Predictors of emotional distress in uveal melanoma survivors: a systematic review.

Uveal melanoma (UM) survivors can experience significant emotional distress, although the factors underpinning this are poorly understood. Systematic reviews of distress in UM only include cross-sectional studies, thereby limiting our understanding of causal factors. This review identified prospective clinical, demographic, social and psychological predictors of distress in UM survivors. A systematic search of the literature for English language prospective studies was conducted. Thirteen papers, reporting data from seven unique datasets were included in a narrative synthesis of the results. Younger age (3 studies from 3 datasets), physical health (including visual impairment, ocular symptoms, and other UM-related factors; 3 studies from 3 datasets), and psychological factors (mainly baseline distress; 3 studies from 3 datasets and worry about recurrence; 2 studies from 2 datasets), significantly predicted distress. There was no consistent evidence for other demographic, clinical or social variables (significant in <50% of datasets). Generally, the quality of the papers was adequate. However, attrition rates were high or not reported in over half of the included studies. The findings of this review emphasise the importance of attempts to prevent and recognise distress immediately post-diagnosis of UM. Particular focus should be given to younger patients, those with physical and psychological health difficulties at the time of diagnosis, and those who develop adverse treatment symptoms during survivorship. More research into potential social and psychological variables and their role in predicting distress in survivors is recommended.

Unusual Presentation of Hodgkin's Lymphoma in Pregnancy: A Case Report and Systematic Review of Literature.

Diseases occurring during pregnancy create a dilemma of managing the patient without causing harm to the unborn child. Three percent of the peak incidence of Hodgkin lymphoma (HL) is congruent with the reproductive period, particularly with pregnancy. Pregnant patients with HL always require a team of medical experts ranging from a medical oncologist, high-risk obstetrician, and neonatologist. Effective communication with both the patient and family is also necessary. The treatment goal for these patients should focus on achieving complete remission for the mother while permitting the delivery of a healthy child. Pregnant patients diagnosed with HL should undergo similar clinical investigations as other non-pregnant patients with accurate disease staging and appropriate non-radiation imaging such as ultrasound while avoiding invasive procedures.

'I Don't Like Uncertainty, I Like to Know': How and why uveal melanoma patients consent to life expectancy prognostication.

BACKGROUND: Technological advances have led to cancer prognostication that is increasingly accurate but often unalterable. However, a reliable prognosis of limited life expectancy can cause psychological distress. People should carefully consider offers of prognostication, but little is known about how and why they decide on prognostication. Using uveal melanoma (UM) patients, we aimed to identify (i) how and why do people with UM decide to accept prognostication and (ii) alignment and divergence of their decision-making from conceptualizations of a 'well-considered' decision. METHODS: UM provides a paradigm to elucidate clinical and ethical perspectives on prognostication, because prognostication is reliable but prognoses are largely nonameliorable. We used qualitative methods to examine how and why 20 UM people with UM chose prognostication. We compared findings to a template of 'well-considered' decision-making, where 'well-considered' decisions involve consideration of all likely outcomes. RESULTS: Participants wanted prognostication to reduce future worry about uncertain life expectancy. They spontaneously spoke of hoping for a good prognosis when making their decisions, but largely did not consider the 50% possibility of a poor prognosis. When pressed, they argued that a poor outcome at least brings certainty. CONCLUSIONS: While respecting decisions as valid expressions of participants' wishes, we are concerned that they did not explicitly consider the realistic possibility of a poor outcome and how this would affect them. Thus, it is difficult to see their decisions as 'well-considered'. We propose that nondirective preference exploration techniques could help people to consider the possibility of a poor outcome. PATIENT OR PUBLIC CONTRIBUTION: This paper is a direct response to a patient-identified and defined problem that arose in therapeutic and conversational discourse. The research was informed by the responses of patient participants, as we used the material from interviews to dynamically shape the interview guide. Thus, participants' ideas drove the analysis and shaped the interviews to come.

Epigenetic drugs induce the potency of classic chemotherapy, suppress post-treatment re-growth of breast cancer, but preserve the wound healing ability of stem cells.

Epigenetic therapy augments neoadjuvant chemotherapy (NACT) in breast cancer and may aid post-surgical wound healing affected by NACT. Our study investigates: (1) The cytotoxicity of classic paclitaxel chemotherapy on triple negative breast cancer (TNBC) independently and in combination with epigenetic drugs. (2) The sustainable inhibition of breast cancer regrowth following paclitaxel and epigenetic therapies. (3) The effects of paclitaxel with and without epigenetic therapy on the post-treatment viability and wound healing potential of adipose stem cells (ASCs). Cytotoxicity assays were performed on TNBC and ASCs. Cells were treated and recovered in drug-free medium. Cell viability was measured via cell counts and MTT assays. W -ound healing was tested with scratch assays. The combination of epigenetic drugs shows increased toxicity against TNBC cells compared to standard chemotherapy alone. Moreover, the combination of paclitaxel with epigenetic treatments causes cancer toxicity that is sustainable to TNBC cells after the drugs' removal with minimal effect on ASCs wound healing ability. The use of epigenetic drugs in addition to standard chemotherapy is cytotoxic to TNBC cells and prevents post-treatment recovery of TNBC while maintaining ASC wound healing ability. This strategy may be useful in maximizing post-surgical wound healing following NACT in TNBC.

Therapists' experiences of remote working during the COVID-19 pandemic.

Objectives: To explore the experiences of therapists who delivered remote psychological therapy during the COVID-19 pandemic. Design: This was a qualitative, phenomenological study. Interpretative Phenomenological Analysis elicited themes from semi-structured interviews. Methods: A purposive sample of eight therapists was recruited from breast cancer services in the United Kingdom. Results: Analysis identified three superordinate themes. Participants spoke about how their experience of remote working changed over time from an initial crisis response to a new status quo. They adapted to the specific practical and personal challenges of remote working and struggled to connect with clients as the use of technology fundamentally changed the experience of therapy. Conclusion: Consideration should be given to the impact of remote working on therapists and the quality of their practise. Adjustments to ways of working can help to maximize the advantages of remote working while minimizing potential issues.

The epidemiology of Plasmodium vivax among adults in the Democratic Republic of the Congo.

Reports of P. vivax infections among Duffy-negative hosts have accumulated throughout sub-Saharan Africa. Despite this growing body of evidence, no nationally representative epidemiological surveys of P. vivax in sub-Saharan Africa have been performed. To overcome this gap in knowledge, we screened over 17,000 adults in the Democratic Republic of the Congo (DRC) for P. vivax using samples from the 2013-2014 Demographic Health Survey. Overall, we found a 2.97% (95% CI: 2.28%, 3.65%) prevalence of P. vivax infections across the DRC. Infections were associated with few risk-factors and demonstrated a relatively flat distribution of prevalence across space with focal regions of relatively higher prevalence in the north and northeast. Mitochondrial genomes suggested that DRC P. vivax were distinct from circulating non-human ape strains and an ancestral European P. vivax strain, and instead may be part of a separate contemporary clade. Our findings suggest P. vivax is diffusely spread across the DRC at a low prevalence, which may be associated with long-term carriage of low parasitemia, frequent relapses, or a general pool of infections with limited forward propagation.

Effect of radiation damage and illumination variability on signal-to-noise ratio in X-ray free-electron laser single-particle imaging.

The deterioration of both the signal-to-noise ratio and the spatial resolution in the electron-density distribution reconstructed from diffraction intensities collected at different orientations of a sample is analysed theoretically with respect to the radiation damage to the sample and the variations in the X-ray intensities illuminating different copies of the sample. The simple analytical expressions and numerical estimates obtained for models of radiation damage and incident X-ray pulses may be helpful in planning X-ray free-electron laser (XFEL) imaging experiments and in analysis of experimental data. This approach to the analysis of partially coherent X-ray imaging configurations can potentially be used for analysis of other forms of imaging where the temporal behaviour of the sample and the incident intensity during exposure may affect the inverse problem of sample reconstruction.

Data-Driven Respiratory Gating Outperforms Device-Based Gating for Clinical 18F-FDG PET/CT.

A data-driven method for respiratory gating in PET has recently been commercially developed. We sought to compare the performance of the algorithm with an external, device-based system for oncologic 18F-FDG PET/CT imaging. Methods: In total, 144 whole-body 18F-FDG PET/CT examinations were acquired, with a respiratory gating waveform recorded by an external, device-based respiratory gating system. In each examination, 2 of the bed positions covering the liver and lung bases were acquired with a duration of 6 min. Quiescent-period gating retaining approximately 50% of coincidences was then able to produce images with an effective duration of 3 min for these 2 bed positions, matching the other bed positions. For each examination, 4 reconstructions were performed and compared: data-driven gating (DDG) (we use the term DDG-retro to distinguish that we did not use the real-time R-threshold-based application of DDG that is available within the manufacturer's product), external device-based gating (real-time position management (RPM)-gated), no gating but using only the first 3 min of data (ungated-matched), and no gating retaining all coincidences (ungated-full). Lesions in the images were quantified and image quality scored by a radiologist who was masked to the method of data processing. Results: Compared with the other reconstruction options, DDG-retro increased the SUVmax and decreased the threshold-defined lesion volume. Compared with RPM-gated, DDG-retro gave an average increase in SUVmax of 0.66 ± 0.1 g/mL (n = 87, P < 0.0005). Although the results from the masked image evaluation were most commonly equivalent, DDG-retro was preferred over RPM-gated in 13% of examinations, whereas the opposite occurred in just 2% of examinations. This was a significant preference for DDG-retro (P = 0.008, n = 121). Liver lesions were identified in 23 examinations. Considering this subset of data, DDG-retro was ranked superior to ungated-full in 6 of 23 (26%) cases. Gated reconstruction using the external device failed in 16% of examinations, whereas DDG-retro always provided a clinically acceptable image. Conclusion: In this clinical evaluation, DDG-retro provided performance superior to that of the external device-based system. For most examinations the performance was equivalent, but DDG-retro had superior performance in 13% of examinations, leading to a significant preference overall.

X-ray diffractive imaging of controlled gas-phase molecules: Toward imaging of dynamics in the molecular frame.

We report experimental results on the diffractive imaging of three-dimensionally aligned 2,5-diiodothiophene molecules. The molecules were aligned by chirped near-infrared laser pulses, and their structure was probed at a photon energy of 9.5 keV (λ ≈ 130 pm) provided by the Linac Coherent Light Source. Diffracted photons were recorded on the Cornell-SLAC pixel array detector, and a two-dimensional diffraction pattern of the equilibrium structure of 2,5-diiodothiophene was recorded. The retrieved distance between the two iodine atoms agrees with the quantum-chemically calculated molecular structure to be within 5%. The experimental approach allows for the imaging of intrinsic molecular dynamics in the molecular frame, albeit this requires more experimental data, which should be readily available at upcoming high-repetition-rate facilities.

Locally Transplanted Adipose Stem Cells Reduce Anastomotic Leaks in Ischemic Colorectal Anastomoses: A Rat Model.

BACKGROUND: Anastomotic leakage remains a dreaded complication after colorectal surgery. Stem-cell-based therapies have been shown to increase angiogenesis and cell proliferation. OBJECTIVE: The purpose of this research was to investigate the use of adipose-derived stem cells on the healing of ischemic colonic anastomoses in a rat model. DESIGN: This is an animal research study using xenotransplantation. SETTINGS: Male Wistar rats (300-400 g, n = 48) were purchased from a licensed breeder. PATIENTS: Adipose stem cells were isolated from the subcutaneous fat of healthy human donors. INTERVENTIONS: The rats underwent laparotomy with creation of an ischemic colorectal anastomosis created by ligation of mesenteric vessels. The animals were divided into 3 groups: control group with an ischemic anastomosis, vehicle-only group in which the ischemic anastomosis was treated with an absorbable gelatin sponge, and a treatment group in which the ischemic anastomosis was treated with an absorbable gelatin sponge plus adipose stem cells. Animals were killed at postoperative days 3 and 7. MAIN OUTCOME MEASURES: Anastomotic leakage was defined as the finding of feculent peritonitis or perianastomotic abscess on necropsy. Rat mRNA expression was measured using real-time polymerase chain reaction. RESULTS: Adipose-derived stem cells significantly decreased anastomotic leakage when compared with control at both postoperative days 3 (25.0% vs 87.5%; p = 0.02) and 7 (25.0% vs 87.5%; p = 0.02). The use of an absorbable gelatin sponge alone had no effect on anastomotic leakage when compared with control and postoperative days 3 or 7. We found that stem cell-treated animals had a 5.9-fold and 7.4-fold increase in the expression of vascular endothelial growth factor when compared with control at 3 and 7 days; however, this difference was not statistically significant when compared with the absorbable gelatin sponge group. LIMITATIONS: This is a preclinical animal research study using xenotransplantation of cultured stem cells. CONCLUSIONS: Locally transplanted adipose stem cells enhance the healing of ischemic colorectal anastomoses and may be a novel strategy for reducing the risk of anastomotic leakage in colorectal surgery. See Video Abstract at http://links.lww.com/DCR/B203. EL TRANSPLANTE LOCAL DE CÉLULAS MADRE ADIPOSAS REDUCE LA FUGA ANASTOMÓTICA EN LAS SUTURAS COLORRECTALES ISQUÉMICAS: MODELO EN RATAS: Las fugas anastomóticas son una complicación pusilánime después de toda cirugía colorrectal. Se ha demostrado que el tratamiento con células madre aumenta la angiogénesis y la proliferación celular.Investigar el uso de células madre derivadas de tejido adiposo en la cicatrización de una anastomosis colónica isquémica basada en ratas como modelo.Estudio de investigación en animales utilizando xenotrasplantes.Adquisición de típicas ratas de laboratorio raza Wistar, todas machos (300-400 g, n = 48) de un criadero autorizado.Aislamiento de células madre de tipo adiposo del tejido celular subcutáneo en donantes humanos sanos.Las ratas se sometieron a laparotomía con la creación de una anastomosis colorrectal isquémica obtenida mediante ligadura controlada de los vasos mesentéricos correspondientes. Los animales se dividieron en tres grupos: grupo de control con anastomosis isquémica, grupo de vehículo único en el que la anastomosis isquémica se trató con una esponja de gelatina absorbible, y un grupo de tratamiento en el que la anastomosis isquémica se trató con una esponja de gelatina absorbible asociada a un vástago adiposo de células madre. Los animales fueron sacrificados el POD3 y el POD7.La fuga anastomótica fué definida como el hallazgo de peritonitis fecaloidea o absceso perianastomótico a la necropsia. La expresión de RNAm de las ratas se midió usando PCR en tiempo real.Las células madre derivadas de tejido adiposo disminuyeron significativamente la fuga anastomótica en comparación con el grupo control tanto en el POD3 (25% frente a 87.5%, p = 0.02) como en el POD7 (25% frente a 87.5%, p = 0.02). El uso de una esponja de gelatina absorbible sola, no tuvo efecto sobre la fuga anastomótica en comparación con los controles el POD3 o el POD7. Descubrimos que los animales tratados con células madre adiposas tenían un aumento de 5,9 y 7,4 veces en la expresión de VEGF en comparación con el control a los 3 y 7 días, respectivamente; sin embargo, esta diferencia no fue estadísticamente significativa en comparación con el grupo de esponja de gelatina absorbible.Este es un estudio preclínico de investigación en animales que utiliza xenotrasplantes de células madre adiposas cultivadas.Las células madre de tipo adiposo trasplantadas localmente mejoran la cicatrisación en casos de anastomosis colorrectales isquémicas, y podrían convertirse en una nueva estrategia para reducir el riesgo de fugas anastomóticas en casos de cirugía colorrectal. Consulte Video Resumen en http://links.lww.com/DCR/B203. (Traducción-Dr Xavier Delgadillo).

Myeloperoxidase-mediated oxidation of edaravone produces an apparent non-toxic free radical metabolite and modulates hydrogen peroxide-mediated cytotoxicity in HL-60 cells.

Edaravone is considered to be a potent antioxidant drug known to scavenge free radical species and prevent free radical-induced lipid peroxidation. In this study, we investigated the effect of edaravone on the myeloperoxidase (MPO) activity, an enzyme responsible for the production of an array of neutrophil-derived oxidants that can cause cellular damage. The addition of edaravone to the reaction of MPO and hydrogen peroxide (H2O2) significantly enhanced the reduction of MPO Compound II back to native MPO. Interestingly, the MPO-mediated production of toxic hypochlorous acid exhibited a concentration-dependent biphasic effect, with the apparent optimal edaravone concentration at 10 µM. Oxidation of edaravone by MPO was examined by various analytical methods. An MPO-catalyzed product(s) of edaravone was identified at 350 nm by kinetic analysis of UV-Vis spectroscopy. Several MPO-catalyzed metabolites of edaravone were proposed from the LC-MS analyses, including oxidized dimers from edaravone radicals. Electron spin resonance (ESR) spin trapping detected a carbon-centred radical metabolite of edaravone. NMR studies revealed that there are two exchangeable hydrogens, one of which is on the α-carbon, justifying the carbon-centred edaravone radical produced from MPO. Despite the formation of an edaravone carbon-radical metabolite, it did not appear to effectively oxidize GSH (in comparison with phenoxyl radicals). Viability (ATP) and cytotoxicity (LDH release) assays showed a concentration-dependent effect of edaravone on HL-60 cells treated with either a bolus concentration of 30 µM H2O2 or a flux of H2O2 generated by 5 mM glucose and 10 mU/mL glucose oxidase. The H2O2-induced toxicity was ameliorated at high edaravone concentrations (100-200 µM). In contrast, low concentrations of edaravone (1-10 µM) exacerbated the H2O2-induced toxicity. However, the effect of edaravone at low concentration (0-10 µM) appeared more prominent with the LDH assay only. The cellular findings correlated with the biochemical studies with respect to hypochlorous acid formation. These findings provide interesting perspectives regarding the duality of edaravone as an antioxidant drug.

Caution for the routine use of phenol red - It is more than just a pH indicator.

Phenol red (PR) is the standard pH indicator in various cell and tissue culture media, as it provides a quick check for the health of the culture. PR has also been used in multiple protocols to detect cellular hydrogen peroxide as well as peroxidase activity from human peroxidase enzymes. The majority of promyelocytic leukemia cell lines (e.g. HL-60 cells) express myeloperoxidase (MPO), which may react with PR, especially as the latter is present in cell culture media at sufficient concentrations (~15 µM) to partake in redox reactions. Moreover, phenolic molecules are often efficient donor substrates for peroxidase enzymes. In this study, we hypothesized that MPO metabolism of PR via MPO-expressing HL-60 cells could result in PR metabolite(s) that could modulate cell viability. We used purified human MPO for UV-visible spectrophotometry, electron paramagnetic resonance (EPR) and LC-MS analyses to investigate PR peroxidation. 2-chloro-5,5-dimethyl-1,3-cyclohexanedione (monochloro-dimedone, MCD) was used to assess the effect of PR on MPO-catalyzed chlorination activity, and we assessed PR uptake by HL-60 cells using LC-MS analysis. Lastly, we investigated the impact of PR metabolism by intracellular MPO on cell viability (ATP, using CellTiter-Glo®), cytotoxicity (using trypan blue), and on reduced and oxidized glutathione (using GSH/GSSG-Glo™). Our results demonstrate that PR undergoes oxidative halogenation via MPO, resulting in its UV-vis spectral changes due to the formation of mono- and di-halogenated products. Moreover, a significant increase in MPO-catalyzed chlorination of MCD and an increase in glutathionyl radical detection (using EPR) were observed in the presence of PR. Our in-vitro studies revealed that PR is readily taken up by HL-60 cells and its metabolism by intracellular MPO leads to a significant decrease in cellular glutathione as well as a significant increase in glutathione disulphide formation. In spite of the latter, PR had no considerable effect on HL-60 cell viability. These results provide evidence that while no overt decrease in cell viability may be observed, PR does impart redox activity, which investigators should be wary of in experimental protocols.

Unravelling the molecular mechanisms of nickel in woodlice.

During the last few years, there has been an alarming increase in the amount of nickel (Ni) being released into the environment, primarily due to its use in the production of stainless steel but also from other sources such as batteries manufacturing and consequent disposal. The established biotic ligand models provide precise estimates for Ni bioavailability, in contrast, studies describing the mechanisms underpinning toxicological effect of Ni are scarce. This study exploits RNA-seq to determine the transcriptomic responses of isopods using Porcellionides pruinosus as an example of a terrestrial metal-resistant woodlouse. Furthermore, the recently proposed model for Ni adverse outcome pathways (Ni-AOP) presents an unprecedented opportunity to fit isopod responses to Ni toxicity and define Porcellionides pruinosus as a metalomic model. Prior to this study, P. pruinosus represented an important environmental sentinel, though lacking genetic/omic data. The reference transcriptome generated here thus represents a major advance and a novel resource. A detailed annotation of the transcripts obtained is presented together with the homology to genes/gene products from Metazoan and Arthropoda phylum, Gene Ontology (GO) classification, clusters of orthologous groups (COG) and assignment to KEGG metabolic pathways. The differential gene expression comparison was determined in response to nickel (Ni) exposure and used to derive the enriched pathways and processes. It revealed a significant impact on ion trafficking and storage, oxidative stress, neurotoxicity, reproduction impairment, genetics and epigenetics. Many of the processes observed support the current Ni-AOP although the data highlights that the current model can be improved by including epigenetic endpoints, which represents key chronic risks under a scenario of Ni toxicity.

Epigenetic therapy can inhibit growth of ovarian cancer cells and reverse chemoresistant properties acquired from metastatic omentum.

OBJECTIVE: To examine the cytotoxicity of epigenetic drugs independently and in combination with chemotherapy on ovarian cancer cells Caov-3, and to investigate their ability to acquire chemoresistance in omental microenvironments and whether epigenetic drugs can counteract this chemoresistance. METHODS: A pilot study was conducted in Cooper University Hospital, NJ, USA from August 1 to October 31, 2017, among women undergoing surgeries for uterine and ovarian cancer. Cytotoxicity assays using IC50 values of epigenetic drugs and paclitaxel and cisplatin were performed on Caov-3. Omental adipose-derived stem cells (OASCs) were isolated from omentum with/without metastases. Caov-3 was cultured with OASCs' conditioned medium and subjected to different drugs. Cell viability and secretome was measured using MTT and Elisa, respectively. RESULTS: Three women met the eligibility criteria and were included in the study. Epigenetic drugs alone or in combination with chemotherapy showed 85%-94% increased cytotoxicity against Caov-3 (P≤0.005). Metastatic OASCs conditioned medium showed up to 27-fold increase in tumorigenic factors and promoted chemoresistance (28%-35%; P < 0.050) against chemotherapy. Epigenetic therapy resulted in up to a 40-fold reversal in this chemoresistance. CONCLUSION: Epigenetic therapies could have an important role in treating a subgroup of ovarian cancer patients that demonstrate resistance to first-line chemotherapy.

Eosinophil peroxidase oxidizes isoniazid to form the active metabolite against M. tuberculosis, isoniazid-NAD.

The formation of isonicotinyl-nicotinamide adenine dinucleotide (INH-NAD+) by the mycobacterial catalase-peroxidase enzyme, KatG, was known to be the major component of the mode of action of isoniazid (INH), an anti-tuberculosis drug. However, there are other enzymes that may catalyze this reaction. We have previously reported that neutrophil myeloperoxidase (MPO) is capable of metabolizing INH through the formation of INH-NAD+ adduct, which could be attributed to being a possible mode of action of INH. However, eosinophilic infiltration of the lungs is more pronounced and characteristic of granulomas in Mycobacterium tuberculosis-infected patients. Thus, the aim of the present study is to investigate the role of eosinophil peroxidase (EPO), a key eosinophil enzyme, during INH metabolism and the formation of its active metabolite, INH-NAD+ using purified EPO and eosinophils isolated from asthmatic donors. UV-Vis spectroscopy revealed INH oxidation by EPO led to a new product (λmax = 326 nm) in the presence of NAD+. This adduct was confirmed to be INH-NAD+ using LC-MS analysis where the intact adduct was detected (m/z = 769). Furthermore, EPO catalyzed the oxidation of INH and formed several free radical intermediates as assessed by electron paramagnetic resonance (EPR) spin-trapping; a carbon-centred radical, which is considered to be the reactive metabolite that binds with NAD+, was found when superoxide dismutase was included in the reaction. Our findings suggest that eosinophilic EPO may also play a role in the pharmacological activity of INH through the formation of INH-NAD+ adduct, and supports further evidence that human cells and enzymes are capable of producing the active metabolite involved in tuberculosis treatment.

Isoniazid induces a monocytic-like phenotype in HL-60 cells.

Isoniazid (INH) is one of the oldest drugs for the treatment of tuberculosis (TB) and is of continual clinical and research interest. The aim of the current study is to investigate the ability of INH to induce monocyte differentiation and the underlying signaling pathway involved in this phenomenon using HL-60 cells. In this study, HL-60 cells were treated with different non-cytotoxic concentrations of INH or vitamin D (a well-known inducer of monocytic differentiation) to determine key functional changes in the phenotype of these cells using several biochemical and cytobiological experiments. HL-60 cells are derived from human promyelocytic leukemia and bear some resemblance to promyelocytes, which differentiate into various cell types. INH-induced differentiation was confirmed to occur in a concentration-dependent manner through several functional markers such as nonspecific esterase activity, NADPH oxidase activity and expression of surface markers CD14 and CD16 (characteristic of monocytes). INH-induced monocytic-like differentiation in HL-60 cells and demonstrated that at least 25% of cells were differentiated within the range of the pharmacological concentrations of INH. To determine the effects of INH on HL-60 cells, we applied quantitative proteomics that revealed 32 proteins were altered significantly in pathways that could involve differentiation signals. Lastly, INH activated the ERK-1/MAPK signaling pathway based on detection of phosphorylated ERK-1. These in vitro findings in HL-60 cells warrant further study using promyelocytes or hematopoietic stem cells to evaluate the physiological capability of INH to induce monocytic differentiation that may aid in host defense against TB.

Wild Mouse Gut Microbiota Promotes Host Fitness and Improves Disease Resistance.

Laboratory mice, while paramount for understanding basic biological phenomena, are limited in modeling complex diseases of humans and other free-living mammals. Because the microbiome is a major factor in mammalian physiology, we aimed to identify a naturally evolved reference microbiome to better recapitulate physiological phenomena relevant in the natural world outside the laboratory. Among 21 distinct mouse populations worldwide, we identified a closely related wild relative to standard laboratory mouse strains. Its bacterial gut microbiome differed significantly from its laboratory mouse counterpart and was transferred to and maintained in laboratory mice over several generations. Laboratory mice reconstituted with natural microbiota exhibited reduced inflammation and increased survival following influenza virus infection and improved resistance against mutagen/inflammation-induced colorectal tumorigenesis. By demonstrating the host fitness-promoting traits of natural microbiota, our findings should enable the discovery of protective mechanisms relevant in the natural world and improve the modeling of complex diseases of free-living mammals. VIDEO ABSTRACT.

An evaluation of myeloperoxidase-mediated bio-activation of NSAIDs in promyelocytic leukemia (HL-60) cells for potential cytotoxic selectivity.

Several lines of evidence have pointed towards the potential therapeutic benefit of NSAIDs in cancer therapy. In this study, we have investigated the acute bio-activation of NSAIDs and their metabolites via myeloperoxidase (MPO), a highly-expressed peroxidase enzyme in acute myeloid leukemia. As bio-activation involves the formation of reactive metabolites, we hypothesized that NSAIDs which produced reactive metabolites would be correlated with leukemia cell toxicity. We tested the enzymatic peroxidation of three NSAIDs, namely diclofenac, indomethacin, and naproxen in comparison with their hepatic metabolites, 4'- hydroxydiclofenac (4'-OHD), 5-hydroxydiclofenac (5-OHD), O-desmethyl-N-deschlorobenzoylindomethacin (DMBI), O-desmethylindomethacin (DMI) and O-desmethylnaproxen (ODN). Firstly, we used purified peroxidases in kinetic UV-vis kinetic spectrophotometry, and electron paramagnetic resonance (EPR) experiments to determine oxidation of ascorbic acid and glutathione (GSH), respectively. We then used HL-60 cells, as a model of acute myelogenous leukemia to carry out trypan blue exclusion, cellular ATP analysis, mitochondrial membrane potential (MMP) and cytofluorometric GSH assays. Our results present evidence that diclofenac, 4'-OHD, 5-OHD, DMBI and DMI demonstrated significant cytotoxic effect in the leukemic cells through oxidation by intracellular MPO. In the same vein, only diclofenac and its two metabolites caused a significant drop in the MMP and cellular ATP level; however, the cell death induced by indomethacin metabolites reflected a subtle effect on MMP or GSH content. Interestingly, only diclofenac and 4'-OHD (and not 5- OHD) caused a significant drop in HL-60 cells' GSH content. Among diclofenac compounds, only 4'-OHD also generated GS radical and caused a significant increase in ascorbate co-oxidation rate. Lastly, even though ODN also generated GS radical and potently cooxidized ascorbate, it showed no significant cytotoxicity. These results provide evidence of a correlation between acute cytotoxicity and MPO-bioactivated NSAIDs, though this was not correlated for all compounds (e.g., ODN). Further studies are required to determine both the MPO-dependent and MPO-independent mechanisms of cytotoxicity.