Persistent Inhibitory Effect Research Articles

Unveiling the inhibitory mechanisms of chromium exposure on microbial reductive dechlorination: Kinetics and microbial responses

Chromium and organochlorine solvents, particularly trichloroethene (TCE), are pervasive co-existing contaminants in subsurface aquifers due to their extensive industrial use and improper disposal practices. In this study, we investigated the microbial dechlorination kinetics under different TCE-Cr(Ⅲ/VI) composite pollution conditions and elucidated microbial response mechanisms based on community shift patterns and metagenomic analysis. Our results revealed that the reductive dechlorinating consortium had high resistance to Cr(III) but extreme sensitivity to Cr(VI) disturbance, resulting in a persistent inhibitory effect on subsequent dechlorination. Interestingly, the vinyl chloride-respiring organohalide-respiring bacteria (OHRB) was notably more susceptible to Cr(III/VI) exposure than the trichloroethene-respiring one, possibly due to inferior competition for growth substrates, such as electron donors. In terms of synergistic non-OHRB populations, Cr(III/VI) exposure had limited impacts on lactate fermentation but significantly interfered with H2-producing acetogenesis, leading to inhibited microbial dechlorination due to electron donor deficiencies. However, this inhibition can be effectively mitigated by the amendment of exogenous H2 supply. Furthermore, being the predominant OHRB, Dehalococcoides have inherent Cr(VI) resistance defects and collaborate with synergistic non-OHRB populations to achieve concurrent bio-detoxication of Cr(VI) and TCE. Our findings expand the understanding of the response patterns of different functional populations towards Cr(III/VI) stress, and provide valuable insights for the development of in situ bioremediation strategies for sites co-contaminated with chloroethene and chromium.

Persistent inhibition of Candida albicans biofilm and hyphae growth on titanium by graphene nanocoating

ObjectivesCandida albicanscolonizes biomaterial surfaces and are highly resistant to therapeutics. Graphene nanocoating on titanium compromises initial biofilm formation. However, its sustained antibiofilm potential is unknown. The objective of this study was to investigate the potential of graphene nanocoating to decrease long-term fungal biofilm development and hyphae growth on titanium. MethodsGraphene nanocoating was deposited twice (TiGD) or five times (TiGV) on grade 4 titanium with vacuum assisted technique and characterized with Raman spectroscopy and atomic force microscope. The biofilm formation and hyphae growth of C. albicans was monitored for seven days by CFU, XTT, confocal, mean cell density and scanning electronic microscopy (SEM). Uncoated titanium was the Control. All tests had three independent biological samples and were performed in independent triplicates. Data was analyzed with one- or two-way ANOVA and Tukey's HSD (α = 0.05). ResultsBoth TiGD and TiGV presented less biofilms at all times points compared with Control. The confocal and SEM images revealed few adhered cells on graphene coated samples, absence of hyphae and no features of a mature biofilm architecture. The increase in number of layers of graphene nanocoating did not improve its antibiofilm potential. SignificanceThe graphene nanocoating exerted a long-term persistent inhibitory effect on the biofilm formation on titanium. The fewer cells that were able to attach on graphene coated titanium were scattered and unable to form a mature biofilm with hyphae elements. The findings open opportunities to prevent microbial attachment and proliferation on implantable materials without the use of antibiotics.

In Vitro Antimicrobial Activity of Fosfomycin, Vancomycin and Daptomycin Alone, and in Combination, Against Linezolid-Resistant Enterococcus faecalis.

IntroductionEnterococcus faecalis is a significant cause of nosocomial infections and is difficult to treat because of intrinsic and acquired resistance to many antibiotics. In addition, the emergence of linezolid-resistant E. faecalis (LZR-Efa) is reducing the choices available for anti-infective therapy. The aim of this study was to examine the in vitro antibacterial effects of fosfomycin (FM), vancomycin (VAN) and daptomycin (DAP), alone and in combination, against LZR-Efa.MethodsFive LZR-Efa strains and E. faecalis ATCC 29212 were studied. The antibacterial effects of FM, and of FM, VAN and DAP, were assessed using the time–kill assay. Biofilm formation and elimination were evaluated by crystal violet staining.ResultsWhen used at concentrations greater than 0.5 × MIC, FM did not produce dose-dependent effects against LZR-Efa isolates. The use of DAP (47.1 mg/L) alone, and FM (83 mg/L) combined with DAP (20.6 mg/L), produced a persistent inhibitory effect against both planktonic LZR-Efa isolates and those forming biofilms. In addition, FM and VAN combined with glucose-6-phosphate produced visible eradication effects against biofilms grown for 24 h, while DAP alone or combined with FM resulted in the best eradication activity against biofilms grown for 72 h prior to exposure.ConclusionThe use of FM combined with DAP provided the best potential therapeutic option for treating LZR-Efa infections out of those tested. In addition, the optimum treatment for biofilm elimination depended on the stage of biofilm formation.Electronic supplementary materialThe online version of this article (10.1007/s40121-020-00342-1) contains supplementary material, which is available to authorized users.

Enhanced and Persistent Inhibition of Organic Cation Transporter 1 Activity by Preincubation of Cyclosporine A.

Recent pharmacogenetic evidence indicates that hepatic organic cation transporter (OCT) 1 can serve as the locus of drug-drug interactions (DDIs) with significant pharmacokinetic and pharmacodynamic consequences. We examined the impact of preincubation on the extent of OCT1 inhibition in transfected human embryonic kidney 293 (HEK293) cells. Following 30-minute preincubation with an inhibitor, approximately 50-fold higher inhibition potency was observed for cyclosporine A (CsA) against OCT1-mediated uptake of metformin compared with coincubation, with IC50 values of 0.43 ± 0.12 and 21.6 ± 4.5 µM, respectively. By comparison, only small shifts (≤2-fold) in preincubation IC50 versus coincubation were observed for quinidine, pyrimethamine, ritonavir, and trimethoprim. The shift in CsA OCT1 IC50 was substrate dependent since it ranged from >1.2- to 50.2-fold using different experimental substrates. The inhibition potential of CsA toward OCT1 was confirmed by fenoterol hepatocyte uptake experiment. Furthermore, no shift in CsA IC50 was observed with HEK293 cells transfected with OCT2 and organic anion transporter (OAT) 1 and OAT3. Short exposure (30 minutes) to 10 µM CsA produced long-lasting inhibition (at least 120 minutes) of the OCT1-mediated uptake of metformin in OCT1-HEK293 cells, which was likely attributable to the retention of CsA in the cells, as shown by the fact that inhibitory cellular concentrations of CsA were maintained long after the removal of the compound from the incubation buffer. The potent and persistent inhibitory effect after exposure to CsA warrants careful consideration in the design and interpretation of clinical OCT1 DDI studies. SIGNIFICANCE STATEMENT: Preincubation of OATP1B1 and OATP1B3 with their inhibitor may result in the enhancement of the inhibitory potency in a cell-based assay. However, limited data are available on potentiation of OCT1 inhibition by preincubation, which is a clinically relevant drug transporter. For the first time, we observed a 50-fold increase in CsA inhibitory potency against OCT1-mediated transport of metformin following a preincubation step. The CsA preincubation effect on OCT1 inhibition is substrate dependent. Moreover, the inhibition potential of CsA toward OCT1 is confirmed by hepatocyte uptake experiment. This study delivers clear evidences about the potent and persistent inhibitory effect on OCT1 after exposure to CsA. Further studies are needed to assess the effect of CsA on OCT1 drug substrates in vivo.

Amphibian toxins from the skin of <i>Bufo regularis</i> have inhibitory effect on electrically invoked contractile response and bimodal effects on tone of longitudinal muscle of guinea pig ileum

The skins of some amphibians contain potentially bioactive principles that may have pharmaceutical, medicinal, toxicological or chemical importance. In addition, such active principles can be used as tools in biomedical research. The present study aims at isolating and purifying bioactive principles from the skin of Bufo regularis and studying their effect on isolated longitudinal smooth muscle strip of guinea pig ileum. High performance liquid chromatography (HPLC) was used to isolate toad toxins. The effects of crude, semi-purified and purified extracts were tested on longitudinal smooth muscle of guinea pig ileum using organ bath method. Effect of the toxins was studied on electrically-induced contractile response and the basal tone of the longitudinal muscle strip. HPLC purification resulted in four different bioactive components with a λmax UV absorbance pattern of around 295 nm. When tested on guinea pig ileum they had persistent inhibitory effect on the electrically-induced contractile responses. The pattern of effect was initial excitatory followed by long lasting inhibitory effect on tone of longitudinal muscle. The HPLC eluate at 79th min in methanol preparative run corresponding to the eluate at 40th min in the acetonitrile run had the maximum bioactivity. Hence, it was concluded that the skin of B. regularis contains four different components which vary in their potency on isolated smooth muscle strip of guinea pig ileum.Keywords: Bufo regularis, organ bath, longitudinal muscle of ileum, toad toxin, electrical field stimulation

Grapefruit seed extract effectively inhibits the Candida albicans biofilms development on polymethyl methacrylate denture-base resin.

This study aimed to investigate the cleansing effects of grapefruit seed extract (GSE) on biofilms of Candida albicans (C. albicans) formed on denture-base resin and the influence of GSE on the mechanical and surface characteristics of the resin. GSE solution diluted with distilled water to 0.1% (0.1% GSE) and 1% (1% GSE) and solutions with Polident® denture cleansing tablet dissolved in distilled water (Polident) or in 0.1% GSE solution (0.1% G+P) were prepared as cleansing solutions. Discs of acrylic resin were prepared, and the biofilm of C. albicans was formed on the discs. The discs with the biofilm were treated with each solution for 5 min at 25°C. After the treatment, the biofilm on the discs was analyzed using a colony forming unit (CFU) assay, fluorescence microscopy, and scanning electron microscopy (SEM). In order to assess the persistent cleansing effect, the discs treated with each solution for 5 min were aerobically incubated in Yeast Nitrogen Base medium for another 24 h. After incubation, the persistent effect was assessed by CFU assay. Some specimens of acrylic resin were immersed in each solution for 7 days, and changes in surface roughness (Ra), Vickers hardness (VH), flexural strength (FS), and flexural modulus (FM) were evaluated. As a result, the treatment with 1% GSE for 5 min almost completely eliminated the biofilm formed on the resin; whereas, the treatment with 0.1% GSE, Polident, and 0.1% G+P for 5 min showed a statistically significant inhibitory effect on biofilms. In addition, 0.1% GSE and 0.1% G+P exerted a persistent inhibitory effect on biofilms. Fluorescence microscopy indicated that Polident mainly induced the death of yeast, while the cleansing solutions containing at least 0.1% GSE induced the death of hyphae as well as yeast. SEM also revealed that Polident caused wrinkles, shrinkage, and some deep craters predominantly on the cell surfaces of yeast, while the solutions containing at least 0.1% GSE induced wrinkles, shrinkage, and some damage on cell surfaces of not only yeasts but also hyphae. No significant changes in Ra, VH, FS, or FM were observed after immersion in any of the solutions. Taken together, GSE solution is capable of cleansing C. albicans biofilms on denture-base resin and has a persistent inhibitory effect on biofilm development, without any deteriorations of resin surface.

Separate and joint eco-toxicological effects of sulfadimidine and copper on soil microbial biomasses and ammoxidation microorganisms abundances

Heavy metals and antibiotics residues in agricultural soils are attracting more and more attention. A laboratory study was conducted to evaluate the single and combined effects of sulfadimidine (SM2) (0.05, 0.20, 0.80 mmol/kg) and copper (Cu) (1.60 mmol/kg) on soil microbial biomasses and ammoxidation microorganisms abundances after 7, 14, 21 and 28 days. The results demonstrated that the single and combined contaminations had a significant and persistent inhibitory effect on soil bacteria, fungi and actinomycetes populations and amoA gene copies of ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB) (Except SM2 0.05 and 0.20 mmol/kg on 7 and 14 d and SM2 0.05 mmol/kg on 21 d led to a stimulatory effect on fungi and AOA-amoA gene, respectively). With higher dosage and longer exposure time, the toxic effect of single and combined contaminants on soil bacteria, fungi and actinomycetes as well as on the amoA gene of AOA and AOB was greatly reinforced. Combined contaminants produced more toxicity than the chemicals were used alone. Overall, the interaction effects of SM2 and Cu on bacteria (on 14, 21 and 28 d), fungi and AOA-amoA were mainly synergism, in contrast, on actinomycetes (on 14, 21 and 28 d) and AOB-amoA were mainly antagonism. The order of the toxic effects of the single Cu and combined contaminants on microbial activity was: bacteria > actinomycetes > fungi. Furthermore, AOB-amoA was more sensitive to both contaminants toxicity than AOA-amoA, while AOA-amoA gene copies were greater than AOB-amoA gene copies about one order of magnitude.

S145. Effect of pulse width and phase on MEP latency and its significance in inducing cortical plastic changes

Repetitive transcranial magnetic stimulation (rTMS) has been found to be a promising non-invasive therapeutic tool for a variety of neuropsychiatric conditions. Variable pulse shapes can now be generated with a controllable pulse stimulator TMS (cTMS). We looked into two main characteristics of the pulse: the pulse width and the pulse direction. The pulse direction was expressed by the M ratio, which refers to the ratio between the amplitude of the main phase and the second phase i.e.: the lowest is monophasic while the highest is biphasic with two equal amplitude phases. to investigate the effect of different pulse widths and phase directions in rTMS on plastic aftereffects, we compared MEP latencies among TMS pulses with different pulse durations (40 μs, 80 μs and 120 μs) using 0.2 M ratio pulses, and with different pulse directions (0.2, 0.6 and 1.0 M ratios) 80 μs wide pulses. We also examined effects of AP 900 pulses 1 Hz rTMS. During AP stimulation, results from 12 subjects showed that the narrower pulse shapes had longer onset and peak latencies while wider pulse shapes showed shorter latencies. rTMS results from 10 subjects showed that shorter pulse widths of 40 and 80 μs were inhibitory with the 40 μs rTMS having more consistent and persistent inhibitory effects. The widest pulse of 120 μs was excitatory in terms of the post stimulation MEP amplitude. The 0.2 M ratio rTMS produced inhibitory after-effects, while the 0.6 and the 1.0 ratio showed almost similar facilitatory effects. The shorter pulse width stimuli caused preferentially inhibition as they stimulated a smaller number of neurons as a portion from the local circuit causing the stimulated neurons to desynchronize the firing of the local neuronal network. In comparison, the wider pulse stimuli likely stimulated a wider area setting the pace for the neuronal network firing rate. As for the phasicity, we believe that the inhibition brought by the monophasic 0.2 M ratio pulses could be accounted to repeated hyperpolarization of pyramidal cell, while the biphasic 0.6 and 1.0 M ratio pulses induce similar hyperpolarization followed by a steeper polarization thus producing facilitation. Figuring out the optimal pulse shape for either excitation or inhibition of the brain would help understand how its effect is actually propagating through local cortical networks and eventually being able to fine-tune its therapeutic efficacy.

P305 Effect of pulse width and phase on mep latency and its significance in inducing cortical plastic changes

Introduction Repetitive transcranial magnetic stimulation (rTMS) has been found to be a promising non-invasive therapeutic tool for a variety of neuropsychiatric conditions. Variable pulse shapes can now be generated with a controllable pulse stimulator TMS (cTMS). We looked into two main characteristics of the pulse: the pulse width and the pulse direction. The pulse direction was expressed by the M ratio, which refers to the ratio between the amplitude of the main phase and the second phase i.e.: the lowest is monophasic while the highest is biphasic with two equal amplitude phases. Methods to investigate the effect of different pulse widths and phase directions in rTMS on plastic aftereffects, we compared MEP latencies among TMS pulses with different pulse durations (40 μs, 80 μs and 120 μs) using 0.2 M ratio pulses, and with different pulse directions (0.2, 0.6 and 1.0 M ratios) 80 μs wide pulses. We also examined effects of AP 900 pulses 1 Hz rTMS. Results During AP stimulation, results from 12 subjects showed that the narrower pulse shapes had longer onset and peak latencies while wider pulse shapes showed shorter latencies. rTMS results from 10 subjects showed that shorter pulse widths of 40 and 80 μs were inhibitory with the 40 μs rTMS having more consistent and persistent inhibitory effects. The widest pulse of 120 μs was excitatory in terms of the post stimulation MEP amplitude. The 0.2 M ratio rTMS produced inhibitory after-effects, while the 0.6 and the 1.0 ratio showed almost similar facilitatory effects. Conclusion The shorter pulse width stimuli caused preferentially inhibition as they stimulated a smaller number of neurons as a portion from the local circuit causing the stimulated neurons to desynchronize the firing of the local neuronal network. In comparison, the wider pulse stimuli likely stimulated a wider area setting the pace for the neuronal network firing rate. As for the phasicity, we believe that the inhibition brought by the monophasic 0.2 M ratio pulses could be accounted to repeated hyperpolarization of pyramidal cell, while the biphasic 0.6 and 1.0 M ratio pulses induce similar hyperpolarization followed by a steeper polarization thus producing facilitation. Figuring out the optimal pulse shape for either excitation or inhibition of the brain would help understand how its effect is actually propagating through local cortical networks and eventually being able to fine-tune its therapeutic efficacy.

Antioxidant, antimicrobial, antiparasitic, and cytotoxic properties of various Brazilian propolis extracts.

Propolis is known for its biological properties and its preparations have been continuously investigated in an attempt to solve the problem of their standardization, an issue that limits the use of propolis in food and pharmaceutical industries. The aim of this study was to evaluate in vitro antioxidant, antimicrobial, antiparasitic, and cytotoxic effects of extracts of red, green, and brown propolis from different regions of Brazil, obtained by ethanolic and supercritical extraction methods. We found that propolis extracts obtained by both these methods showed concentration-dependent antioxidant activity. The extracts obtained by ethanolic extraction showed higher antioxidant activity than that shown by the extracts obtained by supercritical extraction. Ethanolic extracts of red propolis exhibited up to 98% of the maximum antioxidant activity at the highest extract concentration. Red propolis extracts obtained by ethanolic and supercritical methods showed the highest levels of antimicrobial activity against several bacteria. Most extracts demonstrated antimicrobial activity against Staphylococcus aureus. None of the extracts analyzed showed activity against Escherichia coli or Candida albicans. An inhibitory effect of all tested ethanolic extracts on the growth of Trypanosoma cruzi Y strain epimastigotes was observed in the first 24 h. However, after 96 h, a persistent inhibitory effect was detected only for red propolis samples. Only ethanolic extracts of red propolis samples R01Et.B2 and R02Et.B2 showed a cytotoxic effect against all four cancer cell lines tested (HL-60, HCT-116, OVCAR-8, and SF-295), indicating that red propolis extracts have great cytotoxic potential. The biological effects of ethanolic extracts of red propolis revealed in the present study suggest that red propolis can be a potential alternative therapeutic treatment against Chagas disease and some types of cancer, although high activity of red propolis in vitro needs to be confirmed by future in vivo investigations.

P048. Cigarette smoke impairs intestinal epithelial cell polarity development and morphogenesis

Background: Cigarette smoking is emerging as an important lifestylefactor that affects intestinal function. Epidemiological studies indicated that cigarette smoking influences the course of inflammatory bowel disease and is a risk factor for colorectal cancer andintestinal polyp development. The mechanism via which cigarette smoke exerts its effect on the intestinal epithelium is unclear. These may involve indirect effects via alterations in the immune system or direct effects of cigarette smoke on the intestinal epithelium. Little is known about these direct effects of cigarette smoke on intestinal epithelial cells. Here we present an in-vitro study of these direct effects Methods: Human intestinal epithelial Caco-2 cell-matrigel-embedded spheriods were grown for 7 days in the presence of, or pre treated with, cigarette smoke extract (CSE). For pre treatment cell were incubated with CSE for 24 hours, and subsequently plated out in fresh in3D culture. Intestinal epithelial barrier function was measured witha FITC-dextran leakage assay. Cell polarity and morphogenesis were assessed with fluorescently labelled polarity markers Results: We found that CSE did not affect the polarity and barrier function of pre established lumen-forming Caco-2 spheroids.However, when exposed to CSE during the process of 3D morphogenesis,cells showed impaired growth and impaired de novo development of apical-basal cell polarity and tight junctions, resulting in defective self-assembly into single lumen-forming spheroids. This effect was dose-dependent and this phenotype could be induced with pre incubation with CSE as well. However, the administration of the glutathione precursor N-acetyl-L-cysteine (NAC) completely neutralised the inhibitory effects of CSE. The mechanisms by which NAC rescued the phenotype were independent of its capacity to induce glutathione production Conclusions: Our data demonstrate that CSE has direct and persistentinhibitory effects on intestinal epithelial cell polarity developmentand morphogenesis, and provide insight into the cell biologicalmechanisms. NAC supplementation was identified as a protectivestrategy for smoke-induced epithelial dysfunction

Abstract A62: TRX-1 targets chemoresistant tumor-initiating cells and prolongs survival in a recurrent ovarian cancer animal model.

Abstract Background: Ovarian cancer is the leading cause of mortality from gynecologic cancers due to the high incidence of chemoresistance and disease recurrence. Disease recurrence is thought to occur due to the presence of residual disease following first-line standard of care (i.e. optimal debulking and chemotherapy). Residual disease post chemotherapy is composed of a unique population of chemoresistant cancer cells with stemness properties and a high capacity for tumor repair. Currently, there are no available options that can target these cells. The objective of this study was to develop a new therapeutic modality that will target chemoresistant ovarian cancer stem cells (OCSC) and consequently prevent recurrence and improve survival. To achieve this objective we developed a library of super-benzopyran (SBP) analogues and identified TRX-1 as the most potent analogue able to induce OCSC cell death in a short period of time. Moreover, in contrast to Cisplatin and Paclitaxel, TRX-1 is able to induce a persistent growth inhibitory effect both in vitro and in vivo thus improving overall survival in a mouse model of recurrent ovarian cancer. Materials and methods: A panel of SBP analogues were generated and activity was determined by testing against pure clones of CD44+/MyD88+ OCSC. In vitro efficacy was assessed using the IncucyteTM kinetic imaging platform complemented by CelltoxTM dye labeling. In vivo efficacy was tested using an intra-peritoneal (i.p.) a mouse model of recurrent ovarian cancer1. Results: TRX-1 was the most potent analogue identified and is able to induce cell death in all OCSC clones tested (IC50 of 136 nM). We observed that 2h in vitro exposure to 2μM TRX-1 was sufficient to induce a sustained growth inhibitory effect in OCSC, hence the cells were not able to recover growth potential even after removal of the drug. In contrast, OCSC exposed for up to 24h with the same dose of Cisplatin or Paclitaxel were able to recover. In vivo, animals bearing residual tumors after Paclitaxel treatment demonstrated tumor progression when further maintained with vehicle or Paclitaxel indicating Paclitaxel resistance. In contrast, maintenance with TRX-1 was able to effectively decrease tumor burden (p = 0.02) and prevent recurrence. In addition, combination treatment with TRX-1 and Cisplatin was able to significantly improve survival (p<0.001) compared to Cisplatin alone. Conclusion: We describe the in vitro and in vivo anti-tumoral effect of a novel compound, TRX-1, which exhibits significant efficacy against chemoresistant OCSC and is able to prevent recurrence in a chemoresistant in vivo model. Recurrence characterized by chemoresistance is the main cause of mortality in ovarian cancer patients. Previous studies from our laboratory have shown that conventional chemotherapy is not effective against OCSC and cannot prevent recurrence. Our finding that TRX-1, by targeting OCSC can prevent recurrence in vivo as maintenance therapy or in combination with chemotherapy provides a new opportunity for developing new therapeutic strategies that can improve survival in ovarian cancer patients. Citation Format: Ayesha Alvero, Eydis Lima, Mary Pitruzzello, Yang Yang-Hartwich, David Brown, Andrew Heaton, Gil Mor. TRX-1 targets chemoresistant tumor-initiating cells and prolongs survival in a recurrent ovarian cancer animal model. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr A62.

Crossed inhibition of sensory cortex by 0.3 Hz transcranial magnetic stimulation of motor cortex.

Low-frequency repetitive transcranial magnetic stimulation (rTMS) of motor cortex causes persistent inhibitory effects in the targeted area. rTMS of motor cortex impairs sensory perception and results in a persistent change in cortical function at remote sites. The ability of rTMS to induce sustained changes in cortical function has led to studies testing its therapeutic efficacy in neurologic disorders, including epilepsy. Studies on the effect of low-frequency rTMS of motor cortex on the contralateral motor cortex have provided evidence for both inhibitory and excitatory changes. This study was designed to determine the effect of low-frequency rTMS of the right motor cortex on the contralateral sensory cortex. Before and after 0.3-Hz rTMS of right motor cortex, perception of ipsilateral threshold of cutaneous stimuli was assessed and somatosensory evoked potentials (SEPs) recorded after stimulation of the right thumb in eight normal subjects. In a control group of six subjects, sensory responses were assessed after rTMS anterior to the right motor cortex. After rTMS of motor cortex, detection of threshold sensory stimuli decreased by more than 50% compared with pre-rTMS (P < 0.05). The change in sensory perception lasted at least 30 minutes. No change was detected in the control group. Amplitude of the N20-P25 waveform of the SEP decreased from a mean of 0.84 muV before rTMS to 0.54 muV immediately after rTMS of motor cortex (P < 0.05). 0.3 Hz rTMS of motor cortex inhibits the contralateral sensory cortex.

109. Replicating Oncolytic Chimeric Adenovirus Expressing shRNA Improves Inhibition of Human Colorectal Carcinoma (CRC) Growth

RNAi combined with oncolytic viral vector therapy may provide a new approach to control malignant disease since allele specific shRNA to a retrogene that modulates stemness will inhibit growth of human CRC in vivo. Our original delivery vector was nonreplicating Lentiviral vectors (LV) but now oncolytic adenovirus is the vector. We have created a conditionally replicating chimeric oncolytic adenovirus (CRAd) expressing a shRNA to inhibit growth of established tumors in preclinical models. shRNA is directed to NANOGP8, a retrogene that differs from parental NANOG primarily in a single nucleotide variant: c.759G>C, p.Gln253His. Oncolytic adenoviruses were generated with a 5/3 fiber with either a control (Ad5/3-wtE1a-shNEG (Ad5/3shNEG)) or an anti-NANOGP8 shRNA (Ad5/3-wtE1a-shNp8 (Ad5/3shNp8)). LV shNEG or LV shNp8 were added 24 hr after creation of 3-D organoid cultures of human CRC cells (Clone A, CX-1 or LS174T) in serum-free medium. LV transduced 45 – 90% of cells in culture and shNp8 inhibited organoids by ~50% compared to either shNEG or untreated CRC organoids. In addition, LV injected into 7 -9 mm diameter CX-1 tumors in NOD/SCID mice reduced tumor growth in vivo by 40% for 11 days, but then tumors grew at an accelerated rate. NMF cluster analysis reveals that LVshNp8 affects a gene expression profile that is distinct from that of either LVshNEG or untreated controls. In contrast, Ad5/3shNp8 inhibited organoid growth by more than 70% and controlled growth of CX-1 and LS174T in vivo. Infection of cells in vitro was over 90%. Thus, shNp8 alters gene expression and replicating oncolytic virus with shNp8 has a more persistent inhibitory effect than that achieved with non-replicating LV-delivered shRNA.

Accumulation of chlorothalonil and its metabolite, 4-hydroxychlorothalonil, in soil after repeated applications and its effects on soil microbial activities under greenhouse conditions

The effect of repeated chlorothalonil applications on its persistence and soil microbial activities were investigated in planted soil under greenhouse conditions. The results revealed that suppressed dissipation of chlorothalonil and the consequent accumulation of chlorothalonil and its toxic metabolite, 4-hydroxychlorothalonil, resulted from four successive applications at rates of 2, 4, and 10 kg a.i./ha. Soil respiration for 7 h (SR7) was slightly affected by chlorothalonil applications at all three levels, whereas soil respiration for 24 h (SR24) at 10 kg a.i./ha was reduced by 17.6-59.1 %, depending upon the frequency. This harmful effect of chlorothalonil on SR24 persisted throughout the experiment although it gradually weakened with time. A persistent inhibitory effect on soil dehydrogenase activity was also observed with repeated applications of chlorothalonil at 10 kg a.i./ha. This study indicated that repeated chlorothalonil applications may lead to the accumulation of chlorothalonil and its metabolite in soil under greenhouse conditions and thereby alter soil microbial activity.

Influence of a long-term high-fat diet on ghrelin secretion and ghrelin-induced food intake in rats

The aims of this study were: (1) to define the extent to which a high-fat (HF) diet given on a long-term basis reduces resting plasma ghrelin (total [acyl+des-acyl]) levels and the plasma ghrelin (total) response to fasting, (2) to determine whether a chronic HF diet modifies the orexigenic activity of acyl-ghrelin, (3) whether insulin pretreatment inhibits the plasma ghrelin (total) response to fasting, and (4) the extent to which pioglitazone (PIO) treatment will increase stomach and plasma ghrelin (total) levels in rats fed a HF diet. PIO is a drug given to diabetics which improves insulin resistance. Our findings show that a chronic HF diet given for either 10 or 60weeks exerts a persistent inhibitory effect on resting plasma ghrelin (total) levels. Additionally, the plasma ghrelin (total) elevation to overnight fasting is not altered in rats fed a HF diet on a long-term basis. A HF diet does not impair the ingestive response to acyl-ghrelin. Together, these results suggest that acyl-ghrelin serves as an important orexigenic factor. Results show that insulin pretreatment does not inhibit the plasma ghrelin (total) response to fasting suggesting that meal-induced insulin secretion does not have a role in reducing ghrelin (total) secretion. In rats fed a HF diet, PIO administration increases stomach ghrelin (total) levels. Because PIO can reduce systemic glucose and lipid levels, our findings suggest that elevated glucose and lipid levels are part of the inhibitory mechanism behind reduced ghrelin (total) secretion in rats fed a HF diet.

Sphingolipid-mediated Inhibition of Apoptotic Cell Clearance by Alveolar Macrophages

A decreased clearance of apoptotic cells (efferocytosis) by alveolar macrophages (AM) may contribute to inflammation in emphysema. The up-regulation of ceramides in response to cigarette smoking (CS) has been linked to AM accumulation and increased detection of apoptotic alveolar epithelial and endothelial cells in lung parenchyma. We hypothesized that ceramides inhibit the AM phagocytosis of apoptotic cells. Release of endogenous ceramides via sphingomyelinase or exogenous ceramide treatments dose-dependently impaired apoptotic Jurkat cell phagocytosis by primary rat or human AM, irrespective of the molecular species of ceramide. Similarly, in vivo augmentation of lung ceramides via intratracheal instillation in rats significantly decreased the engulfment of instilled target apoptotic thymocytes by resident AM. The mechanism of ceramide-induced efferocytosis impairment was dependent on generation of sphingosine via ceramidase. Sphingosine treatment recapitulated the effects of ceramide, dose-dependently inhibiting apoptotic cell clearance. The effect of ceramide on efferocytosis was associated with decreased membrane ruffle formation and attenuated Rac1 plasma membrane recruitment. Constitutively active Rac1 overexpression rescued AM efferocytosis against the effects of ceramide. CS exposure significantly increased AM ceramides and recapitulated the effect of ceramides on Rac1 membrane recruitment in a sphingosine-dependent manner. Importantly, CS profoundly inhibited AM efferocytosis via ceramide-dependent sphingosine production. These results suggest that excessive lung ceramides may amplify lung injury in emphysema by causing both apoptosis of structural cells and inhibition of their clearance by AM.

Inhibitory effect of emodin and Astragalus polysaccharideon the replication of HBV

To evaluate the anti-viral effect of emodin plus Astragalus polysaccharide (APS) in hepatitis B virus (HBV) transgenic mice. Sixty HBV transgenic mice (HBV TGM) whose weight varied between 18 and 24 g were randomly divided into 3 groups, with 20 mice in each group. Group A was the normal control, where the mice were treated with physiological saline; group B was the positive control where the mice were treated with lamivudine solution (100 mL/kg per day). Group C was the experimental group where the mice were treated with physiological saline containing emodin and APS (57.59 mg/kg per day and 287.95 mg/kg per day, respectively). The mice were treated daily for 3 wk. After 1 wk recovery time, the mice were sacrificed and serum as well as liver tissues were collected for ELISA and histological examination. After 21 d treatment, HBV DNA levels in group B and group C significantly declined when compared with group A (P < 0.05). However, a significant increase in HBV DNA content was observed in group B, whereas this phenomenon was not observed in group C. A reduction in the contents of HBsAg, HBeAg and HBcAg in the mice from group B and C was observed when compared with group A. Emodin and APS have a weak but persistent inhibitory effect on HBV replication in vivo, which may function as a supplementary modality in the treatment of hepatitis B infection.

Role of spinal voltage-dependent calcium channel α 2δ-1 subunit in the expression of a neuropathic pain-like state in mice

The present study was undertaken to investigate the role of spinal voltage-dependent calcium channel α 2δ-1 subunit in the expression of a neuropathic pain-like state induced by partial sciatic nerve ligation in mice. In cultured spinal neurons, gabapentin (GBP), which displays the inhibitory effect of α 2δ-1 subunit, suppressed the extracellular Ca 2+ influx induced by KCl, whereas it failed to inhibit the intracellular Ca 2+ release induced by inositol-1,4,5-triphosphate. Seven days after sciatic nerve ligation, the protein level of α 2δ-1 subunit in the ipsilateral spinal cord was clearly increased compared to that observed in sham-operated mice. In addition, the mRNA level of α 2δ-1 subunit was significantly increased in the dorsal root ganglion, but not in the spinal cord, of nerve-ligated mice. Under these conditions, a marked decrease in the latency of paw-withdrawal against a thermal stimulation and tactile stimulation, induced by sciatic nerve ligation was abolished by repeated intrathecal (i.t.) treatment with GBP. Additionally, the persistent reduction in the nociceptive threshold by i.t. treatment with GBP at the early stage of the neuropathic pain-like state was maintained for 7 days even after GBP withdrawal. It is of interest to note that a single i.t. post-injection of GBP showed a marked and transient inhibitory effect on the developed neuropathic pain-like state, whereas repeated i.t. post-treatment with GBP produced a persistent inhibitory effect during the treatment. In conclusion, we propose here that the neuropathic pain-like state with sciatic nerve ligation is associated with the increased level of the α 2δ-1 subunit of Ca 2+ channels at the sensory nerve terminal in the spinal dorsal horn of mice. Furthermore, the present data provide evidence that the neuropathic pain may be effectively controlled by repeated treatment with GBP at the early stage.

The effect of soil fumigants methyl bromide and metham sodium on the microbial degradation of the nematicide Cadusafos

Rapid biodegradation of Cadusafos in a soil from a previously treated greenhouse site from the area of Vasilika in Northern Greece was observed using both incubation and bioassay studies with nematodes. The slow rates of Cadusafos degradation observed in soils from the previously treated site after sterilization with chloroform and also in soils from an adjacent previously untreated site suggested that soil microorganisms adapted to metabolize Cadusafos were responsible for its rapid dissipation. Fumigation of the soil from the previously treated site with methyl bromide (MB) 9 months prior to the experimental addition of Cadusafos (4 μg g −1) only temporarily inhibited degradation of Cadusafos, unlike metham sodium (MS) whose application significantly hampered degradation of the nematicide. However, when the time interval between fumigation of soil from the previously treated greenhouse with MB and MS and the experimental application of Cadusafos was minimized to 20 d, both fumigants halted the rapid degradation of the nematicide. The transient inhibitory effect of MB on the microbial degradation of subsequent Cadusafos applications was attributed to the recovery of the soil microflora within 9 months. In contrast, the persistent inhibitory effect of MS on the microbial degradation of Cadusafos might have been caused by the adverse effects of the fumigant on the microorganisms, which were responsible for the rapid degradation of Cadusafos in the specific soil, although this was not tested in this study. According to these findings, the combined application of MS as a pre-planting treatment with a post-planting application of Cadusafos would maximize the biological efficacy of the non-fumigant nematicide by extending its persistence and preventing the development of enhanced biodegradation for organophosphorus nematicides such Cadusafos.