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The protozoan parasite Trypanosoma brucei has a complex digenetic lifecycle between a mammalian host and an insect vector, and adaption of its proteome between lifecycle stages is essential to its survival and virulence. We have optimized a procedure for growing Trypanosoma brucei procyclic form cells in conditions suitable for stable isotope labeling by amino acids in culture (SILAC) and report a comparative proteomic analysis of cultured procyclic form and bloodstream form T. brucei cells. In total we were able to identify 3959 proteins and quantify SILAC ratios for 3553 proteins with a false discovery rate of 0.01. A large number of proteins (10.6%) are differentially regulated by more the 5-fold between lifecycle stages, including those involved in the parasite surface coat, and in mitochondrial and glycosomal energy metabolism. Our proteomic data is broadly in agreement with transcriptomic studies, but with significantly larger fold changes observed at the protein level than at the mRNA level.

BACKGROUND: Primary prostate cancers are infiltrated with programmed death-1 (PD-1) expressing CD8+ T-cells. However, in early clinical trials, men with metastatic castrate-resistant prostate cancer did not respond to PD-1 blockade as a monotherapy. One explanation for this unresponsiveness could be that prostate tumors generally do not express programmed death ligand-1 (PD-L1), the primary ligand for PD-1. However, lack of PD-L1 expression in prostate cancer would be surprising, given that phosphatase and tensin homolog (PTEN) loss is relatively common in prostate cancer and several studies have shown that PTEN loss correlates with PD-L1 upregulation—constituting a mechanism of innate immune resistance. This study tested whether prostate cancer cells were capable of expressing PD-L1, and whether the rare PD-L1 expression that occurs in human specimens correlates with PTEN loss. METHODS: Human prostate cancer cell lines were evaluated for PD-L1 expression and loss of PTEN by flow cytometry and western blotting, respectively. Immunohistochemical (IHC) staining for PTEN was correlated with PD-L1 IHC using a series of resected human prostate cancer samples. RESULTS: In vitro , many prostate cancer cell lines upregulated PD-L1 expression in response to inflammatory cytokines, consistent with adaptive immune resistance. In these cell lines, no association between PTEN loss and PD-L1 expression was apparent. In primary prostate tumors, PD-L1 expression was rare, and was not associated with PTEN loss. CONCLUSIONS: These studies show that some prostate cancer cell lines are capable of expressing PD-L1. However, in human prostate cancer, PTEN loss is not associated with PD-L1 expression, arguing against innate immune resistance as a mechanism that mitigates antitumor immune responses in this disease.

Glutathione S-transferase P1-1 (GST P1) is an important drug target as it is implicated in drug resistance. GST P1-1 inhibitors are typically non-productive analogues of glutathione which covers broad chemical space; hence it is likely that several clinically used drugs may unknowingly act as GST P1-1 inhibitors. We developed a high-throughput screening assay for GST P1-1 and screened 5830 compounds. From the screening, 24 potent GST P1-1 inhibitors were identified and assessed for cytotoxicity in MCF-7 and MDA-MB-231 breast cancer cell lines. Ethacrynic acid (a known GST P1-1 inhibitor), ZM 39923, PRT 4165, 10058-F4, and cryptotanshinone were shown to be the most active. A competitive GST P1-1 assay was performed to identify the inhibition type of these five compounds. Another in vitro cytotoxicity experiment was conducted to explore the differences in the cytotoxicity profiles of the combinations tested. Western blot analysis was used to identify the presence of GST P1-1 in the breast cancer cell lines tested. The implications of these results concerning alternative treatment options for breast cancers are discussed.

A type III protein secretion system encoded by Salmonella pathogenicity island 2 (SPI2) has been found to be required for virulence and survival within macrophages. Here, SPI2 was shown to allow Salmonella typhimurium to avoid NADPH oxidase-dependent killing by macrophages. The ability of SPI2-mutant bacteria to survive in macrophages and to cause lethal infection in mice was restored by abrogation of the NADPH oxidase-dependent respiratory burst. Ultrastructural and immunofluorescence microscopy demonstrated efficient localization of the NADPH oxidase in the proximity of vacuoles containing SPI2-mutant but not wild-type bacteria, suggesting that SPI2 interferes with trafficking of oxidase-containing vesicles to the phagosome.

Antimicrobial resistance is a silent pandemic considered a public health concern worldwide. Strategic therapies are needed to replace antibacterials that are now ineffective. One approach entails the use of well-known antibacterials along with adjuvants that possess non-antibiotic properties but can extend the lifespan and enhance the effectiveness of the treatment, while also improving the suppression of resistance. In this regard, a group of uniform materials based on organic salts (GUMBOS) presents an alternative to this problem allowing the combination of antibacterials with adjuvants. Fluoroquinolones are a family of antibacterials used to treat respiratory and urinary tract infections with broad-spectrum activity. Ciprofloxacin and moxifloxacin-based GUMBOS were synthesized via anion exchange reactions with lithium and sodium salts. Structural characterization, thermal stability and octanol/water partition ratios were evaluated. The antibacterial profiles of most GUMBOS were comparable to their cationic counterparts when tested against Gram-positive S. aureus and Gram-negative E. coli, except for deoxycholate anion, which demonstrated the least effective antibacterial activity. Additionally, some GUMBOS were less cytotoxic to L929 fibroblast cells and non-hemolytic to red blood cells. Therefore, these agents exhibit promise as an alternative approach to combining drugs for treating infections caused by resistant bacteria.

Accurate and selective monitoring of thiamine levels in multivitamin supplements is essential for preventing deficiencies and ensuring product quality. To achieve this, a Förster resonance energy transfer (FRET) system using carbon dots (CDs) as energy donors and citrate-stabilized silver nanoparticles (AgNPs) as energy acceptors was developed. The aqueous synthesis of AgNPs using microwave irradiation was optimized to obtain efficient plasmonic nanoparticles for FRET applications, targeting maximal absorbance intensity, stability, and wavelength alignment. Using a central composite orthogonal design (CCOD), the optimal conditions were identified as a 12.5 min microwave reaction time, a Ag molar ratio of 0.72, and a pH of 8.28. The FRET sensing scheme was applied for thiamine determination, where the vitamin’s presence impaired the FRET process, restoring CDs’ photoluminescence (PL) emission in a concentration-dependent manner. To mitigate interference from other vitamins, PL kinetic data and excitation–emission matrix (EEM) data were analyzed using unfolded partial least-squares (U-PLS) with the subsequent application of the residual bilinearization technique (RBL), achieving high sensitivity and specificity for thiamine detection. This method demonstrated its accuracy and robustness by attaining a determination coefficient (R2) of 0.952 and a relative error of prediction (REP%) of 11%. This novel method offers highly sensitive and interference-free thiamine detection, with significant potential for a wide range of analytical applications.

A novel automated method based on sequential injection analysis (SIA), a non-segmented flow injection technique, was developed to evaluate glutathione S-transferase P1-1 (GST P1-1) activity in the presence of organometallic complexes with putative anticancer activity. The assay is based on the reaction of L-glutathione (GSH) and 1-chloro-2,4-dinitrobenzene (CDNB) in the presence of GST P1-1 to afford the GS-DNB conjugate and the reaction may be monitored by an increase in absorbance at 340 nm. A series of ruthenium, iron, osmium and iridium complexes were evaluated as GST P1-1 inhibitors by evaluating their half-maximal inhibitory concentration (IC 50 ). An iridium compound displays the lowest IC 50 value of 6.7 ± 0.7 µM and an iron compound displays the highest IC 50 value of 275 ± 9 µM. The SIA method is simple to use, robust, reliable, and efficient and uses fewer reagents than batch methods and each analysis takes only 5 minutes.

Human neutrophil elastase (HNE) is used as diagnostic biomarker for inflammation/infection. In this work, 10 ionic liquids (ILs) and 11 ionic liquids active pharmaceutical ingredients (ILs-APIs) were tested to evaluate the inhibition effect on the activity of porcine pancreatic elastase enzyme, frequently employed as a model for HNE. The insertion of ionic liquids in some drugs is useful, as the insertion of ILs with inhibitory capacity will also slow down all processes in which this enzyme is involved. Therefore, a spectrophotometric method was performed to the determination of EC50 values of the compounds tested. EC50 values of 124 ± 4 mM to 289 ± 11 mM were obtained, with the most toxic IL for elastase being tetrabutylammonium acetate and the least toxic 1-butyl-3-methylimidazolium acetate. Moreover, sodium salicylate (raw material) presented the lower and benzethonium bistriflimide the higher EC50 when compared with all the IL-APIs tested. This work provides significant information about the effect of the studied IL and IL-APIs in elastase enzyme activity.

The combination of multiple quantum dots (QDs) in a multi-emitter nanoprobe can be envisaged as a promising sensing scheme, as it enables obtaining a collective response of individual emitters towards a given analyte and allows for achieving specific analyte-response profiles. The processing of these profiles using adequate chemometric methods empowers a more sensitive, reliable and selective determination of the target analyte. In this work, we developed a kinetic fluorometric method consisting of a dual CdTe/AgInS2 quantum dots photoluminescence probe for the determination of acetylsalicylic acid (ASA). The fluorometric response was acquired as second-order time-based excitation/emission matrices that were subsequently processed using chemometric methods seeking to assure the second-order advantage. The data obtained in this work are considered second-order data as they have a three-dimensional size, I × J × K (where I represents the samples’ number, J the fluorescence emission wavelength while K represents the time). In order to select the most adequate chemometric method regarding the obtained data structure, different chemometric models were tested, namely unfolded partial least squares (U-PLS), N-way partial least squares (N-PLS), multilayer feed-forward neural networks (MLF-NNs) and radial basis function neural networks (RBF-NNs).

African sleeping sickness or human African trypanosomiasis, caused by Trypanosoma brucei spp., is responsible for ∼30,000 deaths each year. Available treatments for this disease are poor, with unacceptable efficacy and safety profiles, particularly in the late stage of the disease when the parasite has infected the central nervous system. Here we report the validation of a molecular target and the discovery of associated lead compounds with the potential to address this lack of suitable treatments. Inhibition of this target— T. brucei N -myristoyltransferase—leads to rapid killing of trypanosomes both in vitro and in vivo and cures trypanosomiasis in mice. These high-affinity inhibitors bind into the peptide substrate pocket of the enzyme and inhibit protein N -myristoylation in trypanosomes. The compounds identified have promising pharmaceutical properties and represent an opportunity to develop oral drugs to treat this devastating disease. Our studies validate T. brucei N -myristoyltransferase as a promising therapeutic target for human African trypanosomiasis. A new antitrypanosomal African trypanosomiasis, or sleeping sickness, caused by Trypanosoma brucei spp., is responsible for some 30,000 deaths each year. The few treatments that are available tend to involve drugs with poor efficacy and safety profiles. N -myristoyltransferase (NMT), which adds myristate to the N-terminal glycine of many eukaryotic and microbial proteins, has been suggested as a possible target for drugs to treat trypanosomiasis and other parasitic diseases — if selectivity to protect the human enzyme can be achieved. A new study validates T. brucei NMT as a viable protein target for antitrypanosomals, and a potent inhibitor with drug-like properties has been identified. The compound, DDD85646, kills T. brucei in the bloodstream in mouse models. African sleeping sickness, caused by Trypanosoma brucei species, is responsible for some 30,000 human deaths each year. Available treatments are limited by poor efficacy and safety profiles. However, a new molecular target for potential treatments has now been identified. The protein target is T. brucei N -myristoyltransferase. In further experiments, lead compounds have been discovered that inhibit this protein, kill trypanosomes in vitro and in vivo , and can cure trypanosomiasis in mice.