Naringenin Alleviates Radiation-Induced Intestinal Injury by Inhibiting TRPV6 in Mice.

SCOPE: Naringenin (NAR) possesses unique anti-inflammatory, antiapoptosis effects and various bioactivities; however, its role against radiation-induced intestinal injury (RIII) remains unclear. This study aims to investigate whether NAR has protective effects against radiation-induced intestinal injury and the underlying mechanisms. METHODS AND RESULTS: C57BL/6J mice are exposed to a single dose of 13 Gy X-ray total abdominal irradiation (TAI), then gavaged with NAR for 7 days. NAR treatment prolongs the survival rate, protects crypts and villi from damage, alleviates the level of radiation-induced inflammation, and mitigates intestinal barrier damage in the irradiated mice. Additionally, NAR reduces immune cell infiltration and intestinal epithelial cell apoptosis. NAR also shows radioprotective effects in human colon cancer cells (HCT116) and human intestinal epithelial cells (NCM460). It reduces cell damage by reducing intracellular calcium ion levels and reactive oxygen species (ROS) levels. NAR-mediated radioprotection is associated with the downregulation of transient receptor potential vanilloid 6 (TRPV6), and inhibition of apoptosis pathway. Notably, treatment with NAR fails to further increase the protective effects of the TRPV6 inhibitor 2-APB, indicating that TRPV6 inhibition is essential for NAR activity. CONCLUSION: NAR inhibits the apoptosis pathway by downregulating TRPV6 and reducing calcium ion level, thereby alleviating RIII. Therefore, NAR is a promising therapeutic drug for RIII.

Genes and Pathways Underpinning Klinefelter Syndrome at Bulk and Single-Cell Levels.

Klinefelter syndrome (KS) is the most frequent genetic anomaly in infertile men. Given its unclear mechanism, we aim to investigate critical genes and pathways in the pathogenesis of KS based on three bulk and one single-cell transcriptome data sets from Gene Expression Omnibus. We merged two data sets (GSE42331 and GSE47584) with human KS whole blood samples. When comparing the control and KS samples, five hub genes, including defensin alpha 4 (DEFA4), bactericidal permeability increasing protein (BPI), myeloperoxidase (MPO), intelectin 1 (ITLN1), and Xg Glycoprotein (XG), were identified. Besides, infiltrated degree of certain immune cells such as CD56bright NK cell were positively associated with the expression of ITLN1 and XG. Kyoto Encyclopedia of Genes and Genomes analysis identified upregulated phosphatidylinositol 3-kinase (PI3K)/AKT pathway in KS. Gene set enrichment analysis followed by gene set variation analysis confirmed the upregulation of G2M checkpoint and heme metabolism in KS. Thereafter, the GSE200680 data set was used for external validation of the expression variation of hub genes from healthy to KS testicular samples, and each hub gene yielded excellent discriminatory capability for KS without exception. At the single-cell level, the GSE136353 data set was utilized to evaluate intercellular communication between different cell types in KS patient, and strong correlations were detected between macrophages/ dendritic cells/ NK cells and the other cell types. Collectively, we provided hub genes, pathways, immune cell infiltration degree, and cell-cell communication in KS, warranting novel insights into the pathogenesis of this disease.

Plasmon enhanced luminescence of Tb/Eu co-doped film by Au NRs-PVA nanocomposite film.

Plasmonic nanostructures have great potential for improving the radiation properties of emitters. Here, the plasmonic Au nanorods-PVA nanocomposite films are used to uniformly improve the photoluminescence of Tb/Eu co-doped PMMA film within the local micro-region. Under the excitation of 292 nm, the maximum enhancement factor is 37.2-fold for emission at 612 nm and 21.6-fold for emission at 545 nm. Moreover, the finite different time domain simulations are developed to further explain the experimental results. It is indicated that the modulation of luminescence can be attributed to the increase of the local density of optical states through the Purcell effect and the improvement of the energy transfer efficiency between Tb and Eu. Under the excitation of 360 nm, the maximum enhancement factor is about 71.5-fold. In this case, the Au nanorods are mainly used for modulating the emission process at 612 nm, which deduced a greater enhancement factor at 612 nm. This study provides a deep understanding of the interactions between rare earth ions co-doped materials and plasmonic nanostructures, building a bridge to fabricate a useful platform for several applications, such as thin film-based detectors and sensors.

Trends and Characteristics of Terrorist Attacks Against Nightclub Venues Over 5 Decades.

INTRODUCTION: Nightclubs are entertainment and hospitality venues historically vulnerable to terrorist attacks. This study identified and characterized terrorist attacks targeting nightclubs and discotheques documented in the Global Terrorism Database (GTD) over a 50-y period. METHODS: A search of the Global Terrorism Database (GTD) was conducted from 1970 to 2019. Precoded variables for target type "business" and target subtype "entertainment/cultural/stadium/casino" were used to identify attacks potentially involving nightclubs. Nightclub venues were specifically identified using the search terms "club," "nightclub," and "discotheque." Two authors manually reviewed each entry to confirm the appropriateness for inclusion. Descriptive statistics were performed using R (3.6.1). RESULTS: A total of 114 terrorist attacks targeting nightclub venues were identified from January 1, 1970, through December 31, 2019. Seventy-four (64.9%) attacks involved nightclubs, while forty (35.1%) attacks involved discotheques. A bombing or explosion was involved in 84 (73.7%) attacks, followed by armed assault in 14 (12.3%) attacks. The highest number of attacks occurred in Western Europe and Sub-Saharan Africa. In total, 284 persons died, and 1175 persons were wounded in attacks against nightclub venues. CONCLUSIONS: While terrorist attacks against nightclub venues are infrequent, the risk for mass casualties and injuries can be significant, mainly when explosives and armed assaults are used.

Bioassay-guided discovery and identification of new potent α-glucosidase inhibitors from Morus alba L. and the interaction mechanism.

ETHNOPHARMACOLOGICAL RELEVANCE: Morus alba L. (mulberry) is a well-known medicinal species that has been used by herbalist doctors for the treatment of diabetes for a long history, and modern ethnopharmacological studies have demonstrated the ameliorating effects of different mulberry extracts toward diabetes-related symptoms and identified a number of α-glucosidase inhibitors as hypoglycemic ingredients. AIM OF THE STUDY: The present study aims to explore new potent α-glucosidase inhibitors from the root bark of M. alba (known as Sang-Bai-Pi in traditional medicine) based on an in vivo antidiabetic evaluation of its extract fractions and further characterize the preliminary mechanism of the new active constituents. MATERIALS AND METHODS: α-Glucosidase inhibitory assay and diabetic mice model were used to locate and evaluate the active fractions from the extract. Diverse separation techniques (e.g. Sephadex LH-20 column chromatograph (CC) and HPLC) and spectroscopic methods (e.g. MS, NMR and ECD) were employed to isolate and structurally characterize the obtained constituents, respectively. Fluorescence quenching, kinetics and molecular docking experiments were conducted to investigate the enzyme inhibitory mechanism of the active compounds. RESULTS: The 80% ethanol eluate from the macroporous resin CC exerted good antidiabetic effects in the tested mice. Fifty-two flavonoids including 22 new ones were then separated and identified, and most of them showed strong inhibition against α-glucosidase with their structure-activity relationship being also discussed. The four new most active ingredients were further characterized to be mixed type of α-glucosidase inhibitors, and their binding modes with the enzyme were also explored. CONCLUSIONS: Our current work has demonstrated that the root bark of M. alba is an extremely rich source of flavonoids as potent α-glucosidase inhibitors and potential antidiabetic agents, which makes it a promising candidate species to develop new natural remedies for the prevention and treatment of diabetes.

Pregnane steroid glycosides with multidrug resistance reversal activity from the stems of Marsdenia tenacissima.

Eighteen previously unreported pregnane glycosides, namely marsdenosides S1-S18, along with 15 known analogues, have been isolated from the stems of Marsdenia tenacissima. The structures of the undescribed compounds were elucidated by spectroscopic means, and their absolute configurations were established on the basis of time-dependent density functional theory (TD-DFT) based electronic circular dichroism (ECD) calculation, X-ray crystallography and acid hydrolysis. All the isolates were evaluated for their chemo-reversal ability against P-glycoprotein (P-gp)-mediated multidrug resistance (MDR) in MCF-7/ADR cell line, and nine ones displayed moderate MDR reversal activity with reversal folds in the range of 2.45-9.01. The most active 12-O-acetyl-20-O-benzoyl-(14,17,18-orthoacetate)-dihydrosarcostin-3-O-ß-d-thevetopyranosyl-(1 â†’ 4)-O-ß-d-oleandropyranosyl-(1 â†’ 4)-O-ß-d-cymaropyranoside increased the sensitivity of MCF-7/ADR cell to adriamycin comparably to the reference drug verapamil (RF = 8.93).

COVID-19 of differing severity: from bulk to single-cell expression data analysis.

Coronavirus disease 2019 (COVID-19) is raging worldwide and causes an immense disease burden. Despite this, the biomarkers and targeting drugs of COVID-19 of differing severity remain largely unknown. Based on the GSE164805 dataset, we identified modules most critical for mild COVID-19 (mCOVID-19) and severe COVID-19 (sCOVID-19) through WGCNA, respectively. We subsequently constructed a protein-protein interaction network, and detected 16 hub genes for mCOVID-19 and 10 hub genes for sCOVID-19, followed by the prediction of upstream transcription factors (TFs) and kinases. The enrichment analysis then showed downregulation of TNFA signaling via NFKB for mCOVID-19, as well as downregulation of MYC targets V1 for sCOVID-19. Infiltration degrees of many immune cells, such as macrophages, were also sharply different between mCOVID-19 and sCOVID-19 samples. Predicted protein targeting drugs with the highest scores nearly all belong to naturally derived or synthetic glucocorticoids. For the two single-cell RNA-seq datasets, we explored the expression distribution of hub genes for mCOVID-19/sCOVID-19 in each cell type. The expression levels of PRKCA, MCM5, TYMS, RBBP4, BCL6, FLOT1, KDM6B, and TLR2 were found to be cell-type-specific. Furthermore, the expression levels of 10 hub genes for mCOVID-19 were significantly upregulated in PBMCs between eight healthy controls and eight mCOVID-19 patients at our institution. Collectively, we detected critical modules, pathways, TFs, kinases, immune cells, targeting drugs, hub genes, and their expression distributions in different cell types that may involve the pathogenesis of COVID-19 of differing severity, which may propel earlier diagnosis and more effective treatment of this intractable disease in the future.

Pregnane steroids and steroid glycosides with multidrug resistance reversal activity from Marsdenia tenacissima.

Twenty compounds comprising four pregnane steroids (2-4 & 20) and 16 pregnane glycosides (1 & 5-19) have been obtained from the ethanol extract of the roots of a Dai ethnological herb, Marsdenia tenacissima. Their structures were characterized on the basis of comprehensive spectroscopic analyses with 17 ones (1-17) being reported for the first time, including the rare cases (2 & 3) of free C21 steroids with 17α-acetyl substitution, compounds 4-7 bearing an unusual 14α-OH, and the first examples with simultaneous 14α-OH/17α-acetyl substitution (7) and glycosylation at C-12 position (10 & 11). An empirical rule for the identification of C-17 configuration, in C21 steroids incorporating the marsdenin constitution structure, was also proposed. All the isolates, along with an array of previously reported analogues in our compound library, were screened for their chemo-reversal ability against P-glycoprotein (P-gp)-mediated multidrug resistance (MDR) in MCF-7/ADR cell line, and six compounds exhibited moderate MDR reversal activity with reversal folds ranging from 1.92 to 4.44.

Anti-tumor effect of infant-derived Enterococcus via the inhibition of proliferation and inflammation as well as the promotion of apoptosis.

Probiotic Enterococcus hirae WEHI01 and Enterococcus faecium WEFA23 from infants were previously found to effectively inhibit the development of melanoma. In this study, their immunomodulatory and antitumor mechanisms were systemically studied. In vitro assay showed that E. hirae WEHI01 and E. faecium WEFA23 achieved biphasic immune regulation, which was revealed by the activation of resting spleen lymphocytes and RAW264.7 macrophages, as well as the anti-inflammation effect when immune cells were treated with LPS. The antitumor effects of E. hirae WEHI01 and E. faecium WEFA23 in vitro and vivo were then investigated. CCK8 and the cell scratch assay showed that the conditioned media, which were co-incubated with Enterococcus and spleen lymphocytes, significantly inhibited the proliferation and migration of B16F10, HepG-2 and HT-29 cells. The results of the tumor-bearing mice model experiment showed that E. faecium WEFA23 inhibition of the growth of tumors in mice, and the anti-tumor mechanism involved three aspects, namely tumor proliferation (decreasing expressions of LDHA, VEGF, MMP2, MMP9 and HIF-1α), inhibition of the pro-inflammation state (decreasing expressions of IL-6, TGF-ß and IL-17) and the promotion of apoptosis (increasing expression of Bax/Bcl-2, caspase-3 and p53). The results suggest that the two strains of Enterococcus could be promising candidates for treating melanoma with a highly inhibitory effect.

Identification of Small-Molecule Bioactive Constituents from the Leaves of Vaccinium bracteatum Confirms It as a Potential Functional Food with Health Benefits.

The health benefits of Vaccinium bracteatum are well recorded in ancient Chinese medical books and were also demonstrated by modern researches. However, the relationship between its beneficial functions and specific chemical constituents has not been fully characterized. This study investigated the bioactive small-molecule constituents in the leaves of V. bracteatum, which afforded 32 compounds including ten new ones (1-9) and ten pairs of enantiomers (9-18). Their structures with absolute configurations were elucidated by spectroscopic methods, especially nuclear magnetic resonance (NMR) and electronic circular dichroism (ECD) analyses, with 1-4 bearing a novel revolving-door shaped scaffold. While half-compounds exhibited decent antioxidant activity by scavenging 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals, all except 19 and 20 exerted significant capturing activity against diammonium 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) radicals. In addition, the new iridoids 1, 5, 6, and 7 exerted apparent neuroprotective activity toward PC12 cells, with 1 being comparable to the positive control, and selective compounds also displayed anti-diabetic and anti-inflammatory properties by inhibiting α-glucosidase and NO production, respectively. The current work revealed that the bioactive small-molecule constituents could be closely related to the functional food property of the title species.

Terrorist Attacks Against Firefighters, 1970-2019.

Firefighters are a critical component of the emergency response system and therefore a potential target for organizations seeking to disrupt this system. Terrorist organizations may deliberately attack firefighters to both increase the devastation of an attack and impair the affected community's ability to respond to an attack. We performed a focused search of the Global Terrorism Database to identify terrorist attacks against firefighters worldwide. The database includes incidents from 1970 through 2019, with a total of 201,183 entries. These entries were searched for incidents involving firefighters or fire trucks. We analyzed trends in the number of incidents occurring per year, regions of the world impacted, methods employed, and number of casualties inflicted. A total of 42 attacks involving firefighters were identified in the Global Terrorism Database resulting in 26 deaths and 95 wounded. Of the 42 attacks, 12 (28.6%) were secondary attacks, where firefighters responding to an initial attack were themselves targeted. The most common method for both primary and secondary attacks was the use of a bomb or explosive. Although attacks against firefighters are uncommon, they highlight both the strategic value and vulnerability of firefighters to terrorist attacks. Increased efforts must be made to protect firefighters from future terrorist attacks.

Relationships between denitrification rates and functional gene abundance in a wetland: The roles of single- and multiple-species plant communities.

Wetland soil denitrification removes excess inorganic nitrogen (N) and prevents eutrophication in aquatic ecosystems. Wetland plants have been considered the key factors determining the capacity of wetland soil denitrification to remove N pollutants in aquatic ecosystems. However, the influences of various plant communities on wetland soil denitrification remain unknown. In the present study, we measured variations in soil denitrification under different herbaceous plant communities including single Phragmites karka (PK), single Paspalum thunbergia (PT), single Zizania latifolia (ZL), a mixture of Paspalum thunbergia plus Phragmites karka (PTPK), a mixture of Paspalum thunbergia plus Zizania latifolia (PTZL), and bare soil (CK) in the Estuary of Nantiaoxi River, the largest tributary of Qingshan Lake in Hangzhou, China. The soil denitrification rate was significantly higher in the surface (0-10 cm) than the subsurface (10-20 cm) layer. Wetland plant growth increased the soil denitrification rate by significantly increasing the soil water content, nitrate concentration, and ln(nirS) + ln(nirK). A structural equation model (SEM) showed that wetland plants indirectly regulated soil denitrification by altering the aboveground and belowground plant biomass, nitrate concentration, abundances of denitrifying functional genes, and denitrification potential. There was no significant difference in soil denitrification rates among PT, PK and ZL. The soil denitrification rate was significantly lower in PTZL than PTPK. Two-plant communities did not necessarily enhance the denitrification rate compared to single planting, the former had a greater competitiveness on N uptake and consequently reduced the amount of nitrate available for denitrification. As PTPK had the highest denitrification rate, co-planting P. thunbergia and P. karka could effectively improve N removal efficiency and help mitigate eutrophication in adjacent aquatic ecosystems. The results of this investigation provide useful information guiding the selection of appropriate wetland herbaceous plant species for wetland construction and the removal of N pollutants in aquatic ecosystems.

Methyl 2-naphthoates with anti-inflammatory activity from Morinda officinalis.

Chemical fractionation of the EtOH extract of the roots of a traditional Chinese herb, Morinda officinalis, afforded an array of methyl 2-naphthoate derivatives (1-9) including four pairs of enantiomers (1-4), two pimarane diterpenes and two ursane triterpenoids. Among them, eight compounds (1a/1b-3a/3b, 11 and 13) were reported in the current work for the first time. The structures of the new compounds, including their absolute configurations, were defined by spectroscopic analyses in combination with quantum chemical electronic circular dichroism (ECD) and gauge-independent atomic orbital (GIAO) NMR calculations. All the isolates were evaluated for their inhibitory effect on nitric oxide (NO) production induced by lipopolysaccharide (LPS) in murine RAW264.7 macrophage cells, and the enantiomers 1a and 3b exhibited moderate activity with IC50 values of 41.9 and 26.2 µM. Meanwhile, compound 3b also dose-dependently inhibited the secretion of two pro-inflammatory cytokines TNF-α and IL-6 in the same cell model.

Steroid glycosides from the roots of Marsdenia tenacissima.

Eleven undescribed glycosylated C21 steroids and nine known homologous glycosides with diverse acyl substituents, as well as their common steroid aglycone, have been obtained from the roots of Marsdenia tenacissima. Their structures were elucidated mainly by comprehensive spectroscopic analyses and comparison with previously reported analogues, with the absolute configuration assignment being supported by chemical degradation, X-ray crystallography and ECD exciton chirality method. Among them, two pairs of regioisomers were found to exist as inseparable equilibrium mixtures due to an interesting intramolecular transesterification, and nicotinoyl substitution was first reported for metabolites from the title plant. Screening of these compounds in a panel of bioassays revealed that two glycosides displayed mild inhibition against butyrylcholinesterase.

Sludge disintegration and phosphorus migration in anaerobic fermentation of waste activated sludge by the addition of EDTA-2Na.

The effects of the addition of EDTA-2Na on sludge disintegration and phosphorus (P) migration during anaerobic fermentation (AF) of waste activated sludge (WAS) are investigated. The efficiency of sludge disintegration was positively correlated with the dose of EDTA-2Na from 0.5-2.0 g/g SS, and an enormous quantity of P was liberated into the aqueous phase, accompanied by sludge disintegration. The proper dose of EDTA-2Na for P release from WAS was 1.5 g/g SS, with an orthophosphate concentration of 394.72 mg/L. P release was more consistent with the pseudo second-order kinetic model. The migration of P species during AF with EDTA-2Na addition was also studied. Orthophosphate was the main species in both of the liquid phase and the loosely bound extracellular polymeric substances (EPS), but organic P (OP) was much more abundant in tightly bound EPS. Inorganic P (IP) was the dominant P speciation in the solid and was mainly distributed in the fraction of non-apatite IP, which accounted for more than 62.8% of IP in the presence of EDTA-2Na. In addition, both IP and OP in the solid contributed to the accumulation of P and the former was outperformed. Furthermore, the increased total dissolved P mainly came from cells. However, the fermented sludge tended to be smaller and to have low compressibility, which is detrimental to its further treatment.

Retraction Notice to: Long Noncoding RNA OIP5-AS1 Promotes the Progression of Liver Hepatocellular Carcinoma via Regulating the hsa-miR-26a-3p/EPHA2 Axis.

[This retracts the article DOI: 10.1016/j.omtn.2020.05.032.].

Terrorist Attacks Against Performing Arts Venues: Global Trends and Characteristics Spanning 50 Years.

INTRODUCTION: In fostering community and culture through entertainment in shared spaces, performing arts venues have also become targets of terrorism. A greater understanding of these attacks is needed to assess the risk posed to different types of venues, to inform medical disaster preparedness, to anticipate injury patterns, and to reduce preventable deaths. METHODS: A search of the Global Terrorism Database (GTD) was conducted from the year 1970 through 2019. Using pre-coded variables for target/victim type and target subtype, attacks involving "business" and "entertainment/cultural/stadium/casino" were identified. Attacks targeting performing arts venues were selected using the search terms "theater," "theatre," "auditorium," "center," "hall," "house," "concert," "music," "opera," "cinema," and "movie." Manual review by two authors was performed to confirm appropriateness for inclusion of entries involving venues where the primary focus of the audience was to view a performance. Descriptive statistics were performed using R (version 3.6.1). RESULTS: A total of 312 terrorist attacks targeting performing arts venues were identified from January 1, 1970 through December 31, 2019. Two-hundred nine (67.0%) attacks involved cinemas or movie theaters, 80 (25.6%) involved unspecified theaters, and 23 (7.4%) specifically targeted live music performance venues. Two-hundred thirty-four (75.0%) attacks involved a bombing or explosion, 50 (16.0%) damaged a facility or infrastructure, and 17 (5.4%) included armed assault. Perpetrators used explosives in 234 (75.0%) attacks, incendiary weapons in 50 (16.0%) attacks, and firearms in 19 (6.1%) attacks. In total, attacks claimed the lives of 1,307 and wounded 4,201 persons. Though fewer in number, attacks against music venues were responsible for 29.4% of fatalities and 35.0% of those wounded, and more frequently involved the use of firearms. Among 95 attacks falling within the highest quartile for victims killed or wounded (>two killed and/or >ten wounded), 83 (87.4%) involved explosives, seven (7.4%) involved firearms, and three (3.2%) involved incendiary methods. CONCLUSION: While uncommon, terrorist attacks against performing arts venues carry the risk for mass casualties, particularly when explosives and firearms are used.

Aberrant Gene Expression Profiling in Men With Sertoli Cell-Only Syndrome.

Sertoli cell-only syndrome (SCOS) is the most severe and common pathological type of non-obstructive azoospermia. The etiology of SCOS remains largely unknown to date despite a handful of studies reported in this area. According to the gene expression of testicular tissue samples in six datasets from the Gene Expression Omnibus, we detected 1441 differentially expressed genes (DEGs) between SCOS and obstructive azoospermia (OA) testicular tissue samples. Enriched GO terms and KEGG pathways for the downregulated genes included various terms and pathways related to cell cycle and reproduction, while the enrichment for the upregulated genes yielded many inflammation-related terms and pathways. In accordance with the protein-protein interaction (PPI) network, all genes in the most critical module belonged to the downregulated DEGs, and we obtained nine hub genes, including CCNB1, AURKA, CCNA2, BIRC5, TYMS, UBE2C, CDC20, TOP2A, and OIP5. Among these hub genes, six were also found in the most significant SCOS-specific module obtained from consensus module analysis. In addition, most of SCOS-specific modules did not have a consensus counterpart. Based on the downregulated genes, transcription factors (TFs) and kinases within the upstream regulatory network were predicted. Then, we compared the difference in infiltrating levels of immune cells between OA and SCOS samples and found a significantly higher degree of infiltration for most immune cells in SCOS than OA samples. Moreover, CD56bright natural killer cell was significantly associated with six hub genes. Enriched hallmark pathways in SCOS had remarkably more upregulated pathways than the downregulated ones. Collectively, we detected DEGs, significant modules, hub genes, upstream TFs and kinases, enriched downstream pathways, and infiltrated immune cells that might be specifically implicated in the pathogenesis of SCOS. These findings provide new insights into the pathogenesis of SCOS and fuel future advances in its theranostics.

The CLOCK protein regulates insulin secretion related with L-type calcium channels in rat pancreatic beta cells.

BACKGROUND: Circadian locomotor output cycles kaput protein (CLOCK) plays a crucial role in glucose homeostasis and controlling insulin secretion. However, the mechanism of the CLOCK regulating rhythmic insulin secretion has not been fully understood. METHODS: Rhythmic expression of the CLOCK in rat pancreatic beta cell was detected. INS-1 cells were transfected with siRNAs to knockdown the CLOCK before the cells were incubated with different concentrations of glucose. Insulin secretion was analyzed by ELISA method. Expression of the L-type calcium channel protein (Cav1.2, Cacna1c) was determined both in the CLOCK-knockdown cells and the control cells. Calcium influx was probed by fluorescent. Chromatin immunoprecipitation (ChIP) test and dual-luciferase reporter gene experiments were applied to verify the relationship between the CLOCK and Cav1.2. RESULTS: The CLOCK is abundantly expressed in rat pancreatic beta cells. Transcription level of the CLOCK showed rhythmicity in the beta cells. Compared to the control group, insulin release was significantly impaired with 25 mM glucose incubation in the CLOCK-knockdown group, but not showed with 2.5 mM glucose incubation. The expression of Cav1.2 and the influx of calcium were significantly decreased in the CLOCK-knockdown group with 25 mM glucose incubation. ChIP test indicted that the CLOCK bound to -444∼-454 region of the Cacna1c promoter of the INS-1 cells, but the binding was significantly reduced following the CLOCK-knockdown. Luciferase experiment was in accordance with the finding of ChIP. CONCLUSIONS: The CLOCK mediating Cav1.2 expression may point out a potential pathway of circadian rhythm affecting insulin secretion.