BACKGROUND:Ammonia poisoning is considered to be the main hypothesis for the pathogenesis of hepatic encephalopathy.Ammonia can lead to psychiatric and cognitive behavioral disorders,although the specific pathological molecular mechanisms remain unclear.OBJECTIVE:To investigate the effects of ammonia poisoning on cognitive behavior and hippocampal neuronal synapses in mice.METHODS:Thirty-two C57BL/6J mice were randomly divided into a normal control group and an ammonium chloride group,with 16 mice in each group.Normal saline was injected intraperitoneally in the control group,and ammonium chloride(10 mmol/kg)was injected intraperitoneally in the ammonium chloride group to construct a model of ammonia poisoning,once a day.After 7 days of ammonium chloride intervention,blood samples were collected from the hearts of six mice in each group for blood ammonia concentration detection.Behavioral experiments,including the open field test,novel object recognition test,and Y-maze test,were performed to assess mental and cognitive-behavioral changes in mice.Finally,hippocampal tissues were extracted for western blot analysis to detect the expression levels of synaptophysin and postsynaptic density protein-95 in hippocampal neurons.RESULTS AND CONCLUSION:The blood ammonia concentration was significantly elevated in the ammonium chloride group compared with the control group(P＜0.05).Mice in the ammonium chloride group showed anxiety-like behavior and disinhibition phenomenon,and a significant decrease in recognition memory and working memory ability.Western blot results revealed that the expression of synaptophysin and postsynaptic density protein-95 protein in hippocampal neurons in the ammonium chloride group was lower than that in the control group(P＜0.05).To conclude,ammonia poisoning can induce hippocampal neuronal synaptic damage,leading to psychiatric and cognitive behavioral abnormalities in mice.

BACKGROUND:Ammonia poisoning is considered to be the main hypothesis for the pathogenesis of hepatic encephalopathy.Ammonia can lead to psychiatric and cognitive behavioral disorders,although the specific pathological molecular mechanisms remain unclear.OBJECTIVE:To investigate the effects of ammonia poisoning on cognitive behavior and hippocampal neuronal synapses in mice.METHODS:Thirty-two C57BL/6J mice were randomly divided into a normal control group and an ammonium chloride group,with 16 mice in each group.Normal saline was injected intraperitoneally in the control group,and ammonium chloride(10 mmol/kg)was injected intraperitoneally in the ammonium chloride group to construct a model of ammonia poisoning,once a day.After 7 days of ammonium chloride intervention,blood samples were collected from the hearts of six mice in each group for blood ammonia concentration detection.Behavioral experiments,including the open field test,novel object recognition test,and Y-maze test,were performed to assess mental and cognitive-behavioral changes in mice.Finally,hippocampal tissues were extracted for western blot analysis to detect the expression levels of synaptophysin and postsynaptic density protein-95 in hippocampal neurons.RESULTS AND CONCLUSION:The blood ammonia concentration was significantly elevated in the ammonium chloride group compared with the control group(P＜0.05).Mice in the ammonium chloride group showed anxiety-like behavior and disinhibition phenomenon,and a significant decrease in recognition memory and working memory ability.Western blot results revealed that the expression of synaptophysin and postsynaptic density protein-95 protein in hippocampal neurons in the ammonium chloride group was lower than that in the control group(P＜0.05).To conclude,ammonia poisoning can induce hippocampal neuronal synaptic damage,leading to psychiatric and cognitive behavioral abnormalities in mice.

Objective:The variability of the apnea-hypopnea index（AHI） measured in the first and second halves of the night is significant in patients with obstructive sleep apnea hypopnea syndrome（OSAHS）. This variation may be related to fluid redistribution caused by the supine position during sleep. Methods:Eighty-nine adult subjects were enrolled. Circumferences（neck, chest, waist, and calf） were measured before sleep onset and upon awakening. Polysomnography（PSG） was performed, and the night was divided into two halves based on the midpoint of total sleep time to calculate AHI for each half. The correlation between changes in AHI and changes in circumferences was analyzed. Results:Twenty simple snorers and sixty-nine OSAHS patients were included, with a median AHI of 22.6（11.8, 47.3） events/hour. Compared to pre-sleep measurements, there was no significant change in neck circumference upon awakening in the control group（P=0.073）, while reductions were observed in the other three measurements（P=0.006, P=0.038, P<0.001）. In the OSAHS group, neck circumference increased（P<0.001）, and reductions were noted in the other three measurements（P<0.001 for all）, with the most significant change observed in calf circumference 40.0（37.1, 42.0） cm to 38.0（35.8, 40.5） cm. Compared to the first half of the night, total AHI, supine AHI, and NREM AHI significantly decreased in the second half（P=0.010, P=0.031, P=0.001）, while no significant changes were observed in lateral AHI and REM AHI（P=0.988, P=0.530）. Further analysis revealed a significant relationship between increased chest circumference and decreases in NREM AHI, supine AHI, and supine NREM AHI（P=0.036, P=0.072, P=0.034）, as well as between decreased lateral position AHI and increased waist circumference（P=0.048）. Additionally, this study found a negative correlation between changes in calf circumference and changes in AHI（R=-0.24, P=0.048）, while neck circumference changes positively correlated with changes in AHI（R=0.26, P=0.03）. Conclusion:In OSAHS patients during the second half of sleep compared to before sleeping, chest circumference, waist circumference, and calf circumference decrease while neck circumference increases; total AHI, supine position AHI, and NREM period AHI decrease; increases in chest circumference are associated with decreases in NREM period AHI, supine position AHI, supine position NREM period AHI. There is nocturnal variability in AHI among OSAHS patients that may be associated with fluid shifts during sleep.
Humans ; Sleep Apnea, Obstructive/physiopathology* ; Male ; Female ; Polysomnography ; Fluid Shifts/physiology* ; Adult ; Middle Aged ; Neck ; Severity of Illness Index ; Sleep/physiology* ; Snoring/physiopathology*

Anxiety disorder is a major symptom of autism spectrum disorder (ASD) with a comorbidity rate of ~40%. However, the neural mechanisms of the emergence of anxiety in ASD remain unclear. In our study, we found that hyperactivity of basolateral amygdala (BLA) pyramidal neurons (PNs) in Shank3 InsG3680 knock-in (InsG3680^+/+) mice is involved in the development of anxiety. Electrophysiological results also showed increased excitatory input and decreased inhibitory input in BLA PNs. Chemogenetic inhibition of the excitability of PNs in the BLA rescued the anxiety phenotype of InsG3680^+/+ mice. Further study found that the diminished control of the BLA by medial prefrontal cortex (mPFC) and optogenetic activation of the mPFC-BLA pathway also had a rescue effect, which increased the feedforward inhibition of the BLA. Taken together, our results suggest that hyperactivity of the BLA and alteration of the mPFC-BLA circuitry are involved in anxiety in InsG3680^+/+ mice.
Animals ; Prefrontal Cortex/metabolism* ; Basolateral Nuclear Complex/metabolism* ; Mice ; Anxiety/metabolism* ; Nerve Tissue Proteins/genetics* ; Male ; Gene Knock-In Techniques ; Pyramidal Cells/physiology* ; Mice, Transgenic ; Neural Pathways/physiopathology* ; Mice, Inbred C57BL ; Microfilament Proteins

OBJECTIVE This study investigated the clinical implications of endothelial cell-specific molecule 1(ESM-1)in obstructive sleep apnea(OSA)patients,with particular focus on its dynamic correlation with adhesion molecules,aiming to elucidate the regulatory role of ESM-1 in OSA-associated vascular endothelial impairment.METHODS This cross-sectional study enrolled participants undergoing polysomnography(PSG)at the Sleep Medicine Center of Beijing Anzhen Hospital,Capital Medical University between March 2017 and January 2018.Based on the inclusion criteria,161 participants were ultimately included and divided into OSA group(n=118)and control group(n=43).Demographic data and polysomnography parameters were collected.We used a powerful high-throughput Multiplex Immunobead Assay technology to simultaneously test plasm cytokines levels of ESM-1,inter-cellular adhesion molecule 1(ICAM-1),vascular cell adhesion molecule 1(VCAM-1).Circulating C-reactive protein(CRP)and homocysteine(Hcy)were detected by routine blood chemistry panel.RESULTS Circulating ESM-1 levels were significantly elevated in patients with OSA compared with healthy controls[819.73(612.36-1393.47)pg/ml]vs.[286.17(114.48-513.81)pg/ml,P<0.001].After adjusting for confounding factors,we found that circulating ESM-1 levels were an independent risk factor for OSA(odds ratio=2.162,95%CI=1.522-3.072,P<0.001)and circulating ESM-1 levels were positively associated with ICAM-1 and VCAM-1 levels(β=1.977,95%CI=1.429-2.734,P<0.001).CONCLUSION Circulating ESM-1 levels were significantly increased in patients with OSA,which is closely related with adhesion molecules levels.ESM-1 may be a surrogate endothelial dysfunction marker and an independent risk factor for OSA.

BACKGROUND:Solute carrier family 1 member 5(SLC1A5)plays a potential role in a variety of diseases,but the exact mechanism of action is unclear.The construction of stable SLC1A5 overexpression and knockdown cell models can provide a powerful experimental tool for in-depth study of the exact role and mechanism of SLC1A5 in diseases and the discovery of potential therapeutic targets. OBJECTIVE:To construct lentiviral vectors for overexpression and knockdown of mouse SLC1A5 and establish stable transfected RAW264.7 cell lines,so as to provide an experimental foundation for further investigation of the role of SLC1A5 in inflammation. METHODS:Primers were designed and synthesized based on the SLC1A5 gene sequence,and the gene segment was amplified using polymerase chain reaction.Subsequently,the target gene segment was directionally inserted into the GV492 vector plasmid,which had been digested with AgeI/NheI enzymes,to construct recombinant lentiviral plasmids.Positive clones were further selected,and their sequences were confirmed.The pHelper1.0 plasmid vector and pHelper2.0 plasmid vector,along with the target plasmid vector,was co-cultured with 293T cells for transfection,resulting in the production and titration of lentiviral stocks.Furthermore,RAW264.7 cells were cultured in vitro,and the working concentration of puromycin was determined.Lentiviruses were separately co-cultured with RAW264.7 cells,and transfection efficiency was determined by measuring fluorescence intensity.Stable transfected cells were selected using puromycin,and real-time fluorescence quantitative PCR and western blot assay were employed to assess the gene and protein expression levels of SLC1A5 in stably transfected cell lines. RESULTS AND CONCLUSION:(1)Sequencing results indicated a perfect match between the sequencing and target sequences,confirming the successful construction of recombinant lentiviral vectors.(2)The titer for the overexpression SLC1A5 lentivirus was 1×109 TU/mL,while the titer for the knockdown SLC1A5 lentivirus was 3×109 TU/mL.(3)The working concentration of puromycin for RAW264.7 cells was determined to be 3 μg/mL.(4)The optimal conditions for transfecting RAW264.7 cells with overexpression/knockdown expression of SLC1A5 lentivirus involved the use of HiTransG P transfection enhancer with a multiplicity of infection value of 50.(5)A significant upregulation of the gene and protein expression levels of SLC1A5 was detected in cell lines stably overexpressing SLC1A5,while gene and protein expression levels of SLC1A5 were significantly decreased in the knockdown stable cell lines.These findings indicate that lentiviral vectors for mouse SLC1A5 overexpression and knockdown have been successfully constructed and a stably transfected RAW264.7 cell line has been obtained.

Objective:The SARS-CoV-2 has a high natural mutation rate, and dynamic monitoring of virus variants remains a key focus in current COVID-19 prevention and control efforts.Methods:In this study, a sensitive and rapid method for detecting SARS-CoV-2 omicron variant nucleic acid was established based on the BMD-PCR technology.Results:This method showed good specificity, and had no cross-reactivity with 11 common viruses transmitted via the respiratory and gastrointestinal tracts, and the limit of detection is 555 copies/ml. Compared with SARS-CoV-2 whole-genome sequencing result, among 50 samples with original Ct values ≤32 tested for the Omicron variant, 49 samples tested positive for the N679K mutation site using BMD-PCR Omicron variant detection, achieving a concordance rate of 98.00%. For 30 samples JN.1 lineage, 29 samples tested positive for the K356T mutation site using BMD-PCR JN.1 lineage detection, with a concordance rate of 96.67%. For 10 samples with original SARS-CoV-2 detection Ct values between 35 and 32, 7 samples tested positive for the N679K mutation site using BMD-PCR Omicron variant detection, Resultsing in a detection rate of 70.00%. For samples with SARS-CoV-2 nucleic acid detection Ct values>35, the detection rate for the N679K mutation site in the BMD-PCR Omicron variant was 20.00%.Conclusions:This method can serve as a high-throughput supplementary approach for the preliminary identification of SARS-CoV-2 variant genotypes.

Objective:To investigate the effects of musculoskeletal ultrasound(MSUS)-guided shoulder joint capsule hydraulic expansion method combined with rotator interval(RI)injection method on ultrasound imaging indicators,shoulder function,and serum inflammatory factors in treating patients with early primary frozen shoulder(PFS).Methods:A total of 168 patients with early PFS admitted to Jiangbei District of The Frist Affiliated Hospital to Army Medical University between June 2021 and June 2023 were selected.They were randomly divided into a control group and an observation group by using a random number table,with 84 patients in each group.The control group received the therapy with MSUS-guided shoulder joint capsule hydraulic expansion.The observation group received the therapy with RI injection on the basis of the control group.After 5 weeks of treatment,the changes of the following parameters were compared and analyzed between the two groups:Visual Analog Scale(VAS)score for shoulder pain,Simple Shoulder Test(SST)score,Constant-Murley Score(CMS),thickness of the glenohumeral joint capsule at the axilla of ultrasound imaging,thickness of coracohumeral ligament(CHL),RI thickness,positivity rate of RI blood flow,passive range of motion(ROM)of the shoulder joint,and serum inflammatory factor levels including tumor necrosis factor-α(TNF-α),C-reactive protein(CRP)and interleukin-6(IL-6).Results:At 5th week after treatment,both groups showed VAS scores decreased,and SST and CMS scores increased.The VAS score(1.34±0.30)of observation group was significantly lower than(1.97±0.46)of control group,and the SST score and CMS score were significantly higher than those of control group(t=10.514,9.597,7.910,P<0.05),respectively.Both groups showed decreases in thickness of the glenohumeral joint capsule at the axilla,CHL thickness,RI thickness,and positivity rate of RI blood flow,and these indicators of observation group were significantly lower than them of control group,with statistically significant differences(t=5.518,16.106,9.050,25.211,P<0.05).The ranges of backward extension and outward rotation of observation group were larger than those of control group,and the differences were statistically significant(t=9.209,12.447,P<0.05).The serum levels of TNF-α,IL-6 and CRP of observation group were significantly lower than those of control group,and the differences were statistically significant(t=10.523,17.750,19.995,P<0.05),respectively.Conclusion:The treatment of MSUS-guided shoulder joint capsule hydraulic expansion combined with RI injection can effectively alleviate the degrees of shoulder pain and dysfunction in patients with early PFS,and improve indicators of ultrasound imaging,the ROM values of outward rotation and backward extension,and reduce serum levels of inflammatory factors,which have favorable therapeutic effects.

Objective:To establish a fluorescent quantitative RT-PCR assay based on N_sgRNA of SARS-CoV-2 and preliminarily apply it on real samples.Methods:Recombinant plasmid, specific primers and probes of N_sgRNA were designed and synthesized based on Wuhan-Hu-1/2019_MN908947 and synthesis mechanism of subgenomic RNA (sgRNA). Using recombinant plasmid as amplification templates, the optimal reaction conditions and reaction system were screened according to the Ct value, fluorescence intensity, and shape of amplification curve and was evaluated for sensitivity, reproducibility, and specificity. Meanwhile, the possibility of practical application of the method was explored by testing 172 clinical samples and 256 municipal wastewater samples. Results:A qRT-PCR assay for N_sgRNA in SARS-CoV-2 was initially established. The detection limit of the assay was 20 copies/mL, and the variation coefficients of in-batch (0.002%~0.767%) and batch to batch repetition (0.016%~0.752%) were less than 1%. Only N_sgRNA recombinant plasmid was detected in the specificity assay. So the method is more highly sensitive, specific and reproducible. The RatiosgRNA/ gRNA of clinical samples HK.3, EG.5.1, JN.1 and their sub-lineages and their corresponding urban sewage samples in epidemic period were significantly different ( P<0.05). There is a strong correlation between the median of RatiosgRNA/ gRNA in clinical samples and sewage samples in the same period (correlation coefficient r=1.000, P=0.010). Conclusions:In this study, a qRT-PCR method for detecting N_sgRNA of SARS-CoV-2 was established and the method has the characteristics of higher sensitivity, stronger specificity and better repeatability, and it can be used to detect SARS-CoV-2 infectivity.

Methods: This retrospective study analyzed 32 consecutive IDS patients who underwent UEDD surgery. Clinical features, laboratory data (erythrocyte sedimentation rate and C-reactive protein), and treatment outcomes were analyzed. Results: Definite microorganisms were identified in 27 patients (84.3%), with 24 (88.9%) meeting cure criteria. The cure rate was significantly higher in the detected pathogen group compared to the undetected pathogen group (88.9% vs. 80%; χ²=19.36, p<0.0001). Metagenomic next generation sequencing (mNGS) provided faster diagnosis (41.72±6.81 hours) compared to tissue culture (95.74±35.47 hours, p<0.05). The predominant causative pathogen was Mycobacterium tuberculosis, followed by Staphylococcus aureus. Significant improvements were observed in Visual Analog Scale pain scores, from a mean of 7.9 preoperatively to 1.06 at 1 year postoperatively. The Oswestry Disability Index revealed a similar trend, showing significant improvement (p<0.05). Conclusions UEDD is a viable alternative to traditional open surgery for managing IDS in high-risk patients. UEDD offers a dual therapeutic-diagnostic advantage during the initial admission phase, enabling simultaneous debridement, neurological decompression, and targeted biopsy in a single intervention. Compared with traditional tissue culture, mNGS enables rapid microbiological diagnosis and extensive pathogen coverage.