Growth, nutrient removal, and lipid productivity promotion of Chlorella sorokiniana by phosphate solubilizing bacteria Bacillus megatherium in swine wastewater: Performances and mechanisms.

Effects of a phosphorus-solubilizing bacteria (PSB) Bacillus megatherium on growth and lipid production of Chlorella sorokiniana were investigated in synthesized swine wastewater with dissolved inorganic phosphorus (DIP), insoluble inorganic phosphorus (IIP), and organic phosphorus (OP). The results showed that the PSB significantly promoted the algal growth in OP and IIP, by 1.10 and 1.78-fold, respectively. The algal lipid accumulation was also greatly triggered, respectively by 4.39, 1.68, and 1.38-fold in DIP, IIP, and OP. Moreover, compared with DIP, OP improved the oxidation stability of algal lipid by increasing the proportion of saturated fatty acids (43.8 % vs 27.9 %), while the PSB tended to adjust it to moderate ranges (30.2-41.6 %). Further, the transcriptome analysis verified the OP and/or PSB-induced up-regulated genes involving photosynthesis, lipid metabolism, signal transduction, etc. This study provided novel insights to enhance microalgae-based nutrient removal combined with biofuel production in practical wastewater, especially with complex forms of phosphorus.

Enhancing lipid production and sedimentation of Chlorella pyrenoidosa in saline wastewater through the addition of agricultural phytohormones.

In this study, the effect of external agricultural phytohormones (mixed phytohormones) addition (1.0, 5.0, 10.0, and 20.0 mg L-1) on the growth performance, lipid productivity, and sedimentation efficiency of Chlorella pyrenoidosa cultivated in saline wastewater was investigated. Among the different concentrations evaluated, the highest biomass (1.00 g L-1) and lipid productivity (11.11 mg L-1 d-1) of microalgae were obtained at 10.0 mg L-1 agricultural phytohormones addition. Moreover, exogenous agricultural phytohormones also improved the sedimentation performance of C. pyrenoidosa, which was conducive to the harvest of microalgae resources, and the improvement of sedimentation performance was positively correlated with the amount of agricultural phytohormones used. The promotion of extracellular polymeric substances synthesis by phytohormones in microalgal cells could be considered as the reason for its promotion of microalgal sedimentation. Transcriptome analysis revealed that the addition of phytohormones upregulated the expression of genes related to the mitogen-activated protein kinase (MAPK)-mediated phytohormone signaling pathway and lipid synthesis, thereby improving salinity tolerance and lipid production in C. pyrenoidosa. Overall, agricultural phytohormones provide an effective and inexpensive strategy for increasing the lipid productivity and sedimentation efficiency of microalgae cultured in saline wastewater.

Efficient coupling of sulfadiazine removal with microalgae lipid production in a membrane photobioreactor.

This study explored the feasibility of a coupled system for antibiotic removal and biofuel production through microalgae cultivation. Initial, batch culture experiments demonstrated that sulfadiazine (SDZ) had an inhibitory effect on Chlorella sp. G-9, and 100.0 mg L-1 SDZ completely inhibited its growth. In order to improve SDZ removal efficiency by microalgae, three membrane photobioreactors (MPBRs) with different hydraulic retention times (HRTs) were established for continuous microalgae cultivation. The efficient coupling of SDZ removal and microalgal lipid production was achieved through the gradual increment of influent SDZ concentration from 0 to 100.0 mg L-1. The reduction in SDZ ranged between 57.8 and 89.7%, 54.7-91.7%, and 54.6-93.5% for the MPBRs with HRT of 4 d, 2 d, and 1 d, respectively. Chlorella sp. Was found to tolerate higher concentrations of SDZ in the MPBR system, and the resulting stress from high concentrations of SDZ effectively increased the lipid content of microalgae for potential biodiesel production. With the increase of influent SDZ concentration from 0 to 100.0 mg L-1, the lipid content of microalgae increased by 43.5%. Chlorophyll content, superoxide dismutase activity, and malondialdehyde content of microalgae were also evaluated to explore the mechanism of microalgae tolerance to SDZ stress in MPBR.

Comparisons of Supine Roll Test and Alternative Positional Tests in HC-BPPV Lateralization.

OBJECTIVE: The purpose of the study was to evaluate the efficiency of the supine roll test (SRT) and alternative positional tests (APTs) including the bow and lean test (BLT), pseudo-spontaneous nystagmus (PSN), and lying down nystagmus (LDN) to identify the affected side in horizontal canal benign paroxysmal positional vertigo (HC-BPPV). METHODS: In our prospective study, we performed a testing profile (PSN, BLT, LDN, SRT) on 59 HC-BPPV patients using videonystagmography. We compared the accuracy and sensitivity of these tests in HC-BPPV lateralization. Data from 30 healthy patients were collected as the control group. RESULTS: When performing positional tests, the elicited nystagmus coinciding with Ewald's second law was defined as a "positive response". In 44 patients with geotropic nystagmus, the rates of positive response in LDN, PSN, and BLT were 22/44 (50%), 19/44 (43%), and 18/44 (41%), respectively, while in 15 patients with apogeotropic nystagmus, the positive response rates of these three tests were 10/15 (66.7%), 9/15 (60%), and 4/15 (27.00%), respectively. The sensitivity of LDN (54.38%) was higher than that of PSN (47.37%) and BLT (38.60%) but lower than that of SRT (89.47%). Notably, the accuracy rate of PSN (71.8%) was higher than that of the other APTs. In 6 patients with symmetrical nysgtamus during the roll test, 5 patients showed a positive response in both LDN and BLT (83.34%), whereas 4 patients showed a positive response in PSN (66.67%). CONCLUSION: All positional tests are helpful for determining the affected side of HC-BPPV, but SRT carries the highest accuracy of lateralization followed by PSN.

Expression of α2-Na/K-ATPase in C57BL/6J Mice Inner Ear and Its Relationship with Age-related Hearing Loss.

K+ cycling in the cochlea is critical to maintain hearing. Many sodium-potassium pumps are proved to participate in K+ cycling, such as Na/K-ATPase. The α2-Na/K-ATPase is an important isoform of Na/K-ATPase. The expression of α2-Na/K-ATPase in the cochlea is not clear. In this study, we used C57BL/6 mice as a model of presbycusis and implemented immunohistochemistry staining and quantitative real time-PCR, and the α2-Na/K-ATPase expression pattern was confirmed in the inner ear. It was found α2-Na/K-ATPase was expressed widely in cochlea and its mRNA and protein expression was gradually reduced with aging (4-, 14-, 26- and 48-weeks old mice). We suspected that, the down-regulation of α2-Na/K-ATPase expression might be associated with the remodeling of K+ cycling, degeneration of morphological structure and decrease of hearing function in aging C57 mice. In conclusion, we speculated that the reduction of α2-Na/K-ATPase might play an important role in the pathogenesis of age-related hearing loss.

Expression Patterns and Implications of LaminB1 in Rat Cochleae.

LaminB1, a major component of the nuclear lamina, is a potent regulator of cellular proliferation and senescence and also known to be essential for neuronal migration and brain development. However, the expression patterns of LaminB1 in the rat cochleae are still not fully revealed. Utilizing immunofluorescence, Western blotting, and quantitative real-time PCR, we identified the distribution and expression of LaminB1 in the rat cochleae. Immunofluorescence staining indicated that LaminB1 was mainly localized in the auditory hair cells (HCs), spiral ganglion cells (SGC), stria vascularis (STV, including spiral ligament), Reissner's membrane (RM), and limbus laminae spiralis (LLS). Western blotting analysis illustrated that the distribution of LaminB1 in rat cochleae was characterized by tissue specificity. The LaminB1 protein was expressed more in SGC and basilar membrane (BM) than in STV. Meanwhile, the mRNA expression of LaminB1 displayed difference in cochlear tissues. These observations preliminarily revealed the expression patterns of LaminB1, providing a theoretical basis for further study on the role of LaminB1 in auditory function.

Molecular Dynamics Simulations of Molecular Diffusion Equilibrium and Breakdown Mechanism of Oil-Impregnated Pressboard with Water Impurity.

The water molecule migration and aggregation behaviors in oil-impregnated pressboard are investigated by molecular dynamics simulations in combination with Monte Carlo molecular simulation technique. The free energy and phase diagram of H2O-dodecylbenzene (DDB) and H2O-cellulose mixtures are calculated by Monte Carlo technique combined with the modified Flory-Huggins model, demonstrating that H2O molecules can hardly dissolved with infinitesimal content in cellulose system at temperature lower than 650 K, based on which the oil/cellulose layered structure with water impurity representing three-phase coexistence in oil-impregnated pressboard are modeled and performed for molecular dynamics. The molecular dynamics of H2O/DDB/cellulose three-phase mixture simulating oil-paper insulating system with H2O impurity indicates that DDB molecules can thermally intrude into the cellulose-water interface so as to separate the water phase and cellulose fiber. The first-principles electronic structure calculations for local region of H2O/DDB interface show that H2O molecules can introduce bound states to trap electrons and acquire negative charges, so that they will obtain sufficient energy from applied electric field to break DDB molecular chain by collision, which are verified by subsequent molecular dynamics simulations of H2O-/DDB interface model. The electric breakdown mechanism under higher than 100 kV/m electric field is presented based on the further first-principles calculations of the produced carbonized fragments being dissolved and diffusing in DDB phase. The resulted broken DDB fragments will introduce impurity band between valence and conduction bands of DDB system, evidently decreasing bandgap as to that of conducting materials in their existence space. The conductance channel of these carbonized DDB fragments will eventually be formed to initiate the avalanche breakdown process by the cycle-feedback of injected charge carriers with carbonized channels.

Downregulation of inwardly rectifying potassium channel 5.1 expression in C57BL/6J cochlear lateral wall.

Age-related hearing loss (AHL) is one of the most common sensory disorders among elderly persons. The inwardly rectifying potassium channel 5.1 (Kir5.1) plays a vital role in regulating cochlear K(+) circulation which is necessary for normal hearing. The distribution of Kir5.1 in C57BL/6J mice cochleae, and the relationship between the expression of Kir5.1 and the etiology of AHL were investigated. Forty C57BL/6J mice were randomly divided into four groups at 4, 12, 24 and 52 weeks of age respectively. The location of Kir5.1 was detected by immunofluorescence technique. The mRNA and protein expression of Kir5.1 was evaluated in mice cochleae using real-time polymerase-chain reactions (RT-PCR) and Western blotting respectively. Kir5.1 was detected in the type II and IV fibrocytes of the spiral ligament in the cochlear lateral wall of C57BL/6J mice. The expression levels of Kir5.1 mRNA and protein in the cochleae of aging C57BL/6J mice were down-regulated. It was suggested that the age-related decreased expression of Kir5.1 in the lateral wall of C57BL/6J mice was associated with hearing loss. Our results indicated that Kir5.1 may play an important role in the pathogenesis of AHL.

[Association of age-related hearing loss with ion transporter KCNQ1 and NKCC1 in cochlea of C57BL/6J mice].

OBJECTIVE: To investigate the age-related expression of KCNQ1 and NKCC1 ion transporters in the stria vascularis in the cochlea of C57BL/6J mice, and to analyze the relationship between the two ion transporters and age-related hearing loss. METHODS: Auditory function of C57BL/6J mice was measured by auditory brainstem response (ABR) at the ages of 4, 8, 14, 24, 40 weeks old respectively. The location of KCNQ1 and NKCC1 ion transporters in the cochlea of C57BL/6J mice was detected by immunohistochemistry staining. Reverse transcription polymerase chain reaction (RT-PCR) was used to detect the levels of KCNQ1 and NKCC1 mRNA in the cochlea of C57BL/6J mice at different ages. RESULTS: The mean values for ABR thresholds in response to click, 4 kHz and 8 kHz sound stimulus of C57BL/6J mice gradually increased with age. The ABR thresholds of the mice of over 14 weeks age were significantly elevated in comparison with lower ages (P < 0.05). In the lateral wall of C57BL/6J mice cochlea, the KCNQ1 protein was mainly expressed at the apical membrane of the strial marginal cells. The localization of NKCC1 protein was mainly present at the basolateral membrane of the stria marginal cells, spiral ligament and the fibrocytes in the inferior portion of spiral limbus. Expression of KCNQ1 and NKCC1 protein in cochlea of C57BL/6J mice showed age-related decreasing. The level of KCNQ1 and NKCC1 mRNA in cochlea of C57BL/6J also showed a age-related decreasing trend. There was a significant reducing of KCNQ1 mRNA level between C57BL/6J mice of 40 and 4 weeks old (P < 0.05). In comparison with the C57BL/6J mice of 4 weeks old, the NKCC1 mRNA levels of 24 and 40 weeks old also showed significant reducing (P < 0.05). CONCLUSIONS: The mean value for ABR thresholds of C57BL/6J mice gradually increased with age. Expression of KCNQ1 and NKCC1 protein in the stria vascularis of C57BL/6J mice decreases with age. The levels of KCNQ1 and NKCC1 mRNA in cochlea of C57BL/6J showed a age-related reducing trend. Regulating after post-translation may also participate in the adjusting of the age-related decreasing of KCNQ1 and NKCC1 protein in the cochlea of C57BL/6J mice. KCNQ1 and NKCC1 ion transporters may play a critical role in maintaining normal hearing function of inner ear.

[A novel retractor designed for mouse microsurgery].

OBJECTIVE: To introduce a novel retractor with magnetic fixator for mouse microsurgery. METHODS: The retractor was consisted of a magnet, a screw, two screw nuts and a hook made of dental stainless wire. The screw was connected to the magnet with magnetic force, and then was assembled to be a so-called magnetic fixator. The hook was clamped by two screw nuts on the screw, and these makes up of the retractor finally. Comparison has been done between the novel retractor and traditional retractor in the clinical application of the otocyst exposure. RESULTS: The retractor can quickly claw and retract massive tissue like muscles and vessels to the target position, thus, this properties would tend to offer a clear and expanded operative field. In addition, the height, orientation and strength of traction was all adjustable. By Comparison with tradition retractor, the operative incision can be shorten via the application of the retractor, also, it would reduce the trauma of muscles and vessels as well as the accidental rate of bleeding in the process of operation. CONCLUSIONS: The retractor can offer a expanded operative field of the mouse otocyst conveniently. It could be a simple, powerful and minimal invasive tool for mouse microsurgery.

[Rapid cochlea lesion induced by co-administration of kanamycin and furosemide in mouse].

OBJECTIVE: To investigate the ototoxicity of co-administration of kanamycin and furosemide in mouse and establish a reliable model to induce a sensorineural hearing loss. METHODS: CBA/J mice strain was selected, with the age around 3-4 weeks old, to be received a single subcutaneous injection of kanamycin at dose of 1 g/kg and another single intraperitoneal injection of furosemide at dose of 0.4 g/kg 30 - 45 min afterward. The auditory brainstem response (ABR) threshold shift was tested. The series of experimental methods including propidium iodide, phalloidin staining, semithin section toluidine blue staining, TUNEL, scanning electron microscopy and transmission electron microscopy were applied to observe the characteristics of the lesion of cochlea and hair cells. The time course was set as following: before injection, 12, 24, 48 hours and 1, 2, 4, 12 weeks after injections, respectively. RESULTS: The ABR threshold shift was firstly presented a significant increase at 12 h after injection at 2, 4, 8 kHz, then the ABR threshold kept going up during next 36 h until it was presented a stable level around 90 dB. Pathological examination showed an absence of outer hair cells at basal turn rapidly since 12 h after treatment, and then by 48 h the most commonly observed lesion, where all outer hair cells throughout the length of the cochlea were killed, in the contrast, however, the inner hair cells loss were delayed and mild. TUNEL-positive nuclei demonstrated that most hair cells died via an apoptotic pathway. In scanning electron microscopy abundance of necrotic outer hair cells were detected by 24 h after treatment, in which reticular lamina were collapsed. Then all outer hair cells were replaced by expansion of heads of supporting cells. At 48 h after treatment, marginal cells presented a swollen and some of them were observed to be fused. In addition, spherical cell extrusion appeared to leak out from some marginal cells. By 2 weeks, nearly all microvillus were lost and marginal cells presented a shape of stone-like change. A significant and progressive decrease in strial vascularis thickness was found, of which the reason probably related with a reduction in volume of marginal cells. CONCLUSION: This systemic protocol eliminates hair cells extensively in vivo, and it could be a reliable model to examine different aspects of cochlear pathology in transgenic or mutant mice strains.

[Expression of plasma membrane Ca²(+)-ATPase isoform 2 in spiral ganglion cells of newborn rats].

OBJECTIVE: To study the expression of plasma membrane Ca(2+)-ATPase isoform 2 (PMCA2) in spiral ganglion cell (SGC) from inner ear of newborn rats and further check PMCA2 splice variants at site A and C. METHODS: Spiral ganglion tissues isolated from cochlea of newborn rats (P3-P4) were cultured and identified in vitro. The cochlea of newborn rats (P3-P4) were isolated and cut into frozen sections. The expression of PMCA2 was detected by immunofluorescence analyses. The SGC cultured in 4 wells of the 6-well culture plate were collected and the total RNA was extracted by Trizol and reverse transcribed to cDNA. The site A and C splice variants of PMCA2 were respectively checked by nested PCR and common PCR. RESULTS: The SGC grew well with good refraction and showed positive immunoreactivity for neuronal marker NF-200. Strong green fluorescence could be seen in cytomembrane, cytoplasm and neuritis, as well showing SGC immunoreactivity for PMCA2 antibody. In the cochlear sections, the spiral ganglion tissues were strongly stained by PMCA2. PMCA2z was present at splice site A, but PMCA2b and PMCA2c were present at splice site C. CONCLUSION: SGC from newborn rats strongly expresses PMCA2 and different splice variants are present at PMCA2 splice site A and C.

[Application of PCR-dGGE in selective activation of stratal bacteria].

With the development of molecular biology, the research on microflora in the environment is accelerated and a new research method for oil reservoir microflora is provided. Based on the methods of PCR-DGGE, the change of the structure of dominant microbial population in selectiveactivation of stratal bacteria was studied. During activation, the change of samples' DGGE bandings under different pressure conditions (1 MPa and 10 MPa) was analyzed, the PCR alignment analysis of bacteria was studied, the dominant bacteria at different periods were explored, the phylogenetic tree of dominant DGGE banding sequence was build, and the groups of the main indigenous bacteria was also analyzed. The result showed that the structure and the quantity of stratal microorganism indicated obvious differences, under different pressure conditions. The species number of dominant microbial population increased, and the structure of dominant microbial population obviously changed after activation. Before activation, the main bacteria were belonged to Bacillales, and after injecting activation agent, the species of Proteobacteria increased.