Simultaneous determination of 10 kinds of biogenic amines in rat plasma using high-performance liquid chromatography coupled with fluorescence detection.

We describe a simple, rapid, selective and sensitive HPLC method coupled with fluorescence detection for simultaneous determination of 10 kinds of biogenic amines (BAs: tryptamine, 2-phenethylamine, putrescine, cadaverine, histamine, 5-hydroxytryptamine, tyramine, spermidine, dopamine and spermine). BAs and IS were derivated with dansyl chloride. Fluorescence detection (λex /λem = 340/510 nm) was used. A satisfactory result for method validation was obtained. The assay was shown to be linear over the ranges 0.005-1.0 µg/mL for tryptamine, 2-phenethylamine and spermidine, 0.025-1.0 µg/mL for putrescine, 0.001-1.0 µg/mL for cadaverine, 0.25-20 µg/mL for histamine, 0.25-10 µg/mL for 5-hydroxytryptamine and dopamine, and 0.01-1.0 µg/mL for tyramine and spermine. The limits of detection and the limits of quantification were 0.3-75.0 ng/mL and 1.0-250.0 ng/mL, respectively. Relative standard deviations were ≤5.14% for intra-day and ≤6.58% for inter-day precision. The recoveries of BAs ranged from 79.11 to 114.26% after spiking standard solutions of BAs into a sample at three levels. Seven kinds of BAs were found in rat plasma, and the mean values of tryptamine, 2-phenethylamine, putrescine, cadaverine, histamine, spermidine and spermine determined were 52.72 ± 7.34, 11.45 ± 1.56, 162.56 ± 6.26, 312.75 ± 18.11, 1306.50 ± 116.16, 273.89 ± 26.41 and 41.51 ± 2.07 ng/mL, respectively.

Sonodynamic action of hypocrellin B on biofilm-producing Staphylococcus epidermidis in planktonic condition.

Staphylococcus epidermidis is an opportunistic pathogen causing biofilm-associated infections. To investigate sonodynamic action of hypocrellin B on biofilm-producing Staphylococcus epidermidis in planktonic culture, a biofilm-producing strain Staphylococcus epidermidis (ATCC 35984) was incubated with hypocrellin B and then exposed to ultrasound at intensity (ISATA) of 1.56 W/cm(2) with a frequency of 1 MHz in continuous mode for 5 min. After sonodynamic treatment of hypocrellin B, the bacterial growth was measured using the colony counting method. Bacterial membrane integrity was investigated using a flow cytometry with propidium iodide staining. Intracellular reactive oxygen species (ROS) level was measured using a flow cytometry with DCFH-DA staining. The results showed that sonodynamic action of hypocrellin B significantly induced survival reduction of Staphylococcus epidermidis in a hypocrellin B dose-dependent manner, and a 4-log reduction was observed after the combined treatment of hypcorellin B (40 µM) and ultrasound sonication with the intensity of 1.56 W/cm(2) for 5 min. Bacterial membrane integrity was notably damaged and the level of intracellular ROS level was remarkably increased after sonodynamic treatment. The findings demonstrated that sonodynamic action of hypocrellin B had significant antibacterial activity on biofilm-producing Staphylococcus epidermidis in planktonic condition probably through increasing intracellular ROS level to cause damage to bacterial membrane integrity.

Multilayer Porous Fe/Co-N-MWCNT Electrocatalyst For Rechargeable Zinc-Air Batteries.

The design of efficient, stable, low-cost non-precious metal-based electrocatalysts with enhanced oxygen reduction reaction (ORR) activity has garnered significant attention in the scientific community. This study introduces a novel electrocatalyst, Fe/Co-N-MWCNT, synthesized through the in-situ growth of ZIF-8 and Fe/Co-Phen on multi-walled carbon nanotubes (MWCNTs), followed by pyrolysis at varying temperatures to optimize its properties. The inclusion of Fe and Co during the pyrolysis process facilitated the creation of metal active sites and Fe-Co, enhancing electron transfer and ORR activity. Compared to Pt/C (E1/2=0.854 V, JL=4.90 mA cm-2), Fe/Co-N-MWCNT exhibited a similar half-wave potential (E1/2=0.812 V) and an improved limiting current density (JL=5.37 mA cm-2). Moreover, Fe/Co-N-MWCNT displayed remarkable stability, showing only a 7 mV negative shift in E1/2 after 2000 cycles. Ampere response testing indicated a current decay of only 7.8 % for Fe/Co-N-MWCNT after 10000 s, while Pt/C experienced a decay of about 18.4 %. The exceptional catalytic stability of Fe/Co-N-MWCNT positions it as a promising candidate for rechargeable zinc-air batteries, attributed to its high pyridinic nitrogen content, unique structure, and abundant metal active sites.

Efficacy of curculigoside in protecting against ischemic brain injury through regulation of oxidative stress and NF-κB and PI3K/Akt expression.

ETHNOPHARMACOLOGICAL RELEVANCE: The ancient Chinese medicine book "Huangdi Neijing" reports that "the brain is the sea of marrow" and that the kidney "mainly induces bones to produce marrow". Therefore, Chinese medicine has a "kidney-brain axis" theory, but supporting evidence is lacking. In this study, curculigoside, the main component of the kidney-tonifying drug Rhizoma Curculiginis, was used to explore whether a kidney-tonifying drug could regulate the pathological state of the brain. AIM OF THE STUDY: To explore the efficacy of curculigoside in protecting against ischemic brain injury (IBI) through the regulation of oxidative stress and NF-κB and PI3K/Akt expression. MATERIALS AND METHODS: Middle cerebral artery occlusion (MCAO) was used to induce IBI in rats, and curculigoside was administered. The degree of IBI, morphological changes and severity of nerve injury (using neurological severity scores; NSSs) in the rats were assessed. Enzyme-linked immunosorbent assays (ELISAs), Western blotting, and immunohistochemistry were used to evaluate changes in hydrogen peroxide (H2O2), nitric oxide (NO), malondialdehyde (MDA), TNF-α, IL-1ß, catalase (CAT), superoxide dismutase (SOD), nitric oxide synthase (NOS), NF-κB, PI3K and Akt levels. RESULTS: Curculigoside significantly alleviated behavioral deficits and reduced the degree of cerebral ischemia in the rats. After curculigoside treatment, the levels of H2O2, NO, MDA, NOS, iNOS, TNF-α, IL-1ß, intercellular adhesion molecule-1 (ICAM-1) and NF-κB in the ischemic area of the brain were significantly reduced. The activities of CAT, SOD, PI3K and Akt were significantly increased. CONCLUSION: Curculigoside is a potentially effective drug for the treatment of IBI.

The Accumulation of Toxic Elements (Pb, Hg, Cd, As, and Cu) in Red Swamp Crayfish (Procambarus clarkii) in Qianjiang and the Associated Risks to Human Health.

Due to rapidly expanding crayfish consumption worldwide, the food safety of red swamp crayfish (Procambarus clarkii) is of great concern. China is the largest consumer and producer of crayfish globally. As of yet, it is unknown whether the main crayfish production cities in China are within safe levels of toxic heavy metals and metalloids. For 16 consecutive years, Qianjiang city ranked first in China in processing export volumes of red swamp crayfish. This study presents a comprehensive analysis of the enrichment levels and associated health risks of the species in Qianjiang. In our research, samples of four crayfish tissues, including the head, hepatopancreas, gills, and muscles, were collected from 38 sampling sites distributed in Qianjiang to evaluate the concentration levels of five heavy metals (Pb, Hg, Cd, As, and Cu). The concentration levels of all five metals in muscle did not surpass the national standard. Furthermore, eight significant correlations have been found. For further in-depth assess risk of crayfish in Qianjiang, estimated daily intake (EDI), target hazard quotient (THQ), carcinogenic risk (CR), and estimated maximum allowable consumption rates (CRmm) were evaluated in the abdomen muscle and hepatopancreas. The THQ values for each metal were found to be less than 1, while the CR values were below 10-6. Additionally, the CRmm for adults was determined to be 17.2 meals per month. These findings, based on the analysis of five metallic elements included in this study, suggest that the consumption of crayfish abdomen muscle in Qianjiang does not pose any significant health risks. However, it is noteworthy that certain regions exhibit elevated levels of arsenic in the hepatopancreas, surpassing the national standard, thereby rendering them unsuitable for excessive consumption. In general, the findings can be used to provide guidance for safe dietary practices in China.

Effects of Acupuncture, Moxibustion, Cupping, and Massage on Sports Injuries: A Narrative Review.

With the evolution of society, an increasing number of people have realized the importance of sports on human health. However, participation in sports is a double-edged sword as improperly exercising can lead to injury. Many athletes and patients with sports injuries choose traditional Chinese medicine (TCM) when modern medicine fails to relieve their musculoskeletal symptoms. TCM is a splendid legacy of Chinese civilization whose therapies are effective, economical, and convenient, with some administration by trained patients at home. This review analyzes the literature on the application of acupuncture, moxibustion, massage, and cupping in sports injuries to provide novel ideas for the application of TCM in sports medicine.

Network-Based Elaboration of the Efficacy of the Dachangshu (BL25) and Tianshu (ST25) Points in the Treatment of Functional Constipation in Children through Inflammation, Adipocytokine, or Leptin Pathways.

Constipation commonly occurs during childhood, and more than 95% of cases are classified as functional constipation. If not effectively treated, 20% of patients with childhood constipation can continue to exhibit symptoms into adulthood, which seriously affects their mental health and quality of life. The main feature of acupuncture or acupoint stimulation, a special branch of traditional Chinese medicine, is the selection of different acupoints for different diseases, and many worthy guidelines have been established for matching acupoints. The back-shu and front-mu point combination adheres to an important acupoint compatibility law that has been used since its proposal 2,500 years ago but has not yet been verified by the modern evidence-based experiments. This study focused on the back-shu and front-mu point combination using the Dachangshu (BL25) and Tianshu (ST25) points as examples to explore possible research methods for network acupoint-based stimulation based on existing evidence and to elucidate the mechanisms induced by BL25 and ST25 in the treatment of functional constipation in children (FCC). The study found that BL25 and ST25 have 20 common targets, namely, AQP8, DRD2, VIP, TAC1, IL6R, TNF, FOS, KIT, CHAT, HTR3A, GAS8, SOD3, TRPV1, MPO, CALCA, IL1B, P2RX7, NPY2R, IL10RA, and TPH1, and these targets may provide a strategy for the combined usage of BL25 and ST25. In addition, BL25 and ST25 can affect FCC treatment through inflammation-relatedTh17-cell differentiation, the NF-kappa B signaling pathway, and the Toll-like receptor signaling pathway. Adipocytokines or leptin may also comprise the mechanism through which BL25 and ST25 regulate FCC. In addition, BL25 and ST25 regulate FCC through 13 core targets, namely, NFKBIA, RELA, TNF, IKBKB, IRAK1, TLR4, MYD88, TNFRSF1A, IL1R1, TLR2, IL1B, TRAF6, and TNFRSF1B. In short, this study provides new ideas and methods for studying the mechanism of acupuncture points.

A glutathione-responsive silica-based nanosystem capped with in-situ polymerized cell-penetrating poly(disulfide)s for precisely modulating immuno-inflammatory responses.

HYPOTHESIS: Precise modulation of immuno-inflammatory response is crucial to control periodontal diseases and related systemic comorbidities. The present nanosystem with the controlled-release and cell-penetrating manner enhances the inflammation modulation effects of baicalein in human gingival epithelial cells (hGECs) for better oral healthcare. EXPERIMENTS: We constructed a red-emissive mesoporous silica nanoparticle-based nanosystem with cell-penetrating poly(disulfide) (CPD) capping, through a facile in-situ polymerization approach. It was featured with a glutathione-responsive manner and instant cellular internalization capacity for precisely delivering baicalein intracellularly. Laboratory experiments assessed whether and how the nanosystem per se with the delivered baicalein could modulate immuno-inflammatory responses in hGECs. FINDINGS: The in-situ polymerized CPD layer capped the nanoparticles and yet controlled the release of baicalein in a glutathione-responsive manner. The CPD coating could facilitate cellular internalization of the nanosystem via endocytosis and thiol-mediated approaches. Notably, the intracellularly released baicalein effectively downregulated the expression of pro-inflammatory cytokines through inhibiting the NF-κB signaling pathway. The nanosystem per se could modulate immuno-inflammatory responses by passivating the cellular response to interlukin-1ß. This study highlights that the as-synthesized nanosystem may serve as a novel multi-functional vehicle to modulate innate host response via targeting the NF-κB pathway for precision healthcare.

Multiphoton microfabrication and micropatterning (MMM) - An all-in-one platform for engineering biomimetic soluble cell niches.

Engineered biomimetic cell niches represent a valuable in vitro tool for investigating physiological and pathological cellular activities, while developing an all-in-one technology to engineer cell niches, particularly soluble cell niche factors, with retained bioactivities, remains challenging. Here, we report a mask-free, non-contact and biocompatible multiphoton microfabrication and micropatterning (MMM) technology in engineering a spatially and quantitatively controllable bone morphogenetic protein-2 (BMP-2) soluble niche, by immobilizing optimally biotinylated BMP-2 (bBMP-2) on micro-printed neutravidin (NA) micropatterns. Notably, the micropatterned NA bound-bBMP-2 niche elicited a more sustained and a higher level of the downstream Smad signaling than that by free BMP-2, in C2C12 cells, suggesting the advantages of immobilizing soluble niche factors on engineered micropatterns or scaffold materials. This work reports a universal all-in-one cell niche engineering platform and contributes to reconstituting heterogeneous native soluble cell niches for signal transduction modeling and drug screening studies.

Examining the Effector Mechanisms of the Feishu Acupoint (BL13) in the Treatment of Pneumonia Based on Systematic Acupuncture and Moxibustion Research.

BACKGROUND: Pneumonia is a serious global health problem. In traditional Chinese medicine, acupuncture or moxibustion is used to directly stimulate select acupoints on the surface of the human body and produce physical stimulation to further stimulate regulatory functions in the body, strengthening bodily resistance, eliminating disease, and adjusting the viscera. However, this Chinese medicine knowledge does not include the specific mechanisms of action or targets of acupoints. Therefore, an in-depth research is needed. METHODS: An acupoint-element database was constructed, and the target elements of the Feishu point were screened. The UniProt-Swiss-Prot sublibrary was used to obtain correct gene name information. The National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO) database and GEO2R were used to analyze differentially expressed genes in pneumonia. The STRING database was used to analyze interactions, construct a network of the Feishu point efficacy system in pneumonia, and elucidate the mechanisms of action. RESULTS: The Feishu point comprises 34 elements in total. The protein interaction analysis has 38 nodes and 115 edges. The Feishu point efficacy system-pneumonia system network shows that cytokine signaling in the immune system, signaling by interleukins (ILs), IL-4 and IL-13 signaling, and the immune system may be related to immunity and inflammation. The Feishu point efficacy system regulating pneumonia showed that FCER2, IL4R, FASLG, TGFB1, IL6R, STAT6, IL1B, CASP3, IL5RA, IL2RB, MYD88, SQSTM1, IL12RB1, IFNGR1, ADAM17, and CDH1 are the main targets. CONCLUSION: From the perspective of systematic acupuncture and moxibustion, the Feishu point regulates cytokine signaling in the immune system, signaling by ILs, IL-4 and IL-13 signaling, and the immune system by targeting FCER2, IL4R, FASLG, TGFB1, IL6R, STAT6, IL1B, CASP3, IL5RA, IL2RB, MYD88, SQSTM1, IL12RB1, IFNGR1, ADAM17, and CDH1, thereby regulating pneumonia.

Bioinformatics analysis indicates that microRNA­628­5p overexpression may alleviate Alzheimer's disease by targeting TYROBP.

Alzheimer's disease (AD) is a global health issue, but the precise underlying mechanism has not yet been elucidated. The present study aimed to integrate microRNA (miRNA or miR) and mRNA profiles of AD and identify hub genes via bioinformatics analysis. Datasets associated with AD (GSE113141, GSE104249 and GSE138382) were integrated. Bioinformatics analysis was used to identify the hub mRNAs. TargetScan was used to predict miRNAs that have binding sites for the hub genes. Reverse transcription­quantitative (RT­q)PCR and western blot analysis was performed to assess miRNA and mRNA expression levels in APP/PS1 transgenic mice and human U251 cells. Luciferase reporter assay and RNA interference were utilized to verify the functions of these miRNAs in vitro. Bioinformatics analysis demonstrated that expression levels of the gene encoding transmembrane immune signaling adaptor TYROBP were upregulated in both the GSE113141 and GSE104249 datasets; TYROBP also served as the hub gene in AD. miR­628­5p was predicted to have binding sites for TYROBP and was downregulated in GSE138382. RT­qPCR confirmed low miR­628­5p and high TYROBP expression levels in APP/PS1 transgenic mice and human U251 cells. Western blot analysis demonstrated high protein expression levels of amyloid ß (Aß) precursor protein, Aß and TYROBP in APP/PS1 transgenic mice and U251 cells. Dual luciferase reporter assay confirmed that TYROBP was targeted by miR­628­5p. miR­628­5p/TYROBP may inhibit progressive neurodegeneration in AD and could be used as novel biomarkers and candidate drug targets.

Mitochondrial damage in nasopharyngeal carcinoma cells induced by ultrasound radiation in the presence of hypocrellin B.

OBJECTIVE: The mitochondrion is an important target of ultrasound-induced cell death. This study aimed to investigate the mitochondrial damage in nasopharyngeal carcinoma (NPC) cells induced by ultrasound radiation in the presence of hypocrellin B (HB). METHODS: The NPC cell line CNE2 was used to investigate the effect of HB on ultrasonic action with an HB concentration of 2.5 mumol/L and ultrasound exposure for 15 seconds at an intensity of 0.65 W/cm(2). Cytotoxicity was investigated 24 hours after ultrasound exposure. Mitochondrial structure changes were observed by transmission electron microscopy. The mitochondrial membrane potential was evaluated by confocal laser-scanning microscopy with rhodamine 123 staining. RESULTS: The mean death rates of the CNE2 cells +/- SD were 25.14% +/- 1.50% after ultrasound radiation alone and 76.72% +/- 1.13% after ultrasound radiation in the presence of HB. Transmission electron microscopy showed that slightly enlarging mitochondria were found in the ultrasound-treated cells. After treatment with ultrasound and HB together, some cells had seriously damaged mitochondria, namely, obvious swollen mitochondria and mitochondria in which cristae had almost completely disappeared. The mitochondrial membrane potential was more significantly collapsed when the CNE2 cells were exposed to HB for 5 hours and then ultrasound at 0.65 mW/cm(2) than with ultrasound radiation alone (P < .05). CONCLUSIONS: Hypocrellin B significantly enhanced the cytotoxicity of ultrasound radiation in the CNE2 cells. The damage to the mitochondrial structure and function might be an important cause of death in the CNE2 cells induced by treatment with ultrasound radiation and HB together.

Sonodynamic action of pyropheophorbide-a methyl ester in liver cancer cells.

OBJECTIVE: This study aimed to investigate the sonodynamic action of pyropheophorbide-a methyl ester (MPPa) in liver cancer cells to explore a novel therapeutic modality. METHODS: H22 cells were chosen as model cells to investigate the sonodynamic action of MPPa on liver cancer. The MPPa concentration was kept constant at 2 micromol/L, and the cells were subjected to ultrasound exposure at an intensity of 0.97 W/cm(2). Cytotoxicity was investigated 24 hours after ultrasound exposure. Apoptosis was evaluated using flow cytometry with annexin V-fluorescein isothiocyanate and propidium iodine staining and nuclear staining with Hoechst 33258. Reactive oxygen species (ROS) were analyzed using flow cytometry with 2,7-dichlorodihydrofluorescein diacetate staining. RESULTS: No significant dark cytotoxicity of MPPa was shown in the H22 cells at the concentration of 2 micromol/L. The cell death rate induced by ultrasound treatment was significantly higher in the presence of MPPa than in the absence of it (P < .05). Flow cytometry showed that the sonodynamic action of MPPa significantly increased the early and late apoptotic rates of the H22 cells. Nuclear condensation and an ROS increase were found after sonodynamic treatment. CONCLUSIONS: Our findings showed that MPPa-mediated sonodynamic action significantly enhanced death of H22 cells and the ROS level, suggesting that MPPa is a novel sonosensitizer and the sonodynamic action of MPPa might be a potential therapeutic modality in the management of liver cancer.

Hejie Zhitong prescription promotes sleep and inhibits nociceptive transmission-associated neurotransmitter activity in a rodent migraine model.

BACKGROUND: Migraine is painful disease in which neurotransmitters related to pain transmission play an important role. Hejie Zhitong prescription (HJZT) has been used in the clinic as an effective prescription for the treatment of migraine for many years. Our team aimed to further explore its antimigraine mechanism based on previous research results and to explore the inhibitory effect of HJZT on the transmission of pain related to nitroglycerine (NTG)-induced migraine as well as the synergistic effect of HJZT with pentobarbital sodium on promoting sleep. METHODS: Sixty mice were randomly assigned to groups and received the corresponding interventions. Sleep latency and sleep time were recorded to calculate the incidence of sleep. Forty-eight Wistar rats were randomly assigned and administered an intervention corresponding to their group. Calcitonin gene-related peptide (CGRP), serotonin (5-HT), substance P (SP), and cholecystokinin (CCK) levels were measured using ELISAs. Levels of the cannabinoid receptor type 1 (CB1R) and cyclooxygenase-2 (COX-2) protein were assessed using immunohistochemistry. The expression of the CGRP and CCK mRNAs in the midbrain and trigeminal ganglion (TG) were measured using real-time quantitative PCR. RESULTS: HJZT promoted the occurrence of sleep in mice. HJZT downregulated COX-2 expression in the midbrain and TG of rats but upregulated the expression of the CB1R, and decreased the plasma level of the CGRP protein and expression of its mRNA in the midbrain and TG. It also downregulated the expression of the CCK mRNA in the midbrain and TG. The high-dose HJZT treatment increased plasma 5-HT levels, but did not induce changes in the plasma levels of the SP or CCK protein. CONCLUSIONS: HJZT exerts a synergistic effect with pentobarbital sodium on promoting sleep. As for anti-migraine, HJZT can inhibits the expression of nociceptive transmission-associated neurotransmitters, including 5-HT, CGRP and CCK, which may be related to its upregulation of CB1R and downregulation of COX-2.

A Highly Selective and Sensitive Colorimetric Probe for Cu2+ Determination in Aqueous Media Based on Derivative of Tryptanthrin.

A new colorimetric probe, based on tryptanthrin derivative (TR-A), has been successfully synthesized. The probe shows good selectivity and sensitivity for Cu2+ over 12 competing metal ions in a 10 mM Tris-HCl buffer solution (pH 5.5). A significant peak at 623nm appears in the UV-Vis absorption of TR-A-Cu2+, and a noteworthy color change is observed with the naked eye from aquamarine blue to light orange. The interaction of TR-A and Cu2+ are proven to form a 1:1 binding stoichiometry; this identifying is expected to be completed within 1 min. The probe with a limit of detection (16 nM, R2 = 0.9934) shows excellent potential to determine Cu2+ in analysis systems.

Preparation of hypocrellin B nanocages in self-assembled apoferritin for enhanced intracellular uptake and photodynamic activity.

Photodynamic therapy (PDT) is a promising modality, which uses light energy to activate a nontoxic photosensitizer to treat various diseases like cancer and infectious diseases. Hypocrellin B (HB) is a naturally occurring photosensitizer isolated from traditional Chinese medicine Hypocrella bambusea. However, the high hydrophobicity and poor selectivity severely limit its application. Apoferritin, a macromolecular protein, can serve as an attractive nanocage to carry HB while improving its water solubility and tumor selectivity. In this study, novel HB-apoferritin nanoparticles (HB-AFT NPs) were successfully developed by assembling HB within the apoferritin nanocage. The self-assembled HB-AFT NPs have a narrow size distribution and smooth surface with an average diameter of 12 nm and a HB encapsulation efficiency of 85%. The morphological observation and circular dichroism analysis showed that the encapsulation strategy of HB did not impair apoferritin structure, and thus it potentially maintained the tumor targeting ability of apoferritin. Compared with free HB solution, HB-AFT NPs exhibited more pronounced photodynamic activity on MDA-MB-231 cells due to the higher intracellular uptake, increased reactive oxygen species (ROS) production and excellent tumor-targeting. All these results suggest that the self-assembled HB-AFT NPs can be considered as a potential photosensitive drug for tumor targeting photodynamic therapy.

Multiphoton Fabrication of Fibronectin-Functionalized Protein Micropatterns: Stiffness-Induced Maturation of Cell-Matrix Adhesions in Human Mesenchymal Stem Cells.

Cell-matrix adhesions are important structures governing the interactions between cells and their microenvironment at the cell-matrix interface. The focal complex (FC) and focal adhesion (FA) have been substantially investigated in conventional planar culture systems using fibroblasts as an in vitro model. However, the formation of more mature types of cell-matrix adhesion in human mesenchymal stem cells (hMSCs), including fibrillar adhesion (FBA) and 3D matrix adhesion (3DMA), have not been fully elucidated. Here we investigate the niche factor(s) that influence(s) the maturation of FBA and 3DMA by using multiphoton fabrication-based micropatterning. First, the bovine serum albumin (BSA)-made protein micropatterns were functionalized by incorporating various concentrations of fibronectin (FN) in fabrication solution. The amount of cross-linked FN is positively correlated with the initial concentration of FN in the reaction liquid, as verified by immunofluorescence staining. On the other hand, the anisotropic FN-functionalized micropatterns were fabricated by varying the length (i.e., in-plane stiffness) and height (i.e., bending stiffness) of micropatterns, respectively. Finally, hMSCs were cultured on these micropatterns for 2 h and 1 day to determine the formation of FBA and 3DMA, respectively, using immunofluorescence staining. Results demonstrated that FN-functionalized micropatterns with high anisotropy in x-y dimension benefit FBA maturation. Furthermore, niche factors such as higher bending and in-plane stiffness and the presence of abundant fibronectin have a positive effect on the maturation of FN-based cell-matrix adhesion. These findings could provide some new perspectives on designing platforms for further cell niche study and rationalizing scaffold design for tissue engineering.

Sonodynamic action of chlorin e6 on Staphylococcus aureus and Escherichia coli.

Bacteria remain a great threat to human health. In the present study, we examined whether sonodynamic action of chlorin e6 had antibacterial activity on gram-positive bacterial strain Staphylococcus aureus (S. aureus) and gram-negative bacterial strain Escherichia coli (E. coli). Colony forming unit (CFU) assay showed that sonodynamic treatment of chlorin e6 induced a 2-log reduction in CFU of E. coli cells, 7-log reduction in CFU of S. aureus. Fluorescent microscopy observed that dead cells remarkably increased whereas live cells decreased after sonodynamic treatment of chlorin e6 on S. aureus cells. We first demonstrated that sonodynamic action of chlorin e6 has antibacterial effect on both gram-positive and negative bacteria, more powerful on gram-positive bacteria.

Sonodynamic action of hypocrellin B on methicillin-resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) commonly causes refractory infections and has recently become a serious public health concern. The present study was designed to investigate sonodynamic action of hypocrellin B on MRSA. A MRSA strain (ATCC BAA-43) was used in the present study. The dark toxicity of hypocrellin B on MRSA and its uptake in MRSA first were measured. And then bacteria were incubated with hypocrellin B and exposed to ultrasound. After sonodynamic treatment, colony forming unit assay and bacterial viability assay were conducted. Membrane permeability assay, DNA fragmentation assay, and DNA synthesis assay were also performed to examine the underlying mechanism. The results showed that hypocrellin B at concentrations of up to 500 µM had no toxicity to MRSA in the dark. After incubation for 50 min, hypocrellin B could be maximally absorbed by MRSA, and exhibited significant sonodynamic activity in a dose-dependent manner. The 5-log reduction in colony forming unit (CFU) was observed after hypocrellin B (40 µM) treatment at an intensity of 1.38 W/cm(2) ultrasound for 5 min. Compared to the control, hypocrellin B alone and ultrasound sonication alone group, more dead cells were found and bacterial membrane integrity was notably damaged after sonodynamic treatment of hypocrellin B. However, no remarkable DNA damage was found in MRSA after sonodynamic treatment of hypocrellin B. All the findings demonstrated that hypocrellin B could serve as a potential antibacterial sonosensitizer to significantly cause damage to the membrane integrity of MRSA and inhibit its growth under ultrasound sonication.

Hypocrellin B in hepatocellular carcinoma cells: Subcellular localization and sonodynamic damage.

PURPOSE: To study subcellular localization of hypocrellin B in hepatocellular carcinoma cells, and hypocrellin B-mediated sonodynamic action-induced cell damage. MATERIALS AND METHODS: After incubation with 2.5 µM of hypocrellin B, human hepatocellular carcinoma HepG2 cells were exposed to ultrasound waves for 8 sec at an intensity of 0.46 W/cm(2). Clonogenic survival of HepG2 cells was measured using a colony forming assay and light microscope. Ultrastructural morphology was observed using transmission electron microscope (TEM) and mitochondrial membrane potential (MMP) was assessed using confocal laser scanning microcope (CLSM) after rhodamine 123 staining. Additionally, subcellular localization of hypocrellin B in HepG2 cells with organelle probe staining was also observed using CLSM. RESULTS: The colony forming units of HepG2 cells decreased substantially after sonodynamic treatment. The results of TEM showed microvilli disappearance, apoptotic body formation, swollen mitochondria with loss of cristae and mitochondrial myelin-like features (or membrane whorls). Collapse of MMP was found in the treated cells. Hypocrellin B was distributed in mitochondria and lysosomes as well as in endoplasmic reticulum and Golgi apparatus. CONCLUSIONS: The findings demonstrated that sonodynamic action of hypocrellin B induced mitochondrial damage, survival inhibition, and apoptosis of HepG2 cells. Additionally, other subcellular organelles such as endoplasmic reticulum, Golgi apparatus and lysosomes were also the targets of hypocrellin B-mediated sonodynamic action as well as mitochondria.