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author:("Zhang, daping")
1.  Structure basis for the unique specificity of medaka enteropeptidase light chain 
Protein & Cell  2014;5(3):178-181.
PMCID: PMC3967055  PMID: 24481630
2.  Structure basis for the unique specificity of medaka enteropeptidase light chain 
Protein & Cell  2014;5(3):178-181.
PMCID: PMC3967055  PMID: 24481630
3.  Experience of Offering HIV Rapid Testing to At-Risk Patients in Community Health Centers in Eight Chinese Cities 
PLoS ONE  2014;9(1):e86609.
To explore the feasibility of offering HIV counseling and testing in community health centers (CHCs) and to provide evidence for the HIV/AIDS response in China.
Forty-two CHCs were selected from the eight cities that participated in the study. Rapid testing was mainly provided to: clients seeking HIV testing and counseling (HTC); outpatients with high-risk behavior of contracting HIV; inpatients and outpatients of key departments. Aggregate administrative data were collected in CHCs and general hospitals and differences between the two categories were compared.
There were 23,609 patients who underwent HIV testing, accounting for 0.37% of all estimated clinic visits at the 42 sites (0.03%–4.35% by site). Overall, positive screening prevalence was 0.41% (95% confidence interval [CI] 0.33%–0.49%, range 0.00%–0.98%), which is higher than in general hospitals (0.17%). The identification efficiency was 0.22% (95% CI: 0.16%–0.27%) in pilot CHCs, 3.5 times higher than in general hospitals (0.06%) (Chi square test = 95.196, p<0.001). The percentage of those receiving confirmatory tests among those who screened positive was slightly lower in CHCs (73.7%) than in general hospitals (80.1%) (Chi-square test = 17.472, p<0.001). Composition of clients mobilized for testing was consistent with the usage of basic public health and medical services in CHCs. The rate of patients testing HIV positive was higher among patients from key CHC departments (0.68%) than among high-risk Voluntary Counseling and Testing (VCT) clients (0.56%), those participating in outreach activities (0.41%), pregnant women (0.05%), and surgical patients (0.00%).
This project demonstrates that providing HIV testing services for patients who exhibit high risk behavior has a high HIV case detection rate and that CHCs have the capacity to integrate HTC into routine work. It provides concrete evidence supporting the involvement of CHCs in the expansion of HIV/AIDS testing and case finding.
PMCID: PMC3904922  PMID: 24489750
4.  Requirement of PEA3 for Transcriptional Activation of FAK Gene in Tumor Metastasis 
PLoS ONE  2013;8(11):e79336.
Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase critically involved in cancer metastasis. We found an elevation of FAK expression in highly metastatic melanoma B16F10 cells compared with its less metastatic partner B16F1 cells. Down-regulation of the FAK expression by either small interfering RNA or dominant negative FAK (FAK Related Non-Kinase, FRNK) inhibited the B16F10 cell migration in vitro and invasiveness in vivo. The mechanism by which FAK activity is up-regulated in highly metastatic cells remains unclear. In this study, we reported for the first time that one of the Est family proteins, PEA3, is able to transactivate FAK expression through binding to the promoter region of FAK. We identified a PEA3-binding site between nucleotides −170 and +43 in the FAK promoter that was critical for the responsiveness to PEA3. A stronger affinity of PEA3 to this region contributed to the elevation of FAK expression in B16F10 cells. Both in vitro and in vivo knockdown of PEA3 gene successfully mimicked the cell migration and invasiveness as that induced by FAK down-regulation. The activation of the well-known upstream of PEA3, such as epidermal growth factor, JNK, and ERK can also induce FAK expression. Furthermore, in the metastatic human clinic tumor specimens from the patients with human primary oral squamous cell carcinoma, we observed a strong positive correlation among PEA3, FAK, and carcinoma metastasis. Taking together, we hypothesized that PEA3 might play an essential role in the activation of the FAK gene during tumor metastasis.
PMCID: PMC3832605  PMID: 24260201
5.  Bacterial GRAS domain proteins throw new light on gibberellic acid response mechanisms 
Bioinformatics  2012;28(19):2407-2411.
Summary: Gibberellic acids (GAs) are key plant hormones, regulating various aspects of growth and development, which have been at the center of the ‘green revolution’. GRAS family proteins, the primary players in GA signaling pathways, remain poorly understood. Using sequence-profile searches, structural comparisons and phylogenetic analysis, we establish that the GRAS family first emerged in bacteria and belongs to the Rossmann fold methyltransferase superfamily. All bacterial and a subset of plant GRAS proteins are likely to function as small-molecule methylases. The remaining plant versions have lost one or more AdoMet (SAM)-binding residues while preserving their substrate-binding residues. We predict that GRAS proteins might either modify or bind small molecules such as GAs or their derivatives.
Supplementary Information: Supplementary Material for this article is available at Bioinformatics online.
PMCID: PMC3463117  PMID: 22829623
6.  Computational identification of novel biochemical systems involved in oxidation, glycosylation and other complex modifications of bases in DNA 
Nucleic Acids Research  2013;41(16):7635-7655.
Discovery of the TET/JBP family of dioxygenases that modify bases in DNA has sparked considerable interest in novel DNA base modifications and their biological roles. Using sensitive sequence and structure analyses combined with contextual information from comparative genomics, we computationally characterize over 12 novel biochemical systems for DNA modifications. We predict previously unidentified enzymes, such as the kinetoplastid J-base generating glycosyltransferase (and its homolog GREB1), the catalytic specificity of bacteriophage TET/JBP proteins and their role in complex DNA base modifications. We also predict the enzymes involved in synthesis of hypermodified bases such as alpha-glutamylthymine and alpha-putrescinylthymine that have remained enigmatic for several decades. Moreover, the current analysis suggests that bacteriophages and certain nucleo-cytoplasmic large DNA viruses contain an unexpectedly diverse range of DNA modification systems, in addition to those using previously characterized enzymes such as Dam, Dcm, TET/JBP, pyrimidine hydroxymethylases, Mom and glycosyltransferases. These include enzymes generating modified bases such as deazaguanines related to queuine and archaeosine, pyrimidines comparable with lysidine, those derived using modified S-adenosyl methionine derivatives and those using TET/JBP-generated hydroxymethyl pyrimidines as biosynthetic starting points. We present evidence that some of these modification systems are also widely dispersed across prokaryotes and certain eukaryotes such as basidiomycetes, chlorophyte and stramenopile alga, where they could serve as novel epigenetic marks for regulation or discrimination of self from non-self DNA. Our study extends the role of the PUA-like fold domains in recognition of modified nucleic acids and predicts versions of the ASCH and EVE domains to be novel ‘readers’ of modified bases in DNA. These results open opportunities for the investigation of the biology of these systems and their use in biotechnology.
PMCID: PMC3763556  PMID: 23814188
7.  Potent DNA damage by polyhalogenated quinones and H2O2 via a metal-independent and Intercalation-enhanced oxidation mechanism 
Scientific Reports  2013;3:1269.
Polyhalogenated quinones are a class of carcinogenic intermediates. We found recently that the highly reactive and biologically/environmentally important ·OH can be produced by polyhalogenated quinones and H2O2 independent of transition metal ions. However, it is not clear whether this unusual metal-independent ·OH producing system can induce potent oxidative DNA damage. Here we show that TCBQ and H2O2 can induce oxidative damage to both dG and dsDNA; but surprisingly, it was more efficient to induce oxidative damage in dsDNA than in dG. We found that this is probably due to its strong intercalating ability to dsDNA through competitive intercalation assays. The intercalation of TCBQ in dsDNA may lead to ·OH generation more adjacent to DNA. This is the first report that polyhalogenated quinoid carcinogens and H2O2 can induce potent DNA damage via a metal-independent and intercalation-enhanced oxidation mechanism, which may partly explain their potential genotoxicity, mutagenesis, and carcinogenicity.
PMCID: PMC3572447  PMID: 23429247
8.  Tolerability, pharmacokinetics and pharmacodynamics of CMAB007, a humanized anti-immunoglobulin E monoclonal antibody, in healthy Chinese subjects 
mAbs  2012;4(1):110-119.
The goal of the studies presented here was to determine the tolerability, pharmacokinetic and pharmacodynamic profiles of CMAB007, a biosimilar of omalizumab (Xolair; a humanized anti-immunoglobulin E monoclonal antibody), in healthy, male Chinese subjects. Thirty-six healthy Chinese men participated in two open-label, dose-escalation studies: 27 in a single-dose study (150, 300 or 600 mg) and 9 in a multiple-dose study (150 or 300 mg every 4 weeks for 20 weeks). The safety profiles of both studies were generally unremarkable. No drug-related adverse event was observed. CMAB007 exhibited a linear PK profile over the dose range of 150-600 mg. In the single-dose study, maximum concentration (Cmax) was reached within 6–8 d, and Cmax and area under concentration-time curve (AUC) increased linearly with the dose. In the multiple-dose study, steady-state appeared to have been achieved after the third dose. Css-max and AUCτ also showed dose-linearity. A dose-dependent suppression of free IgE was observed during treatment, as a median percentage change from baseline, 91.9–98.8%, in the three single-dose groups. No anti-CMAB007 antibodies were detected after dosing in any subject. Subcutaneous administration of CMAB007 was well-tolerated and seemed to be effective in reducing free IgE in healthy Chinese volunteers, which provides important information for further clinical studies.
PMCID: PMC3338945  PMID: 22327434
pharmacokinetics; pharmacodynamics; safety; IgE; humanized antibody
9.  Discovery of Novel DENN Proteins: Implications for the Evolution of Eukaryotic Intracellular Membrane Structures and Human Disease 
Frontiers in Genetics  2012;3:283.
The tripartite DENN module, comprised of a N-terminal longin domain, followed by DENN, and d-DENN domains, is a GDP-GTP exchange factor (GEFs) for Rab GTPases, which are regulators of practically all membrane trafficking events in eukaryotes. Using sequence and structure analysis we identify multiple novel homologs of the DENN module, many of which can be traced back to the ancestral eukaryote. These findings provide unexpected leads regarding key cellular processes such as autophagy, vesicle-vacuole interactions, chromosome segregation, and human disease. Of these, SMCR8, the folliculin interacting protein-1 and 2 (FNIP1 and FNIP2), nitrogen permease regulator 2 (NPR2), and NPR3 are proposed to function in recruiting Rab GTPases during different steps of autophagy, fusion of autophagosomes with the vacuole and regulation of cellular metabolism. Another novel DENN protein identified in this study is C9ORF72; expansions of the hexanucleotide GGGGCC in its first intron have been recently implicated in amyotrophic lateral sclerosis (ALS) and fronto-temporal dementia (FTD). While this mutation is proposed to cause a RNA-level defect, the identification of C9ORF72 as a potential DENN-type GEF raises the possibility that at least part of the pathology might relate to a specific Rab-dependent vesicular trafficking process, as has been observed in the case of some other neurological conditions with similar phenotypes. We present evidence that the longin domain, such as those found in the DENN module, are likely to have been ultimately derived from the related domains found in prokaryotic GTPase-activating proteins of MglA-like GTPases. Thus, the origin of the longin domains from this ancient GTPase-interacting domain, concomitant with the radiation of GTPases, especially of the Rab clade, played an important role in the dynamics of eukaryotic intracellular membrane systems.
PMCID: PMC3521125  PMID: 23248642
membrane trafficking; evolution; homology detection; DENN domain; longin domain; C9ORF72; ALS; FTD
10.  Novel transglutaminase-like peptidase and C2 domains elucidate the structure, biogenesis and evolution of the ciliary compartment 
Cell Cycle  2012;11(20):3861-3875.
In addition to their role in motility, eukaryotic cilia serve as a distinct compartment for signal transduction and regulatory sequestration of biomolecules. Recent genetic and biochemical studies have revealed an extraordinary diversity of protein complexes involved in the biogenesis of cilia during each cell cycle. Mutations in components of these complexes are at the heart of human ciliopathies such as Nephronophthisis (NPHP), Meckel-Gruber syndrome (MKS), Bardet-Biedl syndrome (BBS) and Joubert syndrome (JBTS). Despite intense studies, proteins in some of these complexes, such as the NPHP1-4-8 and the MKS, remain poorly understood. Using a combination of computational analyses we studied these complexes to identify novel domains in them which might throw new light on their functions and evolutionary origins. First, we identified both catalytically active and inactive versions of transglutaminase-like (TGL) peptidase domains in key ciliary/centrosomal proteins CC2D2A/MKS6, CC2D2B, CEP76 and CCDC135. These ciliary TGL domains appear to have originated from prokaryotic TGL domains that act as peptidases, either in a prokaryotic protein degradation system with the MoxR AAA+ ATPase, the precursor of eukaryotic dyneins and midasins, or in a peptide-ligase system with an ATP-grasp enzyme comparable to tubulin-modifying TTL proteins. We suggest that active ciliary TGL proteins are part of a cilia-specific peptidase system that might remove tubulin modifications or cleave cilia- localized proteins, while the inactive versions are likely to bind peptides and mediate key interactions during ciliogenesis. Second, we observe a vast radiation of C2 domains, which are key membrane-localization modules, in multiple ciliary proteins, including those from the NPHP1-4-8 and the MKS complexes, such as CC2D2A/MKS6, RPGRIP1, RPGRIP1L, NPHP1, NPHP4, C2CD3, AHI1/Jouberin and CEP76, most of which can be traced back to the last eukaryotic ancestor. Identification of these TGL and C2 domains aid in the proper reconstruction of the Y-shaped linkers, which are key structures in the transitional zone of cilia, by allowing precise prediction of the multiple membrane-contacting and protein-protein interaction sites in these structures. These findings help decipher key events in the evolutionary separation of the ciliary and nuclear compartments in course of the emergence of the eukaryotic cell.
PMCID: PMC3495828  PMID: 22983010
ciliogenesis; transglutaminase-like; membrane; tubulin-tyrosine ligase; C2; transition zone; Y-shaped linkers; evolution; origin of eukaryotes; ciliopathy
11.  Potent Neutralization of Botulinum Neurotoxin/B by Synergistic Action of Antibodies Recognizing Protein and Ganglioside Receptor Binding Domain 
PLoS ONE  2012;7(8):e43845.
Botulinum neurotoxins (BoNTs), the causative agents for life-threatening human disease botulism, have been recognized as biological warfare agents. Monoclonal antibody (mAb) therapeutics hold considerable promise as BoNT therapeutics, but the potencies of mAbs against BoNTs are usually less than that of polyclonal antibodies (or oligoclonal antibodies). The confirmation of key epitopes with development of effective mAb is urgently needed.
Methods and Findings
We selected 3 neutralizing mAbs which recognize different non-overlapping epitopes of BoNT/B from a panel of neutralizing antibodies against BoNT/B. By comparing the neutralizing effects among different combination groups, we found that 8E10, response to ganglioside receptor binding site, could synergy with 5G10 and 2F4, recognizing non-overlapping epitopes within Syt II binding sites. However, the combination of 5G10 with 2F4 blocking protein receptor binding sites did not achieve synergistical effects. Moreover, we found that the binding epitope of 8E10 was conserved among BoNT A, B, E, and F, which might cross-protect the challenge of different serotypes of BoNTs in vivo.
The combination of two mAbs recognizing different receptors' binding domain in BoNTs has a synergistic effect. 8E10 is a potential universal partner for the synergistical combination with other mAb against protein receptor binding domain in BoNTs of other serotypes.
PMCID: PMC3430616  PMID: 22952786
12.  Genetic diversity in Capsicum baccatum is significantly influenced by its ecogeographical distribution 
BMC Genetics  2012;13:68.
The exotic pepper species Capsicum baccatum, also known as the aji or Peruvian hot pepper, is comprised of wild and domesticated botanical forms. The species is a valuable source of new genes useful for improving fruit quality and disease resistance in C. annuum sweet bell and hot chile pepper. However, relatively little research has been conducted to characterize the species, thus limiting its utilization. The structure of genetic diversity in a plant germplasm collection is significantly influenced by its ecogeographical distribution. Together with DNA fingerprints derived from AFLP markers, we evaluated variation in fruit and plant morphology of plants collected across the species native range in South America and evaluated these characters in combination with the unique geography, climate and ecology at different sites where plants originated.
The present study mapped the ecogeographic distribution, analyzed the spatial genetic structure, and assessed the relationship between the spatial genetic pattern and the variation of morphological traits in a diverse C. baccatum germplasm collection spanning the species distribution. A combined diversity analysis was carried out on the USDA-ARS C. baccatum germplasm collection using data from GIS, morphological traits and AFLP markers. The results demonstrate that the C. baccatum collection covers wide geographic areas and is adapted to divergent ecological conditions in South America ranging from cool Andean highland to Amazonia rainforest. A high level of morphological diversity was evident in the collection, with fruit weight the leading variable. The fruit weight distribution pattern was compatible to AFLP-based clustering analysis for the collection. A significant spatial structure was observed in the C. baccatum gene pool. Division of the domesticated germplasm into two major regional groups (Western and Eastern) was further supported by the pattern of spatial population structure.
The results reported improve our understanding of the combined effects of geography, ecology and human intervention on organization of the C. baccatum genepool. The results will facilitate utilization of C. baccatum for crop improvement and species conservation by providing a framework for efficient germplasm collection management and guidance for future plant acquisitions.
PMCID: PMC3496591  PMID: 22866868
13.  Polymorphic toxin systems: Comprehensive characterization of trafficking modes, processing, mechanisms of action, immunity and ecology using comparative genomics 
Biology Direct  2012;7:18.
Proteinaceous toxins are observed across all levels of inter-organismal and intra-genomic conflicts. These include recently discovered prokaryotic polymorphic toxin systems implicated in intra-specific conflicts. They are characterized by a remarkable diversity of C-terminal toxin domains generated by recombination with standalone toxin-coding cassettes. Prior analysis revealed a striking diversity of nuclease and deaminase domains among the toxin modules. We systematically investigated polymorphic toxin systems using comparative genomics, sequence and structure analysis.
Polymorphic toxin systems are distributed across all major bacterial lineages and are delivered by at least eight distinct secretory systems. In addition to type-II, these include type-V, VI, VII (ESX), and the poorly characterized “Photorhabdus virulence cassettes (PVC)”, PrsW-dependent and MuF phage-capsid-like systems. We present evidence that trafficking of these toxins is often accompanied by autoproteolytic processing catalyzed by HINT, ZU5, PrsW, caspase-like, papain-like, and a novel metallopeptidase associated with the PVC system. We identified over 150 distinct toxin domains in these systems. These span an extraordinary catalytic spectrum to include 23 distinct clades of peptidases, numerous previously unrecognized versions of nucleases and deaminases, ADP-ribosyltransferases, ADP ribosyl cyclases, RelA/SpoT-like nucleotidyltransferases, glycosyltranferases and other enzymes predicted to modify lipids and carbohydrates, and a pore-forming toxin domain. Several of these toxin domains are shared with host-directed effectors of pathogenic bacteria. Over 90 families of immunity proteins might neutralize anywhere between a single to at least 27 distinct types of toxin domains. In some organisms multiple tandem immunity genes or immunity protein domains are organized into polyimmunity loci or polyimmunity proteins. Gene-neighborhood-analysis of polymorphic toxin systems predicts the presence of novel trafficking-related components, and also the organizational logic that allows toxin diversification through recombination. Domain architecture and protein-length analysis revealed that these toxins might be deployed as secreted factors, through directed injection, or via inter-cellular contact facilitated by filamentous structures formed by RHS/YD, filamentous hemagglutinin and other repeats. Phyletic pattern and life-style analysis indicate that polymorphic toxins and polyimmunity loci participate in cooperative behavior and facultative ‘cheating’ in several ecosystems such as the human oral cavity and soil. Multiple domains from these systems have also been repeatedly transferred to eukaryotes and their viruses, such as the nucleo-cytoplasmic large DNA viruses.
Along with a comprehensive inventory of toxins and immunity proteins, we present several testable predictions regarding active sites and catalytic mechanisms of toxins, their processing and trafficking and their role in intra-specific and inter-specific interactions between bacteria. These systems provide insights regarding the emergence of key systems at different points in eukaryotic evolution, such as ADP ribosylation, interaction of myosin VI with cargo proteins, mediation of apoptosis, hyphal heteroincompatibility, hedgehog signaling, arthropod toxins, cell-cell interaction molecules like teneurins and different signaling messengers.
This article was reviewed by AM, FE and IZ.
PMCID: PMC3482391  PMID: 22731697
14.  Gene flow and biological conflict systems in the origin and evolution of eukaryotes 
The endosymbiotic origin of eukaryotes brought together two disparate genomes in the cell. Additionally, eukaryotic natural history has included other endosymbiotic events, phagotrophic consumption of organisms, and intimate interactions with viruses and endoparasites. These phenomena facilitated large-scale lateral gene transfer and biological conflicts. We synthesize information from nearly two decades of genomics to illustrate how the interplay between lateral gene transfer and biological conflicts has impacted the emergence of new adaptations in eukaryotes. Using apicomplexans as example, we illustrate how lateral transfer from animals has contributed to unique parasite-host interfaces comprised of adhesion- and O-linked glycosylation-related domains. Adaptations, emerging due to intense selection for diversity in the molecular participants in organismal and genomic conflicts, being dispersed by lateral transfer, were subsequently exapted for eukaryote-specific innovations. We illustrate this using examples relating to eukaryotic chromatin, RNAi and RNA-processing systems, signaling pathways, apoptosis and immunity. We highlight the major contributions from catalytic domains of bacterial toxin systems to the origin of signaling enzymes (e.g., ADP-ribosylation and small molecule messenger synthesis), mutagenic enzymes for immune receptor diversification and RNA-processing. Similarly, we discuss contributions of bacterial antibiotic/siderophore synthesis systems and intra-genomic and intra-cellular selfish elements (e.g., restriction-modification, mobile elements and lysogenic phages) in the emergence of chromatin remodeling/modifying enzymes and RNA-based regulation. We develop the concept that biological conflict systems served as evolutionary “nurseries” for innovations in the protein world, which were delivered to eukaryotes via lateral gene flow to spur key evolutionary innovations all the way from nucleogenesis to lineage-specific adaptations.
PMCID: PMC3417536  PMID: 22919680
antibiotics; biological conflict; endosymbiosis; immunity proteins; restriction-modfication; RNAi; selfish elements; toxins
15.  Optimal Design of the Absolute Positioning Sensor for a High-Speed Maglev Train and Research on Its Fault Diagnosis 
Sensors (Basel, Switzerland)  2012;12(8):10621-10638.
This paper studies an absolute positioning sensor for a high-speed maglev train and its fault diagnosis method. The absolute positioning sensor is an important sensor for the high-speed maglev train to accomplish its synchronous traction. It is used to calibrate the error of the relative positioning sensor which is used to provide the magnetic phase signal. On the basis of the analysis for the principle of the absolute positioning sensor, the paper describes the design of the sending and receiving coils and realizes the hardware and the software for the sensor. In order to enhance the reliability of the sensor, a support vector machine is used to recognize the fault characters, and the signal flow method is used to locate the faulty parts. The diagnosis information not only can be sent to an upper center control computer to evaluate the reliability of the sensors, but also can realize on-line diagnosis for debugging and the quick detection when the maglev train is off-line. The absolute positioning sensor we study has been used in the actual project.
PMCID: PMC3472847  PMID: 23112619
high-speed maglev train; absolute positioning sensor; fault diagnosis; position mark plate; support vector machine
16.  Identification of Novel Families and Classification of the C2 domain Superfamily Elucidate the Origin and Evolution of Membrane Targeting Activities in Eukaryotes 
Gene  2010;469(1-2):18-30.
Eukaryotes contain an elaborate membrane system, which bounds the cell itself, nuclei, organelles and transient intracellular structures, such as vesicles. The emergence of this system was marked by an expansion of number of structurally distinct classes of lipid-binding domains that could throw light on the early evolution of eukaryotic membranes. The C2 domain is a useful model to understand these events because it is one of the most prevalent eukaryotic lipid-binding domains deployed in diverse functional contexts. Most studies have concentrated on C2 domains prototyped by those in protein kinase C (PKC-C2) isoforms that bind lipid in a calcium-dependent manner. While two other distinct families of C2 domains, namely those in PI3K-C2 and PTEN-C2 are also recognized, a complete picture of evolutionary relationships within the C2 domain superfamily is lacking. We systematically studied this superfamily using sequence-profile searches, phylogenetic and phyletic-pattern analysis and structure-prediction. Consequently, we identified several distinct families of C2 domains including those respectively typified by C2 domains in the Aida (axin interactor, dorsalization associated) proteins, B9 proteins (e.g. Mks1 (Xbx-7), Stumpy (Tza-1) and Tza-2) involved in centrosome migration and ciliogenesis, Dock180/Zizimin proteins which are Rac/CDC42 GDP exchange factors, the EEIG1/Sym-3, EHBP1 and plant RPG/PMI1 proteins involved in endocytotic recycling and organellar positioning and an apicomplexan family. We present evidence that the last eukaryotic common ancestor (LECA) contained at least 10 C2 domains belonging to 6 well-defined families. Further, we suggest that this pre-LECA diversification was linked to emergence of several quintessentially eukaryotic structures, such as membrane repair and vesicular trafficking system, anchoring of the actin and tubulin cytoskeleton to the plasma and vesicular membranes, localization of small GTPases to membranes and lipid-based signal transduction. Subsequent lineage-specific expansions of Zizimin-type C2 domains and functionally linked CDC42/Rac GTPases occurred independently in eukaryotes that evolved active amoeboid motility. While two lipid-binding regions are likely to be shared by majority of C2 domains, the actual constellation of lipid-binding residues (predominantly basic) are distinct in each family potentially reflective of the functional and biochemical diversity of these domains. Importantly, we show that the calcium-dependent membrane interaction is a derived feature limited to the PKC-C2 domains. Our identification of novel C2 domains offers new insights into interaction between both the microtubular and microfilament cytoskeleton and cellular membranes.
PMCID: PMC2965036  PMID: 20713135
C2 domain; evolution; remote homology detection; structure and function; membranes; lipid recognition; cytoskeleton; GTPase signaling; vesicular trafficking
17.  Evolution of the deaminase fold and multiple origins of eukaryotic editing and mutagenic nucleic acid deaminases from bacterial toxin systems 
Nucleic Acids Research  2011;39(22):9473-9497.
The deaminase-like fold includes, in addition to nucleic acid/nucleotide deaminases, several catalytic domains such as the JAB domain, and others involved in nucleotide and ADP-ribose metabolism. Using sensitive sequence and structural comparison methods, we develop a comprehensive natural classification of the deaminase-like fold and show that its ancestral version was likely to operate on nucleotides or nucleic acids. Consequently, we present evidence that a specific group of JAB domains are likely to possess a DNA repair function, distinct from the previously known deubiquitinating peptidase activity. We also identified numerous previously unknown clades of nucleic acid deaminases. Using inference based on contextual information, we suggest that most of these clades are toxin domains of two distinct classes of bacterial toxin systems, namely polymorphic toxins implicated in bacterial interstrain competition and those that target distantly related cells. Genome context information suggests that these toxins might be delivered via diverse secretory systems, such as Type V, Type VI, PVC and a novel PrsW-like intramembrane peptidase-dependent mechanism. We propose that certain deaminase toxins might be deployed by diverse extracellular and intracellular pathogens as also endosymbionts as effectors targeting nucleic acids of host cells. Our analysis suggests that these toxin deaminases have been acquired by eukaryotes on several independent occasions and recruited as organellar or nucleo-cytoplasmic RNA modifiers, operating on tRNAs, mRNAs and short non-coding RNAs, and also as mutators of hyper-variable genes, viruses and selfish elements. This scenario potentially explains the origin of mutagenic AID/APOBEC-like deaminases, including novel versions from Caenorhabditis, Nematostella and diverse algae and a large class of fast-evolving fungal deaminases. These observations greatly expand the distribution of possible unidentified mutagenic processes catalyzed by nucleic acid deaminases.
PMCID: PMC3239186  PMID: 21890906
18.  Phase I trial of a humanized, Fc receptor nonbinding anti-CD3 antibody, hu12F6mu in patients receiving renal allografts 
mAbs  2011;2(4):449-456.
Hu12F6mu is an Fc-mutated, humanized anti-CD3 antibody developed in our lab. The aim of this study was to assess single dose escalation pharmacokinetics (PK) and safety profile of hu12F6mu and to measure the effects of the antibody on levels of circulating T cells over time. Twenty-seven patients receiving renal allografts were randomized to receive hu12F6mu intravenously at a single-dose of 2.5, 5 or 10 mg. The concentration-time data obtained by a validated ELISA method were subjected to non-compartmental PK analysis by DAS 2.1 software. Subgroups of CD2+, CD3+, CD4+ and CD8+ lymphocytes were monitored periodically by flow cytometry. Our results showed that hu12F6mu exhibited linear PK over the dose range of 2.5–10 mg. A significant decline in the proportion of T cells was observed immediately after the infusion, followed by a progressive increase occurring over the ensuing days of therapy. A significant negative correlation was observed between serum concentration of hu12F6mu and CD3+ cell proportion. Intravenous infusion of hu12F6mu was well-tolerated in patients receiving renal allografts. These results suggest that hu12F6mu may have potential as a therapeutic agent, although further studies are needed.
PMCID: PMC3180091  PMID: 20519962
CD3; humanized antibody; pharmacokinetics; enzyme immunoassay; first dose reaction
19.  A novel immunity system for bacterial nucleic acid degrading toxins and its recruitment in various eukaryotic and DNA viral systems 
Nucleic Acids Research  2011;39(11):4532-4552.
The use of nucleases as toxins for defense, offense or addiction of selfish elements is widely encountered across all life forms. Using sensitive sequence profile analysis methods, we characterize a novel superfamily (the SUKH superfamily) that unites a diverse group of proteins including Smi1/Knr4, PGs2, FBXO3, SKIP16, Syd, herpesviral US22, IRS1 and TRS1, and their bacterial homologs. Using contextual analysis we present evidence that the bacterial members of this superfamily are potential immunity proteins for a variety of toxin systems that also include the recently characterized contact-dependent inhibition (CDI) systems of proteobacteria. By analyzing the toxin proteins encoded in the neighborhood of the SUKH superfamily we predict that they possess domains belonging to diverse nuclease and nucleic acid deaminase families. These include at least eight distinct types of DNases belonging to HNH/EndoVII- and restriction endonuclease-fold, and RNases of the EndoU-like and colicin E3-like cytotoxic RNases-folds. The N-terminal domains of these toxins indicate that they are extruded by several distinct secretory mechanisms such as the two-partner system (shared with the CDI systems) in proteobacteria, ESAT-6/WXG-like ATP-dependent secretory systems in Gram-positive bacteria and the conventional Sec-dependent system in several bacterial lineages. The hedgehog-intein domain might also release a subset of toxic nuclease domains through auto-proteolytic action. Unlike classical colicin-like nuclease toxins, the overwhelming majority of toxin systems with the SUKH superfamily is chromosomally encoded and appears to have diversified through a recombination process combining different C-terminal nuclease domains to N-terminal secretion-related domains. Across the bacterial superkingdom these systems might participate in discriminating `self’ or kin from `non-self’ or non-kin strains. Using structural analysis we demonstrate that the SUKH domain possesses a versatile scaffold that can be used to bind a wide range of protein partners. In eukaryotes it appears to have been recruited as an adaptor to regulate modification of proteins by ubiquitination or polyglutamylation. Similarly, another widespread immunity protein from these toxin systems, namely the suppressor of fused (SuFu) superfamily has been recruited for comparable roles in eukaryotes. In animal DNA viruses, such as herpesviruses, poxviruses, iridoviruses and adenoviruses, the ability of the SUKH domain to bind diverse targets has been deployed to counter diverse anti-viral responses by interacting with specific host proteins.
PMCID: PMC3113570  PMID: 21306995
20.  Evolution of information-driven HIV/AIDS policies in China 
International Journal of Epidemiology  2010;39(Suppl 2):ii4-ii13.
Background As China continues to commit to universal access to HIV/AIDS prevention, treatment and care services, its HIV/AIDS policies have become increasingly information driven. We review China’s key national-level HIV/AIDS policies and discuss policy gaps and challenges ahead.
Methods We conducted a desk review of key national-level policies that have had a major impact on China’s HIV/AIDS epidemic, and examined recent epidemiological data relevant to China’s HIV response.
Results National-level policies that have had a major impact on China’s HIV/AIDS response include: ‘Four Frees and One Care’; 5-year action plans; and HIV/AIDS regulation. These landmark policies have facilitated massive scaling up of services over the past decade. For example, the number of drug users provided with methadone maintenance treatment significantly increased from 8116 in 2005 to 241 975 in 2009; almost a 30-fold increase. The ‘Four Frees and One Care’ policy has increased the number of people living with AIDS on anti-retroviral treatment from some 100 patients in 2003 to over 80 000 in 2009. However, stigma and discrimination remains major obstacles for people living with HIV/AIDS trying to access services.
Conclusions China’s current national policies are increasingly information driven and responsive to changes in the epidemic. However, gaps remain in policy implementation, and new policies are needed to meet emerging challenges.
PMCID: PMC2992621  PMID: 21113036
HIV/AIDS; policy; implementation; challenges; China
21.  Estimating the population of female sex workers in two Chinese cities on the basis of the HIV/AIDS behavioural surveillance approach combined with a multiplier method 
Sexually Transmitted Infections  2006;83(3):228-231.
To estimate the size of the population of female sex workers (FSWs) on the basis of the HIV/AIDS behavioural surveillance approach in two Chinese cities, using a multiplier method.
Relevant questions were inserted into the questionnaires given to two behavioural surveillance groups—female attendees of sexually transmitted disease (STD) clinics and FSWs. The size of the FSW population was derived by multiplying the number of FSWs in selected STD clinics during the study period by the proportion of FSW population who reported having attended the selected STD clinics during the same period.
The size of the FSW population in the urban area of Xingyi, China, was estimated to be about 2500 (95% CI 2000 to 3400). This accounted for 3.6% of the total urban adult female population. There were an estimated 17 500 FSWs in the urban area of Guiyang, China (95% CI 10 300 to 31 900) or about 3.4% of its total urban adult female population (rounded to the nearest 100).
The multiplier method could be a useful and cost‐effective approach to estimate the FSW population, especially suitable in countries where HIV behavioural surveillance has been established in high‐risk populations.
PMCID: PMC2659102  PMID: 17090568
22.  OST-HTH: a novel predicted RNA-binding domain 
Biology Direct  2010;5:13.
The mechanism by which the arthropod Oskar and vertebrate TDRD5/TDRD7 proteins nucleate or organize structurally related ribonucleoprotein (RNP) complexes, the polar granule and nuage, is poorly understood. Using sequence profile searches we identify a novel domain in these proteins that is widely conserved across eukaryotes and bacteria.
Using contextual information from domain architectures, sequence-structure superpositions and available functional information we predict that this domain is likely to adopt the winged helix-turn-helix fold and bind RNA with a potential specificity for dsRNA. We show that in eukaryotes this domain is often combined in the same polypeptide with protein-protein- or lipid- interaction domains that might play a role in anchoring these proteins to specific cytoskeletal structures.
Thus, proteins with this domain might have a key role in the recognition and localization of dsRNA, including miRNAs, rasiRNAs and piRNAs hybridized to their targets. In other cases, this domain is fused to ubiquitin-binding, E3 ligase and ubiquitin-like domains indicating a previously under-appreciated role for ubiquitination in regulating the assembly and stability of nuage-like RNP complexes. Both bacteria and eukaryotes encode a conserved family of proteins that combines this predicted RNA-binding domain with a previously uncharacterized domain (DUF88). We present evidence that it is an RNAse belonging to the superfamily that includes the 5'->3' nucleases, PIN and NYN domains and might be recruited to degrade certain RNAs.
This article was reviewed by Sandor Pongor and Arcady Mushegian.
PMCID: PMC2848206  PMID: 20302647
23.  Two doses of humanized anti-CD25 antibody in renal transplantation 
mAbs  2009;1(1):49-55.
HuCD25mAb is a humanized anti-CD25 antibody which has the same amino acid sequence as daclizumab (Zenapax, Roche). HuCD25mAb is expressed in Chinese hamster ovary (CHO) cells while daclizumab is expressed in the NSO myeloma cell line. A comparative study was performed to evaluate the pharmacokinetics and pharmacodynamics between huCD25mAb and daclizumab in a two-dose regimen incorporating triple immunosuppressant treatment regimens (MMF, CsA and steroids). Fifteen patients were enrolled and randomized to receive intravenous infusion of either huCD25mAb (n = 10) or daclizumab (n = 5) at a dosage of 1 mg·kg−1 on operation day 0 and post-operation day 14. Serum concentrations of huCD25mAb and daclizumab were measured by a validated competitive ELISA. Subgroups of CD3+, CD25+, CD4+ and CD8+ lymphocytes were monitored periodically by flow cytometry. The concentration-time curves of huCD25mAb and daclizumab were found to fit well to a one-compartment model. A significant decline of proportion (%) of CD3-CD25+ and CD3+CD25+ lymphocytes was observed 30 min after first infusion on day 0 (3.40 ± 1.83 to 0.03 ± 0.07, 3.35 ± 2.02 to 0.37 ± 0.49), and these levels remained low for at least 70 days (0.03 ± 0.05, 0.31 ± 0.47). All pharmacokinetic parameters of huCD25mAb seemed similar to those of daclizumab. The two-dose huCD25mAb regimen was as effective as daclizumab in rapidly achieving high therapeutic concentration in the treated patients, and a significant decrease of CD3−CD25+ and CD3+CD25+ lymphocytes was demonstrated. This suggests that two-dose regimen is feasible in maintaining host immunosuppression and may provide an effective and economical strategy for reducing incidence of acute graft rejection.
PMCID: PMC2715186  PMID: 20046574
CD25; pharmacokinetics; kidney transplantation; enzyme immunoassay; flow cytometry; monoclonal antibody
24.  Defining Global Neuroendocrine Gene Expression Patterns Associated with Reproductive Seasonality in Fish 
PLoS ONE  2009;4(6):e5816.
Many vertebrates, including the goldfish, exhibit seasonal reproductive rhythms, which are a result of interactions between external environmental stimuli and internal endocrine systems in the hypothalamo-pituitary-gonadal axis. While it is long believed that differential expression of neuroendocrine genes contributes to establishing seasonal reproductive rhythms, no systems-level investigation has yet been conducted.
Methodology/Principal Findings
In the present study, by analyzing multiple female goldfish brain microarray datasets, we have characterized global gene expression patterns for a seasonal cycle. A core set of genes (873 genes) in the hypothalamus were identified to be differentially expressed between May, August and December, which correspond to physiologically distinct stages that are sexually mature (prespawning), sexual regression, and early gonadal redevelopment, respectively. Expression changes of these genes are also shared by another brain region, the telencephalon, as revealed by multivariate analysis. More importantly, by examining one dataset obtained from fish in October who were kept under long-daylength photoperiod (16 h) typical of the springtime breeding season (May), we observed that the expression of identified genes appears regulated by photoperiod, a major factor controlling vertebrate reproductive cyclicity. Gene ontology analysis revealed that hormone genes and genes functionally involved in G-protein coupled receptor signaling pathway and transmission of nerve impulses are significantly enriched in an expression pattern, whose transition is located between prespawning and sexually regressed stages. The existence of seasonal expression patterns was verified for several genes including isotocin, ependymin II, GABAA gamma2 receptor, calmodulin, and aromatase b by independent samplings of goldfish brains from six seasonal time points and real-time PCR assays.
Using both theoretical and experimental strategies, we report for the first time global gene expression patterns throughout a breeding season which may account for dynamic neuroendocrine regulation of seasonal reproductive development.
PMCID: PMC2686097  PMID: 19503831
25.  Functional insight into Maelstrom in the germline piRNA pathway: a unique domain homologous to the DnaQ-H 3'–5' exonuclease, its lineage-specific expansion/loss and evolutionarily active site switch 
Biology Direct  2008;3:48.
Maelstrom (MAEL) plays a crucial role in a recently-discovered piRNA pathway; however its specific function remains unknown. Here a novel MAEL-specific domain characterized by a set of conserved residues (Glu-His-His-Cys-His-Cys, EHHCHC) was identified in a broad range of species including vertebrates, sea squirts, insects, nematodes, and protists. It exhibits ancient lineage-specific expansions in several species, however, appears to be lost in all examined teleost fish species. Functional involvement of MAEL domains in DNA- and RNA-related processes was further revealed by its association with HMG, SR-25-like and HDAC_interact domains. A distant similarity to the DnaQ-H 3'–5' exonuclease family with the RNase H fold was discovered based on the evidence that all MAEL domains adopt the canonical RNase H fold; and several protist MAEL domains contain the conserved 3'–5' exonuclease active site residues (Asp-Glu-Asp-His-Asp, DEDHD). This evolutionary link together with structural examinations leads to a hypothesis that MAEL domains may have a potential nuclease activity or RNA-binding ability that may be implicated in piRNA biogenesis. The observed transition of two sets of characteristic residues between the ancestral DnaQ-H and the descendent MAEL domains may suggest a new mode for protein function evolution called "active site switch", in which the protist MAEL homologues are the likely evolutionary intermediates due to harboring the specific characteristics of both 3'–5' exonuclease and MAEL domains.
This article was reviewed by L Aravind, Wing-Cheong Wong and Frank Eisenhaber. For the full reviews, please go to the Reviewers' Comments section.
PMCID: PMC2628886  PMID: 19032786

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