Abstract

BACKGROUND:

Metabolic reprogramming in cancer encompasses the insulin receptor (IR) as a player of energy homeostasis and proliferation. We aimed to characterize vascular (VIR) and epithelial (EIR) IR expression in CRC and correlate it with clinico-pathological parameters and survival.

METHODS:

1580 primary CRCs were explored by immunohistochemistry for evaluation of VIR and EIR. Subgroup analyses included in situhybridization for IR isoform A (IR-A) and DNA mismatch repair protein immunohistochemistry. Clinico-pathological and survival parameters were studied.

RESULTS:

High VIR was evident in 63.5% of all CRC samples and was associated with T-stage (P = 0.005). EIR was present in 72.2% and was associated with lower T-stages (P = 0.006) and UICC-stages (P < 0.001). EIR negativity was associated with increased metastasis (P =0.028), nodal spread (P < 0.001), lymphatic invasion (P = 0.008) and a decreased tumor-specific (P = 0.011) and overall survival (P = 0.007; 95%-C.I.: 44.5-84.1). EIR negativity in UICC-stage II was associated with a significantly worse tumor-specific (P = 0.045) and overall (P =0.043) survival. IR-A was expressed in CRC vessels and cells.

CONCLUSIONS:

We demonstrate VIR to be frequent in CRC and characterize EIR negativity as an important prognostic risk factor. The association between EIR negativity and worse survival in UICC-stage II should be prospectively evaluated for an application in therapeutic algorithms.

NUCB2/nesfatin-1 is first known to be expressed in the hypothalamus while controlling appetite and energy metabolism. However, recent studies have shown that NUCB2/nesfatin-1 was expressed in the various organs as well as the hypothalamus. Our previous reports also demonstrated that NUCB2/nesfatin-1 was expressed in the ovary, testis, pituitary gland, lung, kidney, and stomach of fetal and adult mice. However, the role of NUCB2/nesfatin-1 in mouse fetus remains unknown. Thus, the aim of this study was to investigate whether NUCB2/nestatin-1 is expressed in mouse fetus at the developmental stage in which organogenesis begins. To do this, we performed in situhybridization (ISH) and immunohistochemistry (IHC) staining to examine the distribution of NUCB2 mRNA and nesfatin-1 protein in the mouse fetal organs during early developmental stages, especially at embryonic day (E) 10.5. As a result of ISH, NUCB2 mRNA positive signals were more frequent in the liver, but there were relatively few positive signals in heart. On the other hand, no positive signals were detected in other organs. These ISH results were validated by IHC staining and qRT-PCR analysis. Expression of nesfatin-1 protein detected by IHC staining was similar to that of NUCB2 mRNA detected by ISH in the liver and heart. In addition, the levels of NUCB2 mRNA expression analyzed by qRT-PCR were significantly increased in the liver and heart compared to other organs of the mouse fetus at E13.5, whereas its level was extensively decreased in the liver, but increased in the lung, stomach, and kidney of the mouse fetus at E17.5. These results suggest that NUCB2/nesfatin-1 may play an important role in liver and heart development and physiological functions in the developmental process of mouse fetus. Further studies are needed on the function of NUCB2/nesfatin-1, which is highly expressed in the various organs, including liver and heart during mouse development.

The central amygdala (CeA) is important for fear responses to discrete cues. Recent findings indicate that the CeA also contributes to states of sustained apprehension that characterize anxiety, although little is known about the neural circuitry involved. The stress neuropeptide corticotropin releasing factor (CRF) is anxiogenic and is produced by subpopulations of neurons in the lateral CeA and the dorsolateral bed nucleus of the stria terminalis (dlBST). Here we investigated the function of these CRF neurons in stress-induced anxiety using chemogenetics in male rats that express Cre recombinase from a Crh promoter. Anxiety-like behavior was mediated by CRF projections from the CeA to the dlBST and depended on activation of CRF1 receptors and CRF neurons within the dlBST. Our findings identify a CRFCeA→CRFdlBST circuit for generating anxiety-like behavior and provide mechanistic support for recent human and primate data suggesting that the CeA and BST act together to generate states of anxiety.SIGNIFICANCE STATEMENT Anxiety is a negative emotional state critical to survival, but persistent, exaggerated apprehension causes substantial morbidity. Identifying brain regions and neurotransmitter systems that drive anxiety can help in developing effective treatment. Much evidence in rodents indicates that neurons in the bed nucleus of the stria terminalis (BST) generate anxiety-like behaviors, but more recent findings also implicate neurons of the CeA. The neuronal subpopulations and circuitry that generate anxiety are currently subjects of intense investigation. Here we show that CeA neurons that release the stress neuropeptide corticotropin-releasing factor (CRF) drive anxiety-like behaviors in rats via a pathway to dorsal BST that activates local BST CRF neurons. Thus, our findings identify a CeA→BST CRF neuropeptide circuit that generates anxiety-like behavior.

Historically, the rat has been the preferred animal model for behavioral studies. Limitations in genome modification have, however, caused a lag in their use compared to the bevy of available transgenic mice. Here, we have developed several transgenic tools, including viral vectors and transgenic rats, for targeted genome modification in specific adult rat neurons using CRISPR-Cas9 technology. Starting from wild-type rats, knockout of tyrosine hydroxylase was achieved with adeno-associated viral (AAV) vectors expressing Cas9 or guide RNAs (gRNAs). We subsequently created an AAV vector for Cre-dependent gRNA expression as well as three new transgenic rat lines to specifically target CRISPR-Cas9 components to dopaminergic neurons. One rat represents the first knockin rat model made by germline gene targeting in spermatogonial stem cells. The rats described herein serve as a versatile platform for making cell-specific and sequence-specific genome modifications in the adult brain and potentially other Cre-expressing tissues of the rat.

Preclinical studies have demonstrated that the endocannabinoid system (ECS) plays an important role in the protection against intestinal inflammation and colorectal cancer (CRC); however, human data are scarce. We determined members of the ECS and related components of the 'endocannabinoidome' in patients with inflammatory bowel disease (IBD) and CRC, and compared them to control subjects. Anandamide (AEA) and oleoylethanolamide (OEA) were increased in plasma of ulcerative colitis (UC) and Crohn's disease (CD) patients while 2-arachidonoylglycerol (2-AG) was elevated in patients with CD, but not UC. 2-AG, but not AEA, PEA and OEA, was elevated in CRC patients. Lysophosphatidylinositol (LPI) 18:0 showed higher levels in patients with IBD than in control subjects whereas LPI 20:4 was elevated in both CRC and IBD. Gene expression in intestinal mucosal biopsies revealed different profiles in CD and UC. CD, but not UC patients, showed increased gene expression for the 2-AG synthesizing enzyme diacylglycerol lipase alpha. Transcripts of CNR1 and GPR119 were predominantly decreased in CD. Our data show altered plasma levels of endocannabinoids and endocannabinoid-like lipids in IBD and CRC and distinct transcript profiles in UC and CD. We also report alterations for less known components in intestinal inflammation, such as GPR119, OEA and LPI.

Arthrogryposis is a group of phenotypically and genetically heterogeneous disorders characterized by congenital contractures of two or more parts of the body; the pathogenesis and the causative genes of arthrogryposis remain undetermined. We examined a four-generation arthrogryposis pedigree characterized by camptodactyly, limited forearm supination, and loss of myofibers in the forearms and hands. By using whole-exome sequencing, we confirmed MET p.Y1234C mutation to be responsible for arthrogryposis in this pedigree. MET p.Y1234C mutation caused the failure of activation of MET tyrosine kinase. A Met p.Y1232C mutant mouse model was established. The phenotypes of homozygous mice included embryonic lethality and complete loss of muscles that originated from migratory precursors. Heterozygous mice were born alive and showed reduction of the number of myofibers in both appendicular and axial muscles. Defective migration of muscle progenitor cells and impaired proliferation of secondary myoblasts were proven to be responsible for the skeletal muscle dysplasia of mutant mice. Overall, our study shows MET to be a causative gene of arthrogryposis and MET mutation could cause skeletal muscle dysplasia in human beings.

The central melanocortin system plays a crucial role in the control of energy balance. Although the decreased energy expenditure and increased adiposity of melanocortin-3 receptor (Mc3R)-null mice suggest the importance of Mc3R-regulated neurons in energy homeostasis, the roles for specific subsets of Mc3R neurons in energy balance have yet to be determined. Because the lateral hypothalamic area (LHA) contributes to the control of energy expenditure and feeding, we generated Mc3rcre mice to determine the roles of LHA Mc3R (Mc3RLHA) neurons in energy homeostasis. We found that Mc3RLHA neurons overlap extensively with LHA neuron markers that contribute to the control of energy balance (neurotensin, galanin, and leptin receptor) and project to brain areas involved in the control of feeding, locomotion, and energy expenditure, consistent with potential roles for Mc3RLHA neurons in these processes. Indeed, selective chemogenetic activation of Mc3RLHA neurons increased locomotor activity and augmented refeeding after a fast. Although the ablation of Mc3RLHA neurons did not alter food intake, mice lacking Mc3RLHA neurons displayed decreased energy expenditure and locomotor activity, along with increased body mass and adiposity. Thus, Mc3R neurons lie within LHA neurocircuitry that modulates locomotor activity and energy expenditure and contribute to energy balance control.

Colorectal cancer (CRC) is one of the most common and lethal types of cancer worldwide. Multiple lines of evidence have illustrated that long non‑coding RNAs (lncRNAs) are critical molecules in the regulation of CRC development and progression. The identification of new lncRNAs is expected to provide new biomarkers and potential therapeutic targets for CRC diagnosis and prevention. Previous research has revealed that, ENST00000547547, a 434‑bp lncRNA on human chromosome12q15 (RP11‑611E13.3‑001), was found to be downregulated in CRC tissues from lncRNA microarray studies. However, its role in the development and progression of CRC remains largely unknown. In the present study, we demonstrated that the ENST00000547547 level is significantly downregulated in CRC tissues compared to normal tissues. Furthermore, the overexpression of ENST00000547547 inhibited the cell proliferation, invasion and migration of CRC cells in vitro and decreased tumorigenesis ability in vivo. Furthermore, we provided the first evidence that ENST00000547547 inhibited the proliferation of CRC cells by affecting the cell cycle and apoptosis. Finally, we demonstrated that the epithelial‑mesenchymal transition (EMT) process was associated with the inhibitory effects of ENST00000547547 on the invasion and migration of CRC cells. In summary, the present study indicated that lncRNA ENST00000547547 acted as a tumor suppressor in CRC, which may provide us with a new biomarker for CRC prognosis and a potential therapeutic target for molecular cancer therapy.

Taste buds develop in different regions of the mammal oral cavity. Adult stem cells in various organs including the tongue papillae are marked by leucine-rich repeat-containing G protein-coupled receptor 5 (Lgr5) and its homolog, Lgr6. Recent studies have reported that adult taste stem/progenitor cells in circumvallate papilla (CVP) on the posterior tongue are Lgr5-positive. In this study, we confirm the Lgr5 expression pattern during CVP development. A previous study reported that mesenchymal Fgf10 is necessary for maintaining epithelial Lgr5-positive stem/progenitor cells. To confirm the interaction between Lgr5-positive CVP epithelium and mesenchymal factor FGF10, reverse recombination (180-degree) was performed after tongue epithelium detachment. FGF10 protein-soaked bead implantation was performed after reverse recombination to rescue CVP development. Moreover, we reduced mesenchymal Fgf10 by BIO and SU5402 treatment which disrupted CVP morphogenesis. This study suggests that the crosstalk between epithelial Lgr5 and mesenchymal Fgf10 plays a pivotal role in CVP epithelium invagination during mouse tongue CVP development by maintaining Lgr5-positive stem/progenitor cells.

Zoonotic influenza A viruses of avian origin can cause severe disease in individuals, or even global pandemics, and thus pose a threat to human populations. Waterfowl and shorebirds are believed to be the reservoir for all influenza A viruses, but this has recently been challenged by the identification of novel influenza A viruses in bats1,2. The major bat influenza A virus envelope glycoprotein, haemagglutinin, does not bind the canonical influenza A virus receptor, sialic acid or any other glycan1,3,4, despite its high sequence and structural homology with conventional haemagglutinins. This functionally uncharacterized plasticity of the bat influenza A virus haemagglutinin means the tropism and zoonotic potential of these viruses has not been fully determined. Here we show, using transcriptomic profiling of susceptible versus non-susceptible cells in combination with genome-wide CRISPR-Cas9 screening, that the major histocompatibility complex class II (MHC-II) human leukocyte antigen DR isotype (HLA-DR) is an essential entry determinant for bat influenza A viruses. Genetic ablation of the HLA-DR α-chain rendered cells resistant to infection by bat influenza A virus, whereas ectopic expression of the HLA-DR complex in non-susceptible cells conferred susceptibility. Expression of MHC-II from different bat species, pigs, mice or chickens also conferred susceptibility to infection. Notably, the infection of mice with bat influenza A virus resulted in robust virus replication in the upper respiratory tract, whereas mice deficient for MHC-II were resistant. Collectively, our data identify MHC-II as a crucial entry mediator for bat influenza A viruses in multiple species, which permits a broad vertebrate tropism.

The dysregulation of autophagy, an evolutionarily conserved lysosomal degradation process, has been implicated in a wide variety of human diseases, and thus, small chemicals that modulate autophagy have therapeutic potential. Here, we assessed the ability of active components isolated from Bupleurum falcatum, a popular Chinese herb, to modulate autophagy. We found that saikosaponin D (SsD) and A (SsA) but not C (SsC) potently and reversibly inhibited the fusion of autophagosomes and lysosomes, resulting in the accumulation of autophagosomes, an increased lysosomal pH, and TFEB nuclear translocation. RAB5A knockdown or the expression of a dominant-negative RAB5 mutant significantly reduced the ability of SsD or SsA to block autophagy. Enterovirus A71 (EV-A71), the cause of hand-foot-mouth disease, has been shown to induce autophagy. We found that SsD potently inhibited EV-A71 RNA replication and subsequent viral protein synthesis, thereby preventing EV-A71-induced cell death. ATG5 knockdown inhibited EV-A71 viral protein synthesis, whereas autophagy induction by rapamycin promoted synthesis. Taken together, our data indicate that SsD and SsA are potent late-stage autophagy inhibitors that can be used to prevent EV-A71 infection.

Cortical networks are characterized by sparse connectivity, with synapses found at only a subset of axo-dendritic contacts. Yet within these networks, neurons can exhibit high connection probabilities, suggesting that cell-intrinsic factors, not proximity, determine connectivity. Here, we identify ephrin-B3 (eB3) as a factor that determines synapse density by mediating a cell-cell competition that requires ephrin-B-EphB signaling. In a microisland culture system designed to isolate cell-cell competition, we find that eB3 determines winning and losing neurons in a contest for synapses. In a Mosaic Analysis with Double Markers (MADM) genetic mouse model system in vivo the relative levels of eB3 control spine density in layer 5 and 6 neurons. MADM cortical neurons in vitro reveal that eB3 controls synapse density independently of action potential-driven activity. Our findings illustrate a new class of competitive mechanism mediated by trans-synaptic organizing proteins which control the number of synapses neurons receive relative to neighboring neurons.