Proteomic Correlates of Enhanced Daptomycin Activity following ß-Lactam Preconditioning in Daptomycin-Resistant, Methicillin-Resistant Staphylococcus aureus.

Clinical treatment options for daptomycin (DAP)-resistant (DAP-R), methicillin-resistant Staphylococcus aureus (MRSA) infections are relatively limited. Current therapeutic strategies often take advantage of potential synergistic activity of DAP plus ß-lactams; however, the mechanisms underlying their combinatorial efficacy are likely complex and remain incompletely understood. We recently showed that in vitro ß-lactam passaging can resensitize DAP-R strains to a DAP-susceptible (DAP-S) phenotype. To further investigate the implications of selected ß-lactam pretreatments on DAP plus ß-lactam combination efficacy, we utilized DAP-R strain D712. We studied six such combinations, featuring ß-lactams with a broad range of penicillin-binding protein-targeting profiles (PBP-1 to -4), using DAP-R strain D712. Of note, preconditioning with each ß-lactam antibiotic (sequential exposures), followed by DAP exposure, yielded significantly enhanced in vitro activity compared to either DAP treatment alone or simultaneous exposures to both antibiotics. To explore the underpinnings of these outcomes, proteomic analyses were performed, with or without ß-lactam preconditioning. Relative proteomic quantitation comparing ß-lactam pretreatments (versus untreated controls) identified differential modulation of several well-known metabolic, cellular, and biosynthetic processes, i.e., the autolytic and riboflavin biosynthetic pathways. Moreover, these differential proteomic readouts with ß-lactam preconditioning were not PBP target specific. Taken together, these studies suggest that the cellular response to ß-lactam preconditioning in DAP-R MRSA leads to distinct and complex changes in the proteome that appear to resensitize such strains to DAP-mediated killing.

Trehalose Synthesis Contributes to Osmotic Stress Tolerance and Virulence of the Bacterial Wilt Pathogen Ralstonia solanacearum.

The xylem-dwelling plant pathogen Ralstonia solanacearum changes the chemical composition of host xylem sap during bacterial wilt disease. The disaccharide trehalose, implicated in stress tolerance across all kingdoms of life, is enriched in sap from R. solanacearum-infected tomato plants. Trehalose in xylem sap could be synthesized by the bacterium, the plant, or both. To investigate the source and role of trehalose metabolism during wilt disease, we evaluated the effects of deleting the three trehalose synthesis pathways in the pathogen: TreYZ, TreS, and OtsAB, as well as its sole trehalase, TreA. A quadruple treY/treS/otsA/treA mutant produced 30-fold less intracellular trehalose than the wild-type strain missing the trehalase enzyme. This trehalose-nonproducing mutant had reduced tolerance to osmotic stress, which the bacterium likely experiences in plant xylem vessels. Following naturalistic soil-soak inoculation of tomato plants, this triple mutant did not cause disease as well as wild-type R. solanacearum. Further, the wild-type strain out-competed the trehalose-nonproducing mutant by over 600-fold when tomato plants were coinoculated with both strains, showing that trehalose biosynthesis helps R. solanacearum overcome environmental stresses during infection. An otsA (trehalose-6-phosphate synthase) single mutant behaved similarly to ΔtreY/treS/otsA in all experimental settings, suggesting that the OtsAB pathway is the dominant trehalose synthesis pathway in R. solanacearum.

Large-scale label-free comparative proteomics analysis of polo-like kinase 1 inhibition via the small-molecule inhibitor BI 6727 (Volasertib) in BRAF(V600E) mutant melanoma cells.

Polo-like kinase 1 (Plk1) is a serine/threonine kinase that plays a key role during the cell cycle by regulating mitotic entry, progression, and exit. Plk1 is overexpressed in a variety of human cancers and is essential to sustained oncogenic proliferation, thus making Plk1 an attractive therapeutic target. However, the clinical efficacy of Plk1 inhibition has not emulated the preclinical success, stressing an urgent need for a better understanding of Plk1 signaling. This study addresses that need by utilizing a quantitative proteomics strategy to compare the proteome of BRAF(V600E) mutant melanoma cells following treatment with the Plk1-specific inhibitor BI 6727. Employing label-free nano-LC-MS/MS technology on a Q-exactive followed by SIEVE processing, we identified more than 20 proteins of interest, many of which have not been previously associated with Plk1 signaling. Here we report the down-regulation of multiple metabolic proteins with an associated decrease in cellular metabolism, as assessed by lactate and NAD levels. Furthermore, we have also identified the down-regulation of multiple proteasomal subunits, resulting in a significant decrease in 20S proteasome activity. Additionally, we have identified a novel association between Plk1 and p53 through heterogeneous ribonucleoprotein C1/C2 (hnRNPC), thus providing valuable insight into Plk1's role in cancer cell survival.

Novel extended-release transdermal formulations of the psychedelic N,N-dimethyltryptamine (DMT).

There is considerable evidence from the literature that psychedelics, such as N,N-dimethyltryptamine (DMT), are safe and effective treatments for depression. However, clinical administration to induce psychedelic effects and expensive psychotherapy-assisted treatments likely limit accessibility to the average patient. There is emerging evidence that DMT promotes positive behavioral changes in vivo at sub-hallucinogenic dosages, and depending on the target indication, subjecting patients to high, bolus dosages may not be necessary. Due to rapid metabolic degradation, achieving target levels of DMT in subjects is difficult, requiring IV administration, which poses risks to patients during the intense hallucinogenic and subjective drug effects. The chemical and physical properties of DMT make it an excellent candidate for non-invasive, transdermal delivery platforms. This paper outlines the formulation development, in vitro, and in vivo testing of transdermal drug-in-adhesive DMT patches using various adhesives and permeation enhancers. In vivo behavioral and pharmacokinetic studies were performed with lead patch formulation (F5) in male and female Swiss Webster mice, and resulting DMT levels in plasma and brain samples were quantified using LC/MS/MS. Notable differences were seen in female versus male mice during IV administration; however, transdermal administration provided consistent, extended drug release at a non-hallucinogenic dose. The IV half-life of DMT was extended by 20-fold with administration of the transdermal delivery system at sub-hallucinogenic plasma concentrations not exceeding 60 ng/mL. Results of a translational head twitch assay (a surrogate for hallucinogenic effects in non-human organisms) were consistent with absence of hallucinations at low plasma levels achieved with our TDDS. Despite the reported low bioavailability of DMT, the non-invasive transdermal DMT patch F5 afforded an impressive 77 % bioavailability compared to IV at two dosages. This unique transdermal delivery option has the potential to provide an out-patient treatment option for ailments not requiring higher, bolus doses and is especially intriguing for therapeutic indications requiring non-hallucinogenic alternatives.

Quantitative characterization of changes in the cardiac mitochondrial proteome during anesthetic preconditioning and ischemia.

Changes in mitochondrial bioenergetics have been proposed to be critical for triggering and effecting anesthetic-induced preconditioning (APC) against cardiac ischemia and reperfusion injury. The objective of this study was to analyze changes in mitochondrial protein levels and link those changes to potential functional changes. A (18)O-labeling method was applied for relative comparison of cardiac mitochondrial samples from control and isoflurane exposed rats before and after ischemia and reperfusion. Wistar rats were exposed to isoflurane for 30 min (APC) or did not receive the anesthetic (control). Rats were subjected to 30 min coronary occlusion and 15 min reperfusion without (ischemia) or after APC (ischemia + APC). The following comparisons were made: control vs. APC, control vs. ischemia, and APC vs. ischemia + APC. Proteins were analyzed by liquid chromatography-mass spectrometry. A total of 98 proteins currently annotated as mitochondrial proteins in the UniProt database were positively identified from three replicate experiments. Most of the changes during APC and ischemia occur in complexes of the electron transport chain. Overall, fewer changes in ETC complexes were found when comparing APC with APC + ischemia than when comparing control and ischemia. This corresponds to the preservation of bioenergetics due to APC after ischemia and reperfusion as indicated by preserved ATP level and generation. APC itself induced changes in complex I, but those changes were not correlated with activity changes in mitochondria after APC. Thus, a proteomic mass spectral approach does not only assess quantitative changes without prior knowledge of proteins, but also allows insight into the mechanisms of ischemia and reperfusion injury and APC.

Under-Oil Autonomously Regulated Oxygen Microenvironments: A Goldilocks Principle-Based Approach for Microscale Cell Culture.

Oxygen levels in vivo are autonomously regulated by a supply-demand balance, which can be altered in disease states. However, the oxygen levels of in vitro cell culture systems, particularly microscale cell culture, are typically dominated by either supply or demand. Further, the oxygen microenvironment in these systems is rarely monitored or reported. Here, a method to establish and dynamically monitor autonomously regulated oxygen microenvironments (AROM) using an oil overlay in an open microscale cell culture system is presented. Using this method, the oxygen microenvironment is dynamically regulated via the supply-demand balance of the system. Numerical simulation and experimental validation of oxygen transport within multi-liquid-phase, microscale culture systems involving a variety of cell types, including mammalian, fungal, and bacterial cells are presented. Finally, AROM is applied to establish a coculture between cells with disparate oxygen demands-primary intestinal epithelial cells (oxygen consuming) and Bacteroides uniformis (an anaerobic species prevalent in the human gut).

Molecular signatures of sanguinarine in human pancreatic cancer cells: A large scale label-free comparative proteomics approach.

Pancreatic cancer remains one of the most lethal of all human malignancies with its incidence nearly equaling its mortality rate. Therefore, it's crucial to identify newer mechanism-based agents and targets to effectively manage pancreatic cancer. Plant-derived agents/drugs have historically been useful in cancer therapeutics. Sanguinarine is a plant alkaloid with anti-proliferative effects against cancers, including pancreatic cancer. This study was designed to determine the mechanism of sanguinarine's effects in pancreatic cancer with a hope to obtain useful information to improve the therapeutic options for the management of this neoplasm. We employed a quantitative proteomics approach to define the mechanism of sanguinarine's effects in human pancreatic cancer cells. Proteins from control and sanguinarine-treated pancreatic cancer cells were digested with trypsin, run by nano-LC/MS/MS, and identified with the help of Swiss-Prot database. Results from replicate injections were processed with the SIEVE software to identify proteins with differential expression. We identified 37 differentially expressed proteins (from a total of 3107), which are known to be involved in variety of cellular processes. Four of these proteins (IL33, CUL5, GPS1 and DUSP4) appear to occupy regulatory nodes in key pathways. Further validation by qRT-PCR and immunoblot analyses demonstrated that the dual specificity phosphatase-4 (DUSP4) was significantly upregulated by sanguinarine in BxPC-3 and MIA PaCa-2 cells. Sanguinarine treatment also caused down-regulation of HIF1α and PCNA, and increased cleavage of PARP and Caspase-7. Taken together, sanguinarine appears to have pleotropic effects, as it modulates multiple key signaling pathways, supporting the potential usefulness of sanguinarine against pancreatic cancer.

Quantitative proteomic analysis of the chemolithoautotrophic bacterium Nitrosomonas europaea: comparison of growing- and energy-starved cells.

Obligately aerobic ammonia-oxidizing bacteria (AOB) like Nitrosomonas europaea play a pivotal role in the global nitrogen cycle. Although starvation tolerance is a key environmental adaptation, little is known about this response in AOB. The goal of these studies was to compare the composition of the N. europaea proteome in growing- and energy-starved cells using ¹5N labeling and HPLC-ESI-MS/MS. More than 6500 peptides were sequenced with high confidence, and matched to 876 proteins (34% of the protein coding genes). Of these, 126 proteins had two or more peptide forms identified by 10 or more scans, and were used in quantitative analysis and 27 were found to be significantly different in abundance between growing and starved cells. Proteins showing greater abundance in growing cells are geared toward biosynthesis, particularly DNA replication. Energy-starved cells were shifted away from biosynthesis and toward survival functions that included: cell envelope modification, protein protection/degradation, detoxification, and implementation of alternative energy generation mechanisms. Most of these activities have not previously been reported as associated with energy-starvation stress in N. europaea. This study provides insights into the potential effects of fluctuating environmental conditions on the regulation of physiological networks in N. europaea.