Self-assembled core-shell nanoparticles with embedded internal standards for SERS quantitative detection and identification of nicotine released from snus products.

Surface enhanced Raman spectroscopy (SERS) is a unique analytical technique with excellent performance in terms of sensitivity, non-destructive detection and resolution. However, due to the randomness and poor repeatability of hot spot distribution, SERS quantitative analysis is still challenging. Meanwhile, snus is a type of tobacco product that can release nicotine and other components in the mouth without burning, and the rapid detection technique based on SERS can reliably evaluate the amount of nicotine released from snus, which is of great significance for understanding its characteristics and regulating its components. Herein, the strategy was proposed to solve the feasibility of SERS quantitative detection based on self-assembled core-shell nanoparticles with embedded internal standards (EIS) due to EIS signal can effectively correct SERS signal fluctuations caused by different aggregation states and measurement conditions, thus allowing reliable quantitative SERS analysis of targets with different surface affinity. By means of process control, after the Au nanoparticles (Au NPs) were modified with 4-Mercaptobenzonitrile (4-MBN) as internal standard molecules, Ag shell with a certain thickness was grown on the surface of the AuNP@4-MBN, and then the Au@4-MBN@Ag NPs were used to regulate and control the assembly of liquid-liquid interface. The high-density nano-arrays assembled at the liquid-liquid interface ensure high reproducibility as SERS substrates, and which could be used for SERS detection of nicotine released from snus products. In addition, time-mapping research shows that this method can also be used to dynamically monitor the release of nicotine. Moreover, such destruction-free evaluation of the release of nicotine from snus products opens up new perspectives for further research about the impact of nicotinoids-related health programs.

A sensitive AAV transduction inhibition assay assists evaluation of critical factors for detection and concordance of pre-existing antibodies.

Pre-existing antibodies to viral capsids may have a negative impact on the efficacy and safety of adeno-associated virus (AAV)-based gene therapies. Total antibody (TAb) and/or cell-based transduction inhibition (TI) assays have been used to exclude seropositive individuals in clinical studies. Published AAV seroprevalence and patient enrollment criteria regarding antibody status lack comparability between assay formats, hindering a direct cross-study comparison. To identify critical factors impacting TI assay detection of AAV neutralizing antibodies (NAbs), we created a reporter construct expressing NanoLuc® luciferase (Nluc) that enabled a more sensitive and robust detection of AAV6 NAbs than using firefly luciferase. Assessment of additional factors including multiplicity of infection, cell lines, viral production, and capsid purity revealed the reporter is the major determinant of assay sensitivity impacting NAb detection. The Nluc reporter was further used to assess seroprevalence to AAV5, 8, and 9. Last, we compared AAV6 Nluc TI with two TAb assay formats. A higher correlation of Nluc TI was observed with direct binding (90%) than with the more sensitive bridging TAb assay (65%), suggesting both assay sensitivity and TAb formats contribute to AAV seropositivity concordance. Our results support a need to standardize assay formats to ensure proper assessment of pre-existing AAV immunity.

The volatile release evaluation of nicotine from snus products under different storage conditions based on surface-enhanced Raman spectroscopy technology.

Surface enhanced Raman spectroscopy (SERS) is a highly sensitive analytical detection technique that provides unique chemical and structural information on target molecules. Snus is a type of tobacco product that can release nicotine and other components under certain humidity and temperature without burning, and the evaluation of its nicotine release under different storage conditions is very important for understanding its characteristics, regulating its components, and setting reasonable storage conditions. Herein, by means of an artificial climate box and suction extraction device, the volatile release evaluations of nicotine from snus products under different storage conditions were performed based on Fe3O4 microparticles coated with Au nanorods and Au nanoparticles (Fe3O4@AuNRsNPs) as SERS substrates combined with a capillary. The Fe3O4@AuNRsNPs assemblies can be fixed in the inner wall of the capillary with the aid of an external magnetic field, which improved the maneuverability of the SERS substrates. By comparing the intensities of the spectral peaks of the symmetrical breathing of the pyridine moiety of nicotine molecules with increasing temperature and humidity, which could significantly accelerate the volatile release of a small amount of nicotine, the nicotine release under different conditions could be evaluated. Based on this strategy, it was possible to obtain the storage or placement conditions of the product. The results of this study provide a reference to clarify the volatile release of nicotine under various storage conditions, which is helpful for better regulation of the levels of nicotine in snus. Moreover, such destruction-free evaluation of the volatile release of nicotine from snus products under different storage conditions opens up new perspectives for further research about the impact of nicotinoids on smokers' health and cessation programs.

Clinical enrollment assay to detect preexisting neutralizing antibodies to AAV6 with demonstrated transgene expression in gene therapy trials.

Recombinant adeno-associated virus (AAV) vectors are the leading platform for gene delivery for a variety of clinical applications. Patients with preexisting antibodies to AAV are currently excluded from most AAV gene therapy trials to avoid vector neutralization and ensure response to therapy. Anti-AAV neutralizing antibodies (NAbs) are typically assessed by in vitro cell-based transduction inhibition (TI) assays. However, clinical relevance of the determined enrollment cutoff and the inherent variability of a cell-based assay present challenges for use as an enrollment screening test. Here, we describe an enrollment cutoff that was clinically validated and strategies to overcome assay challenges to enable long-term stable performance. A validated anti-AAV6 cell-based TI assay was used to support clinical enrollment across multiple investigational gene therapies and to evaluate AAV6 seroprevalence in healthy and disease populations. The clinical enrollment cutoff was determined statistically using samples collected from healthy donors, applying a 0.1% false error rate with the inclusion of a minimum significant ratio (MSR) metric and in consideration of results from in vivo mouse passive transfer studies. Our strategy for long-term monitoring and control of assay performance employed plate quality control samples flanking the predefined cutoff. An approach using donor samples was implemented to bridge different lots of critical reagents without the need to redefine the cutoff.

Cetirizine dihydrochloride loaded microparticles design using ionotropic cross-linked chitosan nanoparticles by spray-drying method.

To control the release rate and mask the bitter taste, cetirizine dihydrochloride (CedH) was entrapped within chitosan nanoparticles (CS-NPs) using an ionotropic gelation process, followed by microencapsulation to produce CS matrix microparticles using a spray-drying method. The aqueous colloidal CS-NPs dispersions with a drug encapsulation efficiency (EE) of <15%, were then spray dried to produce a powdered nanoparticles-in-microparticles system with an EE of >70%. The resultant spherical CS microparticles had a smooth surface, were free of organic solvent residue and showed a diameter range of 0.5~5 µm. The in vitro drug release properties of CedH encapsulated microparticles showed an initial burst effect during the first 2 h. Drug release from the matrix CS microparticles could be retarded by the crosslinking agent pentasodium tripolyphosphate or the wall material. The technique of 'ionotropic gelation' combined with 'spray-drying' could be applicable for preparation of CS nanoparticlesin-microparticles drug delivery systems. CS-NPs based microparticles might provide a potential micro-carrier for oral administration of the freely water-soluble drug--CedH.

[Effects of liuwei dihuang pills on expressions of apoptosis-related genes bcl-2 and Bax in pancreas of OLETF rats].

OBJECTIVE: To investigate the effects of Liuwei Dihuang Pills (LWDHP) on expressions of apoptosis-related genes bcl-2 and Bax in pancreas of OLETF rats. METHODS: Forty male OLETF rats were randomly divided into LWDHP-treated group and untreated group. Another ten male LETO rats were included in normal control group. OLETF rats in the LWDHP-treated group were given LWDHP (2.4 g.kg(-1).d(-1)) orally since the age of 8 weeks and the rats in the other two groups were given distilled water orally. Body weights of rats were recorded weekly and blood glucose concentration was determined by oral glucose tolerance test (OGTT). Pancreas weights were recorded after rats were killed and the expression levels of bcl-2 mRNA and Bax mRNA were detected by reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS: In the LWDHP-treated group, the expression of bcl-2 mRNA in the pancreas of rats at the age of 40 weeks (1.25+/-0.07) was much higher than that in the untreated group (1.01+/-0.16), P<0.01. Bax mRNA level in the LWDHP-treated group (0.57+/-0.11) was obviously lower than that in the untreated group (1.18+/-0.28), P<0.01. There was no significant difference of pancreas-to-body weight ratios between the LWDHP-treated group and the untreated group. The ability of glucose tolerance was improved in the LWDHP-treated group. CONCLUSION: LWDHP can up-regulate the expression of bcl-2 and down-regulate the expression of Bax at transcription level, which maybe contribute to the anti-apoptosis effects of LWDHP.

gamma-secretase functions through Notch signaling to maintain skin appendages but is not required for their patterning or initial morphogenesis.

The role of Notch signaling during skin development was analyzed using Msx2-Cre to create mosaic loss-of-function alleles with precise temporal and spatial resolution. We find that gamma-secretase is not involved in skin patterning or cell fate acquisition within the hair follicle. In its absence, however, inner root sheath cells fail to maintain their fates and by the end of the first growth phase, the epidermal differentiation program is activated in outer root sheath cells. This results in complete conversion of hair follicles to epidermal cysts that bears a striking resemblance to Nevus Comedonicus. Sebaceous glands also fail to form in gamma-secretase-deficient mice. Importantly, mice with compound loss of Notch genes in their skin phenocopy loss of gamma-secretase in all three lineages, demonstrating that Notch proteolysis accounts for the major signaling function of this enzyme in this organ and that both autonomous and nonautonomous Notch-dependent signals are involved.

Ectodomain shedding and intramembrane cleavage of mammalian Notch proteins is not regulated through oligomerization.

Intramembrane cleaving proteases such as site 2 protease, gamma-secretase, and signal peptide peptidase hydrolyze peptide bonds within the transmembrane domain (TMD) of signaling molecules such as SREBP, Notch, and HLA-E, respectively. All three enzymes require a prior cleavage at the juxtamembrane region by another protease. It has been proposed that removing the extracellular domain allows dissociation of substrate TMD, held together by the extracellular domain or loop. Using gamma-secretase as a model intramembrane cleaving protease and Notch as a model substrate, we investigated whether activating and inactivating mutations in Notch modulate gamma-secretase cleavage through changes in oligomerization. We find that although the Notch epidermal growth factor repeats can promote dimer formation, most surface Notch molecules in mammalian cells are monomeric as are constitutively active or inactive Notch1 proteins. Using a bacterial assay for TM dimerization, we find that the isolated TMD of Notch and amyloid precursor protein self-associate and that mutations affecting Notch cleavage by gamma-secretase cleavage do not alter TMD dimerization. Our results indicate that ligand-induced reversal of controlled TMD dimerization by the Notch extracellular domain is unlikely to underlie the regulatory mechanism of intramembranous cleavage.

Notch pathway is dispensable for adipocyte specification.

In the past decade we have witnessed an epidemic of obesity in developed countries. Therefore, understanding the mechanisms involved in regulation of body weight is becoming an increasingly important goal shared by the public and the scientific community. The key to fat deposition is the adipocyte, a specialized cell that plays a critical role in energy balance and appetite regulation. Much of our knowledge of adipogenesis comes from studies using preadipocytic cell lines that have provided important information regarding molecular control of adipocyte differentiation. However, they fall short of revealing how naive cells acquire competence for adipogenesis. Studies in preadipocytes indicate that the Notch pathway plays a role in regulating adipogenesis (Garces et al.: J Biol Chem 272:29729-29734, 1997). Given the known biological functions of Notch in mediating cell fate decisions (Artavanis-Tsakonas et al.: Science 284:770-776, 1999), we wished to test the hypothesis that the Notch pathway is required for this cellular program by examining adipogenesis in several genetic loss-of-function models that encompass the entire pathway. We conclude that the "canonical" Notch signaling pathway is dispensable for adipocyte specification and differentiation from either mesenchymal or epithelial progenitors.