Rufomycins or Ilamycins: Naming Clarifications and Definitive Structural Assignments.

Rufomycin and ilamycin are synonymous for the same class of cyclopeptides, currently encompassing 33 structurally characterized isolates and 9 semisynthetic derivatives. Elucidation of new structures prioritized the consolidation of the names and established the structures of four diastereoisomeric rufomycins with a 2-piperidinone, named rufomycins 4-7, including full 1H/13C NMR assignments. The characteristic HSQC cross-peak for the CH-5, the hemiaminal carbon in amino acid #5, allows assignment of the stereocenters C-4 and C-5 within this ring. Semisynthetic derivatives (rufomycinSS 1, 2, and 3) were prepared from a rufomycins 4 and 6 mixture to validate the structural assignments. Based on the X-ray crystal structures of rufomycins 2 and 4, considering the NMR differences of rufomycins 7 vs 4-6 compared to rufomycinSS 1 vs 2 and 3, and taking into account that two major conformers, A and B, occur in both rufomycinSS 2 and 3, structural modeling was pursued. Collectively, this paper discusses the NMR spectroscopic differences of the stereoisomers and their possible 3D conformers and correlates these with the anti-Mycobacterium tuberculosis activity. In addition, a look at the history prioritizes names and numbering schemes for this group of antibiotics and leads to consolidated nomenclature for all currently known members, natural and semisynthetic derivatives, and serves to accommodate future discoveries.

Aquaporin 1 alleviates acute kidney injury via PI3K-mediated macrophage M2 polarization.

BACKGROUND: Lipopolysaccharide (LPS)-induced acute kidney injury (AKI) is associated with an abnormal immune response. Accumulating evidence has demonstrated that aquaporin 1 (AQP1) prevents kidney tissue injury in LPS-induced AKI by mediating immune response. However, the underlying mechanisms remain obscure. Macrophages as immune cells with multiple phenotypes are important mediators in tissue homeostasis and host defense. We propose that macrophage polarization is implicated in AQP1-mediated immune response. METHODS: Herein we established sepsis-induced AKI model rats through intraperitoneal injection of LPS into Wistar rats to reveal immune mechanism of damage. We also used LPS-induced mouse RAW264.7 cells to elucidate the molecular mechanism of macropage polarization. RESULTS: Histopathology showed that renal tubular epithelial cells in the model group were swollen, inflammatory exudation was obvious and the inflammatory factors, interleukin-6 (IL-6) and tumor necrosis factor α (TNF-α) were increased. Western blotting showed PI3K was upregulated in the model group. Serum creatinine and urea nitrogen increased after LPS injection. Renal AQP1 mRNA is downregulated and serum AQP1 protein increased first and then decreased in LPS-induced AKI rats. M2 macrophage markers (Arg-1, CD206) were increased in repair stage. In addition, treatment of murine macrophages (RAW264.7) with AQP1 siRNA resulted in decreased PI3K activation and M2 polarization, but increased IL-6 and TNF-α. Moreover, inhibiting PI3K with wortmannin imitated the results of AQP1 silencing. CONCLUSIONS: Macrophage M2 polarization is likely the cellular mechanism underlying the anti-AKI property of AQP1, and PI3K activation is involved in the AQP1-induced M2 phenotype switch.

Antimycobacterial Rufomycin Analogues from Streptomyces atratus Strain MJM3502.

This study represents a systematic chemical and biological study of the rufomycin (RUF) class of cyclic heptapeptides, which our anti-TB drug discovery efforts have identified as potentially promising anti-TB agents that newly target the caseinolytic protein C1, ClpC1. Eight new RUF analogues, rufomycins NBZ1-NBZ8 (1-8), as well as five known peptides (9-13) were isolated and characterized from the Streptomyces atratus strain MJM3502. Advanced Marfey's and X-ray crystallographic analysis led to the assignment of the absolute configuration of the RUFs. Several isolates exhibited potent activity against both pathogens M. tuberculosis H37Rv and M. abscessus, paired with favorable selectivity (selectivity index >60), which collectively underscores the promise of the rufomycins as potential anti-TB drug leads.

Industrial attributes of ß-glucanase produced by Bacillus sp. CSB55 and its potential application as bio-industrial catalyst.

The ß-glucanase produced from Bacillus sp. CSB55 not only depicts the potent industrial characteristics but also relates as bio-industrial catalyst supporting the spontaneous formation of the products, high hydrolytic efficiency, and feasibility of the enzymatic reaction. A homogeneous ß-glucanase (GluB55) was purified via various purification processes resulting in 11.69% yield and 14.24-fold purity. Biochemical characterization of the purified enzyme revealed the molecular mass of approximately 40 kDa, which was verified by zymography. The optimum activity of GluB55 was determined at pH 7.2 and 55 °C. GluB55 could highly hydrolyze carboxymethylcellulose and was stable over a wide range of pH, retaining more than 70% residual activity at pH 5.8-11.0 and carried 100% thermostability as high as 60 °C. In addition, it showed 68% residual activity at 70 °C. The N-terminal amino acid sequence of GluB55 was Ala-Asn-Pro-Glu-Leu-Val-Asn-X-Gln-Ala-X-X-Ala-X-Gln-Gly. The enzyme activity was stimulated by Co2+ (158.6%), Zn2+ (211.1%), Mn2+ (264.4%), and Ba2+ (211.4%). Enzyme kinetics showed Km and Vmax values of 0.022 mg mL-1 and 994.56 ± 3.72 U mg-1, respectively. Q10 was calculated to be 1.12. ∆H, ∆G, and ∆S were low revealing that the formation of the transition phase and conversion to the product is very well organized. The lower the free energy change (∆G), the more feasible is the reaction.

Rufomycin Targets ClpC1 Proteolysis in Mycobacterium tuberculosis and M. abscessus.

ClpC1 is an emerging new target for the treatment of Mycobacterium tuberculosis infections, and several cyclic peptides (ecumicin, cyclomarin A, and lassomycin) are known to act on this target. This study identified another group of peptides, the rufomycins (RUFs), as bactericidal to M. tuberculosis through the inhibition of ClpC1 and subsequent modulation of protein degradation of intracellular proteins. Rufomycin I (RUFI) was found to be a potent and selective lead compound for both M. tuberculosis (MIC, 0.02 µM) and Mycobacterium abscessus (MIC, 0.4 µM). Spontaneously generated mutants resistant to RUFI involved seven unique single nucleotide polymorphism (SNP) mutations at three distinct codons within the N-terminal domain of clpC1 (V13, H77, and F80). RUFI also significantly decreased the proteolytic capabilities of the ClpC1/P1/P2 complex to degrade casein, while having no significant effect on the ATPase activity of ClpC1. This represents a marked difference from ecumicin, which inhibits ClpC1 proteolysis but stimulates the ATPase activity, thereby providing evidence that although these peptides share ClpC1 as a macromolecular target, their downstream effects are distinct, likely due to differences in binding.

Aquaporin-1 attenuates macrophage-mediated inflammatory responses by inhibiting p38 mitogen-activated protein kinase activation in lipopolysaccharide-induced acute kidney injury.

OBJECTIVE: This study was designed to investigate the role of AQP1 in the development of LPS-induced AKI and its potential regulatory mechanisms in the inflammatory responses of macrophages. METHODS: Male Wistar rats were injected intraperitoneally with LPS, and biochemical and histological renal damage was assessed. The levels of inflammatory mediators, macrophage markers and AQP1 in blood and kidney tissues were assessed by ELISA. RTPCR was used to assess changes in the relative levels of AQP1 mRNA induced by LPS. Western blot and immunofluorescence analyses were performed to assay the activation of the p38 MAPK and NF-κB pathways, respectively. The same detection methods were used in vitro to determine the regulatory mechanisms underlying AQP1 function. RESULTS: AQP1 mRNA levels were dramatically decreased in AKI rats following the increased expression of inflammatory factors. In vitro experiments demonstrated that silencing the AQP1 gene increased inflammatory mediator secretion, altered the classical activation of macrophages, greatly enhanced the phosphorylation of p38 and accelerated the translocation of NF-κB. Furthermore, these results were blocked by doramapimod, a p38 inhibitor. Therefore, these effects were mediated by the increased phosphorylation of p38 MAPK. CONCLUSION: Our results suggest that altered AQP1 expression may be associated with the development of inflammation in AKI. AQP1 plays a protective role in modulating acute renal injury and can attenuate macrophage-mediated inflammatory responses by downregulating p38 MAPK activity in LPS-induced RAW264.7 cells. The pharmacological targeting of AQP1-mediated p38 MAPK signalling may provide a novel treatment approach for AKI.

Inhibition of inflammation using diacerein markedly improved renal function in endotoxemic acute kidney injured mice.

BACKGROUND: Inflammation is an important pathogenic component of endotoxemia-induced acute kidney injury (AKI), finally resulting in renal failure. Diacerein is an interleukin-1ß (IL-1ß) inhibitor used for osteoarthritis treatment by exerting anti-inflammatory effects. This study aims to investigate the effects of diacerein on endotoxemia-induced AKI. METHODS: Male C57BL/6 mice were intraperitoneally injected with lipopolysaccharide (LPS, 10 mg/kg) for 24 h prior to diacerein treatment (15 mg/kg/day) for another 48 h. Mice were examined by histological, molecular and biochemical approaches. RESULTS: LPS administration showed a time-dependent increase of IL-1ß expression and secretion in kidney tissues. Diacerein treatment normalized urine volume and osmolarity, reduced blood urea nitrogen (BUN), fractional excretion of sodium (FENa), serum creatinine and osmolarity, and protected renal function in an endotoxemic AKI mice model. In the histopathologic study, diacerein also improved renal tubular damage such as necrosis of the tubular segment. Moreover, diacerein inhibited LPS-induced increase of inflammatory cytokines, such as IL-1ß, tumor necrosis factor-α, monocyte chemoattractant protein-1 and nitric oxide synthase 2. In addition, LPS administration markedly decreased aquaporin 1 (AQP1), AQP2, AQP3, Na,K-ATPase α1, apical type 3 Na/H exchanger and Na-K-2Cl cotransporter expression in the kidney, which was reversed by diacerein treatment. We also found that diacerein or IL-1ß inhibition prevented the secretion of inflammatory cytokines and the decrease of AQP and sodium transporter expression induced by LPS in HK-2 cells. CONCLUSION: Our study demonstrates for the first time that diacerein improves renal function efficiently in endotoxemic AKI mice by suppressing inflammation and altering tubular water and sodium handing. These results suggest that diacerein may be a novel therapeutic agent for the treatment of endotoxemic AKI.

The cyclic peptide ecumicin targeting ClpC1 is active against Mycobacterium tuberculosis in vivo.

Drug-resistant tuberculosis (TB) has lent urgency to finding new drug leads with novel modes of action. A high-throughput screening campaign of >65,000 actinomycete extracts for inhibition of Mycobacterium tuberculosis viability identified ecumicin, a macrocyclic tridecapeptide that exerts potent, selective bactericidal activity against M. tuberculosis in vitro, including nonreplicating cells. Ecumicin retains activity against isolated multiple-drug-resistant (MDR) and extensively drug-resistant (XDR) strains of M. tuberculosis. The subcutaneous administration to mice of ecumicin in a micellar formulation at 20 mg/kg body weight resulted in plasma and lung exposures exceeding the MIC. Complete inhibition of M. tuberculosis growth in the lungs of mice was achieved following 12 doses at 20 or 32 mg/kg. Genome mining of lab-generated, spontaneous ecumicin-resistant M. tuberculosis strains identified the ClpC1 ATPase complex as the putative target, and this was confirmed by a drug affinity response test. ClpC1 functions in protein breakdown with the ClpP1P2 protease complex. Ecumicin markedly enhanced the ATPase activity of wild-type (WT) ClpC1 but prevented activation of proteolysis by ClpC1. Less stimulation was observed with ClpC1 from ecumicin-resistant mutants. Thus, ClpC1 is a valid drug target against M. tuberculosis, and ecumicin may serve as a lead compound for anti-TB drug development.

Rapid construction of a Bacterial Artificial Chromosomal (BAC) expression vector using designer DNA fragments.

Bacterial artificial chromosomal (BAC) vectors are increasingly being used in cloning large DNA fragments containing complex biosynthetic pathways to facilitate heterologous production of microbial metabolites for drug development. To express inserted genes using Streptomyces species as the production hosts, an integration expression cassette is required to be inserted into the BAC vector, which includes genetic elements encoding a phage-specific attachment site, an integrase, an origin of transfer, a selection marker and a promoter. Due to the large sizes of DNA inserted into the BAC vectors, it is normally inefficient and time-consuming to assemble these fragments by routine PCR amplifications and restriction-ligations. Here we present a rapid method to insert fragments to construct BAC-based expression vectors. A DNA fragment of about 130 bp was designed, which contains upstream and downstream homologous sequences of both BAC vector and pIB139 plasmid carrying the whole integration expression cassette. In-Fusion cloning was performed using the designer DNA fragment to modify pIB139, followed by λ-RED-mediated recombination to obtain the BAC-based expression vector. We demonstrated the effectiveness of this method by rapid construction of a BAC-based expression vector with an insert of about 120 kb that contains the entire gene cluster for biosynthesis of immunosuppressant FK506. The empty BAC-based expression vector constructed in this study can be conveniently used for construction of BAC libraries using either microbial pure culture or environmental DNA, and the selected BAC clones can be directly used for heterologous expression. Alternatively, if a BAC library has already been constructed using a commercial BAC vector, the selected BAC vectors can be manipulated using the method described here to get the BAC-based expression vectors with desired gene clusters for heterologous expression. The rapid construction of a BAC-based expression vector facilitates heterologous expression of large gene clusters for drug discovery.

Construction and comparison of short-term prognosis prediction model based on machine learning in acute ischemic stroke.

Objective: To construct and compared the short-term prognosis prediction models of acute ischemic stroke (AIS) by machine learning (ML). Methods: Retrospectively study. The group W (mRS≤3) was clustered, and combined with group P (mRS>3) to form the post-clustering dataset for modeling. The "glmnet", "rpart", "xgboost", "randomForest", "neuralnet" packages were used to construct ML models. The accuracy, sensitivity, specificity, positive predict value (PPV), negative predict value (NPV) among the models were compared. Four external clinical datasets were used for external clinical validation. The optimal prediction model was determined by variable screening ability, model visualization, and external clinical validation performance. Results: The post-clustering dataset contains 139 patients (group W) and 122 patients (group P). The neutrophil multiplied by D-dimer (NDM) has predictive value in all ML prediction models in this study. In the decision tree model, NDMQ occupies the first tree node, When NDM≤5.62 and the age<74.5, the probability of poor prognosis of AIS is less than 20 %. When NDM>5.62 and accompanied by pneumonia, the incidence of poor prognosis of AIS is about 90 %. In the Random Forest (RF) model, NDMQ had the highest Gini index. The variable combination screened by the RF model had the best performance in the neural network, and the accuracy, sensitivity, specificity, PPV, and NPV of the external validation were 0.800, 0.774, 0.833, 0.857, and 0.741, respectively. The RF model had the best performance in the external clinical validation datasets, with accuracies of 0.646, 0.697, 0.695, and 0.713, respectively. Conclusions: NDM shows predictive value for AIS short-term prognosis in all ML models in this study. The optimal model in screening characteristic variables and the performance of in external clinical datasets was RF model. In the analysis of medical data with small sample size and outcome as categorical variables, RF could be used as the main algorithm to build a model.

Aquaporin 1 is renoprotective in septic acute kidney injury by attenuating inflammation, apoptosis and fibrosis through inhibition of P53 expression.

Sepsis associated Acute kidney injury (AKI) is a common clinical syndrome characterized by suddenly decreased in renal function and urinary volume. This study was designed to investigate the role of Aquaporin 1 (AQP1) and P53 in the development of sepsis-induced AKI and their potential regulatory mechanisms. Firstly, transcriptome sequencing analysis of mice kidney showed AQP1 expression was reduced and P53 expression was elevated in Cecal ligation and puncture (CLP)-induced AKI compared with controls. Bioinformatics confirmed that AQP1 expression was remarkably decreased and P53 expression was obviously elevated in renal tissues or peripheral blood of septic AKI patients. Moreover, we found in vivo experiments that AQP1 mRNA levels were dramatically decreased and P53 mRNA significantly increased following the increased expression of inflammation, apoptosis, fibrosis, NGAL and KIM-1 at various periods in septic AKI. Meanwhile, AQP1 and P53 protein levels increased significantly first and then decreased gradually in kidney tissue and serum of rats in different stages of septic AKI. Most importantly, in vivo and vitro experiments demonstrated that silencing of AQP1 greatly exacerbates renal or cellular injury by up-regulating P53 expression promoting inflammatory response, apoptosis and fibrosis. Overexpression of AQP1 prevented the elevation of inflammation, apoptosis and fibrosis by down-regulating P53 expression in Lipopolysaccharide (LPS)-induced AKI or HK-2 cells. Therefore, our results suggested that AQP1 plays a protective role in modulating AKI and can attenuate inflammatory response, apoptosis and fibrosis via downregulating P53 in septic AKI or LPS-induced HK-2cells. The pharmacological targeting of AQP1 mediated P53 expression might be identified as potential targets for the early treatment of septic AKI.

Aquaporin-1 Facilitates Macrophage M1 Polarization by Enhancing Glycolysis Through the Activation of HIF1α in Lipopolysaccharide-Induced Acute Kidney Injury.

This study aimed to investigate how aquaporin 1 (AQP1) modulates hypoxia-inducible factor-1α (HIF1α) to promote glycolysis and drive the M1 polarization of macrophages. Within 12 h post-treatment with LPS to induce acute kidney injury in rats, a significant upregulation of AQP1 and HIF1α protein levels was noted in serum and kidney tissues. This elevation corresponded with a decrease in blood glucose concentrations and an enhancement of glycolytic activity relative to the control group. Furthermore, there was a pronounced reduction in the circulating levels of the anti-inflammatory cytokine IL-10, accompanied by an upregulation in the levels of the pro-inflammatory cytokines IL-6 and TNF-α. The administration of an HIF1α inhibitor reversed these effects, which did not affect the production of AQP1 protein. In cellular assays, AQP1 knockdown mitigated the increase in HIF1α expression induced by LPS. Furthermore, the suppression of HIF1α with PX-478 led to decreased expression levels of Hexokinase 2 (HK2) and Lactate Dehydrogenase A (LDHA), indicating that AQP1 regulates glycolysis through HIF1α. M1 polarization of macrophages was reduced by AQP1 knockdown and was further diminished by the addition of an HIF1α inhibitor. Inhibition of the glycolytic process not only weakened M1 polarization but also promoted M2 polarization, thereby reducing the release of inflammatory cytokines. These findings provide a novel perspective for developing therapeutic strategies that target AQP1 and HIF1α, potentially improving the treatment of sepsis-associated AKI.

Down-regulated expression of AQP5 on lung in rat DIC model induced by LPS and its effect on the development of pulmonary edema.

Disseminated intravascular coagulation (DIC) is an acquired syndrome characterized by the widespread activation of coagulation, which leads to failure of multiple organs in the body. DIC of rat with lipopolysaccharide (LPS) is associated with subsequent pulmonary edema. Lung tissue is highly water permeable and expresses several aquaporins (AQPs). We therefore explored whether AQP5 involved in the pathogenesis of LPS-induced lung edema. The rats were intravenously infused with LPS (30 mg/kg) for 4 h, 6 h, 8 h, 10 h, and 12 h to induce DIC. Platelets count (PLT), D-Dimer (DD), fibrinogen (FIB), prothrombin time (PT), and activated partial thromboplastin time (APTT) were determined. Real-time quantitative PCR and Western blot were used to analyze the mRNA and protein expression of AQP5. Lung samples were stained with hematoxylin-eosin and lung wet/dry weight (W/D) ratios were measured. Here, we demonstrated that PLT and FIB values were significant decreased, the values for DD, PT, and APTT were marked increased, microthrombus was observed in lung specimens, and simultaneously with the AQP5 showed down-regulated expression following LPS infused from 4 h to 12 h. However, histopathological changes such as pulmonary edema and the increased lung W/D weight ratio were observed after LPS infused from 6 h to 12 h. These results indicated that the decreased expression of AQP5 maybe induce liquid transport obstacles between alveolar and capillary, and provides the report of AQP5 gene regulation, revealing the pathogenesis of pulmonary edema in DIC model of rat.

Application of a combined approach involving classical random mutagenesis and metabolic engineering to enhance FK506 production in Streptomyces sp. RM7011.

FK506 production by a mutant strain (Streptomyces sp. RM7011) induced by N-methyl-N'-nitro-N-nitrosoguanidine and ultraviolet mutagenesis was improved by 11.63-fold (94.24 mg/l) compared to that of the wild-type strain. Among three different metabolic pathways involved in the biosynthesis of methylmalonyl-CoA, only expression of propionyl-CoA carboxylase (PCC) pathway led to a 1.75-fold and 2.5-fold increase in FK506 production and the methylmalonyl-CoA pool, respectively, compared to those of the RM7011 strain. Lipase activity of the high FK506 producer mutant increased in direct proportion to the increase in FK506 yield, from low detection level up to 43.1 U/ml (12.6-fold). The level of specific FK506 production and lipase activity was improved by enhancing the supply of lipase inducers. This improvement was approximately 1.88-fold (71.5 mg/g) with the supplementation of 5 mM Tween 80, which is the probable effective stimulator in lipase production, to the R2YE medium. When 5 mM vinyl propionate was added as a precursor for PCC pathway to R2YE medium, the specific production of FK506 increased approximately 1.9-fold (71.61 mg/g) compared to that under the non-supplemented condition. Moreover, in the presence of 5 mM Tween 80, the specific FK506 production was approximately 2.2-fold (157.44 mg/g) higher than that when only vinyl propionate was added to the R2YE medium. In particular, PCC expression in Streptomyces sp. RM7011 (RM7011/pSJ1003) together with vinyl propionate feeding resulted in an increase in the FK506 titer to as much as 1.6-fold (251.9 mg/g) compared with that in RM7011/pSE34 in R2YE medium with 5 mM Tween 80 supplementation, indicating that the vinyl propionate is more catabolized to propionate by stimulated lipase activity on Tween 80, that propionyl-CoA yielded from propionate generates methylmalonyl-CoA, and that the PCC pathway plays a key role in increasing the methylmalonyl-CoA pool for FK506 biosynthesis in RM7011 strain. Overall, these results show that a combined approach involving classical random mutation and metabolic engineering can be applied to supply the limiting factor for FK506 biosynthesis, and vinyl propionate could be successfully used as a precursor of important methylmalonyl-CoA building blocks.

Pure tone audiometry is a new method for evaluating congenital malformation of the middle and outer ear (CMMOE).

Background: The preoperative evaluation of Congenital Malformation of the Middle and Outer Ear (CMMOE) is very important. Jahrsdoerfer score commonly used at present, based on CT scanning images of the temporal bone, is often unable to accurately evaluate deformity and hearing level.Aims/Objectives: To investigate and promote a straightforward and easily accessible assessment method, pure tone audiometry, for the evaluation of CMMOE.Material and Methods: A total of 223 cases (244 ears) CMMOE with hearing data were retrospectively analyzed. Among them, 180 cases (197 ears) underwent exploratory tympanoplasty with clear conditions: ossicle numbers in 136 cases (147 ears) and morphology in 128 cases (138 ears) and vestibular window development in 137 cases (146 ears), and CT scans of temporal bone in 113 cases (120 ears). 1). The correlation was analyzed between ossicle numbers, ossicle morphology, Jahrsdoerfer score groups and their corresponding Average Air-Conduction Threshold of pure tone (AACT) at 0.5-4 KHz. 2) The AACT difference is compared among the above groups respectively and between the developed and undeveloped groups of vestibular window at 0.5-4 KHz and each frequency of 0.125-8 KHz. Spearman method was used for correlation analysis (calculating coefficient r and p values). For the data followed a normal distribution, a one-way analysis of variance (ANOVA) and t-test were employed, otherwise, Kruskal Wallis multiple local rank coincidence test and Wilcoxon rank sum test were used. p <0 .05 was considered statistically significant.Results: 1) The correlation coefficients between the groups of ossicle number scores, ossicle morphology scores, Jahrsdoerfer scores and their corresponding AACT are r = -0.187 (p <0 .05), r = -0.073 (p >0 .05) and r = -0.079 (p > 0.05), respectively. 2) Comparison of AACT difference based on ossicle number or morphological scores and Jahrsdoerfer scores with p > 0.05 among all groups, respectively. The AACT difference between the developed and undeveloped vestibular window groups is 5.5 (63.5/69.0) dB HL(p < .05) at 0.5-4KHz, out of 0.125-8 KHz frequency 1, 2, 4 KHz were 5.7 (65.0/70.7) dB HL, 8.4 (60.7/69.1) dB HL and 2 (61.5/63.5) dB HL, respectively, all p < 0.05, the other frequencies with all p > 0.05.Conclusions and Significance: 1) Ossicle number was correlated with AACT, but not for ossicle morphology and Jahrsdoerfer scores. 2) There was no significant difference in AACT corresponding to ossicle number or morphology scores and Jahrsdoerfer scores groups, but the patients with undeveloped vestibular window had poorer hearing than those with developed ones. Therefore, the AACT can evaluate the development of ossicle and vestibular window, and more directly reflect the hearing level than Jahrsdoerfer score. Pure tone audiometry is simple, widely used, and easily accessible, which making it a new assessment method of CMMOE.

Antioxidant and in vitro cosmeceutical activities of chestnut inner shell fermented by Monascus kaoliang.

Chestnut inner shell (CIS) was fermented at 30 °C for 12 day using Monascus kaoliang, either in solid or submerged state, and alcohol extracts (70% ethanol) of the fermented CIS were examined for their antioxidant (total phenol content and diphenylpicrylhydrazyl radical scavenging activity) and in vitro cosmeceutical activities (tyrosinase and elastase inhibitory activities). Both activities were significantly increased by the M. kaoliang-fermentation, more apparently by submerged fermentation (SMF) than by solid-state fermentation (SSF). The cosmeceutical activity reached its maximum value on the 3rd day of fermentation. The residual amounts of phenolic acids and catechins in the CIS extracts were increased by the fermentation, up to 395.0 and 344.3 µg/g, respectively. More phenolic acids were produced by SMF than SSF, whereas more catechins were produced by SSF than SMF. Therefore, SMF using M. kaoliang was an efficient process for the utilization of CIS as a source of cosmeceuticals.

Aquaporin 1 Facilitates Ferroptosis, M1 Polarization, Mitochondrial Dysfunction, and Autophagy Damage on Lipopolysaccharide-Induced Macrophage Through Down-Regulation of P53 Signaling Pathway.

This study was designed to investigate the role of aquaporin 1 (AQP1) in ferroptosis, macrophage polarization, mitochondrial dysfunction, and impaired autophagy of lipopolysaccharide (LPS)-stimulated RAW264.7 cells and explored the underlying mechanisms. Si-AQP1-mediated AQP1 silencing RAW264.7 cells was constructed. Si-P53-mediated P53 silencing or pcDNA-P53 overexpression RAW264.7 cells was constructed. Assays of ATP, reverse transcription-quantitative polymerase chain reaction (RT-qPCR), and Mitochondrial membrane potential (JC-1) staining were performed to evaluate mitochondrial biological function. Assays of flow cytometry, reactive oxygen species (ROS) staining, western blot (WB), RT-qPCR, malondialdehyde (MDA), glutathione (GSH), and total superoxide dismutase (SOD) were performed to detect cell ferroptosis, macrophage polarization, and impaired autophagy. The involvement of the P53 pathway was revealed by WB. The results showed that LPS (30 µg/mL) could induce ferroptosis, M1 polarization, mitochondrial dysfunction, and autophagy damage in RAW264.7 cells. Meanwhile, the expression of AQP1 was increased and the expression of P53 was decreased. In addition, Pifithrin-α (PIF; 15 µM), a P53 inhibitor, significantly aggravated ferroptosis, M1 polarization, mitochondrial dysfunction, and autophagy damage as well as up-regulation of AQP1 protein expression in LPS-induced RAW264.7 cells. Interestingly, this phenomenon was markedly alleviated by Kevetrin hydrochloride (70 µM), a P53 agonist. Mechanistically, silencing AQP1 significantly alleviated ferroptosis, M1 polarization, mitochondrial dysfunction, and autophagy damage by up-regulating the expression of P53 in LPS-stimulated RAW264.7 cells. Indeed, inhibition of P53 expression by PIF treatment dramatically reversed this effect on the basis of LPS+si-AQP1. Therefore, we concluded for the first time that AQP1 can promote ferroptosis, M1 polarization, mitochondrial dysfunction, and autophagy impairment by inhibiting the expression of P53 in LPS-stimulated RAW264.7 cells, and AQP1 or P53 may be considered as a crucial determiner that can regulate the biological behavior of RAW264.7 cells stimulated by LPS.

Analysis of the effectiveness of hearing solutions for congenital malformation of the middle and outer ear (CMMOE).

BACKGROUND: There are no reports about comprehensive comparative analysis of the effects after various hearing surgery solutions for congenital malformation of the middle and outer ear (CMMOE). AIMS/OBJECTIVES: To analyze the improvement of Average Air-Conduction Threshold (AACT) of pure tone after various hearing surgery solutions for CMMOE and provide a reference for the selection of accurate hearing solutions. MATERIALS AND METHODS: A retrospective analysis of 159 cases (170 ears) with CMMOE submitted to various ear surgery solutions, including: (1) Three situations of outer ear canal (OEC): ① atresia 85 ears, ② stenosis 28 ears, and ③ normal 57 ears. (2) Three commonly used hearing solutions: eardrum repair 53 ears, Porp 44 ears and Piston 32 ears implantation. (3) Three OEC situations with different hearing solutions: type I. Reconstruction of OEC (r-OEC), type II. r-OEC and/or different tympanoplasty, including ① eardrum repair, ② release of ossicular chain, ③ Porp implantation, and ④ Torp implantation, type III. Piston implantation with fenestration of the inner ear. Compare AACT of postoperative short term (0.5 years) or long term (0.5-10 years) and preoperative in the speech frequency range of 0.5-4 kHz to assess efficacy. If the sample number ≥10, and not subject to normal distribution, the Kruskal-Wallis multi-sample rank sum test is used for the comparison of multiple groups and Wilcoxon's rank sum test for two groups, with P < 0.05 being statistically significant. If the sample size <10, the standard of clinical efficacy is one frequency improvement value ≥15 dB HL, or 10 dB HL ≤2 frequency improvements <15 dB HL at 0.125-8 KHz. RESULTS: Intra-group comparison of AACT: (1) three situations of OEC: atresia, stenosis and normal all had P < 0.05 postoperatively in short term, while in long term only the normal group had P < 0.05. (2) Three commonly used hearing solutions: eardrum repair, Porp and Piston implantation all had P < 0.05 in short and long terms, except for eardrum repair P >0 .05 in long term. (3) Three OEC situations with different hearing solutions: 1) Atresia of OEC: Porp and Piston implantation, r-OEC and release of ossicular chain were effective in short term and were not effective in long term, and the eardrum repair was not effective in both short and long term. 2) Stenosis of OEC: eardrum repair, Porp and Piston implantation were effective in short and long term. r-OEC P >0 .05 for short and long term, Torp implantation was not effective in long term, 3) Normal of OEC: Porp, Torp and Piston implantation were all P < 0.05 in short and long term except for Torp >0.05 in long term, and release of ossicular chain is both short and long term clinically effective. The AACT values of postoperative in long term for three groups of atresia, stenosis, normal of OEC are over 58.7 dB HL (except Porp implantation 52.5 dB HL), 51.3 dB HL (except Porp implantation 42.5 dB HL), and 37.5 dB HL (except Torp implantation are 32.6 dB HL), respectively. CONCLUSIONS AND SIGNIFICANCE: Intra-group comparison of AACT. (1) Three groups of the atresia, stenosis and normal of OEC are all effective in short term, while in long term only the normal group is effective. (2) The three most commonly used surgical solutions of eardrum repair, Porp and Piston implantation are effective in short and long terms, except for long term eardrum repair. (3) Three OEC situations with different hearing solutions: some of surgical solutions were effective in short term or long term for CMMOE, but based on the AACT values of postoperative in long term for three OEC situations, it is better to choose a hearing device for atresia of OEC, comprehensive review of surgical or hearing device for stenosis of OEC. Surgery can be considered for normal OEC.

New strategies and methods for reconstruction of the outer ear canal (OEC).

Background: The incidence of re-stenosis or re-atresia after reconstruction of the Outer Ear Canal (OEC) in patients with Congenital Malformation of the Middle and Outer Ear (CMMOE) is very high (up to 48%), and it has been a difficult problem for otologists not being able to solve.Aims/Objectives: To explore new strategies and methods to improve re-stenosis or re-atresia after reconstruction of the OEC in patients with CMMOE.Material and Methods: According to the characteristics of reconstructed OEC (r-OEC) re-stenosis or re-atresia summarized by us, a number of new prevention strategies and methods have been proposed and related patent products have been designed, including the improvement of covering epithelium types and skin grafting methods (7 types), simulated drum ring function to prevent the formation of negative pressure in the cavity, and strengthen postoperative support to reduce skin shrinkage and bone hyperplasia. The postoperative effects of different ages and preoperative OEC malformations are statistically analyzed.Results: The incidence of re-stenosis/re-atresia is 14.3% (5/35) in the thin sectional skin of the temporal scalp overlap splicing skin grafting, which was significantly better than 45.5% (15/33) in the whole piece mosaic splicing and barrel skin grafting from the inner thin sectional thigh skin and overlay splicing other methods, including the inner thigh thin sectional skin, chest medium thick skin and subcutaneous pedicle + chest medium thick skin (p<0.05). The patent artificial drum ring and the model stent of the OEC have obvious effects. The mean operation age of postoperative atresia, stenosis, and good groups are 9.3, 13.1, and 12.5 years old, respectively. The proportion of preoperative atresia is 91.3%, 85.7%, and 57.7%, respectively. The total incidence of re-atresia and re-stenosis of r-OEC for two groups of atresia and stenosis of OEC before surgery is 40.5% (49/121) and 13.3% (8/60), respectively.Conclusions and Significance: The best result is found in overlapping the splicing thin sectional skin of the temporal scalp, combined with artificial drum ring implantation, effective support of postoperative model stent of OEC and post-pubertal surgery selection are new and effective strategies and methods to prevent re-stenosis or re-atresia of r-OEC. Atresia or stenosis of the OEC before the operation is the influence factor of the postoperative effect.