive oxygen species (ROS) as the iron content decreases. We conclude that FPN is required to provide sufficient iron to the SCV, where iron serves as a cofactor for the generation of antimicrobial ROS rather than as a nutrient for Salmonella.

During the peripheral nerve regeneration process, a variety of neurotrophic factors play roles in nerve repair by acting on neuronal or non-neuronal cells. In this report, we investigated the role(s) of hepatocyte growth factor (HGF) and its receptor, c-met, in peripheral nerve regeneration. When mice were subjected to sciatic nerve injury, the HGF protein level was highly increased at the injured and distal sites. The level of both total and phosphorylated c-met was also highly upregulated, but almost exclusively in Schwann cells (SCs) distal from the injury site. When mice were treated with a c-met inhibitor, PHA-665752, myelin thickness and axon regrowth were decreased indicating that re-myelination was hindered. HGF promoted the migration and proliferation of cultured SCs, and also induced the expression of various genes such as GDNF and LIF, presumably by activating ERK pathways. Furthermore, exogenous supply of HGF around the injury site, by intramuscular injection of a plasmid DNA expressing human HGF, enhanced the myelin thickness and axon diameter in injured nerves. Taken together, our results indicate that HGF and c-met play important roles in Schwann cell-mediated nerve repair, and also that HGF gene transfer may provide a useful tool for treating peripheral neuropathy.

A rare variant (BAFF-var) of the tumor necrosis factor superfamily 13b (TNFSF13B) gene has been recently associated with multiple sclerosis (MS) and systemic lupus erythematosus (SLE). The aim of this study was to investigate the association between TNFSF13B BAFF-var and susceptibility to rheumatoid arthritis (RA) and replicate that association in SLE. 6,218 RA patients, 2,575 SLE patients and 4,403 healthy controls from three different countries were included in the study. TNFSF13B BAFF-var was genotyped using TaqMan allelic discrimination assay. PLINK software was used for statistical analyses. TNFSF13B BAFF-var was significantly associated with RA (p = 0.015, OR = 1.21, 95% CI = 1.03-1.41) in the Spanish cohort. A trend of association was observed in the Dutch (p = 0.115) and German (p = 0.228) RA cohorts. A meta-analysis of the three RA cohorts included in this study revealed a statistically significant association (p = 0.002, OR = 1.24, 95% CI = 1.10-1.38). In addition, TNFSF13B BAFF-var was significantly associated with SLE in the Spanish (p = 0.001, OR = 1.41, 95% CI = 1.14-1.74) and the German cohorts (p = 0.030, OR = 1.86, 95% CI = 1.05-3.28), with a statistically significant p-value obtained in the meta-analysis (p = 0.0002, OR = 1.46, 95% CI = 1.09-2.32). The results obtained confirm the known association of TNFSF13B BAFF-var with SLE and, for the first time, demonstrate that this variant contributes to susceptibility to RA.

Inherited retinal dystrophies (IRDs) are caused by mutations in over 200 genes, resulting in a range of therapeutic options. Translational read-through inducing drugs (TRIDs) offer the possibility of treating multiple IRDs regardless of the causative gene. TRIDs promote ribosomal misreading of premature stop codons, which results in the incorporation of a near-cognate amino acid to produce a full-length protein. The IRD choroideremia (CHM) is a pertinent candidate for TRID therapy, as nonsense variants cause 30% of cases. Recently, treatment of the UAA nonsense-carrying CHM zebrafish model with the TRID PTC124 corrected the underlying biochemical defect and improved retinal phenotype. To be clinically relevant, we studied PTC124 efficiency in UAA nonsense-carrying human fibroblasts and induced pluripotent stem cell-derived retinal pigment epithelium, as well as in a UAA-mutated CHM overexpression system. We showed that PTC124 treatment induces a non-significant trend for functional rescue, which could not be improved by nonsense-mediated decay inhibition. Furthermore, it does not produce a detectable CHM-encoded protein even when coupled with a proteasome inhibitor. We suggest that drug efficiency may depend upon on the target amino acid and its evolutionary conservation, and argue that patient cells should be screened in vitro prior to inclusion in a clinical trial.

Homoharringtonine (HHT), an inhibitor of protein synthesis, has been used to treat leukemia. Its therapeutic effects on non-small cell lung adenocarcinoma carrying KRAS mutation and their immune system are less understood. The present study examined the therapeutic efficacy and the immune effects of HHT in two murine lung tumor models, xenograft and transgenic, carrying the Kras mutation G12D and G12C respectively. HHT exhibited efficient anticancer activity, significantly suppressing lung tumor growth in vitro and in vivo. The levels of 22 cytokines and chemokines in splenocytes of tumor-bearing mice were examined. Interleukin-12 expression was lower in splenocytes of HHT-treated mice when compared to the controls as demonstrated by a cytokine array and an enzyme-linked immunosorbent assay. The expression levels of CD80, CD86, and CD69 in B220+ B cells from splenocytes of HHT-treated mice were higher than that of control mice in two mouse tumor models. Furthermore, antitumor effect of HHT was attenuated with depletion of B cells. Increased numbers of CD80+ and CD86+ B cells were observed in the mice treated with narciclasine, another translation inhibitor. In conclusion, HHT changed the features of immune cells, and exhibited efficient anti-tumor activity against lung tumor carrying mutant Kras expression.

α-synuclein plays a key role in the pathogenesis of Parkinson's disease (PD); its deposits are found as amyloid fibrils in Lewy bodies and Lewy neurites, the histopathological hallmarks of PD. Amyloid fibrillation is a progressive polymerization path starting from peptide/protein misfolding and proceeding through the transient growth of oligomeric intermediates widely considered as the most toxic species. Consequently, a promising approach of intervention against PD might be preventing α-synuclein build-up, misfolding and aggregation. A possible strategy involves the use of small molecules able to slow down the aggregation process or to alter oligomer conformation favouring the growth of non-pathogenic species. Here, we show that oleuropein aglycone (OleA), the main olive oil polyphenol, exhibits anti-amyloidogenic power in vitro by interacting with, and stabilizing, α-synuclein monomers thus hampering the growth of on-pathway oligomers and favouring the growth of stable and harmless aggregates with no tendency to evolve into other cytotoxic amyloids. We investigated the molecular basis of such interference by both biophysical techniques and limited proteolysis; aggregate morphology was monitored by electron microscopy. We also found that OleA reduces the cytotoxicity of α-synuclein aggregates by hindering their binding to cell membrane components and preventing the resulting oxidative damage to cells.

Neurodevelopmental disorders, such as ASD and ADHD, affect males about three to four times more often than females. 16p11.2 hemideletion is a copy number variation that is highly associated with neurodevelopmental disorders. Previous work from our lab has shown that a mouse model of 16p11.2 hemideletion (del/+) exhibits male-specific behavioral phenotypes. We, therefore, aimed to investigate with magnetic resonance imaging (MRI), whether del/+ animals also exhibited a sex-specific neuroanatomical endophenotype. Using the Allen Mouse Brain Atlas, we analyzed the expression patterns of the 27 genes within the 16p11.2 region to identify which gene expression patterns spatially overlapped with brain structural changes. MRI was performed ex vivo and the resulting images were analyzed using Voxel-based morphometry for T1-weighted sequences and tract-based spatial statistics for diffusion-weighted images. In a subsequent step, all available in situ hybridization (ISH) maps of the genes involved in the 16p11.2 hemideletion were aligned to Waxholm space and clusters obtained by sex-specific group comparisons were analyzed to determine which gene(s) showed the highest expression in these regions. We found pronounced sex-specific changes in male animals with increased fractional anisotropy in medial fiber tracts, especially in those proximate to the striatum. Moreover, we were able to identify gene expression patterns spatially overlapping with male-specific structural changes that were associated with neurite outgrowth and the MAPK pathway. Of note, previous molecular studies have found convergent changes that point to a sex-specific dysregulation of MAPK signaling. This convergent evidence supports the idea that ISH maps can be used to meaningfully analyze imaging data sets.

Genome editing is the introduction of directed modifications in the genome, a process boosted to therapeutic levels by designer nucleases. Building on the experience of ex vivo gene therapy for severe combined immunodeficiencies, it is likely that genome editing of haematopoietic stem/progenitor cells (HSPC) for correction of inherited blood diseases will be an early clinical application. We show molecular evidence of gene correction in a mouse model of primary immunodeficiency. In vitro experiments in DNA-dependent protein kinase catalytic subunit severe combined immunodeficiency (Prkdc scid) fibroblasts using designed zinc finger nucleases (ZFN) and a repair template demonstrated molecular and functional correction of the defect. Following transplantation of ex vivo gene-edited Prkdc scid HSPC, some of the recipient animals carried the expected genomic signature of ZFN-driven gene correction. In some primary and secondary transplant recipients we detected double-positive CD4/CD8 T-cells in thymus and single-positive T-cells in blood, but no other evidence of immune reconstitution. However, the leakiness of this model is a confounding factor for the interpretation of the possible T-cell reconstitution. Our results provide support for the feasibility of rescuing inherited blood disease by ex vivo genome editing followed by transplantation, and highlight some of the challenges.

Natural biochemical systems are ubiquitously organized both in space and time. Engineering the spatial organization of biochemistry has emerged as a key theme of synthetic biology, with numerous technologies promising improved biosynthetic pathway performance. One strategy, however, may produce disparate results for different biosynthetic pathways. We use a spatially resolved kinetic model to explore this fundamental design choice in systems and synthetic biology. We predict that two example biosynthetic pathways have distinct optimal organization strategies that vary based on pathway-dependent and cell-extrinsic factors. Moreover, we demonstrate that the optimal design varies as a function of kinetic and biophysical properties, as well as culture conditions. Our results suggest that organizing biosynthesis has the potential to substantially improve performance, but that choosing the appropriate strategy is key. The flexible design-space analysis we propose can be adapted to diverse biosynthetic pathways, and lays a foundation to rationally choose organization strategies for biosynthesis.

Microalgae-based metabolic engineering has been proven effective for producing valuable substances such as food supplements, pharmaceutical drugs, biodegradable plastics, and biofuels in the past decade. The ability to accurately visualize and quantify intracellular metabolites in live microalgae is essential for efficient metabolic engineering, but remains a major challenge due to the lack of characterization methods. Here we demonstrate it by synthesizing fluorogenic peptide aptamers with specific binding affinity to a target metabolite and delivering them into live microalgae by femtosecond laser photoporation at single-cell resolution. As a proof-of-principle demonstration of our method, we use it to characterize Euglena gracilis, a photosynthetic unicellular motile microalgal species, which is capable of producing paramylon (a carbohydrate granule similar to starch). Specifically, we synthesize a peptide aptamer containing a paramylon-binding fluorescent probe, 7-nitrobenzofurazan, and introduce it into E. gracilis cells one-by-one by suppressing their mobility with mannitol and transiently perforating them with femtosecond laser pulses at 800 nm for photoporation. To demonstrate the method's practical utility in metabolic engineering, we perform spatially and temporally resolved fluorescence microscopy of single live photoporated E. gracilis cells under different culture conditions. Our method holds great promise for highly efficient microalgae-based metabolic engineering.

Warburg effect is an emerging hallmark of cancer cells with pyruvate kinase M2 (PKM2) as its key regulator. Curcumin is an extensively-studied anti-cancer compound, however, its role in affecting cancer metabolism remains poorly understood. Herein, we show that curcumin inhibits glucose uptake and lactate production (Warburg effect) in a variety of cancer cell lines by down-regulating PKM2 expression, via inhibition of mTOR-HIF1α axis. Stable PKM2 silencing revealed that PKM2 is required for Warburg effect and proliferation of cancer cells. PKM2 over-expression abrogated the effects of curcumin, demonstrating that inhibition of Warburg effect by curcumin is PKM2-mediated. High PKM2 expression correlated strongly with poor overall survival in cancer, suggesting the requirement of PKM2 in cancer progression. The study unravels novel PKM2-mediated inhibitory effect of curcumin on metabolic capacities of cancer cells. To the best of our knowledge, this is the first study linking curcumin with PKM2-driven cancer glycolysis, thus, providing new perspectives into the mechanism of its anticancer activity.

Brown adipose tissue (BAT) is a specialized thermogenic organ in mammals. The ability of BAT mitochondria to generate heat in response to cold-challenge to maintain core body temperature is essential for organismal survival. While cold activated BAT mitochondrial biogenesis is recognized as critical for thermogenic adaptation, the contribution of mitochondrial quality control to this process remains unclear. Here, we show mitophagy is required for brown adipocyte mitochondrial homeostasis during thermogenic adaptation. Mitophagy is significantly increased in BAT from cold-challenged mice (4 °C) and in β-agonist treated brown adipocytes. Blockade of mitophagy compromises brown adipocytes mitochondrial oxidative phosphorylation (OX-PHOS) capacity, as well as BAT mitochondrial integrity. Mechanistically, cold-challenge induction of BAT mitophagy is UCP1-dependent. Furthermore, our results indicate that mitophagy coordinates with mitochondrial biogenesis, maintaining activated BAT mitochondrial homeostasis. Collectively, our in vivo and in vitro findings identify mitophagy as critical for brown adipocyte mitochondrial homeostasis during cold adaptation.