Prolonged Incubation Time Research Articles

Detection, Identification and Diagnostic Characterization of the Staphylococcal Small Colony-Variant (SCV) Phenotype.

While modern molecular methods have decisively accelerated and improved microbiological diagnostics, phenotypic variants still pose a challenge for their detection, identification and characterization. This particularly applies if they are unstable and hard to detect, which is the case for the small-colony-variant (SCV) phenotype formed by staphylococci. On solid agar media, staphylococcal SCVs are characterized by tiny colonies with deviant colony morphology. Their reduced growth rate and fundamental metabolic changes are the result of their adaptation to an intracellular lifestyle, regularly leading to specific auxotrophies, such as for menadione, hemin or thymidine. These alterations make SCVs difficult to recognize and render physiological, biochemical and other growth-based methods such as antimicrobial susceptibility testing unreliable or unusable. Therefore, diagnostic procedures require prolonged incubation times and, if possible, confirmation by molecular methods. A special approach is needed for auxotrophy testing. However, standardized protocols for SCV diagnostics are missing. If available, SCVs and their putative parental isolates should be genotyped to determine clonality. Since their detection has significant implications for the treatment of the infection, which is usually chronic and relapsing, SCV findings should be specifically reported, commented on, and managed in close collaboration with the microbiological laboratory and the involved clinicians.

Spatial mapping and quantitative evaluation of protein corona on PEGylated mesoporous silica particles by super-resolution fluorescence microscopy

Nanoparticles (NPs) adsorb serum proteins when exposed to biological fluids, forming a dynamic protein corona that has a profound impact on their overall biological profile and fate. Polyethylene glycol (PEG) modification is the most widely used strategy to mitigate and inhibit protein corona formation. Nevertheless, the accurate mapping and quantification of PEG inhibition effects on protein corona formation have scarcely been reported. Herein, we demonstrate the direct observation and quantification of protein corona adsorbed onto PEGylated mesoporous silica particles by direct stochastic optical reconstruction microscopy (dSTORM). The variation tendency of protein penetration depth in terms of PEG molecular weights and incubated time is investigated for the first time. The maximum penetration depths present slight increase with the prolonged incubation time, while they tend to remarkably decrease with increased chain length of modified PEG. Moreover, the co-localization of preformed protein corona with lysosomes and the destination of adsorbed protein are demonstrated. Our method provides important technical characterization information and in-depth understanding of protein corona adsorbed onto PEGylated mesoporous silica particles. This shines new light on the behaviors of silica materials in cells and may promote their practical applications in biomedicine.

Comparing cytotoxicity and efficacy of miltefosine and standard antimicrobial agents against Acanthamoeba trophozoites and cyst forms: An in vitro study

Acanthamoeba keratitis (AK) is an eye disease often occurring in contact lens wearers. AK treatment is prolonged and requires multiple drugs, which can lead to adverse effects. Our study aimed to compare the in vitro activities and safety of Miltefosine with that of conventional antimicrobial agents used to treat AK. Acanthamoeba castellanii genotype T4 was obtained from a patient with keratitis and subjected to in vitro susceptibility testing with various antimicrobial agents, including Chlorhexidine (CHX), Pentamidine isethionate (PI)Polyhexamethylene biguanide (PHMB), and Miltefosine to assess their efficacy against Acanthamoeba trophozoites and cyst. The cytotoxicity of the agents was evaluated in Vero cells, and their selectivity indexes (SI) were calculated. Chlorhexidine exhibited the highest amoebicidal activity with the highest selectivity index against the trophozoite and cyst, ranging from 1.17 to 8.35. The selectivity index of PHMB is slightly comparable to Chlorhexidine, exhibiting significant anti-Acanthamoeba activity.On the other hand, Pentamidine isethionate and Miltefosine displayed low SI among the compounds. Pentamidine isethionate was effective at high concentrations, which was toxic. Miltefosine exhibited the lowest cytotoxicity; nevertheless, due to the lowest anti-Acanthamoeba activity presented a low selectivity against the parasite. Further studies on more clinical samples and prolonged incubation time should be done to investigate the effectiveness and toxicity of drugs in both in vitro and in vivo conditions.

Endocytic internalization mechanism of bioactive antibacterial nanoparticles by fibroblasts

Studying the uptake mechanism of photosensitizers is an important step in developing an ideal photosensitizer for use in photodynamic therapy (PDT). Understanding the uptake mechanism can help design novel photosensitizers that are selectively accumulated in the target tissue, with improved pharmacokinetics, and are dosed optimally to maximize the efficacy of the treatment. In our previous studies we synthesized and characterized the use of chitosan nanoparticles functionalized with rose bengal (CSRBnp) as a photosensitizer against dental biofilm. The aim of this study is to analyze the internalization mechanism and cellular proinflammatory activities of CSRBnps on fibroblasts. Fibroblasts (NIH 3T3) were incubated with chlorpromazine (5 µg/ml), nystatin (5 µg/ml), wortmannin (100 ng/ml) and at 4 °C for 30 min followed by CSRBnp (0.3 mg/ml). Cell viability (MTS assay), intracellular adenosine triphosphate content (Luminescence assay), cytokine expression (TNF-α) using ELISA and nitric oxide (NO) production by Griess reaction system were conducted at different time intervals (30 min, 1, 4, and 12 h). The internalization of CSRBnps was analyzed using live cell imaging confocal microscope with excitation wavelengths of 405 and 568 to detect nuclei (Hoechst 33,342) and CSRBnps respectively. CSRBnps and inhibitors at the applied concentrations were not cytotoxic. ATP content in chlorpromazine and without inhibitors groups were significantly lower than the control group at 12 h. All inhibitors showed significantly lower CSRBnps uptake compared to the control group at 30 min, 1 h, and 4 h time. Wortmannin resulted in the most significant inhibition of CSRBnps uptake as compared to chlorpromazine and nystatin (P < 0.05). TNF-α expression and NO production were not significant during the entire CSRBnps uptake. The results showed macropinocytosis was a dominant CSRBnps uptake mechanism by fibroblasts in the early stages and non-specific uptake pathways were activated after prolonged incubation time. CSRBnps uptake by fibroblasts was energy dependent and did not cause any proinflammatory response.

Detection of Cutibacterium acnes in Tissue Samples from Clean Primary Shoulder Surgeries - Part II.

Objective Research and identification of Cutibacterium acnes ( C. acnes ) and other microorganisms in deep tissue samples collected in clean shoulder surgeries of patients who did not undergo any previous invasive joint procedure and who had no clinical history of infection. Methods We analyzed the results of cultures of intraoperative deep tissue samples from 84 patients submitted to primary clean shoulder surgery. Tubes containing culture medium were used for storage and transport of anaerobic agents, prolonged incubation time, and mass spectrometer for diagnosis of bacterial agents. Results Bacteria growth was evidenced in 34 patients (40.4%) of the 84 included in the study. Of these, 23 had growth of C. acnes in at least one sample of deep tissue collected, corresponding to 27.3% of the total patients. The second most common agent was Staphylococcus epidermidis , present in 7.2% of the total individuals included. We showed a higher relationship between sample positivity and males, a lower mean age, absence of diabetes mellitus, ASA I score, and antibiotic prophylaxis in anesthetic induction with cefuroxime. Conclusions A high percentage of isolates of different bacteria was found in shoulder tissue samples of patients undergoing clean and primary surgeries, who had no history of previous infection. Identification of C. acnes was high (27.6%), and Staphylococcus epidermidis was the second most frequent agent (7.2%).

Supramolecular assemblies based on polymeric cyclodextrin nanosponges and a cationic porphyrin with antimicrobial photodynamic therapy action

Within of the increasing requirement of alternative approaches to fight emerging infections, nano-photosensitisers (nanoPS) are currently designed with the aim to optimize the antimicrobial photodynamic (aPDT) efficacy. The utilize of less expensive nanocarriers prepared by simple and eco-friendly methodologies and commercial photosensitisers are highly desiderable. In this direction, here we propose a novel nanoassembly composed of water soluble anionic polyester β-CD nanosponges (β-CD-PYRO hereafter named βNS) and the cationic 5,10,15,20-tetrakis(1-methylpyridinium-4- yl)porphine (TMPyP). Nanoassemblies were prepared in ultrapure water by mixing PS and βNS, by exploiting their mutual electrostatic interaction, and characterized by various spectroscopic techniques such as UV/Vis, Steady-State and Time Resolved Fluorescence, Dynamic Light Scattering and ζ-potential. NanoPS produce appreciable amount of single oxygen similar to free porphyrin and a prolonged stability after 6 days of incubations in physiological conditions and following photoirradiation. Antimicrobial photodynamic action against fatal hospital-acquired infections such as P. aeruginosa and S. aureus was investigated by pointing out the ability of cationic porphyrin loaded- CD nanosponges to photo-kill bacterial cells at prolonged time of incubation and following irradiation (MBC99 = 3.75 µM, light dose = 54.82 J/cm2).

Endophytic bacteria as biological agents to control fusarium wilt disease and promote tomato plant growth

Plant endophytic bacteria are symbiotic bacteria that live in plant tissues and may play a role in disease control and/or plant growth promotion. In this study, six plant endophytic bacterial isolates, Arthrobacter sp. AM08, Pseudomonas aeruginosa AJ14, P. mosselii AB06, Bacillus cereus AP12, B. thuringiensis AK08 and Serratia marcescens AS09, were tested for their ability to control Fusarium oxysporum f. sp. lycopersici (FOL) and to promote the growth of tomato plant. The isolates were evaluated for their ability to produce hydrolytic enzymes (cellulase, protease and chitinase), hydrogen cyanide (HCN), indole 3-acetic acid (IAA), 1-aminocyclopropane 1-carboxylic acid (ACC) deaminase, and siderophores. The activity of cellulase, protease and chitinase enzymes was detected in all the isolates studied, whereas the highest HCN activity was observed in P. aeruginosa. The activity of IAA, ACC deaminase, and siderophore was also observed in all isolates, with B. cereus, P. mosselii, and S. marcescens being the highest producers of IAA and B. thuringiensis being the highest producer of siderophores. All isolates produced ACC deaminase at almost the same level. Application of the isolates to tomato seeds prolonged FOL incubation time, reduced disease incidence, and shortened xylem discoloration length compared to the controls, confirming the ability of the isolates to control FOL. In addition, all tested isolated showed plant growth promoting activity, as indicated by increased plant height and root length. The study showed that the endophytic bacteria are capable of controlling FOL and promoting the growth of tomato plant, revealing their potential to be developed as biocontrol agents.

Morphology-Dependent Interactions between α-Synuclein Monomers and Fibrils.

Amyloid fibrils may adopt different morphologies depending on the solution conditions and the protein sequence. Here, we show that two chemically identical but morphologically distinct α-synuclein fibrils can form under identical conditions. This was observed by nuclear magnetic resonance (NMR), circular dichroism (CD), and fluorescence spectroscopy, as well as by cryo-transmission electron microscopy (cryo-TEM). The results show different surface properties of the two morphologies, A and B. NMR measurements show that monomers interact differently with the different fibril surfaces. Only a small part of the N-terminus of the monomer interacts with the fibril surface of morphology A, compared to a larger part of the monomer for morphology B. Differences in ThT binding seen by fluorescence titrations, and mesoscopic structures seen by cryo-TEM, support the conclusion of the two morphologies having different surface properties. Fibrils of morphology B were found to have lower solubility than A. This indicates that fibrils of morphology B are thermodynamically more stable, implying a chemical potential of fibrils of morphology B that is lower than that of morphology A. Consequently, at prolonged incubation time, fibrils of morphology B remained B, while an initially monomorphic sample of morphology A gradually transformed to B.

Advances in the Microbiological Diagnosis of Prosthetic Joint Infections

A significant number of prosthetic joint infections (PJI) are culture-negative and/or misinterpreted as aseptic failures in spite of the correct implementation of diagnostic culture techniques, such as tissue sample processing in a bead mill, prolonged incubation time, or sonication of removed implants. Misinterpretation may lead to unnecessary surgery and needless antimicrobial treatment. The diagnostic value of non-culture techniques has been investigated in synovial fluid, periprosthetic tissues, and sonication fluid. Different feasible improvements, such as real-time technology, automated systems and commercial kits are now available to support microbiologists. In this review, we describe non-culture techniques based on nucleic acid amplification and sequencing methods. Polymerase chain reaction (PCR) is a frequently used technique in most microbiology laboratories which allows the detection of a nucleic acid fragment by sequence amplification. Different PCR types can be used to diagnose PJI, each one requiring the selection of appropriate primers. Henceforward, thanks to the reduced cost of sequencing and the availability of next-generation sequencing (NGS), it will be possible to identify the whole pathogen genome sequence and, additionally, to detect all the pathogen sequences present in the joint. Although these new techniques have proved helpful, strict conditions need to be observed in order to detect fastidious microorganisms and rule out contaminants. Specialized microbiologists should assist clinicians in interpreting the result of the analyses at interdisciplinary meetings. New technologies will gradually be made available to improve the etiologic diagnoses of PJI, which will remain an important cornerstone of treatment. Strong collaboration among all specialists involved is essential for the correct diagnosis of PJI.

Optimization of the Hemolysis Assay for the Assessment of Cytotoxicity.

In vitro determination of hemolytic properties is a common and important method for preliminary evaluation of cytotoxicity of chemicals, drugs, or any blood-contacting medical device or material. The method itself is relatively straightforward, however, protocols used in the literature vary substantially. This leads to significant difficulties both in interpreting and in comparing the obtained values. Here, we examine how the different variables used under different experimental setups may affect the outcome of this assay. We find that certain key parameters affect the hemolysis measurements in a critical manner. The hemolytic effect of compounds tested here varied up to fourfold depending on the species of the blood source. The use of different types of detergents used for generating positive control samples (i.e., 100% hemolysis) produced up to 2.7-fold differences in the calculated hemolysis ratios. Furthermore, we find an expected, but substantial, increase in the number of hemolyzed erythrocytes with increasing erythrocyte concentration and with prolonged incubation time, which in turn affects the calculated hemolysis ratios. Based on our findings we propose an optimized protocol in an attempt to standardize future hemolysis studies.

Can Prolonged Incubation of Negative Blood Cultures Show Fungal Positivity?

Objective: Standard duration of one set blood culture (BC) is a maximum of 5-7 days. The aim of this study was to evaluate “culture-negative” vials with a prolonged incubation (max 30 days) time and to observe any mycological growth. Materials and Methods: Routine BCs optained from adult patients of Balıkesir Atatürk City Hospital for a year period were included. Render BC System BC128 (Render Biotech Co. Ltd., Shenzhen, China) were used. Randomly selected vials were re-incubated for additional three weeks by conventional methods. In case of any growth, identifications were done by PhoenixTM 100 system (Becton Dickinson, MA, USA) with cornmeal tween 80 agar (RTA Laboratories, Kocaeli, Turkey). Antifungal susceptibility testing was applied with CLSI disk diffusion method. Results: A total of 6047 BC sets were obtained and randomly chosen 1040+122 negative sets (A and B groups, respectively) were included. In group A, none of them had fungal growth. In B (ongoing empiric antifungal ≤48h), only 2 sets showed significant fungal growth, which were observed in 7±2 days, and all strains were identified as C.glabrata complex and these patients were on empiric fluconazole (200 mg/day). One isolate was susceptible dose dependent, the other one was resistant for fluconazole. Latter sets of these fungemia patients showed positive signals in routine incubation period. Conclusion: Invasive fungal infections are increasingly encountered and isolation capacity and optimization of BCs are crucial. In this study, it was obviously observed that standard incubation period is satisfactory in order to detect almost all fungemia.

Antimicrobial and Antibiofilm Effect of Commonly Used Disinfectants on Salmonella Infantis Isolates.

Salmonella enterica subsp. enterica serovar Infantis is the most prevalent serovar in broilers and broiler meat in the European Union. The aim of our study was to test the biofilm formation and antimicrobial effect of disinfectants on genetically characterized S. Infantis isolates from poultry, food, and humans. For the biofilm formation under various temperature conditions (8 °C, 20 °C, and 28 °C) and incubation times (72 h and 168 h), the crystal violet staining method was used. The evaluation of the in vitro antimicrobial effect of Ecocid® S, ethanol, and hydrogen peroxide was determined using the broth microdilution method. The antibiofilm effect of subinhibitory concentration (1/8 MIC) of disinfectants was then tested on S. Infantis 323/19 strain that had the highest biofilm formation potential. Our results showed that the biofilm formation was strain-specific; however, it was higher at 20 °C and prolonged incubation time. Moreover, strains carrying a pESI plasmid showed higher biofilm formation potential. The antibiofilm potential of disinfectants on S. Infantis 323/19 strain at 20 °C was effective after a shorter incubation time. As shown in our study, more effective precautionary measures should be implemented to ensure biofilm prevention and removal in order to control the S. Infantis occurrence.

Fatty acid oxidation fuels agonist-induced platelet activation and thrombus formation: Targeting β-oxidation of fatty acids as an effective anti-platelet strategy.

Platelet mitochondria possess remarkable plasticity for oxidation of energy substrates, where metabolic dependency on glucose or fatty acids is higher than glutamine. Since platelets metabolize nearly the entire pool of glucose to lactate rather than fluxing through mitochondrial tricarboxylic acid cycle, we posit that majority of mitochondrial ATP, which is essential for platelet granule secretion and thrombus formation, is sourced from oxidation of fatty acids. We performed a comprehensive analysis of bioenergetics and function of stimulated platelets in the presence of etomoxir, trimetazidine and oxfenicine, three pharmacologically distinct inhibitors of β-oxidation. Each of them significantly impaired oxidative phosphorylation in unstimulated as well as thrombin-stimulated platelets leading to a small but consistent drop in ATP level in activated cells due to a lack of compensation from glycolytic ATP. Trimetazidine and oxfenicine attenuated platelet aggregation, P-selectin externalization and integrin αIIb β3 activation. Both etomoxir and trimetazidine impeded agonist-induced dense granule release and platelet thrombus formation on collagen under arterial shear. The effect of inhibitors on platelet aggregation and dense granule release was dose- and incubation time- dependent with significant inhibition at higher doses and prolonged incubation times. Neither of the inhibitors could protect mice from collagen-epinephrine-induced pulmonary embolism or prolong mouse tail bleeding times. However, mice pre-administered with etomoxir, trimetazidine and oxfenicine were protected from ferric chloride-induced mesenteric thrombosis. In conclusion, β-oxidation of fatty acids sustains ATP level in stimulated platelets and is therefore essential for energy-intensive agonist-induced platelet responses. Thus, fatty acid oxidation may constitute an attractive therapeutic target for novel antiplatelet agents.

Comparison of in situ ruminal straw fiber degradation and bacterial community between buffalo and Holstein fed with high-roughage diet.

Buffalo exhibits great efficiency in utilizing low-quality roughage, which can be due to the combined effect of host physiological feature and roughage diet fed. The present study was designed to compare the ruminal fiber degradation and the bacterial community attached to straws in buffalo and Holstein when fed with the same high-roughage diet using in situ ruminal incubation technique. Rice and wheat straws were selected as the incubation substrates and sampled at 0, 4, 12, 24, 48, 72, 120, and 216 h of incubation time to measure the kinetics of dry matter (DM) and neutral detergent fiber (NDF) disappearance. Additional two bags were incubated and sampled at 4 and 48 h of incubation time to evaluate the bacterial community attached to straws. The results showed that buffalo exhibited a greater (p ≤ 0.05) fraction of rapidly soluble and washout nutrients and effective ruminal disappearance for both DM and NDF of straw than Holstein, together with a greater (p ≤ 0.05) disappearance rate of potentially degradable nutrient fraction for NDF. Principal coordinate analysis indicated that both host and incubation time altered the bacterial communities attached to straws. Buffalo exhibited greater (p ≤ 0.05) 16S rRNA gene copies of bacteria and greater (p ≤ 0.05) relative abundance of Ruminococcus attached to straw than Holstein. Prolonging incubation time increased (p ≤ 0.05) the 16S rRNA gene copies of bacteria, and the relative abundance of phyla Proteobacteria and Fibrobacters by comparing 4 vs. 48 h of incubation time. In summary, buffalo exhibits greater ruminal fiber degradation than Holstein through increasing bacterial population and enriching Ruminococcus, while prolonging incubation time facilitates fiber degradation through enriching phyla Proteobacteria and Fibrobacteres.

SaKLK1-374 is more difficult to induce KLK1 expression in normal prostate cell lines than that in prostate cancer cell lines: Rethinking the universality of RNA activation

RNA activation, as a method of regulating gene expression at the transcriptional level, is far less widely used than RNA interference because of the insufficient understanding of the mechanism and the unstable success rate. It is necessary to analyze the failure cases of RNA activation to promote the application of RNA activation. When we validated the saRNAs designed to induce KLK1 expression, we found that saKLK1-374 can upregulate KLK1 expression in prostate tumor cell lines, but failed in normal prostate cell lines. To determine whether the RNA activation of normal cells is difficult only when the target gene is KLK1, we tested p21WAF1/CIP1 as the target gene in RNA activation experiments of normal and cancer prostate cells. Next, to determine whether the above phenomenon exists in other tissues, we used normal and cancerous bladder cells to perform RNA activation experiments with KLK1 and p21WAF1/CIP1 as targets. We have also extended the time from transfection to detection to evaluate whether a longer incubation time can make saRNA upregulate the target genes in normal cells. Fluorescently labeled dsRNA was transfected to evaluate the transfection efficiency, and the expression of Ago2 and IPO8 necessary for RNA activation was also detected. The p21WAF1/CIP1 could be significantly upregulated by saRNA in prostate cancer cells, but not in normal prostate cells. The expression of KLK1 in bladder-derived cell lines was extremely low and could not be induced by saRNA. The p21WAF1/CIP1 was upregulated by saRNA to a higher extent in bladder cancer cells but to a lower extent in normal bladder cells. Prolonging incubation time could not make saRNA induce the expression of target genes in normal cells. Compared with tumor cells used in this study, normal cells had lower transfection efficiency or lower expression of Ago2 and IPO8. Although it has been currently found that normal cell lines in the prostate and bladder might be more difficult to be successfully induced target gene expression by exogenous saRNA than tumor cells due to low transfection efficiency or Ago2 and IPO8 expression, it is not certain that this phenomenon occurs in other types of tissue. However, researchers still need to pay attention to the transfection efficiency and/or the expression levels of Ago2 and IPO8 when conducting RNA activation experiments in normal cells.

Climate-Mediated Recruitment Failure in a Turtle Population and Its Bearing on Northern Limits of Distribution

The eruption of Mount Pinatubo in the Philippines in June 1991 reduced global temperatures over the following 2 yrs. The greatest suppression (apart from Antarctica) was centered in the northern Great Plains of North America, directly over my long-term turtle study site. Temperatures at that site in 1992 and 1993 were the coldest in at least 50 yrs. Normal annual hatchling recruitment of yellow mud turtles (Kinosternon flavescens) in the spring following incubation at that site averaged 375; however, only 3 hatchlings emerged in 1993 (1992 incubation cohort), and none emerged in 1994 (1993 incubation cohort). The depressed temperatures apparently prolonged incubation times to such an extent in 1992 and 1993 that hatching was nearly impossible before winter mortality. The result was a gap in the age class structure that was still evident 26 yrs later. This site is at the northern range limit of this species, and this event suggests that incubation temperatures (i.e., summer season length) may be responsible for that limit.

Directional growth of quasi-2D Cu2O monocrystals on rGO membranes in aqueous environments.

The preparation technology of unconventional low-dimensional Cu2O monocrystals, which exhibit specific crystal planes and present significantly unique interfacial and physicochemical properties, is attracting increasing attention and interest. Herein, by integrating a high-temperature oxidation process under vacuum and a pure-water incubation process under ambient conditions, we propose the self-assembled growth and synthesis of quasi-two-dimensional Cu2O monocrystals on reduced graphene oxide (rGO) membranes. The prepared Cu2O crystals have a single (110) crystal plane, regular rectangular morphology, and potentially well conductivity. Experimental and theoretical results suggest that this assembly is attributed to the pre-nucleation clusters aggregation and directional attachment of Cu and O on the rGO membranes in aqueous environment and cation-π interactions between the (110) crystal plane of Cu2O and rGO surface. Our findings offer a potential avenue for the discovery and design of advanced low-dimensional single-crystal materials with specific interfacial properties in a pure aqueous environment.

Prolonged semen incubation alters the biological characteristics of human spermatozoa

The present study assessed the biological characteristics of human spermatozoa at different time intervals (0, 1, 1.5, and 2 hours) after incubation at 37°C. Twenty-five normozoospermic semen samples were incubated at 37°C. Incubation was performed at four time intervals of 0 (after liquefaction), 1, 1.5, and 2 hours. The samples were evaluated for sperm parameters at each time interval. The rate of sperm progressive motility decreased at 1.5 hours compared to 0 hours as well as 2 hours compared to 1 hour and 0 hours. The rate of non-motile spermatozoa also decreased after 2 hours compared to after 0 hours. No significant changes were observed in sperm viability (p=0.98) and non- progressive motility (p=0.48) at any time intervals. Abnormal sperm morphology increased at 1.5 hours of incubation time (p<0.001). No significant changes were observed in DNA fragmentation at 1 hour compared to 0 hours (median [interquartile range]: 19.5 [4] vs. 19 [4]), as well as at 1.5 hours compared to 1 hour (20 [5]). However, a significant increase in DNA fragmentation was observed at 1.5 hours compared to 0 hours. The mitochondrial membrane potential decreased remarkably after 1 hour of incubation time. No significant differences were observed in the acrosome reaction or malonaldehyde levels at any time point (p=0.34 and p=0.98, respectively). The incubation of normozoospermic samples before use in assisted reproductive technology should be less than 1.5 hours to minimize the destructive effects of prolonged incubation time on general and specific sperm parameters.

Photoinduced Carbene for Effective Photodynamic Therapy Against Hypoxic Cancer Cells

Photoinduced Carbene for Effective Photodynamic Therapy Against Hypoxic Cancer Cells

Low-temperature thermal-enhanced anoxic biodegradation of polycyclic aromatic hydrocarbons in aged subsurface soil

Many deep soil environments are anoxic due to the scarcity of O2, wherein NO3–, SO42–, Fe3+, Mn4+, and HCO3– (CO2) can function as terminal electron acceptors (TEAs). Anoxic biodegradation of PAHs using NO3– and SO42– as TEAs has been the subject of extensive research. However, information related to the degradation of PAHs under methanogenic conditions using CO2 as TEA is poorly understood, although it is a critical pathway in elimination of PAHs from anoxic soils. However, the low degradation rate of PAHs under anoxic conditions is the primary bottleneck restricting the promotion and application of this technology. Therefore, in this study, a low-temperature (< 50 °C) thermal enhanced biodegradation microcosm experiment was conducted using three temperatures (15 °C, 30 °C, and 45 °C) under methanogenic conditions. The results revealed that the limited PAH removal was somewhat compensated for by elevated temperature (e.g., 45 °C) compared to lower temperature (e.g., 15 °C or 30 °C), and removal efficiency of 2-, 3- and 4-ring PAHs (except for benz(a)anthracene (BaA)) obeyed the order of 45 °C > 30 °C > 15 °C. Kinetic analysis and t-test confirmed this and indicated that the promotion of PAH removal is more significant when temperature is raised from 30 °C to 45 °C than from 15 °C to 30 °C. Soil microbial communities were significantly affected by both the incubation time and temperature, and the changes in bacterial and archaeal communities were enhanced with increasing temperature. Network analysis demonstrated that soil microbes tended to cooccur rather than coexclude. The bacterial and archaeal cooccurrence network became less complex after incubation. The numbers of nodes, edges, and modules declined after 250 days of incubation, indicating that microbial function tends to be simple with prolonged incubation time. These results provide new insights and a scientific basis for the bioremediation of PAH-contaminated sites.