Development Invivo Research Articles

Resistance to gemcitabine is mediated by the circ_0036627/miR-145/S100A16 axis in pancreatic cancer.

The development of gemcitabine (GEM) resistance severely limits the treatment efficacy in pancreatic cancer (PC) and increasing evidence highlights the vital roles of circular RNAs (circRNAs) in the tumorigenesis, progression and drug resistance of PC. However, the circRNAs underlying GEM resistance development of PC remains to be clarified. The current research aims to unveil the roles of circ_0036627 in dictating the aggressiveness and GEM sensitivity in PC. We reported the increased expression of circ_0036627 in PC tissues and PC cell lines. Elevated circ_0036627 expression level was correlated with advanced tumour grade and poor overall survival in PC patients. Functional assays and in vivo experiments demonstrated that circ_0036627 overexpression was required for the proliferation, migration invasion and GEM resistance in PC cells. circ_0036627 knockdown suppressed tumour development invivo. The molecular analysis further showed that circ_0036627 increased S100A16 expression by sponging microRNA-145 (miR-145), a tumour-suppressive miRNA that could significantly attenuate PC cell proliferation, migration, invasion and GEM resistance. Furthermore, our findings suggested that S100A16 acted as an oncogenic factor to promote aggressiveness and GEM resistance in PC cells. In conclusion, the current findings provide new mechanistic insights into PC aggressiveness and GEM resistance, suggesting the critical role of circ_0036627/miR-145/S100A16 axis in PC progression and drug resistance development and offering novel therapeutic targets for PC therapy.

Targeting HOXA11-AS to mitigate prostate cancer via the glycolytic metabolism: Invitro and invivo.

As oncogenes or oncogene suppressors, long-stranded non-coding RNAs are essential for the formation and progression of human tumours. However, the mechanisms behind the regulatory role of RNA HOXA11-AS in prostate cancer (PCa) are unclear. PCa is a common malignant tumour worldwide, and an increasing number of studies have focused on its metabolic profile. Studies have shown that the long non-coding RNA (lncRNA) HOXA11-AS is aberrantly expressed in many tumours. However, the role of HOXA11-AS in PCa is unclear. This work aimed to determine how HOXA11-AS regulated PCa invitro and invivo. We first explored the clinical role of HOXA11-AS in PCa using bioinformatics methods, including single sample gene set enrichment analysis (ssGSEA), weighted gene co-expression network analysis (WGCNA), and least absolute shrinkage and selection operator (LASSO)-logistics systematically. In this study, PCa cell lines were selected to assess the PCa regulatory role of HOXA11-AS overexpression versus silencing invitro, and tumour xenografts were performed in nude mice to assess tumour suppression by HOXA11-AS silencing invivo. HOXA11-AS expression was significantly correlated with clinicopathological factors, epithelial-mesenchymal transition (EMT) and glycolysis. Moreover, key genes downstream of HOXA11-AS exhibited good clinical diagnostic properties for PCa. Furthermore, we studied both invitro and invivo effects of HOXA11-AS expression on PCa. Overexpression of HOXA11-AS increased PCa cell proliferation, migration and EMT, while silencing HOXA11-AS had the opposite effect on PCa cells. In addition, multiple metabolites were downregulated by silencing HOXA11-AS via the glycolytic pathway. HOXA11-AS silencing significantly inhibited tumour development invivo. In summary, silencing HOXA11-AS can inhibit PCa by regulating glucose metabolism and may provide a future guidance for the treatment of PCa.

PHLDA2-mediated phosphatidic acid peroxidation triggers a distinct ferroptotic response during tumor suppression

Although the role of ferroptosis in killing tumor cells is well established, recent studies indicate that ferroptosis inducers also sabotage anti-tumor immunity by killing neutrophils and thus unexpectedly stimulate tumor growth, raising a serious issue about whether ferroptosis effectively suppresses tumor development invivo. Through genome-wide CRISPR-Cas9 screenings, we discover a pleckstrin homology-like domain family A member 2 (PHLDA2)-mediated ferroptosis pathway that is neither ACSL4-dependent nor requires common ferroptosis inducers. PHLDA2-mediated ferroptosis acts through the peroxidation of phosphatidic acid (PA) upon high levels of reactive oxygen species (ROS). ROS-induced ferroptosis is critical for tumor growth in the absence of common ferroptosis inducers; strikingly, loss of PHLDA2 abrogates ROS-induced ferroptosis and promotes tumor growth but has no obvious effect in normal tissues in both immunodeficient and immunocompetent mouse tumor models. These data demonstrate that PHLDA2-mediated PA peroxidation triggers a distinct ferroptosis response critical for tumor suppression and reveal that PHLDA2-mediated ferroptosis occurs naturally invivo without any treatment from ferroptosis inducers.

Effects of Sulforaphene on the Cariogenic Properties of Streptococcus Mutans In Vitro and Dental Caries Development In Vivo.

Sulforaphene (SFE) is a common nutritional supplement with antibacterial, anti-cancer, and anti-inflammatory effects. However, the effects of SFE on the cariogenicity of Streptococcus mutans and dental caries have not been reported. The objectives of this study were to investigate the caries-controlling potential of SFE. The effects of SFE on S. mutans were investigated using the broth microdilution method, crystal violet staining, SEM observation, acid tolerance assays, lactic acid quantification, and polysaccharide measurements. A rat caries model was established to evaluate the caries-controlling effects and biocompatibility of SFE in vivo. SFE inhibited S. mutans growth and biofilm formation. Furthermore, SFE restrained the cariogenic properties of S. mutans, including its acid production, acid tolerance, and extracellular polysaccharide production, without affecting the bacterial viability at sub-inhibitory levels. In the rat caries model, SFE significantly arrested the onset and development of dental caries. Moreover, no visible hemolytic phenomenon or cytotoxicity was detected in the SFE groups. After four weeks of SFE treatment, all rats remained in apparent good health with no significant differences in weight gain; their hemogram and biochemical parameters were normal; no pathological changes were observed in the oral mucosa, liver, or kidneys. In conclusion, SFE was safe and inhibited the development of caries effectively.

A novel intergenic enhancer that regulates Bdnf expression in developing cortical neurons

Brain-derived neurotrophic factor (BDNF) promotes neuronal differentiation and survival and is implicated in the pathogenesis of many neurological disorders. Here, we identified a novel intergenic enhancer located 170 kb from the Bdnf gene, which promotes the expression of Bdnf transcript variants during mouse neuronal differentiation and activity. Following Bdnf activation, enhancer-promoter contacts increase, and the region moves away from the repressive nuclear periphery. Bdnf enhancer activity is necessary for neuronal clustering and dendritogenesis invitro, and for cortical development invivo. Our findings provide the first evidence of a regulatory mechanism whereby the activation of a distal enhancer promotes Bdnf expression during brain development.

Loss of Apc Cooperates with Activated Oncogenes to Induce Liver Tumor Formation in Mice

Hepatocellular carcinoma (HCC) and hepatoblastoma are the major types of primary liver cancer in adulthood and childhood, respectively. Wnt/β-catenin signaling deregulation is one of the most frequent genetic events in hepatocarcinogenesis. APC regulator of WNT signaling pathway (APC) encodes an inhibitor of the Wnt cascade and acts as a tumor suppressor. Germline defects of the APC gene lead to familial adenomatous polyposis, and its somatic mutations occur in multiple tumor types. However, the contribution of APC in hepatocarcinogenesis remains unclear. Therefore, APC mutations and expression patterns were examined in human HCC and hepatoblastoma samples. Whether loss of Apc alone or in cooperation with other oncogenes triggers liver tumor development invivo was also investigated. sgApc alone could not drive liver tumor formation, but synergized with activated oncogenes (YapS127A, TazS89A, and c-Met) to induce hepatocarcinogenesis. Mechanistically, Apc deletion induced the activation of β-catenin and its downstream targets in mouse liver tumors. Furthermore, Ctnnb1 ablation or TCF4-mediated transcription blockade completely prevented liver tumor formation, indicating the requirement of a functional β-catenin pathway for loss of Apc-driven hepatocarcinogenesis. This study shows that a subset of HCC patients with loss-of-function APC mutations mightbenefit from therapeutic strategies targeting the Wnt/β-catenin pathway.

Magnolol Suppresses Pancreatic Cancer Development In Vivo and In Vitro via Negatively Regulating TGF-β/Smad Signaling.

Magnolol, a hydroxylated biphenyl extracted from Magnolia officinalis, has recently drawn attention due to its anticancer potential. The present study was aimed to explore the effects of Magnolol on restraining the proliferation, migration and invasion of pancreatic cancer in vivo and in vitro. Magnolol showed significant anti-growth effect in an orthotopic xenograft nude mouse model, and immunohistochemical staining of the xenografts revealed that Magnolol suppressed vimentin expression and facilitated E-cadherin expression. The cytoactive detection using CCK-8 assay showed Magnolol inhibited PANC-1 and AsPC-1 concentration-dependently. Scratch healing assay and the Transwell invasion assay proved the inhibiting effects of Magnolol on cellular migration and invasion at a non-cytotoxic concentration. Western blot and rt-PCR showed that Magnolol suppressed epithelial-mesenchymal-transition by increasing the expression level of E-cadherin and decreasing those of N-cadherin and vimentin. Magnolol suppressed the TGF-β/Smad pathway by negatively regulating phosphorylation of Smad2/3. Moreover, TGF-β1 impaired the antitumor effects of Magnolol in vivo. These results demonstrated that Magnolol can inhibit proliferation, migration and invasion in vivo and in vitro by suppressing the TGF-β signal pathway and EMT. Magnolol could be a hopeful therapeutic drug for pancreatic malignancy.

ADORA1 Inhibition Promotes Tumor Immune Evasion by Regulating the ATF3-PD-L1 Axis

Here, we show that tumor ADORA1 deletion suppresses cell growth in human melanoma cell lines invitro and tumor development invivo in immune-deficient xenografts. However, this deletion induces the upregulation of PD-L1 levels, which inactivates cocultured Tcells invitro, compromises anti-tumor immunity invivo, and reduces anti-tumor efficacy in an immune-competent mouse model. Functionally, PD-1 mAb treatment enhances the efficacy of ADORA1-deficient or ADORA1 antagonist-treated melanoma and NSCLC immune-competent mouse models. Mechanistically, we identify ATF3 as the factor transcriptionally upregulating PD-L1 expression. Tumor ATF3 deletion improves the effect of ADORA1 antagonist treatment of melanoma and NSCLC xenografts. We observe higher ADORA1, lower ATF3, and lower PD-L1 expression levels in tumor tissues from nonresponders among PD-1 mAb-treated NSCLC patients.

PSMP/MSMP promotes hepatic fibrosis through CCR2 and represents a novel therapeutic target

C-C motif chemokine receptor 2 (CCR2) has been recognized as a promising target for the treatment of liver fibrosis. PC3-secreted microprotein (PSMP)/microseminoprotein (MSMP) is a novel chemotactic cytokine and its receptor is CCR2. In the present study we investigated the expression and role of PSMP in liver fibrosis/cirrhosis. PSMP expression was studied in patients with fibrosis/cirrhosis and in 3 murine models of liver fibrosis, including mice treated with carbon tetrachloride (CCl4), bile-duct ligation, or a 5-diethoxycarbonyl-1,4-dihydrocollidine diet. The role of PSMP was evaluated in Psmp-/- mice and after treatment with a PSMP antibody in wild-type mice. The direct effects of PSMP on macrophages and hepatic stellate cells were studied invitro. In this study, we found that PSMP was highly expressed in fibrotic/cirrhotic tissues from patients with different etiologies of liver disease and in the 3 experimental mouse models of fibrosis. Damage-associated molecular pattern molecules HMGB-1 and IL-33 induced hepatocytes to produce PSMP. PSMP deficiency resulted in a marked amelioration of hepatic injury and fibrosis. In CCl4-induced hepatic injury, the infiltration of macrophages and CCR2+ monocytes into the liver was significantly decreased in Psmp-/- mice. Consistent with the decreased levels of intrahepatic macrophages, proinflammatory cytokines were significantly reduced. Moreover, adeno-associated virus-8 vectors successfully overexpressing human PSMP in Psmp-/- mouse livers could reverse the attenuation of liver injury and fibrosis induced by CCl4 in a CCR2-dependent manner. Treatment with a specific PSMP-neutralizing antibody, 3D5, prevented liver injury and fibrosis induced by CCl4 in mice. At the cellular level, PSMP directly promoted M1 polarization of macrophages and activation of LX-2 cells. PSMP enhances liver fibrosis through its receptor, CCR2. PSMP is a potentially attractive therapeutic target for the treatment of patients with liver fibrosis. Our present study identifies the essential role of the protein PSMP for the development and progression of liver fibrosis in humans and mice. PSMP promotes liver fibrosis through inflammatory macrophage infiltration, polarization and production of proinflammatory cytokines, as well as direct activation of hepatic stellate cells via its receptor CCR2. A PSMP antibody can significantly reduce liver fibrosis development invivo. These findings indicate that PSMP is a potential therapeutic target and its antibody is a potential therapeutic agent for the treatment of liver fibrosis.

Deciphering the Role of Locally Disordered DNA Methylation on CLL Development In Vivo

Large-scale DNA methylation analysis of chronic lymphocytic leukemia (CLL) has identified a pervasive genome-wide level of discordance in local methylation state in leukemic cells compared to normal B cells. This is associated with variation in gene expression, increased clonal evolution and poorer clinical outcomes. We hypothesized that locally disordered methylation could lead to dysregulation of gene expression and hence contribute to cancer development and progression. To test this, we have engineered mouse lines with B-cell restricted homozygous or heterozygous knock-out of Dnmt3a by crossing Dnmt3a-floxed mice with CD19-Cre mice. Dnmt3a is a DNA methyltransferase, catalyzing the addition of a methyl group to CpG sequences in the DNA and thereby regulating gene expression. Although DNMT3A mutations are only rarely identified in CLL, RNA sequencing and protein expression analysis reveal dysregulation of DNMT3A. We confirmed partial or complete reduction in Dnmt3a protein levels in B cells from CD19-Cre;Dnmt3a heterozygous (Dnmt3a-het) and CD19-Cre;Dnmt3a homozygous mice (Dnmt3a-hom), respectively. These mice therefore provide a unique opportunity to study B cell restricted changes in locally discordant methylation over time. We first assessed the impact of Dnmt3a deletion on normal B cell development, prior to CLL development, by characterizing splenic B cell of CD19-Cre (control) or Dnmt3a-hom mice. Flow cytometry data using B220, CD21 and CD23 markers to identify B220+CD23+CD21- follicular B cells and B220+CD23+CD21high marginal zone B cells revealed elevated levels of follicular B cells (83.1% vs 87.6%, p=0.008) and reduced levels of marginal zone B cells (9.6% vs 4.1%, p=0.001) in Dnmt3a-hom mice in comparison to control mice (n=3 mice per group). These results indicate that mice with Dnmt3a deletion present with massive changes in their B cells, even prior to overt CLL development. We next monitored both Dnmt3a-het and Dnmt3a-hom cohorts over time for CLL development. We observed that 100% Dnmt3a-hom mice developed CLL-like disease by 7 months (n=23), characterized by CD5+B220+;Igk+ expression and evident within the blood, bone marrow (BM), spleen and peritoneum, suggesting a fundamental role of altered DNMT3A expression in generation of CLL. In comparison, 75% of Dnmt3a-het mice developed CLL-like disease by 18 months (n=12), with similar expansion of CD5+C220+ expansion in the BM and spleen. By RNA-sequencing analysis of normal splenic B cells from CD19-Cre and Dnmt3a-hom mice (n=3 mice, 10 weeks old), we detected substantial changes in gene expression, including 113 upregulated genes and 39 downregulated (p<0.05, FC>2). To explore the development of locally disordered methylation following transformation, CLL cells from Dnmt3a-hom mice (n=3) were subjected to reduced representation bisulfite sequencing (RRBS), a high-throughput technique to analyze genome wide methylation patterns. We found that murine CLL-like cells display locally disordered methylation, which was detected in all genomic features covered by this assay, indicating that disordered methylation is broadly affecting the murine CLL cells' epigenome. Additionally, we identified a set of differentially methylated regions (DMRs) between B cells from CD19-Cre vs CLL cells from Dnmt3a-hom (n = 2,839 DMRs), with a minimum difference of 0.2 and a minimum of 10 CpGs per DMR. Interestingly, gene ontology analysis demonstrated strong association with genes hypermethylated in TCL1 mouse model, linking this model with alternative murine models for CLL. In conclusion, we have studied B cell specific deletion of Dntm3a and showed the development of CLL in 100% of the case in Dnmt3a-hom mice. Our data suggest a fundamental role for Dnmt3a in CLL development through increased locally disordered methylation and changes in associated transcriptional signatures. This mouse model provides an exciting experimental model to undertake functional in vivo studies in order to elucidate the contribution of epigenetic changes on CLL development. Disclosures Neuberg: Pharmacyclics: Research Funding; Madrigal Pharmaceuticals: Equity Ownership; Celgene: Research Funding. Wu:Neon Therapeutics: Other: Member, Advisory Board; Pharmacyclics: Research Funding.

Improved Retinal Organoid Differentiation by Modulating Signaling Pathways Revealed by Comparative Transcriptome Analyses with Development InVivo.

SummaryStem cell-derived retinal organoids recapitulate many landmarks of in vivo differentiation but lack functional maturation of distinct cell types, especially photoreceptors. Using comprehensive temporal transcriptome analyses, we show that transcriptome shift from postnatal day 6 (P6) to P10, associated with morphogenesis and synapse formation during mouse retina development, was not evident in organoids, and co-expression clusters with similar patterns included different sets of genes. Furthermore, network analysis identified divergent regulatory dynamics between developing retina in vivo and in organoids, with temporal dysregulation of specific signaling pathways and delayed or reduced expression of genes involved in photoreceptor function(s) and survival. Accordingly, addition of docosahexaenoic acid and fibroblast growth factor 1 to organoid cultures specifically promoted the maturation of photoreceptors, including cones. Our study thus identifies regulatory signals deficient in developing retinal organoids and provides experimental validation by producing a more mature retina in vitro, thereby facilitating investigations in disease modeling and therapies.

The Long Noncoding RNA Pnky Is a Trans-acting Regulator of Cortical Development In Vivo

While it is now appreciated that certain long noncoding RNAs (lncRNAs) have important functions in cell biology, relatively few have been shown to regulate development invivo, particularly with genetic strategies that establish cis versus trans mechanisms. Pnky is a nuclear-enriched lncRNA that is transcribed divergently from the neighboring proneural transcription factor Pou3f2. Here, we show that conditional deletion of Pnky from the developing cortex regulates the production of projection neurons from neural stem cells (NSCs) in a cell-autonomous manner, altering postnatal cortical lamination. Surprisingly, Pou3f2 expression is not disrupted by deletion of the entire Pnky gene. Moreover, expression of Pnky from a BAC transgene rescues the differential gene expression and increased neurogenesis of Pnky-knockout NSCs, as well as the developmental phenotypes of Pnky-deletion invivo. Thus, despite being transcribed divergently from a key developmental transcription factor, the lncRNA Pnky regulates development in trans.

Morphology, developmental stages and quality parameters of in vitro-produced equine embryos.

Intracytoplasmic sperm injection (ICSI) is used to produce equine embryos invitro. The speed of embryo development invitro is roughly equivalent to what has been described for embryos produced invivo. Morphological evaluations of ICSI-produced embryos are complicated by the presence of debris and the dark nature of equine embryo cytoplasm. Morulas and early blastocysts produced invitro appear similar to those produced invivo. However, with expansion of the blastocyst, distinct differences are observed compared with uterine embryos. In culture, embryos do not undergo full expansion and thinning of the zona pellucida (ZP) or capsule formation. Cells of the inner cell mass (ICM) are dispersed, in contrast with the differentiated trophoblast and ICM observed in embryos collected from uteri. As blastocysts expand invitro, embryo cells often escape the ZP as organised or disorganised extrusions of cells, probably through the hole incurred during ICSI. Quality assessment of invitro-produced early stage equine embryos is in its infancy, because limited information is available regarding the relationship between morphology and developmental competence. Early embryo development invivo is reviewed in this paper, with comparisons made to embryo development invitro and clinical assessments from a laboratory performing commercial ICSI for >15 years.

MicroRNA miR-181a/b-1 controls MAIT cell development.

Mucosal-associated invariant T (MAIT) cells constitute a major fraction of innate-like T cells in humans with critical roles in defense against microbial pathogens and in maintaining mucosal integrity. However, the molecular mechanisms underlying MAIT cell development remain largely elusive. Here we investigated the role of miR-181a/b-1, a pair of microRNAs that serve as rheostat of TCR signal strength, in this process. Loss of miR-181a/b-1 in mice resulted in a profound arrest in early MAIT cell development. As a consequence, in the absence of miR-181a/b-1, thymic MAIT cells failed to acquire functional maturity based on expression of transcription factors PLZF, T-bet and RORγt. Temporal analysis of development using a molecular timer in combination with loss of miR-181a/b-1 revealed that MAIT cells complete functional maturation in the periphery and indicates that functionally mature MAIT cells in the thymus are long-term resident cells. Thus, our study provides insight into the dynamics of MAIT cell development invivo. Of note, deletion of miR-181a/b-1 alone completely mirrored loss of all miRNAs.

TFEB protects nucleus pulposus cells against apoptosis and senescence via restoring autophagic flux

Excessive apoptosis and senescence of nucleus pulposus (NP) cells are major pathological changes in intervertebral disc degeneration (IVDD) development; previous studies demonstrated pharmacologically or genetically stimulation of autophagy may inhibit apoptosis and senescence in NP cells. Transcription factor EB (TFEB) is a master regulator of autophagic flux via initiating autophagy-related genes and lysosomal biogenesis. This study was performed to confirm whether TFEB was involved in IVDD development and its mechanism. TFEB activity was detected in NP tissues in puncture-induced rat IVDD model by immunofluorescence as well as in tert-Butyl hydroperoxide (TBHP), the reactive oxygen species (ROS) donor to induce oxidative stress, treated NP cells by western blot. After TFEB overexpression in NP cells with lentivirus transfection, autophagic flux, apoptosis and senescence percentage were assessed. In invivo study, the lentivirus-normal control (LV-NC) or lentivirus-TFEB (LV-TFEB) were injected into the center space of the NP tissue, after 4 or 8 weeks, Magnetic resonance imaging (MRI), X ray, Hematoxylin-Eosin (HE) and Safranin O staining were used to evaluate IVDD grades. The nuclear localization of TFEB declined in degenerated rat NP tissue as well as in TBHP treated NP cells. Applying lentivirus to transfect NP cells, TFEB overexpression restored the TBHP-induced autophagic flux blockage and protected NP cells against apoptosis and senescence; these protections of TFEB are diminished by chloroquine-medicated autophagy inhibition. Furthermore, TFEB overexpression ameliorates the puncture-induced IVDD development in rats. Experimental IVDD inhibited the TFEB activity. TFEB overexpression suppressed TBHP-induced apoptosis and senescence via autophagic flux stimulation in NP cell and alleviates puncture-induced IVDD development invivo.

NEDD4 Is Involved in Inflammation Development during Keloid Formation

Keloids mark a chronic inflammatory disease characterized by a fibroproliferative disorder of the skin. A genome-wide association study showed that single-nucleotide polymorphism rs8032158 in the neural precursor cell-expressed NEDD4 gene, which has six protein-coding transcript variants (TVs), is genetically linked to keloids. Here, we show that the high frequency of risk allele C in rs8032158 in keloid patients is associated with a selectively higher expression of TV3 of NEDD4 to activate the NF-κB pathway. Comparisons of keloid scars with normal skin samples that do not have the single-nucleotide polymorphism allele and were derived from different anatomical sites showed stronger expressions of NEDD4 TV3 and activated forms of NF-κB and STAT3 in keloid scars. Forced expression or selective knockdown of NEDD4 TV3 increased or decreased NF-κB activation invitro. Furthermore, NEDD4 knockdown suppressed NF-κB-dependent inflammation development invivo. Mechanistic analysis showed that NEDD4 TV3 is involved in NF-κB activation through its association with the adaptor protein RIP. These results suggest that NEDD4 TV3 is a potential diagnostic marker and therapeutic target for chronic skin diseases, including keloid.

Targeting the TREK-1 potassium channel via riluzole to eliminate the neuropathic and depressive-like effects of oxaliplatin

Neurotoxicity remains the most common adverse effect of oxaliplatin, limiting its clinical use. In the present study, we developed a mouse model of chronic oxaliplatin-induced neuropathy, which mimics both sensory and motor deficits observed in patients, in a clinically relevant time course. Repeated oxaliplatin administration in mice induced both cephalic and extracephalic long lasting mechanical and cold hypersensitivity after the first injection as well as delayed sensorimotor deficits and a depression-like phenotype. Using this model, we report that riluzole prevents both sensory and motor deficits induced by oxaliplatin as well as the depression-like phenotype induced by cumulative chemotherapeutic drug doses. All the beneficial effects are due to riluzole action on the TREK-1 potassium channel, which plays a central role in its therapeutic action. Riluzole has no negative effect on oxaliplatin antiproliferative capacity in human colorectal cancer cells and on its anticancer effect in a mouse model of colorectal cancer. Moreover, riluzole decreases human colorectal cancer cell line viability invitro and inhibits polyp development invivo. The present data in mice may support the need to clinically test riluzole in oxaliplatin-treated cancer patients and state for the important role of the TREK-1 channel in pain perception.

An Unusual Prohibitin Regulates Malaria Parasite Mitochondrial Membrane Potential

Proteins of the stomatin/prohibitin/flotillin/HfIK/C (SPFH) family are membrane-anchored and perform diverse cellular functions in different organelles. Here, we investigate the SPFH proteins of the murine malaria model parasite Plasmodium berghei, the conserved prohibitin 1, prohibitin 2, and stomatin-like protein and an unusual prohibitin-like protein (PHBL). The SPFH proteins localize to the parasite mitochondrion. While the conserved family members could not be deleted from the Plasmodium genome, PHBL was successfully ablated, resulting in impaired parasite fitness and attenuated virulence in the mammalian host. Strikingly, PHBL-deficient parasites fail to colonize the Anopheles vector because of complete arrest during ookinete development invivo. We show that this arrest correlates with depolarization of the mitochondrial membrane potential (ΔΨmt). Our results underline the importance of SPFH proteins in the regulation of core mitochondrial functions and suggest that fine-tuning of ΔΨmt in malarial parasites is critical for colonization of the definitive host.

Transcription Factor IRF8 Governs Enhancer Landscape Dynamics in Mononuclear Phagocyte Progenitors

Monocytes and dendritic cells (DCs), mononuclearphagocytes essential for immune responses, develop from hematopoietic stem cells via monocyte-DC progenitors (MDPs). The molecular basis of their development remains unclear. Because promoter-distal enhancers are key to cell fate decisions, we analyzed enhancer landscapes during mononuclear phagocyte development invivo. Monocyte- and DC-specific enhancers were gradually established at progenitor stages before the expression of associated genes. Of the transcription factors predicted to bind to these enhancers, IRF8, essential for monocyte and DC development, was found to be required for the establishment of these enhancers, particularly those common to both monocyte and DC lineages. Although Irf8-/- mononuclear phagocyte progenitors, including MDPs, displayed grossly normal gene expression patterns, their enhancer landscapes resembled that of an upstream progenitor population. Our results illustrate the dynamic process by which key transcription factors regulate enhancer formation and, therefore, direct future gene expression to achieve mononuclear phagocyte development.

Natural Parasite Exposure Induces Protective Human Anti-Malarial Antibodies

Antibodies against the NANP repeat of circumsporozoite protein (CSP), the major surface antigen of Plasmodium falciparum (Pf) sporozoites, can protect from malaria in animal models but protective humoral immunity is difficult to induce in humans. Here wecloned and characterized rare affinity-matured human NANP-reactive memory B cell antibodies elicited by natural Pf exposure that potently inhibited parasite transmission and development invivo. We unveiled the molecular details of antibody binding to two distinct protective epitopes within the NANP repeat. NANP repeat recognition was largely mediated by germline encoded and immunoglobulin (Ig) heavy-chain complementarity determining region 3 (HCDR3) residues, whereas affinity maturation contributed predominantly to stabilizing the antigen-binding site conformation. Combined, our findings illustrate the power of exploring human anti-CSP antibody responses to develop tools for malaria control in the mammalian and the mosquito vector and provide a molecular basis for the structure-based design of next-generation CSP malaria vaccines.