[Arterial prophylactic occlusion technique in the application of surgery for locally advanced pancreatic cancer with arterial involvement after conversion therapy].

Objective: To investigate and compare the clinical outcomes of the arterial pre-occlusion technique(APOT) and the traditional technique in the surgery of locally advanced pancreatic cancer with arterial involvement after conversion therapy. Methods: This is a retrospective cohort study. The clinical data of 145 patients with locally advanced pancreatic cancer with arterial involvement admitted to the Department of Hepato-Biliary-Pancreatic Surgery of the First Hospital Affiliated to Naval Medical University,from January 2020 to December 2022 were retrospectively analyzed. All patients completed neoadjuvant therapy for tumors, and the feasibility of radical surgical treatment was determined by a multidisciplinary collaborative team evaluation before surgery. According to whether the intraoperative artery was pre-occluded, 145 patients were divided into two groups, including 28 cases in the APOT group(16 males, 12 females, aged (59.0±9.4) years), and 117 cases in the routine surgery group(76 males, 41 females, aged (55.1±8.2) years). To ensure comparability of baseline data between the APOT group and the routine surgery group,a 1︰2 match was performed using the propensity score matching method,and the caliper value was 0.006 45. The t-test,the Mann-Whitney U test, χ2 test or Fishier's exact test were used to compare the data between the two groups,respectively. Results: After matching the propensity score,there were 28 cases in the APOT group and 56 cases in the routine surgery group. There were no significant differences in gender,age,preoperative comorbidities,preoperative body mass index,surgical approaches,chemotherapy regimen,stereotactic body radiation therapy ratio,tumor markers,and type of invaded artery between the two groups (all P>0.05).The arterial occlusion time M(IQR) in the APOT group was 7.0(3.8)minutes(range:3 to 15 minutes),and no ischemic manifestations were observed in the distal target organs that blocked blood vessels after surgery. The operation time was (170.3±57.7)minutes in the APOT group and (235.0±80.2)minutes in the routine surgery group,and the difference was statistically significant (t=-3.800,P<0.01). The APOT group also experienced less intraoperative blood loss(650(588)ml vs. 800(600)ml;U=1 026.500,P=0.021). No significant differences were found between the groups in combined vein resection and reconstruction,celiac trunk resection,early postoperative complications, readmission rates at 30 days,and postoperative length of stay(all P>0.05). Extra-arterial dissection was performed in all patients,with arterial resection and reconstruction in 3 cases: 2 cases in the APOT group(1 case involving the superior mesenteric artery and 1 case involving the common hepatic artery) and 1 case in the routine group(involving the common hepatic artery). Postoperative abdominal bleeding occurred in 4 cases,with 3 cases in the routine group,1 case in the routine group. The R0 resection rate was 85.7%(24/28)in the APOT group and 80.4%(45/56) in the routine group,without significant differences between the groups(P=0.763). The median overall survival time was 27.6 months for the APOT group and 22.5 months for the routine group,while the median disease-free survival was 11.7 months and 16.8 months,respectively,with no significant differences between the groups(P=0.532,P=0.927). Conclusion: The arterial pre-occlusion technique can be used for extra-arterial dissection in patients with locally advanced pancreatic cancer involving the arteries,reducing surgery time and intraoperative blood loss.

[Effect of pH on the chelation between strontium ions and decellularized small intestinal submucosal sponge scaffolds].

OBJECTIVE: To investigate the preparation of decellularized small intestinal submucosa (dSIS) sponge scaffolds with chelated strontium (Sr) ions at different pH values, and to select the appropriate pH values for synthesizing Sr/dSIS scaffolds using the physicochemical properties and biocompatibility of the scaffolds as evaluation indexes. METHODS: (1) Sr/dSIS scaffolds preparation and grouping: After mixing dSIS solution and strontium chloride solution in equal volumes, adjusting pH of the solution to 3, 5, 7, and 9 respectively, porous scaffolds were prepared by freeze-drying method after full reaction at 37℃, which were named Sr/dSIS-3, -5, -7, and -9 respectively, and the dSIS scaffolds were used as the control group. (2) Physicochemical property evaluation: The bulk morphology of the scaffolds was observed in each group, the microscopic morphology analyzed by scanning electron microscopy, and the porosity and pore size determined, the surface elements analyzed by energy spectroscopy, the structure of functional groups analyzed by infrared spectroscopy, the chelation rate determined by atomic spectrophotometry, the water absorption rate detected by using specific gravity method, and the compression strength evaluated by universal mechanical testing machine.(3) Biocompatibility evaluation: The cytotoxicity and proliferative effect to bone mesenchymal stem cells (BMSCs) of each group were evaluated by Calcein-AM/PI double staining method. RESULTS: Scanning electron microscopy showed that the scaffolds of each group had an interconnected three-dimensional porous structure with no statistical difference in pore size and porosity. Energy spectrum analysis showed that strontium could be detected in Sr/dSIS-5, -7 and -9 groups, and strontium was uniformly distributed in the scaffolds. Functional group analysis further supported the formation of chelates in the Sr/dSIS-5, -7 and -9 groups. Chelation rate analysis showed that the Sr/dSIS-7 group had the highest strontium chelation rate, which was statistically different from the other groups (P < 0.05). The scaffolds in all the groups had good water absorption. The scaffolds in Sr/dSIS-5, -7 and -9 groups showed significantly improved mechanical properties compared with the control group (P < 0.05). The scaffolds in all the groups had good biocompatibility, and the Sr/dSIS-7 group showed the best proliferation of BMSCs. CONCLUSION: When pH was 7, the Sr/dSIS scaffolds showed the highest strontium chelation rate and the best proliferation effect of BMSCs, which was the ideal pH value for the preparation of the Sr/dSIS scaffolds.

[Comparison of intraoperative neurophysiological monitoring between patients with arthrogryposis multiplex congenita and adolescent idiopathic scoliosis].

Objective: To compare the intraoperative neurophysiological monitoring (IONM) results between patients with arthrogryposis multiplex congenita (AMC) and adolescent idiopathic scoliosis (AIS) and to analyze the influence of congenital spinal deformity on IONM in AMC patients, thus to evaluate the efficiency of IONM in AMC patients. Methods: A cross-sectional study. The clinical data of 19 AMC patients underwent correction surgery from July 2013 to January 2022 in Nanjing Drum Tower Hospital were retrospectively reviewed. There were 13 males and 6 females with a mean age of (15.2±5.6) years, and the average Cobb angle of main curve was 60.8°±27.7°. And 57 female AIS patients of similar age and curve type with the AMC patients during the same period were selected as the control group, with an average age of (14.6±4.4) years and a mean Cobb angle of 55.2°±14.2°. The latency and amplitude of samatosensory evoked potentials (SSEPs) and transcranial electric motor evoked potentials (TCeMEPs) were compared between the two groups. The difference in IONM data between AMC patients with and without congenital spinal deformity was also evaluated. Results: The success rates of SSEPs and TCeMEPs were 100% and 14/19 for AMC patients, 100% and 100% for AIS patients. The SSEPs-P40 latency, SSEPs-N50 latency, SSEPs-amplitude, TCeMEPs-latency, TCeMEPs-amplitude showed no significant difference between AMC patients and AIS patients (P>0.05 for all). The side-difference of TCeMEPs-amplitude showed an increasing trend in AMC patients when compared with that in AIS patients, but there was no statistical difference between the two groups [(147.0±185.6) µV vs (68.1±311.4) µV, P=0.198]. The SSEPs-amplitude value was (1.4±1.1) µV on concave side in AMC patients with congenital spinal deformity, and it was (2.6±1.2) µV on concave side in AMC patients without congenital spinal deformity (P=0.041). The SSEPs-amplitude value was (1.4±0.8) µV on convex side in AMC patients with congenital spinal deformity, and it was (2.6±1.3) µV on convex side in AMC patients without congenital spinal deformity (P=0.028). Conclusions: The values of SSEPs-P40 latency, SSEPs-N50 latency, SSEPs-amplitude, TCeMEPs-latency and TCeMEPs-amplitude are similar in AMC and AIS patients. The SSEPs-amplitude of AMC patients with congenital spinal deformity is lower than that of AMC patients without congenital spinal deformity.

[The related factors of postoperative recurrence in trigeminalneuralgia patients undergoing fully neuroendoscopic microvascular decompression].

Objective: To evaluate the related factors of postoperative recurrence intrigeminal neuralgia (TN) patients treated with fully neuroendoscopic microvascular decompression (MVD). Methods: The clinical baseline data and preoperative MRI imaging data of 112 patients with TN treated by neuroendoscopic MVD from December 2008 to December 2020 in the Department of Neurosurgery, Beijing Shijitan Hospital Affiliated to Capital Medical University were retrospectively analyzed, including: area ratio of cerebellopontine area (CPA)(healthy side/affected side), trigeminal nerve(TGN)length ratio(healthy side/affected side), TGN angle ratio(healthy side/affected side), and criminal vessel type. Multivariate Cox proportional hazards model was used to analyze the factors affecting postoperative recurrence. Results: Among the 112 patients in this group, there were 49 males and 63 females. The age ranged from 20 to 82 (59±9) years, and the course of disease was 0.05 to 30.00 (5.60±5.15) years. Pain was located on the left side in 43 cases (38.39%) and on the right side in 69 cases (61.61%), respectively. All patients were followed up for more than 1 year, with an average follow-up time of 21.5 months, and 11 cases recurred. Multivariate Cox regression analysis revealed that disease duration≥3 years(HR=9.34, 95%CI:1.12-39.07), CPA area ratio(healthy side/affected side)>1 (HR=27.47, 95%CI:1.69-44.20), criminal vessel type with vein(HR=35.39, 95%CI:1.26-18.60) and criminal vessel type with arteriovenous (HR=46.07, 95%CI: 2.74-27.75) were the main factors influencing recurrence of MVD surgery (all P<0.05). Conclusion: The disease duration≥3 years, CPA area ratio(healthy side/affected side)>1, and criminal vessel type with vein/arteriovenous are the relevant factors that affect the recurrence rate after the fully neuroendoscopic MVD treatment for trigeminal neuralgia.

[Methylation detection of phosphatase and tensin homolog deleted on chromosome ten gene promoter in hepatocellular carcinoma samples by next-generation sequencing].

Objective: The purpose of this study is to use the next-generation sequencing (NGS) technology platform to detect the methylation rate of phosphatase and tensin homolog deleted on chromosome ten (PTEN) promoter region in hepatocellular carcinoma (HCC) tissue samples, and to analyze the clinical significance of its correlation with the prognosis of patients receiving sorafenib treatment. Methods: The 52 pairs of tumor tissue and para-cancerous tissue samples from HCC patients treated with sorafenib alone, which were collected and preserved in the Liver Tumor Diagnosis and Research Center of the former 302 Hospital of the People's Liberation Army by the National Natural Science Foundation of China Youth Project with the project batch number 81702986 in 2018, were extracted total DNA from the samples. Then the DNA samples were treated with bisulfite and specific primers were designed to amplify the PTEN promoter region. Finally, the amplified products were analyzed by second-generation sequencing. In the analysis of clinical significance of PTEN methylation, log-rank statistical analysis was used to calculate whether there was a statistical difference in survival between the patient groups. Results: The methylation rate of PTEN promoter region in tumor tissues (29.17%±9.58%) was significantly higher than that in paracancer tissues (4.17%±2.86%)(t=19.970,P<0.05). At the same time, in HCC tissues, the methylation rate of the PTEN promoter region is negatively correlated with its expression (F=47.270,P<0.000 1;Y=-1 800×X+38.03), and the PTEN methylation rate is negatively correlated with the prognosis of patients receiving the molecularly targeted drug Sorafenib (χ²=4.313,P<0.05). Conclusion: This study successfully established a new method for detecting methylation in the promoter region of PTEN, and the methylation rate of PTEN can be used as one of the targets of HCC diagnosis and targeted therapy.

[Relationships between serum cystatin C, chemerin levels and subclinical atherosclerosis in type 2 diabetes mellitus patients].

Objective: To investigate the relationships between serum cystatin C (Cys C), chemerin levels and subclinical atherosclerosis in type 2 diabetes mellitus (T2DM) patients. Methods: A cross-sectional study was carried out between January 2016 and January 2018, and T2DM patients with carotid intima-media thickness (IMT) less than 1.1 mm were selected as subjects (100 males and 80 females, aged 40-60 years). The brachial-ankle pulse wave velocity (baPWV) ≥ 1 700 cm/s was set as the observation group (subclinical atherosclerosis) and baPWV<1 700 cm/s as the control group (non-subclinical atherosclerosis). Physical and blood examination were performed in both groups. Serum Cys C and chemerin levels were measured and their relationship with subclinical atherosclerosis was analyzed. Results: There was a statistically significant correlation between serum creatinine (r=0.167, P=0.011) and baPWV in the observation group, but not in the control group (r=0.105, P=0.070). Multiple linear regression analysis showed that age, duration of diabetes, serum creatinine, estimated glomerular filtration rate (eGFR), Cys C and chemerin were independently associated with baPWV, while high sensitive C reactive protein (hsCRP) and glycosylated hemoglobin (HbA1c) were not associated with baPWV. The elevation of serum Cys C (ß'=0.393, P=0.003) and chemokine (ß'=0.340, P=0.007) were correlative factors for atherosclerosis. Conclusion: The level of serum Cys C and chemerin is possibly related to the occurrence and development of subclinical atherosclerosis in T2DM patients.

[Clinical significance of ascitic interleukin-7 expression levels in cirrhotic patients complicated with spontaneous bacterial peritonitis].

Objective: To observe ascitic interleukin-7 expression level in cirrhotic patients complicated with spontaneous bacterial peritonitis, and to detect the effect of recombinant human IL-7 on CD4(+) and CD8(+)T lymphocyte function. Methods: A total of 84 patients with liver cirrhosis who were hospitalized from August 2017 to April 2018 were selected. Among them, 51 cases were complicated with cirrhosis and untainted ascites, and 33 cases were cirrhosis complicated with spontaneous bacterial peritonitis. Peripheral blood and ascites were collected routinely. The levels of IL-7 in peripheral blood and ascites were measured by enzyme-linked immunosorbent assay. CD4(+)T cells and CD8(+)T cells were purified from ascites, and were stimulated with recombinant IL-7. Cellular proliferation, key transcription factors for mRNA, and cytokines production by CD4(+)T cells in response to IL-7 stimulation was measured. mRNA expression corresponding to perforin, granzyme B, and granulysin as well as cytokines production by CD8(+)T cells was also measured in response to IL-7 stimulation. Cytolytic and non-cytolytic activity of CD8(+)T cells in response to IL-7 stimulation was also investigated in both direct and indirect contact co-culture system. Measurement data of the normal distribution were compared between the two groups by Student's t-test and the data before and after stimulation were compared by paired t-test. Measurements that did not conform to normal distribution were compared between the two groups using Mann-Whitney U test, and data before and after stimulation were compared using Wilcoxon paired test. Results: There was no significant statistical difference in serum IL-7 levels between the two groups [(5 001 ± 1 458) pg/ml vs. (4 768 ± 1 128) pg/ml, P = 0.41]. The level of ascitic IL-7 in cirrhotic patients complicated with SBP was significantly lower than cirrhosis patients with untainted ascites [(966.4 + 155.8) pg/ml vs. (792.1 + 126.4) pg/ml, P < 0.01]. Recombinant IL-7 stimulation promoted the proliferation of CD4(+) and CD8(+)T cells from ascites in patients with liver cirrhosis complicated by SBP. T-bet mRNA relative expression and IFN-γ secretion in CD4(+)T cells was also elevated in response to IL-7 stimulation in vitro. Moreover, IL-7 stimulation also increased the mRNA expressions of perforin, granzyme B, and granulysin as well as productions of IFN-γ and TNF-α by CD8(+)T cells. Recombinant IL-7 stimulation elevated cytolytic and non-cytolytic activity of CD8(+)T cells from ascites in patients with liver cirrohosis complicated by SBP, which manifested as increased target cell death and IFN-γ production in both direct and indirect contact co-culture system. Conclusion: Ascitic IL-7 promotes T lymphocyte function in patients with liver cirrhosis complicated with SBP.

[Function and mechanism of zinc finger protein ZNF50 in inhibiting the growth of esophageal squamous cell carcinoma cells].

Objective: To investigate the function and mechanism of zinc finger protein 750 (ZNF750) in esophageal squamous cell carcinoma (ESCC). Methods: Xenograft in nude mice was applied to detect the tumorigenesis of ZNF750-depleted ESCC cells. Western blot was performed to observe the expression of downstream target protein of ZNF750 in ESCC cell lines and xenograft tumor tissues in which ZNF750 was knocked down. 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H tetrazolium bromide (MTT) assay was used to determine the proliferation of ZNF750 stably depleted cells after restoration of its target protein. Results: The tumor weight of blank control, negative control and ZNF750 knockdown groups was 137±26 mg, 161±31 mg and 463±89 mg, respectively, with a statistically significant difference (P<0.01). The expressions of Kruppel-like factor 4 (KLF4) in ZNF750-depleted ESCC cells and its derived tumor tissue xenograft in nude mice were significantly down-regulated. Restoration of KLF4 in ZNF750 stably depleted cells significantly inhibited the cell proliferation (P<0.01). Conclusions: ZNF750 may be a new tumor suppressor in the tumorigenesis of ESCC, and the inhibition of cell proliferation induced by ZNF750 may be partially through the regulation of KLF4 expression.

[Hepatitis B core antigen promotes invasion of hepatocellular carcinoma cell line HepG2.2.15 via Toll-like receptor 4].

Objective: To investigate the effect of hepatitis B core antigen (HBcAg) in promoting the invasion of hepatitis B virus (HBV)-related hepatocellular carcinoma cell line HepG2.2.15 and the role of Toll-like receptor 4 (TLR4) in the mechanism. Methods: TLR4 mRNA and protein expression in HepG2 cells and HepG2.2.15 cells was measured by reverse transcription real-time PCR and Western blot analysis, respectively. HepG2.2.15 cells were transfected with TLR4 specific small interfering RNA (siRNA) to silence TLR4 expression, and stimulated by recombinant HBcAg in culture. The invasion of cells was measured by Transwell invasion assay. The expression of TLR4 signaling pathway-related proteins in the cultured cells and proinflammatory cytokines in the supernatant was also determined. The student's t-test, one-way ANOVA, and SNK-q test were used for statistical analysis. Results: TLR4 mRNA and protein expression in HepG2.2.15 cells was significantly higher than that in HepG2 cells. TLR4 siRNA transfection remarkably down-regulated TLR4 expression in HepG2.2.15 cells. Inhibiting TLR4 expression and/or HBcAg stimulation did not affect the proliferation of HepG2.2.15 cells. However, HBcAg stimulation significantly increased the invasion ability of HepG2.2.15 cells and the secretion of proinflammatory cytokines [including interferon (IFN)-γ, interleukin (IL)-6, IL-8, and tumor necrosis factor (TNF)-α]. Inhibiting TLR4 expression significantly reduced HBcAg-induced cellular invasion. Meanwhile, HBcAg stimulation elevated the expression of MyD88 and TRIF in HepG2.2.15 cells. TLR4 silencing inhibited HBcAg-induced increase in the expression of MyD88, while it showed no significant impact on TRIF expression. Conclusion: HBcAg can promote the invasion of HepG2.2.15 cells. The TLR4/MyD88 signaling pathway may be involved in this process by inducing the expression of proinflammatory cytokines.

Visual working memory deterioration preceding relapse in psychosis.

BACKGROUND: Relapse is distressingly common after the first episode of psychosis, yet it is poorly understood and difficult to predict. Investigating changes in cognitive function preceding relapse may provide new insights into the underlying mechanism of relapse in psychosis. We hypothesized that relapse in fully remitted first-episode psychosis patients was preceded by working memory deterioration. METHOD: Visual memory and verbal working memory were monitored prospectively in a 1-year randomized controlled trial of remitted first-episode psychosis patients assigned to medication continuation (quetiapine 400 mg/day) or discontinuation (placebo). Relapse (recurrence of positive symptoms of psychosis), visual (Visual Patterns Test) and verbal (Letter-Number span test) working memory and stressful life events were assessed monthly. RESULTS: Remitted first-episode patients (n = 102) participated in the study. Relapsers (n = 53) and non-relapsers (n = 49) had similar baseline demographic and clinical profiles. Logistic regression analyses indicated relapse was associated with visual working memory deterioration 2 months before relapse [odds ratio (OR) 3.07, 95% confidence interval (CI) 1.19-7.92, P = 0.02], more stressful life events 1 month before relapse (OR 2.11, 95% CI 1.20-3.72, P = 0.01) and medication discontinuation (OR 5.52, 95% CI 2.08-14.62, P = 0.001). CONCLUSIONS: Visual working memory deterioration beginning 2 months before relapse in remitted first-episode psychosis patients (not baseline predictor) may reflect early brain dysfunction that heralds a psychotic relapse. The deterioration was found to be unrelated to a worsening of psychotic symptoms preceding relapse. Testable predictors offer insight into the brain processes underlying relapse in psychosis.

Estimation of the standardized ileal digestible lysine requirement for primiparous pregnant sows.

This experiment was conducted to determine the optimal standardized ileal digestible lysine (SID Lys) level in diets fed to primiparous sows during gestation. A total of 150 (Landrace × Large White) crossbred gilts (weighing 149.9 ± 3.1 kg) were fed gestation diets (12.55 MJ of ME/kg) containing SID Lys levels of 0.43, 0.52, 0.60, 0.70 or 0.80% respectively. Gilts were fed 2.0 kg/day from day 1 to 80 and 3.0 kg/day from day 80 to 110 of gestation respectively. Gilts were allocated to treatments based on their body weight on the day of breeding. Weight gain from day 80 to 110 increased with increasing dietary SID Lys levels (p = 0.044). Fitted broken-line (p = 0.031) and quadratic plot (p = 0.047) analysis of body weight gain indicated that the optimal SID Lys level for primiparous sows was 0.70 and 0.69% respectively. During gestation, neither backfat thickness nor loin eye area was affected by dietary SID Lys level. Increasing dietary Lys had no effect on the litter size at birth or pigs born alive per litter. Litter weight at birth was not affected by dietary SID Lys level. The litter weight variation at birth quadratically decreased with increasing dietary SID Lys (p = 0.021) and was minimized at 0.70% dietary SID Lys. Gilts fed the 0.70% SID Lys diet had the highest dry matter (p = 0.031) and protein (p = 0.044) content in colostrum. On day 110 of gestation, gilts fed the 0.70% SID Lys diet tended to have the highest serum prolactin (p = 0.085) and serum insulin (p = 0.074) levels. The data demonstrate that the optimal dietary SID Lys was 0.70% for pregnant gilts, which is similar to the recommendation of 0.69% that was estimated by the NRC (2012).

The Nutritive Values in Different Varieties of Corn Planted in One Location Fed to Growing Pigs over Three Consecutive Years.

This experiment was conducted to determine the effect of variety and planting year on the nutritive values of corn fed to growing pigs. Four corn varieties examined in this experiment were planted in the same village located in Longhua County, Heibei Province, China, in 2012, 2013, and 2014, respectively. During each year, corn was hand-harvested in early October and sun dried to about 14% moisture content. Three batches of twenty-four barrows (33.27±4.30, 31.88±2.93, 34.21±3.81 kg body wight [BW] in 2012, 2013, and 2014, respectively) were used and allotted to a complete block design with 4 diets and 6 replicate pigs per diet. Pigs were individually placed in metabolic crates. The four experimental diets were formulated by mixing each variety of corn and vitamins and minerals, respectively. A five-day collection period followed a seven-day diet acclimation period. The results indicated that variety of corn significantly influenced the available energy content (digestible energy [DE] on dry matter basis, p<0.05; metabolizable energy (ME) on dry matter basis, p<0.05, respectively), and the apparent total tract digestibility (ATTD) of organic matter (p<0.01), dry matter (p<0.05), gross energy (p<0.05), neutral detergent fiber (p<0.01), acid detergent fiber and ether extract (p<0.05). The planting year also significantly influenced the available energy contents (DE on dry matter basis, p<0.05; ME on dry matter basis, p<0.01, respectively) and the ATTD of neutral detergent fiber (p<0.01), acid detergent fiber (p<0.01), crude protein (p<0.01), and ether extract (p<0.01). No interaction was observed between the variety and planting year in DE and ME contents in corn. In conclusion, the variety and planting year significantly influenced the available energy and nutrient digestibility of corn fed to growing pigs.

Efficient construction of recombinant adenovirus expression vector of the Qinchuan cattle LYRM1 gene.

In this study, we cloned the coding DNA sequence (CDS) region of Qinchuan cattle LYR motif-containing 1 (LYRM1) and constructed a recombinant adenovirus expression vector to examine the function of LYRM1 on the cellular level. Total RNA was extracted from the adipose tissue of Qinchuan cattle, cDNA was obtained by reverse transcription, and polymerase chain reaction was used to amplify the CDS region of the LYRM1 gene. The CDS-containing fragment was inserted into the shuttle vector pAdTrack-CMV to construct pAdTrack-CMV-LYRM1 vector. After linearization of pAdTrack-CMV-LYRM1 and the negative control vector pAdTrack-CMV by restriction endonuclease PmeI, the vectors were transformed into Escherichia coli BJ5183 containing pAdEasy-1 to obtain the recombinant adenovirus vector pAd-LYRM1 and pAd-CMV through homologous recombination. pAd-LYRM1 and pAd-CMV were then digested by PacI and transfected into the 293A cell line. The recombinant adenovirus Ad-LYRM1 and Ad-CMV was obtained at a concentration of 7 x 108 and 1.3 x 109 green fluorescent units/mL, respectively. Preadipocytes derived from Qinchuan cattle were separately infected with Ad-LYRM1 and Ad- CMV. Quantitative real-time polymerase chain reaction demonstrated that the expression of LYRM1 was increased by approximate 28,000-folds after the infection with recombinant adenovirus for 48 h. In conclusion, we successfully cloned the CDS region of the Qinchuan cattle LYRM1 gene, constructed the recombinant adenovirus expression vector, and obtained the adenovirus with high titer, providing valuable materials for studying the function of LYRM1 at the cellular level.

Novel polymorphisms of the PRKAG2 gene and their association with body measurement and meat quality traits in Qinchuan cattle.

Body measurement and meat quality traits play important roles in the evaluation of productivity and economy in cattle, which are influenced by genes and environmental factors. PRKAG2, which encodes the γ2 regulatory subunit of AMPK, is associated with key metabolic pathways in muscle. We detected bovine PRKAG2 gene polymorphisms and analyzed their associations with body measurement and meat quality traits of cattle. DNA samples were taken from 578 Qinchuan cattle aged 18-24 months. DNA sequencing, polymerase chain reaction-restriction fragment length polymorphism, and time-of-flight mass spectrometry were used to detect PRKAG2 single nucleotide polymorphisms (SNPs). Sequence analysis revealed three SNPs in exon 3 (g.95925G>A, g.95973G>C, and g.95992A>G) and one g.96058T>C mutation in intron 3. g.95973G>C, g.95992A>G, and g.96058T>C each showed 3 genotypes: GG, GC, and CC; AA, AG, and GG; and TT, TC, and CC, respectively. In contrast, g.95925G>A only showed 2 genotypes, GG and GA. Analysis showed that g.95925G>A had no effects on body measurement and meat quality traits, whereas the other 3 polymorphisms were significantly associated with some of the body measurement and meat quality traits in the Qinchuan cattle population. It is inferred that the PRKAG2 gene can be used for marker-assisted selection to improve the body measurement and meat quality traits in the Qinchuan cattle population.

Protective effects of ascorbic acid and vitamin E on antioxidant enzyme activity of freeze-thawed semen of Qinchuan bulls.

The aim of this study was to determine the protective effects of the combination of ascorbic acid (Vc) and vitamin E (VE) on antioxidant enzyme activity, sperm motility, viability, and acrosome integrity of Qinchuan bulls after freeze-thaw. In this study, we determined the effects of Vc and VE on the activity of the antioxidant enzyme defense system comprising glutathione peroxidase (GSH-Px), glutathione reductase (GR), catalase (CAT), and superoxide dismutase (SOD). The combination of Vc and VE had protective effects on sperm motility and viability. With respect to acrosome integrity and the activity of GR and SOD, differences were observed between the experimental groups with added Vc (7 mg/mL) and VE (0.12 IU/mL) and the control group. The activity of GSH-Px in the experimental group (1400 IU/mL Vc and 0.12 IU/mL VE) was not different (P > 0.05) compared with that in the control group, while the activity of CAT showed a significant difference between the 2 groups (P < 0.05). Therefore, we inferred that the combination of Vc (1400 IU/mL) and VE (0.12 IU/mL) protected the sperm quality in the freeze-thaw process.

Effects of bovine SMO gene polymorphisms on the body measurement and meat quality traits of Qinchuan cattle.

Beef cattle breeding programs focus on improving important economic traits, including growth rates, and meat quantity and quality. Molecular marker-assisted selection based on genetic variation represents a potential method for breeding genetically improved livestock with better economic traits. Smoothened (SMO) protein is a signal transducer that contributes to the regulation of both osteogenesis and adipogenesis through the hedgehog pathway. In this study, we detected polymorphisms in the bovine SMO gene of Qinchuan cattle, and we analyzed their associations with body measurement traits (BMTs) and meat quality traits (MQTs). Using DNA sequencing and polymerase chain reaction-restriction fragment length polymorphism, 3 novel single nucleotide polymorphisms were identified in the SMO gene of 562 cattle: 1 G > C mutation on exon 9 (G21234C) and 2 C > T mutations on exon 11 (C22424T and C22481T). Association analysis showed that polymorphisms on both the G21234C and C22424T loci significantly affected certain BMTs and MQTs (P < 0.05 or P < 0.01), whereas those on the C22481T locus did not (P > 0.05). Therefore, the SMO gene could be used as a candidate gene to alter BMTs and MQTs in Qinchuan cattle or for marker-assisted selection to breed cattle with superior BMTs and MQTs.

New polymorphisms in the novel LYRM1 gene are associated with body measurement and meat quality traits in Qinchuan cattle.

Body measurement and meat quality traits play important roles in the evaluation of productivity in cattle; they are influenced by genetic and environmental factors. Recent studies have shown that LYRM1 is a novel gene related to obesity and may influence fat deposition. We screened for new polymorphisms in the bovine LYRM1 gene and analyzed their association with body measurement and meat quality traits in cattle. DNA samples were obtained from 572 Qinchuan cattle aged from 18 to 24 months. DNA sequencing was used to find the LYRM1 single nucleotide polymorphisms (SNPs). Sequence analysis of LYRM1 revealed four novel SNPs in exon 3: G50A in coding region, C126A, A127T, and T128A in a 3'-untranslated region. G50A, A127T and T128A showed two genotypes: AG and GG, AA and AT, AT and TT, respectively; while C126A showed three genotypes: AA, AC and CC. Analysis showed that these four polymorphisms were significantly associated with body measurement and meat quality traits in the Qinchuan cattle population. We suggest that the LYRM1 gene can be used for marker-assisted selection to improve body measurement and meat quality traits in the Qinchuan cattle population.

Polymorphisms in the delta-like 2 homolog gene and their association with growth and meat-quality traits in Qinchuan cattle.

The delta-like 2 homolog (DLK2) modulates adipogenesis, hematopoiesis, osteogenesis, and other cell-differentiation processes. In the present study, we detected potential polymorphisms in the DLK2 gene in 604 individuals of Qinchuan cattle by using PCR-RFLP and DNA-sequencing methods. Herein, we identified five novel single-nucleotide polymorphisms (SNPs) (g.888G>A, g.910A>G, g.995G>A, g.4321A>G, g.4850A>G) and analyzed their association with measured traits. Four of the five analyzed polymorphisms were associated with at least one of the following traits: body weight (BW), chest depth (CD), chest circumference (CC), back fat thickness (BT), and rib-eye area (REA). To the best of our knowledge, our research is the first to report the association of DLK2 gene polymorphisms with growth and meat quality traits in Qinchuan cattle. In summary, the results of our study suggest that the DLK2 gene can be used as a candidate gene in beef cattle breeding.

Effects of transcranial direct current stimulation combined with tinnitus retraining therapy on sleep disorders in patients with chronic tinnitus.

OBJECTIVE: The aim of this study was to investigate the effects of transcranial direct current stimulation (TDCS) combined with tinnitus retraining therapy (TRT) on clinical efficacy and sleep disorder in patients with chronic tinnitus. PATIENTS AND METHODS: 126 patients with chronic tinnitus treated in our hospital from May 2020 to June 2022 were retrospectively analyzed. These subjects were randomly divided into two groups: the electrical stimulation group and the combined group, in line with the random table method, with 63 patients in each group. Patients in the electrical stimulation group received TDCS treatment, and patients in the combined group were given TDCS combined with TRT. The clinical effects, tinnitus severity [Tinnitus Evaluation Questionnaire (TEQ) score and Tinnitus handicap inventory (THI) score], sleep status [Sleep Status Rating Scale (SRSS) score and Pittsburgh Sleep Quality Index (PSQI) score], psychological status [Hamilton Anxiety Scale (HAMA) score and Self Rating Depression Scale (SDS) score] and the quality of life (Quality of Life Scale) of these subjects in two groups were analyzed. RESULTS: The clinical effect of simple TDCS was 82.53%, which was sharply lower compared to 95.24% in the combined group (p<0.05). After the treatment, TEQ score, THI score, SRSS score, PSQI score, HAMA score, and SDS score were decreased in both groups (p<0.05), and the combined group was much lower than the TDCS group (p<0.01). Compared with the pre-treatment period, the scores of restrictions in daily living, medical resource utilization, somatic symptoms, and emotional disturbance were elevated in both groups after treatment, and the combined group had markedly higher scores than the TDCS group (p<0.05). CONCLUSIONS: TDCS combined with TRT had obvious effects in treating chronic tinnitus, which largely reduced the severity of tinnitus, improved patients' sleep quality and psychological status, and improved the quality of life, indicating a certain worthy of clinical application and promotion.

Novel SNPs in the exon region of bovine DKK4 gene and their association with body measurement traits in Qinchuan cattle.

The aim of this study was to determine whether single nucleotide polymorphisms (SNPs) of bovine Dickkopf homolog 4 (DKK4) are associated with body measurement traits in Qinchuan cattle. By using PCR-SSCP technology and DNA sequencing, we discovered 5 DKK4 SNPs in Qingchuan cattle, including -65G>A and -77G>T in the 5'-untranslated region, 1532C>G and 1533T>C in exon 2, and 2088C>T in exon 3. The sequencing map showed that 1532C>G and 1533T>C were in close linkage disequilibrium and were treated as 1532C>G-1533T>C in this study. Allele frequencies were calculated and analyzed by the chi-square test, which showed that -65G>A and 1532C>G-1533T>C were in Hardy-Weinberg equilibrium (P > 0.05), whereas -77G>T and 2088C>T were not in all 633 tested Qinchuan cattle individuals (P < 0.01). Gene heterozygosity (HE), effective allele number (NE), and polymorphism information content (PIC) were 0.407, 1.686, and 0.324 at -65G>A; 0.472, 1.894, and 0.361 at -77G>T; 0.476, 1.908, and 0.363 at 1532C>G-1533T>C; and 0.218, 1.279, and 0.195 at 2088C>T. We also evaluated the potential association of these SNPs with body measurement traits in all 633 individuals; the results suggest that several SNPs in Qinchuan cattle DKK4 were significantly associated with body length, hip height, rump length, hip width, heart girth, and pin bone width (P < 0.05 and P < 0.01). These results suggest that bovine DKK4 could be used as candidate gene for Qinchuan cattle breeding.