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Several studies have shown that tRNAs can be enzymatically cleaved to generate distinct classes of tRNA-derived fragments (tRF). Here, we report that tRF/miR-1280, a 17-bp fragment derived from tRNALeu and pre-miRNA, influences Notch signaling pathways that support the function of cancer stem-like cells (CSC) in colorectal cancer progression. tRF/miR-1280 expression was decreased in human specimens of colorectal cancer. Ectopic expression of tRF/miR-1280 reduced cell proliferation and colony formation, whereas its suppression reversed these effects. Mechanistic investigations implicated the Notch ligand JAG2 as a direct target of tRF/miR-1280 binding through which it reduced tumor formation and metastasis. Notably, tRF/miR-1280–mediated inactivation of Notch signaling suppressed CSC phenotypes, including by direct transcriptional repression of the Gata1/3 and miR-200b genes. These results were consistent with findings of decreased levels of miR-200b and elevated levels of JAG2, Gata1, Gata3, Zeb1, and Suz12 in colorectal cancer tissue specimens. Taken together, our results established that tRF/miR-1280 suppresses colorectal cancer growth and metastasis by repressing Notch signaling pathways that support CSC phenotypes. Furthermore, they provide evidence that functionally active miRNA can be derived from tRNA, offering potential biomarker and therapeutic uses. Cancer Res; 77(12); 3194–206. ©2017 AACR.

Supplementary Figure 1, The expression levels of pre-miRNA-1280 and tRNALeu in CRC patients. Supplementary Figure 2, tRF/miR-1280 inhibits proliferation of Colorectal Cancer Cells in vitro and in vivo. Supplementary Figure 3, Prediction of target candidates for tRF/miR-1280. Supplementary Figure 4, tRF/miR-1280 inhibits Notch pathways and suppresses the stem cell-like phenotype.Supplementary Figure 5,JAG2 knockdown decreases Colorectal Cancer Cell growth.Supplementary Figure 6,tRF/miR-1280 reduces Gata1/3 expression, and knockdown of Gata1 or Gata3 decreases Colorectal Cancer Cells growth. Supplementary Figure 7, miR-200b targets Zeb1 and Suz12.

Supplementary materials and methods

The emergence of superorganisms in ants, termites, bees, and wasps1 represents a major transition in evolution2. Pre-imaginal caste determination, leading to reproductively dominant queens, which mate, store sperm, and lay fertilized eggs, and lifetime unmatedness of worker castes are key features of these animal societies1. While workers in superorganismal species like honey bees and many ant species have consequently either lost, or retain only vestigial spermathecal structures, workers in the bumble bees, wasps and certain ant subfamilies retain complete spermathecae3,4,5. In the case of bumble bees, the retention of these structures in workers and whether they remain reproductively functional despite 25-40 million years of evolution6, is an evolutionary mystery. Here we show that bumble bee workers have retained queen-like reproductive traits. We demonstrate through artificial insemination experiments that worker spermathecae are functional, and that their gene expression post-insemination mirrors that of queens. Further, we show that inseminated workers can rear colonies and produce female offspring throughout the colony life-cycle, an act previously believed to be the sole domain of queens. Mating experiments identified that social isolation is a trigger for successful mating, with social interactions with brood, workers, or queens inhibiting mating. Subsequent semi-field experiments showed that workers can naturally mate in de-queened colonies and go on to produce queen offspring. In the wild, this may act as a back-up strategy to maintain colonies in response to the early loss of the queen. Our results demonstrate that bumble bee workers retain the ability to function as queens, resolving the paradox of why workers in certain taxa maintain intact spermatheca which never mate, and suggesting that worker lifetime unmatedness is not a pre-requisite for the evolutionary transition to superorganisms. Our finding also provides an exciting new tool to combat global declines in bumble bees through artificial insemination of workers from rare and declining species and could therefore have applications in protecting bumble bee biodiversity.

Adipokinetic hormone (AKH) and its receptor (AKHR) are crucial for energy homeostasis in insects, while their identification and functional characterization are controversial in bee species since t...

An increasingly amount of evidence supports that the evolution of eusociality is accompanies by shifts in ancient molecular and physiological pathways. The juvenile hormone, one of the most important hormones in the post-embryonic development of insects, attracts the most attention in the context of social organization. Allatoregulatory neuropeptides (Allatotropin, Allatostatin-A and Allatostatin-C) are known to regulate juvenile hormone synthesis and release in insects. In order to clarify the transitions of juvenile hormone synthesis involved in eusocial evolution, the substitutions of amino acid residues and the complexity of post-translational modifications in allatoregulatory neuropeptide receptors were characterized. Both allatotropin and allatostatin receptors are identified in all examined bee species regardless if they are solitary or eusocial. Although the amino acid sequences are highly conserved, phylogenetic results are consistent with the eusocial status. The abundance of predicted post-translational modifications correlates with social complexity except for that in allatostatin-C receptors. Even though the consequences of these specific amino acid substitutions and various post-translational modification complexity have not been studied, they likely contribute to the localizing, binding and coupling characteristics of the receptor groups.

In this paper, a novel hybrid forecasting model combining modified group method of data handling (GMDH) and back propagation (BP) is introduced for time series oilfield production forecasting. The proposed model takes advantages of both the modified GMDH networks in effective parameter selection and the BP network in excellent nonlinear mapping and provides a robust simulation ability for oilfield production with higher precision. Various production parameters of an actual oilfield were utilized to analyze and test the annual output predicted by proposed model (modified GMDH-BP). The performance of the proposed model was compared with the multiple linear regression (MLR), GMDH, modified GMDH, BP, and the hybrid model combining group method of data handling and back propagation (GMDH-BP) using time series annual production data. The relative error, correlation coefficient (R), root mean square error, mean absolute percentage of error, and scatter index were utilized to investigate the performance of the presented models. The evaluation results indicate that the hybrid model provides more accurate production forecasts compared to other models and exhibits a robust simulation ability for capturing the nonlinear relation of complex production time series prediction of oilfield.

// Meiyu Peng 1, 2, * , Qi Zhang 3, 2, * , Yingnan Cheng 2, * , Shuyu Fu 2, 4 , Huipeng Yang 2 , Xiangdong Guo 2 , Jieyou Zhang 2 , Lina Wang 1 , Lijuan Zhang 2 , Zhenyi Xue 2 , Yan Li 2 , Yurong Da 2 , Zhi Yao 2 , Liang Qiao 5 and Rongxin Zhang 2, 6 1 Department of Immunology, School of Clinical Medicine, Weifang Medical University, Weifang, China 2 Laboratory of Immunology and Inflammation, Department of Immunology, Key Laboratory of Immune Microenvironment and Diseases of Educational Ministry of China, Tianjin Key Laboratory of Molecular and Cellular Immunology, Tianjin Medical University, Tianjin, China 3 Institute of Integrative Medicines for Acute Abdominal Diseases, Nankai Hospital, Tianjin, China 4 Institute of Human Virology, Sun Yat-Sen University, Guangzhou, China 5 Storr Liver Unit, Westmead Institute for Medical Research, the University of Sydney and Westmead Hospital, Westmead, New South Wales, Australia 6 Laboratory of Immunology and Inflammation, School of Life Science and Biopharmaceutics, Guangdong Pharmaceutical University, Guangzhou, China * These authors contributed equally to this work Correspondence to: Rongxin Zhang, email: rongxinz@yahoo.com Liang Qiao, email: liang.qiao@sydney.edu.au Meiyu Peng, email: pmy-107@163.com Yurong Da, email: dayr@tmu.edu.cn Keywords: pancreatic cancer, L-4F, inflammation, tumor-associated macrophages, STAT3 Received: June 09, 2017 Accepted: September 08, 2017 Published: September 22, 2017 ABSTRACT Pancreatic cancer is an aggressive malignancy that is unresponsive to conventional radiation and chemotherapy. Therefore, development of novel immune therapeutic strategies is urgently needed. L-4F, an Apolipoprotein A-I (ApoA-I) mimetic peptide, is engineered to mimic the anti-inflammatory and anti-oxidative functionalities of ApoA-I. In this work, H7 cells were orthotopically implanted in C57BL/6 mice and treated with L-4F. Then, pancreatic cancer progression and the inflammatory microenvironment were investigated in vivo . The cytotoxicity of L-4F toward H7 cells was assessed in vitro . Furthermore, we investigated the effects of L-4F on macrophage polarization by analyzing the polarization and genes of mouse bone marrow-derived macrophages in vitro . The results show that L-4F substantially reduced the tumorigenicity of H7 cells. L-4F inhibited inflammation by reducing the accumulation of inflammatory cells, such as IL-17A-, IL-4-, GM-CSF-, IL-1β-, and IL-6-producing cells and Th1 and Th17. Notably, L-4F also decreased the percentage of macrophages in tumor tissues, especially M2 macrophages (CD11b + F4/80 + CD206 + ), which was also confirmed in vitro . Additionally, the expression of the M2 marker genes Arg1 , MRC1 , and CCL22 and the inflammatory genes IL-6 , iNOS , and IL-12 was decreased by L-4F, indicating that L-4F prevents M2 type macrophage polarization. However, L-4F could not directly attenuate H7 cell invasion or proliferation and did not induce apoptosis. In addition, L-4F potently down-regulated STAT3, JNK and ERK signaling pathways but not affects the phosphorylation of p38 in RAW 264.7 cells. These results suggest that L-4F exhibits an effective therapeutic effect on pancreatic cancer progression by inhibiting tumor-associated macrophages and inflammation.

Adiponectin (APN), also known as apM1, Acrp30, GBP28 and adipoQ, is a circulating hormone that is predominantly produced by adipose tissue. Many pharmacological studies have demonstrated that this protein possesses potent anti-diabetic, anti-atherogenic and anti-inflammatory properties. Although several studies have demonstrated the antioxidative activity of this protein, the regulatory mechanisms have not yet been defined in skeletal muscles. The aim of the present study was to examine the cytoprotective effects of APN against damage induced by oxidative stress in mouse-derived C2C12 myoblasts. APN attenuated H2O2-induced growth inhibition and exhibited scavenging activity against intracellular reactive oxygen species that were induced by H2O2. Furthermore, treating C2C12 cells with APN significantly induced heme oxygenase-1 (HO-1) and nuclear factor-erythroid 2 related factor 2 (Nrf2). APN also suppressed H2O2-induced mitophagy and partially inhibited the colocalization of mitochondria with autophagosomes/lysosomes, correlating with the expression of Pink1 and Parkin and mtDNA. Moreover, APN protected C2C12 myoblasts against oxidative stress-induced apoptosis. Furthermore, APN significantly reduced the mRNA and protein expression levels of Bax. These data suggest that APN has a moderate regulatory role in oxidative stress-induced mitophagy and suppresses apoptosis. These findings demonstrate the antioxidant potential of APN in oxidative stress-associated skeletal muscle diseases.

Agonist-induced internalization plays a key role in the tight regulation of the extent and duration of G protein-coupled receptor signaling. Previously, we have shown that the Bombyx corazonin receptor (BmCrzR) activates both Gαq- and Gαs-dependent signaling cascades. However, the molecular mechanisms involved in the regulation of the internalization and desensitization of BmCrzR remain to be elucidated. Here, vectors for expressing BmCrzR fused with enhanced green fluorescent protein (EGFP) at the C-terminal end were used to further characterize BmCrzR internalization. We found that the BmCrzR heterologously expressed in HEK-293 and BmN cells was rapidly internalized from the plasma membrane into the cytoplasm in a concentration- and time-dependent manner via a β-arrestin (Kurtz)-dependent and clathrin-independent pathway in response to agonist challenge. While most of the internalized receptors were recycled to the cell surface via early endosomes, some others were transported to lysosomes for degradation. Assays using RNA interference revealed that both GRK2 and GRK5 were essentially involved in the regulation of BmCrzR phosphorylation and internalization. Further investigations indicated that the identified cluster of Ser/Thr residues ((411)TSS(413)) was responsible for GRK-mediated phosphorylation and internalization. This is the first detailed investigation of the internalization and trafficking of Bombyx corazonin receptors.

Bias foraging of pollen is general in different pollinators since various nutrition demanding, co-evolution and interaction of insect-plant. To clarify the preference of pollen foraging during sunflower blooming, the pollen foraging behaviors of Apis mellifera Linnaeus and Apis cerana Fabricius were observed. Our results displayed that two summits of pollen foraging occurred in the morning before the ambient temperature climbed up to thirty-one degree centigrade and in the afternoon after the ambient temperature decreased below thirty-one degree centigrade, respectively. Notably, the first foraging summit of Apis cerana emerged one hour earlier than that of Apis mellifera . These results imply that Apis mellifera is less resistant to low temperature but more resistant to high temperature than Apis cerana does. The colonies were surrounded by sunflowers with sporadic weeds, while only few maize dispersed over two hundred meters away. However, no more than forty percent of total pollens foraged by Apis mellifera was from sunflower, and which was no more than twenty percent in Apis cerana group. These results suggest that sunflower pollens are not the prior choice for both honey bee species, while the ratio of sunflower pollens foraged by Apis mellifera is more than that of Apis cerana does.

Adipokinetic hormone receptor (AKHR) was regarded as the crucial regulator of lipid consuming, but now has been renewed as a pluripotent neuropeptide G protein-coupled receptor. It has been identified in all sequenced bee genomes from the solitary to the eusocial. In the current study, we try to clarify the transitions of AKHR on lipid utilization and other potential functions from solitary to eusocial bees. The results showed that the AKHRs were divided into different groups based on their social complexity approximately. Nevertheless, the critical motifs and tertiary structures were highly conserved. As to the post-translational modifications, the eusocial possessed more phosphorylation residues and modification patterns, which might be due to the necessity of more diverse functions. These results suggest that AKHRs are highly conserved on both primary motifs and tertiary structures, but more flexible on posttranslational modifications so as to accommodate to more complicated eusocial life.

CAPA peptides, such as periviscerokinin (PVK), are insect neuropeptides involved in many signaling pathways controlling, for example, metabolism, behavior, and reproduction. They are present in a large number of insects and, together with their cognate receptors, are important for research into approaches for improving insect control. However, the CAPA receptors in the silkworm (Bombyx mori) insect model are unknown. Here, we cloned cDNAs of two putative CAPA peptide receptor genes, BNGR-A27 and -A25, from the brain of B. mori larvae. We found that the predicted BNGR-A27 ORF encodes 450 amino acids and that one BNGR-A25 splice variant encodes a full-length isoform (BNGR-A25L) of 418 amino acid residues and another a short isoform (BNGR-A25S) of 341 amino acids with a truncated C-terminal tail. Functional assays indicated that both BNGR-A25L and -A27 are activated by the PVK neuropeptides Bom-CAPA-PVK-1 and -PVK-2, leading to a significant increase in cAMP-response element-controlled luciferase activity and Ca2+ mobilization in a Gq inhibitor-sensitive manner. In contrast, BNGR-A25S was not significantly activated in response to the PVK peptides. Moreover, Bom-CAPA-PVK-1 directly bound to BNGR-A25L and -A27, but not BNGR-A25S. Of note, CAPA-PVK-mediated ERK1/2 phosphorylation and receptor internalization confirmed that BNGR-A25L and -A27 are two canonical receptors for Bombyx CAPA-PVKs. However, BNGR-A25S alone is a nonfunctional receptor but serves as a dominant-negative protein for BNGR-A25L. These results provide evidence that BNGR-A25L and -A27 are two functional Gq-coupled receptors for Bombyx CAPA-PVKs, enabling the further elucidation of the endocrinological roles of Bom-CAPA-PVKs and their receptors in insect biology.

// Ze-Han Liu 1, * , Qi Zhang 2, 3, * , Yi-Jie Ding 4, * , Ying-Hui Ren 3 , Hui-Peng Yang 3 , Qing Xi 3 , Ying-Nan Cheng 3 , Guo-Lin Miao 3 , Hong-Kun Liu 3 , Cai-Xia Li 2 , Wen-Qiang Yan 5 , Yan Li 3 , Zhenyi Xue 3 , Lijuan Zhang 3 , Xin-Ye Li 6 , Chen-Long Zhao 6 , Yurong Da 3 , Xian-Zhong Wu 2 , Jun-Qiang Chen 7 , Rongxin Zhang 8, 3 and Zhi-Gang Li 9, 6, 2 1 Nankai Clinical College, Tianjin Medical University, Tianjin, P.R. China 2 Institute of Integrative Medicine Therapy for Acute Abdominal Diseases of Tianjin, Nankai Hospital, Tianjin, P. R. China 3 Laboratory of Immunology and Inflammation, Department of Immunology, Key Laboratory of Immune Microenvironment and Diseases of Educational Ministry of China, Basic Medical College, Tianjin Medical University, Tianjin, P.R. China 4 First Central Clinical College, Tianjin Medical University, Tianjin, P.R. China 5 Department of Thoracic Surgery, Nankai Hospital, Nankai District, Tianjin, P. R. China 6 General Hospital, Tianjin Medical University, Tianjin, P.R. China 7 Department of Radiotherapy, Fujian Provincial Tumor Hospital, Affiliated Tumor Hospital of Fujian Medical University, Fuzhou, P.R. China 8 Laboratory of Immunology and Inflammation, Guangdong Pharmaceutical University, Guangzhou, P.R. China 9 Hainan Cancer Hospital, Affiliated Cancer Hospital of Hainan Medical College, Haikou City, P.R. China * These authors have contributed equally to this work Correspondence to: Jun-Qiang Chen, email: junqiangc@163.com Rongxin Zhang, email: rongxinz@yahoo.com , rxzhang@tmu.edu.cn Zhi-Gang Li, email: zhigli38@hotmail.com Keywords: esophageal carcinoma, CHD1L protein, apoptosis, migration, prognosis Received: March 21, 2017 Accepted: June 04, 2017 Published: June 29, 2017 ABSTRACT Esophageal carcinoma (EC) is a malignancy with high metastatic potential. Chromosomal helicase/ATPase DNA binding protein 1-like (CHD1L) gene is a newly identified oncogene located at Chr1q21, and it is amplified in many solid tumors. However, the status of CHD1L protein expression in EC and its clinical significance is uncertain. This study was designed to investigate the significance of CHD1L expression in human EC and its biological function in EC cells. The expression of CHD1L was examined by immunohistochemistry in 191 surgically resected ECs. The associations between CHD1L expression and clinical pathological parameters and the prognostic value of CHD1L were analyzed. Western blot analysis showed that CHD1L was overexpressed in EC cell lines. In addition, positive CHD1L expression was strongly related to advanced clinical stage ( P

Tachykinins constitute one of the largest peptide families in the animal kingdom and exert their diverse actions via G protein-coupled receptors (GPCRs). In this study, the Bombyx tachykinin-related peptides (TKRPs) were identified as specific endogenous ligands for the Bombyx neuropeptide GPCR A24 (BNGR-A24) and thus designated BNGR-A24 as BmTKRPR. Using both mammalian cell line HEK293 and insect cell line Sf21, further characterization demonstrated that BmTKRPR was activated, thus resulting in intracellular accumulation of cAMP, Ca(2+) mobilization, and ERK1/2 phosphorylation in a Gs and Gq inhibitor-sensitive manner. Moreover, quantitative reverse transcriptase polymerase chain reaction analysis and dsRNA-mediated knockdown experiments suggested a possible role for BmTKRPR in the regulation of feeding and growth. Our findings enhance the understanding of the Bombyx TKRP system in the regulation of fundamental physiological processes.

Background: The corazonin system is of importance for insects to control multiple physiological processes. Results: Bombyx BNGR-A21 was activated in signaling and internalization by direct interaction with corazonin. Conclusion: Bombyx corazonin was a specific ligand for dual Gq- and Gs-coupled BmCrzR. Significance: Our findings will lead to a better understanding of the corazonin system in regulation of fundamental physiological processes., Corazonin, an undecapeptide neurohormone sharing a highly conserved amino acid sequence across Insecta, plays different physiological roles in the regulation of heart contraction rates, silk spinning rates, the induction of dark color and morphometric phase changes, and ecdysis. Corazonin receptors have been identified in Drosophila melanogaster, Manduca sexta, and Musca domestica. However, detailed information on the signaling and major physiological functions of corazonin and its receptor is largely unknown. In the current study, using both the mammalian cell line HEK293 and insect cell lines BmN and Sf21, we paired the Bombyx corazonin neuropeptide as a specific endogenous ligand for the Bombyx neuropeptide G protein-coupled receptor A21 (BNGR-A21), and we therefore designated this receptor as BmCrzR. Further characterization indicated that synthetic BmCrz demonstrated a high affinity for and activated BmCrzR, resulting in intracellular cAMP accumulation, Ca2+ mobilization, and ERK1/2 phosphorylation via the Gq- and Gs-coupled signaling pathways. The direct interaction of BmCrzR with BmCrz was confirmed by a rhodamine-labeled BmCrz peptide. Moreover, experiments with double-stranded RNA and synthetic peptide injection suggested a possible role of BmCrz/BmCrzR in the regulation of larval growth and spinning rate. Our present results provide the first in-depth information on BmCrzR-mediated signaling for further elucidation of the BmCrz/BmCrzR system in the regulation of fundamental physiological processes.

Several studies have shown that tRNAs can be enzymatically cleaved to generate distinct classes of tRNA-derived fragments (tRF). Here, we report that tRF/miR-1280, a 17-bp fragment derived from tRNALeu and pre-miRNA, influences Notch signaling pathways that support the function of cancer stem-like cells (CSC) in colorectal cancer progression. tRF/miR-1280 expression was decreased in human specimens of colorectal cancer. Ectopic expression of tRF/miR-1280 reduced cell proliferation and colony formation, whereas its suppression reversed these effects. Mechanistic investigations implicated the Notch ligand JAG2 as a direct target of tRF/miR-1280 binding through which it reduced tumor formation and metastasis. Notably, tRF/miR-1280–mediated inactivation of Notch signaling suppressed CSC phenotypes, including by direct transcriptional repression of the Gata1/3 and miR-200b genes. These results were consistent with findings of decreased levels of miR-200b and elevated levels of JAG2, Gata1, Gata3, Zeb1, and Suz12 in colorectal cancer tissue specimens. Taken together, our results established that tRF/miR-1280 suppresses colorectal cancer growth and metastasis by repressing Notch signaling pathways that support CSC phenotypes. Furthermore, they provide evidence that functionally active miRNA can be derived from tRNA, offering potential biomarker and therapeutic uses. Cancer Res; 77(12); 3194–206. ©2017 AACR.

Adipokinetic hormones (AKHs) are the best studied insect neuropeptides with the function of mobilizing lipids and carbohydrates during energy-expensive activities and modulating fundamental physiological processes, such as sugar homeostasis, lipid metabolism, and reproduction. Three distinct cDNAs encoding the prepro-Bombyx AKH1-3 have been cloned and confirmed by mass spectrometric methods. Our previous research suggested the Bombyx AKH receptor is activated by AKH1 and AKH2 with high affinity but by AKH3 with quite low affinity. In this study, using stable functional expression of the receptors in HEK293 cells, we have now identified AKH3 as a specific ligand for two orphan G-protein-coupled receptors, and we therefore named them AKHR2a and AKHR2b, respectively. We demonstrated that both AKHR2a and AKHR2b were activated by AKH3 at high affinity and by AKH1 and AKH2 at low affinity, leading to an increase of intracellular cAMP levels and activation of ERK1/2 and receptor internalization, but they were not activated by Bombyx corazonin. Conversely, the Bombyx corazonin receptor was activated by corazonin but not by AKH1-3. Quantitative RT-PCR revealed that AKHR2a and AKHR2b were both highly expressed in the testis but were also detected at low levels in other tissues. These results will lead to a better understanding of the AKH/AKHR system in the regulation of fundamental physiological processes.

At present, production split methods mainly include effective thickness method, formation coefficient method and dynamic equation split method. On account of various factors restriction, the applied results are unsatisfactory. In order to fully grasp the multilayer reservoir layering exploitation status, a new production split method is established by utilizing grey relational analysis based on traditional production split methods. The new method divides the influence factors of single layer production split into static geological factor and dynamic development factor. We confirm correlation degree weight of static geological factor and dynamic development factor by utilizing grey relational analysis, which synthetically considers various production split influence factors. And finally a new production split method is established. Through actual applied verification, the new method is accurate and widely adaptable, which provides a new idea for production split work.

Cyclin-dependent protein kinases (CDKs) play key roles at different checkpoint regulations of the eukaryotic cell cycle. However, only few studies of lepidoptera CDK family proteins have been reported so far. In this study, we performed the cDNA sequencing of 10 members of the CDK family in Bombyx mori. Gene structure analysis suggested that CDK12 and CDC2L1 owned two and three isoforms, respectively. Phylogenetic analysis showed that CDK genes in different species were highly conserved, implying that they evolved independently even before the split between vertebrates and invertebrates. We found that the expression levels of BmCDKs in 13 tissues of fifth-instar day 3 larvae were different: CDK1, CDK7, and CDK9 had a high level of expression, whereas CDK4 was low-level expressed and was detected only in the testes and fat body cells. Similar expression profiles of BmCDKs during embryo development were obtained. Among the variants of CDK12, CDK12 transcript variant A had the highest expression, and the expression of CDC2L1 transcript variant A was the highest among the variants of CDC2L1. It was shown from the RNAi experiments that the silencing of CDK1, CDK10, CDK12, and CDC2L1 could influence the cells from G0/G1 to S phase transition.

Heat shock proteins (Hsps) are involved in a variety of critical biological functions, including protein folding, degradation, and translocation and macromolecule assembly, act as molecular chaperones during periods of stress by binding to other proteins. Using expressed sequence tag (EST) and silkworm (Bombyx mori) transcriptome databases, we identified 27 cDNA sequences encoding the conserved J domain, which is found in DnaJ-type Hsps. Of the 27 J domain-containing sequences, 25 were complete cDNA sequences. We divided them into three types according to the number and presence of conserved domains. By analyzing the gene structures, intron numbers, and conserved domains and constructing a phylogenetic tree, we found that the DnaJ family had undergone convergent evolution, obtaining new domains to expand the diversity of its family members. The acquisition of the new DnaJ domains most likely occurred prior to the evolutionary divergence of prokaryotes and eukaryotes. The expression of DnaJ genes in the silkworm was generally higher in the fat body. The tissue distribution of DnaJ1 proteins was detected by western blotting, demonstrating that in the fifth-instar larvae, the DnaJ1 proteins were expressed at their highest levels in hemocytes, followed by the fat body and head. We also found that the DnaJ1 transcripts were likely differentially translated in different tissues. Using immunofluorescence cytochemistry, we revealed that in the blood cells, DnaJ1 was mainly localized in the cytoplasm.

In insects, molting and metamorphosis are strictly regulated by ecdysteroids. Ecdysteroid synthesis is positively or negatively controlled by several neuropeptides. The prothoracicostatic peptide (PTSP) BmPTSP (Bombyx mori prothoracicostatic peptide), isolated from the larval brain of B. mori, has been demonstrated to inhibit ecdysteroid synthesis in the prothoracic glands (PGs) [Hua et al. (1999) J. Biol. Chem. 274, 31169–31173]. More recently, the newly recognized B. mori receptor for Drosophila melanogaster sex peptide (DmSP) has been identified as a receptor for BmPTSP. However, details on the signalling pathways and physiological functions of this receptor have remained elusive. In the present paper, we report the functional characterization of the BmPTSP receptor (BmPTSPR)/sex peptide (SP) receptor (SPR) using both mammalian and insect cells. Synthetic DmSP shows the potential to inhibit forskolin (FSK) or adipokinetic hormone (AKH)-induced cAMP-response element (CRE)-driven luciferase (Luc) activity in a manner comparable with synthetic BmPTSP1. However, DmSP displayed a much lower activity in triggering Ca2+ mobilization and internalization than did BmPTSP1. Additionally, 6-carboxy-fluorescein fluorophore (FAM)-labelled DmSP and BmPTSP3 were found to bind specifically to BmPTSPR/SPR. The binding of FAM–DmSP was displaced by unlabelled DmSP, but not by unlabelled BmPTSP1 and, vice versa, the binding of FAM–BmPTSP3 was blocked by unlabelled BmPTSP3, but not by unlabelled DmSP. Moreover, internalization assays demonstrated that BmPTSP1, but not DmSP, evoked recruitment of the Bombyx non-visual arrestin, Kurtz, to the activated BmPTSPR/SPR in the plasma membrane. This was followed by induction of internalization. This suggests that BmPTSP1 is probably an endogenous ligand specific for BmPTSPR/SPR. We therefore designate this receptor BmPTSPR. In contrast, DmSP is an allosteric agonist that is biased towards Gαi/o-dependent cAMP production and away from Ca2+ mobilization and arrestin recruitment.

Members of the mammalian neuropeptide Y (NPY) family serve as neurotransmitters and contribute to a diversity of physiological functions. Although neuropeptide F (NPF), the NPY-like orthologs from insects, have been identified, the NPF receptors and their signaling and physiological functions remain largely unknown. In this study, we established the stable and transient functional expression of a Bombyx orphan G protein-coupled receptor, BNGR-A4, in both mammalian HEK293 and insect SF21 cells. We identified Bombyx mori NPFs as specific endogenous ligands for the Bombyx Neuropeptide GPCR A4 (BNGR-A4) and, accordingly, named the receptor BomNPFR. Our results demonstrated that BomNPFR was activated by synthetic BomNPF1a and BomNPF1b at a high efficacy and by BomNPF2 at a low efficacy. This activation led to a decrease of forskolin or adipokinetic hormone peptide-stimulated adenylyl cyclase activity, an increase of intracellular Ca(2+), the activation of ERK1/2 signaling and receptor internalization. Moreover, a Rhodamine-labeled BomNPF1a peptide was found to bind specifically to BomNPFR. The results derived from quantitative RT-PCR analysis and dsRNA-mediated knockdown experiments demonstrated the possible role of BomNPFR in the regulation of food intake and growth. Our results provide the first in-depth information on BomNPFR-mediated signaling for the further elucidation of the BomNPF/BomNPFR system in the regulation of fundamental physiological processes.

The cAMP response element binding protein, CREB, is a G protein-coupled receptor (GPCR) signal-activated transcription factor implicated in the control of many biological processes. In the current study, we constructed a cAMP response element (CRE)-driven luciferase assay system for GPCR characterization in insect cells. Our results indicated that Gs-coupled Bombyx adipokinetic hormone receptor (AKHR) and corazonin receptor could effectively initiate CRE-driven luciferase transcription, but forskolin, a reagent widely used to activate adenylyl cyclase in mammalian systems, failed to induce luciferase activity in insect cells co-transfected with a CRE-driven reporter construct upon agonist treatment. Further investigation revealed that the specific protein kinase C (PKC) inhibitors exhibited stimulatory effects on CRE-driven reporter transcription, and blockage of Ca 2+ signals and inhibition of Ca 2+ -dependent calcineurin resulted in a significant decrease in the luciferase activity. Taken together, these results suggest that PKC likely acts as a negative regulator to modulate CREB activation; in contrast, Ca 2+ signals and Ca 2+ -dependent calcineurin, in addition to PKA, essentially contribute to the positive regulation of CREB activity. This study presents evidence to elucidate the underlying molecular mechanism by which CREB activation is regulated in insects.

The domestic silkworm, Bombyx mori, is a model insect with important economic value for silk production that also acts as a bioreactor for biomaterial production. The functional complexity of the silkworm transcriptome has not yet been fully elucidated, although genomic sequencing and other tools have been widely used in its study. We explored the transcriptome of silkworm at different developmental stages using high-throughput paired-end RNA sequencing. A total of about 3.3 gigabases (Gb) of sequence was obtained, representing about a 7-fold coverage of the B. mori genome. From the reads that were mapped to the genome sequence; 23,461 transcripts were obtained, 5,428 of them were novel. Of the 14,623 predicted protein-coding genes in the silkworm genome database, 11,884 of them were found to be expressed in the silkworm transcriptome, giving a coverage of 81.3%. A total of 13,195 new exons were detected, of which, 5,911 were found in the annotated genes in the Silkworm Genome Database (SilkDB). An analysis of alternative splicing in the transcriptome revealed that 3,247 genes had undergone alternative splicing. To help with the data analysis, a transcriptome database that integrates our transcriptome data with the silkworm genome data was constructed and is publicly available at http://124.17.27.136/gbrowse2/. To our knowledge, this is the first study to elucidate the silkworm transcriptome using high-throughput RNA sequencing technology. Our data indicate that the transcriptome of silkworm is much more complex than previously anticipated. This work provides tools and resources for the identification of new functional elements and paves the way for future functional genomics studies.