Nucleic acid strand displacement for indirect determination of foodborne bacteria by capillary electrophoresis and its application in antagonism and bacteriostasis studies.

Foodborne bacteria threaten human's health. Capillary electrophoresis (CE) is a powerful separation means for the determination of bacteria. Direct separation of bacteria suffers from the shortages of low resolution, channel adsorption, and bacterial aggregation. In this work, a method of nucleic acid strand displacement was developed to indirect separate the bacteria by CE. DNA complexes, consisting of probes and aptamers, were mixed with the three bacteria Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa. The aptamers could specifically bond with bacteria and release the probes. Through the separation of the probes, the bacteria could be indirectly determined by CE. This method avoided the shortages of direct separation of bacteria. Under the optimized conditions, the three probes for the bacteria could be separated and detected within 2.5 min by high-speed CE with laser-induced fluorescence detection. The limits of detection for the bacteria were in the range 4.20 × 106 to 1.75 × 107  CFU/mL. Finally, the developed method was applied on the study of antagonism of the coexistent bacteria to reveal the relationship between them. Furthermore, the efficiency of bacteriostasis of three traditional Chinese medicines, Coptis chinensis, Schisandra chinensis, and honeysuckle, was also studied by this method.

Opportunity and challenges in recovering and functionalizing anode graphite from spent lithium-ion batteries: A review.

Recent concerns have emerged regarding the improper disposal of spent lithium-ion batteries (LIBs), which has garnered widespread societal attention. Graphite materials accounted for 12-21 wt % of LIBs' mass, typically contain heavy metals, binders, and residual electrolytes. Regenerating spent graphite not only alleviated the shortage of plumbago, but also contributed to the supports environmental protection as well as national carbon peak and neutrality ("dual carbon" goals). Despite significant advancements in recycling spent LIBs had been made, a comprehensive overview of the processes for pretreatment, regeneration, and functionalization of spent graphite from retired LIBs, along with the associated technical standards and industry regulations enabling their smooth implementation still needed to be mentioned. Hence, we conducted the following research work. Firstly, the pre-treatment process of spent graphite, including discharging, crushing, and screening was summed up. Next,. Subsequently, graphite recovery methods, such as acid leaching, pyrometallurgy, and combined methods were summarized. Moreover, the modification and doping approach was used to enhance the electrochemical properties of graphite. Afterwards, we reviewed the functionalization of anode graphite from an economically and environmentally friendly view. Meanwhile, the technical standards and industry regulations of spent LIBs in domestic and oversea industries were described. Finally, we provided an overview of the technical challenges and development bottlenecks in graphite recycling, along with future prospects Overall, this study outlined the opportunities and challenges in recovering and functionalizing of anode materials via a efficient and sustainable processes.

IL-6/STAT3 axis dictates the PNPLA3-mediated susceptibility to non-alcoholic fatty liver disease.

BACKGROUND & AIMS: A number of genetic polymorphisms have been associated with susceptibility to or protection against non-alcoholic fatty liver disease (NAFLD), but the underlying mechanisms remain unknown. Here, we focused on the rs738409 C>G single nucleotide polymorphism (SNP), which produces the I148M variant of patatin-like phospholipase domain-containing protein 3 (PNPLA3) and is strongly associated with NAFLD. METHODS: To enable mechanistic dissection, we developed a human pluripotent stem cell (hPSC)-derived multicellular liver culture by incorporating hPSC-derived hepatocytes, hepatic stellate cells, and macrophages. We first applied this liver culture to model NAFLD by utilising a lipotoxic milieu reflecting the circulating levels of disease risk factors in affected individuals. We then created an isogenic pair of liver cultures differing only at rs738049 and compared NAFLD phenotype development. RESULTS: Our hPSC-derived liver culture recapitulated many key characteristics of NAFLD development and progression including lipid accumulation and oxidative stress, inflammatory response, and stellate cell activation. Under the lipotoxic conditions, the I148M variant caused the enhanced development of NAFLD phenotypes. These differences were associated with elevated IL-6/signal transducer and activator of transcription 3 (STAT3) activity in liver cultures, consistent with transcriptomic data of liver biopsies from individuals carrying the rs738409 SNP. Dampening IL-6/STAT3 activity alleviated the I148M-mediated susceptibility to NAFLD, whereas boosting it in wild-type liver cultures enhanced NAFLD development. Finally, we attributed this elevated IL-6/STAT3 activity in liver cultures carrying the rs738409 SNP to increased NF-κB activity. CONCLUSIONS: Our study thus reveals a potential causal link between elevated IL-6/STAT3 activity and 148M-mediated susceptibility to NAFLD. IMPACT AND IMPLICATIONS: An increasing number of genetic variants manifest in non-alcoholic fatty liver disease (NAFLD) development and progression; however, the underlying mechanisms remain elusive. To study these variants in human-relevant systems, we developed an induced pluripotent stem cell-derived multicellular liver culture and focused on a common genetic variant (i.e. rs738409 in PNPLA3). Our findings not only provide mechanistic insight, but also a potential therapeutic strategy for NAFLD driven by this genetic variant in PNPLA3. Our liver culture is therefore a useful platform for exploring genetic variants in NAFLD development.

Replication-Dependent Biogenesis of Turnip Crinkle Virus Long Noncoding RNAs.

Long noncoding RNAs (lncRNAs) of virus origin accumulate in cells infected by many positive-strand (+) RNA viruses to bolster viral infectivity. Their biogenesis mostly utilizes exoribonucleases of host cells that degrade viral genomic or subgenomic RNAs in the 5'-to-3' direction until being stalled by well-defined RNA structures. Here, we report a viral lncRNA that is produced by a novel replication-dependent mechanism. This lncRNA corresponds to the last 283 nucleotides of the turnip crinkle virus (TCV) genome and hence is designated tiny TCV subgenomic RNA (ttsgR). ttsgR accumulated to high levels in TCV-infected Nicotiana benthamiana cells when the TCV-encoded RNA-dependent RNA polymerase (RdRp), also known as p88, was overexpressed. Both (+) and (-) strand forms of ttsgR were produced in a manner dependent on the RdRp functionality. Strikingly, templates as short as ttsgR itself were sufficient to program ttsgR amplification, as long as the TCV-encoded replication proteins p28 and p88 were provided in trans. Consistent with its replicational origin, ttsgR accumulation required a 5' terminal carmovirus consensus sequence (CCS), a sequence motif shared by genomic and subgenomic RNAs of many viruses phylogenetically related to TCV. More importantly, introducing a new CCS motif elsewhere in the TCV genome was alone sufficient to cause the emergence of another lncRNA. Finally, abolishing ttsgR by mutating its 5' CCS gave rise to a TCV mutant that failed to compete with wild-type TCV in Arabidopsis. Collectively, our results unveil a replication-dependent mechanism for the biogenesis of viral lncRNAs, thus suggesting that multiple mechanisms, individually or in combination, may be responsible for viral lncRNA production. IMPORTANCE Many positive-strand (+) RNA viruses produce long noncoding RNAs (lncRNAs) during the process of cellular infections and mobilize these lncRNAs to counteract antiviral defenses, as well as coordinate the translation of viral proteins. Most viral lncRNAs arise from 5'-to-3' degradation of longer viral RNAs being stalled at stable secondary structures. Here, we report a viral lncRNA that is produced by the replication machinery of turnip crinkle virus (TCV). This lncRNA, designated ttsgR, shares the terminal characteristics with TCV genomic and subgenomic RNAs and overaccumulates in the presence of moderately overexpressed TCV RNA-dependent RNA polymerase (RdRp). Furthermore, templates that are of similar sizes as ttsgR are readily replicated by TCV replication proteins (p28 and RdRp) provided from nonviral sources. In summary, this study establishes an approach for uncovering low abundance viral lncRNAs, and characterizes a replicating TCV lncRNA. Similar investigations on human-pathogenic (+) RNA viruses could yield novel therapeutic targets.

Application of simplified regional citrate anticoagulation in hemodialysis patients with high risk of bleeding.

AIM: To explore the safety, effectiveness, and dialysis adequacy of simplified regional citrate anticoagulation hemodialysis (SRCA-HD) in hemodialysis patients with high risk of bleeding. MATERIALS AND METHODS: From 64 hemodialysis patients, 400 cases of low blood flow (150 mL/min, dialysate flow 300 mL/min) SRCA-HD were retrospectively analyzed and subsequently referred to as the LBF-SRCA group. Then, a prospective cross-over study was performed in 24 hemodialysis patients with normal blood flow (200 mL/min, dialysate flow 500 mL/min) SRCA-HD, which was called the NBF-SRCA group. Citrate was pumped at the artery pipeline, and calcium-containing dialysate (A group: 1.25 mmol/L, B group: 1.5 mmol/L) was used. The differences in laboratory tests, pipeline and dialyzer clotting, adequacy of dialysis, and adverse events of the groups were compared. RESULTS: 1) In the LBF-SRCA study, the correlation between citrate dosage and serum Ca2+ level at 2 hours post-filter during dialysis was negative (r = -0.228, p < 0.05). Compared with the LBF-SRCA and NBF-SRCA-A group, the pump speed of citrate in the NBF-SRCA-B group was the highest, with 355.0 ± 19.5 mL/h, 396.3 ± 11.9 mL/h, and 407.7 ± 13.0 mL/h, respectively, p < 0.001. 2) The serum Ca2+ at 2 and 4 hours post-filter during dialysis in the NBF-SRCA-B group was closer to the physiological level and significantly higher than in the A group, with 0.80 ± 0.06 vs. 0.68 ± 0.12 mmol/L, p < 0.001; 1.03 ± 0.11 vs. 0.93 ± 0.10 mmol/L, p = 0.005, respectively. 3) Both Kt/V of the NBF-SRCA-A (1.17 ± 0.24) and B (1.22 ± 0.23) group were significantly higher than that of the LBF-SRCA group (0.94 ± 0.02), p = 0.024 and p = 0.005, respectively. 4) The efficiency of anticoagulation was higher than 95% LBF-SRCA, NBF-SRCA-A and NBF-SRCA-B groups. The total clotting in the NBF-SRCA-B group (5/24) was significantly higher than that in the A group (3/24), p = 0.005. CONCLUSION: SRCA is safe, simple, and effective in hemodialysis. The dosage of citrate can be adjusted by monitoring serum Ca2+ at 2 hours post-filter during dialysis. BFR of 200 mL/min, dialysate flow rate of 500 mL/min, and 1.5 mmol/L calcium dialysate are much safer in hemodialysis patients with a high-risk of bleeding.

Transcriptomic and Non-Targeted Metabolomic Analyses Reveal the Flavonoid Biosynthesis Pathway in Auricularia cornea.

Flavonoids, which are abundant in plants, are recognized for their antioxidant and anticancer roles in clinical applications. However, little is known about the molecular basis of flavonoid biosynthesis in fungi. In this study, we found that inclusion of leachate of Korshinsk peashrub (Caragana korshinskii) in the fermentation medium increased the total flavonoid content of the edible fungus Auricularia cornea by 23.6% relative to that grown in a control medium. Combined transcriptomic and non-targeted metabolomic analysis of the flavonoid biosynthesis pathway in A. cornea illustrated that there are important metabolites in the phenylpropanoid, coumarin and isoflavonoid biosynthesis pathways. In addition, we found that certain homologous genes encode phenylalanine ammonia lyase (PAL), polyphenol oxidase (PPO) and chalcone isomerase (CHI) in these biosynthesis pathways. These results, in this study, provide a new line for studying the regulation of flavonoid production in edible fungi.

Study of antagonism between some intestinal bacteria with high-speed micellar electrokinetic chromatography.

In this work, high-speed micellar electrokinetic chromatography with LIF detection was applied to study the antagonism between three intestinal bacteria, Escherichia coli (E. coli), Bacillus licheniformis (B. licheniformis) and Bacillus subtilis (B. subtilis). The fluorescent derivatization for the bacteria was performed by labeling the bacteria with FITC. In a high-speed capillary electrophoresis (HSCE) device, the three bacteria could be completely separated within 4 min under the separation mode MEKC. The BGE was 1 × TBE containing 30 mM SDS and 1.5 × 10-5  g/mL polyethylene oxide. The limits of detection for E. coli, B. licheniformis and B. subtilis were 2.80 × 106 CFU/mL, 1.60 × 106 CFU/mL and 1.90 × 106 CFU/mL respectively. Lastly, the method was applied to investigate the antagonism between the three bacteria. The bacteria were mixed and cultured for 7 days. The samples were separated and determined every day to study the interaction between bacteria. The results showed that B. licheniformis and B. subtilis could not inhibit each other, but they could effectively inhibit the reproduction of E. coli. The method developed in this work was quick, sensitive and convenient, and it had great potential in the application of antagonism study for bacteria.

Bi-directional and temporal relationship between elevated alanine aminotransferase and hypertension in a longitudinal study of Chinese adults.

OBJECTIVE: Despite the strong correlation between elevated alanine aminotransferase (ALT) and hypertension, their bi-directional and temporal relationship are currently unclear. Our study aimed to explore the bi-directional and temporal association between elevated ALT (ALT > 40 U/L) and hypertension. METHODS: Measurements of alanine aminotransferase and blood pressure were obtained twice from 2013 to 2017 in 3314 Chinese adults without cardiovascular disease at baseline. Bi-directional and cross-lagged panel analyses were performed to dissect the temporal relationship between elevated ALT and hypertension. RESULTS: Longitudinally, we found that baseline elevated ALT was strongly correlated with incident hypertension (odds ratios = 2.16, P = .001), and baseline hypertension was also significantly associated with incident elevated ALT (odds ratios = 1.64, P = .026). The cross-lagged path coefficients from baseline ALT to follow-up blood pressure were significantly greater than that from baseline blood pressure to follow-up ALT (ß: 0.043 vs. 0.026, P < .05 for systolic blood pressure and ß: 0.052 vs. 0.024, P < .05 for diastolic blood pressure). CONCLUSION: Our results provide evidence for the bi-directional association of elevated ALT and hypertension among Chinese adults, and elevated ALT probably antedates the development of hypertension.

Superinfection Exclusion by p28 of Turnip Crinkle Virus Is Separable from Its Replication Function.

We recently reported that the p28 auxiliary replication protein encoded by turnip crinkle virus (TCV) is also responsible for eliciting superinfection exclusion (SIE) against superinfecting TCV. However, it remains unresolved whether the replication function of p28 could be separated from its ability to elicit SIE. Here, we report the identification of two single amino acid mutations that decouple these two functions. Using an Agrobacterium infiltration-based delivery system, we transiently expressed a series of p28 deletion and point mutants, and tested their ability to elicit SIE against a cointroduced TCV replicon. We found that substituting alanine (A) for valine (V) and phenylalanine (F) at p28 positions 181 and 182, respectively, modestly compromised SIE in transiently expressed p28 derivatives. Upon incorporation into TCV replicons, V181A and F182A decoupled TCV replication and SIE diametrically. Although V181A impaired SIE without detectably compromising replication, F182A abolished TCV replication but had no effect on SIE once the replication of the defective replicon was restored through complementation. Both mutations diminished accumulation of p28 protein, suggesting that p28 must reach a concentration threshold in order to elicit a strong SIE. Importantly, the severe reduction of F182A protein levels correlated with a dramatic loss in the number of intracellular p28 foci formed by p28-p28 interactions. Together, these findings not only decouple the replication and SIE functions of p28 but also unveil a concentration dependence for p28 coalescence and SIE elicitation. These data further highlight the role of p28 multimerization in driving the exclusion of secondary TCV infections.

AR-12 Exhibits Direct and Host-Targeted Antibacterial Activity toward Mycobacterium abscessus.

Therapeutic options for Mycobacterium abscessus infections are extremely limited. New or repurposed drugs are needed. The anti-M. abscessus activity of AR-12 (OSU-03012), reported to express broad-spectrum antimicrobial effects, was investigated in vitro and in vivo Antimicrobial susceptibility testing was performed on 194 clinical isolates. Minimum bactericidal concentration and time-kill kinetics assays were conducted to distinguish the bactericidal versus bacteriostatic activity of AR-12. Synergy between AR-12 and five clinically important antibiotics was determined using a checkerboard synergy assay. The activity of AR-12 against intracellular M. abscessus residing within macrophage was also evaluated. Finally, the potency of AR-12 in vivo was determined in a neutropenic mouse model that mimics pulmonary M. abscessus infection. AR-12 exhibited high anti-M. abscessus activity in vitro, with an MIC50 of 4 mg/liter (8.7 µM) and an MIC90 of 8 mg/liter (17.4 µM) for both subsp. abscessus and subsp. massiliense AR-12 and amikacin exhibited comparable bactericidal activity against extracellular M. abscessus in culture. AR-12, however, exhibited significantly greater intracellular antibacterial activity than amikacin and caused a significant reduction in the bacterial load in the lungs of neutropenic mice infected with M. abscessus No antagonism between AR-12 and clarithromycin, amikacin, imipenem, cefoxitin, or tigecycline was evident. In conclusion, AR-12 is active against M. abscessusin vitro and in vivo and does not antagonize the most frequently used anti-M. abscessus drugs. As such, AR-12 is a potential candidate to include in novel strategies to treat M. abscessus infections.

Ginkgolide B protects human pulmonary alveolar epithelial A549 cells from lipopolysaccharide-induced inflammatory responses by reducing TRIM37-mediated NF-κB activation.

The treatment options for acute stroke combined with pulmonary infection are limited. Clinically, there are several therapies to promote blood circulation and dissipate blood stasis; these treatment options include ginkgolide B (GB), which has PAF (platelet activating factor)-inhibiting effects. PAF-receptor (PAF-R) antagonists are used to treat a variety of inflammatory diseases; however, the potential of PAF-R antagonists as a treatment for lung infections remains unclear. The aim of the present study is to investigate the protective effect of GB on lipopolysaccharide-induced inflammatory responses in A549 human pulmonary alveolar epithelial cells (HPAEpiC) in vitro. Cell viability and apoptosis were measured by CCK-8 and flow cytometry. TRIM37, Caspase-3, and NF-κBp65 expression levels were measured by real-time PCR and Western blotting. The release of tumor necrosis factor-α and interleukin-1ß was measured by ELISA. The data indicates that GB may reduce TRIM37 expression by antagonizing the PAF-R pathway, thereby inhibiting the activation of nuclear factor-κB and alleviating the inflammatory response of alveolar epithelial cells. This study is the first to provide insight into the therapeutic potential of GB and suggests that clinical application of GB in acute stroke combined with pulmonary inflammation may be efficacious.

Association Between MTHFR Gene Common Variants, Serum Homocysteine, and Risk of Early-Onset Coronary Artery Disease: A Case-Control Study.

The common variants of the methylenetetrahydrofolate reductase (MTHFR) gene are related to the activity of the MTHFR enzyme and the concentrations of blood homocysteine (Hcy). This study was designed to investigate the associations of MTHFR in Chinese populations with early-onset coronary artery disease (EOCAD). The two common variants of the MTHFR gene were genotyped in 875 EOCAD patients and 956 controls using PCR, followed by direct sequencing of the PCR product. Serum levels of Hcy were measured using an automatic biochemistry analyzer. A significant association between the MTHFR-677C/T variant and the risk of EOCAD was detected in CC versus TT (odds ratio (OR) 1.456, 95% confidence interval (CI) 1.120-1.892), dominant genetic model (OR 1.266, 95% CI 1.027-1.546), and recessive genetic model (OR 1.306, 95% CI 1.040-1.639). Hcy was most abundant in TT genotype (18.31 ± 7.22 µmol/L), least abundant in CC genotype (11.37 ± 5.23 µmol/L), and detectable at intermediate levels in heterozygotes (15.25 ± 6.58 µmol/L). Elevated serum Hcy levels were an independent risk factor for EOCAD (ORadjust 1.431, 95% CI 1.135-1.763). Our findings indicated that the T allele of -677C/T MTHFR variant predisposes to high levels of Hcy, and that the T allele is an important risk factor for EOCAD in the Chinese population.

USP9X promotes LPS-induced pulmonary epithelial barrier breakdown and hyperpermeability by activating an NF-κBp65 feedback loop.

NF-κB is a central regulator of inflammatory and immune responses and has been shown to regulate transcription of several inflammatory factors as well as promote acute lung injury. However, the regulation of NF-κB signaling in acute lung injury has yet to be investigated. Human pulmonary alveolar epithelial cells (HPAEpiC) were treated with LPS to establish an acute lung injury model in vitro in which LPS stimulation resulted in pulmonary epithelial barrier breakdown and hyperpermeability. Cell viability was measured by CCK-8, and the transepithelial permeability was examined by measurement of transepithelial electrical resistance (TEER) and the transepithelial flux. Expression of ubiquitin-specific peptidase 9 X-linked (USP9X), zonula occludens (ZO-1), occludin and NF-κBp65, and the secretion of TNF-α and IL-1ß were measured by Western blotting and ELISA, respectively. For in vivo studies, mice were intraperitoneally injected with LPS and/or NF-κB inhibitor pyrrolidine dithiocarbamate (PDTC). Lung tissues were harvested for hematoxylin-eosin staining and Western blotting, and bronchoalveolar lavage fluid (BALF) was harvested for ELISA. We found that treatment with LPS in HPAEpiC inhibited cell viability and induced the expression of USP9X. Interestingly, knockdown of USP9X and treatment with PDTC suppressed LPS-induced HPAEpiC injury. USP9X overexpression promoted NF-κB activation, while NF-κB inactivation inhibited USP9X transcription and HPAEpiC injury induced by USP9X overexpression. Furthermore, LPS also induced the expression of USP9X in lungs, which was inhibited by PDTC. Taken together, these results demonstrate a critical role of USP9X-NF-κBp65 loop in mediating LPS-induced acute lung injury and may serve as a potential therapeutic target in acute lung injury.

Endothelial cell autophagy in chronic intermittent hypoxia is impaired by miRNA-30a-mediated translational control of Beclin-1.

Chronic intermittent hypoxia (CIH) in obstructive sleep apnea causes damage of aortic endothelial cells, which predisposes the development of many cardiovascular diseases. Recently, both altered expression of microRNAs (miRNAs) and impaired autophagy were found to be associated with endothelial cell dysfunction in CIH. However, the exact molecular regulatory pathway has not been determined. Here, we address this question. In a mouse model of CIH, we detected significant upregulation of miR-30a, a miRNA that targets 3'-untranslated region of autophagy-associated protein 6 (Beclin-1) messenger RNA (mRNA) for suppressing the protein translation, which subsequently attenuated the endothelial cell autophagy against cell death. Indeed, unlike Beclin-1 mRNA, the Beclin-1 protein in endothelial cells did not increase after CIH. Suppression of miR-30a by expression of antisense of miR-30a significantly increased Beclin-1 levels to enhance endothelial cell autophagy in vitro and in vivo, which improved endothelial cell survival against CIH. Together, these data suggest that endothelial cell autophagy in CIH may be attenuated by miR-30a-mediated translational control of Beclin-1 as an important cause of endothelial cell dysfunction and damage.

A self-perpetuating repressive state of a viral replication protein blocks superinfection by the same virus.

Diverse animal and plant viruses block the re-infection of host cells by the same or highly similar viruses through superinfection exclusion (SIE), a widely observed, yet poorly understood phenomenon. Here we demonstrate that SIE of turnip crinkle virus (TCV) is exclusively determined by p28, one of the two replication proteins encoded by this virus. p28 expressed from a TCV replicon exerts strong SIE to a different TCV replicon. Transiently expressed p28, delivered simultaneously with, or ahead of, a TCV replicon, largely recapitulates this repressive activity. Interestingly, p28-mediated SIE is dramatically enhanced by C-terminally fused epitope tags or fluorescent proteins, but weakened by N-terminal modifications, and it inversely correlates with the ability of p28 to complement the replication of a p28-defective TCV replicon. Strikingly, p28 in SIE-positive cells forms large, mobile punctate inclusions that trans-aggregate a non-coalescing, SIE-defective, yet replication-competent p28 mutant. These results support a model postulating that TCV SIE is caused by the formation of multimeric p28 complexes capable of intercepting fresh p28 monomers translated from superinfector genomes, thereby abolishing superinfector replication. This model could prove to be applicable to other RNA viruses, and offer novel targets for antiviral therapy.

Enhanced Ganoderic Acids Accumulation and Transcriptional Responses of Biosynthetic Genes in Ganoderma lucidum Fruiting Bodies by Elicitation Supplementation.

Ganoderic acids (GAs) are a type of highly oxygenated lanostane-type triterpenoids that are responsible for the pharmacological activities of Ganoderma lucidum. They have been investigated for their biological activities, including antibacterial, antiviral, antitumor, anti-HIV-1, antioxidation, and cholesterol reduction functions. Inducer supplementation is viewed as a promising technology for the production of GAs. This study found that supplementation with sodium acetate (4 mM) significantly increased the GAs content of fruiting bodies by 28.63% compared to the control. In order to explore the mechanism of ganoderic acid accumulation, the transcriptional responses of key GAs biosynthetic genes, including the acetyl coenzyme A synthase gene, and the expression levels of genes involved in calcineurin signaling and acetyl-CoA content have been analyzed. The results showed that the expression of three key GAs biosynthetic genes (hmgs, fps, and sqs) were significantly up-regulated. Analysis indicated that the acetate ion increased the expression of genes related to acetic acid assimilation and increased GAs biosynthesis, thereby resulting in the accumulation of GAs. Further investigation of the expression levels of genes involved in calcineurin signaling revealed that Na+ supplementation and the consequent exchange of Na+/Ca2+ induced GAs biosynthesis. Overall, this study indicates a feasible new approach of utilizing sodium acetate elicitation for the enhanced production of valuable GAs content in G. lucidum, and also provided the primary mechanism of GAs accumulation.

Investigation of cochlear hair cells and the perception of ultrasound signals in guinea pigs.

We established a specific ultrasound frequency-dependent model of cochlear injury using bone conduction ultrasounds in the inner ear of guinea pigs at 50 kHz and 83 kHz, to explore the effects of bone conduction ultrasound in the cochlea. To establish a unilateral cochlear damage model, the unilateral cochlea was destroyed. The control group consisted of 50 kHz and 83 kHz bone conduction ultrasounds in unaltered guinea pigs. In each group, cerebral blood oxygenation level dependent (BOLD) effects were determined by functional magnetic resonance imaging (fMRI). The cochlear outer hair cell motor protein, Prestin, and the microfilament protein, F-Actin, were detected. We found that bone conduction ultrasound irradiation at 50 kHz and 83 kHz on the guinea pig inner ear for six hours leads to hair cell damage. Furthermore, low frequency bone conduction ultrasound induces major damage to outer hair cells, while high frequency ultrasound damages both internal and external hair cells. fMRI analysis of cerebral BLOD effects revealed an affected cerebral cortex region of interest (ROI) of 4 and 2, respectively, for the normal control group at 50 kHz or 83 kHz, and 2 for the 83 kHz bone conduction ultrasound cochlear injury group, while 50 kHz bone conduction ultrasound failed to induce the cortical ROI within injury model. Results reveal that the spatial location of guinea pig cochlear hair cells determines coding function for lower ultrasound frequencies, and high frequency bone conduction ultrasound may affect the cochlear spiral ganglion or cranial nerve nucleus in bone conduction ultrasound periphery perception.

Establishment of a cochlear injury model using bone-conducted ultrasound irradiation in guinea pigs and investigation on peripheral coding and recognition of ultrasonic signals.

The cochlea of guinea pigs was irradiated with different frequencies of bone-conducted ultrasound (BCU) at a specific dose to induce cochlear hair cell-specific injuries, in order to establish frequency-related cochlear hair cell-specific injury models. Cochlear near-field potentials were then evoked using BCU of different frequencies and intensities to explore the peripheral coding and recognition of BCU by the cochlea. The inner ears of guinea pigs were irradiated by 30 kHz at 100 db and 80 kHz at100 db BCU for 6h to create frequency-related, ultrasound-specific cochlear injury models. Then, 30 kHz and 80 kHz BCU of different intensities were used to evoke auditory brainstem response (ABR) thresholds, compound action potential (CAP) thresholds, and action potential (AP) intensity-amplitude input-output curves in the normal control group and the ultrasonic cochlear injury group. This allowed us to explore the coding and recognition of BCU frequencies and intensities by cochlear hair cells. Immunofluorescence assay of outer hair cell (OHC) Prestin and inner hair cell (IHC) Otofelin was performed to verify the injury models. Irradiation of guinea pig inner ears by 30 kHz and 80 kHz BCU at a specific dose induced hair cell injuries at different sites. Irradiation with low frequency BCU mainly induced OHC injury, whereas irradiation with high frequency BCU induced IHC injury; moreover, IHC injury was more serious than OHC injury. The 30 kHz-evoked ABR threshold was significantly higher in the 30 kHz ultrasonic cochlear injury group compared to the normal control group. The 30 kHz-evoked ABR threshold was significantly higher in the 30 kHz ultrasonic cochlear injury group compared to the 80 kHz ultrasonic cochlear injury group. The difference in the 80 kHz-evoked ABR thresholds were not significant between the 30 kHz and 80 kHz ultrasonic cochlear injury groups. The click- and 30 kHz-evoked AP intensity-amplitude curves for the 30 kHz ultrasonic cochlear injury group indicate that the AP amplitude evoked at the same intensity was higher in the 30 kHz-evoked group than the click-evoked group. The spatial positions of cochlear hair cells in guinea pigs had a coding function for the frequencies of low-frequency ultrasound. OHCs have an amplification effect on the coding of low-frequency ultrasonic intensities. The peripheral perception of high frequency BCU may not require the participation of cochlear hair cells.

Theoretical Study on the Reaction Mechanism and Kinetics of Criegee Intermediate CH2OO with Acrolein.

The detailed reaction mechanism and kinetics of Criegee intermediate CH2OO with acrolein were investigated. CH2OO may add to the C═O or C═C double bond of acrolein to form a five-membered ring adducts, and it may also insert the terminal oxygen atom or insert itself into the C-H bond of acrolein. The addition reactions are more favorable in energy than the insertion reactions. The master equation calculation show that the most competitive reaction channel is the 1,3-cycloaddition of CH2OO across the C═O double bond forming the secondary ozonide (SOZ). The lowest energy pathway for SOZ decomposition involves the formation of the singlet biradical intermediate by ring fission, the H-shift isomerization and the dissociation to products. The calculated overall rate constant decreases as the temperature increases from 200 to 500 K, and at 298 K, it is 4.31 × 10-12 cm3 molecule-1 s-1. The branching ratio of collisionally stabilized SOZ increases with the increase of pressure. At low pressure, some of SOZ decompose to HCOOH + acrolein or HCHO + acrylic acid. The pressure dependence of this reaction is in agreement with the previous theoretical and experimental observations for the reaction of CH2OO with acetaldehyde.