Human Probiotic Lactobacillus paracasei-Derived Extracellular Vesicles Improve Tumor Necrosis Factor-α-Induced Inflammatory Phenotypes in Human Skin.

Lactic acid bacteria (LAB), a probiotic, provide various health benefits. We recently isolated a new Lactobacillus paracasei strain with strong anti-inflammatory effects under lipopolysaccharide-induced conditions and proposed a new mode of action-augmenting the endoplasmic reticulum stress pathway for anti-inflammatory functions in host cells. The beneficial effects of the L. paracasei strains on the skin have been described; however, the effects of L. paracasei-derived extracellular vesicles (LpEVs) on the skin are poorly understood. Herein, we investigated whether LpEVs can improve inflammation-mediated skin phenotypes by determining their effects on primary human skin cells and a three-dimensional (3D) full-thickness human skin equivalent under tumor necrosis factor (TNF)-α-challenged inflammatory conditions. LpEVs were efficiently taken up by the human skin cells and were much less cytotoxic to host cells than bacterial lysates. Furthermore, low LpEV concentrations efficiently restored TNF-α-induced cellular phenotypes, resulting in increased cell proliferation and collagen synthesis, but decreased inflammatory factor levels (matrix metalloproteinase 1, interleukin 6, and interleukin 8) in the human dermal fibroblasts, which was comparable to that of retinoic acid, a representative antiaging compound. The beneficial effects of LpEVs were validated in a 3D full-thickness human skin equivalent model. LpEV treatment remarkably restored the TNF-α-induced epidermal malformation, abnormal proliferation of keratinocytes in the basal layer, and reduction in dermal collagen synthesis. Additionally, LpEVs penetrated and reached the deepest dermal layer within 24 h when overlaid on top of a 3D full-thickness human skin equivalent. Furthermore, they possessed superior antioxidant capacity compared with the human cell-derived EVs. Taken together, the anti-inflammatory probiotic LpEVs can be attractive antiaging and antioxidant substances for improving inflammation-induced skin phenotypes and disorders.

The Effects of Exergaming on Executive and Physical Functions in Older Adults With Dementia: Randomized Controlled Trial.

BACKGROUND: Despite increasing interest in the effects of exergaming on cognitive function, little is known about its effects on older adults with dementia. OBJECTIVE: The purpose of this is to investigate the effects of exergaming on executive and physical functions in older adults with dementia compared to regular aerobic exercise. METHODS: In total, 24 older adults with moderate dementia participated in the study. Participants were randomized into either the exergame group (EXG, n=13, 54%) or the aerobic exercise group (AEG, n=11, 46%). For 12 weeks, EXG engaged in a running-based exergame and AEG performed a cycling exercise. At baseline and postintervention, participants underwent the Ericksen flanker test (accuracy % and response time [RT]) while recording event-related potentials (ERPs) that included the N2 and P3b potentials. Participants also underwent the senior fitness test (SFT) and the body composition test pre- and postintervention. Repeated-measures ANOVA was performed to assess the effects of time (pre- vs postintervention), group (EXG vs AEG), and group×time interactions. RESULTS: Compared to AEG, EXG demonstrated greater improvements in the SFT (F1.22=7.434, P=.01), reduction in body fat (F1.22=6.476, P=.02), and increase in skeletal mass (F1.22=4.525, P=.05), fat-free mass (F1.22=6.103, P=.02), and muscle mass (F1.22=6.636, P=.02). Although there was a significantly shorter RT in EXG postintervention (congruent P=.03, 95% CI 13.581-260.419, incongruent P=.04, 95% CI 14.621-408.917), no changes occurred in AEG. EXG also yielded a shorter N2 latency for central (Cz) cortices during both congruent conditions compared to AEG (F1.22=4.281, P=.05). Lastly, EXG presented a significantly increased P3b amplitude compared to AEG during the Ericksen flanker test (congruent: frontal [Fz] F1.22=6.546, P=.02; Cz F1.22=5.963, P=.23; parietal [Pz] F1.22=4.302, P=.05; incongruent: Fz F1.22=8.302, P=.01; Cz F1.22=15.199, P=.001; Pz F1.22=13.774, P=.001). CONCLUSIONS: Our results suggest that exergaming may be associated with greater improvements in brain neuronal activity and enhanced executive function task performance than regular aerobic exercise. Exergaming characterized by both aerobic exercise and cognitive stimulation can be used as an effective intervention to improve cognitive and physical functions in older adults with dementia. TRIAL REGISTRATION: Clinical Research Information Service KCT0008238; https://cris.nih.go.kr/cris/search/detailSearch.do/24170.

Citrus junos Tanaka Peel Extract and Its Bioactive Naringin Reduce Fine Dust-Induced Respiratory Injury Markers in BALB/c Male Mice.

Particulate matter (PM) 10 refers to fine dust with a diameter of less than 10 µm and induces apoptosis and inflammatory responses through oxidative stress. Citrus junos Tanaka is a citrus fruit and contains bioactive flavonoids including naringin. In the present study, we aimed to identify the preventive effect of Citrus junos Tanaka peel extract (CPE) against PM10-induced lung injury. As a proof of concept, NCI-H460 cells were treated with CPE (800 µg/mL, 12 h) in conjunction with PM10 to examine intracellular antioxidative capacity in the pulmonary system. In an in vivo model, male BALB/c mice (n = 8/group) were randomly assigned into five groups: NEG (saline-treated), POS (PM10 only), NAR (PM10 + naringin, 100 mg/kg), CPL (PM10 + CPE low, 100 mg/kg), and CPH (PM10 + CPE high, 400 mg/kg). Intervention groups received dietary supplementations for 7 days followed by PM10 exposure (100 mg/kg, intranasal instillation). Compared to the NEG, the CPE decreased to 22% of the ROS generation and significantly increased cell viability in vitro. The histological assessments confirmed that pulmonary damages were alleviated in the PM10 + CPL group compared to the POS. Pro-inflammatory cytokines and NF-κB/apoptosis signaling-related markers were decreased in the PM10 + CPL group compared to the POS. These results indicated that CPE showed promising efficacy in preventing pulmonary injuries in vivo. Such protection can be explained by the anti-oxidative capacity of CPE, likely due to its bioactives, including naringin (7.74 mg/g CPE). Follow-up human intervention, as well as population-level studies, will further shed light on the preventive efficacy of CPE against pulmonary damage in humans.

Study on Optimal Position and Covering Pressure of Wearable Neck Microphone for Continuous Voice Monitoring.

Vocal cord disorder is one of the important health problems, especially in noisy industrial sites where excessive voice is required. A convenient and reliable communication method is required in a noisy environment to prevent the related disorders. However, the signal sensitivity of previous neck microphones is still insufficient to accurately convey the voice. In this study, we developed a skin-attachable neck microphone with a lightweight and flexible form factor. Also, we optimized the attachment position and covering pressure to maximize the signal sensitivity. As a result, we obtained the optimal position near the thyroid cartilage and confirmed that the signal sensitivity is the highest when the covering pressure is approximately 4 mmHg.Clinical Relevance- People can measure the voice status using a wearable neck microphone at the optimal position and covering pressure. It provides a solution to keep the vocal cords in good health even in a noisy environment.

A Wearable System for Heart Rate Recovery Evaluation with Real-Time Classification on Exercise Condition.

Heart rate recovery (HRR) is a convenient index to assess a cardiovascular autonomic function response to physical exercise. HRR monitoring during daily exercise can be an effective way to verify cardiorespiratory performance. Because HRR varies depending on exercise intensity and resting condition, an exercise condition needs to be acquired for a reliable HRR analysis. This study presents a wearable system for HRR evaluation with automatic labeling of exercise conditions using real-time activity classification. We developed an activity classification algorithm using two features from accelerometer sensor: an acceleration peak and an angle tilt peak. The classification algorithm was applied to a chest-attached wearable device with an embedded electrocardiogram sensor and accelerometer sensors. We classified daily activities such as running, walking, and postural transitions performed under supervised conditions. The wearable device system accurately detected activities with a sensitivity of 99.2 % and posture transitions with a sensitivity of 92 % and specificity of 93.3 % for seven healthy subjects. The proposed wearable system can help monitor HRR during exercise training by labeling the exercise condition simultaneously.

Isolation of Lactococcus lactis ssp. cremoris LRCC5306 and Optimization of Diacetyl Production Conditions for Manufacturing Sour Cream.

The sensory properties and flavor of sour cream are important factors that influence consumer acceptability. The present study aimed to isolate lactic acid bacteria with excellent diacetyl production ability and to optimize the fermentation conditions for sour cream manufacture. Lactococcus lactis ssp. cremoris was isolated as a lactic acid bacterium derived from raw milk. This strain showed the greatest diacetyl production among other strains and was named LRCC5306. Various culture conditions were optimized to improve the diacetyl production of LRCC5306. The highest diacetyl production was found to be at 105.04±2.06 mg/L, when 0.2% citric acid and 0.001% Fe2+ were added and cultured at 20°C for 15 h. Based on the optimal cultivation conditions, sour cream was manufactured using LRCC5306, with a viable count of 1.04×108 CFU/g and a diacetyl concentration of 106.56±1.53 mg/g. The electronic tongue system was used to compare the sensory properties of the sour cream; the fermented product exhibited sweetness and saltiness which was similar to that of an imported commercial product, but with slightly reduced bitterness and a significantly greater degree of sour taste. Therefore, our study shows that if cream is fermented using the LRCC5306, it is possible to produce sour cream with greatly improved sensory attractiveness, resulting in increased acceptance by consumers. Since this sour cream has a higher viable count of lactic acid bacteria, it is also anticipated that it will have a better probiotic effect.

Isolation of the Cholesterol-Assimilating Strain Pediococcus acidilactici LRCC5307 and Production of Low-Cholesterol Butter.

The objective of the present study was to evaluate the cholesterol-assimilation ability of lactic acid bacteria (LAB), which were isolated from kimchi, a Korean traditional fermented cabbage. The isolated strain, using modified MRS medium, showed 30.5% cholesterol assimilation activity and was named Pediococcus acidilactici LRCC5307. Types and concentrations of bile were investigated for their effects on increasing the cholesterol assimilation ability of the LRCC5307 strain, a 74.5% decrease in cholesterol was observed when 0.2% bile salts were added. In addition, the manufacture of low-cholesterol butter using LRCC5307 was examined. After fermentation, LRCC5307 with butter showed 8.74 Log CFU/g viable cells, pH 5.43, and a 11% decrease in cholesterol. These results suggest that LRCC5307 could help in the production of healthier butter by decreasing cholesterol and including living LAB.

Temperature-specific vsiRNA confers RNAi-mediated viral resistance at elevated temperature in Capsicum annuum.

Resistance (R) gene-mediated resistance is a robust and efficient antiviral immune system in the plants. Thus, when R-mediated resistance was suppressed at elevated temperatures, resistance towards viruses was expected to be completely collapsed. Nonetheless, the multiplication of Tobacco mosaic virus pathotype P0 (TMV-P0) was inhibited, and TMV-P0 particles were only occasionally present in the systemic leaves of pepper plants (Capsicum annuum). RNAi-mediated RNA silencing is a well-known antiviral immune mechanism. At elevated temperatures, RNAi-mediated antiviral resistance was induced and virus-derived siRNAs (vsiRNAs) were dramatically increased. Through sRNA-sequencing (sRNA-Seq) analysis, we revealed that vsiRNAs derived from TMV-P0 were greatly increased. Intriguingly, virus-infected plants could select the temperature-specific vsiRNAs for antiviral resistance from the amplified vsiRNAs at elevated temperatures. Pre-application of these temperature-specific vsiRNAs endowed antiviral resistance of the plants. Therefore, plants sustain antiviral resistance by activating RNAi-mediated resistance, based on temperature-specific vsiRNAs at elevated temperatures.

Effect of a new Lactobacillus plantarum product, LRCC5310, on clinical symptoms and virus reduction in children with rotaviral enteritis.

BACKGROUND: Rotavirus is one of the most common causes of infantile enteritis. In common enterocolitis, probiotic organisms, including Lactobacilli, are effective in treating diarrhea. A new species, Lactobacillus plantarum (LRCC5310), which was shown to inhibit the adherence and proliferation of rotavirus in the small intestine through animal experiments, was investigated for the efficacy and safety of patients with rotaviral enteritis. METHODS: LRCC5310 (Group I) and control (Group II) groups consisting of children who were hospitalized for rotaviral enteritis were compared, and the medical records of patients (Group III) who were hospitalized for rotaviral enteritis during the same study period were retrospectively analyzed. Clinical symptoms were compared and stool samples were collected to compare changes in virus multiplication between Groups I and II. RESULTS: Groups I, II, and III comprised 15, 8, and 27 children, respectively. There were no differences in clinical information among the groups at admission. In Group I, a statistically significant improvement was noted in the number of patients with diarrhea, number of defecation events on Day 3, and total diarrhea period as opposed to Group II (P = .033, P = .003, and P = .012, respectively). The improvement of Vesikari score in Group I was greater than that in the other groups (P = .076, P = .061, and P = .036, respectively). Among rotavirus genotypes, 9 (22.5%) strains and 8 (20.0%) strains belonged to the G9P8 and G1P8 genotypes, respectively. The virus reduction effect, as confirmed via stool specimens, was also greater in Group I. No significant side effects were noted in infants. CONCLUSION: LRCC5310 improved clinical symptoms, including diarrhea and Vesikari score, and inhibited viral proliferation in rotaviral gastroenteritis.

Can 24 weeks strength training reduce feelings of depression and increase neurotransmitter in elderly females?

BACKGROUND: Geriatric depression is common due to the high rate of chronic diseases suffered by the older population. There is a well-established effect of exercise on one's wellbeing, however, for the elderly females there is dearth in research addressing exercise and its effect on their neurotransmitters and depression score. Therefore, this study aimed to investigate the effects of 24 weeks of the Growing Stronger program on neurotransmitter and depression of older women. METHODS: Twenty-one older women, aged between 67 and 81, participated in this study. The participants were randomly assigned into a strength exercise (n = 11) and control group (n = 10) (age 76.40 ±â€¯3.27, height 152.77 ±â€¯5.63 kg, Weight 52.35 ±â€¯2.86Kg, BMI 22.50 ±â€¯1.88). The strength exercise group (age 76.10 ±â€¯3.85, height 151.14 ±â€¯5.42 kg, Weight 54.74 ±â€¯6.73Kg, BMI 23.96 ±â€¯2.70) participated for 50-80 min a day three times per week for a total of 24 weeks. All participants had their height, weight, blood testing and depression score tested before and after the training program. Two-way ANOVA with effect sizes was used to identify differences between times in each group and the statistical significance was set at 0.05. RESULTS: In neurotransmitter factor, serotonin, dopamine, epinephrine, and norepinephrine significantly decreased in the strength exercise group but not for the control group. Notably, one participant whose serotonin level was lower than normal range before the exercise program showed normal level in serotonin after the program. In depression factor, there were no significant differences for both the strength exercise group and control group. CONCLUSION: The results suggest that the effect of strength exercise on neurotransmitter and depression score remain unclear, but highlight the need for future studies investigating the relationship between strength training and depression.

Capsicum annuum transcription factor WRKYa positively regulates defense response upon TMV infection and is a substrate of CaMK1 and CaMK2.

Plants are constantly exposed to pathogens and environmental stresses. To minimize damage caused by these potentially harmful factors, plants respond by massive transcriptional reprogramming of various stress-related genes via major transcription factor families. One of the transcription factor families, WRKY, plays an important role in diverse stress response of plants and is often useful to generate genetically engineered crop plants. In this study, we carried out functional characterization of CaWRKYa encoding group I WRKY member, which is induced during hypersensitive response (HR) in hot pepper (Capsicum annuum) upon Tobacco mosaic virus (TMV) infection. CaWRKYa was involved in L-mediated resistance via transcriptional reprogramming of pathogenesis-related (PR) gene expression and affected HR upon TMV-P0 infection. CaWRKYa acts as a positive regulator of this defense system and could bind to the W-box of diverse PR genes promoters. Furthermore, we found Capsicum annuum mitogen-activated protein kinase 1 (CaMK1) and 2 (CaMK2) interacted with CaWRKYa and phosphorylated the SP clusters but not the MAPK docking (D)-domain of CaWRKYa. Thus, these results demonstrated that CaWRKYa was regulated by CaMK1 and CaMK2 at the posttranslational level in hot pepper.

Deubiquitinating activity of Sdu1, a putative member of the PPPDE peptidase family, in Schizosaccharomyces pombe.

The Schizosaccharomyces pombe sdu⁺ gene encoding a putative member of the PPPDE (Permuted Papain fold Peptidases of DsRNA viruses and Eukaryotes) superfamily was cloned into an Escherichia coli - yeast shuttle vector pRS316, resulting in the recombinant plasmid pYSTP. The determined nucleotide sequence carries 1207 bp, which would encode a protein of 201 amino acid residues. The S. pombe cells harboring pYSTP contained higher sdu1⁺ mRNA and deubiquitinating activity levels than the vector control cells, indicating that the sdu1⁺ gene is functioning. They exhibited a better growth in normal rich medium than the vector control cells. When shifted into the fresh medium containing hydrogen peroxide, menadione, or sodium nitroprusside, the S. pombe cells harboring pYSTP were able to grow reasonably well, while the growth of the vector control cells was arrested. The reactive oxygen species and total glutathione levels of the S. pombe cells harboring pYSTP were lower and higher than those of the vector control cells under the same stressful conditions, respectively. They exhibited a lower nitric oxide level than the vector control cells when subjected to sodium nitroprusside. Taken together, the sdu1⁺ gene encodes an actual protein having deubiquitinating activity and is involved in the response against oxidative and nitrosative stresses in S. pombe.

Effects of intronic and exonic polymorphisms of paraoxonase 1 (PON1) gene on serum PON1 activity in a Korean population.

Paraoxonase 1 (PON1) hydrolyzes a number of toxic organophosphorous compounds and reduces lipid peroxide accumulation, and PON1 genetic polymorphisms in the coding region modulate serum PON1 activity. In this study, we investigated the association between 3 polymorphisms of PON1 located in intron 5 (17899insdelTT and 17974CT) and exon 6 (192QR) and serum PON1 activity. The genetic polymorphisms and serum activity of PON1 were analyzed in 153 healthy Koreans by using a direct sequencing assay and spectrophotometric method, respectively. A significant linkage disequilibrium (LD) was observed between all tested single nucleotide polymorphisms, with the strongest LD observed between 17899insdelTT and 192QR (D' = 0.984). The 17899insdelTT, 17974CT and 192QR genetic polymorphisms were associated with significant differences in serum paraoxonase activity. In multiple regression analyses, smoking, triglyceride level, high-density lipoprotein (HDL) level, and the 17899insdelTT and 192QR genetic polymorphisms were significant determinants of serum paraoxonase activity, while age, smoking, triglyceride level, HDL level, and the 192QR genetic polymorphism were significant determinants of serum arylesterase activity. These results suggest that although the 192QR genetic polymorphism in the coding region of PON1 is primarily associated with serum PON1 activity, the intronic polymorphisms are also involved in serum PON1 activity, and this association may be mediated by LD.

Characterization of chromosome ends in the filamentous fungus Neurospora crassa.

Telomeres and subtelomere regions have vital roles in cellular homeostasis and can facilitate niche adaptation. However, information on telomere/subtelomere structure is still limited to a small number of organisms. Prior to initiation of this project, the Neurospora crassa genome assembly contained only 3 of the 14 telomeres. The missing telomeres were identified through bioinformatic mining of raw sequence data from the genome project and from clones in new cosmid and plasmid libraries. Their chromosomal locations were assigned on the basis of paired-end read information and/or by RFLP mapping. One telomere is attached to the ribosomal repeat array. The remaining chromosome ends have atypical structures in that they lack distinct subtelomere domains or other sequence features that are associated with telomeres in other organisms. Many of the chromosome ends terminate in highly AT-rich sequences that appear to be products of repeat-induced point mutation, although most are not currently repeated sequences. Several chromosome termini in the standard Oak Ridge wild-type strain were compared to their counterparts in an exotic wild type, Mauriceville. This revealed that the sequences immediately adjacent to the telomeres are usually genome specific. Finally, despite the absence of many features typically found in the telomere regions of other organisms, the Neurospora chromosome termini still retain the dynamic nature that is characteristic of chromosome ends.

Organization of chromosome ends in the rice blast fungus, Magnaporthe oryzae.

Eukaryotic pathogens of humans often evade the immune system by switching the expression of surface proteins encoded by subtelomeric gene families. To determine if plant pathogenic fungi use a similar mechanism to avoid host defenses, we sequenced the 14 chromosome ends of the rice blast pathogen, Magnaporthe oryzae. One telomere is directly joined to ribosomal RNA-encoding genes, at the end of the approximately 2 Mb rDNA array. Two are attached to chromosome-unique sequences, and the remainder adjoin a distinct subtelomere region, consisting of a telomere-linked RecQ-helicase (TLH) gene flanked by several blocks of tandem repeats. Unlike other microbes, M.oryzae exhibits very little gene amplification in the subtelomere regions-out of 261 predicted genes found within 100 kb of the telomeres, only four were present at more than one chromosome end. Therefore, it seems unlikely that M.oryzae uses switching mechanisms to evade host defenses. Instead, the M.oryzae telomeres have undergone frequent terminal truncation, and there is evidence of extensive ectopic recombination among transposons in these regions. We propose that the M.oryzae chromosome termini play more subtle roles in host adaptation by promoting the loss of terminally-positioned genes that tend to trigger host defenses.

Growth-promoting activity of Hominis Placenta extract on regenerating sciatic nerve.

AIM: Extract of Hominis Placenta (HP) has been used in oriental medicine as an agent for improving physiological function. The present study was conducted to investigate whether HP treatment in an experimental sciatic nerve injury animal model produces growth-promoting effects on regenerating peripheral nerve fibers after injury. METHODS: After HP was injected into a sciatic nerve injury site, changes in protein levels were analyzed in the regenerating nerve area by Western blotting and immunofluorescence staining analyses. For quantitative assessment of axonal regeneration, a retrograde tracing technique was used to identify the neuronal cell bodies corresponding to regenerating axons, and the extent of neurite outgrowth in cultured dorsal root ganglia (DRG) sensory neurons prepared from animals that had experienced a sciatic nerve crush injury 7 d before neuron collection was analyzed. RESULTS: Induction levels of axonal growth-associated protein (GAP-43) in the injured sciatic nerves were elevated by HP treatment. HP treatment also upregulated cell division cycle 2 (Cdc2) protein levels in the distal stump of the injured sciatic nerve. Induced Cdc2 protein was detected in Schwann cells, suggesting that Cdc2 kinase activity may be involved in the growth-promoting activity of regenerating axons via Schwann cell proliferation. Cell body measurement by retrograde tracing indicated that HP treatment produced significant increases in regenerating motor axons. Finally, HP treatment of cultured DRG sensory neurons significantly increased neurite arborization and elongation. CONCLUSION: HP promotes the regeneration of injured sciatic axons by upregulating the synthesis of regeneration-related protein factors such as GAP-43 and Cdc2.

Telomere hypervariability in Magnaporthe oryzae.

SUMMARY The gray leaf spot disease of perennial ryegrass and tall fescue is caused by the fungus Magnaporthe oryzae (anamorph = Pyricularia oryzae). A collection of single-copy and repetitive DNA markers was used to investigate genetic diversity among 22 isolates of the gray leaf spot pathogen. The single-copy DNA markers revealed only three polymorphisms among 95 restriction fragments spanning approximately 0.6% of the genome. In addition, Southern hybridization analysis and mating tests revealed that all isolates possessed the MAT1-2 mating-type allele. Fingerprinting of repetitive DNA loci using the Pot2 and MGR583 probes also revealed a high degree of genetic similarity (> 85%) among isolates. These data are consistent with the gray leaf spot pathogens having a recent evolutionary origin. In contrast to the results obtained with probes for internal chromosome loci, a telomere probe revealed that the chromosome ends of the very same isolates are highly divergent, with most isolates sharing less than 20% fingerprint similarity with any other isolate. Telomere mutations arise extremely frequently and changes in telomere fingerprint profiles were readily observed during vegetative growth and among cultures derived from single spores isolated from agar medium and from lesions on perennial ryegrass leaves.

Field Resistance to Strobilurin (Q(o)I) Fungicides in Pyricularia grisea Caused by Mutations in the Mitochondrial Cytochrome b Gene.

ABSTRACT Gray leaf spot caused by Pyricularia grisea is a highly destructive disease of perennial ryegrass turf. Control of gray leaf spot is dependent on the use of preventative fungicide treatments. Strobilurin-based (Q(o)I) fungicides, which inhibit the cytochrome bc(1) respiratory complex, have proven to be very effective against gray leaf spot. However, in August 2000, disease was diagnosed in Q(o)I-treated perennial ryegrass turf on golf courses in Lexington, KY, Champaign, IL, and Bloomington, IL. To determine if resistance was due to a mutation in the fungicide target, the cytochrome b gene (CYTB) was amplified from baseline and resistant isolates. Nucleotide sequence analysis revealed an intronless coding region of 1,179 bp. Isolates that were resistant to Q(o)I fungicides possessed one of two different mutant alleles, each of which carried a single point mutation. The first mutant allele had a guanine-to-cytosine transition at nucleotide position +428, resulting in a replacement of glycine 143 by alanine (G143A). Mutant allele two exhibited a cytosine-to-adenine transversion at position +387, causing a phenylalanine-to-leucine change (F129L). Cleavable amplified polymorphic sequence analysis revealed that neither mutation was present in a collection of baseline isolates collected before Q(o)I fungicide use and indicated that suspected Q(o)I- resistant isolates found in 2001 in Indiana and Maryland possessed the F129L mutation. The Pyricularia grisea isolates possessing the G143A substitution were significantly more resistant to azoxystrobin and trifloxystrobin, in vitro, than those having F129L. DNA fingerprinting of resistant isolates revealed that the mutations occurred in just five genetic backgrounds, suggesting that field resistance to the Q(o)I fungicides in Pyricularia grisea is due to a small number of ancestral mutations.