Synthesis of α-Difluoromethylene Ethers via Photoredox-Induced Hyperconjugative Ring Opening of gem-Difluorocyclopropanes.

Fluorinated compounds have found widespread applications in pharmaceuticals, agrochemicals, and materials science. Precise construction of α-difluoromethylene ether (CF2-O) moiety in organic molecules is of high demand. Herein, a visible light-promoted reaction protocol for the synthesis of α-difluoromethylene ether from gem-difluorocyclopropane is described. The key ring-opening step is induced by hyperconjugative interaction of cyclopropane with photo-oxidized aromatic rings. This reaction is easy scale-up, and the products bearing a synthetic handle enable their further manipulation.

Iminyl Radical-Triggered 1,5-Hydrogen-Atom Transfer/Heck-Type Coupling by Visible-Light Photoredox Catalysis.

An efficient iminyl radical-triggered 1,5-hydrogen-atom transfer/Heck-type coupling cascade has been achieved through visible-light photoredox catalysis. A variety of unactivated C(sp3)-H bonds have been alkenylated efficiently and selectively with easily available alkenes, providing an elegant route to γ-alkenylated ketone.

Kv1.3 potassium channel mediates macrophage migration in atherosclerosis by regulating ERK activity.

Ion channels expressed in macrophages have been tightly related to atherosclerosis by coupling cellular function. How the voltage-gated potassium channels (Kv) affect macrophage migration remain unknown. The aim of our study is to investigate whether Kv1.3-ERK signaling pathway plays an important role in the process. We explored the expression of Kv1.3 in coronary atherosclerotic heart disease and found Kv1.3 channel was increased in acute coronary syndrome patients. Treatment of RAW264.7 cells with Kv1.3 small interfering RNA, suppressed cell migration. The expression of phosphorylated ERK1/2 also decreased after knockdown of Kv1.3. On the other hand, overexpression of Kv1.3 channel promoted cell migration and ERK1/2 phosphorylation. U-0126, the mitogen-activated protein kinase inhibitors, could reverse macrophage migration induced by Kv1.3 channel overexpression. Downregulation of Kv1.3 channel by siRNA could not further inhibit cell migration when cells were treated with U-0126. It means that ERK is downstream signal of Kv1.3 channel. We concluded that Kv1.3 may stimulate macrophage migration through the activation of ERK.

BP5 regulated B cell development promoting anti-oxidant defence.

Bursa of Fabricius is the humoral immune system for B cell differentiation and antibody production. Bursopentine (BP5) is a novel immunomodulatory peptide and significantly stimulated an antigen-specific immune response in mice. BP5 was also found to protect LPS-activated murine peritoneal macrophages from oxidative stress. In this study, the effects of BP5 on B cell development were examined. The results suggested that BP5 markedly promoted B cell development by increasing CFU-pre B, and affected the redox homeostasis regulation of B cells. To study the molecular mechanism of effect of bursal-derived BP5, this research utilized 2D-E and MALDI-TOF/TOF to analyze the differentially expressed proteins of BP5-treated WEHI-231 cells. The results showed that BP5 affected the redox homeostasis regulation of WEHI-231 cells and induced alterations in the protein expression profiles related to the oxidoreduction coenzyme metabolic process, precursor metabolites and energy, proteolysis, RNA splicing and translation and cellular process, respectively. BP5 also induced glucose-6-phosphate dehydrogenase (G6PD) activity, an essential anti-oxidant cofactor. We found that the redox homeostasis regulation effect of BP5 was reduced in G6PD-deficient cells. These data suggested that BP5 affected the redox balance toward reducing conditions by promoting the expression of G6PD, which in turn regulated the glutathione redox cycle and other processes.

Involvement of VKORC1 in the inhibition of calcium oxalate crystal formation in HK-2 cells.

The vitamin K epoxide reductase complex subunit 1 (VKORC1), the rate-limiting enzyme for vitamin K recycling, is significantly down-regulated in the kidneys of urolithiasis patients. This study searched for direct evidence to define the inhibitory activity of VKORC1 against calcium oxalate (CaOx) crystal formation. In the experiment of VKORC1 overexpression, HK-2 cells were transfected with the pFLAG-CMV-7.1-VKORC1 plasmid as a pFLAG-CMV-7.1-VKORC1 transfection group or the pFLAG-CMV-7.1 plasmid as a pFLAG-CMV-7.1 control group. In the experiment of VKORC1 knockdown, HK-2 cells were transfected with the PGPU6/GFP/Neo-VKORC1shRNA-2 as a PGPU6/GFP/Neo-VKORC1shRNA-2 transfection group or the PGPU6/GFP/Neo-shRNA-NC plasmid as a PGPU6/GFP/Neo-shRNA-NC control group. The expression of VKORC1 in HK-2 cells was detected by real-time quantitative PCR and Western blotting. The CaOx crystal formation was observed under the laser-scanning confocal microscope. It was found that the expression levels of VKORC1 mRNA and protein were significantly higher in the pFLAG-CMV-7.1-VKORC1 transfection group than in the pFLAG-CMV-7.1 control group (P<0.01). The number of CaOx crystals in HK-2 cells incubated in fluorescently labeled CaOx monohydrate (COM) crystal medium for 48 h was 14±4 per field (100×) in the pFLAG-CMV-7.1-VKORC1 transfection group and 26±5 per field (100×) in the pFLAG-CMV-7.1 control group respectively under the laser-scanning confocal microscope. The amount of CaOx crystal aggregation and formation in the pFLAG-CMV-7.1-VKORC1 transfection group was significantly reduced as compared with the pFLAG-CMV-7.1 control group (P<0.05). The expression levels of VKORC1 mRNA and protein were significantly lower in the PGPU6/GFP/Neo-VKORC1shRNA-2 transfection group than in the PGPU6/GFP/Neo-shRNA-NC control group (P<0.05). The number of CaOx crystals in HK-2 cells incubated in fluorescently labeled COM crystal medium was 65±11 per field (100×) in the PGPU6/GFP/Neo-VKORC1shRNA-2 transfection group and 24±6 per field (100×) in the PGPU6/GFP/Neo-shRNA-NC control group respectively under the laser-scanning confocal microscope. The amount of CaOx crystal aggregation and formation in the PGPU6/GFP/Neo-VKORC1shRNA-2 transfection group was significantly increased as compared with the PGPU6/GFP/Neo-shRNA-NC control group (P<0.05). These findings suggested that the VKORC1 protein could inhibit CaOx salt crystallization, adhesion and aggregation. This research would help us to understand the mechanisms involving the interaction between crystallization and epithelial cells and the formation of CaOx.

Identification and characterization of novel immunomodulatory bursal-derived pentapeptide-II (BPP-II).

The bursa of Fabricius, the acknowledged central humoral immune organ, plays a vital role in B lymphocyte differentiation. However, there are few reports of the molecular basis of the mechanism on immune induction and potential antitumor activity of bursal-derived peptides. In this paper, a novel bursal-derived pentapeptide-II (BPP-II, MTLTG) was isolated and exerted immunomodulatory functions on antibody responses in vitro. Gene microarray analyses demonstrated that BPP-II regulated expression of 2478 genes in a mouse-derived hybridoma cell line. Immune-related gene ontology functional procedures were employed for further functional analysis. Furthermore, the majority of BPP-II-regulated pathways were associated with immune responses and tumor processes. Moreover, BPP-II exhibited immunomodulatory effects on antigen-specific immune responses in vivo, including enhancement of avian influenza virus (H9N2 subtype)-specific antibody and cytokine production and modification of T cell immunophenotypes and lymphocyte proliferation. Finally, BPP-II triggered p53 expression and stabilization and selectively inhibited tumor cell proliferation. These data identified the multifunctional factor, BPP-II, as a novel biomaterial representing an important linking between the humoral central immune system and immune induction, including antitumor. Information generated in this study elucidates further the mechanisms involved in humoral immune system and represents the potential basis of effective immunotherapeutic strategies for treating human tumors and immune improvement.

Characterization of dissolved organic matter in groundwater from the Coastal Dagu River watershed, China using fluorescence excitation-emission matrix spectroscopy.

The fluorescent properties of dissolved organic matter (DOM) from groundwater in the coastal Dagu River watershed, North China were determined using excitation-emission matrix spectroscopy (EEMs) analysis. Surface water DOM samples were also investigated for comparison. Two humic-like components (C1: 250, 355/472 nm; C2: < 240, 325/400 nm) and one protein-like component (C3: <240, 280/340 nm) were identified using parallel factor analysis. Low intensities for all components were observed in groundwater DOM from the upper and lower reaches of the study area. However, higher abundances of these components occurred in the middle reaches, reflecting the combined effect of seepage of surface water with strong anthropogenic pollution and the alteration of groundwater circulation due to cutoff as a result of the construction of a cutoff wall since the late 1990s. The humic-like components were dominant in groundwater DOM, with the average percentage of the protein-like component being only 15%, which was less than half of the corresponding percentage in surface water DOM. The freshness index in groundwater DOM was lower than the surface water samples, while the fluorescence index and humification index were higher than in the latter. These indices demonstrated the much higher degree of humification for groundwater DOM, which may be related to the longer residence time of groundwater and greater contribution of microbial degradation in the aquifer environment. This study demonstrated that EEMs could distinguish between the effects of natural background and human activities on the quantity and characteristics of the groundwater DOM, and thus could be a useful tool for studing the carbon dynamics and the controlling factors in groundwater systems.

Crosstalk between PML and p53 in response to TGF-ß1: A new mechanism of cardiac fibroblast activation.

Cardiac fibrosis is a common pathological cardiac remodeling in a variety of heart diseases, characterized by the activation of cardiac fibroblasts. Our previous study uncovered that promyelocytic leukemia protein (PML)-associated SUMO processes is a new regulator of cardiac hypertrophy and heart failure. The present study aimed to explore the role of PML in cardiac fibroblasts activation. Here we found that PML is significantly upregulated in cardiac fibrotic tissue and activated cardiac fibroblasts treated with transforming growth factor-ß1 (TGF-ß1). Gain- and loss-of-function experiments showed that PML impacted cardiac fibroblasts activation after TGF-ß1 treatment. Further study demonstrated that p53 acts as the transcriptional regulator of PML, and participated in TGF-ß1 induced the increase of PML expression and PML nuclear bodies (PML-NBs) formation. Knockdown or pharmacological inhibition of p53 produced inhibitory effects on the activation of cardiac fibroblasts. We further found that PML also may stabilize p53 through inhibiting its ubiquitin-mediated proteasomal degradation in cardiac fibroblasts. Collectively, this study suggests that PML crosstalk with p53 regulates cardiac fibroblasts activation, which provides a novel therapeutic strategy for cardiac fibrosis.

Angiotensin-converting enzyme (ACE) 2 overexpression ameliorates glomerular injury in a rat model of diabetic nephropathy: a comparison with ACE inhibition.

The reduced expression of angiotensin-converting enzyme (ACE) 2 in the kidneys of animal models and patients with diabetes suggests ACE2 involvement in diabetic nephrology. To explore the renoprotective effects of ACE2 overexpression, ACE inhibition (ACEI) or both on diabetic nephropathy and the potential mechanisms involved, 50 Wistar rats were randomly divided into a normal group that received an injection of sodium citrate buffer and a diabetic model group that received an injection of 60 mg/kg streptozotocin. Eight wks after streptozotocin injection, the diabetic rats were divided into no treatment group, adenoviral (Ad)-ACE2 group, Ad-green flurescent protein (GFP) group, ACEI group receiving benazepril and Ad-ACE2 + ACEI group. Four wks after treatment, physical, biochemical, and renal functional and morphological parameters were measured. An experiment in cultured glomerular mesangial cells was performed to examine the effects of ACE2 on cellular proliferation, oxidative stress and collagen IV synthesis. In comparison with the Ad-GFP group, the Ad-ACE2 group exhibited reduced systolic blood pressure, urinary albumin excretion, creatinine clearance, glomeruli sclerosis index and renal malondialdehyde level; downregulated transforming growth factor (TGF)-ß1, vascular endothelial growth factor (VEGF) and collagen IV protein expression; and increased renal superoxide dismutase activity. Ad-ACE2 and ACEI had similar effects, whereas combined use of Ad-ACE2 and ACEI offered no additional benefits. ACE2 transfection attenuated angiotensin (Ang) II-induced glomerular mesangial cell proliferation, oxidative stress and collagen IV protein synthesis. In conclusion, ACE2 exerts a renoprotective effect similar to that of ACEI treatment. Decreased renal Ang II, increased renal Ang-(1-7) levels, and inhibited oxidative stress were the possible mechanisms involved.

Serum positive for the autoantibody against the ß(1)-adrenoceptor from Chinese patients with congestive heart failure decreases I(ss) in mouse cardiac myocytes.

Autoantibodies targeting the ß(1)-adrenergic receptor (AAB-ß(1)) display agonist-like effects, which may have a pathogenic role in the progression of heart failure. Here, we used the electrophysiological recordings to explore the effects of AAB-ß(1)-positive serum from Chinese patients with heart failure on the activity of the peak transient outward potassium current (I(to)) and the end 50 ms steady-state potassium current (I(ss)) in mouse cardiac myocytes. We found that the AAB-ß(1)-positive serum had no effect on the activity of I(to), but it produced a decrease in the currents of I(ss). A low concentration of positive serum (1/100) had a small inhibitory effect on I(ss). However, positive serum at 1 : 10, 1 : 20, and 1 : 50 significantly decreased I(ss). The concentration-dependence analysis showed that the EC(50) of AAB-ß(1)-positive serum was 1/60.24 and its nH was 2.86. It indicated that the AAB-ß(1) could inhibit I(ss) in mouse cardiomyocyte in a concentration-dependent manner.

Glycyrrhetinic acid extracted from Glycyrrhiza uralensis Fisch. induces the expression of Toll-like receptor 4 in Ana-1 murine macrophages.

Glycyrrhetinic acid (GA) is an active component of licorice root that has long been used as a herbal medicine for the treatment of peptic ulcer, hepatitis, and pulmonary and skin diseases in Asia and Europe. In this study, we analyzed the effect of GA extracted from Glycyrrhiza uralensis Fisch. on the expression of Toll-like receptors (TLRs) that play key roles in regulating the innate immune response against invading pathogens. Stimulation of Ana-1 murine macrophages with GA induced a significant dose-dependent expression of TLR-4, and its mRNA expression that increased from 3-h post-treatment was approximately fivefold over the level in the mock-treated cells. No endotoxin contamination contributed to the GA-induced TLR-4 expression, because polymyxin B treatment did not alter the upregulated expression of TLR-4 in GA-treated cells. Several molecules, such as myeloid differentiation factor 88, interferon-ß, and interleukin-6, which are involved in the TLR-4 downstream signaling pathway, were upregulated significantly in response to GA stimulation. Our findings demonstrate that GA is able to induce the expression of TLR-4 and activate its downstream signaling pathway.

Sulfonamide as Photoinduced Hydrogen-Atom Transfer Catalyst for Regioselective Alkylation of C(sp3)-H Bonds Adjacent to Heteroatoms.

Based on the DFT calculations, the sulfonamide was explored as an efficient hydrogen-atom transfer catalyst for the C(sp3)-H alkylation. The combination of a metal-free photoredox catalyst and a sulfonamide catalyst enables highly regioselective alkylation of the C-H bonds adjacent to heteroatoms, which features broad substrate scope and excellent functional group compatibility. Remarkably, the sulfonamide catalyst was also applicable to the C(sp3)-C(sp3) couplings through the merger of photoredox, nickel, and HAT catalysis.

Bevacizumab plus erlotinib in Chinese patients with untreated, EGFR-mutated, advanced NSCLC (ARTEMIS-CTONG1509): A multicenter phase 3 study.

Dual inhibition of epidermal growth factor receptor (EGFR) and vascular endothelial growth factor (VEGF) pathways may delay therapeutic resistance in advanced non-small cell lung cancer (NSCLC). This phase 3 study investigated the efficacy and safety of an erlotinib plus bevacizumab regimen in untreated patients with advanced NSCLC. In total, 311 patients received bevacizumab plus erlotinib (n = 157) or erlotinib only (n = 154). Progression-free survival (PFS) was 17.9 months (95% confidence interval [CI], 15.2-19.9) for bevacizumab plus erlotinib and 11.2 months (95% CI, 9.7-13.8) for erlotinib only (hazard ratio [HR] = 0.55; 95% CI, 0.41-0.73; p < 0.001). A brain metastases subgroup treated with bevacizumab plus erlotinib also showed improved PFS (HR = 0.48; 95% CI, 0.27-0.84; p = 0.008). Grade ≥3 treatment-related adverse events occurred in 86 (54.8%) and 40 (26.1%) patients, respectively. Bevacizumab plus erlotinib significantly improved PFS in patients with untreated metastatic EGFR-mutated NSCLC, including those with brain metastases at baseline.

Interactions, functions, and independence of plasma membrane STIM1 and TRPC1 in vascular smooth muscle cells.

Stromal interaction molecule 1 (STIM1) is a predicted single membrane-spanning protein involved in store-operated calcium entry and interacting with ion channels including TRPC1. Here, we focus on endogenous STIM1 of modulated vascular smooth muscle cells, which exhibited a nonselective cationic current in response to store depletion despite strong buffering of intracellular calcium at the physiological concentration. STIM1 mRNA and protein were detected and suppressed by specific short interfering RNA. Calcium entry evoked by store depletion was partially inhibited by STIM1 short interfering RNA, whereas calcium release was unaffected. STIM1 short interfering RNA suppressed cell migration but not proliferation. Antibody that specifically bound STIM1 revealed constitutive extracellular N terminus of STIM1 and extracellular application of the antibody caused fast inhibition of the current evoked by store depletion. The antibody also inhibited calcium entry and cell migration but not proliferation. STIM1 interacted with TRPC1, and TRPC1 contributed partially to calcium entry and cationic current. However, the underlying processes could not be explained only by a STIM1-TRPC1 partnership because extracellular TRPC1 antibody suppressed cationic current only in a fraction of cells, TRPC1-containing channels were important for cell proliferation as well as migration, and cell surface localization studies revealed TRPC1 alone, as well as with STIM1. The data suggest a complex situation in which there is not only plasma membrane-spanning STIM1 that is important for cell migration and TRPC1-independent store-operated cationic current but also TRPC1-STIM1 interaction, a TRPC1-dependent component of store-operated current, and STIM1-independent TRPC1 linked to cell proliferation.

Differential expression of TRPC channels in the left ventricle of spontaneously hypertensive rats.

This study was designed to identify the differential expression of the canonical transient receptor potential (TRPC) channels in the left ventricle of spontaneously hypertensive rats (SHR). Echocardiography studies were performed to compare the left ventricular function in SHR vs. Wistar-Kyoto rats (WKY), and the mRNA level of the TRPC channels was determined by quantitative real-time RT-PCR (qRT-PCR). Western blots were performed to examine whether the mRNA expression corresponded with the protein expression. Compared with the WKY, the mRNA expression of TRPC4 and TRPC5 was significantly increased in the 10-week-old SHR (P = 0.032 for TRPC4 and P = 0.043 for TRPC5), so did the TRPC4/5 protein content. The midwall fractional shortening (mFS) of SHR was lower than WKY (P = 0.016). Furthermore, increased expression of TRPC4/5 was correlated with both increased blood pressure and decreased mFS. These findings suggest that TRPC4 and 5 seem to be the main subtypes expressed in the heart of the SHR at the beginning period of hypertension. Theses channels may participate in the development of left ventricular systolic dysfunction.

Two single nucleotide polymorphisms in ALOX15 are associated with risk of coronary artery disease in a Chinese Han population.

Arachidonate 12/15-lipoxygenase (12/15-LOX) has been implicated in the pathogenesis of atherosclerosis, but with contradicting results. The aim of this study was to investigate the association of two polymorphisms in ALOX15 and the risk of coronary artery disease (CAD) in a Chinese Han population. A total of 519 unrelated CAD patients and 608 unrelated control subjects of the Chinese Han population were recruited in the case-control study. Two tagSNPs, rs7217186:T>C and rs2619112:G>A, were selected and genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). The carriers of the C allele (the CC homozygote and the CT heterozygote) of rs7217186:T>C and the carriers of the A allele (the AA homozygote and the GA heterozygote) of rs2619112:G>A displayed elevated odds ratios (ORs) for CAD compared with the TT homozygotes and GG homozygotes, respectively, after adjusting for other potential confounders including age, sex, body mass index, systolic blood pressure, diastolic blood pressure, glucose, triglyceride, total cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, and smoking status (adjusted odds ratio [OR] = 3.2, 95% confidence interval [CI]: 1.335-7.665, P = 0.009 and adjusted OR = 3.5, 95% CI: 1.343-9.330, P = 0.011). In stratified analyses, after adjusting those aforementioned confounders, the CC and CT genotypes of rs7217186:T>C were associated with a greater risk of CAD in subjects <60 years (adjusted OR = 5.7, 95% CI: 1.557-21.097, P = 0.009) and in females (adjusted OR = 9.3, 95% CI: 1.048-82.213, P = 0.045). For rs2619112:G>A, subjects (<60 years) carrying the A allele had a greater risk of CAD than the GG homozygotes (adjusted OR = 4.9, 95% CI: 1.215-19.547, P = 0.025); the male carriers of A allele also had a greater risk (adjusted OR = 3.5, 95% CI: 1.136-11.006, P = 0.029). In summary, the present study shows that after adjustment for other confounding CAD factors, rs7217186:T>C and rs2619112:G>A of ALOX15 are associated with increased risk of CAD in this Chinese Han population.

Estrogen analogues: promising target for prevention and treatment of esophageal squamous cell carcinoma in high risk areas.

Esophageal cancer (EC) is one of the six most common cancers across the world. The striking 3-4: 1male predominance of esophageal squamous cell carcinoma (ESCC) has not yet been well explained. Our hypothesis is that the changes in level of estrogen and/or subtype of estrogen receptor (ER) may exert a protective factor in esophageal carcinogenesis and thus estrogen analogues may represent a promising target for prevention and treatment of ESCC. Several lines of evidence in a mouse ESCC model have suggested an inhibitory role of estrogen in ESCC growth and development. Consistent with this, our results showed that male and female counterparts from a high incidence area (HIA) for EC had significantly decreased serum estradiol compared to healthy controls from a low incidence area (LIA). Moreover, serum level of estradiol of ESCC patients from the HIA were significantly lower compared to healthy controls from both HIA and LIA. Numerous studies indicate that relatively low androgen level, high estrogen level (environmental and endogenous) and ratio alteration of sex hormones are important factors explaining decreased ESCC incidence. Both ERalpha and ERbeta are ligands to estradiol with different effects on transcription at activator protein-1 sites. Estrogen exerts a suppressive effect, mainly through ERalpha in ESCC, and an accelerative function, mainly through ERbeta. Our hypothesis suggests that administration of novel potent estrogen analogues might be an effective measure for prevention and treatment of ESCC in HIA.

Clinical characteristics of four cancer patients with SARS-CoV-2 infection in Wuhan, China.

BACKGROUND: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) led to the outbreak of pneumonia in Wuhan. The virus is highly infectious. Patients with cancer might be susceptible to the viral infection because of the immunosuppressive state cause by therapies on tumors. CASE PRESENTATION: We present the clinical features of four cancer patients who were infected with SARS-CoV-2 in late January of 2020 in our hospital. Cases 1 and 3 were diagnosed as mild and common type of coronavirus disease 2019 (COVID-2019) and survived from the viral infection. They acquired SARS-CoV-2 infection during their staying in hospital under radiotherapy and surgery of the tumors. Cases 2 and 4 suffered from severe type of COVID-19, and Case 2 was dead owning to the advanced age, uncontrolled chronic B cell lymphocytic leukemia and many other underlying diseases. The immunosuppressive state induced by liver transplantation and anti-rejection therapy might contribute to the severity of COVID-19 in Case 4, who suffered from hepatitis B related hepatocellular carcinoma. However, Case 4 was recovered from COVID-19 after a combination therapy against virus, bacteria and fungi, and also respiratory support. Nearly all patients showed a decrease in lymphocytes including total CD3+ T cells, B cells, and natural killer cells after infection of the virus. CONCLUSIONS: The severity of COVID-19 might be influenced by immune system state and underlying diseases in cancer patients. And the treatment of SARS-CoV-2 infection in cancer patients is challenged by the immunosuppressive state of these patients under chemotherapy or surgery.

Tandem repeat sequence of duck circovirus serves as downstream sequence element to regulate viral gene expression.

Duck circovirus (DuCV) has a small, single-stranded circular DNA genome of approximately 1.99 kb. Through a genome sequence analysis using the dottup program, we found that a quadruple tandem repeat sequence (QTR) in the intergenic region between the rep and cap genes of the DuCV genome, but not in other circoviruses. The QTR was also substantially different and evolutionarily conserved in the genotype 1 and 2 DuCV strains. Furthermore, a luciferase reporter assay demonstrated that QTR functioned as a downstream sequence element (DSE) of polyadenylation signals to enhance mRNA stability, which was dependent on four copies but not the QTR direction. Cap and Rep expression derived by subgenomic constructs also revealed a critical role of QTR in regulating viral gene expression. Finally, a reverse genetic study of a DuCV-based minicircle DNA technique found that a deletion of QTR induced a significant deficiency in viral genes transcription and replication. Our findings were the first to report that QTR only exists in the DuCV genome and serves as a novel molecular marker of DuCV genotyping, and has revealed its crucial biological function in regulating viral gene expression.