Atmospheric Oxygen Conditions Research Articles

Low Oxygen Concentration Reduces Neisseria gonorrhoeae Susceptibility to Resazurin.

Neisseria gonorrhoeae has developed resistance to every antibiotic currently approved for the treatment of gonorrhea, prompting the development of new therapies. The phenoxazine dye resazurin exhibits robust antimicrobial activity against N. gonorrhoeae in vitro but fails to limit vaginal colonization by N. gonorrhoeae in a mouse model. The lack of in vivo efficacy may be due to oxygen limitation as in vitro susceptibility assays with resazurin are conducted under atmospheric oxygen while a microaerophilic environment is present in the vagina. Here, we utilized broth microdilution assays to determine the susceptibility of N. gonorrhoeae to resazurin under low and atmospheric oxygen conditions. The minimal inhibitory concentration of resazurin for multiple N. gonorrhoeae clinical isolates was significantly higher under low oxygen. This effect was specific to resazurin as N. gonorrhoeae was equally susceptible to other antibiotics under low and atmospheric oxygen conditions. The reduced susceptibility of N. gonorrhoeae to resazurin under low oxygen was largely attributed to reduced oxidative stress, as the addition of antioxidants under atmospheric oxygen mimicked the reduced susceptibility to resazurin observed under low oxygen. Together, these data suggest oxygen concentration is an important factor to consider when evaluating the efficacy of new antibiotics against N. gonorrhoeae in vitro.

Blood Clots Are Mechanically Weak in Patients with Sickle Cell Disease

Introduction: Sickle cell disease (SCD) causes a hypercoagulable state characterized by frequent thrombotic sequelae including pulmonary embolism (PE) (Connes et al. Blood. Rev. 2016). Although the coagulation cascade is well characterized in SCD, the engineering-based, biomechanical structural properties of sickle blood clots are poorly understood. Furthermore, the role of clot mechanics in both traditional thrombotic disease manifestations (in situ blood clot, PE) but also common sickle cell related sequelae (pain, stroke) is unknown. Since blood clot embolization is inherently a mechanical process, where clots embolize or “fracture” when hemodynamic forces exceed the internal “strength” of the clot (Tutwiler et al. Sci. Adv. 2020), we submit that investigating the mechanical properties of sickle cell blood clots will reveal insights into the disease-specific factors at play. We hypothesize that SCD clots are not only structurally abnormal, but that this abnormality informs an increased propensity for clot embolization and causes severe vascular complications. Methods: Under the guidance and approval of the IRB, we acquired venipuncture-derived human blood samples from 10 healthy control subjects (14.3±0.9 g/dL Hgb) and 10 patients with sickle cell disease (7 HbSS, 2 HbSC, 1 HbS beta; 10.5±2.3 g/dL Hgb). We coagulated blood into 3D-printed molds designed for uniaxial extension on our mechanical testing machine, see figure 1. To do so, we added calcium chloride to a concentration of 20mM to reverse the ACD anticoagulant and incubated samples for 60 minutes at 37°C and atmospheric oxygen conditions (Sugerman et al. Curr. Protoc. 2021). Samples were then mounted to our tensile testing machine (Instron, Norwood, MA, USA) and two tests were conducted: extension-to-failure in a simple pure shear geometry and in a mode-I fracture geometry. From this data, we calculated various mechanical metrics by quantifying the structural abnormalities and propensity for embolization of SCD blood clots. Results and Conclusion: We primarily compared the stiffness and fracture toughness of sickle cell and healthy blood clots. Stiffness measures how the blood clot deforms in response to force, while fracture toughness measures whether a blood clot is strong or weak (i.e its resistance to rupture). The measured stiffness of SCD clots is 7.27±1.68 kPa, which is significantly higher than the stiffness of healthy clots, 5.22±1.09 kPa (p < 0.001). Additionally, the blood from SCD patients makes clots with a fracture toughness of 2.91±0.85 J/m 2, which is significantly lower than the fracture toughness of healthy clots, 4.43±1.11 J/m 2 (p < 0.0001), see figure 2. The high stiffness of sickle cell blood clots suggests that they are more resistant to deformation than healthy clots. However, the low fracture toughness of sickle cell blood clots suggests that they are more likely to break or embolize when compared to healthy clots. In other words, sickle blood clots exhibit mechanical properties similar to ceramic which has high stiffness and low toughness as opposed to leather (low stiffness, high toughness) or diamond (high stiffness, high toughness). In conclusion, our biomechanical findings of higher stiffness and lower toughness in sickle blood clots are well aligned with established rheological data showing that SCD red blood cells are less deformable than healthy red blood cells (Lu et al. Exp. Biol. Med. 2020). That is, the low deformability of red blood cells in SCD is apparent even at the clot level, which may inform novel disease mechanisms. The sickle cell related determinants of these biomechanical abnormalities, their role in clinical outcomes (PE, pain and stroke) and their response to therapy is the object of future study.

Recording of Low-Oxygen Stress Response Using Chlorophyll Fluorescence Kinetics in Apple Fruit

AbstractLong-term storage of apples (Malus x domestica, Borkh.) is increasingly taking place under Dynamic Controlled Atmosphere (DCA). The oxygen level is lowered to ≤ 1 kPa O2 and the apples are stored just above the Lower Oxygen Limit (LOL). Low oxygen stress during controlled atmosphere storage can lead to fermentation in apples if oxygen levels are too low. Chlorophyll fluorescence can be used to detect low-oxygen stress at an early stage during storage. The currently available non-imaging fluorescence systems often use the minimal fluorescence (Fo) parameter. In contrast, the use of chlorophyll fluorescence kinetics is insufficiently described. Therefore, this study aimed to gain more knowledge about the response of chlorophyll fluorescence kinetics to low oxygen stress in apples using a fluorescence imaging system. The results show that the kinetic fluorescence curves differ under aerobic and fermentation conditions. The fermentative conditions initiated a decrease in fluorescence intensity upon application of the saturation pulses during exposure to actinic light. This result was made at 18 °C and 2 °C ambient temperatures. Interestingly, the kinetic curve changed at 2 °C before fermentation products accumulated in the apples. Non-photochemical quenching (NPQ) decreased under fermentation conditions in the dark phase after relaxation. Upon entering the dark relaxation phase after Kautsky induction, ɸPSII began to increase. Under atmospheric oxygen conditions, ɸPSII reached values of 0.81 to 0.76, while under fermentation, ɸPSII values ranged from 0.57 to 0.44.

Modulating tumour metabolism enhances gold nanoparticle radiosensitisation in HPV-negative head and neck cancer

BackgroundRadiotherapy is a major therapeutic modality for locally advanced head and neck cancer. However, the effectiveness of radiotherapy is hindered by resistance mechanisms, most notably hypoxia, leading to unfavourable treatment outcomes. In this study, we investigate the radiosensitising potential of AuNPs in combination with the complex III electron transport chain inhibitor, using models of head and neck cancer.ResultsAuNP intracellular accumulation occurred in a concentration-dependent manner and was not influenced by microenvironmental oxygen levels, with citrate capped 15 nm AuNPs readily internalised, accumulating primarily within the cytoplasmic compartment. Pre-treatment with atovaquone had a profound and rapid impact on oxygen consumption, promoting a glycolytic switch under both normoxic and hypoxic conditions, a finding underlined by the concurrent increase in extracellular acidification. AuNPs alone sensitised FaDu cells to radiation under atmospheric oxygen conditions, producing a sensitiser enhancement ratio (SER) of 1.37. In combination with atovaquone, maximum dose enhancements were achieved yielding a SER value of 1.43 and 2.1 under normoxic and hypoxic conditions, respectively. Studies to elucidate the underlying mechanism of radiosensitisation revealed S-phase accumulation and a significant increase in apoptosis. Additionally, combined treatment significantly increased yields of unrepaired DNA double strand breaks, indicating increased yields of DNA double strand break damage.ConclusionsTaken together, we believe this to be the first work providing evidence that AuNP radiosensitisation can be enhanced via metabolic modulation. This study reveals the dual action of both physical and biological pathways of AuNPs radiosensitisation, resulting in superior radiotherapeutic effects.

Limited access to oxygen reduces the release of harmful trace elements from submerged alum shale debris

Construction and mining activities in acid-producing alum shale regions often produce large volumes of crushed rock. Disposal under groundwater level (e.g., a bog) may minimize oxygen access. In this study, the effect of varying oxygen access on the leaching potential of alum shale was investigated by submerging tunnel construction rock debris in synthetic rainwater under atmospheric (AOC) and low oxygen conditions (LOC) for 52 weeks. The sulphate increase and nitrate decrease in the leachates suggested that pyrite (FeS2) in the alum shale was oxidized, but carbonates originating from calcite dissolution provided sufficient buffering capacity (leachate pH ~7.7 over 52 weeks), resulting in neutral rock drainage. Less available oxygen led to significantly lower production of sulphate and acid from pyrite oxidation, reducing the release of harmful elements. Under LOC, the leaching of Mo, Co, Ni, Zn and Cd was 2–4 times lower than under AOC and the lower buffering requirement diminished the release of Ca as well as divalent cations (Mg, Sr, Mn) likely present as impurities in calcite. Contrastingly, limited pyrite oxidation led to less oversaturation with respect to BaSO4 and lower release of Fe in the LOC leachates. Thus, co-precipitation of 226Ra was inhibited and scavenging of leached V, As and Sb by newly formed Fe(OH)3 was not as dominant as in the AOC systems. Leaching of U was ~20 % higher under LOC likely due to enhanced complexation by dissolved carbonate. In general, element leaching rates were slower under low O2 levels. Characterization of water collected at the disposal site after ~1.2 years of discarding tunnel materials showed that the weathering of debris submerged in the open, water-filled pond occurred similarly to leaching under low oxygen conditions. Overall, these results highlight the importance of minimal oxygen access or anaerobic conditions when acid-producing rock waste is stored under water.

P-230 Senescence in mammalian embryos as a novel assay for quality control in the IVF laboratory

Abstract Study question Can senescence biomarkers be used as a more sensitive assay for IVF laboratory quality control than a conventional mouse embryo assay (MEA)? Summary answer Senescence biomarkers are more sensitive than a MEA and should be considered as a novel quality control assay in assessing overall IVF laboratory environment. What is known already An MEA is a bioassay that is utilized to detect toxicity and suboptimal conditions, but only assesses the ability of the embryos to develop to the blastocyst stage. The majority of human embryos created during IVF do not result in a live birth, most either arrest or degenerate. Suboptimal culture conditions and accompanying environmental stressors may be responsible or some of this lack of embryo development. Senescence or developmental arrest, like apoptosis, is a defensive mechanism that halts further development of stress, poor quality or abnormal embryos. Senescence should be considered a possible reason for a high embryos loss rate. Study design, size, duration Murine zygotes from the strain B6C3F1 x B6D2F1 (N = 263) were exposed to 12.5nM Doxorubicin (DOX), a known inducer of senescence as a positive control, either 250nM or 500nM H2O2 or atmospheric oxygen conditions (20%) to mimic oxidative stress. These treatment groups were compared to embryos cultured without H2O2 in a low oxygen (5% 02) environment. The zygotes were cultured for 72 hours, and biomarkers of senescence were assessed. Participants/materials, setting, methods Embryos were assessed at 72 hours with a confocal microscope (Zeiss) and blastocyst conversion rate and number of cells/blastocyst was calculated. The following senescence markers were examined, SAβ-gal (SPiDER-β-Gal), IL-6 mRNA (RT-qPCR) and CellRox Deep Red, a fluorogenic reactive oxygen species (ROS) indicator was also evaluated. Fiji Image J software was used to calculate Corrected Total Cell Fluorescence (CTCF) as a percentage of the low oxygen group and GraphPad Prism was used for data analysis. Main results and the role of chance When comparing the blastocyst conversion rate, all treatment groups showed a blastocyst conversion rate of > 80% after 72 hours of exposure, indicating that all would have “passed” assessment with a conventional MEA (p > 0.05). However, when the average number of cells per blastocyst was compared to the low oxygen group, all groups had a significantly lower average number of cells per blastocyst (112±26.65 for low oxygen, 74±7.95 for high oxygen, 82± 24.54 for 500 nM H2O2, 89 ± 34 for 250 nM H2O2 and 63± 20.01 for 12.5 nM DOX, p < 0.05). All of the treatment groups had a significant increase in the relative fold change of IL-6 mRNA compared to the low oxygen environment (2.2 for the high oxygen, 2.5 for 500 nM H2O2, 2.9 for 250 nM H2O2, and 7.4 for 12.5 nM DOX, p < 0.05). The CTCF for SAβ-Gal using the SPIDER-β- Gal assay was significantly elevated in all treatment groups (145% for high oxygen, 162% for H2O2250 nM, 193% for 12.5 nM DOX and 196% for 500 nM H2O2, p < 0.05). A significant increase in the CTCF for CellRox Deep Red was also observed (289% for 12.5 nM DOX, 492% for 250 nM H2O2, 763% for 500 nM H2O2, and 925% for high oxygen, p < 0.05). Limitations, reasons for caution This analysis is currently limited by the lack of a specific senescence marker. This approach is also invasive, destroying the embryo, not lending itself to clinical human embryo selection. Although the mouse model has advantages, translating these results into human embryos is a limitation of this study. Wider implications of the findings This optimized novel assay can be used to provide great insight about the subtle laboratory culture/environmental conditions in the IVF laboratory. The results support the hypothesis that environmental stressors can induce senescence biomarkers and implicate their participation in the degeneration of previously viable embryos. Trial registration number not applicable

Oxygen as an important factor modulating in vitro MeHgCl toxicity associated with mitochondrial genes in hiPSCs

Mitochondria are energy factories of cells and important targets for methylmercury chloride (MgHgCl). Methylmercury (MeHg) is a well-known environmental toxicant that bioaccumulates in fish and shellfish. It readily crosses the placental barrier, making it a threat to correct fetal development. Despite being comprehensively investigated for years, this compound has not been assessed for its in vitro mitochondrial toxicity under different oxygen conditions. In this study, human induced pluripotent stem cells (hiPSCs) were used to evaluate the dependence of the expression of genes associated with pluripotency and mitochondria on atmospheric (21% O2) and low (5% O2) oxygen concentrations upon MeHgCl treatment. We showed that the toxicity of MeHgCl was strongly related to an increased mtDNA copy number and downregulation of the expression of an mtDNA replication and damage repair-associated gene POLG1 (Mitochondrial Polymerase Gamma Catalytic Subunit) in both tested oxygen conditions. In addition, the viability and mitochondrial membrane potential of hiPSCs were significantly lowered by MeHgCl regardless of the oxygen concentration. However, reactive oxygen species accumulation significantly increased only under atmospheric oxygen conditions; what was associated with increased expression of TFAM (Transcription Factor A, Mitochondrial) and NRF1 (Nuclear Respiratory Factor 1) and downregulation of PARK2 (Parkin RBR E3 Ubiquitin Protein Ligase). Taken together, our results demonstrated that MeHgCl could induce in vitro toxicity in hiPSCs through altering mitochondria-associated genes in an oxygen level-dependent manner. Thus, our work suggests that oxygen should be considered a factor was modulating the in vitro toxicity of environmental pollutants. Typical atmospheric conditions of in vitro culture significantly lower the predictive value of studies of such toxicity.

Lycopene Supplementation to Serum-Free Maturation Medium Improves In Vitro Bovine Embryo Development and Quality and Modulates Embryonic Transcriptomic Profile.

Bovine embryos are typically cultured at reduced oxygen tension to lower the impact of oxidative stress on embryo development. However, oocyte in vitro maturation (IVM) is performed at atmospheric oxygen tension since low oxygen during maturation has a negative impact on oocyte developmental competence. Lycopene, a carotenoid, acts as a powerful antioxidant and may protect the oocyte against oxidative stress during maturation at atmospheric oxygen conditions. Here, we assessed the effect of adding 0.2 μM lycopene (antioxidant), 5 μM menadione (pro-oxidant), and their combination on the generation of reactive oxygen species (ROS) in matured oocytes and the subsequent development, quality, and transcriptome of the blastocysts in a bovine in vitro model. ROS fluorescent intensity in matured oocytes was significantly lower in the lycopene group, and the resulting embryos showed a significantly higher blastocyst rate on day 8 and a lower apoptotic cell ratio than all other groups. Transcriptomic analysis disclosed a total of 296 differentially expressed genes (Benjamini–Hochberg-adjusted p < 0.05 and ≥ 1-log2-fold change) between the lycopene and control groups, where pathways associated with cellular function, metabolism, DNA repair, and anti-apoptosis were upregulated in the lycopene group. Lycopene supplementation to serum-free maturation medium neutralized excess ROS during maturation, enhanced blastocyst development and quality, and modulated the transcriptomic landscape.

Characterization of Oxygen Levels in an Uninfected and Infected Human Blood-Cerebrospinal-Fluid-Barrier Model.

The host–pathogen interaction during meningitis can be investigated with blood-cerebrospinal-fluid-barrier (BCSFB) cell culture models. They are commonly handled under atmospheric oxygen conditions (19–21% O2), although the physiological oxygen conditions are significantly lower in cerebrospinal fluid (CSF) (7–8% O2). We aimed to characterize oxygen levels in a Streptococcus (S.) suis-infected BCSFB model with transmigrating neutrophils. A BCSFB model with human choroid plexus epithelial cells growing on transwell-filters was used. The upper “blood”-compartment was infected and blood-derived neutrophils were added. S. suis and neutrophils transmigrated through the BCSFB into the “CSF”-compartment. Here, oxygen and pH values were determined with the non-invasive SensorDish® reader. Slight orbital shaking improved the luminescence-based measurement technique for detecting free oxygen. In the non-infected BCSFB model, an oxygen value of 7% O2 was determined. However, with S. suis and transmigrating neutrophils, the oxygen value significantly decreased to 2% O2. The pH level decreased slightly in all groups. In conclusion, we characterized oxygen levels in the BCSFB model and demonstrated the oxygen consumption by cells and bacteria. Oxygen values in the non-infected BCSFB model are comparable to in vivo values determined in pigs in the CSF. Infection and transmigrating neutrophils decrease the oxygen value to lower values.

Hypoxia represses early responses of prostate and renal cancer cells to YM155 independent of HIF-1α and HIF-2α

The imidazolium compound Sepantronium Bromide (YM155) successfully promotes tumor regression in various pre-clinical models but has shown modest responses in human clinical trials. We provide evidence to support that the hypoxic milieu of tumors may limit the clinical usefulness of YM155. Hypoxia (1% O2) strongly (>16-fold) represses the cytotoxic activity of YM155 on prostate and renal cancer cells in vitro. Hypoxia also represses all early signaling responses associated with YM155, including activation of AMPK and retinoblastoma protein (Rb), inactivation of the mechanistic target of rapamycin complex 1 (mTORC1), inhibition of phospho-ribosomal protein S6 (rS6), and suppression of the expression of Cyclin Ds, Mcl-1 and Survivin. Cells pre-incubated with hypoxia for 24 ​h are desensitized to YM155 even when they are treated with YM155 under atmospheric oxygen conditions, supporting that cells at least temporarily retain hypoxia-induced resistance to YM155. We tested the role of hypoxia-inducible factor (HIF)-1α and HIF-2α in the hypoxia-induced resistance to YM155 by comparing responses of YM155 in VHL-proficient versus VHL-deficient RCC4 and 786-O renal cancer cells and silencing HIF expression in PC-3 prostate cancer cells. Those studies suggested that hypoxia-induced resistance to YM155 occurs independent of HIF-1α and HIF-2α. Moreover, the hypoxia mimetics deferoxamine and dimethyloxalylglycine, which robustly induce HIF-1α levels in PC-3 ​cells under atmospheric oxygen, did not diminish their early cellular responses to YM155. Collectively, our data support that hypoxia induces resistance of cells to YM155 through a HIF-1α and HIF-2α-independent mechanism. We hypothesize that a hypothetical hypoxia-inducer factor (HIF-X) represses early signaling responses to YM155.

Gaseous environment modulates volatile emission and viability loss during seed artificial ageing

Main conclusionModulation of the gaseous environment using oxygen absorbers and/or silica gel shows potential for enhancing seed longevity through trapping toxic volatiles emitted by seeds during artificial ageing.Volatile profiling using non-invasive gas chromatography–mass spectrometry provides insight into the specific processes occurring during seed ageing. Production of alcohols, aldehydes and ketones, derived from processes such as alcoholic fermentation, lipid peroxidation and Maillard reactions, are known to be dependent on storage temperature and relative humidity, but little is known about the potential modulating role of the gaseous environment, which also affects seed lifespan, on volatile production. Seeds of Lolium perenne (Poaceae), Agrostemma githago (Caryophyllaceae) and Pisum sativum (Fabaceae) were aged under normal atmospheric oxygen conditions and in sealed vials containing either oxygen absorbers, oxygen absorbers and silica gel (equilibrated at 60% RH), or silica gel alone. Seeds of A. githago that were aged in the absence of oxygen maintained higher viability and produced fewer volatiles than seeds aged in air. In addition, seeds of A. githago and L. perenne aged in the presence of silica gel were longer lived than those aged without silica, with no effect on seed moisture content or oxygen concentration in the storage containers, but with silica gel acting as a volatile trap. These results indicate that the use of inexpensive oxygen absorbers and silica gel could improve seed longevity in storage for some species and suggests a potential, and previously unidentified, role for silica gel in ultra-dry storage.

Assessment of the Neuroprotective and Stemness Properties of Human Wharton's Jelly-Derived Mesenchymal Stem Cells under Variable (5% vs. 21%) Aerobic Conditions.

To optimise the culture conditions for human Wharton’s jelly-derived mesenchymal stem cells (hWJ-MSCs) intended for clinical use, we investigated ten different properties of these cells cultured under 21% (atmospheric) and 5% (physiological normoxia) oxygen concentrations. The obtained results indicate that 5% O2 has beneficial effects on the proliferation rate, clonogenicity, and slowdown of senescence of hWJ-MSCs; however, the oxygen level did not have an influence on the cell morphology, immunophenotype, or neuroprotective effect of the hWJ-MSCs. Nonetheless, the potential to differentiate into adipocytes, osteocytes, and chondrocytes was comparable under both oxygen conditions. However, spontaneous differentiation of hWJ-MSCs into neuronal lineages was observed and enhanced under atmospheric oxygen conditions. The cells relied more on mitochondrial respiration than glycolysis, regardless of the oxygen conditions. Based on these results, we can conclude that hWJ-MSCs could be effectively cultured and prepared under both oxygen conditions for cell-based therapy. However, the 5% oxygen level seemed to create a more balanced and appropriate environment for hWJ-MSCs.

A novel high-content screening approach for the elucidation of C. jejuni biofilm composition and integrity

BackgroundCampylobacter jejuni is the leading cause of bacterial gastroenteritis worldwide and the main source of infection is contaminated chicken meat. Although this important human pathogen is an obligate microaerophile, it must survive atmospheric oxygen conditions to allow transmission from contaminated chicken meat to humans. It is becoming increasingly evident that formation of biofilm plays a key role in the survival of this organism for extended periods on poultry products. We have recently demonstrated a novel inducible model for the study of adherent C. jejuni biofilm formation under aerobic conditions. By taking advantage of supercoiling mediated gene regulation, incubation of C. jejuni with subinhibitory concentrations of the Gyrase B inhibitor novobiocin was shown to promote the consistent formation of metabolically active adherent biofilm.ResultsIn this study, we implement this model in conjunction with the fluorescent markers: TAMRA (live cells) and SytoX (dead cells, eDNA) to develop a novel systematic high-content imaging approach and describe how it can be implemented to gain quantifiable information about the integrity and extracellular polymeric substance (EPS) composition of adherent C. jejuni biofilm in aerobic conditions. We show that this produces a model with a consistent, homogenous biofilm that can be induced and used to screen a range of inhibitors of biofilm adherence and matrix formation.ConclusionsThis model allows for the first time a high throughput analysis of C. jejuni biofilms which will be invaluable in enabling researchers to develop mechanisms to disrupt these biofilms and reduce the viability of these bacteria under aerobic conditions.

Physioxia Stimulates Extracellular Matrix Deposition and Increases Mechanical Properties of Human Chondrocyte-Derived Tissue-Engineered Cartilage.

Cartilage tissue has been recalcitrant to tissue engineering approaches. In this study, human chondrocytes were formed into self-assembled cartilage sheets, cultured in physiologic (5%) and atmospheric (20%) oxygen conditions and underwent biochemical, histological and biomechanical analysis at 1- and 2-months. The results indicated that sheets formed at physiological oxygen tension were thicker, contained greater amounts of glycosaminoglycans (GAGs) and type II collagen, and had greater compressive and tensile properties than those cultured in atmospheric oxygen. In all cases, cartilage sheets stained throughout for extracellular matrix components. Type II-IX-XI collagen heteropolymer formed in the neo-cartilage and fibrils were stabilized by trivalent pyridinoline cross-links. Collagen cross-links were not significantly affected by oxygen tension but increased with time in culture. Physiological oxygen tension and longer culture periods both served to increase extracellular matrix components. The foremost correlation was found between compressive stiffness and the GAG to collagen ratio.

Wildfire during deposition of the \u201cIllinger Fl\xf6zzone\u201d (Heusweiler-Formation, \u201cStephanian B\u201d, Kasimovian\u2013Ghzelian) in the Saar-Nahe Basin (SW-Germany)

Wildfires occurred more or less regularly in many Pennsylvanian ecosystems, not only in seasonally dry regions but also in the ever wet tropics. One of the reasons for this was probably the relatively high atmospheric oxygen conditions prevailing during this period. The present study reports evidence for the occurrence of wildfires during deposition of the Upper Pennsylvanian Heusweiler Formation (“Stephanian B”, Kasimovian–Gzhelian) in the intramontane Saar-Nahe (or Saar-Lorraine) Basin in SW-Germany. Based on anatomical features of the charcoal, as well as the co-occurring adpression flora, it seems possible that some of the fires occurred in an ecosystem inhabited by Cordaites. Some of the charcoal fragments exhibit traces of pre-charring decay by fungi, indicating either the consumption of litter by ground or surface fires, or of still standing (partly) dead trees by crown fires.

Susceptibility of Neisseria gonorrhoeae to Resazomycins in Varying Oxygen Concentrations

Antibiotic resistance is a growing issue globally as many pathogenic bacterial species are becoming less susceptible to common antibiotic treatments. Antibiotic resistant pathogens are the cause of 23,000 deaths in the United States each year and increasing health care costs. It is imperative that new antibiotics be developed to combat this public health crisis. We recentlyidentified a novel family of resazurin-based compounds, resazomycins, to have antimicrobial activity in vitro against Neisseria gonorrhoeae, a human pathogen. Resazomycins are capable of killing N. gonorrhoeae in broth culture as well as within human endometrial cells, HEC-1-B. However, when different resazomycins were tested in a mouse model of gonorrhea, none of the compounds were able to completely clear the infection. The difference between in vitro and invivo antibacterial efficacy may be due to differences in oxygen concentration. Atmospheric oxygen levels are typically around 20% while the oxygen concentration of most mammalian tissues is around 3-5%. We hypothesize that the decreased oxygen concentration in vivo may affect the antimicrobial activity of resazomycins. To test this hypothesis, the susceptibility of different clinical isolates of N. gonorrhoeae to resazurin was measured under atmospheric (20%)and physiologic (2-3%) oxygen conditions. To date, certain strains of N. gonorrhoeae showed more growth following exposure to resazomycins under physiologic oxygen than atmospheric oxygen. This suggests N. gonorrhoeae is more resistant to resazomycins under low oxygen conditions. Further experimentation is needed to determine why certain N. gonorrhoeae strainsare showing less susceptibility to resazomycins under physiologic oxygen conditions.

Generation and characterization of human emryonic stem cells with increased expression of HIF-2a

The HIF-2 a subunit is involved in regulation of transcription factors, controlling the self-renewal of human pluripotent stem cells, embryonic development of the cardiovascular system and the regulation of angiogenesis by transcriptional activation of angiogenic cascades in physiological and pathological processes. Currently, modulation of HIF-2a expression is considered as a promising strategy for the treatment of ischemic and cancer diseases. However, the problem of choosing the optimal methods of effective regulation of HIF-2a remains. The aim of this study is to obtain human embryonic stem cells with increased expression of HIF-2a at normal oxygen concentration due to silencing of INT6, the regulator of HIF-2a. In this study, we obtained genetically modified human embryonic stem cells with increased expression of HIF-2a under atmospheric oxygen conditions. The approach used is based on a CRISPR/Cas9-mediated deletion of a part of the INT6 gene, an HIF-2a inhibitor. A study of the resulting genetically modified human embryonic stem cells will contribute to an understanding of the connection between hypoxia and pluripotency. Obtaining endothelial derivatives of pluripotent stem cells with increased expression of HIF-2a and enhanced regenerative potential may become the basis for the development of promising strategies for treatment of ischemic diseases.

Solvation interactions and photostability of tetrakis(1-methylpyridyl)porphyrin derivatives

In this paper, a series of water-soluble meso-tetrakis(1′-methyl-pyrid-4- and -3-yl)porphyrin tetratosylate and meso-tetrakis(1′-methyl-pyrid-4- and -3-yl)porphyrin tetraiodide were synthesized with high yields. All synthesized compounds were characterized by 1Н NMR, FT-IR, UV–vis spectroscopy and elemental analysis. The enthalpy changes of meso-tetrakis(1′-methyl-pyrid-4- and 3-yl)porphyrin tetratosylates dissolution in water, alcohol and phosphate buffer (pH = 6.86) were determined by the calorimetric method at 298.15 K. The effect of peripheral substituents and anion of the porphyrin molecule on the enthalpy of solvation have been studied by calorimetry and UV–vis spectroscopy.To understand more clearly the nature of changes in the meso-substituents and anion of a porphyrin molecule, we have also synthesized N-methyl-pyridinium salts (1-methyl-pyridium tosylate and 1-methyl-pyridium iodide) model. The processes of their dissolution under the same solution conditions have been investigated. The nature of the anion and isomerism of the pyridylporphyrin substituent significantly affects the thermochemical characteristics of the compounds transition into solution. In particular, porphyrin-tosylates were found to be solvated several times better than its iodide analogs.Stability to photooxidative degradation is one of the key characteristics of chromophore and characterizes the kinetic stability of a compound in solution under UV-irradiation influence in atmospheric oxygen conditions. In this regard, the stability to photooxidative destruction of porphyrinic salt derivatives has been also measured in aqueous solutions. The results indicated that N-methyl-pyridylporphyrins were stable and slowly degrade under the influence of light radiation in aqueous solutions. It follows from the observed rate constant values of porphyrins (kobs) photooxidative destruction that 4N-substituted porphyrins are much more stable than their 3N-substituted analogs. A similar dependence was observed for the half-flow time of the studied compounds.

Metabolomic and Transcriptional Analyses Reveal Atmospheric Oxygen During Human Induced Pluripotent Stem Cell Generation Impairs Metabolic Reprogramming.

The transition to pluripotency invokes profound metabolic restructuring; however, reprogramming is accompanied by the retention of somatic cell metabolic and epigenetic memory. Modulation of metabolism during reprogramming has been shown to improve reprogramming efficiency, yet it is not known how metabolite availability during reprogramming affects the physiology of resultant induced pluripotent stem cells (iPSCs). Metabolic analyses of iPSCs generated under either physiological (5%; P-iPSC) or atmospheric (20%; A-iPSC) oxygen conditions revealed that they retained aspects of somatic cell metabolic memory and failed to regulate carbohydrate metabolism with A-iPSC acquiring different metabolic characteristics. A-iPSC exhibited a higher mitochondrial membrane potential and were unable to modulate oxidative metabolism in response to oxygen challenge, contrasting with P-iPSC. RNA-seq analysis highlighted that A-iPSC displayed transcriptomic instability and a reduction in telomere length. Consequently, inappropriate modulation of metabolism by atmospheric oxygen during reprogramming significantly impacts the resultant A-iPSC metabolic and transcriptional landscape. Furthermore, retention of partial somatic metabolic memory in P-iPSC derived under physiological oxygen suggests that metabolic reprogramming remains incomplete. As the metabolome is a regulator of the epigenome, these observed perturbations of iPSC metabolism will plausibly have downstream effects on cellular function and physiology, both during and following differentiation, and highlight the need to optimize nutrient availability during the reprogramming process. Stem Cells 2019;37:1042-1056.

Effect of Oxygen Concentration on Susceptibility of Neisseria Species to Resazomycins

A growing number of human pathogens are becoming resistant to most common antibiotics. Each year, antibiotic-resistant infections are responsible for 23,000 deaths in the United States and billions of dollars in health care costs. To prevent the loss of additional lives from once “curable” diseases, new antibiotics must be developed. We recently identified a novel family of resazurin-based compounds, resazomycins, which exhibit antimicrobial activity against various Neisseria species including the human pathogen N. gonorrhoeae in vitro. However, when these compounds were tested in a mouse model of gonorrhea, few resazomycins exhibited any therapeutic effect. These differences in in vitro and in vivo therapeutic efficacy may be due to differences in oxygen concentration. Most mammalian tissues exist at oxygen concentrations well below atmospheric levels, typically 3-5% instead of 20%. Therefore, we hypothesized that decreased oxygen levels may confer resistance to resazomycins. To test this hypothesis, we cultivated the avirulent Neisseria strain, N.sicca, under atmospheric (20%) and physiologic (2-3%) oxygen conditions and then measured the susceptibility of the bacteria to various resazomycins. To date, more N. sicca growth was observed following exposure to resazurin and resorufin under physiologic oxygen than atmospheric oxygen suggesting oxygen levels effect susceptibility to resazomycins. Further investigation is needed to determine the precise mechanism by which N. sicca is becoming resistant to resazomycins under low oxygen conditions.