Decreased LPS-induced lung injury in pigs treated with a lung surfactant protein A-derived nonapeptide that inhibits peroxiredoxin 6 activity and subsequent NOX1,2 activation.

This study addressed the efficacy of a liposome-encapsulated nine amino acid peptide [peroxiredoxin 6 PLA2 inhibitory peptide-2 (PIP-2)] for the prevention or treatment of acute lung injury (ALI) +/- sepsis. PIP-2 inhibits the PLA2 activity of peroxiredoxin 6 (Prdx6), thereby preventing rac release and activation of NADPH oxidases (NOXes), types 1 and 2. Female Yorkshire pigs were infused intravenously with lipopolysaccharide (LPS) + liposomes (untreated) or LPS + PIP-2 encapsulated in liposomes (treated). Pigs were mechanically ventilated and continuously monitored; they were euthanized after 8 h or earlier if preestablished humane endpoints were reached. Control pigs (mechanical ventilation, no LPS) were essentially unchanged over the 8 h study. LPS administration resulted in systemic inflammation with manifestations of clinical sepsis-like syndrome, decreased lung compliance, and a marked decrease in the arterial Po2 with vascular instability leading to early euthanasia of 50% of untreated animals. PIP-2 treatment significantly reduced the requirement for supportive vasopressors and the manifestations of lung injury so that only 25% of animals required early euthanasia. Bronchoalveolar lavage fluid from PIP-2-treated versus untreated pigs showed markedly lower levels of total protein, cytokines (TNF-α, IL-6, IL-1ß), and myeloperoxidase. Thus, the porcine LPS-induced sepsis-like model was associated with moderate to severe lung pathophysiology compatible with ALI, whereas treatment with PIP-2 markedly decreased lung injury, cardiovascular instability, and early euthanasia. These results indicate that inhibition of reactive oxygen species (ROS) production via NOX1/2 has a beneficial effect in treating pigs with LPS-induced ALI plus or minus a sepsis-like syndrome, suggesting a potential role for PIP-2 in the treatment of ALI and/or sepsis in humans.NEW & NOTEWORTHY Currently available treatments that can alter lung inflammation have failed to significantly alter mortality of acute lung injury (ALI). Peroxiredoxin 6 PLA2 inhibitory peptide-2 (PIP-2) targets the liberation of reactive O2 species (ROS) that is associated with adverse cell signaling events, thereby decreasing the tissue oxidative injury that occurs early in the ALI syndrome. We propose that treatment with PIP-2 may be effective in preventing progression of early disease into its later stages with irreversible lung damage and relatively high mortality.

Is the implant-retained overdenture a solution to improve the patient quality of life?

Design The research was a retrospective cohort study evaluating a study which performed routine control sessions to assess bone loss around the dental implant over five years and 12 and 24 months, after their prosthetic restorations.Aim The study aimed to evaluate patients' dental implant marginal bone loss after oral rehabilitation with bar and locator retainers and their possible complications.Methods A study of 114 patients who had received 283 dental implants from 2013-2018 was conducted. Two follow-up recall sessions were conducted, one at 12 months and one at 24 months, after dentures were placed. Intraoral and extraoral examinations were also conducted in addition to clinical assessments. The patients' prostheses were assessed for occlusion, tissue health and soft-tissue continuity, complaints, implant success rates and marginal bone loss, in addition to prosthetic complications, at follow-up sessions.Results In this study, 94 patients were fitted with implant-supported removable prostheses on locator attachments on both arches and 20 were fitted with removable prostheses using bar attachments. After evaluating the location of the implant, the number of days after implantation and the type of retainer in all patients, both groups showed a significant amount of marginal bone loss in the 12th and 24th months. The presence of complications was significantly associated with both prosthesis types at month 24 (p >0.05). When the relation between the position of the denture and the presence of complications at month 12 was evaluated, 36% of the complications were observed in the maxilla and 21.3% in the mandible. There was no significant difference associated between denture location and complication presence at month 24 (p >0.05).Conclusions Even though complications do not affect marginal bone loss, a patient's failure to maintain adequate oral hygiene results in marginal bone loss. Therefore, it appears that regular post-treatment prosthesis inspections are essential. If the requirements resulting from these inspections are satisfied, future complications may be prevented.

Mutations in Influenza A Virus Neuraminidase and Hemagglutinin Confer Resistance against a Broadly Neutralizing Hemagglutinin Stem Antibody.

Influenza A virus (IAV), a major cause of human morbidity and mortality, continuously evolves in response to selective pressures. Stem-directed, broadly neutralizing antibodies (sBnAbs) targeting the influenza virus hemagglutinin (HA) are a promising therapeutic strategy, but neutralization escape mutants can develop. We used an integrated approach combining viral passaging, deep sequencing, and protein structural analyses to define escape mutations and mechanisms of neutralization escape in vitro for the F10 sBnAb. IAV was propagated with escalating concentrations of F10 over serial passages in cultured cells to select for escape mutations. Viral sequence analysis revealed three mutations in HA and one in neuraminidase (NA). Introduction of these specific mutations into IAV through reverse genetics confirmed their roles in resistance to F10. Structural analyses revealed that the selected HA mutations (S123G, N460S, and N203V) are away from the F10 epitope but may indirectly impact influenza virus receptor binding, endosomal fusion, or budding. The NA mutation E329K, which was previously identified to be associated with antibody escape, affects the active site of NA, highlighting the importance of the balance between HA and NA function for viral survival. Thus, whole-genome population sequencing enables the identification of viral resistance mutations responding to antibody-induced selective pressure.IMPORTANCE Influenza A virus is a public health threat for which currently available vaccines are not always effective. Broadly neutralizing antibodies that bind to the highly conserved stem region of the influenza virus hemagglutinin (HA) can neutralize many influenza virus strains. To understand how influenza virus can become resistant or escape such antibodies, we propagated influenza A virus in vitro with escalating concentrations of antibody and analyzed viral populations by whole-genome sequencing. We identified HA mutations near and distal to the antibody binding epitope that conferred resistance to antibody neutralization. Additionally, we identified a neuraminidase (NA) mutation that allowed the virus to grow in the presence of high concentrations of the antibody. Virus carrying dual mutations in HA and NA also grew under high antibody concentrations. We show that NA mutations mediate the escape of neutralization by antibodies against HA, highlighting the importance of a balance between HA and NA for optimal virus function.

Nanomaterial Synthesis Insights from Machine Learning of Scientific Articles by Extracting, Structuring, and Visualizing Knowledge.

Nanomaterials of varying compositions and morphologies are of interest for many applications from catalysis to optics, but the synthesis of nanomaterials and their scale-up are most often time-consuming and Edisonian processes. Information gleaned from the scientific literature can help inform and accelerate nanomaterials development, but again, searching the literature and digesting the information are time-consuming manual processes for researchers. To help address these challenges, we developed scientific article-processing tools that extract and structure information from the text and figures of nanomaterials articles, thereby enabling the creation of a personalized knowledgebase for nanomaterials synthesis that can be mined to help inform further nanomaterials development. Starting with a corpus of ∼35k nanomaterials-related articles, we developed models to classify articles according to the nanomaterial composition and morphology, extract synthesis protocols from within the articles' text, and extract, normalize, and categorize chemical terms within synthesis protocols. We demonstrate the efficiency of the proposed pipeline on an expert-labeled set of nanomaterials synthesis articles, achieving 100% accuracy on composition prediction, 95% accuracy on morphology prediction, 0.99 AUC on protocol identification, and up to a 0.87 F1-score on chemical entity recognition. In addition to processing articles' text, microscopy images of nanomaterials within the articles are also automatically identified and analyzed to determine the nanomaterials' morphologies and size distributions. To enable users to easily explore the database, we developed a complementary browser-based visualization tool that provides flexibility in comparing across subsets of articles of interest. We use these tools and information to identify trends in nanomaterials synthesis, such as the correlation of certain reagents with various nanomaterial morphologies, which is useful in guiding hypotheses and reducing the potential parameter space during experimental design.

Accelerating the design of lattice structures using machine learning.

Lattices remain an attractive class of structures due to their design versatility; however, rapidly designing lattice structures with tailored or optimal mechanical properties remains a significant challenge. With each added design variable, the design space quickly becomes intractable. To address this challenge, research efforts have sought to combine computational approaches with machine learning (ML)-based approaches to reduce the computational cost of the design process and accelerate mechanical design. While these efforts have made substantial progress, significant challenges remain in (1) building and interpreting the ML-based surrogate models and (2) iteratively and efficiently curating training datasets for optimization tasks. Here, we address the first challenge by combining ML-based surrogate modeling and Shapley additive explanation (SHAP) analysis to interpret the impact of each design variable. We find that our ML-based surrogate models achieve excellent prediction capabilities (R2 > 0.95) and SHAP values aid in uncovering design variables influencing performance. We address the second challenge by utilizing active learning-based methods, such as Bayesian optimization, to explore the design space and report a 5 × reduction in simulations relative to grid-based search. Collectively, these results underscore the value of building intelligent design systems that leverage ML-based methods for uncovering key design variables and accelerating design.

Direct custom implant impression copings for the preservation of the pontic receptor site architecture.

The direct custom implant impression coping technique is designed to record the periimplant gingiva and pontic receptor site after the tissues have been shaped with a provisional restoration. The technique prevents inaccurate recording of the gingival architecture by using a dual polymerizing composite resin placed into the sulcus and pontic receptor sites and adapted to the open tray implant impression copings. This technique may improve soft tissue accuracy between the clinical condition and the laboratory cast.

Fine epitope mapping of monoclonal antibodies against hemagglutinin of a highly pathogenic H5N1 influenza virus using yeast surface display.

Highly pathogenic H5N1 avian influenza viruses pose a debilitating pandemic threat. Thus, understanding mechanisms of antibody-mediated viral inhibition and neutralization escape is critical. Here, a robust yeast display system for fine epitope mapping of viral surface hemagglutinin (HA)-specific antibodies is demonstrated. The full-length H5 subtype HA (HA0) was expressed on the yeast surface in a correctly folded conformation, determined by binding of a panel of extensively characterized neutralizing human monoclonal antibodies (mAbs). These mAbs target conformationally-dependent epitopes of influenza A HA, which are highly conserved across H5 clades and group 1 serotypes. By separately displaying HA1 and HA2 subunits on yeast, domain mapping of two anti-H5 mAbs, NR2728 and H5-2A, localized their epitopes to HA1. These anti-H5 mAb epitopes were further fine mapped by using a library of yeast-displayed HA1 mutants and selecting for loss of binding without prior knowledge of potential contact residues. By overlaying key mutant residues that impacted binding onto a crystal structure of HA, the NR2728 mAb was found to interact with a fully surface-exposed contiguous patch of residues at the receptor binding site (RBS), giving insight into the mechanism underlying its potent inhibition of virus binding. The non-neutralizing H5-2A mAb was similarly mapped to a highly conserved H5 strain-specific but poorly accessible location on a loop at the trimer HA interface. These data further augment our toolchest for studying HA antigenicity, epitope diversity and accessibility in response to natural and experimental influenza infection and vaccines.

Enhancement of salivary IgA response to a DNA vaccine against Streptococcus mutans wall-associated protein A in mice by plasmid-based adjuvants.

A specific salivary IgA (sIgA) response was obtained in mice by intranasal immunization with a naked DNA vaccine consisting of the Streptococcus mutans wall-associated protein A gene (wapA) inserted into the mammalian expression vector pcDNA3.1/V5/His-TOPO. In the present study, the vaccine, referred to as pcDNA-wapA, was administered with or without the cationic lipid DMRIE-C. No mucosal response was observed in mice immunized with the vaccine alone, whereas a weak and temporal sIgA response was obtained when the vaccine was mixed with DMRIE-C. To investigate the use of pcDNA containing the interleukin 5 (IL-5) gene (pcDNA-il-5) or the cholera toxin B gene (pcDNA-ctb) as genetic adjuvants, these constructs were used in co-immunization studies. The enhancement effect was transient with pcDNA-il-5, but longer lasting with pcDNA-ctb, thus supporting the use of the latter as a genetic adjuvant to DNA vaccine.

How do smoking, diabetes, and periodontitis affect outcomes of implant treatment?

PURPOSE: Implant therapy is highly predictable and successful. However, certain risk factors can predispose individuals to lower rates of success. The purpose of this systematic review was to evaluate the available literature to assess whether smoking, diabetes, and periodontitis have an adverse affect on the outcomes of implants placed in patients with these conditions. MATERIALS AND METHODS: The dental literature was searched using the MEDLINE, Cochrane Collaboration, and EMBASE databases. Using specific inclusion and exclusion criteria, 2 reviewers evaluated titles, abstracts, and full articles to identify articles relevant to this review. All searches were conducted for articles published through May 2005. Data from included articles for each of the risk factor groups, smoking, diabetes, and periodontitis, were abstracted and analyzed. RESULTS: A detailed search of the literature and evaluation of relevant articles identified 35 articles for inclusion in this systematic review. Nineteen articles were identified for smoking, 4 articles were identified for diabetes, and 13 articles were identified for periodontitis. One article met the criteria for both smoking and periodontitis. Implant survival and success rates were reported for smokers versus nonsmokers; diabetic patients versus nondiabetic patients; and patients with a history of treated periodontitis versus patients with no history of periodontitis. The findings revealed statistically significant differences in survival and success rates for smokers (better for nonsmokers), with greater differences observed when the data were analyzed according to bone quality (less for loose trabecular bone). No difference in implant survival rate was found between patients with and without diabetes. Likewise, no difference in implant survival rates was found between patients with a history of treated periodontitis compared to patients with no history of periodontitis. CONCLUSIONS: The results of this systematic review of the literature demonstrated that smoking has an adverse affect on implant survival and success. The effect of smoking on implant survival appeared to be more pronounced in areas of loose trabecular bone. Type 2 diabetes may have an adverse effect on implant survival rates, but the limited number of studies included in this review do not permit a definitive conclusion. A history of treated periodontitis does not appear to adversely affect implant survival rates but it may have a negative influence on implant success rates, particularly over longer periods.

Differential immunogenicity of a DNA vaccine containing the Streptococcus mutans wall-associated protein A gene versus that containing a truncated derivative antigen A lacking in the hydrophobic carboxyterminal region.

Two plasmid DNA constructs were obtained by cloning separately into the eukaryotic expression vector pcDNA3.1/V5-His-TOPO the wall-associated protein A (wapA) gene of Streptococcus mutans GS-5 or its truncated derivative antigen A (agA) gene encoding a known candidate antigen for dental caries vaccine. The immunogenicity of the two constructs, designated pcDNA-wapA and pcDNA-agA, was compared by intranasal immunization of two groups of mice using the cationic DMRIE-C (1,2-dimyristyloxypropyl-3-dimethylhydroxy ethyl ammonium bromide-cholesterol) as an adjuvant. Immunization with pcDNA-wapA or pcDNA- agA resulted in specific salivary IgA and systemic IgG antibodies to the target antigens after two doses given at 3-week intervals. Higher salivary IgA level was observed in the mice immunized with the pcDNA-wapA vaccine compared to those immunized with the pcDNA-agA vaccine. Furthermore, anti-WapA antibody inhibited S. mutans sucrose-dependent adherence suggesting a potential protection against S. mutans colonization of the tooth, while anti-AgA had no significant effect. Indeed, prediction and analysis of protein epitopes showed that WapA contains highly promiscuous MHC-II binding motifs in addition to those found in AgA. Immunodot assay confirmed that WapA bound biotin-labeled dextran, whereas AgA did not. These data indicated that full-length WapA is a better candidate vaccine antigen than the soluble AgA, which is truncated in the hydrophobic membrane and wall-spanning region.

Platelet-derived growth factor stimulates bone fill and rate of attachment level gain: results of a large multicenter randomized controlled trial.

BACKGROUND: Growth factors are generally accepted to be essential mediators of tissue repair via well-established mechanisms of action that include stimulatory effects on angiogenesis and cellular proliferation, ingrowth, differentiation, and matrix biosynthesis. The aim of this study was to evaluate in a large-scale, prospective, blinded, and randomized controlled clinical trial the safety and effectiveness of purified recombinant human platelet-derived growth factor (rhPDGF-BB) mixed with a synthetic beta-tricalcium phosphate (beta-TCP) matrix for the treatment of advanced periodontal osseous defects at 6 months of healing. METHODS: Eleven clinical centers enrolled 180 subjects, each requiring surgical treatment of a 4 mm or greater intrabony periodontal defect and meeting all inclusion and exclusion criteria. Subjects were randomized into one of three treatment groups: 1) beta-TCP + 0.3 mg/ml rhPDGF-BB in buffer; 2) beta-TCP + 1.0 mg/ml rhPDGF-BB in buffer; and 3) beta-TCP + buffer (active control). Safety data were assessed by the frequency and severity of adverse events. Effectiveness measurements included clinical attachment levels (CAL) and gingival recession (GR) measured clinically and linear bone growth (LBG) and percent bone fill (% BF) as assessed radiographically by an independent centralized radiology review center. The area under the curve (AUC), an assessment of the rate of healing, was also calculated for CAL measurements. The surgeons, clinical and radiographic evaluators, patients, and study sponsor were all masked with respect to treatment groups. RESULTS: CAL gain was significantly greater at 3 months for group 1 (rhPDGF 0.3 mg/ml) compared to group 3 (beta-TCP + buffer) (3.8 versus 3.3 mm; P = 0.032), although by 6 months, this finding was not statistically significant (P = 0.11). This early acceleration of CAL gain led to group 1 exhibiting a significantly greater rate of CAL gain between baseline and 6 months than group 3 as assessed by the AUC (68.4- versus 60.1-mm weeks; P = 0.033). rhPDGF (0.3 mg/ml)-treated sites also had significantly greater linear bone gain (2.6 versus 0.9 mm, respectively; P < 0.001) and percent defect fill (57% versus 18%, respectively; P < 0.001) than the sites receiving the bone substitute with buffer at 6 months. There was less GR at 3 months in group 1 compared to group 3 (P = 0.04); at 6 months, GR for group 1 remained unchanged, whereas there was a slight gain in gingival height for group 3 resulting in comparable GR. There were no serious adverse events attributable to any of the treatments. CONCLUSIONS: To our knowledge, this study is the largest prospective, randomized, triple-blinded, and controlled pivotal clinical trial reported to date assessing a putative periodontal regenerative and wound healing therapy. The study demonstrated that the use of rhPDGF-BB was safe and effective in the treatment of periodontal osseous defects. Treatment with rhPDGF-BB stimulated a significant increase in the rate of CAL gain, reduced gingival recession at 3 months post-surgery, and improved bone fill as compared to a beta-TCP bone substitute at 6 months.

Human anti-CCR4 minibody gene transfer for the treatment of cutaneous T-cell lymphoma.

BACKGROUND: Although several therapeutic options have become available for patients with Cutaneous T-cell Lymphoma (CTCL), no therapy has been curative. Recent studies have demonstrated that CTCL cells overexpress the CC chemokine receptor 4 (CCR4). METHODOLOGY/PRINCIPAL FINDINGS: In this study, a xenograft model of CTCL was established and a recombinant adeno-associated viral serotype 8 (AAV8) vector expressing a humanized single-chain variable fragment (scFv)-Fc fusion (scFvFc or "minibody") of anti-CCR4 monoclonal antibody (mAb) h1567 was evaluated for curative treatment. Human CCR4+ tumor-bearing mice treated once with intravenous infusion of AAV8 virions encoding the h1567 (AAV8-h1567) minibody showed anti-tumor activity in vivo and increased survival. The AAV8-h1567 minibody notably increased the number of tumor-infiltrating Ly-6G+ FcγRIIIa(CD16A)+ murine neutrophils in the tumor xenografts over that of AAV8-control minibody treated mice. Furthermore, in CCR4+ tumor-bearing mice co-treated with AAV8-h1567 minibody and infused with human peripheral blood mononuclear cells (PBMCs), marked tumor infiltration of human CD16A+ CD56+ NK cells was observed. The h1567 minibody also induced in vitro ADCC activity through both mouse neutrophils and human NK cells. CONCLUSIONS/SIGNIFICANCE: Overall, our data demonstrate that the in vivo anti-tumor activity of h1567 minibody is mediated, at least in part, through CD16A+ immune effector cell ADCC mechanisms. These data further demonstrate the utility of the AAV-minibody gene transfer system in the rapid evaluation of candidate anti-tumor mAbs and the potency of h1567 as a potential novel therapy for CTCL.

Structural basis of influenza virus neutralization.

Although seasonal influenza vaccines play a valuable role in reducing the spread of virus at the population level, ongoing viral evolution to evade immune responses remains problematic. No current vaccines elicit enduring protection in the face of emerging and re-emerging influenza viruses that are rapidly undergoing antigenic drift. Eliciting broadly cross-neutralizing antibody (nAb) responses against influenza virus is a crucial goal for seasonal and pandemic influenza vaccine preparation. Recent three-dimensional structure information obtained from crystallization of influenza antigens in complex with nAbs has provided a framework for interpreting antibody-based viral neutralization that should aid in the design of vaccine immunogens. Here, we will review current knowledge of the structure-based mechanisms contributing to the neutralization and neutralization escape of influenza viruses. We will also explore the potential for this structure-based approach to overcome the obstacles in developing the highly desired "universal" influenza vaccine.

Anti-gp120 minibody gene transfer to female genital epithelial cells protects against HIV-1 virus challenge in vitro.

BACKGROUND: Although cervico-vaginal epithelial cells of the female lower genital tract provide the initial defense system against HIV-1 infection, the protection is sometimes incomplete. Thus, enhancing anti-HIV-1 humoral immunity at the mucosal cell surface by local expression of anti-HIV-1 broadly neutralizing antibodies (BnAb) that block HIV-1 entry would provide an important new intervention that could slow the spread of HIV/AIDS. METHODS AND FINDINGS: This study tested the hypothesis that adeno-associated virus (AAV)-BnAb gene transfer to cervico-vaginal epithelial cells will lead to protection against HIV-1. Accordingly, a recombinant AAV vector that encodes human b12 anti-HIV gp120 BnAb as a single-chain variable fragment Fc fusion (scFvFc), or "minibody" was constructed. The secreted b12 minibody was shown to be biologically functional in binding to virus envelope protein, neutralizing HIV-1 and importantly, blocking transfer and infectivity of HIV-1(bal) in an organotypic human vaginal epithelial cell (VEC) model. Furthermore, cervico-vaginal epithelial stem cells were found to be efficiently transduced by the optimal AAV serotype mediated expression of GFP. CONCLUSION: This study provides the foundation for a novel microbicide strategy to protect against sexual transmission of HIV-1 by AAV transfer of broadly neutralizing antibody genes to cervico-vaginal epithelial stem cells that could replenish b12 BnAb secreting cells through multiple menstrual cycles.

Structural and functional bases for broad-spectrum neutralization of avian and human influenza A viruses.

Influenza virus remains a serious health threat, owing to its ability to evade immune surveillance through rapid genetic drift and reassortment. Here we used a human non-immune antibody phage-display library and the H5 hemagglutinin ectodomain to select ten neutralizing antibodies (nAbs) that were effective against all group 1 influenza viruses tested, including H5N1 'bird flu' and the H1N1 'Spanish flu'. The crystal structure of one such nAb bound to H5 shows that it blocks infection by inserting its heavy chain into a conserved pocket in the stem region, thus preventing membrane fusion. Nine of the nAbs employ the germline gene VH1-69, and all seem to use the same neutralizing mechanism. Our data further suggest that this region is recalcitrant to neutralization escape and that nAb-based immunotherapy is a promising strategy for broad-spectrum protection against seasonal and pandemic influenza viruses.

Bmi-1 cooperates with human papillomavirus type 16 E6 to immortalize normal human oral keratinocytes.

Bmi-1 is a member of the polycomb group (PcG) transcription repressors and is implicated in human carcinogenesis. In normal human oral keratinocytes (NHOK), we found that exogenous Bmi-1 expression significantly extended the replicative life span without causing cellular immortalization. Immortalization of NHOK occurs only in combination with human papillomavirus type 16 E6 (HPV-16 E6) but not with E7. During immortalization of NHOK by sequential expression of exogenous Bmi-1 and E6, telomerase activation was observed only after the cells had overcome crisis. Genetic analysis with E6 deletion mutants revealed that the intact second zinc finger domain (amino acids 118-122) was necessary for its cooperative effects with Bmi-1 in the immortalization process. Using these mutants, we found that the increased telomerase activity was closely associated with cell immortalization by Bmi-1 and E6, whereas p53 degradation was not. Using microarray analysis, we identified genes that are immortalization-specific and may participate in the process of NHOK immortalization by Bmi-1 and HPV-16 E6. Our results provide new information on the roles of Bmi-1 and HPV-16 E6 in the multi-step process of oral epithelial carcinogenesis.

Potential of two-photon-excited fluorescence for distributed fiber sensing.

The design and operating parameters of a new class of continuously distributed optical fiber sensor are described. By use of counterpropagating pulses, two-photon excitation of fluorescence from ions doped into the fiber enables any position to be monitored. By this means temperature and also strain may be sensed with high spatial and temporal resolution. As the doped fiber is transparent for single-photon absorption at the wavelength of the light pulses, attenuation does not set an upper limit to its length.

Identification and characterization of collagen-binding activity in Streptococcus mutans wall-associated protein: a possible implication in dental root caries and endocarditis.

Streptococcus mutans is implicated in coronal and dental root decay, and in endocarditis. Comparative study of the amino acid sequence of S. mutans 47 kDa wall-associated protein A (WapA) revealed a collagen-binding domain (CBD) at the N-terminal region. Recombinant AgA (WapA truncated at the carboxyterminal end) was isolated, biotin-labeled, and analyzed by Solid Phase Binding Assay. The results showed that biotin-labeled AgA bound significantly and in a dose-dependent manner to immobilized collagen type I, and to a lesser extent to fibronectin, but not to collagen type IV or laminin. Binding of biotin-labeled S. mutans cells to collagen-coated surfaces was significantly inhibited by antibody to WapA or AgA (P<0.001). The results obtained confirmed the collagen-binding activity of CBD in AgA and WapA, and suggested that WapA may be used, not only as a vaccine against coronal and dental root caries, but also against S. mutans-mediated endocarditis.

Identification, molecular cloning, and sequence analysis of a deoxyribose aldolase in Streptococcus mutans GS-5.

Bacterial fitness in the environment, where nutrients are limited and competition is intense, plays a central role in survival and virulence of the organisms. Deoxyribose aldolase, found in several species of bacteria, is known to be involved in the catabolism of deoxynucleosides arising from dead cells, thereby giving a selective advantage to the microorganisms with a capability to consume DNA as an alternative carbon and energy source. A gene encoding a deoxyribose aldolase gene ( deoC) was identified in the cariogenic Streptococcus mutans strain GS-5 by comparative sequence analysis and gene cloning. The gene encodes a protein of 220 amino acids, having a predicted molecular weight of 23.3 kDa with a p I of 5.44. The gene was cloned into the expression vector pFLAG-1, and the biological function of the gene product was analyzed by a complementation assay with a deoC(-) Escherichia coli mutant SPhi063. Transformation of the E. coli SPhi063 with the plasmid construct allowed this organism to grow on glucose minimal medium supplemented with 2 mM deoxyadenosine or deoxythymidine. These results showed activity of deoxyribose aldolase, confirming the identity of the gene. Utilization of exogenous deoxynucleotides as a carbon and energy source may confer a survival and growth advantage to S. mutans over other bacteria in dental plaque, suggesting that deoxyribose aldolase may be a contributing factor to virulence.

Effects of brevetoxins on murine myeloma SP2/O cells: aberrant cellular division.

Massive deaths of manatees (Trichechus manatus latirostris) during the red tide seasons have been attributed to brevetoxins produced by the dinoflagellate Karenia brevis (formerly Ptychodiscus breve and Gymnodinium breve). Although these toxins have been found in macrophages and lymphocytes in the lung, liver, and secondary lymphoid tissues of these animals, the molecular mechanisms of brevetoxicosis have not yet been identified. To investigate the effects of brevetoxins on immune cells, a murine myeloma cell line (SP2/O) was used as a model for in vitro studies. By adding brevetoxins to cultures of the SP2/O cells at concentrations ranging from 20 to 600 ng/ml, an apparent increase in proliferation was observed at around 2 hours post challenge as compared to the unchallenged cell cultures. This was followed by a drop in cell number at around 3 hours, suggesting an aberrant effect of brevetoxins on cellular division, the cells generated at 2 hours being apparently short-lived. In situ immunochemical staining of the SP2/O cells at 1 and 2 hour post challenge showed an accumulation of the toxins in the nucleus. A 21-kDa protein was subsequently isolated from the SP2/O cells as having brevetoxin-binding properties, and immunologically identified as p21, a nuclear factor known to down-regulate cellular proliferation through inhibition of cyclin-dependent kinases. These data are the first on a possible effect of brevetoxins on the cell cycle via binding to p21, a phenomenon that needs to be further investigated and validated in normal immune cells.