Genetic manipulation of gut microbes enables single-gene interrogation in a complex microbiome.

Hundreds of microbiota genes are associated with host biology/disease. Unraveling the causal contribution of a microbiota gene to host biology remains difficult because many are encoded by nonmodel gut commensals and not genetically targetable. A general approach to identify their gene transfer methodology and build their gene manipulation tools would enable mechanistic dissections of their impact on host physiology. We developed a pipeline that identifies the gene transfer methods for multiple nonmodel microbes spanning five phyla, and we demonstrated the utility of their genetic tools by modulating microbiome-derived short-chain fatty acids and bile acids in vitro and in the host. In a proof-of-principle study, by deleting a commensal gene for bile acid synthesis in a complex microbiome, we discovered an intriguing role of this gene in regulating colon inflammation. This technology will enable genetically engineering the nonmodel gut microbiome and facilitate mechanistic dissection of microbiota-host interactions.

Gut-innervating nociceptors regulate the intestinal microbiota to promote tissue protection.

Nociceptive pain is a hallmark of many chronic inflammatory conditions including inflammatory bowel diseases (IBDs); however, whether pain-sensing neurons influence intestinal inflammation remains poorly defined. Employing chemogenetic silencing, adenoviral-mediated colon-specific silencing, and pharmacological ablation of TRPV1+ nociceptors, we observed more severe inflammation and defective tissue-protective reparative processes in a murine model of intestinal damage and inflammation. Disrupted nociception led to significant alterations in the intestinal microbiota and a transmissible dysbiosis, while mono-colonization of germ-free mice with Gram+Clostridium spp. promoted intestinal tissue protection through a nociceptor-dependent pathway. Mechanistically, disruption of nociception resulted in decreased levels of substance P, and therapeutic delivery of substance P promoted tissue-protective effects exerted by TRPV1+ nociceptors in a microbiota-dependent manner. Finally, dysregulated nociceptor gene expression was observed in intestinal biopsies from IBD patients. Collectively, these findings indicate an evolutionarily conserved functional link between nociception, the intestinal microbiota, and the restoration of intestinal homeostasis.

Inulin fibre promotes microbiota-derived bile acids and type 2 inflammation.

Dietary fibres can exert beneficial anti-inflammatory effects through microbially fermented short-chain fatty acid metabolites^1,2, although the immunoregulatory roles of most fibre diets and their microbiota-derived metabolites remain poorly defined. Here, using microbial sequencing and untargeted metabolomics, we show that a diet of inulin fibre alters the composition of the mouse microbiota and the levels of microbiota-derived metabolites, notably bile acids. This metabolomic shift is associated with type 2 inflammation in the intestine and lungs, characterized by IL-33 production, activation of group 2 innate lymphoid cells and eosinophilia. Delivery of cholic acid mimics inulin-induced type 2 inflammation, whereas deletion of the bile acid receptor farnesoid X receptor diminishes the effects of inulin. The effects of inulin are microbiota dependent and were reproduced in mice colonized with human-derived microbiota. Furthermore, genetic deletion of a bile-acid-metabolizing enzyme in one bacterial species abolishes the ability of inulin to trigger type 2 inflammation. Finally, we demonstrate that inulin enhances allergen- and helminth-induced type 2 inflammation. Taken together, these data reveal that dietary inulin fibre triggers microbiota-derived cholic acid and type 2 inflammation at barrier surfaces with implications for understanding the pathophysiology of allergic inflammation, tissue protection and host defence.

The Toxoplasma protein phosphatase 6 catalytic subunit (TgPP6C) is essential for cell cycle progression and virulence.

Protein phosphatases are post-translational regulators of Toxoplasma gondii proliferation, tachyzoite-bradyzoite differentiation and pathogenesis. Here, we identify the putative protein phosphatase 6 (TgPP6) subunits of T. gondii and elucidate their role in the parasite lytic cycle. The putative catalytic subunit TgPP6C and regulatory subunit TgPP6R likely form a complex whereas the predicted structural subunit TgPP6S, with low homology to the human PP6 structural subunit, does not coassemble with TgPP6C and TgPP6R. Functional studies showed that TgPP6C and TgPP6R are essential for parasite growth and replication. The ablation of TgPP6C significantly reduced the synchronous division of the parasite's daughter cells during endodyogeny, resulting in disordered rosettes. Moreover, the six conserved motifs of TgPP6C were required for efficient endodyogeny. Phosphoproteomic analysis revealed that ablation of TgPP6C predominately altered the phosphorylation status of proteins involved in the regulation of the parasite cell cycle. Deletion of TgPP6C significantly attenuated the parasite virulence in mice. Immunization of mice with TgPP6C-deficient type I RH strain induced protective immunity against challenge with a lethal dose of RH or PYS tachyzoites and Pru cysts. Taken together, the results show that TgPP6C contributes to the cell division, replication and pathogenicity in T. gondii.

CK2 promotes jasmonic acid signaling response by phosphorylating MYC2 in Arabidopsis.

Jasmonic acid (JA) signaling plays a pivotal role in plant development and defense. MYC2 is a master transcription factor in JA signaling, and was found to be phosphorylated and negatively regulated by MAP kinase and receptor-like kinase. However, the kinases that positively regulate MYC2 through phosphorylation and promote MYC2-mediated activation of JA response have not been identified. Here, we identified CK2 as a kinase that phosphorylates MYC2 and thus regulates the JA signaling. CK2 holoenzyme can interact with MYC2 using its regulatory subunits and phosphorylate MYC2 at multiple sites with its catalytic subunits. Inhibition of CK2 activity in a dominant-negative plant line, CK2mut, repressed JA response. On the other hand, increasing CK2 activity by overexpression of CKB4, a regulatory subunit gene of CK2, enhanced JA response in a MYC2-dependent manner. Substitution of the Ser and Thr residues at phosphorylation sites of MYC2 by CK2 with Ala impaired MYC2 function in activating JA response. Further investigations evidenced that CK2 facilitated the JA-induced increase of MYC2 binding to the promoters of JA-responsive genes in vivo. Our study demonstrated that CK2 plays a positive role in JA signaling, and reveals a previously undiscovered mechanism that regulates MYC2 function.

Organoid forming potential as complementary parameter for accurate evaluation of breast cancer neoadjuvant therapeutic efficacy.

BACKGROUND: 13-15% of breast cancer/BC patients diagnosed as pathological complete response/pCR after neoadjuvant systemic therapy/NST suffer from recurrence. This study aims to estimate the rationality of organoid forming potential/OFP for more accurate evaluation of NST efficacy. METHODS: OFPs of post-NST residual disease/RD were checked and compared with clinical approaches to estimate the recurrence risk. The phenotypes of organoids were classified via HE staining and ER, PR, HER2, Ki67 and CD133 immuno-labeling. The active growing organoids were subjected to drug sensitivity tests. RESULTS: Of 62 post-NST BC specimens, 24 were classified as OFP-I with long-term active organoid growth, 19 as OFP-II with stable organoid growth within 3 weeks, and 19 as OFP-III without organoid formation. Residual tumors were overall correlated with OFP grades (P < 0.001), while 3 of the 18 patients (16.67%) pathologically diagnosed as tumor-free (ypT0N0M0) showed tumor derived-organoid formation. The disease-free survival/DFS of OFP-I cases was worse than other two groups (Log-rank P < 0.05). Organoids of OFP-I/-II groups well maintained the biological features of their parental tumors and were resistant to the drugs used in NST. CONCLUSIONS: The OFP would be a complementary parameter to improve the evaluation accuracy of NST efficacy of breast cancers.

E3 ligase BRG3 persulfidation delays tomato ripening by reducing ubiquitination of the repressor WRKY71.

Hydrogen sulfide (H2S) is a gaseous signaling molecule reported to play multiple roles in fruit ripening. However, the molecular mechanisms underlying H2S-mediated delay in fruit ripening remain to be established. Here, the gene encoding a WRKY transcription factor, WRKY71, was identified as substantially upregulated in H2S-treated tomato (Solanum lycopersicum) via transcriptome profiling. The expression of WRKY71 was negatively associated with that of CYANOALANINE SYNTHASE1 (CAS1). Transient and stable genetic modification experiments disclosed that WRKY71 acts as a repressor of the tomato ripening process. CAS1 appears to play an opposite role, based on the finding that the ripening process was delayed in the cas1 mutant and accelerated in CAS1-OE tomatoes. Dual-luciferase reporter assay, yeast one-hybrid, electrophoretic mobility shift assay, and transient transformation experiments showed that WRKY71 bound to the CAS1 promoter and suppressed its activation. Moreover, the persulfidation of WRKY71 enhanced its binding ability to the CAS1 promoter. Data from luciferase complementation and Y2H assays confirmed that WRKY71 interacts with a BOI-related E3 ubiquitin-protein ligase 3 (BRG3) and is ubiquitinated in vitro. Further experiments showed that modification of BRG3 via persulfidation at Cys206 and Cys212 led to reduced ubiquitination activity. Our findings support a model whereby BRG3 undergoes persulfidation at Cys206 and Cys212, leading to reduced ubiquitination activity and decreased interactions with the WRKY71 transcript, with a subsequent increase in binding activity of the persulfidated WRKY71 to the CAS1 promoter, resulting in its transcriptional inhibition and thereby delayed ripening of tomatoes. Our collective findings provide insights into a mechanism of H2S-mediated regulation of tomato fruit ripening.

The relative and combined ability of stress hyperglycemia ratio and N-terminal pro-B-type natriuretic peptide to predict all-cause mortality in diabetic patients with multivessel coronary artery disease.

BACKGROUND: Stress hyperglycemia ratio (SHR) and N-terminal pro-B-type natriuretic peptide (NT-proBNP) are independently associated with increased mortality risk in diabetic patients with coronary artery disease (CAD). However, the role of these biomarkers in patients with diabetes and multivessel disease (MVD) remains unknown. The present study aimed to assess the relative and combined abilities of these biomarkers to predict all-cause mortality in patients with diabetes and MVD. METHODS: This study included 1148 diabetic patients with MVD who underwent coronary angiography at Tianjin Chest Hospital between January 2016 and December 2016. The patients were divided into four groups according to their SHR (SHR-L and SHR-H) and NT-proBNP (NT-proBNP-L and NT-proBNP-H) levels. The primary outcome was all-cause mortality. Multivariate Cox regression analyses were performed to evaluate the association of SHR and NT-proBNP levels with all-cause mortality. RESULTS: During a mean 4.2 year follow-up, 138 patients died. Multivariate analysis showed that SHR and NT-proBNP were strong independent predictors of all-cause mortality in diabetic patients with MVD (SHR: HR hazard ratio [2.171; 95%CI 1.566-3.008; P < 0.001; NT-proBNP: HR: 1.005; 95%CI 1.001-1.009; P = 0.009). Compared to patients in the first (SHR-L and NT-proBNP-L) group, patients in the fourth (SHR-H and NT-proBNP-H) group had the highest mortality risk (HR: 12.244; 95%CI 5.828-25.721; P < 0.001). The areas under the curve were 0.615(SHR) and 0.699(NT-proBNP) for all-cause mortality. Adding either marker to the original models significantly improved the C-statistic and integrated discrimination improvement values (all P < 0.05). Moreover, combining SHR and NT-proBNP levels into the original model provided maximal prognostic information. CONCLUSIONS: SHR and NT-proBNP independently and jointly predicted all-cause mortality in diabetic patients with MVD, suggesting that strategies to improve risk stratification in these patients should incorporate SHR and NT-porBNP into risk algorithms.

The antioxidant protein glutaredoxin 1 is essential for oxidative stress response and pathogenicity of Toxoplasma gondii.

Glutaredoxins (Grxs) are ubiquitous antioxidant proteins involved in many molecular processes to protect cells against oxidative damage. Here, we study the roles of Grxs in the pathogenicity of Toxoplasma gondii. We show that Grxs are localized in the mitochondria (Grx1), cytoplasm (Grx2), and apicoplast (Grx3, Grx4), while Grx5 had an undetectable level of expression. We generated Δgrx1-5 mutants of T. gondii type I RH and type II Pru strains using CRISPR-Cas9 system. No significant differences in the infectivity were detected between four Δgrx (grx2-grx5) strains and their respective wild-type (WT) strains in vitro or in vivo. Additionally, no differences were detected in the production of reactive oxygen species, total antioxidant capacity, superoxide dismutase activity, and sensitivity to external oxidative stimuli. Interestingly, RHΔgrx1 or PruΔgrx1 exhibited significant differences in all the investigated aspects compared to the other grx2-grx5 mutant and WT strains. Transcriptome analysis suggests that deletion of grx1 altered the expression of genes involved in transport and metabolic pathways, signal transduction, translation, and obsolete oxidation-reduction process. The data support the conclusion that grx1 supports T. gondii resistance to oxidative killing and is essential for the parasite growth in cultured cells and pathogenicity in mice and that the active site CGFS motif was necessary for Grx1 activity.

The Influence of Hyperglycemia on Liver Triglyceride Deposition in Partially Pancreatectomized Rats.

Nonalcoholic fatty liver disease and diabetes always coexist. The relationship of fatty liver and hyperglycemia is not clear. We studied the influence of hyperglycemia on triglyceride (TG) accumulation in the liver and explored its possible mechanisms. SD rats were divided into three groups: Group A (sham operation control), Group B (partially pancreatectomized rats), and Group C (partially pancreatectomized rats treated with insulin). At 4 weeks after surgery, pancreatic weights and liver TG contents were measured. Serum biochemical parameters were determined, and oral glucose tolerance tests (OGTT) were performed. The gene expression of sterol regulatory element-binding protein1c (SREBP-1c), carbohydrate regulatory element-binding protein (ChREBP), fatty acid synthase(FAS), carnitine palmitoyltransferase 1 (CPT-1), and fibroblast growth factor 21 (FGF21) was determined by real-time PCR. Compared with Group A, postprandial glucose increased significantly; the concentrations of insulin and C-peptides, pancreatic weights and serum FGF21 levels were decreased, liver TG was increased significantly in Group B, and insulin treatment improved these changes. Compared with Group A, the gene expressions of FGF21, CPT-1 and FAS in the liver were decreased in Group B (all p<0.05). Compared with Group B, the gene expressions of FGF21, FAS, ChREBP, SREBP-1c and CPT-1 in the liver in Group C were all increased significantly (p<0.05, respectively). Hyperglycemia induced by partial pancreatectomy could lead to increased liver TG. Insulin treatment could decrease glucose levels and improve fatty liver, and genes related to lipid metabolism may play a role in this process.

Hierarchical Superhydrophilic/Superaerophobic Ni(OH)2@NiFe-PBA Nanoarray Supported on Nickel Foam for Boosting the Oxygen Evolution Reaction.

The design of hierarchical electrocatalysts with plentiful active sites and high mass transfer efficiency is critical to efficiently and sustainably carrying out the oxygen evolution reaction (OER), which presents a challenging and pressing need. In this study, a hierarchical Ni(OH)2@NiFe-Prussian blue analogue nanoarray grown on nickel foam (NF) [labeled as Ni(OH)2@NiFe-PBA/NF] was synthesized by combining a mild electrodeposition method with an ion-exchange strategy. The resultant Ni(OH)2@NiFe-PBA/NF displays superhydrophilic/superaerophobic properties that optimize the contact with the electrolyte, improve mass transfer efficiency, and expedite detachment of O2 bubbles during the electrocatalytic OER. Specifically, Ni(OH)2@NiFe-PBA/NF exhibits exceptional capability in the OER with low overpotentials of 224 and 240 mV at the current densities of 50 and 100 mA cm-2, respectively, accompanied by a low Tafel slope of 37.1 mV dec-1 and outstanding stability over 100 h at a fixed potential of 1.78 V vs reversible hydrogen electrode (RHE). Furthermore, Ni(OH)2@NiFe-PBA/NF demonstrates remarkable OER performance even in alkaline simulated seawater. During the OER process, active metal-OOH intermediates were formed by the partial self-reconstruction of NiFe-PBA in the heterostructure, as revealed by in situ Raman spectroscopy.

TM6SF2 reduces lipid accumulation in vascular smooth muscle cells by inhibiting LOX-1 and CD36 expression.

TM6SF2, predominantly expressed in the liver and intestine, is closely associated with lipid metabolism. We have demonstrated the presence of TM6SF2 in VSMCs within human atherosclerotic plaques. Subsequent functional studies were conducted to investigate its role in lipid uptake and accumulation in human vascular smooth muscle cells (HAVSMCs) using siRNA knockdown and overexpression techniques. Our results showed that TM6SF2 reduced lipid accumulation in oxLDL-stimulated VSMCs, likely through the regulation of lectin-like oxLDL receptor 1 (LOX-1) and scavenger receptor cluster of differentiation 36 (CD36) expression. We concluded that TM6SF2 plays a role in HAVSMC lipid metabolism with opposing effects on cellular lipid droplet content by downregulation of LOX-1 and CD36 expression.

Biologically active franchetine-type diterpenoid alkaloids: Isolation, synthesis, anti-inflammatory, agalgesic activities, and molecular docking.

In this study, four franchetine-type diterpenoid alkaloids (1-4) were isolated from Aconitum sinoaxillare, and fourteen diverse franchetine analogs (5-18) were synthesized. Compounds 1, 2, 7 and 16 exhibited stronger inhibitory effects on NO production when compared to celecoxib. Among them, compound 1 had the best inhibitory effect on iNOS and COX-2 inflammatory proteins. The in vitro studies displayed that the anti-inflammatory effect of the most active compound 1 was ascribed to the inhibition of the TLR4-MyD88/NF-κB/MAPKs signalling pathway. Consequently, this led to a inhibition in the expression of inflammatory factors or mediators including NO, ROS, TNF-α, IL-6, IL-1ß, iNOS, and COX-2. Additionally, compound 1 had low toxicity (LD50 > 20 mg/kg) in mice, and it had notable analgesic effects on acetic acid-induced visceral pain (ED50 = 2.15 ± 0.07 mg/kg). Moreover, compound 1 exhibited a distinct reduction in the NaV1.7 and NaV1.8 channel currents during both resting and half-inactivated states at 50 µM. The present study enriches the pharmacological activities of franchetine derivatives and provides valuable insights for the development of novel anti-inflammatory and analgesic agents.

Animal models of heart failure with preserved ejection fraction (HFpEF): from metabolic pathobiology to drug discovery.

Heart failure (HF) with preserved ejection fraction (HFpEF) is currently a preeminent challenge for cardiovascular medicine. It has a poor prognosis, increasing mortality, and is escalating in prevalence worldwide. Despite accounting for over 50% of all HF patients, the mechanistic underpinnings driving HFpEF are poorly understood, thus impeding the discovery and development of mechanism-based therapies. HFpEF is a disease syndrome driven by diverse comorbidities, including hypertension, diabetes and obesity, pulmonary hypertension, aging, and atrial fibrillation. There is a lack of high-fidelity animal models that faithfully recapitulate the HFpEF phenotype, owing primarily to the disease heterogeneity, which has hampered our understanding of the complex pathophysiology of HFpEF. This review provides an updated overview of the currently available animal models of HFpEF and discusses their characteristics from the perspective of energy metabolism. Interventional strategies for efficiently utilizing energy substrates in preclinical HFpEF models are also discussed.

A novel anti-CTLA-4 nanobody-IL12 fusion protein in combination with a dendritic cell/tumour fusion cell vaccine enhances the antitumour activity of CD8+ T cells in solid tumours.

BACKGROUND: We previously developed a nanobody targeting CTLA-4 and demonstrated that it can boost antitumour T-cell responses in vitro; however, the resulting responses after the injection of T cells into cancer models are usually weak and transient. Here, we explored whether fusing our nanobody to IL-12 would enable it to induce stronger, longer-lasting T-cell immune responses after exposure to immature dendritic cell and tumour cell fusions. RESULTS: The fusion protein enhanced the response of CD8+ T cells to tumour antigens in vitro and led to stronger, more persistent immune responses after the T cells were injected into mice bearing different types of xenografts. CONCLUSION: Our in vitro and in vivo results suggest the anticancer potential of our nanobody-interleukin fusion system and support the clinical application of this fusion approach for various nanobodies.

Meta-analysis and systematic review of the relationship between sex and the risk or incidence of poststroke aphasia and its types.

OBJECTIVE: To analyse and discuss the association of gender differences with the risk and incidence of poststroke aphasia (PSA) and its types, and to provide evidence-based guidance for the prevention and treatment of poststroke aphasia in clinical practice. DATA SOURCES: Embase, PubMed, Cochrane Library and Web of Science were searched from January 1, 2002, to December 1, 2023. STUDY SELECTION: Including the total number of strokes, aphasia, the number of different sexes or the number of PSA corresponding to different sex. DATA EXTRACTION: Studies with missing data, aphasia caused by nonstroke and noncompliance with the requirements of literature types were excluded. DATA SYNTHESIS: 36 papers were included, from 19 countries. The analysis of 168,259 patients with stroke and 31,058 patients with PSA showed that the risk of PSA was 1.23 times higher in female than in male (OR = 1.23, 95% CI = 1.19-1.29, P < 0.001), with a prevalence of PSA of 31% in men and 36% in women, and an overall prevalence of 34% (P < 0.001). Analysis of the risk of the different types of aphasia in 1,048 patients with PSA showed a high risk in females for global, broca and Wenicke aphasia, and a high risk in males for anomic, conductive and transcortical aphasia, which was not statistically significant by meta-analysis. The incidence of global aphasia (males vs. females, 29% vs. 32%) and broca aphasia (17% vs 19%) were higher in females, and anomic aphasia (19% vs 14%) was higher in males, which was statistically significant (P < 0.05). CONCLUSIONS: There are gender differences in the incidence and types of PSA. The risk of PSA in female is higher than that in male.

Discovery of 4-(isopentyloxy)-3-nitrobenzamide derivatives as xanthine oxidase inhibitors through a non-anthraquinone exploration.

In our previous study, we reported a series of N-(9,10-anthraquinone-2-carbonyl) amino acid derivatives as novel inhibitors of xanthine oxidase (XO). Recognizing the suboptimal drug-like properties associated with the anthraquinone moiety, we embarked on a nonanthraquinone medicinal chemistry exploration in the current investigation. Through systematic structure-activity relationship (SAR) studies, we identified a series of 4-(isopentyloxy)-3-nitrobenzamide derivatives exhibiting excellent in vitro potency against XO. The optimized compound, 4-isopentyloxy-N-(1H-pyrazol-3-yl)-3-nitrobenzamide (6k), demonstrated exceptional in vitro potency with an IC50 value of 0.13 µM. Compound 6k showed favorable drug-like characteristics with ligand efficiency (LE) and lipophilic ligand efficiency (LLE) values of 0.41 and 3.73, respectively. In comparison to the initial compound 1d, 6k exhibited a substantial 24-fold improvement in IC50, along with a 1.6-fold enhancement in LE and a 3.7-fold increase in LLE. Molecular modeling studies provided insights into the strong interactions of 6k with critical amino acid residues within the active site. Furthermore, in vivo hypouricemic investigations convincingly demonstrated that 6k significantly reduced serum uric acid levels in rats. The MTT results revealed that compound 6k is nontoxic to healthy cells. The gastric and intestinal stability assay demonstrated that compound 6k exhibits good stability in the gastric and intestinal environments. In conclusion, compound 6k emerges as a promising lead compound, showcasing both exceptional in vitro potency and favorable drug-like characteristics, thereby warranting further exploration.

Optimization of fermentation conditions for the production of γ-aminobutyric acid by Lactobacillus hilgardii GZ2 from traditional Chinese fermented beverage system.

γ-Aminobutyric acid (GABA) is a crucial neurotransmitter with wide application prospects. In this study, we focused on a GABA-producing strain from a traditional Chinese fermented beverage system. Among the six isolates, Lactobacillus hilgardii GZ2 exhibited the greatest ability to produce GABA in the traditional Chinese fermented beverage system. To increase GABA production, we optimized carbon sources, nitrogen sources, temperature, pH, and monosodium glutamate and glucose concentrations and conducted fed-batch fermentation. The best carbon and nitrogen sources for GABA production and cell growth were glucose, yeast extract and tryptone. Gradual increases in GABA were observed as the glucose and monosodium glutamate concentrations increased from 10 g/L to 50 g/L. During fed-batch fermentation, lactic acid was used to maintain the pH at 5.56, and after feeding with 0.03 g/mL glucose and 0.4 g/mL sodium glutamate for 72 h, the GABA yield reached 239 g/L. This novel high-GABA-producing strain holds great potential for the industrial production of GABA, as well as the development of health-promoting functional foods and medical fields.

[Effects of different exercise modes on neuromuscular junction and metabolism of skeletal muscle-related proteins in aging rats].

The present study aimed to explore the effects of different exercise modes on neuromuscular junction (NMJ) and metabolism of skeletal muscle-related proteins in aging rats. Ten from 38 male Sprague-Dawley (SD) rats (3-month-old) were randomly selected into young (Y) group, while the rest were raised to 21 months old and randomly divided into elderly control (O), endurance exercise (EN) and resistance exercise (R) groups. After 8 weeks of corresponding exercises training, the gastrocnemius muscles of rats were collected, and the expression of S100B in Schwann cells was detected by immunofluorescence staining. Western blot was used to detect the protein expression levels of agglutinate protein (Agrin), low-density lipoprotein receptor-related protein 4 (Lrp4), muscle- specific kinase protein (MuSK), downstream tyrosine kinase 7 (Dok7), phosphorylated protein kinase B (p-Akt), phosphorylated mammalian target rapamycin (p-mTOR), and phosphorylated forkhead box O1 (p-FoxO1) in rat gastrocnemius muscles. The results showed that, endurance and resistance exercises increased the wet weight ratio of gastrocnemius muscle in the aging rats. The protein expression of S100B in the R group was significantly higher than those in the O and EN groups. Proteins related to NMJ function, including Agrin, Lrp4, MuSK, and Dok7 were significantly decreased in the O group compared with those in the Y group. Resistance exercise up-regulated these four proteins in the aging rats, whereas endurance exercise could not reverse the protein expression levels of Lrp4, MuSK and Dok7. Regarding skeletal muscle-related proteins, the O group showed down-regulated p-Akt, and p-mTOR protein expression levels and up-regulated p-FoxO1 protein expression level, compared to the Y group. Resistance and endurance exercises reversed the changes in p-mTOR and p-FoxO1 protein expression in the aging rats. These findings demonstrate that both exercise modes can enhance NMJ function, increase protein synthesis and reduce the catabolism of skeletal muscle-related proteins in aging rats, with resistance exercise showing a more pronounced effect.

Analgesic Characteristics of Ultrasound-guided Great Auricular Nerve Block in Middle Ear Surgery: A Prospective Randomized Controlled Preliminary Trial.

PURPOSE: To explore the analgesic characteristics of ultrasound-guided great auricular nerve (GAN) block to further improve pain management. DESIGN: Single-center, prospective, randomized, controlled, and double-blind preliminary clinical trial. METHODS: Thirty-seven patients who underwent middle ear surgery were included in this study: 15 in the GAN block group (the large ear nerve block [NB] group) and 22 in the traditional anesthesia group (control [CON] group). After induction of anesthesia, the NB group was given an ultrasound-guided GAN block (0.25 % Ropivacaine 2 mL), while the CON group was exempt from the GAN block. The patient's basic information, perioperative information, the region, and numeric rating scale of postoperative pain (at 1 hour, 6 hours, 12 hours, and 24 hours), and adverse reactions were recorded. Repeated measurement analysis, t test, and Fisher exact probability method were used for statistical analysis. FINDINGS: Compared with the CON group, the numeric rating scale in the NB group was lower after surgery (1 hour: 1.18 ± 0.35 vs 0.27 ± 0.20, P = .023; 6 hours: 1.82 ± 0.37 vs 1.13 ± 0.39, P = .203; 12 hours: 1.05 ± 0.19 vs 0.20 ± 0.10, P < .001; 24 hours: 0.55 ± 0.17 vs 0.13 ± 0.09, P = .029). In the NB group, the region of pain was merely concentrated in the ear canal. In the CON group, the pain extended to areas outside the ear canal, such as tragus and mastoid (at 12 hours, P = .006). There was no significant difference in the risk of postoperative adverse reactions between the two groups. CONCLUSIONS: Ultrasound-guided GAN block can relieve patients' pain after middle ear surgery, especially in the area outside the ear canal.