Objective To explore the polymorphism of tyrosinase (TYR) and melanocortin 1 receptor (MC1R) genes and their mRNA expression levels in relation to coat-color phenotypes in guinea pigs, providing genetic markers for locating dominant traits in guinea pigs. Methods A total of 57 self-bred ordinary-level guinea pigs were selected and divided into three groups based on coat color: white (n=22), variegated (n=22) and black (n=13). The guinea pigs were euthanized with an overdose of pentobarbital sodium via intraperitoneal injection. DNA was then extracted from the dorsal skin tissue. Polymorphism in the coding sequence (CDS) of the exons of the TYR and MC1R genes in each group was detected by cloning and sequencing. The mRNA expression of the two genes in skin tissues was detected by real-time fluorescent quantitative PCR to investigate the relationship between these genes and guinea pig coat color. Results A single nucleotide polymorphism (SNP) site was found in the CDS region of TYR exon Ⅰ, where the base A was replaced by G. All white guinea pigs had the G/G genotype for TYR, while no deep-colored (variegated and black) guinea pigs exhibited the G/G genotype for TYR. Most deep-colored guinea pigs had the A/A genotype, and a few had A/G genotype. The A/A genotype frequency in black guinea pigs was higher than in variegated guinea pigs. A 2 760 bp sequence deletion was identified in the exon of the MC1R gene, marked as the - gene, with non-deleted samples marked as N gene. Most white guinea pigs had the -/- genotype for MC1R, variegated guinea pigs mainly had the -/N genotype, and black guinea pigs mainly had the N/N genotype, with a few showing the -/N. The TYR gene expression level was higher in white guinea pigs, lower in variegated guinea pigs, and intermediate in black guinea pigs, but there was no significant difference among the three groups (P>0.05). The MC1R gene expression level in white guinea pigs was extremely low, while both variegated and black guinea pigs showed significantly higher levels than white guinea pigs (P<0.01). Black guinea pigs showed significantly higher levels than variegated guinea pigs (P<0.05). ConclusionThe TYR and MC1R genes synergistically regulate coat color of guinea pigs. The G-site mutation in the TYR gene may lead to albinism, and the change of N-site in the MC1R gene affects the depth of the coat color.

ObjectiveThe utilization of assisted reproductive technology to rescue genetically modified mouse strains with reproductive disorders provides a reference for improving techniques to preserve valuable experimental mouse strains. MethodsIn vitro fertilization-embryo transfer (IVF-ET) technology was performed on 28 strains of infertile male mice aged 9-18 months. Several indicators such as sperm density and sperm motility in infertile male mice were assessed to select the most viable sperm for IVF-ET experiments. Fertility rate, abnormal egg rate, and birth rate were recorded after the birth of the pups. An optimized ovarian transplantation procedure was applied to 12 strains of infertile female mice aged 8-18 months. 6-week-old female mice with the same genetic background were selected as recipients. One intact ovary was removed from each recipient mouse, and the contralateral oviduct was ligated. An ovary from a donor mouse was isolated and transplanted orthotopically into the side where the ovary had been removed in the recipient mouse. Twenty-one days post-surgery, recipient mice were co-housed with 8-week-old wild type male mice of the same genetic background for breeding. Data such as the pregnancy rate and live birth rate of the recipients were recorded after the birth of the pups. ResultsIVF-ET successfully rescued 28 mouse strains, with the oldest male mice being 18 months old. The success rate of the first round of IVF-ET experiments was 89.29% (25/28). The average fertility rate of IVF in infertile male mice was (51.01±14.97)%, the abnormal egg rate was (9.03±5.28)%, and the birth rate of offspring mice was (18.60±7.03)%. 39 out of 40 ovarian transplant recipient mice survived, with a pregnancy rate of 33.33% (13/39) for ovarian transplant recipients, and a live birth rate of 17.95% (7/39). Four mouse strains were successfully rescued using optimized ovarian transplantation technology, with the oldest female mice being 18 months old. 8 strains were not rescued as they failed to produce offspring that survived to sexual maturity. ConclusionIVF-ET is an effective approach for rescuing mice with reproductive disorders caused by different reasons, especially for those beyond the optimal breeding age. Ovarian transplantation technology can also be used as an alternative for aged female mice. But its success rate is relatively lower than that of IVF-ET, and carries a higher experimental risk.

The purpose of this study was to investigate the anxiety-like behaviors, circadian rhythms and sleep, and to elucidate the possible underlying mechanisms of the abnormal sleep behavior in Shank3 gene knockout (Shank3-KO) mice. The anxiety-like behaviors were detected by elevated plus-maze (EPM) test, open field test (OFT) and tail suspension test (TST). The circadian rhythms were detected by running wheel test. The electroencephalogram (EEG)/electromyogram (EMG) recordings were performed synchronically by polysomnograph. The distribution of SHANK3 in anterior cingulate cortex (ACC), paraventricular thalamus (PVT), nucleus accumbens (NAc), basolateral amygdala (BLA) and hippocampal CA2 region in wild type (WT) mice was detected by immunofluorescence assay. The protein expression of c-Fos in PVT, ACC and NAc was also detected by immunofluorescence assay during light cycle. The colocalization of c-Fos and vesicular glutamate transporter 2 (Vglut2, a marker for glutamatergic neurons) in the PVT was detected by immunofluorescence double labeling experiment. The results of EPM test showed that, compared with the WT mice, the Shank3-KO mice showed less time in open arms and less number of open arm entries. The results of OFT showed that the Shank3-KO mice showed less time in central area and less number of central area entries. The immobility time of Shank3-KO mice was increased in the TST. The results of running wheel rhythm test showed that the phase shift time of Shank3-KO mice in the continuous dark period was increased. The results of EEG/EMG recording showed that, compared with the WT mice, the duration of wakefulness in Shank3-KO mice was increased and the duration of non-rapid eye movement (NREM) sleep was decreased during light phase; The bout number of wakefulness was increased, the bout number of NREM sleep was decreased, NREM-wake transitions were increased, and wake-NREM transitions were decreased during light phase. SHANK3 was expressed in ACC, PVT, NAc and BLA in the WT mice. The expression of c-Fos in the PVT of Shank3-KO mice was up-regulated 2 h after entering the light phase, and majority of c-Fos was co-localized with Vglut2. These results suggest that the anxiety level of Shank3-KO mice is increased, the regulation of the internal rhythms is decreased, and the bout number of wakefulness is increased during light phase. The glutamatergic neurons in PVT may be involved in the regulation of abnormal sleep behavior in Shank3-KO mice during the light phase.
Animals ; Mice, Knockout ; Mice ; Neurons/metabolism* ; Nerve Tissue Proteins/physiology* ; Male ; Midline Thalamic Nuclei/cytology* ; Circadian Rhythm/physiology* ; Sleep/physiology* ; Anxiety/physiopathology* ; Proto-Oncogene Proteins c-fos/metabolism* ; Vesicular Glutamate Transport Protein 2/metabolism* ; Mice, Inbred C57BL ; Microfilament Proteins

Diabetic nephropathy(DN), a prevalent microvascular complication of diabetes, has emerged as a leading cause of end-stage renal disease worldwide. Recent studies on the dual-specific phosphatase (DUSP) family have revealed a significant reduction in DUSP expression levels in renal disease, suggesting that enhancing its expression may mitigate or alleviate the symptoms associated with renal disease. The primary function of DUSP is to mediate the dephosphorylation of mitogen-activated protein kinase (MAPK), which effectively inhibits the activation of the MAPK pathway, thus playing a crucial regulatory role in the onset and progression of DN. This article aims to investigate the correlation between DN and DUSP and to summarize the current research advancements concerning DUSP in the context of DN, providing new insights and essential theoretical foundations for its diagnosis and treatment.

OBJECTIVE: To elucidate the modulation mechanism of Suanzaoren Decoction (SZRD) on basolateral amygdala (BLA) neuronal activity to alleviate chronic restraint stress (CRS)-related behavioral deficits. METHODS: The male C57BL/6J mice were assigned to 4 groups using the complete randomization method, including control (CON, n=19), CRS (n=19), SZRD (n=21), and fluoxetine (Flu, n=22) groups. Mice were restrained for 6 h per day, over a 21-d period to establish CRS models. The CON group remained in their cages without food or water during the 6-h matching period. SZRD and Flu groups received intragastric administration of SZRD (4.68 g/kg) and Flu (20 mg/kg) daily, respectively, 30 min before restraint for 21 consecutive days. The therapeutic effects of SZRD were evaluated using behavioral tests including the tail suspension test, elevated plus maze test, and forced swimming test. The cellular Fletcher B. Judson murine osteosarcoma proto-oncogene (c-Fos) expression in the BLA was measured using immunofluorescence, while action potential (AP) firing and synaptic transmission in BLA pyramidal neurons were evaluated using whole-cell patch-clamp recordings. RESULTS: SZRD administration significantly increased time spent in the open arms and open-arm entries while reducing immobility time (P<0.05 or P<0.01). It downregulated CRS-induced c-Fos expression and AP firing of pyramidal neurons in the BLA (P<0.01). Additionally, SZRD selectively attenuated excitatory (P<0.01), but not inhibitory, synaptic transmission onto BLA pyramidal neurons. CONCLUSION SZRD alleviated CRS-induced anxiety- and depression-like behaviors in mice by modulating the excitability and synaptic transmission of BLA pyramidal neurons.
Animals ; Drugs, Chinese Herbal/therapeutic use* ; Depression/complications* ; Pyramidal Cells/pathology* ; Male ; Mice, Inbred C57BL ; Basolateral Nuclear Complex/pathology* ; Restraint, Physical ; Anxiety/complications* ; Behavior, Animal/drug effects* ; Stress, Psychological/physiopathology* ; Mice ; Proto-Oncogene Proteins c-fos/metabolism* ; Action Potentials/drug effects* ; Synaptic Transmission/drug effects*

Objective To explore the feasibility and corresponding implementation methods of constructing a sample resource bank based on individual-level data of completed clinical trials and using it to construct external controls for drug/device clinical trials.Methods Taking the pre-marketing clinical trial of transcatheter active valve replacement(TAVR)for the treatment of aortic valve stenosis as an example,the individual-level databases of multiple trials were standardized to form a sample bank.The original data of any trial in the sample bank were selected as the experimental group,and the remaining samples were selected as the control group.The potential confounding was handled by using the propensity score matching and stratification methods to clarify the process of constructing external controls based on the sample bank of individual-level data of clinical trials.Results This study included individual-level data of single-group trials of 4 TAVR devices,with a total of 569 subjects(59.2%male).The number of subjects in Trials 1 to 4 was 120,120,163,and 166,respectively.Propensity score matching enabled the matching of 113,117,125,and 147 subjects with comparable or similar characteristics from individual-level data from other trials,respectively,demonstrating a high matching success rate.The PS score distribution plot after stratification showed that the proportions of subjects in the experimental and control groups in strata 1 to 5 in scheme 1 were 4/103,11/103,22/92,32/87,and 51/64,respectively.For all constructed external controlled trials,a certain number of control samples with similar baseline characteristics to the experimental groups were distributed within each propensity score stratum.The results of the simulation test also reflected the potential differences between different devices in the 12-month all-cause mortality rate.Conclusions The sample bank constructed with individual-level data from clinical trials,as a high-quality data source,can serve as a source of external control for single-arm trials in the same field,and as a useful supplement to the external control scenario of real-world evidence to support drug and device development.At the same time,targeted research on research methods and bias control measures in related fields is also needed.

Objective:Analyze the risk factors of gastrointestinal perforation caused by foreign body and summarize the experience of surgical treatment of foreign bodies.Method:From Jan 2008 to Dec 2023, 89 patients with foreign bodies in the digestive tract were admitted to the Department of Gastrointestinal Surgery, Peking University First Hospital. Relevant data were collected and binary logistic regression was used to analyze the independent risk factors for intestinal perforation, resection and anastomosis of intestine or enterostomy/colostomy.Results:The mean age of 89 patients was (60.1±16.2) years old, 65 patients (73%) had unintentionally ingested foreign bodies. The most common foreign bodies were jujube pits (40 cases). Thirty-nine patients diagnosed with gastrointestinal perforation. Binary Logistic regression analysis showed that the total number of leukocytes ( OR=4.085, 95% CI: 1.214-13.745, P=0.023), sharp foreign body ( OR=26.124, 95% CI: 5.194-131.392, P<0.001), and the location of foreign body ( OR=3.980, 95% CI: 1.178-13.465, P=0.026) were the independent risk factors for gastrointestinal perforation. Thirty-three patients underwent gastrointestinal repair surgery, and 36 patients underwent resection and anastomosis of intestine or enterostomy/colostomy. Binary Logistic regression analysis showed that the foreign body located in the colorectum ( OR=71.928, 95% CI: 4.646-1 113.479, P=0.002) and the length of the foreign body ≤2.5 cm ( OR=5.791, 95% CI: 1.606-20.882, P=0.007) were the independent risk factors for resection and anastomosis of intestine or enterostomy/colostomy. Conclusions:Leukocyte count ≥10×10 9/L, sharp foreign body, and location of foreign body are independent risk factors for gastrointestinal perforation. Foreign body located in the colorectum and foreign body length ≤2.5 cm are risk factors for resection and anastomosis of intestine or enterostomy/colostomy.

Objective To explore the surgical treatment experience of primary giant gastrointestinal stromal tumors(GIST)(with isolated lesions with a maximum diameter＞10 cm).Methods A retrospective analysis was conducted on the clinical and pathological data of 67 patients with primary giant GIST admitted from January 2018 to December 2024.Among them,35 cases underwent surgical operations after preoperative neoadjuvant therapy(25 effective cases and 10 ineffective cases)(neoadjuvant therapy group).Due to the initial diagnosis assessment expecting radical(R0)resection(13 cases),or preoperative complications(12 cases),or difficulty in obtaining a pathological diagnosis through puncture biopsy(7 cases),32 cases underwent direct surgery without neoadjuvant therapy(direct surgery group).Compare the general information,tumor condition,surgical condition,postoperative recovery,postoperative pathology,postoperative adjuvant therapy,and recurrence between two groups.Results Comparative analysis revealed that there was no statistically significant difference(P＞0.05)between the neoadjuvant therapy group and the direct surgery group in terms of gender,age,primary tumor location,initial maximum diameter,growth type,localized or locally advanced stage,and postoperative follow-up time.The maximum diameters of the tumors before surgery in the neoadjuvant therapy group and the direct surgery group were(12.4±7.1)cm and(18.2±5.0)cm respectively,and the operation times were(125.4±30.6)minutes and(153.0±31.7)minutes respectively.The intraoperative blood loss was(228.3±76.4)ml and(300.3±67.2)ml,respectively.The postoperative hospital stay was(9.1±2.6)days and(11.1±3.2)days,respectively.There was a statistically significant difference between the two groups(P＜0.05).The proportion of laparoscopic surgery in the neoadjuvant therapy group was 17.1％,which was higher than that in the direct surgery group(0),and the difference was statistically significant(P＜0.05).There was no statistically significant difference between the two groups in terms of the proportion of tumor rupture,combined organ resection,postoperative complications and postoperative recurrence(P＞0.05).Conclusion Primary giant gastrointestinal stromal tumors can mostly be reduced in size and progression through neoadjuvant therapy,improving the chances of minimally invasive surgery.However,there is also a risk of tumor progression during neoadjuvant therapy leading to increased surgical difficulty or even loss of curative surgical opportunities.

Objective To investigate the effect of Eriodictyol(ERI)on the development of metabolic dysfunction-associated steatotic liver disease by regulating the expression of ubiquitin A 52(UBA52)at both in vivo and in vitro levels.Methods A mouse metabolic dysfunction-associated steatotic liver disease model was established using a high-fat diet induction.The mice were randomly separated into the normal control group(normal group),the model group,the low-dose ERI group(ERI-L group,50 mg/kg ERI)and the high-dose ERI group(ERI-H group,100 mg/kg ERI),with 12 mice in each group.Oil red O staining was applied to observe lipid deposition in mouse liver tissue.HE staining was applied to observe pathological changes in mouse liver tissue.ELISA method was applied to detect serum levels of alanine aminotransferase(ALT),aspartate aminotransferase(AST),low-density lipoprotein cholesterol(LDL-C),total cholesterol(TC)and triglycerides(TG)in mice.The expression of UBA52 protein in liver was detected by Western blot assay.HepG2 cells were treated with 0.5 mmol/L oleic acid to induce an in vitro metabolic dysfunction-associated steatotic liver disease model.HepG2 cells were randomly divide into the control group,the oleic acid induced group,the low concentration ERI group(ERI low group,50 μmol/L ERI),the high concentration ERI group(ERI high group,100 μmol/L ERI),the high concentration ERI+si-NC group(ERI high+si-NC group,100 μmol/L ERI+transfected with si-NC)and the high concentration ERI+si-UBA52 group(ERI high+si-UBA52 group,100 μmol/L ERI+transfected with si-UBA52).Oil red O staining was applied to detect lipid deposition in HepG2 cells of each group.ELISA method was applied to detect the levels of TG,TC,SOD and MDA in HepG2 cells in each group.Immunoblotting was used to detect the expression levels of UBA52,p62 and autophagy related proteins in HepG2 cells.Results Compared with the normal group,serum levels of ALT,AST,LDL-C,TC,TG and the expression of UBA52 protein in liver tissue were increased in the model group(P＜0.05),and the lipid deposition in liver increased,pathological damage was severe,and the proportion of lipid deposition area and non-alcoholic fatty liver disease(NAFLD)activity score were also increased(P＜0.05).Changes in the corresponding indicators in the ERI-L group and the ERI-H group were opposite to those of the model group(P＜0.05),and the ERI-H group was even lower(P＜0.05).The lipid deposition in liver decreased and the pathological damage was alleviated.Compared with the control group,the levels of TG,TC,MDA,the proportion of lipid droplet area and the expression of UBA52 protein were increased in HepG2 cells of the oleic acid-induced group,while the levels of SOD,p62 and LC3Ⅱ/LC3Ⅰ decreased(P＜0.05).Changes in the corresponding indicators of the low-concentration ERI group and the high-concentration ERI group were opposite to those of the oleic acid-induced group(P＜0.05),and the therapeutic effect of ERI on metabolic dysfuntion-associated steatotic liver disease was enhanced after knocking down the expression of UBA52.Conclusion ERI may slow down the progression of metabolic dysfuntion-associated steatotic liver disease by down-regulating the expression of UBA52 at both in vivo and in vitro levels.