Absolute Quantification of Dynamic Cellular Uptake of Small Extracellular Vesicles via Lanthanide Element Labeling and ICP-MS.

Small extracellular vesicles (sEVs) are increasingly reported to play important roles in numerous physiological and pathological processes. Cellular uptake of sEVs is of great significance for functional regulation in recipient cells. Although various sEV quantification, labeling, and tracking methods have been reported, it is still highly challenging to quantify the absolute amount of cellular uptake of sEVs and correlate this information with phenotypic variations in the recipient cell. Therefore, we developed a novel strategy using lanthanide element labeling and inductively coupled plasma-mass spectrometry (ICP-MS) for the absolute and sensitive quantification of sEVs. This strategy utilizes the chelation interaction between Eu3+ and the phosphate groups on the sEV membrane for specific labeling. sEVs internalized by cells can then be quantified by ICP-MS using a previously established linear relationship between the europium content and the particle numbers. High Eu labeling efficiency and stability were demonstrated by various evaluations, and no structural or functional alterations in the sEVs were discovered after Eu labeling. Application of this method revealed that 4020 ± 171 sEV particles/cell were internalized by HeLa cells at 37 °C and 61% uptake inhibition at 4 °C. Further investigation led to the quantitative differential analysis of sEV cellular uptake under the treatment of several chemical endocytosis inhibitors. A 23% strong inhibition indicated that HeLa cells uptake sEVs mainly through the macropinocytosis pathway. This facile labeling and absolute quantification strategy of sEVs with ppb-level high sensitivity is expected to become a potential tool for studying the functions of sEVs in intracellular communication and cargo transportation.

Sensitive N-Glycopeptide Profiling of Single and Rare Cells Using an Isobaric Labeling Strategy without Enrichment.

Single-cell omics is critical in revealing population heterogeneity, discovering unique features of individual cells, and identifying minority subpopulations of interest. As one of the major post-translational modifications, protein N-glycosylation plays crucial roles in various important biological processes. Elucidation of the variation in N-glycosylation patterns at single-cell resolution may largely facilitate the understanding of their key roles in the tumor microenvironment and immune therapy. However, comprehensive N-glycoproteome profiling for single cells has not been achieved due to the extremely limited sample amount and incompatibility with the available enrichment strategies. Here, we have developed an isobaric labeling-based carrier strategy for highly sensitive intact N-glycopeptide profiling for single cells or a small number of rare cells without enrichment. Isobaric labeling has unique multiplexing properties, by which the "total" signal from all channels triggers MS/MS fragmentation for N-glycopeptide identification, while the reporter ions provide quantitative information. In our strategy, a carrier channel using N-glycopeptides obtained from bulk-cell samples significantly improved the "total" signal of N-glycopeptides and, therefore, promoted the first quantitative analysis of averagely 260 N-glycopeptides from single HeLa cells. We further applied this strategy to study the regional heterogeneity of N-glycosylation of microglia in mouse brain and discovered region-specific N-glycoproteome patterns and cell subtypes. In conclusion, the glycocarrier strategy provides an attractive solution for sensitive and quantitative N-glycopeptide profiling of single/rare cells that cannot be enriched by traditional workflows.

A three-stage search strategy combining database reduction and retention time filtering to improve the sensitivity of low-input and single-cell proteomic analysis.

When performing proteome profiling of low-input and single-cell samples, achieving deep protein coverage is very challenging due to the sensitivity limitation of current proteomic methods. Herein, we introduce a three-stage search strategy that combines the advantages of database reduction and Δ retention time (ΔRT) filtering. The strategy improves peptide/protein identification and reproducibility by retaining more correct identifications and filtering out incorrect identifications. The raw data were first merged and searched against a Uniprot database with a relaxed false discovery rate (FDR) of 40% to identify the possible detectable proteins. The identified proteins were then used as a new database to search the raw data against with a tighter FDR of 10%. After this, the results were filtered using ΔRT (the difference between the measured and predicted RT) to reduce the incorrect identifications and maintain the FDR below 1%. This strategy resulted in over 30% improvement in proteome coverage for single-cells and samples of similar size. The reproducibility of identification and quantification was also enhanced for the low-input samples. Moreover, the 50% higher number of differential proteins found in the two types of single neurons indicates the application potential of this strategy.

A rapid and sensitive single-cell proteomic method based on fast liquid-chromatography separation, retention time prediction and MS1-only acquisition.

Single-cell analysis has received much attention in recent years for elucidating the widely existing cellular heterogeneity in biological systems. However, the ability to measure the proteome in single cells is still far behind that of transcriptomics due to the lack of sensitive and high-throughput mass spectrometry methods. Herein, we report an integrated strategy termed "SCP-MS1" that combines fast liquid chromatography (LC) separation, deep learning-based retention time (RT) prediction and MS1-only acquisition for rapid and sensitive single-cell proteome analysis. In SCP-MS1, the peptides were identified via four-dimensional MS1 feature (m/z, RT, charge and FAIMS CV) matching, therefore relieving MS acquisition from the time consuming and information losing MS2 step and making this method particularly compatible with fast LC separation. By completely omitting the MS2 step, all the MS analysis time was utilized for MS1 acquisition in SCP-MS1 and therefore led to 65%-138% increased MS1 feature collection. Unlike "match between run" methods that still needed MS2 information for RT alignment, SCP-MS1 used deep learning-based RT prediction to transfer the measured RTs in long gradient bulk analyses to short gradient single cell analyses, which was the key step to enhance both identification scale and matching accuracy. Using this strategy, more than 2000 proteins were obtained from 0.2 ng of peptides with a 14-min active gradient at a false discovery rate (FDR) of 0.8%. Comparing with the DDA method, improved quantitative performance was also observed for SCP-MS1 with approximately 50% decreased median coefficient of variation of quantified proteins. For single-cell analysis, 1715 ± 204 and 1604 ± 224 proteins were quantified in single 293T and HeLa cells, respectively. Finally, SCP-MS1 was applied to single-cell proteome analysis of sorafenib resistant and non-resistant HepG2 cells and revealed clear cellular heterogeneity in the resistant population that may be masked in bulk studies.

Tandem enrichment of serum exosomes and exosomal RNA with titanium dioxide.

Exosomes have great potential as biomarker carriers for disease diagnosis and prognosis. In recent years, exosomal RNA (exoRNA) has become a promising candidate for the early diagnosis and prognosis of cancers, and its pathophysiological roles in various diseases have been revealed. For example, exosome-derived mRNAs, miRNAs, circRNAs, and lncRNAs function as signalling molecules to regulate tumour growth, angiogenesis, invasion, metastasis, and the response to chemotherapy. However, the isolation of exosomes and exoRNA with high quality and purity remains challenging due to the relatively small size of exosomes and the limited amount of RNA in exosomes. In this work, we developed a novel tandem enrichment method to isolate exoRNA from serum based on the specific interaction between titanium dioxide (TiO2) and the phosphate groups on the lipid bilayer of exosomes and of the exoRNA. TiO2-based RNA isolation was first demonstrated and optimized in HeLa cells. A total of 130.9 ± 8.34 µg of RNA was rapidly enriched from approximately 5 × 106 HeLa cells within 10 min. This was a 41.5% higher yield than that using a commercial Ultrapure RNA Kit. TiO2-based tandem enrichment of exoRNA was then performed using human serum, obtaining 64.53±3.41 ng of exoRNA from 500 µL of human serum within 30 min. A total of 2,137,902 reads, including seven types of exoRNAs, were identified from the exosomes. This method is compatible with various downstream RNA processing techniques and does not use toxic or irritating reagents, such as phenol or chloroform, providing a simple, economical, rapid, and safe approach for exoRNA extraction from biological samples.

Case Report: A neurolinguistic and neuroimaging study on a Chinese follow-up case with logopenic-variant of primary progressive aphasia.

Primary progressive aphasia (PPA), typically resulting from a neurodegenerative disease, is characterized by a progressive loss of specific language functions while other cognitive domains are relatively unaffected. The logopenic variant, characterized by impairments of word retrieval and sentence repetition along with preserved semantic, syntactic, and motor speech abilities, is the most recently described and remains less understood than other variants due to a comparatively small number of case studies and a lack of investigations with a thorough specification. In this article, we report a 2-year follow-up case study of a 74-year-old Chinese female patient with a logopenic variant of primary progressive aphasia, including its neurolinguistic study, magnetic resonance imaging (MRI), and 11C-Pittsburgh compound B-Positron emission tomography imaging analyses, as well as gene sequencing. This case confirms that, in addition to word-finding and sentence repetition difficulties, the logopenic variant may also present with mild auditory comprehension and naming deficits attributed to impaired access to lexical representations. The observation of clinical treatment suggests the efficacy of memantine hydrochloride tablet and rivastigmine transdermal patch in slowing down the cognitive deterioration of this patient. The description and exploration of this case may shed new insights into a better understanding of the Chinese logopenic variant of primary progressive aphasia.

Attractive Pickering Emulsion Gels.

Properties of emulsions highly depend on the interdroplet interactions and, thus, engineering interdroplet interactions at molecular scale are essential to achieve desired emulsion systems. Here, attractive Pickering emulsion gels (APEGs) are designed and prepared by bridging neighboring particle-stabilized droplets via telechelic polymers. In the APEGs, each telechelic molecule with two amino end groups can simultaneously bind to two carboxyl functionalized nanoparticles in two neighboring droplets, forming a bridged network. The APEG systems show typical shear-thinning behaviors and their viscoelastic properties are tunable by temperature, pH, and molecular weight of the telechelic polymers, making them ideal for direct 3D printing. The APEGs can be photopolymerized to prepare APEG-templated porous materials and their microstructures can be tailored to optimize their performances, making the APEG systems promising for a wide range of applications.

A novel LC-MS/MS approach to the pharmacokinetic study of free and bound aflibercept simultaneously.

To comprehensively evaluate the pharmacokinetic (PK) characteristics of aflibercept, we established a liquid chromatography with tandem mass spectrometry (LC-MS/MS) method to determine the concentration of vascular endothelial growth factor (VEGF)-A-bound aflibercept and free aflibercept. A specific sample preparation method of nano-surface and molecular-orientation limited (nSMOL) proteolysis was performed to extract both free and bound aflibercept from plasma. The tryptic peptides unique to aflibercept and VEGF-A were selected to quantify the amounts of total aflibercept and aflibercept-VEGF complex, respectively. The method was validated by evaluating its selectivity, linearity, precision, accuracy, extraction recovery, matrix effect, and stability. It was then successfully used to quantify total and bound aflibercept concentrations in cynomolgus monkey plasma, while indirectly obtaining the concentration of free aflibercept by subtraction. The PK results of this LC-MS/MS method are comparable to the traditional enzyme-linked immunosorbent assay (ELISA) results. It is thus a reliable and complementary method for the PK evaluation of aflibercept. Graphical abstract.

Curcumin, as a pleiotropic agent, improves doxorubicin-induced nephrotic syndrome in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Curcumin, a phenolic compound extracted from the rhizome of turmeric (Curcuma longa L.), has been reported to have broad biological functions including potent antioxidant and renoprotective effects. It has been reported that Curcumin has a certain protective effect on the kidney. However, its mechanism of action needs further study. AIM OF THE STUDY: The present research aims at investigating the therapeutic effects and its underlying mechanism of curcumin on NS. MATERIALS AND METHODS: The conditionally immortalized mouse podocyte cell line was utilized to evaluate the podocyte-protective effect of curcumin and its effects on NF-κB pathway and Nrf2/ARE pathway in podocyte in vitro. Furthermore, the DOX-induced NS rats were utilized to investigate the therapeutic effects and its underlying mechanism of curcumin against NS in vivo. RESULTS: The consequences of this study revealed that curcumin activated Nrf2, inhibited NF-κB pathway and up-regulated podocin in DOX-induced podocyte. Further research results showed that curcumin can considerably alleviate proteinuria and improve hypoalbuminemia in NS rats, and lower blood lipid levels to alleviate hyperlipidemia in NS rats, indicating that curcumin has significant therapeutic effects on rat NS. Further observation by electron microscopy and detection showed that curcumin can improve renal function and podocyte injury, which may be related to the repairment of mRNA expression and podocin protein. Interestingly, the results of the blood rheology test showed that curcumin can effectively reduce whole blood viscosity (WBV) and plasma viscosity (PV), and reduce hematocrit (Hct). In addition, the oxidative stress state of kidney in NS rats was considerably reversed by curcumin, which may be achieved by activating Nrf2 and increasing the expression of antioxidant enzymes HO-1, NQO-1. We also found that NF-κB pathway is activated in the kidney of NS rats, and curcumin can inhibit the activation of NF-κB by down-regulating the expression of NF-κB p65, reducing the level of p-IκBα and up-regulating the expression of IκBα. CONCLUSION: These findings suggest that curcumin, as a multifunctional agent, exerts a protective effect on DOX-induced nephrotic syndrome in rats, which provides a pharmacological basis for the further development of curcumin and also provides a basis for the advantages of multi-targeted drugs in the processing of NS.

Application of T1-/T2-Weighted Ratio Mapping to Elucidate Intracortical Demyelination Process in the Alzheimer's Disease Continuum.

Background The biological diagnosis criteria of the Alzheimer's disease (AD) suggests that previous work may misclassify the cognitive impairment caused by other factors into AD. Consequently, re-assessing the imaging profile of AD continuum is needed. Considering the high vulnerability of cortical association fibers, we aimed to elucidate the cortical demyelination process in the AD continuum biologically defined. Methods According to the biological diagnosis criteria, we determined the positive amyloid status (A+) as cerebrospinal fluid (CSF) amyloid1 - 42 < 192 pg/ml, Florbetapir Positron emission tomography (PET) composite standardized uptake value ratio (SUVR) >1.11. Also, the positive Tau status (T+) was determined as p-Tau181 > 23 pg/ml. Based on the cognitive characterization, we further categorized 252 subjects into 27 cognitively unimpaired with normal AD biomarkers (A-T-, controls), 49 preclinical AD (A+T+), 113 AD with mild cognitive impairment (MCI) (A+T+), and 63 AD dementia (A+T+). We estimated the intracortical myelin content used the T1- and T2-weighted (T1W/T2W) ratio mapping. To investigate the sensitivity of the ratio mapping, we also utilized well-validated AD imaging biomarkers as the reference, including gray matter volume and Fludeoxyglucose PET (FDG-PET). Based on the general linear model, we conducted the voxel-wise two-sample T-tests between the controls and each group in the AD continuum. Results Compared to the controls, the results showed that the preclinical AD patients exhibited decreased T1W/T2W ratio value in the right inferior parietal lobule (IPL); as the disease progresses, the prodromal AD patients demonstrated lower ratio value in bilateral IPL, with hippocampus (HP) atrophy. Lastly, the AD dementia patients exhibited decreased ratio value in bilateral IPL and hippocampus; also, we observed the bilateral temporal cortices atrophy and widespread decreased metabolism in the AD dementia patients. After corrected with gray volume, the results remained mostly unchanged. Conclusion Our study implied the decreased right IPL T1W/T2W ratio might represent early AD-related demyelination in disease continuum. Additionally, we demonstrated that the T1W/T2W ratio mapping is an easy-to-implement and sensitive metric to assess the intracortical myelin content in AD, particularly in the early stage.

Controlled co-precipitation of biocompatible colorant-loaded nanoparticles by microfluidics for natural color drinks.

Natural colorants, which impart a vivid color to food and add additional health benefits, are favored over synthetic colorants; however, their applications are limited by their low solubility in water and low stability. Here, we develop a versatile microfluidic strategy to incorporate natural colorants in shellac nanoparticles with controlled physicochemical properties. The rapid mixing in the microfluidic channels ensures that the mixing time is shorter than the aggregation time, thus providing control over the co-precipitation of the colorant and the polymer. By introducing molecular interactions, colorant nanoaggregates are efficiently embedded in the polymer matrix, forming hierarchical colorant-loaded nanoparticles. The colorant-loaded nanoparticles dispersed in water are transparent and stable over a wide pH range and their polymer matrix also provides a favorable microenvironment that greatly improves the shelf life of the colorants. The improved solubility, stability and bioavailability of the natural colorants suggest that shellac nanoparticles are ideal carriers and the stable, transparent dispersions of biocompatible colorant-loaded nanoparticles in water are well-suited for the development of functional foods, such as natural color drinks.

Therapeutic and antiproteinuric effects of salvianolic acid A in combined with low-dose prednisone in minimal change disease rats: Involvement of PPARγ/Angptl4 and Nrf2/HO-1 pathways.

Danshen (Salvia miltiorrhiza) and prednisone are extensively applied in the treatment of kidney disease. Salvianolic acid A (SAA), the major biologically active component of Danshen, which has various biological effects. Our previous findings have demonstrated the renoprotective effect of SAA in various kidney disease rodent models. Here, we explore the therapeutic potential and possible mechanisms of SAA in combination with low-dose prednisone in adriamycin (ADR)-induced minimal change disease (MCD) rat model and mouse podocyte injury cell model. SAA was injected via tail vein at 10 mg/kg/day and prednisone at 5 mg/kg/day via gavage. Each drug was administered daily alone or in combination for 3 weeks. Combination therapy showed significant therapeutic efficacy as manifested by relieved urinary proteins, improved blood biochemical indicators including serum total protein, albumin, triglyceride, cholesterol, the indices of renal function i.e. blood urea nitrogen and serum creatinine levels, and ameliorated pathological lesions. Particularly, co-administration showed a significant anti-proteinuria effect in MCD rats. Further studies suggested that co-administration effectively ameliorated the podocyte injury as indicated by the reduction of podocyte foot processes fusion, up-regulation of synaptopodin and down-regulation of desmin. These beneficial effects are accompanied by activation of the Nrf2/HO-1 and PPARγ/Angptl4 pathways in vivo, and the effect of SAA on PPARγ/Angptl4 is also demonstrated in vitro. These findings suggested that SAA exerted podocyte-protection against MCD injury through PPARγ/Angptl4 and Nrf2/HO-1 pathways, and combined with low-dose prednisone possessed a significant anti-proteinuria and therapeutic effects in MCD rats.

Amyloid ß protein injection into medial septum impairs hippocampal long-term potentiation and cognitive behaviors in rats.

The specific loss of cholinergic neurons and the progressive deficits of cognitive function are the most primary characteristics of Alzheimer's disease (AD). Although the neurotoxicity of amyloid ß protein (Aß) in AD has been investigated extensively, it is still unclear whether the Aß aggregated in the medial septum (MS), a major cholinergic nucleus projecting to the hippocampus, could affect hippocampal synaptic plasticity and further impair the memory behaviors. The present study investigated the effects of Aß injection into the MS on hippocampal long-term potentiation (LTP) and cognitive behaviors of rats by using Morris water maze (MWM), Y maze and in vivo hippocampal LTP recording. The effects of kainic acid (KA), an agent with specific neurotoxicity to GABAergic neurons, were also observed. The results showed that: (1) Intra-MS injection of Aß25-35, not KA, impaired spatial learning and memory of rats in classical and reversal MWM tests; (2) Both Aß25-35 and KA impaired novelty-seeking behavior of rats in Y maze; (3) Intra-MS injection of Aß25-35, not KA, suppressed in vivo hippocampal LTP in the CA1 region of rats; (4) Both Aß25-35 and KA did not affect the motor ability in behavioral tests and the hippocampal paired-pulse facilitation (PPF) in electrophysiological recording. These results indicate that intra-MS injection of Aß could impair spatial memory, cognitive flexibility and exploratory motivation, as well as hippocampal LTP in rats, suggesting that the cholinergic neurons in the MS and the septo-hippocampal projection could be important targets of neurotoxic Aß, and the specific damage of cholinergic neurons in the MS is likely responsible for the impairments of hippocampal synaptic plasticity and cognitive function in AD.

Davunetide improves spatial learning and memory in Alzheimer's disease-associated rats.

Memory loss and cognition decline are the main clinical manifestations of Alzheimer's disease (AD). Amyloid ß protein (Aß) aggregated in the brain is one of the key pathological characteristics of AD and responsible for the deficits in learning and memory. It is reported that davunetide, an octapeptide derived from activity-dependent neuroprotective protein (ADNP), inhibited Aß aggregation and Aß-induced neurotoxicity. To further characterize the neuroprotective roles of davunetide and its possible mechanism, the present study investigated the effects of davunetide on Aß1-42-induced impairments in spatial memory, synaptic plasticity and hippocampal AKT level. In Morris water maze (MWM) test, bilateral intrahippocampal injection of Aß1-42 significantly increased escape latency and decreased target quadrant swimming time of rats, while three weeks of intranasal application of davunetide reversed the Aß1-42-induced learning deficits and memory loss in a dose-dependent manner. In vivo field potentiation recording showed that Aß1-42 suppressed long-term potentiation (LTP) of excitatory postsynaptic potential (fEPSP) in the hippocampal CA1 region of rats, while davunetide effectively blocked the suppression of LTP, without affecting paired-pulse facilitation (PPF). Western blotting experiments showed a significant decrease in the level of hippocampal p-AKT (Ser473), not total AKT, in Aß1-42 only group, which was mostly antagonized by davunetide treatment. These findings demonstrate that davunetide, probably by enhancing PI3K/AKT pathway, plays an important positive role in attenuating Aß1-42-induced impairments in spatial memory and synaptic plasticity, suggesting that davunetide could be an effective therapeutic candidate for the prevention and treatment of neurodegenerative disease such as AD.

Characteristics of Agraphia in Chinese Patients with Alzheimer's Disease and Amnestic Mild Cognitive Impairment.

BACKGROUND: Patients with Alzheimer's disease (AD) manifest progressive decline in writing abilities. Most studies on agraphia in AD have been performed in the alphabetic system, such as English. However, these findings may not be applicable to other written language systems. The unique features of the Chinese written script could affect the patterns of agraphia in Chinese AD patients. The aim of this study was to explore the features of writing errors in Chinese patients with AD and amnestic mild cognitive impairment (a-MCI), as well as to study the relationship between their writing errors and neuropsychological functions. METHODS: In this study, we performed an observational study in a group of subjects including 17 AD patients, 14 patients with a-MCI, and 16 elderly healthy controls. We analyzed the writing errors in these subjects and also studied the relationship between their writing errors and neuropsychological functions. RESULTS: Our study showed that in patients whose mother tongue is Chinese, writing ability was comparatively well preserved in the MCI phase but significantly impaired when the disease progressed to the stage of AD. The writing errors showed corresponding increase with the severity of cognition decline, both in the types of errors and rate of occurrence. Analysis of the writing errors showed that word substitution and unintelligible words were the most frequent error types that occurred in all the three study groups. The occurrence rate of unintelligible words was significantly higher in the AD group compared with the a-MCI group (P = 0.024) and control group (P = 0.018). In addition, the occurrence rates of word substitution were also significantly higher in AD (P = 0.013) and a-MCI groups (P = 0.037) than that of control group. However, errors such as totally no response, visuospatial impairment, paragraph agraphia, ideograph, and perseverative writing errors were only seen in AD group. Besides, we also found a high occurrence rate of visuoconstructional errors (13.3%) in our AD group. CONCLUSIONS: Our study confirmed that agraphia is an important feature in patients with AD. The writing error profile in patients whose native language is Chinese was unique compared to patients using the alphabetic language system.

Binding and Anticancer Properties of Plumbagin with Human Serum Albumin.

Plumbagin has received extensive attention as a promising anticancer drug. Therefore, we investigated the binding and anticancer properties of plumbagin with human serum albumin. Fluorescence results demonstrated that plumbagin interacts with human serum albumin, although its binding affinity may be affected to various extents by different compounds. The human serum albumin-plumbagin complex structure revealed that plumbagin binds to the hydrophobic cavity in the IIA subdomain of human serum albumin through hydrogen bonding and hydrophobic interactions. The plumbagin-human serum albumin complex enhances cytotoxicity by 2- to 3-fold particularly in cancer cells but has no effect on normal cells in vitro. Compared with the unbound drug, the human serum albumin-plumbagin complex promotes HeLa cell apoptosis and has a stronger capacity for cell cycle arrest at the G2/M phase of HeLa cells. In conclusion, this study contributes to the rational design and development of plumbagin-based drugs and a drug-human serum albumin delivery system.

Structural basis and anticancer properties of ruthenium-based drug complexed with human serum albumin.

Ruthenium-based anticancer complexes have become increasingly popular for study over the last two decades. Although ruthenium complexes are currently being investigated in clinical trials, there are still some difficulties with their delivery and associated side effects. Human serum albumin (HSA)-based delivery systems are promising for improving anticancer drug targeting and reducing negative side effects. However, there have been few studies regarding the HSA delivery system for metal-based anticancer compounds and no mention of its structural mechanism. Therefore, we studied the structure and anticancer properties of the ruthenium-based compound [RuCl5(ind)](2-) in complex with HSA. The structure revealed that [RuCl5(ind)](2-) has two binding sites in HSA. In the IB subdomain, [RuCl5(ind)](2-) binds to a new sub-site by coordinating with His-146. In the IIA subdomain, ruthenium (III) of [RuCl5(ind)](2-) binds to the hydrophobic cavity and forms coordination bonds by replacing chlorine atoms with the His-242 and Lys-199 residues of HSA. Interestingly, [RuCl5(ind)](2-), together with HSA, can enhance cytotoxicity by two to five times in cancer cells but has no effect on normal cells in vitro. Compared with unbound drug, the HSA-[RuCl5(ind)](2-) complex promotes MGC-803 cell apoptosis and also has a stronger capacity for cell cycle arrest at the G2 phase in MGC-803. In conclusion, this study will guide the rational design and development of ruthenium-containing or ruthenium-centered drugs and an HSA delivery system for ruthenium-based drugs.

Comparison of binding characterization of two antiviral drugs to human serum albumin.

Ribavirin and lamivudine are representatives of antiviral drugs that are widely used to treat viral infections, especially chronic liver disease. To compare binding mechanism and behavior of antiviral drugs with human serum albumin (HSA), we performed fluorescence spectroscopy and X-ray crystallography to investigate the interactions of ribavirin and lamivudine with HSA. Fluorescence spectroscopy showed ribavirin and lamivudine inhibit binding affinity each other. Our results further demonstrated that ribavirin and lamivdudine interaction with HSA could be affected by the presence of other compounds, including the non-steroidal anti-inflammatory drugs, indometacin. X-ray structures revealed that ribavirin and lamivudine bind in IIA subdomain of HSA mainly by forming hydrogen bond and hydrophobic interactions forces. The carboxamido of ribavirin forms hydrogen bonds with Arg222; Hydroxyl group (6) of ribavirin forms hydrogen bond with Arg257. Hydroxyl group (15) of lamivudine forms hydrogen bond with Arg222; amino group (4) of lamivudine forms hydrogen bond with carbonyl of Arg257. Our results reveal the key biochemical and structural characteristics of the HSA interaction with ribavirin and lamivudine, providing guidance for future development of ribavirin- and lamivudine-based compounds and a drug-HSA delivery system.

What Is Important During the Selection of Traditional Chinese Medicine (TCM) in a Health Care Reimbursement or Insurance System?" Critical Issues of Assessment from the Perspective of TCM Practitioners.

OBJECTIVE: Complementary and alternative medicine (CAM) has gained increasing popularity over the last several decades internationally, leading to an increasing interest from decision makers and researchers as to how to assess the effectiveness of CAM. The attempts, however, have been unsatisfactory. The most important reason is a lack of attention to the theoretical characteristics of CAM, which are completely different from those of allopathic medicine or biomedicine. This study attempted to survey expert Traditional Chinese Medicine (TCM) practitioners in China to elucidate critical issues when assessing the effectiveness of TCM. METHODS: A questionnaire (with 20 close-ended and 2 open-ended questions) about the influencing factors of measuring the cost and effectiveness of TCM was distributed to TCM practitioners who had been working in the field of research for at least 5 years and had published at least one related scientific article in the last 5 years. Internal consistency test was performed for all questions to verify the reliability of the questionnaire. Principal-component analysis was performed for remaining items after Kaiser-Meyer-Olkin (KMO) index and Bartlett's test of sphericity. A linear combination model was then built to evaluate the contribution of various factors involved for the selection of TCM into the health care reimbursement or insurance system. RESULTS: Of 429 questionnaires issued, 137 were returned from respondents from 31 medical and research institutions, giving a recovery rate of 31.93%. Internal consistency coefficient obtained was 0.745, indicating good reliability of this measurement scale, and the data passed the KMO test and Bartlett's test of sphericity (KMO index = 0.691). In addition, eight common factors were extracted after the rotation of principal-component analysis with a cumulative variance of 70.92%. CONCLUSIONS: Our findings suggested that factors to be considered during the selection of TCM in health care reimbursement or insurance system include patient preference, long-term outcomes, comparative study of alternative options between TCM and allopathic medicine or biomedicine, pharmacoeconomic evaluation results and the overall economic burden of patients, and side effects of TCM. In addition, the TCM experts stressed the need of cost-effectiveness assessment of the expensive TCM of similar therapeutic functions during the selection process. Moreover, during the evaluation of health-related quality of life of TCM, they warned to avoid overexaggeration of their roles and that the generic scale should be modified according to the clinical circumstances.

Evaluation of impact on health-related quality of life and cost effectiveness of Traditional Chinese Medicine: a systematic review of randomized clinical trials.

BACKGROUND: Traditional Chinese Medicine (TCM), an important part of health care in China and with increased popularity worldwide, has received extensive attention from governments at all levels. With the current emphasis on clinical efficacy and cost-effectiveness, TCM, as indeed do all other treatments, requires rigorous evidence to be considered in reimbursement decision-making. Nevertheless, despite the fact that TCM treatment has always been considered to possess the advantage of improving the health-related quality of life (HRQOL) of patients, there is a lack of systematic study about available evidence to assess the impact of TCM treatments on HRQOL of patients. OBJECTIVES: The current study aimed to perform a review of available literature to evaluate whether sufficient evidence existed to allow an assessment of the impact on HRQOL and cost effectiveness of TCM treatments. This information would support a recommendation for wider use of TCM in the clinical setting as well as its consideration for reimbursement. METHODS: A structured search was performed using data sources including MEDLINE,(®) Cumulative Index for Allied Health and Nursing (CINAHL), PubMed, Cochrane database, EBSCO, SciSearch, Embase, and Google Scholar from 2000 to 2010. The search was supplemented with manual search after relevant articles were retrieved. RESULTS: After culling, a total 31 articles covering a range of TCM therapies applied to a variety of conditions were retrieved. The measurement tools used in these studies to assess impact in patient's HRQOL were mainly SF-36-based scales, but the results of HRQOL/patient preference studies were inconsistent and inconclusive. Of the 10 articles of cost-effectiveness evaluation of TCM treatments, the majority reported that TCM treatments resulted in better outcomes at a higher cost, but the incremental cost-effectiveness ratio was below the usually recommended thresholds. The overall results showed acupuncture and t'ai chi to be the most studied TCM-related therapies. CONCLUSIONS: The current review showed that there is a relative lack of cost-effectiveness research in TCM. For those few empirical research available, the major emphasis is for acupuncture or t'ai chi showing the acceptance of these branches of TCM that are better understood by the scientific community. The current results also showed the need for studies with better designs and longer duration to ascertain the actual impact of TCM on patients' HRQOL as well as a need for a generic HRQOL instrument that is specific for TCM.