Primary Human M2 Macrophage Subtypes Are Distinguishable by Aqueous Metabolite Profiles.

The complexity of macrophage (MΦ) plasticity and polarization states, which include classically activated pro-inflammatory (M1) and alternatively activated anti-inflammatory (M2) MΦ phenotypes, is becoming increasingly appreciated. Within the M2 MΦ polarization state, M2a, M2b, M2c, and M2d MΦ subcategories have been defined based on their expression of specific cell surface receptors, secreted cytokines, and specialized immune effector functions. The importance of immunometabolic networks in mediating the function and regulation of MΦ immune responses is also being increasingly recognized, although the exact mechanisms and extent of metabolic modulation of MΦ subtype phenotypes and functions remain incompletely understood. In this study, proton (1H) nuclear magnetic resonance (NMR) metabolomics was employed to determine the polar metabolomes of M2 MΦ subtypes and to investigate the relationship between aqueous metabolite profiles and M2 MΦ functional phenotypes. Results from this study demonstrate that M2a MΦs are most distinct from M2b, M2c, and M2d MΦ subtypes, and that M2b MΦs display several metabolic traits associated with an M1-like MΦ phenotype. The significance of metabolome differences for metabolites implicated in glycolysis, the tricarboxylic acid (TCA) cycle, phospholipid metabolism, and creatine-phosphocreatine cycling is discussed. Altogether, this study provides biochemical insights into the role of metabolism in mediating the specialized effector functions of distinct M2 MΦ subtypes and supports the concept of a continuum of macrophage activation states rather than two well-separated and functionally distinct M1/M2 MΦ classes, as originally proposed within a classical M1/M2 MΦ framework.

Pseudomonas aeruginosa Planktonic- and Biofilm-Conditioned Media Elicit Discrete Metabolic Responses in Human Macrophages.

Macrophages (MΦs) are prevalent innate immune cells, present throughout human bodily tissues where they orchestrate innate and adaptive immune responses to maintain cellular homeostasis. MΦs have the capacity to display a wide array of functional phenotypes due to different microenvironmental cues, particularly soluble bacterial secretory products. Recent evidence has emerged demonstrating that metabolism supports MΦ function and plasticity, in addition to energy and biomolecular precursor production. In this study, 1D 1H-NMR-based metabolomics was used to identify the metabolic pathways that are differentially altered following primary human monocyte-derived MΦ exposure to P. aeruginosa planktonic- and biofilm-conditioned media (PCM and BCM). Metabolic profiling of PCM- and BCM-exposed MΦs indicated a significant increase in glycolytic metabolism, purine biosynthesis, and inositol phosphate metabolism. In addition, these metabolic patterns suggested that BCM-exposed MΦs exhibit a hyperinflammatory metabolic profile with reduced glycerol metabolism and elevated catabolism of lactate and amino acids, relative to PCM-exposed MΦs. Altogether, our study reveals novel findings concerning the metabolic modulation of human MΦs after exposure to secretory microbial products and contributes additional knowledge to the field of immunometabolism in MΦs.

Metabolic Implications of Using BioOrthogonal Non-Canonical Amino Acid Tagging (BONCAT) for Tracking Protein Synthesis.

BioOrthogonal Non-Canonical Amino acid Tagging (BONCAT) is a powerful tool for tracking protein synthesis on the level of single cells within communities and whole organisms. A basic premise of BONCAT is that the non-canonical amino acids (NCAA) used to track translational activity do not significantly alter cellular physiology. If the NCAA would induce changes in the metabolic state of cells, interpretation of BONCAT studies could be challenging. To address this knowledge-gap, we have used a global metabolomics analyses to assess the intracellular effects of NCAA incorporation. Two NCAA were tested: L-azidohomoalanine (AHA) and L-homopropargylglycine (HPG); L-methionine (MET) was used as a minimal stress baseline control. Liquid Chromatography Mass Spectrometry (LC-MS) and Nuclear Magnetic Resonance (NMR) were used to characterize intracellular metabolite profiles of Escherichia coli cultures, with multivariate statistical analysis using XCMS and MetaboAnalyst. Results show that doping with NCAA induces metabolic changes, however, the metabolic impact was not dramatic. A second set of experiments in which cultures were placed under mild stress to simulate real-world environmental conditions showed a more consistent and more robust perturbation. Pathways that changed include amino acid and protein synthesis, choline and betaine, and the TCA cycle. Globally, these changes were statistically minor, indicating that NCAA are unlikely to exert a significant impact on cells during incorporation. Our results are consistent with previous reports of NCAA doping under replete conditions and extend these results to bacterial growth under environmentally relevant conditions. Our work highlights the power of metabolomics studies in detecting cellular response to growth conditions and the complementarity of NMR and LCMS as omics tools.

Quantitative 1H NMR Metabolomics Reveal Distinct Metabolic Adaptations in Human Macrophages Following Differential Activation.

Macrophages (MΦs) are phagocytic immune cells that are found in nearly all human tissues, where they modulate innate and adaptive immune responses, thereby maintaining cellular homeostasis. MΦs display a spectrum of functional phenotypes as a result of microenvironmental and stress-induced stimuli. Evidence has emerged demonstrating that metabolism is not only crucial for the generation of energy and biomolecular precursors, but also contributes to the function and plasticity of MΦs. Here, 1D 1H NMR-based metabolomics was employed to identify metabolic pathways that are differentially modulated following primary human monocyte-derived MΦ activation with pro-inflammatory (M1) or anti-inflammatory (M2a) stimuli relative to resting (M0) MΦs. The metabolic profiling of M1 MΦs indicated a substantial increase in oxidative stress as well as a decrease in mitochondrial respiration. These metabolic profiles also provide compelling evidence that M1 MΦs divert metabolites from de novo glycerophospholipid synthesis to inhibit oxidative phosphorylation. Furthermore, glycolysis and lactic acid fermentation were significantly increased in both M1 and M2a MΦs. These metabolic patterns highlight robust metabolic activation markers of MΦ phenotypes. Overall, our study generates additional support to previous observations, presents novel findings regarding the metabolic modulation of human MΦs following activation, and contributes new knowledge to the rapidly evolving field of immunometabolism.

The ClpCP Complex Modulates Respiratory Metabolism in Staphylococcus aureus and Is Regulated in a SrrAB-Dependent Manner.

The staphylococcal respiratory regulator (SrrAB) modulates energy metabolism in Staphylococcus aureus Studies have suggested that regulated protein catabolism facilitates energy homeostasis. Regulated proteolysis in S. aureus is achieved through protein complexes composed of a peptidase (ClpQ or ClpP) in association with an AAA+ family ATPase (typically, ClpC or ClpX). In the present report, we tested the hypothesis that SrrAB regulates a Clp complex to facilitate energy homeostasis in S. aureus Strains deficient in one or more Clp complexes were attenuated for growth in the presence of puromycin, which causes enrichment of misfolded proteins. A ΔsrrAB strain had increased sensitivity to puromycin. Epistasis experiments suggested that the puromycin sensitivity phenotype of the ΔsrrAB strain was a result of decreased ClpC activity. Consistent with this, transcriptional activity of clpC was decreased in the ΔsrrAB mutant, and overexpression of clpC suppressed the puromycin sensitivity of the ΔsrrAB strain. We also found that ClpC positively influenced respiration and that it did so upon association with ClpP. In contrast, ClpC limited fermentative growth, while ClpP was required for optimal fermentative growth. Metabolomics studies demonstrated that intracellular metabolic profiles of the ΔclpC and ΔsrrAB mutants were distinct from those of the wild-type strain, supporting the notion that both ClpC and SrrAB affect central metabolism. We propose a model wherein SrrAB regulates energy homeostasis, in part, via modulation of regulated proteolysis.IMPORTANCE Oxygen is used as a substrate to derive energy by the bacterial pathogen Staphylococcus aureus during infection; however, S. aureus can also grow fermentatively in the absence of oxygen. To successfully cause infection, S. aureus must tailor its metabolism to take advantage of respiratory activity. Different proteins are required for growth in the presence or absence of oxygen; therefore, when cells transition between these conditions, several proteins would be expected to become unnecessary. In this report, we show that regulated proteolysis is used to modulate energy metabolism in S. aureus We report that the ClpCP protein complex is involved in specifically modulating aerobic respiratory growth but is dispensable for fermentative growth.

Characterization of the antibacterial activity of Bald's eyesalve against drug resistant Staphylococcus aureus and Pseudomonas aeruginosa.

Bald's eyesalve is an Anglo-Saxon medicinal remedy that has been used through ancient times to treat eye sty infections and may represent a source of ancientbiotics. This study assessed the efficacy of Bald's eyesalve against several strains of Staphylococcus aureus and Pseudomonas aeruginosa, including a multi-drug resistant phenotype, and identified the principal compound conveying antibacterial activity. Bald's eyesalve formulations were produced by combining garlic, onion or leek, wine, bovine bile, and brass, with specific ingredient omissions in several formulations, followed by incubation at 4 °C for 9 days. Bald's eyesalve formulation ES-GBBr exhibited the greatest antibacterial activity against S. aureus and P. aeruginosa. Fractionation of ES-GBBr using molecular size exclusion and organic solvent partitioning isolated its antibacterial activity to the small molecule nonpolar fraction, and 1D 1H NMR revealed the identity of the antibacterial agent to be allicin. Depletion of allicin from this fraction by addition of exogenous cysteine established that all observable growth inhibition originated from allicin. Quantification of allicin demonstrated that its concentration was significantly greater in ES-GBBr compared to the ES-O formulation; however, this was not due to greater yield. The antibacterial activity of allicin against S. aureus was antagonized by other ingredients within Bald's eyesalve, whereas they were additive or synergistic against P. aeruginosa. These results suggest that neither leek nor onion is necessary for the antibacterial efficacy of Bald's eyesalve against S. aureus or P. aeruginosa, and while allicin was identified as the principal antibacterial agent present, its activity is influenced differentially in the presence of additional Bald's eyesalve ingredients when used against S. aureus compared to P. aeruginosa. Ancientbiotics may provide a source of promising antibacterials; however, identifying the source of activity and assessing distinct formulations for cooperative effects are essential to using ancient remedies, such as Bald's eyesalve, effectively against drug resistant pathogens.

Ecological niche modeling of the genus Papio.

OBJECTIVE: Ecological niche modeling (ENM) has been used to assess how abiotic variables influence species distributions and diversity. Baboons are broadly distributed throughout Africa, yet the degree of climatic specialization is largely unexplored for individual taxa. Also, the influence of climate on baboon phylogenetic divergence is unknown. In this study, we constructed ENMs to investigate how niches vary across Papio species to understand how climatic variables have influenced their biogeography and mode of speciation. MATERIALS AND METHODS: We used Maxent to generate ENMs by collating locality data for six Papio species and climate information from WorldClim. In addition, we examined the degree of niche overlap among all possible pairs of taxa, which can provide insight into patterns of species diversity. Lastly, we conducted a Mantel test to assess the relationship between niche overlap and estimated time since divergence. RESULTS: Our models performed moderately to extremely well, with a mean area under the curve value of 0.868. The species with the best models include P. papio and P. kindae, whereas P. hamadryas had the poorest models. We found that most species pairs exhibited significantly different niches. Lastly, we found no significant correlation between niche overlap and divergence times. DISCUSSION: Niche models had good predictive power, which indicates Papio species distributions are correlated with climatic variables to varying degrees. Significantly little niche overlap and incomplete geographic boundaries suggests these models generally support a parapatric speciation scenario for the genus Papio.

Pediatric Oncology Providers' Attitudes and Practice Patterns Regarding Fertility Preservation in Adolescent Male Cancer Patients.

BACKGROUND: The aim of this study was to evaluate pediatric oncology providers' attitudes toward fertility preservation (FP), their use of educational materials, their approach to FP discussion, and their FP knowledge specifically pertaining to adolescent males. METHODS: A 40-item online survey was distributed to physicians, advanced practice nurses (APN), and nurses within pediatric oncology. RESULTS: About 78.7% of physicians, 81.4% of APN, and 51.9% of nurses reported high levels of comfort in discussing FP options with adolescent males (P<0.05). Fifty-one percent of physicians and 54.2% of APN reported using educational materials, compared with 38.9% of nurses (P<0.05). Regarding knowledge of FP technologies, 48.7% of physicians, 52.5% of APN, and 81.1% of nurses reported being unfamiliar with intracytoplasmic sperm injection (P<0.05). An overwhelming majority (92.9%) of respondents reported having no formal training in discussing FP. Finally, 84.8% of respondents believed formal training on this issue would be useful to them. CONCLUSIONS: This study illustrates an unmet need in the education of pediatric oncology providers, as knowledge gaps and discomfort are common themes reported by health care professionals within the context of adolescent male FP care. In addition, this study reveals a high level of receptiveness to FP training by these same providers.

Optimization of Metabolite Extraction Protocols for the Identification and Profiling of Small Molecule Metabolites from Planktonic and Biofilm Pseudomonas aeruginosa Cultures.

BACKGROUND: Metabolomics aims to characterize the metabolic phenotype and metabolic pathways utilized by microorganisms or other cellular systems. A crucial component to metabolomics research as it applies to microbial metabolism is the development of robust and reproducible methods for extraction of intracellular metabolites. The goal is to extract all metabolites in a non-biased and consistent manner; however, most methods used thus far are targeted to specific metabolite classes and use harsh conditions that may contribute to metabolite degradation. Metabolite extraction methodologies need to be optimized for each microorganism of interest due to different cellular characteristics contributing to lysis resistance. METHODS: Three cell pellet wash solutions were compared for the potential to influence intracellular metabolite leakage of P. aeruginosa. We also compared four different extraction methods using (i) methanol:chloroform (2:1); (ii) 50% methanol; (iii) 100% methanol; or (iv) 100% water to extract intracellular metabolites from P. aeruginosa planktonic and biofilm cultures. RESULTS: Intracellular metabolite extraction efficiency was found to be dependent on the extraction method and varies between microbial modes of growth. Methods using the 60% methanol wash produced the greatest amount of intracellular material leakage. Quantification of intracellular metabolites via 1H NMR showed that extraction protocols using 100% water or 50% methanol achieved the greatest extraction efficiencies, while addition of sonication to facilitate cell lysis to the 50% methanol extraction method resulted in at least a two-fold increase in signal intensities for approximately half of the metabolites identified. Phosphate buffered saline (PBS) was determined to be the most appropriate wash solution, yielding little intracellular metabolite leakage from cells. CONCLUSION: We determined that washing in 1X PBS and extracting intracellular metabolites with 50% methanol is the most appropriate metabolite extraction protocol because (a) leakage is minimal; (b) a broad range of metabolites present at sufficiently high concentrations is detectable by NMR; and (c) this method proved suitable for metabolite extraction of both planktonic and biofilm P. aeruginosa cultures.

Risk of urethral stricture recurrence increases over time after urethroplasty.

OBJECTIVE: To report a single institutional experience with urethroplasty outcomes and success rates at long-term follow up. METHODS: A retrospective review was carried out of all urethroplasties performed by a single surgeon from 2000 to 2010. A total of 347 patients underwent urethroplasty during this time period, of which 227 had minimum 1-year follow-up data available. Demographic, clinical, pathological and outcome data were reviewed. Recurrence was defined by patient reported urinary symptoms or need for subsequent intervention. Statistical analyses were carried out using SPSS statistical software. RESULTS: A total of 26% of all patients had a recurrence at a mean follow up of 62 months (range 13-147 months). The recurrence rate after anastomotic urethroplasty was 18%, as compared with 31% after substitution urethroplasty. Mean time to recurrence was 34 months (range 5-87). On univariate analysis, use of abdominal skin graft, history of prior urethroplasty, lichen sclerosus and length of follow up were statistically significant predictors of recurrence. On multivariate analysis, only history of prior urethroplasty and length of follow-up time exceeding 48 months were statistically significant predictors of recurrence. CONCLUSIONS: Urethroplasty for urethral stricture is the most durable treatment modality, regardless of surgical approach. However, there is an ongoing risk of recurrence with the passage of time. Patients should be counseled appropriately on the potential for late recurrence of stricture disease after urethroplasty.

Long-term outcomes of urethroplasty with abdominal wall skin grafts.

OBJECTIVE: To report the long-term outcomes of urethroplasty using abdominal wall skin (AWS) grafts. Men with long-segment strictures, prior urethroplasty, and lichen sclerosus (LS) pose challenges in surgical management, including the choice of graft tissue for urethral reconstruction. AWS grafts are an alternative when buccal mucosa or penile grafts are not feasible or chosen by the patient. METHODS: We retrospectively reviewed 238 patients who underwent urethroplasty (2000-2010) with at least 1 year of follow-up. Demographics, etiology, comorbidities, prior procedures, and surgical technique were analyzed for correlation with recurrence. RESULTS: Mean age was 42.9 years (range, 15-79 years), mean stricture length 5.6 cm (1-24 cm), and median follow-up of 59.3 months (12.5-147 months). A total of 58.4% patients had prior intervention, of which 15 patients (6.3%) had urethroplasty and 41 patients (17.2%) had hypospadias repair. Twenty-six patients (10.9%) underwent urethroplasty with AWS graft, whereas 107 (45.0%) and 12 (5.0%) patients were augmented with buccal mucosa or genital skin. Sixty-six patients (27.7%) had stricture recurrence at a mean of 34.5 months (range, 1.87-87.1 months). On univariate analysis, patients with AWS graft had longer strictures (P = .0001), were more likely to have LS (P = .0002), prior urethroplasty (P = .007), and recurrence (P = .002). On multivariate analysis, prior urethroplasty (odds ratio [OR], 5.3; P = .009), diabetes (OR, 2.6; P = .04), and LS (OR, 2.8; P = .05) were significantly associated with recurrence, whereas AWS graft was not (OR, 2.0; P = .28). CONCLUSION: AWS grafts are an alternative tissue source for urethral stricture, but may be associated with greater risk of recurrence. This may be secondary to patient selection, with this population often having other risk factors for recurrence.

Diagnosis of varicoceles in men undergoing vasectomy may lead to earlier detection of hypogonadism.

OBJECTIVE: To determine the temporal relationship between vasectomy, varicocele, and hypogonadism diagnosis. Many young men undergo their first thorough genitourinary examination in their adult lives at the time of vasectomy consultation, providing a unique opportunity for diagnosis of asymptomatic varicoceles. Varicoceles have recently been implicated as a possible reversible contributor to hypogonadism. Hypogonadism may be associated with significant adverse effect, including decreased libido, impaired cognitive function, and increased cardiovascular events. Early diagnosis and treatment of hypogonadism may prevent these adverse sequelae. METHODS: Data were collected from the Truven Health Analytics MarketScan database, a large outpatient claims database. We reviewed records between 2003 and 2010 for male patients between the ages of 25 and 50 years with International Classification of Diseases, Ninth Revision codes for hypogonadism, vasectomy, and varicocele, and queried dates of first claim. RESULTS: A total of 15,679 men undergoing vasectomies were matched with 156,790 men with nonvasectomy claims in the same year. Vasectomy patients were diagnosed with varicocele at an earlier age (40.9 vs 42.5 years; P=.009). We identified 224,817 men between the ages of 25 and 50 years with a claim of hypogonadism, of which 5883 (2.6%) also had a claim of varicocele. Men with hypogonadism alone were older at presentation compared with men with an accompanying varicocele (41.3 [standard deviation±6.5] vs 34.9 [standard deviation±6.1]; P<.001). CONCLUSION: Men undergoing vasectomies are diagnosed with varicoceles at a younger age than age-matched controls. Men with varicoceles present with hypogonadism earlier than men without varicoceles. Earlier diagnosis of varicocele at the time of vasectomy allows for earlier detection of hypogonadism.

Pregnancy after anterior spinal surgery: fertility, cesarean-section rate, and the use of neuraxial anesthesia.

BACKGROUND CONTEXT: Previous publications have reported results with respect to functional outcome and sexual function in young women after traumatic injuries to the pelvis. It is well known that anterior spinal surgery has the possibility of causing reproductive dysfunction in men. Little has been described concerning deleterious effects of anterior spinal surgery in women of childbearing age. PURPOSE: The purpose of the study was to determine the fertility, cesarean section rate, and the use of neuraxial anesthesia in a population of women of childbearing age who have undergone anterior spinal surgery. STUDY DESIGN/SETTING: This was a retrospective chart review combined with a telephone questionnaire of the patients who had undergone anterior spinal surgery by a single orthopedic surgeon. METHODS: Demographic data were collected from hospital and office records. Patients were asked if they had tried to become pregnant, if their pregnancy had come to term, the use of spinal or epidural anesthesia for that labor and delivery, and the type of delivery. RESULTS: Seventy-nine potential subjects were identified and 67 patients were contacted to determine their fertility status. Ninteen patients had tried to become pregnant after their anterior spinal surgery. All patients who reported that they tried to become pregnant were successful at having a child. Seven of the 19 women had children who were born by cesarean section (37%). Only two patients received an epidural as analgesia for their delivery. CONCLUSIONS: Although the cesarean-section rate appears high, it is consistent with the current obstetrical trends. Anesthesiologists appear less inclined to offer neuraxial anesthesia to a population which has undergone anterior spinal surgery.