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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.

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.

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 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

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) 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.

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.

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.

This study was designed to evaluate the safety, MR imaging characteristics, and clinical response to intrathecal gadopentetate dimeglumine (gadolinium) administration in human patients.Eleven adult patients were included in this prospective study. Via lumbar puncture, a single dose of either 0.2 ml, 0.5 ml, or 1.0 ml of gadolinium (500 mmol/l) mixed with 5 ml of previously removed CSF was slowly injected into the lumbar subarachnoid space. Immediate and delayed MR imaging were subsequently carried out using a 1.0-T magnet.No patient manifested gross behavioral changes, neurologic alterations, or seizure activity. The intrathecal gadolinium-enhanced MR myelography revealed disk herniation (n = 4), posttraumatic spinal stenosis (n = 3), postsurgical noncommunicating cyst (n = 1), myelitis (n = 1), intradural extramedullary mass formation (n = 1), and intradural vascular malformation (n = 1).This pilot study shows the relative safety and feasibility of low-dose intrathecal gadolinium administration. The potential clinical applications include the evaluation of obstructions and communications of the subarachnoid space, spontaneous or traumatic CSF leaks, and CSF dynamics. Additional animal and human studies must be performed to further evaluate the long-term safety and to prove the clinical applications of this procedure in a larger number of subjects.

The high level of safety and tolerance of IV administrated gadolinium has been proved through extensive clinical use (Goldstein et al. 1990; Niendorf et al. 1991a, 1991b). However, only a limited number of laboratory studies have been performed focusing on the possible usage and neurotoxic or inflammatory reactions of paramagnetic contrast agents injected into the subarachnoid space (Dichiro et al. 1985; Allard et al. 1987; Ray et al. 1996; Skalpe and Tang 1997; Ray et al. 1998; Skalpe 1998; Jinkins et al. 1999). Only one study on two human patients with meningeal carcinomatosis who underwent intraventricular gadolinium injection has been published (Siebner et al. 1997). The present chapter details the results of a controlled pilot medical experiment (Zeng et al. 1999). It should be noted that the intrathecal gadolinium is not presently an approved route of administration of gadolinium worldwide.