Emerging strategies to fabricate polymeric nanocarriers for enhanced drug delivery across blood-brain barrier: An overview.

Blood-brain barrier (BBB) serves as an essential interface between central nervous system (CNS) and its periphery, allowing selective permeation of ions, gaseous molecules, and other nutrients to maintain metabolic functions of brain. Concurrently, it restricts passage of unsolicited materials from bloodstream to CNS which could otherwise lead to neurotoxicity. Nevertheless, in the treatment of neurodegenerative diseases such as Parkinson's, Alzheimer's, diffuse intrinsic pontine glioma, and other brain cancers, drugs must reach CNS. Among various materials developed for this purpose, a few judiciously selected polymeric nanocarriers are reported to be highly prospective to facilitate BBB permeation. However, the challenge of transporting drug-loaded nanomaterials across this barrier remains formidable. Herein a concise analysis of recently employed strategies for designing polymeric nanocarriers to deliver therapeutics across BBB is presented. Impacts of 3Ss, namely, size, shape, and surface charge of polymeric nanocarriers on BBB permeation along with different ligands used for nanoparticle surface modification to achieve targeted delivery have been scrutinized. Finally, we elucidated future research directions in the context of designing smart polymeric nanocarriers for BBB permeation. This work aims to guide researchers engaged in polymeric nanocarrier design, helping them navigate where to begin, what challenges to address, and how to proceed effectively.

pH-responsive polyelectrolyte complexation on upconversion nanoparticles: a multifunctional nanocarrier for protection, delivery, and 3D-imaging of therapeutic protein.

The delicate tertiary structure of proteins, their susceptibility to heat- and enzyme-induced irreversible denaturation, and their tendency to get accumulated at the cell membrane during uptake are daunting challenges in proteinaceous therapeutic delivery. Herein, a polyelectrolyte complex having encapsulated therapeutic protein has been designed on the surface of upconverting luminescent nanoparticles (NaYF4:20%Yb3+,2%Er3+). This nanosized complex system has been found to overcome the challenges of protein aggregation at the cell membrane. It has also defended the cargo from denaturation against (a) enzymatic action of proteinase K and (b) heat (up to 60 °C). Additionally, the nanoparticles at the core of the loaded carrier served as near-infrared (980 nm) responsive probe to accomplish extended-duration 3D imaging during protein delivery. The outer layer of polymer played pivotal role to protect/retrieve the protein structure from denaturation as investigated by circular dichroism studies. Both the masked surface-charges of protein and the nanoscale size of the loaded carrier have facilitated their efficient passage through the cell membrane as observed through 3D images/videos. This nanocarrier is the first of its kind for direct delivery of protein. Thus, the findings can be useful to protect and transport various proteinaceous materials to overcome challenges of accumulation at the cell-membrane and low-temperature storage, as nature does.

Effect of Immersive Virtual Reality-Based Bilateral Arm Training in Patients with Chronic Stroke.

Virtual reality (VR)-based therapies are widely used in stroke rehabilitation. Although various studies have used VR techniques for bilateral upper limb training, most have been only semi-immersive and have only been performed in an artificial environment. This study developed VR content and protocols based on activities of daily living to provide immersive VR-based bilateral arm training (VRBAT) for upper limb rehabilitation in stroke patients. Twelve patients with chronic stroke were randomized to a VRBAT group or a normal bilateral arm training (NBAT) group and attended 30-min training sessions five times a week for four weeks. At the end of the training, there was a significant difference in upper limb function in both groups (p < 0.05) and in the upper limb function sensory test for proprioception in the NBAT group (p < 0.05). There was no significant between-group difference in upper limb muscle activity after training. The relative alpha and beta power values for electroencephalographic measurements were significantly improved in both groups. These findings indicate that both VRBAT and NBAT are effective interventions for improving upper limb function and electroencephalographic activity in patients with chronic stroke.

Uptake of Polyelectrolyte Functionalized Upconversion Nanoparticles by Tau-Aggregated Neuron Cells.

Tauopathy is the aggregation phenomenon of tau proteins and associated with neurodegenerative diseases. It metastasizes via the transfer of tau aggregates to adjacent neuron cells; however, the mechanism has not yet been fully understood. Moreover, if the materials used for designing drug delivery system to treat such neurodegenerative diseases do not undergo biodegradation or exocytosis but remains in cells or tissues, they raise concerns about their possible negative impacts. In this study, the uptake and delivery mechanisms of nano-sized carriers in tau aggregated neuron cells were investigated employing polyelectrolyte-functionalized upconversion nanoparticles (UCNPs) of diameter ~100 nm. Investigation through bioimaging was carried out by irradiating the particles with near-infrared light. Here, forskolin and okadaic acid were employed to induce tau aggregation into healthy neuron cells. It was noticed that the tau-aggregated neuron cells, when treated with relatively large sized UCNPs, showed uptake efficiency similar to that of normal neuron cells however their intracellular transport and exocytosis were impacted, and most of the carriers remained accumulated around lysosome. This demonstrates that metastasis mechanisms of tauopathy can get influenced by the size of carriers and are to be considered during their pharmacokinetic studies which is often not addressed in many drug delivery studies.

Structural basis for the inhibition of PDK2 by novel ATP- and lipoyl-binding site targeting compounds.

Pyruvate dehydrogenase kinase (PDK) controls the activity of pyruvate decarboxylase complex (PDC) by phosphorylating key serine residues on the E1 subunit, which leads to a decreased oxidative phosphorylation in mitochondria. Inhibition of PDK activity by natural/synthetic compounds has been shown to reverse the Warburg effect, a characteristic metabolism in cancer cells. PDK-PDC axis also has been associated with diabetes and heart disease. Therefore, regulation of PDK activity has been considered as a promising strategy to treat related diseases. Here we present the X-ray crystal structure of PDK2 complexed with a recently identified PDK4 inhibitor, compound 8c, which has been predicted to bind at the lipoyl-binding site and interrupt intermolecular interactions with the E2-E3bp subunits of PDC. The co-crystal structure confirmed the specific binding location of compound 8c and revealed the remote conformational change in the ATP-binding pocket. In addition, two novel 4,5-diarylisoxazole derivatives, GM10030 and GM67520, were synthesized and used for structural studies, which target the ATP-binding site of PDK2. These compounds bind to PDK2 with a sub-100nM affinity as determined by isothermal titration calorimetry experiments. Notably, the crystal structure of the PDK2-GM10030 complex displays unprecedented asymmetric conformation of human PDK2 dimer, especially in the ATP-lids and C-terminal tails.

Anomalous Dynamics of in Vivo Cargo Delivery by Motor Protein Multiplexes.

Vesicle transport conducted by motor protein multiplexes (MPMs), which is ubiquitous among eukaryotes, shows anomalous and stochastic dynamics qualitatively different from the dynamics of thermal motion and artificial active matter; the relationship between in vivo vesicle-delivery dynamics and the underlying physicochemical processes is not yet quantitatively understood. Addressing this issue, we perform accurate tracking of individual vesicles, containing upconverting nanoparticles, transported by kinesin-dynein-multiplexes along axonal microtubules. The mean-square-displacement of vesicles along the microtubule exhibits unusual dynamic phase transitions that are seemingly inconsistent with the scaling behavior of the mean-first-passage time over the travel length. These paradoxical results and the vesicle displacement distribution are quantitatively explained and predicted by a multimode MPM model, developed in the current work, where ATP-hydrolysis-coupled motion of MPM has both unidirectional and bidirectional modes.

Two-Dimensional and Three-Dimensional Single Particle Tracking of Upconverting Nanoparticles in Living Cells.

Lanthanide-doped upconversion nanoparticles (UCNPs) are inorganic nanomaterials in which the lanthanide cations embedded in the host matrix can convert incident near-infrared light to visible or ultraviolet light. These particles are often used for long-term and real-time imaging because they are extremely stable even when subjected to continuous irradiation for a long time. It is now possible to image their movement at the single particle level with a scale of a few nanometers and track their trajectories as a function of time with a scale of a few microseconds. Such UCNP-based single-particle tracking (SPT) technology provides information about the intracellular structures and dynamics in living cells. Thus far, most imaging techniques have been built on fluorescence microscopic techniques (epifluorescence, total internal reflection, etc.). However, two-dimensional (2D) images obtained using these techniques are limited in only being able to visualize those on the focal planes of the objective lens. On the contrary, if three-dimensional (3D) structures and dynamics are known, deeper insights into the biology of the thick cells and tissues can be obtained. In this review, we introduce the status of the fluorescence imaging techniques, discuss the mathematical description of SPT, and outline the past few studies using UCNPs as imaging probes or biologically functionalized carriers.

Recent progress on biocompatible nanocarrier-based genistein delivery systems in cancer therapy.

Diets with naturally occuring chemopreventive agents are showing good potentials in serving dual purposes: firstly, for maintaining health, and secondly, for emerging as most puissant cost-effective strategy against chronic diseases like cancer. Genistein, one of the active soy isoflavone, is gaining attention due to its ability to impede carcinogenic processes by regulating wide range of associated molecules and signalling mechanisms. Epidemiologic and preclinical evidences suggest that sufficient consumption of soy-based food having genistein can be correlated to the reduction of cancer risk. However, certain adverse effects like poor oral bioavailability, low aqueous solubility and inefficient pharmacokinetics have pushed it down in the list of phytoconstituents currently undergoing successful clinical trials. In order to maximise the utilisation of therapeutic benefits of this phytoestrogen, suitable drug carrier designs are required. Recently, nanocarriers, mainly composed of polymeric materials, are progressively and innovatively exploited with the aim to improve pharmacokinetics and pharmacodynamics of genistein. Here, we have briefly reviewed (a) the targeted molecular mechanisms of geinstein, (b) nanopolymeric approaches opted so far in designing carriers and (c) the reasons behind their restricted clinical applications. Finally, some mechanism-based approaches are proposed presenting genistein as the future paradigm in cancer therapy.

Cellular uptake efficiency of nanoparticles investigated by three-dimensional imaging.

Understanding the interaction of nanoparticles with living cells on the basis of cellular uptake efficiency is a fundamental requisite in biomedical research. Cellular internalization of nanoparticles takes place by mechanisms like ATP hydrolysis-driven endocytosis that deliver nanoparticles to the cytoplasm, organelles and nuclei. Despite its importance in nanomedicine, this uptake procedure is not understood in-depth because of the complexity of the biochemical mechanisms and the lack of available experimental methods for quantitative analysis. The only breakthrough is likely to be the development of imaging techniques that can visualize, monitor and even count the number of nanoparticles inside the cell. To this end, we report here a new, fast and background-free three-dimensional (3-D) imaging technique with quantitative evaluation of the uptake efficiency for NaYF4:Yb3+,Er3+/NaYF4 core/shell upconversion nanoparticles (UCNPs) functionalized with different chemical and biological groups. Furthermore, the multiple 3-D trajectories of the UCNPs have been analyzed to investigate the cellular dynamics. This study reveals the nuclear uptake of UCNPs to be dependent on the specific chemical groups conjugated to the UCNPs. The developed 3-D imaging technique is of great significance for exploring complex biological systems.

Osteoconductivity of Binary Titanium Alloys with Different Micro/Nanoporous Surfaces.

The surface characteristics and osteoconductivity were evaluated for the micro/nanoporous surfaces of titanium (Ti) alloys prepared by micro-arc oxidation (MAO) and hydrothermal treatment (HT) of binary Ti-5 wt% A alloys (A = Au, Mn, Nb, and Pd). Surface properties were analyzed using X-ray diffractometry, scanning electron microscopy, and energy dispersive X-ray spectroscopy. The osteoconductivity was evaluated by measuring the total protein, ALPase activity, and osteocalcin production. The surface morphologies of MAO/HT specimens mainly affected on their osteoconductivity. Total proteins on Ti alloys (MAO/HT) were slightly lower than that on commercially pure Ti (MAO/HT) after incubation of MG-63 osteoblast-like cells for 14 days. However, better ALPase activity and osteocalcin production were observed on MAO/HT-treated Ti­5Mn, Ti­5Nb, and Ti­5Pd than that on cp-Ti (MAO/HT) after 14 days. Especially, Ti­5Mn (MAO/HT) showed a significant increase of ALPase activity due to its well grown micro/nano structure. Meanwhile, very small nanorods on Ti­5Au (MAO/HT) affected negatively to ALPase activity and osteocalcin production.

Fast and background-free three-dimensional (3D) live-cell imaging with lanthanide-doped upconverting nanoparticles.

We report on the development of a three-dimensional (3D) live-cell imaging technique with high spatiotemporal resolution using lanthanide-doped upconverting nanoparticles (UCNPs). It employs the sectioning capability of confocal microscopy except that the two-dimensional (2D) section images are acquired by wide-field epi-fluorescence microscopy. Although epi-fluorescence images are contaminated with the out-of-focus background in general, the near-infrared (NIR) excitation used for the excitation of UCNPs does not generate any autofluorescence, which helps to lower the background. Moreover, the image blurring due to defocusing was naturally eliminated in the image reconstruction process. The 3D images were used to investigate the cellular dynamics such as nuclear uptake and single-particle tracking that require 3D description.

Surface Characterization and Osteoconductivity Evaluation of Micro/Nano Surface Formed on Titanium Using Anodic Oxidation Combined with H2O2 Etching and Hydrothermal Treatment.

In this study, surface characteristics and osteoconductivity were investigated for the micro/nanostructured oxide layers fabricated on titanium using anodic oxidation (ANO), chemical etching (Et), and hydrothermal treatment (HT). Commercially pure titanium (CP-Ti) disks were anodic-oxidized using DC-type power supply in 1 M phosphoric acid electrolyte (P-ANO group). These specimens were further chemically etched using 30% H2O2 solution at 60 °C for 10 min (P-ANO-Et group). The P-ANO-Et-HT group was fabricated by hydrothermally treating the P-ANO-Et specimens in phosphorus-containing alkaline solution at 190 °C for 8 hrs. The P-ANO group showed a porous surface that was evenly covered with micro- and sub-micro pores. The size of these pores was decreased in the P-ANO-Et group. The P-ANO-Et-HT group showed a porous surface that was covered with nano-sized crystallites. Anatase TiO2 structure was observed in P-ANO-Et-HT group. The results of XPS demonstrated that the P-ANO-Et-HT group had a well-crystallized TiC2 structure, while the P-ANO and P-ANO-Et groups had an amorphous and phosphate-containing structure. Hydrophilicity of the P-ANO-Et-HT group was the highest. After MG63 osteoblast-like cells were cultured on the specimens for 3 hrs, SEM images of the cells cultured on P-ANO-Et-HT group specimens showed low initial adhesion. However, the osteoconductivity of these specimens increased more rapidly compared to that of the micro-structured surfaces. These results could be applied to fabricate titanium implants with an optimum micro/nano-surface for enhancing their osteoconductivity.

Cytotoxicity of soft denture lining materials depending on their component types.

PURPOSE: To evaluate the difference in cytotoxicity of soft denture lining materials depending on their component types. MATERIALS AND METHODS: Ten commercially available soft denture lining materials (SDLM) consisting of five silicone-based materials and five acrylic-based materials were evaluated. For the MTT test, cured SDLM samples were extracted in a culture medium for 24 hours, and L-929 cells were incubated in the extracted medium for 24 hours. Cell viability was determined using a microplate reader and compared with those of the negative control, which were cultured in a culture medium without test material. Agar overlay test was performed for the cured SDLM samples according to International Organization for Standardization (ISO) 7405. RESULTS: Among silicone-based lining materials, GC Reline Soft, Mollosil plus, and Dentusil showed a cell viability of 107.2% ± 4.5%, 102.3% ± 2.84%, and 93.0% ± 8.0%, respectively, compared with the control. Mucopren and Sofreliner Tough displayed significantly lower cell viability (86.4% ± 10.3% and 81.5% ± 4.3%,respectively) compared with the control (P < .05). Among acrylic-based materials, Kooliner, Visco-gel, Soft liner, Dura Base, and Coe-Soft displayed cell viability of 99.2% ± 14.6%, 93.1% ± 9.5%, 89.1% ± 9.8%, 87.6% ± 7.9%, and 75.9% ± 15.7%, respectively, compared with the control. Dura Base and Coe-Soft displayed significantly lower cell viability compared to the control. However, for all tested materials, cell viability exceeded the requirement limit of 70% specified in ISO 10993-5. In the agar overlay test, all five silicone-based materials and acrylic-based Kooliner were ranked as "noncytotoxic." However, Visco-gel was ranked as "mildly cytotoxic," and Soft liner, Coe-Soft, and Dura Base were ranked as "moderately cytotoxic." CONCLUSION: When an acrylic-based soft denture lining material is used, the possibility of a cytotoxic effect should be considered.

Cytotoxicity of alloying elements and experimental titanium alloys by WST-1 and agar overlay tests.

OBJECTIVE: This study was performed to evaluate the biocompatibility of nine types of pure metals using 36 experimental prosthetic titanium-based alloys containing 5, 10, 15, and 20wt% of each substituted metal. METHODS: The cell viabilities for pure metals on Ti alloys that contain these elements were compared with that of commercially pure (CP) Ti using the WST-1 test and agar overlay test. RESULTS: The ranking of pure metal cytotoxicity from most potent to least potent was: Co>Cu>In>Ag>Cr>Sn>Au>Pd>Pt>CP Ti. The cell viability ratios for pure Co, Cu, In, and Ag were 13.9±4.6%, 21.7±10.4%, 24.1±5.7%, and 24.8±6.0%, respectively, which were significantly lower than that for the control group (p<0.05). Pure Pd and Pt demonstrated good biocompatibility with cell viabilities of 93.8±9.6% and 97.2±7.1%, respectively. The Ti-5Pd alloy exhibited the highest cell viability (128.4±21.4%), which was greater than that of CP Ti. By alloying pure Co or Cu with Ti, the cell viabilities for the Ti-xCo and Ti-xCu alloys increased significantly up to 10wt% of the alloying element followed by a gradual decrease with a further increase in the concentration of the alloying element. Based on the agar overlay test, pure Ag, Co, Cr, Cu, and In were ranked as 'moderately cytotoxic', whereas all Ti alloys were ranked as 'noncytotoxic'. SIGNIFICANCE: The cytotoxicity of pure Ag, Co, Cr, Cu, and In suggests a need for attention in alloy design. The cytotoxicity of alloying elements became more biocompatible when they were alloyed with titanium. However, the cytotoxicity of titanium alloys was observed when the concentration of the alloying element exceeded its respective allowable limit. The results obtained in this study can serve as a guide for the development of new Ti-based alloy systems.

Osteoblast cell adhesion and viability on nanostructured surfaces of porous titanium oxide layer.

Surface characteristics and osteoblast cell functions were investigated for the nano-structured oxide layer on commercially pure titanium (CP-Ti) fabricated using microarc oxidation (MAO) and hydrothermal treatment (HT) methods. Ti-MAO-135HT, Ti-MAO-150HT, and Ti-MAO-175HT groups were fabricated by hydrothermally treating the MAO-treated specimens (Ti-MAO) in phosphorus-containing alkaline solution at temperatures of 135, 150, or 175 °C, respectively. After hydrothermal treatment, a nanosheet-shaped morphology, nano-needles and nanorods were observed on the porous surface of the Ti-MAO-135HT, Ti-MAO-150HT and Ti-MAO-175HT groups, respectively. The roughness was not significantly different for all groups. However the contact angle decreased dramatically as the hydrothermal temperature increased. The osteoblastic cell adhesion and viability of the Ti-MAO-150HT and Ti-MAO-175HT groups were significantly lower compared to those of the Ti-MAO group. This study showed that nano-topology formed on micro porous oxide layer was more important than hydrophilicity in its effect upon initial osteoblastic cell functions.

Cytocompatibility of pure metals and experimental binary titanium alloys for implant materials.

OBJECTIVE: This study was performed to evaluate the biocompatibility of nine types of pure metal ingots (Ag, Al, Cr, Cu, Mn, Mo, Nb, V, Zr) and 36 experimental titanium (Ti) alloys containing 5, 10, 15, and 20 wt% of each alloying element. METHODS: The cell viabilities for each test group were compared with that of CP-Ti using the WST-1 test and agar overlay test. RESULTS: The ranking of pure metal cytotoxicity from most potent to least potent was as follows: Cu>Al>Ag>V>Mn>Cr>Zr>Nb>Mo>CP-Ti. The mean cell viabilities for pure Cu, Al, Ag, V, and Mn were 21.6%, 25.3%, 31.7%, 31.7%, and 32.7%, respectively, which were significantly lower than that for the control group (p<0.05). The mean cell viabilities for pure Zr and Cr were 74.1% and 60.6%, respectively (p<0.05). Pure Mo and Nb demonstrated good biocompatibility with mean cell viabilities of 93.3% and 93.0%, respectively. The mean cell viabilities for all the Ti-based alloy groups were higher than 80% except for Ti-20 Nb (79.6%) and Ti-10 V (66.9%). The Ti-10 Nb alloy exhibited the highest cell viability (124.8%), which was higher than that of CP-Ti. Based on agar overlay test, pure Ag, Cr, Cu, Mn, and V were ranked as 'moderately cytotoxic', whereas the rest of the tested pure metals and all Ti alloys, except Ti-10 V (mild cytotoxicity), were ranked as 'noncytotoxic'. SIGNIFICANCE: The results obtained in this study can serve as a guide for the development of new Ti-based alloy implant systems.

Chemical composition and antimicrobial activity of the essential oil of Cryptomeria japonica.

The composition and the antibacterial activity of the essential oil obtained from Cryptomeria japonica D. Don on oral bacteria were studied. The chemical composition of the essential oil was analysed by GC and GC-MS. Sixty-eight compounds accounting for 95.82% of the oil were identified. The main compounds in the oil were alpha-pinene (6.07%), sabinene (8.86%), terpinen-4-ol (9.77%), alpha-terpineol (6.13%), elemol (11.17%) and 10(15)-cadinen-4-ol (7.16%). The essential oil and some of its major compounds were tested for antimicrobial activity against 15 different genera of oral bacteria. The essential oil of C. japonica exhibited considerable inhibitory effects against all bacteria tested (MICs, 0.025-0.05 mg/mL; MBCs, 0.025-0.1 mg/mL), while its major compounds demonstrated various degrees of growth inhibition.

Chemical composition and antimicrobial activity of the essential oil of Artemisia lavandulaefolia.

The chemical components of the essential oil obtained from Artemisia lavandulaefolia DC. were analyzed by GC-MS. Ninety-nine compounds accounting for 94.9 % of the essential oil were identified. The major compounds in the essential oil were beta-caryophyllene (16.1 %), cis-chrysanthenol (7.0 %), 1,8-cineole (5.6 %), borneol (5.3 %), trans-beta-farnesene (5.1 %), camphor (4.9 %), yomogi alcohol (4.5 %), alpha-terpineol (3.9 %), and alpha-humulene oxide (3.3 %). The essential oil and some of its major compounds were tested for antimicrobial activity against 15 different genera of oral bacteria. The essential oil of A. lavandulaefolia exhibited considerable inhibitory effects against all obligate anaerobic bacteria (MIC values, 0.025 to 0.05 mg/mL; MBC values, 0.025 to 0.1 mg/mL) tested, while its major compounds demonstrated different degrees of growth inhibition.

Chemical composition and antimicrobial activity of the essential oils of Artemisia scoparia and A. capillaris.

The chemical composition of the essential oils obtained from Artemisia scoparia Waldst. et Kitamura and Artemisia capillaris Thunb. was analyzed by GC/MS. The essential oil of A. scoparia was rich in camphor (11.0 %), 1,8-cineole (21.5 %), and beta-caryophyllene (6.8 %) as the major compounds, whereas A. capillaris oil was rich in beta-pinene (9.4 %), beta-caryophyllene (11.1 %), and capillene (32.7 %). The essential oils and some of their major compounds were tested for their antimicrobial activity against 15 different genera of oral bacteria. The essential oils of A. scoparia and A. capillaris exhibited considerable inhibitory effects against all oral bacteria tested, while their major components demonstrated various degrees of growth inhibition.

Genes of periodontopathogens expressed during human disease.

BACKGROUND: Since many bacterial genes are environmentally regulated, the screening for virulence-associated factors using classical genetic and molecular biology approaches can be biased under laboratory growth conditions of a given pathogen, because the required conditions for expression of many virulence factors may not occur during in vitro growth. Thus, technologies have been developed during the past several years to identify genes that are expressed during disease using animal models of human disease. However, animal models are not always truly representative of human disease, and with many pathogens, there is no appropriate animal model. METHODS: A new technology, in vivo-induced antigen technology (IVIAT) was thus engineered and tested in our laboratory to screen for genes of pathogenic organisms induced specifically in humans, without the use of animal or artificial models of infection. This technology uses pooled sera from patients to probe for genes expressed exclusively in vivo (or ivi, in vivo-induced genes). IVIAT was originally designed for the study of Actinobacillus actinomycetemcomitans pathogenesis, but we have now extended it to other oral pathogens including Porphyromonas gingivalis. RESULTS: One hundred seventy-one thousand (171,000) clones from P. gingivalis strain W83 were screened and 144 were confirmed positive. Over 300,000 A. actinomycetemcomitans clones were probed, and 116 were confirmed positive using a quantitative blot assay. CONCLUSION: MAT has proven useful in identifying previously unknown in vivo-induced genes that are likely involved in virulence and are thus excellent candidates for use in diagnostic : and therapeutic strategies, including vaccine design.