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1.  Herbal medicines for viral myocarditis 
Herbal medicines are being used for treating viral diseases including viral myocarditis, and many controlled trials have been done to investigate their efficacy.
To assess the effects of herbal medicines on clinical and indirect outcomes in patients with viral myocarditis.
Search strategy
We searched the Cochrane Central Register of Controlled Trials (CENTRAL) in The Cochrane Library Issue 3, 2009, MEDLINE (January 1966 - July 2009), EMBASE (January 1998 - July 2009), Chinese Biomedical Database (1979 - 2009), China National Knowledge Infrastructure (1979 - 2009), Chinese VIP Information (1989 - 2009), Chinese Academic Conference Papers Database and Chinese Dissertation Database (1980 - 2009), AMED (1985 - 2009), LILACS accessed in July 2009 and the trials register of the Cochrane Complementary Medicine Field. We handsearched Chinese journals and conference proceedings. No language restrictions were applied.
Selection criteria
Randomised controlled trials of herbal medicines (with a minimum of seven days treatment duration) compared with placebo, no intervention, or conventional interventions were included. Trials of herbal medicine plus conventional drug versus drug alone were also included. Only trials that reported adequate description of allocation sequence generation were included.
Data collection and analysis
Two review authors independently extracted data and evaluated trial quality. Adverse effects information was collected from the trials.
Main results
Fourteen randomised trials involving 1463 people were included. All trials were conducted and published in China. Quality of the trials was assessed to be low. No trial had diagnosis of viral myocarditis confirmed histologically, and only a few trials attempted to establish viral aetiology. Nine different herbal medicines were tested in the included trials. The trials reported electrocardiogram results, level of myocardial enzymes, cardiac function, symptoms, and adverse effects.
Astragalus membranaceus (either as an injection or granules) showed significant positive effects in symptom improvement, normalisation of electrocardiogram results, CPK levels, and cardiac function. Shengmai injection also showed significant effects in symptom improvement. Shengmai decoction triggered significant improvement in quality of life measured by SF-36. No serious adverse effects were reported.
Authors’ conclusions
Some herbal medicines may lead to improvement of symptoms, ventricular premature beat, electrocardiogram, level of myocardial enzymes, and cardiac function in viral myocarditis. However, interpretation of these findings should be taken with care due to the low methodological quality, small sample size, and limited number of trials on individual herbs. Further robust trials are needed to explore the use of herbal medicines in viral myocarditis.
PMCID: PMC3407372  PMID: 15266498
Astragalus membranaceus; Drug Combinations; Drugs, Chinese Herbal [adverse effects; *therapeutic use]; Myocarditis [*drug therapy; virology]; Phytotherapy [adverse effects; *methods]; Randomized Controlled Trials as Topic; Virus Diseases [*drug therapy]; Humans
2.  A genome-wide linkage scan for bone mineral density in an extended sample: evidence for linkage on 11q23 and Xq27 
Journal of Medical Genetics  2004;41(10):743-751.
Background: Osteoporosis is a major public health problem, mainly quantified by low bone mineral density (BMD). The majority of BMD variation is determined by genetic effects. A pilot whole genome linkage scan (WGS) was previously reported in 53 white pedigrees with 630 subjects. Several genomic regions were suggested to be linked to BMD variation.
Objective: To substantiate these previous findings and detect new genomic regions.
Methods: A WGS was conducted on an extended sample where the size was almost tripled (1816 subjects from 79 pedigrees). All the subjects were genotyped with 451 microsatellite markers spaced ∼8.1 cM apart across the human genome. Two point and multipoint linkage analyses were carried out using the variance component method.
Results: The strongest linkage signal was obtained on Xq27 with two point LOD scores of 4.30 for wrist BMD, and 2.57 for hip BMD, respectively. Another important region was 11q23, which achieved a maximum LOD score of 3.13 for spine BMD in multipoint analyses, confirming the results on this region in two earlier independent studies. Suggestive linkage evidence was also found on 7p14 and 20p12.
Conclusions: Together with the findings from other studies, the current study has further delineated the genetic basis of bone mass and highlights the importance of increasing sample size to confirm linkage findings and to identify new regions of linkage.
PMCID: PMC1735607  PMID: 15466007
3.  A suite of web-based programs to search for transcriptional regulatory motifs 
Nucleic Acids Research  2004;32(Web Server issue):W204-W207.
The identification of regulatory motifs is important for the study of gene expression. Here we present a suite of programs that we have developed to search for regulatory sequence motifs: (i) BioProspector, a Gibbs-sampling-based program for predicting regulatory motifs from co-regulated genes in prokaryotes or lower eukaryotes; (ii) CompareProspector, an extension to BioProspector which incorporates comparative genomics features to be used for higher eukaryotes; (iii) MDscan, a program for finding protein–DNA interaction sites from ChIP-on-chip targets. All three programs examine a group of sequences that may share common regulatory motifs and output a list of putative motifs as position-specific probability matrices, the individual sites used to construct the motifs and the location of each site on the input sequences. The web servers and executables can be accessed at
PMCID: PMC441599  PMID: 15215381
4.  Microglial activation by uptake of fDNA via a scavenger receptor 
Journal of neuroimmunology  2004;147(0):50-55.
The fate of the fragmented DNA (fDNA) observed in neuronal nuclei in Alzheimer brain is unknown. However, its fate is suggested as fDNA is found in the cytoplasm of adjacent activated microglia. After a brief incubation with fDNA, approximately 70% of microglia had fDNA in their cytoplasm, were activated, and overexpressed interleukin-1β. Microglial activation enhanced uptake whereas blocking scavenger receptors suppressed this uptake. These results suggest that the brain rids itself of fDNA from dying neurons through microglial uptake, activation, and overexpression of IL-1. Such overexpression of IL-1 in Alzheimer brain has been linked to Alzheimer pathogenesis.
PMCID: PMC3846353  PMID: 14741427
Alzheimer’s disease; Cytokines flow cytometry; fDNA; Microglia; Scavenger receptor; TUNEL
5.  Genetic Structure of the Endangered Plant Neolitsea sericea (Lauraceae) from the Zhoushan Archipelago Using RAPD Markers 
Annals of Botany  2004;95(2):305-313.
• Background and Aims The Zhoushan archipelago is the largest archipelago in China. It separated from the mainland about 9000 years ago due to rising sea levels and climate change. Because of the long-term influences of human activities, the original forest vegetation on the large islands has been badly damaged and its plant diversity reduced.
• Methods Levels and patterns of genetic diversity in 114 individuals from six natural populations and four cultivated populations of the insular endangered plant Neolitsea sericea (Lauraceae) on the Zhoushan archipelago were assessed using random amplified polymorphic DNA (RAPD) markers.
• Key Results A total of 99 discernible loci were obtained for all populations using ten primers, 50·5 % of which were polymorphic [percentage of polymorphic bands (PPB) = 50·5 %]. Despite being a woody, long-lived, perennial, outcrossing and insect-pollinated plant, N. sericea exhibited low levels of genetic variation. The cultivated populations (PPB = 18·9 %, HE = 0·060, S = 0·092) were genetically less diverse than the natural populations (PPB = 23·1 %, HE = 0·082, S = 0·123). Based on analysis of molecular variance, a high degree of among-population differentiation was revealed for both natural (0·387) and cultivated populations (0·598).
• Conclusions Removal of plants from the wild for horticulture purposes has eroded the level of genetic variation of N. sericea. Low levels of genetic diversity and a high degree of population differentiation indicate that management strategies should include conservation of natural habitats occupied by all six wild populations, and sampling of germplasm resources from multiple seed sources.
PMCID: PMC4246830  PMID: 15546928
Conservation management; endangered plant; genetic structure; Neolitsea sericea; RAPD; Zhoushan archipelago
6.  CD24 Controls Expansion and Persistence of Autoreactive T Cells in the Central Nervous System during Experimental Autoimmune Encephalomyelitis 
In the development of experimental autoimmune encephalomyelitis (EAE), a model for multiple sclerosis (MS), autoreactive T cells must be activated and clonally expand in the lymphoid organs, and then migrate into the central nervous system (CNS) where they undergo further activation. It is unclear whether the autoreactive T cells further expand in the CNS and if so, what interactions are required for this process. We have demonstrated previously that expression by the host cells of the heat-stable antigen (CD24), which was recently identified as a genetic modifier for MS, is essential for their susceptibility to EAE. Here we show that CD24 is essential for local clonal expansion and persistence of T cells after their migration into the CNS, and that expression of CD24 on either hematopoietic cells or nonhematopoietic antigen-presenting cells in the recipient is sufficient to confer susceptibility to EAE.
PMCID: PMC2211938  PMID: 15314074
costimulatory molecules; autoimmune diseases; central nervous system; multiple sclerosis; clonal expansion
7.  MARA: a novel approach for highly multiplexed locus-specific SNP genotyping using high-density DNA oligonucleotide arrays 
Nucleic Acids Research  2004;32(22):e181.
We have developed a locus-specific DNA target preparation method for highly multiplexed single nucleotide polymorphism (SNP) genotyping called MARA (Multiplexed Anchored Runoff Amplification). The approach uses a single primer per SNP in conjunction with restriction enzyme digested, adapter-ligated human genomic DNA. Each primer is composed of common sequence at the 5′ end followed by locus-specific sequence at the 3′ end. Following a primary reaction in which locus-specific products are generated, a secondary universal amplification is carried out using a generic primer pair corresponding to the oligonucleotide and genomic DNA adapter sequences. Allele discrimination is achieved by hybridization to high-density DNA oligonucleotide arrays. Initial multiplex reactions containing either 250 primers or 750 primers across nine DNA samples demonstrated an average sample call rate of ∼95% for 250- and 750-plex MARA. We have also evaluated >1000- and 4000-primer plex MARA to genotype SNPs from human chromosome 21. We have identified a subset of SNPs corresponding to a primer conversion rate of ∼75%, which show an average call rate over 95% and concordance >99% across seven DNA samples. Thus, MARA may potentially improve the throughput of SNP genotyping when coupled with allele discrimination on high-density arrays by allowing levels of multiplexing during target generation that far exceed the capacity of traditional multiplex PCR.
PMCID: PMC545476  PMID: 15601992
8.  Melatonin promoted chemotaxins expression in lung epithelial cell stimulated with TNF-α 
Respiratory Research  2004;5(1):20.
Patients with asthma demonstrate circadian variations in the airway inflammation and lung function. Pinealectomy reduces the total inflammatory cell number in the asthmatic rat lung. We hypothesize that melatonin, a circadian rhythm regulator, may modulate the circadian inflammatory variations in asthma by stimulating the chemotaxins expression in the lung epithelial cell.
Lung epithelial cells (A549) were stimulated with melatonin in the presence or absence of TNF-α(100 ng/ml). RANTES (Regulated on Activation Normal T-cells Expressed and Secreted) and eotaxin expression were measured using ELISA and real-time RT-PCR, eosinophil chemotactic activity (ECA) released by A549 was measured by eosinophil chemotaxis assay.
TNF-α increased the expression of RANTES (307.84 ± 33.56 versus 207.64 ± 31.27 pg/ml of control, p = 0.025) and eotaxin (108.97 ± 10.87 versus 54.00 ± 5.29 pg/ml of control, p = 0.041). Melatonin(10-10 to 10-6M) alone didn't change the expression of RNATES (204.97 ± 32.56 pg/ml) and eotaxin (55.28 ± 6.71 pg/ml). However, In the presence of TNF-α (100 ng/ml), melatonin promoted RANTES (410.88 ± 52.03, 483.60 ± 55.37, 559.92 ± 75.70, 688.42 ± 95.32, 766.39 ± 101.53 pg/ml, treated with 10-10, 10-9, 10-8, 10-7,10-6M melatonin, respectively) and eotaxin (151.95 ± 13.88, 238.79 ± 16.81, 361.62 ± 36.91, 393.66 ± 44.89, 494.34 ± 100.95 pg/ml, treated with 10-10, 10-9, 10-8, 10-7, 10-6M melatonin, respectively) expression in a dose dependent manner in A549 cells (compared with TNF-α alone, P < 0.05). The increased release of RANTES and eotaxin in A549 cells by above treatment were further confirmed by both real-time RT-PCR and the ECA assay.
Taken together, our results suggested that melatonin might synergize with pro-inflammatory cytokines to modulate the asthma airway inflammation through promoting the expression of chemotaxins in lung epithelial cell.
PMCID: PMC533859  PMID: 15537425
melatonin; TNF-α; chemotaxin; lung epithelia cell
9.  Activation of Transcription Factor Nrf-2 and Its Downstream Targets in Response to Moloney Murine Leukemia Virus ts1-Induced Thiol Depletion and Oxidative Stress in Astrocytes 
Journal of Virology  2004;78(21):11926-11938.
The neuroimmunodegenerative syndrome that develops in mice infected with ts1, a mutant of Moloney murine leukemia virus, resembles human AIDS. Both ts1 and human immunodeficiency virus type 1 infect astrocytes, microglia, and oligodendrocytes but do not infect neurons. Oxidative stress has been implicated in the neuropathology of AIDS dementia and other neurodegenerative diseases. We report here that ts1 infection of astrocytes (both transformed C1 cells and primary cultures) also induces thiol (i.e., glutathione and cysteine) depletion and reactive oxygen species (ROS) accumulation, events occurring in parallel with viral envelope precursor gPr80env accumulation and upregulated expression of endoplasmic reticulum chaperones GRP78 and GRP94. Furthermore, ts1-infected astrocytes mobilize their thiol redox defenses by upregulating levels of the Nrf-2 transcription factor, as well its targets, the xCT cystine/glutamate antiporter, γ-glutamylcysteine ligase, and glutathione peroxidase. Depleting intracellular thiols by treating uninfected astrocytes with buthionine sulfoximine (BSO), a glutathione synthesis inhibitor, or by culturing in cystine-deficient medium, also induces ROS accumulation, activates Nrf-2, and upregulates Nrf-2 target gene expression in these astrocytes. Overexpression of Nrf-2 in astrocytes specifically increases expression of the above thiol synthesis-related proteins. Further treatment with BSO or N-acetylcysteine in transfected cells modulates this expression. Thiol depletion also accelerates cell death, while thiol supplementation promotes survival of ts1-infected cells. Together, our results indicate that ts1 infection of astrocytes, along with ts1-induced gPr80env accumulation, endoplasmic reticulum stress, thiol depletion, and oxidative stress, accelerates cell death; in response to the thiol depletion and oxidative stress, astrocytes activate their Nrf-2-mediated thiol antioxidant defenses, promoting cell survival.
PMCID: PMC523278  PMID: 15479833
10.  Mutational dynamics of the SARS coronavirus in cell culture and human populations isolated in 2003 
The SARS coronavirus is the etiologic agent for the epidemic of the Severe Acute Respiratory Syndrome. The recent emergence of this new pathogen, the careful tracing of its transmission patterns, and the ability to propagate in culture allows the exploration of the mutational dynamics of the SARS-CoV in human populations.
We sequenced complete SARS-CoV genomes taken from primary human tissues (SIN3408, SIN3725V, SIN3765V), cultured isolates (SIN848, SIN846, SIN842, SIN845, SIN847, SIN849, SIN850, SIN852, SIN3408L), and five consecutive Vero cell passages (SIN2774_P1, SIN2774_P2, SIN2774_P3, SIN2774_P4, SIN2774_P5) arising from SIN2774 isolate. These represented individual patient samples, serial in vitro passages in cell culture, and paired human and cell culture isolates. Employing a refined mutation filtering scheme and constant mutation rate model, the mutation rates were estimated and the possible date of emergence was calculated. Phylogenetic analysis was used to uncover molecular relationships between the isolates.
Close examination of whole genome sequence of 54 SARS-CoV isolates identified before 14th October 2003, including 22 from patients in Singapore, revealed the mutations engendered during human-to-Vero and Vero-to-human transmission as well as in multiple Vero cell passages in order to refine our analysis of human-to-human transmission. Though co-infection by different quasipecies in individual tissue samples is observed, the in vitro mutation rate of the SARS-CoV in Vero cell passage is negligible. The in vivo mutation rate, however, is consistent with estimates of other RNA viruses at approximately 5.7 × 10-6 nucleotide substitutions per site per day (0.17 mutations per genome per day), or two mutations per human passage (adjusted R-square = 0.4014). Using the immediate Hotel M contact isolates as roots, we observed that the SARS epidemic has generated four major genetic groups that are geographically associated: two Singapore isolates, one Taiwan isolate, and one North China isolate which appears most closely related to the putative SARS-CoV isolated from a palm civet. Non-synonymous mutations are centered in non-essential ORFs especially in structural and antigenic genes such as the S and M proteins, but these mutations did not distinguish the geographical groupings. However, no non-synonymous mutations were found in the 3CLpro and the polymerase genes.
Our results show that the SARS-CoV is well adapted to growth in culture and did not appear to undergo specific selection in human populations. We further assessed that the putative origin of the SARS epidemic was in late October 2002 which is consistent with a recent estimate using cases from China. The greater sequence divergence in the structural and antigenic proteins and consistent deletions in the 3' – most portion of the viral genome suggest that certain selection pressures are interacting with the functional nature of these validated and putative ORFs.
PMCID: PMC517714  PMID: 15347429
11.  Normal reference value of hemoglobin of middleaged women and altitude. 
AIM: In order to supply a scientific basis for uniting the normal reference value standard of hemoglobin of Chinese middlescent women. METHODS: A research is made about the relationship between the normal reference value of 25,917 examples of hemoglobin of middlescent women and altitude in 268 areas in China, the normal reference values of hemoglobin of middlescent women are determined by the hemoglobincyanide method. RESULTS: The correlation between the normal reference value of hemoglobin of middlescent women and altitude is quite significant (r = 0.827). By using the method of univariate linear regression analysis, one regression equation is inferred. CONCLUSION: If the altitude values are obtained in some areas, the normal reference value of hemoglobin of middlescent women of this area can be reckoned by using the regression equation. Furthermore, depending on the altitude, China can be divided into three districts: Qingzang District, Central District, and Eastern District.
PMCID: PMC2259123  PMID: 15989740
12.  BOD: a customizable bioinformatics on demand system accommodating multiple steps and parallel tasks 
Nucleic Acids Research  2004;32(14):4175-4181.
The integration of bioinformatics resources worldwide is one of the major concerns of the biological community. We herein established the BOD (Bioinformatics on demand) system to use Grid computing technology to set up a virtual workbench via a web-based platform, to assist researchers performing customized comprehensive bioinformatics work. Users will be able to submit entire search queries and computation requests, e.g. from DNA assembly to gene prediction and finally protein folding, from their own office using the BOD end-user web interface. The BOD web portal parses the user's job requests into steps, each of which may contain multiple tasks in parallel. The BOD task scheduler takes an entire task, or splits it into multiple subtasks, and dispatches the task or subtasks proportionally to computation node(s) associated with the BOD portal server. A node may further split and distribute an assigned task to its sub-nodes using a similar strategy. In the end, the BOD portal server receives and collates all results and returns them to the user. BOD uses a pipeline model to describe the user's submitted data and stores the job requests/status/results in a relational database. In addition, an XML criterion is established to capture task computation program details.
PMCID: PMC514377  PMID: 15302917
13.  Vibrio vulnificus in Taiwan 
Emerging Infectious Diseases  2004;10(8):1363-1368.
Clinical features of 84 patients with V. vulnificus infection are analyzed and molecular features of isolates are described.
Residents in Taiwan are often exposed to marine microorganisms through seafood and occupational exposure. The number of reported cases of infection attributable to this organism has increased since the first case was reported in 1985. The increasing number of cases may be caused by greater disease activity or improved recognition by clinicians or laboratory workers. We analyze a clinical-case series of 84 patients with Vibrio vulnificus infection from 1995 to 2000 and describe the molecular epidemiologic features of pathogens isolated from these patients. The spectrum of clinical manifestations and outcomes, options of antimicrobial therapy, and virulence mechanisms were investigated. Results of molecular typing of isolates from humans and marine environment in this country had a high genetic divergence among these isolates. Education and measures are needed to prevent this emerging disease.
PMCID: PMC3320410  PMID: 15496235
Vibrio vulnificus; Taiwan; emerging; research
14.  Inhibition of Severe Acute Respiratory Syndrome Coronavirus Replication by Niclosamide 
Antiviral agents are urgently needed to fight severe acute respiratory syndrome (SARS). We showed that niclosamide, an existing antihelminthic drug, was able to inhibit replication of a newly discovered coronavirus, SARS-CoV; viral antigen synthesis was totally abolished at a niclosamide concentration of 1.56 μM, as revealed by immunoblot analysis. Thus, niclosamide represents a promising drug candidate for the effective treatment of SARS-CoV infection.
PMCID: PMC434198  PMID: 15215127
16.  Simultaneous Deficiency in CD28 and STAT6 Results in Chronic Ectoparasite-Induced Inflammatory Skin Disease  
Infection and Immunity  2004;72(7):3706-3715.
A mouse lacking CD28, a T-cell costimulatory molecule, and STAT6, a transcription factor that mediates interleukin-4 (IL-4) signaling, was developed from parental CD28- and STAT6-deficient mice. STAT6/CD28−/− BALB/c mice that were 8 weeks old had a normal phenotype, and IL-4 production was induced following infection with nematode parasites. Unexpectedly, when they were between 4 and 8 months old, all mice examined spontaneously developed severe chronic dermatitis associated with pronounced numbers of Demodex ectoparasites. In addition, pronounced CD4 and CD8 T-cell infiltrates in the dermis and subcutaneous fat, increased serum immunoglobulin G2a levels, and lymphadenopathy associated with increased gamma interferon and IL-12 expression were observed. Single-knockout siblings lacking either CD28 or STAT6 had a phenotype similar to that of BALB/c wild-type controls. To distinguish whether the ectoparasite Demodex or the Th1 immunity was the proximal cause of the inflammatory skin disease, STAT6/CD28−/− mice were treated with a miticide that eliminated the ectoparasites. This treatment markedly reduced the severity of the dermatitis and the associated lymphoid infiltrates. These findings suggest that ubiquitous ectoparasites, which are generally considered to be commensal, may contribute to disease when specific molecules required for an effective Th2 response are blocked.
PMCID: PMC427407  PMID: 15213110
17.  Patterns of linkage disequilibrium and haplotype distribution in disease candidate genes 
BMC Genetics  2004;5:11.
The adequacy of association studies for complex diseases depends critically on the existence of linkage disequilibrium (LD) between functional alleles and surrounding SNP markers.
We examined the patterns of LD and haplotype distribution in eight candidate genes for osteoporosis and/or obesity using 31 SNPs in 1,873 subjects. These eight genes are apolipoprotein E (APOE), type I collagen α1 (COL1A1), estrogen receptor-α (ER-α), leptin receptor (LEPR), parathyroid hormone (PTH)/PTH-related peptide receptor type 1 (PTHR1), transforming growth factor-β1 (TGF-β1), uncoupling protein 3 (UCP3), and vitamin D (1,25-dihydroxyvitamin D3) receptor (VDR). Yin yang haplotypes, two high-frequency haplotypes composed of completely mismatching SNP alleles, were examined. To quantify LD patterns, two common measures of LD, D' and r2, were calculated for the SNPs within the genes. The haplotype distribution varied in the different genes. Yin yang haplotypes were observed only in PTHR1 and UCP3. D' ranged from 0.020 to 1.000 with the average of 0.475, whereas the average r2 was 0.158 (ranging from 0.000 to 0.883). A decay of LD was observed as the intermarker distance increased, however, there was a great difference in LD characteristics of different genes or even in different regions within gene.
The differences in haplotype distributions and LD patterns among the genes underscore the importance of characterizing genomic regions of interest prior to association studies.
PMCID: PMC421754  PMID: 15157284
linkage disequilibrium (LD); haplotype; single nucleotide polymorphism (SNP)
18.  Balanced-PCR amplification allows unbiased identification of genomic copy changes in minute cell and tissue samples 
Nucleic Acids Research  2004;32(9):e76.
Analysis of genomic DNA derived from cells and fresh or fixed tissues often requires whole genome amplification prior to microarray screening. Technical hurdles to this process are the introduction of amplification bias and/or the inhibitory effects of formalin fixation on DNA amplification. Here we demonstrate a balanced-PCR procedure that allows unbiased amplification of genomic DNA from fresh or modestly degraded paraffin-embedded DNA samples. Following digestion and ligation of a target and a control genome with distinct linkers, the two are mixed and amplified in a single PCR, thereby avoiding biases associated with PCR saturation and impurities. We demonstrate genome-wide retention of allelic differences following balanced-PCR amplification of DNA from breast cancer and normal human cells and genomic profiling by array-CGH (cDNA arrays, 100 kb resolution) and by real-time PCR (single gene resolution). Comparison of balanced-PCR with multiple displacement amplification (MDA) demonstrates equivalent performance between the two when intact genomic DNA is used. When DNA from paraffin-embedded samples is used, balanced PCR overcomes problems associated with modest DNA degradation and produces unbiased amplification whereas MDA does not. Balanced-PCR allows amplification and recovery of modestly degraded genomic DNA for subsequent retrospective analysis of human tumors with known outcomes.
PMCID: PMC419625  PMID: 15155823
19.  Comparing gene discovery from Affymetrix GeneChip microarrays and Clontech PCR-select cDNA subtraction: a case study 
BMC Genomics  2004;5:26.
Several high throughput technologies have been employed to identify differentially regulated genes that may be molecular targets for drug discovery. Here we compared the sets of differentially regulated genes discovered using two experimental approaches: a subtracted suppressive hybridization (SSH) cDNA library methodology and Affymetrix GeneChip® technology. In this "case study" we explored the transcriptional pattern changes during the in vitro differentiation of human monocytes to myeloid dendritic cells (DC), and evaluated the potential for novel gene discovery using the SSH methodology.
The same RNA samples isolated from peripheral blood monocyte precursors and immature DC (iDC) were used for GeneChip microarray probing and SSH cDNA library construction. 10,000 clones from each of the two-way SSH libraries (iDC-monocytes and monocytes-iDC) were picked for sequencing. About 2000 transcripts were identified for each library from 8000 successful sequences. Only 70% to 75% of these transcripts were represented on the U95 series GeneChip microarrays, implying that 25% to 30% of these transcripts might not have been identified in a study based only on GeneChip microarrays. In addition, about 10% of these transcripts appeared to be "novel", although these have not yet been closely examined. Among the transcripts that are also represented on the chips, about a third were concordantly discovered as differentially regulated between iDC and monocytes by GeneChip microarray transcript profiling. The remaining two thirds were either not inferred as differentially regulated from GeneChip microarray data, or were called differentially regulated but in the opposite direction. This underscores the importance both of generating reciprocal pairs of SSH libraries, and of real-time RT-PCR confirmation of the results.
This study suggests that SSH could be used as an alternative and complementary transcript profiling tool to GeneChip microarrays, especially in identifying novel genes and transcripts of low abundance.
PMCID: PMC415544  PMID: 15113399
20.  CD4-Independent Infection of Astrocytes by Human Immunodeficiency Virus Type 1: Requirement for the Human Mannose Receptor 
Journal of Virology  2004;78(8):4120-4133.
Human immunodeficiency virus type 1 (HIV-1) infection occurs in the central nervous system and causes a variety of neurobehavioral and neuropathological disorders. Both microglia, the residential macrophages in the brain, and astrocytes are susceptible to HIV-1 infection. Unlike microglia that express and utilize CD4 and chemokine coreceptors CCR5 and CCR3 for HIV-1 infection, astrocytes fail to express CD4. Astrocytes express several chemokine coreceptors; however, the involvement of these receptors in astrocyte HIV-1 infection appears to be insignificant. In the present study using an expression cloning strategy, the cDNA for the human mannose receptor (hMR) was found to be essential for CD4-independent HIV-1 infectivity. Ectopic expression of functional hMR rendered U87.MG astrocytic cells susceptible to HIV-1 infection, whereas anti-hMR serum and hMR-specific siRNA blocked HIV-1 infection in human primary astrocytes. In agreement with these findings, hMR bound to HIV-1 virions via the abundant and highly mannosylated sugar moieties of HIV-1 envelope glycoprotein gp120 in a Ca2+-dependent fashion. Moreover, hMR-mediated HIV-1 infection was dependent upon endocytic trafficking as assessed by transmission electron microscopy, as well as inhibition of viral entry by endosomo- and lysosomotropic drugs. Taken together, these results demonstrate the direct involvement of hMR in HIV-1 infection of astrocytes and suggest that HIV-1 interaction with hMR plays an important role in HIV-1 neuropathogenesis.
PMCID: PMC374297  PMID: 15047828
21.  Genomic and Genetic Analysis of Bordetella Bacteriophages Encoding Reverse Transcriptase-Mediated Tropism-Switching Cassettes 
Journal of Bacteriology  2004;186(5):1503-1517.
Liu et al. recently described a group of related temperate bacteriophages that infect Bordetella subspecies and undergo a unique template-dependent, reverse transcriptase-mediated tropism switching phenomenon (Liu et al., Science 295: 2091-2094, 2002). Tropism switching results from the introduction of single nucleotide substitutions at defined locations in the VR1 (variable region 1) segment of the mtd (major tropism determinant) gene, which determines specificity for receptors on host bacteria. In this report, we describe the complete nucleotide sequences of the 42.5- to 42.7-kb double-stranded DNA genomes of three related phage isolates and characterize two additional regions of variability. Forty-nine coding sequences were identified. Of these coding sequences, bbp36 contained VR2 (variable region 2), which is highly dynamic and consists of a variable number of identical 19-bp repeats separated by one of three 5-bp spacers, and bpm encodes a DNA adenine methylase with unusual site specificity and a homopolymer tract that functions as a hotspot for frameshift mutations. Morphological and sequence analysis suggests that these Bordetella phage are genetic hybrids of P22 and T7 family genomes, lending further support to the idea that regions encoding protein domains, single genes, or blocks of genes are readily exchanged between bacterial and phage genomes. Bordetella bacteriophages are capable of transducing genetic markers in vitro, and by using animal models, we demonstrated that lysogenic conversion can take place in the mouse respiratory tract during infection.
PMCID: PMC344406  PMID: 14973019
22.  Current limitations of SNP data from the public domain for studies of complex disorders: a test for ten candidate genes for obesity and osteoporosis 
BMC Genetics  2004;5:4.
Public SNP databases are frequently used to choose SNPs for candidate genes in the association and linkage studies of complex disorders. However, their utility for such studies of diseases with ethnic-dependent background has never been evaluated.
To estimate the accuracy and completeness of SNP public databases, we analyzed the allele frequencies of 41 SNPs in 10 candidate genes for obesity and/or osteoporosis in a large American-Caucasian sample (1,873 individuals from 405 nuclear families) by PCR-invader assay. We compared our results with those from the databases and other published studies. Of the 41 SNPs, 8 were monomorphic in our sample. Twelve were reported for the first time for Caucasians and the other 29 SNPs in our sample essentially confirmed the respective allele frequencies for Caucasians in the databases and previous studies. The comparison of our data with other ethnic groups showed significant differentiation between the three major world ethnic groups at some SNPs (Caucasians and Africans differed at 3 of the 18 shared SNPs, and Caucasians and Asians differed at 13 of the 22 shared SNPs). This genetic differentiation may have an important implication for studying the well-known ethnic differences in the prevalence of obesity and osteoporosis, and complex disorders in general.
A comparative analysis of the SNP data of the candidate genes obtained in the present study, as well as those retrieved from the public domain, suggests that the databases may currently have serious limitations for studying complex disorders with an ethnic-dependent background due to the incomplete and uneven representation of the candidate SNPs in the databases for the major ethnic groups. This conclusion attests to the imperative necessity of large-scale and accurate characterization of these SNPs in different ethnic groups.
PMCID: PMC395827  PMID: 15113403
SNP Databases; Polymorphism; Obesity; Osteoporosis; Complex diseases; Ethnicity
23.  Development of Proteomic Patterns for Detecting Lung Cancer 
Disease Markers  2004;19(1):33-39.
Lung cancer is at present the number one cause of cancer death and no biomarker is available to detect early lung cancer in serum samples so far. The objective of this study is to find specific biomarkers for detection of lung cancer using Surface Enhanced Laser Desorption/Ionization (SELDI) technology. In this study, serum samples from 30 lung cancer patients and 51 age-and sex-matched healthy were analyzed by SELDI based ProteinChip reader, PBSII-C. The spectra were generated on WCX2 chips and protein peaks clustering and classification analyses were performed utilizing Biomarker Wizard and Biomarker Patterns software packages, respectively. Three protein peaks were automatically chosen for the system training and the development of a decision classification tree. The constructed model was then used to test an independent set of masked serum samples from 15 lung cancer patients and 31 healthy individuals. The analysis yielded a sensitivity of 93.3%, and a specificity of 96.7%. These results suggest that the serum is a capable resource for detection of specific lung cancer biomarkers. SELDI technique combined with an artificial intelligence classification algorithm can both facilitate the discovery of better biomarkers for lung cancer and provide a useful tool for molecular diagnosis in future.
PMCID: PMC3851077  PMID: 14757945
24.  Ciprofloxacin-resistant Salmonella enterica Typhimurium and Choleraesuis from Pigs to Humans, Taiwan 
Emerging Infectious Diseases  2004;10(1):60-68.
We evaluated the disk susceptibility data of 671 nontyphoid Salmonella isolates collected from different parts of Taiwan from March 2001 to August 2001 and 1,261 nontyphoid Salmonella isolates from the National Taiwan University Hospital from 1996 to 2001. Overall, ciprofloxacn resistance was found in 2.7% (18/671) of all nontyphoid Salmonella isolates, in 1.4% (5/347) of Salmonella enterica serotype Typhimurium and in 7.5% (8/107) in S. enterica serotype Choleraesuis nationwide. MICs of six newer fluoroquinolones were determined for the following isolates: 37 isolates of ciprofloxacin-resistant (human) S. enterica Typhimurium (N = 26) and Choleraesuis (N = 11), 10 isolates of ciprofloxacin-susceptible (MIC <1 μg/mL) (human) isolates of these two serotypes, and 15 swine isolates from S. enterica Choleraesuis (N = 13) and Typhmurium (N = 2) with reduced susceptibility to ciprofloxacin (MIC >0.12 μg/mL). Sequence analysis of the gryA, gyrB, parC, parE, and acrR genes, ciprofloxacin accumulation; and genotypes generated by pulsed-field gel electrophoresis with three restriction enzymes (SpeI, XbaI, and BlnI) were performed. All 26 S. enterica Typhimurium isolates from humans and pigs belonged to genotype I. For S. enterica Choleraesuis isolates, 91% (10/11) of human isolates and 54% (7/13) of swine isolates belonged to genotype B. These two genotypes isolates from humans all exhibited a high-level of resistance to ciprofloxacin (MIC 16–64 μg/mL). They had two-base substitutions in the gyrA gene at codons 83 (Ser83Phe) and 87 (Asp87Gly or Asp87Asn) and in the parC gene at codon 80 (Ser80Arg, Ser80Ile, or Ser84Lys). Our investigation documented that not only did these two S. enterica isolates have a high prevalence of ciprofloxacin resistance nationwide but also that some closely related ciprofloxacin-resistant strains are disseminated from pigs to humans.
PMCID: PMC3322755  PMID: 15078598
ciprofloxacin resistance; Salmonella enterica Typhimurium; Salmonella enterica Choleraesuis; Taiwan
25.  Dietary Glycemic Load and Breast Cancer Risk in the Women’s Health Study 
A diet with a high glycemic load (GL) may contribute to a metabolic environment that enhances tumorigenesis. Little is known, however, about whether high glycemic diets increase breast cancer risk in women. We examined the associations between baseline measurements of dietary GL and overall glycemic index (GI) and subsequent breast cancer in a cohort of 39,876 women, ages 45 years or older, participating in the Women’s Health Study. During a mean of 6.8 years of follow-up there were 946 confirmed cases of breast cancer. We found no association between dietary GL [multivariable-adjusted relative risk (RR), 1.01; confidence interval (CI), 0.76–1.35, comparing extreme quintiles; P for trend = 0.96] or overall GI (corresponding RR, 1.03; CI, 0.84–1.28; P for trend = 0.66) and breast cancer risk in the cohort as a whole. Exploratory analyses stratified by baseline measurements of menopausal status, physical activity, smoking history, alcohol use, and history of diabetes mellitus, hypertension, or hypercholesterolemia showed no significant associations, except in the subgroup of women who were premenopausal and reported low levels of physical activity (GL multivariable-adjusted RR, 2.35; CI, 1.03–5.37; P for trend = 0.07; GI multivariable-adjusted RR, 1.56; CI, 0.88–2.78; P for trend = 0.02, comparing extreme quintiles). Although we did not find evidence that a high glycemic diet increases overall breast cancer risk, the increase in risk in premenopausal women with low levels of physical activity suggests the possibility that the effects of a high glycemic diet may be modified by lifestyle and hormonal factors. Prospective studies of a larger sample size and longer duration are warranted to confirm our findings.
PMCID: PMC4166477  PMID: 14744735

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