Objective:To compare the protective effects of the static cold storage (SCS) and normothermic machine perfusion (NMP) on the skeletal muscle of the amputated limbs of pigs.Methods:Four Landrace pigs were selected, from which eight limbs were amputated and divided into SCS group ( n=5) and NMP group ( n=3) according to the random number table method. After blood collection from the carotid artery, an amputated limb model was established by amputating the limbs at the scapulohumeral joints. The limbs in the SCS group were wrapped in sterile cloth and stored at 4 ℃ for 24 hours. In the NMP group, the limbs were mechanically perfused with a red blood cell-containing perfusion fluid at 37 ℃ for 24 hours, with 70% of the perfusion fluid replaced every 6 hours. Before the experiment, cross-matching tests with the saline medium were conducted between donor and recipient pigs to evaluate blood coagulation and blood safety in the NMP group. An allogeneic red blood cell perfusion fluid was prepared and the levels of pH, Na +, K +, Cl -, Ca 2+, glucose (Glu), hematocrit (Hct), lactic acid (Lac) and osmotic pressure of the perfusion fluid were measured. At 0, 6, 12, 18, and 24 hours after perfusion, the skin temperature and oxyhemoglobin saturation (SaO 2) levels in the NMP group were monitored and the levels of pH, Glu, creatine kinase (Ck), K +, Ca 2+, and Na +levels of the perfusion fluid were analyzed to evaluate the metabolism of the skeletal muscle in the amputated limbs. The mean intercellular distance and apoptosis index of the myocytes were quantitatively analyzed and histopathological changes were observed by performing HE staining and TUNEL staining on the skeletal muscle of the amputated limbs in both groups at 0 and 24 hours after perfusion. After perfusion was ended, the weight gain rate and swelling degree of the amputated limbs were compared between the two groups and the overall state of the amputated limbs was evaluated. Results:The result of the cross-matching test between donor and recipient pig blood was negative. The parameters in the prepared red blood cell-containing perfusion fluid generally maintained within a normal range: pH 7.38±0.04, Na + concentration (138.30±4.48)mmol/L, K + concentration (3.50±0.26)mmol/L, Glu concentration (6.11±2.08)mmol/L, and osmotic pressure (305.67±3.79)mmol/L. However, slightly higher Cl - and Ca 2+ concentrations [(118.34±12.00)mmol/L and (2.00±0.15)mmol/L] and lower Hct and lactate concentrations [0.30±0.03 and (1.54±0.38)mmol/L] were detected when compared with the reference range. During the perfusion, the average skin temperature of the amputated limbs in the NMP group was (36.13±0.98)℃, with the skin temperatures at 6, 12, 18, and 24 hours after perfusion being significantly higher than that at 0 hour ( P<0.01), while no significant difference among the skin temperatures at 6, 12, 18, and 24 hours after perfusion was observed ( P>0.05). The SaO 2 levels in the skin of the amputated limbs in the NMP group averaged over 95%, which showed no significant difference at 0, 12, 18, and 24 hours after perfusion ( P>0.05), while a significant elevation was observed at 6 hours compared with that at 0 hour ( P<0.05). There were no significant differences in pH, Glu, Na +, and Ca 2+ levels in the NMP group at 0, 6, 12, 18, and 24 hours after perfusion ( P>0.05), while the Ck levels at 18 and 24 hours were both significantly higher than that at 6 hours after perfusion ( P<0.05), and the Ck levels at 6, 12, 18, and 24 hours were all significantly higher than that at 0 hour ( P<0.05). The K + level progressively increased with the perfusion time, with significant elevations at 18 and 24 hours after perfusion compared with that at 0 hour ( P<0.05). HE staining revealed well-preserved muscle fiber continuity and regular arrangement in the NMP group and the SCS group at 0 hour, with an intercellular distance of (8.95±0.60)μm. At 24 hours, the NMP group exhibited slight skeletal muscle fiber rupture and swelling, with a slightly increased intercellular distance of (14.75±0.90)μm, significantly greater than that at 0 hour ( P<0.01). At 24 hours, the SCS group showed marked skeletal muscle fiber rupture and swelling, with a significantly increased intercellular distance of (23.51±1.49)μm, significantly larger than those at 0 hour in the same group and at 24 hours in the NMP group ( P<0.01). TUNEL immunofluorescence staining indicated a tiny amount of apoptotic cells in the skeletal muscle in both groups at 0 hour, with an apoptotic index of (4.26±1.62)%. There was a small number of apoptotic cells in the skeletal muscle in the NMP group at 24 hours, with an apoptotic index of (25.94±2.69)%, significantly larger than that in the same group at 0 hour ( P<0.01). The SCS group exhibited a large number of apoptotic cells at 24 hours, with an apoptotic index of (62.97±3.22)%, significantly larger than those at 0 hour in the same group and at 24 hours in the NMP group ( P<0.01). In comparison with the SCS group at 24 hours, the amputated limbs in the NMP group showed red color in the appearance, no symptoms of ischemic muscle contracture and good joint movement despite slight edema in the subcutaneous layer. At 24 hours, the weight gain rate of the amputated limbs was (15.82±0.89)% in the NMP group, significantly higher than (0.97±0.28)% in the SCS group ( P<0.01). Conclusion:Compared with SCS, NMP with the red blood cell-containing perfusion fluid prepared with the allogeneic blood for the amputated limbs of pigs can alleviate the ischemic injury of the muscle fibers and inhibit the apoptosis of the muscle cells by sustaining stable energy and oxygen supply and balancing ion homeostasis and pH of the perfusion fluid.

Objective:To compare the protective effects of the static cold storage (SCS) and normothermic machine perfusion (NMP) on the skeletal muscle of the amputated limbs of pigs.Methods:Four Landrace pigs were selected, from which eight limbs were amputated and divided into SCS group ( n=5) and NMP group ( n=3) according to the random number table method. After blood collection from the carotid artery, an amputated limb model was established by amputating the limbs at the scapulohumeral joints. The limbs in the SCS group were wrapped in sterile cloth and stored at 4 ℃ for 24 hours. In the NMP group, the limbs were mechanically perfused with a red blood cell-containing perfusion fluid at 37 ℃ for 24 hours, with 70% of the perfusion fluid replaced every 6 hours. Before the experiment, cross-matching tests with the saline medium were conducted between donor and recipient pigs to evaluate blood coagulation and blood safety in the NMP group. An allogeneic red blood cell perfusion fluid was prepared and the levels of pH, Na +, K +, Cl -, Ca 2+, glucose (Glu), hematocrit (Hct), lactic acid (Lac) and osmotic pressure of the perfusion fluid were measured. At 0, 6, 12, 18, and 24 hours after perfusion, the skin temperature and oxyhemoglobin saturation (SaO 2) levels in the NMP group were monitored and the levels of pH, Glu, creatine kinase (Ck), K +, Ca 2+, and Na +levels of the perfusion fluid were analyzed to evaluate the metabolism of the skeletal muscle in the amputated limbs. The mean intercellular distance and apoptosis index of the myocytes were quantitatively analyzed and histopathological changes were observed by performing HE staining and TUNEL staining on the skeletal muscle of the amputated limbs in both groups at 0 and 24 hours after perfusion. After perfusion was ended, the weight gain rate and swelling degree of the amputated limbs were compared between the two groups and the overall state of the amputated limbs was evaluated. Results:The result of the cross-matching test between donor and recipient pig blood was negative. The parameters in the prepared red blood cell-containing perfusion fluid generally maintained within a normal range: pH 7.38±0.04, Na + concentration (138.30±4.48)mmol/L, K + concentration (3.50±0.26)mmol/L, Glu concentration (6.11±2.08)mmol/L, and osmotic pressure (305.67±3.79)mmol/L. However, slightly higher Cl - and Ca 2+ concentrations [(118.34±12.00)mmol/L and (2.00±0.15)mmol/L] and lower Hct and lactate concentrations [0.30±0.03 and (1.54±0.38)mmol/L] were detected when compared with the reference range. During the perfusion, the average skin temperature of the amputated limbs in the NMP group was (36.13±0.98)℃, with the skin temperatures at 6, 12, 18, and 24 hours after perfusion being significantly higher than that at 0 hour ( P<0.01), while no significant difference among the skin temperatures at 6, 12, 18, and 24 hours after perfusion was observed ( P>0.05). The SaO 2 levels in the skin of the amputated limbs in the NMP group averaged over 95%, which showed no significant difference at 0, 12, 18, and 24 hours after perfusion ( P>0.05), while a significant elevation was observed at 6 hours compared with that at 0 hour ( P<0.05). There were no significant differences in pH, Glu, Na +, and Ca 2+ levels in the NMP group at 0, 6, 12, 18, and 24 hours after perfusion ( P>0.05), while the Ck levels at 18 and 24 hours were both significantly higher than that at 6 hours after perfusion ( P<0.05), and the Ck levels at 6, 12, 18, and 24 hours were all significantly higher than that at 0 hour ( P<0.05). The K + level progressively increased with the perfusion time, with significant elevations at 18 and 24 hours after perfusion compared with that at 0 hour ( P<0.05). HE staining revealed well-preserved muscle fiber continuity and regular arrangement in the NMP group and the SCS group at 0 hour, with an intercellular distance of (8.95±0.60)μm. At 24 hours, the NMP group exhibited slight skeletal muscle fiber rupture and swelling, with a slightly increased intercellular distance of (14.75±0.90)μm, significantly greater than that at 0 hour ( P<0.01). At 24 hours, the SCS group showed marked skeletal muscle fiber rupture and swelling, with a significantly increased intercellular distance of (23.51±1.49)μm, significantly larger than those at 0 hour in the same group and at 24 hours in the NMP group ( P<0.01). TUNEL immunofluorescence staining indicated a tiny amount of apoptotic cells in the skeletal muscle in both groups at 0 hour, with an apoptotic index of (4.26±1.62)%. There was a small number of apoptotic cells in the skeletal muscle in the NMP group at 24 hours, with an apoptotic index of (25.94±2.69)%, significantly larger than that in the same group at 0 hour ( P<0.01). The SCS group exhibited a large number of apoptotic cells at 24 hours, with an apoptotic index of (62.97±3.22)%, significantly larger than those at 0 hour in the same group and at 24 hours in the NMP group ( P<0.01). In comparison with the SCS group at 24 hours, the amputated limbs in the NMP group showed red color in the appearance, no symptoms of ischemic muscle contracture and good joint movement despite slight edema in the subcutaneous layer. At 24 hours, the weight gain rate of the amputated limbs was (15.82±0.89)% in the NMP group, significantly higher than (0.97±0.28)% in the SCS group ( P<0.01). Conclusion:Compared with SCS, NMP with the red blood cell-containing perfusion fluid prepared with the allogeneic blood for the amputated limbs of pigs can alleviate the ischemic injury of the muscle fibers and inhibit the apoptosis of the muscle cells by sustaining stable energy and oxygen supply and balancing ion homeostasis and pH of the perfusion fluid.

Objective To explore the application effect of bedside sitting activity combined with staged breathing training on patients with chronic obstructive pulmonary disease(COPD)complicated with respiratory failure,and to provide references for nurses to carry out pulmonary rehabilitation therapy.Methods A total of 120 COPD patients with respiratory failure in a tertiary A hospital of Beijing City were selected from May 2021 to December 2023 as the study subjects.The study subjects were divided into an experimental group and a control group(each n=60)by the random number table method.The experimental group received bedside sitting activity combined with staged breathing training on the basis of routine lung rehabilitation nursing,while the control group was given routine lung rehabilitation nursing.After intervention,the blood gas indicators,lung function respiratory capacity,exercise capacity,and quality of life were compared between groups after intervention.Results After intervention,the arterial partial blood oxygen saturation and arterial partial pressure of oxygen in the experimental group were better than those in the control group(P＜0.05).The experimental group had higher maximum ventilatory volume,forced expiratory volume in the first second and vital capacity than those in the control group(P＜0.05).After intervention,the lung function respiratory capacity and quality of life in the experimental group were better compared with those in the control group(P＜0.05).The exercise capacity of the experimental group was higher than that in the control group(P＜0.05).Conclusion Bedside sitting activity combined with staged breathing training can effectively improve lung function and enhance exercise tolerance and quality of life in patients with COPD and respiratory failure.

Objective To evaluate the preservation effect of normothermic mechanical perfusion with red blood cells from humanized genetically modified pig on severed human limbs.Methods Severed human limbs were perfused with red blood cells from humanized genetically modified pigs for 6 h.Perfusion solution was taken every hour to measure the oxygen partial pressure,Na+,K+,Ca2+,pH value,glucose,lactic acid and creatine kinase levels.Superficial flexor muscle was sampled to detect the changes of tumor necrosis factor(TNF)-α,interleukin(IL)-2 and IL-1 levels.At 0 and 6 h after perfusion,the superficial flexor muscles of the forearm were taken for pathological examination.Intercellular space and glycogen consumption of skeletal muscles were observed.An appropriate amount of forearm vessels was collected every 2 h to detect the apoptosis of vascular endothelial cells.X-ray angiography was performed before perfusion and 6 h after perfusion to observe the filling degree of finger-tip peripheral vessels.Results The oxygen partial pressure was observed in the normal range throughout the perfusion.Na+concentration peaked at 1 h,reaching 138.7 mmol/L,and then fluctuated within the normal range.K+level peaked at 2 h up to 6.08 mmol/L,then decreased and fluctuated within the normal range.Ca2+concentration reached the peak at 4 h,up to 1.03 mmol/L.Glucose level was gradually decreased at the beginning of perfusion,reaching the lowest value of 17.7 mmol/L at 2 h after perfusion,and then maintained a dynamic balance.The pH value was decreased to 7.28 at 6 h after perfusion.The lactic acid level was increased to 9.6 mmol/L at 1 h after perfusion,and then gradually decreased.The creatine kinase level was increased at the start of perfusion,reached the peak at 2 h up to 20 030 U/L,then decreased and remained stable at the end of perfusion.At the end of perfusion,the morphology of muscle fibers was normal,the gap among muscle fibers was expanded slightly,and the glycogen of skeletal muscles was not significantly accumulated.At 0 h perfusion,the number of apoptotic cells in vascular endothelial cells was large,which was declined at 6 h perfusion.Evident vascular filling was observed at 0 h,and the filling degree of some finger-tip vessels was decreased at 6 h.Conclusions Normothermic mechanical perfusion of severed human limbs with red blood cells from humanized genetically modified pigs may continuously and stably supply energy and oxygen,adjust the ion pH balance of perfusion solution,maintain normal cellular metabolism and exerts certain protective effect upon severed human limbs.

Objective To explore the application effect of bedside sitting activity combined with staged breathing training on patients with chronic obstructive pulmonary disease(COPD)complicated with respiratory failure,and to provide references for nurses to carry out pulmonary rehabilitation therapy.Methods A total of 120 COPD patients with respiratory failure in a tertiary A hospital of Beijing City were selected from May 2021 to December 2023 as the study subjects.The study subjects were divided into an experimental group and a control group(each n=60)by the random number table method.The experimental group received bedside sitting activity combined with staged breathing training on the basis of routine lung rehabilitation nursing,while the control group was given routine lung rehabilitation nursing.After intervention,the blood gas indicators,lung function respiratory capacity,exercise capacity,and quality of life were compared between groups after intervention.Results After intervention,the arterial partial blood oxygen saturation and arterial partial pressure of oxygen in the experimental group were better than those in the control group(P＜0.05).The experimental group had higher maximum ventilatory volume,forced expiratory volume in the first second and vital capacity than those in the control group(P＜0.05).After intervention,the lung function respiratory capacity and quality of life in the experimental group were better compared with those in the control group(P＜0.05).The exercise capacity of the experimental group was higher than that in the control group(P＜0.05).Conclusion Bedside sitting activity combined with staged breathing training can effectively improve lung function and enhance exercise tolerance and quality of life in patients with COPD and respiratory failure.

Humans ; Staphylococcus lugdunensis/genetics* ; Peptides, Cyclic ; Chromosomes ; Operon/genetics* ; Staphylococcal Infections/genetics* ; Bacterial Proteins/genetics* ; Anti-Bacterial Agents

Humans ; COVID-19 ; Quarantine ; Renal Dialysis ; SARS-CoV-2 ; Hospitals

In order to screen African swine fever virus (ASFV) diagnostic antigen with the best enzyme linked immunosorbent assay (ELISA) reactivity. By establishing the ELISA method, the diagnostic antigen of ASFV p30 protein expressed by baculovirus-insect cell expression system as reference, we explored the antigenic properties and diagnostic potential of ASFV p35 protein expressed by prokaryotic expression system as a diagnostic antigen. The results of Western blotting and immunofluorescence show that the molecular weight of the recombinant p35 protein and p30 protein obtained was 40 kDa and 30 kDa, respectively, and these two proteins had good immuno-reactivity with ASFV positive serum. Recombinant p30 and p35 proteins were used as diagnostic antigens to establish ELISA, and the sensitivity and repeatability of these methods were tested. The results show that although the detection sensitivity of the p30-ELISA established in this study was higher than that of the p35-ELISA, the sensitivity of p35-ELISA was 95.8%, and variations in intra- and inter-assay repeatability of the two methods were less than 10%. The coincidence rate between the p35-ELISA and the imported kit was 97.2%. Results show that p35-ELISA was sensitive and stable, and could detect specific antibodies against ASFV.
African Swine Fever/diagnosis* ; African Swine Fever Virus/genetics* ; Animals ; Antibodies, Viral ; Enzyme-Linked Immunosorbent Assay ; Recombinant Proteins/genetics* ; Swine

Objective To investigate the expression of proline rich tyrosine kinase 2 (Pyk2) in non-muscle invasive bladder cancer,and analyze its correlation to clinicopathologic features and prognosis of non-muscle invasive bladder cancer.Methods 114 surgical specimens and 50 normal bladder mucosa specimens were collected from 114 non-muscle invasive bladder cancer patients who underwent TURBT at our hospital,from June 2013 to March 2018.Of the 114 patients,63 were male and 51 were female,aged 42-87 years,average age of (63.6 ± 13.8) years,73 cases of tumor ＜3 cm,41 cases of tumor ≥3 cm,83 cases were single and 31 cases were multiple tumor,53 cases were high grade and 61 cases were low grade,59 cases were Ta and 55 cases were T1 stage.Pyk2 protein expression was detected by immunohistochemistry and western blot.The correlation of the expression of Pyk2 with clinicopathologic features,including gender,age,tumor size,the number of tumors,histological grade and clinical stage were analyzed.Survival analysis was calculated by using the Kaplan-Meier method,and the difference in survival curve was analyzed by using the log-rank test.Association of Pyk2 expression with prognosis of non-muscle invasive bladder cancer analyzed by using the Cox proportional hazards regression model.Results Compared with normal bladder tissues,expression of Pyk2 protein was increased in bladder cancer tissue significantly(0.571 ±0.230 vs.0.253 ± 0.152,P ＜0.01).The expression of Pyk2 protein was closely related to clinical stage(P =0.027) and grade(P =0.010),rather than gender (P =0.275),age (P =0.419),tumor size (P =0.317),and tumor number(P =0.208).The recurrence rate in the Pyk2 positive group and negative group were 46.1％ (35/76)and 28.9％ (11/38)respectively.The progression rate in the Pyk2 positive group and negative group were 35.5％ (27/76) and 10.5 ％ (4/38) respectively.Survival analysis suggested expression of Pyk2 in non-muscle invasive bladder cancer had a significant relation to recurrence-free survival rate(P ＜0.001) and progression-free survival rate(P =0.003).In the multivariable Cox analysis,we found that Pyk2 protein was an independent predictor of recurrence-free survival rate(HR 0.245,95％ CI 0.078-0.768,P =0.016) and progression-free survival rate (HR 0.095,95％ CI 0.012-0.764,P =0.027).Conclusions The expression of Pyk2 in non-muscle invasive bladder cancer was significantly increased.The expression of Pyk2 has a significant relation to recurrence and progression of non-muscle invasive bladder cancer.High Pyk2 expression is an independent prognostic factor in non-muscle invasive bladder cancer.

Objective: Human rhinovirus A21 (HRVA21) with mutations in antigenic gene has been reported causing severe human infections. This study aimed to investigate intensively the pathogenesis of HRVA21 by identifying the characteristics of neutralizing antibodies (NAbs) distribution. Methods: Virus stock was isolated from HRVA21-positive respiratory specimens. The tissue culture infective dose 50 (TCID₅₀) was applied. Sera from healthy volunteers in different age groups were used to analyze the NAbs by using the diluted serum and fixed viral TCID₅₀. Results: We obtained the HRVA21 virus stock and its whole genome sequences shared 100% similarity to the used clinical sample. In the 379 collected serum samples, the positive rates of NAbs were 21.7%, 14.1%, 28.2%, 25.4%, 27.9% and 20.7% in age groups of 0-5 y, 5-14 y, 15-25 y, 26-45 y, 46-60 y and > 60 y, respectively. In age groups of >5-14 y and 15-25 y, the positive rate of NAbs showed significantly different (χ²=4.68454, P=0.03044). Higher geometric mean titers were found in 15-25 y group compared to 5-14 y age groups (P=0.0313). Conclusions The low level of HRVA21 NAbs in different age groups indicated a low epidemic of HRV21 infection and high susceptability to infections by the virus.