Determination of the Interaction and Pharmacological Modulation of MCHR1 Signaling by the C-Terminus of MRAP2 Protein.

Melanin concentrating hormone (MCH), an orexigenic neuropeptide, is primarily secreted by the hypothalamus and acts on its receptor, the melanin-concentrating hormone receptor 1 (MCHR1), to regulate appetite and energy homeostasis. The Melanocortin Receptor Accessory Protein 2 (MRAP2), a small single transmembrane protein broadly expressed in multiple tissues, has been defined as a vital endocrine modulator of five melanocortin receptors (MC1R-MC5R) and several other GPCRs in the regulation of central neuronal activities and peripheral energy balance. Here, we demonstrated the interaction between MRAP2 and MCHR1 by immunoprecipitation and bimolecular fluorescent assay and found that MRAP2 could inhibit MCHR1 signaling in vitro. A series of functional truncations of different regions further identified that the C-terminal domains of MRAP2 protein were required for the pharmacological modulation of intracellular Ca2+ coupled cascades and membrane transport. These findings elucidated the broad regulatory profile of MRAP2 protein in the central nervous system and may provide implications for the modulation of central MCHR1 function in vivo.

Selective Interactions of Mouse Melanocortin Receptor Accessory Proteins with Somatostatin Receptors.

Somatostatin receptors (SSTRs) are G protein-coupled receptors (GPCRs) known to regulate exocrine secretion, neurotransmission, and inhibit endogenous cell proliferation. SSTR subtypes (SSTR1-SSTR5) exhibit homo- or heterodimerization with unique signaling characteristics. Melanocortin receptor accessory protein 1 (MRAP1) functions as an allosteric modulator of melanocortin receptors and some other GPCRs. In this study, we investigated the differential interaction of MRAP1 and SSTRs and examined the pharmacological modulation of MRAP1 on mouse SSTR2/SSTR3 and SSTR2/SSTR5 heterodimerization in vitro. Our results show that the mouse SSTR2 forms heterodimers with SSTR3 and SSTR5 and that MRAP1 selectively interacts with SSTR3 and SSTR5 but not SSTR2. The interactive binding sites of SSTR2/SSTR3 or SSTR2/SSTR5 with MRAP1 locate on SSTR3 and SSTR5 but not SSTR2. The binding sites of MRAP1 to SSTR3 are extensive, while the ones of SSTR5 are restricted on transmembrane region six and seven. The heterodimerization of mouse SSTR2, SSTR3, and SSTR5 can be modulated by binding protein in addition to an agonist. Upregulation of extracellular signal-regulated kinases phosphorylation, p27Kip1, and increased cell growth inhibition with the co-expression of SSTR2/SSTR3 or SSTR2/SSTR5 with MRAP1 suggest a regulatory effect of MRAP1 on anti-proliferative response of two SSTR heterodimers. Taken together, these results provide a new insight of MRAP1 on the maintenance and regulation of mouse SSTR dimers which might be helpful to better understand the molecular mechanism involving SSTRs in tumor biology or other human disorders.

Real-life analysis of neoadjuvant-therapy-associated benefits for pathological complete response and survival in early breast cancer patients - role of trastuzumab in HER2+ BC and platinum in TNBC.

Background: Neoadjuvant therapy, which aims to achieve a pathological complete response (pCR) for better overall survival (OS) has several advantages for patients with early breast cancer (eBC) and subtypes of HER2-positive (HER2+) and triple-negative breast cancer (TNBC). However, there has been no large-scale real-world investigation on the clinical outcomes associated with trastuzumab-based and platinum-based neoadjuvant treatments for patients with HER2+ and TNBC, respectively. Material and methods: Taiwan Cancer Registry and National Health Insurance Research Database were utilized in this study. Patients diagnosed with clinically lymph-node-positive (LN+) HER2+ or TNBC were identified for analysis. Logistic regression and Cox proportional hazard models were employed to estimate the adjusted odds ratios (aOR) of achieving pCR and adjusted hazard ratios (aHR) of overall survival associated with treatment agents, respectively. Results: A total of 1,178 HER2+ eBC and 354 early TNBC patients were identified, respectively. Neoadjuvant trastuzumab significantly increased the pCR rates by 3.87-fold among HER2+ patients. Trastuzumab-associated survival benefit was found in HER2+ patients who achieved pCR (aHR [95% CI]: 0.30 [0.11-0.84]) but not in those without pCR (1.13 [0.77-1.67]). Among the TNBC patients, platinum was associated with a 1.6-fold increased pCR rate; however, it did not improve OS regardless of pCR status. Conclusions: Trastuzumab improved pCR and OS for patients with HER2+ subtype. Using platinum agents for TNBC patients increased pCR rates but was not linked to better survival. Optimal neoadjuvant anti-HER2 therapy for patients with HER2+ eBC and the introduction of novel therapy for patients with TNBC should be considered.

A convenient polyculture system that controls a shrimp viral disease with a high transmission rate.

Developing ecological approaches for disease control is critical for future sustainable aquaculture development. White spot syndrome (WSS), caused by white spot syndrome virus (WSSV), is the most severe disease in cultured shrimp production. Culturing specific pathogen-free (SPF) broodstock is an effective and widely used strategy for controlling WSS. However, most small-scale farmers, who predominate shrimp aquaculture in developing countries, cannot cultivate SPF shrimp, as they do not have the required infrastructure and skills. Thus, these producers are more vulnerable to WSS outbreaks than industrial farms. Here we developed a shrimp polyculture system that prevents WSS outbreaks by introducing specific fish species. The system is easy to implement and requires no special biosecurity measures. The promotion of this system in China demonstrated that it allowed small-scale farmers to improve their livelihood through shrimp cultivation by controlling WSS outbreaks and increasing the production of ponds.

Integrated Multiomic Analysis Reveals the High-Fat Diet Induced Activation of the MAPK Signaling and Inflammation Associated Metabolic Cascades via Histone Modification in Adipose Tissues.

BACKGROUND: The number of diet induced obese population is increasing every year, and the incidence of type 2 diabetes is also on the rise. Histone methylation and acetylation have been shown to be associated with lipogenesis and obesity by manipulating gene expression via the formation of repression or activation domains on chromosomes. OBJECTIVE: In this study, we aimed to explore gene activation or repression and related biological processes by histone modification across the whole genome on a high-fat diet (HFD) condition. We also aimed to elucidate the correlation of these genes that modulated by histone modification with energy metabolism and inflammation under both short-term and long-term HFD conditions. METHOD: We performed ChIP-seq analysis of H3K9me2 and H3K9me3 in brown and white adipose tissues (WATs; subcutaneous adipose tissue) from mice fed with a standard chow diet (SCD) or HFD and a composite analysis of the histone modification of H3K9me2, H3K9me3, H3K4me1 and H3K27ac throughout the whole genome. We also employed and integrated two bulk RNA-seq and a single-nuclei RNA sequencing dataset and performed western blotting (WB) to confirm the gene expression levels in adipose tissue of the SCD and HFD groups. RESULTS: The ChIP-seq and transcriptome analysis of mouse adipose tissues demonstrated that a series of genes were activated by the histone modification of H3K9me2, H3K9me3, H3K4me1, and H3K27ac in response to HFD condition. These genes were enriched in Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways involved in lipogenesis, energy metabolism and inflammation. Several genes in the activated mitogen-activated protein kinase (MAPK) pathway might be related to both inflammation and energy metabolism in mice, rats and humans fed with HFD for a short or long term, as showed by bulk RNA-seq and single nuclei RNA-seq datasets. Western blot analyses further confirmed the increased expression of MET, VEGFA and the enhanced phosphorylation ratio of p44/42 MAPK upon HFD treatment. CONCLUSION: This study expanded our understanding of the influence of eating behavior on obesity and could assist the identification of putative therapeutic targets for the prevention and treatment of metabolic disorders in the future.

Author Correction: The mid-developmental transition and the evolution of animal body plans.

An Amendment to this paper has been published and can be accessed via a link at the top of the paper.

Pharmacological modulation of MRAP2 protein on melanocortin receptors in the sea lamprey.

Melanocortin receptors (MCRs) and their accessory proteins (MRAPs) evolutionarily first appear in the genome of sea lamprey. The most ancient melanocortin system consists of only two melanocortin receptors (slMCa and slMCb) and one MRAP2 (slMRAP2) protein, but the physiological roles have not been fully explored in this primitive species. Here, we synthesize and characterize the pharmacological features of slMRAP2 protein on two slMCRs. Our results show that the slMRAP2 protein lacks the long carboxyl terminus; it directly interacts and decreases the surface expression but enhances the α-MSH-induced agonism of slMCa and slMCb. In comparison with higher organisms such as elephant shark and zebrafish, we also demonstrate the constantly evolving regulatory function of the carboxyl terminus of MRAP2 protein, the unique antiparallel topology of slMRAP2 dimer and the homo- and hetero-dimerization of two slMCRs. This study elucidates the presence and modulation of melanocortin receptor by the accessory protein of the agnathans for the first time, which provides a better insight of the melanocortin system in ancient species of chordates.

Total RNA Isolation from Planarian Tissues.

Isolation of high-quality and nondegraded RNA is a prerequisite for many modern molecular biology applications. A variety of RNA extraction products and protocols are available for the standard model organisms, yet species-specific peculiarities of less well studied organisms often require specific protocol adaptations. Here we describe a robust RNA extraction protocol for planarians that is widely used in the community. The protocol combines tissue homogenization in TRIzol with phenol-chloroform extraction and subsequent purification over commercial columns. The purified RNA can be used for many downstream applications, such as cDNA synthesis and next-generation sequencing (NGS, RNA-seq).

Pharmacological effect of human melanocortin-2 receptor accessory protein 2 variants on hypothalamic melanocortin receptors.

PURPOSE: Melanocortin-3 receptor (MC3R), melanocortin-4 receptor (MC4R), and a recently identified melanocortin-2 receptor accessory protein 2 (MRAP2), are highly expressed in hypothalamus and coordinately regulate energy homeostasis, but the single cellular transcriptome of melanocortin system remains unknown. Several infrequent MRAP2 variants are reported from severe obese human patients but the mechanisms on how they affect melanocortin signaling are unclear. METHODS: First, we performed in silico analysis of mouse hypothalamus RNA sequencing datasets at single-cell resolution from two independent studies. Next, we inspected the three-dimensional conformational alteration of three mutations on MRAP2 protein. Finally, the influence of MRAP2 variants on MC3R and MC4R signaling was analyzed in vitro. RESULTS: (1) We confirmed the actual co-expression of Mrap2 with Mc3r and Mc4r, and demonstrated more broad distribution of Mrap2-positive neuronal populations than Mc3r or Mc4r in mouse hypothalamus. (2) Compared with wild-type MRAP2, MRAP2N88Y, and MRAP2R125C showed impaired α-MSH-induced MC4R or MC3R stimulation. (3) MRAP2N88Yexhibited enhanced interaction with MC4R protein and its own. CONCLUSIONS: This is the first dedicated description of single-cell transcriptome signature of Mrap2, Mc3r, and Mc4r in the central nerve system and the first evidence describing the unique dimer formation, conformational change, and pharmacological effect of MRAP2 mutations on MC3R signaling.

Antagonistic Self-Organizing Patterning Systems Control Maintenance and Regeneration of the Anteroposterior Axis in Planarians.

Planarian flatworms maintain their body plan in the face of constant internal turnover and can regenerate from arbitrary tissue fragments. Both phenomena require self-maintaining and self-organizing patterning mechanisms, the molecular mechanisms of which remain poorly understood. We show that a morphogenic gradient of canonical Wnt signaling patterns gene expression along the planarian anteroposterior (A/P) axis. Our results demonstrate that gradient formation likely occurs autonomously in the tail and that an autoregulatory module of Wnt-mediated Wnt expression both shapes the gradient at steady state and governs its re-establishment during regeneration. Functional antagonism between the tail Wnt gradient and an unknown head patterning system further determines the spatial proportions of the planarian A/P axis and mediates mutually exclusive molecular fate choices during regeneration. Overall, our results suggest that the planarian A/P axis is patterned by self-organizing patterning systems deployed from either end that are functionally coupled by mutual antagonism.

The mid-developmental transition and the evolution of animal body plans.

Animals are grouped into ~35 'phyla' based upon the notion of distinct body plans. Morphological and molecular analyses have revealed that a stage in the middle of development--known as the phylotypic period--is conserved among species within some phyla. Although these analyses provide evidence for their existence, phyla have also been criticized as lacking an objective definition, and consequently based on arbitrary groupings of animals. Here we compare the developmental transcriptomes of ten species, each annotated to a different phylum, with a wide range of life histories and embryonic forms. We find that in all ten species, development comprises the coupling of early and late phases of conserved gene expression. These phases are linked by a divergent 'mid-developmental transition' that uses species-specific suites of signalling pathways and transcription factors. This mid-developmental transition overlaps with the phylotypic period that has been defined previously for three of the ten phyla, suggesting that transcriptional circuits and signalling mechanisms active during this transition are crucial for defining the phyletic body plan and that the mid-developmental transition may be used to define phylotypic periods in other phyla. Placing these observations alongside the reported conservation of mid-development within phyla, we propose that a phylum may be defined as a collection of species whose gene expression at the mid-developmental transition is both highly conserved among them, yet divergent relative to other species.

PlanMine--a mineable resource of planarian biology and biodiversity.

Planarian flatworms are in the midst of a renaissance as a model system for regeneration and stem cells. Besides two well-studied model species, hundreds of species exist worldwide that present a fascinating diversity of regenerative abilities, tissue turnover rates, reproductive strategies and other life history traits. PlanMine (http://planmine.mpi-cbg.de/) aims to accomplish two primary missions: First, to provide an easily accessible platform for sharing, comparing and value-added mining of planarian sequence data. Second, to catalyze the comparative analysis of the phenotypic diversity amongst planarian species. Currently, PlanMine houses transcriptomes independently assembled by our lab and community contributors. Detailed assembly/annotation statistics, a custom-developed BLAST viewer and easy export options enable comparisons at the contig and assembly level. Consistent annotation of all transcriptomes by an automated pipeline, the integration of published gene expression information and inter-relational query tools provide opportunities for mining planarian gene sequences and functions. For inter-species comparisons, we include transcriptomes of, so far, six planarian species, along with images, expert-curated information on their biology and pre-calculated cross-species sequence homologies. PlanMine is based on the popular InterMine system in order to make the rich biology of planarians accessible to the general life sciences research community.

Functional dissection of lysine deacetylases reveals that HDAC1 and p300 regulate AMPK.

First identified as histone-modifying proteins, lysine acetyltransferases (KATs) and deacetylases (KDACs) antagonize each other through modification of the side chains of lysine residues in histone proteins. Acetylation of many non-histone proteins involved in chromatin, metabolism or cytoskeleton regulation were further identified in eukaryotic organisms, but the corresponding enzymes and substrate-specific functions of the modifications are unclear. Moreover, mechanisms underlying functional specificity of individual KDACs remain enigmatic, and the substrate spectra of each KDAC lack comprehensive definition. Here we dissect the functional specificity of 12 critical human KDACs using a genome-wide synthetic lethality screen in cultured human cells. The genetic interaction profiles revealed enzyme-substrate relationships between individual KDACs and many important substrates governing a wide array of biological processes including metabolism, development and cell cycle progression. We further confirmed that acetylation and deacetylation of the catalytic subunit of the adenosine monophosphate-activated protein kinase (AMPK), a critical cellular energy-sensing protein kinase complex, is controlled by the opposing catalytic activities of HDAC1 and p300. Deacetylation of AMPK enhances physical interaction with the upstream kinase LKB1, leading to AMPK phosphorylation and activation, and resulting in lipid breakdown in human liver cells. These findings provide new insights into previously underappreciated metabolic regulatory roles of HDAC1 in coordinating nutrient availability and cellular responses upstream of AMPK, and demonstrate the importance of high-throughput genetic interaction profiling to elucidate functional specificity and critical substrates of individual human KDACs potentially valuable for therapeutic applications.

Clinicopathological characteristics and prognosis of patients with small intestinal stromal tumors.

BACKGROUND AND PURPOSE: Gastrointestinal stromal tumors (GISTs) involving the small intestine are less common than those involving the stomach, and data on small intestinal stromal tumors (SISTs) are more limited. This study investigated the clinicopathological characteristics and prognostic factors of SISTs and compared them with those of gastric stromal tumors (GSTs). METHODS: A total of 82 surgically resected and pathologically diagnosed smooth muscle tumors of small bowel in patients treated from January 1986 to December 2000 were included. Immunohistochemical studies were performed on these tumors with antibodies of CD117, CD34, smooth muscle actin (SMA), desmin and S-100. The results were analyzed and compared with 74 cases of GSTs diagnosed and treated from January 1986 to December 1997. RESULTS: Among the 82 small intestine tumors, 71 were CD117-positive (86.6%) and were classified as SISTs. Of the 71 SISTs, 70.4% were immunoreactive to CD34, 88.7% to SMA, 46.5% to S-100, but none to desmin. Survival analysis demonstrated that tumor size < 5 cm (p = 0.021), mitosis number < 5/50 high-power field (p < 0.001), SMA-positive (p = 0.027), non-epithelioid cell type (p = 0.005) and tumor with skeinoid fibers (p = 0.010) predicted longer disease-free survival after operation. Multivariate analysis revealed that mitotic number (p = 0.001), cell morphology (p = 0.031) and tumor size (p = 0.004) were independent prognostic factors. In comparison to GSTs, SISTs exhibited significantly lower rates of CD34, but significantly higher rates of SMA and S-100 immunoreactivity. CONCLUSIONS: SISTs exhibited a different immunophenotype from GSTs. SMA reactivity is a predictor of benign clinical behavior in SISTs. Tumor mitotic numbers, tumor size, and cell type were independent prognostic factors for patients with SISTs after operation.

Increased endostatin/collagen XVIII expression correlates with elevated VEGF level and poor prognosis in hepatocellular carcinoma.

Liver is the primary source for collagen XVIII, the precursor of angiogenesis inhibitor, endostatin. However, the role of endostatin/collagen XVIII expression during liver carcinogenesis remains elusive. Therefore, we studied its expression in five hepatoma cell lines and 105 hepatocellular carcinoma specimens. The poorly differentiated hepatoma cell lines exhibited increased endostatin/collagen XVIII levels compared with the well-differentiated ones. In hepatoma tissues, endostatin/collagen XVIII expression was detected in various types of liver cells and was significantly stronger in adjacent nontumor tissues than that in tumors (P<0.001). Endostatin/collagen XVIII expression in nontumor tissues correlated with tumor stages (P=0.014) and expression of vascular endothelial growth factor (P=0.007), but not the stages of hepatic fibrosis (P>0.05). Kaplan-Meier analysis showed that patients with higher endostatin/collagen XVIII expression had significantly shorter overall survival (P=0.011) and disease-free survival (P=0.0034). Moreover, endostatin/collagen XVIII level was an independent prognostic factor for tumor recurrence (P=0.034) by multivariate analysis. In conclusion, increased endostatin/collagen XVIII expression correlated with hepatoma progression and predicted poor prognosis for patients with hepatocellular carcinoma.

Immunohistochemical analyses of gastric stromal tumors in Taiwanese.

BACKGROUND AND PURPOSE: Gastrointestinal stromal tumors (GISTs), identified by the presence of CD117 (KIT), were previously classified as gastric and intestinal smooth muscle tumors prior to the availability of immunohistochemical methods. This study evaluated the percentage of GISTs previously diagnosed as gastric smooth muscle tumors in our hospital during an 11-year period. METHODS: A total of 81 surgically resected gastric smooth muscle tumor specimens from 81 patients were collected from January 1986 to December 1997. Immunohistochemical studies were performed on these tumors with antibodies of CD34, CD117, smooth muscle actin (SMA), S-100, and desmin. RESULTS: Among the 81 tumors, 74 (91.4%) were CD117-positive and were classified as GISTs. Among the 74 GISTs, CD34 was positive in 72 tumors (97.3%), SMA was positive in 12 tumors (16.2%), desmin was positive in 5 tumors (6.7%), and S-100 was positive in 4 tumors (5.4%). The 7 tumors classified as non-GISTs had the following immunohistochemical characteristics: 1 was a CD117-negative CD34-positive stromal tumor (GINST) [1/81, 1.2%]; 3 were schwannomas with strong S-100-positive characteristics (3/81, 3.7%); and 3 were smooth muscle tumors with both SMA- and desmin-positive status (3/81, 3.7%). No clear relationship between CD117 or CD34 expression and prognosis was found for these tumors. CONCLUSIONS: The majority (91.4%) of gastric tumors originally diagnosed as gastric smooth muscle tumors were GISTs, except for small groups of smooth muscle tumors and schwannomas.

Expression of hepatoma-derived growth factor in hepatocellular carcinoma.

BACKGROUND: Hepatoma-derived growth factor (HDGF) is a novel growth factor derived from a hepatoma cell line. The current study was designed to elucidate the role of HDGF expression during the pathogenesis of hepatocellular carcinoma (HCC). METHODS: HDGF expression in hepatoma cell lines was analyzed using the reverse transcriptase-polymerase chain reaction (RT-PCR), Western blot analysis, and immunofluorescence analysis. Immunohistochemical studies were performed to examine the intensity and spatial distribution of HDGF immunostaining in 105 HCC specimens. To evaluate its prognostic value, the labeling index of HDGF immunostaining was analyzed for potential correlations with the clinicopathologic characteristics of HCC. RESULTS: RT-PCR and Western blot analysis detected increased HDGF expression in malignant hepatoma cell lines. In resected HCC specimens, HDGF immunostaining was detected in the nuclei and cytoplasm of hepatocytes and hepatoma cells. HDGF levels in hepatoma tissue samples were significantly higher than in adjacent nontumor tissue samples (P < 0.05). Elevated nuclear HDGF levels were found to be correlated with loss of differentiation features (P < 0.05), absence of tumor capsules (P < 0.01), high alpha-fetoprotein levels (P < 0.05), and overexpression of proliferating cell nuclear antigen (P < 0.001). Kaplan-Meier analysis indicated that patients with higher nuclear HDGF levels had a shorter duration of survival and a higher incidence of recurrence (P < 0.001). Multivariate analysis indicated that for patients with HCC, the nuclear HDGF level is an independent prognostic factor for overall and disease-free survival. CONCLUSIONS: Increased HDGF expression is correlated with the proliferating states of HCC and represents a novel prognostic factor for patients with HCC who have undergone surgery.