Structured illumination phase and fluorescence microscopy for bioimaging.

This study presents a dual-modality microscopic imaging approach that combines quantitative phase microscopy and fluorescence microscopy based on structured illumination (SI) to provide structural and functional information for the same sample. As the first imaging modality, structured illumination digital holographic microscopy (SI-DHM) is implemented along the transmission beam path. SI-DHM acts as a label-free, noninvasive approach and provides high-contrast and quantitative phase images utilizing the refractive index contrast of the inner structures of samples against the background. As the second imaging modality, structured illumination (fluorescence) microscopy (SIM) is constructed along the reflection beam path. SIM utilizes fluorescent labeling and provides super-resolution images for specific functional structures of samples. We first experimentally demonstrated phase imaging of SI-DHM on rice leaves and fluorescence (SIM) imaging on mouse kidney sections. Then, we demonstrated dual-modality imaging of biological samples, using DHM to acquire the overall cell morphology and SIM to obtain specific functional structures. These results prove that the proposed technique is of great importance in biomedical studies, such as providing insight into cell physiology by visualizing and quantifying subcellular structures.

Adipose mesenchymal stem cell-derived extracellular vesicles reduce glutamate-induced excitotoxicity in the retina.

Adipose mesenchymal stem cells (ADSCs) have protective effects against glutamate-induced excitotoxicity, but ADSCs are limited in use for treatment of optic nerve injury. Studies have shown that the extracellular vesicles (EVs) secreted by ADSCs (ADSC-EVs) not only have the function of ADSCs, but also have unique advantages including non-immunogenicity, low probability of abnormal growth, and easy access to target cells. In the present study, we showed that intravitreal injection of ADSC-EVs substantially reduced glutamate-induced damage to retinal morphology and electroretinography. In addition, R28 cell pretreatment with ADSC-EVs before injury inhibited glutamate-induced overload of intracellular calcium, downregulation of α-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid receptor (AMPAR) subunit GluA2, and phosphorylation of GluA2 and protein kinase C alpha in vitro. A protein kinase C alpha agonist, 12-O-tetradecanoylphorbol 13-acetate, inhibited the neuroprotective effects of ADSC-EVs on glutamate-induced R28 cells. These findings suggest that ADSC-EVs ameliorate glutamate-induced excitotoxicity in the retina through inhibiting protein kinase C alpha activation.

Pathologically high intraocular pressure disturbs normal iron homeostasis and leads to retinal ganglion cell ferroptosis in glaucoma.

Glaucoma can result in retinal ganglion cell (RGC) death and permanently damaged vision. Pathologically high intraocular pressure (ph-IOP) is the leading cause of damaged vision during glaucoma; however, controlling ph-IOP alone does not entirely prevent the loss of glaucomatous RGCs, and the underlying mechanism remains elusive. In this study, we reported an increase in ferric iron in patients with acute primary angle-closure glaucoma (the most typical glaucoma with ph-IOP damage) compared with the average population by analyzing free iron levels in peripheral serum. Thus, iron metabolism might be involved in regulating the injury of RGCs under ph-IOP. In vitro and in vivo studies confirmed that ph-IOP led to abnormal accumulation of ferrous iron in cells and retinas at 1-8 h post-injury and elevation of ferric iron in serum at 8 h post-injury. Nuclear receptor coactivator 4 (NCOA4)-mediated degradation of ferritin heavy polypeptide 1(FTH1) is essential to disrupt iron metabolism in the retina after ph-IOP injury. Furthermore, knockdown of Ncoa4 in vivo inhibited FTH1 degradation and reduced the retinal ferrous iron level. Elevated ferrous iron induced by ph-IOP led to a marked accumulation of pro-ferroptotic factors (lipid peroxidation and acyl CoA synthetase long-chain family member 4) and a depletion of anti-ferroptotic factors (glutathione, glutathione peroxidase 4, and nicotinamide adenine dinucleotide phosphate). These biochemical changes resulted in RGC ferroptosis. Deferiprone can pass through the blood-retinal barrier after oral administration and chelated abnormally elevated ferrous iron in the retina after ph-IOP injury, thus inhibiting RGC ferroptosis and protecting visual function. In conclusion, this study revealed the role of NCOA4-FTH1-mediated disturbance of iron metabolism and ferroptosis in RGCs during glaucoma. We demonstrate the protective effect of Deferiprone on RGCs via inhibition of ferroptosis, providing a research direction to understand and treat glaucoma via the iron homeostasis and ferroptosis pathways.

HIV-1 gp120 Protein Activates Cyclin-Dependent Kinase 1, a Possible Link to Central Nervous System Cell Death.

Human immunodeficiency virus-1 (HIV-1)-associated neurodegenerative disorder (HAND) is frequently reported in HIV-infected individuals. The gp120 envelope viral protein has been implicated in the pathogenesis of HAND in HIV-1-infected patients; however, its pathogenic mechanism remains unclear. In this study, we first overexpressed gp120 proteins in pc12 cells and used PI staining, a CCK8 assay, a TUNEL assay, and caspase-9/caspase-3-induced apoptosis to ascertain the mediated cell death. Subsequently, the gp120-overexpressed cells were subjected to RNA transcriptomics and mass spectrometry. The obtained results were integrated and validated using a quantitative polymerase chain reaction (qPCR) and the postmortem brain samples with HIV-associated dementia were analyzed against the normal control (using the GSE35864 data set on gene ontology omnibus repository). Upon the integration of the RNA transcriptomic and proteomic results, 78 upregulated genes were revealed. Fut8, Unc13c, Cdk1, Loc100359539, and Hspa2 were the top five upregulated genes. Upon the analysis of the GSE35864 data set, the results indicate that Cdk1 was upregulated in HIV-associated dementia in comparison to the normal control. Moreover, the protein expression of Cdk1 was significantly higher in the gp120 transfected group compared to the normal control and decreased significantly upon inhibition using Roscovitine (a known Cdk1 inhibitor). Taken together, our results provide a possible molecular signature of the neurological impairment secondary to HIV glycoprotein 120.

Public awareness and attitudes toward biobank and sample donation: A regional Chinese survey.

Background: The biobank is an extraordinary aid to research and scientific progress. Public involvement in biobanks, necessary for their development, is limited due to inadequate knowledge of biobanking and concerns about sample donation. This study explores the effectiveness of different publicity methods in improving participants' willingness to donate, and assesses public motivations and concerns. It aims to identify an efficient method of improving participants' awareness of biobanking and promoting sample donation. Methods: A structured 20-item questionnaire was formulated to evaluate participants' knowledge of and attitudes toward biobanks and sample donation. In total, 1,500 questionnaires were disseminated to three groups of 500 participants who received, respectively, picture-based promotional material, text-based promotional material, or who attended a biobank-related lecture. Of these, 945 completed questionnaires were received. All the participants completed the questionnaires twice, before and after the corresponding publicity education. Results: After each of the three methods of publicity based on text, pictures and a lecture, respondents' willingness to donate samples was significantly increased (P < 0.001), the lecture being more effective than the other two methods (P = 0.001). Participants with a medical background were more willing to donate biospecimens after publicity than those without medical backgrounds (P < 0.005) but had common motivations for donation including altruism and aiding medical research. The main concern hindering respondents' willingness to donate was the security of personal information. Conclusion: Different types of biobank-related publicity based on text material, pictorial material and a lecture all improved respondents' willingness to donate and reduced concerns regarding sample donation. Medical background was a critical factor affecting attitudes toward sample donation after publicity. The results of this study suggest strategies that may popularize biobanks and enhance sample donation, further promoting the development of biobanks.

Valdecoxib Protects against Cell Apoptosis Induced by Endoplasmic Reticulum Stress via the Inhibition of PERK-ATF4-CHOP Pathway in Experimental Glaucoma.

The purpose of this study was to investigate the effects of valdecoxib on the retina in retinal ischemia-reperfusion injury (IRI) and R28 cells following oxygen-glucose deprivation/recovery (OGD/R) injury, as well as the underlying mechanisms. Immunofluorescence and Cell Counting Kit-8 (CCK-8) analyses were used to identify the proper timepoint and concentration of valdecoxib's protective effect on the R28 cells in the OGD/R model. Hematoxylin-eosin (HE) staining and immunofluorescence were used to explore valdecoxib's effect on the retina and retina ganglion cell (RGC) in IRI. Cell apoptosis was determined by a TUNEL Apoptosis Detection Kit and Annexin V-FITC/PI flow cytometry. The expression levels of p-PERK, transcription factor 4 (ATF4), GRP78, CHOP, cleaved caspase 3, bax and bcl-2 were measured by Western blot analyses. The valdecoxib protected the R28 cells from OGD/R injury by decreasing the cell apoptosis rate, and it exerted a protective effect on retinas in I/R injury by inhibiting RGC apoptosis. The valdecoxib pretreatment reversed the expression of p-PERK, ATF4, CHOP, GRP78, cleaved caspase 3 and bax induced by the glaucomatous model. Meanwhile, the CCT020312 reversed the valdecoxib's anti-apoptosis effect by activating PERK-ATF4-CHOP pathway-mediated endoplasmic reticulum (ER) stress. These findings suggest that valdecoxib protects against glaucomatous injury by inhibiting ER stress-induced apoptosis via the inhibition of the PERK-ATF4-CHOP pathway.

Autophagy in glaucoma pathogenesis: Therapeutic potential and future perspectives.

Glaucoma is a common blinding eye disease characterized by progressive loss of retinal ganglion cells (RGCs) and their axons, progressive loss of visual field, and optic nerve atrophy. Autophagy plays a pivotal role in the pathophysiology of glaucoma and is closely related to its pathogenesis. Targeting autophagy and blocking the apoptosis of RGCs provides emerging guidance for the treatment of glaucoma. Here, we provide a systematic review of the mechanisms and targets of interventions related to autophagy in glaucoma and discuss the outlook of emerging ideas, techniques, and multidisciplinary combinations to provide a new basis for further research and the prevention of glaucomatous visual impairment.

HIV-Proteins-Associated CNS Neurotoxicity, Their Mediators, and Alternative Treatments.

Human immunodeficiency virus (HIV)-infected people's livelihoods are gradually being prolonged with the use of combined antiretroviral therapy (ART). Conversely, despite viral suppression by ART, the symptoms of HIV-associated neurocognitive disorder (HAND) endure. HAND persists because ART cannot really permanently confiscate the virus from the body. HAND encompasses a variety of conditions based on clinical presentation and severity level, comprising asymptomatic neurocognitive impairment, moderate neurocognitive disorder, and HIV-associated dementia. During the early stages of HIV infection, inflammation compromises the blood-brain barrier, allowing toxic virus, infected monocytes, macrophages, T-lymphocytes, and cellular products from the bloodstream to enter the brain and eventually the entire central nervous system. Since there are no resident T-lymphocytes in the brain, the virus will live for decades in macrophages and astrocytes, establishing a reservoir of infection. The HIV proteins then inflame neurons both directly and indirectly. The purpose of this review is to provide a synopsis of the effects of these proteins on the central nervous system and conceptualize avenues to be considered in mitigating HAND. We used bioinformatics repositories extensively to simulate the transcription factors that bind to the promoter of the HIV-1 protein and possibly could be used as a target to circumvent HIV-associated neurocognitive disorders. In the same vein, a protein-protein interaction complex was also deduced from a Search Tool for the Retrieval of Interacting Genes. In conclusion, this provides an alternative strategy that could be used to avert HAND.

Altered diversity and composition of gut microbiota in patients with allergic rhinitis.

BACKGROUND: Recently, multiple studies have suggested an association between gut dysbiosis and allergic rhinitis (AR) development. However, the role of gut microbiota in AR development remains obscure. METHODS: The goal of this study was to compare the gut microbiota composition and short-chain fatty acid (SCFAs) differences associated with AR (N = 18) and HCs (healthy controls, N = 17). Gut microbiota 16SrRNA gene sequences were analyzed based on next-generation sequencing. SCFAs in stool samples were analyzed by gas chromatography-mass spectrometry (GC-MS). RESULTS: Compared with HCs, the gut microbiota composition of AR was significantly different in diversity and richness. At the phylum level, the abundance of Firmicutes in the AR group were significantly lower than those in the HCs group. At the genus level, the abundance of Blautia, Eubacterium_hallii_group, Romboutsia, Collinsella, Dorea, Subdoligranulum and Fusicatenibacter in the AR group were significantly lower than that in the HCs group. The concentrations of SCFAs were significantly lower in the AR group compared with the HCs group. Correlation analysis showed that the Eubacterium-hallii-group and Blautia correlated positively with SCFAs. CONCLUSION: Our results demonstrate compositional and functional alterations of the gut microbiome in AR.

Structured illumination microscopy with partially coherent illumination for phase and fluorescent imaging.

This study presents a partially coherent illumination based (PCI-based) SIM apparatus for dual-modality (phase and fluorescent) microscopic imaging. The partially coherent illumination (PCI) is generated by placing a rotating diffuser on a monochromatic laser beam, which suppresses speckle noise in the dual-modality images and endows the apparatus with sound sectioning capability. With this system, label-free quantitative phase and super-resolved/sectioned fluorescent images can be obtained for the same sample. We have demonstrated the superiority of the system in phase imaging of transparent cells with high endogenous contrast and in a quantitative manner. In the meantime, we have also demonstrated fluorescent imaging of fluorescent beads, rat tail crosscut, wheat anther, and hibiscus pollen with super-resolution and optical sectioning. We envisage that the proposed method can be applied to many fields, including but not limited to biomedical, industrial, chemistry fields.

Adenosine A3 receptor activated in H2O2 oxidative stress of primary open-angle glaucoma.

BACKGROUND: Primary open-angle glaucoma (POAG), as one of the leading reasons for blindness, is mainly due to trabecular meshwork (TM) dysfunction. Bioinformatics analysis was used to find related genes involved in TM oxidative stress, which is a major cause of TM fibrosis. METHODS: A total of three datasets from the Gene Expression Omnibus (GEO) database were used to identify differentially expressed genes (DEGs). Gene expression relationships were enriched by the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and Gene Ontology (GO) pathways. The interaction network was listed by the protein-protein interaction (PPI) network. The expression of adenosine A3 receptor (ADORA3) was validated in POAG tissue and human trabecular meshwork cells (HTMCs) by western blot (WB) and reverse transcription polymerase chain reaction (RT-PCR). Additionally, WB and RT-PCR were used to measure oxidative stress injury relative protein and gene expression, respectively, such as fibronectin (FN), collagen-I (Col-I), and α-smooth muscle actin (α-SMA). Cell migration function and vitality were tested via transwell migration assay and Cell Counting Kit-8 (CCK-8). The cell vitality was measured using CCK-8. RESULTS: A total of 61 significant DEGs among the three data sources were analyzed. Among all three different datasets, two significant DEGs [ADORA3 and DNA damage-inducible transcript 4 protein (DDIT4)] were identified. The dataset ADORA3 was selected for further analysis. In the POAG TM tissue, ADORA3 was overexpressed at transcriptional and post-transcriptional levels. Overexpression of ADORA3 reduced TMC viability and migration but upregulated the extracellular matrix (ECM) proteins (FN, Col-I, and α-SMA) expression. It was found that ADORA3 can exacerbate oxidative stress injury in normal TMCs. These results indicated that ADORA3 might play an essential role in the occurrence and progression of POAG. CONCLUSIONS: A total of 61 novel common DEGs identified are related to the development and prognosis of POAG. In the POAG, ADORA3 was verified as overexpressed; therefore, it may be associated with an oxidative stress injury in TMCs.

The Xiangya Ocular Tumor Bank: A Disease-Specific Biobank for Advancing Translational Research Into Ocular Tumors.

The pathogenesis and etiology of various ocular tumors remain largely unclear, limiting the development of diagnostic and treatment approaches for such tumors. Tissue samples from patients are also valuable resource to elucidate mechanisms underlying tumorigenesis. Here we present the early phase setup of an ocular tumor biobank at Xiangya Hospital. Blood and tissue samples along with associated clinical data were obtained from patients who underwent surgery in the Department of Ophthalmology of Xiangya Hospital from December 1, 2018 to January 31, 2020. Standardized operating protocols were developed for the collection, transportation, processing and preservation of ocular tumor samples. A total of 92 clinical cases suffered from 21 types of eye tumors and several undiagnosed eye diseases were covered. A total of 846 samples were preserved in the ocular tumor biobank, including 356 blood samples (42.1%), 324 plasma samples (38.3%), and 166 tissue samples (19.6%). Using the clinical data, we analyzed the prevalence of malignant ocular tumors in association with variables of age, gender, tumors' location and size, and presenting complaints of lump and proptosis. The factors predictive of malignant ocular tumors, included gender (B = 1.599; P = 0.025) and the symptom of proptosis (B = -2.534; P = 0.001). Overall, the setup of clinically-based ophthalmologic biobank could support pathological and translational research into ocular tumors.

The cardioprotective effects of the new crystal form of puerarin in isoproterenol-induced myocardial ischemia rats based on metabolomics.

Puerarin has shown unique pharmacological effects on myocardial ischemia (MI). Changing the crystal form is an effective approach to improve the cardioprotective effects of puerarin. However, the mechanisms of the new crystal form of puerarin are unclear. In this study, an electrocardiogram, echocardiography, cardiac marker enzymatic activity, oxidative stress indices, and myocardial histology analysis of cardiac tissues were performed to evaluate the cardioprotective effects of the new crystal form of puerarin. Moreover, serum and cardiac tissue metabolomics based on nuclear magnetic resonance (NMR) were used to investigate the potential mechanism of the new crystal form. The results indicated that the new crystal form of puerarin (30 mg/kg) could improve oxidative stress indices, and these improvements were similar to those of the original crystal form of puerarin (120 mg/kg). The new crystal form of puerarin (30 mg/kg) could effectively improve the activities of cardiac marker enzymes, and the improvement effects were better than those of the original crystal form (120 mg/kg). Moreover, metabolomics analysis showed that amino acid metabolism, oxidative stress and energy metabolism were disturbed after MI and could be improved by puerarin. These results demonstrated that the new crystal form of puerarin was effective in treating MI.

Downregulation of CKS1B restrains the proliferation, migration, invasion and angiogenesis of retinoblastoma cells through the MEK/ERK signaling pathway.

Retinoblastoma (RB) is a common neoplasm that is exhibited in individuals globally. Increasing evidence demonstrated that cyclin­dependent kinase regulatory subunit 1B (CKS1B) may be involved in the pathogenesis of various tumor types, including multiple myeloma and breast cancer. In the present study, the hypothesis that CKS1B downregulation would effectively inhibit the proliferation, invasion and angiogenesis of RB cells through the mitogen­activated protein kinase kinase (MEK)/extracellular signal­regulated kinase (ERK) signaling pathway was examined. Initial investigation of the expression profile of CKS1B in RB and adjacent retina tissues was performed using reverse transcription­quantitative polymerase chain reaction and western blot analysis. A total of three RB cell lines, SO­RB50, Y79 and HXO­RB44, were examined for selection of the cell line with the highest expression of CKS1B, and human normal retinal vascular endothelial cells (ACBRI­181) were also evaluated. CKS1B short hairpin RNA (shRNA) sequences (shRNA CKS1B­1, shRNA CKS1B­2 and shRNA CKS1B­3) and negative control shRNA sequences were constructed and transfected into cells at the third generation to evaluate the role of shCKS1B and the MEK/ERK signaling pathway in RB. Furthermore, the effect of shCKS1B on cell proliferation, migration, invasion, apoptosis and angiogenesis was investigated. CKS1B was determined to be highly expressed in RB tissue, compared with adjacent retina tissue. SO­RB50 and HXO­RB44 cells treated with shRNA CKS1B­1 and shRNA CKS1B­2 were selected for the present experiments. Activation of the MEK/ERK signaling pathway increases the expression of MEK, ERK, B­cell lymphoma 2, proliferating cell nuclear antigen, cyclin D1, vascular endothelia growth factor and basic fibroblast growth factor, enhances cell proliferation, migration, invasion and lumen formation, and decreases apoptosis. Following silencing CKS1B, the aforementioned conditions were reversed. The key observations of the present study demonstrated that shCKS1B can inhibit the proliferation, invasion and angiogenesis of RB cells by suppressing the MEK/ERK signaling pathway. Thus, CKS1B represents a potential research target in the development of therapeutics for RB.

Did you choose appropriate tracer for retrograde tracing of retinal ganglion cells? The differences between cholera toxin subunit B and Fluorogold.

Cholera toxin subunit B (CTB) and Fluorogold(FG) are two widely utilized retrograde tracers to assess the number and function of retinal ganglion cells (RGCs). However, the relative advantages and disadvantages of these tracers remain unclear, which may lead to their inappropriate application. In this study, we compared these tracers by separately injecting the tracer into the superior Colliculi (SC) in rats, one or 2 weeks later, the rats were sacrificed, and their retinas, brains, and optic nerves were collected. From the first to second week, FG displayed a greater number of labeled RGCs and a larger diffusion area in the SC than CTB; The number of CTB labeled RGCs and the diffusion area of CTB in the SC increased significantly, but there was no distinction between FG; Furthermore, CTB exhibited more labeled RGC neurites and longer neurites than FG, but no difference was evident between the same trace; The optic nerves labeled using CTB were much clearer than those labeled using FG. In conclusion, both CTB and FG can be used for the retrograde labeling of RGCs in rats at 1 or 2 weeks. FG achieves retrograde labeling of a greater number of RGCs than CTB, whereas CTB better delineates the morphology of RGCs. Furthermore, CTB seems more suitable for retrograde labeling of some small, non-image forming nuclei in the brain to which certain RGC subtypes project their axons.

MicroRNA-28 potentially regulates the photoreceptor lineage commitment of Müller glia-derived progenitors.

Retinal degenerative diseases ultimately result into irreversible photoreceptor death or loss. At present, the most promising treatment for these diseases is cell replacement therapy. Müller glia are the major glia in the retina, displaying cardinal features of retinal progenitor cells, and can be candidate of seed cells for retinal degenerative diseases. Here, mouse retinal Müller glia dissociated and cultured in vitro amplified and were dedifferentiated into Müller glia-derived progenitors (MGDPs), demonstrating expression of stem/progenitor cell markers Nestin, Sox2 and self-renewal capacity. MicroRNAs (miRNAs) play unique roles in the retinogenesis, so we hypothesized miRNAs would contribute to photoreceptor lineage commitment of MGDPs. By TargetScan, Miranda, and Pictar bioinformatics, gain/loss-of-function models, dual luciferase assay, we identified and validated that miR-28 targeted the photoreceptor-specific CRX transcription factor. Anti-miR-28 could induce MGDPs to differentiate into neurons strongly expressing CRX and Rhodopsin, while miR-28 mimic suppressed CRX and Rhodopsin expression. Knockdown of CRX by siRNA blocked the expression of CRX and Rhodospin upregulated by anti-miR-28, indicating that anti-miR-28 potentially induced photoreceptor commitment of MGDPs by targeting CRX, but more experiments are necessary to confirm their role in differentiation.

Which has more stem-cell characteristics: Müller cells or Müller cells derived from in vivo culture in neurospheres?

OBJECTIVE: Müller cells can be acquired from in vitro culture or a neurosphere culture system. Both culture methods yield cells with progenitor-cell characteristics that can differentiate into mature nervous cells. We compared the progenitor-cell traits of Müller cells acquired from each method. METHODS: Primary murine Müller cells were isolated in serum culture media and used to generate Müller cells derived from neurospheres in serum-free culture conditions. Gene expression of neural progenitor cell markers was examined by Q-PCR in the two groups. Expression of rhodopsin and the cone-rod homeobox protein CRX were assessed after induction with 1 µM all-trans retinoic acid (RA) for 7 days. RESULTS: After more than four passages, many cells were large, flattened, and difficult to passage. A spontaneously immortalized Müller cell line was not established. Three-passage neurospheres yielded few new spheres. Genes coding for Nestin, Sox2, Chx10, and Vimentin were downregulated in cells derived from neurospheres compared to the cells from standard culture, while Pax6 was upregulated. Müller cells from both culture methods were induced into rod photoreceptors, but expression of rhodopsin and CRX was greater in the Müller cells from the standard culture. CONCLUSION: Both culture methods yielded cells with stem-cell characteristics that can be induced into rod photoreceptor neurons by RA. Serum had no influence on the "stemness" of the cells. Cells from standard culture had greater "stemness" than cells derived from neurospheres. The standard Müller cells would seem to be the best choice for transplantation in cell replacement therapy for photoreceptor degeneration.

Development of new reference material neohesperidin for quality control of dietary supplements.

BACKGROUND: Neohesperidin is an important natural flavanone glycoside distributed in several citrus species. This compound is widely used as a raw material for food additives in the food industry. The request for certified reference materials (CRMs) in dietary supplements was stipulated by the National Administrative Committee for CRMs and was underpinned by the need to improve the accuracy and comparability of measurement data and to establish metrological traceability of analytical results. RESULTS: This paper reports the sample preparation methodology, homogeneity and stability studies, value assignment and uncertainty estimation of a new certified reference material of neohesperidin (GBW09522). Differential scanning calorimetry, coulometric titration and mass balance methods proved to be sufficiently reliable and accurate for certification purposes. The certified value of neohesperidin CRM is 994 g kg(-1) with an expanded uncertainty of 4 g kg(-1) (k = 2). The reference material described above was homogeneous and stable for 12 months at a storage temperature of 25 °C. CONCLUSION: The new CRM of neohesperidin can be used to validate analytical methods and improve the accuracy of measurement data as well as quality control of neohesperidin-related dietary supplements, foods, traditional herbs and pharmaceutical formulations.

Development of a new certified reference material of diosgenin using mass balance approach and Coulometric titration method.

Certified reference materials (CRMs) can be used as a valuable tool to validate the trueness of measurement methods and to establish metrological traceability of analytical results. Diosgenin has been selected as a candidate reference material. Characterization of the material relied on two different methods, mass balance method and Coulometric titration method (CT). The certified value of diosgenin CRM is 99.80% with an expanded uncertainty of 0.37% (k=2). The new CRM of diosgenin can be used to validate analytical methods, improve the accuracy of measurement data and control the quality of diosgenin in relevant pharmaceutical formulations.

Characterization and uncertainty evaluation of a new certified reference material of baicalein.

A new certified reference material (CRM) of baicalein was developed aiming to control the quality of baicalein and relative pharmaceuticals. Sample preparation, homogeneity, stability, value assignment, and uncertainty of a new certified reference material (CRM) of baicalein were presented in this paper. Characterization of the material relied on three different methods, which were differential scanning calorimetry (DSC), coulometric titration method (CT) and mass balance method. DSC and CT were used for purity determination of baicalein firstly. The certified value of baicalein CRM is 99.72% with an expanded uncertainty of 0.25% (k=2). The new CRM of baicalein can be used to validate analytical methods, improve the accuracy of measurement data as well as establish meteorological traceability of analytical results.