Efficient Gene Knockout in Salivary Gland Epithelial Explant Cultures.

We have developed methods to achieve efficient CRISPR-Cas9-mediated gene knockout in ex vivo mouse embryonic salivary epithelial explants. Salivary epithelial explants provide a valuable model for characterizing cell signaling, differentiation, and epithelial morphogenesis, but research has been limited by a paucity of efficient gene perturbation methods. Here, we demonstrate highly efficient gene perturbation by transient transduction of guide RNA-expressing lentiviruses into Cas9-expressing salivary epithelial buds isolated from Cas9 transgenic mice. We first show that salivary epithelial explants can be cultured in low-concentration, nonsolidified Matrigel suspensions in 96-well plates, which greatly increases sample throughput compared to conventional cultures embedded in solidified Matrigel. We further show that salivary epithelial explants can grow and branch with FGF7 alone, while supplementing with insulin, transferrin, and selenium (ITS) enhances growth and branching. We then describe an efficient workflow to produce experiment-ready, high-titer lentiviruses within 1 wk after molecular cloning. To track transduced cells, we designed the lentiviral vector to coexpress a nuclear fluorescent reporter with the guide RNA. We routinely achieved 80% transduction efficiency when antibiotic selection was used. Importantly, we detected robust loss of targeted protein products when testing 9 guide RNAs for 3 different genes. Moreover, targeting the ß1 integrin gene (Itgb1) inhibited branching morphogenesis, which supports the importance of cell-matrix adhesion in driving branching morphogenesis. In summary, we have established a lentivirus-based method that can efficiently perturb genes of interest in salivary epithelial explants, which will greatly facilitate studies of specific gene functions using this system.

Rapid susceptibility testing for slowly growing nontuberculous mycobacteria using a colorimetric microbial viability assay based on the reduction of water-soluble tetrazolium WST-1.

Rapid susceptibility testing for slowly growing nontuberculous mycobacteria (NTM) using a colorimetric microbial viability assay based on the reduction of the water-soluble tetrazolium salt {2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt (WST-1)} using 2,3,5,6-tetramethyl-1,4-benzoquinone as an electron mediator was developed. Using the Clinical and Laboratory Standards Institute (CLSI) method, a long-term incubation time (7-14 days) was required to determine the minimum inhibitory concentrations (MICs) of the slowly growing NTM. The MICs for a variety of different antibiotics against the slowly growing NTM were determined by the WST-1 colorimetric method and compared with those obtained using the broth microdilution methods approved by the CLSI. Good agreement was found between the MICs determined after 3-4 days using the WST-1 colorimetric method and those obtained after 10-14 days using the broth microdilution method. The results suggest that the WST-1 colorimetric assay is a useful method for the rapid determination of the MICs for the slowly growing NTM.

Branched-chain amino acid supplementation attenuates muscle soreness, muscle damage and inflammation during an intensive training program.

AIM: The aim of this paper was to assess the effects of branched-chain amino acid (BCAA) supplementation on muscle soreness, muscle damage and inflammation during an intensive training program. METHODS: Twelve long-distance runners (20 + or - 1 year-old) participated in a double-blinded crossover designed study conducted during two intensive training periods (three-day). The subjects were provided either a drink containing BCAA (0.8% BCAA in a 3.5% carbohydrate solution; 2,500 mL/day) or an isocaloric placebo drink during each training period. All subjects completed the same training program (total running distance: males: 86 km, females: 64 km), and ate the same meals during the training period. Whole body muscle soreness and fatigue sensation were measured in the morning before and during the training period by Visual Analogue Scale method. Plasma creatine kinase (CK), lactate dehydrogenase (LDH), and granulocyte elastase (GEL) levels were measured as indicators of muscle damage and inflammation before and after the training period. RESULTS: Muscle soreness and fatigue sensation during the training period in the BCAA trial were lower than those in the placebo trial (-32% and -24%, respectively; P<0.05). The plasma CK, LDH, and GEL levels after the training program in the BCAA trial were lower than those in the placebo trial (-21%, -6%, and -15%, respectively; P<0.05). CONCLUSIONS: These results demonstrate that BCAA supplementation during an intensive training program effectively reduces the muscle soreness and fatigue sensation, and that the perceived changes could be attributed to the attenuation of muscle damage and inflammation.

Branched-chain amino acids supplementation attenuates the accumulation of blood lactate dehydrogenase during distance running.

AIM: We investigated the effect of branched-chain amino acids (BCAA) supplementation on tissue damage during distance running. METHODS: Eight male distance runners (mean +/- standard deviation; age: 20.4+/-1.2 years, body weight: 58.4+/-4.2 kg) participated in a double blinded cross over designed study conducted during training camp. During each intervention period, the subjects were asked to participate in a 25-km run, and the blood BCAA and lactate dehydrogenase (LDH) level, an index of tissue damage, were measured pre- and post-run. Either a drink containing BCAA (0.4% BCAA in a 4% carbohydrate solution) or an iso-calorie placebo drink was provided to the subjects 5 times during the run without any restriction in the volume. RESULTS: The total volume of the drink consumed by the subjects did not differ substantially between the trials: 591+/-188 (2.36 g BCAA) vs 516+/-169 mL in BCAA and placebo trial, respectively. During the run, the blood BCAA concentration was maintained in the BCAA trial. However, the blood BCAA concentration level tended to decrease in the placebo trial (P<0.1). The extent of the blood LDH increase in the BCAA trial was significantly less than that of the placebo trail (48% vs 58%, P<0.05). CONCLUSION: Maintaining the blood BCAA level throughout a long distance run contributes to a reduction in the LDH release and, therefore, the effect of BCAA supplementation is suggested to reduce the degree of muscle damage.

Branched-chain amino acids and arginine supplementation attenuates skeletal muscle proteolysis induced by moderate exercise in young individuals.

This study aimed at evaluating the effect of a single oral intake of branched-chain amino acids (BCAA) with Arg on skeletal muscle protein metabolism during moderate exercise in young individuals. Eight healthy volunteers (4 males and 4 females, means +/- SEM, 26 +/- 1 yrs, 177.8 +/- 3.7 cm, 72.6 +/- 3.9 kg) were studied in a randomized double-blind placebo-controlled cross-over trial. The subjects performed 3 bouts of 20-min cycling exercise (5-min break between each bout) at 126 +/- 13 W corresponding to 50 % of the maximal work intensity. A single oral supplement of either a BCAA drink containing 2 g of BCAA and 0.5 g of Arg or an isocaloric placebo drink was given at 10 min of the 1st exercise bout. Both arterial and venous blood samples were simultaneously taken from the radial artery and the femoral vein, respectively. Blood flow in the femoral artery was determined using the ultrasound Doppler technique. The blood sampling and blood flow measurements were performed at rest, every 10 min during each exercise bout. Net balance of BCAA and Phe across the leg muscles were measured by the arteriovenous difference method. The BCAA ingestion resulted in increases in both the plasma BCAA concentration and BCAA uptake into the working leg. The Phe release from the leg during exercise significantly increased as compared to the basal level in the placebo trial (0.97 +/- 0.28 vs. 0.23 +/- 0.22 micromol/min, p < 0.05). In the BCAA trial, the cumulative Phe release from the leg during the 3rd exercise bout was significantly lower than that in the placebo trial (5.0 +/- 7.4 vs. 35.9 +/- 13.2 micromol/25 min, p < 0.05). These results suggest that endurance exercise at moderate intensity enhances proteolysis in working muscles, and a single oral intake of 2 g of BCAA with Arg at onset of exercise effectively suppresses exercise-induced skeletal muscle proteolysis.

Anti-hyperglycemic Potential of Natural Products.

In order to evaluate the anti-hyperglycemic effect of natural compounds via the inhibition of alpha-glucosidase (AGH), the potential inhibitory effect of anthocyanins, caffeic acid analogs, and caffeoylquinic acid analogs have been reviewed. A new AGH assay system to mimic the membrane-bound AGH at the small intestine was proposed and evaluated.

Analysis of lead content in herbal preparations in Malaysia.

In Malaysia, the phase 3 registration for traditional medicines was implemented on 1 January 1992 under the Control of Drugs and Cosmetics Regulation 1984, emphasizing quality, efficacy and safety (including the detection of the presence of heavy metals) in all pharmaceutical dosage forms of traditional medicine preparations. Therefore, a total of 100 products in various pharmaceutical dosage forms of a herbal preparation, were analysed for lead content using atomic absorption spectrophotometer. Results showed that 8% (eight products) possessed 10.64-20.72 ppm of lead, and therefore, do not comply with the quality requirement for traditional medicines in Malaysia. One of these products, M-Tongkat Ali (exhibited 10.64 +/-0.37 ppm of lead), was in fact already registered with the DCA Malaysia. The rest, Sukarno Tongkat Ali, Eurycoma Madu, Super Pill Tongkat Ali, Force Pill Tongkat Ali, Tender Pill Tongkat Ali, Super Pill Tongkat Ali Plus and Great Pill Tongkat Ali Plus have not registered with the DCA Malaysia and exhibited 12.24-20.72 ppm of lead. Although this study showed that only 92% of the products complied with the quality requirement for traditional medicines in Malaysia, however, they cannot be assumed safe from lead contamination because of batch-to-batch inconsistency.

Homogeneous time-resolved fluorescence DNA hybridization assay by DNA-mediated formation of an EDTA-Eu(III)-beta-diketonate ternary complex.

A sensitive homogenous time-resolved fluorescence DNA hybridization assay method based on the formation of an EDTA-Eu(3+)-beta-diketonate ternary complex in the DNA hybrid was developed. The new approach combined the use of two DNA probes whose sequences compose the whole complementary strand to the target DNA, in which one probe was labeled with an EDTA-Eu(3+) complex on the 5'-terminus and the other, labeled with a bidentate beta-diketone on the 3'-terminus. After hybridization of two DNA probes with target DNA, EDTA-Eu(3+) and beta-diketone come close to each other, and an EDTA-Eu(3+)-beta-diketonate ternary complex with a strong and long-lived fluorescence was formed; thus the target DNA was detected sensitively with a detection limit of 6 pM (0.6 fmol per assay) by time-resolved fluorescence measurement. In the absence of the target DNA, due to the poor stability of bidentate beta-diketonate-Eu(3+) complex in very diluted solution, only a small amount of ternary fluorescence complex was formed.

Contents and uptake rates of Mn, Fe, Co, Zn, and Se in Se-deficient rat liver cell fractions.

The contents of manganese (Mn), iron (Fe), cobalt (Co), zinc (Zn), and selenium (Se) in nuclear (NU), mitochondrial (MT), microsomal (MC), and cytosolic (CS) fractions of liver homogenates of normal and selenium-deficient (SeD) rats were determined by instrumental neutron activation analysis (INAA). The uptake rates of these elements in the liver cell fractions of both groups of rats were determined by multitracer analysis (MTA). The results indicated that Se-deficiency caused a significant increase in the content of Fe in the MC fractions. The MTA showed that the uptake rate of Fe was highest in the MC fraction, and that the uptake rate in the fraction was similar between the SeD and normal rats.

New approach for weight reduction by a combination of diet, light resistance exercise and the timing of ingesting a protein supplement.

We have reported that ingesting a meal immediately after exercise increased skeletal muscle accretion and less adipose tissue accumulation in rats employed in a 10 week resistance exercise program. We hypothesized that a possible increase in the resting metabolic rate (RMR) as a result of the larger skeletal muscle mass might be responsible for the less adipose deposition. Therefore, the effect of the timing of a protein supplement after resistance exercise on body composition and the RMR was investigated in 17 slightly overweight men. The subjects participated in a 12-week weight reduction program consisting of mild energy restriction (17% energy intake reduction) and a light resistance exercise using a pair of dumbbells (3-5 kg). The subjects were assigned to two groups. Group S ingested a protein supplement (10 g protein, 7 g carbohydrate, 3.3 g fat and one-third of recommended daily allowance (RDA) of vitamins and minerals) immediately after exercise. Group C did not ingest the supplement. Daily intake of both energy and protein was equal between the two groups and the protein intake met the RDA. After 12 weeks, the bodyweight, skinfold thickness, girth of waist and hip and percentage bodyfat significantly decreased in the both groups, however, no significant differences were observed between the groups. The fat-free mass significantly decreased in C, whereas its decrease in S was not significant. The RMR and post-meal total energy output significantly increased in S, while these variables did not change in C. In addition, the urinary nitrogen excretion tended to increase in C but not in S. These results suggest that the RMR increase observed in S might be associated with an increase in body protein synthesis.

Effects of erbium,chromium:YSGG laser irradiation on canine mandibular bone.

BACKGROUND: Only relatively few reports have described the morphological effects on bone produced by erbium,chromium: yttrium,scandium,gallium,garnet (Er,Cr:YSGG) laser irradiation, and none has investigated the atomic changes or estimated the temperature increases involved. The objectives of this study were to investigate the morphological, atomic, and temperature changes in irradiated areas during and after laser irradiation, and to evaluate the cutting effect on canine mandibular bone in vitro. METHODS: Two canine mandibular bones were cut into 3 to 5 cm pieces and irradiated by an Er,Cr:YSGG laser utilizing a water-air spray at 5 W and 8 Hz for 10 or 30 seconds. During and after laser irradiation, temperature increases in the irradiated areas were measured by thermography. The samples were then observed by stereoscopy and scanning electron microscopy to determine morphological changes and by energy dispersive x-ray spectroscopy to evaluate atomic alterations. RESULTS: Regular holes or grooves having sharp edges and smooth walls were produced, but no melting or carbonization was observed. The maximum temperature increase was an average 12.6 degrees C for 30-second irradiation. The continuous time of a temperature increase of more than 10 degrees C was consistently less than 10 seconds. An atomic analytical examination revealed that the calcium:phosphorus ratio was not significantly changed between the lased and unlased areas (P>0.0 1). CONCLUSION: These results showed that the Er,Cr:YSGG laser cuts canine mandibular bone effectively without burning, melting, or altering the calcium:phosphorus ratio of the irradiated bone.

Histological changes in monoaminergic neurons of Long-Evans Cinnamon rats.

The Long-Evans Cinnamon rat, an animal model of Wilson's disease, is an inbred mutant strain with spontaneous hepatitis isolated from Long-Evans rats. The copper concentration in the brains of Long-Evans Cinnamon rats at 4 weeks of age was lower than that of controls, but higher than that of controls at 20 weeks of age. We investigated the tyrosine hydroxylase and 5-hydroxytryptamine immunoreactive fiber densities in the brains of Long-Evans Cinnamon rats aged 4, 10, and 20 weeks by immunohistochemistry, comparing them with Long-Evans Agouti rats used as controls. Tyrosine hydroxylase immunoreactive fiber densities in the cingulate cortex, hippocampus and cerebellum in Long-Evans Cinnamon rats were significantly lower than those of Long-Evans Agouti rats at 4 and 10 weeks of age. On the other hand, 5-hydroxytryptamine immunoreactive fiber densities in the cingulate cortex, caudate-putamen, hypothalamus, and hippocampus in Long-Evans Cinnamon rats were significantly higher than those of controls at 4, 10 and 20 weeks of age. In the cingulate cortex and caudate-putamen, 5-hydroxytryptamine immunoreactive fiber densities became gradually higher with age. The number of aberrant 5-hydroxytryptamine immunoreactive fibers in the cingulate cortex, caudate-putamen, hypothalamus and hippocampus in LEC rats was significantly higher than that of controls. The number of another type of aberrant 5-hydroxytryptamine immunoreactive fibers, which were detected only at 20 weeks of age in the caudate-putamen in LEC rats was significantly higher than that of controls. These results suggest that age-dependent changes in copper concentrations of Long-Evans Cinnamon rats were related to changes in monoaminergic neuron systems.

Mechanism of therapeutic effectiveness of cefixime against typhoid fever.

beta-Lactams have been considered ineffective against organisms growing inside mammalian cells because of their poor penetration into cells. However, cefixime has been shown to be clinically effective against typhoid fever. The probable mechanism of therapeutic effectiveness of cefixime against typhoid fever was investigated using Salmonella enterica serovar Typhimurium instead of S. enterica serovar Typhi both in a cellular and in a mouse infection model. Cefixime was able to inhibit the growth of serovar Typhimurium inhabiting monocyte-derived THP-1 cells. Elongation of serovar Typhimurium in THP-1 cells was observed microscopically. Apparent morphological changes of serovar Typhimurium in THP-1 cells were also observed by electron microscopy. The concentration of cefixime inside THP-1 cells was almost half (46 to 48%) of the concentration outside the cells when serovar Typhimurium coexisted in the solution. The length of time after oral dosing (8 mg/kg) that cefixime was present-calculated from levels in serum-at a concentration above the MIC at which 90% of the serovar Typhi organisms inside human cells were inhibited was presumed to be more than 12 h. Cefixime also showed excellent activity in the mouse systemic and oral infection models based on infections caused by serovar Typhimurium. It is concluded that a fair amount of cefixime can enter mammalian cells and inhibit the growth of bacteria inside cells when the bacteria are sensitive enough to cefixime, as are serovars Typhimurium and Typhi.

Effects of erbium, chromium:YSGG laser irradiation on root surface: morphological and atomic analytical studies.

OBJECTIVE: The purpose of this study was to investigate the morphological and atomic changes on the root surface by stereoscopy, field emission-scanning electron microscopy (FE-SEM), and energy dispersive X-ray spectroscopy (SEM-EDX) after erbium, chromium:yttrium, scandium, gallium, garnet (Er,Cr:YSGG) laser irradiation in vitro. SUMMARY BACKGROUND DATA: There have been few reports on morphological and atomic analytical study on root surface by Er,Cr:YSGG laser irradiation. METHODS: Eighteen extracted human premolar and molar teeth were irradiated on root surfaces at a vertical position with water-air spray by an Er,Cr:YSGG laser at the parameter of 5.0 W and 20 Hz for 5 sec while moving. The samples were then morphologically observed by stereoscopy and FE-SEM and examined atomic-analytically by SEM-EDX. RESULTS: Craters having rough but clean surfaces and no melting or carbonization were observed in the samples. An atomic analytical examination showed that the calcium ratio to phosphorus showed no significant changes between the control and irradiated areas (p > 0.01). CONCLUSIONS: These results showed that the Er,Cr:YSGG laser has a good cutting effect on root surface and causes no burning or melting after laser irradiation.

Effects of a dual inhibitor of tumor necrosis factor-alpha and interleukin-1 on lipopolysaccharide-induced lung injury in rats: involvement of the p38 mitogen-activated protein kinase pathway.

OBJECTIVE: Sepsis is a major cause of adult respiratory distress syndrome. In this study, we evaluated the effect of FR167653, which is a potent suppressant of tumor necrosis factor (TNF)-alpha and interleukin (IL)-1 production, on lipopolysaccharide (LPS)-induced lung injury and lethality in rats, and we examined the involvement of p38 mitogen-activated protein (MAP) kinase in the action of FR167653. DESIGN: Prospective, randomized study. SETTING: Animal research facility in a university. SUBJECTS: Male Sprague-Dawley rats weighing 200-270 g. INTERVENTIONS: All the animals were assigned to one of the following four groups: control group, FR-only group, LPS-only group, and LPS/FR group. Animals in the LPS-only and LPS/FR groups received 6 mg/kg of LPS intravenously. The animals in the FR-only and LPS/FR groups also received an infusion of FR167653 at 0.2 mg x kg(-1) x hr(-1), commencing 30 mins before the LPS (or vehicle) injection and continuing for 5.5 hrs. MEASUREMENTS AND MAIN RESULTS: LPS significantly induced the accumulation of pulmonary neutrophils and lung edema, both of which were significantly attenuated by treatment with FR167653. FR167653 also significantly decreased the LPS-induced lethality. Histologically, tissue damage was milder in the LPS/FR group than in the LPS-only group. Serum concentrations of TNF-alpha and IL-1beta and plasma concentrations of thromboxane B2 were all suppressed in the LPS/FR group compared with the LPS-only group. Western blot analysis revealed that FR167653 inhibited the phosphorylation of p38 MAP kinase in lung tissues. CONCLUSIONS: FR167653 administration decreased serum TNF-alpha and IL-1beta concentrations, which was associated with decreased lung injury and lethality. The mechanism responsible for the decreased TNF-alpha and IL-1 may be related to the inhibitory effect of FR167653 on p38 MAP kinase activation.

alpha-Glucosidase inhibitory action of natural acylated anthocyanins. 1. Survey of natural pigments with potent inhibitory activity.

alpha-Glucosidase (AGH) inhibitory study by natural anthocyanin extracts was done. As the result of a free AGH assay system, 12 anthocyanin extracts were found to have a potent AGH inhibitory activity; in particular, Pharbitis nil (SOA) extract showed the strongest maltase inhibitory activity, with an IC(50) value of 0.35 mg/mL, as great as that of Ipomoea batatas (YGM) extract (IC(50) = 0.36 mg/mL). Interestingly, neither extract inhibited the sucrase activity at all. For the immobilized assay system, which may reflect the pharmacokinetics of AGH at the small intestine, SOA and YGM extracts gave more potent maltase inhibitory activities than those of the free AGH assay, with IC(50) values of 0.17 and 0.26 mg/mL, respectively. Both extracts also inhibited alpha-amylase action, indicating that anthocyanins would have a potential function to suppress the increase in postprandial glucose level from starch.

alpha-Glucosidase inhibitory action of natural acylated anthocyanins. 2. alpha-Glucosidase inhibition by isolated acylated anthocyanins.

Four diacylated pelargonidin (Pg: SOA-4 and SOA-6), cyanidin (Cy: YGM-3), and peonidin (Pn: YGM-6) 3-sophoroside-5-glucosides isolated from the red flowers of the morning glory, Pharbitis nil cv. Scarlett O'Hara (SOA), and the storage roots of purple sweet potato, Ipomoea batatas cv. Ayamurasaki (YGM), were subjected to an alpha-glucosidase (AGH) inhibitory assay, in which the assay was performed with the immobilized AGH (iAGH) system to mimic the membrane-bound AGH at the small intestine. As a result, the acylated anthocyanins showed strong maltase inhibitory activities with IC(50) values of <200 microM, whereas no sucrase inhibition was observed. Of these, SOA-4 [Pg 3-O-(2-O-(6-O-(E-3-O-(beta-D-glucopyranosyl)caffeyl)-beta-D-glucopyranosyl)-6-O-E-caffeyl-beta-D-glucopyranoside)-5-O-beta-D-glucopyranoside] possessed the most potent maltase inhibitory activity (IC(50) = 60 microM). As a result of a marked reduction of iAGH inhibitory activity by deacylating the anthocyanins, that is, Pg (or Cy or Pn) sophoroside-5-glucoside, acylation of anthocyanin with caffeic (Caf) or ferulic (Fer) acid was found to be important in the expression of iAGH (maltase) inhibition. In addition, the result that Pg-based anthocyanins showed the most potent maltase inhibition, with an IC(50) value of 4.6 mM, and the effect being in the descending order of potency of Pg > Pn/Cy strongly suggested that no replacement at the 3'(5')-position of the aglycon B-ring may be essential for inhibiting iAGH action.

Analysis of surface roughness of enamel and dentin after Er,Cr:YSGG laser irradiation.

OBJECTIVE: The purpose of this investigation was to compare the surface roughness of enamel and dentin following the Er,Cr:YSGG laser irradiation and acid etching. BACKGROUND DATA: Laser-roughened enamel or dentin surfaces have been expected to enhance restorative materials bond strength. MATERIALS AND METHODS: Er,Cr:YSGG laser irradiation was performed in one half of each polished enamel or dentin sample at 3 W (33.9 J/cm2, with air 70% and water 20%,) pulse energy for 6 sec. Then the other half was treated with 37% phosphoric acid for 30 sec. Surface roughness and morphological studies were performed. RESULTS: It was found that surface roughness was significantly increased with the laser system. Scanning electron microscopy analysis showed that irradiated surface produces a rough surface that was completely lacking of a smear layer; there was also no cracking of enamel or dentin. CONCLUSION: Er,Cr:YSGG laser irradiation could provide an effective and alternative method to the acid etch technique.

Preventive effect of tooth fracture by pulsed Nd:YAG laser irradiation with diamine silver fluoride solution.

OBJECTIVE: The purpose of the present study was to evaluate the preventive effect of pulsed Nd:YAG laser irradiation with 38% diamine silver fluoride [Ag(NH3)2F] solution for the fracture of endodontically treated teeth in vitro. BACKGROUND DATA: There have been no reports on the preventive effect of tooth fracture using Nd:YAG laser with Ag(NH3)2F solution. MATERIALS AND METHODS: Twenty-eight human extracted teeth were used in this study. The teeth were randomly classified into four groups: control group, where tooth surfaces were not submitted to any treatment; group 1, where tooth surfaces were coated with 38% Ag(NH3)2F solution; group 2, where tooth surfaces were coated with Ag(NH3)2F solution and irradiated by pulsed Nd:YAG laser for 2 sec; and group 3, where tooth surfaces were coated with Ag(NH3)2F solution and irradiated by pulsed Nd:YAG laser for 10 sec. After preparation, shear tests were performed and the maximum load for the fracture was measured. Results were analyzed using the Scheffe test, and difference at p < 0.05 was considered significant. RESULTS: The failure load for group 2 (mean, 182.5 kg) had the highest mean value and differed significantly from those for the control group (mean, 146.3 kg) and group 1 (mean, 147.1 kg; p < 0.05). The failure loads for groups 1 and 3 (mean, 150.0 kg) did not differ significantly from that for the control group (p > 0.05). CONCLUSION: The results show that the application of 38% Ag(NH3)2F solution followed by pulsed Nd:YAG laser irradiation for 2 sec is useful for prevention of tooth fracture at endodontically treated teeth.