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Identification, characterization, and distribution of novel amidase gene aphA in sphingomonads conferring resistance to amphenicol antibiotics

Yingying Qian,Lin Lai,Minggen Cheng,Hua Fang,Dandan Fan,Gerben J. Zylstra,Xing Huang

Amphenicol antibiotics, such as chloramphenicol (CHL), thiamphenicol (TAP), and florfenicol (Ff), are high-risk emerging pollutants. Their extensive usage in aquaculture, livestock, and poultry farming has led to an increase in bacterial antibiotic resistance and facilitated the spread of resistance genes. Yet, limited research has been conducted on the co-resistance of CHL, TAP, and Ff. Herein, a novel amidase AphA was identified from a pure cultured strain that can concurrently mediate the hydrolytic inactivation of CHL, TAP, and Ff, yielding products p-nitrophenylserinol, thiamphenicol amine (TAP-amine), and florfenicol amine (Ff-amine), respectively. The antibacterial activity of these antibiotic hydrolysates exhibited a significant reduction or complete loss in comparison to the parent compounds. Notably, AphA shared less than 26% amino acid sequence identity with previously reported enzymes and exhibited high conservation within the sphingomonad species. Through enzymatic kinetic analysis, the AphA exhibited markedly superior affinity and catalytic activity toward Ff in comparison to CHL and TAP. Site-directed mutagenesis analysis revealed the indispensability of catalytic triad residues, particularly serine 153 and histidine 277, in forming crucial hydrogen bonds essential for AphA’s hydrolytic activity. Comparative genomic analysis showed that aphA genes in some species are closely adjacent to various transposable elements, indicating that there is a high potential risk of horizontal gene transfer (HGT). This study established a hydrolysis resistance mechanism of amphenicol antibiotics in sphingomonads, which offers theoretical guidance and a novel marker gene for assessing the prevalent risk of amphenicol antibiotics in the environment.

Poster Session C

ITCH-Mediated Ubiquitination and Degradation of THBS1: A Key Mechanism for Enhancing Mitochondrial Biogenesis and Alleviating Mouse Skeletal Muscle Atrophy

Wan Yu,Yanteng Wang,Na Li,Difei Wang

Aim Skeletal muscle atrophy is tightly associated with maladaptive alterations in mitochondrial function and morphology. Itchy E3 ubiquitin-protein ligase (ITCH) modulates mitochondria, and thrombospondin 1 (THBS1) positively regulates muscle atrophy, but their roles in muscle atrophy are unclear. Methods A muscle atrophy model was established in C57BL/6 mice via daily intraperitoneal injection of dexamethasone (Dex, 20 mg/kg). ITCH overexpression in skeletal muscle was achieved by adeno-associated virus serotype 9 injection. C2C12 cells were treated with 50 μM Dex to mimic in vitro muscle atrophy. Skeletal muscle atrophy in mice was evaluated using hematoxylin–eosin staining and immunofluorescence staining. Mitochondrial damage was assessed via transmission electron microscopy, succinate dehydrogenase staining, and JC-1 staining. Immunoprecipitation-liquid chromatography/mass spectrometry, molecular docking, and co-immunoprecipitation were used to investigate the interaction between ITCH and THBS1. Phosphoproteomics analysis was performed to detect the THBS1 downstream proteins. Results Dex treatment downregulated ITCH expression in skeletal muscle. ITCH overexpression increased body weight, muscle mass, and muscle strength, downregulated the expression of atrophy-related genes (Atrogin-1, Mstn, MuRF-1), and promoted mitochondrial biogenesis. The results of the C2C12 cells were consistent with those obtained in vivo. Proteomic profiling and Co-IP confirmed ITCH–THBS1 interaction and subsequent THBS1 ubiquitination. THBS1 knockdown reduced the expression of Atrogin-1 and MuRF-1 and inhibited the phosphorylation of JUN and Map3k7, whereas THBS1 overexpression reversed the ITCH-mediated improvement in mitochondrial biogenesis. Conclusion ITCH enhances mitochondrial biogenesis and mitigates Dex-induced muscle atrophy by promoting the ubiquitin-dependent degradation of THBS1 and subsequent inhibition of downstream JUN/Map3k7 phosphorylation. Practitioner Points This study clarifies a novel regulatory mechanism of skeletal muscle atrophy, confirming that the E3 ubiquitin ligase ITCH plays a protective role in dexamethasone-induced muscle atrophy. THBS1 acts as a critical downstream effector of ITCH, promoting muscle atrophy via activation of the JUN/Map3k7 phosphorylation signaling pathway. Targeting ITCH-dependent THBS1 ubiquitination may represent a potential strategy to mitigate skeletal muscle loss.

Isoflurane impairs olfaction by increasing neuronal activity in the olfactory bulb

Hanyu Fu,Jingwei Zhou,Shan Li,Ying Zhang,Zhiyun Chen,Yingying Yang,Anan Li,Dejuan Wang

Aim General anesthesia can induce cognitive deficits in both humans and rodents, correlating with pathological alterations in the hippocampus. However, whether general anesthesia affects olfactory behaviors remains controversial as clinical studies have produced inconsistent results. Therefore, we aimed to investigate how olfactory behaviors and neuronal activity are affected by isoflurane exposure in adult mice. Methods The olfactory detection test, olfactory sensitivity test, and olfactory preference/avoidance test were used to examine olfactory function. In vivo electrophysiology was performed in awake, head-fixed mice to record single-unit spiking and local field potentials in the olfactory bulb (OB). We also performed patch-clamp recordings of mitral cell activity. For morphological studies, immunofluorescence and Golgi–Cox staining were applied. Results Repeated exposure to isoflurane impaired olfactory detection in adult mice. The main olfactory epithelium, the first region exposed to anesthetics, displayed increased proliferation of basal stem cells. In the OB, a crucial hub for olfactory processing, repeated isoflurane exposure increased the odor responses of mitral/tufted cells. Furthermore, the odor-evoked high gamma response was decreased after isoflurane exposure. Whole-cell recordings further indicated that repeated isoflurane exposure increased the excitability of mitral cells, which may be due to weakened inhibitory input in isoflurane-exposed mice. In addition, elevated astrocyte activation and glutamate transporter-1 expression in the OB were observed in isoflurane-exposed mice. Conclusions Our findings indicate that repeated isoflurane exposure impairs olfactory detection by increasing neuronal activity in the OB in adult mice.

Tacrolimus Induced Hypertension and Vascular Remodeling Includes Mechanisms of Cellular Senescence—The Protective Effect of Valsartan

Lingyan Fei,Xiaohua Wang,Lingyi Kong,Tianjiao Cui,Pratik H Khedkar,Yunxiu Xiang,Dongliang Zhao,Junxuan Fang,Yulin Liang,Yangyang Zhang,Nan Xu,Xingyu Qiu,Liang Zhao,Gensheng Zhang,Yan Lei,Chun Tang,Kongyang Ma,Liwei Lu,Jin Wei,En Yin Lai,Pontus B Persson,Andreas Patzak,Zhihua Zheng,Shan Jiang

Aim Calcineurin inhibitors (CNI) such as tacrolimus (Tac) are the first‐line treatment to prevent transplant rejection. However, CNI have adverse effects on blood vessels and renal function, which may be linked to cellular senescence. Valsartan, a common angiotensin II type 1 receptor (AT 1 R) blocker, exhibits anti‐senescence properties. We tested the hypothesis that tacrolimus causes hypertension and microvascular remodeling that involves induction of senescence, and that valsartan is protective. Methods Microperfusion and wire myography were employed to assess the contractile and dilatory functions of renal afferent arterioles (Af‐Art) and mesenteric arteries, respectively. The expression of components of the renin‐angiotensin system (RAS) and senescence‐associated biomarkers was investigated using qPCR and immunohistochemistry. Results Long‐term administration of Tac activated the RAS. Tac‐induced microvascular remodeling in mesenteric arteries and Af‐Art was mitigated by treatment with valsartan. Mice treated with Tac exhibited increased vasoconstriction in response to angiotensin II and reduced dilation to acetylcholine. Both effects were abolished by valsartan. Additionally, senescence‐associated biomarkers were upregulated in mesenteric and renal resistance arteries from Tac‐treated mice. Co‐administration of Tac with valsartan or ABT‐263, a senolytic agent, rescued Tac‐induced microvascular injury and reduced hypertension (conscious mice, noninvasive tail‐cuff system). Treatment with the antihypertensive drug amlodipine normalized blood pressure and downregulated senescence‐associated beta‐galactosidase in mesenteric arteries. Conclusion These findings suggest that cellular senescence contributes to Tac‐induced microvascular injury and hypertension and demonstrate the effectiveness of senolytic treatment for protection. Valsartan could reduce senescence indirectly by lowering blood pressure; a direct anti‐senescence effect might also play a role in this context.

EGCG inhibits pressure overload-induced cardiac hypertrophy via the PSMB5/Nmnat2/SIRT6-dependent signalling pathways

Yi Cai,Shan Shan Yu,Yang He,Xue Ying Bi,Si Gao,Ting Dong Yan,Guo Dong Zheng,Ting Ting Chen,Jian Tao Ye,Pei Qing Liu

Aim Epigallocatechin-3-gallate (EGCG), the major polyphenol found in green tea, exerts multiple protective effects against cardiovascular diseases, including cardiac hypertrophy. However, the molecular mechanism underlying its anti-hypertrophic effect has not been clarified. This study revealed that EGCG could inhibit pressure overload-induced cardiac hypertrophy by regulating the PSMB5/Nmnat2/SIRT6-dependent signalling pathway. Methods Quantitative real-time polymerase chain reaction and western blotting were used to determine the expression of mRNA and protein respectively. A fluorometric assay kit was used to determine the activity of SIRT6, a histone deacetylase. Luciferase reporter gene assay and electrophoretic mobility shift assay were employed to measure transcriptional activity and DNA binding activity respectively. Results EGCG could significantly increase Nmnat2 protein expression and enzyme activity in cultured neonatal rat cardiomyocytes stimulated with angiotensin II (Ang II) and heart tissues from rats subjected to abdominal aortic constriction. Nmnat2 knockdown by RNA interference attenuated the inhibitory effect of EGCG on cardiac hypertrophy. EGCG blocked NF-κB DNA binding activity induced by Ang II, which was dependent on Nmnat2 and the subsequent SIRT6 activation. Moreover the activation of PSMB5 (20S proteasome subunit β-5, chymotrypsin-like) was required for EGCG-induced Nmnat2 protein expression. Additionally, we demonstrated that EGCG might interact with PSMB5 and inhibit the activation of the proteasome. Conclusions These findings serve as the first evidence that the effect of EGCG against cardiac hypertrophy may be, at least partially, attributed to the modulation of the PSMB5/Nmnat2-dependent signalling pathway, suggesting the therapeutic potential of EGCG in the prevention and treatment of cardiac hypertrophy.