Theaflavin-3,3'-Digallate from Black Tea Inhibits Neointima Formation Through Suppression of the PDGFRß Pathway in Vascular Smooth Muscle Cells.

The abnormal neointima formation caused by the phenotypic switching of vascular smooth cells (VSMCs) into a synthetic state plays a key role in the pathogenesis of various vascular diseases, including atherosclerosis and postangioplasty restenosis. Theaflavin-3,3'-digallate (TF3) in black tea has been reported to exert antiinflammatory and anticancer effects, but its role in neointima formation remains unclear. Here, we delineated a remarkable effect of TF3 in suppressing neointima formation of VSMCs in vivo as well as the ability of primary rat aortic smooth cells (RASMCs) to proliferate and migrate in vitro. Further study confirmed that the effects of TF3 on PDGF-BB-induced RASMCs were due to reduced phosphorylation of PDGFRß, which led to the repression of downstream pathways. We concluded that TF3 may act as a repressor in the progression of neointima formation and serve as a potential therapeutic candidate for excessive phenotypic switching of VSMCs.

ß2-adrenergic receptor promotes liver regeneration partially through crosstalk with c-met.

The ß2-adrenergic receptor (ß2AR) is a G protein-coupled receptor (GPCR) that mediates the majority of cellular responses to external stimuli. Aberrant expression of ß2AR results in various pathophysiological disorders, including tumorigenesis, but little is known about its role in liver regeneration. This study aims to investigate the impact and the underlying mechanism of ß2AR in liver regeneration. Here, we found that ß2AR was upregulated during liver regeneration induced by 70% PH. Deletion of ß2AR in mice resulted in 62% mortality 2 days post-PH, decreased proliferative marker expression and impaired liver function throughout regeneration. Moreover, AAV8-mediated overexpression of ß2AR in hepatocytes accelerated the regeneration process and increased target gene expression. Mechanistically, ß2AR recruited G-protein-coupled receptor kinase 2 (GRK2) to the membrane and then formed a complex with c-met to transactivate c-met signaling, which triggered downstream extracellular regulated protein kinase (ERK) signaling activation and nuclear translocation. Inhibition of c-met with SU11274 or ERK with U0126 decreased ß2AR overexpression-induced hepatocyte proliferation. Our findings revealed that ß2AR might act as a critical mediator regulating liver regeneration by crosstalk with c-met and activation of ERK signaling.

Germline Mutation of PLCD1 Contributes to Human Multiple Pilomatricomas through Protein Kinase D/Extracellular Signal-Regulated Kinase1/2 Cascade and TRPV6.

Pilomatricoma, a benign skin appendage tumor, also known as calcifying epithelioma, consists of islands of epithelial cells histologically that contain anucleated cells in the center surrounded by basophilic cells and partial calcification. Sporadic pilomatricomas commonly have somatic mutations in the gene CTNNB1, but causative genes from germline and the underlying pathophysiology are unclear. In this study, we identified a germline missense variant of PLCD1 encoding PLCδ1, c.1186G>A (p.Glu396Lys), in a large Chinese family with autosomal dominant multiple pilomatricomas. Phospholipase C, a key enzyme playing critical roles in intracellular signal transduction, is essential for epidermal barrier integrity. The p.Glu396Lys variant increased the enzymatic activity of PLCδ1, leading to protein kinase C/protein kinase D/extracellular signal-regulated kinase1/2 pathway activation and TPRV6 channel closure, which not only resulted in excessive proliferation of keratinocytes in vitro and in vivo but also induced local accumulation of calcium in the pilomatricoma-like tumor that developed spontaneously in the skin of Plcd1E396K/E396K mice. Our results implicate this p.Glu396Lys variant of PLCD1 from germline leading to gain-of-function of PLCδ1 as a causative genetic defect in familial multiple pilomatricomas.

Comparing Mini Sarcopenia Risk Assessment With SARC-F for Screening Sarcopenia in Community-Dwelling Older Adults.

OBJECTIVE: The Mini Sarcopenia Risk Assessment (MSRA), a new sarcopenia screening tool, has 2 versions: MSRA-7 (full version, 7 items) and MSRA-5 (short version, 5 items). We aimed to compare the diagnostic values of MSRA-7 and MSRA-5 to SARC-F for screening sarcopenia. DESIGN: A diagnostic accuracy study. SETTING: A community in Chengdu, China. PARTICIPANTS: Older adults. MEASUREMENTS: Muscle mass, strength, and physical performance were tested using a bioimpedance analysis (BIA) device, handgrip strength, and walking speed, respectively. Using the Asian Working Group for Sarcopenia (AWGS) criteria as the gold standard, the sensitivity/specificity analyses of the 3 scales were assessed. Receiver operating characteristic (ROC) curves and area under the ROC curves (AUC) were used to compare the overall diagnostic accuracy of the 3 scales. RESULTS: We recruited 384 participants. Against the AWGS criteria, SARC-F had a sensitivity of 29.5% and a specificity of 98.1%, and the MSRA-7 had a sensitivity of 86.9% and a specificity of 39.6%, whereas the MSRA-5 had a sensitivity of 90.2% and a specificity of 70.6%. The AUCs of SARC-F, MSRA-7, and MSRA-5 were 0.89 [95% confidence interval (CI), 0.86-0.92], 0.70 (95% CI, 0.65-0.74), and 0.85 (95% CI, 0.81-0.89), respectively. The differences in AUCs between SARC-F and MSRA-7 and in those between MSRA-7 and MSRA-5 were statistically significant (P <.001), but the difference between SARC-F and MSRA-5 was not statistically significant (P = .130). CONCLUSION: MSRA-5 may serve as a novel screening tool for sarcopenia in Chinese community-dwelling older adults. SARC-F, a class screening tool, is also suitable for this population. MSRA-5 and SARC-F demonstrated a similar diagnostic accuracy in our study population. MSRA-5 has better sensitivity, whereas SARC-F has better specificity. However, the diagnostic value of MSRA needs to be further validated in different populations.

A sarcopenia index based on serum creatinine and cystatin C cannot accurately detect either low muscle mass or sarcopenia in urban community-dwelling older people.

The aim of this study was to evaluate the diagnostic value of the sarcopenia index (serum creatinine [mg/dl]/cystatin C [mg/dl] × 100) for estimating low muscle mass and sarcopenia in community-dwelling older adults. We included 371 older adults (≥60 years) with normal kidney function. Four common diagnostic criteria (the European Working Group on Sarcopenia in Older People (EWGSOP), Asia Working Group for Sarcopenia (AWGS), International Working Group on Sarcopenia (IWGS), and Foundation for the National Institutes of Health (FNIH) criteria) were separately applied as the "gold standard". The receiver operating characteristic (ROC) curves and the area under the ROC curves (AUC) were applied to evaluate the overall diagnostic accuracy. For identifying low muscle mass, the AUC ranged from 0.505 (95% confidence interval [CI] 0.453-0.557) to 0.558 (95% CI 0.506-0.609). For identifying sarcopenia, the AUC ranged from 0.555 (95% CI 0.503-0.606) to 0.618 (95% CI 0.566-0.668). Subgroup analyses according to gender showed similar results. In conclusion, the sarcopenia index based on serum creatinine and cystatin C may not serve as biomarkers of either low muscle mass or sarcopenia in urban community-dwelling older people with normal kidney function.

SARC-F for sarcopenia screening in community-dwelling older adults: Are 3 items enough?

A 3-item SARC-F (termed SARC-F-3 in our study) was recently suggested as a screening tool for sarcopenia.The aim of this study was to compare the diagnostic value of SARC-F-3 to SARC-F in community-dwelling older people.We conducted a diagnostic accuracy study in an urban community in Chengdu, China. People aged 60 years or older were included. Muscle mass, strength, and physical performance were measured by a bio-impedance analysis (BIA) device, handgrip strength, and gait speed test, respectively. The Asia Working Group for Sarcopenia (AWGS) criteria were applied as the "gold reference." The sensitivity/specificity analyses of SARC-F and SARC-F-3 were performed. The receiver operating characteristic (ROC) curve and area under the ROC curve (AUC) were applied to compare the overall accuracy of SARC-F and SARC-F-3. The cut-off points of SARC-F-3 for sarcopenia were determined using the Youden index method.A total of 384 older people aged 71.5 ±â€Š5.8 years were included. On the basis of the AWGS criteria, the prevalence of sarcopenia in our study population was 15.9%. The optimal cut-off point of SARC-F-3 for identifying sarcopenia was a total score of ≥ 2. In the whole study population, the sensitivity and specificity of SARC-F were 29.5% [95% confidence interval (95% CI): 18.5-42.6] and 98.1% (95% CI: 96.0-99.3), respectively, whereas the sensitivity and specificity of SARC-F-3 were 13.1% (95% CI: 5.8-24.2) and 97.8% (95% CI: 95.6-99.1), respectively. The AUCs of SARC-F and SARC-F-3 were 0.894 (95% CI: 0.859-0.923) and 0.676 (95% CI: 0.627-0.723), respectively (P < .001).The 3-item SARC-F may not be suitable for screening sarcopenia in community-dwelling older people.

Screening Sarcopenia in Community-Dwelling Older Adults: SARC-F vs SARC-F Combined With Calf Circumference (SARC-CalF).

OBJECTIVES: To compare the diagnostic value of the 5-component questionnaire that measures strength, assistance walking, rise from a chair, climb stairs, and falls (SARC-F) and SARC-F combined with calf circumference (SARC-CalF) for screening sarcopenia in community-dwelling older adults. DESIGN: A diagnostic accuracy study. SETTING: A community in Chengdu, China. PARTICIPANTS: Older adults aged 60 years or older. MEASUREMENTS: Muscle mass, muscle strength, and physical performance were estimated using a bioimpedance analysis device, handgrip strength, and gait speed, respectively. Four commonly used diagnostic criteria [European Working Group on Sarcopenia in Older People (EWGSOP), Asian Working Group for Sarcopenia (AWGS), International Working Group on Sarcopenia (IWGS), and Foundation for the National Institutes of Health (FNIH) criteria] were applied as the reference standard, separately. The sensitivity/specificity analyses of the SARC-F and SARC-CalF methods were evaluated. The receiver operating characteristics curves and the area under the receiver operating characteristics curves were used to compare the overall diagnostic accuracy of the SARC-F and SARC-CalF for identifying sarcopenia. RESULTS: We included 160 men and 224 women. Based on the 4 diagnostic criteria, the prevalence of sarcopenia ranged from 11.7% to 25.0%. Using the AWGS criteria as the reference standard, the SARC-CalF had a sensitivity of 60.7% and a specificity of 94.7% in the whole study population, whereas the SARC-F had a sensitivity of 29.5% and a specificity of 98.1%. The area under the receiver operating characteristics curves for SARC-CalF and SARC-F were 0.92 (95% confidence interval 0.89‒0.94) and 0.89 (95% confidence interval 0.86‒0.92), respectively (P = .003). We obtained similar results when using the other 3 criteria as the reference standard. Subgroup analyses revealed similar results in both men and women. CONCLUSIONS: SARC-CalF significantly improves the sensitivity and overall diagnostic accuracy of SARC-F for screening sarcopenia in community-dwelling older adults.

PiT2 regulates neuronal outgrowth through interaction with microtubule-associated protein 1B.

PiT2 is a member of the inorganic phosphate transporter family, and is extensively expressed in the nervous system. It was found that loop7 domain of PiT2 is not required for retroviral recognition and transport function. The exact functions of loop7 remain poorly understood. Here we show that loop7 of PiT2 is necessary for the transport of PiT2 protein to the cell surface. Further, loop7 is also related to the outgrowth of neurite in Neuro2A cells interacts with the light chain 1 of microtubule-associated protein 1B (MAP1B). PiT2 with mutated MAP1B binding sites affect neurite outgrowth whereas Pi transport function deficient mutants of PiT2 do not. We also show that Drosophila dPiT interacts with microtubule-associated protein Futsch, and dPiT is crucial for the normal development of neuromuscular junctions (NMJs). These results indicate that PiT2 might participate in the regulation of neuronal outgrowth by interacting with MAP1B and independently of its Pi transport function in the nervous system.

MicroRNA-532 and microRNA-3064 inhibit cell proliferation and invasion by acting as direct regulators of human telomerase reverse transcriptase in ovarian cancer.

Human telomerase reverse transcriptase (hTERT) plays a crucial role in ovarian cancer (OC) progression. However, the mechanisms underlying hTERT upregulation in OC, and the specific microRNAs (miRNAs) involved in the regulation of hTERT in OC cells, remains unclear. We performed a bioinformatics search to identify potential miRNAs that bind to the 3'-untranslated region (3'-UTR) region of the hTERT mRNA. We examined the expression levels of miR-532/miR-3064 in OC tissues and normal ovarian tissues, and analyzed the correlation between miRNA expression and OC patient outcomes. The impacts of miR-532/miR-3064 on hTERT expression were evaluated by western blot analysis and hTERT 3'-UTR reporter assays. We investigated the effects of miR-532/miR-3064 on proliferation and invasion in OC cells. We found that miR-532 and miR-3064 are down-regulated in OC specimens. We observed a significant association between reduced miR-532/miR-3064 expression and poorer survival of patients with OC. We confirmed that in OC cells, these two miRNAs downregulate hTERT levels by directly targeting its 3'-UTR region, and inhibited proliferation, EMT and invasion of OC cells. In addition, the overexpression of the hTERT cDNA lacking the 3'-UTR partially restored miR-532/miR-3064-inhibited OC cell proliferation and invasion. The silencing of hTERT by siRNA oligonucleotides abolished these malignant features, and phenocopied the effects of miR-532/miR-3064 overexpression. Furthermore, overexpression of miR-532/miR-3064 inhibits the growth of OC cells in vivo. Our findings demonstrate a miR-532/miR-3064-mediated mechanism responsible for hTERT upregulation in OC cells, and reveal a possibility of targeting miR-532/miR-3064 for future treatment of OC.

CGDB: a database of circadian genes in eukaryotes.

We report a database of circadian genes in eukaryotes (CGDB, http://cgdb.biocuckoo.org), containing ∼73 000 circadian-related genes in 68 animals, 39 plants and 41 fungi. Circadian rhythm is ∼24 h rhythm in behavioral and physiological processes that exists in almost all organisms on the earth. Defects in the circadian system are highly associated with a number of diseases such as cancers. Although several databases have been established for rhythmically expressed genes, a comprehensive database of cycling genes across phyla is still lacking. From the literature, we collected 1382 genes of which transcript level oscillations were validated using methods such as RT-PCR, northern blot and in situ hybridization. Given that many genes exhibit different oscillatory patterns in different tissues/cells within an organism, we have included information regarding the phase and amplitude of the oscillation, as well as the tissue/cells in which the oscillation was identified. Using these well characterized cycling genes, we have then conducted an orthologous search and identified ∼45 000 potential cycling genes from 148 eukaryotes. Given that significant effort has been devoted to identifying cycling genes by transcriptome profiling, we have also incorporated these results, a total of over 26 000 genes, into our database.

Nuclear envelope protein MAN1 regulates clock through BMAL1.

Circadian clocks serve as internal pacemakers that influence many basic homeostatic processes; consequently, the expression and function of their components are tightly regulated by intricate networks of feedback loops that fine-tune circadian processes. Our knowledge of these components and pathways is far from exhaustive. In recent decades, the nuclear envelope has emerged as a global gene regulatory machine, although its role in circadian regulation has not been explored. We report that transcription of the core clock component BMAL1 is positively modulated by the inner nuclear membrane protein MAN1, which directly binds the BMAL1 promoter and enhances its transcription. Our results establish a novel connection between the nuclear periphery and circadian rhythmicity, therefore bridging two global regulatory systems that modulate all aspects of bodily functions.

Dual PDF signaling pathways reset clocks via TIMELESS and acutely excite target neurons to control circadian behavior.

Molecular circadian clocks are interconnected via neural networks. In Drosophila, PIGMENT-DISPERSING FACTOR (PDF) acts as a master network regulator with dual functions in synchronizing molecular oscillations between disparate PDF(+) and PDF(-) circadian pacemaker neurons and controlling pacemaker neuron output. Yet the mechanisms by which PDF functions are not clear. We demonstrate that genetic inhibition of protein kinase A (PKA) in PDF(-) clock neurons can phenocopy PDF mutants while activated PKA can partially rescue PDF receptor mutants. PKA subunit transcripts are also under clock control in non-PDF DN1p neurons. To address the core clock target of PDF, we rescued per in PDF neurons of arrhythmic per°¹ mutants. PDF neuron rescue induced high amplitude rhythms in the clock component TIMELESS (TIM) in per-less DN1p neurons. Complete loss of PDF or PKA inhibition also results in reduced TIM levels in non-PDF neurons of per°¹ flies. To address how PDF impacts pacemaker neuron output, we focally applied PDF to DN1p neurons and found that it acutely depolarizes and increases firing rates of DN1p neurons. Surprisingly, these effects are reduced in the presence of an adenylate cyclase inhibitor, yet persist in the presence of PKA inhibition. We have provided evidence for a signaling mechanism (PKA) and a molecular target (TIM) by which PDF resets and synchronizes clocks and demonstrates an acute direct excitatory effect of PDF on target neurons to control neuronal output. The identification of TIM as a target of PDF signaling suggests it is a multimodal integrator of cell autonomous clock, environmental light, and neural network signaling. Moreover, these data reveal a bifurcation of PKA-dependent clock effects and PKA-independent output effects. Taken together, our results provide a molecular and cellular basis for the dual functions of PDF in clock resetting and pacemaker output.

MicroRNA-23a promotes myelination in the central nervous system.

Demyelinating disorders including leukodystrophies are devastating conditions that are still in need of better understanding, and both oligodendrocyte differentiation and myelin synthesis pathways are potential avenues for developing treatment. Overexpression of lamin B1 leads to leukodystrophy characterized by demyelination of the central nervous system, and microRNA-23 (miR-23) was found to suppress lamin B1 and enhance oligodendrocyte differentiation in vitro. Here, we demonstrated that miR-23a-overexpressing mice have increased myelin thickness, providing in vivo evidence that miR-23a enhances both oligodendrocyte differentiation and myelin synthesis. Using this mouse model, we explored possible miR-23a targets and revealed that the phosphatase and tensin homologue/phosphatidylinositol trisphosphate kinase/Akt/mammalian target of rapamycin pathway is modulated by miR-23a. Additionally, a long noncoding RNA, 2700046G09Rik, was identified as a miR-23a target and modulates phosphatase and tensin homologue itself in a miR-23a-dependent manner. The data presented here imply a unique role for miR-23a in the coordination of proteins and noncoding RNAs in generating and maintaining healthy myelin.

p75 neurotrophin receptor is a clock gene that regulates oscillatory components of circadian and metabolic networks.

The p75 neurotrophin receptor (p75(NTR)) is a member of the tumor necrosis factor receptor superfamily with a widespread pattern of expression in tissues such as the brain, liver, lung, and muscle. The mechanisms that regulate p75(NTR) transcription in the nervous system and its expression in other tissues remain largely unknown. Here we show that p75(NTR) is an oscillating gene regulated by the helix-loop-helix transcription factors CLOCK and BMAL1. The p75(NTR) promoter contains evolutionarily conserved noncanonical E-box enhancers. Deletion mutagenesis of the p75(NTR)-luciferase reporter identified the -1039 conserved E-box necessary for the regulation of p75(NTR) by CLOCK and BMAL1. Accordingly, gel-shift assays confirmed the binding of CLOCK and BMAL1 to the p75(NTR-)1039 E-box. Studies in mice revealed that p75(NTR) transcription oscillates during dark and light cycles not only in the suprachiasmatic nucleus (SCN), but also in peripheral tissues including the liver. Oscillation of p75(NTR) is disrupted in Clock-deficient and mutant mice, is E-box dependent, and is in phase with clock genes, such as Per1 and Per2. Intriguingly, p75(NTR) is required for circadian clock oscillation, since loss of p75(NTR) alters the circadian oscillation of clock genes in the SCN, liver, and fibroblasts. Consistent with this, Per2::Luc/p75(NTR-/-) liver explants showed reduced circadian oscillation amplitude compared with those of Per2::Luc/p75(NTR+/+). Moreover, deletion of p75(NTR) also alters the circadian oscillation of glucose and lipid homeostasis genes. Overall, our findings reveal that the transcriptional activation of p75(NTR) is under circadian regulation in the nervous system and peripheral tissues, and plays an important role in the maintenance of clock and metabolic gene oscillation.