Search

Three-dimensional speckle-tracking echocardiography (3D-STE) has been increasingly used to quantify right ventricular (RV) function. However, direct comparisons of 3D-STE with cardiac magnetic resonance (CMR) imaging for evaluation of RV function are limited. This study aimed to test the feasibility and accuracy of 3D-STE for the quantification of RV volumes, ejection fraction (EF), and longitudinal strain in comparison with CMR imaging and to determine whether 3D-STE for RV strain is superior to two-dimensional (2D) STE in comparison with CMR imaging.A total of 195 consecutive patients referred for both CMR imaging and echocardiography were studied. Right ventricular end-diastolic volume (RVEDV), RV end-systolic volume (RVESV), RVEF, and 3D RV longitudinal strain (3D-RVLS) of the free wall by 3D-STE and 2D-RVLS of the free wall by 2D-STE, were compared with CMR measurements. Pearson correlation and Bland-Altman analyses were used to assess the intertechnique agreement.Right ventricular 3D-STE was feasible in 174 patients (89%). Right ventricular volumes and EF determined by 3D-STE strongly correlated with CMR values (RVEDV, r = 0.94; RVESV, r = 0.96; RVEF, r = 0.91; all P .001). Three-dimensional STE slightly underestimated the RV volumes and longitudinal strain and overestimated the RVEF. The 3D-RVLS values correlated better than 2D-RVLS values with CMR values (0.85 vs 0.64, P .001) with smaller bias and narrower limits of agreement (bias: 2.0 and 2.6; limits of agreement: 8.5 and 12.5, respectively). The bias and limits of agreement for 3D-STE-obtained RVLS were increased in patients with RV dilation, RVEF45%, or lower frame rate compared with those with normal RV size, RVEF ≥ 45%, or higher frame rate, respectively. Right ventricular 3D-STE measurements were highly reproducible.The 3D-STE measurements of RV volumes, EF, and longitudinal strain are highly feasible and reproducible, and data measured by 3D-STE correlate strongly with those determined using CMR imaging. Thus, 3D-STE may be a valid alternative to CMR imaging for the quantification of RV function in everyday clinical practice.

Compatible vial container closure system (CCS) components in combination with a proper capping process are crucial to ensuring reliable performance, maintaining container closure integrity (CCI), and achieving CCS visual acceptance. CCI is essential for parenteral packaging and must be maintained throughout the entire sealed drug product life. In order to build the most robust CCS performance, many variables, including component selection, fit, function, and capping processes, must be set according to the actual dimensions of the CCS components used. However, conventional CCS stack-up calculations are based on dimensional engineering data and its tolerance from CCS component drawings without consideration of the real statistical distributions and their resultant impact on the risk of CCS end performance. CCS dimensional variations may lead to capping failure, resulting in CCS visual defects, CCI failure, and potentially costly destruction of an entire CCS production batch. In this paper, we demonstrated a comprehensive approach utilizing real CCS component dimensional data as a statistical input for CCS dimension stack-up calculations to calculate the actual CCS end performance window and the CCS's quantitative failure risk to determine the CCS's optimal sealing performance and visual acceptance under different stopper compression percentages. We examined two vial CCSs differing by the stopper as a case study. Each component was measured and included in comprehensive dimensional stack-up calculations. The resulting statistical distributions were used to examine component variability and stack-up assemblies at multiple stopper compressions and to identify the optimal CCS based on the performance window generated from the real data. Using this data-driven approach, we quantitatively identified that as little as 5% stopper compression difference could impact the CCS chosen. More importantly, comprehensive dimensional stack-up calculations can assist in selecting the best vial CCS and appropriate stopper compression, as well as troubleshoot processing concerns and ensure operation within the optimal CCS performance window.

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.

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.

Meningiomas are one of the most prevalent primary brain tumors. Our study aims to obtain mechanistic insights of meningioma pathobiology using mass spectrometry-based label-free quantitative proteome analysis to identifying druggable targets and perturbed pathways for therapeutic intervention. Label-free based proteomics study was done from peptide samples of 21 patients and 8 non-tumor controls which were followed up with Phosphoproteomics to identify the kinases and phosphorylated components of the perturbed pathways. In silico approaches revealed perturbations in extracellular matrix remodeling and associated cascades. To assess the extent of influence of Integrin and PI3K-Akt pathways, we used an Integrin Linked Kinase inhibitor on patient-derived meningioma cell line and performed a transcriptomic analysis of the components. Furthermore, we designed a Targeted proteomics assay which to the best of our knowledge for very first-time enables identification of peptides from 54 meningioma patients via SRM assay to validate the key proteins emerging from our study. This resulted in the identification of peptides from CLIC1, ES8L2, and AHNK many of which are receptors and kinases and are difficult to be characterized using conventional approaches. Furthermore, we were also able to monitor transitions for proteins like NEK9 and CKAP4 which have been reported to be associated with meningioma pathobiology. We believe, this study can aid in designing peptide-based validation assays for meningioma patients as well as IHC studies for clinical applications.

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

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.

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.

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.

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.

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) 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.

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.