Whole-body heat exposure induces membrane changes in spermatozoa from the cauda epididymidis of laboratory mice

This study was carried out to determine if exposure to hot environmental temperatures had a direct, detrimental effect on sperm quality. For this the effect of whole-body heat exposure on epididymal spermatozoa of laboratory mice was investigated. C57BL/6 mice （n = 7） were housed in a microclimate chamber at 37℃-38℃ for 8 h per day for three consecutive days, while control mice （n = 7） were kept at 23℃-24℃. Cauda epididymal spermatozoa were obtained 16 h after the last heat treatment. The results showed that sperm numbers were similar in the two groups （P = 0.23）, but after heat treatment, a significant reduction in the percentage of motile sperm was present （P 〈 0.0001）. Membrane changes of the spermatozoa were investigated by staining with phycoerythrin （PE）- conjugated Annexin V, which detects exteriorization of phosphotidylserine from the inner to the outer leaflet of the sperm plasma membrane, and 7-aminoactinomycin D （7-AAD）, which binds to the sperm nucleus when the plasma membrane is damaged. The percentage of spermatozoa showing positive staining with Annexin V-PE or 7-AAD or both, was significantly higher （P 〈 0.05） in heat-exposed mice compared with controls. These results show that whole-body heat exposure to 37℃-38℃ induces membrane changes in the epididymal spermatozoa of mice, which may lead to apoptosis.

Physiological Changes in Leaves of Medicago falcata Under Low-Temperature Stress

Low temperature significantly restricts crop yield and quality.Medicago falcata(M.falcata)is a typical legume species that exhibits great capacity of tolerance to low temperature.To understand the low-temperature responses in M.falcata,the electrolyte leakage and lipid peroxidation level,and activities of superoxide dismutase(SOD),catalase(CAT)and peroxidase(POD),and contents of reduced glutathione(GSH),soluble protein,soluble sugar and proline were investigated in low-temperature-stressed M.falcata leaves.The electrolyte leakage and malondialdehyde(MDA)content increased,and could be used to quantify low-temperature damage at cellular level.And then,the significant change of SOD,POD and CAT activities,and GSH content reflected the higher reactive oxygen species(ROS)scavenging capacity in M.falcata.In addition,the significant change of soluble protein,soluble sugar and proline contents helped to maintain osmotic equilibrium,energy supply and protein functions.These nine physiological traits were analyzed by gray relational grade analysis and ranked from the highest to the lowest as follows:electrolyte leakage,GSH,proline,soluble protein,MDA,soluble sugar,SOD,CAT and POD,and illustrated that the electrolyte leakage level,GSH and proline contents should be selected and measured priority in M.falcata low-temperature tolerance to improve measurement efficiency.

Postharvest Application of 1-Methylcyclopropene Maintains Quality and Extends the Shelf-Life of Zizania latifolia during Storage at 25˚C

The effects of 1-methylcyclopropene (1-MCP) on postharvest quality of Zizania latifolia during storage at 25˚C were investigated. The results pointed out that a postharvest application of 1-MCP maintained the good visual appearance of fresh Z. latifolia, inhibited browning, mildew and weight loss at the bottom of Z. latifolia, and there is no significant changes on L, a*, b* and ΔE during the whole storage period. In addition, 1-MCP treatment inhibited the respiratory intensity of Z. latifolia during the first three days of storage, but it was significantly higher than that of the control on the sixth day of storage. Compared with the control, 1-MCP treatment maintained relatively high SOD, CAT, APX activities and low PAL, POD, PLD, lipase and LOX activities, delayed the decline of AsA content, reduced the accumulation of O2-, H2O2 and MDA, and ultimately maintained the integrity of cell structure and delayed the senescence of Z. latifolia. In addition, positive effects of 1-MCP on maintaining the cell structure integrity were observed in this investigation throughout the storage period at 25˚C.

Mechanisms of ultraviolet disinfection and chlorination of Escherichia coli： Culturability, membrane permeability,metabolism, and genetic damage

Traditional culture methods may underestimate the tolerance of microorganisms to disinfectants because of the existence of viable but nonculturable or sublethally injured cells after disinfection.The selection of a strict method is crucial for the evaluation of disinfection performance.The actions of 2 typical disinfectants–ultraviolet（UV）and chlorine–on the fecal indicator Escherichia coli were investigated by the detection of culturability,membrane permeability,metabolic activity,deoxyribonucleic acid（DNA）,and messenger ribonucleic acid（m RNA）.During UV disinfection,the irreversible damages in the cell membrane and cellular adenosine triphosphate（ATP）were negligible at low UV doses（80 m J/cm^2）.However,membrane permeability was damaged at low doses of chlorine（5 mg/L）,leading to leakage of cellular ATP.Our study showed that a slight lesion in DNA was detected even at high doses of UV（400 m J/cm^2）and chlorine（5 mg/L）treatments.The decay of m RNA was more rapid than that of DNA.The degradation level of m RNA depended on the choice of target genes.After exposure to 50 m J/cm^2UV dose or 5 mg/L chlorine for30 min,the DNA damage repair function（Rec A m RNA）was inhibited.The m RNA involved in the DNA damage repair function can be a potential indicator of bacterial viability.

Effects of High-Temperature Stress on Photosynthetic Characteristics and Antioxidant Enzyme System of Paeonia ostii

Paeonia ostii is an economically important oil crop,which has been widely cultivated in the middle and lower reaches of the Yangtze River in China in recent years.Although P.ostii is highly adaptable to the environment,the prolonged high summer temperature in this region severely inhibits its growth,which adversely affects seed yield and quality.In this study,P.ostii plants were subjected to 20℃/15℃(day/night)and 40℃/35℃(day/night)temperatures for 15 days.The changes in physiological and biochemical indicators of P.ostii under high-temperature stress were initially investigated.The results showed that with the deepening of leaf etiolation,chlorophyll a and chlorophyll b concentration,carotenoid concentration,Soil Plant Analysis Development(SPAD)values and leaf relative water content decreased significantly,while both relative electrical conductivity(REC)and free proline concentration showed an upward trend.Meanwhile,the continuous accumulation of reactive oxygen species(ROS)in P.ostii plants,led to an increased activity of antioxidant enzymes including superoxide dismutase(SOD),peroxidase(POD),catalase(CAT)and ascorbate peroxidase(APX).Moreover,with the extension of the high-temperature treatment,the anatomical structures of P.ostii were destroyed,resulting in a decreased photochemical efficiency of the photosystem II(PSII)reaction center and photosynthesis was inhibited.Taken together,these results provide reference values for understanding the physiological response of P.ostii to hightemperature stress and establish a foundation for further research on the relevant underlying molecular mechanisms.

Sensitivity of Different Cytotoxic Responses of Vero Cells Exposed to Organic Chemical Pollutants and their Reliability in the Bio-toxicity Test of Trace Chemical Pollutants

Objective To find a sensitive cytotoxic response to reflect the bio-toxicity of trace organic pollutants, the sensitivity and reliability of morphological change and proliferation inhibition of Vero cells exposed to 2, 4, 6-trichlorophenol （TCP） and the leachate from products related to drinking water （PRDW） were compared, and the mechanism of the morphological change in Vero cells exposed to chemical pollutants was studied. Methods Vero cells were treated by different concentration of TCP and the leachate from PRDW. Methylthiazol-2-yl-2, 5-diphenyl tetrazolium bromide （MTT） assay was carried out for proliferation inhibition. Bioluminescence method was carried out as another method to test the toxicity of TCP. Flow Cytometry assay was used to test cell Apoptosis and damage of cell-membrane. Results 0.25 mg/L TCP had an effect on cell morphology, and the proportion of morphologically changed cells increased with increasing TCP concentration. At low TCP concentrations, inhibition of cell proliferation did not seem to correlate to TCP concentration, and was negative when TCP concentration was 〈1.0 mg/L. After exposure to leachate from PRDW extracted at different temperatures, the percentage of morphologically changed cells increased with extracting temperature, but the inhibition of cell proliferation failed to reflect the correlation between extracting temperature and proliferation inhibition of Vero cells. Although the Sensitivity of bioluminescence method seems to be similar to morphological change in Veto cells, the bacterial in this method is not homologous enough with human body cells to reflect the toxicity to human body. These imply cell morphological change is a more sensitive and reliable method to reflect bio-toxicity of organic pollutants than proliferation inhibition. Flow cytometry analysis and cell rejuvenation experiments indicated cell membrane damage, which results in cell morphological change, was an early and sensitive cytotoxic response comparing with necrosis. Conelusion These results indicated that the cell membrane toxicity represented by morphological changes is a more sensitive and reliable method to indicate the composite bio-toxicity of trace chemicals than proliferation inhibition, inhibition on bioluminescence and necrosis. Nevertheless, the quantification of morphological change should be studied further.

Antimicrobial activity and mode of action of terpene linalyl anthranilate against carbapenemase-producing Klebsiella pneumoniae

Mining of plant-derived antimicrobials is the major focus at current to counter antibiotic resistance. This study was conducted to characterize the antimicrobial activity and mode of action of linalyl anthranilate(LNA) against carbapenemase-producing Klebsiella pneumoniae(KPC-KP). LNA alone exhibited bactericidal activity at 2.5%(V/V), and in combination with meropenem(MPM) at 1.25%(V/V). Comparative proteomic analysis showed a significant reduction in the number of cytoplasmic and membrane proteins,indicating membrane damage in LNA-treated KPC-KP cells. Up-regulation of oxidative stress regulator proteins and down-regulation of oxidative stress-sensitive proteins indicated oxidative stress. Zeta potential measurement and outer membrane permeability assay revealed that LNA increases both bacterial surface charge and membrane permeability. Ethidium bromide influx/efflux assay showed increased uptake of ethidium bromide in LNA-treated cells, inferring membrane damage. Furthermore, intracellular leakage of nucleic acid and proteins was detected upon LNA treatment. Scanning and transmission electron microscopies again revealed the breakage of bacterial membrane and loss of intracellular materials. LNA was found to induce oxidative stress by generating reactive oxygen species(ROS) that initiate lipid peroxidation and damage the bacterial membrane. In conclusion, LNA generates ROS, initiates lipid peroxidation, and damages the bacterial membrane, resulting in intracellular leakage and eventually killing the KPC-KP cells.

Probing cell membrane integrity using a histone-targeting protein nanocage displaying precisely positioned fluorophores

Cell membrane integrity is fundamental to the normal activities of cells and is involved in both acute and chronic pathologies.Here,we report a probe for analyzing cell membrane integrity developed from a 9 nm-sized protein nanocage named Dps via fluorophore conjugation with high spatial precision to avoid self-quenching.The probe cannot enter normal live cells but can accumulate in dead or live cells with damaged membranes,which,interestingly,leads to weak cytoplasmic and strong nuclear staining.This differential staining is found attributed to the high affinity of Dps for histones rather than DNA,providing a staining mechanism different from those of known membrane exclusion probes(MEPs).Moreover,the Dps nanoprobe is larger in size and thus applies a more stringent criterion for identifying severe membrane damage than currently available MEPs.This study shows the potential of Dps as a new bioimaging platform for biological and medical analyses.

C60(OH)n-loaded nanofibrous membranes protect HaCaT cells from ROS-associated damage

Environmental stress factors could lead to the excess generation of reactive oxygen species（ROS） that induces various forms of skin damage related to oxidative stress. Polyhydroxylated fullerene derivative C（60）（OH）n, acting as an effective agent for prevention of skin aging, is widely used in the lotion and sunscreens in the field of cosmetics, but rarely used in the masks. In this study, we prepared C（60）（OH）n loaded nanofibrous membranes to protect human keratinocyte cells from ROS-associated damage and suppress the elevation of intracellular ROS and Ca（2＋） along with the apoptotic cell death. Two FDAapproved biodegradable polymers, PLGA and PCL, have been used for making the electrospun nanofibers,with C（60）（OH）n added to the polymers as an antioxidant. The nanofibrous membranes with good biocompatibility might be potentially applied in clinical practice to reduce skin aging.

Elevated axonal membrane permeability and its correlation with motor deficits in an animal model of multiple sclerosis

Background:It is increasingly clear that in addition to myelin disruption,axonal degeneration may also represent a key pathology in multiple sclerosis(MS).Hence,elucidating the mechanisms of axonal degeneration may not only enhance our understanding of the overall MS pathology,but also elucidate additional therapeutic targets.The objective of this study is assess the degree of axonal membrane disruption and its significance in motor deficits in EAE mice.Methods:Experimental Autoimmune Encephalomyelitis was induced in mice by subcutaneous injection of myelin oligodendrocyte glycoprotein/complete Freud’s adjuvant emulsion,followed by two intraperitoneal injections of pertussis toxin.Behavioral assessment was performed using a 5-point scale.Horseradish Peroxidase Exclusion test was used to quantify the disruption of axonal membrane.Polyethylene glycol was prepared as a 30%(w/v)solution in phosphate buffered saline and injected intraperitoneally.Results:We have found evidence of axonal membrane disruption in EAE mice when symptoms peak and to a lesser degree,in the pre-symptomatic stage of EAE mice.Furthermore,polyethylene glycol(PEG),a known membrane fusogen,significantly reduces axonal membrane disruption in EAE mice.Such PEG-mediated membrane repair was accompanied by significant amelioration of behavioral deficits,including a delay in the emergence of motor deficits,a delay of the emergence of peak symptom,and a reduction in the severity of peak symptom.Conclusions:The current study is the first indication that axonal membrane disruption may be an important part of the pathology in EAE mice and may underlies behavioral deficits.Our study also presents the initial observation that PEG may be a therapeutic agent that can repair axolemma,arrest axonal degeneration and reduce motor deficits in EAE mice.

The combined toxicity and mechanism of multi-walled carbon nanotubes and nano copper oxide toward freshwater algae:Tetradesmus obliquus

Nanoparticles(NPs)are widely used for their special physical properties and released into the natural environment.When two types of NPs exist in the same environment,the presence of one type of NP may affect the properties of the other type of NP.This study investigated the toxic effects of multi-walled carbon nanotubes(MWCNTs)and copper oxide nanoparticles(Cu O NPs)on Tetradesmus obliquus.Both NPs had toxic effects on algae,and the toxic effects of MWCNTs were significantly stronger than Cu O NPs which the 96-hr median effective concentration to algae were 33.8 and 169.2 mg/L,respectively.Oxidative stress and cell membrane damage were the main reasons for the toxicity of NPs to algae,and they were concentration-dependent,and the existence of Cu O NPs in some groups reduced cell membrane damage caused by MWCNTs which may because that Cu O NPs formed heteroaggregation with MWCNTs,reducing the contact of nanoparticles with cell membranes,then reducing physical damage.Scanning electron microscopy(SEM)and transmission electron microscope(TEM)results indicated cell damage,the heteroaggregation of MWCNTs-Cu O NPs and obvious nanoparticles internalization.In some groups,the presence of Cu O NPs significantly reduced reactive oxygen species(ROS)level induced by MWCNTs.However,for the highest concentration group,the ROS level was much higher than that of the two NPs alone treatment groups,which might be related to the high concentration of MWCNTs promoting the internalization of Cu O NPs.MWCNTs and Cu O NPs affected and interacted with each other,causing more complex toxic effects on aquatic organisms.

Perturbation of clopyralid on bio-denitrification and nitrite accumulation:Long-term performance and biological mechanism

The contaminant of herbicide clopyralid(3,6-dichloro-2-pyridine-carboxylic acid,CLP)poses a potential threat to the ecological system.However,there is a general lack of research devoted to the perturbation of CLP to the bio-denitrification process,and its biological response mechanism remains unclear.Herein,long-term exposure to CLP was systematically investigated to explore its influences on denitrification performance and dynamic microbial responses.Results showed that low-concentration of CLP(<15 mg/L)caused severe nitrite accumulation initially,while higher concentrations(35e60 mg/L)of CLP had no further effect after long-term acclimation.The mechanistic study demonstrated that CLP reduced nitrite reductase(NIR)activity and inhibited metabolic activity(carbon metabolism and nitrogen metabolism)by causing oxidative stress and membrane damage,resulting in nitrite accumulation.However,after more than 80 days of acclimation,almost no nitrite accumulation was found at 60 mg/L CLP.It was proposed that the secretion of extracellular polymeric substances(EPS)increased from 75.03 mg/g VSS at 15 mg/L CLP to 109.97 mg/g VSS at 60 mg/L CLP,which strengthened the protection of microbial cells and improved NIR activity and metabolic activities.Additionally,the biodiversity and richness of the microbial community experienced a U-shaped process.The relative abundance of denitrification-and carbon metabolism-associated microorganisms decreased initially and then recovered with the enrichment of microorganisms related to the secretion of EPS and N-acyl-homoserine lactones(AHLs).These microorganisms protected microbe from toxic substances and regulated their interactions among interand intra-species.This study revealed the biological response mechanism of denitrification after successive exposure to CLP and provided proper guidance for analyzing and treating herbicide-containing wastewater.