Multi-Locus Phylogeny and Morphology Reveal Two New Species of Hypoxylon (Hypoxylaceae, Xylariales) from Motuo, China.

Hypoxylaceous fungi are abundant in China, but their discovery and report are uneven in various provinces, with more fungi in Yunnan and Hainan and fewer fungi in Tibet. During the investigation of macro-fungi in Motuo county, Tibet Autonomous Region, we collected a number of xylarialean specimens. Six hypoxylaceous specimens growing on dead angiosperm were collected from the forests of Motuo county, and they were described and illustrated as two new species in Hypoxylon based on a combination of morphological characters and molecular evidence. Hypoxylon diperithecium was characterized by its bistratal perithecia, purple-brown stromatal granules, citrine to rust KOH-extractable pigments, and light brown to brown ascospores ellipsoid-inequilateral with conspicuous coil-like ornamentation. Hypoxylon tibeticum was distinct from other species by having pulvinate and applanate stromata, surface vinaceous, with orange granules, orange KOH-extractable pigments, and brown ascospores with inconspicuous ornamentation. The multi-gene phylogenetic analyses (ITS-LSU-RPB2-TUB) supported the two new taxa as separate lineages in the genus Hypoxylon. A key to all known Hypoxylon taxa from China is provided.

Severe hyperbilirubinemia in a neonate with hereditary spherocytosis due to a de novo ankyrin mutation: A case report.

BACKGROUND: Hereditary spherocytosis (HS) is a common type of hemolytic anemia caused by a red cell membrane disorder. HS type 1 (HS1) is mostly caused by mutations in ankyrin (ANK1). Newborns with HS1 usually only exhibit anemia and mild jaundice. We herein report a case of HS1 and discuss its clinical characteristics. CASE SUMMARY: A 2-d-old male full-term newborn was admitted to our hospital with severe, intractable neonatal jaundice. Laboratory investigations showed hemolytic anemia and hyperbilirubinemia and excluded immune-mediated hemolysis. The patient underwent two exchange transfusions and one plasmapheresis resulting in significantly reduced serum bilirubin. Hematologic analyses and genomic DNA sequencing studies were performed. The trio clinical exome sequencing revealed a de novo null heterozygous mutation in the patient's ANK1 gene: c.841C > T(p.Arg281Ter). This mutation results in the premature termination of the ANK1 protein. CONCLUSION: Our case demonstrates that genetic analysis can be an essential method for diagnosing HS when a newborn has severe hyperbilirubinemia.

Selective sensing of DNA and live/dead cells and histological imaging based on a perylene derivative.

A mannose-modified perylene monoimide derivative PMI-Man was developed, which shows highly selective binding to double-stranded DNA molecules, potent live/dead cell imaging, and histological imaging via both confocal and light microscopies. This approach can be used to develop a universal colorful staining method for human tissues for both confocal and light microscopies.

Gibberellin repression of axillary bud formation in Arabidopsis by modulation of DELLA-SPL9 complex activity.

The formation of lateral branches has an important and fundamental contribution to the remarkable developmental plasticity of plants, which allows plants to alter their architecture to adapt to the challenging environment conditions. The Gibberellin (GA) phytohormones have been known to regulate the outgrowth of axillary meristems (AMs), but the specific molecular mechanisms remain unclear. Here we show that DELLA proteins regulate axillary bud formation by interacting and regulating the DNA-binding ability of SQUAMOSA-PROMOTER BINDING PROTEIN LIKE 9 (SPL9), a microRNA156-targeted squamosa promoter binding protein-like transcription factor. SPL9 participates in the initial regulation of axillary buds by repressing the expression of LATERAL SUPPRESSOR (LAS), a key regulator in the initiation of AMs, and LAS contributes to the specific expression pattern of the GA deactivation enzyme GA2ox4, which is specifically expressed in the axils of leaves to form a low-GA cell niche in this anatomical region. Nevertheless, increasing GA levels in leaf axils by ectopically expressing the GA-biosynthesis enzyme GA20ox2 significantly impaired axillary meristem initiation. Our study demonstrates that DELLA-SPL9-LAS-GA2ox4 defines a core feedback regulatory module that spatially pattern GA content in the leaf axil and precisely control the axillary bud formation in different spatial and temporal.

Supramolecular nanofiber of pyrene-lactose conjugates and its two-photon fluorescence imaging.

A lactose modified pyrene derivative (Py-Lac) was synthesized, with which novel twisted supramolecular nanofibers in diameter about 20 nm were constructed by self-assembly. The nanofibers showed solid-state fluorescence between 400 nm and 650 nm with the maximum emission at 495 nm. Furthermore, its recognition reaction with PNA lectin was investigated by fluorescence spectra and turbidity assays. It is interesting found that the supramolecular assembly as multivalent glycocluster exhibited unique and selectively binding interactions with PNA lectin with the binding constant of 5.74 × 106 M-1. Moreover, compound Py-Lac showed two-photon fluorescence imaging with Hep G2 cells.

A fluorescent calixarene-based dimeric capsule constructed via a MII-terpyridine interaction: cage structure, inclusion properties and drug release.

Two analogues of capsule-like fluorescent cages have been constructed by dimerization of terpyridine-containing calixarene derivatives utilizing a MII-terpyridine (M = Zn and Cd) interaction. 1H NMR spectral studies show that the self-assembled molecular capsules Zn4L12 and Cd4L12 have a highly symmetrical D 4h-structure. The encapsulation of the anticancer drug mercaptopurine in their cavities has been documented by NMR, ESI-TOF-MS, fluorescence switching, and molecular simulation, indicating that strong S-π and π-π interactions between drug and cage are of importance for the host-guest binding. The nanoscale cages exhibit excellent behaviors to control the release of mercaptopurine in phosphate buffered saline solution (pH = 7.4). These results further highlight the potential of self-assembled Zn4L12 cages for drug-carrier applications.

Immunogenicity and protective efficacy of recombinant Bacille Calmette-Guerin strains expressing mycobacterium antigens Ag85A, CFP10, ESAT-6, GM-CSF and IL-12p70.

OBJECTIVE: This study aimed to evaluate the immunogenicity and protective efficacy of recombinant bacille calmette-guerin (rBCG) strains expressing Ag85A (A), CFP10 (C), ESAT6 (E), IL-12p70 (I), and fusion protein GM-CSF (G). METHOD: rBCGs were established by integrating of A, C, E, I, G, AE, CE, IE, GC, GE and GCE into Mycobacterium bovis BCG-1173 and BCG-SH. The macro-effects of rBCGs on mice were evaluated by phenotype and weight. The immunogenicity of rBCGs was analyzed by lgG, lgG1 and lgG2a antibody titers, and IFN-γ and IL-4 contents through Enzyme-linked immunosorbent assay (ELISA). Meanwhile, the proportions of CD4+ and CD8+ T splenic lymphocytes were determined using flow cytometry. The protective efficacy of rBCGs was evaluated by bacterial load in spleen and lung tissues from immunized mice. RESULTS: rBCGs exhibited no obvious side effects on mice. The antibody titers of lgG, lgG1 and lgG2a, proportion of CD4+ and CD8+ T cells, and concentrations of IFN-γ were found to be significantly higher in multiple-gene rBCGs than that in single-gene rBCGs (P < 0.05). Bacterial load in both spleen and lung tissues from mice infected with M. tuberculosis H37Rv were significantly reduced by rBCGs. A significantly lower bacterial load was revealed in rBCG-1173:A compared with multiple-gene rBCGs (P < 0.05). CONCLUSION: Immunogenicity was better on multiple-gene rBCGs than on single-gene rBCGs, while excellent protective efficacy was exhibited on rBCG-1173:A and BCG-1173.

Commonly administered bacille Calmette-Guerin strains induce comparable immune response.

Bacille Calmette-Guerin (BCG) is currently the only available vaccine against tuberculosis (TB), but its protective efficacy in adults is highly variable. This study aimed to compare the immune response induced by two widely used BCG strains: BCG China strain (derivative of BCG Danish strain) in DU2-III group and BCG Pasteur in DU2 -IV group. Healthy BALB/c mice were immunized with BCG China strain or BCG Pasteur strain. Specific IgG, IgG1, and IgG2a antibodies titers, the proliferation of splenocytes, the percentages of splenocyte subsets and the concentrations of induced IFN-γ and IL-4 at 6(th), 8(th), 10(th), and 12(th) weeks after the immunization were detected. We found that BCG Pasteur strain induced higher specific IgG and IgG1 titers, higher proliferation of splenocytes, higher percentages of CD4(+) or CD8(+) T cells, and higher concentration of secreted IFN-γ than BCG China strain. However, there were no significant differences in IgG2a titer and IL-4 concentration between both strains. In conclusion, our study shows that immune responses to BCG vaccine differ by strain, which may account for variable outcomes of BCG immunization.

Resveratrol synthesis under natural conditions and after UV-C irradiation in berry skin is associated with berry development stages in 'Beihong' (V. vinifera×V. amurensis).

Though UV-C irradiation may induce the synthesis of resveratrol in grape berry skins, it is unclear which developmental stage is most sensitive to UV-C induction. In this study, we investigated resveratrol content and gene expression of 'Beihong' (Vitisvinifera×Vitisamurensis) berries by UV-C irradiation compared with under natural conditions during different developmental stages. Under natural condition, resveratrol synthesis was related to veraison. Before veraison, resveratrol content was very low; however, total resveratrol increased about 500% from veraison to maturity in the main forms of piceids. UV-C irradiation significantly stimulated resveratrol synthesis, mainly in the form of trans-resveratrol. Young berries at 55days after anthesis were most sensitive to UV-C irradiation, the total resveratrol in the skin of UV-C irradiated berry is about 90 times higher than that of the control; however, resveratrol synthesis subsequently declined, which may be attributed to STS regulation by Myb14.

Transforming growth factor-ß (TGF-ß) induces the expression of chondrogenesis-related genes through TGF-ß receptor II (TGFRII)-AKT-mTOR signaling in primary cultured mouse precartilaginous stem cells.

Precartilaginous stem cells (PSCs) are adult stem cells which could initiate chondrocytes and bone growth. In the current study, we purified PSCs from the neonate mice' perichondrial mesenchyme through immunomagnetic beads with the fibroblast growth factor receptor-3 (FGFR-3) antibody. Mouse PSCs were seeded and cultured, and their phenotype was confirmed by FGFR-3 over-expression. Transforming growth factor-ß (TGF-ß) was added to induce PSCs differentiation. TGF-ß increased mRNA expression of chondrogenesis-related genes (collagen type II, Sox 9, and aggrecan) in the cultured PSCs, which was abolished by TGF-ß receptor II (TGFRII) lentiviral shRNA depletion. TGF-ß induced AKT activation in mouse PSCs, while the PI3K/AKT inhibitor (LY294002) and the AKT specific inhibitors (perifosine and MK-2206) largely suppressed TGF-ß-induced collagen II, Sox 9, and aggrecan mRNA expression. Meanwhile, the mTOR complex 1 (mTORC1) blocker RAD001 or the mTORC1/2 dual inhibitor AZD-2014 also alleviated TGF-ß-induced chondrogenesis-associated genes expression. Further, lentiviral shRNA depletion of SIN1 (a mTORC2 component) or mTOR inhibited TGF-ß's effect in the mouse PSCs. In conclusion, our evidence suggests that TGF-ß induces the expression of chondrogenesis-related genes through TGFRII-AKT-mTOR signaling in cultured mouse PSCs.

Analysis of nine compounds from Alpinia oxyphylla fruit at different harvest time using UFLC-MS/MS and an extraction method optimized by orthogonal design.

BACKGROUND: The dried fruits of Alpinia oxyphylla Miq have been widely used as an herbal medicine for the treatment of diarrhea and enuresis in China. Medicinal chemistry studies revealed that the tepenes, diphenylheptanes and flavones were the main components. Therefore, these three kinds of components should be chosen as the bioactive marker compounds for the quality control of A. oxyphylla fruits. Moreover, multiple active components has been widely recognized to be a more feasible method for the quality control of herbal medicines. This study firstly provided a better method for comprehensive component analysis of A. oxyphylla fruits. Meanwhile, the different harvest time was also evaluated. RESULTS: The solvent-to-sample ratio was the most important factor comparing with solvent, extraction time and temperature. The highest yield of nine compounds was achieved with 70% ethanol-water and a solvent-to-sample ratio of 20:1 at 60°C for 30 min. The optimized analytical method for ultra fast high performance liquid chromatography (UFLC) was a gradient elution using water containing 0.04‰ formic acid (A) and methanol containing 0.04‰ formic acid (B), at a flow rate of 0.3 mL/min. Using this optimized method, nine compounds were simultaneously separated and quantified by UFLC coupled with tandem electro-spray ionized mass spectrometry (MS/MS). CONCLUSIONS: The contents of the six bioactive compounds were reported in A. oxyphylla for the first time. The contents of nine compounds of different harvest time fruits of A. oxyphylla were assessed under the optimized extraction and UFLC-MS/MS analytical conditions, and the 45-day culture fruit had the highest content levels.

Transcriptomic analysis of grape (Vitis vinifera L.) leaves during and after recovery from heat stress.

BACKGROUND: Grapes are a major fruit crop around the world. Heat stress can significantly reduce grape yield and quality. Changes at the molecular level in response to heat stress and subsequent recovery are poorly understood. To elucidate the effect of heat stress and subsequent recovery on expression of genes by grape leaves representing the classic heat stress response and thermotolerance mechanisms, transcript abundance of grape (Vitis vinifera L.) leaves was quantified using the Affymetrix Grape Genome oligonucleotide microarray (15,700 transcripts), followed by quantitative Real-Time PCR validation for some transcript profiles. RESULTS: We found that about 8% of the total probe sets were responsive to heat stress and/or to subsequent recovery in grape leaves. The heat stress and recovery responses were characterized by different transcriptional changes. The number of heat stress-regulated genes was almost twice the number of recovery-regulated genes. The responsive genes identified in this study belong to a large number of important traits and biological pathways, including cell rescue (i.e., antioxidant enzymes), protein fate (i.e., HSPs), primary and secondary metabolism, transcription factors, signal transduction, and development. We have identified some common genes and heat shock factors (HSFs) that were modulated differentially by heat stress and recovery. Most HSP genes were upregulated by heat stress but were downregulated by the recovery. On the other hand, some specific HSP genes or HSFs were uniquely responsive to heat stress or recovery. CONCLUSION: The effect of heat stress and recovery on grape appears to be associated with multiple processes and mechanisms including stress-related genes, transcription factors, and metabolism. Heat stress and recovery elicited common up- or downregulated genes as well as unique sets of responsive genes. Moreover, some genes were regulated in opposite directions by heat stress and recovery. The results indicated HSPs, especially small HSPs, antioxidant enzymes (i.e., ascorbate peroxidase), and galactinol synthase may be important to thermotolerance of grape. HSF30 may be a key regulator for heat stress and recovery, while HSF7 and HSF1 may only be specific to recovery. The identification of heat stress or recovery responsive genes in this study provides novel insights into the molecular basis for heat tolerance in grape leaves.

Photosynthetic responses to heat treatments at different temperatures and following recovery in grapevine (Vitis amurensis L.) leaves.

BACKGROUND: The electron transport chain, Rubisco and stomatal conductance are important in photosynthesis. Little is known about their combined responses to heat treatment at different temperatures and following recovery in grapevines (Vitis spp.) which are often grown in climates with high temperatures. METHODOLOGY/FINDINGS: The electron transport function of photosystem II, the activation state of Rubisco and the influence of stomatal behavior were investigated in grapevine leaves during heat treatments and following recovery. High temperature treatments included 35, 40 and 45°C, with 25°C as the control and recovery temperature. Heat treatment at 35°C did not significantly (P>0.05) inhibit net photosynthetic rate (P(n)). However, with treatments at 40 and 45°C, P(n) was decreased, accompanied by an increase in substomatal CO(2) concentration (C(i)), decreases in stomatal conductance (g(s)) and the activation state of Rubisco, and inhibition of the donor side and the reaction center of PSII. The acceptor side of PSII was inhibited at 45°C but not at 40°C. When grape leaves recovered following heat treatment, P(n), g(s) and the activation state of Rubisco also increased, and the donor side and the reaction center of PSII recovered. The increase in P(n) during the recovery period following the second 45°C stress was slower than that following the 40°C stress, and these increases corresponded to the donor side of PSII and the activation state of Rubisco. CONCLUSIONS: Heat treatment at 35°C did not significantly (P>0.05) influence photosynthesis. The decrease of P(n) in grape leaves exposed to more severe heat stress (40 or 45°C) was mainly attributed to three factors: the activation state of Rubisco, the donor side and the reaction center of PSII. However, the increase of P(n) in grape leaves following heat stress was also associated with a stomatal response. The acceptor side of PSII in grape leaves was responsive but less sensitive to heat stress.

[Study on social determinants and health-related quality of life among the 'empty nest' (lonely) elderly Chinese people].

OBJECTIVE: To study the social determinants and health-related quality of life among the Chinese 'empty nest' (lonely) elderly. METHODS: A total of 4995 elderly, aged 60 or over were recruited in rural and urban areas of Zhejiang province, under a cluster sampling method. Stepwise logistic regression analysis was performed to analyze the social determinants on the 'empty nest' elderly. Health-related quality of life was assessed by SF-36 and compared between the 'empty nest' and 'non-empty nest' elderly, using multiple linear regression analysis after adjusting for confounding factors. RESULTS: Comparing with the 'non-empty nest' elderly, the characteristics of the empty nest population were as follows: age at 70 to 79 (P = 0.0417), having higher education level (P < 0.0001), living in rural area (P < 0.0001), lower income level (P = 0.0178, P = 0.0049), with higher level of satisfaction of life (P = 0.0070, P = 0.0035) and lower level of depressive mood (P = 0.0371). 'Empty nest' elderly people had lower scores of SF-36 in BP (P = 0.0032)dimension of physical function, and RE (P = 0.0033) dimension of psychological function as well as MCS (P = 0.0342) after adjusting for age, gender, marriage status, education level, residential areas, personal income, scores for satisfaction of life and depressive mood. CONCLUSION: The 'empty nest' elderly people with the characteristics such as higher education level, lower income level and living in the rural areas should be under greater concern, especially on those people with lower health-related quality of life or poorer psychological function.

[Factors associated with attitudes toward tobacco control policy in public places among adults in three counties of China].

OBJECTIVE: vestigate the attitudes of urban and rural community members toward total banning on smoking in public places and to explore the factors associated with these attitudes, in three counties/cities in China. METHODS: A cross-sectional study was conducted in three counties/cities in 2004, including Xin' an county of Henan province, Anyi county of Jiangxi province, and Mianzhu city of Sichuan province. A total of 5642 residents at age of 18-69 years old were interviewed face-to-face with a uniform questionnaire by locally-trained interviewers, through a random three-stage stratified sampling in each county. Factors were assumed to be associated with attitudes, using chi-square test in univariate analysis and non-conditional logistic regression model in multivariate analysis. RESULTS: The prevalence of current smoking among respondents was 44.3%. 80.7% (1379/1709) of the current smokers reported smoking often or sometimes in public places. Only 9.6% (479/4983) of the respondents reported that their indoor workplaces had totally banned on smoking. 43.5% of the respondents supported a total smoking ban strategy in pubic venues. The results of multivariate logistic regression model showed that eight factors were significantly associated with support for the total smoking ban in public places included region, residency, age, gender, education, smoking status, awareness about passive smoking hazards, and hearing of any message on tobacco control through media differences of rates regarding the factors as: residents in urban to rural areas (OR = 1.29), elderly to youngsters (30-49 vs. 18-29, OR = 1.46; 50-69 vs. 18-29, OR = 1.71), female to male (OR = 1.27), high-educated to less-educated ones, quitters to current smokers (OR = 1.90), nonsmokers to current smokers (OR = 2.01). Those who know messages on health hazards of passive smoking (OR = 2.26), or heard of message on tobacco control through media (OR = 1.43). CONCLUSION: Results from our study revealed that a thorough smoke-free policy in public places should be developed and implemented in these three counties/cities.

[Investigation on emission characteristics of methanol vehicle's aldehyde and ketone pollutants].

Investigation on the character and quantity of aldehydes and ketones emitted from methanol vehicle was implemented respectively by using high performance liquid chromatograph (HPLC) method. Experiments of vehicles equipped with and without three-way catalyst (TWC) under transient and steady mode were carried out. The emission results were compared with that of gasoline vehicle. The data showed that the efficiency of aldehydes and ketones emitted from methanol vehicle and gasoline vehicle were 22.53% and 48.95% with TWC under transient state respectively. When the vehicle is fueled with methanol, the main emissions are formaldehyde, acetaldehyde, acraldehyde + acetone, and these three matters account for 97.18% of the total emission. This proportion is 39.07% when the vehicle is fueled with gasoline. However, the total quantity of aldehydes and ketones from methanol vehicle without TWC was less than that from gasoline vehicle without TWC. Whether with or without TWC, aldehydes and ketones from methanol vehicle were more than that from gasoline vehicle under steady states. When the vehicle is fueled with methanol, the emission of aldehydes and ketones is on the top level at the speed of 60 km/h, and the converting efficiency is also the highest, which is 88.50%. When at the speed of 60 km/h, 90 km/h, 120 km/h, the formaldehyde quantity of methanol vehicle was 332.94%, 374.47% and 357.58% as much as that from gasoline vehicle respectively.

[Unregulated emissions from the gasoline vehicle].

Based on the emission test cycle of China National Regulation Stage III, the aldehyde and alkone emissions and VOCs emissions of three typical gasoline cars were studied with HPLC and TD-GC/MS and the exhausted particulates number and mass concentration were researched using ELPI. The results indicate that the unregulated emissions of different cars is diverse changed, the brake specific emission of the carbonyls in three cars are 36.44, 16.71 and 10.43 mg/km respectively and TVOC are 155.39, 103.75 and 42.29 mg/km respectively. Formaldehyde, acetaldehyde, acrolein, acetone and cyclohexanone are the main compounds in gasoline cars exhaust, which accounted for 77.9%-89.7% of total carbonyl compounds. Aromatic hydrocarbons and alkane are the main part of VOCs, the detected number of which is occupied 31.6%-39.2% and 23.1%-27.9% of VOCs. Toluene, xylene and benzene have high concentration, which are occupied 16.68%, 16.87% and 5.23% of TVOC in average. Ultra-fine particles (< 100 nm) dominate the particulates emission. Exhausted particulate number of high speeds is higher than that of slow and medium speeds.

[The current status of passive smoking in Chinese families and associated factors].

OBJECTIVE: To understand the prevalence of passive smoking in Chinese families and discuss its associated factors, as to providing scientific evidence for establishing tobacco control measures in China. METHOD: Cross-sectional survey: from June to September, 2004, we randomly selected six counties in three different provinces ( Mianzhu and Xichong of Sichuan Province; Anyi and Hukou of Jiangxi Province; Xinan and Yanshi of Henan Province) and performed face-to-face questionnaire survey on citizens between 18 and 69 years old. All the data were double independently input by professional data entry company to ensure data accuracy. The prevalence of home passive smoking exposure in families with different demographic characteristics was described by using prevalence, and the possible correlated factors of home passive smoking exposure as independent variables, multiple factors were analyzed using Logistic Stepwise Regression Analysis method. RESULTS: The analysis on 8142 nonsmokers revealed that the rate of passive smoking was 28.42%, with 27.38% of male and 28.93% of female suffering from passive smoking. All 87.19% of the smokers would smoke in front of their families. As many as 42.14% of the nonsmokers would offer cigarettes to their guests, while about 46.82% of the nonsmokers would suggest smokers to smoke outdoor. Home restriction on tobacco was extremely rare and only 6.33% of all the families completely forbade smoking. Multivariate logistic regression analysis of non-conditions revealed that, there was a lower level of involuntary tobacco smoke exposure in female, older age group, lower education level, divorced, or widowed families. There was no difference in involuntary tobacco smoke exposure between town dwellers and county dwellers, but such difference did exist in different districts. CONCLUSION: The three provinces under investigation should have severe involuntary tobacco smoking exposure. Gender, age, literacy level, occupation and region should be all factors that influence the status of involuntary tobacco smoking exposure in different families. There is a high percentile that smokers would smoke in front of their families and kids and a relative low pressure against smoking from nonsmokers. Cigarette offering is very prevalence. The knowledge and attitude about passive smoking should be separated from the situation of passive smoking exposure.

Validity of diagnostic evidence for deceased cases in hospitals.

OBJECTIVE: To determine the validity of the diagnostic evidence for deceased cases in hospitals. METHODS: All information collected from medical records of the deceased cases in tertiary care health facilities was input into our database. Four diagnosis levels were determined based on level of diagnostic evidence: level I was based on autopsy, pathology or operative exploration, level II on physical and laboratory tests plus expert clinical judgment, level III on expert clinical judgment, level IV on postmortem assumptions. After the diagnostic evidence of each deceased case was reviewed by a panel of three experts, the diagnostic level of each diagnosis was determined. RESULTS: Among the 2102 medical cases for verbal autopsy study, only 26 (1.24%) afforded diagnostic evidence for level III. Among the level III evidence-based cases of death, the major causes of death were cardiovascular diseases, respiratory diseases, and gastroenterological diseases. According to some special symptoms and medical histories, these cases could be diagnosed by comprehensive clinical judgment. Only one case met the criteria for level IV. CONCLUSION: Level I diagnostic evidence is hard to attain in China because of the traditional concept and economic restriction. The causes for 2101 deaths can be validated by level II or III diagnostic evidence.