Separation of proteins by two-dimensional gel electrophoresis (2-DE) coupled with identification of proteins through peptide mass fingerprinting (PMF) by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is the widely used technique for proteomic analysis. This approach relies, however, on the presence of the proteins studied in public-accessible protein databases or the availability of annotated genome sequences of an organism. In this work, we investigated the reliability of using raw genome sequences for identifying proteins by PMF without the need of additional information such as amino acid sequences. The method is demonstrated for proteomic analysis of Klebsiella pneumoniae grown anaerobically on glycerol. For 197 spots excised from 2-DE gels and submitted for mass spectrometric analysis 164 spots were clearly identified as 122 individual proteins. 95% of the 164 spots can be successfully identified merely by using peptide mass fingerprints and a strain-specific protein database (ProtKpn) constructed from the raw genome sequences of K. pneumoniae. Cross-species protein searching in the public databases mainly resulted in the identification of 57% of the 66 high expressed protein spots in comparison to 97% by using the ProtKpn database. 10 dha regulon related proteins that are essential for the initial enzymatic steps of anaerobic glycerol metabolism were successfully identified using the ProtKpn database, whereas none of them could be identified by cross-species searching. In conclusion, the use of strain-specific protein database constructed from raw genome sequences makes it possible to reliably identify most of the proteins from 2-DE analysis simply through peptide mass fingerprinting.

Severe acute respiratory syndrome (SARS) is a novel infectious disease. No information is currently available on host-specific immunity against the SARS coronavirus (CoV), and detailed characteristics of the epidemiology of SARS CoV infection have not been identified.

Studies of SARS coronavirus (SARS-CoV)-the causative agent of severe acute respiratory syndrome (SARS)-have been hampered by its high transmission rate and the pathogenicity of this virus. To permit analysis of the host range and entry mechanism of SARS-CoV, we incorporated the humanized SARS-CoV spike (S) glycoprotein into HIV particles to generate a highly infectious SARS-CoV pseudotyped virus. The infection on Vero E6-a permissive cell line to SARS-CoV-could be neutralized by sera from convalescent SARS patients, and the entry was a pH-dependent process. With these highly infectious SARS-CoV pseudotypes, several cell lines derived from various tissues were revealed as susceptible to SARS-CoV, which were highly corresponding to the expression pattern of virus's receptor angiotensin-converting enzyme 2 (ACE2). In addition, we also demonstrated angiotensin 1 converting enzyme (ACE)-the homologue of ACE2 could not function as a receptor for SARS-CoV.

microRNAs (miRNAs) are endogenous small non-coding RNAs that regulate gene expression at the post-transcriptional level. While the number of known human and murine miRNAs is continuously increasing, information regarding miRNAs from other species such as amphioxus remains limited.

To characterize the disease-causing mutations in a Chinese family with progressive childhood cataracts.

Liuweidihuang wan (LW), initially a well-known formula for curing "wu chi wu ruan", is commonly used nowadays for clinical treatment of postmenopausal osteoporosis (PO), but the identity of the effective substance(s) remains unclear. The present study was designed to evaluate the effects of morroniside and loganin isolated from LW on the proliferation, differentiation and apoptosis of MC3T3-E1 cells, as well as the possible mechanism of action. Morroniside and loganin had no effects on the proliferation of MC3T3-E1 cells, but both susbtances could improve the activity of alkaline phosphatase (ALP), and increase the contents of collagen type I and osteocalcin. Simultaneously, the mRNA expression of caspase-3, capase-9, RANKL was down-regulated and that of bcl-2 was up-regulated, which partially explains the anti-osteoporosis mechanism in MC3T3-E1 cells. In conclusion, morroniside and loganin may directly promote the differentiation and inhibit the apoptosis of MC3T3-E1 cells, and accordingly indirectly reduce bone resorption, which makes them promising natural drugs leads for treating PO in the near future.

The choice of the tumor antigen preparation used for dendritic cell (DC) loading is important for optimizing DC vaccines. In the present study, we compared DCs pulsed with hepatocellular carcinoma (HCC) total RNA or cell lysates for their capacity to activate T cells. We showed here that HCC total RNA pulsed-DCs induced effector T lymphocyte responses which showed higher killing ability to HCC cell lines, as well as higher frequency of IFN-γ producing of CD4+ and CD8+ T cells when compared with lysate pulsed-DCs. Both of RNA and lysate loading did not influence the changes of mature DC phenotype and the capacity of inducing T cell proliferation. However, HCC lysate loading significantly inhibited the production of inflammatory cytokines IL-12p70, IFN-γ and enhanced the secretion of anti-inflammatory cytokines IL-10 of mature DCs. Our results indicated that DCs loaded with HCC RNA are superior to that loaded with lysate in priming anti-HCC CTL response, suggesting that total RNA may be a better choice for DCs-based HCC immunotherapy.

Periodontitis is one of the most widespread infectious diseases in humans. It is the main cause of tooth loss and associated with a number of systemic diseases. Until now, there is no appropriate method for functional periodontal tissue regeneration. Here, we establish a novel approach of using allogeneic periodontal ligament stem cells (PDLSCs) sheet to curing periodontitis in a miniature pig periodontitis model. Significant periodontal tissue regeneration was achieved in both the autologous and the allogeneic PDLSCs transplantation group at 12 weeks post-PDLSCs transplantation. Based on clinical assessments, computed tomography (CT) scanning, and histological examination, there was no marked difference between the autologous and allogeneic PDLSCs transplantation groups. In addition, lack of immunological rejections in the animals that received the allogeneic PDLSCs transplantation was observed. Interestingly, we found that human PDLSCs fail to express human leukocyte antigen (HLA)-II DR and costimulatory molecules. PDLSCs were not able to elicit T-cell proliferation and inhibit T-cell proliferation when stimulated with mismatched major histocompatibility complex molecules. Furthermore, we found that prostaglandin E2 (PGE2) plays a crucial role in PDLSCs-mediated immunomodulation and periodontal tissue regeneration in vitro and in vivo. Our study demonstrated that PDLSCs possess low immunogenicity and marked immunosuppression via PGE2-induced T-cell anergy. We developed a standard technological procedure of using allogeneic PDLSCs to cure periodontitis in swine.

Marine natural products and their synthetic derivatives represent a major source of novel candidate anti-cancer compounds. We have recently tested the anti-cancer activity of more than forty novel compounds based on an iminoquinone makaluvamine scaffold, and have found that many of the compounds exert potent cytotoxic activity against human cancer cell lines. One of the most potent compounds, BA-TPQ [(11,12),7-(benzylamino)-1,3,4,8-tetrahydropyrrolo[4,3,2-de]quinolin-8(1H)-one], was active against a variety of human cancer cell lines, and inhibited the growth of breast and prostate xenograft tumors in mice. However, there was some toxicity noted in the mice following administration of the compound. In order to further the development of BA-TPQ, and in a search for potential sites of accumulation that might underlie the observed toxicity of the compound, we accomplished preclinical pharmacological studies of the compound. We herein report the in vitro and in vivo pharmacological properties of BA-TPQ, including its stability in plasma, plasma protein binding, metabolism by S9 enzymes, and plasma and tissue distribution. We believe these studies will be useful for further investigations, and may be useful for other investigators examining the use of similar compounds for cancer therapy.

Muscle progenitors, labeled by the transcription factor Pax7, are responsible for muscle growth during development. The signals that regulate the muscle progenitor number during myogenesis are unknown. We show, through in vivo analysis, that Bmp signaling is involved in regulating fetal skeletal muscle growth. Ectopic activation of Bmp signaling in chick limbs increases the number of fetal muscle progenitors and fibers, while blocking Bmp signaling reduces their numbers, ultimately leading to small muscles. The Bmp effect that we observed during fetal myogenesis is diametrically opposed to that previously observed during embryonic myogenesis and that deduced from in vitro work. We also show that Bmp signaling regulates the number of satellite cells during development. Finally, we demonstrate that Bmp signaling is active in a subpopulation of fetal progenitors and satellite cells at the extremities of muscles. Overall, our results show that Bmp signaling plays differential roles in embryonic and fetal myogenesis.

The antiproliferative effect of the Hsp90 inhibitor 17-AAG (17-allylamino-17-demethoxygeldanamycin) on human retinal pigment epithelial cells is investigated.

Pandemic influenza A/H1N1 2009 and seasonal influenza viruses are currently co-circulating worldwide. A rapid, sensitive, and specific assay for distinguishing pandemic influenza A/H1N1 2009 from seasonal influenza viruses and for subtyping seasonal influenza A viruses could aid in the surveillance and control of these viral infections. Here, such a multiplex real-time RT-PCR (rRT-PCR) assay for typing influenza A and B viruses and the pandemic influenza A/H1N1 2009 is developed. This assay can also subtype seasonal influenza A viruses simultaneously. The analytical sensitivity is 10-10(4) copies/reaction. The coefficients of variation of inter-assay and intra-assay are 0.04-0.45% and 0.08-0.97%, respectively. The new multiplex rRT-PCR assay is more sensitive in subtyping seasonal influenza viruses than the conventional PCR techniques. Results obtained with this assay for the detection of pandemic influenza A/H1N1 2009 are highly consistent (96.88%) with those achieved using the US CDC's rRT-PCR protocol. A sample identified as "pandemic influenza A/H1N1 2009 positive" by the US CDC's rRT-PCR was reclassified correctly as subtype H3N2 using this assay. Taken together, this new multiplex rRT-PCR protocol could be an important tool for improving diagnosis and management of the pandemic influenza A/H1N1 2009 and seasonal influenza viruses.

The tumor suppressor p53 mainly induces cell cycle arrest/DNA repair or apoptosis in the DNA damage response. How to choose between these two outcomes is not fully understood. We proposed a four-module model of the p53 signaling network and associated the network dynamics with cellular outcomes after ionizing radiation. We found that the cellular response is mediated by both the level and posttranslational modifications of p53 and that p53 is activated in a progressive manner. First, p53 is partially activated by primary modifications such as phosphorylation at Ser-15/20 to induce cell cycle arrest, with its level varying in a series of pulses. If the damage cannot be fixed after a critical number of p53 pulses, then p53 is fully activated by further modifications such as phosphorylation at Ser-46 to trigger apoptosis, with its concentration switching to rather high levels. Thus, p53 undergoes a two-phase response in irreparably damaged cells. Such combinations of pulsatile and switch-like behaviors of p53 may represent a flexible and efficient control mode, avoiding the premature apoptosis and promoting the execution of apoptosis. In our model, p53 pulses are recurrently driven by ataxia telangiectasia mutated (ATM) pulses triggered by DNA damage. The p53-Mdm2 and ATM-p53-Wip1 negative feedback loops are responsible for p53 pulses, whereas the switching behavior occurs when the p53-PTEN-Akt-Mdm2 positive feedback loop becomes dominant. Our results suggest that a sequential predominance of distinct feedback loops may elicit multiple-phase dynamical behaviors. This work provides a new mechanism for p53 dynamics and cell fate decision.

Plexin receptors regulate cell adhesion, migration, and guidance. The Rho GTPase binding domain (RBD) of plexin-A1 and -B1 can bind GTPases, including Rnd1. By contrast, plexin-C1 and -D1 reportedly bind Rnd2 but associate with Rnd1 only weakly. The structural basis of this differential Rnd1 GTPase binding to plexin RBDs remains unclear. Here, we solved the structure of the plexin-A2 RBD in complex with Rnd1 and the structures of the plexin-C1 and plexin-D1 RBDs alone, also compared with the previously determined plexin-B1 RBD.Rnd1 complex structure. The plexin-A2 RBD·Rnd1 complex is a heterodimer, whereas plexin-B1 and -A2 RBDs homodimerize at high concentration in solution, consistent with a proposed model for plexin activation. Plexin-C1 and -D1 RBDs are monomeric, consistent with major residue changes in the homodimerization loop. In plexin-A2 and -B1, the RBD β3-β4 loop adjusts its conformation to allow Rnd1 binding, whereas minimal structural changes occur in Rnd1. The plexin-C1 and -D1 RBDs lack several key non-polar residues at the corresponding GTPase binding surface and do not significantly interact with Rnd1. Isothermal titration calorimetry measurements on plexin-C1 and -D1 mutants reveal that the introduction of non-polar residues in this loop generates affinity for Rnd1. Structure and sequence comparisons suggest a similar mode of Rnd1 binding to the RBDs, whereas mutagenesis suggests that the interface with the highly homologous Rnd2 GTPase is different in detail. Our results confirm, from a structural perspective, that Rnd1 does not play a role in the activation of plexin-C1 and -D1. Plexin functions appear to be regulated by subfamily-specific mechanisms, some of which involve different Rho family GTPases.

Generation of induced pluripotent stem cells (iPSCs) and converting one cell type to another (transdifferentiation) by manipulating the expression of a small number of genes highlight the progress of cellular reprogramming, which holds great promise for regenerative medicine. A key challenge is to find the recipes of perturbing genes to achieve successful reprogramming such that the reprogrammed cells function in the same way as the natural cells.

SHP-1 belongs to the family of non-receptor protein tyrosine phosphatases (PTPs) and generally acts as a negative regulator in a variety of cellular signaling pathways. Previously, the crystal structures of the tail-truncated SHP-1 and SHP-2 revealed an autoinhibitory conformation. To understand the regulatory mechanism of SHP-1, we have determined the crystal structure of the full-length SHP-1 at 3.1 Å. Although the tail was disordered in current structure, the huge conformational rearrangement of the N-SH2 domain and the incorporation of sulfate ions into the ligand-binding site of each domain indicate that the SHP-1 is in the open conformation. The N-SH2 domain in current structure is shifted away from the active site of the PTP domain to the other side of the C-SH2 domain, resulting in exposure of the active site. Meanwhile, the C-SH2 domain is twisted anticlockwise by about 110°. In addition, a set of new interactions between two SH2 domains and between the N-SH2 and the catalytic domains is identified, which could be responsible for the stabilization of SHP-1 in the open conformation. Based on the structural comparison, a model for the activation of SHP-1 is proposed.

Pandemic 2009 H1N1 influenza A virus (2009 H1N1) differs from H1N1 strains that circulated in the past 50 years, but resembles the A/New Jersey/1976 H1N1 strain used in the 1976 swine influenza vaccine. We investigated whether sera from persons immunized with the 1976 swine influenza or recent seasonal influenza vaccines, or both, neutralize 2009 H1N1. Using retroviral pseudovirions bearing hemagglutinins on their surface (HA-pseudotypes), we found that 77% of the sera collected in 1976 after immunization with the A/New Jersey/1976 H1N1 swine influenza vaccine neutralized 2009 H1N1. Forty five percent also neutralized A/New Caledonia/20/1999 H1N1, a strain used in seasonal influenza vaccines during the 2000/01-2006/07 seasons. Among adults aged 48-64 who received the swine influenza vaccine in 1976 and recent seasonal influenza vaccines during the 2004/05-2008/09 seasons, 83% had sera that neutralized 2009 H1N1. However, 68% of age-matched subjects who received the same seasonal influenza vaccines, but did not receive the 1976 swine influenza vaccine, also had sera that neutralized 2009 H1N1. Sera from both 1976 and contemporary cohorts frequently had cross-neutralizing antibodies to 2009 H1N1 and A/New Caledonia/20/1999 that mapped to hemagglutinin subunit 2 (HA2). A conservative mutation in HA2 corresponding to a residue in the A/Solomon Islands/3/2006 and A/Brisbane/59/2007 H1N1 strains that circulated in the 2006/07 and 2007/08 influenza seasons, respectively, abrogated this neutralization. These findings highlight a cross-neutralization determinant influenced by a point mutation in HA2 and suggest that HA2 may be evolving under direct or indirect immune pressure.

Foxj1 is a member of the Forkhead/winged-helix (Fox) family of transcription factors, which is required for postnatal differentiation of ependymal cells and a subset of astrocytes in the subventricular zone. The subpopulation of astrocytes has the ability of self-renew and neurogenic potential differentiated into astrocytes, oligodendrocytes, and neurons. However, its expression and function in the central nervous system lesion are not well understood. In this study, we performed a traumatic brain injury (TBI) model in adult rats and investigated the changed expression of Foxj1 in the brain cortex. Western blot and immunohistochemistry analysis showed that the expression of Foxj1 gradually increased, reached a peak at day 3 after TBI, and declined during the following days. Double immunofluorescence staining revealed that Foxj1 was co-expressed with MAP-2 and GFAP. In addition, we detected that Ki67 had the co-localization with NeuN, GFAP, and Foxj1. All our findings suggested that Foxj1 may be involved in the pathophysiology of brain after TBI.

Introgression plays an important role in evolution of plant species via its influences on genetic diversity and differentiation. Outcrossing determines the level of introgression but little is known about the relationships of outcrossing rates, genetic diversity, and differentiation particularly in a weedy taxon that coexists with its conspecific crop.

Platelets play an essential role in hemostasis and atherothrombosis. Owing to their short storage time, there is constant demand for this life-saving blood component. In this study, we report that it is feasible to generate functional megakaryocytes and platelets from human embryonic stem cells (hESCs) on a large scale. Differential-interference contrast and electron microscopy analyses showed that ultrastructural and morphological features of hESC-derived platelets were indistinguishable from those of normal blood platelets. In functional assays, hESC-derived platelets responded to thrombin stimulation, formed microaggregates, and facilitated clot formation/retraction in vitro. Live cell microscopy demonstrated that hESC-platelets formed lamellipodia and filopodia in response to thrombin activation, and tethered to each other as observed in normal blood. Using real-time intravital imaging with high-speed video microscopy, we have also shown that hESC-derived platelets contribute to developing thrombi at sites of laser-induced vascular injury in mice, providing the first evidence for in vivo functionality of hESC-derived platelets. These results represent an important step toward generating an unlimited supply of platelets for transfusion. Since platelets contain no genetic material, they are ideal candidates for early clinical translation involving human pluripotent stem cells.