Status of Rhipicephalus microplus resistance to ivermectin, fipronil and fluazuron in Mato Grosso do Sul, Brazil / Status da resistência de Rhipicephalus microplus a ivermectina, fipronil e fluazuron em Mato Grosso do Sul

Abstract Southern cattle tick resistance to pour-on and injectable acaricides has yet to be evaluated on a broader scope, and the paucity of information on the subject may hinder efforts to control this parasite. The objective of this study was to evaluate the resistance profile of ten populations of Rhipicephalus microplus to the acaricides fluazuron, fipronil and ivermectin in cattle herds in Mato Grosso do Sul, Brazil. The larval immersion test (LIT) was used to evaluate susceptibility to ivermectin and fipronil and the adult immersion test (AIT) was performed to evaluate fluazuron. Samples were randomly obtained in ten farms, and in general, we found resistance in five samples to fluazuron and in four samples to ivermectin and fipronil. Six samples showed incipient resistance to ivermectin and fipronil. Five of the ten evaluated samples showed resistance and/or incipient resistance to all the active ingredients, and the other five to two active ingredients. Among the samples classified as resistant, the average resistance ratio for ivermectin was 2.75 and 3.26 for fipronil. These results demonstrate the advanced status of resistance to the most modern chemical groups for the control of R. microplus in the state of Mato Grosso do Sul.

Resumo A resistência do carrapato-do-boi a acaricidas com modo de aplicação "pour-on" e injetáveis é pouco avaliada em estudos mais abrangentes, e essa escassez de informação pode resultar falhas no seu controle. Este estudo teve como objetivo avaliar o perfil de resistência em dez populações de Rhipicephalus microplus aos acaricidas fluazuron, fipronil e ivermectina, em rebanhos bovinos em Mato Grosso do Sul, Brasil. A caracterização fenotípica da resistência foi realizada por meio do teste de imersão de adultos (AIT) para o fluazuron, e teste de imersão de larvas (LIT) para fipronil e ivermectina. As amostras foram obtidas aleatoriamente em dez fazendas, sendo diagnosticada resistência em cinco amostras ao fluazuron e em quatro amostras à ivermectina e fipronil. Seis amostras apresentaram resistência incipiente à ivermectina e fipronil. Cinco das dez amostras avaliadas apresentaram resistência e / ou resistência incipiente a todos os princípios ativos, e as outras cinco a dois princípios ativos. Entre as amostras classificadas como resistentes, a média do fator de resistência para ivermectina foi de 2,75 e de 3,26 para fipronil. Esses resultados demonstram o avançado estado de resistência aos mais modernos grupos químicos para o controle de R. microplus em Mato Grosso do Sul.

De novo in vitro shoot morphogenesis from shoot tip-induced callus cultures of Gymnema sylvestre (Retz.) R. Br. ex Sm

BACKGROUND: Gymnema sylvestre is a medicinal woody perennial vine known for its sweetening properties and antidiabetic therapeutic uses in the modern and traditional medicines. Its over-exploitation for the therapeutic uses and to meet the demand of pharmaceutical industry in raw materials supply for the production of anti-diabetic drugs has led to considerable decline in its natural population. RESULTS: An efficient system of shoot bud sprouting from nodal segment explants and indirect plant regeneration from apical meristem-induced callus cultures of G. sylvestre have been developed on Murashige and Skoog (MS) medium amended with concentrations of cytokinins. Of the three growth regulators tested, N6-benzylaminopurine (BAP) was the most efficient and 2.0 mg L-1 gave the best shoot formation efficiency. This was followed by thidiazuron (TDZ) and kinetin (Kin) but, most of the TDZ-induced micro shoots showed stunted growth. Multiple shoot formation was observed on medium amended with BAP or TDZ at higher concentrations. The produced micro shoots were rooted on half strength MS medium amended with auxins and rooted plantlets acclimatized with 87% survival of the regenerates. CONCLUSIONS: The developed regeneration system can be exploited for genetic transformation studies, particularly when aimed at producing its high yielding cell lines for the anti-diabetic phytochemicals. It also offers opportunities for exploring the expression of totipotency in the anti-diabetic perennial vine.

Thidiazuron (TDZ) increases fruit set and yield of 'Hosui' and 'Packham's Triumph' pear trees

ABSTRACT The low fruit set is one of the main factors leading to poor yield of pear orchards in Brazil. The exogenous application of thidiazuron (TDZ) and aminoethoxyvinilglycine (AVG) has shown promising results in some pear cultivars and other temperate fruit trees. The objective of this study was to evaluate the effect of TDZ and AVG on fruit set, yield, and fruit quality of 'Hosui' and 'Packham's Triumph' pears. The study was performed in a commercial orchard located in São Joaquim, SC. Plant material consisted of 'Hosui' and 'Packham's Triumph' pear trees grafted on Pyrus calleryana. Treatments consisted on different rates of TDZ (0 mg L-1, 20 mg L-1, 40 mg L-1 and 60 mg L-1) sprayed at full bloom for both cultivars. An additional treatment of AVG 60 mg L-1 was sprayed one week after full bloom in 'Hosui'. The fruit set, number of fruit per tree, yield, fruit weight, seed number, and fruit quality attributes were assessed. Fruit set and yield of both cultivars are consistently increased by TDZ, within the rates of 20 to 60 mg L-1. Besides, its application increased fruit size of 'Hosui' and did not negatively affect fruit quality attributes of both cultivars.

Interleukin-27 augments the inhibitory effects of sorafenib on bladder cancer cells

Both sorafenib and interleukin-27 (IL-27) are antineoplastic drugs. This study aimed to investigate the synergistic effect of these two drugs on bladder cancer cells. HTB-9 and T24 cells were stimulated with IL-27 (50 ng/mL), sorafenib (2 μM) or the synergistic action of these two drugs. The cells without treatment acted as control. Cell proliferation, apoptosis and invasion were measured by bromodeoxyuridine assay, flow cytometry and modified Boyden chamber, respectively. Simultaneously, both modified Boyden chamber and scratch assay were used to assess cell migration. Finally, the phosphorylation levels of key kinases in the Akt/mechanistic target of rapamycin (mTOR)/mitogen-activated protein kinase (MAPK) pathway, and expression levels of matrix metalloproteinase (MMP)-2 and MMP-9 were detected by western blot analysis. Stimulation with IL-27 or sorafenib repressed proliferation, migration and invasion but promoted apoptosis, and the effects were all enhanced by the combination of these two drugs in HTB-9 cells. The effect of the combined treatment on bladder cancer cells was verified in T24 cells. Additionally, the phosphorylation levels of AKT, mTOR and MAPK as well as the expression levels of MMP-2 and MMP-9 were all decreased by a single treatment of IL-27 or sorafenib, and further decreased by the combined treatment of these two drugs. The combination of IL-27 and sorafenib inhibited proliferation, migration and invasion and promoted apoptosis of bladder cancer cells compared with mono-drug treatment. Additionally, the AKT/mTOR/MAPK pathway might be implicated in the functional effects by down-regulations of MMP-2 and MMP-9.

EGF Induced RET Inhibitor Resistance in CCDC6-RET Lung Cancer Cells

PURPOSE: Rearrangement of the proto-oncogene rearranged during transfection (RET) has been newly identified potential driver mutation in lung adenocarcinoma. Clinically available tyrosine kinase inhibitors (TKIs) target RET kinase activity, which suggests that patients with RET fusion genes may be treatable with a kinase inhibitor. Nevertheless, the mechanisms of resistance to these agents remain largely unknown. Thus, the present study aimed to determine whether epidermal growth factor (EGF) and hepatocyte growth factor (HGF) trigger RET inhibitor resistance in LC-2/ad cells with CCDC6-RET fusion genes. MATERIALS AND METHODS: The effects of EGF and HGF on the susceptibility of a CCDC6-RET lung cancer cell line to RET inhibitors (sunitinib, E7080, vandetanib, and sorafenib) were examined. RESULTS: CCDC6-RET lung cancer cells were highly sensitive to RET inhibitors. EGF activated epidermal growth factor receptor (EGFR) and triggered resistance to sunitinib, E7080, vandetanib, and sorafenib by transducing bypass survival signaling through ERK and AKT. Reversible EGFR-TKI (gefitinib) resensitized cancer cells to RET inhibitors, even in the presence of EGF. Endothelial cells, which are known to produce EGF, decreased the sensitivity of CCDC6-RET lung cancer cells to RET inhibitors, an effect that was inhibited by EGFR small interfering RNA (siRNA), anti-EGFR antibody (cetuximab), and EGFR-TKI (Iressa). HGF had relatively little effect on the sensitivity to RET inhibitors. CONCLUSION: EGF could trigger resistance to RET inhibition in CCDC6-RET lung cancer cells, and endothelial cells may confer resistance to RET inhibitors by EGF. E7080 and other RET inhibitors may provide therapeutic benefits in the treatment of RET-positive lung cancer patients.

Alterations in the oxidative metabolism of Rhipicephalus (Boophilus) microplus ticks in response to exposure to the insect growth regulator fluazuron / Alterações no metabolismo oxidativo de carrapatos Rhipicephalus (Boophilus) microplus em resposta à exposição ao regulador de crescimento fluazuron

Abstract Aiming to characterize the potential off-target effects of fluazuron on ticks, biochemical analyses were conducted to evaluate changes in the carbohydrate metabolism of Rhipicephalus (Boophilus) microplus ticks after exposure to fluazuron. Hemolymph and fat body were collected from female ticks before and after (4, 8 and 15 days) exposure to fluazuron. Spectrophotometric analyses were done to quantify glucose concentration and lactate dehydrogenase (LDH) activity in the hemolymph and the concentration of glycogen in the tick’s fat body. High Performance Liquid Chromatography (HPLC) was employed to determine the concentration of carboxylic acids in the hemolymph and to evaluate changes in intermediary metabolic processes requiring oxygen consumption. Increases in the levels of LDH activity and lactic acid concentration indicated that fluazuron enhanced fermentative metabolism in ticks. Exposure to fluazuron was also found to increase glucose concentrations in the hemolymph over time, although no significant differences were noted daily. In addition to expanding the body of knowledge about the mode of action of fluazuron, investigations into these mechanisms may also be useful in discovering new and as yet unexplored secondary effects.

Resumo Com o objetivo de caracterizar os efeitos não-alvo da ação do fluazuron, foram realizados testes bioquímicos para analisar possíveis alterações no metabolismo de carboidratos em carrapatos Rhipicephalus (Boophilus) microplus após sua exposição ao composto. Foram coletados hemolinfa e corpo gorduroso de fêmeas ingurgitadas antes e após (4, 8 e 15 dias) a exposição ao fluazuron. Análises espectrofotométricas foram usadas para quantificar a concentração de glicose e a atividade da lactato desidrogenase (LDH) na hemolinfa e concentração de glicogênio no corpo gorduroso. Cromatografia Líquida de Alta Eficiência (CLAE) foi usada para determinação das concentrações de ácidos carboxílicos na hemolinfa e avaliar possíveis alterações em metabolismo intermediário em relação ao consumo de oxigênio. Aumento na atividade de LDH e concentração de ácido lático indicaram que o fluazuron pode regular o metabolismo fermentativo em carrapatos. A exposição ao fluazuron também aumentou a concentração de glicose na hemolinfa, apesar de não ter havido diferença significativa na comparação entre as médias no mesmo dia de avaliação. Além de aumentar o conhecimento sobre o modo de ação do fluazuron, investigações sobre tais mecanismos também são úteis no descobrimento de novos efeitos secundários ainda não explorados.

Combined treatment with silibinin and either sorafenib or gefitinib enhances their growth-inhibiting effects in hepatocellular carcinoma cells

BACKGROUND/AIMS: Silibinin, the main component of silymarin, is used as a hepatoprotectant and exhibits anticancer effects against various cancer cells. This study evaluated the effects of a combination of silibinin with either gefitinib or sorafenib on hepatocellular carcinoma (HCC) cells. METHODS: Several different human HCC cell lines were used to test the growth-inhibiting effects and cell toxicity of silibinin both alone and in combination with either gefitinib or sorafenib. The cell viability and growth inhibition were assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, trypan blue staining, and a colony-forming assay. Furthermore, changes in epidermal growth factor receptor (EGFR)-related signals were evaluated by Western blot analysis. RESULTS: Gefitinib, sorafenib, and silibinin individually exhibited dose-dependent antiproliferative effects on HCC cells. Combined treatment with silibinin enhanced the gefitinib-induced growth-inhibiting effects in some HCC cell lines. The combination effect of gefitinib and silibinin was synergistic in the SNU761 cell line, but was only additive in the Huh-BAT cell line. The combination effect may be attributable to inhibition of EGFR-dependent Akt signaling. Enhanced growth-inhibiting effects were also observed in HCC cells treated with a combination of sorafenib and silibinin. CONCLUSIONS: Combined treatment with silibinin enhanced the growth-inhibiting effects of both gefitinib and sorafenib. Therefore, the combination of silibinin with either sorafenib or gefitinib could be a useful treatment approach for HCC in the future.

Mannitol-induced drought stress on calli of Trigonella foenum-graecum L. Var. RMt-303.

Different explants of fenugreek, T. foenum-graecum L. (Var. RMt-303), were compared for their callus induction and subsequent shoot regeneration capabilities on Murashige and Skoog media supplemented with different phytohormones in varying concentration. The highest percentage of callus induction frequency was observed in 1ppm benzylaminopurine (BAP). Maximum shoots were induced on media supplemented with 0.5ppm BAP using leaf and stem tissues as explants. However, root tissues showed only callusing with no subsequent shooting. Cotyledonary node responded better than hypocotyls in terms of shoot induction on media supplemented with thidiazuron (0.1ppm). The callus was subjected to drought stress as simulated by reduced water potential of growth media due to addition of mannitol. Calli could withstand -2 MPa water potential till 30 days indicating that the drought stress tolerance mechanisms are functional in this variety. Chlorophyll a and b and total chlorophyll, proline and total phenolic contents, total peroxidase and catalase activities increased under stress conditions suggesting the tolerance of callus to drought stress. However, ascorbate peroxidase, guaiacol peroxidase activities were found to decrease slightly. Malondialdehyde and H2O2 contents were found to decrease while only a slight disturbance was found in membrane stability index. These results underline the mechanisms that are crucial for drought stress tolerance in fenugreek.

Biochemical markers assisted screening of Fusarium wilt resistant Musa paradisiaca (L.) cv. Puttabale micropropagated clones.

An efficient protocol was standardized for screening of panama wilt resistant Musa paradisiaca cv. Puttabale clones, an endemic cultivar of Karnataka, India. The synergistic effect of 6-benzyleaminopurine (2 to 6 mg/L) and thidiazuron (0.1 to 0.5 mg/L) on MS medium provoked multiple shoot induction from the excised meristem. An average of 30.10 ± 5.95 shoots was produced per propagule at 4 mg/L 6-benzyleaminopurine and 0.3 mg/L thidiazuron concentrations. Elongation of shoots observed on 5 mg/L BAP augmented medium with a mean length of 8.38 ± 0.30 shoots per propagule. For screening of disease resistant clones, multiple shoot buds were mutated with 0.4% ethyl-methane-sulfonate and cultured on MS medium supplemented with Fusarium oxysporum f. sp. cubense (FOC) culture filtrate (5–15%). Two month old co-cultivated secondary hardened plants were used for screening of disease resistance against FOC by the determination of biochemical markers such as total phenol, phenylalanine ammonia lyase, oxidative enzymes like peroxidase, polyphenol oxidase, catalase and PR-proteins like chitinase, β-1-3 glucanase activities. The mutated clones of M. paradisiaca cv. Puttabale cultured on FOC culture filtrate showed significant increase in the levels of biochemical markers as an indicative of acquiring disease resistant characteristics to FOC wilt.

The efficacy of a chitin synthesis inhibitor against field populations of organophosphate-resistant Aedes aegypti in Brazil

The mosquito Aedes aegypti is the main focus of dengue control campaigns. Because of widespread resistance against conventional chemical insecticides, chitin synthesis inhibitors (CSIs) are considered control alternatives. We evaluated the resistance status of four Brazilian Ae. aegypti populations to both the organophosphate temephos and the pyrethroid deltamethrin, which are used in Brazil to control larvae and adults, respectively. All vector populations exhibited high levels of temephos resistance and varying rates of alterations in their susceptibility to pyrethroids. The effect of the CSI novaluron on these populations was also investigated. Novaluron was effective against all populations under laboratory conditions. Field-simulated assays with partial water replacement were conducted to evaluate novaluron persistence. Bioassays were continued until an adult emergence inhibition of at least 70% was attained. We found a residual effect of eight weeks under indoor conditions and novaluron persisted for five-six weeks in assays conducted in an external area. Our data show that novaluron is effective against the Ae. aegypti populations tested, regardless of their resistance to conventional chemical insecticides.

Cytokinin preconditioning enhances multiple shoot regeneration in Pongamia pinnata (L) Pierre - a potential, non-edible tree seed oil source for biodiesel

An efficient, highly reproducible protocol for multiple shoot induction and plant regeneration of Pongamia pinnata has been successfully developed using cotyledonary node explants. This study also demonstrates that preconditioning of explant stimulates production of multiple shoots from cotyledonary nodes of P. pinnata. The highest direct shoot regeneration (90 percent) with an average of 18.4 +/- 3.1 shoots/explant were obtained when cotyledonary node explants were excised from seedlings germinated on Murashige and Skoog (MS) media supplemented with benzyladenine (BA) 1 mg l-1, and subsequently cultured on MS media with 1 mgl-1 thidiazuron (TDZ). Scanning electron microscope observations of cotyledonary node (CN) explants excised from pre-conditioned and normal seedlings, revealed larger buds with rapid development in BA-preconditioned CN explants. The addition of adenine sulphate significantly increased the average number of shoots per explant. The highest direct shoot regeneration (93 percent) with an average of 32.2 +/- 0.93 shoots/explant was obtained from BA-preconditioned CN when cultured on MS media supplemented with 1 mg l-1 TDZ and 200 mg l-1 adenine sulphate (ADS). Repeated shoot proliferation was observed from BA preconditioned CN explants up to 3 cycles with an average of 15 shoots/explant/cycle when cultured on MS media supplemented with 1 mg l-1 TDZ and 150 mg l-1 L-glutamine, thus producing 45 shoots/CN explant. Shoots were elongated on hormone free MS media and rooted on 1/2 MS media supplemented with 1 mg l-1 of IBA. Rooted shoots were successfully acclimatized and established in soil with 80 percent success. The highly regenerative system developed in this investigation for this important tree could be a useful tool for genetic transformation.

The influence of growth regulators on shoot proliferation and rooting of in vitro propagated caper.

The objectives of this study were to examine the effect of TDZ and BAP on shoot proliferation and to develop a satisfactory rooting procedure for in vitro propagation of caper (Capparis spinosa L). TDZ was found to be more effective than BAP in shoot production. The highest shoot number per explant (45.7) was achieved on the media that contained 4.54 microM TDZ in the absence of IAA. TDZ at 4.54 microM alone or in combination with 0.57 microM IAA did not inhibit shoot elongation, although they produced the most shoots. Dipping the caper shoots in the solution of IBA at 24.6 microM for 10 minutes before planting on MS media, improved the rooting percentage up to 80.5% under a 16 hr photoperiod.

A randomised bouble-blind placebo controlled trial of lidamidine HCL in irritable vowel syndrome

BACKGROUND: We have previously shown electro-mechanical recto-anal alterations in irritable bowel syndrome patients (Awad R. Neurogastroenterol Motil 1993; 5; 265-271). To assess whether the alpha 2-agonist lidamidine HCL is able to modify these physiological alterations and alleviate clinical symptoms, 50 patients with irritable bowel syndrome were studied in a random, double blind, placebo-controlled trial. METHODS: Lidamidine HCL (4 mg) or placebo was taken orally t.i.d. with food. Fasting and post-prandial electrical and mechanical activities of rectum and internal anal sphincter were recorded before and at the end of treatment. Recto-anal sensitivity was also tested. RESULTS: After treatment, post-prandial duration of spontaneous recto-anal inhibitory reflex diminished in the lidamidine group (18.9 +/- 1 vs. 15.1 +/- 1.3 sec; p < 0.05). Amplitude of induced rectoanal inhibitory reflex decreased after lidamidine (24.6 +/- 2.9 vs 17.3 +/- 3 mmHg; p = 0.02). Rectal electrical activity showed no changes during basal and post-prandial periods in any group. Rectal painful sensation decreased after treatment with lidamidine (54.8 +/- 5.4 vs 43.6 +/- 3.5 ml; p < 0.05) as well as with placebo (p < 0.05). Abdominal distension and requency, severity and duration of pain diminished in both groups (p < 0.05). CONCLUSION: Lidamidine decreased the augmented mechanical response to food, reduced rectal sensitivity, and relieved symptoms. These facts suggest that in spite of the strong placebo response obtained, lidamidine HCL can become a useful alternative for treatment of patients with irritable bowel syndrome.

Non-competitive inhibition of serum acetylcholinesterase (E.C.3.1.1.7) by vacor (N-3-pyridyl methyl-N'-P-nitrophenyl urea) in bandicoot rats.

Single dose (1 mg/rat) administration of VACOR (N-3-pyridyl methyl-N'-nitrophenyl urea) produced significant paralytic symptoms in rats. Such rats which lived for one week were sacrificed and serum AChE was extracted and purified by Sephadex column chromatography. The serum enzyme from the normal rats showed the existence of two isozymes on polyacrylamide gels which could be resolved through column chromatography. Both the isozymes showed non-competitive inhibition with VACOR in vitro; but to a varied degree. The ACh concentration increased by 70-75% in serum and 95-100% in erythrocytes of the poisoned rats. VACOR thus affects ACh hydrolysis thereby causing an accumulation of ACh in the serum of poisoned rats.