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Candida species are amongst the commensals of the mucosal surfaces of the human body which include the oral cavity, vagina, and intestinal mucosa. Fungal infections are on the rise worldwide. The overall burden of infections due to fungi is difficult to estimate because the majority of them remain undiagnosed. The present study aims to determine the burden of antifungal resistance in low socioeconomic country, Pakistan and the frequency of ERG11 and MDR1 genes involved. A total of 636 Candida isolates were obtained from various tertiary care institutions in Lahore in the form of culture on various culture plates. Sabouraud agar culture plates were used to culture the Candida spp. Antifungal resistance was determined against Fluconazole, Itraconazole, Ketoconazole, and Nystatin via disk diffusion technique. Most resistance was observed against Fluconazole followed by Itraconazole, Ketoconazole, and Nystatin. The Candida isolates recovering from CVP tip and tissue have a high resistance profile. Candida species resistant to at least two antifungals were chosen for further ERG11 and MDR1 detection through real-time PCR. Among 255 Candida isolates, 240 contained ERG11 gene while MDR1 gene is present in 149 Candida isolates. The isolates carrying both genes were tested by the broth microdilution technique for the susceptibility against cycloheximide, all of them were able to grow in cycloheximide. The genetic determinants of antifungal resistance such as ERG11 and MDR1 are as important in the multidrug resistance against a variety of compounds and antifungal drugs.

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Background: Leishmaniasis, caused by the protozoan Leishmania sp., infects phagocyte cells present in lymphatic organs. This study demonstrates the influence of nanostructured lipid carrier-loaded hydroxymethylnitrofurazone (NLC-NFOH) on lymphatic uptake using a chylomicron-blocking flow model in rats. Method: Lymphatic uptake of NFOH was assessed 1 h after oral administration of dimethyl sulfoxide with NFOH or NLC-NFOH with and without cycloheximide pretreatment. Result: Dimethyl sulfoxide with NFOH and NLC-NFOH showed NFOH serum concentrations of 0.0316 and 0.0291 µg/ml, respectively. After chylomicron blocking, NFOH was not detected. Conclusion: Despite log P below 5, NFOH was successfully taken up by the lymphatic system. Long-chain fatty acids and particle size might be main factors in these findings. NLC-NFOH is a promising and convenient platform for treating leishmaniasis via oral administration.

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Oocyte activation via dual inhibition of protein synthesis and phosphorylation has improved in vitro embryo production in different mammalian species. In this study, we evaluated the effects of the combination of cycloheximide (CHX), dimethyl amino purine (DMAP), and anisomycin (ANY) on the activation of bovine oocytes, particularly on dynamics of MPF and MAPKs, embryonic developmental potential, and quality. The results showed that the cleavage and blastocyst rates, as well as levels of CCNB1, CDK1, p-CDK1Thr161, and p-CDK1Thr14-Tyr15, were similar among groups; ANY and ANY + CHX reduced the expression of ERK1/2 compared to DMAP-combinations (p < 0.05), whereas ANY + DMAP, CHX + DMAP, and ANY + CHX + DMAP reduced p-ERK1/2 compared to ANY and ANY + CHX treatments (p < 0.05). The quality of blastocysts in terms of cell counts, their allocation, and the numbers of TUNEL-positive cells did not differ among groups. However, transcript levels of POU5F1 were higher in embryos derived from ANY + CHX + DMAP treatment compared to other groups, while expression levels of CDX2 did not show differences. In addition, the BCL2A1/BAX ratio of the ANY + CHX + DMAP treatment was significantly low compared to the ANY treatment (p < 0.05) and did not differ significantly from the other treatments. In conclusion, oocyte activation by dual inhibition of protein synthesis and phosphorylation induces MPF inactivation without degradation of CCNB1, while MAPK inactivation occurs differentially between these inhibitors. Thus, although the combined use of these inhibitors does not affect early developmental competence in vitro, it positively impacts the expression of transcripts associated with embryonic quality.

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The present study evaluated the mechanism by which protein synthesis inhibitors activate bovine oocytes. The aim was to analyze the dynamics of MPF and MAPKs. MII oocytes were activated with ionomycin (Io), ionomycin+anisomycin (ANY) and ionomycin+cycloheximide (CHX) and by in vitro fertilization (IVF). The expression of cyclin B1, p-CDK1, p-ERK1/2, p-JNK, and p-P38 were evaluated by immunodetection and the kinase activity of ERK1/2 was measured by enzyme assay. Evaluations at 1, 4, and 15 hours postactivation (hpa) showed that the expression of cyclin B1 was not modified by the treatments. ANY inactivated MPF by p-CDK1Thr14-Tyr15 at 4 hpa (P < 0.05), CHX increased pre-MPF (p-CDK1Thr161 and p-CDK1Thr14-Tyr15) at 1 hpa and IVF increased p-CDK1Thr14-Tyr15 at 17 hours postfertilization (hpf) (P < 0.05). ANY and CHX reduced the levels of p-ERK1/2 at 4 hpa (P < 0.05) and its activity at 4 and 1 hpa, respectively (P < 0.05). Meanwhile, IVF increased p-ERK1/2 at 6 hpf (P < 0.05); however, its kinase activity decreased at 6 hpf (P < 0.05). p-JNK in ANY, CHX, and IVF oocytes decreased at 4 hpa (P < 0.05). p-P38 was only observed at 1 hpa, with no differences between treatments. In conclusion, activation of bovine oocytes by ANY, CHX, and IVF inactivates MPF by CDK1-dependent specific phosphorylation without cyclin B1 degradation. ANY or CHX promoted this inactivation, which seemed to be more delayed in the physiological activation (IVF). Both inhibitors modulated MPF activity via an ERK1/2-independent pathway, whereas IVF activated the bovine oocytes via an ERK1/2-dependent pathway. Finally, ANY does not activate the JNK and P38 kinase pathways.

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Fully consolidated associative memories may be altered by alternative retrieval dependent memory processes. While a brief exposure to the conditioned stimulus (CS) can trigger reconsolidation of the original memory, a prolonged CS exposure will trigger memory extinction. The conditioned response is maintained after reconsolidation, but is inhibited after extinction, presumably by the formation of a new inhibitory memory trace. In rats and humans, it has been shown that CS exposure of intermediate duration leave the memory in an insensitive or limbo state. Limbo is characterised by the absence of reconsolidation or extinction. Here we investigated the evolutionary conserved nature of limbo using a contextual Pavlovian conditioning (CPC) memory paradigm in the crab Neohelice granulata. In animals with fully consolidated CPC memory, systemic administration of the protein synthesis inhibitor cycloheximide after 1 CS presentation disrupted the memory, presumably by interfering with memory reconsolidation. The same intervention given after 320 CSs prevented CPC memory extinction. Cycloheximide had no behavioural effect when administered after 80 CS presentations, a protocol that failed to extinguish CPC memory. Also, we observed that a stronger CPC memory engaged reconsolidation after 80 CS instead of limbo, indicating that memory strength affects the parametrical conditions to engage either reconsolidation or limbo. Altogether, these results indicate that limbo is an evolutionary conserved memory process segregating reconsolidation from extinction in the number of CSs space. Limbo appears as an intrinsic component of retrieval dependent memory processing, with a key function in the transition from memory maintenance to inhibition.

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ABSTRACT Leaf-cutting ants live symbiotically with a fungus that they cultivate on the plant leaves that they cut. The innumerous studies on the plant selection mechanism used by leaf-cutting ants show the researchers' interest in this issue. Many classical studies propose that plants are selected according to the fungus garden nutritional needs and the absence of potentially harmful substances. This hypothesis is corroborated by behavioral experiments using cycloheximide (fungicide) with citric pulp or forage plants greatly accepted by leaf-cutting ants. According to this hypothesis, under the action of a fungicide, the fungus emits an allomone that informs worker ants that some food is inadequate to its growth. Although some authors state that the cycloheximide "fungicide" used is specific and non toxic to ants, our findings are distinct. In our study, various concentrations of cycloheximide were administered orally to leaf-cutting worker ants in a citric pulp paste diet. After the ingestion period, the ants were isolated and offered the symbiotic fungus for 21 days and the mortality rate was evaluated. As expected, the treatment with 0.01% cycloheximide showed a low mortality rate (8.86%). At 0.1%, the mortality rate was mild (27.85%), and treatment with 1% cycloheximide resulted in moderate mortality (45.57%). In contrast, the positive control with 0.1% sulfluramid showed a high mortality rate (91.14%). Therefore, we concluded that the ingestion of high concentrations of cycloheximide results in a moderate mortality rate in leaf-cutting worker ants.

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Recombinant Drosophila S2 cells have been used for the expression of many proteins of medical interest. However, membrane-attached glycoproteins, which commonly exhibit lower expression levels compared to soluble proteins, may require special procedures in order to attain high levels of expression. In this study, two S2 cell population enrichment methods (antibiotic and immunomagnetic selection) were evaluated for their ability to enhance expression of the membrane-anchored rabies virus glycoprotein (RVGP). Quantification of RVGP production and determination of its cDNA copy number in transformed cells showed that both enrichment methods increased RVGP expression without significantly affecting its gene copy number. More interestingly, RVGP mRNA levels measured after cycloheximide treatment were poorly correlated with glycoprotein levels. Both enrichment methods enhanced expression of RVGP by recombinant S2 cells, with the highest level of expression achieved using immunomagnetic selection.

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Increasing the efficiency of intracytoplasmic sperm injection (ICSI) in domestic animals has been attempted by many researchers, however embryonic development to the blastocyst stage remains low compared with that of in vitro fertilization (IVF) embryos. One of the main problems observed in cattle is inadequate oocyte activation after ICSI. The present study compared the effect of cycloheximide (CHX), 6-dimethylaminopurine (DMAP), and anisomycin (ANY) on the fertilization rate, development, ploidy and quality of bovine embryos generated by ICSI. Although no differences were observed between treatments in terms of cleavage, higher blastocyst rates were observed for ANY (37.3%) compared with CHX (21.8%, P 0.05) treatments. No differences were observed in the quality of embryos as assessed by the total number of cells, their distribution to the different embryo compartments [inner cell mass (ICM) and trophectoderm (TE)], the proportion of ICM cells to the total cell numbers and terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL)-positive cells. Similarly, no differences were observed in the normal ploidy of embryos (56, 67, and 55%) for ANY, CHX and DMAP, respectively. However, higher fertilization rates were observed for ANY (75%) and CHX (87%) treatments compared with DMAP (35%). In conclusion, ANY showed a superior developmental rate compared with CHX treatment. Although no significant differences were observed compared with an improved protocol of DMAP (2Io-DMAP), the lower fertilization rate recorded with DMAP strongly suggests that ANY could be a better alternative for oocyte activation than traditional chemical compounds used currently in ICSI.

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It has been proposed that polybasic peptides cause slower movement of ribosomes through an electrostatic interaction with the highly negative ribosome exit tunnel. Ribosome profiling data-the sequencing of short ribosome-bound fragments of mRNA-is a powerful tool for the analysis of mRNA translation. Using the yeast Saccharomyces cerevisiae as a model, we showed that reduced translation efficiency associated with polybasic protein sequences could be inferred from ribosome profiling. However, an increase in ribosome density at polybasic sequences was evident only when the commonly used translational inhibitors cycloheximide and anisomycin were omitted during mRNA isolation. Since ribosome profiling performed without inhibitors agrees with experimental evidence obtained by other methods, we conclude that cycloheximide and anisomycin must be avoided in ribosome profiling experiments.

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Mossy fiber sprouting is among the best-studied forms of post-lesional synaptic plasticity and is regarded by many as contributory to seizures in both humans and animal models of epilepsy. It is not known whether mossy fiber sprouting increases the number of synapses in the molecular layer or merely replaces lost contacts. Using the pilocarpine (Pilo) model of status epilepticus to induce mossy fiber sprouting, and cycloheximide (CHX) to block this sprouting, we evaluated at the ultrastructural level the number and type of asymmetric synaptic contacts in the molecular layer of the dentate gyrus. As expected, whereas Pilo-treated rats had dense silver grain deposits in the inner molecular layer (IML) (reflecting mossy fiber sprouting), pilocarpine + cycloheximide (CHX + Pilo)-treated animals did not differ from controls. Both groups of treated rats (Pilo group and CHX + Pilo group) had reduced density of asymmetric synaptic profiles (putative excitatory synaptic contacts), which was greater for CHX-treated animals. For both treated groups, the loss of excitatory synaptic contacts was even greater in the outer molecular layer than in the best-studied IML (in which mossy fiber sprouting occurs). These results indicate that mossy fiber sprouting tends to replace lost synaptic contacts rather than increase the absolute number of contacts. We speculate that the overall result is more consistent with restored rather than with increased excitability.