In Situ and Ex Situ Studies of Ring-Like Assembly of Silica Nanoparticles in the Presence of Poly(propylene oxide)-Poly(ethylene oxide) Block Copolymers.

Block copolymer-mediated self-assembly of colloidal nanoparticles has attracted great attention for fabricating various nanoparticle arrays. We have previously shown that silica nanoparticles (SNPs) assemble into ring-like nanostructures in the presence of temperature-responsive block copolymers poly[(2-ethoxyethyl vinyl ether)-block-(2-methoxyethyl vinyl ether)] (PEOVE-PMOVE) in an aqueous phase. The ring-like nanostructures formed within an aggregate of PEOVE-PMOVE when the temperature was increased to 45 °C, at which the polymer is amphiphilic. Herein, we report that SNPs assemble into ring-like nanostructures even with a different temperature-responsive, amphiphilic block copolymer poly(propylene oxide)-block-poly(ethylene oxide) (PPO-PEO) at 45 °C. Field-emission scanning electron microscopy for SNP assemblies that were spin-coated on a substrate indicated that SNP first assembled into chain-like nanostructures and then bent into closed loops over several days. In contrast, in situ small-angle X-ray diffraction measurements revealed the formation of SNP nanorings within 75 s at 45 °C in the liquid phase. These results indicated that ring-like assembly of SNPs occurs quickly in the liquid phase, but the slow formation of Si-O-Si bonds between SNPs leads to their structure being destroyed by spin-coating. Intriguingly, SNPs with a diameter of 15 nm form a well-defined nanoring structure, with five SNPs located at the vertex points of a regular pentagon. Additionally, small-angle neutron scattering, where the contrast of the solvent (a mixture of H2O and D2O) matches that of SNPs, clarified that SNPs are contained within the spherical micelle formed from PPO-PEO. This work offers a facile and versatile approach to preparing ring-like arrays from inorganic colloidal nanoparticles, leading to applications including sensing, catalysis, and nanoelectronics.

Ferroelectric columnar assemblies from the bowl-to-bowl inversion of aromatic cores.

Organic ferroelectrics, in which the constituent molecules retain remanent polarization, represent an important topic in condensed-matter science, and their attractive properties, which include lightness, flexibility, and non-toxicity, are of potential use in state-of-the-art ferroelectric devices. However, the mechanisms for the generation of ferroelectricity in such organic compounds remain limited to a few representative concepts, which has hitherto severely hampered progress in this area. Here, we demonstrate that a bowl-to-bowl inversion of a relatively small organic molecule with a bowl-shaped π-aromatic core generates ferroelectric dipole relaxation. The present results thus reveal an unprecedented concept to produce ferroelectricity in small organic molecules, which can be expected to strongly impact materials science.

Clinical outcomes of concurrent surgery with weight bearing after modified lasso-loop stitch arthroscopic ankle stabilization.

PURPOSE: To determine the effects of unilateral and bilateral ankle stabilization surgery with or without additional concurrent procedures for other pathologies on return to activity in patients who were allowed unrestricted weight bearing postoperatively. METHODS: Ninety-three athletes underwent 120 ankle stabilization surgeries including 27 that underwent bilateral simultaneous surgery using the all-inside arthroscopy-modified lasso-loop technique and were divided into two groups: arthroscopic ligament repair alone without concurrent procedures (group A) and with simultaneous procedures for other pathologies (group B). Group A was further subdivided into unilateral (group A1) and simultaneous bilateral ankle surgery (group A2), and group B into ankle stabilization surgery with simultaneous procedures not requiring weight bearing postoperatively (Group B1) and with concurrent procedures allowing weight bearing (Group B2). Return to activity postoperatively was assessed by recording the time to walk without any support, jog, and return to full athletic activities. Clinical outcomes were assessed preoperatively and 12 months postoperatively using a subjective clinical score. RESULTS: The average time between surgery and unsupported walk, jog, and return to full athletic activities was 1.6 ± 2.5, 16.9 ± 3.7, and 42.4 ± 19.3 days in group A, 17.2 ± 19.6, 34.5 ± 20.8, and 60.9 ± 22.8 days in group B, 1.7 ± 2.9, 16.1 ± 2.4, and 41.6 ± 18.2 days in group A1, 1.3 ± 0.6, 18.9 ± 5.5, and 44.6 ± 22.5 days in group A2, 25.3 ± 20.2, 43.3 ± 21.1, and 70.7 ± 23.1 days in group B1, and 4.8 ± 11.7, 20.7 ± 11.7, and 45.0 ± 13.7 days in group B2, respectively. These results indicate that the patients in group B2 showed a statistically significant faster time to return to activity than did those restricted from weight bearing. Differences in ankle stabilization alone between patients in groups A1 and A2 as well as groups B2 and A were not statistically significant. Clinical outcomes were similar for patients in groups B2 and A1 versus group A2. CONCLUSION: Time to return to activity and clinical outcomes after ankle stabilization surgery using the modified lasso-loop technique were negatively affected if simultaneous bilateral surgery or simultaneous concurrent procedures were added or if weight bearing was unrestricted. However, a delay in return to athletic activity was observed when ankle stabilization surgery was performed using the modified lasso-loop technique with concurrent procedures that require non-weight bearing postoperatively. LEVEL OF EVIDENCE: Level III.

Thermocells Driven by Phase Transition of Hydrogel Nanoparticles.

Thermoelectric conversion of low temperature, delocalized, and abundant thermal sources is crucial for the development of the Internet of Things (IoT) and/or a carbon-free society. Thermocells are of great interest in thermoelectric conversion of low-temperature heat due to the low cost and flexibility of components. However, significant improvement of the conversion efficiency is required for the practical use of the cells. Here, we report thermo-electrochemical cells driven by volume phase transition (VPT) of hydrogel nanoparticles (NPs). Entropically driven VPT of poly(N-isopropylacrylamide) NPs containing carboxylic acids and amines generates a pH gradient of up to 0.049 and -0.053 pH K-1, respectively, around physiological temperature. The pH gradient triggers the proton-coupled electron transfer (PCET) reactions of quinhydrone on the electrodes, resulting in the highly efficient thermoelectric conversion with a Seebeck coefficient (Se) of -6.7 and +6.1 mV K-1. Thermocells driven by phase transition of hydrogels provide a nontoxic, flexible, and inexpensive charger that harvests carbon-free energy from abundant energy sources such as solar, body and waste heat.

MicroRNA-150 suppresses p27Kip1 expression and promotes cell proliferation in HeLa human cervical cancer cells.

MicroRNAs (miRNAs) exert critical roles in the majority of biological and pathological processes. Recent studies have associated miR-150 with a number of different cancer types. However, little is known about miR-150 targets in cervical cancer. In the present study, the HeLa human cervical cancer cell line was transfected with hsa-miR-150-5p mimics, hsa-miR-150-5p inhibitors or miRNA controls. miR-150 was predicted to bind the 3'untranslated region (3'UTR) of the CDKN1B gene, which encodes the cyclin-dependent kinase inhibitor 1B (p27Kip1). The direct binding between miR-150 and the 3'UTR of CDKN1B was confirmed using dual-luciferase reporter assays. The effects of miR-150 on CDKN1B mRNA expression, p27Kip1 protein expression, cell cycle and cell proliferation were determined using reverse-transcription quantitative PCR, western blot analysis, flow cytometry and WST-8 assays, respectively. miR-150 was demonstrated to directly target the 3'UTR of CDKN1B in transfected HeLa cells. The expression of CDKN1B mRNA and p27Kip1 protein was reduced by miR-150 mimics, and increased by miR-150 inhibitors. Moreover, the overexpression of miR-150 promoted cell cycle progression from the G0/G1 to the S phase and led to a significant increase in HeLa cell proliferation. The results of the present study indicated that miR-150 promotes HeLa cell cycle progression and proliferation via the suppression of p27Kip1 expression.

Influence of measurement principle on total hemoglobin value.

BACKGROUND: Total hemoglobin (tHb) measurement is indispensable for determining the patient's condition (hemorrhagic vs. ischemic) and need for blood transfusion. Conductivity- and absorbance-based measurement methods are used for blood gas analysis of tHb. For conductivity-based measurement, tHb is calculated after converting blood conductivity into a hematocrit value, whereas absorbance measurement is based on light absorbance after red blood cell hemolysis. Due to changes in plasma electrolytes and hemolysis, there is a possibility that conductivity- and absorbance-based measurement methods may cause a difference in tHb. METHODS: In this study, test samples with controlled electrolyte changes and hemolysis were created by adding sodium chloride, distilled water or hemolytic blood to blood samples collected from healthy volunteers, and tHb values were compared between both methods. RESULTS: Conductivity-based measurement revealed reduced tHb value (from 15.49 to 13.05 g/dl) following the addition of 10% sodium chloride, which was also reduced by the addition of hemolysate. Conversely, the addition of distilled water significantly increased tHb value than the expected value. In the absorbance method, there was no significant change in tHb value due to electrolyte change or hemolysis. CONCLUSIONS: We have to recognize unexpected conductivity changes occur at all times when tHb is measured via conductivity- and absorbance-based measurement methods. The absorbance method should be used when measuring tHb in patients with expected blood conductivity changes. However, when using this method, the added contribution of hemoglobin from hemolytic erythrocytes lacking oxygen carrying capacity must be considered. We recognize that discrepancy can occur between conductivity- and absorbance-based measurement methods when tHb is measured.

Changes in Muscle Activity in Response to Assistive Force during Isometric Elbow Flexion.

This study investigated the muscle activity and force variability in response to perturbation of assistive force during isometric elbow flexion. Sixteen healthy right-handed young men (age: 22.0 ± 1.1 years; height: 171.9 ± 4.8 cm; weight 68.4 ± 11.2 kg) were recruited and the muscle activity of biceps brachii and triceps brachii were assessed using surface electromyography. Workload force and assistive force applied on isometric elbow flexion significantly affected the changes in both biceps and triceps muscle activities. A higher assistive force was shown to result in reduced biceps muscle activity compared to the unassisted period. In contrast, the efficiency of the assistive force acting on the biceps decreased as the assistive force increased. In general, the force variability of the biceps muscle remained approximately the same at lower workload force conditions than that at higher workload force conditions. In conclusion, higher assistive force may not yield a higher performance efficiency in human-assistive force interaction.

The measurement of blood pressure by the linear method compared to the deflation methods differently modifies the pulse oximeter alarm frequency.

BACKGROUND: Noninvasive blood-pressure measurement device and pulse oximeter are important for patient monitoring. When these are placed on the same side, cuff inflation sometimes causes measurement failure by pulse oximeter. OBJECTIVE: The present study aimed to compare the pulse oximeter alarm frequency and pulse-wave disappearance duration between noninvasive blood-pressure measurement using the deflation method and that using the linear inflation method. METHODS: The study included 10 healthy subjects. The cuff for automatic sphygmomanometer was wrapped on one side of the upper arm and for pulse oximeter was attached to the thumb of the same side of upper limbs. RESULTS: The alarm frequency was 0 and 26% using the linear inflation and the deflation methods, respectively. Additionally, the pulse-wave disappearance duration was significantly longer using the deflation method than that using the linear inflation method (10.0 ± 1.5 vs 1.7 ± 0.8 s). With the linear inflation method, this duration was or less 3 s. In the deflation method, an excess pressure of 40 mmHg was used, which caused the alarm to turn on. Additionally, the heart rate was found to influence the alarm occurrence during measurement using the deflation method. CONCLUSION: Heart rate may influence alarm occurrence during blood-pressure measurement using the step deflation method. Using the linear inflation method, the risks of alarm occurrence and measurement failure are low, even when the pulse oximeter and blood-pressure measurement cuffs are installed on the same side, suggesting that this method is suitable for clinical use.

Improvement of the Culture Medium for the Dichlorvos-Ammonia (DV-AM) Method to Selectively Detect Aflatoxigenic Fungi in Soil.

The dichlorvos-ammonia (DV-AM) method is a simple but sensitive visual method for detecting aflatoxigenic fungi. Here we sought to develop a selective medium that is appropriate for the growth of aflatoxigenic fungi among soil mycoflora. We examined the effects of different concentrations of carbon sources (sucrose and glucose) and detergents (deoxycholate (DOC), Triton X-100, and Tween 80) on microorganisms in soils, using agar medium supplemented with chloramphenicol. The results demonstrated that 5⁻10% sucrose concentrations and 0.1⁻0.15% DOC concentrations were appropriate for the selective detection of aflatoxigenic fungi in soil. We also identified the optimal constituents of the medium on which the normal rapid growth of Rhizopus sp. was completely inhibited. By using the new medium along with the DV-AM method, we succeeded in the isolation of aflatoxigenic fungi from non-agricultural fields in Fukui city, Japan. The fungi were identified as Aspergillus nomius based on their calmodulin gene sequences. These results indicate that the new medium will be useful in practice for the detection of aflatoxigenic fungi in soil samples including those from non-agricultural environments.

A Significant Association of Malnutrition with Dysphagia in Acute Patients.

Dysphagia and malnutrition seem to be associated, but little research in detail has been reported. We aimed to clarify the association between dysphagia and malnutrition by adopting accurate diagnosis and mathematical evaluation of dysphagia using videofluorography and nutritional assessment calculated by a well-established nutritional risk index. We conducted a retrospective analysis of 165 enrolled patients who were admitted to our hospital for acute diseases and underwent videofluorography on suspicion of dysphagia in the year 2016. We diagnosed high-risk dysphagia in patients with 8-point penetration-aspiration scale (PAS) score over 4. We used the geriatric nutritional risk index (GNRI) as a nutritional assessment tool. A GNRI score less than 91.2 corresponds to malnutrition. The median age of 165 enrolled patients was 76.0, and the number of female patients was 53. The mean GNRI was 81.2, and 134 patients (81.2%) had malnutrition. The number of the patients with a diagnosis of high-risk dysphagia was 54 (32.7%). The GNRI of patients with high-risk dysphagia was significantly less than that of patients without (mean value 77.7 ± 10.5 vs. 83.0 ± 10.5, P = 0.003). GNRI < 91.2 was independently and significantly associated with high-risk dysphagia (OR 3.094; CI 1.057-9.058; P = 0.039). Based on the current study, the authors propose evaluating nutritional status to predict dysphagia risk of patients in the acute phase.

QPY/RAH haplotypes of the GZMB gene are associated with natural killer cell cytotoxicity.

Granzyme B (GzmB) is a component of cytolytic granules within NK cells and is involved in several pathologies. It has previously been reported that there are three non-synonymous coding SNPs (rs8192917; Q48R, rs11539752; P88A, and rs2236338; Y245H) in the GZMB gene and that the QPY/RAH allele was clustered together close to the C-terminal α-helix. However, it is unknown whether the function of GzmB produced from NK cells is influenced by QPY/RAH polymorphism. The authors investigated the distribution of QPY/RAH polymorphism of the GZMB gene in a Japanese population (n = 106), and the involvement of Q48R polymorphism in NK cell cytotoxicity, degranulation, and production of GzmB. A strong linkage disequilibrium was observed among these SNPs, and NK cell cytotoxicity was influenced by rs8192917 (Q48R). Moreover, it found that R48-GzmB is a stable protein that accumulates to similar levels in activated NK cells as Q48-GzmB. rs8192917 polymorphism may influence antitumor activity and the effect of antitumor cellular immunotherapy. The authors expect that these new informations about QPY/RAH polymorphism of the GZMB gene could help to assess the impact of NK cell cytotoxicity in several pathologies and aid their treatment.

Clinical Characteristics and Lesions Responsible for Swallowing Hesitation After Acute Cerebral Infarction.

Some stroke patients with a unilateral lesion demonstrate acute dysphagia characterized by a markedly prolonged swallowing time, making us think they are reluctant to swallow. In order to clarify the clinical characteristics and causative lesions of delayed swallowing, we conducted a retrospective analysis of 20 right-handed patients without a history of swallowing dysfunction who underwent videofluorography on suspicion of dysphagia after a first ischemic stroke. The oral processing time plus the postfaucial aggregation time required to swallow jelly for patients classified as having delayed swallowing was over 10 s. The time required for swallowing jelly was significantly longer than that without the hesitation (median value, 24.1 vs. 8.9 s, P < 0.001). The oral processing time plus the postfaucial aggregation time required for patients with delayed swallowing to swallow thickened water was largely over 5 s and significantly longer than that of patients without swallowing hesitation (median value, 10.2 vs. 3.3 s, P < 0.001). Swallowing hesitation caused by acute unilateral infarction could be separated into two different patterns. Because four of the five patients with a rippling tongue movement in the swallowing hesitation pattern had a lesion in the left primary motor cortex, which induces some kinds of apraxia, swallowing hesitation with a rippling tongue movement seems to be a representative characteristic of apraxia. The patients with swallowing hesitation with a temporary stasis of the tongue in this study tended to have broad lesions in the frontal lobe, especially in the middle frontal gyrus, which is thought to be involved in higher cognition.

Biosynthesis of bacteriochlorophyll c derivatives possessing chlorine and bromine atoms at the terminus of esterifying chains in the green sulfur bacterium Chlorobaculum tepidum.

The green sulfur photosynthetic bacterium Chlorobaculum tepidum newly produced BChl c derivatives possessing a chlorine or bromine atom at the terminus of the esterifying chain in the 17-propionate residue by cultivation with exogenous ω-halo-1-alkanols. The relative ratios of BChl c derivatives esterified with 8-chloro-1-octanol and 10-chloro-1-decanol were estimated to be 26.5% and 33.3% by cultivation with these ω-chloro-1-alkanols at the final concentrations of 300 and 150 µM, respectively. In contrast, smaller amounts of unnatural BChls c esterified with ω-bromo-1-alkanols were biosynthesized than those esterified with ω-chloro-1-alkanols: the ratios of BChl c derivatives esterified with 8-bromo-1-octanol and 10-bromo-1-decanol were 11.3% and 12.2% at the concentrations of 300 and 150 µM, respectively. These indicate that ω-chloro-1-alkanols can be incorporated into bacteriochlorophyllide c more than ω-bromo-1-alkanols in the BChl c biosynthetic pathway. The homolog compositions of the novel BChl c derivatives possessing a halogen atom were analogous to those of coexisting natural BChl c esterified with farnesol. These results demonstrate unique properties of BChl c synthase, BchK, which can utilize unnatural substrates containing halogen in the BChl c biosynthesis of Cba. tepidum.

New screening system for selective blockers of voltage-gated K(+) channels using recombinant cell lines dying upon single action potential.

To develop a simple screening system for blockers of voltage-gated Kv1.3 and Kv1.5 channels, new cell lines co-expressing mutated Nav1.5 (IFM/Q3), Kir2.1 (Kir), and Kv1.3 or Kv1.5 were introduced as IFM/Q3+Kir+Kv1.3 and IFM/Q3+Kir+Kv1.5, respectively. Electrical stimulation (ES) of a cell line, IFM/Q3+Kir, induced prolonged action potentials due to the slow inactivation of IFM/Q3 and subsequent cell death. Additional co-expression of Kv1.3 or Kv1.5 to IFM/Q3+Kir shortened the evoked action potentials and prevented cell death. In the presence of margatoxin, a selective Kv1.3-blocker, ES induced cell death in IFM/Q3+Kir+Kv1.3, but not in IFM/Q3+Kir+Kv1.5. In the presence of 4-aminopyridine, a non-selective Kv-channel blocker, ES application elicited cell death in both cell lines. The IC50s of acacetin, a Kv1.5-blocker, was 10.2 µM in IFM/Q3+Kir+Kv1.3 and almost identical to that in IFM/Q3+Kir+Kv1.5 (7.6 µM). The IC50s of citalopram, a 5-HT uptake-inhibitor, were 1.8 µM in IFM/Q3+Kir+Kv1.3 and 1.5 µM in IFM/Q3+Kir+Kv1.5, respectively. These IC50s were comparable to those determined electrophysiologically. In conclusion, acacetin and citalopram block both Kv1.3 and Kv1.5 without selectivity. The Kv1.3 or Kv1.5 channel inhibition assay using these new cell lines may be applicable to high-throughput screening because of its simplicity, accuracy, and high cost-performance.

Integration of liquid crystal elements for creating an infrared Lyot filter.

An infrared Lyot filter was fabricated by integrating a polarization beam splitter and two retarders into a single device. A liquid crystal layer was constructed between two silicon pentaprisms that were designed suitably so that light was incident on this layer at 28°. At this angle, the liquid crystal transmitted p-polarized light (Brewster's angle) and reflected s-polarized light (total internal reflection). The p- or s-polarized light was directed to another liquid crystal layer (retarder) between the prism and a mirror, which induced a wavelength-dependent retardation in the reflection process. Consequently, the light that returned to the beam splitter was transmitted or reflected depending on wavelength.

Comparison of feeding systems: feed cost, palatability and environmental impact among hay-fattened beef, consistent grass-only-fed beef and conventional marbled beef in Wagyu (Japanese Black cattle).

The objective of this article is to compare feed cost, palatability and environmental impacts among feeding systems of high concentrate (HC), high hay (HH) and grass-only-fed (Gof) groups. Feed cost was the sum of costs paid for feed intake times the price of feed per kilogram. Palatability was measured by a panel taste test using HH and Gof beef and analyzed for differences. Environmental impacts were calculated based on 1 kg of Japanese beef yield of CO(2) equivalents (eq) and animal end weights at each feeding stage. Results showed that the HH and Gof feeding systems could significantly reduce feed costs by approximately 60% and 78%, respectively, from the HC. In the panel taste test, 50% and 47.50% of panelists indicated that HH beef was 'extremely delicious' and 'acceptable,' respectively, while 15% indicated that Gof beef was 'extremely delicious'; 62.50% indicated that Gof beef was 'acceptable.' Environmental impacts of each feeding system in terms of CO(2) equivalents (eq) were 9.32, 6.10 and 2.04 tonnes of eq for the HC, HH and Gof, respectively. The HH was an economical system that produced moderate impacts on the environment and had impressive taste.

Possibility of oral feeding after induction of percutaneous endoscopic gastrostomy.

BACKGROUND AND AIM: Although percutaneous endoscopic gastrostomy (PEG) has become established as a useful enteral nutrition technique, the associated risks must always be kept in mind. Recently, we experienced several patients who could orally ingest after PEG. To avoid unnecessary PEG, we investigated patients who could orally ingest after PEG, and analyzed predictive factors of postoperative oral feeding. METHODS: We retrospectively analyzed data of 302 patients who underwent PEG at our hospital. After all patients were divided according to postoperative oral feeding status, we assessed factors of patients' backgrounds. In patients who could orally ingest after PEG, we investigated the course of oral feeding status. We attempted to identify predictive factors for postoperative oral feeding using logistic regression analysis. RESULTS: Mean age was high in both groups, and overall condition was markedly poor. Forty-four patients (15%) were able to ingest orally after PEG. Enteral nutrition could be avoided during our observation period in 15 cases, because sufficient oral intake was achieved. Conversely, oral feeding was reduced or discontinued in 14 cases. Multivariate analysis identified the following independent predictive factors for postoperative oral feeding: (i) absence of dysphagia or aphagia; (ii) younger age; (iii) favorable performance status; (iv) presence of post-traumatic encephalopathy; and (v) preoperative swallowing training. CONCLUSIONS: A total of 15% of PEG cases were able to ingest orally after PEG. In patients showing positive predictive factors, indications for PEG should be carefully considered.

Impact of fungal drug transporters on fungicide sensitivity, multidrug resistance and virulence.

Drug transporters are membrane proteins that provide protection for organisms against natural toxic products and fungicides. In plant pathogens, drug transporters function in baseline sensitivity to fungicides, multidrug resistance (MDR) and virulence on host plants. This paper describes drug transporters of the filamentous fungi Aspergillus nidulans (Eidam) Winter, Botrytis cinerea Pers and Mycosphaerella graminicola (Fückel) Schroter that function in fungicide sensitivity and resistance. The fungi possess ATP-binding cassette (ABC) drug transporters that mediate MDR to fungicides in laboratory mutants. Similar mutants are not pronounced in field resistance to most classes of fungicide but may play a role in resistance to azoles. MDR may also explain historical cases of resistance to aromatic hydrocarbon fungicides and dodine. In clinical situations, MDR development in Candida albicans (Robin) Berkhout mediated by ABC transporters in patients suffering from candidiasis is common after prolonged treatment with azoles. Factors that can explain this striking difference between agricultural and clinical situations are discussed. Attention is also paid to the risk of MDR development in plant pathogens in the future. Finally, the paper describes the impact of fungal drug transporters on drug discovery.

Modulators of membrane drug transporters potentiate the activity of the DMI fungicide oxpoconazole against Botrytis cinerea.

Modulators known to reduce multidrug resistance in tumour cells were tested for their potency to synergize the fungitoxic activity of the fungicide oxpoconazole, a sterol demethylation inhibitor (DMI), against Botrytis cinerea Pers. Chlorpromazine, a phenothiazine compound known as a calmodulin antagonist, appeared the most potent compound. Tacrolimus, a macrolide compound with immunosuppressive activity, was also active. The synergism of chlorpromazine negatively correlated with the sensitivity of the parent strain and mutants of B. cinerea. The synergism was highest in a mutant that overexpressed the ATP-binding cassette transporter BcatrD, known to transport DMI fungicides such as oxpoconazole. The synergism of chlorpromazine positively correlated with its potency to enhance the accumulation of oxpoconazole in BcatrD mutants. These results indicate that chlorpromazine is a modulator of BcatrD activity in B. cinerea and suggest that mixtures of DMI fungicides with modulators may represent a perspective for the development of new resistance management strategies.

Bcmfs1, a novel major facilitator superfamily transporter from Botrytis cinerea, provides tolerance towards the natural toxic compounds camptothecin and cercosporin and towards fungicides.

Bcmfs1, a novel major facilitator superfamily gene from Botrytis cinerea, was cloned, and replacement and overexpression mutants were constructed to study its function. Replacement mutants showed increased sensitivity to the natural toxic compounds camptothecin and cercosporin, produced by the plant Camptotheca acuminata and the plant pathogenic fungus Cercospora kikuchii, respectively. Overexpression mutants displayed decreased sensitivity to these compounds and to structurally unrelated fungicides, such as sterol demethylation inhibitors (DMIs). A double-replacement mutant of Bcmfs1 and the ATP-binding cassette (ABC) transporter gene BcatrD was more sensitive to DMI fungicides than a single-replacement mutant of BcatrD, known to encode an important ABC transporter of DMIs. The sensitivity of the wild-type strain and mutants to DMI fungicides correlated with Bcmfs1 expression levels and with the initial accumulation of oxpoconazole by germlings of these isolates. The results indicate that Bcmfs1 is a major facilitator superfamily multidrug transporter involved in protection against natural toxins and fungicides and has a substrate specificity that overlaps with the ABC transporter BcatrD. Bcmfs1 may be involved in protection of B. cinerea against plant defense compounds during the pathogenic phase of growth on host plants and against fungitoxic antimicrobial metabolites during its saprophytic phase of growth.