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Conventional ALA-PDT (C-PDT) has limited efficacy in cutaneous squamous cell carcinoma (cSCC), and there is obvious pain during treatment, which limits its clinical application. We sought to modify photodynamic therapy into a more painless and effective treatment.We modified C-PDT by reducing the incubation time of the pro-sensitizer and increasing the light dose; we named this method modified ALA-PDT (M-PDT). We compared the pain response and curative effect between C-PDT and M-PDT in cSCC mouse models. Pain-related proteins were examined by western blot analysis and immunohistochemistry. Tumor progression-associated signaling pathways were analyzed by RNA-seq and western blot analysis. Reactive oxygen species (ROS) generation was measured with a ROS test kit and Microplate reader.M-PDT greatly reduced pain during treatment. Interestingly, when the cSCC tumor volume increased to 150-200 mmOur results suggest that M-PDT, which is more painless and effective than C-PDT, is expected to provide a solution for the treatment of cSCC.

5-Aminolevulinic acid-based photodynamic therapy (ALA-PDT) is an effective therapy in treating inoperable cutaneous squamous cell carcinoma (cSCC) in China. However, its exact mechanism in treating cSCC is still unclear. Here, we analyzed expression profiles of lncRNAs and mRNAs of cSCC after ALA-PDT in a mouse model using Mouse Transcriptome Assay 1.0 chip. A total of 592 and 828 differentially expressed genes were identified in comparisons of ALA-PDT after 3 h vs. control, ALA-PDT after 6 h vs. control respectively. Regarding the lncRNAs analysis, a total of 185 and 410 differentially expressed lncRNAs were sought out. Bioinformatics analyses including Gene Ontology, Kyoto Encyclopedia of Genes and Genomes pathway, pathway relation network, and gene set enrichment analysis revealed that the pathways of ALA-PDT in treating cSCC were mainly associated with immune regulation, NF-κB signaling pathway, TLR signaling pathway, PI3K-Akt signaling pathway, TNF signaling pathway, and MAPK signaling pathway, which was validated by western blot analysis. Furthermore, a lncRNA-mRNA co-expression network revealed 118 network pairs comprising 36 lncRNAs and 68 mRNAs. These findings provide a basis for investigations into the treatment mechanisms of ALA-PDT in treating cSCC.

Background Acne vulgaris is a chronic inflammatory skin disease around pilosebaceous unit. 5-Aminolaevulinic acid photodynamic therapy (ALA-PDT) is an effective therapy for severe acne vulgaris. However, its specific treatment mechanism remains unclear. In the present study, we investigated the potential mechanism of how ALA-PDT induced lipid secretion inhibition in acne vulgaris. Methods Primary human sebocytes and sebaceous gland of golden hamster were treated with/without ALA-PDT. Cell viability was evaluated by Live/Dead Cell assay. Fluorescence microscope was used to observe lipids secretion in sebocytes after Nile red staining. The expression of SREBP-1 after ALA-PDT was evaluated by qRT-PCR. Regulation of ALA-PDT on AMPK/SREBP-1 was evaluated by western blot. Results The results showed that ALA-PDT suppressed lipid secretion of primary human sebocytes. In addition, ALA-PDT could inhibit the expression of SREBP-1 in vitro. We also found that ALA-PDT activated AMPK pathway, down-regulating the expression of SREBP-1 in sebocytes after ALA-PDT. Conclusions These findings elucidate that ALA-PDT suppresses lipid secretion through AMPK/SREBP-1 pathway in treatment of acne vulgaris.

Right ventricular (RV) function is an independent predictor of cardiovascular adverse events in patients with multiple cardiopulmonary disease ([1][1]). Recently, 2-dimensional (2D) speckle-tracking echocardiography (2D-STE) has been demonstrated to be a more sensitive and robust technique for

Tumor-derived exosomes contain informative microRNAs involved in carcinogenesis, cell migration, invasion and epithelial-mesenchymal transition (EMT), eventually contributing to metastasis of cancers. This study aims to clarify which and how exosomal miRNA affects tumor carcinogenesis and metastasis. Among them, miR-499a-5p was upregulated in both highly metastatic lung cancer cell line and their exosomes. MiR-499a-5p overexpression promoted cell proliferation, migration and EMT, while miR-499a-5p knockdown suppressed these processes in vitro. Inhibition of miR-499a-5p by antagomirs administration restrained tumor growth in vivo. Consequently, miR499a-sufficient exosomes, derived from highly metastatic cell line, enhanced cell proliferation, migration and EMT via mTOR pathway, and the effect could be inhibited by miR-499a-5p inhibitor. The study reveals the potential diagnostic and therapeutic value of cancer-derived exosomal miR-499a-5p, and sheds a new insight on a novel molecular mechanism which modulates metastasis.

Background Small-cell lung cancer (SCLC), a malignant tumor, is usually widely metastatic when diagnosed. The lack of important therapeutic clinical advances makes it difficult to treat. Previous studies showed that Adjudin had anticancer effects in many other human cancers, and it was synergetic with cisplatin in non-small cell lung cancer. However, the mechanism on SCLC was unclear. Methods We investigated the potential mechanism and effect of Adjudin on SCLC both in vitro and in vivo. Results An SCLC xenograft model showed that Adjudin inhibited tumor growth and was significantly synergetic with paclitaxel (in vitro as well). Cell Counting Kit-8 assays, flow cytometric analysis and western blotting showed that Adjudin effectively suppressed SCLC cell proliferation by inducing S phase arrest and caspase-dependent apoptosis. Moreover, Transwell and scratch assays showed that Adjudin also effectively inhibited migration and invasion. Furthermore, Adjudin activated the sirtuin 3 (SIRT3)-Forkhead box O3a (FOXO3a) pathway. Downregulating SIRT3 or FOXO3a significantly attenuated Adjudin-induced anticancer effects. Furthermore, higher expression of SIRT3 and FOXO3a were positively correlated, and both were associated with longer survival in lung cancer patients. Conclusion Overall, the present study is the first to show that Adjudin synergizes with paclitaxel and inhibits cell growth and metastasis by regulating the SIRT3-FOXO3a axis in SCLC; thus, Adjudin has great potential to be an anticancer agent.

B cell activating factor from the TNF family (BAFF) is implicated in not only the physiology of normal B cells, but also the pathophysiology of aggressive B cells related to malignant and autoimmune diseases. Autophagy plays a crucial role in balancing the beneficial and detrimental effects of immunity and inflammation. However, little is known about whether and how excessive BAFF mediates autophagy contributing to B-cell proliferation and survival. Here, we show that excessive human soluble BAFF (hsBAFF) inhibited autophagy with a concomitant reduction of LC3-II in normal and B-lymphoid (Raji) cells. Knockdown of LC3 not only potentiated hsBAFF inhibition of autophagy, but also attenuated hsBAFF activation of Akt/mTOR pathway, thereby diminishing hsBAFF-induced B-cell proliferation/viability. Further, we found that hsBAFF inhibition of autophagy was Akt/mTOR-dependent. This is supported by the findings that hsBAFF increased mTORC1-mediated phosphorylation of ULK1 (Ser757); Akt inhibitor X, mTORC1 inhibitor rapamycin, mTORC1/2 inhibitor PP242, expression of dominant negative Akt, or knockdown of mTOR attenuated hsBAFF-induced phosphorylation of ULK1, decrease of LC3-II level, and increase of cell proliferation/viability. Chelating intracellular free Ca2+ ([Ca2+]i) with BAPTA/AM or preventing [Ca2+]i elevation using EGTA or 2-APB profoundly blocked hsBAFF-induced activation of Akt/mTOR, phosphorylation of ULK1 and decrease of LC3-II, as well as increase of cell proliferation/viability. Similar effects were observed in the cells where CaMKII was inhibited by KN93 or knocked down by CaMKII shRNA. Collectively, these results indicate that hsBAFF inhibits autophagy promoting cell proliferation and survival through activating Ca2+-CaMKII-dependent Akt/mTOR signaling pathway in normal and neoplastic B-lymphoid cells. Our findings suggest that manipulation of intracellular Ca2+ level or CaMKII, Akt, or mTOR activity to promote autophagy may be exploited for prevention of excessive BAFF-induced aggressive B lymphocyte disorders and autoimmune diseases.

A distressing issue of diabetic ulcer (DU) is its poor healing feature with limited clinical solutions. We have previously shown that 5-aminolevulinic acid photodynamic therapy (ALA-PDT) is a promising alternative to the currently limited measures for DU. Mesenchymal stem cells (MSCs) transplantation has been believed to impose certain therapeutic effect on restoration of injury. Thus, this study aims to explore whether the combination of MSCs and ALA-PDT will exert a more advanced curative effect on DU.Diabetic mice were induced by intraperitoneal injection of streptozotocin (STZ, 60 mg/kg/d) for consecutive 5 days. A full-thickness skin injury (diameter 6 mm) was created in the center of the back of each mouse, and then 10 μl of methicillin-resistant Staphylococcus aureus (MRSA) suspension was added to establish an infected DU model. All DU models were randomly divided into four groups: Untreated group, MSCs group, ALA-PDT group, and ALA-PDT combined with human umbilical cord mesenchymal stem cells (hUC-MSCs) (ALA-PDT + MSCs) group. The wound sizes were recorded by a digital camera, and the healing rates were calculated using Image J software. Bacterial loads on wounds were measured using CFU (Colony forming units) analysis. The epithelialization, inflammatory cells infiltration and granulation tissue formation were monitored by Haematoxylin and eosin (HE) staining, and the corresponding semi-quantitative score was matched. Growth and pro-inflammatory cytokines were detected by enzyme-linked immunosorbent assay (ELISA).Either ALA-PDT or injection of hUC-MSCs resulted in a rapid wound closure compared with the untreated, while their combination brought about the most prominent healing. On day 12, healing rates of the untreated, MSCs, ALA-PDT and ALA-PDT + MSCs were 40.56% ± 7.06%, 74.23 ± 4.83%, 84.03 ± 3.53%, 99.67 ± 0.49%, respectively. The bacterial burden reductions were approximately 1.58 logs (97.36%, P0.05), 2.34 logs (99.54%, P0.01), 4.50 logs (nearly 100%, P0.001) for MSCs, ALA-PDT and ALA-PDT + MSCs, respectively. Histology revealed reduced inflammatory cells and improved collagen precipitation and angiogenesis after hUC-MSCs and ALA-PDT treatment compared to the untreated. The combined therapy leaded to a more intact epithelium, similar to the healthy. Finally, ELISA revealed that the property of ALA-PDT to stimulate transforming growth factor-β1 (TGF-β1) and vascular endothelial growth factor (VEGF) and inhibit IL (interleukin) -1β and IL-6 outweighed that of hUC-MSCs, and this function of the combination overwhelmed that of any single therapy.Our findings indicated that the strategy of combining ALA-PDT with hUC-MSCs possessed a significantly enhanced therapeutic effect over either single therapy, providing a promising innovative therapeutic candidate for refractory wounds.

This paper presents a new theoretical integrated modeling approach with practical case studies for calculating container closure integrity (CCI) that concurrently accounts for both diffusion and mass/volumetric flow in real time. For pharmaceutical, biological, cell, and gene therapies, container closure systems (CCSs) must ensure drug sterility and stability by safeguarding against microbial contamination and gaseous ingress (e.g., oxygen, carbon dioxide, moisture, etc.) according to product requirements. In addition to the testing approach for evaluating CCI performance, a modeling approach can be an important part of CCI control strategy. Modeling is a powerful tool that provides information in situations where testing is not feasible, technically impossible, too time-consuming, or too expensive. Previously published models have lacked a systematic approach, or the versatility needed to coherently and concurrently integrate both diffusion and effusion to solve problems arising in field applications. The new integrated modeling approach described in this paper applies a robust numerical method to real world applications. The model is based on the law of conservation and continuity for molecular flow, Fick′s law of diffusion, and Darcy-Weisbach theory of frictional mass/volumetric flow. This new integrated modeling approach handles time-dependent diffusion and effusion by combining diffusion and mass/volumetric flow seamlessly in real time. For CCS under vacuum filled with nitrogen, this new modeling approach is able to reveal that oxygen ingress into CCS through a leak path will enter in two phases, starting with effusion and continuously followed by diffusion in a seamless transition. Our integrated modeling approach is capable to calculate and capture exact timing of the phase transition point providing unique understanding of complicated CCS problems. Using the finite difference method, all modeling results are numerically solved from governing equations along with initial and boundary conditions for each individual cases. The modeling results were precise and consistent with previously published testing results. This new integrated modeling approach displayed its capability and versatility to handle complicated leakage scenarios in practical applications. As a part of CCI control strategy, the modeling approach is a powerful tool for evaluating leaks, gauging their leak sizes, determining whether the CCS conforms to product requirements, and making informed decisions accordingly. While additional studies are to be carried out to fully develop the potential of this model, the applications hold great promise and in addition, to providing insight into container closure integrity may also provide a solid foundation for CCIT, and providing a solid foundation for CCI testing method development and validation for CCI performance.

Chronic refractory wounds are generally caused by local tissue defects and necrosis, and they are characterized by delayed wound healing as well as high recurrence, which seriously affects life quality. However, effective therapeutics to treat wounds are currently unavailable. Therapy primarily aims to accelerate generation of granulation tissue and decrease recurrence. The pathogenesis of chronic refractory wounds is closely related to multiple complex signaling pathways and a series of cytokines. Among these signaling pathways, TGF-β/Smad7 axis plays a critical role. Specifically, Smad7 is an antagonist of TGF-β that inhibits activation of TGF-β. Moreover, Smad7 promotes wound healing by regulating cytokines and controlling growth, differentiation and apoptosis of cells, which may be exploited to cure the disease. This review aims to reveal the exact functions and mechanisms of Smad7 in regulation of wound healing.

Acne vulgaris is a chronic inflammatory cutaneous disease. 5-Aminolaevulinic acid photodynamic therapy (ALA-PDT) is a novel and effective approach for severe acne vulgaris treatment. However, its specific treatment mechanism still remains unclear. In the present study, we investigated the potential mechanism of how ALA-PDT regulated intense inflammatory response in acne vulgaris. It appeared that ALA-PDT suppresses proliferation and lipid secretion of primary human sebocytes. Besides, ALA-PDT could up-regulate the expression of CXCL8 in vivo and in vitro, amplifying the inflammatory response by recruiting T cells, B cells, neutrophils and macrophages. We also found that ALA-PDT elevated the expression of CXCL8 via p38 pathway. SB203580, a p38 pathway inhibitor, decreased the expression of CXCL8 in sebocytes after ALA-PDT. These findings indicate that ALA-PDT amplifies the intense inflammatory response in the treatment of acne vulgaris via CXCL8. Our data decipher the mechanism of intense inflammatory response after ALA-PDT for acne vulgaris.

Aims Glioblastoma Multiforme (GBM) is the most aggressive form of primary brain tumour, with a median survival of 12-14 months after diagnosis. Although GBM has been extensively characterised on the molecular level during the past decades, many targeted therapies have been proved ineffective due, in part, to high heterogeneity of GBM. Thus, novel therapies targeting the altered metabolism which is exhibited by all cancer cells have gained much attention. The therapeutic ketogenic diet (KD) is a high fat, low carbohydrate and adequate protein diet. It has been recognized as a treatment for refractory paediatric epilepsy for decades. Recent studies have shown that a KD reduced tumour growth and potentiated the effects of therapy in some glioma animal models. However, the underlying mechanism(s) is still unclear. Thus, the aim of this study was to understand the mechanism of action behind the KD’s effects in inhibiting tumour growth and potentiating chemotherapy and radiotherapy. Method To unravel the mechanism of action, we analyzed the expression of genes encoding chromatin modifying enzymes in brain tumour samples from mice fed either a KD or standard diet (SD), using the Mouse Epigenetic Chromatin Modification Enzyme PCR Array (Qiagen, Germany). The expression of genes of interest selected from the array were validated by qRT-PCR. Human GBM cell lines and primary cells from GBMs were used to validate the results of the GBM mouse model. Beta-hydroxybutyrate, the main physiological ketone body found in the circulation of patients during KD, was used in in vitro experiments to mimic the in vivo physiological effect of a KD. The effect of protein arginine methyltransferase 8 (PRMT8) overexpression in GBM cells was studied using a lentiviral system. Cell proliferation was measured by Sulforhodamine B assay (Sigma, USA). Spheroid growth and invasion was measured in GBM spheroids cultured in Matrigel matrix (Corning, USA). Results Our results highlighted changes in the expression of a number of key chromatin modifying enzymes in mice fed a KD compared to those fed a SD. PRMT8, a gene highly downregulated in GBM, was upregulated in tumors from mice fed a KD, with corresponding downregulation of its target genes, dihydrofolate reductase (DHFR) and C-X-C chemokine receptor type 4 (CXCR4). Our results also showed that overexpression of PRMT8 in GBM cells reduced cell proliferation and invasiveness. Conclusion PRMT8, DHFR and CXCR4 have been shown to play key roles in tumour growth, invasion, migration and chemo/radio-resistance. Moreover, therapeutic strategies to downregulate these genes have been investigated in the form of methotrexate for DHFR inhibition and small molecule inhibitors of CXCR4. Thus, our results suggest that one mechanism through which the KD exerts its therapeutic effects may be through altering the expression of chromatin modifying enzymes. This provides additional support for the use of a KD as an adjuvant in combination with existing therapeutic approaches.

Background: Acne vulgaris is a chronic inflammatory cutaneous disease. 5-Aminolaevulinic acid photodynamic therapy (ALA-PDT) is a novel and effective therapy for severe acne vulgaris. However, the specific mechanism of ALA-PDT for acne still remain unclear. Here, we investigate the possible mechanism of intense inflammatory response of ALA-PDT for acne vulgaris. Results: It appeared that ALA-PDT suppresses proliferation and lipid secretion of primary human sebocytes. And ALA-PDT could up-regulate the expression of CXCL8 in vivo and in vitro, amplifying inflammatory response by recruiting T cells, B cells, neutrophils and macrophages. We also found that ALA-PDT elevated the expression of CXCL8 via p38 pathway. SB203580, a p38 pathway inhibitor, decreased the expression of CXCL8 after ALA-PDT in sebocytes. Conclusion: ALA-PDT amplifies intense inflammatory response in the treatment of acne vulgaris via CXCL8. Our data deciphers the mechanism of intense inflammatory response after ALA-PDT for acne vulgaris.

Background 5-aminolevulinic acid-mediated PDT (ALA-PDT) has been used in a variety of skin diseases including cSCC (cutaneous squamous cell carcinoma). Halofuginone (HL) is a less-toxic febrifugine derivative and has inhibitory effects on a variety of cancer cells. For now, there is no published study focusing on the combination use of ALA-PDT with HL to improve clinical efficacy of cSCC. Objective In this study, we will examine the effectiveness of combined treatment of ALA-PDT and HL in cSCC as well as its underlying mechanism. Methods The human epidermoid carcinoma cell line SCL-1 was treated with ALA-PDT or/ and HL, and cell viability, cell migration, ROS production, apoptosis were evaluated by CCK-8, colony formation, scratch assay, DCFH-DA probe, flow cytometry, respectively. The protein expression of NRF2 signaling was examined by western blot. Results HL strengthened ALA-PDT's inhibition of SCL-1 cell viability, migration, as well as NRF2 related β-catenin, p-Erk1/2, p-Akt and p-S6K1 expression. Overexpression of NRF2 conferred resistance to co-treatment's effects on c-Myc, Cyclin D1, Bcl-2, as well as cell proliferation. HL also strengthened ALA-PDT's inhibition of tumor volume in cSCC mouse model and elevated ROS generation of ALA-PDT. Conclusion HL enhances the anti-tumor effect of ALA-PDT in vitro and in vivo. HL has the potential to enhance the anti-tumor effect of ALA-PDT in cSCC via inhibiting NRF2 signaling.

Hyperlipidemia is considered one of the most important risk factors for erectile dysfunction (ED). To determine the effect of sodium tanshinone IIA sulfonate (STS) as an antioxidant agent on ED in high-fat diet- (HFD-) induced hyperlipidemia in rats and to investigate if STS administration could improve erectile function via hydrogen sulfide (H2S) production by inhibition of oxidative stress. Hyperlipidemia was induced in Sprague-Dawley rats by feeding HFD for 16 weeks. The rats were randomly divided into 3 groups: control, HFD, and HFD treated with STS (10 mg/kg/day for 12 weeks, intraperitoneal injection). Erectile function including intracavernosal pressure (ICP), H2S production, and antioxidant capacity was assessed. In addition, cavernosal smooth muscle cells (CSMC) isolated from SD rats were pretreated with STS in vitro and exposed to H2O2. Expressions of nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1), activity of antioxidant enzymes, and H2S-generating enzymes within CSMC were examined. ICP was significantly decreased in HFD rats compared with control. In addition, decreased H2S production and expression of cystathionine ɣ-lyase (CSE) and cystathionine β-synthase (CBS) associated with increased oxidative stress were observed in the penile tissue of HFD rats. However, all these changes were reversed by 16 weeks after STS administration. STS also increased antioxidant defense as evidenced by increased expression of Nrf2/HO-1 in the penile tissue of HFD rats. In CSMC, pretreatment with STS attenuated the decreased expression of CSE and CBS and H2S production by H2O2. STS exerted similar protective antioxidative effect as shown in the in vivo hyperlipidemia model. The present study demonstrated the redox effect of STS treatment on ED via increased H2S production in HFD-induced hyperlipidemia rat model by increased antioxidant capacity via activation of the Nrf2/HO-1 pathway, which provides STS potential clinical application in the treatment of hyperlipidemia-related ED.

The Hippo pathway plays a critical role in organ size control, tissue homeostasis and tumor genesis through its key transcription regulator Yes-associated protein1 (YAP1), but the mechanism underlying its role in lung cancer is unclear. We hypothesized that YAP1 influences FGFR1 signaling to maintain cancer stem-like cell (CSC) properties in FGFR1-amplified lung cancer. In support of this, our data confirms that expression levels of YAP1 are positively associated with those of FGFR1 in clinical lung carcinoma samples as measured by real-time PCR, western blot, and immunohistochemistry (IHC) staining. Mechanistically, YAP1 up-regulates FGFR1 expression at the level of promoter through the TEAD binding site while bFGF/FGFR1 induces YAP1 expression via large tumor suppressors 1(LATS1). In addition, the absence of YAP1 abolishes self-renewal ability in lung cancer. Furthermore, an orthotropic mouse model highlights the function of YAP1 in the initiation and metastasis of lung cancer. Verteporfin, a YAP1 inhibitor, effectively inhibits both YAP1 and FGFR1 expression in lung cancer. Thus, we conclude that YAP1 is a potential therapeutic target for lung cancer. Combined targeting of YAP1 and FGFR1 may provide benefits to patients with FGFR1-amplified lung cancer.

Aortic stenosis (AS) and aortic regurgitation (AR) were two types of aortic valve defects which had different hemodynamic characteristics. Ultimately, chronic pressure and volume overloads result in different geometrical and functional patterns. The expectation of our study was to compare the impacts of two different types of loading conditions on myocardial deformation in asymptomatic patients with normal ejection fraction in compensatory stage using three-dimensional speckle tracking echocardiography (3D-STE). Among severe aortic valve disease, 36 patients with AR, and 32 patients with AS were enrolled. All subjects underwent conventional two-dimensional echocardiography and 3D-STE. Clinical and echocardiography characteristics were compared between the groups. Compared with control group, left ventricular twist was increased, along with decreased longitudinal, radial, and circumferential strain in the AS group. And AR patients were characterized by a spherical expansion of ventricle, and reduced myocardial longitudinal and radial strain, along with a preserved rotational motion. In univariate linear regression analysis, age, diastolic blood pressure, e’, sphericity index, end-diastolic volume index, stoke volume index, ejection fraction, and left ventricular mass index were independently associated with global longitudinal strain (GLS) in the AR group and relative wall thickness, stroke volume index (SVI), LV mass index, and peak velocity were independently associated with GLS in the AS group. In addition, multiple stepwise regression analysis showed that sphericity index, SVI, and left ventricular ejection fraction (LVEF) were independently associated with GLS in the AR group. And LVEF, peak velocity, and left atrial dimension were independently associated with GLS in the AS group. The affected myocardial layers of AS patients were worse than AR patients, but increased twist was the compensatory mechanism to maintain the effective stroke volume. Multidirectional myocardial damages alert clinicians to be aware that these patients need closer attention.

Background Squamous cell carcinoma (SCC) of the lip removed by surgery may cause lip dysfunction and scar. ALA-PDT (5-Aminolevulinic acid photodynamic therapy) is a minimally invasive treatment for superficial SCC in situ. However, few studies reported the use of topical ALA-PDT to manage lip SCC. Methods Between 2015 and 2017, 6 patients with Tis to T2 lip squamous cell carcinoma without evidence of lymph node spread, were treated with topical ALA-PDT at Shanghai Skin Disease Hospital. Clinical responses and side effect were evaluated according to the Response Evaluation Criteria in Solid Tumors (RECIST) at 1 month, 6 months, 12 months and 24 months after ALA-PDT. Results All of the 6 patients achieved complete response (CR) by 2–7 sessions of ALA-PDT. There was no relapse during 24 months follow up for 4 patients. Two out of 6 patients relapsed at 10 months and 20 months post PDT but achieved CR again by 1–3 more sessions of PDT. There was no functional or aesthetic problem. Conclusion Topical 5-Aminolevulinic acid photodynamic could be considered a potential alternative therapeutic option for early-stage lip squamous cell carcinoma without lymph node spread.

OBJECTIVES A sonographic study was conducted to determine the prevalence of atherosclerosis across multiple arterial beds in an elderly Chinese population and to examine relationships between detected atherosclerosis and traditional risk factors. METHODS A total of 197 participants underwent sonography of the abdominal aorta and bilateral carotid, femoral, and lower limb arteries. Images were reviewed to determine the presence or absence of plaques in each artery. Plaque thickness was measured as the indicator of plaque burden. Plaque prevalence was estimated per site and correlated with age, sex, and the Framingham Risk Score (FRS). Plaque frequency and thickness were compared between different arterial beds. RESULTS Of the 197 participants (54% female; age range, 58-86 years), 90% had plaques present in at least 1 artery, and 55% had plaques present in at least 4 arteries. The most common sites for plaques were the carotid arteries (80%), followed by the lower limb arteries (59%), femoral arteries (57%), and abdominal aorta (37%). Plaque prevalence in each arterial bed except the abdominal aorta was significantly associated with male participants (P

Glioblastoma Multiforme (GBM) is the most aggressive form of primary brain tumour, with a median survival of 12–14 months after diagnosis. Although GBM has been extensively characterised on the molecular level during the past decades, many targeted therapies have been proved to be ineffective due to high heterogeneity of GBM. Thus, novel therapies targeting the altered metabolism which is exhibited by all cancer cells have gained more attentions. Our strategy is the therapeutic ketogenic diet (KD), a high fat, low carbohydrate and adequate protein diet, which has been recognized as a treatment for refractory paediatric epilepsy. Recent studies have shown that KD reduces tumour growth and potentiates the effects of radiotherapy in some glioma animal models. However, the underlying mechanism is still unclear. To unravel the mechanism of action, we carried out small-RNA sequencing and chromatin modifying enzyme analysis using brain tumour samples from GBM mice model, fed with either KD or standard diet (SD). Our results highlighted an overall upregulation of tumour suppressor miRNAs and key chromatin modifying enzymes that are typically downregulated in GBM in mice fed with KD compared with those fed with SD. We also observed a corresponding downregulation in target genes of these miRNAs and chromatin modifying enzymes. These genes have been reported as key regulators in tumour growth, tumour invasion and chemo/radio-resistance in previous publications. Therefore, our study indicates that targeting cancer metabolism using the KD alters the epigenetic landscape of GBM and induces changes in key genes that potentiate the effects of chemo/radiotherapy.

OBJECTIVE: To identify a convergent, multitarget proliferation characteristic for astrocytoma transformation that could be targeted for therapy discovery.METHODS: Using an integrated functional genomics approach, we prioritized networks associated with astrocytoma progression using the following criteria: differential co-expression between grade II and grade III IDH1-mutated and 1p/19q euploid astrocytomas, preferential enrichment for genetic risk to cancer, association with patient survival and sample-level genomic features. Drugs targeting the identified multitarget network characteristic for astrocytoma transformation were computationally predicted using drug transcriptional perturbation data and validated using primary human astrocytoma cells.RESULTS: A single network, M2, consisting of 177 genes, was associated with glioma progression on the basis of the above criteria. Functionally, M2 encoded physically interacting proteins regulating cell cycle processes and analysis of genome-wide gene-regulatory interactions using mutual information and DNA-protein interactions revealed the known regulators of cell cycle processes FoxM1, B-Myb, and E2F2 as key regulators of M2. These results suggest functional disruption of M2 via gene mutation or altered expression as a convergent pathway regulating astrocytoma transformation. By considering M2 as a multitarget drug target regulating astrocytoma transformation, we identified several drugs that are predicted to restore M2 expression in anaplastic astrocytoma toward its low-grade profile and of these, we validated the known antiproliferative drug resveratrol as down-regulating multiple nodes of M2 including at nanomolar concentrations achievable in human cerebrospinal fluid by oral dosing.INTERPRETATION: Our results identify M2 as a multitarget network characteristic for astrocytoma progression and encourage M2-based drug screening to identify new compounds for preventing glioma transformation.

MicroRNA (miRNA) and fibroblast growth factor receptor 1 (FGFR1) dysregulation are considered to play an important role in tumor proliferation, invasion, and metastasis. However, the regulatory mechanism between miRNAs and FGFR1 in lung cancer remains unclear and extremely critical. miR-214-3p was sharply decreased and showed a significantly negative correlation with FGFR1 in lung cancer patients (n = 30). Luciferase reporter assay confirmed that miR-214-3p could downregulate FGFR1 by directly targeting 3′-untranslated region (UTR). miR-214-3p inhibited the processes of epithelial–mesenchymal transition and Wnt/MAPK/AKT (Wnt/mitogen-activated protein kinase/AKT) signaling pathway by targeting FGFR1. Moreover, miR-214-3p not only established a negative feedback regulation loop with FGFR1 through ERK (extracellular signal-regulated kinase) but also developed a synergism with FGFR1 inhibitor AZD4547. In conclusion, our study demonstrated the regulatory mechanism between miR-214-3p and FGFR1 in lung cancer. miR-214-3p acts as a vital target in FGFR1-amplified lung cancer by forming a miR-214-3p-FGFR1-Wnt/MAPK/AKT signaling pathway network. Co-targeting miR-214-3p and FGFR1 could provide greater benefits to patients with FGFR1-amplified lung cancer.

Container closure integrity (CCI) is essential for parenteral packaging. CCI performance must be maintained throughout the entire time temperature transition cycle of a sealed drug′s product lifespan. Prior publications have lacked systematic considerations for the impact of dynamic temperature transition coupled with time dependent transition on sealing performance associated with the viscoelastic characteristics of rubber stoppers used in container closure system (CCS). This paper demonstrates that sealing performance changes inherently and is fundamentally both time- and temperature- dependent. Our research results display this critical time temperature transition impact on CCS sealing performance by applying compression stress relaxation (CSR) on a rubber stopper for experimental data collection and modeling evaluation. The experimental results agree with modeling evaluation following Maxwell-Wiechert theory and the time temperature superposition based on the Arrhenius and Williams-Landel-Ferry methods. Both testing and modeling data show good consistency, demonstrating that the sealing force inevitably changes over time together with temperature transition because of the viscoelastic nature of the rubber stoppers. Our results show that compression seal force decreases quickly as temperature decreases. The significant loss of rubber stopper sealing force at lower temperature transitions could contribute significant risk to CCI at low storage and transport temperatures. By employing both experimental testing and modeling evaluation, an in-depth understanding of the time- and temperature- dependent nature of rubber stopper sealing force was developed. The nonlinearity of CSR is quantitatively predictable, deriving from the viscoelastic characteristics of the rubber stopper material coupled with the time temperature superposition. Modeling evaluation, with a powerful capability to handle actual testing data, can be employed as a predictive tool to evaluate the time- and temperature- dependent sealing force throughout the entire sealed drug product lifespan. The present study is only applicable before reaching the rubber glass transition temperature Tg - a critical transition phase that can′t be skipped/separated from real time temperature transition, and it will further determine the CCS sealing performance while approaching cryogenic temperature. There has been a growing interest in recent years in the assessment of container closure systems for cold storage and shipment. The present work provides a new, integrated methodology framework and some fresh insights to the parenteral packaging industry for practically and proactively considering, designing, setting up, controlling, and managing stopper sealing performance throughout the entire sealed drug product lifespan. Particularly, CCS seal performance at lower temperature needs to be properly addressed, evaluated, calculated, simulated, predicted, tested, and assessed through an integrated system approach for critical data-driven risk management.

The emergence of drug-resistant bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA), has brought great difficulties to clinical treatment. Antibacterial photodynamic therapy (aPDT) is a new non-antibiotic treatment strategy for a variety of drug-resistant bacteria. However, there are few studies on the antimicrobial mechanism of a PDT mediated by 5-aminolevulinic acid (ALA-PDT) for MRSA. In this study, we observed the bactericidal effect of ALA-PDT on MRSA. We found that ALA-PDT had the strongest bactericidal effect when ALA was at 0.05 mM, and the bactericidal activity of aPDT increased with the increase of light dose. MRSA was almost completely eliminated at 0.05 mM and 384 Jcm−2. In addition, the bactericidal morphology was also observed under a fluorescence microscope using a LIVE/DEAD® BacLight™ Bacterial Viability Kit and an electron microscope. It was also found that proteins and DNA were damaged by ALA-PDT. Finally, the transcription level of the specific gene of MRSA, nuc, was decreased by 0.74-fold (P

Progression of chronic aortic regurgitation (CAR) is insidious, and management is challenging. The primary aim of this study was to evaluate left ventricular (LV) remodeling and its progression in asymptomatic patients with CAR and preserved LV ejection fraction by three-dimensional speckle-tracking echocardiography (STE). The secondary aim was to identify the effect of management strategies on LV remodeling in severe CAR.One hundred thirty-five patients and 41 control subjects were enrolled. Patients were divided according to regurgitation degree: mild (n = 48), moderate (n = 40), or severe (n = 47). Routine follow-up was not possible in 13 patients in the severe CAR group. The remaining 34 patients were divided into three groups on the basis of treatment (surgical, n = 13; drug, n = 11; and untreated, n = 10) and followed for 2.1 ± 0.37 years. All subjects underwent three-dimensional STE at baseline and follow-up, while 20 patients with CAR also underwent baseline two-dimensional STE and feature-tracking cardiovascular magnetic resonance imaging. Volumetric and strain parameters were acquired.Compared with global circumferential strain derived from two-dimensional STE and feature-tracking cardiovascular magnetic resonance imaging, three-dimensional global circumferential strain was largest (P .001); however, no significant differences in volumetric parameters, global longitudinal strain (GLS), and global radial strain (GRS) were identified at baseline. GLS, GRS, torsion, apical rotation, and twist were worse in the severe group (P .05). During follow-up, LV volumetric indexes and sphericity indexes increased, while global longitudinal strain, apical rotation, and twist worsened (P .05) in the untreated group. In the surgical group, LV volumetric and sphericity indexes decreased, while GLS and GRS improved (P .05). In the drug group, LV volumetric indexes increased, while LV ejection fraction, GLS, and GRS worsened (P .05).Three-dimensional STE may be a reliable tool to monitor the progression of ventricular remodeling in CAR. Drug therapy may not prevent progressive ventricular dilatation and myocardial depression.

We and others have demonstrated that altering metabolism through the use of a ketogenic diet (KD) has numerous effects in mouse models of malignant glioma including slowed growth, reduced hypoxia and angiogenesis, enhanced survival and potentiation of the effects of radiation and chemotherapy. These effects are recapitulated in vitro when the ketone β-hydroxybutyrate (BHB) is added to mouse and human malignant glioma cells grown under high glucose conditions. BHB has been demonstrated to be an epigenetic modifier, providing a potential mechanism for the pluripotent effects of ketones even in the presence of high glucose. Therefore, we have analyzed alterations in microRNA (miRNA) expression in tumors and cells treated with a KD or BHB, respectively. MicroRNAs are short, single-stranded, noncoding RNAs that posttranscriptionally control gene expression. Each miRNA can affect the expression of many genes and they are implicated in all hallmarks of cancer. We found a number of miRNAs deregulated in tumor tissue from mice fed a KD, particularly mmu-miR-138 which was increased over 30-fold relative to tumors from mice fed a standard diet. MiR-138 is involved in a variety of anti-tumor effects that correlate with our data from KD treated gliomas. We have shown that ketones radiosensitize glioma cells, potentially through a reduction in DNA damage repair. MiR-138 has been shown to reduce the expression of H2AX which plays a key role in sensing and repairing DNA damage. Hypoxia inducible factor-1α (HIF-1α) is also a target of miR-138, and we have demonstrated a reduction in hypoxia, angiogenesis and the expression of HIF-1α in tumors from mice fed a KD. Furthermore, when we genetically engineered tumor cells to express miR-138 and injected them into mice, only necrotic tumor foci were found. Thus, we suggest that miR-138 is an important regulatory mechanism for the anti-tumor actions of metabolic ketosis in glioma.

Solid-state nanopores have shown great potential in investigating salinity gradient energy generation as a renewable power generator. In this work, various diameter silicon nitride (Si3N[Formula: see text] nanopores were fabricated to investigate the power generation between two potassium chloride solutions with different concentration gradient ratios by reverse electrodialysis. The maximal estimated power density of a Si3N4 nanopore measured experimentally can be high to 16[Formula: see text]649Wm[Formula: see text]. To compare with the single Si3N4 nanopore, multiple nanopores array has also been investigated. The equivalent circuit model of multiple Si3N4 nanopores array generator is quantitatively constructed by massive reproducible experimental data and theoretical derivation. For nanopore array, the osmotic current basically keep a linear growth with the number of the nanopores at every concentration ratio. While, the osmotic voltage is basically independent on the number of nanopore. The power generation circuit of the nanopore array can be regarded as a parallel circuit of multiple nanopores. Power generation from concentration gradients in Si3N4 nanopores could be widely used in a variety of applications like ultra-low power devices and micro-nano electromechanical systems.

Background Exosomes are nano-sized extracellular vesicles containing different biomolecules such as proteins and microRNAs (miRNAs) that mediate intercellular communication. Recently, numerous studies have reported the important functions of exosomal miRNAs in disease development and the potential clinical application as diagnostic biomarkers. Up to now, the most commonly used methods to extract exosomes are ultracentrifugation (UC) and precipitation-based commercial kit (e.g., ExoQuick). Generally, both UC and ExoQuick method could co-isolate contaminating proteins along with exosomes, with the UC method yielding even purer exosomes than ExoQuick. However, the comparison of these two methods on co-precipitated free miRNAs is still unknown. Methods In this study, we isolated exosomes from the human serum with exogenously added cel-miR-39 by UC and ExoQuick and compared the proportion of cel-miR-39 co-precipitated with exosomes extracted by these two methods. Results Using exogenous cel-miR-39 as free miRNAs in serum, we concluded that ExoQuick co-isolates a small proportion of free miRNAs while UC hardly precipitates any free miRNAs. We also found that incubation at 37 °C for 1 h could decrease the proportion of free miRNAs, and exosomal miRNAs like miR-126 and miR-152 also decreased when RNase A was used. In conclusion, our findings provide essential information about the details of serum exosome isolation methods for further research on exosomal miRNAs.

B-cell activating factor (BAFF) is a crucial survival factor for B cells, and excess BAFF contributes to development of autoimmune diseases. Recent studies have shown that rapamycin can prevent BAFF-induced B-cell proliferation and survival, but the underlying mechanism remains to be elucidated. Here we found that rapamycin inhibited human soluble BAFF (hsBAFF)-stimulated cell proliferation by inducing G(1)-cell cycle arrest, which was through downregulating the protein levels of CDK2, CDK4, CDK6, cyclin A, cyclin D1, and cyclin E. Rapamycin reduced hsBAFF-stimulated cell survival by downregulating the levels of anti-apoptotic proteins (Mcl-1, Bcl-2, Bcl-xL and survivin) and meanwhile upregulating the levels of pro-apoptotic proteins (BAK and BAX). The cytostatic and cytotoxic effects of rapamycin linked to its attenuation of hsBAFF-elevated intracellular free Ca(2+) ([Ca(2+)](i)). In addition, rapamycin blocked hsBAFF-stimulated B-cell proliferation and survival by preventing hsBAFF from inactivating PTEN and activating the Akt-Erk1/2 pathway. Overexpression of wild type PTEN or ectopic expression of dominant negative Akt potentiated rapamycin’s suppression of hsBAFF-induced Erk1/2 activation and proliferation/viability in Raji cells. Interestingly, PP242 (mTORC1/2 inhibitor) or Akt inhibitor X, like rapamycin (mTORC1 inhibitor), reduced the basal or hsBAFF-induced [Ca(2+)](i) elevations. Chelating [Ca(2+)](i) with BAPTA/AM, preventing [Ca(2+)](i) elevation using EGTA, 2-APB or verapamil, inhibiting CaMKII with KN93, or silencing CaMKII strengthened rapamycin’s inhibitory effects. The results indicate that rapamycin inhibits BAFF-stimulated B-cell proliferation and survival by blunting mTORC1/2-mediated [Ca(2+)](i) elevations and suppressing Ca(2+)-CaMKII-dependent PTEN/Akt-Erk1/2 signaling pathway. Our finding underscores that rapamycin may be exploited for prevention of excessive BAFF-induced aggressive B-cell malignancies and autoimmune diseases.

B cell activating factor from the TNF family (BAFF) is implicated in not only the physiology of normal B cells, but also the pathophysiology of aggressive B cells related to malignant and autoimmune diseases. Autophagy plays a crucial role in balancing the beneficial and detrimental effects of immunity and inflammation. However, little is known about whether and how excessive BAFF mediates autophagy contributing to B-cell proliferation and survival. Here, we show that excessive human soluble BAFF (hsBAFF) inhibited autophagy with a concomitant reduction of LC3-II in normal and B-lymphoid (Raji) cells. Knockdown of LC3 not only potentiated hsBAFF inhibition of autophagy, but also attenuated hsBAFF activation of Akt/mTOR pathway, thereby diminishing hsBAFF-induced B-cell proliferation/viability. Further, we found that hsBAFF inhibition of autophagy was Akt/mTOR-dependent. This is supported by the findings that hsBAFF increased mTORC1-mediated phosphorylation of ULK1 (Ser757); Akt inhibitor X, mTORC1 inhibitor rapamycin, mTORC1/2 inhibitor PP242, expression of dominant negative Akt, or knockdown of mTOR attenuated hsBAFF-induced phosphorylation of ULK1, decrease of LC3-II level, and increase of cell proliferation/viability. Chelating intracellular free Ca(2+) ([Ca(2+)](i)) with BAPTA/AM or preventing [Ca(2+)](i) elevation using EGTA or 2-APB profoundly blocked hsBAFF-induced activation of Akt/mTOR, phosphorylation of ULK1 and decrease of LC3-II, as well as increase of cell proliferation/viability. Similar effects were observed in the cells where CaMKII was inhibited by KN93 or knocked down by CaMKII shRNA. Collectively, these results indicate that hsBAFF inhibits autophagy promoting cell proliferation and survival through activating Ca(2+)-CaMKII-dependent Akt/mTOR signaling pathway in normal and neoplastic B-lymphoid cells. Our findings suggest that manipulation of intracellular Ca(2+) level or CaMKII, Akt, or mTOR activity to promote autophagy may be exploited for prevention of excessive BAFF-induced aggressive B lymphocyte disorders and autoimmune diseases.

Sufficient rubber stopper sealing performance throughout the entire sealed product life cycle is essential for maintaining container closure integrity in the parenteral packaging industry. However, prior publications have lacked systematic considerations for the time-dependent influence on sealing performance that results from the viscoelastic characteristics of the rubber stoppers. In this paper, we report results of an effort to study these effects by applying both compression stress relaxation testing and residual seal force testing for time-dependent experimental data collection. These experiments were followed by modeling fit calculations based on the Maxwell-Wiechert theory modified with the Kohlrausch-Williams-Watts stretched exponential function, resulting in a nonlinear, time-dependent sealing force model. By employing both testing evaluations and modeling calculations, an in-depth understanding of the time-dependent effects on rubber stopper sealing force was developed. Both testing and modeling data show good consistency, demonstrating that the sealing force decays exponentially over time and eventually levels off because of the viscoelastic nature of the rubber stoppers. The nonlinearity of stress relaxation derives from the viscoelastic characteristics of the rubber stoppers coupled with the large stopper compression deformation into restrained geometry conditions. The modeling fit with capability to handle actual testing data can be employed as a tool to calculate the compression stress relaxation and residual seal force throughout the entire sealed product life cycle. In addition to being time-dependent, stress relaxation is also experimentally shown to be temperature-dependent. The present work provides a new, integrated methodology framework and some fresh insights to the parenteral packaging industry for practically and proactively considering, designing, setting up, controlling, and managing stopper sealing performance throughout the entire sealed product life cycle.

To calculate left ventricular (LV) global performance values in heart transplant (HT) recipients by three-dimensional speckle tracking imaging (3D-STI) and to observe the changes in LV global performance over time after HT and investigate the correlated factors. The 30 HT patients were divided into 2 groups according to postoperative time: 1 month postoperatively (HT-1) group and 6 months postoperatively (HT-2) group. Thirty healthy subjects were enrolled as control group. 3D-STI was performed to assess LV torsion, LV systolic dyssynchrony index (SDI), and LV global strain (GS). Global performance index (GPI) was calculated, and correlations factors with GPI were studied. Heart rate (HR), left atrium (LA), interventricular septum thickness (IVST), left ventricular posterior wall thickness (LVPWT), and left ventricular mass (LVM) in both HT groups were higher than those in the control group. Compared with the control group, SDI was significantly higher in both HT groups, and SDI of the HT-1 group was much higher than that of HT-2 group. Compared with the control group, apical rotation (RoA), twist and torsion in the both HT groups decreased significantly. There were no significant differences in these values between the 2 HT groups; Basal rotation (RoB) showed no significantly difference among the 3 groups. GS in the both HT groups decreased significantly compared with the control group, and there were no significant differences in these values between the 2 HT groups. GPI of the both HT groups was significantly lower than that of the control group; however, GPI of HT-2 group was higher than that of HT-1 group. Multivariate stepwise regression analysis identified global left ventricular longitudinal peak systolic strain (GLS), the time length since surgery, left ventricular mass (LVM), and RoA as predictors of LV GPI. GLS was the most influential to GPI. The values of LV rotation, twist and SDI can be used to assess the LV systolic function and dyssynchrony. The GPI value based on 3D-STI may accurately reflect LV performance changes over time after HT. The GPI value has potential applications in clinical practice. GLS, the time length since surgery, LVM and RoA values can be the predictors of LV global performance, and as long as the left ventricular ejection fraction (LVEF) is preserved, the left ventricular global performance of HT recipients remains stable, and tends to improve over time after HT.

B cell activating factor from the TNF family (BAFF) stimulates B-cell proliferation and survival, but excessive BAFF promotes the development of aggressive B cells leading to malignant and autoimmune diseases. Recently, we have reported that rapamycin, a macrocyclic lactone, attenuates human soluble BAFF (hsBAFF)-stimulated B-cell proliferation/survival by suppressing mTOR-mediated PP2A-Erk1/2 signaling pathway. Here, we show that the inhibitory effect of rapamycin on hsBAFF-promoted B cell proliferation/survival is also related to blocking hsBAFF-stimulated phosphorylation of Akt, S6K1, and 4E-BP1, as well as expression of survivin in normal and B-lymphoid (Raji and Daudi) cells. It appeared that both mTORC1 and mTORC2 were involved in the inhibitory activity of rapamycin, as silencing raptor or rictor enhanced rapamycin's suppression of hsBAFF-induced survivin expression and proliferation/viability in B cells. Also, PP242, an mTORC1/2 kinase inhibitor, repressed survivin expression, and cell proliferation/viability more potently than rapamycin (mTORC1 inhibitor) in B cells in response to hsBAFF. Of interest, ectopic expression of constitutively active Akt (myr-Akt) or constitutively active S6K1 (S6K1-ca), or downregulation of 4E-BP1 conferred resistance to rapamycin's attenuation of hsBAFF-induced survivin expression and B-cell proliferation/viability, whereas overexpression of dominant negative Akt (dn-Akt) or constitutively hypophosphorylated 4E-BP1 (4EBP1-5A), or downregulation of S6K1, or co-treatment with Akt inhibitor potentiated the inhibitory effects of rapamycin. The findings indicate that rapamycin attenuates excessive hsBAFF-induced cell proliferation/survival via blocking mTORC1/2 signaling in normal and neoplastic B-lymphoid cells. Our data underscore that rapamycin may be a potential agent for preventing excessive BAFF-evoked aggressive B-cell malignancies and autoimmune diseases.

B-cell activating factor (BAFF) is involved in not only the physiology of normal B cells, but also the pathophysiology of aggressive B cells related to malignant and autoimmune diseases. However, how excessive BAFF promotes aggressive B-cell proliferation and survival is not well understood. Here we show that excessive human soluble BAFF (hsBAFF) enhanced cell proliferation and survival in normal and B-lymphoid (Raji) cells, which was associated with suppression of PP2A, resulting in activation of Erk1/2. This is supported by the findings that pretreatment with U0126 or PD98059, expression of dominant negative MKK1, or overexpression of PP2A prevented hsBAFF-induced activation of Erk1/2 and cell proliferation/viability in the cells. It appears that hsBAFF-mediated PP2A-Erk1/2 pathway and B-cell proliferation/viability was Ca(2+)-dependent, as pretreatment with BAPTA/AM, EGTA or 2-APB significantly attenuated these events. Furthermore, we found that inhibiting CaMKII with KN93 or silencing CaMKII also attenuated hsBAFF-mediated PP2A-Erk1/2 signaling and B-cell proliferation/viability. The results indicate that BAFF activates Erk1/2, in part through Ca(2+)-CaMKII-dependent inhibition of PP2A, increasing cell proliferation/viability in normal and neoplastic B-lymphoid cells. Our data suggest that inhibitors of CaMKII and Erk1/2, activator of PP2A or manipulation of intracellular Ca(2+) may be exploited for prevention of excessive BAFF-induced aggressive B-cell malignancies and autoimmune diseases.

Low-intensity pulsed ultrasound (LIPUS) is a noninvasive therapeutic method which gradually being used in clinic including cancers. Exosomes mediate intercellular communication functions in disease development and the potential clinical applications in diagnosis and therapy. However, few studies have discussed the relationship between LIPUS and exosomes. Herein, we show that low intensity (0.6–2.1 W/cm2 or 0.6–3.4 W/cm2) LIPUS promoted exosomes secretion whereas higher intensity (3.4–5.0 W/cm2 or 5.0 W/cm2) LIPUS inhibited exosomes secretion, and this phenomenon is associated with autophagy. Pretreatment with 3-MA or down-regulation of LC3 potentiated low intensity LIPUS's promotion of exosomes secretion and conferred resistance to higher intensity LIPUS's effects on exosomes secretion. Furthermore, pretreatment with PP242 attenuated LIPUS-influenced exosomes secretion while expression of constitutively active Akt (Ad-myr-Akt) elevated LIPUS-influenced exosomes secretion, implying mTOR-dependent mechanism involved. The findings indicate that LIPUS influences exosomes secretion by targeting mTOR-mediated LC3 signaling in SPC-A1 and SPC-A1-BM cells. Our data provided initial evidence to connect LIPUS and secretion of exosomes, and highlight that LIPUS may be exploited in exosome-related diseases.

B-cell activating factor of the TNF family (BAFF, also called BLyS, TALL-1, THANK, or zTNF4) has revealed its critical function in B lymphocyte proliferation and survival, as well as the pathogenesis of autoimmune disease. However, the molecular mechanisms of excess BAFF-extended aggressive B lymphocytes have not been completely defined. Here we show that excessive hsBAFF-elevated [Ca(2+)]i activated mammalian target of rapamycin (mTOR) signaling pathway, leading to proliferation and survival in B lymphocytes. This is supported by the findings that intracellular Ca(2+) chelator (BAPTA/AM) or mTOR inhibitor (rapamycin) abolished the events. Sequentially, we observed that preventing [Ca(2+)]i elevation using EGTA or 2-APB dramatically inhibited hsBAFF activation of mTOR signaling, as well as cell growth and survival, suggesting that hsBAFF-induced extracellular Ca(2+) influx and ER Ca(2+) release elevates [Ca(2+)]i contributing to B lymphocyte proliferation and survival via activation of mTOR signaling. Further, we noticed that pretreatment with BAPTA/AM, EGTA or 2-APB blocked hsBAFF-increased phosphorylation of calcium/calmodulin-dependent protein kinase II (CaMKII), and inhibiting CaMKII with KN93 attenuated hsBAFF-activated mTOR signaling, as well as cell growth and survival, revealing that the effects of hsBAFF-elevated [Ca(2+)]i on mTOR signaling as well as proliferation and survival in B lymphocytes is through stimulating phosphorylation of CaMKII. The results indicate that hsBAFF activates mTOR pathway triggering B lymphocyte proliferation and survival by calcium signaling. Our findings suggest that manipulation of intracellular Ca(2+) level or CaMKII and mTOR activity may be exploited for the prevention of excessive BAFF-induced aggressive B lymphocyte disorders and autoimmune diseases.

B-cell activating factor of the TNF family (BAFF) has been documented to act as a critical factor in the development of aggressive B lymphocytes and autoimmune diseases. However, the effect of various cytokines on BAFF-elicited neoplastic B-lymphoid cells is not known. In this study, we exhibited that administration of human soluble BAFF (hsBAFF), IL-2, IL-4, IFN-γ, or TNF-α alone increased cell viability and survival in Raji cells concentration-dependently, yet a more robust viability/survival was seen in the cells co-treatment of IL-2, IL-4, IFN-γ, or TNF-α with hsBAFF, respectively. Further research revealed that both Erk1/2 and S6K1 signaling pathways were essential for IL-2, IL-4, IFN-γ, or TNF-α enhancement of the viability/survival in the hsBAFF-stimulated cells, as inhibition of Erk1/2 with U0126 or down-regulation of Erk1/2, or blockage of S6K1 with rapamycin or silencing S6K1, or silencing S6K1/Erk1/2, respectively, reduced the cell viability/survival in the cells treated with/without hsBAFF ± IL-2, IL-4, IFN-γ, or TNF-α. These findings indicate that IL-2, IL-4, IFN-γ or TNF-α enhances BAFF-stimulated cell viability/survival by activating Erk1/2 and S6K1 signaling in neoplastic B-lymphoid cells. Our data suggest that modulation of IL-2, IL-4, IFN-γ and/or TNF-α levels, or inhibitors of Erk1/2 or S6K1 may be a new approach to prevent BAFF-induced aggressive B-cell malignancies.

A sonographic study was conducted to determine the prevalence of atherosclerosis across multiple arterial beds in an elderly Chinese population and to examine relationships between detected atherosclerosis and traditional risk factors.A total of 197 participants underwent sonography of the abdominal aorta and bilateral carotid, femoral, and lower limb arteries. Images were reviewed to determine the presence or absence of plaques in each artery. Plaque thickness was measured as the indicator of plaque burden. Plaque prevalence was estimated per site and correlated with age, sex, and the Framingham Risk Score (FRS). Plaque frequency and thickness were compared between different arterial beds.Of the 197 participants (54% female; age range, 58-86 years), 90% had plaques present in at least 1 artery, and 55% had plaques present in at least 4 arteries. The most common sites for plaques were the carotid arteries (80%), followed by the lower limb arteries (59%), femoral arteries (57%), and abdominal aorta (37%). Plaque prevalence in each arterial bed except the abdominal aorta was significantly associated with male participants (P .05), increasing age (P .003) and FRS (P .04). Male participants were more likely to have carotid (P = .04), femoral (P = .045), and lower limb (P = .006) plaques than female participants, but there was no significant difference in aortic plaque prevalence between male and female participants (P = .9).Plaque prevalence increased significantly in the carotid and peripheral arteries with increasing FRS. These findings should be considered for designing screening programs for stroke and heart attack prevention.

Osteoporosis is a common complication in patients with rheumatoid arthritis (RA). The change of bone mineral density (BMD) in patients with RA is slow, and little data are known about the long-term change of BMD.This study aimed to determine the frequency of osteoporosis and the long-term change on BMD in a cohort of Chinese patients with RA routinely receiving calcium and vitamin D supplementation.A total of 304 consecutive patients with RA were recruited. Bone mineral density measurements of the forearm, lumbar spine, and total hip were performed by dual-energy x-ray absorptiometry and compared with 200 age- and sex-matched healthy controls. Risk factors were analyzed by logistic regression models.The prevalence of osteoporosis at all measured sites in patients with RA was statistically significantly higher than in healthy controls. A total of 107 patients of the cohort had a mean of 4 years of follow-up. More patients with BMD decrease were found without calcium and vitamin D use compared with those who continuously took calcium and vitamin D (64.3% vs 19.8% at the forearm and 28.6% vs 16.1% at the total hip, respectively). Only the use of calcium and vitamin D supplementation was associated with a decreased risk of BMD decrease both at the forearm and at the total hip.Osteoporosis is common in Chinese patients with RA. Routine use of calcium and vitamin D supplementation decreased the risk of BMD decrease and should be recommended for all patients with RA.

Purpose To explore the blood oxygen level dependent (BOLD) response in the posterior cingulate cortex (PCC) and the adjacent precuneus regions in healthy elderly adults during problem solving tasks. Materials and Methods Eighteen participants (7 women, mean age of 63.6 ± 6.0 years old) were analyzed. The functional magnetic resonance imaging (fMRI) tasks were simplified 4 × 4 Sudoku puzzles that were divided into simple tasks (using the row rule or the column rule to solve the puzzle) and complex tasks (using both the row and column rules to solve the puzzle). Results The mean accuracy on the simple task was higher than that on the complex task ( P = 0.04); the reaction time on the simple task was shorter than that on the complex task ( P = 0.001). On both tasks, the participants showed deactivation in the bilateral PCC/precuneus regions. The extent of deactivation on the complex task was greater than that on the simple task (left: P = 0.04; right: P = 0.04). Conclusions Healthy elderly adults showed deactivation in the bilateral PCC and precuneus regions during a problem solving task; in addition, the extent of deactivation was enhanced by increasing the difficulty of the problem solving task.

Methotrexate (MTX) has been widely used at low dose for the treatment of different diseases including rheumatoid arthritis. MTX might be present in plasma in free form, and in blood cells in methotrexate polyglutamate (MTXPG). A rapid and sensitive HPLC method was developed for the determination of plasma MTX level, whole-blood MTX level, and whole-blood total MTX (MTX+MTXPG) level. To determine plasma MTX level or whole-blood MTX level, a 0.2-ml aliquot of plasma or whole blood (after a freeze-thaw cycle to break blood cells) was well mixed with 0.8 ml methanol and centrifuged. To determine whole-blood total MTX level, a 0.1-ml aliquot of whole blood (after a freeze-thaw cycle) was mixed with 80 microl ascorbic acid (114 mM) and incubated at 37 degrees C for 2h to enzymatically convert the MTXPG to MTX. Then 20 microl NaOH solution (0.5M) and 0.8 ml methanol were added and mixed well. After centrifugation, a 0.5-ml aliquot of the supernatant was evaporated to dryness and re-dissolved in 0.2 ml hydrochloric acid (10mM). Methylene chloride (0.2 ml) was added and mixed well. After centrifugation, the top aqueous layer was injected to HPLC for analysis. After the MTX was eluted from the HPLC column, it was electrochemically oxidized and detected by a fluorescence detector. Recoveries of spiked MTX at ppb (ng/ml) level were between 87.9 and 118% with within-day relative standard deviation less than 5.2% and day-to-day relative standard deviation less than 9.8%. The limit of detection (LOD) and limit of quantitation (LOQ) of the described method were 1.2 and 2.6 ng/ml, respectively.

B-cell activating factor (BAFF) is involved in not only physiology of normal B cells, but also pathophysiology of aggressive B cells related to malignant and autoimmune diseases. Rapamycin, a lipophilic macrolide antibiotic, has recently shown to be effective in the treatment of human lupus erythematosus. However, how rapamycin inhibits BAFF-stimulated B-cell proliferation and survival has not been fully elucidated. Here, we show that rapamycin inhibited human soluble BAFF (hsBAFF)-induced cell proliferation and survival in normal and B-lymphoid (Raji and Daudi) cells by activation of PP2A and inactivation of Erk1/2. Pretreatment with PD98059, down-regulation of Erk1/2, expression of dominant negative MKK1, or overexpression of wild-type PP2A potentiated rapamycin's suppression of hsBAFF-activated Erk1/2 and B-cell proliferation/viability, whereas expression of constitutively active MKK1, inhibition of PP2A by okadaic acid, or expression of dominant negative PP2A attenuated the inhibitory effects of rapamycin. Furthermore, expression of a rapamycin-resistant and kinase-active mTOR (mTOR-T), but not a rapamycin-resistant and kinase-dead mTOR-T (mTOR-TE), conferred resistance to rapamycin's effects on PP2A, Erk1/2 and B-cell proliferation/viability, implying mTOR-dependent mechanism involved. The findings indicate that rapamycin inhibits BAFF-stimulated cell proliferation/survival by targeting mTOR-mediated PP2A-Erk1/2 signaling pathway in normal and neoplastic B-lymphoid cells. Our data highlight that rapamycin may be exploited for preventing excessive BAFF-induced aggressive B-cell malignancies and autoimmune diseases.

To analyze the relationship between the pathological features and 64-multislice spiral computed tomography (64-MSCT) findings of pulmonary nodules in autopsies from patients with coal workers' pneumoconiosis (CWP), to investigate the optimal imaging method for the distribution of pulmonary nodules, and to provide data for the establishment of CT diagnostic criteria for CWP.Cadaveric lung specimens were collected from 7 CWP patients. All of them were men, aged 42∼77 years (mean, 60.00±13.00 years), and their dust exposure time was 5∼30 years (mean, 15.4±8.01 years). The cadaveric lung specimens were treated by aeration, sectioning, and immobilization and were then examined by coronary 64-MSCT. The primitive images were reconstructed into the maximumintensity projection (MIP) images (slice thickness: 3 mm, 5 mm, and 8 mm). The sensitivities of imaging methods with different slice thickness were evaluated based on the pathology and anatomy of local pulmonary nodules, and the correlation between pathological results and radiological findings was analyzed.There were significant differences between the stages determined by pathological examination and high-kV chest radiography (before death) (χ(2) = 4.667, P0.05; kappa value = 0.167, P0.05). A total of 271 nodules were found in all pathological sections, including peribronchovascular nodules (27, 9.9%), centrilobular nodules (67, 24.6%), interlobular nodules (65, 24.3%), nodules within 5 mm from the pleura (45, 16.5%), pleural plaque-like nodules on the lateral chest wall (45, 16.5%), and nodules on the interlobar pleura (22, 8.1%). The likelihood ratio was the highest (0.981) between 5-mm MIP images and pathological results according to the chi-square test.The stage of pulmonary nodules determined by pathological examination is significantly different from that determined by high-kV chest radiography. The 5-mm MIP images of 64-MSCT provide a good reflection of the local pathology and anatomy of pulmonary nodules in CWP patients.