Oxygen Stress Research Articles

Comparison of effects of dexmedetomidine and amifostine against X-ray radiation-induced parotid damage.

Radiotherapy can be employed as a therapeutic modality alone in the early stages of cancer and is used together with other treatments such as surgery and chemotherapy in more advanced stages. However, exposure to ionizing radiation in association with radiotherapy affects several organs in the head and neck and can give rise to early and late side effects. Exposure to ionizing radiation used in radiotherapy is known to cause cell damage by leading to oxygen stress through the production of free oxygen radicals (such as superoxide radicals, hydroxyl radical, hydrogen peroxide, and singlet oxygen), depending on the total radiation dosage, the fractionation rate, radiosensitivity, and linear energy transfer. The purpose of the present study was to determine the potential protective role of a powerful and highly selective α2-adrenoreceptor agonist with a broad pharmacological spectrum against salivary gland damage induced by ionizing radiation exposure. Forty Sprague-Dawley rats were divided into five groups-control, ionizing radiation, ionizing radiation + dexmedetomidine (100µg/kg), ionizing radiation + dexmedetomidine (200µg/kg), and ionizing radiation + amifostine (200mg/kg). Following exposure to ionizing radiation, we observed necrosis, fibrosis, and vascular congestions in parotid gland epithelial cells. We also observed increases in malondialdehyde (MDA) and cleaved Caspase-3 levels and a decrease in glutathione (GSH). In groups receiving dexmedetomidine, we observed necrotic epithelial cells, fibrosis and vascular congestion in parotid gland tissue, a decrease in MDA levels, and an increase in GSH. Dexmedetomidine may be a promising antioxidant agent for the prevention of oxidative damage following radiation exposure.

Comparative transcriptomic analysis reveals potential mechanisms for high tolerance to submergence in arbor willows.

BackgroundSubmergence threatens plant growth and survival by decreasing or eliminating oxygen supply. Uncovering the complex regulatory network underlying the tolerance of Salix to submergence and identifying the key regulators are important for molecular-assisted breeding of Salix.MethodsIn this study, we screened germplasm resources of arbor willows and discovered both submergence-tolerant and submergence-sensitive varieties. Then, by performing RNA-seq, we compared the differences between the transcriptomes of two varieties, i.e., the submergence-tolerant variety “Suliu 795” and the submergence-sensitive variety “Yanliu No. 1,” and the different submergence treatment time points to identify the potential mechanisms of submergence in Salix and the unique approaches by which the variety “Suliu 795” possessed a higher tolerance compared to “Yanliu No. 1”.ResultsA total of 22,790 differentially expressed genes were identified from 25 comparisons. Using gene ontology annotation and pathway enrichment analysis, the expression pattern of transcriptional factors, important players in hormone signaling, carbohydrate metabolism, and the anaerobic respiration pathway were found to differ significantly between the two varieties. The principal component analysis and qRT-PCR results verified the reliability of the RNA sequencing data. The results of further analysis indicated that “Suliu 795” had higher submergence tolerant activity than “Yanliu No. 1” because of three characteristics: (1) high sensitivity to the probable low oxygen stress and initiation of appropriate responding mechanisms in advance; (2) maintenance of energy homeostasis to prevent energy depletion under hypoxic stress; and (3) keep “quiescence” through fine-tuning the equilibrium between phytohormones GA, SA and ethylene.

Ferulic acid protects renal tubular epithelial cells against anoxia/reoxygenation injury mediated by AMPKα1

Anoxia/reoxygenation (A/R) injury causes dysfunction of rat renal tubular epithelial cells (NRK-52E), which is associated with excess reactive oxygen species (ROS) generation and eventually leads to apoptosis. Ferulic acid (FA), a phenolic acid, which is abundant in fruits and vegetables. FA possesses the properties of scavenging free radicals and cytoprotection against oxygen stress. In the study, the protective effects of FA against NRK-52E cells damage induced by A/R were explored and confirmed the role of AMP-activated protein kinaseα1 (AMPKα1). We found that after NRK-52E cells suffered A/R damage, FA pretreatment increased the cell viability and decreased LDH activity in culture medium in a concentration-dependent manner, the activities of endogenous antioxidant enzymes such as glutathione peroxidase, superoxide dismutase and catalase improved, intracellular ROS generation and malondialdehyde contents mitigated. In addition, pretreatment of 75 μM FA ameliorated mitochondrial dysfunction by A/R-injury and ultimately decreased apoptosis (25.3 ± 0.61 vs 12.1 ± 0.60), which was evidenced by preventing the release of cytochrome c from mitochondria to the cytoplasm. 75 μM FA pretreatment also significantly upregulated AMPKα1 expression (3.16 ± 0.18 folds) and phosphorylation (2.56 ± 0.13 folds). However, compound C, a specific AMPK inhibitor, significantly attenuated FA pretreatment’s effects, as mentionedabove. These results firstly clarified that FA pretreatment attenuated NRK-52E cell damage induced by A/R via upregulating AMPKα1 expression and phosphorylation.

Extreme altitude induces divergent mass reduction of right and left ventricle in mountain climbers.

Mountain climbing at high altitude implies exposure to low levels of oxygen, low temperature, wind, physical and psychological stress, and nutritional insufficiencies. We examined whether right ventricular (RV) and left ventricular (LV) myocardial masses were reversibly altered by exposure to extreme altitude. Magnetic resonance imaging and echocardiography of the heart, dual x‐ray absorptiometry scan of body composition, and blood samples were obtained from ten mountain climbers before departure to Mount Everest or Dhaulagiri (baseline), 13.5 ± 1.5 days after peaking the mountain (post‐hypoxia), and six weeks and six months after expeditions exceeding 8000 meters above sea level. RV mass was unaltered after extreme altitude, in contrast to a reduction in LV mass by 11.8 ± 3.4 g post‐hypoxia (p = 0.001). The reduction in LV mass correlated with a reduction in skeletal muscle mass. After six weeks, LV myocardial mass was restored to baseline values. Extreme altitude induced a reduction in LV end‐diastolic volume (20.8 ± 7.7 ml, p = 0.011) and reduced E’, indicating diastolic dysfunction, which were restored after six weeks follow‐up. Elevated circulating interleukin‐18 after extreme altitude compared to follow‐up levels, might have contributed to reduced muscle mass and diastolic dysfunction. In conclusion, the mass of the RV, possibly exposed to elevated afterload, was not changed after extreme altitude, whereas LV mass was reduced. The reduction in LV mass correlated with reduced skeletal muscle mass, indicating a common denominator, and elevated circulating interleukin‐18 might be a mechanism for reduced muscle mass after extreme altitude.

Bioremediation of Heavy Metals from Crude 0il Polluted Soil Using Serratia marcescens and its Toxicology Assessment on Aquaculture

Serratia marcescens has been studied to show a higher degradation potential for organo-phosphorus pesticides. This property has made it useful as a potential bioremediation agent. The bacterial cell was bio-stimulated in luria beterni broth at a selected experimental temperature ranging from 25 ºC – 40 ºC. The highest cell biomass was recorded at 35 ºC to range from 7.56 -8.10mg/ml for single strength and double strength yeast extract in luria beterni, respectively. Percentage bio-sorption efficiency of both the cell culture and extracted membrane protein augmented cell culture of Serratia marcescens showed 100% removal in heavy metals such as lead, potassium and manganese, respectively. The cell culture percentage sorption also showed 99.2%, 95.9% and 82% removal of copper, nickel and zinc, respectively, while the protein extract augmented cell culture showed improved corresponding heavy metal removal with 100% removal of copper and nickel and 86.5% removal of zinc. The bacterial cell and corresponding protein extracts tend to create oxygen stress in an aquaculture model, reducing the dissolved oxygen level to 3 mg/l. Lethal dose 50 of the bacterial extracts were high a 14.14%; hence this showed high toxicity or pathogenicity of the bacteria. This showed that the bacterial cell and corresponding proteins extract showed promising in the removal of heavy metals in an aquaculture environment; however, its toxicity effect on the test fishes makes it a potential risk to aquatic life.

Haemin deprivation renders Bacteroides fragilis hypersusceptible to metronidazole and cancels high-level metronidazole resistance.

BackgroundInfections with Bacteroides fragilis are routinely treated with metronidazole, a 5-nitroimidazole antibiotic that is active against most anaerobic microorganisms. Metronidazole has remained a reliable treatment option, but resistance does occur, including in B. fragilis.ObjectivesIn this study we tested whether haemin, a growth supplement for B. fragilis in vivo and in vitro, had an influence on the susceptibility of resistant B. fragilis strains to metronidazole. We further tested whether haemin-deprived B. fragilis would be more susceptible to oxygen and oxidative stress. Metronidazole has been described to cause oxidative stress, which we argued would be exacerbated in haemin-deprived B. fragilis because the bacteria harness haemin, and the iron released from it, in antioxidant enzymes such as catalase and superoxide dismutase.MethodsHaemin was omitted from growth media and the effect on metronidazole susceptibility was monitored in susceptible and resistant B. fragilis strains. Further, haemin-deprived B. fragilis were tested for resistance to aeration and hydrogen peroxide and the capacity for the removal of oxygen.ResultsOmission of haemin from the growth medium rendered metronidazole-resistant B. fragilis strains, including an MDR isolate from the UK, highly susceptible to metronidazole. Haemin deprivation further rendered B. fragilis highly susceptible to oxygen, which was further exacerbated in resistant strains. B. fragilis was incapable of scavenging oxygen when haemin was omitted.ConclusionsWe propose that haemin deprivation overrules resistance mechanisms by rendering B. fragilis hypersusceptible to metronidazole due to a compromised antioxidant defence. Monitoring of haemin concentrations is imperative when conducting metronidazole susceptibility testing in B. fragilis.

Hepatoprotective activity of andrographolide possibly through antioxidative defense mechanism in Sprague-Dawley rats

The aims of this study to assess the efficiency of AGL against acetaminophen (APAP)-induced hepatic toxicity that was generated by mitochondrial oxidative stress and glutathione depletion. Free radical scavenging potentiality was analyzed by using 2, 2-diphenyl-1-picrylhydrazyl (DPPH), hydrogen peroxide, nitric oxide, and hydroxyl radical scavenging assays. APAP-induced liver toxicity was formed at a dose level of 640 mg/kg mg/kg BW each, p.o. for 14 days for all experimental rats except the vehicle control group. AGL (5 and 10 mg/kg) were treated orally with negative control and negative control silymarin (50 mg/kg) group. To assess the protective effect, we looked at the levels of serum biochemical markers, liver histoarchitecture, and hepatic antioxidant enzyme activity. AGL showed in vitro anti-oxidant potentialities by scavenging radicals in the respective assays. As evidenced by serum biochemical indicators and relative liver weight, AGL co-administration substantially reduced toxicant-induced hepatic damage. APAP-intoxication increased the malondialdehyde (MDA) level and declined in cellular endogenous antioxidant enzymes such as reduced catalase, superoxide dismutase, and glutathione, where, AGL treatment amended their level. In the same way, histopathological evaluation further verified that AGL protected the hepatocyte from APAP-induced damage. As AGL scavenges toxic free radicals, thereby protects mitochondria and other organelles from reactive oxygen and nitrogen species-mediated stress and its eventual consequence necrosis. Therefore, we propose the hepatoprotective activity of AGL through its antioxidant mechanism.

Influence of the addition of microcrystalline cellulose (Mcc) on structural properties of cellulose nanofibrils (NFC) suspensions and films

Background: In the study of biologically-based materials, nanocelluloses have been showing great prominence and positioned themselves as promising alternatives for the production of different industrialized materials. This polymer has received significant attention recently because it is produced from renewable sources and has unique properties offered by its organic nature and semi-crystalline structure. This work aimed to study the structural properties of suspensions and films by increasing MCC concentration in the form of powder with variations of 5 % (m/m) from 5 % to 30 %. Results: As expected, incorporating MCC increased the Segal index. The morphological analysis showed an increase in the diameters of the structures (NFC / MCC) in the suspensions when the presence of MCC was more significant, and films with cluster formations were observed. The films showed air permeability. Due to the MCC increase, the surface charge had results close to electrostatically stabilized nanosuspensions. An increase in the resistance to thermal degradation of the films was also observed Conclusion: NFC has promising properties for different applications; it provides a film with a stable structure and is resistant to oxygen and tensile stresses. In addition, MCC has excellent potential due to its high crystallinity, structural characteristics, and nature. The increase of the MCC content altered the properties of the suspensions and films produced with NFC, forming a cohesive and resistant film, and influencing the performance of the different properties of the materials evaluated in this study, like air permeability, suspension stability, and thermal resistance.

Effects of bottom trawling and hypoxia on benthic invertebrate communities

Marine benthic habitats in continental shelf regions are increasingly impacted by hypoxia caused by the combination of eutrophication and climate warming. Many regions that have the potential for hypoxic conditions are being fished by mobile bottom-contacting fishing gears. The combined effects of trawling and hypoxia may be synergistic and disproportionally impact benthic fauna, or they may act antagonistically, leading to smaller trawl impacts in hypoxic areas. Yet, few studies have quantified how bottom trawling and hypoxia interact to affect benthic communities. Here we examine these combined effects on benthic community biomass and abundance, the number of large organisms, the longevity distribution of the community and the vertical position of fauna in the sediment in the southern Baltic Sea. We find large declines in benthic biomass and abundance that co-occur with declines in near-bed oxygen concentrations from 5.8 to 0.8 ml O2 l-1. Conversely, no relationships and weak positive relationships are found between bottom trawl disturbance and benthic community biomass and abundance. No interacting effects between hypoxia and trawling are detected. Our findings therefore highlight a low likelihood of synergistic impacts of bottom trawling and hypoxia on the benthic communities studied. These results suggest that management may prioritize benthic protection from fishing in regions that are not in a state of oxygen stress.

Gene expression patterns of sea urchins (Strongylocentrotus intermedius) exposed to different combinations of temperature and hypoxia

Strongylocentrotus intermedius is one of the most economically valuable sea urchin species in China, and its growth and survival are severely constrained by ocean warming and the hypoxia that often accompanies high water temperatures. To elucidate the molecular mechanisms of S. intermedius that regulate gene expression in response to multi-causal environmental stresses. We performed a de novo transcriptome analysis of coelomocyte from S. intermedius to heat (25 °C), hypoxia (2 mg/L), and the combined stress. We identified 35,635, 29,107, and 29,440 differentially expressed genes (DEGs) in S. intermedius cultured under high temperature, low oxygen, and combined stress, respectively. Further Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways analyses revealed that these DEGs mainly enriched the functional categories of “Protein processing in endoplasmic reticulum,” and “Glutathione metabolism” by heat stress, such as HSP70, GSTO1, PDIA4. After hypoxic stress, “Notch signaling pathway” and metabolism-related pathways such as “Glycerolipid metabolism”, “Pyruvate metabolism” were significantly enriched. Exposure to combined stress resulted in a two-factor additive effect at the transcriptome level and have a more extensive impact on the immune correlated pathways in S. intermedius than single stress, the expression of related immune genes (C3, C5, and AIFM2) were up-regulated. Quantitative real-time PCR (qRT-PCR) analysis of the expression of 18 DEGs confirmed the RNA-Seq results. Observations in the present study will improve the understanding of the molecular mechanism of S. intermedius in response to multi-causal environmental stress.

The N-terminal cysteine is a dual sensor of oxygen and oxidative stress

Cellular homeostasis requires the sensing of and adaptation to intracellular oxygen (O2) and reactive oxygen species (ROS). The Arg/N-degron pathway targets proteins that bear destabilizing N-terminal residues for degradation by the proteasome or via autophagy. Under normoxic conditions, the N-terminal Cys (Nt-Cys) residues of specific substrates can be oxidized by dioxygenases such as plant cysteine oxidases and cysteamine (2-aminoethanethiol) dioxygenases and arginylated by ATE1 R-transferases to generate Arg-CysO2(H) (R-CO2). Proteins bearing the R-CO2 N-degron are targeted via Lys48 (K48)-linked ubiquitylation by UBR1/UBR2 N-recognins for proteasomal degradation. During acute hypoxia, such proteins are partially stabilized, owing to decreased Nt-Cys oxidation. Here, we show that if hypoxia is prolonged, the Nt-Cys of regulatory proteins can be chemically oxidized by ROS to generate Arg-CysO3(H) (R-CO3), a lysosomal N-degron. The resulting R-CO3 is bound by KCMF1, a N-recognin that induces K63-linked ubiquitylation, followed by K27-linked ubiquitylation by the noncanonical N-recognin UBR4. Autophagic targeting of Cys/N-degron substrates is mediated by the autophagic N-recognin p62/SQTSM-1/Sequestosome-1 through recognition of K27/K63-linked ubiquitin (Ub) chains. This Cys/N-degron-dependent reprogramming in the proteolytic flux is important for cellular homeostasis under both chronic hypoxia and oxidative stress. A small-compound ligand of p62 is cytoprotective under oxidative stress through its ability to accelerate proteolytic flux of K27/K63-ubiquitylated Cys/N-degron substrates. Our results suggest that the Nt-Cys of conditional Cys/N-degron substrates acts as an acceptor of O2 to maintain both O2 and ROS homeostasis and modulates half-lives of substrates through either the proteasome or lysosome by reprogramming of their Ub codes.

Transcriptome Analysis of Gills Provides Insights Into Translation Changes Under Hypoxic Stress and Reoxygenation in Golden Pompano, Trachinotus ovatus (Linnaeus 1758)

Golden pompano (Trachinotus ovatus) is one of the most economically critical marine fish in South China. Low oxygen stress has resulted in substantial economic losses to the aquaculture of T. ovatus. However, the molecular responses of fish gills to hypoxia challenge remain unclear. To understand the mechanism underlying adaption to hypoxia, we analyzed the transcriptome of T. ovatus gills in response to hypoxic stress in the normal oxygen group, hypoxic group, and hypoxia treatment after oxygen recovery group. This study found that hypoxia for 8 h was the critical time of hypoxic stress and corresponded to the largest number of differentially expressed genes. After hypoxic stress, genes for chemokines, chemokine receptors, interleukins, complement factors, and other cytokines were significantly downregulated, which may be why fish are vulnerable to pathogen infection in a hypoxic environment. According to a Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, many downregulated genes were significantly enriched in the steroid biosynthesis, focal adhesion, and the extracellular matrix (ECM)-receptor interaction signal pathways, which affected cell signal transduction, adhesion, and apoptosis. Compared with the hypoxic group, the amounts of upregulated genes related to phagocytosis and protein degradation were upregulated in the dissolved oxygen recovery group. These results indicated that after the recovery of dissolved oxygen, the fish body repaired the stress-induced damage by rapidly removing misfolded proteins. These findings provide a better understanding of the hypoxia response mechanism of fish and represent a useful resource for the genetic breeding of T. ovatus.

Perioperative supplemental oxygen and oxidative stress in patients undergoing moderate- to high-risk major abdominal surgery – A subanalysis of randomized clinical trial

Study objectiveOxidative stress plays a pivotal role in the development and aggravation of cardiovascular diseases. The influence of intraoperative inspired oxygen concentrations on oxidative stress is still not entirely known. Therefore, we evaluated in this sub-study if supplemental oxygen affects the oxidation-reduction potential in patients at-risk for cardiovascular complications undergoing moderate- to high-risk major abdominal surgery. DesignSub-study of a prospective parallel-arm double-blinded single-center superiority randomized trial. SettingOperating room and postoperative recovery area. InterventionAdministration of 0.8 FiO2 versus 0.3 FiO2 throughout surgery and for the first two postoperative hours. MeasurementsThe primary outcome was the static oxidation-reduction potential (sORP) and the oxidation-reduction potential capacity (cORP) between both groups. The secondary outcome was the trend of sORP and cORP in the overall study population. We assessed sORP and cORP before induction of anesthesia, 2 h after induction of anesthesia, within 2 h after surgery and on the first and third postoperative day. Main results258 patients were analyzed. 128 patients were randomly assigned to the 80% oxygen group and 130 patients were randomly assigned to the 30% oxygen group. Postoperative sORP values did not differ significantly between the 80% and 30% oxygen group (effect estimate: −1.162 mV,95% CI: −2.584 to 0.260; p = 0.109). On average, we observed a change in sORP of 5.288 mV (95% CI:4.633 to 5.913, p < 0.001) per day. cORP values did not differ significantly between the 80% and 30% oxygen group (effect estimate: −0.015μC, (95%CI: −0.062 to 0.032; p = 0.524). On average, we observed a change in cORP values of −0.170μC (95%CI: −0.194 to −0.147, p < 0.001) per day. ConclusionIn contrast to previous reports, we could not find any evidence of an association between intraoperative supplemental oxygen and perioperative oxidative stress assessed by sORP and cORP. Trial registrationclinicaltrials.gov: NCT03366857https://clinicaltrials.gov/ct2/show/NCT03366857?term=vienna&cond=oxygen&draw=2&rank=1

Effects of pollution-induced changes in oxygen conditions scaling up from individuals to ecosystems in a tropical river network

Anthropogenic inputs of nutrients and organic matter are common in tropical lowland rivers while little is known about the pollution-induced changes in oxygen availability and respiratory performance of ectotherms in these high temperature systems. We investigated the effects of agriculture and urban land-use on river water oxygen levels (diel measurements), decomposition rates (Wettex) and macroinvertebrate assemblages (field studies), as well as the oxy-regulatory capacity of eight riverine macroinvertebrate taxa (laboratory study) from a tropical lowland river network in Myanmar. The highest decomposition rates (0.1–5.5 mg Wettex degree day−1) and oxygen stress (≤91% saturation deficits) were found in reaches draining degraded catchments with elevated concentrations of nutrients. All individual macroinvertebrate taxa investigated were to some extent able to regulate their respiration when placed under oxygen stress in the laboratory (regulation value of 0.74–0.89). The oxy-regulation capacity of macroinvertebrate assemblages in the river network were, as predicted, inversely related to diel oxygen stress (maximum deficit; lm, R2 = 0.69), where taxonomic richness and pollution sensitivity (ASPT metric) also declined sharply (lm, R2 ≥ 0.79). Our study shows that eutrophication and organic pollution induce oxygen deficits in tropical rivers but stimulate decomposition rates, which may further deplete oxygen levels. Furthermore, macroinvertebrate oxy-regulatory capacity predicts assemblage composition along gradients in oxygen stress at the ecosystem level. Our findings suggest that tropical lowland river systems could be highly sensitive to pollution by nutrients and organic matter leading to substantial impacts on ectotherm community composition and ecosystem functioning.

MaNmrA, a Negative Transcription Regulator in Nitrogen Catabolite Repression Pathway, Contributes to Nutrient Utilization, Stress Resistance, and Virulence in Entomopathogenic Fungus Metarhizium acridum.

Simple SummaryNutrient metabolism is closely related to the growth, development, and pathogenicity of pathogenic fungi. The nitrogen catabolite repression (NCR) pathway is a well-known fungal nitrogen source regulation path, in which NmrA plays an important regulatory role. Here, we reported a negative regulatory protein MaNmrA, the NmrA homologous protein, in the entomopathogenic fungus Metarhizium acridum, and found that it played important roles in carbon and nitrogen metabolism, growth, stress tolerance, and virulence of M. acridum. Our work will provide a theoretical basis for further exploring the functions of NCR pathway related genes in entomopathogenic fungi.The NCR pathway plays an important regulatory role in the nitrogen metabolism of filamentous fungi. NmrA, a central negative regulatory protein in the NCR pathway and a key factor in sensing to the carbon metabolism, plays important roles in pathogenic fungal nutrition metabolism. In this study, we characterized the functions of MaNmrA in the insect pathogenic fungus M. acridum. Multiple sequence alignments found that the conserved domain (NAD/NADP binding domain) of MaNmrA was highly conservative with its homologues proteins. Deletion of MaNmrA improved the utilization of multiple carbon sources (such as glucose, mannose, sucrose, and trehalose) and non-preferred nitrogen sources (such as NaNO3 and urea), significantly delayed the conidial germination rate and reduced the conidial yield. The MaNmrA-disruption strain (ΔMaNmrA) significantly decreased tolerances to UV-B and heat-shock, and it also increased the sensitivity to the hypertonic substance sorbitol, oxygen stress substance H2O2, and cell wall destroyer calcofluor white, indicating that loss of MaNmrA affected cell wall integrity, tolerances to hypertonic and oxidative stress. Bioassays demonstrated that disruption of MaNmrA decreased the virulence in both topical inoculation and intrahemocoel injection tests. Further studies revealed that the appressorium formation, turgor pressure, and colonization in hemolymph were significantly reduced in the absence of MaNmrA. Our work will deepen the functional cognition of MaNmrA and make a contribution to the study of its homologous proteins.

Assessment of biofilm formation by Campylobacter spp. isolates mimicking poultry slaughterhouse conditions

This research aimed to assess the biofilm formation ability of Campylobacter strains under temperature and oxygen stress conditions, similar to those found in the industrial environment, to explain the persistence of this pathogen on the poultry slaughter line. A collection of C. jejuni and C. coli isolates (n = 143) obtained from poultry samples (cecal content and neck skin), collected at slaughterhouse level, from diverse flocks, on different working days, was genotyped by flaA-restriction fragment length polymorphism (RFLP) typing method. A clustering analysis resulted in the assignment of 10 main clusters, from which 15 strains with different flaA-RFLP genotypes were selected for the assessment of biofilm formation ability and antimicrobial susceptibility. Biofilm assays, performed by crystal violet staining method, were conducted with the goal of mimicking some conditions present at the slaughterhouse environment, based on temperature, atmosphere, and contamination levels. Results indicated that many C. jejuni strains with similar flaA-RFLP profiles were present at the slaughterhouse on different processing days. All the strains tested (n = 15) were multidrug-resistant except for one. Biofilm formation ability was strain-dependent, and it appeared to have been affected by inoculum concentration, temperature, and tolerance to oxygen levels. At 10°C, adherence levels were significantly lower than at 42°C. Under microaerobic and aerobic atmospheres, at 42°C, 3 strains (C. jejuni 46E, C. jejuni 61C, and C. coli 65B) stood out, exhibiting significant levels of biofilm formation. C. jejuni strains 46E and 61C were inserted in clusters with evidence of persistence at the slaughterhouse for a long period of time. This study demonstrated that Campylobacter strains from broilers are capable of forming biofilms under conditions resembling the slaughterhouse environment. These results should be seen as a cue to improve the programs of hygiene implemented, particularly in those zones that can promote biofilm formation.

Extracellular DEK Treatment Mimics Hypoxic Blockade of Extra Physiologic Oxygen Stress in Human and Mouse Hematopoietic Cells

Hematopoietic stem cell (HSC) and progenitor cell (HPC) self-renewal, proliferation, survival, and function are regulated by extracellular signals from cytokines and chemokines. DEK, a nuclear regulator of chromatin availability, was recently shown to also function as an extracellular protein that regulates HSC and HPC by inducing signaling through the CXC chemokine receptor 2 (CXCR2), enhancing pools of long-term stem cells while decreasing pools of functional HPC [Capitano et al., JCI, 2019, 129(6):2555-70]. Due to differing effects of extracellular (ec)DEK treatment on HSC growth and function compared to the effects on HPC, we hypothesized that DEK differentially regulates HSC compared to HPC. To explore this, we pulse-treated purified human cord blood (CB) CD34+ cells for 18 hours with recombinant human ecDEK protein. We then isolated by flow cytometry stringently immunophenotypically-defined human HSC, multipotent progenitors (MPP), common myeloid progenitors (CMP), and granulocyte-macrophage progenitors (GMP) and performed mRNA-sequencing to profile ecDEK-dependent transcriptomes of these separate populations of HSC/HPC. Interestingly, there are widely different ranges of effects on the transcriptomes of the different isolated HSC/HPC populations. CMP had the most differentially expressed (DE) genes induced by DEK treatment, followed by HSC and MPP, and GMP had relatively few changes, suggesting DEK treatment may primarily affect HSC and earlier HPC. HSC exhibited the strongest magnitude of changes in DE genes. Fast gene set enrichment analysis (FGSEA) revealed that GMP showed upregulation of gene programs associated with leukocyte migration and downregulation of gene programs regulated by MYC, CMP showed upregulation of gene programs associated with myeloid cell development and downregulation of lysine methyltransferase activity, and MPP and HSC both upregulated cytokine signaling responses in response to DEK treatment, though HSC also exhibited a downregulation of genes associated with cell cycle. Despite the identified unique changes upon DEK treatment in these different HSC/HPC populations, we were most interested to discover that all examined cell types demonstrated upregulation of genes associated with hypoxic responses in cells, and a concordant downregulation of genes that are frequently downregulated by hypoxic exposure following DEK treatment. This suggested to us that DEK may stimulate overlapping pathways related to hypoxia responses and/or response to exposure to extra physiologic oxygen. In fact, the effects on HSC and HPC induced by ecDEK is similar with previous work demonstrating that isolating HSC/HPC at 3% O 2 compared to ambient air conditions (~21% O 2) leads to a preserved pool of functional HSC but a lower number of functional HPC, due to a phenomenon termed extracellular physiologic shock/stress (EPHOSS) [Mantel et al., Cell, 2015, 161(7):1553-65]. To explore whether DEK regulates similar stress response pathways that are associated with EPHOSS, we assessed the effects of ecDEK treatment on HSC/HPC isolated at 3% O 2. Mice were treated with DEK in vivo, then bone marrow cells were isolated at 3% O 2 in a hypoxia chamber. Cells were kept at 3% O 2 or split to ambient air to allow equilibration to a higher oxygen tension and then assessed for HSC and functional HPC numbers. In contrast to untreated HSC/HPC isolated at 3% O 2, ecDEK treated cells isolated at 3% O 2 did not exhibit further increases in LT-HSC numbers or decreases in pools of functional HPC over ecDEK treatment alone identified by colony assays. Isolation at 3% O 2 followed by in vitro treatment with DEK showed similar results. This suggests ecDEK may neutralize the effects of EPHOSS by acting on similar gene programs that are induced by ambient air exposure. We also demonstrate that ecDEK prevents the ex vivo apoptosis of CB HSC/HPC populations, again suggesting that ecDEK is preventing the cellular stress associated with ambient air exposure and ex vivo growth, resulting in a preservation of HSC. Taken together, these data show that ecDEK acts as an extracellular signaling protein that prevents cellular stress by activating gene programs that overlap with hypoxia associated gene programs. This has important implications for the role of ecDEK in normal and diseased hematopoiesis, as well as clinical implications wherein ecDEK may be used to mimic the HSC preserving effects of isolation at 3% O 2. DisclosuresMarkovitz: University of Michigan: Patents & Royalties: H84T BanLec and of the H84T-driven CAR construct.

Characterization of hydrodynamic stress in ambr250® bioreactor system and its impact on mammalian cell culture

During the cell culture in stirred aerated vessels, both stirring and sparging is required for proper homogenization of a mixture and oxygen delivery. Quality by design (QbD) in biopharmaceutical industry aims at identification of the impact of mass transfer, dissolved oxygen, pH and hydrodynamic stress on the cell behavior, productivity and product quality. Most of these properties are influenced by impeller motion and sparger aeration. In this research, the shear sensitive poly(methyl methacrylate) (PMMA) aggregates are used as probes capable of determining the maximum hydrodynamic stress (τmax) present in the ambr250® bioreactor. The results from our micro-aggregate probes are connected with the cultivation data using two Chinese hamster ovary K1 glutamine synthetase mediated cell lines (CHO-K1 GS) to determine the stress threshold, below which no significant impact of operating conditions on cell behavior is observed. Such calibrated system can be in the future used as a scale-down model of large-scale bioreactors to test cell robustness during process development.

A PDMS-Based Interdigitated Platform for Trophoblast Invasion Study Under Oxygen Stress Conditions

A PDMS-Based Interdigitated Platform for Trophoblast Invasion Study Under Oxygen Stress Conditions

Is hyaluronic acid production transcriptionally regulated? A transcriptional repressor gene deletion study in Streptococcus zooepidemicus.

Hyaluronic acid (HA) is a multiple-function biopolymer that is widely used in food, cosmetic, and biomedical fields. In group C streptococci, the major workhorse of HA production in industry, the HA biosynthetic pathway has been proposed, while how HA synthesis is regulated is unclear. In this study, we identified twenty-five putative transcriptional repressors in S. zooepidemicus and studied whether they regulate HA synthesis or not. The individual gene deletion strain was firstly constructed, and the phenotypic changes of the corresponding deletion strains in stress tolerance and HA production were detected. The hrcA deletion strain is more sensitive to high temperature, and the rex deletion strain is more resistant to the oxidative stress. Three transcriptional repressor deletions resulted significantly decreased transcriptional levels of hasA, among which the scrR deletion strain shows most dramatical decrease in HA production. The regulatory mechanism of how ScrR affects the production of HA was further explored by transcriptional expression analysis of scrA and scrB, two direct target genes of ScrR regulon. Our results indicates that the deficiency of ScrR results in the unbalanced expression of scrA and scrB, which might also partly account for the decreasing production of HA. In agreement with the speculation, overexpression of scrB in ΔscrR genetic background results in 80% improvement in HA production. Taken together, the systemic genetic study of transcriptional repressors expands our understanding for the physiological regulation process of S. zooepidemicus and should help in the development of high-performance industrial strains for the efficient production of HA. KEY POINTS: • Twenty-two transcriptional repressor genes in S. zooepidemicus were deleted individually, and the phenotypes of corresponding mutants on a variety of conditions were characterized. • HrcA deficiency showed inferior cell tolerance to high temperature, and Rex deficiency showed superior cell tolerance to reactive oxygen stress, and four repressors deficiency showed inferior hyaluronic acid synthesis, among which the transcriptional levels of hasA of three mutants decreased significantly. • Optimizing sucrose metabolic flux can enhance hyaluronic acid synthesis significantly.